Insights into acylation mechanisms: co-expression of serine carboxypeptidase-like acyltransferases and their non-catalytic companion paralogs.

Serine carboxypeptidase-like acyltransferases (SCPL-ATs) play a vital role in the diversification of plant metabolites. Galloylated flavan-3-ols highly accumulate in tea (Camellia sinensis), grape (Vitis vinifera), and persimmon (Diospyros kaki). To date, the biosynthetic mechanism of these compounds remains unknown. Herein, we report that two SCPL-AT paralogs are involved in galloylation of flavan-3-ols: CsSCPL4, which contains the conserved catalytic triad S-D-H, and CsSCPL5, which has the alternative triad T-D-Y. Integrated data from transgenic plants, recombinant enzymes, and gene mutations showed that CsSCPL4 is a catalytic acyltransferase, while CsSCPL5 is a non-catalytic companion paralog (NCCP). Co-expression of CsSCPL4 and CsSCPL5 is likely responsible for the galloylation. Furthermore, pull-down and co-immunoprecipitation assays showed that CsSCPL4 and CsSCPL5 interact, increasing protein stability and promoting post-translational processing. Moreover, phylogenetic analyses revealed that their homologs co-exist in galloylated flavan-3-ol- or hydrolyzable tannin-rich plant species. Enzymatic assays further revealed the necessity of co-expression of those homologs for acyltransferase activity. Evolution analysis revealed that the mutations of the CsSCPL5 catalytic residues may have taken place about 10 million years ago. These findings show that the co-expression of SCPL-ATs and their NCCPs contributes to the acylation of flavan-3-ols in the plant kingdom.

Alternative splicing of CsJAZ1 negatively regulates flavan-3-ol biosynthesis in tea plants.

Flavan-3-ols are abundant in the tea plant (Camellia sinensis) and confer tea with flavor and health benefits. We recently found that alternative splicing of genes is likely involved in the regulation of flavan-3-ol biosynthesis; however, the underlying regulatory mechanisms remain unknown. Here, we integrated metabolomics and transcriptomics to construct metabolite-gene networks in tea leaves, collected over five different months and from five spatial positions, and found positive correlations between endogenous jasmonic acid (JA), flavan-3-ols, and numerous transcripts. Transcriptome mining further identified CsJAZ1, which is negatively associated with flavan-3-ols formation and has three CsJAZ1 transcripts, one full-length (CsJAZ1-1), and two splice variants (CsJAZ1-2 and -3) that lacked 3' coding sequences, with CsJAZ1-3 also lacking the coding region for the Jas domain. Confocal microscopy showed that CsJAZ1-1 was localized to the nucleus, while CsJAZ1-2 and CsJAZ1-3 were present in both the nucleus and the cytosol. In the absence of JA, CsJAZ1-1 was bound to CsMYC2, a positive regulator of flavan-3-ol biosynthesis; CsJAZ1-2 functioned as an alternative enhancer of CsJAZ1-1 and an antagonist of CsJAZ1-1 in binding to CsMYC2; and CsJAZ1-3 did not interact with CsMYC2. In the presence of JA, CsJAZ1-3 interacted with CsJAZ1-1 and CsJAZ1-2 to form heterodimers that stabilized the CsJAZ1-1-CsMYC2 and CsJAZ1-2-CsMYC2 complexes, thereby repressing the transcription of four genes that act late in the flavan-3-ol biosynthetic pathway. These data indicate that the alternative splicing variants of CsJAZ1 coordinately regulate flavan-3-ol biosynthesis in the tea plant and improve our understanding of JA-mediated flavan-3-ol biosynthesis.

Metabolic engineering of the anthocyanin biosynthetic pathway in Artemisia annua and relation to the expression of the artemisinin biosynthetic pathway.

