BACKGROUND
proline  metabolism is sensitive to environmental cues and undergoes particular alterations that contribute to stress tolerance.
plants exposed to dehydration increase pro synthesis and reduce its catabolism, accumulating high levels of the amino acid.
pro increase has beneficial effects on plant cells and acts as energy reserve to be used upon stress release.
pro metabolism can also affect the cellular redox homeostasis and mobilization of nutrients, which also helps in stress alleviation .
plants synthesize pro from ornithine and glutamic acid , involving mostly the second pathway under stress conditions.
this process takes place in the cytosol and plastids, involving the action of ∆ <dig> pyrroline-5-carboxylate  synthase  and p5c reductase   <cit> .
the catabolic route occurs at the mitochondria, where pro is oxidized into glu by two enzymes.
first, pro dehydrogenase  transforms pro into p5c.
then, p5c is nonenzymatically converted into glutamate semi-aldehyde that acts as substrate of p5c dehydrogenase  for generation of glu  <cit> .
oxidation of pro into glu is a well-conserved process mediated by enzymes that adopt different organizations in nature.
gram-negative bacteria combine prodh and p5cdh activities into a single polypeptide, whereas eukaryotes and gram-possitive bacteria contain monofunctional enzymes  <cit> .
prodh catalyzes the rate limiting step in the oxidation pathway, transferring two electrons from pro to the non-covalently bound fad cofactor to subsequently deliver these electrons to a final acceptor  <cit> .
the human prodh has been early recognized as a tumor suppressor protein regulated by p <dig>  which triggers intrinsic and extrinsic apopototic pathways  <cit> .
the bacterial prodhs have been well characterized at the kinetic and structural levels by crystalographic studies  <cit> .
unlike these enzymes, the plant prodh has been mostly studied at the gene expression level, with little functional and biochemical information and no crystal structure being currently available  <cit> .
this enzyme resides at the inner mitochondrial membrane where it sustains oxidative phosphorylation and atp generation  <cit>  using endogenous electron acceptors that still deserve characterization.
several studies described the sensitivity of pro metabolism genes to pathogen infections.
in flax, p5cdh is induced by virulent rust-fungi races that overcome host defense barriers  <cit> .
in arabidopsis, p5cs <dig>  p5cr and prodh, but not p5cdh, are activated by pseudomonas syringae pv.
tomato  strains eliciting effector triggered immunity  and hypersensitive response   <cit> .
under the latter condition, salicylic acid mediates p5cs <dig> activation leading to pro increase at late stages of infection  <cit> .
in nicotiana benthamiana, p5cs <dig>  p5cr, prodh and ornithine δ-amino transferase  genes, but not p5cdh, are induced by p. syringae pv.
tomato t <dig> strain eliciting hr-like lesions  <cit> .
interestingly, prodh and oat were selected by a vigs-based forward genetic screen by looking for genes regulating non-host resistance  <cit> , and the participation of these enzymes in disease resistance was inferred from the reduction of hr markers in infected tissues of prodh- or oat- silenced plants  <cit> .
in animals, yeast, and bacteria, the prodh activity was associated with the increase in reactive oxygen species , and was suspected to generate either cellular damage  <cit>  or protective functions  <cit> , apparently depending on the ros levels.
whereas prodh potentiates ros and cell death in plant hr-developing tissues  <cit> , it extends life span by ros-mediated activation of antioxidant enzymes in caenorhabditis elegans <cit> .
it is unclear how prodh modulates ros levels and is coordinated with other pro metabolizing enzymes under these conditions.
its joint action with p5cdh provides energy by charging electrons into the metc for atp synthesis, and produces glu that enters the tricarboxylic acid cycle via α-ketoglutarate  <cit> .
prodh may also operate without p5cdh.
this is feasible in yeast, plants and animals containing monofunctional enzymes.
in this case, incomplete pro oxidation can led to p5c increase.
several studies suggested that p5c accumulation can result harmful, apparently by enhancement of ros levels  <cit> .
interestingly, arabidopsis plants were found sensitive to exogenous p5c, particularly in the absence of p5cdh where it produced hr-like lesions  <cit> .
however, p5c was also informed as innocuous for this plant  <cit> , raising doubts about the extent and base of its toxicity.
more recently, the compound was proposed as an inhibitor of mitochondrial respiration generating lethal superoxide anion levels in yeasts  <cit> .
functional uncoupling of prodh and p5cdh not always leads to p5c increase  <cit> .
alternatively, the p5c produced by prodh can be used by p5cr to regenerate pro.
moreover, the joint action of prodh and p5cr can stimulate the pro/p5c cycle  <cit> .
as part of this cycle, the human prodh increases mitochondrial ros and induces apoptosis, with these effects being abolished by over-expression of mitochondrial mnsod  <cit> .
at present, several features of this cycle  remain poorly characterized in plants.
however, this circuit seems to operate in the arabidopsis p5cdh mutant, and the prodh-  <cit>  and p5cs1-  <cit>  over-expressing plants.
as part of this cycle, prodh may not only promote mitochondrial ros generation, but also shuttle reducing power from cytosol to mitochondria producing additional redox changes  <cit> .
finally, prodh as a flavoenzyme, can eventually transfer electrons to o <dig> as observed for the monofunctional protein of thermus thermophilus in vitro <cit> .
as described above, many effects could derive from prodh activation in hr.
to start characterizing them, we here analyzed the metabolic context accompanying prodh stimulation at three different hr stages.
we induced hr by inoculating pst-avrrpm <dig> in arabidopsis leaves and isolated samples before, during and after the oxidative burst for assessing pro catabolism, prodh activity and amino acid content.
the studies were performed in wild type plants and the p5cdh mutant, used to evaluate how p5cdh affects the prodh action in hr.
