Invertase Gene Research Articles

Concurrent overexpression of amino acid permease

Using pea as our model crop, we sought to understand the regulatory control over the import of sugars and amino acids into the developing seeds and its importance for seed yield and quality. Transgenic peas simultaneously overexpressing a sucrose transporter and an amino acid transporter were developed. Pod walls, seed coats, and cotyledons were analysed separately, as well as leaves subtending developing pods. Sucrose, starch, protein, free amino acids, and endogenous cytokinins were measured during development. Temporal gene expression analyses (RT-qPCR) of amino acid (AAP), sucrose (SUT), and SWEET transporter family members, and those from cell wall invertase, cytokinin biosynthetic (IPT) and degradation (CKX) gene families indicated a strong effect of the transgenes on gene expression. In seed coats of the double transgenics, increased content and prolonged presence of cytokinin was particularly noticeable. The transgenes effectively promoted transition of young sink leaves into source leaves. We suggest the increased flux of sucrose and amino acids from source to sink, along with increased interaction between cytokinin and cell wall invertase in developing seed coats led to enhanced sink activity, resulting in higher cotyledon sucrose at process pea harvest, and increased seed number and protein content at maturity.

Genome-wide identification and expression pattern of alkaline/neutral invertase gene family in Dendrobium catenatum

Alkaline/neutral invertase (NI) proteins irreversibly break down sucrose into glucose and fructose and play a vital role in carbohydrate and plant growth and development. Currently, a comprehensive understanding of NI genes in Dendrobium catenatum is lacking. After genome-wide identification and prediction, nine DcNI genes were identified and named DcNI1–DcNI9. Through comprehensive analysis of gene structure, amino acid sequence, conserved motif and phylogenetic tree, it was found that the DcNI gene family has a typical conserved region and its amino acid sequence is similar to other plant NI genes. However, DcNI3 lost amino acid sequences at both the N-terminus and C-terminus, while DcNI8 lost amino acids at the C-terminus. Phylogenetic analysis revealed that the DcNI family could be divided into α and β groups. It reveals conservation and divergence of DcNIs. Predictive analysis of cis-acting elements in the promoter region reveals the potential response of DcNI protein to hormones and environmental stress and suggests it to be widely involved in growth and development. DcNIs were differentially expressed in different tissues such as roots, stems and leaves. In particular, DcNI4 was highly expressed in the stem. They also had a differential expression response under drought, cold, P1 fungus infection and jasmonic acid (JA) treatment. To the best of our knowledge, this is the first genome-wide analysis of the DcNI gene family in D. catenatum. This study will provide important and primary information for the functional study of the DcNI gene family and the polysaccharide synthesis pathway in D. catenatum.

Identification and Expression Pattern Analysis of Acid Invertase Gene Family in Pineapple Fruit

Identification and Expression Pattern Analysis of Acid Invertase Gene Family in Pineapple Fruit

Comparison of carbohydrate partitioning and expression patterns of some genes involved in carbohydrate biosynthesis pathways in annual and biennial species of Cichorium spp.

Variation in metabolism and partitioning of carbohydrates, particularly fructans, between annual and perennial Cichorium species remains a challenging topic. To address this problem, an annual (endive, Cichorium endive L. var. Crispum; Asteraceae) and a biennial species (chicory, Cichorium intybus L. var. Witloof; Asteraceae) were compared with in terms of variability in carbohydrate accumulation and expression patterns of fructan-active enzyme genes, as well as sucrose metabolism at various growth and developmental stages. In general, constituents such as 1-kestose, nystose, and inulin were detected only in the root of chicory and were not present in any of the endive tissues. For both species, flower tissue contained maximum levels of both fructose and glucose, while for sucrose, more fluctuations were observed. On the other hand, all the genes under study exhibited variation, not only between the two species but also among different tissues at different sampling times. In endive root compared to endive leaf, the expression of cell wall invertase genes and sucrose accumulation decreased simultaneously, indicating the limited capacity of its roots to absorb sucrose, a precursor to inulin production. In addition, low expression of fructan: fructan fructosyltransferase in endive root compared to chicory root confirmed the inability of endive to inulin synthesis. Overall, annual and biennial species were different in the production of inulin, transport, remobilization, and unloading of sucrose.

