1

Characterization and Functional Analysis of ABSCISIC ACID INSENSITIVE3-like Genes from Physcomitrella patens Heather H. Marella*, Yoichi Sakata1*, and Ralph S. Quatrano2 Department of Biology, Washington University, 1 Brookings Dr., St. Louis, MO 63130, USA * These authors contributed equally to this work 2 Corresponding Author: Ralph Quatrano Address: Washington University, 1 Brookings Dr., Campus Box 1137, St. Louis, MO 63130 Email: rsq@wustl.edu Telephone: 314-935-6850 Fax: 314-935-8137 Email of authors: Heather Marella- hinkle@biology.wustl.edu Yoichi Sakata- sakata@nodai.jp.ac Running title: Physcomitrella patens ABI3

Key Words: ABI3, abscisic acid, Physcomitrella, ABI5, VP1, transcriptional regulation

Accession Numbers: PpABI3A-AB233419, PpABI3B-AB233420, PpABI3C-AB245516

Word Count: Total-9875 Summary-250 Introduction-1083 Results-1547 Discussion-1823 Experimental Procedures-1898 Acknowledgements-118 References-2259 Figure Legends-897 1 Present address; Dept. of Bioscience, Tokyo University of Agriculture Sakuragaoka, Setagaya-ku, Tokyo 156-8502 Japan

2

SUMMARY

Although the moss Physcomitrella patens is known to respond to abscisic acid (ABA) by

activating gene expression, the transcriptional components involved have not been

characterized. Initially, we used the ABA-responsive Em promoter from wheat linked to

β-glucuronidase (GUS) to determine if ABI3/VP1, transcriptional regulators in the ABA

signaling pathway in angiosperms, were similarly active in the ABA response of P.

patens. We show by particle bombardment that ABI3 and VP1 affect Em-GUS

expression in P. patens in a manner similar to angiosperms. We also show the

involvement of ABI1 in the pathway, utilizing the abi1-1 mutant allele. We isolated

three ABI3-like genes from P. patens. Using an Em-like ABA-responsive promoter from

P. patens (PpLea1), we demonstrate that PpABI3A, only in the presence of ABA,

strongly enhances PpLea1-GUS expression in P. patens. PpABI3A also enhances ABA-

induced Em-GUS expression in P. patens. In barley aleurone, PpABI3A transactivates

Em-GUS but to a lesser extent than VP1 and ABI3. PpABI3A:GFP is localized to the

nucleus of both protonemal cells and barley aleurone indicating that the nuclear

localization signals are conserved. We show that at least a part of the inability of

PpABI3A to fully complement the phenotypes of the Arabidopsis abi3-6 mutant is due to

a weak interaction between PpABI3A and the bZIP transcription factor ABI5, as assayed

functionally in barley aleurone and physically in the yeast-two-hybrid assay. Our data

clearly demonstrate that P. patens will be useful for comparative structural and functional

studies of components in the ABA response pathway such as ABI3.

3

INTRODUCTION

The phytohormone abscisic acid (ABA) not only regulates processes occurring during

seed development (e.g. desiccation tolerance), but also controls processes associated with

responses to water stress during vegetative development of seed plants (e.g. stomatal

opening and closing). Since ABA is found in most land plants (Finkelstein and Rock,

2002), and has demonstrated physiological (Goode et al., 1993; Minami et al., 2003;

Minami et al., 2005; Werner et al., 1991) and molecular responses in non-seed plants

such as mosses (Knight et al., 1995), we have decided to take a comparative approach to

determine the evolution of this response pathway as well as the role of specific regulatory

proteins and protein domains that may be conserved.

Genetic approaches in Arabidopsis have been primarily responsible for

identifying several components involved in the ABA response pathway in seed plants.

Arabidopsis ABA-insensitive (abi) mutants were isolated by their ability to germinate in

the presence of ABA (Brocard-Gifford et al., 2004; Finkelstein, 1994; Koornneef et al.,

1984) and have been extensively characterized. The abi genes that have been cloned

revealed a diverse set of proteins including ABI1 and ABI2 which encode type 2C protein

phosphatases (PP2Cs) and are ABA signaling intermediates that act as negative

regulators (Gosti et al., 1999; Leung et al., 1994; Leung et al., 1997). The ABI3

(Giraudat et al., 1992), ABI4 (Finkelstein et al., 1998) and ABI5 (Finkelstein and Lynch,

2000) genes each encode a different type of transcriptional regulator, while ABI8

represents a novel plant specific protein (Brocard-Gifford et al., 2004). We will take a

comparative approach to characterize the ABA response in Physcomitrella patens and

will initially focus on the plant specific transcriptional regulator ABI3 from Arabidopsis,

4

and its ortholog from maize VP1 (Giraudat et al., 1992; McCarty et al., 1991) and

determine if a similar regulator is found in P. patens.

ABI3/VP1-like genes have been found in various seed plants (Bobb et al., 1995;

Chandler and Bartels, 1997; Footitt et al., 2003; Hattori et al., 1994; Lazarova et al.,

2002; Rohde et al., 2002; Shiota et al., 1998), and regulate a set of proteins expressed

during the later stages of seed development. One such gene, the Em gene (Marcotte et

al., 1989), requires both ABA and ABI3/VP1 for expression (Bies-Etheve et al., 1999;

McCarty et al., 1991; Vasil et al., 1995). All of the ABI3/VP1-like genes cloned from

seed plants revealed highly conserved protein domains, designated A1, B1, B2 and B3,

starting from the N-terminal (Suzuki et al., 1997). In fact, maize VP1 can complement

the major phenotypes of the Arabidopsis abi3-6 mutant (Suzuki et al., 2001). The B3

domain has been shown to bind DNA in vitro (Suzuki et al., 1997), whereas the B1

domain is involved in the physical interaction with the bZIP transcription factor, ABI5

(Nakamura et al., 2001). The B2 domain has been shown to be responsible for the ABA-

dependent activation of ABA-regulated genes, like Em, through the ABA-responsive

element (ABRE) (Bies-Etheve et al., 1999; Ezcurra et al., 2000; Hill et al., 1996), and

facilitates the interaction with bZIP transcription factors (Hill et al., 1996) such as ABI5.

However, the mechanism by which this domain functions in seed plants remains to be

elucidated. Until recently, ABI3/VP1 was thought to function exclusively during seed

development, specifically as a component of the ABA signaling pathway involved in the

maturation and germination of seeds (Giraudat et al., 1992; McCarty et al., 1991;

Nambara et al., 1995; Nambara et al., 2000; Parcy et al., 1997). However, recent reports

have pointed out that ABI3 might have broader functions outside of the seed, such as

5

plastid development, flowering time, and outgrowth of axillary meristems (reviewed in

Rohde et al., 2000). These analyses also revealed a novel crosstalk between ABA and

auxin in seed germination and lateral root formation in Arabidopsis (Brady et al., 2003;

Suzuki et al., 2001). These data clearly indicate that ABI3/VP1 is not only involved in

the ABA-regulation of seed development and germination, but also has broader functions

in vegetative growth.

The ABA response pathway in the moss P. patens was demonstrated previously

using the ABA-responsive promoter of the wheat Em gene. The wheat Em promoter can

be activated by exogenous ABA in a transient assay using protonemal tissue as well as in

stable expression lines of P. patens (Knight et al., 1995). Also, the in vitro footprint of

proteins of P. patens on the Em promoter was identical to that of seed plants, indicating

that the moss transcriptional machinery recognizes the same promoter region as the seed

plant factors (Knight et al., 1995). This suggests that higher plants and P. patens share

common ABA regulatory components and as such will be suitable for a comparative

approach to elucidate the mechanism(s) involved and the evolution of the transcriptional

response to ABA. Furthermore, a homolog of the wheat Em gene from P. patens,

PpLEA1, has recently been described (Kamisugi and Cuming, 2005). Similar to the

wheat Em gene, the expression of PpLEA1 is highly inducible by ABA and is mediated

through an ACGT motif in the promoter, a common feature of ABA-inducible genes in

seed plants (Kamisugi and Cuming, 2005). This provides another comparative tool for

the study of the ABA response pathway in P. patens.

We have started to dissect the ABA-regulated transcriptional mechanism in P.

patens by characterizing the structure and function of ABI3-like genes. We show by

6

particle bombardment that the seed plant proteins, ABI1, ABI3, and VP1 can affect Em-

GUS expression in P. patens. We successfully cloned three P. patens cDNAs encoding

ABI3/VP1-like genes and tested their activity in both P. patens and barley aleurone cells

using ABA-responsive Group 1LEA promoters from P. patens and wheat (Em).

PpABI3A enhances ABA-induced PpLea1-GUS and Em-GUS expression in protonemal

tissue and in barley aleurone cells, similar to the response elicited by ABI3/VP1.

However, PpABI3A cannot significantly enhance GUS expression in the absence of ABA

unlike ABI3/VP1. We also demonstrate that PpABI3A is able to function in certain

cellular and molecular functions both in protonemal tissue and in aleurone cells. We

show that at least a part of the inability of PpABI3A to fully complement the molecular

response in the Arabidopsis abi3-6 mutant is due to a weak interaction between

PpABI3A and the bZIP transcription factor ABI5, as assayed functionally in barley

aleurone and physically in the yeast-two-hybrid assay. Our data clearly demonstrate that

P. patens will be useful for comparative structural and functional studies of components

in the ABA response pathway such as ABI3.

7

RESULTS

ABA transcriptional regulation and signaling in P. patens is similar to that of higher

plants

In order to determine if the seed plant proteins ABI1, VP1 and ABI3 can act in the ABA

induced gene expression pathway, we tested their function in P. patens protonemal tissue.

Co-bombardment of the reporter Em-GUS with either Ubi-VP1 or Ubi-ABI3

demonstrated that both VP1 and ABI3 were able to transactivate Em-GUS expression,

four- and ten-fold respectively (Figure 1A). This response to VP1 and ABI3, in the

absence of ABA, is similar to the effect each has in the seed plant ABA response

pathway. In Arabidopsis and barley, ABI3 and VP1 characteristically act downstream

from ABI1 in the ABA signaling pathway (Brady et al., 2003; Casaretto and Ho, 2003).

