Citric Acid Accumulation Research Articles

Citrate exporter enhances both extracellular and intracellular citric acid accumulation in the koji fungi Aspergillus luchuensis mut. kawachii and Aspergillus oryzae

Citrate exporter CexA plays a key role in the production of citric acid in fungi; however, its role in intracellular metabolism has remained unclear. In this study, we comparably characterized homologous cexA genes in the white koji fungus Aspergillus luchuensis mut. kawachii and the yellow koji fungus Aspergillus oryzae, which exhibit high and low abilities, respectively, to produce citric acid. Disruption of cexA caused a significant decline of both extracellular and intracellular citric acid accumulation in Aspergillus kawachii, while overexpression of the A.kawachii cexA gene (AkcexA) into A.oryzae significantly enhanced both extracellular and intracellular citric acid accumulation in A.oryzae to a level comparable to that of A.kawachii. In addition, overexpression of two intrinsic cexA homologs (AocexA and AocexB) in A.oryzae also enhanced its extracellular and intracellular citric acid accumulation. Comprehensive analysis of intracellular metabolites from an AkcexA-overexpressing strain of A.oryzae compared with its control strain identified metabolic changes associated with intracellular citric acid accumulation via the glycolytic pathway, pentose phosphate pathway, and tricarboxylic acid cycle. Our results indicate that citric acid export enhances not only extracellular citric acid accumulation but also intracellular metabolic fluxes to generate citric acid.

Identification and transcript analysis of CsAPD2 reveal its potential role in citric acid accumulation in citrus fruits

Aberrant pollen development gene 2 (APD2) encodes a plant-specific RING-finger ubiquitin-protein, which may play an important role in pollen development or fruit development and ripening. However, the role of ubiquitin-proteins in fruit quality formation is not clear. In this study, we successfully cloned an Arabidopsis APD2 homologous gene (CsAPD2) from the citrus genome. The full-length sequence of CsAPD2 has 1431 nucleotides, encoding a protein of 476 amino acids. This gene contains ten exons and nine introns with two transmembrane domains and one RING-finger domain. The tissue-specific transcript analysis showed that CsAPD2 was predominantly expressed in fruit juice sacs (JS), followed by young leaves. Moreover, temporal expression analysis in the JS of six citrus cultivars displayed a significantly positive correlation coefficient (0.939, P < 0.01) between the change in CsAPD2 transcript level and citric acid content. Additionally, when drought stress was applied to citrus trees, both the CsAPD2 transcript level and the citric acid content were significantly enhanced in fruit JS. Taken together, the present study results suggested that CsAPD2 possibly participates in the dynamic change of citric acid accumulation in citrus fruits.

Biological and functional properties of xylem sap extracted from grapevine (cv. Rosario Bianco)

Xylem sap exudes from grapevine branches and ensures survival during the dormant period. However, the extensive analysis on the biological and functional characterization of xylem sap has not yet been carried out. Therefore, to comprehend the critical role and potential usage of the xylem sap, we identified and quantified the panel of active ingredients, such as protein, amino acids, carbohydrates, saponins, flavonoids, phenolic compounds, alkaloids, and polyphenols. Results indicates that bleeding sap is slightly acidic, with the accumulation of oxalic acid, tartaric acid, malic acid, lactic acid, and citric acid. Identification and quantification of sugars depicted that xylem sap is mainly rich in fructose, glucose, and lactose. Moreover, distinct amino acids (16), hormones (9), and mineral elements are identified in the bleeding sap. These bioactive compounds are vital for vine growth and development. In addition, xylem sap also has the growth-promoting ability and can be used as a tissue culturing medium for plants. Finally, functional studies suggested that xylem sap possesses antifungal effects that can be considered as a bacteriostatic agent against Botrytis cinerea. Overall, these findings suggested that xylem sap possesses the panel of antioxidants, protein, and growth-promoting and antifungal properties.

