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.

Evaluation of reference genes for transcript normalization in Fragaria chiloensis fruit and vegetative tissues.

Quantitative real-time PCR (RT-qPCR) is used extensively in gene expression studies. For adequate comparisons, the identification and use of reliable reference genes are crucial. Nevertheless, the availability of such genes in strawberry species is limited and has yet to be described for the Chilean strawberry, Fragaria chiloensis. In this study, the expression dynamics of a set of 10 candidate reference genes were analyzed in various F. chiloensis vegetative tissues (root, runners, stem, leaf, and flower), and fruits at different ripening stages or subjected to different hormonal treatments (ABA, auxin). The expression stability of candidate genes was examined by a series of algorithms, such as geNorm, NormFinder, BestKeeper, and ΔCt, for comparisons and rankings. Finally, by using RefFinder, a comprehensive and comparative ranking of the four methods was achieved. The results highlight that the expression stability of candidate reference genes fluctuates depending on tissue type, fruit stage, and hormonal treatment. As reference genes, the use of FcCHP2 and FcACTIN1 is recommended for F. chiloensis vegetative tissues; FcDBP and FcCHP1 for fruit ripening stages; FcGAPDH and FcDBP for fruit subjected to ABA and NDGA treatments; FcCHP1 and FcCHP2 for fruit under AUXIN and TIBA treatments; and FcDBP and FcCHP2 when all fruit stages and hormonal treatments are compared. If just one reference gene is employed as a normalizer, FcDBP should be chosen as it is the most stable internal control in most conditions. Therefore, the present study delivers a set of reliable reference genes for RT-qPCR expression analysis in F. chiloensis tissues and fruits subjected to several hormonal treatments. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01227-y.

ABA Speeds Up the Progress of Color in Developing F. chiloensis Fruit through the Activation of PAL, CHS and ANS, Key Genes of the Phenylpropanoid/Flavonoid and Anthocyanin Pathways.

Phenolic compounds with antioxidant properties have risen in interest due to their benefits for human health. Fragaria chiloensis is a native wild berry species from Chile that develops a white/pink receptacle and white flesh at the ripe stage. Changes in color parameters, anthocyanins, secondary metabolites (phenolics, flavonoids), and total antioxidant capacity were followed during the development and ripening of F. chiloensis fruit. The increment in color 'a' index takes place in parallel with anthocyanins rise and the reduction in phenolics, flavonoids, and antioxidant capacity. Good correlations were determined between color development, anthocyanins, and the expression of key phenylpropanoid/flavonoid and anthocyanin pathway genes. To investigate the role of ABA on color development, detached immature fruit (C2 stage) were treated with exogenous ABA and stored at 20 °C. Fruit color development was accelerated by ABA treatment compared to non-treated fruit, and consistent with that, the increment in the accumulation of anthocyanins and transcripts of phenylpropanoid/flavonoid, and anthocyanin pathways genes such as FcPAL, FcCHS, and FcANS were observed. This suggests that ABA promotes transcriptional changes that lead to the color formation on this non-climacteric fruit.

Molecular Events Occurring During Softening of Strawberry Fruit.

Changes in fruit texture taking place during ripening, described as softening, are mainly due to alterations in structure and/or composition of the cell wall. Several non-covalent interactions between the three carbohydrate polymers of the cell wall, cellulose, pectins and hemicellulose, and many structural proteins and ions, enable a complex structure. During softening, the disassembly of the cell wall structure takes place, mediated by a complete set of cell wall degrading enzymes or proteins. Softening is a coordinated event that requires the orchestrated participation of a wide variety of proteins. Plant hormones and a set of transcription factors are the organizers of this multi-protein effort. Strawberry is a non climacteric fruit that softens intensively during the last stages of development. The Chilean strawberry fruit (Fragaria chiloensis), the maternal relative of the commercial strawberry (F. × ananassa), softens even faster than commercial strawberry. Softening of the Chilean strawberry fruit has been studied at different levels: changes in cell wall polymers, activity of cell wall degrading enzymes and transcriptional changes of their genes, providing a general view of the complex process. The search for the 'orchestra director' that could coordinate softening events in strawberry fruit has been focussed on hormones like ABA and auxins, and more precisely the relation ABA/AUX. These hormones regulate the expression of many cell wall degrading enzyme genes, and this massive transcriptional change that takes place involves the participation of key transcriptional factors (TF). This review provides an update of the present knowledge regarding the softening of strawberry fruit. Nevertheless, the entire softening process is still under active research especially for the great influence of texture on fruit quality and its high impact on fruit shelf life, and therefore it is expected that new and promising information will illuminate the field in the near future.