Genome-wide identification and characterization of the sucrose invertase gene family in Hemerocallis citrina.

Background: Sucrose invertase is an important catalytic enzyme that is widely distributed in plants and can irreversibly hydrolyze sucrose into fructose and glucose. Daylily is an important perennial flower worldwide and a traditional vegetable in East Asia. Previous studies have suggested that sucrose invertase is involved in the aging of daylily flowers. However, knowledge about the number, physicochemical properties, and expression patterns of daylily sucrose invertases is still lacking. Identifying the daylily sucrose invertase family genes in the genome is highly important for understanding phylogenetic evolution and determining the genetic function of sucrose invertase. Methods: To obtain basic knowledge about the number, classification, sequence composition, and physicochemical properties of sucrose invertases in daylily, bioinformatics software was used to analyze the genome of Hemerocallis citrina (H. citrina), and the basic properties of sucrose invertase genes and proteins were obtained. Then, combined with transcriptome data from flower organs at different developmental stages, the expression patterns of each gene were clarified. Finally, the reliability of the transcriptome data was verified by quantitative real-time polymerase chain reaction (PCR). Results: Through software analysis, 35 sucrose invertases were identified from the H. citrina genome and named HcINV1-HcINV35; these enzymes belong to three subfamilies: cell wall invertases, vacuolar invertases, and chloroplast invertases. The amino acid composition, motif types, promoter composition, gene structure, protein physicochemical properties, gene chromosomal localization, and evolutionary adaptability of daylily invertases were determined; these results provided a comprehensive understanding of daylily invertases. The transcriptome expression profile combined with fluorescence quantitative reverse transcription-polymerase chain reaction (RT‒PCR) analysis suggested that almost all daylily invertase genes were expressed in flower organs, but even genes belonging to the same subfamily did not exhibit the same expression pattern at different developmental stages, suggesting that there may be redundancy or dissimilation in the function of daylily sucrose invertases.

Experimental evolution of yeast shows that public-goods upregulation can evolve despite challenges from exploitative non-producers.

Microbial secretions, such as metabolic enzymes, are often considered to be cooperative public goods as they are costly to produce but can be exploited by others. They create incentives for the evolution of non-producers, which can drive producer and population productivity declines. In response, producers can adjust production levels. Past studies suggest that while producers lower production to reduce costs and exploitation opportunities when under strong selection pressure from non-producers, they overproduce secretions when these pressures are weak. We challenge the universality of this trend with the production of a metabolic enzyme, invertase, by Saccharomyces cerevisiae, which catalyses sucrose hydrolysis into two hexose molecules. Contrary to past studies, overproducers evolve during evolutionary experiments even when under strong selection pressure from non-producers. Phenotypic and competition assays with a collection of synthetic strains - engineered to have modified metabolic attributes - identify two mechanisms for suppressing the benefits of invertase to those who exploit it. Invertase overproduction increases extracellular hexose concentrations that suppresses the metabolic efficiency of competitors, due to the rate-efficiency trade-off, and also enhances overproducers' hexose capture rate by inducing transporter expression. Thus, overproducers are maintained in the environment originally thought to not support public goods production.

Genome-wide analysis of the passion fruit invertase gene family reveals involvement of PeCWINV5 in hexose accumulation.

