REVIEW ARTICLE
Proline accumulation in plants: a review
Nathalie Verbruggen Æ Christian Hermans
Received: 31 December 2007 / Accepted: 8 March 2008
Ó Springer-Verlag 2008
Abstract Proline (Pro) accumulation is a common
physiological response in many plants in response to a
wide range of biotic and abiotic stresses. Controversy has
surrounded the possible role(s) of proline accumulation. In
this review, knowledge on the regulation of Pro metabo-
lism during development and stress, results of genetic
manipulation of Pro metabolism and current debate on Pro
toxicity in plants are presented.
Keywords Osmotic stress Genetic engineering
Proline toxicity P5CS P5CR P5CDH PDH
Abbreviations
ABA Abscissic acid
ABRE ABA responsive element
AS Antisense
At Arabidopsis thaliana
GFP Green fluorescent protein
GSA Glutamate semialdehyde
Nat-siRNAs Natural silencing RNA
PPP Pentose phosphate pathway
Pro Proline
PDH Pro dehydrogenase
P5C Pyrroline-5-carboxylate
P5CDH P5C dehydrogenase
P5CR P5C reductase
P5CS P5C synthase
RNAi RNA interference
ROS Reactive oxygen species
UTR Untranslated region.
Introduction
Proline (Pro) accumulation occurs in eubacteria, protozoa,
marine invertebrates and plants after various stresses. In
plants, Pro accumulation has been reported to occur after
salt, drought, high temperature, low temperature, heavy
metal, pathogen infection, anaerobiosis, nutrient deficiency,
atmospheric pollution and UV irradiation (Hare and Cress
1997; Saradhi et al. 1995; Siripornadulsil et al. 2002). The
level of Pro accumulation in plants varies from species to
species and can be 100 times greater than in control situa-
tion. Osmotic stress, which include treatments lowering the
osmotic potential component of the water potential, are by
far the most studied ones because they represent a major
concern in agriculture. Pro metabolism in plants has mainly
been studied in response to osmotic stress.
Pro accumulation is believed to play adaptive roles in
plant stress tolerance. Pro has been proposed to act as a
compatible osmolyte and to be a way to store carbon and
nitrogen (Hare and Cress 1997). Salinity and drought are
known to induce oxidative stress. Early in vitro studies
showed that Pro can be a ROS scavenger (Smirnoff and
Cumbes 1989). Pro has also been proposed to function as
molecular chaperone stabilizing the structure of proteins,
and Pro accumulation can provide a way to buffer cytosolic
pH and to balance cell redox status. Finally Pro accumu-
lation may be part of the stress signal influencing adaptive
responses (Maggio et al. 2002).
Pro accumulation during osmotic stress is mainly due to
increased synthesis and reduced degradation. Although Pro
N. Verbruggen (&) C. Hermans
Laboratoire de Physiologie et de Ge
´
ne
´
tique mole
´
culaire des
Plantes, Universite
´
Libre de Bruxelles,
Campus Plaine—CP242, Bd du Triomphe,
1050 Brussels, Belgium
e-mail: nverbru@ulb.ac.be
123
Amino Acids
DOI 10.1007/s00726-008-0061-6
transport certainly plays an important role in Pro distribution,
its role during stress has been poorly studied (Rentsch et al.
1996).
In plants, there are two different precursors for Pro. The
first pathway is from glutamate, which is converted to Pro
by two successive reductions catalyzed by pyrroline-5-
carboxylate synthase (P5CS) and pyrroline-5-carboxylate
reductase (P5CR), respectively. P5CS is a bifunctional
enzyme catalyzing first the activation of glutamate by
phosphorylation and second the reduction of the labile
intermediate c-glutamyl phosphate into glutamate semial-
dehyde (GSA), which is in equilibrium with the P5C form
(Hu et al. 1992). An alternative precursor for Pro biosyn-
thesis is ornithine (Orn), which can be transaminated to
P5C by Orn-d-aminotransferase (OAT), a mitochondrial
located enzyme. Glutamate pathway is the main pathway
during osmotic stress. However in young Arabidopsis
plants, the ornithine pathway seems also to contribute and
d-OAT activity is enhanced (Roosens et al. 1998). The Pro
degradation is the reverse process of Pro biosynthesis and
catalyzed by Pro dehydrogenase (PDH) and P5C dehy-
drogenase (P5CDH). Pro biosynthesis occurs in the cytosol
and in the plastids (like chloroplasts in green tissues) while
Pro degradation takes place in mitochondria (Elthon and
Stewart 1981; Rayapati et al. 1989; Szoke et al. 1992).
