Mol. Cells 2015; 38(7): 597-603
Published online June 22, 2015
https://doi.org/10.14348/molcells.2015.0152
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: yoong@purdue.edu
Biosynthesis of the phytohormone ethylene is under tight regulation to satisfy the need for appropriate levels of ethylene in plants in response to exogenous and endogenous stimuli. The enzyme 1-aminocyclopropane-1-carboxylic acid synthase (ACS), which catalyzes the rate-limiting step of ethylene biosynthesis, plays a central role to regulate ethylene production through changes in ACS gene expression levels and the activity of the enzyme. Together with molecular genetic studies suggesting the roles of post-translational modification of the ACS, newly emerging evidence strongly suggests that the regulation of ACS protein stability is an alternative mechanism that controls ethylene production, in addition to the transcriptional regulation of ACS genes. In this review, recent new insight into the regulation of ACS protein turnover is highlighted, with a special focus on the roles of phosphorylation, ubiquitination, and novel components that regulate the turnover of ACS proteins. The prospect of cross-talk between ethylene biosynthesis and other signaling pathways to control turnover of the ACS protein is also considered.
Keywords 14-3-3, ACS, ethylene, phosphorylation, protein turnover
The simple gas ethylene has been recognized as a plant growth regulator for a century (Crocker and Knight, 1908; Knight et al., 1910; Neljubov, 1901). Ethylene influences many aspects of plant growth and developmental processes, including germination, fruit ripening, flower senescence, leaf abscission, nodulation, lateral root initiation, and the response to a variety of abiotic and biotic stresses (Abeles et al., 1992; Mattoo and Suttle, 1991). The Russian scientist Neljubov first demonstrated that ethylene is the responsible component caused the early defoliation of the tree nearby a leaking illuminating gas main in a small German town in the late1800’s (Neljubov, 1901). Neljubov used pea seedlings to determine that ethylene is the active component of the illuminating gas by exposing the filtered illuminating gas to pea seedlings. The pea seedlings exhibited distinctive morphology changes which include shortening of hypocotyl and root, swelling and thickening of hypocotyl, and formation of exaggerated hook. This seedling phenotype was later defined as the triple response, which is a hallmark of the ethylene response of dark-grown seedlings (Knight et al., 1910). 30 years later, Gane showed that ethylene is naturally produced by plants (Gane, 1934).
Through the efforts of Yang and co-workers, the biosynthesis of ethylene was fully elucidated in late 1980’s (Fig. 1) (Kende, 1993; Yang and Hoffman, 1984; Zarembinski and Theologis, 1994). The pathway of ethylene biosynthesis is simple and straightforward. Ethylene is synthesized from the amino acid methionine via two intermediates, S-adenosyl methionine (SAM) and 1-aminocyclopropane-1-carboxylic acid (ACC) (Adams and Yang, 1977; Lieberman and Mapson, 1964). In the first step, methionine is converted to SAM by SAM synthases. Conversion of SAM to ACC, which is catalyzed by a family of ACC synthase (ACS) enzymes, is the first committed and rate-limiting step in ethylene biosynthesis (Boller et al., 1979; Yang and Hoffman, 1984). During this conversion, 5-methylthioadenosine (MTA) is generated as a by-product which is readily recycled back to the Yang cycle to conserve the methlythio group of MTA into the methionine (Murr and Yang, 1975). This salvage step enables plants to maintain a constant level of cellular methionine, which can be utilized during rapid ethylene production (Sauter et al., 2013). ACC is then converted to ethylene by ACC oxidase (ACO), a member of the oxygenase/oxidase superfamily of enzymes (Dong et al., 1992). The levels of free ACC are regulated by formation of ACC derivatives, malonyl-ACC, γ-glutamyl-ACC, and jasmonyl-ACC, which likely affect the pools of free ACC for ethylene biosynthesis (Van de Poel and Van Der Straeten, 2014).
As gaseous ethylene is diffusible and not degraded in plant cells, strict regulation of ethylene biosynthesis is necessary to its diverse function in plant growth and development. Due to its role in the rate-limiting step of the ethylene biosynthesis pathway, ACS has been considered as a major point of the regulation in the pathway. Regulation of the transcript levels of ACS genes appears to be a key mechanism to control changes in ethylene production in plants (Argueso et al., 2007; Harpaz-Saad et al., 2012). However, recent studies suggest that post-translational modifications, such as phosphorylation and ubiquitination, serve as an important mechanism to regulate the stability of the ACS proteins, thus controlling the levels of ethylene in plants (Argueso et al., 2007; Chae and Kieber, 2005; Chae et al., 2003).
