Effects of Iron and Aluminum Oxides and Kaolinite on Adsorption and Activities of Invertase *1

Experiments were conducted to study the influences of synthetic bayerite, non crystalline aluminum oxide (N AlOH), goethite, non crystalline iron oxide (N FeOH) and kaolinite on the adsorption, activity, kinetics and thermal stability of invertase. Adsorption of invertase on iron, aluminum oxides fitted Langmuir equation. The amount of invertase held on the minerals followed the sequence kaolinite > goethite > N AlOH > bayerite > N FeOH. No correlation was found between enzyme adsorption and the specific surface area of minerals examined. The differences in the surface structure of minerals and the arrangement of enzymatic molecules on mineral surfaces led to the different capacities of minerals for enzyme adsorption. The adsorption of invertase on bayerite, N AlOH, goethite, N FeOH and kaolinite was differently affected by pH. The order for the activity of invertase adsorbed on minerals was N FeOH > N AlOH > bayerite > reak goethite > kaolinite. The inhibition effect of minerals on enzyme activity was kaolinite > crystalline oxides > non crystalline oxides. The pH optimum of iron oxide and aluminum oxide invertase complexes was similar to that of free enzyme (pH 4.0), whereas the pH optimum of kaolinite inv ertase complex was one pH unit higher than that of free enzyme. The affinity to substrate and the maximum reaction velocity as well as the thermal stability of combined invertase were lower than those of the free enzyme.

Vacuolar invertase genes SbVIN1 and SbVIN2 are differently associated with stem and grain traits in sorghum (Sorghum bicolor)

In higher plants, vacuolar invertases play essential roles in sugar metabolism, organ development, and sink strength. In sorghum(Sorghum bicolor), two vacuolar invertase genes,Sb VIN1(Sobic. 004 G004800) and Sb VIN2(Sobic. 006 G160700) have been reported, but their enzymatic properties and functional differences are largely unknown. We combined molecular, biochemical and genomic approaches to investigate their roles in sorghum stem and grain traits. Sb VIN1 and Sb VIN2 showed different expression levels in internodes,leaves, and panicles, indicating that their importance in each organ was different. In an in vitro sucrose hydrolysis assay, proteins of both genes heterologously expressed in Pichia pastoris displayed similar enzyme properties including the same optimum reaction p H(5)and similar Kmfor sucroe(49 mmol L-1 and 45 mmol L-1 for Sb VIN1 and Sb VIN2,respectively). The optimum reaction temperatures of Sb VIN1 and Sb VIN2 were 45 °C and65 °C, respectively. Sb VIN2 showed higher tolerance to high temperature than Sb VIN1. We characterized the sequence variation of these two vacuolar invertase genes in a panel of 216 diverse inbred lines of sweet and grain sorghum and performed gene-based association analysis. Sb VIN1 showed significant associations with stem traits including stem length,stem diameter, internode number, stem fresh weight, and Brix, as well as grain traits including hundred-grain weight and grain width. Significantly associated variation sites were mainly in 5′ upstream and intron regions. Sb VIN2 only associated with grain width and stem water-soluble carbohydrates(WSCs) content. We conclude that the vacuolar invertase genes Sb VIN1 and Sb VIN2 are differently associated with stem and grain traits in sorghum.

Research Progress in Plant Invertase

Invertase is a key enzyme in sucrose catabolism and crucial for plant assimilate distribution. With the development of molecularbiology, a lot of invertsae genes were cloned recently, and significant progress have been made in regulators on the expression of invertase genes.Thus, this article summarized theresearch progress of invertase in biological characteristics, molecular characteristics and expression regulation.

Partition coefficient prediction of Baker's yeast invertase in aqueous two phase systems using hybrid group method data handling neural network

A hybrid GMDH neural network model has been developed in order to predict the partition coefficients of invertase from Baker's yeast. ATPS experiments were carried out changing the molar average mass of PEG(1500–6000 Da), p H(4.0–7.0), percentage of PEG(10.0–20.0 w/w), percentage of MgSO_4(8.0–16.0 w/w), percentage of the cell homogenate(10.0–20.0 w/w) and the percentage of MnSO_4(0–5.0 w/w) added as cosolute. The network evaluation was carried out comparing the partition coefficients obtained from the hybrid GMDH neural network with the experimental data using different statistical metrics. The hybrid GMDH neural network model showed better fitting(AARD = 32.752%) as well as good generalization capacity of the partition coefficients of the ATPS than the original GMDH network approach and a BPANN model. Therefore hybrid GMDH neural network model appears as a powerful tool for predicting partition coefficients during downstream processing of biomolecules.

