Genome-Wide Identification, Expression, and Functional Analysis of the Alkaline/Neutral Invertase Gene Family in Pepper.

Alkaline/neutral invertase (NINV) proteins irreversibly cleave sucrose into fructose and glucose, and play important roles in carbohydrate metabolism and plant development. To investigate the role of NINVs in the development of pepper fruits, seven NINV genes (CaNINV1-7) were identified. Phylogenetic analysis revealed that the CaNINV family could be divided into α and ß groups. CaNINV1-6 had typical conserved regions and similar protein structures to the NINVs of other plants, while CaNINV7 lacked amino acid sequences at the C-terminus and N-terminus ends. An expression analysis of the CaNINV genes in different tissues demonstrated that CaNINV5 is the dominant NINV in all the examined tissues (root, stem, leaf, bud, flower, and developmental pepper fruits stage). Notably, the expression of CaNINV5 was found to gradually increase at the pre-breaker stages, followed by a decrease at the breaker stages, while it maintained a low level at the post-breaker stages. Furthermore, the invertase activity of CaNINV5 was identified by functional complementation of the invertase-deficient yeast strain SEY2102, and the optimum pH of CaNINV5 was found to be ~7.5. The gene expression and enzymatic activity of CaNINV5 suggest that it might be the main NINV enzyme for hydrolysis of sucrose during pepper fruit development.

PtrA/NINV, an alkaline/neutral invertase gene of Poncirus trifoliata, confers enhanced tolerance to multiple abiotic stresses by modulating ROS levels and maintaining photosynthetic efficiency.

BACKGROUND: Alkaline/neutral invertase (A/N-INV), an enzyme that hydrolyzes sucrose irreversibly into glucose and fructose, is essential for normal plant growth,development, and stress tolerance. However, the physiological and/or molecular mechanism underpinning the role of A/N-INV in abiotic stress tolerance is poorly understood. RESULTS: In this report, an A/N-INV gene (PtrA/NINV) was isolated from Poncirus trifoliata, a cold-hardy relative of citrus, and functionally characterized. PtrA/NINV expression levels were induced by cold, salt, dehydration, sucrose, and ABA, but decreased by glucose. PtrA/NINV was found to localize in both chloroplasts and mitochondria. Overexpression of PtrA/NINV conferred enhanced tolerance to multiple stresses, including cold, high salinity, and drought, as supported by lower levels of reactive oxygen species (ROS), reduced oxidative damages, decreased water loss rate, and increased photosynthesis efficiency, relative to wild-type (WT). The transgenic plants exhibited higher A/N-INV activity and greater reducing sugar content under normal and stress conditions. CONCLUSIONS: PtrA/NINV is an important gene implicated in sucrose decomposition, and plays a positive role in abiotic stress tolerance by promoting osmotic adjustment, ROS detoxification and photosynthesis efficiency. Thus, PtrA/NINV has great potential to be used in transgenic breeding for improvement of stress tolerance.

Sugar suppresses cell death caused by disruption of fumarylacetoacetate hydrolase in Arabidopsis.

MAIN CONCLUSION: Sugar negatively regulates cell death resulting from the loss of fumarylacetoacetate hydrolase that catalyzes the last step in the Tyr degradation pathway in Arabidopsis . Fumarylacetoacetate hydrolase (FAH) hydrolyzes fumarylacetoacetate to fumarate and acetoacetate, the final step in the tyrosine (Tyr) degradation pathway that is essential to animals. Previously, we first found that the Tyr degradation pathway plays an important role in plants. Mutation of the SSCD1 gene encoding FAH in Arabidopsis leads to spontaneous cell death under short-day conditions. In this study, we presented that the lethal phenotype of the short-day sensitive cell death1 (sscd1) seedlings was suppressed by sugars including sucrose, glucose, fructose, and maltose in a dose-dependent manner. Real-time quantitative PCR (RT-qPCR) analysis showed the expression of Tyr degradation pathway genes homogentisate dioxygenase and maleylacetoacetate isomerase, and sucrose-processing genes cell-wall invertase 1 and alkaline/neutral invertase G, was up-regulated in the sscd1 mutant, however, this up-regulation could be repressed by sugar. In addition, a high concentration of sugar attenuated cell death of Arabidopsis wild-type seedlings caused by treatment with exogenous succinylacetone, an abnormal metabolite resulting from the loss of FAH in the Tyr degradation pathway. These results indicated that (1) sugar could suppress cell death in sscd1, which might be because sugar supply enhances the resistance of Arabidopsis seedlings to toxic effects of succinylacetone and reduces the accumulation of Tyr degradation intermediates, resulting in suppression of cell death; and (2) sucrose-processing genes cell-wall invertase 1 and alkaline/neutral invertase G might be involved in the cell death in sscd1. Our work provides insights into the relationship between sugar and sscd1-mediated cell death, and contributes to elucidation of the regulation of cell death resulting from the loss of FAH in plants.

