Publications by authors named "Sitakanta Pattanaik"

40 Publications

is a member of a bHLH gene cluster regulating terpenoid indole alkaloid biosynthesis in .

Plant Direct 2021 Jan 25;5(1):e00305. Epub 2021 Jan 25.

Kentucky Tobacco Research & Development Center University of Kentucky Lexington KY USA.

Basic helix-loop-helix (bHLH) transcription factors (TFs) are key regulators of plant specialized metabolites, including terpenoid indole alkaloids (TIAs) in . Two previously characterized subgroup-IVa bHLH TFs, BIS1 (bHLH Iridoid Synthesis 1) and BIS2 regulate iridoid biosynthesis in the TIA pathway. We reanalyzed the recently updated genome sequence and discovered that and are clustered on the same genomic scaffold with a previously uncharacterized bHLH gene, designated as . Only a few bHLH gene clusters have been studied to date. Comparative analysis of 49 genome sequences from different plant lineages revealed the presence of analogous bHLH clusters in core angiosperms, including the medicinal plants (giant milkweed) and (yellow jessamine), but not in the analyzed basal angiosperm and lower plants. Similar to the iridoid pathway genes, is highly expressed in roots and induced by methyl jasmonate. BIS3 activates the promoters of iridoid branch genes, (), (), (), (), (), and (), but not (). Transactivation of the promoters was abolished when BIS3 is converted to a dominant repressor by fusing with the ERF-associated amphiphilic repression (EAR) sequence. In addition, BIS3 acts synergistically with BIS1 and BIS2 to activate the promoter in tobacco cells. Mutation of the known bHLH TF binding motif, G-box (CACGTG) in the promoter significantly reduced but did not abolish the transactivation by BIS3. Promoter deletion analysis of suggests that the sequences adjacent to the G-box are also involved in the regulation by BIS3. Overexpression of in flower petals significantly upregulated the expression of iridoid biosynthetic genes and increased loganic acid accumulation. expression was significantly induced by BIS3 although BIS3 did not directly activate the promoter. Our results advance our understanding of the regulation of plant specialized metabolites by bHLH TF clusters.
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http://dx.doi.org/10.1002/pld3.305DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7833464PMC
January 2021

Protein phosphatase NtPP2C2b and MAP kinase NtMPK4 act in concert to modulate nicotine biosynthesis.

J Exp Bot 2021 Feb;72(5):1661-1676

Department of Plant and Soil Sciences, and the Kentucky Tobacco Research and Development Center, University of Kentucky, University Drive, Lexington, KY USA.

Protein phosphatases (PPs) and protein kinases (PKs) regulate numerous developmental, defense, and phytohormone signaling processes in plants. However, the underlying regulatory mechanism governing biosynthesis of specialized metabolites, such as alkaloids, by the combined effects of PPs and PKs, is insufficiently understood. Here, we report the characterization of a group B protein phosphatase type 2C, NtPP2C2b, that likely acts upstream of the NICOTINE2 locus APETALA 2/Ethylene Response Factors (AP2/ERFs), to regulate nicotine biosynthesis in tobacco. Similar to the nicotine pathway genes, NtPP2C2b is highly expressed in roots and induced by jasmonic acid (JA). Overexpression of NtPP2C2b in transgenic hairy roots or stable transgenic tobacco plants repressed nicotine pathway gene expression and reduced nicotine accumulation. Additionally, transient overexpression of NtPP2C2b, together with the NtERF221, repressed transactivation of the quinolinate phosphoribosyltransferase promoter in tobacco cells. We further demonstrate that the JA-responsive tobacco mitogen-activated protein kinase (MAPK) 4 interacts with NtPP2C2b in yeast and plant cells. Conditional overexpression of NtMPK4 in tobacco hairy roots up-regulated nicotine pathway gene expression and increased nicotine accumulation. Our findings suggest that a previously uncharacterized PP-PK module acts to modulate alkaloid biosynthesis, highlighting the importance of post-translational control in the biosynthesis of specialized plant metabolites.
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http://dx.doi.org/10.1093/jxb/eraa568DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7921305PMC
February 2021

Maleic hydrazide elicits global transcriptomic changes in chemically topped tobacco to influence shoot bud development.

Planta 2020 Sep 24;252(4):64. Epub 2020 Sep 24.

Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY, 40546, USA.

Main Conclusion: Transcriptomic analysis revealed maleic hydrazide suppresses apical and axillary bud development by altering the expression of genes related to meristem development, cell division, DNA replication, DNA damage and recombination, and phytohormone signaling. Topping (removal of apical buds) is a common agricultural practice for some crop plants including cotton, cannabis, and tobacco. Maleic hydrazide (MH) is a systemic suckercide, a chemical that inhibits shoot bud growth, used to control the growth of apical (ApB) and axillary buds (AxB) following topping. However, the influence of MH on gene expression and the underlying molecular mechanism of controlling meristem development are not well studied. Our RNA sequencing analysis showed that MH significantly influences the transcriptomic landscape in ApB and AxB of chemically topped tobacco. Gene ontology (GO) enrichment analysis revealed that upregulated genes in ApB were enriched for phosphorelay signal transduction, and the regulation of transition timing from vegetative to reproductive phase, whereas downregulated genes were largely associated with meristem maintenance, cytokinin metabolism, cell wall synthesis, photosynthesis, and DNA metabolism. In MH-treated AxB, GO terms related to defense response and oxylipin metabolism were overrepresented in upregulated genes. GO terms associated with cell cycle, DNA metabolism, and cytokinin metabolism were enriched in downregulated genes. Expression of KNOX and MADS transcription factor (TF) family genes, known to be involved in meristem development, were affected in ApB and AxB by MH treatment. The promoters of MH-responsive genes are enriched for several known cis-acting elements, suggesting the involvement of a subset of TF families. Our findings suggest that MH affects shoot bud development in chemically topped tobacco by altering the expression of genes related to meristem development, DNA repair and recombination, cell division, and phytohormone signaling.
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http://dx.doi.org/10.1007/s00425-020-03460-9DOI Listing
September 2020

TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR4 Interacts with WRINKLED1 to Mediate Seed Oil Biosynthesis.

Plant Physiol 2020 10 6;184(2):658-665. Epub 2020 Jul 6.

