BACKGROUND
cytochrome p <dig> monooxygenases  form a large superfamily composed of many genes from many organisms.
the reactions catalyzed by these enzymes are extremely diverse, but generally involve the transfer of one atom from molecular oxygen to a substrate and reduction of the other atom to form water at the expense of nadph or nadh  <cit> .
cyps are therefore classified as monooxygenases, but in addition to hydroxylation  <cit> , cyps can catalyze oxidation  <cit> , dealkylation  <cit> , deamination, dehalogenation and sulfoxide formation  <cit> .
arabidopsis thaliana has  <dig> predicted cyp genes  making it one of the largest gene families in higher plants.
the encoded enzymes participate in the anabolism or catabolism of membrane sterols, structural polymers, hormones and many secondary metabolites functioning as pigments, antioxidants and defense compounds.
cyp enzymes can also detoxify exogenous molecules such as pesticides and pollutants  <cit> .
cyp enzymes are important regulators of plant growth because they catalyze the synthesis or degradation of several hormones including gibberellins, auxin and brassinosteroids  <cit> .
brassinosteroids are key hormones involved in cell division and expansion, and are derived from the 30-carbon triterpenoid squalene  <cit> .
cyps are in this pathway converting squalene to the common membrane sterol campesterol, and also in the brassinosteroid-specific branch pathway that converts campesterol to brassinolide  <cit> .
specifically, the hydroxylations at c- <dig> and c- <dig> have been demonstrated to be catalyzed by cyp90b <dig>  encoded by dwarf <dig>   <cit>  and cyp90a <dig>  <cit> , encoded by constitutive photomorphogenesis and dwarfism , respectively, by genetic, biochemical, and molecular analyses in arabidopsis.
auxins also regulate many aspects of growth and development, and cyp79b <dig> and cyp83b <dig> participate in tryptophan-dependent indole acetic acid  synthesis  <cit> .
gibberellins  are tetracyclic diterpenoid compounds which play important roles in germination, stem elongation and reproductive development  <cit> .
gas are synthesized by a pathway involving three enzyme classes spanning different subcellular compartments  <cit> .
the steps of the pathway from ent-kaurene to ga <dig> are catalyzed by cyp88a and cyp701a family members, and cyp714d <dig> participates in ga deactivation  <cit> .
cyp enzymes are also involved in detoxifying exogenous molecules.
this is best studied in animal systems where cyps have significant pharmaceutical impact, but action against xenobiotics is also observed in bacteria, fungi and plants  <cit> .
in plants, commonly used herbicides such as prosulfuron, diclofop and chlortoluron can be detoxified by cyps.
in weeds, herbicide resistance can arise from elevated cyp activity, which is particularly problematic because it can increase resistance to a broad class of related molecules  <cit> .
in the case of the phenylurea herbicide, chlortoluron, cyp-mediated detoxification is achieved either by hydroxylation of the ring-methyl or by di-n-demethylation  <cit> .
in addition, cyp genes from other organisms have been used for engineering herbicide resistance in plants, as well as for developing new herbicides in conjunction with cognate antidote genes conferring resistance.
understanding and manipulating the association between herbicides and herbicide-resistance genes is therefore a prominent goal for agricultural biotechnology  <cit> .
the p450su <dig> gene from the soil bacteria streptomyces griseolus encodes an inducible cytochrome p <dig>  cyp105a <dig>  capable of metabolizing sulfonylurea herbicides via dealkylation  <cit> .
however, the activity of cyp105a <dig> also results in the metabolism of the sulfonylurea pro-herbicide 2-methylethyl- <dig>  3-dihydro-n-- <dig>  2-benzoisothiazole-7-sulfonamide- <dig>  1-dioxide  to a highly phytotoxic metabolite, such that plants expressing p450su <dig> are killed by r <dig> treatment at levels that are benign to plants without p450su <dig> expression.
this has allowed p450su <dig> to be used in conjunction with r <dig> as a negative-selection marker to select for plants that lack p450su <dig> as a transgene  <cit> .
negative selection markers like p450su <dig> are useful in experiments where selecting for the loss of genes linked to the marker is desired.
for example p450su <dig> has been used in ac/ds transposon-mediated mutagenesis screens to select for progeny in which the ac transposase gene had segregated away from the ds element, thereby ensuring that the location of the ds element was stable after the initial ac-mediated transposition event  <cit> .
in addition, negative-selection markers are commonly used in combination with site-specific recombinases and serve as a screening tool for selecting the desired recombinase-mediated excision event.
for example, to demonstrate the utility of the p450su1/r <dig> negative-selection system for crop plants and biotechnology, it was used to select transgenic barley in which the transgene of interest was retained, but the gene encoding antibiotic resistance was linked to p450su <dig> and lost by recombinase-mediated excision  <cit> .
the work reported in this study initiated as an effort to select for plants that had lost a cdna sequence encoding a suc/h+ symporter necessary for efficient suc transport through the phloem  <cit> .
the cdna for atsuc <dig> and p450su <dig> were placed between target sequences for saccharomyces cereviseae flp recombinase, with the intention of using r <dig> to select for efficient flp-mediated excision of the cassette.
however transgenic arabidopsis plants transformed with this construct displayed a range of aberrant growth phenotypes, with more extreme lines exhibiting dwarfing, rosettes with a distinctive spiral-growth habit, delayed transition to flowering, low pollen yields and fecundity, and delayed senescence.
these phenotypes have not been described in plants with altered atsuc <dig> expression but resemble those described for plants with disrupted brassinosteroid signaling  <cit> .
we describe experiments correlating the severity of the phenotypes with p450su <dig> expression levels and not atsuc <dig> expression levels, and report on further experiments indicating that cyp105a <dig> from s. griseolus disrupts brassinosteroid homeostasis in these transgenic plants.
