The SlIMP3 has potential as an important candidate gene for molecular breeding with the goal of improving shelf-life of tomato fruit. Interestingly, treating tomato fruits with myoinositol also improved cell wall bio-genesis, delayed softening, and extended shelf-life in fruits. Litchi fruit lost less water after myoinositol treatment . Collectively, all these results demonstrate that myoinositol has strong potential for improving tomato post harvest life. Does the increased AsA biosynthesis contribute to the cell wall bio-genesis and delayed fruit softening? The SlDHAR gene, that is responsible for recycling of AsA, was overexpressed in AC tomato. The SlDHAR-overexpressed plants exhibited higher AsA contents compared with the WT plants. However, cell wall thickness, fruit firmness, wall loss, and softening time in the SlDHAR-overexpressed plants were similar to those in WT plants . The data presented here corroborates that the increased AsA content does not delay softening and or prolonged shelf-life in tomato fruit.B. cinerea is a necrotrophic fungal pathogen leading to grey mould rot. It is among the most destructive postharvest pathogens of fruit .
Causing huge economic losses, plant plastic pots genetic modification has been attempted to control this postharvest pathogen. The cell wall is an important barrier to pathogen infections . Simultaneous downregulation of PG and Exp1 genes in tomato fruit reduced cell wall breakdown and susceptibility to B. cinerea . Suppression of SlPL in tomato resulted in increased fruit firmness and reduced susceptibility to B. cinerea . Overexpression of the carbohydratebinding module of expansin 1 in tomato increased fruit firmness and decreased susceptibility to B. cinerea . In this study, overexpression of SlIMP3 increased cell wall thickness and improved fruit resistance to B. cinerea . The increased cell wall thickness serves to retain inhibition of B. cinerea infection. Our data corroborated that the cell wall modification is an effective strategy for improving fruit tolerance of postharvest pathogens. In addition, the increased cell wall thickness was not only found in SlIMP3-overexpressed fruit, but also in SlIMP3-overexpressed leaf and stem . Therefore, we speculate that SlIMP3 may confer resistance to broad-spectrum pathogens in tomato. In conclusion, SlIMP3 is a bifunctional enzyme with the ability to regulate AsA and myoinositol biosynthesis. Overexpressing SlIMP3 in tomato not only increased AsA accumulation, but also delayed the fruit softening and enhanced tolerance to B. cinerea, suggesting the potential value of SlIMP3 in plant improvement programmes with the goal of improving postharvest fruit life.
Citrus is one of the most important and widely grown commodity fruit crops . Citrus has a non-climacteric fruit maturation behaviour and a unique anatomical fruit structure . The fruit contains two peel tissues, flavedo and albedo. The flavedo accumulates pigments and compounds which contribute to the fruit aroma, while the albedo comprises spongy cells rich in pectin. During the early stages of fruit development the albedo occupies most of the fruit volume and it becomes gradually thinner during fruit development as the juice cells in the pulp grow . Growth and development of the citrus fruit can be divided into three major stages . Stage I starts immediately after fruit set and is characterized by extensive cell division. During the transition to stage II, cell division ceases in all fruit tissues except the outermost flavedo layers and the tips of the juice sacs. During this stage, citrus fruit grows through cell expansion. Juice sac cell enlargement is mostly driven by the expansion of the vacuole, which occupies most of the cell volume. Stage III is the fruit maturation and ripening stage when fruit growth slows down and the pulp reaches its final size. Citrus fruit development is characterized by changes in primary and secondary metabolite content, with sugars and citric acid being the major components of the juice sac cells. Sucrose is translocated to the fruits from the leaves throughout fruit development, and constitutes about 50% of the total soluble sugars.
The anatomy of the citrus fruit, where the juice sacs are disconnected from the vascular bundles present in the albedo, suggest apoplastic sucrose downloading . Sucrose can then be hydrolysed by cytosolic invertases or stored inCitrus is one of the most important and widely grown commodity fruit crops . Citrus has a non-climacteric fruit maturation behaviour and a unique anatomical fruit structure . The fruit contains two peel tissues, flavedo and albedo. The flavedo accumulates pigments and compounds which contribute to the fruit aroma, while the albedo comprises spongy cells rich in pectin. During the early stages of fruit development the albedo occupies most of the fruit volume and it becomes gradually thinner during fruit development as the juice cells in the pulp grow . Growth and development of the citrus fruit can be divided into three major stages . Stage I starts immediately after fruit set and is characterized by extensive cell division. During the transition to stage II, cell division ceases in all fruit tissues except the outermost flavedo layers and the tips of the juice sacs. During this stage, citrus fruit grows through cell expansion. Juice sac cell enlargement is mostly driven by the expansion of the vacuole, which occupies most of the cell volume. Stage III is the fruit maturation and ripening stage when fruit growth slows down and the pulp reaches its final size. Citrus fruit development is characterized by changes in primary and secondary metabolite content, with sugars and citric acid being the major components of the juice sac cells. Sucrose is translocated to the fruits from the leaves throughout fruit development, and constitutes about 50% of the total soluble sugars. The anatomy of the citrus fruit, where the juice sacs are disconnected from the vascular bundles present in the albedo, suggest apoplastic sucrose downloading . Accumulation of citric acid in the vacuole of the juice sac cells is correlated with vacuole acidification mediated by the proton pumping activity of the tonoplastic H+ -ATPase. Citrate begins to accumulate during the second phase of fruit development. The accumulation continues for a few weeks, reaching a peak when the fruit volume is about 50% of its final value and then acid declines gradually as the fruit matures . Citrate decline during the second half of fruit development is associated with the activity of CsCit1, a H+ /citrate symporter . It has been suggested that some of the citrate is targeted for amino acid biosynthesis generally induced during the second half of fruit development . Indeed, black plastic pots there is an increase in some amino acid metabolizing genes, including those of the GABA shunt, and their corresponding enzymes during the citrate decline stage . In the last few years, studies using transcriptome analysis and metabolite profiling demonstrated a tight regulation of fruit metabolism during fruit maturation . However, comparison of mRNA expression levels, proteins amounts, and enzymatic activities have revealed low correlations between metabolome and transcriptome, indicating that transcriptome analysis was not sufficient to understand protein dynamics or biochemical regulation . A more direct correlation is expected for proteins and metabolites and, therefore, quantitative mass spectrometric proteomics and metabolomics are becoming attractive approaches. Quantitative proteomics has been used for the quantification of complex biological samples . Previously, LC-MS/MS was used to identify the proteome of various cellular fractions of the juice sac cell . More recently, a label-free differential quantitative mass spectrometry method was developed to follow protein changes in citrus juice sac cells. Two alternative methods, differential mass-spectrometry and spectral counting were used to analyse the protein changes occurring during the earlier and late stages of fruit development . Along with the generation of a novel bio-informatics tool, iCitrus, the above method enabled the identification of approximately 1500 citrus proteins expressed in fruit juice sac cells and the quantification of changes in their expression during fruit development. In this study, label-free LC-MS/MS-based shot-gun proteomic and metabolomic approaches were utilized to investigate citrus fruit development.
