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

Gusti A, Baumberger N, Nowack M, Pusch S, Eisler H, Potuschak T, De Veylder L, Schnittger A, Genschik P - PLoS ONE (2009)

Bottom Line: FBL17 expression is strongly enhanced in plants co-expressing E2Fa and DPa, transcription factors that promote S-phase entry.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.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie Moléculaire des Plantes du CNRS, Université de Strasbourg, Strasbourg, France.

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.

Show MeSH
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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2651519&req=5

pone-0004780-g004: 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.

Mentions: Since FBL17 carries an F-box motif, we next examined whether this protein forms an SCF-type complex. Yeast two hybrid assays were conducted with the different A. thaliana SKP1-like proteins, called ASKs [38]. In yeast, several ASKs were able to interact with FBL17 when fused to the GAL4 DNA binding domain while failing to allow yeast growth on selective medium when fused to the activating domain (Figure 4A). In contrast, ASK11 displayed a robust interaction with FBL17 in both fusions. Interestingly, ASK11 is among the members of the A. thaliana ASK family that are strongly expressed in pollen [39], [40] and constitutes therefore a strong candidate for participating in an SCFFBL17 complex. It is noteworthy that the most predominant ASK, ASK1, was not found to interact with FBL17 in our yeast-two hybrid experiment. To further test a possible interaction between FBL17 and ASK11, we performed bimolecular fluorescence complementation (BiFC) experiments. As a control we fused to the N- and C-terminal half of YFP to the C-terminus of FBL17 and GUS, respectively. After co-injection of these fusion constructs into Nicotiana benthamiana leaf cells no YFP fluorescence could be found (data not shown). In contrast, when split-YFP fusions with FBL17 and ASK11 were co-injected, a strong fluorescence signal was recovered corroborating an interaction between these two components of an SCF-type complex in vivo (Figure 4B–G). Moreover transient expression of YFP fusions revealed that the FBL17 protein is essentially nuclear, whereas ASK11 localizes both to the cytoplasm and the nucleus (Figure 4B–E). Consequently, an interaction between ASK11 and FBL17 in the BiFC assays was restricted to nuclei (Figure 4F,G).


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

Gusti A, Baumberger N, Nowack M, Pusch S, Eisler H, Potuschak T, De Veylder L, Schnittger A, Genschik P - PLoS ONE (2009)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2651519&req=5

pone-0004780-g004: 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.
Mentions: Since FBL17 carries an F-box motif, we next examined whether this protein forms an SCF-type complex. Yeast two hybrid assays were conducted with the different A. thaliana SKP1-like proteins, called ASKs [38]. In yeast, several ASKs were able to interact with FBL17 when fused to the GAL4 DNA binding domain while failing to allow yeast growth on selective medium when fused to the activating domain (Figure 4A). In contrast, ASK11 displayed a robust interaction with FBL17 in both fusions. Interestingly, ASK11 is among the members of the A. thaliana ASK family that are strongly expressed in pollen [39], [40] and constitutes therefore a strong candidate for participating in an SCFFBL17 complex. It is noteworthy that the most predominant ASK, ASK1, was not found to interact with FBL17 in our yeast-two hybrid experiment. To further test a possible interaction between FBL17 and ASK11, we performed bimolecular fluorescence complementation (BiFC) experiments. As a control we fused to the N- and C-terminal half of YFP to the C-terminus of FBL17 and GUS, respectively. After co-injection of these fusion constructs into Nicotiana benthamiana leaf cells no YFP fluorescence could be found (data not shown). In contrast, when split-YFP fusions with FBL17 and ASK11 were co-injected, a strong fluorescence signal was recovered corroborating an interaction between these two components of an SCF-type complex in vivo (Figure 4B–G). Moreover transient expression of YFP fusions revealed that the FBL17 protein is essentially nuclear, whereas ASK11 localizes both to the cytoplasm and the nucleus (Figure 4B–E). Consequently, an interaction between ASK11 and FBL17 in the BiFC assays was restricted to nuclei (Figure 4F,G).

Bottom Line: FBL17 expression is strongly enhanced in plants co-expressing E2Fa and DPa, transcription factors that promote S-phase entry.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.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie Moléculaire des Plantes du CNRS, Université de Strasbourg, Strasbourg, France.

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.

Show MeSH