AtCYT-INV1 in Arabidopsis Sugar Signaling

Sugar acts as a signal molecule and plays a pivotal role in plant development and stress response. Neutral/alkaline invertases found only in photosynthetic bacteria and plants is sucrose-specific enzymes cleave sucrose into glucose and fructose. We have identified a gene for neutral/alkaline invertase in Arabidopsis designated as AtCYT-INV1 which is involved in sugar/ABA signaling and plays multiple roles in plant development and osmotic stress-induced inhibition on lateral root growth.Key Words: Arabidopsis thaliana, AtCTY-INV1, sugar signaling     

Copper-induced alteration in sucrose partitioning and its relationship to the root growth of two Elsholtzia haichowensis Sun populations

Hydroponic culture was used to comparatively investigate the copper (Cu)-induced alteration to sucrose metabolism and biomass allocation in two Elsholtzia haichowensis Sun populations with one from a Cu-contaminated site (CS) and the other from a non-contaminated site (NCS). Experimental results revealed that biomass allocation preferred roots over shoots in CS population, and shoots over roots in NCS population under Cu exposure. The difference in biomass allocation was correlated with the difference in sucrose partitioning between the two populations. Cu treatment (45 μM) significantly decreased leaf sucrose content and increased root sucrose content in CS population as a result of the increased activities of leaf sucrose synthesis enzymes (sucrose phosphate synthetase and sucrose synthase) and root sucrose cleavage enzyme (vacuolar invertase), which led to increased sucrose transport from leaves to roots. In contrast, higher Cu treatment increased sucrose content in leaves and decreased sucrose content in roots in NCS population as a result of the decreased activities of root sucrose cleavage enzymes (vacuolar and cell wall invertases) that led to less sucrose transport from leaves to roots. These results provide important insights into carbon resource partitioning and biomass allocation strategies in metallophytes and are beneficial for the implementation of phytoremediation techniques.     

Acid and Neutral Invertases in the Mesocarp of Developing Muskmelon (Cucumis melo L. cv Prince) Fruit

Acid and neutral invertases were found in the mesocarp of developing muskmelon (Cucumis melo L. cv Prince) fruit and the activities of these enzymes declined with maturation of the fruit, concomitantly with the accumulation of sucrose. Neutral invertase was only present in the soluble fraction and acid invertase was present in both the soluble and cell-wall fractions. The cell-wall fraction contained three types of acid invertase: a NaCl-released invertase; an EDTA-released invertase, and a tightly bound invertase that still remained on the cell wall after treatment with NaCl and EDTA. The soluble acid and neutral invertases could be separated from one another by chromatography on DEAE-cellulose and they exhibited clear differences in their properties, namely, in their pH optima, substrate specificity, K_m values for sucrose, and inhibition by metal ions. The EDTA-released invertase and the soluble acid invertase were similar with regard to their chromatographic behavior on DEAE-cellulose, but the NaCl-released invertase was different because it was adsorbed to a column of CM-cellulose. The soluble acid invertase and two cell-wall bound invertases had very similar characteristics with regard to optimal pH and temperature, K_m value for sucrose, and substrate specificity.     

Sink development, sucrose metabolising enzymes and carbohydrate status in turnip (Brassica rapa L.)

Accumulation of 60–70 % of biomass in turnip root takes place between 49–56 days after sowing. To understand the phenomenon of rapid sink filling, the activities of sucrose metabolising enzymes and carbohydrate composition in leaf blades, petiole and root of turnip from 42–66 days of growth were determined. An increase (2–3 folds) in glucose and fructose contents of roots accompanied by an increase in activities of acid and alkaline invertases was observed during rapid biomass accumulating phase of roots. The observed decrease in the activities of acid and alkaline invertases along with sucrose synthase (cleavage) in petiole during this period could facilitate unrestricted transport of sucrose from leaves to the roots. During active root filling period, a decrease in sucrose synthase (cleavage) and alkaline invertase activities was also observed in leaf blades. A rapid decline in the starch content of leaf blades was observed during the phase of rapid sink filling. These metabolic changes in the turnip plant led to increase in hexose content (35–37 %) of total dry biomass of roots at maturity. High hexose content of the roots appears to be due to high acid invertase activity of the root.     

Antisense repression of vacuolar and cell wall invertase in transgenic carrot alters early plant development and sucrose partitioning.

