A lily pollen-specific cDNA encoding the Cdc42/Rac-interactive-binding motif-containing protein associated with pollen tube growth

Abstract

This work characterizes a pollen-specific and desiccation-associated transcript that encodes the CRIB (Cdc42/Rac-interactive-binding) motif-containing protein in lily (Lilium longiflorum Thunb. cv. Snow Queen) pollen during development and stress. The transcript, designated LLP12-2, encodes a gene product having a sequence of 222 amino acids, a calculated molecular mass of 24 kDa, and a calculated pI of 9.1. The protein contains a high content of glycine, serine, and proline (11-16%) with relatively high amounts (8%) of arginine and lysine. Sequence analysis revealed that the LLP12-2 is a Rho of plants- interactive CRIB motif-containing protein (RIC), sharing 40-41% identities with RIC6, RIC7, and RIC8, all of which belong to Group II RICs in Arabidopsis. Antiserum was raised against the overexpressed LLP12-2 protein in Escherichia coli. Affinity-purified LLP12-2 antibodies were prepared from antiserum to investigate the specificity and distribution of the protein during development. The affinity-purified LLP12-2 antibodies recognized both the 32- and 35-kDa pollen proteins, and the two polypeptides were immunologically related. Immunoblot analyses of total protein from floral and vegetative organs confirmed that LLP12-2 proteins accumulated to detectable levels only in a discrete stage of anther development. The level of LLP12-2 protein remained for 24 h during germination in vitro, and they were eventually degraded thereafter. Premature drying of developing pollen indicated that the accumulation of LLP12-2 proteins was associated with desiccation. Subcellular fractionation of pollen proteins revealed that the LLP12-2 was located in the cytosolic fraction, correlated to the observation of the overexpressed GFP-LLP12-2 in pollen tubes. Nevertheless, it is preferably associated with tube plasma membrane, particularly at the apical region of pollen tubes. Overexpression of GFP-LLP12-2 in lily pollen tubes causes distinct tip-growth phenotypes.