Randeep Rakwal

Plant secretome: Unlocking secrets of the secreted proteins

Plant secretomics is a newly emerging area of the plant proteomics field. It basically describes the global study of secreted proteins into the extracellular space of plant cell or tissue at any given time and under certain conditions through various secretory mechanisms. A combination of biochemical, proteomics and bioinformatics approaches has been developed to isolate, identify and profile secreted proteins using complementary in vitro suspension‐cultured cells and in planta systems. Developed inventories of secreted proteins under normal, biotic and abiotic conditions revealed several different types of novel secreted proteins, including the leaderless secretory proteins (LSPs). On average, LSPs can account for more than 50% of the total identified secretome, supporting, as in other eukaryotes, the existence of novel secretory mechanisms independent of the classical endoplasmic reticulum‐Golgi secretory pathway, and suggesting that this non‐classical mechanism of protein expression is, for as yet unknown reasons, more massively used than in other eukaryotic systems. Plants LSPs, which seem to be potentially involved in the defense/stress responses, might have dual (extracellular and/or intracellular) roles as most of them have established intracellular functions, yet presently unknown extracellular functions. Evidence is emerging on the role of glycosylation in the apical sorting and trafficking of secretory proteins. These initial secretome studies in plants have considerably advanced our understanding on secretion of different types of proteins and their underlying mechanisms, and opened a door for comparative analyses of plant secretomes with those of other organisms. In this first review on plant secretomics, we summarize and discuss the secretome definition, the applied approaches for unlocking secrets of the secreted proteins in the extracellular fluid, the possible functional significance and secretory mechanisms of LSPs, as well as glycosylation of secreted proteins and challenges involved ahead. Further improvements in existing and developing strategies and techniques will continue to drive forward plant secretomics research to building comprehensive and confident data sets of secreted proteins. This will lead to an increased understanding on how cells couple the concerted action of secreted protein networks to their internal and external environments.

Proteome analysis of differentially displayed proteins as a tool for investigating ozone stress in rice (Oryza sativa L.) seedlings

Employing classical two‐dimensional electrophoresis (2‐DE), amino acid sequencing and immunoblot analysis, we examine for the first time the effect of ozone, a highly notorious environmental pollutant, on rice seedling proteins. Drastic visible necrotic damage to leaf by ozone and consequent increase in ascorbate peroxidase protein(s) was accompanied by rapid changes in the 2‐DE protein profiles, over controls. Out of a total of 56 proteins investigated, which were reproducible in repeated experiments, 52 protein spots were visually identified as differentially expressed over controls. Six proteins were N‐terminally blocked, and the sequence of 14 proteins could not be determined, whereas 36 proteins were N‐terminally and one was internally sequenced. Ozone caused drastic reductions in the major leaf photosynthetic proteins, including the abundantly present ribulose‐1, 5‐bisphosphate carboxylase/oxygenase, and induction of various defense/stress related proteins. Most prominent change in leaves, within 24 h post‐treatment with ozone, was the induced accumulation of a pathogenesis related (PR) class 5 protein, three PR 10 class proteins, ascorbate peroxidase(s), superoxide dismutase, calcium‐binding protein, calreticulin, a novel ATP‐dependent CLP protease, and an unknown protein. Present results demonstrate the highly damaging effect of ozone on rice seedlings at the level of the proteome.

Chitosan activates defense/stress response(s) in the leaves of Oryza sativa seedlings

Abstract In this study, we examined the response(s) of rice (Oryza sativa L. japonica-type cv. Nipponbare) seedling leaves treated with a fungal elicitor chitosan (CT). Small brownish necrotic spots (streaks) appeared in the interveinal regions on the leaf surface after treatment by 0.1% CT, over the cut control. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis further revealed strong induction of ascorbate peroxidase, and changes in “phytocystatins” (cysteine proteinase inhibitors). Using two-dimensional polyacrylamide gel electrophoresis, evidence is provided for the accumulation of two major classes of pathogenesis-related (PR) proteins, namely OsPR5 and OsPR10 in the leaves. In parallel, northern analyses revealed potent accumulation of the OsPR5 and OsPR10 mRNAs; a time- and dose-dependent expression, and a requirement for de novo protein synthesis was observed. Furthermore, CT-elicited changes were also accompanied by production of anti-fungal phytoalexins, the flavonoid sakuranetin and the diterpenoid lactone momilactone A, as determined by liquid chromatography-mass spectrometry/mass spectrometry analysis. Present results reveal for the first time the potency of CT in initiating multiple events linked with defense/stress response(s) in the leaves of whole rice plants.

