UNIVERSITY OF HAWAI'I LIBRARY

CHARACTERIZATION OF SENESCENCE REGULATEDGENE EXPRESSSION IN ANTHURIUM

A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THEUNIVERSITY OF HAWAI'I IN PARTIAL FULFILLMENT OF THE

REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

IN

MOLECULAR BIOSCIENCES AND BIOENGINEERING

MAY 2003

By

Daniel Maida Hayden

Dissertation Committee:

David Christopher, ChairpersonDulal Borthakur

John HuMonto Kumagai

Wei-Wen Winston SuRichard Manshardt

©Daniel Maida Hayden 2003

ALL RIGHTS RESERVED

iii

Dedicated to an thosewho listen to myaspirations,

those who have helpedme reach for them,and those in the future

who will assist me in completing them

!V

ABSTRACT

Leaf senescence is a genetically regulated and orderly series of events signifying

the final stage of leaf development prior to organ death. During senescence, the loss of

photosynthesis is associated with the breakdown of chlorophyll and the disassembly of the

photosynthetic apparatus. Cell structure changes, organelles are disassembled and proteins,

lipids, nucleic acids and carbohydrates are hydrolyzed while the nutrients released by

hydrolysis are transported and recycled to growing tissues or are stored in reserve for latter

use. The factors regulating leaf senescence are complex and involve both external and

internal stimuli, such as darkness, pathogens, developmental signals and hormones. These

factors initiate signal transduction pathways that repress photosynthesis and activate the

expression of genes involved in cellular disassembly, recycling and integral defense-related

processes. The genes for mRNAs that increase markedly in abundance during senescence

have been designated as "senescence-associated genes" or SAGs.

This work characterizes senescence inAnthurium andraeanum through expression

analysis of theArabidopsis senescence-upreguiated promoter SAG12, isolation of a new

senescence-associated gene (anthl7), and expression profiling under dark induced and age

dependent senescence. Analysis of Pr-SAG1zrevealed a 20 fold increase in expression

when transiently bombarded into senescent leaves vs. healthy leaves. This shows the

senescence-dependent activation of the Arabidopsis Pr-SAG1Z in Anthurium implying the

existence of an orthologous senescence-regulated system with associated genes, signal

v

transduction pathways, and transcription factors in this plant. Two cysteine protease

cDNAs from Anthurium, anth16 and anth17, were sequenced and found to be expressed

differently throughout development and differ structurally in comparison with known

cysteine protease genes. These cDNAs are the first to be cloned and characterized from

this plant. Anth16 is expressed in immature leaves and is structurally similar to a thiol

protease sequenced from Matricaria chamomilla, possibly defining an alternate role in

development for these proteases. Anth17 is characterized as a senescence-associated gene

based on Northern blot analysis of developmental leaf stages in comparison with psbA, and

cab genes as indicators of photosynthetic gene expression. Furthermore, cytokinin

treatments decreased significantly anth17 mRNA levels during dark-induced senescence of

dark detached whole leaves, and leaf discs.

V1

TABLE OF CONTENTS

ABSTRACT v

LIST OF TABLES x

LIST OF FIGURES xi

LIST OF ABBREVIATIONS xiii

CHAPTER I. INTRODUCTION 1

Current Research Involving Senescence Gene Expression 2

Classification of senescence associated genes 2

Typical senescence up-regulated genes 4

Genes involved with protein degradation 8

Hormones Involved in the Regulation of Senescence 11

Honnones that accelerate senescence 11

Honnones that decrease or delay senescence 14

Specific roles of cytokinins in plant development 15

Anthurium Molecular Physiology 17

Anthutium senescence biochemistry 18

Anthutium flower senescence control 20

Autoregulatory Senescence-Inhibition System 21

CHAPTER II. SIGNIFIGANCE OF RESEARCH. 26

The Senescence-Activated Signaling Pathway That Activates the DieotPromoter, SAG12, May Be Evolutionarily Conserved In Anthurium 28

Expression and Proper Post-Translational Modification of GFP inAnthurium, Can Provide A Necessary Reporter Gene Tool ForAnthurium Molecular Studies 30

v11

A Senescence-Regulated Cysteine Protease Gene, Orthologous to sagl2,Exists In Anthurium 31

The Effect of Cytokinin and Sucrose on SAG Expression DuringSenescence 33

CHAPTER III. REGULATION OF THE SAG12 PROMOTER INANTHURIUM USING TRANSIENT BIOLISTIC

PARTICLE BOMBARDMENT 36

Introduction 36

Materials and Methods 38

Construction of Pr-SAGl2 reporter vectors 39

Particle bombardment of Anthurium leaves 41

GUS detennination 41

Visualization and detection of GFP 42

Results 43

Pr-SAG12 transient bombardment assays for expression of GFP 43

Discussion 46

CHAPTER IV. ISOLATION AND CHARACTERIZATION OF TWOCYSTEINE PROTEASE GENES FROMANTHURIUM AND EXPRESSION PROFILINGDURING DEVELOPMENT AND IN RESPONSE TOHORMONE TREATMENTS 54

Introduction 54

Materials and Methods 57

Plant material and growth conditions 57

RNA isolation 57

Hormone treatments and chlorophyll detennination 58

viii

RT-PCR, RACE, cDNA library construction, screening and cDNAcharacterization 58

Nortbem blot analysis, probe construction, and band quantification 60

Results 61

Isolation and characterization of two cDNAs encoding cysteineproteases from Anthuriwn 61

Synthetic cytokinin, BA, represses the expression of anth 17 68

Discussion 71

RNA isolation and manipulation 71

Analysis of Anth16 and Anth17 protein structure 76

Expression analysis of cysteine proteases during development 82

Expression analysis of anth 17 mRNA under dark induced senescenceand hormone treatment 84

CHAPTER V. CONCLUSION AND PROSPECTIVE 90

Establishing Molecular Techniques to Deal withRecalcitrant Tropical Species 90

Contribution to the Fields of Post-Harvest Physiology and MolecularBiology of Leaf Senescence 92

Cysteine Proteases and Their Role in Commercial and IndustrialApplications 93

CHAPTER VI. LITERATURE CITED 96

IX

LIST OF TABLES

TABLE PAGE

1.1 Cysteine protease genes that are characterized asbeing senescence up-regulated 24

3.1 Quantitation of PSAGldJP4 and p35S-gjp4expression in senescent and healthy tissues ..46

4.1 Percent identity and similarity (in parenthesis)between the deduced amino acid sequences ofcysteine proteases from 8 plant species and withanth16 and anth/7 fromAnthutium andraeanum 65

x

LIST OF FIGURES

FIGURE PAGE

3.1 P,,,,12-GFP4 in pBLUESCRIPT II KS ..40

3.2 P,,,,12-IPT in a binary vectot.. ..40

3.3 P"glZ-IPT in pUC18 termed pSGS16 40

3.4 pBIN 3SS-mGFP4 40

3.5 GFP in senescing leaf tissues expressed with Pr"g12 .44

3.6 GFP in senescing leaf tissues expressed with Pr35S 44

3.7 GFP in senescing leaf tissues expressed with Pr3SS(lower magnification) 44

3.8 Transient expression of GFP4 and GFPS asregulated by Pr-SAG12 and Pr-3SS in tissue cultureleaf samples ofvarious developmental stages .45

3.9 Comparison of the FITC and wtGFP filter cubeswith wound response to Pr-SAG12 in healthy tissues ..46

4.1 Nucleotide and deduced amino acid sequence ofanthl6 62

4.2 Nucleotide and deduced amino acid sequence ofanthI7 63

4.3 Alignment of amino acid sequences derived fromanth16 and anth17 with those from 8 differentplant proteases 64

4.4 Expression of anthl7, anthl6, cab, psbA, and ubimRNA in leaves at different developmental stages 67

4.5 Expression of anth17 in comparison withphotosynthetic transcript psbA under cytokinintreatments using dark detached whole leaves 69

Xl

4.6 Expression of anth/7 in comparison withphotosynthetic transcript psbA under cytokinintreatments using dark detatched leaf discs 70

4.7 10 I-'ll of Total RNA from developmental leavesloaded on a 1.5% Formaldehyde MOPS gel andvisualized with EtBr 75

4.8 5 f'l of Total RNA from leaf disc treatments loadedon a 1.5% Formaldehyde MOPS gel and visualizedwith EtBr 76

4.9 Hydropathy profile of the deduced amino acidsequences of Anth16 and Anth17 predicted usingthe Kyle and Doolitde method 78

Xll

ABA

ACC

ADP

Ala

anth/6

anth/7

Asn

Asp

ATP

BA

bp

cab

cDNA

ChI

COOH

Cys

DLU

DMF

DNA

ECM

ER

EtBr

EXT

FITC

GAL

GCG

LIST OF ABBREVIATIONS

Absorbance at 260 run

Absorbance at 280 nm

Abscisic Acid

aminocyclopropane carboxylate

adenosine di-phosphate

alanine

Anthurium cysteine protease gene

Anthunum SAG cysteine protease gene

asparagme

aspartic acid

adenosine tri-phosphate

benzyladenine

base pair

Chlorophyll AlB binding protein gene

complementary DNA

chlorophyll

carboxyl terminus

cysteine

digital light unit

dimethyl formamide

deoxyribonucleic acid

extracellular matrix

endoplasmic reticulum

ethidium bromide

endoxyloglucan transferases

fluorescein isothiocyanate

~-galactosidase

genetics computer group

XI11

GENBANK

GFP

Gly

GPI

GST

GUS

His

HPPD

IPT

JAKB

Luc

Lue

Met

MG

MOPS

mRNA

NH

nos

PCD

PCR

PDS

PEP

Pr-3SS

Pr-ANTH17

Pro

Pr-SAG12

psbA

PVP

PVPP

RACE

NIH genetic sequence database

green fluorescent prorein

glycine

glycosylphosphatidylinositol

glutathione S-transferase

~-glucuronidase

histidine

4-hydroxyphenylpyruvate dioxygenase

isopentyl transferase

jasmonic acid

kilobase

Iuciferase

luecine

methionine

mature green leaf

3-(N-Morpholino)-propanesulfonic acid

messenger RNA

amino terminus

nopaline synthase

programmed cell death

polymerase chain reaction

particle delivery system

phosphoenOlpyruvate

cauliflower mosaic 3SS viral promoter

anth/7 promoter

proline

sag!2 promoter

Dl protein of PSI!

Polyvinylpyrrolidone

Polyvinylpolypyrrolidone

Rapid amplification of cDNA endsXlv

REDOX

RNA

rpPCR

RT-PCR

RUBISCO

S1

S2

S3

SAG

SARI<.

SDS

Ser

T-SrAR

Tyr

~E

ubi

USDA

UV

Val

wrGFP

X-glue

YG

oxidation reduction

ribonucleic acid

reverse primer PCR

reverse transcription PCR

ribulose bisphosphate carboxylase/oxygenase

senescence stage one

senescence stage two

senescence stage three

senescence-associated gene

senescence associated receptor-like protein kinase

sodium dodecyl sulfate

senne

Tropical & Subtropical Agricultural Research

Tyrosine

micro Einstein

ubiquitin gene

United States Department ofAgriculture

Ultra Violet

Valine

wild type GFP

S-bromo-4-chloro-3-indoyl glucuronide

young green (immature)

xv

CHAPTER I

INTRODUCTION

Senescence is a complex, highly controlled developmental phase in the final stage

of the leaf life cycle that results in the coordinated degradation of macromolecules and

the subsequent mobilization of components to other parts of the plant. Cells remain

viable during the process and new gene expression is required. Physiological studies

show that the cell structure changes, chloroplasts break down, but mitochondria remain

intact until late senescence. Biochemical studies show an increase in pH, metabolic

enzyme activities, the degradation of chlorophylls and the synthesis of anthocyanins and

phenolics. Molecular studies show an increase in the expression of genes encoding

enzymes for protein degradation (proteases), nitrogen remobilization, lipid and

carbohydrate metabolism, and nucleic acid degradation (nucleases). While these genes

are enhanced during senescence, the expression of photosynthesis genes that encode light

harvesting components and Calvin cycle enzymes, are decreased. Analysis of gene

expression during distinct developmental stages shows a concise pattern of gene

regulation enabling us to predict the function of the proteins produced and their

importance to the life cycle of the plant.

1

Current Research Involving Senescence Gene Expression

Classification ofsenescence associated genes

Senescence in plants can be stimulated by a variety of environmental and genetic

controls, such as development, wounding, heat stress, light stress, salt stress, and

pathogen attack. It is also coordinated on a molecular level by natural ripening responses

(ethylene), or nutrient relocation involved in seed production, nutrient storage, flower

production, changes in source-sink relationships, and the removal of leaves that no longer

receive sufficient light. Natural senescence includes genes that influence protein

degradation, nucleic acid breakdown, lipid remobilization, chlorophyll breakdown, and

nitrogen remobilization. Several reviews of leaf senescence, (Sancher 1973; Beevers

1976; Thimann 1980; Thompson and Platt-Aloia 1980; Biswal and Biswal 1984; Sexton

and Woolhouse 1984; Kelly and Davies 1988; Matile 1992; Lohman et al. 1994; Smart

1994; Buchanan-Wollaston and Ainsworth 1997; Nooden et at. 1997; Lee et at. 2001)

describe the leaf senescence of cereal, tobacco, some legumes, and a few flower model

systems. Leaf senescence has been extensively studied, but its mechanisms are complex

and consist of multiple pathways. By analyzing gene expression under natural and

induced conditions, we can gain a better understanding of the role of each gene in leaf

development.

Gene expression in leaf senescence is defined by a number of classes labeled

senescence activated genes (SAGs) (Smart 1994). There are 6 separate classes of genes

characterized by their pattern of expression and by the function of the proteins produced

during plant leaf senescence. Four additional gene classes have been added as a result of

SAG expression analysis during senescence in Brassica napus (Buchanan-Wollaston

2

1994). Class 1 contains "housekeeping" genes that control essential metabolic activities

of the cell and are expressed at a constant level throughout the life of the leaf. Classes 2

and 3 are genes expressed in green leaves, but their effect is seen at a later stage. Class 2

involves proteins encoded during leaf growth which are activated during senescence long

after their gene is turned off. Class 3 genes are also turned off and their inactivation

causes the initiation of senescence. These genes encode growth and carbon assimilation

components. Class 6 genes encode proteins that remobilize storage products. These

genes are also expressed during other developmental stages.

Further research on Brassica napus gene expression has identified 4 more classes

of genes with SAG functions (Buchanan-Wollaston and Ainsworth 1997). Class 7 genes

include genes that show a gradual increase in mRNA levels, from low expression in

young leaves, to a high level of expression increasing throughout senescence. Class 8

genes are similar to class 7, but differ in the respect that mRNA levels are low in early

stages of development, but increase dramatically at a particular stage of senescence.

Class 9 genes are expressed at a specific stage in senescence, but unlike class 5 genes,

their expression does not continue throughout late senescence. Class 10, the final class,

is a set of genes that show strong expression in early leaf development and again during

senescence

Classes 4 and 5 genes have some involvement in senescence and their expression

can be identified during a senescence inducing, or repressing event. Class 4 type genes

include regulatory genes expressed for a short period of time at the initiation of

senescence and control its timing and rate of progress. Class 5 genes are involved with

mobilization processes specifically expressed during senescence. SAG genes include

3

genes involved in defense, sugar remobilization, amino acid metabolism, fatty acid

metabolism, oxidation protection, sequesterization and transport of molecules and

macromolecules, cell wall disassembly, and phytohormone production. It is beyond the

scope of this report to list every gene involved with the coordinated process of recycling

cell components for export to healthy tissues, however some genes that have been

characterized through transcript analysis as being senescence-associated will be

discussed.

Typical senescence up-regulated genes

Chitinases are important for the degradation of chitin, the most abundant organic

nitrogen-bearing compound in nature. Chitin is most common in insects and in fungal cell

walls, but is also found in many other organisms. An ethylene-induced chitin-binding

protein has been purified from rose leaves (Yang and Gong 2002). It is possible that a

senescent tissue synthesizes proteins to defend against pathogenic attack while in a

weakened senescent state, but may also synthesis chitinases to degrade cell walls or be

involved in signal transduction by acting on endogenous substrates (Hanfrey et al. 1996).

Chitinases commonly induced during pathogenic attack have been found to be inducible

during natural and artificial leaf senescence further implicating pathogen-related proteins

role in the senescence process (Lers et al. 1998).

Pyruvate a-phosphate dikinase catalyzes the reversible conversion of ATP to

AMP, pyrophosphate and phosphoenolpyruvate (PEP). Its potential role in senescence

may involve converting the pyruvate produced from amino acid breakdown into PEP

(Smart et aI. 1995). PEP is also an intermediate in gluconeogenesis, active cycle in a

4

senescing leaf, turning lipids and photosynthetic products into sucrose for use in

respiration or export from the senescing leaf. Many genes implicated in lipid metabolism

have been characterized as SAGs, including an in-chain fatty acid hydroxylase bound to

cytochrome P450 (panavas et al. 1999). Interestingly enough, this hydroxylase is also

up-regulated in the presence of certain herbicides and MnCh (Cabello-Hurtado et al.

1998). Cytochorome P450 includes a class of key enzymes involved in the synthesis of a

large variety of secondary plant metabolites including flavonoids, phytoalexins and lignin

(Butt and Lamb 1981). The role of these enzymes in plant senescence is not understood.

Metallothioneins are small proteins that bind heavy metals such as zinc, copper,

and magnesium, through clusters of thiolate bonds. During senescence it may be

required to bind metal ions as either a detoxification role or a sequestering role for

storage or mobilization. The metallothioneins may also protect DNA from damaging

oxidative effects caused by chlorophyll and membrane breakdown and subsequent free

radical production (Chubatsu and Meneghini 1993). A metallothionein gene is shown to

be upreguiated during senescence (Buchanan-Wollaston 1994) and also shows some

sensivity to pathogenic attack (Butt et al. 1998).

Many genes SAGs are involved in the modification and mobilization of amino

acids. Genes that encode enzymes aminotransferase, branched-chain a-keto

dehydrogenase, and ~-methylcrotonyl-CoA carboxylase for amino acid metabolism and

4-hydroxyphenylpyruvate dioxygenase (HPPD) for aromatic amino acid degradation are

upregulated at some point during senescence (Lee et aI. 2001). The function of

glutathione S-transferase (GST) is the conjugation of reduced glutathione to a wide

number of exogenous and endogenous hydrophobic electrophiles. GST could also have a

5

role in sulfur storage and mobilization released during macromolecule degradation

(Smart et al. 1995). GST also retains a detoxification role with herbicides and its

senescence-related expression may be related to stress. Glutamine synthase has many

functions and isoforms located in the cytosolic and chloroplastic regions of the cell. A

glutamine synthase like protein has been shown to be senescence upregulated in Brassica

napus (Buchanan-Wollaston and Ainsworth 1997). This coincides with the need to

transport nitrogen in the form of glutamine from senescing cells (Feller and Fischer

1994). Many genes that involve the recycling amino acids are upregulated during

senescence to provide easy transport to sinks located in other parts of the plant.

Endoxyloglucan transferases (EXTs) have two distinct activities, the endo-type

splitting of xyloglucans and transglycosylation. These processes are suggested to modify

a major hemicellulose of the cell wall (Okazawa et al. 1993; Xu et al. 1996). Due to their

expression during senescence, it is considered that the EXTs carry out hydrolysis of

xyloglucan rather than transglycosylation, and is involved in cell wall degradation (Park

et al. 1998).

