Introduction

Cell wall is a network of polysaccharides mainly composed by a complex matrix of cellulose, hemicellulose, and pectin, which together are fruit firmness responsible (Harker et al. 1997; Cosgrove 2000; Brummell and Harpster 2001; Brummell 2006). Across the ripening, genes encoding enzymes which catalyze polysaccharides, an increasing in relative expression and their enzymatic activity are observed on cell wall polysaccharides (Cosgrove et al. 2002; Ramos et al. 2018). Expansins are not classified as enzymes; this fact has been demonstrated observing the mechanism of action: (1) they do not alter the molecular composition of cell wall; (2) the activity does not produce a weaking of the cell wall as hydrolases; (3) they have affinity to the insoluble cell wall fraction, preferably to cellulose attached to hemicellulose (McQueen-Mason and Cosgrove 1995). The classification of expansins has been reported in several studies (Cosgrove 2000, 2015; Sampedro and Cosgrove 2005), but essentially exists five classification groups described as alpha-expansin (EXPA) beta-expansin (EXPB), expansin-like A (EXLA), expansin-like B (EXLB) and expansin-like X (EXLX) (Kende et al., 2004; Choi et al. 2008), whose differences have been discussed in the literature (Sampedro and Cosgrove 2005). Alpha-expansins are related to acid growing with pH dependence, producing stress relaxation and wall creep, cleaving non-covalent bonds between cellulose and xyloglucan polysaccharides (Cosgrove 2015). This phenomenon that must occur for cell enlargement in plant development and organogenesis (Cosgrove 1997; McQueen-Mason and Rochange 1998). Marowa et al. (2016) and Samalova et al. (2022), describes an extensive list with alpha-expansin involved in development and stress response in plants (Table 1). However, has been demonstrated that expansins are related with the fruit ripening of apricot, banana, mango, pear, mountain papaya, Chilean and commercial strawberries (Mbéguié-A-Mbéguié et al. 2002; Hiwasa et al. 2003; Sane et al. 2005, 2007; Gaete-Eastman et al. 2009; Valenzuela-Riffo et al. 2019; Ren et al. 2023). Today, in front to the climatic change and increasing of world population, researchers must be developing new strategies and solutions to prolong fruit shelf life and to help to increase the tolerance to environmental changes. For this reason, this review provides an integrative view of alpha-expansins involved in fruit ripening, describing experimental and in silico studies and phytohormones dependence-response.

Table 1 Research of the last ten years with alpha-expansins related in another biological process in plants different to ripening

