Woodland Strawberry Fragaria Vesca Research Articles

Genome-Wide Identification and Analysis of the Nuclear Factor Y Gene Family in the Woodland Strawberry Fragaria vesca

Nuclear factor Ys (NF-Ys) are heterotrimeric transcription factors that specifically bind to CCAAT boxes present in numerous eukaryotic promoters. In plants, NF-Y proteins consist of the following three subunits: NF-YA, NF-YB, and NF-YC, each encoded by a gene family. Accumulating evidence underscores the crucial roles of NF-Y proteins in various plant development processes and stress responses, such as embryogenesis, flowering time control, drought tolerance, and heat tolerance. Despite this, a comprehensive genome-wide overview of the NF-Y gene family in strawberries is lacking. To bridge this gap, this study was conducted to identify and characterize the NF-Ys in Fragaria vesca. The investigation revealed the presence of six NF-YA, twelve NF-YB, and five NF-YC members in F. vesca. Furthermore, a comprehensive analysis of the FveNF-Ys was performed, including their phylogenetic relationships, gene structures, chromosomal locations, and conserved domains. MiRNA target site prediction found that there were 30 miRNA target sites in 12 (52.2%) FveNF-Y genes. Additionally, the expression profiles of different tissues and developmental stages demonstrated tissue-specific expression patterns among certain members of each NF-Y subfamily. This observation suggests that specific NF-Y subfamily members may play unique roles in different tissues or stages of development. Furthermore, the transient expression assay demonstrated that three selected FveNF-Ys were localized in the nucleus. Our study represents a pioneering effort in the systemic analyses of FveNF-Y genes and will be useful in understanding the functional characterization of NF-Y genes in Fragaria species.

The Short Inflorescence Mutation in Diploid Strawberry Fragaria vesca Affects Inflorescence Architecture and Runner Elongation

Mutants are useful for determining the genes that underlie a given trait. This information is highly useful for developing molecular markers for breeding and is the foundational knowledge required for future genomic crop improvements. The dessert strawberry, Fragaria ×ananassa, is a valuable crop with high potential for increased use in controlled environment agriculture. The genome of the woodland strawberry Fragaria vesca is the dominant genome of the four diploid strawberry subgenomes that contribute to the octoploid F. ×ananassa genome. F. vesca is therefore a useful reference system for determining gene function and should be a useful source of gene diversity for breeding of F. ×ananassa. Chemical mutagenesis of the inbred F. vesca line H4 F7-3 resulted in one M2 line with a smaller stature overall and which produces flowers on very short peduncles close to the crown. This line was named short inflorescence (sin). The sin phenotype results from a single gene recessive mutation that is pleiotropic in that the mutation also affects internode lengths of runners as well as petiole elongation of sin plants. Microscopic characterization revealed that sin peduncles are most likely short because of a failure of cells to elongate. Inflorescences, runners, and petioles of sin plants were found to elongate in response to exogenous gibberellin, indicating that sin could be a gibberellin biosynthesis or transport mutant. A brief characterization of sin plants is presented to facilitate collaborative studies of the line.

DNA methylation and histone modification patterns around Agrobacterium T-DNA integrations in woodland strawberry Fragaria vesca

Agrobacterium transformation is widely used for plant genome manipulation, which is completed by integration of transfer DNA (T-DNA) into the plant genome. Application of new biotechnology such as genome editing in many horticultural crops is mediated by Agrobacterium transformation. Currently, our knowledge on the chromatin environment around the T-DNA integration sites is limited. In this study, we mapped the genomic locations of T-DNA integrations in woodland strawberry (Fragaria vesca) to the telomere-to-telomere genome, and found that T-DNA integrations recovered under antibiotic selections were biased towards TE-poor euchromatic sequences. We further performed bisulfite sequencing and chromatin immunoprecipitation to analyze DNA methylation and histone modifications of the integration sites. T-DNA integration sites had low levels of DNA methylation, as well as low levels of 24-nt small RNAs which were associated with RNA-dependent DNA methylation. In addition, the sequences flanking T-DNA integration sites had low levels of a silent mark histone 3 lysine 27 trimethylation, and high levels of an active mark histone 3 lysine 9/lysine 14 acetylation. Such structure indicates an open chromatin environment favored by T-DNA integration and/or antibiotic selection in strawberries. This study provides clues for increasing the transformation efficiency for molecular breeding in Rosaceae species in the future.

