Like IAA, GA1 levels are low during ripening stages. Analysis of spatial expression in F. vesca showed that genes encoding AUX/IAA transcriptional coregulators and auxin response factors , are highly expressed in the expanding receptacle, as expected if IAA is being released from the achenes to stimulate receptacle growth prior to ripening. How, and in what form, auxin travels from the fertilized achenes to the receptacle is still unknown, as are the signals that turn on auxin biosynthesis in precise tissues in response to fertilization. Auxin transport might be most easily addressed in the larger, octoploid organs. As indicated by the diploid transcriptome studies, such analyses should include measurements of conjugated IAA biosynthesis and possible movement using mass labeled compounds. GA receptor protein genes are also highly expressed in expanding F. vesca fruit, as are DELLA repressor genes. Transcripts from genes encoding homologs of GA transporter proteins were found in achenes postfertilization, however, direct evidence of GA transport and responses is lacking. Other hormones likely play roles in early fruit development, and their contributions require further study in octoploid strawberry. Cytokinin signaling genes appear to be active in young seed tissue of F. vesca. Castasterone, a bio-active brass inosteroid ,10 liter drainage collection pot was detected in young developing octoploid fruit in concordance with transcriptome data indicating biosynthesis and signaling in the receptacle of the diploid.
The transferase that methylates Jasmonic Acid to form the volatile compound Methyl Jasmonate was recently characterized from F. vesca and F. × ananass but may not be important for ripening. Clearly, there is a need for further investigations into the possible roles of these compounds in the developing achenes and receptacle in order to understand fruit set and growth. Unlike what is found in climacteric fruits, experiments with F. × ananassa have shown that ABA is clearly a promoter of ripening in strawberry. VIGS of FaNCED1, encoding a key enzyme in ABA biosynthesis, produced white fruits, and this phenotype was rescued by exogenous ABA. RNAi mediated down regulation of an ABA receptor, FaCHLH/ABAR in fruits, resulted in upregulation of the negative signaling regulator ABI1 and down regulation of positive ABA signaling regulators. Sucrose appears to act as a signal upstream of the ABA signaling pathway in regulating strawberry fruit ripening. Recently, auxin was unambiguously detected in the ripe receptacles of “Camarosa” at about the same low levels reported earlier in “Red Gauntlet”. These levels are about tenfold lower than what is commonly found in leaves. However, the increase in expression of FaTAR2 encoding the auxin biosynthesis enzyme tryptophan amino transferase, and genes encoding proteins involved in auxin perception together with expression of genes involved in auxin signaling in ripening receptacles strongly suggests cell-autonomous auxin synthesis and cell-specific response in the receptacle at ripening Laser capture microdissection and newer methodologies in mass spectral analysis of very small amounts of tissues, for example, are needed to specify which types of cells are engaged in hormone metabolism in the ripening receptacle. The critical importance of studying development and ripening in achene and receptacle separately is discussed in Merchante et al., who found that in F. × ananassa, expression of ethylene biosynthesis gene families was temporally and organ specific, and this applied as well to which members of a given gene family were expressed.
Their results support many earlier reports implicating ethylene as playing a role in strawberry ripening. The most recent support for a role for ethylene in strawberry ripening comes from global analysis of transcriptomic changes in the achene and receptacle during ripening. Analysis of the FaERF gene family identified three members, FaERF3, FaERF6, and FaERF71a, that are significantly expressed in the receptacle and upregulated upon ripening. Down regulating FaSAMS1 or FaCTR1 using the VIGS technique in the receptacle inhibited fruit red color formation. Ethephon application promoted natural red color development in white fruits and partially rescued FaSAM1-RNAi and FaCTR1-RNAi fruit. The results implicate FaCTR1 as a positive regulator and ethylene as a required signaling molecule in strawberry fruit ripening. Therefore, ethylene appears to be required for the normal development of the strawberry fruit, where it acts differently in the achenes and the receptacle. In achenes, it acts at the green and red stages, while in the receptacle it acts at the green/white stages. In these organs, ethylene selectively appears to influence the expression of genes involved in ethylene reception, phenylpropanoid metabolism, cell wall degradation, and strawberry aroma production. The expression pattern of the gene encoding the BR receptor, FaBRI1, in F. × ananassa receptacle suggests that BR may also play a role in ripening, and VIGS of this gene results in failure to redden. However, a direct role for this class of hormone in strawberry ripening is in question, as neither active BR castasterone nor brassinolide were detected by the end of the white stage using unambiguous and sensitive analytical techniques.
