Inhibitory effect of arginine on proline utilization in Saccharomyces cerevisiae.

Proline is a predominant amino acid in grape must, but it is poorly utilized by the yeast Saccharomyces cerevisiae in wine-making processes. This sometimes leads to a nitrogen deficiency during fermentation and proline accumulation in wine. Although the presence of other nitrogen sources under fermentation conditions is likely to interfere with proline utilization, the inhibitory mechanisms of proline utilization remain unclear. In this study, we examined the effect of arginine on proline utilization in S. cerevisiae. We first constructed a proline auxotrophic yeast strain and identified an inhibitory factor by observing the growth of cells when proline was present as a sole nitrogen source. Intriguingly, we found that arginine, and not ammonium ion, clearly inhibited the growth of proline auxotrophic cells. In addition, arginine prevented the proline consumption of wild-type and proline auxotrophic cells, indicating that arginine is an inhibitory factor of proline utilization in yeast. Next, quantitative polymerase chain reaction (PCR) analysis showed that arginine partially repressed the expression of genes involved in proline degradation and uptake. We then observed that arginine induced the endocytosis of the proline transporters Put4 and Gap1, whereas ammonium induced the endocytosis of only Gap1. Hence, our results may involve an important mechanism for arginine-mediated inhibition of proline utilization in yeast. The breeding of yeast that utilizes proline efficiently could be promising for the improvement of wine quality.

Design and Synthesis of a Series of l-trans-4-Substituted Prolines as Selective Antagonists for the Ionotropic Glutamate Receptors Including Functional and X-ray Crystallographic Studies of New Subtype Selective Kainic Acid Receptor Subtype 1 (GluK1) Antagonist (2S,4R)-4-(2-Carboxyphenoxy)pyrrolidine-2-carboxylic Acid.

Ionotropic glutamate receptor antagonists are valuable tool compounds for studies of neurological pathways in the central nervous system. On the basis of rational ligand design, a new class of selective antagonists, represented by (2S,4R)-4-(2-carboxyphenoxy)pyrrolidine-2-carboxylic acid (1b), for cloned homomeric kainic acid receptors subtype 1 (GluK1) was attained (Ki = 4 µM). In a functional assay, 1b displayed full antagonist activity with IC50 = 6 ± 2 µM. A crystal structure was obtained of 1b when bound in the ligand binding domain of GluK1. A domain opening of 13-14° was seen compared to the structure with glutamate, consistent with 1b being an antagonist. A structure-activity relationship study showed that the chemical nature of the tethering atom (C, O, or S) linking the pyrrolidine ring and the phenyl ring plays a key role in the receptor selectivity profile and that substituents on the phenyl ring are well accommodated by the GluK1 receptor.

Conformational changes in human prolyl-tRNA synthetase upon binding of the substrates proline and ATP and the inhibitor halofuginone.

Aminoacyl-tRNA synthetases recognize cognate amino acids and tRNAs from their noncognate counterparts and catalyze the formation of aminoacyl-tRNAs. Halofuginone (HF), a coccidiostat used in veterinary medicine, exerts its effects by acting as a high-affinity inhibitor of the enzyme glutamyl-prolyl-tRNA synthetase (EPRS). In order to elucidate the precise molecular basis of this inhibition mechanism of human EPRS, the crystal structures of the prolyl-tRNA synthetase domain of human EPRS (hPRS) at 2.4 Šresolution (hPRS-apo), of hPRS complexed with ATP and the substrate proline at 2.3 Šresolution (hPRS-sub) and of hPRS complexed with HF at 2.62 Šresolution (hPRS-HF) are presented. These structures show plainly that motif 1 functions as a cap in hPRS, which is loosely opened in hPRS-apo, tightly closed in hPRS-sub and incorrectly closed in hPRS-HF. In addition, the structural analyses are consistent with more effective binding of hPRS to HF with ATP. Mutagenesis and biochemical analysis confirmed the key roles of two residues, Phe1097 and Arg1152, in the HF inhibition mechanism. These structures will lead to the development of more potent and selective hPRS inhibitors for promoting inflammatory resolution.

Proline-induced changes in acetylcholinesterase activity and gene expression in zebrafish brain: reversal by antipsychotic drugs.

