Citrate Secretion Research Articles

Illumina sequencing revealed roles of microRNAs in different aluminum tolerance of two citrus species.

Self-germinated seedlings of Citrus sinensis and C. grandis were supplied with nutrient solution with 0mM AlCl3·6H2O (control, -Al) or 1mM AlCl3·6H2O (+Al) for 18weeks. The DW(Dry weights) of leaf, stem, shoot and the whole plant of C. grandis were decreased and the ratio of root DW to shoot DW in C. grandis were increased by Al, whereas these parameters of C. sinensis were not changed by Al. Al treatment dramatically decreased the sulfur (S) content in C. grandis roots and the phosphorus (P) content in both C. sinensis and C. grandis roots. More Al was transported to shoots and leaves in C. grandis than in C. sinensis under Al treatment. Al treatment has more adverse effects on C. grandis than on C. sinensis, as revealed by the higher production of superoxide anion (O2 ·-), H2O2 and thiobarbituric acid reactive substace (TBARS) content in C. grandis roots. Via the Illumina sequencing technique, we successfully identified and quantified 12 and 16 differentially expressed miRNAs responding to Al stress in C. sinensis and C. grandis roots, respectively. The possible mechanism underlying different Al tolerance of C. sinensis and C. grandis were summarized as having following aspects: (a) enhancement of adventitious and lateral root development (miR160); (b) up-regulation of stress and signaling transduction related genes, such as SGT1, PLC and AAO (miR477, miR397 and miR398); (c) enhancement of citrate secretion (miR3627); (d) more flexible control of alternative glycolysis pathway and TCA cycle (miR3627 and miR482); (e) up-regulation of S-metabolism (miR172); (f) more flexible control of miRNA metabolism. For the first time, we showed that root development (miR160) and cell wall components (cas-miR5139, csi-miR12105) may play crucial roles in Al tolerance in citrus plants. In conclusion, our study provided a comprehensive profile of differentially expressed miRNAs in response to Al stress between two citrus plants differing in Al tolerance whichfurther enriched our understanding of the molecular mechanism underlying Al tolerance in plants.

Plasma membrane proteomic analysis by TMT-PRM provides insight into mechanisms of aluminum resistance in tamba black soybean roots tips.

Aluminum (Al) toxicity in acid soil is a worldwide agricultural problem that inhibits crop growth and productivity. However, the signal pathways associated with Al tolerance in plants remain largely unclear. In this study, tandem mass tag (TMT)-based quantitative proteomic methods were used to identify the differentially expressed plasma membrane (PM) proteins in Tamba black soybean (TBS) root tips under Al stress. Data are available via ProteomeXchange with identifier PXD017160. In addition, parallel reaction monitoring (PRM) was used to verify the protein quantitative data. The results showed that 907 PM proteins were identified in Al-treated plants. Among them, compared to untreated plants, 90 proteins were differentially expressed (DEPs) with 46 up-regulated and 44 down-regulated (fold change > 1.3 or < 0.77, p < 0.05). Functional enrichment based on GO, KEGG and protein domain revealed that the DEPs were associated with membrane trafficking and transporters, modifying cell wall composition, defense response and signal transduction. In conclusion, our results highlight the involvement of GmMATE13, GmMATE75, GmMATE87 and H+-ATPase in Al-induced citrate secretion in PM of TBS roots, and ABC transporters and Ca2+ have been implicated in internal detoxification and signaling of Al, respectively. Importantly, our data provides six receptor-like protein kinases (RLKs) as candidate proteins for further investigating Al signal transmembrane mechanisms.

Protective Role of Melatonin Against Postmenopausal Bone Loss via Enhancement of Citrate Secretion From Osteoblasts.

