Comparative transcriptome analysis identifies candidate genes related to sucrose accumulation in longan (Dimocarpus longan Lour.) pulp.

Sucrose content is one of the important factors to determine longan fruit flavor quality. To gain deep insight of molecular mechanism on sucrose accumulation in longan, we conducted comparative transcriptomic analysis between low sucrose content longan cultivar 'Qingkebaoyuan' and high sucrose content cultivar 'Songfengben'. A total of 12,350 unique differentially expressed genes (DEGs) were detected across various development stages and different varieties, including hexokinase (HK) and sucrose-phosphate synthase (SPS), which are intricately linked to soluble sugar accumulation and metabolism. Weighted gene co-expression network analysis (WGCNA) identified magenta module, including DlSPS gene, was significantly positively correlated with sucrose content. Furthermore, transient expression unveiled DlSPS gene play crucial role in sucrose accumulation. Moreover, 5 transcription factors (MYB, ERF, bHLH, C2H2, and NAC) were potentially involved in DlSPS regulation. Our findings provide clues for sucrose metabolism, and lay the foundation for longan breeding in the future.

A 224-bp Indel in the Promoter of PeMYB114 Accounts for Anthocyanin Accumulation of Skin in Passion Fruit (Passiflora spp.).

Passion fruit (Passiflora spp.) is an important fruit tree in the family Passifloraceae. The color of the fruit skin, a significant agricultural trait, is determined by the content of anthocyanin in passion fruit. However, the regulatory mechanisms behind the accumulation of anthocyanin in different passion fruit skin colors remain unclear. In the study, we identified and characterized a R2R3-MYB transcription factor, PeMYB114, which functions as a transcriptional activator in anthocyanin biosynthesis. Yeast one-hybrid system and dual-luciferase analysis showed that PeMYB114 could directly activate the expression of anthocyanin structural genes (PeCHS and PeDFR). Furthermore, a natural variation in the promoter region of PeMYB114 alters its expression. PeMYB114purple accessions with the 224-bp insertion have a higher anthocyanin level than PeMYB114yellow accessions with the 224-bp deletion. The findings enhance our understanding of anthocyanin accumulation in fruits and provide genetic resources for genome design for improving passion fruit quality.

The haplotype-resolved autotetraploid genome assembly provides insights into the genomic evolution and fruit divergence in wax apple (Syzygium samarangense (Blume) Merr. and Perry).

Wax apple (Syzygium samarangense) is an economically important fruit crop with great potential value to human health because of its richness in antioxidant substances. Here, we present a haplotype-resolved autotetraploid genome assembly of the wax apple with a size of 1.59 Gb. Comparative genomic analysis revealed three rounds of whole-genome duplication (WGD) events, including two independent WGDs after WGT-γ. Resequencing analysis of 35 accessions partitioned these individuals into two distinct groups, including 28 landraces and seven cultivated species, and several genes subject to selective sweeps possibly contributed to fruit growth, including the KRP1-like, IAA17-like, GME-like, and FLACCA-like genes. Transcriptome analysis of three different varieties during flower and fruit development identified key genes related to fruit size, sugar content, and male sterility. We found that AP2 also affected fruit size by regulating sepal development in wax apples. The expression of sugar transport-related genes (SWEETs and SUTs) was high in 'ZY', likely contributing to its high sugar content. Male sterility in 'Tub' was associated with tapetal abnormalities due to the decreased expression of DYT1, TDF1, and AMS, which affected early tapetum development. The chromosome-scale genome and large-scale transcriptome data presented in this study offer new valuable resources for biological research on S. samarangense and shed new light on fruit size control, sugar metabolism, and male sterility regulatory metabolism in wax apple.

Genome-wide identification of the AOMT gene family in wax apple and functional characterization of SsAOMTs to anthocyanin methylation.