Genome-Wide Identification and Expression Profiling Suggest that Invertase Genes Function in Silique Development and the Response to Sclerotinia sclerotiorum in Brassica napus

Invertase(INV),a key enzyme in sucrose metabolism,irreversibly catalyzes the hydrolysis of sucrose to glucose and fructose,thus playing important roles in plant growth,development,and biotic and abiotic stress responses.In this study,we identified 27 members of the BnaINV family in Brassica napus.We constructed a phylogenetic tree of the family and predicted the gene structures,conserved motifs,cis-acting elements in promoters,physicochemical properties of encoded proteins,and chromosomal distribution of the BnaINVs.We also analyzed the expression of the BnaINVs in different tissues and developmental stages in the B.napus cultivar Zhongshuang 11 using qRT-PCR.In addition,we analyzed RNA-sequencing data to explore the expression patterns of the BnaINVs in four cultivars with different harvest indices and in plants inoculated with the pathogenic fungus Sclerotinia sclerotiorum.We used WGCNA(weighted coexpression network analysis)to uncover BnaINV regulatory networks.Finally,we explored the expression patterns of several BnaINV genes in cultivars with long(Zhongshuang 4)and short(Ningyou 12)siliques.Our results suggest that BnaINVs play important roles in the growth and development of rapeseed siliques and the defense response against pathogens.Our findings could facilitate the breeding of high-yielding B.napus cultivars with strong disease resistance.

Genome-wide analysis of the invertase genes in strawberry(Fragaria×ananassa)

Sugar is an important material basis in fruit development,and strawberry fruit flavour and sweetness largely depend on the sugar content and variety.Invertases(INVs)play an important role in the regulation of sugar accumulation because they irreversibly catalyse the hydrolysis of sucrose into the corresponding nucleoside diphosphate-glucose,glucose or fructose in fruit.In this work,we provided a comprehensive analysis of the INV gene family in octoploid strawberry(Fragaria×ananassa),including the gene structure,chromosomal locations,conserved domains,and gene evolution and expression profiles during strawberry fruit development.Our study revealed that polyploid events resulted in the abundant amplification(almost three-or four-fold)of the INV gene in the F.×ananassa genome,and these amplified INV genes showed dominant expression in strawberry fruit.More than half of the FaINVs transcripts with low expression had incomplete coding sequences by alternative splicing.Previous studies have shown that cell wall invertases(CWINV)are involved in the regulation of phloem unloading and sink strength establishment.The expression of FaCWINV1 was markedly upregulated during fruit development and strongly expressed in ripe fruit.Moreover,a significant correlation was observed between the total sugar content and the FaCWINV1 expression level.These findings suggest that FaCWINV1 may be involved in sugar accumulation in strawberry fruit.Taken together,the results of our study will be beneficial for further research into the functions of INVs in the regulation of fruit ripening.

Expression of Invertase Genes in Virus-free Sugarcane

Virus-free sugarcane seedlings have improved biomass and sucrose content compared with ordinary seedlings, and sucrose invertases are key enzymes regulating sugarcane growth and sucrose accumulation. In this study, the differences in the expression levels of 3 invertase genes, CWI, SAI and NI, between virus- free and ordinary sugarcane seedlings were analyzed. Compared with ordinary sugarcane plants, the expression of CWI was mainly up-regulated in immature leaves and stems at elongation stage and leaves and immature internodes at maturation stage, and especially, greatly up-regulated in immature interuedes at maturation stage of virus-free plants. The expression of SAI and NI were mainly up-regnlated in leaves and immature internedes of virus-free plants at maturation stage, which might be beneficial to sugar accumulation and rapid utilization of monosaccharide in the stalks of virus-free plants. It is further indicated that virus-free treatment could significantly improve the expression of sucrose invertases at late growth period, and might facilitate the increase of plant biomass.

