Tobacco Flowers Research Articles

Ppbbx24-del mutant positively regulates light-induced anthocyanin accumulation in the ‘Red Zaosu’ pear (Pyrus pyrifolia White Pear Group)

Red fruit peel is one of the most valuable economic traits in pear and is mainly determined by anthocyanins. Many pear cultivars with a red peel originated from bud sports; however, little is known about the genetic mechanisms underlying this trait. We have previously identified a mutant PpBBX24 containing a 14-nucleotide deletion in the coding region (Ppbbx24-del) as the only known variant associated with the red coloration of the mutant ‘Red Zaosu’ pear (Pyrus pyrifolia White Pear Group). Herein, we analyzed the role of the mutant gene in red coloration and its mechanism of action. The results showed that light promoted red <pg=>peel coloration in ‘Red Zaosu’ pear, and Ppbbx24-del had positive effects on light-induced anthocyanin biosynthesis, while normal PpBBX24 had the opposite effects. Transient and stable <pg=>transformation experiments confirmed that Ppbbx24-del could promote anthocyanin accumulation in pear fruit peels, calli, and tobacco flowers. Due to the loss of NLS and VP domains, Ppbbx24-del co-localized in the nucleus and cytoplasm, whereas PpBBX24 localized only in the nucleus. Real-time PCR and transcriptome analyses indicated that PpMYB10 and PpHY5 is highly expressed in ‘Red Zaosu’ pear. In yeast one-hybrid and dual luciferase assays, Ppbbx24-del and PpHY5 independently promoted the expression of PpCHS, PpCHI, and PpMYB10 by binding to their promoters; however, PpBBX24 did not affect the expression of these genes. Additionally, we found that Ppbbx24-del and PpHY5 had additive effects on the expression of PpCHS, PpCHI, and PpMYB10, as they promote the expression of anthocyanin synthesis genes separately. The co-expression of PpBBX24 and PpHY5 inhibited the activation of downstream genes by PpHY5, and this was attributed to the interaction between the two loci. In conclusion, our results clarify the molecular mechanism by which mutant Ppbbx24-del and PpBBX24 exert opposite effects in the regulation of anthocyanin accumulation in pear. These findings lay an important theoretical foundation for the use of Ppbbx24-del to create red pear cultivars.

AINTEGUMENTA-LIKE genes regulate reproductive growth and bud dormancy in Platanus acerifolia.

Platanus acerifolia AIL genes PaAIL5a/b and PaAIL6b participate in FT-AP1/FUL-AIL pathways to regulate bud dormancy. In addition, PaAIL6a/b can promote flowering, and PaAIL5b and PaAIL6b affect floral development. Bud dormancy and floral induction are essential processes for perennial plants, they are both regulated by photoperiod, temperature, and hormones, indicating the existence of common regulators for both processes. AINTEGUMENTA-LIKE (AIL) genes regulate reproductive growth of annual plants, including floral induction and flower development, and their homologs in poplar and grape act downstream of the florigen gene FT and the floral meristem identity genes AP1/FUL and function to maintain growth and thus inhibit dormancy induction. However, it is not known whether AIL homologs participate in the reproduction processes in perennials and whether the Platanus acerifolia AIL genes are involved in dormancy. P. acerifolia is a perennial woody plant whose reproductive growth is strongly associated with dormancy. Here, we isolated four AIL homologs from P. acerifolia, PaAIL5a, PaAIL5b, PaAIL6a, and PaAIL6b, and systematically investigated their functions by ectopic-overexpression in tobacco. The findings demonstrate that PaAIL5a/b and PaAIL6b respond to short day, low temperature, and hormone signals and act as the components of the FT-AP1/FUL-AIL pathway to regulate the bud dormancy in P. acerifolia. Notably, PaAIL5a/b and PaAIL6b function downstream of PaFTL-PaFUL1/2/3 to inhibit the dormancy induction and downstream of PaFT-PaFUL2/3 to promote the dormancy release. In addition, PaAIL6a/b were found to accelerate flowering in transgenic tobacco, whereas PaAIL5b and PaAIL6b affected the flower development. Together, our results suggest that PaAIL genes may act downstream of different PaFT/PaFTL and PaFUL proteins to fulfill conservative and diverse roles in floral initiation, floral development, and dormancy regulation in P. acerifolia.

