【作者】 任立刚；

【导师】 郭蔼光；

【作者基本信息】 西北农林科技大学 ， 生物化学与分子生物学， 2012， 博士

【摘要】 在对模式植物拟南芥的研究中发现,蔗糖可以诱导miR398的大量表达,而且在铜缺乏的情况下miR398的产生受到了SQUAMOSA promoter binding protein-like7(SPL7)转录因子的调控。蔗糖在植物体中是光合作用产生的糖类的主要运输形式,不仅仅作为植物重要的碳源和能量来源,而且也被认为在植物的信号传导和分子应答中起到了关键作用。在植物中,蔗糖从产生部位运输到储存部位需要两种蔗糖降解酶的参与和驱动,它们分别是蔗糖转化酶和蔗糖合成酶。这两类蔗糖降解酶广泛的分布在特定的植物亚细胞结构中。蔗糖通过这两类酶的催化作用能够转化为葡萄糖或尿苷二磷酸葡萄糖(glucose/UDP-glucose)。这种从蔗糖到葡萄糖的快速转变有利于蔗糖信号转换为葡萄糖信号,使得蔗糖信号效应和葡萄糖信号效应变得难以区分。更加普遍的称谓,糖信号更为广泛地和优先地被用来描述植物蔗糖或葡萄糖信号。被蔗糖调控的基因广泛的参与了植物生命周期的各个阶段,例如：种子萌发,营养生长,生殖生长,植物衰老和对生物胁迫与非生物胁迫的应答等。内源的蔗糖主要产生于植物光合作用,然后从成熟的叶片中运输到储存器官或者生长旺盛的部位。内源蔗糖能够通过激活或减慢光合作用速率来自发调节体内的蔗糖水平。低浓度的蔗糖激活光合作用和光合产物的运输,而高浓度的蔗糖促进光合产物的储藏,抑制光合产物的运输。植物除了可以依赖光合作用产生的蔗糖作为能量来源进行生长和发育外,还能够依靠在培养基中添加的蔗糖来生长。在外加蔗糖的条件下,植物选择性地激活或者抑制某些基因的表达以更加有效和精细的方式进行生长发育和代谢。细胞内的铜离子对光合作用来说是不可或缺的微量元素。铜离子也可作为清除活性氧自由基(reactive oxygen species)的超氧化物歧化酶的辅酶,有研究发现铜离子也能参与乙烯信号的转导。拟南芥体内含铜离子最丰富的蛋白质是位于内囊体腔中的质体蓝素(PC),它是光合电子传递链所必需的蛋白。而另外一类主要的含铜蛋白是铜／锌超氧化物歧化酶(CSD),它对于植物各类代谢所产生的活性氧自由基的脱毒和清除超氧离子非常重要。拟南芥体内含有三类超氧化物歧化酶同工酶：细胞质超氧化物歧化酶Ⅰ,叶绿体超氧化物歧化酶Ⅱ以及过氧化物酶体中的超氧化物酶Ⅲ。为了有效地利用铜离子,植物在面对铜缺乏时重新分配和定位有限的铜离子。而铜缺乏响应的分子机制已经分别在单细胞生物莱茵衣藻(Chlamydomonas)和高等植物拟南芥中得到了广泛的研究。在莱茵衣藻中,转录因子铜响应调控子Crrl(Copper response regulator1)在对铜缺乏响应时调控了光合作用装置的开闭。而在拟南芥中,SQUAMOSA启动子结合蛋白(SQUAMOSApromoter binding protein) SPL7在铜缺乏条件下调控多类基因的表达。糖信号与铜代谢平衡对植物的生长和发育来说都是非常重要的。在植物通过光合作用进而产生蔗糖的过程中,在植物叶片细胞中伴随着大量有毒的活性氧自由基的产生,包括了超氧离子。植物借助于CSD1和CSD2这两种超氧化物歧化酶清除植株体内产生的超氧离子。另外一方面CSD1和CSD2的表达又是通过对CSD1和CSD2转录本的特异性切割而被miR398紧密地调控着的。对拟南芥来讲,CSD1和CSD2的功能可以被不受miRNAs调控的铁超氧化物歧化酶所替代(Fe-SOD)。生物胁迫和非生物胁迫,例如强光照,盐胁迫,UV-B,细菌与真菌的浸染都会特异性的使miR398的表达量下降,同时激活CSD1和CSD2的表达。而相比较,铜元素缺乏或高含量的蔗糖能够极强地诱导miR398的表达,同时抑制CSD1和CSD2的水平。在这些例子中,通过后转录基因沉默机制(post-transcriptional gene silencing mechanism),CSD1和CSD2的表达量都严格的受到了miR398的调控。抑制CSD1和CSD2的表达量可以释放更多的铜离子,借此来调控细胞内的铜离子平衡。铜离子更容易被转运及进一步作为辅酶与质体蓝素蛋白结合,形成光合作用装置的重要组成部分。虽然miR398被蔗糖诱导表达导致miR398的两个靶标基因CSD1和CSD2的(?)nRNA在植物中的累积量下降,以及铜缺乏对铜代谢平衡相关基因的表达调控已经被广泛地进行了研究,但建立在蔗糖信号和铜代谢平衡之间联系的分子机制却很少有研究涉及到。本研究借助于近年来建立的植物miRNAs芯片检测技术在拟南芥中筛选蔗糖响应的miRNAs,进而利用Northern印迹杂交方法验证对蔗糖响应的候选(?)niRNAs以及应用实时定量荧光PCR(qRT-PCR)检测候选(?)niRNAs的靶标基因的表达量。除了已经验证并且发表的miR398之外,miR408,miR319和miR160在本研究中也被证实为蔗糖响应的miRNAs。更进一步的结论是蔗糖对miR398和miR408的诱导表达依赖于转录因子SPL7,且这种诱导表达可被培养基中加入的高浓度的铜离子所抑制。另外一个有趣的发现是培养基中存在的高浓度的蔗糖能够强烈的抑制拟南芥植株对铜离子的累积。而通过对使用人工miRNAs方法得到的spl7录因子沉默植株进行研究后发现,在外源蔗糖浓度较低时,转基因spl7默植株较正常野生型植株对铜离子的累积量大为减少,但这种效应在外源蔗糖浓度较高时并不明显。以上的结果证明在植物中蔗糖信号和铜代谢平衡之间存在着一定的联系,蔗糖对拟南芥铜离子累积的调控是以部分依赖于SPL7转录因子的方式进行的。更多还原

【Abstract】 Sucrose induces the expression of miR398and the production of miR398is controlled by SQUAMOSA promoter binding protein-like7(SPL7) transcription factor under copper deficiency in Arabidopsis thaliana. Little else, however, is known about the sucrose-regulated copper homeostasis in Arabidopsis. Here, we employed the microarray technology to screen for sucrose-responsive microRNAs (miRNAs), and identified several candidates using northern blotting. In addition to miR398, miR408, miR319, and miR160are also responsive to sucrose. Furthermore, the