E. Prinsen

Agrobacterium T-DNA gene 1 codes for tryptophan 2-monooxygenase activity in tobacco crown gall cells

Cloned tobacco crown gall tissue induced by the Agrobacterium tumefaciens C58 T‐DNA mutant pGV3132, defective for the T‐DNA‐encoded amihydrolase (iaaH), accumulates about 1000‐times more indole‐3‐acet‐amide (IAM) when compared to untransformed tobacco callus and crown gall tissue induced by a T‐DNA mutant defective for gene 1. In vitro experiments demonstrated that this IAM accumulation is correlated with the active conversion of Trp into IAM. The results presented in this report provide biochemical evidence that the T‐DNA gene 1 encodes a tryptophan 2‐monooxygenase (iaaM) activity in transformed plant cells. This gene cooperates with the gene 2‐encoded amidohydrolase (iaaH) in the T‐DNA‐controlled indole‐3‐acetic acid (IAA) biosynthesis pathway in crown gall cells.

Anther-specific expression of the rolB gene of Agrobacterium rhizogenes increases IAA content in anthers and alters anther development and whole flower growth

SummaryThe promoter activity of a 2.2-kb DNA fragment from the 5′ flanking sequence of the tap1 gene of snapdragon has been characterised in tobacco with the β-glucuronidase reporter gene. The tap1 promoter conferred to the reporter gene an expression that was limited to the early stages of anther development. Expression of the rolB gene of Agrobacterium rhizogenes under the control of the tap1 promoter impaired the development of tobacco flowers. Tobacco plants transgenic for the tap1-rolB chimeric gene showed altered anthers and a reduction in whole flower growth. Such growth alterations were correlated with an increase in free IAA content and a decrease in gibberellin activity present in anthers. Since the rolB gene codes for an indole β-glucosidase (Estruch et al. 1991), we interpret the phenotypic alterations to be a consequence of the increased content and activity of auxin in anthers. The perturbation of anther development would in turn affect gibberellin production and content, which is reflected in a reduced elongation of flowers.

Transcriptional Analysis of the Azospirillum brasilense Indole-3-Pyruvate Decarboxylase Gene and Identification of a cis-Acting Sequence Involved in Auxin Responsive Expression

Expression of the Azospirillum brasilense ipdC gene, encoding an indole-3-pyruvate decarboxylase, a key enzyme in the production of indole-3-acetic acid (IAA) in this bacterium, is upregulated by IAA. Here, we demonstrate that the ipdC gene is the promoter proximal gene in a bicistronic operon. Database searches revealed that the second gene of this operon, named iaaC, is well conserved evolutionarily and that the encoded protein is homologous to the Escherichia coli protein SCRP-27A, the zebrafish protein ES1, and the human protein KNP-I/GT335 (HES1), all of unknown function and belonging to the DJ-1/PfpI superfamily. In addition to this operon structure, iaaC is also transcribed monocistronically. Mutation analysis of the latter gene indicated that the encoded protein is involved in controlling IAA biosynthesis but not ipdC expression. Besides being upregulated by IAA, expression of the ipdC-iaaC operon is pH dependent and maximal at acidic pH. The ipdC promoter was studied using a combination of deletion analyses and site-directed mutagenesis. A dyadic sequence (ATTGTTTC(GAAT)GAAACAAT), centered at -48 was demonstrated to be responsible for the IAA inducibility. This bacterial auxin-responsive element does not control the pH-dependent expression of ipdC-iaaC.

The ORF8 gene product of Agrobacterium rhizogenes TL-DNA has tryptophan 2-monooxygenase activity.

The open reading frame 8 (ORF8) is located on the TL-DNA of the phytopathogenic soil bacterium Agrobacterium rhizogenes strain A4. The predicted ORF8 protein has a particular structure and is possibly a natural fusion protein. The N-terminal domain shows homology to the A. rhizogenes rolB protein and may modulate the auxin responsiveness of host cells. The C terminus has up to 38% homology to tryptophan 2-monooxygenases (t2m). We show that ORF8 overexpressing plants contain a fivefold higher concentration of indole-3-acetamide (IAM) than untransformed plants. Protein extracts from seedlings and Escherichia coli overexpressing ORF8 show significantly higher turnover rates of tryptophan to IAM than negative controls. We conclude that the ORF8 gene product has tryptophan 2-monooxygenase activity.

