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TCA cycle, “GABA shunt”, valine, and tyrosine catabolisms: main reactions and their links that illustrate possible molecular strategies employed by Faris non-acid lemon to reduce the TCA cycle flux. The genes encoding TAT, BCAT, BCKD, BDH, and GAD (white boxes) showed higher expression levels in FNA than sour genotypes suggesting a higher activity of “GABA shunt”, valine, and tyrosine catabolisms in sweet lemons. The three pathways utilize, directly or indirectly, oxoglutarate in their early steps and their enhanced activities could explain the reduced citric acid amount observed in sweet lemons. The numbers in the boxes refer to enzyme IUBMB nomenclature
Source publication
The sour taste of lemons (Citrus limon (L.) Burm.) is determined by the amount of citric acid in vacuoles of juice sac cells. Faris is a "sweet" lemon variety since it accumulates low levels of citric acid. The University of California Riverside Citrus Variety Collection includes a Faris tree that produces sweet (Faris non-acid; FNA) and sour fruit...
Contexts in source publication
Context 1
... checked the pathways corresponding to the nine amino acids indicated by FunCat analysis, focusing on probe sets related to the TCA cycle that showed high fold changes. A probe set (Cit.9469.1.S1_at) coding for gluta- mate decarboxylase (EC.4.1.1.15), the enzyme converting glutamate into γ-aminobutyrate (GABA) through a proton- consuming reaction (Fig. 5) (Bown and Shelp 1997), was highly expressed in FNA (pH ∼6) and not expressed in FA or L (pH ∼3-4). When non-acid and acid samples were compared, the fold change was 67.2 for the FNA young vs FA young comparison, 60.5 for the FNA young vs L young, 90.8 for FNA mature vs FA mature, and 75.4 for FNA mature vs L mature (Table 2). ...
Context 2
... four enzymes of the tyrosine degradation pathway were not differentially expressed between acid and non-acid lemon fruit (Table S5). Leucine, isoleucine, and valine degradation pathways share the first two steps catalyzed by the branched-chain-amino acid transaminase (EC 2.6.1.42) and branched-chain keto-acid dehydrogenase (BCKD) (EC 1.2.1.25; Fig. 5, Figure S2). The former enzyme is represented by seven probe sets, four of which were not expressed, Cit.11674.1.S1_s_at was equally expressed in all samples, Cit.3629.1.S1_s_at was differentially expressed and belonged to cluster 13 (a maturation-related cluster, Table S5), while Cit.31144.1. S1_at was differentially expressed with a ...
Context 3
... non-acid lemons leads to a reduced (or absent) citric acid accumulation. As a consequence, non-acid lemons might have a partially different citric acid metabolism. This hypothesis is supported by the higher expression levels in non-acid samples observed for several genes involved in amino acid pathways such as GABA, valine, and tyrosine pathways (Fig. 5). Since all these pathways require 2- oxoglutarate for their early reaction steps, they represent an efficient way to redirect TCA cycle products in FNA where citric acid is not transported into the vacuole. Moreover, GAD catalyzes a proton-consuming reaction (Bown and Shelp 1997) that could be sufficient to explain the higher pH ...
Similar publications
Citrus fruits are characterized by the accumulation of high levels of citric acid, which account for 90% of the total organic acids. Citric acid is an important determinant of fruit taste because it affects the sourness of the fruit as well as the perceived sweetness, by masking the taste of sugars. To study the role of gene expression in citric ac...
Citations
... Many reports over the past two decades have revealed that citrate concentration in citrus fruit is influenced by biosynthesis (Schmidtmann et al., 2014;Verschueren et al., 2019), catabolism (Sadka et al., 2000a,b;Hu et al., 2015;Huang et al., 2016), and transportation (Huang et al., 2016;Liu et al., 2022). In recent years, it has been demonstrated that gene encoding P-ATPase (PH genes) is involved in citrate transport and contributes to citrate accumulation in citrus fruits (Aprile et al., 2011;Shi et al., 2015;Guo et al., 2016). Transcription factors including bHLH, bZIP, MYB, and ERF also participate in the regulation of citrate levels (Li et al., 2015;S. ...
... 'Faris' lemon yields sweet and sour fruits on distinct branches. Transcriptional analysis reveals that AHA10 (homolog of CsPH8) is not expressed in Faris sweet lemon but is highly expressed in Faris sour lemon (Aprile et al., 2011). Similarly, 'Qianyang Bingtang' navel orange and 'Jinxiu Bingtang' sweet orange are bud mutants of 'Bingtangcheng', characterized by the trait of higher and lower fruit acidity, respectively. ...
