Transcriptomic Characterization of Novel Fruit Ripening Pathways

Ripening of climacteric fruits is characterized by a rise in respiration and concomitant burst of ethylene biosynthesis. While this general pattern is shared among climacteric fruits, there are a few exceptions. European pears (Pyrus communis) require a genetically pre-determined amount of cold exposure, called conditioning, to transition from System 1 (S1) to System 2 (S2) ethylene biosynthesis. Recent research from the lab has implicated pre-climacteric alternative oxidase activity in this transition. In addition to the need for cold exposure, European pear cultivars exhibit variability in response to ethylene receptor antagonist 1-methylcyclopropene (1-MCP). Unlike apples and other climacteric fruits, pears do not regain their ripening capacity following 1-MCP treatment despite the conditioning treatment. A chemical genomics study aimed at activating alternative oxidase in 1-MCP treated pears identified glyoxylic acid for its ability to override the metabolic blockage of ethylene biosynthesis caused by 1-MCP.This dissertation characterizes a natural variation in climacteric ripening represented by European pear, by (1) Summarizing and distilling published information regarding the role of respiration in climacteric fruit. Many of the physiological and phenotypic changes that occur during ripening are attributed to ethylene, however the molecular and metabolic crosstalk between autocatalytic ethylene production and respiration are less understood. Results of recent work implicate the alternative oxidase (AOX) respiratory pathway in crosstalk between ethylene, respiration, and other ripening-associated processes; (2) Conducting comparative physiological and transcriptomics analyses of two pear genotypes that require different durations of cold temperature exposure in order to ripen. Transcriptomic analysis revealed that cold temperature conditioning leads to heightened expression of vernalization-associated genes, homologs of which are necessary for other cold-dependent processes like seed germination and flowering. These genes may serve as regulatory points in the initiation of ripening in fruit that require cold conditioning; (3) Assessing the impact of glyoxylic acid in chemical induction of ripening in 1-MCP treated 'D'Anjou' pear fruit using physiological and transcriptomics approaches. Results demonstrate that glyoxylic acid effectively elicits physiological responses as well as significant changes in global gene expression, including that of AOX, in 1-MCP treated pear fruit that would be otherwise incapable of ripening normally

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Genome editing in fruit, ornamental, and industrial crops

The advent of genome editing has opened new avenues for targeted trait enhancement in fruit, ornamental, industrial, and all specialty crops. In particular, CRISPR-based editing systems, derived from bacterial immune systems, have quickly become routinely used tools for research groups across the world seeking to edit plant genomes with a greater level of precision, higher efficiency, reduced off-target effects, and overall ease-of-use compared to ZFNs and TALENs. CRISPR systems have been applied successfully to a number of horticultural and industrial crops to enhance fruit ripening, increase stress tolerance, modify plant architecture, control the timing of flower development, and enhance the accumulation of desired metabolites, among other commercially-important traits. As editing technologies continue to advance, so too does the ability to generate improved crop varieties with non-transgenic modifications; in some crops, direct transgene-free edits have already been achieved, while in others, T-DNAs have successfully been segregated out through crossing. In addition to the potential to produce non-transgenic edited crops, and thereby circumvent regulatory impediments to the release of new, improved crop varieties, targeted gene editing can speed up trait improvement in crops with long juvenile phases, reducing inputs resulting in faster market introduction to the market. While many challenges remain regarding optimization of genome editing in ornamental, fruit, and industrial crops, the ongoing discovery of novel nucleases with niche specialties for engineering applications may form the basis for additional and potentially crop-specific editing strategies.The authors would like to acknowledge funding from MINECO, Spain (PGC2018-097655-B-I00 to P Christou), Generalitat de Catalunya Grant 2017 SGR 828 to the Agricultural Biotechnology and Bioeconomy Unit (ABBU). Work in the Dhingra lab in crop improvement is supported in part by Washington State University Agriculture Research Center Hatch grant WNP00011. ES and FR acknowledge the support received from the Department of Horticulture, BW was supported in part by a Research Assistantship from the Washington State University Graduate School. The authors would also like to thank Drs A. McHughen and H. Quemada for input and clarifications on US genome editing regulations. We would also like to thank the anonymous reviewers for their insightful comments

Impact of heat stress, water stress, and their combined effects on the metabolism and transcriptome of grape berries

Abstract Recurring heat and drought episodes present challenges to the sustainability of grape production worldwide. We investigated the impacts of heat and drought stress on transcriptomic and metabolic responses of berries from two wine grape varieties. Cabernet Sauvignon and Riesling grapevines were subjected to one of four treatments during early fruit ripening: (1) drought stress only, (2) heat stress only, (3) simultaneous drought and heat stress, (4) no drought or heat stress (control). Berry metabolites, especially organic acids, were analyzed, and time-course transcriptome analysis was performed on samples before, during, and after the stress episode. Both alone and in conjunction with water stress, heat stress had a much more significant impact on berry organic acid content, pH, and titratable acidity than water stress. This observation contrasts with previous reports for leaves, which responded more strongly to water stress, indicating that grape berries display a distinct, organ-specific response to environmental stresses. Consistent with the metabolic changes, the global transcriptomic analysis revealed that heat stress had a more significant impact on gene expression in grape berries than water stress in both varieties. The differentially expressed genes were those associated with the tricarboxylic acid cycle and glyoxylate cycle, mitochondrial electron transport and alternative respiration, glycolysis and gluconeogenesis, carbohydrate allocation, ascorbate metabolism, and abiotic stress signaling pathways. Knowledge regarding how environmental stresses, alone and in combination, impact the berry metabolism of different grape varieties will form the basis for developing recommendations for climate change mitigation strategies and genetic improvement

Evidence for pre-climacteric activation of AOX transcription during cold-induced conditioning to ripen in European pear (Pyrus communis L.).