School of Biological Sciences, Nanyang Technological University, Singapore 637551, Singapore

Cross-family transcription factor (TF) interactions play critical roles in the regulation of plant developmental and metabolic pathways. WRINKLED1 (WRI1) is a key TF governing oil biosynthesis in plants. However, little is known about WRI1-interacting factors and their roles in oil biosynthesis. We screened a TF library using Arabidopsis () WRI1 (AtWRI1) as bait in yeast two-hybrid assays and identified three TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) family TFs, namely TCP4, TCP10, and TCP24, as AtWRI1-interacting partners. The physical interaction between AtWRI1 and TCPs was further validated using bimolecular fluorescence complementation assays. TCPs play important roles in various plant developmental processes; however, their involvement in fatty acid biosynthesis was not previously known. Coexpression of TCP4, but not TCP10 or TCP24, with AtWRI1 reduced AtWRI1-mediated oil biosynthesis in leaves. Transcriptomic analysis in transgenic Arabidopsis plants with enhanced TCP4 activity engineered by expressing (i.e. miR319-resistant ) revealed that AtWRI1 target genes were significantly repressed. TCP4 expression is strongly correlated with AtWRI1 during embryo development. A loss-of-function mutant, the mutant with a strong reduction of expression, and a triple mutant accumulated more seed oil than wild-type Arabidopsis. In addition, TCP4 repressed the AtWRI1-mediated transactivation of the promoters of fatty acid biosynthetic genes. Collectively, our findings suggest that TCP4 represses fatty acid biosynthetic gene expression through interaction with AtWRI1, leading to a reduction of AtWRI1-mediated seed oil accumulation.
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http://dx.doi.org/10.1104/pp.20.00547DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7536675PMC
October 2020

Revisiting the ORCA gene cluster that regulates terpenoid indole alkaloid biosynthesis in Catharanthus roseus.

Plant Sci 2020 Apr 9;293:110408. Epub 2020 Jan 9.

Department of Plant and Soil Sciences and the Kentucky Tobacco Research and Development Center, University of Kentucky, 1401 University Drive, Lexington, KY 40546 USA; South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, China. Electronic address:

Transcription factor (TF) gene clusters in plants, such as tomato, potato, petunia, tobacco, and almond, have been characterized for their roles in the biosynthesis of diverse array of specialized metabolites. In Catharanthus roseus, three AP2/ERF TFs, ORCA3, ORCA4, and ORCA5, have been shown to be present on the same genomic scaffold, forming a cluster that regulates the biosynthesis of pharmaceutically important terpenoid indole alkaloids (TIAs). Our analysis of the recently updated C. roseus genome sequence revealed that the ORCA cluster comprises two additional AP2/ERFs, the previously characterized ORCA2 and a newly identified member designated as ORCA6. Transcriptomic analysis revealed that the ORCAs are highly expressed in stems, followed by leaves, roots and flowers. Expression of ORCAs was differentially induced in response to methyl-jasmonate and ethylene treatment. In addition, ORCA6 activated the strictosidine synthase (STR) promoter in tobacco cells. Activation of the STR promoter was significantly higher when ORCA2 or ORCA6 was coexpressed with the mitogen-activated protein kinase kinase, CrMPKK1. Furthermore, transient overexpression of ORCA6 in C. roseus flower petals activated TIA pathway gene expression and TIA accumulation. The results described here advance our understanding of regulation of TIA pathway by the ORCA gene cluster and the evolution for plant ERF gene clusters.
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http://dx.doi.org/10.1016/j.plantsci.2020.110408DOI Listing
April 2020

Mutually Regulated AP2/ERF Gene Clusters Modulate Biosynthesis of Specialized Metabolites in Plants.

Plant Physiol 2020 02 14;182(2):840-856. Epub 2019 Nov 14.

Department of Plant and Soil Sciences and Kentucky Tobacco Research and Development Center, University of Kentucky, Lexington, Kentucky 40546

APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) gene clusters regulate the biosynthesis of diverse specialized metabolites, including steroidal glycoalkaloids in tomato () and potato (), nicotine in tobacco (), and pharmaceutically valuable terpenoid indole alkaloids in Madagascar periwinkle (). However, the regulatory relationships between individual AP2/ERF genes within the cluster remain unexplored. We uncovered intracluster regulation of the AP2/ERF regulatory circuit, which consists of , , and ORCA3 and ORCA5 activate by directly binding to a GC-rich motif in the promoter. ORCA5 regulates its own expression through a positive autoregulatory loop and indirectly activates In determining the functional conservation of AP2/ERF clusters in other plant species, we found that GC-rich motifs are present in the promoters of analogous AP2/ERF clusters in tobacco, tomato, and potato. Intracluster regulation is evident within the tobacco () cluster. Moreover, overexpression of in tobacco and of in hairy roots activates nicotine and terpenoid indole alkaloid pathway genes, respectively, suggesting that the AP2/ERFs are functionally equivalent and are likely to be interchangeable. Elucidation of the intracluster and mutual regulation of transcription factor gene clusters advances our understanding of the underlying molecular mechanism governing regulatory gene clusters in plants.
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http://dx.doi.org/10.1104/pp.19.00772DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6997685PMC
February 2020

GATA and Phytochrome Interacting Factor Transcription Factors Regulate Light-Induced Vindoline Biosynthesis in .

Plant Physiol 2019 07 13;180(3):1336-1350. Epub 2019 May 13.

Key Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, China 510650

is the exclusive source of an array of terpenoid indole alkaloids including the anticancer drugs vincristine and vinblastine, derived from the coupling of catharanthine and vindoline. Leaf-synthesized vindoline is regulated by light. A seven-step enzymatic process is involved in the sequential conversion of tabersonine to vindoline; however, the regulatory mechanism controlling the expression of genes encoding these enzymes has not been elucidated. Here, we identified CrGATA1, an Leu-Leu-Met domain GATA transcription factor that regulates light-induced vindoline biosynthesis in seedlings. Expression of and the vindoline pathway genes , , , , and was significantly induced by light. In addition, CrGATA1 activated the promoters of five light-responsive vindoline pathway genes in plant cells. Two GATC motifs in the promoter were critical for CrGATA1-mediated transactivation. Transient overexpression of in seedlings resulted in up-regulation of vindoline pathway genes and increased vindoline accumulation. Conversely, virus-induced gene silencing of in young leaves significantly repressed key vindoline pathway genes and reduced vindoline accumulation. Furthermore, we showed that a Phytochrome Interacting Factor, CrPIF1, is a repressor of and vindoline biosynthesis. Transient overexpression or virus-induced gene silencing of in seedlings altered and vindoline pathway gene expression in the dark. CrPIF1 repressed and promoter activity by binding to G/E-box/PBE elements. Our findings reveal a regulatory module involving Phytochrome Interacting Factor -GATA that governs light-mediated biosynthesis of specialized metabolites.
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http://dx.doi.org/10.1104/pp.19.00489DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6752914PMC
July 2019

Cross-family transcription factor interaction between MYC2 and GBFs modulates terpenoid indole alkaloid biosynthesis.

