Performing Department
(N/A)
Non Technical Summary
The intermediates and immediate end-products of the shikimate pathway serve in plants as precursors to thousands of compounds with agronomic, nutritional, and industrial relevance. A complete set of enzymes necessary for the shikimate pathway is known to be located in the subcellular compartment known as the plastid. While this compartment is generally accepted as the sole site of synthesis of shikimate pathway products, it is necessary for plant cells to maintain pools of the key intermediate shikimate and the closely related compound quinate outside of the plastids for the production of several major metabolites. This apparent contradiction in our current understanding of plant metabolism prevents effective targeting of enhanced production of valuable shikimate pathway-derived phytochemicals via either traditional breeding or bioengineering strategies.DHDSDH enzymes with apparent extra-plastidial localizationsmay help resolve this apparent contradiction in our current understanding of plant metabolism. We will use a combination of in vitro biochemistry, reverse genetics, and metabolic analysis to explore the link between different DHDSDH isoforms and downstream phytochemicals that influence food quality and/or have agronomic and industrial relevance. Using tomato and beet as our experimental systems, we will determine the biochemical properties and verify the subcellular localization of DHDSDH enzymes, including those presumed to be cytosolic, thereby determining the metabolic potential of these enzymes. This will be complemented by examining the real in planta impact of alternative DHDSDH isoforms in tomato and beet, including determining whether the metabolic roles of these isozymes may be integrated with a more complete cytosolic shikimate pathway, as posited nearly four decades ago. This will involve the creation of genetically modified plants in which the levels of these enzymes have been altered and measuring the impact of these alterations on shikimate pathway intermediates and products, including several chemicals that confer desirable characteristics on crop species. Results generated from this work will impact our fundamental understanding of phytochemical metabolism, providing targets for future development of enhanced crop varieties. This is expected to ultimately facilitate develop of crops with better resistance to pests and abiotic stresses, improved nutritional value, and flavors and colors that are more desireable to consumers. Due to the industrial potential of many shikimate pathway-derived compounds as lubricants, plastic alternatives, and fuels, this research will also build on existing groundwork intended to develop plant-based platforms for sustainable production of petrochemical replacements as society moves towards the goal of post-petroleum lifestyles.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
We seek to understand the physiological role of putative DHDSDHs in the context of cytosolic modulation of agriculturally-relevant metabolites. Thus, the specific objectives of this project are as follows:1) Fully biochemically characterize the DHDSDH isoforms from Arabidopsis, tomato, and beet;2) Determine the in planta metabolic role of cytosolic DHDSDH isoforms using reverse genetic strategies; and3) Assess the non-canonical beet DHDSDH contribution to betalain metabolism
Project Methods
Objective 1Sequences of full-length cDNAs will be determined by Rapid Amplification of cDNA Ends (RACE) using a commercially available kit (GeneRacer, Invitrogen) and appropriate gene-specific primers. RACE amplicons will be gel cloned into a plasmid for sequence determination by Sanger sequencing. Once the full-length coding sequences of all transcript variants are determined, the full sequence will be PCR-amplified and subcloned into pET32a for recombinant expression in E coli. Recombinant proteins will be purified by IMAC chromatography and the fusion tag will be cleaved by enterokinase digestion to leave the native protein. All purified recombinant proteins will be assayed for DHD, QDT, SDH, and QDH activities under optimized reaction conditions, and the kinetic parameters determinedTo determined subcellular localization of proteins, the full-length open reading frames will be cloned into pK7WGF2 and pK7FWG2 with and without, respectively, the genes' native stop codons, to allow for strong plant expression of the proteins with green fluorescence protein fused to the N- or C- terminus, respectively. The expression constructs will be agro-infiltrated into Nicotiana benthamiana leaves following standard protocols. Subcellular distribution of GFP fluorescence will be observed by confocal microscopy using chlorophyll autofluorescence as a plastidial marker.Objective 2Null mutants will be created by transforming tomato cotyledons with CRISPR/Cas9 constructs using a standard protocol for agrobacterium-mediated transformation. Double mutants will subsequently be generated by crossing a confirmed null mutant for each individual gene. We will conduct targeted metabolic analysis in both fruits and vegetative tissues of all transgenic lines using established protocols. To assess herbivory impacts, we will perform no-choice feeding assays using the wildtype and mutant lines. To assess gain-of-function, we have already used a construct that overexpresses SlDHDSDH2 under the control of the CMV 35s promoter to transform two genetic backgrounds of Arabidopsis: wild-type Columbia and a CM2 null mutant. The BASTA resistance phenotype will be used to ensure segregation consistent with single-copy insertion and ultimately for selection of homozygous transformants in the T3 generation. Expression will be assessed qRT-PCR with biological replication in the homozygous T3 plants. Homozygous lines will be used in metabolite analysis.Objective 3We will collect multiple tissues from greenhouse-grown beet plants (inbred line W357B) and analyze gene expression by qRT-PCR. DHDSDH expression will be compared to that of known to be involved in post-chorismate AAA and betalain metabolism. All tissues will also be analyzed for content of betalain pigments and AAAs.An RNAi strategy will be used for reverse genetic analysis. RNAi hairpin constructs specific to the target genes will be generated in the Gateway destination vector pH7GWIWG2(II). The final RNAi constructs will used to transform beet via an established agrobacterium-mediated protocol. Roots and leaves of transgenic plants and non-transformed controls will be used in metabolic analyses. If a metabolic effect is observed upon RNAi suppression, we will perform genetic complementation experiments in that background using synthetic, recoded sequences of the open reading frames of relevant expressed under the control of the strong constitutive CMV p35s promoter. Intact leaves from RNAi beet lines will be transiently infiltrated with agrobacterium strains carrying the pB2GW7 constructs or a control empty vector, and change in metabolite levels will be monitored.