An arginine-to-histidine mutation in flavanone-3-hydroxylase results in pink strawberry fruits.

Fruit color is a very important external commodity factor for consumers. Compared to the most typical red octoploid strawberry (Fragaria × ananassa), the pink strawberry often sells for a more expensive price and has a higher economic benefit due to its outstanding color. However, few studies have examined the molecular basis of pink-colored strawberry fruit. Through an EMS mutagenesis of woodland strawberry (Fragaria vesca), we identified a mutant with pink fruits and green petioles. Bulked-segregant analysis sequencing analysis and gene function verification confirmed that the responsible mutation resides in a gene encoding flavanone-3-hydroxylase (F3H) in the anthocyanin synthesis pathway. This nonsynonymous mutation results in an arginine-to-histidine change at position 130 of F3H. Molecular docking experiments showed that the arginine-to-histidine mutation results in a reduction of intermolecular force-hydrogen bonding between the F3H protein and its substrates. Enzymatic experiments showed a greatly reduced ability of the mutated F3H protein to catalyze the conversion of the substrates and hence a blockage of the anthocyanin synthesis pathway. The discovery of a key residue in the F3H gene controlling anthocyanin synthesis provides a clear target of modification for the molecular breeding of strawberry varieties with pink-colored fruits, which may be of great commercial value.

Arabidopsis cryptochrome 1 controls photomorphogenesis through regulation of H2A.Z deposition.

Light is a key environmental cue that fundamentally regulates plant growth and development, which is mediated by the multiple photoreceptors including the blue light (BL) photoreceptor cryptochrome 1 (CRY1). The signaling mechanism of Arabidopsis thaliana CRY1 involves direct interactions with CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1)/SUPPRESSOR OF PHYA-105 1 and stabilization of COP1 substrate ELONGATED HYPOCOTYL 5 (HY5). H2A.Z is an evolutionarily conserved histone variant, which plays a critical role in transcriptional regulation through its deposition in chromatin catalyzed by SWR1 complex. Here we show that CRY1 physically interacts with SWC6 and ARP6, the SWR1 complex core subunits that are essential for mediating H2A.Z deposition, in a BL-dependent manner, and that BL-activated CRY1 enhances the interaction of SWC6 with ARP6. Moreover, HY5 physically interacts with SWC6 and ARP6 to direct the recruitment of SWR1 complex to HY5 target loci. Based on previous studies and our findings, we propose that CRY1 promotes H2A.Z deposition to regulate HY5 target gene expression and photomorphogenesis in BL through the enhancement of both SWR1 complex activity and HY5 recruitment of SWR1 complex to HY5 target loci, which is likely mediated by interactions of CRY1 with SWC6 and ARP6, and CRY1 stabilization of HY5, respectively.

Establishment of a Cas12a-Based Visual Detection Method Involving PMNT for the Colletotrichum gloeosporioides Species Complex.

Strawberry anthracnose, caused by Colletotrichum spp., is a devastating disease that significantly reduces strawberry yield and quality. This study aimed to develop a simple diagnostic method to detect infection by the Colletotrichum gloeosporioides complex (CGSC), the most predominant and virulent Colletotrichum species complex causing strawberry anthracnose in China. In this study, a Cas12aVIP diagnostic method was developed for the rapid detection of CGSC in strawberry seedlings. This method targets the ß-tubulin gene and combines recombinase polymerase amplification (RPA), the CRISPR/Cas12a system, and a cationic-conjugated polythiophene derivative [poly(3-(3'-N,N,N-triethylamino-1'-propyloxy)-4-methyl-2,5-thiophene hydrochloride) (PMNT)] mixed with single-stranded DNA (ssDNA). This method shows high sensitivity (ten copies per reaction) and no cross-reactivity against related pathogens. The entire procedure, from sample to result, can be completed within 50 min, including simplified DNA extraction (15 min), RPA reaction (37°C for 20 min), CRISPR/Cas12a detection (37°C for 10 min), and visual detection by the naked eye (1-2 min). Furthermore, the Cas12aVIP assay successfully detected CGSC in naturally infected strawberry seedling samples in field conditions. Asymptomatic infected plants and plant residues have been identified as primary inoculum sources for CGSC. This method enables visible detection without the need for expensive equipment or specialized technical skills, thereby offering an efficient and straightforward approach for detecting CGSC in strawberries. The newly developed detection method can be used to promote healthier strawberry production.