J Exp Bot 2018 08;69(18):4267-4281

Department of Plant and Soil Sciences and Kentucky Tobacco Research and Development Center, University of Kentucky, Lexington, KY, USA.

Biosynthesis of medicinally valuable terpenoid indole alkaloids (TIAs) in Catharanthus roseus is regulated by transcriptional activators such as the basic helix-loop-helix factor CrMYC2. However, the transactivation effects are often buffered by repressors, such as the bZIP factors CrGBF1 and CrGBF2, possibly to fine-tune the accumulation of cytotoxic TIAs. Questions remain as to whether and how these factors interact to modulate TIA production. We demonstrated that overexpression of CrMYC2 induces CrGBF expression and results in reduced alkaloid accumulation in C. roseus hairy roots. We found that CrGBF1 and CrGBF2 form homo- and heterodimers to repress the transcriptional activities of key TIA pathway gene promoters. We showed that CrGBFs dimerize with CrMYC2, and CrGBF1 binds to the same cis-elements (T/G-box) as CrMYC2 in the target gene promoters. Our findings suggest that CrGBFs antagonize CrMYC2 transactivation possibly by competitive binding to the T/G-box in the target promoters and/or protein-protein interaction that forms a non-DNA binding complex that prevents CrMYC2 from binding to its target promoters. Homo- and heterodimer formation allows fine-tuning of the amplitude of TIA gene expression. Our findings reveal a previously undescribed regulatory mechanism that governs the TIA pathway genes to balance metabolic flux for TIA production in C. roseus.
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http://dx.doi.org/10.1093/jxb/ery229DOI Listing
August 2018

Correction to: Genome-wide identification of hexokinase gene family in Brassica napus: structure, phylogenetic analysis, expression, and functional characterization.

Planta 2018 07;248(1):183

School of Life Sciences, Shanxi University, Taiyuan, 030006, Shanxi, China.

In the original version of this article the name of the second author was misspelled. The correct name is: Xiaomin Wang.
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http://dx.doi.org/10.1007/s00425-018-2895-9DOI Listing
July 2018

Genome-wide identification of hexokinase gene family in Brassica napus: structure, phylogenetic analysis, expression, and functional characterization.

Planta 2018 Jul 11;248(1):171-182. Epub 2018 Apr 11.

School of Life Sciences, Shanxi University, Taiyuan, 030006, Shanxi, China.

Main Conclusion: Genome-wide identification, expression analysis, and functional characterization of previously uncharacterized hexokinase family of oil crop, Brassica napus, underscore the importance of this gene family in plant growth and development. In plants, the multi-gene family of dual-function hexokinases (HXKs) plays important roles in sugar metabolism and sensing that affect growth and development. Rapeseed (Brassica napus L.) is an important oil crop; however, little is known about the B. napus HXK gene family. We identified 19 putative HXKs in B. napus genome. B. rapa and B. oleracea, the two diploid progenitors of B. napus, contributed almost equally to the BnHXK genes. Phylogenetic analysis divided the 19 BnHXKs into four groups. The exon-intron structures of BnHXKs share high similarity to those of HXKs in Arabidopsis and rice. The group III and IV BnHXKs are highly expressed in roots, whereas group I members preferentially express in leaves. Analysis of seed transcriptomes at different developmental stages showed that most of group I and IV HXKs are highly expressed 2-weeks after pollination (2WAP), compared to 4WAP for group III. BnHKXs are differentially expressed in susceptible and tolerant B. napus cultivars after fungal infection, suggesting the possible involvement in defense response. We generated rapeseed RNAi lines for BnHXK9, a member of relatively less characterized group IV, by pollen-mediated gene transformation. The seedlings of BnHXK9-RNAi lines showed delayed growth compared to the wild type. The RNAi plants were dwarf with curly leaves, suggesting the involvement of BnHXK9 in plant development. Collectively, our findings provides a comprehensive account of BnHXK gene family in an important crop and a starting point for further elucidation of their roles in sugar metabolism and sensing, as well as plant growth and development.
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http://dx.doi.org/10.1007/s00425-018-2888-8DOI Listing
July 2018

A network of jasmonate-responsive bHLH factors modulate monoterpenoid indole alkaloid biosynthesis in Catharanthus roseus.

New Phytol 2018 03 27;217(4):1566-1581. Epub 2017 Nov 27.

Department of Plant and Soil Sciences, Kentucky Tobacco Research and Development Center, University of Kentucky, 1401 University Drive, Lexington, KY, 40546, USA.

The pharmaceutically valuable monoterpene indole alkaloids (MIAs) in Catharanthus roseus are derived from the indole and iridoid pathways that respond to jasmonate (JA) signaling. Two classes of JA-responsive bHLH transcription factor (TF), CrMYC2 and BIS1/BIS2, are known to regulate the indole and iridoid pathways, respectively. However, upregulation of either one of the TF genes does not lead to increased MIA accumulation. Moreover, little is known about the interconnection between the CrMYC2 and BIS transcriptional cascades and the hierarchical position of BIS1/BIS2 in JA signaling. Here, we report that a newly identified bHLH factor, Repressor of MYC2 Targets 1 (RMT1), is activated by CrMYC2 and BIS1, and acts as a repressor of the CrMYC2 targets. In addition, we isolated and functionally characterized the core C. roseus JA signaling components, including CORONATINE INSENSITIVE 1 (COI1) and JASMONATE ZIM domain (JAZ) proteins. CrMYC2 and BIS1 are repressed by the JAZ proteins in the absence of JA, but de-repressed by the SCF complex on perception of JA. Our findings suggest that the repressors, JAZs and RMT1, mediate crosstalk between the CrMYC2 and BIS regulatory cascades to balance the metabolic flux in MIA biosynthesis.
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http://dx.doi.org/10.1111/nph.14910DOI Listing
March 2018

The miRNAome of Catharanthus roseus: identification, expression analysis, and potential roles of microRNAs in regulation of terpenoid indole alkaloid biosynthesis.

Sci Rep 2017 02 22;7:43027. Epub 2017 Feb 22.

Department of Plant and Soil Sciences, University of Kentucky, 1401 University Drive, Lexington, KY 40546, USA.