Predictably, over-expression of the dominant negative allele of ABI1, 35S-abi1-1

(Armstrong et al., 1995), with Em-GUS completely repressed induction of Em-GUS

expression by ABA (Figure 1B). Furthermore, Ubi-VP1 could overcome the repression

effect of abi1-1 placing VP1 also downstream from ABI1 in the ABA signaling pathway

(Figure 1B). These results suggest the involvement of molecules in P. patens with the

same or similar functions as the regulatory ABI3/VP1 proteins from angiosperms.

8

Figure 1

Seed plant ABA response factors function in P. patens.

A) Maize VP1 and Arabidopsis ABI3 transactivate Em-GUS expression in P. patens.

Em-GUS and Ubi-LUC were co-bombarded into P. patens protonemal tissue with or

without the effector constructs, Ubi-VP1 or Ubi-ABI3, using 0.2µg of each construct.

Bars indicate the relative GUS activities ±SE after 48h incubation (n=4).

B) Em-GUS expression in P. patens is repressed by abi1-1 and restored by VP1.

Em-GUS and Ubi-LUC were co-bombarded into P. patens protonemal tissue with or

without the effector constructs, 35S-abi1-1 and Ubi-VP1, using 0.2µg of each construct.

Bars indicate the relative GUS activities ±SE after 48h incubation with (black bars) or

without (white bars) 10µM ABA (n=4).

0

30

60

90

120

150

180

GU

S/L

UC

+ VP1 Em +abi1-1 +abi1-1

GUSEm 35S 3’

Reporter

Effector

abi1-135S NOS 3’

VP1Ubi NOS 3’

Em +VP1 +ABI30

10

20

30

40

50

60

GU

S/L

UC

GUSEm 35S 3’

Reporter

Effector

VP1Ubi NOS 3’

ABI3Ubi NOS 3’

A B

9

Identification of ABI3/VP1-like proteins from P. patens

By searching public EST and genomic databases, we cloned three PpABI3 genes. The

PpABI3A gene encodes a 658 amino acid product, while the PpABI3B gene and PpABI3C

gene encode 515 and 539 amino acid products, respectively. The domain structure of

PpABI3A, PpABI3B, PpABI3C, AtABI3, and VP1 is relatively conserved (Figure 2A).

However, PpABI3B is missing the acidic activation and the serine-rich domains, while

PpABI3C contains a serine-rich region, but lacks the activation domain (Figure 2A). The

position of three of the introns of the B3 domain is conserved across all species, but

PpABI3C has all five of the intron positions found in the higher plant ABI3/VP1 (Figure

2A).

The amino acid sequence alignment of PpABI3A, PpABI3B, PpABI3C, AtABI3,

and VP1 reveals that the B3 domain, underlined in blue, is the most highly conserved

with 50% identity in all five sequences (Figure 2B). The B1 domain, underlined in red,

and the B2 domain, underlined in green, have 23% and 40% identity in the five proteins,

respectively (Figure 2B). The P. patens PpABI3 sequences are more closely related to

each other than the higher plant sequences and share 34% identity over their entirety.

PpABI3A, PpABI3B and PpABI3C share higher identity in the conserved basic domains;

B1 is 68% identical, while B2 and B3 are 60% and 64% identical, respectively. These

data strongly suggest that higher plant VP1/ABI3 and P. patens ABI3A, ABI3B, and

ABI3C originated from a common ancestor gene.

10

Figure 2

Comparison of the PpABI3 proteins with Arabidopsis ABI3 and maize VP1.

A) Schematic representation of PpABI3A, PpABI3B, PpABI3C, ABI3 and VP1 proteins.

Conserved basic regions (B1-B3) and the Serine-rich regions (S-rich) are indicated by

boxes. Asterisks indicate the position of introns.

PpABI3A :

PpABI3B :

PpABI3C :

ABI3 :

VP1 :

* 20 * 40 * 60 * 80

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

MKSLHVAANAGDLAEDCGILGGDADDTVLMDGIDEVGREIWLDDHGGDNNHVHGHQDDDLIVHHDPSIFYGDLPTLPDFP

-MEASSGSSPPHSQENPPEHGG--------------------DMGGAPAEEIGGEAADDFMFAEDT------FPSLPDFP

: -

: -

: -

: 80

: 53

PpABI3A :

PpABI3B :

PpABI3C :

ABI3 :

VP1 :

* 100 * 120 * 140 * 160

-----------------------MVLLSSVEAYQQDRLGLLKRSEPLREP-----------------------------T

--------------------------------------------------------------------------------

--------------------------------------------------------------------------------

CMSSSSSSSTSPAPVNAIVSSASSSSAASSSTSSAASWAILRSDGEDPTPNQNQYASGNCDDSSGALQSTASMEIPLDSS

CLSSPSSSTFS---------SNSSSNSSSAYTNTAG-----RAGGEPSEP------------------------------

: 28

: -

: -

: 160

: 89

PpABI3A :

PpABI3B :

PpABI3C :

ABI3 :

VP1 :

* 180 * 200 * 220 * 240

GLRSWIDIPLGLEDYRHCASTFACNKDDQNFDMRGLSRIGFPNPTINLEIQGIGDFYSQARSPVASYSSGTSNDCNSAGT

--------------------------------------------------------------------------------

-----------------------------------------------MVNQGSGDFSSQDGS-LPSCSSTSSERNQSSGS

QGFGCGEGGGDCIDMMETFGYMDLLDSNEFFDTSAIFSQDDDTQNPNLMDQTLERQEDQVVVPMMENNSGGDMQMMNSSL

ASAGEGFDALDDIDQLLDFASLSMPWDSEPFPGVSMMLENAMSAPPQPVGDGMSEEK-----AVPEGTTGGEEACMDAS-

: 108

: -

: 32

: 240

: 163

PpABI3A :

PpABI3B :

PpABI3C :

ABI3 :

VP1 :

* 260 * 280 * 300 * 320

STVDSTEDASLSSYLMDGN--VRAMLKKVTVNEGVLNQTIQSLGGGEKGLKELMAYIMAWVKEQNGE-NCDKLFLSQNL-

---------------MDGN--VRAVLKKVTVNEDSLSQAIQALGGGEKGLKELIGHIMLWVKQHNGEMDYGKTSLSQKL-

GLGEG------CSLLMDDE--VRAMLKKVTVNEAVLNRAIQSLGGGEKGLKSLMGYIMIWVKQQKE----CKSARSNSG-

EQDDDLAAVFLEWLKNNKETVSAEDLRKVKIKKATIESAARRLGGGKEAMKQLLKLILEWVQTNHLQRRRTTTTTTNLSY

-EGEELPRFFMEWLTSNRENISAEDLRGIRLRRSTIEAAAARLGGGRQGTMQLLKLILTWVQNHHLQRKRPRDVMEEEAG

: 184

: 62

: 99

: 320

: 242

PpABI3A :

PpABI3B :

PpABI3C :

ABI3 :

VP1 :

* 340 * 360 * 380 * 400

------EKLNPPQMNPTFGFAPSLANNIPTCNLTLGTAACDQRSDNLRRTFDTVQSSDDTQDLNQVIQCSKSRRLMVE-G

------EKLHPPQMNHALEFASGKGISGTVYNPNLASEGYDQRNEKSPCPFVRVQSSEDLQDMNHLFQCIKSRRLMGE-G

------EQSSGSPFNFSMLGAEAAAEAEAIV-LQHTSDAFQSLNGILQP--SATQSPVNLPDTQHQ-QRFKSRRLMMEDA

QQSFQQDPFQNPNPNNNNLIPPSDQTCFSPSTWVPPPPQQQAFVSDPGFG----YMPAP----NYPPQPEFLPLLES-PP

LHVQLPSPVANP-PGYEFPAGGQDMAAGGGTSWMP---HQQAFTPPAAYGGDAVYPSAAGQQYSFHQGPSTSSVVVN-SQ

: 257

: 135

: 169

: 391

: 317

PpABI3A :

PpABI3B :

PpABI3C :

ABI3 :

VP1 :

* 420 * 440 * 460 * 480

EVDSSYRADAYPRQGTFLNARGSVSED-FHSTKLESNMTNPSPIDAVVLQGMSSELGTLTQQAPCSQFGLSFNPGQNELN

GLDINFPRDVHSGSGTFVDVRGSVPDV-LHFPSGGMGPRSPNHIGPAMLQGMPCELDIQRPLIPCAQAGLSFSGEHDGLS

EVDAGLKVDAGFCPSNFMAVGVPPGDYSMYMPAMAGPAVSPYMPRLNYDSGMQQPVCNMQSMIPSTTSQNSMQP----IE

SWPPPPQ------SGPMPHQQFPMPPTSQYN-QFGDPTGFNGYNMNPYQYPYVPAGQMRD----------------QRLL

PFSPPPVGDMHGANMAWPQQYVPFPPPGASTGSYPMPQPFSPGFGGQYAGAGAGHLSVAP----------------QRMA

: 336

: 214

: 245

: 448

: 381

PpABI3A :

PpABI3B :

PpABI3C :

ABI3 :

VP1 :

* 500 * 520 * 540 * 560

RTLAASTRAARRNRLSRQRQSVQ---------------------QSHRR-SPTSGTTVTSGGCGLWSASPPALDFMNTRW

RIFTATTRAARRNQISRQRQSGL---------------------QSNSTFTPSNTVTAVSSECGLWSVAPPAWD-MSVGV

QTPAMATKAARRNRMARQRQSMM---------------------KQHAR--ATNQANPVSVGFWVWNGAPPAGGTKKTEI

RLCSSATKEARKKRMARQRRFLSHHHRHNNNNNNNNNNQQNQTQIGETCAAVAPQLNPVAT-----TATGGTWMYWPNVP

GVEASATKEARKKRMARQRRLSCLQQQRSQQLSLGQIQTSVHLQEPSPRSTHSGPVTPSAGGWGFWSPSSQQQVQNPLSK

: 394

: 272

: 302

: 523

: 461

PpABI3A :

PpABI3B :

PpABI3C :

ABI3 :

VP1 :

