Unraveling the key step in the aroma puzzle: Insights into alcohol acyltransferases in strawberries.

Alcohol acyltransferases (AATs) play a crucial role in catalyzing the transfer of acyl groups, contributing to the diverse aroma of fruits, including strawberries. In this research we identified nine AAT genes in strawberries through a comprehensive analysis involving phylogenetics, gene structure, conserved motifs, and structural protein model examinations. The study used the 'Camarosa' strawberry genome database, and experiments were conducted with fruits harvested at different developmental and ripening stages. The transcriptional analysis revealed differential expression patterns among the AAT genes during fruit ripening, with only four genes (SAAT, FaAAT2, FaAAT7, and FaAAT9) showing increased transcript accumulation correlated with total AAT enzyme activity. Additionally, the study employed in silico methods, including sequence alignment, phylogenetic analysis, and structural modeling, to gain insights into the AAT protein model structures with increase expression pattern during fruit ripening. The four modeled AAT proteins exhibited structural similarities, including conserved catalytic sites and solvent channels. Furthermore, the research investigated the interaction of AAT proteins with different substrates, highlighting the enzymes' promiscuity in substrate preferences. The study contributes with valuable information to unveil AAT gene family members in strawberries, providing scientific background for further exploration of their biological characteristics and their role in aroma biosynthesis during fruit ripening.

Involvement of the GH38 Family Exoglycosidase α-Mannosidase in Strawberry Fruit Ripening.

Exoglycosidase enzymes hydrolyze the N-glycosylations of cell wall enzymes, releasing N-glycans that act as signal molecules and promote fruit ripening. Vesicular exoglycosidase α-mannosidase enzymes of the GH38 family (EC 3.2.1.24; α-man) hydrolyze N-glycans in non-reduced termini. Strawberry fruit (Fragaria × ananassa) is characterized by rapid softening as a result of cell wall modifications during the fruit ripening process. Enzymes acting on cell wall polysaccharides explain the changes in fruit firmness, but α-man has not yet been described in F. × ananassa, meaning that the indirect effects of N-glycan removal on its fruit ripening process are unknown. The present study identified 10 GH38 α-man sequences in the F. × ananassa genome with characteristic conserved domains and key residues. A phylogenetic tree built with the neighbor-joining method and three groups of α-man established, of which group I was classified into three subgroups and group III contained only Poaceae spp. sequences. The real-time qPCR results demonstrated that FaMAN genes decreased during fruit ripening, a trend mirrored by the total enzyme activity from the white to ripe stages. The analysis of the promoter regions of these FaMAN genes was enriched with ripening and phytohormone response elements, and contained cis-regulatory elements related to stress responses to low temperature, drought, defense, and salt stress. This study discusses the relevance of α-man in fruit ripening and how it can be a useful target to prolong fruit shelf life.

Transcriptional and structural analysis of non-specific lipid transfer proteins modulated by fungal endophytes in Antarctic plants under drought.

Lipid transfer proteins (LTPs) play crucial roles in various biological processes in plants, such as pollen tube adhesion, phospholipid transfer, cuticle synthesis, and response to abiotic stress. While a few members of the non-specific LTPs (nsLTPs) have been identified, their structural characteristics remain largely unexplored. Given the observed improvement in the performance of Antarctic plants facing water deficit when associated with fungal endophytes, this study aimed to assess the role of these symbiotic organisms in the transcriptional modulation of putative nsLTPs. The study focused on identifying and characterizing two nsLTP in the Antarctic plant Colobanthus quitensis that exhibit responsiveness to drought stress. Furthermore, we investigated the influence of Antarctic endophytic fungi on the expression profiles of these nsLTPs, as these fungi have been known to enhance plant physiological and biochemical performance under water deficit conditions. Through 3D modeling, docking, and molecular dynamics simulations with different substrates, the conducted structural and ligand-protein interaction analyses showed that differentially expressed nsLTPs displayed the ability to interact with various ligands, with a higher affinity towards palmitoyl-CoA. Overall, our findings suggest a regulatory mechanism for the expression of these two nsLTPs in Colobanthus quitensis under drought stress, further modulated by the presence of endophytic fungi.