MicroRNAs (miRNAs) regulate numerous crucial biological processes in plants. However, information is limited on their involvement in the biosynthesis of specialized metabolites in plants, including Catharanthus roseus that produces a number of pharmaceutically valuable, bioactive terpenoid indole alkaloids (TIAs). Using small RNA-sequencing, we identified 181 conserved and 173 novel miRNAs (cro-miRNAs) in C. roseus seedlings. Genome-wide expression analysis revealed that a set of cro-miRNAs are differentially regulated in response to methyl jasmonate (MeJA). In silico target prediction identified 519 potential cro-miRNA targets that include several auxin response factors (ARFs). The presence of cleaved transcripts of miRNA-targeted ARFs in C. roseus cells was confirmed by Poly(A) Polymerase-Mediated Rapid Amplification of cDNA Ends (PPM-RACE). We showed that auxin (indole acetic acid, IAA) repressed the expression of key TIA pathway genes in C. roseus seedlings. Moreover, we demonstrated that a miRNA-regulated ARF, CrARF16, binds to the promoters of key TIA pathway genes and repress their expression. The C. roseus miRNAome reported here provides a comprehensive account of the cro-miRNA populations, as well as their abundance and expression profiles in response to MeJA. In addition, our findings underscore the importance of miRNAs in posttranscriptional control of the biosynthesis of specialized metabolites.
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http://dx.doi.org/10.1038/srep43027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5320439PMC
February 2017

A differentially regulated AP2/ERF transcription factor gene cluster acts downstream of a MAP kinase cascade to modulate terpenoid indole alkaloid biosynthesis in Catharanthus roseus.

New Phytol 2017 Feb 1;213(3):1107-1123. Epub 2016 Nov 1.

Department of Plant and Soil Sciences and Kentucky Tobacco Research Development Center, University of Kentucky, 1401 University Drive, Lexington, KY, 40546, USA.

Catharanthus roseus produces bioactive terpenoid indole alkaloids (TIAs), including the chemotherapeutics, vincristine and vinblastine. Transcriptional regulation of TIA biosynthesis is not fully understood. The jasmonic acid (JA)-responsive AP2/ERF transcription factor (TF), ORCA3, and its regulator, CrMYC2, play key roles in TIA biosynthesis. ORCA3 forms a physical cluster with two uncharacterized AP2/ERFs, ORCA4 and 5. Here, we report that (1) the ORCA gene cluster is differentially regulated; (2) ORCA4, while overlapping functionally with ORCA3, modulates an additional set of TIA genes. Unlike ORCA3, ORCA4 overexpression resulted in dramatic increase of TIA accumulation in C. roseus hairy roots. In addition, CrMYC2 is capable of activating ORCA3 and co-regulating TIA pathway genes concomitantly with ORCA3. The ORCA gene cluster and CrMYC2 act downstream of a MAP kinase cascade that includes a previously uncharacterized MAP kinase kinase, CrMAPKK1. Overexpression of CrMAPKK1 in C. roseus hairy roots upregulated TIA pathways genes and increased TIA accumulation. This work provides detailed characterization of a TF gene cluster and advances our understanding of the transcriptional and post-translational regulatory mechanisms that govern TIA biosynthesis in C. roseus.
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http://dx.doi.org/10.1111/nph.14252DOI Listing
February 2017

Comparative Transcriptomic Analysis of Two Near-Isogenic Lines Reveals a Network of Genes That Influences Seed Oil Accumulation.

Front Plant Sci 2016 29;7:1498. Epub 2016 Sep 29.

College of Life Sciences, Shanxi UniversityTaiyuan, China; Department of Plant and Soil Sciences, University of Kentucky, LexingtonKY, USA.

Rapeseed () is an important oil seed crop, providing more than 13% of the world's supply of edible oils. An in-depth knowledge of the gene network involved in biosynthesis and accumulation of seed oil is critical for the improvement of . Using available genomic and transcriptomic resources, we identified 1,750 acyl-lipid metabolism (ALM) genes that are distributed over 19 chromosomes in the . genome. and , two diploid progenitors of , contributed almost equally to the ALM genes. Genome collinearity analysis demonstrated that the majority of the ALM genes have arisen due to genome duplication or segmental duplication events. In addition, we profiled the expression patterns of the ALM genes in four different developmental stages. Furthermore, we developed two near isogenic lines (NILs). The high oil NIL, YC13-559, accumulates significantly higher (∼10%) seed oil compared to the other, YC13-554. Comparative gene expression analysis revealed upregulation of lipid biosynthesis-related regulatory genes in YC13-559, including , and , as well as structural genes, such as , and -. We observed that several genes related to the phytohormones, gibberellins, jasmonate, and indole acetic acid, were differentially expressed in the NILs. Our findings provide a broad account of the numbers, distribution, and expression profiles of acyl-lipid metabolism genes, as well as gene networks that potentially control oil accumulation in . seeds. The upregulation of key regulatory and structural genes related to lipid biosynthesis likely plays a major role for the increased seed oil in YC13-559.
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http://dx.doi.org/10.3389/fpls.2016.01498DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5040705PMC
September 2016

Altered Phenylpropanoid Metabolism in the Maize Lc-Expressed Sweet Potato (Ipomoea batatas) Affects Storage Root Development.

Sci Rep 2016 Jan 4;6:18645. Epub 2016 Jan 4.

National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Science, Shanghai 200032, China.

There is no direct evidence of the effect of lignin metabolism on early storage root development in sweet potato. In this study, we found that heterologous expression of the maize leaf color (Lc) gene in sweet potato increased anthocyanin pigment accumulation in the whole plant and resulted in reduced size with an increased length/width ratio, low yield and less starch content in the early storage roots. RT-PCR analysis revealed dramatic up-regulation of the genes involved in the lignin biosynthesis pathway in developing storage roots, leading to greater lignin content in the Lc transgenic lines, compared to the wild type. This was also evidenced by the enhanced lignification of vascular cells in the early storage roots. Furthermore, increased expression of the β-amylase gene in leaves and storage roots also accelerated starch degradation and increased the sugar use efficiency, providing more energy and carbohydrate sources for lignin biosynthesis in the Lc transgenic sweet potato. Lesser starch accumulation was observed in the developing storage roots at the initiation stage in the Lc plants. Our study provides experimental evidence of the basic carbohydrate metabolism underlying the development of storage roots, which is the transformation of lignin biosynthesis to starch biosynthesis.
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http://dx.doi.org/10.1038/srep18645DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4698713PMC
January 2016

RNA-sequencing Reveals Global Transcriptomic Changes in Nicotiana tabacum Responding to Topping and Treatment of Axillary-shoot Control Chemicals.

Sci Rep 2015 Dec 16;5:18148. Epub 2015 Dec 16.

Kentucky Tobacco Research and Development Center , University of Kentucky, Lexington, KY 40546, U.S.A.

