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.

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.

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.

Genome-Wide Characterization and Expression Profiling of HD-Zip Genes in ABA-Mediated Processes in Fragaria vesca.

Members of homeodomain-leucine zipper (HD-Zip) transcription factors can play their roles by modulating abscisic acid (ABA) signaling in Arabidopsis. So far, our knowledge of the functions of HD-Zips in woodland strawberries (Fragaria vesca), a model plant for studying ABA-mediated fruit ripening, is limited. Here, we identified a total of 31 HD-Zip genes (FveHDZ1-31) in F. vesca, and classified them into four subfamilies (I to IV). Promoter analyses show that the ABA-responsive element, ABRE, is prevalent in the promoters of subfamily I and II FveHDZs. RT-qPCR results demonstrate that 10 of the 14 investigated FveHDZs were consistently >1.5-fold up-regulated or down-regulated in expression in response to exogenous ABA, dehydration, and ABA-induced senescence in leaves. Five of the six consistently up-regulated genes are from subfamily I and II. Thereinto, FveHDZ4, and 20 also exhibited significantly enhanced expression along with increased ABA content during fruit ripening. In yeast one-hybrid assays, FveHDZ4 proteins could bind the promoter of an ABA signaling gene FvePP2C6. Collectively, our results strongly support that the FveHDZs, particularly those from subfamilies I and II, are involved in the ABA-mediated processes in F. vesca, providing a basis for further functional characterization of the HD-Zips in strawberry and other plants.

High mobility group A3 enhances transcription of the DNA demethylase gene SlDML2 to promote tomato fruit ripening.

DNA methylation plays an important role in regulating tomato (Solanum lycopersicum) fruit ripening. Although SlDML2, a DNA demethylase (DML) gene, is critically involved in tomato fruit ripening, little is known about genes that regulate its expression. Using yeast one-hybrid screening, we identified a High Mobility Group A protein, named SlHMGA3, and demonstrated its binding activity to the AT-rich region of the SlDML2 promoter. We produced slhmga3 tomato mutants using CRISPR/Cas9 and observed that slhmga3 fruit reached the breaker stage much later than fruit from the wild-type. We further demonstrated that at the initiation stage of fruit ripening, the increased expression of SlDML2 and ethylene biosynthetic and signaling genes was significantly delayed in slhmga3 fruit, along with delays in ethylene production and demethylation and activation of ripening-associated transcription factor genes. Our results demonstrate that SlHMGA3 plays a role in enhancing SlDML2 expression, and its effects on tomato fruit ripening are largely through DNA demethylation of ripening-associated transcription factor genes.

Down-Regulation of Cytokinin Receptor Gene SlHK2 Improves Plant Tolerance to Drought, Heat, and Combined Stresses in Tomato.

Environmental stresses negatively affect the growth and development of plants. Several previous studies have elucidated the response mechanisms of plants to drought and heat applied separately; however, these two abiotic stresses often coincide in environmental conditions. The global climate change pattern has projected that combined drought and heat stresses will tend to increase in the near future. In this study, we down-regulated the expression of a cytokinin receptor gene SlHK2 using RNAi and investigated the role of this gene in regulating plant responses to individual drought, heat, and combined stresses (drought + heat) in tomato. Compared to the wild-type (WT), SlHK2 RNAi plants exhibited fewer stress symptoms in response to individual and combined stress treatments. The enhanced abiotic stress tolerance of SlHK2 RNAi plants can be associated with increased membrane stability, osmoprotectant accumulation, and antioxidant enzyme activities. Furthermore, photosynthesis machinery was also protected in SlHK2 RNAi plants. Collectively, our results show that down-regulation of the cytokinin receptor gene SlHK2, and consequently cytokinin signaling, can improve plant tolerance to drought, heat, and combined stress.

Identification and Expression Analysis of Hormone Biosynthetic and Metabolism Genes in the 2OGD Family for Identifying Genes That May Be Involved in Tomato Fruit Ripening.

Phytohormones play important roles in modulating tomato fruit development and ripening. The 2-oxoglutarate-dependent dioxygenase (2OGD) superfamily containing several subfamilies involved in hormone biosynthesis and metabolism. In this study, we aimed to identify hormone biosynthesis and metabolism-related to 2OGD proteins in tomato and explored their roles in fruit development and ripening. We identified nine 2OGD protein subfamilies involved in hormone biosynthesis and metabolism, including the gibberellin (GA) biosynthetic protein families GA20ox and GA3ox, GA degradation protein families C19-GA2ox and C20-GA2ox, ethylene biosynthetic protein family ACO, auxin degradation protein family DAO, jasmonate hydroxylation protein family JOX, salicylic acid degradation protein family DMR6, and strigolactone biosynthetic protein family LBO. These genes were differentially expressed in different tomato organs. The GA degradation gene SlGA2ox2, and the auxin degradation gene SlDAO1, showed significantly increased expression from the mature-green to the breaker stage during tomato fruit ripening, accompanied by decreased endogenous GA and auxin, indicating that SlGA2ox2 and SlDAO1 were responsible for the reduced GA and auxin concentrations. Additionally, exogenous gibberellin 3 (GA3) and indole-3-acetic acid (IAA) treatment of mature-green fruits delayed fruit ripening and increased the expression of SlGA2ox2 and SlDAO1, respectively. Therefore, SlGA2ox2 and SlDAO1 are implicated in the degradation of GAs and auxin during tomato fruit ripening.

