Reversible assembly and disassembly of V-ATPase during the lysosome regeneration cycle.

Regulation of the luminal pH of late endocytic compartments in continuously fed mammalian cells is poorly understood. Using normal rat kidney fibroblasts, we investigated the reversible assembly/disassembly of the proton pumping V-ATPase when endolysosomes are formed by kissing and fusion of late endosomes with lysosomes and during the subsequent reformation of lysosomes. We took advantage of previous work showing that sucrosomes formed by the uptake of sucrose are swollen endolysosomes from which lysosomes are reformed after uptake of invertase. Using confocal microscopy and subcellular fractionation of NRK cells stably expressing fluorescently tagged proteins, we found net recruitment of the V1 subcomplex during sucrosome formation and loss during lysosome reformation, with a similar time course to RAB7a loss. Addition of invertase did not alter mTORC1 signalling, suggesting that the regulation of reversible V-ATPase assembly/disassembly in continuously fed cells differs from that in cells subject to amino acid depletion/refeeding. Using live cell microscopy, we demonstrated recruitment of a fluorescently tagged V1 subunit during endolysosome formation and a dynamic equilibrium and rapid exchange between the cytosolic and membrane bound pools of this subunit. We conclude that reversible V-ATPase assembly/disassembly plays a key role in regulating endolysosomal/lysosomal pH in continuously fed cells.

The Complex GH32 Enzyme Orchestra from Priestia megaterium Holds the Key to Better Discriminate Sucrose-6-phosphate Hydrolases from Other ß-Fructofuranosidases in Bacteria.

Inulin is widely used as a prebiotic and emerging as a priming compound to counteract plant diseases. We isolated inulin-degrading strains from the lettuce phyllosphere, identified as Bacillus subtilis and Priestia megaterium, species hosting well-known biocontrol organisms. To better understand their varying inulin degradation strategies, three intracellular ß-fructofuranosidases from P. megaterium NBRC15308 were characterized after expression in Escherichia coli: a predicted sucrose-6-phosphate (Suc6P) hydrolase (SacAP1, supported by molecular docking), an exofructanase (SacAP2), and an invertase (SacAP3). Based on protein multiple sequence and structure alignments of bacterial glycoside hydrolase family 32 enzymes, we identified conserved residues predicted to be involved in binding phosphorylated (Suc6P hydrolases) or nonphosphorylated substrates (invertases and fructanases). Suc6P hydrolases feature positively charged residues near the structural catalytic pocket (histidine, arginine, or lysine), whereas other ß-fructofuranosidases contain tryptophans. This correlates with our phylogenetic tree, grouping all predicted Suc6P hydrolases in a clan associated with genomic regions coding for transporters involved in substrate phosphorylation. These results will help to discriminate between Suc6P hydrolases and other ß-fructofuranosidases in future studies and to better understand the interaction of B. subtilis and P. megaterium endophytes with sucrose and/or fructans, sugars naturally present in plants or exogenously applied in the context of defense priming.

A review of fructosyl-transferases from catalytic characteristics and structural features to reaction mechanisms and product specificity.

Carbohydrate-active enzymes are accountable for the synthesis and degradation of glycosidic bonds among diverse carbohydrates. Fructosyl-transferases represent a subclass of these enzymes, employing sucrose as a substrate to generate fructooligosaccharides (FOS) and fructan polymers. This category primarily includes levansucrase (LS, EC 2.4.1.10), inulosucrase (IS, EC 2.4.1.9), and ß-fructofuranosidase (Ffase, EC 3.2.1.26). These three enzymes possess a similar five-bladed ß-propeller fold and employ an anomer-retaining reaction mechanism mediated by nucleophiles, transition state stabilizers, and general acids/bases. However, they exhibit distinct product profiles, characterized by variations in linkage specificity and molecular mass distribution. Consequently, this article comprehensively explores recent advancements in the catalytic characteristics, structural features, reaction mechanisms, and product specificity of levansucrase, inulosucrase, and ß-fructofuranosidase (abbreviated as LS, IS, and Ffase, respectively). Furthermore, it discusses the potential for modifying catalytic properties and product specificity through structure-based design, which enables the rational production of custom fructan and FOS.