Alpha-expansins on fruit ripening and postharvest

Different experimental strategies have made it possible to elucidate through the years that alpha-expansins has a principal role in fruit ripening, such as relative expression, genetic modification, SNPs, lncRNA and genomic analysis, among others (Table 2). For example, relative expression of alpha-expansins genes is different between cultivars of Fragaria x ananassa (Dotto et al. 2006; Ramos et al. 2018). Dotto et al. (2006) showed that expansin genes have differential expression level during fruit ripening in the different developmental stage, contrasting firmness strawberry. The authors found a correlation between mRNA expression levels and fruit firmness for FaEXPA1, FaEXPA2 and FaEXPA5. For these three mRNAs, higher expression levels were observed in the softest ‘Toyonaka’ than in the other two firmer ‘Selva’ and ‘Camarosa’ at the beginning of ripening. Similar results were observed by Ren et al. (2023), where FaEXPA5 display higher level of transcripts in nearly red and red developmental stages in the cultivar ‘Yuexiu’ in comparison of ‘Hongjia’ and ‘Yuexin’ cultivars; but in the case of ‘Yuexin’ the authors suggest that FaEXP7 could be a potential softening activator, expressed in initial red developmental stage. This information could be related with the firmness changes, where strawberry ‘Yuexiu’ is the firmest and ‘Yuexin’ is the softest. In the parental mother of the commercial strawberry, Fragaria chiloensis three alpha-expansin were described, even though the three expansins genes that show an increase throughout fruit ripening, the expression profile increases at different times and developmental stage (Valenzuela-Riffo et al. 2019). In another fruit such as Punica granatum L. ‘Taishanhong’, 33 expansins were found, and 25 are classified as alpha-expansins. However, only PgEXP5, PgEXP23 and PgEXP31, are related with cell wall and fruit ripening (Xu et al. 2023). Similar results were found in Solanum lycopersicum, and only SlEXPA1 is expressed mainly in fruits (Lu et al. 2016). In Ziziphus jujuba Mill., only ZjEXPA4 and ZjEXPA6 displays a high relative expression in pre-ripe fruit stage (Hou et al. 2019). In Fragaria vesca 35 FvEXPs were found, which 27 are alpha-expansins, but only FvEXPA9, FvEXPA12 and FvEXPA27 are highly expressed during turning and red developmental stage of fruit (Dong et al. 2022). In some cases, alpha-expansins genes can be in QTL associated to ripening characteristics, such as sugar total content as in the case of PpEXP5 and PpEXP6 in four cultivars of Pyrus pyrifolia. These genes were upregulated, relating this fact with the increase in sugar content during the fruit ripening process (Jiang et al. 2023). Relative expression of alpha-expansins in fruit ripening is regulated by cis elements principally in response to phytohormones such as ABA (ABRE), auxin (ARFAT and SAUR) (Valenzuela-Riffo et al. 2020). Ethylene response has been demonstrated experimentally by Gaete-Eastman et al. (2009) in fruits treated with the phytohormone, where an increase of relative expression until 86 times were observed in VpEXPA2, and not relative expression were observed in fruit treated with ethylene inhibitor, 1-MCP. Similar results were observed in banana where MaERF11 binds to MaEXP2, MaEXP7 and MaEXP8 (Han et al. 2016), demonstrating ERFs acting as cis-regulatory elements. In Rubus chingii has been described five alpha-expansins regulated by MYBs and MYCs in wound or drought stress conditions (Chen et al. 2024). Li et al. (2014) suggests the presence of MYB and MYC elements in drought, low temperature, salt, ABA and GA stress responses, demonstrating cell wall remodeling under stress conditions. the above described is affirmed in the publication of Nardi et al. (2016), where WRKY transcription factors, linked to the presence of gibberellins, ABA and wound stress, are also added. Coincidence has been observed in the cis-regulatory elements between fruit ripening and another process such as seed germination in A. thaliana (ABA and gibberellins, light, and specific elements responses), fiber cell growth in Gossypium hirsutum (light, ABA, methyl jasmonate, ethylene, gibberellins, auxin, and wound), and drought tolerance in Triticum aestivum (light, ABA, methyl jasmonate, gibberellins, drought inducibility, and circadian control responses) (Chen et al., 2020; Ferreira Ribas et al. 2019; Lv et al. 2020).

Table 2 Reports of alpha-expansins related in ripening and postharvest in the last ten years

Genetic modification also is an experimental technique used to demonstrate the relevance of expansins in fruit ripening. For example, in tomatoes, suppression of Exp1 is related to fruit firmness, and overexpression with softening from green stage, which Exp1 could help to relax the wall (Brummell et al. 1999). Because the fruit ripening process is orchestrated by the activity of different enzymes, Powell et al. (2023) and Su et al. (2024) has been demonstrated a synergistic effect between expansin and another cell wall enzymes. Building of a knockdown of SlExp1 and SlCel2 increase fruit firmness, and their overexpression promote an early softening. In another hand, knockdown of LeExp1 and LePg produces firmer and less susceptible to microorganisms fruits. In both publications, it was shown that knockdown or overexpression of genes separately does not generate the same impact on the fruit.

In Rubus genus was found four SNPs in Ra_g7125 gene (Arabidopsis thaliana homolog ID At4g38210). This gene was related to firmness and red drupelet reversion disorder in blackberry (Chizk et al. 2023). SNPs are useful to select candidate markers in ‘Hanuko’ peaches; ppa017982m and ppa010443m can be useful to select fruit with a good weight and size (Cao et al. 2016). Similar results were found by Huang et al. (2023), where alpha-expansins has a positive effect on fruit size of Prunus armeniaca ‘Sungold’. Tiny molecules such as non-coding RNA (lncRNA) and circular RNA (circRNA) were evaluated in two genotypes, DC-48 and DC-83 of Cucumis melo, demonstrating that lncRNA and circRNAs acting as regulators of CmEXPA1 and CmEXPA3, because this molecule has a target gene related with fruit firmness and in DC-48 the shelf-life is extended until 10–15 days (Dey et al. 2022).

