Arabidopsis HY1-Modulated Stomatal Movement: An Integrative Hub Is Functionally Associated with ABI4 in Dehydration-Induced ABA Responsiveness.

Heme oxygenase (HO; EC 1.14.99.3) has recently been proposed as a novel component in mediating wide ranges of the plant adaptive signaling processes. However, the physiological significance and molecular basis underlying Arabidopsis (Arabidopsis thaliana) HO1 (HY1) functioning in drought tolerance remained unclear. Here, we report that mutation of HY1 promoted, but overexpression of this gene impaired, Arabidopsis drought tolerance. This was attributed to the abscisic acid (ABA)-hypersensitive or -hyposensitive phenotypes, with the regulation of stomatal closure in particular. However, comparative transcriptomic profile analysis showed that the induction of numerous ABA/stress-dependent genes in dehydrated wild-type plants was differentially impaired in the hy1 mutant. In agreement, ABA-induced ABSCISIC ACID-INSENSITIVE4 (ABI4) transcript accumulation was strengthened in the hy1 mutant. Genetic analysis further identified that the hy1-associated ABA hypersensitivity and drought tolerance were arrested in the abi4 background. Moreover, the promotion of ABA-triggered up-regulation of RbohD abundance and reactive oxygen species (ROS) levels in the hy1 mutant was almost fully blocked by the mutation of ABI4, suggesting that the HY1-ABI4 signaling in the wild type involved in stomatal closure was dependent on the RbohD-derived ROS production. However, hy1-promoted stomatal closure was not affected by a nitric oxide scavenger. Correspondingly, ABA-insensitive behaviors in rbohD stomata were not affected by either the mutation of HY1 or its ectopic expression in the rbohD background, both of which responded significantly to exogenous ROS. These data indicate that HY1 functioned negatively and acted upstream of ABI4 in drought signaling, which was casually dependent on the RbohD-derived ROS in the regulation of stomatal closure.

Rapid regulation of the plasma membrane H⁺-ATPase activity is essential to salinity tolerance in two halophyte species, Atriplex lentiformis and Chenopodium quinoa.

BACKGROUND AND AIMS: The activity of H(+)-ATPase is essential for energizing the plasma membrane. It provides the driving force for potassium retention and uptake through voltage-gated channels and for Na(+) exclusion via Na(+)/H(+) exchangers. Both of these traits are central to plant salinity tolerance; however, whether the increased activity of H(+)-ATPase is a constitutive trait in halophyte species and whether this activity is upregulated at either the transcriptional or post-translation level remain disputed. METHODS: The kinetics of salt-induced net H(+), Na(+) and K(+) fluxes, membrane potential and AHA1/2/3 expression changes in the roots of two halophyte species, Atriplex lentiformis (saltbush) and Chenopodium quinoa (quinoa), were compared with data obtained from Arabidopsis thaliana roots. KEY RESULTS: Intrinsic (steady-state) membrane potential values were more negative in A. lentiformis and C. quinoa compared with arabidopsis (-144 ± 3·3, -138 ± 5·4 and -128 ± 3·3 mV, respectively). Treatment with 100 mm NaCl depolarized the root plasma membrane, an effect that was much stronger in arabidopsis. The extent of plasma membrane depolarization positively correlated with NaCl-induced stimulation of vanadate-sensitive H(+) efflux, Na(+) efflux and K(+) retention in roots (quinoa > saltbush > arabidopsis). NaCl-induced stimulation of H(+) efflux was most pronounced in the root elongation zone. In contrast, H(+)-ATPase AHA transcript levels were much higher in arabidopsis compared with quinoa plants, and 100 mm NaCl treatment led to a further 3-fold increase in AHA1 and AHA2 transcripts in arabidopsis but not in quinoa. CONCLUSIONS: Enhanced salinity tolerance in the halophyte species studied here is not related to the constitutively higher AHA transcript levels in the root epidermis, but to the plant's ability to rapidly upregulate plasma membrane H(+)-ATPase upon salinity treatment. This is necessary for assisting plants to maintain highly negative membrane potential values and to exclude Na(+), or enable better K(+) retention in the cytosol under saline conditions.

Endogenous hydrogen sulfide enhances salt tolerance by coupling the reestablishment of redox homeostasis and preventing salt-induced K⁺ loss in seedlings of Medicago sativa.