ACC oxidase is involved with the biosynthetic pathway of a common senescence­

inducing hormone, ethylene. Ethylene up-regulates its own production during senescence

but also is implicating in up-regulating genes such as a calmodulin-binding protein,

(Yang and Poovaiah 2000) a lipolytic acyl hydrolase, (Hong et al. 2000) and a

glutathione S-transferase (Meyer et al. 1991). Most genes that are ethylene-induced

during senescence were isolated from flowers such as rose, carnation, and daylily.

Another phytohormone related gene induced during senescence is involved with signal

transduction. A senescence associated receptor-like protein kinase (SARK) cloned from

6

Phaseolous vulgaris shows up-regulation slightly before senescence begins and is present

throughout (Hajouj et al. 2000). This may suggest that SARK expression may regulate

some pathways of the senescence program.

Genes that exhibit SAG characteristics are identified through Northern Blot

analysis using cDNA clones to detect increased mRNA transcription levels in senescing

leaf tissue. In order to identify the precise time during leaf senescence at which the

expression of a specific gene is induced, it is essential to characterize the biochemical and

physiological changes that map each stage during leaf development and senescence

(Buchanan-Wollaston and Ainsworth 1997). Some SAGs are expressed in pre-senescent

tissue and their expression simply increases during senescence, while other SAGs are

more specific to senescence and are expressed only during senescence. This indicates the

presence of multiple pathways in the regulation of senescence (Gan and Amasino 1995;

Weaver et al. 2001). Based on this analysis, senescence includes pathways that are

exclusively activated by developmental, environmental, or hormonal stimuli (Noh and

Amasino 1999a). In order to gain a complete understanding of a particular gene and its

senescence associated expression, we must expose the tissue to several experimental

treatments. Through this method we are able to establish a consistent measure of SAG

expression throughout the life of the plant, under hormonal control, and in response to

stress conditions such as desiccation, abscission, and darkness. This enables the proper

characterization of gene function and contributes to the identification of a variety of

senescence-related promoters.

7

Genes involved with protein degradation

Senescence activated protein degradation is the most significant breakdown

process that occurs in an aging leaf. The degradation of proteins to amino acids allows

them to be recycled for use in other tissues. This remobilization of amino acids from

senescing tissues is essential in the formation of new organs in other areas of the plant.

Identification of key enzymes involved in senescence is difficult due to the high

concentration already present in the vacuole of the developing leaf. Therefore, the

identification of genes involved in senescence-enhanced protein degradation is

particularly important and gives insight into the biochemical and physiological changes

in the plant on a molecular level. Many genes encoding proteases, whose expression is

enhanced in senescence, have been isolated and characterized including aspartic

proteases and cysteine proteases.

The LSC760 aspartic protease gene of B. napus is expressed in young leaves and

mature green leaves, but expression increased markedly during senescence (Buchanan­

Wollaston and Ainsworth 1997). Cysteine proteases have been isolated from a variety of

species and their genes vary in expression during senescence. Oryzain y protease from

rice is a seed-specific protease that is expressed in germinating seeds. It functions in the

remobilization of storage proteins to supply developing seedlings (Watanabe and Imasek

1982). This gene may perform a similar function in senescing leaves. The levels of other

cysteine proteases are enhanced in senescing leaves. The sag12 gene, from Arabidopsis,

encodes a similar protein sequence to papain-like proteases and is specifically expressed

during the onset of senescence in leaves (Lohman et al. 1994; Gan and Amasino 1995).

A third type of cysteine protease from B. napus is expressed at high levels in young green

8

leaves, decreases in mature leaves, and increases significantly in senescing tissues

(Buchanan-WOllaston and Ainsworth 1997). This gene is expressed in all developmental

stages in all plant organs and has a high homology to a drought-induced cysteine protease

in Arabidopsis (Koizumi et al. 1993).

Many cysteine proteases, active in both developmental and senescence stages of

plant organs, have been isolated and characterized in a variety of different species.

Protein degradation and remobilization during senescence involves a complex set of

proteases that differ in organelle location and level of expression. This regulation may be

so complex that it includes protease inhibitors that act to repress proteases until the

precise stage. Chloroplast proteases are extremely important in the remobilization of

stored nitrogen to other parts of the plant. The chloroplast is considered to be an

important storage organelle, as 70%-80% of the nitrogen in mature leaves is located here.

Roughly 90% of the nitrogen exported from senescing leaves comes from the

chloroplasts (Morita 1980). Nuclear expressed genes encoding chloroplast-targeted

proteases have been isolated (Mitsuhashi and Feller 1992) but senescence-specific

protease activity involved with the dismantling and transport of the photosynthetic

apparatus has yet to be characterized. A senescence-enhanced gene isolated from maize

appears to encode a protein similar to a vacuolar enzyme present in castor bean seeds,

which functions in the conversion of vacuolar proteins to their mature forms (Smart et al.

1995). This enzyme is up-regulated during senescence and may indicate that some

enzymes involved in senescence are expressed during leaf development and stored in the

vacuole where they await processing into a functional enzyme during the onset of

senescence.

9

The ubiquitin pathway for targeted protein degradation is important in the

elimination of abnormal cytosolic proteins and also the rapid turnover of short-lived

proteins (Buchanan-Wollaston and Ainsworth 1997). Proteins destined for degradation

are 'tagged' by ubiquitination in a series of ordered events and require ubiquitin­

activating enzymes. These multi-ubiquitinated complexes are then recognized by ATP­

dependent proteases which degrade the target proteins (Rechsteiner 1991). Although it

appears that most of the proteases in the ubiquitin pathway are not influenced by

senescence, an ubiquitin carrier protein gene has been identified showing enhanced

expression in senescing leaves of the potato (Garbarino et al. 1995). The SEN3 gene of

arabidopsis, a poly-ubiquitin encoding gene, is also upregulated in senescing leaves

induced by age-dependent, dark detachment, light detachment, and phytohormone

treatment methods (Park et al. 1998). This may indicate that the ubiquitin-dependent

degradation of proteins does occur during senescence, but probably directed toward

specific cytosolic proteins (Buchanan-Wollaston and Ainsworth 1997). It has also been

shown that proteins that promote leaf longevity are targeted for degradation during

senescence by ORE9, an pobox protein, which may form a complex with its substrate for

subsequent ubiquitin tagging (yVoo et al. 2001).

Although numerous research studies have been performed on isolating,

sequencing, and characterizing protease genes with increased expression during

senescence, little is known about the exact locations of the synthesis of protease proteins.

RNA levels measured during developmental stages in plant organs are crucial in

characterizing senescence-activated genes. This is seen with a Northern Blot of mRNA

transcripts throughout the plants life cycle and through later stages of necrosis. However,

10

the exact location of where protein degradation takes place and how the encoded proteins

function remain to be demonstrated.

Hormones Involved in the Regulation of Senescence

Many hormones are involved in growth, development, nutrient translocation and

eventual!y senescence. Al though senescence is a complex molecular and biochemical

process, it appears to be initiated by changes in localized hormone concentrations.

Numerous environmental switches have been observed to induce organ senescence

including, temperature, light, wounding, pathogen attack, drought, and inadequate

nutrients. Most of these factors occur with less frequency when compared with hormone­

controlled age-dependent senescence. Information about the exact biochemical and

molecular effects of hormones is incomplete, but current research demonstrates that

hormones delay or interrupt senescence, accelerate senescence, or trigger it by a decline

in concentration, or location.

Hormones that accelerate senescence

Ethylene, abscisic acid (ABA), and jasmonic acid can induce senescence.

Ethylene is shown to increase senescence dramatically and coincides with the rapid phase

of chlorophyll degradation and increased respiration in dicots (Aharoni and Richmond

1978). Ethylene also plays a key role in the progression of senescence in some species.

Transgenic tomato containing an anti-sense gene leading to a reduction of

polygalacturonase mRNA, showed a delayed senescence period by one week through

11

inhibition of ethylene biosynthesis (Picton et al. 1993). An ethylene-insensitive mutant

of arabidopsis, compared with wild type, had a higher level of gene expression of

photosynthetic-associated genes, while the expression of senescence-activated genes was

delayed (Grbic and Bleecker 1995). The current proposal is that ethylene hastens the

progress of senescence by activating senescence-associated genes while repressing

photosynthesis-associated genes (Smart et al. 1995) thereby promoting membrane

breakdown, loss of chlorophyll, and positive feedback responses. These responses create

more ethylene that leads to a more rapid effect.

Ethylene induces the expression of genes, such as those encoding a lipase (Hong

et al. 2000) and a calmodulin-binding protein, (Yang and Poovaiah 2000) which are

identified as triggers of senescence. It has been demonstrated that protein

phosphorylation via protein kinase up-regulates ethylene production, ethylene

biosynthetic gene expression, and senescence acceleration in orchid flowers, while

serine/threonine protein phosphates reverse the processes (Wang et al. 2001). The

production of ethylene is primarily associated with cut flowers, fruits, and vegetables.

Flowers that exhibit ethylene induced expression show increased transcription of

aminocyclopropane carboxylate (ACe) and ACC oxidase genes, which encode enzymes

involved in ethylene biosynthesis. Most flowers that exhibit ethylene inducible gene

expression are dicots such as carnations, (Roberts et al. 1983) geranium, (Clark et al.

1997) and roses. Monocot flowers, such as anthuriums, iris, lily, and orchids, display

little sensitivity to ethylene. Anthurium flowers are sensitive to lowered levels of

cytokinins and daylily petals experience a rapid increase in abscisic acid content,

contributing to the initiation of senescence.

12

Abscisic Acid (ABA) accelerates senescence in a wide variety of species but is

less effective when applied to attached leaves, except at relatively high levels (50-100mg)

(El-Antably and Wareing 1967). The endogenous level of ABA increases in early stages

of senescence of detached tobacco leaves and declines rapidly later into the process

(Even-Chen and Atsmon 1978). The ethylene-insensitive daylily flower is very sensitive

to endogenous ABA application. ABA accelerates petal death and prematurely induces

many of the same biochemical changes that occur during natural senescence, such as loss

of differential membrane permeability, increase in lipid peroxidation, and increase in

activity of proteases and nucleases (Panavas et al. 1999). Much of ABAs role in

senescence is unknown. ABA in leaves increases when plants are exposed to stresses

such as drought, low temperature, and high salt concentrations.

The senescence-accelerating hormone jasmonic acid (JA), has been shown to

promote senescence when applied to excised oat leaves (Veda and Kato 1981). It

promotes senescence by decreasing the expression of nuclear and chloroplast genes

involved in photosynthesis (Creelman and Mullet 1997). Jasmonates appear to be

derived from fatty acid biosynthesis, (Vick 1983) primarily lipoxygenase generation. The

proteins stimulated by jasmonates degrade and transport vegetative storage proteins

(Anderson and Spilatro 1989) and lipids. Jasmonates increase in concentration during

wounding (Farmer and Ryan 1990), fungal, and bacterial pathogenic attacks (Anderson

and Spilatro 1989). Although these reports suggest jasmonic acid is only involved with

senescence, JA is also found in high levels in zones of cell division, young leaves, and

reproductive structures (Creelman and Mullet 1997). One hypothesis is that JA works by

decreasing or inhibiting the expression of photosynthetic genes. This is important in

13

vascular areas of the plant where the photosynthetic apparatus is not needed, and during

senescence where photosynthetic nuclear and chloroplast genes are down-regulated. JA

may also be induced by the energy quenching mechanisms of the chloroplasts to down­

regulate chlorophyll genes. In effect, the down-regulation of chlorophyll decreases the

excess energy absorbed and prevents further photochemical damage (Creelman and

Mullet 1997).

Genes involved with JA biosynthesis include the plastid-derived lipoxygenase:

LOX2, LOX3, LOX4, and !be cytoplasmic LOX]. LOX2 plays a role in wounding and

defense related senescence (Creelman and Mullet 1997) but is unlikely to be related to JA

biosynthesis during senescence because its expression is turned off at the onset of leaf

senescence (He et at. 2002). In contrast, LOX] is strongly activated during leaf

senescence and is likely responsible for the increased JA production in senescing leaves

(He et at. 2002). There appears to be a strong evidence to support a role for JA in

senescence. An exogenous application of JA induces leaf senescence and causes an

increase in endogenous JA levels of senescing tissues to nearly 500% of those found in a

non-senescing counterpart. It is also important to note that the JA biosynthetic pathway

in senescing leaves is mediated by genes other than !bose involved in the wounding and

defense-related JA biosynthetic pathways involving chloroplast-targeted enzymes.

Hormones that decrease or delay senescence

Cytokinins, and to some extent gibberellins and auxins are able to retard

senescence. This section will detail the role of these hormones in senescence. Little is

14

known about the role of gibberellins in the plant life cycle. Gibberellins are involved in a

wide range of functions that vary among species. In one case, the application of

gibberellins to leaves strongly inhibited senescence where cytokinins had little effect

(Fletcher and Osbourn 1966). In other species both seem to work effectively and show

that senescence is coupled with a decrease in gibberellin concentration at the site.

Gibberellins cause an increased of gene expression in stem elongation (Chory and Voytas

1987) and remobilization of nutrients during seed germination (Baulcombe and BlIffard

1983). However, the effect of gibberellins on senescence-related gene expression has yet

to be determined.

Auxins are another class of hormones that have various functions in diverse

species. Although some reports indicate that they may delay senescence, there are

numerous cases in which high concentrations of auxins are needed to retard senescence.

Studies suggest auxins are not very powerful in suppressing senescence and high

concentrations have to be used in comparison with cytokinins (Smart 1994). One

complication is that auxins stimulate the production of ethylene when the concentration is

only slightly above the physiological level (Thimann 1980). The production of ethylene

accelerates senescence producing the opposite reaction desired. Auxin research shows a

wide range of results in various species; therefore, it is extremely difficult to form

universal generalizations about the role of auxins in the inhibition of senescence.

Specific roles ofcytokinins in plant development

Cytokinins are very important in the healthy development and growth of young

and mature plant organs. Cytokinins have been shown to prevent the natural senescence

15

of leaves or in some cases promote the re-greening of leaves (Van Staden 1988). During

the senescence of a leaf, the tissue has no ability to generate cytokinins whereas young

leaves have the ability to synthesize and accumulate cytokinins. Without the source of

cytokinins, a leaf will begin to go through its natural senescence program and recycle its

nutrients to supplement the generation of healthy and developing plant organs.

Therefore, it has been concluded that if the proper level of cytokinins are made available

to a senescing leaf, its senescence program will be interrupted indefinitely or delayed

through a number of molecular and metabolic processes (Gan and Amasino 1995).

Current research has shown that exogenous cytokinin application to senescing leaves

delays chlorophyll breakdown, stimulates protein and mRNA synthesis, and stimulates

chlorophyll synthesis. Cytokinins prevent the transient increase in respiratory rate seen in

senescence (Tetley and Thimann 1974).

Cytokinins seem to operate at both the transcriptional and post-translational level.

The presence of cytokinins inhibit the expression of senescence-related genes (Teramoto

et al. 1995; Buchanan-Wollaston and Ainsworth 1997) and contribute to the activation of

developmental genes (Flores and Tobin 1988; Chen et al. 1993). Cytokinin modulates

genes and proteins by a variety of unknown signaling mechanisms. One postulated

cytokinin signal transduction mechanism could be mediated by a histidine kinase

analogous to two of the component regulators (Kakimoto 1996). It can also be elucidated

that cytokinin regulates protein kinases/phosphatases which contribute to altering

transcription factors, nucleic acid binding proteins, and mature proteins.

The tissue and organ-specific overproduction of cytokinins produced a number of

morphological and physiological changes, including stunting, loss of apical dominance,

16

and reduction in root initiation and growth. Abnormal cytokinin concentrations can

accelerate or prolong senescence in leaves depending on the growth conditions, initiate

adventitious shoot formation from unwounded leaf veins and petioles, alter nutrient

distribution and abnormal tissue development in stems (Li et al. 1992). While some of

these morphological changes result directly from the localized overproduction of

cytokinins, other changes probably result from the mobilization of plant nutrients to

tissues rich in cytokinins. Therefore a system that reduces senescence using cytokinin

concentrations must be engineered to regulate an effective concentration of cytokinin to a

specific location. This method of regulation is crucial in the development of a senescence

inhibition system.

Anthurium Molecular Physiology

Anthurium andraeanum, is one of the Hawaiian islands' principal ornamental

exports to the U.S. mainland, Canada, Japan, Italy, Germany and many other countries.

The anthurium belongs to the family Araceae, a family chiefly of tropical plants, and is a

perennial herbaceous plant usually cultivated for its attractive, long-lasting flowers

(Higaki et al. 1979). The anthurium plant consists of many different species which all

produce a flower that is a complex of a modified leaf (spathe) and hundreds of tiny

flowers on the pencil-like protrusion (spadix) rising from the base of the spathe. The

anthurium plant produces flowers that emerge from each new leaf axil, and follow a

sequence of leaf-flower-Ieaf year-round throughout the entire life of the plant. Intervals

between leaf emergences are shortened or lengthened depending on environmental

conditions and usually grow more vigorously in the summer months (Higaki et al. 1979).17

The anthurium flower is usually harvested when three-quarters mature and the cut flower

will last from 7-10 days.

Anthurium senescence biochemistry

To establish a connection between physiological changes during senescence of

anthurium flowers and a molecular explanation, data concerning physiological attributes

of anthurium flowers (paull et al. 1985) was analyzed and correlated with known

molecular data on senescence. The first indication of senescence in anthurium flowers is

marked by a 250% increase in respiration 8-12 days after harvest preceded by the first

noticeable bluing of the spathe (Paull et al. 1985). A respiration increase during

senescence is a molecular flag that identifies the transition from photosynthetic energy

production, to sugar respiration involving the mitochondria that is functional until late

senescence. Enzymes produced in early senescence include two key enzymes, isocitrate

lyase and malate synthase, involved in the glyoxylic acid cycle. These enzymes drive

sugar respiration to enable several processes of senescence, including nutrient

remobilization (Gut and Matile 1988). This respiration increase is paralleled with a

decrease in photosynthetic enzyme production including ribulose bisphosphate

carboxylase (RUBISCO) and components of the light harvesting complex (Grover 1993).

Basic preparations involved in chloroplast breakdown. A gradual decrease in total sugars

and starch is observed during the period when respiration in the senescing anthurium

flower plateaus confirming the use of anabolic substrates in the respiration process (Paull

et al. 1985).

18

A dramatic increase of ammonium ions in senescing flowers is observed which is

paralleled with spathe bluing (Paull et al. 1985). This is because the ammonium ion is a

key ion in the isoenzyme of glutamate dehydrogenase, involved in packaging nitrogen for

transport (Lauriere and Daussant 1983). The senescing flower also shows a small

increase in alpha amino acids and proteins (Paull et al. 1985). This increase is consistent

with increased levels of amides glutamine and asparagine, which are products of protein

degradation and major organic forms of nitrogen able to be translocated in plants

(Simpson and Dalling 1981).

All physiological and biochemical changes described in senescing anthurium

flowers are consistent with known changes in the molecular physiology of senescing

plants. The ammonium ion increase is believed to react by copigmentation with

anthocyanins in senescing anthurium flowers, resulting in an increase in phenols and a

visible bluing of the spathe (Paull et aI. 1985). The blue color can also be attributed to an

increase in pH caused by the activation of proteases involved in the remobilization of

nutrients during senescence. These changes occur first in the spadix and are seen by the

early browning. Finally, flower bluing can be related to peak respiration, ammonium ion

concentration, pH elevation, and nitrogen remobilization marking the senescence of the

anthurium flower, which occurs prior to the final increase in respiration as the flower

begins to senesce rapidly (Paull et al. 1985).

