Cytochemical methods for the localization of invertase activity in plant tissues

Two cytochemical methods for the localization of acid and alkaline invertases are given. The first is based upon the reduction of a silver complex at two different pH ranges, whilst the second is based upon the tetrazolium reaction and permits quantification of the rate of activity of alkaline invertase activity. The distribution of alkaline invertase activity throughout the root apex of Pisum sativum and the cell wall localization of acid invertase for material excised from tuber tissue of Helianthus tuberosus are both confirmed.     

Properties of Invertases in Sugar Storage Tissues of Citrus Fruit and Changes in Their Activities during Maturation

Both acid and alkaline invertases were present in immature juice sacs of satsuma mandarin (Citrus‘Unshu Marcovitch”) fruit, in which sugar content was low. Maturing and mature juice sacs, in which sugar content increased steadily with time, were characterized by the presence of alkaline invertase and the absence of acid invertase. When the immature juice sacs were homogenized with 0.2 M sodium phosphate-citrate buffer (pH 8.0), almost all of the acid invertase activity was found in the solubilized fraction, whereas almost all of the alkaline invertase activity was present in the insoluble fraction. The distribution of alkaline invertase between the solubilized and insoluble fractions changed with the development of fruit. The acid invertase had a molecular weight of 69,000, optimum pH of 4.8–5.3, and K_m value for sucrose of 7.3 mM. The alkaline invertase had a molecular weight of 200,000, pH optimum of 7.2–7.7, and K_m value of 35.7 mM. The hydrolysing activities of both enzymes for raffinose were considerably less than those for sucrose. The alkaline invertase had lower activity for raffinose than the acid invertase.     

Alkaline β-fructofuranosidases of tuberous roots: Possible physiological function

Summary Alkaline invertase of roots of carrot (Daucus carota L.) did not hydrolyze raffinose while the acid invertase from the same tissue showed with this sugar ca. 60% of the activity found with sucrose. The activity of the two invertases was inhibited by fructose to a different extent, the K _i value being ca. 4×10^–2 M and 3×10^–1M, respectively, for the alkaline and the acid invertases from the roots of both carrot and turnip (Brassica rapa L.). It is proposed that fructose inhibition of acid invertase is of no physiological significance but that, in contrast, hexoses might regulate the activity of alkaline invertase.Comparing several species and cultivars, it was found that the content of reducing sugars and the activity of alkaline invertase of mature tuberous roots showed a positive correlation. This indicates that alkaline invertase may participate in the regulation of the hexose level of the cell, as was previously suggested for sugar-cane. A scheme is presented which proposes a way of participation of alkaline invertase in such a regulation, assuming that this enzyme is located in the cytoplasm and acid invertase is membrane-bound and mainly located at the cell surface.     

Acid and alkaline invertases in roots and nodules of Lupinus angustifolius infected with Rhizobium lupini

Summary Both acid and alkaline invertase activity were found in tips and cortical tissue of Lupinus angustifolius L. roots infected with Rhizobium lupini NZP 2257. Only the alkaline invertase was detected in the nodule cytoplasm. Weak invertase activity found in the bacteroids was probably a contamination from plant invertase. The alkaline invertase activity in the nodule cytoplasm was 250 times that detected in the bacteroids and 8 times that detected in cortical tissue. No intracellular or extracellular invertase was detected in R. lupini cultured in liquid medium containing sucrose.     

