The lemon vacuolar H+-ATPase was purified and characterized by Taiz’s group . They revealed that, in fact, two tonoplast-bound ATPase activities exist, a nitrate-sensitive V-type ATPase that is partially inhibited by vanadate, and a vanadate-sensitive ATPase that is partially inhibited by nitrate . These results should be taken with caution because of the possible cross-contamination of the tonoplast vesicles with other membrane vesicles. Nitrate inhibition seemed to be dependent on the time of tonoplast vesicle preparation; for the same phenological stage, inhibition peaked during the spring and was minimal during the autumn–winter, suggesting an environmental effect resulting in seasonal changes in membrane lipid composition . Moreover, the H+/ATP coupling ratio varied between 1 to 2 as the DpH increased, displaying a pH-dependent slippage, where the hydrolytic activity and the H+ transport are partially uncoupled. Further, pot with drainage holes the fruit V-ATPase reconstituted into artificial proteoliposomes showed a steeper pH gradient than the corresponding reconstituted epicotyl enzyme .
Overall, the following characteristics seem to allow lemon fruit V-ATPase to generate a steep pH gradient: variable coupling, low pH-dependent slip rate, low proton permeability of the membrane, lower H+/ATP stoichiometry, and improved coupling by citrate, the major accumulated organic acid, which also enhance the enzyme’s ability to generate a pH gradient. The pyrophosphatase activity in acid lime fruit was much lower than that of H+-ATPase, suggesting the latter as the major mechanism for proton influx . Tonoplast vesicles isolated from juice cells of ‘Valencia’ oranges displayed similar V-type ATPase and V-PPiase activities, although a steady-state was reached faster with ATP as substrate. At a DpH of 3 units, V-PPiase synthesized PPi in the presence of Pi, indicating that mature orange juice cells acted as a source of PPi, providing a mechanism for recovery of stored energy in the form of the pH gradient across the vacuole during later stages of development and postharvest storage . In summary, in light of the possible presence of an additional tonoplastic H+ transport mechanism, P-ATPase, vacuolar proton homeostasis and transport across the tonoplast require further biochemical research. A vacuolar citrate/H+ symporter, CsCit1 , homologous to the Arabidopsis decarboxylate transporter, was characterized in orange fruit; its mRNA and protein levels coincided with the acid-decline stage, suggesting its role in citrate efflux .
Yeast cells expressing the CsCit1 displayed electroneutral coupled citrate–H+ cotransport with a stoichiometry of 1citrate/2H+.Amino acids have been studied in citrus fruit in relation to the nutritional value of the juice provided the motivation, mostly for early workers, to analyze the levels of free amino acids and their patterns of accumulation during fruit development and storage . The exposure of fruit to stress on-the tree and cold or heat treatments during storage was associated with the accumulation of several amino acids. Glycolysis and the tricarboxylic acid cycle are metabolically associated to amino acid metabolism , its relation to citrate decline and the induction of a γ-aminobutyric acid shunt during the second half of fruit development. Moreover, the possible relationships between amino acid accumulation and Huanglongbing resistance/tolerance mechanisms have been recently investigated .In general, all of the amino acids are detected in the juice of mature fruit, with aspartic acid, asparagine, serine, glutamic acid, proline and GABA being the more abundant . A gradual increase in most of the free amino acids was detected during fruit development and toward maturation of Valencia orange .
