Functional analysis of cyclin-dependent kinase inhibitors of Arabidopsis

Abstract

Cyclin-dependent kinase inhibitors, such as the mammalian p27(Kip1) protein, regulate correct cell cycle progression and the integration of developmental signals with the core cell cycle machinery. These inhibitors have been described in plants, but their function remains unresolved. We have isolated seven genes from Arabidopsis that encode proteins with distant sequence homology with p27(Kip1), designated Kip-related proteins (KRPs). The KRPs were characterized by their domain organization and transcript profiles. With the exception of KRP5, all presented the same cyclin-dependent kinase binding specificity. When overproduced, KRP2 dramatically inhibited cell cycle progression in leaf primordia cells without affecting the temporal pattern of cell division and differentiation. Mature transgenic leaves were serrated and consisted of enlarged cells. Although the ploidy levels in young leaves were unaffected, endoreduplication was suppressed in older leaves. We conclude that KRP2 exerts a plant growth inhibitory activity by reducing cell proliferation in leaves, but, in contrast to its mammalian counterparts, it may not control the timing of cell cycle exit and differentiation.

Figure 1.

Structural Organization of KRP1, KRP2, KRP3, KRP4, KRP5, KRP6, KRP7, and Chenopodium KRP. Conserved sequence boxes are indicated (1 to 6). N, nuclear localization signal; P, CDK consensus phosphorylation site; striped boxes, PEST domains. The predicted molecular masses (kD) are indicated at right.

Figure 2.

In Vitro CDKA;1 Binding by KRP3. Protein extracts of 3-day-old cell suspensions of Arabidopsis were loaded onto a KRP3-Sepharose column, and bound and unbound fractions were tested for the presence of CDKA;1 or CDKB1;1 with specific antibodies.

Figure 3.

Differential Expression of KRP Genes in Various Arabidopsis Organs and a 3-Day-Old Cell Suspension Culture. cDNA prepared from the indicated organs and the suspension cell culture were subjected to semiquantitative reverse transcriptase–mediated PCR analysis using gene-specific primers (see Methods). The actin 2 gene (ACT2) was used as a loading control.

Figure 4.

KRP2 Inhibition of Advanced Mitosis in Yeast Caused by the CDKA;1.A14F15 Gene. (A) Wild-type fission yeast cells transformed with empty control vectors. (B) Yeast cells expressing the KRP2 gene. (C) Yeast cells expressing the dominant positive CDKA;1.A14F15 gene. (D) Yeast cells coexpressing the CDKA;1.A14F15 and KRP2 genes. (E) Protein gel blot analysis of protein levels of CDKA;1 and KRP2 in the cells from (A) to (D). Bars in (A) to (D) = 30 μm.

Figure 5.

KRP2 Transgene Expression and CDK Histone H1 Activity in Untransformed and Four Independent Transgenic Arabidopsis Plants. (A) KRP2 mRNA levels. (B) KRP2 protein levels. (C) CDKA;1 protein levels. (D) Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) pro-tein levels visualized by Ponceau S staining (loading control). (E) CDK histone H1 activity bound to p10^CKS1At beads. (F) Quantification of signals in (E).

Figure 6.

Phenotypic Analysis of KRP2-Overproducing Lines. (A) Four-week-old soil-grown control plant. The inflorescence was removed to visualize the rosette leaves. (B) KRP2-overproducing plant with removed inflorescence. (C) Fifth leaf of a control plant grown in vitro. (D) Fifth leaf of a KRP2-overproducing plant. (E) Adaxial epidermal cells of the fifth leaf of a control plant. (F) Adaxial epidermal cells of a KRP2-overproducing plant. (G) Palisade cells of the fifth leaf of a control plant. (H) Palisade cells of a KRP2-overproducing plant. Bars in (A) and (B) = 2 mm; bars in (C) and (D) = 5 mm; bars in (E) to (H) = 50 μm.

Figure 7.

Transverse Sections through the Central Part of the First Leaf. (A) Sixteen-day-old wild-type leaf grown in vitro. (B) Sixteen-day-old KRP2-overproducing leaf grown in vitro. Bar = 500 μm.

Figure 8.

Kinematic Analysis of Leaf Growth of the First Leaf Pair of Wild-Type (Col-0) and KRP2-Overproducing (35S-KRP2) Plants. (A) Leaf blade area. (B) Epidermal cell number on the abaxial side of the leaf. (C) Average cell division rates of the epidermal cells on the abaxial side of the leaf. (D) Epidermal cell size on the abaxial side of the leaf.

Figure 9.

Ploidy Distribution Diagrams of Leaves of Wild-Type (WT) and KRP2-Overexpressing Lines. Values are means of two independent measurements. Maximum differences found between two samples were 4.0, 3.0, and 2.0% for 2C, 4C, and 8C, respectively.