Plenty of rainfall but unevenly seasonal distribution happens regularly in southern China. Seasonal drought from summer to early autumn leads to citrus fruit acidification, but how seasonal drought regulates citrate accumulation remains unknown.
Herein, we employed a set of physiological, biochemical, and molecular approaches to reveal that CsABF3 responds to seasonal drought stress and modulates citrate accumulation in citrus fruits by directly regulating CsAN1 and CsPH8.
Here, we demonstrated that irreversible acidification of citrus fruits is caused by drought lasting for > 30 d during the fruit enlargement stage. We investigated the transcriptome characteristics of fruits affected by drought and corroborated the pivotal roles of a bHLH transcription factor (CsAN1) and a P3A‐ATPase gene (CsPH8) in regulating citrate accumulation in response to drought. Abscisic acid (ABA)‐responsive element binding factor 3 (CsABF3) was upregulated by drought in an ABA‐dependent manner. CsABF3 activated CsAN1 and CsPH8 expression by directly and specifically binding to the ABA‐responsive elements (ABREs) in the promoters and positively regulated citrate accumulation.
Taken together, this study sheds new light on the regulatory module ABA‐CsABF3‐CsAN1‐CsPH8 responsible for citrate accumulation under drought stress, which advances our understanding of quality formation of citrus fruit.
... However, whether this enzyme is critical for PA biosynthesis or is instead involved in detoxification of excess epicatechin is still unresolved. In Arabidopsis, the TT12 MATE transporter binds to and transports epicatechin-3 0 -O-glucoside to vacuoles, dependent on a proton gradient generated by TT13 (AHA10) (Zhao and Dixon, 2009;Aprile et al., 2011;Appelhagen et al., 2015). The tt13 (aha10) mutant phenocopies the tt12 (mate) mutant, and neither is significantly affected in anthocyanin accumulation (Debeaujon et al., 2001;Appelhagen et al., 2015), consistent with the observation that cyanidin-3-O-glucoside is not a preferred ligand for TT12 MATE (Zhao and Dixon, 2009). ...
Proanthocyanidins (PAs) are natural flavan-3-ol polymers, contributing protection for plants under biotic and abiotic stress, benefits to human health, and bitterness and astringency to food products. They are also potential targets for carbon sequestration for climate mitigation. In recent years, from model species to commercial crops, research has moved closer to elucidating the flux control and channeling, subunit biosynthesis and polymerization, transport mechanisms, and regulatory networks involved in PA metabolism in plants. This review extends the conventional understanding with recent findings that provide new insights to address lingering questions and focus strategies for manipulating PA traits in plants.
... Relevant upregulated genes, for instance, were an aquaporin (LOC18045515), a calciumtransporting ATPase (LOC18042874), a potassium transporter (LOC18043735), and two different sugar transporters ERD6like (LOC18040371 and LOC18046355), which are vacuolar H + /glucose symporters involved in the export of glucose to cytosol (Klemens et al., 2014). Downregulated genes were a boron transporter (LOC18036378), a nitrate reductase (LOC18041814), a zinc transporter (LOC18050857), and overall, an ATPase 10, plasma membrane-type (LOC18035736), that has been previously associated with citric acid accumulation in lemon juice (Aprile et al., 2011). There were also two members of the sodium/hydrogen exchanger gene family (LOC18040436 and LOC18039966), involved in acidity regulation in oranges , expressed in opposite directions. ...
... The finding that ATPase 10, plasma membrane-type (Aprile et al., 2011), a pivotal component of the vacuolar proton-pumping P-ATPase complex that regulates acidity in citrus (Strazzer et al., 2019), was downregulated in the three segregants, prompted us to focus our attention on the expression of the rest of components of this complex and of other related genes previously associated with this process . This set of genes, listed in Supplementary Table 6, included CitPH1 (LOC18037376, magnesium-transporting ATPase, P-type 1), two existing versions of CitPH5; namely, CitPH5.2 ...
... Relative expression of genes reported to be involved in acid regulation of citrus fruits (Aprile et al., 2011;Butelli et al., 2019;Strazzer et al., 2019;Huang et al., 2021), determined by RT-qPCR in juice vesicles of ripening fruits (November) of ICH, SCM, WMU, CLM and S1, S2, and S3 segregants. Vertical bars represent standard error from two technical replicates. ...