Another important aspect involving anthurium flower production is the

mechanism by which the flowers are produced. Anthurium flowers and leaves are born

at a 1:1 ratio. The newly emerged immature leaf develops a negative net photosynthetic

19

rate, depriving the young flower bud nutrients for its cell division and growth phase.

Removal of the new immature leaf 7 days after emergence results in delayed senescence

of mature leaves, increased flower production, a larger flower, and an advanced rate of

flower emergence (Dai and Paull 1990). It is predicted that if this practice could be

followed in collaboration with the engineered auto-regulatory senescence inhibition

system, that the anthurium plant could be a more hearty producer of flowers. This

created by the longer lasting photosynthetically productive mature leaves supporting the

plant with energy without the immediate need for fresh leaves. This allows them to be

excised to allow for faster flower production increasing the economic value of each

individual plant. Whether or not this remains a viable option remains to be seen.

Anthurium flower senescence control

Many attempts have been made to preserve the cut anthurium flowers life beyond

the natural senescence period. These methods include temperature and fertilizer

variations during a yearly growing season (Akamine and Goo 1975). Post-harvest

treatments include pulsing stems with silver nitrate (Paull and Goo 1982), benzyl

adenine (BA-cytokinin) drip treatments (Paull and Chantrachit 2001), commercial

floral preservatives, sodium benzoate, glucose, sodium hypochlorite, and carbonated

water (Akamine and Goo 1975). After many trials, the best method for floral

preservation of cut anthurium, involves a benzyladenine drip treatment after cutting,

and a silver nitrate pulsing after shipping of the flowers. In most cases, BA drip

treatments increase flower life significantly (42%-142%) although, in one cultivar

senescence is increased (Paull and Chantrachit 2001). It is concluded that an

20

application of exogenous cytokinin (BA dip treatment), delays the onset of molecular

senescence in cut flowers, and silver nitrate pulsing, increases flower life by inhibiting

bacterial growth that leads to blocking of the stem.

Through the analysis of anthurium flower senescence, it is reasonable to deduce

that the flower functions much like other species during senescence and may have an

advantage over other ornamentals because of having a flower comprised of a modified

leaf. This includes a non-ethylene controlled flower and molecular senescent responses

similar to that of a leaf. Engineering a molecular system into anthuriums that delays

senescence by synthesizing endogenous cytokinin makes sense. Data showing delayed

senescence due to exposure with cytokinins suggest that the main method of flower

preservation is to interrupt the senescence cycle with a steady flow of regulated cytokinin

production. This type of system may decrease the time between each new flower,

maintain flower color, luster, and spadix life, or interrupt senescence of cut flowers

indefinitely, but has to be examined for its impact on all developmental phases.

Autoregulatory Senescence-Inhibition System

The discovery and identification of senescence enhanced genes and promoters

enable us to chart the progression of senescence and manipulate its interruption using

cytokinins. The construct of this auto-regulatory system consists of the senescence-

regulated SAG12 cysteine protease promoter, which is upregulated during age-dependent

senescence, transcribing the ipt gene to creating the IPT enzyme. This enzyme is the rate-

limiting factor of the cytokinin biosynthesis pathway and when available results in an21

increase of cytokinins to the senescing tissue. This construct is crucial in providing

precise amounts of cytokinin to senescing tissues to keep them healthy without exceeding

normal cytokinin levels that result in altered morphology as seen in overproduction

reports. This system is defined as autoregulatory because as the cytokinin levels in the

leaf decline, genes are switched on to initiate senescence. The PSAG12-ipt construct is now

a member of the senescence regulated genes, and is also transcribed providing the leaf

with the slow production of cytokinin that will interrupt the senescence program once it

reaches a threshold level. Cytokinin production will never reach abnormal levels since as

the threshold level is reached, senescence related genes are switched off including the

PSAG12-ipt construct. This construct will cycle in this fashion to interrupt senescence

indefinitely and the leaves will exhibit a prolonged, photosynthetically active life-span.

The system has been tested in only a few plant species. To date, the effect of ipt

expression in transgenic plants has been assessed mainly in a limited number of

solanaceous species (Gan and Amasino 1995; Jordi et al. 2000). However, there are brief

reports of the introduction of PSAG12-ipt into rice, (Fu et al. 1998) cauliflower, (Nguyen et

al. 1998) and lettuce (McCabe et al. 1998). Its introduction into tobacco resulted in more

green leaves, no sign of senescence, a two fold increase in flower and seed production

and an increase in biomass for PSAG12-ipt plants versus wild type (Gan and Amasino

1995). When introduced into lettuce, it seemed to be regulated by a positive instead of

negative feedback loop in the panicles during bolting (McCabe et al. 2001). An

interaction between high concentrations of hexoses and cytokinins in these tissues may

have repressed photosynthesis and stimulated senescence. Although this displays a

negative impact of the system on specialized tissues, the leaves of the lettuce stayed

22

green for an increased period of time post-harvest, and also allowed growers to reduce

nitrogen fertilization applied to the plant prior to harvest.

When studying the impact of delayed leaf senescence on the functioning of plants

growing under conditions of nitrogen remobilization, potential consequences of modified

sink-source relations may affect plant productivity and the efficiency of light and mineral

utilization. Expression of ipt in older leaves causes maintenance of chlorophyll content

and PPFD absorption but is less effective in delaying other aspects of senescence such as

loss of soluble protein, and still less effective in maintaining RUBISCO and net

photosynthetic energy levels (Jordi et al. 2000). Younger developing leaves in the IPT

system showed lower contents of chlorophyll, Rubisco, and protein than those leaves in

corresponding wild type plants. This is caused by the inhibition of remobilization of

nutrients from older leaves to younger leaves and may seriously limit potential increases

in biomass of these plants under limited Nitrogen nutrition (Jordi et al. 2000). It is

apparent, that at some capacity, nutrients are exported from older to younger leaves prior

to Pr-SAG12 initiation, or that remobilization is not completely inhibited by the auto­

regulatory senescence inhibition system.

Many genes are classified as senescence-associated depending only on the

increase in abundance of transcript levels during the onset and throughout senescence. A

few SAGs are characterized thorough expression analysis as being senescence­

upregulated. The expression of such genes is absent during germination, early

development and maturity but show a distinct increase at during some point during the

senescence phase. SAG12 is a cysteine protease with an expression pattern that comes

the closest to having the specificity for natural senescence. It is undetectable in younger

23

leaves and is not induced by any senescence-inducing treatment that does not cause

visible yellowing to the leaves (Weaver et al. 1998). This makes SAG12 the best

molecular marker of senescence to date and suggests a specific role for senescence-

specific cysteine proteases. The successful implementation of the SAG12 promoter in

Table 1: Cysteine protease genes that are characterized as being senescence up-regulated.

Gene nameGenbank

PlantCommon

ReferenceAccession name

SAG12 U37336Arabidopsis

Arabidopsis Lohman el. al., 1994Ihaliana

SAG12-1 AF089848 Brassica napus Rape Noh and Amasino, 1999

See1 X99936 Zeas mays Corn Griffiths el. al.,1997

See1 AJ249847Lolium

Ryegrass Li el.al., 2000mulliflorum

NTH1 U44947 Pisum sativum Garden PeaKardailsky and Brewin,1996

Tpp X66061 Pisum sativum Garden Pea Granell el. 01. 1992

NTCP-23 AB032168Nicotiana

Tobacco Ueda el.al., 2000tabacum

SENU3 Z48736Lycopersicon

Tomato Drake el.al.,1996esculentum

SmCP AF082181Solanum Eggplant

Xu and Chye, 1999melongena (Brinjal)

SPG31 AF242185 Ipomoea balalas Sweet Potato Chen et.al., 2002

SEN102 X74406 Hermocallis sp. Daylily Valpuesta et.al., 1995

BoCPS AF4S4960Brassica

Broccoli Coupe et.al. (unpublished)oleracea

ALSCYP1 AAK6312SAistroemeria

N/AWagstaff et.al.,

hybrid (Samora) (unpublished)

PRT5 AF133839Sandersonia Chinese-

Eason, (unpublished)aurantiaca lantern Iil y

the autoregulatory senescence inhibition system has lead to the pursuit of orthologous

SAG12 type cysteine proteases from a number of other species. Table 1 shows cysteine

proteases from a variety of plants that have been characterized as senescence-upregulated

through a combination of molecular techniques. Regulating cytokinin biosynthesis under

24

the control of a native promoter dedicated to expression under natural senescence is the

most preferable from an engineering standpoint. This type of promoter is less likely to be

induced by stresses, environmental pressures, and the intricacies of development thereby

establishing a system that will work as desired without adverse affects.

There is promise for this system to work in the anthurium species because the

flower is actually a modified leaf and should react as a leaf would respond when involved

with the auto-regulatory system. It must be considered that the plant is reliant on the

production of new flowers and leaves to be commercially productive and that this system

may limit the production of quality flowers, especially under nutrient deprived

conditions. However, with the proper nutrients and pruning techniques, the system may

perform better than the results seen in tobacco.

25

CHAPTER II

SIGNIFIGANCE OF RESEARCH

Senescence is relatively slow and ordered, maximizes reallocation of nutrients

and metabolites, and always has the potential to be reversible until the later stages.

Programmed Cell Death (PCD), commonly termed apoptosis or hypersensitive response,

is relatively rapid and usually reserved for responses to pathogen attack or developmental

disposal of unwanted cells. These two terms are not interchangeable, however may share

common pathways and gene expression. PCD is essential for normal reproductive and

vegetative development, such as floral development, seed maturation, tracheary element

formation, trichrome development, and root development. PCD also involves responses

to pathogenic attack, protecting the plant against systemic infection by minimizing the

pathogens opportunity to gain a foothold. Senescence on the other hand is an extremely

coordinated process that progresses in metabolically active cells.

Senescence is initiated by an environmental or developmental initiator, which

induces a hormonal response and/or signal transduction events. This coordinates gene

activation or inactivation that occurs on the transcriptional, translational, and post­

translational level. Signal transduction also includes protein modification, mobilization,

activation or inactivation. These events create a positive feedback loop, increasing the

efficiency of the senescence process, by expressing genes encoding proteins involved

with DNA and RNA binding, receptors, kinases, phosphatases, growth regulator

synthesis, and metabolite/nutrient mobilization.

26

Subsequent physiological events after the onset of senescence-associated gene

expression include a chlorophyll degradation pathway, accompanied by the unmasking or

accumulation of carotenoids and other pigments. This process can be quantified to

observe the progression of senescence through chlorophyll concentration of leaf tissue.

Proteins are broken down, and the mobilized organic nitrogen and organic sulfur are

exported from senescing leaves. Catabolism of nucleic acids releases inorganic

phosphate. Magnesium ions are captured, chelated, and mobilized as well leaving

chlorophyll inactive. Photosynthesis declines, and peroxisomes are re-differentiated into

g1yoxysomes, which convert lipids to sugars. Metabolic regulation during senescence

involves responses to cellular REDOX conditions, compartmentalization, and enzyme

specificity involving degradation. Where the great energy producing and preserving

Calvin cycle of photosynthesis once predominated, gluconeogenesis spurred into action

by senescence, now converts intermediates into sucrose through glycolysis.

Senescence involves plant molecular, physiological, and developmental aspects

that can be examined through modern techniques. This process gives us one of the best

tools to examine the complex structure of the plant cell, through its organized degradation

and remobilization. Biochemistry, enzymology, proteomics, genomics, signal

transduction, photosynthesis, gene expression, REDOX regulation, vacuolar

compartmentalization and differentiation, and growth regulators can all be successfully

studied during the slow moving senescence program. The results of this research will

expand our knowledge of cellular biology paving the way for impacting successful

changes to harness the power of the plant cell. Every piece of evidence we gather when

studying senescence, especially in unique species, is an important contribution, not only

27

to our scientific understanding of plant systems, but also in increasing the quality of

genetic manipulations resulting in better agricultural performance.

The Senescence-Activated Signaling Pathway That Activates the Dicot

Promoter, SAG12, May Be Evolutionarily Conserved In Anthurium

Senescence involves a highly regulated, co-coordinated series of events

accompanied by repression and up-regulation of gene expression. Senescence-enhanced

genes encode degradative enzymes that include proteases, ribonucleases, lipases and

nucleases. Senescence-repressed genes include those that encode the photosynthetic

apparatus. Senescence can also be characterized by phenotypic changes in plant organs

including chlorophyll loss and changes in pigmentation eventually leading to necrosis.

Protein activation, modification, degradation, and mobilization are all actively changing

events during senescence. A receptor-like protein kinase induced during senescence

may have a regulatory function of some senescence pathways (Hajouj et al. 2000).

Proteins that promote leaf longevity are targeted for degradation during senescence by

ORE9, an F-box protein, which may form a complex with its substrate for subsequent

ubiquitin tagging (Woo et al. 2001). Both external and internal stimuli can trigger

changes in plant hormone concentrations thereby initiating signaling mechanisms that

modify gene expression at transcriptional and translational levels, as well as post­

translationally by modifying proteins.

Modification of gene expression involves promoters that serve as switches,

turning genes on and off in response to signals. Senescence-associated promoters are

28

comprised of conserved cis-acting elements and enhancer regions (Noh and Amasino

1999a) necessary for senescence-specific expression. DNA gel-shift mobility assays

show these regions form different mobility complexes with DNA-binding proteins in

young versus senescent leaves. The research suggests that a transcriptional repressor or

inactive form of a transcriptional activator is present in the healthy state, while during

senescence a new transcriptional activator is produced or the repressor/inactive

transcriptional factor is modified to an active form (Noh and Amasino 1999a).

Research involving senescence mechanisms in arabidopsis has shown a complex

series of events involving gene and protein modification in an effort to recycle nutrients

for mobilization to healthy portions of the plant. This process is thought to be conserved

throughout all higher plant species, but a question still remains: Are the molecular

signaling pathways, transcriptions factors, and promoters interchangeable in evolutionary

divergent species? The successful senescence induced expression of the arabidopsis

senescence-activated promoter in anthurium suggests that cis-acting and enhancer regions

of Pr-sag12 interact with transcriptional factors present in anthurium making an expression

system using a senescence-enhanced promoter from arabidopsis a viable option in

anthurium. This also suggests that anthurium has senescence-activated promoters and

associated transcription factors that are homologous to arabidopsis and identification and

characterization would prove to be highly useful for biotechnology.

29

Expression and Proper Post-Translational Modification of GFP in

Anthurium, Can Provide A Necessary Reporter Gene Tool For

Anthurium Molecular Studies.

Reporter genes are an important tool in evaluating promoter expreSSIOn,

successful transformations, and mutation analysis. They can be used in both stable

transformation and transient expression analyses. In order to determine that Pr-SAG12 is

regulated in anthurium, there must be a reliable reporter system. Important criteria of a

reporter system includes !be successful production of the protein in the host, quantifiable

expression, and non-invasive analysis. The protein must not be confused with

background proteins native to the host. Several reporter genes are available and

examples include uidA (encoding ~-glucuronidase 'GUS'), luc (encoding Luciferase), gal

(encoding ~-galactosidase), and gfp (encoding green florescent protein). Most reporter

genes encode an enzyme that is foreign to the host that can catalyze a reaction when in

the presence of the correct substrate. However, for quantification analysis, the plant tissue

must be destroyed by homogenation and/or exposure with an infiltration buffer. One

unique protein, gfp, encodes a functional protein that can be visualized through excitation

with a non-invasive wavelength of light. The gfp protein emits a different wavelength of

light that is easily quantifiable in living cells (Schenk et aJ. 1998).

In order to conduct molecular studies in anthurium, it is necessary to identify a

reporter system that is effective, and provides for non-invasive analysis. Anthurium

tissues contain heavy amounts of phenolic and polysaccharide compounds !bat inhibit

pure extraction and analysis of cellular proteins. Previous research shows that the uidA

30

or "gus" gene is effectively expressed in stably transformed anthurium tissues, but has

improper post-translational modification and folding, making the reporter enzyme non­

functional on the GUS substrate (Kuehnle and Chen 1994). Our research also shows that

the GUS system is not an effective reporter system for transient bombardment expression

analysis in anthurium (Hayden and Christopher, unpublished).

Chapter 3 will focus on determining the transient expression of Pr-SAG12 during different

stages of development using GFP as a reporter gene. This work will identify the gfp4 and

gfp5-ER genes as useful reporter systems for further research on promoter assays, tissue­

regulated expression and protein targeting studies in anthurium.

A Senescence-Regulated Cysteine Protease Gene, Orthologous to sag12,

Exists In Anthurium

Chapter 4 will focus on the identification and characterization of cysteine protease

genes in anthurium. Two cysteine proteases anth16 and anth17 will be studied in detail

ultimately determining the role of each gene as it applies to development. Understanding

gene regulation during senescence begins with isolating and characterizing senescence­

associated genes. These genes give us insight into the complex molecular process

involved during senescence. The factors regulating leaf senescence are complex and

involve both external and internal stimuli, such as darkness, pathogens, developmental

signals and hormones. These factors initiate signal transduction pathways that repress

photosynthesis gene expression and activate the expression of genes involved in cellular

disassembly, recycling and integral defense-related processes (Quirino et al. 2000). The

genes for mRNAs that increase markedly in abundance during senescence have been

31

designated as "senescence-associated genes" or SAGs (Lohman et a!., 1994) and encode

chitinases, cysteine proteases, lipases, aspartic proteases, and metalloproteases from

plants such as rape seed (Hanfrey et a!., 1996), rice (Lee et a!., 2001), cucumber

(Delorme et a!., 2000); carnation (Hong et a!., 2000), tomato (Davies and Grierson, 1989;

John et aI., 1997), parsley (Lers et aI., 1998), arabidopsis (Gan and Amasino, 1995;

Lohman et aI., 1994; Yoshida et al., 2001), daylily (Valpuesta et a!., 1995), ryegrass (Li

et aI., 2000), sweet potato Huang et al. 2001), and eggplant (Xu and Chye, 1999) and

enzymes involved in phosphonate biosynthesis in carnation (Wang et aI., 1993).

Cysteine protease genes expressed during senescence are of particular interest since they

fall under class 5 SAG expression. This stage indicates expression at the extreme

initiation of senescence that is sustained throughout the later stages. There also exists

many isoforms of cysteine proteases with different expression patterns suggesting unique

and specific roles in the developmental cycle.

One benefit that aids in the identification of these genes is the extent of homology

contained in specific regions representing enzymatic functionality. This enables the

creation of degenerate primer sequences that can produce the successful cloning of small

portions of DNA from cysteine protease cDNAs synthesized from unique developmental

mRNA populations. Once a native anthurium fragment is isolated, it can serve as a

useful tool to identify full-length cDNAs from libraries or mRNA populations. After the

full-length cDNA sequence is verified, many tools are available for full identification.

The deduced amino acid sequence allows the comparison of the anthurium cysteine

proteases to other homologous proteases, whose sequences are available in online

databases. Careful analysis of differences in deduced amino acid composition can help

32

predict unique features of the proteins. These include: secondary and tertiary structure

prediction, signaling peptides to determine cellular location, peptide cleavage sites to

predict mature protein length, and common features that characterize enzymes such as

active domains and critical bonding locations.

