High Concentrations Of Malate Research Articles

Using Low-Calorie Orange Juice as a Dietary Alternative to Alkali Therapy.

Purpose: The pursuit of a dietary source to increase urine pH and citrate in stone formers has been ongoing for >30 years. Early evidence showed that orange juice (OJ) contains alkali and citrate, but high sugar and ascorbic acid content limited the use of OJ as a viable daily source of alkali. Recently, novel low-calorie OJs have emerged and could potentially be a better option. Methods: Beverages with high concentrations of alkali citrate and malate were identified using ion chromatography. Two low-calorie OJ beverages, in addition to crystal light lemonade beverage (CLLB), were chosen. Healthy volunteers (5 men, 5 women) drank 1 L of OJ or CLLB with 1 L water daily for 7 days, and then completed a 24-hour urinalysis. A washout week was instituted between trial weeks. The study design is a prospective randomized crossover control trial. A paired analysis using comparison of means was used to evaluate low-calorie OJ and CLLB. Volunteers had no prior history of kidney stones and maintained a journal with beverage compliance, side effect (SE), and dietary consumption data. Results: Tropicana 50 (TRP50), Kroger low-calorie OJ (KLCO), and CLLB were found to have a total alkali content of 56.60, 47.9, and 17.3 mEq/L, respectively, based on ion chromatography. Consumption of all three beverages raised urinary citrate (116.6 [-118 to 373, 177.9 [-3 to 359], 155.6 [-4 to 237] ▵mg/day 95% confidence interval) and urinary pH (0.25 [0.08-0.53], 0.74 [0.41-1.07 p < 0.05], 0.25 [0.25-0.64]), respectively, compared with water phase. Based on journal entries by volunteers, TRP50 had the most SEs (90% participants) felt to be a result of the artificial sweetener (Stevia®). Conclusion: Low-calorie OJs, and to a lesser extent CLLB, have alkali and citrate based on ion chromatography. Daily consumption by healthy volunteers of KLCO can raise urinary pH.

Malate Content in Wild Vitis spp. Demonstrates a Range of Behaviors during Berry Maturation

Wild <i>Vitis</i> spp. and their interspecific hybrids are known to have high malate concentrations at sugar maturity as compared to domesticated <i>Vitis vinifera</i>, but it is unknown if differences in malate at harvest among species arise from differences in malate accumulation or degradation. Over two years, fruit from <i>Vitis riparia</i> and <i>Vitis cinerea</i> accessions along with commercial <i>V. vinifera</i> and interspecific hybrid cultivars were collected at multiple time points. In contrast to the well-known biphasic behavior of malate in <i>V. vinifera</i> (preveraison accumulation, postveraison degradation), we observed a range of behaviors for malate in wild species. On average, <i>V. riparia</i> accessions had malate per berry comparable to <i>V. vinifera</i> just prior to veraison, but degraded malate to a much lesser extent. <i>V. cinerea</i> accessions had lower malate prior to veraison than other species but showed an increase in malate from pre- to postveraison. Variation in postveraison malate behavior appears related to diminished malate degradation in the mesocarp of wild <i>Vitis</i> spp. Our results indicate that studies of malate behavior in <i>Vitis</i> spp. and their hybrids should include both pre- and postveraison time points.

Plastidial NADP-malic enzymes from grasses: Unraveling the way to the C4 specific isoforms

Malic enzyme is present in many plant cell compartments such as plastids, cytosol and mitochondria. Particularly relevant is the plastidial isoform that participates in the C(4) cycle providing CO(2) to RuBisCO in C(4) species. This type of photosynthesis is more frequent among grasses where anatomical preconditioning would have facilitated the evolution of the C(4) syndrome. In maize (C(4) grass), the photosynthetic NADP dependent Malic enzyme (ZmC(4)-NADP-ME, l-malate:NADP oxidoreductase, E.C. 1.1.1.40) and the closest related non-photosynthetic isoform (ZmnonC(4)-NADP-ME, l-malate:NADP oxidoreductase, E.C. 1.1.1.40) are both plastidial but differ in expression pattern, kinetics and structure. Features like high catalytic efficiency, inhibition by high malate concentration at pH 7.0, redox modulation and tetramerization are characteristic of the photosynthetic NADP-ME. In this work, the proteins encoded by sorghum (C(4) grass) and rice (C(3) grass) NADP-ME genes, orthologues of the plastidial NADP-MEs from maize, were recombinantly expressed, purified and characterized. In a global comparison, we could identify a small group of residues which may explain the special features of C(4) enzymes. Overall, the present work presents biochemical and molecular data that helps to elucidate the changes that took place in the evolution of C(4) NADP-ME in grasses.

