Cloning the Arabidopsis GA1 Locus by Genomic Subtraction

Arabidopsis thaliana ga1 mutants are gibberellin-responsive dwarfs. We used the genomic subtraction technique to clone DNA sequences that are present in wild-type Arabidopsis (ecotype Landsberg erecta, Ler) but are missing in a presumptive ga1 deletion mutant, ga1-3. The cloned sequences correspond to a 5.0-kb deletion in the ga1-3 genome. Three lines of evidence indicated that the 5.0-kb deletion in the ga1-3 mutant is located at the GA1 locus. First, restriction fragment length polymorphism mapping showed that DNA sequences within the 5.0-kb deletion map to the GA1 locus. Second, cosmid clones that contain wild-type DNA inserts spanning the deletion in ga1-3 complemented the dwarf phenotype when integrated into the ga1-3 genome by Agrobacterium tumefaciens-mediated transformation. Third, we identified molecular lesions in four additional ga1 alleles within the 5.0-kb region deleted in mutant ga1-3. One of these lesions is a large insertion or inversion located within the most distal intron encoded by the GA1 locus. The three other lesions are all single base changes located within the two most distal exons. RNA gel blot analysis indicated that the GA1 locus encodes a 2.8-kb mRNA. We calculated a recombination rate of 10-5 cM per nucleotide for the GA1 region of the Arabidopsis genome.     

GA3-regulated cDNAs from Hordeum vulgare leaves

The barley mutant dbg 576 shows an extreme vegetative dwarf phenotype. This is reversed after the application of GA₃ which also induces the mutant florets to become partly fertile. cDNA clones ES1A and ES2A were isolated by differential screening with subtracted probes from a DNA library prepared from mutant leaf blades after GA₃ treatment. Both the ES1A and the ES2A mRNA level increases as early as 30 min after GA₃ treatment.and decreases later. Accumulation of ES1A and ES2A mRNAs is leaf blade-specific and both are ca. 750 nucleotides long. ES1A encodes a protein of approximately 6 kD which shows a significant homology with mammalian epidermal growth fractors (EGFs). ES2A encodes a protein of 22 kDa with homology, in regions with potential amphiphilic helices, with the D7 family of late embryogenesis-abundant proteins (LEA).     

New insight in the Gibberellin biosynthesis and signal transduction

Gibberellin (GA) plays important roles through plant growth and development. However, where GA is synthesized inside a cell and how it regulates sex determination is obscure. We analyzed the classic dwarf1 (d1) mutant in maize and revealed that D1 encodes GA 3-oxidase converting inactive GA intermediates to bioactive GA. As such, the D1 protein marks the sites where GA is potentially synthesized. Interestingly, the D1 protein was found to localize in the cytosol and nucleus, a dual-localization coinciding with the GA receptor. The same result was found for GA 20-oxidase catalyzing the upstream reaction. These results suggest that GA can be synthesized in the cytosol and nucleus. The D1 protein was highly and specifically expressed in the stamen primordia in the ear florets, but low in the whole tassel. Hence it is possible that low level of GA in the tassel is insufficient to suppress stamen development. As jasmonic acid (JA) plays antagonistic role to GA in the tassel florets, here we propose a model to explain this antagonism effect on the regulation of the stamen and pistil organ development in the tassel florets in maize.     

nolC, a Rhizobium fredii gene involved in cultivar-specific nodulation of soybean, shares homology with a heat-shock gene

Rhizobium fredii strain USDA257 does not nodulate soybean (Glycine max (L.) Merr.) cultivar McCall. Mutant 257DH5, which contains a Tn5 insert in the bacterial chromosome, forms nodules on this cultivar, but acetylene-reduction activity is absent. We have sequenced the region corresponding to the site of Tn5 insertion in this mutant and find that it lies within a 1176bp open reading frame that we designate nolC. nolC encodes a protein of deduced molecular weight 43564. Nucleotide sequences homologous to nolC are present in several other Rhizobium strains, as well as Agrobacterium tumefaciens, but not in Pseudomonas syringae pathovar glycinea. nolC lacks significant sequence homology with known genes that function in nodulation, but is 61% homologous to dnaJ, an Escherichia coli gene that encodes a 41 kDa heat-shock protein. Both R. fredii USDA257 and mutant 257DH5 produce heat-shock proteins of 78, 70, 22, and 16kDa. A 4.3kb EcoRI-HindIII subclone containing nolC expresses a single 43kDa polypeptide in mini-cells. A longer, 9.4kb EcoRI fragment expresses both the 43kDa polypeptide and a 78kDa polypeptide that corresponds in size to that of the largest heat-shock protein. Thus, although nolC has strong sequence homology to dnaJ and appears to be linked to another heat-shock gene, it does not directly function in the heat-shock response.     

A third genetic locus required for the formation of heterocysts in Anabaena sp. strain PCC 7120.

Mutagenesis of Anabaena sp. strain PCC 7120 with a derivative of transposon Tn5 led to the isolation of a mutant strain, P6, in which heterocysts are not formed (A. Ernst, T. Black, Y. Cai, J.-M. Panoff, D. N. Tiwari, and C. P. Wolk, J. Bacteriol. 174:6025-6032, 1992). Reconstruction of the transposon mutation of P6 in the wild-type strain reproduced the phenotype of the original mutant. Analysis by pulsed-field gel electrophoresis localized the transposition at ca. 3.44 Mb on the physical map of the chromosome of wild-type Anabaena sp. The transposon was situated within an open reading frame (ORF), which we denote hetP, whose wild-type form was cloned and also sequenced. The predicted HetP protein was not found to show significant sequence similarity to other proteins. The mutation in strain P6 could be complemented by a clone of a fragment of wild-type DNA that includes hetP and at least one additional ORF 3' from hetP, but not by a clone that includes hetP as its only ORF. The latter clone proved highly toxic. The phenotype of the P6 mutant may, therefore, be due to a polar effect of the insertion of the transposon. Filaments of strain P6 and of the wild-type strain, when bearing the complementing fragment on a pDU1-based plasmid, showed an increased frequency of clustered heterocysts compared with that of the wild-type strain.     

Characterization of a cDNA encoding a proline-rich 14 kDa protein in developing cortical cells of the roots of bean (Phaseolus vulgaris) seedlings

A cDNA clone, corresponding to mRNAs preferentially expressed in the roots of bean (Phaseolus vulgaris L.) seedlings, was isolated. This clone contains a 381 bp open reading frame encoding a polypeptide of 13.5 kDa, designated PVR5 (Phaseolus vulgaris root 5). The amino acid sequence of this clone is rich in proline (13.5%) and leucine (12.7%) and shares significant amino acid sequence homology with root-specific and proline-rich proteins from monocots (maize and rice), and proline-rich proteins from dicots (carrot, oilseed rape, and Madagascar periwinkle). The precise biological roles of these polypeptides are unknown. PVR5 mRNA accumulation is developmentally regulated within the root, with high levels at the root apex and declining levels at distances further from the root tip. In situ hybridization shows that PVR5 mRNA specifically accumulates in the cortical ground meristem in which maximal cell division occurs. Southern blot analysis suggests that genomic DNA corresponding to PVR5 cDNA is encoded by a single gene or a small gene family.