Identification of early and late flowering time candidate genes in endodormant and ecodormant almond flower buds.

Flower bud dormancy in temperate fruit tree species, such as almond [Prunus dulcis (Mill.) D.A. Webb], is a survival mechanism that ensures that flowering will occur under suitable weather conditions for successful flower development, pollination and fruit set. Dormancy is divided into three sequential phases: paradormancy, endodormancy and ecodormancy. During the winter, buds need cultivar-specific chilling requirements (CRs) to overcome endodormancy and heat requirements to activate the machinery to flower in the ecodormancy phase. One of the main factors that enables the transition from endodormancy to ecodormancy is transcriptome reprogramming. In this work, we therefore monitored three almond cultivars with different CRs and flowering times by RNA sequencing during the endodormancy release of flower buds and validated the data by quantitative real-time PCR in two consecutive seasons. We were thus able to identify early and late flowering time candidate genes in endodormant and ecodormant almond flower buds associated with metabolic switches, transmembrane transport, cell wall remodeling, phytohormone signaling and pollen development. These candidate genes were indeed involved in the overcoming of the endodormancy in almond. This information may be used for the development of dormancy molecular markers, increasing the efficiency of temperate fruit tree breeding programs in a climate-change context.

SHORT-ROOT Deficiency Alleviates the Cell Death Phenotype of the Arabidopsis catalase2 Mutant under Photorespiration-Promoting Conditions.

Hydrogen peroxide (H2O2) can act as a signaling molecule that influences various aspects of plant growth and development, including stress signaling and cell death. To analyze molecular mechanisms that regulate the response to increased H2O2 levels in plant cells, we focused on the photorespiration-dependent peroxisomal H2O2 production in Arabidopsis thaliana mutants lacking CATALASE2 (CAT2) activity (cat2-2). By screening for second-site mutations that attenuate the PSII maximum efficiency (Fv'/Fm') decrease and lesion formation linked to the cat2-2 phenotype, we discovered that a mutation in SHORT-ROOT (SHR) rescued the cell death phenotype of cat2-2 plants under photorespiration-promoting conditions. SHR deficiency attenuated H2O2-dependent gene expression, oxidation of the glutathione pool, and ascorbate depletion in a cat2-2 genetic background upon exposure to photorespiratory stress. Decreased glycolate oxidase and catalase activities together with accumulation of glycolate further implied that SHR deficiency impacts the cellular redox homeostasis by limiting peroxisomal H2O2 production. The photorespiratory phenotype of cat2-2 mutants did not depend on the SHR functional interactor SCARECROW and the sugar signaling component ABSCISIC ACID INSENSITIVE4, despite the requirement for exogenous sucrose for cell death attenuation in cat2-2 shr-6 double mutants. Our findings reveal a link between SHR and photorespiratory H2O2 production that has implications for the integration of developmental and stress responses.

Lack of GLYCOLATE OXIDASE1, but Not GLYCOLATE OXIDASE2, Attenuates the Photorespiratory Phenotype of CATALASE2-Deficient Arabidopsis.

The genes coding for the core metabolic enzymes of the photorespiratory pathway that allows plants with C3-type photosynthesis to survive in an oxygen-rich atmosphere, have been largely discovered in genetic screens aimed to isolate mutants that are unviable under ambient air. As an exception, glycolate oxidase (GOX) mutants with a photorespiratory phenotype have not been described yet in C3 species. Using Arabidopsis (Arabidopsis thaliana) mutants lacking the peroxisomal CATALASE2 (cat2-2) that display stunted growth and cell death lesions under ambient air, we isolated a second-site loss-of-function mutation in GLYCOLATE OXIDASE1 (GOX1) that attenuated the photorespiratory phenotype of cat2-2 Interestingly, knocking out the nearly identical GOX2 in the cat2-2 background did not affect the photorespiratory phenotype, indicating that GOX1 and GOX2 play distinct metabolic roles. We further investigated their individual functions in single gox1-1 and gox2-1 mutants and revealed that their phenotypes can be modulated by environmental conditions that increase the metabolic flux through the photorespiratory pathway. High light negatively affected the photosynthetic performance and growth of both gox1-1 and gox2-1 mutants, but the negative consequences of severe photorespiration were more pronounced in the absence of GOX1, which was accompanied with lesser ability to process glycolate. Taken together, our results point toward divergent functions of the two photorespiratory GOX isoforms in Arabidopsis and contribute to a better understanding of the photorespiratory pathway.

Spreading the news: subcellular and organellar reactive oxygen species production and signalling.

