Research Interests: I. Molecular mechanism in the regulation of ethylene biosynthesis and signalling
II. Chemical genetics to study the functional role of plant hormones in growth and development
The long-term goal of my research is to understand the physiological role and regulatory mechanism of phytohormones in plant growth and development. We are particularly interested in plant hormones responding to environmental signals for adaptation and survival. Our current research focus is to study the molecular mechanism in regulation of ethylene biosynthesis and signaling in Arabidopsis thaliana. In spite of its simple structure, ethylene has been shown to play a crucial role in various stages of the plant life cycle, from seed germination, flower development, fruit ripening, and the induction of flower senescence and leaf abscission. Moreover, by interaction with other "stress" hormones, such as salicylic acid (SA), jasmonates (JA), and abscisic acid (ABA), ethylene actively participates in the signaling networks involved in plant responses to a wide range of biotic and abiotic stresses. Genetic and biochemical approaches have been successfully used to uncover the key components of ethylene pathway over the last two decades, and a nearly linear signaling pathway starting from membrane receptors to nuclear transcriptional factors has been established based on studies primarily in the reference plant, Arabidopsis thaliana.
I. Molecular mechanism in the regulation of ethylene biosynthesis and signalling
The biosynthetic pathway of ethylene has been studied for years, however, it is still not clear exactly how ethylene biosynthesis is regulated at the molecular level. My previous work has uncovered a molecular mechanism in the regulation of ethylene biosynthesis in plants. Mutations in ETHYLENE-OVERPRODUCER1-3 (ETO1-ETO3) result in an overproduction of ethylene in Arabidopsis thaliana. The recessive nature of all eto1 mutations indicates that ETO1 encodes a negative regulator of ethylene biosynthesis. On the other hand, the dominant mutations in eto2-1 and eto3-1 were mapped to two closely related genes encoding ACC synthases (1-aminocyclopropane-1-carboxylate synthase, ACS), ACS5/ETO2 and ACS9/ETO3, respectively. There are nine functional ACS enzymes in Arabidopsis thaliana. ETO1 interacts specifically with type II ACS, which includes ACS4, ACS5, ACS8, and ACS9. The ACS5eto2-1 and ACS9eto3-1 are mutated proteins that no longer interact with ETO1 and therefore, escape from the negative regulation by ETO1. Because ACS enzymes catalyze the rate-limiting step of ethylene biosynthesis pathway in plants, ETO1 appears to be the first component identified as a key regulator in ethylene evolution.
ETO1 represents a member of a novel gene family unique in plants with BTB (Broad-Complex, Tramtrack, and Bric a brac) and TPR (Tetratricopeptide Repeat) domains, which both have been implicated in protein-protein interactions. The expression sequence tags (EST) of ETO1-Like homologs (EOL) are present in different species, suggesting a common regulatory mechanism is conserved in the plant kingdom. ETO1 interacts directly with ACS5 and suppresses its enzymatic activity. In addition, ETO1 promotes ACS5 degradation in planta, which depends on the ubiquitin-mediated proteolysis. These results indicate that ETO1 functions as a negative regulator of type II ACS (represented by the ACS5) by a dual mechanism, a direct inhibition of enzyme activity and the promotion of protein degradation (Fig. 1B). Further investigation reveals that ETO1 is a novel adaptor in the Culllin 3 (CUL3)-based SCF (Skp1-Cullin-F box) ubiquitin E3 ligase. The BTB domain of ETO1 interacts specifically with Arabidopsis CUL3, but not CUL1, while the TPR domain interacts with ACS5 and presumably, other type II ACC synthases. A proposed structure of SCFETO1 associated with ACS5 is superimposed on a hypothetic CUL3-BTB complex shown in Figure 1A. We are currently using molecular genetics, genomics, and proteomics tools for research aimed to answer specific questions regarding to the molecular mechanism of how members of ETO1 family regulate ethylene evolution in responding to developmental signals and environmental stimuli in Arabidopsis thaliana (see model in Fig. 1B)
Figure 1. A model for the regulation of ethylene biosynthesis by the SCFETO1 ubiquitin E3 ligase complex
(A) The proposed SCFETO1 complex associated with ACS5 was superimposed on a hypothetic model for CUL3-BTB ubiquitin E3 ligase complex (Stogios, et al., Genome Biology 2005, 6:R82). (B) ETO1 interacts with ACS5 and inhibits enzyme activity (inactivation mechanism, Low C2H4). ACS5 is also subjected to the ubiquitin/proteasome-dependent protein degradation via the interaction with a CUL3-based SCFETO1 ubiquitin E3 ligase (degradation mechanism, No C2H4). Note that ACS5 forms a homodimer with two catalytic sites shared by the two monomers in the current model. The red dots in the active sites indicate the essential co-factor, pyridoxal-5'-phosphate.
