Insights into the Genes Involved in ABA Biosynthesis and Perception during Development and Ripening of the Chilean Strawberry Fruit.

Hormones act as master ripening regulators. In non-climacteric fruit, ABA plays a key role in ripening. Recently, we confirmed in Fragaria chiloensis fruit that in response to ABA treatment the fruit induces ripening-associated changes such as softening and color development. In consequence of these phenotypic changes, transcriptional variations associated with cell wall disassembly and anthocyanins biosynthesis were reported. As ABA stimulates the ripening of F. chiloensis fruit, the molecular network involved in ABA metabolism was analyzed. Therefore, the expression level of genes involved in ABA biosynthesis and ABA perception was quantified during the development of the fruit. Four NCED/CCDs and six PYR/PYLs family members were identified in F. chiloensis. Bioinformatics analyses confirmed the existence of key domains related to functional properties. Through RT-qPCR analyses, the level of transcripts was quantified. FcNCED1 codifies a protein that displays crucial functional domains, and the level of transcripts increases as the fruit develops and ripens, in parallel with the increment in ABA. In addition, FcPYL4 codifies for a functional ABA receptor, and its expression follows an incremental pattern during ripening. The study concludes that FcNCED1 is involved in ABA biosynthesis; meanwhile, FcPYL4 participates in ABA perception during the ripening of F. chiloensis fruit.

RNAseq, transcriptome analysis and identification of DEGs involved in development and ripening of Fragaria chiloensis fruit.

Fragaria chiloensis (Chilean strawberry) is a native species that produces fruit with an exotic pinkish color and a fruity aroma. It has a non-climacteric pattern of fruit ripening, and it is the mother of the commercial Fragaria x ananassa. The ripening of F. chiloensis fruit seems stimulated by ABA, and a complete set of genes participate in its softening, color, and aroma development. In addition, a set of transcription factors regulate the entire process, but few of them have been described. Over the last two decades, RNA-seq was used to identify genes at three fruit development/ripening stages, named C2 (unripe, large green) to C4 (full ripe), in whole fruit and fruit without achenes. A total of 204,754 contigs were assembled considering all samples, obtaining an N50 of 1.125 bp. Differentially expressed genes (DEGs) between two samples were identified, obtaining a total of 77,181 DEGs. Transcripts for genes involved in ABA biosynthesis present high and differential expression during the C2, C3, and C4 stages. Besides, contigs corresponding to ABA receptors, which interact with a regulatory network, are also differentially expressed. Genes associated with cell wall remodeling and those involved in flavonoid synthesis were also differentially expressed. An interaction network was built considering differentially expressed genes for the phenylpropanoid and flavonoid molecular pathways and having FcMYB1 as a transcription factor regulator. Identifying key genes could give an option to control the ripening of this non-climacteric fruit.

Differential Expression of Arabinogalactan in Response to Inclination in Stem of Pinus radiata Seedlings.

Arabinogalactan proteins (AGPs) are members of a family of proteins that play important roles in cell wall dynamics. AGPs from inclined pines were determined using JIM7, LM2, and LM6 antibodies, showing a higher concentration in one side of the stem. The accumulation of AGPs in xylem and cell wall tissues is enhanced in response to loss of tree stem verticality. The differential gene expression of AGPs indicates that these proteins could be involved in the early response to inclination and also trigger signals such as lignin accumulation, as well as thicken cell wall and lamella media to restore stem vertical growth. A subfamily member of AGPs, which is Fasciclin-like has been described in angiosperm species as inducing tension wood and in some gymnosperms. A search for gene sequences of this subfamily was performed on an RNA-seq library, where 12 sequences were identified containing one or two fasciclin I domains (FAS), named PrFLA1 to PrFLA12. Four of these sequences were phylogenetically classified in group A, where PrFLA1 and PrFLA4 are differentially expressed in tilted pine trees.

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.

Comparative in silico study of the differences in the structure and ligand interaction properties of three alpha-expansin proteins from Fragaria chiloensis fruit.

Expansins are cell wall proteins associated with several processes, including changes in the cell wall during ripening of fruit, which matches softening of the fruit. We have previously reported an increase in expression of specific expansins transcripts during softening of Fragaria chiloensis fruit. Here, we characterized three α-expansins. Their full-length sequences were obtained, and through qRT-PCR (real-time PCR) analyses, their transcript accumulation during softening of F. chiloensis fruit was confirmed. Interestingly, differential but overlapping expression patterns were observed. With the aim of elucidating their roles, 3D protein models were built using comparative modeling methodology. The models obtained were similar and displayed cellulose binding module(CBM ) with a ß-sandwich structure, and a catalytic domain comparable to the catalytic core of protein of the family 45 glycosyl hydrolase. An open groove located at the central part of each expansin was described; however, the shape and size are different. Their protein-ligand interactions were evaluated, showing favorable binding affinity energies with xyloglucan, homogalacturonan, and cellulose, cellulose being the best ligand. However, small differences were observed between the protein-ligand conformations. Molecular mechanics-generalized Born-surface area (MM-GBSA) analyses indicate the major contribution of van der Waals forces and non-polar interactions. The data provide a dynamic view of interaction between expansins and cellulose as putative cell wall ligands at the molecular scale. Communicated by Ramaswamy H. Sarma.

XTHs from Fragaria vesca: genomic structure and transcriptomic analysis in ripening fruit and other tissues.

BACKGROUND: Fragaria vesca or 'woodland strawberry' has emerged as an attractive model for the study of ripening of non-climacteric fruit. It has several advantages, such as its small genome and its diploidy. The recent availability of the complete sequence of its genome opens the possibility for further analysis and its use as a reference species. Fruit softening is a physiological event and involves many biochemical changes that take place at the final stages of fruit development; among them, the remodeling of cell walls by the action of a set of enzymes. Xyloglucan endotransglycosylase/hydrolase (XTH) is a cell wall-associated enzyme, which is encoded by a multigene family. Its action modifies the structure of xyloglucans, a diverse group of polysaccharides that crosslink with cellulose microfibrills, affecting therefore the functional structure of the cell wall. The aim of this work is to identify the XTH-encoding genes present in F. vesca and to determine its transcription level in ripening fruit. RESULTS: The search resulted in identification of 26 XTH-encoding genes named as FvXTHs. Genetic structure and phylogenetic analyses were performed allowing the classification of FvXTH genes into three phylogenetic groups: 17 in group I/II, 2 in group IIIA and 4 in group IIIB. Two sequences were included into the ancestral group. Through a comparative analysis, characteristic structural protein domains were found in FvXTH protein sequences. In complement, expression analyses of FvXTHs by qPCR were performed in fruit at different developmental and ripening stages, as well as, in other tissues. The results showed a diverse expression pattern of FvXTHs in several tissues, although most of them are highly expressed in roots. Their expression patterns are not related to their respective phylogenetic groups. In addition, most FvXTHs are expressed in ripe fruit, and interestingly, some of them (FvXTH 18 and 20, belonging to phylogenic group I/II, and FvXTH 25 and 26 to group IIIB) display an increasing expression pattern as the fruit ripens. CONCLUSION: A discrete group of FvXTHs (18, 20, 25 and 26) increases their expression during softening of F. vesca fruit, and could take part in cell wall remodeling required for softening in collaboration with other cell wall degrading enzymes.