Diurnal fluctuations of endogenous IAA content in aralia leaves

Diurnal variations in endogenous IAA levels inFatsia japonica leaves, maintaining constant other external factors such as temperature and relative humidity, were studied. Plants were cultivated in a growth chamber (20 °C, 75 % RH, 16 h photoperiod, 400 µmol m-2 s-1 PAR). IAA analyses were carried out by analytical IP-HPLC with on-line spectrofluorimetry. Rhythmic variation of endogenous IAA levels was found. At the onset of the light period the IAA concentration dropped very rapidly from 1070 pmol g-1 (fr.m.) to 144 pmol g-1 (fr.m.). This concentration was nearly constant throughout the entire light period. During the subsequent dark period the IAA levels increased again to about 1000 pmol g-1 (fr.m.) at the end of the dark phase. These results were not confirmed in open field conditions where many other external factors probably influence the endogenous IAA content.

The Pseudomonas savastanoi tryptophan-2-mono-oxygenase is biologically active in Nicotiana tabacum

It has been proposed that the “eukaryotic” T-DNA-encoded indole-3-acetic acid (IAA) biosynthesis genes of Agrobacterium tumefaciens and their prokaryotic counterpart in Pseudomonas savastanoi originated from common ancestor genes. This paper provides additional evidence for the functional similarity between the gene products. We have demonstrated that a chimeric gene consisting of the coding sequence of the P. savastanoi tryptophan-2-mono-oxygenase (iaaM gene) and a plant promoter encodes an active enzyme in Nicotiana tabacum. Transformants obtained with this chimeric gene grew as a callus on hormone-free media. No stably transformed plantlets could be isolated. The callus tissues contained extremely high levels of indole-3-acetamide and slightly elevated levels of IAA. Either indole-3-acetamide by itself has a low auxin activity or, alternatively, it is converted aspecifically and at low rates into IAA. The P. savastanoi tryptophan-2-mono-oxygenase activity in plants is also able to detoxify the amino-acid analogue 5-methyltryptophan. This property can be used for positive selection of transformed calli.

Azospirillum-Plant Root Associations: Genetics of IAA Biosynthesis and Plant Cell Wall Degradation

The genus Azospirillum comprises free-living N2 fixing rhizosphere bacteria that have been isolated from different soil types and from the roots of numerous wild and cultivated plants all over the world. Field trials, carried out at different locations, have demonstrated that under certain environmental and soil conditions, inoculation with Azospirillum has beneficial effects on plant yields. Bacterial phytohormone biosynthesis has often been proposed as being responsible for the observed plant growth promotion upon Azospirillum inoculation.

Azospirillum-Cereals: An Intriguing Partnership

Azospirillum is probably the best studied example of beneficial plant rhizosphere bacteria. Studies in our laboratory focus on the identification of bacterial genes and gene products that are of importance in the physical and metabolic interaction of Azospirillum brasilense with plant roots. Here we report for Azospirillum brasilense, flagellation, motility, the physical interaction with plant roots, the synthesis of indole-3-acetic acid, the expression of nif genes in plant-root associated bacteria, and the induction of gene expression with plant root exudates.

Transgenic plants and transgenic plant mosaics for the expression of pathogen derived genes able to affect phytohormone activity

Plant development is greatly influenced by several plant growth regulators including two major classes, cytokinins and auxins. Most experimental systems studied to date with respect to hormonal effects on plant development make use of exogenously applied hormones, and consequently contain uncontrolled variables related to uptake and transport of these compounds. Plant genetic engineering allows us to circumvent this problem by constructing plants transgenic for genes able to modify phytohormone content and activity.