As plants are sessile organisms that have to attune their physiology and morphology continuously to varying environmental challenges in order to survive and reproduce, they have evolved complex and integrated environment-cell, cell-cell, and cell-organelle signalling circuits that regulate and trigger the required adjustments (such as alteration of gene expression). Although reactive oxygen species (ROS) are essential components of this network, their pathways are not yet completely unravelled. In addition to the intrinsic chemical properties that define the array of interaction partners, mobility, and stability, ROS signalling specificity is obtained via the spatiotemporal control of production and scavenging at different organellar and subcellular locations (e.g. chloroplasts, mitochondria, peroxisomes, and apoplast). Furthermore, these cellular compartments may crosstalk to relay and further fine-tune the ROS message. Hence, plant cells might locally and systemically react upon environmental or developmental challenges by generating spatiotemporally controlled dosages of certain ROS types, each with specific chemical properties and interaction targets, that are influenced by interorganellar communication and by the subcellular location and distribution of the involved organelles, to trigger the suitable acclimation responses in association with other well-established cellular signalling components (e.g. reactive nitrogen species, phytohormones, and calcium ions). Further characterization of this comprehensive ROS signalling matrix may result in the identification of new targets and key regulators of ROS signalling, which might be excellent candidates for engineering or breeding stress-tolerant plants.

Activation of auxin signalling counteracts photorespiratory H2O2-dependent cell death.

The high metabolic flux through photorespiration constitutes a significant part of the carbon cycle. Although the major enzymatic steps of the photorespiratory pathway are well characterized, little information is available on the functional significance of photorespiration beyond carbon recycling. Particularly important in this respect is the peroxisomal catalase activity which removes photorespiratory H2O2 generated during the oxidation of glycolate to glyoxylate, thus maintaining the cellular redox homeostasis governing the perception, integration and execution of stress responses. By performing a chemical screen, we identified 34 small molecules that alleviate the negative effects of photorespiration in Arabidopsis thaliana mutants lacking photorespiratory catalase (cat2). The chlorophyll fluorescence parameter photosystem II maximum efficiency (Fv'/Fm') was used as a high-throughput readout. The most potent chemical that could rescue the photorespiratory phenotype of cat2 is a pro-auxin that contains a synthetic auxin-like substructure belonging to the phenoxy herbicide family, which can be released in planta. The naturally occurring indole-3-acetic acid (IAA) and other chemically distinct synthetic auxins also inhibited the photorespiratory-dependent cell death in cat2 mutants, implying a role for auxin signalling in stress tolerance.

Cryptogein-induced transcriptional reprogramming in tobacco is light dependent.

The fungal elicitor cryptogein triggers a light-dependent hypersensitive response in tobacco (Nicotiana tabacum). To assess the effect of light on this nonhost resistance in more detail, we studied various aspects of the response under dark and light conditions using the tobacco-cryptogein experimental system. Here, we show that light drastically alters the plant's transcriptional response to cryptogein, notably by dampening the induction of genes involved in multiple processes, such as ethylene biosynthesis, secondary metabolism, and glutathione turnover. Furthermore, chlorophyll fluorescence measurements demonstrated that quantum yield and functioning of the light-harvesting antennae decreased simultaneously, indicating that photoinhibition underlies the observed decreased photosynthesis and that photooxidative damage might be involved in the establishment of the altered response. Analysis of the isomer distribution of hydroxy fatty acids illustrated that, in the light, lipid peroxidation was predominantly due to the production of singlet oxygen. Differences in (reduced) glutathione concentrations and the rapid development of symptoms in the light when cryptogein was coinfiltrated with glutathione biosynthesis inhibitors suggest that glutathione might become a limiting factor during the cryptogein-induced hypersensitive response in the dark and that this response might be modified by an increased antioxidant availability in the light.

A subcellular localization compendium of hydrogen peroxide-induced proteins.

The signal transduction mechanisms of the oxidative stress response in plants remain largely unexplored. Previously, increased levels of cellular hydrogen peroxide (H(2)O(2)) had been shown to drastically affect the plant transcriptome. Genome-wide transcriptome analyses allowed us to build a comprehensive inventory of H(2)O(2)-induced genes in plants. Here, the primary objective was to determine the subcellular localization of these genes and to assess potential trafficking during oxidative stress. After high-throughput cloning in Gateway-derived vectors, the subcellular localization of 49 proteins fused to the green fluorescent protein (GFP) was identified in a transient assay in tobacco (Nicotiana benthamiana) by means of agro-infiltration and confirmed for a selection of genes in transgenic Arabidopsis thaliana plants. Whereas eight of the GFP-tagged proteins are exclusively localized in the nucleus, 23 reside both in the nucleus and cytosol, in which several classes of known transcription factors and proteins of unknown function can be recognized. In this study, the mapping of the subcellular localization of H(2)O(2) -induced proteins paves the way for future research to unravel the H(2)O(2) responses in plants. Furthermore, the effect of increased H(2)O(2) levels on the subcellular localization of a subset of proteins was assessed.

