PGR5-LIKE PHOTOSYNTHETIC PHENOTYPE1A redox states alleviate photoinhibition during changes in light intensity.

Plants have evolved photosynthetic regulatory mechanisms to maintain homeostasis in response to light changes during diurnal transitions and those caused by passing clouds or by wind. One such adaptation directs photosynthetic electron flow to a cyclic pathway to alleviate excess energy surges. Here, we assign a function to regulatory cysteines of PGR5-like protein 1A (PGRL1A), a constituent of the PROTON GRADIENT REGULATION5 (PGR5)-dependent cyclic electron flow (CEF) pathway. During step increases from darkness to low light intensity in Arabidopsis (Arabidopsis thaliana), the intermolecular disulfide of the PGRL1A 59-kDa complex was reduced transiently within seconds to the 28-kDa form. In contrast, step increases from darkness to high light stimulated a stable, partially reduced redox state in PGRL1A. Mutations of 2 cysteines in PGRL1A, Cys82 and Cys183, resulted in a constitutively pseudo-reduced state. The mutant displayed higher proton motive force (PMF) and nonphotochemical quenching (NPQ) than the wild type (WT) and showed altered donor and acceptor dynamic flow around PSI. These changes were found to correspond with the redox state of PGRL1A. Continuous light regimes did not affect mutant growth compared to the WT. However, under fluctuating regimes of high light, the mutant showed better growth than the WT. In contrast, in fluctuating regimes of low light, the mutant displayed a growth penalty that can be attributed to constant stimulation of CEF under low light. Treatment with photosynthetic inhibitors indicated that PGRL1A redox state control depends on the penultimate Fd redox state. Our results showed that redox state changes in PGRL1A are crucial to optimize photosynthesis.

Multiple paths of plant host toxicity are associated with the fungal toxin cercosporin.

The Cercospora species of fungi are responsible for leaf spot disease affecting many key economic crops. Most of these fungi secrete a toxic photodynamic molecule, cercosporin, that reacts with light and oxygen to produce reactive singlet oxygen (1 O2 ) contributing to fungal virulence. We show similar cellular localization and aetiology of cercosporin in the non-host Arabidopsis and the host Nicotiana benthamiana. Cercosporin accumulates in cell membranes in an oxidized state and in plastids in a mixture of redox states in a manner that is dependent on ongoing photosynthetic processes. We observed that cercosporin rapidly compromised photosynthesis as measured by Fv /Fm , NPQ, and photosystem I (PSI) parameters. Stomatal guard cells in particular demonstrated rapid light-dependent membrane permeabilization that led to changes in leaf conductance. We showed that cercosporin-mediated 1 O2 generation oxidized RNA to form 8-oxoguanosine (8-oxoG), leading to translational attenuation and induction of 1 O2 signature gene transcripts. We also identified a subset of cercosporin-induced transcripts that were independent of the photodynamic effect. Our results point to the multimodal action of cercosporin that includes the inhibition of photosynthesis, the direct oxidation of nucleic acid residues and the elicitation of complex transcriptome responses.

Double-stranded RNA targeting fungal ergosterol biosynthesis pathway controls Botrytis cinerea and postharvest grey mould.

Pathogenic fungi cause major postharvest losses. During storage and ripening, fruit becomes highly susceptible to fungi that cause postharvest disease. Fungicides are effective treatments to limit disease. However, due to increased public concern for their possible side effects, there is a need to develop new strategies to control postharvest fungal pathogens. Botrytis cinerea, a common postharvest pathogen, was shown to uptake small double-stranded RNA (dsRNA) molecules from the host plant. Such dsRNA can regulate gene expression through the RNA interference system. This work aimed to develop a synthetic dsRNA simultaneously targeting three essential transcripts active in the fungal ergosterol biosynthesis pathway (dsRNA-ERG). Our results show initial uptake of dsRNA in the emergence zone of the germination tube that spreads throughout the fungus and results in down-regulation of all three targeted transcripts. Application of dsRNA-ERG decreased B. cinerea germination and growth in in vitro conditions and various fruits, leading to reduce grey-mould decay. The inhibition of growth or decay was reversed by the addition of ergosterol. While dual treatment with dsRNA-ERG and ergosterol-inhibitor fungicide reduced by 100-fold the required amount of fungicide to achieve the same protection rate. The application of dsRNA-ERG induced systemic protection as shown by decreased decay development at inoculation points distant from the treatment point in tomato and pepper fruits. Overall, this study suggests that dsRNA-ERG can effectively control B. cinerea growth and grey-mould development suggesting its efficacy as a future method for postharvest control of fungal pathogens.

