Cytochemical methods for the localization of invertase activity in plant tissues

Summary 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 raction 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.     

Developmental changes in carbohydrate content and sucrose degrading enzymes in tuberising stolons of potato (Solanum tuberosum)

Tuberising stolon tips of potato ( Solanum tuberosum L. cv. Record) accumulate starch and sucrose but the hexose content, particularly fructose, declines rapidly. Similar changes occur in the region 2 cm behind the swelling apex but the decline in glucose is far more pronounced than in the developing tuber. Tuberisation is characterised by an apparent switch from an invertase-dominated sucrolytic system (both acid and alkaline invertases [EC 3.2.1.26] are present) to one dominated by sucrose synthase (EC 2.4.1.13). Sucrose synthase and fructokinase (EC 2.7.1.4) activities were, at a maximum, ca 10- and 5-fold higher, respectively in the swelling stolon tip compared with the non-tuberising region. At the highest starch contents attained, the starch level in the young developing tuber was approximately double that in the adjacent non-tuberising stolon region. Immunoblots revealed that developmental changes in sucrose synthase. fructokinase and alkaline invertase polypeptides corresponded with enzyme activities. Antibodies raised against the N-terminal amino acid sequence of a soluble invertase purified from mature tubers did not detect significant quantities of a polypeptide in stolons and young, developing tubers. Antibodies raised against an in vitro expression product of an apoplastic invertase cloned from a leaf cDNA library detected a polypeptide in developing tubers but not in mature ones. However, expression of the protein did not correlate well with acid invertase activity during early tuber formation.     

Biochemical and immunological properties of alkaline invertase isolated from sprouting soybean hypocotyls.

Alkaline invertase from sprouting soybean (Glycine max) hypocotyls was purified to apparent electrophoretic homogeneity by consecutive use of DEAE-cellulose, green 19 dye, and Cibacron blue 3GA dye affinity chromatography. This protocol produced about a 100-fold purification with about a 11% yield. The purified protein had a specific activity of 48 mumol of glucose produced mg-1 protein min-1 (pH 7.0) and showed a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) (58 kDa) and in native PAGE, as indicated by both protein and activity staining. The native enzyme molecular mass was about 240 kDa, suggesting a homotetrameric structure. The purified enzyme exhibited hyperbolic saturation kinetics with a Km (sucrose) near 10 mM and the enzyme did not utilize raffinose, maltose, lactose, or cellibose as a substrate. Impure alkaline invertase preparations, which contained acid invertase activity, on contrast, showed biphasic curves versus sucrose concentration. Combining equal activities of purified alkaline invertase with acid invertase resulted in a biphasic response, but there was a transition to hyperbolic saturation kinetics when the activity ratio, alkaline: acid invertase, was increased above unity. Alkaline invertase activity was inhibited by HgCl2, pridoxal phosphate, and Tris with respective Ki values near 2 microM, 5 microM, and 4 mM. Glycoprotein staining (periodic acid-Schiff method) was negative and alkaline invertase did not bind to two immobilized lectins, concanavalin A and wheat germ agglutinin; hence, the enzyme apparently is not a glycoprotein. The purified alkaline invertase, and a purified soybean acid invertase, was used to raise rabbit polyclonal antibodies. The alkaline invertase antibody preparation was specific for alkaline invertase and cross-reacted with alkaline invertases from other plants. Neither purified soybean alkaline invertases nor the crude enzyme from several plants cross-reacted with the soybean acid invertase antibody.     

Sugar metabolism in developing tubers of Solanum tuberosum

Regulation of the sugar content of the developing tubers of three varieties (King Edward, Maris Bard, Pentland Javelin) of Solanum tuberosum was investigated. Sucrose, glucose, fructose, UDP-glucose and fructose-2,6-bispbosphate were measured during tuber development as were the maximum catalytic activities of acid invertase, alkaline invertase, sucrose synthase, α-glucan phosphorylase, hexokinase, phospbofructokinase and pyrophosphate: fructose 6-phosphate 1-phosphotransferase [PFK(PPi)]. Sucrose was the dominant sugar and there was less fructose than glucose; the amounts of all three per gram fresh weight fell during tuber development. The activity of hexokinase per gram fresh weight declined during development but those of the other enzymes listed did not alter significantly. No change in the amounts of fructose-2,6-bisphosphate or UDP-glucose per gram fresh weight were found. The above measurements suggest that much of the sucrose translocated to the developing tuber is metabolized via sucrose syntbase to UDP-glucose that is converted to glucose 1-phosphate by UDP-glucose pyrophosphorylase using pyrophosphate generated by PFK (PPi).     

