Invertase Inhibitor Research Articles

CRISPR-Cas9-Mediated Reduced Expression of Potato Apoplastic Invertase Inhibitor Gene and Analysis of Transformation Efficiency Parameters

CRISPR-Cas9-Mediated Reduced Expression of Potato Apoplastic Invertase Inhibitor Gene and Analysis of Transformation Efficiency Parameters

Phenotypic and transcriptomics characterization uncovers genes underlying tuber yield traits and gene expression marker development in potato under aeroponics.

Transcriptome analysis in potato varieties revealed genes associated with tuber yield-related traits and developed gene expression markers. This study aimed to identify genes involved in high tuber yield and its component traits in test potato varieties (Kufri Frysona, Kufri Khyati, and Kufri Mohan) compared to control (Kufri Sutlej). The aeroponic evaluation showed significant differences in yield-related traits in the varieties. Total RNA sequencing was performed using tuber and leaf tissues on the Illumina platform. The high-quality reads (QV > 25) mapping with the reference potato genomes revealed statistically significant (P < 0.05) differentially expressed genes (DEGs) into two categories: up-regulated (> 2 Log2 fold change) and down-regulated (< -2 Log2 fold change). DEGs were characterized by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Collectively, we identified genes participating in sugar metabolism, stress response, transcription factors, phytohormones, kinase proteins, and other genes greatly affecting tuber yield and its related traits. A few selected genes were UDP-glucose glucosyltransferase, glutathion S-transferase, GDSL esterase/lipase, transcription factors (MYB, WRKY, bHLH63, and BURP), phytohormones (auxin-induced protein X10A, and GA20 oxidase), kinase proteins (Kunitz-type tuber invertase inhibitor, BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1) and laccase. Based on the selected 17 peptide sequences representing 13 genes, a phylogeny tree and motifs were analyzed. Real time-quantitative polymerase chain reaction (RT-qPCR) analysis was used to validate the RNA-seq results. RT-qPCR based gene expression markers were developed for the genes such as 101kDa heat shock protein, catechol oxidase B chloroplastic, cysteine protease inhibitor 1, Kunitz-type tuber invertase inhibitor, and laccase to identify high yielding potato genotypes. Thus, our study paved the path for potential genes associated with tuber yield traits in potato under aeroponics.

LcINH1 as an inhibitor of cell wall invertase LcCWIN5 regulates early seed development in Litchi chinensis Sonn

Sugar signal mediated by Cell wall invertase (CWIN) plays a central role in seed development. In higher plants, invertase inhibitors (INHs) suppress CWIN activities at a post-translational level. In Litchi chinensis cultivar ‘Nuomici’, impaired CWIN expression is associated with seed abortion. Here, the expression of LcINH1 was significantly higher in the funicle of seed-aborting cultivar ‘Nuomici’ than big-seeded cultivar ‘Heiye’. Promoter analyses found LcINH1 contained a 404 bp repeat fragment with an endosperm regulatory element of Skn-1_motif. LcINH1 and LcCWIN2/5 were located in plasma membrane. LcINH1 was able to interact with LcCWIN5, but not with LcCWIN2. In vitro enzyme activity assay demonstrated that LcINH1 could inhibit CWIN activity. Silencing LcINH1 in ‘Nuomici’ resulted in normal seed development, paralleled increased CWIN activities and glucose levels. Transcriptome analysis identified 1079 differentially expressed genes (DEGs) in LcINH1-silenced fruits. KEGG analysis showed significant enrichment of DEGs in pathways related to transporters and plant hormone signal transduction. Weighted gene co-expression network analysis indicated that the turquoise module was highly correlated with fructose content, and LcSWEET3b was closely associated with early seed development. These findings suggest that LcINH1 regulate LcCWIN5 activity at the post-translational level to alter sucrose metabolism, thereby affecting early seed development in litchi.

