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Physiologia Plantarum 2012
Copyright © Physiologia Plantarum 2012, ISSN 0031-9317
Root proteases: reinforced links between nitrogen uptake
and mobilization and drought tolerance
Ajay Kohlia,∗ , Joan Onate Narcisoa , Berta Mirob and Manish Raoranea
Plant Breeding, Genetics, and Biotechnology Division, International Rice Research Institute, DAPO, Metro Manila, Philippines
Crop and Environmental Sciences Division, International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines
*Corresponding author,
e-mail: [email protected]
Received 22 June 2011;
revised 15 November 2011
Integral subcellular and cellular functions ranging from gene expression,
protein targeting and nutrient supply to cell differentiation and cell death
require proteases. Plants have unique organelles such as chloroplasts
composed of unique proteins that carry out the unique process of
photosynthesis. Hence, along with proteases common across kingdoms,
plants contain unique proteases. Improved knowledge on proteases can
lead to a better understanding of plant development, differentiation and
death. Because of their importance in multiple processes, plant proteases
are actively studied. However, root proteases specifically are not as well
studied. The associated rhizosphere, organic matter and/or inorganic matter
make roots a difficult system. Yet recent research conclusively demonstrated
the occurrence of endocytosis of proteins, peptides and even microbes by
root cells, which, hitherto known for specialized pathogenesis or symbiosis,
was unsuspected for nutrient uptake. These results reinforced the importance
of root proteases in endocytosis or root exudate-mediated nutrient uptake.
Rhizoplane, rhizosphere or in planta protease action on proteins, peptides
and microbes generates sources of nitrogen, especially during abiotic stresses
such as drought. This article highlights the recent research on root proteases
for nitrogen uptake and the connection of the two to drought-tolerance
mechanisms. Drought-induced proteases in rice roots, as known from rice
expression databases, are discussed for future research on certain M50,
Deg, FtsH, AMSH and deubiquitination proteases. The recent emphasis on
linking drought and plant hydraulics to nutrient metabolism is illustrated
and connected to the value of a systematic study of root proteases in crop
Abbreviations – AA, amino acid; ABA, abscissic acid; AMSH, associated molecule with the SH3 domain of STAM;
ATP, adenosine triphosphate; BNF, biological nitrogen fixation; C, carbon; ClpP1, caseinolytic protease P1; CP, cysteine
protease; Deg, degradation of periplasmic protein; ER, endoplasmic reticulum; FC, field capacity; FtsH, filamentation
temperature-sensitive H; GFP, green fluorescent protein; IPT, isopentenyltransferase; LHT, lysine histidine transporter; MT,
membrane-tethered; N, nitrogen; OAA, oxaloacetate; OTU, ovarian tumor; PAGE, polyacrylamide gel electrophoresis; PDZ,
post synaptic density protein (PSD95), drosophila disc large tumor suppressor (DlgA) and zonula occludens-1 protein (zo-1);
PSII, photosystem II; PTR, peptide transporter; RIP, regulated intramembrane proteolysis; SREBP, sterol-regulated element
binding protein; SUMO, small ubiquitin-related modifier; TF, transcription factor; Ulp, ubiquitin-like protease; UPR, unfolded
protein response.
Physiol. Plant. 2012
Proteases cleave peptide bonds, leading to protein breakdown. Thus, the general perception on biological functions of proteases was largely restricted to degradation
of toxic, undesirable and damaged proteins, releasing
amino acids (AAs) that could be reused in protein synthesis. However, proteases are now known to regulate
various biological processes spanning the life cycle of
plants, starting with cell division to specialized tissue,
organ or organelle formation and development such as
gametes, embryo, seed coat, stomata and chloroplast
(Van der Hoorn 2008). Their activity in response to
external stimuli such as pest and pathogen attack (Xia
2004, Baek and Choi 2008) or in response to internal
developmental stimuli such as senescence has also been
characterized (Woltering 2004).
Sequenced genomes of Arabidopsis and rice reveal
close to 700 and 800 proteases, respectively, which are
classified into clans and families, while members within
the families also exhibit functional variation (Van der
Hoorn 2008). Plants with larger genomes are likely to
have a higher number of proteases. However, the biological function of only around 40 proteases is known.
Considering the involvement of proteases in various
aspects of plant growth, development, survival and
senescence, a better understanding of their phenotypic
effects and molecular mechanisms is imperative. This
is especially true for plant-specific members of some
families of proteases such as the plastidial proteases. For
example, chloroplast biogenesis and function involve
the chloroplast genome-encoded caseinolytic protease
P1 (ClpP1) whose mutation arrests etiolated shoot development in tobacco plants (Kuroda and Maliga 2003).
Nuclear-encoded Clp proteases could not complement
ClpP1 mutation, thus suggesting that ClpP1 performs
more than the housekeeping protein breakdown function. Kuroda and Maliga (2003) speculated that ClpP1
may act on a specific regulatory protein. Similarly, the
plastidial filamentation temperature-sensitive H (FtsH)
protease complex is involved in the critical function of
turnover of the photosystem II (PSII) reaction center D1
protein. It is also involved in other processes required for
the development and maintenance of the photosynthetic
apparatus (Liu et al. 2010). Studies on plant proteases
have advanced substantially over the past few years and
an ever-increasing understanding of the role of proteases
in the life cycle of plants is emerging. The regulatory
role of plant proteases in post-translational modification of proteins by limited proteolysis at specific sites is
being recognized as a key route to involvement in different functions. Limited and site-specific proteolysis gives
rise to functional enzymes and regulatory proteins and
peptides, facilitates subcellular targeting, and is required
for protein assembly (Schaller 2004). In a recent review,
Van der Hoorn (2008) collated information on 40 plant
proteases with known phenotypes and described some
of the well-characterized proteases representing different
functional aspects in different cellular processes. Only
two of the plant proteases described were concerned
with roots. One affected root meristem maintenance in
Arabidopsis (Casamitjana-Martinez et al. 2003) and the
other had a role in Medicago truncatula root infection
by Sinorhizobium meliloti (Combier et al. 2007). In the
last 3 years, only one more protease in roots has since
been assigned a phenotype, a cysteine protease (CP)
that is a negative regulator of nodules and bacteroid
senescence in Astragalus sinicus (Li et al. 2008). A relative lack of detailed characterization of root proteases
is perhaps indicative of the difficulties associated with
working with roots under natural environments.