RESULTS
selection of three hr stages for evaluation of prodh action
we evaluated prodh action at different hr stages.
in particular, before and during the maximum ros accumulation that precedes cell death, and at a late hr phase already manifesting cell death.
to select these stages, we used conditions that slow hr development, such as infiltration of a moderate dose of bacteria  into adult leaves of short-day grown plants.
leaves were excised at  <dig>   <dig>   <dig>   <dig>   <dig>   <dig> hours post-inoculation  with pathogen or mock solution , and monitored for ros and cell death, as described in additional file 1a.
mock inoculations generated a transient and mild increase in ros before  <dig> hpi and did not affect cell viability at any analyzed stage .
pathogen treatment began to increase ros at  <dig> hpi to produce maximum ros accumulation at  <dig> hpi, and trigger cell death since  <dig> hpi .
based on these responses, we selected  <dig>   <dig>  and  <dig> hpi for further studies, defining these stages as phases i, ii and iii of hr.
pro metabolic enzymes in hr developing tissues
total protein extracts from untreated and pst-avrrpm1-treated leaves were analyzed by western blot assays with anti-prodh  <cit>  and anti-p5cdh polyclonal antibodies .
a net prodh increase was evidenced at phase i, and maintained until phase iii .
unlike prodh, p5cdh only showed slight increases during hr.
these protein levels were consistent with the abundance of prodh and p5cdh transcripts observed under the same conditions .
since pst-avrrpm <dig> is able to induce the p5cs <dig> gene expression at advanced hr stages , we monitored its expression in our samples.
as observed in figure  <dig> and additional file 1b, we did not detect p5cs <dig> transcript accumulation early in hr .
in turn, expression of p5cr and prodh <dig> were found enhanced in hr tissues  as reported previously  <cit> .
pro catabolism in hr in vivo
we investigated how prodh impacts on pro catabolism before and during ros accumulation.
to this purpose, we fed plants with 14c-pro and monitored its consumption in vivo.
a pro molecule labelled at all c residues was used to improve detection of the pro derivatives .
uninfected  and bacterial-infected leaves excised at phases i and ii of hr, were fed with 14c-pro , transferred to water for different time periods , and sampled as outlined in figure 2b.
total amino acids were extracted from these samples and analyzed by tlc .
consumption of 14c-pro during the water incubation period was evaluated by quantifying the labelled amino acid in this set of samples .
healthy tissues had little capacity to catabolize exogenous pro, as they conserved 83% and 77% of the initial 14c-pro after  <dig> and 60 min of incubation in water, respectively .
in contrast, tissues at hr phase i showed a proficient 14c-pro consumption, since they preserved 34% and 26% of the amino acid at  <dig> or 60 min of incubation, respectively .
this increase in pro catabolism paralleled prodh accumulation , suggesting it may result from prodh activation.
at phase i, reduction of 14c-pro was not accompanied by appearance of 14c-p5c, 14c-glu or other derivatives.
given that the mass of these compounds is directly proportional to their labelling, the 14c-p5c and 14c-glu levels would be substantially lower than those of 14c-pro.
therefore, the results suggested that at phase i, prodh acted with p5cdh to generate 14c-glu which was rapidly metabolized.
this possibility was further supported by quantification of amino acids .
surprisingly, the in vivo14c-pro consumption was not maintained at hr phase ii .
at this stage, 90% of the amino acid persisted after 60 min incubation .
prodh activity remained enhanced , but had little effect on the pro levels.
therefore, during maximun oxidative burst p5cdh did not seem to accompany prodh, suggesting prodh could act with p5cr under this condition.
in vitro
prodh activity at different hr phases
p5cdh
prodh activity was analyzed on protein extracts of uninoculated  and pst-avrrpm1-inoculated leaves of wild type and p5cdh plants.
infected leaves were evaluated at  <dig>   <dig> and  <dig> hours post-infection .
enzyme activity was expressed in nmol of pro min-1 mg of total protein- <dig>  each value is mean ± sd of at least two independent experiments with  <dig> independent plants.
t4c: l -thiazolidine-4-carboxylic acid, inhibitor of prodh.
*significant differences respect to uninfected condition  of the same genotype .
in vitro prodh activity
we tested if the lack of 14c-pro consumption at phase ii resulted from a reduction in prodh activity.
we quantified this activity by using an in vitro assay that prevents native conditions favoring functional coupling of prodh with other enzymes.
to this end, the assay previously used to analyze cultured cell extracts  <cit>  was adapted to evaluate photosynthetic tissue samples.
the new system included two modifications.
first, the prodh activity was determined by monitoring 3h-pro consumption instead of nad+ reduction.
the pro radiolabelled molecule that we selected as substrate contained high specific activity .
second, the reaction was developed at neutral instead of alkaline ph, to avoid the reverse p5cr activity  <cit> , preserving the prodh activity  <cit> .
the new assay, conserved the use of l-thiazolidine-4-carboxylic acid  as competitive prodh inhibitor, and resulted suitable to determine prodh activity in arabidopsis leaf extracts .
as expected, wild type plants increased the prodh activity at hr phase i .
clearly, the enhancement of enzyme activity was preserved at phase ii, and slightly increased at phase iii.
therefore, the results suggested that in vivo prodh remained active at all three hr phases.
changes in amino acid levels
to further evaluate the coordinated action of prodh with p5cdh or p5cr, we quantified the content of pro and its derivatives at each hr stage.
alterations in levels of these compounds were indicative of the plant enzyme activities under stress  <cit> .
we included in this study the a. thaliana t-dna insertion line salk_ <dig> previously described as a p5cdh knock-out mutant plant  <cit> .
as expected, this plant neither accumulated p5cdh protein nor p5cdh transcripts in leaf or floral tissues .
interestingly, the p5cdh infected tissues retained the capacity to activate prodh in response to pst-avrrpm <dig> reaching similar activity levels than infected wild type tissues .
therefore, this plant resulted useful to detect responses dependent on p5cdh, but not prodh, activity.
by hplc assays we quantified  <dig> amino acids on naive and infected tissues of wild type and the p5cdh plants.
at basal conditions, all amino acids had similar levels in both plants , except pro which was increased in the mutant .
after bacterial treatment all amino acids, except ser, altered their content in at least one hr phase in one genotype, showing significant differences respect to the uninfected condition  .
most of them  displayed a progressive increase in both plants, which was generally lower in the mutant, suggesting that p5cdh may contribute to accumulation of these amino acids in infected wild type tissues.
gaba was included in this group based on its large increase at hr phase ii, which was more pronounced in the mutant.
in contrast, glu and asp were reduced by infection in both plants.
in turn, p5c, pro and gln exhibited different alterations in both genotypes.
p5cdh
c:uninfected samples ; i, ii, iii: pst-avrrpm1-infected samples isolated at phases i, ii or iii of hr.
*significant differences between infected and control samples of the same genotype .
