FvemiR160-FveARF18A-FveAP1/FveFUL module regulates flowering time in woodland strawberry.

Flowering is an indicator of plant transformation from vegetative to reproductive growth. miR160 has been shown to have a significant effect on the growth and development of fruits, leaves, and roots of plants or their stress response to environment, but the participation of miR160 in regulating flowering time in plants is unclear. In this study, we found that two FvemiR160s (FvemiR160a/FvemiR160b) mature sequences in strawberry (Fragaria vesca) were consistent. It was displayed that the miR160 mature sequence is highly conserved in various species, and the miR160 mature sequence formed by the 5' arm of the MIR160 precursor was more conserved. Three FveARFs in woodland strawberry were negatively regulated by FvemiR160a, among which FveARF18A was the most significant. Phylogenetic analysis indicated that FvemiR160 is closely related to apple (Malus domestica), grape (Vitis vinifera), and Arabidopsis thaliana, while FveARF18A is closely related to RcARF18. Subsequently, we demonstrated that FvemiR160a can target cutting FveARF18A to negatively regulate its expression by RLM-5' RACE, cleavage site mutation, and GFP fluorescence assay. Moreover, we observed that FveMIR160a overexpressed plants have advanced flowering, while mFveARF18A overexpressed plants have delayed flowering. We also verified that FveARF18A negatively regulates the expression of FveAP1 and FveFUL by binding their promoters by yeast one-hybrid, LUC, and GUS assay, and FveAP1 and FveFUL transgenic Arabidopsis showed early flowering phenotype. In addition, the expression level of FvemiR160a was decreased obviously while that of FveARF18A was increased obviously by MeJA, GA and IAA. In conclusion, our study reveals the important role of the FvemiR160-FveARF18A-FveAP1/FveFUL module in the flowering process of woodland strawberry and provides a new pathway for studying flowering.

FaMYB123 interacts with FabHLH3 to regulate the late steps of anthocyanin and flavonol biosynthesis during ripening.

In this work, we identified and functionally characterized the strawberry (Fragaria × ananassa) R2R3 MYB transcription factor FaMYB123. As in most genes associated with organoleptic properties of ripe fruit, FaMYB123 expression is ripening-related, receptacle-specific, and antagonistically regulated by ABA and auxin. Knockdown of FaMYB123 expression by RNAi in ripe strawberry fruit receptacles downregulated the expression of enzymes involved in the late steps of anthocyanin/flavonoid biosynthesis. Transgenic fruits showed a parallel decrease in the contents of total anthocyanin and flavonoid, especially malonyl derivatives of pelargonidin and cyanidins. The decrease was concomitant with accumulation of proanthocyanin, propelargonidins, and other condensed tannins associated mainly with green receptacles. Potential coregulation between FaMYB123 and FaMYB10, which may act on different sets of genes for the enzymes involved in anthocyanin production, was explored. FaMYB123 and FabHLH3 were found to interact and to be involved in the transcriptional activation of FaMT1, a gene responsible for the malonylation of anthocyanin components during ripening. Taken together, these results demonstrate that FaMYB123 regulates the late steps of the flavonoid pathway in a specific manner. In this study, a new function for an R2R3 MYB transcription factor, regulating the expression of a gene that encodes a malonyltransferase, has been elucidated.

Diversity of the volatilome and the fruit size and shape in European woodland strawberry (Fragaria vesca).

Woodland strawberry (Fragaria vesca subsp. vesca) is a wild relative of cultivated strawberry (F. × ananassa) producing small and typically conical fruits with an intense flavor and aroma. The wild strawberry species, F. vesca, is a rich resource of genetic and metabolic variability, but its diversity remains largely unexplored and unexploited. In this study, we aim for an in-depth characterization of the fruit complex volatilome by GC-MS as well as the fruit size and shape using a European germplasm collection that represents the continental diversity of the species. We report characteristic volatilome footprints and fruit phenotypes of specific geographical areas. Thus, this study uncovers phenotypic variation linked to geographical distribution that will be valuable for further genetic studies to identify candidate genes or develop markers linked to volatile compounds or fruit shape and size traits.