Regulation of Pro metabolism during development
and stress
Many studies have been performed in Arabidopsis thali-
ana. A. thaliana is the most studied model plant because it
has the smallest genome size, was the first available plant
genome sequence, has a short life cycle (2 months 1/2), a
small size, is easy to culture and to transform and is
amenable for genetic studies.
In A. thaliana there are two P5CS isoenzymes which
play specific roles in the control of Pro biosynthesis (Fabro
et al. 2004; Sze
´
kely et al. 2008). In other plant species
P5CS is also encoded by two genes (Ginzberg et al. 1998;
Fujita et al. 1998). P5CS represents a rate-limiting step of
pro synthesis and is controlled by feed-back inhibition and
transcriptional regulation (Savoure
´
et al. 1995; Yoshiba
et al. 1995; Zhang et al. 1995). P5CR is encoded by only
one gene but the enzyme seems to be active in chloroplasts
and cytosol (Rayapati et al. 1989; Szoke et al. 1992;
Verbruggen et al. 1993).
During development of A. thaliana and in the absence of
stress, levels of free Pro vary among plant organs, inde-
pendently of the amino acid pool size. Highest Pro levels
are found in flowers, especially in pollen grains, and in
seeds, and lowest levels in roots. The Pro content is also
dependent on plant age, leaf age, position or leaf part
(Verbruggen et al. 1993; Chiang and Dandekar 1995). The
levels of P5CS and P5CR transcripts and P5CR protein are
correlated with the Pro content in the different organs of A.
thaliana, except in roots. The apparent discrepancy in roots
between low Pro content and high P5CS/P5CR transcripts
levels can be explained by Pro export via xylem to the
shoot. Pro synthesis and degradation seem to be both
induced in reproductive organs and seeds. Since the two
pathways take place in different cell compartments, Pro
metabolism may serve to exchange redox potential
(Verbruggen et al. 1996). Moreover Pro transport to flow-
ers is also active. Pro can be a metabolic compatible solute
to transfer nitrogen, carbon and reducing potential to
developing flowers and seeds.
Studies of transcriptional regulation of genes involved in
Pro synthesis confirmed developmental regulation. In
young Arabidopsis plants, GUS analysis of AtP5R (Ara-
bidopsis P5CR gene) promoter revealed high expression in
apical meristem and young leaf, in root meristem, sec-
ondary root primordia and root vascular cylinder. In young
leaf high P5R expression could be detected all over the leaf
blade, while in old leaves, expression was restricted to the
veins, hydathodes, guard cells and base of trichomes (Hua
et al. 1997). In flowering plants, high AtP5R expression
could be detected in rapidly dividing cells, such as root
meristem, and cells or tissues undergoing changes in water
potential, such as hydathode, guard cell, ovule, developing
seed and pollen grain (Hua et al. 1997).
Arabidopsis accumulates Pro upon osmotic stress
(Verbruggen et al. 1993; Yoshiba et al. 1997). During
stress, the expression of P5CS, but not of P5CR gene, is
well correlated with Pro content (Yoshiba et al. 1995;
Savoure
´
et al. 1995). During heat for example, AtP5R
transcripts accumulate without further protein level
enhancement or Pro accumulation (Hua et al. 2001). Post-
transcriptional regulation of AtP5R occurs during stress.
The role of the 5
0
UTR leader sequence of AtP5R in mRNA
stabilisation and translation inhibition was demonstrated
(Hua et al. 2001).
More recently two closely related P5CS genes have been
identified in Arabidopsis thaliana. During stress P5CS1 but
not P5CS2 gene is required for Pro accumulation (Fabro
et al. 2004; Sze
´
kely et al. 2008). During osmotic and salt
stress there are different signalling pathways responsible
for the up-regulation of P5CS1 gene. Induction of P5CS1
expression in A. thaliana depends on phospholipase C
during salt stress but not during drought (Parre et al. 2007).
