Opaque1 encodes a myosin XI motor protein that is required for endoplasmic reticulum motility and protein body formation in maize endosperm

Abstract

Myosins are encoded by multigene families and are involved in many basic biological processes. However, their functions in plants remain poorly understood. Here, we report the functional characterization of maize (Zea mays) opaque1 (o1), which encodes a myosin XI protein. o1 is a classic maize seed mutant with an opaque endosperm phenotype but a normal zein protein content. Compared with the wild type, o1 endosperm cells display dilated endoplasmic reticulum (ER) structures and an increased number of smaller, misshapen protein bodies. The O1 gene was isolated by map-based cloning and was shown to encode a member of the plant myosin XI family (myosin XI-I). In endosperm cells, the O1 protein is associated with rough ER and protein bodies. Overexpression of the O1 tail domain (the C-terminal 644 amino acids) significantly inhibited ER streaming in tobacco (Nicotiana benthamiana) cells. Yeast two-hybrid analysis suggested an association between O1 and the ER through a heat shock protein 70-interacting protein. In summary, this study indicated that O1 influences protein body biogenesis by affecting ER morphology and motility, ultimately affecting endosperm texture.

Figure 1.

Phenotypic Features of Maize o1 Mutants. (A) Light transmission by mature kernels. Wild-type and mutant kernels were randomly selected from BC ears of o1-ref and o1-N1478A and viewed on a light box. Bars = 1 cm. (B) Scanning electron microscopy analysis of the peripheral regions of mature wild-type (WT) and o1 endosperm. Bars = 30 μm. (C) Ultrastructure of developing endosperms of the wild type (WT) and o1. Top (25 DAP): Low-magnification images of endosperm cells in the wild type and o1. Bars = 10 μm. Middle (25 DAP): Smaller and misshapen protein bodies in o1. Bars = 1 μm. Bottom (20 DAP): Dilated ER (blue arrows) and polygonal ring structures (red arrowheads) in o1. Bars = 2 μm. PB, protein body; SG, starch granules.

Figure 2.

Comparison of Zein Biosynthesis and Accumulation in Wild-Type and o1-ref during Kernel Development. (A) RNA expression profiles of representative zein genes. WT, the wild type. (B) SDS-PAGE analysis of total protein extracted from endosperm. (C) Immunoblot comparing 16- and 10-kD zein accumulation in wild-type and o1-ref developing endosperms.

Figure 3.

Map-Based Cloning and Identification of O1. (A) The o1 locus was mapped to a 250-kb region on chromosome 4 with four candidate genes. See Supplemental Table 1 online for detailed information. (B) The candidate gene GRMZM2G449909 is downregulated in o1. WT, the wild type. (C) Structure and mutation sites of the O1 gene. Lines represent introns, and black boxes represent exons. (D) Immunoblot comparing accumulation of O1 protein in endosperms of the wild type, o1 alleles, and other opaque mutants at 20 DAP. Lanes: 1, the wild type; 2, o1-ref; 3, o1-N1478A; 4, o1-N1243; 5, o5; 6, o7; 7, W22. (E) Schematic diagram of maize O1 protein structure. aa, amino acids.

Figure 4.

Phylogenetic Analysis of Plant Myosins. Maize O1 and currently identified myosins in rice (monocot) and Arabidopsis (dicot) plants were aligned by ClustalW. The phylogenetic tree was constructed using MEGA 5.0, and yeast class I and V myosins were used as outgroups (see Methods). At, Arabidopsis thaliana; Os, Oryza sativa; Sc, Saccharomyces cerevisiae. See Supplemental Data Set 1 online for the sequences and alignment used.

Figure 5.

Expression Pattern of O1. (A) RNA expression level of O1 in various tissues. Ubiquitin was used as an internal control. For each RNA sample, three technical replicates were performed. Representative results from two biological replicates are shown. Error bars represent sd. Transcript abundance is indicated relative to tassel. (B) Expression profiles of O1 during maize kernel development. Ubiquitin was used an internal control. For each RNA sample, three technical replicates were performed. Representative results from two biological replicates are shown. Error bars represent sd. Transcript abundance is indicated relative to 24-DAP kernels. (C) Immunoblot analysis of O1 protein accumulation during kernel development.

Figure 6.

O1 Is Associated with the ER Membrane. (A) Immunoblotting of O1 in fractionated plant proteins with anti-O1 antibody. (B) Immunoblots showing that O1 is preferentially associated with the ER membrane fraction. Fractions were subjected to immunoblot analysis with antibodies against O1, BIP (ER marker), or 16-kD zein (protein body [PB] marker). DEX, the dextran T500 phase containing the endomembrane fraction; PEG, the polyethylene glycol 3350 phase containing the plasma membrane fraction; TM, total membranes.

Figure 7.

Stimulated UPR Was Not Observed in the o1 Mutant. (A) RT-PCR analysis of ER stress sensors Zm BIP1, Zm BIP2, and Zm PDI transcript in the wild type (WT) and o1 during kernel development. Ubiquitin was used as an internal control. (B) Immunoblot comparing BIP accumulation in wild-type and o1 developing kernels. Anti-TUB was used as a sample loading control.

Figure 8.

ER Streaming Is Suppressed in the Presence of a Dominant-Negative O1 Tail Construct Fused to eYFP. (A) to (D) N. benthamiana leaves were infiltrated with the ER marker, mCherry-HDEL, alone. (A) and (B) The first (A) and last frame (B) of a 50-frame 80-s movie (see Supplemental Movie 2 online). (C) Merger of the first (colored red) and last frame (colored green) showing the total motility pattern. (D) Three random, successive frames are colored green, red, and blue, respectively, and then overlapped together to generate an integrity image. (A’) to (D’) N. benthamiana leaves were infiltrated with the ER marker, mCherry-HDEL, with eYFP-O1t. One image was acquired including the two channels to ensure that both reporters were present in the same cell, and a time-lapse movie was acquired using only the mCherry channel. (A’) and (B’) The first (A’) and last frame (B’) of a 50-frame 80-s movie (see Supplemental Movie 4 online). (C’) Merger of the first (colored red) and last frame (colored green) showing total motility pattern. (D’) Three random, successive frames are colored green, red, and blue, respectively, and then overlapped together to generate an integrity image. The brighter color indicates the slower movement. Bars = 10 μm.

Figure 9.

Interaction between O1 and HSP70-Interacting Protein, HIP. (A) Schematic diagram of maize HIP protein structure. (B) Yeast two-hybrid interactions between the O1 tail domain and HIP. a68 is one of the identified clones only containing HIP C terminus (from Glu-445 to stop codon). Interaction between T-antigen and P53 and 50-kD zein itself were used as positive control. AD, activating domain; BD, binding domain. [See online article for color version of this figure.]

Figure 10.

Model Depicting the Function of Myosin in Protein Body Formation. In the wild type, zeins are synthesized and stored in the ER as protein bodies, accompanying normal ER morphology and streaming mainly driven by a myosin motor. In the o1 mutant, ER dynamics were suppressed due to the O1 (myosin) defect, giving rise to the enlarged ER lumen, in which the zein protein bodies assemble and are stored. Therefore, there are more loci for zein aggregation in o1. Given that the total zein protein synthesis is not altered, the protein bodies reasonably become smaller. Moreover, because the ER motor system is out of homeostasis, this could affect the normal zein assembly, generating the misshapen protein bodies.