Construction and evaluation of cDNA libraries for large-scale expressed sequence tag sequencing in wheat (Triticum aestivum L.).

A total of 37 original cDNA libraries and 9 derivative libraries enriched for rare sequences were produced from Chinese Spring wheat (Triticum aestivum L.), five other hexaploid wheat genotypes (Cheyenne, Brevor, TAM W101, BH1146, Butte 86), tetraploid durum wheat (T. turgidum L.), diploid wheat (T. monococcum L.), and two other diploid members of the grass tribe Triticeae (Aegilops speltoides Tausch and Secale cereale L.). The emphasis in the choice of plant materials for library construction was reproductive development subjected to environmental factors that ultimately affect grain quality and yield, but roots and other tissues were also included. Partial cDNA expressed sequence tags (ESTs) were examined by various measures to assess the quality of these libraries. All ESTs were processed to remove cloning system sequences and contaminants and then assembled using CAP3. Following these processing steps, this assembly yielded 101,107 sequences derived from 89,043 clones, which defined 16,740 contigs and 33,213 singletons, a total of 49,953 "unigenes." Analysis of the distribution of these unigenes among the libraries led to the conclusion that the enrichment methods were effective in reducing the most abundant unigenes and to the observation that the most diverse libraries were from tissues exposed to environmental stresses including heat, drought, salinity, or low temperature.

Accumulation of a Brazil nut albumin in seeds of transgenic canola results in enhanced levels of seed protein methionine.

We have increased the methionine content of the seed proteins of a commercial winter variety of canola by expressing a chimeric gene encoding a methionine-rich seed protein from Brazil nut in the seeds of transgenic plants. Transgenic canola seeds accumulate the heterologous methionine-rich protein at levels which range from 1.7% to 4.0% of the total seed protein and contain up to 33% more methionine. The precursor of the methionine-rich protein is processed correctly in the seeds, resulting in the appearance of the mature protein in the 2S protein fraction. The 2S methionine-rich protein accumulates in the transgenic seeds at the same time in development as the canola 11S seed proteins and disappears rapidly upon germination of the seed. The increase in methionine in the canola seed proteins should increase the value of canola meal which is used in animal feed formulations.

Enhancement of the methionine content of seed proteins by the expression of a chimeric gene encoding a methionine-rich protein in transgenic plants.

We have constructed a chimeric gene encoding a Brazil nut methionine-rich seed protein which contains 18% methionine. This gene has been transferred to tobacco and expressed in the developing seeds. Tobacco seeds are able to process the methionine-rich protein efficiently from a larger precursor polypeptide of 17 kDa to the 9 kDa and 3 kDa subunits of the mature protein, a procedure which involves three proteolytic cleavage steps in the Brazil nut seed. The accumulation of the methionine-rich protein in the seeds of tobacco results in a significant increase (30%) in the levels of the methionine in the seed proteins of the transgenic plants. Our data indicate that the introduction of a chimeric gene encoding a methionine-rich seed protein into crop plants, particularly legumes whose seeds are deficient in the essential sulfur-containing amino acids, represents a feasible method for improving the nutritional quality of seed proteins.

Properties, biosynthesis and processing of a sulfur-rich protein in Brazil nut (Bertholletia excelsa H.B.K.).

An abundant seed protein, which is exceptionally rich in the sulfur-containing amino acids, methionine (18%) and cysteine (8%), is synthesized in Brazil nut embryos about 9 months after flowering. This sulfur-rich protein consists of two low-molecular-mass polypeptide components, a 9-kDa polypeptide and a 3-kDa polypeptide. The two-subunit polypeptides associate through disulfide linkage(s) to form a 12-kDa protein molecule. We have demonstrated through in vitro translation studies, using RNA from 9-month-old embryos, that the sulfur-rich protein is synthesized as a larger precursor polypeptide of 18 kDa. In addition, data from in vivo labelling studies of 9-month-old Brazil nuts suggest that there are two intermediate precursors of the sulfur-rich protein, one of 15 kDa and another of 12 kDa. One of these precursors, the 12-kDa polypeptide, accumulates for a 2-month period in the developing embryos. From these data we infer that at least three stepwise cleavages are involved in the maturation of the sulfur-rich protein from its 18-kDa precursor.