* 580 * 600 * 620 * 640

NHPGTQMPL---------------------------GASVKSEKKGNTTDMLTFLLQKELRPSDVGNLGRIILPKKEAEV

FLSRTQSPS---------------------------CGPDISDPK-----MLTFLLQKELRPSDVGNLGRIILPKKEAEA

SHSGAQPAQ---------------------------GTAVNAEQKGRNMDSLTFLLQKELRPSDVGSLGRIILPKKEAEQ

AVPPQLPPVMETQLPTMDRAGSASAMPRQ-------QVVPDRRQGWKPEKNLRFLLQKVLKQSDVGNLGRIVLPKKEAET

SNSSRAPPSSLEAAAAAPQTKPAPAGARQDDIHHRLAAASDKRQGAKADKNLRFLLQKVLKQSDVGSLGRIVLPKKEAEV

: 447

: 320

: 355

: 596

: 541

PpABI3A :

PpABI3B :

PpABI3C :

ABI3 :

VP1 :

* 660 * 680 * 700 * 720

HLPILALREGVSLLMEDFDSGYCWNIRYRFWPNNKSRMYLLENTGEFVKSHHLKEGDLLILYRN-EQGNYVLRGKKKVPS

HLPILALREGILLQMEDFDSGHCWKIRYWFWPNNKSRMYLLENTGEFVKSHRLEEGDLLVLYKI-QEGNYVLRAQKKVHS

HMPFLSMRGGVCIQVEDFDSGHIWNLRYRFWPNNKSRMYLLENTGDFVKSHRLVEGDLLIIYRS-QQGDYVMRGKKR-KT

HLPELEARDGISLAMEDIGTSRVWNMRYRFWPNNKSRMYLLENTGDFVKTNGLQEGDFIVIYSDVKCGKYLIRGVKVRQP

HLPELKTRDGISIPMEDIGTSRVWNMRYRFWPNNKSRMYLLENTGEFVRSNELQEGDFIVIYSDVKSGKYLIRGVKVRPP

: 526

: 399

: 433

: 676

: 621

PpABI3A :

PpABI3B :

PpABI3C :

ABI3 :

VP1 :

* 740 * 760 * 780 * 800

ETRVAYGSQHRTAHSLACRFSEGIPNKIEESFKDRDFGGGLTETASGKLKIEVKAEPLVVQPSTDGDKFEDVMSNIGSSF

ESSGAPGSQQKNAYSLARDFPEA--SKTEEPLKEKDIVG-FAEWSCG----KVNEERNILVRDDDPFFKDDQKHKISSSF

QRLETESVKIQFAHSSARDSNEEHTSPKGNGLPMERLYG-GGPPEK-----FVKEEP--PNLDDDPFL-KDMMMSMGPSF

SGQKPEAPPSSAATKR---QNKSQRNINNNSPSANVVVA------------------------------SPTSQTVK---

PAQEQGSGSSGGGKHRPLCPAGPERAAAAGAPEDAVVDG------------------------------VSGACKGRSPE

: 606

: 472

: 504

: 720

: 671

PpABI3A :

PpABI3B :

PpABI3C :

ABI3 :

VP1 :

* 820 * 840 *

ATEG-PLQPLERFPSLNLDFPLDEIMAGIPKEEDNDLTIEPHMDSKVKTEPAG

SPE--PLQSLERFPSLSIDFPLDEMMP---WDDPDEFNTDSELAELVG-----

GSDATSLQPLERFPSLNLDFPLDEIMAVNPKTE---------LE---------

-----------------------------------------------------

GVRRVRQQGAGAMSQMAVSI---------------------------------

: 658

: 515

: 539

: -

: 691

A

B

B1 B2 B3S-rich

ABI3

720* * ** *VP1

691* * ** *

PpABI3A

* * ** 658

* * * 515

PpABI3B

PpABI3C

539* * ** *

11

B) Comparison of amino acid sequences of PpABI3A, PpABI3B, PpABI3C, ABI3 and

VP1. The alignment was made by the T-coffee program. Identical residues between

PpABI3A, PpABI3B, PpABI3C, and ABI3 or VP1 are shaded black and similar residues

are shaded gray. The Bl, B2, and B3 domains are indicated respectively by the red,

green, or blue underlines.

Does PpABI3A act as a transcriptional enhancer of ABA responsive promoters?

In order to address the question of whether the PpABI3 proteins from P. patens were

active, we utilized the ABA-responsive promoter of the P. patens Late Embyrogenesis

Abundant (PpLEA1) gene linked to GUS (Kamisugi and Cuming, 2005). Only PpABI3A

strongly enhanced Lea1-GUS expression in the presence of ABA (Figure 3A) comparable

to that of VP1 (Figure 3B). All three PpABI3 proteins had little or no activity in

activating GUS expression in the absence of ABA. Interestingly, when comparing the

level of enhancement between VP1 and PpABI3A with and without ABA, PpABI3

exhibits a ten-fold increase in GUS activity versus a two-fold increase with VP1 (Figure

3B). VP1, however, clearly shows an enhancement of the ABA response in the absence

of ABA (Figure 3B). These results clearly show that the PpABI3A gene can act as a

strong transcriptional enhancer of an ABA response from a P. patens promoter of an

ABA responsive gene.

12

Figure 3 PpABI3A enhances ABA-regulated gene expression in P. patens.

A) The PpLea1-GUS reporter construct and each of the PpABI3 effector constructs were

introduced into P. patens protonemal tissue by particle bombardment. The protonemal

tissue was allowed to recover 24h untreated and then subsequently incubated with (black

bars) or without (white bars) 10µM ABA for 24h. Protein extracts were made from the

treated tissue for GUS and LUC assays. Bars indicate the relative GUS activities ±SE

(n=4).

B) The PpLea1-GUS reporter construct and each of the effector constructs were

introduced into P. patens protonemal tissue by particle bombardment. The protonemal

tissue was allowed to recover 24h untreated and then subsequently incubated with (black

bars) or without (white bars) 10µM ABA for 24h. Protein extracts were made from the

0

200

400

600

800

1000

1200

PpLea1-

GUS

PpABI3A PpABI3B PpABI3C

GUS/LUC

w/o ABA

10uM ABA

0

200

400

600

800

1000

1200

1400

PpLea1-GUS PpABI3A VP1

GU

S/L

UC

w/o ABA

10uM ABA

GUSPpLea1 NOS 3’

Reporter

Effector

PpABI3AUbi NOS 3’

VP1Ubi NOS 3’

GUSPpLea1 NOS 3’

Reporter

Effector

PpABI3AUbi NOS 3’

PpABI3BUbi NOS 3’

PpABI3CUbi NOS 3’

A B

13

treated tissue for GUS and LUC assays. Bars indicate the relative GUS activities ±SE

(n=4).

Using the Em promoter (Group 1 LEA from wheat), we observed a similar pattern

with PpABI3A, with an enhancement effect only when ABA is present in P. patens

(Figure 4A). However, neither VP1 nor ABI3 showed an enhancement of GUS activity

in combination with ABA (Figure 4A). Comparing these results with those observed

with the PpLea1 promoter, we see that the enhancement effect is much greater (ten-fold)

with the endogenous PpLea1 promoter (Figure 3B) than with the wheat Em promoter

(three-fold) (Figure 4A).

To determine if PpABI3A could function in higher plants, we analyzed the ability

of PpABI3A, VP1, and ABI3 to transactivate Em-GUS in barley aleurone cells (Figure

4B). Co-bombardment of Ubi-PpABI3A and Em-GUS resulted in a three-fold activation

of expression. Em-GUS is more strongly activated by VP1 and ABI3 (four- and thirteen-

fold, respectively) in barley aleurone cells.

14

Figure 4

PpABI3A functions as a transcriptional activator in P. patens and barley.

A) PpABI3A functions as a transcriptional activator in P. patens.

The Em-GUS reporter construct was introduced into P. patens protonemal tissue with

each of the effector constructs by particle bombardment. P. patens protonemal tissue was

incubated with (black bars) or without (white bars) 10µM ABA for 48h and protein

extracts were made for GUS and LUC assays. Bars indicate the relative GUS activities

±SE (n=4).

B) PpABI3A functions as a transcriptional activator in barley aleurone.

GUSEm 35S 3’

Reporter

Effector

PpABI3AUbi NOS 3’

VP1Ubi NOS 3’

ABI3Ubi NOS 3’

0

50

100

150

200

250

300

Em PpABI3A VP1 ABI3

GU

S/L

UC

-ABA

+ABA

0

10

20

30

40

50

60

GU

S/L

UC

Em PpABI3A VP1 ABI30

50

100

150

200

250

300

Em PpABI3A VP1 ABI3

GUS/LUC

BA

15

The Em-GUS reporter construct was introduced into barley aleurone cells with each of

the effector constructs by particle bombardment. Barley half seeds were incubated for

24h and protein extracts were made for GUS and LUC assays. Bars indicate the relative

GUS activities ±SE (n=4).

What higher plant functions are conserved in PpABI3A?

For analysis of the sub-cellular of localization of PpABI3A, a GFP fusion construct

(PpABI3A:GFP) was introduced into cells via particle bombardment. In both barley

aleurone cells and P. patens protonemal cells bombarded with PpABI3A:GFP, the GFP

signal localizes at the nucleus (Figures 5B and 5E). The AtABI3:GFP control also

localizes to the nucleus of both barley aleurone cells and P. patens protonemal cells

(Figures 5A and 5D). The GFP control shows a diffuse localization, in both the

cytoplasm and the nucleus (Figures 5C and 5F). These results demonstrate that the

localization of PpABI3A is consistent with its function as a transcriptional activator and

that the nuclear localization signals of PpABI3A and AtABI3 are recognized in both P.

patens and barley cells.

16

Figure 5

A PpABI3A:GFP fusion protein localizes to the nucleus in barely aleurone and P. patens

cells.

Constructs for the over-expression of AtABI3:GFP (A and D), PpABI3A:GFP (B and E),

and GFP (C and F) protein were delivered into barley aleurone cells (A-C) and P. patens

protonemal tissue (D-F) by particle bombardment. Images of transformed cells were

taken by confocal microscopy. The GFP signal and auto-fluorescence of chloroplasts are

shown by green and red color, respectively.