Molecular and genetic studies on roots have largely
concentrated on root development, morphology and
architecture (reviewed by Rost 2011). The other popularly explored aspect of roots is the root–microbe symbiosis for nitrogen fixation. Murray (2011) and Yokota
and Hayashi (2011) reviewed the genes involved in
root–microbe symbiosis in legumes. Traditionally, roots
have been considered little more than a conduit for water
and dissolved minerals. Genetic controls and molecular
biology of mineral acquisition by roots, including both
macronutrients such as nitrogen, phosphorus, potassium, sulfur and calcium and micronutrients such as
iron, molybdenum, boron, copper, manganese, zinc,
chloride, silicon, sodium and nickel, etc., are now
being elucidated. Kraiser et al. (2011) presented an integrated view on different mechanisms that help plants
to acquire and maintain an adequate nitrogen supply. These included root-specific gene expression and
function modulation, root growth and development
modulation and root–microbe association modulation.
Similarly, the state of knowledge on root uptake and
use of phosphorus (Panigrahy et al. 2009), potassium
(Szczerba et al. 2009) and sulfate (Rouached et al.
2009) has also been reviewed, while Chen et al. (2008)
reviewed membrane transporters involved in the uptake
of the three macronutrients, i.e. nitrogen, phosphate and
The three main aspects of root research – root development and symbiotic and non-symbiotic nutrient acquisition – were reviewed for an integrated understanding
of root adaptations to biotic and abiotic constraints on
root function and plant health (Hodge et al. 2009). Such
integrative studies on nutrient acquisition are increasing
due to an imperative need to optimize nutrient application, uptake and use as an important aspect of precision
Physiol. Plant. 2012
farming in the future so as to increase land and plant
yield potential under normal and/or stress conditions.
Improving the yields of major cereal crops is a key to
future food security under population and climatic pressure. As cereals do not depend on nutrient availability
through symbiosis, an improved understanding of nonsymbiotic nutrient uptake and mobilization is desirable.
Interestingly, recent reports on two modes of nutrient
uptake have added to our knowledge on the mechanisms that roots employ for the purpose. One is root
exudate-mediated nutrient uptake, which was known for
some time, but more details of exudate components and
their roles are emerging now. The second is endocytosis
of proteins and intact bacteria by root cells. Endocytosis by roots was known in connection with symbiotic or
pathogenic relationships, but clear demonstrations of the
process being used for nutrient acquisition have been
provided recently. Both mechanisms invoke a crucial
role for root proteases, exuded or internal, in order to
break down, use and remobilize the scavenged AAs,
peptides and proteins and thus maintain the nitrogen
status of plants.
The nitrogen status of plants under stress is a critical
factor in stress tolerance. Under water-deficit conditions,
for example, nitrogen content can decline drastically
(Gonzalez-Dugo et al. 2010). A corollary to this is that
increased root proteases would have a critical role in
stress tolerance because they can mobilize nitrogen
from external or internal sources. This is somewhat a
different view on the role of plant proteases under stress,
specifically wheat CPs, whereby an increase in their
stress-associated expression in leaves was proposed as a
marker of susceptibility to water-deficit (Simova-Stoilova
et al. 2010). A similar study conducted on the roots of
red clover did not show an increase in CPs till after
21 days of stress, after which the roots were terminally
consigned to senescence (Webb et al. 2010). Nevertheless, preliminary results from our laboratory combined
with extensive information gleaned from rice expression
databases clearly implicate a role for rice root proteases
in compensating for the nitrogen stress concomitant to
drought. It is argued that proteases that are seen to be
specifically upregulated in roots under drought belong
to two categories, cell death and nutrient mobilization/regulation. It is further suggested that during drought
the balance may shift more toward the latter category
in tolerant accessions of rice, conferring a better ability
to uptake and remobilize nitrogen under stress. Such a
shift from vacuolar degradative protease activity to nonvacuolar energy-dependent regulatory protease activity
was shown in wheat leaves in relation to developmental
stage and water saturation deficit (Wisniewski and Zagdanska 2001), although total protease activity was not
Physiol. Plant. 2012
substantially different. The importance of this shift under
stress was not established and such observations have
not been made in roots yet.
We suggest that further research on stress-responsive
proteases in roots and their specific roles would add
valuable information on integrating the root–leaf and/or
root–tiller response in terms of hydraulics and nutrient
re-mobilization. This would be particularly useful for
rice, whose roots seem to survive long after the aerial
parts have wilted. The return of favorable conditions
supports a fast-tracked growth of new tillers that could
bear seeds. This is reminiscent of red clover roots surviving for a long time after defoliation or grazing (Bingham
and Rees 2008, Webb et al. 2010).
Root proteases and nitrogen uptake
The root–soil interface is an active site for the exchange
of organic and inorganic C and N, the two major
elements of plant nutrition, whereas other macroand micronutrients are also sourced from the rhizosphere–soil continuum. The C and N exchange at the
roots driven by variables of soil, microbial biomass
and plants is highly complex and is spatiotemporally
regulated. Jones et al. (2009) presented four possible
utilities for such exchanges, two of which centered
on root–root and root–microbe interactions and the
other two on nutrient uptake and distribution by plants.
