A full-length cDNA clone for a novel glutathione S-transferase was isolated from Arabidopsis thaliana and characterized. The cDNA encodes a polypeptide of 218 amino acids with a calculated molecular mass of 24363 Da. The sequence was most related to the theta class within the glutathione-S-transferase superfamily of enzymes. The protein encoded by the cDNA was functionally expressed and enzymically active in Escherichia coli ; glutathione-S-transferase activity with the standard enzyme substrate 1 -chloro-2,4-dinitrobenzene was demonstrated (apparent K,, 10 mM ; apparent K,, for glutathione, 0.08 mM). The enzyme is substrate specific and did not use several electrophilic reduced-glutathione acceptor molecules for conjugation. However, it efficiently catalyzed the conversion of 13-hydroperoxy-9,11,15-octadecatrienoic acid (Km, 0.67 mM) as well as 13-hydroperoxy-9,ll -0ctadecadienoic acid (K,,, 0.79 mM) to the corresponding hydroxy derivatives with concomitant formation of oxidized glutathione. The enzyme did not use H,O, as substrate. Thus, the cloned A. thaliana enzyme functions as glutathione peroxidase and, in the plant cell, may be involved in the removal of reactive organic hydroperoxides, such as the products of lipid peroxidation. The enzyme is structurally and enzymically, however, unrelated to the selenium-containing glutathione peroxidases. Enzymic and immunoblotting data suggest that the A. thaliana enzyme is soluble and constitutively expressed in vegetative rosettes, but is under developmental control during the transition to bolting and flowering.
From an Arahidopsis thuliana cDNA expression library, a cDNA clone was isolated, characterized and sequenced which, at the amino acid level, resembled the Klebsiellu ozuenue bromoxynil nitrilase encoded by the hxn gene. The cDNA contained a long open reading frame, starting from two possible neighbouring ATG codons and capable of encoding 340 or 346 amino acids with calculated molecular masses of 37526 Da or 381 76 Da, respectively. The sequence similarity between the deduced polypeptides from the Aruhidopsis cDNA and bxn was clustered in three domains, one at the C-terminus, one in the center and one near the N-terminus of the two proteins, suggesting important functional elements in these parts of the proteins. The cDNA was cloned into different vectors under the control of the lacZ promotor and was functionally expressed by induction with isopropyl-fi-u-thiogalactoside. Using a combination of high-performance liquid chromatography, monoclonal-antibody based enzyme-linked immunosorbent assay and mass spectroscopy, it was shown that the isolated cDNA clone encodes an enzymatically active nitrilase which is able to convert indole-3-acetonitrile to the plant growth hormone, indole-3-acetic-acid.Biogenic nitriles such as cyanogenic glycosides [l] occur widespread in plants or may be released in large quantities from the breakdown of glucosinolates [2] upon wounding or drying of plant tissues. Over 3000 cyanogenic, and thus potentially toxic, plant species, belonging to 110 families, are now known, including many economically important food plants [3]. In addition to their role as cyanogens, which help to protect plants from herbivors, nitriles are biosynthetic intermediates in plant metabolism. One of these, indole-3-acetonitrile, is particularly important as a presumed precursor for the plant hormone, indole-3-acetic acid.Indole-3-acetic acid has long been recognized as one of the major plant growth-promoting hormones, or auxins, involved in the regulation of nearly all aspects of plant life [4]. On a cellular basis, indole-3-acetic acid controls plant cell elongation [5]. At the plant level, the hormone promotes fruit growth [6], establishes apical dominance [7], induces lateral as well as adventitious root formation [8], inhibits fruit and kdf abscission [9] and, together with the cytokinins, controls the key morphogenetic process in all higher plants, the differentiation into root and shoot [lo]. These processes, to a large extent, also determinc plant productivity, and thus understanding indole-3-acetic acid biosynthesis, distribution and action has far-reaching consequences for the basic as well as applied plant sciences.Several pathways for the biosynthesis of indole-3-acetic acid in higher plants have been discussed (see 11 11, for a recent review), but there is considerable uncertainty as to their relative importance. In the majority of plants, indole-3-pyruvate, originating from tryptophan, is the presumed key intermediate in the biosynthesis of indole-3-acetic-acid 1121. In the Rrussicaceue, indole-3-ac...