Effects of heat stress on the biological Maillard reaction, oxidative stress, and occurrence of internal browning in Japanese radish (Raphanus sativus L.)

Internal browning (IB) of Japanese radish, in which the entire central part of the root changes from light tan to dark brown, is a physiological disorder caused by heat stress at the root maturation stage and represents significant economic losses to farmers. In this study, we cultivated three cultivars with different tolerance to IB disorder under high-temperature conditions, and examined the physiological factors involved in IB disorder differences among cultivars. There was a close correlation between the severity of IB disorder among cultivars and the size of root parenchymal cells. Significantly smaller cells were observed in the IB-resistant cultivars compared to the IB-susceptible one. Glucose concentration and invertase gene expression level tended to be higher in the normal roots of the IB-susceptible cultivar than in those of the resistant cultivars. Results of immunohistochemical analysis revealed that hexanoyl-lysine and acrolein, which are indicators of oxidative stress, in parenchymal cells were significantly elevated in the normal roots of the IB-susceptible cultivar than in those of the resistant ones. Similarly, the normal roots of the IB-susceptible cultivar showed elevated levels of Nε-(carboxymethyl)lysine, which is an indicator of the Maillard reaction, compared with those of the resistant cultivars. In addition, the immunohistochemistry of these indicators of oxidative stress and the Maillard reaction were more strongly observed in the damaged roots of the IB-susceptible cultivar. These results indicate that the occurrence of IB in Japanese radish is closely related to the increase in cell size, enhancement of the Maillard reaction by elevation in reducing sugar concentration, and increase in intracellular oxidative stress.

Enhancing the Heterologous Fructosyltransferase Activity of Kluyveromyces lactis: Developing a Scaled-Up Process and Abolishing Invertase by CRISPR/Cas9 Genome Editing.

The enzymatic production of prebiotic fructo-oligosaccharides (FOS) from sucrose involves fructosyltransferases (FFTs) and invertases, both of which catalyze forward (transferase) and reverse (hydrolysis) reactions. FOS yields can therefore be increased by favoring the forward reaction. We investigated process conditions that favored transferase activity in the yeast strain Kluyveromyces lactis GG799, which expresses a native invertase and a heterologous FFT from Aspergillus terreus. To maximize transferase activity while minimizing native invertase activity in a scaled-up process, we evaluated two reactor systems in terms of oxygen input capacity in relation to the cell dry weight. In the 0.5-L reactor, we found that galactose was superior to lactose for the induction of the LAC4 promoter, and we optimized the induction time and induction to carbon source ratio using a response surface model. Based on the critical parameter of oxygen supply, we scaled up the process to 7 L using geometric similarity and a higher oxygen transport rate, which boosted the transferase activity by 159%. To favor the forward reaction even more, we deleted the native invertase gene by CRISPR/Cas9 genome editing and compared the ΔInv mutant to the original production strain in batch and fed-batch reactions. In fed-batch mode, we found that the ΔInv mutant increased the transferase activity by a further 66.9%. The enhanced mutant strain therefore provides the basis for a highly efficient and scalable fed-batch process for the production of FOS.

Evaluation of RNA Interference for Control of the Grape Mealybug Pseudococcus maritimus (Hemiptera: Pseudococcidae).

