Identification in silico and expression analysis of a β-1-4-endoglucanase and β-galactosidase genes related to ripening in guava fruit

Background Guava fruit softening is a crucial process during ripening and this process involves a number of enzymes that modifies the cell wall. Two of the enzymes that regulate this process are (a) the β-1, 4-endoglucanase 17 (BEG) which hydrolyze β-1, 4 bonds from cellulose and hemicellulose, and (b) β-galactosidase (BGA) that hydrolyzes pectin chains. Bioinformatics and expression analysis information on these genes is limited in guava fruit. Results A fragment of a β-1, 4-endoglucanase 17 (PgE17), and another of a β-galactosidase (PgGa1) were identified. These sequences have a similarity of more than 85% with those reported in the NCBI database. In the guava genome, one homologous sequence was found for PgE17 in Chr 4 and two homologous to PgGa1: one in Chr 3 and the other one in Chr 6. Putative protein PgE17 contains part of the glyco_hydro_9 domain. Putative protein PgGa1 has a part of the glyco_hydro_35 domain. Phylogenetic analysis of PgE17 and PgGa1 revealed that both are highly conserved inside the Myrtaceae family. In silico expression analysis showed that both PgE17 and PgGa1 work in a coordinated way with other cell wall modifier enzymes. Expression of these genes was found in all the guava samples analyzed. However, the highest expression was found in the fruit in the breaking and ripe states. Conclusions A β-1, 4-endoglucanase 17, and β-galactosidase 1 sequences were identified. PgE17 and PgGa1 are expressed in all the plant tissues, and fruit ripening states. Although, the highest expression was on breaker and ripe states.

FvMYB79 Positively Regulates Strawberry Fruit Softening via Transcriptional Activation of FvPME38

Strawberry is a soft fruit with short postharvest life, due to a rapid loss of firmness. Pectin methylesterase (PME)-mediated cell wall remodeling is important to determine fruit firmness and softening. Previously, we have verified the essential role of FvPME38 in regulation of PME-mediated strawberry fruit softening. However, the regulatory network involved in PME-mediated fruit softening is still largely unknown. Here, we identified an R2R3-type MYB transcription factor FvMYB79, which activates the expression level of FvPME38, thereby accelerating fruit softening. During fruit development, FvMYB79 co-expressed with FvPME38, and this co-expression pattern was opposite to the change of fruit firmness in the fruit of ‘Ruegen’ which significantly decreased during fruit developmental stages and suddenly became very low after the color turning stage. Via transient transformation, FvMYB79 could significantly increase the transcriptional level of FvPME38, leading to a decrease of firmness and acceleration of fruit ripening. In addition, silencing of FvMYB79 showed an insensitivity to ABA-induced fruit ripening, suggesting a possible involvement of FvMYB79 in the ABA-dependent fruit softening process. Our findings suggest FvMYB79 acts as a novel regulator during strawberry ripening via transcriptional activation of FvPME38, which provides a novel mechanism for improvement of strawberry fruit firmness.

Inhibitory Effects of CaCl2 and Pectin Methylesterase on Fruit Softening of Raspberry during Cold Storage

Quality of raspberry fruit experiences a rapid decline after harvest due to its vulnerable texture and high moisture content. Application of calcium chloride (CaCl2) combined with pectin methylesterase (PME) is efficient in delaying fruit softening. In this study, the effects of exogenous CaCl2 alone or in combination with PME on the structure of the cell wall, the molecular properties of pectin, and the amount of free water of raspberry during postharvest storage were investigated. The results showed that CaCl2 combined with PME treatment could maintain fruit firmness and inhibit weight loss. The treatment of CaCl2+PME maintained the cell wall structure via sustaining middle lamella integrity and reducing the activities of cell wall-degrading enzymes, such as polygalacturonase, pectin methylesterase, β-galactosidase, α-L-arabinofuranosidase, and β-xylosidase. In addition, CaCl2+PME treatment could effectively increase the content of chelate-soluble pectin (CSP) and develop a cross-linked structure between Ca2+ and CSP. Moreover, CaCl2+PME treatment was of benefit in maintaining free water content. CaCl2 in combination with PME treatment could be a promising method for inhibiting softening and maintaining the quality of postharvest raspberry during cold storage.