Lipoxygenase functions in 1O2 production during root responses to osmotic stress.

Drought induces osmotic stress in roots, a condition simulated by the application of high-molecular-weight polyethylene glycol. Osmotic stress results in the reduction of Arabidopsis thaliana root growth and production of 1O2 from an unknown non-photosynthetic source. Reduced root growth can be alleviated by application of the 1O2 scavenger histidine (HIS). Here, we examined the possibility that 1O2 production involves Russell reactions occurring among the enzymatic products of lipoxygenases (LOXs), the fatty acid hydroperoxides. LOX activity was measured for purified soybean (Glycine max) LOX1 and in crude Arabidopsis root extracts using linoleic acid as substrate. Formation of the 13(S)-Hydroperoxy-9(Z),11(E)-octadecadienoic acid product was inhibited by salicylhdroxamic acid, which is a LOX inhibitor, but not by HIS, whereas 1O2 production was inhibited by both. D2O, which specifically extends the half-life of 1O2, augmented the LOX-dependent generation of 1O2, as expected from a Russell-type reaction. The addition of linoleic acid to roots stimulated 1O2 production and inhibited growth, suggesting that the availability of LOX substrate is a rate-limiting step. Indeed, water stress rapidly increased linoleic and linolenic acids by 2.5-fold in roots. Mutants with root-specific microRNA repression of LOXs showed downregulation of LOX protein and activity. The lines with downregulated LOX displayed significantly less 1O2 formation, improved root growth in osmotic stress, and an altered transcriptome response compared with wild type. The results show that LOXs can serve as an enzymatic source of "dark" 1O2 during osmotic stress and demonstrate a role for 1O2 in defining the physiological response.

Enhanced proteostasis, lipid remodeling, and nitrogen remobilization define barley flag leaf senescence.

Leaf senescence is a developmental process allowing nutrient remobilization to sink organs. We characterized flag leaf senescence at 7, 14, and 21 d past anthesis in two near-isogenic barley lines varying in the allelic state of the HvNAM1 transcription factor gene, which influences senescence timing. Metabolomics and microscopy indicated that, as senescence progressed, thylakoid lipids were transiently converted to neutral lipids accumulating in lipid droplets. Senescing leaves also exhibited an accumulation of sugars including glucose, while nitrogen compounds (nucleobases, nucleotides, and amino acids) decreased. RNA-Seq analysis suggested lipid catabolism via ß-oxidation and the glyoxylate cycle, producing carbon skeletons and feeding respiration as a replacement of the diminished carbon supply from photosynthesis. Comparison of the two barley lines highlighted a more prominent up-regulation of heat stress transcription factor- and chaperone-encoding genes in the late-senescing line, suggesting a role for these genes in the control of leaf longevity. While numerous genes with putative roles in nitrogen remobilization were up-regulated in both lines, several peptidases, nucleases, and nitrogen transporters were more highly induced in the early-senescing line; this finding identifies processes and specific candidates which may affect nitrogen remobilization from senescing barley leaves, downstream of the HvNAM1 transcription factor.

Attenuation of cytosolic translation by RNA oxidation is involved in singlet oxygen-mediated transcriptomic responses.

Singlet oxygen (1 O2 ) production is associated with stress signalling. Here, using Arabidopsis as a model system, we study the effects of the accumulation of 8-hydroxyguanosine (8-oxoG), a major product of 1 O2 -mediated RNA oxidation. We show that 8-oxoG can accumulate in vivo when 1 O2 is produced in the cytoplasm. Conditions for such production include the application of RB in the light, dark-to-light transitions in the flu mutant, or subjecting plants to combined dehydration/light exposure. Transcriptomes of these treatments displayed a significant overlap with transcripts stimulated by the cytosolic 80S ribosomal translation inhibitors, cycloheximide and homoharringtonine. We demonstrate that 8-oxoG accumulation correlates with a decrease in RNA translatability, resulting in the rapid decrease of the levels of labile gene repressor elements such as IAA1 and JAZ1 in a proteasome-dependent manner. Indeed, genes regulated by the labile repressors of the jasmonic acid signalling pathway were induced by cycloheximide, RB or dehydration/light treatment independently of the hormone. The results suggest that 1 O2 , by oxidizing RNA, attenuated cellular translatability and caused specific genes to be released from the repression of their cognate short half-life repressors. The findings here describe a novel means of gene regulation via the direct interaction of 1 O2 with RNA.

