Regulation of source-sink development and leaf homeostasis in vascular plants that translocate monoterpenes as photo-assimilates in addition to sugars such as sucrose

除了蔗糖等糖之外，维管束植物还以光同化物形式转运单萜，对源库发育和叶片稳态的调节

• 批准号: 7188-2011
• 负责人: Grodzinski, Bernard
• 金额: $ 1.75万
• 依托单位: University of Guelph
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2015
• 资助国家: 加拿大
• 起止时间: 2015-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=568505
• 关键词: Regulation; source; sink; development; leaf

Vascular plants are complex eukaryotes that are photoautotrophs. They are desirable bio-reactors because they offer a sustainable source of organic compounds essential to our survival and well being. Plants have evolved multiple survival mechanisms for dealing with environmental stress. Their spatial and temporal, phenotype plasticity is unique in biology as plants regulate energy flow, initially solar energy trapped by the photosynthetic canopy into stable and mobile forms of reduced C, N and S. A primary source of photoassimilates is the leaf canopy, but, during vegetative and reproductive cycles contrary to popular belief other organs also contribute photoassimilates. If we are to exploit plants as bio-reactors for traditional (e.g., food, starch, sugars, amino acids, vitamins, etc) and novel bio-products (e.g., fuel, pharmaceuticals) our understanding of assimilate fluxes from source organs/tissues to developing sinks must improve. For example, the forms of reduced-C that are translocated via vascular phloem vary depending on the environmental stress and status of canopy development. Source-sink interactions determine productivity yet metabolite fluxes remain one of poorest understood plant processes. Selecting genes that can be targeted by either classical breeding protocols or using molecular bio-technologies to improve productivity of herbaceous and woody crops requires quantification of transport fluxes. In the Biotron facilities at Guelph we have engineered new analytical tools that are required to quantify phenotype plasticity at the organism, organ and organelle levels. In this project we will test new breeding lines, novel ecotypes and transgenics each with different genetic abilities to synthesize and transport large amounts of newly reduced C as monoterpenes (glucose esters such iridoids) as well as the better recognized sugar disaccaride, sucrose. Of particular interest as a model botanical family is the "newly" reclassified botanic family, the Plantaginaceae. These will be compared to selected members of the Leguminosea and the Brassicaeae as we have an opportunity to also test how targeted genetic modification of leaf shape and sink respiratory activity has altered efflux of reduced-C from leaves. Our experiments will integrate 3 levels of complexity that reflect translocate these intermediates are selected members of t both spatially (anatomical) and temporally (metabolism and regulation). The three levels are loosely defined as, 1) the organism or whole plant level that are in vivo; 2) the intact organ or tissue