Pollen Germination and Pollen Tube Elongation of Tomato （Lycopersicum esculentum L.） Regulated by Cell Wall Invertase through Sucrose Hydrolysis

Sucrose is a sugar required for pollen germination and pollen tube elongation. It is little known on the regulation mechanism. As such, this research was conducted to reveal mechanism pathway of the sugar in regulating pollen germination and pollen tube elongation by cell wall invertase. The pollen grains, respectively originated from wild type and transgenic tomato plants, which had been silenced their inhibitor gene （INVINH1） of the cell wall invertase were used in this study. The pollen grains were cultured in media containing glucose, fi＇uctose or sucrose. Results of the study showed that percentage of the pollen germination derived from transgenic plants was much higher than that from wild type plants. Moreover, pollen tube elongation was longer in transgenic plants compared with wild type plants. Interestingly, these results were observed in medium containing sucrose only, but not in glucose and fructose. This result suggests that cell wall invertase activity regulates pollen germination and pollen tube elongation through sucrose hydrolysis.

Using a Membrane Reactor for Sucrose Hydrolysis： The Effect of Reactor＇s Design on Invertase Stability

Invertase hydrolyses sucrose, produces inverted sugar syrup, which is used, mainly, as a food composition in industries. To carry out the hydrolysis properly, the invertase should be stable in the soluble form through a considerable reaction period and recovered afterwards. The chosen reactor was a CSTR-type （continuous stirred tank reactor-type） coupled with an UFM （ultrafiltration-membrane）, the so-called MR （membrane reactor）. The varied parameters were： sucrose concentration （10-300 mM）, temperature （5-65 ℃）, reaction volume （14 mL and 65 mL）, stirring （100-500 rpm）, volumetric feeding rate （2.2-12 mL/h） and UFM MWCO （molecular weight cut off） （10, 20, 30, 50 and 100 kDa）. The invertase kinetic constants （KM = 23.5 mM; Vmax = 2,758 μmolgluJmin-mgprot; Ea = 9.1 kcal/mol） and the temperature deactivation energy （Ead= 20 kcal/mol） were calculated. Moreover, the invertase was unstable as the MR capacity diminished and the agitation increased up to 500 rpm most likely due to the damaging effect of shearing forces （present inside the MR） on the invertase molecule. Finally, both the MWCO and the chemical nature of the UFM affected the invertase stability along the hydrolysis. The enzyme stability increased as the UFM cut-off decreased, the highest value being observed with the 10 kDa-UFM.

Low Peptone Dose as Inductor of Alkaline Protease Promoter Used for Invertase Gene Expression in Yarrowia lipolytica

According literature, the induction of Yarrowia lipolytica alkaline protease promoter （PXPR2） is efficient in pH 〉 6.0 and with high peptone dose. To establish optimal pH and peptone concentration for induction of invertase gene （suc2 of Saccharomyces cerevisaie） under PXPR2 in new Y. lipolytica A-101 invertase positive （Suc＋） transformants their growth on Bioscreen C was analyzed. Minimal mineral medium with thiamine （MMT） and sucrose （1%）, adjusted to pH from 5.8 to 7.6 and supplemented by 0-0.1% of peptone was used. Biomass （OD）, maximal specific growth rate （μmax） and consumed sucrose were measured. Maximal yeasts growth, resulting from the optimal PXPR2 induction, was observed at pH 7.2 and with very low peptone doses （0.0025% and 0.01%）. For five clones （A-101 1356-5; A-101 B54-6; A-101 B57-4; A-101 A18 and W29 ura3-302） only 0.005% of peptone was needed. Amount of hydrolyzed sucrose varied from 24% to 83% and μmax from 0.06 to 0.28 hl. Suc^＋ clones differ in growth parameters, so the site of yeast cassette integration into genome influences expression level of suc2 under PXPR2. Designing large scale processes with Y. lipolytica Suc^＋ clones peptone concentration has to be 100 times smaller than recommended so far.

