The Regulation of Ethylene Biosynthesis
Hormones influence virtually every aspect of plant growth and development. The elucidation of the molecular mechanisms that control the biosynthesis and perception of these hormonal signals, and how these signals are integrated with each other and with other developmental and environmental cues remain fundamental goals in plant biology. The simple gas ethylene has been recognized as a plant hormone for almost a century (See: History of ethylene). It has been shown to influence a diverse array of plant growth and developmental processes, including germination, leaf and flower senescence and abscission, fruit ripening, nodulation and the response to a wide variety of stresses. In order to understand how ethylene or any signaling molecule affects development, one must consider how its biosynthesis is controlled, the molecular mechanisms governing its transport and perception, and finally, how the signal is removed from the target. The fact that ethylene is a gaseous compound under physiological conditions has clear implications for its transport both throughout the plant and across cellular membranes and also for its diffusion away from its site of action. Significant progress has been made in analyzing the molecular mechanisms underlying the ethylene signal transduction pathway. This project deals with an investigation of the remaining aspect, the regulation of ethylene biosynthesis. We have chosen Arabidopsis seedlings as a model system to begin to understand the circuitry underlying the regulation of ethylene biosynthesis.
Almost all plant tissues have the capacity to make ethylene, although in most cases the amount of ethylene produced is very low. Ethylene production increases dramatically during a number of developmental events such as germination, leaf and flower senescence and abscission, and fruit ripening. There is a diverse group of stimuli that can increase the level of ethylene biosynthesis, including a wide variety of stresses, light and the application of other plant hormones, such as auxins and cytokinins. We have focused on the induction of ethylene by cytokinin.
Biosynthetic pathway for ethylene. The ethylene biosynthetic pathway has been elucidated in a series of elegant studies by a number of laboratories. ACC synthase (ACS) catalyzes the conversion of AdoMet to 1-aminocyclopropane-1-carboxylic acid (ACC), which is the first committed and in most instances the rate-limiting step in ethylene biosynthesis.
ACS protein stability is highly regulated: Recent molecular genetic studies in Arabidopsis have provided compelling evidence that ACS protein stability is regulated and have converged on a common mechanism. Mutants that affect ethylene biosynthesis been identified using the triple response morphology as a screen (see below). these mutants fall into two classes: ethylene-overproducer (Eto) mutants display a constitutive ethylene response as etiolated seedlings due to elevated ethylene biosynthesis; and the cytokinin-insensitive (Cin) mutants make reduced levels of ethylene in response to cytokinin, a second phytohormone that increases ethylene biosynthesis in various plant tissues. The gene corresponding to the recessive cin5 mutation was the first of these mutants to be cloned, and the mutation was found to be the result of loss-of-function alleles in the ACS5 gene. This suggests that ACS5 is the primary target for cytokinin-induction of ethylene in etiolated seedlings. However, unlike other hormonal triggers such as auxin, which induce ethylene biosynthesis via increased ACS transcription, cytokinin regulates ACS5 function by increasing its protein stability.
Left: The triple response. Arabidopsis seedlings were grown for three days in the dark in the absence (left) or presence of 10 ppm ethylene (right). Right: Cartoon depicting Cin and Eto mutations identified in ACS5. Note the the cin5 mutations are recessive while the eto2 mutation is dominant.
Further evidence that ACS protein stability is regulated comes from the analysis of the Eto mutants. Three Eto mutants have been identified: eto1 is inherited as a recessive mutation and the eto2 and eto3 mutations are dominant. The eto2 mutation is the result of a single base pair insertion that is predicted to disrupt the C-terminal twelve amino acids of ACS5 (see above). The eto2 mutation increases the stability of the ACS5 protein. In a similar manner, the ethylene-overproducing phenotype of eto3 is the result of a single amino acid change, V457D, at the C-terminal of ACS9, which is the closest homolog of ACS5 in the Arabidopsis genome. This suggests that the C-terminal domains of ACS5 and ACS9 act to target the proteins for rapid degradation. This targeting domain interacts with the ETO1, which belongs to a class of proteins bridge a Cullin3 (CUL3)-based ubiquitin ligase to substrate proteins. Ubiquitin ligases transfer ubiquitin (Ub) moieties to specific substrate proteins, which targets them for degradation by the 26S proteasome.
Left: System for ACS protein analysis. ACS5 protein (WT or Eto) is expressed from a Dex-inducible promoter as a myc-epitope tag fusion protein in stable transgenic plants. Top is a cartoon of the construct used to express the fusion protein; Lower left is an analysis of the ethylene level (graph, in pl•seedling-1•3 days-1),in response to increasing Dex concentrations, with the inset showing the level of myc-ACS5 fusion protein. Right: The eto2 mutation results in an increase in ACS5 protein stability. WT and eto2 myc-ACS5 protein levels various times following inhibition of protein synthesis by cycloheximide. The top shows a Western blot and the bottom is a quantification derived from the Western.
Research in our lab curently focuses on unraveling the mechanism of the regulation of ACS protein stability. To these ends, we are exploring the role of protein phosphorylation in regulating ACS protein stability. We are examining if the stability of ACS protein increases during developmental events that are associated with a rise in ethylene biosynthesis or in response to exogenous cues such as light. We are screening for and subsequently cloning mutations that alter the ACS protein stability. Finally, we have used the yeast two-hybrid screen to identify proteins that directly interact with ACS, and have characterized the role of several of these in ACS protein turnover. These studies will shed light on the mechanism regulating the regulation of the stability of ACC synthase proteins and how this contributes to the control of the biosynthesis of ethylene.
Personnel: Joe Kieber, PI
Maureen Hansen, graduate student
Gyeong Mee Yoon, postdoctoral fellow
Publications:
Vogel,
J. P., Schuerman, P., Woeste, K., Brandstatter, I. and J. J. Kieber (1998) Isolation
and characterization of Arabidopsis mutants defective in the induction of ethylene
biosynthesis by cytokinin. Genetics 149, 417-427.
