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. Whilethis 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.We have identified a DHDSDHenzymewith apparent extra-plastidial localization that may help resolve this apparent contradiction in our current understanding of plant metabolism. We willusea 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 tomatoand 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 ofpost-petroleum lifestyles.
Animal Health Component
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Research Effort Categories
Basic
100%
Applied
0%
Developmental
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Goals / Objectives
Goal 1:Fully biochemically characterize the DHDSDH isoforms from Arabidopsis, tomato, and beetObjective 1.1:Determine the full sequence of all transcripts of beet BvDHDSDH2Objective 1.2:Biochemically characterize DHDSDH isoformsObjective 1.3:Determine subcellular localization of DHDSDH isoformsGoal 2: Determine the in planta metabolic role of cytosolic DHDSDH isoforms using reverse genetic strategiesObjective 2.1: Determing metabolic impact of SlDHDSDH2 loss of functionObjective 2.2: Determine contribution of SlDHDSDH2 to plant herbivory resistanceObjective 2.3: Assess gain of function in a type-b-negative species3) Assess the non-canonical beet DHDSDH contribution to betalain metabolismObjective 3.1:Analyze DHDSDH gene expression in beetObjective 3.2: Test loss of function of BvDHDSDH isoforms
Project Methods
Objective 1To determine the full-length protein coding sequence(s) of any and all BvDHDSDH2 transcripts, we will perform 5'- and 3'-RNA Ligase Mediated-Rapid Amplification of cDNA Ends (RLM-RACE) on the encoding gene using the standard protocols developed for the GeneRacer kit (Invitrogen). Briefly, total RNA will be extracted from beet seedlings and an RNA oligonucleotide will be selectively ligated to the 5'-ends of uncapped mRNAs. Subsequent, the full-length mRNAs, including the ligated oligonucleotide, will be reverse transcribed, and the resulting cDNA will be subject to PCR amplification of the 5'- and 3'- ends of the BvDHDSDH2 cDNA using gene-specific primers in conjunction with the GeneRacer 5' (homologous to the RNA oligonucleotide) and 3' (homologous to the oligo dT reverse transcription primer) primers, respectively, and a proofreading DNA polymerase. Amplicons will be gel purified and cloned using the Zero Blunt TOPO cloning kit.The coding sequences of AtDHDSDH and all transcript variants of BvDHDSDH2 will be cloned into the E coli expression vector pET32a, as has already been done for all tomato DHDSDHs. The constructs will be used for recombinant expression of all proteins using Rosetta 2 (DE3) E coli. 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. Conditions will be optimized (i.e. buffer composition, ionic strength, metal cofactors) to maximize activity within the constraints of maintaining physiological relevance.The open reading frames of all DHDSDH genes characterized above will be cloned into pK7WGF2 and pK7FWG2 with and without, respectively, the genes' native stop codons, toallow 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 an standard protocols. Subcellular distribution of GFP fluorescence will be observed by confocal microscopy using chlorophyll autofluorescence as a plastidial marker.Objective 2SlDHDSDH2 and SlCM2 null mutants will be created by transforming tomato cotyledons using a standard protocol for agrobacterium-mediated transformation.Plantlets will be regenerated under kanamycin selection. Regenerated shoots that are successfully rooted will be screened for CRISPR/Cas9-induced mutations by PCR amplification of the respective genes from genomic DNA and subsequent Sanger sequencing. A SlDHDSDH2/CM2 double mutant will subsequently be generated by crossing a confirmed null mutant for each of the two genes.We will conduct targeted metabolic analysis in both fruits and vegetative tissues of all transgenic lines.To directly test for a roll of SlDHDSDH2 in sustaining synthesis of aromatic amino acids in the cytosol, we will measure total Phe, Tyr, and Trp levels. We will directly measure shikimate, quinate, and chlorogenic acid in mutants using established HPLC based methods. We will use a previously described LC-MS/MS method to simultaneously measure the organic acids, aldehydes, and alcohols of the phenylpropanoid network, as well as the coumaroyl and caffeoyl shikimate esters.Additionally, we will measure anthocyanin and condensed tannin levels.To assess herbivory impacts, we will perform no-choice feeding assays using the wildtype and mutant tomato lines.Briefly, tobacco hornworm larvae will be allowed to feed on the plants, and hornworm mass gain and development speed will be monitored.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-PCRwith biological replication in the homozygous T3 plants. Homozygous lines will be used toanalyze shikimate, quinate, and the three AAAs, as well as intermediates and products of the general phenylpropanoid pathway, including monolignols, anthocyanins, and tannins, as described above.Objective 3We will collect root peel, root flesh, hypocotyls, leaf petioles, and leaf lamina from greenhouse-grown beet plants (inbred line W357B), with a minimum of five biological replicates per tissue. Each tissue will be analyzed by qRT-PCR for expression of both BvDHDSDH1 and BvDHDSDH2. If 5'-RLM-RACE experimentsreveal the presence of multiple transcripts of BvDHDSDH2, we will utilize separate primer pairs specific to each transcript in order to determine expression of each transcript variant individually.As a positive control, we will also measure expression of genes known to be involved in betalain production, including ADHα, ADHβ, MYB1, DODAα, and BvCYPAD1α (36). Finally, expression of the single beet CM2 homolog (BvCM2, XP_019102621), shown in other species to be involved in a cytosolic post-chorismate pathway for production of AAAs, will also be measured to further examine the possibility of a cytosolic contribution to synthesis of the betalain precursor Tyr. All tissues will also be analyzed for content of betalain pigments by a spectrophotometric method, and for Tyr via an HPLC-based method.If the unique biochemistry of BvDHDSDH2 enables high production of betalains, we expect expression to correlated with betalain content and with genes previously shown to facilitate betalain production in beet roots.We will pursue an RNAi strategy to downregulate expression of BvDHDSDH2 in beets. We will create an RNAi hairpin constructs in the Gateway destination vector pH7GWIWG2(II). The hairpin will target nucleotides 480-780 and the corresponding nucleotides (528-828) of the predicted full lengther BvDHDSDH1 and BvDHDSDH2, respectively, full-length coding sequences.The final RNAi constructs will used to transform beet via an established agrobacterium-mediated protocol shown to be efficient across multiple beet cultivars. For each construct, the three lines showing the greatest, specific repression of the target gene's expression will be propagated by seed and used in downstream analyses.Roots and leaves of transgenic plants and non-transformed controls will be used in metabolic analyses. Betalain pigments will be measured spectrophotometrically as described above. Tyr, as well as Phe, Trp, and shikimate, will be measured by HPLC as described above.If a metabolic effect is observed upon RNAi suppression of either DHDSDH isoform, we will perform genetic complementation experiments in that background. Synthetic, recoded sequences of the open reading frames of all DHDSDH isoforms, including any alternative transcripts identified for BvDHDSDH2, will produced by Twist Bioscience. The synthetic genes will be subcloned into pB2GW7 to drive expression of the proteins 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 betalain levels will be monitored as described above.