Characterization of two full-length anthurium cysteine protease clones by

Northern Blot analysis of developmental RNA populations indicates differential

expression patterns confirming senescence-associated gene expression. Developmental

populations include: YG (immature); MG (Mature Green); and Sl, S2, S3 (Senescent

leaves, progression characterized by chlorophyll content). Once the anthurium SAG is

identified it can be used in correlation with the non-senescence associated anthurium

cysteine protease gene, photosynthetic genes (cab and psbA), and a constitutively

expressed recycling gene (ubi), to determine expression under senescence controlling

conditions.

The Effect of Cytokinin and Sucrose on SAG Expression During

Senescence

A comparison of expression patterns in response to stress and hormone

treatments using Northern blot analysis, is an excellent indicator for characterizing

senescence-associated genes (Weaver et al. 2001). Certain stresses and hormones are

able to hasten or repress senescence as seen visually by leaf yellowing and detected on

the molecular level by responses in gene expression. Cytokinin is a well-known

senescence-delaying phytohormone (Gan and Amasino 1997; Nooden et al. 1997).

33

Exogenous treatments of the synthetic cytokinin benzyladenine (SA) show a repression

of senescence characteristics when applied on anthurium leaves and flowers (paull and

Chantrachit 2001). Cytokinins can be used to repress many SAG transcripts, and

occassionally reverse the senescence of a leaf in the later stages. Considering sugars are

the main product of photosynthesis, certain sugars can serve as signaling molecules,

changing sugar levels during photosynthetic decline may act as a senescing-inducing

signal (Jang et al. 1997). Sucrose can effectively repress the accumulation of certain SAG

transcripts when applied to detached leaves (Weaver et al. 1998).

Although these treatments are somewhat effective in reversing the effects of

senescence and SAG transcripts, inducing senescence in leaves can result in a different

pattern of SAG transcripts from the observed expression during developmentally-induced

senescence. The most common techniques of inducing senescence include detachment

and incubation of leaves in water under dark and light conditions, desiccation, and

hormone treatments with ABA and ethylene. Certain SAG transcripts may not respond to

this type of induced senescence even though the visual indicator (chlorophyll loss) of

senescence progresses rapidly (Noh and Amasino 1999a).

Senescence is a complex process in which SAG genes encode unique transcripts

having specific functions. Each of the techniques listed above can give insightful clues in

characterizing a SAG gene in relationship to the many already isolated and characterized.

It is necessary to include the proper controls to identify the SAG expression pattern of

isolated clones, and understand that only the combination of mUltiple treatments in

comparison to developmental populations will give a firm conclusion. This will provide

valid data in the pursuit of identifying a native senescence-upregulated promoter, to

34

better the auto-regulatory senescence inhibition system, for future applications in

engineering the Anthurium species for increased production and improved post-harvest

floral vase life.

35

CHAPTER III

REGULATION OF THE SAG12 PROMOTER INANTHURIUM USING TRANSIENT BIOLISTIC

PARTICLE BOMBARDMENT

Introduction

Modification of gene expression involves promoters that serve as switches,

turning genes on and off in response to signals. Both external and internal stimuli can

trigger changes in plant hormone concentrations thereby initiating signaling mechanisms

that modify gene expression at transcriptional and translational levels, as well as post-

translationally by modifying proteins. Senescence-associated promoters are comprised of

conserved cis-acting elements and enhancer regions (Noh and Amasino 1999b) necessary

for senescence-specific expression. It is proposed that a transcriptional repressor or

inactive form of a transcriptional activator is present in the healthy state, while during

senescence a new transcriptional activator is produced or the repressor/inactive

transcriptional factor is modified to an active form. DNA gel-shift mobility assays show

that regions of the Pr-SAG12 form different mobility complexes with DNA-binding

proteins in young versus senescent leaves. Two orthologs of SAG12 isolated from

Brassica napus show sequence similarities to SAG12 and expression analysis consistent

with that of SAG genes. When the promoters of the three genes are compared, two

distinct regions show over 75% identity with one another. The regions from the Brassica

genes also bound to arabidopsis DNA-binding proteins as shown in a gel-shift assay

using extracts from senescent arabidopsis leaves (Noh and Amasino 1999b). This

36

suggests these two regions are necessary for the senescence-specific regulation of these

particular SAG genes. Another study compares the upstream region of a SAG gene from

sweet potato. It was concluded that the promoter did not include any regions similar to

those in sag12 however it did include putative ethylene-responsive elements.

Research involving senescence mechanisms in arabidopsis has shown a complex

series of events involving gene and protein modification in an effort to recycle nutrients

for mobilization to healthy portions of the plant. The successful senescence induced

expression of the arabidopsis senescence-activated promoter in anthurium would suggest

that cis-acting and enhancer regions of Pr-SAG12 interact with transcriptional factors

present in anthurium making a expression system using a senescence-enhanced promoter

from arabidopsis a viable option in anthurium. This would also suggest anthuriurn has

senescence-activated promoters and associated transcription factors that are homologous

to arabidopsis and identification and characterization would prove to be highly useful for

biotechnology.

Understanding the regulation of a foreign promoter in a host of interest is

necessary to confirming the efficacy of a gene manipulation system prior to stable

transformation. In this case, Pr-SAG12 is evaluated by regulating the expression of a

reporter gene, when introduced via particle bombardment in anthurium leaf tissues.

Reporter genes are an important tool in evaluating promoter expression, successful

transformations, and mutation analysis. They can be used in both stable transformation

and transient expression analyses. The important criteria of a reporter protein include

successful production in the host, quantifiable expression, and non-invasive analysis.

The protein must not be confused with background proteins native to the host. Several

37

reporter genes are available and examples include uidA (encoding ~-glucuronidase

'GUS'), luc (encoding Luciferase), gal (encoding ~-galactosidase), and gfp (encoding

green florescent protein). Most reporter genes encode an enzyme that is foreign to the

host that can catalyze a reaction when in the presence of the correct substrate. However,

for quantifiable analysis, the plant tissue must be destroyed by homogenation and/or

exposed to an infiltration buffer. The unique reporter, GFP, encodes a protein that can be

visualized through excitation with a non-invasive wavelength of light. The GFP protein

emits a different wavelength of light that is easily quantifiable in living cells (Schenk et

al. 1998).

In order to conduct molecular studies in anthurium it is necessary to identify a

reporter protein system that is successfully produced and also provides for non-invasive

analysis. Anthurium tissues contain heavy amounts of phenolic and polysaccharide

compounds that inhibit pure extraction and analysis of cellular proteins. This physiology

can also interfere with substrate/enzyme interaction that is necessary in most reporter

systems. This chapter will focus on the transient regulation of Pr-SAGl2 in anthurium and

also include data verifying the successful implementation of the GFP reporter system in

anthurium tissues.

Materials and Methods

To determine whether the senescence-associated promoter, Pr-SAG12 (from the

dicot Arabidopsis thaliana) is up-regulated by senescence in Anthurium tissues, a

transient expression assay was conducted using the reporter genes of either uidA or gfp

38

depending on the plasmid vector introduced to the plant tissue. The following controls

include: a) uncoated beads to determine the effects of particle bombardment wounding

for a negative control, b) reporter gene expression with a constitutive promoter for a

positive control, c) Pr-SAG12 expression in tissues of all developmental stages, d) reporter

gene without a promoter for a negative control.

Construction ofPr-SAG12 reporter vectors

The first plasmid used to test for senescence-upregulated promoter expression was

PSAG1Z-gUS, containing, a promoter isolated from a cysteine protease and a gus reporter

gene. The plasmid PSAG12-gUS (provided by Rick Amasino, University of Wisconsin),

was used to transform XU-blue Ecoli cells by the standard heat shock method (Sambrook

et al. 1989). After an overnight incubation at 37QC, the plasmid was isolated using a midi­

prep plasmid isolation kit (Qiagen, Valencia, CA). The control plasmids, p35S-gfp4,

p35S-gfp5-ER (Jim Hasselhoff, University of Cambridge, UK) and pBI221 (Clontech,

Palo Alto, CA) were obtained through the UH College of Tropical Agriculture and

contain the constitutive 35S CAMV promoter that drives the expression of either the

green fluorescent protein (p35S-gfp) or the beta-glucuronidase enzyme (pBI426). A new

plasmid, PSAG1z-gfp4, was constructed from p35S-gfp4 (Fig. 3.4) and Pr-SAG12 from

pSG516 (Fig. 3.3) were excised and subcloned into pBLUESCRIPT KS II (Sambrook et

at 1989) to create PSAG1z-gfp4 (Fig. 3.1). Restriction enzyme cloning sites were available

in both original vectors so a unique subcloning scheme was established as follows: a)

gfp4-nos was excised from 35S-gfp4 and cloned into pBSII KS; b) Pr-SAG12 was excised

from PSAG12-GUS; c) sag12-nos was cloned into pBSIIKS-GFP4. This formed PSAG12-

39

gfp4 (Fig. 3.1). Scale-up and isolation of all plasmids were performed using identical

methods as previously described for PSAG12-guS. Plasmids were concentrated or diluted to

give a solution of I J.lg/J.lI for biolistic bombardment procedures. Plasmid PSAG12-ipt in a

binary vector (Fig. 3.2) was also provided by Richard Amasino for future experiments

with stable transformation

pSaa12-GFP46.2kb r"'"

11."",111.•0/1>,.,1$o,rlf'~IU

pSagl2-IPT(in binary vector)

17.60kb

Figure 3.1: psag12-GFP4 in pBLUESCRIPT II KS. Figure 3.2: PsagI2-IPT in a binary vector

pSaal2-IPTpSG5165.88 Kb

pBIN 3S8-mGFP4

E<GIf.I

'11/­...,"Sp</01"_

_ HI­J:fMI­E<GIf.I

Figure 3.3: psaglz-IPT in pUC18 termed pSG516 Figure 3.4: pBIN 35S-mGFP4.

40

Particle bombardment ofAnthurium leaves.

Tissue cultured leaves from anthurium varieties Marian Seefurth and Southern

Blush were excised halfway down the petiole and incubated immediately on sterile

deionized H20 for 5 minutes. The leaves were then placed on agar plates, with the

petiole submerged. A control was set up to bombard leaves of whole tissue culture

plantlets to determine if detachment induced expression of sag12 in healthy leaves.

Microprojectile bombardment of anthurium leaves was conducted with the Biolistic PDS

1000/He System (BioRad, La Jolla, CA) at 1,550 psi with 28 Hg vacuum in the chamber,

macrocarrier level 3, and leaf tissue level 4. 3)lg of plasmid DNA was combined with 1

mg of 1.6,ttm gold particles (BioRad). 50 ,ttl of 2.5 M CaCl2 and 20 ,ttl of 0.1 M

spermidine were added simultaneously and vortexed for 3 min. The plamsid DNA-coated

gold particles were washed with 70% ethanol and suspended in 60,tt1 of absolute ethanol.

Ten microliters of this mixture was loaded on carrier disk and used for bombardment of

detached anthurium leaves (Itaya et al. 1997). Gold particles without plasmid were

washed in 70% ethanol 2X, resuspended in absolute ethanol and loaded directly on the

macrocarriers.

GUS determination

After bombardment, leaves were incubated for 3-5 days at room temperature

under low light before visualization was detected. Staining protocols for GUS activity

were performed according to Jefferson (1987). X-glue buffer (50 mM phosphate buffer

(pH 7.2) containing 0.05% [beta]-mercaptoethanol and 0.0025% Triton X-IOO, 0.1 mM

41

potassium ferricyanide and 0.1 mM potassium ferrocyanide. 0.5 mg/ml 5-bromo-4­

chloro-3-indoyl glucuronide (X-glue) was added fresh in a DMF solution prior to staining

leaf tissue. The bombarded leaf tissue was incubated at 24QC for 2-3 days, then incubated

in the X-glue solution (pH 7.0) for 4 hours (arabidopsis) to 24 hours (anthurium).

Visualization and detection ofGFF

Photo-documentation was obtained by using the Olympus BX60 uv­

microscope with a "wild-type" filter set (Omega Optical cat # XF1076, #XF2040,

#XF3003) that effectively excites at ~'f.. 400nm and detects emissions at -'f.. 505-540 from

wtGFP (mGFP4). The second filter set is the FITC cube filter for visual detection of

GFP5 (Omega Optical cat # XFI063, #XF2054, #XF3067) excitation -'f.. 488nm,

emission -'f.. 505-530nm. Both filter sets can detect GFP4 and GFP5. Pictures were

documented with the SPOT 1.5.0 camera (Diagnostic Instruments Inc., Sterling Heights,

Michigan) and SPOT software v.3.0. PCR amplification of GFP cDNA was verified via

gel electrophoresis. The eDNA was constructed from mRNA fractions of pooled leaves 5

days post-bombardment. The mRNA was treated with RQl DNase (Promega) to avoid

contamination from plasmid constructs.

42

Results

Pr-SAG12 transient bombardment assays for expression ofGFP

This experiment sought to determine whether a senescence-activated promoter

(Pr-SAG12) from the dicot arabidopsis is regulated in a senescence-dependent manner in

the monocot anthurium. The second purpose of this experiment was to develop a suitable

reporter gene system for this plant because GUS was not expressed in anthurium under a

variety of conditions (Hayden and Christopher, data not shown). Therefore, we

conducted transient assays using biolistic bombardments of healthy and senescent leaves

with Pr-SAG12 and the constitutive 35S-Pr fused to both green fluorescent protein genes,

gfp4 and gfp5-ER. The FITC filter cube, which allows orange chlorophyll

autofluorescence, and the wild type GFP (wt-GFP) filter cube, which eliminates it, were

used. In Figures 3.6, 3.7, 3.8A, 3.8B and Table 3.1, the positive control, 35SPr-gfp4, is

robustly expressed in both healthy and senescing anthurium leaves, while Pr-SAG12-gfp4

(Fig. 3.5, 3.8C, 3.8D) was expressed 20-fold higher in senescing relative to healthy

tissue. The 35SPr-gfp4 was expressed lA-fold higher in healthy relative to senescing

tissues (Table 3.1). Both gfp4 and gfp5-ER were expressed markedly (Figs. 3.8E and

3.8F), which demonstrated the suitability of these reporters for gene expression studies in

anthurium. Figures 3.9A and 3.9B show the wound-induced expression of Pr-SAGI2-gfp4

in healthy tissues. This also serves as a comparison of the wtGFP (Fig. 3.9B) and the

F1TC (Fig. 3.9A) filter sets.

43

Figure 3.5: GFP in senescing leaftissues expressed with Prsagl2 Bar scale=l OO~m

Figure 3.6: GFP in senescing leaf tissues expressed with Pr35S Bar scale=lOO~m

Figure 3.7: GFP in senescing leaf tissues expressed with Pr35S (lower mag.) Bar scale=1 OOJ1m

44

Figure 3.8. Representative examples of GFP expression driven by the 35S promoter or Pr-SAGl2 asmeasured via transient assays after biolistic bombardment of healthy and senescing leaves (Table 3.1)(A,B) mgfp4 expression with the 35S promoter, properly folds and can be visualized in anthurium tissue.Visualization demonstrated with the FITC filter cube, excitation 370-420nm, emission 505-540nm. (C,D)Two focal view points of pSAG 12-mgfp4 plasmid expression in senescing tissue demonstrated with thewtGFP filter cube.(E,F) A comparison of mgfp4 and mgfp5-ER transient expression in similar tissues afterbombardment. (E) GFP4 localizes around nucleosomes and the cytoplasm which in epidermal leaf cells.(B) GFP5-ER seems to localize in cytoplamsic organelles including proplastids and the endoplasmicreticulum. All photographs taken with the SPOT camera system, and are at lOX magnification.

45

Figure 3.9: (A,B) pSAG 12-mgfp4 expression in healthy tissue, small localization of areas possibly causedby wounding taken with the FITC (A)and wt-GFP (B) filter cubes. Photographs taken with the SPOTcamera system, and are at lOX magnification.

Table 3.1: Quantitation OfpSAG12-gfjJ4 and p35S-gfjJ4 expression in senescent and healthy tissues.

Discussion

sag12-gfp4Healthy Leaf

6.8 (± 3)

sag12-gfp4Senescent Leaf135.2 (± 71.5)

35S-gfp4Healthy Leaf

267.6 (± 74.2)

35S-gfp4Senescent Leaf180.3 (±44.8)

Plasmids PsAGI2-gfp4, p35S-gfp4, p35S-gfp5-E~ pBlue-gfp4 were used to test

transient expression in developmental stages of leaf tissue in anthurium. Preliminary

experiments focused on transiently expressing the uitlA or ~-glucuronidase(GUS) gene in

both arabidopsis and anthurium Leaf tissue. These experiments show that the

bombardment and staining procedures were correct as evidence of the of arabidopsis leaf

tissue showing the enzymatic cleavage of -Bromo-4-chloro-3-indoxyl-beta-D-

glucuronide into its colorimetric product (data not shown). It was also confrrmed that the

PSAGI2-guS vector was regulated in a senescence-associated manner, when transiently

46

bombarded into arabidopsis tissues. The constitutive promoter 35S also successfully

expressed gus in arabidopsis leaves. Variations of incubation time and temperature,

staining concentrations, and infiltration techniques were attempted for anthurium GUS

assays with no success.

This is consistent with previous research determining that the gus gene is

effectively expressed in stably transformed anthurium tissues, but GUS protein was not

detected by Western blot analysis (Kuehnle and Chen 1994). According to Kuehnle and

Chen, modifications at the gene level contribute to the inactivation of GUS. This is not

entirely consistent with the data presented in the research because key experiments were

missing that would effectively deduce this postulation. The authors performed PCR on

genomic DNA extracted from transformed tissues and confirmed the presence of the

inserted gene sequences. The second experiment showed that a Western blot of proteins

extracted from the transgenic plants did not successfully respond to the anti- ~-

glucuronidase antibody. More research is needed to come to a firm conclusion on this

data. First!y a genomic restriction digest can be performed and run on an agarose gel, the

southern of this gel can confirm the presence of multiple copies of the transgene in the

genome. The presence of multiple transgene copies can lead to promoter methylation and

result in gene silencing (Hobbs et al. 1990). Direct or inverted repeats can cause hairpins

also noted as triggers of transgene silencing. A reverse primer PCR (rpPCR) experiment

is commonly used to determine if inverted or tandem repeats of T-DNA inserts occur in

the transgenic genome (Kumar and Fladung 2000). These techniques are in addition to

an RT-PCR that is used to identify a mRNA transcript of the gus gene consequently

determining the problem to be transcriptional or translational.

47

In our transient assays no study was conducted to determine if mRNA

transcripts of gus were present. However, it was determined that there was no interaction

of GUS with the substrate resulting in the colorimetric product. When both GFP and

GUS were used as reporter genes, we successfully visualized GFP in parallel with the

unsuccessful production of GUS. GFP mRNA transcripts were confirmed via RT-PCR,

after digestion with DNAse to eliminate any contaminating genomic or plasmid DNA.

This may indicate that unsuccessful GUS production occurs at the translational or post­

translational level. Improper folding, post-translational modification, or interaction with

other cell components may be the primary cause. Anthurium tissues contain a unique

ratio of phenolic compounds which could irreversible bind to a foreign protein. Thionins

(small defense proteins) have also been suggested as inactivators of the GUS protein

(Diaz et al. 1992). It has been determined that the GUS reporter enzyme system is an

unreliable, if not unusable, system for expression analysis studies in anthurium. We

report here that GFP is an effective reporter gene system for anthurium. The benefits of

the GFP system include non-invasive and non-destructive analysis of transformed and

transiently expressing tissues, ease of documentation, and clear differentiation from

endogenous proteins. To understand GFP and how it interacts with anthurium physiology

we conducted several bombardments with 35S-gfp4 and 35S-gfp5-ER. GFP4 was chosen

for experiments involving the characterization of Pr-SAG12 due to ease of sub-cloning

procedures with available Pr-SAG12 vectors.