Auxin biosynthesis gene FveYUC4 is critical for leaf and flower morphogenesis in woodland strawberry.

Auxin plays an essential role in plant growth and development, particularly in fruit development. The YUCCA (YUC) genes encode flavin monooxygenases that catalyze a rate-limiting step in auxin biosynthesis. Mutations that disrupt YUC gene function provide useful tools for dissecting general and specific functions of auxin during plant development. In woodland strawberry (Fragaria vesca), two ethyl methanesulfonate mutants, Y422 and Y1011, have been identified that exhibit severe defects in leaves and flowers. In particular, the width of the leaf blade is greatly reduced, and each leaflet in the mutants has fewer and deeper serrations. In addition, the number and shape of the floral organs are altered, resulting in smaller fruits. Mapping by sequencing revealed that both mutations reside in the FveYUC4 gene, and were therefore renamed as yuc4-1 and yuc4-2. Consistent with a role for FveYUC4 in auxin synthesis, free auxin and its metabolites are significantly reduced in the yuc4 leaves and flowers. This role of FveYUC4 in leaf and flower development is supported by its high and specific expression in young leaves and flower buds using GUS reporters. Furthermore, germline transformation of pYUC4::YUC4, which resulted in elevated expression of FveYUC4 in yuc4 mutants, not only rescued the leaf and flower defects but also produced parthenocarpic fruits. Taken together, our data demonstrate that FveYUC4 is essential for leaf and flower morphogenesis in woodland strawberry by providing auxin hormone at the proper time and in the right tissues.

Overexpression of transcription factor FaMYB63 enhances salt tolerance by directly binding to the SOS1 promoter in Arabidopsis thaliana.

Salinity is a pivotal abiotic stress factor with far-reaching consequences on global crop growth, yield, and quality and which includes strawberries. R2R3-MYB transcription factors encompass a range of roles in plant development and responses to abiotic stress. In this study, we identified that strawberry transcription factor FaMYB63 exhibited a significant upregulation in its expression under salt stress conditions. An analysis using yeast assay demonstrated that FaMYB63 exhibited the ability to activate transcriptional activity. Compared with those in the wild-type (WT) plants, the seed germination rate, root length, contents of chlorophyll and proline, and antioxidant activities (SOD, CAT, and POD) were significantly higher in FaMYB63-overexpressing Arabidopsis plants exposed to salt stress. Conversely, the levels of malondialdehyde (MDA) were considerably lower. Additionally, the FaMYB63-overexpressed Arabidopsis plants displayed a substantially improved capacity to scavenge active oxygen. Furthermore, the activation of stress-related genes by FaMYB63 bolstered the tolerance of transgenic Arabidopsis to salt stress. It was also established that FaMYB63 binds directly to the promoter of the salt overly sensitive gene SOS1, thereby activating its expression. These findings identified FaMYB63 as a possible and important regulator of salt stress tolerance in strawberries.

Recombination Variation Shapes Phylogeny and Introgression in Wild Diploid Strawberries.

Introgressive hybridization is widespread in wild plants and has important consequences. However, frequent hybridization between species makes the estimation of the species' phylogeny challenging, and little is known about the genomic landscape of introgression as it results from complex interactions of multiple evolutionary processes. Here, we reconstructed the phylogeny of ten wild diploid strawberries with whole genome resequencing data and then investigated the influence of recombination rate variation on phylogeny and introgression. We found that genomic regions with low recombination showed reduced levels of incomplete lineage sorting and introgression, and concentrated phylogenetic signals, thus contributing to the most likely species tree of wild diploid strawberries. We revealed complex and widespread introgression across the genus Fragaria, with an average proportion of approximately 4.1% of the extant genome. Introgression tends to be retained in the regions with high recombination rates and low gene density. Furthermore, we identified four SLF genes under selective sweeps that may play potential roles in the possible regain of self-incompatibility by ancient introgression. Altogether, our study yielded novel insights into the evolutionary history and genomic characteristics of introgression in wild diploid strawberries and provides evidence for the role of introgression in plant mating system transitions.

