post-ingestion metabolism of fresh forage

7/28/2019 Post-Ingestion Metabolism of Fresh Forage

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REVIEW New Phytol. (2000), 148, 3755

Tansley Review No. 118

Post-ingestion metabolism of fresh forage

A . H . K I N G S T O N -S M I T H * M . K . T H E O D O R O U

Department of Animal Science and Microbiology, Institute of Grassland and

Environmental Research, Plas Gogerddan, Aberystwyth, Ceredigion SY23 3EB, UK

Received 28 October 1999; accepted 12 June 2000

Summary 37

I. 37

II.

:

39

III. - :

41

1. Summary of plant cell death processes 41

2. Anaerobic stress and flooding tolerance of

plants 42

It is generally assumed that breakdown of plant material in the rumen is a process mediated by gut

microorganisms. This view arose because of the identification of a pre-gastric fermentation in the rumen, brought

about by a large and diverse microbial population. The extensive use of dried and ground feed particles in forage

evaluation might have helped to promote this assumption. However, although the assumption might be correctin animals feeding on conserved forage (hay and silage) where the cells of ingested forage are dead, it is possible

that with grazed (living) forage, the role played by plant enzymes in the rumen has been overlooked. In a grazing

situation, plant cells that remain intact on entering the rumen are not inert, but will respond to the perceived

stresses of the rumen environment for as long as they are metabolically viable. Metabolic adjustments could

include anaerobic and heat-shock responses that could promote premature senescence, leading to remobilization

of cell components, especially proteins. Moreover, contact of plant cells with colonizing microorganisms in the

rumen might promote a type of hypersensitive response, in much the same way as it does outside the rumen. After

fresh plant material enters the rumen and prior to extensive plant cell-wall degradation, there is often a phase of

rapid proteolysis providing N in excess of that required to maintain the rumen microbial population. The

inefficient use of this ingested N results in generation of ammonia and urea in exhaled breath and urine, which

promotes welfare and environmental pollution concerns. Therefore an important research goal in livestock

agriculture is to find ways of decreasing this initial rate of proteolysis in the rumen. This will benefit the farmer

financially (through decreased use of feed supplements), but will also benefit the environment, as N pollution can

adversely affect pasture diversity and ecology. This review considers the possible responses of plant metabolism

to the rumen environment, and how such considerations could alter current thinking in ruminant agriculture.

Key words: forage, proteolysis, protease, heat shock, anaerobic, senescence, rumen.

I.

Livestock farming is essential in sustaining tra-

ditional landscapes and wildlife, particularly in

Europe. Large areas of land exist as permanent or

*Author for correspondence (fax j44 1970 828357; e-mailalison.kingston-smith!bbrsc.ac.uk).

3. Plant cell responses to elevated

temperatures 45

4. Wounding responses\pathogen attachment 455. Senescence in the rumen? 47

IV. 50

Acknowledgements 51

References 51

improved pastures and are sown with forage crops

such as grasses, clovers and other forage legumes to

feed ruminants throughout the production cycle. In

response to market pressure towards acceptable

environmental practices, improved traceability and

food safety, farming practices are becoming less

intensive. Although there are now financial

incentives, such as premium prices for organically


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38 REVIEW A. H. Kingston-Smith and M. K. Theodorou

Time

Protein

Energy

Fig. 1. Asynchrony in forage degradation by ruminants. Aschematic representation of the mismatch in the timing ofpeaks of protein degradation (indicated by release ofammonia) and energy (indicated by breakdown of cell-walloligosaccharides).

produced meat and milk, new and improved grass-

land technologies are required to facilitate the

changes back to more sustainable systems of ru-

minant agriculture.

Grassland-based systems of ruminant production

are characterized by the biological inefficiency of

converting plant biomass (relatively low in protein)

to animal product (relatively high in protein). In

addition, only c. 2030% of the ingested N can be

traced through to meat or milk (Dewhurst et al.,

1996; Firkins, 1996). Ruminant farming generates

vast quantities of ammonia (Siddons et al., 1985)

through an imbalance of release of proteolytic

products from the plant biomass, and the availability

of microbial energy generated by cellulolysis (the

asynchrony hypothesis) (Fig. 1). This means that,

in an environment where energy is limiting but thereis an excess of peptides and amino acids of plant

origin, the rumen microbial population uses amino

acids for energy and liberates ammonia through

deamination. In grazing ruminants, the first few

hours after feeding are characterized by rapid and

extensive proteolysis in the rumen, liberating plen-

tiful peptides and amino acids for uptake by the

rumen microbial population. According to Beever et

al. (1986), up to 35% of N lost from the rumen can

be accounted for by ammonia production in excess of

that assimilated by rumen microorganisms. Am-

monia cannot be used by the animal for growth, andis therefore eliminated in faeces, urine and exhaled

breath (Wetherall et al., 1995). The basic inefficiency

of N use is often overcome on farms by protein

supplementation. In the past, bone or fish meal was

used in addition to sources of plant proteins, but

following bovine spongiform encephalopathy (BSE)

the industry is searching for more natural ways to

improve protein supply, such as the use of high-

protein leguminous forage crops in addition to grass

and cereals. However, supplementation in any form

exacerbates the problems of pollution of land,

watercourses and air that are direct consequences of

intensive systems of livestock agriculture.

Management of grassland and rumen efficiency

have consequences beyond the welfare and per-

formance of the animal. Poor grassland management

can disrupt native ecosystems, whereas good man-

agement can resist the appearance of invasive species.

Long-term N deposition, as manure or through

application of chemical fertilizer, results in the

presence of excessive nitrate and ammonia in grazing

land. This has a negative effect on the diversity ofplant communities because selection pressure is

exerted in favour of species which are tolerant of

high N, but which are not necessarily characteristic

of the pasture (Bobbink et al., 1998). This can be

prevented to some extent by a change in grassland

management practice, for instance the use of mixed

grass\clover swards to decrease nitrate run-off

(Cuttle & Scholefield, 1995). Atmospheric ammonia

can result in plant damage and contributes to

reduced plant biodiversity in natural systems

(McGinn & Janzen, 1998). Ammonia is considered

to be an acidifying pollutant to which some plant

species are naturally more susceptible than others.

The exact mechanism of ammonia toxicity is still not

clear, although assimilation of ammonia by leaves

releases protons which can cause cellular acidosis

(Pearson & Stewart, 1993). In addition, ammonia

emissions from manure coincide with odours, which

can be a problem in areas of intensive livestock

farming (McGinn & Janzen, 1998). Minimizing the

emissions of ammonia from manure will substan-

tially decrease the impact of agriculture on the

surrounding environment.

According to Nugent & Mangan (1981), increasing

the stability of plant protein over the first 2 h ofincubation in the rumen would noticeably improve

N-use efficiency. Numerous attempts have been

made to manipulate protein and energy supply,

usually directed at the microbial population

(Broderick & Buxton, 1991; Flint, 1997; Rooney et

al., 1997; Gordon & Phillips, 1998; Forsberg et al.,

1999; Wallace et al., 1999), and rarely at the plant.

One exception is the potential use of tannins as

protein protectants during feeding (Theodorou et

al., 2000). Many plant species contain intrinsically

differing amounts and types of hydrolysable and

condensed tannin. Tannin in feedstuff is believed tocomplex with proteins inhibiting their degradation

in the rumen (Feeney, 1969), although an excess of

tannin can result in inhibition of microbial and

mammalian digestive enzymes (Swain, 1979;

Theodorou et al., 2000). The precise action of

tannins during digestion or protection of forage

protein is unclear at present. As discussed by

Theodorou et al. (2000), it is theoretically possible

for tannins to bind to forage proteins, extracellular

microbial proteases or plant proteases. Tannins are

located in vacuoles, as is the majority of the protease

pool. Assuming that they are both in the same type

of vacuole (Swanson et al., 1998), the formation of

tanninprotease complexes could occur before ac-


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REVIEW Post-ingestion metabolism of forage 39

tivation or access of vacuolar proteases to protein

substrates (sections III.1, .4 and .5). Regulation of

the formation of proteintannin complexes is com-

plicated, involving the size, conformation and

amino-acid content of proteins, and the molecular

weight, stereochemistry and composition of the

tannins (Mehansho et al., 1983 ; Spencer et al., 1988;

Butler, 1989). Future research in this area willundoubtedly be important in manipulating protein

stability in the rumen.

Enhancing rumen efficiency will result in effec-

tively converting more plant protein to animal

protein per unit of livestock, and this will bring

considerable benefits to the environment, helping to

maintain rural infrastructure and a countryside that

is attractive to visitors. By identifying and using

novel forages in grassland-based systems, farming

practices could be developed to significantly improve

the efficiency of meat and milk production from

grazed and conserved forage. Thus the correctidentification of factors determining rates of degra-

dation of cell-wall and protein components of plant

cells is of vital importance. We discuss the currently

accepted model of plant-cell degradation in the

rumen, and suggestions for modifications to the

model in the light of experimental evidence. Our

conclusions support a role for plant enzymes in the

processes culminating in release of plant-cell com-

ponents within the rumen.