The family of ACS proteins can be grouped into 3 types, type-1, type-2, and type-3, based on the presence of distinct consensus sequences including phosphorylation target sites near the non-catalytic C-termini (Fig. 2) (Chae and Kieber, 2005; McClellan and Chang, 2008; Yoshida et al., 2005). Type-1 ACS proteins contain a relatively long C-terminal domain that shares highly conserved sequences and the target sites for a mitogen-activated protein kinase (MAPK) and calcium-dependent protein kinase (CDPK) (Hern?ndez Sebasti? et al., 2004; Kim et al., 2003; Liu and Zhang, 2004). Type-2 ACS proteins have only the predicted CDPK phosphorylation site. However, type-2 ACS proteins contain a unique regulatory motif called a target of ethylene overproducer 1 (ETO1) (TOE) which overlaps with the CDPK target site. TOE motif mediates interaction with ETO1 E3 ligase and its two paralogs, ETO1-Like (EOL1 and EOL2) and is required for degradation of type-2 ACS (Chae et al., 2003; Christians et al., 2009; Wang et al., 2004; Yoshida et al., 2005; 2006); type-3 ACS contains no target sites for a CDPK and MAPK, with only a short stretch of amino acids in the C-terminal extension (Chae and Kieber, 2005).
Here, I focus on recent advances and current knowledge on the protein turnover regulation of ACS in ethylene biosynthesis. New insights into the role of phosphorylation, ubiquitination and recently identified novel components governing the stability of ACS proteins are discussed. Finally, I conclude with the prospects regarding the cross-talk between ethylene and other cellular biosynthetic/signaling pathways. Transcriptional regulation in ethylene biosynthesis has been well documented, therefore not discussed in this review.
Treatment of Arabidopsis etiolated seedlings with ethylene results in the triple response (Guzman and Ecker, 1990). The triple response has been extensively used to identify Arabidopsis mutants with defects in ethylene perception and signaling, as well as mutants affecting ethylene biosynthesis. The ethylene biosynthesis mutants can be further categorized into two groups: (1) cytokinin-insensitive (
Analysis of
Characterization of the
Other factors that modify E3 ligase function involved in ethylene biosynthesis are the Related to the Ubiquitin (RUB) and RUB1 Conjugating Enzyme (RCE1) (Bostick et al., 2004; Larsen and Cancel, 2004). Like ubiquitin, RUB functions through a covalent attachment to target proteins. In Arabidopsis, RUB attaches to the cullins, thereby promoting the activity of the SCF (for Skp, Cdc53p/Cul1, and F-box protein) ubiquitin ligase complex for polyubiquitination of target proteins. Interestingly, RNA interference lines of RUB exhibit the partial triple response in etiolated seedlings due to the increase in ethylene biosynthesis, implying that conjugation of RUB to CUL3 is required for the activation of ETO1 containing CUL3 E3 ligase complex (Bostick et al., 2004). Analysis of the
XBAT32, a RING domain-containing ankyrin repeat subfamily of E3 ligases, also plays a role in ethylene biosynthesis by controlling the turnover of ACS4 (type-2) and ACS7 (type-3) (Lyzenga et al., 2012). XBAT32 was previously identified as a positive regulator of lateral root development and the
Phosphorylation is one of the most abundant post-translational modifications which affect many important aspects of protein function, including activity, stability, subcellular localization and protein-protein interaction (Holt et al., 2009). Mounting evidence suggests that ethylene biosynthesis is regulated by phosphorylation events which likely influence ACS protein turnover. Studies from the application of kinase and phosphatase inhibitors to tomato suspension cell cultures and tissues indicated that phosphorylation regulates the activity and/or turnover of ACS (Kamiyoshihara et al., 2010). A CDPK present in the extracts of wounded tomato fruits phosphorylates LeACS2 (Mayfield et al., 2007). The extract containing CDPK activity phosphorylates the C-terminal domain of LeACS2 in vitro, but the activity of the LeACS2 does not show a significant increase, suggesting phosphorylation regulates the turnover of ACS rather than affecting the activity. The C-terminus of LeACS2 contains a consensus phosphorylation target site for a CDPK, and this CDPK recognition site is present in a subset of ACS isoforms (Fig. 2) (Kamiyoshihara et al., 2010). Unlike the target sites of MAPK in the C-terminal domain of the type-1 ACS proteins, phosphorylation of the CDPK target site, which lies immediately upstream from the target site for MAPK has not been shown to be phosphorylated
