BACKGROUND
the plant catharanthus roseus  g. don  produces a large number of terpenoid indole alkaloids , some of which are of substantial pharmacological interest.
vinblastine and vincristine have been used as chemotherapeutics in the treatment of lymphoma and leukemia  <cit> .
ajmalicine and serpentine are sometimes used as anti-hypertensive agents.
most of these alkaloids are produced in extremely low amounts in planta, limiting the usage of these chemicals.
substantial efforts to use chemical syntheses, in vitro cell cultures or bacterial cells for large-scale production of these alkaloids have proven ineffective  <cit> .
one of the difficulties in developing methods for large-scale production of tias is the complexity of both the tia biosynthetic pathway and of the regulatory pathways governing tia production.
despite the complexity of these pathways, significant progress in our understanding of the biochemistry and regulation of the tia pathway in c. roseus has been made in recent years.
in particular, many of the genes coding for the tia biosynthetic enzymes and tia transcriptional activators and repressors have been identified  <cit> .
tia biosynthesis is a tightly coordinated process involving at least  <dig> intermediates,  <dig> biosynthetic enzymes and several intra and inter cellular compartments  <cit> .
tia biosynthesis in c. roseus starts with the condensation of tryptamine, which is derived from the indole pathway, and secologanin, which is derived from the monoterpenoid pathway, to form strictosidine .
this reaction is catalyzed by strictosidine synthase .
strictosidine is then deglucosylated by strictosidine β-d-glucosidase  to form cathenamine and an equilibrium between cathenamine and  <dig> -dehydrogeissoschizine is established  <cit> .
different branches of the tia pathway then lead to the production of numerous tias, including ajmalicine and serpentine, lochnericine and hörhammericine, and catharanthine and vindoline.
vindoline and catharanthine are the precursors of vinblastine and vincristine.
the genes encoding the enzymes catalyzing at least four of the reactions in the six-step pathway leading to production of vindoline from tabersonine have been cloned and characterized.
these genes include tabersonine 16-hydroxylase   <cit> , 16-hydroxytabersonine-16-o-methyltransferase   <cit> , desacetoxyvindoline 4-hydroxylase   <cit> , and deacetylvindoline acetyltransferase   <cit> .
an enzyme with anhydrovinblastine synthase activity that catalyzes the synthesis of α-3′, 4′-anhydrovinblastine from vindoline and catharanthine has also been identified and characterized as the major class iii peroxidase  in c. roseus leaves  <cit> .
tia biosynthesis is a highly regulated process that involves a number of transcriptional activators and repressors.
to date, seven putative activators  and five putative repressors  have been implicated as regulators of the tia pathway.
however, very few studies have been done on any of these regulators, with the exception of orca <dig>  <cit> .
both orca <dig>   <cit>  and orca <dig>  <cit>  are ap2-domain transcription factors that are proposed to activate str expression by binding to the jasmonate and elicitor-responsive element  in the str promoter  <cit> .
crbpf <dig> is also proposed to activate str transcription by binding to a separate element in the str promoter  <cit> .
crmyc <dig>  <cit>  and crmyc <dig>  <cit>  are basic helix-loop-helix transcription factors.
crmyc <dig> has been shown to act upstream of orca <dig> and orca <dig>  activating their transcription  <cit> .
crwrky <dig> and crwrky <dig> are jasmonate responsive wrky transcription factors that positively regulate expression of several genes involved in tia biosynthesis  <cit> .
overexpression of crwrky <dig> also leads to increased transcript levels of the tia transcriptional repressors zct <dig>  zct <dig> and zct <dig> and decreased transcript levels of the tia transcriptional activators orca <dig>  orca <dig> and crmyc <dig> <cit> .
in contrast, overexpression of crwrky <dig> leads to increased expression of both specific tia transcriptional activators  and repressors   <cit> .
the three zinc finger proteins, zct <dig>  zct <dig>  and zct <dig>  were found to bind specifically to the tryptophan decarboxylase  and str promoters in vitro, inhibiting their activities.
in addition, the zct proteins repress the activation of the str promoter by the orcas  <cit> .
two g-box-binding factors, gbf <dig> and gbf <dig>  were found to repress str transcription by binding to the g-box sites in the str promoter region  <cit> .
the identification of multiple transcriptional activators and repressors for the tia pathway suggests that regulation of this pathway is a complex process.
however, to date there have been very few published studies describing in depth characterization of tia regulators other than orca <dig>  orca <dig> has been the focus of several studies in recent years.
the results of these studies indicate that orca <dig> acts as a positive regulator of many tia biosynthetic genes  <cit> .
for these studies orca <dig> was overexpressed in either suspension cells or hairy root cultures.
in c. roseus cell cultures, overexpression of orca <dig> increases the transcript levels of tdc, str, cytochrome p <dig> reductase , 1-deoxy-d-xylulose 5-phosphate synthase , anthranilate synthase α subunit  and d4h.
in contrast, overexpression of orca <dig> in c. roseus cell suspension cultures has no significant effects on transcript levels of geraniol 10-hydroxylase , sgd or dat <cit> .
in c. roseus hairy root cultures, overexpression of orca <dig> induces expression of asα, dxs, secologanin synthase , str and zct <dig>  zct <dig> and zct <dig>  but represses sgd expression.
the transcript levels of tdc, cpr, g10h, gbf <dig>  gbf <dig> and orca <dig> were not significantly affected by orca <dig> overexpression  <cit> .
although several studies have characterized the role of orca <dig> in regulating the tia pathway, little work has been done to characterize the role of the related orca <dig> transcriptional activator.
recently liu et al.
reported the generation of c. roseus transgenic hairy root lines that exhibit constitutive overexpression of orca <dig> and stated that catharanthine and vindoline concentrations increased in these lines, but only hplc and an authentic standard were used for the identification of vindoline  <cit> .
vinblastine levels in these lines were below detection limits and other tia metabolites were not assayed as part of this study.
the expression levels of tia biosynthetic genes and of other tia regulatory genes were also not characterized by liu and colleagues, leaving the role of orca <dig> in regulating tia metabolism unknown.
here we describe the generation, metabolic and molecular characterization of a transgenic c. roseus hairy root line that expresses orca <dig> under the control of an ethanol-inducible promoter.
the transcript levels of a total of  <dig> tia biosynthetic and regulatory genes were tracked over a period of 72 h following induction of orca <dig> overexpression.
the levels of seventeen tia and related metabolites were also investigated over the same time period, with thirteen of those metabolites being present at detectable levels in at least some of the samples analyzed.
the results of these experiments indicate that orca <dig> plays an important role in regulating the tia pathway, particularly the downstream portions of this pathway.
based on the results of these experiments, a model for regulation of the tia pathway by orca <dig> and other tia transcriptional regulators is presented.
RESULTS
generation of c. roseus transgenic hairy root lines expressing orca <dig> under the control of an ethanol-inducible promoter
to date, only a few studies have characterized orca <dig>  <cit>  and no published studies have determined which tia genes, other than str, are regulated by orca <dig> or how orca <dig> overexpression affects the levels of a broad group of tia metabolites.
as a result, determining the role of orca <dig> in regulating the tia pathway has not been possible.
to address this deficiency, transgenic hairy root lines that overexpress orca <dig> under the control of an ethanol-inducible promoter were generated.
the ethanol-inducible system offers significant advantages over constitutive expression systems in that the ethanol-inducible system allows the timing and level of transgene expression to be controlled  <cit> .
as a result, studies on the transient effects of transgene expression are made possible.
in addition, the potentially deleterious effects of constitutive overexpression of transgenes on tissue growth and development may be mitigated.
to generate transgenic hairy roots, approximately 6-week-old c. roseus seedlings were co-transformed using a mixture of agrobacterium tumefaciens gv <dig> carrying the binsrna-orca <dig> construct  and gv <dig> carrying the ppzprol plasmid.
the ppzprol plasmid was previously engineered to carry the rola, rolb and rolc genes that are sufficient to induce hairy root production  <cit> .
a total of  <dig> hairy root lines were screened for genomic integration of orca <dig> by assaying for kanamycin resistance and using pcr to identify a dna fragment spanning the orca <dig> gene and alca promoter.
five of the  <dig> hairy root lines were found to carry orca <dig> under the control of the alca promoter.
these positive lines were transferred to liquid culture for adaptation.
following adaptation, the lines were screened for ethanol-inducible overexpression of orca <dig>  figure  <dig> shows orca <dig> relative transcript levels from the five orca <dig> transgenic hairy root lines.
the results indicate that the transgenic lines express orca <dig> transcripts at approximately three to  <dig> times higher levels than the control line 10 h after induction by  <dig> %  ethanol.