Differential gene expression in irradiated potato tubers contributed to sprout inhibition and quality retention during a commercial scale storage

Current study is the first ever storage cum market trial of radiation processed (28 tons) of potato conducted in India at a commercial scale. The objective was to affirm the efficacy of very low dose of gamma radiation processing of potato for extended storage with retained quality and to understand the plausible mechanism at the gene modulation level for suppression of potato sprouting. Genes pertaining to abscisic acid (ABA) biosynthesis were upregulated whereas its catabolism was downregulated in irradiated potatoes. Additionally, genes related to auxin buildup were downregulated in irradiated potatoes. The change in the endogenous phytohormone contents in irradiated potato with respect to the control were found to be correlated well with the differential expression level of certain related genes. Irradiated potatoes showed retention of processing attributes including cooking and chip-making qualities, which could be attributed to the elevated expression of invertase inhibitor in these tubers. Further, quality retention in radiation treated potatoes may also be related to inhibition in the physiological changes due to sprout inhibition. Ecological and economical analysis of national and global data showed that successful adoption of radiation processing may gradually replace sprout suppressants like isopropyl N-(3-chlorophenyl) carbamate (CIPC), known to leave residue in the commodity, stabilize the wholesale annual market price, and provide a boost to the industries involved in product manufacturing.

Genome-Wide Identification and Expression Patterns of Cucumber Invertases and Their Inhibitor Genes.

Invertases and their inhibitors play important roles in sucrose metabolism, growth and development, signal transduction, and biotic and abiotic stress tolerance in many plant species. However, in cucumber, both the gene members and functions of invertase and its inhibitor families remain largely unclear. In this study, in comparison with the orthologues of Citrullus lanatus (watermelon), Cucumis melo (melon), and Arabidopsis thaliana (Arabidopsis), 12 invertase genes and 12 invertase inhibitor genes were identified from the genome of Cucumis sativus (cucumber). Among them, the 12 invertase genes were classified as 4 cell wall invertases, 6 cytoplasmic invertases, and 2 vacuolar invertases. Most invertase genes were conserved in cucumber, melon, and watermelon, with several duplicate genes in melon and watermelon. Transcriptome analysis distinguished these genes into various expression patterns, which included genes CsaV3_2G025540 and CsaV3_2G007220, which were significantly expressed in different tissues, organs, and development stages, and genes CsaV3_7G034730 and CsaV3_5G005910, which might be involved in biotic and abiotic stress. Six genes were further validated in cucumber based on quantitative real-time PCR (qRT-PCR), and three of them showed consistent expression patterns as revealed in the transcriptome. These results provide important information for further studies on the physiological functions of cucumber invertases (CSINVs) and their inhibitors (CSINHs).

IbInvInh2, a novel invertase inhibitor in sweet potato, regulates starch content through post-translational regulation of vacuolar invertase IbβFRUCT2

As a key enzyme in the starch and sugar metabolic pathways in sweet potato (Ipomoea batatas (L.) Lam.), the vacuolar invertase (EC 3.2.1.26) IbβFRUCT2 is involved in partitioning and modulating the starch and sugar components of the storage root. However, the post-translational regulation of its invertase activity remains unclear. In this study, we identified three invertase inhibitors, IbInvInh1, IbInvInh2, and IbInvInh3, as potential interaction partners of IbβFRUCT2. All were found to act as vacuolar invertase inhibitors (VIFs) and belonged to the plant invertase/pectin methyl esterase inhibitor superfamily. Among the three VIFs, IbInvInh2 is a novel VIF in sweet potato and was confirmed to be an inhibitor of IbβFRUCT2. The N-terminal domain of IbβFRUCT2 and the Thr39 and Leu198 sites of IbInvInh2 were predicted to be engaged in their interactions. The transgenic expression of IbInvInh2 in Arabidopsis thaliana plants reduced the starch content of leaves, while its expression in the Ibβfruct2-expressing Arabidopsis plants increased the starch content of leaves, suggesting that the post-translational inhibition of IbβFRUCT2 activity by IbInvInh2 contributes to the regulation of the plant starch content. Taken together, our findings reveal a novel VIF in sweet potato and provide insights into the potential regulatory roles of the VIFs and invertase–VIF interaction in starch metabolism. These insights lay the foundation for using VIFs to improve the starch properties of crops.

Genotype-Specific Features of Cold-Induced Sweetening Process Regulation in Potato Varieties Nikulinsky, Symphony, Nevski

Increasing the shelf life of potato tubers demands their storage under low temperature. However, storage at low temperatures causes activation of cold-protective mechanism, so-called cold-induced sweetening (CIS). During this process, the starch is converted into reducing sugars such as glucose and fructose. Upon processing at high-temperature, these sugars react with free amino acids, resulting in brown, bitter-tasting products, which reduces the consumer value of potatoes. In this study, we performed expression analysis of genes associated with cold-induced sweetening in potato tubers: vacuolar invertase (Pain-1), sucrose-synthase (SUS4), invertase inhibitor (InvInh2). Potato varieties Nikulinsky, Symphony, Nevski were used. All three varieties were found to accumulate sugars at low temperatures, the maximum accumulation of reducing sugars being observed at 4°C. It was found that the expression pattern of genes associated with cold-induced sweetening differs depending on the variety and storage duration. The increased expression of vacuolar invertase and its inhibitor is more pronounced at the beginning of storage period while the increased expression of sucrose synthase is more pronounced after three months of storage. At early storage periods, high expression of invertase and low expression of inhibitor is observed in Dutch variety Symphony, and vice versa in Russian varieties Nikulinsky and Nevsky. The participation of the studied genes in the process of cold-induced sweetening is discussed.