Removal of terminal buds (topping) and control of the formation of axillary shoots (suckers) are common agronomic practices that significantly impact the yield and quality of various crop plants. Application of chemicals (suckercides) to plants following topping is an effective method for sucker control. However, our current knowledge of the influence of topping, and subsequent suckercide applications, to gene expression is limited. We analyzed the differential gene expression using RNA-sequencing in tobacco (Nicotiana tabacum) that are topped, or treated after topping by two different suckercides, the contact-localized-systemic, Flupro(®) (FP), and contact, Off-Shoot-T(®). Among the differentially expressed genes (DEGs), 179 were identified as common to all three conditions. DEGs, largely related to wounding, phytohormone metabolism and secondary metabolite biosynthesis, exhibited significant upregulation following topping, and downregulation after suckercide treatments. DEGs related to photosynthetic processes were repressed following topping and suckercide treatments. Moreover, topping and FP-treatment affect the expression of auxin and cytokinin signaling pathway genes that are possibly involved in axillary shoot formation. Our results provide insights into the global change of plant gene expression in response to topping and suckercide treatments. The regulatory elements of topping-inducible genes are potentially useful for the development of a chemical-free sucker control system.
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http://dx.doi.org/10.1038/srep18148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4680964PMC
December 2015

Small tandem target mimic-mediated blockage of microRNA858 induces anthocyanin accumulation in tomato.

Planta 2015 Jul 28;242(1):283-93. Epub 2015 Apr 28.

College of Life Science, Shanxi Agricultural University, Taigu, 030801, Shanxi, China.

Main Conclusion: Our work strongly suggests that microRNA858 regulates anthocyanin biosynthesis in tomato by modulating the expression of two R2R3 MYB transcription factors, underscoring the importance of microRNAs in the gene regulatory network controlling specialized metabolism in plants. The biological functions of microRNA858 (miR858), a recently identified small RNA, are not well understood. Here, we identified miR858 as a negative regulator of anthocyanin biosynthesis in tomato (Solanum lycopersicum). RNA ligase-mediated 5'RACE cleavage assay showed that miR858 mediates the cleavage of SlMYB7-like and SlMYB48-like transcripts in tomato. Expression analysis revealed an inverse correlation between the accumulation of miR858 and its target SlMYB7-like mRNA, in different tissues of tomato. Subsequently, a small tandem target mimic construct for blocking miR858 (STTM858) was generated and transformed into tomato. The majority of endogenous miR858 was blocked in STTM858 over-expressing tomato plants, whereas SlMYB7-like transcripts increased significantly. Concomitantly, upregulated expression was detected for several anthocyanin biosynthetic genes, including PAL, CHS, DFR, ANS and 3GT. As a result, anthocyanins were highly accumulated in young seedlings, leaves, stems and leaf buds of the transgenic plants under normal growth conditions. In addition, over-expression of STTM858 in tomato activated another MYB transcription factor, SlMYB48, implicating the possible involvement of SlMYB48 in anthocyanin biosynthesis.
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http://dx.doi.org/10.1007/s00425-015-2305-5DOI Listing
July 2015

An overview of the gene regulatory network controlling trichome development in the model plant, Arabidopsis.

Front Plant Sci 2014 5;5:259. Epub 2014 Jun 5.

Department of Plant and Soil Sciences, Kentucky Tobacco Research and Development Center, University of Kentucky Lexington, KY, USA.

Trichomes are specialized epidermal cells located on aerial parts of plants and are associated with a wide array of biological processes. Trichomes protect plants from adverse conditions including UV light and herbivore attack and are also an important source of a number of phytochemicals. The simple unicellular trichomes of Arabidopsis serve as an excellent model to study molecular mechanism of cell differentiation and pattern formation in plants. The emerging picture suggests that the developmental process is controlled by a transcriptional network involving three major groups of transcription factors (TFs): the R2R3 MYB, basic helix-loop-helix (bHLH), and WD40 repeat (WDR) protein. These regulatory proteins form a trimeric activator complex that positively regulates trichome development. The single repeat R3 MYBs act as negative regulators of trichome development. They compete with the R2R3 MYBs to bind the bHLH factor and form a repressor complex. In addition to activator-repressor mechanism, a depletion mechanism may operate in parallel during trichome development. In this mechanism, the bHLH factor traps the WDR protein which results in depletion of WDR protein in neighboring cells. Consequently, the cells with high levels of bHLH and WDR proteins are developed into trichomes. A group of C2H2 zinc finger TFs has also been implicated in trichome development. Phytohormones, including gibberellins and jasmonic acid, play significant roles in this developmental process. Recently, microRNAs have been shown to be involved in trichome development. Furthermore, it has been demonstrated that the activities of the key regulatory proteins involved in trichome development are controlled by the 26S/ubiquitin proteasome system (UPS), highlighting the complexity of the regulatory network controlling this developmental process. To complement several excellent recent relevant reviews, this review focuses on the transcriptional network and hormonal interplay controlling trichome development in Arabidopsis.
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http://dx.doi.org/10.3389/fpls.2014.00259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4071814PMC
July 2014

Analyses of Catharanthus roseus and Arabidopsis thaliana WRKY transcription factors reveal involvement in jasmonate signaling.

BMC Genomics 2014 Jun 20;15:502. Epub 2014 Jun 20.

Department of Plant and Soil Science, University of Kentucky, Lexington, KY 40546, USA.

Background: To combat infection to biotic stress plants elicit the biosynthesis of numerous natural products, many of which are valuable pharmaceutical compounds. Jasmonate is a central regulator of defense response to pathogens and accumulation of specialized metabolites. Catharanthus roseus produces a large number of terpenoid indole alkaloids (TIAs) and is an excellent model for understanding the regulation of this class of valuable compounds. Recent work illustrates a possible role for the Catharanthus WRKY transcription factors (TFs) in regulating TIA biosynthesis. In Arabidopsis and other plants, the WRKY TF family is also shown to play important role in controlling tolerance to biotic and abiotic stresses, as well as secondary metabolism.

Results: Here, we describe the WRKY TF families in response to jasmonate in Arabidopsis and Catharanthus. Publically available Arabidopsis microarrays revealed at least 30% (22 of 72) of WRKY TFs respond to jasmonate treatments. Microarray analysis identified at least six jasmonate responsive Arabidopsis WRKY genes (AtWRKY7, AtWRKY20, AtWRKY26, AtWRKY45, AtWRKY48, and AtWRKY72) that have not been previously reported. The Catharanthus WRKY TF family is comprised of at least 48 members. Phylogenetic clustering reveals 11 group I, 32 group II, and 5 group III WRKY TFs. Furthermore, we found that at least 25% (12 of 48) were jasmonate responsive, and 75% (9 of 12) of the jasmonate responsive CrWRKYs are orthologs of AtWRKYs known to be regulated by jasmonate.

