Su Tao, Han Mei, Min Jie, Zhou Huaiye, Zhang Qi, Zhao Jingyi, Fang Yanming
Co-Innovation Center for Sustainable Forestry in Southern China, College of Biology and the Environment, Nanjing Forestry University, Nanjing, China.
Key Laboratory of State Forestry Administration on Subtropical Forest Biodiversity Conservation, Nanjing Forestry University, Nanjing, China.
Front Plant Sci. 2020 Jan 8;10:1654. doi: 10.3389/fpls.2019.01654. eCollection 2019.
In higher plants, cell wall invertase (CWI) and vacuolar invertase (VI) were considered to be essential coordinators in carbohydrate partitioning, sink strength determination, and stress responses. An increasing body of evidence revealed that the tight regulation of CWI and VI substantially depends on the post-translational mechanisms, which were mediated by small proteinaceous inhibitors (C/VIFs, Inhibitor of β-Fructosidases). As yet, the extensive survey of the molecular basis and biochemical property of C/VIFs remains largely unknown in black cottonwood ( Torr. & A. Gray), a model species of woody plants. In the present work, we have initiated a systematic review of the genomic structures, phylogenies, -regulatory elements, and conserved motifs as well as the tissue-specific expression, resulting in the identification of 39 genes encoding C/VIF in poplar genome. We characterized two putative invertase inhibitors and , showing predominant transcript levels in the roots and highly divergent responses to the selected stress cues including fusarium wilt, drought, ABA, wound, and senescence. prediction of the signal peptide hinted us that they both likely had the apoplastic targets. Based on the experimental visualization the transient and stable transformation assays, we confirmed that PtC/VIF1 and 2 indeed secreted to the extracellular compartments. Further validation of their recombinant enzymes revealed that they displayed the potent inhibitory affinities on the extracted CWI, supporting the patterns that act as the typical apoplastic invertase inhibitors. To our knowledge, it is the first report on molecular characterization of the functional C/VIF proteins in poplar. Our results indicate that PtC/VIF1 and 2 may exert essential roles in defense- and stress-related responses. Moreover, novel findings of the up- and downregulated C/VIF genes and functional enzyme activities enable us to further unravel the molecular mechanisms in the promotion of woody plant performance and adapted-biotic stress, underlying the homeostatic control of sugar in the apoplast.
在高等植物中，细胞壁转化酶（CWI）和液泡转化酶（VI）被认为是碳水化合物分配、库强度决定和应激反应中的重要协调因子。越来越多的证据表明，CWI和VI的严格调控在很大程度上依赖于翻译后机制，这些机制由小蛋白质抑制剂（C/VIFs，β-果糖苷酶抑制剂）介导。迄今为止，在木本植物的模式物种黑杨（Torr. & A. Gray）中，对C/VIFs的分子基础和生化特性的广泛研究仍基本未知。在本研究中，我们对基因组结构、系统发育、调控元件、保守基序以及组织特异性表达进行了系统综述，从而在杨树基因组中鉴定出39个编码C/VIF的基因。我们对两个假定的转化酶抑制剂 和 进行了表征，它们在根中显示出主要的转录水平，并且对包括枯萎病、干旱、脱落酸、伤口和衰老在内的选定应激信号有高度不同的反应。信号肽预测提示我们它们都可能具有质外体靶点。基于实验可视化以及瞬时和稳定转化分析，我们证实PtC/VIF1和2确实分泌到细胞外区室。对其重组酶的进一步验证表明，它们对提取的CWI表现出强大的抑制亲和力，支持其作为典型质外体转化酶抑制剂的模式。据我们所知，这是关于杨树中功能性C/VIF蛋白分子表征的首次报道。我们的结果表明，PtC/VIF1和2可能在防御和应激相关反应中发挥重要作用。此外，C/VIF基因上调和下调以及功能酶活性的新发现使我们能够进一步揭示促进木本植物性能和适应生物胁迫的分子机制，这是质外体中糖稳态控制的基础。