Singlet Oxygen Plays an Essential Role in the Root's Response to Osmotic Stress.

The high osmotic potentials in plants subjected to drought stress can be mimicked by the application of high molecular weight polyethylene glycol. Here, we quantified the effects of exposure to polyethylene glycol on the growth of the main and lateral roots of Arabidopsis (Arabidopsis thaliana) seedlings. The effects on root growth were highly correlated with the appearance of singlet oxygen, as visualized using the singlet oxygen-specific probe singlet oxygen sensor green. The production of singlet oxygen was followed by cell death, as indicated by the intracellular accumulation of propidium iodide due to the loss of membrane integrity. Cell death began in the epidermal region of the root tip and spread in a dynamic manner to meristematic sections. In parallel, gene expression changes specific to the presence of singlet oxygen were observed. The accumulation of other reactive oxygen species, namely hydrogen, peroxide, nitric oxide, and superoxide, did not correlate with cell death. In addition, both the singlet oxygen scavenger His and the lipoxygenase inhibitor salicylhydroxamic acid specifically inhibited singlet oxygen accumulation and cell death. These results suggest a light-independent, type-I source of singlet oxygen production. Serpin-protease interactions were used as a model to assess the possibility of vacuolar-type cell death. Osmotic stress induced the accumulation of complexes between the cytoplasmic serpin AtSERPIN1 and its cognate vacuolar proteases, indicating that vacuolar integrity was compromised. These findings imply that singlet oxygen plays an essential role in conveying the root response to osmotic stress.

Early Senescence in Older Leaves of Low Nitrate-Grown Atxdh1 Uncovers a Role for Purine Catabolism in N Supply.

The nitrogen (N)-rich ureides allantoin and allantoate, which are products of purine catabolism, play a role in N delivery in Leguminosae. Here, we examined their role as an N source in nonlegume plants using Arabidopsis (Arabidopsis thaliana) plants mutated in XANTHINE DEHYDROGENASE1 (AtXDH1), a catalytic bottleneck in purine catabolism. Older leaves of the Atxdh1 mutant exhibited early senescence, lower soluble protein, and lower organic N levels as compared with wild-type older leaves when grown with 1 mm nitrate but were comparable to the wild type under 5 mm nitrate. Similar nitrate-dependent senescence phenotypes were evident in the older leaves of allantoinase (Ataln) and allantoate amidohydrolase (Ataah) mutants, which also are impaired in purine catabolism. Under low-nitrate conditions, xanthine accumulated in older leaves of Atxdh1, whereas allantoin accumulated in both older and younger leaves of Ataln but not in wild-type leaves, indicating the remobilization of xanthine-degraded products from older to younger leaves. Supporting this notion, ureide transporter expression was enhanced in older leaves of the wild type in low-nitrate as compared with high-nitrate conditions. Elevated transcripts and proteins of AtXDH and AtAAH were detected in low-nitrate-grown wild-type plants, indicating regulation at protein and transcript levels. The higher nitrate reductase activity in Atxdh1 leaves compared with wild-type leaves indicated a need for nitrate assimilation products. Together, these results indicate that the absence of remobilized purine-degraded N from older leaves of Atxdh1 caused senescence symptoms, a result of higher chloroplastic protein degradation in older leaves of low-nitrate-grown plants.

Plant serpin protease inhibitors: specificity and duality of function.

The serpins are a family of structurally conserved protease inhibitors found in all animal and plant kingdoms. After interaction with their cognate substrate(s), their native energetically stressed state is relaxed by hydrolysis, resulting in a semi-stable covalent bond that disables the protease. The inherent flexible serpin structure supports additional non-inhibitory functions. This review will focus on several biological functions attributed to plant serpins, ranging from specific cell death protease inhibitors to a stabilizing role for ß-amylase in seeds. Functional conservation of a particular serpin type, the LR serpins, is suggested by its compelling ubiquity throughout the plant kingdom. The multiple target specificity of plant serpins including the LR serpins enables them to perform dual functions that are not mutually exclusive both as a regulator of cell death and as a protective anti-pathogenic protein.