To determine if PpABI3A is capable of functioning in higher plants, the

Arabidopsis abi3-6 mutant, a severe allele of abi3 (Nambara et al., 1994), was stably

transformed with PpABI3A under the control of the Arabidopsis ABI3 promoter. As a

control, abi3-6 plants were also transformed with a PAtABI3-AtABI3 construct. The seeds

of abi3-6 plants are green due to a failure to degrade chlorophyll and appear shriveled up

because of the inability to undergo desiccation, as previously reported (Figure 6B)

(Nambara et al., 1994). In complementation lines expressing PAtABI3-PpABI3A the seeds

A

FED

CB

17

are brown (Figure 6C), just like Columbia wild type and the PAtABI3-AtABI3 control

(Figure 6A and 6D respectively). Interestingly, in the PAtABI3-PpABI3A complementation

lines, even though the seeds are brown, they are not desiccation tolerant (data not shown).

Figure 6

PpABI3A complements the green seed phenotype of abi3-6.

Seeds from mature siliques were harvested from (A) Columbia wild type, (B) abi3-6, and

the transgenic lines of (C) PAtABI3-PpABI3A/abi3-6 and (D) PAtABI3-AtABI3/abi3-6.

The germination of abi3-6 seeds is insensitive to ABA, however, ABA inhibits

germination at levels higher than 3µM in Columbia wild type. At 100µM ABA, 100% of

the abi3-6 mutant seeds germinate, while a 40% reduction in germination is seen in the

PAtABI3-PpABI3A complementation line (Figure 7A). The germination of the PAtABI3-

18

AtABI3 control is sensitive to ABA like Columbia wild type (Figure 7A). Therefore,

expression of PpABI3A can partially complement the ABA insensitivity of germination

of the abi3-6 mutant.

Figure 7

PpABI3A partially complements the abi3-6 mutant.

A) PpABI3A partially complements the ABA insensitivity of abi3-6 seed germination.

Seeds of Columbia wild type (red circle), abi3-6 (green square), PAtABI3-PpABI3A/abi3-6

(orange cross), and PAtABI3-AtABI3/abi3-6 (blue triangle) were plated on MS media

containing 0, 0.1, 1, 10, or 100 µM ABA and cold treated for 4 days at 4°C in the dark.

The plates were then moved to 22°C with a 16-hour light and 8-hour dark light regime.

The germination rate was measured after 4 days using at least 40 seeds per assay. Bars

indicate ±SE of three independent replicates.

B) PpABI3A partially complements the expression of AtABI3 regulated genes in abi3-6

seeds.

0

20

40

60

80

100

120

0 0.1 1 10 100

ABA (µM)

% G

erm

inat

ion

Ubq10

Rab18

Oleosin2

Napin

CruciferinC

Em6

Em1

Colum

bia W

TP AB

I3-A

tABI

3/abi3

-6P AB

I3-P

pABI

3A/ab

i3-6

abi3-

6

A B

19

RNA was extracted from siliques 11 days after pollination of Columbia wild type,

PAtABI3-AtABI3/abi3-6, PAtABI3-PpABI3A/abi3-6, and abi3-6. RT-PCR was performed

using 0.5 µg of total RNA and the expression of various seed genes controlled by ABI3

was analyzed.

What molecular responses are not complemented by PpABI3A?

Since the PAtABI3-PpABI3A/abi3-6 lines show partial complementation of the abi3-6

phenotypes, we tested for the expression of several seed genes controlled by ABI3 to

determine if PpABI3A could properly regulate their expression (see Table 1). Semi-

quantitative RT-PCR was performed on RNA extracted from siliques of Columbia wild

type, PAtABI3-AtABI3/abi3-6, PAtABI3-PpABI3A/abi3-6, and abi3-6 harvested 11 days after

pollination. In abi3-6 mutant seeds, the expression of these genes is reduced (Figure 7B).

In the PAtABI3-PpABI3A/abi3-6 complementation lines, the expression level of

CruciferinC, Napin, Oleosin2, and Rab18 is complemented to near wild type levels

(Figure 7B). However, the expression of AtEm1 and AtEm6 is not complemented in the

PAtABI3-PpABI3A/abi3-6 siliques (Figure 7B). The gene expression in the PAtABI3-

AtABI3/abi3-6 line behaves similarly to Columbia wild type (Figure 7B). These data

suggest that PpABI3A is able to partially complement gene expression in the abi3-6

mutant.

Because PpABI3A appeared to have a defect in the complementation of ABI5

dependent gene expression (i.e. AtEm1 and AtEm6, see Table 1) in the abi3-6

background, we tested for a functional interaction between PpABI3A and barley ABI5

(HvABI5) in barley aleurone cells. It is known that ABI5 alone is not sufficient to

20

completely activate ABA-responsive promoters, but co-expression of ABI3/VP1 with

ABI5 compensates for the effect of ABA (Casaretto and Ho, 2003; Hobo et al., 1999). In

the case of the Em promoter in barley aleurone cells, HvABI5 alone also did not show

strong activation (1.5-fold) (Figure 8A). When HvABI5 was over-expressed with VP1 or

ABI3 in the absence of ABA, Em promoter activity was almost the same level with that

of ABA-induction (Figure 8A). In the presence of ABA, HvABI5 co-expressed with

either VP1 or ABI3 enhanced the Em-GUS transactivation. PpABI3A in combination

with HvABI5 also showed synergistic enhancement (11.5-fold) in the absence of ABA,

however, the activation level was far lower than that of ABA induction of the Em

promoter (Figure 8A). Also, co-expression of PpABI3A and HvABI5 in the presence of

ABA lacked the enhancement of Em-GUS transactivation (Figure 8A). These data show

that PpABI3A is capable of a basal interaction with HvABI5 on the transactivation of

Em-GUS, however it is not functionally equivalent to the interaction between ABI3/VP1

and HvABI5 in barley aleurone cells.

We performed yeast-two-hybrid assays to further test the physical interaction

between PpABI3A and HvABI5 in a more quantitative manner. In the nutrient-requiring

growth assay, the colony size of the yeast expressing AD-PpABI3A with BD-HvABI5 is

smaller than the control yeast expressing AD-AtABI3 with BD-HvABI5 (Figure 8B). In

the β-galactosidase assay, the yeast expressing AD-PpABI3A with BD-HvABI5 yielded

25% of the activity of the AD-AtABI3 control (Figure 8C). These data reveal that

PpABI3A is able to interact with HvABI5 although the interaction is weak.

21

Figure 8

PpABI3A interacts weakly with HvABI5.

A) PpABI3A interacts weakly with HvABI5 in barley aleurone cells.

The Em-GUS reporter construct was introduced into barley aleurone cells in various

combinations with each of the effector constructs. Barley half seeds were incubated with

(black bars) or without (white bars) 20µM ABA for 24h. Bars indicate the relative GUS

activities ±SE (n=4).

B) PpABI3A interacts weakly with HvABI5 in yeast-two-hybrid plate assay for analysis

of reporter genes, GAL2-ADE2, GAL1-HIS3. The diploids harboring indicated BD and

AD constructs were grown for one week on SD-LW medium lacking adenine and

histidine containing 35mM 3-AT (see Materials and Methods). Bar=50mm.

GUSEm 35S 3’ Ubi NOS 3’

Reporter Effector

PpABI3AUbi NOS 3’

VP1Ubi NOS 3’

ABI3Ubi NOS 3’

HvABI5

0

100

200

300

400

500

Em

HvABI5

PpABI3A

VP1

ABI3

PpABI3A+HvABI5

VP1+HvABI5

ABI3+HvABI5

GUS/LUC

!-g

ala

cto

sid

ase a

ctivity

(Mill

er

units)

0

50

100

150

200

250

pGAD AD-PpABI3A AD-ABI3

pGBD

BD-HvABI5

pGBD

BD-HvABI5

pGAD AD-PpABI3A AD-ABI3

A

CB

22

C) β-galactosidase activity of the diploids indicated in (B). Black bars represent the BD-

HvABI5 interaction and white bars represent the pGBD control. The individual data is

the average ± SD (n=3). pGBD (pGBTK) and pGAD (pGAD424) are vector controls.

[The differences between pGAD/ABI5 and PpABI3/ABI5, pGAD/BD-ABI5 and

ABI3/ABI5, and PpABI3/HvABI5 and ABI3/HvABI5 were significant (P<0.05, P<0.01

and P<0.01. respectively)]

23

DISCUSSION

ABA signaling pathway in P. patens

The wheat Em promoter was previously shown to respond to ABA in P. patens

protonemal tissue, similar to the response in seed plants (Knight et al., 1995). They

showed that the ABA response was dependent on the ABRE [two ACGT-boxes separated

by 46-bp (Marcotte et al., 1989)]. The activation of genes in seed plants by ABA required

not only the ABRE and its bZIP transcription factor but also the transcriptional activator

VP1. VP1 can transactivate the Em promoter in a transient assay in the absence of ABA

and further enhance expression in the presence of ABA (Vasil et al., 1995). Mutants of

VP1, and its ortholog from Arabidopsis ABI3, failed to accumulate the Em transcript

during seed development (Bies-Etheve et al., 1999; McCarty et al., 1991; Parcy et al.,

1994). In this study, we confirmed the results of Knight et al. (1995), and further showed

that both VP1 and ABI3 can transactivate this promoter in P. patens without exogenous

ABA but unlike the response in angiosperms, they failed to enhance expression with

ABA.

The activation of ABA-regulated genes in seed plants also requires the signaling

intermediate ABI1, which encodes a PP2C phosphatase and acts as a negative regulator of

ABA signaling in Arabidopsis. The dominant negative abi1-1 allele was shown to inhibit

the ABA signaling not only in Arabidopsis but other seed plants such as barley, rice, and

tomato (Carrera and Prat, 1998; Casaretto and Ho, 2003; Gampala et al., 2002; Grabov et

al., 1997; Hagenbeek et al., 2000). We showed that abi1-1 blocks the ABA induction of

Em-GUS expression in P. patens. In addition, this negative regulation was overcome by

24

the co-expression of VP1, showing that abi1-1 acts upstream of the transcriptional

activator VP1. The involvement of abi1-1 in the ABA signaling pathway in P. patens

and its relationship with VP1 appears identical to its role in seed plants (Gampala et al.,

2002). At least five ESTs show significant similarity to ABI1 and ABI2 in the P. patens

database (data not shown), allowing for future functional comparisons of this class of

phosphatases in ABA signaling. Hence, results using the ABA signaling intermediate

ABI1, and the transcription factors ABI3/VP1, demonstrate that at least these

components of the angiosperm ABA signaling pathway function in a similar manner in a

bryophyte.

