Cytosolic proline is required for basal freezing tolerance in Arabidopsis.

The amino acid proline accumulates in many plant species under abiotic stress conditions, and various protective functions have been proposed. During cold stress, however, proline content in Arabidopsis thaliana does not correlate with freezing tolerance. Freezing sensitivity of a starchless plastidic phosphoglucomutase mutant (pgm) indicated that localization of proline in the cytosol might stabilize the plasma membrane during freeze-thaw events. Here, we show that re-allocation of proline from cytosol to vacuole was similar in the pyrroline-5-carboxylate synthase 2-1 (p5cs2-1) mutant and the pgm mutant and caused similar reduction of basal freezing tolerance. In contrast, the starch excess 1-1 mutant (sex1-1) had even lower freezing tolerance than pgm but did not affect sub-cellular localization of proline. Freezing sensitivity of sex1-1 mutants affected primarily the photosynthetic electron transport and was enhanced in a sex1-1::p5cs2-1 double mutant. These findings indicate that several independent factors determine basal freezing tolerance. In a pgm::p5cs2-1 double mutant, freezing sensitivity and proline allocation to the vacuole were the same as in the parental lines, indicating that the lack of cytosolic proline was the common cause of reduced basal freezing tolerance in both mutants. We conclude that cytosolic proline is an important factor in freezing tolerance of non-acclimated plants.

Probing the function of a ligand-modulated dynamic tunnel in bifunctional proline utilization A (PutA).

In many bacteria, the reactions of proline catabolism are catalyzed by the bifunctional enzyme known as proline utilization A (PutA). PutA catalyzes the two-step oxidation of l-proline to l-glutamate using distinct proline dehydrogenase (PRODH) and l-glutamate-γ-semialdehyde dehydrogenase (GSALDH) active sites, which are separated by over 40 Å and connected by a complex tunnel system. The tunnel system consists of a main tunnel that connects the two active sites and functions in substrate channeling, plus six ancillary tunnels whose functions are unknown. Here we used tunnel-blocking mutagenesis to probe the role of a dynamic ancillary tunnel (tunnel 2a) whose shape is modulated by ligand binding to the PRODH active site. The 1.90 Å resolution crystal structure of Geobacter sulfurreducens PutA variant A206W verified that the side chain of Trp206 cleanly blocks tunnel 2a without perturbing the surrounding structure. Steady-state kinetic measurements indicate the mutation impaired PRODH activity without affecting the GSALDH activity. Single-turnover experiments corroborated a severe impairment of PRODH activity with flavin reduction decreased by nearly 600-fold in A206W relative to wild-type. Substrate channeling is also significantly impacted as A206W exhibited a 3000-fold lower catalytic efficiency in coupled PRODH-GSALDH activity assays, which measure NADH formation as a function of proline. The structure suggests that Trp206 inhibits binding of the substrate l-proline by preventing the formation of a conserved glutamate-arginine ion pair and closure of the PRODH active site. Our data are consistent with tunnel 2a serving as an open space through which the glutamate of the ion pair travels during the opening and closing of the active site in response to binding l-proline. These results confirm the essentiality of the conserved ion pair in binding l-proline and support the hypothesis that the ion pair functions as a gate that controls access to the PRODH active site.

Identification of C3H2C3-type RING E3 ubiquitin ligase in grapevine and characterization of drought resistance function of VyRCHC114.

BACKGROUND: RING is one of the largest E3 ubiquitin ligase families and C3H2C3 type is the largest subfamily of RING, which plays an important role in plant growth and development, and growth and responses to biotic and abiotic stresses. RESULTS: A total of 143 RING C3H2C3-type genes (RCHCs) were discovered from the grapevine genome and separated into groups (I-XI) according to their phylogenetic analysis, and these genes named according to their positions on chromosomes. Gene replication analysis showed that tandem duplications play a predominant role in the expansion of VvRCHCs family together. Structural analysis showed that most VvRCHCs (67.13 %) had no more than 2 introns, while genes clustered together based on phylogenetic trees had similar motifs and evolutionarily conserved structures. Cis-acting element analysis showed the diversity of VvRCHCs regulation. The expression profiles of eight DEGs in RNA-Seq after drought stress were like the results of qRT-PCR analysis. In vitro ubiquitin experiment showed that VyRCHC114 had E3 ubiquitin ligase activity, overexpression of VyRCHC114 in Arabidopsis improved drought tolerance. Moreover, the transgenic plant survival rate increased by 30 %, accompanied by electrolyte leakage, chlorophyll content and the activities of SOD, POD, APX and CAT were changed. The quantitative expression of AtCOR15a, AtRD29A, AtERD15 and AtP5CS1 showed that they participated in the response to drought stress may be regulated by the expression of VyRCHC114. CONCLUSIONS: This study provides valuable new information for the evolution of grapevine RCHCs and its relevance for studying the functional characteristics of grapevine VyRCHC114 genes under drought stress.

