Preliminary Study On The Function And Mechanism Of Maize Potassium Channel Gene Add1 On Heat Tolerance

Global warming increases the frequency of high temperature stress,which seriously affects maizegrowth and development.In the whole growth period of maize,the flowering stage is the most sensitive to heat stress.Heat stress hinders the differentiation of maize reproductive organs and the development of maize tassel.Heat stress also increases the degree of pollen abortion,shortens the filaments,influences the pollination,and results in a decrease of seed setting rate and a severe yield loss.Therefore,Identification of genes related to heat tolerance in maize and analysis of their mechanism are important for genetic improvement of heat tolerance and reduction of yield loss by heat stress in maize.In this study,a mutant with a potassium channel gene,add1,and Add1 overexpression lines were used as experimental materials to analyse the function and mechanism of heat tolerance of Add1 in maize by cytological,physiological and gene expression assays.The main results are as follows.1.Add1 has 7 transcripts in the B73 genome,and the 6^thtranscript is identified in maize anthers by RT-PCR.Mutation of add1 was detected at the amino acid 370 by sequencing analysis.Add1 was constitutively expressed in maize tissues analysed by QRT-PCR.Under normal conditions,anther exsertion and pollen shedding was normal in the add1mutants and wild type plants.Under high temperature conditions,no significant difference was detected for the growth vigour and several plant related traits between the wild type plants and the mutant.Compared with the wild type,fewer anthers in the homozygous add1 mutant exserted and no pollen was shedding at all.In addition,compared with the wild type,the pollen fertility of add1 homozygous mutant was significantly decreased under high temperature.This indicated that pollen shedding ability and pollen fertility in the add1 mutant were sensitive to high temperature.2.Under normal conditions,the structure of the anthers and their vascular bundles between Add1 and mutant add1 was no difference.Under high temperature,the vascular bundle cells of the mutant arranged disorderly and became smaller,the sheath cells of vascular bundle degenerated seriously,and the boundary between xylem and phloem was unclear.These implied that the decrease in pollen fertility of add1 mutant under high temperature may be related to the abnormal vascular bundle of anther.There was no difference in water content of anthers between the wild type plants and the add1 mutant under normal environment.However,water content and antioxidant enzyme activity in the anthers of add1 homozygous mutant decreased significantly under high temperature.These results indicated that pollen fertility reduction in add1 mutant under high temperature might be related to the decrease of water content and antioxidant enzyme activity in anthers.It is speculated that high temperature caused a significant decrease in anther water content and antioxidant enzyme activity of mutant add1,and further led to poor development of anther vascular bundle,resulting in a significant decrease in fertility.In addition,compared with normal conditions,the expression of PYL,genes involved in reactive oxygen species homeostasis and MYB in add1 mutant anthers and wild type anthers significantly decreased,while the expression of Sn RK2 in add1 mutant anthers and wild type anthers significantly increased under high temperature,gene involved in regulating flowering timein wild type anthers increased.Therefore,it is speculated that the significant reduction of pollen fertility in mutant add1 caused by high temperature is mainly related to the inactivation of potassium channel Add1,and Add1 improves the heat resistance of maize anther through Sn RK.3.High temperature tests were conducted on Add1 overexpression materials at seedling stage and flowering stage,respectively.The results showed that compared with the negative materials,the damage degree of positive plants at seedling stage was significantly decreased,and the pollen fertility at flowering stage was significantly increased.In addition,the antioxidant enzyme activity of positive plants was significantly increased,and the expression of Sn RK2 and gene involved in regulating floral organ development was significantly upregulated.The overexpression results further indicated that Add1 was related to heat tolerance of maize under high temperature,suggesting that Add1 positively regulated heat tolerance of maize at seedling and flowering stages,and Add1 overexpression lines may have negative feedback mechanism.4.The mutation effect of add1 under different genetic backgrounds was analyzed for heat tolerance.The results showed that the add1 mutation could cause a significant decrease in pollen dispersal ability of anthers under different genetic backgrounds.This indicated that add1 mutation affected the pollen dispersal ability of maize anther under high temperature at flowering stage.It is speculated that Add1 provides new resources for improvement of heat resistant varietiesIn conclusion,it is the first time that potassium channel genes are involved in maize flowering heat tolerance.Under normal conditions,there was no significant difference in pollen shedding and pollen fertility between Add1 and add1.Under high temperature conditions,the pollen shedding and pollen fertility of add1 mutant were significantly decreased.After overexpression of Add1,the leaf damage degree of seedlings decreased and pollen fertility increased significantly under high temperature.Under different genetic backgrounds,the pollen shedding after add1 mutation decreased significantly.Cytological observation,physiological and biochemical analysis and gene expression analysis showed that Add1 was involved in regulating maize flowering heat tolerance.This study provides a new idea for analyzing the mechanism of heat tolerance and genetic improvement of maize.