I I. :

Grazing cattle and sheep excise the leaves, and

portions of stem, from forage crops. These are

chewed briefly, rolled into a bolus and swallowed

into the rumen. Plant material entering the rumen is

not a homogeneous mass, although it undergoes

significant processing after excision from the main

plant. In sheep feeding on grass, half the ingested

matter has a particle size of4 mm, whereas cattle

often ingest entire, intact leaf blades (Fraser &

Baker, 1998; Davies et al., 2000). Particle size also

depends on plant species. Swallowed particles of

grass are generally larger than those of dicoty-ledonous plants (Penning et al., 1995 ; Baumont,

1996), but even the relatively small particle size

allows many cells to be intact and in communication

with neighbouring cells and their environment

(Wilson & Mertens, 1995). Our recent work with

cattle suggests that, although there is extensive

processing to form a feed bolus (usually 510 g fresh

grass per bolus), 50% of the cells are intact (A.

Kingston-Smith, unpublished), with the residual

plant cellular material having been released into the

rumen milieu. Under normal conditions, plant

biomass can reside in the rumen for up to 24 h,

depending on particle size (Theodorou & France,

1993). During the course of this incubation, cell

walls are degraded and proteins are cleaved to

peptides and amino acids. The size of the forage

particles is decreased through mechanical forces of

movement of the rumen walls, but largely through

the process of rumination, the regurgitation and

rechewing of ingested plant biomass.

Dogma in ruminant science states that the rumen

microflora, a diverse population of anaerobic bac-teria, fungi and protozoa, colonize newly ingested

plant material, degrading plant cell walls and

proteins to liberate building blocks for microbial

growth (Hobson, 1988; Flint, 1997). It is this

microbial population which ultimately provides

much of the energy and N requirements of the

animal: energy being derived from the volatile fatty

acid fermentation products of plant biomass (ab-

sorbed from the rumen), and N from the degradation

of microbial biomass in the abomasum (the true

stomach and site of gastric digestion in ruminants).

Hence digestion of plant material in the rumen feedsthe microbial population and provides the animal

with an energy supply, whereas the digestion of

predominantly microbial protein in the true stomach

provides the animal with protein (Theodorou et al.,

1996).

Proteolytic enzymes have been identified in the

rumen microbial population, and in numerous cases

their production has been ascribed to certain rumen

microorganisms (Wallace & Cotta, 1988; Attwood &

Reily, 1995; Forsberg et al., 1999; Wallace et al.,

1999). According to Wallace & Cotta (1988), between

30 and 50% of the bacterial species of the rumen

ecosystem show proteolytic activity towards extra-

cellular protein, yet proteolytic bacteria constitute

only 2226% of the entire population of rumen

microorganisms (Dehority & Orpin, 1988). Rumen

microorganisms can use either organic or inorganic

forms of N for growth, and one of the main enzyme

activities recognized is the dipeptidase found in the

genus Prevotella (Flint, 1997; Wallace et al., 1999),

species of which preferentially take up peptides

rather than amino acids. Thus, to satisfy the needs of

the animal, the rumen microbial population relies on

a plentiful supply of ammonia and pre-formed amino

acids for rapid growth (Wallace et al ., 1999).However, although initial chewing will damage some

plant cells, the majority of the cells entering the

rumen are contained in large particles in which a

significant proportion of cells remain intact. Thus

for microbially mediated proteolysis to occur, plant

cell walls must first be breached in order to provide

the microbial enzymes access to otherwise seques-

tered plant proteins. An association between mi-

crobial enzyme and plant substrate is made all the

more difficult because microbial proteolytic activity

is located on the microbial cell surface, thereby

requiring close contact between microorganism and

plant cell for proteolysis to proceed (Wallace et al.,

1999). Variations in the susceptibility of plant


4/19


Fig. 2. Potential effectors of protein turnover in forage cells entering the rumen.

proteins to degradation by proteolytic enzymes are

recognized, and are believed to be due to the type

and extent of cross-linking, complexing with tannins,

and other chemical modifications (Wallace et al.,

1999; Theodorou et al., 2000). However, protection

afforded by the effective sequestration of proteins

within cells is not usually considered. In a recent

review of microscopic studies, Grenet (1997) esti-mated that colonization of newly ingested feed took

place by bacteria at 8 h, by protozoa after 2 h, and by

fungal spores after 15 min, but these spores then

took 3 h or more to germinate and produce hyphae to

degrade plant cell walls, depending on the degree of

lignification of the forage. These observations on the

length of time taken for microbial colonization are in

line with the general view that cellulolysis in the

rumen is not as rapid as proteolysis. Several potential

plant responses to the rumen environment are likely

to occur within these time scales. For instance,

closure of stomata in response to perceived floodingwould occur in a matter of minutes (Vartapetian &

Jackson, 1997). This would prevent access of

invasive microorganisms through stomata, although

access through cut edges is still possible. The

invasion route (stomata or cut ends) probably

depends on the type of grazing animal: the pro-

portion of cut ends in the feed bolus of cattle would

be substantially lower than the number of cut ends in

plant material ingested by sheep (Fraser & Baker,

1998; D. Davies, A. Kingston-Smith, unpub-

lished).

In addition to the physical considerations of

microbial invasion of ingested plant material, the

effectiveness of microbially mediated proteolysis

needs to be examined. In comparison with the

animals own gastric secretions of proteases, the

proteolytic activity of the rumen microbial popu-

lation is low, and the majority is intracellular

(Wallace & Cotta, 1988; Asoa et al., 1993; Falconer

& Wallace, 1998). In the case of the protozoa,

proteolytic activity is directed against the degra-

dation of ingested bacterial cells (Wallace & Cotta,1988). Moreover, microbial proteolytic activity is

routinely measured in vitro by assessing growth on,

and\or degradation of, either dried, ground plant

substrates or purified proteins, rather than proteins

sequestered in living plant cells. According to

Wallace et al . (1999), the majority of rumen

proteolytic activity is exopeptidase in nature,

whereby cleavage is via the terminal amino acid only.

Thus the values obtained by supplying microbial

enzymes with a purified, soluble (artificial ) sub-

strate such as casein might overestimate the true

value when supplied with mixed plant protein inwhich N-termini for peptidase attack might be

protected and not accessible. Nevertheless, on the

basis of such evidence it was concluded that

degradation of plant biomass in the presence of

rumen fluid was mediated by microorganisms. This

might well be the case for conserved (dead) feeds

such as hay and silage, where much of the degra-

dation of plant proteins has already occurred under

the influence of plant proteases in the silo (for silage)

or standing crop (for hay; Merry et al., 2000); but it

probably underestimates the contribution of the

plant enzymes ingested with fresh forage

(Theodorou et al., 1996).

There is increasing evidence that plant enzymes


5/19


within the rumen make an active contribution to the

initial stages of protein degradation. Even where

plant cells are ruptured during chewing and initial

digestion, their enzymes might still contribute to the

degradative processes in the rumen. Indeed,

although the survival of liberated plant enzymes

might be short-lived in the rumen, the elevated

temperatures of the rumen environment mightenhance their activities. Plants have evolved a range

of responses to a fluctuating environment. The main

stresses perceived by plant cells in the rumen are lack

of oxygen, elevated temperature, darkness and the

presence of a large microbial population (Fig. 2). It

is reasonable to expect that plant cell death, as a

response to these stresses, influences protein turn-

over rate through de novo synthesis of proteases or

post-translational modification of existing proteases.

Initial evidence in favour of plant-mediated pro-

teolysis in the rumen comes from the work of Zhu et

al. (1999). When grass was incubated anaerobicallyat 39mC for several hours in the presence of an active

population of rumen microorganisms, there was a

decrease in abundance of ribulose-1,5-bisphosphate

carboxylase\oxygenase (Rubisco) large subunit,

similar to that observed after similar incubations in

which microorganisms were absent (Zhu et al.,

1999). This implies that the common factor, the

plant enzymes of the fresh grass, were responsible

for the major portion of proteolysis.

Plants contain a wide array of proteases, par-

ticularly aminopeptidases (and often the respective

inhibitors) for different functions and in different

locations (Feller, 1986; Moslov, 1995 ; Vierstra,

1996; Distefano et al., 1997 ; Clarke, 1999). Protease

activity is required by plants for a variety of

functions, dissolution of storage proteins during

seed germination, protein turnover, degradation of

misformed or damaged proteins (the constitutive

housekeeping proteases which might occur in

organelles or within lytic vacuoles), and finally the

induction of proteases to remobilize valuable N

during senescence. These processes are controlled

both during transcription, and later through post-

translational modification. Hence it is important to

understand the metabolic processes occurring withinthe newly ingested plant cells in order to identify

factors responsible for plant-mediated proteolysis in

the rumen.

