Novel anther-specific myb genes from tobacco as putative regulators of phenylalanine ammonia-lyase expression

Abstract

Two cDNA clones (NtmybAS1 and NtmybAS2) encoding MYB-related proteins with strong sequence similarity to petunia (Petunia hybrida) PhMYB3 were isolated from a tobacco (Nicotiana tabacum cv Samsun) pollen cDNA library. Northern blot and in situ hybridization revealed that NtmybAS transcripts are specifically expressed in both sporophytic and gametophytic tissues of the anther including tapetum, stomium, vascular tissue, and developing pollen. Random binding site selection assays revealed that NtMYBAS1 bound to DNA sequences closely resembling consensus MYB binding sites MBSI and MBSIIG, with a higher affinity for MBSI. Transient expression analyses of the N-terminal MYB domain demonstrated the presence of functional nuclear localization signals, and full-length NtMYBAS1 was able to activate two different phenylalanine ammonia-lyase promoters (PALA and gPAL1) in tobacco leaf protoplasts. Similar analysis of truncated NtmybAS1 cDNAs identified an essential, C-terminal trans-activation domain. Further in situ hybridization analyses demonstrated strict co-expression of NtmybAS and gPAL1 in the tapetum and stomium. Despite abundant expression of NtmybAS transcripts in mature pollen, gPAL1 transcripts were not detectable in pollen. Our data demonstrate that NtMYBAS1 is a functional anther-specific transcription factor, which is likely to be a positive regulator of gPAL1 expression and phenylpropanoid synthesis in sporophytic, but not in gametophytic, tissues of the anther.

Figure 1

Comparison of the amino acid sequence of NtMYBAS1/2 and PhMYB3. The R2 and R3 repeats of the MYB domains are boxed. Arrowheads correspond to conserved Trp residues in the MYB domain. The MYB protein subgroup 18 consensus motif (QRaGLPxYPxE/S; Kranz et al., 1998) is highlighted in bold. Identical amino acids are indicated below with asterisks.

Figure 2

Alignment (a) and dendrogram (b) of the deduced amino acid sequence of the MYB domain of NtMYBAS1/2 with those of related MYB proteins. Gene, genus nomenclatures, and accession nos. are as follows: NtMYBAS1, tobacco, AF198499; NtMYBAS2, tobacco, AF198498; PhMYB3, petunia, Z13998; AtMYB101, Arabidopsis, X90379; OsMYBGa, Oryza sativa, X98355; HvMYBGa, Hordeum vulgare, X87690; AtMYB33, Arabidopsis, AF062875; AtMYB65, Arabidopsis, AF062899; AtMYB81, Arabidopsis, AF062911; AtMYB103, Arabidopsis, AF048839; HsMYBC, Homo sapiens, M15024; and DmMYB, Drosophila melanogaster, X05939. Asterisks correspond to positions of conserved Trp residues. Boxes beneath the sequences indicate the three helices in each repeat including the recognition helix (black box). Sequence alignment and dendrogram were performed by the neighbor-joining algorithm of CLUSTALW, where the scale bar represents the degree of base pair substitution per million years (Thomson et al., 1994).

Figure 3

mRNA expression analysis of NtmybAS. Northern blots of tobacco total RNA probed with radiolabeled NtmybAS1 probe. a, Spatial expression. 13D and 17D, Seedlings 13 and 17 d after germination; Rt, root; Yl, young leaf; Sp, sepal; Pt, petal; Pst, pistil; Stm, stamen; MP, mature pollen; GP, germinating pollen. b, Temporal expression in anthers. WL, Wild- type leaf; MP, mature pollen. Flower bud lengths shown in millimeters. c, Temporal expression in isolated spores. MP, Mature pollen; GP, germinating pollen.

Figure 4

Localization of NtmybAS mRNA during tobacco anther development. Paraffin-embedded sections were hybridized with digoxygenin-labeled NtmybAS1 probes and viewed under bright field. Scale bar = 400 μm. Magnification ×100 in 8- to 9-mm and 10- to 12-mm buds; and ×40 in 13- to 15-mm, 16- to 20-mm, and 21- to 30-mm buds. a through e, NtmybAS1 antisense probe with anthers from: a, 8 to 9 mm; b, 10 to 12 mm; c, 13 to 15 mm; d, 16 to 20 mm; and e, 21- to 30-mm buds. Staining represents hybridization signal. f through j, NtmybAS1 sense probe with anthers from: f, 8 to 9 mm; g, 10 to 12 mm; h, 13 to 15 mm; i, 16 to 20 mm; and j, 21- to 30-mm buds.

