Water deficit or dehydration is the most crucial environmental factor that limits crop productivity and influences geographical distribution of many crop plants. It is suggested that dehydration-responsive changes in expression of proteins may lead to cellular adaptation against water deficit conditions. Most of the earlier understanding of dehydration-responsive cellular adaptation has evolved from transcriptome analyses. By contrast, comparative analysis of dehydration-responsive proteins, particularly proteins in the subcellular fraction, is limiting. In plants, cell wall or extracellular matrix (ECM) serves as the repository for most of the components of the cell signaling process and acts as a frontline defense. Thus, we have initiated a proteomics approach to identify dehydrationresponsive ECM proteins in a food legume, chickpea. Several commercial chickpea varieties were screened for the status of dehydration tolerance using different physiological and biochemical indexes. Dehydration-responsive temporal changes of ECM proteins in JG-62, a relatively tolerant variety, revealed 186 proteins with variance at a 95% significance level statistically. The comparative proteomics analysis led to the identification of 134 differentially expressed proteins that include predicted and novel dehydration-responsive proteins. This study, for the first time, demonstrates that over a hundred ECM proteins, presumably involved in a variety of cellular functions, viz. cell wall modification, signal transduction, metabolism, and cell defense and rescue, impinge on the molecular mechanism of dehydration tolerance in plants.

The circadian clock provides adaptive advantages to an organism, resulting in increased fitness and survival. The phosphorylation events that regulate circadian-dependent signaling and the processes which post-translationally respond to clock-gated signals are largely unknown. To better elucidate post-translational events tied to the circadian system we carried out a survey of circadianregulated protein phosphorylation events in Arabidopsis seedlings. A large-scale mass spectrometry-based quantitative phosphoproteomics approach employing TiO2-based phosphopeptide enrichment techniques identified and quantified 1586 phosphopeptides on 1080 protein groups. A total of 102 phosphopeptides displayed significant changes in abundance, enabling the identification of specific patterns of response to circadian rhythms. Our approach was sensitive enough to quantitate oscillations in the phosphorylation of low abundance clock proteins (EARLY FLOWERING4; ELF4 and PSEUDORESPONSE REGULATOR3; PRR3) as well as other transcription factors and kinases. During constant light, extensive cyclic changes in phosphorylation status occurred in critical regulators, implicating direct or indirect regulation by the circadian system. These included proteins influencing transcriptional regulation, translation, metabolism, stress and phytohormones-mediated responses. We validated our analysis using the elf4 -211 allele, in which an S45L transition removes the phosphorylation herein identified. We show that removal of this phosphorylatable site diminishes interaction with EARLY FLOWERING3 (ELF3), a key partner in a tripartite evening complex required for circadian cycling. elf4 -211 lengthens period, which increases with increasing temperature, relative to the wild type, resulting in a more stable temperature compensation of circadian period over a wider temperature range. Molecular

Water deficit or dehydration is the most crucial environmental constraint on plant growth and development and crop productivity. It has been postulated that plants respond and adapt to dehydration by altering their cellular metabolism and by activating various defense machineries. The nucleus, the regulatory hub of the eukaryotic cell, is a dynamic system and a repository of various macromolecules that serve as modulators of cell signaling dictating the cell fate decision. To better understand the molecular mechanisms of dehydration-responsive adaptation in plants, we developed a comprehensive nuclear proteome of rice. The proteome was determined using a sequential method of organellar enrichment followed by two-dimensional electrophoresis-based protein identification by LC-ESI-MS/MS. We initially screened several commercial rice varieties and parental lines and established their relative dehydration tolerance. The differential display of nuclear proteins in the tolerant variety under study revealed 150 spots that showed changes in their intensities by more than 2.5-fold. The proteomics analysis led to the identification of 109 differentially regulated proteins presumably involved in a variety of functions, including transcriptional regulation and chromatin remodeling, signaling and gene regulation, cell defense and rescue, and protein degradation. The dehydration-responsive nuclear proteome revealed a coordinated response involving both regulatory and functional proteins, impinging upon the molecular mechanism of dehydration adaptation. Furthermore a comparison between the dehydrationresponsive nuclear proteome of rice and that of a legume, the chickpea, showed an evolutionary divergence in dehydration response comprising a few conserved proteins, whereas most of the proteins may be involved in cropspecific adaptation. These results might help in understanding the spectrum of nuclear proteins and the biological processes they control under dehydration as well as having implications for strategies to improve dehydration tolerance in plants. Molecular

The extracellular matrix (ECM) or cell wall is a dynamic system and serves as the first line mediator in cell signaling to perceive and transmit extra- and intercellular signals in many pathways. Although ECM is a conserved compartment ubiquitously present throughout evolution, a compositional variation does exist among different organisms. ECM proteins account for 10% of the ECM mass, however, comprise several hundreds of different molecules with diverse functions. To understand the function of ECM proteins, we have developed the cell wall proteome of a crop legume, chickpea (Cicer arietinum). This comprehensive overview of the proteome would provide a basis for future comparative proteomic efforts for this important crop. Proteomic analyses revealed new ECM proteins of unknown functions vis-à-vis the presence of many known cell wall proteins. In addition, we report here evidence for the presence of unexpected proteins with known biochemical activities, which have never been associated with ECM.

