Invertase Levels Research Articles

Comparative study of root growth and sucrose-cleaving enzymes in metallicolous and non-metallicolous populations of Rumex dentatus under copper stress

Sucrose metabolism in roots of metallophytes is very important for root growth and maintenance of heavy metal tolerance. However, rare researches have been carried out on this topic so far. We tested here a hypothesis that roots of copper-tolerant plants should manifest higher activities of sucrose-cleaving enzymes than non-tolerant plants for maintaining root growth under Cu stress. Plants of two contrasting populations of metallophyte Rumex dentatus, one from an ancient Cu mine (MP) and the other from a non-mine site (NMP), were treated with Cu in controlled experiments. Cu treatment resulted in a higher root biomass and root/shoot biomass ratio in MP compared to NMP. More complicated root system architecture was showed in MP under Cu stress. Activities and transcript levels of acid invertase as well as contents of sucrose and reducing sugar in MP were elevated under Cu treatment, while activities of neutral/alkaline invertase and sucrose synthase showed no significant differences between two populations. The results indicate important roles of acid invertase in governing root growth under Cu stress.

Cytokinin, auxin, and abscisic acid affects sucrose metabolism conduce to de novo shoot organogenesis in rice (Oryza sativa L.) callus.

BackgroundShoot regeneration frequency in rice callus is still low and highly diverse among rice cultivars. This study aimed to investigate the association of plant hormone signaling and sucrose uptake and metabolism in rice during callus induction and early shoot organogenesis. The immatured seeds of two rice cultivars, Ai-Nan-Tsao 39 (ANT39) and Tainan 11 (TN11) are used in this study.ResultsCallus formation is earlier, callus fresh weight is higher, but water content is significant lower in ANT39 than in TN11 while their explants are inoculated on callus induction medium (CIM). Besides, the regeneration frequency is prominently higher in ANT39 (~80%) compared to TN11 callus (0%). Levels of glucose, sucrose, and starch are all significant higher in ANT39 than in TN11 either at callus induction or early shoot organogenesis stage. Moreover, high expression levels of Cell wall-bound invertase 1, Sucrose transporter 1 (OsSUT1) and OsSUT2 are detected in ANT39 at the fourth-day in CIM but it cannot be detected in TN11 until the tenth-day. It suggested that ANT39 has higher callus growth rate and shoot regeneration ability may cause from higher activity of sucrose uptake and metabolism. As well, the expression levels of ORYZA SATIVA RESPONSE REGULATOR 1 (ORR1), PIN-formed 1 and Late embryogenesis-abundant 1, representing endogenous cytokinin, auxin and ABA signals, respectively, were also up-regulated in highly regenerable callus, ANT39, but only ORR1 was greatly enhanced in TN11 at the tenth-day in CIM.ConclusionThus, phytohormone signals may affect sucrose metabolism to trigger callus initiation and further de novo shoot regeneration in rice culture.Electronic supplementary materialThe online version of this article (doi:10.1186/1999-3110-54-5) contains supplementary material, which is available to authorized users.

Patterns of enzyme activities and gene expressions in sucrose metabolism in relation to sugar accumulation and composition in the aril of Litchi chinensis Sonn.

Sucrose metabolism enzymes, including invertase (EC 3.2.1.26), sucrose synthase (SS, EC 2.4.1.13), and sucrose phosphate synthase (SPS, EC 2.4.1.14), are key factors that determine fruit sugar accumulation and composition. Sugar concentration and sugar composition in the arils of 42 litchi cultivars were determined at maturity. The cultivars were grouped into three types according to their hexose/sucrose ratio. Five cultivars of each type were selected to monitor the activities and gene expressions of enzymes related to sucrose metabolism. Pattern changes in the arils of four cultivars with different sugar concentrations and compositions were traced from around 40 d after anthesis to full maturity. Highly significant positive correlations were observed between hexose/sucrose ratios and the activities and expression levels of soluble acid invertase (SAI) and SS among the 15 cultivars tested. The increase in hexose/sucrose ratio was accompanied by enhanced acid invertase (AI) and SS activities and the expression of their genes in Feizixiao (FZX) and Heiye (HY). By contrast, no significant correlation was observed between hexose/sucrose ratio and SPS. These results indicate that the sugar composition in litchi aril depends mainly on the sucrose cleavage enzymes AI and SS and not on the sucrose synthetic enzyme SPS. The cultivar Nuomici, which had the highest sugar content among the cultivars studied, displayed significantly lower activities of cell wall acid invertase, SAI, neutral invertase, and SS and lower expression levels of SAI and SS compared with HY, the cultivar with the lowest sugar content. The inconsistent patterns of sugar accumulation and activities and expressions of sucrose metabolism enzymes suggest that these sucrose metabolism enzymes are not necessarily related to sugar accumulation.