MAIN CONCLUSION: Four types of cells were engineered from Artemisia annua to produce approximately 17 anthocyanins, four of which were elucidated structurally. All of them expressed the artemisinin pathway. Artemisia annua is the only medicinal crop to produce artemisinin for the treatment of malignant malaria. Unfortunately, hundreds of thousands of people still lose their life every year due to the lack of sufficient artemisinin. Artemisinin is considered to result from the spontaneous autoxidation of dihydroartemisinic acid in the presence of reactive oxygen species (ROS) in an oxidative condition of glandular trichomes (GTs); however, whether increasing antioxidative compounds can inhibit artemisinin biosynthesis in plant cells is unknown. Anthocyanins are potent antioxidants that can remove ROS in plant cells. To date, no anthocyanins have been structurally elucidated from A. annua. In this study, we had two goals: (1) to engineer anthocyanins in A. annua cells and (2) to understand the artemisinin biosynthesis in anthocyanin-producing cells. Arabidopsis Production of Anthocyanin Pigment 1 was used to engineer four types of transgenic anthocyanin-producing A. annua (TAPA1-4) cells. Three wild-type cell types were developed as controls. TAPA1 cells produced the highest contents of total anthocyanins. LC-MS analysis detected 17 anthocyanin or anthocyanidin compounds. Crystallization, LC/MS/MS, and NMR analyses identified cyanidin, pelargonidin, one cyanin, and one pelargonin. An integrative analysis characterized that four types of TAPA cells expressed the artemisinin pathway and TAPA1 cells produced the highest artemisinin and artemisinic acid. The contents of arteannuin B were similar in seven cell types. These data showed that the engineering of anthocyanins does not eliminate the biosynthesis of artemisinin in cells. These data allow us to propose a new hypothesis that enzymes catalyze the formation of artemisinin from dihydroartemisinic acid in non-GT cells. These findings show a new platform to increase artemisinin production via non-GT cells of A. annua.

Molecular understanding of anthocyanin biosynthesis activated by PAP1 and regulated by 2, 4-dichlorophenoxyacetic acid in engineered red Artemisia annua cells.

MAIN CONCLUSION: Eight promoters were cloned, from which AC and G-box cis-elements were identified. PAP1 enhanced the promoter activity. 2,4-D reduced the anthocyanin biosynthesis via downregulating the expression of the PAP1 transgene. Artemisia annua is an effective antimalarial medicinal crop. We have established anthocyanin-producing red cell cultures from this plant with the overexpression of Production of Anthocyanin Pigment 1 (PAP1) encoding a R2R3MYB transcription factor. To understand the molecular mechanism by which PAP1 activated the entire anthocyanin pathway, we mined the genomic sequences of A. annua and obtained eight promoters of the anthocyanin pathway genes. Sequence analysis identified four types of AC cis-elements from six promoters, the MYB response elements (MRE) bound by PAP1. In addition, six promoters were determined to have at least one G-box cis-element. Eight promoters were cloned for activity analysis. Dual luciferase assays showed that PAP1 significantly enhanced the promoting activity of seven promoters, indicating that PAP1 turned on the biosynthesis of anthocyanins via the activation of these pathway gene expression. To understand how 2,4-dichlorophenoxyacetic acid (2,4-D), an auxin, regulates the PAP1-activated anthocyanin biosynthesis, five different concentrations (0, 0.05, 0.5, 2.5, and 5 µM) were tested to characterize anthocyanin production and profiles. The resulting data showed that the concentrations tested decreased the fresh weight of callus growth, anthocyanin levels, and the production of anthocyanins per Petri dish. HPLC-qTOF-MS/MS-based profiling showed that these concentrations did not alter anthocyanin profiles. Real-time RT-PCR was completed to characterize the expression PAP1 and four representative pathway genes. The results showed that the five concentrations reduced the expression levels of the constitutive PAP1 transgene and three pathway genes significantly and eliminated the expression of the chalcone synthase gene either significantly or slightly. These data indicate that the constitutive PAP1 expression depends on gradients added in the medium. Based on these findings, the regulation of 2,4-D is discussed for anthocyanin engineering in red cells of A. annua.

TAA1-mediated auxin biosynthesis is essential for hormone crosstalk and plant development.

