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REVIEW New Phytol.

(2000), 148, 3755


Tansley Review No. 118
Post-ingestion metabolism of fresh forage
A. H. KINGSTON-SMITH* \i M. K. THEODOROU
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
o1i1s
Summary 37
I. 1noii1o 37
II. iois1o oi ii\1s 1ii nii: oii
\i iv oii1s 39
III. nii-iiii ii\1 i1\vois: iii
iion\i\1o \i ii\1i 41
1. Summary of plant cell death processes 41
2. Anaerobic stress and ooding tolerance of
plants 42
si\nv
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 identication 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 correct
in 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
inecient 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 nd ways of decreasing this initial rate of proteolysis in the rumen. This will benet the farmer
nancially (through decreased use of feed supplements), but will also benet the environment, as N pollution can
adversely aect 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. 1noii1 o
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-mail
alison.kingston-smith!bbrsc.ac.uk).
3. Plant cell responses to elevated
temperatures 45
4. Wounding responses\pathogen attachment 45
5. Senescence in the rumen? 47
IV. ii1ini inosii1s 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 nancial
incentives, such as premium prices for organically
38 REVIEW A. H. Kingston-Smith and M. K. Theodorou
Time
P
r
o
t
e
i
n
E
n
e
r
g
y
Fig. 1. Asynchrony in forage degradation by ruminants. A
schematic representation of the mismatch in the timing of
peaks of protein degradation (indicated by release of
ammonia) and energy (indicated by breakdown of cell-wall
oligosaccharides).
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 ineciency 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 there
is 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 rst 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, and
is therefore eliminated in faeces, urine and exhaled
breath (Wetherall et al., 1995). The basic ineciency
of N use is often overcome on farms by protein
supplementation. In the past, bone or sh 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 eciency
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 eect on the diversity of
plant 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-o
(Cuttle & Scholeeld, 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 rst 2 h of
incubation in the rumen would noticeably improve
N-use eciency. 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
diering amounts and types of hydrolysable and
condensed tannin. Tannin in feedstu is believed to
complex 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-
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 will
undoubtedly be important in manipulating protein
stability in the rumen.
Enhancing rumen eciency will result in eec-
tively converting more plant protein to animal
protein per unit of livestock, and this will bring
considerable benets 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 signicantly improve
the eciency of meat and milk production from
grazed and conserved forage. Thus the correct
identication 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 modications 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.
II. i ois1 o oi ii\1s 1ii nii:
oii \i iv oii1s
Grazing cattle and sheep excise the leaves, and
portions of stem, from forage crops. These are
chewed briey, rolled into a bolus and swallowed
into the rumen. Plant material entering the rumen is
not a homogeneous mass, although it undergoes
signicant processing after excision from the main
plant. In sheep feeding on grass, half the ingested
matter has a particle size of 4 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
microora, 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 feeds
the 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 identied 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
signicant proportion of cells remain intact. Thus
for microbially mediated proteolysis to occur, plant
cell walls must rst 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 dicult 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
40 REVIEW A. H. Kingston-Smith and M. K. Theodorou
Fig. 2. Potential eectors 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 modications (Wallace et al.,
1999; Theodorou et al., 2000). However, protection
aorded by the eective 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
lignication 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 ooding
would 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
eectiveness 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 puried 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 puried, soluble ( articial ) sub-
strate such as casein might overestimate the true
value when supplied with mixed plant protein in
which 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 uid 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 inuence 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
REVIEW Post-ingestion metabolism of forage 41
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 might
enhance their activities. Plants have evolved a range
of responses to a uctuating 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, inuences protein turn-
over rate through de novo synthesis of proteases or
post-translational modication 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 anaerobically
at 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 dierent functions and in dierent
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 nally the
induction of proteases to remobilize valuable N
during senescence. These processes are controlled
both during transcription, and later through post-
translational modication. Hence it is important to
understand the metabolic processes occurring within
the newly ingested plant cells in order to identify
factors responsible for plant-mediated proteolysis in
the rumen.
