- Chemistry Of Fruit Ripening
- Pictures Of Artificial Ripening Of Fruits
- What Is Ripening
- Fruit Ripening Experiment
- Ethylene And Fruit Ripening
- Stages Of Fruit Ripening
Abstract
This review is concerned with the mechanisms controlling fruit softening. Master genetic regulators switch on the ripening programme and the regulatory pathway branches downstream, with separate controls for distinct quality attributes such as colour, flavour, texture, and aroma. Ethylene plays a critical role as a ripening hormone and is implicated in controlling different facets of ripening, including texture change, acting through a range of transcriptional regulators, and this signalling can be blocked using 1-methylcyclopropene. A battery of at least seven cell-wall-modifying enzymes, most of which are synthesized de novo during ripening, cause major alterations in the structure and composition of the cell wall components and contribute to the softening process. Significant differences between fruits may be related to the precise structure and composition of their cell walls and the enzymes recruited to the ripening programme during evolution. Attempts to slow texture change and reduce fruit spoilage by delaying the entire ripening process can often affect negatively other aspects of quality, and low temperatures, in particular, can have deleterious effects on texture change. Gene silencing has been used to probe the function of individual genes involved in different aspects of ripening, including colour, flavour, ethylene synthesis, and particularly texture change. The picture that emerges is that softening is a multi-genic trait, with some genes making a more important contribution than others. In future, it may be possible to control texture genetically to produce fruits more suitable for our needs.
- Ethylene and fruit ripening. Early examples of the human utilisation of ethylene to enhance fruit ripening include the ancient Egyptian practice of gashing figs to enhance ripening responses. The ethylene produced by the injured fruit tissue triggers a broader ripening response.
- Ethylene is plants hormone [26], It acts as stimulating or regulating the ripening of fruit. Crockerin [27] reported that it is plant hormone responsible for fruit ripening. It is produced in all parts of higher plants, including leaves, stems, roots, flowers, fruits, tubers and seedlings.
Cell walls, Ethene, Pectate lyase, Polygalacturonase, Tomato
Introduction
Fruit ripening involves a series of changes in colour, flavour, texture, aroma, and nutrient content, which affect quality, post-harvest life, and value. Most fleshy fruits undergo some, if not all, of these changes during ripening, which, in evolutionary terms, are designed to make them attractive to eat and therefore aid seed dispersal. Perhaps, the best-studied fruit is tomato, where it has been established that ripening is controlled by the interaction between genetic and hormonal factors, and the general features of this ripening model seem applicable to many fruits. Essential features are the involvement of both transcriptional regulators, such as RIN, which was discovered by analysis of the rin tomato mutant (Vrebalov et al., 2002), and structural genes that control biochemical changes. In addition, the action of hormones, particularly ethylene, is essential, and downstream of ethylene the signalling pathway branches to control different facets of ripening separately (Figure 1; Grierson, 2013). Textural change is one of the major characteristic features of fruit ripening and, although the underlying mechanisms are complex, and the details vary between different fruits, textural change is generally recognized to involve both softening and changes in crispness and juiciness of the fruit (Harker et al., 1997; Chaïb et al., 2007). These changes result from major alterations in the structure and composition of the cell wall components during ripening, which are considered as major contributory factors in the softening process (Brummell, 2006; Vicente et al., 2007). However, other mechanisms may also be active in determining the overall textural characteristics of the fruit. Banana, for example, is packed with starch, and part of the texture change during ripening is also due to the conversion of insoluble starch to soluble sugars (see Kojima et al., 1994). In addition, partly because of starch solubilization and changes in cell wall structure, and perhaps also due to other factors, such as changes in activity of water channels and cuticle properties, there can be substantial alterations in the distribution of water molecules within fruits during ripening. All of these changes can have a profound effect on juiciness, texture, and overall softening. Various aspects of ripening, including softening, can also be influenced by the post-harvest environment, for example high and low temperature, the presence of applied or naturally occurring gases, all of which can influence changes in gene expression and the activities of enzymes determining overall ripening behaviour.
