HHP1, a novel signalling component in the cross-talk between the cold and osmotic signalling pathways in Arabidopsis

Chin-Chung Chen 0 Ching-Shin Liang 0 Ai-Ling Kao 0 Chien-Chih Yang 0 1 0 Institute of Microbiology and Biochemistry, National Taiwan University , 1 Sec. 4, Roosevelt Road, Taipei, Taiwan 1 Department of Biochemical Science and Technology, National Taiwan University , 1 Sec. 4, Roosevelt Road, Taipei, Taiwan Heptahelical protein 1 (HHP1) is a negative regulator in abscisic acid (ABA) and osmotic signalling in Arabidopsis. The physiological role of HHP1 was further investigated in this study using transgenic and knock-out plants. In HHP1::GUS transgenic mutants, GUS activity was found to be mainly expressed in the roots, vasculature, stomata, hydathodes, adhesion zones, and connection sites between septa and seeds, regions in which the regulation of turgor pressure is crucial. By measuring transpiration rate and stomatal closure, it was shown that the guard cells in the hhp1-1 mutant had a decreased sensitivity to drought and ABA stress compared with the WT or the c-hhp1-1 mutant, a complementation mutant of HHP1 expressing the HHP1 gene. The N-terminal fragment (amino acids 1-96) of HHP1 was found to interact with the transcription factor inducer of CBF expression-1 (ICE1) in yeast two-hybrid and bimolecular fluorescence complementation (BiFC) studies. The hhp1-1 mutant grown in soil showed hypersensitivity to cold stress with limited watering. The expression of two ICE1-regulated genes (CBF3 and MYB15) and several other cold stress-responsive genes (RD29A, KIN1, COR15A, and COR47) was less sensitive to cold stress in the hhp1-1 mutant than in the WT. These data suggest that HHP1 may function in the cross-talk between cold and osmotic signalling. - Plants use an interconnected signalling network to cope with the abiotic stresses of drought, high salt, and cold/ low temperature (Chinnusamy et al., 2005; YamaguchiShinozaki and Shinozaki, 2006). Stresses can occur at different growth stages during development and more than one stress can affect the plant simultaneously. Drought stress restricts the growth of plants due to the stress of osmosis (osmotic stress), leading to a lack of nutrients and reduced photosynthesis (Zhu, 2002). Salt stress leads to physiological drought and ion toxicity, which limit plant growth (Zhu, 2002). Osmotic stress is also brought about by chilling (low temperatures above freezing) and freezing temperatures (Thomashow, 1999). Osmotic stresses resulting from drought, high salt, and cold are transduced by plants by either an abscisic acid (ABA)-dependent or an ABA-independent signalling pathway (Shinozaki and Yamaguchi-Shinozaki, 2007). These pathways lead to the expression of some common downstream stress-responsive proteins, such as RD29A, RD29B, COR15A, COR47, KIN1, and ADH1 (Yamaguchi-Shinozaki and Shinozaki, 1993, 1994; Stockinger et al., 1997). These stress-responsive proteins can be classified into two categories, those functioning in stress tolerance and those involved in signal transduction. The proteins that are directly involved in tolerance include chaperones, late embryogenesis abundant proteins, osmotin, anti-freeze proteins, mRNA-binding proteins, water channel proteins, and several key enzymes involved in the biosynthesis of osmolytes, such as proline and sugar (Fowler and Thomashow, 2002; Kreps et al., 2002; Seki et al., 2002). The proteins involved in signal transduction and the regulation of gene expression include various transcription factors, which may act co-operatively (Yamaguchi-Shinozaki and Shinozaki, 2006; Chinnusamy et al., 2007). Due to the complex environment that plants face, it is expected that more signalling components involved in plant responses to abiotic stresses remain to be discovered. One candidate is heptahelical protein 1 (HHP1), which may function as a negative regulator in ABA and osmotic signalling (Chen et al., 2009). HHP1 is a member of the HHP family in Arabidopsis that consists of at least five members HHP1, HHP2, HHP3, HHP4, and HHP5 (Hsieh and Goodman, 2005). HHP family proteins are homologous to PAQR family proteins, which include the membrane progestin receptor from fish, the adiponectin receptor from mouse, and YOL002c from yeast (Yamauchi et al., 2003; Zhu et al., 2003; Lyons et al., 2004; Tang et al., 2005). As shown in our previous study (Chen et al., 2009), HHP1 may be involved in stress sensitivity and act as a negative regulator in response to ABA and osmotic stress. The HHP1 T-DNA insertion mutant hhp1-1 shows a higher sensitivity to ABA and osmotic stress than the wild-type (WT), as shown by the germination rate and post-germination growth rate, and the induced expression of stress-responsive genes (RD29A, RD29B, ADH1, KIN1, COR15A, and COR47) is more sensitive to exogenous ABA and osmotic stress in the hhp1-1 mutant than in the WT. The hypersensitivity of the hhp1-1 mutant is reversed in c-hhp1-1, a complementation mutant of HHP1 expressing the HHP1 gene. These data show that mutation of HHP1 renders plants hypersensitive to ABA and osmotic stress and that HHP1 might be a negative regulator in ABA and osmotic signalling. A gene network that can collect and interpret abiotic stresses, including drought, salt, and cold, has been described (Shinozaki et al., 2003; Yamaguchi-Shinozaki and Shinozaki, 2006; Chinnusamy et al., 2007; Shinozaki and Yamaguchi-Shinozaki, 2007; Tran et al., 2007). Central to this network are several transcription factors, including MYB2, NAC, ABF, DREB2, CBF, and ICE1 (YamaguchiShinozaki and Shinozaki, 2006; Chinnusamy et al., 2007), which are regulated through ABA-independent and ABAdependent pathways (Shinozaki and Yamaguchi-Shinozaki, 2007). It is expected that more signalling components will be found. ICE1 encodes a MYC-like basic helix-loop-helix transcription factor that regulates the expression of CBF3/ DREB1A-controlled genes responsible for cold tolerance (Chinnusamy et al., 2003). The activity of ICE1 is regulated by two opposing processes, sumoylation by SIZ1, which activates ICE1, and ubiquitination by HOS1, which causes degradation of ICE1 (Dong et al., 2006; Miura et al., 2007). Cold stress limits the normal growth development of plants directly by the inhibition of metabolic reactions and indirectly through cold-induced osmotic (chilling-induced inhibition of water uptake and freezing-induced cellular dehydration) and oxidative stresses (Chinnusamy et al., 2007). On exposure to low temperatures above freezing, plants can acquire freezing tolerance and this process is called cold acclimation (Chinnusamy et al., 2007). Coldstress signalling in plants is considered to be ABAindependent, although HOS10 is speculated to regulate ABA-mediated cold acclimation (Zhu et al., 2005). ICE1 and C-repeat-binding factors (CBFs), also known as dehydration-responsive element-binding protein 1s (DREB1s), form a transcriptional cascade that is the best-characterized of the transcription factor pathways involved in cold stressresponsive mechanisms (Lee et al., (...truncated)