PwHAP5, a CCAAT-binding transcription factor, interacts with PwFKBP12 and plays a role in pollen tube growth orientation in Picea wilsonii

Abstract

The HAP complex occurs in many eukaryotic organisms and is involved in multiple physiological processes. Here it was found that in Picea wilsonii, HAP5 (PwHAP5), a putative CCAAT-binding transcription factor gene, is involved in pollen tube development and control of tube orientation. Quantitative real-time reverse transcription-PCR showed that PwHAP5 transcripts were expressed strongly in germinating pollen and could be induced by Ca(2+). Overexpression of PwHAP5 in pollen altered pollen tube orientation, whereas the tube with PwHAP5RNAi showed normal growth without diminishing pollen tube growth. Furthermore, PwFKBP12, which encodes an FK506-binding protein (FKBP) was screened and a bimolecular fluorescence complementation assay performed to confirm the interaction of PwHAP5 and PwFKBP12 in vivo. Transient expression of PwFKBP12 in pollen showed normal pollen tube growth, whereas the tube with PwFKBP12RNAi bent. The phenotype of overexpression of HAP5 on pollen tube was restored by FKBP12. Altogether, our study supported the role of HAP5 in pollen tube development and orientation regulation and identified FKBP12 as a novel partner to interact with HAP5 involved in the process.

Fig. 1.

HAP5 gene of P. wilsonii. (A) Alignment of the HAP5 proteins, sequences correspond to the conserved regions in HAP5 proteins across various lineages. Dc, Daucus carota; Hs, Homo sapiens; Os, Oryza sativa; Sc, Saccharomyces cerevisiae. Note that the HAP2 interaction domain extends across two separate regions. The DNA-binding domain in HAP5 consists of the two amino acids AR (found in most HAP5 homologues). (B) Phylogenetic tree of P. wilsonii HAP5 (PwHAP5) and other HAP5 proteins previously characterized. A neighbor-joining tree based on the deduced amino acid sequences of the conserved domains in HAP5s. This bootstrap consensus tree was based on 1000 replicates. Numbers on nodes are bootstrap values. The accession numbers in GenBank and sources of the protein are as follows: AtNF-YC1(At3g48590), AtNF-YC2(At1g56170), AtNF-YC3(At1g54830), AtNF-YC4(At5g63470), AtNF-YC5 (At5g50490), AtNF-YC6(At5g50480), AtNF-YC7(At5g50470), AtNF-YC8(At5g27910), AtNF-YC9(At1g08970), AtNF-YC10(At1g07980), AtNF-YC11(At3g12480), AtNF-YC12(At5g38140), AtNF-YC13(At5g43250) from Arabidopsis thaliana; DcHAP5(AB104612) from D. carota; HsNF-YC(U78774) from H. sapiens; OsHAP5A(AB288041), OsHAP5B(AB288042), OsHAP5C(AB288043), OsHAP5D(AB288044), OsHAP5E(AB288045), OsHAP5F(AB288046), OsHAP5G(AB288047) from O. sativa; ScHAP5(U19932) from S. cerevisiae.

Fig. 2.

Expression of PwHAP5 in different tissues and in developing pollen tubes of P. wilsonii. (A) Tissue-specific expression of PwHAP5 in P. wilsonii. Total RNA was isolated from needles, stems, roots, and pollen (incubated after 0, 6, 12, 18, and 24 h). Above the graph is shown semi-quantitative RT-PCR analysis of expression of PwHAP5. The EF1-α gene was amplified as an internal control. The graph shows quantification of PwHAP5 expression. Quantitative real-time RT-PCR was performed using PwHAP5-specific primers. Column heights represent expression levels relative to the EF1-α gene. The values are means ±SD (n = 3) from three independent experiments. (B) Expression of PwHAP5 in pollen at different development stages (incubated after 0, 6, 12, 18, 24, 30, and 36 h). Above the graph is shown semi-quantitative RT-PCR analysis of expression of PwHAP5; the graph represents the quantification of PwHAP5 expression. The control and values are as in (A). (C) Semi-quantitative RT-PCR analysis of expression of PwHAP5 in pollen 0, 12, 24, and 36 h after incubation, induced by 0.1% (w/v) Ca²⁺ or 0.1% (w/v) H₃BO₃. (D) Quantification of PwHAP5 expression in P. wilsonii pollen at the same intervals after incubation as in (C). The control and values are as in (A).

Fig. 3.