Introduction: Anthocyanins are major pigments in the peels of red-series wax apple fruits, and two principal components of them, namely, the cyanin and the peonidin, are non-methoxylated and methoxylated anthocyanins, respectively. Anthocyanin O-methyltransferases (AOMTs) are an important group of enzymes that have the ability to catalyze anthocyanins methylation to promote the solubility, stability, and bioactivity of anthocyanins. Although AOMT genes have been studied in a variety of plants, the function of them in wax apple is generally not well understood. Methods: The anthocyanin composition in peels of two wax apple cultivars was determined by High Performance Liquid Chromatography Tandem Mass Spectrometry (HPLS-MS). The genome-wide analysis of the AOMT genes was performed with bioinformatics technology, and the expression patterns of different plant tissues, cultivars, fruit ripening stages, and exogenous abscisic acid (ABA) treatments were analyzed by transcriptome sequencing analysis and real-time quantitative PCR verification. An initial functional evaluation was carried out in vitro using recombinant the Anthocyanin O-methyltransferase Gene 5 of S. samarangense (SsAOMT5) protein. Results: Only two main compositions of anthocyanin were found in peels of two wax apple cultivars, and it was worth noting that Tub Ting Jiang cultivar contained non-methoxylated anthocyanin (Cy3G) only, whereas Daye cultivar contained both non-methoxylated and methoxylated (Pn3G) anthocyanins. A total of six SsAOMT genes were identified in the whole genome of wax apple, randomly distributing on three chromosomes. A phylogenic analysis of the protein sequences divided the SsAOMT gene family into three subgroups, and all SsAOMTs had highly conserved domains of AOMT family. In total, four types of stress- related and five types of hormone- related cis-elements were discovered in the promoter region of the SsAOMTs. Expression pattern analysis showed that SsAOMT5 and SsAOMT6 were expressed in all tissues to varying degrees; notably, the expression of SsAOMT5 was high in the flower and fruit and significantly higher in Daye peels than those of other cultivars in the fruit ripening period. Exogenous ABA treatment significantly increased anthocyanin accumulation, but the increase of methoxylated anthocyanin content did not reach significant level compared with those without ABA treatment, whereas the expression of SsAOMT5 upregulated under ABA treatment. We identified two homologous SsAOMT5 genes from Daye cultivar (DSsAOMT5) and Tub Ting Jiang cultivar (TSsAOMT5); the results of functional analyses to two SsAOMT5 recombinant proteins in vitro demonstrated that DSsAOMT5 showed methylation modification activity, but TSsAOMT5 did not. Conclusion: In conclusion, SsAOMT5 was responsible for methylated anthocyanin accumulation in the peels of wax apple and played an important role in red coloration in wax apple peels.

High-Throughput Sequencing Reveals Novel microRNAs Involved in the Continuous Flowering Trait of Longan (Dimocarpus longan Lour.).

A major determinant of fruit production in longan (Dimocarpus longan Lour.) is the difficulty of blossoming. In this study, high-throughput microRNA sequencing (miRNA-Seq) was carried out to compare differentially expressed miRNAs (DEmiRNAs) and their target genes between a continuous flowering cultivar 'Sijimi' (SJ), and a unique cultivar 'Lidongben' (LD), which blossoms only once in the season. Over the course of our study, 1662 known miRNAs and 235 novel miRNAs were identified and 13,334 genes were predicted to be the target of 1868 miRNAs. One conserved miRNA and 29 new novel miRNAs were identified as differently expressed; among them, 16 were upregulated and 14 were downregulated. Through the KEGG pathway and cluster analysis of DEmiRNA target genes, three critical regulatory pathways, plant-pathogen interaction, plant hormone signal transduction, and photosynthesis-antenna protein, were discovered to be strongly associated with the continuous flowering trait of the SJ. The integrated correlation analysis of DEmiRNAs and their target mRNAs revealed fourteen important flowering-related genes, including COP1-like, Casein kinase II, and TCP20. These fourteen flowering-related genes were targeted by five miRNAs, which were novel-miR137, novel-miR76, novel-miR101, novel-miR37, and csi-miR3954, suggesting these miRNAs might play vital regulatory roles in flower regulation in longan. Furthermore, novel-miR137 was cloned based on small RNA sequencing data analysis. The pSAK277-miR137 transgenic Arabidopsis plants showed delayed flowering phenotypes. This study provides new insight into molecular regulation mechanisms of longan flowering.

