Heritable Alterations In DNA Methylation Pattern And Mobilization Of Transposons MPing And Pong Induced By Hydrostatic Pressurization In Rice

Intrinsic DNA methylation pattern is an integral component of the epigenetic network inmany eukaryotes. We found in this study that DNA methylation patterns of mobile elementsbut not of cellular genes were specifically altered in rice (cv. Nipponbare) plants followinghydrostatic-pressurization on germinating seeds. This was evidenced by methylation-sensitivegel blot analysis, which showed that 10 out of 10 studied low-copy transposns andretrotransposons manifested methylation alteration in at least one of the 8 randomly chosenpressure-treated plants, whereas none of the 20 studied low-copy cellular genes showed anychange. Both gel-blotting and genome-wide fingerprinting indicated that the methylationalteration in mobile elements was not accompanied by a general genetic instability. Progenyanalysis indicated retention of the altered methylation patterns in most progeny plants,underscoring early occurrence of the alterations, and their faithful epigenetic inheritance.Because the known correlation between DNA methylation and transposon activity, we alsoanalyzed several endogeneous rice transposable elements that are potentially active.Nonetheless, none of the elements showed apparent transpositional activation.To further test the possibility for pressure-induced transposable activity, we pressurizedtwo more rice cultivars, JL307 and JL01-124.It was found that mPing, a recently discoveredendogenous MITE (miniature inverted repeat transposable element) in rice, together with oneof its putative transposase-encoding partners, Pong, was efficiently mobilized in somatic cellsof intact rice plants, of both cultivars, whereas the other autonomous element of mPing, Ping,remained static in the plants studied. Southern blot analysis and transposon-display assay onselfed progenies (P1 generation) of two selected P0 plants, each from one of the cultivars,revealed polymorphic banding patterns consistent with mobilization of mPing and Pong.Various mPing excisions and de novo insertions, as detected by element-bracketing,locus-specific PCR assays, occurred in the different P1 plants of both cultivars. Pongexcision at one locus for each cultivar was also detected in the P1 plants. Sequencing at 18mPing empty donor sites isolated from the pressurized plants indicated propertiescharacteristic of the element excision. Sequence-based mapping of 10 identified mPing denovo insertions from P1 progenies of pressurized plants indicated all being in unique orlow-copy regions, conform with the targeting propensity of mPing. No evidence for furthermPing activity was detected in tested P2 plants. In spite of the high activity of mPing andPong in the pressurized plants, amplified fragment length polymorphism (AFLP) analysisdenoted their general genomic stability, and several potentially active retrotransposons alsoremained largely immobile. Thus, we have established a simple and robust approach for inplanta MITE -mobilization in rice by using high hydrostatic pressure treatment, which may beuseful as an alternative for gene-tagging and functional genomic studies in this important cropplant.