A Positive Feedback Loop Mediated by CsERF31 Initiates Female Cucumber Flower Development: ETHYLENE RESPONSE FACTOR31 mediates a positive feedback loop that initiates female cucumber flower development.

Sex determination is a crucially important developmental event that is pervasive throughout nature and enhances the adaptation of species. Among plants, cucumber (Cucumis sativus L.) can generate both unisexual and bisexual flowers, and the sex type is mainly controlled by several 1-aminocyclopropane-1-carboxylic acid (ACC) synthases. However, the regulatory mechanism of these synthases remains elusive. Here, we used gene expression analysis, protein-DNA interaction assays and transgenic plants to study the function of a gynoecium-specific gene, ETHYLENE RESPONSE FACTOR31 (CsERF31), in female flower differentiation. We found that in a predetermined female flower, ethylene signalling activates CsERF31 by CsEIN3, and then CsERF31 stimulates CsACS2, which triggers a positive feedback loop to ensure female rather than bisexual flower development. A similar interplay is functionally conserved in melon (Cucumis melo L.). Knockdown of CsERF31 by RNAi causes defective bisexual flowers to replace female flowers. Ectopic expression of CsERF31 suppresses stamen development and promotes pistil development in male flowers, demonstrating that CsERF31 functions as a sex switch. Taken together, our data confirm that CsERF31 represents the molecular link between female-male determination and female-bisexual determination, and provide mechanistic insight into how ethylene promotes female flowers, rather than bisexual flowers, in cucumber sex determination.

A positive feedback loop mediated by CsERF31 initiates female cucumber flower development.

Sex determination is a crucially important developmental event that is pervasive throughout nature and enhances the adaptation of species. Among plants, cucumber (Cucumis sativus L.) can generate both unisexual and bisexual flowers, and the sex type is mainly controlled by several 1-aminocyclopropane-1-carboxylic acid synthases (CsACSs). However, the regulatory mechanism of these synthases remains elusive. Here, we used gene expression analysis, protein-DNA interaction assays, and transgenic plants to study the function of a gynoecium-specific gene, ETHYLENE RESPONSE FACTOR31 (CsERF31), in female flower differentiation. We found that in a predetermined female flower, ethylene signaling activates CsERF31 by CsEIN3, and then CsERF31 stimulates CsACS2, which triggers a positive feedback loop to ensure female rather than bisexual flower development. A similar interplay is functionally conserved in melon (Cucumis melo L.). Knockdown of CsERF31 by RNAi causes defective bisexual flowers to replace female flowers. Ectopic expression of CsERF31 suppresses stamen development and promotes pistil development in male flowers, demonstrating that CsERF31 functions as a sex switch. Taken together, our data confirm that CsERF31 represents the molecular link between female-male determination and female-bisexual determination, and provide mechanistic insight into how ethylene promotes female flowers, rather than bisexual flowers, in cucumber sex determination.

Photoexcited CRYPTOCHROME1 Interacts with Dephosphorylated BES1 to Regulate Brassinosteroid Signaling and Photomorphogenesis in Arabidopsis.

Cryptochromes (CRYs) are blue light photoreceptors that mediate a variety of light responses in plants and animals, including photomorphogenesis, flowering, and circadian rhythms. The signaling mechanism by which Arabidopsis thaliana cryptochromes CRY1 and CRY2 promote photomorphogenesis involves direct interactions with COP1, a RING motif-containing E3 ubiquitin ligase, and its enhancer SPA1. Brassinosteroid (BR) is a key phytohormone involved in the repression of photomorphogenesis, and here, we show that the signaling mechanism of Arabidopsis CRY1 involves the inhibition of BR signaling. CRY1 and CRY2 physically interact with BES1-INTERACTING MYC-LIKE1 (BIM1), a basic helix-loop-helix protein. BIM1, in turn, interacts with and enhances the activity of BRI1-EMS SUPPRESSOR1 (BES1), a master transcription factor in the BR signaling pathway. In addition, CRY1 and CRY2 interact specifically with dephosphorylated BES1, the physiologically active form of BES1 that is activated by BR in a blue light-dependent manner. The CRY1-BES1 interaction leads to both the inhibition of BES1 DNA binding activity and the repression of its target gene expression. Our study suggests that the blue light-dependent, BR-induced interaction of CRY1 with BES1 is a tightly regulated mechanism by which plants optimize photomorphogenesis according to the availability of external light and internal BR signals.

