Recipient Organization
VIRGINIA POLYTECHNIC INSTITUTE
(N/A)
BLACKSBURG,VA 24061
Performing Department
School of Plant and Environmental Sciences
Non Technical Summary
According to the Intergovernmental Panel on Climate Change (IPCC), an increase in global mean temperature of 1.5 to 4 oC is predicted to occur over the next century. Increases in temperature will have beneficial effects on some regions and harmful effects on others (IPCC, 2013). The extent of temperature increase depends mainly on the amounts of heat-trapping gases emitted globally and how sensitive the climate is to those gases. CO2 is one of the important heat-trapping gases. Fortunately, plants help reduce atmospheric CO2 levels through photosynthesis, a biochemical process that captures CO2 and incorporates it into simple sugars that are the building blocks for the production of edible (fruits and grains) and nonedible (wood and other fibers) biomass. While rising levels of CO2 can in theory improve plant growth and productivity by accelerating the rate of photosynthesis, plants have complex regulatory mechanisms that limit photosynthesis. Moreover, rising global temperatures increase the risk of drought, a factor that leads to plant stress and crop losses. Together, intrinsic limitations to photosynthesis and sensitivity to drought are major impediments to optimizing yield in the face of rising CO2 levels and climate change. In addition to using CO2 to produce sugars for energy and growth, plants also produce special regulatory sugars that control the rates of growth and photosynthesis, resource allocation, and stress response. Trehalose-6-phosphate (T6P) is one of the most important regulatory sugars in plants. Recent work with maize showed that reducing the levels of T6P in the ear caused the plant to transport more sugar to the kernel, increasing kernel growth under both well-watered and drought conditions. These are exciting results that point to powerful new ways to engineer or breed plants for increased yield and resilience. The effectiveness of targeted T6P reduction has not yet been tested using multiple food crops or biomass crops. Therefore, we propose to genetically engineer poplar trees for reduction of T6P levels for the purpose of increasing yield and drought tolerance in a biomass crop.It is convenient to think of plants as consisting of two main components: sources and sinks. Sources are the green, photosynthetic, sugar-producing organs (leaves) and sugar-transporting tissues (phloem). Sinks, the consumers of sugars, are organs or tissues that conduct little or no photosynthesis, e.g., seeds, fruits, roots, buds, and woody tissue (xylem). Reducing the amount of T6P in source tissues can increase the amount of sugars transported to sink tissues, thereby increasing the growth of those tissues under both well-watered and drought conditions.For this project, we will prepare transgenic poplar plants that produce trehalose-phosphate phosphatases (TPPs), enzymes that can degrade and reduce the levels of T6P. The TPPs to be tested include all 11 distinct TPP genes that naturally occur in poplar, one TPP from rice, and one TPP from Escherichia coli. The rice and E. coli TPPs have been extensively studied by other researchers and will thus serve as benchmarks for comparison with the activities of the poplar TPP enzymes. The TPPs under investigation will be used to prepare transgenic poplar trees with increased TPP activity in selected source tissues. The impact of TPPs on sink tissues and on drought tolerance of the resulting transgenic trees will be evaluated
Animal Health Component
25%
Research Effort Categories
Basic
75%
Applied
25%
Developmental
(N/A)
Goals / Objectives
The overall goal of this project is to investigate the value of targeted reduction in the levels of the signaling sugar T6P as a tool for improving yield and resilience in biomass crops. We hypothesize that T6P acts as a signaling molecule coordinating the availability of sucrose to axillary buds, wood, shoots, and roots for control of poplar growth. We propose to test this hypothesis by targeted expression of TPP genes in source tissues of transgenic poplar followed by evaluation of growth and resilience. This work will provide a greater understanding of molecular mechanisms linking T6P signaling with carbon availability in growing sink tissues. Such efforts will lead to the ultimate goal of modulating T6P levels for tailoring poplar growth and development to maximize its use and minimize waste, as well as increase yields of other biomass and food crops under both well-watered and drought conditions.
