Citrate Secretion Research Articles

Identification of early Al‐responsive genes in rice bean (Vigna umbellata) roots provides new clues to molecular mechanisms of Al toxicity and tolerance

Significant secretion of citrate from root apex of rice bean (Vigna umbellata) is delayed by several hours under aluminium (Al) stress. However, the molecular basis of regulation of VuMATE1, a gene encoding an Al-activated citrate transporter, remains unclear. In this study, we used suppression subtractive hybridization together with reverse northern blot analysis and qRT-PCR to identify genes with altered transcript levels in the root apex after treatment with low (5 μm) or high (25 μm) concentration of AlCl(3) for a short time (4 h). We found that in addition to VuMATE1, 393 genes showed an early response to Al. Among functionally annotated genes, those related to 'metabolism and energy', 'signal transduction and transcription' and 'transport' was predominantly up-regulated, whereas those associated with 'protein translation, processing and degradation' was predominantly down-regulated. Comparative analysis of transcriptional profiles highlighted candidate genes associated with citrate secretion and revealed several new aspects of the molecular processes underlying Al toxicity and tolerance. Based on the data, it is proposed that metabolic changes represent adaptive mechanisms to Al stress, whereas inhibition of both cell elongation and cell division underlies Al-induced root growth inhibition.

Differences in physiological features associated with aluminum tolerance in Tibetan wild and cultivated barleys

Aluminum (Al) toxicity is a major limiting factor for plant production in acid soils. Wild barley germplasm is a treasure trove of useful genes and offers rich sources of genetic variation for crop improvement. Al-stress-hydroponic-experiments were performed, and the physiochemical characteristic of two contrasting Tibetan wild barley genotypes (Al-resistant XZ16 and Al-sensitive XZ61) and Al-resistant cv. Dayton were compared. Ultrastructure of chloroplasts and root cells in XZ16 was less injured than that in Dayton and XZ61. Moreover, XZ16 secreted significantly more malate besides citrate and exhibited less Al uptake and distribution than both of XZ61 and Dayton in response to Al stress, simultaneously maintained higher H+-, Ca2+Mg2+- and total-ATPase activities over XZ61. The protein synthesis inhibitor cycloheximide reduced citrate secretion from XZ16, but not from Dayton. In Tibetan wild barley, our findings highlight the significant correlations between Al tolerance, ATPase activity and citrate secretion, providing some insights into the physiological basis for Al-detoxification.

Physiological and transcriptional responses of broad bean (Vicia faba L.) leaves to aluminium stress

SummaryAluminium (Al) toxicity is the major factor-limiting crop productivity in acid soils. In the present study, physiological and transcriptional responses of broad bean leaves to Al stress were investigated. Malondialdehyde (MDA) content, H2O2 content and protein carbonyls (PC) level in leaves were increased after 100 μm AlCl3 stress treatment, whereas the total protein content was decreased, compared with the plants without Al treatment. Stomatal closure in leaves of broad bean was increased after Al stress, suggesting that the photosynthesis rate might be affected by Al stress. The relative citrate secretion in leaves was decreased after Al treatment for 24 h according to the 13C-NMR analysis, indicating that citrate in leaves might be transported to the root to chelate Al3+. To investigate the molecular mechanisms of Al toxicity in leaves of broad bean, a suppression subtractive hybridization (SSH) library was constructed to identify up-regulated genes: cDNA from leaves subjected to 12, 24, 48 and 72 h of 50 and 100 μm AlCl3 stress were used as testers and cDNA from leaves subjected to 0 μm AlCl3 treatment for the same lengths of time as above were used as a driver. The SSH analysis identified 156 non-redundant putative Al stress-responsive expressed sequence tags (ESTs) out of 960 clones. The ESTs were categorized into ten functional groups, which were involved in metabolism (0·21), protein synthesis and protein fate (0·10), photosynthesis and chloroplast structure (0·09), transporter (0·08), cell wall related (0·06), signal transduction (0·05), defence, stress and cell death (0·05), energy (0·03), transcription factor (0·03) and unknown proteins (0·30). The effect of Al treatment on expression of 15 selected genes was investigated by reverse transcription polymerase chain reaction (RT–PCR), confirming induction by Al stress. The results indicated that genes involved in organic acid metabolism, transport, photosynthesis and chloroplast structure, defence, stress and cell death might play important roles under Al stress.