III. - :

1. Summary of plant cell death processes

Cell death in plants is a valuable mechanism for the

survival of the whole organism, just as unchecked

cell proliferation in animals is prevented by apoptosis

(Vaux, 1993). Whereas apoptosis in mammals is now

a well understood phenomenon, it is less so in plants,

mainly because there are various forms of cell death,

for example, programmed cell death in organo-

genesis, necrosis and senescence (Beers, 1997;

Pennell & Lamb, 1997; Buckner et al., 1998). Using

the animal system as a model, homologues to the

defender against apoptotic death (dad) gene have been

discovered in pea and maize (Gray et al., 1997;

Orzaez & Granell, 1997). The presence of this genein both animals and plants is not surprising, as this

gene has been shown to code for an oligosaccharide

transferase (Kelleher & Gilmore, 1997) essential for

glycoprotein synthesis. However, the functional

significance ofdad, and glycosylation, in cell death in

plants remains to be elucidated. Programmed cell

death induced by ageing, stress and pathogen attack

results in common responses such as upregulation of

cytochrome P%&!

expression (Godiard et al., 1998)

and increased protease activity (Minami & Fukuda,

1995; Drake et al., 1996; Ye & Varner, 1996 ; Beers

& Freeman, 1997; Gan & Amasino, 1997). Inanimals, the caspase subfamily of the cysteine

proteases plays a key role in apoptotic cascade

signalling, together with the appropriate regulatory

signals (Vaux, 1993; Noode! n et al., 1997). Caspases

are a group of cysteine proteases which cleave

exclusively after aspartic acid (Cohen, 1997).

Cysteine proteases are implicated in cell death in

senescent tissues (Smart, 1994; Morris et al., 1996;

Buchanan-Wollaston, 1997; Del Pozo & Lam, 1998;

Guerrero et al., 1998; Xu & Chye, 1999), but only

limited evidence in favour of involvement of

caspases. Thomas & Donnison (2000) have reported

evidence that the maize SEE2, a senescence-

enhanced cysteine protease of the legumain family, is

part of a proteolytic cascade. Peptide inhibitors of

caspases have varying effectiveness in inhibition of

hypersensitive cell death in plants (Pontier et al.,

1999; Yano et al., 1999). Chemical inhibition of

serine proteases has been shown to inhibit a specific

type of cell death (a nonpathogen-induced hyper-

sensitive response) that would otherwise result from

application of exogenous hydrogen peroxide (Yano

et al., 1999). This work also showed that the serine

proteases were involved during the first 4 h of

programmed cell death, and suggested a regulatoryfunction for this class of enzymes (Yano et al., 1999).

Likewise, cells in which cysteine proteases have been

induced as a result of oxidative stress can be rescued

from death by cystatin (cysteine protease inhibitor)

treatment (Solomon et al., 1999). Hence the balance

between protease activity and the presence of

inhibitors must be a vital factor in determining the

onset and progression of cell death. Other elements

must be common, given recent evidence in which

transgenic plants were constructed to express bcl-xL,

an inhibitor of apoptosis, which also conferred

protection against senescence and oxidative cell

death in tobacco (Mitsuhara et al., 1999). In the

converse experiment, plants expressing Bax, a pro-


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moter of animal cell death, appeared to activate an

endogenous cell death response (Lacomme & Santa

Cruz, 1999). Also, plant cells undergoing hyper-

sensitive cell death can cleave poly(ADP-ribose)

polymerase (PARP), an indicator of apoptotic cell

death, through induction of cysteine protease activity

(DSilva et al., 1998). Visual similarities between

apoptosis and programmed cell death in plantsinclude changes to cytoplasmic morphology

(vacuolarization) and cleavage of nuclear DNA

(Thelen & Northcote, 1989; Ryerson & Heath,

1996; Mittler et al., 1997; OBrien et al., 1998; Yen

& Yang, 1998). The involvement of calcium as an

intermediate in the signal transduction pathway also

appears to be a common theme amongst cell death

pathways.

The role of oxidative signalling in plant cell death

processes has been well established, especially in the

area of pathogen infection and the hypersensitive

response (Mittler & Lam, 1995; Allan & Fluhr,

1997; Morel & Dangl, 1997). Certain enzymic

components of the antioxidant defences (most no-

tably catalase and glutathione-S-transferase) are

increased during senescence (Buchanan-Wollaston,

1997) and could be important in tolerance to

waterlogging-induced anaerobic stress in roots

(Chirkova et al., 1998). The active synthesis of

proteases is a rapid response which occurs approx.

30 min after application of exogenous hydrogen per-

oxide (Solomon et al., 1999). This suggests a role for

oxidative signalling in the regulation of senescence.

This is entirely consistent with observations of

increased lipid peroxidation which preceded visiblesigns of senescence in detached and darkened maize

leaves (Hung & Kao, 1997). The significance of

oxidative signalling in the current context is unclear,

however. Plant tissues entering the rumen are

aerated, containing oxygen within and between cells,

and also some air must be taken into the animals

mouth with the forage. This oxygen will surely be

available for utilization by plant metabolic processes

(section III.2). On the other hand, if the generation

of active oxygen is a prerequisite for cell death, a

proportion of the available oxygen might be used in

this manner, and hence increased antioxidant statusin plants might extend the interval before cell death

in the rumen occurs. According to Solomon et al.

(1999), the extent of oxidative stress in plants might

determine the type of cell death ensuing. A rapid and

extensive accumulation of active oxygen species

results in uncontrolled (necrotic) cell death, whereas

developmental or adaptive cell death (such as occurs

in the formation of xylem) ensues after a slower

accumulation of active oxygen species that never-

theless exceeds a threshold level. It follows that the

persistence of oxygen within plant tissue after entry

into the rumen will largely determine the efficacy of

oxidative signalling and hence the induction of cell

death in ingested forage. Some recent evidence

challenges this view, since it has revealed that cell

death can occur even when the generation of an

oxidative burst has been suppressed (Pallardy et al.,

1997; Yano et al., 1999). Likewise, in the presence of

xylanase, cultured tobacco cells undergo a hyper-

sensitive response and subsequent cell death (note

that rumen bacteria posses many forms of xylanase

activity, Forsberg et al., 1999). Although the oxi-dative burst maxima at 23 h post-elicitation were

prevented by treatment of cell cultures with catalase,

cell death still ensued (Yano et al., 1999).

2. Anaerobic stress and flooding tolerance of plants

Probably the most significant difference between the

normal environment of plants and the rumen is the

lack of oxygen. Rumen microorganisms are pre-

dominantly obligate anaerobes, so are killed on

exposure to oxygen. Thus it is essential for the

rumen to be highly anoxic. This is achieved largely

through the fermentative metabolism of the mi-

crobial population and their production of highly

reduced end products, and possibly also through the

action of facilitative anaerobes which scavenge

available oxygen. In this respect, the newly ingested

plant tissues do not undergo a gradual loss of

environmental oxygen, but are subjected to a sudden

transfer to an anoxic atmosphere, the only oxygen

present being that contained within the tissues. As

noted by Ricard et al. (1994), situations in which

plant material truly undergoes a rapid switch from

aerobic to anaerobic conditions are rare. The most

studied anaerobic situation is waterlogging (Ricardet al., 1994), in which oxygen is gradually depleted

(hypoxia) prior to complete reliance on fermentative

generation of ATP (anoxia). It has been known for a

long time that exposure of plant tissues (especially

roots) to hypoxia can precondition plant cells, thus

improving metabolic tolerance to a subsequent

period of anoxic treatment when compared with

tissues exposed directly to anoxia (reviewed by

Drew, 1997). Therefore the lack of the hypoxic

preconditioning period for plant tissue entering the

rumen is likely to result in a rapid loss of viability.

Continued energy supply is crucial to cell survival.Plants are adapted to an aerobic environment and

rely on respiration to supply ATP from glucose.

However, approx. 15 min after transfer to anaerobic

conditions, roots become ATP-limited (Raymond et

al., 1985). Damage to mitochondria induced by

anaerobic conditions included swelling (in a few

minutes) and changes in membrane lipid compo-

sition, although mitochondria maintained anaer-

obically were capable of respiration if supplied with

ATP (Andreev et al., 1991; Couee et al., 1992).

When the oxygen supply is exhausted, metabolism

switches to fermentation via substrate-level

phosphorylation in an attempt to maintain ATP

production (Perata & Alpi, 1993; Drew, 1997).


7/19


Theoretically, 32 mol ATP can be supplied from

each mol glucose, but recent estimates suggest that

only half of the mitochondrial electron transport is

actually used for glycolysis (Rawyler et al., 1999).

Even this lowest estimate exceeds the capacity of

ATP generation through ethanolic fermentation

(2 mol ATP per mol glucose), implemented due to

lack of oxygen. In practice this means that cells cansurvive for a certain period before metabolism is

seriously hampered. For example, potato cells in

culture showed no membrane damage for the first 12 h

after imposition of anaerobic stress (Rawyler et al.,

1999). Thereafter, free fatty acids accumulated in the

potato cells coincident with the calculated energy

supply decreasing to 12% of the aerobic maximum.