A negative correlation exists between the severity of osteoporosis and citrate levels in bone. Our previous research found that melatonin can significantly improve bone mass in mice with osteoporosis, but the underlying mechanism involving citrate remains unknown. Herein, we demonstrated that melatonin increased bone volume and citrate levels in ovariectomized osteoporosis mice. Melatonin increased citrate and mineralized nodules in osteoblasts induced from primary mouse bone marrow mesenchymal stem cells in vitro. ZIP-1 knockdown and overexpression confirmed that melatonin specifically upregulated ZIP-1 to rescue citrate levels and bone mass. In general, we verified that melatonin can improve bone mass by enhancing matrix mineralization, which is highly related to increased citrate secretion from osteoblasts, and that ZIP-1 is the target of melatonin. These findings reveal another role of melatonin in regulating bone remodeling and provide a research base for its possible application in the treatment of clinical osteoporosis in the future.

Short bowel upregulates oxalate and citrate transporters and increases CaOx crystal deposition in the rat hyperoxaluric kidney

Short bowel (SB) increases the risk of kidney stones. However, the underlying mechanism related to intrarenal changes is unclear. Here we examine SB may affect oxalate and citrate handlings on calcium oxalate (CaOx) crystal formation in the hyperoxaluric kidney. SB was induced by small intestine resection in male Wistar rats. Sham‐operation was performed the same surgery without resection. After 7 days of recovery, rats were then fed with 0.75% ethylene glycol (EG and SB+EG groups) for 28 days as a hyperoxaluric induction. The corresponding controls were fed with distilled water (control and SB groups). Blood and 24 h of urine were collected for analysis. Renal CaOx deposition was examined by Pizzolato’s staining. Oxalate transporters Slc26a2 and Slc26a6, and citrate transporter sodium‐dependent dicarboxylate cotransporter NaDC‐1 at their mRNA levels were examined (n=6 in each group). After 7 days of SB, hypocalciuria and hypocitraturia were found when compared with those in sham‐operated control rats. These were associated with hypocalcemia and low renal NaDC‐1 expression after 28 days. SB aggravated EG‐mediated CaOx crystal nephropathy by fostering hyperoxaluria, degree of supersaturation, and CaOx crystal deposition. Renal expression of Slc26a6 but not Slc26a2 was increased in SB+EG group when compared to those in EG or SB group. SB lowered NaDC‐1 expression, but it was reversed in SB+EG group. Intestinal expression of these transporters was similar among groups. We conclude that SB enhances CaOx crystal formation in the hyperoxaluric kidney. More oxalate and less citrate secretion due to Slc26a6 upregulation and insufficient NaDC‐1 expression, respectively, possibly contribute to the aggravated effect of SB on crystal formation.Support or Funding InformationThis research was supported by grants of the Far Eastern Memorial Hospital (FEMH‐2018‐C‐046, Tseng YS and Ma MC) and the Ministry of Science and Technology (MOST108‐2314‐B‐030‐004‐MY3, Ma MC), Taiwan.Hyperoxaluria and hypocitraturia in the short bowl (SB)‐treated hyperoxlauric rats.More prominent hyperoxaluria (A) and oxalate transporter Slc26a6 upregulation (B) was seen in the SB+EG group. Hypocitraturia were found in the SB group (C), this associated with citrate transporter NaDC‐1 downregulation (D). SB increased CaOx formation in the hyperoxaluric kidney (E). N=6 in each group. *P&lt;0.05, EG vs. C group; #P&lt;0.05, SB+EG vs. EG group; @P&lt;0.05, SB+EG vs. SB groupFigure 1

Tumour metabolism and its unique properties in prostate adenocarcinoma.

Anabolic metabolism mediated by aberrant growth factor signalling fuels tumour growth and progression. The first biochemical descriptions of the altered metabolic nature of solid tumours were reported by Otto Warburg almost a century ago. Now, the study of tumour metabolism is being redefined by the development of new molecular tools, tumour modelling systems and precise instrumentation together with important advances in genetics, cell biology and spectroscopy. In contrast to Warburg's original hypothesis, accumulating evidence demonstrates a critical role for mitochondrial metabolism and substantial variation in the way in which different tumours metabolize nutrients to generate biomass. Furthermore, computational and experimental approaches suggest a dominant influence of the tissue-of-origin in shaping the metabolic reprogramming that enables tumour growth. For example, the unique metabolic properties of prostate adenocarcinoma are likely to stem from the distinct metabolism of the prostatic epithelium from which it emerges. Normal prostatic epithelium employs comparatively glycolytic metabolism to sustain physiological citrate secretion, whereas prostate adenocarcinoma consumes citrate to power oxidative phosphorylation and fuel lipogenesis, enabling tumour progression through metabolic reprogramming. Current data suggest that the distinct metabolic aberrations in prostate adenocarcinoma are driven by the androgen receptor, providing opportunities for functional metabolic imaging and novel therapeutic interventions that will be complementary to existing diagnostic and treatment options.