Conclusion: Overall, the CrWRKY family, ascertained from transcriptome sequences, contains approximately 75% of the number of WRKYs found in other sequenced asterid species (pepper, tomato, potato, and bladderwort). Microarray and transcriptomic data indicate that expression of WRKY TFs in Arabidopsis and Catharanthus are under tight spatio-temporal and developmental control, and potentially have a significant role in jasmonate signaling. Profiling of CrWRKY expression in response to jasmonate treatment revealed potential associations with secondary metabolism. This study provides a foundation for further characterization of WRKY TFs in jasmonate responses and regulation of natural product biosynthesis.
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http://dx.doi.org/10.1186/1471-2164-15-502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4099484PMC
June 2014

Efficient chimeric plant promoters derived from plant infecting viral promoter sequences.

Planta 2014 Feb 1;239(2):381-96. Epub 2013 Nov 1.

Division of Gene Function and Regulation, Department of Biotechnology, Institute of Life Sciences, Government of India, Nalco Square, Chandrasekharpur, Bhubaneswar, 751 023, Odisha, India.

In the present study, we developed a set of three chimeric/hybrid promoters namely FSgt-PFlt, PFlt-UAS-2X and MSgt-PFlt incorporating different important domains of Figwort Mosaic Virus sub-genomic transcript promoter (FSgt, -270 to -60), Mirabilis Mosaic Virus sub-genomic transcript promoter (MSgt, -306 to -125) and Peanut Chlorotic Streak Caulimovirus full-length transcript promoter (PFlt-, -353 to +24 and PFlt-UAS, -353 to -49). We demonstrated that these chimeric/hybrid promoters can drive the expression of reporter genes in different plant species including tobacco, Arabidopsis, petunia, tomato and spinach. FSgt-PFlt, PFlt-UAS-2X and MSgt-PFlt promoters showed 4.2, 1.5 and 1.2 times stronger GUS activities compared to the activity of the CaMV35S promoter, respectively, in tobacco protoplasts. Protoplast-derived recombinant promoter driven GFP showed enhanced accumulation compared to that obtained under the CaMV35S promoter. FSgt-PFlt, PFlt-UAS-2X and MSgt-PFlt promoters showed 3.0, 1.3 and 1.0 times stronger activities than the activity of the CaMV35S² (a modified version of the CaMV35S promoter with double enhancer domain) promoter, respectively, in tobacco (Nicotiana tabacum, var. Samsun NN). Alongside, we observed a fair correlation between recombinant promoter-driven GUS accumulation with the corresponding uidA-mRNA level in transgenic tobacco. Histochemical (X-gluc) staining of whole transgenic seedlings and fluorescence images of ImaGene Green™ treated floral parts expressing the GUS under the control of recombinant promoters also support above findings. Furthermore, we confirmed that these chimeric promoters are inducible in the presence of 150 μM salicylic acid (SA) and abscisic acid (ABA). Taken altogether, we propose that SA/ABA inducible chimeric/recombinant promoters could be used for strong expression of gene(s) of interest in crop plants.
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http://dx.doi.org/10.1007/s00425-013-1973-2DOI Listing
February 2014

Transcriptional regulation of secondary metabolite biosynthesis in plants.

Biochim Biophys Acta 2013 Nov 7;1829(11):1236-47. Epub 2013 Oct 7.

Department of Plant and Soil Sciences, Kentucky Tobacco Research and Development Center, University of Kentucky, Lexington, KY 40546, USA.

Plants produce thousands of secondary metabolites (a.k.a. specialized metabolites) of diverse chemical nature. These compounds play important roles in protecting plants under adverse conditions. Many secondary metabolites are valued for their pharmaceutical properties. Because of their beneficial effects to health, biosynthesis of secondary metabolites has been a prime focus of research. Many transcription factors have been characterized for their roles in regulating biosynthetic pathways at the transcriptional level. The emerging picture of transcriptional regulation of secondary metabolite biosynthesis suggests that the expression of activators and repressors, in response to phytohormones and different environmental signals, forms a dynamic regulatory network that fine-tune the timing, amplitude and tissue specific expression of pathway genes and the subsequent accumulation of these compounds. Recent research has revealed that some metabolic pathways are also controlled by posttranscriptional and posttranslational mechanisms. This review will use recent developments in the biosynthesis of flavonoids, alkaloids and terpenoids to highlight the complexity of transcriptional regulation of secondary metabolite biosynthesis.
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http://dx.doi.org/10.1016/j.bbagrm.2013.09.006DOI Listing
November 2013

Promoter analysis reveals cis-regulatory motifs associated with the expression of the WRKY transcription factor CrWRKY1 in Catharanthus roseus.

Planta 2013 Dec 27;238(6):1039-49. Epub 2013 Aug 27.

Department of Plant and Soil Sciences, Kentucky Tobacco Research and Development Center, University of Kentucky, Lexington, KY, 40546, USA.

WRKY transcription factors (TFs) are emerging as an important group of regulators of plant secondary metabolism. However, the cis-regulatory elements associated with their regulation have not been well characterized. We have previously demonstrated that CrWRKY1, a member of subgroup III of the WRKY TF family, regulates biosynthesis of terpenoid indole alkaloids in the ornamental and medicinal plant, Catharanthus roseus. Here, we report the isolation and functional characterization of the CrWRKY1 promoter. In silico analysis of the promoter sequence reveals the presence of several potential TF binding motifs, indicating the involvement of additional TFs in the regulation of the TIA pathway. The CrWRKY1 promoter can drive the expression of a β-glucuronidase (GUS) reporter gene in native (C. roseus protoplasts and transgenic hairy roots) and heterologous (transgenic tobacco seedlings) systems. Analysis of 5'- or 3'-end deletions indicates that the sequence located between positions -140 to -93 bp and -3 to +113 bp, relative to the transcription start site, is critical for promoter activity. Mutation analysis shows that two overlapping as-1 elements and a CT-rich motif contribute significantly to promoter activity. The CrWRKY1 promoter is induced in response to methyl jasmonate (MJ) treatment and the promoter region between -230 and -93 bp contains a putative MJ-responsive element. The CrWRKY1 promoter can potentially be used as a tool to isolate novel TFs involved in the regulation of the TIA pathway.
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http://dx.doi.org/10.1007/s00425-013-1949-2DOI Listing
December 2013

A R2R3-MYB transcription factor from Epimedium sagittatum regulates the flavonoid biosynthetic pathway.

