Mechanism Of H₂S Signal Resistance To Drought Stress Through Alternative Splicing Of ETFQO

Drought stress has become one of the major adversities limiting crop production worldwide.Hydrogen sulfide（H₂S）,known as the third class of gas signaling molecules,plays an important role in plant resistance to drought stress,and many research have been achieved.Alternative splicing（AS）is an important mechanism for plants functional diversity,which arises from adversity stress.In this paper,using the model plant Arabidopsis thaliana as experimental material,the regulation of alternative splicing patterns of electron transfer flavoprotein-ubiquinone oxdioreductase coding gene（ETFQO）by drought and exogenous fumigation H₂S was verified by RT-PCR.Additionally,the different roles of three transcripts in the process of plant drought resistance were proved.Finally,different types of AS of Chinese cabbage,a cruciferous plant,were predicted by transcriptome sequencing analysis under H₂S signal.It provides a new insight for studying the mechanism of H₂S signal transduction.The main results are as follows:（1）Three transcripts of ETFQO were obtained.There were two transcripts of ETFQO（shown as ETFQO.1 and ETFQO.2）searched in NCBI website.Under the combined treatment of H₂S and PEG,a variety of alternative splicing of ETFQO occurred through RT-PCR verified.First,transcripts ETFQO.1 and ETFQO.2 were obtained by primer amplification,with sizes of 1901 bp and 1755 bp,respectively.Furthermore,the new transcript ETFQO.3 was recovered by glue cutting recycling,the size was 991 bp and the AS type was exon-skipping.（2）The relationship between different transcripts and H₂S and drought resistance were analyzed in vivo.XF350-ETFQO.1,XF350-ETFQO.2 and PCAM2300-ETFQO.3were constructed and transformed under the background of WT and H₂S double mutant material lcd/des1.Through experimental analysis,the H₂S and chlorophyll contents of ETFQO.1 and ETFQO.3 were higher than those of wild type under WT background,and the H₂S and chlorophyll contents of ETFQO.1 and ETFQO.3 recovered to the level of wild type after overexpression of each transcript on lcd/des1 background.In addition,the stomatal aperture size of ETFQO.1 and ETFQO.3 also decreased significantly,indicating that the transcripts ETFQO.1 and ETFQO.3 related to resistance to drought stress closely,while ETFQO.2 is not sensitive to drought.（3）Three transcripts were constructed as prokaryotic expression vectors in vitro,p Cold ^TMTF-ETFQO.1/2/3,approximately.The corresponding recombinant proteins were obtained by IPTG induction expression and purification.The recombinant protein sizes were approximately 124 k Da,118 k Da and 87 k Da,respectively.The optimal IPTG induction concentrations were 0.5 m M,0.3 m M and 0.3 m M,respectively.After incubating with H₂S respectively,the sulfhydrylation level was detected by biotin switch method.The results showed that the transcript ETFQO.1 and ETFQO.3 proteins could be modified by H₂S,while ETFQO.2 did not detect the signal of sulfhydrylation.（4）Through transcriptomic analysis of Chinese cabbage,also a cruciferous family,it was predicted that both H₂S and H₂S inhibitor HA would cause variable splicing of Chinese cabbage,and the proportion of each cutting type was different,and the most frequent AS types was intron retention.In addition,genes related to oxidative phosphorylation,photosynthesis,plant hormone signal transduction,plant pathogen interaction and other pathways were differentially expressed.Through GO and KEGG enrichment analysis,it was concluded that the differential expression was mainly enriched in various metabolic pathways and biological processes.12 genes were randomly selected for q RT-PCR verification,and the experimental results were consistent with the sequencing results.In summary,H₂S signal regulate the AS mode of ETFQO;the three transcripts of ETFQO show functional differentiation,among them,ETFQO.1 and ETFQO.3 have the ability to resist drought stress;mechanism of H₂S signal against drought stress can be achieved through the sulfhydrylation modification of target proteins ETFQO.1 and ETFQO.3 in vitro,which is consistent with the drought resistance results in vivo.H₂S signal is universal for plants with the regulation of AS.