For root–microbe interaction between Medicago and
Sinorhizobiummeliloti, peptidases were found to be an
important component of the root exudates (De-la-Pena
and Vivanco 2010). Interestingly, however, Godlewski
and Adamczyk (2007) found that proteases were secreted
by the roots of 15 different agricultural and wild plant
species without any microbial or other artificial means
of inducing protease secretion. Their studies with wheat
(Adamczyk et al. 2008) and Allium (Adamczyk et al.
2009) suggested that such protease secretion was part of
the plants’ strategy to increase free AAs as a source of N.
Thus, AAs in soil could result from the activity of such
exuded root proteases on proteins and peptides outside
of the roots. Alternately, increased root protease activity
within the roots under stress could be the source of AAs
in the soil, again as part of the root exudate. Indeed, it
was established that increased root exudates of Festuca
rubra under stress were richer in AAs (Paterson et al.
2003), while Lesuffleur et al. (2007) reported that root
exudates of different plants under stress were rich in
different AAs. These and other studies (Schobert and
Komor 1987, Persson and Nasholm 2002) lend credibility to the proposition by Jones et al. (2009) that one
possible utility of exudates is re-absorption of some of
their components by roots to maintain C and N balance.
That roots do uptake AAs from soil and use them in
vivo was demonstrated through a combined proteomic
and isotope tracer approach, whereby labeled glycine
supplied as an N source was traced in plant metabolites in Lolium perenne (Thornton et al. 2007). In this
study, root proteome analysis with and without glycine
exhibited upregulation of a glycine metabolism enzyme,
among others, in the former case.
The proteases in the rhizosphere may equally come
from microbes. Vágnerová and Macura (1974) demonstrated that protease activity because of microbes was
higher in the wheat rhizoplane than in the rhizosphere
and surrounding soil. However, plant root exudates
exercise a selective influence so that only a small and
specific proportion of microbial diversity can inhabit the
rhizosphere and/or the rhizoplane, and the rhizosphere
inhabitants may vary depending also on environmental and plant growth variables (Hartmann et al. 2009).
Thus, proteases in root exudates or within the roots or
those from rhizoplane non-symbiotic bacteria may be
responsible for protein breakdown, facilitating N uptake
in the form of AAs. Indeed, a root–microbe symbiotic
association is another mechanism of organic N availability to plants. Although there was evidence that plants
could use AA from soil or culture media, these were
artificially supplied. Paungfoo-Lonhienne et al. (2008)
demonstrated that axenically cultivated non-symbiotic
Hakeaactites (a woody plant) as well as the herbaceous
Arabidopsis could use protein as a source of nitrogen
in the absence of any other symbiotic or non-symbiotic
organism. Their results validated a role for root proteases
in the absence of any other microbe, when the root surface fluoresced from proteolysis of an externally supplied
protein–chromophore complex capable of fluorescence
only upon proteolysis. More importantly, it was also
shown with the use of green fluorescent protein (GFP)
and through the lack of protein degradation products
in the incubation media that intact protein was taken
up by the roots as long as root hair were present. The
same group further elegantly demonstrated that roots
of tomato and Arabidopsis were capable of taking up
intact Escherichia coli and yeast cells, which were then
degraded within the root cells and were at least a source
of nitrogen, as detected by an increase in 15 N concentrations in the leaves after incubation of tomato roots with
N-labeled E. coli (Paungfoo-Lonhienne et al. 2010).
Such consumption of E. coli N clearly implicated root
proteases. There was evidence through induction of specific genes and structures that the process of taking up
intact microbes by roots involves root outgrowths that
surround E. coli cells stuck to the root surface through
mucilage secreted either from the roots or from the bacteria. Endocytosis was also implicated as a possible process
that could facilitate microbe uptake because induction
of genes for cytoskeleton structure and re-organization
was noticed. The importance of endocytosis in different
aspects of plant–microbe interactions, microbe recognition and colonization of plant cells by endosymbionts
has been recently reviewed with a special emphasis on clathrin-mediated endocytosis (Leborgne-Castel
et al. 2010).
These results clearly linking N uptake and use to
root proteases, apart from the uptake of inorganic N or
symbiotic N in plants, are from studies over the past
4 years only on a limited number of species. Although
not much information is available on any such parallels
in crop plants in field conditions, the stage is set for such
a search.
Drought and nitrogen uptake
The importance of the relationship between drought and
N uptake by plants is becoming steadily clear. Drought
invariably reduces biological nitrogen fixation (BNF) and
consequently the N content, biomass and yield of plants.
In peanut, BNF and biomass increasingly declined under
2/3 of field capacity (FC) water and 1/3 of FC for 12
different lines (Pimratch et al. 2008). However, there
was a positive correlation between BNF and biomass
under both water-deficit conditions and the relationship
was stronger under severe water-deficit. The results
suggested that maintenance of high BNF under drought
would be useful in maintaining high yield under waterdeficit conditions. Indeed, this approach was earlier
used to select Glycine max genotypes with high ability
to fix N under drought, which led to the release of
drought-tolerant varieties (Chen et al. 2007). However,
it is not only the symbiosis-dependent plants that cope
better with drought under improved N content, but also
the symbiosis-independent plants. For example, in rice,
various physiological parameters such as photosynthesis,
relative chlorophyll content, water use efficiency,
transpiration and overall growth were significantly
higher with the application of N during drought than
under drought with no N application (Suralta 2010).
Similarly, under drought stress applied to barley at three
growth stages, tillering, shooting and earing, the negative
effect on grain yield could be relatively relieved with N
fertilizer application (Krcek et al. 2008).
