Search

Endosperm is a filial structure resulting from a second fertilization event in angiosperms. As an absorptive storage organ, endosperm plays an essential role in support of embryo development and seedling germination. The accumulation of carbohydrate and protein storage products in cereal endosperm provides humanity with a major portion of its food, feed, and renewable resources. Little is known regarding the regulatory gene networks controlling endosperm proliferation and differentiation. As a first step toward understanding these networks, we profiled all mRNAs in the maize kernel and endosperm at eight successive stages during the first 12 d after pollination. Analysis of these gene sets identified temporal programs of gene expression, including hundreds of transcription-factor genes. We found a close correlation of the sequentially expressed gene sets with distinct cellular and metabolic programs in distinct compartments of the developing endosperm. The results constitute a preliminary atlas of spatiotemporal patterns of endosperm gene expression in support of future efforts for understanding the underlying mechanisms that control seed yield and quality.

Lysine is the most limiting essential amino acid in cereals, and for many years plant breeders have attempted to increase its concentration to improve the nutritional quality of these grains. The opaque2 mutation in maize doubles the lysine content in the endosperm, but the mechanism by which this occurs is unknown. We show that elongation factor 1 alpha (EF-1 alpha) is overexpressed in opaque2 endosperm compared with its normal counterpart and that there is a highly significant correlation between EF-1 alpha concentration and the total lysine content of the endosperm. This relationship is also true for two other cereals, sorghum and barley. It appears that genetic selection for genotypes with a high concentration of EF-1 alpha can significantly improve the nutritional quality of maize and other cereals.

We provide evidence that a plant rhabdovirus, sonchus yellow net virus (SYNV), is similar to most animal rhabdoviruses in the order of structural genes and in the nucleotide sequences at the gene junctions but that it differs in the presence and location of a putative nonstructural gene. From the patterns of hybridization of a library of recombinant DNA clones, we have shown that the SYNV genome is transcribed into a short 3'-terminal "leader RNA" and six mRNAs. The proteins encoded by the SYNV mRNAs, in order of the appearance of their genes in the SYNV genome, are designated 3'-N-M2-sc4-M1-G-L-5' (N, nucleoprotein; M, matrix protein; sc, protein encoded by SYNV complementary RNA; G, glycoprotein; L, large protein). The intergenic and flanking gene sequences are conserved and consist of a central core of 14 nucleotides (3'-UUCUUUUUGGUUGU/A-5') whose sequence is similar to the sequence at the gene junctions of vesicular stomatitis and rabies viruses. The SYNV core consists of an 8-nucleotide (3'-UUCUUUUU-5') transcription termination signal at the 5' terminus of each gene, a dinucleotide (GG) spacer whose complement does not appear in mRNA, and a tetranucleotide (3'-UUGU/A-5') that is complementary to the first four nucleotides at the 5' terminus of the SYNV mRNAs. These results, when compared with structural information available on animal rhabdoviruses, suggest that organization of structural genes and maintenance of signals thought to play important roles in regulation of transcription have been conserved during evolution in plant, insect, and vertebrate hosts. However, differences in number and location of putative nonstructural genes reveal some flexibility in genome organization that may be important in deducing taxonomic and evolutionary relationships among viruses causing diseases in phylogenetically diverse hosts.

The relatedness of the genomes of satellite panicum mosaic virus (SPMV) and its helper virus, panicum mosaic virus (PMV), were investigated by nucleic acid hybridization. The results show that the satellite and helper virus RNAs have no appreciable homology or complementarity as assessed by hybridization with cDNA probes derived from the genomes of PMV and SPMV and with a probe complementary to the 3′ terminus of SPMV RNA. The complete nucleotide sequence of SPMV RNA reveals that the genome is 826 nucleotides (nt) long. The ability to label SPMV RNA with polynucleotide kinase only after phosphatase treatment suggests that the 5′ terminus is phosphorylated, but the extent of phosphorylation was not determined. The first open reading frame (ORF), encountered after an 88-nt 5′-untranslated region, encodes a 17,000 mol wt protein of a size and amino acid composition that are consistent with analysis of SPMV coat protein. An additional short ORF, located near the 3′ end of the RNA, could encode a 6300 mol wt polypeptide. The minus strand also contains two ORFs that could potentially encode polypeptides of 7100 and 11,000 mol wt. No evidence is available to determine whether the second positive-strand ORF or the two minus-strand ORFs are expressed. The data presented here clearly show the SPMV RNA is distinct from the RNAs of other satellite viruses, in both size and nucleotide sequence. However, the 5′-untranslated portions of SPMV and satellite tobacco mosaic virus RNAs share some structural features that may be important in initiation of translation.

