Brassinosteroid-mediated reactive oxygen species are essential for tapetum degradation and pollen fertility in tomato.

Phytohormone brassinosteroids (BRs) are essential for plant growth and development, but the mechanisms of BR-mediated pollen development remain largely unknown. In this study, we show that pollen viability, pollen germination and seed number decreased in the BR-deficient mutant d^im , which has a lesion in the BR biosynthetic gene DWARF (DWF), and in the bzr1 mutant, which is deficient in BR signaling regulator BRASSINAZOLE RESISTANT 1 (BZR1), compared with those in wild-type plants, whereas plants overexpressing DWF or BZR1 exhibited the opposite effects. Loss or gain of function in the DWF or BZR1 genes altered the timing of reactive oxygen species (ROS) production and programmed cell death (PCD) in tapetal cells, resulting in delayed or premature tapetal degeneration, respectively. Further analysis revealed that BZR1 could directly bind to the promoter of RESPIRATORY BURST OXIDASE HOMOLOG 1 (RBOH1), and that RBOH1-mediated ROS promote pollen and seed development by triggering PCD and tapetal cell degradation. In contrast, the suppression of RBOH1 compromised BR signaling-mediated ROS production and pollen development. These findings provide strong evidence that BZR1-dependent ROS production plays a critical role in the BR-mediated regulation of tapetal cell degeneration and pollen development in Solanum lycopersicum (tomato) plants.

BZR1 Mediates Brassinosteroid-Induced Autophagy and Nitrogen Starvation in Tomato.

Autophagy, an innate cellular destructive mechanism, plays crucial roles in plant development and responses to stress. Autophagy is known to be stimulated or suppressed by multiple molecular processes, but the role of phytohormone signaling in autophagy is unclear. Here, we demonstrate that the transcripts of autophagy-related genes (ATGs) and the formation of autophagosomes are triggered by enhanced levels of brassinosteroid (BR). Furthermore, the BR-activated transcription factor brassinazole-resistant1 (BZR1), a positive regulator of the BR signaling pathway, is involved in BR-induced autophagy. Treatment with BR enhanced the formation of autophagosomes and the transcripts of ATGs in BZR1-overexpressing plants, while the effects of BR were compromised in BZR1-silenced plants. Yeast one-hybrid analysis and chromatin immunoprecipitation coupled with quantitative polymerase chain reaction revealed that BZR1 bound to the promoters of ATG2 and ATG6 The BR-induced formation of autophagosomes decreased in ATG2- and ATG6-silenced plants. Moreover, exogenous application of BR enhanced chlorophyll content and autophagosome formation and decreased the accumulation of ubiquitinated proteins under nitrogen starvation. Leaf chlorosis and chlorophyll degradation were exacerbated in BZR1-silenced plants and the BR biosynthetic mutant d^im but were alleviated in BZR1- and BZR1-1D-overexpressing plants under nitrogen starvation. Meanwhile, nitrogen starvation-induced expression of ATGs and autophagosome formation were compromised in both BZR1-silenced and d^im plants but were increased in BZR1- and BZR1-1D-overexpressing plants. Taken together, our results suggest that BZR1-dependent BR signaling up-regulates the expression of ATGs and autophagosome formation, which plays a critical role in the plant response to nitrogen starvation in tomato (Solanum lycopersicum).

Heat Shock Factor HsfA1a Is Essential for R Gene-Mediated Nematode Resistance and Triggers H2O2 Production1.

Plants generate reactive oxygen species (ROS) in the apoplast in response to pathogen attack, especially following resistance (R) gene-mediated pathogen recognition; however, the mechanisms activating ROS generation remain unknown. Here, we demonstrate that RKN (Meloidogyne incognita) infection rapidly induces ROS accumulation in the roots of tomato (Solanum lycopersicum) plants that contain the R gene Mi-1.2 but rarely induces ROS accumulation in the susceptible or Mi-1.2-silenced resistant genotypes. RNK also induces the hypersensitive response, a form of programmed cell death, in Mi-1.2 plants. RKN induces the expression of numerous class-A heat shock factor (HsfA) genes in resistant tomato plants. Silencing HsfA1a compromises Mi-1.2-mediated resistance, apoplastic H2O2 accumulation, and the transcription of whitefly induced 1 (Wfi1), which encodes a respiratory burst oxidase homolog. HsfA1a regulates Wfi1 transcription by binding to the Wfi1 promoter, and silencing of Wfi1 compromises Mi-1.2-mediated resistance. HsfA1a and Wfi1 are involved in Mi-1.2-triggered Hsp90 accumulation and basal defense in susceptible tomato. Thus, HsfA-1aWfi1-dependent ROS signaling functions as a crucial regulator of plant defense responses.