Arabidopsis TSO1 and MYB3R1 form a regulatory module to coordinate cell proliferation with differentiation in shoot and root.

Fundamental to plant and animal development is the regulated balance between cell proliferation and differentiation, a process intimately tied to cell cycle regulation. In Arabidopsis, mutations in TSO1, whose animal homolog is LIN54, resulted in severe developmental abnormalities both in shoot and root, including shoot meristem fasciation and reduced root meristematic zone. The molecular mechanism that could explain the tso1 mutant phenotype is absent. Through a genetic screen, we identified 32 suppressors that map to the MYB3R1 gene, encoding a conserved cell cycle regulator. Further analysis indicates that TSO1 transcriptionally represses MYB3R1, and the ectopic MYB3R1 activity mediates the tso1 mutant phenotype. Since animal homologs of TSO1 and MYB3R1 are components of a cell cycle regulatory complex, the DREAM complex, we tested and showed that TSO1 and MYB3R1 coimmunoprecipitated in tobacco leaf cells. Our work reveals a conserved cell cycle regulatory module, consisting of TSO1 and MYB3R1, for proper plant development.

FvbHLH9 Functions as a Positive Regulator of Anthocyanin Biosynthesis by Forming a HY5-bHLH9 Transcription Complex in Strawberry Fruits.

Anthocyanin accumulation is transcriptionally regulated by the MYB-bHLH-WD40 complex. Light is indispensable for anthocyanin accumulation, and light-inducible MYB and HY5 were considered to promote anthocyanin accumulation in many fruits. Whether and how light-inducible bHLH transcription factor and HY5 regulate anthocyanin synthesis in strawberry is unknown. In this study, we identified a bHLH transcription factor, FvbHLH9, which was induced by light as well as FvHY5, and found that, similar to FvHY5, the transient overexpression and interference FvbHLH9 in strawberry fruits can promote and decrease anthocyanin accumulation, respectively, indicating FvbHLH9 functions as a positive regulator of anthocyanin biosynthesis. Furthermore, we confirmed that both FvHY5 and FvbHLH9 specifically bind to the promoter region of some key enzyme genes, including FvDFR, and the expression of FvDFR was activated through the heterodimer formation between FvHY5 and FvbHLH9. Finally, we confirmed that FvbHLH9-promoted anthocyanin accumulation is dependent on HY5-bHLH heterodimerisation in Arabidopsis. Our findings provide insights into a mechanism involving the synergistic regulation of light-dependent coloration and anthocyanin biosynthesis via a HY5-bHLH heterodimer formed by the interaction of FvHY5 and FvbHLH9 in strawberry fruits.

Photoexcited CRY1 and phyB interact directly with ARF6 and ARF8 to regulate their DNA-binding activity and auxin-induced hypocotyl elongation in Arabidopsis.

Arabidopsis CRY1 and phyB are the primary blue and red light photoreceptors mediating blue and red light inhibition of hypocotyl elongation, respectively. Auxin is a pivotal phytohormone involved in promoting hypocotyl elongation. CRY1 and phyB interact with and stabilize auxin/indole acetic acid proteins (Aux/IAAs) to inhibit auxin signaling. The present study investigated whether photoreceptors might interact directly with Auxin Response Factors (ARFs) to regulate auxin signaling. Protein-protein interaction studies demonstrated that CRY1 and phyB interact physically with ARF6 and ARF8 through their N-terminal domains in a blue and red light-dependent manner, respectively. Moreover, the N-terminal DNA-binding domain of ARF6 and ARF8 is involved in mediating their interactions with CRY1. Genetic studies showed that ARF6 and ARF8 act partially downstream from CRY1 and PHYB to regulate hypocotyl elongation under blue and red light, respectively. Chromatin immunoprecipitation-PCR assays demonstrated that CRY1 and phyB mediate blue and red light repression of the DNA-binding activity of ARF6 and ARF6-target gene expression, respectively. Altogether, the results herein suggest that the direct repression of auxin-responsive gene expression mediated by the interactions of CRY1 and phyB with ARFs constitutes a new layer of the regulatory mechanisms by which light inhibits auxin-induced hypocotyl elongation.

phyB Interacts with BES1 to Regulate Brassinosteroid Signaling in Arabidopsis.

