Inhibition of aconitase in citrus fruit callus results in a metabolic shift towards amino acid biosynthesis

Citrate, a major determinant of citrus fruit quality, accumulates early in fruit development and declines towards maturation. The isomerization of citrate to isocitrate, catalyzed by aconitase is a key step in acid metabolism. Inhibition of mitochondrial aconitase activity early in fruit development contributes to acid accumulation, whereas increased cytosolic activity of aconitase causes citrate decline. It was previously hypothesized that the block in mitochondrial aconitase activity, inducing acid accumulation, is caused by citramalate. Here, we investigated the effect of citramalate and of another aconitase inhibitor, oxalomalate, on aconitase activity and regulation in callus originated from juice sacs. These compounds significantly increased citrate content and reduced the enzyme’s activity, while slightly inducing its protein level. Citramalate inhibited the mitochondrial, but not cytosolic form of the enzyme. Its external application to mandarin fruits resulted in inhibition of aconitase activity, with a transient increase in fruit acidity detected a few weeks later. The endogenous level of citramalate was analyzed in five citrus varieties: its pattern of accumulation challenged the notion of its action as an endogenous inhibitor of mitochondrial aconitase. Metabolite profiling of oxalomalate-treated cells showed significant increases in a few amino acids and organic acids. The activities of alanine transaminase, aspartate transaminase and aspartate kinase, as well as these of two γ-aminobutyrate (GABA)-shunt enzymes, succinic semialdehyde reductase (SSAR) and succinic semialdehyde dehydrogenase (SSAD) were significantly induced in oxalomalate-treated cells. It is suggested that the increase in citrate, caused by aconitase inhibition, induces amino acid synthesis and the GABA shunt, in accordance with the suggested fate of citrate during the acid decline stage in citrus fruit.     

Recent Advances in the Regulation of Citric Acid Metabolism in Citrus Fruit

The regulation of citric acid metabolism during fruit ripening has a major impact on the production of high-quality fruit. The impact of citric acid on organoleptic fruit quality attributes, fruit storage performance and the synthesis of several secondary metabolites has led to an exponential increase in research efforts during the last two decades. Recent research has focused on the relationship among citric acid biosynthesis, transportation, storage, and utilization. Among citrate metabolic processes, activities of a proton pump, especially the plasma membrane H⁺-ATPase on tonoplast and citrate catabolism in cytosol play important roles in the regulation of citrate accumulation in cell vacuoles. Moreover, we highlight recent advances and provide an overview of citrate metabolism, postharvest physiology of citrate metabolism, and the influence of agro-climatic factors on citrus fruits. It is the first review that provides a comprehensive model for citrate metabolism in citrus fruit juice sacs. We anticipate that this model for the regulation of citrate metabolism will facilitate the study of fruit acidity in citrus and other nonclimacteric fruits.     

Iron-shortage-induced increase in citric acid content and reduction of cytosolic aconitase activity in Citrus fruit vesicles and calli

Aconitase, which catalyses the conversion of citrate into isocitrate, requires Fe for its activity. The yeast and animal enzyme loses its enzymatic activity under Fe shortage and binds to RNA of genes involved in Fe homeostasis, altering their expression. Thus, the enzyme provides a regulatory link between organic acid metabolism and Fe cellular status. Roots and leaves of Fe-deficient plants show induction in organic acids, especially citrate. Although no RNA-binding activity has been so far demonstrated for the plant aconitase, whether alternations in enzyme activity by Fe could play a role in this induction remain unanswered. This question was investigated in lemon fruit [ Citrus limon (L.) Burm var Eureka ], characterized by the accumulation of citrate to about 0.3 M in the juice vesicles cells (pulp). Calli and isolated juice vesicles showed two- to three-fold induction in citrate level when subjected to Fe shortage. The mRNA level of aconitase exhibited no changes under reduced Fe concentrations. Analysis of aconitase isozymes demonstrated that out of two aconitase isozymes, typically detected in citrus fruit, only the cytosolic form displayed a reduced activity under low Fe concentrations. Our data support the notion of a limited Fe-availability-induced reduction in cytosolic aconitase, resulting in a slower rate of citrate breakdown and a concomitant increase in citrate levels.     

Proteome of the phytopathogen <Emphasis Type="Italic">Xanthomonas citri</Emphasis> subsp. <Emphasis Type="Italic">citri</Emphasis>: a global expression profile

Background  

Citrus canker is a disease caused by Xantomonas citri subsp.citri (Xac), and has emerged as one of the major threats to the worldwide citrus crop because it affects all commercial citrus varieties, decreases the production and quality of the fruits and can spread rapidly in citrus growing areas. In this work, the first proteome of Xac was analyzed using two methodologies, two-dimensional liquid chromatography (2D LC) and tandem mass spectrometry (MS/MS).     

