Performing Department
Horticulture
Non Technical Summary
Sorbitol metabolism plays important roles in key events in many plant species. In apple, the most important temperate zone tree fruit in the world, all aspects of fruit development and quality are dependent on the main product of photosynthesis - sorbitol - and its accumulation by fruit. Inefficient utilization of sorbitol at the key step of conversion of sorbitol to fructose by SORBITOL DEHYDROGENASE (SDH) in the fruit can adversely affect the fruit. Knowledge of apple SDH response to the environment and to horticultural practices could lead to improvements in yield and fruit quality.SDH also appears to play a novel role in stress tolerance in many species for which sorbitol is only a minor compound, including the model plant species Arabidopsis thaliana. We discovered that SDH also plays a key role in metabolism of ribitol that seems directly related to drought stress. Improving abiotic stress tolerance of crop species is critical in the face of the anticipated long-term effects of climate change. Seasonal temperatures, rainfall patterns, etc., are expected to change in ways challenging to crop production, while the global human population is expected to increase substantially. Thus, crop yields will need to increase, by some estimates up to 70% more than present levels, under more adverse conditions. All avenues of crop plant improvement will need to be exploited by plant breeding and by genetic engineering to keep pace, and new avenues, exemplified by the direction of the proposed work, will need to be developed to the fullest. The basic knowledge gained by studying the role of SDH in metabolism of sorbitol and ribitol in Arabidopsis could lead to new horticultural and/or biochemical/genetic strategies to optimize plant abiotic stress tolerance in crop plant species.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Goals / Objectives
The long term goal of the project is to understand the role(s), and the factors regulating expression and activity, of SORBITOL DEHYDROGENASE (SDH) in apple and other plant species. The objectives of the current proposal are two-fold:The regulation of expression of 17 known SDH isomers in apple as well as total SDH activity will be studied by:Characterizing tissue-specific expression of the SDH isomers in 'Golden Delicious' apple by quantitative RT-PCR in immature and mature leaves, cambium, and roots for comparison to fruit cortex and seed.Determining which SDH isomers are transcriptionally-induced or inhibited in apple tissue by sorbitol or other sugars, and by cultural practices such as pruning and fruit thinning.Assessing the possibility of posttranslational modification by phosphorylation of SDH in response to inductive treatments.Determining the relationship between xylem sorbitol content and fruit cortex SDH expression and activity during fruit set and early fruit growth.The novel role of SDH in stress tolerance of Arabidopsis as a model for species not commonly producing sorbitol will be studied by:Analyzing SDH expression and activity and measuring content of sorbitol and ribitol in all tissues of wild type and sdh knockout mutants in response to abiotic stresses in time course studies for characterization of the development of the mutant phenotype.Assessing the role of ribitol metabolism by SDH in a putative riboflavin biosynthetic cycle during plant stresses.Determining if toxic metabolites are derived from ribitol and if altered content of reactive oxygen species contribute to the sdh knockout mutant phenotype. Determining if UV-C irradiation can be used to elicit the unique mutant phenotype as a more rapid and easily-regulated treatment than drought stress.
