Nitric oxide on/off in fruit ripening

Fruit ripening is a complex physiological process involving significant external and internal modifications. Classic edible fleshy fruits have been classified as climacteric or non‐climacteric according to their dependence on the phyto hormone ethylene; however, data have increasingly confirmed the involvement of the free radical nitric oxide (NO) in this process. Moreover, the exogenous application of NO demonstrates its beneficial effects on fruit quality.     

A role for jasmonates in climacteric fruit ripening

Jasmonates are a class of oxylipins that induce a wide variety of higher-plant responses. To determine if jasmonates play a role in the regulation of climacteric fruit ripening, the effects of exogenous jasmonates on ethylene biosynthesis and color, as well as the endogenous concentrations of jasmonates were determined during the onset of ripening of apple (Malus domestica Borkh. cv. Golden Delicious) and tomato (Lycopersicon esculentum Mill. cv. Cobra) fruit. Transient (12 h) treatment of pre-climacteric fruit discs with exogenous jasmonates at low concentration (1 or 10 μM) promoted ethylene biosynthesis and color change in a concentration-dependent fashion. Activities of both 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase and ACC synthase were stimulated by jasmonate treatments in this concentration range. The endogenous concentration of jasmonates increased transiently prior to the climacteric increase in ethylene biosynthesis during the onset of ripening of both apple and tomato fruit. The onset of tomato fruit ripening was also preceded by an increase in the percentage of the cis-isomer of jasmonic acid. Inhibition of ethylene action by diazocyclopentadiene negated the jasmonate-induced stimulation of ethylene biosynthesis, indicating jasmonates act at least in part via ethylene action. These results suggest jasmonates may play a role together with ethylene in regulating the early steps of climacteric fruit ripening. Received: 14 August 1997 / Accepted: 4 October 1997     

Facultative ripening in Hamelia patens (Rubiaceae): effects of fruit removal and rotting

Summary In Costa Rica individual Hamelia patens trees produce fruit throughout the year and experience dramatic changes in rates of fruit removal and rotting. During some moths, most fruits rot because they are not removed. Rotting fruits increase the probability that other fruits on the same infructescence will rot. When removal rates are high, fruits are taken as soon as their seeds become viable but before the fruit is completely ripe. Experimental removal of fruits produced significantly higher ripening rates than on control infructescences. This response allows Hamelia to ripen more fruit and increase the number of fruits taken when dispersers are abundant (e.g., during migration). The proximate mechanism of this response probably includes reallocation of energy conserved when partially ripe fruits are removed. Responding to fluctuating disperser populations likely increases dispersal success and may function as the ultimate cause.     

Genetic mapping of ripening and ethylene-related loci in tomato

 The regulation of tomato fruit development and ripening is influenced by a large number of loci as demonstrated by the number of existing non-allelic fruit development mutations and a multitude of genes showing ripening-related expression patterns. Furthermore, analysis of transgenic and naturally occurring tomato mutants confirms the pivotal role of the gaseous hormone ethylene in the regulation of climacteric ripening. Here we report RFLP mapping of 32 independent tomato loci corresponding to genes known or hypothesized to influence fruit ripening and/or ethylene response. Mapped ethylene-response sequences fall into the categories of genes involved in either hormone biosynthesis or perception, while additional ripening-related genes include those involved in cell-wall metabolism and pigment biosynthesis. The placement of ripening and ethylene-response loci on the tomato RFLP map will facilitate both the identification and exclusion of candidate gene sequences corresponding to identified single gene and quantitative trait loci contributing to fruit development and ethylene response. Received: 26 October 1998 / Accepted: 13 November 1998     

