DNA Methylation Regulation Mechanism Of Chemically-induced Male Sterility In Wheat(Triticum Aestivum. L.)

Because wheat hybrids have significantly higher yield and better adaptation to adverseenvironments than the best homozygous genotypes, hybrid wheat has been attractedconsiderable efforts spanning several decades. Chemically-induced male sterility (CIMS)system in wheat is one of the male sterility types used for hybrid wheat (Triticum aestivum. L.)production. Especially, chemical hybridizing agents (CHAs) could provide an alternative,workable system for inducing male sterility, which is rapid, flexible and would not requirefertility restoration during hybrid seed production. Furthermore, the major advantage ofemploying CHAs is that almost any inbred line may be used as a female parent. Thus,exploitation of chemically-induced male sterility is the best available strategy to overcomethe existing yield barriers in wheat. SQ-1is a new type of CHAs in wheat, which hasbroad-spectrum property and can lead to complete male sterility through pharmacodynamictest in experimental field, and has no side effects on the agronomic traits. Thus， SQ-1hasbeen applied to produce hybrid seeds on a large scale in China. Although there is a dearth ofinformation on the interceptive mechanism of chemically-interfered male sterility, themolecular switch system for their concerted mechanism is currently undeciphered. Thus, thispaper firstly employed the methylation sensitive amplification polymorphism (MSAP)technology to study DNA methylation level and DNA methylation patterns in differentdevelopmental periods of wheat, and select some different genes in DNA methylation patternclosely involving the male fertility transition; then, explored the effects of the alteration ofNOX(NADPH oxidase) gene methylation pattern on active oxygen metabolism; finally,discussed the effects of chemical hybridizing agents on anther tapetum development and lipidmetabolism. In brief, the main results were as follows:1. In chemically-induced male sterility line1376from uninucleate microspores throughbicellular pollen,to tricellular pollen, the total number of sites, the total of methylated sites,and fully methylated sites were1346,373and298, respectively. And the ratio of methylatedsites and ratio of fully methylated sites were27.7%and22.1%, respectively. In fertile line1376from the three stages, the total number of sites, the total of methylated sites, and fully methylated sites were1342,357and291, respectively. And the ratio of methylated sites andratio of fully methylated sites were26.6%and21.7%, respectively. In a word,46of1,342bands (3.42%) showed methylation alterations in1376-CIMS compared with its fertile line1376, moreover, the rate of demethylation and methylation rate were1.12%and2.31%,respectively. which, implied more extensive de novo methylation in1376-CIMS was than thatin1376.2. Twenty-six upon46fragments showing changes in methylation status in1376-CIMSand its fertility line1376were randomly selected, cloned, and sequenced. About9sequenceshad no gene annotation in GenBank with BLASTX. The candidate fragments encode a varietyof proteins, including metabolic enzymes, F-box proteins, leucine-rich-like protein, hormonalregulation, and bHLH proteins, retroelement, and proteins of unknown functions. Notably,15.4%(4of26) of the sequenced fragments that undergo underwent methylation changes in1376-CIMS showed similarity to retroelements sequences.3. NOX is a prime source of ROS production in the oxidative burst. In1376-CIMS, themethylation levels of NOX gene core promoter region declined, caused elevated expressionlevel of NOX gene. What is more, the transcripts and protein activities of SOD, POD, CAT andAPX in1376-CIMS were lower than those in1376. Furthermore, the transcripts and proteinactivities of SOD, POD, CAT and APX are too low to scavenge the toxicity of excessive ROSin the sterile line.Combining these results directly leads to the fact that anthers of1376-CIMShave higher contents of O2.–and H2O2compared with1376during the development of theanthers. Therefore, it is possible that the descending methylation levels of NOX gene corepromoter region caused rective oxygen metabolism of serious imbalances and membrane lipidperoxidation, leading to the sterility in1376-CIMS.4. Very-long-chain fatty acids (VLCFAs) play an important role in anther development.VLCFAs is catalyzed by the fatty acyl-CoA elongase, a membrane-bound enzymatic complexcontaining trans-2,3-enoyl-CoA reductase(ECR). Primers were designed based on siliconcloning sequence of ECR gene sequences from Brachypodium distachyon, and the openreading frame of the cDNA were obtained, here as designated TaECR, which had beenregistered under GenBank(No. KC222053). Sequence analysis showed the open readingframe of this gene putatively encoding310amino acids, and had classical domains of ECR.Analysis showed that TaECR was ubiquitously expressed in anther, glume, leaf and root inwheat, but it was less expressed in root.5. ROS has been proposed as a signal for programmed cell death. These elevated levelsof ROS in1376-CIMS is not scavenged, it may be a signal for aberrant programmed celldeath. Cytological observation of the tapetum and DNA ladder fingerprints from the anther at different stages also reveaved tapetum degradated in advance in1376-CIMS. In1376-CIMSanther, fatty metabolism related genes expression involved in fatty acid synthesis, transportand conversion, sharply declined, leading to the reduction in the anther aliphatic compoundscontent, and caused the deformity of the anther and pollen wall, ultimately resulted in malesterility.