This increase is associated with citrate decline and it is common to all citrus cultivars . However, different trends were detected in Navel oranges , with most amino acids and their metabolites decreasing from stage II to III of fruit development . A comparative analysis of total amino acid contents among various citrus cultivars showed lemon and mandarin with overall higher contents of essential amino acids than pomelo, grapefruit or sweet orange . Moreover, lemon displayed higher levels of amino acids with bitter taste, such as histidine, phenylalanine and valine, as well as acidic amino acids, aspartic acid and glutamic acid. Following harvest, citrus fruit are usually subjected to relatively long storage periods at low temperatures. However, heat treatments, which vary from 37°C for 24 h to ~50°C for a few minutes, prior to storage, are common to reduce pathogenic agents, as well as to induce resistance to chilling and pathogens. The effects of such treatments on amino acid contents and metabolism were investigated, with conflicting results. In Satsuma mandarins, the contents of most amino acids were reduced or remained unchanged following heat treatment and only ornithine showed a consistent increase following the treatment . On the other hand, Matsumoto and Ikoma found that most Satsuma mandarin amino acids were heat-responsive, showing a remarkable contents increase during postharvest storage at 20°C or 30°C, but not at 5°C or 10°C. However, two amino acids, ornithine and glutamine, were cold-responsive, suggesting active metabolism during postharvest cold storage. Changes in amino acid metabolism during fruit development of various cultivars and in the presence of external stimuli have been studied mostly by transcriptomic and metabolomic analyses. The activation of the GABA shunt, a major route for citrate catabolism , was identified in a transcriptomic analysis and confirmed by proteomics ; these analyses identified an increase in the transcript of glutamate dehydrogenase, aspartate/alanine aminotransferase, glutamate dehydrogenase, glutamine synthase, GABA amino transferase and succinate semialdehyde dehydrogenase during fruit development, and the presence of their corresponding proteins during the declining-citrate stage of fruit development . Moreover, use of an aconitase inhibitor, which induces citrate accumulation, resulted in induced activities of some of the enzymes of the GABA shunt . In addition, proteins of most amino acid-synthesis enzymes were induced either from early stage II to stage II or from stage II to stage III of fruit development, including pathways leading to the synthesis of cysteine, glycine, serine, leucine, valine, asparagine, aspartate, alanine, ornithine and glutamine . Induction of amino acid metabolism was suggested to play a role in the accumulation flavor-associated volatiles . Comparative transcriptomic analysis of high- and low-citrate oranges showed elevated transcript levels of phenylalanine-, arginine-, proline-, cysteine- and methionine-metabolism genes in the high-citrate orange . Cold storage of mandarins resulted in major alterations in amino acid metabolism, including the biosynthesis of proline and arginine, and significant enhancement of the catabolism of branchedchain amino acids . Catabolism of the branched-chain amino acids leucine, isoleucine, and valine releases acetyl-CoA, providing a precursor for amino acid-derived volatiles that are associated with off-flavor development during fruit storage . Water stress also induced alterations in the amino acid metabolism suggested to be involved in defense mechanisms against stress .Citrus HLB, caused by the phloem sap-restricted bacterium Candidatus Liberibacter, is a serious production threat to the citrus industry in various regions of the world. The bacteria are transmitted by phloem sap-piercing citrus psyllids while they feed, mostly on young expanding vegetative shoots. Different citrus cultivars show varied susceptibility/tolerance to HLB. The differential response seems to be associated with psyllid feeding preferences and with plant tolerance to the bacteria. Based on controlled graft-inoculation experiments, cultivars were classified into three major groups, sensitive, moderately tolerant and tolerant, each showing different symptoms, from severe leaf chlorosis, large pot with drainage depressed growth and death in the sensitive cultivars, to fewer and lesser severe symptoms in the tolerant cultivars. The bacteria appeared to be auxotrophic for a few amino acids, supplied by their host. The bacteria were suggested to affect free amino acid availability by altering the expression of amino acid storage proteins, at least in the insect host.