Light modulated activity of root alkaline/neutral invertase involves the interaction with 14-3-3 proteins.

Alkaline/neutral invertases (A/N-Invs) are now recognized as essential proteins in plant life. They catalyze the irreversible breakdown of sucrose into glucose and fructose and thus supply the cells with energy as well as signaling molecules. In this study we report on a mechanism that affects the activity of the cytosolic invertase AtCINV1 (At-A/N-InvG or AT1G35580). We demonstrate that Ser547 at the extreme C-terminus of the AtCINV1 protein is a substrate of calcium-dependent kinases (CPK3 and 21) and that phosphorylation creates a high-affinity binding site for 14-3-3 proteins. The invertase as such has basal activity, but we provide evidence that interaction with 14-3-3 proteins enhances its activity. The analysis of three quadruple 14-3-3 mutants generated from six T-DNA insertion mutants of the non-epsilon family shows both specificity as well as redundancy for this function of 14-3-3 proteins. The strong reduction in hexose levels in the roots of one 14-3-3 quadruple mutant plant is in line with the activating function of 14-3-3 proteins. The physiological relevance of this mechanism that affects A/N-invertase activity is underscored by the light-induced activation and is another example of the central role of 14-3-3 proteins in mediating dark/light signaling. The nature of the light-induced signal that travels from the shoot to root and the question whether this signal is transmitted via cytosolic Ca(++) changes that activate calcium-dependent kinases, await further study.

HbNIN2, a cytosolic alkaline/neutral-invertase, is responsible for sucrose catabolism in rubber-producing laticifers of Hevea brasiliensis (para rubber tree).

In Hevea brasiliensis, an alkaline/neutral invertase (A/N-Inv) is responsible for sucrose catabolism in latex (essentially the cytoplasm of rubber-producing laticifers, the source of natural rubber) and implicated in rubber yield. However, neither the gene encoding this enzyme nor its molecular and biochemical properties have been well documented. Three Hevea A/N-Inv genes, namely HbNIN1, 2 and 3, were first cloned and characterized in planta and in Escherichia coli. Cellular localizations of HbNIN2 mRNA and protein were probed. From latex, active A/N-Inv proteins were purified, identified, and explored for enzymatic properties. HbNIN2 was identified as the major A/N-Inv gene functioning in latex based on its functionality in E. coli, its latex-predominant expression, the conspicuous localization of its mRNA and protein in the laticifers, and its expressional correlation with rubber yield. An active A/N-Inv protein was partially purified from latex, and determined as HbNIN2. The enhancement of HbNIN2 enzymatic activity by pyridoxal is peculiar to A/N-Invs in other plants. We conclude that HbNIN2, a cytosolic A/N-Inv, is responsible for sucrose catabolism in rubber laticifers. The results contribute to the studies of sucrose catabolism in plants as a whole and natural rubber synthesis in particular.

Down-regulation of a wheat alkaline/neutral invertase correlates with reduced host susceptibility to wheat stripe rust caused by Puccinia striiformis.

Numerous studies have found that sucrose (Suc) metabolism plays a crucial role in the environmental stress response of many plant species. The majority of Suc metabolism-associated reports refer to acid invertases (Ac-Invs). However, alkaline/neutral Invs (A/N-Invs) have been poorly studied. In this study, a wheat A/N-Inv gene, Ta-A/N-Inv1, with three copies located on chromosomes 4A, 4B, and 4D, was cloned from a wheat-Puccinia striiformis f. sp. tritici (Pst) interaction cDNA library. Transcripts of the three Ta-A/N-Inv1 copies were up-regulated in wheat leaves that were infected by Pst or had experienced certain abiotic treatments. Furthermore, the expression of Ta-A/N-Inv1 was decreased by treatment with exogenous hormones. Heterologous mutant complementation and subcellular localization revealed that Ta-A/N-Inv1 is a cytoplasmic invertase. Knocking down all three copies of Ta-A/N-Inv1 using the barley stripe mosaic virus-induced gene silencing system reduced the susceptibility of wheat to the Pst virulent pathotype CYR31, which is associated with pathogen-induced H2O2 accumulation and enhanced necrosis. Interestingly, 48h dark treatment of the Ta-A/N-Inv1-knockdown plants immediately after inoculation abrogated their enhanced resistance, suggesting that H2O2 production and its associated cell death and resistance in the Ta-A/N-Inv1-silenced plants require light. Consistent with this observation, photosynthesis and reactive oxygen species (ROS)-related genes were significantly up-regulated in the Ta-A/N-Inv1-knockdown plants infected by CYR31 under light exposure. These results suggest that Ta-A/N-Inv1 might act as a negative regulator in wheat disease resistance to Pst by increasing cytoplasmic hexose accumulation and downregulating photosynthesis of the leaves to avoid cell death due to excessive ROS production.