Studies in transcription factors also allow to understand the relationship between alpha-expansins and fruit ripening. MaERF11 of Musa acuminata negatively regulates the ripening process through the transcriptional regulation of MaEXP2, MaEXP7 and MaEXP8 (Han et al. 2016). Genome analysis of gene family was chosen to support the predictions on the genome sequence of Pyrus communis L. ‘Bartlett’. The authors found that PcEXP1, PcEXP2, and PcEXP3 are involved in fruit softening (Spera et al. 2023).

Response to phytohormones

Phytohormones are important regulator of the fruit ripening process because their concentration in the different developmental stages can promote plant responses and physiological changes (Chagné et al. 2014). There are publications demonstrating the relevance of phytohormones in ripening and loss of firmness, and their relationship with the expression levels of alpha-expansin genes. For example, four response elements to abscisic acid were found in the promoter region of FaEXPA5 from F. x ananassa. This information could be related to changes in the fruit firmness and relative expression in the six developmental stages, which increase in turning, 75%R and R (Valenzuela-Riffo et al. 2020). In silico analysis of FaEXP2 promoter revealed response elements to circadian control, light, drought, abscisic acid, gibberellin. Treatments with hormones has a relevant response with changes in the relative expression with abscisic acid and auxin with fruits without achenes (Nardi et al. 2016). Another studies in Fragaria genus, showed that abscisic acid applications to F. chiloensis fruit trigger an increase in the expression of FcEXP5 after 24 h of treatment, contrarily to FcEXP2, which increase their expression under fluridone treatment at 24 h. Abscisic acid vs control treatments were evaluated until the fourth day, observing an increase in the expression of FcEXPA2 and FcEXPA5, concluding a direct relationship with the ripening and loss of firmness (Mattus-Araya et al. 2022). Unfavorable conditions such as fruit cracking have been evaluated in Ziziphus jujuba Mill.’Pingshunbenzao’. Treatments with abscisic acid and methyl jasmonate in the white ripening stage, showed an increased in the cracking index after 48 h, concomitantly with differential expression of genes involved in cell wall modification such as ZjEXPA (Contig12.0.7) (Liu et al. 2023). In contrast, applications of the ethylene perception blocker (1-methylcyclopropene; 1-MCP), extend the shelf-life fruit in Psidium guajava L. and produce higher firmness and down regulation of genes such as PgEXP1 (Sachin et al. 2021). Other example of the effect of the ethylene and 1-MCP treatments in Mountain papaya fruits was described by Gaete-Eastman et al. (Gaete-Eastman et al. 2009). The authors observed a reduction in transcription level of VpEXPA2 in fruit treated with the 1-MCP, while an earlier and higher transcript level was observed in ethylene-treated fruit (after 1 day of treatment), suggesting that VpEXPA2 expression is regulated by ethylene.

