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Strawberry fruits greatly reduce their quality due to softening during ripening with economically important losses. Texture changes of fleshy fruits during ripening are mainly due to middle lamellae dissolution, cell-to-cell adhesion losses and wall weakening of parenchyma cells by the coordinated action of several cell wall enzymes. Pectin degradation has been proven a key factor in strawberry softening by functional analysis of several pectinase genes (polygalacturonase, pectate lyase and -galactosidase). The complexity and highly dynamic nature of pectins remains a challenge to fully elucidate structure-function relationships of pectins. In this work, we present the functional analysis of two independent strawberry transgenic lines with more than 95% silencing of a rhamnogalacturonate lyase gene (FaRGLyase1). Firmness of ripe fruit was significantly higher in both transgenic lines than in the control. Cell walls from these fruits were extracted and analyzed by glycan microarray profiling. This high‐throughput technique allows a wide screening of cell-wall glycan occurrence based on the detection of specific cell wall oligosaccharide epitopes by monoclonal antibodies and reveals profiles which can be used as potential fingerprints specific for a singular organ and/or developmental stage. Our microarray results showed that the silencing of FaRGLyase1 reduced degradation of several rhamnogalacturonan-I related epitopes, as expected. Additionally, comparison of transgenic cell walls from ripe fruits with those extracted from control fruits at different developmental stages (green, white and red) by hierarchical clustering, demonstrated a higher similarity of transgenic fruit cell walls with the control cell walls from fruits at the white stage. Glycan microarray profiles revealed less degraded fruit cell walls as result of FaRGLyase1 down-regulation which could contribute to the increased firmness of transgenic fruits Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.

The strawberry is a soft fruit with a very short post-harvest shelf life. The changes in texture during fruit ripening are mainly due to the dissolution of the middle lamellae, reducing cell-to-cell adhesion, and the weakening of parenchymal cell walls as result of the action of cell wall modifying enzymes. At present, no master regulator of this process has been discovered yet. NAC transcription factors have been involved in numerous physiological processes, including fruit ripening. In strawberry, the NAC family comprises more than 110 genes, and at least 6 of them are expressed during fruit development. In this research, we performed a functional analysis of two ripening-related NAC genes, FaNAC2 and FaNAC3, in Fragaria x ananassa Duch. cv. Chandler. Several RNAi transgenic lines showing low FaNAC2 or FaNAC3 mRNA levels in fruit were obtained through Agrobacterium-mediated transformation. These lines produced fruits significantly firmer than control at the ripe stage, being the increase in firmness higher in FaNAC2 silenced plants. Cell walls were extracted from ripe transgenic fruits and characterized by ELISA and Epitope Detection Chromatography (EDC), using monoclonal antibodies against different polysaccharide epitopes. FaNAC2 transgenic lines showed more extensive changes than FaNAC3; these modifications involved increased amounts of demethylated pectins (LM19) in water and CDTA fractions and an alteration of the lateral branches of RG-I, decreasing the amount of arabinan epitopes and increasing galactan epitopes detected by LM6 and LM5, respectively. The amount of arabinogalactan proteins recognized by the JIM13 antibody was also affected, decreasing in the Na2CO3 fraction and increasing in the 4M KOH and cellulase fraction of the transgenic lines.The results obtained indicate that NAC genes could be involved in the regulation of cell wall disassembly associated to strawberry fruit softening. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.