#samples analyzed in different experiments containing similar values similar to those of wild type plants.
asignificant differences regarding the wild type control value .
values are expressed as nmol/g fresh weight.
each value is mean ± sd of  <dig> independent experiments .
to analyze in greater depth the behavior of pro, p5c and glu at phases i and ii of hr, we compared their alterations in wild type and mutant plants .
glu is a central molecule in plant amino acid metabolism contributing to the synthesis of several of these compounds.
its α-amino group can be transferred to oxalic acid to form asp, a precursor of asn and the aspartate family of amino acids  <cit> .
therefore, we included asn in the analysis, as a terminal amino acid indirectly derived from glu.
at phase i, wild type plants reduced pro and increased asn without altering p5c or glu .
once again, the results suggested that prodh and p5cdh acted together at phase i, generating glu and thus contributing to asn increase .
consistently with this possibility, pro consumption was not accompanied by glu accumulation at phase i .
moreover, p5cdh plants were unable to reduce pro or increase asn at this stage .
at phase ii, wild type tissues recovered their basal pro content and maintained asn at similar levels than phase i, while they preserved basal p5c and glu content.
meanwhile, p5cdh plants showed just a modest increase of p5c and asn .
as observed in our previous studies, phases i and ii exhibited differences in pro catabolism , with the responses observed in wild type plants at phase ii  being consistent with stimulation of the pro/p5c cycle.
importantly, wild type plants did not accumulate p5c during the initial  <dig> hpi with pst-avrrpm <dig> .
as described, the oxidative burst and cell death were produced during this period .
prodh action in the p5cdh mutant
we wondered if prodh requires p5cdh to potentiate hr.
to evaluate this issue, we monitored hr features in pst-avrrpm1-infected p5cdh leaves.
these tissues lacked p5cdh protein and transcripts but accumulated prodh transcripts and protein as infected wild type tissues .
as mentioned before, the p5cdh plant also preserved the capacity to activate prodh in response to pst-avrrpm <dig> .
therefore, this mutant was suitable to test the effect of prodh in hr, in the absence of p5cdh.
as in previous studies, we evaluated ros and cell death as hr markers, and susceptibility to avirulent bacteria as parameter of plant resistance  <cit> .
the increase of ros was higher in the mutant , which triggered cell death before wild type plants .
in addition, the p5cdh plants showed enhanced pathogen resistance at  <dig> hpi .
however, at later infection stages these differences were no longer observed .
therefore, the absence of p5cdh affected the timing, but not strength, of hr producing a premature increase of ros and cell death, and transient enhancement of pathogen resistance.
discussion
prodh activation in hr
we used an in vitro assay to quantify prodh activity in arabidopsis leaf tissues.
the methods monitoring pro-dependent nad+ reduction by spectrophotometry were not useful to this end since pigments from photosynthetic tissues interfered with nadh detection  <cit> .
we overcome this limitation by following 3h-pro consumption instead of nad+ reduction.
under basal conditions wild type plants consumed  <dig>  cpm 3h-pro min-1 mg protein- <dig>  equivalent to 33 nmol 3h-pro min-1 mg protein- <dig>  this value was in agreement to that reported for the enzyme in liver mitochondrial extracts , where 3h-pro labelled at the c- <dig> was used as substrate, and transference of 3h to water was monitored  <cit> .
the increase of prodh activity detected in the infected leaves  far exceeded the one observed in extracts of cultured cells challenged with the same pathogen .
this difference was probably due to greater responsiveness of leaf tissues to pathogen, and improvement of the assay.
strong and persistent prodh activation  coexisted with transient pro decrease  in infected tissues, indicating that the greater enzyme capacity did not substantially impact on the pro levels.
interestingly, these tissues induced prodh without a previous pro increase.
something similar was observed for the p5cs <dig> plants that even with low pro levels, retained the ability to activate prodh after stress release  <cit> .
moreover, under cold acclimation, prodh induction was accompanied by pro increase  <cit> .
one interesting observation derived from our studies was the maintenance of prodh activation in the absence of p5cdh , which was already noticed on p5cdh plants recovering from drought stress  <cit> .
therefore, prodh activation occuring after stress release would not always be accompanied by p5cdh and could have minor effects on the pro content.
although pst-avrrpm <dig> induced prodh and p5cs <dig> genes at advanced stages of hr , it only activated prodh at the early hr phases .
thus, in contrast with rehydration, the hr condition stimulates pro catabolism before pro synthesis.
how prodh remains active without de novo pro synthesis under these circumstances is unknown.
transport of pro from healthy tissues  <cit> , stimulation of the pro/p5c cycle  <cit> , or both responses can take place there.
restoration of basal pro  without p5cs induction  observed at phase ii in wild type plants was consistent with the first possibility.
as discussed below, several additional results supported the latter one.
p5c levels in the hr lesion
the functional uncoupling of prodh and p5cdh may generate p5c accumulation with toxic effects for plants  <cit> .
in yeast, the p5c increase from  <dig> to 300 μm results harmful for stressed cells  <cit> .
our previous finding that pst-avrrpm <dig> activated prodh but not p5cdh, suggested that p5c could accumulate and signal hr cell death  <cit> .
in previous studies, we did not detect p5c increase by spectrophotometric assays in wild type tissues developing hr  <cit> .
in the current work, we used hplc to unequivocally determine the p5c content in hr.
in addition, we analyzed p5cdh infected tissues that are prone to accumulate p5c due to prodh activation.
we detected a transient increase of p5c at hr phase ii in p5cdh plants  that paralleled cell death initiation .
how p5c contributes to hr cell death in p5cdh plants is unknown.
however, it is clear that p5c does not signal ros generation or cell death initiation in the hr lesion of wild type plants, since its levels remain almost constant at all phases of this response .
pro catabolism at phases i and ii of hr
in vitro assays evidenced similar prodh activation at all hr stages .
at difference, in vivo studies, and amino acid quantification, showed variations in pro catabolism at phases i and ii.
at phase i, pro consumption  was accompanied by increase of the terminal glu-derivative asn , suggesting that basal pro was transformed into glu.
at phase ii, pro did not continue to decline, and asn was not further increased .
the alteration of pro catabolism manifested in phase ii may have different causes.
one possibility is that enzyme and substrate have different partitioning.
this is, however, unlikely since mitochondrial pro depletion has not been reported to date  <cit> .
alternatively, plant- or bacterial-derived compounds may inhibit prodh at phase ii.
nevertheless, the existence of such products has not been reported.
p5c, which may eventually lead to product inhibition, did not increase under this condition in wild type plants .