Genotype-Specific Features of Cold-Induced Sweetening Process Regulation in Potato Varieties Nikulinsky, Symfonia, and Nevsky

In this study, we performed expression analysis of genes associated with cold-induced sweetening in potato tubers: vacuolar invertase (Pain-1), sucrose synthase (SUS4), and invertase inhibitor (InvInh2). Potato varieties Nikulinsky, Symfonia, and Nevsky were used. All three varieties were found to accumulate sugars at low temperatures; the maximum accumulation of reducing sugars was observed at 4°C. It was found that the expression pattern of genes associated with cold-induced sweetening differs depending on the variety and storage duration. The increased expression of vacuolar invertase and its inhibitor is more pronounced at the beginning of storage period, whereas the increased expression of sucrose synthase is more pronounced after 3 months of storage. At early storage periods, high expression of invertase and low expression of inhibitor is observed in the Dutch variety Symfonia, and vice versa in the Russian varieties Nikulinsky and Nevsky. The involvement of the studied genes in the process of cold-induced sweetening is discussed.

Two vacuolar invertase inhibitors PpINHa and PpINH3 display opposite effects on fruit sugar accumulation in peach.

Soluble sugars are an important determinant of fruit taste, but their accumulation mechanisms remain elusive. In this study, we report two vacuolar invertase inhibitor genes involved in sugar accumulation in peach, PpINHa and PpINH3. Transient overexpression of PpINH3 in peach fruits resulted in an increase in sugar content, while the opposite trend was detected for PpINHa. Unexpectedly, PpINH3 and PpINHa both had no physical interaction with vacuolar invertase (VIN). Moreover, the PpVIN genes had no or extremely low expression in fruits at the ripening stage. These results suggested that the regulatory role of PpINHa and PpINH3 in sugar accumulation is unlikely due to their interaction with PpVINs. Additionally, overexpression of PpINHa and PpINH3 had an impact on transcription of genes related to fruit sugar metabolism and transport, which is likely responsible for their regulatory role in fruit sugar accumulation. Altogether, these results indicated an important role of PpINHs in fruit accumulation in peach. Our study provides new insights into molecular mechanisms underlying sugar accumulation, which could be useful for genetic improvement of fruit taste in breeding programs of peach and other fruit crops.

Characterization of invertase inhibitors (InvInhs) in tea plant, and their potential roles in participating in growth, development and cold response

Invertase inhibitor (InvInh) represses acid invertase (AI) activity though interacting with AI at post-translation level, which plays important roles in vegetative and reproductive processes and stress responses of plant. Our previous studies revealed that CsAI genes were involved in regulating cold response of tea plant, while the post-translation regulation mechanism of AI and the roles of InvInh in tea plant have not been reported yet. Herein, we identified 4 CsInvInhs, including 2 tandem repeat genes (CsInvInh1/2) and 2 segmental duplication genes (CsInvInh3/4), from three sequenced tea plant genomes, and further purified CsInvInh1/2 proteins. Bioinformatics analysis results showed that both of CsInvInh1/2 were located on chromosome (Chr) 5, while CsInvInh3/4 were respectively located on Chr9 and Chr12, which was consistent with the results of inter-species collinearity analysis. Under various abiotic stress conditions, the transcription levels of CsInvInhs were induced with different extent at different treatment time points. In addition, all of CsInvInhs differentially expressed among different tea plant cultivars under drought stress condition. Moreover, CsInvInh1/2 shared similar expression patterns in different tea plant tissues, and under cold stress or cold acclimation conditions, suggesting that CsInvInh1/2 may have functional redundancy in tea plant. Correlation analysis results showed that the changes of CWIN and VIN activities were not significantly correlated with the expression patterns of 5 CsAI genes, while negatively correlated with the expression patterns of 3 CsInvInhs in different tea plant tissues, and under cold stress or cold acclimation conditions, indicating that CsInvInhs participate in growth, development and cold response of tea plant mainly through inhibiting AI activity. These results will provide theoretical foundation for the cold-resistant molecular mechanism of tea plant, and also provide new genetic resources and approaches for cold-resistant molecular breeding of tea plant.