A proteomics approach to investigate the process of Zn hyperaccumulation in Noccaea caerulescens (J &amp; C. Presl) F.K. Meyer

Zinc (Zn) is an essential trace element in all living organisms, but is toxic in excess. Several plant species are able to accumulate Zn at extraordinarily high concentrations in the leaf epidermis without showing any toxicity symptoms. However, the molecular mechanisms of this phenomenon are still poorly understood. A state-of-the-art quantitative 2D liquid chromatography/tandem mass spectrometry (2D-LC-MS/MS) proteomics approach was used to investigate the abundance of proteins involved in Zn hyperaccumulation in leaf epidermal and mesophyll tissues of Noccaea caerulescens. Furthermore, the Zn speciation in planta was analyzed by a size-exclusion chromatography/inductively coupled plasma mass spectrometer (SEC-ICP-MS) method, in order to identify the Zn-binding ligands and mechanisms responsible for Zn hyperaccumulation. Epidermal cells have an increased capability to cope with the oxidative stress that results from excess Zn, as indicated by a higher abundance of glutathione S-transferase proteins. A Zn importer of the ZIP family was more abundant in the epidermal tissue than in the mesophyll tissue, but the vacuolar Zn transporter MTP1 was equally distributed. Almost all of the Zn located in the mesophyll was stored as Zn-nicotianamine complexes. In contrast, a much lower proportion of the Zn was found as Zn-nicotianamine complexes in the epidermis. However, these cells have higher concentrations of malate and citrate, and these organic acids are probably responsible for complexation of most epidermal Zn. Here we provide evidence for a cell type-specific adaptation to excess Zn conditions and an increased ability to transport Zn into the epidermal vacuoles.

Root release and metabolism of organic acids in tea plants in response to phosphorus supply

Self-rooted, 10-month-old, uniform tea [Camellia sinensis (L.) O. Kuntze cv. Huangguanyin] plants were supplied for 17 weeks with 0, 40, 80, 160, 400, or 1000μM phosphorus (P) to investigate the effects of P supply on root citrate and malate release, the concentrations of malate and citrate and the activities of acid-metabolizing enzymes in leaves and roots. Root malate release and accumulation was induced by both 0 and 40μM P, while root citrate release and accumulation was induced only by 0μM P. Phosphorus-deficiency-induced malate and citrate release coincided with higher concentrations of root malate and citrate. The higher concentrations of malate and citrate were accompanied by increased activities of phosphoenolpyruvate carboxylase (PEPC), phosphoenolpyruvate phosphatase (PEPP), citrate synthase (CS) and NAD-malic enzyme (NAD-ME) and decreased activities of pyruvate kinase (PK), NADP-ME and NADP-isocitrate dehydrogenase (NADP-IDH) in roots. In contrast to roots, malate accumulated in the leaves only in response to 0μM P, and no change was observed in citrate levels. The P-deficiency-induced leaf malate accumulation coincided with increased activities of NADP-ME, NAD-ME and PK. Overall, the P-deficiency-induced changes in organic acid (OA) metabolism differed between roots and leaves. The high tolerance of tea plants to P-deficiency might be involved in two major processes: (a) increasing the availability of P by inducing root release of OA anions; and (b) improving the ability to use P efficiently by inducing bypass enzymes involved in tissue P economy.