This was despite increases in ethanol and lactate

which indicated active fermentation (Rawyler et al.,

1999). From this, and studies with inhibitors, it was

suggested that there is a threshold of ATP pro-

duction of approx. 10 mol g"

f. wt h"

, below whichmembrane structure cannot be preserved (Rawyler

et al., 1999). The restricted ATP supply also has

direct implications for proteolysis. The cytosolic

serine protease from lettuce (LoPiero & Petrone,

1999), the 26S proteosome which appears to operate

in cytoplasm and the nucleus of most eukaryotes

(Vierstra, 1996), and the ClpP homologues in the

cytosol (Clarke, 1999) all require ATP to function.

Hence the major protease activities in ingested plant

cells in the rumen are likely to be ATP-independent.

Induction of anaerobic responses involves de novo

synthesis of the anaerobic response proteins, most

notably isoforms of lactate dehydrogenase (LDH)

and alcohol dehydrogenase (ADH) specific to an-

aerobic stress (Perata & Alpi, 1993 ; Drew, 1997 ;

Vartapetian & Jackson, 1997; Kato-Noguchi, 1999).

Synthesis of these specific proteins increases despite

an initial decrease in total protein synthesis caused

by disassociation of ribosomes (Bailey-Serres &

Freeling, 1990). In addition to ADH and LDH,

other proteins and transcripts are upregulated in

response to anaerobic stress, but with roles in the

anaerobic stress response that have yet to be clearly

defined. Different isoforms can be expressed selec-

tively in roots or leaves in response to anaerobicstress, and these activities appear to be an intrinsic

and essential part of the anaerobic response mech-

anism. Interestingly, mRNA accumulation often

exceeds that accounted for by amino-acid flux

through the protein (Ricard et al., 1994), and does

not prevent significant accumulation of potentially

toxic ethanol (Good & Muench, 1993; Ricard et al.,

1994; Dolferus et al., 1997; Kato-Noguchi, 1998 ;

Matsumura et al., 1998).

The best documented pathway of the change from

aerobic to anaerobic metabolism is given by the

DaviesRoberts pH stat theory. Increased activity of

lactate dehydrogenase converts pyruvate (last step in

glycolysis not requiring oxygen) to lactate. The

accumulation of lactate, and ATP restriction of the

operation of the tonoplast proton pumps, results in a

pH decline from 7n3 t o 6n8 over approx. 20 min

(Drew, 1997). This effectively decreases the cytosolic

pH below the pH optimum of LDH, but within the

range of pyruvate decarboxylase (PDC), the first

enzyme of ethanolic fermentation (Fox et al., 1995).

There might also be some form of negative feedbackin which acidosis is prevented by gradual loss of

affinity of LDH for NADH as pH drops (Drew,

1997; OCarra & Mulcahy, 1997). This converts

pyruvate to acetaldehyde, which is then converted to

ethanol by alcohol dehydrogenase. Both LDH and

ADH require NADH as a cofactor in the reactions.

A recent estimate puts the flux of carbon through

LDH as accounting for only about 12% of the total

flux in carrot roots under anoxia (Kato-Noguchi,

1998). PDC is regarded as the rate-limiting step of

ethanolic fermentation, although the pH optimum

for ADH is approx. 8n5 (Sachs etal., 1980). Attemptsto alter rates of ethanolic fermentation have been

addressed by manipulating the PDC-catalysed re-

action. Transgenic tobacco overexpressing PDC did

not show increased ethanol and acetaldehyde in the

roots (Tadege et al., 1998), although activity was

altered in both leaves and roots (20- and twofold,

respectively). The DaviesRoberts pH stat is an

attractive and widely supported hypothesis, although

its applicability might vary depending on the

flooding tolerance of the species under investigation

(Vartapetian & Jackson, 1997). It has been suggested

that flux through lactate is in fact low, despite

induction of the enzyme, such that the observed

increases in lactate, although correlative, are not

causal in the switch to ethanolic fermentation (Kato-

Noguchi, 1998; Tadege et al., 1998). This is due to

the relative affinities of pyruvate dehydrogenase

(PDH) and PDC for substrate (Kml 5779 M and

0n251n0 mM, respectively). There is a noticeable

increase in cytosolic pyruvate when cells enter

anaerobic conditions from 0n1 to 1 mM, allowing

PDC, with the higher Km

, to compete with PDH for

substrate. Thus the lactate accumulation represents

a competitive reaction, not a direct link (Tadege et

al., 1998). Without the appropriate kinetic exper-imentation, it is not yet possible to be sure of the

effect of the three competitive reactions on pyruvate

in vivo. If correct, it means that acidosis is a

consequence, not a cause of anaerobic metabolism.

Hence, attempts to limit anaerobically induced cell

death by addressing cytoplasmic acidosis might be

futile in comparison to increasing flux through PDC,

widely agreed to be the rate-limiting step in ethanolic

fermentation. It is interesting, though, that in-

creasing ethanol production did not increase anoxia

tolerance in these plants, but rather promoted

increased membrane leakage, ethanol and acetalde-

hyde production, and earlier cell death (Tadege et

al., 1998). Thus it is likely that anoxia tolerance is


8/19


multifactorial, involving fermentation rates, sub-

strate availability, and toxicity of fermentation

products.

A role of calcium in anaerobic stress signalling has

also been proposed, following small but significant

changes in mitochondrial calcium content from 109

to120 nMCa#+ after only 10 min of anoxia (Subbaiah

et al., 1998). This work with confocal imaging ofcalcium-sensitive dyes also suggests that there is a

heterogeneity of response of the mitochondria to

anoxia, even within one cell, the reason for which

remains unresolved (Subbaiah et al ., 1998). It

appears that, as with apoptotic signalling, after onset

of anoxia there is a rapid calcium efflux response

from the mitochondria, not the vacuole, which could

be the initiator of the cell death cascade\prevention

systems (Subbaiah et al., 1998).

Plant cells can survive anoxia for a limited time,

and this appears to be genetically predetermined

(Ricard et al., 1994; Drew, 1997). This might be due

to intolerance of one or more of the products of

ethanolic fermentation (Crawford, 1982; Kimmerer

& MacDonald, 1987; Perata & Alpi, 1993), or the

lack of ATP which can occur within 15 min

(Raymond et al., 1985). Differential accumulation of

ethanol has been proposed as a potential cause of

observed species-specific differences in flooding

tolerance (Crawford, 1982). Although plants respond

to physiologically relevant ethanol concentrations

(Perata & Alpi, 1993), these effects are now believed

to be due to conversion of ethanol to acetaldehyde

(Perata & Alpi, 1993, Kreuzwieser et al., 1999). Plant

cells can generate ethanol under field conditions dueto flooding of roots (Crawford, 1982; Kreuzwieser et

al., 1999) or when stomata are closed in the dark and

respiration consumes available oxygen (MacDonald

& Kimmerer, 1993). It has been suggested that

ethanol is toxic to plant cells, but the evidence is

equivocal. In leaves, ethanol has been observed

at concentrations of 250 mM (Kimmerer &

MacDonald, 1987; Kato-Noguchi, 1998; Rawyler et

al., 1999), although in vitro exogenous application

requires 100600 mM to induce death (Perata &

Alpi, 1993). Metabolic attempts to remove ethanol

involve translocation in the xylem and volatilization.Ethanol fed to leaves, or translocated from the roots,

results in release of volatile acetaldehyde

(Kreuzwieser et al., 1999). However, ethanol in the

xylem increases from 0 to 12 mM during 6 h

flooding, but only approx. 0n1% of that delivered is

metabolized to acetaldehyde, the remainder appar-

ently being converted to acetate, which is accumu-

lated (MacDonald & Kimmerer, 1993; Kreuzwieser

et al., 1999).

It is well known that both monocot and dicot

plants differ in their tolerance to flooding (for review

see Crawford, 1982), and their survival mechanisms

might give an insight into the potential ways in

which forage can remain viable for longer in the

rumen. The formation of aerenchyma is an obvious

phenotypic adaptation (Vartapetian & Jackson,

1997), but is relatively long term and so is unhelpful

as an analogy to the rumen situation. Rice is a

flooding-tolerant species, whereas maize is intolerant

of short periods of waterlogging. Different rice

cultivars can vary in their flooding tolerance, and this

has been linked to the onset of senescence processesduring prolonged flooding (Krishnan et al., 1999).

This has some support from observations that foliar

protein remobilization is decreased in flooding-

tolerant compared with intolerant lines. In the latter,

a mere 4 d submergence of plants produces a foliar

senescent state resembling that of natural senescence

in attached leaves after 40 d (Krishnan et al., 1999).

Lipoxygenase activity is largely responsible for

anaerobic degradation of membranes, and activity

has been shown to increase in both shoots and roots

of anaerobically maintained rice and wheat

(Chirkova et al., 1998). These results suggested that

enough oxygen was generated internally for con-

tinued lipid peroxidation over 3 d (Chirkova et al.,

1998). The persistence of oxygen in anaerobically

stressed plant tissue is important, as plant material

entering the rumen could likewise be forced to

undergo premature senescence through water-

logging.