A Defective Vacuolar Proton Pump Enhances Aluminum Tolerance by Reducing Vacuole Sequestration of Organic Acids.

Plants cope with aluminum (Al) toxicity by secreting organic acids (OAs) into the apoplastic space, which is driven by proton (H+) pumps. Here, we show that mutation of vacuolar H+-translocating adenosine triphosphatase (H+-ATPase) subunit a2 (VHA-a2) and VHA-a3 of the vacuolar H+-ATPase enhances Al resistance in Arabidopsis (Arabidopsis thaliana). vha-a2 vha-a3 mutant plants displayed less Al sensitivity with less Al accumulation in roots compared to wild-type plants when grown under excessive Al3+ Interestingly, in response to Al3+ exposure, plants showed decreased vacuolar H+ pump activity and reduced expression of VHA-a2 and VHA-a3, which were accompanied by increased plasma membrane H+ pump (PM H+-ATPase) activity. Genetic analysis of plants with altered PM H+-ATPase activity established a correlation between Al-induced increase in PM H+-ATPase activity and enhanced Al resistance in vha-a2 vha-a3 plants. We determined that external OAs, such as malate and citrate whose secretion is driven by PM H+-ATPase, increased with PM H+-ATPase activity upon Al stress. On the other hand, elevated secretion of malate and citrate in vha-a2 vha-a3 root exudates appeared to be independent of OAs metabolism and tolerance of phosphate starvation but was likely related to impaired vacuolar sequestration. These results suggest that coordination of vacuolar H+-ATPase and PM H+-ATPase dictates the distribution of OAs into either the vacuolar lumen or the apoplastic space that, in turn, determines Al tolerance capacity in plants.

Functional characterization of three MATE genes in relation to aluminum-induced citrate efflux from soybean root

Citrate transporters responsible for Aluminum (Al)-induced citrate efflux have not been identified in soybean. Three soybean multi-drug and toxic compound extrusion (MATE) family genes were characterized by transcriptional expression, subcellular localization and overexpression experiments. GmMATE75, GmMATE79 and GmMATE87 are localized to plasma membrane. Their over-expression respectively resulted in more citrate efflux and less Al contents in soybean hairy roots, alleviated root elongation inhibition in Arabidopsis and partially restored root growth in atmate mutant under Al stress. Al increased their transcriptional expression at either the root apex or the base zone. Cu2+, Cd2+, La3+ increased the expression of GmMATE79 and GmMATE87. Ten day of –Fe culture increased the expression of GmMATE75 and GmMATE79. Al treatment extended β-glucuronidase (GUS) staining from central cylinder to cortical and epidermis cells for pGmMATE75::GUS or pGmMATE79::GUS soybean hairy roots transformation. But GUS staining restricted within central cylinder for pGmMATE87::GUS transformation with or without Al treatment. GmMATE75, GmMATE79 and GmMATE87 are plasma-membrane-localized citrate transporters and have capabilities to increase citrate efflux. They played different role in Al-induced citrate secretion from soybean because their different tissue localization and expression patterns.

Interactions between hydrogen sulphide and nitric oxide regulate two soybean citrate transporters during the alleviation of aluminium toxicity.