Vacuolar invertases in potato (Solanum tuberosum L.): molecular cloning, characterization, sequence comparison, and analysis of gene expression in the cultivars

In plants, vacuolar invertase (β-fructofuranosidase, EC 3.2.1.26) is known to play as a key modulator for hexose accumulation and cell expansion. In this study, two cDNA clones (2,013 and 1,945 bp, with 99 % sequence identity) encoding vacuolar invertase isoforms were isolated from a commercially important Indian potato cultivar, Kufri Chipsona-1 by RT-PCR. The corresponding predicted proteins consisted of 635 amino acids (designated as KC-VIN1, lacking a few amino acids at N-terminus) and 639 amino acids (designated as KC-VIN2), respectively. They showed 99 % identity, and found to vary at several locations with mostly non-conservative substitutions. Multiple sequence alignment of vacuolar invertase homologs covering four Solanaceae family members revealed some notable distinguishing sequence features (signature-type sequences). A consensus sequence was predicted using 45 vacuolar invertase sequences from 27 taxonomically different plant species, and a phylogenetic tree was generated to know the evolutionary relation between them. Hydrophobic characters were predicted, and compared in different plant species. All these data are presented in a comprehensive manner which were not documented in the earlier reports. As a preliminary study, vacuolar invertase expression patterns in the tubers of some Indian potato cultivars were analyzed by semi-quantitative RT-PCR and extractable enzyme assay. In all the potato cultivars, the overall expression level of invertase was found to be considerably higher after storage at low temperature as compared to the freshly harvested tubers.

Effects of drought on gene expression in maize reproductive and leaf meristem tissue revealed by RNA-Seq.

Drought stress affects cereals especially during the reproductive stage. The maize (Zea mays) drought transcriptome was studied using RNA-Seq analysis to compare drought-treated and well-watered fertilized ovary and basal leaf meristem tissue. More drought-responsive genes responded in the ovary compared with the leaf meristem. Gene Ontology enrichment analysis revealed a massive decrease in transcript abundance of cell division and cell cycle genes in the drought-stressed ovary only. Among Gene Ontology categories related to carbohydrate metabolism, changes in starch and Suc metabolism-related genes occurred in the ovary, consistent with a decrease in starch levels, and in Suc transporter function, with no comparable changes occurring in the leaf meristem. Abscisic acid (ABA)-related processes responded positively, but only in the ovaries. Related responses suggested the operation of low glucose sensing in drought-stressed ovaries. The data are discussed in the context of the susceptibility of maize kernel to drought stress leading to embryo abortion and the relative robustness of dividing vegetative tissue taken at the same time from the same plant subjected to the same conditions. Our working hypothesis involves signaling events associated with increased ABA levels, decreased glucose levels, disruption of ABA/sugar signaling, activation of programmed cell death/senescence through repression of a phospholipase C-mediated signaling pathway, and arrest of the cell cycle in the stressed ovary at 1 d after pollination. Increased invertase levels in the stressed leaf meristem, on the other hand, resulted in that tissue maintaining hexose levels at an "unstressed" level, and at lower ABA levels, which was correlated with successful resistance to drought stress.