furthermore, since prodh preserved its catalytic capacity in vitro until phase iii  irreversible enzyme inhibition can be discarded.
another possibility is that prodh acts with p5cr recycling pro and p5c.
recent studies propose that the pro/p5c cycle operates in arabidopsis tissues exposed to abiotic stress, and probably contributes to exogenous pro toxicity  <cit> .
the results that suggest stimulation of this cycle at hr phase ii are: i) co-existence of prodh activation , and unaltered pro and p5c levels ; ii) absence of p5cs induction at the time of prodh activation ; iii) premature accumulation of ros in p5cdh infected tissues able to induce prodh .
moreover, the joint activation of prodh and p5cr genes here observed  was consistent with previous results  <cit>  and also supported this possibility.
therefore, although the pro/p5c cycle requires formal demonstration in plants, the current knowledge about pro metabolism  <cit>  and our results are consistent with stimulation of this cycle in hr.
prodh action at hr phase ii
arabidopsis transgenic plants that reduced, but not eliminated, prodh expression attenuated the oxidative burst, hypersensitive cell death, and resistance to pst-avrrpm <dig> <cit> .
in contrast, the null p5cdh mutant did not reduce these responses .
this suggests that prodh does not require p5cdh to potentiate those hr features.
consistently, prodh had no effect on pro consumption at the time of maximum ros accumulation .
moreover, p5cdh may even prevent or reduce the capacity of prodh to produce ros and associated defenses.
in this sense, infected p5cdh plants prematurely stimulated ros burst and cell death .
moreover, induction of p5cdh was observed in diseased, but not resistant, cultivars of flax  <cit> , nor in arabidopsis or n. benthamiana plants triggering eti or hr non-host resistance  <cit> .
curiously, p5cdh was not required for salt stress tolerance in arabidopsis  <cit> , suggesting this enzyme would neither accompany prodh under such condition.
the participation of prodh in the pro/p5c cycle may have several implications for hr.
the cycle shuttles reducing power into mitochondria, coupling cytosolic nadph oxidation with metc activity.
under an excess of electrons, it may potentiate mitochondrial ros accumulation, as occurring in human cells over-expressing prodh  <cit> .
the cycle may also alter the cytosolic nadph/nadp+ ratio and therefore affect other redox buffers, such as glutathione and ascorbate, intimately associated to biotic defenses  <cit> .
indeed, cytosolic redox alterations in concert with plasma membrane nadph oxidase define the output of pathogen resistance responses  <cit> .
therefore, as part of the pro/p5c cycle, prodh may potentiate plant immunity by supplying energy or altering the mitochondrial or cytosolic redox homeostasis.
changes in the metabolism of other amino acids may also affect hr development
several studies evaluated the amino acid changes occurring in arabidopsis leaf tissues infected with virulent pst, virulent pseudomonas syringae pv maculicola es <dig>  and other pathogens  <cit> , but none of them described the effects of pst-avrrpm <dig> which were reported in the current work.
reduction of glu and increase of gly, thr, arg, val, ile, lys, leu and phe, his, and gaba , are triggered by virulent pst dc <dig>  the ttss-deficient strain pst dc <dig> hrpa  <cit> , and pst-avrrpm <dig> .
similar changes, excepting those affecting glu, thr and arg, are induced by psm <cit> , suggesting their association with plant basal defenses.
in contrast, tyr accumulation was stimulated by pst-avrrpm <dig> , pst dc <dig>  <cit> , psm <cit> , but not by pst dc <dig> hrpa  <cit> , probably involving eti.
the lys and asp alterations here described  are similar to those accompanying the accumulation of pipecolic acid in psm-infected tissues  <cit> .
this lys derivative functions as inducer of systemic acquired resistance, basal resistance, and resistance to psm-avrrpm <dig>  interestingly, pathways affecting the metabolism of pro, lys, asp, and pipecolic acid have been associated with regulation of plant immunity and pipecolic acid  and pro are proposed to involve similar biosynthetic pathways  <cit> .
one of the most notorious changes involved gaba.
this glu derivative, as pro, is a compatible osmolyte that accumulates under different stress conditions, including microbial infections  <cit> .
gaba is a source of n and c for the pathogen, and an inducer of plant antioxidant enzymes  <cit> .
conversely, accumulation of the gaba derivatives succinic semialdehyde and γ-hydroxybutyrate has been associated to ros increase  <cit> .
in the interaction of arabidopsis with pst dc <dig>  gaba represses the expression of pathogen effectors weakening pathogen virulence  <cit> .
in response to pst-avrrpm <dig>  a strong and transient gaba increase paralleled the oxidative burst .
such increase was more pronounced in the p5cdh mutant  where gaba may help to counteract bacteria with little or no effect on anti-oxidant responses, since this plant reduced pathogen propagation displaying enhanced ros .
interestingly, in infections by agrobacterium tumefaciens, pro antagonizes the gaba-mediated effects on quorum-sensing and pathogen virulence  <cit> , whereas their coordinated effects on other plant-pathogen interactions remain to be studied.
CONCLUSIONS
this is the first study describing the effects derived from prodh activation in plant tissues generating hr.
we report that prodh remains activated during hr progression in arabidopsis leaves treated with pst-avrrpm <dig>  to generate different pro metabolic changes before and after ros burst.
in the first case, prodh contributes to pro consumption by acting with p5cdh .
during maximal ros accumulation, prodh has minor effect on the pro, p5c, glu or asn levels, indicating its functional uncoupling to p5cdh.
interestingly, the p5cdh null mutant retains the capacity to activate prodh in hr, offering a suitable model to study functional uncoupling of prodh and p5cdh in plants.
p5cdh was found dispensable for generation of hr cell death and oxidative burst, but required for normal timing of defense activation.
in addition, p5c accumulation was not involved in hr cell death signaling, at least in wild type plants.
therefore, based on the current knowledge about plant pro metabolic enzymes, our results suggest that the main effect of prodh in hr can derive from its joint action with p5cr.

