Mechanism Of Cysteine Desulfhydrylase LCD1 And Nuclear Importin α3 In Regulating Tomato Fruit Ripening And Senescence

Tomato（Solanum lycopersicum）is a fruit and vegetable crop that is widely grown in China and rich in various nutrients.Due to the advantages of clear genetic background and short growth cycle,tomato is a model plant for studying plant growth and fruit ripening and senescence.In recent years,studies have found that H₂S,as a gas signal molecule in plants,can participate in various physiological processes in plants,and can delay the ripening and senescence process of various fruits and vegetables.Preliminary studies in our laboratory have shown that the cysteine desulfhydrase LCD1 is the key enzyme that mediates the generation of endogenous H₂S in tomato.LCD1 deletion accelerates the process of tomato fruit ripening and senescence,but whether LCD1 and H₂S are involved in regulating tomato leaf senescence is still unclear.Preliminary studies have found that LCD1 is localized in the nucleus,and yeast two-hybrid experiments have found that there is an interaction between LCD1 and nuclear importinα3,but whether the nuclear importinα3 is involved in mediating LCD1 into the nucleus still needs to be further explored.Besides,H₂S is an enzyme that generates H₂S in tomato,and H₂S signaling can mediate the sulfhydration of proteins.However,whether the interaction between LCD1 and importinα3 can lead to the sulfhydration of importinα3 is unclear.In addition,whether importinα3 can regulate tomato fruit ripening and senescence by mediating the nuclear localization of LCD1 is still unclear and further research is needed.In this paper,the function of LCD1 in regulating leaf senescence was studied.Wild-type tomato（Micro Tom）,LCD1 overexpression,and LCD1 gene-edited tomato plants were used as experimental materials.It was found that LCD1 overexpression delayed the senescence process of tomato conjoined leaves and the rate of H₂S production and the amount of H₂S production in tomato leaves were increased,whereas the absence of LCD1 resulted in accelerated leaf senescence.In order to explore whether LCD1 is involved in the senescence process of detached leaves under dark conditions,the detached leaves of the above three plants were used as materials.The results showed that LCD1 deletion caused acceleratated leaf yellowing,while LCD1 overexpression leaves were still in green color on day 8 in darkness,suggesting that LCD1 overexpression delayed the process of leaf chlorophyll degradation.In the leaf senescence process of the above three plant materials,by using spectrophotometry,it was found that LCD1 overexpression reduced the accumulation of malondialdehyde（MDA）and hydrogen peroxide（H₂O₂）;by RT-q PCR,it was found that LCD1overexpression down-regulated the expression of key chlorophyll degradation genes（PPH,SGR1,NYC1,PAO）and senescence-related genes（SAGs）,while LCD1 deletion up-regulated the expression of leaf senescence-related genes.In order to study the function of exogenous H₂S signal in regulating leaf senescence,wild-type tomato leaves were treated with 50μM Na HS as H₂S donor.It was found that in the process of dark-induced leaf senescence,H₂S treatment delayed chlorophyll degradation and meanwhile inhibited the accumulation H₂O₂ and MDA,and reduced the expression level of chlorophyll degradation and senescence-related genes（SAGs）.In order to explore the molecular mechanism of tomato leaves in response to H₂S signal to delay senescence,transcriptome sequencing was performed on leaves in the control group and H₂S treatment group under dark conditions,and by KEGG analysis,it was found that H₂S signal may delay the process of leaf senescence under dark-induced conditions by regulating various pathways such as“plant hormone signal transduction”,“porphyrin and chlorophyll metabolism”,and“secondary metabolite biosynthesis”.The previous experimental results showed that LCD1 has a nuclear localization,and the nuclear import protein importinα3 may be the key protein that mediates the nuclear localization of LCD1.In this paper,luciferase complementation experiments were used to further verify the interaction between importinα3 and LCD1;in addition,by bioinformatics analysis,it was found that importinα3 has an IBB domain and multiple ARM domains,and it has a potential nuclear localization signal sequence.By constructing importinα3-GFP vector and using laser confocal microscopy,it was found that importinα3 has significant nuclear localization,and it is also distributed in the cytoplasm.At the same time,in order to explore whether importinα3 can undergo sulfhydration modification,the importinα3-his prokaryotic expression vector was constructed and the importinα3 protein was expressed and purified.Under the condition of H₂S signal treatment,using biotin switch method combined with Western blot technology,it was found that nuclear import protein importinα3 has a sulfhydryl modification signal.In order to further clarify the cysteine（Cys）sites of importinα3protein that may undergo thiolation modification,the importinα3 protein was subjected to mass spectrometry analysis,and the results showed that the Cys351 of importinα3 protein had a sulfhydration modification signal.To explore the function of importinα3 in tomato fruit ripening and senescence,virus-induced gene silencing（VIGS）and Crispr/Cas9 gene editing technology were used to construct importinα3-silenced fruits and importinα3 gene-edited plants.The study found that importinα3-silenced tomato fruits and gene-edited tomato fruits entered the red ripening stage earlier and the chlorophyll degradation process accelerated,which accelerated the ripening and senescence process of tomato fruits.By RT-q PCR,it was found that importinα3-silenced fruits and importinα3-deficient fruits showed higher expression levels of middle chlorophyll degradation genes（PPH,SGR1,NYC1,PAO）,carotenoid synthesis genes（PSY,PDS,ZDS）and ethylene pathway genes（ACO3,ACO1,ACS2,RIN,E8,E4,NOR）,indicating that importinα3is a negative regulator of tomato fruit ripening and senescence.In addition,importinα3deletion leads to dwarf tomato plants,reduced fruit weight,and accelerated fruit water loss.In order to study the mediating effect of importinα3 on the nuclear localization of LCD1,importinα3 gene-edited plants and wild-type tomato plants were used as experimental materials.It was found that LCD1-GFP exhibited obvious nuclear localization in wild-type tomato leaves,while in the leaves of importinα3 gene-edited plants,LCD1 was found to have both nuclear and cytoplasmic localization,indicating that importinα3 has a mediating effect on the nuclear localization of LCD1.In summary,LCD1 regulates the senescence process of tomato leaves by affecting H₂S generation,pigment metabolism,and reactive oxygen species accumulation.Through luciferase complementation experiments,sulfhydration experiments and subcellular localization experiments,it was found that importinα3 interacts with LCD1 to mediate its entrance into the nucleus,and importinα3 can undergo sulfhydration modification in response to H₂S signal.In addition,using VIGS and gene editing technology,it was found that importinα3 silencing or deletion resulted in accelerated chlorophyll degradation and accelerated ethylene synthesis in tomato fruit,indicating that importinα3 regulates the process of tomato fruit ripening and senescence by modulating ethylene signal transduction,pigment metabolism and other pathways.