The identification of a pollen‐specific LEA‐like protein in Lilium longiflorum

Abstract

A novel pollen-specific LEA-like protein, LP28, was detected in Lilium longiflorum using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Immunoblot analysis using antiserum raised against LP28 revealed that the protein was not found in somatic tissues or uninucleate microspores, but accumulated gradually in developing pollen following microspore mitosis. Furthermore, LP28 was abundant in germinated pollen after hydration. The cDNA clone corresponding to LP28 encoded a putative protein of 238 amino acids with a calculated molecular mass of 24·2 kDa and a pI of 4·7. The amino acid sequence is highly hydrophilic except for the N-terminal hydrophobic signal peptide. The sequence has similarities with group 3 LEA (late embryogenesis abundant) proteins. Immunocytochemical analyses demonstrated that LP28 was mainly found in cytoplasmic granules of the vegetative cell until pollen maturation, but after hydration it appeared in the elongating pollen tube wall. LP28 might be a unique pollenspecific protein that is transported to the pollen tube wall after germination. Therefore, it is assumed that LP28 plays a role not only in pollen maturation, but also in the growth of the pollen tube, which penetrates the stylar matrix.

LP28, a lily pollen-specific LEA-like protein, is located in the callosic cell wall during male gametogenesis

“…When the callosic cell wall was digested to free the microspores, LP28 labeling was no longer detected during microspore development. This result agrees with an immunoblot analysis described previously (Mogami et al 2002). It has also been suggested that callose is present in the generative cell wall (Heslop-Harrison 1968;Tanaka 1988).…”

“…Recently, using 2D-PAGE, we detected a novel LEA (late embryogenesis abundant)-like protein, LP28 (lily pollen protein 28), which is abundant in mature pollen (Mogami et al 2002). Here, we report the localization of LP28 during male gametogenesis by immuno-electron microscopy.…”

“…The sections on the grids were incubated for 30 min at RT with 20 mM Tris-HCl buffer (pH 7.4) containing 5% normal goat serum, 1% BSA, 0.1% Tween 20, and 150 mM NaCl. The grids were incubated in either anti-LP28 antiserum, which had been raised in rats (Mogami et al 2002), at a final dilution of 1:800 in the buffer or anti-(1,3)-β-glucan monoclonal antibody (Biosupplies Australia, Melbourne, Australia), diluted to 1:100 in the buffer, and were left overnight in a moist chamber at 4°C. After several washes in the buffer, the grids were then incubated in either colloidal gold (15 nm) goat anti-rat IgG complex (1:100) (British Biocell, Cardiff, UK) or colloidal gold (5 nm) goat antimouse IgG complex (1:100) (British Biocell) for 50 min at RT.…”

“…During pollen development, the callose in the generative cell wall disappeared, and LP28 was no longer found in the wall (data not shown). Instead, LP28 gradually accumulated in the cytoplasmic granules of the vegetative cell until pollen maturation (Mogami et al 2002).…”

Bipolar Localization of Putative Photoreceptor Protein for Phototaxis in Thermophilic Cyanobacterium Synechococcus elongatus

“…Before use in the experiments the rabbit anti serum was purified by antigen-affinity chromatography using the antigen conjugated to HiTrap NHS-activated HP (Amersham Biosciences AB, Uppsala, Sweden). OM, CM, TA and CP represent outer membrane, cytoplasmic membrane, thylakoid area and centroplasm, respectively.The immuno-electronmicroscopy was performed according to previously described method (Mogami et al 2002) using S. elongatus cells harvested at late log phase. amino acid sequence.…”