Tomato is a globally produced and consumed vegetables, valued for its nutritional benefits, sensory qualities, and cultural importance. However, its relatively short shelf-life poses a challenge, as extending it through postharvest techniques often reduces fruit quality, leading to consumer dissatisfaction. Addressing this issue requires understanding how postharvest treatments affect the physiological and molecular pathways involved in tomato fruit ripening and senescence. Furthermore, developing diverse tomato germplasm may enhance fruit quality during postharvest storage.
My first aim was to investigate the effects of common industry practices, such as early harvest and low-temperature storage, on tomato fruit ripening and quality, with a focus on the underlying molecular mechanisms. DNA demethylation plays a critical role in governing tomato fruit ripening, raising the question of whether DNA methylation dynamics are associated with postharvest treatments and the fruit quality reduction. I generated molecular and physiological data on postharvest fruit related to their transcriptome, methylome, quality parameters, and ripening hormones (ethylene and ABA) (Chapter 1; Zhou et al., 2024). My analysis revealed a marked differentiation between postharvest and fresh-harvest fruit. Strikingly, there was altered DNA methylation status and downregulation of gene expression levels related to photosynthesis, accompanied by reduced chlorophyll contents in stored fruit. This raises the question as to whether photosynthetic activities contribute to fruit quality difference in dark-stored postharvest fruit vs. those vine-ripened exposed to sunlight. Another key finding was that the low but non-chilling temperature (12.5°C) stored fruit had the most distinct epigenetic marks, and remarkably high ABA levels and lacked an ethylene burst peak during ripening, which may be related to their unique hormone regulation patterns.
Chapter 2 addressed the remaining questions on whether changes in the expression levels of the photosynthesis genes reflect differences in fruit photosynthetic activities between postharvest and on-the-vine ripening fruit. In the second part, the work was carried to investigate which genes are suppressed or activated in green fruit during chilling and rewarming, and how these changes relate to DNA methylation status. Green fruit photosynthetic capacity during postharvest chilling and rewarming was assessed by chlorophyll fluorescence and gas change measurements using the LICOR-6400, alongside its transcriptome and methylome analyses used samples collected at harvest, after chilling, and after chilling followed by rewarming. The results revealed two main findings: first, the chilling treatment reduced green fruit chlorophyll content, maximum photochemical efficiency, and percentage of net photosynthesis, while the fruit rewarmed after chilling had a higher percentage of net photosynthesis than the fruit ripened on-the-vine. Various photosynthesis parameters showed different correlations with the gene expression levels. Second, diverse gene expression-methylation patterns were observed under the chilling and rewarming conditions. These findings may help identify novel genes or regions highly sensitive to postharvest chilling and rewarming as potential new targets for ameliorating postharvest-induced fruit quality loss (Chapters 1 and 2).
My second objective aimed to fine-tune tomato fruit ripening and quality development utilizing CRISPR/Cas9 genome editing (Chapters 3 and 4). Specifically, the ripening inhibitor (RIN), a key master ripening regulator gene, was studied. RIN plays an important role in activating numerous ripening-associated transcription factors and genes in tomato fruit. The RIN promoter contains a series of Cis-regulatory elements (CREs) and differentially methylated regions (DMRs) that could influence RIN transcriptional regulation, and subsequently affect tomato ripening and quality. However, these regulatory regions in the RIN promoter remain understudied. Furthermore, it’s unknown whether manipulation of the RIN promoter would alter its expression patterns and lead to changes in the fruit ripening process. Therefore, this work employed a promoter editing approach to generate a population of tomato allelic mutations of varying severity, with potential effects on the RIN-induced gene regulatory network and fruit ripening traits. I generated over a hundred first-generation (T0) transgenic lines with various RIN promoter mutations. My findings revealed a non-linear relationship between RIN promoter mutations and its gene expression, underscoring the role of specific mutation types in influencing RIN transcription in ripened fruit (Chapter 3).
Furthermore, changes in RIN transcription don’t necessarily correlate with changes in fruit ripening. I hypothesized that specific mutations within the RIN promoter would lead to differential regulation of RIN-induced ripening-associated genes, resulting in varied fruit quality traits. Therefore, fruit quality traits were evaluated in the next generations (T1 and T2) of selected mutants with distinct mutation types. Assessments included fruit color, firmness, total soluble solids (TSS), acid content, and postharvest chilling injury response (Chapter 4). Transcriptomic analysis was performed to explore the mechanisms underlying alterations in the regulation of fruit ripening due to promoter editing. My results indicated that RIN promoter mutants had mild but distinct phenotypical traits. Surprisingly, mutations at the HY5 binding sites in the RIN gene promoter were found to potentially promote fruit ripening, through the upregulation of many ripening-associated transcription factors. In addition, the two distinct mutants had unique responses to postharvest chilling injury. This work enhanced our understanding of how RIN transcriptional regulation contributes to fruit quality, shelf-life, and response to postharvest chilling and rewarming.
In Chapter 5, postharvest chilling injury (PCI), a physiological disorder affecting tropical or subtropical crops stored at temperatures below the chilling threshold, was studied. I hypothesized that there are specific molecular targets involved in the PCI response that could be leveraged to develop strategies for reducing PCI. A comprehensive review paper that connected cellular processes with gene regulation responses induced by PCI in horticultural crops was published (Albornoz et al., 2022). In collaboration with other authors, I further pointed out potential genes to target using advanced biotechnology. Ultimately, this knowledge could help improve the efficiency and sustainability of the tomato industry while also enhancing consumer satisfaction.
