Identification And Functional Characterization Of Two Genes Associated With Pollen Germination And Tube Growth In Picea Wilsonii

Pollen tube, a carrier of sperm nuclei during the process of sexual plant reproduction in flowering plants, is a highly polarized, rapidly tip-growing plant cell, and thus is an ideal system for studying the molecular mechanism involved in the regulation of cell growth. As compared with angiosperm, pollen tubes of coniferous species grow slowly, tend to ramify, and lack a tip-to-base zonation of organelles, implying that tubes of the two types of plant differ in developmental mechanism and wall construction. However, the mechnisms of pollen germination and tube growth in coniferous species remain enigmatic. Thus, in this paper, we constructed pollen and pollen tubes cDNA library and cloned a cDNA fragment ofα-tubulin gene (TUA1) in coniferous species (Picea wilsonii). In addition, a Ca2+-induced cDNA library of Picea wilsonii pollen was constructed and a CCAAT-box binding factor gene (HAP5) was obtained by differential hybridization. A series of studies have been conduced on the sequence and expression analysis and functional identification of PwTUA1 and PwHAP5.The obtained results are as follows:1. A cDNA library of Picea wilsonii pollen and pollen tubes was constructed with Clontech SmartTM cDNA Library Construction Kit. Two mixed primers were designed to amplify specific DNA fragment using cDNA library on the homologous sequences from other plants. The middle fragment of interested cDNA was obtained by RT-PCR. The full length cDNA was further isolated by 5'-RACE and 3'-RACE. The isolated cDNA is 1721 bp in length and harbors an opening reading frame of 1353 bp encoding a 49.58 kDa protein of 451 amino acids. A Ca2+-induced cDNA library of Picea wilsonii pollen was constructed and a CCAAT-box binding factor gene (HAP5) was obtained by differential hybridization. The isolated cDNA is 985bp in length with an open reading frame (ORF) of 603bp comprising 201 amino acid residues with the predicted molecular mass of 22.44kDa. 2. Multi-alignment analysis revealed that PwTUA1 was highly related to other plant TUAs, sharing a homology of 88.47% to AtTUA1, 97.78% to GhTUA1, 96.9% toOsTUA1, and 99.78% to PmTUA1. The phylogenetic analysis of various TUAs also indicated that PwTUA1 was attributed to class I . The main functional domains of PwHAP5 were conserved compared with other HAP5s from Arabidopsis, rice, yeast and human. The phylogenetic analysis also indicated that the kinship of PwHAP5 was close to AtNF-YC9(AtHAP5C) and OsHAP5A/B.3. Semi-quantitative RT-PCR was used to examine the expression of PwTUA1 and PwHAP5. The results showed that PwTUA1 was expressed specifically in germinating pollen but was not detected in needles, stems, and roots. PwTUA1 was first detected at 6 h after incubation (just germinated); it was abundant during middle developmental stages (12, 18, and 24 h after incubation) and then continued weakly at later stages (30 and 36 h after incubation). The expression of PwHAP5 was detected in all of the tissues and increased during the pollen germination stages till reached the peak after incubation for 18h and keep to the tubes stop growing.4. Transient expression of PwTUA1 improved pollen germination and pollen tube growth in Picea wilsonii, while transient expression of PwHAP5 changed the orientation of pollen tubes growth.5. Arabidopsis overexpressing PwTUA1 driven by the CaMV 35S promoter showed growth retardation and helical growth to some degree. Some of them were unable to complete morphogenesis, resulting in death. When PwTUA1 was expressed in Arabidopsis pollen exclusively by using the pollen-specific promoter Lat52, both pollen germination and pollen tube growth were enhanced in the transgenic lines when compared to wild-type Arabidopsis, even at the nonoptimal boron or Ca2+ concentrations present in the culture medium. PwTUA1-overexpression changed the subcellular localization of theα-tubulin protein and affected the ultrastructure of pollen tubes in Arabidopsis. Theα-tubulin protein existed throughout the tubes of wild-type Arabidopsis, excluding the tip or apical region. However, highly intense fluorescence was observed in the apical region of transgenic Arabidopsis pollen tubes. The pollen tube wall of transgenic Arabidopsis was only slightly detectable, while the wall of wild-type Arabidopsis was much thicker. Additionally, many short, solitary cytoplasmic MTs were observed in the tip-most portion of the transgenic pollen tubes where the vesicles accumulated, but were rarely observed in the tip region of wild-type pollen tubes.6. Overexpressing PwHAP5 changed the arrangement of transgenic Arabidopsis siliques instead of the orient of pollen tubes growth. Interactive proteins were screened from Picea wilsonii pollen cDNA library with the N, C terminal and full-length of PwHAP5 as bait protein by Yeast Two-Hybrid System. More than 100 positive clones were identified by PCR colony screening and the representative clones were sequenced. Among them 7 clones were selected which interacted with C, N terminal and full-length PwHAP5. By blast in GenBank, the prey cDNA inserts were homologous to mRNA sequences in Arabidopsis. PwFKBP12 protein was selected and we tested its interaction with PwHAP5 in vivo by using bimolecular fluorescenc complementation (BiFC) analysis.