ZbMYB111 Expression Positively Regulates ZbUFGT-Mediated Anthocyanin Biosynthesis in Zanthoxylum bungeanum with the Involvement of ZbbHLH2.

Anthocyanin (ACN)-derived pigmentation in the red Zanthoxylum bungeanum peel is an essential commercial trait. Therefore, exploring the metabolic regulatory networks involved in peel ACN levels in this species is crucial for improving its quality. However, its underlying transcriptional regulatory mechanisms are still unknown. This transcriptomic and bioinformatics study not only discovered a new TF (ZbMYB111) as a potential regulator for ACN biosynthesis in Z. bungeanum peel, but also deciphered the underlying molecular mechanisms of ACN biosynthesis. Overexpression of ZbMYB111 and flavonoid 3-O-glucosyltransferase (ZbUFGT) induced ACN accumulation in both Z. bungeanum peels and callus along with Arabidopsis thaliana and tobacco flowers, whereas their silencing impaired ACN biosynthesis. Therefore, the dual-luciferase reporter, yeast-one-hybrid, and electrophoretic mobility shift assays showed that ZbMYB111 directly interacted with the ZbUFGT promoter to activate its expression. This diverted the secondary metabolism toward the ACN pathway, thereby promoting ACN accumulation.

ZmD11 Gene Regulates Tobacco Plant Floral Development under Drought Stress

Maize is most sensitive to drought stress at the floral stage by reducing tassel and silk quality, and thus improving drought tolerance at this stage may help preserve yield. It has been reported that BRs (brassinosteroids) promote floral development under drought stress. However, the function of the brassinosteroid biosynthesis gene ZmDWARF11 (ZmD11) on floral growth under drought stress has not been elucidated. This study found that under normal growth conditions, the heterologous over-expression of ZmD11 significantly enhanced both the vegetative growth and floral development of tobacco. Under drought stress, overexpressing ZmD11 reduced stress-induced tobacco flower size reduction, while it did not affect vegetative growth. After drought treatment, the activities of protective enzymes, including CAT (Catalase), SOD (Superoxide Dismutase), and POD (Peroxidase), were higher, while the content of MDA (Malondialdehyde) was lower in ZmD11 over-expression tobacco lines than that in the wild type control. The relative expression of dehydrin-related genes NtLeat5 and NtERD10 was increased in ZmD11 over-expression tobacco lines compared to that in the control. In summary, we reported that ZmD11 plays a role in tobacco floral development under drought stress. Our data are valuable in understanding the functions of BRs in regulating plant floral development under drought stress.

Phytochemical Profile and Antioxidant Potential of Tobacco Flower Extract in Different Solvents

Tobacco (Nicotiana tabacum) is a commercial plant that is extensively used, particularly for its leaves. Thus far, exploration on the flower part is still limited. The objective of this study was to evaluate the phytochemical profile and antioxidant activity of tobacco flower extracts in different solvents. Tobacco flowers were macerated separately in methanol, ethanol, and n-hexane. Each extract was assayed by color reaction test to detect the phytochemical content. Total phenolic content (TPC) and antioxidant activity were determined by Folin-Ciocalteu and DPPH methods, respectively. Flavonoids, saponins, alkaloids, and triterpenoids are detected in methanol and ethanol extract, anthraquinones is only detected in methanol, while glycosides and steroids are only detected in n-hexane extracts. Among the other extracts, the methanol extract was the richest in phytochemical compounds and had the highest TPC (132.589±0.662 mgGAE/g). However, tobacco flower extract in all solvents were less effective as antioxidant due to very high IC50 values (&gt;500 µg/mL).

Chalcone-Synthase-Encoding RdCHS1 Is Involved in Flavonoid Biosynthesis in Rhododendron delavayi.

