Fruit softening and pectin disassembly: an overview of nanostructural pectin modifications assessed by atomic force microscopy

Background

One of the main factors that reduce fruit quality and lead to economically important losses is oversoftening. Textural changes during fruit ripening are mainly due to the dissolution of the middle lamella, the reduction of cell-to-cell adhesion and the weakening of parenchyma cell walls as a result of the action of cell wall modifying enzymes. Pectins, major components of fruit cell walls, are extensively modified during ripening. These changes include solubilization, depolymerization and the loss of neutral side chains. Recent evidence in strawberry and apple, fruits with a soft or crisp texture at ripening, suggests that pectin disassembly is a key factor in textural changes. In both these fruits, softening was reduced as result of antisense downregulation of polygalacturonase genes. Changes in pectic polymer size, composition and structure have traditionally been studied by conventional techniques, most of them relying on bulk analysis of a population of polysaccharides, and studies focusing on modifications at the nanostructural level are scarce. Atomic force microscopy (AFM) allows the study of individual polymers at high magnification and with minimal sample preparation; however, AFM has rarely been employed to analyse pectin disassembly during fruit ripening.

Scope

In this review, the main features of the pectin disassembly process during fruit ripening are first discussed, and then the nanostructural characterization of fruit pectins by AFM and its relationship with texture and postharvest fruit shelf life is reviewed. In general, fruit pectins are visualized under AFM as linear chains, a few of which show long branches, and aggregates. Number- and weight-average values obtained from these images are in good agreement with chromatographic analyses. Most AFM studies indicate reductions in the length of individual pectin chains and the frequency of aggregates as the fruits ripen. Pectins extracted with sodium carbonate, supposedly located within the primary cell wall, are the most affected.     

Proteomics as an approach to the understanding of the molecular physiology of fruit development and ripening

Fruit ripening is a developmental complex process which occurs in higher plants and involves a number of stages displayed from immature to mature fruits that depend on the plant species and the environmental conditions. Nowadays, the importance of fruit ripening comes mainly from the link between this physiological process in plants and the economic repercussions as a result of one of the human activities, the agricultural industry. In most cases, fruit ripening is accompanied by colour changes due to different pigment content and increases in sugar levels, among others. Major physiological modifications that affect colour, texture, flavour, and aroma are under the control of both external (light and temperature) and internal (developmental gene regulation and hormonal control) factors. Due to the huge amount of metabolic changes that take place during ripening in fruits from higher plants, the accomplishment of new throughput methods which can provide a global evaluation of this process would be desirable. Differential proteomics of immature and mature fruits would be a useful tool to gain information on the molecular changes which occur during ripening, but also the investigation of fruits at different ripening stages will provide a dynamic picture of the whole transformation of fruits. This subject is furthermore of great interest as many fruits are essential for human nutrition. Thus far different maturation profiles have been reported specific for each crop species. In this work, a thorough review of the proteomic database from fruit development and maturation of important crop species will be updated to understand the molecular physiology of fruits at ripening stages.     

In vivo and in vitro swelling of cell walls during fruit ripening

Swelling properties of the cell walls of nine temperate fruit species, selected for their different ripening and textural characteristics, were studied during ripening. Cell wall swelling was examined in intact fruit using microscopy techniques and in vitro, using cell wall material isolated from fruit tissue. In fruit which ripened to a soft melting texture (persimmon, avocado, blackberry, strawberry, plum), wall swelling was pronounced, particularly in vitro. In-vivo swelling was marked only in avocado and blackberry. Fruit which ripened to a crisp, fracturable texture [apple (two cultivars), nashi pear, watermelon] did not show either in-vivo or in-vitro swelling of the cell wall. There was a correlation between swelling and the degree of pectin solubilisation, suggesting that wall swelling occurred as a result of changes to the viscoelastic properties of the cell wall during pectin solubilisation. Chemical and enzymatic removal of pectin from kiwifruit cell wall material supported the idea that swelling is associated with movement of water into voids left in the cellulose-hemicellulose network by the solubilised pectin. However, the results also suggested that swelling in vivo was more complex than this, and that the physicochemical changes which led to swelling included other elements of cell wall modification involving the site and mechanism of pectin solubilisation and-or the cellulose-xyloglucan complex. Received: 28 January 1997 / accepted: 11 March 1997     

Pectin esterase gene family in strawberry fruit: study of FaPE1, a ripening-specific isoform

Pectin esterases (PE, EC 3.1.1.11) catalyse the demethylation of pectin. As a result of its activity, structural interactions among cell wall components during cell wall turnover and loosening are affected. In plants, PEs are typically encoded by a gene family. This family has been studied in strawberry (Fragaria x ananassa Duch.) in order to investigate the role of distinct PE genes during fruit ripening and senescence. By a combination of a PCR-based library screening and RT-PCR four different strawberry PE cDNAs, termed FaPE1 to FaPE4, have been isolated. Differential expression of each FaPE gene in various organs and during fruit development was revealed by northern blot. FaPE1 is specifically expressed in fruit, showing an increasing expression during the ripening process up to a maximum in the turning stage. Concerning hormone regulation, auxin treatment increased FaPE1 mRNA levels in green fruit, whereas exogenous ethylene decreased FaPE1 mRNA levels in ripe and senescing fruits. It is proposed that this repression of FaPE1 expression could be involved in textural changes occurring during fruit senescence.     

