Biomedical subjects

Susan I Gibson

Publications and source records attributed to Susan I Gibson.

12 recordsLinked to original sources

PubMed2006

Effects of terpenoid precursor feeding on Catharanthus roseus hairy roots over-expressing the alpha or the alpha and beta subunits of anthranilate synthase.

Among the pharmacologically important terpenoid indole alkaloids produced by Catharanthus roseus are the anti-cancer drugs vinblastine and vincristine. These two drugs are produced in small yields within the plant, which makes them expensive to produce commercially. Metabolic engineering has focused on increasing flux through this pathway by various means such as elicitation, precursor feeding, and introduction of genes encoding specific metabolic enzymes into the plant. Recently in our lab, a feedback-resistant anthranilate synthase alpha subunit was over-expressed in C. roseus hairy roots under the control of a glucocorticoid inducible promoter system. Upon induction we observed a large increase in the indole precursors, tryptophan, and tryptamine. The current work explores the effects of over-expressing the anthranilate synthase alpha or alpha and beta subunits in combination with feeding with the terpenoid precursors 1-deoxy-D-xylulose, loganin, and secologanin. In feeding 1-deoxy-D-xylulose to the hairy root line expressing the anthranilate synthase alpha subunit, we observed an increase of 125% in hörhammericine levels in the induced samples, while loganin feeding increased catharanthine by 45% in the induced samples. Loganin feeding to the hairy root line expressing anthranilate synthase alpha and beta subunits increases catharanthine by 26%, ajmalicine by 84%, lochnericine by 119%, and tabersonine by 225% in the induced samples. These results suggest that the terpenoid precursors to the terpenoid indole alkaloids are important factors in terpenoid indole alkaloid production.

Anthranilate Synthase↗

PubMed2006

Terpenoid indole alkaloid production by Catharanthus roseus hairy roots induced by Agrobacterium tumefaciens harboring rol ABC genes.

We have established Catharanthus roseus hairy root cultures transgenic for the rol ABC genes from T(L)-DNA of the agropine-type Agrobacterium rhizogenes strain A4. The rol ABC hairy root lines exhibit a wild-type hairy root syndrome in terms of growth and morphology on solid medium. However, they differ from wild-type hairy root lines in that they more frequently have excellent adaptability to liquid medium and do not appear to form calli during cultivation. Moreover, they do not produce detectable levels of mannopine and agropine which, in contrast, are often synthesized abundantly in wild-type hairy root lines. The absence of these opines does not appear to cause the rol ABC lines to have higher levels of terpenoid indole alkaloids than wild-type hairy root lines. Unlike wild-type lines, rol ABC lines produce very similar levels of total alkaloids despite wide variations in individual alkaloid contents. This work demonstrates that the three genes rol ABC are sufficient to induce high-quality hairy roots in Catharanthus roseus.

Agrobacterium tumefaciens↗

PubMed2005

Expression of the Arabidopsis feedback-insensitive anthranilate synthase holoenzyme and tryptophan decarboxylase genes in Catharanthus roseus hairy roots.