Characterization of the SWEET Gene Family in Longan (Dimocarpus longan) and the Role of DlSWEET1 in Cold Tolerance.

Sugars will eventually be exported transporters (SWEET), a group of relatively novel sugar transporters, that play important roles in phloem loading, seed and fruit development, pollen development, and stress response in plants. Longan (Dimocarpus longan), a subtropic fruit tree with high economic value, is sensitive to cold. However, whether the SWEET gene family plays a role in conferring cold tolerance upon longan remains unknown. Here, a total of 20 longan SWEET (DlSWEET) genes were identified, and their phylogenetic relationships, gene structures, cis-acting elements, and tissue-specific expression patterns were systematically analyzed. This family is divided into four clades. Gene structures and motifs analyses indicated that the majority of DlSWEETs in each clade shared similar exon-intron organization and conserved motifs. Tissue-specific gene expression suggested diverse possible functions for DlSWEET genes. Cis-elements analysis and quantitative real-time PCR (qRT-PCR) analysis revealed that DlSWEET1 responded to cold stress. Notably, the overexpression of DlSWEET1 improved cold tolerance in transgenic Arabidopsis, suggesting that DlSWEET1 might play a positive role in D. longan's responses to cold stress. Together, these results contribute to a better understanding of SWEET genes, which could serve as a foundation for the further functional identification of these genes.

Integrative mRNA and Long Noncoding RNA Analysis Reveals the Regulatory Network of Floral Bud Induction in Longan (Dimocarpus longan Lour.).

Longan (Dimocarpus longan Lour.) is a tropical/subtropical fruit tree of significant economic importance. Floral induction is an essential process for longan flowering and plays decisive effects on the longan yield. Due to the instability of flowering, it is necessary to understand the molecular mechanisms of floral induction in longan. In this study, mRNA and long noncoding RNA (lncRNA) transcriptome sequencing were performed using the apical buds of fruiting branches as materials. A total of 7,221 differential expressions of mRNAs (DEmRNAs) and 3,238 differential expressions of lncRNAs (DElncRNAs) were identified, respectively. KEGG enrichment analysis of DEmRNAs highlighted the importance of starch and sucrose metabolic, circadian rhythms, and plant hormone signal transduction pathways during floral induction. Combining the analysis of weighted gene co-expression network (WGCNA) and expression pattern of DEmRNAs in the three pathways, specific transcriptional characteristics at each stage during floral induction and regulatory network involving co-expressed genes were investigated. The results showed that sucrose metabolism and auxin signal transduction may be crucial for the growth and maturity of autumn shoots in September and October (B1-B2 stage); starch and sucrose metabolic, circadian rhythms, and plant hormone signal transduction pathways participated in the regulation of floral bud physiological differentiation together in November and December (B3-B4 stage) and the crosstalk among three pathways was also found. Hub genes in the co-expression network and key DEmRNAs in three pathways were identified. The circadian rhythm genes FKF1 and GI were found to activate SOC1gene through the photoperiod core factor COL genes, and they were co-expressed with auxin, gibberellin, abscisic acid, ethylene signaling genes, and sucrose biosynthesis genes at B4 stage. A total of 12 hub-DElncRNAs had potential for positively affecting their distant target genes in three putative key pathways, predominantly in a co-transcriptional manner. A hypothetical model of regulatory pathways and key genes and lncRNAs during floral bud induction in longan was proposed finally. Our studies will provide valuable clues and information to help elucidate the potential molecular mechanisms of floral initiation in longan and woody fruit trees.

Complete chloroplast genome sequence of Syzygium samarangense (Myrtaceae) and phylogenetic analysis.

