Suppressing the rhamnogalacturonan lyase gene FaRGLyase1 preserves RGI pectin degradation and enhances strawberry fruit firmness.

Plant rhamnogalacturonan lyases (RGLyases) cleave the backbone of rhamnogalacturonan I (RGI), the "hairy" pectin and polymer of the disaccharide rhamnose (Rha)-galacturonic acid (GalA) with arabinan, galactan or arabinogalactan side chains. It has been suggested that RGLyases could participate in remodeling cell walls during fruit softening, but clear evidence has not been reported. To investigate the role of RGLyases in strawberry softening, a genome-wide analysis of RGLyase genes in the genus Fragaria was performed. Seventeen genes encoding RGLyases with functional domains were identified in Fragaria × ananassa. FaRGLyase1 was the most expressed in the ripe receptacle of cv. Chandler. Transgenic strawberry plants expressing an RNAi sequence of FaRGLyase1 were obtained. Three transgenic lines yielded ripe fruits firmer than controls without other fruit quality parameters being significantly affected. The highest increase in firmness achieved was close to 32%. Cell walls were isolated from ripe fruits of two selected lines. The amount of water-soluble and chelated pectins was higher in transgenic lines than in the control. A carbohydrate microarray study showed a higher abundance of RGI epitopes in pectin fractions and in the cellulose-enriched fraction obtained from transgenic lines. Sixty-seven genes were differentially expressed in transgenic ripe fruits when compared with controls. These genes were involved in various physiological processes, including cell wall remodeling, ion homeostasis, lipid metabolism, protein degradation, stress response, and defense. The transcriptomic changes observed in FaRGLyase1 plants suggest that senescence was delayed in transgenic fruits.

Strawberry fruit FanCXE1 carboxylesterase is involved in the catabolism of volatile esters during the ripening process.

Volatile compounds produced during ripening of strawberry are key determinants of fruit quality and consumer preference. Strawberry volatiles are largely esters which are synthesized by alcohol acyltransferases (AATs) and degraded by carboxylesterases (CXEs). Although CXE activity can have a marked influence on volatile contents in ripe strawberry fruits, CXE function and regulation in them are poorly known. Here, we report the biochemical and functional characterization of the fruit receptacle-specific and ripening-related carboxylesterase FanCXE1. The expression of the corresponding gene was found to be antagonistically regulated by auxins and abscisic acid, key hormones that regulate fruit growth and ripening in strawberry. In vitro, FanCXE1 was able to hydrolyze artificial ester substrates similar to those produced by ripe strawberry fruits. Transient suppression of the FanCXE1 gene by RNAi resulted in an increase of important volatile esters such as methyl hexanoate, methyl butanoate and ethyl hexanoate as well as a decrease of the alcohols hexenol and linanool. The results of this work enhance our understanding of the molecular basis for volatile syntheses and facilitate production of better flavored strawberry fruits by introduction of the relevant alleles into common cultivars.

The Intragenesis and Synthetic Biology Approach towards Accelerating Genetic Gains on Strawberry: Development of New Tools to Improve Fruit Quality and Resistance to Pathogens.

Under climate change, the spread of pests and pathogens into new environments has a dramatic effect on crop protection control. Strawberry (Fragaria spp.) is one the most profitable crops of the Rosaceae family worldwide, but more than 50 different genera of pathogens affect this species. Therefore, accelerating the improvement of fruit quality and pathogen resistance in strawberry represents an important objective for breeding and reducing the usage of pesticides. New genome sequencing data and bioinformatics tools has provided important resources to expand the use of synthetic biology-assisted intragenesis strategies as a powerful tool to accelerate genetic gains in strawberry. In this paper, we took advantage of these innovative approaches to create four RNAi intragenic silencing cassettes by combining specific strawberry new promoters and pathogen defense-related candidate DNA sequences to increase strawberry fruit quality and resistance by silencing their corresponding endogenous genes, mainly during fruit ripening stages, thus avoiding any unwanted effect on plant growth and development. Using a fruit transient assay, GUS expression was detected by the two synthetic FvAAT2 and FvDOF2 promoters, both by histochemical assay and qPCR analysis of GUS transcript levels, thus ensuring the ability of the same to drive the expression of the silencing cassettes in this strawberry tissue. The approaches described here represent valuable new tools for the rapid development of improved strawberry lines.

