The cyclin-dependent kinase inhibitor Orysa;KRP1 plays an important role in seed development of rice

Abstract

Kip-related proteins (KRPs) play a major role in the regulation of the plant cell cycle. We report the identification of five putative rice (Oryza sativa) proteins that share characteristic motifs with previously described plant KRPs. To investigate the function of KRPs in rice development, we generated transgenic plants overexpressing the Orysa;KRP1 gene. Phenotypic analysis revealed that overexpressed KRP1 reduced cell production during leaf development. The reduced cell production in the leaf meristem was partly compensated by an increased cell size, demonstrating the existence of a compensatory mechanism in monocot species by which growth rate is less reduced than cell production, through cell expansion. Furthermore, Orysa;KRP1 overexpression dramatically reduced seed filling. Sectioning through the overexpressed KRP1 seeds showed that KRP overproduction disturbed the production of endosperm cells. The decrease in the number of fully formed seeds was accompanied by a drop in the endoreduplication of endosperm cells, pointing toward a role of KRP1 in connecting endocycle with endosperm development. Also, spatial and temporal transcript detection in developing seeds suggests that Orysa;KRP1 plays an important role in the exit from the mitotic cell cycle during rice grain formation.

Figure 1.

Analysis of the amino acid sequences of rice KRPs. A, Amino acid sequence alignment of the predicted KRPs from rice. Identical and conserved amino acids are indicated by dark gray and light gray shading, respectively. Putative nuclear localization signals are underlined. B, Neighbor-joining tree of the C-terminal conserved region of plant KRPs, illustrating the relationship among these proteins. The tree branches including rice KRPs are circled. For simplification, the species names are designated by the first letters of the genus and species names. At, A. thaliana; Cr, Chenopodium rubrum; Ee, Euphorbia esula; Gh, Gossypium hirsutum; Gm, Glycine max; Le, L. esculentum; Ns, Nicotiana sylvestris; Nta, N. tabacum; Nto, Nicotiana tomentosiformis; Os, O. sativa; Ps, Pisum sativum; Zm, Z. mays.

Figure 2.

Phenotypic analysis of Orysa;KRP1-overexpressing leaves. A, Flowering KRP1^OE plants (right) and corresponding controls (left). B and C, Epidermal cells of the mature regions of the sixth leaf from control and KRP1^OE plants, respectively. In both images, cell walls were highlighted by a white pencil to make the cell boundaries more clearly visible. Bar = 50 μm. D, Growth curves of the sixth leaf of KRP1^OE (black) and control plants (gray). E, LERs of transgenic KRP1^OE and corresponding control plants. Error bars represent se (n = 15). F, Mature cell length of KRP1^OE and control leaves (n = 100). G, Rates of cell production per meristematic cell file of KRP1^OE (black) and control (gray) leaves (n = 15).

Figure 3.

Phenotypic analysis of homozygous Orysa;KRP1-overexpressing seeds. A, Analysis of number and weight of filled and nonfilled seeds from KRP1^OE T₂ plants and corresponding controls. For each parameter, the average values for the homozygous KRP1^OE and control lines are shown. Bars indicate se (n = 10). B, Seed populations resulting from a KRP1^OE plant and corresponding control. C, Detailed image of the KRP1^OE seed progeny. D, Microtome section of a KRP1^OE seed showing a partially filled endosperm. Bar = 300 μm.

Figure 4.

Effect of Orysa;KRP1 overexpression on nuclear ploidy of rice endosperm. A and B, Flow cytometric analysis of nuclei from control and KRP1^OE seeds, respectively. C, Percentage of endoreduplicated nuclei calculated by dividing the total number of nuclei with a ploidy equal to or greater than 12C by the total number of nuclei and multiplying by 100. Error bars denote se (n = 13).

Figure 5.

Microscopic analysis of the pollen produced by KRP1^OE flowers. A and B, DAPI staining of pollen grains from a control plant showing two well-defined nuclei and of nonviable pollen grains from homozygous Orysa;KRP1-overexpressing plant, respectively. C and D, Mature pollen grains from control plants and from homozygous KRP1^OE plants, respectively.

Figure 6.

Rice KRP transcript accumulation. A, Expression profiles of Orysa;KRP1 (KRP1) transcript in rice tissues whose levels were measured in roots, stems, leaves, and shoot apical meristems by semiquantitative RT-PCR. cDNA prepared from the indicated tissues were subjected to semiquantitative RT-PCR analysis with gene-specific primers (see “Materials and Methods”). B, Expression analysis of rice KRP1, KRP3, KRP4, and KRP5 during seed development, as determined by qPCR analysis. cDNA prepared from developing seeds were subjected to qPCR analysis using gene-specific primers (see “Materials and Methods”). The rice actin1 gene (ACT1) was used as a loading control (A and B). The values represent expression fold change compared to the time point with lowest transcript level. C and D, Expression pattern of Orysa;KRP1 in developing rice seeds as revealed by in situ hybridization, using dark-field and bright-field optics, respectively. Both micrographs show longitudinal sections of rice caryopsis at 8 DAP. En, Endosperm; Pe, pericarp. Bar = 250 μm.