Polygalacturonase,biomass, and ascospore production by byssochlamys fulva. I. Effects of acids found in fruits

Polygalacturonase, biomass, and ascospore production by four strains of Byssochlamys fulva cultured in laboratory media supplemented with citric, malic, or tartaric acids was determined over a 20-day incubation period at 30°C. Polygalacturonase activity was higher in tartaric acid media than in malic or citric acid media, with 1 % acid supporting the greatest activity in media initially at pH 4 or 5; at 0.1 % acid, highest activity was noted in media initially at pH 3. Most activity was produced between 4 and 8 days of incubation. Malic acid supported greater biomass production than did citric or tartaric acids. Media containing tartaric acid was the best for the production of ascospores whereas citric acid was the poorest. Higher numbers of ascospores generally were produced in media initially at pH 3 as compared to pH 4 or 5 after 10 days of incubation.     

Production of Leuconostoc oenos Biomass under pH Control

Leuconostoc oenos was grown on apple juice-based media. The effect of pH control on metabolism and biomass production was studied. Without pH control, L. oenos acidified the apple juice media to approximately pH 3.6. More than 75% of the malic acid was used under these conditions, but less than half of the carbohydrates was assimilated. Under pH control, biomass yields increased by 60%; most of the malic acid was used, but high levels of unfermented carbohydrates remained. The addition of tomato juice, vitamins, nucleotides, Mn⁺, and malic acid did not permit further increases in the cell counts; however, malic acid did induce further acidification. Growth without pH control favored a more homofermentative metabolism. Biomass production was higher in filter-sterilized apple juice media compared with that in the autoclaved media.     

Effects of Low-Molecular-Weight Organic Acids on Gadolinium Accumulation and Transportation in Tomato Plants

Effects of low-molecular-weight organic acids on the accumulation and transportation of gadolinium (Gd) in tomato plants were studied under hydroponic condition. The results indicated that changes of organic acids occurred in the processes of Gd accumulation and transportation in tomato plants which were treated with extraneous Gd solutions. Malic, citric, and succinic acids contributed to both Gd accumulation in roots and transportation in xylem vessels. When Gd was unloaded from the xylem to the leaf cells, formic, lactic, citric, and succinic acids played important roles in Gd accumulation in leaves. When tomato plants were cultured in the uptake solution of Gd-containing malic, citric, or succinic acid for 48 h, the succinic acid in roots and leaves and the malic acid in xylem saps both increased obviously. From the results above, we can conclude that succinic acid had the most important role in Gd accumulation in tomato roots and leaves, while malic acid transported Gd via xylem vessels more effectively.     

The Effect of CO2-Concentration on the Occurrence of a Number of Acids from the Citric Acid Cycle in Tomato Leaves

Examination of the effect of CO₂-concentration and time of day on the content of malic acid, citric acid, aconitic acid, isocitric acid, succinic acid and fumaric acid in tomato leaves, revealed that the total content of these acids will rise with the CO₂-concentration up to 0.10 vol% CO₂. In the morning up to 0.22 vol% CO₂ was needed for optimal effect. Samples of leaves picked at 1 a.m. showed the lowest content of these acids. At 9 a.m. the content had increased, and at 4 p.m. the increase was considerable. The content of malic and citric acid constituted 36 and 34% of the total acid content. In the afternoon and the night the aconitic acid represented 14% and in the morning 20% of the total acid content. Isocitric acid, fumaric acid and succinic acid occurred only in relatively small concentrations.     

Production of Cutin Hydrolyzing Enzymes by Botrytis cinerea in vitro

The production in vitro of cutin hydrolyzing enzymes by five isolates of B. cinerea was studied, using cutin of tomato fruits as a carbon source. Chemical depolymerisation of the cutin yielded 10,16-dihydroxyhexadecanoic acid as the main component. The same fatty acid was found after incubation of cutin with a crude enzyme preparation from a culture filtrate of B. cinerea. Hydrolysis was optimal at pH 5.5–6.0. In cultures with glucose as the only carbon source no cutinase activity was detected. Crude enzyme preparations which hydrolyzed cutin, also hydrolyzed para-nitrophenylbutyrate, with an optimum activity at pH 8. All five isolates showed para-nitrophenylbutyrate hydrolyzing activity when grown on tomato cutin, but the activity varied with the isolate used. No correlation was found between para-nitrophenylbutyrate-hydrolyzing activity of an isolate and its production of small lesions on young tomato fruits.     