It has been suggested several times that carbo-

hydrate status influences tolerance by providing

a ready and plentiful supply of respiratory\

fermentable substrate. Substrates proposed include

sucrose, glucose and fructans (Albrecht et al., 1997;

Vartapetian & Jackson, 1997). It would also appearthat, as with much of plant metabolism, the sugar

status represents an important signalling component

of anaerobic metabolism (Zeng et al., 1998). Trans-

genic root tips with increased rates of ethanolic

fermentation die more quickly than roots from

untransformed plants, but this can be prevented if

the transgenic plants are supplied with exogenous

glucose (Tadege et al., 1998). Plants in which

hexokinase has been manipulated showed altered

response to anaerobic stress and change in tran-

scription of the anaerobic response proteins. Ni-

trogen metabolism and exogenous nitrate supplymight also be important in anoxia tolerance (Van

Lerberghe et al., 1991; Fan et al., 1997 ; Vartapetian

& Polyakova, 1999). Intolerant plants show NADH

accumulation within a few minutes of the stress

(Roberts et al ., 1984). By maintaining nitrate

reduction to ammonium, and subsequent reassimi-

lation, an extra sink for six protons and four NADH

per reaction cycle during anoxia become available.

This pathway avoids generation of the toxic or acidic

products of lactate and ethanolic fermentation lead-

ing to cell damage and death (Fan et al., 1997;

Vartapetian & Polyakova, 1999). Work with

flooding-tolerant rice showed uptake of nitrate or

ammonium ions from the bathing solution, and


9/19


incorporation of "&N into amino acids (specifically

glutamine and alanine) was enhanced under an-

aerobic conditions relative to aerobic conditions

(Fan et al., 1997). It was suggested that continued N

assimilation maintained the flux of electrons and

decreased accumulation of NADH, enabling con-

tinued fermentation. This is intriguing in the context

of the rumen, in which ammonium concentrationsprior to feeding might be low, thereby allowing the

possibility that forage with high rates of N as-

similation might survive longer in the rumen than

those with poor assimilation rates.

3. Plant cell responses to elevated temperatures

Plants are exposed to varying temperatures in the

field and have well characterized responses to periods

of cold and heat (reviewed by Howarth & Ougham,

1993). The rumen is maintained at c. 39mC, which is

similar to the acclimation temperatures used in heat-shock experiments on temperate species. At the

metabolic level the immediate response to elevated

temperature would be an increase in reaction rate. In

the rumen, this would very probably contribute to an

increased rate of consumption of metabolic oxygen,

exacerbating the onset of anaerobic responses as

already discussed. However, few data exist relating

to the effect of a temperature shift to 39mC on the

biology of plants. Hence the results from exposure to

higher temperatures must be used in the first

instance to identify potential responses of plant

tissues to the rumen temperature. Experiments with

cell cultures showed that treatment at 44mC induced

symptoms of apoptosis and cell death in 50% of

cells after 24 h treatment, compared with death of

5% of cells maintained at ambient temperature

(Chen et al., 1999). A heat treatment lasting only 4 h

was sufficient to result in cytoplasmic condensation

and DNA fragmentation (Chen et al., 1999). A

temperature of 39mC might be sufficient to induce

similar responses, albeit observed over a longer time

or to a lesser extent.

Increased temperature will also affect rates of

protein turnover. For example, Rubisco is sus-

ceptible to both heat-induced proteolysis anddecreased synthesis (Vierling & Key, 1985; Bose et

al., 1999). The majority of plant protease activity in

mature leaves is endoproteinase (cleaving within the

peptide chain) and resides in the vacuole (Matile,

1997). Thus, theoretically, plant enzymes have an

advantage over those of the rumen microbes in being

closer to their substrates, especially should the

tonoplast (and other organelle membranes) be

damaged in response to the elevated temperature of

the rumen. It should be noted, however, that

proteases released from the vacuole might require

processing to become functional, and it is not known

at present how, or if, this would occur in damaged

cells. Likewise, the pH within the plant cells in the

rumen is unclear at present, as is the timing of

processes such as cytoplasmic acidosis and infil-

tration of rumen fluid (buffered to approx. pH 6n8).

This could affect protease activity in vivo and cause

errors in the estimation of activity by in vitro

measurements. Although genotypic variations in

heat tolerance exist (Howarth & Ougham, 1993),

there is a threshold in the response which couldpotentially be utilized through plant breeding, for

example to limit heat-induced proteolysis in a

susceptible variety. The combination of increased

proteolytic activity and decreased energy restricting

the capacity for protein synthesis could therefore

result in a net decrease in cellular protein.

Exposure to temperatures above ambient induces

de novo synthesis of heat-shock proteins (HSP).

Synthesis is transient, detectable minutes after

imposition of heat stress, but occurring only during

the first 2 h at 40mC (Kimpel et al., 1990). These

proteins are thought to participate in cellularattempts to prevent heat-induced misfolding of

proteins (Boston et al., 1996). Cellular localization

and precise functions of the HSPs varies. For

example, the HSP70 family of the cytosol and the

cpn family in the chloroplast aid correct protein

folding, whereas the HSP100 family functions to

promote disaggregation of protein, hence enabling

degradation and remobilization (reviewed by Boston

et al., 1996). Under aerobic conditions these proteins

can account for up to 2% of the cellular protein. In

relation to plant cells in the rumen, these two factors

suggest that the balance between protein degradation

and synthesis favours degradation, especially as

incubation in the rumen progresses. We have

observed net loss of protein from grass incubated

under anaerobic conditions at 39mC in the absence of

microorganisms (Beha et al., 1998; Kingston-Smith

& Theodorou, 2000).

Other metabolic changes in response to heat

involve lipid and membrane chemistry (Blum &

Ebercon, 1980; Howarth & Ougham, 1993; Mittler

& Lam, 1995). Heat stress promotes lipid turnover,

increased fluidity and membrane permeability simi-

lar to that observed during senescence (section

III.5). In the presence of oxygen, such as thatentering the rumen within or between plant tissues,

increased lipid peroxidation might also occur

(Thompson et al., 1998). However, this might be a

transient event as oxygen is consumed by plant

respiration or by the facultative anaerobes of the

rumen. The significance of lipid peroxidation in

plant tissues in the rumen remains to be demon-

strated.

4. Wounding responses\pathogen attachment

Studies of wounding responses following herbivory

are usually conducted on remaining plant parts, with

little attention being given to the ingested portions.

Although herbivory damage usually concerns aphid


10/19


or caterpillar attack, the generation of wound

responses could be the same after cattle or sheep

damage. Plant parts damaged by herbivory can

produce a systemic wound response, localized pro-

teolysis and necrosis (Jakobek et al., 1999; Rhodes

et al., 1999; Suh et al., 1999). The response to

wounding in detached plant parts might differ from

that observed in intact plant parts. For example,systemic responses might occur in the long leaf

lengths ingested by cattle as part of the feed bolus

(Davies et al., 2000), but more localized responses

would occur, of necessity, in the smaller sections

ingested by sheep (Fraser & Baker, 1998).

Investigations into plantmicrobe interactions in

the rumen are generally exemplified by attachment

and plant degradation studies, rather than by studies

of the plants response to sudden exposure to a large

microbial population (estimates between 10) and

10"# colony-forming units (cfu) ml"). In the field,

plants are exposed to a variety of potentially

pathogenic microorganisms and have developed

species-specific degrees of tolerance and resistance

(Greenberg, 1997; Morel & Dangl, 1997). The

hypersensitive response can be generated in vitro by

incubation of plant material with 10& cells ml" of

bacteria, with lesions appearing after 72 h (Jakobek

et al., 1999). The observation that hypersensitive cell

death does not occur in cell cultures (Honee et al.,

1998) could be interpreted to dismiss the significance

of hypersensitive response in plant tissue in the

rumen. However, plant material entering the rumen

is not in the form of single cells, but is in tissue pieces

ranging from 0n5 cm to an entire leaf lamina in sheepand cattle, respectively. It is therefore possible that

plant cells entering the rumen react to rumen

microorganisms, or certain species of them (the

primary colonizers), as pathogens. It is also possible

that extracellular compounds secreted by micro-

organisms act as elicitors of a hypersensitive-like

response, or simply induce expression of defence

genes without hypersensitive cell death (Mittler &

Lam, 1995; Greenberg, 1997). The hypersensitive

response is an active cell death processes that also

requires protein synthesis (Mittler & Lam, 1995 ;

Morel & Dangl, 1997). The signalling pathwaysinvolve an active oxygen burst from NADPH oxidase

on the cell wall. Cell contents are degraded without

extensive remobilization towards other cells, the

main objective being the formation of a barrier of

dead cells between pathogen and host. Although

hypersensitive cell death is a severe response,

specificity is retained, and induced proteases and

inhibitors are not necessarily the same as those

involved in the wounding response (Suh et al.,

1999). It is possible, but as yet untested, that plant

cells entering the rumen perceive the microbial

population as pathogens and undergo some form of

cell death involving rapid degradation of proteins

and lipids.

