Invertase Isoenzymes Research Articles

Screening of Barley Resistance Against Powdery Mildew by Simultaneous High-Throughput Enzyme Activity Signature Profiling and Multispectral Imaging.

Molecular marker analysis allow for a rapid and advanced pre-selection and resistance screenings in plant breeding processes. During the phenotyping process, optical sensors have proved their potential to determine and assess the function of the genotype of the breeding material. Thereby, biomarkers for specific disease resistance traits provide valuable information for calibrating optical sensor approaches during early plant-pathogen interactions. In this context, the combination of physiological, metabolic phenotyping and phenomic profiles could establish efficient identification and quantification of relevant genotypes within breeding processes. Experiments were conducted with near-isogenic lines of H. vulgare (susceptible, mildew locus o (mlo) and Mildew locus a (Mla) resistant). Multispectral imaging of barley plants was daily conducted 0–8 days after inoculation (dai) in a high-throughput facility with 10 wavelength bands from 400 to 1,000 nm. In parallel, the temporal dynamics of the activities of invertase isoenzymes, as key sink specific enzymes that irreversibly cleave the transport sugar sucrose into the hexose monomers, were profiled in a semi high-throughput approach. The activities of cell wall, cytosolic and vacuole invertase revealed specific dynamics of the activity signatures for susceptible genotypes and genotypes with mlo and Mla based resistances 0–120 hours after inoculation (hai). These patterns could be used to differentiate between interaction types and revealed an early influence of Blumeria graminis f.sp. hordei (Bgh) conidia on the specific invertase activity already 0.5 hai. During this early powdery mildew pathogenesis, the reflectance intensity increased in the blue bands and at 690 nm. The Mla resistant plants showed an increased reflectance at 680 and 710 nm and a decreased reflectance in the near infrared bands from 3 dai. Applying a Support Vector Machine classification as a supervised machine learning approach, the pixelwise identification and quantification of powdery mildew diseased barley tissue and hypersensitive response spots were established. This enables an automatic identification of the barley-powdery mildew interaction. The study established a proof-of-concept for plant resistance phenotyping with multispectral imaging in high-throughput. The combination of invertase analysis and multispectral imaging showed to be a complementing validation system. This will provide a deeper understanding of optical data and its implementation into disease resistance screening.

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.

Copper Affects the Cotyledonary Carbohydrate Status During the Germination of Bean Seed

Seeds of bean (Phaseolus vulgaris L.) were germinated by soaking in distilled water or copper chloride solution. The relationships among copper excess treatment, germination rate, dry weight, sugar contents, and carbohydrase activities in cotyledon were investigated. Heavy metal stress provoked a diminution in germination rate and biomass mobilization, as compared with the control. A drastic disorder in soluble sugars export, especially glucose and fructose liberation, was also imposed after exposure to excess copper. This restricted the starch and sucrose breakdown in reserve tissue, as evidenced by the inhibition in the activities of alpha-amylase and invertase isoenzymes (soluble acid, soluble neutral, cell wall-bound acid).

Respiratory metabolism in the embryonic axis of germinating pea seed exposed to cadmium

Seeds of pea (Pisum sativum L.) were germinated for 5d by soaking in distilled water or 5mM cadmium nitrate. The relationships among cadmium stress, germination rate, changes in respiratory enzyme activities and carbohydrates mobilization were studied. Two cell fractions were obtained from embryonic axis: (1) mitochondria, used to determine enzyme activities of citric acid cycle and electron transport chain, and (2) soluble, to measure some enzyme activities involved in fermentation and pentose phosphate pathway. Activities of malate- and succinate-dehydrogenases (MDH, SDH) and NADH- and succinate-cytochrome c reductases (NCCR, SCCR) were rapidly inhibited, while cytochrome c oxidase (CCO) was unaltered by cadmium treatment. However, this stimulated the NADPH-generating enzyme activities of the pentose phosphate pathway, glucose-6-phosphate- and 6-phosphogluconate-dehydrogenases (G6PDH, 6PGDH), as well as enzyme activity of fermentation, alcohol dehydrogenase (ADH), with concomitant inhibition in the capacity of enzyme inactivator (INADH). Moreover, Cd restricted carbohydrate mobilization in the embryonic axis. Almost no glucose and less than 7% of control fructose and total soluble sugars were available in the embryo tissues after 5d of exposure to cadmium. Cotyledonary invertase isoenzyme activity was also inhibited by Cd. The results indicate that cadmium induces disorder in the resumption of respiration in germinating pea seeds. The contribution of Cd-stimulated alternative metabolic pathways to compensate for the failure in mitochondrial respiration is discussed in relation to the delay in seed germination and embryonic axis growth.