Hydrogen sulphide (H2 S) is emerging as an important signalling molecule involved in plant resistance to various stresses. However, the underlying mechanism of H2 S in aluminium (Al) resistance and the crosstalk between H2 S and nitric oxide (NO) in Al stress signalling remain elusive. Citrate secretion is a wide-spread strategy for plants against Al toxicity. Here, two citrate transporter genes, GmMATE13 and GmMATE47, were identified and characterized in soybean. Functional analysis in Xenopus oocytes and transgenic Arabidopsis showed that GmMATE13 and GmMATE47 mediated citrate exudation and enhanced Al resistance. Al treatment triggered H2 S generation and citrate exudation in soybean roots. Pretreatment with an H2 S donor significantly elevated Al-induced citrate exudation, reduced Al accumulation in root tips, and alleviated Al-induced inhibition of root elongation, whereas application of an H2 S scavenger elicited the opposite effect. Furthermore, H2 S and NO mediated Al-induced GmMATE expression and plasma membrane (PM) H+ -ATPase activity and expression. Further investigation showed that NO induced H2 S production by regulating the key enzymes involved in biosynthesis and degradation of H2 S. These findings indicate that H2 S acts downstream of NO in mediating Al-induced citrate secretion through the upregulation of PM H+ -ATPase-coupled citrate transporter cotransport systems, thereby conferring plant resistance to Al toxicity.

Metabolic Phenotyping of Adipose-Derived Stem Cells Reveals a Unique Signature and Intrinsic Differences between Fat Pads.

White adipose tissues are functionally heterogeneous and differently manage the excess of energy supply. While the expansion of subcutaneous adipose tissues (SAT) is protective in obesity, that of visceral adipose tissues (VAT) correlates with the emergence of metabolic diseases. Maintained in fat pads throughout life, adipose stem cells (ASC) are mesenchymal-like stem cells with adipogenesis and multipotent differentiation potential. ASC from distinct fat pads have long been reported to present distinct proliferation and differentiation potentials that are maintained in culture, yet the origins of these intrinsic differences are still unknown. Metabolism is central to stem cell fate decision in line with environmental changes. In this study, we performed high-resolution nuclear magnetic resonance (NMR) metabolomic analyses of ASC culture supernatants in order to characterize their metabolic phenotype in culture. We identified and quantified 29 ASC exometabolites and evaluated their consumption or secretion over 72 h of cell culture. Both ASC used glycolysis and mitochondrial metabolism, as evidenced by the high secretions of lactate and citrate, respectively, but V-ASC mostly used glycolysis. By varying the composition of the cell culture medium, we showed that glutaminolysis, rather than glycolysis, supported the secretion of pyruvate, alanine, and citrate, evidencing a peculiar metabolism in ASC cells. The comparison of the two types of ASC in glutamine-free culture conditions also revealed the role of glutaminolysis in the limitation of pyruvate routing towards the lactate synthesis, in S-ASC but not in V-ASC. Altogether, our results suggest a difference between depots in the capacity of ASC mitochondria to assimilate pyruvate, with probable consequences on their differentiation potential in pathways requiring an increased mitochondrial activity. These results highlight a pivotal role of metabolic mechanisms in the discrimination between ASC and provide new perspectives in the understanding of their functional differences.

SUN-543 Myostatin Inhibits IGF1-Dependent Citrate Production during Osteogenesis in a Mouse Mesenchymal Stem Cell Line