Simple SummaryRNA interference (RNAi) is a defense mechanism that protects insects from viruses by targeting and degrading RNA. This feature has been exploited to reduce the expression of endogenous RNA for determining functions of various genes and for killing insect pests by targeting genes that are vital for insect survival. When dsRNA matching perfectly to the target RNA is administered, the RNAi machinery dices the dsRNA into ~21 bp fragments (known as siRNAs) and one strand of siRNA is employed by the RNAi machinery to target and degrade the target RNA. In this study we used a cocktail of dsRNAs targeting grape mealybug’s aquaporin and sucrase genes to kill the insect. Aquaporins and sucrases are important genes enabling these insects to maintain water relations indispensable for survival and digest complex sugars in the diet of plant sap-feeding insects, including mealybugs. In our experiments, administration of dsRNA caused a reduction in expression of the target genes and an increase in insect mortality. These results provide support for the application of RNAi to control the grape mealybug.The grape mealybug Pseudococcus maritimus (Ehrhorn, 1900) (Hemiptera: Pseudococcidae) is a significant pest of grapevines (Vitis spp.) and a vector of disease-causing grape viruses, linked to its feeding on phloem sap. The management of this pest is constrained by the lack of naturally occurring resistance traits in Vitis. Here, we obtained proof of concept that RNA interference (RNAi) using double-stranded RNA (dsRNA) molecules against essential genes for phloem sap feeding can depress insect survival. The genes of interest code for an aquaporin (AQP) and a sucrase (SUC) that are required for osmoregulation in related phloem sap-feeding hemipteran insects (aphids and whiteflies). In parallel, we investigated the grape mealybug genes coding non-specific nucleases (NUC), which reduce RNAi efficacy by degrading administered dsRNA. Homologs of AQP and SUC with experimentally validated function in aphids, together with NUC, were identified in the published transcriptome of the citrus mealybug Planococcus citri by phylogenetic analysis, and sequences of the candidate genes were obtained for Ps. maritimus by PCR with degenerate primers. Using this first sequence information for Ps. maritimus, dsRNA was prepared and administered to the insects via an artificial diet. The treatment comprising dsRNA against AQP, SUC and NUC significantly increased insect mortality over three days, relative to dsRNA-free controls. The dsRNA constructs for AQP and NUC were predicted, from sequence analysis to have some activity against other mealybugs, but none of the three dsRNA constructs have predicted activity against aphids. This study provides the basis to develop in planta RNAi strategies against Ps. maritimus and other mealybug pests of grapevines.

Nitric Oxide and Its Interaction with Hydrogen Peroxide Enhance Plant Tolerance to Low Temperatures by Improving the Efficiency of the Calvin Cycle and the Ascorbate–Glutathione Cycle in Cucumber Seedlings

The present study was aimed to assess the effect of nitric oxide (NO) and the interaction of NO with hydrogen peroxide (H2O2) on plant tolerance to low temperatures in cucumber seedlings. Exogenous NO significantly increased the endogenous NO content, initial and total activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), RuBisCO carboxylation rate (Vc,max), RuBP regeneration rate (Jmax) and the transcript levels of related genes in cucumber seedlings under low temperatures (11 °C/7 °C); however, the effect of NO was blocked by PTIO (NO scavenger). In addition, the SNP treatment significantly improved the contents of glucose, fructose, sucrose, starch, and the activities of sucrose phosphate synthase (SPS), acid invertase (AI), sucrose synthase (SS), as well as the expression levels of SUCROSE PHOSPHATE SYNTHASE 1&2 (SPS1, SPS2), SUCROSE TRANSPORTER 1&2 (SUT2, SUT4), β-starch hydrolase (BAM), and invertase gene (INVERTASE) in cucumber leaves under low temperatures, and the positive effect of NO was impaired by PTIO. Furthermore, we found that the H2O2, induced by NO, participated in NO-induced elevation of ascorbic acid (AsA), glutathione (GSH), and increased activities of related enzymes in the AsA-GSH cycle at low temperatures. However, the positive effect of NO was blocked by l-NAME (NOS inhibitor), PTIO, DPI (inhibitor of NADPH oxidase), and DMTU (reactive oxygen species scavenger). Taken together, our findings indicate that NO increased the low temperature tolerance of cucumber seedlings via H2O2 by improving the efficiency of the Calvin cycle, which in turn increased the carbohydrates content and accelerated the AsA-GSH cycle to enhance ROS scavenging.

PpINH1, an invertase inhibitor, interacts with vacuolar invertase PpVIN2 in regulating the chilling tolerance of peach fruit

Sucrose metabolism, particularly the decomposition of sucrose by invertase, plays a central role in plant responses to cold stress. Invertase inhibitors (INHs) evolved in higher plants as essential regulators of sucrose metabolism. By limiting invertase activity, INHs keep cellular sugar levels elevated, which provides enhanced protection to plants under stress. Our results showed that the expression of PpVIN2, the only vacuolar invertase (VIN) gene in peach fruit sensitive to chilling temperatures, increases significantly during cold storage, while VIN enzyme activity increases more modestly. We also found that peach fruit transiently overexpressing PpINH1 had decreased VIN activity. Interactions of PpINH1 and PpVIN2 with recombinant proteins were shown by yeast two-hybrid assays and bimolecular fluorescence complementation assays, as well as in vitro. During cold storage, trehalose-treated peach fruit had significantly increased PpINH1 expression, decreased VIN enzyme activity, and significantly higher sucrose content than did untreated fruit. As a result, the treated fruit had enhanced resistance to chilling injury. Collectively, our data show that the post-translational repression of VIN enzyme activity by PpINH1 helps maintain sucrose levels in peach fruit during cold storage, thereby improving resistance to chilling injury.

