Journal of Experimental Botany, Vol. 53, No. 376, pp. 1891-1897,
September 1, 2002
© 2002 Oxford University Press
Isolation and characterization of the CYP71D16 trichome-specific promoter from Nicotiana tabacum L.
Received 18 January 2002; Accepted 11 June 2002
Plant Physiology/Biochemistry/Molecular Biology Program, Agronomy Department, 200L THRI, University of Kentucky, Lexington, KY40546-0236, USA
1 To whom correspondence should be addressed. Fax: +1 859 323 1077. E-mail: gwagner{at}uky.edu
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Abstract |
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Trichomes are specialized epidermal cells that produce secretions that are thought to provide a first line of defence against pests and pathogens. Many trichome-secreted compounds are used commercially as flavourings, medicines, etc. Described here is the cloning and characterization of the promoter of a tobacco trichome-specific P450 gene, CYP71D16. This promoter is shown to direct the specific expression of the reporter gene, ß-glucuronidase (GUS), in glandular trichomes of Nicotiana tabacum cv. T.I. 1068 at all developmental stages. With the full promoter, GUS activity was predominantly in the gland cell, with less in the stalk cell adjacent to the gland, and in lower stalk cells. GUS staining was also observed in the most distal trichome stalk cells of non-glandular trichomes found on variety T.I. 1112. Promoter deletion analysis revealed that the region from –223 to +111 bp is sufficient to direct trichome-specific expression, but not strong gland expression. Examination of the literature suggests that this is the first characterized trichome-specific-promoter shown to function at all stages of plant development. This promoter may provide efficient bioengineering to enhance pest and pathogen resistance, and for molecular farming based on the trichome gland system.
Key words: Key words: P450 gene, secretion, trichome-specific-promoter.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionSummaryReferences |
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Many flowering plants bear trichomes, modified epidermal structures on their aerial surfaces. Trichomes frequently function as a first line of defence against insect attack, either by spatial hindrance, physical entrapment or by secreting toxic or behaviour modifying chemicals (Duffey, 1986; Wagner, 1991). This trichome-based defence plays an important role in the development of sustainable pest control strategies (Lewis et al., 1997). Glandular trichomes often secrete diverse natural products that are economically important (Lange and Croteau, 1999). These natural compounds include products such as cosmetic ingredients, flavour and aroma substances, essential oils and resins, and other chemicals having a specific industrial use (McCaskill and Croteau, 1999). Several terpenes that are thought to be secreted by trichomes have been implicated in allelopathic processes (Macias et al., 1999). To exploit the plant trichome system fully for enhancing natural-product-based resistance to pests and pathogens, and for the synthesis of commercially useful natural products (molecular farming), it would be desirable to bioengineer the trichome system only, through the use of a trichome-specific promoter having high activity in glands. Such bioengineering would be advantageous in several ways. First, the fact that plant trichomes are not essential and that the deposition of trichome secretions at the plant surface would allow the production of chemicals that are toxic or growth inhibiting if accumulated in the body of the plant. Second, trichome-specific bioengineering would restrict the expression of foreign genes to trichomes, thereby avoiding the accumulation of foreign proteins in organs used for human consumption (e.g. grains, fruits). Third, trichome-specific-expression and surface accumulation of products might allow very high-level product accumulation, because there would be minimal physical limitation to product storage capacity. Finally, surface-deposited secretions would be easily recovered in a relatively pure form.
In a previous paper, a tobacco trichome-specific P450 gene (CYP71D16) was described, and its function was established using antisense and sense co-suppression strategies (Wang et al., 2001). This gene catalyses the hydroxylation of cembratriene-ol to form cembratriene-diol, a diterpene that constitutes about 60% of trichome exudate weight in the tobacco cultivar, T.I. 1068. The study reported here describes the isolation and characterization of its trichome-specific promoter. Two reports in the literature describe the promoter analysis of the cotton lipid transfer protein genes LTP3 and LTP6 (Hsu et al., 1999; Liu et al., 2000). In tobacco, at the seedling stage, these promoters can direct GUS expression in trichomes, but not in non-trichome cells of leaves (Hsu et al., 1999; Liu et al., 2000; DP Ma, personal communication). The spatial and temporal expression patterns of these promoters have not been described in detail. Also, several promoters which direct specific expression, primarily in plant epidermal cells and trichomes have been reported, including the regulatory elements of the Arabidopsis thaliana GLABROUS1 gene (Larkin et al., 1993), the promoter of the Brassica oleracea wax 9D gene (Pyee and Kolattukudy, 1995), and the promoter of the tobacco Itp1 gene (Canevascini et al., 1996). This study is the first detailing the spatial and temporal expression pattern of a plant trichome-specific promoter.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionSummaryReferences |
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Plant growth
Nicotiana tabacum L. cultivars T.I. 1068 and T.I. 1112 were grown in a greenhouse without supplemented light in 8" pots and fertilized once a week with 20-20-20 fertilizer.