Characterization of the PpABI3 proteins

Since VP1 and ABI3 were active in P. patens, we proceeded to isolate three ABI3-like

genes from P. patens: PpABI3A, PpABI3B, and PpABI3C. Three copies of ABI3 are

present in the P. patens genome as compared to one in angiosperms, and although smaller

than their seed plant counterparts, the overall structure of the PpABI3 proteins follows

the same pattern with the three basic domains: B1, B2, and B3. Variation in the structure

occurs primarily at the N-terminus, where the PpABI3 proteins have either a shorter

acidic activation domain (PpABI3A) or lack this domain (PpABI3B and PpABI3C). The

serine-rich region found in the seed plant orthologs is also found in PpABI3A and

PpABI3C. The intron-exon junctions between the angiosperm orthologs and PpABI3C

are perfectly conserved. In contrast, PpABI3A and PpABI3B only share three of the five

intron positions in the B3 domain, suggesting that these paralogs were lost in the seed

25

plant lineage. These observations strongly suggest that the VP1/ABI3 gene family arose

prior to the divergence between mosses and seed plants.

The B1 and B2 domains of ABI3 and VP1 are responsible for some unique

functions of these proteins (Bies-Etheve et al., 1999; Ezcurra et al, 2000; Hill et al., 1996;

Nakamura et al., 2001). For example, the Arabidopsis abi3-7 mutant has a point

mutation that converts an alanine into threonine in the B2 domain and prevents the

expression of AtEm1 and AtEm6 (Bies-Etheve et al., 1999). This alanine residue is

perfectly conserved not only in all known ABI3/VP1s from seed plants, but also in the

PpABI3 proteins. Similarly, the abi3-8 mutation alters a conserved leucine in B1, which

is possibly involved with ABI5 interaction (Nambara et al., 2002). However, this leucine

is not conserved in P. patens. Alignments such as these will target those amino

acids/domains for further analysis in determining their conserved function during the

evolution of land plants.

Functional analysis of PpABI3A

We demonstrate that like VP1/ABI3 in angiosperms (Gampala et al., 2002; Vasil et al.,

1995), PpABI3A functions in the ABA-response pathway as a transcriptional enhancer of

ABA-responsive promoters. When PpABI3A is over-expressed in the presence of

exogenous ABA, enhanced expression from both P. patens (PpLea1) and wheat (Em)

promoters is observed in P. patens protonemal tissue. This enhancement is similar to

what has been reported in angiosperms, when VP1 is over-expressed in ABA-treated

cells. PpABI3A also functions weakly (compared to ABI3 and VP1) as a transcriptional

activator of the Em promoter in barley aleurone cells. In fact, the length of the activation

26

domain of these three proteins is proportional to their enhancement of the ABA response.

We also showed that PpABI3A and AtABI3 localize to the nucleus of both barley

aleurone and P. patens protonemal cells. The fact that PpABI3A localizes to the nucleus

and enhances expression in both P. patens and barley aleurone cells indicates that the

core transcriptional regulatory properties of PpABI3A are at least partially conserved.

We also show that PpABI3A, but not PpABI3B and PpABI3C, enhances

expression from a P. patens ABA responsive promoter, PpLea1-GUS (Kamisugi and

Cuming, 2005). PpABI3A contains all the conserved domains found in the seed plant

orthologs, including the activation domain, while PpABI3B and PpABI3C lack the N-

terminal activation domain. All three ABI3 proteins from P. patens either lack the ability

or show weak transactivation in the absence of ABA. Interestingly, these results in

protonemal tissues are specific to PpLea1-GUS promoter, since all three PpABI3 proteins

are able to transactivate the Em-GUS promoter (data not shown). Hence, the

structure/function relationship of the ABI3 proteins from P. patens reflects not only their

domain structure but also their interaction with specific ABA-responsive promoters. This

will allow further understanding of this interaction by the use of domain swaps of the

PpABI3 proteins with the angiosperm orthologs, along with the construction of specific

promoter sequences.

With respect to the specific ABRE in each of these promoters, PpLEA1 contains a

single ACGT motif while the ABRE from angiosperms have either two ACGT motifs or

one ACGT motif and a coupling element (Kamisugi and Cuming, 2005; Shen et al.,

2004). While we demonstrate that the wheat Em promoter functions in an ABA-

responsive manner in both barley and P. patens, the same cannot be said of the PpLea1

27

promoter, which fails to express in barley aleurone cells (Kamisugi and Cuming, 2005).

These studies appear to indicate that the ABA-responsive promoter in P. patens

(PpLEA1) represents the ancestral state, while ABA-responsive promoters from

angiosperms (wheat Em) have additional factors/sequences involved in the response.

(Kamisugi and Cuming, 2005). A further comparative analysis of the cis-elements in the

wheat Em and PpLea1 promoters which are required in aleurone and protonemal cells for

the level of ABA-responsiveness will aid in our understanding of the evolution of the

response elements and the factors that bind to them.

Complementation of abi3-6

Given the transcriptional activation and nuclear localization properties of PpABI3A in

barley we further showed that PpABI3A could partially complement the Arabidopsis

abi3-6 mutant. Similar results have been reported for ABI3/VP1 orthologs from seed

plants; abi3-6 lines expressing maize VP1 were unable to properly express CruciferinC

(Suzuki et al., 2001) while the yellow cedar ABI3 failed to fully complement the ABA

sensitivity of germination (Zeng and Kermode, 2004). However, in both of these

complementation experiments, ABI3/VP1 expression was driven by the enhanced 35S

promoter, which is known to have weak promoter activity during seed development

(Benfey et al., 1990; Sunilkumar et al., 2002; Terada and Shimamoto, 1990). Hence, the

failure of these higher plant proteins to fully complement may be due to improper spatial,

temporal, or level of expression of ABI3 or VP1. In our hands, complementation of the

abi3-6 mutant using 35S-PpABI3A or 35S-AtABI3 showed no detectable

complementation of the abi3-6 phenotypes (data not shown). However, when the AtABI3

28

promoter drives expression of PpABI3A or AtABI3, AtABI3 fully complemented the

mutant while PpABI3A only partially complemented.

The seed color phenotype of the abi3-6 was fully complemented (from green to

brown) in lines expressing PpABI3A. However, full ABA sensitivity was not observed

in PAtABI3-PpABI3A/abi3-6 seeds, where only 60% of the seeds germinate on 100µM

ABA. Seeds from abi3-6 germinated at 100µM ABA, whereas seeds from wild type and

AtABI3 complemented lines did not germinate above 10µM ABA. This partial

complementation of ABA sensitivity by PpABI3A may be due to a failure of PpABI3A

to interact with the Arabidopsis ABA response factors, such as ABI5, since certain

conserved amino acids in the B1 domain are lacking. The PpABI3 proteins lack the

conserved leucine of the abi3-8 mutation, which alters the B1 domain and possibly

the interaction with ABI5 (Nambara et al., 2002).

Transcript levels of several ABI3-regulated, seed-specific genes suggest that this

may indeed be the case. Expression of AtEm1 and AtEm6, both of which require ABI5

for expression (Finkelstein and Lynch, 2000), are not complemented in the PAtABI3-

PpABI3A lines. However, other seed-specific genes that are not regulated by ABI5 are

fully complemented by PpABI3A, such as Napin and CruciferinC, which require another

member of the bZIP family (OPAQUE2-like) (Lara et al., 2003), and Oleosin2 and

Rab18 (see Table 1).

29

PpABI3A interaction with ABI5

In this study, we showed that PpABI3A and HvABI5 functionally interact in aleurone

cells but it was not functionally equivalent to the interaction of ABI3/VP1 with HvABI5

in the same cells. This conclusion is also supported by our demonstration of a weak

physical interaction between PpABI3A and HvABI5 in the yeast-two-hybrid assays;

AtABI3 and HvABI5 have a strong interaction even though one protein is from a dicot

and the other from a monocot. The B1 domain of AtABI3 is necessary for the interaction

with AtABI5 in yeast two hybrid assays (Nakamura et al., 2001) and now targets this

domain for functional comparisons of ABI5 interactions. Several sequences for bZIP

transcription factors are in the P. patens genome, at least two of which fall into the

ABI5 subfamily. This will enable a functional comparison of the interaction

between ABI3 and ABI5 in P. patens. In this same regard, ABI3/VP1 are able to

physically interact not only with ABI5 and OPAQUE2-related bZIP transcription factors,

but also other proteins such as 14-3-3 (Schultz et al., 1998), the transcription factor

CONSTANS, a protein involved in the cell cycle regulation, and an E3 ligase with a

RING motif (Jones et al., 2000; Kurup et al., 2000; Zhang et al., 2005). It will be

interesting to determine how the composition of the ABI3/VP1 transcriptional complex

varies and how this relates to the different functions of the members of the ABA

signaling networks in P. patens and higher plants.

30

EXPERIMENTAL PROCEDURES

Plant material

P. patens subspecies patens (Gransden) was used as the wild type strain. The strain was

maintained as previously described. Briefly, protonemal tissue were ground with a

PowerGen 125 homogenizer (Fisher Science, Hampton, NH) and inoculated onto

cellophane overlaid on 0.7 % agar plates of PpNH4 medium (1.8 mM KH2PO4, pH7.0,

3.4 mM Ca(NO3)2, 1 mM MgSO4, 45mM FeSO4, 0.22 mM CuSO4, 0.19 µM ZnSO4, 9.9

µM H3BO3, 2 µM MnCl2, 0.23 µM CoCl2, 0.17 µM KI, 0.1 µM Na2MoO4, 2.7 µM

ammonium tartrate). The tissue was cultured at 25°C under the cycle of 16 hours light

(90 µmol m-2s-1) and 8 hours dark.

Barley seeds (Hordeum vulgare cv Himalaya) from 1998 harvests at Washington

State University in Pullman were used in all experiments.

Arabidopsis thaliana ecotype Columbia was used as the wild-type. The abi3-6

mutant was a gift of Dr. Peter McCourt. Plants were grown in a mixture of 60% Redi-

Earth and 40% vermiculite at 22°C under a 16-hour light and 8-hour dark light regime in

a growth chamber. Seeds were surface sterilized for 5 minutes in 95% ethanol, dried, and

plated on MS media containing 0.8% agar and subjected to 4 days of cold-treatment in

the dark before being moved to a growth chamber at 22°C under a 16-hour light and 8-

hour dark light regime.