Adaptation to acidic soil is achieved by increased numbers of cis‐acting elements regulating ALMT1 expression in Holcus lanatus

Yorkshire fog (Holcus lanatus), which belongs to the Poaceae family and is a close relative of the agronomic crop oat (Avena sativa), is a widely adaptable grass species that is able to grow on highly acidic soils with high levels of Al, but the mechanism underlying the high Al tolerance is unknown. Here, we characterized two accessions of H.lanatus collected from an acid plot (soil pH 3.6, HL-A) and a neutral plot (pH 7.1, HL-N) in terms of Al tolerance, organic acid anion secretion and related gene expression. In response to Al (pH 4.5), the HL-A roots secreted approximately twice as much malate as the HL-N roots, but there was no difference in citrate secretion. Cloning of the gene HlALMT1 responsible for malate secretion showed that the encoded amino acid sequence did not differ between two accessions, but the expression level in the outer cell layers of the HL-A roots was twice as high as in the HL-N roots. This difference was not due to the genomic copy number, but was due to the number of cis-acting elements for an Al-responsive transcription factor (HlART1) in the promoter region of HlALMT1, as demonstrated by both a yeast one-hybrid assay and a transient assay in tobacco protoplasts. Furthermore, introduction of HlALMT1 driven by the HL-A promoter into rice resulted in significantly more Al-induced malate secretion than introduction of HlALMT1 driven by the HL-N promoter. These findings indicate that the adaptation of H.lanatus to acidic soils may be achieved by increasing number of cis-acting elements for ART1 in the promoter region of the HlALMT1 gene, enhancing the expression of HlALMT1 and the secretion of malate.

Up-regulation and interaction of the plasma membrane H+-ATPase and the 14-3-3 protein are involved in the regulation of citrate exudation from the broad bean (Vicia faba L.) under Al stress

Our previous study showed that citrate excretion coupled with a concomitant release of protons was involved in aluminum (Al) resistance in the broad bean. Furthermore, genes encoding plasma membrane (PM) H(+)-ATPase (vha2) and the 14-3-3 protein (vf14-3-3b) were up-regulated by Al in Al-resistant (YD) broad bean roots. In this study, the roles of PM H(+)-ATPase (E.C. 3.6.3.6) and the 14-3-3 protein in the regulation of citrate secretion were further investigated in Al-resistant (YD) and Al-sensitive (AD) broad bean cultivars under Al stress. The results showed that greater citrate exudation was positively correlated with higher activities of PM H(+)-ATPase in roots of YD than AD. Real-time RT-PCR analysis revealed that vha2 was clearly up-regulated by Al in YD but not in AD roots, whereas the transcription levels of vf14-3-3b were elevated in a time-dependent manner in both YD and AD roots. Immunoprecipitation and Western analysis suggested that phosphorylation and interaction with the vf14-3-3b protein of the VHA2 were enhanced in YD roots but not in AD roots with increasing Al treatment time. Fusicoccin or adenosine 5'-monophosphate increased or decreased the interaction between the phosphorylated VHA2 and the vf14-3-3b protein, followed by an enhancement or reduction of the PM H(+)-ATPase activity and citrate exudation in both cultivars under Al stress conditions, respectively. Taken together, these results suggested that Al enhanced the expression and interaction of the PM H(+)-ATPase and the 14-3-3 protein, which thereby led to higher activity of the PM H(+)-ATPase and more citrate exudation from YD plants.

A combination of five mechanisms confers a high tolerance for aluminum to a wild species of Poaceae, Andropogon virginicus L.