Arabidopsis OSMOTIN 34 Functions in the ABA Signaling Pathway and Is Regulated by Proteolysis.

Plants have evolutionarily established resistance responses to a variety of abiotic stress conditions, in which ABA mediates the integrated regulation of these stress responses. Numerous proteins function at the transcription level or at the protein level when contributing to controls of the ABA signaling process. Although osmotin is identified as a salt-inducible protein, its function in the abiotic stress response is yet to be elucidated. To examine the role of Arabidopsis OSMOTIN 34 (OSM34) in the ABA signaling pathway, a deletion mutant osm34 generated by a CRISPR/Cas9 system and the double mutant osm34 osml (osmotin 34-like) were analyzed for various ABA responses. Both osm34 and osm34 osml showed reduced levels of ABA responses in seeds and leaves. Moreover, proline level and expression of the proline biosynthesis gene P5CS1 was significantly reduced in osm34 osml. Interestingly, OSM34 binds to SKP2A, an F-Box protein whose transcription is induced by ABA. The protein stability of OSM34 was determined to be under the control of the 26S proteasome. In conclusion, our data suggest that OSM34 functions as a positive regulator in the generation of ABA responses and is under post-translational control.

Abscisic acid-responsive element binding transcription factors contribute to proline synthesis and stress adaptation in Arabidopsis.

Proline accumulation is one of the most common adaptive responses of higher plants against abiotic stresses like drought. It plays multiple roles in osmotic adjustment, cell homeostasis and stress recovery. Genetic regulation of proline accumulation under drought is complex, and transcriptional cascades modulating proline is poorly understood. Here, we employed quadruple mutant (abf1 abf2 abf3 abf4) to dissect the role of ABA-responsive elements (ABREs) binding transcription factors (ABFs) in modulating proline accumulation across varying stress scenarios. ABREs are present across the promoter of the P5CS1 gene, whose upregulation is considered a hallmark for drought inducible proline accumulation. Upon ABA treatment, P5CS1 mRNA expression and proline content in the shoot were significantly higher in Col-0 compared to the quadruple mutant. Similar results were found at 2 h and 3 h after acute dehydration. We quantified proline at different time points after drought stress treatment. The proline content was higher in wild type (Col-0) than the quadruple mutant at the early stage of drought. Notably, the proline accumulation in wild type increased at a slower rate than the quadruple mutant 7 d after drought stress. Besides, the quadruple mutant displayed significant oxidative damage, low tissue turgidity and higher membrane damage under terminal drought stress. Both terminal drought stress and long-term constant water stress revealed substantial differences in growth rate between wild type and quadruple mutant. The study provides evidence that ABFs are involved in drought stress response, such as proline biosynthesis in Arabidopsis.

Overexpression of Isoprene Synthase Affects ABA- and Drought-Related Gene Expression and Enhances Tolerance to Abiotic Stress.

Isoprene is the most abundant single biogenic volatile compound emitted by plants. Despite the relevance of this molecule to plant abiotic resistance and its impact on global atmospheric chemistry, little is known about the details of its mechanism of action. Here, we characterized through both physiological and molecular methods the mechanisms of action of isoprene using model transgenic arabidopsis lines overexpressing a monocot isoprene synthase gene. Our results demonstrated the effect that isoprene had on ABA signaling at different tissue-specific, spatial, and temporal scales. In particular, we found that isoprene enhanced stomatal sensitivity to ABA through upregulation of RD29B signaling gene. By contrast, isoprene decreased sensitivity to ABA in germinating seeds and roots, suggesting tissue-specific mechanisms of action. In leaves, isoprene caused the downregulation of COR15A and P5CS genes, suggesting that the enhanced tolerance to water-deprivation stress observed in isoprene-emitting plants may be mediated chiefly by an enhanced membrane integrity and tolerance to osmotic stress.