An atypical HLH transcriptional regulator plays a novel and important role in strawberry ripened receptacle.

BACKGROUND: In soft fruits, the differential expression of many genes during development and ripening is responsible for changing their organoleptic properties. In strawberry fruit, although some genes involved in the metabolic regulation of the ripening process have been functionally characterized, some of the most studied genes correspond to transcription factors. High throughput transcriptomics analyses performed in strawberry red receptacle (Fragaria x ananassa) allowed us to identify a ripening-related gene that codes an atypical HLH (FaPRE1) with high sequence homology with the PACLOBUTRAZOL RESISTANCE (PRE) genes. PRE genes are atypical bHLH proteins characterized by the lack of a DNA-binding domain and whose function has been linked to the regulation of cell elongation processes. RESULTS: FaPRE1 sequence analysis indicates that this gene belongs to the subfamily of atypical bHLHs that also includes ILI-1 from rice, SlPRE2 from tomato and AtPRE1 from Arabidopsis, which are involved in transcriptional regulatory processes as repressors, through the blockage by heterodimerization of bHLH transcription factors. FaPRE1 presented a transcriptional model characteristic of a ripening-related gene with receptacle-specific expression, being repressed by auxins and activated by abscisic acid (ABA). However, its expression was not affected by gibberellic acid (GA3). On the other hand, the transitory silencing of FaPRE1 transcription by agroinfiltration in receptacle produced the down-regulation of a group of genes related to the ripening process while inducing the transcription of genes involved in receptacle growth and development. CONCLUSIONS: In summary, this work presents for the first time experimental data that support an important novel function for the atypical HLH FaPRE1 during the strawberry fruit ripening. We hypothesize that FaPRE1 modulates antagonistically the transcription of genes related to both receptacle growth and ripening. Thus, FaPRE1 would repress the expression of receptacle growth promoting genes in the ripened receptacle, while it would activate the expression of those genes related to the receptacle ripening process.

Genome-wide analysis of the NAC transcription factor family and their expression during the development and ripening of the Fragaria × ananassa fruits.

NAC proteins are a family of transcription factors which have a variety of important regulatory roles in plants. They present a very well conserved group of NAC subdomains in the N-terminal region and a highly variable domain at the C-terminus. Currently, knowledge concerning NAC family in the strawberry plant remains very limited. In this work, we analyzed the NAC family of Fragaria vesca, and a total of 112 NAC proteins were identified after we curated the annotations from the version 4.0.a1 genome. They were placed into the ligation groups (pseudo-chromosomes) and described its physicochemical and genetic features. A microarray transcriptomic analysis showed six of them expressed during the development and ripening of the Fragaria x ananassa fruit. Their expression patterns were studied in fruit (receptacle and achenes) in different stages of development and in vegetative tissues. Also, the expression level under different hormonal treatments (auxins, ABA) and drought stress was investigated. In addition, they were clustered with other NAC transcription factor with known function related to growth and development, senescence, fruit ripening, stress response, and secondary cell wall and vascular development. Our results indicate that these six strawberry NAC proteins could play different important regulatory roles in the process of development and ripening of the fruit, providing the basis for further functional studies and the selection for NAC candidates suitable for biotechnological applications.

The fruit-specific transcription factor FaDOF2 regulates the production of eugenol in ripe fruit receptacles.