One response of plant cells to pathogens involves

synthesis of PR proteins and protease inhibitors

(Mittler & Lam, 1995; Morel & Dangl, 1997;

Krishnaveni et al., 1999; Pernas et al., 1999). The

exact function of these compounds is unclear at

present, although options include direct damage to

the invading fungus, or production of metabolites

leading to inhibition of fungal growth. One group ofcysteine protease inhibitors, cystatins, are particu-

larly effective against the latter; the cystatin from

chestnut stopped growth of phytopathic fungi at

9 M and inhibited proteases from Botrytis cinerea at

5 M (Pernas et al., 1999). It should also be noted

that as part of the senescence disassembly procedure

chitinase activity increases, presumably as a defence

against potential pathogens that could enter weak-

ened, senescent tissues and spread to healthy parts

(Buchanan-Wollaston, 1997; Lers et al., 1998). It is

therefore also a possibility that plant cells entering

the rumen might themselves affect the ability of

rumen fungi to colonize the ingested plant material.

Presumably, putative PR protein expression during

senescence also fulfils a protective role (Tamas et al.,

1998). The source of extracellular proteases in the

rumen is always considered to be microbial. How-

ever, preliminary results indicate that extractable

proteases of ryegrass and clover show a similar

activity and isoform profile at acidic pH, assumed to

be optimal for vacuolar proteases, and the more

neutral pH of the rumen fluid (A. Kingston-Smith,

unpublished). As up to half of the cells could be

disrupted through mechanical processing prior to

ingestion, this is a potentially large source ofnonmicrobial, extracellular protease activity.

Investigations have revealed that plant responses

to wounding and pathogenic attack share some

features, such as transcription of common coding

regions, but the genes involved respond differentially

during wounding and hypersensitive responses

(Jakobek et al., 1999). The regulatory molecule

jasmonate is derived from fatty acid breakdown, so

could reasonably be expected to occur in ingested

portions of leaf tissue as well as the remainder.

Changes in transcript accumulation in response to

jasmonate can be rapid, for example the accumu-lation of the transcript of the PINII protease

inhibitor within 3 h (Suh et al., 1999). It has been

suggested that whereas jasmonate signals during the

wounding response, salicylate is the signal molecule

during infection by pathogens (Suh et al., 1999).

The role of abscisic acid as the primary signalling

molecule of the wound response has been considered

(Birkenmeier & Ryan, 1998; Jakobek et al., 1999).

Recent evidence casts doubt on this, as no general

increase in abscisic acid was found after wounding,

but it was concentrated at the site of wounding itself,

suggesting that desiccation at the wound site led to

an artefact of measurement (Birkenmeier & Ryan,

1998). In addition, the induction of a wound-specific


11/19


protease inhibitor correlated better with rapid

jasmonate production (1 h post-wounding) than with

the peak of abscisic acid accumulation (6 h post-

wounding). Thus although signalling mechanisms in

wounding might be different from those of the

hypersensitive response and of senescence, it is

possible that common features exist in the cell death

pathways (Butt et al., 1998; Pontier et al., 1999).The oxidative burst is a common feature of both

the hypersensitive response and wound damage, but

can be induced apoplastically after wounding, or

intracellularly in response to fungal elicitors (Allan &

Fluhr, 1997). With bacterial infection, a first oxi-

dative burst occurs approx. 1 h after infection, but

cell death only comes about as a result of a second,

larger oxidative burst approx. 6 h after infection

(Lamb & Dixon, 1997). This is considered to be

brought about by the action of NADPH oxidase on

the plasma membrane, the resultant hydrogen per-

oxide acting as a diffusable signal to neighbouringcells. Interestingly, hydrogen peroxide also stimu-

lates a rapid influx of calcium ions to the cytosol

(Lamb & Dixon, 1997). This could explain why

there is induction of peroxidase genes after both

wounding and pathogen infection (Curtis et al.,

1997; Mittler et al., 1999). However, reactive oxygen

does not appear to be an absolute requirement for

hypersensitive cell death (Yano et al., 1999). In

tobacco, inhibitor studies suggested that in the

absence of an oxidative burst, an elicitor-mediated

hypersensitive cell death was brought about by a

serine protease (Yano et al., 1999).

Proteinase inhibitors and polyphenol oxidase

genes (Constabel & Ryan, 1998) appear several hours

after wounding and are part of the late responsive

genes. By contrast the early response genes appear

approx. 30 min after wounding, as for example in

expression of the lipoxygenase gene in tomatoes

(Heitz et al., 1997). In this case wound signalling was

mediated by the octadecanoid (jasmonate-forming)

pathway, but expression appeared to be light-

dependent and targeted towards the chloroplast

(Heitz et al., 1997). Therefore wound responses in

the rumen might be attenuated due to aspects of

light\dark regulation.

5. Senescence in the rumen?

Senescence is a genetically programmed process, the

rate of which depends on the organ and species

under consideration (Thomas & Stoddart, 1980 ;

Thomas & Smart, 1993; Buchanan-Wollaston, 1997 ;

Gan & Amasino, 1997; Thomas & Howarth, 2000).

During senescence, N stored in mature leaves is

remobilized for transport to younger or more

demanding plant parts. Hence the observation of

increased protease activity, increased content of the

readily transportable amino acids asparagine and

glutamine, and appearance of ammonia in senescent

tissues (Thomas, 1978; Vaux, 1993; Buchanan-

Wollaston, 1997; Distefano et al., 1997; Jiang et al.,

1999). The cells around the vascular tissues are the

last to senesce (Gan & Amasino, 1997), and this

might be related to the requirement for prolonged

transport ability. Within cells undergoing sen-

escence, losses of chlorophyll and chloroplast in-

tegrity are the earliest symptoms and breakdown ofthe nucleus is the final event (Thomas & Stoddart,

1980; Smart, 1994 ; Buchanan-Wollaston, 1997; Gan

& Amasino, 1997; Matile, 1997). Chloroplast break-

down is under nuclear control (Smart, 1994;

Buchanan-Wollaston, 1997; Gan & Amasino, 1997;

Matile, 1997; Noode! n et al., 1997), and the chloro-

plasts can remain intact until up to 80% of the

chlorophyll has been removed (Wittenbach, 1978 ;

Zavaleta-Mancera et al., 1999b). The exceptions to

this are the stay-green mutants in which normal

senescence is impaired by a lesion in chlorophyll

catabolism, such that senescent leaves mobilizesoluble protein but the leaves remain green (Thomas,

1987; Thomas et al., 1992; Thomas & Howarth,

2000). The final stage in chloroplast degradation is

membrane degradation. This involves phase sep-

aration, which is a change in membrane lipid

composition leading to leaky membranes and loss of

ion gradients. Phase separation of the lipid bilayers

of chloroplast membranes does not occur until the

final stages of chloroplast disassembly (Thompson et

al., 1997, 1998).

Mitochondrial function is required until fairly late

in senescence to provide energy to drive the

senescence machinery (Thomas, 1978 ; Hillman

et al., 1994). It is also possible that membrane

degradation contributes to energy generation via the

glyoxylate cycle and gluconeogenic production of

carbohydrates from acetyl CoA liberated through

lipid breakdown (Thomas, 1986; Graham et al.,

1992; Thompson et al., 1998). Membrane break-

down can occur through blebbing of peroxidized

lipids and lipidprotein complexes into the cytosol.

However, during senescence this does not appear to

be the main route, and at this stage peroxidized

lipids remain integrated within the body of the

membrane to a much greater extent than in non-senescent tissue (Thomas, 1986; Thompson et al.,

1997, 1998). Membrane peroxidation and the

associated enzyme activities increase in senescent

tissue (Thompson et al., 1998), which could indicate

that membrane turnover in plant cells in the rumen

might be impaired because of the anaerobic con-

ditions. Membrane deterioration during senescence

results in poor integration of membrane proteins

(Thompson et al., 1997, 1998). Proteins could also

become available for proteolysis through dissociation

from chlorophyll (Thomas, 1997). Membrane phase

separation could account for observed increases in

calcium release from organelles into cytoplasm

associated with senescence (Huang et al., 1997;


12/19


Thompson et al., 1998), and release of lipid degra-

dation products has been suggested as part of the cell

death signal transduction pathway (Thomas, 1986;

Thompson et al., 1997).