Hormonal changes during non-climacteric ripening in strawberry

In contrast to climacteric fruits, where ethylene is known to be pivotal, the regulation of ripening in non-climacteric fruits is not well understood. In the non-climacteric strawberry (Fragaria anannassa), auxin and abscisic acid (ABA) are thought to be important, but the roles of other hormones suggested to be involved in fruit development and ripening are not clear. Here changes in the levels of indole-3-acetic acid (IAA), ABA, GA(1), and castasterone from anthesis to fully ripened fruit are reported. The levels of IAA and GA(1) rise early in fruit development before dropping to low levels prior to colour accumulation. Castasterone levels are highest at anthesis and drop to very low levels well before ripening commences, suggesting that brassinosteroids do not play an important role in ripening in strawberry. ABA levels are low at anthesis and gradually rise through development and ripening. The synthetic auxin, 1-naphthaleneacetic acid (NAA), can delay ripening, but the application of GA(3), the gibberellin biosythesis inhibitor paclobutrazol, and ABA had no significant effect. IAA and ABA levels are higher in the developing achenes than in the receptacle tissue and may be important for receptacle enlargement and ripening, and seed maturation, respectively. Contrary to a recent report, the biologically active GA(4) was not detected. The pattern of changes in the levels of the hormones are different from those reported in another well studied non-climateric fruit, grape, suggesting that a single consistent pattern of hormone changes does not occur in this group of fruit during ripening.     

Electron-energy-loss spectroscopic imaging of calcium and nitrogen in the cell walls of apple fruits

Changes in texture are an integral part of ripening in most fleshy fruits and these changes are thought to be determined, primarily, by alterations in cell wall structure. Electron energy loss spectroscopy (EELS) imaging was used to obtain quantitative information on the levels of calcium and nitrogen in the cell walls of apple (Malus domestica Borkh. cv. Cox's Orange Pippin) fruits. Samples of fruit cortex were prepared for EELS by high-pressure freezing and molecular distillation drying to minimize loss and redistribution of soluble cell wall components such as calcium. The EELS imaging successfully resolved calcium and nitrogen levels in the middle lamella and primary cell wall. When the elemental compositions of the cell walls of Cox's apples from two sites in the UK were compared at harvest or after 6 months storage, the orchard which always produced consistently firmer fruit had significantly lower levels of cell wall calcium and higher levels of cell wall nitrogen. This result was unexpected since firm texture in apples and other fruits has been commonly associated with elevated levels of fruit calcium. The nitrogen-rich material in the sections used for EELS was insoluble in acidified methanol, indicating that it represented a high-molecular-weight component in the cell wall. Furthermore, total tissue hydroxyproline levels were greatest in material with elevated cell wall nitrogen, suggesting enhanced levels of wall structural proteins in the tissue. These data indicate a correlation between increased amounts of cell wall nitrogen and firm fruit texture. The possible role of cell wall proteins in determining the textural properties of fruit tissue is discussed. Received: 19 November 1998 / Accepted: 28 January 1999     

Cloning of cDNAs encoding cell-wall hydrolases from pear (Pyrus communis) fruit and their involvement in fruit softening and development of melting texture

'La France' pear ( Pyrus communis L.) fruit stored at 1°C for 1 month (short-term storage) before transfer to 20°C softened and developed a melting texture during ripening, whereas fruit stored for 5 months (long-term storage) before transfer to 20°C softened but did not develop a melting texture. To clarify the mechanisms involved in fruit softening and textural changes, the cDNAs encoding cell-wall hydrolases were isolated by RT-PCR, and their expression and localization were investigated in 'La France' pears. Genes encoding three polygalacturonases (PG; EC 3.2.1.15), four pectin methylesterases (PME; EC 3.1.1.11), one α -arabinofuranosidase (ARF; EC 3.2.1.55), three β -galactosidases (GAL; EC 3.2.1.23), and two endo-1,4- β - d -glucanases (Cel; EC 3.2.1.4) were isolated. Among these 13 isolated genes, PcPG1 was the only gene for which the mRNA expression levels increased in both the short- and long-term stored fruits. This suggested that PcPG1 is involved in fruit softening rather than in the development of the melting texture. In contrast, the expression levels of PcPG3 , PcPME1 , PcPME2 , PcPME3 , PcGAL1 , PcGAL2 , and PcCel2 increased during ripening only in the short-term stored fruit. These genes might thus be involved in the development of the melting texture.     