As in maize [Wright, A. D (5) and, recently, for Arabidopsis thaliana (6), are not yet known. A. thaliana lends itself particularly to the study of IAA biosynthesis because of the availability of several mutants in the tryptophan biosynthetic pathway (6-8). From precursor feeding experiments using the A. thaliana trp2-1 (tryptophan synthase (3 deficient) and trp3-1 (tryptophan synthase a deficient) mutants, Normanly et al. (6) showed recently that anthranilate, but not L-tryptophan, was a major precursor to IAA. The pool of endogenous indole-3-acetonitrile (IAN) as well as that of IAA was increased in these mutants, and IAN carried label from anthranilate, as expected for the IAA precursor (6). Minor contributions to the pool of IAA from tryptophan via the indoleacetaldoxime pathway proposed earlier (9) could not be excluded completely in this study (6). IAN is also a proposed intermediate in this pathway. A third route to IAA may lead from myrosinase-catalyzed degradation ofindole-3-methyl glucosinolate (glucobrassicin) via IAN to the auxin, but this pathway may occur only at specific stages in plant development (10). Glucobrassicin is a major glucosinolate in A. thaliana, especially in the seeds (11).These data suggest multiple pathways to IAA in A. thaliana, all involving IAN as the direct auxin precursor. Nitrilase (nitrile aminohydrolase, EC 3.5.5.1) must thus be regarded as the key enzyme in the biosynthesis ofIAA in this species. Nitrilase I has been cloned in our laboratory (12), but Southern hybridizations showed the presence of a second nitrilase gene in this plant. We have now cloned and functionally expressed a cDNA encoding this second enzyme, and we show that the two nitrilases, while similar in their enzymatic properties, are localized in different intracellular compartments and that their expression is differentially regulated during plant development.
The primary structure of thioredoxin f from spinach chloroplasts was determined by standard amino acid sequencing and furthermore by sequencing the corresponding nuclear genome region. The protein, with a calculated molecular mass of 12564 Da and a molar absorption coefficient at 280 nm of 17700 M−1 cm−1, consists of 113 residues and exhibits 24% residue identities with spinach chloroplast thioredoxin mb or Escherichia coli thioredoxin. A monospecific antibody elicited against thioredoxin f has been used to select recombinant phage from spinach cDNA libraries in λgt11. The inserts of positive clones were sequenced. They code for a polypeptide of 190 amino acids, composed of the thioredoxin f sequence (113 residues) and an upstream element (77 residues) which most probably consitutes the N‐terminal transit peptide that directs the polypeptide into chloroplasts. In vitro transcription and translation of this construct generates a polypeptide of approximately 21 kDa, which is imported by isolated spinach chloroplasts and processed to the mature 12.5‐kDa protein.
Summary Nitrilase (E.C. 3.5.5.1) cloned from Arabidopsis thaliana converts indole‐3‐acetonitrile to the plant growth hormone, indole‐3‐acetic acid in vitro. To probe the capacity of this enzyme under physiological conditions in vivo, the cDNA PM255, encoding nitrilase II, was stably integrated into the genome of Nicotiana tabacum by direct protoplast transformation under the control of the CaMV‐35S promotor. The regenerated plants appeared phenotypically normal. Nitrilase II was expressed, based on the occurrence of its mRNA and polypeptide. The enzyme was catalytically active, when extracted from leaf tissue of transgenic plants (specific activity: 25 fkat mg−1 protein with indole3‐acetonitrile as substrate). This level of activity was lower than that found in A. thaliana, and this was deemed essential for the in vivo analysis. Leaf tissue from the transgenic plants converted 1‐[13C]‐indole‐3‐acetonitrile to 1‐[13C]‐indole‐3‐acetic acid in vivo as determined by HPLC/ GC‐MS analysis. Untransformed tobacco was unable to catalyze this reaction. When transgenic seeds were grown on medium in the absence of indole‐3‐acetonitrile, germination and seedling growth appeared normal. In the presence of micromolar levels of exogenous indole‐3‐acetonitrile, a strong auxin‐overproducing phenotype developed resulting in increased lateral root formation (at 10 µM indole‐3‐acetonitrile) or stunted shoot growth, excessive lateral root initiation, inhibition of root out‐growth and callus formation at the root/shoot interface (at 100 µM indole‐3‐acetonitrile). Collectively, these data prove the ability of nitrilase II to convert low micromolar levels of indole‐3‐acetonitrile to indole‐3‐acetic acid in vivo, even when expressed at subphysiological levels thereby conferring a high‐auxin phenotype upon transgenic plants. Thus, the A. thaliana nitrilase activity, which exceeds that of the transgenic plants, would be sufficient to meet the requirements for auxin biosynthesis in vivo.
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