Cullin 4-ring finger-ligase plays a key role in the control of endoreplication cycles in Arabidopsis trichomes

Abstract

One of the predominant cell-cycle programs found in mature tissues is endoreplication, also known as endoreduplication, that leads to cellular polyploidy. A key question for the understanding of endoreplication cycles is how oscillating levels of cyclin-dependent kinase activity are generated that control repeated rounds of DNA replication. The APC/C performs a pivotal function in the mitotic cell cycle by promoting anaphase and paving the road for a new round of DNA replication. However, using marker lines and plants in which APC/C components are knocked down, we show here that outgrowing and endoreplicating Arabidopsis leaf hairs display no or very little APC/C activity. Instead we find that RBX1-containing Cullin-RING E3 ubiquitin-Ligases (CRLs) are of central importance for the progression through endoreplication cycles; in particular, we have identified CULLIN4 as a major regulator of endoreplication in Arabidopsis trichomes. We have incorporated our findings into a bio-mathematical simulation presenting a robust two-step model of endoreplication control with one type of cyclin-dependent kinase inhibitor function for entry and a CRL-dependent oscillation of cyclin-dependent kinase activity via degradation of a second type of CDK inhibitor during endoreplication cycles.

Fig. 1.

Destruction box reporter gene expression. (A–C) Trichome plants expressing PRO_GL2:YFP:DB in a WT background show YFP fluorescence in all trichomes throughout trichome development. (A) Bright field. (B) Fluorescence. (C) Overlay. (D–F) In contrast to WT, YFP fluorescence accumulates in a patchy pattern in only a few trichomes or cells of a multicellular trichome of sim mutant plants expressing PRO_GL2:YFP:DB. (D) Bright field. (E) Fluorescence. (F) Overlay.

Fig. 2.

Scanning electron micrographical analyses. (A) Plants expressing PRO_GL2:APC11-RNAi display unicellular and endoreplicated trichomes that are indistinguishable from WT trichomes. (B) Multicellular sim mutant trichome. (C) Expression of PRO_GL2:APC11-RNAi in sim mutant largely repressed the formation of multicellular trichomes. (D) Number of trichome clusters in sim and sim mutants expressing APC10-RNAi and APC11-RNAi. Red number of clusters, i.e., more than one trichome per trichome initiation site, blue trichome initiation sites with only one trichome. 11#1 and 11#2 refer to two independent sim lines expressing PRO_GL2:APC11-RNAi. Similarly, 10#1 and 10#2 refer to two independent PRO_GL2:APC10-RNAi in a sim mutant background. (Scale bars: A and B, 100 μm; C, 200 μm.)

Fig. 3.

DNA content. Box–whisker plot (after Tukey) of the DNA content in trichomes of Col-0 (Col), PRO_GL2:APC11-RNAi line 1 and line 2 (11#1, 11#2), glabra 3 (gl3), siamese (sim), PRO_GL2:APC11-RNAi in sim line 1 and line 2 (sim-11#1, sim-11#2), PRO_GL2:RBX1-RNAi line 1 and line 2 (R1, R2), PRO_35S:CUL4-RNAi (C4). Data were normalized to an assumed WT mean of 32C for Col and 16C for gl3 trichomes. Thus, one RFU approximately represents 1C. Boxes encompass 50% of all data points (25th through 75th percentile of data); lines within the boxes are the medians; and error bars represent 5th (lower bar) and 95th (upper bar) percentiles. R1, R2, and C4 have significantly reduced DNA levels in comparison with Col (P < 0.0001 for all three). In contrast, PRO_GL2:APC11-RNAi line 1 and 2 displayed no significant reduction of DNA levels when compared with WT (P < 0.0001). Both sim-11#1 and sim-11#2 have significantly higher DNA levels than sim mutant trichomes (P < 0.0001 for both).

Fig. 4.

Scanning electron micrographical analyses. (A) Expression of PRO_GL2:RBX1-RNAi results in plants with trichomes displaying strongly reduced growth and reduction of branches. (B) In addition, to growth defects PRO_GL2:RBX1-RNAi plants also display dead trichomes. (C) The phenotypes of PRO_GL2:RBX1-RNAi are reminiscent of plants expressing PRO_GL2:YFP:KRP1. (D) Coexpression of PRO_GL2:YFP:KRP1 and PRO_GL2:KRP x RBX1-RNAi causes a very strong additive phenotype and almost all trichomes on these plants die. (E) Trichomes on plant expression a PRO_35S:CUL4-RNAi construct are reduced in size. (Inset) Close-up of a rudimentary trichome found in PRO_35S:CUL4-RNAi plants. (F) Quantification of trichome morphologies found on PRO_GL2:YFP:KRP1 and PRO_GL2:RBX1-RNAi plants. Dark red refers to aborting trichomes; light red signifies unbranched trichomes; light blue indicates two-branched trichomes; dark blue symbolizes three-branched trichomes. R3 denotes reference plant number 3 expressing PRO_GL2:RBX1-RNAi; likewise, R4 denotes reference plant number 4. Both R3 and R4 display only a weak RBX1 knock-down phenotype. (Scale bars: A, B, E, and F, 100 μm; C and D, 200 μm.)

Fig. 5.

Cell-cycle simulation. (A) Diagram of the in the simulation used wiring of M-phase (MPF) and S-phase (SPF) CDK activity. (B) The simulation was run for 80 min with a low rate of synthesis for CDK inhibitor SIM (k_SIM = 0.01) characteristic for the mitotic cycle. At 80 min, the rate of SIM synthesis has been increased 10-fold (k_SIM = 0.1, indicated by black arrow). Details of simulations are given in SI Methods.