Light is an important environmental factor, which mainly inhibits hypocotyl elongation through various photoreceptors. In contrast, brassinosteroids (BRs) are major hypocotyl elongation-promoting hormones in plants, which could optimize photomorphogenesis concurrent with external light. However, the precise molecular mechanisms underlying the antagonism of light and BR signaling remain largely unknown. Here we show that the Arabidopsis red light receptor phyB is involved in inhibition of BR signaling via its direct interaction with the BR transcription factor BES1. In our study, the phyB mutant displays BR hypersensitivity, which is repressed in transgenic plants overexpressing phyB, suggesting that phyB negatively regulates the BR signaling pathway. In addition, protein interaction results show that phyB directly interacts with dephosphorylated BES1, the physiologically active form of BES1 induced by BR, in a red light-dependent manner. Genetic analyses suggest that phyB may act partially through BES1 to regulate BR signaling. Transcriptomic data and quantitative real-time PCR assay further show that phyB-mediated red light inhibits BR signaling by repressing expression of BES1 target genes, including the BR biosynthesis genes DWF4, the SAUR family and the PRE family genes required for promoting cell elongation. Finally, we found that red light treatment inhibits the DNA-binding activity of BES1 and photoactivated phyB represses the transcriptional activity of BES1 under red light. Taken together, we suggest that the interaction of phyB with dephosphorylated BES1 may allow plants to balance light and BR signaling by repressing transcriptional activity of BES1 to regulate expression of its target genes.

Mapping and identification of CsUp, a gene encoding an Auxilin-like protein, as a putative candidate gene for the upward-pedicel mutation (up) in cucumber.

BACKGROUND: Pedicel orientation can affect the female flower orientation and seed yield in cucumber. A spontaneous mutant possessing upward growth of pedicels was identified in the wild type inbred strain 9930 and named upward-pedicel (up). The morphological and genetic analyses of up were performed in this study. In order to clone the up gene, 933 F2 individuals and 524 BC1 individuals derived from C-8-6 (WT) and up were used for map-based cloning. RESULTS: up was mapped to a 35.2 kb physical interval on chromosome 1, which contains three predicted genes. Sequencing analysis revealed that a 5-bp deletion was found in the second exon of Csa1G535800, and it led to a frameshift mutation resulting in a premature stop codon. The candidate gene of CsUp (Csa1G535800) was further confirmed via genomic and cDNA sequencing in biparental and natural cucumber populations. Sequencing data showed that a 4-bp deletion was found in the sixth exon of Csa1G535800 in CGN19839, another inbred line, and there was also a mutation of an amino acid in Csa1G535800 that could contribute to the upward growth of pedicels in CGN19839. Moreover, it was found that Csa1G535800 exhibited strong expression in the pedicel of WT, suggesting its important role in development of pedicel orientation. Thus, Csa1G535800 was considered to be the candidate gene of CsUp. CONCLUSIONS: CsUp encodes an Auxilin-like protein and controls pedicel orientation in cucumber. The identification of CsUp may help us to understand the mechanism of pedicel orientation development and allow for investigation of novel functions of Auxilin-like proteins in cucumber.

Blue-light-dependent interaction of cryptochrome 1 with SPA1 defines a dynamic signaling mechanism.

Cryptochromes (CRYs) are blue-light photoreceptors that mediate various light responses in plants and animals. The signaling mechanism by which CRYs regulate light responses involves their physical interactions with COP1. Here, we report that CRY1 interacts physically with SPA1 in a blue-light-dependent manner. SPA acts genetically downstream from CRYs to regulate light-controlled development. Blue-light activation of CRY1 attenuates the association of COP1 with SPA1 in both yeast and plant cells. These results indicate that the blue-light-triggered CRY1-SPA1 interaction may negatively regulate COP1, at least in part, by promoting the dissociation of COP1 from SPA1. This interaction and consequent dissociation define a dynamic photosensory signaling mechanism.