Among three types of ACS, protein stability of the type-1 and type-2 ACS proteins has been shown to be directly regulated by phosphorylation. Genetic and biochemical studies of a MAPK pathway have revealed that pathogen-activated Arabidopsis MPK6 phosphorylates the type-1 ACS2 and ACS6, which leads to increased accumulation of these ACS proteins and, hence, increases ethylene production (Joo et al., 2008; Liu and Zhang, 2004). MPK6 phosphorylates 3 serine residues residing within the consensus MAPK target site in the C-terminus
Until recently, the effect of phosphorylation on type-2 ACS protein stability was not clear; neither direct phosphorylation nor responsible kinase has been identified. However, a recent study demonstrated that a Casein Kinase isoform 1.8 (CK 1.8) phosphorylates the type-2 ACS5 protein, which in turn promotes the interaction between ETO1 and ACS5, resulting in the degradation of ACS5 protein (Tan and Xue, 2014). The
Several recent studies have identified novel regulatory factors which target multiple ACS isoforms belonging in different types of ACS (Fig. 3). This regulatory feature is distinct from that of previous known regulatory proteins with a type-specific targeting (e.g. ETO1/EOL E3 ligase for type-2 and MAPK3/6 for type-1 ACS). 14-3-3 proteins, novel regulator of ethylene biosynthesis, target all three types of ACS (Fig. 3) (Yoon and Kieber, 2013a). 14-3-3 proteins are a family of evolutionarily well-conserved dimer proteins that specifically interact with phosphoproteins and are involved in a diverse array of physiological processes (Darling et al., 2005; Dougherty and Morrison, 2004; Freeman and Morrison, 2011). Upon interaction with target proteins, 14-3-3 proteins change their localization, stability, and activity, resulting in changes in physiological responses (Freeman and Morrison, 2011). There are 13 functional 14-3-3 genes in
14-3-3 interacts with all three types of ACS proteins via a non-C-terminal domain of the proteins and there is no specificity in the interaction between 14-3-3 isoforms and ACS proteins in bimolecular fluorescence complementation assay (Yoon and Kieber, 2013a). 14-3-3 stabilizes ACS protein by direct interaction and by negatively regulating the stability of the E3 ligases, ETO1/EOLs, which specifically target the type-2 ACS proteins for degradation (Yoon and Kieber, 2013a). Studies from mammalian and yeast systems have suggested that the stability of F-box proteins which promote ubiquitination in the ubiquitinproteasome pathway, is regulated based on the availability of substrates through an autocatalytic process (Ho et al., 2008; Wee et al., 2005). It is possible that 14-3-3 proteins preferentially interact with type-2 ACS proteins, which in turn leads to the depletion of the ACS proteins for the ETO1/EOLs, thus regulating the turnover of both sets of proteins. Alternatively, the interaction with dimeric 14-3-3 proteins may cause the ETO1/EOLs to dimerize, thereby promoting self-ubiquitination and subsequent degradation. Finally, the interaction with 14-3-3 proteins could enhance the interaction with distinct E3 ligases, such as XBAT32, leading to the ubiquitination and subsequent degradation of the ETO1/EOLs. Intriguingly, in mammalian cells, 14-3-3σ interacts with and regulates the protein stability of a short-lived p53 tumor suppressor protein and its cognate E3 ligases COP1 and MDM2 (Su et al., 2011; Yang et al., 2007). 14-3-3σ stabilizes p53 by down-regulation of MDM2 and COP1 protein stability. This 14-3-3-mediated inverse stability regulation on p53 and MDM2 and COP shows a similar regulatory mechanism by which Arabidopsis 14-3-3 ω control the protein stability of ACS5 and ETO1/EOLs, suggesting that the function of a subset of 14-3-3 isoforms in protein stability regulation is evolutionarily conserved between mammalian and plants. Several findings imply that 14-3-3 also regulates ACS stability independently of ETO1/EOLs (Yoon and Kieber, 2013a; 2013b). First, 14-3-3 interacts with ACS5eto2, a type-2 ACS with a lack of TOE motif for ETO1/EOL interaction. Secondly, 14-3-3 directly interacts and stabilizes the sole type-3 ACS7 and type-1 ACS2, whose protein stability is not regulated by the ETO1/EOLs. Finally, 14-3-3 increases ACS stability in
