Pollen phenotypes of mutants for components of the G1/S phase control module.

<p>(A) Tricellular mature wild-type DAPI-stained pollen at anthesis (one vegetative cell enclosing two sperm cells). (B) DAPI-stained pollen at anthesis from heterozygous <i>cdka;1</i> mutant plants (similar to pollen from heterozygous <i>fbl17</i> mutants, data not shown) containing approximately 43% bicellular pollen (one vegetative cell and one sperm-cell-like cell) and 57% tricellular, wild-type-like pollen. (C) DAPI-stained pollen at anthesis from double heterozygous <i>cdka;1 fbl17</i> mutant plants carrying a hemizygous <i>Pro_CDKA;1:CDKA;1:YFP</i> rescue construct (similar to pollen from <i>e2fa^−/− fbl17^+/−</i> mutants, data not shown) and containing single-celled pollen grains (only one vegetative-like cell), in addition to bicellular (<i>cdka;1</i>/<i>fbl17</i>-like) and tricellular (wild-type-like) pollen. (D) Close-up of bicellular pollen as found in <i>cdka;1</i> or <i>fbl17</i> heterozygous plants. (E) Close-up of monocellular pollen grains as found in <i>cdka;1 fbl17</i> or <i>e2fa fbl17</i> double heterozygous mutants. (F) Quantification of DAPI-stained pollen. The DNA content of the single-celled pollen from <i>cdka;1^+/− fbl17^+/−</i> or <i>e2fa^−/− fbl17^+/−</i> double mutants reaches 1C, similarly to the vegetative nucleus in wild-type pollen and, thus, resides in a G1 phase.</p

A General G1/S-Phase Cell-Cycle Control Module in the Flowering Plant <em>Arabidopsis thaliana</em>

<div><p>The decision to replicate its DNA is of crucial importance for every cell and, in many organisms, is decisive for the progression through the entire cell cycle. A comparison of animals versus yeast has shown that, although most of the involved cell-cycle regulators are divergent in both clades, they fulfill a similar role and the overall network topology of G1/S regulation is highly conserved. Using germline development as a model system, we identified a regulatory cascade controlling entry into S phase in the flowering plant <em>Arabidopsis thaliana</em>, which, as a member of the <em>Plantae</em> supergroup, is phylogenetically only distantly related to <em>Opisthokonts</em> such as yeast and animals. This module comprises the <em>Arabidopsis</em> homologs of the animal transcription factor E2F, the plant homolog of the animal transcriptional repressor Retinoblastoma (Rb)-related 1 (RBR1), the plant-specific F-box protein F-BOX-LIKE 17 (FBL17), the plant specific cyclin-dependent kinase (CDK) inhibitors KRPs, as well as CDKA;1, the plant homolog of the yeast and animal Cdc2⁺/Cdk1 kinases. Our data show that the principle of a double negative wiring of Rb proteins is highly conserved, likely representing a universal mechanism in eukaryotic cell-cycle control. However, this negative feedback of Rb proteins is differently implemented in plants as it is brought about through a quadruple negative regulation centered around the F-box protein FBL17 that mediates the degradation of CDK inhibitors but is itself directly repressed by Rb. Biomathematical simulations and subsequent experimental confirmation of computational predictions revealed that this regulatory circuit can give rise to hysteresis highlighting the here identified dosage sensitivity of CDK inhibitors in this network.</p> </div

Interaction assays of dominant negative versus wild-type CDKA;1 variants.

<p>Positive BiFC assays showing the interaction of CDKA;1 with KRP1 under bright field (A) and epifluorescence (B). (C) The dominant-negative CDKA;1 variant CDKA;1^D146N interacting with both positive (cyclins) and negative (KRPs) regulators similar to the wild-type CDKA;1 form. The CDKA;1^PSTAIRE-dead variant shows neither interaction with cyclins nor with KRPs, but can still bind to the cofactor CKS. The interactions are presented in a semiquantitative manner based on the number of positive cells obtained and the observed fluorescent intensities. All interactions were at least three times independently tested.</p

General G1/S phase cell-cycle control module.

<p>(A) The transcription factor E2F activates the expression of <i>FBL17</i>, which is repressed by RBR1. FBL17 targets the CDKA;1 inhibitors KRP1, KRP3, KRP4, KRP6 and KRP7 for proteasome-dependent degradation, enabling the germ cell to progress through S phase. Phosphorylation of RBR by the CDKA;1-cyclin complex will relieve the inhibition of the S-phase genes and allows transcription of the <i>FBL17</i> gene. Promoters and genes are depicted in light sand color; proteins in dark sand; transcription is indicated by a grey arrow; negative regulation, i.e. at the transcriptional or protein level, is shown by rust-colored lines with a blunt end; positive regulation by a green line with a green arrowhead. Receiving input is placed above and executing output under the respective gene/protein. (B) The model presented in A gives rise to a bistable switch controlling the G1-to-S transition in the plant cell cycle. KRPs inhibit the CDKA;1-cyclin complexes, which in turn downregulate the levels of KRPs by phosphorylating and inhibiting RBR1, thereby activating E2F-dependent FBL17 synthesis leading to the degradation of KRPs. The antagonistic interaction between CDKA;1 and KRP is illustrated by the two curves (red and green for KRP and for CDKA;1, respectively) along which the rates of synthesis and degradation of KRPs and the CDKA;1-cyclin complexes are exactly balanced. The KRP balance curve has an inverse S-shape with high and low levels, depending on the CDKA;1-cyclin values. The dashed branch of the balance curve represents unstable steady states. At low CDKA;1-cyclin levels, only one steady state exists with high KRP levels and low CDKA;1 activity. At intermediate CDKA;1 levels, the system is bistable with three steady states. At high CDKA;1-cyclin values, the steady-state level of KRP is low and the CDKA;1-cyclin complexes are fully active. The transition from high to low KRP values corresponds to the G1-to-S transition. (C) Quantitative expression analyses of <i>CDKA;1</i> and <i>FBL17</i> in wild type and heterozygous <i>cdka;1</i> mutants. The mean plus standard deviation of the normalized relative quantities (NRQ) of three biological replicates are shown. The stars indicate statistically significant differences based on a t-test of log-transformed data with a p<0.05. As expected, the expression of <i>CDKA;1</i> drops by approximately 50% in the mutant. In addition, <i>FBL17</i> expression declines, consistent with a prediction of the model presented in A and B.</p