Hyperprolinemia is an inherited disorder of proline metabolism and hyperprolinemic patients can present neurological manifestations, such as seizures, cognitive dysfunctions, and schizoaffective disorders. However, the mechanisms related to these symptoms are still unclear. In the present study, we evaluated the in vivo and in vitro effects of proline on acetylcholinesterase (AChE) activity and gene expression in the zebrafish brain. For the in vivo studies, animals were exposed at two proline concentrations (1.5 and 3.0mM) during 1h or 7 days (short- or long-term treatments, respectively). For the in vitro assays, different proline concentrations (ranging from 3.0 to 1000 µM) were tested. Long-term proline exposures significantly increased AChE activity for both treated groups when compared to the control (34% and 39%). Moreover, the proline-induced increase on AChE activity was completely reverted by acute administration of antipsychotic drugs (haloperidol and sulpiride), as well as the changes induced in ache expression. When assessed in vitro, proline did not promote significant changes in AChE activity. Altogether, these data indicate that the enzyme responsible for the control of acetylcholine levels might be altered after proline exposure in the adult zebrafish. These findings contribute for better understanding of the pathophysiology of hyperprolinemia and might reinforce the use of the zebrafish as a complementary vertebrate model for studying inborn errors of amino acid metabolism.

Synthesis and evaluation of effective inhibitors of plant δ1-pyrroline-5-carboxylate reductase.

Analogues of previously studied phenyl-substituted aminomethylene-bisphosphonic acids were synthesized and evaluated as inhibitors of Arabidopsis thaliana δ(1)-pyrroline-5-carboxylate reductase. With the aim of improving their effectiveness, two main modifications were introduced into the inhibitory scaffold: the aminomethylenebisphosphonic moiety was replaced with a hydroxymethylenebisphosphonic group, and the length of the molecule was increased by replacing the methylene linker with an ethylidene chain. In addition, chlorine atoms in the phenyl ring were replaced with various other substituents. Most of the studied derivatives showed activity in the micromolar to millimolar range, with two of them being more effective than the lead compound, with concentrations inhibiting 50% of enzyme activity as low as 50 µM. Experimental evidence supporting the ability of these inhibitors to interfere with proline synthesis in vivo is also shown.

Impact of the proline residue on ligand binding of neurotensin receptor†2 (NTS2)-selective peptide-peptoid hybrids.

To investigate the binding mode and structure-activity relationships (SARs) of selective neurotensin receptor 2 (NTS2) ligands, novel peptide-peptoid hybrids that simulate the function of the endogenous ligand were developed. Starting from our recently described NTS2 ligands of type 1, structural variants of type 2 and the metabolically stable analogues 3 a,b were developed. Replacement of the proline unit by a collection of structural surrogates and evaluation of the respective molecular probes for NTS2 affinity and selectivity indicated similar SARs as described for NT(8-13) derivatives bound to the subtype NTS1. Peptide-peptoid hybrids 2 d, 3 a,b showed substantial NTS2 binding affinity (Ki =8.1-16 nM) and 2400-8600-fold selectivity over NTS1. The thiazolidine derivative 3 b showed metabolic stability over 32 h in a serum degradation assay. In an inositol phosphate accumulation assay, the neurotensin mimetics 3 a and 3 b displayed an inhibition of constitutive activity exceeding 1.7-2.0 times the activity of NT(8-13). The fluorinated derivative 3 a could afford attractive opportunities to detect NTS2 by (19) F magnetic resonance imaging.

Behavioral changes induced by long-term proline exposure are reversed by antipsychotics in zebrafish.

Hyperprolinemia is an inherited disorder of proline metabolism and patients affected by this disease may present neurological manifestations, including seizures and cognitive dysfunctions. Moreover, an association between adulthood schizoaffective disorders and moderate hyperprolinemia has been reported. However, the mechanisms underlying these behavioral phenotypes still remain unclear. In the present study, we investigated the effect of proline treatments on behavioral parameters in zebrafish, such as locomotor activity, anxiety, and social interaction. Adult zebrafish (Danio rerio) were exposed to proline (1.5 and 3.0 mM) during 1h or 7 days (short- or long-term treatments, respectively). Short-term proline exposure did not promote significant changes on the behavioral parameters observed. Long-term exposure at 1.5 mM proline significantly increased the number of line crossing (47%), the total distance (29%), and the mean speed (33%) when compared to control group. A significant increase in the time spent in the upper portion of the test tank was also observed after this treatment (91%), which may be interpreted as an indicator of anxiolytic behavior. Proline at 1.5 mM also induced social interaction impairment (78%), when compared to the untreated group after long-term treatment. Moreover, these proline-induced behavioral changes in zebrafish were completely reversed by acute administration of an atypical antipsychotic drug (sulpiride), but not by a typical (haloperidol). These findings demonstrate that proline is able to induce schizophrenia-like symptoms in zebrafish, which reinforce the use of this species as a complementary vertebrate model for studying behavioral phenotypes associated with neurological dysfunctions characteristic of metabolic diseases.