Plants have evolved a tremendous ability to respond to environmental changes by adapting their growth and development. The interaction between hormonal and developmental signals is a critical mechanism in the generation of this enormous plasticity. A good example is the response to the hormone ethylene that depends on tissue type, developmental stage, and environmental conditions. By characterizing the Arabidopsis wei8 mutant, we have found that a small family of genes mediates tissue-specific responses to ethylene. Biochemical studies revealed that WEI8 encodes a long-anticipated tryptophan aminotransferase, TAA1, in the essential, yet genetically uncharacterized, indole-3-pyruvic acid (IPA) branch of the auxin biosynthetic pathway. Analysis of TAA1 and its paralogues revealed a link between local auxin production, tissue-specific ethylene effects, and organ development. Thus, the IPA route of auxin production is key to generating robust auxin gradients in response to environmental and developmental cues.

Molecular and biochemical characterization of two 4-coumarate: CoA ligase genes in tea plant (Camellia sinensis).

KEY MESSAGE: Two 4-coumarate: CoA ligase genes in tea plant involved in phenylpropanoids biosynthesis and response to environmental stresses. Tea plant is rich in flavonoids benefiting human health. Lignin is essential for tea plant growth. Both flavonoids and lignin defend plants from stresses. The biosynthesis of lignin and flavonoids shares a key intermediate, 4-coumaroyl-CoA, which is formed from 4-coumaric acid catalyzed by 4-coumaric acid: CoA ligase (4CL). Herein, we report two 4CL paralogs from tea plant, Cs4CL1 and Cs4CL2, which are a member of class I and II of this gene family, respectively. Cs4CL1 was mainly expressed in roots and stems, while Cs4CL2 was mainly expressed in leaves. The promoter of Cs4CL1 had AC, nine types of light sensitive (LSE), four types of stress-inducible (SIE), and two types of meristem-specific elements (MSE). The promoter of Cs4CL2 also had AC and nine types of LSEs, but only had two types of SIEs and did not have MSEs. In addition, the LSEs varied in the two promoters. Based on the different features of regulatory elements, three stress treatments were tested to understand their expression responses to different conditions. The resulting data indicated that the expression of Cs4CL1 was sensitive to mechanical wounding, while the expression of Cs4CL2 was UV-B-inducible. Enzymatic assays showed that both recombinant Cs4CL1 and Cs4CL2 transformed 4-coumaric acid (CM), ferulic acid (FR), and caffeic acid (CF) to their corresponding CoA ethers. Kinetic analysis indicated that the recombinant Cs4CL1 preferred to catalyze CF, while the recombinant Cs4CL2 favored to catalyze CM. The overexpression of both Cs4CL1 and Cs4CL2 increased the levels of chlorogenic acid and total lignin in transgenic tobacco seedlings. In addition, the overexpression of Cs4CL2 consistently increased the levels of three flavonoid compounds. These findings indicate the differences of Cs4CL1 and Cs4CL2 in the phenylpropanoid metabolism.

Transcriptional and Metabolic Characterization of Feeding Ramie Growth Enhanced by a Combined Application of Gibberellin and Ethrel.

Feeding ramie cultivars (Boehmaria nivea L.) are an important feedstock for livestock. Increasing their biomass and improving their nutritional values are essential for animal feeding. Gibberellin (GA3) and ethylene (ETH) are two plant hormones that regulate the growth, development, and metabolism of plants. Herein, we report effects of the GA3 and ETH application on the growth and plant metabolism of feeding ramie in the field. A combination of GA3 and ETH was designed to spray new plants. The two hormones enhanced the growth of plants to produce more biomass. Meanwhile, the two hormones reduced the contents of lignin in leaves and stems, while increased the content of flavonoids in leaves. To understand the potential mechanisms behind these results, we used RNA-seq-based transcriptomics and UPLC-MS/MS-based metabolomics to characterize gene expression and metabolite profiles associated with the treatment of GA3 and ETH. 1562 and 2364 differentially expressed genes (DEGs) were obtained from leaves and stems (treated versus control), respectively. Meanwhile, 99 and 88 differentially accumulated metabolites (DAMs) were annotated from treated versus control leaves and treated versus control stems, respectively. Data mining revealed that both DEGs and DAMs were associated with multiple plant metabolisms, especially plant secondary metabolism. A specific focus on the plant phenylpropanoid pathway identified candidates of DEGs and DEMs that were associated with lignin and flavonoid biosynthesis. Shikimate hydroxycinnamoyl transferase (HCT) is a key enzyme that is involved in the lignin biosynthesis. The gene encoding B. nivea HCT was downregulated in the treated leaves and stems. In addition, genes encoding 4-coumaryl CoA ligase (4CL) and trans-cinnamate 4-monooxygenase (CYP73A), two lignin pathway enzymes, were downregulated in the treated stems. Meanwhile, the reduction in lignin in the treated leaves led to an increase in cinnamic acid and p-coumaryl CoA, two shared substrates of flavonoids that are enhanced in contents. Taken together, these findings indicated that an appropriate combination of GA3 and ETH is an effective strategy to enhance plant growth via altering gene expression and plant secondary metabolism for biomass-enhanced and value-improved feeding ramie.

