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杨树根特异性表达β-果糖苷酶抑制子的功能性验证
苏涛, 周怀烨, 周碧瑶, 石婉婷, 张琪
南京林业大学学报（自然科学版） ›› 2020, Vol. 44 ›› Issue (6) : 169-174.
PDF(4593 KB)
PDF(4593 KB)
杨树根特异性表达β-果糖苷酶抑制子的功能性验证
The enzyme purification and functional evaluation of a root-expressed invertase inhibitor in poplar
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【目的】植物细胞壁转化酶(CWI)和液泡转化酶(VI)是分配碳水化合物、维持库器官强度和环境胁迫应答的重要因子。大量证据表明CWI和VI由β-果糖苷酶蛋白抑制子的翻译后调控机制介导。然而,关于C/VIFs家族的分子和遗传学基础以及生化特性研究目前还不清楚。【方法】应用生物信息学分析基因结构特征和系统进化关系,并利用实时荧光定量PCR研究转录本在组织中的丰富度。同时,结合烟草叶片瞬时和拟南芥遗传转化法的荧光表达,以及重组蛋白系统分别对PtC/VIF1亚细胞定位特征和体外抑制活性进行深入鉴定。【结果】共39个PtC/VIF候选编码基因家族被筛选出来。PtC/VIF1在根中具有高转录水平;共聚焦显微镜观察证实PtC/VIF1是分泌到细胞外空间。重组蛋白活性检测表明其对CWI蛋白具有特异的亲和抑制活性,说明PtC/VIF1是定位于质外体的β-果糖苷酶抑制子。【结论】对C/VIF编码基因家族生物信息学分析和PtC/VIF1在杨树中的功能性鉴定属于首次报道,可为后期进一步深入探索该基因的生理学作用和参与抗病防御路径以及环境胁迫响应的潜在功能提供理论基础和研究依据。
【Objective】Cell wall invertase (CWI) and vacuolar invertase (VI) act as the essential regulators in photosynthate distribution, maintenance of sink strength and stress responses. A large body of evidence prompted that the tight control of CWI and VI substantially subjects to the post-translational mechanisms mediated by small proteinaceous inhibitors (C/VIFs, inhibitor of β-fructosidases). However, the survey of the molecular information and gene transcript abundance, as well as biochemical characteristics of C/VIFs is largely unknown in a woody model plant, black cottonwood (Populus trichocarpa). 【Method】In this work, the bioinformatics and qRT-PCR were performed to analyze the phylogenetic tree and gene tissue-specific expression profiling. The transient and stable transformation systems and enzyme assays were conducted to evaluate the subcellular target and inhibitory function of PtC/VIF1. 【Result】A total of 39 genes encoding C/VIF were identified. PtC/VIF1 showing root-specific expression was characterized and its extracellular localization was verified. Evaluation of the recombinant enzyme suggested that PtC/VIF1 showed patterns of the typical apoplastic invertase inhibitor. 【Conclusion】It is the first report on the functional isolation of the C/VIF in poplar. These results pointed out that PtC/VIF1 may play potential roles in defense- and stress-related responses.
杨树 / β-果糖苷酶抑制子; / 翻译后调控 / 细胞壁和液泡转化酶 / 蔗糖 / 质外体 / 生物胁迫
poplar / β-fructosidases invertase inhibitor; / post-translational regulation mechanism / cell wall invertase and vacuolar invertase / sucrose / apoplast / biotic stress
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In higher plants, invertases hydrolyze sucrose (Suc), the major end product of photosynthesis, into glucose (Glc) and fructose (Fru), which are used as nutrients, energy sources, and signaling molecules for plant growth, yield formation, and stress responses. The invertase enzymes, named CWINs, VINs, and CINs, are located in the cell wall, vacuole, and cytosol, respectively. We hypothesize, based on their distinctive subcellular locations and physiological roles, that invertases may have undergone different modes during evolution with important functional implications. Here, we provide phylogenetic and functional genomic evidence that CINs are evolutionarily and functionally more stable compared with CWINs and VINs, possibly reflecting their roles in maintaining cytosolic sugar homeostasis for cellular function, and that CWINs have coevolved with the vasculature, likely as a functional component of phloem unloading.
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Overexpression of the Gossypium hirsutum sucrose synthase (SuSy) gene under the control of 2 promoters was examined in hybrid poplar (Populus alba x grandidentata). Analysis of RNA transcript abundance, enzyme activity, cell wall composition, and soluble carbohydrates revealed significant changes in the transgenic lines. All lines showed significantly increased SuSy enzyme activity in developing xylem. This activity manifested in altered secondary cell wall cellulose content per dry weight in all lines, with increases of 2% to 6% over control levels, without influencing plant growth. The elevated concentration of cellulose was associated with an increase in cell wall crystallinity but did not alter secondary wall microfibril angle. This finding suggests that the observed increase in crystallinity is a function of altered carbon partitioning to cellulose biosynthesis rather than the result of tension wood formation. Furthermore, the augmented deposition of cellulose in the transgenic lines resulted in thicker xylem secondary cell wall and consequently improved wood density. These findings clearly implicate SuSy as a key regulator of sink strength in poplar trees and demonstrate the tight association of SuSy with cellulose synthesis and secondary wall formation.
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Sucrose and monosaccharide transporters mediate long distance transport of sugar from source to sink organs and constitute key components for carbon partitioning at the whole plant level and in interactions with fungi. Even if numerous families of plant sugar transporters are defined; efflux capacities, subcellular localization and association to membrane rafts have only been recently reported. On the fungal side, the investigation of sugar transport mechanisms in mutualistic and pathogenic interactions is now emerging. Here, we review the essential role of sugar transporters for distribution of carbohydrates inside plant cells, as well as for plant fungal interaction functioning. Altogether these data highlight the need for a better comprehension of the mechanisms underlying sugar exchanges between fungi and their host plants.
