Cell Wall Enzymatic Activity Control: A Reliable Technique in the Fruit Ripening Process

Abstract

The softening and structural changes that occur during fruit ripening are characteristic of specific species and can be attributed primarily to cell wall composition and cell swelling. Cell wall modifications are thought to result in changes in stiffness and texture, but the nature and extent of changes that occur during maturation vary widely. While some cell wall changes associated with ripening, such as depolymerization of matrix glycans, appear to be universal, other changes are highly variable in degree or present in different fruit types. However, the common point in all species is the involvement of the activities of enzymes linked to maturation in all these modifications, in particular the pectinolytic enzymes, namely polygalacturonase (PG), β-galactosidase (β-Gal) and pectin methyl esterase (SME). For good management of these changes, which have considerable consequences on the quality of fruits and their fate in post-harvest, the control of the activities of pectinolytic enzymes seems essential, which is what we propose to study in this chapter.

1. Introduction

Ripening of fruits and vegetables is one of the final development stages of product ontogeny and involves many genetic, biochemical, and physiological changes. These changes include pigment and sugar accumulation, aromatic compound production, and meat tenderization [1]. These changes evolved to make fruits more attractive and edible to seed-dispersing organisms. Ripening also improves the sensory properties of the fruit, making it suitable for human consumption. However, once an advanced stage of ripening is reached, fruit quality deteriorates, mainly due to excessive fruit softening, increased susceptibility to pathogens, development of undesirable taste and skin color, etc., resulting in significant fruit management challenges and economic loss. Not only post-harvest longevity but also other economically important aspects determine yield [2]. Therefore, the rate of softening depends on handling procedures, harvest frequency, and the distance the fruit can be transported. Indeed, slowing down fruit softening is one of the main objectives and challenges of most product selection programs. This delay requires effective control of the factors involved in fruit ripening, especially softening.

2. Texture, a key criterion of ripening

Hardness and juiciness are the most important structural elements of fleshy fruits [3]. Both properties are primarily determined by parenchymal cell properties (cell wall thickness, shape, size, strength, and cell turgor pressure) and the extent and strength of adhesion zones between adjacent cells. During ripening, the parenchymal cell wall undergoes significant changes, altering its mechanical properties and greatly reducing cell adhesion due to the dissolution of the intermediate lamellae. Changes in the cell wall and intermediate lamellae that lead to fruit softening are usually caused by the activity of cell wall-modifying enzymes (e.g., polygalacturonase, pectin methylesterase, pectate lyase, β-galactosidase, and cellulase) encoded by ripening-related genes [4, 5]. Other cell wall proteins without hydrolase activity, such as B. expansin, also play a softening role [4]. In general, the cell wall degradation processes responsible for softening include depolymerization of matrix glycans, solubilization and depolymerization of pectin, and loss of neutral sugars from pectin side chains [5, 6]. The extent of these changes varies considerably between species. Recently, it has been suggested that the structural integrity of the xyloglucan network maintained by xyloglucosyltransferase/endohydrolase (XTH) may be important during fruit softening. This activity is usually higher during fruit development and then decreases or remains constant during ripening [5]. Miedes and Lorences [7] suggested that the XTH gene may be involved in the maintenance of cell wall structure rather than its degradation, and therefore, decreased expression and activity of the XTH gene may contribute to cell wall softening. This hypothesis is supported by the fact that overexpression of the SlXTH1 gene in tomatoes reduces fruit softening [7]. On the other hand, although less studied than cell wall degradation, cell swelling also affects fruit tenderness. During fruit ripening, a decrease in turgor pressure is often observed as the accumulation of dissolved apoplast is regulated. Water loss by transpiration through the cuticle may also be relevant, especially in fruits with thick, well-developed cuticles such as tomato [8]. Cell turgor pressure can also be influenced by cell wall changes that occur during fruit softening, so active changes in turgor pressure can be combined with passive moisture. It is difficult to distinguish from effects due to loss or changes in cell wall mechanical properties of fruit.

3. Main factors of softening during ripening

One of the main factors that reduce the quality of fruits and vegetables and cause significant economic losses is excessive softening. Changes in texture and changes during fruit ripening are mainly due to the dissolution of the interlayer due to the action of enzymes that modify the cell wall, the reduction of cell–cell adhesion, and the weakening of the parenchymal cell wall. Pectin, the main component of fruit cell walls, undergoes significant changes during ripening. These changes include solubilization, depolymerization, and loss of neutral side chains. Our work on apricot fruit [9, 10] and and recent evidence on strawberries [11] and apples [12, 13] characterized by a soft or crispy texture at maturity suggest that pectin disassembly is a key factor in texture changes during ripening. This change is mainly due to a commonality of active biomolecules, namely pectinolytic or pectic enzymes.

