Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments

Nour-Eldin, Hussam Hassan; Hansen, Bertel; Nørholm, Morten H. H.; Jensen, Jacob Krüger; Halkier, Barbara Ann

doi:10.1093/nar/gkl635

Cited by 445 publications

(452 citation statements)

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“…The cDNA of SLAC1, CPK3, 6, 21, 23, 31, ABI1, ABI2, HAB1, and HAB2 were cloned into oocyte (BIFC) expression vectors by an advanced uracil-excision-based cloning technique described by Nour-Eldin et al (41). Oocyte preparation and cRNA generation and injection have been described elsewhere (42).…”

Section: Methodsmentioning

confidence: 99%

Guard cell anion channel SLAC1 is regulated by CDPK protein kinases with distinct Ca ²⁺ affinities

Geiger¹,

Scherzer²,

Mumm³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

507

563

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In response to drought stress, the phytohormone abscisic acid (ABA) induces stomatal closure. Thereby the stress hormone activates guard cell anion channels in a calcium-dependent, as well as -independent, manner. Open stomata 1 protein kinase (OST1) and ABI1 protein phosphatase (ABA insensitive 1) represent key components of calcium-independent ABA signaling. Recently, the guard cell anion channel SLAC1 was identified. When expressed heterologously SLAC1 remained electrically silent. Upon coexpression with Ca 2+ -independent OST1, however, SLAC1 anion channels appear activated in an ABI1-dependent manner. Mutants lacking distinct calcium-dependent protein kinases (CPKs) appeared impaired in ABA stimulation of guard cell ion channels, too. To study SLAC1 activation via the calcium-dependent ABA pathway, we studied the SLAC1 response to CPKs in the Xenopus laevis oocyte system. Split YFP-based protein-protein interaction assays, using SLAC1 as the bait, identified guard cell expressed CPK21 and 23 as major interacting partners. Upon coexpression of SLAC1 with CPK21 and 23, anion currents document SLAC1 stimulation by these guard cell protein kinases. Ca 2+ -sensitive activation of SLAC1, however, could be assigned to the CPK21 pathway only because CPK23 turned out to be rather Ca 2+ -insensitive. In line with activation by OST1, CPK activation of the guard cell anion channel was suppressed by ABI1. Thus the CPK and OST1 branch of ABA signal transduction in guard cells seem to converge on the level of SLAC1 under the control of the ABI1/ABA-receptor complex.abscisic acid signaling | drought stress | guard cell | S-type anion channel T he drought hormone abscisic acid (ABA) triggers release of K + and anions from guard cells and thereby causes stomatal closure (1, 2). Recently, SLAC1, a guard cell anion channel, was identified (3-5). In guard cells of these ABA-and CO 2 /O 3 -insensitive mutant plants, anion currents appeared largely suppressed. When SLAC1 was expressed with the open stomata 1 protein kinase (OST1) in Xenopus oocytes, SLAC1-related anion currents, similar to those observed in guard cells, appeared (6). The presence of ABI1, however, prevented SLAC1 activation. This ABA pathway resembles the Ca 2+ -independent activation of SLAC-type anion currents in guard cells. ABA signal transduction, however, has been shown to activate guard cell anion channels in a calcium-independent as well as -dependent manner (7-10). This became evident in abi1-1 mutant plants, where anion channels do not respond to ABA anymore (11) but still activate with calcium (12). Furthermore the described mutant growth controlled by abscisic acid (gca2) (13-14), isolated from the Arabidopsis ecotype Landsberg erecta, was shown to be impaired in ABA-induced stomatal closure in a Ca 2+ -dependent manner. Moreover [Ca 2+ ] cyt elevation was shown to result in activation of S-type anion channels via phosphorylation (12, 15), suggesting a role of phosphorylation events in [Ca 2+ ] cyt signaling.CDPKs resemble Ca 2+ -dependent Ser/Thr pr...