Production of Invertases by Anamorphic(Aspergillus nidulans)and Teleomorphic(Emericela nidulans)Fungi under Submerged Fermentation Using Rye Flour as Carbon Source

The production of invertases by anamorph (A. nidulans) and teleomorph (E. nidulans) was investigated. The best level of extracellular enzymatic production for anomorph was obtained in Khanna medium containing sucrose as carbon source, whereas for teleomorph the best production was archived using M5 medium containing inulin as carbon source. Despite this, rye flour was selected as carbon source. The extracellular enzyme production was higher for teleomorph than that observed for anomorph for all carbon sources used. The enzyme production was inhibited by the addition of fructose and glucose in the medium containing rye flour as carbon source. The best conditions to recover the higher enzymatic activity were temperature of 54℃ - 62℃ and pH of 4.8 5.6 for both enzymes determined by experimental design (CCRD). The stability of the temperatures at 40℃ and 50℃were similar for both enzymes. The invertases from the anomorph and teleomorph were activated by Mn2+, but the response of each one towards the presence of this cation was different with best activation observed for the anomorph enzyme (+80%). The extracellular enzymes were able to hydrolyze inulin, sucrose and raffinose. However, the affinity was higher for sucrose than inulin. In conclusion, the carbon source assimilation and the invertase production, as well as the enzymes properties, were different for the anomorph and teleomorph mycelia.

Effects of Straw Processing Methods and Irrigation Sources on Enzymatic Activity of Soils under Winter Wheat

[Objective] The aim was to study on effects of "straw returning and ir-returning" and "irrigation with ground water and water in the Yellow River" on changes of enzyme activity in soils under wheat at different developmental stages. [Method] Jimai 22, a kind of winter wheat, was made use of in fields to study on effects of "straw returning and ir-returning" and "irrigation with ground water and water in the Yellow River" on changes of enzyme activity in soils under wheat in different developmental stages. [Result] With advancement of developmental stage, urease activity of wheat in the four groups all showed the trend of "increasing-decreasing-increasing" and activities of invertase and phosphatase both changed from increasing to decreasing. In addition, urease activities of soils in wheat roots were improved by straw returning in four developmental stages. Meanwhile, activity of soil enzyme was better promoted by irrigation with ground water than with water in the Yellow River, except in grain-filling stage. Before developmental stage, different processing methods had a significant effect on phosphatase activity, for example, straw returning and ground water significantly enhanced activities of two kinds of phosphatase and promoted P absorption and transferring by plants and microorganisms in jointing stage; activity of acid phosphatase was higher in the group where irrigation with ground water and straw returning were adopted than those in the rest three groups in booting stage. [Conclusion] The research laid a foundation for dynamic relationship among activity of soil enzyme, crop growth and microorganisms.

Responses of soil enzymes to long-term CO_2 enrichment in forest ecosystems of Changbai Mountains

A study was conducted to determine the responses of soil enzymes （invertase, polyphenol oxidase, catalase, and dehydrogenase） to long-term CO2 enrichment at the Research Station of Changbai Mountain Forest Ecosystems, Chinese Academy of Sciences （42°24＇N, 128°28＇E; 738 m in elevation） in the northeast China during 1999-2006. Three treatments of the CO2 enrichment, designed as 500 μmol·mol-1 CO2 open-top chamber （OTC）, ambient control chamber and unchambered field （approx. 370 μmol·mol^-1CO2）, were conducted with Pinus koraiensis and Pinus sylvestriformis tree species. Soil sampling was made and analyzed separately in spring, summer and autumn in 2006 after the soil enzymes were exposed to elevated CO2 concentration （500 μmol·mol^-1） for eight growing seasons. Results showed that, at elevated CO2 concentration （500 μmol·mol^-1）, the activities of invertase （except for the summer samples of P. koraiensis） presented a remarkable decline in all growing seasons, while the activities of dehydrogenase had an increase but only part of the results was remarkable; the activities of polyphenol oxidase in P. sylvestriformis rhizosphere showed a remarkable decrease; the catalase activities increased in spring, while in turn were decline in other seasons. This study also revealed that the soil enzyme activities are significantly correlated with the tree species under the CO2 enhancement.