4. Pectinolytic enzymes

Pectinolytic enzymes or pectinases are a heterogeneous group of enzymes that hydrolyze pectic substances and are widespread in higher plants and microorganisms. This family of enzymes is able to attack a variety of chemical bonds in pectins. The term “pectinolytic enzyme” relates only to enzymes that act on the galacturonic part of pectic substances (Figure 1), and the enzymes capable of degrading the side chains are not classified among the pectolytic enzymes.

Figure 1.

Pectinase interaction model: (a) pectin esterase interaction model, (b) polygalacturonase [14].

The enzymatic activity of the cell walls is linked above all to the pectolytic enzymes, which take part in the structural evolution of the wall by ensuring rearrangements or the degradation of the parietal polysaccharides and modifying and/or hydrolyzing the main components of the matrix. Different pectolytic enzymes are involved in these modifications. Pectinases are involved in the modification of parietal polysaccharides. For the majority of fruits, most of the cell wall changes are explained by the increased enzymatic activities of polygalacturonase (PG), β-galactosidase (β-Gal), and pectin methyl esterase (PME) [10, 15, 16].

4.1 Polygalacturonase

Polygalacturonases (PG) are glycosidases that hydrolyze the α-(1 → 4) glycosidic bond between unesterified galacturonic acid residues and in homogalacturonic chains (Figure 1). Recent works [16, 17] have shown differences in PG activity in different varieties of apricot but with low levels compared to the activities found in other fruits and vegetables [18]. Cardarelli et al. [19] did not detect polygalacturonase activity in apricots. Endo-polygalacturonase (endoPG) activity has been associated with loss of firmness in several species, but this has never been confirmed in apricots. In tomatoes, the observation of a strong endo-PG activity in ripe fruits showed that PGs play an important role in the loss of firmness. For peaches, the activity of polygalacturonases in different cultivars is positively correlated with the loss of firmness and is only induced when the fruit ripens.

Two methods have been developed to determine the activity of PGs. This activity can be monitored by measuring the decrease in viscosity or the increase in the reducing power of the substrate (pectic acid or pectin). The comparison of viscosity and reducing power measurements during the depolymerization of pectins and pectic acids makes it possible to distinguish between “endo” and “exo” PG activities.

4.2 Pectin methylesterase

Pectin methylesterase (PME, EC 3.1.1.11) is an enzyme ubiquitous inside the plant kingdom. However, its function in plant increase and improvement continues to be unclear. Pectin methylesterase (PME) is involved in the loss of firmness by demethylating pectin (Figure 1), making it sensitive to the activity of PGs. Ünal and Şener [20] studied the biochemical properties of MSY in Alyanak apricots (an important variety in the Malatya region of Turkey). This PME has a high activity for a pH between 7.0 and 8.0 with a maximum activity at pH 7.5, knowing that the pH of an apricot fruit for example generally varies between 3.3 and 4.0. The enzyme is stable at temperatures between 30 and 40°C for 10 min and loses all its activity after 10 min at 80°C, which implies that PME can easily be inactivated by a pasteurization process during the processing of mumps apricots in syrup or jam. Other recent studies have reported PME activity in apricot fruit [10, 16, 17, 19, 21].

The activity of the SME is influenced by several factors, in particular, the stage of maturity of the fruits. PG and PME activities increase significantly (p < 0.05) with maturity (green, green mature, and ripe) [17]. In all the apricot cultivars studied, the PG activity increased from 713 to 14,286 nkat mg⁻¹. The highest PME activity was found at the ripe stage: green (45 nkat mg⁻¹) and ripe (128 nkat mg⁻¹) fruits. However, Ribas-Agusti et al. [16] reported that MSY activity tends to decrease with ripening for the studied apricot varieties.

The activity of PE can be monitored either by assaying the methanol released, or by determining the increase in the number of free carboxyls, or even by using a pH regulator. Indeed, the ionization of the carboxyl group produces a proton in the medium, which causes a variation in pH. PE is inhibited by the increase in the number of free carboxyls along the progressively demethylated polygalacturonic chains. This inhibition is due to the repulsion exerted by the negative charge of the ionized carboxyls. The presence of cations (Ca²+ and Na+) could counteract this inhibition. This inhibition of PEs would also be due to the side chains of neutral sugars in the pectin molecule [22].