Bone formation and osteoblast activation require glucose. Citrate is a key intermediate in the tricarboxylic acid (TCA) cycle that is the major energy-yielding metabolic pathway in cells. Citrate homeostasis, therefore, is critically important for glucose metabolism and energy production. In addition, citrate is an indispensable component of bone. The vast amount of citrate with its carboxylate groups provides a huge capacity for calcium binding on apatite that stabilizes the size of nanocrystal of bone. Reduced citrate levels in bone and serum are reported in elderly compared to young subjects, as well as in osteoporotic patients. IGF1, a growth factor that elicits downstream signaling events similar to insulin and also controls TCA activity, is essential in bone matrix mineralization. However, the source of citrate in bone and what roles that IGF1 plays in regulating citrate metabolism and deposition during osteogenesis are largely unknown. Myostatin (Mstn), a muscle cell-secreted cytokine, is involved in regulating energy metabolism, however, its effect on osteogenesis and bone formation are not well delineated. In this study, the interactive effect of Mstn and IGF1 on citrate production during osteogenesis was examined in C3H10T1/2 mouse mesenchymal stem cells (MSC). MSCs cultured in osteogenic medium induced high levels of citrate secretion with the peak (5.6-fold) was observed at a later stage (14-21d) of osteogenic differentiation. The levels of IGF1 mRNA and protein were also upregulated with a similar pattern, but of reduced magnitude than that of citrate secretion. In contrast, cells treated with Mstn showed a significant inhibition in both citrate secretion and IGF1 expression during the same period of osteogenesis. Alizarin Red staining (ARs) that was used to confirm the biological effect of Mstn showed a significant decrease in ARs in Mstn-treated cells compared to that of control cells. In addition, alkaline phosphatase (ALP) staining revealed a dramatical decrease in ALP expression and activity. These results suggest that inhibited citrate levels by Mstn led to defects in osteogenic differentiation and mineralization. To determine whether IGF1 plays role in these processes, an anti-IGF1 neutralizing antibody that inhibits IGF1 expression was employed. MSCs treated with IGF1 antibody showed a 35% reduction in citrate secretion compared to that of control cells. Moreover, the addition of exogenous IGF1 partially reversed the inhibitory effect of Mstn on citrate secretion, indicating that increased levels of citrate during osteogenesis is partially IGF1-dependent and that Mstn-inhibited citrate production is, at least in part, mediated by reduced IGF1 levels. These results imply a novel effect of Mstn on modulating osteogenesis of bone marrow MSCs by preventing IGF1-dependent citrate production that leads to defects in osteogenic differentiation and mineralization.

Involvement of phosphatidylinositol metabolism in aluminum-induced malate secretion in Arabidopsis.

To identify the upstream signaling of aluminum-induced malate secretion through aluminum-activated malate transporter 1 (AtALMT1), a pharmacological assay using inhibitors of human signal transduction pathways was performed. Early aluminum-induced transcription of AtALMT1 and other aluminum-responsive genes was significantly suppressed by phosphatidylinositol 4-kinase (PI4K) and phospholipase C (PLC) inhibitors, indicating that the PI4K-PLC metabolic pathway activates early aluminum signaling. Inhibitors of phosphatidylinositol 3-kinase (PI3K) and PI4K reduced aluminum-activated malate transport by AtALMT1, suggesting that both the PI3K and PI4K metabolic pathways regulate this process. These results were validated using T-DNA insertion mutants of PI4K and PI3K-RNAi lines. A human protein kinase inhibitor, putatively inhibiting homologous calcineurin B-like protein-interacting protein kinase and/or Ca-dependent protein kinase in Arabidopsis, suppressed late-phase aluminum-induced expression of AtALMT1, which was concomitant with the induction of an AtALMT1 repressor, WRKY46, and suppression of an AtALMT1 activator, Calmodulin-binding transcription activator 2 (CAMTA2). In addition, a human deubiquitinase inhibitor suppressed aluminum-activated malate transport, suggesting that deubiquitinases can regulate this process. We also found a reduction of aluminum-induced citrate secretion in tobacco by applying inhibitors of PI3K and PI4K. Taken together, our results indicated that phosphatidylinositol metabolism regulates organic acid secretion in plants under aluminum stress.

AhFRDL1-mediated citrate secretion contributes to adaptation to iron deficiency and aluminum stress in peanuts.

Although citrate transporters are involved in iron (Fe) translocation and aluminum (Al) tolerance in plants, to date none of them have been shown to confer both biological functions in plant species that utilize Fe-absorption Strategy I. In this study, we demonstrated that AhFRDL1, a citrate transporter gene from peanut (Arachis hypogaea) that is induced by both Fe-deficiency and Al-stress, participates in both root-to-shoot Fe translocation and Al tolerance. Expression of AhFRDL1 induced by Fe deficiency was located in the root stele, but under Al-stress expression was observed across the entire root-tip cross-section. Overexpression of AhFRDL1 restored efficient Fe translocation in Atfrd3 mutants and Al resistance in AtMATE-knockout mutants. Knocking down AhFRDL1 in the roots resulted in reduced xylem citrate and reduced concentrations of active Fe in young leaves. Furthermore, AhFRDL1-knockdown lines had reduced root citrate exudation and were more sensitive to Al toxicity. Compared to an Al-sensitive variety, enhanced AhFRDL1 expression in an Fe-efficient variety contributed to higher levels of Al tolerance and Fe translocation by promoting citrate secretion. These results indicate that AhFRDL1 plays a significant role in Fe translocation and Al tolerance in Fe-efficient peanut varieties under different soil-stress conditions. Given its dual biological functions, AhFRDL1 may serve as a useful genetic marker for breeding for high Fe efficiency and Al tolerance.

