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. 2008 Jan;20(1):88-100.
doi: 10.1105/tpc.107.054676. Epub 2008 Jan 25.

Requirement of B2-type Cyclin-Dependent Kinases for Meristem Integrity in Arabidopsis Thaliana

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Free PMC article

Requirement of B2-type Cyclin-Dependent Kinases for Meristem Integrity in Arabidopsis Thaliana

Stig Uggerhøj Andersen et al. Plant Cell. .
Free PMC article

Abstract

To maintain proper meristem function, cell division and differentiation must be coordinately regulated in distinct subdomains of the meristem. Although a number of regulators necessary for the correct organization of the shoot apical meristem (SAM) have been identified, it is still largely unknown how their function is integrated with the cell cycle machinery to translate domain identity into correct cellular behavior. We show here that the cyclin-dependent kinases CDKB2;1 and CDKB2;2 are required both for normal cell cycle progression and for meristem organization. Consistently, the CDKB2 genes are highly expressed in the SAM in a cell cycle-dependent fashion, and disruption of CDKB2 function leads to severe meristematic defects. In addition, strong alterations in hormone signaling both at the level of active hormones and with respect to transcriptional and physiological outputs were observed in plants with disturbed CDKB2 activity.

Figures

Figure 1.
Figure 1.
Relative Expression Levels of CDKA;1, CDKB2;1, and CDKB2;2. (A) The CDKB2s were highly expressed in the apex, whereas CDKA;1 was expressed at similar levels across all tissues. Expression levels were normalized to the per-gene average across all samples. (B) CDKB2 expression was reduced in stm and wus mutants, which have impaired meristem function, while their transcript levels were increased in clv3 mutants with enlarged meristems. Expression levels were normalized to the wild-type control. (C) The CDKB2s showed a peak in expression level at the G2/M transition, while CDKA;1 expression did not change in response to the cell cycle phase. Transcript abundance was normalized to the expression level at the time of cell cycle block release. Microarray data were extracted from the AtGenExpress compendium (Menges et al., 2003; Schmid et al., 2005) and represent biological triplicates in (A) or biological duplicates in (B) and (C).
Figure 2.
Figure 2.
Phenotypes of AM1-2 Double Knockdown and 35S:CDKB2;1 (OE1) Plants. The genotype is indicated at the lower left of each picture. (A) Apices of 15-d-old plants. At this age, the emergence of multiple rosettes from the disorganized apex was observed in both AM1-2 and OE1 plants. Bars = 500 μm. (B) Scanning electron micrographs of apices from 12-d-old seedlings. The strict organization seen at the wild-type apex was disrupted in both AM1-2 and OE1 plants. Bars = 90 μm. (C) Overlays of differential interference contrast bright-field images and 4′,6-diamidino-2-phenylindole nuclear stainings. Nuclei appear blue. The dome-shaped meristem-like structures found in AM1-2 and OE1 plants contained fewer cells than wild-type meristems. In addition, several nuclei of AM1-2 and OE1 plants were abnormally expanded, and in OE1 plants, these were accompanied by abnormally large cells. Bar = 100 μm. (D) FM4-64 staining of root tips. The organization of the root meristem is maintained in both AM1-2 and OE1 plants. Bar = 50 μm. (E) Lugol staining of root tips. Starch grains are deposited normally in the columella cells in both transgenic lines. Bar = 50 μm. (F) Phenotype of wus/35S:CDKB2;1 (OE1) double mutant plants. Homozygous wus mutant plants produced the first set of fully developed true leaves before terminating in a flat apex with no discernible meristem. Homozygous OE1 plants produced the first set of true leaves, followed by swelling of the apex and initiation of multiple irregularly spaced rosettes. wus/OE1 double homozygous plants displayed developmental arrest and completely failed to produce organs. Bars = 200 μm.
Figure 3.
Figure 3.