Regulation of ripening in tomato. The branched nature of the pathway, with a range of regulatory (transcription factors) and structural genes (encoding enzymes) acting downstream of ethylene to control colour, flavour (taste and aroma), texture, and vitamins, is supported by studies on mutants and using gene silencing to test the role of specific genes. Available knowledge indicates that the general principles of this scheme are applicable to all climacteric fruits; at least some aspects apply also to non-climacteric fruits. Although ripening of non-climacteric fruits has been considered to occur independently of ethylene, and there may be other regulators, recent evidence indicates that ethylene may be important in non-climacteric fruits, although this still requires clarification. What is clear, however, is that similar regulator and structural genes are expressed during ripening of both types of fruits. Modified from Grierson (1986 and 2013).
Regulation of ripening in tomato. The branched nature of the pathway, with a range of regulatory (transcription factors) and structural genes (encoding enzymes) acting downstream of ethylene to control colour, flavour (taste and aroma), texture, and vitamins, is supported by studies on mutants and using gene silencing to test the role of specific genes. Available knowledge indicates that the general principles of this scheme are applicable to all climacteric fruits; at least some aspects apply also to non-climacteric fruits. Although ripening of non-climacteric fruits has been considered to occur independently of ethylene, and there may be other regulators, recent evidence indicates that ethylene may be important in non-climacteric fruits, although this still requires clarification. What is clear, however, is that similar regulator and structural genes are expressed during ripening of both types of fruits. Modified from Grierson (1986 and 2013).
In this review, we shall focus on fruit cell wall structure, the mechanisms that bring about texture- and softening-related changes during ripening and post-harvest storage, and consider the contribution and role of gene expression, ethylene, and other factors in controlling these changes.
Cell wall composition and structure
Plant cell walls are very diverse and complex structures, and there are significant differences between different groups of plants, and between different cells within a plant. Growing plant cells are surrounded by a dynamic, carbohydrate-rich primary cell wall and, in dicots, in particular, the space between adjacent cells is occupied by a pectin-rich middle lamella (Popper et al., 2011). In those tissues that have ceased growth (such as the woody tissue of trees), the cell walls undergo a process of lignification in which an aromatic polymer (lignin) is laid down to form a relatively inert secondary cell wall.
Fruit in general have primary walls with very little, if any, lignification (Toivonen and Brummell, 2008; but see the later discussion on loquat fruit post-harvest lignification).
Use of Calcium Carbide for Artificial Ripening of Fruits -Its Application and Hazards. Ethylene's role as a potent plant growth regulator affecting many phases of plant growth and development was established only in the last 50 years, but its effect has been known for centuries. Artificial fruit ripening. Download PDF; Full Text. ARTIFICIAL RIPENING OF FRUITS BY ETHYLENE. Download citation file: RIS. Borland developer studio 2006 professional keygen generator software. ARTIFICIAL RIPENING OF SWEETGUM SEEDS F. BONNER 1 Southern Forest Experiment Station Forest Service, USDA. Fruits collected on this date. How to artificially ripe a banana(or.
Several general models of the primary cell wall have been constructed during the last 30 years (Carpita and Gibeaut 1993; Carpita, 1996; Somerville et al., 2004; Cosgrove, 2005; Doblin et al., 2010; Albersheim et al., 2011). These all have the general feature of cellulose fibrils being embedded in a matrix of hemicellulose and pectic polymers (Figure 2). The wall also contains a certain amount of structural proteins, such as extensin, hydroxyproline-rich, and arabinogalactan proteins. Cell walls are generally assigned to one of the two general groups—type I or type II. Type I cell walls are normally associated with dicotyledonous plants and type II are associated with monocotyledonous species. Cellulose fibrils are a feature of both types of wall with the major differentiating factors being the nature of hemicellulosic polymers present and the relative proportion of pectic polymers, type I walls having a much higher proportion of pectin than type II. There are, however, a number of plants that appear to have cell wall compositions that are intermediate between these two types, one example being the pineapple (Smith and Harris, 1995).