RESULTS
arabidopsis lines overexpressing p450su <dig> show abnormal growth
the plasmid part-p450-csuc2-bar  was used to create transgenic plants with an excisable atsuc <dig> cdna  adjacent to the negative selection marker p450su <dig>  atsuc <dig> encodes the predominant suc/h+ symporter required for efficient phloem loading and transport, and plants harboring a homozygous mutation are severely debilitated  <cit> .
transgenic plants with an excisable csuc <dig> cassette would be a valuable research tool and alleviate some of the difficulties associated with null mutants.
the negative-selection gene p450su <dig> was incorporated into the excisable cassette as a marker for effective excision.
p450su <dig> encodes cyp105a <dig>  a cyp from streptomyces griseolus which converts the relatively benign pro-herbicide r <dig> into a highly phytotoxic product.
in the presence of r <dig>  whole plants or tissues expressing p450su <dig> die while those having lost the sequences retain viability  <cit> .
similarly, plasmids part-csuc2-bar and part-uida-bar  were used to create transgenic plants used as controls in the experiments.
growth aberrations on sterile media during selection on kanamycin and in potting mix were noted among a large proportion of independent t <dig> seedlings harboring part-p450-csuc2-bar .
in plants displaying the most severe phenotype, these aberrations included severe stunting, darker green and purplish leaves characteristic of anthocyanin accumulation, thicker leaves in the abaxial/adaxial orientation, delayed flowering, shortened inflorescence internodes, reduced apical dominance , and numerous unexpanded siliques with no or very few seeds.
in addition, plants with the most severe phenotype demonstrated counter-clockwise leaf curling that gave rosettes a distinctive 'twirled' appearance .
similar phenotypes were not observed in t <dig> plants  harboring part-csuc2-bar or part-uida-bar, or in any wt plants.
the two antibiotic genes, nptii and bar, are common markers that are present in all three t-dna sequences: they are unlikely to be responsible for the growth abnormalities observed in plants transformed with part-p450-csuc2-bar.
reduced or ectopic expression of genes encoding suc/h+ symporters can disrupt patterns of carbon partitioning and cause growth anomalies, such as stunting, anthocyanin accumulation, and low seed yield  <cit> .
however, growth aberrations were not observed among part-csuc2-bar plants , and altered carbon partitioning does not account for the full spectrum of phenotypes observed among part-p450-csuc2-bar plants.
p450su <dig> has been used as a negative-selection marker in tobacco, arabidopsis and barley  <cit> .
in barley, "striking morphological differences" were observed in transgenic plants compared to non-transgenic plants  <cit> .
however, elaboration of those differences was not provided, and no morphological changes are described for arabidopsis or tobacco.
transcript levels of p450su <dig> correlate with the aberrant phenotype
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 p450su <dig> but possibly atsuc <dig>  p450su <dig> and atsuc <dig> transcript levels were analyzed relative to ubq <dig> transcripts  by semi-quantitative rt-pcr in  <dig> ocp lines, as well as in wt and csuc <dig> lines, and those transformed with part-uida-bar  .
in figure  <dig>  the ocp lines were ranked by height for severity of phenotype in 50-day old plants and there is a strong correlation between p450su <dig> transcript level and phenotype: lines with the most severe phenotype had the highest levels of p450su <dig> transcript while those with intermediate and no phenotype had lesser and no transcript, respectively .
conversely, atsuc <dig> and csuc <dig> transcript levels  showed variation among lines with no obvious correlation to phenotype.
these findings strongly suggest that expression levels of p450su <dig>  and thus levels of cyp105a <dig> protein, interfere with plant growth and development.
over expression of p450su <dig> affects vegetative and reproductive growth
having established a correlation between p450su <dig> 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, csuc <dig> and uida lines as controls.
as shown in table  <dig>  the reproductive phase of the ocp lines was significantly delayed: under long-day conditions, wt, csuc <dig> and uida lines had visible floral organs within 24- <dig> days while p450su <dig> expression associated with delayed transition to flowering .
plants overexpressing p450su <dig> also had fewer siliques and individual siliques had fewer seeds, resulting in an overall lower seed yield .