Reviewing water-deficit and nitrogen nutrition of
crops, Gonzalez-Dugo et al (2010) elaborated that
mineral N from the rhizosphere is largely made available
through the transpiration stream and that under waterdeficit mineral N fluxes decrease in the xylem. The
concomitant N assimilation is also more sensitive to
water scarcity. Why does increased N, organic or
Physiol. Plant. 2012
inorganic, through transpiration pull or through exudates
and/or endocytosis, help to relieve the effects of drought?
Plants under stress tend to maintain the internal nutrient
status of the C–N ratio either by modulating the
partitioning of nutrients or by nutrient re-mobilization.
Vegetative growth may be controlled to limit the tissues
that require balanced nutrient status. For example,
Norway spruce under drought but with an N source
available exhibited less new shoot development than
this species under drought, but with no N source
available (Rosengren-Brinck and Nihlgard 1995). This
suggested preferential allocation of available N to
maintain the nutrient status of a limited body of the plant.
In rice, reproductive tiller formation may be favored
over new leaf development under drought and with N
Under progressive drought, an imbalance in the internal nutrient status of aerial parts is more likely to occur
before terminal water-deficit per se. This is because the
perception of water-deficit initiates hydraulic and hormonal changes that restrict stomatal opening, in turn
restricting transpiration pull and carbon dioxide entry,
both of which initiate changes in C and N metabolism
and assimilation. In tomato, root restriction stress, which
mimics water stress, affected xylem sap ABA concentration, which in turn directly affected aerial characteristics
such as stomatal conductance, leaf water potential,
intercellular CO2 concentration, accumulation of carbohydrates, maximum carboxylation rate of Rubisco,
ribulose-1,5-bisphosphate regeneration and quantum
yield of PSII electron transport (Shi et al. 2008). Hence,
under drought, along with setting in motion the physiological and molecular machinery for efficient water
uptake and use, the plant’s effort is also toward activating mechanisms that ensure efficient nutrient uptake,
re-mobilization and use. For nutrient uptake and mobilization by roots the activity of exuded or internal root
may be critical. Proteases in the aerial parts may also
be activated for the purpose of leaf N re-mobilization.
However, root proteases are likely to play a more important role as being at the site of N uptake. Our preliminary
results discussed below demonstrate much higher protease activity in the roots of different rice genotypes than
in the shoots, both with and without drought.
Relation between root proteases, nutrient
status and drought
Between ammonium and nitrate as an N source under
drought, rice showed a preference for the former. A
direct link with increased photosynthesis in the presence
of ammonium nutrition was noticed (Guo et al. 2007).
Rice is special in that flooded field and drought situation
Physiol. Plant. 2012
change the main source of N from ammonium to
nitrate, as also the degree of non-symbiotic BNF, in
turn influencing the relative importance of ammonium
and nitrate (Buresh et al. 2008). However, uptake of
AAs by plant roots as additional or alternate source of
N through AA transporters has been known (Nasholm
et al. 2009). Interestingly, it was shown by carbon and
nitrogen isotope labelling that wheat roots took up
AAs in the form of tripeptides in preference to nitrates
and single AAs. Uptake of nitrates was inferior to that
of ammonium, L-trialanine and even the biologically
unutilized D-trialanine (Hill et al. 2011). Also, as
illustrated above, peptides, proteins and/or microbes
of the rhizosphere/rhizoplane could be used as a source
of N under drought and root proteases may have an
important role in hydrolyzing soil proteins or in digesting
those taken up by roots.
Drought-induced proteases are known from the aerial
parts of plants such as Arabidopsis (Koizumi et al. 1993),
pea (Jones and Mullet 1995), Phaseolus (Hieng et al.
2004) and wheat (Zagdanska and Wisniewski 1996,
1998, Demirevska et al. 2008, Zang et al. 2010). Such
induction correlates positively with drought-sensitive
or drought-tolerant cultivars. This confusion exists if
detailed characterization of the proteases involved is
not available because senescence-related proteases as
well as nutrient re-mobilization and regulatory proteases
both can be induced under drought. It is possible that the
former class of proteases is induced more in droughtsensitive genotypes while the latter class is induced
more in tolerant genotypes. For example, increased
protease induction in drought-sensitive genotypes was
noticed in Phaseolus (Roy-Macauley et al. 1992, Hieng
et al. 2004), while in wheat the drought-tolerant varieties exhibited increased induction (Zagdanska and
Wisnievski 1998, Demirevska et al. 2008). Importantly,
the proteases studied in wheat were ATP-dependent
regulatory proteases while in Phaseolus they were vacuolar serine and/or CPs. For wheat under water-deficit,
total protease activity increased and CPs largely contributed to the increase (Zagdanska and Wisnievski
1996). While Simova-Stoilova et al. (2006) associated
increased CP activity with sensitive wheat genotypes,
Zang et al. (2010) recently associated another wheat
CP (TaCP) with drought tolerance through transgenic
validation in Arabidopsis, whereby constitutive expression of TaCP enhanced the drought tolerance of the
transgenic Arabidopsis. Indeed, different CPs are associated with senescence and drought (Khanna-Chopra
et al. 1999) and both categories, degradative and processive CPs, can be upregulated. Depending on which
CPs were characterized, the association with tolerant or
susceptible genotypes could vary.
Despite evidence for the importance of intracellular
proteolysis during drought, most likely in re-organizing
cellular metabolism, the role of specific proteases under
stress remains uncertain. This is even more the case
for root proteases. The limited information available on
root proteases is mostly from work on isolated root tips
and largely in relation to C starvation. For example,
a root protease upregulated in maize root tips under
glucose starvation was described (Chevalier et al. 1995)
and it was shown that protein catabolism in maize root
tips followed fatty acid catabolism under C starvation
(Dieuaide-Noubhani et al. 1997). In general, endopeptidase and protease expression increased in excised maize
root tips under C-starvation conditions (Brouquisse et al.