The potential roles of acid invertase family in Dendrobium huoshanense: Identification, evolution, and expression analyses under abiotic stress.

Dendrobium huoshanense, a traditional Chinese medicine prized for its horticultural and medicinal properties, thrives in an unfavorable climate and is exposed to several adverse environmental conditions. Acid invertase (AINV), a widely distributed enzyme that has been demonstrated to play a significant role in response to environmental stresses. However, the identification of the AINV gene family in D. huoshanense, the collinearity between relative species, and the expression pattern under external stress have yet to be resolved. We systematically retrieved the D. huoshanense genome and screened out four DhAINV genes, which were further classified into two subfamilies by the phylogenetic analysis. The evolutionary history of AINV genes in D. huoshanense was uncovered by comparative genomics investigations. The subcellular localization predicted that the DhVINV genes may be located in the vacuole, while the DhCWINV genes may be located in the cell wall. The exon/intron structures and conserved motifs of DhAINV genes were found to be highly conserved in two subclades. The conserved amino acids and catalytic motifs in DhAINV proteins were determined to be critical to their function. Notably, the cis-acting elements in all DhAINV genes were mainly relevant to abiotic stresses and light response. In addition, the expression profile coupled with qRT-PCR revealed the typical expression patterns of DhAINV in response to diverse abiotic stresses. Our findings could be beneficial to the characterization and further investigation of AINV functions in Dendrobium plants.

Molecular physiology for the increase of soluble sugar accumulation in citrus fruits under drought stress.

To investigate the mechanism for drought promoting soluble sugar accumulation will be conducive to the enhancement of citrus fruit quality as well as stress tolerance. Fruit sucrose mainly derives from source leaves. Its accumulation in citrus fruit cell vacuole involves in two processes of unloading in the fruit segment membrane (SM) and translocating to the vacuole of fruit juice sacs (JS). Here, transcript levels of 47 sugar metabolism- and transport-related genes were compared in fruit SM or JS between drought and control treatments. Results indicated that transcript levels of cell wall invertase genes (CwINV2/6) and sucrose synthase genes (SUS2/6) in the SM were significantly increased by the drought. Moreover, transcript levels of SWEET genes (CsSWEET1/2/4/5/9) and monosaccharide transporter gene (CsPMT3) were significantly increased in SM under drought treatment. On the other hand, SUS1/3 and vacuolar invertase (VINV) transcript levels were significantly increased in JS by drought; CsPMT4, sucrose transporter gene 2 (CsSUT2), tonoplast monosaccharide transporter gene 2 (CsTMT2), sugar transport protein gene 1 (CsSTP1), two citrus type I V-PPase genes (CsVPP1, and CsVPP2) were also significantly increased in drought treated JS. Collectively, the imposition of drought stress resulted in more soluble sugar accumulation through enhancing sucrose download by enhancing sink strength- and transport ability-related genes, such as CwINV2/6, SUS2/6, CsSWEET1/2/4/5/9, and CsPMT3, in fruit SM, and soluble sugar storage ability by increasing transcript levels of genes, such as CsPMT4, VINV, CsSUT2, CsTMT2, CsSTP1, CsVPP1, and CsVPP2, in fruit JS.

Multiscale QM/MM Simulations Identify the Roles of Asp239 and 1-OH···Nucleophile in Transition State Stabilization in Arabidopsis thaliana Cell-Wall Invertase 1.