Functional demonstration of plant flavonoid carbocations proposed to be involved in the biosynthesis of proanthocyanidins.

The plant flavonoid dogma proposes that labile plant flavonoid carbocations (PFCs) play vital roles in the biosynthesis of proanthocyanidins (PAs). However, whether PFCs exist in plants and how PFCs function remain unclear. Here, we report the use of an integrative strategy including enzymatic assays, mutant analysis, metabolic engineering, isotope labeling and metabolic profiling to capture PFCs and demonstrate their functions. In anthocyanidin reductase (ANR) assays, an (-)-epicatechin conjugate was captured in protic polar nucleophilic methanol alone or methanol-HCl extracts. Tandem mass spectrum (MS/MS) analysis characterized this compound as an (-)-epicatechin-4-O-methyl (EOM) ether, which resulted from (-)-epicatechin carbocation and the methyl group of methanol. Acid-based catalysis of procyanidin B2 and B3 produced four compounds, which were annotated as two EOM and two (+)-catechin-4-O-methyl (COM) ethers. Metabolic profiling of seven PA pathway mutants showed an absence or reduction of two EOM ether isomers in seeds. Camellia sinensis ANRa (CsANRa), leucoanthocyanidin reductase c (CsLARc), and CsMYB5b (a transcription factor) were independently overexpressed for successful PA engineering in tobacco. The EOM ether was remarkably increased in CsANRa and CsMYB5b transgenic flowers. Further metabolic profiling for eight green tea tissues revealed two EOM and two COM ethers associated with PA biosynthesis. Moreover, an incubation of (-)-epicatechin or (+)-catechin with epicatechin carbocation in CsANRa transgenic flower extracts formed dimeric procyanidin B1 or B2, demonstrating the role of flavan-3-ol carbocation in the formation of PAs. Taken together, these findings indicated that flavan-3-ol carbocations exist in extracts and are involved in the biosynthesis of PAs of plants.

Overexpression of a type-I isopentenyl pyrophosphate isomerase of Artemisia annua in the cytosol leads to high arteannuin B production and artemisinin increase.

We recently characterized a gene-terpene network that is associated with artemisinin biosynthesis in self-pollinated (SP) Artemisia annua, an effective antimalarial plant. We hypothesize that an alteration of gene expression in the network may improve the production of artemisinin and its precursors. In this study, we cloned an isopentenyl pyrophosphate isomerase (IPPI) cDNA, AaIPPI1, from Artemisia annua (Aa). The full-length cDNA encodes a type-I IPPI containing a plastid transit peptide (PTP) at its amino terminus. After the removal of the PTP, the recombinant truncated AaIPPI1 isomerized isopentenyl pyrophosphate (IPP) to dimethyl allyl pyrophosphate (DMAPP) and vice versa. The steady-state equilibrium ratio of IPP/DMAPP in the enzymatic reactions was approximately 1:7. The truncated AaIPPI1 was overexpressed in the cytosol of the SP A. annua variety. The leaves of transgenic plants produced approximately 4% arteannuin B (g g-1 , dry weight, dw) and 0.17-0.25% artemisinin (g g-1 , dw), the levels of which were significantly higher than those in the leaves of wild-type plants. In addition, transgenic plants showed an increase in artemisinic acid production of more than 1% (g g-1 , dw). In contrast, isoprene formation was significantly reduced in transgenic plants. These results provide evidence that overexpression of AaIPPI1 in the cytosol can lead to metabolic alterations of terpenoid biosynthesis, and show that these transgenic plants have the potential to yield high production levels of arteannuin B as a new precursor source for artemisinin.

