Developmental and transgenic analysis of two tomato fruit enhanced genes.

Tomato fruit development is characterized by distinct developmental stages: fruit set, periods of rapid cell division and cell expansion, and the period where processes associated with ripening are dominant. During each of these stages, different aspects of cellular metabolism are favored. Accompanying these developmental changes are dramatic differences in gene expression, with a subset of genes being expressed early and a subset being expressed later in development. We have isolated and characterized several sequences from tomato that are expressed primarily in immature green fruit. Two of these genes (Tfm7 and Tfm5) have been characterized more extensively and their sequence indicates that they encode proteins corresponding to a proline-rich protein (PRP) and a glycine-rich protein (GRP). RNA blot analysis indicates that the transcripts from these genes are present at the earliest stages of fruit development, and continue to be expressed throughout the growth period of the fruit. Expression analysis during development indicates that the gene encoding the PRP may be down-regulated by ethylene. As a means to understanding the functional significance and the transcriptional contribution of these tissue-limited proteins during development, we constructed promoter-reporter gene fusions to identify which cell types express each of these sequences. GUS protein produced in transgenic plants by both promoter-reporter gene constructs was detected in most tissues of the fruit including the pericarp, columella, and placental tissues of young immature fruit through the mature green stage. However, only one of the promoter sequences conferred expression in the fruit locular tissue.

Genetic Transformation of Wheat Mediated by Agrobacterium tumefaciens.

A rapid Agrobacterium tumefaciens-mediated transformation system for wheat was developed using freshly isolated immature embryos, precultured immature embryos, and embryogenic calli as explants. The explants were inoculated with a disarmed A. tumefaciens strain C58 (ABI) harboring the binary vector pMON18365 containing the [beta]-glucuronidase gene with an intron, and a selectable marker, the neomycin phosphotransferase II gene. Various factors were found to influence the transfer-DNA delivery efficiency, such as explant tissue and surfactants present in the inoculation medium. The inoculated immature embryos or embryogenic calli were selected on G418-containing media. Transgenic plants were regenerated from all three types of explants. The total time required from inoculation to the establishment of plants in soil was 2.5 to 3 months. So far, more than 100 transgenic events have been produced. Almost all transformants were morphologically normal. Stable integration, expression, and inheritance of the transgenes were confirmed by molecular and genetic analysis. One to five copies of the transgene were integrated into the wheat genome without rearrangement. Approximately 35% of the transgenic plants received a single copy of the transgenes based on Southern analysis of 26 events. Transgenes in T1 progeny segregated in a Mendelian fashion in most of the transgenic plants.

Identification of mRNAs with enhanced expression in ripening strawberry fruit using polymerase chain reaction differential display.

Fruit ripening is a complex developmental process that involves specific changes in gene expression and cellular metabolism. In climateric fruits these events are coordinated by the gaseous hormone ethylene, which is synthesized autocatalytically in the early stages of ripening. Nonclimacteric fruits do not synthesize or respond to ethylene in this manner, yet undergo many of the same physiological and biochemical changes associated with the production of a ripe fruit. To gain insight into the molecular determinants associated with nonclimacteric fruit ripening, we examined mRNA populations in ripening strawberry fruit using polymerase chain reaction (PCR) differential display. Five mRNAs with ripening-enhanced expression were identified using this approach. Three of the mRNAs appear to be fruit-specific, with little or no expression detected in vegetative tissues. Sequence analysis of cDNA clones revealed positive identities for three of the five mRNAs based on homology to known proteins. These results indicate that the differential display technique can be a useful tool to study fruit ripening and other developmental processes in plants at the RNA level.

Expression of the Arabidopsis AtAux2-11 auxin-responsive gene in transgenic plants.

Five constructions containing deletions of the promoter from an auxin-inducible gene of Arabidopsis thaliana, AtAux2-11, were fused to the coding region of the reporter gene LacZ, which encodes beta-galactosidase, and a polyadenylation 3'-untranslated nopaline synthase sequence from Agrobacterium. These chimeric genes were introduced into Arabidopsis by Agrobacterium tumefaciens-mediated transformation, and expression of the gene was examined by spectrophotometric and histochemical analyses. A 600 bp fragment from the AtAux2-11 promoter conferred histochemical patterns of staining similar to the longest 5' promoter tested, a 3.0 kb fragment. Localization of AtAux2-11/LacZ activity in the transgenic plants revealed spatial and temporal expression patterns that correlated with tissues and cells undergoing physiological processes modulated by auxin. LacZ activity was expressed in the elongating region of roots, etiolated hypocotyls, and anther filaments. Expression was detected in the vascular cylinder of the root and the vascular tissue, epidermis, and cortex of the hypocotyl, and filament. The AtAux2-11/LacZ gene was preferentially expressed in cells on the elongating side of hypocotyls undergoing gravitropic curvature. Expression of the chimeric gene in the hypocotyls of light-grown seedlings was less than that in etiolated seedling hypocotyls. The AtAux2-11/LacZ gene was active in the root cap, and expression in the root stele increased at sites of lateral root initiation. Staining was evident in cell types that develop lignified cell walls, e.g. trichomes, anther endothecial cells, and especially developing xylem. The chimeric gene was not expressed in primary meristems. While the magnitude of expression increased after application of exogenous auxin (2,4-D), the histochemical localization of AtAux2-11/LacZ remained unchanged. Transgenic plants with a 600 bp promoter construct (-0.6 kb AtAux2-11/LacZ) had higher levels of basal and auxin-inducible expression than plants with a 3.0 kb promoter construct. Transgenic plants with a -500 bp promoter had levels of expression similar to the -3.0 kb construct. The -0.6 kb AtAux2-11/LacZ gene responded maximally to a concentration of 5 x 10(-6) to 5 x 10(-5) M 2,4-D and was responsive to as little as 5 x 10(-8) M. The evidence presented here suggests that this gene may play a role in several auxin-mediated developmental and physiological processes.

