The CDK inhibitor NtKIS1a is involved in plant development, endoreduplication and restores normal development of cyclin D3; 1-overexpressing plants
Sophie Jasinski1,
,
Catherine Riou-Khamlichi2,*,
Odile Roche3,
Claudette Perennes1,
Catherine Bergounioux1 and
Nathalie Glab1
1 Laboratoire Cycle Cellulaire, Institut de Biotechnologie des Plantes, CNRS
UMR8618, Université Paris-Sud, 91405 Orsay
Cedex, France
2 Institute of Biotechnology, University of Cambridge, Tennis Court Road,
Cambridge, CB2 1QT, UK
3 Service de Cytologie, Institut de Biotechnologie des Plantes, CNRS UMR8618,
Université Paris-Sud, 91405 Orsay Cedex,
France
* Present address: Institut des Sciences de la Vie et de la
Santé, EA 3176,
Université de Limoges, 87060 Limoges Cedex,
France
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Fig. 1. Gain of function of NtKIS1a produces serrated leaves, abnormal flowers and
reduces plant size. (A) Rosettes from three T1 35S::NtKIS1a independent lines,
displaying respectively a weak (a), medium (b) and strong (c) serrated leaf
phenotype, are compared with 35S::NtKIS1b (d) or WT (e) rosette. Abnormal
flowers from 35S::NtKIS1a line displaying an extreme phenotype (f,g; see text)
are compared with WT flower (h). Bars, 5 mm (a-e); 0.5 mm(f-h). (B) The
rosette diameter of 24 plants (5-week-old) from 2 weak, 2 medium and 2 strong
lines were measured. The graph represents the average diameter ± s.d.
(C) Three cases were met according to the number of T2 plants displaying a
serrated leaf phenotype in 12 KmR plants analysed. For each case, a
serrated plant is shown. Bar, 10 mm.
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Fig. 2. NtKIS1a overexpression induces anther and gynoecium disorganised
structure. Transverse sections of flowers from wildtype (WT) or a 35S::NtKIS1a
line displaying an extreme phenotype (NtKIS1a) were performed. a and b
corresponded to general views. More detailed views of anthers (c,d) and
ovaries (e,f) are shown. In WT ovary, exocarp (ex), mesocarp (ms), and
endocarp (en) layers could be distinguished. The dark staining cells of the
transmitting tract (tt) occupied the inner portion of the ovary. Inside the
locules (lc), ovules (ov) were visible in WT and NtKIS1a.
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Fig. 3. The CDK kinase activity of 35S::NtKIS1a lines is reduced. Protein extracts
from wildtype (WT), two 35S::NtKIS1a Arabidopsis lines, one strong
(S) and one medium (M), or buffer (C) were added to
p9CKSHs1 beads to purify CDK/cyclin complexes. Histone H1
phosphorylation (H1-P) was monitored and equal loading of histone H1 (H1) was
controlled. Proteins bound to p9CKSHs1 beads were
recovered and immunoblotted with an antibody raised against the conserved CDK
PSTAIR motif (PSTAIR). The histone H1 phosphorylation and the PSTAIR signals
were quantified with NIH image 1.62 software. Their ratio was calculated and
presented in the graph. The maximal value obtained in WT was referred as
100%.
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Fig. 4. Overexpression of NtKIS1a decreases organ size in increasing cell
size. (A) A photograph of flowers from an extreme 35S::NtKIS1a line (NtKIS1a)
and from wildtype (WT) indicating the three regions (I, II, III) of the petal
that were used for microscopic analysis. (B) Light microscopic analysis of
outer epidermal cells at three positions (I, II and III) of 35S::NtKIS1a and
WT petal. (C) Cell size in the three regions of 35S::NtKIS1a and WT petals.
Bars indicate s.d. (D) Scanning electron microscopy of the leaf lower
epidermis of WT and two 35S::NtKIS1a Arabidopsis lines, one medium
(M) and one strong (S). Bars, 100 µm. (E) From several scanning electron
microscopy images of the lines described in D, cell areas were measured using
the Optimas 6.0 software. The mean cell areas were reported in three
categories: areas less than 300 µm2, areas between 300 and 3000
µm2 and areas greater than 3000 µm2 (n.o.
correspond to categories not observed). The number of cells (n) recorded in
each categories is indicated in brackets.
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Fig. 5. Overexpression of NtKIS1a inhibits rosette leaf cell
endoreduplication. Ploidy distributions for nuclei isolated from old (RL2) and
young (RL1) rosette leaves, cauline leaves (CL) and flower buds (FB) of A.
thaliana wildtype (WT), strong 35S::NtKIS1a (NtKIS1a) or 35S::NtKIS1b
(NtKIS1b) lines. For each sample, 5000 DAPI-stained nuclei were analysed. The
x-axis represents DNA fluorescence in logarithmic scale and DNA
content is indicated. The y-axis represents frequency.
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Fig. 6. A. thaliana plants overexpressing NtKIS1a and
AtCycD3;1 display a wildtype phenotype. (A) The interaction between
NtKIS1a and AtCycD3;1 using the two-hybrid system is presented. BD-NtKIS1a
corresponds to the DNA binding domain of GAL4 fused to NtKIS1a and
AD-AtCycD3;1 corresponds to the activation domain of GAL4 fused to AtCycD3;1.
AD and BD correspond respectively to the activation and DNA binding domains of
GAL4. (B) RT-PCR analysis was performed as described in Materials and Methods.
Lanes WT, AtCycD3;1, NtKIS1a, AtCycD3;1xNtKIS1a and AtCycD3;1+NtKIS1a
are RT-PCR products obtained from RNA samples of the corresponding plants.
AtCycD3;1xNtKIS1a correspond to the F1 plants of the cross between the
AtCycD3;1 and NtKIS1a-overexpressing lines. AtCycD3;1+NtKIS1a correspond to T1
plants issued from the transformation of the AtCycD3;1-overexpressing line by
the NtKIS1a-containing transgene. Three independent plants were tested for
AtCycD3;1xNtKIS1a. PCR was performed with specific primers from
NtKIS1a (first row), AtCycD3;1 (second row) and
Actin2 (third row; used as control), respectively. (C) Top row: view
of a 6-week-old plantlet from the AtCycD3;1-overexpressing line
(AtCycD3;1), and view of 4-week-old plantlets from 35S::NtKIS1a (NtKIS1a) and
AtCycD3;1xNtKIS1a lines. Bottom row: view of 3-week-old plantlets from
AtCycD3;1-overexpressing line (AtCycD3;1), AtCycD3;1+NtKIS1a and WT.
(D) From several scanning electron microscopy images of the lines described in
C, bottom row, cell areas were measured using the Optimas 6.0 software. The
averages of cell areas were reported in three categories, as described in the
legend of Fig. 4. The
measurement presented here for WT is an independent measurement of the WT
measurement shown in Fig.
4.
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© The Company of Biologists Ltd 2002