III. nii- iiii ii\1 i1\voi s:
iii iion\i\1 o \i ii\1i
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 gene
in 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
signicance of dad, 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). In
animals, 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 eectiveness 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 specic
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 rst 4 h of
programmed cell death, and suggested a regulatory
function 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-x
L,
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-
42 REVIEW A. H. Kingston-Smith and M. K. Theodorou
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 plants
include 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 visible
signs of senescence in detached and darkened maize
leaves (Hung & Kao, 1997). The signicance 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 status
in 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 ecacy 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 ooding tolerance of plants
Probably the most signicant dierence 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 (Ricard
et 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).
REVIEW Post-ingestion metabolism of forage 43
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 can
survive for a certain period before metabolism is
seriously hampered. For example, potato cells in
culture showed no membrane damage for the rst 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 which
membrane 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) specic to an-
aerobic stress (Perata & Alpi, 1993; Drew, 1997;
Vartapetian & Jackson, 1997; Kato-Noguchi, 1999).
Synthesis of these specic 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
dened. Dierent isoforms can be expressed selec-
tively in roots or leaves in response to anaerobic
stress, 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 ux
through the protein (Ricard et al., 1994), and does
not prevent signicant 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 to 6n8 over approx. 20 min
(Drew, 1997). This eectively decreases the cytosolic
pH below the pH optimum of LDH, but within the
range of pyruvate decarboxylase (PDC), the rst
enzyme of ethanolic fermentation (Fox et al., 1995).
There might also be some form of negative feedback
in which acidosis is prevented by gradual loss of
anity 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 ux of carbon through
LDH as accounting for only about 12% of the total
ux in carrot roots under anoxia (Kato-Noguchi,
1998). PDC is regarded as the rate-limiting step of
ethanolic fermentation, although the pH optimum
for ADHis approx. 8n5 (Sachs et al., 1980). Attempts
to 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
ooding tolerance of the species under investigation
(Vartapetian & Jackson, 1997). It has been suggested
that ux 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 anities of pyruvate dehydrogenase
(PDH) and PDC for substrate (K
m
l5779 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 K
m
, 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
eect 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 ux 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
44 REVIEW A. H. Kingston-Smith and M. K. Theodorou
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 signicant
changes in mitochondrial calcium content from 109
to 120 nMCa#+
after only 10 min of anoxia (Subbaiah
et al., 1998). This work with confocal imaging of
calcium-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 eux 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). Dierential accumulation of
ethanol has been proposed as a potential cause of
observed species-specic dierences in ooding
tolerance (Crawford, 1982). Although plants respond
to physiologically relevant ethanol concentrations
(Perata & Alpi, 1993), these eects 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 eld conditions due
to ooding 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
ooding, 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 dier in their tolerance to ooding (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
ooding-tolerant species, whereas maize is intolerant
of short periods of waterlogging. Dierent rice
cultivars can vary in their ooding tolerance, and this
has been linked to the onset of senescence processes
during prolonged ooding (Krishnan et al., 1999).
This has some support from observations that foliar
protein remobilization is decreased in ooding-
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 inuences 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 appear
that, 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 supply
might 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
ooding-tolerant rice showed uptake of nitrate or
ammonium ions from the bathing solution, and
REVIEW Post-ingestion metabolism of forage 45
incorporation of "&N into amino acids (specically
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 ux of electrons and
decreased accumulation of NADH, enabling con-
tinued fermentation. This is intriguing in the context
of the rumen, in which ammonium concentrations
prior 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
eld 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 eect of a temperature shift to 39mC on the
biology of plants. Hence the results from exposure to
higher temperatures must be used in the rst
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 sucient to result in cytoplasmic condensation
and DNA fragmentation (Chen et al., 1999). A
temperature of 39mC might be sucient to induce
similar responses, albeit observed over a longer time
or to a lesser extent.
Increased temperature will also aect rates of
protein turnover. For example, Rubisco is sus-
ceptible to both heat-induced proteolysis and
decreased 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 inl-
tration of rumen uid (buered to approx. pH 6n8).