The trithorax group factor ULTRAPETALA1 controls flower and leaf development in woodland strawberry

The trithorax group (TrxG) factors play a critical role in the regulation of gene transcription by modulating histone methylation. However, the biological functions of the TrxG components are poorly characterized in different plant species. In this work, we identified three allelic ethyl methane-sulfonate-induced mutants P7, R67 and M3 in the woodland strawberry Fragaria vesca. These mutants show an increased number of floral organs, a lower pollination rate, raised achenes on the surface of the receptacle and increased leaf complexity. The causative gene is FvH4_6g44900, which contains severe mutations leading to premature stop codons or alternative splicing in each mutant. This gene encodes a protein with high similarity to ULTRAPETALA1, a component of the TrxG complex, and is therefore named as FveULT1. Yeast-two-hybrid and split-luciferase assays revealed that FveULT1 can physically interact with the TrxG factor FveATX1 and the PcG repressive complex 2 (PRC2) accessory protein FveEMF1. Transcriptome analysis revealed that several MADS-box genes, FveLFY and FveUFO were significantly up-regulated in fveult1 flower buds. The leaf development genes FveKNOXs, FveLFYa and SIMPLE LEAF1 were strongly induced in fveult1 leaves, and their promoter regions showed increased H3K4me3 levels and decreased H3K27me3 levels in fveult1 compared to WT. Taken together, our results demonstrate that FveULT1 is important for flower, fruit and leaf development and highlight the potential regulatory functions of histone methylation in strawberry.

TEMPRANILLO homologs in apple regulate flowering time in the woodland strawberry Fragaria vesca

The long juvenile period of fruit trees makes their breeding costly and time-consuming. Therefore, flowering time engineering and shortening the juvenile phase have become a breeding priority for the genetic improvement of fruit tree crops. Many economically valuable fruit trees belong to the Rosaceae family including apples and strawberries. TEMPRANILLO (TEM) acts as a key player in flowering time control through inhibiting FT function. Two genes with high sequence similarity with the Arabidopsis TEM genes were isolated from apple (Malus domestica). Due to the complexity of carrying out functional studies in apple, we characterized their function in woodland strawberry as well as their expression in apple. The expression of MdTEM genes in apple tissues from juvenile plants was dramatically higher than that in the tissues from adult trees. In woodland strawberry, the overexpression of MdTEM genes down-regulated FvFT1, FvGA3OX1, and FvGA3OX2 genes in strawberry. The MdTEM-overexpressing lines exhibited delayed flowering, in terms of days to flowering and the number of leaves at flowering. While, RNAi-mediated silencing of TEM resulted in five days earlier flowering, with a lower number of leaves, a higher trichome density, and in some cases, caused in vitro flowering. According to these results and in silico analyses, it can be concluded that MdTEM1 and MdTEM2 can be considered as orthologs of FvTEM and probably AtTEM genes, which play an important role in regulating the juvenile phase and flowering time through regulating FT and GA biosynthetic pathway.

Characterization of expansin genes and their transcriptional regulation by histone modifications in strawberry.

The possible candidate expansin genes, which may be important for strawberry fruit softening, have been identified in the diploid woodland strawberry Fragaria vesca and the octoploidcultivated strawberry Fragaria × ananassa and their transcriptional regulation by histone modifications has been studied. Softening process greatly affects fruit texture and shelf life. Expansins (EXPs) are a group of structural proteins participating in cell wall loosening, which break the hydrogen bonding between cellulose microfibrils and hemicelluloses. However, our knowledge on how EXP genes are regulated in fruit ripening, especially in non-climacteric fleshy fruits, is limited. Here, we have identified the EXP genes in both the octoploidcultivated strawberry (Fragaria × ananassa) and one of its diploid progenitor species, woodland strawberry (Fragaria vesca). We found that EXP proteins in F. × ananassa were structurally more divergent than the ones in F. vesca. Transcriptome data suggested that FaEXP88, FaEXP114, FveEXP11 and FveEXP33 were the four candidate EXP genes more likely involved in fruit softening, whose transcript levels dramatically increased when firmness decreased during fruit maturation. Phylogenetic analyses showed that those candidate genes were closely clustered, indicating the presence of homoeolog expression dominance in the EXP gene family in strawberry. Moreover, we have performed chromatin immunoprecipitation (ChIP) experiments to investigate the distribution of histone modifications along the promoters and genic regions of the EXP genes in F. vesca. ChIP data revealed that the transcript levels of EXP genes were highly correlated with the enrichment of H3K9/K14 acetylation and H3K27 tri-methylation. Collectively, this study identifies the key EXP genes involved in strawberry fruit softening and reveals a regulatory role of histone modifications in their transcriptional regulation, which would facilitate functional studies of the EXP genes in the ripening of non-climacteric fruits.