GFP analysis was performed multiple times with healthy and senescing tissues.

In this report we show that Pr-35S promotes GFP expression in anthurium leaf tissues,

and Pr-SAG12 promotes GFP in a senescence-upregulated manner. Observing GFP4 in

48

anthurium tissues was performed with two different confocal microscopes and two

different tissues. The materials and methods section lists the primary method, tissue, and

equipment. The first experiment focused on GFP4 visualization in senescing leaves,

driven by both Pr-SAG12 and Pr-35S, from mature plants cut into 10 cm2 sections. These

tissues were incubated post bombardment one month to maximize expression potential

and GFP accumulation. The senescing leaf squares had minimal amounts of chlorophyll

left providing a source free of background fluorescence, making it easy to visualize GFP

fluorescence. Figure 3.6 and 3.7 show good examples of GFP, establishing that Pr-SAG12

(Fig. 3.5.) is regulated in anthurium tissues but visually appears to be less active than the

GFP regulated by the constitutive Pr-35S (Fig. 3.6). At a reduced magnification (Fig.

3.7), it is easy to see a pattern of fluorescent vs. non-fluorescent sections of the anthurium

leaf square.

Further analysis was conducted on a late model confocal microscope (Olympus

BX60) fitted with a digital camera for documentation. This camera allows the use of

multiple filter cubes, to reduce background, and gain a different perspective on

fluorescent bodies within the cell. These experiments were conducted with whole

anthurium leaves (Marian Seefurth and Southern Blush cultivars) that were harvested

from tissue culture plantlets. The size of tissue culture leaves are significantly smaller

than whole leaves which results in a slightly smaller overall cell size. No significant

difference was observed between leaves that were excised from tissue culture plants and

placed on agar, and those leaves that were bombarded while still attached to the tissue

culture plantlet.

49

Figure 3.8A and 3.8B demonstrate GFP4 fluorescence visualization, with a FITC

cube, in anthurium leaves. The two pictures represent two different focal points and GFP

intensity in the epidermal layers of the leaf tissue. GFP4 was detected in the perimeter of

the cells and seemed to interact closely with the cell wall or plasma membrane creating a

distinct hexagonal shaped outline. This is consistent with previous reports involving the

visualization of hypocotyl epidermal cells in Arabidopsis showing cytoplasmic localized

GFP pressed in a thin layer between the cell wall and vacuole (Haseloff et al. 1997). The

visualization usually occurs in the nucleosome or cytoplasm with little or no association

with other organelles in the cell. Epidermal cells commonly referred to as "pavement

cells" commonly consist of large vacuoles, few organs, and no chloroplasts. These cells

have a polygonal shape and are simply required for turgor and water retention on the

surface of the leaf.

As depicted in Figures 3.8A, 3.8B, 3.8C and 3.8D the GFP4 fluorescence appears

in a polygonal shape and is associated with these cells easily visualized on the surface of

the leaf. The mesophyll layer is clearly present below this translucent cell layer. The

mesophyll cells are filled with many chloroplasts that appear as autofluorescing,

characterized by reddish/orange appearance. The FITC filter cube captured this distinct

difference between fluorescence and background for healthy tissue GFP analysis. GFP5­

ER has an ER retention signal. The presence of this signal peptide tends to direct it to

accumulate in proplastids and other cytoplasmic organelles such as the endoplasmic

reticulum, but not in the cytoplasm or nucleosome. Figure 3.8E and 3.8F demonstrate the

difference between GFP4 (E) and GFP5-ER (F). These photos are consistent with the

two types of patterns we see with GFP4 and GFP5-ER in our transient assays. GFP5

50

seems to be associated with structural organelles at or below epidermal layers and

typically associated with the mesophyll cell, whereas GFP4 is easier to visualize in the

cytoplasmic contents of the epidermal cells, making it more readily detectable when

using the FITC filter cube, which also captures the auto-fluorescence from chlorophyll.

The GFP5 is slightly difficult to visualize with the FITC filter cube because of the

interfering fluorescence from chlorophyll. The Olympus microscope is limited to the

photo-documentation of an area no larger than the 120 cell area we obtained at lOX. The

actual image observed in the microscope optical at this magnification is nearly 5-8 times

this amount. Overall, it was easier to visualize GFP4 in anthurium tissues, both healthy

and senescent with the FITC filter cube. Senescent tissue had a slightly more

greenish/yellow auto-fluorescence making the wtGFP cube a better choice for

documentation because of it eliminated this backround. Healthy tissues having GFP4

present on the epidermal portion of leaf tissue showed extremely clear results.

The main purpose of developing a reporter gene system that functions in

anthurium is to evaluate the senescence-associated promoter, Pr-SAG12, from arabidopsis.

Such regulation suggests that anthurium has similar developmental mechanisms involved

with senescence. These include regulatory elements, transcription factors, enhancer

regions, and a myriad of proteins designed to initiate, delay, or prevent the development

in senescence. SAG12 is a cysteine protease isolated from arabidopsis that shows a

distinct expression pattern during development. This pattern shows no expression during

non-senescent conditions, but a steady increase throughout senescence, until it is one of

the most abundant proteins during late senescence (Lohman et aI. 1994). The SAG12

promoter was cloned from an arabidopsis genomic library and was further determined to

51

be regulated in a senescence-upregulated manner through reporter gene expression (Gan

and Amasino 1997).

More recent research on Pr-SAG12 has identified the specific region that confers

senescence specificity, and enhancer region, and a transcription factor binding site (Noh

and Amasino 1999a). When these regions were compared with two SAG12 homolog

promoters isolated from Brassica napus, two highly conserved regions were detected

(Noh and Amasino 1999b). Both these regions are necessary for senescence specificity

in both Brassica and Arabidopsis. Furthermore, transcription factors from arabidopsis

senescing leaf fractions bound to both Arabidopsis and Brassica promoters when radio­

labeled probes of the essential -607 to -565 promoter region were incubated in whole cell

extract of arabidopsis senescent leaves. This, along with Pr-SAG12 successful regulation in

tobacco, establishes that transcription factors and promoter binding locations are

conserved in related species.

The research we present indicates that senescence-specific transcription factors

are also present in anthurium, and may be distinctly similar to those from arabidopsis.

Figures 3.6, 3.Se and 3.SD show GFP4 expression in anthurium regulated by Pr-SAG12.

These pictures clear!y show two unique focal points of GFP4 expression in senescing

tissues with the wt-GFP filter cube. Table 3.1 shows the average expression patterns of

multiple leaf bombardments using the vectors listed. Each leaf was observed post­

bombardment and regions of contiguous cells were identified and individual cells that

make up that region were counted and recorded. Table 3.1 shows the average number of

cells within a contiguous region for each bombardment series. A distinct difference was

52

observed between healthy and senescing leaf tissue when bombarded with PSAG1z-gfp4.

Expression was broad in senescing tissues, however it was also identified in small regions

in healthy tissues. Figure 3.9A and 3.9B show the character of the localized region of

GFP expression in healthy tissues. This expression might be associated with cells

damaged when the biolistic particle penetrated the epidermal cells. GFP visualization

was never widespread and not present outside the area of damage. Figure 3.9 also

displays the difference between the FlTC filter cube (A) and the wt-GFP filter cube (B)

on an identical sample. Based on these results we feel confident that Pr-SAG1Z is regulated

in a senescence-associated manner in anthurium tissues, suggesting orthologous

mechanisms that regulate molecular mechanisms of senescence between anthurium and

Arabidopsis are present. It is also evident that the promoter may be regulated, to some

extent, in response to wounding caused by the biolistic bombardment consistent with a

cell autonomous view of senescence proposed by Weaver and Amasino (1998).

However, this regulation is localized and sag12 only shows a broad overall pattern of

expression in leaf senescent tissues.

53

CHAPTER IV

ISOLATION AND CHARACTERIZATION OF TWOCYSTEINE PROTEASE GENES FROM

ANTHURIUM AND THE EXPRESSION PATTERNOF EACH DURING DEVELOPMENT AND INRESPONSE TO HORMONE TREATMENTS

Introduction

Understanding gene regulation during senescence begins with isolating and

characterizing senescence-associated genes (SAG's). These genes give us insight into the

complex molecular process involved during senescence. Cysteine protease genes

expressed during senescence are of particular interest since they fall under class 5 SAG

expression. This stage indicates expression at the extreme initiation of senescence that is

sustained throughout the later stages. There also exists many isoforms of cysteine

proteases with different expression patterns suggesting unique and specific roles in the

developmental cycle.

One benefit that aids in the characterization of these genes is the extent of

homology contained in specific regions representing enzymatic functionality. This

enables the creation of degenerate primer sequences that can produce the successful

cloning of small portions of DNA from cysteine protease cDNAs synthesized from

unique developmental mRNA populations. Once a native fragment is isolated, it can

serve as a useful tool to identify full-length cDNAs. This can be accomplished by using

it as a probe to identify larger fragments within a cDNA library, or can simply be used to

develop primers and amplify cDNA ends within any given eDNA population. The eDNA

54

fragment may also be useful in developing an early expression pattern using Northern

Blot analysis. This analysis must be double checked for accuracy after the full-length

transcript is isolated.

Analysis of SAG gene expression by Northern Blot analysis of developmental

RNA populations indicates differential expression patterns confirming the senescence­

associated gene expression. Once the SAG is identified it can be used in correlation with

the non-senescence associated cysteine protease, photosynthetic genes (Lhcb and psbA),

and a constitutively expressed gene (ubi), to determine expression under senescence

controlling conditions.

A comparison of expression patterns in response to stress and hormone treatments

is an excellent indicator in characterizing senescence-associated genes (Weaver et aI.

2001). Certain stresses and hormones are able to hasten or repress senescence as seen

visually by leaf yellowing and detected on the molecular level by responses in gene

expression. Cytokinin is a well-known senescence-delaying phytohormone (Gan and

Amasino 1997; Nooden et al. 1997). Endogenous treatments of the synthetic cytokinin

benzyladenine (BA) show a repression of senescence characteristics when applied on

anthurium leaves and flowers (Paull and Chantrachit 2001). Cytokinin can be used to

repress many SAG transcripts, and actually reverse the senescence of a leaf in the later

stages. Considering sugars are the main product of photosynthesis and certain sugars can

serve as signaling molecules, changing sugar levels during photosynthetic decline may

act as a senescing-inducing signal (Jang et aI. 1997). Sucrose can effectively repress the

accumulation of certain SAG transcripts when applied to detached leaves (Noh and

Amasino 1999a).

55

Although these treatments are somewhat effective in reversing the effects of

senescence and SAG transcripts, inducing senescence in leaves can result in a different

pattern of SAG transcripts from the observed expression during developmentally-induced

senescence. The most common techniques of inducing senescence include detachment

and incubation of leaves in water under dark and light conditions; desiccation; and

hormone treatments with ABA and ethylene. Certain SAG transcripts may not respond to

this type of induced senescence although the visual indicator (chlorophyll loss) of

senescence progresses rapidly (Noh and Amasino 1999a).

This chapter presents the details of two unique cysteine proteases isolated from

the anthurium species. The genes are characterized through comparison with like genes

from the GENBANK data base, the deduced amino acid sequence, and expression level

during development and hormone treatment.

56

Materials and Methods

Plant material and growth conditions

Tissue culture plantlets of Southern Blush (graciously provided by Agri-Starts

Micro, Mt. Dora, Florida.) and Marian Seefurth (University of Hawaii) were grown in

Magenta® GA-7 vessels (20-40,uE m-2s-\ 12 h light/12 h dark cycle) at 25°C and 35%

Humidity. Greenhouse-grown Marian Seefurth was grown under 78% shade (20-28QC,

80-95% humidity) year round at the University of Hawaii, Manoa campus (Honolulu,

Hawaii).

RNA isolation

Leaves from greenhouse grown anthurium plants (Marian Seefurth) were

collected and developmental stages were characterized by visual inspection and

chlorophyll determination. After collection or treatment of leaves, they were directly

frozen in liquid nitrogen and total cellular RNA was extracted using the protocol

developed by Champagne and Kuenhle (2000) with a few modifications. PVPP (sigma

P-6755) was substituted in place of PVP-40 and all extractions were performed at 13" C.

In addition, a low salt, low ethanol precipitation was included prior to the final ethanol

precipitation step to remove polysaccharide contamination from older tissue (Asif et al.

2000). A full protocol is located in the Appendix.

57

Hormone treatments and chlorophyll determination

Cut leaf sections (40 mm X 40 mm) from a mature healthy leaf, were submerged

in sterile H20 for 30 seconds, then floated on either 1 flM N6- Benzylaminopurine (BA,

Sigma B-3408) or 3% Sucrose (Sigma S·5390) solutions or sterile deionized water

(controls). All samples were incubated in darkness for the duration of the experiment.

Hormone treatments of whole leaves derived from greenhouse-grown plants were

conducted by submerging the petioles in either sterile H20 (controls), or 3% sucrose or 1

flM BA. For the BA treatments, a 1 mM BA solution was also sprayed on the leaves and

allowed to dry. Chlorophyll was determined as flg chi! g fresh weight of tissue for all

samples according to Lichtenthaler (1987).

RT-PCR, RACE, eDNA library construction, screening and eDNA characterization

Total cellular RNA isolated from greenhouse-grown leaves at various stages of

development was enriched for poly(Ar RNA using the PolyATract mRNA Isolation

System III (Promega Co., Madison, WI). A mixture of poly(A+) mRNA was prepared

from the different developmental stages at a ratio of 4:2:2, senescent, mature and

immature leaves, respectively. Reverse transcription was performed on this RNA with

100 units M-MLV Reverse Transcriptase (New England Biolabs, Beverly, MA). An

additional tracer reaction with [u32p]dCTP (lCN, Costa MESA, CA) was performed to

assess the purity, quality and size of eDNA products, via alkaline agarose gel

electrophoresis and autoradiography (Sambrook et al. 1989). The remaining RNA was

removed with 0.2M NaOH and the single-stranded eDNA products were used directly in

58

PCR. Degenerate primers with the sequences, forward 5'

TGYGGNAGYTGYTGGGCNTIY 3' and reverse 5'

TGGATHGTNAARAAKAGYTGGGGN 3' [Y(CT), N(ACGT), H(ACT), R(AG),

K(GT)] (lOT, Coralville, IA, USA) were constructed based on conserved regions in

known cysteine proteases from other plants. They were used in PCR to amplify two

different products of 500 bp that were cloned into the TOPO TA vector Invitrogen

(Carlsbad, CA). Sequence analysis (BMBITF, University of Hawaii at Manoa) confirmed

that two different PCR products, that shared high sequence identity with cysteine

proteases, were cloned. The inserts were used as probes to screen the eDNA library

according to Sambrook et at. (1989). The library was constructed from the mixture of

leaf poly(A)+ mRNA (ratio described above) using the ZAP-eDNA synthesis kit from

Stratagene (La Jolla, CA). Two different cysteine protease cDNAs were isolated,

sequenced, and designated, anth16 and anth17. Rapid amplification of 5' cDNA ends

(RACE, GeneRacer, Invitrogen, Inc.) was conducted to complete the full eDNA

sequences and verify the original eDNA clones. In addition, a cDNA encoding cab was

isolated via RACE.

All sequences were analyzed using the GCG package (Madison, WI), BLAST

(www.ncbi.nlm.nih.gov/BLAST), Prosite (www.expasy.ch/tools/scnpsitl.htmI), SignalP

(www.cbs.dtu.dk/services/SignaIP). ClustalW (dotimgen.bcm.tmc.edu:93311multi­

aIign/Options/clustaIw.html), Swiss EMBnet (www.ch.embnet.org/software/BOX_form)

(Boxshade) and links on the CMS MBR homepage (restools.sdsc.edu/).

59

Northern blot ana(vsis, probe construction, and band quantification.

RNA concentrations were determined spectrophotometrically and verified using

the Kodak 1D scientific imaging system (Kodak, New Haven, C1). Total cellular RNA

(3-5 ug) was denatured and loaded on a 0.75% formaldehyde agarose gels as described

(Hoffer and Christopher 1997). After electrophoresis and transfer to Genescreen plus

(NEN, Boston, MA), (Sambrook et al. 1989) the blots were prehybridized for 6 hours

and then hybridized to [a32p] dCTP-labeled probes. The probes were assembled as

follows: Anth17 full-length cDNA clone, Anth16 overlapping 5' and 3' regions from

RACE,psbA 1.3 KB fragment from arabidopsis, cab overlapping 5' and 3' regions from

RACE, ubi 1.3KB eDNA clone from Pineapple Library. The photosynthetic genes were

used as a reference for nuclear and chloroplast photosynthetic gene activity during

development (Rapp et al. 1992; Oh et al. 1996; Hajouj et al. 2000). Labeling was

performed with the decaprime kit (Ambion, Austin, TX) and unincorporated radioisotope

was removed with ProbeQuant G-50 Microcolumns (Amersham Pharmacia, Little

Chalfont, UK). Hybridization and prehybridization were done in 50% deionized

formamide, 5x SSPE and 2.5% SDS at 42°C for 24 hours. After hybridization, blots

were washed 3X for 30 minutes in 0.5X SSPE/1% SDS at 42°C before exposure to

Classic blue sensitive X-ray film (Molecular Technologies, St. Louis, MO). The blots

were exposed to a Cyclone storage phosphor screen, and radioactive bands were

quantified using the Optiquant imaging system (Packard Instrument Company, Inc.

Meriden, C1). Data (digital light units, DLUomm-2) was normalized to uniform probe

length and by subtracting background DLU. The mean ± standard error was calculated

for three replicated experiments using the highest value for each probe as 100%.

60

Results

Isolation and characterization of two cDNAs encoding cysteine proteases from anthurium

We sought to determine whether senescence-dependent cysteine protease genes

that were homologous to arabidopsis sag12 were present in anthurium. Primers were

designed, based on conserved amino acid sequences in plant cysteine proteases, and used

in RT-PCR of a mixture of poly(A)+ mRNA from different tissues at a ratio of 4:2:2,

senescent, mature and immature leaves, respectively. The mixture was used to increase

the probability of obtaining more than one developmentally expressed gene family

member. Two different PCR products were obtained that were homologous to cysteine

proteases. They were cloned and used as probes to screen a cDNA library made from the

same mixture of poly(A)+ mRNA. The resulting nucleotide and deduced amino acid

sequences of the full-length cDNAs corresponding to the two different cysteine proteases

(termed anth16 and anthl7), are shown in Figures 4.1 and 4.2 respectively.

The boxshade analysis of the deduced amino acid sequences of anth17 and anth16

shows highly conserved regions with both dieot and monocot cysteine proteases (Fig.

4.3). Anth16 and Anth17 contain several characteristic regions, such as the highly

conserved Cys-His-Asn catalytic triad, type II Gly residues, and several other conserved

cysteine residues (Fig. 4.3) (Kamphuis et al. 1985; Cohen et al. 1986; Watanabe et al.