In plants, the indole pathway provides precursors for a variety of secondary metabolites. In Catharanthus roseus, a decarboxylated derivative of tryptophan, tryptamine, is a building block for the biosynthesis of terpenoid indole alkaloids. Previously, we manipulated the indole pathway by introducing an Arabidopsis feedback-insensitive anthranilate synthase (AS) alpha subunit (trp5) cDNA and C. roseus tryptophan decarboxylase gene (TDC) under the control of a glucocorticoid-inducible promoter into C. roseus hairy roots [Hughes, E.H., Hong, S.-B., Gibson, S.I., Shanks, J.V., San, K.-Y. 2004a. Expression of a feedback-resistant anthranilate synthase in Catharanthus roseus hairy roots provides evidence for tight regulation of terpenoid indole alkaloid levels. Biotechnol. Bioeng. 86, 718-727; Hughes, E.H., Hong, S.-B., Gibson, S.I., Shanks, J.V., San, K.-Y. 2004b. Metabolic engineering of the indole pathway in Catharanthus roseus hairy roots and increased accumulation of tryptamine and serpentine. Metabol. Eng. 6, 268-276]. Inducible expression of either or both transgenes did not lead to significant increases in overall alkaloid levels despite the considerable accumulation of tryptophan and tryptamine. In an attempt to more successfully engineer the indole pathway, a wild type Arabidopsis ASbeta subunit (ASB1) cDNA was constitutively expressed along with the inducible expression of trp5 and TDC in C. roseus hairy roots. Transgenic hairy roots expressing both trp5 and ASB1 show a significantly greater resistance to feedback inhibition of AS activity by tryptophan than plants expressing only trp5. In fact, a 4.5-fold higher concentration of tryptophan is required to achieve 50% inhibition of AS activity in plants overexpressing both genes than in plants expressing only trp5. In addition, upon a 3 day induction during the exponential phase, a trp5:ASB1 hairy root line produced 1.8 times more tryptophan (specific yield ca. 3.0 mg g(-1) dry weight) than the trp5 hairy root line. Concurrently, tryptamine levels increase up to 9-fold in the induced trp5:ASB1 line (specific yield ca. 1.9 mg g(-1) dry weight) as compared with only a 4-fold tryptamine increase in the induced trp5 line (specific yield ca. 0.3 mg g(-1) dry weight). However, endogenous TDC activities of both trp5:ASB1 and trp5 lines remain unchanged irrespective of induction. When TDC is ectopically expressed together with trp5 and ASB1, the induced trp5:ASB1:TDC hairy root line accumulates tryptamine up to 14-fold higher than the uninduced line. In parallel with the remarkable accumulation of tryptamine upon induction, alkaloid accumulation levels were significantly changed depending on the duration and dosage of induction.

Anthranilate Synthase↗

PubMed2005

Control of plant development and gene expression by sugar signaling.

Coordination of development with the availability of nutrients, such as soluble sugars, may help ensure an adequate supply of building materials and energy with which to carry out specific developmental programs. For example, in-vivo and in-vitro experiments suggest that increasing sugar levels delay seed germination and stimulate the induction of flowering and senescence in at least some plant species. Higher sugar concentrations can also increase the number of tubers formed by potatoes and can stimulate the formation of adventitious roots by Arabidopsis. New insights into the mechanisms by which sugar-response pathways interact with other response pathways have been provided by microarray experiments examining sugar-regulated gene expression under different light and nitrogen conditions.

Carbohydrates↗

PubMed2004

Expression of a feedback-resistant anthranilate synthase in Catharanthus roseus hairy roots provides evidence for tight regulation of terpenoid indole alkaloid levels.

Different plant species produce a variety of terpenoid indole alkaloids, which are of interest as plant defensive secondary metabolites and as valuable pharmaceuticals. Although significant progress has been made, the mechanisms regulating the levels of this important class of compounds require continued elucidation. Previous precursor feeding studies have indicated that alkaloid accumulation can be improved during the exponential growth phase of hairy root cultures through enhanced tryptophan availability. To test this relationship, transgenic hairy root cultures of Catharanthus roseus were established with a glucocorticoid-inducible promoter controlling the expression of an Arabidopsis feedback-resistant anthranilate synthase alpha subunit. Enzyme assays demonstrated that the Arabidopsis alpha subunit is compatible with the native beta subunit and that anthranilate synthase activity is more resistant to tryptophan inhibition in induced than in uninduced extracts. The metabolic effects of expressing the feedback-resistant anthranilate synthase alpha subunit were also dramatic. Over a 6-day induction period during the late exponential growth phase, tryptophan and tryptamine specific yields increased from almost undetectable levels to 2.5 mg/g dry weight and from 25 microg/g to 267 microg/g dry weight, respectively. The greater than 300-fold increase in tryptophan levels observed in these studies under certain induction conditions compares favorably with the fold increases obtained in previous constitutive expression studies. Despite the large increases in tryptophan and tryptamine, the levels of most terpenoid indole alkaloids were not significantly altered, with the exception of lochnericine, which increased 81% after a 3-day induction period. These results suggest that terpenoid indole alkaloid levels are tightly controlled.

