Characterization of FchAGL9 and FchSHP, two MADS-boxes related to softening of Fragaria chiloensis fruit.

Fragaria chiloensis is a Chilean native species that softens intensively during its ripening. Its softening is related to cell wall disassembly due to the participation of cell wall degrading enzymes. Softening of F. chiloensis fruit can be accelerated by ABA treatment which is accompanied by the increment in the expression of key cell wall degrading genes, however the molecular machinery involved in the transcriptional regulation has not been studied until now. Therefore, the participation of two MADS-box transcription factors belonging to different subfamilies, FchAGL9 and FchSHP, was addressed. Both TFs are members of type-II MADS-box family (MIKC-type) and localized in the nucleus. FchAGL9 and FchSHP are expressed only in flower and fruit tissues, rising as the fruit softens with the highest expression level at C3-C4 stages. EMSA assays demonstrated that FchAGL9 binds to CArG sequences of RIN and SQM, meanwhile FchSHP interacts only with RIN. Bimolecular fluorescence complementation and yeast two-hybrid assays confirmed FchAGL9-FchAGL9 and FchAGL9-FchSHP interactions. Hetero-dimer structure was built through homology modeling concluding that FchSHP monomer binds to DNA. Functional validation by Luciferase-dual assays indicated that FchAGL9 transactivates FchRGL and FchPG's promoters, meanwhile FchSHP transactivates those of FchEXP2, FchRGL and FchPG. Over-expression of FchAGL9 in C2 F. chiloensis fruit rises FchEXP2 and FchEXP5 transcripts, meanwhile the over-expression of FchSHP also increments FchXTH1 and FchPL; in both cases there is a down-regulation of FchRGL and FchPG. In summary, we provided evidence of FchAGL9 and FchSHP participating in the transcription regulation associated to F. chiloensis's softening.

The Characterization of a Novel PrMADS11 Transcription Factor from Pinus radiata Induced Early in Bent Pine Stem.

A novel MADS-box transcription factor from Pinus radiata D. Don was characterized. PrMADS11 encodes a protein of 165 amino acids for a MADS-box transcription factor belonging to group II, related to the MIKC protein structure. PrMADS11 was differentially expressed in the stems of pine trees in response to 45° inclination at early times (1 h). Arabidopsis thaliana was stably transformed with a 35S::PrMADS11 construct in an effort to identify the putative targets of PrMADS11. A massive transcriptome analysis revealed 947 differentially expressed genes: 498 genes were up-regulated, and 449 genes were down-regulated due to the over-expression of PrMADS11. The gene ontology analysis highlighted a cell wall remodeling function among the differentially expressed genes, suggesting the active participation of cell wall modification required during the response to vertical stem loss. In addition, the phenylpropanoid pathway was also indicated as a PrMADS11 target, displaying a marked increment in the expression of the genes driven to the biosynthesis of monolignols. The EMSA assays confirmed that PrMADS11 interacts with CArG-box sequences. This TF modulates the gene expression of several molecular pathways, including other TFs, as well as the genes involved in cell wall remodeling. The increment in the lignin content and the genes involved in cell wall dynamics could be an indication of the key role of PrMADS11 in the response to trunk inclination.

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.

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.

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.

Postharvest Treatment of Hydrogen Sulfide Delays the Softening of Chilean Strawberry Fruit by Downregulating the Expression of Key Genes Involved in Pectin Catabolism.

Hydrogen sulfide (H2S) plays several physiological roles in plants. Despite the evidence, the role of H2S on cell wall disassembly and its implications on fleshy fruit firmness remains unknown. In this work, the effect of H2S treatment on the shelf-life, cell wall polymers and cell wall modifying-related gene expression of Chilean strawberry (Fragaria chiloensis) fruit was tested during postharvest storage. The treatment with H2S prolonged the shelf-life of fruit by an effect of optimal dose. Fruit treated with 0.2 mM H2S maintained significantly higher fruit firmness than non-treated fruit, reducing its decay and tripling its shelf-life. Additionally, H2S treatment delays pectin degradation throughout the storage period and significantly downregulated the expression of genes encoding for pectinases, such as polygalacturonase, pectate lyase, and expansin. This evidence suggests that H2S as a gasotransmitter prolongs the post-harvest shelf-life of the fruit and prevents its fast softening rate by a downregulation of the expression of key pectinase genes, which leads to a decreased pectin degradation.

Southern Species From the Biodiversity Hotspot of Central Chile: A Source of Color, Aroma, and Metabolites for Global Agriculture and Food Industry in a Scenario of Climate Change.