Cloning of PpABI3 cDNAs

The B3 domain of ABI3 from Arabidopsis was used as a query sequence for the BLAST

program (tblastn) (Altschul et al., 1990) against Physcobase (http://moss.nibb.ac.jp/)

31

(Nishiyama et al., 2003) to retrieve cDNAs encoding the B3 domain protein. As a result,

three independent cDNA clones were identified as candidates. From the sequence

information of the contigs, we set up primers to amplify the full-length cDNAs. The

primers to amplify the cDNA clone pphn12b05, which we named PpABI3A, are 5’-ATG

TGA GAG TGA GCC TGC AG-3’ and 5’-TCC ACC CCT GTA ACA GAA AA-3’ for

5’ and 3’ end, respectively. PCR of P. patens against the full-length cDNA library, a gift

from Dr. M. Hasebe (Nishiyama et al., 2003) was performed with KlenTaq LA

(Clontech, Mountain View, CA) and the supplied buffer with 25 µM each dNTP and

3.5mM MgCl2 by 35 cycles of 96°C for 1 minute, 56°C for 1 minute, and 68°C for 2

minutes. The DNA fragment was subcloned into pGEM-T Easy (Promega, Madison,

WI) and subjected to the DNA sequencing using the M13 forward and reverse primers

and Big Dye Terminator mix (Applied Biosystems, Foster City, CA). The amino acid

sequence was analyzed using DNASIS software (Hitachi Software Engineering, Tokyo,

Japan).

PpABI3B was cloned from a partial EST sequence (Frank et al., 2005) using 5’

and 3’ RACE. RNA was extracted from P. patens gametophore tissue using the RNeasy

Kit (Qiagen, Valencia, CA) for use with the 5’ and 3’ RACE kit (Invitrogen, Carlsbad,

CA) to amplify the remaining sequence. Three sequential rounds of 5’RACE were

required to reach the 5’ end of the PpABI3B mRNA. The full-length coding sequence of

PpABI3B was amplified by RT-PCR and cloned into the pCR 2.1-TOPO vector

(Invitrogen, Carlsbad, CA). The DNA sequence was checked using M13 forward and

reverse primers with the Big Dye Terminator mix (Applied Biosystems, Foster City, CA).

32

PpABI3C was obtained by a BLAST search (tblastx) (Altschul et al., 1997) of the

P. patens JGI raw sequences in Physcobase (Nishiyama et al., 2003) using the

Arabidopsis ABI3 coding sequence from the start codon through the B2 domain (bases 1-

1401) as the query. This search identified P. patens sequences with B1 and B2 domains

that did not match to either PpABI3A or PpABI3B. We assembled a contig from the

available genomic sequences (ASYA169080.bl, ASYA 384419.gl, AXOS216104.xl, and

AXOS216373.yl) and used this to design primers for 5’ and 3’ RACE based on predicted

amino acid conservation with the other PpABI3 proteins. One round of 3’RACE and two

rounds of 5’RACE were required to obtain the full cDNA. RNA for the RACE reactions

was extracted from protonema treated with 10µM ABA for two hours using the RNeasy

Kit (Qiagen, Valencia, CA) for use with the 5’ and 3’ RACE kit (Invitrogen, Carlsbad,

CA). The coding sequence of PpABI3C was amplified by RT-PCR and cloned into the

pCR 2.1-TOPO vector (Invitrogen, Carlsbad, CA). The DNA sequence was checked

using M13 forward and reverse primers with the Big Dye Terminator mix (Applied

Biosystems, Foster City, CA).

Amino acid alignments were created by the T-coffee program (Notredame et al.,

2000).

DNA constructs

The effector constructs of Ubi-PpABI3A, Ubi-PpABI3B, Ubi-PpABI3C, Ubi-VP1 and

Ubi-ABI3 were made as follows. The coding sequence of PpABI3A, PpABI3B,

PpABI3C, VP1 and ABI3 were amplified by PCR from the cDNAs, and subcloned into

pGEM-Teasy (Promega, Madison, WI) or pCR 2.1-TOPO vector (Invitrogen, Carlsbad,

33

CA). Accuracy of the amplified fragments was checked by sequencing, then each

fragment was placed downstream of the maize Ubi1 promoter (Christensen and Quail,

1996), followed by the terminator of nopalin synthetase gene. The GFP fusions were

generated by PCR to add GFP to the C-terminal end as a translational fusion in the

respective Ubi-ABI3/PpABI3A-nos vector. Em-GUS [pBM113KP, (Marcotte et al.,

1989)], Ubi-LUC [pAHC18, (Bruce et al., 1989)], Ubi-HvABI5 (Casaretto and Ho, 2003),

Ubi-GFP (Zentella et al., 2002), PpLea1-GUS (Kamisugi and Cuming, 2005) and 35S-

abi1-1 (Armstrong et al., 1995) were described previously.

Transient assays

In principle, DNA delivery to barley aleurone cells was performed as described

previously (Shen et al., 1993). Two µg of each reporter constructs (Em-GUS and Ubi-

LUC) and effector constructs were used to prepare tungsten particles for three shots.

Bombarded seeds were treated with or without 20µM ABA for 24 h at room temperature.

Transient transformation of P. patens by particle bombardment was carried out as

described (Bezanilla et al., 2003). We used 0.8 µg of each reporter construct (Em-

GUS/PpLea1-GUS and Ubi-LUC) and effector construct to prepare gold particles for four

shots. One-week old protonemal tissue was used for bombardment, and then incubated

on PpNH4 agar media with or without 10µM ABA for 48 h under the conditions

described above.

Four barley seeds or moss protonemal tissue were ground in extraction buffer

(Lanahan et al., 1992) and cell debris was spun down. GUS and luciferase activities were

measured as described (Lanahan et al., 1992). GUS activity was normalized by the

34

luciferase activity and represented as relative GUS activity (GUS/LUC) ± SE. All the

experiments consisted of four replicates.

Microscopy

Confocal microscopy was performed using barley aleurone tissue or protonemal tissue of

P. patens placed in water on a glass slide and covered with a cover slip. A Leica

Confocal System TCS SP2 (Mannheim, Germany) was used for imaging GFP signal with

an argon laser.

Light microscopy of Arabidopsis seeds and siliques was performed using an

Olympus SZX12 system (Melville, NY).

Arabidopsis Complementation

The vector pCHF3 (Jarvis et al., 1998), a gift from Michael Neff, which contains the

CaMV 35S promoter and pea RBCS terminator, was used to express PpABI3A or ABI3

in Arabidopsis. The abi3-6 mutant plants were transformed by Agrobacterium

tumefaciens strain GV3101 using the floral dip technique (Clough and Bent, 1998).

Transformants were obtained by selection on MS plates containing kanamycin (50 mg/L).

35S-ABI3 and 35S-PpABI3A plants in the T3 generation, which were segregating 3:1 for

kanamycin resistance, failed to show any complementation of the abi3-6 phenotypes.

Therefore, we removed the CaMV 35S promoter from pCHF3 and replaced it by EcoRI

and BamHI digestion with the first 1752 bp of the Arabidopsis ABI3 promoter, which we

amplified by PCR from the genomic sequence of the At3g24650 locus. The primers used

to amplify the ABI3 promoter were 5’ GAATTCCGTGTACGTTTAGGTGGCATG-3

35

and 5’-GGATCCAGAAGACTCTCTCTTTCTTTC-3’. The PAtABI3-PpABI3A and the

PAtABI3-AtABI3 constructs were then transformed into the abi3-6 mutant as described

above. We obtained four independent PAtABI3-PpABI3A complementation lines and seven

of the PAtABI3-AtABI3 lines, which had a single T-DNA insertion as determined by

kanamycin segregation. Seed color was scored after the siliques had turned yellow,

approximately 18 days after flowering.

ABA germination assay

Seeds were harvested at 15 days after flowering from Columbia wildtype, abi3-6, PAtABI3-

PpABI3A/abi3-6 and PAtABI3-AtABI3/abi3-6 complementation lines and surface sterilized.

Seeds were plated on MS media containing 0, 0.1, 1, 10, or 100 µM ABA and cold

treated for 4 days at 4°C in the dark. The plates were then moved to 22°C with a 16-hour

light and 8-hour dark light regime. The germination rate was scored after 4 days using at

least 40 seeds for each assay condition. The assay was performed in triplicate.

RT-PCR analysis

RNA was extracted from 11-day-old siliques using RNAqueous kit with the Plant RNA

isolation aid (Ambion, Austin, TX). 500 ng of total RNA was first treated with DNaseI

(Amplification grade, Invitrogen, Carlsbad, CA) for 15 minutes at room temperature.

The enzyme was inactivated by heating at 65°C for 10 minutes in the presence of 2.5 mM

EDTA. The RNA was used for making cDNA by the ThermoScript RT-PCR system

(Invitrogen, Carlsbad, CA). The RNA was first pre-incubated with oligo(dT)20 and

dNTPs at 65°C for 5 minutes, then placed on ice. The cDNA Synthesis Mix was added

36

to the RNA and incubated at 55°C for 1 hour. The reaction was terminated by heating at

85°C for 5 minutes and then RNaseH was added and incubated at 37°C for 20 minutes to

remove the RNA template. PCR was carried out using 2 µL of the cDNA with 0.4 µL of

Taq polymerase (Invitrogen, Carlsbad, CA) in a 50 µL reaction. The primer sequences

are AtEm1 (5’-CGTCAAAGCAACTGAGCA-3’ and 5’-TCCATCGTACTGAGTCCT-

3’); AtEm6 (5’-TGGCGTCTCAACAAGAGAAG-3’ and 5’-

TCTCCGGTGCTAAGACCA-3’); Napin (5’-GCAATCACAACACCTAAGAGCT-3’

and 5’-CTGGATTGGAATGGTCCGTG-3’); CruciferinC (5’ -

AGCTCAGCAATCTCCTCGTT-3’and 5’-TTTCCGGCCAAATGGAACAC-3’); Rab18

(5’-ATCCAGCAGCAGTATGACGAGT-3’ and 5’-ACCGGGAAGCTTTTCTTGATC-

3’); Oleosin2 (5’-AGGTCAGTATGAAGGTGACGGTG-3 and 5’-

TGCTCAGGCACAGTTCTCCTT-3’); Ubiquitin10 (5’-

AAGATCCAGGACAAGGAGGTATTCCT-3’ and 5’-

CATAACAGAGACGAGATTTAGAAACC-3’). The cycling parameters for AtEm1 and

AtEm6 were 35 cycles of 94°C for 30 seconds, 52°C for 45 seconds and 72°C for 1

minute. The cycling parameters for Ubq10, Oleosin2, and Rab18 were 21 cycles of 94°C

for 30 seconds, 58°C for 45 seconds and 72°C for 1 minute. The cycling parameters for

Napin and CruciferinC were 28 cycles of 94°C for 30 seconds, 56°C for 45 seconds and

72°C for 1 minute. Ten µL of each PCR reaction was run out on a 1.5% agarose gel.