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 , 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 csuc <dig> lines had completely senesced .
seed size was not affected but germination varied among the ocp lines whereas it was consistently high among wt, csuc <dig>  and uida lines .
data represents mean values ± standard deviation of  <dig> plants from different ocp and control lines.
a student's t-test, p <  <dig> , relative to wild type .
overexpression of p450su <dig> impacts brassinosteroid homeostasis
the morphological and developmental anomalies observed among ocp lines are characteristic of plants defective in brassinosteroid  synthesis and signaling.
plants defective in br synthesis and signaling display characteristic phenotypes that include severe stunting, darker color from anthocyanin accumulation, epinastic round leaves, delayed flowering, late senescence, reduced male fertility, and compromised germination  <cit> .
seedlings deficient in br signaling also undergo abnormal skotomorphogenesis  <cit> .
unlike the elongated hypocotyls, closed cotyledons and prominent apical hooks of wt arabidopsis seedlings germinated and grown in the dark, br-deficient seedlings exhibit short and thickened hypocotyls, open and expanded cotyledons, and the emergence of true leaves characteristic of the de-etiolation that occurs during photomorphogenesis  <cit> .
exogenous br can stimulate cell division and expansion and rescue biosynthetic mutants.
in wt plants, exogenous br can cause supraoptimal effects and result in abnormal development from chaotic growth  <cit> .
to test if p450su <dig> expression in the ocp lines affects br signaling, the impact of exogenous 24-epibrassinolide  on skotomorphogenesis was analyzed in dark grown seedlings.
in the absence of 24-epibl, severe ocp lines showed moderate reductions in hypocotyl elongation relative to less severe lines and controls .
in the presence of supraoptimal  <dig> μm 24-epibl, importantly, severe ocp lines showed no significant alteration in growth while wt and other control seedlings displayed substantial morphological disruptions including chaotic growth in hypocotyls and cotyledons  and generally shorter hypocotyls .
br levels are also known to impact root development.
mutants deficient in br or br signaling have shorter roots than wt and in the presence of supraoptimal exogenous br, root development can be severely impaired  <cit> .
root growth was measured in ocp and wt lines on vertically-oriented sterile media.
in the absence of exogenous 24-epibl, ocp lines had shorter roots than wt but this did not correlate strongly with the severity of the above-ground phenotype .
in the presence of  <dig> μm 24-epibl, the length of wt roots was reduced to 22% of roots grown in the absence of 24-epibl, whereas roots of the most severe ocp lines were reduced to only 65% to 75% relative to those grown without exogenous 24-epibl .
these findings that exogenous 24-epibl severely affects wt root and aerial growth, but has little impact on the most severe ocp lines, combined with a growth pattern that phenocopies br deficient mutants , strongly suggests that the cyp105a <dig> enzyme encoded by the p450su <dig> gene is affecting br homeostasis directly or indirectly.
overexpression of p450su <dig> does not impact gibberellin or auxin mediated growth characteristics
gibberellin and auxin metabolism are also impacted by cyp activity, and hypocotyl- and root-growth experiments were conducted to test if cyp105a <dig> visually affects growth responses to these hormones.
exogenous application of ga <dig> or iaa is known to modestly increase hypocotyl length of etiolated seedlings  <cit> .
this was observed in wild type and control plants, but the effect was identical among even the most severe ocp lines .
conversely, exogenous ga <dig> or iaa treatment is known to result in decreased root elongation in etiolated seedlings  <cit> .
in our experiments with  <dig> μm of either hormone, ocp and control lines showed identical extents of reduced root elongation .
these results show that p450su <dig> expression does not mitigate the influence of exogenous ga <dig> or iaa  as it did for exogenous 24-epibl , and argues that the cyp105a <dig> enzyme impacts br homeostasis, but not that of iaa or ga <dig>
discussion
this study initiated as an effort to create a vector system in which a cdna sequence of interest could be excised upon delivery or activation of a site-specific recombinase.
it was designed with dual selection for recombination.
after flp-mediated recombination at the frt sites, the positive selection marker bar  was to be activated by being placed adjacent to a camv 35s promoter  <cit>  and the negative selection marker p450su <dig> was to be inactivated by being excised from the genome along with the cdna of interest .
independent transgenic lines harboring this construct displayed a range of phenotypes with the most severe lines resembling plants with disrupted br synthesis or perception  <cit> .
this included stunted rosettes and inflorescences with short internodes and reduced apical dominance, thicker leaves with dark coloration characteristic of anthocyanin accumulation, leaf curling that gave rosettes a distinctive twirled appearance , reduced male fertility and seed yields , and delayed senescence .
the severity of these characteristics showed a high correlation with p450su <dig> expression levels , and on sterile media these lines showed the least response to supraoptimal levels of 24-epibl .
as controls, plants transformed with t-dna that retained the atsuc <dig> cdna but had p450su <dig> deleted were phenotypically normal, as were plants lacking both atsuc <dig> cdna and p450su <dig> and instead expressing uida encoding β-glucuronidase.
the combined results of  the close correlation between p450su <dig> expression and a phenotype resembling a deficiency in br synthesis or perception,  p450su <dig> expression mitigating the effects of exogenous 24-epibl, and  the process of eliminating other candidate genes indicate that the cyp105a <dig> enzyme is acting on exogenous br and affects endogenous br by altering br homoeostasis.
a t-dna construct harboring only p450su <dig> was not tested.
expression of p450su <dig> did not modify the growth of etiolated seedlings in the presence of iaa or ga <dig>  indicating that it does not act on these hormones .