2001, 2007). Sugar starvation in tomato roots led to a
reduction in root carbohydrate as well as in root protein
content (Devaux et al. 2003) implicating increased protease activity in the latter case. In the case of intact roots
of Phaseolus vulgaris under stress, protease activity did
not increase in the first 3 days of stress and AA levels
decreased, including that of asparagine (Bathellier et al.
2009). However, intact roots within 3 days of stress may
have still not reached a point of critical stress more easily
achieved in excised roots as exemplified by the increase
in AAs after excision of red clover roots from shoots
(Bingham and Rees 2008). Interestingly, asparagine synthetase – the enzyme for synthesis of asparagine, the
major N transport molecule in plants during stress – was
highly upregulated in Arabidopsis under sugar stress
imposed by darkness (Gaufichon et al. 2010). This suggests that during C starvation the plant prepares for N
What is the relationship between drought, C starvation
and protease upregulation? The water deficit-mediated
hydraulic signal from roots to shoots results in changes in
leaf cell turgor leading to hormonal signaling-mediated
stomatal closure (Christmann et al. 2007). Stomatal closure is useful in limiting transpiration under drought, but
it also limits carbon dioxide intake, which in turn affects
net photosynthesis (McDowell et al. 2008, Pinheiro and
Chaves 2011). A change in photosynthesis leads to
the initiation of drought-induced C starvation, which
results in a sugar-mediated signaling cascade for the
re-organization of cellular metabolism. Overall, droughtinduced C starvation is seen to be the major threat to
plant survival concomitant to changes in C–N ratio
and in plant hydraulics (McDowell, 2011). Recent findings suggest that a number of late molecular responses
are more to changes in carbohydrate availability than to
environmental cues (Smith and Stitt 2007) and that plants
may survive some period of complete loss of conductivity
(McDowell 2011). Additionally, during drought, nutrient and mineral uptake decline (Sardans and Penuelas
2005), transport to the aerial parts decreases (Hu
and Schmidhalter 2005) and energy costs of converting nitrogen into usable forms increase (Farooq et al.
2009, Pinheiro and Chaves 2011). Under such nitrogendeficient conditions, the action of proteases may supply the AAs, such as asparagine and glutamine for
N re-mobilization. Additionally, root exudate-mediated
and/or root endocytosis-mediated uptake of water, nutrients, organic molecules and microbes can provide a
lifeline to the plant, depending on the timing and severity of drought. It is to be noted that the organic molecules
sourced through the activity of proteases are not just N
sources, but also C sources as observed with asparagine
breakdown into oxaloacetate (OAA) through the activity of the amidohydrolase asparaginase. The OAA can
be used in the Krebs cycle, which produces the reducing power as hydrogen atoms for the electron transport
chain and intermediates that can be further anabolized
(Simpson 2011). Additional root growth observed under
nutrient deficiency was proposed to be important in
sourcing additional soil area for nutrient uptake (LopezBucio et al. 2003). Hence, drought-induced root growth
is opposite to shoot growth retardation under drought.
Nutrients for root growth are sourced as organic matter
from the soil, thus saving energy in converting inorganic
N into an AA (Nasholm et al. 2009). Another source of
water and nutrients is the process of autophagy. Cells
respond to stress with a burst of autophagic activity
through co-ordinated upregulation of autophagy genes
at the onset of starvation in Arabidopsis and proteases
are part of the autophagic gene complement (Rose et al.
2006). A lack of these proteases makes Arabidopsis
mutants hypersensitive to C and N starvation (Thompson
et al. 2005). Indeed, root tips of Arabidopsis undergoing
water stress exhibited autophagy (Duan et al. 2010).
During drought, nutrients and even water sourced
through different mechanisms by roots, either from outside or within, need to be transported to the aerial
parts. The uptake of AAs and their transfer into the
root xylem have been described in Ricinus communis
(Schobert and Komor 1990), and the corresponding AA
transporters in this plant have been characterized (Bick
et al. 1998). Overall importance of organic N (AAs,
peptides and proteins) uptake by roots and associated
transporters were reviewed by Nasholm et al. (2009)
and Tegeder and Rentsch (2010). Nitrogenous nutrients in xylem can flow to phloem and to the shoots
(Atkins 2000, Zhang et al. 2010) through specific transporters, including peptide transporters (PTRs) (Lubkowitz
2011). Proton-coupled, independent or pH-gradientdriven AA and sugar influx, efflux and bidirectional
transporters at the plasma membrane and tonoplast
have been described (Yang et al. 2010, Okumoto and
Physiol. Plant. 2012
Pilot 2011). The lysine histidine transporter (LHT1) is a
high affinity broad-spectrum AA transporter expressed
in the leaf and root epidermis and it directly affects
AA uptake and distribution in plants (Hirner et al.
2006). Upregulation of LHT1 was recently shown in
drought-tolerant rice plants generated by overexpression
of the cytokinin synthesis gene, isopentenyltransferase
(IPT), under the stress- and maturation-induced
promoter PSARK (Peleg et al. 2011). Root-specific expression of the Arabidopsis LHT1, amino acid permease
(AtAAP) type, proline and glycine betaine (ProT) and
PTR among others, under abiotic and biotic stresses, was
recently reviewed by Tegeder and Rentsch (2010).
These studies on the relationship between changing
nutrient status under drought suggest that maintaining the
nutrient status of plants may improve drought tolerance.
Indeed, survival of maize plants under drought by
addition of exogenous sugars revealed that the main
cause for tissue mortality was not low water potential,
but low carbon availability (Boyle et al. 1991). Our
contention is that root proteases play an important
role in nutrient uptake and mobilization through the
processes discussed above, to maintain the critical
nutrient balance as long as possible and thus contribute
to drought tolerance. We support this hypothesis through
analyzing the rice expression database for the expression
of proteases in roots under drought at the critical
developmental stages, as described below.
