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Gene Expression of Hsp90 Members and HOP2 in Response to Light Exposure in Arabidopsis Thaliana

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Gene Expression of Hsp90 Members and HOP2 in Response to Light Exposure in Arabidopsis Thaliana
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    #$% &$'() *++),) -)%./$%/0% 1$220% Gene expression of Hsp90 members and HOP2 in response to light exposure in  Arabidopsis thaliana   Abstract Hsp90 is involved in responses to environmental stressors, and functions as a buffering mechanism that permits the concealment and retention of mutations by maintaining homeostatic stability. Hsp90 is highly conserved across species and  A. thaliana  has seven homolog members. The role of Hsp90 members in maintaining molecular stability and retaining variation, and the role of HOP2 during stressful conditions may be involved in gene expression and phenotypic changes in response to light exposure. However, research to determine the impact of light variation on the differential expression of Hsp90 and HOP2 is scant. We hypothesized that different lengths of light exposure would induce gene expression differences in Hsp90-5, Hsp90-6, Hsp90-7, and HOP2. The parental generation of  A. thaliana  grew under low light or high light exposure. Nine replicates of sixteen genotypes were planted. Three seeds were taken from three replicates of each genotype and transplanted into four possible offspring chambers: [high to high], [high to low], [low to low], [low to high]. We compared gene expression between the groups. Differences in length of light exposure of the parents or the offspring did not produce differences in the expression of HOP2 or any of the Hsp90 members. However, HOP2 expression was found to be strongly associated with Hsp90-6 expression using bivariate models. This study did not account for timing, intensity and spectrum of light exposure, or gene expression in different tissues. Future research should evaluate these variables. Introduction As an essential component to plant functions, light exposure mediates a myriad of factors  paramount to organismal viability. Studies have shown that variant lighting schemes in plants have produced differential phenotypic expression (Stanton, Weeks, Dana, & Mickelbart, 2010). Transitively, studies have also shown that variable light exposure produces differential gene expression in  A. Thaliana  (Rossel, Wilson, & Pogson, 2002). Light induced loci expression has even been shown to reposition light-reactive proteins after transcription (Miao & Qui, 2013). Photoreactive proteins are some of the more light-sensitive gene transcripts relative to the  A. Thaliana  genome, supplementary proteins such as heat shock protein 90 and histidine  phototransfer protein HOP2 may also exhibit differential gene expression as a result of light differences.  Hsp90: A Possible Link to Light Stress Response  Heat shock protein 90 (Hsp90) is a member of the chaperone system, which is involved in  protein assembly and conformation, and molecular stability under heat and oxidative stress (Wang, Vinocur, Shoseyov, & Altman, 2004). Seven members of the Hsp90 family (also known as Hsp81) have been identified in  Arabidopsis thaliana (A. thaliana) . The cytoplasmic subfamily is composed of AtHsp90-1 to AtHsp90-4; AtHsp90-5 is the plastid member, AtHsp90-6 is the mitochondrial member, and AtHsp90-7 is the endoplasmic reticulum member (Cha et al., 2013; Krishna & Gloor, 2001).      #$% &$'() *++),) -)%./$%/0% 1$220% The Hsp90 family is involved in pathogen and disease resistance in  A. thaliana  (Takahashi, Casais, Ichimura, & Shirasu, 2003). Exposure to cantharidin , from the blister beetle (Epicauta spp.) causes a hypersensitive response to the pathogen in  A. thaliana , and activates the expression of    Hsp81-1 (Bajsa, Pan, & Duke, 2011). Protein RPM1 is a client protein of Hsp90 in  A. thaliana , and is involved in disease resistance to  Pseudomonas syringae  (Hubert et al., 2003). Rhizosphere interactions between  A. thaliana  and fungus  Paraphaeosphaeria quadriseptata  via cocultivation influence environmental responses to heat stress and growth in  A. thaliana . Hsp90 inhibitors produced by  P. quadriseptata increase tolerance to heat stress in  A. thaliana  (McLellan et al., 2007). The Hsp90 family is regulated during growth and development, and its expression in  A. thaliana  is influenced by cold, heat, salt stress, hormones, oxidative stress and light exposure (Haralampidis, Milioni, Rigas, & Hatzopoulos, 2002; Krishna & Gloor, 2001; Milioni & Hatzopoulos, 1997; Yamada et al., 2007). Excessive light exposure in plants elicit oxidative stress due to reactive oxygen species buildup (Carvalho, Vilela, Mullineaux, & Amâncio, 2011). The CR88 gene is essential for chloroplast biogenesis and encodes for Hsp90 in the chloroplasts of  A. thaliana . In  A. thaliana  CR88 is highly expressed in leaves, reproductive organs, and during post germination (Cao, Froehlich, Zhang, & Cheng, 2003; Lin & Cheng, 1997).  AtHsp90-5 Heat shock protein 90-5 (Hsp90-5) is localized in plastids. Evidence indicates that the protein is an integral part of a chaperone complex (containing Tic110, Tic40, Toc75, Tic22, and the stromal chaperones, Hsp93 and Hsp70) in the chloroplast stroma that facilitates membrane translocation during protein import into the organelle and is thusly essential to viability (Inoue, Li, & Schnell, 2013). The proteins association with the photosynthetic complex makes it a worthwhile candidate for light reactive experimentation as studies have shown that  A. Thaliana Hsp90-5 mutants (G646R) respond differentially to red light, resulting in delays in chloroplast development (Cao et al., 2003). Additional studies have proposed that, as a function of stress response, Hsp90-5 functions in the transduction of photo-regulative signaling cascades (Altieri, Stein, Lian, & Languino, 2012; M Marzec, D, & Argon, 2012; Michal Marzec, Eletto, & Argon, 2012; Taipale, Jerosz, & Lindquist, 2010).  