Isolation of novel rice (Oryza sativa L.) multiple stress responsive MAP kinase gene, OsMSRMK2, whose mRNA accumulates rapidly in response to environmental cues

In search for components of MAPK (mitogen-activated protein kinase) cascades in rice (Oryza sativa L. cv. Nipponbare), we identified a single copy gene called OsMSRMK2 from jasmonic acid (JA) treated rice seedling leaf cDNA library. This gene has a conserved protein kinase domain, including a MAPK family signature, and encodes a 369 amino acid polypeptide with a predicted molecular mass of 42995.43 and a pI of 5.48. OsMSRMK2 did not show constitutive expression in leaves and was induced within 15 min in response to wounding by cut. Using in vitro system, we show that the expression of OsMSRMK2 mRNA was potently enhanced within 15 min by signalling molecules, protein phosphatase inhibitors, ultraviolet irradiation, fungal elicitor, heavy metals, high salt and sucrose, and drought. OsMSRMK2 expression was further modulated by co-application of JA, salicylic acid, and ethylene and required de novo synthesized protein factor(s) in its transient regulation. Moreover, high (37 degrees C) and low temperatures (12 degrees C) and environmental pollutants-ozone and sulfur dioxide-differentially regulate the OsMSRMK2 mRNA accumulation in leaves of intact plants. Present results demonstrating dramatic transcriptional and transient regulation of the OsMSRMK2 expression by diverse biotic/abiotic stresses, a first report for any rice (or plant) MAPK to date, suggest a role for OsMSRMK2 in rice defense/stress response pathways.

Role of jasmonate in the rice (Oryza sativa L.) self-defense mechanism using proteome analysis.

Exogenously applied jasmonic acid (JA) was used to study changes in protein patterns in rice (Oryza sativa L.) seedling tissues, to classify these changes, and to assign a role for these changes, in order to define the role of JA in the rice self‐defense mechanism. High resolution two‐dimensional polyacrylamide gel electrophoretic analysis revealed induction of new proteins in both leaf and stem tissues after JA treatment, with the major protein spots further analyzed through N‐terminal and internal amino acid sequencing, purification, antibody production, and immunoblot analysis. JA treatment results in necrosis in these tissues, which is accompanied by drastic reductions in ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RuBisCO) subunits, and was confirmed using immunoblotting. Induction of novel proteins was found particularly in the stem tissues, including a new basic 28 kDa Bowman‐Birk proteinase inhibitor protein (BBPIN; jasmonate‐induced stem protein, JISP 6), and acidic 17 kDa pathogenesis‐related class 1 protein (PR‐1, JISP 9). This induction of proteins was blocked by a protein synthesis inhibitor cycloheximide (CHX) indicating de novo protein synthesis. Kinetin (KIN), a cytokinin and free radical scavenger reversed RuBisCO decreases, but not induction of proteins. Immunoblot analysis using antibodies generated against these purified proteins revealed a tissue‐specific expression pattern and time‐dependent induction after JA treatment. Our results indicate that jasmonate affects defense‐related gene expression in rice seedlings, as evidenced by de novo synthesis of novel proteins with potential roles in plant defense.

Physiology and proteomics of the water-deficit stress response in three contrasting peanut genotypes.

Peanut genotypes from the US mini-core collection were analysed for changes in leaf proteins during reproductive stage growth under water-deficit stress. One- and two-dimensional gel electrophoresis (1- and 2-DGE) was performed on soluble protein extracts of selected tolerant and susceptible genotypes. A total of 102 protein bands/spots were analysed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and by quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS) analysis. Forty-nine non-redundant proteins were identified, implicating a variety of stress response mechanisms in peanut. Lipoxygenase and 1l-myo-inositol-1-phosphate synthase, which aid in inter- and intracellular stress signalling, were more abundant in tolerant genotypes under water-deficit stress. Acetyl-CoA carboxylase, a key enzyme of lipid biosynthesis, increased in relative abundance along with a corresponding increase in epicuticular wax content in the tolerant genotype, suggesting an additional mechanism for water conservation and stress tolerance. Additionally, there was a marked decrease in the abundance of several photosynthetic proteins in the tolerant genotype, along with a concomitant decrease in net photosynthesis in response to water-deficit stress. Differential regulation of leaf proteins involved in a variety of cellular functions (e.g. cell wall strengthening, signal transduction, energy metabolism, cellular detoxification and gene regulation) indicates that these molecules could affect the molecular mechanism of water-deficit stress tolerance in peanut.