37

Yeast Two Hybrid

Plasmid construction

For constructs used in yeast two hybrid experiments, the entire coding sequence of

HvABI5, ABI3 and PpABI3A were amplified by PCR. The HvABI5 fragment was cloned

into the multi-cloning site of pGBTK (Clontech, Mountain View, CA) in frame with

GAL4 binding domain sequence (BD-HvABI5), and fragments of ABI3 and PpABI3A

were cloned into the multi-cloning site of pGAD424 (Clontech, Mountain View, CA) in

frame with GAL4 activation domain sequence (AD-ABI3 and AD-PpABI3A).

Yeast two hybrid assays

Media was prepared as described (Sambrook and Russell, 2001). Yeast two hybrid

assays were carried out as described before (James et al., 1996). In this system three

different reporter genes, GAL2-ADE2, GAL1-HIS3 and GAL7-LacZ are available. A host

strain PJ69-4A-a was used for BD constructs, whereas PJ69-4A-AD-a was used for AD

constructs. Mating was carried out by co-incubation of a strain harboring the BD

construct and a strain harboring the AD construct. The diploid strains were selected on

SD media without leucine and tryptophan (SD-LW), and used for analyses of the

interaction between the transcription factors. For the plate assay, SD-LW medium

without histidine and adenine (SD-LWHA) with 35mM 3-aminotriazole (3-AT) was used

for culture. For the quantification assay, GAL4-BD driven β-galactosidase gene

expression was analyzed using ONPG (O-nitrophenylgalactoside) as the substrate

according to the protocol described at

http://www.fhcrc.org/labs/gottschling/yeast/Bga37.html.

38

ACKNOWLEDGEMENTS

We thank Dr. Peter McCourt for the abi3-6 seeds, Dr. Andrew Cuming for the PpLea1-

GUS construct, and Dr. Tuan-hua David Ho and Dr. Jose Casaretto for providing DNA

constructs of Ubi-HvABI5, Ubi-GFP, and Ubi-LUC. We thank Jana Hakenjos, Julie

Thole and Paul Klueh for their technical assistance and Drs. Ho and Casaretto for their

help with the particle bombardment of barley aleurone cells. We thank Yoshinori Murai

and Takahiro Kawato for their technical assistance with the yeast-two-hybrid

experiments. Some of the sequence data were produced by the U.S. Department of

Energy Joint Genome Institute http://www.jgi.doe.gov/. This work was supported

by a JSPS Postdoctoral Fellowship for Research Abroad to Y.S. and by The Danforth

Foundation/Washington University to R.S.Q.

39

REFERENCES Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990). Basic local alignment search tool. J. Mol. Biol. 215, 403-410. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389-3402. Armstrong, F., Leung, J., Grabov, A., Brearley, J., Giraudat, J., and Blatt, M. R. (1995). Sensitivity to abscisic acid of guard-cell K+ channels is suppressed by abi1-1, a mutant Arabidopsis gene encoding a putative protein phosphatase. Proc. Natl. Acad. Sci. USA 92, 9520-9524. Benfey, P. N., Ren, L., Chua, N. H. (1990). Tissue-specific expression from CaMV 35S enhancer subdomains in early stages of plant development. EMBO J. 9, 1677-1684. Bezanilla, M., Pan, A., and Quatrano, R. S. (2003). RNA interference in the moss Physcomitrella patens. Plant Physiol. 133, 470-474. Bies-Etheve, N., da Silva Conceicao, A., Giraudat, J., Koornneef, M., Leon-Kloosterziel, K., Valon, C., and Delseny, M. (1999). Importance of the B2 domain of the Arabidopsis ABI3 protein for Em and 2S albumin gene regulation. Plant Mol. Biol. 40, 1045-1054. Bobb, A. J., Eiben, H. G., and Bustos, M. M. (1995). PvAlf, an embryo-specific acidic transcriptional activator enhances gene expression from phaseolin and phytohemagglutinin promoters. Plant J. 8, 331-343. Brady, S. M., Sarkar, S. F., Bonetta, D., and McCourt, P. (2003). The ABSCISIC ACID INSENSITIVE 3 (ABI3) gene is modulated by farnesylation and is involved in auxin signaling and lateral root development in Arabidopsis. Plant J. 34, 67-75. Brocard-Gifford, I., Lynch, T. J., Garcia, M. E., Malhotra, B., and Finkelstein, R. R. (2004). The Arabidopsis thaliana ABSCISIC ACID-INSENSITIVE8 locus encodes a novel protein mediating abscisic acid and sugar responses essential for growth. Plant Cell 16, 406-421. Bruce, W. B., Christensen, A. H., Klein, T., Fromm, M., and Quail, P. H. (1989). Photoregulation of a phytochrome gene promoter from oat transferred into rice by particle bombardment. Proc. Natl. Acad. Sci. USA 86, 9692-9696. Carrera, E., and Prat, S. (1998). Expression of the Arabidopsis abi1-1 mutant allele inhibits proteinase inhibitor wound-induction in tomato. Plant J. 15, 765-771.

40

Casaretto, J., and Ho, T. H. (2003). The transcription factors HvABI5 and HvVP1 are required for the abscisic acid induction of gene expression in barley aleurone cells. Plant Cell 15, 271-284. Chandler, J. W., and Bartels, D. (1997). Structure and function of the vp1 gene homologue from the resurrection plant Craterostigma plantagineum Hochst. Mol. Gen. Genet. 256, 539-546. Christensen, A. H., and Quail, P. H. (1996). Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgenic Res. 5, 213-218. Clough, S. J., Bent, A. F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735-743. Ezcurra, I., Wycliffe, P., Nehlin, L., Ellerstrom, M., and Rask, L. (2000). Transactivation of the Brassica napus napin promoter by ABI3 requires interaction of the conserved B2 and B3 domains of ABI3 with different cis-elements: B2 mediates activation through an ABRE, whereas B3 interacts with an RY/G-box. Plant J. 24, 57-66. Finkelstein, R. R. (1994). Mutations at two new Arabidopsis ABA response loci are similar to the abi3 mutations. Plant J. 5, 765-771. Finkelstein, R. R., Wang, M. L., Lynch, T. J., Rao, S., and Goodman, H. M. (1998). The Arabidopsis abscisic acid response locus ABI4 encodes an APETALA 2 domain protein. Plant Cell 10, 1043-1054. Finkelstein, R. R., and Lynch, T. J. (2000). The Arabidopsis abscisic acid response gene ABI5 encodes a basic leucine zipper transcription factor. Plant Cell 12, 599-609. Finkelstein, R. R., and Rock, C. D. (2002). Abscisic acid biosynthesis and response. The Arabidopsis Book, eds. C. R. Somerville and E. M. Meyerowitz, American Society of Plant Biologists, pp.1-48. Footitt. S., Ingouff, M., Clapham, D., von Arnold, S. (2003). Expression of the viviparous 1 (Pavp1) and p34cdc2 protein kinase (cdc2Pa) genes during somatic embryogenesis in Norway spruce (Picea abies [L.] Karst). J. Exp. Bot. 54, 1711-1719. Frank, W., Ratnadewi, D., Reski, R. (2005). Physcomitrella patens is highly tolerant against drought, salt and osmotic stress. Planta 220, 384-394. Gampala, S. S., Finkelstein, R. R., Sun, S. S., and Rock, C. D. (2002). ABI5 interacts with abscisic acid signaling effectors in rice protoplasts. J. Biol. Chem. 277, 1689-1694.

41

Giraudat, J., Hauge, B. M., Valon, C., Smalle, J., Parcy, F., and Goodman, H. M. (1992). Isolation of the Arabidopsis ABI3 gene by positional cloning. Plant Cell 4, 1251-1261. Goode, J. A., Stead, A. D., and Duckett, J. G. (1993). Redifferentiation of moss protonemata: an experimental and immunofluorescence study of brood cell formation. Can. J. Bot. 71, 1510-1519. Gosti, F., Beaudoin, N., Serizet, C., Webb, A. A., Vartanian, N., Giraudat, J. (1999). ABI1 protein phosphatase 2C is a negative regulator of abscisic acid signaling. Plant Cell 11, 1897-1910. Grabov, A., Leung, J., Giraudat, J., and Blatt, M. R. (1997). Alteration of anion channel kinetics in wild-type and abi1-1 transgenic Nicotiana benthamiana guard cells by abscisic acid. Plant J. 12, 203-213. Hagenbeek, D., Quatrano, R. S., and Rock, C. D. (2000). Trivalent ions activate abscisic acid-inducible promoters through an ABI1-dependent pathway in rice protoplasts. Plant Physiol. 123, 1553-1560. Hattori, T., Terada, T., and Hamasuna, S. T. (1994). Sequence and functional analyses of the rice gene homologous to the maize Vp1. Plant Mol. Biol. 24, 805-810. Hill, A., Nantel, A., Rock, C. D., and Quatrano, R. S. (1996). A conserved domain of the viviparous-1 gene product enhances the DNA binding activity of the bZIP protein EmBP-1 and other transcription factors. J. Biol. Chem. 271, 3366-3374. Hobo, T., Kowyama, Y., and Hattori, T. (1999). A bZIP factor, TRAB1, interacts with VP1 and mediates abscisic acid-induced transcription. Proc. Natl. Acad. Sci. USA 96, 15348-15353. James, P., Halladay, J., and Craig, E. A. (1996). Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics 144, 1425-1436. Jarvis, P., Chen, L. J., Li, H., Peto, C. A., Fankhauser, C., and Chory, J. (1998). An Arabidopsis mutant defective in the plastid general protein import apparatus. Science 282, 100-103. Jones, H. D., Kurup, S., Peters, N. C., and Holdsworth, M. J. (2000). Identification and analysis of proteins that interact with the Avena fatua homologue of the maize transcription factor VIVIPAROUS 1. Plant J. 21, 133-142. Kamisugi, Y. and Cuming, A.C. (2005). The evolution of the Abscisic acid-response in land plants: comparative analysis of Group 1 LEA gene expression in moss and cereals. Plant Mol. Biol. 59, 723-737.

