To better define the still unclear role of proline (Pro) metabolism in drought resistance, we analyzed Arabidopsis (Arabidopsis thaliana) Ɗ1-pyrroline-5-carboxylate synthetase1 (p5cs1) mutants deficient in stress-induced Pro synthesis as well as proline dehydrogenase (pdh1) mutants blocked in Pro catabolism and found that both Pro synthesis and catabolism were required for optimal growth at low water potential (ψw). The abscisic acid (ABA)-deficient mutant aba2-1 had similar reduction in root elongation as p5cs1 and p5cs1/aba2-1 double mutants. However, the reduced growth of aba2-1 but not p5cs1/aba2-1 could be complemented by exogenous ABA, indicating that Pro metabolism was required for ABA-mediated growth protection at low ψw. PDH1 maintained high expression in the root apex and shoot meristem at low ψw rather than being repressed, as in the bulk of the shoot tissue. This, plus a reduced oxygen consumption and buildup of Pro in the root apex of pdh1-2, indicated that active Pro catabolism was needed to sustain growth at low ψw. Conversely, P5CS1 expression was most highly induced in shoot tissue. Both p5cs1-4 and pdh1-2 had a more reduced NADP/NADPH ratio than the wild type at low ψw. These results indicate a new model of Pro metabolism at low ψw whereby Pro synthesis in the photosynthetic tissue regenerates NADP while Pro catabolism in meristematic and expanding cells is needed to sustain growth. Tissue-specific differences in Pro metabolism and function in maintaining a favorable NADP/NADPH ratio are relevant to understanding metabolic adaptations to drought and efforts to enhance drought resistance.

Drought-induced proline accumulation is widely observed in plants but its regulation and adaptive value are not as well understood. Proline accumulation of the Arabidopsis accession Shakdara (Sha) was threefold less than that of Landsberg erecta (Ler) and quantitative trait loci mapping identified a reduced function allele of the proline synthesis enzyme Δ 1 -pyrroline-5-carboxylate synthetase1 (P5CS1) as a basis for the lower proline of Sha. Sha P5CS1 had additional TA repeats in intron 2 and a G-to-T transversion in intron 3 that were sufficient to promote alternative splicing and production of a nonfunctional transcript lacking exon 3 (exon 3-skip P5CS1). In Sha, and additional accessions with the same intron polymorphisms, the nonfunctional exon 3-skip P5CS1 splice variant constituted as much as half of the total P5CS1 transcript. In a larger panel of Arabidopsis accessions, low water potential-induced proline accumulation varied by 10-fold and variable production of exon 3-skip P5CS1 among accessions was an important, but not the sole, factor underlying variation in proline accumulation. Population genetic analyses suggest that P5CS1 may have evolved under positive selection, and more extensive correlation of exon 3-skip P5CS1 production than proline abundance with climate conditions of natural accessions also suggest a role of P5CS1 in local adaptation to the environment. These data identify a unique source of alternative splicing in plants, demonstrate a role of exon 3-skip P5CS1 in natural variation of proline metabolism, and suggest an association of P5CS1 and its alternative splicing with environmental adaptation.amino acid metabolism | drought adaptation | stress gene expression | osmoprotectant | compatible solute P roline acts as an osmoprotectant and cryoprotectant in organisms as diverse as bacteria, plants, and insects (1, 2). Many plants accumulate proline in response to low water potential (ψ w ) and dehydration caused by drought or freezing. The mechanisms by which proline may promote drought resistance include osmoprotectant functions, as well as newly emerging functions of proline metabolism in NADP/NADPH balance and transfer or storage of energy and reducing potential (1-3). However, the overall importance of proline in drought adaptation is not as firmly established. In Arabidopsis thaliana, transcriptional up-regulation of Δ 1 -pyrroline-5-carboxylate synthetase1 (P5CS1) is essential for low ψ w -induced proline accumulation, and proline accumulation of p5cs1 mutants is only 15-20% of the wild-type level (4-6). Additional regulation of proline metabolism is likely but not understood. The timing and duration of water limitation, as well as other environmental variables, all influence drought-adaptation strategies used by plants. Thus, we may expect substantial natural variation of these traits within a widely distributed species, such as Arabidopsis (7-10), with many of these differences related to the local conditions to which a particular accession has adapted (11-13). Several studies ha...

Chemical probes have great potential for identifying functional residues in proteins in crude proteomes. Here we studied labeling sites of chemical probes based on sulfonyl fluorides (SFs) on plant and animal proteomes. Besides serine proteases and many other proteins, SF-based probes label Tyr residues in glutathione transferases (GSTs). The labeled GSTs represent four different GST classes that share less than 30% sequence identity. The targeted Tyr residues are located at similar positions in the promiscuous substrate binding site and are essential for GST function. The high selectivity of SF-based probes for functional Tyr residues in GSTs illustrates how these probes can be used for functional studies of GSTs and other proteins in crude proteomes.