Syzygium samarangense (Blume) Merr. et Perry, 1938, commonly known as wax apple, is a Myrtaceae species that is known for its unique fruit shape, flavorful and colorful fruits, medicinal value and increasing economic relevance. In this study, we reported the complete chloroplast genome sequence of S. samarangense. The complete genome is 159,109 bp in length with a quadripartite structure containing two single copy regions, a Large Single Copy region (LSC, 88,155 bp) and a Small Single Copy region (SSC, 18,796 bp) separated by Inverted Repeat regions (IRs, 26,079 bp). The GC content was 37.0%. It encoded 126 genes, including 81 protein-coding genes, 37 transfer RNA genes, and 8 ribosomal RNA genes. The phylogenetic relationships of 20 species inferred that all Syzygium species formed a single cluster belonging to Syzygieae tribe. Our results offer insights into the evolutionary relationship of S. samarangense within Myrtaceae, indicating a closer relationship between S. samarangense and S. forrestii.

The red flesh of kiwifruit is differentially controlled by specific activation-repression systems.

Anthocyanins are visual cues for pollination and seed dispersal. Fruit containing anthocyanins also appeals to consumers due to its appearance and health benefits. In kiwifruit (Actinidia spp.) studies have identified at least two MYB activators of anthocyanin, but their functions in fruit and the mechanisms by which they act are not fully understood. Here, transcriptome and small RNA high-throughput sequencing were used to comprehensively identify contributors to anthocyanin accumulation in kiwifruit. Stable overexpression in vines showed that both 35S::MYB10 and MYB110 can upregulate anthocyanin biosynthesis in Actinidia chinensis fruit, and that MYB10 overexpression resulted in anthocyanin accumulation which was limited to the inner pericarp, suggesting that repressive mechanisms underlie anthocyanin biosynthesis in this species. Furthermore, motifs in the C-terminal region of MYB10/110 were shown to be responsible for the strength of activation of the anthocyanic response. Transient assays showed that both MYB10 and MYB110 were not directly cleaved by miRNAs, but that miR828 and its phased small RNA AcTAS4-D4(-) efficiently targeted MYB110. Other miRNAs were identified, which were differentially expressed between the inner and outer pericarp, and cleavage of SPL13, ARF16, SCL6 and F-box1, all of which are repressors of MYB10, was observed. We conclude that it is the differential expression and subsequent repression of MYB activators that is responsible for variation in anthocyanin accumulation in kiwifruit species.

Recent Advanced Metabolic and Genetic Engineering of Phenylpropanoid Biosynthetic Pathways.

The MYB transcription factors (TFs) are evolving as critical role in the regulation of the phenylpropanoid and tanshinones biosynthetic pathway. MYB TFs relate to a very important gene family, which are involved in the regulation of primary and secondary metabolisms, terpenoids, bioactive compounds, plant defense against various stresses and cell morphology. R2R3 MYB TFs contained a conserved N-terminal domain, but the domain at C-terminal sorts them different regarding their structures and functions. MYB TFs suppressors generally possess particular repressive motifs, such as pdLNLD/ELxiG/S and TLLLFR, which contribute to their suppression role through a diversity of complex regulatory mechanisms. A novel flower specific "NF/YWSV/MEDF/LW" conserved motif has a great potential to understand the mechanisms of flower development. In the current review, we summarize recent advanced progress of MYB TFs on transcription regulation, posttranscriptional, microRNA, conserved motif and propose directions to future prospective research. We further suggest there should be more focus on the investigation for the role of MYB TFs in microalgae, which has great potential for heterologous protein expression system for future perspectives.

NtbHLH1, a JAF13-like bHLH, interacts with NtMYB6 to enhance proanthocyanidin accumulation in Chinese Narcissus.