Physiological responses of pressed baker’s yeast cells pre-treated with citric, malic and succinic acids

Summary The dough-leavening power of baker’s yeast, Saccharomyces cerevisiae, is strongly influenced by conditions under which the pressed yeast is maintained prior to bread dough preparation. In this study, the influence of the yeast cell’s pre-treatment with organic acids (malic, succinic, and citric acids) was investigated at a wide range of pH values when the pressed yeast samples were exposed to 30 °C. Increased fermentative activity was observed immediately after pre-treatment of the cells with organic acids. When the pH of the pressed yeast containing added citric acid was raised from 3.5 to 7.5, increases in both fermentative and maltase activities were obtained. Improvements in viability and levels of total protein were also observed during storage in the presence of citric acid, notably at pH 7.5. Glycerol-3-phosphate dehydrogenase activity and levels of internal glycerol also increased in the presence of citrate. On the other hand, pressed yeast samples containing succinic acid at pH 7.5 showed decreased viability during storage despite the maintenance of high levels of fermentative activity, similar to pressed yeast containing malic acid at pH 4.5 and 7.5. Decreases in intracellular levels of trehalose were observed during storage in all cases. Overall, the results of this study revealed the potential benefits of adding organic acids to pressed yeast preparations for baking purposes.     

Carbon requirements of Colletotrichum gloeosporioides PENZ

Monosporic isolates of Colletotrichum gloeosporioides were made from Punica grantum and one suitable isolate was selected for further work on carbon nutrition. Preliminary experiments showed that a pH value of 5, a temperature of 32°C and a period of 14 days were optimum conditions for the growth of this pathogen. Out of 41 carbon compounds tested, the pathogen showed excellent growth on starch, maltose, melibiose, dextrose, sucrose, raffinose, and dulcitol; good on tartaric acid, mannose, galactose, fructose, mannitol, and castor oil; fair on inulin, isopropyl alcohol, coconut oil, and pectin; poor on sorbose, n-butyl alcohol, arabionose, maleic acid, ethyl alcohol, succinic acid, citric acid, ribose, and malic acid, and no growth on the rest of the carbon compounds. In general, compounds which supported the best mycelial growth, yielded excellent or good sporulation of C. gloeosporioides and vice versa.     

Organic Acids of Ditylenchus triformis and Turbatrix aceti

Pyruvic acid, lactic acid and several tricarboxylic acid cycle acids were extracted from Ditylenchus triformis and Turbatrix aceti and identified. Fumaric acid was predominant in both nematodes. Small amounts o f malic and α-ketoglutaric acids and intermediate quantities o f lactic, citric, succinic, and pyruvic acids occurred in D. triformis. In T. aceti citric, lactic, and α-ketoglutaric acids were less abundant than succinic, malic and pyruvic acids. Only traces of aconitic and oxalacetic acids occurred in both nematodes. All the organic acids detected accounted for only about one per cent of the dry weight of nematodes o f the two species.     

