Abstract：
Reactive oxygen species (ROS) play important roles during anther and pollen development. DNA damage may cause chromosome fragmentation that is considered to underlie chromosome elimination for haploid induction by matrilineal pollen, a key step in MATRILINEAL-based double haploid breeding technology. But when and how DNA damage occurs is unknown. We performed comparative studies of wheat pollens from the wild-type and the CRISPR/Cas9 edited matrilineal mutant (mMTL). Chemical assays detected a second wave of ROS in mMTL pollen at the three-nuclei-stage and subsequently, along with reduced antioxidant enzyme activities. RNA-seq analysis revealed disturbed expression of genes for fatty acid biosynthesis and ROS homoeostasis. Gas chromatography-mass spectrometry measurement identified abnormal fatty acid metabolism that may contribute to defective mMTL pollen walls as observed using electron microscopy, consistent with the function of MTL as a phospholipase. Moreover, DNA damage was identified using TdT-mediated dUTP nick-end labelling and quantified using comet assays. Velocity patterns showed that ROS increments preceded that of DNA damage over the course of pollen maturation. Our work hypothesises that mMTL-triggered later-stage-specific ROS causes DNA damage that may contribute to chromosome fragmentation and hence chromosome elimination during haploid induction. These findings may provide more ways to accelerate double haploid-based plant breeding.
摘要：
活性氧（ROS）在花药和花粉发育中起重要作用。DNA损伤可能导致染色体断裂，这被认为是母系花粉单倍体诱导的染色体消除的基础，基于MATRILINEAL的双单倍体育种技术的关键步骤。但是DNA损伤何时以及如何发生尚不清楚。我们对野生型小麦花粉和CRISPR/Cas9编辑的母系突变体（mMTL）进行了比较研究。化学分析在三核阶段检测到mMTL花粉中的第二波ROS，随后，随着抗氧化酶活性的降低。RNA-seq分析揭示了脂肪酸生物合成和ROS同源稳态的基因表达受到干扰。气相色谱-质谱测量确定了异常的脂肪酸代谢，这可能导致使用电子显微镜观察到的mMTL花粉壁缺陷。与MTL作为磷脂酶的功能一致。此外，使用TdT介导的dUTP缺口末端标记鉴定DNA损伤，并使用彗星测定进行定量。速度模式表明，在花粉成熟过程中，ROS的增量先于DNA损伤。我们的工作假设mMTL触发的后期特异性ROS会导致DNA损伤，这可能导致染色体片段化，从而导致单倍体诱导过程中染色体消除。这些发现可能提供更多的方法来加速基于双单倍体的植物育种。