Epigenomic and transcriptomic persistence of heat stress memory in strawberry (Fragaria vesca).

BACKGROUND: In plants, epigenetic stress memory has so far been found to be largely transient. Here, we wanted to assess the heritability of heat stress-induced epigenetic and transcriptomic changes following woodland strawberry (Fragaria vesca) reproduction. Strawberry is an ideal model to study epigenetic inheritance because it presents two modes of reproduction: sexual (self-pollinated plants) and asexual (clonally propagated plants named daughter plants). Taking advantage of this model, we investigated whether heat stress-induced DNA methylation changes can be transmitted via asexual reproduction. RESULTS: Our genome-wide study provides evidence for stress memory acquisition and maintenance in F. vesca. We found that specific DNA methylation marks or epimutations are stably transmitted over at least three asexual generations. Some of the epimutations were associated with transcriptional changes after heat stress. CONCLUSION: Our findings show that the strawberry methylome and transcriptome respond with a high level of flexibility to heat stress. Notably, independent plants acquired the same epimutations and those were inherited by their asexual progenies. Overall, the asexual progenies can retain some information in the genome of past stresses encountered by their progenitors. This molecular memory, also documented at the transcriptional level, might be involved in functional plasticity and stress adaptation. Finally, these findings may contribute to novel breeding approaches for climate-ready plants.

Cuticle deposition ceases during strawberry fruit development.

BACKGROUND: Ideally, the barrier properties of a fruit's cuticle persist throughout its development. This presents a challenge for strawberry fruit, with their rapid development and thin cuticles. The objective was to establish the developmental time course of cuticle deposition in strawberry fruit. RESULTS: Fruit mass and surface area increase rapidly, with peak growth rate coinciding with the onset of ripening. On a whole-fruit basis, the masses of cutin and wax increase but on a unit surface-area basis, they decrease. The decrease is associated with marked increases in elastic strain. The expressions of cuticle-associated genes involved in transcriptional regulation (FaSHN1, FaSHN2, FaSHN3), synthesis of cutin (FaLACS2, FaGPAT3) and wax (FaCER1, FaKCS10, FaKCR1), and those involved in transport of cutin monomers and wax constituents (FaABCG11, FaABCG32) decreased until maturity. The only exceptions were FaLACS6 and FaGPAT6 that are presumably involved in cutin synthesis, and FaCER1 involved in wax synthesis. This result was consistent across five strawberry cultivars. Strawberry cutin consists mainly of C16 and C18 monomers, plus minor amounts of C19, C20, C22 and C24 monomers, ω-hydroxy acids, dihydroxy acids, epoxy acids, primary alcohols, carboxylic acids and dicarboxylic acids. The most abundant monomer is 10,16-dihydroxyhexadecanoic acid. Waxes comprise mainly long-chain fatty acids C29 to C46, with smaller amounts of C16 to C28. Wax constituents are carboxylic acids, primary alcohols, alkanes, aldehydes, sterols and esters. CONCLUSION: The downregulation of cuticle deposition during development accounts for the marked cuticular strain, for the associated microcracking, and for their high susceptibility to the disorders of water soaking and cracking.

The growth, nutrient uptake and fruit quality in four strawberry cultivars under different Spectra of LED supplemental light.