Singlet Oxygen-Induced Membrane Disruption and Serpin-Protease Balance in Vacuolar-Driven Cell Death.

Singlet oxygen plays a role in cellular stress either by providing direct toxicity or through signaling to initiate death programs. It was therefore of interest to examine cell death, as occurs in Arabidopsis, due to differentially localized singlet oxygen photosensitizers. The photosensitizers rose bengal (RB) and acridine orange (AO) were localized to the plasmalemma and vacuole, respectively. Their photoactivation led to cell death as measured by ion leakage. Cell death could be inhibited by the singlet oxygen scavenger histidine in treatments with AO but not with RB In the case of AO treatment, the vacuolar membrane was observed to disintegrate. Concomitantly, a complex was formed between a vacuolar cell-death protease, RESPONSIVE TO DESSICATION-21 and its cognate cytoplasmic protease inhibitor ATSERPIN1. In the case of RB treatment, the tonoplast remained intact and no complex was formed. Over-expression of AtSerpin1 repressed cell death, only under AO photodynamic treatment. Interestingly, acute water stress showed accumulation of singlet oxygen as determined by fluorescence of Singlet Oxygen Sensor Green, by electron paramagnetic resonance spectroscopy and the induction of singlet oxygen marker genes. Cell death by acute water stress was inhibited by the singlet oxygen scavenger histidine and was accompanied by vacuolar collapse and the appearance of serpin-protease complex. Over-expression of AtSerpin1 also attenuated cell death under this mode of cell stress. Thus, acute water stress damage shows parallels to vacuole-mediated cell death where the generation of singlet oxygen may play a role.

Organelle redox autonomy during environmental stress.

Oxidative stress is generated in plants because of inequalities in the rate of reactive oxygen species (ROS) generation and scavenging. The subcellular redox state under various stress conditions was assessed using the redox reporter roGFP2 targeted to chloroplastic, mitochondrial, peroxisomal and cytosolic compartments. In parallel, the vitality of the plant was measured by ion leakage. Our results revealed that during certain physiological stress conditions the changes in roGFP2 oxidation are comparable to application of high concentrations of exogenous H2 O2 . Under each stress, particular organelles were affected. Conditions of extended dark stress, or application of elicitor, impacted chiefly on the status of peroxisomal redox state. In contrast, conditions of drought or high light altered the status of mitochondrial or chloroplast redox state, respectively. Amalgamation of the results from diverse environmental stresses shows cases of organelle autonomy as well as multi-organelle oxidative change. Importantly, organelle-specific oxidation under several stresses proceeded cell death as measured by ion leakage, suggesting early roGFP oxidation as predictive of cell death. The measurement of redox state in multiple compartments enables one to look at redox state connectivity between organelles in relation to oxidative stress as well as assign a redox fingerprint to various types of stress conditions.

A Role of AREB in the Regulation of PACC-Dependent Acid-Expressed-Genes and Pathogenicity of Colletotrichum gloeosporioides.

Gene expression regulation by pH in filamentous fungi and yeasts is controlled by the PACC/RIM101 transcription factor. In Colletotrichum gloeosporioides, PACC is known to act as positive regulator of alkaline-expressed genes, and this regulation was shown to contribute to fungal pathogenicity. PACC is also a negative regulator of acid-expressed genes, however; the mechanism of downregulation of acid-expressed genes by PACC and their contribution to C. gloeosporioides pathogenicity is not well understood. RNA sequencing data analysis was employed to demonstrate that PACC transcription factor binding sites (TFBS) are significantly overrepresented in the promoter of PACC-upregulated, alkaline-expressed genes. In contrast, they are not overrepresented in the PACC-downregulated, acid-expressed genes. Instead, acid-expressed genes showed overrepresentation of AREB GATA TFBS in C. gloeosporioides and in homologs of five other ascomycetes genomes. The areB promoter contains PACC TFBS; its transcript was upregulated at pH 7 and repressed in ΔpacC. Furthermore, acid-expressed genes were found to be constitutively upregulated in ΔareB during alkalizing conditions. The areB mutants showed significantly reduced ammonia secretion and pathogenicity on tomato fruit. Present results indicate that PACC activates areB expression, thereby conditionally repressing acid-expressed genes and contributing critically to C. gloeosporioides pathogenicity.

