Post-translational inhibitory regulation of acid invertase induced by fructose and glucose in developing apple fruit

Acid invertase (EC 3.2.1.26) is one of the key enzymes involved in the carbohydrate sinkorgan development and the sink strength modulation in crops. The experiment conducted with ‘Starkrimson’ apple (Malus domestica Borkh) fruit showed that, during the fruit development, the activity of acid invertase gradually declined concomitantly with the progressive accumulation of fructose, glucose and sucrose, while Western blotting assay of acid invertase detected a 30 ku peptide of which the immuno-signal intensity increased during the fruit development. The immunolocalization via immunogold electron microscopy showed that, on the one hand, acid invertase was mainly located on the flesh cell wall with numbers of the immunosignals present in the vacuole at the late stage of fruit development; and on the other hand, the amount of acid invertase increased during fruit development, which was consistent with the results of Western blotting. The in vivo preincubation of fruit discs with soluble sugars showed that the activity of extractible acid invertase was inhibited by fructose or glucose, while Western blotting did not detect any changes in apparent quantity of the enzyme nor other peptides than 30 ku one. So it is considered that fructose and glucose induced the post-translational or translocational inhibitory regulation of acid invertase in developing apple fruit. The mechanism of the post-translational inhibition was shown different from both the two previously reported ones that proposed either the inhibition by hexose products in the in vitro chemical reaction equilibrium system or the inhibition by the proteinaceous inhibitors. It was hypothesized that fructose and glucose might induce acid invertase inhibition by modulating the expression of some inhibition-related genes or some structural modification of acid invertase.     

Heterologous expression of the apple hexose transporter MdHT2.2 altered sugar concentration with increasing cell wall invertase activity in tomato fruit

Sugar transporters are necessary to transfer hexose from cell wall spaces into parenchyma cells to boost hexose accumulation to high concentrations in fruit. Here, we have identified an apple hexose transporter (HTs), MdHT2.2, located in the plasma membrane, which is highly expressed in mature fruit. In a yeast system, the MdHT2.2 protein exhibited high ¹⁴C‐fructose and ¹⁴C‐glucose transport activity. In transgenic tomato heterologously expressing MdHT2.2, the levels of both fructose and glucose increased significantly in mature fruit, with sugar being unloaded via the apoplastic pathway, but the level of sucrose decreased significantly. Analysis of enzyme activity and the expression of genes related to sugar metabolism and transport revealed greatly up‐regulated expression of SlLIN5, a key gene encoding cell wall invertase (CWINV), as well as increased CWINV activity in tomatoes transformed with MdHT2.2. Moreover, the levels of fructose, glucose and sucrose recovered nearly to those of the wild type in the sllin5‐edited mutant of the MdHT2.2‐expressing lines. However, the overexpression of MdHT2.2 decreased hexose levels and increased sucrose levels in mature leaves and young fruit, suggesting that the response pathway for the apoplastic hexose signal differs among tomato tissues. The present study identifies a new HTs in apple that is able to take up fructose and glucose into cells and confirms that the apoplastic hexose levels regulated by HT controls CWINV activity to alter carbohydrate partitioning and sugar content.     

Distribution of acid invertase in the tomato plant

Acid invertase activity in Lycopersicon esculentum was highest in the locular wall of ripe fruit and lowest in roots. Activity was greater in leaf laminae than in petiole tissue and increased with leaf age, whereas there was more invertase in the upper part of the stem compared with the older portion. Activity in whole fruit increased with increasing ripeness and was greatest in overripe fruit. Of various tissues from a number of wild tomato species examined, the fruit of L. pimpinellifolium were particularly rich in the enzyme, in contrast to the fruit of L. hirsutum, L. hirsutum, var. glabratum and L. peruvianum which had low activity.     

Changes in jasmonic acid concentration during early development of apple fruit

Apple fruits ( Malus domestica Borkh.) were harvested from 24 to 136 days after full bloom (DAFB) and endogenous jasmonic acid was analyzed by GC-MS. There were two isomers of jasmonic acid in apple fruit with a ratio of 37:63 ( cis:trans ). The cis:trans ratio remained relatively constant throughout this period of fruit development. The endogenous jasmonic acid concentration was 138 ng g⁻¹ fresh weight 24 DAFB and decreased as fruit developed. Changes in jasmonic acid concentration were coincident with those of respiration, ethylene production, and anthocyanin accumulation in patterns consistent with the reported responses to exogenous jasmonates. Possible roles for jasmonic acid during early fruit development are discussed.     

