MJ potentiates secondary metabolite accumulation in a variety of plant suspension cell culture systems, and is now a widely-used augmenting tool for bio-active molecule production
. Accordingly, paclitaxel accumulation is significantly enhanced with MJ elicitation in T. cuspidata suspension cultured cells
[27, 37–40]. MJ is also implicated in specific induction of a wide range of secondary metabolite genes involved in biotic-stress response and wounding
. Conversely, primary metabolic genes such as those involved in photosynthesis, electron transport and cytoskeletal organization are either down-regulated or unaffected by MJ elicitation
. MJ elicitation and targeted cloning approaches have been effectively used in Taxus to identify specific taxane biosynthetic genes
[43, 44]. Therefore, an up-regulated cDNA library of MJ treated cells will presumably contain genes related to secondary metabolism, including paclitaxel biosynthesis.
SSH is a powerful genomics technique that enriches differentially regulated genes and has mostly been used to identify transcripts expressed in contrasting environmental conditions. SSH is a suitable approach in Taxus where the genomic information is currently scarce. We used the PCR-based SSH approach to identify MJ responsive (up- and down-regulated) transcripts that precede paclitaxel accumulation in the paclitaxel accumulating T. cuspidata P991 cell line. Putative up-regulation of as many as 155 unigenes and down-regulation of 176 unigenes of diverse functional groups suggested involvement of a spectrum of genes regulating paclitaxel biosynthesis, MJ-signaling and response, and potentially, taxane transport and taxane degradation. The up-regulated SSH library contained several previously known positively regulated genes that are likely to be directly involved in paclitaxel synthesis ( Additional file
2: Table S2). For instance, putative taxadiene synthase, taxane hydroxylase and taxane acyl transferases having strong sequence similarity with the corresponding known paclitaxel biosynthetic pathway genes were detected in the up-regulated library. Taxadiene synthase catalyses the first committed step of paclitaxel biosynthesis in Taxus by cyclization of the linear isoprenoid substrate geranyl geranyl diphosphate (GGPP) to form taxa-4(5),11(12)diene
. Four unigenes representing different isoforms of putative taxadiene synthase were up-regulated in the library. It will be interesting to unravel the individual contribution of each of these taxadiene synthase genes towards paclitaxel production. Another paclitaxel biosynthetic gene, taxoid 2-α-hydroxylase, produces 2α-hydroxy taxoids as starting materials for subsequent acylation at the C2-position of the taxane core that ultimately bears a benzoyl group, an important paclitaxel pharmacophore
. Five novel putative taxoid hydroxylases that are similar but not identical to known taxoid hydrolases (taxoid 2-alpha hydroxylase, taxoid 5-alpha hydroxylase, and taxoid 7-beta hydroxylase) were detected in the up-regulated library. Each might function to hydroxylate taxoids at specific positions. Similarly, novel taxane acyl transferases are not only important targets for genetic manipulation to improve paclitaxel production in genetically engineered host systems but also provides a means of attaching modified aroyl groups to taxoid precursors to improve drug efficacy
[18, 21, 25, 46]. Characterization of the two newly identified candidate acyl transferases in the library will further suggest roles for these novel enzymes in paclitaxel biosynthesis.
Apart from the paclitaxel biosynthesis genes, up-regulated genes that are potential candidates for regulation of the MJ response were also obtained. One such gene encodes ACC oxidase (1-aminocyclopropane-1-carboxylate oxidase),which is the final step in the synthesis of the plant hormone ethylene
. Ethylene is known to be produced following MJ elicitation, and to function in suppression of the MJ signal. An increased production of paclitaxel upon application of ethylene synthesis inhibitors to elicited Taxus cultures was reported by Zhang et al., (2003)
. Disruption of this ACC oxidase gene may allow the MJ signal to be strengthened, which may lead to increased paclitaxel accumulation.