p450su <dig> and the encoded enzyme cyp105a <dig> were originally identified from the soil bacterium streptomyces griseolus as being able to degrade sulfonylurea herbicides  <cit> .
in transgenic plants, cyp105a <dig> converted the relatively benign compound r <dig> into a highly phytotoxic herbicide and could thus be used for negative selection: plants or individual tissues expressing p450su <dig> were ablated by r <dig> application, while plants or tissues not expressing the gene were spared  <cit> .
p450su <dig> was used previously in several studies, but we are aware of only one were growth aberrations in the absence of r <dig> were noted.
specifically, koprek and colleagues  <cit>  compared the efficacy of p450su <dig> and the coda gene, which converts non-toxic 5-fluorocytosine to toxic 5-fluorouracil  <cit> , as negative-selection tools in transgenic barley.
the abstract of  <cit>  notes growth anomalies with p450su <dig> but did not elaborate, and the authors concluded that despite these anomalies, p450su <dig> along with r <dig> was suitable for negative selection among plants grown in soil.
based on our findings, the growth anomalies reported in barley  <cit>  are likely the result of perturbed brassinosteroid signaling.
there are several explanations as to why a link between p450su <dig> and growth aberrations from perturbed brassinosteroid signaling have not been reported.
first, the system is used for negative selection in conjunction with r <dig> and production of the phytotoxic byproduct results in rapid death of plants or tissues.
therefore, the effects of p450su <dig> in the absence of r <dig> are mild compared to the effects in the presence of r <dig>  second, since the system is used for negative selection, most attention has focused on characteristics of plants or tissues after loss of the gene by segregation, transposition, or recombination  <cit> .
third, in the unique vector system used here, a strong camv 35s promoter was placed upstream of a strong rubisco promoter , and this combination may result in expression levels higher than those obtained in studies where growth anomalies were not reported.
this is supported by the strong correlation between transcript abundance and phenotype severity.
lines with moderate to low p450su <dig> transcript levels displayed moderate to mild symptomology in the absence of r <dig>  but were still highly sensitive to r <dig> and suitable for negative selection .
in addition, cyp105a <dig> as used here is targeted to plastids  <cit>  and expression from a dual promoter system may overwhelm plastid targeting and result in more enzyme mislocalized to the cytosol for acting on brs.
potential mislocalization of plastid-targeted cyp105a <dig> was previously reported  <cit> .
the dual promoters may also explain discrepancies between the phenotypes of our most severe lines and mutants defective in br synthesis.
for example, in the cpd mutant which is disrupted in br synthesis, dark-grown seedlings show photomorphogenesis and have short, thickened hypocotyls  <cit>  but our most severe ocp line showed normal skotomorphogenesis and differed only moderately from wt.
the rubisco small subunit promoter is light-activated, and in dark-grown seedlings expression would have been minimal.
under these conditions, p450su <dig> expression from the more distal camv 35s promoter alone may have been insufficient to cause a more severe phenotype.
however, in the presence of 24-epibl, ocp seedlings likely had sufficient p450su <dig> expression to bring brassinosteroid levels into a range that allowed relatively normal development.
as described above, cyp105a <dig> metabolizes sulfonylurea herbicides by dealkylation.
sulfonylurea herbicides are agricultural soil additives, and the natural target and substrate specificity of cyp105a <dig> is not known.
in transgenic plants, cyp105a <dig> disrupts brassinosteroid homeostasis to give a phenotype, but the full range of potential substrates and the extent to which their levels are altered is not known.
work by others has shown that cyp105a <dig> can hydroxylate vitamin d <dig> and d <dig> at multiple positions  <cit>  and can catalyze the conversion of 7-ethoxycoumarin to 7-hydroxycoumarin by o-dealkylation  <cit> .
detoxification of sulfonylurea herbicides and n-dealkylation of the pro-herbicide r <dig> to produce a toxic metabolite are additional activities  <cit> , and collectively, these reactions suggest that cyp105a <dig> substrate selection and mode of action may be quite broad, but does not extend to iaa or ga <dig>
it is now apparent that the development of herbicide resistance in several weeds is the result of enhanced detoxification associated with elevated levels of cyp activity.
weeds with enhanced cyp-mediated detoxification can be difficult to control because resistance can develop against multiple, unrelated classes of herbicide  <cit> .
however, in the limited species that have been subjected to analysis, there is a fitness cost associated with elevated cyp levels: in the absence of the selective pressure imparted by the herbicide, herbicide-resistant varieties of lolium rigidum showed up to 30% reduced vitality relative to their herbicide-sensitive counterparts  <cit> .
the development of cyps from both plant and non-plant origins for engineering herbicide resistance in biotechnology has garnered substantial interest.
however, the reduced vitality of plants that have naturally developed resistance and the undesired effects on plant-hormone homeostasis observed here with overexpression of p450su <dig> highlight some of the potential deleterious outcomes that will need to be addressed for successful exploitation of this promising area.