Simultaneous transcriptome analysis of Colletotrichum gloeosporioides and tomato fruit pathosystem reveals novel fungal pathogenicity and fruit defense strategies.

The fungus Colletotrichum gloeosporioides breaches the fruit cuticle but remains quiescent until fruit ripening signals a switch to necrotrophy, culminating in devastating anthracnose disease. There is a need to understand the distinct fungal arms strategy and the simultaneous fruit response. Transcriptome analysis of fungal-fruit interactions was carried out concurrently in the appressoria, quiescent and necrotrophic stages. Conidia germinating on unripe fruit cuticle showed stage-specific transcription that was accompanied by massive fruit defense responses. The subsequent quiescent stage showed the development of dendritic-like structures and swollen hyphae within the fruit epidermis. The quiescent fungal transcriptome was characterized by activation of chromatin remodeling genes and unsuspected environmental alkalization. Fruit response was portrayed by continued highly integrated massive up-regulation of defense genes. During cuticle infection of green or ripe fruit, fungi recapitulate the same developmental stages but with differing quiescent time spans. The necrotrophic stage showed a dramatic shift in fungal metabolism and up-regulation of pathogenicity factors. Fruit response to necrotrophy showed activation of the salicylic acid pathway, climaxing in cell death. Transcriptome analysis of C. gloeosporioides infection of fruit reveals its distinct stage-specific lifestyle and the concurrent changing fruit response, deepening our perception of the unfolding fungal-fruit arms and defenses race.

Singlet oxygen signatures are detected independent of light or chloroplasts in response to multiple stresses.

The production of singlet oxygen is typically associated with inefficient dissipation of photosynthetic energy or can arise from light reactions as a result of accumulation of chlorophyll precursors as observed in fluorescent (flu)-like mutants. Such photodynamic production of singlet oxygen is thought to be involved in stress signaling and programmed cell death. Here we show that transcriptomes of multiple stresses, whether from light or dark treatments, were correlated with the transcriptome of the flu mutant. A core gene set of 118 genes, common to singlet oxygen, biotic and abiotic stresses was defined and confirmed to be activated photodynamically by the photosensitizer Rose Bengal. In addition, induction of the core gene set by abiotic and biotic selected stresses was shown to occur in the dark and in nonphotosynthetic tissue. Furthermore, when subjected to various biotic and abiotic stresses in the dark, the singlet oxygen-specific probe Singlet Oxygen Sensor Green detected rapid production of singlet oxygen in the Arabidopsis (Arabidopsis thaliana) root. Subcellular localization of Singlet Oxygen Sensor Green fluorescence showed its accumulation in mitochondria, peroxisomes, and the nucleus, suggesting several compartments as the possible origins or targets for singlet oxygen. Collectively, the results show that singlet oxygen can be produced by multiple stress pathways and can emanate from compartments other than the chloroplast in a light-independent manner. The results imply that the role of singlet oxygen in plant stress regulation and response is more ubiquitous than previously thought.

Impairment in Sulfite Reductase Leads to Early Leaf Senescence in Tomato Plants.

Sulfite reductase (SiR) is an essential enzyme of the sulfate assimilation reductive pathway, which catalyzes the reduction of sulfite to sulfide. Here, we show that tomato (Solanum lycopersicum) plants with impaired SiR expression due to RNA interference (SIR Ri) developed early leaf senescence. The visual chlorophyll degradation in leaves of SIR Ri mutants was accompanied by a reduction of maximal quantum yield, as well as accumulation of hydrogen peroxide and malondialdehyde, a product of lipid peroxidation. Interestingly, messenger RNA transcripts and proteins involved in chlorophyll breakdown in the chloroplasts were found to be enhanced in the mutants, while transcripts and their plastidic proteins, functioning in photosystem II, were reduced in these mutants compared with wild-type leaves. As a consequence of SiR impairment, the levels of sulfite, sulfate, and thiosulfate were higher and glutathione levels were lower compared with the wild type. Unexpectedly, in a futile attempt to compensate for the low glutathione, the activity of adenosine-5'-phosphosulfate reductase was enhanced, leading to further sulfite accumulation in SIR Ri plants. Increased sulfite oxidation to sulfate and incorporation of sulfite into sulfoquinovosyl diacylglycerols were not sufficient to maintain low basal sulfite levels, resulting in accumulative leaf damage in mutant leaves. Our results indicate that, in addition to its biosynthetic role, SiR plays an important role in prevention of premature senescence. The higher sulfite is likely the main reason for the initiation of chlorophyll degradation, while the lower glutathione as well as the higher hydrogen peroxide and malondialdehyde additionally contribute to premature senescence in mutant leaves.