Ripening of Nectarine Fruit (Changes in the Cell Wall,Vacuole, and Membranes Detected Using Electrical Impedance Measurements)

Electrical impedance measurements were used to characterize changes in intracellular and extracellular resistance as well as changes in the condition of membranes during ripening of nectarines (Prunus persica [L.] Batsch cv Fantasia). These measurements were related to changes in fruit texture assessed by flesh firmness and apparent juice content. An electrical model indicated that, during ripening (d 1-5) of freshly harvested fruit, the resistance of the cell wall and vacuole declined by 60 and 26%, respectively, and the capacitance of the membranes decreased by 9%. Accurate modeling of the impedance data required an additional resistance component. This resistance, which declined by 63% during ripening, was thought to be associated with either the cytoplasmic or membrane resistance. Changes in tissue resistance measured using low frequencies of alternating current were closely related to flesh firmness. After storage at 0[deg]C for 8 weeks, the nectarines developed a woolly (dry) texture during ripening at 20[deg]C. The main difference between these chilling-injured nectarines and fruit ripened immediately after harvest was the resistance of the cell wall, which was higher in woolly tissue (4435 [omega] after 5 d at 20[deg]C) than in nonwoolly tissue (2911 [omega] after 5 d at 20[deg]C). The results are discussed in relation to physiological changes that occur during the ripening and development of chilling injury in nectarine fruit.     

Genetic identification and genomic organization of factors affecting fruit texture

Fleshy fruits are an essential part of the human diet providing vital vitamins, minerals and other health-promoting compounds. The texture of the ripe fruit has a significant effect on quality and influences consumer acceptance, shelf-life, resistance, and transportability. The development of rational approaches to improve texture and shelf-life depend on understanding the biological basis of fruit ripening. Until recently, work has focused on the isolation of ripening-related genes from a variety of fleshy fruits. However, little is known about the genes that regulate this complex developmental process or whether similar regulatory genes are active in all fruiting species. A major breakthrough would be the identification of generic genes associated with texture and other aspects of ripening in fleshy fruits. In tomato, a small number of single gene mutations exist, such as ripening-inhibitor (rin), non-ripening (nor), Never-ripe (Nr), and Colourless non-ripening (Cnr) which have pleiotropic effects resulting in the reduction or almost complete abolition of ripening. These mutations probably represent lesions in regulatory genes. The cloning of the wild-type alleles of RIN and NOR is reported by Moore et al. in this issue. This review focuses on the texture characteristics of the Cnr mutant. A possible framework for the molecular regulation of fruit texture is discussed and quantitative genetic approaches to determining the generic attributes of fruit texture are explored.     

Effects of chilling on tomato fruit texture

The effects of chilling on tomato ( Lycopersicon esculentum Mill cv. Caruso) texture were investigated using fruit stored at 22°C (nonchilled) or 5°C (chilled) for 28 days. or at 5°C for 15 days before transfer to 22°C to facilitate ripening during and additional 13 days (prechilled). Prechilled fruit exhibited symptoms of slight chilling injury, i.e. development of mealiness, accelerated softening relative to that of nonchilled fruit and nonuniform surface colour development. The firmness of all fruit decreased during ripening and chilled storage when measured by flat plate compression and puncture, especially during the early stages of ripening of nonchilled and prechilled fruit. The compression firmness of pericarp tissue similarly decreased during ripening of nonchilled and prechilled fruit, but was maintained during chilling. Total moisture content (ca 94%) of tissue, uronide content (32-35% w/w) and extracted β-galactosidase activity did not differ significantly ( P > 0.05) among fruit during ripening and chilled storage. The degree of uronide methyl esterification in ethanol-insoluble solids prepared from pericarp tissue (EIS) was relatively low for all fruit. i.e. <40%. EIS from which greater levels of pectinesterase were extracted (i.e. nonchilled>chilled>prechilled) exhibited decreased levels of uronide methyl esterification. Markedly elevated levels of β-glucosidase activity were extracted from prechilled EIS. Total polygalacturonase activity (mainly as PGI) and autolysis of enzyme-extracted EIS were inversely correlated ( P ≤ 0.05) only with the loss of nonchilled fruit and tissue firmness and prechilled fruit firmness. Results suggest a possible role for β-glucosidase in textural changes of prechilled fruit and tissue (e.g. loss of firmness, development of mealiness) and also implicate loss of skin strength in the softening of whole fruit during chilling.     

猕猴桃品种果实性状特征和主成分分析研究

为深入了解猕猴桃不同品种(系)的果实性状与其倍性的相关性,本研究对国家猕猴桃种质资源圃保存的44个栽培品种(系)进行了果实性状分析,结果表明,44个栽培品种(系)的果实性状具有丰富的遗传多样性并且果实重量、果面毛被、果肉颜色和质地、果实维生素C含量、果实后熟天数和软熟果硬度、果实成熟期等与品种(系)倍性呈显著相关。对相关性状采用主成分分析表明,果实成熟时间、果肉质地、果面毛被和果实后熟天数、果肉颜色是区分品种(系)的主要特征。由于品种(系)的倍性与主要性状特征关联,品种(系)按倍性相对聚类,且二倍体品种(系)群和六倍体品种(系)群间无重叠,而四倍体品种(系)群与相邻的二倍体和六倍体品种(系)群均有一定重叠。