The ability to induce senescence by placing

excised leaves in the dark has been an experimental

tool for some time (Thimann et al ., 1974;

Wittenbach, 1978; Krishnan et al., 1999). There isjustifiable concern about the relevance of obser-

vations in detached plant parts to events occurring

during natural senescence of attached leaves, not

least because of the possibility of erroneously

attributing wound-inducible gene expression as

senescence-specific gene expression (Becker & Apel,

1993; Kleber-Janke & Krupinska, 1997). However

this procedure is especially relevant to investigations

of post-ingestion metabolism, for which one of the

working hypotheses is that plant cells entering the

rumen undergo similar processes to those occurring

during artificially induced senescence. In both cases

leaves are detached and held in the dark, but as other

conditions differ the responses are not likely to be

identical. In detached oats, the accumulation of free

amino acids was observed after 6 h, and 65% of the

leaf protein had been degraded after 72 h (Thimann

et al., 1974). Especially interesting in the current

context is the ability to break down Rubisco in the

dark (Wittenbach, 1978; Thomas et al., 1992); this is

the most abundant of the soluble proteins in C$

plants. Hence any proteolysis in the rumen en-

vironment which is directed against the Rubisco

contained within ingested cells is likely to be of

consequence simply because of the amount ofavailable substrate. The function of dark-induced

catabolism of Rubisco in vivo might be to prevent

excessive catalytic misfiring, which could lead to the

formation of inhibitory sugar phosphate molecules

(Portis, 1992). This process is likely to be distinct

from the light- and metalloproteinase-dependent

Rubisco catabolism observed in intact chloroplasts

(Roulin & Feller, 1998).

There are both similarities and differences be-

tween artificial and natural senescence. In both cases

chlorophyll is broken down (Matile et al., 1999), and

there is the appearance of new mRNA, induction ofproteases and de novo protein synthesis (Thomas

et al., 1992). However, the genetic material and

proteins involved can be specific to induced or

natural senescence (Smart, 1994 ; Morris et al., 1996;

Buchanan-Wollaston, 1997; Gan & Amasino, 1997).

It is clear that senescence is a genetically determined

process that can be modulated by exogenous ap-

plication of plant growth factors such as ethylene and

cytokinin, or by altered cytokinin synthesis within

the plant through genetic manipulation (Smart et al.,

1991 ; Smart, 1994 ; Buchanan-Wollaston, 1997 ; Gan

& Amasino, 1997; Hung & Kao, 1997; Park et al.,

1998 ; Ori et al., 1999; Thomas & Donnison, 2000).

The cytokinin response itself can be modulated

through sugar signalling (Wingler et al., 1998) such

that increased starch breakdown can overcome

cytokinin-mediated repression of senescence. Eth-

ylene could be a common signal both in wounding

response and in inducing senescence (Thomas &

Stoddart, 1980 ; Hillman et al., 1994 ; Buchanan-

Wollaston, 1997; Fluhr, 1998). However, its role in

the rumen is dubious because the terminal stagein ethylene production, 1-aminocyclopropane-1-

carboxylic acid (ACC) oxidase, is unlikely to function

under the anaerobic conditions of the rumen.

However, ACC itself could act as a diffusable signal,

as it appears to during submergence (Bradford &

Yang, 1981). It seems unlikely that increased

cytoplasmic calcium is the primary signal promoting

senescence. Imaging studies have shown that protein

degradation has begun before any increase in

cytoplasmic calcium signal, which does coincide

with or slightly precede chlorophyll breakdown and

lipid peroxidation (Huang et al., 1997).

Induced senescence often proceeds faster than

natural senescence, although there is a large variation

in the progression of senescence between different

plant species and even cultivars (Thomas &

Stoddart, 1980; Thomas, 1987; Hensel et al., 1993;

Morris et al., 1996; Thomas & Howarth, 2000).

Induction of natural senescence by nutrient star-

vation or water deficit has been reported, and

coincides with molecular evidence for sugar-

mediated regulation of senescence processes. It has

been proposed that senescence is induced when the

photosynthetic rate drops below a threshold (Dai et

al., 1999), although it is possible for photosyntheticrate to fall without loss of chlorophyll (Smart, 1994 ;

Thomas & Howarth, 2000). When the hexose

products of photosynthesis were consumed rapidly

as a result of increased hexokinase activity, sen-

escence was accelerated in both old and newly

mature leaves (Dai et al ., 1999). Some, albeit

correlative, evidence from tobacco leaves also points

to the possible involvement of a threshold cellular

sugar content which regulates the activity of certain

senescence-related promoter regions (Chung et al.,

1997). This would explain the onset of senescence in

responise to nutrient and water stress when photo-synthate is limiting (Gan & Amasino, 1997), and is

one explanation for differential gene expression

during natural and dark-induced senescence. Such a

signalling response would therefore be important in

plant responses in the rumen. Hence plant cells

entering the rumen could be forced to undergo an

early senescence as a result of anaerobic consumption

of carbohydrate during metabolic attempts to main-

tain ATP generation.

Although it is recognized that nuclease activity

increases during senescence, resulting in DNA

cleavage and degradation (Gan & Amasino, 1997 ;

Yen & Yang, 1998), various senescence-associated

genes have been identified and their function has


13/19


recently been reviewed (Buchanan-Wollaston, 1997,

Gan & Amasino, 1997). Notably, the expression of

the pea homologue of dadis downregulated with the

onset of natural or induced senescence (Orzaez &

Granell, 1997). Increasing use of differential

sequencing and subtractive hybridization has made

it possible to begin to identify both high- and low-

abundance genes involved with the senescenceprogramme (Smart et al ., 1995). Those genes

identified as senescence-enhanced to date include

proteases, RNAses, lipases and some enzymes of N

metabolism, as well as the housekeeping genes which

continue to be expressed during senescence as well as

in nonsenescent tissues (Thomas et al., 1992; Smart,

1994; Buchanan-Wollaston, 1997 ; Quirino et al.,

1999). However, during dark-induced senescence,

accumulation of the transcripts of the photosynthetic

genes rbcL, psaA, psaB and aptB decreases (Thomas

et al., 1992; Kleber-Janke & Krupinska, 1997;

Krause et al., 1998). However, transcript levels ofpsbA and psbD are unaffected, even though after 8 h

in the dark chloroplast transcriptional activity was

estimated (from UTP incorporation) to be only 30%

of that observed in the light (Krause et al., 1998).

Downregulation is reversible, but there is discrep-

ancy between separate experiments in estimating the

point at which complete chloroplast disassembly

comes about. Chloroplasts can become substantially

dismantled, yet still be capable of renewed synthesis

of chlorophyll and associated proteins (Zavaleta-

Mancera et al., 1999a,b). Re-illumination of excised

barley leaves stimulated a return of transcription

even after 6 d dark treatment (Kleber-Janke and

Krupinska, 1997), but other work suggested that 56 h

dark treatment was sufficient for complete chloro-

plast dismantling (Krause et al., 1998). Dismantling

of the chloroplast is a critical process in terms of

plant metabolism in the rumen as most of the soluble

protein is confined within this organelle, and

while it remains intact the question of microbial

access remains.

In determining the role that senescence processes

play in cell death and proteolysis in ingested plant

material in the rumen, the potential activation\

production of senescence-related proteases is ofprimary importance. Likewise, the observation that

cellulases can be induced as part of senescence

(Pavanas et al., 1998) might also be important in the

ability of rumen microorganisms to colonize ingested

material, as this would aid cell-wall loosening. A

recent estimate suggested that due to the physical

nature of plant cell walls, only c. 20% would be

digested during the average rumen incubation

(Wilson & Mertens, 1995). Senescence-related

proteases are believed to be primarily associated with

lytic vacuoles and the endoplasmic reticulum

(Thomas & Stoddart, 1980; Feller, 1986; Buchanan-

Wollaston, 1997; Matile, 1997; Rogers, 1998).

Proteases do also exist in the cytosol and chloroplast,

although these are primarily concerned with removal

of structurally deficient proteins, processing transit

peptides, and degrading damaged proteins of the

photosystems (Adam, 1996; Andersson & Aro, 1997;

Clarke, 1999). Specific proteases are upregulated

during senescence (Feller, 1986). There are ever-

increasing examples of the involvement of cysteine

proteases during senescence and other cell deathpathways (Linthorst et al., 1993; Smart, 1994;

Valpuesta et al ., 1995; Griffiths et al ., 1997;

Guerrero et al ., 1998; Popovic et al ., 1998;

Sokolenko et al., 1998; Thomas & Donnison, 2000).

It is interesting to note that cysteine proteases from

plants are often inactivated on extraction, due to the

presence of air (Yonezawa et al., 1997); this is not

a problem if cysteine proteases are liberated into

the anaerobic rumen fluid. The influence of the

ubiquitin system of protein degradation (Hershko

& Ciechanover, 1998) has been demonstrated for

cytosolic proteins through the upregulation ofubiquitin-carrier proteins during senescence (Callis

& Vierstra, 1989). There are currently two views as to

the mechanism of degradation of chloroplast proteins

during senescence: this could occur in the chloro-

plast or after transport of proteins to the vacuole;

or in situ mediated by chloroplast proteases. Pro-

teasesin the vacuole are capable of degrading Rubisco

in vitro (Miyadai et al., 1990; Yoshida & Minimaka,

1996; Bose et al., 1999). However, it has been

suggested that observations of chloroplast-associated

protease activity (Liu & Jagendorf, 1986, Mae et al.,

1989; Bushnell et al., 1993) represent carry-over

contamination (Bose et al., 1999). The persistence ofthe hypothesis that proteins must be translocated to

lytic vacuoles for breakdown is largely due to the lack

of chloroplast-targeting leader sequences on sene-

scence-induced proteases, the apparent absence of

ubiquitin, and the equivocal nature of the evidence

from the ClpP protease system in the chloroplast.