BACKGROUND: Flavonoid biosynthesis in plants is primarily regulated at the transcriptional level by transcription factors modulating the expression of genes encoding enzymes in the flavonoid pathway. One of the most studied transcription factor complexes involved in this regulation consists of a MYB, bHLH and WD40. However, in Chinese Narcissus (Narcissus tazetta L. var. chinensis), a popular monocot bulb flower, the regulatory mechanism of flavonoid biosynthesis remains unclear. RESULTS: In this work, genes related to the regulatory complex, NtbHLH1 and a R2R3-MYB NtMYB6, were cloned from Chinese Narcissus. Phylogenetic analysis indicated that NtbHLH1 belongs to the JAF13 clade of bHLH IIIf subgroup, while NtMYB6 was highly homologous to positive regulators of proanthocyanidin biosynthesis. Both NtbHLH1 and NtMYB6 have highest expression levels in basal plates of Narcissus, where there is an accumulation of proanthocyanidin. Ectopic over expression of NtbHLH1 in tobacco resulted in an increase in anthocyanin accumulation in flowers, and an up-regulation of expression of the endogenous tobacco bHLH AN1 and flavonoid biosynthesis genes. In contrast, the expression level of LAR gene was significantly increased in NtMYB6-transgenic tobacco. Dual luciferase assays showed that co-infiltration of NtbHLH1 and NtMYB6 significantly activated the promoter of Chinese Narcissus DFR gene. Furthermore, a yeast two-hybrid assay confirmed that NtbHLH1 interacts with NtMYB6. CONCLUSIONS: Our results suggest that NtbHLH1 may function as a regulatory partner by interacting directly with NtMYB6 to enhance proanthocyanidin accumulation in Chinese Narcissus.

Discovery of genes involved in anthocyanin biosynthesis from the rind and pith of three sugarcane varieties using integrated metabolic profiling and RNA-seq analysis.

BACKGROUND: Sugarcane (Saccharum officinarum) is one of the most valuable feedstocks for sugar production. In addition to the production of industrial raw materials such as alcohol, papermaking, the fiber of livestock feed, respectively, sugarcane can produce bioactive compounds such as anthocyanins. Elucidation of the anthocyanin biosynthesis pathway is critical for the molecular breeding of sugarcane varieties with favorable traits. We aimed to identify candidate genes involved in anthocyanin biosynthesis by transcriptomic and metabolomic analyses. RESULTS: Three varieties of sugarcane displaying different colors were used in this study: FN15 (greed rind), ROC22 (red rind), and Badila (purple rind). Sample materials were subjected to metabolomic analysis using UPLC-Q-TOF/MS and RNA-seq analysis. The metabolomic profiling results showed Cyanidin, Cyanidin (6'-malonylglucoside), Cyanidin O-glucoside, and Peonidin O-glucoside were the main components responsible for the rind color. Then, through RNA-seq analysis, we identified a total of 3137, 3302, 3014 differentially expressed genes (DEGs) between the rind and pith tissues for the corresponding varieties Badila rind, ROC22, and FN15. We then compared the expression levels of genes among the rind tissues from the three varieties. We identified 2901, 2821, and 3071 DEGs between Badila rind vs. ROC22 rind, Badila rind vs. FN15 rind, ROC22 rind vs. FN15 rind, respectively. We identified two enriched pathways, including phenylpropanoid biosynthesis and flavonoid biosynthesis. Sequencing similarity search identified a total of 50 unigenes belonging to 15 enzyme families as putative genes involved in anthocyanin biosynthesis in sugarcane rind. Seven of them were identified as candidate genes related to anthocyanin biosynthesis in the rind of sugarcane through co-localization analysis with the anthocyanin content in sugarcane. In total, 25 unigenes were selected and subjected to RT-qPCR analysis, and qRT-PCR results were consistent with those obtained with the RNA-Seq experiments. CONCLUSIONS: We proposed a pathway for anthocyanin biosynthesis in sugarcane rind. This is the first report on the biosynthesis of anthocyanin in sugarcane using the combined transcriptomic and metabolomic methods. The results obtained from this study will lay the foundation for breeding purple pith sugarcane varieties with high anthocyanin contents.

The Critical Role of miRNAs in Regulation of Flowering Time and Flower Development.