Changes in invertase activities precede ovary growth induced by gibberellic acid in

Developing pods of pea ( Pisum sativum L. cv. Alaska no 7) were used to study the enzymes of sucrose metabolism. Acid and neutral invertase (EC 3.2.1.26). sucrose synthase (SS, EC 2.4.1.13) and sucrose phosphate synthase (SPS, EC 2.4.1.14) have been localized in the soluble fraction. Acid invertase activity was also present in the insoluble fraction and in pea ovary apoplast. In pea pods, sucrose breakdown was dominated by the invertase pathway. SS specific activity only increased at late stages of parthenocarpic pod development, while SPS did so in pods obtained by pollination. Changes in time course of invertase activities have been correlated with the growth rate of fruits induced to develop either by fertilization or by exogenous application of giberellic acid (GA), 2,4-dichloro-phenoxy acetic acid (2,4-D) or 6-benzylaminopurine (6-BAP). The soluble neutral activities might be associated with pod elongation, while the acid ones were rather related to assimilate import by the induced fruits. Application of gibberellic acid to non-pollinated ovaries significantly enhanced the soluble neutral invertase activity before any ovary outgrowth was detected (up to 2 h after treatment). Within the same period of time. GA-treated ovaries showed a decrease in the acid invertase activity of the soluble fraction and an increase of the acid invertase activity in the apopiast. preceding in time the increment of the acid invertase activity associated with the insoluble fraction. Our results suggest that the early GA response may be mediated through a promotion of processes of protein secretion.     

Soluble and wall-bound glycoproteins of apple fruit tissue

Apple fruit tissue contains small amounts of readily soluble glycoproteins, rich in hydroxyproline; polymethylgalacturonide is not covalently bound to the soluble glycoproteins. Barium hydroxide hydrolysis of apple fruit cell walls liberated glycopeptides containing 4 arabinosyl residues per hydroxyprolyl residue, which were attacked very slowly by α-l-arabinofuranosidase. Hydrazinolysis liberated similar glycopeptides, which were difficult to separate from a polysaccharide containing galactose residues. Protease treatment of walls also released glycopeptides containing hydroxyproline, and a small proportion of these were associated with polyuronide. Polygalacturonase pretreatment of walls led to increased release of hydroxyprolyl residues by protease. Susceptibility of the hydroxyproline containing glycoprotein in the cell wall to attack by protease and arabinosidase did not change during fruit ripening. The amount of an unknown hexosamine associated with the wall was less in ripethan in unripe fruit.     

Purification and characterization of two soluble acid invertase isozymes from Japanese pear fruit

Two isozymes (AIV I and AIV II) of soluble acid invertase (EC 3.2.1.26) were purified from Japanese pear fruit through procedures including (NH(4))(2)SO(4) precipitating, DEAE-Sephacel column chromatography, Concanavalin A (ConA)-Sepharose affinity chromatography, hydroxyapatite column chromatography and Mono Q HR 5/5 column chromatography. The specific activities of purified AIV I and AIV II were 2670 and 2340 (nkat/mg protein), respectively. AIV I was a monomeric enzyme of 80 kDa, while AIV II may be also a monomeric enzyme, which is easy to be cleaved to 52 kDa and 34 kDa polypeptide during preparation by SDS-PAGE. The Km values for sucrose of AIV I and AIV II were 3.33 and 4.58 mM, respectively, and optimum pH of both enzyme activities was pH 4.5.     

Effects of sugars,gibberellic acid and kinetin on acid invertase of developing carrot roots

The activities of acid invertase carrot roots 32, 50 and 60 days old were, respectively, 5.7, 1.4 and 0.5 nkat/g fr. wt. When portions of such roots were excised and incubated in water for 20 hr the activities of the enzyme rose, respectively, to 9.7, 14.4 and 18.4. Fructose (50 mM), GA (30 μM) and kinetin (50 μM) affected the rise in invertase activity, GA stimulating it and fructose and kinetin decreasing it. The magnitude of these effects varied, however, with the age of the roots. Fructose had the highest effect in young non-tuberized roots while the effects of kinetin and GA were highest in mature tuberous roots. A 48 hr incubation of discs from mature roots in fructose plus kinetin reduced the rise in invertase activity by 75%; nevertheless, fructose plus kinetin could not abolish, even after 66 hr of incubation, the ca 10% increase in invertase activity produced by a 1 hr GA pulse treatment applied at 0 hr.     