Plants produce hundreds of glycosidases. Despite their importance in cell wall (re)modeling, protein and lipid modification, and metabolite conversion, very little is known of this large class of glycolytic enzymes, partly because of their post-translational regulation and their elusive substrates. Here, we applied activity-based glycosidase profiling using cell-permeable small molecular probes that react covalently with the active site nucleophile of retaining glycosidases in an activity-dependent manner. Using mass spectrometry we detected the active state of dozens of myrosinases, glucosidases, xylosidases, and galactosidases representing seven different retaining glycosidase families. The method is simple and applicable for different organs and different plant species, in living cells and in subproteomes. We display the active state of previously uncharacterized glycosidases, one of which was encoded by a previously declared pseudogene. Interestingly, glycosidase activity profiling also revealed the active state of a diverse range of putative xylosidases, galactosidases, glucanases, and heparanase in the cell wall of Nicotiana benthamiana. Our data illustrate that this powerful approach displays a new and important layer of functional proteomic information on the active state of glycosidases. Molecular & Cellular Proteomics

BackgroundThe herbicides glyphosate (Gly) and imazamox (Imx) inhibit the biosynthesis of aromatic and branched-chain amino acids, respectively. Although these herbicides inhibit different pathways, they have been reported to show several common physiological effects in their modes of action, such as increasing free amino acid contents and decreasing soluble protein contents. To investigate proteolytic activities upon treatment with Gly and Imx, pea plants grown in hydroponic culture were treated with Imx or Gly, and the proteolytic profile of the roots was evaluated through fluorogenic kinetic assays and activity-based protein profiling.ResultsSeveral common changes in proteolytic activity were detected following Gly and Imx treatment. Both herbicides induced the ubiquitin-26 S proteasome system and papain-like cysteine proteases. In contrast, the activities of vacuolar processing enzymes, cysteine proteases and metacaspase 9 were reduced following treatment with both herbicides. Moreover, the activities of several putative serine protease were similarly increased or decreased following treatment with both herbicides. In contrast, an increase in YVADase activity was observed under Imx treatment versus a decrease under Gly treatment.ConclusionThese results suggest that several proteolytic pathways are responsible for protein degradation upon herbicide treatment, although the specific role of each proteolytic activity remains to be determined.

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The Arabidopsis thaliana accession Shahdara (Sha) differs from Landsberg erecta (Ler) and other accessions in its responses to drought and low water potential including lower levels of proline accumulation. However, Sha maintained greater seedling root elongation at low water potential and a higher NADP/NADPH ratio than Ler. Profiling of major amino acids and organic acids found that Sha had reduced levels of all glutamate family amino acids metabolically related to proline, but increased levels of aspartate-derived amino acids (particularly isoleucine), leucine and valine at low water potential. Although Sha is known for its different abiotic stress response, RNA sequencing and co-expression clustering found that Sha differed most from Ler in defence/ immune response and reactive oxygen-related gene expression. HVA22B and Osmotin34 were two of the relatively few abiotic stress-associated genes differentially expressed between Ler and Sha. Insensitivity to exogenous glutamine and a different expression profile of glutamate receptors were further factors that may underlie the differing metabolism and low water potential phenotypes of Sha. These data define the unique environmental adaptation and differing metabolism of Sha including differences in defence gene expression, and will facilitate further analysis of Sha natural variation to understand metabolic regulation and abiotic/ biotic stress interaction.

Contents Summary936I.Introduction936II.The quest for plant protease substrates – proteomics to the rescue?937III.Quantitative proteome comparison reveals candidate substrates938IV.Dynamic metabolic stable isotope labeling to measure protein turnover in vivo938V.Terminomics – large‐scale identification of protease cleavage sites939VI.Substrate or not substrate, that is the question940VII.Concluding remarks941Acknowledgements941References941 Summary Proteolysis is a central regulatory mechanism of protein homeostasis and protein function that affects all aspects of plant life. Higher plants encode for hundreds of proteases, but their physiological substrates and hence their molecular functions remain mostly unknown. Current quantitative mass spectrometry‐based proteomics enables unbiased large‐scale interrogation of the proteome and its modifications. Here we provide an overview of proteomics techniques that allow profiling of changes in protein abundance, measurement of proteome turnover rates, identification of protease cleavage sites in vivo and in vitro and determination of protease sequence specificity. We discuss how these techniques can help to reveal protease substrates and determine plant protease function, illustrated by recent studies on selected plant proteases.