BackgroundGrapes (Vitis vinifera L.) are economically the most important fruit crop worldwide. However, the complexity of molecular and biochemical events that lead to the onset of ripening of nonclimacteric fruits is not fully understood which is further complicated in grapes due to seasonal and cultivar specific variation. The Portuguese wine variety Trincadeira gives rise to high quality wines but presents extremely irregular berry ripening among seasons probably due to high susceptibility to abiotic and biotic stresses.ResultsRipening of Trincadeira grapes was studied taking into account the transcriptional and metabolic profilings complemented with biochemical data. The mRNA expression profiles of four time points spanning developmental stages from pea size green berries, through véraison and mature berries (EL 32, EL 34, EL 35 and EL 36) and in two seasons (2007 and 2008) were compared using the Affymetrix GrapeGen® genome array containing 23096 probesets corresponding to 18726 unique sequences. Over 50% of these probesets were significantly differentially expressed (1.5 fold) between at least two developmental stages. A common set of modulated transcripts corresponding to 5877 unigenes indicates the activation of common pathways between years despite the irregular development of Trincadeira grapes. These unigenes were assigned to the functional categories of "metabolism", "development", "cellular process", "diverse/miscellanenous functions", "regulation overview", "response to stimulus, stress", "signaling", "transport overview", "xenoprotein, transposable element" and "unknown". Quantitative RT-PCR validated microarrays results being carried out for eight selected genes and five developmental stages (EL 32, EL 34, EL 35, EL 36 and EL 38). Metabolic profiling using 1H NMR spectroscopy associated to two-dimensional techniques showed the importance of metabolites related to oxidative stress response, amino acid and sugar metabolism as well as secondary metabolism. These results were integrated with transcriptional profiling obtained using genome array to provide new information regarding the network of events leading to grape ripening.ConclusionsAltogether the data obtained provides the most extensive survey obtained so far for gene expression and metabolites accumulated during grape ripening. Moreover, it highlighted information obtained in a poorly known variety exhibiting particular characteristics that may be cultivar specific or dependent upon climatic conditions. Several genes were identified that had not been previously reported in the context of grape ripening namely genes involved in carbohydrate and amino acid metabolisms as well as in growth regulators; metabolism, epigenetic factors and signaling pathways. Some of these genes were annotated as receptors, transcription factors, and kinases and constitute good candidates for functional analysis in order to establish a model for ripening control of a non-climacteric fruit.

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Moderate heat response involves proteins related to lipid biogenesis, cytoskeleton structure, sulfate assimilation, thiamine and hydrophobic amino acid biosynthesis, and nuclear transport. Photostasis is achieved through carbon metabolism adjustment, a decrease of photosystem II (PSII) abundance and an increase of PSI contribution to photosynthetic linear electron flow. Thioredoxin h may have a special role in this process in P. euphratica upon moderate heat exposure.

Summary Growing pollen tubes of Agapanthus umbellatus exhibited a tip‐to‐base gradient in cytosolic free calcium ([Ca2+]c). Although this gradient is believed to be involved in pollen tube growth, its role in specifying reorientation is unknown. The direction of pollen tube growth could be modified by iontophoretic micro‐injection, electrical fields (EFs) or photolysis of caged Ca2+. Iontophoretic injection resulted in a temporary cessation of growth, an increase in [Ca2+]c and, upon recovery, reoriented growth. Weak EFs also elevated [Ca2+]c, reduced growth rates and resulted in the reorientation of pollen tubes towards the cathode. Treatment with very low concentrations of the Ca2+‐channel blocker lanthanum chloride, prior to exposure to an EF, inhibited both the increase in [Ca2+]c and reorientation whilst only slightly affecting growth rates. The responses of growth inhibition and reorientation were mimicked when [Ca2+]c was artificially elevated by photoactivating caged Ca2+ (Nitr‐5). Our data suggest that [Ca2+]c is part of a transduction mechanism which enables growing pollen tubes to successfully reorient to directional signals in the style and thus accomplish eventual fertilization of the egg.

We have shown previously that the inhibition of pollen tube growth and its subsequent reorientation in Agapanthus umbellatus are preceded by an increase in cytosolic free calcium ([Ca2+]c), suggesting a role for Ca2+ in signaling these processes. In this study, a novel procedure was used to measure Ca2+ channel activity in living pollen tubes subjected to various growth reorienting treatments (electrical fields and ionophoretic microinjection). The method involves adding extracellular Mn2+ to quench the fluorescence of intracellular Indo-1 at its ca2+-insensitive wavelength (isosbestic point). The spatial and temporal kinetics of Ca2+ channel activity correlated well with measurements of [Ca2+]c dynamics obtained by fluorescence ratio imaging of Indo-1. Tip-focused gradients in Ca2+ channel activity and [Ca2+]c were observed and quantified in growing pollen tubes and in swollen pollen tubes before reoriented growth. In nongrowing pollen tubes, Ca2+ channel activity was very low and [Ca2+]c gradients were absent. Measurements of membrane potential indicated that the growth reorienting treatments induced a depolarization of the plasma membrane, suggesting that voltage-gated Ca2+ channels might be activated.