Expression variation in rice root proteases
under drought
For total soluble protein extracts following different
extraction protocols, total protease activity was apparently higher in rice roots than in shoots as seen by
the extent of degradation of high-molecular-weight proteins after polyacrylamide gel electrophoresis (PAGE)mediated separation (Raorane et al. 2011). Fig. 1 shows
more degradation of proteins if leaf and root tissue (L/R)
proteins from one genotype were extracted together
than when only leaf tissue (L) was used. The L/R samples
were far more degraded than L samples to begin with and
became progressively more degraded with time. That the
root extract per se was responsible for the degradation of
leaf proteins was demonstrated by leaf extract degradation on the addition of root extract (Raorane and Narciso,
unpublished data). The addition of a protease inhibitor
cocktail in the extraction buffer did not substantially
alter the scenario (Raorane et al. 2011). This was true
for plants at the 15-day-old seedling stage as well as at
tillering stage for 12 different rice genotypes under normal non-stress conditions in hydroponics and soil-grown
plants (Raorane and Narciso, unpublished data). Under
Physiol. Plant. 2012
drought stress imposed on 15-day-old seedlings of the 12
rice genotypes varying for drought tolerance, preliminary
results suggested a trend toward higher total root protease
activity in the tolerant genotypes. This was also noticed
for salinity treatment whereby a tolerant genotype such
as Pokkali exhibited stronger upregulation of proteases
compared to the intolerant IR29 (Miro and Raorane,
unpublished data). It was not clear if this was because
of more proteases in the roots of more tolerant plants.
To obtain an idea on whether or not that could be the
case, the rice expression database (
was queried for changes in expression pattern of 672
proteases in roots and leaves at tillering and panicle
elongation stages under control and drought conditions
(Experiment GSE26280: Genome-wide temporal–spatial
gene expression profiling of drought responsiveness in
rice; Wang et al. 2011). The two stages were chosen
for comparison because of recent trends in screening
for reproductive stage drought tolerance (Serraj et al.
2009). Fig. 2 shows that in leaves and roots at the
tillering or panicle development stage under drought
stress, 32 proteases were upregulated twofold or more
in comparison to the control plants. Of these, 11 were
uniquely upregulated in roots, 5 at tillering stage and
6 at panicle elongation stage. Similarly, 15 proteases
were unique to leaves, 2 at tillering stage and 13 at
panicle elongation stage. Upregulation of one protease
was common to roots at both stages of development
L1 L/R1 L2 L//R2 L3 L/R
Fig. 1. PAGE of total soluble protein of leaves (L) or leaves and roots
(L/R) after incubation at 37◦ C for 10, 20 and 30 min (1, 2 and 3),
respectively. The leaf extracts (L1, L2 and L3) are not affected while
a progressive degradation of proteins with time is obvious in leaf and
root extracts (L/R1, L/R2 and L/R3), especially in the region between the
RT: 8
Total:: 20
RE: 12
LT: 6
otal: 21
LE: 1
Fig. 2. Rice proteases showing more than twofold induction under
drought. RT, root at tillering stage; RE, root at panicle elongation stage;
LT, leaves at tillering stage; LE, leaves at panicle elongation stage. In
total, 21 proteases (15 + 6) were upregulated in leaves and 20 (12 +
8) in roots. Six proteases were common to more than one box and, of
those, only one was common to leaves and roots at both developmental
and the other 5 were upregulated in roots and leaves
both at the same or different stages of development
(Fig. 2). Only one protease was more than twofold
upregulated in all four tissues under drought. This
was an ATP-dependent La protease. The importance
of such regulatory proteases in wheat under drought
was described earlier (Zagdanska and Wisnievski
1998, Demirevska et al. 2008) and a distinction was
made between the common vacuolar degradative
proteases and the regulatory processive proteases. In
our analysis, rice leaves exhibited a larger number
of uniquely upregulated proteases. These comprised
several aspartic proteases, including nepenthesin and the
deubiquitination CPs such as a small ubiquitin-related
modifier (SUMO) protease, an ovarian tumor (OTU)-like
protease and four ubiquitin-like protease (Ulp) proteases.
These proteases serve a regulatory function by activating
the proteins that are deubiquitinated, including histones
(H2B), which affects the transcriptional activation of
genes such as those involved in flowering (Schmitz
et al. 2009). Similarly, two serine carboxypeptidases
were uniquely upregulated in leaves, which also
generally help in protein maturation through post
translational modifications. Other proteases were nonspecific subtilisins and CPs most likely involved
in general protein degradation. In roots also, a
couple of unique nepenthesins, subtilisins, serine
carboxypeptidases, a Ulp protease and a CP (EP-B1) were
upregulated. However, more than twofold upregulation
of an M50 protease, a degradation of periplasmic protein
(Deg) protease and an FtsH protease was unique to roots.
In fact, these proteases were among the most highly
upregulated ones in roots.
The M50 proteases are a class of membrane-bound
metalloproteases. Their active sites lie in transmembrane
domains that participate in regulated intramembrane
proteolysis (RIP). They perform site-specific proteolysis
within the membranes, of other membrane-tethered (MT)
proteins, to liberate the cytosolic fragment for signaling
or transcription (Brown et al. 2000). RIP proteases are
special in that they apparently use water to hydrolyze the
peptide bond despite the hydrophobic intramembrane
environment (Wolfe and Kopan 2004). RIP can drive
processes ranging from differentiation, development,
sterol/lipid metabolism and response to unfolded
proteins. Drought conditions do induce an unfolded
protein response (UPR) pathway (Zhang et al. 2008). RIP
participates in this induction by liberating the cytosolic
fragment of the MT transcription factor (TF) bZIP60
and bZIP28, which induce the transcription of proteinfolding chaperones, called binding proteins [(BiP);
Iwata and Koizumi (2005), Liu et al. (2007)]. Also, the
connection between drought, sterol levels in roots and
stomatal responses has been established in Arabidopsis
(Pose et al. 2009). Changes in root sterol levels can affect
the sterol-regulated element binding protein (SREBP) TF.