An experiment was conducted in a greenhouse to determine the effects of different supplemental light spectra on the growth, nutrient uptake, and fruit quality of four strawberry cultivars. The plants were grown under natural light and treated with blue (460 nm), red (660 nm), and red/blue (3:1) lights. Results showed that the "Parous" and "Camarosa" had higher fresh and dry mass of leaves, roots, and crowns compared to the "Sabrina" and "Albion". The use of artificial LED lights improved the vegetative growth of strawberry plants. All three supplemental light spectra significantly increased the early fruit yield of cultivars except for "Parous". The red/blue supplemental light spectrum also increased the fruit mass and length of the "Albion". Supplemental light increased the total chlorophyll in "Camarosa" and "Albion", as well as the total soluble solids in fruits. The "Albion" had the highest concentration of fruit anthocyanin, while the "Sabrina" had the lowest. The use of supplemental light spectra significantly increased the fruit anthocyanin concentration in all cultivars. Without supplemental light, the "Camarosa" had the lowest concentration of K and Mg, which increased to the highest concentration with the use of supplemental light spectra. All three spectra increased Fe concentration to the highest value in the "Sabrina", while only the red/blue light spectrum was effective on the "Camarosa". In conclusion, the use of supplemental light can increase the yield and fruit quality of strawberries by elevating nutrients, chlorophyll, and anthocyanin concentrations in plants.

Genomic analyses provide insights into sex differentiation of tetraploid strawberry (Fragaria moupinensis).

The strawberry genus, Fragaria, exhibits a wide range of sexual systems and natural ploidy variation. Nearly, all polyploid strawberry species exhibit separate sexes (dioecy). Research has identified the sex-determining sequences as roughly conserved but with repeatedly changed genomic locations across octoploid strawberries. However, it remains unclear whether tetraploid wild strawberries evolved dioecy independently or shared a common origin with octoploid strawberries. In this study, we investigated the sex determinants of F. moupinensis, a dioecious plant with heterogametic females (ZW). Utilizing a combination of haplotype-resolved genome sequencing of the female F. moupinensis, k-mer-based and coverage-based genome-wide association studies (GWAS), and transcriptomic analysis, we discovered a non-recombining, approximately 33.6 kb W-specific region on chromosome 2a. Within this region, only one candidate sex-determining gene (FmoAFT) was identified. Furthermore, an extensive resequencing of the entire Fragaria genus indicated that the W-specific region displays conservative female specificity across all tetraploid species. This observation suggests that dioecy evolved independently in tetraploid and octoploid strawberries. Moreover, employing virus-induced gene silencing (VIGS), we knocked down the expression of the FmoAFT homologue transcript in cultivated strawberries, revealing its potential role in promoting female functions during early carpel development. We also applied DNA affinity purification sequencing (DAP-seq) and yeast one-hybrid assays to identify potential direct targets of FmoAFT. These insights shed new light on the genetic basis and evolutionary history of sex determination in strawberries, thereby facilitating the formulation of strategies to manipulate sex determination in breeding programs.

Cold tolerance of woodland strawberry (Fragaria vesca) is linked to Cold Box Factor 4 and the dehydrin Xero2.

Domesticated strawberry is susceptible to sudden frost episodes, limiting the productivity of this cash crop in regions, where they are grown during early spring. In contrast, the ancestral woodland strawberry (Fragaria vesca) has successfully colonised many habitats of the Northern Hemisphere. Thus, this species seems to harbour genetic factors promoting cold tolerance. Screening a germplasm established in frame of the German Gene Bank for Crop Wild Relatives we identified, among 70 wild accessions, a pair contrasting with respect to cold tolerance. By following the physiological, biochemical, molecular, and metabolic responses of this contrasting pair, we identified the transcription factor Cold Box Factor 4 and the dehydrin Xero-2 as molecular markers associated with superior tolerance to cold stress. Overexpression of GFP fusions with Xero-2 in tobacco BY-2 cells conferred cold tolerance to these recipient cells. A detailed analysis of the metabolome for the two contrasting genotypes allows to define metabolic signatures correlated with cold tolerance versus cold stress. This work provides a proof-of-concept for the value of crop wild relatives as genetic resources to identify genetic factors suitable to increase the stress resilience of crop plants.