Internodal soluble invertase isoenzymes in relation to sucrose storage and maturity behaviour in sugarcane cultivars

Changes in activities of internodal soluble invertases in relation to sucrose storage in two sugarcane cultivars viz. Co 89003 (early maturing) and Co 1148 (late maturing) have been studied at different physiological stages of cane development. The higher rate of decline in the activity of soluble acid invertase with ageing of the cane coupled with rapid rise in sucrose/reducing sugars ratio indicated better sink strength and higher sucrose storage in Co 89003. Six isoenzymes of soluble acid invertase in Co 89003 and five isoforms of this enzyme in Co 1148 were identified at internodal elongation stage. However, no neutral invertase isoenzyme was detected in either cultivar at this stage. At maturity stage, three isoenzymes of neutral and four isoenzymes of acid invertase were identified in Co 89003 whereas five isoenzymes of neutral and two isoenzymes of acid invertase were identified in Co 1148. These internodal soluble invertase isoenzymes differed with respect to their catalytic efficiencies for sucrose mobilization in storage tissue. Relative to Co 1148, soluble acid invertase isoenzymes of Co 89003 were less efficient catalytically because of their low Vmax/Km values. Marked differences were also observed in the inhibitory action of Mn++ on various isoenzymes of invertase.

Infection with virulent and avirulent P. syringae strains differentially affects photosynthesis and sink metabolism in Arabidopsis leaves

Infection of plants with pathogens leads not only to the induction of defence reactions but also to changes in carbohydrate metabolism. In this study, the effects of infection by a virulent and an avirulent strain of P. syringae on spatio-temporal changes in photosynthesis were compared using chlorophyll fluorescence imaging. The maximum PSII quantum yield, effective PSII quantum yield and nonphotochemical quenching were decreased in Arabidopsis leaves infected with either strain. At the same time, the quantum yield of nonregulated energy dissipation was increased. These changes could be detected by chlorophyll fluorescence imaging before symptoms were visible by eye. The effects were restricted to the vicinity of the infection site and did not spread to uninfected areas of the leaf. Qualitatively similar changes in photosynthetic parameters were observed in both interactions. Major differences between the responses to both strains were evident in the onset and time course of changes. A decrease in photosynthesis was detectable already at 3 h only after challenge with the avirulent strain while after 48 h the rate of photosynthesis was lower with the virulent strain. In contrast to photosynthesis, the regulation of marker genes for source/sink relations and the activities of invertase isoenzymes showed qualitative differences between both interactions. Inoculation of the virulent but not the avirulent strain resulted in downregulation of photosynthetic genes and upregulation of vacuolar invertases. The activity of vacuolar invertases transiently increased upon infection with the virulent strain but decreased with the avirulent strain while extracellular invertase activity was downregulated in both interactions.

Gibberellin-dependent induction of tomato extracellular invertase Lin7 is required for pollen development.