Characterization and expression analysis of P5CS (Δ1-pyrroline-5-carboxylate synthase) gene in two distinct populations of the Atlantic Forest native species Eugenia uniflora L.

Eugenia uniflora is an Atlantic Forest native species, occurring in contrasting edaphoclimatic environments. The identification of genes involved in response to abiotic factors is very relevant to help in understanding the processes of local adaptation. 1-Pyrroline-5-carboxylate synthetase (P5CS) is one interesting gene to study in this species since it encodes a key enzyme of proline biosynthesis, which is an osmoprotectant during abiotic stress. Applying in silico analysis, we identified one P5CS gene sequence of E. uniflora (EuniP5CS). Phylogenetic analysis, as well as, gene and protein structure investigation, revealed that EuniP5CS is a member of P5CS gene family. Plants of E. uniflora from two distinct environments (restinga and riparian forest) presented differences in the proline accumulation and P5CS expression levels under growth-controlled conditions. Both proline accumulation and gene expression level of EuniP5CS were higher in the genotypes from riparian forest than those from restinga. When these plants were submitted to drought stress, EuniP5CS gene was up-regulated in the plants from restinga, but not in those from riparian forest. These results demonstrated that EuniP5CS is involved in proline biosynthesis in this species and suggest that P5CS gene may be an interesting candidate gene in future studies to understand the processes of local adaptation in E. uniflora.

Overexpression of the Lolium perenne L. delta1-pyrroline 5-carboxylate synthase (LpP5CS) gene results in morphological alterations and salinity tolerance in switchgrass (Panicum virgatum L.).

In plants, Δ1-pyrroline- 5-carboxylate synthase (P5CS) is the rate-limiting enzyme in proline biosynthesis. In this study, we introduced the LpP5CS (Lolium perenne L.) gene into switchgrass by Agrobacterium-mediated transformation. The transgenic lines (TG) were classified into two groups based on their phenotypes and proline levels. The group I lines (TG4 and TG6) had relatively high proline levels and improved biomass yield. The group II lines (TG1 and TG2) showed low proline levels, severely delayed flowering, stunted growth and reduced biomass yield. Additionally, we used RNA-seq analysis to detect the most significant molecular changes, and we analyzed differentially expressed genes, such as flowering-related and CYP450 family genes. Moreover, the biomass yield, physiological parameters, and expression levels of reactive oxygen species scavenger-related genes under salt stress all indicated that the group I plants exhibited significantly increased salt tolerance compared with that of the control plants, in contrast to the group II plants. Thus, genetic improvement of switchgrass by overexpressing LpP5CS to increase proline levels is feasible for increasing plant stress tolerance.

Proline synthesis in developing microspores is required for pollen development and fertility.

BACKGROUND: In many plants, the amino acid proline is strongly accumulated in pollen and disruption of proline synthesis caused abortion of microspore development in Arabidopsis. So far, it was unclear whether local biosynthesis or transport of proline determines the success of fertile pollen development. RESULTS: We analyzed the expression pattern of the proline biosynthetic genes PYRROLINE-5-CARBOXYLATE SYNTHETASE 1 & 2 (P5CS1 & 2) in Arabidopsis anthers and both isoforms were strongly expressed in developing microspores and pollen grains but only inconsistently in surrounding sporophytic tissues. We introduced in a p5cs1/p5cs1 p5cs2/P5CS2 mutant background an additional copy of P5CS2 under the control of the Cauliflower Mosaic Virus (CaMV) 35S promoter, the tapetum-specific LIPID TRANSFER PROTEIN 12 (Ltp12) promoter or the pollen-specific At5g17340 promoter to determine in which site proline biosynthesis can restore the fertility of proline-deficient microspores. The specificity of these promoters was confirmed by ß-glucuronidase (GUS) analysis, and by direct proline measurement in pollen grains and stage-9/10 anthers. Expression of P5CS2 under control of the At5g17340 promoter fully rescued proline content and normal morphology and fertility of mutant pollen. In contrast, expression of P5CS2 driven by either the Ltp12 or CaMV35S promoter caused only partial restoration of pollen development with little effect on pollen fertility. CONCLUSIONS: Overall, our results indicate that proline transport is not able to fulfill the demand of the cells of the male germ line. Pollen development and fertility depend on local proline biosynthesis during late stages of microspore development and in mature pollen grains.