Figure 5

Nuclear localization of NtMYBAS1-sGFP fusion proteins. Tobacco pollen grains bombarded with pLAT52-sGFP control plasmid (a) and pLAT52-sGFPM1-3 plasmids harboring NtMYBAS1-sGFP fusion constructs (b–d). a, The control, sGFP alone, remains predominantly in the cytoplasm. b, pLAT52-sGFPM1. N-terminal 126 amino acids of NtMybAS1 result in localization of sGFP exclusively to the pollen nucleus. c, pLAT52-sGFPM2. N-terminal 119 amino acids of NtMybAS1 result in a dramatic reduction in nuclear localization. d, pLAT52-sGFPM3. The MTRLKRRQRA motif alone is not sufficient for exclusive nuclear targeting of sGFP. R2R3, MYB repeats; TEV-L, tobacco etch virus 5′-untranslated region; C3′, cauliflower mosaic virus 35S transcript polyadenylation sequence; n, NcoI; B, BspHI; NB, NcoI/BsphI junction.

Figure 6

Oligonucleotide sequences selected by NtMYBASΔC1 and relative binding affinity. Sequences of 20 oligonucleotides isolated after six rounds of selection with NtMYBASΔC1. The core binding sequence is in bold; lowercase letters represent the conserved oligonucleotide ends. Asterisks indicate that the sequence has been inverted. The percentage of bound oligonucleotide was quantified using a phosphorimager and expressed as the percentage of bound/total radioactivity in each lane.

Figure 7

EMSA with purified recombinant NtMYBASΔC1 protein. a, The sequences of double-stranded oligonucleotides used as probes. b, The NtMYBASΔC1 protein binds preferentially to MBSI (GTAACCG). The binding reaction fractionated in the first lane did not contain NtMYBASΔC1, whereas those in other lanes contained 200 ng NtMYBASΔC1. c, Competition for binding of NtMYBASΔC1 to the MBSI probe. The binding assay was performed by pre-incubating with unlabeled competitor before addition of 1 ng of ³²P-labeled MBSI probe. The reaction fractionated in lane 1 contained no competitor, whereas those in lanes 2 through 7 contained 200 ng of competitor.

Figure 8

EMSA with plant promoter fragments. Binding of the NtMYBASΔC1 to PALA (a) and gPAL1 promoter fragments (b). The locations of the fragments in the promoters relative to the transcriptional start site are given as follows: PALA P1, −1,115 to −876; P2, −875 to −626; P3, −625 to −366; P4, −365 to −356; gPAL1 T1, −658 to −475; T2, −474 to −204; and T3, −203 to −42. ψ represents the presence of MBSs.

Figure 9

Trans-activation of tobacco promoters by NtMYBAS1. a, Effector and reporter constructs used in cotransfection experiments. The 35S::NtMYBAS1 effector plasmid consisted of the coding region of NtmybAS1 under control of the CaMV 35S promoter. The reporter plasmids consist of two tobacco PAL promoters, PALA (Pellegrini et al., 1994) and gPAL1 (Tomoko et al., 1996); tobacco polygalacturonase NPG1 promoter (Tebbutt et al., 1994) and tobacco neIF4A8 promoter (Brander and Kuhlemeier, 1995) fused to the GUS reporter gene. b, Response of reporter constructs to NtMYBAS1 in transient expression assays. Tobacco leaf protoplasts were cotransfected with each reporter plasmid together with 35S::NtMYBAS2. Error bars represent se (n > 7).

Figure 10

Effect of C-terminal deletions on NtMYBAS1 trans-activation of PAL genes. a, The NtmybAS1 cDNAs containing deleted regions, as indicated by numbers of amino acid residues, were inserted between the CaMV 35S promoter and the NOS terminator. Effector plasmids were cotransfected into tobacco leaf protoplasts with reporter plasmids, containing gPAL1 and PALA, respectively. b, Transfection and assay of the reporter activity are as in Figure 9. The error bars represent the se (n > 10).

Figure 11

Expression of gPAL1 mRNA during tobacco anther development. a, Temporal northern analysis of gPAL1 expression in tobacco anthers. WL, Wild-type leaf; MP, mature pollen. Flower bud lengths shown in millimeters. b through k, In situ hybridization with gPAL1 antisense (b–f) and sense (g–k) probes. Bud lengths: b and g, 8 to 9 mm; c and h, 10 to 12 mm; d and i, 13 to 15 mm; e and j, 16 to 20 mm; f and k, 21 to 30 mm. Black bar = 400 μm. b, c, g, and h, magnification ×100; d through f and i through k, magnification ×40.