CONCLUSIONS
the p450su <dig> gene from streptomyces griseolus has been used as a negative-selection marker in conjunction with the pro-herbicide r <dig> since plants expressing the gene are killed by r <dig> while those not expressing it retain viability.
however, in the absence of r <dig>  plants with high p450su <dig> expression show aberrant growth characteristic of defects in brassinosteroid synthesis and perception.
when exposed to supraoptimal exogenous brassinosteroids, the growth habit of these plants is relatively normal compared to wild type.
together, these results indicate that both endogenous and exogenous brassinosteroids are a target of the p450su <dig> encoded cyp105a <dig> monooxygenase.

 cytochrome p monooxygenases form a large superfamily composed of many genes from many organisms. the reactions catalyzed by these enzymes are extremely diverse, but generally involve the transfer of one atom from molecular oxygen to a substrate and reduction of the other atom to form water at the expense of nadph or nadh. cyps are therefore classified as monooxygenases, but in addition to hydroxylation, cyps can catalyze oxidation, dealkylation, deamination, dehalogenation and sulfoxide formation. arabidopsis thaliana has predicted cyp genes making it one of the largest gene families in higher plants. the encoded enzymes participate in the anabolism or catabolism of membrane sterols, structural polymers, hormones and many secondary metabolites functioning as pigments, antioxidants and defense compounds. cyp enzymes can also detoxify exogenous molecules such as pesticides and pollutants. cyp enzymes are important regulators of plant growth because they catalyze the synthesis or degradation of several hormones including gibberellins, auxin and brassinosteroids. brassinosteroids are key hormones involved in cell division and expansion, and are derived from the 30-carbon triterpenoid squalene. cyps are in this pathway converting squalene to the common membrane sterol campesterol, and also in the brassinosteroid-specific branch pathway that converts campesterol to brassinolide. specifically, the hydroxylations at c- and c- have been demonstrated to be catalyzed by cyp90b encoded by dwarf and cyp90a, encoded by constitutive photomorphogenesis and dwarfism, respectively, by genetic, biochemical, and molecular analyses in arabidopsis. auxins also regulate many aspects of growth and development, and cyp79b and cyp83b participate in tryptophan-dependent indole acetic acid synthesis. gibberellins are tetracyclic diterpenoid compounds which play important roles in germination, stem elongation and reproductive development. gas are synthesized by a pathway involving three enzyme classes spanning different subcellular compartments. the steps of the pathway from ent-kaurene to ga are catalyzed by cyp88a and cyp701a family members, and cyp714d participates in ga deactivation. cyp enzymes are also involved in detoxifying exogenous molecules. this is best studied in animal systems where cyps have significant pharmaceutical impact, but action against xenobiotics is also observed in bacteria, fungi and plants. in plants, commonly used herbicides such as prosulfuron, diclofop and chlortoluron can be detoxified by cyps. in weeds, herbicide resistance can arise from elevated cyp activity, which is particularly problematic because it can increase resistance to a broad class of related molecules. in the case of the phenylurea herbicide, chlortoluron, cyp-mediated detoxification is achieved either by hydroxylation of the ring-methyl or by di-n-demethylation. in addition, cyp genes from other organisms have been used for engineering herbicide resistance in plants, as well as for developing new herbicides in conjunction with cognate antidote genes conferring resistance. understanding and manipulating the association between herbicides and herbicide-resistance genes is therefore a prominent goal for agricultural biotechnology. the p450su gene from the soil bacteria streptomyces griseolus encodes an inducible cytochrome p cyp105a capable of metabolizing sulfonylurea herbicides via dealkylation. however, the activity of cyp105a also results in the metabolism of the sulfonylurea pro-herbicide 2-methylethyl- 3-dihydro-n-- 2-benzoisothiazole-7-sulfonamide- 1-dioxide to a highly phytotoxic metabolite, such that plants expressing p450su are killed by r treatment at levels that are benign to plants without p450su expression. this has allowed p450su to be used in conjunction with r as a negative-selection marker to select for plants that lack p450su as a transgene. negative selection markers like p450su are useful in experiments where selecting for the loss of genes linked to the marker is desired. for example p450su has been used in ac/ds transposon-mediated mutagenesis screens to select for progeny in which the ac transposase gene had segregated away from the ds element, thereby ensuring that the location of the ds element was stable after the initial ac-mediated transposition event. in addition, negative-selection markers are commonly used in combination with site-specific recombinases and serve as a screening tool for selecting the desired recombinase-mediated excision event. for example, to demonstrate the utility of the p450su1/r negative-selection system for crop plants and biotechnology, it was used to select transgenic barley in which the transgene of interest was retained, but the gene encoding antibiotic resistance was linked to p450su and lost by recombinase-mediated excision. the work reported in this study initiated as an effort to select for plants that had lost a cdna sequence encoding a suc/h+ symporter necessary for efficient suc transport through the phloem. the cdna for atsuc and p450su were placed between target sequences for saccharomyces cereviseae flp recombinase, with the intention of using r to select for efficient flp-mediated excision of the cassette. however transgenic arabidopsis plants transformed with this construct displayed a range of aberrant growth phenotypes, with more extreme lines exhibiting dwarfing, rosettes with a distinctive spiral-growth habit, delayed transition to flowering, low pollen yields and fecundity, and delayed senescence. these phenotypes have not been described in plants with altered atsuc expression but resemble those described for plants with disrupted brassinosteroid signaling. we describe experiments correlating the severity of the phenotypes with p450su expression levels and not atsuc expression levels, and report on further experiments indicating that cyp105a from s. griseolus disrupts brassinosteroid homeostasis in these transgenic plants.  