SIAMESE, a plant-specific cell cycle regulator, controls endoreplication onset in Arabidopsis thaliana

Abstract

Recessive mutations in the SIAMESE (SIM) gene of Arabidopsis thaliana result in multicellular trichomes harboring individual nuclei with a low ploidy level, a phenotype strikingly different from that of wild-type trichomes, which are single cells with a nuclear DNA content of approximately 16C to 32C. These observations suggested that SIM is required to suppress mitosis as part of the switch to endoreplication in trichomes. Here, we demonstrate that SIM encodes a nuclear-localized 14-kD protein containing a cyclin binding motif and a motif found in ICK/KRP (for Interactors of Cdc2 kinase/Kip-related protein) cell cycle inhibitor proteins. Accordingly, SIM was found to associate with D-type cyclins and CDKA;1. Homologs of SIM were detected in other dicots and in monocots but not in mammals or fungi. SIM proteins are expressed throughout the shoot apical meristem, in leaf primordia, and in the elongation zone of the root and are localized to the nucleus. Plants overexpressing SIM are slow-growing and have narrow leaves and enlarged epidermal cells with an increased DNA content resulting from additional endocycles. We hypothesize that SIM encodes a plant-specific CDK inhibitor with a key function in the mitosis-to-endoreplication transition.

Figure 1.

sim Loss-of-Function Phenotype. (A) Scanning electron micrograph of a wild-type trichome. (B) Scanning electron micrograph of sim mutant trichome. Arrows indicate cell junctions. (C) Light micrograph of sim loss-of-function phenotype. (D) Complementation of sim loss-of-function phenotype by pGL2:SIM. (E) Complementation of sim loss-of-function phenotype by genomic fragment. (F) GUS staining pattern of sim-2 enhancer trap. (G) pCYCB1;1:GUS expression in sim mutant trichomes. Bars in (A) and (B) = 200 μm.

Figure 2.

SIM Encodes a Small Protein of Unknown Function Defining a Small Gene Family in Arabidopsis and Other Plants. (A) The SIM locus (At5g04470). Sequence changes in mutant alleles are indicated; the sim-2 gene contains an insertion of the pD991 enhancer trap T-DNA that deletes 1461 bp, including 272 bp of coding sequence and 1189 bp of upstream sequence. simL and simR indicate the primers used for RT-PCR (see Supplemental Figure 1 online). (B) Alignment of conceptual translation of SIM reading frame and related plant proteins. The regions numbered 1 to 5 denote conserved domains referred to in the text. Sl, Solanum lycopersicum; St, Solanum tuberosum; Zm, Zea mays; Os, Oryza sativa; Pt, Populus tremula; Gm, Glycine max. (C) Similarity between SIM and D-type cyclin binding domain of ICK/KRPs.

Figure 3.

The SIM Family Localizes to the Nucleus. Expression of EYFP fusion constructs in leaves was examined after introduction by biolistic bombardment of the DNA. EYFP alone (A), cytoplasmically localized; EYFP:TGA5 (B), TGA5 is a nuclear-localized trancription factor (Zhang et al., 1993; Kato et al., 2002); EYFP:SIM (C); EYFP:SMR1 (D); EYFP:SMR2 (E); EYFP:SMR3 (F). Bars = 18.75 μm; arrows indicate nuclei.

Figure 4.

Expression of the SIM Gene Family in Various Arabidopsis Tissues. (A) Absolute quantification of SIM family transcript levels by quantitative RT-PCR. (B) Increase of SIM transcript levels in response to increasing levels of GL3 function. Expression of SIM transcripts in leaves of a gl3 egl3 line that lacks GL3 function, a Columbia (Col) wild-type line with normal GL3 function, and a line overexpressing GL3 was compared by quantitative RT-PCR. The values shown represent averages of three separate biological replicates ± sd.

Figure 5.

Expression Pattern of SIM. In situ RNA hybridizations to longitudinal sections of the shoot apex probed with DIG-labeled single-stranded antisense SIM (A) and sense probe of Ceratopteris richardii gene of unknown function ([B]; accession number CV735270). Closed arrow indicates a developing trichome. Open arrows indicate procambial strands and developing vasculature. Bars = 200 μm.

Figure 6.

Phenotypic Analysis of SIM-Overexpressing Plants. (A) Four-week-old wild-type (left) and 35S:SIM (right) plants with inflorescences removed. (B) Second leaf of 35S:SIM plant shown in (A). (C) Adaxial epidermal pavement cells of wild-type first leaf. (D) Adaxial epidermal pavement cells of 35S:SIM first leaf. (E) Cross section through wild-type first leaf. (F) Cross section through 35S:SIM first leaf. (G) DAPI-stained epidermal pavement cell nuclei of wild-type first leaf. Arrows indicate nuclei. (H) DAPI-stained epidermal pavement cell nuclei of 35S:SIM first leaf. Arrows indicate nuclei. All analyses were done using 4-week-old wild-type and 35S:SIM plants. Bars = 1 cm in (A), 1 mm in (B), 200 μm in (C) and (D), 50 μm in (E) and (F), and 22 μm in (G) and (H).

Figure 7.

DNA Contents of SIM-Overexpressing Plants. (A) Ploidy level distribution of the first leaves of wild-type (Col) and 35S:SIM plants at 9, 15, and 21 DAS as measured by flow cytometry. The indicated values are means ± sd (n = 3 to 5). (B) Relative DNA contents of DAPI-stained epidermal pavement cell nuclei of wild-type (Col) and 35S:SIM enlarged cells of plants at 28 DAS. The degree of fluorescence is expressed as RFUs normalized to the mean fluorescence of Col. These RFU values have been adjusted to be comparable to published C values (see Methods), but they should only be interpreted as relative comparisons, not as measurements of absolute DNA contents.