Crassulacean acid metabolism enhances underwater photosynthesis and diminishes photorespiration in the aquatic plant Isoetes australis

• Underwater photosynthesis by aquatic plants is often limited by low availability of CO(2), and photorespiration can be high. Some aquatic plants utilize crassulacean acid metabolism (CAM) photosynthesis. The benefits of CAM for increased underwater photosynthesis and suppression of photorespiration were evaluated for Isoetes australis, a submerged plant that inhabits shallow temporary rock pools. • Leaves high or low in malate were evaluated for underwater net photosynthesis and apparent photorespiration at a range of CO(2) and O(2) concentrations. • CAM activity was indicated by 9.7-fold higher leaf malate at dawn, compared with at dusk, and also by changes in the titratable acidity (μmol H(+) equivalents) of leaves. Leaves high in malate showed not only higher underwater net photosynthesis at low external CO(2) concentrations but also lower apparent photorespiration. Suppression by CAM of apparent photorespiration was evident at a range of O(2) concentrations, including values below air equilibrium. At a high O(2) concentration of 2.2-fold the atmospheric equilibrium concentration, net photosynthesis was reduced substantially and, although it remained positive in leaves containing high malate concentrations, it became negative in those low in malate. • CAM in aquatic plants enables higher rates of underwater net photosynthesis over large O(2) and CO(2) concentration ranges in floodwaters, via increased CO(2) fixation and suppression of photorespiration.

Organic acid metabolism and root excretion of malate in wheat cultivars under aluminium stress

The effects of aluminium (Al) on the metabolism of organic acids synthesised via nonphotosynthetic carbon fixation in the roots and on malate exudation were investigated in Al-tolerant Shirosanjyaku (SH) and Al-sensitive Chikushikomugi (CK) wheat cultivars labelled with bicarbonate-(14)C. Aluminum triggered the excretion of (14)C into the solution, especially in the SH that excreted 2.5 times more (14)C than the CK. The loss of radioactivity (about 10%) into the solution represented a small drain in the (14)C reserve found in the roots. In the organic acid fraction within the roots, malate contained the greatest amount of (14)C, and this amount decreased rapidly with time in both cultivars. The disappearance of radioactivity in the malate resulted from metabolism and translocation rather than to root efflux. Aluminium decreased the malate concentrations in roots of both cultivars. The Al-sensitive cultivar had higher concentrations of malate regardless of the presence of Al. It was therefore assumed that the decrease of malate concentration in roots under Al stress did not result from the decline in malate synthesis but due to an increase in malate decomposition. This response was interpreted as the result of the Al-induced stress and not as the cause of a differential Al-tolerance between the wheat cultivars. An important component of the differential Al tolerance between SH and CK is the greater ability of the Al-tolerant cultivar to excrete malate from the roots, which is independent of its internal concentration in the roots.

Identification of domains involved in the allosteric regulation of cytosolic Arabidopsis thaliana NADP‐malic enzymes

The Arabidopsis thaliana genome contains four genes encoding NADP-malic enzymes (NADP-ME1-4). Two isoenzymes, NADP-ME2 and NADP-ME3, which are shown to be located in the cytosol, share a remarkably high degree of identity (90%). However, they display different expression patterns and show distinct kinetic properties, especially with regard to their regulation by effectors, in both the forward (malate oxidative decarboxylation) and reverse (pyruvate reductive carboxylation) reactions. In order to identify the domains in the primary structure that could be responsible for the regulatory differences, four chimeras between these isoenzymes were constructed and analysed. All chimeric versions exhibited the same native structures as the parental proteins. Analysis of the chimeras constructed indicated that the region from amino acid residue 303 to the C-terminal end of NADP-ME2 is critical for fumarate activation. However, the region flanked by amino acid residues 303 and 500 of NADP-ME3 is involved in the pH-dependent inhibition by high malate concentration. Furthermore, the N-terminal region of NADP-ME2 is necessary for the activation by succinate of the reverse reaction. Overall, the results show that NADP-ME2 and NADP-ME3 are able to distinguish and interact differently with similar C(4) acids as a result of minimal structural differences. Therefore, although the active sites of NADP-ME2 and NADP-ME3 are highly conserved, both isoenzymes acquire different allosteric sites, leading to the creation of proteins with unique regulatory mechanisms, probably best suited to the specific organ and developmental pattern of expression of each isoenzyme.