arabidopsis lines overexpressing p450su show abnormal growth the plasmid part-p450-csuc2-bar was used to create transgenic plants with an excisable atsuc cdna adjacent to the negative selection marker p450su atsuc encodes the predominant suc/h+ symporter required for efficient phloem loading and transport, and plants harboring a homozygous mutation are severely debilitated. transgenic plants with an excisable csuc cassette would be a valuable research tool and alleviate some of the difficulties associated with null mutants. the negative-selection gene p450su was incorporated into the excisable cassette as a marker for effective excision. p450su encodes cyp105a a cyp from streptomyces griseolus which converts the relatively benign pro-herbicide r into a highly phytotoxic product. in the presence of r whole plants or tissues expressing p450su die while those having lost the sequences retain viability. similarly, plasmids part-csuc2-bar and part-uida-bar were used to create transgenic plants used as controls in the experiments. growth aberrations on sterile media during selection on kanamycin and in potting mix were noted among a large proportion of independent t seedlings harboring part-p450-csuc2-bar. in plants displaying the most severe phenotype, these aberrations included severe stunting, darker green and purplish leaves characteristic of anthocyanin accumulation, thicker leaves in the abaxial/adaxial orientation, delayed flowering, shortened inflorescence internodes, reduced apical dominance, and numerous unexpanded siliques with no or very few seeds. in addition, plants with the most severe phenotype demonstrated counter-clockwise leaf curling that gave rosettes a distinctive'twirled' appearance. similar phenotypes were not observed in t plants harboring part-csuc2-bar or part-uida-bar, or in any wt plants. the two antibiotic genes, nptii and bar, are common markers that are present in all three t-dna sequences: they are unlikely to be responsible for the growth abnormalities observed in plants transformed with part-p450-csuc2-bar. reduced or ectopic expression of genes encoding suc/h+ symporters can disrupt patterns of carbon partitioning and cause growth anomalies, such as stunting, anthocyanin accumulation, and low seed yield. however, growth aberrations were not observed among part-csuc2-bar plants, and altered carbon partitioning does not account for the full spectrum of phenotypes observed among part-p450-csuc2-bar plants. p450su has been used as a negative-selection marker in tobacco, arabidopsis and barley. in barley, "striking morphological differences" were observed in transgenic plants compared to non-transgenic plants. however, elaboration of those differences was not provided, and no morphological changes are described for arabidopsis or tobacco. transcript levels of p450su correlate with the aberrant phenotype 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 p450su but possibly atsuc p450su and atsuc transcript levels were analyzed relative to ubq transcripts by semi-quantitative rt-pcr in ocp lines, as well as in wt and csuc lines, and those transformed with part-uida-bar. in figure the ocp lines were ranked by height for severity of phenotype in 50-day old plants and there is a strong correlation between p450su transcript level and phenotype: lines with the most severe phenotype had the highest levels of p450su transcript while those with intermediate and no phenotype had lesser and no transcript, respectively. conversely, atsuc and csuc transcript levels showed variation among lines with no obvious correlation to phenotype. these findings strongly suggest that expression levels of p450su and thus levels of cyp105a protein, interfere with plant growth and development. over expression of p450su affects vegetative and reproductive growth having established a correlation between p450su 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, csuc and uida lines as controls. as shown in table the reproductive phase of the ocp lines was significantly delayed: under long-day conditions, wt, csuc and uida lines had visible floral organs within 24- days while p450su expression associated with delayed transition to flowering. plants overexpressing p450su also had fewer siliques and individual siliques had fewer seeds, resulting in an overall lower seed yield. 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, 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 csuc lines had completely senesced. seed size was not affected but germination varied among the ocp lines whereas it was consistently high among wt, csuc and uida lines. data represents mean values ± standard deviation of plants from different ocp and control lines. a student's t-test, p <, relative to wild type. overexpression of p450su impacts brassinosteroid homeostasis the morphological and developmental anomalies observed among ocp lines are characteristic of plants defective in brassinosteroid synthesis and signaling. plants defective in br synthesis and signaling display characteristic phenotypes that include severe stunting, darker color from anthocyanin accumulation, epinastic round leaves, delayed flowering, late senescence, reduced male fertility, and compromised germination. seedlings deficient in br signaling also undergo abnormal skotomorphogenesis. unlike the elongated hypocotyls, closed cotyledons and prominent apical hooks of wt arabidopsis seedlings germinated and grown in the dark, br-deficient seedlings exhibit short and thickened hypocotyls, open and expanded cotyledons, and the emergence of true leaves characteristic of the de-etiolation that occurs during photomorphogenesis. exogenous br can stimulate cell division and expansion and rescue biosynthetic mutants. in wt plants, exogenous br can cause supraoptimal effects and result in abnormal development from chaotic growth. to test if p450su expression in the ocp lines affects br signaling, the impact of exogenous 24-epibrassinolide on skotomorphogenesis was analyzed in dark grown seedlings. in the absence of 24-epibl, severe ocp lines showed moderate