Functional disruption of cell wall invertase inhibitor by genome editing increases sugar content of tomato fruit without decrease fruit weight

Sugar content is one of the most important quality traits of tomato. Cell wall invertase promotes sucrose unloading in the fruit by maintaining a gradient of sucrose concentration between source leaves and fruits, while invertase inhibitor (INVINH) regulates this process. In this study, knock-out of cell wall INVINH in tomato (SlINVINH1) was performed by genome editing using, CRISPR/Cas9 and Target-AID technologies. Most of the genome-edited lines set higher soluble solid content (SSC) fruit than the original cultivar ‘Suzukoma’, while fruit weight was different among the genome-edited lines. From these genome-edited lines, three lines (193–3, 199–2, and 247–2), whose SSC was significantly higher than ‘Suzukoma’ and fruit weight were almost the same as the original cultivar, were selected. The fruit weight and overall plant growth of the two lines were comparable to those of the original cultivar. In contrast, the fructose and glucose contents in the mature fruits of the two lines were significantly higher than those of the original cultivar. The mature fruits of genome edited line 193–3 showed the highest sugar content, and the fructose and glucose contents were 29% and 36% higher than that of the original cultivar, respectively. Whole genome sequence data showed no off-target mutations in the genome-edited lines. Non-target metabolome analysis of mature fruits revealed that fructose was the highest loading factor in principal component analysis (PCA) between the genome-edited line and the original cultivar, and no unexpected metabolites appeared in the genome-edited line. In this study, we succeeded in producing tomato lines with high sugar content without a decrease in fruit weight and deterioration of plant growth by knock-out of SlINVINH1 using genome editing technology. This study showed that functional disruption of SlINVINH1 is an effective approach to produce tomato cultivars with high sugar content.

StRAP2.3, an ERF‐VII transcription factor, directly activates StInvInh2 to enhance cold-induced sweetening resistance in potato

Potato invertase inhibitor (StInvInh2) positively regulates cold-induced sweetening (CIS) resistance by inhibiting the activity of vacuolar invertase. The distinct expression patterns of StInvInh2 have been thoroughly characterized in different potato genotypes, but the related CIS ability has not been characterized. The understanding of the regulatory mechanisms that control StInvInh2 transcription is unclear. In this study, we identified an ERF‐VII transcription factor, StRAP2.3, that directly regulates StInvInh2 to positively modulate CIS resistance. Acting as a nuclear-localized transcriptional activator, StRAP2.3 directly binds the ACCGAC cis-element in the promoter region of StInvInh2, enabling promoter activity. Overexpression of StRAP2.3 in CIS-sensitive potato tubers induced StInvInh2 mRNA abundance and increased CIS resistance. In contrast, silencing StRAP2.3 in CIS-resistant potato tubers repressed the expression of StInvInh2 and decreased CIS resistance. We conclude that cold-responsive StInvInh2 is due to the binding of StRAP2.3 to the ACCGAC cis-element in the promoter region of StInvInh2. Overall, these findings indicate that StRAP2.3 directly regulates StInvInh2 to positively modulate CIS resistance, which may provide a strategy to improve the processing quality of potatoes.

Transcriptomic Profiling of Populus Roots Challenged with Fusarium Reveals Differential Responsive Patterns of Invertase and Invertase Inhibitor-Like Families within Carbohydrate Metabolism

Fusarium solani (Fs) is one of the notorious necrotrophic fungal pathogens that cause root rot and vascular wilt, accounting for the severe loss of Populus production worldwide. The plant–pathogen interactions have a strong molecular basis. As yet, the genomic information and transcriptomic profiling on the attempted infection of Fs remain unavailable in a woody model species, Populus trichocarpa. We used a full RNA-seq transcriptome to investigate the molecular interactions in the roots with a time-course infection at 0, 24, 48, and 72 h post-inoculation (hpi) of Fs. Concomitantly, the invertase and invertase inhibitor-like gene families were further analyzed, followed by the experimental evaluation of their expression patterns using quantitative PCR (qPCR) and enzyme assay. The magnitude profiles of the differentially expressed genes (DEGs) were observed at 72 hpi inoculation. Approximately 839 genes evidenced a reception and transduction of pathogen signals, a large transcriptional reprogramming, induction of hormone signaling, activation of pathogenesis-related genes, and secondary and carbohydrate metabolism changes. Among these, a total of 63 critical genes that consistently appear during the entire interactions of plant–pathogen had substantially altered transcript abundance and potentially constituted suitable candidates as resistant genes in genetic engineering. These data provide essential clues in the developing new strategies of broadening resistance to Fs through transcriptional or translational modifications of the critical responsive genes within various analyzed categories (e.g., carbohydrate metabolism) in Populus.

