Contents Introduction 250 Increases in free cellular [Ca2+]: an early and key event in plant defence signalling 251 Relationships between [Ca2+]cyt increases and defence‐signalling pathways 256 Calcium targets involved in plant defence reactions 258 Concluding remarks 262 Acknowledgements 263 References 263 Summary In plant cells, the calcium ion is a ubiquitous intracellular second messenger involved in numerous signalling pathways. Variations in the cytosolic concentration of Ca2+ ([Ca2+]cyt) couple a large array of signals and responses. Here we concentrate on calcium signalling in plant defence responses, particularly on the generation of the calcium signal and downstream calcium‐dependent events participating in the establishment of defence responses with special reference to calcium‐binding proteins.

Plant pathogen attacks are perceived through pathogen-issued compounds or plant-derived molecules that elicit defense reactions. Despite the large variety of elicitors, general schemes for cellular elicitor signaling leading to plant resistance can be drawn. In this article, we review early signaling events that happen after elicitor perception, including reversible protein phosphorylations, changes in the activities of plasma membrane proteins, variations in free calcium concentrations in cytosol and nucleus, and production of nitric oxide and active oxygen species. These events occur within the first minutes to a few hours after elicitor perception. One specific elicitor transduction pathway can use a combination or a partial combination of such events which can differ in kinetics and intensity depending on the stimulus. The links between the signaling events allow amplification of the signal transduction and ensure specificity to get appropriate plant defense reactions. This review first describes the early events induced by cryptogein, an elicitor of tobacco defense reactions, in order to give a general scheme for signal transduction that will be use as a thread to review signaling events monitored in different elicitor or plant models.

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Nitric oxide (NO) has recently emerged as an important cellular mediator in plant defense responses. However, elucidation of the biochemical mechanisms by which NO participates in this signaling pathway is still in its infancy. We previously demonstrated that cryptogein, an elicitor of tobacco defense responses, triggers a NO burst within minutes in epidermal sections from tobacco leaves (Nicotiana tabacum cv Xanthi). Here, we investigate the signaling events that mediate NO production, and analyze NO signaling activities in the cryptogein transduction pathway. Using flow cytometry and spectrofluorometry, we observed that cryptogein-induced NO production in tobacco cell suspensions is sensitive to nitric oxide synthase inhibitors and may be catalyzed by variant P, a recently identified pathogen-inducible plant nitric oxide synthase. NO Moreover, NO appears to be involved in the pathway(s) leading to the accumulation of transcripts encoding the heat shock protein TLHS-1, the ethyleneforming enzyme cEFE-26, and cell death. In contrast, NO does not act upstream of the elicitor-induced activation of mitogenactivated protein kinase, the opening of anion channels, nor expression of GST, LOX-1, PAL, and PR-3 genes. Collectively, our data indicate that NO is intimately involved in the signal transduction processes leading to cryptogein-induced defense responses.Plants are frequently challenged by potential pathogens and have therefore evolved inducible defense mechanisms to survive in their environment. The activation of plant defense responses is initiated through the recognition of microorganism-derived molecules called elicitors, which trigger rapid defense responses via complex signal transduction pathways (Scheel, 1998). Plant defense responses classically include the production of active oxygen species (AOS), reinforcement of cell walls, and enhanced expression of a large number of defense-related genes, including those encoding cell wall proteins, enzymes involved in the phenylpropanoid biosynthetic pathway, and pathogenesis-related (PR) proteins (Fritig et al., 1998). Plants resisting pathogen attack sometimes develop a hypersensitive response (HR) in which rapid, localized cell death is associated with the restriction of invaders to the infection site (Lam et al., 2001). The HR shares some features of the apoptotic cell death process in animals, and it may likewise represent a type of programmed cell death (PCD; Lam et al., 2001). Moreover, plants usually develop a long-lasting resistance to diverse pathogens via systemic acquired resistance (SAR) or the less well understood induced systemic resistance (ISR; Pieterse et al., 1998).Cryptogein is a basic 10-kD proteinaceous elicitor secreted by the hemibiotrophic oomycete Phytophthora cryptogea (Ricci, 1997). The application of nanomolar concentrations of cryptogein to tobacco plants triggers expression of defense-related genes, induces an HR-like response, and inaugurates the protection of plants against invasion by a broad spectrum of microorganisms including...