New insights into the evolution and expression dynamics of invertase gene family in Solanum lycopersicum

Invertases catalyze the irreversible hydrolysis of sucrose into glucose and fructose and thus play key roles in carbon metabolism and plant development. To gain insights into their evolutional and functional relationships, we conducted genome-wide analyses of invertase genes in tomato and other species, focusing on their evolution and expression dynamics. The analyses unexpectedly identified in the tomato genome 5 pseudo invertsase sequences and 5 non-functional cell wall invertases (CWINs) lacking the critical β-fructosidase motif or other amino acids required for hydrolyzing sucrose. Based on their phylogeny relationship and exon–intron structure, we speculated that the invertase gene family could arose from different ancestral genes. The acid invertase gene family, comprised of CWIN and vacuolar invertase (VIN), expanded through segmental and tandem duplication. Analysis of functional divergence suggests site-specific shifted evolutionary rate (Type-I) have played an important role in evolutionary novelties after acid invertase gene duplication in plants. Finally, paralogs within each of the CWIN, VIN and CIN subfamilies exhibited diverse expression responses to the same set of stress treatments including salt and temperature stresses, probably reflecting functional adaptability of the invertase genes during evolution.

Deletion of beta-fructofuranosidase (invertase) genes is associated with sucrose content in Date Palm fruit.

The fruit of date palm trees are an important part of the diet for a large portion of the Middle East and North Africa. The fruit is consumed both fresh and dry and can be stored dry for extended periods of time. Date fruits vary significantly across hundreds of cultivars identified in the main regions of cultivation. Most dried date fruit are low in sucrose but high in glucose and fructose. However, high sucrose content is a distinctive feature of some date fruit and affects flavor as well as texture and water retention. To identify the genes controlling high sucrose content, we analyzed date fruit metabolomics for association with genotype data from 120 date fruits. We found significant association of dried date sucrose content and a genomic region that contains 3 tandem copies of the beta‐fructofuranosidase (invertase) gene in the reference Khalas genome, a low‐sucrose fruit. High‐sucrose cultivars including the popular Deglet Noor had a homozygous deletion of two of the 3 copies of the invertase gene. We show the deletion allele is derived when compared to the ancestral allele that retains all copies of the gene in 3 other species of Phoenix. The fact that 2 of the 3 tandem invertase copies are associated with dry fruit sucrose content will assist in better understanding the distinct roles of multiple date palm invertases in plant physiology. Identification of the recessive alleles associated with end‐point sucrose content in date fruit may be used in selective breeding in the future.

The acid invertase gene family is involved in internode elongation in Phyllostachys heterocycla cv. pubescens.

Acid invertases (INVs) play a pivotal role in both vegetative and reproductive growth of plants. However, their possible functions in fast-growing plants such as bamboo are largely unknown. Here, we report the molecular characterization of acid INVs in Phyllostachys heterocycla cv. pubescens, a fast-growing bamboo species commercially grown worldwide. Nine acid INVs (PhINVs), including seven cell wall INVs (PhCWINV1, PhCWINV2, PhCWINV3, PhCWINV4, PhCWINV5, PhCWINV6 and PhCWINV7) and two vacuolar INVs (PhVINV11 and PhVINV12) were isolated. Bioinformatic analyses demonstrated that they all share high amino acid identity with other INVs from different plant species and contain the motifs typically conserved in acid INV. Enzyme activity assays revealed a significantly higher INV activity in the fast-growing tissues, such as the elongating internodes of stems. Detailed quantitative reverse-transcription PCR analyses showed various expression patterns of PhINVs at different developmental stages of the elongating stems. With the exception of PhCWINV6, all PhINVs were ubiquitously expressed in a developmental-specific manner. Further studies in Arabidopsis exhibited that constitutive expression of PhCWINV1, PhCWINV4 or PhCWINV7 increased the biomass production of transgenic plants, as indicated by augmented plant heights and shoot dry weights than the wild-type plants. All these results suggest that acid INVs play a crucial role in the internode elongation of P. heterocycla cv. pubescens and would provide valuable information for the dissection of their exact biological functions in the fast growth of bamboo.