The tomato extracellular invertase family comprises four members with different expression patterns. Among the three invertase isoenzymes expressed in floral tissues, Lin5, Lin6 and Lin7, the expression of Lin7 was previously shown to be restricted to the tapetum and pollen. Histochemical analysis of β-glucuronidase (GUS) reporter activity shows Lin7 expression in pollen and pollen tubes of corresponding transgenic plants. The physiological relevance of the identification of gibberellin-responsive cis-acting elements for induction of the Lin7 promoter is supported by the repression of Lin7 expression in pollen grains by the gibberellin biosynthesis inhibitor paclobutrazol. Functional approaches with transgenic tomato plants establish a link between gibberellin action and invertase function in the tapetum for pollen development: both tissue-specific antisense repression of extracellular Lin7 and ectopic inactivation of the biologically active GAs by expression of a GA2-oxidase under control of the Lin7 promoter result in germination deficient pollen. These complementary findings support the idea that the GA requirement of pollen development, pollen germination and pollen tube growth are linked to energy metabolism via the regulation of an extracellular invertase as a key enzyme for carbohydrate supply via an apoplasmic pathway.

Tissue-specific induction of the mRNA for an extracellular invertase isoenzyme of tomato by brassinosteroids suggests a role for steroid hormones in assimilate partitioning.

Brassinosteroids (BRs) induce various growth responses when applied exogenously to plant tissues, and the analysis of biosynthetic mutants reveals an essential role for plant growth and development. Only a few BR-regulated genes have been identified so far, and the corresponding gene products are assumed to be involved in cell elongation. The present study shows that BR growth responses are linked to the regulation of carbohydrate metabolism by induction of the mRNA for the key enzyme of an apoplastic phloem-unloading pathway. Addition of BRs to autotrophic tomato suspension culture cells specifically elevates the activity of cell-wall-bound invertase, whereas the intracellular invertase activities were not affected. This enhanced enzyme activity was shown to correlate with the induction of the mRNA of extracellular invertase Lin6, whereas the mRNA levels of the other three extracellular invertase isoenzymes were not affected. The induction level induced by different BRs correlates with their growth-promoting activity. The physiological significance of this regulation is further supported by the low concentrations and short incubation times required to induce Lin6 mRNA. This regulatory mechanism results in an elevated uptake of sucrose via the hexose monomers, and thus an increased supply of carbohydrates to the BR-treated cells. Experiments with tomato seedlings showed that the localized BR-dependent growth response of the hypocotyl elongation zone was accompanied by a specific induction of Lin6 mRNA that is restricted to the corresponding tissues. This study demonstrates a role of BRs in tissue-specific source/sink regulation.

Invertases and life beyond sucrose cleavage

In most species, the long distance transport of assimilates is in the form of sucrose. This process is driven by a concentration gradient between source and sink tissues. Sucrose cleavage steepens the gradient; therefore sucrolytic enzymes are thought to contribute to carbohydrate partitioning. Invertases catalyse the irreversible hydrolysis of sucrose. The different invertase isoenzymes can be distinguished on the basis of their intracellular location, isoelectric points and pH optima. Our current understanding of the diverse roles of invertases was discussed recently at the first meeting**Workshop: The Role of Invertases in Carbohydrate Partitioning and Beyond, University of Regensburg, Germany, 3–6 October 1999. to be devoted entirely to this topic.

Regulation and function of extracellular invertase from higher plants in relation to assimilate partitioning, stress responses and sugar signalling

This paper originates from a presentation at the International Conference on Assimilate Transport and Partitioning, Newcastle, NSW, August 1999 Carbohydrates are synthesised in photosynthetically active source tissues and exported, in most species in the form of sucrose, to photosynthetically less active or inactive sink tissues. Sucrose hydrolysis at the site of utilisation contributes to phloem unloading. This phenomenon links sink metabolism with phloem transport to, and partitioning between, sinks. Invertases catalyse the irreversible hydrolysis of sucrose and thus are expected to contribute to carbohydrate partitioning. Different invertase isoenzymes may be distinguished based on their intracellular location, their isoelectric points and pH optima. Extracellular, cell-wall-bound invertase is uniquely positioned to supply carbohydrates to sink tissues via an apoplasmic pathway, and links the transport sugar sucrose to hexose transporters. A number of studies demonstrate an essential function of this invertase isoenzyme for phloem unloading, carbohydrate partitioning and growth of sink tissues. Extracellular invertases were shown to be specifically expressed under conditions that require a high carbohydrate supply to sink tissues. Further, their expression is upregulated by a number of stimuli that affect source–sink relations. Substrate and reaction products of invertases are not only nutri-ents, but also signal molecules. Like hormones and in combination with hormones and other stimuli, they can regu-late many aspects of plant development from gene expression to long-distance nutrient allocation. Based on studies in Chenopodium rubrum, tomato (Lycopersicon esculentum) and tobacco (Nicotiana tabacum), the regulation of extracellular invertase and its function in assimilate partitioning, defence reactions and sugar signal transduction pathways are discussed.