Set-point control of RD21 protease activity by AtSerpin1 controls cell death in Arabidopsis.

Programmed cell death (PCD) in plants plays a key role in defense response and is promoted by the release of compartmentalized proteases to the cytoplasm. Yet the exact identity and control of these proteases is poorly understood. Serpins are an important group of proteins that uniquely curb the activity of proteases by irreversible inhibition; however, their role in plants remains obscure. Here we show that during cell death the Arabidopsis serpin protease inhibitor, AtSerpin1, exhibits a pro-survival function by inhibiting its target pro-death protease, RD21. AtSerpin1 accumulates in the cytoplasm and RD21 accumulates in the vacuole and in endoplasmic reticulum bodies. Elicitors of cell death, including the salicylic acid agonist benzothiadiazole and the fungal toxin oxalic acid, stimulated changes in vacuole permeability as measured by the changes in the distribution of marker dye. Concomitantly, a covalent AtSerpin1-RD21 complex was detected indicative of a change in protease compartmentalization. Furthermore, mutant plants lacking RD21 or plants with AtSerpin1 over-expression exhibited significantly less elicitor-stimulated PCD than plants lacking AtSerpin1. The necrotrophic fungi Botrytis cinerea and Sclerotina sclerotiorum secrete oxalic acid as a toxin that stimulates cell death. Consistent with a pro-death function for RD21 protease, the growth of these necrotrophs was compromised in plants lacking RD21 but accelerated in plants lacking AtSerpin1. The results indicate that AtSerpin1 controls the pro-death function of compartmentalized protease RD21 by determining a set-point for its activity and limiting the damage induced during cell death.

Sulfite reductase protects plants against sulfite toxicity.

Plant sulfite reductase (SiR; Enzyme Commission 1.8.7.1) catalyzes the reduction of sulfite to sulfide in the reductive sulfate assimilation pathway. Comparison of SiR expression in tomato (Solanum lycopersicum 'Rheinlands Ruhm') and Arabidopsis (Arabidopsis thaliana) plants revealed that SiR is expressed in a different tissue-dependent manner that likely reflects dissimilarity in sulfur metabolism between the plant species. Using Arabidopsis and tomato SiR mutants with modified SiR expression, we show here that resistance to ectopically applied sulfur dioxide/sulfite is a function of SiR expression levels and that plants with reduced SiR expression exhibit higher sensitivity than the wild type, as manifested in pronounced leaf necrosis and chlorophyll bleaching. The sulfite-sensitive mutants accumulate applied sulfite and show a decline in glutathione levels. In contrast, mutants that overexpress SiR are more tolerant to sulfite toxicity, exhibiting little or no damage. Resistance to high sulfite application is manifested by fast sulfite disappearance and an increase in glutathione levels. The notion that SiR plays a role in the protection of plants against sulfite is supported by the rapid up-regulation of SiR transcript and activity within 30 min of sulfite injection into Arabidopsis and tomato leaves. Peroxisomal sulfite oxidase transcripts and activity levels are likewise promoted by sulfite application as compared with water injection controls. These results indicate that, in addition to participating in the sulfate assimilation reductive pathway, SiR also plays a role in protecting leaves against the toxicity of sulfite accumulation.

An essential role for tomato sulfite oxidase and enzymes of the sulfite network in maintaining leaf sulfite homeostasis.