1991). Both proteins also contain a hydrophobic signal peptide (Fig. 4.1 and 4.2), a

positively charged hydrophilic propeptide sequence (Fig. 4.9), and a C-terminal region

61

CGACTGGAGCACGAGGACACTGACATGGACTGAAGGAGTCGACTGGAGCACGAGGACACTGTACATGGACTGAAGGAGTAGAAAAAGA

100GCCATCGGTCTACAGCTTCCCACGATGGGCAATGGAAGGATCACAGTGCTACTCCTTTCCGTCGTGTTGCTCTCAGTTGCTCCCTCTGGG

MGNGRITVLLLSVVLLSVAPSG

200~TTGCAGCGCTGGGGAGTGGCCGAGCTCCGGGCAGGGGCCGGAGGACGTGGGGGCCGGTGGTGTGGAGGGAGGGCAGGAGCTGTTCGAG

SCSAGEWPSSGQGPEDVGAGGVEGGQELFE300 50

CGGTGGATGGAGAAGCACCGCAAGGTGTACGCCCACCCGGGGGAGAAGGCCAGGAGGTACGCCAACTTCCTGAGCAACCTCGCGTTCGTGR W M E K H R K V YAH P G E K A R R Y A NFL S N L A F V

400AGGAAGCGGAACGCCGAGGGGAGGCGGGCGCCGTCGTCGGGGCAGGGCGTGGGGATGAACGTGTTCGCGGACCTGAGCAACGAGGAGTTCR K RNA E G R RAP SSG Q G V G M N V FAD L S NEE F

AGGGAGGTGTACTCGTCGAGGGTGCTGAGGAAGAAGGCCGCGGAGGGGAJaaAGCGAGGCGGAGAGCCGGGGAGGGGAGAGTGGTGGCGREVYSSRVLRKKAAEGRGARRRAGEGRVVA

600GGGTGCGACGCGCCGGCTTCGCTGGATTGGAGGAAGCGGGGGGCTGTCACGGCGGTGAAAAACCAAGGCGATTGTGGGAGTTGCTGGGCAG C 0 A PAS LOW R K R G A V T A V K N Q G D C G S C VI A

150 700 •TTCTCTTCCACGGGAGCAATGGAAGGGATAAACGCCATTACCACAGGGGAGCTCATCAGCCTCTCCGAGCAAGAACTGGTCGACTGTGACF SST GAM E GIN A ITT GEL I S L SEQ E L V 0 C 0

~88ACCACCAACGAAGGCTGCGACGGGGGTTACATGGACTACGCATTCGAGTGGGTCATCAACAATGGAGGAATCGATTCAGAAGCCAACTACT T NEG COG GYM 0 Y A F E VI V INN G G IDS E ANY

CCCTACACCGGCCAGGCGGACAGCGTCTGCAACACCACAAAGGAGGAGATTAAAGTGGTGTCAATCGATGGCTACGAAGATGTTGCAACAPYTGQADSVCNTTKEEIKVVSIDGYEDVAT900 250AGCGAAAGCGCTCTCCTTTGTGCAGCCGTTCAGCAACCGGTCAGCGTGGGGATCGACGGATCGTCACTAGACTTCCAATTATACGCCGGASESALLCAAVQQPVSVGIDGSSLDFQLYAG

1000GGGATCTACGACGGCGACTGCTCGGGAAATCCCGACGACATCGACCACGCCGTGCTGGTCGTCGGCTACGGGCAGCAGGGAGGCACCGAC

G I Y 0 G D C [POOO GNP 0 DID ~ A V L V V G Y G Q Q G G T D

TACTGGATCGTGAAGAACTCGTGGGGAACCGATTGGGGGATGCAGGGATACATCTACATAAGGAGGAACACCGGGTTGCCCTACGGCGTCY VI I V K N S W G T D VI G M Q G Y I Y I R R N T G L P Y G V

TGCGCCATCGACGCC:TGGCCTCCTATCCc!i&~~GCAATTCGCACCCGCCGCCACTCCCCCGTCACCAGCGCCACCGC~~~GTCGCCGC A I DAM A S Y P T K Q F A P A A T P P SPA P P P P S P

1300CCGCCGCCGCCAACTCCCCCTAGCCCTTCACCATCTCAGTGCGGAGACTACTCATACTGCCCGTCGGATGAGACTTGCTGCTGCCTCGTGPPPPTPPSPSPSQCGDYSYCPSDETCCCLV

fJl&GAGTTGGGTGGCTTCTGTCTCATCTATGGCTGCTGCGCTTACCAGAATGCGGTGTGTTGCACCGGGACTGTCTACTGTTGCCCACAAGACELGGFCLIYGCCAYQNAVCCTGTVYCCPQD

1500TACCCAATCTGTGATGTGCCAGATGGGCTCTGCCTCCAGCACCTTGGAGATGTTGTGGGTGTGGCGGCAAGGAAGCGGAAGCTGGCCAAAYPICDVPDGLCLQHLGDVVGVAARKRKLAK

450 1600CACAAGTTCCCATGGACGAAAGCTGGAGACACGCCCCAGCAGTACTACCAGCCATTGCTGTGGAAGAGAGATGGAGTTGCAGCACTGCGGHKFPWTKAGDTPQQYYQPLLVlKRDGVAALR

NdbTGAAAACCGAAGACAGATAATTCTATCTGTTTTCACTTTGGATCAATGCTTGTTCCGGGGAATAACTATGATGCTCGTTGTCTTGATGGC

CATATTCTTTCCTGGGTCTGGAGTTTTTGGACACCATATGTAACTTATATTAGAACTCATCCTTGCGGATCTCTCTCTTATCTAAATAAA

1800ATTTCCAAACGTCGA

Figure 4.1. Nucleotide and deduced amino acid sequence of anth16. Computer analysis and assembly ofcDNA clone sequences was performed with the GCC package. Marked features include the predictedsignal peptide sequence (underline) and catalytic triad residues (Cys-Ris-Asn) marked with a circle.

62

CMGrPCN:J'{fCCCCCGGGCCCTCTCTCACCTTTGAGCCTTCGACGCCGCTCGGGGAGGAACGAGATGGGTGTGCTCAG=CCGATGAM G V L RAP M M

100TGGTCCTCCTCCTCCTCCTGGCCCTGGTGGCCGCGAGTTCCAGGGGGATCGTGGCGGAGCGGACGGAGGAGGAGGTGAGGCTGCTGTPCGVLLLLLALVAASSRGIVAERTEEEVRLLYE

200AGGGGTGGCTGGTGGGAAACGGCAAGGCGTACAACCTGCTGGGTGAGAAGGAACGCCGCTTCGAGATCTTCTGGGACAACCTCCGCTPCA

G W L V G N G KAY N L L G EKE R R F ElF WON L R Y I50

300TCGPCGACCACAACCGGGCGGAGAACAACCACTCCTACACACTCGGCCTCACCCGCTTCGCCGACCTCACCAACGAGGAGrACCGCTCCA

DOH N RAE N N H S Y T L G L T R FAD L T NEE Y R S T

400=ACCTGGGGGTGAAGCCCGGCCAGGTGCGGCCCCGCAGGGCGAACCGGGCCCCCGGCCGCGGCCGCGACCTGTCCGCCAACGGCGACGJOL G V K P G Q V R P R RAN RAP G R G R 0 L SAN GOD

500ACCTGCCGCAGAAGGTGGACTGGAGGGAGAAGGGGGCCGTCGCPCCCATCAAGGATCAGGGCGGCTGTGGGAGCTGCTGGGCCTTCTCCA

L P Q K VOW R E K G A V A P I K 0 Q ~gG C G S ~ WAF S T

CTGTGGCTGCAGTGGAAGGCATCAACCAGATCGTTACGGGGGACTTGATAGTCTTGTCTGAGCAGGAGCTAGTTGATTGTGACACTGCCTV A A V E GIN Q I V T G 0 L I V L SEQ E L V 0 COT A Y

700ACAPCGAGGGCTGCAPCGGAGGGCTCATGGACTATGCCTTCCAGTTCATCATCTCCAATGGTGGCATCGACACCGAGGAGGATTACCCCT

NEG C N G G L M 0 Y A F Q F I I S N G G I D TEE 0 Y P Y200

ACAAGGAACGCGATGGGCTGTGCGATCCCAACAGGAAAAACGCCAAGGTTGTCTCCATTGATTCGTACGAGGATGTAT~ggAGAACGATGK E R D G LCD P N R K N A K V V SID S YEO V LEN 0 E

900AGCATGCCCTCAAMCAGCAGTCGCTCATCAPCCTGTTAGTGrTGCTATTGAAGGTGGTGGCCGGAGTTTCCAGCTCTACAAATCGGGCA

HAL K T A V A H Q P V S V A lEG G G R S F Q L Y K S G I250

TNrTCGATGGGAGNrGTGGCATAGATCTTGATCATGGTGTTGTTGCAGrAGGATATGGCACGGAGAGTGGGMGGPCTACTGGATCGTGAFDGRCGIDLDHGVVAVGYGTESGKDYWIVR

• 3001000

GGAACTCGTGGGGTAAAAGCTGGGGAGAGGCTGGATACATCAGMTGGAGCGTAATCTTCCTAGCTCCAGCAGTGGCMGTGCGGGATCGNSWGKSWGEAGYIRMERNLPSSSSGKCGIA

• 1100CCATTGAACCCTCGTACCCCATAMGAAGGGCCAAMCCCACCCAAACCTGCACCATCCCCTCCATCACCTGTGAAGCCTCCCACTGMT

I E P S Y P I K K G Q N P P K PAP S P P S P V K P PTE C1201jl50

GCGACAACTACTACTCCTGTCCAGAGAGCACTACTTGCTGCTGTGTCTATGAGTATGGCAMTACTGCTTTGCATGGGGNrGCTGTCCTCON Y Y S C PES T T C C C V Y E Y G K Y C F AW G C C P L

1300TAGTAAATGCTGTCTGCTGTGPCGATCATAGCAGCTGCTGCCCTCATGACTACCCAGTTTGCANrGTTAAGCAGGGAATATGCCTCGCTA

V N A V C COD H S S C C PHD Y PVC N V K Q G I C LAS400 1400

GCAAAAATAACCCACTTGGAGTGMGATGCTGAAACGCACTCCTGCAAAACCCCACCAGGCTTTCTCCGGAGCGGAGGGTGGAAGGAGCAK N N P L G V K M L K R T P A K P H Q A 4~0 S G A EGG R S S

1500GCATATGATMGTAGGTGCTTTCAAATTAAGCCATCGTACTAGTATATGCCTTTAGATGGTAGCTGGGCGCATAGTAGATGGCTTCGCCA

I1600

TGATTGAAGTGCACTGCCCCATCCATGGCTGCCACAAAGTAMCTATCGNrAGTGTCTGCTCAGrTTATTGGATCTGTGTATATCCTTAT

TTTCCATCGCAAGMGGATAGTACTTCAATTTTATTTCCMTGTACATTAAACATMTTCATCTATGTGCTTTCTMJJ:,~~k

Figure 4.2. Nucleotide and deduced amino acid sequence ofan/h] 7. Computer analysis and assembly ofcDNA clone sequences was performed with the GCC package. Matked features include the predictedsignal peptide sequence (underline) and catalytic triad residues (Cys-His-Asn) matked with a circle.

63

A17PVZXOZBVBBATA16XC

1 U8LALVAASSRGIVARR1 KSIISYDNABODKATMRT1 S RGGGLMSIVSTGSRT1 LL SLAAADKSIVSYGBRS1 LAPAPALGVPLTBIDLAS1 --------·-)(AKPIPIALALVA SP SIAOS-IPPTBlDLAS1 -·····-------)(ALKBHOIPLPVAlPSSPC.SITLSRPLD1 MGBGRITVLLLSVVLLS PSGSCSAGBWPSSGOGPBDVGAGQ1 -KAT8BSXITILIPLTTV TSISTK PSBPSILBGQBBDILSS

A

A17 163PV 160ZM 166OZ 162BV 165BB 163AT 163A16 179MC 176

DRSSDBYSDBYS

.. ~ .

•• ••

IPGQVRPRRANRAPGR . LSABGD~~~~rD'--PRRRL PS APRVGB

TR--··-POR - LGA' AADNBRRI·--·-PRRB' VSD TLAADNB

SRVRBBLSLSGG DGGPRTGDADHLPIIQRBRSQRGIQ NTGSrKTBNVGSLPIGVSALSSOSQTIMSP QNVSSG

SSRVLRlIAA.GRG~RAG.GRVVAGCD

SIVIGSRSNILIMGGVKRRMSVSSRTCD

PSPAPPPPSPP~~PISPSISOIGDlSTlPSDB.~IG~I.YQNlMllTGTV.1PPPPAPPSPP~PIIPTPPAlSIIGDPB~ADQ I~YR~I YSDlYJlKRSA S

76 ·--BNNBIT76 ···AIJf1lTT82 A-DAGVBSP78 A-DAGVB8'R79 - - - KRDMP r76 ---IKDAPYli.74 S-IPAGRTn89 GRRAPSSGQG88 --RKSBLDRT

A17PVZKOZBVBBATA16MC

A17 250PV 247ZM 253OZ 2UBV 251BB 2UAT 249A16 265 SMC 264

A17 336PV 333ZM 338OZ 334RV 337BR 335AT 335A16 353MC 352

443 IIIIDIPD~BLGDVV~AR~LIlaIPP.TIAGDTPOO~RDGVAALR--­442 IIIIDIOAlYlYIHSAITP~AI~LIlaIMP.BIIBBTIIBBPQPLA.HRRPPAAAA---

A17PVZMOZBVBBATA16MC

416413418414

8l.-PL KL p. BOAPSGABGGRSSI----·····_-·-----·SIRNP. ALR P BGAPAGRKVSNA-·-···-------------·GIDSPLS SVlATKRTLAIRTOLSPATQLTALISSALRIRSVALORQQPO

~a~trl~~RDSPLAlXAL~LllPHLS'LPOHOIISSA------------------

Figure 4.3. Alignment of amino acid sequences derived from anth16 and anth 17 (Anthurium andraeanum)with those from 8 different plant proteases. Black boxshade denotes complete conserved amino acids,while grey boxshade denotes conservative amino acid replacements. Anth 16 and anth 17 are labeled alongwith other plant species abbreviations. The GENBANK Accession numbers, [PY] kidney bean (T12041),[ZM] com (TO I207), [OS] rice (P25776), [HY] barley (AADI0337), [HH] daylily (P43 156), [AT] sagl2(AAC49135), [Me] Matricaria chamomil/a (AAD54424). Key features * cysteine disulfide bonds ­Type II glycine residues C Type II glycine residues missing from anth16 • amino acids that form thecatalytic enzyme site. important tryptophan residues. A proline-rich segment is indicated by black bar,including the additional inserted region found in anth16 identified with a thicker black bar region. APotential targeting signal is boxed at the C-terminus of anthl6.

64

also predicted to be subject to post-translational cleavage (Abe et aJ. 1987; Watanabe et

aJ. 1991; Yamada et aJ. 2000).The cysteine proteases compared (Fig. 4.3) can be divided

into two groups based on the presence of an -100 amino acid c-terminal extension in the

predicted polypeptides from anthurium, kidney bean, corn, rice and chamomile. The

enzymes from arabidopsis, daylily and barley lack the c-terminal extension. Of the two

clones, anth17 is most closely related to seven out of eight of the plant cysteine proteases

analyzed, such as maize, rice, bean and, of special note, arabidopsis SAG12 (Table 4.1).

Anth16 is more closely related to a cysteine protease from chamomile. Interestingly, the

polypeptides for ChaTP and Anth16 contain a novel 10 residue proline-rich insertion

beginning at positions 372 and 373, respectively.

Anthurium 17 Anthurium 16Zea maize 77 58

mir3 cysteine proteasePhaseolus vulgaris 76 59

cysteine protease precursorOryza sativa 76 57

Alpha chain cysteine pro.Brassica napus 67 56

COT44 cysteine proteaseHordeum vulgare 62 58

cysteine protease precursorHemerocallis ssp. 64 54

Thiol protease Sen102Matricaria chamomilla 52 60

Thiol proteaseArabidopsis thaliana 58 51

SAG12 protein

Table 4.1. Percent identity and similarity (in parenthesis) between the deduced amino acid sequences ofcysteine proteases from 8 plant species and with anth16 and anth17 from Anthurium andraeanum.

65

Because a senescence-regulated system was identified in the transient reporter gene

expression assays (Table 3.1), we conducted northern blot hybridization analysis to

determine if either anth16 or anth17mRNA levels were regulated during senescence. As

a control for photosynthesis genes that are expected to be down-regulated during

senescence (Rapp et al. 1992; Dh et al. 1996; Buchanan-Wollaston and Ainsworth 1997;

Hajouj et al. 2000), we also isolated a cab cDNA, which encodes a light harvesting

chlorophyll-a,b-binding protein. It was used in conjunction with a heterologous barley

chloroplast psbA gene (encodes D1 subunit of P5II) and pineapple ubi cDNA (ubiquitin).

The samples consisted of total cellular RNAs from five different stages of leaf

development, including young (YG) and mature healthy (MG) leaves and leaves from

three stages of senescence (51, 52, 53). The three stages of anthurium leaf senescence

were defined based on chlorophyll levels, as shown in Figure 6 (51: 60%-90%, 52: 30%­

60%,53: 5%-30%). In Figure 7, anth17 mRNA levels are low in healthy green leaves,

while they increase during senescence, reaching peak levels at 53. In contrast, anth16

mRNA levels are undetected during senescence and are highest in young leaves. The

mRNA levels for genes encoding the photosynthetic apparatus (nuclear-encoded cab and

chloroplast-encoded psbA) are high in healthy leaves and decrease markedly during

senescence. Cab mRNA levels decrease markedly compared to psbA mRNA levels. The

psbA mRNA levels may be maintained longer during senescence than cab because the

highly stable psbA mRNA has a half life of 48 hrs (Kim et al. 1993). Ubiquitin gene

expression increased markedly from MG to 53 and this may be associated with the

numerous proteolytic processes occurring during senescence.

66

535251MGYG

B

.--f--

rt- - -

r-- -l-

I-

f- r-ho

800

700

600

500

400

300

200

100

EtBr

AYG

anthl7

anthl601

........01::l

cab >.~

Q.0...

psbA .Q~

U

ubi

.VG _MG &lS1 DS2 DS3

100%

80%

60%

40% I

20%

0%

[

[

nanth17 anth16 cab ubi psbA

Figure 4.4. Expression of anth17, anth16, cab, psbA, and ubi mRNA in leaves at different developmentalstages (A). Northern blots carrying RNA isolated from leaf tissue were hybridized with the indicated [32p]_labeled probes. (B) Histogram displaying chlorophyll content ofdevelopmental leaves. YG refers to youngleaves, fully expanded, but slightly fleshy of a brownish-green color. MG refers to mature leaves, fullyridged of a rich green color. 8 I, 82, and 83 indicate senescing leaves determined by chlorophyllconcentration (81:90-60%, 82 60-30%, 83 30-5%). (C) Histograms of gene expression duringdevelopment for each probe set. The mean ± standard error was calculated for three replicated experimentsusing the highest value for each probe as 100%.

67

Synthetic cytokinin, BA, represses the expression ofanth17

One of the defining characteristics of sag12 expression is its repression upon

treatment with cytokinin. Sucrose also can affect senescence (Sheen 1990; Krapp et al.

1991; Jang et al. 1997b; Noh and Amasino 1999a). Therefore, Figure 4.5 provides the

analysis of synthetic cytokinin treatment (BA, 1,uM solution/1mM spray) and sucrose on

anth17 expression relative to psbA expression in excised whole leaves placed in darkness

for 10, 20, 30 and 40 days. Leaves incubated in water served as a control. Whole leaves

were selected 10-15 days after full expansion. The BA-treated leaves were also sprayed

with a 1 mM BA solution, similar to the treatment commercial cut anthurium flowers

received prior to sale to increase shelf life (paull and Chantrachit 2001). Anth17 mRNA

levels were significantly lower in BA treated leaves during all time periods (Fig. 4.5).

