Izp58-1 mutant. Forty-four independent transgenic lines have been obtained, 20 of which exhibited a nearly wild-type seed phenotype. Two complemented lines (CL1 and CL2) with single insertions (Supplementary Fig. S1C) have been selected for further analysis. The two CL set seeds had typical sizes and shapes (Figs 2B and 3M, Q). Transverse sections of CL grains revealed standard to slight chalkiness within the ventral region (Fig. 3N, R). SEM of transverse sections of CL grains inside the ventral region showed that most of the starch granules have been densely packed and routinely polyhedral (Fig. 3P, T), which was related to these on the wild-type Dongjin (Fig. 3C, D). The expression of OsbZIP58 in the CL lines was also restored to wild-type levels (Supplementary Fig. S1D). These final results indicated that the defective seed phenotype was triggered by the OsbZIP58 mutation.Seeds of osbzip58s show altered starch accumulationTo determine the function of these four OsbZIPs in seed starch accumulation, we searched the T-DNA insertion mutant database (Jeong et al., 2002) plus the rice Tos17 retrotransposon insertion database (Miyao et al., 2007) and obtained six mutant lines (Table 2). Among these, two T-DNA insertion lines of OsbZIP58, osbzip58-1 (PFG_1B-15317.R) and osbzip58-2 (PFG_3A-09093.R), both harboured a pGA2715 T-DNA insertion within the 1st intron of OsbZIP58 (Fig. 2A). Homozygotes of those two mutants had been isolated by PCR screening from the segregating progeny populations (Fig. 2A). Southern blot analysis revealed the presence of a single T-DNA insertion in homozygous plants (Supplementary Fig. S1A at JXB on the net), and all of those plants exhibited white, floury RIP kinase site endosperm (Fig. 3E, I). No transcripts from OsbZIP58 were detected by RT-PCR in 7 DAF seeds from the homozygous mutants, though they were detected within the heterozygous and in wild-type plants (Supplementary Fig. S1B), suggesting that the expression of OsbZIP58 was entirely abolished by the T-DNA insertion within the two mutant lines. The two osbzip58 mutants showed quite a few defective seed phenotypes, including reduced mass per 1000 seeds, reduced grain width, abnormal seed shape, along with a white belly, which can be a floury-white core that occupies the centre for the ventral area on the seed; (Figs 2B and 3F, J). The osbzip58-1 mutant also had an apparently shrunken belly inside the grain (Fig. 3E). SEM photos of transverse sections of osbzip58-1 and osbzip58-2 grains indicated that the dorsal endosperm consisted of densely packed, polyhedral starch granules (Fig. 3G, K), which were similar to those of the wild-type Dongjin (Fig. 3C, D), even though the ventral endosperm was filled with loosely packed, spherical starch granules with substantial air spaces (Fig. 3H, L), corresponding towards the chalky region of endosperm. The morphology of starch granules in the ventral regions of the immature osbzip58-1 seeds was analysed in semi-thin sections. Endosperm cells in the wild form were full of amyloplasts, and every amyloplast consisted of denselyDisruption of OsbZIP58 alters the starch content material and chain length distribution of amylopectinTo comprehend additional the part of OsbZIP58 in starch synthesis, we measured the seed starch content and also the chain length distribution of amylopectin. Total starch content material and AAC in the osbzip58-1 and osbzip58-2 mutants had been slightly decreased compared with those within the wild form (Fig. 5A, B), although the soluble sugar content material was significantly PIM3 Biological Activity increased within the mutants (Fig. 5C). The total starch content, AA.