Differences in invertase activity in embryogenic and non-embryogenic calli from Medicago arborea

The different invertase activities in embryogenic and non-embryogenic calli induced from explants (cotyledons, petioles, hypocotyls and leaves) obtained from Medicago arborea L. subsp. arborea seedlings were evaluated. Total invertase activity was lower in the calli with the greatest embryogenic capacity. The greatest fraction of this activity corresponded to soluble invertase. Wall-bound invertase showed maximum activity during the first two months of culture and the highest activities of this type were found in non-embryogenic calli. Extracellular invertase formed the smallest fraction of the total invertase activity evaluated. Acid and alkaline invertase activities were found in all calli but differences were detected between the embryogenic and non-embryogenic calli. In the former, the activity of both types of invertase exhibited a similar type of behaviour but different from that observed in the non-embryogenic calli. The calli with the greatest embryogenic capacity had very low levels of acid invertase and very high levels of the alkaline form. Soluble invertase – both acid and alkaline – accounted for the highest fraction after the first two months of culture and was present in lower amounts in the embryogenic than in the non-embryogenic calli. Regarding bound invertase, the highest production was seen to correspond to acid invertase. The extracellular invertase evaluated corresponded to the acid form since the alkaline extracellular invertase did not show any physiologically significant activity.     

Invertase activity in sinks undergoing cell expansion

During the development of roots, internodes and leaves, closely correlated changes occur in the rates of cell expansion, specific activities of acid invertase and concentrations of hexose sugars and sucrose. Rates of cell growth and acid invertase activities frequently exhibit closely coupled responses to environmental changes and to growth regulator treatments. The possibility is considered that, by controlling the availability of hexose substrates for cellular metabolism, acid invertase may regulate cell growth. Potential mechanisms regulating the in vivo activity of acid invertases are reviewed and attention is drawn to the need for more information on the sub-cellular localization of the enzyme.     

Changes in the forms of invertase during germination of mung bean seeds

High levels of ‘alkaline’ invertase activity occur in dormant mung bean seeds. During germination this activity decreases rapidly and is replaced by high ‘acid’ invertase activity. Cycloheximide prevented the formation of the latter activity and also inhibited germination. It is suggested that de novo synthesis of ‘acid’ invertase occurs during germination. Both enzymes bind to concanavalin A and, hence, are presumed to be glycoproteins. Affinity-purified enzyme samples show similar ratios of ‘acid’ and ‘alkaline’ invertase activities to the crude preparations indicating that specific enzyme inhibitors or activators are probably not involved in controlling the activities in vivo.     

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.     

Analysis of acid invertase and comparison with acid phosphatase in the ericoid mycorrhizal fungus Hymenoscyphus ericae (Read) Korf and Kernan

Fractions of acid invertase and acid phosphatase of the ericoid mycorrhizal fungus Hymenoscyphus ericae (Read) Korf & Kernan were compared by column chromatography and polyacrylamide gel electrophoresis. Acid invertase levels were measured during the exponential phase after 14 days growth in pure culture. Most acid invertase was wall associated (50%) with 41% forming an extracellular fraction and 9% a soluble, cytoplasmic fraction. The wall-bound fraction was partially solubilized by 1 M NaCl, bulked with the extracellular fraction and separated by gel filtration into two acid invertase activity peaks. These peaks corresponded closely to two acid phosphatase activity peaks measured in the same eluates. Anion exchange chromatography under a continuous salt gradient separated the invertase and phosphatase isoforms from each other. Non-denaturing polyacrylamide gel electrophoresis demonstrated that the more active isoforms of each enzyme have different electrophoretic properties and are high mannose-type glycoproteins with a high affinity for the lectin, concanavalin A. The results are discussed in terms of the functional aspects of the two enzymes and their cytochemical localization.     

Wall-bound Invertase Activity in Convolvulus Callus: Increase after Subculturing, and Paradoxical Effects of Actinomycin D, Cycloheximide, and Thienylalanine

The wall-bound invertase activity increased 3.3-fold upon transfer of fragments of Convolvulus callus to fresh solid nutrient medium and 7.7-fold upon transfer to liquid nutrient medium. Addition of actinomycin D, cycloheximide or the amino acid analogue thienylalanine brought about a further stimulation of the invertase content of the cell walls. The rise of wall-bound invertase activity was not due to redistribution of invertase activity between cytoplasm and cell walls, and appeared to be dependent on metabolic energy. An equation is presented to calculate the half-life of enzymes from their time-course. Applied on the time-courses of wall-bound invertase activity, a half-life of about 12 h was obtained in callus transferred to fresh solid medium and of about 5.4 h in tissue transferred to liquid medium. It is argued that the increase of invertase content of the cell walls is due to an enhanced rate of invertase synthesis.     