Brassinosteroids act as a positive regulator for resistance against root-knot nematode involving RESPIRATORY BURST OXIDASE HOMOLOG-dependent activation of MAPKs in tomato.

Interplay of hormones with reactive oxygen species (ROS) fine-tunes the response of plants to stress; however, the crosstalk between brassinosteroids (BRs) and ROS in nematode resistance is unclear. In this study, we found that low BR biosynthesis or lack of BR receptor increased, whilst exogenous BR decreased the susceptibility of tomato plants to Meloidogyne incognita. Hormone quantification coupled with hormone mutant complementation experiments revealed that BR did not induce the defence response by triggering salicylic acid (SA), jasmonic acid/ethylene (JA/ET) or abscisic acid (ABA) signalling pathway. Notably, roots of BR-deficient plants had decreased apoplastic ROS accumulation, transcript of RESPIRATORY BURST OXIDASE HOMOLOG1 (RBOH1) and WHITEFLY INDUCED1 (WFI1), and reduced activation of mitogen-activated protein kinase 1/2 (MPK1/2) and MPK3. Silencing of RBOH1, WFI1, MPK1, MPK2 and MPK3 all increased the root susceptibility to nematode and attenuated BR-induced resistance against the nematode. Significantly, suppressed transcript of RBOH1 compromised BR-induced activation of MPK1/2 and MPK3. These results strongly suggest that RBOH-dependent MPK activation is involved in the BR-induced systemic resistance against the nematode.

Brassinosteroid-mediated apoplastic H2 O2 -glutaredoxin 12/14 cascade regulates antioxidant capacity in response to chilling in tomato.

Brassinosteroids (BRs) regulate plant development and stress response. Although much has been learned about their roles in plant development, the mechanisms by which BRs regulate plant stress tolerance remain unclear. Chilling is a major stress that adversely affects plant growth. Here, we report that BR positively regulates chilling tolerance in tomato. BR partial deficiency aggravated chilling-induced oxidized protein accumulation, membrane lipid peroxidation, and decrease of maximum quantum efficiency of photosystem II (Fv/Fm). By contrast, overexpression of BR biosynthetic gene Dwarf or treatment with 24-epibrassinolide (EBR) attenuated chilling-induced oxidative damages and resulted in an increase of Fv/Fm. BR increased transcripts of RESPIRATORY BURST OXIDASE HOMOLOG1 (RBOH1) and GLUTAREDOXIN (GRX) genes, and BR-induced chilling tolerance was associated with an increase in the ratio of reduced/oxidized 2-cysteine peroxiredoxin (2-Cys Prx) and activation of antioxidant enzymes. However, RBOH1-RNAi plants failed to respond to EBR as regards to the induction of GRX genes, activation of antioxidant capacity, and attenuation of chilling-induced oxidative damages. Furthermore, silencing of GRXS12 and S14 compromised EBR-induced increases in the ratio of reduced/oxidized 2-Cys Prx and activities of antioxidant enzymes. Our study suggests that BR enhances chilling tolerance through a signalling cascade involving RBOH1, GRXs, and 2-Cys Prx in tomato.

Involvement of an ethylene response factor in chlorophyll degradation during citrus fruit degreening.

Chlorophyll degradation naturally occurs during plant senescence. However, in fruit such as citrus, it is a positive characteristic, as degreening is an important colour development contributing to fruit quality. In the present work, Citrus sinensis Osbeck, cv. Newhall fruit was used as a model for chlorophyll degradation. An ethylene response factor, CitERF13, was isolated and its transcriptional changes were closely correlated with fruit peel degreening during development or in response to ethylene. Dual-luciferase and yeast one-hybrid assays, as well as motif mutation, indicated that CitERF13 directly binds to the CitPPH promoter and enhances its activity. Transient and stable over-expression of CitERF13 resulted in rapid chlorophyll degradation in Nicotiana tabacum leaves and led to accumulation of pheophorbide (Pheide) a, a metabolite of pheophorbide hydrolase (PPH). Similar results were observed from transient transformation of CitERF13 in citrus fruit peel. Moreover, this function of CitERF13 was conserved within Arabidopsis and tomato, as the homologs AtERF17 and SlERF16 similarly acted as activators of PPH genes and accelerators of chlorophyll degradation.

