Arabidopsis thaliana expresses three divergent Srp54 genes.

The Arabidopsis thaliana Srp54 gene family was determined to consist of three genes, all of which were cloned and sequenced. In addition, cDNAs corresponding to two of the genes were obtained. To our knowledge this is the first description of multiple Srp54 genes within an organism. In contrast to the situation in mammals, where there are only three amino acid differences between the mouse and canine sequences, there was significant amino acid sequence diversity among the genes, particularly in the methionine-rich region of the protein, which is the region responsible for binding to the 7S RNA of the signal recognition particle and to the signal sequence of newly synthesized proteins. The amino acid sequences of the GTP-binding domains of the three clones were 86% identical, whereas the methionine-rich domains were only 65% identical. RNA gel blots of various tissues and developmental stages hybridized with gene-specific probes revealed that all three genes were expressed in all the tissues investigated. There were, however, quantitative differences in expression levels.

Purification and Characterization of an Endophytic Fungal Proteinase That Is Abundantly Expressed in the Infected Host Grass.

A novel Acremonium typhinum proteinase that is expressed during endophytic infection of the grass Poa ampla Merr. was purified from endophyte-infected leaf sheath tissue. It is a thiol-containing serine alkaline endoproteinase with bound carbohydrate. In the infected host tissue, this proteinase is an abundant protein localized within fungal membrane vesicles and in the plant and/or fungal cell walls. This proteinase was not expressed constitutively during fungus culture. Rather, its expression appeared to be induced by nutrient depletion. Expression of an antigenically similar proteinase was detected in five other endophyte-infected Poa species. The regulated expression of the proteinase in culture and its abundance in infected plant tissue suggest that its expression may be involved in the symbiotic interaction of the plant and the fungus.

Genetic length polymorphisms create size variation in proline-rich proteins of the cell wall.

Two genes, Prp1 and Prp2, encode proline-rich proteins that are found in different stages of developing seed coats, hypocotyls, and roots of soybeans (Glycine max (L.) Merr.). PRP1 is found in young seed coats and PRP2 is found later during seed desiccation. In some soybean varieties, both proteins are smaller as determined by immunoblotting seed coat proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It was found that Prp1 and Prp2 genes are linked to each other by testing seed coat protein extracts from an F2 population of a cross between the cultivar Richland, which exhibits the larger PRP proteins, and Blackhawk, which has the smaller PRP proteins. The Prp1 and Prp2 genes were separated by approximately 13% recombination. Simultaneous expression of soluble PRP2 polypeptides in the maternal seed coat and underlying aleurone layer of the embryo was found. The molecular basis for the size difference between the two varieties was examined using polymerase chain reaction to isolate the Prp genes from Blackhawk, the variety that exhibited the smaller proteins. Both of the genes from Blackhawk contained length polymorphisms that result in omission of some of the repeat units (pro pro val tyr lys) from the proteins. In Prp1, there were two separate deletions in different parts of the gene, each being two tandem repeats in length. In Prp2, there was only one deletion of two tandem repeats. These deletions occur within the coding regions in a manner that conserves the reading frame. The results are the first description of genetic variation in cell wall proteins and its molecular basis.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and characterization of an Arabidopsis thaliana gene for the 54 kDa subunit of the signal recognition particle.

The first step in the routing of newly synthesized proteins into the secretory pathway is the binding of the nascent signal sequence to the signal recognition particle. The mammalian signal recognition particle is a complex consisting of 6 proteins and a single 7S RNA molecule. Signal recognition particle-like complexes have been described from wheat and maize but none of the protein components have yet been described from any plant species. Here we report the cloning and characterization of an Arabidopsis thaliana gene encoding the 54 kDa protein subunit of the signal recognition particle. This is the first report of a SRP-54 sequence for any plant species and the first genomic sequence for any multicellular organism.

A Novel Fungal Protease Expressed in Endophytic Infection of Poa Species.

The fungus Acremonium typhinum produces a novel endoprotease during symbiotic endophytic infection of the grass, Poa ampla. This protease is unusual because it is highly active in the presence of sodium dodecyl sulfate. The enzyme is a thiol-containing serine protease and is localized to a crude membrane fraction. Similar protease activity has been detected in endophyte-infected Poa autumnalis and Poa sylvestris plants. Expression of this protease may be important in endophytic infection of Poa spp., because similar activity has not been detected in endophyte-infected Festuca arundinacea or Lolium perenne.