Comparison of metabolic changes for stone risks in 24-hour urine between non- and postmenopausal women.

BackgroundTo explore the differences of 24-hour urine compositions associated with urolithiasis between non- and postmenopausal females.MethodsThe 24-hour urine samples of female participants were collected from May 2013 to July 2014 along with national cross-sectional study of urolithiasis among adults aged ≥18 years in China. The exclusion criteria for the participants were: serum creatinine > 133μmol/L, with urinary tract infection, gout, hyperthyroidism, malignancy, had a history of cancer, kidney stones, enterectomy, had taken thiazide diuretics, allopurinol, vitamin supplement, potassium citrate or calcium supplements during the past two weeks. The compositions associated with urinary stone in 24-hour urine were measured and compared between non-and postmenopausal women.ResultsA total of 603 24-hour urine samples of female participants were analyzed. 354 women with a mean age of 52.5± 14.03 (range 19–84) years met the criteria, including 160 non-menopausal women and 194 postmenopausal women. Compared to the non-menopausal women, postmenopausal women had a lower secretion of citrate (p = 0.043), magnesium (p = 0.001) and creatinine (p = 0.001) in 24h urine. Multivariate linear regression analysis showed that the menopause status was associated with the changes in magnesium (p = 0.003) and creatinine (p = 0.002) secretion, whereas not with the changes in citrate (p = 0.402) secretion.ConclusionsPostmenopausal women have a significant lower secretion of magnesium in their 24-hour urine than non-menopausal ones. We suppose that might be associated with increased risk of urinary stone formation among postmenopausal women.

Intracellular expression of Vitreoscilla haemoglobin improves lipid production in Yarrowia lipolytica.

Genetic modification by overexpressing Vitreoscilla haemoglobin (VHb) is an efficient and economic method for improving O2 utilization for microbial fermentation. In this study, VHb was expressed in oleaginous yeast, Yarrowia lipolytica, and its effect on lipid accumulation was analysed. During fermentation, the dissolved oxygen (DO) concentration was controlled at 5, 10, 20 and 30 of full O2 saturation and also uncontrolled by varying the stirring speed and aeration rate. Yarrowia lipolytica harbouring the VHb gene (VHb+ strain) displayed more pseudomycelium than the control strain (VHb- strain), and VHb expression also enhanced the cell density. When DO level was controlled at 30%, the biomass of VHb+ strains reached about 19gl-1 and increased by 27% compared to VHb- strains. Total fatty acid contents, although, were higher in VHb+ strains than in VHb- strains under all DO levels, and maximally increased by c. 40% (from 10·5 to 14·5% of the biomass) when the DO concentration was controlled at 30%. VHb overexpression, however, markedly suppressed citrate secretion. In addition, expression of VHb also induced significant changes in fatty acid composition and increased the oleic acid content. SIGNIFICANCE AND IMPACT OF THE STUDY: Genetic modification by overexpressing Vitreoscilla haemoglobin (VHb) is an efficient and economic method for improving O2 utilization for microbial fermentation. In this study, VHb was expressed in Yarrowia lipolytica, which is a most attractive model for microbial oil production because of its excellent lipid accumulation capacity and convenient genetic tools. Expression of VHb resulted in lipid accumulation increased and cell morphology shift, however, markedly suppressed citrate secretion. This study provided the new strategy for fermentation technology to improve lipid production in oleaginous micro-organisms.