HsfA1a upregulates melatonin biosynthesis to confer cadmium tolerance in tomato plants.

Melatonin regulates broad aspects of plant responses to various biotic and abiotic stresses, but the upstream regulation of melatonin biosynthesis by these stresses remains largely unknown. Herein, we demonstrate that transcription factor heat-shock factor A1a (HsfA1a) conferred cadmium (Cd) tolerance to tomato plants, in part through its positive role in inducing melatonin biosynthesis under Cd stress. Analysis of leaf phenotype, chlorophyll content, and photosynthetic efficiency revealed that silencing of the HsfA1a gene decreased Cd tolerance, whereas its overexpression enhanced plant tolerance to Cd. HsfA1a-silenced plants exhibited reduced melatonin levels, and HsfA1a overexpression stimulated melatonin accumulation and the expression of the melatonin biosynthetic gene caffeic acid O-methyltransferase 1 (COMT1) under Cd stress. Both an in vitro electrophoretic mobility shift assay and in vivo chromatin immunoprecipitation coupled with qPCR analysis revealed that HsfA1a binds to the COMT1 gene promoter. Meanwhile, Cd stress induced the expression of heat-shock proteins (HSPs), which was compromised in HsfA1a-silenced plants and more robustly induced in HsfA1a-overexpressing plants under Cd stress. COMT1 silencing reduced HsfA1a-induced Cd tolerance and melatonin accumulation in HsfA1a-overexpressing plants. Additionally, the HsfA1a-induced expression of HSPs was partially compromised in COMT1-silenced wild-type or HsfA1a-overexpressing plants under Cd stress. These results demonstrate that HsfA1a confers Cd tolerance by activating transcription of the COMT1 gene and inducing accumulation of melatonin that partially upregulates expression of HSPs.

Overexpression of a brassinosteroid biosynthetic gene Dwarf enhances photosynthetic capacity through activation of Calvin cycle enzymes in tomato.

BACKGROUND: Genetic manipulation of brassinosteroid (BR) biosynthesis or signaling is a promising strategy to improve crop yield and quality. However, the relationships between the BR-promoted growth and photosynthesis and the exact mechanism of BR-regulated photosynthetic capacity are not clear. Here, we generated transgenic tomato plants by overexpressing Dwarf, a BR biosynthetic gene that encodes the CYP85A1, and compared the photosynthetic capacity with the BR biosynthetic mutant d (im) and wild type. RESULTS: Overexpression of Dwarf promoted net photosynthetic rate (P N), whereas BR deficiency in d (im) led to a significant inhibition in P N as compared with WT. The activation status of RuBisCO, and the protein content and activity of RuBisCO activase, but not the total content and transcripts of RuBisCO were closely related to the endogenous BR levels in different genotypes. However, endogenous BR positively regulated the expression and activity of fructose-1,6-bisphosphatase. Dwarf overexpression enhanced the activity of dehydroascorbate reductase and glutathione reductase, leading to a reduced redox status, whereas BR deficiency had the contrasting effects. In addition, BR induced a reduction of 2-cystein peroxiredoxin without altering the protein content. CONCLUSIONS: BR plays a role in the regulation of photosynthesis. BR can increase the photosynthetic capacity by inducing a reduced redox status that maintains the activation states of Calvin cycle enzymes.

DWARF overexpression induces alteration in phytohormone homeostasis, development, architecture and carotenoid accumulation in tomato.