Two interesting plants within the Chilean flora (wild and crop species) can be found with a history related to modern fruticulture: Fragaria chiloensis subsp. chiloensis (Rosaceae) and Vasconcellea pubescens (Caricaceae). Both species have a wide natural distribution, which goes from the Andes mountains to the sea (East-West), and from the Atacama desert to the South of Chile (North-South). The growing locations are included within the Chilean Winter Rainfall-Valdivian Forest hotspot. Global warming is of great concern as it increases the risk of losing wild plant species, but at the same time, gives a chance for usually longer term genetic improvement using naturally adapted material and the source for generating healthy foods. Modern agriculture intensifies the attractiveness of native undomesticated species as a way to provide compounds like antioxidants or tolerant plants for climate change scenario. F. chiloensis subsp. chiloensis as the mother of commercial strawberry (Fragaria × ananassa) is an interesting genetic source for the improvement of fruit flavor and stress tolerance. On the other hand, V. pubescens produces fruit with high level of antioxidants and proteolytic enzymes of interest to the food industry. The current review compiles the botanical, physiological and phytochemical description of F. chiloensis subsp. chiloensis and V. pubescens, highlighting their potential as functional foods and as source of compounds with several applications in the pharmaceutical, biotechnological, and food science. The impact of global warming scenario on the distribution of the species is also discussed.

Photooxidative stress activates a complex multigenic response integrating the phenylpropanoid pathway and ethylene, leading to lignin accumulation in apple (Malus domestica Borkh.) fruit.

Photooxidative stress, when combined with elevated temperatures, triggers various defense mechanisms leading to physiological, biochemical, and morphological changes in fruit tissue. Furthermore, during sun damage, apple fruit undergo textural changes characterized by high flesh firmness compared to unexposed fruit. Fuji and Royal Gala apples were suddenly exposed to sunlight on the tree and then sampled for up to 29 days. Cell wall components and lignin biosynthetic pathway analyses were carried out on the fruit tissue. At harvest, Fuji apples with different sun exposure levels, such as exposed to direct sunlight (Exp), shaded (Non-Exp), and with severe sun damage (Sev), were also characterized. In fruit suddenly exposed to sunlight, the expression levels of phenylpropanoid-related genes, phenylalanine ammonia lyase (MdPAL), chalcone synthase (MdCHS), and flavanone-3-hydroxylase (MdF3H), were upregulated in the skin and flesh of Exp and Sev. Exposure had little effect on the lignin-related genes caffeic acid O-methyltransferase 1 (MdCOMT1) and cinnamyl alcohol dehydrogenase (MdCAD) in the skin; however, the expression of these genes was highly induced in the flesh of Exp and Sev in both cultivars. Lignin deposition increased significantly in skin with sun injury (Sev); in flesh, this increase occurred late during the stress treatment. Additionally, the ethylene biosynthesis genes 1-aminocyclopropane-1-carboxylate synthase (MdACS) and 1-aminocyclopropane-1-carboxylate oxidase (MdACO) were highly expressed in the skin and flesh tissues but were more upregulated in Sev than in Exp during the time-course experiment, which paralleled the induction of the phenylpropanoid pathway and lignin accumulation. At harvest, flesh from Sev fruit exhibited higher firmness than that from Non-Exp and Exp fruit, although no differences were observed in the alcohol-insoluble residues (AIR) among groups. The fractionation of cell wall polymers revealed an increase in the uronic acid contents of the water-soluble pectin fraction (WSF) in Exp and Sev tissues compared to Non-Exp tissues, while the other pectin-rich fractions, that is, CDTA-soluble (CSF) and Na2CO3-soluble (NSF), were increased only in Sev. The amount of hemicellulose and cellulose did not differ among fruit conditions. These findings suggest that increases in the flesh firmness of apples can be promoted by photooxidative stress, which is associated with the induction of lignin accumulation in the skin and flesh of stressed fruit, with the involvement of stress phytohormones such as ethylene.

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.

Induction of PrMADS10 on the lower side of bent pine tree stems: potential role in modifying plant cell wall properties and wood anatomy.

The molecular mechanisms underlying inclination responses in trees are unclear. In this study, we identified a MADS-box transcription factor differentially expressed early after inclination in the stems of Pinus radiata D. Don. PrMADS10 has a CDS of 582 bp and encodes a group II MADS-box transcription factor. We measured highest accumulation of this transcript on the lower side of inclined pine stems. In an effort to identify putative targets, we stably transformed Arabidopsis thaliana with a 35S::PrMADS10 construct. Transcriptome analysis revealed 1,219 genes differentially-expressed, with 690 and 529 genes up- and down-regulated respectively, when comparing the transgenic and wild-type. Differentially-expressed genes belong to different biological processes, but were enriched in cell wall remodeling and phenylpropanoid metabolic functions. Interestingly, lignin content was 30% higher in transgenic as compared to wild-type plants consistent with observed changes in gene expression. Differentially expressed transcription factors and phenylpropanoid genes were analyzed using STRING. Several MYB and NAC transcription factors showed interactions with genes of the phenylpropanoid pathway. Together, these results implicate PrMADS10 as a regulatory factor, triggering the expression of other transcription factors and genes involved in the synthesis of lignin.