In Situ Hybridizations. All images were taken at the same magnification. Bar = 100 μm. (A) In the wild type, CDKB2;1 was expressed at the apex in a spotty pattern characteristic of a cell cycle–regulated gene. Expression could not be detected in AM1-2 plants, whereas OE1 plants showed a weaker expression in a larger number of cells. (B) HISTONE H4 was expressed in many cells of wild-type apices, but its expression was attenuated in both AM1-2 and OE1 plants. (C) to (E) A single center of meristematic activity with WUS, STM, and CLV3 expression was seen in wild-type apices. By contrast, AM1-2 and OE1 plants contained multiple foci of WUS, STM, and CLV3 expression, consistent with the emergence of multiple rosettes observed. See Supplemental Figure 8 online for images of serial sections. (F) In roots, CDKB2;1 was expressed in a spotty pattern in cells near the root tip. This is consistent with the root digital in situ data available (Brady et al., 2007; see Supplemental Figure 2 online). Like in the shoot, expression could not be detected in AM1-2 plants, whereas OE1 plants showed a weaker expression in a larger number of cells.
Figure 4.
Figure 4.
Nuclear DNA Content of AM1-2 Double Knockdown and 35S:CDKB2;1 (OE1) Plants. Ploidy of AM1-2 and OE1 plants was compared with wild-type control seedlings with (wt_whole) and without (wt_young) the oldest set of leaves. The horizontal axis indicates the genome copy number, and the vertical axis shows the percentage of nuclei counted. Error bars represent se. In both AM1-2 and OE1 plants, the relative number of 2n nuclei was reduced, while the number of 16n nuclei was increased compared with either wild-type sample, indicating a shift toward higher genome copy numbers in plants with altered CDKB2 activity.
Figure 5.
Figure 5.
Molecular Phenotypes of AM1-2 Double Knockdown and 35S:CDKB2;1 (OE1) Plants by Global Expression Analysis. The horizontal axis shows the log2-transformed OE1/wild type expression ratio, and the vertical axis indicates the log2-transformed AM1-2/wild type expression ratio. (A) Expression ratios of all genes (gray circles). The 219 genes that changed significantly in both conditions (percentage of false positives < 10%) are highlighted in black. (B) Expression ratios of A-type ARRs, JA biosynthesis genes (JA biosynthesis), and THIONIN 2.1.
Figure 6.
Figure 6.
Hormone Response of Wild-Type and 35S:CDKB2;1 (OE1) Tissue. (A) to (J) Wild-type and OE1 root explants were subjected to hormone treatments to assess their regeneration capacity. Plant genotype and growth medium are indicated. CIM, callus induction medium; RIM, root induction medium; SIM_0.5, shoot induction medium with 0.5 μM 2-iP; SIM_5, shoot induction medium with 5 μM 2-iP; SIM_50, shoot induction medium with 50 μM 2-iP. OE1 roots were recalcitrant to tissue regeneration and, in contrast with wild-type explants, did not form calli, roots, or green foci in response to hormone treatment. (K) and (L) Wild-type root explants after 5 d of incubation on either hormone-free (control) or CIM plates. Whereas CDKB2 expression in the control explants could only be detected in root tips, CDKB2 expression was detected throughout proliferating callus tissue in the explants incubated on CIM.
Figure 7.
Figure 7.
Hormone Content of 35S:CDKB2;1 (OE1) and AM1-2 Double Knockdown Plants. Hormone levels of AM1-2 and OE1 plants were compared with wild-type control seedlings with (wt_whole) and without (wt_young) the oldest set of leaves. (A) Quantification of trans-zeatin (tZ), its precursors tZ riboside (tZR) and tZ riboside 5′ monophospate (tZR5′MP), and conjugates tZ O-glucoside (tZOG), tZ riboside O-glucoside (tZROG), and tZ 9-glucoside (tZ9G). The bioactive nonglycosylated forms tZ, tZR, and tZR5′MP were present at lower levels in OE1 plants than in the wild-type controls, whereas the inactive O-glycosylated forms tZOG and tZROG were increased. (B) Quantification of indole-3-acetic-acid (IAA). Both AM1-2 and OE1 plants contained significantly more IAA than either wild-type control. Error bars indicate sd. Cytokinin quantifications were performed on biological quadruplicates, and biological triplicates were used for IAA measurements.

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