Functional characterization of a cell wall invertase inhibitor StInvInh1 revealed its involvement in potato microtuber size in vitro.

Cell wall invertase (CWI) is as an essential coordinator in carbohydrate partitioning and sink strength determination, thereby playing key roles in plant development. Emerging evidence revealed that the subtle regulation of CWI activity considerably depends on the post-translational mechanism by their inhibitors (INHs). In our previous research, two putative INHs (StInvInh1 and StInvInh3) were expected as targets of CWI in potato (Solanum tubersum), a model species of tuberous plants. Here, transcript analysis revealed that StInvInh1 showed an overall higher expression than StInhInh3 in all tested organs. Then, StInvInh1 was further selected to study. In accordance with this, the activity of StInvInh1 promoter increased with the development of leaves in plantlets but decreased with the development of microtubers in vitro and mainly appeared in vascular bundle. The recombinant protein StInvInh1 displayed inhibitory activities on the extracted CWI in vitro and StInvInh1 interacted with a CWI StcwINV2 in vivo by bimolecular fluorescence complementation. Furthermore, silencing StInvInh1 in potato dramatically increased the CWI activity without changing activities of vacuolar and cytoplasmic invertase, indicating that StInvInh1 functions as a typical INH of CWI. Releasing CWI activity in StInvInh1 RNA interference transgenic potato led to improvements in potato microtuber size in coordination with higher accumulations of dry matter in vitro. Taken together, these findings demonstrate that StInvInh1 encodes an INH of CWI and regulates the microtuber development process through fine-tuning apoplastic sucrose metabolism, which may provide new insights into tuber development.

Genome-Wide Analysis of the RNase T2 Family and Identification of Interacting Proteins of Four ClS-RNase Genes in 'XiangShui' Lemon.

S-RNase plays vital roles in the process of self-incompatibility (SI) in Rutaceae plants. Data have shown that the rejection phenomenon during self-pollination is due to the degradation of pollen tube RNA by S-RNase. The cytoskeleton microfilaments of pollen tubes are destroyed, and other components cannot extend downwards from the stigma and, ultimately, cannot reach the ovary to complete fertilisation. In this study, four S-RNase gene sequences were identified from the ‘XiangShui’ lemon genome and ubiquitome. Sequence analysis revealed that the conserved RNase T2 domains within S-RNases in ‘XiangShui’ lemon are the same as those within other species. Expression pattern analysis revealed that S3-RNase and S4-RNase are specifically expressed in the pistils, and spatiotemporal expression analysis showed that the S3-RNase expression levels in the stigmas, styles and ovaries were significantly higher after self-pollination than after cross-pollination. Subcellular localisation analysis showed that the S1-RNase, S2-RNase, S3-RNase and S4-RNase were found to be expressed in the nucleus according to laser confocal microscopy. In addition, yeast two-hybrid (Y2H) assays showed that S3-RNase interacted with F-box, Bifunctional fucokinase/fucose pyrophosphorylase (FKGP), aspartic proteinase A1, RRP46, pectinesterase/pectinesterase inhibitor 51 (PME51), phospholipid:diacylglycerol acyltransferase 1 (PDAT1), gibberellin receptor GID1B, GDT1-like protein 4, putative invertase inhibitor, tRNA ligase, PAP15, PAE8, TIM14-2, PGIP1 and p24beta2. Moreover, S3-RNase interacted with TOPP4. Therefore, S3-RNase may play an important role in the SI of ‘XiangShui’ lemon.