Metabolic viability assessment of cystic echinococcosis using high‐field 1H MRS of cyst contents

Cystic echinococcosis is a worldwide disease caused by larval stages of the parasite Echinococcus granulosus (canine tapeworm). In clinical practice, staging of cyst development by ultrasonography (US) has allowed treatment options to be tailored to individual patient needs. However, the empirical correlation between cyst morphology and parasite viability is not always dependable and has, until now, required confirmation by invasive assessment of cyst content by light microscopy (LM), for example. Alternatively, high-field 1H MRS may be used to examine cyst fluid ex vivo and prepare detailed quantitative metabolite profiles, enabling a multivariate metabolomics approach to cyst staging. One-dimensional and two-dimensional 1H and 1H/13C MRS at 600 MHz (14.1 T) was used to analyze 50 cyst aspirates of various US and LM classes. MR parameters and concentrations relative to internal valine were determined for 44 metabolites and four substance classes. The high concentrations of succinate, fumarate, malate, acetate, alanine, and lactate found in earlier studies of viable cysts were confirmed, and additional metabolites such as myo-inositol, sorbitol, 1,5-anhydro-D-glucitol, betaine, and 2-hydroxyisovalerate were identified. Data analysis and cyst classification were performed using univariate (succinate), bivariate (succinate vs fumarate), and multivariate partial least squares discriminant analysis (PSL-DA) methods (with up to 48 metabolite variables). Metabolic classification of 23 viable and 18 nonviable cysts on the basis of succinate alone agreed with LM results. However, for seven samples, LM and MRS gave opposing results. Reclassification of these samples and two unclassified samples by PLS-DA prediction techniques led to a set of 50 samples that could be completely separated into viable and nonviable MRS classes with no overlap, using as few as nine variables: succinate, formate, malate, 2-hydroxyisovalerate, acetate, total protein content, 1,5-anhydro-D-glucitol, alanine, and betaine. Thus, future noninvasive in vivo applications of MRS would appear promising.

Concentrations of Nitrate, Organic Acids, Free Amino Acids, Cations and Sugars in Komatsuna (Brassica campestris var. perviridis) Grown with Carbonate, Sulfate and Chloride Application

This study compared the concentrations of components such as nitrate, organic acids, free amino acids, cations and sugars in komatsuna (Brassica campestris var. perviridis) grown with carbonate, sulfate and chloride application (CO3-TK, SO4-TK and Cl-TK, respectively). The komatsuna was cultivated in 1/2000-a Wagner pots (6 hills per pot) filled with light-colored Andosol in a glasshouse for 37 days at soil water potential (SWP) right before harvesting of −6.2 and −62 kPa. And 26.5 mmolc of carbonate, sulfate or chloride in the form of potassium salts was applied to each pot. Chloride application to the komatsuna induced relatively high concentrations of potassium, calcium and magnesium ions, and the low concentrations of malate, glucose and fructose on a dry weight basis (DWB). Carbonate application induced relatively high concentrations of malate and low concentrations of nitrate. Sulfate was almost between chloride and carbonate. Concentration variation was not substantial in free amino acids. Those tendencies were almost the same in both SWPs. Most of the variations in component concentrations were attributed to the regulation of ionic and osmotic balance to respond to chloride, nitrate and sulfate absorption, as judged from quantitative relationships among the components and water in the komatsuna. It seems that the absorption of nitrate is influenced by the pH of the soil. There were clear differences in glucose, fructose and malate concentrations among CO3-TK, SO4-TK and Cl-TK on a fresh weight basis just as on a DWB.

Identification of Domains Involved in Tetramerization and Malate Inhibition of Maize C4-NADP-Malic Enzyme

C(4) photosynthetic NADP-malic enzyme (ME) has evolved from non-C(4) isoforms and gained unique kinetic and structural properties during this process. To identify the domains responsible for the structural and kinetic differences between maize C(4) and non-C(4)-NADP-ME several chimeras between these isoforms were constructed and analyzed. By using this approach, we found that the region flanked by amino acid residues 102 and 247 is critical for the tetrameric state of C(4)-NADP-ME. In this way, the oligomerization strategy of these NADP-ME isoforms differs markedly from the one that present non-plant NADP-ME with known crystal structures. On the other hand, the region from residue 248 to the C-terminal end of the C(4) isoform is involved in the inhibition by high malate concentrations at pH 7.0. The inhibition pattern of the C(4)-NADP-ME and some of the chimeras suggested an allosteric site responsible for such behavior. This pH-dependent inhibition could be important for regulation of the C(4) isoform in vivo, with the enzyme presenting maximum activity while photosynthesis is in progress.