A helix-breaking mutation in the epithelial Ca(2+) channel TRPV5 leads to reduced Ca(2+)-dependent inactivation.

TRPV5, a member of transient receptor potential (TRP) superfamily of ion channels, plays a crucial role in epithelial calcium transport in the kidney. This channel has a high selectivity for Ca(2+) and is tightly regulated by intracellular Ca(2+) concentrations. Recently it was shown that the molecular basis of deafness in varitint-waddler mouse is the result of hair cell death caused by the constitutive activity of transient receptor potential mucolipin 3 (TRPML3) channel carrying a helix breaking mutation, A419P, at the intracellular proximity of the fifth transmembrane domain (TM5). This mutation significantly elevates intracellular Ca(2+) concentration and causes rapid cell death. Here we show that substituting the equivalent location in TRPV5, the M490, to proline significantly modulates Ca(2+)-dependent inactivation of TRPV5. The single channel conductance, time constant of inactivation (τ) and half maximal inhibition constant (IC(50)) of TRPV5(M490P) were increased compared to TRPV5(WT). Moreover TRPV5(M490P) showed lower Ca(2+) permeability. Out of different point mutations created to characterize the importance of M490 in Ca(2+)-dependent inactivation, only TRPV5(M490P)-expressing cells showed apoptosis and extremely altered Ca(2+)-dependent inactivation. In conclusion, the TRPV5 channel is susceptible for helix breaking mutations and the proximal intracellular region of TM5 of this channel plays an important role in Ca(2+)-dependent inactivation.

Enhancement of the citrulline–nitric oxide cycle inastroglioma cells by the proline-rich peptide-10c from Bothrops jararaca venom

The biological activity of the proline-rich decapeptide Bj-PRO-10c, a processing product of the C-type natriuretic peptide precursor protein, expressed in the brain and the venom gland of the pit viper Bothrops jararaca, was originally attributed to the inhibition of the somatic angiotensin-converting enzyme activity with subsequent anti-hypertensive effect. However, recent results suggest broader biological activity may also be involved in the cardiovascular effects of this peptide. Here we show that Bj-PRO-10c enhances and sustains the generation of nitric oxide (NO) by regulating argininosuccinate synthase activity and thereby velocity of the citrulline–NO cycle. Bj-PRO-10c-mediated effects not restricted to thecardiovascular system, since NO production was also induced in cells of astroglial origin. Bj-PRO-10c was internalized by C6 astroglioma cells where it induces NO production and upregulation of the citrulline–NO cycle cells in a dose-dependent fashion. In view of that, astroglial cells function as L-arginine pool for NO production in neighboring neurons, we suggest a regulatory function for Bj-PRO-10c on the metabolism of this gaseous neurotransmitter in the CNS. Moreover, proliferation of astroglial cells was reduced in the presence of Bj-PRO-10c; however, cell death was not induced. Since NO donors have been studied for the treatment of solid cancers, Bj-PRO-10c may serve as structural model fordeveloping drugs to improve the effects of cancer therapy based on the peptide's ability to augment NO production.

The Central Nervous System as Target forAntihypertensive Actions of a Proline-Rich Peptide from Bothrops jararaca Venom

Pyroglutamyl proline-rich oligopeptides, present in the venom of the pit viper Bothrops jararaca (Bj-PROs), are the first described naturally occurring inhibitors of the angiotensin I-converting enzyme (ACE). The inhibition of ACE by the decapeptide Bj-PRO- 10c (

L-arginine-threonine-arginine (RTR) tetramer peptide inhibits ulceration in the alkali-injured rabbit cornea.

