L-Cysteine desulfhydrase-dependent hydrogen sulfide is required for methane-induced lateral root formation.

KEY MESSAGE: Methane-triggered lateral root formation is not only a universal event, but also dependent on L-cysteine desulfhydrase-dependent hydrogen sulfide signaling. Whether or how methane (CH4) triggers lateral root (LR) formation has not been elucidated. In this report, CH4 induction of lateral rooting and the role of hydrogen sulfide (H2S) were dissected in tomato and Arabidopsis by using physiological, anatomical, molecular, and genetic approaches. First, we discovered that CH4 induction of lateral rooting is a universal event. Exogenously applied CH4 not only triggered tomato lateral rooting, but also increased activities of L-cysteine desulfhydrase (DES; a major synthetic enzyme of H2S) and induced endogenous H2S production, and contrasting responses were observed in the presence of hypotaurine (HT; a scavenger of H2S) or DL-propargylglycine (PAG; an inhibitor of DES) alone. CH4-triggered lateral rooting were sensitive to the inhibition of endogenous H2S with HT or PAG. The changes in the transcripts of representative cell cycle regulatory genes, miRNA and its target genes were matched with above phenotypes. In the presence of CH4, Arabidopsis mutant Atdes1 exhibited defects in lateral rooting, compared with the wild-type. Molecular evidence showed that the transcriptional profiles of representative target genes modulated by CH4 in wild-type plants were impaired in Atdes1 mutant. Overall, our data demonstrate the main branch of the DES-dependent H2S signaling cascade in CH4-triggered LR formation.

Nitric oxide contributes to methane-induced osmotic stress tolerance in mung bean.

BACKGROUND: Osmotic stress is a major abiotic stress limiting crop production by affecting plant growth and development. Although previous reports discovered that methane (CH4) has a beneficial effect on osmotic stress, the corresponding downstream signal(s) is still elusive. RESULTS: Polyethylene glycol (PEG) treatment progressively stimulated the production of CH4 in germinating mung bean seeds. Exogenous CH4 and sodium nitroprusside (SNP) not only triggered nitric oxide (NO) production in PEG-stressed plants, but also alleviated the inhibition of seed germination. Meanwhile, amylase activity was activated, thus accelerating the formation of reducing sugar and total soluble sugar. Above responses could be impaired by NO scavenger(s), suggesting that CH4-induced stress tolerance was dependent on NO. Subsequent tests showed that CH4 could reestablish redox balance in a NO-dependent fashion. The addition of inhibitors of the nitrate reductase (NR) and NO synthase in mammalian (NOS), suggested that NR and NOS-like protein might be partially involved in CH4-alleviated seed germination inhibition. In vitro and scavenger tests showed that NO-mediated S-nitrosylation might be associated with above CH4 responses. CONCLUSIONS: Together, these results indicated an important role of endogenous NO in CH4-enhanced plant tolerance against osmotic stress, and NO-regulated redox homeostasis and S-nitrosylation might be involved in above CH4 action.

Hydrogen peroxide is involved in hydrogen sulfide-induced lateral root formation in tomato seedlings.

BACKGROUND: Both hydrogen sulfide (H2S) and hydrogen peroxide (H2O2) are separately regarded as a highly reactive molecule involved in root morphogenesis. In this report, corresponding causal link governing lateral root formation was investigated. METHODS: By using pharmacological, anatomic, and molecular approaches, evidence presented here revealed the molecular mechanism underlying tomato lateral root development triggered by H2S. RESULTS: A H2S donor sodium hydrosulfide (NaHS) triggered the accumulation of H2O2, the up-regulation of RBOH1 transcript, and thereafter tomato lateral root formation. Above responses were sensitive to the H2O2 scavenger (dimethylthiourea; DMTU) and the inhibitor of NADPH oxidase (diphenylene idonium; DPI), showing that the accumulations of H2O2 and increased RBOH1 transcript were respectively prevented. Lateral root primordial and lateral root formation were also impaired. Further molecular evidence revealed that H2S-modulated gene expression of cell cycle regulatory genes, including up-regulation of SlCYCA2;1, SlCYCA3;1, and SlCDKA1, and the down-regulation of SlKRP2, were prevented by the co-treatment with DMTU or DPI. Above mentioned inducing phenotypes were consistent with the changes of lateral root formation-related microRNA transcripts: up-regulation of miR390a and miR160, and with the opposite tendencies of their target genes (encoding auxin response factors). Contrasting tendencies were observed when DMTU or DPI was added together. The occurrence of H2S-mediated S-sulfhydration during above responses was preliminarily discovered. CONCLUSIONS: Overall, these results suggested an important role of RBOH1-mediated H2O2 in H2S-elicited tomato lateral root development, and corresponding H2S-target proteins regulated at transcriptional and post-translational levels.

Potential Succinate Dehydrogenase Inhibitors Bearing a Novel Pyrazole-4-sulfonohydrazide Scaffold: Molecular Design, Antifungal Evaluation, and Action Mechanism.