Anthranilate Synthase↗

PubMed2004

Metabolic engineering of the indole pathway in Catharanthus roseus hairy roots and increased accumulation of tryptamine and serpentine.

Transgenic hairy roots of Catharanthus roseus were established with glucocorticoid inducible tryptophan decarboxylase (TDC) expression alone or in combination with inducible expression of a feedback-resistant anthranilate synthase alpha subunit (ASalpha) from Arabidopsis. Northern blot analysis confirmed transcription of the anthranilate synthase gene upon induction in the double line (TDC+ASalpha) and in vitro enzyme assays confirmed increased resistance to feedback inhibition by tryptophan. In TDC enzyme assays, increases of 48% and 87% in the TDC and double lines, respectively, were noted. Although the TDC line showed no significant increase in tryptamine levels on induction, induction of the double line resulted in increases in tryptamine levels of as much as six-fold for a 3 day late exponential induction. Downstream effects on alkaloids were noted in the TDC line where serpentine specific yields increased as much as 129% on induction. No effects on measured alkaloids were noted in the double line, but the two clones have very different basal alkaloid biosynthetic capacities. Within this study, the engineering of the indole pathway in C. roseus hairy roots is reported, and the role of the indole pathway in alkaloid biosynthesis explored.

Catharanthus↗

PubMed2003

Sugar and phytohormone response pathways: navigating a signalling network.

Many plant developmental, physiological and metabolic processes are regulated, at least in part, by nutrient availability. In particular, alterations in the availability of soluble sugars, such as glucose and sucrose, help regulate a diverse array of processes. Multiple lines of evidence indicate that many of these processes are also regulated in response to other signalling molecules, such as phytohormones. This review draws examples from a variety of plant systems, including bean, Arabidopsis, potato, and cereals. Five of the most interesting and best developed examples of processes regulated via 'interactions' or 'crosstalk' between sugars and phytohormones are described, including embryogenesis, seed germination, early seedling development, tuberization, and the regulation of alpha-amylase activity. The types of mechanisms by which different response pathways are known or postulated to interact are also described. These mechanisms include regulation of the metabolism and/or transport of a signalling molecule by a different response pathway. For example, sugars have been postulated to help regulate the synthesis, conjugation and/or transport of phytohormones, such as gibberellins and abscisic acid. Conversely, phytohormones, such as abscisic acid, gibberellins and cytokinins have been shown to help regulate sugar metabolism and/or transport. Similarly, sugars have been shown to regulate the expression of components of phytohormone-response pathways and phytohormones regulate the expression of some genes encoding possible components of sugar-response pathways. Examples of proteins and second messengers that appear to act in multiple response pathways are also described.

Arabidopsis↗

PubMed2002

Mobilization of seed storage lipid by Arabidopsis seedlings is retarded in the presence of exogenous sugars.

BACKGROUND: Soluble sugar levels must be closely regulated in germinating seeds to ensure an adequate supply of energy and building materials for the developing seedling. Studies on germinating cereal seeds indicate that production of sugars from starch is inhibited by increasing sugar levels. Although numerous studies have focused on the regulation of starch metabolism, very few studies have addressed the control of storage lipid metabolism by germinating oilseeds. RESULTS: Mobilization of storage lipid by germinating seeds of the model oilseed plant Arabidopsis thaliana (L.) Heynh. occurs at a greatly reduced rate in the presence of exogenous glucose or mannose, but not in the presence of equi-molar 3-O-methylglucose or sorbitol. The sugar-insensitive5-1/abscisic acid-insensitive4-101 (sis5-1/abi4-101) mutant is resistant to glucose inhibition of seed storage lipid mobilization. Wild-type seedlings become insensitive to glucose inhibition of storage lipid breakdown within 3 days of the start of imbibition. CONCLUSIONS: Growth in the presence of exogenous glucose significantly retards mobilization of seed storage lipid in germinating seeds from wild-type Arabidopsis. This effect is not solely due to the osmotic potential of the media, as substantially higher concentrations of sorbitol than of glucose are required to exert significant effects on lipid breakdown. The inhibitory effect of glucose on lipid breakdown is limited to a narrow developmental window, suggesting that completion of some critical metabolic transition results in loss of sensitivity to the inhibitory effect of glucose on lipid breakdown.