Flowering is an important biological process for plants that ensures reproductive success. The onset of flowering needs to be coordinated with an appropriate time of year, which requires tight control of gene expression acting in concert to form a regulatory network. MicroRNAs (miRNAs) are non-coding RNAs known as master modulators of gene expression at the post-transcriptional level. Many different miRNA families are involved in flowering-related processes such as the induction of floral competence, floral patterning, and the development of floral organs. This review highlights the diverse roles of miRNAs in controlling the flowering process and flower development, in combination with potential biotechnological applications for miRNAs implicated in flower regulation.

Genome-Wide Identification and Expression Analysis of Sugar Transporter (ST) Gene Family in Longan (Dimocarpus longan L.).

Carbohydrates are nutrients and important signal molecules in higher plants. Sugar transporters (ST) play important role not only in long-distance transport of sugar, but also in sugar accumulations in sink cells. Longan (Dimocarpus longan L.) is one of the most important commercial tropical/subtropical evergreen fruit species in Southeast Asia. In this study, a total of 52 longan sugar transporter (DlST) genes were identified and they were divided into eight clades according to phylogenetic analysis. Out of these 52 DlST genes, many plant hormones (e.g., MeJA and gibberellin), abiotic (e.g., cold and drought), and biotic stress responsive element exist in their promoter region. Gene structure analysis exhibited that each of the clades have closely associated gene architectural features based on similar number or length of exons. The numbers of DlSTs, which exhibited alternative splicing (AS) events, in flower bud is more than that in other tissues. Expression profile analysis revealed that ten DlST members may regulate longan flowerbud differentiation. In silico expression profiles in nine longan organs indicated that some DlST genes were tissue specificity and further qRT-PCR analysis suggested that the transcript level of seven DlSTs (DlINT3, DlpGlcT1, DlpGlcT2, DlPLT4, DlSTP1, DlVGT1 and DlVGT2) was consistent with sugar accumulation in fruit, indicating that they might be involved in sugar accumulations during longan fruit development. Our findings will contribute to a better understanding of sugar transporters in woody plant.

Genome-Wide Identification and Expression Analysis of Auxin Response Factor (ARF) Gene Family in Longan (Dimocarpus longan L.).

Auxin response factor (ARF) is the key regulator involved in plant development. Despite their physiological importance identified in various woody plants, the functions of ARF genes in longan were still not clear. In this study, 17 longan ARF genes (DlARF) were identified using the reference longan genome data. According to the phylogenetic relationships among longan, Arabidopsis and apple, DlARFs were divided into four classes. Most DlARFs showed a closer relationship with ARFs from apple than those from Arabidopsis. The analysis of gene structure and domain revealed high similarity of different ARF genes in the same class. Typical features of B3-type DNA binding domain (DBD) motif, Auxin Resp motifs, and a highly conserved C-terminal Phox and Bem1 (PB1) domain were present in all DlARFs except for DlARF-2,-3,-13 which lacked PBI domain. Expression profiles of 17 DlARF genes in longan different tissues showed that some DlARF genes were tissues-specific genes. Analysis of three longan transcriptomes showed seven DlARFs (DlARF-1,-2,-6,-8,-9,-11,-16) had higher expression levels during floral bud differentiation of common longan and in the buds of 'Sijimi', suggesting these genes may promote floral bud differentiation in longan. Further qPCR analysis showed that among seven DlARF genes, the expression levels of DlARF-2,-6,-11,-16 increased significantly during the physiological differentiation stage of longan floral buds, confirming that they may play a role in flowering induction. Promoter sequence analysis revealed cis-elements related to flowering induction such as low-temperature responsiveness motif and circadian control motif. Motifs linked with hormone response for instance Auxin, MeJA, Gibberellin, and Abscisic acid were also found in promoters. This study provides a comprehensive overview of the ARF gene family in longan. Our findings could provide new insights into the complexity of the regulation of ARFs at the transcription level that may be useful to develop breeding strategies to improve development or promote flowering in longan.