II. Chemical genetics to study the functional role of plant hormones in growth and development
We are also interested in the hormone interaction network of ethylene in plants. To further identify new components in the functional interaction between ethylene and other phytohormones, we have utilized the chemical genetics approach to explore the possibilities. The concept of chemical genetics is similar to what pharmaceutical companies have been doing for drug screening on specific targets related to diseases. Because of the availability of chemical compounds with highly diverse structures, an increasing number of researchers have used collections of drug-like small molecules to probe various biological processes, including those in plant biology. By using chemical compounds, we can circumvent the difficulty of conventional mutagenesis methods to acquire null phenotypes by inactivating a group of proteins with functional redundancy. Moreover, the effect of small molecule compounds is reversible that it solves the issue of cell lethality from knocking out essential genes. Finally, small molecule compounds are cell permeable and therefore, have the potential utility to apply on non-transgenic plants.
We have screened a collection of 10,000 small molecules chemical compounds with unspecified structures to identify modulators that interfere with ethylene response. We used three-day old etiolated seedlings of eto1-4 plants as the material for phenotype-based screening. In our initial screening, we have identified potential compounds that suppressed the triple response of eto1-4, exemplified by the compound 19 in Figure 2A. We also found compounds that affected only part of the triple response in root (5, 8, and 14) or in hook (20) (Fig. 2A). In addition, we found compounds that resulted in anthocyanin accumulation (Fig. 2B, 2C, and 2E) or viable albino seedlings (Fig.2D). We also have identified several compounds that generate a delayed germination phenotype of light-grown seedlings (Fig. 2H). With many unexpected phenotypes identified from our small molecule compound screening originally aimed to study the regulation of ethylene response, the chemical genetics strategy holds the potential to be a promising tool to add new information in hormone interaction network and to dissect complex physiological program in plant development. We are currently combining both genetics and biochemistry approaches to characterize the biological property of hit compounds and to identify the cellular targets in Arabidopsis thaliana.
Figure 2. Examples of using small molecule compounds to interrogate ethylene biology and plant development in Arabidopsis thaliana
(A) Representative results from screening with a collection of 10,000 small molecule compounds in etiolated eto1-4 seedlings to score for suppression of triple response. Etiolated seedlings (eto1-4) in the absence of chemical compound (1, with 0.5% DMSO) and in the presence of 10 µm of STS (★, silver thiosulfate), an antagonist of ethylene receptor, were used as the controls. Compound #19 is a representative that shows suppression of eto1-4 phenotype comparable with the effect of STS. While some candidates elicit visible phenotypes in root development (5, 8, and 14), some causes a hookless phenotype (20). Other chemical induced phenotypes that are not obviously related to ethylene response were also scored, such as partial (B) and strong (C) accumulation of anthocyanin in cotyledons, viable albino in cotyledons (D), and pigmentation near the junction of hypocotyl and root (E). Compounds that result in delayed germination of light-grown seedlings were also identified (H). Wild type (Columbia) seedlings in the absence (G) or presence (F) of 1 µm of ABA were used as controls.