Metabolism of Slices of the Tomato Stem

The rate of respiration of tomato stem slices varied considerably,the highest values (Qo₂, 2–3) being obtained for plantsin a good nutritional state, and the lowest (Qo₂, 1) in starvedplants. The respiratory quotient of 1.0 remained constant. Glucose fermentation was found to follow both glycolytic andalcohol fermentation pathways, the ratio of ethyl alcohol: lacticacid being 6.6:1. Fermentation seems to take place accordingto the Embden-Meyerhof scheme, as shown by the presence of someof these enzymes operative in this scheme and by inhibitionexperiments. In the presence of oxygen there was no formationof alcohol or lactic acid. Pyruvate added to tomato stem slices was metabolized by directoxidation to acetic acid and by dismutation to lactic and aceticacids and CO₂ The metabolism of acetic acid was demonstratedby its condensation with oxaloacetic acid to form citrate, thisbeing the second time that synthesis of citric acid by thismechanism has been found in plants. The presence of aconitase,of isocitric dehydrogenase, of succinic dehydrogenase, and ofmalic dehydrogenase, as well as the inhibition of respirationby malonic acid, favour the hypothesis that oxidation of carbohydratein tomato stem slices proceeds via the citric acid cycle. Thepossibility of an auxiliary route, the malic acid oxidationpathway, also was demonstrated. Tomato stem tissue anaerobicallysplit malic acid into glycolic acid. The further oxidation ofglycolic, glyoxylic, and formic acid was demonstrated. In experiments with C¹⁴-labelled acetate and butyrate a dilutionof the C¹⁴-labelled acids was found after incubation indicatingnew formation of these acids and of active participation offatty acid metabolism in the metabolic activities of the tissue. With the exception of alanine, added amino acids produced adefinite increase in O₂ uptake without extra formation of ammonia. Experimental demonstration of the possibility of electron transportfrom substrate to molecular oxygen in respiration via polyphenoloridase was provided by the attainment in a tomato tissue homogenateof a coupled oxidation-reduction between -ketoglutarate andcatechol with DPN and tyrosinase as the catalysts. The presenceof cytochrome oxidase was also demonstrated. Thus both systemspossibly may take part in the respiration of tomato stem slices.     

Enhanced production of succinic acid by metabolically engineered<Emphasis Type="Italic">Escherichia coli</Emphasis> with amplified activities of malic enzyme and fumarase

Apfl ldhA double mutantEscherichia coli strain NZN111 was used to produce succinic acid by overexpressing theE. coli malic enzyme gene (sfcA). This strain, however, produced a large amount of malic acid as well as succinic acid. After the analyses of the metabolic pathways, thefumB gene encoding the anaerobic fumarase ofE. coli was co-amplified to solve the problem of malic acid accumulation. A plasmid, pTrcMLFu, was constructed, which contains an artificial operon (sfcA-fumB) under the control of the inducibletrc promoter. From the batch culture of recombinantE. coli NZN111 harboring pTrcMLFu, 7 g/L of succinic acid was produced from 20 g/L of glucose, with no accumulation of malic acid. From the metabolic flux analysis the strain was found under reducing power limiting conditions by severe reorientation of metabolic fluxes.     

Effect of Pseudomonas fluorescens on the root exudates of two tomato mutants differently sensitive to Fe chlorosis

Plants with different Fe-mobilization properties are known to differ in the amount and kind of Fe-reducing and Fe-chelating compounds exuded by their roots. Although rhizosphere bacteria are known to affect the exudation of organic compounds by the plant roots, their effect on the root exudates of plants differing in Fe-mobilization properties is not known. We studied the effect of Pseudomonas fluorescens, on the exudation of sugars and organic and amino acids by roots of an iron chlorosis-resistant (T3238FER) and a chlorosis-susceptible (T3238fer) tomato mutant. Under sterile conditions two tomato mutants grew equally well and did not differ in the total amount of sugars and organic acid exuded by their roots. More amino acids, however, were exuded by the roots of T3238FER than T323fer. Mutants differed in the amount of oxalic acid and the amino acids Ala, Asp, Gaba, Gln, Gly, His, Hyl, Ile, Leu, Lys, Phe, Pro, and Val exuded by their roots into sterile rooting media. Addition of P. fluorescens to the rooting medium did not affect the growth of T3238FER but stimulated the root growth of chlorosis-susceptible T3238fer, reduced the amounts of glucose, arabinose and fructose but increased the amount of sucrose, reduced the amounts of fumaric, malic and oxalic acid but increased the amounts of citric and succinic acid in the rooting media of both mutants. P. fluorescens resulted in the following changes in the amino acids in the rooting media: reduced the amounts of Gly, Leu, and Lys in T3238FER, and of Asp, Gln, Hyp, and Ile in T3238fer, and increased the amounts of Cys, Glu, His, Hyp, Ile, Phe and Tyr in T3238FER and of Ala, Glu, His, Phe, and Ser in T323fer—in cases more than 40-fold. These differential effects of P. fluorescens in altering the pattern of organic and amino acids compounds with some Fe-chelating properties detected in the rooting medium of these two mutants may indicate that the differences in Fe-chlorosis susceptibility of these tomato mutants may be the result of, or modified by, the interactions between plant roots and rhizosphere microorganisms. We postulate that the Fe-chlorosis susceptibility in plants may be the product of the interactions between soil microorganisms and plant roots, and may not be solely related to the plant per se.     