Accumulation and localization of CDKA;1-YFP fusion protein during female and male gametophyte development.

<p>(A-A^VI) Expression of a <i>PRO_CDKA;1:CDKA;1-YFP</i> construct completely rescues <i>cdka;1</i> pollen resulting in wild-type-like pollen with one vegetative cell (arrowhead in A^V) and two sperm cells (triangle in A^V and A^VI, see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002847#pgen-1002847-g001" target="_blank">Figure 1</a>). <i>CDKA;1</i> is expressed throughout male gametophyte development but becomes restricted at anthesis to the two sperm cells as revealed by YFP accumulation. (B-B^VI) A hemizygous <i>PRO_CDKA;1:CDKA;1-YFP^+/−</i> allele in homozygous mutant <i>cdka;1^−/−</i> plants mimics heterozygous <i>cdka;1^+/−</i> mutant plants that produce pollen of which half resembles wild-type pollen but half comprises one vegetative cell and only one instead of two sperm cells at anthesis (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002847#pgen-1002847-g001" target="_blank">Figure 1</a>). Continuous observation of the CDK-YFP fusion protein during male gametophyte development showed that the CDKA;1 protein concentration gradually decreased in 50% of pollen (marked by an asterisk) and that around the bicellular stage, clearly one pollen population can be identified that shows no or very little YFP fluorescence; occasionally, residual CDKA;1-YFP protein could be detected in the single sperm pollen at anthesis (red arrowhead in B^VI). A and B, Microspore mother cell; A^I and B^I, tetrads; A^II and B^II, monocellular stage; A^III and B^III, early bicellular stage; A^IV and B^IV, late bicellular stage; A^V and B^V, tricellular stage; A^VI and B^VI, anthesis. (C) Cartoon summarizing the decrease in CDKA;1 concentration as seen in B-B^VI. At the bicellular stage, CDKA;1 levels in mutant pollen (dashed yellow line) drop below an assumed threshold (dashed red line) for executing mitosis. Consistent with the disappearance of the YFP fluorescence at this stage, <i>cdka;1</i> mutant pollen typically arrest before PMII. (D-D^VI) CDKA;1-YFP signal appears in all nuclei at all developmental stages of the developing embryo sac (MMC to FG7, arrowheads mark the nuclei in the embryo sac). At maturity, YFP fluorescence is only present in the gametic cells, the egg cell and the central cell (marked by a white triangle in D^VI). MMC, megaspore mother cell/microspore mother cell; MC, monocellular; BC, bicellular; TC, tricellular; FG, female gametophyte stage.</p

Mature ovules and seed development in wild type and <i>cdka;1 fbl17</i> double mutant.

<p>(A–C) Wild-type embryo sac and seed development; (D–F) aberrant development in a class of <i>cdka;1 fbl17</i> mutant plants. (A) Wild type showing a typical cellular morphology (arrowheads pointing to the central cell nucleus and the egg cell nucleus from top to bottom, respectively). Aberrant morphologies in the <i>cdka;1^+/− fbl17^+/−</i> double mutant with one (D), or two (G) nuclei in the absence of a cellularized egg apparatus. (B) While the wild-type seed 3 days after pollination has a normal embryo and endosperm development, the double mutant seed development collapsed after pollination, with only one (E) or two (H) nuclei staying in the middle of the empty embryo sac. (C) Wild type showing normal seed development 6 days after pollination, while <i>cdka;1^+/− fbl17^+/−</i> double mutants show approximately 24% seed abortion (F) after pollination with the wild-type pollen.</p

Pollen phenotypes.

<p>Pollen from anthers just before flowering of wild type and the indicated genotypes was stained with DAPI and epifluorescence was observed under UV illumination. n = total number of pollen analyzed.</p

Interaction of FBL17 with KRPs in BiFC assays and degradation promotion of KRP <i>in vivo</i>.

<p>Two nonfluorescent fragments (YN and YC) of the yellow fluorescent protein (YFP) fused to FBL17 (YN-FBL17) and seven different CDK inhibitors (YC-KRP1–7). Co-production of FBL17 and KRP1-KRP7 fusions in tobacco leaves reconstituted the expected yellow fluorescence for all seven protein combinations. Exemplarily, one interaction in the BiFC assays is shown in (A–B) displaying the interaction of FBL17 with KRP1 under bright field (A) and epifluorescence (B). (C) Transient expression assays were conducted in tobacco leaves to determine whether FBL17 can target CDK inhibitors for degradation. Among the seven KRPs, FBL17 can especially reduce the fluorescence, implying protein degradation, for KRP3, KRP4, KRP5 and KRP7, whereas the fluorescence intensity of KRP2 and KRP6 diminished only moderately after co-infiltration with FBL17. CKS1 was used as a reference.</p