The role of K(+) and H(+) transport systems during glucose- and H(2)O(2)-induced cell death in Saccharomyces cerevisiae.

Glucose, in the absence of additional nutrients, induces programmed cell death in yeast. This phenomenon is independent of yeast metacaspase (Mca1/Yca1) and of calcineurin, requires ROS production and it is concomitant with loss of cellular K(+) and vacuolar collapse. K(+) is a key nutrient protecting the cells and this effect depends on the Trk1 uptake system and is associated with reduced ROS production. Mutants with decreased activity of plasma membrane H(+)-ATPase are more tolerant to glucose-induced cell death and exhibit less ROS production. A triple mutant ena1-4 tok1 nha1, devoid of K(+) efflux systems, is more tolerant to both glucose- and H(2)O(2)-induced cell death. We hypothesize that ROS production, activated by glucose and H(+)-ATPase and inhibited by K(+) uptake, triggers leakage of K(+), a process favoured by K(+) efflux systems. Loss of cytosolic K(+) probably causes osmotic lysis of vacuoles. The nature of the ROS-producing system sensitive to K(+) and H(+) transport is unknown.

Singlet oxygen is the major reactive oxygen species involved in photooxidative damage to plants.

Reactive oxygen species act as signaling molecules but can also directly provoke cellular damage by rapidly oxidizing cellular components, including lipids. We developed a high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry-based quantitative method that allowed us to discriminate between free radical (type I)- and singlet oxygen ((1)O(2); type II)-mediated lipid peroxidation (LPO) signatures by using hydroxy fatty acids as specific reporters. Using this method, we observed that in non-photosynthesizing Arabidopsis (Arabidopsis thaliana) tissues, nonenzymatic LPO was almost exclusively catalyzed by free radicals both under normal and oxidative stress conditions. However, in leaf tissues under optimal growth conditions, (1)O(2) was responsible for more than 80% of the nonenzymatic LPO. In Arabidopsis mutants favoring (1)O(2) production, photooxidative stress led to a dramatic increase of (1)O(2) (type II) LPO that preceded cell death. Furthermore, under all conditions and in mutants that favor the production of superoxide and hydrogen peroxide (two sources for type I LPO reactions), plant cell death was nevertheless always preceded by an increase in (1)O(2)-dependent (type II) LPO. Thus, besides triggering a genetic cell death program, as demonstrated previously with the Arabidopsis fluorescent mutant, (1)O(2) plays a major destructive role during the execution of reactive oxygen species-induced cell death in leaf tissues.

A Temperature-sensitive mutation in the Arabidopsis thaliana phosphomannomutase gene disrupts protein glycosylation and triggers cell death.

Eukaryotic phosphomannomutases (PMMs) catalyze the interconversion of mannose 6-phosphate to mannose 1-phosphate and are essential to the biosynthesis of GDP-mannose. As such, plant PMMs are involved in ascorbic acid (AsA) biosynthesis and N-glycosylation. We report on the conditional phenotype of the temperature-sensitive Arabidopsis thaliana pmm-12 mutant. Mutant seedlings were phenotypically similar to wild type seedlings when grown at 16-18 degrees C but died within several days after transfer to 28 degrees C. This phenotype was observed throughout both vegetative and reproductive development. Protein extracts derived from pmm-12 plants had lower PMM protein and enzyme activity levels. In vitro biochemical analysis of recombinant proteins showed that the mutant PMM protein was compromised in its catalytic efficiency (K cat/K m). Despite significantly decreased AsA levels in pmm-12 plants, AsA deficiency could not account for the observed phenotype. Since, at restrictive temperature, total glycoprotein patterns were altered and glycosylation of protein-disulfide isomerase was perturbed, we propose that a deficiency in protein glycosylation is responsible for the observed cell death phenotype.

Conditional oxidative stress responses in the Arabidopsis photorespiratory mutant cat2 demonstrate that redox state is a key modulator of daylength-dependent gene expression, and define photoperiod as a crucial factor in the regulation of H2O2-induced cell death.

