Fu Ningning, Wang Li, Sun Qinglin, Wang Qiguang, Zhang Yuting, Han Xiao, Yang Qi, Ma Wenjun, Tong Zaikang, Zhang Junhong
State Key Laboratory of Subtropical Silviculture, School of Forestry & Biotechnology, Zhejiang A&F University, Lin'an, Hangzhou 311300, Zhejiang, PR China.
State Key Laboratory of Subtropical Silviculture, School of Forestry & Biotechnology, Zhejiang A&F University, Lin'an, Hangzhou 311300, Zhejiang, PR China; Zhejiang Key Laboratory of Forest Genetics and Breeding, Zhejiang A&F University, Lin'an, Hangzhou 311300, Zhejiang, PR China.
Int J Biol Macromol. 2024 Dec;283(Pt 2):137760. doi: 10.1016/j.ijbiomac.2024.137760. Epub 2024 Nov 16.
Phoebe species constitute a large portion of subtropical forestry, which are key players in biomass resources. However, abiotic stresses such as drought stress severely limit the growth and development of P. bournei, and even lead to its death. It has been shown that basic helix-loop-helix (bHLH) as the second largest transcription factor family plays essential roles in response to multiple stresses in plants. However, little information of bHLH family is available in P. bournei. In this study, 130 PbbHLHs were identified and classified into 24 subfamilies. Then, the bHLH domain, conserved motifs and gene structures, evolutionary patterns and protein structural features were probed. The expression levels of 17 PbbHLHs were differentially induced by PEG and ABA by RT-qPCR analysis, indicating that they may be involved in drought stress response. Characterization of the drought candidate gene PbbHLH74 showed that it was transcriptionally active and localized in the nucleus. Heterologous transformation of PbbHLH74 into yeast improved cellular tolerance to drought stress. Meanwhile, overexpression of PbbHLH74 in Arabidopsis showed higher seed germination, plant biomass and expression levels of stress-related genes under drought conditions. Through the hairy root technique, overexpression of PbbHLH74 in P. bournei improved drought tolerance by enhancing root development and expression levels of genes involved in ABA-dependent and ROS scavenging pathways. Moreover, PbbHLH74 might positively regulate the expression of PbPOD by Y1H and dual-luciferase reporter assays. Overall, these results elucidated the structure and evolution of the PbbHLH family, in which PbbHLH74 could be applied to molecular assisted breeding for drought tolerance in P. bournei.
楠木属物种在亚热带森林资源中占很大比例，是生物质资源的关键组成部分。然而，干旱胁迫等非生物胁迫严重限制了闽楠的生长发育，甚至导致其死亡。研究表明，作为植物中第二大转录因子家族的碱性螺旋-环-螺旋（bHLH）在植物应对多种胁迫中发挥着重要作用。然而，关于闽楠bHLH家族的信息却很少。本研究鉴定了130个PbbHLH基因，并将其分为24个亚家族。随后，对bHLH结构域、保守基序和基因结构、进化模式以及蛋白质结构特征进行了探究。通过RT-qPCR分析发现，17个PbbHLH基因的表达水平受到聚乙二醇（PEG）和脱落酸（ABA）的差异诱导，表明它们可能参与干旱胁迫响应。对干旱候选基因PbbHLH74的特性分析表明，它具有转录活性且定位于细胞核。将PbbHLH74异源转化到酵母中可提高细胞对干旱胁迫的耐受性。同时，在拟南芥中过表达PbbHLH74，在干旱条件下种子发芽率更高、植株生物量更大且胁迫相关基因的表达水平更高。通过发根技术，在闽楠中过表达PbbHLH74可通过增强根系发育以及ABA依赖和活性氧清除途径相关基因的表达水平来提高耐旱性。此外，通过酵母单杂交（Y1H）和双荧光素酶报告基因分析表明，PbbHLH74可能正向调控过氧化物酶（PbPOD）的表达。总体而言，这些结果阐明了PbbHLH家族的结构和进化，其中PbbHLH74可应用于闽楠耐旱性的分子辅助育种。