AtHsp90-6 Heat shock protein 90-6 (Hsp90-6) is the mitochondrial member of the Hsp90 family. In  A. thaliana , AtHsp90-6 is most closely related to AtHsp90-5 than to any of the other AtHsp90 family members (Krishna & Gloor, 2001). The AtHsp90-6 and the AtHsp90-5 genes include  between 18 and 19 introns, and AtHsp90-6 expression is minimally influenced by exposure to heat (Milioni & Hatzopoulos, 1997). Plastid activity is related to ATP production in the mitochondria via oxidative phosphorylation (Dat et al., 2000). Overexposure to UV light increases mitochondrial activity and the production of reactive oxygen species in  A. thaliana  (Gao, Xing, Li, & Zhang, 2008). Therefore, length of light exposure is likely to influence mitochondrial function and the expression of the AtHsp90-6 gene.    #$% &$'() *++),) -)%./$%/0% 1$220%  AtHsp90-7 Heat shock protein 90-7 (Hsp90-7) is the endoplasmic reticulum member of the Hsp90 family. Mutations in the Hsp90-7 gene have been reported to produce phenotypic changes in floral and shoot structures (Sangster & Queitsch, 2005). The AtHsp90-7 sequence is similar to the sequence of Catharanthus roseus  and    Hordeum vulgare  (Schröder, Beck, Eichel, Vetter, & Schröder, 1993; Walther-Larsen, Brandt, Collinge, & Thordal-Christensen, 1993). Plant cells have strategies for coping with stress in the endoplasmic reticulum by increasing activities such as protein folding, unfolded protein degradation, and apoptosis, without changes in gene regulation (Kamauchi, Nakatani, Nakano, & Urade, 2005). However, changes in AtHsp90-7 gene expression can be elicited by endoplasmic reticulum stress (Bruhat et al., 2002; Koizumi et al., 2001). If length of light exposure can influence protein folding activities in the endoplasmic reticulum, it is plausible that it can have an effect on AtHsp90-7 gene expression.  Histidine Phototransfer Protein HOP2: Mediating Abiotic Stress Response  Also known as histidine    phosphotransfer protein AHP2, HOP2 is an essential component of cytokinin (plant growth hormone) signaling and shuttling from the cytoplasm to the nucleus. Histidine protein kinases (AHKs) receive cytokinin, the signal is subsequently transmitted via AHPs to nuclear response regulators which influence transcription. It has been implicated in mediating drought response in a negative and redundant manner —down-regulated under conditions of severe dehydration (Nishiyama, Watanabe, Leyva, & Ha, 2013) and facilitating expression of protein Arabidopsis Response Regulator (ARR) to negatively regulate cytokinin signaling during cold response (J. P. To et al., 2007). The protein has also been found to play a central role in gametogenesis by facilitating the segregation of homologous chromosomes during meiosis (Schommer, Beven, Lawrenson, Shaw, & Sablowski, 2003). Hop2’s role as a manifold mediator in abiotic response makes it a possible candidate for photoreactive expression studies. Aims and Hypotheses Previous research has shown that Hsp90 is highly preserved across species, and strongly suggests that Hsp90 can function as a capacitor for morphological evolution by maintaining cryptic variation (Gibson & Dworkin, 2004; Rohner et al., 2013; Rutherford & Lindquist, 1998). In  A. thaliana , the Hsp90 members function as a buffering mechanism that permit the concealment and retention of mutations by maintaining homeostatic stability. When environmental stressors surpass its buffering effects, hidden variability can become expressed. In this sense, Hsp90 is likely to be an important mediator of phenotypic plasticity and evolutionary adaptation in  A. thaliana  (Queitsch, Sangster, & Lindquist, 2002; Sangster et al., 2007; Sangster & Queitsch, 2005; Sangster et al., 2008; Wang et al., 2004). As a function of Hsp90s role in maintaining molecular stability and retaining variation, responses to different light exposures may be accompanied by changes in Hsp90 gene expression in    A. thaliana . In addition, Hsp90 might be involved in epigenetic inheritance by providing molecular stability to phenotypic plasticity that has resulted from different light exposures. In the present study with  A. thaliana , we aimed to: 1) Assess the effect of parental light exposure on gene    #$% &$'() *++),) -)%./$%/0% 1$220% expression in the offspring under differential light exposure. 2) Measure gene expression differences in the offspring in response to different lengths of light exposure. Based on the aforementioned observations, we hypothesize that different lengths of light exposure in the parental population can induce gene expression differences in AtHsp90-5, AtHsp90-6, AtHsp90-7, and HOP 2  in the offspring. We also posit that different lengths of light exposure in the offspring can induce gene expression differences in AtHsp90-5, AtHsp90-6, AtHsp90-7, and HOP2. These postulates also presume that differences between genotypes will also yield significant variation amongst the experimental groups. Methods Utilizing a transplant design, the parent generation was grown in low light and high light chambers. We exposed the “low light” green group to 10 hours of light and 14 hours of darkness, and the “high light” orange group to 14 hours of light and 10 hours of darkness. Nine replicates of sixteen genotypes were planted. Three seeds were taken from three replicates of each genotype and transplanted into four possible offspring chambers: [high light to high light], [high light to low light], [low light to low light], [low light to high light] (figure 1.1) Figure 1.1:  A. Thaliana Experimental Design   Figure 1.1: The parent generation consisted of 9 replicates of 16 genotypes in high light (yellow) and low light (purple) environments. Three plants were chosen from each genotype and 3 seeds were harvested in each plant and cultivated according to the above scheme.

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