Salinity is an important limiting environmental factor for rapeseed production worldwide. In this study, we assessed the extent and pattern of DNA damages caused by salt stress in rapeseed plants. Amplified fragment length polymorphism （AFLP） analysis revealed dose-related increases in sequence alterations in plantlets exposed to 10-1000 mmol/L sodium chloride. In addition, individual plantlets exposed to the same salt concentration showed different AFLP and selected region amplified polymorphism banding patterns. These observations suggested that DNA mutation in response to salt stress was random in the genome and the effect was dose-dependant. DNA methylation changes in response to salt stress were also evaluated by methylation sensitive amplified polymorphism （MSAP）. Three types of MSAP bands were recovered. Type Ⅰ bands were observed with both isoschizomers Hpa Ⅱ and Msp Ⅰ, while type Ⅱ and type Ⅲ bands were observed only with Hpa Ⅱ and Msp Ⅰ, respectively. Extensive changes in types of MSAP bands after NaCI treatments were observed, including appearance and disappearance of type Ⅰ, Ⅱ and Ⅲ bands, as well as exchanges between either type Ⅰand type Ⅱ or type Ⅰ and type Ⅲ bands. An increase of 0.2-17.6% cytosine methylated CCGG sites were detected in plantlets exposed to 10- 200 mmol/L salt compared to the control, and these changes included both de novo methylation and demethylation events. Nine methylation related fragments were also recovered and sequenced, and one sharing a high sequence homology with the ethylene responsive element binding factor was identified. These results demonstrated clear DNA genetic and epigenetic alterations in planUets as a response to salt stress, and these changes may suggest a mechanism for plants adaptation under salt stress.
Guangyuan Lu Xiaoming Wu Biyun Chen Guizhen Gao Kun Xu
DNA methylation is known to play a crucial role in regulating plant development and or- gan or tissue differentiation. In this study, we as- sessed the extent and pattern of cytosine methylation during rapeseed (Brassica napus L.) seed germina- tion, and compared the methylation level of various tissues in seedling, using the techniques of methyla- tion-sensitive amplified polymorphism (MSAP) and HPLC separation and quantification of nucleosides. In all, 484 bands, each representing a recognition site cleaved by either or both of the isoschizomers, were amplified by 12 pairs of selective primers in DNA ob- tained from dry seeds. A total of 76 sites were found to be differentially digested by the isoschizomers, indicating that approximately 15.7% of 5′-CCGG-3′ sites in the genome were cytosine methylated. Four classes of patterns were observed in a comparative assay of cytosine methylation in the dry and germi- nating seeds; a small number of hypermethylation events occurred at 5′-CCGG-3′ sites in germinating seeds compared with dry seeds, while many more hypomethylation events were detected after seed germination. Differences in DNA methylation level in various tissues were also detected; radicel was less methylated than hypocotyl and cotyledon. These observations were further confirmed by HPLC analy- sis. In addition, sequencing of eleven differentially methylated fragments and the subsequent blast search revealed that cytosine methylated 5′-CCGG- 3′ sequences were equally distributed between cod- ing and non-coding regions. These results clearly demonstrate the power of MSAP technique for large-scale DNA methylation detection in rapeseed genome, and the complexity of DNA methylationchange during seed germination. DNA Hypomethyla- tion going with seed germination appears to be a necessary step toward transcriptional activation in gene expression, and may well contribute to the de- velopmental gene regulation.
LU Guangyuan WU Xiaoming CHEN Biyun GAO Guizhen XU Kun LI Xiangzhi