BACKGROUND: Invertases (INVs) are key enzymes in sugar metabolism, cleaving sucrose into glucose and fructose and playing an important role in plant development and the stress response, however, the INV gene family in passion fruit has not been systematically reported. RESULTS: In this study, a total of 16 PeINV genes were identified from the passion fruit genome and named according to their subcellular location and chromosome position. These include six cell wall invertase (CWINV) genes, two vacuolar invertase (VINV) genes, and eight neutral/alkaline invertase (N/AINV) genes. The gene structures, phylogenetic tree, and cis-acting elements of PeINV gene family were predicted using bioinformatics methods. Results showed that the upstream promoter region of the PeINV genes contained various response elements; particularly, PeVINV2, PeN/AINV3, PeN/AINV5, PeN/AINV6, PeN/AINV7, and PeN/AINV8 had more response elements. Additionally, the expression profiles of PeINV genes under different abiotic stresses (drought, salt, cold temperature, and high temperature) indicated that PeCWINV5, PeCWINV6, PeVINV1, PeVINV2, PeN/AINV2, PeN/AINV3, PeN/AINV6, and PeN/AINV7 responded significantly to these abiotic stresses, which was consistent with cis-acting element prediction results. Sucrose, glucose, and fructose are main soluble components in passion fruit pulp. The contents of total soluble sugar, hexoses, and sweetness index increased significantly at early stages during fruit ripening. Transcriptome data showed that with an increase in fruit development and maturity, the expression levels of PeCWINV2, PeCWINV5, and PeN/AINV3 exhibited an up-regulated trend, especially for PeCWINV5 which showed highest abundance, this correlated with the accumulation of soluble sugar and sweetness index. Transient overexpression results demonstrated that the contents of fructose, glucose and sucrose increased in the pulp of PeCWINV5 overexpressing fruit. It is speculated that this cell wall invertase gene, PeCWINV5, may play an important role in sucrose unloading and hexose accumulation. CONCLUSION: In this study, we systematically identified INV genes in passion fruit for the first time and further investigated their physicochemical properties, evolution, and expression patterns. Furthermore, we screened out a key candidate gene involved in hexose accumulation. This study lays a foundation for further study on INV genes and will be beneficial on the genetic improvement of passion fruit breeding.

Sucrose as an electron source for cofactor regeneration in recombinant Escherichia coli expressing invertase and a Baeyer Villiger monooxygenase.

BACKGROUND: The large-scale biocatalytic application of oxidoreductases requires systems for a cost-effective and efficient regeneration of redox cofactors. These represent the major bottleneck for industrial bioproduction and an important cost factor. In this work, co-expression of the genes of invertase and a Baeyer-Villiger monooxygenase from Burkholderia xenovorans to E. coli W ΔcscR and E. coli BL21 (DE3) enabled efficient biotransformation of cyclohexanone to the polymer precursor, ε-caprolactone using sucrose as electron source for regeneration of redox cofactors, at rates comparable to glucose. E. coli W ΔcscR has a native csc regulon enabling sucrose utilization and is deregulated via deletion of the repressor gene (cscR), thus enabling sucrose uptake even at concentrations below 6 mM (2 g L-1). On the other hand, E. coli BL21 (DE3), which is widely used as an expression host does not contain a csc regulon. RESULTS: Herein, we show a proof of concept where the co-expression of invertase for both E. coli hosts was sufficient for efficient sucrose utilization to sustain cofactor regeneration in the Baeyer-Villiger oxidation of cyclohexanone. Using E. coli W ΔcscR, a specific activity of 37 U gDCW-1 was obtained, demonstrating the suitability of the strain for recombinant gene co-expression and subsequent whole-cell biotransformation. In addition, the same co-expression cassette was transferred and investigated with E. coli BL21 (DE3), which showed a specific activity of 17 U gDCW- 1. Finally, biotransformation using photosynthetically-derived sucrose from Synechocystis S02 with E. coli W ΔcscR expressing BVMO showed complete conversion of cyclohexanone after 3 h, especially with the strain expressing the invertase gene in the periplasm. CONCLUSIONS: Results show that sucrose can be an alternative electron source to drive whole-cell biotransformations in recombinant E. coli strains opening novel strategies for sustainable chemical production.

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.

Mutant ß-fructofuranosidase synthesizing blastose [ß-d-Fruf-(2→6)-d-Glcp].

Fructooligosaccharides (FOS) are leading prebiotics that help keep the gut healthy and aid wellness by stimulating the growth and activity of beneficial intestinal bacteria. The best-studied FOS are inulin-type FOS, mainly oligosaccharides with ß-Fruf-(2→1)-Fruf linkages, including 1-kestose [ß-Fruf-(2→1)-ß-Fruf-(2↔1)-α-Glcp] and nystose [ß-Fruf-(2→1)-ß-Fruf-(2→1)-ß-Fruf-(2↔1)-α-Glcp]. However, the properties of other types of FOS-levan-type FOS with ß-Fruf-(2→6)-Fruf linkages and neo-type FOS with ß-Fruf-(2→6)-Glcp linkages-remain ambiguous because efficient methods have not been established for their synthesis. Here, using site-saturation mutation of residue His79 of ß-fructofuranosidase from Zymomonas mobilis NBRC13756, we successfully obtained a mutant ß-fructofuranosidase that specifically produces neo-type FOS. The H79G enzyme variant loses the native ß-Fruf-(2→1)-Fru-transfer ability (which produces 1-kestose), and instead has ß-Fruf-(2→6)-Glc-transfer ability and produces neokestose. Its hydrolytic activity specific to the ß-Fruf-(2↔1)-α-Glcp bond of neokestose then yields blastose [ß-Fruf-(2→6)-Glcp]. The enzyme produces 0.4 M blastose from 1.0 M sucrose (80 % of the theoretical yield). The production system for blastose established here will contribute to the elucidation of the physiological functions of this disaccharide.