PURPOSE: Proline-glycine-proline (PGP) peptides have been identified as inflammatory mediators initiating neutrophil invasion into alkali-injured cornea. The complementary peptide, arginine-threonine-arginine (RTR), has been shown to bind to the PGP sequence and impede neutrophil infiltration. A prior study showed that L-RTR tetramer and D-RTR tetramer, used alternately (14 times a day), resulted in significantly reduced incidences of corneal ulceration and severity. The purpose of this experiment is to determine the effectiveness of both tetramers, used separately, compared with control. METHODS: Rabbit corneas were exposed to 1 N NaOH for 35 seconds. Sixteen animals were randomly assigned to each of 3 groups: 1) phosphate-buffered saline (PBS), 2) 1.5 mM L-RTR, or 3) 800 microM D-RTR. One drop of each was administered hourly (14 times a day) for 36 days. Additional studies were done to assess neutrophil infiltration into corneas with and without RTR treatment. RESULTS: The severity of corneal ulceration in both RTR groups was statistically significantly different from the 21st day of the experiment to the end. As a result of ulcers healing in the L-RTR group, there was a statistically significant reduction in the number of ulcers beginning on day 22 versus control. Although there was healing in the D-RTR group, the incidence of ulcers was not significantly different from control or L-RTR. Morphometric analysis revealed decreased neutrophil (PMN) invasion with RTR treatment compared with PBS control. CONCLUSIONS: Binding of the PGP molecules by RTR tetramer seems to deprive the cornea of this neutrophilic chemotactic stimulus, leading to a reduction in the severity and incidence of corneal ulceration.

Inhibition of protein aggregation in vitro and in vivo by a natural osmoprotectant.

Small organic molecules termed osmolytes are harnessed by a variety of cell types in a wide range of organisms to counter unfavorable physiological conditions that challenge protein stability and function. Using a well characterized reporter system that we developed to allow in vivo observations, we have explored how the osmolyte proline influences the stability and aggregation of a model aggregation-prone protein, P39A cellular retinoic acid-binding protein. Strikingly, we find that the natural osmolyte proline abrogates aggregation both in vitro and in vivo (in an Escherichia coli expression system). Importantly, proline also prevented aggregation of constructs containing exon 1 of huntingtin with extended polyglutamine tracts. Although compatible osmolytes are known to stabilize the native state, our results point to a destabilizing effect of proline on partially folded states and early aggregates and a solubilizing effect on the native state. Because proline is believed to act through a combination of solvophobic backbone interactions and favorable side-chain interactions that are not specific to a particular sequence or structure, the observed effect is likely to be general. Thus, the osmolyte proline may be protective against biomedically important protein aggregates that are hallmarks of several late-onset neurodegenerative diseases including Huntington's, Alzheimer's, and Parkinson's. In addition, these results should be of practical importance because they may enable protein expression at higher efficiency under conditions where aggregation competes with proper folding.

Dissection of Escherichia coli glutamate 5-kinase: functional impact of the deletion of the PUA domain.

Glutamate 5-kinase (G5K) catalyzes the controlling first step of the synthesis of the osmoprotective amino acid proline, which feed-back inhibits G5K. Microbial G5K generally consists of one amino acid kinase (AAK) and one PUA (named after pseudo uridine synthases and archaeosine-specific transglycosylases) domain. To investigate the role of the PUA domain, we have deleted it from Escherichia coli G5K. We show that wild-type G5K requires free Mg for activity, it is tetrameric, and it aggregates to higher forms in a proline-dependent way. G5K lacking the PUA domain remains tetrameric, active, and proline-inhibitable, but the Mg requirement and the proline-triggered aggregation are greatly diminished and abolished, respectively, and more proline is needed for inhibition. We propose that the PUA domain modulates the function of the AAK domain, opening the way to potential PUA domain-mediated regulation of G5K; and that this domain moves, exposing new surfaces upon proline binding.

Proline reduces acetylcholinesterase activity in cerebral cortex of rats.