To unravel the functions of cell wall and vacuolar invertases in carrot, we used an antisense technique to generate transgenic carrot plants with reduced enzyme activity. Phenotypic alterations appeared at very early stages of development; indeed, the morphology of cotyledon-stage embryos was markedly changed. At the stage at which control plantlets had two to three leaves and one primary root, shoots of transgenic plantlets did not separate into individual leaves but consisted of stunted, interconnected green structures. When transgenic plantlets were grown on media containing a mixture of sucrose, glucose, and fructose rather than sucrose alone, the malformation was alleviated, and plantlets looked normal. Plantlets from hexose-containing media produced mature plants when transferred to soil. Plants expressing antisense mRNA for cell wall invertase had a bushy appearance due to the development of extra leaves, which accumulated elevated levels of sucrose and starch. Simultaneously, tap root development was markedly reduced, and the resulting smaller organs contained lower levels of carbohydrates. Compared with control plants, the dry weight leaf-to-root ratio of cell wall invertase antisense plants was shifted from 1:3 to 17:1. Plants expressing antisense mRNA for vacuolar invertase also had more leaves than did control plants, but tap roots developed normally, although they were smaller, and the leaf-to-root ratio was 1.5:1. Again, the carbohydrate content of leaves was elevated, and that of roots was reduced. Our data suggest that acid invertases play an important role in early plant development, most likely via control of sugar composition and metabolic fluxes. Later in plant development, both isoenzymes seem to have important functions in sucrose partitioning.     

Purification, Biochemical and Immunological Characterization of Acid Invertases from Apple Fruit

The soluble acid invertase (SAI) and cell wall-bound invertase (CWI) were purified from apple fruit to apparent electrophoretic homogeneity. Based on sequencing, substrate specificity, and immunoblotting assay, the purified enzymes were identified to be two isoforms of acid invertase (β-fructosidase; EC 3.2.1.26). The SAI and CWI have the same apparent molecular mass with a holoenzyme of molecular mass of 220 kDa composed of 50 kDa subunits. The SAI has a lower Km value for sucrose and higher Km for raffinose compared with CWI. These acid invertases differ from those in other plants in some of their biochemical properties, such as the extremely high Km value for raffinose, no hydrolytic activity for stachyose, and a mixed form of inhibition by fructose to their activity. The antibodies directed against the SAI and CWI recognized, from the crude extract, three polypeptides with a molecular mass of 50, 68, and 30 kDa, respectively.These results provide a substantial basis for the further studies of the acid invertases in apple fruit.     

Invertases in Oat Seedlings: SEPARATION, PROPERTIES, AND CHANGES IN ACTIVITIES IN SEEDLING SEGMENTS

The soluble invertase activity in etiolated Avena seedlings was highest at the apex of the coleoptile and much lower in the primary leaf, mesocotyl, and root. The activity in all parts of the seedling consisted of two invertases (I and II) which were separated by chromatography on diethylaminoethylcellulose. Both enzymes appeared to be acid invertases, but they differed in molecular size, pH optimum, and the kinetic parameters K_m and V_max of their action on sucrose, raffinose, and stachyose. Invertase II had low stability at pH 3.5 and below, and exhibited high sensitivity to Hg²⁺, with complete inhibition by 2 micromolar HgCl₂. Segments of coleoptiles incubated in water lost about two-thirds of the total invertase activity after 16 hours. The loss of activity was due primarily to a decrease in the level of invertase II. The loss of invertase was decreased by indoleacetic acid, 2,4-dichlorophenoxyacetic acid, and α-naphthaleneacetic acid but not by β-naphthaleneacetic acid and p-chlorophenoxyisobutyric acid. Conditions that inhibited auxin-induced growth of the segments (20 millimolar CaCl₂ and 200 millimolar mannitol) also blocked the auxin effect on invertase loss.     

Purification, Biochemical and Immunological Characterization of Acid Invertases from Apple Fruit

The soluble acid invertase (SAI) and cell wall-bound invertase (CWI) were purified from apple fruit to apparent electrophoretic homogeneity. Based on sequencing, substrate specificity, and immunoblotting assay, the purified enzymes were identified to be two isoforms of acid invertase (β-fructosidase; EC 3.2.1.26). The SAI and CWI have the same apparent molecular mass with a holoenzyme of molecular mass of 220 kDa composed of 50 kDa subunits. The SAI has a lower Km value for sucrose and higher Km for raffinose compared with CWI. These acid invertases differ from those in other plants in some of their biochemical properties, such as the extremely high Km value for raffinose, no hydrolytic activity for stachyose, and a mixed form of inhibition by fructose to their activity. The antibodies directed against the SAI and CWI recognized, from the crude extract, three polypeptides with a molecular mass of 50, 68, and 30 kDa, respectively.These results provide a substantial basis for the further studies of the acid invertases in apple fruit.     