Identification and importance of mitochondrial citrate carriers and ATP citrate lyase for glycolipid production in Starmerella bombicola.

Starmerella bombicola is a non-conventional yeast commercially used as a microbial cell factory for sophorolipid production. Sophorolipids are glycolipid biosurfactants composed of a glucose disaccharide sophorose and a fatty acid. In de novo sophorolipid synthesis, the fatty acid moiety is derived from the fatty acid synthesis (FAS) complex; therefore, the yeast's lipid metabolism plays a crucial role in sophorolipid biosynthesis. As a fatty acid precursor, citric acid is a key primary metabolite that connects carbohydrate and lipid metabolism, and in S. bombicola, it also has a regulatory effect on sophorolipid composition and productivity. We aimed to identify the mitochondrial transporters involved in citrate shuttling and the ATP citrate lyase (Acl), the enzyme that converts citric acid into acetyl-CoA. Subsequently, we studied their role in the citric acid shuttle and glycolipid synthesis and the potential of citrate metabolism as a genetic manipulation target for increased glycolipid synthesis. Bioinformatics analyses predicted 32 mitochondrial carriers of which two were identified as citrate transporters, named SbCtp1 and SbYhm2. Deletion of these mitochondrial carriers led to a lesser sophorolipid yield and a shift in the lactonic/acidic sophorolipid ratio. However, only the knockout of SbYhm2 caused a decrease of citric and an increase of malic acid extracellular concentrations. Additionally, deletion of SbAcl1 had a negative effect on S. bombicola's specific growth rate and sophorolipid synthesis and contributed to extra- and intracellular citric acid accumulation. Unexpectedly, SbAcl1 overexpression also decreased glycolipid production.Key Points• Starmerella bombicola is an industrially relevant microbial cell factory for biosurfactant production.• There are 32 predicted mitochondrial carriers in S. bombicola.• Citrate mitochondrial carriers SbYhm2 and SbCtp1 are essential for glycolipid synthesis in S. bombicola.• Deletion of SbAcl1 negatively affects growth and sophorolipid production in S. bombicola. Graphical abstract.

Improved Production of Malic Acid in Aspergillus niger by Abolishing Citric Acid Accumulation and Enhancing Glycolytic Flux.

Microbial fermentation was widely explored to produce malic acid. Previously, Aspergillus niger has been successfully engineered, and a high titer of malic acid was achieved with strain S575, but it also produced a high level of byproduct citric acid. Here, the capability of A. niger in malic acid biosynthesis was further improved by eliminating the accumulation of citric acid and enhancing glycolytic flux. Characterization of variant mutants suggested that disruption of cexA, a gene encoding citric acid transporter located on cell membrane, abolished citric acid accumulation. However, cexA-deficient strain S895 showed significantly decreased malic acid production. Further analysis of S895 indicated that the transcription level of genes involved in glucose transportation and glycolytic pathway was significantly reduced, and the corresponding enzyme activity was also lower than those of S575. Individual overexpression of genes encoding glucose transporter MstC and key enzymes (hexokinase HxkA, 6-phosphofructo-2-kinase PfkA, and pyruvate kinase PkiA) involved in irreversible reactions of glycolic pathway increased malic acid production. Accordingly, genes of mstC, hxkA, pfkA, and pkiA were overexpressed altogether in S895, and the resultant strain S1149 was constructed. The titer of malic acid in fed-batch fermentation with S1149 reached 201.13 g/L. Compared with S575, the byproduct of citric acid was completely abolished in S1149, and the ratio of malic acid/glucose was increased from 1.27 to 1.64 mol/mol, the highest yield reported so far, and the fermentation period was shortened from 9 to 8 days. Thus, a strain with great industrial application potential was developed by engineering nine genes in A. niger, and a pilot fermentation technology was exploited.

Characterization of Organic Acid Metabolism and Expression of Related Genes During Fruit Development of Actinidia eriantha 'Ganmi 6'.