The levels of psbA mRNAs were higher in the SA treatments for 10 and 40 days relative

to controls, but were not significantly different at 30 and 40 days. Sucrose repressed

psbA expression, but did not repress anth17 expression in whole leaves. This is

consistent with previous studies that showed sugars repress photosynthetic gene

expression (Sheen 1990; Jang et al. 1997).

In the experiment in Figure 4.5, it was not possible to use leaves at the exact

same stage of development. Thus, an additional approach was conducted using leaf

disks, which were harvested from a single leaf at the mature phase in each trial. A

second advantage of the leaf disks is a more uniform treatment of cells within the disk.

The leaf disks were incubated by floating on a 1,uM SA, sucrose, or an H20 solution for

3, 8, 14 and 20 days. Anth17 and psbA mRNA levels were measured via northern blot

hybridization analysis. In the SA treatments, anth17 mRNA levels were 50 to 75% lower

68

A BA treated whole leaf Sucrose treated whole leaf Control

10d 20d 30d 40d

anth171 ",.......

psbA

B psbA snth17

100% 1~

cS"80%]

80% a$UCIro..

1 00" 1_w_

a6ucro.e

I 80%_w_

I 80%

j 40% I 40"'-

20%120%

0% 0%10d 20d 30d 40d 10d 20d 30d 40d

Whole Leaf Hormone Chlorophyll

300

o 10d 0 20d 0 30d • 40d

200

100

o

400

800

700

600

~5' 500

i

c

Water Sucrose SA

Figure 4.5. Expression of anth17 in comparison with photosynthetic transcrifl psbA under cytokinintreatments. (A) Whole leaves were excised with petiole intact and incubated in darkness for 40 days withwater serving as a control. Leaves were harvested at corresponding intervals and RNA was extracted.Expression of anthl7 and psbA is indicated. Northern blots carrying RNA isolated from leaf tissue werehybridized with the indicated [32P]-labeled probes. (B) Histogram analysis of anth17 and psbA asexpressed in specified treatments.

69

A

anth17

psbA

EtBr

B

BA Sucrose

snth17 ".bA

100% 100%

lICJ'l(, 80%

1 1I 80"" : I

lICJ'l(,

I 40%~ j 40%1I

20%1 20%

0%' 0%W_ SA Sucro.. Wat« SA SUCI'08e

Figure 4.6. Leaf discs were excised from a single leaf and incubated for a time interval of 3, 8, 14, and 20days in 1uM BA, 3% sucrose, with water serving as a control. Expression of anthl 7 and psbA is indicated.EtBr staining is included to verii)' equal loading. (B) Histogram analysis of anth17 andpsbA as expressedin specified treatments. Histograms ofexpression data were calculated using the PhosphoImager.

70

relative to water controls for 3, 8 and 14 days (Fig. 4.6). No difference was observed

after 20 days, suggesting the effect of BA was breaking down. In the sucrose treatments,

anth17 mRNA levels were lower in the 3, 8 and 14 day periods relative to water controls.

The levels of psbA mRNA were not significantly different in the BA compared to the

water treatments, but decreased somewhat (25%) in the sucrose treatment (Fig. 4.6). The

lack of a marked effect of the treatments on psbA mRNA levels could be related to the

shorter time intervals (3-20 days, Fig. 9) relative to 10-40 days (Fig. 4.5) used to measure

this highly stable mRNA (Kim et al. 1993).

Discussion

RNA Isolation and Manipulation

Studying the molecular genetics of anthurium has been hampered by a lack of

suitable protocols for the extraction and analysis of DNA, RNA, and proteins. In order to

effectively research genes and promoters of any anthurium species, a suitable RNA and

DNA extraction method was developed to deal with the high concentration of phenolics,

polyphenolics, and polysaccharides located in all anthurium tissues. Phenolics are

compartmentalized in intact cells. During homogenization for nucleic acid recovery,

these compounds are oxidized and then interact irreversibly with proteins and nucleic

acids (Schneiderbauer et al. 1991).

Phenolics and polyphenols are characterized as having a high oxidation potential.

Phenolics most commonly oxidize to form quinones. These compounds are then

covalently coupled with nucleic acids preventing their isolation and extraction. The

nitrogen of the pyrimidine and the hydroxyl group of the carboxylic acid component of

71

RNA is highly reactive with phenolic components forming molecular aggregates. These

interactions can also occur with "face to face" and "edge to face" interaction of aromatic

surfaces, protonation of amides, hydrogen bonding, dipole-dipole, and dipole-induced

dipole interactions leading to the crystallization of the structures. These structures can

exist alone preventing separation or can form large molecular aggregates that sequester

proteins, carbohydrates, phenolics and nucleic acids (Sanenger 1984; Haslam 1998). The

resultant solution we observed in our attempts to extract RNA appeared in many forms.

It has resembled a dark brown mixture that becomes extremely fluffy during re­

precipitation with salts, extremely viscous solutions containing many polysaccharides,

and a solution containing no RNA at all due to its removal during purification. These

types of solutions contained little or no recoverable nucleic acids available for analysis.

Many attempts were conducted to isolate total RNA from anthurium using

traditional extraction methods (Sambrook et al. 1989), guanidinium isothiocyanate­

phenol-chloroform (Chomczynski and Sacchi 1987), high phenolics extractions

(Schneiderbauer et al. 1991; Levi et al. 1992), removal of polysaccharides, additional

precipitation steps, and commercially available RNA extraction kits (Qiagen, Valencia

CA; Ambion, Austin TX; Promega, Madison WI). None of these methods was suitable

for isolating high quality DNNRNA. It was determined that a broad variety of

secondary compounds, especially phenolics found in anthurium leaves, reacted with the

RNA and bound irreversibly, resulting in low 260/280 ratios. The RNA was also un­

suitable for any further manipulation such a template for cDNA synthesis and subsequent

polymerase chain reactions. This is a typical result presented other attempts which

72

extract RNA or DNA from recalcitrant tissues (Schneiderbauer et al. 1991; Levi et al.

1992).

To extract high quality RNA from anthurium tissues of all developmental stages,

we assembled a variety of methods to create a reproducible technique. The core

extraction is based on a method published from researchers at the University of Hawaii

(Champagne and Kuehnle 2000). It is suggested that introducing phenol during the early

part of the extraction procedure may itself be responsible for an adverse reaction (Levi et

al. 1992). The extraction does not introduce phenol until after the cellular material and

non-nucleic acids have been removed by replacing early phenol/chloroforms steps with

pure chloroform extractions. Chloroform extractions are sufficient enough in removing

proteins and denaturizing ribonucleases (Murray and Thompson 1980). 13­

mercaptoethanol and PVPP are two components present in the extraction buffer that

make a significant difference in preventing the oxidation of phenolic compounds.

Polyvinylpolypyrrolidone (PVPP) and Polyvinylpyrrolidone (PVP) have been

used in multiple RNA isolation protocols to reduce oxidation, to bind phenolics, and to

reduce contaminating substances. In an aqueous solution, PVP has a loose, random-coil

type of conformation analogous to that of proline rich proteins and displays a strong

affinity towards dissolved aromatic compounds including phenols (Molyneaux and Frank

1961). PVPP is an insoluble cross-linked polymer of PVP and resembles similar

properties to PVP, but differing in solubility. PVP is used in a many extractions to bind

contaminating phenolics by forming a complex by hydrogen bonding thereby protecting

nucleic acids. This generally allows the phenolics to be separated from the RNA when

combined with a phenol extraction of acidic pH. Insoluble PVP is preferred in extractions

73

since soluble PVP might obstruct or co-precipitate with the RNA. PVP may also be

incompatible with phenol and require an ethanol precipitation prior to the introduction of

phenol (Salzman et ai. 1999). p-mercaptoethanol is a well known and well used strong

reducing agent. In the absence of PVP, it has been shown to directly affect the quality of

extracted RNA when present in high enough concentrations (Lal et ai. 2001). Both

compounds were used to facilitate the rapid removal of phenolic compounds during

extraction, inhibiting oxidation reactions, which cause irreversible damage to the RNA.

Three versions of PVP/PVPP were used, a high molecular weight PVP (360,00

kDa), a low molecular weight PVP (40,000 kDa) and PVPP. p-mercaptoethanol was

added at a concentration of 550mM and the extraction was conducted in 15 ml to

maximize the dilution of polyphenolic compounds. Both PVP extractions seemed to

produce RNA at a mediocre quality, contaminated with polysaccharides and genomic

DNA but free of phenolics. Occasionally interfaces became unclear and a high loss of

RNA occurred during phenol/chloroform extractions. The substitution of PVP with

PVPP resulted in cleaner extractions and higher quality RNA. This method was also

reproducible. It appeared most PVPP was transferred to the organic phase in the first few

steps but was highly effective in eliminating phenolic contamination. PVPP also did not

interact with phenol when it was introduced. A white pellet may form following the first

precipitation, (2-propanol and Sodium Acetate) depending upon the preciseness of the

phase transfers during the extraction, and contains insoluble material and RNA.

However, if the pellet is exposed to gentle agitation at room temperature for a few hours,

high quality RNA is obtained after the insoluble fraction is centrifuged and removed.

This insoluble precipitate may be residual PVPP carried over in the precipitation. If in

74

fact this precipitate is PVPP it is able to be effectively removed from the RNA when

water is added. This is most likely due to its reversible binding to RNA which enables

PVPP to release the RNA when in a lower ionic solution. RNA yields are reasonable

(50-1001lg per gram of tissue), pure, and reproducible. Figure 4.7 shows 10Ilg of

developmental RNA fractions extracted from anthurium leaf tissue run on a 1.5%

FonnaidehydelMOPS gel and stained with ethidium bromide. It clearly shows the

quality of RNA does not diminish until the leaf is severely senescent and the low

presence of genomic DNA contamination. Figure 4.8 shows 14 separate leaf of hormone

treated RNA. This RNA was loaded directly on the gel (Sill) to show the reproducibility

of the extraction. This extraction is not efficient, fast, or cost effective but it has worked

on all tissues ofanthurium and Pineapple plants.

Figure 4.7. lOJ.1g ofTotaJ RNA loaded on a 1.5% Fonnaldehyde MOPS gel and stained with EthidiumBromide. RNA samples are from Immature, Healthy, Sl, S2, and S3 leaves (left to right).

75

Figure 4.8. 5J1l ofTotal RNA loaded on a 1.5% Fonnaldehyde MOPS gel and stained with EthidiurnBromide. RNA samples were extracted from 1 gram of leaf disc tissue.

Analysis ofAnth16 and Anth17protein sequences

Two genes were sequenced from cDNA created using mRNA isolated from

anthurium leaves. Comparisons of the deduced amino acid sequences with others that

have been reported indicate high homology with monocot cysteine proteases (Table 3.1).

The deduced polypeptide sequence of both cysteine proteases contain several regions

characteristic of cysteine proteases including a highly hydrophobic signal peptide, a

positively charged hydrophilic propeptide sequence, a mature protein, and a C-terminal

region also predicted to be subject to post-translational cleavage. In addition both

proteins contain the highly conserved Cys-His-Asn catalytic triad, type II Gly residues,

and cysteine residues. Figure 7 and 8 show the nucleotide and deduced amino acid

sequence of anth17 and anth16, respectively and also shows marked features that will be

discussed. Figure 4.3 shows boxshade analysis with other closely related cysteine

proteases including sag12 a known senescence-associated cysteine protease from

arabidopsis. Both clones were sequenced from cDNA created from mRNA extracted

from all developmental stages of anthurium leaf tissue. Based on the deduced amino acid

sequence, several attributes about the mature proteins can be predicted.

Anthl7 and Anthl6 have hydrophobic signal peptides located at Metl - Ala27 and

Metl-SecS respectively, which indicates the encoded polypeptides enter the secretory

76

pathway. The algorithms of (Nielsen et al. 1997; Emanuelsson et al. 2000) predict

Anth17 to be involved in the secretory pathway with a final destination outside the cel!.

The same algorithm predicts Anth16 to also enter the secretory pathway and may

associate with the plasma membrane to some degree. The prediction for Anth16 is at a

lower level of confidence than Anth17. Several papers indicate common regions that are

unique identifiers of cysteine proteases. Anth17 and Anth16 contain the conserved

catalytic triad (17:Cys154_HiS290

_Asn31 C)(16:Cys170_His30B-Asn328

) considered to be

integral to the active site of cysteine proteases. 6 Cys residues integral for the formation

of disulfide bridges are also present in their respective locations (17: 151,185,284,336)

(16:167,201,299,353). A conserved glutamine (17:148)(16:164) has been suggested to be

the proton donor necessary for cleavage of the peptide bond. When the hydropathy

profiles (Fig. 4.9) of both proteins are compared, the longer Anth16 seems to have

increased regions of hydrophobicity over Anth17.

Kamphuis, Drenth and Baker (1985) performed comparative studies based on

high-resolution structures of several thiol proteases and defined several key structural

attributes necessary for proper activity. Cysteine proteases contain specific glycine

residues that are usual!y associated with conformational flexibility of the protein that are

involved mainly in secondary structure turns. Type I residues provide conformational

flexibility and facilitate insertions and deletions in the sequence while type II are less

flexible. Serious alterations of the main-chain conformation through steric interactions

caused by the C~ of amino acids other than glycine.

77

4

3

- Anth17- Anth16

-3

I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I Io 20 40 &0 00 100 120 1+0 1&0 100 200 220 240 2&0 200 300 320 340 3&0 3eo 400 420 440 4&0 4eo SOO

I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I t Io 20 4' D' 0 100 120 140 1&0 100 200 220 240 2&0 200 300::120 040 0&0 000 400 420 440

Figure 4.9. Hydropathy profile of the deduced amino acid sequences of Anth 16 and Anth17 predictedusing the Kyle and Doolittle method. Plot was derived to compare the two cysteine protease sequenceswith each other, substituting gaps in areas to increase alignment. Gaps are indicated below the region ofintroduction with the line color corresponding to the protein with the missing segment.

Position 284 may not be serious because of the presence of glycine residues

upstream of 284 and directly following a conserved Glu-Pro-Val-Ser-Val segment.

Position 300 is of particular interest because it is directly following an important Cys

residue, and includes a 3 amino acid insertion increasing the distance between the Cys

residue and the integral His involved in the catalytic triad. This substitution increases the

distance between this highly conserved region from 5 to 8 amino acids. There is a small

number of single amino acid insertions/deletions in the mature protein region of various

cysteine proteases, however generally the interior portion of the polypeptide is extremely

conserved. Anth16 and a thiol protease isolated from cultured shoot primordia of

Matricaria chamomilla (termed ChaTP for identification purposes in this report) both

exhibit this 3 amino acid insertion as well as the missing glycine residues.

78

Mature cysteine proteases are translated into long polypeptides and then cleaved,

generally at both the NH and COOH termini, to produce a functionally active enzyme.

Oryzain, a family of rice seed cysteine proteases, follows this methodology, while some

cysteine proteases lack an extended COOH terminus tail (Abe et al. 1987; Watanabe et al.

1991; Yamada et al. 2000). Anth17 exhibits similar properties to these types of cysteine

protease and it is most likely that the two termini tails are processed to yield a fUlly

functional protein. The cleavage in the NH termini typically occurs before the first

conserved Leucine (17: Lue l3C) and near the Asp-Pro-Pro (17: Asp 351) sequence at the

COOH termini. This cleavage would produce a mature fully functional cysteine protease

of about 23.5 kilodaltons (Abe et al. 1987).

Anth16 and ChaTP exhibit slightly different properties at these two cleavage sites.

At the NH termini cleavage site, they are missing the characteristic luecine which is

replaced by a Cys-Asp-Ala motif before the highly conserved region begins at Pro147•

The COOH terminus includes a 10 amino acid, proline-rich, addition that includes the

absence of the Asn-Pro-Pro conserved motif. No conserved or special sequence has been

identified that regulates cleavage at these locations, however the Asn-Pro-Pro sequence

of long tailed COOH cysteine proteases seems to be followed by a proline-rich segment

that includes as many as 10 proline residues within a 15 amino acid segment. Anth16

and ChaTP have 17 and 19 proline residues, respectively, within a 26 amino acid

segment.

Anth16 is unique in that the inserted 10 amino acid proline-rich segment contains

a possible palindrome-like sequence that includes a Ser-Pro4 segment. The Ser-Pro4,

generally referred as Ser-Hyp4 due to the post-translational hydroxylation of proline

79

residues in specific amino acid combinations, is present in almost all types of extensin

peptides in repeats of three or more (Kieliszewski 94 for review). Extensin modules can

also be found in chimeric proteins where the protein is homologous to a certain family,

and a module, such as a tail region, is homologous to extensin. Such examples include

cysteine-rich lectins (Kieliszewski and Lamport 1994), stylar transmitting tissue proteins

(Baldwin et al. 1992), and chitinases (Sticher et al. 1992) as well as specialist proteins

involved in defense (Kurata et al. 1993), wounding (Sheng et al. 1991), and lateral root

initiation (Keller and Lamb 1989). Anth16 contains a proline-rich region that is longer

slightly more organized than most long-tailed cysteine protease propeptides. It is unclear

whether this region is of appropriate size or configuration to be considered a proline-rich

region, however TARGETP analysis of the deduced amino acid sequence suggests that

Anth16 may be specifically targeted towards the plasma membrane or cell wall, in

comparison with the more conserved Anth17 which is predicted to go outside or to

vacuole locations. Both proteins are predicted to be secreted.

Anth16 contains a COOH terminus tail sequence that includes a di-luecine (LL)

and the Tyr-based sorting motif, YXXlD (where Y referes to tyrosine, X refers to any

amino acid residues, and lD refers to hydrophobic residues with a bulky side-chain). Both

motifs regulate specific sorting of proteins through intracellular vesicle compartments by

associating with coat complexes such as clathrin (Sandoval and Bakke 1994; Mellman

1996). Although Anth16 contains both the YXXlD and the LL signals, its is not known

whether these motifs or the proline-rich sequence serve as anything that is functionally

significant for differences in targeting, location, or unique function from other cysteine

proteases.

80

It is interesting to note the inclusion of three potential signal or structural motifs

in Anth16 and characterize it as having a potential function unique in comparison with

typical cysteine proteases. Several proteins with similar signaling motifs have been

identified or suggested to exhibit protease like functions. A class of groupI allergens from

grass pollen has the characteristic catalytic triad and exhibits some protease activity after

the propeptide sequence is cleaved (Grobe et at. 2002). An arabidopsis drought-inducible

cysteine protease-like protein was identified as putatively GPI

(glycosylphosphatidylinositol) anchored (Borner et at. 2002). GPI anchors provide a

potential mechanism for targeting the protein to the plant plasma membrane and cell wall

(Morita et at. 1996; Youl et at. 1998; Sherrier et at. 1999). Extracellular proteases could

be involved in the remodeling and degradation of plant extracellular matrix (ECM)

proteins and are also involved in pathogen or wound response such as a matrix

metalloproteinase that is activated in response to pathogen infection in soybean (Uu et al.

2001).

Both Anthurium andraeanum and Matricaria chamomilla are plant species that

exhibit unique secondary metabolites. Anthurium includes a diverse combination of

phenolics, polysaccharides, and cell wall components. This type of cysteine protease

may playa role in negotiating interactions between these compounds during development

as shown by the Northern analysis. Anth16 is expressed at low levels in immature tissues

and not later in development. Analysis of the mature protein size, post-translational

modification, and sub-cellular location would help further characterize this type of

cysteine protease in anthurium.

