DNA糖基化酶通过催化从DNA中去除受损或错配的碱基来启动碱基切除修复(BER)途径。拟南芥DNA糖基化酶甲基-CpG结合域蛋白4样(MBD4L)是响应基因毒性剂5-氟尿嘧啶和5-溴尿嘧啶而触发BER的核酶。迄今为止,尚未分析MBD4L在植物生理过程中的参与。为了解决这个问题,我们研究了种子中的酶功能。我们发现吸胀诱导MBD4L基因表达通过产生两个替代转录本,MBD4L.3和MBD4L.4.老化种子的基因激活强于未老化种子。mbd4l-1突变体的种子在控制或老化条件下保持发芽失败,35S:MBD4L.3/mbd4l-1和35S:MBD4L.4/mbd4l-1种子逆转了这些表型。种子核DNA修复,通过彗星试验评估,在吸入后24小时以MBD4L依赖性方式加剧。在这种情况下,BER基因ARP,APE1L,LIG1在35S:MBD4L.3/mbd4l-1和35S:MBD4L.4/mbd4l-1种子中的表达高于mbd4l-1种子中的表达,表明这些成分可以与MBD4L协调以修复种子中受损的DNA碱基。有趣的是,ATM,ATR,与DNA损伤反应(DDR)途径相关的BRCA1,RAD51和WEE1基因在mbd4l-1中被激活,但在35S:MBD4L.3/mbd4l-1或35S:MBD4L.4/mbd4l-1种子中未被激活。这些结果表明,MBD4L是在种子吸胀过程中运作的BER级联的关键酶,其缺陷会导致DDR检测到的基因组损伤,产生发芽延迟或减少。
DNA glycosylases initiate the base excision repair (BER) pathway by catalyzing the removal of damaged or mismatched bases from DNA. The Arabidopsis DNA glycosylase methyl-CpG-binding domain protein 4 like (MBD4L) is a nuclear enzyme triggering BER in response to the genotoxic agents 5-fluorouracil and 5-bromouracil. To date, the involvement of MBD4L in plant physiological processes has not been analyzed. To address this, we studied the enzyme functions in seeds. We found that imbibition induced the MBD4L gene expression by generating two alternative transcripts, MBD4L.3 and MBD4L.4. Gene activation was stronger in aged than in non-aged seeds. Seeds from mbd4l-1 mutants displayed germination failures when maintained under control or ageing conditions, while 35S:MBD4L.3/mbd4l-1 and 35S:MBD4L.4/mbd4l-1 seeds reversed these phenotypes. Seed nuclear DNA repair, assessed by comet assays, was exacerbated in an MBD4L-dependent manner at 24 h post-imbibition. Under this condition, the BER genes ARP, APE1L, and LIG1 showed higher expression in 35S:MBD4L.3/mbd4l-1 and 35S:MBD4L.4/mbd4l-1 than in mbd4l-1 seeds, suggesting that these components could coordinate with MBD4L to repair damaged DNA bases in seeds. Interestingly, the ATM, ATR, BRCA1, RAD51, and WEE1 genes associated with the DNA damage response (DDR) pathway were activated in mbd4l-1, but not in 35S:MBD4L.3/mbd4l-1 or 35S:MBD4L.4/mbd4l-1 seeds. These results indicate that MBD4L is a key enzyme of a BER cascade that operates during seed imbibition, whose deficiency would cause genomic damage detected by DDR, generating a delay or reduction in germination.