The Clp family of proteases was first identified in

bacteria and requires both a catalytic (ClpP) and

ATP hydrolytic (called ClpA in bacteria and ClpC in

plants) subunit (reviewed by Clarke, 1999). Homo-

logues have been identified in plants in both cytosolic

and chloroplastic compartments in mature leaves(Desimone et al., 1997; Nakabaya et al., 1999;

Weaver et al., 1999). The thylakoid-associated ATP-

dependent protease discovered by Liu & Jagendorf

(1984) might also be an example of a ClpP homo-

logue. The presence of functional ClpP and ClpC in

the chloroplast supports the idea that degradation of

chloroplast proteins can occur in situ (Desimone et

al., 1997). Recent investigations into the expression

of the transcripts of plastidic ClpP (pClpP) and

ClpC identified in Arabidopsis thaliana show ex-

tensive downregulation in darkness during natural

and induced senescence (Humbeck & Krupinska,

1996; Nakabaya et al., 1999). Initially this suggested

that this protease complex had little role during


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senescence, but further investigation revealed that,

although transcript level decreased, the pre-existing

proteins of the plastidic subunits (ClpC and pClpP)

were stable during induced senescence, although less

so during natural senescence (Nakabaya et al., 1999 ;

Weaver et al., 1999). This poses the interesting

possibility that Clp protease activity might not be of

primary importance during natural senescence, butcould function during the initial stages of incubation

of fresh forage in the rumen (at least until ATP

became limiting through anaerobic stress), and could

contribute to breakdown of chloroplastic proteins

such as Rubisco. Stromal proteases have also been

implicated in degradation of chlorophyll-associated

proteins such as LHCPII (Thomas & Stoddart,

1980 ; Noode! n et al., 1997; Thomas, 1997) in a

process that might also interact with membrane

degradation to form blebs containing lipid, chloro-

phyll and proteinaceous degradation products.

These might or might not be transported to the

vacuole or endoplasmic reticulum for complete

catabolism (Noode! n et al., 1997; Thompson et al.,

1997, 1998).

There is a high degree of similarity between

proteases of senescence and those associated with

mobilization of seed-storage proteins, such as

oryzain and aleurain, which are active during

germination (Buchanan-Wollaston, 1997, Griffiths

et al., 1997). Also, specific transcripts for proteases

which increase in response to pathogen infection can

be upregulated during senescence in the absence of

the pathogen, suggesting considerable cross-talk

between the cell death pathways (Quirino et al.,1999). Post-translational activation of proteases

might be under the control of growth regulators such

as jasmonate, itself produced through lipid degra-

dation (Thomas, 1986; Solomon et al ., 1999).

Peptide-processing enzymes also become active

during senescence, although these might actually be

synthesized in the mature green leaves, stored as

precursor peptides, and cleaved to the mature length

polypeptide only during senescence (Smart et al.,

1995; Solomon et al., 1999). Of particular interest in

relation to rumen conditions is the recent identifi-

cation of a serine protease in parsley, which increasesin activity during 5 d of induced senescence. This

enzyme shows a temperature optimum of 37mC and

a neutral pH optimum (Jiang et al., 1999) consistent

with rumen conditions. Likewise, a cysteine protease

with a neutral pH optimum also exists, and has been

identified in kidney bean leaves (Sokolenko et al.,

1998).

Although senescence takes place over many days,

analogous reactions could occur in plant tissue in the

rumen over a shorter time scale due to the influence

of heat and oxygen stress. Senescence is an active

process in which specific senescence-associated or

senescence-enhanced genes accumulate, unless

blocked in vitro by treatment of leaves with cyclo-

heximide or 2-methyl-2,6-dinitroanilino-N-methyl-

propionamide (MDMP) (Thomas & Stoddart,

1980; Smart, 1994 ; Buchanan-Wollaston, 1997; Gan

& Amasino, 1997; Pontier et al., 1999). So it is worth

noting that if plant tissue in the rumen does undergo

induced senescence, it might be attenuated by the

lack of an adequate energy supply to drive tran-

scription and protein synthesis. Therefore, despiteinduction of various stress responses on entering the

rumen, it is possible that the ultimate cause of cell

death will be due to the anaerobic stress of the rumen

and consequent metabolic shutdown.

I V.

The discussion in section III describes some of the

processes that might occur in newly ingested plant

material entering the rumen. It is clear that to

construct a more realistic hypothesis of protein

digestion in ruminants, the amount and activity of

microbial protease should be considered alongside

enzymes arriving in the plant material itself. We

consider that plant biomass entering the rumen

through grazing is subject initially to autoproteolysis

(Fig. 3). Over a period of several hours, plant-

mediated proteolysis cleaves tertiary structures,

releasing small peptides and amino acids to the

rumen fluid through diffusion and as a result of

cellulolytic (microbial) degradation of the cell walls.

The combination of available amino acid and

carbohydrate promotes growth of the rumen mi-

crobial population, and in subsequent hours the

microbial proteolytic enzymes can access plantprotein substrates.

Recurrent themes of plant metabolism include a

role for calcium signalling and induction of proteases

in response to a wide variety of cell death signals that

could occur in plant cells within the rumen. It

remains possible that there is a major route for cell

death on which all forms of cell death (hypersensitive

response, senescence, anaerobic stress) converge,

albeit entering at various points. Given the mul-

tiplicity of signals received by plant cells entering the

rumen, it is highly unlikely that there is no metabolic

response within ingested plant cells; the questionsare (1) what is the response and (2) what is the

timing? If the first phase of rapid proteolysis occurs

2 h after a ruminant has fed, and it takes 3 h for the

primary colonizers to begin breaking down the cell

walls, there is an obvious window in which plant-

mediated post-ingestion metabolism could play a

significant role. It is less clear what the exact

response(s) to multiple stresses are, which takes

precedence, and which is the lethal factor, if indeed

this takes place before significant microbial degra-

dation of both cell walls and cell interior. This forms

a basis from which we can try to elucidate the

relationship between plant and microbial processes

in the rumen of animals grazing at pasture. It is


15/19


Fig. 3. Revised model of forage protein degradation in the rumen: the involvement of plant and microbialprocesses.

hoped that identification of key factors\processes

will enable plant breeding programmes of the future

to incorporate quality considerations into the evol-

ution of new forage lines. Regulation of plantprotease activity in the rumen and protein com-

plexing with components such as the tannins will be

the primary targets. There is also the interesting

observation that rumen-stable proteins (which pass

undegraded to the abomasum) tend to be enriched in

proline, glycine and aspartate (Wallace et al., 1999).

Through a deeper understanding of the interactions

between the rumen microorganisms and plant cells

that contribute to plant cell degradation in the

rumen, it might be possible to breed forage crops for

decreased rates of proteolysis, targeted for ruminant

agriculture. The converse might also be achieved:forage with rapid proteolysis characteristics of

potential value for monogastric nutrition. Address-

ing the problem from both microbial and plant

perspectives might yet prove the most successful

way to improve protein nutrition in ruminants and

reduce the accumulation of environmentally dam-

aging by-products.

The authors wish to thank Prof. H. Thomas and Dr R.

Merry for useful discussions, and the Biotechnology and

Biological Sciences Research Council (BBSRC), theMinistry of Agriculture, Fisheries and Food (MAFF) and

Milk Development Council (MDC) for funding.

Adam Z. 1996. Protein stability and degradation in chloroplasts.Plant Molecular Biology 32 : 773783.

Albrecht G, Biemelt S, Baumgartner S. 1997. Accumulation offructans following oxygen deficiency stress in related plantspecies with different flooding tolerances. New Phytologist 136:137144.

Allan AC, Fluhr R. 1997. Two distinct sources of elicitedreactive oxygen species in tobacco epidermal cells. Plant Cell9 :15591572.

Andersson B, Aro EM. 1997. Proteolytic activities and proteasesof plant chloroplasts. Physiologia Plantarum 100: 780793.

Andreev VY, Generozova IP, Vartapetian BB. 1991. Energystatus and mitochondrial ultrastructure of excised pea root at

anoxia and postanoxia. Plant Physiology and Biochemistry 29 :171176.

Asoa N, Ushida K, Kojima Y. 1993. Proteolytic activity ofrumen fungi belonging to the genera Neocallimastix and

Piromyces. Letters in Applied Microbiology 16 : 247250.

Attwood GT, Reily K. 1995. Identification of proteolytic rumenbacterial isolated from New Zealand cattle. Journal of AppliedBacteriology 79 : 2229.

Bailey-Serres J, Freeling M. 1990. Hypoxic stress-inducedchanges in ribosomes of maize seedling roots. Plant Physiology94: 12371243.

Baumont R. 1996. Palatability and feeding behaviour inruminants. A review. Annales De Zootechnie 45 : 385400.

Becker W, Apel K. 1993. Differences

post-ingestion metabolism of fresh forage

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