Identification and expression analysis of the sucrose synthase gene family in pomegranate (Punica granatum L.)

Background Sucrose synthase (SUS, EC 2.4.1.13) is one of the major enzymes of sucrose metabolism in higher plants. It has been associated with C allocation, biomass accumulation, and sink strength. The SUS gene families have been broadly explored and characterized in a number of plants. The pomegranate (Punica granatum) genome is known, however, it lacks a comprehensive study on its SUS genes family. Methods PgSUS genes were identified from the pomegranate genome using a genome-wide search method. The PgSUS gene family was comprehensively analyzed by physicochemical properties, evolutionary relationship, gene structure, conserved motifs and domains, protein structure, syntenic relationships, and cis-acting elements using bioinformatics methods. The expression pattern of the PgSUS gene in different organs and fruit development stages were assayed with RNA-seq obtained from the NCBI SRA database as well as real-time quantitative polymerase chain reaction (qPCR). Results Five pomegranate SUS genes, located on four different chromosomes, were divided into three subgroupsaccording to the classification of other seven species. The PgSUS family was found to be highly conserved during evolution after studying the gene structure, motifs, and domain analysis. Furthermore, the predicted PgSUS proteins showed similar secondary and tertiary structures. Syntenic analysis demonstrated that four PgSUS genes showed syntenic relationships with four species, with the exception of PgSUS2. Predictive promoter analysis indicated that PgSUS genes may be responsive to light, hormone signaling, and stress stimulation. RNA-seq analysis revealed that PgSUS1/3/4 were highly expressed in sink organs, including the root, flower, and fruit, and particularly in the outer seed coats. qPCR analysis showed also that PgSUS1, PgSUS3, and PgSUS4 were remarkably expressed during fruit seed coat development. Our results provide a systematic overview of the PgSUS gene family in pomegranate, developing the framework for further research and use of functional PgSUS genes.

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

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

Enhance Fruit Ripening Uniformity and Accelerate the Rutab Stage by Using ATP in ‘Zaghloul’ Dates during the Shelf Life

The Rutab date involves a physiological process by which the fruit turns completely ripe. The objective of this study was to research the effect of ATP-treated fruit to improve their biologically active compounds of the Rutab process of the ‘Zagloul’ date during shelf-life. Fruits at full color (red) were dipped in 0, 1, 1.5 mmol L−1 ATP solution for 10 min, and then stored at room temperature (27 ± 1 °C) with a relative humidity of (67 ± 4 RH%) for 12 d. We found that ATP treatment, especially at 1.5 mM, enhances the Rutab stage of date fruit, and certain biologically active compounds such as total phenols and flavonoids, in all ATP treatments compared to untreated fruits. ATP enhanced the loss of tannin compounds in fruit but had no impact on the change in fruit moisture percentage of fruit during storage. The treatments did affect the changes in total sugar content and activated the sucrose enzymes, i.e., acid invertase (AI), neutral invertase (NI), sucrose synthase-cleavage (SS-c), and sucrose synthase-synthesis (SS-s) during storage. Interestingly, immersion in 1.5 mM ATP forced the date fruit to reach the Rutab stage during storage. These results indicated that the dose of ATP (1.5 mM) is a new potential tool that pushes the fruits to regular ripening after harvest, thus reducing the losses in the fruits during the production process. A linear model could be satisfactorily used for predicting the properties of the treated date with ATP 1.5 mM at different shelf-life durations.

Olive Varieties under UV-B Stress Show Distinct Responses in Terms of Antioxidant Machinery and Isoform/Activity of RubisCO

In recent decades, atmospheric pollution led to a progressive reduction of the ozone layer with a consequent increase in UV-B radiation. Despite the high adaptation of olive trees to the Mediterranean environment, the progressive increase of UV-B radiation is a risk factor for olive tree cultivation. It is therefore necessary to understand how high levels of UV-B radiation affect olive plants and to identify olive varieties which are better adapted. In this study we analyzed two Italian olive varieties subjected to chronic UV-B stress. We focused on the effects of UV-B radiation on RubisCO, in terms of quantity, enzymatic activity and isoform composition. In addition, we also analyzed changes in the activity of antioxidant enzymes (SOD, CAT, GPox) to get a comprehensive picture of the antioxidant system. We also evaluated the effects of UV-B on the enzyme sucrose synthase. The overall damage at biochemical level was also assessed by analyzing changes in Hsp70, a protein triggered under stress conditions. The results of this work indicate that the varieties (Giarraffa and Olivastra Seggianese) differ significantly in the use of specific antioxidant defense systems, as well as in the activity and isoform composition of RubisCO. Combined with a different use of sucrose synthase, the overall picture shows that Giarraffa optimized the use of GPox and opted for a targeted choice of RubisCO isoforms, in addition to managing the content of sucrose synthase, thereby saving energy during critical stress points.