Brassinosteroids (BRs) play a critical role in plant growth, development and stress response; however, genetic evidence for the BR-mediated integrated regulation of plant growth still remains elusive in crop species. Here, we clarified the function of DWARF (DWF), the key BR biosynthetic gene in tomato, in the regulation of plant growth and architecture, phytohormone homeostasis and fruit development by comparing wild type, d^(im), a weak allele mutant impaired in DWF, and DWF-overexpressing plants in tomato. Results showed that increases in DWF transcripts and endogenous BR level resulted in improved germination, lateral root development, CO2 assimilation and eventually plant growth as characterized by slender and compact plant architecture. However, an increase in DWF transcript down-regulated the accumulation of gibberellin, which was associated with decreases in leaf size and thickness. BRs positively regulated lateral bud outgrowth, which was associated with decreased transcript of Aux/IAA3, and the ethylene-dependent petiole bending and fruit ripening. Notably, overexpression of DWF did not significantly alter fruit yield per plant; however, increases by 57.4% and 95.3% might be estimated in fruit yield per square metre in two transgenic lines due to their compact architecture. Significantly, BR level was positively related with the carotenoid accumulation in the fruits. Taken together, our results demonstrate that BRs are actively involved in the regulation of multiple developmental processes relating to agronomical important traits.

Apoplastic H2 O2 plays a critical role in axillary bud outgrowth by altering auxin and cytokinin homeostasis in tomato plants.

Although phytohormones such as indole-3-acetic acid (IAA), cytokinin (CK) and strigolactone are important modulators of plant architecture, it remains unclear whether reactive oxygen species are involved in the regulation of phytohormone-dependent axillary bud outgrowth in plants. We used diverse techniques, including transcriptional suppression, HPLC-MS, biochemical methodologies and gene transcript analysis to investigate the signaling pathway for apoplastic hydrogen peroxide (H2 O2 )-induced axillary bud outgrowth. Silencing of tomato RESPIRATORY BURST OXIDASE HOMOLOG 1 (RBOH1) and WHITEFLY INDUCED 1 (WFI1), two important genes involved in H2 O2 production in the apoplast, enhanced bud outgrowth, decreased transcript of FZY - a rate-limiting gene in IAA biosynthesis and IAA accumulation in the apex - and increased the transcript of IPT2 involved in CK biosynthesis and CK accumulation in the stem node. These effects were fully abolished by the application of exogenous H2 O2 . Both decapitation and the silencing of FZY promoted bud outgrowth, and downregulated and upregulated the transcripts for IAA3 and IAA15, and IPT2, respectively. However, these effects were not blocked by treatment with exogenous H2 O2 but by napthaleneacetic acid (NAA) treatment. These results suggest that RBOHs-dependent apoplastic H2 O2 promotes IAA biosynthesis in the apex, which, in turn, inhibits CK biosynthesis and subsequent bud outgrowth in tomato plants.

Microarray and genetic analysis reveals that csa-miR159b plays a critical role in abscisic acid-mediated heat tolerance in grafted cucumber plants.

Root-shoot communication plays a vital role in plant growth, development and adaptation to environmental stimuli. Grafting-induced stress tolerance is associated with the induction of plentiful stress-related genes and proteins; the mechanism involved, however, remains obscure. Here, we show that the enhanced tolerance against heat stress in cucumber plants with luffa as rootstock was accompanied with an increased accumulation of abscisic acid (ABA), down-regulation of a subset of microRNAs (miRNAs) but up-regulation of their target genes and CsHSP70 accumulation in the shoots. Significantly, luffa rootstock and foliar application of ABA both down-regulated csa-miR159b and up-regulated its target mRNAs CsGAMYB1 and CsMYB29-like and CsHSP70 accumulation in cucumber, while ectopic expression of csa-miR159b led to decreased heat tolerance, AtMYB33 transcript and AtHSP70 accumulation in Arabidopsis plants. Taken together, our results suggest that root-originated signals such as ABA could alter miRNAs in the shoots, which have a major role in the post-transcriptional regulation of the stress-responsive genes.

Interactions between 2-Cys peroxiredoxins and ascorbate in autophagosome formation during the heat stress response in Solanum lycopersicum.