CRY1 interacts directly with HBI1 to regulate its transcriptional activity and photomorphogenesis in Arabidopsis.

Cryptochromes (CRYs) are blue light photoreceptors that mediate various light responses in plants and animals. In Arabidopsis, there are two homologous CRYs, CRY1 and CRY2, which mediate blue light inhibition of hypocotyl elongation. It is known that CRY2 interacts with CIB1, a basic helix-loop-helix (bHLH) transcriptional factor, to regulate transcription and floral induction. In this study, we performed yeast two-hybrid screening and identified CIB1 as a CRY1-interacting protein. Moreover, we demonstrated that CRY1 physically interacted with the close homolog of CIB1, HBI1, which is known to act downstream of brassinosteroid (BR) and gibberellin acid (GA) signaling pathways to promote hypocotyl elongation, in a blue light-dependent manner. Transgenic and genetic interaction studies showed that overexpression of HBI1 resulted in enhanced hypocotyl elongation under blue light and that HBI1 acted downstream of CRYs to promote hypocotyl elongation. Genome-wide gene expression analysis indicated that CRYs and HBI1 antagonistically regulated the expression of genes involved in regulating cell elongation. Moreover, we demonstrated that CRY1-HBI1 interaction led to inhibition of HBI1's DNA binding activity and its target gene expression. Together, our results suggest that HBI1 acts as a new CRY1-interacting protein and that the signaling mechanism of CRY1 involves repression of HBI1 transcriptional activity by direct CRY1-HBI1 interaction.

Identification and mapping of ts (tender spines), a gene involved in soft spine development in Cucumis sativus.

KEY MESSAGE: Using map-based cloning of ts gene, we identified a new sort of gene involved in the initiation of multicellular tender spine in cucumber. The cucumber (Cucumis sativus L.) fruit contains spines on the surface, which is an extremely valuable quality trait affecting the selection of customers. In this study, we elaborated cucumber line NC072 with wild type (WT) hard fruit spines and its spontaneous mutant NC073, possessing tender and soft spines on fruits. The mutant trait was named as tender spines (ts), which is controlled by a single recessive nuclear gene. We identified the gene ts by map-based cloning with an F2 segregating population of 721 individuals generated from NC073 and WT line SA419-2. It was located between two markers Indel6239679 and Indel6349344, 109.7 kb physical distance on chromosome 1 containing fifteen putative genes. With sequencing and quantitative reverse transcription-polymerase chain reaction analysis, the Csa1G056960 gene was considered as the most possible candidate gene of ts. In the mutant, Csa1G056960 has a nucleotide change in the 5' splicing site of the second intron, which causes different splicing to delete the second exon, resulting in a N-terminal deletion in the predicted amino acid sequence. The gene encodes a C-type lectin receptor-like tyrosine-protein kinase which would play an important role in the formation of cucumber fruit. This is firstly reported of a receptor kinase gene regulating the development of multicellular spines/trichomes in plants. The ts allele could accelerate the molecular breeding of cucumber soft spines.

COP1 and phyB Physically Interact with PIL1 to Regulate Its Stability and Photomorphogenic Development in Arabidopsis.

In Arabidopsis thaliana, the cryptochrome and phytochrome photoreceptors act together to promote photomorphogenic development. The cryptochrome and phytochrome signaling mechanisms interact directly with CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1), a RING motif-containing E3 ligase that acts to negatively regulate photomorphogenesis. COP1 interacts with and ubiquitinates the transcription factors that promote photomorphogenesis, such as ELONGATED HYPOCOTYL5 and LONG HYPOCOTYL IN FAR-RED1 (HFR1), to inhibit photomorphogenic development. Here, we show that COP1 physically interacts with PIF3-LIKE1 (PIL1) and promotes PIL1 degradation via the 26S proteasome. We further demonstrate that phyB physically interacts with PIL1 and enhances PIL1 protein accumulation upon red light irradiation, probably through suppressing the COP1-PIL1 association. Biochemical and genetic studies indicate that PIL1 and HFR1 form heterodimers and promote photomorphogenesis cooperatively. Moreover, we demonstrate that PIL1 interacts with PIF1, 3, 4, and 5, resulting in the inhibition of the transcription of PIF direct-target genes. Our results reveal that PIL1 stability is regulated by phyB and COP1, likely through physical interactions, and that PIL1 coordinates with HFR1 to inhibit the transcriptional activity of PIFs, suggesting that PIL1, HFR1, and PIFs constitute a subset of antagonistic basic helix-loop-helix factors acting downstream of phyB and COP1 to regulate photomorphogenic development.