4.3 β: D-galactosidase

The hydrolysis of cell wall pectins during maturation is also linked to the activity of glycosidases and especially β-D-Galactosidase which hydrolyzes galactosyl polymers. β-galactosidase leads to the loss of galactose units from the side chains [23]. Galactose was found as a product of this activity during tomato ripening. Kovács and Szerdahelyi [24] reported that the stage of maturity greatly influences the activity of apricot galactosidases. Ribas-Agusti et al. [16] also reported that β-Gal activity tended to increase during maturation for all cultivars analyzed. Other work has shown that the solubilization of pectins by β-galactosidase accelerates the softening of fruits in general [4] and melon in particular.

5. Ripening management and enzymatic activity control

Recent studies [10, 12, 13, 16, 25] have shown that the degradation of polysaccharides in the cell wall results in A synergistic effect occurs between several enzymes that modify the cell wall, including PME, PG, and β-GAL. Consistent with our study and other reports [26, 27], apricot fruit wall enzyme activity increased during the fruit ripening process. Indeed, these enzymatic activities represent a major asset for managing the ripening of fruits and vegetables and determining the stages of development, the optimal stage of harvesting, and post-harvest management.

Fan et al. [25], recently, reported that the use of suppressed NFT storage can inhibit the enzymatic activities. Indeed, the PME activity of apricots increased rapidly from the beginning of storage at 5°C, but this increase in enzyme activity was effectively inhibited by storage at 0°C and NFT. Compared to storage at 0°C, NFT storage reduced the enzyme activity of apricots to a lower level. The PME activity of apricots stored in NFT was 88% of that of apricots stored at 0°C on day 60.

Moreover, the changes in apricot enzyme activities showed similar trends for all cell wall enzymes.

Genetically, in fruits, softening was reduced due to the antisense downregulation of polygalacturonase genes [28]. Indeed, changes in pectic polymer size, composition, and structure have traditionally been studied by conventional techniques; other studies focusing on changes at the nanostructural and genetic level have reported that gene regulation of enzymes is a solution for a better management of their activity during maturation and indeed consequences on the harvest [29].

Pectin methylesterase (PME) activity is controlled by a family of protein inhibitors called pectin methylesterase inhibitors (PMEIs) (Figure 2). Therefore, the interaction of PME and PMEI is considered not only as a determinant of cell adhesion, cell wall porosity and elasticity, but also as a source of release of signaling molecules during cell wall stress during fruit development stages. Wormit and Usadel [29], highlighted the importance of the PMEI gene family, its regulation and structure, its interaction with PMEI, and its function in response to stress during fruit and vegetable development and crop management.

Figure 2.

Schematic diagram of HG demethylesterification and its effect on structure. HG is highly methylesterified when deposited on the cell wall. PME can demethylate HG in blocks, resulting in multiple contiguous GalA residues without methyl ester groups. Because these HG scaffolds are negatively charged, they can cross-link with cations such as calcium ions, resulting in the formation of so-called “egg-crate” structures that are responsible for gel formation. On the other hand, PME can demethylate individual HisGalA residues, causing random methyl esterification patterns. Low-level HG methyl ester is depolymerized by pectinolytic enzymes such as pectin/pectate lyase (PL) and polygalacturonase (PG), leading to the formation of oligogalacturonides (OG). In contrast, PME activity is inhibited by the protein inhibitor PMEI.

6. Conclusion

Textural changes in fruits during ripening are a result of cell wall changes implied by enzymatic activity. These modifications have considerable consequences on the quality of the fruits and their fate after harvest; in fact, controlling the activities of pectinolytic enzymes is a key to managing the harvest and optimizing fruit quality.

Indeed, control tools have been developed and tested, controlling the activity of pectinolytic enzymes, specifically PME, PG, and β-GAL, during ripening is a preventive measure in the management of the fruit harvest. The choice of fruit storage conditions after harvest, such as NFT storage, is a solution that makes it possible to inhibit the activity of enzymes. Other studies are underway on the genetic regulation of pectinolytic enzymes to manage and modify their activity. This will make it possible to optimize the quality of the fruits, both for the consumer market and also for industrial processing.