Interestingly, RIP proteases such as M50 are implicated in
SREBP site 2 proteolysis (S2P) to activate the transcription
factor (Rawson et al. 1997). It is not difficult to further
imagine a relationship between sterol level and the UPR
due to changes in sterols affecting changes in glycans,
in turn affecting protein glycosylation, and thus leading
to ER stress-mediated induction of UPR during drought.
The upregulation of an M50 RIP protease in roots under
drought thus seems logical and important.
The Deg proteases are ATP-independent serine
endopeptidases, which contain one or more [post synaptic density protein (PSD95), drosophila disc large tumor
suppressor (DlgA) and zonula occludens-1 protein (zo1)] (PDZ) protein–protein interaction domains (Kieselbach and Funk 2003). The PDZ domain regulates the
protease activity and oligomerization of Deg proteases.
Deg proteases display dual function, as general proteases
and as specific regulatory proteases in signal transduction or protein maturation (chaperones). This variation
may be attributed to the monomer and oligomer configuration respectively. Also, Deg proteases can either
have a broad substrate range or highly specific substrates. An Arabidopsis glyoxysomal Deg protease was
specifically involved in the maturation of the glyoxysomal malate dehydrogenase (Helm et al. 2007). Deg
proteases have not been characterized in rice and
Physiol. Plant. 2012
root-specific or drought-inducible Deg proteases are not
known. However, a Deg protease was earlier shown
to be relevant to protein quality control in chloroplasts
(Huesgen et al. 2009) through a role in repairing UV-B
damage of PSII (Nixon et al. 2010). In this particular
function the Deg proteases had a complementary role
with the FtsH proteases, which are ATP-dependent, Znmetalloproteases. Both Deg and FtsH proteases are rather
known as plastidial proteases in leaves and not in root.
An FtsH protease was characterized to be responsible for
the variegated phenotype through affecting chloroplast
development (Pogson and Albrecht 2011). Therefore,
root-specific upregulation of rice Deg and FtsH proteases under drought warrants attention. It is likely that
their function in degrading unassembled membrane proteins and/or oxidatively damaged proteins makes them
relevant in drought as part of the UPR pathway similar to
the M50 and rhomboid proteases. It is noteworthy that
proteases highly and uniquely upregulated in rice roots
under drought, although belonging to different classes,
may be concerned with the RIP and UPR pathways both
of which can either affect the C–N pool under stress
by internal proteolysis and/or by facilitating external
nutrient uptake as discussed above or affect protein processing and maturation for signaling and transcription.
Additionally, our in silico analysis revealed some
proteases which, although not twofold upregulated
under drought, were specifically induced in response
to drought in both leaves and roots at all developmental stages assessed by Wang et al. (2011) (Fig. 3,
Table 1). Most of these were similar to proteases discussed above for example, a SUMO protease, a Deg
protease, an SCP, an OTU-like CP, and an M16 domain
containing Zinc peptidase similar to the M50 peptidase.
However, there were four unique proteases, a rhomboid
(serine) protease, an AMSH-like protease, a signal peptide peptidase and a mitochondrial processing peptidase.
Rhomboid proteases are also a part of the RIP arsenal
and are substrate-specific (Kanaoka et al. 2005). The
potential role of these proteases under drought assumes
more importance with the knowledge that plants can
endocytose large organic molecules and microbes
(Paungfoo-Lonhienne et al. 2010), which invokes
mucilage-mediated attraction and receptor-mediated
endocytosis of molecules/microbes. Both of these can
be imagined to be affected by root RIP proteases upregulated under drought. Interestingly, the AMSH protease
is closely associated with clathrin-mediated endosomal
transport of nutrients through symplastic or apoplastic movements from one part of the plant to another.
The other two peptidases are also processing peptidases
that enable protein trafficking to the correct subcellular
Physiol. Plant. 2012
Fig. 3. Drought-induced upregulation of a rhomboid protease designated by the oligo-array element, in leaves and roots at different
developmental stages.
An arbitrary comparative estimate of an increase
in protease activity contributed by the twofold or
more upregulated proteases in roots and leaves (Fig. 2)
was obtained by the sum of fold increases in roots
(20 proteases upregulated 52.57-fold) and leaves (21
proteases upregulated 54.69-fold). The result that a
higher number of proteases were upregulated and to
a larger extent in leaves than in roots was contrary to
the observation of higher and more potent root protease
activity. This suggests that root proteases may be highly
degradative and/or processive. The FtsH protease from
Synechocystis is indeed highly processive (Komenda
et al. 2007) and such an attribute is likely retained in
rice root proteases.
Root proteases and crop improvement
Increasing biomass and seed yield is mostly the ultimate
goal of a large body of agricultural research. However,
plant growth and productivity are directly and strongly
affected by edaphic and other environmental factors,
most of which are perceived by, responded to and
effected on roots. Yet, root responses at the physiological
and/or molecular levels are poorly understood. Although
it is becoming increasingly appreciated that optimal
plant growth and productivity can best be achieved by
Table 1. Proteases specifically induced in response to drought in both leaves and roots at all developmental stages assessed under experiment
GSE26280 by Wang et al. (2011)
Array element ID
Gene ID
Gene name
Serine-type peptidase
Puromycin-sensitive aminopeptidase
AMSH-like protease, putative, expressed
Mitochondrial-processing peptidase
SUMO protease
Signal peptide peptidase-like 2B
OsRhmbd12–putative rhomboid protease
OsSCP56–serine carboxypeptidase
OTU-like cysteine protease family protein
OsDegp3–putative Deg protease
M16 domain containing zinc peptidase
better understanding and leveraging the role of roots,
several issues are emerging in how best to strengthen the
desirable traits of roots while avoiding their undesirable
traits for overall crop improvement (Herder et al. 2010).