Arabidopsis thaliana cell-wall invertase 1 (AtCWIN1), a key enzyme in sucrose metabolism in plants, catalyzes the hydrolysis of sucrose into fructose and glucose. AtCWIN1 belongs to the glycoside hydrolase GH-J clan, where two carboxylate residues (Asp23 and Glu203 in AtCWIN1) are well documented as a nucleophile and an acid/base catalyst. However, details at the atomic level about the role of neighboring residues and enzyme-substrate interactions during catalysis are not fully understood. Here, quantum mechanical/molecular mechanical (QM/MM) free-energy simulations were carried out to clarify the origin of the observed decreased rates in Asp239Ala, Asp239Asn, and Asp239Phe in AtCWIN1 compared to the wild type and delineate the role of Asp239 in catalysis. The glycosylation and deglycosylation steps were considered in both wild type and mutants. Deglycosylation is predicted to be the rate-determining step in the reaction, with a calculated overall free-energy barrier of 15.9 kcal/mol, consistent with the experimental barrier (15.3 kcal/mol). During the reaction, the -1 furanosyl ring underwent a conformational change corresponding to 3E ↔ [E2]⧧ ↔ 1E according to the nomenclature of saccharide structures along the full catalytic reaction. Asp239 was found to stabilize not only the transition state but also the fructosyl-enzyme intermediate, which explains findings from previous structural and mutagenesis experiments. The 1-OH···nucleophile interaction has been found to provide an important contribution to the transition state stabilization, with a contribution of ∼7 kcal/mol, and affected glycosylation more significantly than deglycosylation. This study provides molecular insights that improve the current understanding of sucrose binding and hydrolysis in members of clan GH-J, which may benefit protein engineering research. Finally, a rationale on the sucrose inhibitor configuration in chicory 1-FEH IIa, proposed a long time ago in the literature, is also provided based on the QM/MM calculations.

Evolution of cytosolic and organellar invertases empowered the colonization and thriving of land plants.

The molecular innovation underpinning efficient carbon and energy metabolism during evolution of land plants remains largely unknown. Invertase-mediated sucrose cleavage into hexoses is central to fuel growth. Why some cytoplasmic invertases (CINs) function in the cytosol, whereas others operate in chloroplasts and mitochondria, is puzzling. We attempted to shed light on this question from an evolutionary perspective. Our analyses indicated that plant CINs originated from a putatively orthologous ancestral gene in cyanobacteria and formed the plastidic CIN (α1 clade) through endosymbiotic gene transfer, while its duplication in algae with a loss of its signal peptide produced the ß clade CINs in the cytosol. The mitochondrial CINs (α2) were derived from duplication of the plastidic CINs and coevolved with vascular plants. Importantly, the copy number of mitochondrial and plastidic CINs increased upon the emergence of seed plants, corresponding with the rise of respiratory, photosynthetic, and growth rates. The cytosolic CIN (ß subfamily) kept expanding from algae to gymnosperm, indicating its role in supporting the increase in carbon use efficiency during evolution. Affinity purification mass spectrometry identified a cohort of proteins interacting with α1 and 2 CINs, which points to their roles in plastid and mitochondrial glycolysis, oxidative stress tolerance, and the maintenance of subcellular sugar homeostasis. Collectively, the findings indicate evolutionary roles of α1 and α2 CINs in chloroplasts and mitochondria for achieving high photosynthetic and respiratory rates, respectively, which, together with the expanding of cytosolic CINs, likely underpin the colonization of land plants through fueling rapid growth and biomass production.

IbInvInh2, a novel invertase inhibitor in sweet potato, regulates starch content through post-translational regulation of vacuolar invertase IbßFRUCT2.

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) IbßFRUCT2 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 IbßFRUCT2. 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 IbßFRUCT2. The N-terminal domain of IbßFRUCT2 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 Ibßfruct2-expressing Arabidopsis plants increased the starch content of leaves, suggesting that the post-translational inhibition of IbßFRUCT2 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, 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.

Intracellular invertase hyperproducing strain of Saccharomyces cerevisiae isolated from Abagboro palm wine.

There is an ever-increasing demand for industrial enzyme, necessitating a constant search for its efficient producers. The isolation and characterization of invertase producer yeasts from natural palm wine is reported in this study. Yeasts were isolated from fresh palm wine obtained from Abagboro community Ile-Ife, Nigeria following standard methods. A total of six yeast strains were isolated from the palm wine. The strains were screened for their ability to produce invertase and the most efficient invertase producer was characterized and identified using phenotypic and molecular methods. Isolate C showed the highest invertase activity (34.15 µmole/ml/min), followed by isolate B (18.070 µmole/ml/min) and isolate A (14.385 µmole/ml/min). The identity of isolate C was confirmed by genotypic methods to be Saccharomyces cerevisiae (OL629078.1 accession number on NCBI database). The Saccharomyces cerevisiae strain fermented galactose, arabinose, maltose, glucose, sucrose and raffinose, grew in 50% and 60% glucose and at 25-35 °C. The newly isolated Saccharomyces cerevisiae strain is an efficient producer of invertase and can be exploited for commercial biosynthesis of the enzyme for use in biotechnological applications.