Invertases in young and mature leaves of Citrus sinensis

The sucrose catabolic enzymes acid invertase (EC 3.2.1.26) and alkaline invertase (EC 3.2.1.27) were studied in young and mature Citrus sinensis leaf tissue. In young, expanding leaves (60 % final length) soluble acid invertase activity predominated, while soluble alkaline invertase activity predominated in mature leaves. The acid and alkaline invertase activities were separated on Sephadex G-200. The acid invertase had an M_r of approximately 60 000, pH maximum of 4.5 and apparent K_m of 3.3 mM sucrose. The alkaline invertase had an M_r of approximately 200 000, pH maxima of 6.8 and an apparent K_m of 20 mM sucrose. Alkaline invertase was strongly inhibited by 10 mM Tris while acid invertase was not. Possible physiological roles for the two invertases are discussed.     

Vacuolar Invertase Gene Silencing in Potato (Solanum tuberosum L.) Improves Processing Quality by Decreasing the Frequency of Sugar-End Defects

Sugar-end defect is a tuber quality disorder and persistent problem for the French fry processing industry that causes unacceptable darkening of one end of French fries. This defect appears when environmental stress during tuber growth increases post-harvest vacuolar acid invertase activity at one end of the tuber. Reducing sugars produced by invertase form dark-colored Maillard reaction products during frying. Acrylamide is another Maillard reaction product formed from reducing sugars and acrylamide consumption has raised health concerns worldwide. Vacuolar invertase gene (VInv) expression was suppressed in cultivars Russet Burbank and Ranger Russet using RNA interference to determine if this approach could control sugar-end defect formation. Acid invertase activity and reducing sugar content decreased at both ends of tubers. Sugar-end defects and acrylamide in fried potato strips were strongly reduced in multiple transgenic potato lines. Thus vacuolar invertase silencing can minimize a long-standing French fry quality problem while providing consumers with attractive products that reduce health concerns related to dietary acrylamide.     

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.     

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.     

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.     

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.     

An examination of methods used to assay potato tuber invertase and its naturally occurring inhibitor

Confirming an earlier report, it was shown that the endogenous inhibitor of potato tuber invertase forms an essentially undissociable complex with the enzyme. Consequently, several previous analyses of potato tuber invertase which were based on equations derived for highly dissociable enzyme-inhibitor complexes are presumed to be in serious error. The complex formation proceeded slowly, requiring approximately 1 day to reach completion at 2 C, and 1 hr at 37 C. Allowing complex formation to reach completion before assaying enzyme activity did not affect the noncompetitive nature of the inhibition.     

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.     

Developmental changes of enzymes involved in conversion of sucrose to hexose-phosphate during early tuberisation of potato

A highly synchronised in-vitro tuberisation system, based on single-node cuttings containing an axillary bud, was used to investigate the activity patterns of enzymes involved in the conversion of sucrose to hexose-phosphates during stolon-to-tuber transition of potato (Solanum tuberosum L.). Two different non-tuberising systems were included to distinguish between changes that are or are not tuber-specific. At tuberisation the activity of soluble acid invertase decreased (13-fold) and of sucrose synthase increased (12-fold). The activity of both enzymes remained unchanged in the non-tuberising treatments. Based on the opposite patterns and large difference in activity of these two sucrolytic enzymes, we conclude that sucrose synthase constitutes the predominant route of sucrose breakdown after tuber initiation. During the period before tuberisation, the activity of cell-wall-bound invertase and of hexokinase showed a highly positive correlation (r ² = 0.96 in all the three treatments, suggesting coordinated coarse control of both enzyme activities. After the onset of tuberisation cell-wall-bound invertase activity decreased to a very low level, a change not observed in the non-tuberising systems, indicating that cell-wall-bound invertase is presumably not involved in the unloading mechanism and/or short-distance transport of sucrose within the perimedulla of growing tubers. The overall activity of fructokinase and of hexokinase both showed a fourfold increase after tuber initiation, but remained unchanged in the non-tuberising systems. The increase of fructokinase suggests that the phosphorylation of fructose by fructokinase down-regulates the cytosolic fructose content in order to maintain a high sucrose-synthase-catalysed net flux of sucrose to phosphorylated hexoses during rapid tuber growth. The increase of total glucose-phosphorylating potential could be a response to the tuberisation-related starch accumulation process. The activity of UDP-glucose pyrophosphorylase showed no developmental change. The level of UDP-glucose pyrophosphorylase activity is very likely the result of metabolic regulation. Received: 21 June 1996 / Accepted: 21 October 1996     

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.     

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.     

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.     

Control of the hexose content of potato tubers

The amounts of glucose and fructose in a range of harvested tubers of Solanum tuberosum were compared with the labelling of these hexoses by [U-¹⁴C]sucrose supplied to the tubers. Hexose content varied. Fructose was more heavily labelled than glucose. There was no correlation between the amounts of glucose and fructose in the tuber and their labelling. The maximum catalytic activities of α-glucan phosphorylase, acid invertase, alkaline invertase, sucrose synthase, α-amylase and β-amylase in tubers stored for 17 weeks at 5° and at 10° were estimated. The values showed no clear correlation with hexose content, but provided sound evidence that starch breakdown was phosphorolytic. It is suggested that the amounts of glucose and fructose in mature harvested tubers may be determined more by the partitioning of the translocated sucrose during the development of the tubers than by the metabolism of the harvested tuber.     

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.