The different pH optima and substrate specificities of extracellular and vacuolar invertases from plants are determined by a single amino-acid substitution.

Different plant invertase isoenzymes are characterized by a single amino-acid difference in a conserved sequence, the WEC-P/V-D box. A proline residue is present in this sequence motif of extracellular invertase sequences, whereas a valine is found at the same position of vacuolar invertase sequences. The role of this distinct difference was studied by substituting the proline residue of extracellular invertase CIN1 from Chenopodium rubrum with a valine residue, by site-directed mutagenesis. The mutated gene was heterologously expressed in an invertase-deficient Saccharomyces cerevisiae strain. The single amino-acid difference was shown to be the molecular basis for two enzymatic properties of invertase isoenzymes, for both the pH optimum and the substrate specificity. A proline in the WEC-P/V-D box determines the more acidic pH optimum and the higher cleavage rate of raffinose of extracellular invertases, compared to vacuolar invertases that have a valine residue at this position.

Regulation and tissue-specific distribution of mRNAs for three extracellular invertase isoenzymes of tomato suggests an important function in establishing and maintaining sink metabolism.

The aim of the present study was to gain insight into the contribution of extracellular invertases for sink metabolism in tomato (Lycopersicon esculentum L.). The present study shows that extracellular invertase isoenzymes are encoded by a gene family comprising four members: Lin5, Lin6, Lin7, and Lin8. The regulation of mRNA levels by internal and external signals and the distribution in sink and source tissues has been determined and compared with mRNA levels of the intracellular sucrose (Suc)-cleaving enzymes Suc synthase and vacuolar invertase. The specific regulation of Lin5, Lin6, and Lin7 suggests an important function of apoplastic cleavage of Suc by cell wall-bound invertase in establishing and maintaining sink metabolism. Lin6 is expressed under conditions that require a high carbohydrate supply. The corresponding mRNA shows a sink tissue-specific distribution and the concentration is elevated by stress-related stimuli, by the growth-promoting phytohormone zeatin, and in response to the induction of heterotrophic metabolism. The expression of Lin5 and Lin7 in gynoecia and stamens, respectively, suggests an important function in supplying carbohydrates to these flower organs, whereas the Lin7 mRNA was found to be present exclusively in this specific sink organ.

Differential effect of D-glucose on the level of mRNAs for three invertase isoenzymes of Chenopodium rubrum

The present study shows that invertase isoenzymes of Chenopodium rubrum are encoded by a small gene family comprising at least three members. In addition to the previously described cDNA CIN1 , coding for an extracellular invertase, two further invertase cDNA sequences, CIN2 and CIN3 , have been cloned and characterised from Chenopodium rubrum. CIN2 encodes a putative acidic, vacuolar invertase, whereas the protein encoded by CIN3 has a unique combination of properties from both extracellular and vacuolar invertases cloned so far and thus represents a novel type of invertase isoenzyme. The effect of source-sink transition on the transcript levels of the different invertases has been investigated in suspension-culture cells. The mRNAs for the three isoenzymes are present in photoautotrophically grown suspension culture cells and are differentially regulated in response to induction of heterotrophic metabolism. Whereas the mRNA concentration of CIN1 is induced by the addition of glucose, as shown before, the low transcript levels of both CIN2 and CIN3 are not affected. The physiological significance of the differential regulation pattern is discussed.