How can high tolerance against aluminum (Al) toxicity be obtained in plants? To address this question, tolerant mechanisms were characterized in a highly Al tolerant wild species of Poaceae, Andropogon virginicus L. A. virginicus showed an Al-stress-induced synthesis and secretion of citrate and malate in roots. This mechanism may help to suppress an increase of toxic Al ions in the root region. Microscopic observation of the morin-stained leaves indicated that the Al transferred to shoots was specifically accumulated in the trichomes and spikes of the leaves and that some portion of the accumulated Al was furthermore secreted as sap from the tips of trichomes. Al-induced synthesis of poly-phenolic compounds including anthocyanin also occurred in roots as a long term response to Al toxicity and anthocyanin production did not co-localize with either Al accumulation, nitric oxide (NO) production or lipid peroxides production in the roots. It was suggested that oxidative damage caused by Al stress was suppressed in these areas where anthocyanin was localized. Moreover, induction of NO production occurred in roots within 24h of Al treatment. Our results suggested that NO could not efficiently ameliorate the Al-dependent nuclei deformation and DNA fragmentation, but could function as a trigger to stimulate anti-peroxidation enzymes under Al stress. Collectively the results suggested that A. virginicus manifests its high Al tolerance by a unique combination of effective mechanisms.

Al-enhanced Expression and Interaction of 14-3-3 Protein and Plasma Membrane H+-ATPase is Related to Al-induced Citrate Secretion in an Al-resistant Black Soybean

The up-regulated expression of plasma membrane (PM) H+-ATPase is related to Al-enhanced citrate exudation in soybean. Moreover, the interaction with a 14-3-3 protein can activate PM H+-ATPase. This study investigated if the expression and interaction level of the 14-3-3 protein and PM H+-ATPase is associated with PM H+-ATPase activity as well as citrate secretion in Al-tolerant (RB) and Al-sensitive (SB) black soybeans, respectively, under 50 mM Al stress. The results showed that Al stress significantly induced transcription of the 14-3-3a gene in RB but not in SB roots. The Al-enhanced transcription of the PM H+-ATPase encoding gene (gha2) was higher in RB than in SB roots. The translation and phosphorylation of PM H+-ATPase, the interaction between the 14-3-3 protein and the phosphorylated PM H+-ATPase all showed an increasing pattern in RB but displayed a descending pattern in SB during the whole Al stress period. Consistently, H+ pump activity and citrate secretion were also higher in RB than in SB under Al stress. Furthermore, citrate secretion was correlated positively with PM H+-ATPase activity in RB and SB under Al stress. These results indicated that Al-enhanced expression of 14-3-3 protein and PM H+-ATPase and the interaction between 14-3-3 protein and phosphorylated PM H+-ATPase increased the activity of PM H+-ATPase and the H+ pump, thereby augmenting citrate secretion in RB. This might be an important molecular mechanism underlying the higher Al-tolerance in RB compared to SB.

The role of VuMATE1 expression in aluminium-inducible citrate secretion in rice bean (Vigna umbellata) roots

Aluminium (Al)-activated citrate secretion plays an important role in Al resistance in a number of plant species, such as rice bean (Vigna umbellata). This study further characterized the regulation of VuMATE1, an aluminium-activated citrate transporter. Al stress induced VuMATE1 expression, followed by the secretion of citrate. Citrate secretion was specific to Al stress, whereas VuMATE1 expression was not, which could be explained by a combined regulation of VuMATE1 expression and Al-specific activation of VuMATE1 protein. Pre-treatment with a protein translation inhibitor suppressed VuMATE1 expression, indicating that de novo biosynthesis of proteins is required for gene expression. Furthermore, post-treatment with a protein translation inhibitor inhibited citrate secretion, indicating that post-transcriptional regulation of VuMATE1 is critical for citrate secretion. Protein kinase and phosphatase inhibitor studies showed that reversible phosphorylation was important not only for transcriptional regulation of VuMATE1 expression but also for post-translational regulation of VuMATE1 protein activity. These results suggest that citrate secretion is dependent on both transcriptional and post-transcriptional regulation of VuMATE1. Additionally, VuMATE1 promoter–β-glucuronidase fusion lines revealed that VuMATE1 expression was restricted to the root apex and was entirely Al induced, indicating the presence of cis-acting elements regulating root tip-specific and Al-inducible gene expression, which will be an important resource for genetic improvement of plant Al resistance.

