論文の公開元へFunctional Analysis of MCAs and PIF4 in Plant Cold Signaling
概要
Calcium ions are used as secondary messengers in eukaryotic cells. The cytosolic Ca2+ concentration, [Ca2+]cyt, fluctuates in response to a variety of stimuli, including mechanical stimulation, hormones, pathogens, light, and abiotic stresses such as low temperature (Sanders et al., 2002; White and Broadley, 2003; Dodd et al., 2010). The stimulus-specific spatiotemporal patterning of [Ca2+]cyt dynamics is called the Ca2+ signature (Monshausen, 2012), and to create these signatures, Ca2+ influx channels and Ca2+ efflux transporters that permit transient increases in [Ca2+]cyt are required (McAinsh and Pittman, 2009).
How plant cells generate stimulus-specific Ca2+ signals remains unknown. To identify the spatiotemporal patterning of [Ca2+]cyt dynamics, recombinant aequorin has been introduced as a reporter of [Ca2+]cyt changes in plant systems (Knight et al., 1991). In Arabidopsis plants expressing aequorin in the cytoplasm, low temperature triggers an immediate and transient rise in [Ca2+]cyt (Knight et al., 1991; Knight et al., 1996; Carpaneto et al., 2007). The final temperature and cooling rate are important for sensing low temperature in Arabidopsis (Knight, 2002). In mammals, many TRP (transient receptor potential) channels, which are a specific class of ion channels, function as intracellular Ca2+ release channels (Gees et al., 2010). Some of these channels also function as thermosensors (Gees et al., 2010), and TRPA1 seems to act as a sensor for cold (Karashima et al., 2009; del Camino et al., 2010; Aubdool et al., 2014). Although no proteins with high similarity to TRP channels have been identified in land-plant genomes, the genes for Cr-TRP proteins are encoded in the genomic sequence of the alga Chlamydomonas reinhardtii and show functional properties that are similar to those of mammalian TRP channels (Arias-Darraz et al., 2015).
Two Ca2+-permeable mechanosensitive channels, named MCA1 and MCA2 (mid1- complementing activity 1 and 2), have been identified in Arabidopsis (Nakagawa et al., 2007; Yamanaka et al., 2010; Nakano et al., 2011; Furuichi et al., 2012; Kamano et al., 2015). Both MCA1 and MCA2 complement deficiency of Ca2+ uptake in yeast cells lacking a Ca2+ channel composed of the Mid1 and Cch1 subunits (Nakagawa et al., 2007; Yamanaka et al., 2010). It should be noted that this complementation activity is detected under conditions that allow the Mid1/Cch1 channel to function as the sole Ca2+ influx system in yeast cells, suggesting that MCA1 and MCA2 can directly mediate Ca2+ influx in the cells lacking both Mid1 and Cch1. Electrophysiological studies have shown that both MCA1 and MCA2 produce stretch-activated currents when expressed in Xenopus laevis oocytes (Furuichi et al., 2012). These results with yeast cells and Xenopus oocytes suggest that MCA1 and MCA2 mediate Ca2+ influx as mechanosensitive channels and are not accessory factors that facilitate Ca2+ influx. Overexpression of MCA1 enhances an increase in [Ca2+]cyt upon hypoosmotic shock (Nakagawa et al., 2007). The mca2 mutant exhibits a defect in Ca2+ uptake from the roots (Yamanaka et al., 2010). Structurally, MCA1 and MCA2 have 74% identity and 89% similarity in amino acid sequences (Nakagawa et al., 2007). Both have a single transmembrane segment and an EF-hand-like motif and coiled-coil motif in the N-terminal region, as well as a plac8 motif in the C-terminal region (Nakagawa et al., 2007; Kamano et al., 2015). MCA1-GFP and MCA2-GFP are localized to the plasma membrane (Nakagawa et al., 2007). MCA1 and MCA2 form a homotetramer (Nakano et al., 2011; Shigematsu et al., 2014). Topological analysis has indicated that the EF-hand-like motif, the coiled-coil motif, and the plac8 motif are present in the cytoplasm (Kamano et al., 2015), suggesting that both channels recognize intracellular Ca2+. The MCA genes are conserved in the plant kingdom (Kurusu et al., 2013), and an increase in [Ca2+]cyt as a result of hypo-osmotic shock is mediated by MCA proteins in rice and tobacco (Kurusu et al., 2012).
Application of the patch-clamp technique has demonstrated that Ca2+-permeable channels are transiently activated by cold shock in Arabidopsis mesophyll cells (Carpaneto et al., 2007). In plants, extracellular freezing causes dehydration and mechanical stresses on the plasma membrane, and cold-acclimated plant plasma membranes become resistant to mechanical stress (Yamazaki et al., 2008). Expression of CBF2 is induced not only by cold, but also by mechanical stress (Zarka et al., 2003). Therefore, it is assumed that mechanical stress may be one of the factors involved in cold acclimation.
Three CBF/DREB1 (C-repeat binding factor/DRE binding factor 1) transcription factors have been extensively studied. CBF/DREB1, belonging to the AP2/ERF (apetala/ethylene-responsive factor) superfamily, are important factors for cold acclimation in plants (Miura and Furumoto, 2013). CBF/DREB1 genes are rapidly and transiently induced after cold treatment (Gilmour et al., 1998), and overexpression of CBF/DREB1 constitutively enhances freezing tolerance (Stockinger et al., 1997; Liu et al., 1998). Under cold stress, CBF/DREB1 proteins bind to CRT/DRE cis-elements in the promoter of cold-regulated (COR) genes and induce transcription (Stockinger et al., 1997). However, gene expression analyses reveals that only 6.5% of the total COR genes are regulated by CBF/DREB1 (Park et al., 2015). In addition to CBF/DREB1 genes, 27 transcription factors that were up-regulated at an early stage after cold treatment were considered as first-wave transcription factors (Park et al., 2015). Use of the cbf1/2/3 triple mutant showed that six first-wave transcription factors are partially regulated by CBF/DREB1, whereas the transcription factors HSFC1, ZAT12, and CZF1, which regulate cold-regulated genes (Vogel et al., 2005; Park et al., 2015), are not regulated by CBF/DREB1 (Zhao et al., 2016). As acclimated cbf1/2/3 triple mutants are more tolerant to freezing stress than non-acclimated ones (Jia et al., 2016), and the expression of a large number of cold-regulated genes is not affected by the cbf1/2/3 triple mutation (Zhao et al., 2016), a CBF/DREB1-independent pathway may control cold tolerance. Overexpression of HSFC1 enhances cold tolerance without an increase in expression of CBF1, CBF2, or CBF3 (Park et al., 2015), suggesting that HSFC1 is one of the important transcription factors controlling non-CBF/DREB1 regulons and cold tolerance.
This research demonstrates that MCA1 and MCA2 are involved in a transient rise in [Ca2+]cyt upon cold shock. The cold-induced increase in [Ca2+]cyt was smaller in the mca1 and mca2 mutants than in the Col-0 wild type. The mca1 mca2 double mutant exhibited increased sensitivity to chilling and freezing stresses. These results suggest that MCA1 and MCA2 are involved in cold-induced Ca2+ influx and that the reduced [Ca2+]cyt increase caused by the mca1 and mca2 mutations affects cold acclimation. As the CBF/DREB1 genes and their regulon genes were not down-regulated in the mca1 mca2 mutant, MCA may not be involved in the regulation of CBF/DREB1-dependent cold signaling.
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