Chilling Tolerance of Potato Plants Transformed with a Yeast-Derived Invertase Gene under the Control of the B33 Patatin Promoter

Tolerance to chilling was compared under in vitro conditions in potato plants (Solanum tuberosum L., cv. Désirée) transformed with a yeast-derived invertase gene under the control of the B33 class 1 tuber-specific promoter (the B33-inv plants) and potato plants transformed only with a reporter gene (the control plants). The expression of the inserted yeast invertase gene was proved by following the acid and alkaline invertase activities and sugar contents in the leaves under the optimum temperature (22°C). The total activities of acid and alkaline invertases in the B33-inv plants exceeded those in the control plants by the factors of 2–3 and 1.3, respectively. In the B33-inv plants, the activity of acid invertase twice exceeded that of the alkaline invertase, whereas the difference equaled 12% in the control plants. The contents of sucrose and glucose increased in the B33-inv plants by 21 and 13%, respectively, as compared to the control. Chilling at +3 and –1°C for 1, 3, and 6 h did not affect the rate of lipid peroxidation, as measured by the content of malonic dialdehyde (MDA) in the leaves of the genotypes under study. Only the longer exposures (24 h at +3 and –1°C and 7 days at +5°C) produced a significant decline in the MDA content in the B33-invplants, as compared to the control. Following short freezing (20 min at –9°C), the content of MDA increased by 50% in the leaves of the control plants, while in the B33-inv plants, cold-treated and control plants did not differ in the MDA content. The authors presume that the potato plants transformed with the yeast invertase gene acquire a higher tolerance to low temperatures as compared to the control plants, apparently due to the changes in sugar ratio produced by the foreign invertase.     

Change in invertase activity of sweet potato in response to wounding and purification and properties of its invertases

When root tissue of sweet potato (Ipomoea batatas Lam.) was sliced, acid invertase activity, initially absent in freshly sliced tissue, appeared after a 3- to 6-hour lag phase, rapidly reached a maximum in 18 hours, and thereafter decreased. The increase in invertase activity was accompanied by a decrease in sucrose content of the root tissue. Alkaline invertase activity was present in fresh root tissue, but changed little after wounding. Acid invertase in wounded tissue and alkaline invertase in fresh tissue were purified and their properties were investigated. The acid invertase was a ß-fructofuranosidase and was unaffected by substrate or by any of the cations and several metabolites. The alkaline invertase was more specific for sucrose, was inhibited by glucose and glucose 6-phosphate, and displayed non-Michaelis-Menten kinetics.     

Isolation and characterization of a cDNA clone from Lolium temulentum L. encoding for a sucrose hydrolytic enzyme which shows alkaline/neutral invertase activity

A novel cDNA clone, functionally expressed in E. coli, was isolated from a L. temulentum L. cDNA library. The expressed protein hydrolysed sucrose with an apparent Km of approximately 18 mM, and produced equi-molar concentrations of glucose and fructose. Optimum activity was observed at pH 7-7.5; there was little or no activity at pH 5.5. The expressed protein did not hydrolyse raffinose, stachyose or maltose. The activity of the expressed protein was inhibited by fructose (50% at 15 mM) and TRIS (50% at 2.5 mM), but was not affected by MgCl2, CaCl2 or MnCl2. These findings suggest that this cDNA clone encodes for an alkaline/neutral invertase. Sequence analysis revealed little homology with published sequences for acid invertase, however the invertase motif (NDPN) identified in other invertases was present. Expression studies show that the gene encoding for this enzyme is not regulated by sucrose accumulation in leaf tissue.     