Correction to: Cloning and characterization of a monoterpene synthase gene from flowers of Camelina sativa.

In the original publication, the order of figures and citations was incorrect. The corrections are listed below.

Cloning and characterization of a monoterpene synthase gene from flowers of Camelina sativa.

MAIN CONCLUSION: CsTPS1 encodes for a monoterpene synthase that contributes to the emission of a blend of volatile compounds emitted from flowers of Camelina sativa. The work describes the in vitro characterization of a monoterpene synthase and its regulatory region that we cloned from Camelina sativa (Camelina). Here, we named this gene as C. sativa terpene synthase 1 (CsTPS1). In vitro experiments performed with the CsTPS1 protein after expression and purification from Escherichia coli (E. coli) showed production of a blend of monoterpene volatile organic compounds, of which the emission was also detected in the floral bouquet of wild-type Camelina plants. Quantitative-PCR measurements revealed a high abundance of CsTPS1 transcripts in flowers and experiments performed with the GUS reporter showed high CsTPS1 expression in the pistil, in the cells of the wall of the ovary and in the stigma. Subcellular localization of the CsTPS1 protein was investigated with a GFP reporter construct that showed expression in plastids. The CsTPS1 gene identified in this study belongs to a mid-size family of 60 genes putatively codifying for TPS enzymes. This enlarged family of TPS genes suggests that Camelina has the structural framework for the production of terpenes and other secondary metabolites of relevance for the consumers.

Molecular Cloning and Functional Characterization of a Dihydroflavonol 4-Reductase from Vitis bellula.

Vitis bellula is a new grape crop in southern China. Berries of this species are rich in antioxidative anthocyanins and proanthocyanidins. This study reports cloning and functional characterization of a cDNA encoding a V. bellula dihydroflavonol reductase (VbDFR) involved in the biosynthesis of anthocyanins and proanthocyanidins. A cDNA including 1014 bp was cloned from young leaves and its open reading frame (ORF) was deduced encoding 337 amino acids, highly similar to V. vinifera DFR (VvDFR). Green florescence protein fusion and confocal microscopy analysis determined the cytosolic localization of VbDFR in plant cells. A soluble recombinant VbDFR was induced and purified from E. coli for enzyme assay. In the presence of NADPH, the recombinant enzyme catalyzed dihydrokaempferol (DHK) and dihydroquercetin (DHQ) to their corresponding leucoanthocyanidins. The VbDFR cDNA was introduced into tobacco plants via Agrobacterium-mediated transformation. The overexpression of VbDFR increased anthocyanin production in flowers. Anthocyanin hydrolysis and chromatographic analysis revealed that transgenic flowers produced pelargonidin and delphinidin, which were not detected in control flowers. These data demonstrated that the overexpression of VbDFR produced new tobacco anthocyanidins. In summary, all data demonstrate that VbDFR is a useful gene to provide three types of substrates for metabolic engineering of anthocyanins and proanthocyanidins in grape crops and other crops.

Metabolic engineering of anthocyanins in dark tobacco varieties.