Production and partial characterization of invertase from Mucor geophillus EFRL 03

In the present study, cultural conditions for invertase production from Mucor geophillus using Brassica niger (oilcake) in batch wise submerged fermentation was investigated. The effects of time period (24 to 240 h), carbon sources [ Brassica campestris, B. niger , pomegranate peel, coconut, malta peel, apple pulp and glucose (control)] and nitrogen sources (peptone, yeast extract, corn steep liquor, ammonium chloride, sodium nitrates and potassium nitrate) were checked on invertase production. The highest level of invertase was achieved using mineral medium containing 1.0% B. niger hydrolyzed with 0.3 N HCl and 0.5% yeast extract as carbon and nitrogen sources, respectively, and after 48 h of incubation at 45°C, initial pH was adjusted to 6.5. Invertase exhibited the maximum stability in the range of 25 to 50°C temperature and 4 to 6 pH, respectively, within 10 min of incubation. The enzyme retained more than 60 and 50% activities at 8 and 70°C pH and temperature, respectively. In this study, cost effective substrate was utilized for invertase production. The pH and thermostable invertase can be utilized in industrial process. Key words : Mucor geophillus, Brassica niger, submerged fermentation, invertase.

Production and partial characterization of invertase from Mucor geophillus EFRL 03

In the present study, cultural conditions for invertase production from Mucor geophillus using Brassica niger (oilcake) in batch wise submerged fermentation was investigated. The effects of time period (24 to 240 h), carbon sources [ Brassica campestris, B. niger , pomegranate peel, coconut, malta peel, apple pulp and glucose (control)] and nitrogen sources (peptone, yeast extract, corn steep liquor, ammonium chloride, sodium nitrates and potassium nitrate) were checked on invertase production. The highest level of invertase was achieved using mineral medium containing 1.0% B. niger hydrolyzed with 0.3 N HCl and 0.5% yeast extract as carbon and nitrogen sources, respectively, and after 48 h of incubation at 45°C, initial pH was adjusted to 6.5. Invertase exhibited the maximum stability in the range of 25 to 50°C temperature and 4 to 6 pH, respectively, within 10 min of incubation. The enzyme retained more than 60 and 50% activities at 8 and 70°C pH and temperature, respectively. In this study, cost effective substrate was utilized for invertase production. The pH and thermostable invertase can be utilized in industrial process. Key words : Mucor geophillus, Brassica niger, submerged fermentation, invertase.

Tapetal development and abiotic stress: a centre of vulnerability.

Many self-fertilising crops are particularly sensitive to abiotic stress at the reproductive stage. In rice (Oryza sativa L.) and wheat (Triticum aestivum L.), for example, abiotic stress during meiosis and the young microspore stage indicates the tapetum is highly vulnerable and that the developmental program appears to be compromised. Tapetal hypertrophy can occur as a consequence of cold and drought stress, and programmed cell death (PCD) is delayed or inhibited. Since the correct timing of tapetal PCD is essential for pollen reproduction, substantial losses in grain yield occur. In wheat and rice, a decrease in tapetal cell wall invertase levels is correlated with pollen abortion and results in the amount of hexose sugars reaching the tapetum, and subsequently the developing microspores, being severely reduced ('starvation hypothesis'). ABA and gibberellin levels may be modified by cold and drought, influencing levels of cell wall invertase(s) and the tapetal developmental program, respectively. Many genes regulating tapetal and microspore development have been identified in Arabidopsis thaliana (L.) Heynh. and rice and the specific effects of abiotic stresses on the program and pathways can now begin to be assessed.

Developmentally regulated temporal expression and differential acid invertase activity in differentiating cotyledonary explants of mungbean [Vigna radiata (L.) Wilczek

Invertases hydrolyze disaccharide-sucrose into glucose and fructose, and are involved in various metabolic processes in plants as key modulators of source/sink activities. Cotyledons of Vigna radiata seeds are photosynthetically inactive sink tissues that store sucrose. However, a sink to source transition occurs when de-embryonated cotyledons are grown under in vitro conditions to undergo differentiation. In this study, two cotyledon types, Cot and Cot E based on a unique differential regeneration response reported earlier, are grown separately in vitro on Gamborg’s B5 basal nutrient medium (Gamborg et al. 1968), 2% sucrose, and supplemented with 5, 10, 15, or 20 μM N6-benzyladenine (BA). Temporal differences in acid invertase enzyme activity are observed in differentiating explants. In general all BA levels and both in the presence and/or absence of sucrose, acid invertase levels are lower in Cot than Cot E explants. A higher frequency of regeneration of Cot E explants is positively correlated with higher invertase activity. A partial acid invertase (VrAI1) gene has been isolated, cloned, and sequenced (GenBank ID. GQ227550). Using RT–PCR, temporal variations in VrAI1 gene expression are observed in differentiating cotyledons and the transcripts are higher in Cot E than in Cot explants, up to 9 days following culture. Moreover, invertase gene expression is highest in Cot explants at 12 and 15 days following culture. Thus, temporal variations in both amounts of transcripts and in enzyme activity between these two explant types correspond to in vitro responses to shoot differentiation. It is suggested that acid invertase may be involved in temporal differential regeneration patterns observed in Cot and Cot E explants of V. radiata.