Transcriptional regulation of fruit ripening in tomato is well characterized, and although strawberry is non-climacteric it is likely that there are conserved pathways and regulatory mechanisms in common with the ripening achene or ripening receptacle. In tomato fruit, chromatin remodeling activities as well as changes in DNA methylation influence normal ripening in maturing fruit tissues. Such studies in strawberry lag well behind. Recently, 71 genes encoding enzymes responsible for histone lysine methylation modifications were identified and characterized in the F. vesca genome. qPCR showed that, in the receptacle, expression of some of the SET methyltransferase genes peak at turning stage; an indication of a role for chromatin remodeling in strawberry fruit ripening. In addition, nine DNA methyltransferase genes and four demethylase genes were identified in the F. vesca genome. These reports indicate that DNA methylation changes dramatically at the onset of ripening, warranting a detailed investigation of the roles of epigenomics in development and ripening in octoploid fruit. Recent comparative transcriptome analysis of developing fruit of two wild selections of F. pentaphylla that differ in ripe fruit color demonstrated a key role of long noncoding RNAs in fruit development and fruit color formation. Future studies must elucidate the functions of the genes targeted by these differentially expressed lnRNAs and their roles in the cultivated strawberry. Superimposing transcriptomics, proteomics, and metabolomics in the same tissues will allow for a more precise determination of how fruit set, development, and ripening are regulated, pointing to the most productive areas for genetic manipulation to improve fruit growth and quality.External quality characteristics of ripe strawberry fruit including size, color, and absence of surface defects have always been a focus of research and genetic improvement. In the last decade, flavor has gained increased importance as a quality attribute demanded by consumers. Strawberry flavor is imparted by sugars , acids , and an unknown number of over 360 reported volatiles. A comprehensive study, using psychophysics to determine attributes that influence pleasure and sensory perception of strawberry fruit, found that overall liking was most greatly influenced by sweetness and strawberry flavor intensity,10 liter drainage pot which are affected by environmental pressures that reduce sucrose and total volatile content. While sucrose was the single metabolite with the most significant contribution to overall liking, it was found that volatiles influence perception of both flavor and sweetness through retronasal olfaction. Thirty-eight volatile compounds significantly enhanced the perceived intensity of sweetness and may be worthwhile targets for molecular study. Four of these volatiles are common to most studies: 3,7-dimethyl-1,6-octadien-3-ol ; the methyl and ethyl esters of butanoic acid; and 2,5-dimethyl-4-methoxy-3-furanone . Linalool imparts a sweet, floral, citrus-like note to strawberries, while the closely related terpene nerolidol imparts a rose/ apple/green note. Mesifurane is said to have a sherry-like or fusty aroma, while its precursor, furaneol -furanone imparts caramel and sweet notes at high concentrations. How confident can we be in these results, and are there other fruit compounds with measurable effects on consumer perception and liking? Further research combining sensory and fruit chemical analyses, including more germplasm and environments, would be valuable for answering these questions. Several strawberry genes involved with production of compounds contributing to flavor/aroma were identified relatively early, including FaSAAT, encoding a fruitspecific ALCOHOL ACYLTRANSFERASE that is exclusively expressed in receptacle tissue; FaOMT, encoding an O-methyltransferase catalyzing the formation of mesifurane from furaneol; and FaNES1 encoding a nerolidol synthase capable of generating linalool or nerolidol with geranyl diphosphate or farnesyl diphosphate, respectively, as substrates.