5' Rapid Amplification of cDNA Ends-PCR (RACE-PCR)
Total RNA was isolated from tobacco trichomes, prepared as previously described by Wang et al. (2001) using a RNeasy Plant Mini kit (QIAGEN, 74903). 5' RACE-PCR was carried out using a First ChoiceTM RNA Ligase Mediated Rapid Amplification of cDNA Ends (RLM-RACE) kit (Ambion, 1700). Briefly, total RNA from trichomes was treated with calf intestinal phosphatase to remove free 5' phosphates. The RNA was then treated with tobacco acid pyrophosphatase to remove the cap structure from full-length mRNAs, leaving 5' monophosphates. Next, a synthetic RNA adapter was ligated to mRNAs using T4 RNA ligase. Lastly, the randomly-primed reverse transcription reaction and nested PCR was made to amplify the 5' end of specific transcripts. Two gene-specific primers GSP-1 (5'-GCAAAAGAGGTAGTGGAGGATGCAGTCTGAA-3') and GSP-2 (5'-GGCACTGAGCAATTCCAAGAGACA-3') were used for the nested PCR. The final PCR product was cloned into pGEM®-T vector (Promega, A3600), and M13 forward and reverse primers were used to sequence the cloned fragment. The sequence of the full promoter is shown in Fig. 1.
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Histochemical and fluorometric GUS assay
Leaf discs and root, stem, and flower petal pieces of both transgenic and non-transformed T.I. 1068 were stained in a 0.1% X-Gluc solution according to the method of Jefferson (1987). After incubation at 37 °C for a period of 3 h to overnight, tissues were destained in 70% ethanol until chlorophyll pigments were completely bleached.
Three fully-expanded, size-matched leaves (about 30x10 cm) from about 80 cm tall, greenhouse-grown transgenic plants were used for fluorometric GUS assays. Trichomes from the leaves (all surfaces) were removed using the cold-brushing method (Wang et al., 2001), ground into a fine powder with liquid nitrogen, and then vortexed with GUS extraction buffer containing 50 mM sodium phosphate (pH 7.0), 10 mM ß-mercaptoethanol, 10 mM Na2-EDTA (pH 8.0), 0.1% SDS, and 0.1% Triton X-100. The extracts were centrifuged for 10 min in a microcentrifuge at 4 °C, and the supernatants were collected for GUS assay according to Jefferson (1987) using 4-methylumbelliferyl-ß-D-glucuronide (MUG) as a substrate. The amount of methyl-umbelliferone (MU) production was determined using a luminescence spectrometer (Perkin Elmer, LS50B), and MUG activity was expressed as fluorescence units h–1 mg–1 trichome protein.
Constructs for promoter–GUS deletion analyses and transformations
The full CYP71D16 promoter and a series of 5' progressive deletions of it were generated using PCR. The positions of the forward primers for deletion constructs were: EW1 (from nt –1852 to –1832), DEL-1 (–1724 to –1704), DEL-2 (–1413 to –1393), DEL-3 (–1273 to –1253), DEL-4 (–1123 to –1102), DEL-5 (–973 to –953), DEL-6 (–823 to –803), DEL-7 (–673 to –653), DEL-8 (–523 to –503), DEL-9 (–373 to –353), and DEL-10 (–223 to –202). A SpeI restriction enzyme site was created at the 5' end of each of the 11 forward primers. An NcoI restriction site at the 3' end of the promoter (from +106 to +111, Fig. 1) was incorporated into the design of the reverse primer EW-5. To ensure in-frame fusion with the GUS gene, one nucleotide (G) was added just 5' to this NcoI site. Thus, the whole sequence of EW-5 is as follows: 5'-CTCCATGGCACCTGGA GGCAATCTTTTG-3' (from +111 to +87, Fig. 1, the NcoI site is underlined above). Each of the 11 promoter PCR products was double digested with SpeI and NcoI, and ligated into the SpeI and NcoI sites of the vector pSG506 (Gan, 1995), immediately upstream of the ATG start codon for ß-glucuronidase. The expression cassettes (CYP71D16 promoter+GUS+MAS terminator) were released and subsequently cloned into the binary vector pPZP 211 at PstI and XbaI sites (Hajdukiewicz et al., 1994). Each deletion was verified by sequencing. Constructs were introduced into Agrobacterium tumefaciens strain ABI, and used to transform tobacco T.I. 1068 using the leaf disc method (Horsch et al., 1985).
