A novel lily anther-specific gene encodes adhesin-like proteins associated with exine formation during anther development

Abstract

The anther-specific gene LLA1271 isolated from lily (Lilium longiflorum Thunb.) anthers is novel and exists in two forms. The protein encoded by LLA1271 may represent an adhesin-like protein first found in higher plants. The protein contains a typical N-terminal signal peptide followed by a highly conserved repeat domain. The LLA1271 gene is temporally expressed at the phase of microspore development. RNA blot and RNA in situ hybridization analyses demonstrated that the gene was expressed both in the tapetum and in the microspore. The gene is endo- and exogenously induced by gibberellin. Studies with the gibberellin biosynthesis inhibitor uniconazole and an inhibitor of ethylene activity, 2,5-norbornadien (NBD), revealed that LLA1271 is negatively regulated by ethylene, and a cross-talk of regulation between gibberellin and ethylene occurs in young anthers. The treatment with NBD caused the tapetum to become densely cytoplasmic and highly polarized, whereas uniconazole arrested tapetal development in a state close to that of a tapetum without treatment. The LLA1271 protein is heat stable and heterogeneous. An immunoblot of separated protein fractions of the anther revealed that the LLA1271 protein was detected in protein fraction of the microspore released from the cell wall by treatment with either 0.5% or 2% Triton X-100. Ectopic expression of LLA1271 resulted in impaired stamen and low pollen germination. Scanning electron microscopy of TAP::LLA1271 pollen showed distorted exine formation and patterning. The LLA1271 protein once synthesized in both the tapetum and microspore is secreted and deposited on the surface of microspores, moderately affecting exine formation and patterning.

Keywords: Adhesin; anther; exine; hormone; lily (Lilium longiflorum Thunb.); microspore; tapetum..

Fig. 1.

Hydropathy profile and alignment of eight repeat sequences of LLA1271a. (A) Hydropathy profiles of LLA1271a protein sequence. The black line indicates a hydrophobic sequence at the N-terminus of the sequence. The eight repeat sequences are indicated as R1–R8. (B) Sequence alignment of eight repeats. The box indicates the putative phosphorylation (S/T-X-K/R) sites.

Fig. 2.

Alignment of lily LLA1271a with Os adhesin, a GLEYA adhesin domain protein. The deduced amino acid sequence of LLA1271a from Lilium longiflorum (accession no. EF026009) is aligned with a GLEYA adhesin domain protein (Os adhesin, accession no. EQL00008.1) of Ophiocordyceps sinesis C018 at the C-terminus. The amino acid residues that are identical between the two sequences are highlighted by black blocks. The black and grey lines indicate the conserved tandem repeats in LLA1271a and Os adhesin, respectively.

Fig. 3.

Organ specificity and temporal expression of LLA1271 in the anther of L. longiflorum. (A) Total RNA (20 μg) was isolated from various vegetative organs and floral organs of 34–46mm buds. (B) Total RNA (20 μg) was isolated from stamen/anthers of different sizes of buds: 1, <15mm buds; 2, 24–26mm buds; 3, 34–36mm buds; 4, 44–46mm buds; 5, 60–65mm buds; 6, 90–95mm buds; 7,120–125mm buds; 8,150–155mm buds. (C) Total RNA (20 μg) was isolated from anthers of 24–26mm buds (1) and from microspores of various sizes of buds: 2, 34–36mm buds; 3, 44–46mm buds; 4, 60–65mm buds; 5, 70–75mm buds; and from anthers (6) and anther wall (7) of 44–46mm buds. Total RNA was denatured, fractionated on formaldehyde–agarose gels, transferred to nylon membranes, and hybridized to the ³²P-labelled LLA1271a cDNA insert. Almost equal amounts of total RNA were loaded in each lane, as determined by ethidium bromide staining of the gel.

Fig. 4.

In situ hybridization of LLA1271 transcripts in the developing anther of L. longiflorum. Expression of LLA1271 was analysed in bright-field illuminated 7–10 μm cross-sections of anthers of 20–25mm (A and B), 35–40mm (C-F), 45–50mm (G-J), and 55–60mm (K and L) buds using DIG-labelled sense (E, F, I, and J) and antisense (A–D, G, H, K, and L) LLA1271a RNA probes. E, epidermis; En, endothecium; M, middle layer; T, tapetum. Bar=100 μm.

Fig. 5.

