Publications by authors named "Barunava Patra"

26 Publications

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The Pumilio RNA-binding protein APUM24 regulates seed maturation by fine-tuning the BPM-WRI1 module in Arabidopsis.

J Integr Plant Biol 2021 Mar 17. Epub 2021 Mar 17.

Guangdong Key Laboratory of Biotechnology for Plant Development, College of Life Sciences, South China Normal University, Guangzhou, 510631, China.

Pumilio RNA-binding proteins participate in mRNA degradation and translational repression, but their roles in plant development are largely unclear. Here, we show that Arabidopsis PUMILIO PROTEIN24 (APUM24), an atypical Pumilio-homology domain-containing protein, plays an important part in regulating seed maturation, a major stage of plant development. APUM24 is strongly expressed in maturing seeds. Reducing APUM24 expression resulted in abnormal seed maturation, wrinkled seeds, and lower seed oil contents, and APUM24 knockdown resulted in lower levels of WRINKLED 1 (WRI1), a key transcription factor controlling seed oil accumulation, and lower expression of WRI1 target genes. APUM24 reduces the mRNA stability of BTB/POZMATH (BPM) family genes, thus decreasing BPM protein levels. BPM is responsible for the 26S proteasome-mediated degradation of WRI1 and has important functions in plant growth and development. The 3'UTRs of BPM family genes contain putative Pumilio response elements (PREs), which are bound by APUM24. Reduced BPM or increased WRI1 expression rescued the deficient seed maturation of apum24-2 knockdown mutants, and APUM24 overexpression resulted in increased seed size and weight. Therefore, APUM24 is crucial to seed maturation through its action as a positive regulator fine-tuning the BPM-WRI1 module, making APUM24 a promising target for breeding strategies to increase crop yields. This article is protected by copyright. All rights reserved.
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http://dx.doi.org/10.1111/jipb.13092DOI Listing
March 2021

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

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

VvWRKY8 represses stilbene synthase genes through direct interaction with VvMYB14 to control resveratrol biosynthesis in grapevine.

J Exp Bot 2019 01;70(2):715-729

Beijing Key Laboratory of Grape Sciences and Enology, CAS Key Laboratory of Plant Resources, Institute of Botany, Chinese Academy of Sciences, Beijing, China.

Resveratrol (Res) is a stilbenoid, a group of plant phenolic metabolites derived from stilbene that possess activities against pests, pathogens, and abiotic stresses. Only a few species, including grapevine (Vitis), synthesize and accumulate Res. Although stilbene synthases (STSs) have been isolated and characterized in several species, the gene regulatory mechanisms underlying stilbene biosynthesis are still largely unknown. Here, we characterize a grapevine WRKY transcription factor, VvWRKY8, that regulates the Res biosynthetic pathway. Transient and stable overexpression of VvWRKY8 in grapevine results in decreased expression of VvSTS15/21 and VvMYB14, as well as in a reduction of Res accumulation. VvWRKY8 does not bind to or activate the promoters of VvMYB14 and VvSTS15/21; however, it physically interacts with VvMYB14 proteins through their N-terminal domains to prevent them from binding to the VvSTS15/21 promoter. Application of exogenous Res results in the stimulation of VvWRKY8 expression and in a decrease of VvMYB14 and VvSTS15/21 expression in grapevine suspension cells, and in the activation of the VvWRKY8 promoter in tobacco leaves. These results demonstrate that VvWRKY8 represses VvSTS15/21 expression and Res biosynthesis through interaction with VvMYB14. In this context, the VvMYB14-VvSTS15/21-Res-VvWRKY8 regulatory loop may be an important mechanism for the fine-tuning of Res biosynthesis in grapevine.
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http://dx.doi.org/10.1093/jxb/ery401DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6322584PMC
January 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

Isolation and characterization of a salt stress-responsive betaine aldehyde dehydrogenase in Lycium ruthenicum Murr.

Physiol Plant 2018 May 18;163(1):73-87. Epub 2018 Jan 18.

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

As compatible solute, glycine betaine (GB) plays a significant role in salinity tolerance in GB accumulating plants. Solanaceous crops such as tomato (Solanum lycopersicum) and tobacco (Nicotiana tabacum) are salt sensitive and naturally GB non-accumulators. In Solanaceae, only the Lycium genus has been recorded as halophytes in China, and several Lycium species have been reported as GB accumulators. The last biosynthetic step of GB is catalyzed by aminoaldehyde dehydrogenase (AMADH) with betaine aldehyde dehydrogenase (BADH) activities. Failure of GB synthesis in tomato and tobacco was attributed to lack of BADH activity. Here, by comparing the BADH functional residues of AMADHs between the Lycium genus and solanaceous crops, we predict that all studied AMADH1s have low BADH activities while only LbAMADH2 from L. barbarum has high BADH activity. For two AMADHs in L. ruthenicum, results from substrate enzyme assays confirmed low BADH activity of LrAMADH1 and no BADH activity of LrAMADH2. Despite the very low GB contents in L. ruthenicum seedlings (< 0.5 μmol g fresh weight), GB contents in fruits are up to 150 μmol g FW, inferring fruits of L. ruthenicum as good GB sources. In NaCl treated seedlings, accompanied by elevated GB accumulation, expression of LrAMADH1 was up-regulated, indicating response of LrAMADH1 to salt stress in L. ruthenicum. Virus-induced silence of LrAMADH1 leads to less GB accumulation than control, revealing that LrAMADH1 participates in GB synthesis in planta. Collectively, our results show that LrAMADH1 is the bona fide BADH, which responds to salt stress in L. ruthenicum.
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http://dx.doi.org/10.1111/ppl.12669DOI Listing
May 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

