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Sulfide or sulfur metabolism plays an important role in the growth and development of plants. Cystathionine β-lyase (CBL) is an important enzyme in methionine synthesis, but a comprehensive understanding of CBL functions is limited. As the third gasotransmitter, hydrogen sulfide (H 2 S) plays important physiological roles in plants. In this study, we found that the endogenous H 2 S content in Arabidopsis thaliana cbl mutants was lower than that in the wild type. Under PEG-based osmotic stress conditions, the H 2 S contents of CBL -overexpression (OE-CBL) plants increased significantly compared with the wild type. Additionally, the OE-CBL plants increased their tolerance to osmotic stress by increasing the transcription levels of drought-related genes and their relative water-loss rates. Compared with cbl and wild type, OE-CBL plants resisted drought stress by significantly closing their stomata, resulting in improved survival rates. Root tip-bending experiments showed that CBL overexpression relieved osmotic, heavy metal and cold stresses in Arabidopsis. The recombinant CBL activity in vitro revealed that CBL produced H 2 S using L-cysteine as a substrate. Thus, CBL had a very strong cysteine desulfhydrase activity that could produce endogenous H 2 S using L-cysteine as a substrate, and it played an important role in plant abiotic stress resistance. [Display omitted] • The H 2 S concentration of cbl plants were lower than that of Col-0. • The activity of recombinant CBL in vitro revealed that CBL produced H 2 S using L-Cys as a substrate. • CBL enhanced the ability of plants to mitigate drought stress. • Root tip-bending experiment confirmed CBL responses to osmotic, heavy metals or cold stress. [ABSTRACT FROM AUTHOR]

Key message: Hydrogen sulfide closed Arabidopsis thaliana stomata by increasing the transcription of melatonin-producing enzymes and the post-translational modification levels to combat osmotic stress. Hydrogen sulfide (H2S) and melatonin (MEL) reportedly have similar functions in many aspects of plant growth, development and stress response. They regulate stomatal movement and enhance drought resistance. However, their physiological relationship is not well understood. Here, their crosstalk involved in osmotic stress resistance in Arabidopsis thaliana was studied. Exogenous H2S and MEL closed stomata under normal or osmotic stress conditions and increased the relative water contents of plants under osmotic stress conditions. At the same time, exogenous H2S and MEL responded to osmotic stress by increasing the content of proline and soluble sugar, and reducing malondialdehyde (MDA) content and relative conductivity. Using mutants in the MEL-associated production of serotonin N-acetyltransferase (snat), caffeic acid O-methyltransferase (comt1) and N-acetylserotonin methyltransferase (asmt), we determined that H2S was partially dependent on MEL to close stomata. Additionally, the overexpression of ASMT promoted stomatal closure. Exogenous H2S increased the transcription levels of SNAT, ASMT and COMT1. Furthermore, exogenous H2S treatments increased the endogenous MEL content significantly. At the post-translational level, H2S sulfhydrated the SNAT and ASMT, but not COMT1, enzymes associated with MEL production. Thus, H2S appeared to promote stomatal closure in response to osmotic stress by increasing the transcription levels of MEL synthesis-related genes and the sulfhydryl modification of the encoded enzymes. These results increased our understanding of H2S and MEL functions and interactions under osmotic stress conditions. [ABSTRACT FROM AUTHOR]

The roles of the gasotransmitter hydrogen sulfide (H 2 S) and the phytohormone melatonin (MEL) in enhancing plant drought resistance are increasingly understood, but the specific regulatory mechanisms in response to drought remain unclear. This study investigated their relationship in the regulation of stomatal movement in response to drought stress. Under normal and drought stress conditions, exogenous MEL increased the H 2 S content, production rate and the transcription level of H 2 S production-related encoding genes LCD and DES1. The ability of MEL to increase drought stress tolerance was inhibited significantly in lcd , des1 and lcd/des1 mutants, which implied that MEL was partially dependent on H 2 S in this process. Exogenous MEL and H 2 S increased the transcriptional level of drought related transcription factors CBF2 , CBF3 , RD29A , DREB2A and DREB2B in response to drought stress and H 2 S and MEL affected the transcription level of K+ channel-related genes KCO1 , GORK , AKT1 , AKT2 , KAT1 , and KC1 to regulate stomatal movement. At the post-translational level, H 2 S increased the persulfidation levels of KCO1 and AKT1 and reduced their degradation rate. It was also found that endogenous H 2 S and MEL content had no effect on the mutants of kco1 , kat1 , akt1 and gork. It was concluded that H 2 S mediated the MEL induced stoma closure by regulating the K+ channel to enhance plant drought resistance in Arabidopsis. [Display omitted] ● Exogenous MEL increased the H 2 S biosynthetic enzymes. ● H 2 S generated after MEL treatment triggered stomatal closure. ● MEL and H 2 S closed the stomata by regulating the expression of K+ channel-related genes. ● H 2 S improved proteins stability by persulfidating KCO1 and AKT1. [ABSTRACT FROM AUTHOR]

Asarum sieboldii Miq. possesses remarkable medicinal value due to its essential oil enriched with phenylpropenes (e.g., methyleugenol and safrole). Although the biosynthesis of phenylpropenes shares a common pathway with lignin, the regulation mechanisms in carbon flux allocation between them are unclear. This study is the first to genetically verify the carbon flux regulation mechanism in A. sieboldii roots. We regulated the expression of Caffeoyl-coenzyme A O -methyltransferase (CCoAOMT), an essential enzyme in the common pathway, to investigate carbon flux allocation in vegetative organs. Here, the lignin and phenylpropene content fluctuation was analyzed by wet chemistry and GC-MS methods. A bona fide CCoAOMT gene from A. sieboldii was firstly cloned and verified. Preliminary heterologous expression validation in transgenic Arabidopsis thaliana showed that RNAi-induced CCoAOMT down-regulation significantly decreased lignin content by 24% and increased the S/G ratio by 30%; however, AsCCoAOMT over-expression in A. thaliana resulted in a 40% increase in lignin content and a 20% decrease in the S/G ratio when compared to the wild type. Similar trends were noted in homologous transformation in A. sieboldii , although the variations were not conspicuous. Nevertheless, the transgenic A. sieboldii plants displayed substantial differences in the level of phenylpropene compounds methyleugenol and safrole leading to a 168% increase in the methyleugenol/safrole ratio in the over-expression line and a 73% reduction in RNAi-suppression line. These findings suggest that the biosynthesis of phenylpropene constituents methyleugenol and safrole seems to be prioritized over lignin. Furthermore, this study indicated that suppression of AsCCoAOMT resulted in marked root susceptibility to pathogenic fungal disease, implying a significant additional role of CCoAOMT in protecting plant vegetative parts from diseases. Overall, the present study provides important references and suggests that future research should be aimed at elucidating the detailed mechanisms of the carbon flux allocation between phenylpropenes and lignin biosynthesis, as well as the disease resistance competency. • This study explores carbon flux regulation between phenylpropenes and lignin biosynthesis in plant vegetative organs. • The phenylpropenes biosynthesis seems to be prioritized over lignin biosynthesis in Asarum sieboldii Miq. roots. • Caffeoyl-coenzyme A O -methyltransferase gene was firstly isolated and functionally characterized in Asarum plants. • Caffeoyl-coenzyme A O -methyltransferase regulates phenylpropenes/lignin biosynthesis, and root resistance to fungal disease. [ABSTRACT FROM AUTHOR]