The Reasearch On Heritable Epigenetic Variation Induced By Drought Stress And Changes Relative To Physiological Metabolism

Cytosine DNA methylation plays crucial roles in multiple fundamental cellular activities,including control of gene expression, genome defense, and growth and developmental controlin plants. Existing studies showed that environmental stresses often are associated withchanges in DNA methylation levels and patterns in plants. In this study, we subjected rice (cv.Matsumae) to a stress of15%PEG treatment for four consecutive days. We selected on arandom basis14treated rice plants and performed methylation-senstive amplifiedpolymorphism (MSAP) analysis, and identified one individual that showed clear changes inDNA methylation. We discussed the correlation between DNA methylation variations andgene expression in S0and14rice plants of S1by methylation-sensitive Southern blot andrealtime-PCR analysis. Next, we investigated physiological characteristics of S1and S2（theprogenies that have possible correlation between DNA methylation variations and geneexpression）as well alteration in gene expression patterns and their inheritance.We obtainedthe following major results.1. Changes were found in both DNA methylation level and pattern at CHG sites in S0but nochange was observed at CG sites. The range of variation was0-3.8%for CHGhypermethylation and0.2-4.2%for CHG hypomethylation.2. Methylation-sensitive Southern blotting indicated that no obvious alteration in DNAmethylation were detected in S0，and a key gene in the glutamate pathway of prolinebiosynthesis, P5CS, exhibited markedly demethylation at CHG sites in all14selfedprogenies from S1, the variation ratio was100%,another key gene in the ornithine pathwayδ-OAT, showed demethylation at CHG sites in3of the14selfed progenies, the variationratio was21%. In contrast, two genes, P5CDH and P5CR, did not show changes in DNAmethylation. Although P5CDH showed no obvious alteration in DNA methylation, thevariation ratio was0%. Realtime-PCR indicated that up-regulation ratio of P5CS was100%,up-regulation ratio ofδ-OAT and P5CR was71.4%, down-regulation ratio of P5CDH was7.1%in leaf tissue of selfed progenies, which suggested that DNA methylation and prolinebiosynthesis were probably related only in some of the genes.3. The expression of all studied genes related to nitrogen metabolism and ion absorbtionshowed alteration, and magnitudes of alteration were different among the self-pollinatedprogenies. But the altered expression of a given gene family in a given tissue showed similar trend. For example, all three members of the GS1gene family in S2-2showedup-regulation. The expression of genes related to nitrogen metabolism showed that theup-regulation of some genes might help to accumulate the proline of the plants. Theoffspring could inherit altered pattern of gene expression from their parent. This indicatesthat offspring could inherit parental gene expression patterns after secondary drought stress.4. Self progenies plants (S1) showed much higher concentrations of proline than that ofcontrol plant after drought stress for six hours. The cocentrations of proline in S1and selfedprogenies plants（S2-3） were higher than that of control after being stressed for four days.S1and S2-3showed similar physiological characteristics, suggesting that the tolerance todrought stress of self-pollinated progenies could be enhanced after stress.5. We investigated the content of anion and cation in drought-stressed plants and their selfedprogenies. Selfed progenies revealed a different Na/K ion balance and absorption to anionespecially H2PO4—after secondary drought stress probably as a result of inheritance ofaltered expression of the relevant genes, We infer that this is likely an adaptive response tothe stress, but we can not exclude the possibility that this is merely a passive reaction.6. Three of the14selfed progenies showed different phenotypes. Under the drought stresscondition, S2-3has the same height of the unstressed control plant, but S2-1and S2-2weresignificantly smaller. This suggested S2-3grew faster under drought stress. We infer thatthis alteration in phenotype was likely due to heritable changes in gene expression relatedalteration in DNA methylation, which results in a series of physiological and metabolicchanges.Together, our results showed that drought stress could induce changes in of DNAmethylation of some key genes in the proline metabolism pathway, which results inconstitutive higher expression of these genes and accumulation of proline. Thus, epigeneticmodifications in the form of altered DNA methylation may have played a role in theaccumulation of proline. For example, DNA demethylation of the P5CS and δ-OAT genesmay have activated their up-regulation, enhanced the proline synthesis, and consequentlyled to accumulation of free proline in the leaf tissue. However, the exact mechanistic linksbetween DNA methylation and proline synthesis needs further elucidation. The relatedgenes expression of proline metabolism, nitrogen metabolism elated genes showed thatthese nitrogen metabolism might help to accumulate the proline of the plants，We detectedthe gene expression and the content of ion, which showed the alteration in ion absorbtionwas due to the altered ion expression patterns, and at least some of the offsprings could inherit the altered expression patterns of their parent. Given that concomitant changes inDNA methylation were detected in these plants, we consider that it is reasonable toconclude that DNA methylation alteration underlies the heritable changes in geneexpression and which is then responsible for the altered phenotypes seen in progenies of thestressed rice plants.