While 14-3-3 positively regulates ACS protein stability (Yoon and Kieber, 2013a; 2013b), studies from characterization of the
Identification and characterization of the role of CK1.8 have also brought new insights into the post-translational regulation in ethylene biosynthesis (Tan and Xue, 2014). CK1.8 is a conserved serine/threonine protein kinase that plays role in various physiological processes, including blue light signaling, flowering, microtubule organization and brassinosteroid signaling in rice (Ben-Nissan et al., 2008; Dai and Xue, 2010; Liu et al., 2003; Tan et al., 2013). As briefly discussed in the previous section, the
The
Due to the lack of regulatory motifs in the C-terminal domain, including phosphorylation sites, it was considered that ACS7, the sole type-3 ACS, may not be subjected to proteasome-mediated degradation pathway, and that it may be more stable than other ACS proteins (Chae and Kieber, 2005). However, Lyzenga et al., recently showed that the protein stability of ACS7 is also governed by the ubiquitin-mediated proteasomal degradation, and that degradation requires the Ring-type E3 ligase XBAT32 (Lyzenga et al., 2012). Interestingly, XBAT32 also confers protein instability to the type-2 ACS4, suggesting XBAT32-mediated degradation mechanism is not specific for the type-3 ACS. A cell-free degradation assay shows that changes in 4 lysine residues in the C-terminal domain of ACS4 results in accelerating degradation of ACS4 protein. This result is similar to the observation that K435R in the C-terminus of Flag-ACS7 promotes the turnover rate of the ACS7, suggesting the C-terminal lysine residues are not for ubiquitination, but for stabilization of ACS4 and ACS7. Shortly after, Xiong et al. raise interesting aspects of the protein stability regulation of ACS7. They showed that destabilization sequences of the ACS7 are located in the N-terminus of ACS7. The N-terminal 54 residues of the ACS7 confer significant instability to ACS71?54-GUS and first 14 amino acids are responsible for negative regulation of the ACS7 protein stability (Xiong et al., 2014). One possible explanation for this may be due to the nature of the C-terminal fusion of the ACS7-GUS used in the study. Traditionally, the N-terminal fusion of ACS has been routinely utilized for studying the turnover of ACS to avoid masking the C-terminal regulatory domain and this may be blamed for concealing the destabilization signals located at the N-termini and making only the C-terminal signals available to degradation machinery. It is interesting to further study the role of the N-terminal domain of other types of ACS whether they also contain putative degradation sequences in their N-termini.
Several studies indicate that there are molecular components acing on the non-C-terminal domain of ACS proteins to regulate their stability. Cytokinin and brassinosteroid stabilize ACS5eto2 and ACS9eto3 and the effects of these two hormones on the protein stability are additive, suggesting cytokinin and brassinosteroid act through distinct TOE-independent mechanisms on these ACS proteins (Hansen et al., 2009). Genetic studies showed that cytokinin-mediated ACS stabilization requires a functional cytokinin signaling pathway (Hansen et al., 2009). Mutation in the signaling components, cytokinin receptors, AHPs, and type-A and type-B transcription factors, in the cytokinin signaling pathway produce reduced amounts of ethylene in response to exogenous cytokinin. The effect of brassinosteroid in ethylene biosynthesis is somewhat distinguished from the typical triple response that has been observed with cytokinin treatment; BR treatment results in a shortened and thickened hypocotyl formation; but it does not induce an exaggerated hook formation; and shortening of the root and hypocotyl is less severe than for cytokinin-treated seedlings. Interestingly, the ethylene-insensitive mutant
Together, these studies indicate that there are molecular components that act as the points of cross-talk between ethylene biosynthesis and other hormonal signaling pathways. Identification of these elements will bring new insights into understanding the mechanism by which protein turnover of ACS is regulated to coordinate and merge different hormonal inputs to regulate ethylene production which effects on many diverse ranges of plant growth and development.