Organic Acid Characteristics and Tolerance of Sengon (Paraserianthes falcataria L Nielsen) to Lead

This study aimed to find out the lead tolerance of sengon (Paraserianthes falcataria) seedling based on growth performance, tolerance index, and ecretion and accumulation of organic acids content. Seedlings were exposed to lead (Pb) with the concentration of 0, 0.5, 1, 1.5, 5, and 10 mM in liquid nutrient culture for 4 days in order to investigate secretion and accumulation of oxalic, malic, and citric content, and for 15 days to examine growth performance and olerance index. The result showed that tolerance index and growth performance of engon seedling were insignificant (p > 0.05) to the rising of Pb concentration up to 1.5 mM with tolerance index at least 95%, and even caused an increase of fresh weight. However, the tolerance index and growth of sengon decreased significantly due to Pb exposure of 5 and 10 mM. Among the three organic acids, citrate was most dominant as compared to malate and oxalate. Secretion of citrate increased significantly (p < 0.05) with the rising concentration of Pb 0.5, 1 and 1.5 mM, reaching to 0.464, 0.540, and 0.587 µg mℓ-1, respectively, or rising according linear line (r = 0.9, p < 0.5). Citrate accumulation showed inconsistent pattern with the rising Pb exposure. The result suggested that sengon seedling have a slightly tolerance to lead by secretion of organic acid especially citric acid.

Involvement of putrescine and nitric oxide in aluminum tolerance by modulating citrate secretion from roots of red kidney bean

Background and Aims Polyamines and nitric oxide (NO) are two important molecules modulating numerous environment stresses in plants. This study was to investigate the roles of polyamines and NO in aluminum (Al) tolerance in red kidney bean.

Aluminum tolerance in Centipedegrass (Eremochloa ophiuroides [Munro] Hack.): Excluding Al from root

Centipedegrass (Eremochloa ophiuroides [Munro] Hack.) is a C4 perennial grass and well adapted to acid soils. Centipedegrass accession E041 (Lingchuan, Guangxi Province, China) was found to be more tolerant to Aluminum (Al) than E006 (Nanjing, Jiangsu Province, China). The objective of this study was to identify possible mechanisms associated with centipedegrass. After exposure to Al, citrate was detected in both E041 and E006 roots secretion. Phosphorous (P) starvation and La3+ (Lanthanum) treatment did not induce organic acid secretion. Secretion of organic acid of both E041 and E006 need an initial lag phase of 6h, and increased significantly after 6h exposure to Al, which suggested that centipedegrass showed a pattern II-type organic acid secretion. Secretion of citrate increased with increasing external Al concentration, but the amount of citrate secreted was higher in E041 than in E006 which also accumulated more Al than E041. Al accumulated in the epidermal and sub epidermal cortical of root apices. The results suggested that secretion of citrate may be one of Al exclusion mechanisms in centipedegrass, and other mechanism may help to prevent Al from entering into central cylinder.

Physiological and molecular responses of broad bean (Vicia faba L.) to aluminum stress

In this study, the responses of broad bean cultivars resistant (YD) and sensitive (AD) to aluminum (Al) stress were investigated at physiological and molecular levels. The results showed that Al induced more citrate exudation in YD roots than that in AD roots, suggesting that citrate exudation is involved in broad bean Al resistance. The analyses for oxidative stress levels and antioxidant enzyme activities indicated that YD had a strong ability to cope with the oxidative stress induced by Al. To investigate the molecular responses of broad bean to Al stress further, a forward suppression subtractive hybridization cDNA library was constructed to identify Al-responsive genes in YD roots treated with 50-μM Al for a 24-h period. Of the obtained 162 high-quality ESTs, genes related to antioxidant enzymes including copper-zinc superoxide dismutase (SOD), class III peroxidase (POD) and germin-like protein (GEP) were up-regulated. Higher transcription levels of SOD and POD were observed in YD but not in AD roots, which is in agreement with the enhanced activities of antioxidant enzymes in YD roots under Al stress conditions. Furthermore, the up-regulated expression of vha2, encoding a plasma membrane (PM) H+-ATPase, and 14-3-3b in YD roots under Al stress were also detected and confirmed by RT-PCR analysis. Western and immunoprecipitation analyses indicated that Al-enhanced expressions and interactions of the PM H+-ATPase and 14-3-3 proteins might be involved in the regulation of citrate secretion in YD roots under Al stress.

