Dieter Bartling

Structural analysis of the nit2/nit1/nit3 gene cluster encoding nitrilases, enzymes catalyzing the terminal activation step in indole-acetic acid biosynthesis in Arabidopsis thaliana

A 13.8 kb DNA sequence containing the promoters and the structural genes of the Arabidopsis thaliana nit2/nit1/nit3 gene cluster has been isolated and characterized. The coding regions of nit2, nit1 and nit3 spanned 1.9, 1.8 and 2.1 kb, respectively. The architecture of the three genes is highly conserved. Each isoform consists of five exons separated by four introns. The introns are very similar with respect to size and position, but differ considerably in sequence composition. In contrast to the coding sequences the three promoters are very different in sequence, size and in their repertoire of cis elements, suggesting differential regulation of the three nitrilase isoenzymes by the developmental program of the plant and by diverse environmental factors. The nit1 promoter was subjected to analysis in planta. Translational fusions placing the nit1 full-length promoter and a series of 5′-deletion fragments in front of the uidA gene encoding β-glucuronidase (GUS) were used for Agrobacterium tumefaciens-mediated transformation of Nicotiana tabacum. GUS expression was highest in fully expanded leaves and in the shoot apex as well as in the apices of developing lateral buds, whereas the GUS activity displayed by developing younger leaflets was restricted to the tips of the expanding leaves. Within the root tissue GUS expression was restricted to the root tips and the tips of newly forming lateral roots. Structural features of the nitrilase gene family and nitrilase gene expression patterns are discussed in context with current knowledge of auxin biosynthesis and auxin effects on different tissues.

Molecular and immunological characterization of leucine aminopeptidase in Arabidopsis thaliana : a new antibody suggests a semi-constitutive regulation of a phylogenetically old enzyme

Plants of Arabidopis thaliana probably contain only a single leucine aminopeptidase (LAP) gene and are therefore good candidates for investigating the still unknown function of LAPs in plants. The cDNA clone PM25 [1], encoding a A. thaliana leucine aminopeptidase of 520 amino acids with a calculated molecular mass of 54 506 Da, was expressed in Escherichia coli with a N-terminal sequence extension as a protein of molecular mass 57 kDa. This polypeptide was purified and used to raise an anti-LAP antiserum. The antiserum recognized a single band of 54 kDa on nitrocellulose-blots following denaturating gel electrophoresis of A. thaliana protein. Zymogram analysis of the native enzyme using l-leucine β-napthylamide as substrate indicated a hexameric structure of 320 kDa for the active complex in plants. Immunoblot analysis of LAPs in several di- and monocotyledonous species indicated a subgroup of a phylogenetically old enzyme. LAP protein in A. thaliana can be detected at different developmental stages, in different organs and after treatment of plants with phytohormones or by wounding. Except for slight variations in the accumulation of LAP during development and organ specificity, the data indicate that LAP is present during all stages of development in A. thaliana.

Functional expression and molecular characterization of AtUBC2-1, a novel ubiquitin-conjugating enzyme (E2) from Arabidopsis thaliana

The first member of a novel subfamily of ubiquitin-conjugating E2-proteins was cloned from a cDNA library of Arabidopsis thaliana. Genomic blots indicate that this gene family (AtUBC2) consists of two members and is distinct from AtUBC1, the only other E2 enzyme known from this species to date (M.L. Sullivan and R.D. Vierstra, Proc. Natl. Acad. Sci. USA 86 (1989) 9861-9865). The cDNA sequence of AtUBC2-1 extends over 794 bp which would encode a protein of 161 amino acids and a calculated molecular mass of 18.25 kDa. The protein encoded by AtUBC2-1 is shown to accept 125I-ubiquitin from wheat E1 enzymes, when expressed from Escherichia coli hosts as fusion protein carrying N-terminal extensions. It is deubiquitinated in the presence of lysine and, by these criteria, is considered a functional E2 enzyme.

An Arabidopsis thaliana cDNA clone encoding a 17.6 kDa class II heat shock protein

High-temperature treatment of plants induces the synthesis of a set of proteins referred to as heat shock proteins (HSPs) ranging in size from 15 to 110 kDa. Several of the proteins induced by elevated temperatures are also synthesized in response to a variety of other stresses and some are present at normal environmental conditions [7]. These findings suggest a role of some HSPs or highly conserved close relatives in normal cell function. We have isolated and characterized a eDNA clone (Ats213) from poly(A) + mRNA from leaves ofArabidopsis thaliana grown at 21 °C (i.e. not heat-stressed) which shares high homology to the low-molecular-weight HSPs (LMW HSPs) encoded by the class VI HSP gene family, first described in soybean [5]. Members of this gene family are included in the class II HSP genes according to latest terminology [7]. The deduced sequence of 155 amino acids shows, in the Cterminal part of the protein, homology to the soybean HSP 17.9-D [5], maize Gmhsp 18.3 [2], pea Pshsp 17.7 [4], wheat Tshsp 17.3 [8] and maize MHSP 17.9 [1], all of them class VI (now class II) HSPs to which Ats 213 shows 65, 64, 66, 65 and 68~o, respectively, overall amino acid identity (Fig. 1). To indicate the homology and calculated molecular mass of 17622 Da, clone Ats 213 was renamed Athsp 17.6-II. The amino acid sequence of Athsp 17.6-II shows only 31.6 ~o homology to a 17.4 kDa HSP [6] and 44.3?o homology to a 17.6 kDa HSP [3] ofA. thaliana which both belong to the class I LMW HSPs [5]. Northern blot analysis indicates a strong increase of Athsp 17.6-II transcripts at 35 °C, compared to 21 °C (not shown). Our findings suggest that the protein encoded by Athsp 17.6-II, the first LMW HSP cloned from mRNA isolated from a non-heat-stressed plant, may have functions additional to heat stress related ones in the plant. To gain insights into the function, expression and intracellular location of this protein, we are now conducting protein expression and isolation studies with clone Athsp 17.6-II to raise monospecific antibodies.