Biotic and abiotic stress response of alpha-expansins

It has been observed that alpha-expansins genes are induces in biotic stress conditions to remodelling cell wall in response tobacco mosaic virus, potato virus Y (Chen et al. 2017; Otulak-Koziel et al. 2019). Samalova et al. (2022) describes a summary of alpha-expansins in response to abiotic stress remodelling cell wall, where demonstrated participation in tolerance to cadmium, cold, drought, H2O2, heat, osmotic stress, and salt, in plants as well as Nicotiana tabacum, T. aestivum, Camellia sinensis, Craterostigma plantagineum, Erianthus arundinaceus, Saccharum spp. hybrid, Zea mays, A. thaliana, Agrostis scabra, and Agrostis stolonifera. Another works is related with O2 deficiency in Rumex palustris, aluminum tolerance in Oryza sativa, (Colmer et al. 2004; Tsutsui et al. 2012; Tenhaken 2014). Relative expression of expansin genes has been described in Cucumis pepo and Cucumis sativus, in root and leaves under different stress conditions: salt, cold, drought, abscisic acid applications, and heat stress, where CpEXPA1, CpEXPA18, and CpEXPA20, and CsEXPA8 and CsEXPA11, increase the relative expression in these stress conditions (Arslan et al. 2021). In a similar study, ClaEXPA4, ClaEXPA6, and ClaEXPA9 from Citrullus lanatus and CmEXPA10, CmEXPA12, and CmEXPA13, from Cucumis melo respond positively in the same stress conditions and tissues (Symons et al. 2012). Pyrus communis displays changes in their cell wall and expression levels of genes such as PcEXP1 in sunburn injury (Spera et al. 2023). Cold stress causes stony hard in Prunus persica L. Batsch showing 91 differential expressed genes. Transcript levels of two genes that coding of expansin Prupe.5G047300 and Prupe.5g057900, was upregulated after 20- and 40-days storage to 4 °C. These results were correlated with loss of firmness and texture changes, where soft pulp and decreasing juice content has been observed (Wang et al. 2021).

In biotic stress, alpha-expansins are relevant too. Grapevine red blotch virus disease (GRBV) causes damages from $2213/ha to $68,548/ha in the United States. Two alpha-expansins and other genes of cell wall-related enzymes were upregulated in the harvest stage in fruits with GRBV, relating the increase of transcript levels with the cell wall metabolism in infected tissues (Rumbaugh et al. 2023).

Bioinformatics approaches as a source of knowledge

Various studies have described crystallographic structures of beta-expansin proteins (Incili et al. 2022), and expansins-like (Yennawar et al. 2006; Georgelis et al. 2011; Incili et al. 2022). However, unfortunately, it has not been possible to obtain crystallographic structures of alpha-expansins. For this reason, structural bioinformatics approaches have had a positive impact trying to understand the molecular mechanisms by which alpha-expansins interact with several putative ligands. Additionally, because it is still unknown how to quantify the expansins activity, structural bioinformatics offers approaches to understand their action mechanisms and interaction with different cell wall polysaccharides. In this context, protein–ligand interaction a molecular modelling of VpEXPA2 from Vasconcellea pubescens was conducted using as reference structure the crystallographic beta-expansin from Phleum pratense showed that in the active site the acid residue (Asp104, member of the HFD motif) occupies the same position and orientation that the template. The hydrophobic environment facilitating Asp104 comprises the residues Ala41, Ala42, and Tyr13. This setting is expected to stabilize the protonated state of Asp104, enabling its role as a catalytic acid. Its function involves protonating the glycosidic oxygens in polysaccharides, such as cellulose, as elucidated in endoglucanase V from Humicola insolens (EGV) (PDB: 2ENG) (Davies et al. 1995). However, the authors propose that in VpEXPA2 lacks the residue corresponding to the basic Asp10 (Gaete-Eastman et al. 2015). The nearest Asp residue (Asp17) is situated too far away to directly partake in the hydrolytic reaction, with 16.7 ± 2 Å in VpEXPA2. The molecular modeling of the VpEXPA2 showed a notable difference arises in the distance between catalytic Asp10 and Tyr8 in EGV (4.8 Å) compared to the distance between Asp17 and Tyr13 in VpEXPA2 (14.8 Å) (Gaete-Eastman et al. 2015). When aligning and scrutinizing the sequences of EXPA, EXPB, and EGV, it becomes apparent that the catalytic Asp is positioned two residues away from the Tyr residue in EGV. In contrast, in VpEXPA2 and other EXPA sequences, this spacing between Tyr and Asp consists of four residues. Interestingly, this four-residue pattern is conserved in EXPA sequences, but the corresponding Asp residue is notably absent in EXPB sequences (Gaete-Eastman et al. 2015; Valenzuela-Riffo et al. 2019, 2020).