Mol. Cells 2015; 38(7): 597-603
Published online July 31, 2015 https://doi.org/10.14348/molcells.2015.0152
Copyright © The Korean Society for Molecular and Cellular Biology.
Gyeong Mee Yoon*
Department of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana 47907, USA
Correspondence to:*Correspondence: yoong@purdue.edu
Biosynthesis of the phytohormone ethylene is under tight regulation to satisfy the need for appropriate levels of ethylene in plants in response to exogenous and endogenous stimuli. The enzyme 1-aminocyclopropane-1-carboxylic acid synthase (ACS), which catalyzes the rate-limiting step of ethylene biosynthesis, plays a central role to regulate ethylene production through changes in ACS gene expression levels and the activity of the enzyme. Together with molecular genetic studies suggesting the roles of post-translational modification of the ACS, newly emerging evidence strongly suggests that the regulation of ACS protein stability is an alternative mechanism that controls ethylene production, in addition to the transcriptional regulation of ACS genes. In this review, recent new insight into the regulation of ACS protein turnover is highlighted, with a special focus on the roles of phosphorylation, ubiquitination, and novel components that regulate the turnover of ACS proteins. The prospect of cross-talk between ethylene biosynthesis and other signaling pathways to control turnover of the ACS protein is also considered.
Keywords: 14-3-3, ACS, ethylene, phosphorylation, protein turnover
The simple gas ethylene has been recognized as a plant growth regulator for a century (Crocker and Knight, 1908; Knight et al., 1910; Neljubov, 1901). Ethylene influences many aspects of plant growth and developmental processes, including germination, fruit ripening, flower senescence, leaf abscission, nodulation, lateral root initiation, and the response to a variety of abiotic and biotic stresses (Abeles et al., 1992; Mattoo and Suttle, 1991). The Russian scientist Neljubov first demonstrated that ethylene is the responsible component caused the early defoliation of the tree nearby a leaking illuminating gas main in a small German town in the late1800’s (Neljubov, 1901). Neljubov used pea seedlings to determine that ethylene is the active component of the illuminating gas by exposing the filtered illuminating gas to pea seedlings. The pea seedlings exhibited distinctive morphology changes which include shortening of hypocotyl and root, swelling and thickening of hypocotyl, and formation of exaggerated hook. This seedling phenotype was later defined as the triple response, which is a hallmark of the ethylene response of dark-grown seedlings (Knight et al., 1910). 30 years later, Gane showed that ethylene is naturally produced by plants (Gane, 1934).
Through the efforts of Yang and co-workers, the biosynthesis of ethylene was fully elucidated in late 1980’s (Fig. 1) (Kende, 1993; Yang and Hoffman, 1984; Zarembinski and Theologis, 1994). The pathway of ethylene biosynthesis is simple and straightforward. Ethylene is synthesized from the amino acid methionine via two intermediates, S-adenosyl methionine (SAM) and 1-aminocyclopropane-1-carboxylic acid (ACC) (Adams and Yang, 1977; Lieberman and Mapson, 1964). In the first step, methionine is converted to SAM by SAM synthases. Conversion of SAM to ACC, which is catalyzed by a family of ACC synthase (ACS) enzymes, is the first committed and rate-limiting step in ethylene biosynthesis (Boller et al., 1979; Yang and Hoffman, 1984). During this conversion, 5-methylthioadenosine (MTA) is generated as a by-product which is readily recycled back to the Yang cycle to conserve the methlythio group of MTA into the methionine (Murr and Yang, 1975). This salvage step enables plants to maintain a constant level of cellular methionine, which can be utilized during rapid ethylene production (Sauter et al., 2013). ACC is then converted to ethylene by ACC oxidase (ACO), a member of the oxygenase/oxidase superfamily of enzymes (Dong et al., 1992). The levels of free ACC are regulated by formation of ACC derivatives, malonyl-ACC, γ-glutamyl-ACC, and jasmonyl-ACC, which likely affect the pools of free ACC for ethylene biosynthesis (Van de Poel and Van Der Straeten, 2014).
As gaseous ethylene is diffusible and not degraded in plant cells, strict regulation of ethylene biosynthesis is necessary to its diverse function in plant growth and development. Due to its role in the rate-limiting step of the ethylene biosynthesis pathway, ACS has been considered as a major point of the regulation in the pathway. Regulation of the transcript levels of ACS genes appears to be a key mechanism to control changes in ethylene production in plants (Argueso et al., 2007; Harpaz-Saad et al., 2012). However, recent studies suggest that post-translational modifications, such as phosphorylation and ubiquitination, serve as an important mechanism to regulate the stability of the ACS proteins, thus controlling the levels of ethylene in plants (Argueso et al., 2007; Chae and Kieber, 2005; Chae et al., 2003).