2-Cys peroxiredoxins (2-CPs) function in the removal of hydrogen peroxide and lipid peroxides but their precise roles in the induction of autophagy have not been characterized. Here we show that heat stress, which is known to induce oxidative stress, leads to the simultaneous accumulation of transcripts encoding 2-CPs and autophagy proteins, as well as autophagosomes, in tomato (Solanum lycopersicum) plants. Virus-induced gene silencing of the tomato peroxiredoxin genes 2-CP1, 2-CP2, and 2-CP1/2 resulted in an increased sensitivity of tomato plants to heat stress. Silencing 2-CP2 or 2-CP1/2 increased the levels of transcripts associated with ascorbate biosynthesis but had no effect on the glutathione pool in the absence of stress. However, the heat-induced accumulation of transcripts associated with the water-water cycle was compromised by the loss of 2-CP1/2 functions. The transcript levels of autophagy-related genes ATG5 and ATG7 were higher in plants with impaired 2-CP1/2 functions, and the formation of autophagosomes increased, together with an accumulation of oxidized and insoluble proteins. Silencing of ATG5 or ATG7 increased the levels of 2-CP transcripts and protein but decreased heat stress tolerance. These results demonstrate that 2-CPs fulfil a pivotal role in heat stress tolerance in tomato, via interactions with ascorbate-dependent pathways and autophagy.

Melatonin enhances thermotolerance by promoting cellular protein protection in tomato plants.

Melatonin is a pleiotropic signaling molecule that provides physiological protection against diverse environmental stresses in plants. Nonetheless, the mechanisms for melatonin-mediated thermotolerance remain largely unknown. Here, we report that endogenous melatonin levels increased with a rise in ambient temperature and that peaked at 40°C. Foliar pretreatment with an optimal dose of melatonin (10 µmol/L) or the overexpression of N-acetylserotonin methyltransferase (ASMT) gene effectively ameliorated heat-induced photoinhibition and electrolyte leakage in tomato plants. Both exogenous melatonin treatment and endogenous melatonin manipulation by overexpression of ASMT decreased the levels of insoluble and ubiquitinated proteins, but enhanced the expression of heat-shock proteins (HSPs) to refold denatured and unfolded proteins under heat stress. Meanwhile, melatonin also induced expression of several ATG genes and formation of autophagosomes to degrade aggregated proteins under the same stress. Proteomic profile analyses revealed that protein aggregates for a large number of biological processes accumulated in wild-type plants. However, exogenous melatonin treatment or overexpression of ASMT reduced the accumulation of aggregated proteins. Aggregation responsive proteins such as HSP70 and Rubisco activase were preferentially accumulated and ubiquitinated in wild-type plants under heat stress, while melatonin mitigated heat stress-induced accumulation and ubiquitination of aggregated proteins. These results suggest that melatonin promotes cellular protein protection through induction of HSPs and autophagy to refold or degrade denatured proteins under heat stress in tomato plants.

Melatonin mediates selenium-induced tolerance to cadmium stress in tomato plants.

Both selenium (Se) and melatonin reduce cadmium (Cd) uptake and mitigate Cd toxicity in plants. However, the relationship between Se and melatonin in Cd detoxification remains unclear. In this study, we investigated the influence of three forms of Se (selenocysteine, sodium selenite, and sodium selenate) on the biosynthesis of melatonin and the tolerance against Cd in tomato plants. Pretreatment with different forms of Se significantly induced the biosynthesis of melatonin and its precursors (tryptophan, tryptamine, and serotonin); selenocysteine had the most marked effect on melatonin biosynthesis. Furthermore, Se and melatonin supplements significantly increased plant Cd tolerance as evidenced by decreased growth inhibition, photoinhibition, and electrolyte leakage (EL). Se-induced Cd tolerance was compromised in melatonin-deficient plants following tryptophan decarboxylase (TDC) gene silencing. Se treatment increased the levels of glutathione (GSH) and phytochelatins (PCs), as well as the expression of GSH and PC biosynthetic genes in nonsilenced plants, but the effects of Se were compromised in TDC-silenced plants under Cd stress. In addition, Se and melatonin supplements reduced Cd content in leaves of nonsilenced plants, but Se-induced reduction in Cd content was compromised in leaves of TDC-silenced plants. Taken together, our results indicate that melatonin is involved in Se-induced Cd tolerance via the regulation of Cd detoxification.

RNA-seq analysis reveals the role of red light in resistance against Pseudomonas syringae pv. tomato DC3000 in tomato plants.