Neopolyploidy has variable effects on the diversity and composition of the wild strawberry microbiome.

PREMISE: Whole-genome duplication (neopolyploidy) can instantly differentiate the phenotype of neopolyploids from their diploid progenitors. These phenotypic shifts in organs such as roots and leaves could also differentiate the way neopolyploids interact with microbial species. While some studies have addressed how specific microbial interactions are affected by neopolyploidy, we lack an understanding of how genome duplication affects the diversity and composition of microbial communities. METHODS: We performed a common garden experiment with multiple clones of artificially synthesized autotetraploids and their ancestral diploids, derived from 13 genotypes of wild strawberry, Fragaria vesca. We sequenced epiphytic bacteria and fungi from roots and leaves and characterized microbial communities and leaf functional traits. RESULTS: Autotetraploidy had no effect on bacterial alpha diversity of either organ, but it did have a genotype-dependent effect on the diversity of fungi on leaves. In contrast, autotetraploidy restructured the community composition of leaf bacteria and had a genotype-dependent effect on fungal community composition in both organs. The most differentially abundant bacterial taxon on leaves belonged to the Sphingomonas, while a member of the Trichoderma was the most differentially abundant fungal taxon on roots. Ploidy-induced change in leaf size was strongly correlated with a change in bacterial but not fungal leaf communities. CONCLUSIONS: Genome duplication can immediately alter aspects of the plant microbiome, but this effect varies by host genotype and bacterial and fungal community. Expanding these studies to wild settings where plants are exposed continuously to microbes are needed to confirm the patterns observed here.

RNA interference-based strategies to control Botrytis cinerea infection in cultivated strawberry.

KEY MESSAGE: Gene silencing of BcDCL genes improves gray mold disease control in the cultivated strawberry. Gene silencing technology offers new opportunities to develop new formulations or new pathogen-resistant plants for reducing impacts of agricultural systems. Recent studies offered the proof of concept that the symptoms of gray mold can be reduced by downregulating Dicer-like 1 (DCL1) and 2 (DCL2) genes of Botrytis cinerea. In this study, we demonstrate that both solutions based on dsRNA topical treatment and in planta expression targeting BcDCL1 and BcDCL2 genes can be used to control the strawberry gray mold, the most harmful disease for different fruit crops. 50, 70 and 100 ng µL-1 of naked BcDCL1/2 dsRNA, sprayed on plants of Fragaria x ananassa cultivar Romina in the greenhouse, displayed significant reduction of susceptibility, compared to the negative controls, but to a lesser extent than the chemical fungicide. Three independent lines of Romina cultivar were confirmed for their stable expression of the hairpin gene construct that targets the Bc-DCL1 and 2 sequences (hp-Bc-DCL1/2), and for the production of hp construct-derived siRNAs, by qRT-PCR and Northern blot analyses. In vitro and in vivo detached leaves, and fruits from the hp-Bc-DCL1/2 lines showed significantly enhanced tolerance to this fungal pathogen compared to the control. This decreased susceptibility was correlated to the reduced fungal biomass and the downregulation of the Bc-DCL1 and 2 genes in B. cinerea. These results confirm the potential of both RNAi-based products and plants for protecting the cultivated strawberry from B. cinerea infection, reducing the impact of chemical pesticides on the environment and the health of consumers.

Biocontrol of strawberry Botrytis gray mold and prolong the fruit shelf-life by fumigant Trichoderma spp.