81

Expression analysis ofcysteine proteases during development

Gene expression throughout plant development tends to fluctuate and is greatly

influenced by environmental factors. Any gene that plays a role in senescence can be

considered as Senescence-Associated Gene or SAG. These genes can be present during

other developmental stages, be induced by pathogen attack, or extremely sensitive to

hormone interactions. A gene only needs to be regulated to some extent during

senescence to be classified as a SAG. Other genes are senescence-enhanced, or

senescence-upregulated, due to increased mRNA accumulation during senescence.

However, only a select few show increasing expression from the precise onset of

senescence through its conclusion, without being present during development. These

genes are extremely important to any program which uses very specifically regulated

promoters to manipulate senescence. In general these genes are not induced by pathogen

attack, wounding, or desiccation. Overall a SAG gene is characterized by observing

enhanced transcript abundance coupled with chlorophyll loss. Many SAG mRNAs

appear to increase only after decreases in the nUclear-encoded cab mRNA, an important

protein involved with photosynthesis (Rapp et al. 1992; Oh et al. 1996; Hajouj et al.

2000). Many factors can classify genes as senescence-associated, nevertheless a

combination of factors needs to be analyzed to determine to what extent a gene is age­

dependently senescence-upregulated or regulated by other senescing inducing pressures.

It can be reasonably concluded that the anthurium cysteine protease gene, anth17,

has an expression pattern consistent with that of a senescence-associated gene. It is also

determined that anth17 is senescence upregulated and shows little or no expression

during early development and maturity. Anth17 expression increases during

82

developmental senescence and is present in increasing quantities even as total RNA

levels decline 90% as senescence progresses (Fig. 4.4A). S3 stage RNA, -10%-30%

chlorophyll content compared to healthy leaves, show this expression pattern. A

histogram of chlorophyll levels throughout development is provided (Fig. 4.4B). Anth16

shows relatively light expression and is present only in the early development of leaves.

Photosynthetic gene expression is a good indicator of senescence initiation (Hensel et al.

1993). We show that the expression of the nuclear encoded gene for light-harvesting

chlorophyll alb binding protein (cab) is down-regulated rapidly in correlation to

chlorophyll levels in senescing tissues (SI, S2, S3, Fig. 4.4). In comparison, expression

of the chloroplast encoded gene psbA shows a more progressive decline during

developmental phases SI, S2 and S3. This is consistent with the longevity of chloroplast

transcripts and a potential delayed response to signaling events involved with senescence.

The psbA mRNA levels may be maintained longer during senescence than cab because

the highly stable psbA mRNA has a half life of 48 hrs (Kim et al. 1993).

The ubiquitin gene shows baseline expression during early development and

increases expression throughout senescence. Thought to be constitutive, ubiquitin

pathway genes have been shown to increase during senescence, since the massive

degradation of proteins during leaf senescence likely involves ubiquitin tagging and

proteolysis (Park, Oh et al 1998). Northern analysis of developmental leaf RNA was

conducted several times with consistent results. The combination of many trials is

displayed in a histogram of gene expression (Fig. 4.4C). The average percent is

calculated as a function of the highest expression within each gene transcript listed.

Some radiolabeled probes were stronger than others and certain mRNA levels

83

represented stronger than others in each developmental fraction. Figure 4.4C is presented

to give an indication of overall change of expression throughout development for each

individual transcript, rather than show exact expression intensity in comparison with the

entire group. The histogram accurately reflects by providing the highest level for each

transcript sample as 100%.

This research has concluded some rules and exceptions for SAG characterization

compared to other SAG research. Detaching leaves from the plant and incubating them

in total darkness is a popular technique first reported by Thimann (1980). This technique

has been extensively researched (Becker 1993; Oh et al. 1996; Weaver et al. 1998) and

concluded to be a controlled method to induce senescence as determined by chlorophyll

concentrations, but includes mixed results. One result shows an increase in SAGs

transcripts of detached leaves incubated in darkness but not observed in natural

senescence. (Becker 1993). Another shows that 9 out of 10 SAGs are induced in older

leaves, however 2 out of 10 SAGs were induced when younger leaves were detached and

placed in darkness (Weaver et al. 1998). Most studies that use the dark detached leaf

treatment conclude that senescence-associated genes are differentially regulated when

leaf senescence is induced by different factors. Factors which induce chlorophyll loss are

most likely to mimic gene expression of natural age-induced leaf senescence.

Expression analysis of anth17 mRNA under dark induced senescence and hormone

treatment.

Cytokinins have long been associated as a senescence-retarding hormone that

delays the loss of chlorophyll and proteins (Gan and Amasino 1997; Nooden et al. 1997).

84

Cytokinin has also been shown to reestablish leaves to a pre-senescent state after they

have begun to exhibit senescent characteristics. It is considered that tissues high in

cytokinins compete more effectively as nutrient and sugar sinks. This is apparent in

developing tissues and those with apical dominance. Tissues rich in nutrients and sugars

have a longer lifespan in addition to cytokinins concentrations that regulate molecular

and signal transduction events to keep leaves actively developing and metabolizing,

preventing senescence. Cytokinins are more likely to keep pre-senescent leaves in a pre­

senescent state than to prevent SAG induction in older leaves. Cytokinins are able to

frequently block visible senescence but do not necessarily block expression of all SAGs

(Oh et al. 1996). The common idea is that cytokinins may only block a portion of

senescence progression. This is reflected by a loss in photochemical efficiency detected

in dark detached BA treated leaves (Oh et al. 1996). Although only a portion of the

senescence program is blocked, most SAG transcripts are retarded by endogenously

applied cytokinins. Most importantly for this work, exogenous treatments of the synthetic

cytokinin benzyladenine show a repression of senescence characteristics when applied on

anthurium leaves and flowers (Paull and Chantrachit 2001).

True senescence, induced by age, is a finely tuned process involving delicate

regulation of gene expression. It is clear that many different pathways may be involved

in inducing senescence. Commonly suggested methods include: drought or desiccation;

dark incubated whole plants; detached leaves in light or darkness; abscisic acid treatment;

ethylene treatment; cold treatment; wounding; and pathogen attack. A particularly

interesting report describes the induction of senescence through the placement of

"mittens" on individual leaves still attached to the plant. This type of senescence actually

85

increases if re-exposed to light. Interestingly, if a hole is punched in the mittens,

yellowing occurs in all covered areas of the leaf but not the punched portion that is

exposed to light (Weaver et al. 1998).

This result is consistent with that of Rouseaux et al. (1997) that showing far-red

induced chlorophyll loss is extremely localized. This may suggest that the senescence

response is highly localized, and possibly cell autonomous (Weaver et al. 2001). This

type of example solidifies the suggestion that a phytochrome may be directly or indirectly

involved with the light-mediated inhibition of senescence. Both continuous white light

and pulses of red light are able to inhibit senescence in detached leaves and that the red­

light effect is reversible by far-red light (Tucker 1981; Biswal et aI. 1983; Biswal and

Biswal 1984). More recently this result was reconfirmed in soybean (Guiamet et al.

1989), sunflower (Rousseaux et al. 1996), and tobacco (Rousseaux et al. 1997) when the

plants senesced more rapidly when the red: far-red ratio was decreased. These results

suggest that phytochrome involvement may be and important inducer of the senescence

pathway which has many different signifigant branches. Weaver and Amasino (1998)

present a model stating that senescence induced by age occurs in a "leaf autonomous"

fashion, while senescence induced by darkness occurs in a "cell autonomous" fashion. If

this is correct it may be possible for senescence to occur in a cell autonomous fashion in

response to wounding, stress or even pathogen attack. This remains to be elucidated but

it also suggests that phytochrome involvement may be a cellular or subcellular inducer of

senescence in all plant tissues.

Considering sugars are the main product of photosynthesis and certain sugars can

serve as signaling molecules, changing sugar levels during photosynthetic decline may

86

act as a senescing-inducing signal (lang et al. 1997). Sucrose can effectively repress the

accumulation of certain SAG transcripts when applied to detached leaves (Noh and

Amasino 1999a). However, in nonsenescent leaves, sugar accumulation can lead to a

decline in chlorophyll and photosynthetic proteins (Stitt et al. 1990; Von Schaewen et al.

1990; Krapp et al. 1991; Krapp and Stitt 1994). Sugars can repress the transcription of

photosynthetic genes (Sheen 1990) and tend to increase during leaf senescence (Crafts­

Brandner et al. 1984). The accumulation of sugars can both accelerate and delay

senescence. This is as a result of other factors in the leaf including Nitrogen and Carbon

ratios (Paul and Driscoll 1997), light (Dijkwel et al. 1997), and plant growth regulators

(Koch 1996).

In an effort to further characterize anth17 as a true senescence-upregulated gene,

a few previously described methods were attempted. Anthurium leaves 10 days after full

expansion were cut at the base of the stem and incubated in 12 hours light and 12 hours

of darkness. The leaves incubated in light seemed to senesce at variable rates lasting

from 15 to 40 days. Leaves incubated in darkness senesced in a more controlled fashion.

Leaves covered with a "mitten" type covering failed to induce senescence even after 30

days. A whole healthy anthurium leaf was cut into sections and incubated in darkness

floating on appropriate solutions. This experiment was successful but the leaf discs

showed no chlorophyll loss over 28 days. Leaves were also desiccated, wounded, and

treated with cytokinin (Benzyladenine). It was determined that dark incubation of

detached whole leaves provided the best method for inducing senescence similar to age­

dependent senescence. Extreme care was exercised in choosing anthurium leaves of an

identical developmental stage even though anthurium plants have many variable

87

attributes. Leaf discs may have been more reliable because all of the treated tissue was

provided from a single leaf, assuring a controlled developmental phase as a starting point.

Whole leaves might have suffered from "stem plugging" (PaUll and Goo 1982) resulting

in an accelerated senescence state and also differ in developmental attributes caused by

differences in age of the anthurium plant, time of harvest, and environmental pressures

during leaf maturity.

Leaf disc experiments show that anthi7 expression was moderately inhibited by

cytokinin and sucrose treatments for 14 days in comparison with the water control.

Visible markers of senescence such as a sharp decline in Chlorophyll levels were not

detected during the experiment. The leaves were incubated in the dark for 20 days

without any appreciable difference in chlorophyll concentration. The expression of

anthi7 was inhibited by cytokinins and sucrose in these treatments for up to 18 days.

Leaves floated only on water and incubated in the dark showed a consistently high level

of anthi7 mRNA throughout the experiment. Baseline levels of anthi7 mRNA in all

samples may suggest a type of wound response. Northern analysis of cab expression in

leaf disc samples showed no transcripts. This is consistent with reports that show cab

mRNA transcripts and proteins nearly disappear after 2 days in dark incubated leaves (Oh

et al. 1996; Weaver et al. 1998; Hajouj et al. 2000; Weaver et al. 2001). Northern

analysis of psbA mRNA was also inconclusive due to the stable nature of the transcript.

Whole leaf treatments show a similar pattern with developmental populations when

whole leaves are excised and incubated in darkness for 40 days. Leaves incubated in a

cytokinin solution, in combination with a cytokinin spray treatment, show delayed

expression of anthi7 and prolonged cab and psbA expression. These leaves also showed

88

a delay in chlorophyll loss compared with the control. Leaves incubated in a sucrose

solution senesced extremely rapidly as indicated in the histogram of chlorophyll levels.

Both treatments confirm that cytokinin inhibits the expression of anth17. Whole leaf

treatments may be a more reliable indicator of inhibition due to chlorophyll loss, and

comparable to developmental expression of leaves from plants. It is not surprising that

the addition of a sucrose solution to incubated leaves in the dark increased the rate of

senescence. Many studies show sugar accumulation in non-senescent leaves can lead to a

decline in chlorophyll, photosynthetic proteins (Krapp et al. 1991), and photosynthetic

gene transcripts (Sheen 1990). Both results are consistent with research that shows

cytokinins are frequently able to block visible senescence and tend to retard the

expression of some SAGs depending on developmental stage and treatment parameters

(Becker 1993; Dh et al. 1996; Weaver et al. 1998).

The results presented here are consistent with previous studies on SAG type

genes. Anth17 is a SAG cysteine protease from the Anthurium species. This protease is

most likely senescence-upregulated. Anth17 occasionally shows very slight expression

in healthy leaves, but this may be as a result of cell autonomous expression, localized

wounding, or curling of the leaf perimeter. An anthurium leaf can still be productive and

green even if wounded, torn, or under pathogenic attack, making it plausible that

senescence conditions in anthurium leaves can both be cell and leaf autonomous. Anth17

increases dramatically throughout senescence and the transcript is at its highest levels as

the leaf nears necrosis. This result is consistent with that of sag12 expression from

arabidopsis.

89

CHAPTER V

CONCLUSION AND PROSPECTIVE

The research contained in this dissertation was funded by a special grant of the

USDA tenned Tropical & Subtropical Agriculture Research (T-STAR) awarded to Dr.

David A. Christopher. Some of the program goals include providing research that

maintains and enhances production of established tropical and subtropical agriculture

products; enhancing the role of value-added agriculture in tropical island ecosystems;

developing agricultural practices in the tropics and subtropics that are environmentally

acceptable through an agro-ecosystems approach; expanding tropical and subtropical

agricultures linkages to related industries and economic sectors. This research provides a

foundation for enhancement in post-harvest quality of Anthuirium flowers and a more

productive crop. We also harvest molecular resources from anthurium in an effort to

design an auto-regulatory senescence inhibition system from anthurium components. The

research also contributes to some fundamental questions of molecular biology.

Establishing Molecular Techniques to Deal with Recalcitrant Tropical

Species

Plant Molecular Biology and Physiology are advancing fields of science that

require a thorough understanding of molecular methods unique to each individual species

being researched. Molecular methods involved with researching tropical species are

90

more difficult because of the recalcitrant nature of the plant materials and the uniqueness

of the evolutionary divergent molecular physiology. This research defines techniques

that are new developments in the field concerning the anthurium species and can serve as

a reference for future studies involving anthurium molecular biology. These techniques

include RNA and DNA extraction; successful reporter gene introduction and analysis;

transient expression; cDNA library construction; and foreign promoter evaluation, SAG

characterization, and hormone and stress treatments. These molecular tools can also be

used to successfully evaluate other economically valuable tropical ornamentals for

increased yield and post-harvest quality. The research has characterized the SAG gene

anth17 and establishes the effective regulation of Pr-SAF12 in anthurium tissues

confirming that the molecular events of senescence do occur supporting the claim that

orthologous mechanisms exist between monocots and dicots.

Our understanding of anth17, its role during development, and regulation by

cytokinins, will lead to the isolation of a SAG promoter and the eventual development of

pANTII17-ipt, an auto-regulatory senescence inhibition system that is regulated by an

anthurium SAG promoter. This system is considered a more ideal approach to molecular

engineering since the regulatory element is native to the plant. The research enables the

isolation of Pr-ANTHI7 and its comparison to other SAG promoters to determine its relative

homology and conserved regions. This type of analysis helps to further our

understanding of the regulatory elements, transcription factors and enhancer regions

involved with SAG expression.

91

Contribution to the Fields of Post-Harvest Physiology and Molecular

Biology of Leaf Senescence

Post-harvest physiology is an important science concerning many agricultural

crops. These concerns range from preserving the quality and life of a commodity from

grower to consumer, increasing the commodities resistance to the stresses of shipping and

storage, and manipulation to increase or produce beneficial compounds upon harvest.

Senescence is an extensively investigated field that includes the study of plant growth

regulators, gene expression, proteins, and metabolites contributing to a better

understanding of plant molecular physiology. Many of these processes contribute to the

overall knowledge of all plant cell and molecular systems. This research provides

additional resources to the overall goal of understanding leaf senescence by providing

information on two novel Anthurium cysteine proteases, their role in developmental,

hormonal, and stress induced senescence, and the comparison and addition of them to the

GENBANK database. This research also defines the senescence-upregulated

performance of a dicot promoter in a monocot system suggesting similarities between

dicot and monocot senescence transcription machinery. Publishing the information

obtained through this research contributes to the molecular analysis of senescence and

post-harvest physiology of tropical ornamentals. Finally, research on senescence­

regulated gene expression in a tropical species may be valuable for studies on other

tropical species involved with food, medicine, and ecosystem management fulfilling

many of the T-STAR program goals and improving the future of tropical plant research.

92

Cysteine Proteases: Form and Function for Commercial and Industrial

Applications

Hydrolases which cleave peptide bonds are generally known as protease,

peptidases, proteinases or proteolytic enzymes. Enzymatic studies on such enzymes

usually focus on those directly involved in the digestion of dietary protein, such as

trypsin and chymotrypsin, which are completely characterised. Proteases are amongst the

most studied proteins. It is through detailed characterization of the structure and function

of several proteases that they are also used as models in explaining the basics of enzyme

function. Cysteine proteases are actively researched for expression profiling during

senescence because some of the most specific age-dependent SAG genes are cysteine

proteases. In this sense, gene expression and promoter regulation of cysteine proteases is

very important for models explaining senescence.

Other proteases with varying functions, especially those pertaining to the

regulation of physiological processes, have also been studied. Proteolytic activity

involved in the processing and regulation of enzymes and hormones, cascade reactions in

the blood coagulating process, fertilization, embryo development and industrial processes

for food and feed production.

Proteases are found in all forms of organisms regardless of kingdom. Some

examples include plant proteases include papain of papaya, commonly used as a meat

tenderizer, and bromalein a mixture of several proteases of pineapple used in meat

tenderizers, chill-proofing beer, manufacturing precooked cereals, certain cosmetics, and

in preparations to treat edema and inflammation. Bromelain is also nematicidal. Animal

proteases include rennin, used in dairy industry to produce a stable curd with good flavor,

93

chymotrypsin, used extensively in deallergenizing of milk protein hydrolysates, and

trypsin, used for the biocontrol of insects and in the preparation of bacteriological media.

Proteases of bacteria, fungi and viruses are of interest due to the importance and

subsequent application of those enzymes in industry and biotechnology. Examples of

these include application of bacterial neutral and alkaline proteases from the Bacillus sp.

in fermentation and detergent industry; acid protease of Aspergillus sp. in food industry

namely, the production of cheese. Commercial application of microbial proteases is

attractive due to the relative ease of large scale production as compared to proteases from

plants and animals. However, some proteases from plants have specific functions,

substrates, and inhibitors, making them more attractive for use in unique applications.

Microbial proteases have been studied for their role in the development and

manifestation of diseases such as AIDS, cancer and acute and chronic systemic

infections. Characterization of these proteases, as with other virulence factors, provides

vital information to design new approaches in treatment, therapy and control of infectious

diseases, which include designing and production of vaccines, synthetic protease

inhibitors and pharmaceutical research. One such success story is of the HIV protease.

Structural studies of HIV protease have enabled the successful development of synthetic

HIV protease inhibitors for the treatment of AIDS patients. Similar studies are being

undertaken to develop synthetic inhibitors/2nd generation antibiotics for bacterial

proteases.

For each application there is a different protease that performs the work, or is

stable in the environment. Unique proteases may increase the efficiency or lower the cost

of production of a particular product. By performing knockout experiments with recently

94

isolated proteases, we can beller understand its role in development and subcellular

metabolism. Characterization of two cysteine protease genes, anth16 and anth17 from

Anthurium andraeanum contributes to this knowledge.

95

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