Studies on organic acid metabolism have been mainly concentrated on the fruit, whereas, few have focused on the mechanism of high organic acids content in the fruit of Actinidia eriantha. Fruits of ‘Ganmi 6’ harvested at eleven developmental periods were used as materials. The components and content of organic acids were determined by high-performance liquid chromatography (HPLC) system, the activities of the related enzyme were detected, and gene expression levels were measured by quantitative real-time PCR (qRT-PCR). Components of ascorbic acid (AsA) and eight kinds of organic acids were detected. These results showed that quinic acid and citric acid were the main organic acids in the fruit of ‘Ganmi 6’. Correlation analysis showed that NADP-Quinate dehydrogenase (NADP-QDH), NADP-Shikimate dehydrogenase (NADP-SDH), and Cyt-Aconitase (Cyt-Aco) may be involved in regulating organic acids biosynthesis. Meanwhile, the SDH gene may play an important role in regulating the accumulation of citric acid. In this study, the activities of NADP-SDH, Mit-Aconitase (Mit-Aco), and NAD-Isocitrate dehydrogenase (NAD-IDH) were regulated by their corresponding genes at the transcriptional level. The activity of Citrate synthase (CS) may be affected by post-translational modification. Our results provided new insight into the characteristics of organic acid metabolism in the fruit of A. eriantha.

Evaluation of the comparative morphological and quality in fig (Sichuan, China) with different colors under different ripening stages

Fig (Ficus carica L.) has been used in China as food and medicine for millennia, and globally has been consumed increasingly as a healthy food. Sichuan, especially the Weiyuan area, always had the reputation of producing best fig quality. The comparative morphological and quality of 6 fig cultivars were correlated with the main planting fig with three different colors in Sichuan, China: Yellow (Orphan, Browns Wick), Red (Bpjihon, Masui Dauphine, Wild-type), Purple (Japanese Purple Fruit). This study aimed to explore the morphological characteristics in fig, and composition, content and dynamic changes rule of the organic acids and anthocyanins in different parts (peel and pulp) of fruits under different ripening stages (eight mature and full mature). These provide important theoretical bases for the improvement, production, and quality assessment of fig cultivars. In this experiment, high performance liquid chromatography (HPLC) for the determination of organic acids and anthocyanins in figs was established for the first time. Each organic acid and anthocyanin component had a linear coefficient of >0.996, and good values were obtained for linear correlation, precision, and reproducibility. During the ripening of fig, malic acid, quinic acid, and citric acid were the main organic acids of fig, followed by tartaric acid and succinic acid, with the lowest content found for fumaric acid. The highest anthocyanin content was observed for cyanidin-3-O-glucoside, followed by cyanidin-3-O-rutinoside, and cyanidin-3,5-diglucoside had the least content. The accumulation pattern of organic acids and anthocyanins was observed to change. The organic acids accumulation pattern changed from malic acid and quinic acid to malic acid, citric acid accumulation or malic acid, citric acid and quinic acid to the accumulation of the three organic acids. The accumulation pattern of anthocyanins was mainly composed of cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside accumulation, and the change during ripening was not significant. This pattern may play an important role in the determination of acidity, sweetness, and flavor quality of fig fruits in Sichuan, China.

Valorization of Crude Glycerol, Residue Deriving from Biodiesel- Production Process, with the Use of Wild-type New Isolated Yarrowia lipolytica Strains: Production of Metabolites with Pharmaceutical and Biotechnological Interest.