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Plant family 32 glycoside hydrolase enzymes include hydrolases (cell wall invertases, fructan exohydrolases, vacuolar invertases) and fructosyltransferases. These enzymes are very similar at the molecular and structural levels but are functionally different. Understanding the basis of the functional diversity in this family is a challenging task. By combining structural and site-directed mutagenesis data, Asp239 in AtcwINV1 was identified as an amino acid critical for binding and stabilizing sucrose. Plant fructan exohydrolases lack such an Asp239 equivalent. Substitution of Asp239 led to the loss of invertase activity, while its introduction in fructan exohydrolases increased invertase activity. Some fructan exohydrolases are inhibited by sucrose. The difference between the inhibitor (fructan exohydrolase) and the substrate (invertase) binding configurations of sucrose can be explained by the different orientation of Trp82. Furthermore, the evolutionary hydrolase/transferase transition could be mimicked and the difference between S-type fructosyltransferases (sucrose as donor) and F-type fructosyltransferases (fructan as donor) could be unravelled.
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Fruit quality formation involves a series of physiological and biochemical changes during fruit ripening. Sucrose metabolism plays not only important roles in fruit ripening to establish energy status and nutritional quality but also a non-nutritive role in gene expression. In carbon metabolism and fruit ripening, cell wall invertases (CWINs) perform essential regulatory functions. Knowledge regarding the gene expression changes that occur following the repression of CWIN activity in fruit through the overexpression of a cell-wall inhibitor of beta-fructosidase (CIF) under a fruit-specific promoter is limited. To further explore the molecular mechanism of sucrose regulation, global expression profiling of the fruits of transgenic tomato (Solanum lycopersicum) plants carrying a cell wall invertase inhibitor (SlCIF1) gene was performed using a microarray. In total, 622 and 833 differentially expressed genes (DEGs) were identified. The expression of the SlHSP17.7 gene was increased by thousands of times in the transgenic-SlCIF1 tomato. Then, SlHSP17.7-RNA interference (RNAi) lines were generated by introducing pB7GWIWG2 (I)-SlHSP17.7 into wild-type chmielewskii tomatoes (WT). The sucrose and fructose contents significantly decreased in the RNAi fruits compared with those in the WT. Furthermore, 14 sugar metabolism related genes were also decreased synergistically by silencing SlHSP17.7 gene. Our data indicate that the posttranslational modulation of CWIN activity by SlCIF1 contributes to earlier bloom times. SlHSP17.7 and sugar can interact to regulate the development of tomato fruit and affect the quality of tomato, providing a different insight into improving the quality of tomato.
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Fruit ripening is a complex process that involves a series of physiological and biochemical changes that ultimately influence fruit quality traits, such as color and flavor. Sugar metabolism is an important factor in ripening, and there is evidence that it influences various aspects of ripening, although the associated mechanism is not well understood. In this study, we identified and analyzed the expression of 36 genes involved in Suc metabolism in ripening tomato (Solanum lycopersicum) fruit. Chromatin immunoprecipitation and gel mobility shift assays indicated that SlVIF, which encodes a vacuolar invertase inhibitor, and SlVI, encoding a vacuolar invertase, are directly regulated by the global fruit ripening regulator RIPENING INHIBITOR (RIN). Moreover, we showed that SlVIF physically interacts with SlVI to control Suc metabolism. Repression of SlVIF by RNA interference delayed tomato fruit ripening, while overexpression of SlVIF accelerated ripening, with concomitant changes in lycopene production and ethylene biosynthesis. An isobaric tags for relative and absolute quantification-based quantitative proteomic analysis further indicated that the abundance of a set of proteins involved in fruit ripening was altered by suppressing SlVIF expression, including proteins associated with lycopene generation and ethylene synthesis. These findings provide evidence for the role of Suc in promoting fruit ripening and establish that SlVIF contributes to fruit quality and the RIN-mediated ripening regulatory mechanisms, which are of significant agricultural value.
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| [17] |
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Proper seed development requires coordinated growth among the three genetically distinct components, the embryo, the endosperm, and the seed coat. In Arabidopsis, embryo growth rate accelerates after endosperm cellularization, which requires a chromatin-remodeling complex, the FIS2-Polycomb Repressive Complex 2 (PRC2). After cellularization, the endosperm ceases to grow and is eventually absorbed by the embryo. This sequential growth pattern displayed by the endosperm and the embryo suggests a possibility that the supply of sugar might be shifted from the endosperm to the embryo upon endosperm cellularization. Since invertases and invertase inhibitors play an important role in sugar partition, we investigated their expression pattern during early stages of seed development in Arabidopsis. Two putative invertase inhibitors (InvINH1 and InvINH2) were identified as being preferentially expressed in the micropylar endosperm that surrounds the embryo. After endosperm cellularization, InvINH1 and InvINH2 were down-regulated in a FIS2-dependent manner. We hypothesized that FIS2-PRC2 complex either directly or indirectly represses InvINH1 and InvINH2 to increase invertase activity around the embryo, making more hexose available to support the accelerated embryo growth after endosperm cellularization. In support of our hypothesis, embryo growth was delayed in transgenic lines that ectopically expressed InvINH1 in the cellularized endosperm. Our data suggested a novel mechanism for the FIS2-PRC2 complex to control embryo growth rate via the regulation of invertase activity in the endosperm.
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