Lacticaseibacillus paracasei completely utilizes fructooligosacchrides in the human gut through ß-fructosidase (FosE).

The fecal microbiota of two healthy adults was cultivated in a medium containing commercial fructooligosaccharides [FOS; 1-kestose (GF2), nystose (GF3), and 1F-fructofuranosylnystose (GF4)]. Initially, the proportions of lactobacilli in the two feces samples were only 0.42% and 0.17%; however, they significantly increased to 7.2% and 4.8%, respectively, after cultivation on FOS. Most FOS-utilizing isolates could utilize only GF2; however, Lacticaseibacillus paracasei strain Lp02 could fully consume GF3 and GF4 too. The FOS operon (fosRABCDXE) was present in Lc. paracasei Lp02 and another Lc. paracasei strain, KCTC 3510T, but fosE was only partially present in the non-FOS-degrading strain KCTC 3510T. In addition, the top six upregulated genes in the presence of FOS were fosABCDXE, particularly fosE. FosE is a ß-fructosidase that hydrolyzes both sucrose and all three FOS. Finally, a genome-based analysis suggested that fosE is mainly observed in Lc. paracasei, and only 13.5% (61/452) of their reported genomes were confirmed to include it. In conclusion, FosE allows the utilization of FOS, including GF3 and GF4 as well as GF2, by some Lc. paracasei strains, suggesting that this species plays a pivotal role in FOS utilization in the human gut.

Oligomeric Structure of Yeast and Other Invertases Governs Specificity.

Invertases, or ß-fructofuranosidases, are metabolic enzymes widely distributed among plants and microorganisms that hydrolyze sucrose and release fructose from various substrates. Invertase was one of the earliest discovered enzymes, first investigated in the mid-nineteenth century, becoming a classical model used in the primary biochemical studies on protein synthesis, activity, and the secretion of glycoproteins. However, it was not until 20 years ago that a member of this family of enzymes was structurally characterized, showing a bimodular arrangement with a ß-propeller catalytic domain, and a ß-sandwich domain with unknown function. Since then, many studies on related plant and fungal enzymes have revealed them as basically monomeric. By contrast, all yeast enzymes in this family that have been characterized so far have shown sophisticated oligomeric structures mediated by the non-catalytic domain, which is also involved in substrate binding, and how this assembly determines the particular specificity of each enzyme. In this chapter, we will review the available structures of yeast invertases to elucidate the mechanism regulating oligomer formation and compare them with other reported dimeric invertases in which the oligomeric assembly has no apparent functional implications. In addition, recent work on a new family of invertases with absolute specificity for the α-(1,2)-bond of sucrose found in cyanobacteria and plant invertases is highlighted.

Genome-wide identification and functional analysis of the SiCIN gene family in foxtail millet (Setaria italica L.).