Acid invertase in Nicotiana tabacum crown-gall cells: Molecular properties of the cell-wall isoform

Cell-wall invertase (CWI; EC 3.2.1.26) was salt-eluted from non-disrupted Agrobacterium tumefaciens-transformed Nicotiana tabacum L. cells and purified to homogeneity. More than 90% of total cellular invertase activity (measured at pH 4.8) was recovered in the NaCl-eluted fraction whereas for the cytoplasmic marker glucose-6-phosphate dehydrogenase 96% of total activity could be extracted from the tissue after salt-elution, indicating absence of appreciable stress-induced cell disruption. Likewise, appreciable contamination of CWI with vacuolar acid invertase could be excluded. Tobacco CWI cross-reacted with an antiserum directed against deglycosylated carrot CWI; however, during some purification steps CWI enzyme activity did not correlate with CWI immunosignal. In fractions of low CWI activity and strong immunosignal, a putative inhibitor peptide with an apparent Mr of 17 kDa was detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining (Weil et al. 1994, Planta 193, 438–445). The CWI of transformed tobacco cells has an apparent Mr of 69 kDa (SDS-PAGE) and is a basic (pI 9.5) glycoprotein. Gel-permeation chromatography indicated that enzymatically active CWI is a monomer. Deglycosylation of the denatured CWI by treatment with endo-β-N-acetylglucosaminidase, peptide-N-glycosidase F and trifluoromethanesulfonic acid indicated the presence of two high-mannose and two complex glycans. In partially purified CWI fractions the carrot CWI antiserum cross-reacted with one other tobacco cell-wall peptide (Mr 28 kDa). To address the possibility of a second invertase isoenzyme cross-reacting with the carrot antiserum, intact CWI and the 28-kDa peptide were digested in vitro with Staphylococcus aureus V8 protease and cyanogen bromide. A comparison of the resulting peptide patterns identified the 28-kDa polypeptide as a cleavage product of CWI. Running electroeluted CWI (69 kDa) on a second SDS-polyacrylamide gel led to substantial formation of the 28-kDa peptide. This suggests that the intrinsic 28-kDa cleavage product is the result of an intrinsic lability of tobacco CWI, rather than being a proteolytic degradation product.

CDNA cloning and expression of a potato (Solanum tuberosum) invertase

A cDNA clone encoding an invertase isoenzyme has been isolated from a potato leaf cDNA library. The deduced amino acid sequence shows significant similarities to previously characterised invertases. The highest degree of overall similarity, including the signal peptide sequence, is to carrot cell wall invertase, suggesting that the potato gene encodes an apoplastic enzyme. Expression of the gene, as determined by RT-PCR, is detected in stem and leaf tissue, and at lower levels in tuber, but is absent from roots.

Multiple forms of invertase from Daucus carota cell cultures

Abstract A 400-fold purification of two isoenzymes of acid invertase (pH optima 4.5) and a two- to three-fold purification of alkaline (pH optima 7.5) invertase from suspension cultured carrot cells was obtained using ion-exchange (DEAE cellulose) and affinity (concanavalin-A) column chromatography. SDS polyacrylamide gel electrophoresis resolved major polypeptide bands at 61 000 and 49000 corresponding to the two isoenzymes of acid invertase, designated acid- 1 and acid-2, respectively. The acid invertase isoenzymes, in contrast with alkaline invertase, are likely glycoproteins based on their ability to bind the lectin concanavalin-A.

Subcellular distribution of yeast invertase isoenzymes.

Homogenates from yeast cells contain 1% or less of sedimentable invertase activity. Sedimentability is equally low in homogenates from cells repressed or derepressed with regard to invertase secretion. Intracellulary, the mannanprotein form of invertase is largely localized in vacuoles whereas the small isoenzyme is largely present in the soluble cell fraction. These findings indicate that vesicles are not involved in the secretion of invertase. A soluble mode of invertase secretion is discussed.