Insight into metabolism of CHO cells at low glucose concentration on the basis of the determination of intracellular metabolites

Glucose pulse culture was employed to characterize the growth and metabolism of Chinese hamster ovary (CHO) cells at low glucose concentration. Cell growth was gradually limited when the glucose concentration decreased to below 1.22 mmol/l. Correspondingly, in comparison with the culture at above 4.8 mmol/l glucose, the intracellular concentrations of ATP decreased by 8%, 19% and 47% at the glucose concentrations of 1.2, 0. 54, and 0.25 mmol/l, respectively, showing the shortage of energy supply might result in growth limitation at low glucose concentration. The specific consumption rates of Gln, Ser, His, Thr, Arg, Tyr, Cys2, Val, Met, Phe, Ile, Leu and Lys increased at glucose concentration below 0.54 mmol/l, implying that these amino acids were mainly catabolized instead of used as “building bricks” in protein synthesis under glucose limitation. Both intracellular Asp and Glu are always accumulated under any conditions, while intracellular concentrations of other amino acids, such as Ser, Thr, Arg, Tyr, Ala, Val, Met, Phe, Ile and Leu, increased with the decrease in the glucose concentration. There was no obvious difference in the intracellular concentrations of organic acids at different glucose concentrations. Moreover, high concentrations of intracellular malate and undetectable iso-citrate were observed, implying that citrate might be intercepted from the TCA cycle for other anabolisms.

Malate accumulation in different organs of Mesembryanthemum crystallinum L. following age-dependent or salinity-triggered CAM metabolism.

Different organs of Mesembryanthemum crystallinum exhibit differing levels of CAM (Crassulacean acid metabolism), identifiable by quantification of nocturnal malate accumulation. Shoots and also basal parts of young leaves were observed to accumulate high concentrations of malate. It was typically found in mature leaves and especially prominent in plants subjected to salt stress. Small amount of nocturnal malate accumulation was found in roots of M. crystallinum plants following age-dependent or salinity-triggered CAM. This is an indication that malate can be also stored in non-photosynthetic tissue. Measurements of catalase activity did not produce evidence of the correlation between activity of this enzyme and the level of malate accumulation in different organs of M. crystallinum although catalase activity also appeared to be dependent on the photoperiod. In all material collected at dusk catalase activity was greater than it was observed in the organs harvested at dawn.

Organic Acid Metabolism in Roots of Various Grapevine (Vitis) Rootstocks Submitted to Iron Deficiency and Bicarbonate Nutrition

The effects of Fe limitation and bicarbonate addition to the nutrient medium on the organic acid metabolism were investigated in the root tips of various grapevine genotypes. Cuttings of two limestone‐tolerant and two limestone‐susceptible Vitis genotypes were grown for four weeks in nutrient solutions containing 10 or 0.5 µM Fe. The effect of bicarbonate addition (5 mM) was studied for two of these genotypes. Compared to 10 µM, Fe limitation (0.5 µM) significantly increased citrate concentration in root tips after 2 weeks, and malate concentration after 4 weeks. When Fe limitation and bicarbonate addition were combined, citrate and malate concentrations were significantly increased after 2 weeks. Fe limitation or addition of 5 mM bicarbonate had a larger effect on citrate than on malate concentrations. Addition of 5 mM bicarbonate discriminated more clearly tolerant and susceptible genotypes than Fe limitation. High malate and citrate concentrations in the roots were associated to high PEPC activities. These results confirm that root organic acid metabolism is involved in grapevine response to Fe deficiency stress. If verified on a larger range of genotypes, a procedure using bicarbonate effect on root tip citrate concentration could be proposed to screen limestone‐tolerant Vitis rootstocks.

