Recipient Organization
UNIVERSITY OF KENTUCKY
500 S LIMESTONE 109 KINKEAD HALL
LEXINGTON,KY 40526-0001
Performing Department
Horticulture
Non Technical Summary
Improving crop plant tolerance to abiotic stresses has become all the more urgent in the face of an uncertain future climate due to climate change. Investigation of the novel role of SDH and ribitol metabolism in stress tolerance in crop plant species could provide a new platform upon which to improve abiotic stress tolerance. In addition, elucidating the role of SDH in a putative riboflavin regeneration cycle would add a new dimension to a broader knowledge of plant physiology and biochemistry.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Tomato (Solanum lycopersicum L.) is one of the most important crop species worldwide. Studying the novel role of sorbitol biosynthesis and metabolism in stress tolerance in tomato could have broad application for the rational breeding of stress-resistant varieties of tomato and perhaps other crops. Furthermore, a detailed, coherent picture of the source, metabolism, and fate of ribitol has not been reported for higher plants. We have hypothesized that ribitol conversion to ribulose is critical as the first step in recycling it for regeneration and maintenance of the riboflavin pool during and following drought and salt stress. This project will determine if the ribitol pathway is altered in SDH anti-sense plants of this important agricultural species.
Project Methods
Plant material. Wild-type (WT) tomato and tomato transformed with control vector (CV) and with antisense SDH (TR), kindly provided by Dr. Yoshi Kanayama, Tohoku University, Japan, will be studied. For plants grown in media, each container will contain four sections, each section bearing a single plant, one with a WT plant, one with a CV plant, and two with different TR genotypes. Two TR genotypes with very low to no detectable SDH activity have been identified in preliminary studies of several TR genotypes provided to us. Seeds of each will be germinated in MetroMix 360 (Scotts, Marysville, OH, USA) in the containers, and grown in a greenhouse with natural and supplemental light, or in a growth room under fluorescent and incandescent lighting, with periodic fertilization and pest management treatment as needed. For studies with rooted plants, stem pieces with 2-4 leaves will be excised from stock plants, grown in containers as above, on the same day and rooted in water for up to 14 d, until adventitious roots are abundant.Abiotic stress by withholding water or irrigation with NaCl solution. Plants with 3 to 5 mature leaves will be continuously watered or subjected to drought stress by withholding water, or to salt stress by subirrigation with 50 mM NaCl solution (determined in preliminary studies to elicit stress symptoms such as leaf curling but not plant death), followed by post-watering recovery experiments during which leaf samples will be collected for tissue analyses (see below). The appropriate time intervals for the stress and post-watering recovery will be determined in preliminary studies. Leaf tissues will be collected from 3 containers per sampling day (for example at 0, 4, 8, 12, and 16, if water is withheld for 14 days), from each group of plants within a container, and from each treatment group (control, stress). The tissues will be immediately frozen in liquid N2 and stored at -80 °C until analyzed. Each experiment will be performed at least twice.Drought stress using PEG. Three replicate rooted cuttings of each genotype, WT, CV, and TR plants, will be grown in the growth room for 5 d in water or in 25 mM PEG 6000 solutions (-0.15 mPa) in 50 mL tubes, chosen through preliminary studies which allows stress to develop slowly with no injury evident. These plants and controls will be sampled after 5 d. Leaves from each replicate plant will be collected, immediately frozen in liquid N2, and stored at -80? for analysis. The experiment will be performed at least twice.Tissue analysesTissue polyol content. For measurement of sorbitol and ribitol content, frozen leaf tissues will be extracted, and extracts prepared for analysis by gas chromatography using an internal standard (Wu et al., 2010). Quantitative values will be derived from peak areas relative to the 2-deoxy-D-glucose internal standard, and tissue concentrations will calculated.Tissue reactive oxygen species and flavin content. The effect of stress on leaf content of reactive oxygen species, including H2O2, ascorbic acid, and glutathione will also be measured (Deng and Dong, 2013; Galli et al, 2009; Ouyang et al., 2010). Leaf tissues will be analyzed for riboflavin, lumichrome, FAD, and FMN content by HPLC methods we have recently developed and tested, modified from Deng et al. (2011).SDH activity assays. The SDH enzyme will be assayed as described by Nosarzewski et al. (2004). The enzyme activity will be followed by the reduction of NAD+ at 340 nm, and calculated as nmol NADH per mg protein per min. AR activity assays. AR will extracted from frozen tissue by the method of Tari et al. (2010). The enzyme activity will be followed by the oxidation of NADPH at 340 nm, and calculated as nmol NADPH per mg protein per min..S6PDH activity assays. S6PDH will be assayed by the method of Negm and Loescher (1981). The enzyme activity will be followed by the oxidation of NADPH at 340 nm, and calculated as nmol NADPH per mg protein per min.RT-PCR analysis of SDH and S6PDH expression To determine the expression of SDH and S6PDH genes, procedures based on Nosarzewski et al. (2012) will be used. Sequences of primer pairs will be developed from the genes for each identified in the tomato genome database along with a primer for a control gene.Western analysesSDH and S6PDH protein in tomato leaves will detected using SDH and S6PDH purified antibody according to the protocol described in Nosarzewski et al. (2004). SDH antibody is our own, and S6PDH antibody is a gift from Dr. Yoshinori Kanayama, Tohoku University, Japan.