Crude glycerol (Glol), used as substrate for screening eleven natural Yarrowia lipolytica strains in shake-flask experiments. Aim of this study was to assess the ability of the screened strains to produce biomass (dry cell weight; X), lipid (L), citric acid (Cit), mannitol (Man), arabitol (Ara) and erythritol (Ery), compounds presenting pharmaceutical and biotechnological interest, in glycerol-based nitrogen-limited media, in which initial glycerol concentration had been adjusted to 40 g/L. Citric acid may find use in biomedical engineering (i.e. drug delivery, tissue engineering, bioimaging, orthopedics, medical device coating, wound dressings). Polyols are considered as compounds with non-cariogenic and less calorigenic properties as also with low insulin-mediated response. Microbial lipids containing polyunsaturated fatty acids (PUFA) are medically and dietetically important (selective pharmaceutical and anticancer properties, aid fetal brain development, the sight function of the eye, hormonal balance and the cardio-vascular system, prevent reasons leading to type-2 diabetes, present healing and anti-inflammatory effects). All strains presented satisfactory microbial growth (Xmax=5.34-6.26 g/L) and almost complete substrate uptake. The principal metabolic product was citric acid (Citmax=8.5-31.7 g/L). Production of cellular lipid reached the values of 0.33-0.84 g/L. Polyols were also synthesized as strain dependent compounds (Manmax=2.8-6.1 g/L, Aramax ~2.0 g/L, Erymax= 0.5-3.8 g/L). The selected Y. lipolytica strain ACA-DC 5029 presented satisfactory growth along with synthesis of citric acid and polyols, thus, was further grown on media presenting an increased concentration of Glol~75 g/L. Biomass, lipid and citric acid production presented significant enhancement (Xmax=11.80 g/L, Lmax=1.26 g/L, Citmax=30.8 g/L), but conversion yield of citric acid produced per glycerol consumed was decreased compared to screening trials. Erythritol secretion (Erymax=15.6 g/L) was highly favored, suggesting a shift of yeast metabolism from citric acid accumulation towards erythritol production. Maximum endopolysaccharides (IPS) concentration was 4.04 g/L with yield in dry weight 34.2 % w/w. Y. lipolytica strain ACA-YC 5029 can be considered as a satisfactory candidate grown in high concentrations of crude glycerol to produce added-value compounds that interest pharmaceutical and biotechnology industries.

CsPH8, a P-type proton pump gene, plays a key role in the diversity of citric acid accumulation in citrus fruits

Citric acid homeostasis patterns and its content are diversified among the fruits of citrus cultivars, but the cause remained unclear. In this study we showed that changes of citric acid content were highly associated with the expression profiles of a P-type proton pump gene (CsPH8) in the fruits of six citrus cultivars; moreover, analysis of 21 different fruit samples indicated that the correlation coefficient between titratable acid content and CsPH8 transcript level was 0.5837 with a significant level (P < 0.05). Overexpression of CsPH8 in acidless pumelo juice sacs, strawberry fruit, and tomato fruit significantly increased the titratable acid or citric acid content besides the gene transcript level. On another hand, RNA interference of CsPH8 in acidic pumelo juice sacs significantly decreased the CsPH8 transcript level and the titratable acid or citric acid content as well. In addition, severe drought significantly increased the CsPH8 transcript level besides the titratable acid content. Taken together, these findings address the function of CsPH8 in citrus vacuolar acidification, confirm that CsPH8 plays a key role in the variation of citric acid content, and supported that the acid fluctuation influenced by drought, is at least partly due to the change of CsPH8 transcript level.

Effect of Light-Emitting Diodes and Ultraviolet Irradiation on the Soluble Sugar, Organic Acid, and Carotenoid Content of Postharvest Sweet Oranges (Citrus sinensis (L.) Osbeck).