Cell wall invertase (CIN) is a vital member of plant invertase (INV) and plays a key role in the breakdown of sucrose. This enzyme facilitates the hydrolysis of sucrose into glucose and fructose, which is crucial for various aspects of plant growth and development. However, the function of CIN genes in foxtail millet (Setaria italica) is less studied. In this research, we used the blast-p of NCBI and TBtools for bidirectional comparison, and a total of 13 CIN genes (named SiCINs) were identified from foxtail millet by using Arabidopsis and rice CIN sequences as reference sequences. The phylogenetic tree analysis revealed that the CIN genes can be categorized into three subfamilies: group 1, group 2, and group 3. Furthermore, upon conducting chromosomal localization analysis, it was observed that the 13 SiCINs were distributed unevenly across five chromosomes. Cis-acting elements of SiCIN genes can be classified into three categories: plant growth and development, stress response, and hormone response. The largest number of cis-acting elements were those related to light response (G-box) and the cis-acting elements related to seed-specific regulation (RY-element). qRT-PCR analysis further confirmed that the expression of SiCIN7 and SiCIN8 in the grain was higher than that in any other tissues. The overexpression of SiCIN7 in Arabidopsis improved the grain size and thousand-grain weight, suggesting that SiCIN7 could positively regulate grain development. Our findings will help to further understand the grain-filling mechanism of SiCIN and elucidate the biological mechanism underlying the grain development of SiCIN.

Genomic identification and expression analysis of acid invertase (AINV) gene family in Dendrobium officinale Kimura et Migo.

BACKGROUND: Dendrobium officinale Kimura et Migo, a renowned traditional Chinese orchid herb esteemed for its significant horticultural and medicinal value, thrives in adverse habitats and contends with various abiotic or biotic stresses. Acid invertases (AINV) are widely considered enzymes involved in regulating sucrose metabolism and have been revealed to participate in plant responses to environmental stress. Although members of AINV gene family have been identified and characterized in multiple plant genomes, detailed information regarding this gene family and its expression patterns remains unknown in D. officinale, despite their significance in polysaccharide biosynthesis. RESULTS: This study systematically analyzed the D. officinale genome and identified four DoAINV genes, which were classified into two subfamilies based on subcellular prediction and phylogenetic analysis. Comparison of gene structures and conserved motifs in DoAINV genes indicated a high-level conservation during their evolution history. The conserved amino acids and domains of DoAINV proteins were identified as pivotal for their functional roles. Additionally, cis-elements associated with responses to abiotic and biotic stress were found to be the most prevalent motif in all DoAINV genes, indicating their responsiveness to stress. Furthermore, bioinformatics analysis of transcriptome data, validated by quantitative real-time reverse transcription PCR (qRT-PCR), revealed distinct organ-specific expression patterns of DoAINV genes across various tissues and in response to abiotic stress. Examination of soluble sugar content and interaction networks provided insights into stress release and sucrose metabolism. CONCLUSIONS: DoAINV genes are implicated in various activities including growth and development, stress response, and polysaccharide biosynthesis. These findings provide valuable insights into the AINV gene amily of D. officinale and will aid in further elucidating the functions of DoAINV genes.

Evaluation of different glycerol fed-batch strategies in a lab-scale bioreactor for the improved production of a novel engineered ß-fructofuranosidase enzyme in Pichia pastoris.

The ß-fructofuranosidase enzyme from Aspergillus niger has been extensively used to commercially produce fructooligosaccharides from sucrose. In this study, the native and an engineered version of the ß-fructofuranosidase enzyme were expressed in Pichia pastoris under control of the glyceraldehyde-3-phosphate dehydrogenase promoter, and production was evaluated in bioreactors using either dissolved oxygen (DO-stat) or constant feed fed-batch feeding strategies. The DO-stat cultivations produced lower biomass concentrations but this resulted in higher volumetric activity for both strains. The native enzyme produced the highest volumetric enzyme activity for both feeding strategies (20.8% and 13.5% higher than that achieved by the engineered enzyme, for DO-stat and constant feed, respectively). However, the constant feed cultivations produced higher biomass concentrations and higher volumetric productivity for both the native as well as engineered enzymes due to shorter process time requirements (59 h for constant feed and 155 h for DO-stat feed). Despite the DO-stat feeding strategy achieving a higher maximum enzyme activity, the constant feed strategy would be preferred for production of the ß-fructofuranosidase enzyme using glycerol due to the many industrial advantages related to its enhanced volumetric enzyme productivity.

Metabolism of Inulin via Difructose Anhydride I Pathway in Microbacterium flavum.