Hydrogen sulfide alleviates aluminum toxicity in barley seedlings

Aluminum (Al) toxicity is one of the major factors that limit plant growth. Low concentration of hydrogen sulfide (H2S) has been proven to function in physiological responses to various stresses. The objective of this study is to investigate the possible role of H2S in Al toxicity in barley (Hordeum vulgare L) seedlings. Barley seedlings pre-treated with sodium hydrosulfide (NaHS), a H2S donor, and subsequently exposed to Al treatment were studied for their effects on root elongation, Al accumulation in seedlings, Al-induced citrate secretion and oxidative stress, and plasma membrane (PM) H+-ATPase expression. Our results showed that H2S had significant rescue effects on Al-induced inhibition of root elongation which was correlated well with the decrease of Al accumulation in seedlings. Meanwhile, Al-induced citrate secretion was also significantly enhanced by NaHS pretreatment. Al-induced oxidative stress as indicated by lipid peroxidation and reactive oxygen species burst was alleviated by H2S through the activation of the antioxidant system. Moreover, Al-induced reduction in PM H+-ATPase expression was reversed by exogenous NaHS. Altogether, our results suggest H2S plays an ameliorative role in protecting plants against Al toxicity by inducing the activities of antioxidant enzymes, increasing citrate secretion and citrate transporter gene expression, and enhancing the expression of PM H+-ATPase.

Nitric oxide protects sour pummelo (Citrus grandis) seedlings against aluminum-induced inhibition of growth and photosynthesis

Limited data are available on the amelioration of nitric oxide (NO) on aluminum (Al)-toxicity. Sour pummelo (Citrus grandis) seedlings were irrigated for 18 weeks with nutrient solution containing 0 and 1.2mM AlCl3·6H2O×0 and 10μM sodium nitroprusside (SNP, an NO donor). Under Al stress, SNP increased root phosphorus (P) and Al, but decreased shoot Al. Al decreased photosynthesis, maximum quantum yield of primary photochemistry (Fv/Fm) and total performance index (PItot,sbs), but increased inactivation of oxygen-evolving complex (OEC), K-band and relative variable fluorescence at I-steps (VI). SNP alleviated Al-induced changes for all these parameters. SNP stimulated Al-induced secretion of malate and citrate by excised roots from Al-treated seedlings, while Al did not increase their contents in roots. Antioxidant system in leaves and roots was up- and down-regulated by Al, respectively. SNP prevented Al-induced accumulation of malondialdehyde (MDA) in roots and leaves. In conclusion, SNP alleviates Al-induced inhibition of growth and impairment of the whole photosynthetic electron transport chain. This occurs through increasing Al-immobilization and P level in roots and Al-induced secretion of malate and citrate from roots, and decreasing Al accumulation in shoots. Thus, the decrease of photosynthesis is prevented. Increased P level and Al-immobilization in roots through SNP may be effected through enhanced secretion of malate and citrate.