Efficient production of trans-4-Hydroxy-l-proline from glucose by metabolic engineering of recombinant Escherichia coli.

Trans-4-Hydroxy-l-proline (trans-Hyp) is a valuable chiral building block for the synthesis of pharmaceutical intermediates. Bioconversion of l-proline using recombinant strain with proline-4-hydroxylase (P4H) is a preferred biocatalytic process in the economical production of trans-Hyp. In this study, a recombinant E. coli overexpressing hydroxylase (P4H), γ-glutamyl kinase and glutamate-semialdehyde dehydrogenase (ProBA) genes were constructed by knocking out the key genes in the metabolism. These key genes contained putA encoding proline dehydrogenase (PutA) in the l-proline metabolism and other catalytic enzyme genes, sucAB encoding α-ketoglutarate dehydrogenase (SucAB), aceAK encoding isocitratelyase (AceA) and isocitrate dehydrogenase kinase/phosphatase (AceK) in the TCA cycle. This recombinant strain coupled the synthetic pathway of trans-Hyp with TCA cycle of the host strain. It inhibited the consumption of l-proline completely and promoted the accumulation of 2-oxoglutarate (2-OG) as a co-substrate, which realized the highest conversion of glucose to trans-Hyp. A fed-batch strategy was designed, capable of producing 31·0 g l-1 trans-Hyp from glucose. It provided a theoretical basis for commercial conversion of glucose to trans-Hyp. SIGNIFICANCE AND IMPACT OF THE STUDY: Trans-4-Hydroxy-l-proline (trans-Hyp) is a valuable chiral building block for the synthesis of pharmaceutical intermediates. Unsatisfactory microbial bioconversion resulted in a low yield of trans-Hyp. In this study, we blocked the unwanted blunting pathways of host strain and make the cell growth couple with the trans-Hyp synthesis from glucose. Finally, a recombinant Escherichia coli with short-cut and efficient trans-Hyp biosynthetic pathway was obtained. It provided a theoretical basis for commercial production of trans-Hyp.

Functional FRIGIDA allele enhances drought tolerance by regulating the P5CS1 pathway in Arabidopsis thaliana.

Flowering at the right time is important for the reproductive success of plants and their response to environmental stress. In Arabidopsis, a major determinant of natural variation in flowering time is FRIGIDA (FRI). In the present study, we show that overexpression of the functional FRIGIDA gene in wild-type Col background (ColFRI) positively enhances the drought tolerance by activating P5CS1 expression and promoting proline accumulation during water stress. Furthermore, no significant changes in FRI gene and protein expression levels were observed with drought treatment, whereas P5CS1 protein expression significantly increased. In contrast, vernalization treatment efficiently reduced P5CS1 expression levels and resulted in a decrease in drought tolerance in the ColFRI plants. The flc mutants with a functional FRI background also relieved FRI-mediated activation of P5CS1 during drought tolerance. Taken together, our findings reveal the novel function of FRI in enhancing drought resistance through its downstream P5CS1 pathway during water-deficit stress, which is dependent on its target, the FLC gene.

Control of proline accumulation under drought via a novel pathway comprising the histone methylase CAU1 and the transcription factor ANAC055.

Proline plays a crucial role in the drought stress response in plants. However, there are still gaps in our knowledge about the molecular mechanisms that regulate proline metabolism under drought stress. Here, we report that the histone methylase encoded by CAU1, which is genetically upstream of P5CS1 (encoding the proline biosynthetic enzyme Δ1-pyrroline-5-carboxylate synthetase 1), plays a crucial role in proline-mediated drought tolerance. We determined that the transcript level of CAU1 decreased while that of ANAC055 (encoding a transcription factor) increased in wild-type Arabidopsis under drought stress. Further analyses showed that CAU1 bound to the promoter of ANAC055 and suppressed its expression via H4R3sme2-type histone methylation in the promoter region. Thus, under drought stress, a decreased level of CAU1 led to an increased transcript level of ANAC055, which induced the expression of P5CS1 and increased proline level independently of CAS. Drought tolerance and the level of proline were found to be decreased in the cau1 anac055 double-mutant, while proline supplementation restored drought sensitivity in the anac055 mutant. Our results reveal the details of a novel pathway leading to drought tolerance mediated by CAU1.

