| Taihangia rupestris Yu et Li is an endemic to the Taihang Mountains in China,which is currently only distributed in the narrow strip on the eastern edge of the mountains.Due to the influence of regional climate change,T.rupestris has evolved into two subspecies,which are T.rupestris var.rupestris and T.rupestris var.ciliate.In this study,the two subspecies of T.rupestris were used as experimental materials.Through temperature stress(42℃,5℃),the differences in transcriptome and physiological,biochemical levels between the two subspecies of T.rupestris were compared.We have disscussed the potential of extreme temperature adaptation and the genetic mechanism of adaptive differentiation between the two subspecies of T.rupestris.The main results are as follows:1.Transcriptome analysis of the two subspecies of T.rupestris in response to heatstressRNA-seq was used to sequence and analyze the leaves of the two subspecies treated with high temperature,and the adjusted p value(padj)satisfies padj<0.01 and the difference multiple(Fold change)satisfies |log2 Fold change|≥1 as the screening criteria for differentially expressed genes.Under heat stress,7531 DEGs(3430 up-regulated genes,4101 down-regulated genes)were screened in the leaves of T.rupestris var.rupestris.A total of 6326 DEGs(2917 up-regulated genes,3409 down-regulated genes)were screened from the leaves of the T.rupestris var.ciliate.KEGG Pathway analysis showed that there were 122 KEGG pathways in the DEGs of the two subspecies of T.rupestris.There were 6 pathways(galactose metabolism,ascorbic acid and alginate metabolism,porphyrin and chlorophyll metabolism,carbon fixation in photosynthetic organisms,plant hormone signal transduction and photosynthesis-antenna protein)that were significantly concentrated in T.rupestris var.rupestris,and the number of genes involved in each pathway ranged from 22 to 93.There were 5 pathways(glutathione metabolism,porphyrin and chlorophyll metabolism,carbon metabolism,plant hormone signal transduction and photosynthesis-antenna protein)that were significantly enriched in T.rupestris var.ciliate,and the number of genes involved in each pathway ranged from 23 to 100.In a high temperature environment,the number of DEGs detected in the roots of T.rupestris var.rupestris(6101)is less than that of T.rupestris var.ciliate(6216),and the number of up-regulation(3145)in T.rupestris var.rupestris is higher than the number of down-regulation(2956).The up-regulation number(2785)is lower than the down-regulation number(3431),indicating that the roots of under stress,T.rupestris var.rupestris adapt to the environment through active adaptation strategies,while the T.rupestris var.ciliate under stress adapt to the environment by reducing metabolism and inhibiting growth.The KEGG Pathway enrichment results of the two subspecies of T.rupestris are not significant,but the KEGG Pathway analysis results of the common DEGs in the roots of the two subspecies of T.rupestris shows that a total of 117 pathways are enriched,and there are two significantly enriched:plant hormone signal transduction(48 genes),phenylpropane biosynthesis(45 genes).The DEGs in the leaves of the two subspecies of T.rupestris are higher than that in the roots,indicating that high temperature stress mainly affects the leaves of the two subspecies of T.rupestris,and they are more aggressively regulated than the roots to resist adversity.The down-regulated expression of a large number of genes is a stress adaptation mechanism,which adapts to high temperature environment by reducing metabolism and inhibiting growth,while up-regulating the expressed genes may be an active adaptation strategy.The total number of DEGs,up-regulated and down-regulated genes in T.rupestris var.rupestrisr are all higher than those of the margin.It is speculated that T.rupestris var.rupestrisr have better adaptability to high temperature than T.rupestris var.ciliate.2.Transcriptome analysis of two subspecies of T.rupestris under low temperature stressA total of 9640 DEGs were screened from the leaves of the two subspecies T.rupestris under low temperature stress,among which 7557(4008 up-regulated and 3549 down-regulated)of T.rupestris var.rupestris and 6898(3519 up-regulated and 3379 down-regulated)of T.rupestris var.ciliate.The GO function and KEGG Pathway enrichment analysis of the DEGs showed that the two subspecies T.rupestris have different responses to low temperature.In GO enrichment analysis of the common DEGs in leaves of two subspecies T.rupestris,there was found that low temperature stress caused significant enrichment