Expression and regulation of LLA1271 by GA and ethylene in young anthers of L. longiflorum. (A) The 18–22mm buds dissected from lily plants were dipped in solutions without GA₃ (1) or containing 0.1 μM (2) or 1.0 μM GA₃ (3) for 24h. (B) The 24–26mm buds dissected from lily plants were dipped in a solution containing 1 μM GA₃ for (1) 0h, (2) 3h, (3) 8h, (4) 24h, and (5) 36h. (C) The 17–20mm buds dissected from lily plants were dipped in a solution with or without treatment with 100 μM uniconazole and/or 100 μM NBD for 4 d, after which the bud size grew to 21–24mm. Total RNA (20 μg) was isolated from anthers, denatured, fractionated on formaldehyde–agarose gels, transferred to nylon membranes, and hybridized to the ³²P-labelled LLA1271a cDNA insert (upper panel). Almost equal amounts of total RNA were loaded in each lane, as determined by ethidium bromide staining of the gel. The enlarged transverse sections (lower panel) of anthers around 20mm buds with or without treatment were stained with 1% safranine O in 50% ethanol. T, tapetum. Bar=200 μm.

Fig. 6.

Immunological characterization of the LLA1271 protein in the anther of L. longiflorum. (A) Total protein (40 μg) was isolated from various vegetative organs of lily pants and floral organs of 34–46mm buds. (B) Total protein (40 μg) was isolated from stamen/anthers of different sizes of buds: 1, 10–20mm buds; 2, 20–30mm buds; 3, 30–40mm buds; 4, 40–50mm buds; 5, 50–60mm buds; 6, 70–80mm buds; 7, 90–100mm buds; 8, 110–120mm buds; 9, 130–140mm buds; 10, 150–160mm buds. (C) Total protein was isolated from anthers of 34–46mm buds and heat treated at 90 ºC for 10min. The resolubilized pellet (precipitation, lane 1) and heat-soluble protein (supernatant, lane 2) were fractionated by SDS–PAGE. (D) Total protein (1.2mg) extracted from anthers of 34–46mm buds was electrophoresed by 2D-PAGE. The gels were either stained with Coomassie blue or electroblotted onto nitrocellulose and immunologically detected using affinity-purified LLA1271 or pre-immune antibodies at a 1:10 000 dilution. The LLA1271 polypeptides are indicated by arrows. Marker proteins are indicated on the left.

Fig. 7.

Distribution of the LLA1271 protein in fractions of distinct origins separated from anthers of L. longiflorum. SDS–PAGE of proteins in the anther of 34–46mm buds (total) and separated fractions including the anther wall, exine-released, and microspore fractions. Proteins were released from the exine layer of microspores by an aqueous solution of sodium acetate with or without the addition of either 0.5% or 2% Triton X-100. The gel was either stained with silver (A) or electroblotted onto nitrocellulose and immunologically detected using affinity-purified LLA1271 antibodies at a 1:20 dilution (C). Molecular mass markers in kDa are indicated on the left side. Different proportions of individual samples were applied to the lanes, and these proportions relative to an equal amount of the anther are shown in the gel. (B) Light microscopic photographs of fresh microspores, and microspores after treatment with an aqueous solution with or without 2% Triton X-100. The scale bar represents 100 μm.

Fig. 8.

Generation and phenotype analysis of TAP::LLA1271 transgenic lines. (A) Construct of LLA1271 fused with the RTS gene regulatory region (TAP) for plant transformation. RB, right T-DNA border; LB, left T-DNA border; NPTII, neomycin phosphotransferase II; NOS-p, NOS promoter; NOS-t, NOS terminator. Phenotypes of the wild type (B–D) and two TAP::LLA1271 transgenic lines (E–J) were observed using a dissection microscope. Pollen grains of the wild type (D) and TAP::LLA1271 transgenic lines (G, J) were stained with Alexander’s staining for viability testing. A, anther; S, stigma; Pa, papillae; P, pollen. Scale bars=200 μm (B, C, E, F, H, I) and 100 μm (D, G, J).

Fig. 9.

Germination of TAP::LLA1271 pollen grains. Pollen of the wild type and two TAP::LLA1271 transgenic lines was germinated in vitro in the germination buffer for 16h. At least 500 pollen grains were scored in duplicate for each germination test. The data were obtained from three independent experiments. Error bars represent the SD (t-test: **P<0.01).

Fig. 10.

Scanning electron micrographs of TAP::LLA1271 pollen grains. SEM micrographs of pollen grains from the wild type (A-C) and the two TAP::LLA1271 transgenic lines 11 (D–F) and 25 (G–I). The two TAP::LLA1271 pollen grains show defects in tectum formation. Two independent experiments were carried out, both with similar results. Arrowheads indicate limited areas without a visible exine network. Arrows indicate spherical or amorphous extrabacular protrusions in disconnected or smooth exine. Scale bars=10 μm (A, B, D, E, G, H) and 1 μm (C, F, I).