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

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

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

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

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

Enhanced salt tolerance of transgenic tobacco plants by co-expression of PcINO1 and McIMT1 is accompanied by increased level of myo-inositol and methylated inositol.

Protoplasma 2010 Sep 4;245(1-4):143-52. Epub 2010 Jun 4.

Plant Molecular and Cellular Genetics, Bose Institute (Centenary Building), P1/12, C.I.T Scheme-VIIM, Kolkata, 700054, India.

Introgression and functional expression of either the PcINO1 (L: -myo-inositol 1-phosphate synthase or MIPS coding gene from the wild halophytic rice, Porteresia coarctata) or McIMTI (inositol methyl transferase, IMTI coding gene from common ice plant Mesembryanthemum crystallinum) has earlier been shown to confer salt tolerance to transgenic tobacco plants (Sheveleva et al., Plant Physiol 115:1211-1219, 1997; Majee et al., J Biol Chem 279:28539-28552, 2004). In this communication, we show that transgenic tobacco plants co-expressing PcINO1 and McIMT1 gene either in cytosol or in chloroplasts accumulate higher amount of total inositol (free and methyl inositol) compared to non-transgenic plants. These transgenic plants were more competent in terms of growth potential and photosynthetic activity and were less prone to oxidative stress under salt stress. A positive correlation between the elevated level of total inositol and methylated inositol and the capability of the double transgenic plants to withstand a higher degree of salt stress compared to the plants expressing either PcINO1 or McIMT1 alone is inferred.
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http://dx.doi.org/10.1007/s00709-010-0163-3DOI Listing
September 2010

Identification and organization of chloroplastic and cytosolic L-myo-inositol 1-phosphate synthase coding gene(s) in Oryza sativa: comparison with the wild halophytic rice, Porteresia coarctata.

Planta 2010 Apr 6;231(5):1211-27. Epub 2010 Mar 6.

Plant Molecular and Cellular Genetics, Bose Institute (Centenary Campus), Kolkata, India.

The gene coding for rice chloroplastic L-myo-inositol-1-phosphate synthase (MIPS; EC 5.5.1.4) has been identified by matrix-assisted laser desorption time-of-flight mass spectrometry analysis of the purified and immunologically cross-reactive approximately 60 kDa chloroplastic protein following two-dimensional polyacrylamide gel electrophoresis, which exhibited sequence identity with the cytosolic MIPS coded by OsINO1-1 gene. A possible chloroplastic transit peptide sequence was identified upstream of the OsINO1-1 gene upon analysis of rice genome. RT-PCR and confocal microscope studies confirmed transcription, effective translation and its functioning as a chloroplast transit peptide. Bioinformatic analysis mapped the chloroplastic MIPS (OsINO1-1) gene on chromosome 3, and a second MIPS gene (OsINO1-2) on chromosome 10 which lacks conventional chloroplast transit peptide sequence as in OsINO1-1. Two new PcINO1 genes, with characteristic promoter activity and upstream cis-elements were identified and cloned, but whether these proteins can be translocated to the chloroplast or not is yet to be ascertained. Electrophoretic mobility shift assay carried out with nuclear extract of Porteresia coarctata leaves grown under both control and stressed condition shows binding of nuclear proteins with the upstream elements. Nucleotide divergence among the different Oryza and Porteresia INO1 genes were calculated and compared.
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http://dx.doi.org/10.1007/s00425-010-1127-8DOI Listing
April 2010

Isolation and functional characterization of a floral tissue-specific R2R3 MYB regulator from tobacco.

Planta 2010 Apr 16;231(5):1061-76. Epub 2010 Feb 16.