The library was also enriched with up-regulated genes that presumably have no direct role in paclitaxel accumulation. Good examples are the putative class I chitinase, thaumatin (also known as PR-5); chalcone synthase, and defensin, all of which are defense-related genes that have well-characterized roles in model plant species
[49, 50]. These genes are known to be induced by MJ in other plant species and thus are unlikely to be related to the synthesis of taxanes, which are unique to Taxus species. For several transcripts, a reliable prediction of general biochemical activity can be made, but predictions neither suggest nor challenge a role in paclitaxel synthesis. Genes in this class include three cytochrome P450 genes that lack significant similarity to the several known taxane biosynthetic pathway P450s, two distinct lipoxygenase genes, and a few other enzyme encoding genes. Any of these genes could participate in the uncharacterized steps of the paclitaxel biosynthetic pathway (e.g., hydroxylation reactions and oxetane ring formation), but could equally well be unrelated to paclitaxel synthesis. Further characterization will be necessary to determine whether these genes have direct relevance to paclitaxel biosynthesis.
A large class of genes (59%) have functions that cannot be predicted based on sequence similarity information. These genes may be involved in important but uncharacterized processes of MJ-signaling, taxane transport or taxane degradation. As many as 47% of these ESTs showing no sequence homology with any of the available Genbank sequences may be specific to Taxus species. The other 12% of sequences are similar to genes described as encoding hypothetical proteins or proteins with unknown function. Enrichment of unigenes belonging to various biological process categories (GO), such as oxidation-reduction process (GO:0055114), response to stimulus (GO:0050896), small molecule biosynthetic process (GO:0044283) and paclitaxel biosynthetic process (GO:0042617), provide strong molecular evidence that the SSH-based MJ elicited transcript screening is a good method to identify genes related to paclitaxel metabolism. Nevertheless, this strategy did not enable the isolation of all the known paclitaxel metabolism genes indicating that the collection of MJ-inducible genes identified here is not comprehensive. Sequencing of the total MJ-induced transcriptome will be required to identify transcripts for every gene related to paclitaxel accumulation.
In addition to confirming up-and down-regulated genes, macroarrays identified false positive clones that appeared in the libraries but were not actually differentially regulated in response to MJ. Although most genes identified as being up-regulated were confirmed as such by macroarray analysis, the false positive rate in the down-regulated libraries was 92.2%. SSH libraries of highly complex eukaryotes have often been reported to contain high percentages (30-50%) of false positive clones that escaped subtraction
[51, 52]. These high false positive rates are usually attributed to non-specific annealing of primers, partial hybridization of driver that allows tester amplification and non-specific annealing of suppression adaptors
. While any of these could have been factors in producing the high false positive rate in our down-regulated libraries, we also note the possibility that there could simply be very few genes that are down-regulated by MJ in the Taxus cultures. If few or no down-regulated transcripts occur, then the SSH approach is certain to be unsuccessful, and will identify a random set of highly expressed (but not differentially regulated) genes. Genes encoding cell wall proteins, ribosomal proteins and other highly expressed genes are well represented in the down-regulated libraries. The high false positive rate in the down-regulated libraries here indicates that the genes observed were not actually down-regulated by MJ.
The gene expression kinetics revealed differential temporal regulation of MJ responsive genes. Up-regulation of many of the MJ induced genes at early time points (6h) was highest, followed by middle time point (18h). MJ-responsive up-regulation of most genes, however, did not persist into the latest time point 5 day (Figure
2). However, a subset of late-induced genes was observed. In agreement to this study, similar expression trend was also observed previously in MJ-induced temporal expression kinetics of taxol biosynthesis genes in P991 cultured cells
. This trend was also observed in the paclitaxel-producing T. cuspidata P93AF cell line, as semi-quantitative RT-PCR showed that all 12 genes were up-regulated at 6h but transcripts of only 5 of them were detected on day 5. These 12 genes tested here by RT-PCR do not represent any known genes related to taxol biosynthesis, but rather were selected as novel transcripts identified in this study. Furthermore, use of a second paclitaxel-producing cell line (P93AF) demonstrates the reproducibility of the gene expression changes occurring in Taxus cultured cells. Paclitaxel and other taxanes accumulate in the cultured cells at time points after 24h
. Therefore, persistent translated gene products of early MJ induced genes appear to play a pivotal role in taxane biosynthesis, while late MJ induced genes may be facilitating other functions such as taxane transport and degradation. Genes involved either in intracellular or extracellular paclitaxel transport and degradation in Taxus have not yet been characterized
, but may be present among the ESTs identified here that could not be assigned functions based on molecular similarity to known proteins.