Flower color is an important ornamental feature that is often modulated by the contents of flavonoids. Chalcone synthase is the first key enzyme in the biosynthesis of flavonoids, but little is known about the role of R. delavayi CHS in flavonoid biosynthesis. In this paper, three CHS genes (RdCHS1-3) were successfully cloned from R. delavayi flowers. According to multiple sequence alignment and a phylogenetic analysis, only RdCHS1 contained all the highly conserved and important residues, which was classified into the cluster of bona fide CHSs. RdCHS1 was then subjected to further functional analysis. Real-time PCR analysis revealed that the transcripts of RdCHS1 were the highest in the leaves and lowest in the roots; this did not match the anthocyanin accumulation patterns during flower development. Biochemical characterization displayed that RdCHS1 could catalyze p-coumaroyl-CoA and malonyl-CoA molecules to produce naringenin chalcone. The physiological function of RdCHS1 was checked in Arabidopsis mutants and tobacco, and the results showed that RdCHS1 transgenes could recover the color phenotypes of the tt4 mutant and caused the tobacco flower color to change from pink to dark pink through modulating the expressions of endogenous structural and regulatory genes in the tobacco. All these results demonstrate that RdCHS1 fulfills the function of a bona fide CHS and contributes to flavonoid biosynthesis in R. delavayi.

Recovery of Cembratrien-Diols from Waste Tobacco (Nicotiana tabacum L.) Flowers by Microwave-Assisted Deep Eutectic Solvent Extraction: Optimization, Separation, and In Vitro Bioactivity.

Deep eutectic solvents (DESs) are novel solvents with physicochemical properties similar to those of ionic liquids, and they have attracted extensive attention for the extraction of bioactive compounds from different plant materials in the context of green chemistry and sustainable development. In this study, seven DESs with different polarities were explored as green extraction solvents for cembratrien-diols (CBT-diols) from waste tobacco flowers. The best solvent, DES-3 (choline chloride: lactic acid (1:3)), which outperformed conventional solvents (methanol, ethanol, and ethyl acetate), was selected and further optimized for microwave-assisted DES extraction using the response surface methodology. The maximum yield of CBT-diols (6.23 ± 0.15 mg/g) was achieved using a microwave power of 425 W, microwave time of 32 min, solid/liquid ratio of 20 mg/mL, and microwave temperature of 40 °C. Additionally, the isolated CBT-diols exhibited strong antimicrobial activity against Salmonella, Staphylococcus aureus, Escherichia coli, Bacillus subtilis, and Pseudomonas aeruginosa and antitumor activity in the human liver cancer HepG2 and SMMC-7721 cell lines. This study highlights the feasibility of recovering CBT-diols from tobacco flower waste using DESs and provides opportunities for potential waste management using green technologies.

Influence of Leaf Harvesting Methods on Tobacco Seed Production and Quality

To produce high-quality seeds, the harvest must be carried out on time so that the seed maturity is almost uniform. Like wise to produce seeds of high physiological quality. The study was conducted at the Karangploso Experimental Garden, Balittas Malang, in January-October 2017. The varieties used were Prancak-N1, with 6 treatments namely: (1) Leaves harvested and shredded, (2) Leaves harvested and shredded are not discarded, (3) Leaves not harvested and suckers not removed (4) Leaves not harvested and shredded (5 koseran leaves not harvested shingles disposed and (6) Koseran leaves harvested and shredded discarded.The treatment was planted as many as 10 polybags with a size of 20 kg of soil, one plant per polybag, population of each treatment as many as 10 plants with 3 replications. The fertilizer used was NPK fertilizer with a dose of 15 grams per plant. Analysis used Randomized Group Design repeated 3 times, to calculate the variance using the Duncan Distance Test (DMRT) with a level of 5%. The purpose of the study was to determine the effect of the treatment of harvesting leaves and tobacco flowers on the production and quality of the seeds produced. The results showed that the highest seed production from the six treatments was treated with leaves not harvested and shredded, the highest production of seeds reached 17.4 grams, but not significantly different from the treatment of koseran leaves not the harvested sirung was disposed of (16.10 grams) and the coseran leaves were harvested, the flush was disposed of (15.70 grams). For the highest seed vigor test, the leaves were not harvested and the flush was disposed (17.40%), while the highest germination test for the flush treatment was reached (93.33%), but not significantly different from the treatment of the leaf leaves not harvested by sirung discarded (91.33%) and the treatment of koseran leaves was harvested, flush was disposed (84.68%). Observation of abnormal sprouts from the six treatments was not significantly different between 1.33%-2.33%