Stylized general structure of a plant primary cell wall. The precise structure of the plant cell wall varies between species and tissues and has yet to be fully elucidated. However, it is generally agreed that the cell wall is composed of cellulose fibrils embedded in a matrix of hemicelluloses, pectin, and structural protein (Carpita and Gibeaut 1993, Somerville et al., 2004; Cosgrove, 2005; Carpita, 1996; Doblin et al., 2010; Albersheim et al., 2011). The hemicellulose polymers, in particular, are very diverse in composition but are thought to be able to hydrogen bond to the surface of the cellulose fibrils and also to span the gaps between adjacent fibrils (Scheller and Ulvskov, 2010). This cellulose/hemicellulose framework is then embedded in a matrix of pectin and structural proteins. The relative amounts of the various components of the wall can vary significantly between species. Most fruits, which are dicotyledonous species, are relatively rich in pectin compared with, for instance, the grasses (Toivonen and Brummell, 2008). The cell wall also contains a number of enzymes and one role for these is presumably the turnover of the wall components to accommodate the growth of the cell and the changes in texture associated with ripening of the fruit.
Stylized general structure of a plant primary cell wall. The precise structure of the plant cell wall varies between species and tissues and has yet to be fully elucidated. However, it is generally agreed that the cell wall is composed of cellulose fibrils embedded in a matrix of hemicelluloses, pectin, and structural protein (Carpita and Gibeaut 1993, Somerville et al., 2004; Cosgrove, 2005; Carpita, 1996; Doblin et al., 2010; Albersheim et al., 2011). The hemicellulose polymers, in particular, are very diverse in composition but are thought to be able to hydrogen bond to the surface of the cellulose fibrils and also to span the gaps between adjacent fibrils (Scheller and Ulvskov, 2010). This cellulose/hemicellulose framework is then embedded in a matrix of pectin and structural proteins. The relative amounts of the various components of the wall can vary significantly between species. Most fruits, which are dicotyledonous species, are relatively rich in pectin compared with, for instance, the grasses (Toivonen and Brummell, 2008). The cell wall also contains a number of enzymes and one role for these is presumably the turnover of the wall components to accommodate the growth of the cell and the changes in texture associated with ripening of the fruit.
The cellulose fibril structure is relatively conserved. Cellulose polymers occur as long (up to 5000 residues) linear chains of β-1,4 linked glucose residues. These are found grouped together in fibrils (Somerville, 2006). The precise number of polymers in each fibril is still a matter of debate and may vary between cell walls, but is generally considered to be between 24 and 36. The polymers are aligned parallel to each other, held together by hydrogen bonds to give a crystalline structure that is very resistant to degradation. The degree of crystallinity may vary along the length of the fibril (Viëtor et al., 2002; Šturcová et al., 2004) and again between cell walls. The fibrils themselves also tend to be aligned parallel to each other within each plane of the cell wall.
Hemicelluloses are represented by a very diverse range of structural polymers (Scheller and Ulvskov, 2010). In type I walls of dicotyledonous species, a major hemicellulose is xyloglucan. This polymer also has a backbone of β-1,4 linked glucose, but in this instance, this is substituted by single xylose residues, some of which may be further substituted by galactose and fucose residues (Figure 3). In contrast, the predominant hemicelluloses in type II cell walls are based on xylans. In this case, there is often a backbone of β-1,4 linked xylose residues again, with substitutions, except that a major sugar substituent is arabinose. The xylose residues can also be acetylated in some cases. Other hemicelluloses often present in type II walls include galactomannans. Although these hemicelluloses have very different monomeric compositions, they often seem to share a common structural feature in that they are thought to be capable of hydrogen bonding to the surface of the cellulose fibrils and also embedding themselves in the para-crystalline regions of the cellulose fibril (Pauly et al., 1999; Rose and Bennett, 1999). It has also been shown that, in some instances, the hemicellulose can traverse the gap between adjacent cellulose fibrils and may serve to act as tethers between the fibrils.
Stylized xyloglucan structure. Xyloglucan is a major hemicellulose polymer found in many fruits of dicotyledonous species. It is composed, like cellulose, of a backbone of β-1,4 linked glucose residues and this is thought to allow it to hydrogen bond to the surface of cellulose fibrils. The backbone, unlike cellulose, is substituted with α-1,6 linked xylose residues, these being thought to be present in a repeat sequence as shown above. These xylose residues may be further substituted with β-1,2 linked galactose and α-1,2 linked fucose residues to form short side chains attached to the glucan backbone. These side chains are thought to prevent further hydrogen bonding between polymers (Scheller and Ulvskov, 2010).