as the orca2-oe line exhibited one of the highest levels of orca <dig> induction and adapted well to liquid culture, this line was retained for further study.
a control line was also generated by transforming c. roseus seedlings with gv <dig> cells carrying the ppzprol construct alone.
to determine if ethanol-induced overexpression of orca <dig> in the orca2-oe line is maintained for a substantial period of time, a 72-h time course was performed.
orca <dig> transcripts generated from the orca <dig> transgene and by the endogenous orca <dig> gene were measured separately in this experiment.
an ethanol concentration of  <dig> % was used for this experiment after this concentration of ethanol was found to be sufficient to cause increases in orca <dig> transcript levels similar to those caused by the  <dig> % ethanol used in earlier experiments .
figure 4a shows that orca <dig> transgene transcript levels increase dramatically after adding ethanol to orca2-oe root cultures, rising over seven fold within 6 h. orca <dig> transgene transcript levels remained high in the induced cultures over the 72-h course of the experiment.
in contrast, orca <dig> transgene transcript levels decrease in uninduced orca2-oe root cultures upon transfer to fresh media at the 0-h time point.
this decrease in orca <dig> transgene transcript levels is likely due to the fact that plant cultures grown in liquid typically produce small amounts of ethanol.
thus, the 35-d old cultures from which the 0-h time point samples were collected contained small amounts of ethanol, which likely caused a modest induction of orca <dig> transgene expression.
transferring additional 35-d old cultures to fresh media with no added ethanol at the 0-h time point thus caused a slight decrease in ethanol concentration and, consequently, in orca <dig> transgene expression in those cultures.
as a result, orca <dig> transgene transcript levels are approximately 100-fold higher in the induced than in the uninduced orca2-oe cultures within approximately 12 h after induction.
the levels of orca <dig> transcripts generated from the endogenous orca <dig> gene increase approximately three fold after addition of ethanol to the orca2-oe cultures and then decline back to starting levels by the end of the 72-h time course .
in contrast, orca <dig> endogenous gene transcript levels decline in the orca2-oe cultures transferred to fresh media with no added ethanol.
these results suggest that orca <dig>  directly or indirectly, induces expression of the orca <dig> endogenous gene, as cultures with high levels of expression of the orca <dig> transgene express the endogenous orca <dig> gene at higher levels than cultures with low levels of expression of the orca <dig> transgene.
consistent with this hypothesis, orca <dig> endogenous gene transcript levels are significantly higher in orca2-oe cultures than in control cultures at the 0 h time point, as orca <dig> transgene expression is modestly induced in these orca2-oe cultures by plant-produced ethanol.
orca <dig> endogenous gene transcript levels are not significantly different in uninduced versus induced cultures of the control line.
effects of orca <dig> overexpression on the indole and terpenoid pathways
to determine if orca <dig> plays an important role in regulation of the tia pathway, transcript levels of  <dig> genes encoding key enzymes and regulators of the tia and related pathways were analyzed in the orca2-oe and control hairy root lines.
the genes analyzed include asα and tdc, two key genes from the indole pathway that produces tryptamine; dxs, cpr, g10h and loganic acid o-methyltransferase  from the monoterpenoid pathway which leads to the formation of secologanin; and str, sgd, t16h, 16omt, d4h, dat and prx <dig> from the tia pathway which catalyzes the condensation of tryptamine and secologanin and ultimately leads to the production of vinblastine and vincristine.
transcript levels of four transcriptional activators  and five repressors  that regulate the tia pathway were also analyzed.
to analyze the downstream effects of orca <dig> overexpression, the levels of  <dig> tia and related metabolites were also investigated, with  <dig> of those metabolites found to be present at detectable levels in at least some of the samples analyzed.
both transcript and metabolite levels were tracked over a 72-h period following ethanol induction of orca <dig> overexpression, to allow analysis of both transient and relatively prolonged effects.
to characterize the effects of orca <dig> overexpression on the indole pathway, asα and tdc transcript levels were analyzed.
asα encodes the α subunit of anthranilate synthase, which catalyzes the first committed step in the synthesis of tryptophan, the formation of chorismate from anthranilate.
tdc encodes tryptophan decarboxylase, which catalyzes the conversion of tryptophan to tryptamine.
both asα and tdc transcript levels are significantly higher in the orca2-oe line after ethanol induction than in the uninduced orca2-oe cultures or in the induced or uninduced cultures of the control line .
asα expression in the orca2-oe line increases within 12 h of the start of orca <dig> induction and then decreases slightly, but remains significantly higher than in the uninduced orca2-oe cultures or in the induced or uninduced cultures of the control line.
tdc transcript levels increase within 24 h and increase further within 48 h after the start of orca <dig> induction.
the levels of tryptamine, the product of the indole pathway that is combined with the terpenoid secologanin to form the first tia, strictosidine, were also analyzed in samples from the same hairy root cultures.
tryptamine levels increase significantly within 24 h of induction and remain increased through 72 h. tryptamine levels remain unchanged in the uninduced orca2-oe cultures and the induced and uninduced cultures of the control line .
levels of tryptophan were below detection limits.
to characterize the effects of orca <dig> overexpression on the terpenoid pathway, the transcript levels of dxs, g10h, cpr and lamt were analyzed .
transcript levels of dxs and cpr are not significantly different in the induced orca2-oe line than in the uninduced orca2-oe line .
in contrast, g10h transcript levels in the orca2-oe line increase within 48 h after the start of orca <dig> induction.
expression of lamt increases sharply in the orca2-oe line after induction, but then decreases within less than 24 h, reaching levels that are lower than those seen in uninduced orca2-oe cultures within 72 h .
addition of ethanol to the “induced” control cultures had little affect on gene expression, indicating that differences in ethanol concentration per se are not responsible for the differences in gene expression observed for the induced versus uninduced orca2-oe cultures.
the differences in gene expression observed between the control and orca2-oe cultures at the 0-h time point may be due to the fact that different clonal root cultures have somewhat different growth characteristics.
alternatively, some of these differences may be due to the fact that the 35-d old orca2-oe cultures from which the 0-h time point samples were collected likely contained small amounts of root-produced ethanol, which led to a modest induction of orca <dig> transgene expression.
the effects of orca <dig> overexpression on loganin and secologanin levels were also determined.
loganin is the precursor for secologanin, which is one of the precursors for formation of the first tia, strictosidine.
loganin levels in induced and uninduced orca2-oe cultures are similar .
in contrast, secologanin levels are somewhat variable in the orca2-oe line.
secologanin levels are significantly decreased in induced orca2-oe cultures relative to uninduced orca2-oe cultures at  <dig> and 72 h after induction, but are not significantly different at the other time points assayed .
effects of orca <dig> overexpression on the tia pathway
the first step in tia synthesis, the condensation of tryptamine and secologanin to form strictosidine, is catalyzed by str.
sgd then uses strictosidine as a substrate to form cathenamine, which serves as a substrate for the formation of several tias via different branches of the tia pathway.
to test if orca <dig> plays an important role in regulating these processes, transcript levels for str, sgd, t16h, 16omt, d4h, dat and prx <dig> were analyzed in the orca2-oe and control hairy root lines for a 72-h period following orca <dig> induction .
the results of these experiments indicate that transcript levels of six of the seven genes tested are significantly affected by orca <dig> overexpression.
str, t16h and prx <dig> are strikingly induced by orca <dig> overexpression, although with somewhat different timing and magnitudes.
str transcript levels increase significantly in the orca2-oe cultures within 24 h of the start of orca <dig> induction, reaching a maximum of approximately three times higher levels at the 48-h time point than in the orca2-oe cultures at the zero time point.
t16h transcript levels also increase significantly within 24 h of the start of induction, reaching levels by the 48 h time point that are  <dig> to  <dig> times higher in the induced orca2-oe cultures than in the uninduced orca2-oe cultures at the same time point.
prx <dig> transcript levels increase approximately  <dig> fold in the orca2-oe cultures within 12 h after the start of induction and then decline slightly.
d4h transcript levels are significantly higher in the induced than in the uninduced orca2-oe cultures at  <dig> and 72 h after the start of induction, but are only increased by approximately two to four fold.
interestingly, transcript levels of both sgd and dat decrease in response to orca <dig> overexpression.
the timing of the declines in sgd and dat transcript levels are similar, with both genes exhibiting significantly lower transcript levels in the induced versus uninduced orca2-oe cultures within 12 h of the start of induction.
however, the magnitudes of the changes in transcript levels are different, as sgd transcript levels are only approximately two fold lower in the induced versus uninduced orca2-oe cultures, whereas dat transcript levels are approximately  <dig> to  <dig> fold lower in the induced versus uninduced orca2-oe cultures.
