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.     

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.     

Polysaccharides and glycoproteins of apple fruit cell walls

A proportion of the polysaccharides and glycoproteins of apple fruit cell walls can be readily extracted in neutral buffer at or below 20°. Removal of more material was not achieved with a wide range of dissociative aqueous reagents or non-aqueous solvents. Thus traditional degradative extractants were used to obtain soluble components for further characterization. Polysaccharides and glycoproteins were separated and purified by chromatography on DEAE-cellulose columns and by gel filtration. Purified components were hydrolysed and analysed for neutral sugar and uronic acid content and for their amino acid and hydroxyproline content. The possibility of linkages existing in the cell wall between polyuronide and glycoproteins containing hydroxyproline, arabinose and galactose residues is discussed. Because of aggregation between these components, which occurs after extraction, the presence of such linkages in vivo is difficult to establish. Other cell wall glycoproteins containing xylose and glucose residues are thought to have a possible role in stabilizing hemicellulose structure.     

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.     

Exo-polygalacturonase of apple

Exo-polygalacturonase was extracted from apple cortical tissue. The enzyme hydrolyses polygalacturonic acid and has a pH optimum of 4.5–5 with this substrate. It is inhibited by EDTA and citrate and is activated by Ca²⁺ and to a lesser extent by Sr²⁺. The enzyme which has a MW of 58 000 degrades apple cortical cell wall preparations releasing low MW uronic acid residues and polyuronide.     

β-Galactosidase activity in ripening apples

β-Galactosidase activity has been identified in soluble and cell wall preparations from apple cortex tissue. The enzyme degrades pectin galactan and has a pH optimum of 4·0 with p-nitrophenyl-β, d-galactopyranoside as substrate. Soluble polygalacturonide increased as the applies softened with ripening and these changes were preceded by loss of galactose residues from the cell wall and an increase in β-galactosidase activity.     

Calcium ion transport through tissue discs of the cortical flesh of apple fruit

Tissue discs cut from the cortical flesh of apple fruit (Malus domestica Borkh. ev. Granny Smith) were clamped between two chambers, and the transport of ⁴⁵Ca²⁺ from one chamber to the other was followed. After initial transport associated with partial infiltration of air spaces by the Ca²⁺ -containing solution, steady-state transport rates were achieved over several hours. Transprt was by diffusion through the apoplast, faciliated by exchange with binding sites on the cell walls. Cation competition was observed during Ca²⁺ loading, transport and unloading, suggesting that the presence of other cations and pH will be important in modifying Ca²⁺ transport through non-vascular tissue and in xylem unloading. Modification of the extracellular volume of solution by vacuum infiltration increased Ca²⁺ transport at high concentrations, suggesting that diffusion is the prime motive force when Ca²⁺ is abundant. When low concentrations were infiltrated, there was little effect on Ca²⁺ transport, and exchange had a strong influence. Transport was reduced at 1°C but this could be accounted for by physical effects of low temperature on diffusion and viscosity. The results are discussed in relation to the nature of the apoplast and the transport of Ca²⁺ in non-vascular plant tissue.     

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.     

Ripening-related perturbations in apple cell wall nuclear spin dynamics

Spin-lattice (¹HT₁, ²³Na⁺T₁) and rotating frame spin-lattice (¹HT_1p, ¹³CT_1p) relaxation times were measured on intact, critical point dried apple tissue at various degrees of ripeness using cross polarization and magic angle spinning (CPMAS) NMR techniques. Solid state carbonyl (δ172)¹³CT_1p and ²³Na⁺-carboxylate anion T₁ values, which are inversely proportional to carboxylate reorientation rates, decreased 15–19% during the time course study. Carbonyl resonance ¹HT₁s diminished by 63% as the tissue softened; a maximal decline of 42% was also observed in the ¹HT₁s of nonspecific carbohydrate ring carbon signals (δ74) indicating an increase in both acidic and neutral polymer motion. Treatment of the cell wall with polygalacturonase resulted in a significant decrease in both carbonyl and ring carbon ¹HT₁s (57 and 42%, respectively) demonstrating the important structural function of polyuronides not only in the middle lamellae but also in the primary cell wall.     

Changes in cell wall neutral sugar composition during fruit ripening: a species survey

Non-cellulosic neutral sugar composition of cell walls from seventeen fruit types were analysed during ripening. Galactose was the major non-cellulosic neutral sugar in cell walls of cucurbit and solanaceous fruit, xylose was the predominant non-cellulosic neutral component of berries, and arabinose was the major non-cellulosic component of pome fruits. The major non-cellulosic neutral sugar residue in cell walls of stone fruits varied. In nectarine and peach, plum, and apricot, the major sugar was arabinose, galactose, and xylose, respectively. In 15 of the 17 types of fruit, a net loss of non-cellulosic neutral sugar residues occurred during ripening. No net loss occurred in plums and cucumbers. A net loss of cell wall galactose and/or arabinose occurred in 14 of the types of fruit. Xylose was the major neutral sugar residue lost from walls of apricot during ripening. In general, berry cell walls were comparatively low in galactose and arabinose content.     

Polysaccharide changes in cell walls of ripening apples

Changes in the polysaccharide composition of apple fruits ripening on and off the tree were compared. Polysaccharide fractions defined by their method of extraction were analysed colorimetrically, and the monosaccharide composition of total acetone insoluble material was analysed. Neutral carbohydrate associated with pectic extractives decreased; correspondingly galactose residues were lost in detached fruit, while galactose and arabinose residues were lost in fruit on the tree. Decreases in hemicellulose were correlated with losses of wall glucan; xylose contents did not change. Soluble polyuronide increased especially in detached fruit. DEAE-cellulose chromatography showed that this polyuronide was free from neutral sugar residues. Amounts of soluble neutral polysaccharides and glycoproteins did not change during fruit ripening.     