 proline metabolism is sensitive to environmental cues and undergoes particular alterations that contribute to stress tolerance. plants exposed to dehydration increase pro synthesis and reduce its catabolism, accumulating high levels of the amino acid. pro increase has beneficial effects on plant cells and acts as energy reserve to be used upon stress release. pro metabolism can also affect the cellular redox homeostasis and mobilization of nutrients, which also helps in stress alleviation. plants synthesize pro from ornithine and glutamic acid, involving mostly the second pathway under stress conditions. this process takes place in the cytosol and plastids, involving the action of ∆ pyrroline-5-carboxylate synthase and p5c reductase. the catabolic route occurs at the mitochondria, where pro is oxidized into glu by two enzymes. first, pro dehydrogenase transforms pro into p5c. then, p5c is nonenzymatically converted into glutamate semi-aldehyde that acts as substrate of p5c dehydrogenase for generation of glu. oxidation of pro into glu is a well-conserved process mediated by enzymes that adopt different organizations in nature. gram-negative bacteria combine prodh and p5cdh activities into a single polypeptide, whereas eukaryotes and gram-possitive bacteria contain monofunctional enzymes. prodh catalyzes the rate limiting step in the oxidation pathway, transferring two electrons from pro to the non-covalently bound fad cofactor to subsequently deliver these electrons to a final acceptor. the human prodh has been early recognized as a tumor suppressor protein regulated by p which triggers intrinsic and extrinsic apopototic pathways. the bacterial prodhs have been well characterized at the kinetic and structural levels by crystalographic studies. unlike these enzymes, the plant prodh has been mostly studied at the gene expression level, with little functional and biochemical information and no crystal structure being currently available. this enzyme resides at the inner mitochondrial membrane where it sustains oxidative phosphorylation and atp generation using endogenous electron acceptors that still deserve characterization. several studies described the sensitivity of pro metabolism genes to pathogen infections. in flax, p5cdh is induced by virulent rust-fungi races that overcome host defense barriers. in arabidopsis, p5cs p5cr and prodh, but not p5cdh, are activated by pseudomonas syringae pv. tomato strains eliciting effector triggered immunity and hypersensitive response. under the latter condition, salicylic acid mediates p5cs activation leading to pro increase at late stages of infection. in nicotiana benthamiana, p5cs p5cr, prodh and ornithine δ-amino transferase genes, but not p5cdh, are induced by p. syringae pv. tomato t strain eliciting hr-like lesions. interestingly, prodh and oat were selected by a vigs-based forward genetic screen by looking for genes regulating non-host resistance, and the participation of these enzymes in disease resistance was inferred from the reduction of hr markers in infected tissues of prodh- or oat- silenced plants. in animals, yeast, and bacteria, the prodh activity was associated with the increase in reactive oxygen species, and was suspected to generate either cellular damage or protective functions, apparently depending on the ros levels. whereas prodh potentiates ros and cell death in plant hr-developing tissues, it extends life span by ros-mediated activation of antioxidant enzymes in caenorhabditis elegans. it is unclear how prodh modulates ros levels and is coordinated with other pro metabolizing enzymes under these conditions. its joint action with p5cdh provides energy by charging electrons into the metc for atp synthesis, and produces glu that enters the tricarboxylic acid cycle via α-ketoglutarate. prodh may also operate without p5cdh. this is feasible in yeast, plants and animals containing monofunctional enzymes. in this case, incomplete pro oxidation can led to p5c increase. several studies suggested that p5c accumulation can result harmful, apparently by enhancement of ros levels. interestingly, arabidopsis plants were found sensitive to exogenous p5c, particularly in the absence of p5cdh where it produced hr-like lesions. however, p5c was also informed as innocuous for this plant, raising doubts about the extent and base of its toxicity. more recently, the compound was proposed as an inhibitor of mitochondrial respiration generating lethal superoxide anion levels in yeasts. functional uncoupling of prodh and p5cdh not always leads to p5c increase. alternatively, the p5c produced by prodh can be used by p5cr to regenerate pro. moreover, the joint action of prodh and p5cr can stimulate the pro/p5c cycle. as part of this cycle, the human prodh increases mitochondrial ros and induces apoptosis, with these effects being abolished by over-expression of mitochondrial mnsod. at present, several features of this cycle remain poorly characterized in plants. however, this circuit seems to operate in the arabidopsis p5cdh mutant, and the prodh- and p5cs1- over-expressing plants. as part of this cycle, prodh may not only promote mitochondrial ros generation, but also shuttle reducing power from cytosol to mitochondria producing additional redox changes. finally, prodh as a flavoenzyme, can eventually transfer electrons to o as observed for the monofunctional protein of thermus thermophilus in vitro. as described above, many effects could derive from prodh activation in hr. to start characterizing them, we here analyzed the metabolic context accompanying prodh stimulation at three different hr stages. we induced hr by inoculating pst-avrrpm in arabidopsis leaves and isolated samples before, during and after the oxidative burst for assessing pro catabolism, prodh activity and amino acid content. the studies were performed in wild type plants and the p5cdh mutant, used to evaluate how p5cdh affects the prodh action in hr.  selection of three hr stages for evaluation of prodh action we evaluated prodh action at different hr stages. in particular, before and during the maximum ros accumulation that precedes cell death, and at a late hr phase already manifesting cell death. to select these stages, we used conditions that slow hr development, such as infiltration of a moderate dose of bacteria into adult leaves of short-day grown plants. leaves were excised at hours post-inoculation with pathogen or mock solution, and monitored for ros and cell death, as described in additional file 1a. mock inoculations generated a transient and mild increase in ros before hpi and did not affect cell viability at any analyzed stage. pathogen treatment began to increase ros at hpi to produce maximum ros accumulation at hpi, and trigger cell death since hpi. based on these responses, we selected and hpi for further studies, defining these stages as phases i, ii and iii of hr. pro metabolic enzymes in hr developing tissues total protein extracts from untreated and pst-avrrpm1-treated leaves were analyzed by western blot assays with anti-prodh and anti-p5cdh polyclonal antibodies. a net prodh increase was evidenced at phase i, and maintained until phase iii. unlike prodh, p5cdh only showed slight increases during hr. these protein levels were consistent with the abundance of prodh and p5cdh transcripts observed under the same conditions. since pst-avrrpm is able to induce the p5cs gene expression at advanced hr stages, we monitored its expression in our samples. as observed in figure  and additional file 1b, we did not detect p5cs transcript accumulation early in