Calcineurin B-Like Protein-Interacting Protein Kinase CIPK21 Regulates Osmotic and Salt Stress Responses in Arabidopsis

The role of calcium-mediated signaling has been extensively studied in plant responses to abiotic stress signals. Calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs) constitute a complex signaling network acting in diverse plant stress responses. Osmotic stress imposed by soil salinity and drought is a major abiotic stress that impedes plant growth and development and involves calcium-signaling processes. In this study, we report the functional analysis of CIPK21, an Arabidopsis (Arabidopsis thaliana) CBL-interacting protein kinase, ubiquitously expressed in plant tissues and up-regulated under multiple abiotic stress conditions. The growth of a loss-of-function mutant of CIPK21, cipk21, was hypersensitive to high salt and osmotic stress conditions. The calcium sensors CBL2 and CBL3 were found to physically interact with CIPK21 and target this kinase to the tonoplast. Moreover, preferential localization of CIPK21 to the tonoplast was detected under salt stress condition when coexpressed with CBL2 or CBL3. These findings suggest that CIPK21 mediates responses to salt stress condition in Arabidopsis, at least in part, by regulating ion and water homeostasis across the vacuolar membranes.Drought and salinity cause osmotic stress in plants and severely affect crop productivity throughout the world. Plants respond to osmotic stress by changing a number of cellular processes (Xiong et al., 1999; Xiong and Zhu, 2002; Bartels and Sunkar, 2005; Boudsocq and Lauriére, 2005). Some of these changes include activation of stress-responsive genes, regulation of membrane transport at both plasma membrane (PM) and vacuolar membrane (tonoplast) to maintain water and ionic homeostasis, and metabolic changes to produce compatible osmolytes such as Pro (Stewart and Lee, 1974; Krasensky and Jonak, 2012). It has been well established that a specific calcium (Ca²⁺) signature is generated in response to a particular environmental stimulus (Trewavas and Malhó, 1998; Scrase-Field and Knight, 2003; Luan, 2009; Kudla et al., 2010). The Ca²⁺ changes are primarily perceived by several Ca²⁺ sensors such as calmodulin (Reddy, 2001; Luan et al., 2002), Ca²⁺-dependent protein kinases (Harper and Harmon, 2005), calcineurin B-like proteins (CBLs; Luan et al., 2002; Batistič and Kudla, 2004; Pandey, 2008; Luan, 2009; Sanyal et al., 2015), and other Ca²⁺-binding proteins (Reddy, 2001; Shao et al., 2008) to initiate various cellular responses.Plant CBL-type Ca²⁺ sensors interact with and activate CBL-interacting protein kinases (CIPKs) that phosphorylate downstream components to transduce Ca²⁺ signals (Liu et al., 2000; Luan et al., 2002; Batistič and Kudla, 2004; Luan, 2009). In several plant species, multiple members have been identified in the CBL and CIPK family (Luan et al., 2002; Kolukisaoglu et al., 2004; Pandey, 2008; Batistič and Kudla, 2009; Weinl and Kudla, 2009; Pandey et al., 2014). Involvement of specific CBL-CIPK pair to decode a particular type of signal entails the alternative and selective complex formation leading to stimulus-response coupling (D’Angelo et al., 2006; Batistič et al., 2010).Several CBL and CIPK family members have been implicated in plant responses to drought, salinity, and osmotic stress based on genetic analysis of Arabidopsis (Arabidopsis thaliana) mutants (Zhu, 2002; Cheong et al., 2003, 2007; Kim et al., 2003; Pandey et al., 2004, 2008; D’Angelo et al., 2006; Qin et al., 2008; Tripathi et al., 2009; Held et al., 2011; Tang et al., 2012; Drerup et al., 2013; Eckert et al., 2014). A few CIPKs have also been functionally characterized by gain-of-function approach in crop plants such as rice (Oryza sativa), pea (Pisum sativum), and maize (Zea mays) and were found to be involved in osmotic stress responses (Mahajan et al., 2006; Xiang et al., 2007; Yang et al., 2008; Tripathi et al., 2009; Zhao et al., 2009; Cuéllar et al., 2010).In this report, we examined the role of the Arabidopsis CIPK21 gene in osmotic stress response by reverse genetic analysis. The loss-of-function mutant plants became hypersensitive to salt and mannitol stress conditions, suggesting that CIPK21 is involved in the regulation of osmotic stress response in Arabidopsis. These findings are further supported by an enhanced tonoplast targeting of the cytoplasmic CIPK21 through interaction with the vacuolar Ca²⁺ sensors CBL2 and CBL3 under salt stress condition.     