European pears (Pyrus communis L.) require a range of cold-temperature exposure to induce ethylene biosynthesis and fruit ripening. Physiological and hormonal responses to cold temperature storage in pear have been well characterized, but the molecular underpinnings of these phenomena remain unclear. An established low-temperature conditioning model was used to induce ripening of 'D'Anjou' and 'Bartlett' pear cultivars and quantify the expression of key genes representing ripening-related metabolic pathways in comparison to non-conditioned fruit. Physiological indicators of pear ripening were recorded, and fruit peel tissue sampled in parallel, during the cold-conditioning and ripening time-course experiment to correlate gene expression to ontogeny. Two complementary approaches, Nonparametric Multi-Dimensional Scaling and efficiency-corrected 2-(ΔΔCt), were used to identify genes exhibiting the most variability in expression. Interestingly, the enhanced alternative oxidase (AOX) transcript abundance at the pre-climacteric stage in 'Bartlett' and 'D'Anjou' at the peak of the conditioning treatments suggests that AOX may play a key and a novel role in the achievement of ripening competency. There were indications that cold-sensing and signaling elements from ABA and auxin pathways modulate the S1-S2 ethylene transition in European pears, and that the S1-S2 ethylene biosynthesis transition is more pronounced in 'Bartlett' as compared to 'D'Anjou' pear. This information has implications in preventing post-harvest losses of this important crop

Evaluation of multiple approaches to identify genome-wide polymorphisms in closely related genotypes of sweet cherry (Prunus avium L.)

Identification of genetic polymorphisms and subsequent development of molecular markers is important for marker assisted breeding of superior cultivars of economically important species. Sweet cherry (Prunus avium L.) is an economically important non-climacteric tree fruit crop in the Rosaceae family and has undergone a genetic bottleneck due to breeding, resulting in limited genetic diversity in the germplasm that is utilized for breeding new cultivars. Therefore, it is critical to recognize the best platforms for identifying genome-wide polymorphisms that can help identify, and consequently preserve, the diversity in a genetically constrained species. For the identification of polymorphisms in five closely related genotypes of sweet cherry, a gel-based approach (TRAP), reduced representation sequencing (TRAPseq), a 6k cherry SNParray, and whole genome sequencing (WGS) approaches were evaluated in the identification of genome-wide polymorphisms in sweet cherry cultivars. All platforms facilitated detection of polymorphisms among the genotypes with variable efficiency. In assessing multiple SNP detection platforms, this study has demonstrated that a combination of appropriate approaches is necessary for efficient polymorphism identification, especially between closely related cultivars of a species. The information generated in this study provides a valuable resource for future genetic and genomic studies in sweet cherry, and the insights gained from the evaluation of multiple approaches can be utilized for other closely related species with limited genetic diversity in the breeding germplasm

Plastid transit peptides—where do they come from and where do they all belong? Multi-genome and pan-genomic assessment of chloroplast transit peptide evolution

Subcellular relocalization of proteins determines an organism’s metabolic repertoire and thereby its survival in unique evolutionary niches. In plants, the plastid and its various morphotypes import a large and varied number of nuclear-encoded proteins to orchestrate vital biochemical reactions in a spatiotemporal context. Recent comparative genomics analysis and high-throughput shotgun proteomics data indicate that there are a large number of plastid-targeted proteins that are either semi-conserved or non-conserved across different lineages. This implies that homologs are differentially targeted across different species, which is feasible only if proteins have gained or lost plastid targeting peptides during evolution. In this study, a broad, multi-genome analysis of 15 phylogenetically diverse genera and in-depth analyses of pangenomes from Arabidopsis and Brachypodium were performed to address the question of how proteins acquire or lose plastid targeting peptides. The analysis revealed that random insertions or deletions were the dominant mechanism by which novel transit peptides are gained by proteins. While gene duplication was not a strict requirement for the acquisition of novel subcellular targeting, 40% of novel plastid-targeted genes were found to be most closely related to a sequence within the same genome, and of these, 30.5% resulted from alternative transcription or translation initiation sites. Interestingly, analysis of the distribution of amino acids in the transit peptides of known and predicted chloroplast-targeted proteins revealed monocot and eudicot-specific preferences in residue distribution

Concomitant phytonutrient and transcriptome analysis of mature fruit and leaf tissues of tomato (Solanum lycopersicum L. cv. Oregon Spring) grown using organic and conventional fertilizer.

Enhanced levels of antioxidants, phenolic compounds, carotenoids and vitamin C have been reported for several crops grown under organic fertilizer, albeit with yield penalties. As organic agricultural practices continue to grow and find favor it is critical to gain an understanding of the molecular underpinnings of the factors that limit the yields in organically farmed crops. Concomitant phytochemical and transcriptomic analysis was performed on mature fruit and leaf tissues derived from Solanum lycopersicum L. 'Oregon Spring' grown under organic and conventional fertilizer conditions to evaluate the following hypotheses. 1. Organic soil fertilizer management results in greater allocation of photosynthetically derived resources to the synthesis of secondary metabolites than to plant growth, and 2. Genes involved in changes in the accumulation of phytonutrients under organic fertilizer regime will exhibit differential expression, and that the growth under different fertilizer treatments will elicit a differential response from the tomato genome. Both these hypotheses were supported, suggesting an adjustment of the metabolic and genomic activity of the plant in response to different fertilizers. Organic fertilizer treatment showed an activation of photoinhibitory processes through differential activation of nitrogen transport and assimilation genes resulting in higher accumulation of phytonutrients. This information can be used to identify alleles for breeding crops that allow for efficient utilization of organic inputs