Ethylene Lab Report
The gaseous hormone ethylene has been shown to influence a diverse array of plant growth and developmental processes, thus it is critical for plants to optimize the ethylene production at developmental transitions as well as during stress response. Ethylene is derived from the amino acid methionine, which in the first step is converted to S-adenosyl-methionine (AdoMet) by AdoMet synthetase3,4. The 1-Aminocyclopropane-1-Carboxylic acid Synthase (ACS) catalyzes the conversion of AdoMet to 1-aminocyclopropane-1-carboxylic acid (ACC)5, which is the rate-limiting step in ethylene biosynthesis. ACC is then converted to ethylene by ACC oxidase (ACO), a member of the oxygenase/oxidase superfamily of enzymes6.The ACS plays a central role to regulate ethylene production through changes in ACS gene expression levels and the stability/activity of the enzyme. ACS proteins can be divided into 3 groups (type-1, 2 & 3) based on their C-terminal domain and the presence of phosphorylation sites. Type-1 and -2 ACS isoforms contain phosphorylation sites for kinases, but type-3 lacks of the phosphorylation sites.
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Interestingly, type-2 ACS has a unique regulatory motif called Target of ETO1 which serves as a binding site for Ethylene Overproducer 1 (ETO1) E3 ligase. ETO1 and its two paralogs ETO1-like 1 and 2 (EOL1 & EOL2) play a role in the ubiquitination of type-2 ACS isoforms via the 26S proteasome.
The protein stability of ACS is regulated by phosphorylation.
Pathogen-activated Mitogen Activated Protein Kinase 6 phosphorylates type-1 ACS (ACS2 & ACS6), which lead to increased accumulation of these ACS isoforms and, hence, increased ethylene production 20. A Casein Kinase 1 phosphorylates type-2 ACS5, resulting in the degradation of ACS5 protein 11. Our previous studies also suggested that phosphorylation is likely involved in ACS stabilization. We demonstrated that 14-3-3 is a novel regulatory element in ethylene biosynthesis21. 14-3-3 proteins are evolutionally well-conserved proteins that interact specifically with phosphorylated proteins to regulate their localization, stability, or activity21,23. 14-3-3 proteins interact with all three types of ACS and, surprisingly, with ETO1/EOL as well, and as results, control the protein turnover of both ACS and
ETO1/EOL21.
Ethylene biosynthesis is regulated by multiple stimuli including developmental/environmental inputs or other phytohormone. Among several phytohormone regulating ethylene biosynthesis, BR and cytokinin regulate the ethylene biosynthesis by stabilizing ACS proteins at the post-translational levels, yet our understanding in the mechanism underlying the crosstalk between the hormones to control the stability of ACS remain rudimentary. Recently, our lab has identified a SINA as a novel molecular component regulating ethylene biosynthesis in a BR-dependent manner. The SINA proteins is a family of E3 ligases that contains a RING domain and was first identified in Drosophila melanogaster and subsequently identified in mammals and plants. Mammalian and vertebrate SINA proteins function in a wide range of cellular processes including photoreceptor development, tumor progression, and responses to hypoxia and stress27-30. However, the role of SINA in plants has just begun to be understood. Plant SINA families consist of highly conserved proteins with 5 and 6 members in Arabidopsis thaliana and rice, respectively, and 10 in poplar. Our preliminary results suggested that Arabidopsis SINA1 and its closest homolog SINA2 play a positive role in BR-mediated ethylene biosynthesis, likely via downregulating ETO1/EOL and through 14-3-3-regulated SINA function (Fig.1). Interestingly, a previous 14-3-3 tandem affinity purification study has demonstrated that SINA, ETO1/EOL, and ACS form a complex with 14-3-3 in vivo, implying these proteins could have a functional relationship. Here we propose a series of experiments to delineate the mechanism by which SINA regulates ethylene biosynthesis in response to BR. We will also test the hypothesis that 14-3-3 controls the SINA function in a BR-dependent manner.
Interestingly, type-2 ACS has a unique regulatory motif called Target of ETO1 which serves as a binding site for Ethylene Overproducer 1 (ETO1) E3 ligase. ETO1 and its two paralogs ETO1-like 1 and 2 (EOL1 & EOL2) play a role in the ubiquitination of type-2 ACS isoforms via the 26S proteasome.
The protein stability of ACS is regulated by phosphorylation.
Pathogen-activated Mitogen Activated Protein Kinase 6 phosphorylates type-1 ACS (ACS2 & ACS6), which lead to increased accumulation of these ACS isoforms and, hence, increased ethylene production 20. A Casein Kinase 1 phosphorylates type-2 ACS5, resulting in the degradation of ACS5 protein 11. Our previous studies also suggested that phosphorylation is likely involved in ACS stabilization. We demonstrated that 14-3-3 is a novel regulatory element in ethylene biosynthesis21. 14-3-3 proteins are evolutionally well-conserved proteins that interact specifically with phosphorylated proteins to regulate their localization, stability, or activity21,23. 14-3-3 proteins interact with all three types of ACS and, surprisingly, with ETO1/EOL as well, and as results, control the protein turnover of both ACS and
ETO1/EOL21.
Ethylene biosynthesis is regulated by multiple stimuli including developmental/environmental inputs or other phytohormone. Among several phytohormone regulating ethylene biosynthesis, BR and cytokinin regulate the ethylene biosynthesis by stabilizing ACS proteins at the post-translational levels, yet our understanding in the mechanism underlying the crosstalk between the hormones to control the stability of ACS remain rudimentary. Recently, our lab has identified a SINA as a novel molecular component regulating ethylene biosynthesis in a BR-dependent manner. The SINA proteins is a family of E3 ligases that contains a RING domain and was first identified in Drosophila melanogaster and subsequently identified in mammals and plants. Mammalian and vertebrate SINA proteins function in a wide range of cellular processes including photoreceptor development, tumor progression, and responses to hypoxia and stress27-30. However, the role of SINA in plants has just begun to be understood. Plant SINA families consist of highly conserved proteins with 5 and 6 members in Arabidopsis thaliana and rice, respectively, and 10 in poplar. Our preliminary results suggested that Arabidopsis SINA1 and its closest homolog SINA2 play a positive role in BR-mediated ethylene biosynthesis, likely via downregulating ETO1/EOL and through 14-3-3-regulated SINA function (Fig.1). Interestingly, a previous 14-3-3 tandem affinity purification study has demonstrated that SINA, ETO1/EOL, and ACS form a complex with 14-3-3 in vivo, implying these proteins could have a functional relationship. Here we propose a series of experiments to delineate the mechanism by which SINA regulates ethylene biosynthesis in response to BR. We will also test the hypothesis that 14-3-3 controls the SINA function in a BR-dependent manner.