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionSummaryReferences |
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Isolation of the trichome-specific promoter
A P450-like cDNA was isolated using a PCR-based cDNA subtraction strategy (Wang et al., 2001). This clone was used to probe a Nicotiana tabacum cv. Xanthi-nc genomic library (Clontech, 1071d). Two identical genomic clones of different length were isolated. The complete sequence of one of these was determined, and a full-length P450 gene was thereby identified (Genbank accession number AF166332), designated as CYP71D16 (Nelson, 2001). Northern analysis showed that this gene hybridized to trichome total RNA, but not leaf-minus-trichome, stem-minus-trichome, flower petal or root total RNAs (Wang et al., 2001). The sequence of 1852 bp upstream to its putative translation start site was further studied. A putative TATA box sequence was found at –72 to –78. Also, two putative CAAT box sequences were located at –99 to –102 and –181 to –184, respectively (+1 was assigned to the A of the first codon ATG, Fig. 1). RLM-RACE PCR (Ambion, 1700) was used to locate the transcriptional start site of CYP71D16. Two gene-specific primers for the CYP71D16 gene, GSP1 and GSP2, were combined with the adaptor primers (AP1 and AP2, provided in the kit), for nested PCR. In the final INNER PCR, a DNA fragment of about 550 bp was amplified. This fragment was cloned, and five independent clones were sequenced. The sequences were then compared with the CYP71D16 genomic sequence, and all five independent 5' RACE clones indicated the same transcriptional start site, which is at –47 (Fig. 1).
A search for regulatory elements in the CYP71D16 promoter revealed that, in addition to the ‘TATA’ box and ‘CAAT’ boxes, six MYB-like recognition sites (–844 to –839, TAACTG; –554 to –549, TAACTG; –416 to –411, CTGTTA; –408 to –403, TAACTG; –289 to –285, GGATA; and –56 to –51, CAACAG, Fig. 1) and two HD-ZIP-like protein biding sites (–1381 to –1373, TAATCATTA, and –1193 to –1185, TAATCATTA, Fig. 1) were present. MYB recognition sites were also identified in the promoter region of a tobacco trichome-specific cyclase gene (E Wang, unpublished data), and in the trichome-specific promoters of both the LTP3 and LTP6 genes from cotton (Hsu et al., 1999; Liu et al., 2000). The MYB gene family represents one of the largest regulatory factor families in plants, and one of the important functions for MYB factors is to control development and determination of cell fate and identity (Stracke et al., 2001). In Arabidopsis thaliana, the GLABROUS1 (GL1) governs leaf trichome formation (Oppenheimer et al., 1990) while its equivalent, WEREWOLF (WER), determines root epidermal cell patterning (Lee and Schiefelbein, 2001). The MIXTA gene from Antirrhinum majus, when ectopically expressed in tobacco, can promote trichome differentiation, producing excess numbers of multicellullar trichomes on leaves and floral organs (Glover et al., 1998).
Spatial and temporal expression patterns of the CYP71D16 promoter–GUS fusion
The 1963 bp genomic fragment containing the 5' promoter region (from –48 to –1852), the 5' untranslated region (from –1 to –47), and the first 37 amino acids of the first exon (from +1 to +111) of CYP71D16 (Fig. 1) was PCR amplified and fused in frame to the GUS coding region. This construct, named TSP-W, was used to transform tobacco (T.I. 1068 and T.I. 1112) using Agrobacterium tumefaciens-mediated transformation. Primary transformants were selected on kanamycin, and leaf discs, stem, flower petal, and root pieces were tested for histochemical expression of GUS. Tissues of different developmental stages were tested. For tobacco cultivar T.I. 1068 that possesses glandular trichomes, three independent experiments (Experiments 1 to 3, Table 1) were carried out using 10, 13 and 9 transgenic plants, respectively. Trichome-specific expression of GUS was clearly found in four, five, and four plants of experiments 1 to 3, respectively. The remaining plants did not express GUS in trichomes or other tissues (Table 1).