Hormonal regulation of ripening in the strawberry,a non-climacteric fruit

Anthocyanin accumulation is one measure of ripening in the strawberry (Fragaria ananassa Duch.), a non-climacteric fruit. Neither aminoethoxyvinylglycine, an inhibitor of 1-aminocyclopropane carboxylic acid synthase, nor inhibitors of ethylene action (silver, norbornadiene) affected anthocyanin accumulation in ripening fruit. When the achenes were removed from one half of an unripe fruit there was an accelerated accumulation of anthocyanin and induction of phenylalanine ammonia lyase on the de-achened portion of the ripening fruit. These effects of achene removal could be prevented by the application of the synthetic auxins 1-naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid to the de-achened surface. The introduction of 1-naphthalene acetic acid into intact unripe strawberry fruit through the peduncle delayed their subsequent ripening, as measured by the accumulation of anthocyanin, loss of chlorophyll and decrease in firmness. These findings suggest that the decline in the concentration of auxin in the achenes as strawberry fruit mature modulates the rate of fruit ripening.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - NAA 1-naphthaleneacetic acid - PA1 phenylalanine ammonia-lyase - POA phenoxyacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Gene expression during fruit ripening in avocado

The poly(A) ⁺RNA populations from avocado fruit (Persea americana Mill cv. Hass) at four stages of ripening were isolated by two cycles of oligo-dT-cellulose chromatography and examined by invitro translation, using the rabbit reticulocyte lysate system, followed by two-dimensional gel electrophoresis (isoelectric focusing followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis) of the resulting translation products. Three mRNAs increased dramatically with the climacteric rise in respiration and ethylene production. The molecular weights of the corresponding translation products from the ripening-related mRNAs are 80,000, 36,000, and 16,500. These results indicate that ripening may be linked to the expression of specific genes.     

Enzyme activities in mitochondria isolated from ripening tomato fruit

Mitochondria were isolated from tomato (Lycopersicon esculentum L.) fruit at the mature green, orange-green and red stages and from fruit artificially suspended in their ripening stage. The specific activities of citrate synthase (EC 4.1.3.7), malate dehydrogenase (EC 1.1.1.37), NAD-linked isocitrate dehydrogenase (EC 1.1.1.41) and NAD-linked malic enzyme (EC 1.1.1.38) were determined. The specific activities of all these enzymes fell during ipening, although the mitochondria were fully functional as demonstrated by the uptake of oxygen. The fall in activity of mitochondrial malate dehydrogenase was accompanied by a similar fall in the activity of the cytosolic isoenzyme. Percoll-purified mitochondria isolated from mature green fruit remained intact for more than one week and at least one enzyme, citrate synthase, did not exhibit the fall in specific activity found in normal ripening fruit.     

Timing of ethylene and polygalacturonase synthesis in relation to the control of tomato fruit ripening

A critical role in the initiation of ripening has been proposed for pectolytic enzymes which are known to be involved in fruit softening. The hypothesis that tomato (Lycopersicon esculentum Mill.) ripening is controlled by the initial synthesis of the cell-wall-degrading enzyme polygalacturonase (EC 3.2.1.15), which subsequently liberates cell-wall-bound enzymes responsible for the initiation of ethylene synthesis and other ripening events, has been examined. A study of kinetics of ethylene evolution and polygalacturonase synthesis by individual fruits in a ripening series, employing an immunological method and protein purification to identify and measure polygalacturonase synthesis, showed that ethylene evolution preceded polygalacturonase synthesis by 20h. Exogenous ethylene stimulated the synthesis of polygalacturonase and other ripening events, when applied to mature green fruit, whereas the maintenance of fruits in a low ethylene environment delayed ripening and polygalacturonase synthesis. It is concluded that enhanced natural ethylene synthesis occurs prior to polygalacturonase production and that ethylene is responsible for triggering polygalacturonase synthesis indirectly. Possible mechanisms for ethylene action are discussed.     

