Mol. Cells 2015; 38(7): 651-656
Published online June 17, 2015
https://doi.org/10.14348/molcells.2015.0055
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: hrlee1375@duksung.ac.kr
Plant growth and development are coordinately orchestrated by environmental cues and phytohormones. Light acts as a key environmental factor for fundamental plant growth and physiology through photosensory phytochromes and underlying molecular mechanisms. Although phytochromes are known to possess serine/threonine protein kinase activities, whether they trigger a signal transduction pathway via an intracellular protein kinase network remains unknown. In analyses of
Keywords Arabidopsis, gibberellin, light, MAP kinase kinase, MAPK cascade
Light is one of the most important environmental factors affecting the physiological and developmental growth of plants, which is the major source of energy for plant life. Phytochrome (phy) is a photoreceptor that senses light signals and regulates light-mediated plant growth. Once phytochromes sense light signals, they translocate immediately from the cytoplasm to the nucleus and make complexes with certain proteins in subnuclear photobodies (speckles), where light-induced phys regulate positive or negative regulatory transcription factors (TFs) of photomorphogenesis (Chen and Chory, 2011; Leivar and Quail, 2011). Positively acting TFs including ELONGATED HYPOCOTYL 5 (HY5), LONG HYPOCOTYL IN FAR-RED 1 (HFR1), and LONG AFTER FAR-RED LIGHT 1 (LAF1) are stabilized by the inhibition of COP1 E3 ubiquitin ligase-mediated degradation (Jang et al., 2005; Saijo et al., 2003; Seo et al., 2003; Yang et al., 2005). Negatively acting TFs such as PHYTOCHROME-INTERACTING FACTORs (PIFs), which promote skotomorphogenic growth, are degraded through the direct interaction with light-induced phys (Leivar and Quail, 2010). Notably, PIFs have recently been reported to be regulated by other phytohormones such as gibberellin (GA) and brassinosteroid (BR), because PIFs directly interact with DELLA and BZR1 proteins in each signaling pathway, respectively (de Lucas et al., 2008; Feng et al., 2008; Oh et al., 2012), suggesting that PIFs integrate multiple signaling pathways for the downstream morphogenesis (Leivar and Quail, 2010).
Both positive and negative regulatory TFs involved in light signaling are commonly regulated by ubiquitin-proteasome-mediated degradation and phosphorylation is one of the important post-translational modifications in this process. Recently, casein kinase II (CK2), a ubiquitous Ser/Thr protein kinase, has been reported to phosphorylate both PIF1 at multiple sites and also the positively acting regulator HFR1 (Bu et al., 2011; Park et al., 2008). Although phosphorylation is known to be crucial to proteasome-mediated degradation for the promotion of positive regulator’s stabilities and the repression of negative regulators, the kinases that phosphorylate the regulatory TFs involved in light signaling have not been completely investigated.
The mitogen-activated protein kinase (MAPK) cascade usually functions as an important signaling network involved in physiological and developmental processes in eukaryotes. This MAPK cascade is usually composed of sequential activations of three classes of Ser/Thr protein kinases: 20 MAPK (MPK), 10 MAPK Kinase (MKK), and 60 MAPK Kinase Kinase (MKKK) (Hamel et al., 2006; Ichimura et al., 2002). Since plants cannot move to avoid environmental damage such as biotic and abiotic stresses, they have evolved with complex signaling networks to deal with various environmental challenges. Indeed, plant genomes encode the largest number of MAPK cascade genes in all sequenced eukaryotes (Hamel et al., 2006; Ichimura et al., 2002), suggesting that plant MAPK cascade genes have functional redundancies.
The MAPK Kinase (MKK) gene family has relatively few members in canonical MAPK cascade gene families, which are classified into four groups (A to D) based on the sequence similarities.
Columbia-0 (Col-0) was used as the wild-type (WT) Arabidopsis plant. The
To harvest tissues, seedlings were instantaneously frozen by liquid nitrogen in 1.5 ml tubes. Frozen tissues were ground using a blue pestle and a motor-driven grinder. Total RNA was isolated from ground powder of tissues with TRIzol reagent (Life Technologies, USA). First strand cDNA was synthesized from 1 μg of total RNA with M-MLV reverse transcriptase (Promega, USA). For analyzing the knock-out state in the
For an in-gel kinase assay, seedling extracts containing 300 μg of proteins were fractionated in 10 % SDS-PAGE gel containing 0.25 mg ml?1 of myelin basic protein (MBP), a general MAPK substrate. The protein denaturing, renaturing, and kinase activity assay in the gel were performed as previously described (Zhang and Klessing, 1997). For an immunoblot assay, proteins were fractionated in 10 % SDS-PAGE gel. Phosphorylated MAPKs were detected using an anti-phospho-MAP kinase (p42/44) antibody (Cell Signaling Technology, USA) as previously described (Sharma and Carew, 2002).