Photorespiration is a light-dependent source of H(2)O(2) in the peroxisomes, where concentrations of this signalling molecule are regulated by catalase. Growth of Arabidopsis knock-out mutants for CATALASE2 (cat2) in ambient air caused severely decreased rosette biomass, intracellular redox perturbation and activation of oxidative signalling pathways. These effects were absent when cat2 was grown at high CO(2) levels to inhibit photorespiration, but were re-established following a subsequent transfer to air. Growth of cat2 in air at different daylengths revealed that photoperiod is a critical determinant of the oxidative stress response. Decreased growth was observed in 8-h, 12-h and 16-h photoperiods, but lesion development was dependent on long days. Experiments at different light fluence rates showed that cell death in cat2 was linked to long days and not to total light exposure or the severity of oxidative stress. Perturbed intracellular redox state and oxidative signalling pathway induction were more prominent in short days than in long days, as evidenced by glutathione status and induction of defence genes and oxidative stress-responsive transcripts. Similar daylength-dependent effects were observed in the response of mature plants transferred from short days in high CO(2) conditions to ambient air conditions. Prior growth of plants with short days in air alleviated the cat2 cell-death phenotype in long days. Together, the data reveal the influence of photoperiodic events on redox signalling, and define distinct photoperiod-dependent strategies in the acclimation versus cell-death decision in stress conditions.

Sucrose prevents up-regulation of senescence-associated genes in carnation petals.

cDNA microarrays were used to characterize senescence-associated gene expression in petals of cut carnation (Dianthus caryophyllus) flowers, sampled from anthesis to the first senescence symptoms. The population of PCR fragments spotted on these microarrays was enriched for flower-specific and senescence-specific genes, using subtractive hybridization. About 90% of the transcripts showed a large increase in quantity, approximately 25% transiently, and about 65% throughout the 7 d experiment. Treatment with silver thiosulphate (STS), which blocks the ethylene receptor and prevented the normal senescence symptoms, prevented the up-regulation of almost all of these genes. Sucrose treatment also considerably delayed visible senescence. Its effect on gene expression was very similar to that of STS, suggesting that soluble sugars act as a repressor of ethylene signal transduction. Two fragments that encoded a carnation EIN3-like (EIL) protein were isolated, some of which are key transcription factors that control ethylene response genes. One of these (Dc-EIL3) was up-regulated during senescence. Its up-regulation was delayed by STS and prevented by sucrose. Sucrose, therefore, seems to repress ethylene signalling, in part, by preventing up-regulation of Dc-EIL3. Some other transcription factors displayed an early increase in transcript abundance: a MYB-like DNA binding protein, a MYC protein, a MADS-box factor, and a zinc finger protein. Genes suggesting a role in senescence of hormones other than ethylene encoded an Aux/IAA protein, which regulate transcription of auxin-induced genes, and a cytokinin oxidase/dehydrogenase, which degrades cytokinin. Taken together, the results suggest a master switch during senescence, controlling the co-ordinated up-regulation of numerous ethylene response genes. Dc-EIL3 might be (part of) this master switch.

A tomato metacaspase gene is upregulated during programmed cell death in Botrytis cinerea-infected leaves.

Programmed cell death (PCD) in plant cells is often accompanied by biochemical and morphological hallmarks similar to those of animal apoptosis. However, orthologs of animal caspases, cysteinyl aspartate-specific proteases that constitute the core component of animal apoptosis, have not yet been identified in plants. Recent studies have revealed the presence of a family of genes encoding proteins with distant homology to mammalian caspases, designated metacaspases, in the Arabidopsis thaliana genome. Here, we describe the isolation of LeMCA1, a type-II metacaspase cDNA clone from tomato (Lycopersicon esculentum Mill.). BLAST analysis demonstrated that the LeMCA1 gene is located in close vicinity of several genes that have been linked with PCD. Southern analysis indicated the existence of at least one more metacaspase in the tomato genome. LeMCA1 mRNA levels rapidly increased upon infection of tomato leaves with Botrytis cinerea, a fungal pathogen that induces cell death in several plant species. LeMCA1 was not upregulated during chemical-induced PCD in suspension-cultured tomato cells.

Multiple mediators of plant programmed cell death: interplay of conserved cell death mechanisms and plant-specific regulators.

Programmed cell death (PCD) is a process aimed at the removal of redundant, misplaced, or damaged cells and it is essential to the development and maintenance of multicellular organisms. In contrast to the relatively well-described cell death pathway in animals, often referred to as apoptosis, mechanisms and regulation of plant PCD are still ill-defined. Several morphological and biochemical similarities between apoptosis and plant PCD have been described, including DNA laddering, caspase-like proteolytic activity, and cytochrome c release from mitochondria. Reactive oxygen species (ROS) have emerged as important signals in the activation of plant PCD. In addition, several plant hormones may exert their respective effects on plant PCD through the regulation of ROS accumulation. The possible plant PCD regulators discussed in this review are integrated in a model that combines plant-specific regulators with mechanisms functionally conserved between animals and plants.