Study On The Regulatory Roles Of Cytokinin Receptor Gene SlHK4 In Growth And Development And Drought Tolerance Of Tomato

Cytokinins are one of the most important phytohormones in plants,and are widely involved in various processes of plant growth and development.Cytokinin receptors(histidine kinases,HKs)sense and bind to cytokinins,transmit signals downward,activate/inhibit expression of downstream response genes,and play a key role in the cytokinin regulation of plant growth and development.Tomato is a global economic crop as both vegetable and fruit.It is favored by consumers because of its juicy fruit and unique flavor.China is the country with the largest tomato planting area and the most production in the world.The growth and development of tomatoes require high environmental moisture.However,about half of China’s land area is under arid/semi-arid conditions.Coupled with the uneven distribution of water resources in time and space,the lack of water resources seriously affects the growth,yield and quality of tomatoes in China.Therefore,enhancing drought tolerance of tomatoes is of great significance for improving tomato yield and quality in arid/semi-arid regions in China.In this study,the cytokinin receptor gene SlHK4 was identified in tomato.Its expression was analyzed,and the changes in the growth and development and drought tolerance of its CRISPR/Cas mutant plants were examined for the investigation of its functions.The main findings are as follows:(1)Based on homology search and alignment,the orthologous gene of Arabidopsis cytokinin receptor CRE1/AHK4 in tomato,SlHK4,was identified.By analyzing the data of SlHK4 gene expression in the tomato public database,it is found that SlHK4 is expressed in various organs,such as roots,leaves,flowers,fruits,and developing seeds.The expression levels of SlHK4 in meristems are significantly higher than those in other tissues/organs.Additionally,the expression of SlHK4 is induced by exogenous cytokinin treatment,but not affected by the treatment of other hormones,such as auxin,ethylene,and abscisic acid.(2)A CRISPR/Cas editing vector of the SlHK4 gene was constructed,and genetically transformed into tomato.A total of five homozygous CRISPR/Cas mutant lines with different mutation types were obtained.Phenotypic observation of two homozygous mutant lines,slhk4-4 and slhk4-118,and wild-type plants(WT)was performed.Compared with the WT,the slhk4-4 and slh4-118 plants show lower seed germination rates,smaller cotyledon and leaf areas,thinner stems,significantly shorter shoots,enhanced root systems,larger numbers of fruits,smaller fruits with fewer seeds inside,and delayed fruit ripening.Therefore,the SlHK4 gene likely has regulatory roles in these growth and development processes of tomato.(3)The slhk4-4,slhk4-118 and WT plants grown for 1 month were subjected to drought treatment without watering for 30 days.The leaf relative water content(RWC),drought marker gene expression,as well as main parameters and key gene expression of photosynthesis and oxidative stress,were analyzed under drought conditions.Besides,the survival rate of the plants was evaluated after 7 days of rewatering.The results show that compared with the WT,the slhk4 mutant lines have delayed appearance of drought stress phenotypes such as leaf wilting,higher leaf RWC,and delayed increase of drought marker gene SlDREB1 expression in the drought treatment.Further,the survival rates of slhk4-4 and slhk4-118 plants were significantly increased after rewatering,which were 6.0 and 6.3 times that of WT,respectively,indicating that drought tolerance of the slhk4 lines was significantly higher than that of WT.Along with the increase of drought,the chlorophyll content,photosystem II operating efficiency(ΦPSII),electron transport rate(ETR)and other chlorophyll fluorescence parameters,as well as expression of the key gene SlPsb Q of photosystem II,dropped significantly in the WT plants.In contrast,in the slhk4-4 and slhk4-118 plants,the chlorophyll content and the maximum photochemical yield of photosystem II(Fv/Fm)remained almost unchanged,and the decline speed and amplitude of other parameters were significantly lower than WT,indicating that the photosynthesis of slhk4 lines was less affected by drought stress.In addition,compared with WT,the slhk4 leaves have lower malondialdehyde content,higher antioxidant enzyme glutathione S transferase(GST)content,and higher expression levels of the peroxidase gene SlCEVI-1,indicating that the slhk4 lines have a less damaged membrane system,and a improved capacity to scavenge excessive reactive oxygen species.In summary,the cytokinin receptor SlHK4 plays a regulatory role in both vegetative and reproductive growth of tomato.Under drought stress,the slhk4 mutant lines have higher relative water content and plant survival rate,enhanced photosynthesis,lower membrane damage,improved capacity to scavenge excessive reactive oxygen species,and thus higher drought tolerance than the WT plants.Therefore,the cytokinin receptor SlHK4 has a negative regulatory role in tomato drought tolerance.This study lays the foundation for further analyses of molecular mechanisms of cytokinins and their receptor SlHK4 in the regulation of tomato growth and development and drought tolerance,and provides available genic resources for the cultivation of new drought-tolerant tomato varieties.