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.

In maize, a series of seed mutants with starchy endosperm could increase the lysine content by decreased amount of zeins, the main storage proteins in endosperm. Cloning and characterization of these mutants could reveal regulatory mechanisms for zeins accumulation in maize endosperm. Opaque7 (o7) is a classic maize starchy endosperm mutant with large effects on zeins accumulation and high lysine content. In this study, the O7 gene was cloned by map-based cloning and confirmed by transgenic functional complementation and RNAi. The o7-ref allele has a 12-bp in-frame deletion. The four-amino-acid deletion caused low accumulation of o7 protein in vivo. The O7 gene encodes an acyl-activating enzyme with high similarity to AAE3. The opaque phenotype of the o7 mutant was produced by the reduction of protein body size and number caused by a decrease in the a-zeins concentrations. Analysis of amino acids and metabolites suggested that the O7 gene might affect amino acid biosynthesis by affecting a-ketoglutaric acid and oxaloacetic acid. Transgenic rice seeds containing RNAi constructs targeting the rice ortholog of maize O7 also produced lower amounts of seed proteins and displayed an opaque endosperm phenotype, indicating a conserved biological function of O7 in cereal crops. The cloning of O7 revealed a novel regulatory mechanism for storage protein synthesis and highlighted an effective target for the genetic manipulation of storage protein contents in cereal seeds.T HE texture and protein quality of maize (Zea mays L.) endosperm are important factors affecting grain shipping, insect and fungal pathogen resistance, and nutritional quality. Much evidence indicates that the reduction in the amount of zeins in the endosperm leads to a decrease in the endosperm hardness and an increase in lysine content (Mertz et al. 1964;Misra et al. 1972;Schmidt et al. 1987;Dombrink-Kurtzman and Bietz 1993;Holding and Larkins 2006;. Maize have a number of opaque or floury endosperm mutants that affect the texture and protein quality of endosperm by altering zeins accumulation. Our understanding of the underlying mechanisms determining zeins accumulation comes from the study of seed mutants.There are .18 mutants that can exhibit an opaque or floury endosperm (Thompson and Larkins 1994;Hunter et al. 2002). Among them are the recessive opaque mutants (o1, o2, o5, o7, o9-o11, and o13-o17), the semidominant floury mutants (fl1, fl2, and fl3), and the dominant mutants Mucronate (Mc) and Defective endosperm B30 (De-B30) (Motto et al. 1996;Gibbon and Larkins 2005). The cloning and characterization of some of the opaque mutants has revealed important regulatory mechanisms for zeins accumulation in maize endosperm. The O2 gene, which encodes a defective basic-domain-leucine-zipper transcription factor, regulates several endosperm-expressed genes, in particular the 22-kDa a-zeins (Schmidt et al. 1987(Schmidt et al. , 1990Damerval and De Vienne

Defective interfering (DI) RNAs are often associated with transmission-defective isolates of wound tumor virus (WTV), a plant virus member of the Reoviridae. We report here the cloning and characterization of WTV genome segment S5 [2613 base pairs (bp)] and three related DI RNAs (587-776 bp). Each DI RNA was generated by a simple internal deletion event that resulted in no sequence rearrangement at the deletion boundaries. Remarkably, although several DI RNAs have been in continuous passage for more than 20 years, their nucleotide sequences are identical to that of corresponding portions of segment S5 present in infrequently passaged, standard, transmission-competent virus. The positions of the deletion breakpoints indicate that the minimal sequence information required for replication and packaging of segment S5 resides within 319 bp from the 5' end of the (+)-strand and 205 bp from the 3' end of the (+)-strand. The terminal portions of segment S5 were found to contain a 9-bp inverted repeat immediately adjacent to the conserved terminal 5'-hexanucleotide and 3'-tetranucleotide sequences shared by all 12 WTV genome segments. The presence of a 6-to 9-nucleotide segmentspecific inverted repeat immediately adjacent to the conserved terminal sequences was found to be a feature common to all WTV genome segments. These results reveal several basic principles that govern the replication and packaging of a segmented double-stranded RNA genome.Viruses classified as members of the family Reoviridae have been isolated from a wide range of hosts including vertebrates, insects, mollusks, and plants (1). Several members of this virus group are of medical and economic importance; e.g., rotaviruses are a major cause of infant mortality in many parts of the world, and cytoplasmic polyhedrosis viruses are potential insect-control agents (1). Other members provide valuable model systems for molecular, pathogenicity, and epidemiologic studies (1). The genomes of these viruses consist of 10-12 segments of double-stranded RNA (1). Available evidence suggests that each virus particle contains one copy of each segment comprising the genome (2-5). The mechanism responsible for the selective sorting of individual genome segments remains an intriguing unsolved problem presumably involving complex protein-RNA and RNA-RNA interactions. Characterization of functional remnants of genome RNAs generated by deletion events has provided insights into the mechanism of genome replication and encapsidation for members of other virus families (6-9). Although there exists little information regarding remnant RNAs associated with the genome of mammalian reoviruses, there have been several reports describing remnant RNAs associated with isolates of wound tumor virus (WTV), the type member of the genus Phytoreovirus (10-12).WTV replicates both in plant hosts and in several species of leafhoppers, which act as vectors. Maintenance of WTV exclusively in vegetatively propagated plant hosts results in virus populations that are defective in the abil...

Dunaliella, a unicellular green alga, has the unusual ability to survive dramatic osmotic stress by accumulating high concentrations of intracellular glycerol as a compatible solute. The chloroplastic glycerol-3-phosphate dehydrogenase (GPDH) has been considered to be the key enzyme that produces glycerol for osmoregulation in Dunaliella. In this study, we cloned the two most prominent GPDH cDNAs (DvGPDH1 and DvGPDH2) from Dunaliella viridis, which encode two polypeptides of 695 and 701 amino acids, respectively. Unlike higher plant GPDHs, both proteins contained extra phosphoserine phosphatase (SerB) domains at their N-termini in addition to C-terminal GPDH domains. Such bi-domain GPDHs represent a novel type of GPDH and are found exclusively in the chlorophyte lineage. Transient expression of EGFP fusion proteins in tobacco leaf cells demonstrated that both DvGPDH1 and DvGPDH2 are localized in the chloroplast. Overexpression of DvGPDH1 or DvGPDH2 could complement a yeast GPDH mutant (gpd1Delta), but not a yeast SerB mutant (ser2Delta). In vitro assays with purified DvGPDH1 and DvGPDH2 also showed apparent GPDH activity for both, but no SerB activity was detected. Surprisingly, unlike chloroplastic GPDHs from plants, DvGPDH1 and DvGPDH2 could utilize both NADH and NADPH as coenzymes and exhibited significantly higher GPDH activities when NADH was used as the coenzyme. Q-PCR analysis revealed that both genes exhibited transient transcriptional induction of gene expression upon hypersalinity shock, followed by a negative feedback of gene expression. These results shed light on the regulation of glycerol synthesis during salt stress in Dunaliella.