Genome-wide identification, characterization and evolutionary dynamic of invertase gene family in apple, and revealing its roles in cold tolerance.

Invertases are ubiquitous enzymes that catalyze the unalterable cleavage of sucrose into glucose and fructose, and are crucially involved in plant growth, development and stress response. In this study, a total of 17 putative invertase genes, including 3 cell wall invertases, 3 vacuolar invertases, and 11 neutral invertases were identified in apple genome. Subcellular localization of MdNINV7 and MdNINV11 indicated that both invertases were located in the cytoplasm. Comprehensive analyses of physicochemical properties, chromosomal localization, genomic characterization, and gene evolution of MdINV family were conducted. Gene duplication revealed that whole-genome or segmental duplication and random duplication might have been the major driving force for MdINVs expansion. Selection index values, ω, showed strong evidence of positive selection signatures among the INV clusters. Gene expression analysis indicated that MdNINV1/3/6/7 members are crucially involved in fruit development and sugar accumulation. Similarly, expression profiles of MdCWINV1, MdVINV1, and MdNINV1/2/7/11 suggested their potential roles in response to cold stress. Furthermore, overexpression of MdNINV11 in apple calli at least in part promoted the expression of MdCBF1-5 and H2O2 detoxification in response to cold. Overall, our results will be useful for understanding the functions of MdINVs in the regulation of apple fruit development and cold stress response.

Organ-specific expression of genes associated with the UDP-glucose metabolism in sugarcane (Saccharum spp. hybrids).

BACKGROUND: The importance of uridine 5'-diphosphate glucose (UDP-G) synthesis and degradation on carbon (C) partitioning has been indicated in several studies of plant systems, whereby the kinetic properties and abundance of involved enzymes had a significant effect upon the volume of C moving into the hemicellulose, cellulose and sucrose pools. In this study, the expression of 136 genes belonging to 32 gene families related to UDP-G metabolism was studied in 3 major sugarcane organs (including leaf, internode and root) at 6 different developmental stages in 2 commercial genotypes. RESULTS: Analysis of the genes associated with UDP-G metabolism in leaves indicated low expression of sucrose synthase, but relatively high expression of invertase genes, specifically cell-wall invertase 4 and neutral acid invertase 1-1 and 3 genes. Further, organs that are primarily responsible for sucrose synthesis or bioaccumulation, i.e., in source organs (mature leaves) and storage sink organs (mature internodes), had very low expression of sucrose, cellulose and hemicellulose synthesis genes, specifically sucrose synthase 1 and 2, UDP-G dehydrogenase 5 and several cellulose synthase subunit genes. Gene expression was mostly very low in both leaf and mature internode samples; however, leaves did have a comparatively heightened invertase and sucrose phosphate synthase expression. Major differences were observed in the transcription of several genes between immature sink organs (roots and immature internodes). Gene transcription favoured utilisation of UDP-G toward insoluble and respiratory pools in roots. Whereas, there was comparatively higher expression of sucrose synthetic genes, sucrose phosphate synthase 1 and 4, and comparatively lower expression of many genes associated with C flow to insoluble and respiratory pools including myo-Inositol oxygenase, UDP-G dehydrogenase 4, vacuolar invertase 1, and several cell-wall invertases in immature internodes. CONCLUSION: This study represents the first effort to quantify the expression of gene families associated with UDP-G metabolism in sugarcane. Transcriptional analysis displayed the likelihood that C partitioning in sugarcane is closely related to the transcription of genes associated with the UDP-G metabolism. The data presented may provide an accurate genetic reference for future efforts in altering UDP-G metabolism and in turn C partitioning in sugarcane.

Protein engineering of invertase for enhancing yeast dough fermentation under high-sucrose conditions.

