Chen Xiaoyi, Zhang Luyao, Liu Chengbi, Wang Rui, Dai Jianfeng, Kong Lisheng, Zhang Jinfeng, Zhao Jian
State Key Laboratory of Tree Genetics and Breeding, National Engineering Research Center of Tree Breeding and Ecological Restoration, Key Laboratory of Genetics and Breeding in Forest Trees and Ornamental Plants, Ministry of Education, the Tree and Ornamental Plant Breeding and Biotechnology Laboratory of National Forestry and Grassland Administration, College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing 100083, China.
Hebei Technical Innovation Center for Forest Improved Variety, Hebei Academy of Forestry and Grassland Sciences, Shijiazhuang, Hebei 050061, China.
Plant Physiol. 2025 Jul 3;198(3). doi: 10.1093/plphys/kiaf286.
Somatic embryogenesis is a powerful system for studying embryo development and scaling up the production of elite genetic material. Somatic embryogenesis has been well established in Larix principis-rupprechtii, a Chinese larch species dominant in the world's largest man-made forest. However, genotype-dependent embryogenic variations hinder large-scale forestry, and the molecular mechanisms remain unclear. Here, we constructed stage-specific developmental transcriptomes of the somatic embryogenesis process using 2 lines with contrasting embryogenic capacities. Clustering and coexpression analyses identified LpWRKY65 as a central hub gene highly expressed in early somatic embryogenesis stages and with significantly higher expression in the high-embryogenic-capacity cell line (HEL) compared to the low-embryogenic-capacity cell line (LEL). Overexpressing LpWRKY65 significantly increased somatic embryo yield and quality. DNA affinity purification sequencing (DAP-seq) and RNA-seq were combined to identify a set of target genes downstream of and responsive to LpWRKY65, particularly including genes involved in reactive oxygen species (ROS) scavenging. We identified LpHmgB10 as a critical downstream regulator of LpWRKY65. LpWRKY65 directly binds to the W-box in the promoter of LpHmgB10, markedly enhancing its transcriptional activity. ROS profiling further demonstrated that overexpression of LpWRKY65 or LpHmgB10 enhances ROS scavenging and promotes a stable redox environment, which is crucial for improving embryogenic capacity. These findings suggest that LpWRKY65 regulates the cellular redox environment to promote embryogenic differentiation and somatic embryo development, advancing somatic embryogenesis research in conifers.
体细胞胚胎发生是研究胚胎发育和扩大优良遗传材料生产的有力系统。在华北落叶松（一种在中国最大人工林中占主导地位的落叶松树种）中，体细胞胚胎发生已得到充分确立。然而，基因型依赖性的胚胎发生变异阻碍了大规模林业发展，其分子机制仍不清楚。在此，我们使用两条胚胎发生能力不同的品系构建了体细胞胚胎发生过程的阶段特异性发育转录组。聚类和共表达分析确定LpWRKY65为一个核心枢纽基因，在体细胞胚胎发生早期阶段高度表达，并且在高胚胎发生能力细胞系（HEL）中的表达明显高于低胚胎发生能力细胞系（LEL）。过表达LpWRKY65显著提高了体细胞胚胎的产量和质量。结合DNA亲和纯化测序（DAP-seq）和RNA-seq来鉴定LpWRKY65下游的一组靶基因及其响应基因，特别是包括参与活性氧（ROS）清除的基因。我们确定LpHmgB10是LpWRKY65的关键下游调节因子。LpWRKY65直接结合到LpHmgB10启动子中的W-box，显著增强其转录活性。ROS分析进一步表明，过表达LpWRKY65或LpHmgB10可增强ROS清除并促进稳定的氧化还原环境，这对于提高胚胎发生能力至关重要。这些发现表明，LpWRKY65调节细胞氧化还原环境以促进胚胎发生分化和体细胞胚胎发育，推动了针叶树体细胞胚胎发生的研究。