of functional genes in biofilm system,ATP binding and protein metamorphism and phosphorylation during catabolism.The GO enrichment of the two subspecies T.rupestris shows that the two subspecies T.rupestris are involved in molecular functions,cellular components and biological processes,and the T.rupestris var.rupestris was the most enriched in molecular functions and cellular components,while the T.rupestris var.ciliate was the most enriched in molecular functions and biological processes.The common DEGs of the blade under two subspecies T.rupestris stress KEGG Pathway enrichment analysis results show that the total concentration to 123 metabolic pathways,including five significant enrichment:protease(33),phagosome(31),phosphatidyl inositol signal system(22),galactose metabolism genes(24)and photosynthesis antenna(17),metabolic pathways involved in all phases of the life activities,which show that these metabolic pathways in the two subspecies T.rupestris when subjected to low temperature stress may play an important role.KEGG Pathway enrichment was conducted on the leaf differential genes of the two subspecies T.rupestris,and the results showed that 124 and 125 KEGG pathways were mainly enriched in the two subspecies T.rupestris,respectively.Among them,6 pathways(porphyrin and chlorophyll metabolism,carbon metabolism,autophagy regulation,phosphatidylinositol signaling system,calcium metabolism and photosynthesis-antenna protein)were significantly concentrated in the T.rupestris var.rupestris,and the number of genes involved in each pathway ranged from 20 to 113.There were 5 pathways(amino acid biosynthesis,phagocytes,phosphatidylinositol signaling system,calcium metabolism,and photosynthesis-antenna protein)that were significantly enriched in the T.rupestris var.ciliate,and the number of genes involved in each pathway ranged from 19 to 89.Under low temperature stress,the T.rupestris var.rupestris had more genes involved in the response and more significantly enriched pathways,which could adapt to the environment through more complex regulatory mechanisms.Therefore,it was speculated that the T.rupestris var.rupestris had stronger ability to adapt to low temperature environment.3.Physiological and biochemical changes in two subspecies of T.rupestris in response to heat stressUnder high temperature stress,the chlorophyll content of the two subspecies of T.rupestriswas lower than that of the control group,and the chlorophyll content of T.rupestris var.rupestris recovered with the extension of stress time,while that of T.rupestris var.ciliate decreased continuously.The contents of proline and soluble sugar increased in both subspecies.A large amount of proline was preferentially accumulated in T rupestris var.ciliate,while a large amount of soluble sugar was preferentially accumulated in T.rupestris var.rupestris.The maximum photochemical quantum yield,the potential photochemical activity of PSⅡ and the variable fluorescence of the two subspecies decreased with the increase of the time of high temperature stress.The intercellular CO2 concentration,transpiration,rate,net photosynthetic rate and stomatal conductance of both subspecies decreased,but the net photosynthetic rate of T.rupestris var.rupestris was higher than that of T.rupestris var.ciliate.The above results indicated that the two subspecies showed heterotrophic responses in several physiological and biochemical indexes under high temperature stress,and the comprehensive expression was that T.rupestris var.rupestris had higher adaptability or potential to high temperature than T.rupestris var.ciliate.In conclusion,under temperature stress,the gene expression,physiology and biochemistry of the two subspecies had corresponding changes to adapt to the adverse environment.Through these changes,T.rupestris var.rupestris can maintain the balance of substance synthesis and energy metabolism in the body,and thus improve the survival ability of plants under temperature stress.The gene expression and physiological and biochemical changes of the two subspecies under temperature stress indicated that the two subspecies had similar responses and their respective specific regulatory mechanisms.In conclusion,T.rupestris var.rupestris can better adapt to the stress of ambient temperature.These results can provide theoretical guidance and technical support for the conservation of T.rupestris,enrich the understanding of adaptive genetic mechanisms,and provide research examples for environmental effects on plant distribution and speciation. |
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