FaNES1 is present and highly expressed in the fruit of 112 F. × ananassa cultivars as well as in all but three of 46 octoploid wild F. virginiana and F. chiloensis progenitor species accessions. Conversely, FaNES1 was not present in any diploid, tetraploid, or hexaploid accession tested. Instead, the olefinic monoterpenes, namely, α-pinene, β-phellandrene, and β-myrcene are produced, which contribute to turpentinelike, woody, resinous, and unpleasant odors that are selected against by commercial strawberry breeders. QTL analysis in a population segregating for production of mesifurane and other volatiles identified a homoeolog of FaOMT as the locus responsible for natural variation of mesifurane content. Mesifurane nonproducers lack a 30 bp promoter sequence containing putative binding sites for basic/helix–loop–helix, MYB and BZIP transcription factors. This polymorphism fully cosegregates with both the presence of mesifurane and the high expression of FaOMT during ripening. The amount of the volatile γ-decalactone, which is associated with “peach-like” aroma in strawberry fruit, is highly environmentally influenced. Using a metabolomics approach combined with RNAseq, Chambers et al.identified the fatty acid desaturase gene essential to its biosynthesis. In parallel, Sánchez-Sevilla et al.discovered the same locus by combining transcriptome analysis with a map-based approach. Interestingly, about half of cultivars tested were non-producers of this volatile and had a deletion of this gene, pointing to FaFAD1 as a potential target for breeding or engineering back into desirable cultivars. Methyl anthranilate contributes to the fruity, flowery, and aromatic flavor of the woodland strawberry, F. vesca. MA was only found in a few old strawberry cultivars such as “Mieze Schindler” and “Mara des Bois” , and a gene encoding ANTHRANILIC ACID METHYL TRANSFERASE was recently isolated from the latter using transcriptome bulksegregant analysis. While FanAAMT modulates the amplitude of MA accumulation, additional genes hypothesized to be required for basal MA production have yet to be identified. Consumer preference and yield penalties or advantages will help determine the balance of volatiles in cultivars of the future. The availability of high-quality octoploid and diploid strawberry genomes opens opportunities to manipulate known genes involved in volatile production and identify genes required for production of the many other volatiles involved in strawberry aroma. Texture is another strawberry fruit quality attribute of great importance, both for consumer sensory preference and quality after cold storage. Several strawberry genes encoding enzymes involved in the disassembly of fruit cell walls and the solubilization of pectins in the middle lamella that results in softening during fruit ripening have been identified. In the past 5 years, genes encoding pectin solubilizing enzymes: pectate lyase ; endo-β-1,4- glucanase ; β-galactosidase; and polygalacturonase have been implicated in fruit softening during ripening. Nardi et al.have demonstrated a role for expansins, the nonenzymatic cell wall proteins that are associated with cell wall loosening, in strawberry ripening by functional analysis of the promoter of Expansin 2 . Recently, the xyloglucan endotransglycolase/hydrolase gene family was characterized in F. vesca. These enzymes modify xyloglucans that cross-link cellulose microfibrils. Most of the 26 genes identified are expressed in fruit, but expression of a subset of four genes increases during fruit softening. Fruit texture affects not only sensory perception, but also the ability of strawberry to withstand packing and long distance shipping without bruising. Additional information regarding the identity and regulation of genes encoding enzymes that contribute to fruit softening in strawberry can be mined from the existing Fragaria transcriptome data, and their function analyzed using stable transformation, transient expression, and CRISPR technology. Cultivars or wild accessions with the desired firmness qualities that satisfy producers and consumers could help mitigate fruit losses postharvest. Not to be ignored is the role of small noncoding RNAs such as microRNAs in fruit quality, since these are known effectors of regulatory pathways underlying plant development including fruit ripening. In tomato, miRNAs were differentially expressed in the developing fruit, and one miRNA, sly-miR1917, targets a transcription factor that negatively regulates ethylene responses during ripening. Almost 200 miRNAs were identified in F. × ananassa. In F. vesca, one of these, miR399, appears to be involved in soluble solids content and in fructose and glucose content. The molecular mechanism of miRNA399 action is not known and roles of miRNAs in regulating various aspects of F. × ananassa fruit quality require further investigation.