Linking the platelet antiaggregation effect of different strawberries species with antioxidants: metabolomic and transcript profiling of polyphenols / Asociando el efecto de antiagregación plaquetaria de distintas especies de frutillas con antioxidantes: perfil metabolómico y transcriptómico de polifenoles

A comparative study of antioxidant properties, platelet antiaggregation activity and transcriptional analysis of flavonoid biosynthesis genes were performed in Fragaria x ananassa, F. vesca and F. chiloensis subsp chiloensis f. chiloensis and f. patagonica. Furthermore, differences in flavonoid content were found by UHPLC-MS. The highest free radical scavenging activity by DPPH assay was observed in F. chiloensis f. chiloensis, meanwhile, F. vesca presented the highest antioxidant capacity by FRAP. Biosynthetic flavonoids- related transcripts were higher abundant in F. x ananassa and lower in F. vesca. Additionally, all strawberry extracts showed antiaggregant effect (1 mg mL-1), but F. vesca and F. chiloensis subsp. chiloensis f. patagonica were still active at lower concentration. This study suggests that platelet antiaggregation effect of different strawberries could be due to isoflavones and flavonoids precursors in addition to anthocyanins. Results could usefully to take decisions in future breeding programs to improve the content of healthy compounds in strawberry fruits.

Se realizó un estudio comparativo de propiedades antioxidantes, actividad de antiagregacion plaquetaria, análisis transcripcional de genes de biosíntesis de flavonoides y contenido de estos en Fragaria x ananassa, F. vesca and F. chiloensis subsp chiloensis f. chiloensis and f. patagonica. La mayor actividad removedora de radicales libres por DPPH se observó en F. chiloensis f. chiloensis, mientras F. vesca presentó la mayor capacidad antioxidante mediante FRAP. Transcritos relacionados con biosíntesis de flavonoides fueron mas abundantes en F. x ananassa y menores en F. vesca. Adicionalmente, todos los extractos de frutillas mostraron efectos antiagregante (1 mg mL-1), pero F. vesca and F. chiloensis subsp. chiloensis f. patagonica fueron activos a concentraciones menores. Este estudio sugiere que efectos de antiagregación plaquetaria en distintas frutillas podría deberse a isoflavonas y precursores de flavonoides además de antocianinas. Los resultados podrían ser útiles en programas de mejoramiento genético para mejorar el contenido de compuestos saludables en frutilla.

Characterization of two divergent cDNAs encoding xyloglucan endotransglycosylase/hydrolase (XTH) expressed in Fragaria chiloensis fruit.

Chilean strawberry (Fragaria chiloensis), the maternal progenitor of Fragaria×ananassa, has emerged as a new berry fruit with excellent organoleptic characteristics. The fast softening of strawberries is a limiting step for their commercialization. Fruit softening has been shown to be related to cell wall degradation. Several enzymatic activities related to this process have been isolated in strawberry fruit, however xyloglucan endotransglycosylase/hydrolase (XTH) enzymes have not been identified or characterized so far. Two XTH genes were identified in an EST database of F. chiloensis fruit with high homology to other plant XTHs. We isolated the full-length cDNAs associated to these ESTs in F. chiloensis (Fc-XTH1, Fc-XTH2). Phylogenetic analysis suggests that both F. chiloensis XTH genes belong to distant phylogenetic groups of XTHs. Moreover, DNA gel-blot analysis indicates different genomic organization between the two genes. By means of Real Time qPCR analysis, gene expression profiles show a transcriptional profile of Fc-XTH1 transcripts congruent with a probable role during strawberry ripening, while that exhibited by Fc-XTH2 could be related with vegetative processes like leaf growth. On the other hand, immunodetection and enzyme activity assays allow the detection of XTH-related proteins and high xyloglucan transglycosylating (XETA) and degrading (XDA) activities at the turning stage. The data presented confirms the existence of two divergent XTH genes, and XET and XEH activities, in F. chiloensis fruit.

Differential expression levels of aroma-related genes during ripening of apricot (Prunus armeniaca L.).

Fruit aroma is a complex trait, particularly in terms of the number of different biosynthetic pathways involved, the complexity of the final metabolites, and their regulation. In order to understand the underlying biochemical processes involved in apricot aroma, four cDNAs (Pa-aat, EU784138; Pa-adhEU395433; Pa-pdcEU395434; and Pa-loxEU439430) encoding an alcohol acyl transferase (AAT), alcohol dehydrogenase (ADH), pyruvate decarboxylase (PDC), and lipoxygenase (LOX), respectively, were isolated and characterized at four stages of maturity in Prunus armeniaca L. cv. Modesto. We observed a reduction in aldehyde and alcohol production between early-harvested fruit and late-harvest fruit, concomitant with an increase in ester production. qPCR analyses showed that the expression levels of the adh gene and the lox gene stayed constant at all stages. Interestingly, aat levels showed a sharp increase in the late-harvest stages concurrent with the changes observed in ester levels. The significance of these changes in relation to aroma production in apricot is discussed.