Arabidopsis CYCD3 D-type cyclins link cell proliferation and endocycles and are rate-limiting for cytokinin responses - PubMed (original) (raw)

Walter Dewitte et al. Proc Natl Acad Sci U S A. 2007.

Abstract

Current understanding of the integration of cell division and expansion in the development of plant lateral organs such as leaves is limited. Cell number is established during a mitotic phase, and subsequent growth into a mature organ relies primarily on cell expansion accompanied by endocycles. Here we show that the three Arabidopsis cyclin D3 (CYCD3) genes are expressed in overlapping but distinct patterns in developing lateral organs and the shoot meristem. Triple loss-of-function mutants show that CYCD3 function is essential neither for the mitotic cell cycle nor for morphogenesis. Rather, analysis of mutant and reciprocal overexpression phenotypes shows that CYCD3 function contributes to the control of cell number in developing leaves by regulating the duration of the mitotic phase and timing of the transition to endocycles. Petals, which normally do not endoreduplicate, respond to loss of CYCD3 function with larger cells that initiate endocycles. The phytohormone cytokinin regulates cell division in the shoot meristem and developing leaves and induces CYCD3 expression. Loss of CYCD3 impairs shoot meristem function and leads to reduced cytokinin responses, including the inability to initiate shoots on callus, without affecting endogenous cytokinin levels. We conclude that CYCD3 activity is important for determining cell number in developing lateral organs and the relative contribution of the alternative processes of cell production and cell expansion to overall organ growth, as well as mediating cytokinin effects in apical growth and development.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Arabidopsis CYCD3 genes display overlapping and distinct expression in proliferating tissues of the vegetative and floral shoot. CYCD3;1 expression is detected in young organs (A, D, and G), the periphery of the SAM, and the rib zone (D); CYCD3;2 and CYCD3;3 expression persists longer through lateral organ development (B, C, H, and I) and is detected throughout the vegetative SAM (E and F). In developing flowers CYCD3;2 (H) and CYCD3;3 (I) are maintained until opening. CYCD3;1 is detected only in very young flowers, most persistently in the petal (G).

Fig. 2.

Fig. 2.

Cellular development in cycd3;1-3 mutant and WT leaves and petals. (A) CYCD3 transcript levels in successive rosette leaves of a plant with seven visible leaves. L1 and L2 are the pair of juvenile leaves, and L3–L6 are sequential adult phase leaves (L6 being the youngest dissectible leaf). Relative levels are scaled to expression in the youngest leaf and show a faster decrease in the levels of CYCD3;1 mRNA in the older leaves. (B) Transcript levels of the mitotic gene CYCB1;1 in sequential leaves of cycd3;1-3 (d3;1-3) and WT, consistent with reduced cell division in young cycd3;1-3 leaves. (C–E) Adult phase leaves of cycd3;1-3 have fewer larger cells in both the adaxial epidermis and palisade mesophyll, whereas similar numbers were found in the juvenile leaves (L1+L2). (E) Traces of cell outlines for the adaxial epidermis (Upper) and photographs of palisade mesophyll (Lower) for WT (Left) and cycd3;1-3 (Right). (Scale bar: 50 μm.) (F) Cells in the adaxial epidermis of WT and cycd3 mutant petals. The number of cells in the adaxial epidermis is indicated for cycd3;1, cycd3;1-3, and WT. Loss of CYCD3;1 has the strongest effect. (Scale bar: 60 μm.) (G and H) Ploidy histogram for nuclei isolated from rosette leaves L3 (G) and petal (H) of WT and cycd3;1-3 at the time of flowering. The proportion of nuclei with 16C DNA content is increased in the cycd3;1-3 L3 (G), and a proportion of nuclei with 8C was detected in the mutant petals (H). (I) The growth rate of leaf 3 is similar for cycd3;1-3 and WT (Left), whereas the average ploidy in the mutant is higher (Right).

Fig. 3.

Fig. 3.

cycd3;1-3 mutants have SAM defects and reduced cytokinin responses. (A) Longitudinal sections of the SAM of WT (Left) and cycd3;1-3 (d3;1-3; Right); the mutant SAM contains fewer cells. (Scale bar: 50 μm.) (B) Callus induction on hypocotyl segments of triple cycd3 mutants. Unlike cycd3;1-3, WT explants form calli in the absence of cytokinin (kinetin) on both ends (Left). At 1,000 ng·ml−1 kinetin WT explants form green calli (Right), whereas cycd3;1-3 hypocotyls develop yellow root-like tissue [Upper, 3,000 ng·ml−1 NAA (auxin); Lower, 1,000 ng·ml−1 NAA]. (C) cycd3;1-3 floral shoots (Right) display reduced branching of axillary shoots. WT calli maintained for several weeks on 3,000 ng·ml−1 auxin and kinetin develop shoot-like structures of friable green cells (Left), whereas cycd3;1-3 develops yellow callus covered in roots (Right). (D) floral shoots of triple cycd3 mutants have a reduced density of siliques. (E) Dissected rosette leaves produced by WT and cycd3;1-3 before bolting. The floral stalk is presented at the time of analysis to illustrate late flowering.

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