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A similar spatial expression pattern was observed in all 13 of the above plants expressing GUS. In primary transgenic explants, as early as at the rooting stage, strong GUS expression appeared in trichome glands (Fig. 2A). By contrast, no GUS activity was detected in non-trichome cells of the leaf epidermis, on other leaf cells (Fig. 2A), or in roots (data not shown). GUS assays were subsequently made with samples from the transgenic plants derived from explants after their growth in the greenhouse to about 30 cm height. Intensive blue staining was observed in leaf trichome glands, and much less staining was seen in the first stalk cell next to the gland of these early mature stage plants (Fig. 2B, C). In some trichomes lower stalk cells were also stained (Fig. 2G, F). As with rooting-stage tissue, no GUS staining occurred in non-trichome tissues of leaf blades of 30 cm high plants. However, after overnight incubation with GUS substrate, faint GUS staining was found in guard cells and veins in some areas of some leaves, and at the cut edges of some leaf blades (data not shown). Further study is needed to explore these observations. Intensive GUS staining also occurred in stem trichome glands of 30 cm high plants (Fig. 2D). Similar strong GUS staining appeared in trichome glands of stem (Fig. 2E), flower petal (Fig. 2F), and leaf (Fig. 2G) from 100 cm (flowering) T.I. 1068 plants. No GUS activity was detected in roots of 30 cm high (Fig. 2H, upper), or flowering-stage (Fig. 2H, lower) plants. Figure 2I shows an amplified view of a portion of the root shown in the upper portion of Fig. 2H. Note the lack of staining in root hairs (arrows). On leaves, stems and flower petals, the stalk cell adjacent to the gland was generally stained (Fig. 2C, D, E, F, G). It is also noteworthy that some unstained trichomes were often observed that were adjacent to those with intensely stained glands (Fig. 2A, E, F, G), but overall, it is estimated that >70% of trichomes were stained on trichome bearing tissues. The absence of staining in some trichomes raises the question of whether all glanded trichomes on a tissue are involved in synthesizing the same exudate compounds or mixtures. It is noted that the gene directed by the promoter characterized here is one involved in diterpene production. Trichome glands of T.I.1068 also produce substantial amounts of sucrose esters via pathways not thought to involve P450 modification (Wagner, 1991). Also, one sometimes observes trichomes lacking an exudate droplet around the gland in the midst of most trichomes having large droplets (E Wang, unpublished data). This suggests that some glands may not be producing substantial exudates while neighbouring ones are very active in secretion. No GUS activity was found in anthers, stigmas, ovules or pollen, but glands of trichomes on sepals were highly stained (data not shown).
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0.1 mm.Trichome-specific expression was also unambiguously observed in three of the four transgenic plants from tobacco cultivar T.I. 1112 (Table 1), a line in which virtually all trichomes are non-glandular. T.I. 1112 produces little trichome exudate, and that formed is thought to be synthesized by rare glanded trichomes (Fig. 2J). As shown in Fig. 2K, weak GUS staining only occurred in the uppermost trichome stalk cells of the common non-glanded trichomes of this cultivar. No GUS activity was observed in the lowest stalk cells, in epidermal cells, or other leaf cells of T.I. 1112 trichomes. The finding that unambiguous expression of GUS occurs in the distal stalk cells of non-glandular trichomes of T.I. 1112 raises the possibility that these cells are also capable of synthesizing low levels of cembratriene-diol, a diterpene typically found in glandular trichome exudates of tobacco. By analogy, one may question if certain cells of non-glandular trichomes that are not morphologically recognizable as glands on other plants may also produce secretions.
Taken together, the results shown in Fig. 2 indicate that the 1963 bp genomic fragment can drive specific and strong GUS expression in trichomes on stems, leaves and flowers at all stages of development in tobacco. For T.I. 1068 with glandular trichomes, GUS expression occurred primarily in the gland cells. However, the behaviour of the promoter described here (in both T.I. 1068 and T.I. 1112) suggests that the stalk cells can express genes active in specialized gland cells. Initiation of trichome development is thought to involve the formation of an individual trichome precursor cell, followed by initiation of additional cells at the tip of the precursor cell. Glands are formed from the newest stalk cell (Marks, 1994).