of the seven tia biosynthetic genes characterized, only 16omt does not show significant differences in expression levels between uninduced and induced orca2-oe hairy root cultures.
the differences in expression of some of the genes between the control and orca2-oe cultures at the 0-h time point may be due to different clonal root cultures having somewhat different growth characteristics and/or to the fact that the orca2-oe cultures likely expressed the orca <dig> transgene at modest levels at the 0-h time point due to the presence of small amounts of root-produced ethanol in the 35-d old cultures from which those samples were collected.
as orca <dig> overexpression causes significant alterations in the transcript levels of a high percentage of the tia biosynthetic genes tested, it was of interest to determine if orca <dig> overexpression also alters tia levels.
to address this issue, the levels of  <dig> tias from different branches of the tia pathway were investigated in aliquots of the same tissue samples used to analyze transcript levels.
nine of these tias were present at detectable levels, whereas the levels of the other three tias  were below detection thresholds.
in addition, a tabersonine-like compound, designated unk <dig>  was detected in the samples .
the levels of strictosidine, the first tia to be formed from secologanin and tryptamine, are significantly reduced in the induced versus uninduced orca2-oe cultures within 24 h after the start of induction and remain reduced throughout the remainder of the 72-h time course.
ajmalicine and serpentine are formed via the same branch of the tia pathway .
both ajmalicine and serpentine levels exhibit modestly significant increases in the induced versus uninduced orca2-oe cultures 48 h after the start of induction, but are not significantly different at the other time points assayed.
catharanthine, the precursor for vinblastine production, also exhibits modestly significantly higher levels 48 h after induction, but not at the other time points assayed.
the levels of tabersonine, which is the starting material for multiple branches of the tia pathway, are significantly reduced in induced versus uninduced orca-oe cultures within 6 h of the start of orca <dig> induction.
tabersonine is converted via one branch of the tia pathway to lochnericine and hörhammericine.
lochnericine levels are not significantly altered by orca <dig> overexpression, but hörhammericine levels are significantly reduced in the induced versus uninduced orca2-oe cultures at  <dig> and 72 h after the start of induction.
tabersonine is converted via a different branch of the tia pathway to 16-hydroxytabersonine .
16ohtab levels exhibit modestly significant increases in the induced versus uninduced orca2-oe cultures at  <dig> and 72 h after the start of orca <dig> induction.
tabersonine is also converted to 19-hydroxytabersonine .
19ohtab levels increase within 12 h of the start of orca <dig> induction and remain elevated for at least 72 h. levels of vindoline, the end metabolite of this branch of the tia pathway and precursor to vinblastine production, are below detection limits.
an un-identified metabolite, designated unk <dig>  was also detected in the tissue extracts.
based on its uv absorption spectra between  <dig> and 400 nm , unk <dig> is a tabersonine-like compound.
unk <dig> levels exhibit modestly significant increases in the induced versus uninduced orca2-oe cultures at 48 h after the start of induction, but not at the other time points assayed.
the levels of the pharmaceutically important tias vinblastine and vincristine, formed by combining catharanthine and vindoline, were below detection limits.
effects of orca <dig> overexpression on regulators of the tia pathway
tia production appears to be regulated via a complex process.
a number of transcriptional activators and repressors have been identified  <cit> , including the transcriptional activators orca <dig>  orca <dig>  crbpf <dig>  crmyc <dig>  crmyc <dig>  crwrky <dig> and crwrky <dig> and the transcriptional repressors zct <dig>  zct <dig>  zct <dig>  gbf <dig> and gbf <dig>  to determine if orca <dig> regulates tia transcriptional regulators in addition to tia biosynthetic genes, transcript levels of orca <dig>  crbpf <dig>  crmyc <dig>  zct <dig>  zct <dig>  zct <dig>  gbf <dig> and gbf <dig> were tracked for a 72-h time period in ethanol-induced and uninduced orca2-oe and control hairy root cultures.
the results of these assays indicate that overexpression of orca <dig> leads to increased transcript levels of the tia transcriptional activator orca <dig> in the induced versus uninduced orca2-oe cultures .
orca <dig> transcript levels in the induced orca2-oe cultures increase significantly within 12 h of the start of induction and remain elevated throughout the 72-h time course point, compared with the uninduced orca2-oe cultures.
overexpression of orca <dig> had minimal effects on crmyc <dig> transcript levels, with the only significant difference between the orca2-oe induced versus uninduced cultures being a very modest decrease in crmyc <dig> transcript levels at the 72-h time point.
interestingly, overexpression of orca <dig> also causes increases in the steady-state mrna levels of the tia transcriptional repressors zct <dig>  zct <dig> and zct <dig>  the transcript levels of these three zinc finger genes are significantly higher in the induced versus uninduced orca2-oe cultures within 12 h of the start of induction and remain elevated through the 72-h time point for zct <dig> and zct <dig> and through the 48-h time point for zct <dig>  in contrast, overexpression of orca <dig> has no significant effects on transcript levels of crbpf <dig>  gbf <dig> or gbf <dig>
discussion
orca <dig> is a key regulator of the tia pathway
tia biosynthesis in c. roseus is a complex, multi-step process that is regulated by at least twelve transcriptional regulators.
the roles of most of these transcriptional regulators, particularly with regards to the downstream branches of the tia pathway, remain to be determined  <cit> .
as the downstream branches of the tia pathway are responsible for production of several pharmaceutically important compounds, this lack of information limits attempts to develop models for regulation of tia production.
therefore, the effects of overexpressing orca <dig> on transcript levels of  <dig> tia biosynthetic and regulatory genes were characterized.
the results of the research described here indicate that orca <dig> overexpression in c. roseus hairy roots significantly affects the transcript levels of several key genes from both pathways leading to production of tia precursors, as well as six of the seven genes characterized from the tia pathway .
overexpression of orca <dig> causes increased expression of both genes  characterized from the indole pathway.
of the four genes from the monoterpenoid pathway that were characterized, overexpression of orca <dig> causes increased expression of g10h and lamt, but does not significantly affect transcript levels of dxs or cpr.
perhaps most interestingly, overexpression of orca <dig> leads to significantly altered transcript levels for almost all of the genes from the tia pathway that were characterized, including four out of five genes characterized from the downstream branches of the tia pathway .
overexpression of orca <dig> results in significantly increased transcript levels for str, which encodes the enzyme that catalyzes the first step in tia biosynthesis.
transcript levels of t16h, d4h and prx <dig> also increase in response to orca <dig> overexpression.
in contrast, transcript levels of 16omt are not significantly affected by orca <dig> overexpression.
somewhat surprisingly, transcript levels of sgd and dat decrease, rather than increase, in response to orca <dig> overexpression.
the effects of orca <dig> overexpression on dat transcript levels are particularly striking, as dat transcript levels are more than  <dig> times lower in the orca2-oe line 12 h after ethanol induction than in the control line at the zero time point and  <dig> times lower than in the orca2-oe line at the zero time point.
these findings that overexpression of orca <dig> causes significant alterations in transcript levels for key genes from both feeder pathways, the gene that encodes the enzyme that catalyzes the combination of the products of those feeder pathways to form the first tia, the gene that encodes the enzyme that catalyzes the conversion of the first tia and four out of five genes characterized from the downstream branches of the tia pathway indicates that orca <dig> plays a critical role in regulation of tia metabolism.
“+” = up-regulation ; “-” = down-regulation .
“=” = no change .
orca <dig> overexpression affects the levels of tia and related metabolites
overexpression of orca <dig> also affects the levels of multiple tias and related metabolites .
in characterizing the indole pathway, tryptophan levels were found to be below detection limits and tryptamine levels were found to increase within 24 h in response to orca <dig> overexpression.
higher tryptamine levels are consistent with the increased asα and tdc transcript levels caused by orca <dig> overexpression.
asα encodes the alpha subunit of anthranilate synthase, which catalyzes the first committed step in the tryptophan biosynthetic pathway and is feedback-inhibited by tryptophan  <cit> .
the concurrent increase in tdc expression , however, increases the metabolic flux from tryptophan to tryptamine thus reducing the feedback inhibition of anthranilate synthase by tryptophan.
in contrast, of the two metabolites from the terpenoid pathway that were characterized, loganin levels are not significantly affected and secologanin levels are reduced at both the 24- and 72-h time points in response to orca <dig> overexpression.
two factors may account for the reduced secologanin concentration in response to orca <dig> overexpression.
one factor is that orca <dig> overexpression has a smaller effect on the genes in the terpenoid pathway  than on the genes in the indole pathway .
the other factor is that the increase in str expression  may result in more rapid use of secologanin to form strictosidine.