Sucrose Accumulation and Internodal Soluble Invertase Isoenzymes in Plant and Ratoon Crops of Sugarcane

Accumulation pattern of sucrose and hexoses in relation to activities of invertases in stem tissue of plant and ratoon crops of CoJ88 (mid maturing variety) has been investigated at different physiological stages of cane growth. At each stage of crop development, sucrose content was relatively more in ratoon crop than plant crop, whereas the reducing sugar content was relatively less in ratoon crop than plant crop. The activity of soluble acid invertase in stem was found to be highest at stem elongation stage and declined thereafter in both plant and ratoon crops. The activity of soluble acid invertase (pH 5.5) was markedly lower in ratoon crop as compared to plant crop at each stage of crop development. Higher level of sucrose and lower level of acid invertase in ratoon crop indicates higher sink strength and better juice quality of ratoon crop. The activity of neutral invertase (pH 7.0) increased with the advancement of the cane growth in both the crops. Three isoenzymes of soluble acid invertase in plant crop namely A-I, A-II and A-III and two isoforms of this enzyme in ratoon crop namely A-I and A-II were identified at stem elongation stage. Neutral invertase isoenzyme was not detected in both the crops at this stage. At maturation stage, one acid invertase isoform i.e. A-I was obtained in both the crops along with four neutral invertase isoenzymes in plant crop i.e. N-I, N-II, N-III and N-IV and two isoforms of this enzyme in ratoon crop i.e. N-I and N-II. These invertase isoforms differed with respect to their Km and Vmax values. From the temperature effect on Km and Vmax values, the energy of activation (Ea) and enthalpy change (∆H) of invertase isoenzymes were calculated at stem elongation and maturity stages and compared among isoenzymes of plant and ratoon crops in relation to sucrose accumulation.

Involvement of abscisic acid in the coordinated regulation of a stress-inducible hexose transporter (VvHT5) and a cell wall invertase in grapevine in response to biotrophic fungal infection.

Biotrophic fungal and oomycete pathogens alter carbohydrate metabolism in infected host tissues. Symptoms such as elevated soluble carbohydrate concentrations and increased invertase activity suggest that a pathogen-induced carbohydrate sink is established. To identify pathogen-induced regulators of carbohydrate sink strength, quantitative real-time polymerase chain reaction was used to measure transcript levels of invertase and hexose transporter genes in biotrophic pathogen-infected grapevine (Vitis vinifera) leaves. The hexose transporter VvHT5 was highly induced in coordination with the cell wall invertase gene VvcwINV by powdery and downy mildew infection. However, similar responses were also observed in response to wounding, suggesting that this is a generalized response to stress. Analysis of the VvHT5 promoter region indicated the presence of multiple abscisic acid (ABA) response elements, suggesting a role for ABA in the transition from source to sink under stress conditions. ABA treatment of grape leaves was found to reproduce the same gene-specific transcriptional changes as observed under biotic and abiotic stress conditions. Furthermore, the key regulatory ABA biosynthetic gene, VvNCED1, was activated under these same stress conditions. VvHT5 promoter::beta-glucuronidase-directed expression in transgenic Arabidopsis (Arabidopsis thaliana) was activated by infection with powdery mildew and by ABA treatment, and the expression was closely associated with vascular tissue adjacent to infected regions. Unlike VvHT1 and VvHT3, which appear to be predominantly involved in hexose transport in developing leaves and berries, VvHT5 appears to have a specific role in enhancing sink strength under stress conditions, and this is controlled through ABA. Our data suggest a central role for ABA in the regulation of VvcwINV and VvHT5 expression during the transition from source to sink in response to infection by biotrophic pathogens.