hr. in turn, expression of p5cr and prodh were found enhanced in hr tissues as reported previously. pro catabolism in hr in vivo we investigated how prodh impacts on pro catabolism before and during ros accumulation. to this purpose, we fed plants with 14c-pro and monitored its consumption in vivo. a pro molecule labelled at all c residues was used to improve detection of the pro derivatives. uninfected and bacterial-infected leaves excised at phases i and ii of hr, were fed with 14c-pro, transferred to water for different time periods, and sampled as outlined in figure 2b. total amino acids were extracted from these samples and analyzed by tlc. consumption of 14c-pro during the water incubation period was evaluated by quantifying the labelled amino acid in this set of samples. healthy tissues had little capacity to catabolize exogenous pro, as they conserved 83% and 77% of the initial 14c-pro after and 60 min of incubation in water, respectively. in contrast, tissues at hr phase i showed a proficient 14c-pro consumption, since they preserved 34% and 26% of the amino acid at or 60 min of incubation, respectively. this increase in pro catabolism paralleled prodh accumulation, suggesting it may result from prodh activation. at phase i, reduction of 14c-pro was not accompanied by appearance of 14c-p5c, 14c-glu or other derivatives. given that the mass of these compounds is directly proportional to their labelling, the 14c-p5c and 14c-glu levels would be substantially lower than those of 14c-pro. therefore, the results suggested that at phase i, prodh acted with p5cdh to generate 14c-glu which was rapidly metabolized. this possibility was further supported by quantification of amino acids. surprisingly, the in vivo14c-pro consumption was not maintained at hr phase ii. at this stage, 90% of the amino acid persisted after 60 min incubation. prodh activity remained enhanced, but had little effect on the pro levels. therefore, during maximun oxidative burst p5cdh did not seem to accompany prodh, suggesting prodh could act with p5cr under this condition. in vitro prodh activity at different hr phases p5cdh prodh activity was analyzed on protein extracts of uninoculated and pst-avrrpm1-inoculated leaves of wild type and p5cdh plants. infected leaves were evaluated at and hours post-infection. enzyme activity was expressed in nmol of pro min-1 mg of total protein- each value is mean ± sd of at least two independent experiments with independent plants. t4c: l -thiazolidine-4-carboxylic acid, inhibitor of prodh. *significant differences respect to uninfected condition of the same genotype. in vitro prodh activity we tested if the lack of 14c-pro consumption at phase ii resulted from a reduction in prodh activity. we quantified this activity by using an in vitro assay that prevents native conditions favoring functional coupling of prodh with other enzymes. to this end, the assay previously used to analyze cultured cell extracts was adapted to evaluate photosynthetic tissue samples. the new system included two modifications. first, the prodh activity was determined by monitoring 3h-pro consumption instead of nad+ reduction. the pro radiolabelled molecule that we selected as substrate contained high specific activity. second, the reaction was developed at neutral instead of alkaline ph, to avoid the reverse p5cr activity, preserving the prodh activity. the new assay, conserved the use of l-thiazolidine-4-carboxylic acid as competitive prodh inhibitor, and resulted suitable to determine prodh activity in arabidopsis leaf extracts. as expected, wild type plants increased the prodh activity at hr phase i. clearly, the enhancement of enzyme activity was preserved at phase ii, and slightly increased at phase iii. therefore, the results suggested that in vivo prodh remained active at all three hr phases. changes in amino acid levels to further evaluate the coordinated action of prodh with p5cdh or p5cr, we quantified the content of pro and its derivatives at each hr stage. alterations in levels of these compounds were indicative of the plant enzyme activities under stress. we included in this study the a. thaliana t-dna insertion line salk_ previously described as a p5cdh knock-out mutant plant. as expected, this plant neither accumulated p5cdh protein nor p5cdh transcripts in leaf or floral tissues. interestingly, the p5cdh infected tissues retained the capacity to activate prodh in response to pst-avrrpm reaching similar activity levels than infected wild type tissues. therefore, this plant resulted useful to detect responses dependent on p5cdh, but not prodh, activity. by hplc assays we quantified amino acids on naive and infected tissues of wild type and the p5cdh plants. at basal conditions, all amino acids had similar levels in both plants, except pro which was increased in the mutant. after bacterial treatment all amino acids, except ser, altered their content in at least one hr phase in one genotype, showing significant differences respect to the uninfected condition. most of them displayed a progressive increase in both plants, which was generally lower in the mutant, suggesting that p5cdh may contribute to accumulation of these amino acids in infected wild type tissues. gaba was included in this group based on its large increase at hr phase ii, which was more pronounced in the mutant. in contrast, glu and asp were reduced by infection in both plants. in turn, p5c, pro and gln exhibited different alterations in both genotypes. p5cdh c:uninfected samples; i, ii, iii: pst-avrrpm1-infected samples isolated at phases i, ii or iii of hr. *significant differences between infected and control samples of the same genotype. #samples analyzed in different experiments containing similar values similar to those of wild type plants. asignificant differences regarding the wild type control value. values are expressed as nmol/g fresh weight. each value is mean ± sd of independent experiments. to analyze in greater depth the behavior of pro, p5c and glu at phases i and ii of hr, we compared their alterations in wild type and mutant plants. glu is a central molecule in plant amino acid metabolism contributing to the synthesis of several of these compounds. its α-amino group can be transferred to oxalic acid to form asp, a precursor of asn and the aspartate family of amino acids. therefore, we included asn in the analysis, as a terminal amino acid indirectly derived from glu. at phase i, wild type plants reduced pro and increased asn without altering p5c or glu. once again, the results suggested that prodh and p5cdh acted together at phase i, generating glu and thus contributing to asn increase. consistently with this possibility, pro consumption was not accompanied by glu accumulation at phase i. moreover, p5cdh plants were unable to reduce pro or increase asn at this stage. at phase ii, wild type tissues recovered their basal pro content and maintained asn at similar levels than phase i, while they preserved basal p5c and glu content. meanwhile, p5cdh plants showed just a modest increase of p5c and asn. as observed in our previous studies, phases i and ii exhibited differences in pro catabolism, with the responses observed in wild type plants at phase ii being consistent with stimulation of the pro/p5c cycle. importantly, wild type plants did not accumulate p5c during the initial hpi with pst-avrrpm. as described, the oxidative burst and cell death were produced during this period. prodh action in the p5cdh mutant we wondered if prodh requires p5cdh to potentiate hr. to evaluate this issue, we monitored hr