Structural and transcriptional characterization of pyruvate decarboxylase (PDC) gene family during strawberry fruit ripening process.

Strawberry is one of the most popular fruits in the world, because their high fruit quality, especially with respect to the combination of aroma, flavor, color, and nutritional compounds. Pyruvate decarboxylase (PDC) is the first of two enzymes specifically required for ethanolic fermentation and catalyzes the decarboxylation of pyruvate to yield acetaldehyde and CO2. The ethanol, an important alcohol which acts as a precursor for the ester and other alcohols formation in strawberry, is produced by the PDC. The objective was found all different PDCs genes present in the strawberry genome and investigate PDC gene expression and ligand-protein interactions in strawberry fruit. Volatile organic compounds were evaluated during the development of the fruit. After this, eight FaPDC were identified with four genes that increase the relative expression during fruit ripening process. Molecular dynamics simulations were performed to analyze the behavior of Pyr and TPP ligands within the catalytic and regulatory sites of the PDC proteins. Results indicated that energy-restrained simulations exhibited minor fluctuations in ligand-protein interactions, while unrestrained simulations revealed crucial insights into ligand affinity. TPP consistently displayed strong interactions with the catalytic site, emphasizing its pivotal role in enzymatic activity. However, FaPDC6 and FaPDC9 exhibited decreased pyruvate affinity initially, suggesting unique binding characteristics requiring further investigation. Finally, the present study contributes significantly to understanding PDC gene expression and the intricate molecular dynamics underlying strawberry fruit ripening, shedding light on potential targets for further research in this critical biological pathway.

α-Mannosidase and ß-D-N-acetylhexosaminidase outside the wall: partner exoglycosidases involved in fruit ripening process.

Cell wall is a strong and complex net whose function is to provide turgor, pathogens attack protection and to give structural support to the cell. In growing and expanding cells, the cell wall of fruits is changing in space and time, because they are changing according to stage of ripening. Understand which mechanisms to produce significant could help to develop tools to prolong the fruit shelf life. Cell wall proteins (CWPs) with enzymatic activity on cell wall polysaccharides, have been studied widely. Another investigations take place in the study of N-glycosylations of CWPs and enzymes with activity on glycosidic linkages. α-mannosidase (α-Man; EC 3.2.1.24) and ß-D-N-acetylhexosaminidase (ß-Hex; EC 3.2.1.52), are enzymes with activity on mannose and N-acetylglucosamine sugar presents in proteins as part of N-glycosylations. Experimental evidence indicate that both are closely related to loss of fruit firmness, but in the literature, there is still no review of both enzymes involved fruit ripening. This review provides a complete state-of-the-art of α-Man and ß-Hex enzymes related in fruit ripening. Also, we propose a vesicular α-Man (EC 3.2.1.24) name to α-Man involved in N-deglycosylations of CWPs of plants.

Do fungal-endosymbionts improve crop nutritional quality and tolerance to stress by boosting flavonoid-mediated responses?

Climate change is threatening human activities, but the combination of water scarcity and heat waves are particularly challenging agriculture. Accumulating literature shows that beneficial fungal endophytes improve plant performance, a condition that seems to be magnified in presence of stress. Because evidence points out to an endophytic mediation of antioxidant activity in plants, we here focused on flavonoids for two main reasons: (i) they are involved in plant tolerance to abiotic stress, and (ii) they are known to be healthy for human consumption. With these two premises as guidance, we explored the literature trying to link mechanistically the relationship between endophytes and plant responses to stress as well as identifying patterns and knowledge gaps. Overall, fungal endophytes improve plant growth and tolerance to environmental stresses. However, evidence for endophytes boosting flavonoid mediated responses in plants is relatively scarce. Reports showing endophytes promoting flavonoid contents in grains and fresh fruits are rather limited which may be related to (long) length of the required experiments for testing it. The use of endophytes isolated from extreme environments (e.g., dry and cold deserts, acid lakes, etc.) is proposed to be better in conferring tolerance to plants under very stressful conditions. However, the real challenge is to test the capacity of these endophytes to established and maintain persistent and functional symbiosis under productive conditions. In summary, there is a clear potential for symbiotically modifying crop plants as a strategy to develop more tolerant varieties to face the stress and eventually increase the quality of the agricultural products.