Aiming to develop novel antifungal agents with a distinctive molecular scaffold targeting succinate dehydrogenase (SDH), 24 N'-phenyl-1H-pyrazole-4-sulfonohydrazide derivatives were first devised, synthesized, and verified by 1H NMR, 13C NMR, high-resolution mass spectrometry (HRMS), and single-crystal X-ray diffraction analysis. The bioassays revealed that the target compounds possessed highly efficient and broad-spectrum antifungal activities against four tested plant pathogenic fungi Rhizoctonia solani (R. solani), Botrytis cinerea, Fusarium graminearum, and Alternaria sonali. Strikingly, compound B6 was assessed as the selective inhibitor against R. solani, with an in vitro EC50 value (0.23 µg/mL) that was similar to that of thifluzamide (0.20 µg/mL). The in vivo preventative effect of compound B6 (75.76%) at 200 µg/mL against R. solani was roughly comparable to thifluzamide (84.31%) under the same conditions. The exploration of morphological observations indicated that compound B6 could strongly damage the mycelium morphology, obviously increase the permeability of the cell membrane, and dramatically increase the number of mitochondria. Compound B6 also significantly inhibited SDH enzyme activity with an IC50 value of 0.28 µg/mL, and its fluorescence quenching dynamic curves were similar to that of thifluzamide. Molecular docking and molecular dynamics simulations demonstrated that compound B6 could strongly interact with similar residues around the SDH active pocket as thifluzamide. The present study revealed that the novel N'-phenyl-1H-pyrazole pyrazole-4-sulfonohydrazide derivatives are worthy of being further investigated as the promising replacements of traditional carboxamide derivatives targeting SDH of fungi.

Hydrogen Peroxide Is Involved in ß-Cyclodextrin-hemin Complex-Induced Lateral Root Formation in Tomato Seedlings.

Although previous results showed that ß-cyclodextrin-hemin complex (ß-CDH) could induce tomato lateral root (LR) formation, the corresponding downstream messengers are still not fully understood. In this report, similar to the inducing effects of exogenously applied hydrogen peroxide (H2O2), we discovered that ß-CDH elicited RBOH1 transcript upregulation, endogenous H2O2 accumulation, and thereafter tomato LR development. Above responses were sensitive to dimethylthiourea (DMTU) and ascorbic acid (AsA), two membrane-permeable scavengers of H2O2, showing that accumulation of H2O2 and LR formation were significantly blocked. The test with diphenyleneiodonium (DPI; the inhibitor of NADPH oxidase) revealed that H2O2 mainly produced by NADPH oxidase, might be involved in LR formation triggered by ß-CDH. qPCR combined with pharmacological and anatomical analyses showed that ß-CDH-modulated several marker genes responsible for LR formation, such as CYCA3;1, CYCA2;1, CYCD3;1, and CDKA1 (four cell cycle regulatory genes), ARF7 and RSI-1 (two auxin signaling genes), LAX3 (an auxin influx carrier), IAA14 (encoding a member of the Aux/IAA protein family), PIN3 and PIN7 (two auxin efflux carriers), isocitrate dehydrogenase [NADP], NADH-cytochrome b5 reductase 1, and L-ascorbate oxidase homolog genes (two reactive oxygen species-associated genes and one LR formation-related gene), were causally related to above H2O2 signaling. Particularly, representative proteins related to H2O2 metabolism and lateral rooting, were specifically induced in ß-CDH-treated tomato seedlings. Overall, the results clearly suggested a vital role of H2O2 in the ß-CDH-induced tomato LR formation, and ß-CDH-elicited H2O2-related target proteins responsible for LR formation might be, at least partially, regulated at transcriptional and translational levels.

Involvement of glutathione in ß-cyclodextrin-hemin complex-induced lateral root formation in tomato seedlings.

ß-cyclodextrin-hemin complex (ß-CDH) was shown to induce lateral root (LR) formation in tomato. However, the molecular mechanism is still elusive. In this report, the role of reduced glutathione (GSH) in the induction of lateral root triggered by ß-CDH was investigated. Similar to the responses of ß-CDH, exogenously applied with 0.1 mΜ GSH not only increased endogenous GSH content determined by spectrophotography and the monochlorobimane (MCB)-dependent fluorescent analysis, but also induced, thereafter, LR formation. Meanwhile, both ß-CDH- and GSH-induced lateral root primordia (LRP) exhibited a similar accelerated anatomic structure. Above inducible responses were blocked significantly when the L-buthionine-(S,R)-sulfoximine (BSO), a potent and specific inhibitor of the enzyme catalyzing the first step of GSH biosynthesis, was separately applied. Upon ß-CDH treatment, the changes of endogenous GSH content determined by spectrophotography and fluorescent analysis were consistent with the transcripts of two GSH synthetic genes, GSH1 and GSH2 encoding γ-glutamyl cysteine synthetase and glutathione synthetase, respectively. Exogenously applied with ß-CDH could rescue N-1-naphthylphthalamic acid (NPA; IAA depletion)-triggered inhibition of LR formation. Further molecular evidence revealed that both ß-CDH and GSH modulated gene expression of cell cycle regulatory genes (CYCA2;1, CYCA3;1, CYCD3;1, and CDKA1) and auxin signaling genes (ARF7 and RSI-1), six marker genes responsible for LR formation. By contrast, above changes were sensitive to the co-treatment with BSO. All together, these results suggest a role for GSH in the regulation of tomato LR development triggered by ß-CDH.