Project Methods
Objective 1'Golden Delicious' apple trees in the UK South Farm orchard will be used. In addition, young 'Golden Delicious' trees will be grown in containers if necessary. We are currently developing probes to identify and differentiate among the 17 SDH isomers in the apple genome of this cultivar, the only fully-sequences apple genome available. Preliminary analyses using probes from our prior work with 'Mutsu' apple indicated that the probes used in that work cannot be used to distinguish among the isomers in 'Golden Delicious' (data unpublished), suggesting the partial sequences upon which the probes are based differ among cultivars.Shoots and spurs bearing apple fruit will be defoliated and girdled as described in Archbold (1999) for depriving fruit, immature leaves, and shoot tips of sorbitol. Fruit, shoot tips, cambium, and mature and immature leaves will be collected 2-3 weeks after treatment from control and defoliated/girdled shoots/spurs. Tissues may be frozen immediately in liquid N2 or used in induction studies. Fruit will be sectioned into cortex sections and seed. Roots will be collected from air-layered shoot sections, for induction of adventitious root development, and frozen in liquid N2 also. Frozen tissues will be stored at -80 ºC for subsequent analyses.Tissue-specific and induced expression of SDH isomers by Real Time PCRTissue specific and induced expression of the 17 known SDH isomers will be characterized by RT-PCR by methods described by Nosarzewski and Archbold (2007), using tissues from apple fruit cortex, cambium, roots, immature and mature leaves, and shoot tips following treatments to trees or limbs, and after incubation in control versus polyol-containing solutions for 24 h (Archbold, 1999; Nosarzewski et al, 2004).SDH activity measurementSDH will be extracted and assayed as described (Archbold, 1999; Nosarzewski et al., 2004, 2012).Determination of possible posttranslational modification of SDH If SDH expression and protein are found but activity is not, the possibility of posttranslational modification via phosphorylation will be studied. Protein will be extracted from frozen tissues using either dephosphorylation inhibitors or phosphatase to preserve or remove phosphate groups, respectively, and separated by two-dimensional gel electrophoresis. Protein from these 2D gels will be transferred to PVDF membrane followed by detection by SDH antibody developed in our lab (Nosarzewski et al., 2004) to identify complementary SDH protein spots on the gel (Nosarzewski and Archbold, 2007). On a sister gel, protein spots will be imaged using SPYRO ruby, aligned with the immunoreactive gel, and the spots corresponding to SDH will be precisely excised. Gel fragments will be sent to the Center for Structural Biology proteomic facility at UK. The same analysis will be performed using Arabidopsis SDH protein extracted from leaves prior to and after SDH induction with sorbitol. By comparing SDH isomers obtained from tissues with high SDH activity to those from tissue without SDH activity, a shift in pI values may be evidence of possible phosphorylation. In addition, the separated SDH isomeric proteins will be sent to the proteomic facility at UK to confirm their phosphorylation status and to identify modified residues.Manipulation of xylem collection sap sorbitol content Trees, or branches isolated by girdling at their base, will be defoliated in the fall after harvest and at intervals during spring bud break and early shoot growth to determine if a range of xylem concentrations of sorbitol can be generated. Xylem sap content will be collected from shoot or spur pieces at weekly intervals in the month preceding bloom and ending 2 weeks afterwards. The sap sugar and polyol content will be measured by gas chromatography (Wu et al., 2010). Growth rates of fruit on the trees or branches will be measured, and fruit will be collected for measurement of cortex SDH isomer expression and activity (Wu et. al, 2010).Objective 2Characterizing the development of the unique mutant phenotypeArabidopsis plants will be grown as described by Villadsen and Smith (2004). Wild type and sdh knockout plants will be drought-stressed (Nosarzewski et al., 2012), and sampled at 2-day intervals leading up to the development of the mutant phenotype. Leaf tissues will be analyzed for content of sorbitol and ribitol, expression of the single isomer of SDH present in Arabidopsis, and SDH activity (Nosarzewski et al., 2012).The unique sensitivity of the sdh mutants to ribitol will be studied. During seed germination/incubation on ribitol-containing media (Nosarzewski et al., 2012), seed will be moved at intervals to ribitol-free media to determine if plants can recover and resume germination. Concurrently, seeds will be collected for analysis to determine if unique ribitol-derived and/or ribitol-induced compounds are produced.Assessing the role of SDH in a putative riboflavin cyclePlants of the sdh knockout mutants collected as above in time course studies will be analyzed for riboflavin, lumichrome, FAD, and FMN content by HPLC methods recently developed and tested, modified from Deng et al. (2011).Unique ribitol metabolites and reactive oxygen species in mutant plantsPlants will be fed 14C-ribitol by exogenous application to the leaf surface and/or through the transpiration stream of excised shoots. The fed tissue will be extracted, and the extracts analyzed by HPLC to separate 14C-labelled compounds. Unique compounds will be analyzed by GC-MS. Results using wild type plants will be compared to those using sdh knockout plants.The effect of drought stress on leaf content of reactive oxygen species, including H2O2, ascorbic acid, and glutathione, and related enzymatic activities, including superoxide dismutase, ascorbate reductase, and glutathione reductase, in control versus sdh knockout plants will be measured (Deng and Dong, 2013), using plants collected in time course studies as above.Assessment of UV-C exposure as a treatment to elicit the phenotypePreliminary work indicated that the mutant phenotype was elicited after a 24 h UV-C exposure, although wild-type plants also suffered some injury. This technique would be a much more rapid treatment to use in place of drought stress, but it first must be carefully studied to refine the treatment technique and compared to the drought-induced phenotype, at the whole plant, biochemical, and molecular levels.