Metabolic adaptation of Phytophthora infestans during growth on leaves, tubers and artificial media

Efficient nutrient acquisition is critical to the fitness of plant pathogens. To address how the late blight agent Phytophthora infestans adapts to nutrients offered by its hosts, genes in glycolytic, gluconeogenic and amino acid pathways were mined from its genome and their expression in different plant tissues and artificial media was measured. Evidence for conventional glycolytic and gluconeogenic processes was obtained, although several steps involved pyrophosphate-linked transformations which are uncommon in eukaryotes. In media manipulation studies, nearly all genes in the pathways were subject to strong transcriptional control. However in rye-sucrose media, tomato leaflets, potato tubers and, at both early and late stages of infection, most glycolytic genes were expressed similarly, which indicated that each plant tissue presented a nutrient-rich environment. Biochemical analyses also demonstrated that sporulation occurred from host material in which sugars were abundant, with fructose and glucose increasing at the expense of sucrose late in the disease cycle. The expression of only a few genes changed late in infection, with the most notable example being lower invertase levels in the sucrose-reduced leaves. Interestingly, most gluconeogenic genes were up-regulated in tubers compared with other tissues. Rather than reflecting a starvation response, this probably reveals the role of such enzymes in converting carbon skeletons from the abundant free amino acids of tubers into citric acid cycle and glycolysis intermediates, as genes involved in amino acid catabolism were also more highly expressed in tubers. The corresponding enzymes also displayed higher activities in defined media when amino acids were abundant, as in tubers.

Urease, invertase, dehydrogenase and polyphenoloxidase activities in paddy soil influenced by allelopathic rice variety

Allelopathic rice variety can release allelochemicals from roots to soil in which interfere with neighboring plant and microbial species, but little is currently known about their effect on enzyme activities of paddy soils. This study showed that enzyme activities and concentrations of allelochemicals in paddy field varied with rice varieties, growth stages and moisture regime. Allelopathic rice variety PI312777 paddy fields had higher levels of soil urease, invertase, dehydrogenase and polyphenoloxidase activities when compared to non-allelopathic rice variety Liaojing-9. Furthermore, allelopathic PI312777 released much higher concentrations of allelochemical 5,7,4′-trihydroxy-3′,5′-dimethoxyflavone than non-allelopathic Liaojing-9 during the whole growth stages. Both rice root exudates and the flavone changed soil enzyme activities, but their effects could be distinguished by allelopathic PI312777 and non-allelopathic Liaojing-9 varieties tested. There was a correlation with concentrations of the flavone and enzyme activities in rice soil. The flavone increased enzyme activities, but all test enzyme activities were less responsive to the flavone in flooded soil than in non-flooded soil. The flavone given the short half-life easily degraded in rice soil, but the flavone was more resistant toward degradation in non-flooded soil. These results suggest that the urease, invertase, dehydrogenase and polyphenoloxidase activities of paddy soils may be positively influenced by allelopathic rice variety through the release of allelochemicals.

Post harvest quality deterioration in sugarcane under different environmental conditions

Post harvest changes in the juice quality parameters in relation to storage time were investigated in three sugarcane genotypes varying in maturity behaviour viz.CoJ83 (early), CoJ88 (mid) and S70/00(late) under different environmental conditions i.e in the month of November, January and March. A gradual decrease in cane weight, % juice extraction, sucrose (% juice), purity (% juice) and pH with simultaneous increase in TSS%, titrable acidity, dextran, reducing sugars and activities of acid and neutral invertases was found in juice during 12 days of cane storage in all genotypes under all environmental conditions. Irrespective of the genotype and environmental condition, the level of neutral invertase was found to be higher as compared to acid invertase except in genotype S70/00, where higher activity of acid invertase was found as compared to neutral invertase during the month of November. The rate of decrease/increase per day of all the quality parameters on staling was highest during the late crushing period i.e. March than during November and January in all genotypes. More deterioration in quality parameters was found in genotype CoJ83 during March and in genotype S70/00 during November, which may be due to their over mature and immature conditions, respectively, at that time. Hence, the variation in the rate of change in quality parameters during staling may be attributed to the difference in maturity level among genotypes during these three months.