Enzymative activity of technogenic surface formations of Kuzbass

The coal mining is one of the leading industries in the global energy balance. Kuzbass is the main region of Russia that specializes in coal mining. About 60 % of the country's coal is mined there. Coal mining is carried out mainly by the open-pit method. As a result, some 178 thousand hectares ha of disturbed land are formed. Enzymatic activity is an indicator of soil self-repair. The aim of the work was to study the enzymatic activity of technogenic surface formations of the Kuzbass to assess their toxicity and further selection of destructor microorganisms, rhizobacteria and hyperaccumulator plants, which will be further used at the biological stage of recultivation. As objects of research, the samples of technogenic surface formations taken on the territory of the Barzassky and Mokhovsky coal dumps. Enzymatic activity of technogenic surface formations of dumps was: invertase – 2,24 and 2,12 mg of sucrose split 1 g soil in 1 h; nitrite reductase – 0,57 and 0,07 mg reduced NO2- per 1 g soil in 24 h; asparaginase – 71,22 and 60,63 mg NH3 per 1 g soil in 24 h, respectively. When studying the enzymatic activity, it was assumed that the native microflora uses low- and high-molecular hydrocarbons (alkanes, polycyclic aromatic hydrocarbons (PAH), etc.) as carbon sources. The content of mobile forms of heavy metals (HM) in the studied samples exceeds the maximum allowable concentration by 1,2-2,6 times. In the course of statistical analysis, it was revealed that the gross and mobile forms of zinc and copper are nitrite reductase inhibitors, the gross and mobile form of nickel is an asparaginase activator, in technogenic disturbed formations of the studied coal dumps. Nickel is also an invertase inhibitor in the Mokhovsky coal dump.

The transcription factor StTINY3 enhances cold-induced sweetening resistance by coordinating starch resynthesis and sucrose hydrolysis in potato.

The accumulation of reducing sugars in cold-stored tubers, known as cold-induced sweetening (CIS), negatively affects potato processing quality. The starch to sugar interconversion pathways that are altered in cold-stored CIS tubers have been elucidated, but the mechanism that regulates them remains largely unknown. This study identified a CBF/DREB transcription factor (StTINY3) that enhances CIS resistance by both activating starch biosynthesis and repressing the hydrolysis of sucrose to reducing sugars in detached cold-stored tubers. Silencing StTINY3 in a CIS-resistant genotype decreased CIS resistance, while overexpressing StTINY3 in a CIS-sensitive genotype increased CIS resistance, and altering StTINY3 expression was associated with expression changes in starch resynthesis-related genes. We showed first that overexpressing StTINY3 inhibited sucrose hydrolysis by enhancing expression of the invertase inhibitor gene StInvInh2, and second that StTINY3 promoted starch resynthesis by up-regulating a large subunit of the ADP-glucose pyrophosphorylase gene StAGPaseL3, and the glucose-6-phosphate transporter gene StG6PT2. Using electrophoretic mobility shift assays, we revealed that StTINY3 is a nuclear-localized transcriptional activator that directly binds to the dehydration-responsive element/CRT cis-element in the promoters of StInvInh2 and StAGPaseL3. Taken together, these findings established that StTINY3 influences CIS resistance in cold-stored tubers by coordinately modulating the starch to sugar interconversion pathways and is a good target for improving potato processing quality.

The identification of type I MADS box genes as the upstream activators of an endosperm-specific invertase inhibitor in Arabidopsis

BackgroundNuclear endosperm development is a common mechanism among Angiosperms, including Arabidopsis. During nuclear development, the endosperm nuclei divide rapidly after fertilization without cytokinesis to enter the syncytial phase, which is then followed by the cellularized phase. The endosperm can be divided into three spatial domains with distinct functions: the micropylar, peripheral, and chalazal domains. Previously, we identified two putative small invertase inhibitors, InvINH1 and InvINH2, that are specifically expressed in the micropylar region of the syncytial endosperm. In addition, ectopically expressing InvINH1 in the cellularized endosperm led to a reduction in embryo growth rate. However, it is not clear what are the upstream regulators responsible for the specific expression of InvINHs in the syncytial endosperm.ResultsUsing protoplast transient expression system, we discovered that a group of type I MADS box transcription factors can form dimers to activate InvINH1 promoter. Promoter deletion assays carried out in the protoplast system revealed the presence of an enhancer region in InvINH1 promoter, which contains several consensus cis-elements for the MADS box proteins. Using promoter deletion assay in planta, we further demonstrated that this enhancer region is required for InvINH1 expression in the syncytial endosperm. One of the MADS box genes, AGL62, is a key transcription factor required for syncytial endosperm development. Using promoter-GFP reporter assay, we demonstrated that InvINH1 and InvINH2 are not expressed in agl62 mutant seeds. Collectively, our data supports the role of AGL62 and other type I MADS box genes as the upstream activators of InvINHs expression in the syncytial endosperm.ConclusionsOur findings revealed several type I MADS box genes that are responsible for activating InvINH1 in the syncytial endosperm, which in turn regulates embryo growth rate during early stage of seed development.

A Vacuolar Invertase CsVI2 Regulates Sucrose Metabolism and Increases Drought Tolerance in Cucumis sativus L.