Effect of GA3, kinetin and indole acetic acid on carbohydrate metabolism in chickpea seedlings germinating under water stress

Enhanced amylase activity was observed during a 7-day-growth period in the cotyledons of PEG imposed water stressed chickpea seedlings grown in the presence of GA₃ and kinetin, when compared with stressed seedlings. During the first 5 days of seedling growth, the seedlings growing under water deficit conditions as well as those growing in the presence of PGRs had a higher amylase activity in shoots than that of control seedlings. Neither GA₃ nor kinetin increased the amylase activity of roots whereas IAA reduced root amylase activity. Activity of acid and alkaline invertases was maximum in shoots and at a minimum in cotyledons. Compared with alkaline invertase, acid invertase activity was higher in all the tissues. The reduced acid and alkaline invertase activities in shoots of stressed seedlings were enhanced by GA₃ and kinetin. Roots of stressed seedlings had higher alkaline invertase activity and GA₃ and IAA helped in bringing the level near to those in the controls. GA₃ and kinetin increased the sucrose synthase (SS) and sucrose phosphate synthase (SPS) activities in cotyledons of stressed seedlings, whereas they brought the elevated level of SPS of stressed roots to near normal level. The higher level of reducing sugars in the shoots of GA₃ and kinetin treated stressed seedlings could be due to the high acid invertase activity observed in the shoots, and the high level of bound fructose in the cotyledons of stressed seedlings could be due to the high activity of SPS in this tissue.     

Sucrose mobilization in relation to essential oil biogenesis during palmarosa (<Emphasis Type="Italic">Cymbopogon martinii</Emphasis> Roxb. Wats. var.<Emphasis Type="Italic">motia</Emphasis>) inflorescence development

Palmarosa inflorescence with partially opened spikelets is biogenetically active to incorporate [U-¹⁴C]sucrose into essential oil. The percent distribution of¹⁴C-radioactivity incorporated into geranyl acetate was relatively higher as compared to that in geraniol, the major essential oil constituent of palmarosa. At the partially opened spikelet stage, more of the geraniol synthesized was acetylated to form geranyl acetate, suggesting that majority of the newly synthesized geraniol undergoes acetylation, thus producing more geranyl acetate.In vitro development of palmarosa inflorescence, fed with [U-¹⁴C]sucrose, resulted in a substantial reduction in percent label from geranyl acetate with a corresponding increase in free geraniol, thereby suggesting the role of an esterase in the production of geraniol from geranyl acetate. At time course measurement of¹⁴CO₂ incorporation into geraniol and geranyl acetate substantiated this observation. Soluble acid invertase was the major enzyme involved in the sucrose breakdown throughout the inflorescence development. The activities of cell wall bound acid invertase, alkaline invertase and sucrose synthase were relatively lower as compared to the soluble acid invertase. Sucrose to reducing sugars ratio decreased till fully opened spikelets stage, concomitant with increased acid invertase activity and higher metabolic activity. The phenomenon of essential oil biosynthesis has been discussed in relation to changes in these physiological parameters.     

The naturally occurring silent invertase structural gene suc2 zero contains an amber stop codon that is occasionally read through

Summary The yeast invertase structural gene SUC2 has two naturally occurring alleles, the active one and a silent allele called suc2°. Strains carrying suc2° are unable to ferment sucrose and do not show detectable invertase activity. We have isolated suc2° and found an amber codon at position 232 of 532 amino acids. However, transformants carrying suc2° on a multicopy plasmid were able to ferment sucrose and showed detectable invertase activity. Full-length invertase was found in gels stained for active invertase and in immunoblots. Therefore we concluded that the amber codon is occasionally read as an amino acid. The calculated frequency of read-through is about 4% of all translation events.     