During yeast dough fermentation, such as the high-sucrose bread-making process, the yeast cells are subjected to considerable osmotic stress, resulting in poor outcomes. Invertase is important for catalyzing the irreversible hydrolysis of sucrose to free glucose and fructose, and decreasing the catalytic activity of the invertase may reduce the glucose osmotic stress on the yeast. In this study, we performed structural design and site-directed mutagenesis (SDM) on the Saccharomyces cerevisiae invertase (ScInV) in an Escherichia coli expression system to study the catalytic activity of ScInV mutants in vitro. In addition, we generated the same mutation sites in the yeast endogenous genome and tested their invertase activity in yeast and dough fermentation ability. Our results indicated that appropriately reduced invertase activity of yeast ScInV can enhance dough fermentation activity under high-sucrose conditions by 52%. Our systems have greatly accelerated the engineering of yeast endogenous enzymes both in vitro and in yeast, and shed light on future metabolic engineering of yeast.

Biosynthesis of lactosucrose by a new source of ß-fructofuranosidase from Bacillus methanolicus LB-1.

Lactosucrose (LS) is a prebiotic trisaccharide enzymatically synthesized by transglycosylation from lactose and sucrose with beneficial health effect. The ß-fructofuranosidase used for synthesis of LS was produced from Bacillus methanolicus LB-1, which was isolated from traditional rice wine. A maximal yield of 8.63 U/mL of the enzyme was obtained by fermentation with B. methanolicus LB-1 under the optimized conditions: 10 g/L of glucose, 5 g/L of yeast extract, initial medium pH at 7.0, 37 °C, 24 h. The enzyme was purified and identified by ammonium sulfate fractional precipitation, Sephadex G-75 gel filtration chromatography and LC-MS, and SDS-PAGE of the purified enzyme showed a major protein band at 45 kDa. Biosynthesis of LS was performed using the purified ß-fructofuranosidase, and production of LS reached 110 g/L under the optimized reaction conditions: pH at 7.0, 37 °C, 6.0 U/g sucrose of enzyme, 15% of sucrose, 15% of lactose, 28 h. HPLC analysis of the reaction products showed a distinct peak for LS at about 30 min of elution, confirming that B. methanolicus LB-1 ß-fructofuranosidase had effective transfructosylation activity. Therefore, this new microbial source of ß-fructofuranosidase may be a candidate with potential application prospect in biosynthesis of prebiotic LS.

Mepiquat chloride application combined with high plant population density promotes carbon remobilization in the roots of upland cotton.

Carbon reserves in cotton roots can be remobilized to support reproductive growth, thus potentially affecting cotton yield. However, the regulation of carbon remobilization in cotton roots and its relationship with cotton yield are still poorly understood. Plant population density (PPD) and mepiquat chloride (MC) have been hypothesized to affect the dynamics of nonstructural carbohydrate content and the resulting carbon remobilization in cotton roots through the regulation of carbohydrate metabolism enzyme activities. A mid-maturation cotton line 4003-6 was field-grown in 2019 and 2020. Three different levels of PPD (D1: 2.25 plants m-2, D2: 4.5 plants m-2, and D3: 6.75 plants m-2) and two levels of MC dosage (M0: 0 g hm-2, M1: 82.5 g hm-2) were combined to create six populations differing in terms of the source-sink relationship. The changes in the hexose, sucrose, and starch contents and the key carbon metabolic enzyme activities in cotton roots were examined during peak squaring (PS) to late boll opening (LB). The combination of the PPD of 6.75 plants m-2 and MC application (M1D3) exhibited the greatest cotton yield and reproductive biomass-to-leaf area ratio from peak flowering (PF) onwards. M1D3 presented the greatest total nonstructural carbohydrate (TNC) remobilization amount of 2.96 and 3.80 g m-2, the highest efficiency of 39.11% and 48.39%, and the largest gross contribution to seed cotton yield of 0.66% and 0.79% in 2019 and 2020, respectively. The three parameters were positively correlated with the seed cotton yield except for the remobilization rate in 2019. Unlike the other treatments, the greater carbohydrate content per unit ground area in M1D3 prior to the PF stage was attributed to the higher sucrose phosphate synthase (SPS) and ADP-glucose pyrophosphorylase (AGPase) activities during the PS to first flowering (FF) stages. Conversely, the greater α-amylase and ß-amylase activities in M1D3 at the PF stage accounted for the lower starch content at the EB stage, and the smaller vacuolar invertase (VIN) and cell wall invertase (CWIN) activities at the EB stage could be responsible for the lower hexose concentration at that time. The TNC remobilization amount had a positive association with the AGPase and SPS activities at the FF stage and with ß-amylase activity at the PF stage in cotton tap roots in 2019 and 2020. This study provides a cotton yield-improving alternative through the promotion of carbon remobilization in roots using certain agronomic practices.