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

Tobacco is a commonly used heterologous system for studying combinatorial regulation of the flavonoid biosynthetic pathway by the bHLH-MYB transcription factor (TF) complex in plants. However, little is known about the endogenous tobacco bHLH and MYB TFs involved in the pathway. Ectopic expression in tobacco of heterologous bHLH TF genes, such as maize Lc, leads to increased anthocyanin production in the reproductive tissues, suggesting the presence of a reproductive tissue-specific MYB TF that interacts with the Lc-like bHLH TFs. We isolated a gene (NtAn2) encoding a R2R3 MYB TF from developing tobacco flowers. NtAn2 shares high sequence homology with other known flavonoid-related MYB TFs and is mostly expressed in developing flowers. Constitutive ectopic expression of NtAn2 induces whole-plant anthocyanin production in tobacco and Arabidopsis. In transgenic tobacco and Arabidopsis expressing NtAn2, both subsets of early and late flavonoid pathway genes are up-regulated. Suppression of NtAn2 by RNAi in tobacco resulted in a white-flowered phenotype and the inhibition of the late pathway genes. Yeast two-hybrid assays demonstrated that NtAn2 can interact with five heterologous bHLH TFs known to induce anthocyanin synthesis in other species including maize, perilla, snapdragon and Arabidopsis. Bimolecular fluorescent complementation using split YFP demonstrated that NtAn2 interacts with Lc in tobacco cells and that the complex is localized to nuclei. Transient co-expression of NtAn2 and Lc or Arabidopsis TT8 in tobacco protoplasts activated the promoters of two key flavonoid pathway genes, chalcone synthase and dihydroflavonol reductase. These results suggest that NtAn2 is a key gene controlling anthocyanin production in reproductive tissues of tobacco.
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http://dx.doi.org/10.1007/s00425-010-1108-yDOI Listing
April 2010

Inositol methyl tranferase from a halophytic wild rice, Porteresia coarctata Roxb. (Tateoka): regulation of pinitol synthesis under abiotic stress.

Plant Cell Environ 2008 Oct 14;31(10):1442-59. Epub 2008 Jul 14.

Plant Molecular and Cellular Genetics, Bose Institute, CIT Scheme VIIM, Kolkata 700054, India.

Methylated inositol D-pinitol (3-O-methyl-D-chiro-inositol) accumulates in a number of plants naturally or in response to stress. Here, we present evidence for accumulation and salt-enhanced synthesis of pinitol in Porteresia coarctata, a halophytic wild rice, in contrast to its absence in domesticated rice. A cDNA for Porteresia coarctata inositol methyl transferase 1 (PcIMT1), coding for the inositol methyl transferase implicated in the synthesis of pinitol has been cloned from P. coarctata, bacterially overexpressed and shown to be functional in vitro. In silico analysis confirms the absence of an IMT1 homolog in Oryza genome, and PcIMT1 is identified as phylogenetically remotely related to the methyl transferase gene family in rice. Both transcript and proteomic analysis show the up-regulation of PcIMT1 expression following exposure to salinity. Coordinated expression of L-myo-inositol 1-phosphate synthase (PcINO1) gene along with PcIMT1 indicates that in P. coarctata, accumulation of pinitol via inositol is a stress-regulated pathway. The presence of pinitol synthesizing protein/gene in a wild halophytic rice is remarkable, although its exact role in salt tolerance of P. coarctata cannot be currently ascertained. The enhanced synthesis of pinitol in Porteresia under stress may be one of the adaptive features employed by the plant in addition to its known salt-exclusion mechanism.
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http://dx.doi.org/10.1111/j.1365-3040.2008.01850.xDOI Listing
October 2008

Functional identification of sll1383 from Synechocystis sp PCC 6803 as L-myo-inositol 1-phosphate phosphatase (EC 3.1.3.25): molecular cloning, expression and characterization.

Planta 2007 May 23;225(6):1547-58. Epub 2006 Nov 23.

Plant Molecular and Cellular Genetics, Bose Institute, P-1/12. CIT Scheme VII M, Kolkata, 700054, India.

The genome sequence of the cyanobacterium Synechocystis sp. PCC6803 revealed four Open reading frame (ORF) encoding putative inositol monophosphatase or inositol monophosphatase-like proteins. One of the ORFs, sll1383, is approximately 870 base pair long and has been assigned as a probable myo-inositol 1 (or 4) monophosphatase (IMPase; EC 3.1.3.25). IMPase is the second enzyme in the inositol biosynthesis pathway and catalyses the conversion of L-myo-inositol 1-phosphate to free myo-inositol. The present work describes the functional assignment of ORF sll1383 as myo-inositol 1-phosphate phosphatase (IMPase) through molecular cloning, bacterial overexpression, purification and biochemical characterization of the gene product. Affinity (K (m)) of the recombinant protein for the substrate DL-myo-inositol 1-phosphate was found to be much higher (0.0034 +/- 0.0003 mM) compared to IMPase(s) from other sources but in comparison V (max) ( approximately 0.033 mumol Pi/min/mg protein) was low. Li(+) was found to be an inhibitor (IC(50) 6.0 mM) of this enzyme, other monovalent metal ions (e.g. Na(+), K(+) NH (4) (+) ) having no significant effect on the enzyme activity. Like other IMPase(s), the activity of this enzyme was found to be totally Mg(2+) dependent, which can be substituted partially by Mn(2+). However, unlike other IMPase(s), the enzyme is optimally active at approximately 42 degrees C. To the best of our knowledge, sll1383 encoded IMPase has the highest substrate affinity and specificity amongst the known examples from other prokaryotic sources. A possible application of this recombinant protein in the enzymatic coupled assay of L-myo-inositol 1-phosphate synthase (MIPS) is discussed.
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http://dx.doi.org/10.1007/s00425-006-0441-7DOI Listing
May 2007