Plant genomes contain a huge number of MAPK cascade genes compared to those of other eukaryotes including, yeast and human. Although the plant MKK gene family has relatively few members from
Phytohormone GA regulates hypocotyl elongation and the repression of light-regulated genes, which usually resembles the etiolated responses of seedlings grown in darkness (Alabad? et al., 2004). In addition to the long petiole phenotype shown in
Because the phenotypes of elongated hypocotyls and petioles usually resemble those of the
To assess the possible role of MAPK cascades in light signaling, I first tested the MAPK phosphorylation activated by light signals. For this, seedlings were grown in darkness for 7 days and exposed to white light for 10, 30, 60, and 90 min (Fig. 4A). Interestingly, endogenous MAPKs were phosphorylated from 10 min and gradually disappeared, which is the general behavior of the MAPK phosphorylation stimulated by input signals (Fig. 4B). Consistent with a previous report (Sethi et al., 2014), MPK6 seemed to be mainly activated and MPK3 and MPK4 were also slightly phosphorylated by light signals (Fig. 4B). In the same context, five other
Meanwhile, MPK3 was also constitutively phosphorylated in the
Although the MKK3-MPK6 cascade module has recently been reported to be specifically activated by blue light signals (Sethi et al., 2014), as shown in this study, MKK3 also seems to play a crucial role in red light signaling (Fig. 3) and in the negative regulation of MAPK phosphorylation in darkness (Fig. 4C). Moreover, we still cannot rule out the redundant involvement of other MKKs in addition to MKK3. Therefore, this finding sheds new light on the complicated function of MAPK cascades during dark-light transition.
In 2008, Deng’s and Prat’s groups showed that the crosstalk between light and GA is regulated by the destabilization of PIF proteins that bind to their target promoter and regulate gene expression related with the cell elongation (de Lucas et al., 2008; Feng et al., 2008). Interestingly, since the
Mol. Cells 2015; 38(7): 651-656
Published online July 31, 2015 https://doi.org/10.14348/molcells.2015.0055
Copyright © The Korean Society for Molecular and Cellular Biology.
Horim Lee*
Department of Pre-PharmMed, College of Natural Sciences, Duksung Women’s University, Seoul 132-714, Korea
Correspondence to:*Correspondence: hrlee1375@duksung.ac.kr
Plant growth and development are coordinately orchestrated by environmental cues and phytohormones. Light acts as a key environmental factor for fundamental plant growth and physiology through photosensory phytochromes and underlying molecular mechanisms. Although phytochromes are known to possess serine/threonine protein kinase activities, whether they trigger a signal transduction pathway via an intracellular protein kinase network remains unknown. In analyses of
Keywords: Arabidopsis, gibberellin, light, MAP kinase kinase, MAPK cascade
Light is one of the most important environmental factors affecting the physiological and developmental growth of plants, which is the major source of energy for plant life. Phytochrome (phy) is a photoreceptor that senses light signals and regulates light-mediated plant growth. Once phytochromes sense light signals, they translocate immediately from the cytoplasm to the nucleus and make complexes with certain proteins in subnuclear photobodies (speckles), where light-induced phys regulate positive or negative regulatory transcription factors (TFs) of photomorphogenesis (Chen and Chory, 2011; Leivar and Quail, 2011). Positively acting TFs including ELONGATED HYPOCOTYL 5 (HY5), LONG HYPOCOTYL IN FAR-RED 1 (HFR1), and LONG AFTER FAR-RED LIGHT 1 (LAF1) are stabilized by the inhibition of COP1 E3 ubiquitin ligase-mediated degradation (Jang et al., 2005; Saijo et al., 2003; Seo et al., 2003; Yang et al., 2005). Negatively acting TFs such as PHYTOCHROME-INTERACTING FACTORs (PIFs), which promote skotomorphogenic growth, are degraded through the direct interaction with light-induced phys (Leivar and Quail, 2010). Notably, PIFs have recently been reported to be regulated by other phytohormones such as gibberellin (GA) and brassinosteroid (BR), because PIFs directly interact with DELLA and BZR1 proteins in each signaling pathway, respectively (de Lucas et al., 2008; Feng et al., 2008; Oh et al., 2012), suggesting that PIFs integrate multiple signaling pathways for the downstream morphogenesis (Leivar and Quail, 2010).