The family of ACS proteins can be grouped into 3 types, type-1, type-2, and type-3, based on the presence of distinct consensus sequences including phosphorylation target sites near the non-catalytic C-termini (Fig. 2) (Chae and Kieber, 2005; McClellan and Chang, 2008; Yoshida et al., 2005). Type-1 ACS proteins contain a relatively long C-terminal domain that shares highly conserved sequences and the target sites for a mitogen-activated protein kinase (MAPK) and calcium-dependent protein kinase (CDPK) (Hern?ndez Sebasti? et al., 2004; Kim et al., 2003; Liu and Zhang, 2004). Type-2 ACS proteins have only the predicted CDPK phosphorylation site. However, type-2 ACS proteins contain a unique regulatory motif called a target of ethylene overproducer 1 (ETO1) (TOE) which overlaps with the CDPK target site. TOE motif mediates interaction with ETO1 E3 ligase and its two paralogs, ETO1-Like (EOL1 and EOL2) and is required for degradation of type-2 ACS (Chae et al., 2003; Christians et al., 2009; Wang et al., 2004; Yoshida et al., 2005; 2006); type-3 ACS contains no target sites for a CDPK and MAPK, with only a short stretch of amino acids in the C-terminal extension (Chae and Kieber, 2005).
Here, I focus on recent advances and current knowledge on the protein turnover regulation of ACS in ethylene biosynthesis. New insights into the role of phosphorylation, ubiquitination and recently identified novel components governing the stability of ACS proteins are discussed. Finally, I conclude with the prospects regarding the cross-talk between ethylene and other cellular biosynthetic/signaling pathways. Transcriptional regulation in ethylene biosynthesis has been well documented, therefore not discussed in this review.
Treatment of Arabidopsis etiolated seedlings with ethylene results in the triple response (Guzman and Ecker, 1990). The triple response has been extensively used to identify Arabidopsis mutants with defects in ethylene perception and signaling, as well as mutants affecting ethylene biosynthesis. The ethylene biosynthesis mutants can be further categorized into two groups: (1) cytokinin-insensitive (
Analysis of
Characterization of the
Other factors that modify E3 ligase function involved in ethylene biosynthesis are the Related to the Ubiquitin (RUB) and RUB1 Conjugating Enzyme (RCE1) (Bostick et al., 2004; Larsen and Cancel, 2004). Like ubiquitin, RUB functions through a covalent attachment to target proteins. In Arabidopsis, RUB attaches to the cullins, thereby promoting the activity of the SCF (for Skp, Cdc53p/Cul1, and F-box protein) ubiquitin ligase complex for polyubiquitination of target proteins. Interestingly, RNA interference lines of RUB exhibit the partial triple response in etiolated seedlings due to the increase in ethylene biosynthesis, implying that conjugation of RUB to CUL3 is required for the activation of ETO1 containing CUL3 E3 ligase complex (Bostick et al., 2004). Analysis of the
XBAT32, a RING domain-containing ankyrin repeat subfamily of E3 ligases, also plays a role in ethylene biosynthesis by controlling the turnover of ACS4 (type-2) and ACS7 (type-3) (Lyzenga et al., 2012). XBAT32 was previously identified as a positive regulator of lateral root development and the
Phosphorylation is one of the most abundant post-translational modifications which affect many important aspects of protein function, including activity, stability, subcellular localization and protein-protein interaction (Holt et al., 2009). Mounting evidence suggests that ethylene biosynthesis is regulated by phosphorylation events which likely influence ACS protein turnover. Studies from the application of kinase and phosphatase inhibitors to tomato suspension cell cultures and tissues indicated that phosphorylation regulates the activity and/or turnover of ACS (Kamiyoshihara et al., 2010). A CDPK present in the extracts of wounded tomato fruits phosphorylates LeACS2 (Mayfield et al., 2007). The extract containing CDPK activity phosphorylates the C-terminal domain of LeACS2 in vitro, but the activity of the LeACS2 does not show a significant increase, suggesting phosphorylation regulates the turnover of ACS rather than affecting the activity. The C-terminus of LeACS2 contains a consensus phosphorylation target site for a CDPK, and this CDPK recognition site is present in a subset of ACS isoforms (Fig. 2) (Kamiyoshihara et al., 2010). Unlike the target sites of MAPK in the C-terminal domain of the type-1 ACS proteins, phosphorylation of the CDPK target site, which lies immediately upstream from the target site for MAPK has not been shown to be phosphorylated
