A D-cysteine desulfhydrase, SlDCD2, participates in tomato fruit ripening by modulating ROS homoeostasis and ethylene biosynthesis

Hydrogen sulfide(H_(2)S)is involved in multiple processes during plant growth and development.D-cysteine desulfhydrase(DCD)can produce H_(2)S with D-cysteine as the substrate;however,the potential developmental roles of DCD have not been explored during the tomato lifecycle.In the present study,SlDCD2 showed increasing expression during fruit ripening.Compared with the control fruits,the silencing of SlDCD2 by pTRV2-SlDCD2 accelerated fruit ripening.A SlDCD2 gene-edited mutant was constructed by CRISPR/Cas9 transformation,and the mutant exhibited accelerated fruit ripening,decreased H_(2)S release,higher total cysteine and ethylene contents,enhanced chlorophyll degradation and increased carotenoid accumulation.Additionally,the expression of multiple ripening-related genes,including NYC1,PAO,SGR1,PDS,PSY1,ACO1,ACS2,E4,CEL2,and EXP was enhanced during the dcd2 mutant tomato fruit ripening.Compared with the wild-type fruits,SlDCD2 mutation induced H_(2)O_(2) and malondialdehyde(MDA)accumulation in fruits,which led to an imbalance in reactive oxygen species(ROS)metabolism.A correlation analysis indicated that H_(2)O_(2) content was strongly positively correlated with carotenoids content,ethylene content and ripening-related gene expression and negatively correlated with the chlorophyll content.Additionally,the dcd2 mutant showed earlier leaf senescence,which may be due to disturbed ROS homeostasis.In short,our findings show that SlDCD2 is involved in H_(2)S generation and that the reduction in endogenous H_(2)S production in the dcd2 mutant causes accelerated fruit ripening and premature leaf senescence.Additionally,decreased H_(2)S in the dcd2 mutant causes excessive H_(2)O_(2) accumulation and increased ethylene release,suggesting a role of H_(2)S and SlDCD2 in modulating ROS homeostasis and ethylene biosynthesis.

PyWRKY26 and PybHLH3 cotargeted the PyMYB114 promoter to regulate anthocyanin biosynthesis and transport in red-skinned pears

Red pear is favored because of its bright appearance and abundant anthocyanins.Anthocyanin biosynthesis is controlled by transcription factors(TFs)forming regulatory complexes.In red-skinned pears,the WRKY TFs have a significant relationship with anthocyanin biosynthesis,but the molecular mechanism of the WRKY TFs involved in regulating color formation in red-skinned pear is unclear.In this study,the TFs PyWRKY31 and PyWRKY26 were screened as candidate genes for controlling anthocyanin biosynthesis by transcriptome data and bioinformatics analysis.The effect of anthocyanin accumulations after cotransformation of PyWRKY31 or PyWRKY26 with its partners PyMYB10,PyMYB114,and PybHLH3 was verified in tobacco leaves and strawberry receptacles by a transient expression system.RT-qPCR analysis and a dual-luciferase reporter system further confirmed that this cotransformation activated the expression of PyDFR,PyANS,and PyUFGT in anthocyanin biosynthesis and PyGST in anthocyanin transport instead of the PyABC transporter and PyAVP.Furthermore,the cotransformed PyWRKY26 and PybHLH3 could bind to the PyMYB114 promoter,and PyWRKY26 directly activated the transcription of PyMYB114.In addition,the TF PyWRKY26 could interact with PybHLH3,as confirmed by firefly luciferase complementation and yeast two-hybrid(Y2H)assays.These results showed that the interaction of PyWRKY26 and PybHLH3 could cotarget the PyMYB114 promoter,which resulted in anthocyanin accumulation in red-skinned pear.This study further strengthened the understanding of the regulatory mechanism of anthocyanin accumulation and contributed to improving the appearance of red-skinned pears.

A nuclear-localized cysteine desulfhydrase plays a role in fruit ripening in tomato

Hydrogen sulfide(H_(2)S)is a gaseous signaling molecule that plays multiple roles in plant development.However,whether endogenous H_(2)S plays a role in fruit ripening in tomato is still unknown.In this study,we show that the H_(2)S-producing enzyme L-cysteine desulfhydrase SILCD1 localizes to the nucleus.By constructing mutated forms of SILCD1,we show that the amino acid residue K24 of SILCD1 is the key amino acid that determines nuclear localization.Silencing of SILCD1 by TRV-SILCD1 accelerated fruit ripening and reduced H_(2)S production compared with the control.A SILCD1 gene-edited mutant obtained through CRISPR/Cas9 modification displayed a slightly dwarfed phenotype and accelerated fruit ripening.This mutant also showed increased cysteine content and produced less H_(2)S,suggesting a role of SILCD1 in H_(2)S generation.Chlorophyll degradation and carotenoid accumulation were enhanced in the SILCD1 mutant.Other ripening-related genes that play roles in chlorophyll degradation,carotenoid biosynthesis,cell wall degradation,ethylene biosynthesis,and the ethylene signaling pathway were enhanced at the transcriptional level in the lcd1 mutant.Total RNA was sequenced from unripe tomato fruit treated with exogenous H_(2)S,and transcriptome analysis showed that ripening-related gene expression was suppressed.Based on the results for a SILCD1 gene-edited mutant and exogenous H_(2)S application,we propose that the nuclear-localized cysteine desulfhydrase SILCD1 is required for endogenous H_(2)S generation and participates in the regulation of tomato fruit ripening.