Stylized xyloglucan structure. Xyloglucan is a major hemicellulose polymer found in many fruits of dicotyledonous species. It is composed, like cellulose, of a backbone of β-1,4 linked glucose residues and this is thought to allow it to hydrogen bond to the surface of cellulose fibrils. The backbone, unlike cellulose, is substituted with α-1,6 linked xylose residues, these being thought to be present in a repeat sequence as shown above. These xylose residues may be further substituted with β-1,2 linked galactose and α-1,2 linked fucose residues to form short side chains attached to the glucan backbone. These side chains are thought to prevent further hydrogen bonding between polymers (Scheller and Ulvskov, 2010).
The structure and synthesis of pectins has been reviewed (Mohnen, 2008). The three major pectic polymers homogalacturonan (HMG), rhamnogalacturonan I (RhaI), and rhamnogalacturonan II (RhaII) have a generally conserved structure (Figure 4). HMG consists of chains of α-1,4 linked galacturonic acids, the individual monomers of which may exist as either a the free acid or a methyl ester. Where there are blocks of de-esterified residues on adjacent polymers, these may interact through calcium chelation to form so-called egg box structures (Grant et al., 1973). RhaI consists of a backbone of alternate galacturonic acid and rhamnose monomers, with rhamnose being substituted by side chains of galactose and arabinose residues. Individual walls can vary in the proportion and fine structure of these polymers. RhaII is a very complex polymer consisting of 12 sugars and 20 different glycosidic bonds. It is only present in small amounts compared with HMG and RhaI, and seems to be highly conserved. More recently, a fourth pectic polymer, xylogalacturonan, has been identified, and this has been found in several fruits, including watermelon and apple (Thibault and Ralet, 2001).
Stylized structures for homogalacturonan and rhamnogalacturonan I. The precise structure of pectin is yet to be fully elucidated. However, it is generally agreed that there are four types of pectic polymer commonly found in plant cell walls. The two major forms are probably homogalacturonic acid (HMG) and rhamnogalacturonic acid-1 (rha-1). HMG consists of a backbone of α-1,4 linked galacturonic acids. These may contain either a free or methylated carboxyl group at the C6 position. The degree of methylation (or esterification) can vary between tissues and indeed appears to decrease during ripening of many fruits. Similarly, the pattern of methylation (either single residues or blockwise) may vary between tissues and stages of development. The rha-1 consists of an alternating backbone of galacturonic acid and rhamnose residues. The rhamnose residues may be substituted with side chains of arabinose and galactose residues. Again the proportion of rha-1 and the side chain prevalence and composition may vary with species, tissue, and stage of development (Mohnen, 2008).
Stylized structures for homogalacturonan and rhamnogalacturonan I. The precise structure of pectin is yet to be fully elucidated. However, it is generally agreed that there are four types of pectic polymer commonly found in plant cell walls. The two major forms are probably homogalacturonic acid (HMG) and rhamnogalacturonic acid-1 (rha-1). HMG consists of a backbone of α-1,4 linked galacturonic acids. These may contain either a free or methylated carboxyl group at the C6 position. The degree of methylation (or esterification) can vary between tissues and indeed appears to decrease during ripening of many fruits. Similarly, the pattern of methylation (either single residues or blockwise) may vary between tissues and stages of development. The rha-1 consists of an alternating backbone of galacturonic acid and rhamnose residues. The rhamnose residues may be substituted with side chains of arabinose and galactose residues. Again the proportion of rha-1 and the side chain prevalence and composition may vary with species, tissue, and stage of development (Mohnen, 2008).
