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.

Comparison and Characterization of a Cell Wall Invertase Promoter from Cu-Tolerant and Non-Tolerant Populations of Elsholtzia haichowensis

Cell wall invertase (CWIN) activity and the expression of the corresponding gene were previously observed to be significantly elevated in a Cu-tolerant population of Elsholtzia haichowensis relative to a non-tolerant population under copper stress. To understand the differences in CWIN gene regulation between the two populations, their CWIN promoter β-glucuronidase (GUS) reporter vectors were constructed. GUS activity was measured in transgenic Arabidopsis in response to copper, sugar, and phytohormone treatments. Under the copper treatment, only the activity of the CWIN promoter from the Cu-tolerant population was slightly increased. Glucose and fructose significantly induced the activity of CWIN promoters from both populations. Among the phytohormone treatments, only salicylic acid induced significantly higher (p < 0.05) activity of the Cu-tolerant CWIN promoter relative to the non-tolerant promoters. Analysis of 5′-deletion constructs revealed that a 270-bp promoter fragment was required for SA induction of the promoter from the Cu-tolerant population. Comparison of this region in the two CWIN promoters revealed that it had 10 mutation sites and contained CAAT-box and W-box cis-elements in the Cu-tolerant promoter only. This work provides insights into the regulatory role of SA in CWIN gene expression and offers an explanation for differences in CWIN expression between E. haichowensis populations.

A Fructan Exohydrolase from Maize Degrades Both Inulin and Levan and Co-Exists with 1-Kestotriose in Maize

Enzymes with fructan exohydrolase (FEH) activity are present not only in fructan-synthesizing species but also in non-fructan plants. This has led to speculation about their functions in non-fructan species. Here, a cell wall invertase-related Zm-6&1-FEH2 with no “classical” invertase motif was identified in maize. Following heterologous expression in Pichia pastoris and in Nicotiana benthamiana leaves, the enzyme activity of recombinant Zm-6&1-FEH2 displays substrate specificity with respect to inulin and levan. Subcellular localization showed Zm-6&1-FEH2 exclusively localized in the apoplast, and its expression profile was strongly dependent on plant development and in response to drought and abscisic acid. Furthermore, formation of 1-kestotriose, an oligofructan, was detected in vivo and in vitro and could be hydrolyzed by Zm-6&1-FEH2. In summary, these results support that Zm-6&1-FEH2 enzyme from maize can degrade both inulin-type and levan-type fructans, and the implications of the co-existence of Zm-6&1-FEH2 and 1-kestotriose are discussed.

A cell wall invertase controls nectar volume and sugar composition

ABSTRACTFlowering plants produce nectar to attract pollinators. The main nectar sugars are sucrose, glucose, and fructose, which can vary widely in ratio and concentration across species.Brassicaspp. produce a hexose-dominant nectar (high in the monosaccharides glucose and fructose) with very low levels of the disaccharide sucrose. Cell wall invertases (CWINVs) catalyze the irreversible hydrolysis of sucrose into glucose and fructose in the apoplast. We found thatBrCWINV4Ais highly expressed in the nectaries ofBrassica rapa. Moreover, abrcwinv4anull mutant has (1) greatly reduced cell wall invertase activity in the nectaries, and (2) produces a sucrose-rich nectar with little hexose content, but (3) with significantly less volume. These results were recapitulated via exogenous application of an invertase inhibitor to wild-type flowers. Honeybees prefer nectars with some sucrose, but wild-typeB. rapaflowers were much more heavily visited than those ofbrcwinv4a, suggesting that the potentially attractive sucrose-rich nectar ofbrcwinv4acould not compensate for its low volume. These results cumulatively indicate that BrCWINV4A is not only essential for producing a hexose-rich nectar, but also support a model of nectar secretion in which its hydrolase activity is required for maintaining a high intracellular-to-extracellular sucrose ratio that facilitates the continuous export of sucrose into the apoplast via SWEET9. Extracellular hydrolysis of each sucrose into two hexoses byBrCWINV4Aalso likely creates the osmotic potential required for nectar droplet formation. In summary, modulation of CWINV activity can at least partially account for naturally occurring differences in nectar volume and sugar composition.