Identification and Immobilization of an Invertase With High Specific Activity and Sucrose Tolerance Ability of Gongronella sp. w5 for High Fructose Syrup Preparation.

Invertases catalyze the hydrolysis of sucrose into fructose and glucose and can be employed as an alternative in producing high fructose syrup. In this study, we reported the heterologous expression of an invertase gene (GspInv) of Gongronella sp. w5 in Komagataella pastoris. GspInv activity reached 147.6 ± 0.4 U/mL after 5 days of methanol induction. GspInv is invertase with a high specific activity of 2,776.1 ± 124.2 U/mg toward sucrose. GspInv showed high tolerance to sucrose (IC50 = 1.2 M), glucose (IC50 > 2 M), fructose (IC50 = 1.5 M), and a variety of metal ions that make it an ideal candidate for high fructose syrup production. A carbohydrate-binding module was sequence-optimized and fused to the N-terminus of GspInv. The fusion protein had the highest immobilization efficiency at room temperature within 1 h adsorption, with 1 g of cellulose absorption up to 8,000 U protein. The cellulose-immobilized fusion protein retained the unique properties of GspInv. When applied in high fructose syrup preparation by using 1 M sucrose as the substrate, the sucrose conversion efficiency of the fused protein remained at approximately 95% after 50 h of continuous hydrolysis on a packed bed reactor. The fused protein can also hydrolyze completely the sucrose in sugarcane molasses. Our results suggest that GspInv is an unusual invertase and a promising candidate for high fructose syrup preparation.

Primer Design for Isolation of Sucrose:Sucrose 1-Fructosyltransferase (1-SST) Gene from Gembili (Dioscorea esculenta)

Recent studies have reported the presence of inulin, a prebiotic polysaccharide, in gembili (Dioscorea esculenta). Sucrose:sucrose 1-fructosyltransferase (1-SST) is an enzyme that catalyzes the first step of inulin biosynthesis. The identification of this enzyme would be the foundation to improve the yield of inulin in gembili; to modify 1-SST gene for this purpose, its sequence must first be determined. This study aimed to design primers to isolate 1-SST gene from D. esculenta. The primers were designed by using the whole-genome sequence of Dioscorea rotundata due to the lack of genomic information on D. esculenta. Sequences from chromosome 6 and 11 were used as the template of primer design in which four pairs of primers were selected. Amplification products with expected size were gel-purified, then the targets were sequenced and analyzed in-silico. As a result, one of the primer pairs had successfully isolated vacuolar invertase gene, which is closely related to 1-SST gene. On the other hand, the other primer pairs showed either negative or false-positive result. Using the current strategy, 1-SST gene sequence from D. esculenta has not been successfully isolated, thus other approaches in primer design should be considered in further studies.
 Keywords: 1-SST, Dioscorea esculenta, Gembili, Inulin, Primer design

Role and mechanisms of callose priming in mycorrhiza-induced resistance.

Mycorrhizal plants display enhanced resistance to several pathogens. However, the molecular mechanisms regulating mycorrhiza-induced resistance (MIR) are still elusive. We aim to study the mechanisms underlying MIR against Botrytis cinerea and the role of callose accumulation during this process. Mycorrhizal tomato plants inoculated with Rhizoglomus irregularis displayed callose priming upon B. cinerea infection. The callose inhibitor 2-deoxy-d-glucose abolished MIR, confirming the relevance of callose in the bioprotection phenomena. While studying the mechanisms underlying mycorrhiza-induced callose priming, we found that mycorrhizal plants display an enhanced starch degradation rate that is correlated with increased levels of β-amylase1 transcripts following pathogen infection. Starch mobilization in mycorrhizal plants seems coordinated with the increased transcription of sugar transporter and invertase genes. Moreover, the expression levels of genes encoding the vesicular trafficking proteins ATL31 and SYP121 and callose synthase PMR4 were higher in the mycorrhizal plants and further boosted by subsequent pathogen infection. All these proteins play a key role in the priming of callose accumulation in Arabidopsis, suggesting that callose priming is an induced resistance mechanism conserved in different plant species. This evidence highlights the importance of sugar mobilization and vesicular trafficking in the priming of callose as a defence mechanism in mycorrhiza-induced resistance.

