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.