Regulation of the expression of lipoxygenase genes in <Emphasis Type="Italic">Prunus persica</Emphasis> fruit ripening

Three genes of the lipoxygenase (LOX) family in peach (Prunus persica var. compressa cv. Ruipan 4) were cloned, and their expression patterns during fruit ripening were analyzed using real-time quantitative PCR. All of the three peach LOX genes had been expressed during fruit ripening; however, their expression patterns were significantly different. During the normal ripening of peach fruits, the expression levels of PpLox1, PpLox2 and PpLox3 increased in varying degrees accompanying upsurge of ethylene evolution. After treated by methyl jasmonic acid (MeJA), the peak of ethylene releasing occurred in advance, and the declining rate of fruit hardness was accelerated, the expression level of the three peach LOX genes in fruits markedly enhanced at the early stage of storage, but significantly decreased at the late storage stage. So, it could be suggested that all three LOXs relate to fruit ripening; however, their functions might be different. PpLox1 expression increase along with the upsurge of ethylene evolution in both control and MeJA-treated peach fruits suggested that PpLox1 probably played a major role in the peach fruit ripening. Expression peak of PpLox2 appeared at the 1 DAH (days after harvest) in both control and MeJA-treated peach fruits, while obvious changes in ethylene evolution and fruit hardness was not observed, which suggested that the rise of PpLox2 expression can be induced by certain stimulation related to ripening, such as harvesting stress and MeJA treatment. The expression of PpLox3 kept a lower level in the natural ripening fruits, whereas raced up at the early stage of storage in the fruits treated with MeJA, which indicated that PpLox3 was expressed inductively and had minor roles during the normal ripening of peach fruits, but when encountered with external stimulation, its expression level would rapidly enhance and accelerate the ripening of peach fruit.     

Downregulation of RdDM during strawberry fruit ripening

Background

Recently, DNA methylation was proposed to regulate fleshy fruit ripening. Fleshy fruits can be distinguished by their ripening process as climacteric fruits, such as tomatoes, or non-climacteric fruits, such as strawberries. Tomatoes undergo a global decrease in DNA methylation during ripening, due to increased expression of a DNA demethylase gene. The dynamics and biological relevance of DNA methylation during the ripening of non-climacteric fruits are unknown.

Results

Here, we generate single-base resolution maps of the DNA methylome in immature and ripe strawberry. We observe an overall loss of DNA methylation during strawberry fruit ripening. Thus, ripening-induced DNA hypomethylation occurs not only in climacteric fruit, but also in non-climacteric fruit. Application of a DNA methylation inhibitor causes an early ripening phenotype, suggesting that DNA hypomethylation is important for strawberry fruit ripening. The mechanisms underlying DNA hypomethylation during the ripening of tomato and strawberry are distinct. Unlike in tomatoes, DNA demethylase genes are not upregulated during the ripening of strawberries. Instead, genes involved in RNA-directed DNA methylation are downregulated during strawberry ripening. Further, ripening-induced DNA hypomethylation is associated with decreased siRNA levels, consistent with reduced RdDM activity. Therefore, we propose that a downregulation of RdDM contributes to DNA hypomethylation during strawberry ripening.

Conclusions

Our findings provide new insight into the DNA methylation dynamics during the ripening of non-climacteric fruit and suggest a novel function of RdDM in regulating an important process in plant development.

     

A transcriptomic approach highlights induction of secondary metabolism in citrus fruit in response to <Emphasis Type="Italic">Penicillium digitatum</Emphasis>infection

Background  

Postharvest losses of citrus fruit due to green mold decay, caused by the fungus Penicillium digitaum, have a considerable economic impact. However, little is known about the molecular processes underlying the response of citrus fruit to P. digitatum.     

PineappleDB: An online pineapple bioinformatics resource

Background  

A world first pineapple EST sequencing program has been undertaken to investigate genes expressed during non-climacteric fruit ripening and the nematode-plant interaction during root infection. Very little is known of how non-climacteric fruit ripening is controlled or of the molecular basis of the nematode-plant interaction. PineappleDB was developed to provide the research community with access to a curated bioinformatics resource housing the fruit, root and nematode infected gall expressed sequences.     

Regulation of tomato fruit ascorbate content is more highly dependent on fruit irradiance than leaf irradiance

Background and Aims

The mechanisms involving light control of vitamin C content in fruits are not yet fully understood. The present study aimed to evaluate the impact of fruit and leaf shading on ascorbate (AsA) accumulation in tomato fruit and to determine how fruit sugar content (as an AsA precursor) affected AsA content.

Methods

Cherry tomato plants were grown in a glasshouse. The control treatment (normally irradiated fruits and irradiated leaves) was compared with the whole-plant shading treatment and with leaf or fruit shading treatments in fruits harvested at breaker stage. In a second experiment, the correlation between sugars and AsA was studied during ripening.