Objectives To screen high active volatile organic compounds (VOCs)-producing Trichoderma isolates against strawberry gray mold caused by Botrytis cinerea, and to explore their antagonistic mode of action against the pathogen. VOCs produced by nine Trichoderma isolates (Trichoderma atroviride T1 and T3; Trichoderma harzianum T2, T4 and T5; T6, T7, T8 and T9 identified as Trichoderma asperellum in this work) significantly inhibited the mycelial growth (13.9-63.0% reduction) and conidial germination (17.6-96.3% reduction) of B. cinerea, the highest inhibition percentage belonged to VOCs of T7; in a closed space, VOCs of T7 shared 76.9% and 100% biocontrol efficacy against gray mold on strawberry fruits and detached leaves, respectively, prolonged the fruit shelf-life by 3 days in presence of B. cinerea, completely protected the leaves from B. cinerea infecting; volatile metabolites of T7 damaged the cell membrane permeability and integrity of B. cinerea, thereby inhibiting the mycelial growth and conidial germination. Gas chromatography-mass spectrometry (GC-MS) analysis revealed the VOCs contain 23 potential compounds, and the majority of these compounds were categorised as alkenes, alcohols, and esters, including PEA and 6PP, which have been reported as substances produced by Trichoderma spp. T. asperellum T7 showed high biofumigant activity against mycelial growth especially conidial germination of B. cinerea and thus protected strawberry fruits and leaves from gray mold, which acted by damaging the pathogen's plasma membrane and resulting in cytoplasm leakage, was a potential biofumigant for controlling pre- and post-harvest strawberry gray mold.

Evolutionary history and pan-genome dynamics of strawberry (Fragaria spp.).

Strawberry (Fragaria spp.) has emerged as a model system for various fundamental and applied research in recent years. In total, the genomes of five different species have been sequenced over the past 10 y. Here, we report chromosome-scale reference genomes for five strawberry species, including three newly sequenced species' genomes, and genome resequencing data for 128 additional accessions to estimate the genetic diversity, structure, and demographic history of key Fragaria species. Our analyses obtained fully resolved and strongly supported phylogenies and divergence times for most diploid strawberry species. These analyses also uncovered a new diploid species (Fragaria emeiensis Jia J. Lei). Finally, we constructed a pan-genome for Fragaria and examined the evolutionary dynamics of gene families. Notably, we identified multiple independent single base mutations of the MYB10 gene associated with white pigmented fruit shared by different strawberry species. These reference genomes and datasets, combined with our phylogenetic estimates, should serve as a powerful comparative genomic platform and resource for future studies in strawberry.

First report of Stagonosporopsis pogostemonis causing root rot on Strawberry in China.

