Comparative Analysis Of Thermotolerance In Tulip Varieties And Preliminary Function Identification Of TgHSFA2C

Tulip(Tulipa gesneriana L.)is a bulbous plant with autumm planting feature,belongs to the genus Tulip in the Liliaceae.Its origin is in the coastal areas of the Mediterranean,Central Asia,And Xinjiang,China.is also one of the distribution areas of wild tulips.Its suitable temperature for growth and flowering is 16 ℃～22 ℃,and the flowering period is extremely sensitive to high temperature.In central China,the florescence of tulip is between March and April.And then the rapidly rising temperature in late spring and early summer leads to the florescence shortened;At the same time,the short post-anthesis period makes it unable to provide sufficient nutrients for the expansion of underground bulbs through adequate photosynthesis,thus affecting the growth of the second-generation bulbs.At present,there are few researches on the mechanism of how tulip incense responds to heat stress at home and abroad.Our project systematically evaluated 56 tulip varieties during the flowering and postflowering periods,and discussed the differences of different varieties and tissues in response to high temperature from physiological and biochemical levels,primary metabolites and transcriptome levels in tulips under high temperature stress.These results provide new evidence for the selection of tulip resources and the breeding of new varieties resistant to high temperature.At the same time,the screening and breeding of high temperature resistant varieties also provides a new way to solve the problems of short flowering period and tulip bulb degradation.The main conclusions of this study are listed as follows:1.We evaluated the heat resistance of 56 cultivars,and obtained two significantly different varieties in heat resistance(High temperature tolerance variety Ver and sensitive variety WF).The results showed that WF lost their "cup shape" more quickly than Ver after high temperature treatment,and the petals lost water more quicklythe and the electrical conductivity of petal increased obviously.Meanwhile,the stomatal density of the lower epidermis of WF petals was significantly higher than that of Ver varieties.Under the condition of natural high temperature,the senescence rate of WF leaves was faster,and the chlorophyll content of WF leaves was significantly lower than that of Ver.2.We analyzed the transcriptome and primary metabolite levels of tulip petals and leaves in response to high temperature.the results showed that after high temperature treatment,there are 11210 differentially expressed genes,Among them,there were 4192 and 5480 differentially expressed genes specifically responding to heat stress in Flowers and leaves,And there were 1538 common differentially expressed genes responding to heat stress in Flowers and leaves,In addition,the expression level of a large number of HSF-HSP pathway genes were changed significantly;At the same time,through the analysis of differences in gene enrichment of KEGG pathways,Flavonoid biosynthesis of pathways with specific differentially expressed genes in petals and carbohydrate metabolism pathways with specific differentially expressed genes in leaves were analyzed by clustering and statistical analysis.The long and short time differentially expressed genes were enriched in the pathways of endoplasmic reticulum protein processing in Flowers and Leaves.In addition,by analyzing the primary metabolites qualitative determination of the petals and leaves using GC-MS,We found that 12 substances,such as Valine,Alanine,Glycine,Aspartic acid,Citrulline,Fructose and Vitamin C were only detected in the petals,while 14 substances,such as Hexenic acid,Hydroxylysine,5-Oxyproline,Xylose and Galactose were only detected in the leaves,It is suggested that the different accumulation of primary metabolites in different tissues may affect their sensitivity to high temperature.3.We studied the heat resistance mechanism of HSF-HSP pathway in tulip.According to the changes of transcriptome expression,We analyzed the expression of three HSFs genes,TgHSFA2C,TgHSFB2B and TgHSFB4C,and two HSPs genes,TgHSP70-1 and TgHSP7090.qTR-PCR results showed that the expression of TgHSFA2C in WF petals and leaves was higher than that of Ver after high temperature treatment.The expression of TgHSFB4C in Ver petals and leaves was higher than that of WF.The expression trend of TgHSFB2B,TgHSP70-1 and TgHSP7090 in petals and leaves of the two cultivars was the same,which showed an "up-down" trend.The amino acid sequences of TgHSFA2C and TgHSFB4C genes in different cultivars were highly similar,and the similarity between TgHSFA2C and TgHSFB4C genes in banana and Dendrobium candidum was the highest respectively.4.Functional analysis of TgHSFA2C gene.TgHSFA2C gene has the typical AHA motif of class A HSFs genes and has transcriptional activator activity in yeast.The results of yeast two-hybrid and Bi FC assay showed that there was a protein level interaction between TgHSFA2C and TgHSP7090.The function of TgHSFA2C gene was investigated by heterotransformation in Arabidopsis thaliana.We found that TgHSFA2C overexpression in Arabidopsis thaliana exhibited stronger heat resistance than wild-type,and that overexpression of TgHSFA2C gene enhanced the expression of downstream target genes.In conclusion,We evaluated the heat resistance of 56 cultivars and screened two varieties ’Ver’ and ’WF’ with significant difference in high temperature tolerance,Which provides resource information for high temperature resistance breeding.At the same time,We systematically comparedthe the physiological,metabolic and transcriptional levels of petals and leaves of ’Ver’ and ’WF’ in high temperature tolerance during the process of high temperature stress.In addition,we also preliminarily analyzed the regulatory mechanism of TgHSFA2C in tulip response to high temperature.Through gene transcription,protein interaction level and heat resistance of transgenic Arabidopsis thaliana,Found that TgHSFA2C may be involved in the regulation of tulip response to high temperature.