Among three types of ACS, protein stability of the type-1 and type-2 ACS proteins has been shown to be directly regulated by phosphorylation. Genetic and biochemical studies of a MAPK pathway have revealed that pathogen-activated Arabidopsis MPK6 phosphorylates the type-1 ACS2 and ACS6, which leads to increased accumulation of these ACS proteins and, hence, increases ethylene production (Joo et al., 2008; Liu and Zhang, 2004). MPK6 phosphorylates 3 serine residues residing within the consensus MAPK target site in the C-terminus
Until recently, the effect of phosphorylation on type-2 ACS protein stability was not clear; neither direct phosphorylation nor responsible kinase has been identified. However, a recent study demonstrated that a Casein Kinase isoform 1.8 (CK 1.8) phosphorylates the type-2 ACS5 protein, which in turn promotes the interaction between ETO1 and ACS5, resulting in the degradation of ACS5 protein (Tan and Xue, 2014). The
Several recent studies have identified novel regulatory factors which target multiple ACS isoforms belonging in different types of ACS (Fig. 3). This regulatory feature is distinct from that of previous known regulatory proteins with a type-specific targeting (e.g. ETO1/EOL E3 ligase for type-2 and MAPK3/6 for type-1 ACS). 14-3-3 proteins, novel regulator of ethylene biosynthesis, target all three types of ACS (Fig. 3) (Yoon and Kieber, 2013a). 14-3-3 proteins are a family of evolutionarily well-conserved dimer proteins that specifically interact with phosphoproteins and are involved in a diverse array of physiological processes (Darling et al., 2005; Dougherty and Morrison, 2004; Freeman and Morrison, 2011). Upon interaction with target proteins, 14-3-3 proteins change their localization, stability, and activity, resulting in changes in physiological responses (Freeman and Morrison, 2011). There are 13 functional 14-3-3 genes in
14-3-3 interacts with all three types of ACS proteins via a non-C-terminal domain of the proteins and there is no specificity in the interaction between 14-3-3 isoforms and ACS proteins in bimolecular fluorescence complementation assay (Yoon and Kieber, 2013a). 14-3-3 stabilizes ACS protein by direct interaction and by negatively regulating the stability of the E3 ligases, ETO1/EOLs, which specifically target the type-2 ACS proteins for degradation (Yoon and Kieber, 2013a). Studies from mammalian and yeast systems have suggested that the stability of F-box proteins which promote ubiquitination in the ubiquitinproteasome pathway, is regulated based on the availability of substrates through an autocatalytic process (Ho et al., 2008; Wee et al., 2005). It is possible that 14-3-3 proteins preferentially interact with type-2 ACS proteins, which in turn leads to the depletion of the ACS proteins for the ETO1/EOLs, thus regulating the turnover of both sets of proteins. Alternatively, the interaction with dimeric 14-3-3 proteins may cause the ETO1/EOLs to dimerize, thereby promoting self-ubiquitination and subsequent degradation. Finally, the interaction with 14-3-3 proteins could enhance the interaction with distinct E3 ligases, such as XBAT32, leading to the ubiquitination and subsequent degradation of the ETO1/EOLs. Intriguingly, in mammalian cells, 14-3-3σ interacts with and regulates the protein stability of a short-lived p53 tumor suppressor protein and its cognate E3 ligases COP1 and MDM2 (Su et al., 2011; Yang et al., 2007). 14-3-3σ stabilizes p53 by down-regulation of MDM2 and COP1 protein stability. This 14-3-3-mediated inverse stability regulation on p53 and MDM2 and COP shows a similar regulatory mechanism by which Arabidopsis 14-3-3 ω control the protein stability of ACS5 and ETO1/EOLs, suggesting that the function of a subset of 14-3-3 isoforms in protein stability regulation is evolutionarily conserved between mammalian and plants. Several findings imply that 14-3-3 also regulates ACS stability independently of ETO1/EOLs (Yoon and Kieber, 2013a; 2013b). First, 14-3-3 interacts with ACS5eto2, a type-2 ACS with a lack of TOE motif for ETO1/EOL interaction. Secondly, 14-3-3 directly interacts and stabilizes the sole type-3 ACS7 and type-1 ACS2, whose protein stability is not regulated by the ETO1/EOLs. Finally, 14-3-3 increases ACS stability in
