| Water spinach(Ipomoea aquatica)belongs to convolvulaceae family.It can grow rapidly under conditions of high temperature and humidity environment and always served as an important leafy vegetable in summer when it is too hot to grow vegetables.Under stress condition,the fiber content of heat-sensitive vegetables increased,secondary metabolism was vigorous,and the aging speed was accelerated,leading to changes in morphological and physiological indexes,which eventually led to poor quality and low yield of vegetables,and once damaged,it was difficult to recover.High temperatures,heavy rains and droughts can also trigger disease and insect pests,resulting in reduced vegetable production or even total crop failure.In this paper,through screening heat-resistant TG and BD,as well as heat-sensitive LY and ZY,physiological and morphological changes of the water spinach under high temperature conditions were analyzed,and heat-resistant mechanism was studied using transcriptome and sRNA sequencing,providing theoretical basis for elucidating the heat-resistant mechanism of water spinach.The results are as follows:(1)Effects of long-time high temperature on the morphology and physiological indexes of water spinachAfter long-time heat exposure,heat-sensitive LY and ZY appeared irregular white spots,especially LY,which leaves showed significant bleaching.The content of chlorophyll a,b and carotenoid in four varieties decreased.Long-time high temperature also influenced the activity of PS|| reaction center,with an increase of initial fluorescence(FO),decrease the maximum quantum efficiency(Fv/Fm).N on-photochemical quenching(NPQ)of BD decreased significantly after high temperature treatment,while the NPQ values of TG,ZY and LY increased.The polyphenol content and antioxidant capacity were all reduced after treatment with high temperature,and heat-resistant BD and heat-sensitive LY down the most degree,other heat-resistant TG and heat-sensitive ZY decline less,indicating that the gain of heat resistance of water spinach is not completely depends on its antioxidant ability.(2)Effects of long-time high temperature on gene expression of water spinachs with different heat resistanceTranscriptome sequencing was used to analyze gene expression in heat-resistant and sensitive TG and LY after exposure to high temperature for 15 days.A total of 4145 differential expressed genes were identified,of which 2420 were up-regulated and 1,725 were down-regulated.After enrichment analysis,the main differences between the heat-resistant and sensitive water spinach are starch and sugar metabolism and phenylpropane biosynthesis metabolism.Further analysis of sugar content in water spinach with different heat resistance showed that soluble sugar content changed after long-time high temperature treatment and the change trend of sugar content in BD and ZY was different.During the whole high temperature process,the soluble sugar content of BD was higher than that of ZY at all time points except the 9d,and the content of fructose in BD was always higher than that in ZY.qRT-PCR was used to analyze the expression patterns of genes related to starch and glucose metabolism at different time points after high-temperature treatment.The expression of genes related to starch and glucose metabolism,BDx5,Psps4 and Sus,in BD were induced higher than those in ZY.The phenylpropane metabolic pathway is the junction of the primary and secondary metabolic pathways,and the expression of the phenylpropane biosynthetic genes Ant-3 and Pmd in are both higher than those in ZY.AF1,AF2 and BDx4 were involved in the biosynthesis of starch and sugar as well as phenylpropane.The expression of AF1 in BD and ZY was low at the early stage of high-temperature treatment,but increased sharply at the later stage,reaching the peak at 72h.The expression of AF-2 in BD varieties was higher than that of ZY varieties.BDx4 expressed fluctuate during high temperature treatment,and the peak value appeared at 48h in BD and 24h in ZY.(3)Analysis of miRNA in water spinach under long-time high temperatureIn recent years,more and more studies have found that non-coding RNA plays an important role in plant stress resistance.In this paper,taking highly heat-resistant TG and poorly heat-resistant LY as material,two high-quality sRNA pools,S03 and S04,were constructed after treatment with 42? for 15 days.The number of high-quality sequences both exceeded 17 M,and the number of 24 nt clean reads in TG was much higher than that of LY.After comparison with water spinach's transcriptome,1,363,258 and 1,629,209 sequences were obtained in S03 and S04 respectively.The two libraries shared 1,047,133(11.36%)unique reads,indicating that sRNAs varied in cultivars with different heat resistance.A total of 71 miRNAs were identified in Water spinach,of which 21 were differentially expressed.The target genes were predicted by TargetFinder and a sum of 233 target genes were obtained.In addition,Water spinach BD with strong-heat-resistance and weakly heat-resistant ZY were used as materials to study the expression levels of pre-miR160,pre-miR166,pre-miR172,pre-miR393,and pre-miR3627 that were differentially expressed after long-time high temperature treatment.The results showed that the expression of pre-miR160-2 in BD and ZY increased significantly,but in different levels;the expression of miR166 was decreased in BD but increased significantly in ZY;miR172 could be induced by high temperature,however,there was no difference in the two varieties with different heat tolerance;the expression of miR3627 was decreased in BD after high temperature treatment while there is no obvious change in ZY.The difference in the expression patterns of miRNA precursors suggests that the mechanism of miRNA is different during long-term high temperature in water spinach. |
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