Highly Efficient Biosynthesis of Glycyrrhetinic Acid Glucosides by Coupling of Microbial Glycosyltransferase to Plant Sucrose Synthase

Glycyrrhetinic acid (GA) is a principal bioactive pentacyclic triterpenoid from Glycyrrhiza uralensis. Uridine diphosphate-dependent glycosyltransferases (UGTs) have been widely used to catalyze glycosylation of diverse nature products for the development of potential therapeutic compounds. In this study, we have characterized a UGT109A3 from Bacillus subtilis, which can glycosylate both the free C3 hydroxyl and C30 carboxyl groups of GA to yield a unique 3, 30-O-β-D-diglucoside-GA. By coupling the microbial UGT109A3 to plant sucrose synthase (SUS), GA-diglucoside could be biosynthesized in an efficient and economical way. With a fed-batch glycosylation, a large scale of GA-diglucoside (6.26 mM, 4.98 g/L in 8 h) could be enzymatically transformed from GA. The obtained GA-diglucoside showed a significant water solubility improvement of around 3.4 × 103 fold compared with that of the parent GA (29 μM). Moreover, it also exhibited dose-dependent cytotoxicity toward human colon carcinoma Caco-2 cell line according to MTT assay, having an IC50 at 160 μM. This study not only establishes efficient platform for producing GA-glucosides, but is also valuable for developing further the biosynthesis of other complex glycosylated natural products.

Structure and Expression Analysis of Sucrose Phosphate Synthase, Sucrose Synthase and Invertase Gene Families in Solanum lycopersicum

Sucrose phosphate synthase (SPS), sucrose synthase (SUS) and invertase (INV) are all encoded by multigene families. In tomato (Solanum lycopersicum), a comprehensive analysis of structure characteristics of these family genes is still lacking, and the functions of individual isoforms of these families are mostly unclear under stress. Here, the structure characteristics of the three families in tomato were analyzed; moreover, as a first step toward understanding the functions of isoforms of these proteins under stress, the tissue expression pattern and stress response of these genes were also investigated. The results showed that four SPS genes, six SUS genes and nineteen INV genes were identified in tomato. The subfamily differentiation of SlSPS and SlSUS might have completed before the split of monocotyledons and dicotyledons. The conserved motifs were mostly consistent within each protein family/subfamily. These genes demonstrated differential expressions among family members and tissues, and in response to polyethylene glycerol, NaCl, H2O2, abscisic acid or salicylic acid treatment. Our results suggest that each isoform of these families may have different functions in different tissues and under environmental stimuli. SlSPS1, SlSPS3, SlSUS1, SlSUS3, SlSUS4, SlINVAN5 and SlINVAN7 demonstrated consistent expression responses and may be the major genes responding to exogenous stimuli.

Comparative transcriptome analysis reveals key genes potentially related to organic acid and sugar accumulation in loquat

Organic acids and sugars are the primary components that determine the quality and flavor of loquat fruits. In the present study, major organic acids, sugar content, enzyme activities, and the expression of related genes were analyzed during fruit development in two loquat cultivars, ’JieFangZhong’ (JFZ) and ’BaiLi’ (BL). Our results showed that the sugar content increased during fruit development in the two cultivars; however, the organic acid content dramatically decreased in the later stages of fruit development. The differences in organic acid and sugar content between the two cultivars primarily occured in the late stage of fruit development and the related enzymes showed dynamic changes in activies during development. Phosphoenolpyruvate carboxylase (PEPC) and mNAD malic dehydrogenase (mNAD-MDH) showed higher activities in JFZ at 95 days after flowering (DAF) than in BL. However, NADP-dependent malic enzyme (NADP-ME) activity was the lowest at 95 DAF in both JFZ and BL with BL showing higher activity compared with JFZ. At 125 DAF, the activity of fructokinase (FRK) was significantly higher in JFZ than in BL. The activity of sucrose synthase (SUSY) in the sucrose cleavage direction (SS-C) was low at early stages of fruit development and increased at 125 DAF. SS-C activity was higher in JFZ than in BL. vAI and sucrose phosphate synthase (SPS) activities were similar in the two both cultivars and increased with fruit development. RNA-sequencing was performed to determine the candidate genes for organic acid and sugar metabolism. Our results showed that the differentially expressed genes (DEGs) with the greated fold changes in the later stages of fruit development between the two cultivars were phosphoenolpyruvate carboxylase 2 (PEPC2), mNAD-malate dehydrogenase (mNAD-MDH), cytosolic NADP-ME (cyNADP-ME2), aluminum-activated malate transporter (ALMT9), subunit A of vacuolar H+-ATPase (VHA-A), vacuolar H+-PPase (VHP1), NAD-sorbitol dehydrogenase (NAD-SDH), fructokinase (FK), sucrose synthase in sucrose cleavage (SS-C), sucrose-phosphate synthase 1 (SPS1), neutral invertase (NI), and vacuolar acid invertase (vAI). The expression of 12 key DEGs was validated by quantitative reverese transcription PCR (RT-qPCR). Our findings will help understand the molecular mechanism of organic acid and sugar formation in loquat, which will aid in breeding high-quality loquat cultivars.