Joachim Feldwisch

T-DNA gene 5 of Agrobacterium modulates auxin response by autoregulated synthesis of a growth hormone antagonist in plants.

Oncogenes carried by the transferred DNA (T‐DNA) of Agrobacterium Ti plasmids encode the synthesis of plant growth factors, auxin and cytokinin, and induce tumour development in plants. Other T‐DNA genes regulate the tumorous growth in ways that are not yet understood. To determine the function of T‐DNA gene 5, its coding region was expressed in Escherichia coli. Synthesis of the gene 5 encoded protein (26 kDa) correlated with a 28‐fold increase in conversion of tryptophan to indole‐3‐lactate (ILA), an auxin analogue. Expression of chimeric gene 5 constructs in transgenic tobacco resulted in overproduction of ILA that enhanced shoot formation in undifferentiated tissues and increased the tolerance of germinating seedlings to the inhibitory effect of externally supplied auxin. Promoter analysis of gene 5 in plants revealed that its expression was inducible by auxin and confined to the vascular phloem cells. cis‐regulatory elements required for auxin regulation and phloem specific expression of gene 5 were mapped to a 90 bp promoter region that carried DNA sequence motifs common to several auxin induced plant promoters, as well as a binding site for a nuclear factor, Ax‐1. ILA was found to inhibit the auxin induction of the gene 5 promoter and to compete with indole‐3‐acetic acid (IAA) for in vitro binding to purified cellular auxin binding proteins. It is suggested therefore that ILA autoregulates its own synthesis and thereby modulates a number of auxin responses in plants.

Hormonal modulation of plant growth: the role of auxin perception

The organisation of growth and development in vascular plants appears to be highly adapted to meet the specific demands of a sessile, autotrophic habit. Many of the characteristic features of plant development are associated with the activities of five groups of phytohormones. Each of the phytohormones has the ability to influence fundamentally a remarkable variety of developmental and physiological processes. This ability has been widely documented but remains to be explained. Here we describe how recent breakthroughs in the analysis and understanding of eucaryotic signal transduction are being applied, in conjunction with technical advances in molecular genetics, to elucidate the molecular basis of the phytohormonal properties of auxin. Both auxin concentration, and the sensitivity of plant cells to this phytohormone have been implicated as important parameters in auxin action. We describe recent molecular biological approaches to assess the contribution made by each of these parameters. Emphasis is given to a description of recent genetic and biochemical progress towards identification of the molecular targets of the auxin signal and the molecular components involved in its subsequent transduction.

Auxin binding proteins are located in the ER and in the plasma membrane: Identification by photoaffinity-labelling and characterization

Plants modify gene expression and metabolism in response to a large variety of exogenous and endogenous signals. Among the various signals sensed by plants the phytohormones e.g. auxins, cytokinins, ethylene, abscisic acid and gibberellins, have received particular attention. It has been argued that, similar to hormone action in `vertebrates’ the first step in phytohormone action is the interaction of a ligand with binding sites, most likely proteins, located either at the plasma membrane or at various other intracellular locations. It is thought that binding of phytohormones to such proteins should be specific, reversible, of high affinity and saturable and result in a defined biological response.

Signalling elements in higher plants: identification and molecular analysis of an auxin-binding protein, GTP-binding regulatory proteins and calcium sensitive proteins

In eucaryotic cells external signals detected by receptors are translated into a limited repertoire of intracellular second messengers. Occupancy of these receptors initiates the production of active messengers, including the well studied cyclic adenosine monophosphate (cAMP) as well as the recently discovered messenger molecules that are derived from phosphoinositides such as arachidonic acid, inositol-l,4,5-triphosphate, and 1,2-diacylglycerol (for review see: Berridge 1986; Newton and Brown 1986; Boss and Morre 1989). These messengers are capable of regulating a vast array of physiological and biochemical processes either by direct interaction with distinct proteins or indirectly by activating enzymes which trigger conformational changes in the final target proteins. However, the number of second messengers in eucaryotic cells appears to be surprisingly small, indicating that most probably only a limited number of internal signal pathways are needed, albeit remarkably universally in all eucaryotes analyzed up to now, to transduce these signals to their final biological destination.

Photoaffinity labeling and strategies for plasma membrane protein purification.

Publisher Summary This chapter discusses the photoaffinity labeling and strategies for plasma-membrane-protein purification. For the purification of membrane proteins solubilized with detergents, the same methods can be applied as those used to purify soluble proteins; these include gel filtration, ion-exchange, reversed-phase, and affinity chromatography. Auxins play a major role in the regulation of plant cell elongation, cell division, morphogenesis, and differentiation. The molecular sequence of events responsible for the mode of action of auxins is not well understood. Receptor-like proteins that bind auxin and transmit the auxin signal have been postulated. Because of the low concentration of most of these proteins, conventional approaches to identify and characterize them for molecular studies have been fraught with technical difficulties. To overcome problems associated with traditional auxin-binding studies, photolabile synthetic auxins may provide a valuable tool with which to tag auxin-binding proteins. Photoaffinity probes have contributed greatly to the identification of receptor proteins and have aided in structural studies.