Micro-trichome as a class I homeodomain-leucine zipper gene regulates multicellular trichome development in Cucumis sativus.

Plant trichomes serve as a highly suitable model for investigating cell differentiation at the single-cell level. The regulatory genes involved in unicellular trichome development in Arabidopsis thaliana have been intensively studied, but genes regulating multicellular trichome development in plants remain unclear. Here, we characterized Cucumis sativus (cucumber) trichomes as representative multicellular and unbranched structures, and identified Micro-trichome (Mict), using map-based cloning in an F2 segregating population of 7,936 individuals generated from a spontaneous mict mutant. In mict plants, trichomes in both leaves and fruits, are small, poorly developed, and denser than in the wild type. Sequence analysis revealed that a 2,649-bp genomic deletion, spanning the first and second exons, occurred in a plant-specific class I homeodomain-leucine zipper gene. Tissue-specific expression analysis indicated that Mict is strongly expressed in the trichome cells. Transcriptome profiling identified potential targets of Mict including putative homologs of genes known in other systems to regulate trichome development, meristem determinacy, and hormone responsiveness. Phylogenic analysis charted the relationships among putative homologs in angiosperms. Our paper represents initial steps toward understanding the development of multicellular trichomes.

Jasmonic acid enhancement of anthocyanin accumulation is dependent on phytochrome A signaling pathway under far-red light in Arabidopsis.

Anthocyanins are critical for plants. It is shown that the expression of genes encoding the key enzymes such as dihydroflavonol 4-reductase (DFR), UDP-Glc: flavonoid 3-O-glucosyltransferase (UF3GT), and leucoanthocyanidin dioxygenase (LDOX) in anthocyanin biosynthesis pathway is regulated by MYB75, a R2R3 MYB transcription factor. The production of anthocyanin is known to be promoted by jasmonic acid (JA) in light but not in darkness. The photoreceptors cryptochrome 1 (CRY1), phytochrome B (phyB), and phytochrome A (phyA) are also shown to mediate light promotion of anthocyanin accumulation, respectively, whereas their downstream factor COP1, a master negative regulator of photomorphogensis, represses anthocyanin accumulation. However, whether JA coordinates with photoreceptors in the regulation of anthocyanin accumulation is unknown. Here, we show that under far-red light, JA promotes anthocyanin accumulation in a phyA signaling pathway-dependent manner. The phyA mutant is hyposensitive to jasmonic acid analog methyl jasmonic acid (MeJA) under far-red light. The dominant mutant of MYB75, pap1-D, accumulates significantly higher levels of anthocyanin than wild type under far-red light, whereas knockdown of MYBs (MYB75, MYB90, MYB113, and MYB114) through RNAi significantly reduces MeJA promotion of anthocyanin accumulation. The phyA pap1-D double mutant shows reduced responsiveness to MeJA, similar to phyA mutant under far-red light. In darkness, a mutant allele of cop1, cop1-4, shows enhanced responsiveness to MeJA, but pap1-D mutant is barely responsive to MeJA. Upon MeJA application, the cop1-4 pap1-D double mutant accumulates considerably higher levels of anthocyanin than cop1-4 in darkness. Protein studies indicate that MYB75 protein is stabilized by white light and far-red light. Further gene expression studies suggest that MeJA promotes the expression of DFR, UF3GT, and LDOX genes in a phyA- and MYB75-dependent manner under far-red light. Our findings suggest that JA promotion of anthocyanin accumulation under far-red light is dependent on phyA signaling pathway, consisting of phyA, COP1, and MYB75.