PLoS One 2013 1;8(8):e70778. Epub 2013 Aug 1.

Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan, Hubei, China.

Herba epimedii (Epimedium), a traditional Chinese medicine, has been widely used as a kidney tonic and antirheumatic medicine for thousands of years. The bioactive components in herba epimedii are mainly prenylated flavonol glycosides, end-products of the flavonoid pathway. Epimedium species are also used as garden plants due to the colorful flowers and leaves. Many R2R3-MYB transcription factors (TFs) have been identified to regulate the flavonoid and anthocyanin biosynthetic pathways. However, little is known about the R2R3-MYB TFs involved in regulation of the flavonoid pathway in Epimedium. Here, we reported the isolation and functional characterization of the first R2R3-MYB TF (EsMYBA1) from Epimedium sagittatum (Sieb. Et Zucc.) Maxim. Conserved domains and phylogenetic analysis showed that EsMYBA1 belonged to the subgroup 6 clade (anthocyanin-related MYB clade) of R2R3-MYB family, which includes Arabidopsis AtPAP1, apple MdMYB10 and legume MtLAP1. EsMYBA1 was preferentially expressed in leaves, especially in red leaves that contain higher content of anthocyanin. Alternative splicing of EsMYBA1 resulted in three transcripts and two of them encoded a MYB-related protein. Yeast two-hybrid and transient luciferase expression assay showed that EsMYBA1 can interact with several bHLH regulators of the flavonoid pathway and activate the promoters of dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS). In both transgenic tobacco and Arabidopsis, overexpression of EsMYBA1 induced strong anthocyanin accumulation in reproductive and/or vegetative tissues via up-regulation of the main flavonoid-related genes. Furthermore, transient expression of EsMYBA1 in E. sagittatum leaves by Agrobacterium infiltration also induced anthocyanin accumulation in the wounded area. This first functional characterization of R2R3-MYB TFs in Epimedium species will promote further studies of the flavonoid biosynthesis and regulation in medicinal plants.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0070778PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3731294PMC
March 2014

Proteolytic degradation of the flavonoid regulators, TRANSPARENT TESTA8 and TRANSPARENT TESTA GLABRA1, in Arabidopsis is mediated by the ubiquitin/26Sproteasome system.

Plant Signal Behav 2013 Oct;8(10):doi: 10.4161/psb.25901

Regulated proteolysis by the ubiquitin/26S proteasome system (UPS) has emerged as a major posttranslational control mechanism regulating transcription factor (TF) activity in plants. Anthocyanin biosynthesis in Arabidopsis is regulated by a ternary complex comprised of basic helix-loop-helix (bHLH), R2R3MYB and WD-repeat (WDR) proteins. The bHLH TF, TRAN SPAR ENT TESTA 8 (TT 8), and the WDR protein, TRAN SPAR ENT TESTA GLABRA 1 (TT G1), are essential for expression of late flavonoid biosynthesis genes. Previous studies have demonstrated that the turnover of several anthocyanin pathway regulators is controlled by the UPS. Here, we show that TT 8 and TT G1 are short-lived and targeted by the UPS for degradation. Our findings further extend our understanding of the role of the UPS in the regulation of anthocyanin biosynthesis in plants.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4091075PMC
http://dx.doi.org/10.4161/psb.25901DOI Listing
October 2013

Ubiquitin protein ligase 3 mediates the proteasomal degradation of GLABROUS 3 and ENHANCER OF GLABROUS 3, regulators of trichome development and flavonoid biosynthesis in Arabidopsis.

Plant J 2013 May 25;74(3):435-47. Epub 2013 Mar 25.

Kentucky Tobacco Research and Development Center, University of Kentucky, Lexington, KY 40546, USA.

Ubiquitin/26S proteasome (UPS)-dependent proteolysis of a variety of cellular proteins plays an essential role in many basic cellular processes. UPS impacts transcriptional regulation by controlling the stability, and thus the activity, of numerous transcription factors (TFs). In Arabidopsis, trichome development and flavonoid metabolism are intimately connected, and several TFs have been identified that simultaneously control both processes. Here we show that UPS-dependent proteolysis of two of these TFs, GLABROUS 3 (GL3) and ENHANCER OF GL3 (EGL3), is mediated by ubiquitin protein ligase 3 (UPL3). Cell-free degradation and in planta stabilization assays in the presence of MG132, an inhibitor of proteasome activity, demonstrated that the degradation of GL3 and EGL3 proteins is 26S UPS-dependent. Yeast- or protoplast-based two-hybrid and bimolecular fluorescent complementation assays showed that GL3 and EGL3 interact via their C-terminal domains with the N-terminal portion of UPL3. Moreover, both TFs are stabilized and show increased activities in a upl3 mutant background. Gene expression analyses revealed that UPL3 expression is negatively affected by mutation in the gl3 locus, but is moderately upregulated by the overexpression of GL3, suggesting the presence of a regulatory loop involving GL3 and UPL3. Our findings underscore the importance of post-translational controls in epidermal cell differentiation and flavonoid metabolism.
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http://dx.doi.org/10.1111/tpj.12132DOI Listing
May 2013

Regulatory switch enforced by basic helix-loop-helix and ACT-domain mediated dimerizations of the maize transcription factor R.

Proc Natl Acad Sci U S A 2012 Jul 9;109(30):E2091-7. Epub 2012 Jul 9.

Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY 40546, USA.

The maize R2R3-MYB regulator C1 cooperates with the basic helix-loop-helix (bHLH) factor R to activate the expression of anthocyanin biosynthetic genes coordinately. As is the case for other bHLH factors, R harbors several protein-protein interaction domains. Here we show that not the classical but rather a briefly extended R bHLH region forms homodimers that bind canonical G-box DNA motifs. This bHLH DNA-binding activity is abolished if the C-terminal ACT (aspartokinase, chorismate, and TyrA) domain is licensed to homodimerize. Then the bHLH remains in the monomeric form, allowing it to interact with R-interacting factor 1 (RIF1). In this configuration, the R-RIF1 complex is recruited to the promoters of a subset of anthocyanin biosynthetic genes, such as A1, through the interaction with its MYB partner C1. If, however, the ACT domain remains monomeric, the bHLH region dimerizes and binds to G-boxes present in several anthocyanin genes, such as Bz1. Our results provide a mechanism by which a dimerization domain in a bHLH factor behaves as a switch that permits distinct configurations of a regulatory complex to be tethered to different promoters. Such a combinatorial gene regulatory framework provides one mechanism by which genes lacking obviously conserved cis-regulatory elements are regulated coordinately.
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http://dx.doi.org/10.1073/pnas.1205513109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3409794PMC
July 2012

The transcription factor CrWRKY1 positively regulates the terpenoid indole alkaloid biosynthesis in Catharanthus roseus.