features in pst-avrrpm1-infected p5cdh leaves. these tissues lacked p5cdh protein and transcripts but accumulated prodh transcripts and protein as infected wild type tissues. as mentioned before, the p5cdh plant also preserved the capacity to activate prodh in response to pst-avrrpm. therefore, this mutant was suitable to test the effect of prodh in hr, in the absence of p5cdh. as in previous studies, we evaluated ros and cell death as hr markers, and susceptibility to avirulent bacteria as parameter of plant resistance. the increase of ros was higher in the mutant, which triggered cell death before wild type plants. in addition, the p5cdh plants showed enhanced pathogen resistance at hpi. however, at later infection stages these differences were no longer observed. therefore, the absence of p5cdh affected the timing, but not strength, of hr producing a premature increase of ros and cell death, and transient enhancement of pathogen resistance. discussion prodh activation in hr we used an in vitro assay to quantify prodh activity in arabidopsis leaf tissues. the methods monitoring pro-dependent nad+ reduction by spectrophotometry were not useful to this end since pigments from photosynthetic tissues interfered with nadh detection. we overcome this limitation by following 3h-pro consumption instead of nad+ reduction. under basal conditions wild type plants consumed  cpm 3h-pro min-1 mg protein- equivalent to 33 nmol 3h-pro min-1 mg protein- this value was in agreement to that reported for the enzyme in liver mitochondrial extracts, where 3h-pro labelled at the c- was used as substrate, and transference of 3h to water was monitored. the increase of prodh activity detected in the infected leaves far exceeded the one observed in extracts of cultured cells challenged with the same pathogen. this difference was probably due to greater responsiveness of leaf tissues to pathogen, and improvement of the assay. strong and persistent prodh activation coexisted with transient pro decrease in infected tissues, indicating that the greater enzyme capacity did not substantially impact on the pro levels. interestingly, these tissues induced prodh without a previous pro increase. something similar was observed for the p5cs plants that even with low pro levels, retained the ability to activate prodh after stress release. moreover, under cold acclimation, prodh induction was accompanied by pro increase. one interesting observation derived from our studies was the maintenance of prodh activation in the absence of p5cdh, which was already noticed on p5cdh plants recovering from drought stress. therefore, prodh activation occuring after stress release would not always be accompanied by p5cdh and could have minor effects on the pro content. although pst-avrrpm induced prodh and p5cs genes at advanced stages of hr, it only activated prodh at the early hr phases. thus, in contrast with rehydration, the hr condition stimulates pro catabolism before pro synthesis. how prodh remains active without de novo pro synthesis under these circumstances is unknown. transport of pro from healthy tissues, stimulation of the pro/p5c cycle, or both responses can take place there. restoration of basal pro without p5cs induction observed at phase ii in wild type plants was consistent with the first possibility. as discussed below, several additional results supported the latter one. p5c levels in the hr lesion the functional uncoupling of prodh and p5cdh may generate p5c accumulation with toxic effects for plants. in yeast, the p5c increase from to 300 μm results harmful for stressed cells. our previous finding that pst-avrrpm activated prodh but not p5cdh, suggested that p5c could accumulate and signal hr cell death. in previous studies, we did not detect p5c increase by spectrophotometric assays in wild type tissues developing hr. in the current work, we used hplc to unequivocally determine the p5c content in hr. in addition, we analyzed p5cdh infected tissues that are prone to accumulate p5c due to prodh activation. we detected a transient increase of p5c at hr phase ii in p5cdh plants that paralleled cell death initiation. how p5c contributes to hr cell death in p5cdh plants is unknown. however, it is clear that p5c does not signal ros generation or cell death initiation in the hr lesion of wild type plants, since its levels remain almost constant at all phases of this response. pro catabolism at phases i and ii of hr in vitro assays evidenced similar prodh activation at all hr stages. at difference, in vivo studies, and amino acid quantification, showed variations in pro catabolism at phases i and ii. at phase i, pro consumption was accompanied by increase of the terminal glu-derivative asn, suggesting that basal pro was transformed into glu. at phase ii, pro did not continue to decline, and asn was not further increased. the alteration of pro catabolism manifested in phase ii may have different causes. one possibility is that enzyme and substrate have different partitioning. this is, however, unlikely since mitochondrial pro depletion has not been reported to date. alternatively, plant- or bacterial-derived compounds may inhibit prodh at phase ii. nevertheless, the existence of such products has not been reported. p5c, which may eventually lead to product inhibition, did not increase under this condition in wild type plants. furthermore, since prodh preserved its catalytic capacity in vitro until phase iii irreversible enzyme inhibition can be discarded. another possibility is that prodh acts with p5cr recycling pro and p5c. recent studies propose that the pro/p5c cycle operates in arabidopsis tissues exposed to abiotic stress, and probably contributes to exogenous pro toxicity. the results that suggest stimulation of this cycle at hr phase ii are: i) co-existence of prodh activation, and unaltered pro and p5c levels; ii) absence of p5cs induction at the time of prodh activation; iii) premature accumulation of ros in p5cdh infected tissues able to induce prodh. moreover, the joint activation of prodh and p5cr genes here observed was consistent with previous results and also supported this possibility. therefore, although the pro/p5c cycle requires formal demonstration in plants, the current knowledge about pro metabolism and our results are consistent with stimulation of this cycle in hr. prodh action at hr phase ii arabidopsis transgenic plants that reduced, but not eliminated, prodh expression attenuated the oxidative burst, hypersensitive cell death, and resistance to pst-avrrpm. in contrast, the null p5cdh mutant did not reduce these responses. this suggests that prodh does not require p5cdh to potentiate those hr features. consistently, prodh had no effect on pro consumption at the time of maximum ros accumulation. moreover, p5cdh may even prevent or reduce the capacity of prodh to produce ros and associated defenses. in this sense, infected p5cdh plants prematurely stimulated ros burst and cell death. moreover, induction of p5cdh was observed in diseased, but not resistant, cultivars of flax, nor in arabidopsis or n. benthamiana plants triggering eti or hr non-host resistance. curiously, p5cdh was not required for salt stress tolerance in arabidopsis, suggesting this enzyme would neither accompany prodh under such condition. the participation of prodh in the pro/p5c cycle may have several implications for hr. the cycle shuttles reducing power into mitochondria, coupling cytosolic nadph oxidation with metc activity. under an excess of electrons, it may potentiate mitochondrial ros accumulation, as occurring in human