Integrated transcriptomic and metabolomic analysis revealed altitude-related regulatory mechanisms on flavonoid accumulation in potato tubers

Not least because it is adaptable to a variety of geographies and climates, potato (Solanum tuberosum L.) is grown across much of the world. Pigmented potato tubers have been found to contain large quantities of flavonoids, which have various functional roles and act as antioxidants in the human diet. However, the effect of altitude on the biosynthesis and accumulation of flavonoids in potato tubers is poorly characterized. Here we carried out an integrated metabolomic and transcriptomic study in order to evaluate how cultivation at low (800 m), moderate (1800 m), and high (3600 m) altitude affects flavonoid biosynthesis in pigmented potato tubers. Both red and purple potato tubers grown at a high altitude contained the highest flavonoid content, and the most highly pigmented flesh, followed by those grown at a low altitude. Co-expression network analysis revealed three modules containing genes which were positively correlated with altitude-responsive flavonoid accumulation. The anthocyanin repressors StMYBATV and StMYB3 exhibited a significant positive relationship with altitude-responsive flavonoid accumulation. The repressive function of StMYB3 was further verified in tobacco flowers and potato tubers. The results presented here add to the growing body of knowledge regarding the response of flavonoid biosynthesis to environmental conditions, and should aid in efforts to develop novel varieties of pigmented potatoes for use across different geographies.

NataMYB4, a flower specific gene, regulates the flavonoid biosynthesis in Chinese Narcissus

R2R3 MYBs play a crucial role in flower development and its associated flavonoid biosynthesis, but their regulatory network in Chinese Narcissus is not fully understood. Anthocyanin, flavonol and proanthocyanin are very important secondary metabolites with various biological functions and their biosynthesis is controlled by MYB transcriptional factors. NataMYB4, as a member of the R2R3-MYB gene family, isolated from Chinese Narcissus, was found mainly expressed in flowers, particularly in petals and corona. In addition to the R2-R3 region, NataMYB4 displays a conserved motif “NF/YWSV/MEDF/LW” at the end of the C-terminal. Transient expression in tobacco leaves showed that NataMYB4 significantly decreased the red pigmentation that was induced by an anthocyanin activator StMYB. Ectopic overexpression of NataMYB4 in tobacco altered the flower color by reducing the pigmentation and anthocyanin contents in transgenic flowers. However, the contents of total phenolic acid and total flavonol were increased in the transgenic tobacco lines. qPCR analysis indicated that transcript levels of anthocyanin biosynthesis genes were down-regulated in transgenic flowers carrying the NataMYB4 gene, while the flavonol biosynthetic pathway key gene FLS showed higher expression level in transgenic tobacco flowers. Dual luciferase assays showed that co-infiltration of NataMYB4 and NatabHLH1 significantly activated the promoter of the Chinese Narcissus FLS gene. Furthermore, a yeast two-hybrid assay confirmed that NataMYB4 interacts with NatabHLH1. The outcomes of our effort in this study provide novel insights into the regulation of flavonoid biosynthetic pathways in Chinese Narcissus.

Morphological Characterization of Nicotiana tabacum Inflorescences and Chemical-Functional Analysis of Extracts Obtained from Its Powder by Using Green Solvents (NaDESs)