AtCYT-INV1, a neutral invertase,is involved in osmotic stress-induced inhibition on lateral root growth in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Neutral/Alkaline invertases are unique to plant and photosynthetic bacteria. The function of Neutral/Alkaline invertases in plant development is not clear so far. In this study, we isolated an Arabidopsis (Col-0) mutant insensitive to osmotic stress-induced inhibition on lateral root growth. Map-based cloning reveals that a neutral invertase gene (AtCYT-INV1) was point-mutated. The mutant Atcyt-inv1 showed short primary root, smaller size of leaves and siliques, and promotion of the reproductive compared to the wild type (WT). Carbohydrate measurement showed that sucrose is accumulated and glucose is reduced in the mutant Atcyt-inv1 under normal and 3% mannitol treatments. Taken together, AtCYT-INV1 plays multiple roles in plant development and is involved in osmotic stress-induced inhibition on lateral root growth by controlling the concentration of hexose in cells. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization and localization of three soluble invertase forms from Lilium longiflorum flower buds

Three invertase forms (EC 3.2.1.26) were identified in soluble extracts from developing flower buds of Lilium longiflorum Thunb. cv. Nellie White. The enzymes were separable on a diethylaminoethyl (DEAE)-Sephacel column and designated invertase I. II or III according to the order of elution from Sephacel. To determine tissue specificity of these floral invertases, anthers were separated from tepal. pistil and filament tissue, and analyzed for invertase activity. Invertase I was localized primarily in anthers, with invertases II and III being present in much smaller amounts (less than 5% of the invertase I activity). Much higher levels of invertases II and III were found in the nonanther organs of the flower, where essentially no invertase 1 was detectable. Further purification of each form (using gel filtration. Con-A-Sepharose affinity chromatog-raphy and hydrophobic interaction chromatography on phenyl-agarose) resulted in 135- 189- and 202-fold purification of pooled fractions from DEAE-Sephacel. respectively, and established that each invertase form is a glycoprotein. Each was an acid invertase. with pH optima between 4.0 and 5.0 and an apparent molecular mass of 77 500 Da (as determined by Sephadex gel filtration). The invertases had sucrose K_m values of 1.0. 6.4 and 6.6 m M . and temperature optima of 40. 50 and 45°C. respectively. A temperature stability study revealed that invertase III was the most thermostable, followed by II and I. Invertases II and III had lower affinity to raffinose and stachyose than invertase I. All three enzymes were completely inhibited by Hg²⁺ or Ag⁺ ions at 1.7 m M . At this concentration. Cu^2- showed differential partial inhibition . Although fructan was shown to be present in both anther and nonanther tissues of Lilium flower buds, these invertases showed no sucrose:sucrose fructosyltransferase (EC 2.4.1.99) activity.     

Acid and alkaline invertases in suspension cultures of sugar beet cells

Alkaline invertase was induced during the initiation of suspension cultures of single cells from leaf explants of sugar beets in Murashige-Skoog liquid medium which contained benzyladenine. This activity was barely detectable in the leaves themselves. In suspension cultures, the presence of both acid and alkaline invertases was detected; alkaline invertase was only present in the cytoplasm of the cultured cells, whereas acid invertase was present in the cytoplasm and cell walls, and was also detected in the culture medium. The cell wall contained at least three types of acid invertase; two of these activities were solubilized by saline (saline-released) and EDTA (EDTA-released), respectively, and the third remained tightly associated with the cell wall. Saline-released and EDTA-released invertases from the cell wall showed the significant differences in their properties: the saline-released enzyme had the highest affinity for sucrose among the invertases tested, and was easily bound to cell walls, to DNA, and to a cation exchanger, unlike the EDTA-released enzyme. Sucrose is the source of carbon for plant cells in suspension culture and is probably degraded in the cell wall by the saline-released invertase, which had the highest activity and the highest affinity for sucrose. Hexose products of this degradation would be transported to cytoplasm. Soluble invertase, EDTA-released invertase from the cell wall, and one of two extracellular invertases behaved similarly upon chromatography on DEAE-cellulose. They had similar activity profiles with changing pH, and similar K_m values for sucrose. Thus it appears that they are identical. Two extracellular invertases found in the growth medium of the suspension cultures were probably identical with those in the soluble fraction of callus and seedlings of sugar beets, because they showed similar behaviors during chromatography on DEAE-cellulose, and had similar activity profiles with changing pH and K_m values for sucrose.     