Soil Enzyme Activity Changes in Different-Aged Spruce Forests of the Eastern Qinghai-Tibetan Plateau

Activities of selected soil enzymes (invertase, acid phosphatase, proteinase,catalase, peroxidase and polyphenoloxi-dase) were determined under different spruce forests withrestoration histories of 5, 13, 18, 23, 27 years and an old growth forest over 400 years old in theeastern Qinghai-Tibetan Plateau, China, and their possible use as indicators of ecosystems healthwere analyzed. Plots 10 X 10 m with 4 replications were established to investigate three hypotheses:soil enzyme activities a) would increase with the restoration process; b) would be greater insurface soils than at lower depths; and c) would be correlated to selected physicochemicalproperties. Results showed that as the forests developed after restoration, invertase and peroxidaseactivities usually increased up to the 23 year point. Also soil enzyme activities were associatedwith surface soils and decreased with depths, suggesting that in earlier restoration stages surfaceaddition of organic fertilizer to soils might be more effective than additions at depth. In the 0-20cm soil, there were significant correlations (P < 0.01 or < 0.05) between some soil enzymeactivities and some selected chemical properties. Therefore, temporal changes in enzyme activitiesshould be included as an indicator when evaluating sustainable forest management practices.

Effects of Variation in Activities of Starch-Sugar Metabolic Enzymes on Reducing Sugar Accumulation and Processing Quality of Potato Tubers

The experiment was designed, via storing potato tubers of cv. E-Potato1 and E-Potato3 indifferent temperatures, to explore the variation patterns of reducing sugar (RS) andtotal sugar (TS) contents and enzyme activities that are involved in the pathway ofstarch-sugar metabolism aiming at identifying the main factors that influence the chipcolor. The results showed that low temperature in storage was a main factor thataccelerated the accumulation of RS of the stored tubers and a very significant linearrelationship existed between RS content and chip color index (CCI) of the tubers. Furtheranalysis elucidated that when tubers stored at 4℃, the activities of ADP glucosepyrophosphorylase (AGPase), UDP glucose pyrophosphorylase (UGPase) and sucrose synthase(SuSy) were negatively exponential to the RS content significantly while that of acidinvertase and alkaline invertase was significantly linear to RS content. It suggestedthat these enzymes could play main roles in the cold sweetening of potato tubers throughregulating starch-sugar metabolism.

Stem Reserve Mobilization and Sink Activity in Wheat under Drought Conditions

The effect of water deficit on stem reserve mobilization and sink activity in wheat (Triticum aestivum L.) cultivars, viz., C306 (drought tolerant) and PBW343 (drought sensitive) was studied. Drought was maintained in pot raised plants by withholding irrigation at 95 days after sowing (DAS), i.e. just five days before the initiation of anthesis. Drought induced a significant reduction in mean biomass of all the internodes of sensitive cultivar as compared to those of tolerant one. Mobilized dry matter and mobilization efficiency were observed to be higher in the internodes of tolerant cultivar, both under control and stress conditions, which resulted in enhanced translocation of stem reserves to the grains. Water soluble carbohydrates (WSC), which mainly occur as fructans, were observed to be higher in the internodes of tolerant cultivar than those of sensitive one. When drought was applied, fructans were mobilized more effectively from the internodes of tolerant cultivar. A significantly higher sucrose synthase activity in the grains of tolerant cultivar, under drought conditions, increased the sink strength by unloading the assimilates in the sink, thereby increasing further mobilization of assimilates to the grains. Grains of sensitive cultivar attained maturity much earlier as compared to the tolerant one, both under control and stress conditions. The longer duration of grain maturation in tolerant cultivar supported enhanced mobilization of stem reserves, thus restricting heavy decrease in grain yield, under stress conditions, as compared to the sensitive cultivar. It may, therefore, be concluded that certain characteristics viz., enhanced capability of fructan storage, higher mobilization efficiency, stronger sink activity and longer duration of grain maturation might help the drought tolerant cultivar in coping the stress

Effect of High Temperature on Sucrose Content and Sucrose Cleaving Enzyme Activity in Rice Grain During the Filling Stage

Dynamic changes of sucrose, fructose, glucose contents and differences in activities of sucrose synthase, vacuolar invertase, and cell wall bound invertase in rice grain after flowering stage were studied under natural and high temperatures by using two japonica rice varieties Koshihikari and Sasanishiki. In rice grains, the sucrose synthase activity was higher than that of invertase, which was significantly correlated with starch accumulation rate, indicating that the sucrose synthase played an important role in sucrose degradation and starch synthesis. Under high temperature, the significant increase in grain sucrose content without any increase in fructose and glucose contents, suggested that the high temperature treatment enhanced sucrose accumulation, while diminished sucrose degradation in rice grains. Compared with the control plants, the decrease in activities of sucrose synthase, vacuolar invertase, and cell wall bound invertase with high temperature treated plants indicated that the deceleration of sucrose-degradation was related to the decrease in activities of sucrose synthase and invertase.