cells over-expressing prodh. the cycle may also alter the cytosolic nadph/nadp+ ratio and therefore affect other redox buffers, such as glutathione and ascorbate, intimately associated to biotic defenses. indeed, cytosolic redox alterations in concert with plasma membrane nadph oxidase define the output of pathogen resistance responses. therefore, as part of the pro/p5c cycle, prodh may potentiate plant immunity by supplying energy or altering the mitochondrial or cytosolic redox homeostasis. changes in the metabolism of other amino acids may also affect hr development several studies evaluated the amino acid changes occurring in arabidopsis leaf tissues infected with virulent pst, virulent pseudomonas syringae pv maculicola es and other pathogens, but none of them described the effects of pst-avrrpm which were reported in the current work. reduction of glu and increase of gly, thr, arg, val, ile, lys, leu and phe, his, and gaba, are triggered by virulent pst dc the ttss-deficient strain pst dc hrpa, and pst-avrrpm. similar changes, excepting those affecting glu, thr and arg, are induced by psm, suggesting their association with plant basal defenses. in contrast, tyr accumulation was stimulated by pst-avrrpm, pst dc, psm, but not by pst dc hrpa, probably involving eti. the lys and asp alterations here described are similar to those accompanying the accumulation of pipecolic acid in psm-infected tissues. this lys derivative functions as inducer of systemic acquired resistance, basal resistance, and resistance to psm-avrrpm interestingly, pathways affecting the metabolism of pro, lys, asp, and pipecolic acid have been associated with regulation of plant immunity and pipecolic acid and pro are proposed to involve similar biosynthetic pathways. one of the most notorious changes involved gaba. this glu derivative, as pro, is a compatible osmolyte that accumulates under different stress conditions, including microbial infections. gaba is a source of n and c for the pathogen, and an inducer of plant antioxidant enzymes. conversely, accumulation of the gaba derivatives succinic semialdehyde and γ-hydroxybutyrate has been associated to ros increase. in the interaction of arabidopsis with pst dc gaba represses the expression of pathogen effectors weakening pathogen virulence. in response to pst-avrrpm a strong and transient gaba increase paralleled the oxidative burst. such increase was more pronounced in the p5cdh mutant where gaba may help to counteract bacteria with little or no effect on anti-oxidant responses, since this plant reduced pathogen propagation displaying enhanced ros. interestingly, in infections by agrobacterium tumefaciens, pro antagonizes the gaba-mediated effects on quorum-sensing and pathogen virulence, whereas their coordinated effects on other plant-pathogen interactions remain to be studied.  this is the first study describing the effects derived from prodh activation in plant tissues generating hr. we report that prodh remains activated during hr progression in arabidopsis leaves treated with pst-avrrpm to generate different pro metabolic changes before and after ros burst. in the first case, prodh contributes to pro consumption by acting with p5cdh. during maximal ros accumulation, prodh has minor effect on the pro, p5c, glu or asn levels, indicating its functional uncoupling to p5cdh. interestingly, the p5cdh null mutant retains the capacity to activate prodh in hr, offering a suitable model to study functional uncoupling of prodh and p5cdh in plants. p5cdh was found dispensable for generation of hr cell death and oxidative burst, but required for normal timing of defense activation. in addition, p5c accumulation was not involved in hr cell death signaling, at least in wild type plants. therefore, based on the current knowledge about plant pro metabolic enzymes, our results suggest that the main effect of prodh in hr can derive from its joint action with p5cr. 
they did not consume pro during maximal ros accumulation, and maintained almost basal p5c levels at all conditions. control and infected leaves of wild type and p5cdh plants were used to monitor prodh activity, in vivo pro catabolism, amino acid content, and gene expression. in addition, we tested if prodh requires p5cdh to potentiate hr. prodh converts pro into ∆ pyrroline-5-carboxylate and can act together with p5c dehydrogenase to produce glu, or with p5c reductase to regenerate pro and thus stimulate the pro/p5c cycle. in turn, our results suggest that prodh may sustain hr by participating in the pro/p5c cycle, whose action on hr must be formally evaluated in a future. proline dehydrogenase potentiates the oxidative burst and cell death of the plant hypersensitive response by mechanisms not yet elucidated. to better understand the effects of prodh in hr, we studied the enzyme at three stages of the defense response differing in their ros and cell death levels. they achieved maximum oxidative burst and cell death levels producing normal hr lesions, but evidenced premature defense activation. the absence of p5cdh does not reduce ros, cell death, or pathogen resistance, indicating this enzyme is not accompanying prodh in the potentiation of these defense responses. in contrast, p5cdh infected plants displayed increased ros burst and earlier initiation of hr cell death. proline metabolismproline dehydrogenase/oxidasep5cstress responsesarabidopsispseudomonas syringaehypersensitive responsecell deathreactive oxygen species before the oxidative burst it leads to pro consumption involving the action of p5cdh. wild type plants activated prodh at all hr stages. p5cdh mutants activated prodh as wild type plants. prodh activation has different effects on hr.
 proline dehydrogenase potentiates the oxidative burst and cell death of the plant hypersensitive response by mechanisms not yet elucidated. prodh converts pro into ∆ pyrroline-5-carboxylate and can act together with p5c dehydrogenase to produce glu, or with p5c reductase to regenerate pro and thus stimulate the pro/p5c cycle. to better understand the effects of prodh in hr, we studied the enzyme at three stages of the defense response differing in their ros and cell death levels. in addition, we tested if prodh requires p5cdh to potentiate hr.  control and infected leaves of wild type and p5cdh plants were used to monitor prodh activity, in vivo pro catabolism, amino acid content, and gene expression. wild type plants activated prodh at all hr stages. they did not consume pro during maximal ros accumulation, and maintained almost basal p5c levels at all conditions. p5cdh mutants activated prodh as wild type plants. they achieved maximum oxidative burst and cell death levels producing normal hr lesions, but evidenced premature defense activation.  prodh activation has different effects on hr. before the oxidative burst it leads to pro consumption involving the action of p5cdh. during the oxidative burst, prodh becomes functionally uncoupled to p5cdh and apparently works with p5cr. the absence of p5cdh does not reduce ros, cell death, or pathogen resistance, indicating this enzyme is not accompanying prodh in the potentiation of these defense responses. in contrast, p5cdh infected plants displayed increased ros burst and earlier initiation of hr cell death. in turn, our results suggest that prodh may sustain hr by participating in the pro/p5c cycle, whose action on hr must be formally evaluated in a future. proline metabolismproline dehydrogenase/oxidasep5cstress responsesarabidopsispseudomonas syringaehypersensitive responsecell deathreactive oxygen species 