“+” = level of indicated metabolite was higher at one or more time points when orca <dig> was overexpressed; “-” = level of indicated metabolite was lower at one or more time points when orca <dig> was overexpressed; “=” indicates no significant change in metabolite level when orca <dig> was overexpressed; “bd” indicates the metabolite was below detection levels.
the transgenic hairy root samples were also analyzed for the levels of  <dig> tias or tia-related compounds.
three of these tias, vindoline, vinblastine and vincristine, were present at levels below the detection threshold.
the levels of strictosidine, the first tia produced by combining the products of the indole and terpenoid pathways, are significantly lower within 24 h of the beginning of orca <dig> overexpression.
this result was somewhat unexpected as the gene  that encodes the enzyme that catalyzes the formation of strictosidine is induced by orca <dig> overexpression and the gene  that encodes the enzyme that converts strictosidine to cathenamine/ <dig> -dehydrogeissoschizine/epicathenamine  <cit>  is inhibited by orca <dig> overexpression.
however, decreased strictosidine levels might be explained by the increased expression of genes encoding enzymes for downstream steps, in the tia pathway, such as t16h and prx <dig>  causing an increased rate of metabolite flux to the downstream part of the pathway, in combination with the possibility of sgd not being rate limiting for strictosidine metabolism.
alternatively, the reduced sgd transcript levels caused by orca <dig> overexpression may not lead to similar decreases in sgd activity levels.
consistent with the possibilities that sgd activity levels are either not rate limiting or are not decreased, despite the decrease in sgd transcript levels, are the findings that ajmalicine and serpentine are transiently increased in response to orca <dig> overexpression.
both ajmalicine and serpentine, which are made via the same branch of the tia pathway, are present in significantly higher concentrations 48 h after the start of orca <dig> induction, but are not present at significantly altered levels at the other time points assayed.
also consistent with these possibilities are findings that orca <dig> overexpression leads to transient increases in the levels of catharanthine, which is made via a different branch of the tia pathway, as catharanthine levels are significantly increased 48 h after the start of orca <dig> induction.
the levels of tabersonine, which is used as the starting material for multiple branches of the tia pathway , are decreased in response to orca <dig> overexpression.
decreased tabersonine levels in response to orca <dig> overexpression could be the result of a decreased rate of tabersonine synthesis or an increased rate of tabersonine metabolism.
consistent with the first possibility is the finding that sgd transcript levels decrease in response to orca <dig> overexpression, as sgd activity is necessary for tabersonine production.
however, the decrease in sgd transcript levels first occurs at a later time point than the decrease in tabersonine levels, making the extent to which decreases in sgd transcript levels are responsible for the decreased tabersonine levels unclear.
increases in t16h transcript levels in response to orca <dig> overexpression are consistent with the possibility that decreased tabersonine levels are due to a higher rate of tabersonine metabolism, as t16h catalyzes the first step for one branch of the tia pathway, namely the conversion of tabersonine to 16-hydroxytabersonine.
however, t16h transcript levels first increase at a later time point than the decrease in tabersonine levels, making the extent to which increases in t16h transcript levels cause decreased tabersonine levels unclear.
16omt catalyzes the conversion of 16-hydroxytabersonine to 16-methoxytabersonine.
16omt transcript levels are not significantly affected by orca <dig> overexpression.
as 16-hydroxytabersonine levels increase within 48 h of the start of orca <dig> overexpression, these results suggest that 16omt activity levels may be rate limiting in hairy roots where orca <dig> is overexpressed.
interestingly, the increase in 16-hydroxytabersonine levels accounts for only a small portion of the substantial decrease in tabersonine levels, raising the possibility that the levels of other tabersonine-related metabolites are raised in response to orca <dig> overexpression.
consistent with this possibility is the finding that the levels of 19ohtab, which is also made from tabersonine, increase significantly in response to induction of orca <dig> overexpression.
in addition, the identification of a tabersonine-like compound, here designated unk <dig>  that is present at significantly higher levels in orca2-oe induced versus uninduced cultures is also consistent with the idea that the levels of tabersonine-related metabolites increase in response to orca <dig> overexpression.
lochnericine and hörhammericine are also made from tabersonine, via a different branch of the tia pathway.
overexpression of orca <dig> has no significant effects on lochnericine levels, but leads to decreased hörhammericine levels at the 24- and 72-h time points, suggesting that overexpression of orca <dig> causes an overall decrease in metabolic flux to this branch of the tia pathway.
orca <dig> regulates other tia transcriptional regulators
as orca <dig> is believed to act as a transcriptional activator  <cit> , findings that overexpression of orca <dig> causes significant decreases in sgd and dat transcript levels are somewhat surprising and suggest that regulation of the tia pathway is complex.
a possible explanation for these results is that overexpression of orca <dig> may cause increased expression of one or more tia repressors.
to examine this possibility and to gain an increased understanding of tia metabolic regulation, the transcript levels of eight tia regulatory genes, including all five of the known tia repressor genes, were examined .
orca <dig> overexpression causes increased transcript levels of orca <dig>  interestingly, although overexpression of orca <dig> causes increased orca <dig> transcript levels, overexpression of orca <dig> has no significant affects on orca <dig> transcript levels  <cit> .
these results suggest that, directly or indirectly, orca <dig> regulates orca <dig> at the steady-state mrna level but that orca <dig> does not similarly regulate orca <dig>  orca <dig> also appears to, directly or indirectly, induce its own expression.
hairy root cultures with higher levels of orca <dig> transgene expression also have higher transcript levels from the endogenous orca <dig> gene.
in contrast, orca <dig> overexpression has little effect on crmyc <dig> transcript levels and no significant effects on crbpf <dig> transcript levels.
in addition to regulating some of the tia transcriptional activator genes, orca <dig> regulates the tia transcriptional repressor genes zct <dig>  zct <dig> and zct <dig>  but not gbf <dig> or gbf <dig>  these findings provide a possible explanation for how overexpression of the orca <dig> transcriptional activator gene may lead indirectly to decreased sgd and dat transcript levels by causing overexpression of the zct <dig>  zct <dig> and zct <dig> tia transcriptional repressors, one or more of which may then turn down expression of sgd and dat.
if this model is correct, one might also expect overexpression of orca <dig> to lead eventually to a decrease in expression of tdc and str, as the zcts have been shown to repress expression of both these genes  <cit> .
although expression of tdc and str did decrease between the 48-h and 72-h time points, these decreases were very slight.
these results may be due to the competing effects of orca <dig> induction and zct repression on expression of tdc and str.
in addition, differences in the half-lives of the transcripts produced by different genes will cause differences in the timing with which decreases in gene transcript levels are observed.
induction of zct <dig>  zct <dig> and zct <dig> expression in response to orca <dig> overexpression may help explain why most of the observed alterations in tia metabolite levels in the orca2-induced cultures are transient.
interestingly, overexpression of orca <dig> also results in increased transcript levels of specific tia repressor genes  <cit> .
model for orca <dig> regulation of the tia pathway
based on the results of this study and of other studies, a possible model for regulation of the tia pathway by orca <dig> has been developed .
it should be noted that the available data might also be consistent with other models.
in addition, orca <dig> might induce expression of the indicated genes either directly or indirectly.
for example, orca <dig> might affect expression of some genes indirectly, such as via effects on orca <dig> expression.
in the model presented, orca <dig> expression is induced via the jasmonate-signaling pathway as a defense mechanism against environmental stresses, such as insect attack or fungal elicitation.
increased expression of orca <dig>  as well as signaling via the jasmonate-signaling pathway, leads to increased expression of the orca <dig> transcriptional activator.
whether induction of orca <dig> expression via the jasmonate-signaling pathway occurs independently of orca <dig> induction by orca <dig> is currently unclear.
increased orca <dig>  and orca <dig>  <cit>  activity levels then induce expression of partially overlapping sets of key genes from the tia pathway and both tia feeder pathways.
the increased expression of the tia biosynthetic genes in turn affects the levels of several tias and related metabolites, which may provide protection against the triggering environmental stress.
at approximately the same time that orca <dig> and orca <dig> are inducing expression of tia biosynthetic genes, they are also  inducing expression of the tia transcriptional repressors zct <dig>  zct <dig> and zct <dig>  as the levels of these tia transcriptional repressors rise, they begin to decrease transcription of specific tia biosynthetic genes.
as a result, induction of orca <dig> leads to alterations in the transcript levels of tia biosynthetic genes and in tia metabolite levels that are often transient.
orca <dig> has been proposed to act in a similar manner  <cit> .
this model for orca <dig> action is partially based on previous findings on the role of zcts in repressing activation of str and tdc expression by the orcas  <cit> , the fact that the zcts are transiently induced by fungal elicitors or methyl jasmonate  <cit>  and characterization of the effects of orca <dig> overexpression on expression of tia regulatory genes  <cit> .
this model describes a mechanism whereby alterations in tia metabolite levels in response to environmental stresses may be regulated in such a way as to be transient and of limited magnitude.
