Effect Of Exogenous Hydrogen Sulphide On Tobacco Growth And Lateral Root Development

Hydrogen sulphide（H₂S）is the third multifunctional gas signalling molecule to be discovered after carbon monoxide（CO）and nitric oxide（NO）,and it is only recently that the physiological functions of endogenous H₂S gas in plants have been uncovered.In plants,appropriate concentrations of H₂S have been shown to increase seed germination,improve photosynthesis,promote adventitious root formation,delay plant senescence and alleviate stresses.In order to elucidate the molecular mechanism of H₂S-induced root formation in tobacco,We used transcriptome sequencing,transgenic technology and real-time fluorescence quantitative PCR to investigate the regulatory network of H₂S-induced root formation in tobacco,with a view to providing theoretical references for H₂S regulation of plant root growth.The main results of the study are as follows:1.The exogenous H₂S donor sodium sulfide（Na HS）at low concentrations（0.05 mmol/L,0.1 mmol/L）promoted seed germination;high Na HS treatments（0.25mmol/L,0.5 mmol/L）significantly inhibited seed germination.After 0.05 mmol/L Na HS treatment for 14 d,the fresh and dry weights of tobacco seedlings increased by36.97% and 50.58% in leaves and 35.18% and 31.51% in roots,respectively,with no significant difference in plant height compared to the control;it also induced an increase in endogenous H₂S production and chlorophyll in tobacco seedlings.HA treatment decreased the fresh and dry weight of tobacco leaves compared to the control;the height of tobacco seedlings was inhibited compared to the control;the MDA and chlorophyll content of HA treated tobacco seedlings increased;while there was a significant presence of superoxide anion and cell death in tobacco seedlings;and the POD and CAT activities increased,by 2.9 and 1.39 times respectively compared to the control.2.The root vigour of tobacco seedlings treated with exogenous addition of 0.05mmol/L Na HS increased by 97.56%;their root length,surface area,root volume and number of lateral roots increased by 56.41%,79.84%,102.94% and 18.54%,respectively,compared with the control;HA treatment reduced the total root length,number of lateral roots and total root surface area of seedlings compared with the control by The results indicated that HA treatment not only eliminated the promotion effect of H₂S on lateral roots,but also inhibited the root development.3.Targeted metabolomic analysis of tobacco roots after 14 d of treatment revealed that H₂S regulated 42 plant hormones,including Auxin（growth hormone）,ABA（abscisic acid）,ETH（ethylene）,CK（cytokinin）,SA（salicylic acid）,JA（jasmonic acid）and SL（solanum lactone）,with the most significant effect on growth hormones and increased the content of growth hormones,allowing growth hormones to accumulate in the root system and thus promoting lateral root development.4.The RNA-seq results of the different treated tobacco root systems showed that 1568 differentially expressed genes（R-CK vs R-H₂S）and 7790 differentially expressed genes（R-CK vs R-HA）were expressed in the root systems.By transcriptome analysis of the KEGG pathway,the differentially expressed genes in the KEGG pathway were mainly annotated to the metabolic pathway,the secondary metabolite biosynthesis pathway and the phytohormone signalling pathway by transcriptome analysis of R-CK VS R-H₂S.The KEGG pathway is mainly annotated to the metabolic pathway,secondary metabolite biosynthesis pathway and phytohormone signaling pathway.The transcriptome analysis of R-CK VS R-HA showed that the differential genes in the KEGG pathway were mainly annotated to the secondary metabolite biosynthesis pathway,the phytohormone signaling pathway and the MAPK signaling pathway-plant pathway.5.H₂S treatment was found to be significantly associated with the phytohormone signalling pathway,with 46 differentially expressed genes in the phytohormone pathway involved in growth hormone transport and signalling.Of these,three differentially expressed genes were associated with growth hormone transport and 43 differentially expressed genes were associated with growth hormone signaling,including 31 early growth hormone response genes（GH3,11 genes;SAUR,9 genes;Aux/IAA,11 genes）,10 growth hormone response factors（ARF）,and two genes encoding growth hormone-inducible proteins,22 D and 15 A.6.Transcriptome analysis and Q-PCR results showed that exogenous H₂S i ncre-ased endogenous H₂S content by increasing the expression of the H₂S synt hase-encoding genes L-DCD 220（Nitab4.5₀000391g0220）and CYSM（Nitab4.5₀000013g0360）,and regulated the growth hormone synthesis genes YUCCA 4（Nitab4.5₀007173g0010）,YUCCA 8（Nitab4.5₀000326g0350）and YUCCA 9（Nitab4.5₀001557g0030）,thereby promoting lateral root development in tobacco seedlings.