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Section: Methodsmentioning

confidence: 99%

Guard cell anion channel SLAC1 is regulated by CDPK protein kinases with distinct Ca ²⁺ affinities

Geiger¹,

Scherzer²,

Mumm³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

507

563

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“…Full-length CfTPS cDNAs were cloned into the Agrobacterium tumefaciens binary vector for plant transformation pCAMBIA1300_35Su with genespecific primers (Supplemental Table S1) by USER cloning described in Nour-Eldin et al (2006). Transient expression of CfTPS genes with the genesilencing suppressor p19 protein (Voinnet et al, 2003) in N. benthamiana leaves and extraction of diterpenes were performed as recently described (Spanner et al, 2013).…”

Section: Functional Characterization Of Cftps: Transient Expression Imentioning

confidence: 99%

Manoyl Oxide (13R), the Biosynthetic Precursor of Forskolin, Is Synthesized in Specialized Root Cork Cells in Coleus forskohlii

et al. 2014

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“…The PCR fragments of CYP76C1, CYP76C2, CYP76C5, CYP76C7, and CYP76B1 were integrated into the yeast expression vector pYeDP60. The constructs for CYP76C3, CYP76C4, CYP76C6, and CYP76B6 were prepared using the Uracil-Specific Excision Reagent (New England Biolabs) cloning technique according to Nour-Eldin et al (2006) and the PCR fragments were integrated into the yeast expression plasmid pYeDP60u2. For plant expression constructs, CYP76C1 was cloned similarly and integrated in the plant expression vector pCAMBIA2300u.…”

Section: Generation Of Expression Vectorsmentioning

confidence: 99%

Dual Function of the Cytochrome P450 CYP76 Family from Arabidopsis thaliana in the Metabolism of Monoterpenols and Phenylurea Herbicides

et al. 2014

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Comparative genomics analysis unravels lineage-specific bursts of gene duplications related to the emergence of specialized pathways. The CYP76C subfamily of cytochrome P450 enzymes is specific to Brassicaceae. Two of its members were recently associated with monoterpenol metabolism. This prompted us to investigate the CYP76C subfamily genetic and functional diversification. Our study revealed high rates of CYP76C gene duplication and loss in Brassicaceae, suggesting the association of the CYP76C subfamily with species-specific adaptive functions. Gene differential expression and enzyme functional specialization in Arabidopsis thaliana, including metabolism of different monoterpenols and formation of different products, support this hypothesis. In addition to linalool metabolism, CYP76C1, CYP76C2, and CYP76C4 metabolized herbicides belonging to the class of phenylurea. Their ectopic expression in the whole plant conferred herbicide tolerance. CYP76Cs from A. thaliana. thus provide a first example of promiscuous cytochrome P450 enzymes endowing effective metabolism of both natural and xenobiotic compounds. Our data also suggest that the CYP76C gene family provides a suitable genetic background for a quick evolution of herbicide resistance.Although extensive monoterpenol (especially linalool) oxidative metabolism has been described in many plant species, leading to fragrant and bioactive compounds as diverse as alcohols, aldehydes, acids, and epoxides

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Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments

Cited by 445 publications

References 22 publications

Guard cell anion channel SLAC1 is regulated by CDPK protein kinases with distinct Ca ²⁺ affinities

Guard cell anion channel SLAC1 is regulated by CDPK protein kinases with distinct Ca ²⁺ affinities

Manoyl Oxide (13R), the Biosynthetic Precursor of Forskolin, Is Synthesized in Specialized Root Cork Cells in Coleus forskohlii

Dual Function of the Cytochrome P450 CYP76 Family from Arabidopsis thaliana in the Metabolism of Monoterpenols and Phenylurea Herbicides

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Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments

Cited by 445 publications

References 22 publications

Guard cell anion channel SLAC1 is regulated by CDPK protein kinases with distinct Ca 2+ affinities

Guard cell anion channel SLAC1 is regulated by CDPK protein kinases with distinct Ca 2+ affinities

Manoyl Oxide (13R), the Biosynthetic Precursor of Forskolin, Is Synthesized in Specialized Root Cork Cells in Coleus forskohlii

Dual Function of the Cytochrome P450 CYP76 Family from Arabidopsis thaliana in the Metabolism of Monoterpenols and Phenylurea Herbicides

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Guard cell anion channel SLAC1 is regulated by CDPK protein kinases with distinct Ca ²⁺ affinities

Guard cell anion channel SLAC1 is regulated by CDPK protein kinases with distinct Ca ²⁺ affinities