In the present study we investigated the in vivo and in vitro effect of proline (Pro) on acetylcholinesterase (AChE) activity in rat cerebral cortex. The action of vitamins E and C on the effects produced by Pro was also tested. Twelve-day-old rats received one s.c. injection of Pro (12.8 micromol/g body weight) or an equivalent volume of 0.9% saline solution (control) and were killed 1 h later. In another set of experiments, 5-day-old rats were pretreated for 1 week with daily i.p. administration of saline (control) or vitamins E (40 mg/kg) and C (100 mg/kg). Twelve hours after the last injection the rats received one s.c. injection of Pro (12.8 micromol/g body weight) or saline (control) and were killed 1 h later. For the in vitro studies, cerebral cortex homogenates of 12-day-old untreated rats were incubated for 1 h with various concentrations of Pro (3.0 microM-1.0 mM) or with 1.0 mM Pro, 1.0 mM trolox, or 1.0 mM Pro plus 1.0 mM trolox. Controls did not contain Pro in the incubation medium. Our results showed that the AChE activity significantly decreased (25%) in rat brain subjected to Pro administration and that the pretreatment with vitamins E and C prevented this effect. Furthermore, Pro (0.5 and 1.0 mM) also inhibits AChE activity in vitro and trolox prevented this effect. The data suggest that the inhibitory effect of Pro on AChE activity is associated with oxidative stress. Although it is difficult to extrapolate our findings to the human condition, our results may be relevant to explain, at least in part, the neurologic dysfunction associated with hyperprolinemia type II.

Inhibitory effect of a complementary peptide on ulceration in the alkali-injured rabbit cornea.

PURPOSE: Two tripeptide chemoattractants, acetyl-proline-glycine-proline (Ac-PGP) and methyl-proline-glycine-proline (Me-PGP), are the primary triggers for early neutrophil invasion into the alkali-injured cornea. In the present study the effectiveness of a complementary peptide designed to inhibit the PGP chemoattractants (arginine-threonine-arginine [RTR] tetrameric peptide) and an apo A-1 mimicking peptide (5F) was investigated in the alkali-injured rabbit eye. METHODS: (L)-RTR tetramer, (D)-RTR tetramer, and 5F were tested in vitro for their effects on neutrophil polarization. Synthetic 5F was also tested in vitro for its effect on the neutrophil respiratory burst. In the alkali-injured rabbit eye model, the right corneas of 48 rabbits were exposed to 1 N NaOH for 35 seconds. Sixteen animals were randomly assigned to each of three groups: phosphate-buffered saline (PBS) control; 800 microM RTR (dextrorotatory) tetramer in PBS alternating each hour with 1.5 mM RTR (levorotatory) tetramer in PBS; and 12 microM 5F in PBS. One topical drop of each substance was administered hourly (14 times per day) for 33 days. The experiment was continued until day 42 with no additional drops administered. RESULTS: (L)-RTR tetramer and (D)-RTR tetramer inhibited neutrophil polarization activated by the PGP chemoattractants in vitro. Synthetic 5F did not inhibit neutrophil polarization in the presence of Ac-PGP or the respiratory burst of neutrophils in the presence of a metabolic stimulant derived from alkali-degraded corneas. During the entire animal experiment, statistically fewer ulcers occurred in the RTR tetramer group than in the PBS control group (43.8% vs. 87.5%, P = 0.0046). The frequency of ulceration in the 5F group (68.8%) was not significantly different from the PBS control group. CONCLUSIONS: The reduction in the frequency of corneal ulceration by the RTR tetramer possibly resulted from its complementary binding to Ac-PGP and Me-PGP in the cornea shortly after alkali injury, leading to a reduction in the early and late infiltration of neutrophils. RTR tetramer appears to hold enough promise to warrant additional study as a therapeutic drug for the alkali-injured eye.

A nuclear gene encoding mitochondrial Delta-pyrroline-5-carboxylate dehydrogenase and its potential role in protection from proline toxicity.