Project Methods
Through in vitro analysis of recombinant TPP proteins (Objective 1), and subsequent testing of the efficacy of selected TPPs in transgenic poplar, we will identify those TPPs capable of increasing yield (Objective 2) and resilience (Objective 3) of poplar trees.Objective 1: Clone and express all 11 poplar TPP genes and characterize each TPP enzyme for basic kinetic properties and substrate specificity.The Populus trichocarpa genome includes 11 genes predicted to code for TPP enzymes (PtrTPP1 through PtrTPP11). All 11 TPP genes will be cloned in vectors optimized for expression in E. coli and purification via affinity tags, i.e., as glutathione S-transferase (GST)-PtrTPP fusion proteins. Enzymatic properties of each recombinant TPP will be determined as described by Shima et al. (2007) and will include analysis of pH optimum, substrate specificity, heat tolerance. Km, and Kcat will be determined for all recombinant PtrTPPs as well as for recombinant OtsB and OsTPP1 proteins for comparison with poplar TPPs. Poplar TPPs will also be tested for their ability to complement the yeast tps2 mutant (Shima et al., 2007). It is possible that some of the TPP genes will not express at high levels in E. coli or will be difficult or impossible to purify as soluble, active enzymes. Thus, the final number of proteins available for characterization may be less than 13 (11 poplar TPPs genes plus OtsB and OsTPP1). However, Shima et al. (2007) were able to produce sufficient quantities of recombinant rice OsTPP1 and OsTPP2 for analysis, demonstrating the feasibility of producing recombinant plant TPP proteins.Objective 2. Test efficacy of E. coli OtsB, rice OsTPP1, and selected poplar TPP genes under control of selected cell-type or tissue-specific promoters for increasing growth of sink tissues.For this objective, the overall goal is to produce transgenic poplar with altered expression of selected PtrTPP genes in distinct source tissues for enhanced sylleptic branch growth or growth of other sinks, i.e., wood, roots, or the main stem. As noted above, use of the LMX5 promoter to drive OtsB expression resulted in activation of sylleptic branching in multiple transgenic events and is thus a reliable positive control for testing efficacy of other TPP genes and their impact on this phenotype as a model for other source-sink targets. We will prepare transgenic LMX5:OsTPP1 poplar and compare the resulting phenotype with that of the LMX5:OtsB plants described here. Additionally, we will select three PtrTPP genes representing a range of characteristic (e.g., differing substrate specificity and Km) for expression in poplar under control of the LMX5 promoter, again, for comparison with the benchmark LMX5:OtsB plants. The PtrTPP genes that yield the most vigorous sylleptic branch growth will be further tested for their impact on additional poplar sinks as described below.For increased wood production, we will prepare transgenic poplar for expression of the top performing PtrTPP gene under the control of three promoters targeting tissues within the vascular tissue system: PttANT (Potri.002G114800), PtPP2 (Potri.015G120200), and Pt4CL1 (Genbank accession No. for promoter, AF041051; Hu et al., 1998). The PttANT, PttPP2, and Pt4CL1 promoters drive expression in dividing cambium cells, phloem, and differentiating xylem cells, respectively, according to Etchells et al. (2015). While we expect modulation of activity of TPPs in phloem to be the most critical and effective for enhancing growth of adjacent sinks, we cannot rule out the possibility that beneficial outcomes can be achieved through reduction in T6P levels in neighboring cambium or differentiating xylem cells. Mature, photosynthetic leaves are the ultimate carbon source. However, in contrast to the set of vascular tissue-specific promoters listed above, leaf-specific promoters have not been extensively tested in poplar. Studies with rice show that genes for the small subunit of RUBISCO (rbcS) and chlorophyll a-b binding proteins (cab) are expressed in mesophyll cells of mature leaves (Tsutsumi et al., 2006). Both rice and poplar are C3 plants, indicating that promoters for the poplar orthologs of the rice rbcS and cab genes are good candidates for leaf mesophyll-targeted expression of PtrTPP genes. We will test the putative promoters (2 kb upstream of transcriptional start site) of rbcS gene Potri.017G114600 and cab gene Potri.005G239300, both of which show high level expression in mature leaves (https://phytozome.jgi.doe.gov/), as promoter-reporter fusions in transgenic poplar to confirm that one or both of these will be useful for leaf-associated expression of PtrTPP genes in transgenic poplar. We expect that targeting expression of PtrTPP genes to photosynthesizing leaves will increase growth of poplar shoots and roots through enhanced growth of shoot and root apical meristems, i.e., increased meristem sink strength.Measure growth, biomass, and xylem cell number. All transgenic and 717 hybrid control (WT) poplar plants will be grown in a greenhouse under long-day conditions (16:8, light:dark) to prevent bud dormancy using a maintenance level of nitrogen that is sufficient to promote rapid growth (approximately 1.5 m tall), without occurrence of nutrient deficiency symptoms during a six-week trial period. For the transgenics listed above and WT trees, we will determine dry weight, number of internodes, internode length, and stem diameter for trees grown in soil in six-week greenhouse trials. If sylleptic branching is induced, we will determine total number of sylleptic branches and sylleptic leaves. For xylem production measurements, stem tissue will be fixed in paraformaldehyde/glutaraldehyde followed by ethanol dehydration series and embedding in LR White resin. Following thin sectioning via ultramicrotome and staining with toluidine blue/boric acid, images are captured using a Zeiss Axioscope 40 microscope and xylem cells quantified using CellProfiler image analysis. We have published similar analyses of transgenic poplar growth and wood formation (Grant et al., 2010). For each transgenic line we generate, we will use quantitative PCR to identify at least five independent transgenic events with TPP transgene expression. RT-qPCR will be conducted according to our published methods for poplar (Grant et al., 2010). At least three independent events per transgene and five replicates per event will be evaluated in greenhouse trials.Objective 3. Characterization of effects of drought on biomass yield.Both WT controls and TPP transgenics will be subjected to acute drought conditions according to (Xue et al., 2016). Briefly, water will be withheld for two to three days until visible-yet-reversible turgor loss of sink-source transitional leaves (leaf plastochron index 3-5) is observed. Source leaves exhibit normal turgor at this acute drought point. The acute drought treatment will be followed by rewatering to restore full leaf turgor. The acute drought/rewatering cycle will be performed once a week for three weeks. Growth and biomass of trees will be measured as described for Objective 2.