induction of miR398and miR408by sucrose depends on the presence of SPL7. MiR398and miR408induction by high sucrose was eliminated by high copper. Meanwhile, high sucrose treatment strongly suppressed copper accumulation. Knock-down of SPL7dramatically reduced copper accumulation in plants under low sucrose, but this effect was compromised in the presence of high levels of sucrose. Taken together, these results suggest a connection between sucrose signaling and copper accumulation in cells, and that sucrose-regulated copper accumulation is both SPL7-dependent and SPL7-independent in Arabidopsis.Sucrose (C12H22O11), the major sugar transportation molecule in plants, not only serves as an essential carbon and energy resource for plant growth, but also plays a central role in plant sensing and signaling (disaccharide sensing/signaling. In plants, the transport of sucrose from sources to sinks is largely driven by sucrose-cleaving enzymes. Two types of sucrose-cleaving enzymes are widespread in specific plant cellular compartments:invertases and sucrose synthases. Sucrose is frequently converted into glucose/UDP-glucose and fructose by these two types of enzymes. Fast conversion of sucrose into glucose may facilitate the rapid switching of sucrose to glucose signaling, making it difficult to distinguish the effects of sucrose from glucose. A more general term, sugar signaling, is preferably used to describe the sucrose or glucose signaling in plants.Genes regulated by sugar are involved in many aspects of plant life cycle, such as seed germination, vegetative and reproductive growth, plant senescence, and in response to abiotic and biotic stresses. Endogenous sucrose produced mainly by photosynthesis, can be transported from sources (mature leaves) to sinks (growing and expanding roots, stems, meristems, flowers, and immature seeds). The endogenous sucrose auto-regulate its production and/or storage during photosynthesis. For example, low levels of sucrose stimulate photosynthesis and the transport of photosynthetic products, while high levels of sucrose repress sucrose transport and lead to sucrose storage. The coordination of photosynthesis and sucrose storage is well controlled through both metabolic regulation and specific sugar-signaling mechanisms. In addition to the endogenous sucrose produced by photosynthesis, plants can also grow by feeding on exogenous sucrose. In response to exogenous sucrose, plants selectively activate or suppress specific genes to use this exogenous carbon resource efficiently.Cellular copper is an indispensable micronutrient element and is also essential for photosynthesis. Copper acts as a cofactor of scavenger of reactive oxygen species (ROS), and participates in ethylene signaling. The most abundant copper containing protein plastocyanin (PC) is required for photosynthetic electron transport within the thylakoid lumen. Another major copper containing protein, Cu/Zn superoxide