Respect to the protein–ligand interaction, the alpha-expansin (VpEXPA2) and cellodextrin 8-mer (as a cellulose representative ligand) displayed a better interaction (GGGGGGGG) compared to hemicellulose octasaccharides (XXXGXXXG and XXFGXXFG) (Gaete-Eastman et al. 2015), in silico approaches showed that the cellodextrin 8-mer was located on the open groove, a small groove that forms on the surface of the protein and passes through both domains of the protein, and interacts with key amino acids in the active site motif (residues HFD). Other similar study showed that the Domain 2 of FaEXPA1 interacts first with the cellodextrin 8-mer and after this the ligand interact with the domain 1 of the FaEXPA1 (Valenzuela-Riffo et al. 2018). In contrast with described to VpEXPA2 and FcEXPA2 and FcEXPA5 complexes with same ligand, the ligand interacts first with the domain 1 of the protein (Gaete-Eastman et al. 2015; Valenzuela-Riffo et al. 2020) therefore, VpEXPA2 has a better interaction with hemicellulose ligand in comparison to cellulose docking. Narayan et al. (2019), in a docking between EaEXPA1 and xylose of sweet cane (Erianthus arundinaceus), observed that the protein form hydrogen bond with Gly193 and Leu215. An exhaustive work of alpha-expansin in Fragaria x ananassa was recently conducted, where was observed that amino acids Asn104, Phe137 and Asn234 of FaEXPA1 interacts with the cellulose ligand. Mutations of the amino acids Asn104Ala, Phe137Ala and Asn114Ala, demonstrated a reduction in the number of hydrogen bonds, suggesting that these amino acids are key in the establishment of hydrogen bonds between active site and cellulose as substrate (Valenzuela-Riffo et al. 2018). Similar results were found in F. chiloensis and V. pubescens, where Asn104 is the key amino acid to stablish H-bonds in FcEXPA1, FcEXPA2, FcEXPA5, and VpEXPA2 (Gaete-Eastman et al. 2015; Valenzuela-Riffo et al. 2020). Studies in FaEXPA2 demonstrates that alpha-expansin does not interact with pectins, which as reported experimentally by McQueen-Mason and Cosgrove (1995), where pectins does not hydrolyzes by these proteins, and depending on ligand attached to active site, different amino acids establish hydrogen bonds (Valenzuela-Riffo et al. 2020).

Regarding to the structural aspects of the alpha-expansin models, an important observation are two flanking insertions to HFD motif, where in EXPA are located at N-terminal and in EXPB at C-terminal. In expansin-like proteins has been observed lack of the HFD motif (Sampedro and Cosgrove 2005; Gaete-Eastman et al. 2015). All alpha-expansin models show the catalytic motif oriented to the open groove: a binding site to the polysaccharide (Valenzuela-Riffo et al. 2020; Incili et al. 2022).

Conducting evolutionary studies on plant alpha-expansins and correlating the findings with structural bioinformatics analysis, the sequence of FaEXPA5 was grouped alongside FaEXPA2, along with alpha-expansins from rice, mango, and tobacco. A similar analysis was carried out on alpha-expansins from F. chiloensis, a fruit known for rapid softening. The resulting phylogenetic analysis revealed that FcEXPA1, FcEXPA2, and FcEXPA5 fell into three distinct subgroups (Valenzuela-Riffo et al. 2019). The authors concluded that there exists functional redundancy among FcEXPA1, FcEXPA2, and FcEXPA5. However, subtle differences were observed in their energy of interaction with various substrates, including hemicellulose, cellulose, and pectins. This underscores the nuanced variations in the roles played by these expansins in the softening process, providing valuable insights into their functional distinctions (Valenzuela-Riffo et al. 2019).

Conclusions and remarks

In conclusion, the review underscores the pivotal role of alpha-expansins in the intricate processes of cell wall disassembly and loosening, particularly during fruit ripening. Over the past three decades since their discovery, alpha-expansins have been associated with diverse biological phenomena, including development, stress response, and seed germination. Despite lacking catalytic activity, these proteins demonstrate a remarkable ability to extend plant cell walls, influencing fruit firmness. The comprehensive overview delves into recent research, exploring the involvement of alpha-expansins in fruit ripening across various plant species. Additionally, the review highlights the significance of bioinformatics approaches in elucidating structural insights, demonstrating the protein’s interaction with cell wall polysaccharides, and emphasizing its potential applications in addressing challenges related to climatic changes and global food demands.