In Henan, strawberry cultivation occurs on approximately 10,000 hectares, with annual production approaching 230,000 tons. In April 2022, a root rot disease with a 10% incidence rate was observed on the strawberry cultivars 'Ningyu' and 'Sweet Charlie' grown in plastic greenhouses (0.7 ha) located in Xingyang (113.39°E, 34.79°N), Henan, China. Disease symptoms included yellowing of the outer mature leaves, stunted growth, and subsequent wilting of the entire plant. The roots developed dark brown spots, which gradually turned necrotic (Figures 1a, 1b). To determine the causal agent, four symptomatic plants (two plants per cultivar) were collected. Twelve symptomatic root tissues (three root tissue samples per plant) were surface sterilized with 75% ethanol and 0.1% mercuric chloride, washed thrice in sterile water, air dried, and then placed on PDA at 25°C for 3 days. Eight pure isolates were obtained by hyphal-tip isolation (Fang 2007). Each colony had a dark olivaceous green to brown, cottony appearance with a round margin, and the reverse side was grey-black near the center (Figure 1c). Conidia were ellipsoidal, aseptate, with rounded ends, and 3.1 to 4.8 µm × 1.0 to 2.5 µm in size (Figure 1d). Chlamydospores were ellipsoidal, pale brown, and 7.9 to 11.9 µm × 7.6 to 10.7 µm in size (Figure 1e). A representative fungus isolate, designated as Z5, was selected for further molecular identification. Genomic DNA was extracted from the mycelia of isolate Z5, and four gene partial regions (ITS, TUB2, RPB2, and LSU) were amplified using the primer pairs ITS1/ITS4, Bt-2a/Bt-2b, RPB2-5F/RPB2-7CR and LROR/LR5, respectively (White et al.1990, O'Donnell et al.1997, Reeb et al. 2004, Rehner and Samuels 1994). PCR products were sequenced and submitted to GenBank with the following accession numbers OQ130480 (ITS), OQ190093 (TUB2), OQ190092 (RPB2), and OQ255570 (LSU). BLASTn search revealed that the ITS, TUB2, RPB2, and LSU gene sequences of isolate Z5 showed 99.42% (513/516 bp), 99.69% (320/321 bp), 100% (1071/1071 bp), and 100% (857/857 bp) identity with those of ex-type S. pogostemonis stain ZHKUCC 21-0001 (Dong et al. 2021), respectively. A phylogenetic tree was constructed showing that Z5 clustered with S. pogostemonis (Figure 2). The isolates in this study were identified as S. pogostemonis based on morphological and molecular evidence. To confirm pathogenicity, five one-month-old 'Ningyu' cultivar strawberry seedlings were planted in sterilized nursery soil mixed with wheat grains (0.5% w/w) coated with Z5 mycelia (Fang 2007). An equal number of strawberry seedlings were placed in pots filled with non-infected potting mix to serve as controls. The seedlings were kept in a greenhouse under a 12 h light/dark photoperiod at 25°C. After two weeks, the inoculated seedlings displayed symptoms such as leaf wilting and root necrosis, similar to those observed in the greenhouses, while the control seedlings showed no symptoms (Figures 1f, 1g). The experiment was performed thrice. The identical fungus was re-isolated from the symptomatic roots and identified as S. pogostemonis based on morphological characteristics and molecular analysis, thus fulfilling Koch's postulates. This is the first report of S. pogostemonis causing root rot on strawberries worldwide. Our findings will contribute to a more comprehensive study on investigating and managing this disease.

Genome-Wide Characterization and Expression Profiling of the AP2/ERF Gene Family in Fragaria vesca L.

The wild strawberry (Fragaria vesca L.; F. vesca) represents a resilient and extensively studied model organism. While the AP2/ERF gene family plays a pivotal role in plant development, its exploration within F. vesca remains limited. In this study, we characterized the AP2/ERF gene family in wild strawberries using the recently released genomic data (F. vesca V6.0). We conducted an analysis of the gene family expansion pattern, we examined gene expression in stem segments and leaves under cold conditions, and we explored its functional attributes. Our investigation revealed that the FvAP2/ERF family comprises 86 genes distributed among four subfamilies: AP2 (17), RAV (6), ERF (62), and Soloist (1). Tandem and segmental duplications significantly contributed to the growth of this gene family. Furthermore, predictive analysis identified several cis-acting elements in the promoter region associated with meristematic tissue expression, hormone regulation, and resistance modulation. Transcriptomic analysis under cold stress unveiled diverse responses among multiple FvAP2/ERFs in stem segments and leaves. Real-time fluorescence quantitative reverse transcription PCR (RT-qPCR) results confirmed elevated expression levels of select genes following the cold treatment. Additionally, overexpression of FvERF23 in Arabidopsis enhanced cold tolerance, resulting in significantly increased fresh weight and root length compared to the wild-type control. These findings lay the foundation for further exploration into the functional roles of FvAP2/ERF genes.

Involvement of the GH38 Family Exoglycosidase α-Mannosidase in Strawberry Fruit Ripening.