Vacuolar invertase (VI) can irreversibly degrade sucrose into glucose and fructose and involve in plants abiotic-stress-tolerance. Cucumber (Cucumis sativus L.) is susceptible to drought stress, especially during the seedling stage. To date, the involvement of VI in drought tolerance in cucumber seedlings is in urgent need of exploration. In the present study, a cucumber vacuolar invertase gene, CsVI2, was isolated and functionally characterized. The results showed that (1) CsVI2 showed vacuolar invertase activity both in vivo and in vitro; (2) the transcript level of CsVI2, along with VI activity, was significantly induced by drought stress. Moreover, the expression of sucrose synthase 3 (CsSUS3) was increased and that of sucrose phosphate synthase 1 (CsSPS1) was decreased after exposure to drought stress, which was followed by an increase in sucrose synthase activity and a decrease in sucrose phosphate synthase activity; (3) CsVI2-overexpressing transformed cucumber seedlings showed enhanced vacuolar invertase activity and drought tolerance and 4) protein–protein interaction modelling indicated that a cucumber invertase inhibitor, CsINVINH3, can interact with CsVI2. In summary, the results indicate that CsVI2 as an invertase can regulate sucrose metabolism and enhance drought stress in cucumber seedlings.

Role of alkaline/neutral invertases in postharvest storage of potato

Potatoes stored at cold temperatures undergo undesirable sweetening due to the cleavage of sucrose into reducing sugars by invertases. Although significant research has focused on vacuolar invertase (StVAC) during cold storage of potatoes, little is known about the role of alkaline/neutral invertases (StNI). We identified eight members in the NI family from the potato genome, and phylogenetic analysis suggested their probable subcellular location as plastid/mitochondria (group α) or cytosol (group β). Using three commercially important potato cultivars with known differences in cold storage responses (‘Russet Burbank’, ‘Bintje’, and ‘Shepody’), we compared the expression of the eight StNI genes to StVAC and vacuolar invertase inhibitor (StINH2) genes during harvest, and after one and five months of storage at 4 °C or 22 °C. The activity of neutral invertase (NI), soluble acid invertase activity (SAI), and the levels of reducing sugars were also determined. Tubers of ‘Russet Burbank’ displayed high transcript abundance of StNI5, high reducing sugar content and more NI activity than SAI activity. This was associated with a high level of StINH2 transcripts and low StVAC transcript levels suggesting the susceptibility of these tubers to cold-induced sweetening could be due to cytoplasmic cleavage of sucrose by NI. In ‘Bintje’, the induction of StNI5 and StVAC transcripts and NI and SAI activities indicate that sucrose degradation is carried out by both NI and SAI. In ‘Shepody’, a higher abundance of the StNI4 and StNI6 transcripts indicates different isoforms of NI are required for sucrose degradation. This study reveals significant contribution of NI in the regulation of reducing sugars and the process of cold-induced sweetening. Additional studies are needed to determine the in vivo function of NI in cold-stored potato tubers.

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.

Functional Characterization of a Cucumber (Cucumis sativus L.) Vacuolar Invertase, CsVI1, Involved in Hexose Accumulation and Response to Low Temperature Stress.

Cucumber (Cucumis sativus L.), an important vegetable plant species, is susceptible to low temperature stress especially during the seedling stage. Vacuolar invertase (VI) plays important roles in plant responses to abiotic stress. However, the molecular and biochemical mechanisms of VI function in cucumber, have not yet been completely understood and VI responses to low temperature stress and it functions in cold tolerance in cucumber seedlings are also in need of exploration. The present study found that hexose accumulation in the roots of cucumber seedlings under low temperature stress is closely related to the observed enhancement of invertase activity. Our genome-wide search for the vacuolar invertase (VI) genes in cucumber identified the candidate VI-encoding gene CsVI1. Expression profiling of CsVI1 showed that it was mainly expressed in the young roots of cucumber seedlings. In addition, transcriptional analysis indicated that CsVI1 expression could respond to low temperature stress. Recombinant CsVI1 proteins purified from Pichia pastoris and Nicotiana benthamiana leaves could hydrolyze sucrose into hexoses. Further, overexpression of CsVI1 in cucumber plants could increase their hexose contents and improve their low temperature tolerance. Lastly, a putative cucumber invertase inhibitor was found could form a complex with CsVI1. In summary, these results confirmed that CsVI1 functions as an acid invertase involved in hexose accumulation and responds to low temperature stress in cucumber seedlings.

StRAP2.3, an ERF\u2010VII 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.