In this study, we investigate the metabolic engineering of anthocyanins in two dark tobacco crops (Narrow Leaf Madole and KY171) and evaluate the effects on physiological features of plant photosynthesis. Arabidopsis PAP1 (production of anthocyanin pigment 1) gene (AtPAP1) encodes a R2R3-type MYB transcript factor that is a master component of regulatory complexes controlling anthocyanin biosynthesis. AtPAP1 was introduced to Narrow Leaf Madole and KY171 plants. Multiple transgenic plants developed red/purple pigmentation in different tissues. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that the expression levels of six pathway genes were increased two- to eight-fold in AtPAP1 transgenic plants compared with vector control plants. Dihydroflavonol reductase and anthocyanidin synthase genes that were not expressed in wild-type plants were activated. Spectrophotometric measurement showed that the amount of anthocyanins in AtPAP1 transgenic plants were 400-800 µg g-1 fresh weight (FW). High-performance liquid chromatography (HPLC) analysis showed that one main anthocyanin molecule accounted for approximately 98% of the total anthocyanins. Tandem MS/MS analysis using HPLC coupled to electrospray ionization and quadrupole time-of-flight mass spectrometry identified the main anthocyanin as cyanidin 3-O-rutinoside, an important medicinal anthocyanin. Analysis of photosynthesis rate, chlorophylls and carotenoids contents showed no differences between red/purple transgenic and control plants, indicating that this metabolic engineering did not alter photosynthetic physiological traits. This study shows that AtPAP1 is of significance for metabolic engineering of anthocyanins in crop plants for value-added traits.

Metabolic Characterization of the Anthocyanidin Reductase Pathway Involved in the Biosynthesis of Flavan-3-ols in Elite Shuchazao Tea (Camellia sinensis) Cultivar in the Field.

Anthocyanidin reductase (ANR) is a key enzyme in the ANR biosynthetic pathway of flavan-3-ols and proanthocyanidins (PAs) in plants. Herein, we report characterization of the ANR pathway of flavan-3-ols in Shuchazao tea (Camellia sinesis), which is an elite and widely grown cultivar in China and is rich in flavan-3-ols providing with high nutritional value to human health. In our study, metabolic profiling was preformed to identify two conjugates and four aglycones of flavan-3-ols: (-)-epigallocatechin-gallate [(-)-EGCG], (-)-epicatechin-gallate [(-)-ECG], (-)-epigallocatechin [(-)-EGC], (-)-epicatechin [(-)-EC], (+)-catechin [(+)-Ca], and (+)-gallocatechin [(+)-GC], of which (-)-EGCG, (-)-ECG, (-)-EGC, and (-)-EC accounted for 70-85% of total flavan-3-ols in different tissues. Crude ANR enzyme was extracted from young leaves. Enzymatic assays showed that crude ANR extracts catalyzed cyanidin and delphinidin to (-)-EC and (-)-Ca and (-)-EGC and (-)-GC, respectively, in which (-)-EC and (-)-EGC were major products. Moreover, two ANR cDNAs were cloned from leaves, namely CssANRa and CssANRb. His-Tag fused recombinant CssANRa and CssANRb converted cyanidin and delphinidin to (-)-EC and (-)-Ca and (-)-EGC and (-)-GC, respectively. In addition, (+)-EC was observed from the catalysis of recombinant CssANRa and CssANRb. Further overexpression of the two genes in tobacco led to the formation of PAs in flowers and the reduction of anthocyanins. Taken together, these data indicate that the majority of leaf flavan-3-ols in Shuchazao's leaves were produced from the ANR pathway.

Tissue-specific production of limonene in Camelina sativa with the Arabidopsis promoters of genes BANYULS and FRUITFULL.

MAIN CONCLUSION: Arabidopsis promoters of genes BANYULS and FRUITFULL are transcribed in Camelina. They triggered the transcription of limonene synthase and induced higher limonene production in seeds and fruits than CaMV 35S promoter. Camelina sativa (Camelina) is an oilseed crop of relevance for the production of biofuels and the plant has been target of a recent and intense program of genetic manipulation aimed to increase performance, seed yield and to modify the fatty acid composition of the oil. Here, we have explored the performance of two Arabidopsis thaliana (Arabidopsis) promoters in triggering transgene expression in Camelina. The promoters of two genes BANYULS (AtBAN pro ) and FRUITFULL (AtFUL pro ), which are expressed in seed coat and valves of Arabidopsis, respectively, have been chosen to induce the expression of limonene synthase (LS) from Citrus limon. In addition, the constitutive CaMV 35S promoter was utilized to overexpress LS in Camelina . The results of experiments revealed that AtBAN pro and AtFUL pro are actively transcribed in Camelina where they also retain specificity of expression in seeds and valves as previously observed in Arabidopsis. LS induced by AtBAN pro and AtFUL pro leads to higher limonene production in seeds and fruits than when the CaMV 35S was used to trigger the expression. In conclusion, the results of experiments indicate that AtBAN pro and AtFUL pro can be successfully utilized to induce the expression of the transgenes of interest in seeds and fruits of Camelina.