A native chromatin immunoprecipitation (ChIP) protocol for studying histone modifications in strawberry fruits

BackgroundCovalent modifications of histones and histone variants have great influence on chromatin structure, which is involved in the transcriptional regulation of gene expression. Chromatin immunoprecipitation (ChIP) is a powerful tool for studying in vivo DNA-histone interactions. Strawberry is a model for Rosaceae and non-climacteric fruits, in which histone modifications have been implicated to affect fruit development and ripening. However, a validated ChIP method has not been reported in strawberry, probably due to its high levels of polysaccharides which affect the quality of prepared chromatin and the efficiency of immunoprecipitation.ResultsWe describe a native chromatin immunoprecipitation (N-ChIP) protocol suitable for strawberry by optimizing the parameters for nuclei isolation, chromatin extraction, DNA fragmentation and validation analysis using quantitative real-time PCR (qRT-PCR). The qRT-PCR results show that both the active mark H3K36me3 and the silent mark H3K9me2 are efficiently immunoprecipitated for the enriched regions. Compared to X-ChIP (cross-linked chromatin followed by immunoprecipitation), our optimized N-ChIP procedure has a higher signal-to-noise ratio and a lower background for both the active and the silent histone modifications. Furthermore, high-throughput sequencing following N-ChIP demonstrates that nearly 90% of the enriched H3K9/K14ac peaks are overlapped between biological replicates, indicating its remarkable consistency and reproducibility.ConclusionsAn N-ChIP method suitable for the fleshy fruit tissues of woodland strawberry Fragaria vesca is described in this study. The efficiency and reproducibility of our optimized N-ChIP protocol are validated by both qRT-PCR and high-throughput sequencing. We conclude that N-ChIP is a more suitable method for strawberry fruit tissues relative to X-ChIP, which could be combined with high-throughput sequencing to investigate the impact of histone modifications in strawberry and potentially in other fruits with high content of polysaccharides.

Identification of reference genes for transcript normalization in various tissue types and seedlings subjected to different abiotic stresses of woodland strawberry Fragaria vesca

Quantitative real-time PCR (qRT-PCR) is one of the most popular methods for gene expression studies. However, data on the usage of reference genes in strawberry are limited. In this study, we assayed expression dynamics of 20 candidate reference genes across various organs (root, leaf and flower), different developmental stages of fruits, and in seedlings under abiotic stresses (heat, cold, drought and salt) in woodland strawberry Fragaria vesca. Expression stability of those genes was analyzed by four commonly used statistical algorithms (geNorm, NormFinder, BestKeeper and dalteCt) for comparisons and rankings. Our results indicate that the expression stability of reference genes varies according to tissue types and experimental conditions. Furthermore, two reference genes are sufficient for normalization in tissues and seedlings under the four stress conditions in Fragaria vesca. We recommend FveSAND and FveMT1, FveCHC1 and FveGAPDH as reference genes in organs and fruits, FveCHC1 and FveTIM1, FveSAND and FveTIM1, FveCHP1 and FveAP1, FveCHC1 and FveEF 1a under heat, cold, drought and salt stress conditions, respectively. Our study provides a set of reliable standard reference genes for expression analyses of genes in various tissue types and seedlings under different environment conditions of Fragaria vesca.

Transcriptome Profiles of Strawberry (Fragaria vesca) Fruit Interacting With Botrytis cinerea at Different Ripening Stages.