Difructose anhydride I (DFA-I) can be produced from inulin, with DFA-I-forming inulin fructotransferase (IFTase-I). However, the metabolism of inulin through DFA-I remains unclear. To clarify this pathway, several genes of enzymes related to this pathway in the genome of Microbacterium flavum DSM 18909 were synthesized, and the corresponding enzymes were encoded, purified, and investigated in vitro. After inulin is decomposed to DFA-I by IFTase-I, DFA-I is hydrolyzed to inulobiose by DFA-I hydrolase. Inulobiose is then hydrolyzed by ß-fructofuranosidase to form fructose. Finally, fructose enters glycolysis through fructokinase. A ß-fructofuranosidase (MfFFase1) clears the byproducts (sucrose and fructo-oligosaccharides), which might be partially hydrolyzed by fructan ß-(2,1)-fructosidase/1-exohydrolase and another fructofuranosidase (MfFFase2). Exploring the DFA-I pathway of inulin and well-studied enzymes in vitro extends our basic scientific knowledge of the energy-providing way of inulin, thereby paving the way for further investigations in vivo and offering a reference for further nutritional investigation of inulin and DFA-I in the future.

Cell wall invertase 3 plays critical roles in providing sugars during pollination and fertilization in cucumber.

In plants, pollen-pistil interactions during pollination and fertilization mediate pollen hydration and germination, pollen tube growth, and seed set and development. Cell wall invertases (CWINs) help provide the carbohydrates for pollen development; however, their roles in pollination and fertilization have not been well established. In cucumber (Cucumis sativus), CsCWIN3 showed the highest expression in flowers, and we further examined CsCWIN3 for functions during pollination to seed set. Both CsCWIN3 transcript and CsCWIN3 protein exhibited similar expression patterns in the sepals, petals, stamen filaments, anther tapetum, and pollen of male flowers, as well as in the stigma, style, transmitting tract, and ovule funiculus of female flowers. Notably, repression of CsCWIN3 in cucumber did not affect the formation of parthenocarpic fruit but resulted in an arrested growth of stigma integuments in female flowers and a partially delayed dehiscence of anthers with decreased pollen viability in male flowers. Consequently, the pollen tube grew poorly in the gynoecia after pollination. In addition, CsCWIN3-RNA interference plants also showed affected seed development. Considering that sugar transporters could function in cucumber fecundity, we highlight the role of CsCWIN3 and a potential close collaboration between CWIN and sugar transporters in these processes. Overall, we used molecular and physiological analyses to determine the CsCWIN3-mediated metabolism during pollen formation, pollen tube growth, and plant fecundity. CsCWIN3 has essential roles from pollination and fertilization to seed set but not parthenocarpic fruit development in cucumber.

A novel ß-cyclodextrin-assisted enhancement strategy for portable and sensitive detection of miR-21 in human serum.

Benefiting from our discovery that ß-cyclodextrin (ß-CD) could enhance the catalytic activity of invertase through hydrogen bonding to improve detection sensitivity, a highly sensitive and convenient biosensor for the detection of miR-21 was proposed, which is based on the simplicity of reading signals from a personal glucose meter (PGM), combined with self-assembled signal amplification probes and the performance of ß-CD as an enhancer. In the presence of miR-21, magnetic nanoparticle coupled capture DNA (MNPs-cDNA) could capture it and then connect assist DNA/H1-invertase (aDNA/H1) and self-assembled signal amplification probes (H1/H2) in turn. As a result, a "super sandwich" structure was formed. The invertase on MNPs-cDNA could catalyze the hydrolysis of sucrose to glucose and this catalytic process could be enhanced by ß-CD. The PGM signal exhibited a linear correlation with miR-21 concentration within the range of 25 pmol L-1 to 3 nmol L-1, and the detection limit was as low as 5 pmol L-1 with high specificity. Moreover, the recoveries were 103.82-124.65% and RSD was 2.59-6.43%. Furthermore, the biosensor was validated for the detection of miR-21 in serum, and the results showed that miR-21 levels in serum samples from patients with Diffuse Large B-Cell Lymphoma (DLBCL) (n = 12) were significantly higher than those from healthy controls (n = 12) (P < 0.001). Therefore, the ingenious combination of PGM-based signal reading, self-assembled signal amplification probes and ß-CD as an enhancer successfully constructed a convenient, sensitive and specific biosensing method, which is expected to be applied to clinical diagnosis.

Molecular dissection of an intronic enhancer governing cold-induced expression of the vacuolar invertase gene in potato.