The abscissic acid (ABA) hormone and salt stress also
induce Arabidopsis P5CS1 expression through ABA
responsive (ABRE) element (Strizhov et al. 1997; Savoure
´
et al. 1997; Abraham et al. 2003). The role of ABA in Pro
accumulation was dissected by Verslues and Bray (2006).
These authors used ABA biosynthetic and signalling
Amino Acids
123
mutants to show dependency of Pro accumulation not only
on the ABA amount but also on the plant sensitivity, or
competency, to respond to the ABA. Further results by
Verslues et al. (2007) support the idea that H
2
O
2
is part of
ABA signaling and ABA-regulated responses like Pro
accumulation.
Recently Sze
´
kely et al. (2008) provided first evidence
about the specificity of cell type and subcellular localiza-
tion patterns of P5CS1 and P5CS2 proteins in Arabidopsis.
This study highlighted the non-redundancy of the two
P5CS isoenzymes and the importance of compartmentali-
zation of Pro metabolism. Depending on the developmental
stage, organs and growth conditions, P5CS1- and P5CS2-
GFP fusions displayed different localization in the cell
(plastids, vesicles or in the cytoplasm). In mesophyll cells
of mature leaves, salt and osmotic stress stimulate the
import of P5CS1 to chloroplasts, while the distribution of
the P5CS2 pool in the cytoplasm and chloroplasts does not
change (Sze
´
kely et al. 2008).
During osmotic stress, availability of atmospheric CO
2
is reduced because of increased stomatal closure and con-
sumption of NADPH by the Calvin Cycle is decreased.
Activity of P5CS in the chloroplasts can recycle NADP
+
,
the last acceptor of the photosynthetic electron transfer
chain, which may reduce ROS production at the photo-
system I. Also glucose-6-phosphate dehydrogenase, the
first and rate-limiting enzyme of the pentose phosphate
pathway (PPP) requires NADP
+
and is inhibited by
NADPH. Phang (1985) proposed a model in which Pro/
P5C interconversions modulate NADP
+
/NADPH redox
states. Interestingly the PPP takes place in the cytoplasm
and in the plastids of plant cells, the two compartments
where Pro synthesis takes place.
Kohl et al. (1988, 1990) have applied Phang’s model to
nitrogen-fixing soybean nodules, which accumulate Pro.
Pro accumulates via enhanced synthesis and high Pro
synthesis maintains a high NADP/NADPH ratio in the
nodules. Such a high ratio can drive the PPP, which in turn
supports purine biosynthesis. Purine derivatives are used as
the primary transport molecule for fixed nitrogen. However
in vitro activities could not support the correlation between
P5CR and PPP activities (Kohl et al. 1990). Kohl et al.
(1988, 1990) have proposed that Pro transfers redox
potential from the plant cytoplasm to the bacteroid. This
was supported by high PDH activity in bacteroids of soy-
bean nodules. During stress the link between Pro
accumulation and induction of the PPP was episodically
reported but hardly demonstrated (Hare and Cress 1997).
Pro degradation is also regulated during development
and stress. In A. thaliana, PDH expression is low except in
flowers and young siliques, which also contain higher Pro
concentrations than in roots or leaves. PDH expression is
strongly induced by the addition of exogenously supplied
Pro. During stress both transcript and protein levels of At-
PDH are repressed during stress and induced during
recovery from stress (Kiyosue et al. 1996; Peng et al. 1996;
Verbruggen et al. 1996). There is thus evidence for a
negative transcriptional regulator which overrides the
positive effect of accumulated Pro on AtPDH expression
(Verbruggen et al. 1996). Data on At-PDH expression
during development and stress were confirmed by a gene
reporter–promoter study published by the group of Shino-
zaki (Nakashima et al. 1998). Recently bZIP transcription
factors involved in the up-regulation of At-PDH during
rehydration were identified (Weltmeier et al. 2006).