Gray mold caused by Botrytis cinerea is a major cause of economic losses in strawberry fruit production, limiting fruit shelf life and commercialization. When the fungus infects Fragaria × ananassa strawberry at flowering or unripe fruit stages, symptoms develop after an extended latent phase on ripe fruits before or after harvesting. To elucidate the growth kinetics of B. cinerea on flower/fruit and the molecular responses associated with low susceptibility of unripe fruit stages, woodland strawberry Fragaria vesca flowers and fruits, at unripe white and ripe red stages, were inoculated with B. cinerea. Quantification of fungal genomic DNA within 72 h postinoculation (hpi) showed limited fungal growth on open flower and white fruit, while on red fruit, the growth was exponential starting from 24 hpi and sporulation was observed within 48 hpi. RNA sequencing applied to white and red fruit at 24 hpi showed that a total of 2,141 genes (12.5% of the total expressed genes) were differentially expressed due to B. cinerea infection. A broad transcriptional reprogramming was observed in both unripe and ripe fruits, involving in particular receptor and signaling, secondary metabolites, and defense response pathways. Membrane-localized receptor-like kinases and nucleotide-binding site leucine-rich repeat genes were predominant in the surveillance system of the fruits, most of them being downregulated in white fruits and upregulated in red fruits. In general, unripe fruits exhibited a stronger defense response than red fruits. Genes encoding for pathogenesis-related proteins and flavonoid polyphenols as well as genes involved in cell-wall strengthening were upregulated, while cell-softening genes appeared to be switched off. As a result, B. cinerea remained quiescent in white fruits, while it was able to colonize ripe red fruits.

Conserved and novel roles of miR164‐CUC2 regulatory module in specifying leaf and floral organ morphology in strawberry

MicroRNAs (miRNAs) are a kind of short noncoding RNA (20-24 nt), playing versatile roles in plant growth and development. Strawberry generates leaves and flowers with unique features. However, few miRNAs have been functionally characterised in strawberry, especially for their developmental regulation. Here, we identified one ethyl methanesulfonate (EMS) mutant, deeply serrated (des), in the woodland strawberry Fragaria vesca that has wrinkled leaves with deeper serrations, serrated petals and deformed carpels. The causative mutation occurs in the 19th nucleotide of the FvemiR164a mature sequence. Overexpressing FveMIR164A rescued the phenotypes of des/fvemir164a except the petal serrations. Furthermore, we identified two allelic mutants of FveCUC2a, one target of FvemiR164a, which developed leaves with smooth margins and fused leaflets. Phenotypes of the double mutant fvemir164a fvecuc2a indicated that the two genes act linearly in leaf and carpel development, but synergistically in the development of other floral organs and inflorescence architecture. This work demonstrates the conserved and novel roles of the miR164-CUC2 module in leaf and flower development in different plant species, and reveals that the 19th nucleotide of FvemiR164a is important for its processing.

Transcriptome and hormone analyses provide insights into hormonal regulation in strawberry ripening.

The possible molecular mechanisms regulating strawberry fruit ripening were revealed by plant hormone quantification, exogenous hormone application, and RNA-sequencing. Fruit ripening involves a complex interplay among plant hormones. Strawberry is a model for studies on non-climacteric fruit ripening. However, the knowledge on how plant hormones are involved in strawberry ripening is still limited. To understand hormonal actions in the ripening process, we performed genome-wide transcriptome and hormonal analysis for the five major hormones (abscisic acid and catabolites, auxins, cytokinins, gibberellins, and ethylene) in achenes and receptacles (flesh) at different ripening stages of the woodland strawberry Fragaria vesca. Our results demonstrate that the pre-turning stage (a stage with white flesh and red achenes defined in this study) is the transition stage from immature to ripe fruits. The combinatorial analyses of hormone content, transcriptome data, and exogenous hormone treatment indicate that auxin is synthesized predominantly in achenes, while abscisic acid (ABA), bioactive free base cytokinins, gibberellins, and ethylene are mainly produced in receptacles. Furthermore, gibberellin may delay ripening, while ethylene and cytokinin are likely involved at later stages of the ripening process. Our results also provide additional evidence that ABA promotes ripening, while auxin delays it. Although our hormone analysis demonstrates that the total auxin in receptacles remains relatively low and unchanged during ripening, our experimental evidence further indicates that ABA likely enhances expression of the endoplasmic reticulum-localized auxin efflux carrier PIN-LIKES, which may subsequently reduce the auxin level in nucleus. This study provides a global picture for hormonal regulation of non-climacteric strawberry fruit ripening and also evidence for a possible mechanism of ABA and auxin interaction in the ripening process.

Transient Expression Assay in Strawberry Fruits.