Influence of assimilate and water supply on seasonal variation of acids in peach (cv Suncrest)

AbstractSeasonal variations of acid concentrations in peach fruit (cv Suncrest) were investigated in relation to assimilate and water supply for four years. The leaf/fruit ratio and girdling of the shoots on both sides of the fruit pedicel were used to manipulate the assimilate supply. Irrigation intensity was used to manipulate the water supply. There was a positive relationship between assimilate or water supply and fruit growth. The responses of organic acids to assimilate supply were different with fruit development. At the beginning of growth, fruits with high leaf/fruit ratio had lower malate and higher citrate concentration. Near maturity, greater assimilate supply is related to high malate and low citrate concentration. High leaf/fruit ratio increased quinic acid concentration at the beginning of growth. With development, quinic acid concentration in fruits with more assimilate supply decreased more. Shikimic acid concentration fluctuated differently with fruit development in the four years and was generally lower in fruits with more leaves. Irrigation tended to reduce acid concentrations but did not affect the seasonal patterns of the different acids. There were no interactions between tree irrigation and leaf/fruit ratio.© 2002 Society of Chemical Industry

Atomistic Simulations of Competition between Substrates Binding to an Enzyme

Although the idea that electrostatic potentials generated by enzymes can guide substrates to active sites is well established, it is not always appreciated that the same potentials can also promote the binding of molecules other than the intended substrate, with the result that such enzymes might be sensitive to the presence of competing molecules. To provide a novel means of studying such “electrostatic competition” effects, computer simulation methodology has been developed to allow the diffusion and association of many solute molecules around a single enzyme to be simulated. To demonstrate the power of the methodology, simulations have been conducted on an artificial fusion protein of citrate synthase (CS) and malate dehydrogenase (MDH) to assess the chances of oxaloacetate being channeled between the MDH and CS active sites. The simulations demonstrate that the probability of channeling is strongly dependent on the concentration of the initial substrate (malate) in the solution. In fact, the high concentrations of malate used in experiments appear high enough to abolish any channeling of oxaloacetate. The simulations provide a resolution of a serious discrepancy between previous simulations and experiments and raise important questions relating to the observability of electrostatically mediated substrate channeling in vitro and in vivo.

Metabolic regulation and gene expression of root phosphoenolpyruvate carboxylase by different nitrogen sources

ABSTRACTAlfalfa (Medicago sativa L.) N‐sufficient plants were fed 1·5 mM N in the form of NO3−, NH4+ or NO3− in conjunction with NH4+, or were N‐deprived for 2 weeks. The specific activity of phosphoenolpyruvate carboxylase (PEPC) from the non‐nodulated roots of N‐sufficient plants was increased in comparison with that of N‐deprived plants. The PEPC value was highest with NO3− nutrition, lowest with NH4+ and intermediate in plants that were fed mixed salts. The protein was more abundant in NO3−‐fed plants than in either NH4+‐ or N mixed‐fed plants. Nitrogen starvation decreased the level of PEPC mRNA, and nitrate was the N form that most stimulated PEPC gene expression. The malate content was significantly lower in NO3−‐deprived than in NO3−‐sufficient plants. Root malate accumulation was high in NO3−‐fed plants, but decreased significantly in plants that were fed with NH4+. The effect of malate on the desalted enzyme was also investigated. Root PEPC was not very sensitive to malate and PEPC activity was inhibited only by very high concentrations of malate. Asparagine and glutamine enhanced PEPC activity markedly in NO3−‐fed plants, but failed to affect plants that were either treated with other N types or N starved. Glutamate and citrate inhibited PEPC activity only at optimal pH. N‐nutrition also influenced root nitrate and ammonium accumulation. Nitrate accumulated in the roots of NO3−‐ and (NO3− + NH4+)‐fed plants, but was undetectable in those administered NH4+. Both the nitrate and the ammonium contents were significantly reduced in NO3−‐ and (NO3− + NH4+)‐starved plants. Root accumulation of free amino acids was strongly influenced by the type of N administered. It was highest in NH4+‐fed plants and the most abundant amides were asparagine and glutamine. It was concluded that root PEPC from alfalfa plants is N regulated and that nitrate exerts a strong influence on the PEPC enzyme by enhancing both PEPC gene expression and activity.

Influence of aluminum and phosphorus on growth and xylem sap composition in Melastoma malabathricum L.

