During grain (L. kinase (CDK) inhibitors (CKIs) in cell cycle control

During grain (L. kinase (CDK) inhibitors (CKIs) in cell cycle control was investigated here during the transition between syncytium and cellularization. It was found that one of the rice CKIs is strongly expressed in the caryopsis at 2 d after flowering (DAF) U 95666E and its expression is significantly reduced at 3 DAF. The other CKI transcripts did not show such a shift at 2 DAF. hybridization analysis revealed that is expressed in multinucleate syncytial endosperm at 2 DAF but not in cellularized endosperm at 3 DAF. Two-hybrid assays showed that Orysa;KRP3 binds Orysa;CDKA;1 Orysa;CDKA;2 Orysa;CycA1;1 and Orysa;CycD2;2. By contrast Rabbit Polyclonal to MEOX2. Orysa;CDKB2;1 and Orysa;CycB2;2 do not show binding to Orysa;KRP3. Orysa;KRP3 was able to rescue yeast premature cell division due to the dominant positive expression of mutant rice CDKA;1 indicating that Orysa;KRP3 inhibited rice CDK. These data suggest that Orysa;KRP3 is involved in cell cycle control of syncytial endosperm. L.) endosperm comprises a substantial U 95666E proportion of the mature seed and contains a large amount of carbohydrates. It is an important source of calories for humans and animals and also provides raw materials for goods and biofuels. Extensive research has been directed at improving the grain size quality and yield. Some of the limitations of conventional rice breeding may be overcome by biotechnological engineering. However significant improvements require an understanding of the molecular processes controlling endosperm development. Rice seed development begins with double fertilization in which the haploid egg cell and the two polar nuclei in the central cell are fertilized by haploid sperm cells. After double fertilization the triploid primary endosperm nucleus begins to divide rapidly. Endosperm development proceeds in several distinct phases: syncytium formation during which the endosperm U 95666E nuclei undergo many rounds of mitosis without cytokinesis; cellularization during which cell walls form around the endosperm nuclei; differentiation which includes the formation of transfer cells aleurone and starchy endosperm; and maturation which includes endoreduplication for the accumulation of storage compounds dormancy and desiccation (Hoshikawa 1967 EL2 in rice has been identified (Churchman to and (Wang expression (Wang expression is negatively regulated by auxin during early lateral root initiation (Himanen and were involved in the control of germline proliferation (Kim and was suggested to be U 95666E involved in endoreduplication during the middle stage of endosperm development (Coelho to L. cv. Hitomebore) were grown under field conditions in plastic pots filled with ground at Iwate University (Morioka Japan). Spikelets were marked around the flowering day and subsequently sampled daily following maturity. Different tissues (leaf stem root and panicle) were collected around 10 DAF. RT-PCR Total RNA was isolated from herb tissues by the acid guanidinium thiocyanate-phenol-chloroform extraction method (Chomczynski and Sacchi 1987 First-strand cDNA synthesis was carried out via ReverTra Ace reverse transcriptase (Toyobo Osaka Japan) with oligo (dT)15 and random primers. Semi-quantitative PCR was performed with various forward and reverse primers (Table 1). Quantitative real-time RT-PCR was carried out with SYBR Premix Ex Taq II (Takara Ohtsu Japan). Samples were analysed in triplicate in a Thermal Cycler Dice Real Time System (Takara). In each case dissociation curves confirmed the purity of the amplified products. Relative expression levels were calculated according to the 2-ΔΔCT method (Livak and Schmittgen 2001 using 18S rRNA as the internal control. The primers used for these analyses are listed in Table 1. Table 1. List of primers used in this study In situ hybridization U 95666E of sections through developing rice spikelets was performed according to Hirose (2002) with some modifications. Plant materials were fixed in 2% (w/v) paraformaldehyde and 15% (v/v) saturated picric acid in 50 mM sodium phosphate buffer pH 7.4 overnight at 4 °C dehydrated through an ethanol series and hybridization. The sections were deparaffinized with xylene and rehydrated through an ethanol series.