Mature ‘Hamlin’ sweet oranges (Citrus sinensis (L.) Osbeck) were irradiated using light-emitting diodes (LEDs) and ultraviolet (UV) light for six days after harvest. Based on evaluation of the basic ripening parameters of fruits, the contents of soluble sugars, organic acids, and carotenoids were analyzed (in pulps) on the sixth day by high-performance liquid chromatography (HPLC). The results showed that LED and UV irradiation not only accelerated orange ripening but also caused significant changes in the soluble sugar, organic acid, and carotenoid content. Compared with fruit subjected to dark shade (DS) treatment, the total soluble sugar, fructose, and glucose contents increased significantly in UV-treated (UVA, UVB, and UVC) fruits, while the sucrose content increased remarkably in white light, UVB, and UVC-treated fruits (p < 0.05). UV treatment was associated with inducing the largest effect on the total soluble sugar content. Except for UVB, other types of light notably induced an accumulation of the total organic acid content, none but blue light and red light markedly induced citric acid accumulation (p < 0.05). Interestingly, only the red light and dark shade treatments had markedly positive effects in terms of inducing carotenoid accumulation, including the total carotenoid, isolutein, zeaxanthin, lutein, neoxanthin, all-trans-violaxanthin, phytofluene, cis-ζ-carotene, and β-carotene concentrations. Other light treatments had significantly negative effects on carotenoid accumulation (p < 0.05). Therefore, soluble sugar, organic acid, and carotenoid accumulation in sweet oranges vary depending on the levels of UV and LED irradiation. Appropriate light irradiation is a potentially effective way to maintain or improve postharvest fruit quality.

Evidences of organic acids exudation in aluminium stress responses of two Madeiran wheat (Triticum aestivum L.) landraces

Two wheat (Triticum aestivum L.) Madeiran landraces were subjected to 100 μM and 200 μM of aluminium (Al) in hydroponic culture, assessing the organic acid exudation role in plant’s responses to this metal. Samples of initial landrace populations (F0), F3 and haplodiploid lines (DH) were evaluated using standard tests: eriochrome cyanine R staining, root elongation and callose accumulation in roots. Root exudates were obtained to determine if the accumulation of malic and citric acids in hydroponic medium was a response to Al exposure. Additionally, the presence of ALMT1 gene was determined using five microsatellite markers. Standard tests confirmed that ISOP 76 was Al tolerant and ISOP 239, Al susceptible. ISOP 76, in the presence of 100 μM Al, exuded substantially more malic acid (12.87 to 43.33 mg/L), than ISOP 239 (3.65 to 7.72 mg/L). The levels of both organic acid exudation were substantially lower in ISOP 239 than in the ISOP 76. In the presence of 200 μM Al, ISOP 76 F0 shows a higher root elongation ratio (better tolerates Al), but the DH line was the one that exuded higher content of malic acid. Different gene alleles and promoters were detected in both landraces. Molecular differences could explain the observed dissimilarity in organic acid exudation response to Al stress.

Evaluation of germplasm in Solanum section Lycopersicon for tomato taste improvement

The genetic potential of accessions from Solanum section Lycopersicon (S. lycopersicum L., S. lycopersicum var. cerasiforme, S. pimpinellifolium L., and S. habrochaites Knaap & Spooner) for breeding tomato taste has been studied in three environments with clonal replicates. The environment clearly affected the accumulation and level of variation of sugars and acids and derived variables through a direct effect. It seems that photosynthetically active radiation would exert a major effect on sugar accumulation while in the case of organic acids the effect of temperature might be more important. Even more, important genotype × environment interactions can considerably modify the real value of germplasm, being considerably higher in wild species. The environment affected not only mean contents but also the levels of variation. Thus, the need to develop multienvironmental screening programs is suggested to identify solid sources of variation. An important intraaccession variability was also found in wild germplasm, emphasizing the need to analyze a high number of plants per accession in order to identify sources of variation. Accessions with a significant genotypic contribution to the accumulation of sucrose within the Lycopersicon group were identified and may be interesting to analyze the regulation of vacuolar invertase. Accessions with different genotypic contributions to citric, malic, and glutamic acid accumulation have also been identified. These accessions will be valuable for the development of breeding programs considering the acid component of taste. Additionally, these genetic resources will be interesting to study the regulation of the tricarboxylic acid cycle and the gamma-aminobutyric acid shunt.

