Alpha-expansins: more than three decades of wall creep and loosening in fruits.

Expansins are proteins without catalytic activity, but able to break hydrogen bonds between cell wall polysaccharides hemicellulose and cellulose. This proteins were reported for the first time in 1992, describing cell wall extension in cucumber hypocotyls caused particularly by alpha-expansins. Although these proteins have GH45 and CBM63 domains, characteristic of enzymes related with the cleavage of cell wall polysaccharides, demonstrating in vitro that they extend plant cell wall. Its participation has been associated to molecular processes such as development and growing, fruit ripening and softening, tolerance and resistance to biotic and abiotic stress and seed germination. Structural insights, facilitated by bioinformatics approaches, are highlighted, shedding light on the intricate interactions between alpha-expansins and cell wall polysaccharides. After more than thirty years of its discovery, we want to celebrate the knowledge of alpha-expansins and emphasize their importance to understand the phenomena of disassembly and loosening of the cell wall, specifically in the fruit ripening phenomena, with this state-of-the-art dedicated to them.

Isolation of a rhamnogalacturonan lyase expressed during ripening of the Chilean strawberry fruit and its biochemical characterization.

Fragaria chiloensis (L.) Mill. fruit has exotic organoleptic properties however commercialization is a challenge due to its fast and intensive softening. Texture modifications associated to ripening are related to cell wall metabolism. Main cell wall polysaccharides metabolized in F. chiloensis fruit are pectins, being rhamnogalacturonan I (RG-I) an abundant pectin domain in strawberry. Several enzymes belonging to the fruit molecular machinery have been described to act on different cell wall polysaccharides in F. chiloensis, but none acting on the main chain of RG-I until now. A gene sequence coding for a rhamnogalacturonan endolyase (RG-lyase) (EC 4.2.2.23) was isolated from F. chiloensis. The FchRGL1 sequence belongs to Polysaccharide Lyase family 4 and contains the three functional domains of RG-lyases: RGL4 domain, fibronectin type III and the carbohydrate binding module. In addition, it contains key amino acid residues for activity and Ca2+ coordination. qRT-PCR analyses indicate that FchRGL1 transcripts increase in fruit throughout ripening. RG-lyase activity evidences a remarkable increase as the fruit ripens. The heterologous expression of FchRGL1 in Pichia pastoris provided an active protein that allows its biochemical characterization. RG-lyase activity is optimum at pH 5.0, 25-30 °C and 2 mM Ca2+. A KM of 0.086 mg mL-1 was determined for potato RG-I, and the enzyme undergoes inhibition at high substrate concentration. The enzyme is also able to degrade the mucilage of germinating A. thaliana's seeds. Finally, the properties of FchRGL1 and its expression pattern are congruent with a crucial role in cell wall re-organization during softening of F. chiloensis fruit.

Differential expression after UV-B radiation and characterization of chalcone synthase from the Patagonian hairgrass Deschampsia antarctica.

Deschampsiaantarctica inhabits the maritime territory of Antarctica and South Patagonia. It grows under very harsh environmental conditions. The survival of this species in low freezing temperatures and under high levels of UV-B radiation may constitute some of the most remarkable adaptive plant responses and suggests that this plant possesses genes associated with cold and UV tolerance. Frequently, increased levels of flavonoids have been linked to highly UV-B irradiated plants. Studies examining the biosynthesis of flavonoids in D. antarctica may provide clues to its success in this extreme environment. In this study, we characterized the family of genes encoding chalcone synthase, a key enzyme of the flavonoid biosynthetic pathway. DaCHS was cloned, sequenced and characterized by using software tools. CHS contains two domains, the N-terminal domain ranges from amino acid 8 to 231 and the C-terminal domain ranges from amino acid 241 to 391. Sequence analysis of the three family members revealed a high degree of identity after comparison with other monocotyledons such as Oryza sativa L., Zea mays L. and Hordeum vulgare L. According to these results, DaCHS can be grouped together with H. vulgare CHS1 in the same branch. The phylogenetic tree was built using MEGA software and the neighbour join method with 1000 bootstrap replicates. A model of DaCHS was constructed by way of structural tools and key amino acid residues were identified at the active motif site.

Molecular insights of a xyloglucan endo-transglycosylase/hydrolase of radiata pine (PrXTH1) expressed in response to inclination: Kinetics and computational study.

Xyloglucan endotransglycosylase/hydrolases (XTH) may have endotransglycosylase (XET) and/or hydrolase (XEH) activities. Previous studies confirmed XET activity for PrXTH1 protein from radiata pine. XTHs could interact with many hemicellulose substrates, but the favorite substrate of PrXTH1 is still unknown. The prediction of union type and energy stability of the complexes formed between PrXTH1 and different substrates (XXXGXXXG, XXFGXXFG, XLFGXLFG and cellulose) were determined using bioinformatics tools. Molecular Docking, Molecular Dynamics, MM-GBSA and Electrostatic Potential Calculations were employed to predict the binding modes, free energies of interaction and the distribution of electrostatic charge. The results suggest that the enzyme formed more stable complexes with hemicellulose substrates than cellulose, and the best ligand was the xyloglucan XLFGXLFG (free energy of -58.83 ±â€¯0.8 kcal mol-1). During molecular dynamics trajectories, hemicellulose fibers showed greater stability than cellulose. Aditionally, the kinetic properties of PrXTH1 enzyme were determined. The recombinant protein was active and showed an optimal pH 5.0 and optimal temperature of 37 °C. A Km value of 20.9 mM was determined for xyloglucan oligomer. PrXTH1 is able to interact with different xyloglycans structures but no activity was observed for cellulose as substrate, remodeling cell wall structure in response to inclination.