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

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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.