Analysis of the Rice ADP-Glucose Transporter (OsBT1) Indicates the Presence of Regulatory Processes in the Amyloplast Stroma That Control ADP-Glucose Flux into Starch

Previous studies showed that efforts to further elevate starch synthesis in rice (Oryza sativa) seeds overproducing ADP-glucose (ADPglc) were prevented by processes downstream of ADPglc synthesis. Here, we identified the major ADPglc transporter by studying the shrunken3 locus of the EM1093 rice line, which harbors a mutation in the BRITTLE1 (BT1) adenylate transporter (OsBt1) gene. Despite containing elevated ADPglc levels (approximately 10-fold) compared with the wild-type, EM1093 grains are small and shriveled due to the reduction in the amounts and size of starch granules. Increases in ADPglc levels in EM1093 were due to their poor uptake of ADP-[¹⁴C]glc by amyloplasts. To assess the potential role of BT1 as a rate-determining step in starch biosynthesis, the maize ZmBt1 gene was overexpressed in the wild-type and the GlgC (CS8) transgenic line expressing a bacterial glgC-TM gene. ADPglc transport assays indicated that transgenic lines expressing ZmBT1 alone or combined with GlgC exhibited higher rates of transport (approximately 2-fold), with the GlgC (CS8) and GlgC/ZmBT1 (CS8/AT5) lines showing elevated ADPglc levels in amyloplasts. These increases, however, did not lead to further enhancement in seed weights even when these plant lines were grown under elevated CO₂. Overall, our results indicate that rice lines with enhanced ADPglc synthesis and import into amyloplasts reveal additional barriers within the stroma that restrict maximum carbon flow into starch.Cereal grains contribute a significant portion of worldwide starch production. Unlike other plant tissue, starch biosynthesis in the endosperm storage organ of cereal grains is unique in its dependence on two ADP-Glc pyrophosphorylase (AGPase) isoforms (Denyer et al., 1996; Thorbjørnsen et al., 1996; Sikka et al., 2001), a major cytosolic enzyme and a minor plastidial one, to generate ADP-glucose (ADPglc), the sugar nucleotide utilized by starch synthases in the amyloplast (Cakir et al., 2015). The majority of ADPglc in cereal endosperm is generated in the cytosol from AGPase (Tuncel and Okita, 2013) as well as by Suc synthase (Tuncel and Okita, 2013; Bahaji et al., 2014) and subsequently transported into amyloplasts by the BRITTLE-1 (BT1) protein located at the plastid envelope (Cao et al., 1995; Shannon et al., 1998).The Bt1 gene, first identified in maize (Zea mays; Mangelsdorf, 1926) and isolated by Sullivan et al. (1991), encodes a major amyloplast membrane protein ranging from 39 to 44 kD (Cao et al., 1995). The BT1 protein and its homologs belong to the mitochondrial carrier family (Sullivan et al., 1991; Haferkamp, 2007), which has a diverse range of substrates (Patron et al., 2004; Leroch et al., 2005; Kirchberger et al., 2008). The assignment of BT1 protein as the ADPglc transporter in cereal endosperms was first proposed by Sullivan et al. (1991), and then it was characterized based on the increased ADPglc levels and reduced ADPglc import rate in endosperms of BT1-deficient maize and barley (Hordeum vulgare) mutants (Tobias et al., 1992; Shannon et al., 1996, 1998; Patron et al., 2004). Biochemical transport studies of the maize BT1 showed that it imported ADPglc by counter exchanging with ADP (Kirchberger et al., 2007). The wheat (Triticum aestivum) BT1 homolog also transports ADPglc but has similar affinities for ADP and AMP as the counter-exchange substrate (Bowsher et al., 2007).Evidence from previous studies by our laboratory (Sakulsingharoj et al., 2004; Nagai et al., 2009) suggested the potential role of BT1 as well as other downstream processes as a rate-limiting step in starch biosynthesis in the transgenic rice (Oryza sativa) GlgC (CS8) lines overexpressing an up-regulated AGPase (Escherichia coli glgC-TM). In GlgC (CS8) rice lines, grain weights (starch) are elevated up to 15% compared with wild-type plants, indicating that the AGPase-catalyzed reaction is a rate-limiting step in starch biosynthesis under normal conditions. When transgenic GlgC (CS8) plants were grown under elevated CO₂ levels, no further increases in grain weight were evident compared with those grown at ambient CO₂. As Suc levels are elevated in leaf blades, leaf sheaths, culms (Rowland-Bamford et al., 1990), and peduncle exudates (Chen et al., 1994) in rice plants grown under elevated CO₂, developing GlgC (CS8) grains were unable to convert the increased levels of sugars into starch. This lack of increase indicated that the AGPase-catalyzed reaction (ADPglc synthesis) was no longer rate limiting and that one or more downstream processes regulated carbon flux from source tissues in developing GlgC (CS8) endosperm (Sakulsingharoj et al., 2004). This view is also supported by a subsequent metabolite study in which several GlgC (CS8) lines were found to contain up to 46% higher ADPglc levels than wild-type plants (Nagai et al., 2009). As this increase in ADPglc levels was nearly 3-fold higher than the increase in grain weight, starch biosynthesis is saturated with respect to ADPglc levels and carbon flow into starch is restricted by one or more downstream steps. Potential events that may limit the utilization of ADPglc in starch in GlgC (CS8) lines are the import of this sugar nucleotide via the BT1 transporter into amyloplasts and/or the utilization of ADPglc by starch synthases. Mutant analysis of the two major starch synthases indicated no significant impact on grain weight when one of these starch synthases was nonfunctional, suggesting that this enzyme activity, contributed by multiple enzyme isoforms, is present at excessive levels (Fujita et al., 2006, 2007). Therefore, we suspected that BT1 is the likely candidate limiting carbon flow into starch in GlgC (CS8) endosperms.The aim of this study was to investigate the role of BT1 in mediating the transport of ADPglc into amyloplast and to determine whether this transport activity is rate limiting in rice endosperm. In order to address these questions, we show that BT1 is the major transporter of ADPglc by analysis of the EM1093 rice line, which contains a mutation at the shrunken3 (shr3) locus and, specifically, in the OsBt1-1 gene. Second, we assessed the impact of the expression of the maize ZmBt1 gene in wild-type and GlgC (CS8) seeds to determine the potential limiting role of BT1 transport activity on starch biosynthesis. Our results indicate that BT1 is essential for starch synthesis but is not rate limiting and that one or more stroma-localized processes limit maximum carbon flow into starch.     

Enhanced Photosynthesis and Growth in atquac1 Knockout Mutants Are Due to Altered Organic Acid Accumulation and an Increase in Both Stomatal and Mesophyll Conductance

Stomata control the exchange of CO₂ and water vapor in land plants. Thus, whereas a constant supply of CO₂ is required to maintain adequate rates of photosynthesis, the accompanying water losses must be tightly regulated to prevent dehydration and undesired metabolic changes. Accordingly, the uptake or release of ions and metabolites from guard cells is necessary to achieve normal stomatal function. The AtQUAC1, an R-type anion channel responsible for the release of malate from guard cells, is essential for efficient stomatal closure. Here, we demonstrate that mutant plants lacking AtQUAC1 accumulated higher levels of malate and fumarate. These mutant plants not only display slower stomatal closure in response to increased CO₂ concentration and dark but are also characterized by improved mesophyll conductance. These responses were accompanied by increases in both photosynthesis and respiration rates, without affecting the activity of photosynthetic and respiratory enzymes and the expression of other transporter genes in guard cells, which ultimately led to improved growth. Collectively, our results highlight that the transport of organic acids plays a key role in plant cell metabolism and demonstrate that AtQUAC1 reduce diffusive limitations to photosynthesis, which, at least partially, explain the observed increments in growth under well-watered conditions.Stomata are functionally specialized microscopic pores that control the essential exchange of CO₂ and H₂O with the environment in land plants. Stomata are found on the surfaces of the majority of the aerial parts of plants, rendering them as the main control point regulating the flow of gases between plants and their surrounding atmosphere. Accordingly, the majority of water loss from plants occurs through stomatal pores, allowing plant transpiration and CO₂ absorption for the photosynthetic process (Bergmann