Expression patterns conferred by tyrosine/dihydroxyphenylalanine decarboxylase promoters from opium poppy are conserved in transgenic tobacco

Abstract

Opium poppy (Papaver somniferum) contains a large family of tyrosine/dihydroxyphenylalanine decarboxylase (tydc) genes involved in the biosynthesis of benzylisoquinoline alkaloids and cell wall-bound hydroxycinnamic acid amides. Eight members from two distinct gene subfamilies have been isolated, tydc1, tydc4, tydc6, tydc8, and tydc9 in one group and tydc2, tydc3, and tydc7 in the other. The tydc8 and tydc9 genes were located 3.2 kb apart on one genomic clone, suggesting that the family is clustered. Transcripts for most tydc genes were detected only in roots. Only tydc2 and tydc7 revealed expression in both roots and shoots, and TYDC3 mRNAs were the only specific transcripts detected in seedlings. TYDC1, TYDC8, and TYDC9 mRNAs, which occurred in roots, were not detected in elicitor-treated opium poppy cultures. Expression of tydc4, which contains a premature termination codon, was not detected under any conditions. Five tydc promoters were fused to the beta-glucuronidase (GUS) reporter gene in a binary vector. All constructs produced transient GUS activity in microprojectile-bombarded opium poppy and tobacco (Nicotiana tabacum) cell cultures. The organ- and tissue-specific expression pattern of tydc promoter-GUS fusions in transgenic tobacco was generally parallel to that of corresponding tydc genes in opium poppy. GUS expression was most abundant in the internal phloem of shoot organs and in the stele of roots. Select tydc promoter-GUS fusions were also wound induced in transgenic tobacco, suggesting that the basic mechanisms of developmental and inducible tydc regulation are conserved across plant species.

Figure 1

Schematic representation of the early steps in the biosynthetic pathways leading to benzylisoquinoline alkaloids and hydroxycinnamic acid amides of tyramine showing the sites of action of key gene products. NS, (S)-Norcoclaurine synthase; THT, tyramine hydroxycinnamoylCoA:tyramine hydroxycinnamoyltransferase; Dopa, dihydroxyphenylalanine.

Figure 2

Structural and restriction endonuclease maps for regions of genomic clones containing the tydc3, tydc6, tydc7, tydc8, and tydc9 genes from opium poppy. The open boxes represent ORFs and the bent arrows show the approximate location and direction of transcription initiation. The horizontal brackets show the regions amplified by PCR and used as gene-specific probes. B, BamHI; E, EcoRI; H, HindIII; K, KpnI; P, PstI; S, SalI; Sp, SpeI; Xb, XbaI; Xh, XhoI.

Figure 3

Alignment of predicted amino acid sequences from isolated members of the tydc gene family in opium poppy. Amino acid sequences for TYDC1, TYDC2, and TYDC4 were reported by Facchini and De Luca (1994). The asterisk at position 56 in the TYDC4 sequence represents a premature termination codon. The TYDC5 amino acid sequence was also reported previously (Maldonado-Mendoza et al., 1996).

Figure 4

RNA gel-blot hybridization analysis for various members of the tydc gene family in mature opium poppy organs. Fifteen micrograms of total RNA was fractionated on 1.0% formaldehyde agarose gels, transferred to nylon membranes, and hybridized at high stringency with ³²P-labeled full-length probes for tydc1 and tydc2 or gene-specific probes for tydc1/8, tydc2/7, tydc3, tydc4, tydc6, and tydc9. To ensure equal loading, gels were stained with ethidium bromide before blotting.

Figure 5

RNA gel-blot hybridization analysis for various members of the tydc gene family during opium poppy seedling development. Fifteen micrograms of total RNA was fractionated on 1.0% formaldehyde agarose gels, transferred to nylon membranes, and hybridized at high stringency with ³²P-labeled full-length probes for tydc1 and tydc2 or gene-specific probes for tydc2/7 and tydc3. To ensure equal loading, gels were stained with ethidium bromide before blotting.

Figure 6

RNA gel-blot hybridization analysis for various members of the tydc gene family in elicitor-treated opium poppy cell-suspension cultures. Fifteen micrograms of total RNA was fractionated on 1.0% formaldehyde agarose gels, transferred to nylon membranes, and hybridized at high stringency with ³²P-labeled full-length probes for tydc1 and tydc2 or gene-specific probes for tydc1/8, tydc2/7, tydc3, tydc4, tydc6, and tydc9. To ensure equal loading, gels were stained with ethidium bromide before blotting.

Figure 7

Activity of various tydc gene promoters determined by transient expression of promoter-GUS fusions in opium poppy (A) and tobacco (B) cell cultures. Bars represent normalized GUS activity in cultured cells 48 h after microprojectile bombardment with the following constructs: pBI 102 (promoterless), 35S::GUS (CaMV 35S promoter), TYDC3::GUS (tydc3 promoter), TYDC6::GUS (tydc6 promoter), TYDC7::GUS (tydc7 promoter), TYDC8::GUS (tydc8 promoter), and TYDC9::GUS (tydc9 promoter). Values represent the means ± se of three independent experiments whereby cultured cells were co-bombarded with promoter-GUS and CaMV 35S-luciferase constructs and GUS activity was normalized against luciferase activity. MU, 4-Methylumbelliferone.

Figure 8

GUS activity in mature plant organs and in 10-d-old seedlings of transgenic tobacco expressing tydc promoter-GUS constructs. GUS activity levels in transgenic tobacco expressing a CaMV 35S promoter-GUS fusion are shown for comparison. MU, 4-Methylumbelliferone.

Figure 9

(Figure appears on facing page.) Histochemical localization of GUS activity in transgenic tobacco expressing tydc promoter-GUS constructs. A, Cross-section of a young TYDC7::GUS stem showing GUS activity restricted to internal phloem. B, Cross-section of a young TYDC7::GUS petiole with GUS activity localized in internal phloem and an adaxial layer of cortex. C, TYDC7::GUS roots displaying GUS activity in dividing meristematic tissues and in the stele of elongation and maturation zones. D, TYDC7::GUS root showing GUS activity during the early stages of lateral root bud development. E, Ten-day-old TYDC7::GUS seedling with GUS activity in the cotyledons, shoot apical meristem, root meristem, and vascular tissue of the young root. F, Root-shoot transition zone of a TYDC6::GUS plant showing that GUS activity in the stele ends as vascular bundles emerge in the stem. G, TYDC6::GUS roots showing the restriction of GUS activity to the stele. co, Cotyledons, ep, external phloem; ip, internal phloem; lr, lateral root; rm, root meristem; rc, root cap; sm, shoot apical meristem; st, stele; xy, xylem. All bars represent 1 mm.