The Effect Of Temperature On The Growth Of Apple Dwarf Rootstock M9-T337 And Regulating Mechanism

Temperature is one of the key environmental factors that affect plant growth and geographical distribution.In the context of increased global warming by human activities in recent hundreds of years,the impact of temperature on plants has become more and more obvious,under warm temperature conditions,the plant’s thermal morphology builds up faster.Apple is one of the fruit tree species with the largest planting area and important economic status in China.As a kind of deciduous fruit tree,during the one-year growth cycle,the temperature in the growing season and the dormant season can affect the normal growth and fruiting of apples.However,the mechanism by which temperature affects the growth of apples is not yet fully understood.In this study,the apple dwarf rootstock M9-T337 was used as the test material,and the effects of different temperatures(17℃,25℃ and 33℃)on the growth of apple dwarf rootstock were studied from the aspects of physiology and biochemistry,gene expression and gene function.The function several key genes in the temperature regulation of apple dwarf rootstock plant height growth are analyzed,which provides a theoretical basis for exploring temperature promotion of apple dwarf rootstock growth.The main results are as follows:1.Warm temperature promotes the growth of apple dwarf rootstock M9-T337.In order to explore the effect of temperature on the growth and physiological indexes of apple dwarf rootstocks,the apple dwarf rootstock M9-T337,which had been rooted and refined for 3 months,was pretreated at 17℃ for 14 days,and then respectively placed at 17℃,25℃ and 33 ℃ artificial climate box for 14 days,dynamic sampling,and measurement of various growth and physiological indicators.The results showed that when the apple dwarf rootstock M9-T337 was treated at different temperatures,the phenotype began to show differences in 2 days.Under 25℃ and 33℃,plant height,internode length,internode number,leaf area and shoot dry fresh weight increased significantly,while root shoot ratio decreased significantly.The results of hormone measurement showed that the contents of gibberellin and auxin in leaves increased significantly in 25℃ and 33℃.Paraffin section showed that the length of cortical cells increased significantly under 33℃.The scanning results of electron microscope showed that the stomatal opening and closing degree of leaves increased under mild and high temperature.2.Transcrip tome analysis of stem apex of apple dwarf rootstock M9-T337 under different temperature treatments.Transcriptome sequencing analysis was performed on the shoot tips treated at 17℃,25℃ and 33℃ on the fourth day.The results showed that under different temperature conditions,there were 3275 different expression genes between the 17℃ vs 25℃ treatments,of which 750 genes were only in different expression between 17℃ vs 25℃ treatments;there are 1200 different genes expressed between 25℃ vs 33℃ treatments,of which 243 genes are only different expression between 25℃ vs 33℃ treatments;there were 4778 different expression genes between 17℃ vs 33℃ treatments,of which 1866 genes were only different expression between 17℃ vs 33℃ treatments;there are 248 genes diffeerent expression in samples treated at 33℃,25℃ and 17℃.The GO enrichment analysis of different expression genes showed that genes that respond to ultraviolet,blue,red,far-red light,heat,cold,and temperature changes were significantly enriched.Therefore,the response of apple dwarf rootstock M9-T337 to environmental temperature may be achieved by regulating the differential expression of light signal pathway and temperature signal pathway genes.After analyzing the gene heterologous expression of the genes MdMYB6,MdERF-TINY and MdERF-RAP2 that were significantly down-regulated under mild high temperature in the transcriptome,it was found that the hypocotyls of transgenic Arabidopsis overexpressing MdMYB6,MdERF-TINY and MdERF-RAP2 were significantly shorter than those of the wild type.We preliminarily speculate that MdERF-TINY and MdERF-RAP2 genes may play a feedback inhibitory role in response to warm temperature in apples.3.MdPIF4 gene function verification.Real-Time qRT-PCR analysis of MdPIF4,a key gene of apple dwarf rootstocks in response to temperature changes,showed that under 33℃ conditions,the expression of MdPIF4 gene began to increase at 48h after treatment,and was stable and significantly higher than that of materials treated at 17℃.Yeast self-activation test results show that the bHLH domain of MdPIF4 has no self-activation activity in yeast,and the remaining fragments all have self-activation activity.Using the bHLH domain as the bait protein,the yeast two-hybrid screening library obtained 3 interacting proteins,namely MdWRKY70,MdHAT5 and MdRab11D.Combined with temperature treatment hormone results,it is found that MdPIF4 may regulate the response to warm temperature and growth of plants by regulating the synthesis and transport of IAA.Heterologous expression analysis showed that overexpression of MdPIF4 in the wild type Arabidopsis promoted the growth of hypocotyls.