To identify key traits brought about by citrus domestication, we have analyzed the transcriptomes of the pulp of developing fruitlets of inedible wild Ichang papeda (Citrus ichangensis), acidic Sun Chu Sha Kat mandarin (C. reticulata) and three palatable segregants of a cross between commercial Clementine (C. x clementina) and W. Murcott (C. x reticulata) mandarins, two pummelo/mandarin admixtures of worldwide distribution. RNA-seq comparison between the wild citrus and the ancestral sour mandarin identified 7267 differentially expressed genes, out of which 2342 were mapped to 117 KEGG pathways. From the remaining genes, a set of 2832 genes was functionally annotated and grouped into 45 user-defined categories. The data suggest that domestication promoted fundamental growth processes to the detriment of the production of chemical defenses, namely, alkaloids, terpenoids, phenylpropanoids, flavonoids, glucosinolates and cyanogenic glucosides. In the papeda, the generation of energy to support a more active secondary metabolism appears to be dependent upon upregulation of glycolysis, fatty acid degradation, Calvin cycle, oxidative phosphorylation, and ATP-citrate lyase and GABA pathways. In the acidic mandarin, downregulation of cytosolic citrate degradation was concomitant with vacuolar citrate accumulation. These changes affected nitrogen and carbon allocation in both species leading to major differences in organoleptic properties since the reduction of unpleasant secondary metabolites increases palatability while acidity reduces acceptability. The comparison between the segregants and the acidic mandarin identified 357 transcripts characterized by the occurrence in the three segregants of additional downregulation of secondary metabolites and basic structural cell wall components. The segregants also showed upregulation of genes involved in the synthesis of methyl anthranilate and furaneol, key substances of pleasant fruity aroma and flavor, and of sugar transporters relevant for sugar accumulation. Transcriptome and qPCR analysis in developing and ripe fruit of a set of genes previously associated with citric acid accumulation, demonstrated that lower acidity is linked to downregulation of these regulatory genes in the segregants. The results suggest that the transition of inedible papeda to sour mandarin implicated drastic gene expression reprograming of pivotal pathways of the primary and secondary metabolism, while palatable mandarins evolved through progressive refining of palatability properties, especially acidity.
... However, the rate at which citrate passes through the malate channel under the control of cytoplasmic concentration suggests that citrate accumulation in vacuoles is mainly controlled by metabolism 2,47 . Citrate transport from the cytosol to the vacuole is accompanied by a large influx of protons 48,49 . This proton influx leads to vacuolar acidification and provides a strong driving force for increased vacuolar uptake of citrate, thereby maintaining the vacuolar buffering capacity and acidic pH environment 2,19,50 . ...
... This proton influx leads to vacuolar acidification and provides a strong driving force for increased vacuolar uptake of citrate, thereby maintaining the vacuolar buffering capacity and acidic pH environment 2,19,50 . This process is mainly mediated by V-ATPase 48,49 . Citrate transport to the vacuole is competitively inhibited by other organic acids, such as malate, because organic acids cross the tonoplast by the same channels or transporters 6 . ...
In fleshy fruits, organic acids are the main source of fruit acidity and play an important role in regulating osmotic pressure, pH homeostasis, stress resistance, and fruit quality. The transport of organic acids from the cytosol to the vacuole and their storage are complex processes. A large number of transporters carry organic acids from the cytosol to the vacuole with the assistance of various proton pumps and enzymes. However, much remains to be explored regarding the vacuolar transport mechanism of organic acids as well as the substances involved and their association. In this review, recent advances in the vacuolar transport mechanism of organic acids in plants are summarized from the perspectives of transporters, channels, proton pumps, and upstream regulators to better understand the complex regulatory networks involved in fruit acid formation.
... whereas those of acidic Faris lemon was 4.0, and those of Frost Lisbon lemon were 3.5-3.6 15 . Therefore, there must be specific pathways regulating pH in different fruit varieties. ...
Although multiple microscopic techniques have been applied to horticultural research, few studies of individual organelles in living fruit cells have been reported to date. In this paper, we established an efficient system for the transient transformation of citrus fruits using an Agrobacterium-mediated method. Kumquat (Fortunella crassifolia Swingle) was used; it exhibits higher transformation efficiency than all citrus fruits that have been tested and a prolonged-expression window. Fruits were transformed with fluorescent reporters, and confocal microscopy and live-cell imaging were used to study their localization and dynamics. Moreover, various pH sensors targeting different subcellular compartments were expressed, and the local pH environments in cells from different plant tissues were compared. The results indicated that vacuoles are most likely the main organelles that contribute to the low pH of citrus fruits. In summary, our method is effective for studying various membrane trafficking events, protein localization, and cell physiology in fruit and can provide new insight into fruit biology research.