BACKGROUND: Plants attenuate their responses to a variety of bacterial and fungal pathogens, leading to higher incidences of pathogen infection at night. However, little is known about the molecular mechanism responsible for the light-induced defence response; transcriptome data would likely facilitate the elucidation of this mechanism. RESULTS: In this study, we observed diurnal changes in tomato resistance to Pseudomonas syringae pv. tomato DC3000 (Pto DC3000), with the greatest susceptibility before midnight. Nightly light treatment, particularly red light treatment, significantly enhanced the resistance; this effect was correlated with increased salicylic acid (SA) accumulation and defence-related gene transcription. RNA-seq analysis revealed that red light induced a set of circadian rhythm-related genes involved in the phytochrome and SA-regulated resistance response. The biosynthesis and signalling pathways of multiple plant hormones (auxin, SA, jasmonate, and ethylene) were co-ordinately regulated following Pto DC3000 infection and red light, and the SA pathway was most significantly affected by red light and Pto DC3000 infection. This result indicates that SA-mediated signalling pathways are involved in red light-induced resistance to pathogens. Importantly, silencing of nonexpressor of pathogensis-related genes 1 (NPR1) partially compromised red light-induced resistance against Pto DC3000. Furthermore, sets of genes involved in redox homeostasis (respiratory burst oxidase homologue, RBOH; glutathione S-transferases, GSTs; glycosyltransferase, GTs), calcium (calmodulin, CAM; calmodulin-binding protein, CBP), and defence (polyphenol oxidase, PPO; nudix hydrolase1, NUDX1) as well as transcription factors (WRKY18, WRKY53, WRKY60, WRKY70) and cellulose synthase were differentially induced at the transcriptional level by red light in response to pathogen challenge. CONCLUSIONS: Taken together, our results suggest that there is a diurnal change in susceptibility to Pto DC3000 with greatest susceptibility in the evening. The red light induced-resistance to Pto DC3000 at night is associated with enhancement of the SA pathway, cellulose synthase, and reduced redox homeostasis.

The relationship between the plant-encoded RNA-dependent RNA polymerase 1 and alternative oxidase in tomato basal defense against Tobacco mosaic virus.

Salicylic acid (SA) plays a critical role in plant defense against pathogen attack. The SA-induced viral defense in plants is distinct from the pathways mediating bacterial and fungal defense, which is pathogenesis-related protein-independent but involves an RNA-dependent RNA polymerase 1 (RDR1)-mediated RNA silencing mechanism and/or an alternative oxidase (AOX)-associated defense pathway. However, the relationship between these two viral defense-related pathways remains unclear. In this study, Tobacco mosaic virus (TMV) inoculation onto Solanum lycopersicum (tomato) leaves induced a rapid induction of the SlAOX1a transcript level as well as the total and CN-resistant respiration at 0.5 dpi, followed by an increase in SlRDR1 gene expression at 1 dpi in the upper uninoculated leaves. Silencing SlRDR1 using virus-induced gene silencing system significantly reduced SlRDR1 expression and tomato defense against TMV but had no evident effect on SlAOX1a transcription. Conversely, silencing SlAOX1a not only effectively reduced the AOX1a transcript level, but also blocked the TMV-induced SlRDR1 expression and decreased the basal defense against TMV. Furthermore, the application of an exogenous AOX activator on empty vector-silenced control plants greatly induced the accumulation of SlRDR1 and SlAOX1a transcript and reduced TMV viral RNA accumulation, but failed to have such effects on SlRDR1-silenced plants. Moreover, RDR1-overexpressed transgenic Nicotiana benthamiana plants enhanced defense against TMV than the empty vector-transformed plants, but these effects were not affected by the exogenous AOX activator or inhibitor. These results indicate that RDR1 is involved in the AOX-mediated defense pathway against TMV infection and plays a crucial role in enhancing RNA silencing to limit virus systemic spread.

Interplay between reactive oxygen species and hormones in the control of plant development and stress tolerance.

As a consequence of a sessile lifestyle, plants are continuously exposed to changing environmental conditions and often life-threatening stresses caused by exposure to excessive light, extremes of temperature, limiting nutrient or water availability, and pathogen/insect attack. The flexible coordination of plant growth and development is necessary to optimize vigour and fitness in a changing environment through rapid and appropriate responses to such stresses. The concept that reactive oxygen species (ROS) are versatile signalling molecules in plants that contribute to stress acclimation is well established. This review provides an overview of our current knowledge of how ROS production and signalling are integrated with the action of auxin, brassinosteroids, gibberellins, abscisic acid, ethylene, strigolactones, salicylic acid, and jasmonic acid in the coordinate regulation of plant growth and stress tolerance. We consider the local and systemic crosstalk between ROS and hormonal signalling pathways and identify multiple points of reciprocal control, as well as providing insights into the integration nodes that involve Ca(2+)-dependent processes and mitogen-activated protein kinase phosphorylation cascades.