FvDFR2 rather than FvDFR1 play key roles for anthocyanin synthesis in strawberry petioles.

The accumulation of anthocyanins can be found in both the fruit and petioles of strawberries, but the fruit appears red while the petioles appear purple-red. Additionally, in the white-fruited diploid strawberries, the petioles can accumulate anthocyanins normally, suggesting a different synthesis pattern between the petioles and fruits. We screened the EMS mutagenized population of a red-fruited diploid strawberry 'Ruegen' and discovered a mutant which showed no anthocyanin accumulation in the petioles but normal accumulation in the fruit. Through BSA sequencing and allelic test, it was found that a mutation in FvDFR2 was responsible for this phenotype. Furthermore, the complex formed by the interaction between the petiole-specific FvMYB10L and FvTT8 only binds the promoter of FvDFR2 but not FvDFR1, resulting in the expression of only FvDFR2 in the petiole. FvDFR2 can catalyze the conversion of DHQ and eventually the formation of cyanidin and peonidin, giving the petiole a purplish-red color. In the fruit, however, both FvDFR1 and FvDFR2 can be expressed, which can mediate the synthesis of cyanidin and pelargonidin. Our study clearly reveals different regulation of FvDFR1 and FvDFR2 in mediating anthocyanin synthesis in petioles and fruits.

Loss-of-function mutation in anthocyanidin reductase activates the anthocyanin synthesis pathway in strawberry.

Fruit color substantially affects consumer preferences, with darker red strawberries being economically more valuable due to their higher anthocyanin content. However, the molecular basis for the dark red coloration remains unclear. Through screening of an ethyl methanesulfonate mutant library, we identified a rg418 mutant, that demonstrated anthocyanin accumulation during early fruit development stages. Furthermore, the ripening fruits of this mutant had higher anthocyanin content than wild-type (WT) fruits. An analysis of flavonoid content in WT and rg418 mutant fruits revealed substantial changes in metabolic fluxes, with the mutant exhibiting increased levels of anthocyanins and flavonols and decreased levels of proanthocyanidins. Bulked sergeant analysis sequencing indicated that the mutant gene was anthocyanidin reductase (ANR), a key gene in the proanthocyanidin synthesis pathway. Furthermore, transcriptome sequencing revealed the increased expression of MYB105 during the early development stage of mutant fruits, which promoted the expression of UFGT (UDP-glucose flavonoid 3-O-glucosyltransferase), a key gene involved in anthocyanin synthesis, thus substantially enhancing the anthocyanin content in the mutant fruits. Additionally, mutating ANR in a white-fruited strawberry variant (myb10 mutant) resulted in appealing pink-colored fruits, suggesting the diverse roles of ANR in fruit color regulation. Our study provides valuable theoretical insights for improving strawberry fruit color.

Comprehensive Identification and Expression Analysis of the YTH Family of RNA-Binding Proteins in Strawberry.

Plant growth and development processes are tightly regulated at multiple levels, including transcriptional and post-transcriptional levels, and the RNA-binding protein YTH regulates gene expression during growth and development at the post-transcriptional level by regulating RNA splicing, processing, stability, and translation. We performed a systematic characterization of YTH genes in diploid forest strawberry and identified a total of nine YTH genes. With the help of phylogenetic analysis, these nine genes were found to belong to two different groups, YTHDC and YTHDF, with YTHDF being further subdivided into three subfamilies. Replication analysis showed that YTH3 and YTH4 are a gene pair generated by tandem repeat replication. These two genes have similarities in gene structure, number of motifs, and distribution patterns. Promoter analysis revealed the presence of multiple developmental, stress response, and hormone-response-related cis-elements. Analysis of available transcriptome data showed that the expression levels of most of the YTH genes were stable with no dramatic changes during development in different tissues. However, YTH3 maintained high expression levels in all tissues and during fruit development, and YTH4 was expressed at higher levels in tissues such as flowers, leaves, and seedlings, while it was significantly lower than YTH3 in white fruits and ripening fruits with little fluctuation. Taken together, our study provides insightful and comprehensive basic information for the study of YTH genes in strawberry.