Overexpression of a synthetic insect-plant geranyl pyrophosphate synthase gene in Camelina sativa alters plant growth and terpene biosynthesis.

MAIN CONCLUSION: A novel plastidial homodimeric insect-plant geranyl pyrophosphate synthase gene is synthesized from three different cDNA origins. Its overexpression in Camelina sativa effectively alters plant development and terpenoid metabolism. Geranyl pyrophosphate synthase (GPPS) converts one isopentenyl pyrophosphate and dimethylallyl pyrophosphate to GPP. Here, we report a synthetic insect-plant GPPS gene and effects of its overexpression on plant growth and terpenoid metabolism of Camelina sativa. We synthesized a 1353-bp cDNA, namely PTP-MpGPPS. This synthetic cDNA was composed of a 1086-bp cDNA fragment encoding a small GPPS isomer of the aphid Myzus persicae (Mp), 240-bp Arabidopsis thaliana cDNA fragment encoding a plastidial transit peptide (PTP), and a 27-bp short cDNA fragment encoding a human influenza hemagglutinin tag peptide. Structural modeling showed that the deduced protein was a homodimeric prenyltransferase. Confocal microscopy analysis demonstrated that the PTP-MpGPPS fused with green florescent protein was localized in the plastids. The synthetic PTP-MpGPPS cDNA driven by 2 × 35S promoters was introduced into Camelina (Camelina sativa) by Agrobacterium-mediated transformation and its overexpression in transgenic plants were demonstrated by western blot. T2 and T3 progeny of transgenic plants developed larger leaves, grew more and longer internodes, and flowered earlier than wild-type plants. Metabolic analysis showed that the levels of beta-amyrin and campesterol were higher in tissues of transgenic plants than in those of wild-type plants. Fast isoprene sensor analysis demonstrated that transgenic Camelina plants emitted significantly less isoprene than wild-type plants. In addition, transcriptional analyses revealed that the expression levels of gibberellic acid and brassinosteroids-responsive genes were higher in transgenic plants than in wild-type plants. Taken together, these data demonstrated that this novel synthetic insect-plant GPPS cDNA was effective to improve growth traits and alter terpenoid metabolism of Camelina.

Analysis of two TFL1 homologs of dogwood species (Cornus L.) indicates functional conservation in control of transition to flowering.

MAIN CONCLUSION: Two TFL1 -like genes, CorfloTFL1 and CorcanTFL1 cloned from Cornus florida and C. canadensis, function in regulating the transition to reproductive development in Arabidopsis. TERMINAL FLOWER 1 (TFL1) is known to regulate inflorescence development in Arabidopsis thaliana and to inhibit the transition from a vegetative to reproductive phase within the shoot apical meristem. Despite the importance, TFL1 homologs have been functionally characterized in only a handful eudicots. Here we report the role of TFL1 homologs of Cornus L. in asterid clade of eudicots. Two TFL1-like genes, CorfloTFL1 and CorcanTFL1, were cloned from Cornus florida (a tree) and C. canadensis (a subshrub), respectively. Both are deduced to encode proteins of 175 amino acids. The amino acid sequences of these two Cornus TFL1 homologs share a high similarity to Arabidopsis TFL1 and phylogenetically more close to TFL1 paralogous copy ATC (Arabidopsis thaliana CENTRORADIALIS homologue). Two genes are overexpressed in wild-type and tfl1 mutant plants of A. thaliana. The over-expression of each gene in wild-type Arabidopsis plants results in delaying flowering time, increase of plant height and cauline and rosette leaf numbers, excessive shoot buds, and secondary inflorescence branches. The over-expression of each gene in the tfl1 mutant rescued developmental defects, such as the early determinate inflorescence development, early flowering time, and other vegetative growth defects, to normal phenotypes of wild-type plants. These transgenic phenotypes are inherited in progenies. All data indicate that CorfloTFL1 and CorcanTFL1 have conserved the ancestral function of TFL1 and CEN regulating flowering time and inflorescence determinacy.