Although Pro level has been thought to be regulated
mainly by P5CS and PDH, the regulation by P5CDH seems
to be also important. Arabidopsis P5CDH and SRO5,a
gene of unknown function, form an overlapping antisense
gene pair. These two genes generate convergent transcripts
that overlap by 760 nucleotides (Borsani et al. 2005). SRO5
is not expressed in plants grown under normal conditions,
but its expression is upregulated by NaCl treatment (not by
PEG- or mannitol- treatments, which induce osmotic
stress). Upon salt treatment, the SRO5 and P5CDH mRNAs
can form a dsRNA that is then processed to generate 24-nt
SRO5-P5CDH natural silencing RNS (nat-siRNAs). Those
nat-siRNAs downregulate the expression of P5CDH by
causing mRNA cleavage (Borsani et al. 2005). This in turn
contributes to Pro accumulation but also causes an increase
in ROS production, which is counteracted by the SRO5
protein. This is a nice example of regulation of two genes
in a converse manner.
Genetic manipulation of Pro metabolism
As much as one-half of the irrigated areas of the world are
affected by high salinity. Therefore there is a high interest
to improve plant osmotolerance in agriculture. This has
been achieved by the genetic manipulation of osmolytes,
transcriptions factors and more recently of the cytokinin
hormone (Seki et al. 2007; Rivero et al. 2007). Engineered
transgenic plants have also been useful tools to address
fundamental questions.
Figure 1 summarizes different examples of genetically
modifications of the Pro metabolic pathways. First Pro
transgenic plants were published by the group of Desh Pal
Verma (Kishor et al. 1995). Tobacco plants overexpressing
mothbean P5CS gene, coding for the first enzyme of pro
biosynthesis under the activity of a constitutive promoter,
synthesized 10–18-fold more Pro than control plants and
were better salt stress tolerant. Surprisingly the osmotic
potential of P5CS transgenics was not lower than in control
plants. Removal of feed-back inhibition of P5CS resulted in
higher Pro accumulation and protection of plants from
Amino Acids
123
osmotic stress (Hong et al. 2000). On the contrary P5CS
antisense Arabidopsis lines that were impaired in their
capacity to synthesize Pro were hypersensitive to osmotic
stress (Nanjo et al. 1999a). P5CS antisense lines showed
morphological abnormalities of epidermal and parenchy-
matous cells, underlying the role of pro as major constituent
of cell wall proteins (Nanjo et al. 1999a). Similarly p5cs1
Arabidopsis insertion mutant showed reduced salt tolerance
(Sze
´
kely et al. 2008). Furthermore analysis of Arabidopsis
p5cs insertion mutants confirmed a role in vivo for Pro in
ROS scavenging, which was first postulated by Smirnoff
and Cumbes in (1989). Enzymes of the ROS-scavenging
glutathione-ascorbate cycle showed significantly lower
activities in the p5cs1 mutants compared to wild type under
salt stress suggesting that Pro accumulation is implicated in
the control of either stability or activity of enzymes in the
glutathione-ascorbate cycle (Sze
´
kely et al. 2008).
However high pro levels are not always correlated with
osmotolerance. Mutants displaying higher Pro accumula-
tion can be salt hypersensitive (Lui and Zhu 1997).
Effects of genetic manipulation of Pro synthesis can be
plant species specific. Overexpressing soybean P5CR gene
in transgenic tobacco did not increase osmotolerance
(Szoke et al. 1992). Different results were observed in
soybean by a team of South Africa. The A. thaliana gene
encoding P5CR was overexpressed in soybean in the sense
and antisense orientation into a heat shock cassette con-
taining an inducible heat shock promoter (de Ronde et al.
2000). Soybean production in South Africa is affected by
frequent periods of drought. Field trials in South Africa
with P5CR transgenic soybean lines supported improved
drought performance and higher heat tolerance compared
to wild type cultivars (de Ronde et al. 2004). It is therefore
possible that crops with genetically manipulated Pro syn-
thesis will be commercialised in the future.
Pro degradation was also manipulated. Overexpression
of PDH in Arabidopsis thaliana did not result in morpho-
logical abnormalities, probably because Pro homeostasis
relies on regulated transport between cell compartments
(Nanjo et al. 1999b; Mani et al. 2002). Pro degradation
depends on prior Pro transport to mitochondria. The normal
phenotype of PDH-sense plants is in contrast with the
results of the team of Jim Phang in human cells where
overexpression of Pro oxidase induced Pro-dependent and
mitochondria-mediated apoptosis (Hu et al. 2007).