Reguladores vegetais e atividade de invertases em cana-de-açúcar em meio de safra

O objetivo neste trabalho foi a avaliação da atividade das invertases ácida e neutra e a função que essas exercem no controle do acúmulo de sacarose em cana-de-açúcar, variedade SP80-3280, decorrente da aplicação de reguladores vegetais no meio de safra, em Igaraçu do Tietê (SP). O experimento foi conduzido em cana soca, em safras consecutivas (2004 e 2005). O delineamento experimental utilizado foi em blocos casualizados, com cinco repetições. Os tratamentos foram constituídos da aplicação de quatro reguladores vegetais da classe dos retardadores do crescimento (Etefon, Etil-trinexapac, KNO3 e KNO3 + Boro) e uma testemunha (maturação natural), comercialmente encontrados como Ethrel, Moddus, Krista Kana e Krista Kana Plus, respectivamente, adotando-se a dosagem do produto comercial: 2L ha-1, 0,8L ha-1, 3kg ha-1 e 3kg ha-1, respectivamente, sem a adição de adjuvantes. Os reguladores vegetais promovem alterações de intensidades distintas e significativas nos níveis enzimáticos das invertases ácida e neutra. A eficiência agronômica dos maturadores foi afetada de forma significativa pelo clima e pela época de aplicação. Os tratamentos com maturadores, principalmente em 2004, cujas condições climáticas foram desfavoráveis à maturação natural, favoreceram o processo de maturação da cana-de-açúcar, implicando em melhorias da qualidade tecnológica dos colmos.

Seed Size Is Associated with Sucrose Synthase Activity in Developing Cotyledons of Chickpea

Seed size, a key quality determinant in the marketing of the cool‐season food legume chickpea (Cicer arietinum L.), can be reduced under terminal drought. Sucrose synthase and invertase, important enzymes in sugar metabolism, play an important role in seed filling. This study aimed to determine whether these enzymes in the developing cotyledons are associated with the final seed size of chickpea by following their level of activity during seed filling in large‐ and small‐seeded chickpea genotypes. The level of invertase in the cotyledons was unchanged during seed filling, but sucrose synthase reached a maximum about 500 to 600 growing degree days (GDD) after flowering when cell numbers were no longer increasing, but the cotyledons were increasing rapidly in dry weight. In a range of genotypes differing in seed size, the level of sucrose synthase activity in the developing cotyledons at 500 to 600 GDD was correlated with the final seed size at maturity when measured at a rainfed site in two different years. The level of activity of sucrose synthase at the same stage of development in F2, F3, and F7 populations of crosses between a large‐seeded cultivar and two small‐seeded genotypes was also correlated with final seed size. It is concluded that sucrose synthase, but not invertase, in developing cotyledons is the key enzyme influencing seed size in chickpea.

Antisense suppression of an acid invertase gene (MAI1) in muskmelon alters plant growth and fruit development

To unravel the roles of soluble acid invertase in muskmelon (Cucumis melo L.), its activity in transgenic muskmelon plants was reduced by an antisense approach. For this purpose, a 1038 bp cDNA fragment of muskmelon soluble acid invertase was expressed in antisense orientation behind the 35S promoter of the cauliflower mosaic virus. The phenotype of the antisense plants clearly differed from that of control plants. The transgenic plant leaves were markedly smaller, and the stems were obviously thinner. Transmission electron microscopy revealed that degradation of the chloroplast membrane occurred in transgenic leaves and the number of grana in the chloroplast was significantly reduced, suggesting that the slow growth and weaker phenotype of the transgenic plants may be due to damage to the chloroplast ultrastructure, which in turn resulted in a decrease in net photosynthetic rate. The sucrose concentration increased and levels of acid invertase decreased in transgenic fruit, and the fruit size was 60% smaller than that of the control. In addition, transgenic fruit reached full-slip at 25 d after pollination (DAP), approximately 5 d before the control fruit (full-slip at 30 DAP), and this accelerated maturity correlated with a dramatic elevation of ethylene production at the later stages of fruit development. Together, these results suggest that soluble acid invertase not only plays an important role during muskmelon plant and fruit development but also controls the sucrose content in muskmelon fruit.