Exoglycosidase enzymes hydrolyze the N-glycosylations of cell wall enzymes, releasing N-glycans that act as signal molecules and promote fruit ripening. Vesicular exoglycosidase α-mannosidase enzymes of the GH38 family (EC 3.2.1.24; α-man) hydrolyze N-glycans in non-reduced termini. Strawberry fruit (Fragaria × ananassa) is characterized by rapid softening as a result of cell wall modifications during the fruit ripening process. Enzymes acting on cell wall polysaccharides explain the changes in fruit firmness, but α-man has not yet been described in F. × ananassa, meaning that the indirect effects of N-glycan removal on its fruit ripening process are unknown. The present study identified 10 GH38 α-man sequences in the F. × ananassa genome with characteristic conserved domains and key residues. A phylogenetic tree built with the neighbor-joining method and three groups of α-man established, of which group I was classified into three subgroups and group III contained only Poaceae spp. sequences. The real-time qPCR results demonstrated that FaMAN genes decreased during fruit ripening, a trend mirrored by the total enzyme activity from the white to ripe stages. The analysis of the promoter regions of these FaMAN genes was enriched with ripening and phytohormone response elements, and contained cis-regulatory elements related to stress responses to low temperature, drought, defense, and salt stress. This study discusses the relevance of α-man in fruit ripening and how it can be a useful target to prolong fruit shelf life.

Overexpression of a Fragaria vesca NAM, ATAF, and CUC (NAC) Transcription Factor Gene (FvNAC29) Increases Salt and Cold Tolerance in Arabidopsis thaliana.

The NAC (NAM, ATAF1/2, CUC2) family of transcription factors (TFs) is a vital transcription factor family of plants. It controls multiple parts of plant development, tissue formation, and abiotic stress response. We cloned the FvNAC29 gene from Fragaria vesca (a diploid strawberry) for this research. There is a conserved NAM structural domain in the FvNAC29 protein. The highest homology between FvNAC29 and PaNAC1 was found by phylogenetic tree analysis. Subcellular localization revealed that FvNAC29 is localized onto the nucleus. Compared to other tissues, the expression level of FvNAC29 was higher in young leaves and roots. In addition, Arabidopsis plants overexpressing FvNAC29 had higher cold and high-salinity tolerance than the wild type (WT) and unloaded line with empty vector (UL). The proline and chlorophyll contents of transgenic Arabidopsis plants, along with the activities of the antioxidant enzymes like catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) under 200 mM NaCl treatment or -8 °C treatment, were higher than those activities of the control. Meanwhile, malondialdehyde (MDA) and the reactive oxygen species (ROS) content were higher in the WT and UL lines. FvNAC29 improves transgenic plant resistance to cold and salt stress by regulating the expression levels of AtRD29a, AtCCA1, AtP5CS1, and AtSnRK2.4. It also improves the potential to tolerate cold stress by positively regulating the expression levels of AtCBF1, AtCBF4, AtCOR15a, and AtCOR47. These findings suggest that FvNAC29 may be related to the processes and the molecular mechanisms of F. vesca response to high-salinity stress and LT stress, providing a comprehensive understanding of the NAC TFs.

Optimizing the Extraction of the Polyphenolic Fraction from Defatted Strawberry Seeds for Tiliroside Isolation Using Accelerated Solvent Extraction Combined with a Box-Behnken Design.

Tiliroside is a natural polyphenolic compound with a wide range of biological activity, and defatted strawberry seeds are its rich source. The goal of this study was to optimize accelerated solvent extraction (ASE) conditions, including temperature, solvent composition, and the number of extraction cycles, using Box-Behnken design to maximize the yield of tiliroside. UPLC-DAD-MS was applied to investigate the polyphenolic composition of the extracts, and preparative liquid chromatography (pLC) was used for isolation. All obtained mathematical models generally showed an increase in the efficiency of isolating polyphenolic compounds with an increase in temperature, ethanol content, and the number of extraction cycles. The optimal established ASE conditions for tiliroside were as follows: a temperature of 65 °C, 63% ethanol in water, and four extraction cycles. This allowed for the obtainment of a tiliroside-rich fraction, and the recovery of isolated tiliroside from plant material reached 243.2 mg from 100 g. Our study showed that ASE ensures the isolation of a tiliroside-rich fraction with high effectiveness. Furthermore, defatted strawberry seeds proved to be a convenient source of tiliroside because the matrix of accompanying components is relatively poor, which facilitates separation.