Recent studies have highlighted unconventional
sources and routes of nutrient uptake by roots, while
strong links between drought tolerance and nutrient
status have also been highlighted above. These studies
suggest a central role for root proteases not only in
nutrient uptake, but also in water uptake through
processing aquaporins for their role in maintaining
plant water potential (Sade et al. 2009, Maurel et al.
Fig. 4. A schematic model for the role of proteases in proteinaceous nutrition uptake through different modes by root cells at the soil interface.
Left side (beige) represents the cell cytoplasm, cell membrane ad cell wall, while the right side represents the soil. Plant proteases (brown globules)
and/or microbial proteases (yellow globules) act on bacteria (red rods) and proteins (green zigzags) in the soil to generate peptides (small green rods)
and/or amino acids (green AA). Similarly such peptides and AAs are generated within the plant cell by the action of endogenous plant proteases
on endogenous proteins. Additionally, root exudates (evaginated pustule at cell–soil interface) may comprise plant proteins and peptides further
degraded at the rhizoplane or in the soil into peptides and AAs by exuded plant proteases or microbial proteases. It is known that peptides and
AAs serve as sources of carbon and nitrogen through further breakdown within the plant cell. Peptide and AA uptake is well known. However,
under drought conditions, apparently intact proteins and complete bacterial and yeast cells are also taken up by the root cells through the process of
endocytosis (invaginated pustule). Under well-watered conditions root exudates and other process may facilitate most part of the breakdown through
microbial action in the soil, thus enabling re-uptake of simpler peptides and AAs. However, under drought conditions most breakdown and uptake
may happen at the rhizoplane or within the cells, facilitated by endocytosis of larger proteins and cells.
Physiol. Plant. 2012
2010). Yet, very limited knowledge exists on the
identity and function of root proteases under normal
or stress conditions. A better understanding of root
proteases will lead to designing robust strategies for
crop improvement by using proteases as gene-based
markers for genotyping and population surveys or
through transgenic approaches. Regulatory proteases
can play an important part in crop improvement
because they, like transcription factors and kinases,
can affect multiple cascades through spatio-temporalspecific protein processing, leading perhaps to a burst of
response without necessarily involving transcription for
a part of the response.
Although this article concentrated on establishing the
links between root proteases and water and nutrient
uptake and mobilization during drought stress, proteases
functional in root growth, branching and surface may
have an effect on aerial tissues. Root apical and lateral
meristem activity and root vascular development are
controlled by Cle peptides, which are the result of
specific processing proteases (Ni et al. 2010). Processing
of proteins facilitating symplastic movement or xylem to
phloem transporters may also be achieved by proteases.
Studies on proteases in general and root proteases in
particular are also important to differentiate between
desirable and undesirable proteases under specific
conditions. An understanding of root function for water
and nutrient uptake and mobilization, which is inclusive
of studies on root proteases, would fast-track our
physiological and molecular understanding of stress
tolerance. This would lead to informed strategies for
crop improvement under conditions of adverse climate
and shrinking agricultural lands and help meet the
goals of adequate food and nutrition for the increasing
Conclusions and perspectives
Root proteases may have a role in water and nutrient
uptake and mobilization, the quality and quantity of rhizodeposition, microbial population modulation and root
function as a source and sink for nutrients. Increasing evidence suggests that root proteases may be an important
aspect of root development, differentiation and stress
signaling. Various structural and functional classes of
proteases make it difficult to generalize the role of proteases in a process, for example, drought-tolerant plants
may exhibit upregulation of some proteases and downregulation of others. Tissue-specific protease isozymes
also exhibit functional diversity, for example, chloroplast
proteases such as Deg, Clp, FtsH, etc. are upregulated in
roots, where they may not at all be connected with processing photosynthesis-related proteins. Such inherent
Physiol. Plant. 2012
diversity and the difficulty of working with roots under
natural conditions pose a challenge in characterizing
this important class of proteins. It is certain, however,
that making substantial headway in understanding and
affecting above-ground tissues toward crop improvement will critically rest on our understanding of roots.
In that regard, root proteases will be only a part of
the sum, but a very important part, since their capacity to regulate cascades of reactions places them at par
with other protein modification systems such as kinases.
Recent literature clearly makes a case for the importance
of root proteases in drought tolerance. Fig. 4 presents
a schematic version of the role of proteases in nutrient
uptake especially under drought conditions. Drought is
a major bottleneck in food security at present and even
more so in the climate change-driven future scenarios
of agriculture. It is clear that addressing drought tolerance only in terms of water uptake, movement and
use efficiency will be only half the understanding on
drought tolerance because nutrient status imbalances
under drought apparently affect the plant sooner than
the lack of water. Root proteases can positively affect
nutrient and water status more than any other class of
proteins under drought where anabolic processes in the
above-ground source tissues start suffering even before
critically low levels of water are reached. Research on
drought tolerance must therefore put equal if not more
emphasis on the physiology and molecular biology of the
nutrient status of plants under drought and as a corollary
on proteases in general and root proteases in particular.
Acknowledgements – We thank Drs Akshaya Kumar
Biswal, Abdelbagi Ismail, Roland Buresh and Stephan
Haefele of IRRI for critical comments and suggestions on
improving the manuscript. We thank Dr Bill Hardy of IRRI
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