reductions in hypocotyl elongation relative to less severe lines and controls. in the presence of supraoptimal μm 24-epibl, importantly, severe ocp lines showed no significant alteration in growth while wt and other control seedlings displayed substantial morphological disruptions including chaotic growth in hypocotyls and cotyledons and generally shorter hypocotyls. br levels are also known to impact root development. mutants deficient in br or br signaling have shorter roots than wt and in the presence of supraoptimal exogenous br, root development can be severely impaired. root growth was measured in ocp and wt lines on vertically-oriented sterile media. in the absence of exogenous 24-epibl, ocp lines had shorter roots than wt but this did not correlate strongly with the severity of the above-ground phenotype. in the presence of μm 24-epibl, the length of wt roots was reduced to 22% of roots grown in the absence of 24-epibl, whereas roots of the most severe ocp lines were reduced to only 65% to 75% relative to those grown without exogenous 24-epibl. these findings that exogenous 24-epibl severely affects wt root and aerial growth, but has little impact on the most severe ocp lines, combined with a growth pattern that phenocopies br deficient mutants, strongly suggests that the cyp105a enzyme encoded by the p450su gene is affecting br homeostasis directly or indirectly. overexpression of p450su does not impact gibberellin or auxin mediated growth characteristics gibberellin and auxin metabolism are also impacted by cyp activity, and hypocotyl- and root-growth experiments were conducted to test if cyp105a visually affects growth responses to these hormones. exogenous application of ga or iaa is known to modestly increase hypocotyl length of etiolated seedlings. this was observed in wild type and control plants, but the effect was identical among even the most severe ocp lines. conversely, exogenous ga or iaa treatment is known to result in decreased root elongation in etiolated seedlings. in our experiments with μm of either hormone, ocp and control lines showed identical extents of reduced root elongation. these results show that p450su expression does not mitigate the influence of exogenous ga or iaa as it did for exogenous 24-epibl, and argues that the cyp105a enzyme impacts br homeostasis, but not that of iaa or ga discussion this study initiated as an effort to create a vector system in which a cdna sequence of interest could be excised upon delivery or activation of a site-specific recombinase. it was designed with dual selection for recombination. after flp-mediated recombination at the frt sites, the positive selection marker bar was to be activated by being placed adjacent to a camv 35s promoter and the negative selection marker p450su was to be inactivated by being excised from the genome along with the cdna of interest. independent transgenic lines harboring this construct displayed a range of phenotypes with the most severe lines resembling plants with disrupted br synthesis or perception. this included stunted rosettes and inflorescences with short internodes and reduced apical dominance, thicker leaves with dark coloration characteristic of anthocyanin accumulation, leaf curling that gave rosettes a distinctive twirled appearance, reduced male fertility and seed yields, and delayed senescence. the severity of these characteristics showed a high correlation with p450su expression levels, and on sterile media these lines showed the least response to supraoptimal levels of 24-epibl. as controls, plants transformed with t-dna that retained the atsuc cdna but had p450su deleted were phenotypically normal, as were plants lacking both atsuc cdna and p450su and instead expressing uida encoding β-glucuronidase. the combined results of the close correlation between p450su expression and a phenotype resembling a deficiency in br synthesis or perception, p450su expression mitigating the effects of exogenous 24-epibl, and the process of eliminating other candidate genes indicate that the cyp105a enzyme is acting on exogenous br and affects endogenous br by altering br homoeostasis. a t-dna construct harboring only p450su was not tested. expression of p450su did not modify the growth of etiolated seedlings in the presence of iaa or ga indicating that it does not act on these hormones. p450su and the encoded enzyme cyp105a were originally identified from the soil bacterium streptomyces griseolus as being able to degrade sulfonylurea herbicides. in transgenic plants, cyp105a converted the relatively benign compound r into a highly phytotoxic herbicide and could thus be used for negative selection: plants or individual tissues expressing p450su were ablated by r application, while plants or tissues not expressing the gene were spared. p450su was used previously in several studies, but we are aware of only one were growth aberrations in the absence of r were noted. specifically, koprek and colleagues compared the efficacy of p450su and the coda gene, which converts non-toxic 5-fluorocytosine to toxic 5-fluorouracil, as negative-selection tools in transgenic barley. the abstract of notes growth anomalies with p450su but did not elaborate, and the authors concluded that despite these anomalies, p450su along with r was suitable for negative selection among plants grown in soil. based on our findings, the growth anomalies reported in barley are likely the result of perturbed brassinosteroid signaling. there are several explanations as to why a link between p450su and growth aberrations from perturbed brassinosteroid signaling have not been reported. first, the system is used for negative selection in conjunction with r and production of the phytotoxic byproduct results in rapid death of plants or tissues. therefore, the effects of p450su in the absence of r are mild compared to the effects in the presence of r second, since the system is used for negative selection, most attention has focused on characteristics of plants or tissues after loss of the gene by segregation, transposition, or recombination. third, in the unique vector system used here, a strong camv 35s promoter was placed upstream of a strong rubisco promoter, and this combination may result in expression levels higher than those obtained in studies where growth anomalies were not reported. this is supported by the strong correlation