Genome-wide identification and transcript analysis of vacuolar-ATPase genes in citrus reveal their possible involvement in citrate accumulation

The vacuolar H+-ATPase (V-ATPase) proton pump plays an important role in the acidification of vacuoles; however, genes encoding V-ATPase in the citrus genome and their roles in citric acid accumulation remain unclear in citrus fruit. In this study, we found at least one gene encoding subunit A, B, C, D, G, c’’, d or e; two genes encoding the subunit E, F, H or a; and four genes encoding subunit c in the citrus genome. Spatial expression analysis showed that most genes were predominantly expressed in the mature leaves and/or flowers but were less expressed in root and juice cells. Two sweet orange (Citrus sinensis) cultivars, ‘Anliu’ (AL) and ‘Hong Anliu’ (HAL), which differ in terms of fruit acidity, were used in this study. The citric acid content was significantly higher in ‘AL’ fruits than in ‘HAL’ fruits over the entire experimental period (82 days–236 days after full blossom, DAFB). Transcript analysis showed that the transcript levels of most subunit genes, including V1-A, V1-B, V1-C, V1-E1, V1-G, V1-H2 and V0-a2, V0-c”, V0-c4, and V0-d, were significantly lower in ‘HAL’ than in ‘AL’ fruits during fruit development and ripening. Moreover, ABA injection significantly increased the citric acid content, simultaneously accompanied by the obvious induction of V1-A, V1-C, V1-E1, V1-F1, V1-H2, V0-a1, V0-a2, V0-c1, V0-c2, V0-c4, and V0-d transcription levels. In conclusion, the results demonstrated that V1-A, V1-C, V1-E1, V1-H2, V0-a2, V0-c4, and V0-d may play more roles than other subunit genes in the vacuole acidification of citrus fruits. The lower activity of V-ATPase caused by the transcript reduction of some subunit genes may be one reason for the low citrate accumulation in ‘HAL’ juice sacs.

Pex16 is involved in peroxisome and Woronin body formation in the white koji fungus, Aspergillus luchuensis mut. kawachii

We characterized Pex16 in Aspergillus luchuensis mut. kawachii to examine the role of peroxisomes on citric acid production during the shochu-fermentation process. Rice koji made using a Δpex16 strain exhibited no significant change in citric acid accumulation but a 1.4-fold increase in formic acid production. Microscopic observation of mRFP-SKL (a peroxisome protein marker) showed that pex16 disruption decreased the number of dot-like structures per hyphal cell to 5% of the control. Pex16-GFP exclusively co-localized with mRFP-SKL throughout the hyphae including the very close position to the septal pore. Moreover, the Δpex16 strain was hypersensitive to calcofluor white, which appeared to induce bursting of the hyphal tip and translocation of mRFP-SKL signals to the septal pore. These results indicate that Pex16 does not play a role in citric acid accumulation but is significantly involved in peroxisome and Woronin body formation in Aspergillus kawachii.

Transcriptomic analysis of Aspergillus niger strains reveals the mechanism underlying high citric acid productivity

BackgroundAspergillus niger is a highly important industrial microorganism because of its amazing capacity to produce citric acid (CA). To explore the metabolic mechanism and physiological phenotype associated with high CA productivity, the transcriptomes of high CA-producing A. niger YX-1217 and degenerative strain YX-1217G were investigated using A. niger ATCC1015 as a control.ResultsThese strains showed distinct transcriptional differences in CA production. By contrast, the genes encoding glycoside hydrolases, aspartyl endoproteases, and carboxypeptidases were unusually upregulated in CA-producing strain YX-1217, which involved the carbohydrate hydrolysis and polypeptide degradation pathways, and should be related to its powerful capacity to utilize cornmeal fluidified liquid as raw material for the production of CA. In central metabolism of YX-1217, gene 9.735.1, which encodes glyceraldehyde 3-phosphate dehydrogenase, and two transcriptionally outstanding genes, 6000119 (An15g01920) and 3.2152.1 (An08g10920) that encode citrate synthase, were upregulated, thereby ensuring CA accumulation. In addition, a relatively strong electron transport chain, a regeneration system for NAD+/NADP+, and an efficient resistance mechanism may have contributed to the high CA production rate of YX-1217.ConclusionsThese comparisons have shed light on the mechanism underlying high CA yield in A. niger YX-1217 as well as provide insights into the development of novel strains that produce other organic acids.