A Role for Pectinase and Protease Inhibitors in Fungal Rot Development in Tomato Fruits

B. cinerea and C. atramentarium rotted wound-inoculated green tomato fruits and wounded or intact ripe fruits while G. cingulata developed rots only in ripe fruits. Pectic en-zymes were extracted from the fruit tissue rotted by B. cinerea and C. atramentarium but no pectic enzymes attributable to the fungus were detected in ripe fruits rotted by G. cingulata. G. cingulata produced endo-PG and endo-PL in vitro, C. atramentarium produced endo-PL in vitro and in vivo and B. cinerea produced exo-PG in vitro and in green fruits but endo-PG and endo-PL in ripe fruits. Well ripened tomato fruits contained high levels of endogenous PG. All three fungi produced proteolytic enzymes in vitro and in vivo. Proteases produced by G. cingulata and C. atramentarium had optimum activity at pH 9 to 10 and were not trypsin-like or chymotrypsin-like in nature. Protease produced by B. cinerea had optimum activity at pH 7 and showed both trypsin and chymotrypsin-like activity. Proteins extracted from the cell walls of tomato fruits inhibited both the endo-PG and endo-PL produced by G. cingulata and the endo-PL produced by B. cinerea but did not in-hibit the activity of PGs produced by B. cinerea, the endo-PL produced by C. atramentarium or the endogenous PG from tomato fruits. The cell wall proteins also contained trypsin and chymotrypsin inhibitor activity which inhibited 70 % of the activity of the protease produced by B. cinerea, but had little effect on the proteases produced by G. cingulata, C. atramentarium or the tomato endogenous protease. Enzymes produced in vitro by G. cingulata macerated green tomato tissue more slowly than enzymes produced in vitro by C. atramentarium and B. cinerea and the rate of mation was further reduced in the presence of added cell wall proteins. Excess inhibitor of the little effect on the rate of maceration by the enzymes produced by C. atramentarium of the cinerea.     

紫色小白菜有机酸的提取优化及UPLC定量分析

为建立一种紫色小白菜中精准的有机酸提取及定性定量分析方法。本研究在单因素试验的基础上,采用Box-Behnken的中心组合试验设计原理,设计响应面试验优化紫色小白菜有机酸的提取工艺。结果表明,紫色小白菜有机酸提取的最优工艺参数为:乙醇浓度73%,料液比1∶21,超声时间11 min,超声温度70℃。通过超高效液相色谱法(ultra performance liquid chromatography,UPLC)对紫色小白菜叶片和叶柄部位有机酸进行定性定量分析,检测出苹果酸、柠檬酸、丙二酸、琥珀酸和酒石酸,其中苹果酸含量最高,分别为15.968、5.019 mg/g;其次为柠檬酸,分别为9.293、1.385 mg/g。定性定量结果表明,紫色小白菜叶片及叶柄部位含有丰富有机酸类物质,叶片部位苹果酸、柠檬酸、丙二酸、酒石酸含量均高于叶柄部位,而琥珀酸含量较低。     

红壤中镉在有机酸作用下的解吸行为

采用平衡批处理法,研究了3种有机酸及其两两混合液在序列pH值梯度下（pH 3.0～7.0）对华南山地红壤Cd解吸行为的影响.结果表明,草酸与苹果酸不利于Cd的解吸,反而促进了吸附,其中草酸只是在较高浓度(20 mmol·L^-1)且土壤溶液pH＞5.0时促进解吸.随着pH值升高,草s酸、苹果酸以及不含有机酸的对照溶液对红壤中Cd的解吸率都快速下降.柠檬酸在pH＜5.0时不利于Cd解吸;在pH＞5.0时显著促进Cd解吸,但两种浓度柠檬酸解吸特征有所不同,在低浓度(2 mmol·L^-1)下对镉的解吸率呈降低-升高-降低变化,在高浓度(20 mmol·L^-1)下呈降低-升高变化.在低pH条件下（pH 3.0、4.0）,苹果酸最有利于Cd的解吸,但3种酸对Cd解吸率差别不大,在较高pH条件下(pH 5.0～7.0),柠檬酸最有利于解吸,且解吸率大大高于草酸与苹果酸.有机酸混合没有明显的交互作用,对Cd的解吸率介于相应单独有机酸之间.     