... whereas those of acidic Faris lemon was 4.0, and those of Frost Lisbon lemon were 3.5-3.6 15 . Therefore, there must be specific pathways regulating pH in different fruit varieties. ...
Although multiple microscopic techniques have been applied to horticultural research, few studies of individual organelles in living fruit cells have been reported to date. In this paper, we established an efficient system for the transient transformation of citrus fruits using an Agrobacterium-mediated method. Kumquat (Fortunella crassifolia Swingle) was used; it exhibits higher transformation efficiency than all citrus fruits that have been tested and a prolonged-expression window. Fruits were transformed with fluorescent reporters, and confocal microscopy and live-cell imaging were used to study their localization and dynamics. Moreover, various pH sensors targeting different subcellular compartments were expressed, and the local pH environments in cells from different plant tissues were compared. The results indicated that vacuoles are most likely the main organelles that contribute to the low pH of citrus fruits. In summary, our method is effective for studying various membrane trafficking events, protein localization, and cell physiology in fruit and can provide new insight into fruit biology research.
... This association was attributed to a mutation-led truncation in aluminium activated malate transporter (ALTM) associated with low acidity in apple (Bai et al. 2015). Other QTLs and candidate genes associated with sweetness and acidity have been described for melon, lemon, peach (D locus) and apple (Etienne et al. 2002, Boudehri et al. 2009, Aprile et al. 2011, Cohen et al. 2014. In the case of grapes, only candidate genes under QTLs that control MA concentration, TA, and tartaric/malic ratio have been reported. ...
Background and Aims
Identifying the genes that participate in the sweetness and acidity of the berry is key, because these traits are quintessential to the flavour and quality of fresh grapes and wine. In this study we focused on the identification of genomic regions that host genes associated with sweetness and acidity in tablegrapes.
Methods and Results
A highly saturated genetic map was prepared using a Ruby Seedless × Sultanina cross (RxS; n = 138) genotyped using an SNPlex-type platform, and quantitative trait loci (QTLs) were mapped. In the integrated map of this population, 1731 markers were distributed along the 19 linkage groups (LGs) of the species. Three significant QTLs, two in LG5 and one in LG8, were associated with fructose/glucose ratio, TA and tartaric acid concentration; these QTLs explained up to 20% of the phenotypic variance for these traits. Eight candidate genes located within the confidence intervals of the sugar and TA QTLs were chosen and their expression profiles analysed from flowering to berry ripening. A Genome-Wide Association Study (GWAS) analysis revealed the association of single-nucleotide polymorphisms in the same regions as QTLs.
Conclusions
Quantitative trait loci significant for sugar and acidity were identified on a tablegrape high-quality genetic map; furthermore, an association between acidity or sugar concentration and expression changes for candidate genes was also observed.
Significance of the Study
These findings could become the basis to develop selection tools for the breeding of these traits in tablegrape.
... One group contains AHA4 and AHA11, the second one clusters isoforms similar to AHA1 (AHA2, AHA3, AHA5, AHA12), the third and the fourth only consist of AHA10 and AHA7, respectively, and the last group comprises AHA6, AHA8, and AHA9 [152,153]. Unexpectedly, AHA10-like isoforms target the tonoplast where they possibly assemble as heterooligomers [154,155]. Although this organization seems to be overall conserved across species, some plants were not recognized members for all the five subfamilies [27,153]. ...
Nitrogen nutrition in plants is a key determinant in crop productivity. The availability of nitrogen nutrients in the soil, both inorganic (nitrate and ammonium) and organic (urea and free amino acids), highly differs and influences plant physiology, growth, metabolism, and root morphology. Deciphering this multifaceted scenario is mandatory to improve the agricultural sustainability. In root cells, specific proteins located at the plasma membrane play key roles in the transport and sensing of nitrogen forms. This review outlines the current knowledge regarding the biochemical and physiological aspects behind the uptake of the individual nitrogen forms, their reciprocal interactions, the influences on root system architecture, and the relations with other proteins sustaining fundamental plasma membrane functionalities, such as aquaporins and H+-ATPase. This topic is explored starting from the information achieved in the model plant Arabidopsis and moving to crops in agricultural soils. Moreover, the main contributions provided by proteomics are described in order to highlight the goals and pitfalls of this approach and to get new hints for future studies.
