The Arabidopsis thaliana F-box protein FBL17 is essential for progression through the second mitosis during pollen development

Abstract

In fungi and metazoans, the SCF-type Ubiquitin protein ligases (E3s) play a critical role in cell cycle regulation by degrading negative regulators, such as cell cycle-dependent kinase inhibitors (CKIs) at the G1-to-S-phase checkpoint. Here we report that FBL17, an Arabidopsis thaliana F-box protein, is involved in cell cycle regulation during male gametogenesis. FBL17 expression is strongly enhanced in plants co-expressing E2Fa and DPa, transcription factors that promote S-phase entry. FBL17 loss-of-function mutants fail to undergo pollen mitosis II, which generates the two sperm cells in mature A. thaliana pollen. Nonetheless, the single sperm cell-like cell in fbl17 mutants is functional but will exclusively fertilize the egg cell of the female gametophyte, giving rise to an embryo that will later abort, most likely due to the lack of functional endosperm. Seed abortion can, however, be overcome by mutations in FIE, a component of the Polycomb group complex, overall resembling loss-of-function mutations in the A. thaliana cyclin-dependent kinase CDKA;1. Finally we identified ASK11, as an SKP1-like partner protein of FBL17 and discuss a possible mechanism how SCF(FBL17) may regulate cell division during male gametogenesis.

Figure 1. fbl17 T-DNA insertion mutants.

(A) FBL17 transcript accumulation in plants over-expressing the E2Fa transcription factor and its dimerization partner, DPa. Quantitative RT-PCR on RNA extracted from E2Fa-DPa overexpressing (OE) seedlings show a 15-fold increase in the relative abundance of FBL17 transcript compared to control RNA (Col-0). The experiment was three times repeated. Data are means±SE. (B) Diagram of the genomic locus of FBL17. The two T-DNA insertions disrupt the 7^th exon and the 6^th intron in the fbl17-1 and fbl17-2 allele respectively. Light grey filling indicate non-translated region of the transcript whereas dark grey filling indicates coding sequence. (C) Wild type silique opened to reveal the seed content. (D) Heterozygous fbl17-1^+/− silique displaying a reduced fertility and aborted seeds (marked by white arrows). (E) Homozygous fbl17-1 mutant complemented with the FBL17 genomic clone show wild type siliques and normal seed development. (C, D, E, bar = 500 µm).

Figure 2. Pollen phenotype of fbl17 mutants and FBL17 expression during male gametogenesis.

fbl17 (A) and wild type (B) mature pollen viability test by coloration with Alexander's stain. The purple staining indicates that the grains are viable. Dehiscent pollen of qrt1-1^−/− (C) and qrt1-1^−/−, fbl17-1^+/− (D) stained with DAPI and observed under UV fluorescence. The four pollen grains of the tetrad show two densely stained sperm cell nuclei and one large diffuse vegetative cell nuclei in qrt-1 mutants, whereas two pollen grains of the tetrad show only a single germ cell nuclei in qrt1-1,fbl17-1^+/− double mutants. (E, F) transmitted light picture of C and D. (G–L) Expression of the HT10 gene in the fbl17 mutant pollen. Expression of the HTR10-mRFPprotein under the HTR10 promoter in fbl17-1 (H) and wild type (K) pollen, counterstained with DAPI (G, fbl17-1; J, wild type). (I, L) transmitted light pictures of G and J. (A, B) bar = 100 µm; (C–L) bar = 10 µm. (M–U) Promoter-GUS analysis of FBL17 expression in pollen. (M, P, S) DAPI staining is applied to reveal the developmental stage of the pollen grain. (N–U) X-Gluc histochemical staining of pFBL17∶GUS (N, Q, F) and non-transformed Col-0 (O, R, U) pollen grains. Bars = 10 µm (V) DNA content measurement of wild type germinative cell nuclei at prophase (n = 9; DNA = 2C), wild type sperm cell nuclei at telophase (n = 16), WT sperm cell nuclei at anthesis (n = 111) in comparison to the unique germ-cell like nuclei of fbl17 pollen (n = 77). Error bars = standard error mean.

Figure 3. Fertilization with fbl17 mutant pollen leads to embryo developmental arrest and seed abortion.

Embryo development in wild type plants fertilized with fbl17-1 mutant (A, E) and wild type (B) pollen, 3 days after pollination. Embryo development in wild type plants fertilized with fbl17-1 mutant (C, F) and wild type (D) pollen, 4 days after pollination. Early fertilization-independent development of the endosperm is visible in fbl17-1-fertilized seeds (E) as evidenced by the presence of multiple nuclei (asterisks). Arrested globular embryo in fbl17-1 mutant seeds with degenerated endosperm (F). Bars: 50 µm.

Figure 4. FBL17 interacts with a subset of ASKs.

(A) Yeast-two-hybrid analysis of the interaction between FBL17 and ASKs. Yeast were grown for 3 days at 28°C. LTH- 3AT, low stringency selection, LTA, high stringency selection. Negative controls were done with empty bait vectors (pGBD) or empty prey vectors (pGAD). (B–G) Subcellular interaction of FBL17 with ASK11. Confocal laser-scanning micrographs of the abaxial surface of N. benthamiana leaves. (B, C) Transient expression of ASK11-YFP. The YFP signal is detected both in cytoplasm and nucleus. (D, E) Transient expression of FBL17-YFP. The signal is exclusively nuclear. (F–G) BiFC of FBL17-YN/ASK11-YC. Reconstitution of functional YFP as detected by YFP fluorescence occurs only in the nucleus. (H–K) Subcellular interaction of FBL17 with KRP7. (H, I) Transient expression of KRP7-YFP. A weak YFP signal in the cytoplasm and a strong signal in the nucleus can be detected. (J, K) BiFC of FBL17-YN/KRP7-YC. Reconstitution of functional YFP as detected by YFP fluorescence occurs only in the nucleus. (B, D, F, H, K, J) DIC images of the cells documented. (C, E, G, I, K) laser confocal micrograph of the YFP signal. Scale bars in B to K represent 45 µm.

Figure 5. Model showing how the UPS could regulate the cell cycle during male gametogenesis.

In this model RHF1a and RHF2a mediate KRP6 26S-proteasome-dependent degradation starting from meiosis . RHF1a and RHF2a loss-of-function of leads to KRP6 accumulation and as a consequence blocks both PMI and PMII. The SCF^FBL17 E3, most likely containing ASK11, mediates KRP6/7 (and eventually other proteins) is strictly required to allow PMII to occur ( and our work). The role of UBP3 and UBP4 during pollen development remains to be determined.