Only a few transcription factors have been described in the regulation of the strawberry (Fragaria x ananassa) fruit ripening process. Using a transcriptomic approach, we identified and functionally characterized FaDOF2, a DOF-type ripening-related transcription factor, which is hormonally regulated and specific to the receptacle, though high expression levels were also found in petals. The expression pattern of FaDOF2 correlated with eugenol content, a phenylpropanoid volatile, in both fruit receptacles and petals. When FaDOF2 expression was silenced in ripe strawberry receptacles, the expression of FaEOBII and FaEGS2, two key genes involved in eugenol production, were down-regulated. These fruits showed a concomitant decrease in eugenol content, which confirmed that FaDOF2 is a transcription factor that is involved in eugenol production in ripe fruit receptacles. By using the yeast two-hybrid system and bimolecular fluorescence complementation, we demonstrated that FaDOF2 interacts with FaEOBII, a previously reported regulator of eugenol production, which determines fine-tuning of the expression of key genes that are involved in eugenol production. These results provide evidence that FaDOF2 plays a subsidiary regulatory role with FaEOBII in the expression of genes encoding enzymes that control eugenol production. Taken together, our results provide new insights into the regulation of the volatile phenylpropanoid pathway in ripe strawberry receptacles.

The fruit ripening-related gene FaAAT2 encodes an acyl transferase involved in strawberry aroma biogenesis.

Short-chain esters contribute to the blend of volatiles that define the strawberry aroma. The last step in their biosynthesis involves an alcohol acyltransferase that catalyses the esterification of an acyl moiety of acyl-CoA with an alcohol. This study identified a novel strawberry alcohol acyltransferase gene (FaAAT2) whose expression pattern during fruit receptacle growth and ripening is in accordance with the production of esters throughout strawberry fruit ripening. The full-length FaAAT2 cDNA was cloned and expressed in Escherichia coli and its activity was analysed with acyl-CoA and alcohol substrates. The semi-purified FaAAT2 enzyme had activity with C1-C8 straight-chain alcohols and aromatic alcohols in the presence of acetyl-CoA. Cinnamyl alcohol was the most efficient acyl acceptor. When FaAAT2 expression was transiently downregulated in the fruit receptacle by agroinfiltration, the volatile ester production was significantly reduced in strawberry fruit. The results suggest that FaAAT2 plays a significant role in the production of esters that contribute to the final strawberry fruit flavour.

FaQR, required for the biosynthesis of the strawberry flavor compound 4-hydroxy-2,5-dimethyl-3(2H)-furanone, encodes an enone oxidoreductase.

The flavor of strawberry (Fragaria x ananassa) fruit is dominated by an uncommon group of aroma compounds with a 2,5-dimethyl-3(H)-furanone structure. We report the characterization of an enzyme involved in the biosynthesis of 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF; Furaneol), the key flavor compound in strawberries. Protein extracts were partially purified, and the observed distribution of enzymatic activity correlated with the presence of a single polypeptide of approximately 37 kD. Sequence analysis of two peptide fragments showed total identity with the protein sequence of a strongly ripening-induced, auxin-dependent putative quinone oxidoreductase, Fragaria x ananassa quinone oxidoreductase (FaQR). The open reading frame of the FaQR cDNA consists of 969 bp encoding a 322-amino acid protein with a calculated molecular mass of 34.3 kD. Laser capture microdissection followed by RNA extraction and amplification demonstrated the presence of FaQR mRNA in parenchyma tissue of the strawberry fruit. The FaQR protein was functionally expressed in Escherichia coli, and the monomer catalyzed the formation of HDMF. After chemical synthesis and liquid chromatography-tandem mass spectrometry analysis, 4-hydroxy-5-methyl-2-methylene-3(2H)-furanone was confirmed as a substrate of FaQR and the natural precursor of HDMF. This study demonstrates the function of the FaQR enzyme in the biosynthesis of HDMF as enone oxidoreductase and provides a foundation for the improvement of strawberry flavor and the biotechnological production of HDMF.

A strawberry fruit-specific and ripening-related gene codes for a HyPRP protein involved in polyphenol anchoring.