The production of smokeable tobacco for use in cigarettes is characterized by the production of pre-harvest and post-harvest waste, with ensuing undesirable effects on the environment. The inflorescences of tobacco after blunting, deflowering, or topping are considered pre-harvest waste and left in the field. Using green and ecofriendly solvents such as Natural deep eutectic solvents (NaDESs), these wastes could be used to obtain antioxidant molecules of interest in cosmetics. Taking into account its potential as plant matrix to obtain metabolites of commercial interest, tobacco inflorescences and inflorescence powders of different particle sizes were characterized by optic and electronic microscopy. Thus, the powdered inflorescences were extracted with four conventional solvents, i.e., distilled water (DW), acetone: distilled water (AW), ethanol 70° (EW), methanol (Me), and five NaDESs, i.e., lactic acid: sucrose (LAS), lactic acid: sucrose: distilled water (SALA), fructose: glucose: sucrose: distilled water (FGS), choline chloride: urea: distilled water (CU), and citric acid: propylene glycol (CAP). Among the tested NADESs, SALA was the most promising solvent; higher extraction yields of total phenolic compound (3420.0 ± 9.4 µg GAE/mL) than conventional solvents were attained and it was the only selective solvent to phenolics. CU was the best solvent for flavonoids and alkaloids extraction (215.3 ± 3.2 µg QE/mL and 392.3 ± 8.0 µg ACE/mL, respectively). All extracts showed antioxidant activity. A heatmap with dendrogram and main component analysis showed that acid-based NaDESs are grouped together, this group being the one with the best performance in H2O2 scavenging. The extracts obtained with green solvents could be used directly in cosmetic formulations as antioxidant ingredients because both tobacco flower oil and flower extracts are listed in the cosmetic ingredients database as non-toxic products. Additionally, the demand for sustainable ecological cosmetics is growing. In this sense, NaDESs represent an opportunity to develop innovative extracts with unique phytochemical fingerprints and biological activities.

Transcriptome and functional analysis revealed the intervention of brassinosteroid in regulation of cold induced early flowering in tobacco.

Cold environmental conditions may often lead to the early flowering of plants, and the mechanism by cold-induced flowering remains poorly understood. Microscopy analysis in this study demonstrated that cold conditioning led to early flower bud differentiation in two tobacco strains and an Agilent Tobacco Gene Expression microarray was adapted for transcriptomic analysis on the stem tips of cold treated tobacco to gain insight into the molecular process underlying flowering in tobacco. The transcriptomic analysis showed that cold treatment of two flue-cured tobacco varieties (Xingyan 1 and YunYan 85) yielded 4176 and 5773 genes that were differentially expressed, respectively, with 2623 being commonly detected. Functional distribution revealed that the differentially expressed genes (DEGs) were mainly enriched in protein metabolism, RNA, stress, transport, and secondary metabolism. Genes involved in secondary metabolism, cell wall, and redox were nearly all up-regulated in response to the cold conditioning. Further analysis demonstrated that the central genes related to brassinosteroid biosynthetic pathway, circadian system, and flowering pathway were significantly enhanced in the cold treated tobacco. Phytochemical measurement and qRT-PCR revealed an increased accumulation of brassinolide and a decreased expression of the flowering locus c gene. Furthermore, we found that overexpression of NtBRI1 could induce early flowering in tobacco under normal condition. And low-temperature-induced early flowering in NtBRI1 overexpression plants were similar to that of normal condition. Consistently, low-temperature-induced early flowering is partially suppressed in NtBRI1 mutant. Together, the results suggest that cold could induce early flowering of tobacco by activating brassinosteroid signaling.

Molecular cloning and functional analysis of Chinese bayberry MrSPL4 that enhances growth and flowering in transgenic tobacco.

Chinese bayberry (Myrica rubra) is an important tree in South China, with its fruit being of nutritional and high economic value. In this study, early ripening (ZJ), medium ripening (BQ) and late ripening (DK) varieties were used as test materials. Young leaves of ZJ, BQ and DK in the floral bud morphological differentiation periods were selected for transcriptome sequencing to excavate earliness related genes. A total of 4,538 differentially expressed genes were detected. Based on clustering analysis and comparisons with genes reportedly related to flowering in Arabidopsis thaliana, 25 homologous genes were identified. Of these, one gene named MrSPL4 was determined, with its expression down-regulated in DK but up-regulated in ZJ and BQ. MrSPL4 contained SBP domain and the target site of miR156, and its total and CDS length were 1,664 bp and 555 bp respectively. The overexpression vector of MrSPL4 (35S::35S::MrSPL4-pCambia2301-KY) was further constructed and successfully transfected into tobacco to obtain MrSPL4-positive plants. Based on the results of qRT-PCR, the relative expression of MrSPL4 was up regulated by 3,862.0-5,938.4 times. Additionally, the height of MrSPL4-positive plants was also significantly higher than that of wild-type (WT), with the bud stage occurring 12 days earlier. Altogether, this study identified an important gene -MrSPL4 in Chinese bayberry, which enhanced growth and flowering, which provided important theoretical basis for early-mature breeding of Chinese bayberry.