Nitric oxide synthase: models and mechanisms

The overproduction or underproduction of nitric oxide has been implicated in pathological symptoms such as endotoxic shock, diabetes, allograft rejection, and myocardial ischimia/reperfusion injury. A thorough understanding of the biosynthesis of nitric oxide is necessary to probe and manipulate these signaling events. There is also considerable pharmacological interest in developing selective inhibitors of the several isoforms of nitric oxide synthase. The recently determined crystal structures of complexes between nitric oxide synthase and substrate, the mechanisms of the enzymatic reaction that generate nitric oxide and chemical precedents and models for these reactions are now coming into focus, but there are still numerous fascinating and unanswered questions regarding nitric oxide biosynthesis.     

Calcium in plant senescence and fruit ripening

Abstract. Calcium has long been associated with regulation of the ripening process of fruit and post-harvest storage life. Specifically, maintenance of relatively high calcium concentrations in fruit tissue results in a slower rate of ripening, as seen in lower respiration rates, reduced ethylene production, and slower softening of fruit flesh. There are also some specific fruit disorders such as bitter pit, which can be prevented if sufficient calcium is present. Senescence of other plant tissues such as leaves and flowers has also been shown to be retarded by the application of calcium.
Work leading to the above information is reviewed and discussed in the context of what is currently known of cellular regulation of calcium in plants. The major sites for the action of calcium in senescence and ripening are suggested to be in membrane structure and function, and in cell wall structure. Although high external concentrations of calcium are an advantage in reducing the rate of senescence or ripening, it is emphasized that normal cell function requires the maintenance of low concentrations of free calcium in the cell cytosol. It is suggested that one possible feature of senescence is a breakdown in such cellular regulation.     

Cloning of a cDNA encoding 1-aminocyclopropane-1-carboxylate synthase and expression of its mRNA in ripening apple fruit

1-Aminocyclopropane-1-carboxylate (ACC) synthase (EC 4.4.1.14) purified from apple (Malus sylvestris Mill.) fruit was subjected to trypsin digestion. Following separation by reversed-phase high-pressure liquid chromatography, ten tryptic peptides were sequenced. Based on the sequences of three tryptic peptides, three sets of mixed oligonucleotide probes were synthesized and used to screen a plasmid cDNA library prepared from poly(A)⁺ RNA of ripe apple fruit. A 1.5-kb (kilobase) cDNA clone which hybridized to all three probes were isolated. The clone contained an open reading frame of 1214 base pairs (bp) encoding a sequence of 404 amino acids. While the polyadenine tail at the 3-end was intact, it lacked a portion of sequence at the 5-end. Using the RNA-based polymerase chain reaction, an additional sequence of 148 bp was obtained at the 5-end. Thus, 1362 bp were sequenced and they encode 454 amino acids. The deduced amino-acid sequence contained peptide sequences corresponding to all ten tryptic fragments, confirming the identity of the cDNA clone. Comparison of the deduced amino-acid sequence between ACC synthase from apple fruit and those from tomato (Lycopersicon esculentum Mill.) and winter squash (Cucurbita maxima Duch.) fruits demonstrated the presence of seven highly conserved regions, including the previously identified region for the active site. The size of the translation product of ACC-synthase mRNA was similar to that of the mature protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), indicating that apple ACC-synthase undergoes only minor, if any, post-translational proteolytic processing. Analysis of ACC-synthase mRNA by in-vitro translation-immunoprecipitation, and by Northern blotting indicates that the ACC-synthase mRNA was undetectable in unripe fruit, but was accumulated massively during the ripening proccess. These data demonstrate that the expression of the ACC-synthase gene is developmentally regulated.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AdoMet S-adenosyl-l-methionine - HPLC high-pressure liquid chromatography - kDa kilodalton - kb kilobase - mAb monoclonal antibody - Met methionine - PCR polymerase chain reaction - poly(A)⁺ RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis This work was supported by grants DCB-9004129 and INT-8915155 from the National Science Foundation.     