Late spring cold reduces grain number at various spike positions by regulating spike growth and assimilate distribution in winter wheat

Late spring cold(LSC) occurred in the reproductive period of wheat impairs spike and floret differentiation during the reproductive period,when young spikelets are very cold-sensitive.However,under LSC,the responses of wheat spikelets at various positions,leaves,and stems and the interactions between them at physiological levels remain unclear.In the present study,two-year treatments at terminal spikelet stage under two temperatures(2 C,-2 C) and durations(1,2,and 3 days) were imposed in an artificial climate chamber to compare the effects of LSC on grain number and yield in the wheat cultivars Yannong 19(YN19,cold-tolerant) and Xinmai 26(XM26,cold-sensitive).The night temperature regimes were designed to reproduce natural temperature variation.LSC delayed plant growth and inhibited spike and floret differentiation,leading to high yield losses in both cultivars.LSC reduced dry matter accumulation(DMA,g) in spikes,stems,and leaves,reducing the DMA ratios of the spike to leaf and spike to stem.Plant cell wall invertase(CWINV) activity increased in upper and basal spikelets in YN19,whereas CWINV increased in middle spikelets in XM26.Under LSC,soluble sugar and glucose were transported and distributed mainly in upper and basal spikelets for glume and rachis development,so that spike development was relatively complete in YN19,whereas the upper and basal spikelets were severely damaged and most of the glumes in middle spikelets were relatively completely developed in XM26,resulting in pollen abortion mainly in upper and basal spikelets.The development of glumes and rachides was influenced and grain number per spike was decreased after LSC,with kernels present mainly in middle spikelets.Overall,reduced total DMA and dry matter partitioning to spikes under LSC results in poor spikelet development,leading to high losses of grain yield.

Changes of Soil Enzyme Activities in Different Restoration Ages of Spruce Forests on the Eastern Qinghai-Tibet Plateau

Six soil enzymes (invertase, acid phosphatase, proteinase, catalase,peroxidase and polyphenoloxidase) were chosen for investigation under different spruce forests withrestoration ages of 4, 10, 16 years and an old-growth spruce forest over 400 yearsold in the easternQinghai-Tibet Plateau, China. Results showed that the activities of invertase, phosphatase,proteinase, catalase and peroxidase decreased in newly restored forests except forpholyphenoloxidase. With the development of forests after restoration, the activities of invertase,acid phosphadase, proteinase increased gradually. Our study also indicated that the soil enzymeactivities were associated with surface soils and decreased with depths. This result suggested thatin the earlier restoration stage the application of organic fertilizer may be more effective bysurface addition to soils than deep addition.

Variations in Carbohydrate Content and Sucrose-Metabolizing Enzymes in Tomato (<i>Solanum lycopersicum</i>L.) Stamen Parts during Pollen Maturation

The formation of mature and fertile pollen grains, taking place inside the anther, depends on supply of assimilates, in the form of sucrose, provided mainly by the leaves. Data is limited, however, with respect to the understanding of sucrose metabolism in microspores and the supporting tissues. The aims of the present work were to 1) follow the changes in total and relative concentrations of sucrose, glucose, fructose and starch in the stamen parts and microspores up until anthesis, 2) follow the activities of sucrose-metabolism-related enzymes, in the anther walls fraction and microspores of the crop plant tomato. Sucrose was found to be partially cleaved in the filament, decreasing by more than twofold in the anther wall layers and the locular fluid, and to accumulate in the mature pollen grains, constituting 80% of total soluble sugars. Thus, sucrose was both the starting sugar, supporting microspore development, and the main carbohydrate accumulated at the end of the pollen-development program. The major invertase found to be active in both the anther wall layers and in maturing microspores was cell-wall-bound invertase. High fructokinase 2 and sucrose phosphate synthase activities during pollen maturation coincided with sucrose accumulation. The potential importance of sucrose accumulation during pollen dehydration phase and germination is discussed.