Chilley, P.M., Casson, S.A., Tarkowski, P., Wang, K. L.-C., Hawkins, N., Hussey, P.J., Beale, M., Ecker, J.R., Sandberg, G.K., and Lindsey, K. (2006) The POLARIS peptide of Arabidopsis regulates auxin transport and root growth via effects on ethylene signaling. Plant Cell, 18(11): 3058-72.
Yoshida, H., Wang, K. L.-C., Chang, C.-M., Mori, K., Uchida, E., and Ecker, J.R. (2006) The ACC synthase TOE sequence is required for interaction with ETO1 family proteins and destabilization of target proteins. Plant Molecular Biology, 62(3): 427-437.
Yoshida, H., Nagata, M., Saito, K., Wang, K. L.-C., and Ecker, J.R. (2005) Arabidopsis ETO1 specifically interacts with and negatively regulates type 2 1-aminocyclopropane-1-carboxylate synthases. BMC Plant Biology, 5:14.
Wang, K. L.-C., Yoshida, H., Lurin, C., and Ecker, J.R. (2004) Regulation of ethylene gas biosynthesis by the Arabidopsis ETO1 protein. Nature, 428 (6986): 945-950.
Wang, K. L.-C., Li, H., and Ecker, J.R. (2002) Ethylene biosynthesis and signaling networks. Plant Cell, 14:S131-151.
My research is to investigate the regulatory mechanism of leaf senescence. I am currently studying the function role of a novel senescence-associated gene, AtA15, responding to oxidative stress in Arabidopsis thaliana.
My research aims to figure out what are the components involved in differentially regulated protein degradation by proteasome in phytohormone signaling pathways by proteomics profiling.
Chia-Ping (Rita) LIU, 劉佳苹
likepigno@yahoo.com.tw
Master student, National Central University
Co-advisor: Dr. Shaw-Jye WU, 吳少傑博士
My research is to use activation T-DNA tagging approach to identify suppressors of an ethylene overproducing mutant, eto1, in Arabidopsis thaliana.
Jung-Yun CHUANG, 莊鎔昀
littlebu823@hotmail.com
Master student, National Central University
Co-advisor: Dr. Shaw-Jye WU, 吳少傑博士
I am working on a project to apply an alcohol inducible gene expression system to conditionally regulate ethylene response in plants. The aim of this project is to improve the postharvest quality of crop plants.
I am working on a project to study the role of ADP ribosylation in mitochondria and nucleus responding to stress conditions in budding yeasts and Arabidopsis thaliana. This is a collaborative project with Dr. Wan-Sheng Lo at IPMB.
My project is to use molecular biology and genetic tools to characterize the function of AURORA kinase gene family in budding yeasts and Arabidopsis thaliana. This is a collaborative project with Dr. Wan-Sheng Lo at IPMB.
The long-term goal of my research is to understand the physiological role and regulatory mechanism of phytohormones in plant growth and development. We are particularly interested in plant hormones responding to environmental signals for adaptation and survival. Our current research focus is to study the molecular mechanism in regulation of ethylene biosynthesis and signaling in Arabidopsis thaliana. In spite of its simple structure, ethylene has been shown to play a crucial role in various stages of the plant life cycle, from seed germination, flower development, fruit ripening, and the induction of flower senescence and leaf abscission. Moreover, by interaction with other "stress" hormones, such as salicylic acid (SA), jasmonates (JA), and abscisic acid (ABA), ethylene actively participates in the signaling networks involved in plant responses to a wide range of biotic and abiotic stresses. Genetic and biochemical approaches have been successfully used to uncover the key components of ethylene pathway over the last two decades, and a nearly linear signaling pathway starting from membrane receptors to nuclear transcriptional factors has been established based on studies primarily in the reference plant, Arabidopsis thaliana.