Vacuolar sucrose homeostasis is critical for plant development, seed properties, and night-time survival in Arabidopsis

Most cellular sucrose is present in the cytosol and vacuoles of plant cells; however, little is known about the effect of this sucrose compartmentation on plant properties. Here, we examined the effects of altered intracellular sucrose compartmentation in Arabidopsis thaliana leaves by heterologously expressing the sugar beet (Beta vulgaris) vacuolar sucrose loader BvTST2.1 and by generating lines with reduced vacuolar invertase activity (amiR vi1-2). Heterologous expression of BvTST2.1 led to increased monosaccharide levels in leaves, whereas sucrose levels remained constant, indicating that vacuolar invertase activity in mesophyll vacuoles exceeds sucrose uptake. This notion was supported by analysis of tobacco (Nicotiana benthamiana) leaves transiently expressing BvTST2.1 and the invertase inhibitor NbVIF. However, sucrose levels were strongly elevated in leaf extracts from amiR vi1-2 lines, and experiments confirmed that sucrose accumulated in the corresponding vacuoles. The amiR vi1-2 lines exhibited impaired early development and reduced seed weight. When germinated in the dark, amiR vi1-2 seedlings were less able to convert sucrose into monosaccharides than the wild type. Cold temperatures strongly down-regulated both VI genes, but the amiR vi1-2 lines showed normal frost tolerance. These observations indicate that increased vacuolar sucrose levels fully compensate for the effects of low monosaccharide concentrations on frost tolerance.

Vacuolar sucrose homeostasis is critical for development, seed properties and survival of dark phases of Arabidopsis

Although we know that most of the cellular sucrose is present in the cytosol and vacuole, our knowledge on the impact of this sucrose compartmentation on plant properties is still fragmentary. Here we attempted to alter the intracellular sucrose compartmentation of Arabidopsis mesophyll cells by either, overexpression of the vacuolar sucrose loader BvTST2.1 or by generation of mutants with decreased vacuolar invertase activity (amiR vi1-2). Surprisingly, BvTST2.1 overexpression led to increased monosaccharide levels in leaves, while sucrose remained constant. Latter observation allows the conclusion, that vacuolar invertase activity in mesophyll vacuoles exceeds sucrose uptake in Arabidopsis, which gained independent support by analyses on tobacco leaves transiently overexpressing BvTST2.1 and the invertase inhibitor NbVIF. However, we observed strongly increased sucrose levels in leaf extracts from independent amiR vi1-2 lines and non-aqueous fractionations confirmed that sucrose accumulation in corresponding vacuoles. amiR vi1-2 lines exhibited impaired early development and decreased weight of seeds. When germinated in the dark, mutant seedlings showed problems to convert sucrose into monosaccharides. Cold temperatures induced marked downregulation of the expression of both VI genes, while frost tolerance of amiR vi1-2 mutants was similar to WT indicating that increased vacuolar sucrose levels fully compensate for low monosaccharide concentrations.HighlightVacuolar sucrose accumulation in Arabidopsis is limited by high invertase activity and disturbed vacuolar sucrose homeostasis impairs plant germination, development, seed properties and survival under darkness.

Transcriptome profiling of non-climacteric ‘Yellow’ melon during ripening: insights of sugar metabolism

Background: The non-climacteric ‘Yellow’ melon (Cucumis melo, inodorus group) is an economically important crop and its quality is mainly determined by the sugar content. Thus, the knowledge of sugar metabolism and its related pathways can contribute to development of new field management and post-harvest practices, making it possible to deliver to consumers better quality fruits.Results: The RNA-seq associated with RT-qPCR analyses of four maturation stages were performed to identify important enzymes and pathways that are involved in the sugar metabolism profile in non-climacteric ‘Yellow’ melon fruit. We identified 895 genes 10 DAP-biased and 909 genes 40 DAP-biased. The global analyses evidenced pathways related to sucrose metabolism; hormone signal transduction; DNA and protein processing like significant metabolisms in the melon ripening. For fruit sucrose metabolism, 17 genes were differentially expressed (DE) whose pattern varied throughout fruit development.Conclusions: The results demonstrated important enzymes in the sugar pathway those are responsible for the sucrose content and maturation profile in non-climacteric ‘Yellow’ melon. New DE genes were first detected for melon in this study such as invertase inhibitor LIKE 3 (CmINH3), trehalose phosphate phosphatase (CmTPP1) and trehalose phosphate synthases (CmTPS5, CmTPS7, CmTPS9). Besides that, the results of the protein-protein network interaction demonstrated general characteristics of the transcriptome of young and full-ripe melon. Keywords: Cucumis melo, RNA-seq, sucrose, fruit ripening, gene expression