dismutase (CSD), is essential for detoxifying ROS and scavenging superoxide in plants. Arabidopsis thaliana contains three CSD isozymes cytoplasmic CSD1, chloroplast stromal CSD2, and the peroxisomal CSD3. To effectively utilize copper, plants allocate and redistribute their limited copper in response to copper deficiency. The molecular mechanisms underlying the response to copper deficiency have been investigated both in the unicellular green alga Chlamydomonas reinhardtii and the vascular plant Arabidopsis. In Chlamydomonas, the transcription factor copper response regulator1(Crr1) is a SQUAMOSA promoter binding protein, which regulates the switch of the photosynthesis machinery in response to copper deficiency. In Arabidopsis, an SBP homolog, SPL7, regulates the expression of multiple genes upon copper deficiency.Sucrose and copper homeostasis are closely related to plant growth and development. During the production of sucrose through photosynthesis, highly toxic ROS (Reactive Oxygen Species) are produced in plant cells, including superoxide radicals. Plants employ CSD1and CSD2to detoxify and scavenge superoxide radicals. On the other hand, the expression of CSD1and CSD2is tightly regulated by miR398, through specific cleavage of CSD1and CSD2transcripts. In Arabidopsis, the function of CSD1and CSD2can be replaced by Fe-SOD, which is not controlled by miRNAs. Abiotic and biotic stresses, such as, intense light, salinity, UV-B and infection by bacterial and fungal pathogens, specifically down-regulated miR398and activated CSD1and CSD2in Arabidopsis. In contrast, copper starvation and/or high sucrose strongly induced the expression of miR398and suppressed the expression of CSD1and CSD2. In both cases, the expression of CSD1and CSD2is strictly controlled by miR398through a post-transcriptional gene silencing (PTGS) mechanism. Down-regulation of CSD1and CSD2releases copper ions and thus modulates cellular copper homeostasis. The copper ions are preferably transported and incorporated into plastocyanin (PC) proteins as a form of copper storage as well as an essential component of the photosynthetic apparatus.Although the induction of miR398and the down-regulation of CSD1and CSD2by high sucrose and the impacts of copper deficiency on the expression of genes involved in copper homeostasis have been extensively studied, the molecular mechanism underlying the intersection of sucrose signaling and copper homeostasis has not been fully revealed. In this study, we present evidence that sucrose is related strongly to copper homeostasis. High levels of sucrose suppress the accumulation of copper in Arabidopsis, while high copper can fully eliminate miR398and miR408induction by high sucrose. SPL7is the key transcription factor that activates the expression of miR398and miR408in response to high sucrose in Arabidopsis. However, the regulation of copper accumulation by sucrose is not fully controlled by SPL7.更多还原