42

Knight, C. D., Sehgal, A., Atwal, K., Wallace, J. C., Cove, D. J., Coates, D., Quatrano, R. S., Bahadur, S., Stockley, P. G., and Cuming, A. C. (1995). Molecular responses to abscisic acid and stress are conserved between moss and cereals. Plant Cell 7, 499-506. Koornneef, M., Reuling, G., and Karssen, C. M. (1984). The isolation and characterization of abscisic acid-insensitive mutants of Arabidopsis thaliana. Physiol. Plant 61, 377-383. Kurup, S., Jones, H. D., and Holdsworth, M. J. (2000). Interactions of the developmental regulator ABI3 with proteins identified from developing Arabidopsis seeds. Plant J. 21, 143-155. Lanahan, M. B., Ho, T. H., Rogers, S. W., and Rogers, J. C. (1992). A gibberellin response complex in cereal alpha-amylase gene promoters. Plant Cell 4, 203-211. Lara, P., Onate-Sanchez, L., Abraham, Z., Ferrandiz, C., Diaz, I., Carbonero, P., and Vicente-Carbajosa, J. (2003). Synergistic activation of seed storage protein gene expression in Arabidopsis by ABI3 and two bZIPs related to OPAQUE2. J. Biol. Chem. 278, 21003-21011. Lazarova, G., Zeng, Y., and Kermode, A. R. (2002). Cloning and expression of an ABSCISIC ACID-INSENSITIVE 3 (ABI3) gene homologue of yellow-cedar (Chamaecyparis nootkatensis). J. Exp. Bot. 53, 1219-1221. Leung, J., Bouvier-Durand, M., Morris, P.C., Guerrier, D., Chefdor, F., and Giraudat, J. (1994). Arabidopsis ABA response gene ABI1: features of a calcium-modulated protein phosphatase. Science 264, 1448-1452. Leung, J., Merlot, S., and Giraudat, J. (1997). The Arabidopsis ABSCISIC ACID- INSENSITIVE2 (ABI2) and ABI1 genes encode homologous protein phosphatases 2C involved in abscisic acid signal transduction. Plant Cell 9, 759-771. Marcotte, W. R., Jr., Russell, S. H., and Quatrano, R. S. (1989). Abscisic acid-responsive sequences from the em gene of wheat. Plant Cell 1, 969-976. McCarty, D. R., Hattori, T., Carson, C. B., Vasil, V., Lazar, M., and Vasil, I. K. (1991). The Viviparous-1 developmental gene of maize encodes a novel transcriptional activator. Cell 66, 895-905. Minami, A., Nagao, M., Arakawa, K., Fujikawa, S., and Takezawa, D. (2003). Abscisic acid-induced freezing tolerance in the moss Physcomitrella patens is accompanied by increased expression of stress-related genes. J. Plant Physiol. 160, 475-483. Minami, A., Nagao, M., Ikegami, K., Koshiba, T., Arakawa, K., Fujikawa, S.,

43

Takezawa, D. (2005). Cold acclimation in bryophytes: low-temperature-induced freezing tolerance in Physcomitrella patens is associated with increases in expression levels of stress-related genes but not with increase in level of endogenous abscisic acid. Planta 220, 414-423. Nakamura, S., Lynch, T. J., and Finkelstein, R .R. (2001). Physical interactions between ABA response loci of Arabidopsis. Plant J. 26, 627-635. Nambara, E., Keith, K., McCourt, P., and Naito, S. (1994). Isolation of an internal deletion mutant of the Arabidopsis thaliana ABI3 gene. Plant Cell Physiol. 35, 509-513. Nambara, E., Keith, K., McCourt, P., and Naito, S. (1995). A regulatory role for the ABI3 gene in the establishment of embryo maturation in Arabidopsis thaliana. Development 121, 629-636. Nambara, E., Hayama, R., Tsuchiya, Y., Nishimura, M., Kawaide, H., Kamiya, Y., and Naito, S. (2000). The role of ABI3 and FUS3 loci in Arabidopsis thaliana on phase transition from late embryo development to germination. Dev. Biol. 220, 412-423. Nambara, E., Suzuki, M., Abrams, S., McCarty, D. R., Kamiya, Y., and McCourt, P. (2002).A screen for genes that function in abscisic acid signaling in Arabidopsis thaliana. Genetics 161, 1247-1255. Nishiyama, T., Fujita, T., Shin, I.T., Seki, M., Nishide, H., Uchiyama, I., Kamiya, A., Carninci, P., Hayashizaki, Y., Shinozaki, K., Kohara, Y., and Hasebe, M. (2003). Comparative genomics of Physcomitrella patens gametophytic transcriptome and Arabidopsis thaliana: implication for land plant evolution. Proc. Natl. Acad. Sci. USA 100, 8007-8012. Notredame, C., Higgins, D., and Heringa, J. (2000). T-Coffee: a novel method for multiple sequence alignments. J. Mol. Biol. 302, 205-217. Parcy, F., Valon, C., Raynal, M., Gaubier-Comella, P., Delseny, M., Giraudat, J. (1994). Regulation of gene expression programs during Arabidopsis seed development: roles of the ABI3 locus and of endogenous abscisic acid. Plant Cell 6, 1567-1582. Parcy, F., Valon, C., Kohara, A., Misera, S., and Giraudat, J. (1997). The ABSCISIC ACID-INSENSITIVE3, FUSCA3, and LEAFY COTYLEDON1 loci act in concert to control multiple aspects of Arabidopsis seed development. Plant Cell 9, 1265-1277. Rohde, A., Kurup, S., and Holdswoth, M. (2000). ABI3 emerges from the seed. Trends Plant Sci. 5, 418-419. Rohde, A., Prinsen, E., De Rycke, R., Engler, G., Van Montagu, M., and Boerjan, W. (2002). PtABI3 impinges on the growth and differentiation of embryonic leaves during bud set in poplar. Plant Cell 14, 1885-1901.

44

Sambrook, J., and Russell, D.W. (2001). Molecular Cloning: A Laboratory Manual. (Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press). Schultz, T. F., Medina, J., Hill, A., and Quatrano, R. S. (1998). 14-3-3 proteins are part of an abscisic acid-VIVIPAROUS1 (VP1) response complex in the Em promoter and interact with VP1 and EmBP1. Plant Cell 10, 837-847. Shen, Q.J., Casaretto, J.A., Zhang, P., and Ho, T-H.D. (2004). Functional definition of ABA-response complexes: the promoter units necessary and sufficient for ABA induction of gene expression in barley (Hordeum vulgare L.). Plant Mol. Biol. 54, 111-124. Shen, Q., Uknes, S.J., and Ho, T. H. (1993). Hormone response complex in a novel abscisic acid and cycloheximide-inducible barley gene. J. Biol. Chem. 268, 23652-23660. Shiota, H., Satoh, R., Watabe, K., Harada, H., and Kamada, H. (1998). C-ABI3, the carrot homologue of the Arabidopsis ABI3, is expressed during both zygotic and somatic embryogenesis and functions in the regulation of embryo-specific ABA-inducible genes. Plant Cell Physiol. 39, 1184-1193. Sunilkumar, G., Mohr, L., Lopata-Finch, E., Emani, C., Rathore, K. S. (2002). Developmental and tissue-specific expression of CaMV 35S promoter in cotton as revealed by GFP. Plant Mol Biol. 50, 463-474. Suzuki, M., Kao, C. Y., and McCarty, D. R. (1997). The conserved B3 domain of VIVIPAROUS1 has a cooperative DNA binding activity. Plant Cell 9, 799-807. Suzuki, M., Kao, C.Y., Cocciolone, S., and McCarty, D. R. (2001). Maize VP1 complements Arabidopsis abi3 and confers a novel ABA/auxin interaction in roots. Plant J. 28, 409-418. Terada, R., Shimamoto, K. (1990). Expression of CaMV 35S-Gus gene in transgenic rice plants. Mol. Gen. Genet. 220, 389-392. Vasil, V., Marcotte, W. R., Jr., Rosenkrans, L., Cocciolone, S. M., Vasil, I. K., Quatrano, R. S., and McCarty, D. R. (1995). Overlap of Viviparous1 (VP1) and abscisic acid response elements in the Em promoter: G-box elements are sufficient but not necessary for VP1 transactivation. Plant Cell 7, 1511-1518. Werner, O., Ros Espin, R. M., Bopp, M., and Atzorn, R. (1991). Abscisic-acid-induced drought tolerance in Funaria hygrometrica Hedw. Planta 186, 99–103. Zeng, Y., Kermode, A. R. (2004). A gymnosperm ABI3 gene functions in a severe abscisic acid-insensitive mutant of Arabidopsis (abi3-6) to restore the wild-type

45

phenotype and demonstrates a strong synergistic effect with sugar in the inhibition of post-germinative growth. Plant Mol. Biol. 56, 731-746. Zentella, R., Yamauchi, D., and Ho, T. H. (2002). Molecular dissection of the gibberellin/abscisic acid signaling pathways by transiently expressed RNA interference in barley aleurone cells. Plant Cell 14, 2289-2301. Zhang, X., Garreton, V., Chua, N. H. (2005). The AIP2 E3 ligase acts as a novel negative regulator of ABA signaling by promoting ABI3 degradation. Genes Dev. 1, 1532-1543.

46

Table 1. AtABI3 regulated genes grouped by co-regulator for RT-PCR analysis.

* Not ABI5

Co-Regulator Gene Reference

ABI5 AtEm1

(At3g51810)

Finkelstein and Lynch, 2000

AtEm6

(At2g40170)

OPAQUE2-like bZIPs CruciferinC

(At4g28520)

Lara et al., 2003

Napin

(At4g27160)

Unknown* Oleosin2

(At5g40420)

Finkelstein and Lynch, 2000

Rab18

(At5g66400)