An alternative pathway to plant cold tolerance in the absence of vacuolar invertase activity.

To cope with cold stress, plants have developed antioxidation strategies combined with osmoprotection by sugars. In potato (Solanum tuberosum) tubers, which are swollen stems, exposure to cold stress induces starch degradation and sucrose synthesis. Vacuolar acid invertase (VInv) activity is a significant part of the cold-induced sweetening (CIS) response, by rapidly cleaving sucrose into hexoses and increasing osmoprotection. To discover alternative plant tissue pathways for coping with cold stress, we produced VInv-knockout lines in two cultivars. Genome editing of VInv in 'Désirée' and 'Brooke' was done using stable and transient expression of CRISPR/Cas9 components, respectively. After storage at 4°C, sugar analysis indicated that the knockout lines showed low levels of CIS and maintained low acid invertase activity in storage. Surprisingly, the tuber parenchyma of vinv lines exhibited significantly reduced lipid peroxidation and reduced H2 O2 levels. Furthermore, whole plants of vinv lines exposed to cold stress without irrigation showed normal vigor, in contrast to WT plants, which wilted. Transcriptome analysis of vinv lines revealed upregulation of an osmoprotectant pathway and ethylene-related genes during cold temperature exposure. Accordingly, higher expression of antioxidant-related genes was detected after exposure to short and long cold storage. Sugar measurements showed an elevation of an alternative pathway in the absence of VInv activity, raising the raffinose pathway with increasing levels of myo-inositol content as a cold tolerance response.

Changes in sucrose metabolism patterns affect the early maturation of Cassava sexual tetraploid roots.

BACKGROUND: Cassava (Manihot esculenta Crantz) is an important multiuse crop grown for economic and energy purposes. Its vegetative organs are storage roots, in which the main storage material is starch. The accumulation characteristics of starch in cassava roots can directly affect the yield, starch content and maturation of cassava storage roots. In this study, we used a cassava sexual tetraploid (ST), which showed early maturation heterosis in previous work, as the main test material. We analyzed the sucrose metabolism and starch accumulation characteristics of the ST and its parents from the leaf "source" to the storage root "sink" during different developmental stages and explored the regulatory mechanisms of ST storage root early maturation by combining the transcriptome data of the storage roots during the expansion period. RESULTS: The results showed that the trends in sucrose, glucose and fructose contents in the ST leaves were similar to those of the two parents during different stages of development, but the trends in the ST storage roots were significantly different from those of their parents, which showed high sucrose utilization rates during the early stage of development and decreased utilization capacity in the late developmental stage. Transcriptome data showed that the genes that were expressed differentially between ST and its parents were mainly involved in the degradation and utilization of sucrose in the storage roots, and four key enzyme genes were significantly upregulated (Invertase MeNINV8/MeVINV3, Sucrose synthase MeSuSy2, Hexokinase MeHXK2), while the expressions of key enzyme genes involved in starch synthesis were not significantly different. CONCLUSIONS: The results revealed that the pattern of sucrose degradation and utilization in the cassava ST was different from that of its parents and promoted early maturation in its tuberous roots. Starch accumulation in the ST from sucrose mainly occurred during the early expansion stage of the storage roots, and the starch content during this period was higher than that of both parents, mainly due to the regulation of invertase and hexokinase activities during sucrose metabolism. This study provides a basis for further genetic improvements to cassava traits and for breeding varieties that mature early and are adapted well to provide starch supply requirements.

Insights into the Structure of the Highly Glycosylated Ffase from Rhodotorula dairenensis Enhance Its Biotechnological Potential.