Key Results

Fruit shading was the most effective treatment in reducing fruit AsA content. Under normal conditions, AsA and sugar content were correlated and increased with the ripening stage. Reducing fruit irradiance strongly decreased the reduced AsA content (−74 %), without affecting sugars, so that sugar and reduced AsA were no longer correlated. Leaf shading delayed fruit ripening: it increased the accumulation of oxidized AsA in green fruits (+98 %), whereas it decreased the reduced AsA content in orange fruits (−19 %), suggesting that fruit AsA metabolism also depends on leaf irradiance.

Conclusions

Under fruit shading only, the absence of a correlation between sugars and reduced AsA content indicated that fruit AsA content was not limited by leaf photosynthesis or sugar substrate, but strongly depended on fruit irradiance. Leaf shading most probably affected fruit AsA content by delaying fruit ripening, and suggested a complex regulation of AsA metabolism which depends on both fruit and leaf irradiance and fruit ripening stage.Key words: Ascorbate, fruit quality, irradiance, shading, Solanum lycopersicon, sugars, tomato, vitamin C     

Transformation of a marker-free and vector-free antisense ACC oxidase gene cassette into melon via the pollen-tube pathway

Purpose of work  

Melons have short shelf-lives due to fruit ripening caused by ethylene production. The 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase gene is essential for ethylene biosynthesis. As fruit ripening in other fruit crops can be deterred by down-regulation of ACC oxidase expression, we have carried out similar work to improve fruit quality and shelf-life of the melon Cucumis melo.     

Tissue-specific organic acid metabolism in reproductive and non-reproductive parts of the fig fruit is partially induced by pollination

Organic acids are important components of overall fruit quality through flavor, taste, nutritional and medicinal values. Pollinated fig (Ficus carica L.) fruit quality is enhanced by increased acidity. We quantified the major organic acids and characterized the expression pattern of organic acid metabolic pathway-related genes in the reproductive part – inflorescence and non-reproductive part – receptacle of parthenocarpic and pollinated fig fruit during ripening. Essentially, pollinated fruit contains seeds in the inflorescence, as opposed to no seeds in the parthenocarpic inflorescence. The major organic acids – citrate and malate – were found in relatively high quantities in the inflorescence compared to the receptacle of both parthenocarpic and pollinated fig fruit. Notably, pollination increased citric acid content significantly in both inflorescence and receptacle. Genes related to the phosphoenolpyruvate carboxylase (PEPC) cycle, tricarboxylic acid cycle, citrate catabolism and glyoxylate cycle were identified in fig fruit. Expression levels of most of these genes were higher in inflorescences than in receptacles. In particular, FcPEPC and FcFUM (encoding fumarase) had significantly higher expression in the inflorescence of pollinated fruit. Most importantly, expression of the glyoxylate cycle genes FcMLS and FcICL (encoding malate synthase and isocitrate lyase, respectively) was induced to strikingly high levels in the inflorescence by pollination, and their expression level was highly positively correlated with the contents of all organic acids. Therefore, the glyoxylate cycle may be responsible for altering the accumulation of organic acids to upgrade the fruit taste during ripening, especially in the pollinated, seeded inflorescence.     

Fruit softening and pectin disassembly: an overview of nanostructural pectin modifications assessed by atomic force microscopy

Background

One of the main factors that reduce fruit quality and lead to economically important losses is oversoftening. Textural changes during fruit ripening are mainly due to the dissolution of the middle lamella, the reduction of cell-to-cell adhesion and the weakening of parenchyma cell walls as a result of the action of cell wall modifying enzymes. Pectins, major components of fruit cell walls, are extensively modified during ripening. These changes include solubilization, depolymerization and the loss of neutral side chains. Recent evidence in strawberry and apple, fruits with a soft or crisp texture at ripening, suggests that pectin disassembly is a key factor in textural changes. In both these fruits, softening was reduced as result of antisense downregulation of polygalacturonase genes. Changes in pectic polymer size, composition and structure have traditionally been studied by conventional techniques, most of them relying on bulk analysis of a population of polysaccharides, and studies focusing on modifications at the nanostructural level are scarce. Atomic force microscopy (AFM) allows the study of individual polymers at high magnification and with minimal sample preparation; however, AFM has rarely been employed to analyse pectin disassembly during fruit ripening.

Scope

In this review, the main features of the pectin disassembly process during fruit ripening are first discussed, and then the nanostructural characterization of fruit pectins by AFM and its relationship with texture and postharvest fruit shelf life is reviewed. In general, fruit pectins are visualized under AFM as linear chains, a few of which show long branches, and aggregates. Number- and weight-average values obtained from these images are in good agreement with chromatographic analyses. Most AFM studies indicate reductions in the length of individual pectin chains and the frequency of aggregates as the fruits ripen. Pectins extracted with sodium carbonate, supposedly located within the primary cell wall, are the most affected.