Assessing prostate cancer growth with citrate measured by intact tissue proton magnetic resonance spectroscopy

Annual PSA tests have led to a significant increase in the number of prostate cancer (PCa) cases diagnosed. This increased incidence has led to overtreatment of many patients, as current pathology often cannot distinguish latent from aggressive PCa. Studies have shown that the depletion of zinc in prostate cells correlated with cell-line growth rates, and may therefore relate to the progression of PCa. Furthermore, as zinc is normally an inhibitor of citrate oxidation, the reduction of zinc in PCa may cause a decrease in citrate secretion levels in the glandular epithelia of PCa patients. Using high-resolution magic angle spinning proton magnetic resonance spectroscopy followed by quantitative histopathology, we investigate unit histo-benign prostate epithelial citrate concentrations in intact tissue samples obtained from 18 patients with pre-surgical PSA values less than 20 ng/ml. Using these data, we evaluate correlations between citrate concentrations and PSA velocities, densities and blood percent-free PSA. We observe different linear patterns between citrate concentrations and histo-benign glandular epithelia from patients of different PSA velocities. More importantly, we obtain a significant correlation between PSA velocity, density and percent-free PSA, and citrate concentrations in unit volume of histo-benign epithelial glands of the peripheral zone. Low levels of citrate in unit volume represent rapidly increasing PSA values, and, therefore, may be used as an indicator of fast-growing PCa. Thus, tissue samples obtained at the time of biopsy may be evaluated for their citrate concentrations for the prediction of PCa growth rates, allowing for the implementation of alternative treatment options and reducing overtreatment.

Aluminum-induced secretion of organic acid by cowpea (Vigna unguiculata L.) roots

Cowpea (Vigna unguiculata L. cv. Lulutong 1) is a leguminous vegetable which is well adapted to infertile acid soil. The mechanisms of aluminum (Al) resistance in cowpea are poorly understood. This study aimed to analyze the possible Al resistance mechanisms of cowpea. After exposure to Al, both malate and citrate were secreted from cowpea roots, but the secretion of these organic acids was not specific to Al. Potassium (P) starvation also induced the secretion of malate and citrate. The secretion of malate was the dominant response to Al stress. Exposure to 50μM La3+ (Lanthanum) did not induce organic acid secretion. Secretion of organic acid was detected after 3–6h exposure to Al, and increased significantly after 6h exposure, which suggested that this plant showed a pattern II-type organic acid secretion. This is supported by the finding that a protein-synthesis inhibitor, cycloheximide (CHM), significantly inhibited secretion. Two types of anion-channel inhibitors had different effects on Al-induced secretion of organic acids, 9-anthracene carboxylic acid (A-9-C), which completely inhibited secretion whereas niflumic acid (NIF) had no effect. In the presence of A-9-C, Al inhibited root elongation more significantly. Compared with the control, the root tips showed no significant change in organic acid content or in citrate synthase (CS), malate dehydrogenase (MDH) and succinate dehydrogenase (SDH) activities. Taken together, these results indicated that the secretion of malate played a dominant role in the response to Al stress by cowpea plants. The secretion pattern was type II, and protein synthesis might be involved in the secretion process.

Transcriptomic responses to aluminum stress in soybean roots

Aluminum (Al) toxicity is the primary limitation to crop production and plant growth in acid soils. Soybean has multiple mechanisms of Al resistance including the complexing and exclusion of Al in root apices by Al-induced citrate secretion. Microarray analysis is available for the identification of genes in soybean. In the present study, Affymetrix soybean genome array was used to identify the Al-induced differentially expressed genes in Al-resistant genotype Jiyu 70. With a cutoff of > 2.0-fold (p < 0.05) between non Al-treated and Al-treated root apices, 561 genes were upregulated and 78 genes were downregulated when roots were exposed to 30 μmol/L AlCl(3) for 4 h. Quantitative real-time PCR was used to test the microarray data. The analysis showed that nearly half of the Al-responsive genes were of unknown biological function. A higher proportion of genes related to transcription regulation and cell wall processes were observed in Al-induced up- and downregulated genes, respectively. Some genes homologous to the citrate transporter MATE family gene or C(2)H(2) family transcription factor gene, STOP1, were detected in our analysis. Some genes related to lignin deposition were upregulated, which might be related to Al-induced root elongation inhibition.