Morphological regulation of Aspergillus niger to improve citric acid production by chsC gene silencing.

The mycelial morphology of Aspergillus niger, a major filamentous fungus used for citric acid production, is important for citric acid synthesis during submerged fermentation. To investigate the involvement of the chitin synthase gene, chsC, in morphogenesis and citric acid production in A. niger, an RNAi system was constructed to silence chsC and the morphological mutants were screened after transformation. The compactness of the mycelial pellets was obviously reduced in the morphological mutants, with lower proportion of dispersed mycelia. These morphological changes have caused a decrease in viscosity and subsequent improvement in oxygen and mass transfer efficiency, which may be conducive for citric acid accumulation. All the transformants exhibited improvements in citric acid production; in particular, chsC-3 showed 42.6% higher production than the original strain in the shake flask. Moreover, the high-yield strain chsC-3 exhibited excellent citric acid production potential in the scale-up process.The citric acid yield and the conversion rate of glucose of chsC-3 were both improved by 3.6%, when compared with that of the original strain in the stirred tank bioreactor.

Fruit sugar and organic acid were significantly related to fruit Mg of six citrus cultivars

The fruit quality of 6 citrus cultivars growing in the same orchard was determined at ripening stage in both 2014 and 2015. We further measured the components of sugar (sucrose, fructose and glucose), organic acid (citric, malate and quinic acid), enzymes related to Glycolysis and Krebs cycle and mineral elements at 5 stages of fruit development in the second year. The results showed that at ripening stage of both years, ‘Newhall’ cultivar had higher TSS concentration and the TSS/TA ratio but lower TA concentration, while ‘Flame’ cultivar was exactly opposite. Sucrose and citric acid were the most accumulated compounds in fruit during the fruit development of 6 citrus cultivars. Fruit sucrose concentration increased from 9.26 mg·kg−1 at 60 DAFB to 50.92 mg·kg−1 at 180 DAFB, and the citric acid concentration increased from 1.41 mg·kg−1 at 60 DAFB to 29.87 mg·kg−1 at 90 DAFB or 29.02 mg·kg−1 at 120 DAFB then decreased till ripening (5.47 mg·kg−1). We found ACO was the key enzyme resulting in the difference of citric acid accumulation, but not quite clear in sucrose metabolism. The fruit mineral nutrient concentrations of 6 cultivars during the fruit development were 0.94–1.92% of N, 0.11–0.23% of P, 1.03–1.37% of K, 0.31–1.15% of Ca, 0.11–0.29% of Mg, 3.97–72.34 mg·Fe kg−1, 1.93–10.64 mg·Mn kg−1, 1.56–10.73 mg·Cu kg−1, and 0.90–16.80 mg·Zn kg−1. We also analyzed the relationship among each sugar, organic acid component and mineral nutrient in this study by curve estimation and PCA analysis. The results indicated that only Mg was significantly correlated with both sugar and organic acid component, negative and positive respectively. It suggested that the accumulation of sugar and organic acid might be related to the dynamic changes of fruit Mg concentrations of 6 citrus cultivars.

Mitochondria dysfunctions under Fe and S deficiency: is citric acid involved in the regulation of adaptive responses?