Metabolic Discrimination of Catharanthus roseus Calli According to Their Relative Locations Using 1H-NMR and Principal Component Analysis

Creating a plant-cell suspension culture involves first transferring the callus into liquid media, but there are no objective criteria for selecting the location of the callus to be transferred. In this study, inner and outer cells of Catharanthus roseus with various elicitors in solid-state cultures were differentiated by ¹H NMR (nuclear magnetic resonance) spectrometry and principal component analysis (PCA). It was found that the samples of various elicitors and relative locations could be separated in PCA-derived score plots. Especially, there was a clear separation between nontreated samples and those cotreated with silver nitrate and methyl jasmonate. Loading-plot analysis was therefore applied to data obtained from nontreated samples and those cotreated with silver nitrate and methyl jasmonate to determine the separation of major metabolites on score plots. The levels of valine, lactic acid, threonine, alanine, arginine, acetic acid, malic acid, succinic acid, citric acid, asparagine, choline, lactose, fumaric acid, phenylalanine, tryptophan, and formic acid were higher in the inner callus than in the outer callus, whereas 2-oxoglutaric acid, oxalacetic acid, sucrose, and glucose dominated in the outer callus. The results obtained in this study suggest that inner and outer calli can be differentiated by ¹H-NMR-based metabolomic analysis.     

Engineering of unconventional yeast Yarrowia lipolytica for efficient succinic acid production from glycerol at low pH

Yarrowia lipolytica is considered as a potential candidate for succinic acid production because of its innate ability to accumulate citric acid cycle intermediates and its tolerance to acidic pH. Previously, a succinate-production strain was obtained through the deletion of succinate dehydrogenase subunit encoding gene Ylsdh5. However, the accumulation of by-product acetate limited further improvement of succinate production. Meanwhile, additional pH adjustment procedure increased the downstream cost in industrial application. In this study, we identified for the first time that acetic acid overflow is caused by CoA-transfer reaction from acetyl-CoA to succinate in mitochondria rather than pyruvate decarboxylation reaction in SDH negative Y. lipolytica. The deletion of CoA-transferase gene Ylach eliminated acetic acid formation and improved succinic acid production and the cell growth. We then analyzed the effect of overexpressing the key enzymes of oxidative TCA, reductive carboxylation and glyoxylate bypass on succinic acid yield and by-products formation. The best strain with phosphoenolpyruvate carboxykinase (ScPCK) from Saccharomyces cerevisiae and endogenous succinyl-CoA synthase beta subunit (YlSCS2) overexpression improved succinic acid titer by 4.3-fold. In fed-batch fermentation, this strain produced 110.7 g/L succinic acid with a yield of 0.53 g/g glycerol without pH control. This is the highest succinic acid titer achieved at low pH by yeast reported worldwide, to date, using defined media. This study not only revealed the mechanism of acetic acid overflow in SDH negative Y. lipolytica, but it also reported the development of an efficient succinic acid production strain with great industrial prospects.     

Breeding for flavour of fresh market tomato: sources for increasing acid content

Breeding programs in tomato for fresh consumption have concluded in very productive varieties/hybrids with an extraordinary external quality. However, internal quality has not been a priority objective in these breeding programs, so present products have a lack of important internal quality properties. Thus, internal and nutritional improvement including taste and organoleptyc characteristics are important breeding objectives at present. A screening trial to identify sources for internal quality improvement by increasing acid content was carried out. 12 accessions of Lycopersicon esculentum and 8 of L. pimpinellifolium were tested with that purpose. Content in citric, malic, oxalic and fumaric acid by HPLC, soluble solids content (SSC)(1 Brix) by refractometry and total acidity by titration with NaOH were measured. Sources for high citric, malic and fumaric acid content were found to begin those breeding programs. Results could possibly suggest an independent genetic control for every acid.     