An Al‐inducible MATE gene is involved in external detoxification of Al in rice

A number of plant species, including rice, secretes citrate from roots in response to Al stress. Here we characterized the functions of a gene, OsFRDL4 (Os01g0919100) that belongs to the multidrug and toxic compound extrusion (MATE) family in rice (Oryza sativa). Heterologous expression in Xenopus oocyte showed that the OsFRDL4 protein was able to transport citrate and was activated by Al. The expression level of the OsFRDL4 gene in roots was very low in the absence of Al, but was greatly enhanced by Al after short exposure. Furthermore, the OsFRDL4 expression was regulated by ART1, a C2H2-type zinc finger transcription factor for Al tolerance. Transient expression of OsFRDL4 in onion epidermal cells showed that it localized to the plasma membrane. Immunostaining showed that OsFRDL4 was localized in all cells in the root tip. These expression patterns and cell specificity of localization of OsFRDL4 are different from other MATE members identified previously. Knockout of OsFRDL4 resulted in decreased Al tolerance and decreased citrate secretion compared with the wild-type rice, but did not affect citrate concentration in the xylem sap. Furthermore, there is a positive correlation between OsFRDL4 expression level and the amount of citrate secretion in rice cultivars that are differing in Al tolerance. Taken together, our results show that OsFRDL4 is an Al-induced citrate transporter localized at the plasma membrane of rice root cells and is one of the components of high Al tolerance in rice.

Lipopolysaccharide challenge of the mammary gland in bovine induced a transient glandular shift to anaerobic metabolism

Support of milk production in modern dairy cows demands a large proportion of its own metabolic resources, such as glucose, which might be required under stressful situations. The aim of the experiment was to test the hypothesis that acute immune stress shifts oxidative metabolism to glycolysis. Two mammary quarters in 6 Holstein cows were infused with lipopolysaccharide (LPS), whereas the 2 counter quarters served as controls to the treatment. An additional 6 cows were infused with saline and served as running controls. The LPS challenge induced dramatic transient increases in milk lactate (75-fold) and malate (11-fold) concentrations (both markers of glycolysis) at 24h posttreatment. No significant changes in lactate and malate concentrations were recorded in control quarters and control animals, indicating that the effect of LPS was restricted to the treated gland. The LPS challenge induced a dramatic transient decrease in milk yield, and lactose and citrate (a marker of mitochondrial metabolism) secretion at 24h posttreatment. The kinetics were inversely proportional to those of lactate and malate concentrations. Thus, our data suggest that LPS challenge induces acute conversion of epithelial cell metabolism from principally mitochondrial-oxidative to principally cytosolic (glycolytic), which allows the diversion of metabolic resources normally used to synthesize milk to support the immune system. An in vitro bacterial growth test showed that concentrations of lactate, malate, and lactose equivalent to those found in the in vivo experiment delayed and reduced the growth of a pathogenic Escherichia coli strain, suggesting that they play a role in diminution of bacterial multiplication in the mammary gland.

Citrate transporters play an important role in regulating aluminum-induced citrate secretion in Glycine max

To further understand the process of Al-induced citrate secretion from soybean roots, the effect of protein synthesis inhibitor, anion channel blockers, and citrate carrier inhibitors on Al-induced citrate exudation was investigated in Al-resistant soybean cultivar PI 416937. Citrate exudation from roots increased with the increase of Al concentration from 10 to 50 μM and initiated after 4 h of Al exposure. Protein synthesis inhibitor, cycloheximide (CHM; 25 μM) completely inhibited Al-induced citrate secretion during 12-h exposure, suggesting that novel protein synthesis was necessary in Al-induced citrate efflux. Also both anion channel blocker anthracene-9-carboxylic acid (A-9-C) and citrate carrier inhibitor mersalyl acid (Mersalyl) significantly reduced citrate secretion, suggesting that both anion channels in plasma membrane and citrate carriers in mitochondria membrane were the rate limiting factors of Al dependent citrate release. However, Al-induced citrate secretion was insensitive to anion channel blockers phenylglyoxal (PG), 4,4′-diisothiocyanostibene-2,2′-disulfonat (DIDS) and citrate carrier inhibitor pyridoxal 5′-P (PP).