Within the last years, extensive information has been accumulated on the reciprocal influence between S and Fe nutrition at both physiological and molecular level in several plant species, but the mechanisms regulating S and Fe sensing and signaling are not fully understood. Fe and S interact for the building of Fe–S clusters, and mitochondria is one of the cellular compartments where Fe–S cluster assembly takes place. Therefore, it would be expected that mitochondria might play a central role in the regulation of Fe and S interaction. The Fe deficiency-induced alteration in the synthesis of mitochondria-derived carboxylic acids, such as citric acid, and the evidence that such molecules have already been identified as important players of metabolite signaling in several organisms, further support this hypothesis.Tomato plants were grown under single or combined Fe and S deficiency with the aim of verifying whether mitochondria activities played a role in Fe/S interaction. Both Fe and S deficiencies determined similar alteration of respiratory chain activity: a general decrease of Fe-S containing complexes as well as an increase of alternative NAD(P)H activities was observed in both Fe and S deficient-plants. However, the content of Krebs cycle-related organic acids in roots was substantially different in response to treatments, being the accumulation of citric acid always increased, while the others (i.e. succinic, malic, fumaric acids) always decreased. Interestingly, citric acid levels significantly correlated with the expression of some Fe and S deficiency induced genes.Our results contribute to existing knowledge on the complexity of the S/Fe interaction, suggesting a model in which endogenous alteration of citric acid content in plant tissues might act as signal molecule for the regulation of some nuclear-encoded and nutrient-responsive genes and also provide a basis for further study of the mechanism underlying S and Fe sensing and signalling.

Early Embryogenesis of Brown Alga Fucus vesiculosus L. is Characterized by Significant Changes in Carbon and Energy Metabolism.

Brown algae have an important role in marine environments. With respect to their broad distribution and importance for the environment and human use, brown algae of the order Fucales in particular became a model system for physiological and ecological studies. Thus, several fucoids have been extensively studied for their composition on the molecular level. However, research of fucoid physiology and biochemistry so far mostly focused on the adult algae, so a holistic view on the development of these organisms, including the crucial first life stages, is still missing. Therefore, we employed non-targeted metabolite profiling by gas chromatography coupled to mass spectrometry to create a non-biased picture of the early development of the fucoid alga Fucus vesiculosus. We found that embryogenic physiology was mainly dominated by a tight regulation of carbon and energy metabolism. The first dramatic changes of zygote metabolism started within 1 h after fertilization, while metabolism of 6–9 days old embryos appeared already close to that of an adult alga, indicated by the intensive production of secondary metabolites and accumulation of mannitol and citric acid. Given the comprehensive description and analysis we obtained in our experiments, our results exhibit an invaluable resource for the design of further experiments related to physiology of early algal development.

Influence of oxygen availability on the metabolism and morphology of Yarrowia lipolytica: insights into the impact of glucose levels on dimorphism.

Dynamic behavior of Yarrowia lipolytica W29 strain under conditions of fluctuating, low, and limited oxygen supply was characterized in batch and glucose-limited chemostat cultures. In batch cultures, transient oscillations between oxygen-rich and -deprived environments induced a slight citric acid accumulation (lower than 29mgL-1). By contrast, no citric acid was detected in continuous fermentations for all stress conditions: full anoxia (zero pO2 value, 100% N2), limited (zero pO2 value, 75% of cell needs), and low (pO2 close to 2%) dissolved oxygen (DO) levels. The macroscopic behavior (kinetic parameters, yields, viability) of Y. lipolytica was not significantly affected by the exposure to DO fluctuations under both modes of culture. Nevertheless, conditions of oxygen limitation resulted in the destabilization of the glucose-limited growth during the continuous cultivations. Morphological responses of Y. lipolytica to DO oscillations were different between batch and chemostat runs. Indeed, a yeast-to-mycelium transition was induced and progressively intensified during the batch fermentations (filamentous subpopulation reaching 74% (v/v)). While, in chemostat bioreactors, the culture consisted mainly of yeast-like cells (mean diameter not exceeding 5.7μm) with a normal size distribution. During the continuous cultures, growth at low DO concentration did not induce any changes in Y. lipolytica morphology. Dimorphism (up to 80.5% (v/v) of filaments) was only detected under conditions of oxygen limitation in the presence of a residual glucose excess (more than 0.75gL-1). These data suggest an impact of glucose levels on the signaling pathways regulating dimorphic responses in Y. lipolytica.