The seed coat and embryo were washed with RNase-free water three times to remove fragile endosperm tissues. RT-PCR and real-time RT-PCR Total RNA was extracted from cells using the RNeasy Flower Mini Kit (QIAGEN Inc., Valencia, CA, USA) relating to protocols provided by the manufacturer. LPA1 mutant offers less InsP6, this ABC transporter may be associated with InsP6 storage. To clarify InsP6 synthesis during AG-120 seed maturation, MIPS localization was examined using immunohistology, and it is demonstrated here that MIPS proteins localize to the cytosol of the seed endosperm. Moreover, the presence of InsP6 within both embryo and endosperm cells suggests an connection between the cells in the synthesis and subsequent storage of InsP6 in developing seeds of (Columbia accession) were surface sterilized with 95% ethanol and then sown onto 0.2% gellan gum (Wako, Tokyo, Japan) in 1/2 MS medium (Wako) with 3?mg l?1 thiamine-HCl, AG-120 0.5?mg l?1 pyridoxine, and 5?mg l?1 nicotinic acid. After incubation at 4?C for 4?d to break dormancy, the seeds were germinated and grown at 23?C less than continuous light. After 14?d the seedlings were transferred into vermiculite medium for subsequent growth. Developing seeds were harvested from vegetation having 10C12 siliques. Seeds harvested from your sixth to eighth siliques were separated into seed coating and embryo using tweezers under a binocular (SZX16, Olympus). The seed coating and embryo were washed with RNase-free water three times to remove fragile endosperm cells. RT-PCR and real-time RT-PCR Total RNA was extracted from cells using the RNeasy Flower Mini Kit (QIAGEN Inc., Valencia, CA, USA) relating to protocols provided by the manufacturer. First-strand cDNA was generated by reverse transcription with reverse transcriptase XL (AMV) (Takara Bio Inc., Shiga, Japan) using oligo(dT primer), 5- CTGATCTAGAGGTACCGGATCCTTTTTTTTTTTTTTTTTTTT. Real-time PCR amplification was performed using the SYBR? Premix Ex lover Taq? (TaKaRa Bio Inc.,) and a real-time PCR detector (TaKaRa Wise Cycler II system). PCR was performed using gene-specific oligonucleotide primer pairs based on unique sequences for each gene and an Actin-2 (control) gene. The primer sequences used were: for AtMIPS1 (At2g22240), 5-GCGGGATCCCATGGAGTACAAGTGAAGGATGAG-3 and 5-GCGGAATTCGAAAATCCATATTCATAGATCATAAG-3; AtMIPS2 (At4g39800), 5-GCGGAATTCAAGTGAACATGAAGAAGCATGAAC-3 and 5-GCGATCGATGGAACCAAAACCATGATTATATATCTC-3; AtMIPS3 (At5g10170), 5-GCGATCGATTCTCGAGTACAAGTGATCAAAGAGAC-3 and 5-GCGCTCGAGCCCAAATATATATTATAGTTTGAAATG-3; and for Actin-2 (At3g18780), 5-TTTGTTCCAGCCCTCGTTTGT-3 and 5-TCATGCTGCTTGGTGCAAGT-3. In both PCR methods, the same primers units were used for each gene. Preparation of antibodies against MIPS MIPS antibody was prepared relating to Mitsuhashi (2005). An indicated sequence tag (EST) clone (accession no. “type”:”entrez-nucleotide”,”attrs”:”text”:”AV525103″,”term_id”:”8684631″AV525103) for the gene (At4g39800) was provided by Kazusa DNA Study Institute. Oligonucletide primers 5-GAATTCATGTTTATTGAGAGCTTCAAAGTT-3 and 5-CTCGAGCTTGAACTCCATGATCATGTTGTT-3 were designed on the basis of N- and C-terminal sequences of the gene, respectively. The amplified DNA was digested with strain BL21(DE3) (EMD Biosciences). The recombinant protein was purified via a 6His definitely tag by using a HiTrap Chelating HP Column (Amersham Biosciences, Piscataway, NJ, USA) and used as antigen. Specific antisera raised in rabbit were provided by Shibayagi Co., Ltd (Gunma, Japan). Preparation of thin sections Developing seeds with torpedo-shaped embryos were vacuum infiltrated for 1?h having a fixative that consisted of 4% paraformaldehyde, 1% glutaraldehyde, and 0.06?M sucrose in 0.05?M cacodylate buffer, pH 7.4. The cells were cut into slices of <1?mm in thickness having a razor knife and treated for another 2?h with freshly prepared fixative. Immunoelectron microscopy Immunogold labelling methods were basically the same as explained previously (Hara-Nishimura were fixed for 40?min AG-120 in 7.2% (w/v) formaldehyde, 0.1% (v/v) Nonidet P-40, 10% (v/v) dimethylsulphoxide, and 50?mM Na-phosphate buffer, pH 7.2. Seeds were then washed twice with Tris-buffered salineCTween (TBS-T) for 5?min, incubated in TBS-T containing 5% (w/v) Cellulase Onozuka R-10 (Yakult, Tokyo, Japan) and 2% (w/v) Pectolyase Y-23 (Kikkoman, Tokyo, Japan) for 20?min at 30C, AG-120 washed twice with TBS-T, MRX30 incubated in blocking buffer [2% (w/v) AG-120 BSA and TBS-T] for 30?min, and then incubated with anti-AtMIPS2 or pre-immune antibodies in the blocking buffer for 40?min. After this the seeds were washed three times for 5?min each, incubated for 1?h with goat anti-rabbit IgG antibodies conjugated with Alexa Fluor 488 (absorbance, 495?nm; emission, 519?nm; Molecular Probes, Eugene, OR), washed three times for 5?min with TBS-T, and mounted. Immunoblot analysis Seed cells of were homogenized in 10 mM Tris-HCl, pH 7.5, before centrifugation to collect soluble proteins. Proteins (5?g per lane) were subjected to SDSCPAGE and were transferred electrophoretically to a polyvinylidene difluoride.