Nuclear proteins constitute a highly organized, complex network that plays diverse roles during cellular development and other physiological processes. The yeast nuclear proteome corresponds to about one-fourth of the total cellular proteins, suggesting the involvement of the nucleus in a number of diverse functions. In an attempt to understand the complexity of plant nuclear proteins, we have developed a proteome reference map of a legume, chickpea, using two-dimensional gel electrophoresis (2-DE). Approximately, 600 protein spots were detected, and LC-ESI-MS/MS analyses led to the identification of 150 proteins that have been implicated in a variety of cellular functions. The largest percentage of the identified proteins was involved in signaling and gene regulation (36%), while 17% were involved in DNA replication and transcription. The chickpea nuclear proteome indicates the presence of few new nuclear proteins of unknown functions vis-à-vis many known resident proteins. To the best of our knowledge, this is the first report of a nuclear proteome of an unsequenced genome.

Clock-generated biological rhythms provide an adaptive advantage to an organism, resulting in increased fitness and survival. To better elucidate the plant response to the circadian system, we surveyed protein oscillations in Arabidopsis seedlings under constant light. Using large-scale two-dimensional difference in gel electrophoresis (2D-DIGE) the abundance of more than 1000 proteins spots was reproducibly resolved quantified and profiled across a circadian time series. A comparison between phenol-extracted samples and RuBisCO-depleted extracts identified 71 and 40 rhythmically-expressed proteins, respectively, and between 30 and 40% of these derive from non-rhythmic transcripts. These included proteins influencing transcriptional regulation, translation, metabolism, photosynthesis, protein chaperones, and stress-mediated responses. The phasing of maximum expression for the cyclic proteins was similar for both datasets, with a nearly even distribution of peak phases across the time series. STRING clustering analysis identified two interaction networks with a notable number of oscillating proteins: plastid-based and cytosolic chaperones and 10 proteins involved in photosynthesis. The oscillation of the ABA receptor, PYR1/RCAR11, with peak expression near dusk adds to a growing body of evidence that intimately ties ABA signaling to the circadian system. Taken together, this study provides new insights into the importance of post-transcriptional circadian control of plant physiology and metabolism.

BackgroundUbiquitination is a major post-translational protein modification that regulates essentially all cellular and physiological pathways in eukaryotes. The ubiquitination process typically involves three distinct classes of enzymes, ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin ligase (E3). To date, a comprehensive identification and analysis of core components comprising of the whole soybean (Glycine max) ubiquitin system (UBS) has not been reported.ResultsWe performed a systematic, genome-wide analysis of genes that encode core members of the soybean UBS in this study. A total of 1431 genes were identified with high confidence to encode putative soybean UBS components, including 4 genes encoding E1s, 71 genes that encode the E2s, and 1356 genes encoding the E3-related components. Among the E3-encoding genes, 760 encode RING-type E3s, 124 encode U-box domain-containing E3s, and 472 encode F-box proteins. To find out whether the identified soybean UBS genes encode active enzymes, a set of genes were randomly selected and the enzymatic activities of their recombinant proteins were tested. Thioester assays indicated proteins encoded by the soybean E1 gene GmUBA1 and the majority of selected E2 genes are active E1 or E2 enzymes, respectively. Meanwhile, most of the purified RING and U-box domain-containing proteins displayed E3 activity in the in vitro ubiquitination assay. In addition, 1034 of the identified soybean UBS genes were found to express in at least one of 14 soybean tissues examined and the transcript level of 338 soybean USB genes were significantly changed after abiotic or biotic (Fusarium oxysporum and Rhizobium strains) stress treatment. Finally, the expression level of a large number of the identified soybean UBS-related genes was found significantly altered after soybean cyst nematode (SCN) treatment, suggesting the soybean UBS potentially plays an important role in soybean immunity against SCN.ConclusionsOur findings indicate the presence of a large and diverse number of core UBS proteins in the soybean genome, which suggests that target-specific modification by ubiquitin is a complex and important part of cellular and physiological regulation in soybean. We also revealed certain members of the soybean UBS may be involved in immunity against soybean cyst nematode (SCN). This study sets up an essential foundation for further functional characterization of the soybean UBS in various physiological processes, such as host immunity against SCN.Electronic supplementary materialThe online version of this article (10.1186/s12870-018-1365-7) contains supplementary material, which is available to authorized users.

Dehydration is the most crucial environmental factor that considerably reduces the crop harvest index, and thus has become a concern for global agriculture. To better understand the role of nuclear proteins in water-deficit condition, a nuclear proteome was developed from a dehydration-sensitive rice cultivar IR-64 followed by its comparison with that of a dehydration-tolerant c.v. Rasi. The 2DE protein profiling of c.v. IR-64 coupled with MS/MS analysis led to the identification of 93 dehydration-responsive proteins (DRPs). Among those identified proteins, 78 were predicted to be destined to the nucleus, accounting for more than 80% of the dataset. While the detected number of protein spots in c.v. IR-64 was higher when compared with that of Rasi, the number of DRPs was found to be less. Fifty-seven percent of the DRPs were found to be common to both sensitive and tolerant cultivars, indicating significant differences between the two nuclear proteomes. Further, we constructed a functional association network of the DRPs of c.v. IR-64, which suggests that a significant number of the proteins are capable of interacting with each other. The combination of nuclear proteome and interactome analyses would elucidate stress-responsive signaling and the molecular basis of dehydration tolerance in plants.