While 14-3-3 positively regulates ACS protein stability (Yoon and Kieber, 2013a; 2013b), studies from characterization of the
Identification and characterization of the role of CK1.8 have also brought new insights into the post-translational regulation in ethylene biosynthesis (Tan and Xue, 2014). CK1.8 is a conserved serine/threonine protein kinase that plays role in various physiological processes, including blue light signaling, flowering, microtubule organization and brassinosteroid signaling in rice (Ben-Nissan et al., 2008; Dai and Xue, 2010; Liu et al., 2003; Tan et al., 2013). As briefly discussed in the previous section, the
The
Due to the lack of regulatory motifs in the C-terminal domain, including phosphorylation sites, it was considered that ACS7, the sole type-3 ACS, may not be subjected to proteasome-mediated degradation pathway, and that it may be more stable than other ACS proteins (Chae and Kieber, 2005). However, Lyzenga et al., recently showed that the protein stability of ACS7 is also governed by the ubiquitin-mediated proteasomal degradation, and that degradation requires the Ring-type E3 ligase XBAT32 (Lyzenga et al., 2012). Interestingly, XBAT32 also confers protein instability to the type-2 ACS4, suggesting XBAT32-mediated degradation mechanism is not specific for the type-3 ACS. A cell-free degradation assay shows that changes in 4 lysine residues in the C-terminal domain of ACS4 results in accelerating degradation of ACS4 protein. This result is similar to the observation that K435R in the C-terminus of Flag-ACS7 promotes the turnover rate of the ACS7, suggesting the C-terminal lysine residues are not for ubiquitination, but for stabilization of ACS4 and ACS7. Shortly after, Xiong et al. raise interesting aspects of the protein stability regulation of ACS7. They showed that destabilization sequences of the ACS7 are located in the N-terminus of ACS7. The N-terminal 54 residues of the ACS7 confer significant instability to ACS71?54-GUS and first 14 amino acids are responsible for negative regulation of the ACS7 protein stability (Xiong et al., 2014). One possible explanation for this may be due to the nature of the C-terminal fusion of the ACS7-GUS used in the study. Traditionally, the N-terminal fusion of ACS has been routinely utilized for studying the turnover of ACS to avoid masking the C-terminal regulatory domain and this may be blamed for concealing the destabilization signals located at the N-termini and making only the C-terminal signals available to degradation machinery. It is interesting to further study the role of the N-terminal domain of other types of ACS whether they also contain putative degradation sequences in their N-termini.
Several studies indicate that there are molecular components acing on the non-C-terminal domain of ACS proteins to regulate their stability. Cytokinin and brassinosteroid stabilize ACS5eto2 and ACS9eto3 and the effects of these two hormones on the protein stability are additive, suggesting cytokinin and brassinosteroid act through distinct TOE-independent mechanisms on these ACS proteins (Hansen et al., 2009). Genetic studies showed that cytokinin-mediated ACS stabilization requires a functional cytokinin signaling pathway (Hansen et al., 2009). Mutation in the signaling components, cytokinin receptors, AHPs, and type-A and type-B transcription factors, in the cytokinin signaling pathway produce reduced amounts of ethylene in response to exogenous cytokinin. The effect of brassinosteroid in ethylene biosynthesis is somewhat distinguished from the typical triple response that has been observed with cytokinin treatment; BR treatment results in a shortened and thickened hypocotyl formation; but it does not induce an exaggerated hook formation; and shortening of the root and hypocotyl is less severe than for cytokinin-treated seedlings. Interestingly, the ethylene-insensitive mutant
Together, these studies indicate that there are molecular components that act as the points of cross-talk between ethylene biosynthesis and other hormonal signaling pathways. Identification of these elements will bring new insights into understanding the mechanism by which protein turnover of ACS is regulated to coordinate and merge different hormonal inputs to regulate ethylene production which effects on many diverse ranges of plant growth and development.
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