Capturing Environmental Plant Memories in DNA, with a Little Help from Chromatin.

Plants are eukaryotes living mostly immotile in harsh environments. On occasion, it is beneficial for their survival to maintain a transcriptional response to an environmental stress longer than the stress lasts (transcriptional memory) and even to reiterate such a response more quickly or more strongly when the same stress is re-encountered (priming memory). In eukaryotes, transcription takes place in the context of chromatin, the packaging material of DNA. Chromatin regulation is often invoked when it comes to environmental transcriptional and priming memory in plants, but rarely chromatin-based regulation can be accurately assigned to a given aspect of transcription in vivo. The conserved eukaryotic chromatin-modifying system Polycomb/Trithorax can support both long-term stability and flexibility of gene expression in Drosophila. The main principles of Polycomb/Trithorax regulation will be outlined and illustrated with the best-studied case of environmental memory from Arabidopsis. Despite being complex, the Polycomb/Trithorax system relies on experimentally tractable elements in the form of DNA, termed Polycomb/Trithorax Responsive Elements. PREs/TREs are essentially memory DNA elements. Here, relevant information to identify PRE/TRE-like elements in plants is highlighted. Examples of priming memory in plants are discussed in relation to the first two reported putative memory DNA elements. Arguably, similar cases from plants can be conducive in dissecting the contribution of DNA-based from chromatin-based regulation of transcription, when two types of DNA elements are defined: those representing binding sites for the transcription factors determining the environmental response and those controlling memory by regulating chromatin modification dynamics, ultimately maintaining the corresponding transcriptional state.

Proline Accumulation Is Regulated by Transcription Factors Associated with Phosphate Starvation.

Pro accumulation in plants is a well-documented physiological response to osmotic stress caused by drought or salinity. In Arabidopsis (Arabidopsis thaliana), the stress and ABA-induced Δ1-PYRROLINE-5-CARBOXYLATE SYNTHETASE1 (P5CS1) gene was previously shown to control Pro biosynthesis in such adverse conditions. To identify regulatory factors that control the transcription of P5CS1, Y1H screens were performed with a genomic fragment of P5CS1, containing 1.2-kB promoter and 0.8-kb transcribed regions. The myeloblastosis (MYB)-type transcription factors PHOSPHATE STARVATION RESPONSE1 (PHR1) and PHR1-LIKE1 (PHL1) were identified to bind to P5CS1 regulatory sequences in the first intron, which carries a conserved PHR1-binding site (P1BS) motif. Binding of PHR1 and PHL1 factors to P1BS was confirmed by Y1H, electrophoretic mobility assay and chromatin immunoprecipitation. Phosphate starvation led to gradual increase in Pro content in wild-type Arabidopsis plants as well as transcriptional activation of P5CS1 and PRO DEHYDROGENASE2 genes. Induction of P5CS1 transcription and Pro accumulation during phosphate deficiency was considerably reduced by phr1 and phl1 mutations and was impaired in the ABA-deficient aba2-3 and ABA-insensitive abi4-1 mutants. Growth and viability of phr1phl1 double mutant was significantly reduced in phosphate-depleted medium, while growth was only marginally affected in the aba2-3 mutants, suggesting that ABA is implicated in growth retardation in such nutritional stress. Our results reveal a previously unknown link between Pro metabolism and phosphate nutrition and show that Pro biosynthesis is target of cross talk between ABA signaling and regulation of phosphate homeostasis through PHR1- and PHL1-mediated transcriptional activation of the P5CS1 gene.

Genetic variations of HvP5CS1 and their association with drought tolerance related traits in barley (Hordeum vulgare L.).