Plant Physiol 2011 Dec 11;157(4):2081-93. Epub 2011 Oct 11.

Department of Plant and Soil Sciences, University of Kentucky, Lexington, Kentucky 40546, USA.

Catharanthus roseus produces a large array of terpenoid indole alkaloids (TIAs) that are an important source of natural or semisynthetic anticancer drugs. The biosynthesis of TIAs is tissue specific and induced by certain phytohormones and fungal elicitors, indicating the involvement of a complex transcriptional control network. However, the transcriptional regulation of the TIA pathway is poorly understood. Here, we describe a C. roseus WRKY transcription factor, CrWRKY1, that is preferentially expressed in roots and induced by the phytohormones jasmonate, gibberellic acid, and ethylene. The overexpression of CrWRKY1 in C. roseus hairy roots up-regulated several key TIA pathway genes, especially Tryptophan Decarboxylase (TDC), as well as the transcriptional repressors ZCT1 (for zinc-finger C. roseus transcription factor 1), ZCT2, and ZCT3. However, CrWRKY1 overexpression repressed the transcriptional activators ORCA2, ORCA3, and CrMYC2. Overexpression of a dominant-repressive form of CrWRKY1, created by fusing the SRDX repressor domain to CrWRKY1, resulted in the down-regulation of TDC and ZCTs but the up-regulation of ORCA3 and CrMYC2. CrWRKY1 bound to the W box elements of the TDC promoter in electrophoretic mobility shift, yeast one-hybrid, and C. roseus protoplast assays. Up-regulation of TDC increased TDC activity, tryptamine concentration, and resistance to 4-methyl tryptophan inhibition of CrWRKY1 hairy roots. Compared with control roots, CrWRKY1 hairy roots accumulated up to 3-fold higher levels of serpentine. The preferential expression of CrWRKY1 in roots and its interaction with transcription factors including ORCA3, CrMYC2, and ZCTs may play a key role in determining the root-specific accumulation of serpentine in C. roseus plants.
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http://dx.doi.org/10.1104/pp.111.181834DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3327198PMC
December 2011

Directed evolution through DNA shuffling for the improvement and understanding of genes and promoters.

Methods Mol Biol 2011 ;754:325-42

Department of Plant and Soil Sciences, Kentucky Tobacco Research & Development Center, University of Kentucky, Lexington, KY, USA.

Unlike rational protein engineering, directed evolution provides an a priori approach toward the engineering of improved proteins and novel promoters. This minimally recursive technique builds upon small improvements by selecting and combining the best changes. Protein-protein/DNA interactions, catalytic efficiency, or resilience to inhibitors can be improved by thousands of times. By working within a subspace of homologous sequences, DNA shuffling recombines that subspace. Individuals are screened for a particular trait or two and selected for when they meet a set threshold. Here we explain basic principles to follow and provide procedures for the preparation, fragmentation, efficient size fractionation, and purification of parental material, as well as for the reassembly and rescue polymerase chain reactions (PCRs).
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http://dx.doi.org/10.1007/978-1-61779-154-3_19DOI Listing
December 2011

Bimolecular fluorescence complementation as a tool to study interactions of regulatory proteins in plant protoplasts.

Methods Mol Biol 2011 ;754:185-93

Department of Plant and Soil Sciences, Kentucky Tobacco Research & Development Center, University of Kentucky, Lexington, KY, USA.

Protein-protein interactions are an important aspect of the gene regulation process. The expression of a gene in response to certain stimuli, within a specific cell type or at a particular developmental stage, involves a complex network of interactions between different regulatory proteins and the cis-regulatory elements present in the promoter of the gene. A number of methods have been developed to study protein-protein interactions in vitro and in vivo in plant cells, one of which is bimolecular fluorescence complementation (BiFC). BiFC is a relatively simple technique based upon the reconstitution of a fluorescent protein. The interacting protein complex can be visualized directly in a living plant cell when two non-fluorescent fragments, of an otherwise fluorescent protein, are fused to proteins found within that complex. Interaction of tagged proteins brings the two non-fluorescent fragments into close proximity and reconstitutes the fluorescent protein. In addition, the subcellular location of an interacting protein complex in the cell can be simultaneously determined. Using this approach, we have successfully demonstrated a protein-protein interaction between a R2R3 MYB and a basic helix-loop-helix MYC transcription factor related to flavonoid biosynthetic pathway in tobacco protoplasts.
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http://dx.doi.org/10.1007/978-1-61779-154-3_10DOI Listing
December 2011

Flavonoid-related basic helix-loop-helix regulators, NtAn1a and NtAn1b, of tobacco have originated from two ancestors and are functionally active.

Planta 2011 Aug 12;234(2):363-75. Epub 2011 Apr 12.

College of Agronomy, Northwest A&F University, Yangling, 712100, Shaanxi, People's Republic of China.

The basic helix-loop-helix (bHLH) transcription factors (TFs) comprise one of the largest families of TFs involved in developmental and physiological processes in plants. Here, we describe the functional characterization of two bHLH TFs (NtAn1a and NtAn1b) isolated from tobacco (Nicotiana tabacum) flowers. NtAn1a and NtAn1b originate from two ancestors of tobacco, N. sylvestris and N. tomentosiformis, respectively. NtAn1a and NtAn1b share high sequence similarity with other known flavonoid-related bHLH TFs and are predominantly expressed in flowers. GUS expression driven by the NtAn1a promoter is consistent with NtAn1 transcript profile in tobacco flowers. Both NtAn1a and NtAn1b are transcriptional activators as demonstrated by transactivation assays using yeast cells and tobacco protoplasts. Ectopic expression of NtAn1a or NtAn1b enhances anthocyanin accumulation in tobacco flowers. In transgenic tobacco expressing NtAn1a or NtAn1b, both subsets of early and late flavonoid pathway genes were up-regulated. Yeast two-hybrid assays showed that NtAn1 proteins interact with the previously characterized R2R3-MYB TF, NtAn2. The NtAn1-NtAn2 complex activated the promoters of two key anthocyanin pathway genes, dihydroflavonol reductase and chalcone synthase. The promoter activation is severely repressed by dominant repressive forms of either NtAn1a or NtAn2, created by fusing the SRDX repressor domain to the TFs. Our results show that NtAn1 and NtAn2 act in concert to regulate the anthocyanin pathway in tobacco flowers and NtAn2 up-regulates NtAn1 gene expression.
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http://dx.doi.org/10.1007/s00425-011-1407-yDOI Listing
August 2011