Ectopic D-type cyclin expression induces not only DNA replication but also cell division in Arabidopsis trichomes

Abstract

Although the mechanisms controlling the two cell-cycle checkpoints G(1)-S and G(2)-M are well studied, it remains elusive how they are linked in higher eukaryotes. In animals, D-type cyclins have been implicated in the control of cell-cycle progression in mitotic as well as in endoreduplicating cells. By contrast, we show that the expression of the D-type cyclin CYCD3;1 in endoreduplicating Arabidopsis trichome cells not only induced DNA replication but also cell divisions.

Figure 1

Morphological analysis. (a) Scanning electron micrograph of single-celled mature wild-type trichomes. (b) Scanning electron micrograph of multicellular pGL2∷CYCD3;1#1 trichomes. (c) Four consecutive sections through a multicellular pGL2∷CYCD3;1#1 trichome revealing many separate cells. (d) Scanning electron micrograph of pGL2∷CYCD3;1#2 trichomes that have a lower rate of multicellular trichomes in comparison to line 1. (e) Light micrograph of 4′,6-diamidino-2-phenylindole-stained trichomes of pGL2∷CYCD3;1–pGL2∷CYCB1;2 plants showing a strong synergistic phenotype with up to 80 nuclei per trichome initiation site (TIS); similar trichomes arise by the expression of pGL2∷CYCD3;1 in a homozygous sim mutant background. (f) Staining of pGL2∷CYCD3;1 trichomes revealing the expression of the trichome marker line pGL2∷GUS. (g) Staining of pGL2∷CYCD3;1 trichomes (arrows) revealing the expression of the trichome marker line pGL1∷GUS.

Figure 2

Analysis of pGL2∷CYCD3;1 trichome development. (a–d) Scanning electron micrographs of pGL2∷CYCD3;1 trichomes. (a) Very young dividing trichomes giving rise to trichome clusters. (b and c) Further cell divisions take place as trichomes grow out and elongate. (d) Mature multicellular trichome comprising many cells. Note that the individual cells form papillae.

Figure 3

Transgene expression analysis. Semiquantitative RT-PCR showing the relative expression strength of pGL2∷CYCD3;1 in lines 1 (D3;1#1) and #2 (D3;1#2) and pGL2∷CYCD2;2 (D2;2) in comparison with endogenous GLABRA 2 (GL2) expression; 18, 21, 24, and 27 indicate the RT-PCR cycle numbers. In pGL2∷CYCD3;1#1, CYCD3;1 is expressed at least 10-fold stronger than in line 2. The expression strength of pGL2∷CYCD2;2 is slightly less than pGL2∷CYCD3;1#1 but ≈10-fold stronger than in pGL2∷CYCD3;1#2.

Figure 4

Analysis of DNA content. (a–f) Distribution of DNA contents given in relative fluorescence units of the single nuclei (black bar) and the sum of all nuclei per TIS (light bar). The relative, fluorescence units are calibrated with wild-type, triptychon, and glabra 3 trichome nuclei such that 2 relative fluorescence units roughly represent 2C by defining the major peak in the wild-type trichomes as 32C and the major peak in glabra 3 as 16C in accordance to previously measured trichome nuclei (9, 23, 34). The mean (m) is given for the single nuclei (m^s) and the total DNA contents as the sum of all nuclei per TIS (m^t). (a) Wild type. (b) triptychon (try). (c) glabra3 (gl3). (d) Single-nucleated pGL2∷CYCD3;1#2 trichomes. (e) Two-nucleated pGL2∷CYCD3;1#2 trichomes. (f) Three-nucleated pGL2∷CYCD3;1#2 trichomes. (g) pGL2∷CYCD3;1#2 trichomes with more than three nuclei. (h) pGL2∷CYCD3;1#1 trichomes with more than three nuclei.

Figure 5

Expression analysis. (a) Strong staining with the CYCD3;1 antisense probe in pGL2∷CYCD3;1 trichomes (arrows); no signal was obtained with the CYCD3;1 sense probe (data not shown). (b) Detection of CYCD3;1 mRNA in young leaves with no staining in trichomes (arrows). (c) Detection of CYCB1;1 mRNA in pGL2∷CYCD3;1 trichomes (arrows). (d) Staining of trichomes expressing pCYCB1;1∷GUS in pGL2∷CYCD3;1 plants. (e) Detection of CYCB1;2 mRNA in pGL2∷CYCD3;1 trichomes (arrows). (f) Staining of trichomes expressing pCYCB1;2∷GUS in pGL2∷CYCD3;1 plants. (g) No detection of CYCD3;1 mRNA in sim mutant trichomes (arrows). (h) Detection of GL2 mRNA in sim mutant trichomes (arrows).