Xyloglucan endotransglycosylase activity during fruit development and ripening of apple and kiwifruit

Xyloglucan endotransglycosylase (XET) activity was measured in apple (Malus domestica Borkh. cv. Braeburn) pericarp and kiwifruit (Actinidia deliciosa [A. Chev.] C. F. Liang et A. R. Ferguson var. deliciosa cv. Hayward) outer pericarp and core tissues in order to establish whether a correlation exists between the activity of the enzyme and different stages of fruit development Whereas the growth rate of kiwifruit paralleled changes in XET activity throughout fruit growth, that of apple did not. Both fruits showed the highest XET activity, on a fresh weight basis, in the first two weeks after anthesis when cell division was at its highest. XET activity then decreased sharply, but as the fruit increased in size (4–8 weeks after anthesis) there was a concomitant increase in XET activity in both fruits. In the latter stage of fruit development (16–26 weeks after anthesis) XET activity increased to peak at harvest in apple fruit. During this time there was relatively little increase in fruit size and presumably therefore minimal cell expansion. XET activity then declined as fruit softened after harvest. In core tissue from kiwifruit, XET activity increased throughout the later stages of fruit growth to harvest maturity in a similar manner to apple, but continued to increase after harvest until fruit were ripe. In contrast, XET activity in the outer pericarp of kiwifruit did not increase until ripening after harvest. In apple tissue up to 30% of the XET activity was cell wall bound and could not be solubilised, even in buffer containing 2 M NaCl. The results implicate XET in cell wall assembly during cell division and expansion early in apple and kiwifruit growth. However, the disparity between apple and kiwifruit with respect to XET activity late in fruit development and ripening and the different affinities of the enzyme for the cell wall in each fruit, suggest that XET has several roles in plant development, not all of which are related to cell wall loosening during periods of accelerated growth.     

Changes in the glucans of ripening apples

The noncellulosic glucose content, like the xylose content, of cell walls of cortex tissue of apples showed little change as the fruit ripened and the cellulosic glucose also remained constant. There was a considerable loss of galactose residues from the walls, however, whilst only a small change in arabinose was observed. The starch was rapidly hydrolysed.     

Calcium ion transport across discs of the cortical flesh of apple fruit in relation to fruit development

Transport of Ca²⁺ through discs of apple fruit tissue was examined in tissue taken at different stages of fruit development. Transport rates decreased with fruit development when cation exchange was the predominant influence on transport (with 10⁻⁶ M ⁴⁵CaCl₂ as the source solution). This decrease was associated with a reduction in relative cell wall surface area, cation exchange capacity and cell wall yield that occurred during fruit growth. When diffusion was the major transport force, and when transport was influenced by solution infiltration of the tissue disc (10⁻² M ⁴⁵CaCl₂ in the source solution), transport rates increased during fruit growth. This increment was related to increases in air space of the tissue. Ca²⁺ transport through apple fruit tissue is influenced by the extent and nature of the cell wall, changing proportions of air space and Ca²⁺ concentration in the extracellular solution.     

套袋微域环境对富士苹果果皮结构的影响

对富士苹果果实进行双层纸袋套袋处理,通过分析袋内微域环境变化,研究套袋微域环境对果皮扫描与透射结构的影响。结果表明:套袋内微域的黑暗环境导致果皮的光合能力缺失,表皮第一层细胞内容物减少,缺少脂质体,取而代之的是游离的脂体小球,说明表皮细胞形成角质层的物质来源缺乏,导致角质层变薄。与外界比较,套袋内昼夜具较高的温度与湿度,形成所谓的"小温室"环境,而且,由于受套袋的保护,使果实免受外界环境的直接刺激,导致套袋果实的果面光洁平滑,果点小且色淡,蜡质层龟裂均匀,且裂口深度远小于未套袋果;但摘袋后果面龟裂产生的裂纹频度增大,说明环境的改变影响蜡质层龟裂。研究结果从果皮结构的变化为套袋果实外观品质的变化提供了理论依据,同时为果实摘袋后的补钙实施提供了参考依据。     

The carbohydrate constituents of the cell wall of suspension cultures of Rosa glauca

Sequential extractions of 14-day-old Rosa glauca cell walls cultured in vitro showed that two different types of acidic polysaccharide were present. One was extracted with EDTA or ammonium oxalate solutions, and the other remained in close association with cellulose even after 4.3 N NaOH extractions or 2 N H₂SO₄ hydrolysis. The cell wall has a low content in structural protein. The behaviour of each constituent sugar was followed during the course of the various extraction steps, and a complete quantitative account of the protein, uronic acid and neutral sugar components is given at each stage.     

Variability of cell wall polysaccharides composition and hemicellulose enzymatic profile in an apple progeny

The genetic variability of apple cell walls polysaccharides chemical composition and structure was assessed in a progeny of 141 individuals harvested over 2 years. The variability of the hemicelluloses oligosaccharides released by glucanase was analyzed by MALDI-TOF MS. The genetic contribution was distinguished from harvest year as well as from parental crossing patterns and scab resistance selection. Results showed that harvest year had a major impact on cell wall polysaccharide composition and structure. Within each harvest, genetic effect impact more significantly cell wall polysaccharide chemistry than does reciprocal crossing or early scab selection. Uronic acids, glucose, galactose and xylose contents as well as some glucomannan and xyloglucan structures have a high heritability. This first cell wall chemotyping of an apple progeny opens the way for future searches of genetic markers for the chemical variability of cell wall polysaccharides.