Decreasing Pro catabolism by PDH antisense strategy
did not alter plant development (Nanjo et al. 1999b; Mani
et al. 2002). A slight decrease in seed germination was
observed in PDH RNAi tobacco plants (Ribarits et al.
2007). Higher tolerance to salt stress or to drought was
sometimes observed in some PDH antisense lines but not
always (Nanjo et al. 1999b; Mani et al. 2002). Differences
in observed tolerance between laboratories can lie in the
levels of Pro dehydrogenase inhibition and corresponding
increase in Pro content, as well as applied stress conditions.
Furthermore PDH sense lines, overexpressing
PDH under a
constitutive promoter, showed lower Pro accumulation
during osmotic stress but no change in osmotolerance.
However these PDH-sense lines were better osmotolerant
than WT in the presence of exogenously supplied Pro. In
the latter conditions, chromatography after radiolabelled
Pro supply showed that degradation in glutamate was
increased (Mani et al. 2002). These results suggest that Pro
can be a good source of energy during stress too, and that
the second step of the oxidation pathway is not rate-
limiting.
Pro toxicity
Pro accumulation is a common response of plants to stress.
However external supply of pro in control conditions is
toxic. There is a debate whether Pro or its degradation
L-Pro
P5C
spontaneous
P5CR
PDH
GSA
P5CS
P5CDH
L-Glu
Proline DEGRADATION (mitochondria)
Proline SYNTHESIS (cytosol and plastids)
[1,2,3]
[4,5]
[6,7,8]
[9,10]
Fig. 1 Genetic manipulations of Pro metabolic pathways in plants. The
main precursor of Pro synthesis is L-glutamic acid (L-Glu). L-Glu is
first reduced to glutamate semialdehyde, which spontaneously cyclizes
to pyrroline-5-carboxylate (P5C), by P5C synthase (P5CS). The second
reduction, of P5C to Pro, is catalyzed by P5C reductase (P5CR). This
pathway is found in the cytosol and in plastids. Pro is catabolized to Glu
in mitochondria by Pro dehydrogenase (PDH) and P5C dehydrogenase
(P5CDH). Examples of genetic manipulations of Pro metabolic
pathway and impact on osmotolerance: [1] Overexpression of moth-
bean P5CS in tobacco plants. Those transgenics were better salt tolerant
(Kishor et al. 1995); [2] Overexpression of P5CS in antisense
orientation in Arabidopsis (Nanjo et al. 1999a) and [3] p5cs1 insertional
mutation (Sze
´
kely et al. 2008) resulted in reduced osmotolerance; [4]
Overexpression of soybean P5CR gene in tobacco (Szoke et al. 1992)
did not modify osmotolerance; [5] Overexpression of Arabidopsis
P5CR (AtP5R) gene in soybean improved drought and heat stress (de
Ronde et al. 2000, 2004); [6] Overexpression of PDH did not change
osmotolerance in Arabidopsis, except in the presence of exogenously
supplied Pro. In these conditions PDH-sense lines were better
osmotolerant than WT. [7] Higher salt tolerance was observed in
Arabidopsis plants overexpressing PDH in antisense orientation by
Nanjo et al. (1999b), but not by Mani et al. (2002). Study of
modifications of Pro catabolism in Arabidopsis in relation to P5/GSA
and/or Pro toxicity: [6] Overexpression of PDH decreased sensitivity to
externally supplied Pro, [7] decrease of PDH activity by antisense
strategy (Mani et al. 2002)or[8] knock-out mutation (Nanjo et al. 2003)
increased sensitivity to Pro; [9] P5CDH overexpression decreased
sensitivity to externally supplied Pro (Deuschle et al. 2004); [10] p5cdh
knock-out mutants were hypersensitive to Pro (Deuschle et al. 2004)
Amino Acids
123
product P5C is the cause of toxicity (Hellman et al. 2000;
Deuschle et al. 2001; Mani et al. 2002; Ayliffe et al. 2002).
Application of both Pro and P5C cause cell death in
plants (Deuschle et al. 2004). Data of Deuschle et al.
(2004) support that Pro toxicity is mediated by GSA/P5C
accumulation. Exogenously applied P5C increases ROS
production, reduces growth and induces a number of stress-
responsive genes. Furthermore P5CDH overexpression
decreased sensitivity to externally supplied Pro while
p5cdh knock-out mutant was hypersensitive to Pro (Deu-
schle et al. 2004).