Little is known about the homeostasis of sulfite levels, a cytotoxic by-product of plant sulfur turnover. By employing extended dark to induce catabolic pathways, we followed key elements of the sulfite network enzymes that include adenosine-5'-phosphosulfate reductase and the sulfite scavengers sulfite oxidase (SO), sulfite reductase, UDP-sulfoquinovose synthase, and ß-mercaptopyruvate sulfurtransferases. During extended dark, SO was enhanced in tomato (Solanum lycopersicum) wild-type leaves, while the other sulfite network components were down-regulated. SO RNA interference plants lacking SO activity accumulated sulfite, resulting in leaf damage and mortality. Exogenous sulfite application induced up-regulation of the sulfite scavenger activities in dark-stressed or unstressed wild-type plants, while expression of the sulfite producer, adenosine-5'-phosphosulfate reductase, was down-regulated. Unstressed or dark-stressed wild-type plants were resistant to sulfite applications, but SO RNA interference plants showed sensitivity and overaccumulation of sulfite. Hence, under extended dark stress, SO activity is necessary to cope with rising endogenous sulfite levels. However, under nonstressed conditions, the sulfite network can control sulfite levels in the absence of SO activity. The novel evidence provided by the synchronous dark-induced turnover of sulfur-containing compounds, augmented by exogenous sulfite applications, underlines the role of SO and other sulfite network components in maintaining sulfite homeostasis, where sulfite appears to act as an orchestrating signal molecule.

ROSMETER: a bioinformatic tool for the identification of transcriptomic imprints related to reactive oxygen species type and origin provides new insights into stress responses.

The chemical identity of the reactive oxygen species (ROS) and its subcellular origin will leave a specific imprint on the transcriptome response. In order to facilitate the appreciation of ROS signaling, we developed a tool that is tuned to qualify this imprint. Transcriptome data from experiments in Arabidopsis (Arabidopsis thaliana) for which the ROS type and organelle origin are known were compiled into indices and made accessible by a Web-based interface called ROSMETER. The ROSMETER algorithm uses a vector-based algorithm to portray the ROS signature for a given transcriptome. The ROSMETER platform was applied to identify the ROS signatures profiles in transcriptomes of senescing plants and of those exposed to abiotic and biotic stresses. An unexpected highly significant ROS transcriptome signature of mitochondrial stress was detected during the early presymptomatic stages of leaf senescence, which was accompanied by the specific oxidation of mitochondria-targeted redox-sensitive green fluorescent protein probe. The ROSMETER analysis of diverse stresses revealed both commonalties and prominent differences between various abiotic stress conditions, such as salt, cold, ultraviolet light, drought, heat, and pathogens. Interestingly, early responses to the various abiotic stresses clustered together, independent of later responses, and exhibited negative correlations to several ROS indices. In general, the ROS transcriptome signature of abiotic stresses showed limited correlation to a few indices, while biotic stresses showed broad correlation with multiple indices. The ROSMETER platform can assist in formulating hypotheses to delineate the role of ROS in plant acclimation to environmental stress conditions and to elucidate the molecular mechanisms of the oxidative stress response in plants.

Global aspects of pacC regulation of pathogenicity genes in Colletotrichum gloeosporioides as revealed by transcriptome analysis.

Colletotrichum gloeosporioides alkalinizes its surroundings during colonization of host tissue. The transcription factor pacC is a regulator of pH-controlled genes and is essential for successful colonization. We present here the sequence assembly of the Colletotrichum fruit pathogen and use it to explore the global regulation of pathogenicity by ambient pH. The assembled genome size was 54 Mb, encoding 18,456 genes. Transcriptomes of the wild type and ΔpacC mutant were established by RNA-seq and explored for their global pH-dependent gene regulation. The analysis showed that pacC upregulates 478 genes and downregulates 483 genes, comprising 5% of the fungal genome, including transporters, antioxidants, and cell-wall-degrading enzymes. Interestingly, gene families with similar functionality are both up- and downregulated by pacC. Global analysis of secreted genes showed significant pacC activation of degradative enzymes at alkaline pH and during fruit infection. Select genes from alkalizing-type pathogen C. gloeosporioides and from acidifying-type pathogen Sclerotinia sclerotiorum were verified by quantitative reverse-transcription polymerase chain reaction analysis at different pH values. Knock out of several pacC-activated genes confirmed their involvement in pathogenic colonization of alkalinized surroundings. The results suggest a global regulation by pacC of key pathogenicity genes during pH change in alkalinizing and acidifying pathogens.