Phosphoprotein Sak1 Is a Regulator of Acclimation to Singlet Oxygen in 1

32 Singlet oxygen is a highly toxic and inevitable byproduct of oxygenic photosynthesis. 33 The unicellular green alga Chlamydomonas reinhardtii is capable of acclimating 34 specifically to singlet oxygen stress, but the retrograde signaling pathway from the 35 chloroplast to the nucleus mediating this response is unknown. Here we describe a mutant, 36 singlet oxygen acclimation knocked-out 1 (sak1), that lacks the acclimation response to 37 singlet oxygen. Analysis of genome-wide changes in RNA abundance during acclimation 38 to singlet oxygen revealed that SAK1 is a key regulator of the gene expression response 39 during acclimation. The SAK1 gene encodes an uncharacterized protein with a domain 40 conserved among chlorophytes and present in some bZIP transcription factors. The SAK1 41 protein is located in the cytosol, and it is induced and phosphorylated upon exposure to 42 singlet oxygen, suggesting that it is a critical intermediate component of the retrograde 43 signal transduction pathway leading to singlet oxygen acclimation. 44 45 46 47 Introduction 48 Growth of photosynthetic organisms depends on light energy, which in turn can cause 49 oxidative damage to the cell if not managed properly (Li et al., 2009). Light intensity is 50 highly dynamic in terrestrial and aquatic environments, and the cell must constantly 51 control the dissipation of light energy to avoid photo-oxidative stress while maximizing 52 productivity. In addition to being the site of photosynthesis, the chloroplast houses many 53 essential biochemical reactions such as fatty acid and amino acid biosynthesis, but most 54 of its proteins are encoded in the nucleus and must be imported after translation. 55 Therefore the nucleus must monitor the status of the chloroplast and coordinate gene 56 expression and synthesis of proteins to maintain healthy chloroplast functions. 57 It is known that signals originating from a stressed or dysfunctional chloroplast 58 modulate nuclear gene expression, a process that is called retrograde signaling (Nott et 59 al., 2006; Chi et al., 2013). In Arabidopsis thaliana the gun mutants have helped to define 60 the field of chloroplast retrograde signaling, leading to the identification of GUN1, a 61 pentatricopeptide repeat protein that is a regulator of this process (Koussevitzky et al., 62 2007), and pointing to the involvement of the tetrapyrrole biosynthetic pathway (Vinti et 63 al., 2000; Mochizuki et al., 2001; Larkin et al., 2003; Strand et al., 2003; Woodson and 64 Chory, 2008). A role for heme in retrograde signaling has been shown in 65 Chlamydomonas reinhardtii as well (von Gromoff et al., 2008). Many of the gun studies 66 were conducted in context of a dysfunctional chloroplast treated with norflurazon, an 67 inhibitor of carotenoid biosynthesis. More recently a number of exciting advances have 68 shed light on small molecules playing roles in retrograde stress signaling, including 69 methylerythritol cyclodiphosphate, an intermediate of isoprenoid biosynthesis in the 70 chloroplast (Xiao et al., 2012), 3-phosphoadenosine 5-phosphate (PAP) (Estavillo et al., 71 2011), as well as a chloroplast envelope transcription factor PTM (Sun et al., 2011). 72 Plastid gene expression involving sigma factors has been implicated in affecting nuclear 73 gene expression, although the mechanism is unknown (Coll et al., 2009; Woodson et al., 74 2012). 75 Activation of gene expression by reactive oxygen species (ROS) has been well 76 documented (Apel and Hirt, 2004; Mittler et al., 2004; Gadjev et al., 2006; Li et al., 77 2009). Thus ROS have been proposed as a means for chloroplasts to signal stress to the 78 nucleus and many examples of global gene expression changes in response to ROS have 79 been described (Desikan et al., 2001; Vandenabeele et al., 2004; Vanderauwera et al., 8