Strawberry, including the woodland strawberry Fragaria vesca (2x) and the cultivated strawberry (Fragaria × ananassa, 8x), has emerged as a model system for studying fruit development and ripening. Transient expression provides a quick assay for gene functions or gene interactions. In strawberry, virus-induced gene silencing (VIGS) and Agrobacterium tumefaciens-mediated transformation in fruit have been widely used as the transient expression approaches. Unlike VIGS, the latter one can be utilized not only for gene knock-down, but also for overexpression and knock-out. Here, we show the procedures of transiently expressing the 35S::FveMYB10 construct into fruit of the white-fruited F. vesca accession Yellow Wonder. As a master regulator of anthocyanin production, overexpressing FveMYB10 will cause fruit coloration, which was observed at one week post infiltration. We also exhibit the previous results of knocking down Reduced Anthocyanin in Petioles (RAP), encoding an anthocyanin transporter, by RNAi in fruit of the strawberry cultivar 'Sweet Charlie'. Overall, Agrobacterium-mediated transient transformation in strawberry fruit is a quick and versatile approach for studying gene functions in fruit ripening.

Characterization and expression analysis of circadian clock genes in the diploid woodland strawberry Fragaria vesca

Strawberry is an economically important fruit crop worldwide. Circadian clock genes are endogenous timers that regulate a wide range of metabolic processes and consequently plant development. However, little is known about the circadian clock genes in strawberry. In the present work, we identified 12 primary circadian clock genes from the diploid woodland strawberry (Fragaria vesca L.) genome. Phylogenetic, conserved motif, and gene structure analyses revealed the evolutionary relationships of strawberry circadian clock genes with homologous genes from other species. Promoter analysis revealed different regulatory elements responding to abiotic and biotic stresses and phytohormones. We characterized the transcript patterns of strawberry circadian clock genes over a 48-h period. The expression patterns of seven circadian clock genes displayed circadian rhythms. We also examined the expression patterns of these genes in response to low-temperature stress and six of them showed an upregulated expression. Interestingly, most of these upregulated genes were highly expressed during the day. Our study reveals the characteristics of primary circadian clock components in diploid woodland strawberry and their responses to low-temperature stress and lays a foundation for future functional studies of these circadian clock genes during the growth and development of diploid woodland strawberry.

Identification of basic/helix-loop-helix transcription factors reveals candidate genes involved in anthocyanin biosynthesis from the strawberry white-flesh mutant

As the second largest transcription factor family in plant, the basic helix-loop-helix (bHLH) transcription factor family, characterized by the conserved bHLH domain, plays a central regulatory role in many biological process. However, the bHLH transcription factor family of strawberry has not been systematically identified, especially for the anthocyanin biosynthesis. Here, we identified a total of 113 bHLH transcription factors and described their chromosomal distribution and bioinformatics for the diploid woodland strawberry Fragaria vesca. In addition, transcription profiles of 113 orthologous bHLH genes from various tissues were analyzed for the cultivar ‘Benihoppe’, its white-flesh mutant ‘Xiaobai’, and the ‘Snow Princess’ from their fruit development to the ripening, as well as those under either the ABA or Eth treatment. Both the RT-PCR and qRT-PCR results show that seven selected FabHLH genes (FabHLH17, FabHLH25, FabHLH27, FabHLH29, FabHLH40, FabHLH80, FabHLH98) are responsive to the fruit anthocyanin biosynthesis and hormone signaling according to transcript profiles where three color modes are observed for strawberry’s fruit skin and flesh. Further, prediction for the protein interaction network reveals that four bHLHs (FabHLH25, FabHLH29, FabHLH80, FabHLH98) are involved in the fruit anthocyanin biosynthesis and hormone signaling transduction. These bioinformatics and expression profiles provide a good basis for a further investigation of strawberry bHLH genes.

Bioinformatics identification and transcript profile analysis of the mitogen-activated protein kinase gene family in the diploid woodland strawberry Fragaria vesca.