3-O-Methylglucose↗

PubMed2002

ABA and sugar interactions regulating development: cross-talk or voices in a crowd?

Plant growth and development are controlled by the concerted action of many signaling pathways that integrate information from environmental signals with that from developmental and metabolic cues. Physiological studies have demonstrated that abscisic acid and sugars have both similar and antagonistic effects on diverse processes, including seed development, germination, and seedling growth. Recent genetic studies have identified several loci that are involved in both sugar and hormonal responses. It is rarely clear whether these apparent linkages reflect direct or indirect interactions between sugar and hormone signaling pathways, but the identification of gene products that are encoded at these loci is allowing these possibilities to be tested.

Abscisic Acid↗

PubMedDate not supplied

Characterization of an inducible promoter system in Catharanthus roseus hairy roots.

Transgenic hairy root cultures of Catharanthus roseus were established with a glucocorticoid-inducible promoter controlling the expression of green fluorescent protein (GFP), and GFP expression was characterized. The inducible system shows a tightly controlled, reversible, and dosage-dependent response to the glucocorticoid dexamethasone in C. roseus hairy roots. Full induction was noted after 12-18 h in the mature regions of the root tips and after 6 h in the meristem tissue. Upon removal of the inducing agent, GFP expression declined to undetectable levels in the mature tissues after 24 h and in the meristem after 48 h. Although no dosage-dependent response was noted in the meristem region, such a response was apparent in the mature region of the tip and verified by quantitative GFP analysis. The inducible promoter system allowed quantitative control of GFP expression between 0.01 and 10 microM dexamethasone with saturation occurring at higher levels. Using GFP as a model system allowed demonstration of the ability to control temporal and quantitative gene expression with the glucocorticoid-inducible promoter in transgenic C. roseus hairy roots.

Catharanthus↗

PubMedDate not supplied

Role of the non-mevalonate pathway in indole alkaloid production by Catharanthus roseus hairy roots.

The 1-deoxy-D-xylulose-5-phosphate (DXP) pathway (non-mevalonate pathway) leading to terpenoids via isopentenyl diphosphate (IPP) has been shown to occur in most bacteria and in all higher plants. Treatment with the antibiotic fosmidomycin, a specific inhibitor of DXP reductoisomerase, considerably inhibited the accumulation of the alkaloids ajmalicine, tabersonine, and lochnericine by Catharanthus roseus hairy root cultures in the exponential growth phase. However, fosmidomycin did not significantly affect alkaloid levels in stationary phase hairy root cultures. Feeding with 1-deoxy-D-xylulose, 10-hydroxygeraniol, or loganin resulted in significant increases in alkaloid production by exponential phase hairy root cultures. These results suggest that the DXP pathway is a major provider of carbon for the monoterpenoid pathway leading to the formation of indole alkaloids in C. roseus hairy roots in the exponential phase.

Catharanthus↗

PubMedDate not supplied

Transient effects of overexpressing anthranilate synthase alpha and beta subunits in Catharanthus roseus hairy roots.

Catharanthus roseus produces two economically valuable anticancer drugs, vinblastine and vincristine. These drugs are members of the terpenoid indole alkaloids and accumulate in small quantities within the plant; thus these two drugs are expensive to produce. Metabolic engineering efforts have focused on increasing the alkaloids in this pathway through various means such as elicitation, precursor feeding, and gene overexpression. Recently we successfully expressed Arabidopsis genes encoding a feedback-insensitive anthranilate synthase alpha subunit under the control of the glucocorticoid-inducible promoter system and the anthranilate synthase beta subunit under the control of a constitutive promoter in C. roseus hairy roots. In this work we look at the transient behaviors of terpenoid indole alkaloids over a 72 h induction period in late exponential growth phase cultures. Upon induction, the tryptophan, tryptamine, and ajmalicine pools accumulated over 72 h. In contrast, the lochnericine, hörhammericine, and tabersonine pools decreased and leveled out over the 72 h induction period. Visible changes within the individual compounds usually took from 4 to 12 h.

Anthranilate Synthase↗