Biotic strategies to increase plant availability of sewage sludge ash phosphorus

AbstractSewage sludge incineration‐ash (FB‐I) represents a potential alternative phosphorus (P) fertiliser with a high concentration of P, although with relatively low crop availability. In this study, we investigated two P‐solubilisation approaches (acidification and P mobilisation by citrate) to enhance plant P uptake from the FB‐I ash in a pot study by using various biotic strategies: (1) a pre‐treatment of ash with a Penicillium bilaiae inoculum, (2) an isogenic line of wheat that excretes citrate from the root tip, (3) nitrogen (N) provided as combined with nitrification inhibitor dicyandiamide (DCD). All strategies were tested combined with each other and with different methods for ash application: (1) completely mixed within the top one third of soil in a pot, or (2) applied as distinct band at 10 cm depth. Triple super phosphate (TSP) at a rate of 15 mg P kg−1 soil per pot was sufficient to support maximum shoot growth. Ash mixed into the first top third part of soil in the pot at a rate of 180 mg P kg−1 soil (equivalent to 60 mg P kg−1 soil throughout the pot) significantly increased the soil water‐extractable P and the subsequent shoot P uptake and shoot biomass for both wheat lines and microbial pre‐treatment to support maximum plant performance. Shoot P concentration in these treatments was further enhanced when the plants received and DCD, although not leading to a significant increase in shoot biomass. The citrate secretion by the root tips and pre‐inoculation with P. bilaiae of the ash did not influence plant growth. In conclusion, root‐zone soil acidification by nutrition is regarded as a promising strategy to improve the fertilising effect of such alternative P fertilisers originating from urban waste streams.

Nitric oxide mediates aluminum-induced citrate secretion through regulating the metabolism and transport of citrate in soybean roots

Citrate secretion is a kind of typical strategy for plant against aluminum (Al) toxicity. However, the signaling process in Al-activated citrate secretion needs to be clarified. Physiological and biochemical methods as well as gene expression analysis were employed to examine the regulatory roles of nitric oxide (NO) in Al-activated citrate secretion in soybean roots. Application of NO donor alleviated root growth inhibition and decreased Al content in Al-treated root apices. Al-induced NO production and citrate secretion were further elevated by NO donor, but inhibited by NO scavenger. Inhibition of citrate synthase (CS) or plasma membrane (PM) H+-ATPase activity significantly decreased Al-induced secretion of citrate, but inhibition of aconitase (ACO) activity enhanced citrate secretion under Al stress. Furthermore, NO mediated Al-stimulated CS and PM H+-ATPase activities, but decreased ACO activity under Al stress. Further investigation showed that NO modulated Al-activated transcriptional expression of CS and PM H+-ATPase as well as GmMATE. Overexpression of GmMATE in soybean hairy roots caused an enhanced Al-induced citrate efflux and Al resistance. Our findings suggest that NO-dependent up-regulation of citrate synthesis and activation of PM H+-ATPase-coupled MATE transporter co-transport system participates in Al-activated citrate exudation, thus conferring plant resistance to Al toxicity.

Retrotransposon Insertion and DNA Methylation Regulate Aluminum Tolerance in European Barley Accessions.

Aluminum (Al) toxicity is a major stress factor limiting crop productivity in acid soil. Although there is great genotypic variation in tolerance to Al toxicity, the underlying molecular mechanisms are poorly understood. Here, we report that, in barley (Hordeum vulgare), the fourth largest cereal crop produced in the world, both retrotransposon insertion and DNA methylation are involved in regulating differential Al tolerance. HvAACT1 is a major gene responsible for citrate secretion from the roots for external detoxification of Al. A multiretrotransposon-like (MRL) sequence insertion at least 15.3 kb in length was detected in the upstream genomic region of HvAACT1 that displayed promoter activity and significantly enhanced HvAACT1 expression, especially in the root tips of Al-tolerant accessions. Furthermore, in a number of accessions with low levels of HvAACT1 expression, this MRL insertion was present but highly methylated. Geographical analysis showed that accessions with this MRL insertion are distributed mainly in European areas with acid soils. Two wild barley accessions were found to possess this MRL insertion, but with a high degree of methylation. These results indicate that the MRL insertion and its degree of DNA methylation influence HvAACT1 expression and that demethylation of this MRL insertion, which facilitates adaptation to acid soils, occurred following barley domestication. Moreover, our results indicate that barley accessions in East Asia and Europe have developed independent but equivalent strategies to withstand Al toxicity in acid soils.