Gln3p and Nil1p regulation of invertase activity and expression in

In Saccharomyces cerevisiae, sensing and signalling pathways regulate gene expression in response to quality of carbon and nitrogen sources. One such system, the target of rapamycin (Tor) proteins, senses nutrients and uses the GATA activators Gln3p and Nil1p to regulate translation in response to low-quality carbon and nitrogen. The signal transduction, triggered in response to nitrogen nutrition that is sensed by the Tor proteins, operates via a regulatory pathway involving the cytoplasmic factor Ure2p. When carbon and nitrogen are abundant, the phosphorylated Ure2p anchors the also phosphorylated Gln3p and Nil1p in the cytoplasm. Upon a shift from high- to low-quality nitrogen or treatment with rapamycin all three proteins are dephosphorylated, causing Gln3p and Nil1p to enter the nucleus and promote transcription. The genes that code for yeast periplasmic enzymes with nutritional roles would be obvious targets for regulation by the sensing and signalling pathways that respond to quality of carbon and nitrogen sources. Indeed, previous results from our laboratory had shown that the GATA factors Gln3p, Nil1p, Dal80p, Nil2p and also the protein Ure2 regulate the expression of asparaginase II, coded by ASP3. We also had observed that the activity levels of the also periplasmic invertase, coded by SUC2, were 6-fold lower in ure2 mutant cells in comparison to wild-type cells collected at stationary phase. These results suggested similarities between the signalling pathways regulating the expression of ASP3 and SUC2. In the present work we showed that invertase levels displayed by the single nil1 and gln3 and by the double gln3nil1 mutant cells, cultivated in a sucrose-ammonium medium and collected at the exponential phase, were 6-, 10- and 60-fold higher, respectively, in comparison to their wild-type counterparts. RT-PCR data of SUC2 expression in the double-mutant cells indicated a 10-fold increase in the mRNA(SUC2) levels.

Effect of water activity on invertase production in solid state fermentation by improved diploid strains of Aspergillus niger

Invertase production under solid state fermentation (SSF) was determined using two overproducing mutants (Aw96-3 and Aw96-4) isolated previously from the wild type strain Aspergillus niger C28B25, as well as one diploid (DAR1) and two autodiploid strains (AD96-3 and AD96-4) constructed by parasexual crossings among these mutants. Using polyurethane foam (PUF) as an inert carrier, two initial water activity (Aw) values were evaluated (0.99 and 0.96). At Aw=0.99, maximal activity was reached by diploid AD96-4 (48.91 IU/ml) representing 30- and 13-fold increases with respect to maximal values achieved by the wild type and the haploid parental mutant (Aw96-4), respectively. Similar levels were achieved by this strain at Aw=0.96. However, diploid DAR1 only produced high levels of invertase at Aw=0.96 (43.90 IU/ml), whereas strain AD96-3 reached its highest production (31.10 IU/ml) at Aw=0.99. Both productivity and yields were also analysed for every strain at each Aw value.

Metabolizable and non-metabolizable sugars activate different signal transduction pathways in tomato.

To gain insight into the regulatory mechanisms of sugar signaling in plants, the effect of derivatives of the transport sugar sucrose (Suc), the Suc isomers palatinose and turanose, and the Suc analog fluoro-Suc were tested. Photo-autotrophic suspension culture cells of tomato (Lycopersicon peruvianum) were used to study their effect on the regulation of marker genes of source and sink metabolism, photosynthesis, and the activation of mitogen-activated protein kinases (MAPKs). Suc and glucose (Glc) resulted in reverse regulation of source and sink metabolism. Whereas the mRNA level of extracellular invertase (Lin6) was induced, the transcript level of small subunit of ribulose bisphosphate carboxylase (RbcS) was repressed. In contrast, turanose, palatinose, and fluoro-Suc only rapidly induced Lin6 mRNA level, whereas the transcript level of RbcS was not affected. The differential effect of the metabolizable and non-metabolizable sugars on RbcS mRNA regulation was reflected by the fact that only Suc and Glc inhibited photosynthesis and chlorophyll fluorescence. The activation of different signal transduction pathways by sugars was further supported by the analysis of the activation of MAPKs. MAPK activity was found to be strongly activated by turanose, palatinose, and fluoro-Suc, but not by Suc and Glc. To analyze the role of sugars in relation to pathogen perception, an elicitor preparation of Fusarium oxysporum lycopersici was used. The strong activation of MAPKs and the fast and transient induction of Lin6 expresssion by the fungal elicitor resembles the effect of turanose, palatinose, and fluoro-Suc and indicates that non-metabolizable sugars are sensed as stress-related stimuli.