Volatile composition and classification of Lilium flower aroma types and identification, polymorphisms, and alternative splicing of their monoterpene synthase genes.

Lily is a well-known ornamental plant with a diversity of fragrant types. Basic information on lily floral scent compounds has been obtained for only a few accessions, and little is known about Lilium aroma types, the terpene synthase genes that may play roles in the production of key volatiles, or the range of monoterpenes that these genes produce. In this study, 41 cultivars were analyzed for volatile emissions, and a total of 46 individual volatile compounds were identified, 16 for the first time in lilies. Lily accessions were classified into six groups according to the composition of major scent components: faint-scented, cool, fruity, musky, fruity-honey, and lily. Monoterpenes were one of the main groups of volatiles identified, and attention was focused on terpene synthase (TPS) genes, which encode enzymes that catalyze the last steps in monoterpene synthesis. Thirty-two candidate monoterpene synthase cDNAs were obtained from 66 lily cultivars, and 64 SNPs were identified. Two InDels were also shown to result from variable splicing, and sequence analysis suggested that different transcripts arose from the same gene. All identified nucleotide substitution sites were highly correlated with the amounts of myrcene emitted, and InDel site 230 was highly correlated with the emission of all major monoterpenoid components, especially (E)-ß-ocimene. Heterologous expression of five cDNAs cloned from faint-scented and strong-scented lilies showed that their corresponding enzymes could convert geranyl diphosphate to (E)-ß-ocimene, α-pinene, and limonene. The findings from this study provide a major resource for the assessment of lily scent volatiles and will be helpful in breeding of improved volatile components.

Identification of differentially expressed genes in flower, leaf and bulb scale of Lilium oriental hybrid 'Sorbonne' and putative control network for scent genes.

BACKGROUND: Lily is an economically important plant, with leaves and bulbs consisting of overlapping scales, large ornamental flowers and a very large genome. Although it is recognized that flowers and bulb scales are modified leaves, very little is known about the genetic control and biochemical differentiation underlying lily organogenesis and development. Here we examined the differentially expressed genes in flower, leaf and scale of lily, using RNA-sequencing, and identified organ-specific genes, including transcription factors, genes involved in photosynthesis in leaves, carbohydrate metabolism in bulb scales and scent and color production in flowers. RESULTS: Over 11Gb data were obtained and 2685, 2296, and 1709 differentially expressed genes were identified in the three organs, with 581, 662 and 977 unique DEGs in F-vs-S, L-vs-S and L-vs-F comparisons. By functional enrichment analysis, genes likely to be involved in biosynthetic pathways leading to floral scent production, such as 1-deoxy-D-xylulose-5-phosphate synthase (DXS), 3-ketoacyl-CoA thiolase (KAT), hydroperoxide lyase (HPL), geranylgeranyl pyrophosphate (GGPP) 4-hydroxy-3-methylbut-2-en-1-yl diphosphate (HDS) and terpene synthase (TPS), and floral color genes, such as dihydroflavonol 4-reductase (DFR), chalcone synthase (CHS), chalcone isomerase (CHI), flavonol synthase (FLS) were identified. Distinct groups of genes that participate in starch and sucrose metabolism, such as sucrose synthase (SS), invertase (INV), sucrose phosphate synthase (SPS), starch synthase (SSS), starch branching enzyme (SBE), ADP-glucose pyrophosphorylase (AGP) andß-amylase (BAM) and photosynthesis genes (Psa, Psb, Pet and ATP) were also identified. The expression of six floral fragrance-related DGEs showed agreement between qRT-PCR results and RPKM values, confirming the value of the data obtained by RNA-seq. We obtained the open reading frame of the terpene synthase gene from Lilium 'Sorbonne', designated LsTPS, which had 99.55% homology to transcript CL4520.Contig5_All. In addition, 54, 48 and 50 differently expressed transcription factor were identified by pairwise comparisons between the three organs and a regulatory network for monoterpene biosynthesis was constructed. CONCLUSIONS: Analysis of differentially expressed genes in flower, leaf and bulb scale of lily, using second generation sequencing technology, yielded detailed information on lily metabolic differentiation in three organs. Analysis of the expression of flower scent biosynthesis genes has provided a model for the regulation of the pathway and identified a candidate gene encoding an enzyme catalyzing the final step in scent production. These digital gene expression profiles provide a valuable and informative database for the further identification and analysis of structural genes and transcription factors in different lily organs and elucidation of their function.