Full-length genomic cDNA clones of the Type and ND18 strains of barley stripe mosaic virus (BSMV) were transcribed in vitro using T7 RNA polymerase. The combination of RNAs alpha, beta, and gamma synthesized in the presence of 5' cap analogs was infectious after inoculation onto barley plants, conclusively demonstrating the tripartite nature of the BSMV genome. Transcripts synthesized in the absence of cap analogs were not infectious. A gamma-specific subgenomic RNA which is normally present in BSMV virions was not required to establish a systemic infection. In vitro transcripts of variant cDNA clones which were isolated from the ND18 strain, containing either a simple nucleotide substitution or a 372-nucleotide duplication similar to one found in the genome of the Type strain, were also found to be biologically active. Two dicotyledonous hosts which have a differential response to infection with the Type and ND18 strains of BSMV were identified and these phenotypes were shown to be faithfully reproduced by inoculation with in vitro transcripts derived from the appropriate full-length cDNA clones.

We have devised a novel vector-primer strategy for cloning of full-length (FL) cDNA which can be applied to non-polyadenylated RNA species. Single-stranded plasmid DNA is used to prime first-strand synthesis by reverse transcriptase, and plasmids covalently linked to FL cDNA are then circularized by the annealing of a specific oligodeoxyribonucleotide (band-aid oligo). Only limited nucleotide sequence data are required from the termini of each RNA species to be cloned to design the plasmid primer and band-aid oligo. The band-aid strategy has been applied to the cloning of barley stripe mosaic virus genomic RNAs, and found to be both rapid and efficient. A strategy for the preparation of linear double-stranded plasmid DNA templates (suitable for run-off in vitro transcription) which is independent of restriction sites present within the cloned cDNA is also described.

The complete nucleotide sequences of RNA gamma from the Type and ND18 strains of barley stripe mosaic virus (BSMV) have been determined. The sequences are 3164 (Type) and 2791 (ND18) nucleotides in length. Both sequences contain a 5'-noncoding region (87 or 88 nucleotides) which is followed by a long open reading frame (ORF1). A 42-nucleotide intercistronic region separates ORF1 from a second, shorter open reading frame (ORF2) located near the 3'-end of the RNA. There is a high degree of homology between the Type and ND18 strains in the nucleotide sequence of ORF1. However, the Type strain contains a 366 nucleotide direct tandem repeat within ORF1 which is absent in the ND18 strain. Consequently, the predicted translation product of Type RNA gamma ORF1 (mol wt 87,312) is significantly larger than that of ND18 RNA gamma ORF1 (mol wt 74,011). The amino acid sequence of the ORF1 polypeptide contains homologies with putative RNA polymerases from other RNA viruses, suggesting that this protein may function in replication of the BSMV genome. The nucleotide sequence of RNA gamma ORF2 is nearly identical in the Type and ND18 strains. ORF2 codes for a polypeptide with a predicted molecular weight of 17,209 (Type) or 17,074 (ND18) which is known to be translated from a subgenomic (sg) RNA. The initiation point of this sgRNA has been mapped to a location 27 nucleotides upstream of the ORF2 initiation codon in the intercistronic region between ORF1 and ORF2. The sgRNA is not coterminal with the 3'-end of the genomic RNA, but instead contains heterogeneous poly(A) termini up to 150 nucleotides long (J. Stanley, R. Hanau, and A. O. Jackson, 1984, Virology 139, 375-383). In the genomic RNA gamma, ORF2 is followed by a short poly(A) tract and a 238-nucleotide tRNA-like structure.