H. Van Onckelen

Construction of a heat-inducible chimaeric gene to increase the cytokinin content in transgenic plant tissue

The ipt gene of Agrobacterium tumefaciens T‐DNA encodes an isopentenyltransferase which causes cytokinin overproduction and developmental alterations in transformed plants. A chimaeric gene constructed by positioning the ipt coding region under the control of the hsp70 gene from Drosophila melanogaster allows heat‐regulated expression in transgenic plant tissue. Heat‐shock treatment of tobacco calli transgenic for the chimaeric hsipt gene increases the endogenous cytokinin concentration and enables these calli to grow on cytokinin‐free medium. Transgenic plants regenerated from calli transformed with the hsipt gene and grown at normal temperature are phenotypically normal.

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.

Tobacco plants transformed with the Agrobacterium T-DNA gene 1 contain high amounts of indole-3-acetamide

The T‐DNA genes 1 and 2 of the Ti plasmid of Agrobacterium tumefaciens are involved in the biosynthesis of IAA in transformed plant cells. Previously, it has been shown that gene 2 codes for an amidohydrolase able to convert IAM into IAA. We have isolated Nicotiana tabacum regenerates transformed with either gene 1 or genes 6a and 6b of the T‐DNA. The tobacco plants transformed with gene 1 contain 500–1000‐times more IAM as compared to plants transformed with genes 6a and 6b, and as compared to untransformed control plants. No drastic differences in endogenous IAA concentrations were observed between the three plant types analyzed.

Isolation and assay of a cytokinin from barley

Abstract A specific method for the extraction and purification of cytokinins was tested with zeatin and zeatin riboside. For the quantitative determination of cytokinin activity, a bioassay is proposed based upon two different effects of cytokinins on barley during its germination. With the aid of these methods, a barley cytokinin, which resembles zeatin riboside, was isolated and quantitatively determined from 3-day-old barley seedlings.

La formation des isozymes de l' α-amylase durant la germination de l'orge

The α-amylase formed in germinating barley has been separated into six isozymes by means of polyacrylamide gel electrophoresis. These isozymes do not appear from the beginning of germination but are formed gradually so that after six days all six α-amylase isozymes are present.When gibberellic acid is added to the culture medium the production of the α-amylase isozymes is accelerated considerably, whereas the addition of kinetin has no influence at all on the formation of the α-amylase isozymes.The α-amylase induced by gibberellic acid in the aleurone layers of isolated barley endosperms apparently consists of five isozymes, a number that does not change upon further incubation.The action of phytohormones such as gibberellic acid and kinetin on the formation of α-amylase and its isozymes during the germination of barley is discussed.SummaryThe α-amylase formed in germinating barley has been separated into six isozymes by means of polyacrylamide gel electrophoresis. These isozymes do not appear from the beginning of germination but are formed gradually so that after six days all six α-amylase isozymes are present.When gibberellic acid is added to the culture medium the production of the α-amylase isozymes is accelerated considerably, whereas the addition of kinetin has no influence at all on the formation of the α-amylase isozymes.The α-amylase induced by gibberellic acid in the aleurone layers of isolated barley endosperms apparently consists of five isozymes, a number that does not change upon further incubation.The action of phytohormones such as gibberellic acid and kinetin on the formation of α-amylase and its isozymes during the germination of barley is discussed.

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.

Phytohormone-receptors from tobacco crown gall tissues

Plant cell and tissue cultures are useful tools for the study of plant hormone receptors as it is clearly illustrated by the work of the ‘Leiden-group’ on different aspects of auxin binding in tobacco callus tissue. In this contribution we will review some physiological and genetical aspects of the tobacco crown gall system, illustrating not only the possibilities offered by this model to investigate the early mode of action of phytohormones, but also the necessity to integrate the study of plant hormone receptors as an essential complementation of data on the endogenous phytohormonal levels.

La formation des isozymes de l' α-amylase durant la germination de l'orge@@@Formation of α-amylase isozymes during germination of barley

The α-amylase formed in germinating barley has been separated into six isozymes by means of polyacrylamide gel electrophoresis. These isozymes do not appear from the beginning of germination but are formed gradually so that after six days all six α-amylase isozymes are present.When gibberellic acid is added to the culture medium the production of the α-amylase isozymes is accelerated considerably, whereas the addition of kinetin has no influence at all on the formation of the α-amylase isozymes.The α-amylase induced by gibberellic acid in the aleurone layers of isolated barley endosperms apparently consists of five isozymes, a number that does not change upon further incubation.The action of phytohormones such as gibberellic acid and kinetin on the formation of α-amylase and its isozymes during the germination of barley is discussed.SummaryThe α-amylase formed in germinating barley has been separated into six isozymes by means of polyacrylamide gel electrophoresis. These isozymes do not appear from the beginning of germination but are formed gradually so that after six days all six α-amylase isozymes are present.When gibberellic acid is added to the culture medium the production of the α-amylase isozymes is accelerated considerably, whereas the addition of kinetin has no influence at all on the formation of the α-amylase isozymes.The α-amylase induced by gibberellic acid in the aleurone layers of isolated barley endosperms apparently consists of five isozymes, a number that does not change upon further incubation.The action of phytohormones such as gibberellic acid and kinetin on the formation of α-amylase and its isozymes during the germination of barley is discussed.