To assess whether amino acid metabolism plays a role in the variable citrus tolerance to HLB, metabolomics analyses were performed in various cultivars on healthy and infected trees. Although most of the analyses were performed with phloem sap, and not the fruit, we include their brief description, as some fruit symptoms might also be associated with changes in amino acid metabolism. In a metabolic survey of phloem sap and leaves of citrus cultivars showing varied sensitivity/tolerance to HLB, the levels of all amino acids were elevated in the tolerant cultivars . Comparative analyses of amino acid contents in the phloem sap of bacterium-permissive and non-permissive hosts showed that seven amino acids, mostly of the glutamate family, were associated with susceptibility, whereas five amino acids, mostly of the serine family, were associated with tolerance/resistance . Moreover, high proline-to-glycine ratios were associated with bacterium-permissive hosts. Overall, the level of consistency in these studies in relation to amino acid composition in sensitive/tolerant plant species was not high. HLB-symptomatic Valencia orange fruits showed an overall increase in the level of most detected amino acids as compared to no symptomatic fruit, possibly due to protein degradation .Disease resistance or susceptibility of a plant depends not only on the specific plant–pathogen combination, but also on the developmental stage of the host tissues. The ripening process of fleshy fruit is an example of a developmental transition that coincides with increased susceptibility to pathogens. Ripening involves a complex network of regulatory and hormone-mediated pathways leading to significant changes in the physiological and biochemical properties of the fruit . Among the ripening events, modifications in cell wall structure and composition, conversion of starch into simple sugars, changes in apoplastic pH and redox state, and decline in the concentration of antimicrobial metabolites contribute to susceptibility of fruit to pathogens . The enhanced susceptibility of ripe fruit to pathogens could be a default outcome of ripening or, alternatively, could be promoted by some, but not all, ripening processes . Fruit pathogens exhibit necrotrophic, biotrophic, or hemibiotrophic lifestyles , categories that reflect different infection strategies . Necrotrophs, such as the ascomycete, Botrytis cinerea, cause necrosis by deploying hydrolytic enzymes , secreting toxins and/or hijacking the plant’s enzymatic machinery . Biotrophs depend on the integrity of plant host tissues and have developed strategies to deceive the host to obtain nutrients without inducing plant defenses or cell death . Hemibiotrophs are those pathogens that switch lifestyles at different developmental phases and/or in certain environmental conditions . Therefore, the infection strategies of different pathogens challenge the competency of the plant host to respond and deploy effective defense mechanisms. Tomato has served as a model organism to study fruit ripening and has emerged as an informative experimental system to characterize the molecular regulation of the ripening-related susceptibility to pathogens, in particular to necrotrophic fungi, such as B. cinerea . B. cinerea fails to develop in unripe tomato fruit, but as fruit start their ripening program and become ripe , concurrently they become more susceptible to infections, which lead to rapid breakdown of host tissues and extensive microbial colonization . The roles of the plant stress hormones, ethylene , salicylic acid , jasmonic acid , and abscisic acid , in the control of plant developmental processes and the initiation of defense mechanisms against necrotrophic, biotrophic, or hemibiotrophic pathogens have been documented mostly for vegetative tissues . However, our understanding of how these hormones influence plant–pathogen interactions in fruit is still limited. The gaseous hormone, ET, is involved in the control of terminal developmental programs, such as organ abscission, leaf and flower senescence, and fleshy fruit ripening . ET also modulates plant resistance and susceptibility to pathogens. Thus, from one point of view, ET controls a variety of immune responses in conjunction with other signaling networks; but from another perspective, it promotes senescence or ripening, processes which facilitate infection by pathogens . JA influences flower development and may be involved in some ripening processes, depending on the plant species . The best-known function of JA is to regulate plant immune responses against insects and pathogens, particularly necrotrophs . JA may also play a role in resistance against abiotic stresses, including mechanical stress, salinity, and UV irradiation . SA is a phenolic compound with hormonal features that is crucial for the establishment of basal defenses, effector-triggered immunity, and both local and systemic acquired resistance . SA is typically involved in the activation of plant defenses against biotrophs and hemibiotrophs, but it also appears to enhance susceptibility to necrotrophs by antagonizing the JA signaling pathway through the regulatory protein NPR1 and by inhibition of auxin signaling . ABA regulates many aspects of plant development, including seed dormancy and germination, and plays a significant role in tolerance to abiotic stress . Negative and positive roles have been described for this hormone depending on the pathosystem, developmental stage of the host, and/or the environmental conditions in which the plant–pathogen interaction occurs .