Cloning and sequence analysis of a cDNA encoding a Brazil nut protein exceptionally rich in methionine.

The primary amino acid sequence of an abundant methionine-rich seed protein found in Brazil nut (Bertholletia excelsa H.B.K.) has been elucidated by protein sequencing and from the nucleotide sequence of cDNA clones. The 9 kDa subunit of this protein was found to contain 77 amino acids of which 14 were methionine (18%) and 6 were cysteine (8%). Over half of the methionine residues in this subunit are clustered in two regions of the polypeptide where they are interspersed with arginine residues. In one of these regions, methionine residues account for 5 out of 6 amino acids and four of these methionine residues are contiguous. The sequence data verifies that the Brazil nut sulfur-rich protein is synthesized as a precursor polypeptide that is considerably larger than either of the two subunits of the mature protein. Three proteolytic processing steps by which the encoded polypeptide is sequentially trimmed to the 9 kDa and 3 kDa subunit polypeptides have been correlated with the sequence information. In addition, we have found that the sulfur-rich protein from Brazil nut is homologous in its amino acid sequence to small water-soluble proteins found in two other oilseeds, castor bean (Ricinus communis) and rapeseed (Brassica napus). When the amino acid sequences of these three proteins are aligned to maximize homology, the arrangement of cysteine residues is conserved. However, the two subunits of the Brazil nut protein contain over 19% methionine whereas the homologous proteins from castor bean and rapeseed contain only 2.1% and 2.6% methionine, respectively.

Nucleic acid species related to the satellite RNA of turnip crinkle virus in turnip plants and virus particles.

Multiple RNA species which hybridize to a cloned cDNA probe of the turnip crinkle virus satellite RNA (S-TCV) are found both in virions and in plants infected with virus particles containing S-TCV. At least six of these RNAs appear to be multimeric forms of the satellite RNA based on their migration in denaturing gels. Hybridization to strand-specific probes indicates that most of the RNAs present in both infected cells and encapsidated in the virus particles represent strands of the same polarity as S-TCV. We have detected a small RNA species which is homologous to the satellite RNA that appears in plants inoculated with the gel-purified viral genomic RNA. In addition, we have found homology between the cloned satellite probe and DNA sequences in the genome of the uninfected plant.

In vitro translation products of turnip crinkle virus RNA.

Turnip leaves infected with turnip crinkle virus accumulate a 35-kd polypeptide which comigrates with the major protein from isolated virions. RNA from TCV virions directs the synthesis of a number of polypeptides in vitro including a 35-kd protein which is immunoprecipitable with antiserum to virus particles. Translation of viral RNA size-fractionated on sucrose gradients or methyl mercurichydroxide-containing gels shows that the TCV coat protein is synthesized primarily from RNA fragments which are smaller than the genomic RNA. A satellite RNA species found in virions does not direct the synthesis in vitro of any identifiable protein.

Identification of a satellite RNA associated with turnip crinkle virus.

Turnip plants infected with turnip crinkle virus (TCV) contain four major RNA species which are not found in uninfected plants (A = 1.3 x 10(6) MW, B = 0.28 x 10(6) MW, C = 0.17 x 10(6) MW, and D = 0.13 x 10(6) MW). At least two of these RNAs, RNA A and RNA C, are packaged in the mature virion, but only the large RNA A is required for infection. Plants infected with RNA A alone produce neither the small virion RNA C nor the small nonvirion RNAs B and D. The small virion RNA C is not infective by itself, but requires coinfection with RNA A to replicate in plants. RNA C increases the severity of symptoms in plants infected with RNA A and restores the production of the nonvirion RNAs B and D. T1 RNase oligonucleotide mapping and copy DNA hybridization analysis indicate that the virion RNAs A and C do not have extensive homology. These data suggest that the large virion RNA A contains the full TCV genome and that the smaller virion RNA C is a dispensible satellite, designated S-TCV.