level studies on the primary r photosynthetic structures that involve mainly in vivo but some in situ studies, and, 3) a series of integrate in vitro analyses of cellular, sub-cellular (organelle level) and biochemical level studies. Together these data reflect day-night patterns of primary metabolic regulation, assimilate fluxes and C-partitioning, enzyme regulation and gene involvement that linked to development in real-time. Understanding the genetics/ biochemistry and physiology of energy trapping and energy Exp processes is primary if new cultivars are to be bred and selected for efficient production in a range of environmental challenges. 1) Organism-Level-(in vivo): Plant canopy anatomy defines Ps and metabolism at all levels of organization. Consider canopy architecture, a complex 3D- trait. Leaf area, LA, is dynamic. Even transient, subtle alterations have a huge impact on light trapping and growth. For example, we have shown by photography validated with diel measurements of whole plant CO2 exchange that what was thought to be a direct effect of the regulator, ethylene (C2H4) on specific enzyme reactions (RUBISCO) was an indirect, reversible effect on leaf presentation. The capacity for photosynthesis, Ps, per se was not changed. We will determine direct and indirect effects of varying light and CO2 levels on plant form as well as acclimation at the tissue, cellular and sub-cellular levels. In addition, to using displacement transducers to measure growth we are developing a non-invasive technique using near infrared 3D imagery. An advantage of 3D imagery is that a great deal data regarding spatial and temporal parameters such as leaf orientation branching and expansion rates of specific leaves and sinks is gained in real-time . These data will be used to analyze allocation patterns of labelled (14C, 13C and 15N ) assimilates from leaves to sinks helping differentiate between course and fine control of energy capture, C and N reduction and partitioning. 2) Organ (in vivo): We developed new equipment to quantify both immediate and diel 14C fluxes from leaves. Our analyses of immediate Exp (i.e., sugars in isotopic-equilibrium with 14CO2) showed that when photorespiration, Pr, was suppressed Exp increased. Leaf warming reduced Exp prior to inhibition of photosystems and/or C-fixation per se, showing that light trapping and C-reduction (i.e., chloroplast reactions) were not the rate limiting steps. Our studies indicate that the optimum temperature window for Exp is narrower than that for Ps meaning that Exp might be limiting growth even during CO2 enrichment that suppress Pr and photoxidative stress. We will measure development and Exp of plants with different abilities to synthesize, transport and/or store different sugars (e.g., mannitol, raffinose, etc) besides sucrose by varying light, CO2 and N levels. For example, we have identified specific low and high light tolerant lines of snapdragons that seem to metabolize, store and Exp sucrose, mannitol and starch differently. 