Role of Glycoproteins during Fruit Ripening and Seed Development.

Approximately thirty percent of the proteins synthesized in animal or plant cells travel through the secretory pathway. Seventy to eighty percent of those proteins are glycosylated. Thus, glycosylation is an important protein modification that is related to many cellular processes, such as differentiation, recognition, development, signal transduction, and immune response. Additionally, glycosylation affects protein folding, solubility, stability, biogenesis, and activity. Specifically, in plants, glycosylation has recently been related to the fruit ripening process. This review aims to provide valuable information and discuss the available literature focused on three principal topics: (I) glycosylations as a key posttranslational modification in development in plants, (II) experimental and bioinformatics tools to analyze glycosylations, and (III) a literature review related to glycosylations in fruit ripening. Based on these three topics, we propose that it is necessary to increase the number of studies related to posttranslational modifications, specifically protein glycosylation because the specific role of glycosylation in the posttranslational process and how this process affects normal fruit development and ripening remain unclear to date.

Characterization of FcXTH2, a Novel Xyloglucan Endotransglycosylase/Hydrolase Enzyme of Chilean Strawberry with Hydrolase Activity.

Xyloglucan endotransglycosylase/hydrolases (XTHs) are cell wall enzymes with hydrolase (XEH) and/or endotransglycosylase (XET) activities. As they are involved in the modification of the xyloglucans, a type of hemicellulose present in the cell wall, they are believed to be very important in different processes, including growth, development, and fruit ripening. Previous studies suggest that XTHs might play a key role in development and ripening of Fragaria chiloensis fruit, and its characterization is pending. Therefore, in order to provide a biochemical characterization of the FcXTH2 enzyme to explain its possible role in strawberry development, the molecular cloning and the heterologous expression of FcXTH2 were performed. The recombinant FcXTH2 was active and displayed mainly XEH activity. The optimal pH and temperature are 5.5 and 37 °C, respectively. A KM value of 0.029 mg mL-1 was determined. Additionally, its protein structural model was built through comparative modeling methodology. The model showed a typically ß-jelly-roll type folding in which the catalytic motif was oriented towards the FcXTH2 central cavity. Using molecular docking, protein-ligand interactions were explored, finding better interaction with xyloglucan than with cellulose. The data provided groundwork for understanding, at a molecular level, the enzymatic mechanism of FcXTH2, an important enzyme acting during the development of the Chilean strawberry.

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.

Glycosylation is important for FcXTH1 activity as judged by its structural and biochemical characterization.

Xyloglucan endotransglycosylase/hydrolases (XTH) may have endotransglycosylase (XET) and/or hydrolase (XEH) activities. Previous studies suggest that XTHs might play a key role in ripening of Fragaria chiloensis fruit as FcXTH1 transcripts increase as fruit softens. FcXTH1 protein sequence contains a conserved N-glycosylation site adjacent to catalytic residues. The FcXTH1 structure was built through comparative modeling methodology, the structure displays a ß-jellyroll-type folding with a curvature generated by eight antiparallel ß-sheets that holds the catalytic motif that is oriented towards the central cavity of the protein. Through Molecular Dynamic Simulations (MDS) analyses the protein-ligand interactions of FcXTH1 were explored, finding a better interaction with xyloglucans than cellulose. Nevertheless, the stability of the protein-ligand complex depends on the glycosylation state of FcXTH1: better energy interactions were determined for the glycosylated protein. As a complement, the molecular cloning and heterologous expression of FcXTH1 in Pichia pastoris was performed, and the recombinant protein was active and displayed strict XET activity. A KM value of 17.0 µM was determined for xyloglucan oligomer. The deglycosylation of FcXTH1 by PNGase-F treatment affects its biochemical properties (increase KM and reduce kcat/KM ratio) and reduces its stability. As a conclusion, glycosylation of FcXTH1 is important for its biological function.