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A cytochrome P450 monooxygenase commonly used for negative selection in transgenic plants causes growth anomalies by disrupting brassinosteroid signaling.

Dasgupta K, Ganesan S, Manivasagam S, Ayre BG - BMC Plant Biol. (2011)

Bottom Line: However, unexpected and prominent developmental aberrations resembling those described for mutants defective in brassinosteroid signaling were observed in many of the lines.Phenotype severity correlated with P450 SU1 transcript abundance, but not with transcript abundance of other experimental genes, strongly implicating CYP105A1 as responsible for the defects.We show that this gene can cause aberrant growth by disrupting brassinosteroid signaling and affecting homeostasis.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of North Texas, Department of Biological Sciences, 1155 Union Circle #305220, Denton, TX 76203-5017, USA.

ABSTRACT

Background: Cytochrome P450 monooxygenases form a large superfamily of enzymes that catalyze diverse reactions. The P450 SU1 gene from the soil bacteria Streptomyces griseolus encodes CYP105A1 which acts on various substrates including sulfonylurea herbicides, vitamin D, coumarins, and based on the work presented here, brassinosteroids. P450 SU1 is used as a negative-selection marker in plants because CYP105A1 converts the relatively benign sulfonyl urea pro-herbicide R7402 into a highly phytotoxic product. Consistent with its use for negative selection, transgenic Arabidopsis plants were generated with P450 SU1 situated between recognition sequences for FLP recombinase from yeast to select for recombinase-mediated excision. However, unexpected and prominent developmental aberrations resembling those described for mutants defective in brassinosteroid signaling were observed in many of the lines.

Results: The phenotypes of the most affected lines included severe stunting, leaf curling, darkened leaves characteristic of anthocyanin accumulation, delayed transition to flowering, low pollen and seed yields, and delayed senescence. Phenotype severity correlated with P450 SU1 transcript abundance, but not with transcript abundance of other experimental genes, strongly implicating CYP105A1 as responsible for the defects. Germination and seedling growth of transgenic and control lines in the presence and absence of 24-epibrassinolide indicated that CYP105A1 disrupts brassinosteroid signaling, most likely by inactivating brassinosteroids.

Conclusions: Despite prior use of this gene as a genetic tool, deleterious growth in the absence of R7402 has not been elaborated. We show that this gene can cause aberrant growth by disrupting brassinosteroid signaling and affecting homeostasis.

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Related in: MedlinePlus

Relationships between aberrant growths, represented as plant height, and AtSUC2 and P450SU1 transcript abundance. (A) OCP, WT, cSUC2, and uidA lines arranged by phenotype severity, with plant height of the indicated lines at full maturity (i.e., senescent and ready for seed harvesting), n = 6, variation is expressed as standard deviation. (B) Semi-quantitative RT-PCR of P450SU1 (black bars) and AtSUC2 (white bars) transcript levels relative to UBQ10 transcript, encoding ubiquitin, n = 3, variation is expressed as standard deviation. (C) Representative gel used to calculate transcript abundance. See Materials and Methods for details.
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Figure 2: Relationships between aberrant growths, represented as plant height, and AtSUC2 and P450SU1 transcript abundance. (A) OCP, WT, cSUC2, and uidA lines arranged by phenotype severity, with plant height of the indicated lines at full maturity (i.e., senescent and ready for seed harvesting), n = 6, variation is expressed as standard deviation. (B) Semi-quantitative RT-PCR of P450SU1 (black bars) and AtSUC2 (white bars) transcript levels relative to UBQ10 transcript, encoding ubiquitin, n = 3, variation is expressed as standard deviation. (C) Representative gel used to calculate transcript abundance. See Materials and Methods for details.

Mentions: The extent of the phenotype varied among OCP lines independently transformed with pART-P450-cSUC2-BAR and suggested a correlation with expression of one of the transgene: most likely P450SU1 but possibly AtSUC2. P450SU1 and AtSUC2 transcript levels were analyzed relative to UBQ10 transcripts (encoding ubiquitin) by semi-quantitative RT-PCR in 17 OCP lines, as well as in WT and cSUC2 lines, and those transformed with pART-uidA-BAR (uidA lines) (Figure 2). In Figure 2, the OCP lines were ranked by height for severity of phenotype in 50-day old plants and there is a strong correlation between P450SU1 transcript level and phenotype: Lines with the most severe phenotype had the highest levels of P450SU1 transcript while those with intermediate and no phenotype had lesser and no transcript, respectively (Figure 2). Conversely, AtSUC2 and cSUC2 transcript levels (the oligonucleotides used for qRT-PCR detect transcript from both) showed variation among lines with no obvious correlation to phenotype. These findings strongly suggest that expression levels of P450SU1, and thus levels of CYP105A1 protein, interfere with plant growth and development.