... Citrate accumulation in the vacuole depends on the balance of citrate synthesis, transport and degradation or utilization (Cercos et al., 2006a;Sadka et al., 2000). Previous studies (Aprile et al., 2011;Muller and Taiz, 2002;Shi et al., 2015) indicated that vacuolar-type and p-type ATPases play important roles in citrate uptake into the vacuole, the citrate/ H + symporter CsCit1 (Shimada et al., 2006) mediates citrate efflux from the vacuole into the cytoplasm, and citrate is utilized through the Aco-GABA and/or ACL-degradation pathways (Cercos et al., 2006b;Guo et al., 2016;Hu et al., 2015). ...
... Hence, we identified the genes in the four fruit tissues that were involved in citric acid biosynthesis, catabolism and transport (Table S10). Previous studies showed that the influx and efflux of organic acid from vacuoles and mitochondria were important for organic acid accumulation in citrus fruit (Aprile et al., 2011;Hussain et al., 2017;Shi et al., 2015;Shimada et al., 2006). Therefore, we specifically excavated the genes related to citrate and malate transport (Figure 7b; Table S10). ...
... The CsPH8 gene was differentially expressed among the four varieties of fruit, and its expression was positively related to acidity (Figure 7c). Aprile et al. (2011) found that the AHA10 gene was not expressed in no-acid lemon but highly expressed in the wildtype sour lemon. Shi et al. (2015) also found that the expression of CsPH8 in the 'Honganliu' orange (low acid) was lower than that in the 'Anliu' orange (normal acid), and overexpression of CsPH8 in acidless pumelo JSs, strawberry fruit, and tomato fruit significantly increased the titratable acid or citric acid content (Shi et al., 2019). ...
Citrus fruit has a unique structure with soft leathery peel and pulp containing vascular bundles and several segments with many juice sacs. The function and morphology of each fruit tissue are different. Therefore, analysis at the organ‐wide or mixed‐tissue level inevitably obscures many tissue‐specific phenomena. High‐throughput RNA sequencing was used to profile Citrus sinensis fruit development based on four fruit tissue types and six development stages from young fruits to ripe fruits. Using a coexpression network analysis, modules of coexpressed genes and hub genes of tissue‐specific networks were identified. Of particular importance is the discovery of the regulatory network of phytohormones during citrus fruit development and ripening. A model was proposed to illustrate how ABF2 mediates the ABA signalling involved in sucrose transport, chlorophyll degradation, auxin homeostasis, carotenoid and ABA biosynthesis, and cell wall metabolism during citrus fruit development. Moreover, we depicted the detailed spatiotemporal expression patterns of the genes involved in sucrose and citric acid metabolism in citrus fruit and identified several key genes that may play crucial roles in sucrose and citric acid accumulation in the juice sac, such as SWEET15 and CsPH8. The high spatial and temporal resolution of our data provide important insights into the molecular networks underlying citrus fruit development and ripening.
... Sugar is an important biomolecule that contributes to the sweetness, energy accumulation, and respiratory metabolism of harvested pomelo fruit and is the raw material for the synthesis of organic acids, vitamins, and other nutrients (Fernie et al., 2004;Matsumoto & Ikoma, 2012). Organic acids not only affect the taste but also participate in the synthesis of amino acids and provide a carbon skeleton for nitrogen assimilation in the TCA cycle (Aprile et al., 2011;Sun et al., 2013). Limonoids are a prominent group of secondary metabolites in citrus fruits (Chaudhary et al., 2015) and their bitterness can affect the sensory enjoyment of consumers. ...
... The high hydrogen ion concentration pools, formed by a large number of organic acids in vacuoles, can result in the loss of cell membrane permeability and turn to affect the locally distributed enzymes and substrates. Low temperature alleviates these effects and in turn, the "acidification" phenomenon and the senescence process(Aprile et al., 2011;Chaudhary et al., 2015;Shi et al., 2019). Finally, we observed that different air conditioning treatments had different effects on the organic acid metabolism of postharvest pomelo fruit. ...
This research examined the influence of the Pomelo fruit flavor characteristics of four storage methods—room temperature (CK), low‐temperature storage (LTS), modified storage of the atmosphere (MA), and controlled storage atmosphere (CA). The contents, type, and changes in the composition of the taste determining substances have been identified, including sugars, organic acids, and bitter substances. In addition, a sensory assessment was used to calculate the relation between storage and sweetness, acidity, and intensity changes. Results showed the content and change in each taste substance could not be directly related to the taste intensity and fruit flavor. A new taste intensity scale, with a significant correlation between sensory attributes (R2 = .991) was developed for taste assessment. Results indicated that CA storage could effectively control the reduction in sweetness and acidity and reduce the increase in bitterness. The taste intensity scale can be used as an indicator for screening and selecting good‐quality storage methods. CA storage technology is a suitable method for improving the taste of stored pomelo fruits.