CONCLUSIONS
although  <dig> tia transcriptional regulators have been identified, very few studies have been done on any of these regulators, with the exception of orca <dig>  <cit> .
results presented here indicate that orca <dig> plays a critical role in regulation of tia metabolism.
orca <dig> regulates key genes from both feeder pathways, as well as the genes  encoding the enzymes that catalyze the first two steps in tia biosynthesis.
orca <dig> may play an especially important role in regulation of the downstream branches of the tia pathway as it regulates four out of five genes characterized from this part of the pathway.
based on an analysis of the effects of orca <dig> overexpression on transcript levels of other tia regulators, a possible model for the mechanism by which orca <dig> helps regulate tia metabolism has been developed .
this model, together with previous work  <cit> , describes a mechanism that allows alterations in tia metabolite levels in response to external stimuli to be transient and limited in magnitude.

 the plant catharanthus roseus g. don produces a large number of terpenoid indole alkaloids, some of which are of substantial pharmacological interest. vinblastine and vincristine have been used as chemotherapeutics in the treatment of lymphoma and leukemia. ajmalicine and serpentine are sometimes used as anti-hypertensive agents. most of these alkaloids are produced in extremely low amounts in planta, limiting the usage of these chemicals. substantial efforts to use chemical syntheses, in vitro cell cultures or bacterial cells for large-scale production of these alkaloids have proven ineffective. one of the difficulties in developing methods for large-scale production of tias is the complexity of both the tia biosynthetic pathway and of the regulatory pathways governing tia production. despite the complexity of these pathways, significant progress in our understanding of the biochemistry and regulation of the tia pathway in c. roseus has been made in recent years. in particular, many of the genes coding for the tia biosynthetic enzymes and tia transcriptional activators and repressors have been identified. tia biosynthesis is a tightly coordinated process involving at least intermediates, biosynthetic enzymes and several intra and inter cellular compartments. tia biosynthesis in c. roseus starts with the condensation of tryptamine, which is derived from the indole pathway, and secologanin, which is derived from the monoterpenoid pathway, to form strictosidine. this reaction is catalyzed by strictosidine synthase. strictosidine is then deglucosylated by strictosidine β-d-glucosidase to form cathenamine and an equilibrium between cathenamine and -dehydrogeissoschizine is established. different branches of the tia pathway then lead to the production of numerous tias, including ajmalicine and serpentine, lochnericine and hörhammericine, and catharanthine and vindoline. vindoline and catharanthine are the precursors of vinblastine and vincristine. the genes encoding the enzymes catalyzing at least four of the reactions in the six-step pathway leading to production of vindoline from tabersonine have been cloned and characterized. these genes include tabersonine 16-hydroxylase, 16-hydroxytabersonine-16-o-methyltransferase, desacetoxyvindoline 4-hydroxylase, and deacetylvindoline acetyltransferase. an enzyme with anhydrovinblastine synthase activity that catalyzes the synthesis of α-3′, 4′-anhydrovinblastine from vindoline and catharanthine has also been identified and characterized as the major class iii peroxidase in c. roseus leaves. tia biosynthesis is a highly regulated process that involves a number of transcriptional activators and repressors. to date, seven putative activators and five putative repressors have been implicated as regulators of the tia pathway. however, very few studies have been done on any of these regulators, with the exception of orca. both orca and orca are ap2-domain transcription factors that are proposed to activate str expression by binding to the jasmonate and elicitor-responsive element in the str promoter. crbpf is also proposed to activate str transcription by binding to a separate element in the str promoter. crmyc and crmyc are basic helix-loop-helix transcription factors. crmyc has been shown to act upstream of orca and orca activating their transcription. crwrky and crwrky are jasmonate responsive wrky transcription factors that positively regulate expression of several genes involved in tia biosynthesis. overexpression of crwrky also leads to increased transcript levels of the tia transcriptional repressors zct zct and zct and decreased transcript levels of the tia transcriptional activators orca orca and crmyc. in contrast, overexpression of crwrky leads to increased expression of both specific tia transcriptional activators and repressors. the three zinc finger proteins, zct zct and zct were found to bind specifically to the tryptophan decarboxylase and str promoters in vitro, inhibiting their activities. in addition, the zct proteins repress the activation of the str promoter by the orcas. two g-box-binding factors, gbf and gbf were found to repress str transcription by binding to the g-box sites in the str promoter region. the identification of multiple transcriptional activators and repressors for the tia pathway suggests that regulation of this pathway is a complex process. however, to date there have been very few published studies describing in depth characterization of tia regulators other than orca orca has been the focus of several studies in recent years. the results of these studies indicate that orca acts as a positive regulator of many tia biosynthetic genes. for these studies orca was overexpressed in either suspension cells or hairy root cultures. in c. roseus cell cultures, overexpression of orca increases the transcript levels of tdc, str, cytochrome p reductase, 1-deoxy-d-xylulose 5-phosphate synthase, anthranilate synthase α subunit and d4h. in contrast, overexpression of orca in c. roseus cell suspension cultures has no significant effects on transcript levels of geraniol 10-hydroxylase, sgd or dat. in c. roseus hairy root cultures, overexpression of orca induces expression of asα, dxs, secologanin synthase, str and zct zct and zct but represses sgd expression. the transcript levels of tdc, cpr, g10h, gbf gbf and orca were not significantly affected by orca overexpression. although several studies have characterized the role of orca in regulating the tia pathway, little work has been done to characterize the role of the related orca transcriptional activator. recently liu et al. reported the generation of c. roseus transgenic hairy root lines that exhibit constitutive overexpression of orca and stated that catharanthine and vindoline concentrations increased in these lines, but only hplc and an authentic standard were used for the identification of vindoline. vinblastine levels in these lines were below detection limits and other tia metabolites were not assayed as part of this study. the expression levels of tia biosynthetic genes and of other tia regulatory genes were also not characterized by liu and colleagues, leaving the role of orca in regulating tia metabolism unknown. here we describe the generation, metabolic and molecular characterization of a transgenic c. roseus hairy root line that expresses orca under the control of an ethanol-inducible promoter. the transcript levels of a total of tia biosynthetic and regulatory genes were tracked over a period of 72 h following induction of orca overexpression. the levels of seventeen tia and related metabolites were also investigated over the same time period, with thirteen of those metabolites being present at detectable levels in at least some of the samples analyzed. the results of these experiments indicate that orca plays an important role in regulating the tia pathway, particularly the downstream portions of this pathway. based on the results of these experiments, a model for regulation of the tia pathway by orca and other tia transcriptional regulators is presented.  generation of c. roseus transgenic hairy root lines expressing orca under the control of an ethanol-inducible promoter to date, only a few studies have characterized orca and no published studies have determined which tia genes, other than str, are regulated by orca or how orca overexpression affects the levels of a broad group of tia metabolites. as a result, determining the role of orca in regulating the tia pathway has not been possible. to address this deficiency, transgenic hairy root lines that overexpress orca under the control of an ethanol-inducible promoter were generated. the ethanol-inducible system offers significant advantages over constitutive expression systems in that the ethanol-inducible system allows the timing and level of transgene expression to be controlled. as a result, studies on the transient effects of transgene expression are made possible. in addition, the potentially deleterious effects of constitutive overexpression of transgenes on tissue growth and development may be mitigated. to generate transgenic hairy roots, approximately 6-week-old c. roseus seedlings were co-transformed using a mixture of agrobacterium tumefaciens gv carrying the binsrna-orca construct and gv carrying the ppzprol plasmid. the ppzprol plasmid was previously engineered to carry the rola, rolb and rolc genes that are sufficient to induce hairy root production. a total of hairy root lines were screened for genomic integration of orca by assaying for kanamycin resistance and using pcr to identify a dna fragment spanning the orca gene and alca promoter. five of the hairy root lines were found to carry orca under the control of the alca promoter. these positive lines were transferred to liquid culture for adaptation. following adaptation, the lines were screened for ethanol-inducible overexpression of orca figure  shows orca relative transcript levels from the five orca transgenic hairy root lines. the results indicate that the transgenic lines express orca transcripts at approximately three to times higher levels than the control line 10 h after induction by % ethanol. as the orca2-oe line exhibited one of the highest levels of orca induction and adapted well to liquid culture, this line was retained for further study. a control line was also generated by transforming c. roseus seedlings with gv cells carrying the ppzprol construct alone. to determine if ethanol-induced overexpression of orca in the orca2-oe line is maintained for a substantial period of time, a 72-h time course was performed. orca transcripts generated from the orca transgene and by the endogenous orca gene were measured separately in this experiment. an ethanol concentration of % was used for this experiment after this concentration of ethanol was found to be sufficient to cause increases in orca transcript levels similar to those caused by the % ethanol used in earlier