Potato (Solanum tuberosum) is the third most important food crop in the world. Potato tubers must be stored at cold temperatures to minimize sprouting and losses due to disease. However, cold temperatures strongly induce the expression of the potato vacuolar invertase gene (VInv) and cause reducing sugar accumulation. This process, referred to as "cold-induced sweetening," is a major postharvest problem for the potato industry. We discovered that the cold-induced expression of VInv is controlled by a 200 bp enhancer, VInvIn2En, located in its second intron. We identified several DNA motifs in VInvIn2En that bind transcription factors involved in the plant cold stress response. Mutation of these DNA motifs abolished VInvIn2En function as a transcriptional enhancer. We developed VInvIn2En deletion lines in both diploid and tetraploid potato using clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9)-mediated gene editing. VInv transcription in cold-stored tubers was significantly reduced in the deletion lines. Interestingly, the VInvIn2En sequence is highly conserved among distantly related Solanum species, including tomato (Solanum lycopersicum) and other non-tuber-bearing species. We conclude that the VInv gene and the VInvIn2En enhancer have adopted distinct roles in the cold stress response in tubers of tuber-bearing Solanum species.

Increased privatization of a public resource leads to spread of cooperation in a microbial population.

The phenomenon of cooperation is prevalent at all levels of life. In one such manifestation of cooperation in microbial communities, some cells produce costly extracellular resources that are freely available to others. These resources are referred to as public goods. Saccharomyces cerevisiae secretes invertase (public good) in the periplasm to hydrolyze sucrose into glucose and fructose, which are then imported by the cells. After hydrolysis of sucrose, a cooperator retains only 1% of the monosaccharides, while 99% of the monosaccharides diffuse into the environment and can be utilized by any cell. The non-producers of invertase (cheaters) exploit the invertase-producing cells (cooperators) by utilizing the monosaccharides and not paying the metabolic cost of producing the invertase. In this work, we investigate the evolutionary dynamics of this cheater-cooperator system. In a co-culture, if cheaters are selected for their higher fitness, the population will collapse. On the other hand, for cooperators to survive in the population, a strategy to increase fitness would likely be required. To understand the adaptation of cooperators in sucrose, we performed a coevolution experiment in sucrose. Our results show that cooperators increase in fitness as the experiment progresses. This phenomenon was not observed in environments which involved a non-public good system. Genome sequencing reveals duplication of several HXT transporters in the evolved cooperators. Based on these results, we hypothesize that increased privatization of the monosaccharides is the most likely explanation of spread of cooperators in the population.IMPORTANCEHow is cooperation, as a trait, maintained in a population? In order to answer this question, we perform a coevolution experiment between two strains of yeast-one which produces a public good to release glucose and fructose in the media, thus generating a public resource, and the other which does not produce public resource and merely benefits from the presence of the cooperator strain. What is the outcome of this coevolution experiment? We demonstrate that after ~200 generations of coevolution, cooperators increase in frequency in the co-culture. Remarkably, in all parallel lines of our experiment, this is obtained via duplication of regions which likely allow greater privatization of glucose and fructose. Thus, increased privatization, which is intuitively thought to be a strategy against cooperation, enables spread of cooperation.

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.

Identification, characterisation and expression analysis of peanut sugar invertase genes reveal their vital roles in response to abiotic stress.

KEY MESSAGE: Sucrose invertase activity is positively related to osmotic and salt stress resistance in peanut. Sucrose invertases (INVs) have important functions in plant growth and response to environmental stresses. However, their biological roles in peanut are still not fully revealed. In this research, we identified 42 AhINV genes in the peanut genome. They were highly conserved and clustered into three groups with 24 segmental duplication events occurred under purifying selection. Transcriptional expression analysis exhibited that they were all ubiquitously expressed, and most of them were up-regulated by osmotic and salt stresses, with AhINV09, AhINV23 and AhINV19 showed the most significant up-regulation. Further physiochemical analysis showed that the resistance of peanut to osmotic and salt stress was positively related to the high sugar content and sucrose invertase activity. Our results provided fundamental information on the structure and evolutionary relationship of INV gene family in peanut and gave theoretical guideline for further functional study of AhINV genes in response to abiotic stress.

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.

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.