Application of melatonin and PGPR alleviates thiamethoxam induced toxicity by regulating the TCA cycle in Brassica juncea L

Thiamethoxam, a broad spectrum, neonicotinoid insecticide, is used on various crops including Brassica juncea L. to protect from intruding insects such as leaf-hoppers, aphids, thrips and white-flies. Exposure to thiamethoxam causes acute malady such as tumour development, cell apoptosis, liver damage and neurotoxicity. Melatonin is entailed in umpteen developmental processes of plants, including stress responses. The pleiotropic effects of melatonin in modulating plant growth validate it’s imperative contribution as multi-regulatory substance. Exiguous information is known about the role of Pseudomonas putida in improving plant growth under thiamethoxam stress. Taking these aspects into consideration the contemporary study investigates the role of melatonin and Pseudomonas putida strain MTCC 3315 in alleviating the thiamethoxam induced toxicity in B. juncea plant. Fourier Transform Infrared Spectroscopy (FTIR) analysis uncloaked that thiamethoxam induced stress primarily affects the protein content of plant as compared to lipids, carbohydrates and cell wall components. Organic acid profiling of the treated samples carried-out by High-Performance Liquid Chromatography (HPLC), reported an upregulation in the level of organic acids, malic acid (110%), citric acid (170%), succinic acid (81%), fumaric acid (40%) and ascorbic acid (55%) in thiamethoxam treated plants compared to the investigational untreated plants. The melatonin treated seedlings grown under thiamethoxam stress, exhibit increased level of malic acid, citric acid, succinic acid, fumaric acid and ascorbic acid by 81%, 0.94%, 11%, 21% and 6% respectively. Further, thiamethoxam stressed plants inoculated with Pseudomonas putida showed stupendous up-regulation by 161% (malic acid), by 14% (citric acid), by 33% (succinic acid), by 30% (fumaric acid), by 100% (oxalic acid) respectively. Lastly, the combinatorial application of melatonin and Pseudomonas putida resulted in prodigious upsurge of malic acid by 165%, succinic acid by 69%, fumaric acid by 42% respectively in contrast to distinct melatonin and Pseudomonas putida treatments. The accumulation of organic acids ascertains the defence against thiamethoxam stress and corresponds to meet the energy generation requirement to skirmish thiamethoxam mediated abiotic stress in Brassica juncea plant.     

低分子量有机酸对红壤无机态磷转化及酸度的影响

以鄂南、赣北两红壤样品为材料,加入不同有机酸并经室温培养后,测定不同P组分、pH及活性Al含量的变化。结果表明,供试有机酸均使土壤Ca2－P含量增高,增幅大小依次为柠檬酸＞苹果酸＞琥珀酸＞乙酸;2种土壤的Ca8－P和Ca10－P含量无明显变化规律,Fe-P、Al-P和O－P含量有所下降,除乙酸处理的土壤pH值无显著变化外,其它有机酸的加入使pH下降0．65－1．96;有机酸引起活性Al量增多,除乙酸处理的变化较小外,其它有机酸或混合物的加入使土壤中0．02mol.L^-1CaCl2提取Al增加4．7－50．3倍,1mol.L^-1提取Al增加4．0－67．3倍。可见,有机酸具有双重作用,既增加P的有效性,又增加土壤酸度和Al毒。     

Organic acids production byStreptomyces spp. isolated from soil,rhizosphere and mycorrhizosphere of pine (Pinus sylvestris L.)

Summary The streptomycetes studied released into the medium the following organic acids: pyruvic, α-ketoglutaric, lactic, malic (or citric), succinic and oxalic. Soil streptomycetes produced more α-ketoglutaric-, lactic- and succinic acid than the root zone microorganisms. Mean indices of the total production of organic acids were in the following order: soil>rhizosphere>mycorrhizosphere. Amounts of pyruvic acid excreted by the soil streptomycetes were inversely proportional to their biomass, whereas those of α-ketoglutaric acid were directly proportional to dry weight. Small repeatability of the results of α-keto acids determinations in these organisms was stated.