The GLABRA2 gene (GL2) is one of several genes known to have a role in trichome development in Arabidopsis. Mutations at this locus result in abnormal trichome expansion. We have identified several gl2 mutants from a T-DNA-mutagenized population of plants. The T-DNA insert in one of the mutant lines cosegregated with the recessive gl2 phenotype and thus served as a molecular tag to isolate genomic DNA at the putative GL2 locus. RFLP analysis of the segregating population and subsequent molecular complementation experiments established that the GL2 gene had been cloned. The predicted polypeptide from one of the ORFs contained on this fragment showed significant identity to the homeo domain sequence. The construction of a full-length cDNA by RT-PCR confirmed the presence of a homeo box in the GL2 gene and showed that it is substantially different from other recently cloned homeo box genes in plants. The expression pattern of GL2, as demonstrated by in situ hybridization, indicated that the gene is expressed in trichome progenitor cells and at stages associated with trichome development. This suggests that GL2 may regulate events required for the directional cell expansion observed during trichome formation.
The role of the Arabidopsis homeobox gene, GLABRA 2 (GL2), in the development of the root epidermis has been investigated. The wild-type epidermis is composed of two cell types, root-hair cells and hairless cells, which are located at distinct positions within the root, implying that positional cues control cell-type differentiation. During the development of the root epidermis, the differentiating root-hair cells (trichoblasts) and the differentiating hairless cells (atrichoblasts) can be distinguished by their cytoplasmic density, vacuole formation, and extent of elongation. We have determined that mutations in the GL2 gene specifically alter the differentiation of the hairless epidermal cells, causing them to produce root hairs, which indicates that GL2 affects epidermal cell identity. Detailed analyses of these differentiating cells showed that, despite forming root hairs, they are similar to atrichoblasts of the wild type in their cytoplasmic characteristics, timing of vacuolation, and extent of cell elongation. The results of in situ nucleic acid hybridization and GUS reporter gene fusion studies show that the GL2 gene is preferentially expressed in the differentiating hairless cells of the wild type, during a period in which epidermal cell identity is believed to be established. These results indicate that the GL2 homeodomain protein normally regulates a subset of the processes that occur during the differentiation of hairless epidermal cells of the Arabidopsis root. Specifically, GL2 appears to act in a cell-position-dependent manner to suppress hair formation in differentiating hairless cells.
Two distinct legumin genes (LegA1 and LegA2) which encode a major class of seed storage protein in pea were isolated from a genomic library. The cloned fragments were introduced into tobacco via Agrobacterium-mediated transformation and the regenerated plants were used to study the expression characteristics of the genes in a heterologous host. It was found that both LegA1 and LegA2 were functional members of the pea legumin gene family and that their expression was similar in both pea and transgenic tobacco. Legumin was detected only in the seed of tobacco where the primary translation products were processed in a manner analogous to that which occurs in pea. Legumin gene expression was also shown to be temporally regulated during seed development. Legumin polypeptides and mRNA began to accumulate 16 days after flowering (DAF), in contrast to the endogenous tobacco storage proteins which were apparent at 13 DAF. It was also demonstrated that the legumin genes in tobacco were environmentally regulated to the nutritional status of the plant. As has been previously shown in pea, legumin accumulation in transgenic tobacco seed was progressively reduced when the plants were grown under conditions of increasing severity of sulphur-nutrient stress. The reduced accumulation of protein was correlated with lower levels of legumin mRNA in the developing seed. Despite encoding nearly identical subunits, nucleotide sequence data for LegA1 and LegA2 showed that the similarity of their respective 5'-flanking regions was restricted to several short elements mostly within 240 bp from the start of transcription. However, a deletion series using the LegA1 gene demonstrated that 237 bp of 5'-flanking sequence was insufficient to permit the expression of the legumin gene in tobacco. The data indicated that an as yet unidentified sequence element(s) located between positions -668 and -237 was essential in re-establishing the high level of regulated gene expression observed with the full-length LegA1 gene.
In this review the role of sulfur in regulating the expression of genes for pea seed storage proteins in both peas and transgenic tobacco is discussed. The levels of the sulfur-containing proteins, legumin and pea albumin 1 (PA1), are reduced in the seeds of peas grown under mild sulfur nutrient stress. In contrast, the levels of sulfur-poor proteins such as pea lectin and vicilin are either unaffected or increased slightly under the same conditions. The levels of all the proteins are a direct reflection of the levels of their respective mRNAs. The reduced levels of legumin and PA1 mRNAs under sulfur stress is known to be due largely to increased mRNA turnover rather than decreased transcription. The advent of gene transfer procedures for plants has allowed re-examination of the mechanism of regulation of mRNA stability under conditions of sulfur stress. A pea albumin 1 gene was engineered for leaf expression and transferred to tobacco and the transgenic plants were grown on normal and low levels of sulfur. Sulfur stress reduces total leaf protein in tobacco by about 20% and there are minor qualitative changes in the total protein profile. In contrast, PA1 levels are reduced by over 90% compared with the controls when the transgenic tobaccos are grown under sulfur stress. Thus, it is clear that sequences responsible for recognising the sulfur status have been included in the transgene. A number of gene constructs have been designed to test where the sulfur-responsive sequences are located in the PA1 gene and some of the preliminary findings are reported.
The A-subfamily of legumin genes encodes the predominant I IS-underlined and include the TATA (-26 to-32) and the class of seed storage proteins in pea (Pisum sativwn L.). We have 'legumin' (-100 to-130) boxes in the 5' region and overlapping determined the complete nucleotide sequence of one such gene polyadenylation signals (+ 1969, + 1973) in the 3' region. The (LegA2) located on a 4.2 kb EcoR1 fragment and encoding a proposed signal peptide is delineated by boldface type preceding mature subunit with a deduced molecular weight of 56,929 the processing site (I I). The post-translational processing site Daltons. The coding region spans 1,825 bp (including three short between Asn and Gly residues is also indicated. introns) and exhibits a high degree of identity to LegA (1), but diverges from this gene in both the immediate 5'and 3'-flanking REFERENCE regions. Nucleotide positions are numbered relative to the proposed cap-site (+ 1). Potential regulatory sequences are 1. Lycett,G. et al. (1984) Nucl. Acids Res. 11, 4493-4506.
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