Changes in gene expression during strawberry fruit ripening and their regulation by auxin

Changes in messenger RNA during the development of the strawberry (Fragaria ananassa Duch.), a non-climacteric fruit, were analysed by extracting total RNA and separating the in-vitro translated products by two-dimensional polyacrylamide gel electrophoresis. Alterations in numerous messenger RNAs accompanied fruit development between the immature green stage and the overripe stage, with prominent changes detected at or before the onset of ripening. A number of messenger RNAs undetectable in immature green fruit increased as the fruit matured and ripened. Others showed a marked decrease in advance of the ripening phase. A further group of messenger RNAs was prominent in immature and ripe fruit but absent just prior to the turning stage. Removing the achenes from a segment of the fruit accelerated anthocyanin accumulation in the de-achened portion and produced a pattern of translated polypeptides similar to normal ripe fruit. Application of the synthetic auxin 1-naphthaleneacetic acid to the de-achened receptacle produced a translation pattern similar to that in mature green fruit. These findings indicate that ripening in strawberry is associated with the expression of specific genes.     

Nitric oxide and nitric oxide synthase activity in plants

Research on NO in plants has gained considerable attention in recent years mainly due to its function in plant growth and development and as a key signalling molecule in different intracellular processes in plants. The NO emission from plants is known since the 1970s, and now there is abundant information on the multiple effects of exogenously applied NO on different physiological and biochemical processes of plants. The physiological function of NO in plants mainly involves the induction of different processes, including the expression of defence-related genes against pathogens and apoptosis/programmed cell death (PCD), maturation and senescence, stomatal closure, seed germination, root development and the induction of ethylene emission. NO can be produced in plants by non-enzymatic and enzymatic systems. The NO-producing enzymes identified in plants are nitrate reductase, and several nitric oxide synthase-like activities, including one localized in peroxisomes which has been biochemically characterized. Recently, two genes of plant proteins with NOS activity have been isolated and characterized for the first time, and both proteins do not have sequence similarities to any mammalian NOS isoform. However, different evidence available indicate that there are other potential enzymatic sources of NO in plants, including xanthine oxidoreductase, peroxidase, cytochrome P450, and some hemeproteins. In plants, the enzymatic production of the signal molecule NO, either constitutive or induced by different biotic/abiotic stresses, may be a much more common event than was initially thought.     

Papaya fruit ripening: ROS metabolism,gene cloning,characterization and molecular docking of peroxidase

Climacteric fruit ripening has been characterized by oxidative burst and involve active oxidative metabolism with generation of reactive oxygen species (ROS). In the present paper, the papaya fruit ripening was found to be associated with increase in polygalacturonase (PG), pectate lyase (PEL), catalase (CAT), ascorbate peroxidase (APX), H₂O₂ and lipid peroxidation concomitant with decrease in the activities of superoxide dismutase (SOD) and guaiacol peroxidase (GPX). Furthermore, a cDNA (903 bp) of GPX from unripe papaya fruit pulp was isolated and cloned. On BLAST analysis, the deduced protein exhibited homology with various peroxidases and specific hits for plant heme peroxidase family namely heme and calcium binding domains. GPX of papaya was modeled and docked with various substrates and inhibitors among which guaiacol and cysteine were found to be the best substrate and inhibitor, respectively.     

Nitric oxide in invertebrates

Nitric oxide (NO) is considered an important signaling molecule implied in different physiological processes, including nervous transmission, vascular regulation, immune defense, and in the pathogenesis of several diseases. The presence of NO is well demonstrated in all vertebrates. The recent data on the presence and roles of NO in the main invertebrate groups are reviewed here, showing the widespread diffusion of this signaling molecule throughout the animal kingdom, from higher invertebrates down to coelenterates and even to prokaryotic cells. In invertebrates, the main functional roles described for mammals have been demonstrated, whereas experimental evidence suggests the presence of new NOS isoforms different from those known for higher organisms. Noteworthy is the early appearance of NO throughout evolution and striking is the role played by the nitrergic pathway in the sensorial functions, from coelenterates up to mammals, mainly in olfactory-like systems. All literature data here reported suggest that future research on the biological roles of early signaling molecules in lower living forms could be important for the understanding of the nervous-system evolution.