A strawberry (Fragaria x ananassa cv. Chandler) fruit cDNA (Fahyprp -cDNA) and its corresponding gene (Fahyprp) showing sequence homology to higher plant hyprp genes have been isolated. The cDNA contains an open reading frame encoding a 16 kDa protein with 156 amino acids. The peptide has an amino-terminal signal sequence, a repetitive proline-rich sequence, and a cysteine-rich carboxy-terminal region homologous to other HyPRP proteins. Northern blot and QRT-PCR analysis have shown that the strawberry transcript is specifically expressed in fruit, not being detected in other plant tissues. " In situ " hybridization and immunolocalization studies have indicated that the Fahyprp gene is strongly expressed in achene sclerenchyma cells, in the vascular and receptacle cells of immature green fruit and in the vascular cells of mature red fruits. The achenes removal from unripe green fruits induced the expression of this Fahyprp gene. This induction was reverted by treatment of deachened fruit with the auxin NAA, supporting the idea that Fahyprp gene expression is regulated by auxins. Furthermore, the HyPRP protein has been localized in parenchymatic cells of immature fruits associated to structures containing condensed tannins. The results are discussed supporting a putative role of this protein in the anchoring of polymeric polyphenols in the strawberry fruit during growth and ripening.

Identification of a strawberry gene encoding a non-specific lipid transfer protein that responds to ABA, wounding and cold stress.

cDNA and genomic clones encoding a strawberry (Fragariaxananassa cv. Chandler) non-specific lipid transfer protein (Fxaltp gene) were isolated and characterized. The spatio-temporal expression pattern and structural features of this gene were studied for the first time in strawberry, a non-climacteric fruit of agricultural importance. The architecture and the encoded amino acid sequence of this non-climacteric fruit ltp gene were similar to those of other plant LTPs previously reported, and presents the eight cysteine residues and other features characteristic of plant LTPs. In addition, the deduced protein posseses an N-terminal signal peptide and lacks the K/HDEL retention signal, indicating that the strawberry LTP protein would enter the secretory pathway. In situ studies have shown that the Fxaltp gene is expressed in the epidermal cell layer of the strawberry fruit receptacle and achenes, flowers, and within the cell layer surrounding the endosperm. These results suggest that this Fxaltp gene promoter could be used as an endogenous promoter for biotechnological purposes in strawberry. Computer analysis using the PLACE database predicted the presence of several putative cis-regulatory sequences in response to abscisic acid and cold or wounding stresses within the Fxaltp 5'-flanking region. Accordingly, the strawberry gene responds to ABA and SA, but not to salt and heat stresses. It is also reported that ltp gene expression in strawberry is stimulated by wounding and repressed by cold stresses.

Cloning and characterization of two ripening-related strawberry (Fragaria x ananassa cv. Chandler) pectate lyase genes.

Two genomic clones corresponding to putative pectate lyase genes (plA and plB) were isolated and characterized in strawberry (Fragaria x ananassa cv. Chandler). The corresponding ORFs for the plA and plB genes revealed deduced proteins of 451 and 439 amino acids, respectively, that differ from that of the previously isolated strawberry plC gene. Southern blot analysis has shown that while the plB gene is a single copy gene, the plA gene is probably encoded by a small multigene family. By using specific probes corresponding to the untranslated 3' terminal region of the pl genes, and QRT-PCR methodology, the spatio-temporal expression pattern of both strawberry pl genes have been compared with that of the plC gene. The three transcripts were specifically expressed only in fruit and mainly during the ripening stages. Moreover, the expression of the plA and plB genes was induced in green de-achened fruit, but this increase was reduced by the external application of auxins as was the expression of plC. The expression of both pl genes was also strongly reduced in harvested fruit kept in controlled atmosphere (CA) containing high CO(2) levels. Immunolocalization studies using antibodies raised against the strawberry PL proteins placed the proteins in the cell wall of parenchymatic cells of the fruit receptacle. The role of pl genes in cell-wall disassembly and fruit ripening softening is discussed.