The Initial Mechanism Study Of SlARF4 In Regulating Drought Resistance In Tomato

Drought is one of the major natural disasters affecting agricultural production.China is a relatively arid country.Due to the effects of extreme arid weather,the number of arid regions in Guangdong has increased.Therefore,it is particularly important to carry out research on plant drought resistance mechanisms to reduce adverse environmental impacts.Tomato is one of the most widely cultivated vegetable and fruit horticultural crops in the world.Studying the effects of drought stress on tomatoes will help to improve tomato drought resistance,create new varieties of drought resistance,deal better with global environmental changes,and produce better tomato,which has important economic value.In recent years,more and more studies have focused on the signal interactions among various hormones in plants under stress to elucidate the mechanism of molecular-level gene networks.Among them,the auxin response factor(ARF)in the classic auxin signal transduction pathway is also involved in the self-regulation of plants under drought stress,but the specific regulation mechanism is still unknown.In this paper,the CRISPR / Cas9 mutant plant of SlARF4(arf4)was selected as the research material.The physiological and biochemical tests and molecular biological methods were used to study the drought resistance and mechanism of the mutant plants.The main findings are as follows:1.The arf4 plant is characterized by curl leaf,thickness stem,bigger single fruit and seed,less seeds,high chlorophyll content in the MG fruit,and delayed flowering time;2.Drought resistance of arf4 tomato plants was enhanced,and rewatering ability was enhanced after stress.Under drought stress,the maximum photosynthetic efficiency Fv/Fm of Photosystem II(PSII)was significantly higher than that of wild type.The MDA content of arf4 leaves was significantly lower than that of wild type.Under drought stress,arf4 had higher content of superoxide dismutase(SOD),peroxidase(POD),catalase activity(CAT),and glutathione(GSH)content and ascorbic acid(Vc).After drought stress,the antioxidant enzyme activity of rewatering treatment returned to normal levels relatively quickly in arf4.The production rate of superoxide anion in arf4 is significantly higher than that in WT.At the same time,the drought resistance of arf4 tomato seedlings was enhanced,showing higher germination rates and longer hypocotyls and roots under simulated stress;3.Real-time fluorescence quantitative PCR and GUS staining showed that drought stress and exogenous ABA treatment can both reduce the expression of SlARF4.SlARF4 is expressed in the vascular bundles and guard cells of the stomata of tomato;The stomatal morphology of the lower epidermis of arf4 leaves changed,and the stomatal closure was not sensitive under drought stress.It was found that arf4 had more developed xylem by paraffin section observation on the stem transection;4.Transcriptome analysis showed that comparing to WT,arf4 has fewer differentially expressed genes respond to drought stress.There are 628 specifically expressed genes in arf4 under drought stress,which are mainly concentrated in glutathione metabolism,Phenylpropane synthesis,amphetamine metabolism,hormone signaling pathway,linoleic acid metabolism,a-linolenic acid metabolism(JA synthesis)pathway.Among them,86 gene were differentially expressed between WT and arf4 under drought stress,and they were mainly enriched in phenylpropane synthesis,hormone signaling pathway,and a-linolenic acid metabolism(JA synthesis).There are 7 genes that are differentially expressed in wild type and arf4 mutant under drought and normal conditions.Except for two unknown genes,the remaining genes all contain Aux RE(auxin regulation element)binding elements,indicating that the expression of these five genes can be directly regulated by ARF4.Sl ABI5 and Sl ABA4 participate in the ABA synthesis and signaling pathway.The reliability of the RNA-Seq data was verified by real-time PCR;5.Select the Sl ABA4 gene as the research object,and verify the binding and regulation of SlARF4 to Sl ABA4 through yeast one-hybrid test and double luciferase test.The results show that there is interaction between SlARF4 and the Sl ABA4 promoter sequence.SlARF4 can bind to the Sl ABA4 promoter and suppress its expression;In summary,SlARF4 is involved in the growth and development of tomato plants.Under drought stress,SlARF4 can participate in ABA synthesis and signaling pathways by regulating Sl ABI5/ABF and Sl ABA4 exprssion,through operating the opening and closing of stomata and the development of vascular bundles to improve plant drought resistance.