Mitogen-activated protein kinases (MAPKs) play essential roles in mediating biotic and abiotic stress responses in plants. However, the MAPK gene family in strawberry has not been systematically characterized. Here, we performed a genome-wide survey and identified 12 MAPK genes in the Fragaria vesca genome. Protein domain analysis indicated that all FvMAPKs have typical protein kinase domains. Sequence alignments and phylogenetic analysis classified the FvMAPK genes into four different groups. Conserved motif and exon-intron organization supported the evolutionary relationships inferred from the phylogenetic analysis. Analysis of the stress-related cis-regulatory element in the promoters and subcellular localization predictions of FvMAPKs were also performed. Gene transcript profile analysis showed that the majority of the FvMAPK genes were ubiquitously transcribed in strawberry leaves after Podosphaera aphanis inoculation and after treatment with cold, heat, drought, salt and the exogenous hormones abscisic acid, ethephon, methyl jasmonate, and salicylic acid. RT-qPCR showed that six selected FvMAPK genes comprehensively responded to various stimuli. Additionally, interaction networks revealed that the crucial signaling transduction controlled by FvMAPKs may be involved in the biotic and abiotic stress responses. Our results may provide useful information for future research on the function of the MAPK gene family and the genetic improvement of strawberry resistance to environmental stresses.

Identification and Transcript Analysis of the TCP Transcription Factors in the Diploid Woodland Strawberry Fragaria vesca.

Plant-specific TEOSINTE BRANCHED 1, CYCLOIDEA, and PROLIFERATING CELL FACTORS (TCP) transcription factors play versatile functions in multiple processes of plant growth and development. However, no systematic study has been performed in strawberry. In this study, 19 FvTCP genes were identified in the diploid woodland strawberry (Fragaria vesca) accession Heilongjiang-3. Phylogenetic analysis suggested that the FvTCP genes were classified into two main classes, with the second class further divided into two subclasses, which was supported by the exon-intron organizations and the conserved motif structures. Promoter analysis revealed various cis-acting elements related to growth and development, hormone and/or stress responses. We analyzed FvTCP gene transcript accumulation patterns in different tissues and fruit developmental stages. Among them, 12 FvTCP genes exhibited distinct tissue-specific transcript accumulation patterns. Eleven FvTCP genes were down-regulated in different fruit developmental stages, while five FvTCP genes were up-regulated. Transcripts of FvTCP genes also varied with different subcultural propagation periods and were induced by hormone treatments and biotic and abiotic stresses. Subcellular localization analysis showed that six FvTCP-GFP fusion proteins showed distinct localizations in Arabidopsis mesophyll protoplasts. Notably, transient over-expression of FvTCP9 in strawberry fruits dramatically affected the expression of a series of genes implicated in fruit development and ripening. Taken together, the present study may provide the basis for functional studies to reveal the role of this gene family in strawberry growth and development.

The WRKY transcription factors in the diploid woodland strawberry Fragaria vesca: Identification and expression analysis under biotic and abiotic stresses

WRKY proteins comprise a large family of transcription factors that play important roles in response to biotic and abiotic stresses and in plant growth and development. To date, little is known about the WRKY gene family in strawberry. In this study, we identified 62 WRKY genes (FvWRKYs) in the wild diploid woodland strawberry (Fragaria vesca, 2n = 2x = 14) accession Heilongjiang-3. According to the phylogenetic analysis and structural features, these identified strawberry FvWRKY genes were classified into three main groups. In addition, eight FvWRKY-GFP fusion proteins showed distinct subcellular localizations in Arabidopsis mesophyll protoplasts. Furthermore, we examined the expression of the 62 FvWRKY genes in ‘Heilongjiang-3’ under various conditions, including biotic stress (Podosphaera aphanis), abiotic stresses (drought, salt, cold, and heat), and hormone treatments (abscisic acid, ethephon, methyl jasmonate, and salicylic acid). The expression levels of 33 FvWRKY genes were upregulated, while 12 FvWRKY genes were downregulated during powdery mildew infection. FvWRKY genes responded to drought and salt treatment to a greater extent than to temperature stress. Expression profiles derived from quantitative real-time PCR suggested that 11 FvWRKY genes responded dramatically to various stimuli at the transcriptional level, indicating versatile roles in responses to biotic and abiotic stresses. Interaction networks revealed that the crucial pathways controlled by WRKY proteins may be involved in the differential response to biotic stress. Taken together, the present work may provide the basis for future studies of the genetic modification of WRKY genes for pathogen resistance and stress tolerance in strawberry.

Identification, isolation, and expression analysis of heat shock transcription factors in the diploid woodland strawberry Fragaria vesca.