Delta1-pyrroline-5-carboxylate (P5C), an intermediate in biosynthesis and degradation of proline (Pro), is assumed to play a role in cell death in plants and animals. Toxicity of external Pro and P5C supply to Arabidopsis suggested that P5C dehydrogenase (P5CDH; EC 1.2.1.12) plays a crucial role in this process by degrading the toxic Pro catabolism intermediate P5C. Also in a Deltaput2 yeast mutant, lacking P5CDH, Pro led to growth inhibition and formation of reactive oxygen species (ROS). Complementation of the Deltaput2 mutant allowed identification of the Arabidopsis P5CDH gene. AtP5CDH is a single-copy gene and the encoded protein was localized to the mitochondria. High homology of AtP5CDH to LuFIS1, an mRNA up-regulated during susceptible pathogen attack in flax, suggested a role for P5CDH in inhibition of hypersensitive reactions. An Arabidopsis mutant (cpr5) displaying a constitutive pathogen response was found to be hypersensitive to external Pro. In agreement with a role in prevention of cell death, AtP5CDH was expressed at a basal level in all tissues analysed. The highest expression was found in flowers that are known to contain the highest Pro levels under normal conditions. External supply of Pro induced AtP5CDH expression, but much more slowly than Pro dehydrogenase (AtProDH) expression. Uncoupled induction of the AtProDH and AtP5CDH genes further supports the hypothesis that P5C levels have to be tightly controlled. These results indicate that, in addition to the well-studied functions of Pro, for example in osmoregulation, the Pro metabolism intermediate P5C also serves as a regulator of cellular stress responses.

Synthetic complementary peptides inhibit a neutrophil chemoattractant found in the alkali-injured cornea.

PURPOSE: We have previously presented evidence that the neutrophil chemoattractant, N-acetyl-proline-glycine-proline (N-acetyl-PGP), triggers the initial polymorphonuclear leukocyte (PMN) invasion into the alkali-injured eye. In this study, sense-antisense methodology was used to develop novel complementary peptides to be potential inhibitors of N-acetyl-PGP. METHODS: The polarization assay was used to measure the potential chemotactic response of PMNs to synthetic N-acetyl-PGP, the ultrafiltered tripeptide chemoattractants obtained from alkali-degraded rabbit corneas, or leukotriene B4 (LTB4). Inhibition was expressed as the peptide concentration producing 50% inhibition (ID50) of polarization. Five complementary peptides were tested as potential inhibitors of N-acetyl-PGP: arginine-threonine-arginine (RTR), RTR-glycine-glycine (RTRGG), RTR dimer, RTR tetramer, and alanine-serine-alanine (ASA) tetramer. In addition, the RTR tetramer and both monomeric peptides (RTR and RTRGG) were separately tested for inhibition of the ultrafiltered tripeptide chemoattractants or LTB4. RESULTS: The complementary RTR tetrameric peptide was a powerful antagonist of N-acetyl-PGP-induced PMN polarization (ID50 of 200 nM). The RTR dimer was much less potent (ID50 of 105 microM). Both monomeric peptides, RTR and RTRGG, were only antagonistic at millimolar concentrations. The ASA tetramer showed no capacity to inhibit N-acetyl-PGP. The RTR tetramer also inhibited PMN activation by the ultrafiltered tripeptide chemoattractants (ID50 of 30 microM) but had no effect on LTB4. CONCLUSIONS: A complementary peptide (RTR) was designed which is an effective inhibitor of the neutrophil chemoattractant, N-acetyl-PGP. The potency of the RTR complementary peptide is dramatically enhanced by tetramerization. Inhibition of N-acetyl-PGP by complementary peptides offers great promise for control of the inflammatory response in the alkali-injured eye.

Hypersensitivity of an Arabidopsis sugar signaling mutant toward exogenous proline application.

In transgenic Arabidopsis a patatin class I promoter from potato is regulated by sugars and proline (Pro), thus integrating signals derived from carbon and nitrogen metabolism. In both cases a signaling cascade involving protein phosphatases is involved in induction. Other endogenous genes are also regulated by both Pro and carbohydrates. Chalcone synthase (CHS) gene expression is induced by both, whereas the Pro biosynthetic Delta(1)-pyrroline-5-carboxylate synthetase (P5CS) is induced by high Suc concentrations but repressed by Pro, and Pro dehydrogenase (ProDH) is inversely regulated. The mutant rsr1-1, impaired in sugar dependent induction of the patatin promoter, is hypersensitive to low levels of external Pro and develops autofluorescence and necroses. Toxicity of Pro can be ameliorated by salt stress and exogenously supplied metabolizable carbohydrates. The rsr1-1 mutant shows a reduced response regarding sugar induction of CHS and P5CS expression. ProDH expression is de-repressed in the mutant but still down-regulated by sugar. Pro toxicity seems to be mediated by the degradation intermediate Delta(1)-pyrroline-5-carboxylate. Induction of the patatin promoter by carbohydrates and Pro, together with the Pro hypersensitivity of the mutant rsr1-1, demonstrate a new link between carbon/nitrogen and stress responses.