Deciphering Ascorbic Acid Regulatory Pathways in Ripening Tomato Fruit Using a Weighted Gene Correlation Network Analysis Approach

Genotype is generally determined by the co-expression of diverse genes and multiple regulatory pathways in plants. Gene co-expression analysis combining with physiological trait data provides very important information about the gene function and regulatory mechanism. L-Ascorbic acid （AsA）, which is an essential nutrient component for human health and plant metabolism, plays key roles in diverse biological processes such as cell cycle, cell expansion, stress resistance, hormone synthesis, and signaling. Here, we applied a weighted gene correlation network analysis approach based on gene expression values and AsA content data in ripening tomato （Solanum lycopersicum L.） fruit with different AsA content levels, which leads to identification of AsA relevant modules and vital genes in AsA regulatory pathways. Twenty- four modules were compartmentalized according to gene expression profiling. Among these modules, one negatively related module containing genes involved in redox processes and one positively related module enriched with genes involved in AsA biosynthetic and recycling pathways were further analyzed. The present work herein indicates that redox pathways as well as hormone-signal pathways are closely correlated with AsA accumulation in ripening tomato fruit, and allowed us to prioritize candidate genes for follow-up studies to dissect this interplay at the biochemical and molecular level.     

High Temperature Inhibits Ascorbate Recycling and Light Stimulation of the Ascorbate Pool in Tomato despite Increased Expression of Biosynthesis Genes

Understanding how the fruit microclimate affects ascorbate (AsA) biosynthesis, oxidation and recycling is a great challenge in improving fruit nutritional quality. For this purpose, tomatoes at breaker stage were harvested and placed in controlled environment conditions at different temperatures (12, 17, 23, 27 and 31°C) and irradiance regimes (darkness or 150 µmol m^-2 s^-1). Fruit pericarp tissue was used to assay ascorbate, glutathione, enzymes related to oxidative stress and the AsA/glutathione cycle and follow the expression of genes coding for 5 enzymes of the AsA biosynthesis pathway (GME, VTC2, GPP, L-GalDH, GLDH). The AsA pool size in pericarp tissue was significantly higher under light at temperatures below 27°C. In addition, light promoted glutathione accumulation at low and high temperatures. At 12°C, increased AsA content was correlated with the enhanced expression of all genes of the biosynthesis pathway studied, combined with higher DHAR and MDHAR activities and increased enzymatic activities related to oxidative stress (CAT and APX). In contrast, at 31°C, MDHAR and GR activities were significantly reduced under light indicating that enzymes of the AsA/glutathione cycle may limit AsA recycling and pool size in fruit pericarp, despite enhanced expression of genes coding for AsA biosynthesis enzymes. In conclusion, this study confirms the important role of fruit microclimate in the regulation of fruit pericarp AsA content, as under oxidative conditions (12°C, light) total fruit pericarp AsA content increased up to 71%. Moreover, it reveals that light and temperature interact to regulate both AsA biosynthesis gene expression in tomato fruits and AsA oxidation and recycling.     

Ascorbate biosynthesis during early fruit development is the main reason for its accumulation in kiwi

Background

Ascorbic acid (AsA) is a unique antioxidant as well as an enzyme cofactor. Although it has multiple roles in plants, it is unclear how its accumulation is controlled at the expression level, especially in sink tissues. Kiwifruit (Actinidia) is well-known for its high ascorbate content. Our objective was to determine whether AsA accumulates in the fruits primarily through biosynthesis or because it is imported from the foliage.

Methodology/Principal Findings

We systematically investigated AsA levels, biosynthetic capacity, and mRNA expression of genes involved in AsA biosynthesis in kiwi (A. deliciosa cv. Qinmei). Recycling and AsA localization were also monitored during fruit development and among different tissue types. Over time, the amount of AsA, with its capacity for higher biosynthesis and lower recycling, peaked at 30 days after anthesis (DAA), and then decreased markedly up to 60 DAA before declining more slowly. Expression of key genes showed similar patterns of change, except for L-galactono-1,4-lactone dehydrogenase and L-galactose-1-phosphate phosphatase (GPP). However, GPP had good correlation with the rate of AsA accumulation. The expression of these genes could be detected in phloem of stem as well as petiole of leaf and fruit. Additionally, fruit petioles had greater ascorbate amounts, although that was the site of lowest expression by most genes. Fruit microtubule tissues also had higher AsA. However, exogenous applications of AsA to those petioles did not lead to its transport into fruits, and distribution of ascorbate was cell-specific in the fruits, with more accumulation occurring in larger cells.