Despite the external application of hydrogen sulfide (H2S) conferring plant tolerance against various environmental cues, the physiological significance of l-cysteine desulfhydrase (L-DES)-associated endogenous H2S production involved in salt-stress signaling was poorly understood. To address this gap, the participation of in planta changes of H2S homeostasis involved in alfalfa salt tolerance was investigated. The increasing concentration of NaCl (from 50 to 300 mM) progressively caused the induction of total l-DES activity and the increase of endogenous H2S production. NaCl-triggered toxicity symptoms (175 mM), including seedling growth inhibition and lipid peroxidation, were alleviated by sodium hydrosulfide (NaHS; 100 µM), a H2S donor, whereas aggravated by an inhibitor of l-DES or a H2S scavenger. A weaker or negative response was observed in lower or higher dose of NaHS. Further results showed that endogenous l-DES-related H2S modulated several genes/activities of antioxidant defence enzymes, and also regulated the contents of antioxidant compounds, thus counterbalancing the NaCl-induced lipid peroxidation. Moreover, H2S maintained K(+)/Na(+) homeostasis by preventing the NaCl-triggered K(+) efflux, which might be result form the impairment of SKOR expression. Together, our findings indicated that endogenous H2S homeostasis enhance salt tolerance by coupling the reestablishment of redox balance and restraining K(+) efflux in alfalfa seedlings.

Reactive Oxygen Species-Dependent Nitric Oxide Production Contributes to Hydrogen-Promoted Stomatal Closure in Arabidopsis.

The signaling role of hydrogen gas (H2) has attracted increasing attention from animals to plants. However, the physiological significance and molecular mechanism of H2 in drought tolerance are still largely unexplored. In this article, we report that abscisic acid (ABA) induced stomatal closure in Arabidopsis (Arabidopsis thaliana) by triggering intracellular signaling events involving H2, reactive oxygen species (ROS), nitric oxide (NO), and the guard cell outward-rectifying K+ channel (GORK). ABA elicited a rapid and sustained H2 release and production in Arabidopsis. Exogenous hydrogen-rich water (HRW) effectively led to an increase of intracellular H2 production, a reduction in the stomatal aperture, and enhanced drought tolerance. Subsequent results revealed that HRW stimulated significant inductions of NO and ROS synthesis associated with stomatal closure in the wild type, which were individually abolished in the nitric reductase mutant nitrate reductase1/2 (nia1/2) or the NADPH oxidase-deficient mutant rbohF (for respiratory burst oxidase homolog). Furthermore, we demonstrate that the HRW-promoted NO generation is dependent on ROS production. The rbohF mutant had impaired NO synthesis and stomatal closure in response to HRW, while these changes were rescued by exogenous application of NO. In addition, both HRW and hydrogen peroxide failed to induce NO production or stomatal closure in the nia1/2 mutant, while HRW-promoted ROS accumulation was not impaired. In the GORK-null mutant, stomatal closure induced by ABA, HRW, NO, or hydrogen peroxide was partially suppressed. Together, these results define a main branch of H2-regulated stomatal movement involved in the ABA signaling cascade in which RbohF-dependent ROS and nitric reductase-associated NO production, and subsequent GORK activation, were causally involved.

Hydrogen sulfide delays GA-triggered programmed cell death in wheat aleurone layers by the modulation of glutathione homeostasis and heme oxygenase-1 expression.

Hydrogen sulfide (H2S) is considered as a cellular signaling intermediate in higher plants, but corresponding molecular mechanisms and signal transduction pathways in plant biology are still limited. In the present study, a combination of pharmacological and biochemical approaches was used to study the effect of H2S on the alleviation of GA-induced programmed cell death (PCD) in wheat aleurone cells. The results showed that in contrast with the responses of ABA, GA brought about a gradual decrease of l-cysteine desulfhydrase (LCD) activity and H2S production, and thereafter PCD occurred. Exogenous H2S donor sodium hydrosulfide (NaHS) not only effectively blocked the decrease of endogenous H2S release, but also alleviated GA-triggered PCD in wheat aleurone cells. These responses were sensitive to hypotaurine (HT), a H2S scavenger, suggesting that this effect of NaHS was in an H2S-dependent fashion. Further experiment confirmed that H2S, rather than other sodium- or sulphur-containing compounds derived from the decomposing of NaHS, was attributed to the rescuing response. Importantly, the reversing effect was associated with glutathione (GSH) because the NaHS triggered increases of endogenous GSH content and the ratio of GSH/oxidized GSH (GSSG) in GA-treated layers, and the NaHS-mediated alleviation of PCD was markedly eliminated by l-buthionine-sulfoximine (BSO, a selective inhibitor of GSH biosynthesis). The inducible effect of NaHS was also ascribed to the modulation of heme oxygenase-1 (HO-1), because the specific inhibitor of HO-1 zinc protoporphyrin IX (ZnPP) significantly suppressed the NaHS-related responses. By contrast, the above inhibitory effects were reversed partially when carbon monoxide (CO) aqueous solution or bilirubin (BR), two of the by-products of HO-1, was added, respectively. NaHS-triggered HO-1 gene expression in GA-treated layers was also confirmed. Together, the above results clearly suggested that the H2S-delayed PCD in GA-treated wheat aleurone cells was associated with the modulation of GSH homeostasis and HO-1 gene expression.

Roles of NIA/NR/NOA1-dependent nitric oxide production and HY1 expression in the modulation of Arabidopsis salt tolerance.