experiments. figure 4a shows that orca transgene transcript levels increase dramatically after adding ethanol to orca2-oe root cultures, rising over seven fold within 6 h. orca transgene transcript levels remained high in the induced cultures over the 72-h course of the experiment. in contrast, orca transgene transcript levels decrease in uninduced orca2-oe root cultures upon transfer to fresh media at the 0-h time point. this decrease in orca transgene transcript levels is likely due to the fact that plant cultures grown in liquid typically produce small amounts of ethanol. thus, the 35-d old cultures from which the 0-h time point samples were collected contained small amounts of ethanol, which likely caused a modest induction of orca transgene expression. transferring additional 35-d old cultures to fresh media with no added ethanol at the 0-h time point thus caused a slight decrease in ethanol concentration and, consequently, in orca transgene expression in those cultures. as a result, orca transgene transcript levels are approximately 100-fold higher in the induced than in the uninduced orca2-oe cultures within approximately 12 h after induction. the levels of orca transcripts generated from the endogenous orca gene increase approximately three fold after addition of ethanol to the orca2-oe cultures and then decline back to starting levels by the end of the 72-h time course. in contrast, orca endogenous gene transcript levels decline in the orca2-oe cultures transferred to fresh media with no added ethanol. these results suggest that orca directly or indirectly, induces expression of the orca endogenous gene, as cultures with high levels of expression of the orca transgene express the endogenous orca gene at higher levels than cultures with low levels of expression of the orca transgene. consistent with this hypothesis, orca endogenous gene transcript levels are significantly higher in orca2-oe cultures than in control cultures at the 0 h time point, as orca transgene expression is modestly induced in these orca2-oe cultures by plant-produced ethanol. orca endogenous gene transcript levels are not significantly different in uninduced versus induced cultures of the control line. effects of orca overexpression on the indole and terpenoid pathways to determine if orca plays an important role in regulation of the tia pathway, transcript levels of genes encoding key enzymes and regulators of the tia and related pathways were analyzed in the orca2-oe and control hairy root lines. the genes analyzed include asα and tdc, two key genes from the indole pathway that produces tryptamine; dxs, cpr, g10h and loganic acid o-methyltransferase from the monoterpenoid pathway which leads to the formation of secologanin; and str, sgd, t16h, 16omt, d4h, dat and prx from the tia pathway which catalyzes the condensation of tryptamine and secologanin and ultimately leads to the production of vinblastine and vincristine. transcript levels of four transcriptional activators and five repressors that regulate the tia pathway were also analyzed. to analyze the downstream effects of orca overexpression, the levels of tia and related metabolites were also investigated, with of those metabolites found to be present at detectable levels in at least some of the samples analyzed. both transcript and metabolite levels were tracked over a 72-h period following ethanol induction of orca overexpression, to allow analysis of both transient and relatively prolonged effects. to characterize the effects of orca overexpression on the indole pathway, asα and tdc transcript levels were analyzed. asα encodes the α subunit of anthranilate synthase, which catalyzes the first committed step in the synthesis of tryptophan, the formation of chorismate from anthranilate. tdc encodes tryptophan decarboxylase, which catalyzes the conversion of tryptophan to tryptamine. both asα and tdc transcript levels are significantly higher in the orca2-oe line after ethanol induction than in the uninduced orca2-oe cultures or in the induced or uninduced cultures of the control line. asα expression in the orca2-oe line increases within 12 h of the start of orca induction and then decreases slightly, but remains significantly higher than in the uninduced orca2-oe cultures or in the induced or uninduced cultures of the control line. tdc transcript levels increase within 24 h and increase further within 48 h after the start of orca induction. the levels of tryptamine, the product of the indole pathway that is combined with the terpenoid secologanin to form the first tia, strictosidine, were also analyzed in samples from the same hairy root cultures. tryptamine levels increase significantly within 24 h of induction and remain increased through 72 h. tryptamine levels remain unchanged in the uninduced orca2-oe cultures and the induced and uninduced cultures of the control line. levels of tryptophan were below detection limits. to characterize the effects of orca overexpression on the terpenoid pathway, the transcript levels of dxs, g10h, cpr and lamt were analyzed. transcript levels of dxs and cpr are not significantly different in the induced orca2-oe line than in the uninduced orca2-oe line. in contrast, g10h transcript levels in the orca2-oe line increase within 48 h after the start of orca induction. expression of lamt increases sharply in the orca2-oe line after induction, but then decreases within less than 24 h, reaching levels that are lower than those seen in uninduced orca2-oe cultures within 72 h. addition of ethanol to the “induced” control cultures had little affect on gene expression, indicating that differences in ethanol concentration per se are not responsible for the differences in gene expression observed for the induced versus uninduced orca2-oe cultures. the differences in gene expression observed between the control and orca2-oe cultures at the 0-h time point may be due to the fact that different clonal root cultures have somewhat different growth characteristics. alternatively, some of these differences may be due to the fact that the 35-d old orca2-oe cultures from which the 0-h time point samples were collected likely contained small amounts of root-produced ethanol, which led to a modest induction of orca transgene expression. the effects of orca overexpression on loganin and secologanin levels were also determined. loganin is the precursor for secologanin, which is one of the precursors for formation of the first tia, strictosidine. loganin levels in induced and uninduced orca2-oe cultures are similar. in contrast, secologanin levels are somewhat variable in the orca2-oe line. secologanin levels are significantly decreased in induced orca2-oe cultures relative to uninduced orca2-oe cultures at and 72 h after induction, but are not significantly different at the other time points assayed. effects of orca overexpression on the tia pathway the first step in tia synthesis, the condensation of tryptamine and secologanin to form strictosidine, is catalyzed by str. sgd then uses strictosidine as a substrate to form cathenamine, which serves as a substrate for the formation of several tias via different branches of the tia pathway. to test if orca plays an important role in regulating these processes, transcript levels for str, sgd, t16h, 16omt, d4h, dat and prx were analyzed in the orca2-oe and control hairy root lines for a 72-h period following orca induction. the results of these experiments indicate that transcript levels of six of the seven genes tested are significantly affected by orca overexpression. str, t16h and prx are strikingly induced by orca overexpression, although with somewhat different timing and magnitudes. str transcript levels increase significantly in the orca2-oe cultures within 24 h of the start of orca induction, reaching a maximum of approximately three times higher levels at the 48-h time point than in the orca2-oe cultures at the zero time point. t16h transcript levels also increase significantly within 24 h of the start of induction, reaching levels by the 48 h time point that are to times higher in the induced orca2-oe cultures than in the uninduced orca2-oe cultures at the same time point. prx transcript levels increase approximately fold in the orca2-oe cultures within 12 h after the start of induction and then decline slightly. d4h transcript levels are significantly higher in the induced than in the uninduced orca2-oe cultures at and 72 h after the start of induction, but are only increased by approximately two to four fold. interestingly, transcript levels of both sgd and dat decrease in response to orca overexpression. the timing of the declines in sgd and dat transcript levels are similar, with both genes exhibiting significantly lower transcript levels in the induced versus uninduced orca2-oe cultures within 12 h of the start of induction. however, the magnitudes of the changes in transcript levels are different, as sgd transcript levels are only approximately two fold lower in the induced versus uninduced orca2-oe cultures, whereas dat transcript levels are approximately to fold lower in the induced versus uninduced orca2-oe cultures. of the seven tia biosynthetic genes characterized, only 16omt does not show significant differences in expression levels between uninduced and induced orca2-oe hairy root cultures. the differences in expression of some of the genes between the control and orca2-oe cultures at the 0-h time point may be due to different clonal root cultures having somewhat different growth characteristics and/or to the fact that the orca2-oe cultures likely expressed the orca transgene at modest levels at the 0-h time point due to the presence of small amounts of root-produced ethanol in the 35-d old cultures from which those samples were collected. as orca overexpression causes significant alterations in the transcript levels of a high percentage of the tia biosynthetic genes tested, it was of interest to determine if orca overexpression also alters tia levels. to address this issue, the levels of tias from different branches of the tia pathway were investigated in aliquots of the same tissue samples used to analyze transcript levels. nine of these tias were present at detectable levels, whereas the levels of the other three tias were below detection thresholds. in addition, a tabersonine-like compound, designated unk was detected in the samples. the levels of strictosidine, the first tia to be formed from secologanin and tryptamine, are significantly reduced in the induced versus uninduced orca2-oe cultures within 24 h after the start of induction and remain reduced throughout the remainder of the 72-h time course. ajmalicine and serpentine are formed via the same branch of the tia pathway. both ajmalicine and serpentine levels exhibit modestly significant increases in the induced versus uninduced orca2-oe cultures 48 h after the start of induction, but are not significantly different at the other time points assayed. catharanthine, the precursor for vinblastine production, also exhibits modestly significantly higher levels 48 h after induction, but not at the other time points assayed. the levels of tabersonine, which is the starting material for multiple branches of the tia pathway, are significantly reduced in induced versus uninduced orca-oe cultures within 6 h of the start of orca induction. tabersonine is converted via one branch of the tia pathway to lochnericine and hörhammericine. lochnericine levels are not significantly altered by orca overexpression, but hörhammericine levels are significantly reduced in the induced versus uninduced orca2-oe cultures at and 72 h after the start of induction. tabersonine is converted via a different branch of the tia pathway to 16-hydroxytabersonine. 