Changes in cell walls during ripening
There have been several reviews covering the structural and compositional changes that occur in the cell walls of ripening fruits (Rose et al., 2003; Brummell, 2006; Vicente et al., 2007; Toivonen and Brummell, 2008; Li et al., 2010; Ruiz-May and Rose, 2012). There are changes to the structure and chemical composition of most of the fruit cell wall polymers during ripening (Brummell, 2006), and although these may vary between species (Toivonen and Brummell, 2008), there are some common features. These changes affect the cell wall ultrastructure and can be detected by light and electron microscopy (Figure 5). During ripening of fruit, the middle lamella can be seen to swell (Redgwell, 1997), indicating hydration. This is thought to be brought about by Donan forces resulting from increases in the fixed charge on the pectin polymers.
Ultrastructural changes caused by pectolytic enzymes during ripening of tomatoes. A) Ethylene-induced ripening: Transmission electron micrograph of walls of two adjacent cells from a mature green tomato incubated in ethylene for four days. Note the swelling and appearance of fibrils in the central region (middle lamella) Scale bar 1 micrometre. Transmission electron micrograph of walls of green tomato cells (B) and similar tissue incubated overnight in purified tomato polygalacturonase from ripening fruit. Note the swelling and the appearance of fibrils in the middle lamella region. Scale bar 1 micrometre. (Photographs A, B, and C from P. Crookes, PhD thesis 1985; see also Crookes and Grierson 1983. D and E.) Transmission electron micrograph images of tomato parenchyma cells from a GM tomato line in which PL has been inhibited by RNAi. (D) showing individual cells and a tricellar junction boxed (centre). (E) Higher magnification view showing fibrous material in the tricellular junctions labelled with JIM5, a monoclonal antibody to demethylesterified homogalacturonan. Scale bars 200nm. See also Uluisik et al., 2016.
Ultrastructural changes caused by pectolytic enzymes during ripening of tomatoes. A) Ethylene-induced ripening: Transmission electron micrograph of walls of two adjacent cells from a mature green tomato incubated in ethylene for four days. Note the swelling and appearance of fibrils in the central region (middle lamella) Scale bar 1 micrometre. Transmission electron micrograph of walls of green tomato cells (B) and similar tissue incubated overnight in purified tomato polygalacturonase from ripening fruit. Note the swelling and the appearance of fibrils in the middle lamella region. Scale bar 1 micrometre. (Photographs A, B, and C from P. Crookes, PhD thesis 1985; see also Crookes and Grierson 1983. D and E.) Transmission electron micrograph images of tomato parenchyma cells from a GM tomato line in which PL has been inhibited by RNAi. (D) showing individual cells and a tricellar junction boxed (centre). (E) Higher magnification view showing fibrous material in the tricellular junctions labelled with JIM5, a monoclonal antibody to demethylesterified homogalacturonan. Scale bars 200nm. See also Uluisik et al., 2016.
At a molecular level, the earliest detectable change seems to be a loss of neutral sugars, galactose, and arabinose, (Gross and Wallner, 1979) presumably from the side chains of RhaI. This can occur slightly before the onset of other aspects of ripening. There is also a decrease in the degree of methylation of the pectin. In tomato, this reduces from 90% in green fruit down to about 35% in ripe fruit (Koch and Nevins, 1989). This reduction in methylation increases the negative charge on the pectin and is presumably the major cause of the Donan forces that result in swelling of the middle lamella. There is also a significant depolymerization of the pectin polymers.
These changes are brought about by enzymatic action within the wall; indeed, Hobson (1964) demonstrated that ripe tomato fruit contained sufficient enzyme activity to completely de-esterify and depolymerize the pectin in the cell wall within about 4 min of the fruit pericarp being homogenized. This suggests that the enzyme activities in situ must be very tightly regulated and suppressed.
Enzymes involved in cell wall metabolism during ripening
These ultrastructural changes are due to the activity of a range of enzymes that modify the structure, physical, and chemical properties of various cell wall components. It would appear that a similar range of wall-modifying enzymes are present in most fleshy fruit, but that their relative activities may vary (Brummell, 2006). A number of enzyme activities have been identified as being associated with the cell walls of ripening fruit and some key examples are shown in Table 1 along with their proposed modes of action. This list is not exhaustive but represents the most intensively studied enzymes believed to have a potential role in fruit softening.
Cell wall modifying enzymes associated with fruit ripening.