3) Organelle to Leaf Tissue Studies (in vivo and in situ): We will measure diel patterns of intra-cellular and intercellular fluxes of key assimilates in the symplasm (chlorenchyma) and apoplasm. Non-invasive scanning leaf chlorophyll fluorimaging techniques and chloroplast (fluorescence )imaging will be compared. In parallel experiments a new multi-barreled microelectrode injection proctocol will be used to sample specific tissue fluids and determine labelled sugars, amino acids and several key inorganic ion levels (K+). Preliminary data show that changes in apoplast [H+] appear to proceed a transient inhibition of Exp in pea leaves during CO2 treatment. These in situ techniques will be applied to leaves challenged with different environmental conditions (eg., light, CO2, and temperature). Of particular interest is the relation of diel functions of auxillary transport sugar formation (eg., mannitol) in snapdragon and sucrose in transgenics with altered apoplast or vacuolar invertases of "plant" origin. Ps and Exp traits in specific transgenics will be determined.

维管植物是复杂的真核生物，是光合自养生物。它们是理想的生物反应器，因为它们提供了对我们的生存和福祉至关重要的有机化合物的可持续来源。植物进化出了多种应对环境压力的生存机制。它们的空间和时间、表型可塑性在生物学中是独特的，因为植物调节能量流，最初太阳能被光合作用冠层捕获，转化为稳定和移动形式的减少的碳、氮和硫。光同化物的主要来源是叶冠，但是，与普遍看法相反，在营养和生殖周期中，其他器官也贡献光同化物。如果我们要利用植物作为传统生物产品（例如食品、淀粉、糖、氨基酸、维生素等）和新型生物产品（例如燃料、药品）的生物反应器，我们必须提高对从源器官/组织到发展汇的同化通量的理解。例如，通过维管韧皮部转运的还原碳的形式根据环境压力和冠层发育状态而变化。 源库相互作用决定了生产力，但代谢通量仍然是人们最不了解的植物过程之一。选择可以通过经典育种方案或使用分子生物技术来提高草本和木本作物生产力的目标基因需要量化运输通量。在圭尔夫市的 Biotron 设施中，我们设计了新的分析工具，用于量化生物体、器官和细胞器水平的表型可塑性。 在这个项目中，我们将测试新的育种系、新的生态型和转基因品系，它们各自具有不同的遗传能力，以合成和运输大量新还原的碳作为单萜（葡萄糖酯，如环烯醚萜）以及更被认可的糖二糖，蔗糖。作为模式植物科，特别令人感兴趣的是“新”重新分类的植物科——车前草科。这些将与豆科和十字花科的选定成员进行比较，因为我们还有机会测试叶形状和库呼吸活动的定向基因改造如何改变叶中还原碳的流出。 我们的实验将整合 3 个级别的复杂性，反映这些中间体在空间（解剖学）和时间（代谢和调节）上的选定成员的易位。这三个水平被粗略地定义为：1）体内的生物体或整个植物水平； 2）对初级光合结构的完整器官或组织水平研究，主要涉及体内研究，但也有一些原位研究，以及，3）细胞、亚细胞（细胞器水平）和生化水平研究的一系列体外综合分析。 这些数据共同反映了与实时发育相关的主要代谢调节、同化通量和 C 分配、酶调节和基因参与的昼夜模式。 了解能量捕获和能量 Exp 过程的遗传学/生物化学和生理学是 如果要培育和选择新品种以在一系列环境条件下进行高效生产，这是首要的 挑战。 1) 生物体水平（体内）：植物冠层解剖学定义了各个水平的 Ps 和代谢 组织。考虑冠层建筑，这是一种复杂的 3D 特征。洛杉矶的叶面积是动态的。即使是短暂的， 微妙的改变对光捕获和生长有巨大影响。例如，我们已经证明了 通过对整个植物 CO2 交换的昼夜测量进行摄影验证，被认为是调节剂乙烯 (C2H4) 对特定酶反应 (RUBISCO) 的直接影响是间接的， 对叶子呈现的可逆影响。光合作用能力 Ps 本身没有改变。我们将确定不同的光和二氧化碳水平对植物形态以及组织、细胞和亚细胞水平的适应的直接和间接影响。 此外，除了使用位移传感器来测量生长情况外，我们还在开发一种使用近红外 3D 图像的非侵入性技术。 3D 图像的优点是可以实时获得大量有关空间和时间参数的数据，例如叶子方向分枝以及特定叶子和汇的扩展率。这些数据将用于分析标记（14C、13C 和 15N）同化物从叶子到库的分配模式，有助于区分能量捕获、C 和 N 减少和分配的过程和精细控制。 2) 器官（体内）：我们开发了新设备来量化直接和昼夜 14C 通量 树叶。我们对即时 Exp（即与 14CO2 处于同位素平衡的糖）的分析表明，当 光呼吸，Pr，被抑制，Exp 增加。抑制叶面变暖之前，Exp 会降低 光系统和/或 C-固定本身，表明光捕获和 C-还原（即叶绿体反应）不是速率限制步骤。我们的研究表明，Exp 的最适温度窗口比 Ps 的更窄，这意味着即使在 CO2 富集抑制 Pr 和光氧化应激的情况下，Exp 也可能限制生长。我们将通过改变光、二氧化碳和氮水平来测量具有不同合成、运输和/或储存除蔗糖之外的不同糖（例如甘露醇、棉子糖等）能力的植物的发育和Exp。例如，我们已经确定了特定的耐低光和高光品系的金鱼草，它们似乎以不同的方式代谢、储存和消耗蔗糖、甘露醇和淀粉。 3) 细胞器到叶组织研究（体内和原位）：我们将测量细胞内和叶组织的昼夜模式 共质（绿藻）和质外质中关键同化物的细胞间通量。非侵入式扫描 将比较叶片叶绿素荧光成像技术和叶绿体（荧光）成像。并联 实验将使用新的多管微电极注射协议对特定组织进行采样 液体并测定标记糖、氨基酸和几种关键无机离子水平 (K+)。初步数据 结果表明，在 CO2 处理期间，质外体 [H+] 的变化似乎对豌豆叶片中的 Exp 进行了短暂的抑制。这些原位技术将应用于遭受不同环境挑战的叶子 条件（例如光、CO2 和温度）。特别令人感兴趣的是金鱼草中辅助运输糖形成（例如甘露醇）的昼夜功能和转基因中蔗糖与“植物”来源的改变的质外体或液泡转化酶的关系。将确定特定转基因中的 Ps 和 Exp 性状。