Os03g0758100
Plastidial phosphorylase (Pho1) accounts for ~96% of the total phosphorylase activity in developing rice (Oryza sativa) seeds[1].
Contents
Annotated Information
Function
- Pho1 plays a crucial role in starch synthesis in plastids of nonphotosynthetic storage tissue of higher plants. The study describes a novel rice grain phenotype, distinct from other cereal starch mutants, that implicates Pho1 in starch biosynthesis in the rice endosperm[1].
- The early temporal expression of Pho1 during seed development together with the starch deficient phenotype of the shrunken seeds indicate that pho1 is essential for one or more early steps of starch biosynthesis in the rice endosperm[1].
- First, Pho1 might play a crucial role in starch granule formation at a very early stage of the grain filling process[2]. Second, Pho1 might play some crucial role in starch biosynthesis by forming a functional protein–protein complex with other carbohydrate metabolizing enzymes such as BE or SS isoforms.
Mutation
- Mature seeds of >1700 mutant lines generated by MNU treatments of the Kinmaze and Taichung 65 (T65) japonica rice cultivars were screened for Pho1 mutants by SDS-PAGE analysis. Many mutant lines were identified that lacked the 106-kD polypeptide completely (Figure 3A) but did not exhibit any significant alterations in the expression of the other major endosperm proteins when assessed by SDS-PAGE. The complete loss of Pho1 was verified by protein gel blot analysis using anti- Pho1 (Figure 3A). Native-PAGE/activity staining analysis also showed the absence of the Pho1 activity band in the mutant lines, while the activity levels of Pho2 were comparative to that of the wild type (Figure 3B), indicating that expression of Pho1 and Pho2 are regulated independently[1].
- More than 15 plastidial Pho1-deficient mutants were isolated by independentmutation treatments of the rice cultivars Kinmaze and T65 (representative examples of someof these are shown in Figure 3 and Supplemental Figure 3 online). Surprisingly, in the 15 lines examined, all produced seeds with marked variation in morphology, ranging from a pseudonormal phenotype, comparable in appearance but much lower in weight than the wild type, to a shrunken phenotype with extremely reduced starch levels. Examples of seed variability are depicted in Figure 3C for themutant line EM755. Most of the seeds (65%) exhibited a white-core endosperm that varied in size. Approximately 23% were of the pseudonormal type, displaying a vitreous endosperm but with a grain weight of;8%less than seeds containing awhitecore endosperm, which, in turn, were 10% lighter than wild-type seeds. A smaller percentage (13%) of the seeds were severely shrunken (Figure 3D). The shrunken seeds were randomly distributed along the panicle length, indicating that this extreme phenotype was not due to possible differences in nutrient partitioning along the panicle[1].
Figure 3. Effects of pho1 Mutation on Pho1 Content and Activity in Rice Endosperm and on Morphology of the Kernel. [1].
- Scanning electron microscopy revealed that the pho1 mutation resulted in modification in starch granule morphology (Figure 7). Starch granules from plump pho1 seeds from pho1 mutant lines BMF136 and EM640 were slightly smaller than wild-type seeds, and some granules were more spherical than the irregular polyhedron-shaped granules typical of wild-type starch grains. Starch granules from shrunken seeds were markedly smaller than those from wild-type seeds, with the larger granules having an irregular spherical shape instead of the normal irregular polyhedron shape evident for wild-type large starch granules. The x-ray diffraction patterns of endosperm starch granules were similar between the pho1 mutants and the wild-type T65[1].
Figure 7. Effects of pho1 Mutation on Starch Granules as Viewed by Scanning Electron Microscopy. Starch granules were prepared from plump or shrunken seeds from the pho1 mutant lines BMF136 and EM640 and their wild-type parental cultivar, T65. Bars ¼ 5 mm. [1].
- In spite of large differences in seed weight (Figure 3), the chain length distribution pattern of amylopectin was nearly the same for the pseudonormal seed and the shrunken and white-core seed of the pho1 mutant lines BMF134 and BMF136. In the range of DP # 25, amylopectin chain length differences between the pho1 mutants and T65 were somewhat more significant in the shrunken seed than the pseudonormal seed (Figure 8). Collectively, these results suggest that Pho1 operates at two distinct phases of starch biosynthesis, one phase consisting of starch initiation and a second phase encompassing starch elongation[1].
Figure 8. The Change in Chain Length Distribution of Amylopectin in Rice Endosperm between Various pho1 Mutants and Their Wild-Type Parents (T65 or Kinmaze) as Determined by the APTS-Capillary Electrophoresis Method. [1].
Subcellular localization
- Unlike the amyloplast resident marker enzymes BEI and BEIIb, which were detected in both the stroma and starch fractions or the starchlocalized GBSSI, only trace amounts of Pho1 were starch bound (Figure 2). Hence, Pho1 is restricted to the stromacompartment of the amyloplasts. A stroma location was also observed for maize endosperm Pho1[3].
Figure 2. Subcellular Localization of Pho1 in Developing Endosperm Cells of Rice. Total endosperm proteins and proteins associated with starch granules of mid-developing seeds were resolved by SDS-PAGE and then subjected to immunoblotting for Pho1, BEI, BEIIb, and GBSSI. ‘‘Endosperm’’ denotes the total proteins extracted from the endosperm of developing seeds. [1].
Expression
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Evolution
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Labs working on this gene
- Plant Genetic Resources, Institute of Genetic Resources, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan
- Institute of Biological Chemistry, Washington State University, Pullman, Washington, 99164-6340
- Faculty of Bioresource Science, Akita Prefectural University, Akita 010-0195, Japan
References
- ↑ 1.00 1.01 1.02 1.03 1.04 1.05 1.06 1.07 1.08 1.09 1.10 Satoh H, Shibahara K, Tokunaga T, et al. Mutation of the plastidial α-glucan phosphorylase gene in rice affects the synthesis and structure of starch in the endosperm[J]. The Plant Cell, 2008, 20(7): 1833-1849.
- ↑ Tsai, C.Y., and Nelson, O.E. (1969). Mutations at the shrunken-4 locus in maize that produce three altered phosphorylases. Genetics 61: 813–821.
- ↑ Yu, Y., Mu, H.H., Wassermanm, B.P., and Carman, G.M. (2001). Identification of the maize amyloplast stromal 112-kD protein as a plastidic starch phosphorylase. Plant Physiol. 125: 351–359.