Grapevine species (Vitis sp.) are prone to several diseases, fungi being the major pathogens compromising its cultivation and economic profit around the world. Knowledge of the complexity of mechanisms responsible for resistance to fungus infection of cultivars, such as Regent, is necessary for strategies to be defined which will improve resistance in highly susceptible crop species. Transcript and metabolic profiles of the Vitis vinifera cultivars Regent and Trincadeira (resistant and susceptible to fungi, respectively) were analysed by cDNA microarray, quantitative real-time PCR, and nuclear magnetic resonance spectroscopy. The integration of datasets obtained through transcriptome and metabolome analysis revealed differences in transcripts and metabolites between both cultivars. These differences are probably associated with the innate resistance of Regent towards the mildews. Several transcripts related to stress and defence, namely a subtilisin-like protease, phenylalanine ammonia lyase, S-adenosylmethionine synthase, WD-repeat protein like, and J2P, were up-regulated in Regent suggesting an intrinsic resistance capability of this cultivar. A metabolic profile revealed an accumulation of compounds such as inositol and caffeic acid, which are known to confer resistance to fungi. The differences in transcripts and metabolites detected are discussed in terms of the metabolic pathways and their possible role in plant defence against pathogen attack, as well as their potential interest to discriminate among resistant and susceptible grapevine cultivars.

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Arabidopsis PDK1 activity is regulated by binding to the lipid phosphatidic acid (PA) resulting in activation of the oxidative stress-response protein kinase OXI1/AGC2-1. Thus there is an inferred link between lipid signaling and oxidative stress signaling modules. Among a panel of hormones and stresses tested, we found that, in addition to PA, the fungal elicitor xylanase activated PDK1, suggesting that PDK1 has a role in plant pathogen defense mechanisms. The downstream OXI1 was activated by additional stress factors, including PA, H 2 O 2 , and partially by xylanase. We have isolated an interacting partner of OXI1, a Ser/Thr kinase (PTI1-2), which is downstream of OXI1. Its sequence closely resembles the tomato Pti kinase, which has been implicated in the hypersensitive response, a localized programmed cell death that occurs at the site of pathogen infection. PTI1-2 is activated by the same stresses/elicitors as OXI1 and additionally flagellin. We have used RNA interference to knock out the expression of PDK1 and OXI1 and to study the effects on PTI1-2 activity. We show that specific lipid signaling pathways converge on PTI1-2 via the PDK1-OXI1 axis, whereas H 2 O 2 and flagellin signals to OXI1-PTI1-2 via a PDK1-independent pathway. PTI1-2 represents a new downstream component that integrates diverse lipid and reactive oxygen stress signals and functions closely with OXI1. It is now clear that reactive oxygen species (ROS)2 play an important signaling role in plants controlling processes such as growth, development, programmed cell death, and responses to biotic and abiotic environmental stimuli (1). Current evidence supports the concept that ROS represent a significant point of convergence between pathways that respond to biotic and abiotic stresses (2). Nevertheless, our current understanding of ROS participation in cross-talk between these pathways is very limited (2). OXI1/AGC2-1 is a protein kinase that was identified as a downstream signaling component to the PDK1 (3-phosphinositide-dependent protein kinase 1) and as a protein kinase that is required for oxidative burst-mediated signaling in Arabidopsis, hereafter referred to as OXI1 (3, 4). OXI1 is critical for at least two very different ROS-mediated processes, basal resistance to Peronospora parasitica infection and root hair growth (3, 4). OXI1 is a member of the AGC family of protein kinases, and we have reported previously that OXI1 is activated by PDK1-mediated phosphorylation. In addition PDK1 acts upstream of other AGC kinases and regulates diverse signaling pathways, involved in root hair growth, auxin regulation, and plant cell death (3, 5, 6). PDK1 enzyme activity is regulated by binding the lipid signaling molecule, phosphatidic acid (PA), to its pleckstrin homology (PH) domain (3). PA is produced in response to many different stresses, including absisic acid (ABA)-induced stomatal closure, pathogen attack, and oxidative stress (7,8). It is generated via two distinct phospholipase pathways, either directly by phospholipase D (PLD) or the s...