Sequence and Expression of a HSP83 from Arabidopsis thaliana.

A full-length cDNA encoding a heat shock protein (hsp) belonging to the 83 to 90 kilodalton hsp family of Arabidopsis thaliana has been isolated and sequenced. Truncated cDNA clones were isolated by nucleic acid hybridization to a truncated soybean HSP83 cDNA probe and a fragment generated from a Drosophila HSP83 gene. A single strand DNA vector/primer based extension procedure was employed to obtain the full-length cDNA. The level of transcripts homologous to this cDNA (AtHS83) is low in 2-week-old Arabidopsis plants but is rapidly enhanced by elevated temperatures. DNA sequence comparison between this cDNA and hsp83-90 sequences from human, yeast and Drosophila reveal amino acid identities of 63 to 69%, typical identities for interspecies comparisons between hsp83 to 90 kilodalton proteins. Genomic DNA blot analysis performed with probes derived from AtHS83 indicate the presence of a HSP83 gene family estimated to be comprised of at least three genes.

Structure and expression of two auxin-inducible genes from Arabidopsis.

Two genes from Arabidopsis thaliana related to the auxin-inducible Aux28 and Aux22 genes of soybean have been isolated. These genes belong to a small multi-gene family and are similar to the soybean Aux gene family in the sequence of the predicted proteins, intron/exon locations, and auxin-enhanced expression of their transcripts. Application of auxin to 8-day old Arabidopsis plants, 4-day old etiolated seedlings, and suspension culture cells all resulted in enhanced Aux transcript levels. Comparison of the promoter sequences from the soybean and Arabidopsis genes yielded no significant sequence conservation; however, three regions of near sequence identity are present between the two Arabidopsis Aux genes.

Structure of the three beta-tubulin-encoding genes of the unicellular alga, Polytomella agilis.

The quadriflagellate, unicellular, colorless alga, Polytomella agilis, contains several distinct microtubule arrays. To study the genetic basis of microtubule heterogeneity in P. agilis, we characterized its tubulin(Tub)-encoding genes (tub). The three beta tub genes detected in blots of P. agilis DNA were isolated from a genomic library. The structure and organization of the genes were examined by restriction mapping and nucleotide (nt) sequencing. S1 nuclease protection studies showed that all three genes are expressed. The predicted amino acid (aa) sequences are more than 98% conserved with the Chlamydomonas reinhardtii and Volvox carteri beta-Tubs, underscoring the close phylogenetic relationship of these species. Evolutionary divergence among the P. agilis genes is demonstrated by differences in intron number, nt sequences in noncoding regions, and silent nt substitutions in the coding regions. However, the proteins encoded by the beta 1 and beta 3 tub genes are identical; the beta 2 gene product differs by one conservative aa substitution. These results are in striking contrast to the C-terminal aa diversity reported within beta tub gene families in animal, higher plant and fungal systems. The data support the hypothesis that those tub genes whose products assemble into axonemal microtubules are subject

The two alpha-tubulin genes of Chlamydomonas reinhardi code for slightly different proteins.

Full-length cDNA clones corresponding to the transcripts of the two alpha-tubulin genes in Chlamydomonas reinhardi were isolated. DNA sequence analysis of the cDNA clones and cloned gene fragments showed that each gene contains 1,356 base pairs of coding sequence, predicting alpha-tubulin products of 451 amino acids. Of the 27 nucleotide differences between the two genes, only two result in predicted amino acid differences between the two gene products. In the more divergent alpha 2 gene, a leucine replaces an arginine at amino acid 308, and a valine replaces a glycine at amino acid 366. The results predicted that two alpha-tubulin proteins with different net charges are produced as primary gene products. The predicted amino acid sequences are 86 and 70% homologous with alpha-tubulins from rat brain and Schizosaccharomyces pombe, respectively. Each gene had two intervening sequences, located at identical positions. Portions of an intervening sequence highly conserved between the two beta-tubulin genes are also found in the second intervening sequence of each of the alpha genes. These results, together with our earlier report of the beta-tubulin sequences in C. reinhardi, present a picture of the total complement of genetic information for tubulin in this organism.

Analysis of the phenotypes exhibited by rudimentary-like mutants of Drosophila melanogaster.

Flies mutant for one or both of the last two enzymes of de novo pyrimidine biosynthesis express a number of phenotypes that are also expressed by mutants of the first four pathway enzymes (r and Dhod-null mutants). However, r-1 flies also express two phenotypes, mottled eyes and poor viability, that are not usually expressed by r and Dhod-null flies. Chemical determinations show that orotic acid, a substrate for the fifth pathway enzyme, accumulates in r-1 individuals but not in r and wild-type individuals. Moreover, flies simultaneously mutant for r and r-1 do not express the mottled-eye phenotype, showing that r is epistatic to r-1 for this r-1-specific phenotype. When genotypically wild-type flies are cultured on a medium containing 6-azauracil, the base of a potent inhibitor of the last enzyme of de novo pyrimidine biosynthesis, phenocopies are obtained that include the mottled-eye as well as the wing phenotypes of r-1 flies. These results support hypotheses that the phenotypes common to r, Dhod-null, and r-1 flies are consequences of uridylic acid deficiency, whereas the r-1-specific phenotypes result from orotic acid accumulation in flies lacking either or both of the last two enzymes of de novo pyrimidine biosynthesis.