Cloning and Functional Identification of SlPG49 in Solanum lycopersicum

The modification and degradation of pectin in cell walls are necessary for the fruit softening process, which involves a series of pectin-modifying enzymes. Polygalacturonases (PGs) are a major group of pectin-hydrolyzing enzymes, which participate in fruit maturation, organ shedding, pollen development, and other processes by catalyzing the degradation of polygalacturonic acid. However, their function in plants has not yet been fully elucidated. In this paper, a full-length cDNA encoding SlPG49 was cloned from a tomato. Sequence alignment and phylogenetic analysis demonstrated that SlPG49 contains four typical conserved domains and belongs to clade E in PG classification. Quantitative real-time PCR analysis showed that SlPG49 was highly expressed in fruits during the softening stage, indicating that SlPG49 may be involved in fruit softening. Subcellular localization results revealed that SlPG49 was located in the cell membrane and the cell wall. In addition, an in vitro enzymatic activity assay confirmed that SlPG49 does have the ability to catalyze the hydrolysis of polygalacturonic acid. These results indicate that SlPG49 is a newly discovered PG gene involved in tomato fruit softening, and provide an experimental basis for elucidating the biological functions of plant PGs during fruit softening.

Interaction of two MADS-box genes leads to growth phenotype divergence of all-flesh type of tomatoes

All-flesh tomato cultivars are devoid of locular gel and exhibit enhanced firmness and improved postharvest storage. Here, we show that SlMBP3 is a master regulator of locular tissue in tomato fruit and that a deletion at the gene locus underpins the All-flesh trait. Intriguingly, All-flesh varieties lack the deleterious phenotypes reported previously for SlMBP3 under-expressing lines and which preclude any potential commercial use. We resolve the causal factor for this phenotypic divergence through the discovery of a natural mutation at the SlAGL11 locus, a close homolog of SlMBP3. Misexpressing SlMBP3 impairs locular gel formation through massive transcriptomic reprogramming at initial phases of fruit development. SlMBP3 influences locule gel formation by controlling cell cycle and cell expansion genes, indicating that important components of fruit softening are determined at early pre-ripening stages. Our findings define potential breeding targets for improved texture in tomato and possibly other fleshy fruits.

Genome-Wide Identification of the Xyloglucan endotransglucosylase/Hydrolase (XTH) and Polygalacturonase (PG) Genes and Characterization of their Role in Fruit Softening of Sweet Cherry

Fruit firmness is an important economical trait in sweet cherry (Prunus avium L.) where the change of this trait is related to cell wall degradation. Xyloglucan endotransglycosylase/hydrolase (XTH) and polygalacturonases (PGs) are critical cell-wall-modifying enzymes that occupy a crucial position in fruit ripening and softening. Herein, we identified 18 XTHs and 45 PGs designated PavXTH1-18 and PavPG1-45 based on their locations in the genome of sweet cherry. We provided a systematical overview of PavXTHs and PavPGs, including phylogenetic relationships, conserved motifs, and expression profiling of these genes. The results showed that PavXTH14, PavXTH15 and PavPG38 were most likely to participated in fruit softening owing to the substantial increment in expression during fruit development and ripening. Furthermore, the phytohormone ABA, MeJA, and ethephon significantly elevated the expression of PavPG38 and PavXTH15, and thus promoted fruit softening. Importantly, transient expression PavXTH14, PavXTH15 and PavPG38 in cherry fruits significantly reduced the fruit firmness, and the content of various cell wall components including hemicellulose and pectin significantly changed correspondingly in the transgenic fruit. Taken together, these results present an extensive analysis of XTHs and PGs in sweet cherry and provide potential targets for breeding softening-resistant sweet cherry cultivars via manipulating cell wall-associated genes.

A phylogenetic and transcriptomic study of the β-1,3-glucanase family in tomato identifies candidate targets for fruit improvement

Tomato, Solanum lycopersicum, is one of the most cultivated fruits. However, between one-quarter and half of their production is lost during transport and storage. Modifications in cell walls, and specifically pectin composition, delay fruit softening but, so far, the impact of callose metabolism in this process has not been investigated. Callose accumulates in cell walls around plasmodesmata to modify symplasmic transport. It also plays a role in reinforcing cell walls in response to bruising or pathogen invasion. The aim of this work is to identify cell wall β-1,3-glucanases expressed in tomato fruit that can be used as targets to modify callose accumulation during ripening. A phylogenetic analysis identified fifty candidate β-1,3-glucanases in tomato distributed in three clusters (α, β and γ) with evolutionary relations previously characterised in the model Arabidopsis thaliana. Analysis of tomato microarray data indicates different regulatory patterns: the expression of a subset of enzymes in cluster α decreased during ripening, while enzymes in cluster β and γ displayed higher expression in white-red stages. qRT-PCR experiments confirm the differential regulation of enzymes in different clusters suggesting evolutionary divergences that correlate with differences in their predicted localization and function. The potential to exploit this information in the selection of targets to modify cell walls and fruit development is discussed.