MeNINV1: An Alkaline/Neutral Invertase Gene of Manihot esculenta, Enhanced Sucrose Catabolism and Promoted Plant Vegetative Growth in Transgenic Arabidopsis.

Alkaline/neutral invertase (A/N-INV) is an invertase that irreversibly decomposes sucrose into fructose as well as glucose and plays a role in plant growth and development, starch synthesis, abiotic stress, and other plant-life activities. Cassava is an economically important starch crop in tropical regions. During the development of cassava tuber roots, A/N-INV activity is relatively high, which indicates that it may participate in sucrose metabolism and starch synthesis. In this study, MeNINV1 was confirmed to function as invertase to catalyze sucrose decomposition in yeast. The optimal enzymatic properties of MeNINV1 were a pH of 6.5, a reaction temperature of 40 °C, and sucrose as its specific catalytic substrate. VB6, Zn2+, and Pb2+ at low concentrations as well as EDTA, DTT, Tris, Mg2+, and fructose inhibited A/N-INV enzymic activity. In cassava, the MeNINV1 gene was mainly expressed in the fibrous roots and the tuber root phloem, and its expression decreased as the tuber root grew. MeNINV1 was confirmed to localize in chloroplasts. In Arabidopsis, MeNINV1-overexpressing Arabidopsis had higher A/N-INV activity, and the increased glucose, fructose, and starch content in the leaves promoted plant growth and delayed flowering time but did not change its resistance to abiotic stress. Our results provide new insights into the biological function of MeNINV1.

Systematic Analysis of Alkaline/Neutral Invertase Genes Reveals the Involvement of Smi-miR399 in Regulation of SmNINV3 and SmNINV4 in Salvia miltiorrhiza.

Alkaline/neutral invertases (NINVs), which irreversibly catalyze the hydrolysis of sucrose into fructose and glucose, play crucial roles in carbohydrate metabolism and plant development. Comprehensive insights into NINV genes are lacking in Salvia miltiorrhiza, a well-known traditional Chinese medicinal (TCM) plant with significant medicinal and economic value. Through genome-wide prediction, nine putative SmNINV genes, termed SmNINV1-SmNINV9, were identified. Integrated analysis of gene structures, sequence features, conserved domains, conserved motifs and phylogenetic trees revealed the conservation and divergence of SmNINVs. The identified SmNINVs were differentially expressed in roots, stems, leaves, flowers, and different root tissues. They also responded to drought, salicylic acid, yeast extract, and methyl jasmonate treatments. More importantly, computational prediction and experimental validation showed that SmNINV3 and SmNINV4 were targets of Smi-miR399, a conserved miRNA previously shown to affect Pi uptake and translocation through the cleavage of PHOSPHATE2 (PHO2). Consistently, analysis of 43 NINV genes and 26 miR399 sequences from Arabidopsis thaliana, Populus trichocarpa, Manihot esculenta, and Solanum lycopersicum showed that various AtNINV, PtNINV, MeNINV, and SlNINV genes were regulated by miR399. It indicates that the miR399-NINV module exists widely in plants. Furthermore, Smi-miR399 also cleaved SmPHO2 transcripts in S. miltiorrhiza, suggesting the complexity of NINVs, PHO2, and miR399 networks.

Sucrose and invertases, a part of the plant defense response to the biotic stresses.

Sucrose is the main form of assimilated carbon which is produced during photosynthesis and then transported from source to sink tissues via the phloem. This disaccharide is known to have important roles as signaling molecule and it is involved in many metabolic processes in plants. Essential for plant growth and development, sucrose is engaged in plant defense by activating plant immune responses against pathogens. During infection, pathogens reallocate the plant sugars for their own needs forcing the plants to modify their sugar content and triggering their defense responses. Among enzymes that hydrolyze sucrose and alter carbohydrate partitioning, invertases have been reported to be affected during plant-pathogen interactions. Recent highlights on the role of invertases in the establishment of plant defense responses suggest a more complex regulation of sugar signaling in plant-pathogen interaction.