Properties of polygalacturonate and cell cohesion in apple fruit cortical tissue

Examination of the hydrodynamic properties of polygalacturonate fractions from unripe and ripe apple tissue suggested that the wall bound fraction was degraded during ripening but that the soluble fraction was not. Esterification of cell wall preparations with CH₂N₂ caused solubilisation of polygalacturonate. Acid MeOH caused more extensive solubilization, but this reagent hydrolysed arabinofuranosyl linkages. Both reagents reduced the cohesion of EtOH extracted apple tissue. This effect could also be achieved by treatment with sodium polyphosphate at pH 4 but not by EDTA or chaotropic agents. Free carboxyl groups on polygalacturonate probably maintain cell cohesion through co-operative binding of Ca²⁺ ions. The integrity of primary wall structure is thought to depend upon non-covalent bonding between cellulose, protein and polygalacturonate.     

Isolation and characterization of acid invertase cDNA clone in hot pepper (Capsicum annuum L.) fruits

An acid invertase (EC 3.2.1.26.) cDNA clone,CaAIV-18, was isolated from the red pericarp cDNA library of the hot pepper (Capsicum annuum L.) fruit. TheCaAIV-18 clone has 2223 nucleotides and one open reading frame encoding 641 amino acid residues. Analysis of deduced amino acid sequences reveals thatCaAIV-18 has a 24-amino acid transmembrane anchor region in its N-terminal, implying acid invertase in hot pepper may be localized in the membrane and not in the cytosol. This clone showed high homology to tomato acid invertase,Aiv1, in nucleotide and deduced amino acid sequences. In the Southern blot analysis, this clone proved to exist as single or low copy numbers on the genome of hot pepper. The clones had two well-conserved regions which appears in acid invertase of other plant species (eg. tomato,Arabidopsis, etc.) and yeasts. During fruit development,CaAIV-18 was expressed preferentially in the ripe red stage.     

Mutual influence of invertase and acid phosphatase of the yeastSaccharomyces cerevisiae on their secretion into the cultivation medium

The hypothesis that various extracellular enzymes produced by the yeastSaccharomyces cerevisiae exert a mutual influence on their secretion into the cultivation medium was tested experimentally. The statistically processed results indicate that extracellular invertase affects the secretion of acid phosphatase, and acid phosphatase affects the secretion of invertase. In addition, the secretion of each of these enzymes was shown to be subject to autoregulation.     

Metabolism of polymethylgalacturonate in apple fruit cortical tissue during ripening

Changes in the composition and metabolism of polymethylgalacturonate were followed in ripening apples. After the onset of ethylene production and fruit softening total polygalacturonate decreased and the water soluble fraction increased. No change was detected in the overall degree of esterification but the esterification of the water soluble fraction increased. Incorporation of radioactivity from methionine-[¹⁴C] into Me groups on polygalacturonate continued during ripening but incorporation from inositol-[³H] decreased sharply. Cell separation probably depends upon the removal of low ester polygalacturonate from the middle lamella by exopolygalacturonase; the continued incorporation from methionine-[¹⁴C] is probably due to synthesis of new polymethylgalacturonate.     

Soluble acid invertase activity in leaves is independent of species differences in leaf carbohydrates, diurnal sugar profiles and paths of phloem loading

Leaf sucrose, starch, hexose and maximum extractable soluble acid invertase activity were compared throughout the day in source leaves of 13 plant species chosen for their putative phloem-loading type (apoplastic or symplastic). Four species which represent the different phloem-loading types (tomato, barley, maize and Fuchsia ) were studied in detail. Using this information we wished to determine whether a positive correlation between foliar carbohydrates and acid invertase activity exists in leaves from different species and, furthermore, whether this relationship is determined by phloem-loading type. Acid invertase activity was relatively constant throughout the day in all species. The extent of sucrose, hexose and starch accumulation and the sucrose: starch ratio measured at a given time were species-dependent. No correlations were found between foliar soluble acid invertase activity and the hexose, sucrose or starch content of the leaves in any of the species, regardless of phloem-loading type. The species examined could be divided into three distinct groups: (1) high sucrose, low invertase; (2) low sucrose, low invertase; and (3) low sucrose, high invertase. The absence of an inverse relationship between leaf sucrose, hexose or starch contents and endogenous soluble acid invertase suggests that this enzyme is not directly involved in carbon partitioning in leaves but serves an auxiliary function.     