This could aect 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 could
potentially 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 rst 2 h at 40mC (Kimpel et al., 1990). These
proteins are thought to participate in cellular
attempts 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 uidity and membrane permeability simi-
lar to that observed during senescence (section
III.5). In the presence of oxygen, such as that
entering 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 signicance 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
46 REVIEW A. H. Kingston-Smith and M. K. Theodorou
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 dier 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 exemplied 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 eld,
plants are exposed to a variety of potentially
pathogenic microorganisms and have developed
species-specic 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 signicance
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 sheep
and 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 pathways
involve an active oxygen burst fromNADPHoxidase
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,
specicity 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 of
cysteine protease inhibitors, cystatins, are particu-
larly eective 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 aect the ability of
rumen fungi to colonize the ingested plant material.
Presumably, putative PR protein expression during
senescence also fulls 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 prole at acidic pH, assumed to
be optimal for vacuolar proteases, and the more
neutral pH of the rumen uid (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 of
nonmicrobial, 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 dierentially
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-specic
REVIEW Post-ingestion metabolism of forage 47
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 dierent 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 rst 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 diusable signal to neighbouring
cells. Interestingly, hydrogen peroxide also stimu-
lates a rapid inux 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 of
the nucleus is the nal 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 mobilize
soluble protein but the leaves remain green (Thomas,
1987; Thomas et al., 1992; Thomas & Howarth,
2000). The nal 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
nal 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;
48 REVIEW A. H. Kingston-Smith and M. K. Theodorou
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 is
justiable 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-specic 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 articially induced senescence. In both cases
leaves are detached and held in the dark, but as other
conditions dier 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 of
available substrate. The function of dark-induced
catabolism of Rubisco in vivo might be to prevent
excessive catalytic misring, 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 dierences be-
tween articial and natural senescence. In both cases
chlorophyll is broken down (Matile et al., 1999), and
there is the appearance of new mRNA, induction of
proteases and de novo protein synthesis (Thomas
et al., 1992). However, the genetic material and
proteins involved can be specic 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 stage
in 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 diusable 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 dierent
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 decit 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 photosynthetic
rate 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 dierential 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 identied and their function has
REVIEW Post-ingestion metabolism of forage 49
recently been reviewed (Buchanan-Wollaston, 1997,
Gan & Amasino, 1997). Notably, the expression of
the pea homologue of dad is downregulated with the
onset of natural or induced senescence (Orzaez &
Granell, 1997). Increasing use of dierential
sequencing and subtractive hybridization has made
it possible to begin to identify both high- and low-
abundance genes involved with the senescence
programme (Smart et al., 1995). Those genes
identied 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 of
psbA and psbD are unaected, 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 sucient 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 conned 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 of
primary 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 decient proteins, processing transit
peptides, and degrading damaged proteins of the
photosystems (Adam, 1996; Andersson &Aro, 1997;
Clarke, 1999). Specic proteases are upregulated
during senescence (Feller, 1986). There are ever-
increasing examples of the involvement of cysteine
proteases during senescence and other cell death
pathways (Linthorst et al., 1993; Smart, 1994;
Valpuesta et al., 1995; Griths 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 uid. The inuence of the
ubiquitin system of protein degradation (Hershko
& Ciechanover, 1998) has been demonstrated for
cytosolic proteins through the upregulation of
ubiquitin-carrier proteins during senescence (Callis
&Vierstra, 1989). There are currently two views as to
the mechanismof 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-
teases inthe 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 of
the 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 rst identied 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 identied 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 identied 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
50 REVIEW A. H. Kingston-Smith and M. K. Theodorou
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, but
could 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, Griths
et al., 1997). Also, specic 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 identi-
cation of a serine protease in parsley, which increases
in 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
identied 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 inuence
of heat and oxygen stress. Senescence is an active
process in which specic 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, despite
induction 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.
IV. ii1ini inosii1s
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 uid through diusion 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 plant
protein 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 questions
are (1) what is the response and (2) what is the
timing? If the rst 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
signicant 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 signicant 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
REVIEW Post-ingestion metabolism of forage 51
Fig. 3. Revised model of forage protein degradation in the rumen: the involvement of plant and microbial
processes.
hoped that identication 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 plant
protease 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.
\ioviiioii1s
The authors wish to thank Prof. H. Thomas and Dr R.
Merry for useful discussions, and the Biotechnology and
Biological Sciences Research Council (BBSRC), the
Ministry of Agriculture, Fisheries and Food (MAFF) and
Milk Development Council (MDC) for funding.
niiiniis
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