Identification of microRNAs in response to aluminum stress in the roots of Tibetan wild barley and cultivated barley

BackgroundBarley is relatively sensitive to Aluminum (Al) toxicity among cereal crops, but shows a wide genotypic difference in Al tolerance. The well-known Al-tolerant mechanism in barley is related to Al exclusion mediated by a citrate transporter HvAACT1 (Al-activated citrate transporter 1). A 1-kb insertion in the promoter region of HvAACT1 gene results in a dramatic increase of its expression level, which only occurs in some Al-tolerant cultivars. However, Al-tolerant Tibetan wild barley accession XZ29 did not have the 1-kb insertion.ResultsWe confirmed that the expression of HvAACT1 and secretion of citrate and other organic acids did not explain the difference in Al-tolerant wild barley XZ29 and Al-sensitive cultivated barley Golden Promise. To identify microRNAs (miRNAs) and their target genes responsive to Al stress in barley roots, eight small RNA libraries with two biological replicates from these two genotypes exposed to control and Al-treated conditions were constructed and submitted to deep sequencing. A total of 342 miRNAs were identified in Golden Promise and XZ29, with 296 miRNAs being commonly shared in the two genotypes. Target genes of these miRNAs were obtained through bioinformatics prediction or degradome identification. Comparative analysis detected 50 miRNAs responsive to Al stress, and some of them were found to be exclusively expressed in XZ29 and associated with Al tolerance.ConclusionsmiRNAs exclusively expressing in the wild barley were identified and found to be associated with Al stress tolerance. The current results provide a model of describing the roles of some special miRNAs associated with Al tolerance in the Tibetan wild barley.

Bone and plasma citrate is reduced in osteoporosis.

High concentration of citrate exists in bone of humans and all osteo-vertebrates, and citrate incorporation imparts important biomechanical and other functional properties to bone. However, which cells are responsible for citrate production in bone remains unclear and whether the citrate component changes with bone loss during osteoporosis is also not known. Here, we show that the citrate content is markedly reduced in the bone of mice or rats with age-related, ovariectomy-induced or retinoic acid-induced bone loss. Plasmic citrate is also downregulated in osteoporotic animals. Importantly, the plasmic citrate level of aged osteoporotic males is significantly lower than that of young healthy males and positively correlates with human lumbar spine bone mineral density (BMD) and total hip BMD. Furthermore, citrate production increases with in vitro osteoblastic differentiation, accompanied by upregulation of proteins involved in citrate secretion, suggesting that osteoblasts are highly specialized cells that produce citrate in bone. Our findings establish a novel relationship between citrate content and bone loss-related diseases such as osteoporosis, suggesting a critical role of bone citrate in the maintenance of the citrate balance in the circulation. Serum citrate level may thus represent a novel marker for osteoporosis.

Insights into aluminum-tolerance pathways in Stylosanthes as revealed by RNA-Seq analysis

Stylo has a great potential for Al3+ resistance in acidic soils through secretion of citrate from the roots. To get insight into the molecular mechanisms responsible, transcriptomic changes were investigated in the roots after treatment with T01 (−Al3+, pH6.0), T02 (−Al3+, pH4.3) and T03 (50 µM AlCl3, pH4.3). In total, 83,197 unigenes generated from 130,933 contigs were obtained. Of them, 282, 148 and 816 differentially expressed unigenes (DEGs) were revealed in T01_vs_T02, T02_vs_T03 and T01_vs_T03 comparison, respectively (FDR < 0.001, log2FC > 2). DEGs by Al3+ were related to G-proteins, diacyglycerol and inositol metabolism, calcium-signaling, transcription regulation, protein modification and transporters for detoxification of Al3+. Additionally, Al3+ facilitates citrate synthesis via modifying gene expression of pathways responsible for citrate metabolism. Overall, Al3+ resistance in stylo involves multiple strategies and enhancement of citrate anabolism. The Al3+ signal transmits through heterotrimeric G-proteins, phospholipase C, inositol triphosphate, diacylglycerol, Ca2+ and protein kinases, thereby activating transcription and anion channels in plasma membrane, and resulting in citrate secretion from stylo roots.