Optimization of Ultrasound Assisted Extraction (UAE) of Kinsenoside Compound from Anoectochilus roxburghii (Wall.) Lindl by Response Surface Methodology (RSM).

The purpose of this study was to establish an extraction method for the kinsenoside compound from the whole plant Anoectochilus roxburghii. Ultrasound assisted extraction (UAE) and Ultra-high performance liquid chromatography (UPLC) method were used to extract and determine the content of kinsenoside, while response surface method (RSM) was used to optimize the extraction process. The best possible range for methanol concentration (0-100%), the liquid-solid ratio (5:1-30:1 mL/g), ultrasonic power (240-540 W), duration of ultrasound (10-50 min), ultrasonic temperature (10-60 °C), and the number of extractions (1-4) were obtained according to the single factor experiments. Then, using the Box-Behnken design (BBD) of response surface analysis, the optimum extraction conditions were obtained with 16.33% methanol concentration, the liquid-solid ratio of 10.83:1 mL/g and 35.00 °C ultrasonic temperature. Under these conditions, kinsenoside extraction yield reached 32.24% dry weight. The best conditions were applied to determine the kinsenoside content in seven different cultivation ages in Anoectochilus roburghii.

NtMYB3, an R2R3-MYB from Narcissus, Regulates Flavonoid Biosynthesis.

R2R3-MYB transcription factors play important roles in the regulation of plant flavonoid metabolites. In the current study, NtMYB3, a novel R2R3-MYB transcriptional factor isolated from Chinese narcissus (Narcissus tazetta L. var. chinensis), was functionally characterized. Phylogenetic analysis indicated that NtMYB3 belongs to the AtMYB4-like clade, which includes repressor MYBs involved in the regulation of flavonoid biosynthesis. Transient assays showed that NtMYB3 significantly reduced red pigmentation induced by the potato anthocyanin activator StMYB-AN1 in agro-infiltrated leaves of tobacco. Over-expression of NtMYB3 decreased the red color of transgenic tobacco flowers, with qRT-PCR analysis showing that NtMYB3 repressed the expression levels of genes involved in anthocyanin and flavonol biosynthesis. However, the proanthocyanin content in flowers of transgenic tobacco increased as compared to wild type. NtMYB3 showed expression in all examined narcissus tissues; the expression level in basal plates of the bulb was highest. A 968 bp promoter fragment of narcissus FLS (NtFLS) was cloned, and transient expression and dual luciferase assays showed NtMYB3 repressed the promoter activity. These results reveal that NtMYB3 is involved in the regulation of flavonoid biosynthesis in narcissus by repressing the biosynthesis of flavonols, and this leads to proanthocyanin accumulation in the basal plate of narcissus.

StMYB44 negatively regulates anthocyanin biosynthesis at high temperatures in tuber flesh of potato.

High temperatures are known to reduce anthocyanin accumulation in a number of diverse plant species. In potato (Solanum tuberosum L.), high temperature significantly reduces tuber anthocyanin pigment content. However, the mechanism of anthocyanin biosynthesis in potato tuber under heat stress remains unknown. Here we show that high temperature causes reduction of anthocyanin biosynthesis in both potato tuber skin and flesh, with white areas forming between the vasculature and periderm. Heat stress reduced the expression of the R2R3 MYB transcription factors (TFs) StAN1 and StbHLH1, members of the transcriptional complex responsible for coordinated regulation of the skin and flesh pigmentation, as well as anthocyanin biosynthetic pathway genes in white regions. However, the core phenylpropanoid pathway, lignin, and chlorogenic acid (CGA) pathway genes were up-regulated in white areas, suggesting that suppression of the anthocyanin branch may result in re-routing phenylpropanoid flux into the CGA or lignin biosynthesis branches. Two R2R3 MYB TFs, StMYB44-1 and StMYB44-2, were highly expressed in white regions under high temperature. In transient assays, StMYB44 represses anthocyanin accumulation in leaves of Nicotiana tabacum and N. benthamiana by directly suppressing the activity of the dihydroflavonol reductase (DFR) promoter. StMYB44-1 showed stronger repressive capacity than StMYB44-2, with both predicted proteins containing the repression-associated EAR motif with some variation. StMYB44-1 conferred repression without a requirement for a basic helix-loop-helix (bHLH) partner, suggesting a different repression mechanism from that of reported anthocyanin repressors. We propose that temperature-induced reduction of anthocyanin accumulation in potato flesh is caused by down-regulation of the activating anthocyanin regulatory complex, by enhancing the expression of flesh-specific StMYB44 and alteration of phenylpropanoid flux.

