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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

Delayed senescence in OCP lines relative to WT and cSUC2 lines. 60-day old representative plants of the indicated lines. Note the shortened internodes and lack of senescence among the OCP plants; OCP-1 still has active blooms. Scale bar is 5 cm.
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Figure 4: Delayed senescence in OCP lines relative to WT and cSUC2 lines. 60-day old representative plants of the indicated lines. Note the shortened internodes and lack of senescence among the OCP plants; OCP-1 still has active blooms. Scale bar is 5 cm.

Mentions: Having established a correlation between P450SU1 expression and phenotype, a more detailed analysis of OCP growth and development was conducted. Representative lines demonstrating severe, intermediate, and mild phenotypes were analyzed relative to WT, cSUC2 and uidA lines as controls. As shown in Table 1, the reproductive phase of the OCP lines was significantly delayed: Under long-day conditions, WT, cSUC2 and uidA lines had visible floral organs within 24-26 days while P450SU1 expression associated with delayed transition to flowering (Table 1). Plants overexpressing P450SU1 also had fewer siliques and individual siliques had fewer seeds, resulting in an overall lower seed yield (Figure 3A, B). To gain insight into why fecundity in OCP lines was compromised, scanning electron microscopy was used to analyze flower development. Most conspicuous was the near absence of pollen in severe OCP lines (Figure 3C, D), which may account partially or entirely for the reduced seed yield. Additionally, OCP lines had delayed senescence: 60-day old OCP plants had green leaves and siliques while WT and cSUC2 lines had completely senesced (Figure 4). Seed size was not affected but germination varied among the OCP lines whereas it was consistently high among WT, cSUC2, and uidA lines (data not shown).


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)

Delayed senescence in OCP lines relative to WT and cSUC2 lines. 60-day old representative plants of the indicated lines. Note the shortened internodes and lack of senescence among the OCP plants; OCP-1 still has active blooms. Scale bar is 5 cm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Delayed senescence in OCP lines relative to WT and cSUC2 lines. 60-day old representative plants of the indicated lines. Note the shortened internodes and lack of senescence among the OCP plants; OCP-1 still has active blooms. Scale bar is 5 cm.
Mentions: Having established a correlation between P450SU1 expression and phenotype, a more detailed analysis of OCP growth and development was conducted. Representative lines demonstrating severe, intermediate, and mild phenotypes were analyzed relative to WT, cSUC2 and uidA lines as controls. As shown in Table 1, the reproductive phase of the OCP lines was significantly delayed: Under long-day conditions, WT, cSUC2 and uidA lines had visible floral organs within 24-26 days while P450SU1 expression associated with delayed transition to flowering (Table 1). Plants overexpressing P450SU1 also had fewer siliques and individual siliques had fewer seeds, resulting in an overall lower seed yield (Figure 3A, B). To gain insight into why fecundity in OCP lines was compromised, scanning electron microscopy was used to analyze flower development. Most conspicuous was the near absence of pollen in severe OCP lines (Figure 3C, D), which may account partially or entirely for the reduced seed yield. Additionally, OCP lines had delayed senescence: 60-day old OCP plants had green leaves and siliques while WT and cSUC2 lines had completely senesced (Figure 4). Seed size was not affected but germination varied among the OCP lines whereas it was consistently high among WT, cSUC2, and uidA lines (data not shown).

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