Other data in mutants with impaired Pro catabolism
clearly support that Pro toxicity is not, or not only
mediated by P5C/GSA. Pro external concentrations higher
than 10 mM are toxic to wild-type Arabidopsis plants. In
PDH-antisense plants that displayed a lower Pro catabo-
lism Pro toxicity was observed at lower Pro
concentrations than in wild-type (Mani et al. 2002). PDH-
sense transgenic plants, with a higher Pro catabolism
capacity, displayed wild-type Pro sensitivity. Pdh knock-
out mutants were even more sensitive to Pro than PDH
antisense plants, most probably because the mutation
results in a more severe inhibition of Pro catabolism
(Nanjo et al. 2003). Transcriptomic analysis of pdh
mutants treated with Pro revealed the repression of genes
involved in photosynthesis and genes involved in the
synthesis of cell-wall associated proteins, which are
believed to account for Pro toxicity.
Hypersensitivity of pdh KO and of PDH-AS plants to
Pro is against the theory that not Pro but only P5C causes
toxicity.
Conclusion
In short, Pro accumulation is a common physiological
response to various stresses but is also part of the
developmental program in generative tissues like pollen
for example. Transgenic approaches have confirmed the
beneficial effect of Pro overproduction during stress.
However consensus was not achieved on the exact roles
of Pro accumulation. Classical gain or loss of function
strategies could not bring clear answers, probably because
Pro also displays the essential role of being a protein
component. Stresses like drought or salt stress have
multiple targets and Pro is also believed to play different
roles. Moreover the balance between biosynthesis and
degradation of Pro is also thought to be essential in the
determination of the osmoprotective and developmental
functions of Pro.
Acknowledgments The authors thank the Belgian Science Policy
for financial support (project PAIVI/33, return grant of C.H).
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![Fig. 1 Genetic manipulations of Pro metabolic pathways in plants. The main precursor of Pro synthesis is L-glutamic acid (L-Glu). L-Glu is first reduced to glutamate semialdehyde, which spontaneously cyclizes to pyrroline-5-carboxylate (P5C), by P5C synthase (P5CS). The second reduction, of P5C to Pro, is catalyzed by P5C reductase (P5CR). This pathway is found in the cytosol and in plastids. Pro is catabolized to Glu in mitochondria by Pro dehydrogenase (PDH) and P5C dehydrogenase (P5CDH). Examples of genetic manipulations of Pro metabolic pathway and impact on osmotolerance: [1] Overexpression of mothbean P5CS in tobacco plants. Those transgenics were better salt tolerant (Kishor et al. 1995); [2] Overexpression of P5CS in antisense orientation in Arabidopsis (Nanjo et al. 1999a) and [3] p5cs1 insertional mutation (Székely et al. 2008) resulted in reduced osmotolerance; [4] Overexpression of soybean P5CR gene in tobacco (Szoke et al. 1992) did not modify osmotolerance; [5] Overexpression of Arabidopsis P5CR (AtP5R) gene in soybean improved drought and heat stress (de Ronde et al. 2000, 2004); [6] Overexpression of PDH did not change osmotolerance in Arabidopsis, except in the presence of exogenously supplied Pro. In these conditions PDH-sense lines were better osmotolerant than WT. [7] Higher salt tolerance was observed in Arabidopsis plants overexpressing PDH in antisense orientation by Nanjo et al. (1999b), but not by Mani et al. (2002). Study of modifications of Pro catabolism in Arabidopsis in relation to P5/GSA and/or Pro toxicity: [6] Overexpression of PDH decreased sensitivity to externally supplied Pro, [7] decrease of PDH activity by antisense strategy (Mani et al. 2002) or [8] knock-out mutation (Nanjo et al. 2003) increased sensitivity to Pro; [9] P5CDH overexpression decreased sensitivity to externally supplied Pro (Deuschle et al. 2004); [10] p5cdh knock-out mutants were hypersensitive to Pro (Deuschle et al. 2004)](/figures/fig-1-genetic-manipulations-of-pro-metabolic-pathways-in-3jkcpi47.webp)