These tools were used to identify and evaluate changes occurring in the metabolic pathways of juice sac cells which affect citrus fruit development and quality. Integration of proteomic and metabolomic analyses created a more comprehensive overview of changes in protein expression and metabolite composition of primary metabolism during citrus fruit development and maturation.An extensive comparative proteomics study was conducted in order to identify protein changes occurring during citrus fruit growth and development. Samples were collected from three developmental stages; early stage II, stage II, and stage III . For proteomics analysis, two biological repetitions from two consecutive years were collected from at least 20 pooled fruits for each stage . For gene expression, enzyme activities, and metabolome analysis, three biological repetitions of three consecutive years were analysed. For better identification of differentially expressed proteins during fruit development and to decrease sample complexity, the juice sac cells were fractionated into soluble and membrane-bound proteins . Changes in protein expression were revealed by comparisons between spectra originated from fruit juice sac cells at different stages: stage II versus early stage II and stage III versus stage II. The complete data of the differential proteins detected can be found in Supplementary Tables S1 and S2 at JXB online. The analysis revealed a significant metabolic change occurring during the transition from early stage II to stage II and from stage II to stage III . Although these changes involved a wide range of processes, this study focuses on protein changes related to primary metabolism. Processes involving sugar metabolism, the TCA cycle, amino acid metabolism, energy production, and cell wall related metabolism changed significantly in citrus juice sac cells during fruit development. Citrus fruit accumulate large amounts of sugars, mainly sucrose, glucose, and fructose. Enzymes participating in sucrose metabolism were highly represented in the proteome analysis of citrus fruit juice sac cells. Most of the enzymes involved in sucrose degradation and glycolytic pathways were up-regulated during the transition from early stage II to stage II and were upregulated toward maturation, emphasizing the regulatory role of glycolysis in sugar utilization to drive fruit growth during citrus fruit development . Hexokinase, fructokinase, glucose-6-phosphate isomerase, fructosebisphosphate aldolase, ATP-dependent 6-phosphofructose- 1-kinase, triosephosphate isomerase, and enolase protein expression did not change significantly during the early stages and were up-regulated during the transition from stage II to stage III. UDP-glucose pyrophosphorylase, phosphoglucomutase, glyceraldehyde-3-phosphate dehydrogenase, 2,3-biphosphoglycerate-independent phosphoglycerate mutase, phosphoglycerate kinase, phosphoenolpyruvate carboxylase, and phosphoenolpyruvate carboxykinase were up-regulated throughout fruit development. Two pyruvate kinases were identified: iCitrus ID 52671 that did not change during the transition from early stage II to stage II and iCitrus ID 28935 that was up-regulated at stage II compared witho early stage II. Both proteins were upregulated during the transition from stage II to stage III. Sucrose synthase was found to be an interesting exception, since it was down-regulated during the transition from early stage II to stage II, and was up-regulated nearer to maturation . Four citrus sucrose synthase isoforms derived from four different unigenes were identified and clustered into three groups according to their sequence homology. Group 1 consisted of isoforms with homology to unigenes related to CitSUSA , group 2 consisted of proteins derived from unigenes related to CitSUS1 , and group 3 comprised CitSUS4, shown in this study to be expressed in the fruit. The expression patterns of CitSUS1 and CitSUSA were in agreement with their corresponding transcripts and with previously characterized enzymatic activities . The CitSUS1 gene was shown to be expressed in the early stages of fruit development and its expression decreased towards maturation, while the CitSUSA gene was up-regulated towards maturation.In this study, it is shown that the amounts of both CitSUS1 isoforms decreased in the transition from early stage II to stage II while that of iCitrus ID 33038 increased during the transition from stage II to stage III similar to CitSUSA which was up-regulated towards maturation, in agreement with the gene expression profiles and enzyme activity . In addition, CitSUS4 was found to be significantly down-regulated between early stage II and stage II and was not detected in the later stage of fruit development , thus indicating that its amounts remained constant. This reaction catalyses the interconversion of fructose-6-phosphate and fructose-1,6-bisphosphate. While most glycolytic enzymes are highly conserved between organisms, two types of phosphofructokinase isoforms exist in plants . In addition to the ATP-dependent phosphofructokinase , a pyrophosphate-fructose-6-phosphate-phosphotransferase uses pyrophosphate as the phosphoryl donor.