Cloning, molecular characterisation, and expression of putative invertase inhibitor genes and their potential relationship with the activity of vacuolar acid invertase in peach fruit

ABSTRACTWe previously reported that the acid invertase gene PpVIN2 appears to be induced by chilling and may play an important role in sucrose metabolism in peach fruit subjected to cold storage. We also hypothesised that an invertase inhibitor (INH) affects vacuolar acid invertase (VIN) activity at the post-translational level. To determine which INH gene correlates with VIN activity, five INH genes (designated PpINH1–5) were identified and cloned from ‘Yulu’ peach. Relative expression for each gene was then measured in ‘Yulu’ peaches stored at 0, 5, 10, and 20°C and in ‘Hujingmilu’ peaches stored at 5°C. Each INH gene contains four conserved cysteine residues and four asymmetric alpha-helices characteristic of inhibitor protein. PpINH1–4 are members of the INH family while PpINH5 is classified as a pectin methylesterase inhibitor (PMEI). Transcript analyses showed that the PpINHs in ‘Yulu’ peaches have lower expression during storage at 5°C vs. 10°C, indicating that they are not low temperature-induced genes. However, PpINH1 expression is negatively correlated with VIN activity in both ‘Yulu’ and ‘Hujingmilu’ peaches stored at 5°C, suggesting PpINH1 is involved in the post-translational regulation of VIN under chilling conditions.

The Pyrus bretschneideri invertase gene family: identification, phylogeny and expression patterns

Plant invertases comprise a family of enzymes that catalyze the irreversible hydrolysis of sucrose into glucose and fructose, which plays a crucial role in carbohydrate partitioning and regulating plant growth and development. However, currently, there is little information on the roles of invertases in the development of pear trees. In this study, we performed genome-wide identification of a total of 23 invertases from the predicted genome data of the pear plant (Pyrus bretschneideri R.). Based on phylogenetic analysis, the pear invertases could be clustered into 13 acid invertase genes and 10 alkaline/neutral invertase genes. Chromosome location showed that tandem duplication and segmental duplication have played an important role in the expansion of acid and neutral/alkaline invertases in pear plants. Evolutionary analysis of invertases from pear and two model plant species (Arabidopsis thaliana and Oryza sativa) indicated that the evolutionary divergence of invertases occurred before the common ancestor of dicots and monocots. Tissue-specific expression analysis showed that the transcripts of all the identified invertase genes were detectable in various tissues. We hypothesize that the identified invertase genes all participate in regulating pear growth and development.

Evolutionary Conservation and Expression Patterns of Neutral/Alkaline Invertases in Solanum.

The invertase gene family in plants is composed of two subfamilies of enzymes, namely, acid- and neutral/alkaline invertases (cytosolic invertase, CIN). Both can irreversibly cleave sucrose into fructose and glucose, which are thought to play key roles in carbon metabolism and plant growth. CINs are widely found in plants, but little is reported about this family. In this paper, a comparative genomic approach was used to analyze the CIN gene family in Solanum, including Solanum tuberosum, Solanum lycopersicum, Solanum pennellii, Solanum pimpinellifolium, and Solanum melongena. A total of 40 CINs were identified in five Solanum plants, and sequence features, phylogenetic relationships, motif compositions, gene structure, collinear relationship, and expression profile were further analyzed. Sequence analysis revealed a remarkable conservation of CINs in sequence length, gene number, and molecular weight. The previously verified four amino acid residues (D188, E414, Arg430, and Ser547) were also observed in 39 out of 40 CINs in our study, showing to be deeply conserved. The CIN gene family could be distinguished into groups α and β, and α is further subdivided into subgroups α1 and α2 in our phylogenetic tree. More remarkably, each species has an average of four CINs in the α and β groups. Marked interspecies conservation and collinearity of CINs were also further revealed by chromosome mapping. Exon–intron configuration and conserved motifs were consistent in each of these α and β groups on the basis of in silico analysis. Expression analysis indicated that CINs were constitutively expressed and share similar expression profiles in all tested samples from S. tuberosum and S. lycopersicum. In addition, in CIN genes of the tomato and potato in response to abiotic and biotic stresses, phytohormones also performed. Overall, CINs in Solanum were encoded by a small and highly conserved gene family, possibly reflecting structural and functional conservation in Solanum. These results lay the foundation for further expounding the functional characterization of CIN genes and are also significant for understanding the evolutionary profiling of the CIN gene family in Solanum.