Functional characterization of an anthocyanidin reductase gene from the fibers of upland cotton (Gossypium hirsutum).

MAIN CONCLUSION: Metabolic profiling, gene cloning, enzymatic analysis, ectopic expression, and gene silencing experiments demonstrate that the anthocyanidin reductase (ANR) pathway is involved in the biosynthesis of proanthocyanidins in upland cotton. Proanthocyanidins (PAs) are oligomeric or polymeric flavan-3-ols, however, the biosynthetic pathway of PAs in cotton remains to be elucidated. Here, we report on an anthocyanidin reductase (ANR) gene from cotton fibers and the ANR pathway of PAs. Phytochemical analysis demonstrated that leaves, stems, roots, and early developing fibers produced PAs and their monomers, including (-)-epicatechin, (-)-catechin, (-)-epigallocatechin, and (-)-gallocatechin. Crude PA extractions from different tissues were boiled in Butanol:HCl. Cyanidin, delphinidin, and pelargonidin were produced, indicating that cotton PAs include diverse extension unit structures. An ANR cDNA homolog (named GhANR1) was cloned from developing fibers. The open reading frame, composed of 1,011 bp nucleotides, was expressed in E. coli to obtain a recombinant protein. In the presence of NADPH, the recombinant enzyme catalyzed cyanidin, delphinidin, and pelargonidin to (-)-epicatechin and (-)-catechin, (-)-epigallocatechin and (-)-gallocatechin, and (-)-epiafzelechin and (-)-afzelechin, respectively. The ectopic expression of GhANR11 in an Arabidopsis ban mutant allowed for the reconstruction of the ANR pathway and PA biosynthesis in the seed coat. Virus-induced gene silencing (VIGS) of GhANR11 led to a significant increase in anthocyanins and a decrease in the PAs, (-)-epicatechin, and (-)-catechin in the stems and leaves of VIGS-infected plants. Taken together, these data demonstrate that the ANR pathway contributes to the biosynthesis of flavan-3-ols and PAs in cotton.

Enhanced diversity and aflatoxigenicity in interspecific hybrids of Aspergillus flavus and Aspergillus parasiticus.

Aspergillus flavus and A. parasiticus are the two most important aflatoxin-producing fungi responsible for the contamination of agricultural commodities worldwide. Both species are heterothallic and undergo sexual reproduction in laboratory crosses. Here we examine the possibility of interspecific matings between A. flavus and A. parasiticus. These species can be distinguished morphologically and genetically, as well as by their mycotoxin profiles. Aspergillus flavus produces both B aflatoxins and cyclopiazonic acid (CPA), B aflatoxins or CPA alone, or neither mycotoxin; Aspergillus parasiticus produces B and G aflatoxins or the aflatoxin precursor O-methylsterigmatocystin, but not CPA. Only four of forty-five attempted interspecific crosses between opposite mating types of A. flavus and A. parasiticus were fertile and produced viable ascospores. Single ascospore strains from each cross were shown to be recombinant hybrids using multilocus genotyping and array comparative genome hybridization. Conidia of parents and their hybrid progeny were haploid and predominantly monokaryons and dikaryons based on flow cytometry. Multilocus phylogenetic inference showed that experimental hybrid progeny were grouped with naturally occurring A. flavus L strain and A. parasiticus. Higher total aflatoxin concentrations in some F1 progeny strains compared to midpoint parent aflatoxin levels indicate synergism in aflatoxin production; moreover, three progeny strains synthesized G aflatoxins that were not produced by the parents, and there was evidence of allopolyploidization in one strain. These results suggest that hybridization is an important diversifying force resulting in the genesis of novel toxin profiles in these agriculturally important fungi.