Deletion analysis
In order to characterize the cis-elements that are critical to trichome-specific expression, a series of 5' deletion constructs were made (defined in the Materials and methods). For each of the 11 constructs, four to ten transgenic plants were generated, and GUS activity for each plant was determined fluorometrically using the MUG assay. To establish a control, the MU values were determined for ten randomly selected, non-transformed plants of T.I. 1068. For convenience in comparing the relative GUS activities of each construct, the average GUS activity of the full-length promoter was set at 100% and used to define relative GUS activities of the deletions. As Fig. 3 shows, the relative GUS activities for most individual plants from each of the constructs were noticeably higher than the control value. Very high MUG hydrolysis in certain individual TSP-D3 and TSP-D6 plants may be due to positional effects. Overall, as deletions progressed, GUS activities decreased, but not dramatically. The majority of the plants from the last deletion (TSP-D10) still maintained a considerably higher GUS activity than the control (Fig. 3).
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All of the primary transformants derived from the 11 deletions were also subjected to histochemical GUS assay. For all constructs, except for two deletions (TSP-D6 and TSP-D8), at least two individual plants showed distinctive and consistent trichome-specific GUS staining patterns. TSP-D6 and TSP-D8 had only one plant each that showed clear trichome-specific GUS staining. None of the individual plants from each of the 11 constructs showed GUS staining in tissues other than trichomes. A similar pattern of staining to that seen with the full promoter (Fig. 2) was found in TSP-D1, -D2, and -D3 plants (Fig. 4A, TSP-D3 shown), but with less intensity of staining. TSP-D4 to TSP-D10 plants gave the pattern of staining shown in Fig. 4B for TSP-D10, where the lowest gland cell and stalk cells show activity. Thus, staining is trichome specific in all deletions (found in trichome stalk and/or gland cells, but not in non-trichome cells), but the full promoter is required for strong gland expression. It is speculated that the MYB-like domain at –56 to –51 may be important for trichome specificity, and that some or all of the other putative regulatory domains (HD-ZIP, other MYBs, and other domains not identified) regulate, at least, strong gland expression. To summarize, the deletion analysis indicated that the region from –223 to +111 contains all cis-elements required for trichome-specific expression in T.I. 1068.
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The CYP71D16 promoter is not induced by high light, high temperature, drought or cold stress
Tobacco trichome exudate content is said to be increased in the field by drought stress and in the greenhouse by high light. Therefore, the possible inducibility of the CYP71D16 promoter by these and other stressors was tested using 100 cm tall greenhouse-grown plants transformed with the TSP-W construct. Promoter activity was analysed by monitoring MUG hydrolysis in homogenates of leaves. GUS activity was similar in plants subjected to 4 °C under fluorescent lamps, or constant high light (1000 W high pressure sodium lamp about 1 m from the plant tops) at room temperature for 0, 1, 3, 6, and 12 h. Both high temperature (42 °C and constant fluorescent light, monitored at 0, 2, 6, 12, and 24 h) and drought (monitored at 0 and 10 d post-watering, leaves flaccid at 10 d) caused a decrease in GUS activity, perhaps due to a general deterioration of the plants under these extreme stresses. Thus, no clear evidence for the induction of this promoter by these stress conditions was found.
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Summary |
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TopAbstractIntroductionMaterials and methodsResults and discussionSummaryReferences |
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In summary, the full CYP71D16 promoter can direct GUS expression specific to both glandular and non-glandular trichomes, and is primarily restricted to gland cells in the glandular type, T.I. 1068. This specificity was maintained throughout all developmental stages. Deletion analysis indicated that the region from –223 to +111 is sufficient to confer trichome-specific expression, but upstream regions are needed for strong gland expression. More research is needed to delineate the cis-elements responsible for trichome-specific expression. This promoter has promise for use in molecular farming and for enhancing trichome-based pest/disease resistance in plants with glandular trichomes. In addition, based on observations with cultivar T.I. 1112, it may also be applicable to genetic modification of plants that do not have obvious trichome glands.
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Acknowledgements |
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We thank Mr James T Hall for his expert help in maintaining tobacco materials, and preparing trichome tissues for GUS assays. The work was supported by a grant to GJW and SG from the Kentucky Tobacco Research and Development Center and, a grant to GJW from The National Science Foundation/US Department of Agriculture-National Research Initiative, Interagency Metabolic Engineering 90.0 Program.
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References |
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