Rhodotorula dairenensis ß-fructofuranosidase is a highly glycosylated enzyme with broad substrate specificity that catalyzes the synthesis of 6-kestose and a mixture of the three series of fructooligosaccharides (FOS), fructosylating a variety of carbohydrates and other molecules as alditols. We report here its three-dimensional structure, showing the expected bimodular arrangement and also a unique long elongation at its N-terminus containing extensive O-glycosylation sites that form a peculiar arrangement with a protruding loop within the dimer. This region is not required for activity but could provide a molecular tool to target the dimeric protein to its receptor cellular compartment in the yeast. A truncated inactivated form was used to obtain complexes with fructose, sucrose and raffinose, and a Bis-Tris molecule was trapped, mimicking a putative acceptor substrate. The crystal structure of the complexes reveals the major traits of the active site, with Asn387 controlling the substrate binding mode. Relevant residues were selected for mutagenesis, the variants being biochemically characterized through their hydrolytic and transfructosylating activity. All changes decrease the hydrolytic efficiency against sucrose, proving their key role in the activity. Moreover, some of the generated variants exhibit redesigned transfructosylating specificity, which may be used for biotechnological purposes to produce novel fructosyl-derivatives.

CRISPR/Cas-mediated knockdown of vacuolar invertase gene expression lowers the cold-induced sweetening in potatoes.

MAIN CONCLUSION: VInv gene editing in potato using CRISPR/Cas9 resulted in knockdown of expression and a lower VInv enzymatic activity resulting in a decrease in post-harvest cold-storage sugars formation and sweetening in potatoes. CRISPR-Cas9-mediated knockdown of vacuolar invertase (VInv) gene was carried out using two sgRNAs in local cultivar of potato plants. The transformation efficiency of potatoes was found to be 11.7%. The primary transformants were screened through PCR, Sanger sequencing, digital PCR, and ELISA. The overall editing efficacy was determined to be 25.6% as per TIDE analysis. The amplicon sequencing data showed maximum indel frequency for potato plant T12 (14.3%) resulting in 6.2% gene knockout and 6% frame shift. While for plant B4, the maximum indel frequency of 2.0% was found which resulted in 4.4% knockout and 4% frameshift as analyzed by Geneious. The qRT-PCR data revealed that mRNA expression of VInv gene was reduced 90-99-fold in edited potato plants when compared to the non-edited control potato plant. Following cold storage, chips analysis of potatoes proved B4 and T12 as best lines. Reducing sugars' analysis by titration method determined fivefold reduction in percentage of reducing sugars in tubers of B4 transgenic lines as compared to the control. Physiologically genome-edited potatoes behaved like their conventional counterpart. This is first successful report of knockdown of potato VInv gene in Pakistan that addressed cold-induced sweetening resulting in minimum accumulation of reducing sugars in genome edited tubers.

Alterations in Primary Carbon Metabolism in Cucumber Infected with Pseudomonas syringae pv lachrymans: Local and Systemic Responses.

The reconfiguration of the primary metabolism is essential in plant-pathogen interactions. We compared the local metabolic responses of cucumber leaves inoculated with Pseudomonas syringae pv lachrymans (Psl) with those in non-inoculated systemic leaves, by examining the changes in the nicotinamide adenine dinucleotides pools, the concentration of soluble carbohydrates and activities/gene expression of carbohydrate metabolism-related enzymes, the expression of photosynthesis-related genes, and the tricarboxylic acid cycle-linked metabolite contents and enzyme activities. In the infected leaves, Psl induced a metabolic signature with an altered [NAD(P)H]/[NAD(P)+] ratio; decreased glucose and sucrose contents, along with a changed invertase gene expression; and increased glucose turnover and accumulation of raffinose, trehalose, and myo-inositol. The accumulation of oxaloacetic and malic acids, enhanced activities, and gene expression of fumarase and l-malate dehydrogenase, as well as the increased respiration rate in the infected leaves, indicated that Psl induced the tricarboxylic acid cycle. The changes in gene expression of ribulose-l,5-bis-phosphate carboxylase/oxygenase large unit, phosphoenolpyruvate carboxylase and chloroplast glyceraldehyde-3-phosphate dehydrogenase were compatible with a net photosynthesis decline described earlier. Psl triggered metabolic changes common to the infected and non-infected leaves, the dynamics of which differed quantitatively (e.g., malic acid content and metabolism, glucose-6-phosphate accumulation, and glucose-6-phosphate dehydrogenase activity) and those specifically related to the local or systemic response (e.g., changes in the sugar content and turnover). Therefore, metabolic changes in the systemic leaves may be part of the global effects of local infection on the whole-plant metabolism and also represent a specific acclimation response contributing to balancing growth and defense.