Role of H2O2 dynamics in brassinosteroid-induced stomatal closure and opening in Solanum lycopersicum.

Brassinosteroids (BRs) are essential for plant growth and development; however, their roles in the regulation of stomatal opening or closure remain obscure. Here, the mechanism underlying BR-induced stomatal movements is studied. The effects of 24-epibrassinolide (EBR) on the stomatal apertures of tomato (Solanum lycopersicum) were measured by light microscopy using epidermal strips of wild type (WT), the abscisic acid (ABA)-deficient notabilis (not) mutant, and plants silenced for SlBRI1, SlRBOH1 and SlGSH1. EBR induced stomatal opening within an appropriate range of concentrations, whereas high concentrations of EBR induced stomatal closure. EBR-induced stomatal movements were closely related to dynamic changes in H(2)O(2) and redox status in guard cells. The stomata of SlRBOH1-silenced plants showed a significant loss of sensitivity to EBR. However, ABA deficiency abolished EBR-induced stomatal closure but did not affect EBR-induced stomatal opening. Silencing of SlGSH1, the critical gene involved in glutathione biosynthesis, disrupted glutathione redox homeostasis and abolished EBR-induced stomatal opening. The results suggest that transient H(2)O(2) production is essential for poising the cellular redox status of glutathione, which plays an important role in BR-induced stomatal opening. However, a prolonged increase in H(2)O(2) facilitated ABA signalling and stomatal closure.

Hydrogen peroxide mediates abscisic acid-induced HSP70 accumulation and heat tolerance in grafted cucumber plants.

Root-shoot communications play important roles in plant stress responses. Here, we examined the roles of root-sourced signals in the shoot response to heat in cucumber plants. Cucumber plants grafted onto their own roots and luffa roots were exposed to aerial and root-zone heat to examine their tolerance by assessing the levels of oxidative stress, PSII photoinhibition, accumulation of abscisic acid (ABA), H2 O2 and heat shock protein (HSP) 70 using immunoblotting, chlorophyll fluorescence, immunoassay, CeCl3 staining and Western blotting, respectively. Grafting onto the luffa rootstock enhanced the shoot tolerance to the heat. This enhanced tolerance was associated with increased accumulation of ABA and apoplastic H2 O2 , RBOH transcripts and HSP70 expression and a decrease in oxidative stress in the shoots. The increases in the ABA and H2 O2 concentrations in the shoots were attributed to an increase in ABA transport from roots and an increase in ABA biosynthesis in the shoots when the root-zone and shoots were heat stressed, respectively. Inhibition of H2 O2 accumulation compromised luffa rootstock-induced HSP70 expression and heat tolerance. These results suggest that, under heat stress, ABA triggers the expression of HSP70 in an apoplastic H2 O2 -dependent manner, implicating the role of an ABA-dependent H2 O2 -driven mechanism in a systemic response involving root-shoot communication.

RBOH1-dependent H2O2 production and subsequent activation of MPK1/2 play an important role in acclimation-induced cross-tolerance in tomato.

H2O2 and mitogen-activated protein kinase (MAPK) cascades play important functions in plant stress responses, but their roles in acclimation response remain unclear. This study examined the functions of H2O2 and MPK1/2 in acclimation-induced cross-tolerance in tomato plants. Mild cold, paraquat, and drought as acclimation stimuli enhanced tolerance to more severe subsequent chilling, photooxidative, and drought stresses. Acclimation-induced cross-tolerance was associated with increased transcript levels of RBOH1 and stress- and defence-related genes, elevated apoplastic H2O2 accumulation, increased activity of NADPH oxidase and antioxidant enzymes, reduced glutathione redox state, and activation of MPK1/2 in tomato. Virus-induced gene silencing of RBOH1, MPK1, and MPK2 or MPK1/2 all compromised acclimation-induced cross-tolerance and associated stress responses. Taken together, these results strongly suggest that acclimation-induced cross-tolerance is largely attributed to RBOH1-dependent H2O2 production at the apoplast, which may subsequently activate MPK1/2 to induce stress responses.