between transcript abundance and phenotype severity. lines with moderate to low p450su transcript levels displayed moderate to mild symptomology in the absence of r but were still highly sensitive to r and suitable for negative selection. in addition, cyp105a as used here is targeted to plastids and expression from a dual promoter system may overwhelm plastid targeting and result in more enzyme mislocalized to the cytosol for acting on brs. potential mislocalization of plastid-targeted cyp105a was previously reported. the dual promoters may also explain discrepancies between the phenotypes of our most severe lines and mutants defective in br synthesis. for example, in the cpd mutant which is disrupted in br synthesis, dark-grown seedlings show photomorphogenesis and have short, thickened hypocotyls but our most severe ocp line showed normal skotomorphogenesis and differed only moderately from wt. the rubisco small subunit promoter is light-activated, and in dark-grown seedlings expression would have been minimal. under these conditions, p450su expression from the more distal camv 35s promoter alone may have been insufficient to cause a more severe phenotype. however, in the presence of 24-epibl, ocp seedlings likely had sufficient p450su expression to bring brassinosteroid levels into a range that allowed relatively normal development. as described above, cyp105a metabolizes sulfonylurea herbicides by dealkylation. sulfonylurea herbicides are agricultural soil additives, and the natural target and substrate specificity of cyp105a is not known. in transgenic plants, cyp105a disrupts brassinosteroid homeostasis to give a phenotype, but the full range of potential substrates and the extent to which their levels are altered is not known. work by others has shown that cyp105a can hydroxylate vitamin d and d at multiple positions and can catalyze the conversion of 7-ethoxycoumarin to 7-hydroxycoumarin by o-dealkylation. detoxification of sulfonylurea herbicides and n-dealkylation of the pro-herbicide r to produce a toxic metabolite are additional activities, and collectively, these reactions suggest that cyp105a substrate selection and mode of action may be quite broad, but does not extend to iaa or ga it is now apparent that the development of herbicide resistance in several weeds is the result of enhanced detoxification associated with elevated levels of cyp activity. weeds with enhanced cyp-mediated detoxification can be difficult to control because resistance can develop against multiple, unrelated classes of herbicide. however, in the limited species that have been subjected to analysis, there is a fitness cost associated with elevated cyp levels: in the absence of the selective pressure imparted by the herbicide, herbicide-resistant varieties of lolium rigidum showed up to 30% reduced vitality relative to their herbicide-sensitive counterparts. the development of cyps from both plant and non-plant origins for engineering herbicide resistance in biotechnology has garnered substantial interest. however, the reduced vitality of plants that have naturally developed resistance and the undesired effects on plant-hormone homeostasis observed here with overexpression of p450su highlight some of the potential deleterious outcomes that will need to be addressed for successful exploitation of this promising area.  the p450su gene from streptomyces griseolus has been used as a negative-selection marker in conjunction with the pro-herbicide r since plants expressing the gene are killed by r while those not expressing it retain viability. however, in the absence of r plants with high p450su expression show aberrant growth characteristic of defects in brassinosteroid synthesis and perception. when exposed to supraoptimal exogenous brassinosteroids, the growth habit of these plants is relatively normal compared to wild type. together, these results indicate that both endogenous and exogenous brassinosteroids are a target of the p450su encoded cyp105a monooxygenase. 
consistent with its use for negative selection, transgenic arabidopsis plants were generated with p450su situated between recognition sequences for flp recombinase from yeast to select for recombinase-mediated excision. germination and seedling growth of transgenic and control lines in the presence and absence of 24-epibrassinolide indicated that cyp105a disrupts brassinosteroid signaling, most likely by inactivating brassinosteroids. the p450su gene from the soil bacteria streptomyces griseolus encodes cyp105a which acts on various substrates including sulfonylurea herbicides, vitamin d, coumarins, and based on the work presented here, brassinosteroids. phenotype severity correlated with p450su transcript abundance, but not with transcript abundance of other experimental genes, strongly implicating cyp105a as responsible for the defects. 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. p450su is used as a negative-selection marker in plants because cyp105a converts the relatively benign sulfonyl urea pro-herbicide r into a highly phytotoxic product.
 cytochrome p monooxygenases form a large superfamily of enzymes that catalyze diverse reactions. the p450su gene from the soil bacteria streptomyces griseolus encodes cyp105a which acts on various substrates including sulfonylurea herbicides, vitamin d, coumarins, and based on the work presented here, brassinosteroids. p450su is used as a negative-selection marker in plants because cyp105a converts the relatively benign sulfonyl urea pro-herbicide r into a highly phytotoxic product. consistent with its use for negative selection, transgenic arabidopsis plants were generated with p450su 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.  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 p450su transcript abundance, but not with transcript abundance of other experimental genes, strongly implicating cyp105a as responsible for the defects. germination and seedling growth of transgenic and control lines in the presence and absence of 24-epibrassinolide indicated that cyp105a disrupts brassinosteroid signaling, most likely by inactivating brassinosteroids.  despite prior use of this gene as a genetic tool, deleterious growth in the absence of r has not been elaborated. we show that this gene can cause aberrant growth by disrupting brassinosteroid signaling and affecting homeostasis. 