Melastoma (Melastoma malabathricum L.) is an aluminum-accumulating woody plant that accumulates more than 10 000 mg kg−1 of aluminum (Al) in mature leaves. The influence of Al and phosphorus (P) applications on plant growth and xylem sap was examined in the present study in order to elucidate the interaction between Al-induced growth enhancement and P nutrition, and to determine the form of Al for translocation from roots to shoots. Although the Al application significantly increased the growth of Melastomaseedlings with the high P pre-treatment, and P concentrations in the leaves and Pi concentrations in the xylem sap regardless of the P pre-treatment, we could not come to the conclusion that a primary cause of the Al-induced growth enhancement in Melastoma is the stimulation of P uptake. The degree of Al-induced growth enhancement corresponded not with the P concentrations but with the Al concentrations in the plant tissue, suggesting that the Al-induced growth enhancement in Melastoma is primarily caused by Al itself in the plant tissue rather than by the stimulation of P uptake. Through the analysis of organic acids and Al in the xylem sap and plant tissue, the form of Al for translocation from roots to shoots was shown to be an Al-citrate complex that was transformed into Al-oxalate complex for Al storage in the leaves. In addition, the xylem sap of Melastoma seedlings grown in the absence of Al contained higher concentrations of malate. In the presence of Al, however, higher concentrations of citrate were found, indicating that Melastoma changes its organic acid metabolism in the presence or absence of Al; more specifically, it increases the synthesis of citrate.

Isolation of High-Malate-Producing Sake Yeasts from Low-Maltose-Assimilating Mutants.

High-malate-producing sake yeasts were isolated from low-maltose-assimilating mutants. A sake yeast Kyokai no. 701 (K-701) was mutagenized with ethyl methanesulfonate. After nystatin treatment, low-maltose-assimilating mutants were selected using a replica plating method. The forty-two mutants obtained were fermented in koji extract medium and almost all of them produced more malate than the parental strain. In a small-scale sake brewing test, several of these mutants produced 2.3 to 6.7 times higher concentrations of malate and 1.5 to 2.1 times higher total acidity than the parental strain. However, these mutants exhibited neither resistance to cycloheximide nor sensitivity to dimethyl succinate, which are used as the phenotypes for isolation of high-malate-producing sake yeasts. The expression levels of the MDH and FUM1 genes in one mutant (M20), which produced the highest amount of malate among the mutants obtained, were analyzed by Northern hybridization. The transcriptional level of the FUM1 gene in strain M20 during sake brewing had a similar profile to that in strain K-701. However, the transcriptional level of the MDH2 gene in strain M20 for days 4 and 8 of malate formation during sake brewing was higher than that in strain K-701.

Tissue distribution of primary metabolism between epidermal, mesophyll and parenchymatous bundle sheath cells in barley leaves

This paper originates from a presentation at the International Conference on Assimilate Transport and Partitioning, Newcastle, NSW, August 1999 In order to investigate the roles of different cell types, metabolite compartmentation in barley (Hordeum vulgare L.) leaf tissue was mapped at the single-cell level, using single-cell sampling and analysis (SiCSA) techniques. The partitioning of recently fixed photoassimilate was investigated for the first time at single-cell resolution, using BAMS (biological accelerator mass spectroscopy) for precise measurement of 14C in femtomole quantities. The data obtained by BAMS qualitatively reflect concentrations of sugars in different cell types measured by SiCSA. Calculation of 14C-specific activities showed that the radioactive label saturated the mesophyll and parenchymatous bundle sheath (PBS) pools within the 45-min labelling period. During the photoperiod, sucrose concentration increased to 200 mM in mesophyll cells. The concentration of malate also increased during the photoperiod in mesophyll and PBS cells. Epidermal cells contained very low concentrations of sugar but high concentrations of malate (120–180 mM) and did not show significant diurnal changes. Accumulation of sugars and fructan synthesis could be induced in mesophyll and PBS cells by reduced export of sugars from leaves or, alternatively, when sugars were supplied from excised leaf blade bases immersed in a sucrose solution in the dark. The epidermis accumulated additional malate in step with the accumulation of sugar by the mesophyll/PBS cells during the long-term reduction of export. Immunolocalisation of Rubisco and cytochrome oxidase proteins was used to analyse the distribution of enzymes of photoassimilation and respiration between functionally different cells in mature leaves of barley.