Plant phosphoproteomics: An update

Phosphoproteomics involves identification of phosphoproteins, precise mapping, and quantification of phosphorylation sites, and eventually, revealing their biological function. In plants, several systematic phosphoproteomic analyses have recently been performed to optimize in vitro and in vivo technologies to reveal components of the phosphoproteome. The discovery of novel substrates for specific protein kinases is also an important issue. Development of a new tool has enabled rapid identification of potential kinase substrates such as kinase assays using plant protein microarrays. Progress has also been made in quantitative and dynamic analysis of mapped phosphorylation sites. Increased quantity of experimentally verified phosphorylation sites in plants has prompted the creation of dedicated web‐resources for plant‐specific phosphoproteomics data. This resulted in development of computational prediction methods yielding significantly improved sensitivity and specificity for the detection of phosphorylation sites in plants when compared to methods trained on less plant‐specific data. In this review, we present an update on phosphoproteomic studies in plants and summarize the recent progress in the computational prediction of plant phosphorylation sites. The application of the experimental and computed results in understanding the phosphoproteomic networks of cellular and metabolic processes in plants is discussed. This is a continuation of our comprehensive review series on plant phosphoproteomics.

Small GTPase ‘Rop’: molecular switch for plant defense responses

The conserved Rho family of GTPases (Rho, Rac, and Cdc42) in fungi and mammals has emerged as a key regulator of diverse cellular activities, such as cytoskeletal rearrangements, programmed cell death, stress‐induced signaling, and cell growth and differentiation. In plants, a unique class of Rho‐like proteins, most closely related to mammalian Rac, has only been found and termed ‘Rop’ (Rho‐related GTPase from plant [Li et al. (1998) Plant Physiol. 118, 407–417; Yang (2002) Plant Cell 14, S375–S388]). ROPs have been implicated in regulating various plant cellular responses including defense against pathogens. It has been shown that ROPs, like mammalian Rac, trigger hydrogen peroxide production and hence the ‘oxidative burst’, a crucial component associated with the cell death, most likely via activation of nicotinamide adenine dinucleotide phosphate oxidase in both monocotyledonous and dicotyledonous species. Recent studies have established that ROPs also function as a molecular switch for defense signaling pathway(s) linked with disease resistance. As discerning the defense pathway remains one of the priority research areas in the field of plant biology, this review is therefore particularly focused on recent progresses that have been made towards understanding the plant defense responses mediated by ROPs.

Separation and characterization of proteins from green and etiolated shoots of rice (Oryza sativaL.): Towards a rice proteome

Proteins extracted from green and etiolated shoots of rice were separated by two‐dimensional polyacrylamide gel electrophoresis and relative molecular weights and isoelectric points were determined. The separated proteins were electroblotted onto a polyvinylidene difluoride membrane and 85 proteins were analyzed by a gas‐phase protein sequencer. The N‐terminal amino acid sequences of 21 out of 85 proteins were determined in this manner. N‐terminal regions of the remaining proteins could not be sequenced. The internal amino acid sequences of proteins were determined by sequence analysis of peptides obtained by the Cleveland peptide mapping method and compared with those of known plant and animal protein sequences to understand the nature of the proteins. Green shoots revealed the presence of photosynthetic proteins as expected; however, as etiolated shoots were not photosynthetic, only precursors of the photosynthetic proteins were identified. Interestingly, the presence of L‐ascorbate peroxidase only in etiolated shoots suggests a cellular protectant function for this antioxidant enzyme in the etiolating shoots. Using this experimental approach, we could identify the major proteins involved in growth regulation in photosynthetic green shoots as well as in etiolating rice seedlings.

Novel rice MAP kinases OsMSRMK3 and OsWJUMK1 involved in encountering diverse environmental stresses and developmental regulation

We report isolation of two novel rice (Oryza sativa L.) mitogen-activated protein kinases (MAPKs), OsMSRMK3 (multiple stress responsive) and OsWJUMK1 (wound- and JA-uninducible) that most likely exist as single copy genes in its genome. OsMSRMK3 and OsWJUMK1 encode 369 and 569 amino acid polypeptides having the MAPK family signature and phosphorylation activation motifs TEY and TDY, respectively. Steady state mRNA analyses of these MAPKs with constitutive expression in leaves of two-week-old seedlings revealed that OsMSRMK3 was up-regulated upon wounding (by cut), jasmonic acid (JA), salicylic acid (SA), ethylene, abscisic acid, hydrogen peroxide (H(2)O(2)), protein phosphatase inhibitors, chitosan, high salt/sugar, and heavy metals, whereas OsWJUMK1 not induced by either wounding, JA or SA, showed up-regulation only by H(2)O(2), heavy metals, and cold stress (12 degrees C). Moreover, these MAPKs were developmentally regulated. These results strongly suggest a role for OsMSRMK3 and OsWJUMK1 in both stress-signalling pathways and development in rice.