Enzymic analysis of cell wall structure in apple fruit cortical tissue

Galactanase from Phytophthora infestans and an arabinosidase isoenzyme from Sclerotinia fructigena attacked the cortical cell walls of apple fruits liberating galactose and arabinose residues, respectively. Other arabinosidase isoenzymes from S. fructigena attacked cell walls very slowly. A S. fructigena polygalacturonase isoenzyme liberated half of the uronic acid residues with few associated neutral residues, while a second polygalacturonase isoenzyme released more uronic acid with a substantial proportion of arabinose and galactose and lesser amounts of xylose, rhamnose and glucose; reaction products of this enzyme could be further degraded by the first isoenzyme to give high MW fragments, rich in arabinose with most of the xylose, rhamnose and glucose, and low MW fragments rich in galactose and uronic acid. Endoglucanase from Trichoderma viride released a small proportion of the glucose residues from cell walls together with uronic acid, arabinose, xylose and galactose; more extensive degradation occurred if walls were pre-treated with the second polygalacturonase isoenzyme. Endoglucanase reaction products were separated into a high MW fraction, rich in arabinose, and lower MW fractions rich in galactose and glucose residues. The high MW polygalacturonase and endoglucanase products could be degraded with an arabinosidase isoenzyme to release about 75% of their arabinose. Cell walls from ripe fruit showed similar susceptibility to arabinosidase and galactanase to those from unripe apples. Cell walls from fruit, ripened detached from the tree were more susceptible to degradation by polygalacturonase than walls from unripe fruit or fruit ripened on the tree. Endoglucanase released less carbohydrate from ripe fruit cell walls than from unripe fruit cell walls.     

Relationship between sucrose accumulation and activities of sucrose-phosphatase, sucrose synthase, neutral invertase and soluble acid invertase in micropropagated sugarcane plants

The activities of sucrose-phosphate synthase (SPS), sucrose synthase (SUSY), neutral invertase (NI) and soluble acid invertase (SAI) regulates sucrose activity in sugarcane were studied. Micropropagated sugarcane plants were obtained from callus cultures of four Mexican commercially available sugarcane varieties characterized by differences in sugar production, and activities of SPS, SUSY, NI, SAI and concentrations of sucrose were monitored in the sugarcane stem. The results indicated that sucrose accumulation was positively and significantly related to an increase in activity of SPS and SUSY and negatively to a reduction in activity of SAI and NI (P<0.05). SPS explained most of the variations found for sucrose accumulation and least for NI. The relationship between activity of SPS, SUSY, NI and SAI in sugarcane stem was similar in each variety.     

Possible role of soluble invertase in the gibberellic acid berry-sizing effect in Sultana grape

It is well known that post-bloom applications ofgibberellic acid (GA₃) increase seedless grapeberry size by enhancing cell division, or cellenlargement, or both. As a consequence, total waterand sugar per berry are increased. Soluble invertaseis considered to be one of the key enzymes in theaccumulation of sugar in grape berries. To study apossible role of invertase in the GA₃berry-sizing effect, different rates of post-bloomGA₃ were applied to seedless grape cv. Sultanaand hexose concentration and invertase activity weremeasured. GA₃ stimulated both parameters as earlyas 24 and 32 h after applications, respectively.Moreover, the increment in sugar content and enzymeactivity remained throughout the growing of the berries period and, at ripening, increases in hexosescontent (102%) and invertase activity (60%) weredetected when GA₃ was applied at a rate of 45 ppm.At the same GA₃ rate the pericarp cellsdoubled in size. Furthermore, positive correlationswere found between berry-size, invertase activity andhexose content, suggesting that GA₃ stimulationof invertase could be one of the factors involved in theberry sizing-effect of GA₃.