Effect of Kinetin on Starch and Sucrose Metabolising Enzymes in Salt Stressed Chickpea Seedlings

Higher amylase activity in cotyledons of kinetin treated salt stressed (75 mM NaCl) chickpea (Cicer arietinum L. cv. PBG-1) seedlings, as compared to salt stressed seedlings was observed during a growth period of 7 d. The activities of acid and alkaline invertases were maximum in shoots and minimum in cotyledons under all conditions. The reduced shoot invertase activities under salt stress were enhanced by kinetin with a simultaneous increase in reducing sugar content. Kinetin increased the activities of sucrose synthase (SS) and sucrose phosphate synthase (SPS) in both the cotyledons and shoots of stressed seedlings. Kinetin appears to increase the turnover of sucrose in the shoots of stressed seedlings.     

Photosynthate Partitioning and Enzymes of Sucrose Metabolism in Sugarbeet Roots

Populations of sugarbeet (Beta vulgaris L.) plants that differed in taproot/leaf weight ratio and in photosynthate partitioning between taproots and fibrous roots did not differ in root/shoot ratio as indicated by relative dry weight distribution. Based on the hypothesis that dry weight distribution is influenced by the metabolism of imported sucrose, we examined the relationships between the activity of the enzymes of sucrose metabolism and dry weight distribution as a function of genotype and ontogeny. A decreased specific activity of acid invertase in taproots was associated with increased taproot/fibrous root weight ratio at 21 and at 28 days post-emergence. Alkaline invertase activity was negatively correlated with taproot/fibrous root weight ratio at 28 days. Sucrose synthetase specific activities of taproots were not correlated with dry matter distribution. Acid invertase may influence photosynthate partitioning between the taproot and fibrous roots via regulation of sucrose levels in the region of fibrous root initiation.     

A balanced polymorphism in biomass resource allocation controlled by phosphate in grasses screened through arsenate tolerance

The response of arsenate and non-tolerant Holcus lanatus L. phenotypes, where tolerance is achieved through suppression of high affinity phosphate/arsenate root uptake, was investigated under different growth regimes to investigate why there is a polymorphism in tolerance found in populations growing on uncontaminated soil. Tolerant plants screened from an arsenic uncontaminated population differed, when grown on the soil from the populations origin, from non-tolerants, in their biomass allocation under phosphate fertilization: non-tolerants put more resources into tiller production and down regulated investment in root production under phosphate fertilization while tolerants tillered less effectively and did not alter resource allocation to shoot biomass under phosphate fertilization. The two phenotypes also differed in their shoot mineral status having higher concentrations of copper, cadmium, lead and manganese, but phosphorus status differed little, suggesting tight homeostasis. The polymorphism was also widely present (40%) in other wild grass species suggesting an important ecological role for this gene that can be screened through plant root response to arsenate.     

Growth regulator effects on storage root development in carrot

The major carbohydrates stored in carrots are sucrose, glucose and fructose. The ratio of sucrose to reducing sugars varies between cultivars, with early forcing types generally having a higher level of reducing sugars while storage types have a greater proportion of sucrose.In an early forcing cultivar, Super Sprite, high acid invertase activity was correlated with low levels of stored sucrose. As acid invertase activity decreased, the levels of reducing and non-reducing sugars appeared to be related to a balance between alkaline invertase and sucrose synthetase activities.Foliar applications of gibberellic acid at 35 and 42 days after sowing reduced the root/shoot ratio while similar applications of chlormequat chloride marginally increased the ratio. Both growth regulators temporarily increased sucrose stograge, but only gibberellic acid consistently reduced hexose accumulation.Gibberellic acid reduced acid invertase activity following both applications while only reducing the activities of sucrose synthetase after the first application and alkaline invertase after the second application, respectively. Chloremequat chloride increased acid invertase activity after the first application but otherwise has no effect on the activities of the enzymes studied. The significance of changes in assimilate partitioning are discussed in relation to published schemes on carbohydrate storage in root vegetables.