Variation of proline rich cell wall proteins in soybean lines with anthocyanin mutations.

The I locus controls inhibition of anthocyanin accumulation in the epidermal cells of the soybean seed coat and affects abundance of PRP1, a proline-rich cell wall protein in the seed coat. Saline-soluble PRP1 is abundant in the developing seed coats of cultivar Richland (homozygous I, yellow), while it is significantly decreased in the pigmented isogenic mutant T157 (homozygous i, imperfect black). In this report, we examined soluble PRP1 in several cultivars containing alleles of the I locus which affect spatial distribution of pigmentation in the seed coat. We also characterized PRP1 in isolines with allelic variants of several other loci involved in seed coat pigmentation, including T and Im. The T gene is pleiotropic and affects both pubesence color and seed coat pigmentation and structure. Soluble PRP1 was abundant in the developing seed coats of lines with yellow seed (I or ii alleles) regardless of pubescence color, just as in Richland. Likewise, soluble PRP1 was decreased in pigmented seed coats (ik or i alleles) with grey (t) pubescence, as in T157. However, the total seed coat proteins were not extractable from pigmented seed coats with tawny pubescence (i, T genotypes) because they have proanthocyanidins that exhibit tannin properties. The dominant Im allele inhibits seed coat mottling (irregular patches of pigmentation) that occurs if plants are infected with soybean mosaic virus. PRP1 was 35 kDa in mottled (im) isolines and 34 kDa in non-mottled (Im) isolines. PRP2, which is expressed later in seed coat development and in the hypocotyl hooks of soybean seedlings, was also smaller in Im isolines. In summary, some of the anthocyanin mutations affect the quantity of soluble PRP1 polypeptides. while others correlate with structural changes in developmentally regulated proline-rich proteins.

A soybean cell wall protein is affected by seed color genotype.

The dominant I gene inhibits accumulation of anthocyanin pigments in epidermal cells of the soybean seed coat. We compared saline-soluble proteins extracted from developing seed coats and identified a 35-kilodalton protein that was abundant in Richland (genotype I/I, yellow) and much reduced in an isogenic mutant line T157 (genotype i/i, imperfect black seed coats). We purified the 35-kilodalton protein by a novel procedure using chromatography on insoluble polyvinylpolypyrrolidone. The 35-kilodalton protein was composed primarily of proline, hydroxyproline, valine, tyrosine, and lysine. Three criteria (N-terminal amino acid sequence, amino acid composition, and sequence of a cDNA) proved that the seed coat 35-kilodalton protein was PRP1, a member of a proline-rich gene family expressed in hypocotyls and other soybean tissues. The levels of soluble PRP1 polypeptides and PRP1 mRNA were reduced in young seed coats with the recessive i/i genotype. These data demonstrated an unexpected and novel correlation between an anthocyanin gene and the quantitative levels of a specific, developmentally regulated cell wall protein. In contrast, PRP2, a closely related cell wall protein, was synthesized later in seed coat development and was not affected by the genotype of the I locus.

Expression of soybean lectin gene deletions in tobacco.

A series of constructs containing the developmentally regulated soybean lectin gene (Le1) were used to transform tobacco plants in order to assess developmental and quantitative regulation conferred by flanking sequences. The largest of the lectin constructs contained approximately 3,000 base pairs (bp) of Le1 5 flanking region and 1,500 bp of the 3 flanking region. The smallest construct contained no 5 flanking region and 194 bp of the 3 flanking region. ELISA assays of lectin in individual tobacco seeds and Southern blot analyses confirmed that most constructs were inherited as unique insertion events. Maximal expression of Le1 required more than 338 bp of 5 sequence, indicating that far upstream factors are involved in quantitative control of lectin expression. Lectin expression declined more than 80% between deletions with 1,700 versus 338 bp of 5 flanking sequence. In contrast, developmental control of lectin expression was maintained by Le1 inserts with only 190 bp of 5 sequence. The lectin promoter offers a potential means to target high levels of gene expression to the developing seeds of soybean or other dicotyledonous plants.