Delta-1-pyrroline-5-carboxylate synthase gene1 (P5CS1) is the key gene involved in the biosynthesis of proline and is significantly induced by drought stress. The exploration of genetic variation in HvP5CS1 may facilitate a better understanding of the mechanism of drought adaptation in barley. In the current study, 41 polymorphisms including 16 single nucleotide polymorphisms (SNPs) and 25 insertions/deletions (indels) were detected in HvP5CS1 among 287 barley (Hordeum vulgare L.) accessions collected worldwide, with 13 distinct haplotypes identified in the barley collection. Five polymorphisms in HvP5CS1 were significantly (P < 0.001) associated with drought tolerance related traits in barley. The phenotypic variation of a given trait explained by each associated polymorphism ranged from 4.43% to 9.81%. Two sequence variations that were significantly (P < 0.0001) associated with grain yield had marginally significant positive Tajima's D values in the sliding window, so they might have been selected for environmental adaptation. Meanwhile, two haplotypes HvP5CS1_H1 and HvP5CS1_H4, which contained desired alleles of the two variations mentioned above, were significantly (P < 0.001) associated with drought tolerance related traits, and explained 5.00~11.89% of the phenotypic variations. These variations associated with drought tolerance related traits can be used as potential markers for improving drought tolerance in barley.

Synthesis of the compatible solute proline by Bacillus subtilis: point mutations rendering the osmotically controlled proHJ promoter hyperactive.

The ProJ and ProH enzymes of Bacillus subtilis catalyse together with ProA (ProJ-ProA-ProH), osmostress-adaptive synthesis of the compatible solute proline. The proA-encoded gamma-glutamyl phosphate reductase is also used for anabolic proline synthesis (ProB-ProA-ProI). Transcription of the proHJ operon is osmotically inducible whereas that of the proBA operon is not. Targeted and quantitative proteome analysis revealed that the amount of ProA is not limiting for the interconnected anabolic and osmostress-responsive proline production routes. A key player for enhanced osmostress-adaptive proline production is the osmotically regulated proHJ promoter. We used site-directed mutagenesis to study the salient features of this stress-responsive promoter. Two important features were identified: (i) deviations of the proHJ promoter from the consensus sequence of SigA-type promoters serve to keep transcription low under non-inducing growth conditions, while still allowing a finely tuned induction of transcriptional activity when the external osmolarity is increased and (ii) a suboptimal spacer length for SigA-type promoters of either 16-bp (the natural proHJ promoter), or 18-bp (a synthetic promoter variant) is strictly required to allow regulation of promoter activity in proportion to the external salinity. Collectively, our data suggest that changes in the local DNA structure at the proHJ promoter are important determinants for osmostress-inducibility of transcription.

Overexpression of a novel MYB-related transcription factor, OsMYBR1, confers improved drought tolerance and decreased ABA sensitivity in rice.

The MYB proteins play important roles in regulating plant responses to environmental stresses. We cloned and functionally characterized a novel MYB-related gene, OsMYBR1, from rice. Our microarray and qRT-PCR analyses showed that its expression was induced by drought and cold in different tissues at various developmental stages. This gene encodes a putative MYB-related protein of 463 amino acid residues. Compared with wild-type (WT) plants, transgenic plants over-expressing OsMYBR1 exhibited much greater tolerance to drought stress and decreased sensitivity to abscisic acid (ABA). Under drought treatment, levels of free proline and soluble sugar were higher in transgenic plants than in the WT. Furthermore, transcriptional expression of four stress-related genes -- OsP5CS1, OsProt, OsLEA3, and OsRab16 -- was significantly increased in transgenic plants under drought stressed conditions and ABA. Our results provide evidence that OsMYBR1 is involved in mediating plant responses to ABA and drought.

Robust root growth in altered hydrotropic response1 (ahr1) mutant of Arabidopsis is maintained by high rate of cell production at low water potential gradient.