Conclusions

These results suggest that AsA biosynthesis in kiwi during early fruit development is the main reason for its accumulation in the fruits. We also postulate here that GPP is a good candidate for regulating AsA biosynthesis whereas GDP-L-galactose-1-phosphate phosphorylase is not.     

Ethylene and fruit ripening

The latest advances in our understanding of the relationship between ethylene and fruit ripening are reviewed. Considerable progress has been made in the characterisation of genes encoding the key ethylene biosynthetic enzymes, ACC synthase (ACS) and ACC oxidase (ACO) and in the isolation of genes involved in the ethylene signal transduction pathway, particularly those encoding ethylene receptors ( ETR ). These have allowed the generation of transgenic fruit with reduced ethylene production and the identification of the Nr tomato ripening mutant as an ethylene receptor mutant. Through these tools, a clearer picture of the role of ethylene in fruit ripening is now emerging. In climacteric fruit, the transition to autocatalytic ethylene production appears to result from a series of events where developmentally regulated ACO and ACS gene expression initiates a rise in ethylene production, setting in motion the activation of autocatalytic ethylene production. Differential expression of ACS and ACO gene family members is probably involved in such a transition. Finally, we discuss evidence suggesting that the NR ethylene perception and transduction pathway is specific to a defined set of genes expressed in ripening climacteric fruit and that a distinct ETR pathway regulates other ethylene-regulated genes in both immature and ripening climacteric fruit as well as in non-climacteric fruit. The emerging picture is one where both ethylene-dependent and -independent pathways coexist in both climacteric and non-climacteric fruits. Further work is needed in order to dissect the molecular events involved in individual ripening processes and to understand the regulation of the expression of both ethylene-dependent and -independent genes.     

Proanthocyanidin synthesis and expression of genes encoding leucoanthocyanidin reductase and anthocyanidin reductase in developing grape berries and grapevine leaves

Proanthocyanidins (PAs), also called condensed tannins, can protect plants against herbivores and are important quality components of many fruits. Two enzymes, leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR), can produce the flavan-3-ol monomers required for formation of PA polymers. We isolated and functionally characterized genes encoding both enzymes from grapevine (Vitis vinifera L. cv Shiraz). ANR was encoded by a single gene, but we found two highly related genes encoding LAR. We measured PA content and expression of genes encoding ANR, LAR, and leucoanthocyanidin dioxygenase in grape berries during development and in grapevine leaves, which accumulated PA throughout leaf expansion. Grape flowers had high levels of PA, and accumulation continued in skin and seeds from fruit set until the onset of ripening. VvANR was expressed throughout early flower and berry development, with expression increasing after fertilization. It was expressed in berry skin and seeds until the onset of ripening, and in expanding leaves. The genes encoding LAR were expressed in developing fruit, particularly in seeds, but had low expression in leaves. The two LAR genes had different patterns of expression in skin and seeds. During grape ripening, PA levels decreased in both skin and seeds, and expression of genes encoding ANR and LAR were no longer detected. The results indicate that PA accumulation occurs early in grape development and is completed when ripening starts. Both ANR and LAR contribute to PA synthesis in fruit, and the tissue and temporal-specific regulation of the genes encoding ANR and LAR determines PA accumulation and composition during grape berry development.     

