Chemocyanin, a small basic protein from the lily stigma, induces pollen tube chemotropism

Abstract

In plant reproduction, pollination is an essential process that delivers the sperm through specialized extracellular matrices (ECM) of the pistil to the ovule. Although specific mechanisms of guidance for pollen tubes through the pistil are not known, the female tissues play a critical role in this event. Many studies have documented the existence of diffusible chemotropic factors in the lily stigma that can induce pollen tube chemotropism in vitro, but no molecules have been isolated to date. In this study, we identified a chemotropic compound from the stigma by use of biochemical methods. We purified a lily stigma protein that is active in an in vitro chemotropism assay by using cation exchange, gel filtration, and HPLC. Tryptic digestion of the protein yielded peptides that identified the protein as a plantacyanin (basic blue protein), and this was confirmed by cloning the cDNA from the lily stigma. Plantacyanins are small cell wall proteins of unknown function. The measured molecular mass by electrospray ionization ion source MS is 9898 Da, and the molecular mass of the mature protein (calculated from the cDNA) is 9900.2 Da. Activity of the lily plantacyanin (named chemocyanin) is enhanced in the presence of stigma/stylar cysteine-rich adhesin, previously identified as a pollen tube adhesin in the lily style.

Fig. 1.

Chemotropism of lily pollen tubes toward stigma diffusates. Stigma with exudate (a dashed line indicated by an arrow shows the border of stigma exudate) (A) or a stem cross section (B) were placed on a 1% agarose growth medium for 1 h and removed before pollen grains were applied 2–5 mm from the tissue imprint. pt, pollen tubes; pg, pollen grains; sg, stigma imprint; sm, stem imprint. (Bar = 1 mm.)

Fig. 2.

Chemotropism assay in a field of lily pollen. (A) SPs. (B) BSA. (C) Proteinase K-treated SPs. In A–C, 2.5 μg/μl protein was applied to wells. Pollen tubes were stained with Coomassie blue at 6-h treatment. (D) Quantification method showing positive chemotropism. pg, pollen grain; pt, pollen tube. (Bar = 2 mm.)

Fig. 3.

Chemotropism assay showing pollen tube reorientation over time (A and B) and quantification method (C). Wells containing 1.5 μg/μl SPs (A) or water (B) were prepared 40 min before application at0hof pregerminated pollen with their tubes manually oriented away from the well. At 5 h, some pollen tubes in the control were out of the field (data not shown). A single reorienting pollen tube (indicated with arrows) monitored over time is enlarged (Inset). (C) Reorientation of a pollen tube by 90° or more was counted as positive chemotropism. (Bar = 2 mm.)

Fig. 4.

SDS/PAGE and Western blot of SPs (A), HPLC profile of SPs (B), and chemotropism assays (C–E). (A) SDS/PAGE (lane 1) and Western blot (lane 2) of SPs. Proteins were revealed by silver staining after SDS/PAGE. The polyclonal antibody used was produced against SCA (12). (B) SPs were separated into seven peaks. (C) Peak 7 mixed with the combined SCA peaks 2–4. (D) Peak 7 alone (0.23 μg/μl). (E) Peak 7 alone (0.69 μg/μl). (Bar = 2 mm.) Note that the lower amount (0.23 μg) of peak 7 showed full activity when it was mixed with the combined SCA peaks 2–4 in C.

Fig. 5.

Amino acid sequence alignments of lily chemocyanin with plantacyanin (basic blue protein) from other plant species. The deduced amino acid sequence was aligned with plantacyanin proteins from rice, Arabidopsis, and cucumber. The peptides identified by nano-ESI-MS/MS in chemocyanin are underlined. The arrowhead indicates the putative signal peptide cleavage site. Identical amino acids are indicated by asterisks. Colons show the conserved residues, and similar amino acids are indicated by dots. Amino acids involved in copper binding are shaded.