Identification of Stigma Specific Expression Fragment in the Promoter of a Soybean Chitinase Class I Gene

The expression level of heterologous genes in transgenic plants serves as an important indicator of gene efficiency. The small number of currently known effective promoters, limits the possibilities in fine-tuning the expression of transgenes. We cloned and characterized a tissue-specific promoter fragment of the soybean chitinase class I gene (GmChi1). The GmChi1 promoter (GmChi1P) was cloned from Jungery soybean. The promoter sequence contains a number of putative cis-acting elements, including tissue-specific and stress-regulated motifs. By histochemical analysis, the GmChi1P-controlled β-glucuronidase (GUS) reporter enzyme activity was shown to be highest in the roots of transgenic Nicotiana tabacum cv. NC89 at the four-leaf sprout formation stage. Interestingly, the high GUS activity in transgenic tobacco roots was effectively suppressed by salicylic acid (SA) treatment. Deletion analysis of GmChi1P revealed that the sequences located between positions -719 and -382 contain key cis-elements responsible for the reporter uidA gene expression (encoding GUS) in leaves, roots, and wounds of Nicotiana tabacum. In addition, fluorometric analysis showed that the activity of the shortened ChiP(-1292) to ChiP(-719) promoters in the roots of transgenic tobacco was significantly suppressed by abscisic acid and completely suppressed by SA. The ChiP(-382) promoter was also found to be expressed exclusively in the stigma of transgenic tobacco flowers. Using the GUS reporter enzyme, no staining was detected in other flower organs in transgenic Nicotiana tabacum, including sepals, petals, anthers, filaments, and ovaries, or in any vegetative tissues. The results indicate that the promoter fragment ChiP(-382) can be used in tissue-specific regulation of gene expression and plant genetic engineering.

A bZIP transcription factor (CiFD) regulates drought- and low-temperature-induced flowering by alternative splicing in citrus.

Drought and low temperature are two key environmental factors that induce adult citrus flowering. However, the underlying regulation mechanism is poorly understood. The bZIP transcription factor FD is a key component of the florigen activation complex (FAC) which is composed of FLOWERING LOCUS T (FT), FD, and 14-3-3 proteins. In this study, isolation and characterization of CiFD in citrus found that there was alternative splicing (AS) of CiFD, forming two different proteins (CiFDα and CiFDβ). Further investigation found that their expression patterns were similar in different tissues of citrus, but the subcellular localization and transcriptional activity were different. Overexpression of the CiFD DNA sequence (CiFD-DNA), CiFDα, or CiFDβ in tobacco and citrus showed early flowering, and CiFD-DNA transgenic plants were the earliest, followed by CiFDβ and CiFDα. Interestingly, CiFDα and CiFDβ were induced by low temperature and drought, respectively. Further analysis showed that CiFDα can form a FAC complex with CiFT, Ci14-3-3, and then bind to the citrus APETALA1 (CiAP1) promoter and promote its expression. However, CiFDβ can directly bind to the CiAP1 promoter independently of CiFT and Ci14-3-3. These results showed that CiFDβ can form a more direct and simplified pathway that is independent of the FAC complex to regulate drought-induced flowering through AS. In addition, a bHLH transcription factor (CibHLH96) binds to CiFD promoter and promotes the expression of CiFD under drought condition. Transgenic analysis found that CibHLH96 can promote flowering in transgenic tobacco. These results suggest that CiFD is involved in drought- and low-temperature-induced citrus flowering through different regulatory patterns.

Tree peony PsMYB44 negatively regulates petal blotch distribution by inhibiting dihydroflavonol-4-reductase gene expression.