Characterization of mitochondrial dicarboxylate/tricarboxylate transporters from grape berries

Grape berries (Vitis vinifera L fruit) exhibit a double-sigmoid pattern of development that results from two successive periods of vacuolar swelling during which the nature of accumulated solutes changes significantly. Throughout the first period, called green or herbaceous stage, berries accumulate high levels of organic acids, mainly malate and tartrate. At the cellular level fruit acidity comprises both metabolism and vacuolar storage. Malic acid compartmentation is critical for optimal functioning of cytosolic enzymes. Therefore, the identification and characterization of the carriers involved in malate transport across sub-cellular compartments is of great importance. The decrease in acid content during grape berry ripening has been mainly associated to mitochondrial malate oxidation. However, no Vitis vinifera mitochondrial carrier involved in malate transport has been reported to date. Here we describe the identification of three V. vinifera mitochondrial dicarboxylate/tricarboxylate carriers (VvDTC1-3) putatively involved in mitochondrial malate, citrate and other di/tricarboxylates transport. The three VvDTCs are very similar, sharing a percentage of identical residues of at least 83 %. Expression analysis of the encoding VvDTC genes in grape berries shows that they are differentially regulated exhibiting a developmental pattern of expression. The simultaneous high expression of both VvDTC2 and VvDTC3 in grape berry mesocarp close to the onset of ripening suggests that these carriers might be involved in the transport of malate into mitochondria.     

Ascorbic acid formation and profiling of genes expressed in its synthesis and recycling in apple leaves of different ages

Ascorbic acid (AsA), as a unique antioxidant and enzyme cofactor, has multiple roles in plants. However, there is very limited information on the mechanism of AsA accumulation and controlling in leaves. In this study, we determined AsA accumulation levels, analyzed expression patterns of the genes involved in synthesizing via l-galactose pathway and recycling as well as enzyme activities in apple (Malus domestica Borkh) leaves with different age. AsA content was found to increase with leaf development, reaching the highest level in 20-day-old leaves. This level was maintained in mature leaves until the dropping in senescent leaves. Comparing with young and senescent leaves, mature leaves had higher capability for AsA synthesis with high expression levels and activity of l-galactose dehydrogenase and l-galactono-1,4-lactone dehydrogenase. The mRNA expression of genes involved in AsA synthesis also showed highest abundance in 20-day-old leaves, though GDP-mannose-3′,5′-epimerase and l-galactose-1-phosphate phosphatase expression reached the highest levels before 20 days old. These results suggest that AsA accumulation in apple leaves mainly occurs during the transition phase from young to mature leaves with high rates of synthesis and recycling, and that l-galactose-1-phosphate phosphatase could play an important role in regulating AsA biosynthesis via the l-galactose pathway.     

Application of Pseudomonas fluorescens to Blackberry under Field Conditions Improves Fruit Quality by Modifying Flavonoid Metabolism

Application of a plant growth promoting rhizobacterium (PGPR), Pseudomonas fluorescens N21.4, to roots of blackberries (Rubus sp.) is part of an optimised cultivation practice to improve yields and quality of fruit throughout the year in this important fruit crop. Blackberries are especially rich in flavonoids and therefore offer potential benefits for human health in prevention or amelioration of chronic diseases. However, the phenylpropanoid pathway and its regulation during ripening have not been studied in detail, in this species. PGPR may trigger flavonoid biosynthesis as part of an induced systemic response (ISR) given the important role of this pathway in plant defence, to cause increased levels of flavonoids in the fruit. We have identified structural genes encoding enzymes of the phenylpropanoid and flavonoid biosynthetic pathways catalysing the conversion of phenylalanine to the final products including flavonols, anthocyanins and catechins from blackberry, and regulatory genes likely involved in controlling the activity of pathway branches. We have also measured the major flavonols, anthocyanins and catechins at three stages during ripening. Our results demonstrate the coordinated expression of flavonoid biosynthetic genes with the accumulation of anthocyanins, catechins, and flavonols in developing fruits of blackberry. Elicitation of blackberry plants by treatment of roots with P.fluorescens N21.4, caused increased expression of some flavonoid biosynthetic genes and an accompanying increase in the concentration of selected flavonoids in fruits. Our data demonstrate the physiological mechanisms involved in the improvement of fruit quality by PGPR under field conditions, and highlight some of the genetic targets of elicitation by beneficial bacteria.