BackgroundGlobal climate change will noticeably affect plant vegetative and reproductive development. The recent increase in temperatures has already impacted yields and composition of berries in many grapevine-growing regions. Physiological processes underlying temperature response and tolerance of the grapevine fruit have not been extensively investigated. To date, all studies investigating the molecular regulation of fleshly fruit response to abiotic stress were only conducted during the day, overlooking possible critical night-specific variations. The present study explores the night and day transcriptomic response of grapevine fruit to heat stress at several developmental stages. Short heat stresses (2 h) were applied at day and night to vines bearing clusters sequentially ordered according to the developmental stages along their vertical axes. The recently proposed microvine model (DRCF-Dwarf Rapid Cycling and Continuous Flowering) was grown in climatic chambers in order to circumvent common constraints and biases inevitable in field experiments with perennial macrovines. Post-véraison berry heterogeneity within clusters was avoided by constituting homogenous batches following organic acids and sugars measurements of individual berries. A whole genome transcriptomic approach was subsequently conducted using NimbleGen 090818 Vitis 12X (30 K) microarrays.ResultsPresent work reveals significant differences in heat stress responsive pathways according to day or night treatment, in particular regarding genes associated with acidity and phenylpropanoid metabolism. Precise distinction of ripening stages led to stage-specific detection of malic acid and anthocyanin-related transcripts modulated by heat stress. Important changes in cell wall modification related processes as well as indications for heat-induced delay of ripening and sugar accumulation were observed at véraison, an effect that was reversed at later stages.ConclusionsThis first day - night study on heat stress adaption of the grapevine berry shows that the transcriptome of fleshy fruits is differentially affected by abiotic stress at night. The present results emphasize the necessity of including different developmental stages and especially several daytime points in transcriptomic studies.

Reproductive development of grapevine and berry composition are both strongly influenced by temperature. To date, the molecular mechanisms involved in grapevine berries response to high temperatures are poorly understood. Unlike recent data that addressed the effects on berry development of elevated temperatures applied at the whole plant level, the present work particularly focuses on the fruit responses triggered by direct exposure to heat treatment (HT). In the context of climate change, this work focusing on temperature effect at the microclimate level is of particular interest as it can help to better understand the consequences of leaf removal (a common viticultural practice) on berry development. HT (+ 8°C) was locally applied to clusters from Cabernet Sauvignon fruiting cuttings at three different developmental stages (middle green, veraison and middle ripening). Samples were collected 1, 7, and 14 days after treatment and used for metabolic and transcriptomic analyses. The results showed dramatic and specific biochemical and transcriptomic changes in heat exposed berries, depending on the developmental stage and the stress duration. When applied at the herbaceous stage, HT delayed the onset of veraison. Heating also strongly altered the berry concentration of amino acids and organic acids (e.g., phenylalanine, γ-aminobutyric acid and malate) and decreased the anthocyanin content at maturity. These physiological alterations could be partly explained by the deep remodeling of transcriptome in heated berries. More than 7000 genes were deregulated in at least one of the nine experimental conditions. The most affected processes belong to the categories “stress responses,” “protein metabolism” and “secondary metabolism,” highlighting the intrinsic capacity of grape berries to perceive HT and to build adaptive responses. Additionally, important changes in processes related to “transport,” “hormone” and “cell wall” might contribute to the postponing of veraison. Finally, opposite effects depending on heating duration were observed for genes encoding enzymes of the general phenylpropanoid pathway, suggesting that the HT-induced decrease in anthocyanin content may result from a combination of transcript abundance and product degradation.

An estimated one-third of all proteins in higher eukaryotes are regulated by phosphorylation by protein kinases (PKs). Although plant genomes encode more than 1000 PKs, the substrates of only a small fraction of these kinases are known. By mass spectrometry of peptides from cytoplasmic- and nuclear-enriched fractions, we determined 303 in vivo phosphorylation sites in Arabidopsis proteins. Among 21 different PKs, 12 were phosphorylated in their activation loops, suggesting that they were in their active state. Immunoblotting and mutational analysis confirmed a tyrosine phosphorylation site in the activation loop of a GSK3/shaggy-like kinase. Analysis of phosphorylation motifs in the substrates suggested links between several of these PKs and many target sites. To perform quantitative phosphorylation analysis, peptide arrays were generated with peptides corresponding to in vivo phosphorylation sites. These peptide chips were used for kinome profiling of subcellular fractions as well as H 2O 2-treated Arabidopsis cells. Different peptide phosphorylation profiles indicated the presence of overlapping but distinct PK activities in cytosolic and nuclear compartments. Among different H 2O 2-induced PK targets, a peptide of the serine/arginine-rich (SR) splicing factor SCL30 was most strongly affected. SRPK4 (SR protein-specific kinase 4) and MAPKs (mitogen-activated PKs) were found to phosphorylate this peptide, as well as full-length SCL30. However, whereas SRPK4 was constitutively active, MAPKs were activated by H 2O 2. These results suggest that SCL30 is targeted by different PKs. Together, our data demonstrate that a combination of mass spectrometry with peptide chip phosphorylation profiling has a great potential to unravel phosphoproteome dynamics and to identify PK substrates.

In addition to their role as a source of reduced carbon, sugars may directly or indirectly control a wide range of activities in plant cells, through transcriptional and post-translational regulation. This control has been studied in detail using Arabidopsis thaliana, where genetic analysis offers many possibilities. Much less is known about perennial woody species. For several years, various aspects of sugar sensing and signalling have been investigated in the grape (Vitis vinifera L.) berry, an organ that accumulates high concentrations of hexoses in the vacuoles of flesh cells. Here we review various aspects of this topic: the molecular basis of sugar transport and its regulation by sugars in grapevine; the functional analysis of several sugar-induced genes; the effects of some biotic and abiotic stresses on the sugar content of the berry; and finally the effects of exogenous sugar supply on the ripening process in field conditions. A picture of complex feedback and multiprocess regulation emerges from these data.