Both positive and negative regulatory TFs involved in light signaling are commonly regulated by ubiquitin-proteasome-mediated degradation and phosphorylation is one of the important post-translational modifications in this process. Recently, casein kinase II (CK2), a ubiquitous Ser/Thr protein kinase, has been reported to phosphorylate both PIF1 at multiple sites and also the positively acting regulator HFR1 (Bu et al., 2011; Park et al., 2008). Although phosphorylation is known to be crucial to proteasome-mediated degradation for the promotion of positive regulator’s stabilities and the repression of negative regulators, the kinases that phosphorylate the regulatory TFs involved in light signaling have not been completely investigated.
The mitogen-activated protein kinase (MAPK) cascade usually functions as an important signaling network involved in physiological and developmental processes in eukaryotes. This MAPK cascade is usually composed of sequential activations of three classes of Ser/Thr protein kinases: 20 MAPK (MPK), 10 MAPK Kinase (MKK), and 60 MAPK Kinase Kinase (MKKK) (Hamel et al., 2006; Ichimura et al., 2002). Since plants cannot move to avoid environmental damage such as biotic and abiotic stresses, they have evolved with complex signaling networks to deal with various environmental challenges. Indeed, plant genomes encode the largest number of MAPK cascade genes in all sequenced eukaryotes (Hamel et al., 2006; Ichimura et al., 2002), suggesting that plant MAPK cascade genes have functional redundancies.
The MAPK Kinase (MKK) gene family has relatively few members in canonical MAPK cascade gene families, which are classified into four groups (A to D) based on the sequence similarities.
Columbia-0 (Col-0) was used as the wild-type (WT) Arabidopsis plant. The
To harvest tissues, seedlings were instantaneously frozen by liquid nitrogen in 1.5 ml tubes. Frozen tissues were ground using a blue pestle and a motor-driven grinder. Total RNA was isolated from ground powder of tissues with TRIzol reagent (Life Technologies, USA). First strand cDNA was synthesized from 1 μg of total RNA with M-MLV reverse transcriptase (Promega, USA). For analyzing the knock-out state in the
For an in-gel kinase assay, seedling extracts containing 300 μg of proteins were fractionated in 10 % SDS-PAGE gel containing 0.25 mg ml?1 of myelin basic protein (MBP), a general MAPK substrate. The protein denaturing, renaturing, and kinase activity assay in the gel were performed as previously described (Zhang and Klessing, 1997). For an immunoblot assay, proteins were fractionated in 10 % SDS-PAGE gel. Phosphorylated MAPKs were detected using an anti-phospho-MAP kinase (p42/44) antibody (Cell Signaling Technology, USA) as previously described (Sharma and Carew, 2002).
Plant genomes contain a huge number of MAPK cascade genes compared to those of other eukaryotes including, yeast and human. Although the plant MKK gene family has relatively few members from
Phytohormone GA regulates hypocotyl elongation and the repression of light-regulated genes, which usually resembles the etiolated responses of seedlings grown in darkness (Alabad? et al., 2004). In addition to the long petiole phenotype shown in
Because the phenotypes of elongated hypocotyls and petioles usually resemble those of the
To assess the possible role of MAPK cascades in light signaling, I first tested the MAPK phosphorylation activated by light signals. For this, seedlings were grown in darkness for 7 days and exposed to white light for 10, 30, 60, and 90 min (Fig. 4A). Interestingly, endogenous MAPKs were phosphorylated from 10 min and gradually disappeared, which is the general behavior of the MAPK phosphorylation stimulated by input signals (Fig. 4B). Consistent with a previous report (Sethi et al., 2014), MPK6 seemed to be mainly activated and MPK3 and MPK4 were also slightly phosphorylated by light signals (Fig. 4B). In the same context, five other
Meanwhile, MPK3 was also constitutively phosphorylated in the
Although the MKK3-MPK6 cascade module has recently been reported to be specifically activated by blue light signals (Sethi et al., 2014), as shown in this study, MKK3 also seems to play a crucial role in red light signaling (Fig. 3) and in the negative regulation of MAPK phosphorylation in darkness (Fig. 4C). Moreover, we still cannot rule out the redundant involvement of other MKKs in addition to MKK3. Therefore, this finding sheds new light on the complicated function of MAPK cascades during dark-light transition.
In 2008, Deng’s and Prat’s groups showed that the crosstalk between light and GA is regulated by the destabilization of PIF proteins that bind to their target promoter and regulate gene expression related with the cell elongation (de Lucas et al., 2008; Feng et al., 2008). Interestingly, since the
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