Hydrotropism is the directional root growth response determined by water stimulus. In a water potential gradient system (WPGS) the roots of the Arabidopsis wild type have a diminished root growth compared to normal medium (NM). In contrast, the altered hydrotropic response1 (ahr1) mutant roots maintain their robust growth in the same WPGS. The aims of this work were to ascertain how ahr1 roots could sustain growth in the WPGS, with a special focus on the integration of cellular processes involved in the signaling that determines root growth during abiotic stress and their relation to hydrotropism. Cellular analysis of the root apical meristem of ahr1 mutant contrary to the wild type showed an absence of changes in the meristem length, the elongation zone length, the length of fully elongated cells, and the cell cycle duration. The robust and steady root growth of ahr1 seedlings in the WPGS is explained by the mutant capacity to maintain cell production and cell elongation at the same level as in the NM. Analysis of auxin response at a transcriptional level showed that roots of the ahr1 mutant had a lower auxin response when grown in the WPGS, compared to wild type, indicating that auxin signaling participates in attenuation of root growth under water stress conditions. Also, wild type plants exhibited a high increase in proline content while ahr1 mutants showed minimum changes in the Normal Medium→Water Stress Medium (NM→WSM), a lower water potential gradient system than the WPGS. Accordingly, in this condition, gene expression of Δ1-6 Pyrroline-5-Carboxylate Synthetase1 (P5CS1) involved in proline synthesis strongly increased in wild type but not in ahr1 seedlings. The ahr1 phenotype shows unique features since the mutant root cells continue to proliferate and grow in the presence of a progressively negative water potential gradient at a level comparable to wild type growing in the NM. As such, it represents an exceptional resource for understanding hydrotropism.

Light affects salt stress-induced transcriptional memory of P5CS1 in Arabidopsis.

To cope with environmental stresses, plants often adopt a memory response upon primary stress exposure to facilitate a quicker and stronger reaction to recurring stresses. However, it remains unknown whether light is involved in the manifestation of stress memory. Proline accumulation is a striking metabolic adaptation of higher plants during various environmental stresses. Here we show that salinity-induced proline accumulation is memorable and HY5-dependent light signaling is required for such a memory response. Primary salt stress induced the expression of Δ1-pyrroline-5-carboxylate synthetase 1 (P5CS1), encoding a proline biosynthetic enzyme and proline accumulation, which were reduced to basal level during the recovery stage. Reoccurring salt stress-induced stronger P5CS1 expression and proline accumulation were dependent upon light exposure during the recovery stage. Further studies demonstrated that salt-induced transcriptional memory of P5CS1 is associated with the retention of increased H3K4me3 level at P5CS1 during the recovery stage. HY5 binds directly to light-responsive element, C/A-box, in the P5CS1 promoter. Deletion of the C/A-box or hy5 hyh mutations caused rapid reduction of H3K4me3 level at P5CS1 during the recovery stage, resulting in impairment of the stress memory response. These results unveil a previously unrecognized mechanism whereby light regulates salt-induced transcriptional memory via the function of HY5 in maintaining H3K4me3 level at the memory gene.

A Synonymous Mutation Upstream of the Gene Encoding a Weak-Link Enzyme Causes an Ultrasensitive Response in Growth Rate.

UNLABELLED: When microbes are faced with an environmental challenge or opportunity, preexisting enzymes with promiscuous secondary activities can be recruited to provide newly important functions. Mutations that increase the efficiency of a new activity often compromise the original activity, resulting in an inefficient bifunctional enzyme. We have investigated the mechanisms by which growth of Escherichia coli can be improved when fitness is limited by such an enzyme, E383A ProA (ProA*). ProA* can serve the functions of both ProA (required for synthesis of proline) and ArgC (required for synthesis of arginine), albeit poorly. We identified four genetic changes that improve the growth rate by up to 6.2-fold. Two point mutations in the promoter of the proBA* operon increase expression of the entire operon. Massive amplification of a genomic segment around the proBA* operon also increases expression of the entire operon. Finally, a synonymous point mutation in the coding region of proB creates a new promoter for proA* This synonymous mutation increases the level of ProA* by 2-fold but increases the growth rate by 5-fold, an ultrasensitive response likely arising from competition between two substrates for the active site of the inefficient bifunctional ProA*. IMPORTANCE: The high-impact synonymous mutation we discovered in proB is remarkable for two reasons. First, most polar effects documented in the literature are detrimental. This finding demonstrates that polar effect mutations can have strongly beneficial effects, especially when an organism is facing a difficult environmental challenge for which it is poorly adapted. Furthermore, the consequence of the synonymous mutation in proB is a 2-fold increase in the level of ProA* but a disproportionately large 5.1-fold increase in growth rate. While ultrasensitive responses are often found in signaling networks and genetic circuits, an ultrasensitive response to an adaptive mutation has not been previously reported.