Transient expression of PwHAP5 alters the orientation of P. wilsonii pollen tube growth. (A) Diagram of plant expression vectors. The pollen-specific expression promoter Lat52 was used to drive the expression of PwHAP5 in pollen tube. (B) The effect of PwHAP5 on pollen germination and tube length using microprojectile bombardment. After 12 h and 24 h of incubation, the percentage germination and tube length of pollen bombarded with the GFP-only, PwHAP5 overexpression, or RNAi construct showed no significant difference compared with the untreated control pollen (P<0.05). Error bars indicate averages from the number of pollen tubes measured (n≥50). (C) Transient expression of PwHAP5 in P. wilsonii pollen tube growth. PwHAP5–GFP represents overexpression of PwHAP5 in P. wilsonii pollen tube. PwHAP5RNAi+GFP represents interference of PwHAP5. GFP was used as control. The data were obtained from three independent experiments, and every condition was tested three times. Bars 20 μm.

Fig. 4.

Interaction of PwHAP5 with FKBP12. (A) Amino acid alignment of FKBP12 isoforms from different species. H. sapiens (Hs; GenBank protein accession number AAI19733), S. cerevisiae (Sc; AAA03564), Drosophila melanogaster (Dm; AAF57582), Caenorhabditis elegans (Ce; CAA22330), A. thaliana (At; AAB57847), and O. sativa (Os; NP_001048188.1) aligned with P. wilsonii (Pw). Asterisks above residues denote residues that are important for peptidyl prolyl cis/trans isomerase activity in mammalian FKBP12 (DeCenzo et al., 1996; Tradler et al., 1997). Residues in boxes are involved in FK506 and/or rapamycin binding in mammalian FKBP12 (Van Duyne et al., 1993). (B) The interactions were assayed in the GAL4 (a regulator of galactose-induced genes) Y2H system to re-examine the interactions of full-length PwHAP5 (H), N77 (N), and C130 (C) proteins with the pGBDKT7 vector alone (BD) and with PwFKBP12. As a control, combinations of the pGADT7-Rec vector (AD) with the pGBDKT7 vector (BD) and PwTUA1 are also shown. Yeast cells expressing AD–H, AD–N, AD–C, or AD alone and each of BD fusion proteins or BD alone were grown on medium-selective plates (SD/–Leu–Trp–His–Ade). The blue represents growth and interaction between PwHAP5 and PwFKBP. (C) Liquid β-galactosidase assay using o-NPG as a substrate. β-galactosidase activity is expressed in U (=nmol min⁻¹). The values displayed are the average β-galactosidase activities for three individual double transformants, with standard deviations indicated by error bars.

Fig. 5.

In vivo interaction of PwHAP5 with PwFKBP in the BiFC system. BiFC in A. tumefaciens-infiltrated tobacco (N. benthamiana) leaves was visualized by laser confocal microscopy. The laser-scanning confocal microscopy images show fluorescence (indicated by YFP) and merged images of double transformed tobacco leaves with the YFP_N–PwHAP5 (full-length PwHAP5; H), N77 (N), and C130 (C), respectively) and PwFKBP12–YFP_C fusions (YFP_N–PwHAP5/ PwFKBP12–YFP_C). PwHAP5 interacts with PwFKBP12 in the cytoplasm, and does not interact with the negative control PwTUA1 or empty vector. The YFP fluorescence of the positive control vectors, bZIP63-pSPYNE-35S and bZIP63-pSPYCE-35S, was detected only in the nucleus. Arrows indicate the nucleus. Bar 25 μm.

Fig. 6.

Transient expression of PwHAP5 or PwFKBP12 in P. wilsonii pollen tube. The overexpression vectors PwHAP5 or PwFKBP12 fused to CHERRY or GFP, respectively, were constructed and expressed transiently alone or used to co-transform P. wilsonii pollen by microprojectile bombardment. The pollen-specific expression promoter Lat52 was used to drive the expression of PwHAP5 or PwFKBP12 in pollen tube. Microscopic analysis of pollen tube expression was performed 24 h after gene transfer of PwFKBP12 and PwHAP5 in pollen. Transient expression of PwHAP5 alone affects P. wilsonii pollen tube growth orientation, whereas both transient expression of PwFKBP12 and co-expression of PwHAP5 and PwFKBP12 do not. Left panel: transmitted light images. Right panel: fluorescence images. The yellow fluorescence is the overlay of GFP and CHERRY proteins. The data were obtained from three independent experiments, and each condition was tested three times. Bars, 20 μm.