Heat shock transcription factors (Hsfs) are known to play dominant roles in plant responses to heat, as well as other abiotic or biotic stress stimuli. While the strawberry is an economically important fruit plant, little is known about the Hsf family in the strawberry. To explore the functions of strawberry Hsfs in abiotic and biotic stress responses, this study identified 17 Hsf genes (FvHsfs) in a wild diploid woodland strawberry (Fragaria vesca, 2n = 2x = 14) and isolated 14 of these genes. Phylogenetic analysis divided the strawberry FvHsfs genes into three main groups. The evolutionary and structural analyses revealed that the FvHsf family is conserved. The promoter sequences of the FvHsf genes contain upstream regulatory elements corresponding to different stress stimuli. In addition, 14 FvHsf-GFP fusion proteins showed differential subcellular localization in Arabidopsis mesophyll protoplasts. Furthermore, we examined the expression of the 17 FvHsf genes in wild diploid woodland strawberries under various conditions, including abiotic stresses (heat, cold, drought, and salt), biotic stress (powdery mildew infection), and hormone treatments (abscisic acid, ethephon, methyl jasmonate, and salicylic acid). Fifteen of the seventeen FvHsf genes exhibited distinct changes on the transcriptional level during heat treatment. Of these 15 FvHsfs, 8 FvHsfs also exhibited distinct responses to other stimuli on the transcriptional level, indicating versatile roles in the response to abiotic and biotic stresses. Taken together, the present work may provide the basis for further studies to dissect FvHsf function in response to stress stimuli.

Light quality regulates flowering in FvFT1/FvTFL1 dependent manner in the woodland strawberry Fragaria vesca.

Control of flowering in the perennial model, the woodland strawberry (Fragaria vesca L.), involves distinct molecular mechanisms that result in contrasting photoperiodic flowering responses and growth cycles in different accessions. The F. vesca homolog of TERMINAL FLOWER1 (FvTFL1) functions as a key floral repressor that causes short-day (SD) requirement of flowering and seasonal flowering habit in the SD strawberry. In contrast, perpetual flowering F. vesca accessions lacking functional FvTFL1 show FLOWERING LOCUS T (FvFT1)-dependent early flowering specifically under long-days (LD). We show here that the end-of-day far-red (FR) and blue (B) light activate the expression of FvFT1 and the F. vesca homolog of SUPPRESSOR OF THE OVEREXPRESSION OF CONSTANS (FvSOC1) in both SD and LD strawberries, whereas low expression levels are detected in red (R) and SD treatments. By using transgenic lines, we demonstrate that FvFT1 advances flowering under FR and B treatments compared to R and SD treatments in the LD strawberry, and that FvSOC1 is specifically needed for the B light response. In the SD strawberry, flowering responses to these light quality treatments are reversed due to up-regulation of the floral repressor FvTFL1 in parallel with FvFT1 and FvSOC1. Our data highlights the central role of FvFT1 in the light quality dependent flower induction in the LD strawberry and demonstrates that FvTFL1 reverses not only photoperiodic requirements but also light quality effects on flower induction in the SD strawberry.

Genetic Variation in Plant CYP51s Confers Resistance against Voriconazole, a Novel Inhibitor of Brassinosteroid-Dependent Sterol Biosynthesis

Brassinosteroids (BRs) are plant steroid hormones with structural similarity to mammalian sex steroids and ecdysteroids from insects. The BRs are synthesized from sterols and are essential regulators of cell division, cell elongation and cell differentiation. In this work we show that voriconazole, an antifungal therapeutic drug used in human and veterinary medicine, severely impairs plant growth by inhibiting sterol-14α-demethylation and thereby interfering with BR production. The plant growth regulatory properties of voriconazole and related triazoles were identified in a screen for compounds with the ability to alter BR homeostasis. Voriconazole suppressed growth of the model plant Arabidopsis thaliana and of a wide range of both monocotyledonous and dicotyledonous plants. We uncover that voriconazole toxicity in plants is a result of a deficiency in BRs that stems from an inhibition of the cytochrome P450 CYP51, which catalyzes a step of BR-dependent sterol biosynthesis. Interestingly, we found that the woodland strawberry Fragaria vesca, a member of the Rosaceae, is naturally voriconazole resistant and that this resistance is conferred by the specific CYP51 variant of F. vesca. The potential of voriconazole as a novel tool for plant research is discussed.