A cytochrome P450 monooxygenase commonly used for negative selection in transgenic plants causes growth anomalies by disrupting brassinosteroid signaling.

Dasgupta K, Ganesan S, Manivasagam S, Ayre BG - BMC Plant Biol. (2011)

Relationships between aberrant growths, represented as plant height, and AtSUC2 and P450SU1 transcript abundance. (A) OCP, WT, cSUC2, and uidA lines arranged by phenotype severity, with plant height of the indicated lines at full maturity (i.e., senescent and ready for seed harvesting), n = 6, variation is expressed as standard deviation. (B) Semi-quantitative RT-PCR of P450SU1 (black bars) and AtSUC2 (white bars) transcript levels relative to UBQ10 transcript, encoding ubiquitin, n = 3, variation is expressed as standard deviation. (C) Representative gel used to calculate transcript abundance. See Materials and Methods for details.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3094276&req=5

Figure 2: Relationships between aberrant growths, represented as plant height, and AtSUC2 and P450SU1 transcript abundance. (A) OCP, WT, cSUC2, and uidA lines arranged by phenotype severity, with plant height of the indicated lines at full maturity (i.e., senescent and ready for seed harvesting), n = 6, variation is expressed as standard deviation. (B) Semi-quantitative RT-PCR of P450SU1 (black bars) and AtSUC2 (white bars) transcript levels relative to UBQ10 transcript, encoding ubiquitin, n = 3, variation is expressed as standard deviation. (C) Representative gel used to calculate transcript abundance. See Materials and Methods for details.
Mentions: The extent of the phenotype varied among OCP lines independently transformed with pART-P450-cSUC2-BAR and suggested a correlation with expression of one of the transgene: most likely P450SU1 but possibly AtSUC2. P450SU1 and AtSUC2 transcript levels were analyzed relative to UBQ10 transcripts (encoding ubiquitin) by semi-quantitative RT-PCR in 17 OCP lines, as well as in WT and cSUC2 lines, and those transformed with pART-uidA-BAR (uidA lines) (Figure 2). In Figure 2, the OCP lines were ranked by height for severity of phenotype in 50-day old plants and there is a strong correlation between P450SU1 transcript level and phenotype: Lines with the most severe phenotype had the highest levels of P450SU1 transcript while those with intermediate and no phenotype had lesser and no transcript, respectively (Figure 2). Conversely, AtSUC2 and cSUC2 transcript levels (the oligonucleotides used for qRT-PCR detect transcript from both) showed variation among lines with no obvious correlation to phenotype. These findings strongly suggest that expression levels of P450SU1, and thus levels of CYP105A1 protein, interfere with plant growth and development.

Bottom Line: However, unexpected and prominent developmental aberrations resembling those described for mutants defective in brassinosteroid signaling were observed in many of the lines.Phenotype severity correlated with P450 SU1 transcript abundance, but not with transcript abundance of other experimental genes, strongly implicating CYP105A1 as responsible for the defects.We show that this gene can cause aberrant growth by disrupting brassinosteroid signaling and affecting homeostasis.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of North Texas, Department of Biological Sciences, 1155 Union Circle #305220, Denton, TX 76203-5017, USA.

ABSTRACT

Background: Cytochrome P450 monooxygenases form a large superfamily of enzymes that catalyze diverse reactions. The P450 SU1 gene from the soil bacteria Streptomyces griseolus encodes CYP105A1 which acts on various substrates including sulfonylurea herbicides, vitamin D, coumarins, and based on the work presented here, brassinosteroids. P450 SU1 is used as a negative-selection marker in plants because CYP105A1 converts the relatively benign sulfonyl urea pro-herbicide R7402 into a highly phytotoxic product. Consistent with its use for negative selection, transgenic Arabidopsis plants were generated with P450 SU1 situated between recognition sequences for FLP recombinase from yeast to select for recombinase-mediated excision. However, unexpected and prominent developmental aberrations resembling those described for mutants defective in brassinosteroid signaling were observed in many of the lines.

Results: The phenotypes of the most affected lines included severe stunting, leaf curling, darkened leaves characteristic of anthocyanin accumulation, delayed transition to flowering, low pollen and seed yields, and delayed senescence. Phenotype severity correlated with P450 SU1 transcript abundance, but not with transcript abundance of other experimental genes, strongly implicating CYP105A1 as responsible for the defects. Germination and seedling growth of transgenic and control lines in the presence and absence of 24-epibrassinolide indicated that CYP105A1 disrupts brassinosteroid signaling, most likely by inactivating brassinosteroids.

Conclusions: Despite prior use of this gene as a genetic tool, deleterious growth in the absence of R7402 has not been elaborated. We show that this gene can cause aberrant growth by disrupting brassinosteroid signaling and affecting homeostasis.

Show MeSH
Related in: MedlinePlus