The tree peony (Paeonia suffruticosa Andr.) has been widely cultivated as a field plant, and petal blotch is one of its important traits, which not only promotes proliferation but also confers high ornamental value. However, the regulatory network controlling blotch formation remains elusive owing to the functional differences and limited conservation of transcriptional regulators in dicots. We performed phylogenetic analysis to identify MYB44-like transcription factors in P. suffruticosa blotched cultivar 'High noon' petals. A candidate MYB44-like transcription factor, PsMYB44, was analysed via expression pattern analysis, subcellular localization, target gene identification, gene silencing in P. suffruticosa petals and heterologous overexpression in tobacco. A blotch formation-related MYB44-like transcription factor, PsMYB44, was cloned. The C-terminal of the PsMYB44 amino acid sequence had a complete C2 motif that affects anthocyanin biosynthesis, and PsMYB44 was clustered in the MYB44-like transcriptional repressor branch. PsMYB44 was located in the nucleus, and its spatial and temporal expression patterns were negatively correlated with blotch formation. Furthermore, a yeast one-hybrid assay showed that PsMYB44 could target the promoter of the late anthocyanin biosynthesis-related dihydroflavonol-4-reductase (DFR) gene, and a dual-luciferase assay demonstrated that PsMYB44 could repress PsDFR promoter activity. On the one hand, overexpression of PsMYB44 significantly faded the red colour of tobacco flowers and decreased the anthocyanin content by 42.3 % by downregulating the expression level of the tobacco NtDFR gene. On the other hand, PsMYB44-silenced P. suffruticosa petals had a redder blotch colour, which was attributed to the fact that silencing PsMYB44 redirected metabolic flux to the anthocyanin biosynthesis branch, thereby promoting more anthocyanin accumulation in the petal base. These results demonstrated that PsMYB44 negatively regulated the biosynthesis of anthocyanin by directly binding to the PsDFR promoter and subsequently inhibiting blotch formation, which helped to elucidate the molecular regulatory network of anthocyanin-mediated blotch formation in plants.

Transgenic tobacco plant overexpressing ginkgo dihydroflavonol 4-reductase gene GbDFR6 exhibits multiple developmental defects.

Dihydroflavonol Q 4-reductase (DFR), a key enzyme in the flavonoid biosynthetic pathway in plants, significantly influences plant survival. However, the roles of DFR in the regulation of plant development are largely unknown. In the present study, phenotypes of transgenic tobacco plants overexpressing the Ginkgo biloba DFR gene, GbDFR6, were investigated. Transgenic tobacco seedlings exhibited relatively low fresh weights, long primary roots, decreased lateral root numbers, and impaired root gravitropic responses when compared to wild-type tobacco plants. Adult transgenic tobacco plants exhibited a considerably high percentage of wrinkled leaves when compared to the wild-type tobacco plants. In addition to the auxin-related phenotypic changes, transgenic tobacco plants exhibited delayed flowering phenotypes under short-day conditions. Gene expression analysis revealed that the delayed flowering in transgenic tobacco plants was caused by the low expression levels of NtFT4. Finally, variations in anthocyanin and flavonoid contents in transgenic tobacco plants were evaluated. The results revealed that the levels of most anthocyanins identified in transgenic tobacco leaves increased. Specifically, cyanidin-3,5-O-diglucoside content increased by 9.8-fold in transgenic tobacco plants when compared to the wild-type tobacco plants. Pelargonidin-3-O-(coumaryl)-glucoside was only detected in transgenic tobacco plants. Regarding flavonoid compounds, one flavonoid compound (epicatechin gallate) was upregulated, whereas seven flavonoid compounds (Tamarixetin-3-O-rutinoside; Sexangularetin-3-O-glucoside-7-O-rhamnoside; Kaempferol-3-O-neohesperidoside; Engeletin; 2'-Hydoxy,5-methoxyGenistein-O-rhamnosyl-glucoside; Diosmetin; Hispidulin) were downregulated in both transgenic tobacco leaves and roots. The results indicate novel and multiple roles of GbDFR6 in ginkgo and provide a valuable method to produce a late flowering tobacco variety in tobacco industry.

Root-secreted (-)-loliolide modulates both belowground defense and aboveground flowering in Arabidopsis and tobacco.