Drought-induced disturbance of carbohydrate metabolism in anthers and male abortion of two Gossypium hirsutum cultivars differing in drought tolerance.

Cotton pollen abortion, under drought stress, was closely associated with changes in anther carbohydrate metabolism, and pollen abortion rate due to drought was higher in drought-sensitive cultivars than drought-tolerant cultivars. Cotton reproductive failure under drought stress is intrinsically connected with altered male fertility, however, studies investigating the effect of drought stress on cotton male fertility are nonexistent. Thus, a drought stress experiment was conducted with two cotton cultivars, differing in drought tolerance, to study pollen fertility and anthers' physiology. Results indicated that drought stress reduced pollen fertility of both cultivars due to decreases in anther starch and adenosine triphosphate (ATP) synthesis. Lower assimilate supply capacity in conjunction with impaired activities of ADP-glucose pyrophosphorylase and soluble starch synthase were the main reasons for the decreased starch levels in drought-stressed anthers. The decreased activities of sucrose synthetase and acid invertase were responsible for the higher sucrose level in drought-stressed anthers than well-watered anthers and the changing trend of sucrose was intensified by the decreased expressions of sucrose synthase genes (GhSusA, GhSusB, GhSusD) and acid invertase genes (GhINV1, GhINV2). However, despite sucrose degradation being limited in drought-stressed anthers, glucose level was higher in droughted anthers than well-watered ones, and that might be attributed to the down-regulated respiration since decreased anther ATP levels were detected in drought-stressed plants. Furthermore, compared to the drought-tolerant cultivar, pollen fertility was more suppressed by drought stress for the drought-sensitive cultivar, and that was attributed to the larger decrease in starch and ATP contents.

Vacuolar invertase genes SbVIN1 and SbVIN2 are differently associated with stem and grain traits in sorghum (Sorghum bicolor)

In higher plants, vacuolar invertases play essential roles in sugar metabolism, organ development, and sink strength. In sorghum (Sorghum bicolor), two vacuolar invertase genes, SbVIN1 (Sobic. 004G004800) and SbVIN2 (Sobic. 006G160700) have been reported, but their enzymatic properties and functional differences are largely unknown. We combined molecular, biochemical and genomic approaches to investigate their roles in sorghum stem and grain traits. SbVIN1 and SbVIN2 showed different expression levels in internodes, leaves, and panicles, indicating that their importance in each organ was different. In an in vitro sucrose hydrolysis assay, proteins of both genes heterologously expressed in Pichia pastoris displayed similar enzyme properties including the same optimum reaction pH (5) and similar Km for sucroe (49 mmol L−1 and 45 mmol L−1 for SbVIN1 and SbVIN2, respectively). The optimum reaction temperatures of SbVIN1 and SbVIN2 were 45 °C and 65 °C, respectively. SbVIN2 showed higher tolerance to high temperature than SbVIN1. We characterized the sequence variation of these two vacuolar invertase genes in a panel of 216 diverse inbred lines of sweet and grain sorghum and performed gene-based association analysis. SbVIN1 showed significant associations with stem traits including stem length, stem diameter, internode number, stem fresh weight, and Brix, as well as grain traits including hundred-grain weight and grain width. Significantly associated variation sites were mainly in 5′ upstream and intron regions. SbVIN2 only associated with grain width and stem water-soluble carbohydrates (WSCs) content. We conclude that the vacuolar invertase genes SbVIN1 and SbVIN2 are differently associated with stem and grain traits in sorghum.