Ectopic Overexpression of a Novel R2R3-MYB, NtMYB2 from Chinese Narcissus Represses Anthocyanin Biosynthesis in Tobacco.

R2R3 MYB transcription factors play key functions in the regulation of secondary metabolites. In the present study, a R2R3 MYB transcriptional factor NtMYB2 was identified from Chinese narcissus (Narcissus tazetta L. var. Chinensis Roem) and functionally characterized. NtMYB2 belongs to subgroup 4 of the R2R3 MYB transcription factor family that are related to repressor MYBs involved in the regulation of anthocyanin and flavonoids. Transient expression confirmed that NtMYB2 strongly reduced the red pigmentation induced by MYB- anthocyanin activators in agro-infiltrated tobacco leaves. Ectopic expression of NtMYB2 in tobacco significantly reduced the pigmentation and altered the floral phenotypes in transgenic tobacco flowers. Gene expression analysis suggested that NtMYB2 repressed the transcript levels of structural genes involved in anthocyanin biosynthesis pathway, especially the UFGT gene. NtMYB2 gene is expressed in all examined narcissus tissues; the levels of transcription in petals and corona is higher than other tissues and the transcription level at the bud stage was highest. These results show that NtMYB2 is involved in the regulation of anthocyanin biosynthesis pathway and may act as a repressor by down regulating the transcripts of key enzyme genes in Chinese narcissus.

Identification of regulatory genes implicated in continuous flowering of longan (Dimocarpus longan L.).

Longan (Dimocarpus longan L.) is a tropical/subtropical fruit tree of significant economic importance in Southeast Asia. However, a lack of transcriptomic and genomic information hinders research on longan traits, such as the control of flowering. In this study, high-throughput RNA sequencing (RNA-Seq) was used to investigate differentially expressed genes between a unique longan cultivar 'Sijimi'(S) which flowers throughout the year and a more typical cultivar 'Lidongben'(L) which flowers only once in the season, with the aim of identifying candidate genes associated with continuous flowering. 36,527 and 40,982 unigenes were obtained by de novo assembly of the clean reads from cDNA libraries of L and S cultivars. Additionally 40,513 unigenes were assembled from combined reads of these libraries. A total of 32,475 unigenes were annotated by BLAST search to NCBI non-redundant protein (NR), Swiss-Prot, Clusters of Orthologous Groups (COGs) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Of these, almost fifteen thousand unigenes were identified as significantly differentially expressed genes (DEGs) by using Reads Per kb per Million reads (RPKM) method. A total of 6,415 DEGs were mapped to 128 KEGG pathways, and 8,743 DEGs were assigned to 54 Gene Ontology categories. After blasting the DEGs to public sequence databases, 539 potential flowering-related DEGs were identified. In addition, 107 flowering-time genes were identified in longan, their expression levels between two longan samples were compared by RPKM method, of which the expression levels of 15 were confirmed by real-time quantitative PCR. Our results suggest longan homologues of SHORT VEGETATIVE PHASE (SVP), GIGANTEA (GI), F-BOX 1 (FKF1) and EARLY FLOWERING 4 (ELF4) may be involved this flowering trait and ELF4 may be a key gene. The identification of candidate genes related to continuous flowering will provide new insight into the molecular process of regulating flowering time in woody plants.