16ohtab levels exhibit modestly significant increases in the induced versus uninduced orca2-oe cultures at and 72 h after the start of orca induction. tabersonine is also converted to 19-hydroxytabersonine. 19ohtab levels increase within 12 h of the start of orca induction and remain elevated for at least 72 h. levels of vindoline, the end metabolite of this branch of the tia pathway and precursor to vinblastine production, are below detection limits. an un-identified metabolite, designated unk was also detected in the tissue extracts. based on its uv absorption spectra between and 400 nm, unk is a tabersonine-like compound. unk levels exhibit modestly significant increases in the induced versus uninduced orca2-oe cultures at 48 h after the start of induction, but not at the other time points assayed. the levels of the pharmaceutically important tias vinblastine and vincristine, formed by combining catharanthine and vindoline, were below detection limits. effects of orca overexpression on regulators of the tia pathway tia production appears to be regulated via a complex process. a number of transcriptional activators and repressors have been identified, including the transcriptional activators orca orca crbpf crmyc crmyc crwrky and crwrky and the transcriptional repressors zct zct zct gbf and gbf to determine if orca regulates tia transcriptional regulators in addition to tia biosynthetic genes, transcript levels of orca crbpf crmyc zct zct zct gbf and gbf were tracked for a 72-h time period in ethanol-induced and uninduced orca2-oe and control hairy root cultures. the results of these assays indicate that overexpression of orca leads to increased transcript levels of the tia transcriptional activator orca in the induced versus uninduced orca2-oe cultures. orca transcript levels in the induced orca2-oe cultures increase significantly within 12 h of the start of induction and remain elevated throughout the 72-h time course point, compared with the uninduced orca2-oe cultures. overexpression of orca had minimal effects on crmyc transcript levels, with the only significant difference between the orca2-oe induced versus uninduced cultures being a very modest decrease in crmyc transcript levels at the 72-h time point. interestingly, overexpression of orca also causes increases in the steady-state mrna levels of the tia transcriptional repressors zct zct and zct the transcript levels of these three zinc finger genes are significantly higher in the induced versus uninduced orca2-oe cultures within 12 h of the start of induction and remain elevated through the 72-h time point for zct and zct and through the 48-h time point for zct in contrast, overexpression of orca has no significant effects on transcript levels of crbpf gbf or gbf discussion orca is a key regulator of the tia pathway tia biosynthesis in c. roseus is a complex, multi-step process that is regulated by at least twelve transcriptional regulators. the roles of most of these transcriptional regulators, particularly with regards to the downstream branches of the tia pathway, remain to be determined. as the downstream branches of the tia pathway are responsible for production of several pharmaceutically important compounds, this lack of information limits attempts to develop models for regulation of tia production. therefore, the effects of overexpressing orca on transcript levels of tia biosynthetic and regulatory genes were characterized. the results of the research described here indicate that orca overexpression in c. roseus hairy roots significantly affects the transcript levels of several key genes from both pathways leading to production of tia precursors, as well as six of the seven genes characterized from the tia pathway. overexpression of orca causes increased expression of both genes characterized from the indole pathway. of the four genes from the monoterpenoid pathway that were characterized, overexpression of orca causes increased expression of g10h and lamt, but does not significantly affect transcript levels of dxs or cpr. perhaps most interestingly, overexpression of orca leads to significantly altered transcript levels for almost all of the genes from the tia pathway that were characterized, including four out of five genes characterized from the downstream branches of the tia pathway. overexpression of orca results in significantly increased transcript levels for str, which encodes the enzyme that catalyzes the first step in tia biosynthesis. transcript levels of t16h, d4h and prx also increase in response to orca overexpression. in contrast, transcript levels of 16omt are not significantly affected by orca overexpression. somewhat surprisingly, transcript levels of sgd and dat decrease, rather than increase, in response to orca overexpression. the effects of orca overexpression on dat transcript levels are particularly striking, as dat transcript levels are more than times lower in the orca2-oe line 12 h after ethanol induction than in the control line at the zero time point and times lower than in the orca2-oe line at the zero time point. these findings that overexpression of orca causes significant alterations in transcript levels for key genes from both feeder pathways, the gene that encodes the enzyme that catalyzes the combination of the products of those feeder pathways to form the first tia, the gene that encodes the enzyme that catalyzes the conversion of the first tia and four out of five genes characterized from the downstream branches of the tia pathway indicates that orca plays a critical role in regulation of tia metabolism. “+” = up-regulation; “-” = down-regulation. “=” = no change. orca overexpression affects the levels of tia and related metabolites overexpression of orca also affects the levels of multiple tias and related metabolites. in characterizing the indole pathway, tryptophan levels were found to be below detection limits and tryptamine levels were found to increase within 24 h in response to orca overexpression. higher tryptamine levels are consistent with the increased asα and tdc transcript levels caused by orca overexpression. asα encodes the alpha subunit of anthranilate synthase, which catalyzes the first committed step in the tryptophan biosynthetic pathway and is feedback-inhibited by tryptophan. the concurrent increase in tdc expression, however, increases the metabolic flux from tryptophan to tryptamine thus reducing the feedback inhibition of anthranilate synthase by tryptophan. in contrast, of the two metabolites from the terpenoid pathway that were characterized, loganin levels are not significantly affected and secologanin levels are reduced at both the 24- and 72-h time points in response to orca overexpression. two factors may account for the reduced secologanin concentration in response to orca overexpression. one factor is that orca overexpression has a smaller effect on the genes in the terpenoid pathway than on the genes in the indole pathway. the other factor is that the increase in str expression may result in more rapid use of secologanin to form strictosidine. “+” = level of indicated metabolite was higher at one or more time points when orca was overexpressed; “-” = level of indicated metabolite was lower at one or more time points when orca was overexpressed; “=” indicates no significant change in metabolite level when orca was overexpressed; “bd” indicates the metabolite was below detection levels. the transgenic hairy root samples were also analyzed for the levels of tias or tia-related compounds. three of these tias, vindoline, vinblastine and vincristine, were present at levels below the detection threshold. the levels of strictosidine, the first tia produced by combining the products of the indole and terpenoid pathways, are significantly lower within 24 h of the beginning of orca overexpression. this result was somewhat unexpected as the gene that encodes the enzyme that catalyzes the formation of strictosidine is induced by orca overexpression and the gene that encodes the enzyme that converts strictosidine to cathenamine/ -dehydrogeissoschizine/epicathenamine is inhibited by orca overexpression. however, decreased strictosidine levels might be explained by the increased expression of genes encoding enzymes for downstream steps, in the tia pathway, such as t16h and prx causing an increased rate of metabolite flux to the downstream part of the pathway, in combination with the possibility of sgd not being rate limiting for strictosidine metabolism. alternatively, the reduced sgd transcript levels caused by orca overexpression may not lead to similar decreases in sgd activity levels. consistent with the possibilities that sgd activity levels are either not rate limiting or are not decreased, despite the decrease in sgd transcript levels, are the findings that ajmalicine and serpentine are transiently increased in response to orca overexpression. both ajmalicine and serpentine, which are made via the same branch of the tia pathway, are present in significantly higher concentrations 48 h after the start of orca induction, but are not present at significantly altered levels at the other time points assayed. also consistent with these possibilities are findings that orca overexpression leads to transient increases in the levels of catharanthine, which is made via a different branch of the tia pathway, as catharanthine levels are significantly increased 48 h after the start of orca induction. the levels of tabersonine, which is used as the starting material for multiple branches of the tia pathway, are decreased in response to orca overexpression. decreased tabersonine levels in response to orca overexpression could be the result of a decreased rate of tabersonine synthesis or an increased rate of tabersonine metabolism. consistent with the first possibility is the finding that sgd transcript levels decrease in response to orca overexpression, as sgd activity is necessary for tabersonine production. however, the decrease in sgd transcript levels first occurs at a later time point than the decrease in tabersonine levels, making the extent to which decreases in sgd transcript levels are responsible for the decreased tabersonine levels unclear. increases in t16h transcript levels in response to orca overexpression are consistent with the possibility that decreased tabersonine levels are due to a higher rate of tabersonine metabolism, as t16h catalyzes the first step for one branch of the tia pathway, namely the conversion of tabersonine to 16-hydroxytabersonine. however, t16h transcript levels first increase at a later time point than the decrease in tabersonine levels, making the extent to which increases in t16h transcript levels cause decreased tabersonine levels unclear. 