Plant defense, growth, and reproduction can be modulated by chemicals emitted from neighboring plants, mainly via volatile aboveground signals. However, belowground signals and their underlying control mechanisms are largely unknown. Here, we experimentally demonstrate that the root-secreted carotenoid (-)-loliolide mediates both defensive and reproductive responses in wild-type Arabidopsis, a carotenoid-deficient Arabidopsis mutant (szl1-1), and tobacco (Nicotiana benthamiana). Wild-type Arabidopsis plants flower later than szl1-1, and they secrete (-)-loliolide into the soil, whereas szl1-1 roots do not. When Arabidopsis and tobacco occur together, wild-type Arabidopsis induces nicotine production and defense-related gene expression in tobacco, whereas szl1-1 impairs this induction but accelerates tobacco flowering. Furthermore, nicotine production and the expression of the key genes involved in nicotine biosynthesis (QPT, PMT1), plant defense (CAT1, SOD1, PR-2a, PI-II, TPI), and flowering (AP1, LFY, SOC1, FT3, FLC) are differently regulated by incubation with wild-type Arabidopsis and szl1-1 root exudates or (-)-loliolide. In particular, (-)-loliolide up-regulated flowering suppressors (FT3 and FLC) and transiently down-regulated flowering stimulators (AP1 and SOC1), delaying tobacco flowering. Therefore, root-secreted (-)-loliolide modulates plant belowground defense and aboveground flowering, yielding critical insights into plant-plant signaling interactions.

Insights into the Cytochrome P450 Monooxygenase Superfamily in Osmanthus fragrans and the Role of OfCYP142 in Linalool Synthesis.

Osmanthus fragrans flowers have long been used as raw materials in food, tea, beverage, and perfume industries due to their attractive and strong fragrance. The P450 superfamily proteins have been reported to widely participate in the synthesis of plant floral volatile organic compounds (VOCs). To investigate the potential functions of P450 superfamily proteins in the fragrance synthesis of O. fragrans, we investigated the P450 superfamily genome wide. A total of 276 P450 genes were identified belonging to 40 families. The RNA-seq data suggested that many OfCYP genes were preferentially expressed in the flower or other organs, and some were also induced by multiple abiotic stresses. The expression patterns of seven flower-preferentially expressed OfCYPs during the five different flower aroma content stages were further explored using quantitative real-time PCR, showing that the CYP94C subfamily member OfCYP142 had the highest positive correlation with linalool synthesis gene OfTPS2. The transient expression of OfCYP142 in O. fragrans petals suggested that OfCYP142 can increase the content of linalool, an important VOC of the O. fragrans floral aroma, and a similar result was also obtained in flowers of OfCYP142 transgenic tobacco. Combined with RNA-seq data of the transiently transformed O. fragrans petals, we found that the biosynthesis pathway of secondary metabolites was significantly enriched, and many 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway genes were also upregulated. This evidence indicated that the OfCYP proteins may play critical roles in the flower development and abiotic response of O. fragrans, and that OfCYP142 can participate in linalool synthesis. This study provides valuable information about the functions of P450 genes and a valuable guide for studying further functions of OfCYPs in promoting fragrance biosynthesis of ornamental plants.

PlZFP mediates the combinatorial interactions of abscisic acid with gibberellin and ethylene during flower senescence in cut herbaceous peony

Short vase life is a main factor that limits herbaceous peony’s economic value. We found that in cut herbaceous peony, flower senescence was regulated by the combinatorial interactions among plant endogenous hormones. However, the molecular mechanisms underlying these interactive effects are not well understood. In this study, we describe the identification of PlZFP, which influences flower senescence in herbaceous peony. PlZFP was upregulated during herbaceous peony flower senescence and its expression level was influenced by abscisic acid (ABA), ethylene and gibberellin (GA). PlZFP-silenced petal discs had longer lives while PlZFP-overexpressing transgenic tobacco had short flower longevity. Silencing PlZFP in petal discs reduced the transcript abundances of the endogenous hormone biosynthesis-related genes PlACO1, PlACO3, PlACS1, PlNCED2, and PlAAO and increased the transcript abundance of PlGA3ox1. Overexpression of PlZFP in tobacco flowers showed the opposite influences on these genes. Furthermore, PlZFP directly bound to the PlNCED2 promoter, and silencing PlNCED2 delayed senescence in discs. Silencing of PlZFP reduced ethylene production and ABA levels while increased GA levels in peony petal discs. Moreover, exogenous ABA, ethephon and paclobutrazol (PAC, a GA inhibitor), accelerate senescence in PlZFP-silenced discs. Taken together, the PlZFP-PlNCED2 regulatory checkpoint may positively affect the production of ABA and mediates the combinatorial interactions of ABA with GA and ethylene during flower senescence in cut herbaceous peony.