Activities of sucrolytic enzymes in developing pods of lentil genotypes differing in seed size

The activities of sucrolytic enzymes viz. sucrose synthase and invertases were compared in developing pods of two genotypes of lentil differing in seed weight. Biomass accumulation of both the podwall and seed of ‘large’ genotype was higher during development as compared to the ‘small’ genotype. High activity of acid invertase together with prolonged activity of alkaline invertase in podwall of ‘large’ genotype may lead to longer cell division phase resulting in its larger size and biomass. Greater biomass of podwall could be responsible for providing more reserves for the developing seed hence determining its size. Higher alkaline invertase activity in ‘large’ seed from 15–20 DAF can be correlated to the sustained sucrolytic conditions for producing more cells required for its larger size. Increased levels of sucrose synthase in ‘large’ seed especially during maturation phase suggest the role of this enzyme in enhancing the seed sink strength.     

Genome-Wide Identification of the Invertase Gene Family in Populus

Invertase plays a crucial role in carbohydrate partitioning and plant development as it catalyses the irreversible hydrolysis of sucrose into glucose and fructose. The invertase family in plants is composed of two sub-families: acid invertases, which are targeted to the cell wall and vacuole; and neutral/alkaline invertases, which function in the cytosol. In this study, 5 cell wall invertase genes (PtCWINV1-5), 3 vacuolar invertase genes (PtVINV1-3) and 16 neutral/alkaline invertase genes (PtNINV1-16) were identified in the Populus genome and found to be distributed on 14 chromosomes. A comprehensive analysis of poplar invertase genes was performed, including structures, chromosome location, phylogeny, evolutionary pattern and expression profiles. Phylogenetic analysis indicated that the two sub-families were both divided into two clades. Segmental duplication is contributed to neutral/alkaline sub-family expansion. Furthermore, the Populus invertase genes displayed differential expression in roots, stems, leaves, leaf buds and in response to salt/cold stress and pathogen infection. In addition, the analysis of enzyme activity and sugar content revealed that invertase genes play key roles in the sucrose metabolism of various tissues and organs in poplar. This work lays the foundation for future functional analysis of the invertase genes in Populus and other woody perennials.     

Enzymatic studies during spontaneous cell rupture of Saccharomyces cerevisiae

Summary Some of the extract and intracellular enzyme activities in K2nB strain of Saccharomyces cerevisiae that growing in the condition which induce spontaneous cell rupture, were measured. B-1-3-glucanase, invertase, acid phosphatase and active chitin synthetase zymogen showed a reduced activity in ruptured cell while alkaline phosphatase shows no differences in its activity.     

Stimulation of acid invertase activity by indol-3yl-acetic acid in tissues undergoing cell expansion

The soluble acid invertase activity of young, excised P. vulgaris internodal segments fell when they were incubated in water, and their elongation ceased within 6–7 h. IAA (10 M) promoted segment elongation and stimulated an increase in the specific activity of acid invertase to a level greater than that originally present. The rate of segment elongation in the presence of IAA was closely and positively correlated with the specific activity of the enzyme. Optimum concentration of IAA for both elongation and stimulation of invertase activity was 10 M. Concurrent protein synthesis was necessary for these responses to IAA. Segments cut from mature, fully-elongated internodes did not responsd to IAA.Inclusion of Ca²⁺, vanadate or mannitol in the incubation medium abolished IAA-induced segment elongation but did not inhibit the stimulation of acid invertase activity by IAA. Auxin-induced elongation and acid invertase activity were both substantially increased in the presence of up to 25 mM D-glucose or up to 50 mM sucrose. Inclusion of either sugar in the medium considerably increased tissue hexose concentrations. Under some circumstances cell growth and invertase synthesis may compete for available hexose substrate.It is concluded that IAA-induced promotion of acid invertase in P. vulgaris internodal segments is not simply an indirect consequence of removal of end-product (hexose) during IAA-induced cell growth and that a more direct action of IAA on enzyme turnover is involved.