16omt catalyzes the conversion of 16-hydroxytabersonine to 16-methoxytabersonine. 16omt transcript levels are not significantly affected by orca overexpression. as 16-hydroxytabersonine levels increase within 48 h of the start of orca overexpression, these results suggest that 16omt activity levels may be rate limiting in hairy roots where orca is overexpressed. interestingly, the increase in 16-hydroxytabersonine levels accounts for only a small portion of the substantial decrease in tabersonine levels, raising the possibility that the levels of other tabersonine-related metabolites are raised in response to orca overexpression. consistent with this possibility is the finding that the levels of 19ohtab, which is also made from tabersonine, increase significantly in response to induction of orca overexpression. in addition, the identification of a tabersonine-like compound, here designated unk that is present at significantly higher levels in orca2-oe induced versus uninduced cultures is also consistent with the idea that the levels of tabersonine-related metabolites increase in response to orca overexpression. lochnericine and hörhammericine are also made from tabersonine, via a different branch of the tia pathway. overexpression of orca has no significant effects on lochnericine levels, but leads to decreased hörhammericine levels at the 24- and 72-h time points, suggesting that overexpression of orca causes an overall decrease in metabolic flux to this branch of the tia pathway. orca regulates other tia transcriptional regulators as orca is believed to act as a transcriptional activator, findings that overexpression of orca causes significant decreases in sgd and dat transcript levels are somewhat surprising and suggest that regulation of the tia pathway is complex. a possible explanation for these results is that overexpression of orca may cause increased expression of one or more tia repressors. to examine this possibility and to gain an increased understanding of tia metabolic regulation, the transcript levels of eight tia regulatory genes, including all five of the known tia repressor genes, were examined. orca overexpression causes increased transcript levels of orca interestingly, although overexpression of orca causes increased orca transcript levels, overexpression of orca has no significant affects on orca transcript levels. these results suggest that, directly or indirectly, orca regulates orca at the steady-state mrna level but that orca does not similarly regulate orca orca also appears to, directly or indirectly, induce its own expression. hairy root cultures with higher levels of orca transgene expression also have higher transcript levels from the endogenous orca gene. in contrast, orca overexpression has little effect on crmyc transcript levels and no significant effects on crbpf transcript levels. in addition to regulating some of the tia transcriptional activator genes, orca regulates the tia transcriptional repressor genes zct zct and zct but not gbf or gbf these findings provide a possible explanation for how overexpression of the orca transcriptional activator gene may lead indirectly to decreased sgd and dat transcript levels by causing overexpression of the zct zct and zct tia transcriptional repressors, one or more of which may then turn down expression of sgd and dat. if this model is correct, one might also expect overexpression of orca to lead eventually to a decrease in expression of tdc and str, as the zcts have been shown to repress expression of both these genes. although expression of tdc and str did decrease between the 48-h and 72-h time points, these decreases were very slight. these results may be due to the competing effects of orca induction and zct repression on expression of tdc and str. in addition, differences in the half-lives of the transcripts produced by different genes will cause differences in the timing with which decreases in gene transcript levels are observed. induction of zct zct and zct expression in response to orca overexpression may help explain why most of the observed alterations in tia metabolite levels in the orca2-induced cultures are transient. interestingly, overexpression of orca also results in increased transcript levels of specific tia repressor genes. model for orca regulation of the tia pathway based on the results of this study and of other studies, a possible model for regulation of the tia pathway by orca has been developed. it should be noted that the available data might also be consistent with other models. in addition, orca might induce expression of the indicated genes either directly or indirectly. for example, orca might affect expression of some genes indirectly, such as via effects on orca expression. in the model presented, orca expression is induced via the jasmonate-signaling pathway as a defense mechanism against environmental stresses, such as insect attack or fungal elicitation. increased expression of orca as well as signaling via the jasmonate-signaling pathway, leads to increased expression of the orca transcriptional activator. whether induction of orca expression via the jasmonate-signaling pathway occurs independently of orca induction by orca is currently unclear. increased orca and orca activity levels then induce expression of partially overlapping sets of key genes from the tia pathway and both tia feeder pathways. the increased expression of the tia biosynthetic genes in turn affects the levels of several tias and related metabolites, which may provide protection against the triggering environmental stress. at approximately the same time that orca and orca are inducing expression of tia biosynthetic genes, they are also inducing expression of the tia transcriptional repressors zct zct and zct as the levels of these tia transcriptional repressors rise, they begin to decrease transcription of specific tia biosynthetic genes. as a result, induction of orca leads to alterations in the transcript levels of tia biosynthetic genes and in tia metabolite levels that are often transient. orca has been proposed to act in a similar manner. this model for orca action is partially based on previous findings on the role of zcts in repressing activation of str and tdc expression by the orcas, the fact that the zcts are transiently induced by fungal elicitors or methyl jasmonate and characterization of the effects of orca overexpression on expression of tia regulatory genes. this model describes a mechanism whereby alterations in tia metabolite levels in response to environmental stresses may be regulated in such a way as to be transient and of limited magnitude.  although tia transcriptional regulators have been identified, very few studies have been done on any of these regulators, with the exception of orca. results presented here indicate that orca plays a critical role in regulation of tia metabolism. orca regulates key genes from both feeder pathways, as well as the genes encoding the enzymes that catalyze the first two steps in tia biosynthesis. orca may play an especially important role in regulation of the downstream branches of the tia pathway as it regulates four out of five genes characterized from this part of the pathway. based on an analysis of the effects of orca overexpression on transcript levels of other tia regulators, a possible model for the mechanism by which orca helps regulate tia metabolism has been developed. this model, together with previous work, describes a mechanism that allows alterations in tia metabolite levels in response to external stimuli to be transient and limited in magnitude. 
orca may play an especially important role in regulation of the downstream branches of the tia pathway, as it regulates four out of five genes characterized from this part of the pathway. regulation of tia transcriptional repressors by orca may provide a mechanism whereby increases in tia metabolite levels in response to external stimuli are transient and limited in magnitude. transcriptional profiling experiments revealed that overexpression of orca results in altered expression of key genes from the indole and terpenoid pathways, which produce precursors for the tia pathway, and from the tia pathway itself. orca regulates expression of key genes from both feeder pathways, as well as the genes encoding the enzymes that catalyze the first two steps in tia biosynthesis. orca overexpression also causes significant increases in transcript levels of several tia regulators, including tia transcriptional repressors. results presented here indicate that orca plays a critical role in regulation of tia metabolism. in addition, metabolite-profiling experiments revealed that overexpression of orca significantly affects the levels of several tia metabolites.
 the terpenoid indole alkaloid pathway leads to the production of pharmaceutically important drugs, such as the anticancer compounds vinblastine and vincristine. unfortunately, these drugs are produced in trace amounts, causing them to be very costly. to increase production of these drugs, an improved understanding of the tia regulatory pathway is needed. towards this end, transgenic catharanthus roseus hairy roots that overexpress the orca tia transcriptional activator were generated and characterized.  transcriptional profiling experiments revealed that overexpression of orca results in altered expression of key genes from the indole and terpenoid pathways, which produce precursors for the tia pathway, and from the tia pathway itself. in addition, metabolite-profiling experiments revealed that overexpression of orca significantly affects the levels of several tia metabolites. orca overexpression also causes significant increases in transcript levels of several tia regulators, including tia transcriptional repressors.  results presented here indicate that orca plays a critical role in regulation of tia metabolism. orca regulates expression of key genes from both feeder pathways, as well as the genes encoding the enzymes that catalyze the first two steps in tia biosynthesis. orca may play an especially important role in regulation of the downstream branches of the tia pathway, as it regulates four out of five genes characterized from this part of the pathway. regulation of tia transcriptional repressors by orca may provide a mechanism whereby increases in tia metabolite levels in response to external stimuli are transient and limited in magnitude. terpenoid indole alkaloidsorca2catharanthus roseushairy root cultures 