Despite substantial evidence on the separate roles of Arabidopsis nitric oxide-associated 1 (NOA1)-associated nitric oxide (NO) production and haem oxygenase 1 (HY1) expression in salt tolerance, their integrative signalling pathway remains largely unknown. To fill this knowledge gap, the interaction network among nitrate reductase (NIA/NR)- and NOA1-dependent NO production and HY1 expression was studied at the genetic and molecular levels. Upon salinity stress, the majority of NO production was attributed to NIA/NR/NOA1. Further evidence confirmed that HY1 mutant hy1-100, nia1/2/noa1, and nia1/2/noa1/hy1-100 mutants exhibited progressive salt hypersensitivity, all of which were significantly rescued by three NO-releasing compounds. The salinity-tolerant phenotype and the stronger NO production in gain-of-function mutant of HY1 were also blocked by the NO synthetic inhibitor and scavenger. Although NO- or HY1-deficient mutants showed a compensatory mode of upregulation of HY1 or slightly increased NO production, respectively, during 2 d of salt treatment, downregulation of ZAT10/12-mediated antioxidant gene expression (cAPX1/2 and FSD1) was observed after 7 d of treatment. The hypersensitive phenotypes and stress-related genes expression profiles were differentially rescued or blocked by the application of NO- (in particular) or carbon monoxide (CO)-releasing compounds, showing a synergistic mode. Similar reciprocal responses were observed in the nia1/2/noa1/hy1-100 quadruple mutant, with the NO-releasing compounds exhibit the maximal rescuing responses. Overall, the findings present the combination of compensatory and synergistic modes, linking NIA/NR/NOA1-dependent NO production and HY1 expression in the modulation of plant salt tolerance.

Molecular cloning, characterization, and expression analysis of a novel gene encoding L-cysteine desulfhydrase from Brassica napus.

L-Cysteine desulfhydrase (DES; EC 4.4.1.1) is the most important enzyme that catalyzes the decomposition of L-cysteine to pyruvate, ammonia, and hydrogen sulfide (H2S), the latter of which has recently been recognized as the third gasotransmitter for multiple signaling events in plants. Previous results showed the existence of DES activity in Brassica napus; however, the gene encoding the true DES protein has not been characterized yet. Here, a rapeseed DES gene was isolated and sequenced. It shared high homology with Arabidopsis DES1, and encodes a polypeptide with 323 amino acids of 34.5 kDa. Subsequently, prokaryotic expression and biochemical analysis demonstrated that this protein predominantly catalyzes the breakdown of L-cysteine with the side reaction of L-cysteine synthesis [O-acetyl-L-serine(thiol)lyase activity], and was designated as BnDES1. Corresponding analysis of structural features was also in agreement with the above proposition. Molecular evidence showed that BnDES1 mRNA was widely expressed, but with the higher expression level in flowers. Further results showed that the BnDES1 transcripts were differentially up-regulated by several plant growth regulators and chemicals. Overall, the above findings provide evidence showing that BnDES1 is a potentially important enzyme responsible for the H2S production, and may play an important role in plant growth regulators and chemical stimuli responses.

H(2) enhances arabidopsis salt tolerance by manipulating ZAT10/12-mediated antioxidant defence and controlling sodium exclusion.

BACKGROUND: The metabolism of hydrogen gas (H(2)) in bacteria and algae has been extensively studied for the interesting of developing H(2)-based fuel. Recently, H(2) is recognized as a therapeutic antioxidant and activates several signalling pathways in clinical trials. However, underlying physiological roles and mechanisms of H(2) in plants as well as its signalling cascade remain unknown. METHODOLOGY/PRINCIPAL FINDINGS: In this report, histochemical, molecular, immunological and genetic approaches were applied to characterize the participation of H(2) in enhancing Arabidopsis salt tolerance. An increase of endogenous H(2) release was observed 6 hr after exposure to 150 mM NaCl. Arabidopsis pretreated with 50% H(2)-saturated liquid medium, mimicking the induction of endogenous H(2) release when subsequently exposed to NaCl, effectively decreased salinity-induced growth inhibition. Further results showed that H(2) pretreatment modulated genes/proteins of zinc-finger transcription factor ZAT10/12 and related antioxidant defence enzymes, thus significantly counteracting the NaCl-induced reactive oxygen species (ROS) overproduction and lipid peroxidation. Additionally, H(2) pretreatment maintained ion homeostasis by regulating the antiporters and H(+) pump responsible for Na(+) exclusion (in particular) and compartmentation. Genetic evidence suggested that SOS1 and cAPX1 might be the target genes of H(2) signalling. CONCLUSIONS: Overall, our findings indicate that H(2) acts as a novel and cytoprotective regulator in coupling ZAT10/12-mediated antioxidant defence and maintenance of ion homeostasis in the improvement of Arabidopsis salt tolerance.