and Sack, 2007; Kim et al., 2010). The maintenance of an adequate water balance through stomatal control is crucial to plants because cell expansion and growth require tissues to remain turgid (Sablowski and Carnier Dornelas, 2014), and minor reductions in cell water volume and turgor pressure will therefore compromise both processes (Thompson, 2005). As a result, the high sensitivity of plant tissues to turgor has prompted the use of reverse genetic studies in attempt to engineer plants with improved performance (Cowan and Troughton, 1971; Xiong et al., 2009; Borland et al., 2014; Franks et al., 2015).In most land plants, not only redox signals invoked by shifts in light quality (Busch, 2014) but also the transport of inorganic ions (e.g. K⁺, Cl⁻, and NO⁻₃) as well as metabolites such as the phytohormone abscisic acid (ABA), Suc, and malate, are important players controlling stomatal movements (Hetherington, 2001; Roelfsema and Hedrich, 2005; Pandey et al., 2007; Blatt et al., 2014; Kollist et al., 2014). In this context, although organic acids in plants is known to support numerous and diverse functions both within and beyond cellular metabolism, only recently have we obtained genetic evidence to support that modulation of guard cell malate and fumarate concentration can greatly influence stomatal movements (Nunes-Nesi et al., 2007; Araújo et al., 2011b; Penfield et al., 2012; Medeiros et al., 2015). Notably malate, in particular, has been considered as a key metabolite and one of the most important organic metabolites involved in guard cell movements (Hedrich and Marten, 1993; Fernie and Martinoia, 2009; Meyer et al., 2010). During stomatal aperture, the flux of malate into guard cells coupled with hexoses generated on starch breakdown lead to decreases in the water potential, and consequently, water uptake by the guard cells ultimately opens the stomata pore (Roelfsema and Hedrich, 2005; Vavasseur and Raghavendra, 2005; Lee et al., 2008). On the other hand, during stomatal closure, malate is believed to be converted into starch, which has no osmotic activity (Penfield et al., 2012) or, alternatively, is released from the guard cells to the surrounding apoplastic space (Lee et al., 2008; Negi et al., 2008; Vahisalu et al., 2008; Meyer et al., 2010).The role of organic acids on the stomatal movements has been largely demonstrated by studies related to malate transport (Lee et al., 2008; Meyer et al., 2010; Sasaki et al., 2010). In the last decade, two protein families were identified and functionally characterized to be directly involved with organic acid transport at the guard cell plasma membrane and to be required for stomatal functioning (Lee et al., 2008; Meyer et al., 2010; Sasaki et al., 2010). In summary, AtABCB14, a member of the ABC (ATP binding cassette) family, which is involved in malate transport from apoplast to guard cells, was described as a negative modulator of stomatal closure induced by high CO₂ concentration; notably, exogenous application of malate minimizes this response (Lee et al., 2008). In addition, members of a small gene family, which encode the anion channels SLAC1 (slow anion channel 1) and four SLAC1-homologs (SLAHs) in Arabidopsis (Arabidopsis thaliana), have been described to be involved in stomatal movements. SLAC1 is a well-documented S-type anion channel that preferentially transports chloride and nitrate as opposed to malate (Vahisalu et al., 2008, 2010; Geiger et al., 2010; Du et al., 2011; Brandt et al., 2012; Kusumi et al., 2012). Lack of SLAC1 in Arabidopsis and rice (Oryza sativa) culminated in a failure in stomatal closure in response to high CO₂ levels, low relative humidity, and dark conditions (Negi et al., 2008; Vahisalu et al., 2008; Kusumi et al., 2012). Although mutations in AtSLAC1 impair S-type anion channel functions as a whole, the R-type anion channel remained functional (Vahisalu et al., 2008). Indeed, a member of the aluminum-activated malate transporter (ALMT) family, AtALMT12, an R-type anion channel, has been demonstrated to be involved in malate transport, particularly at the plasma membrane of guard cells (Meyer et al., 2010; Sasaki et al., 2010). Although AtALMT12 is a member of ALMT family, it is not activated by aluminum, and therefore Meyer et al. (2010) proposed to rename it as AtQUAC1 (quick-activating anion channel 1; Imes et al., 2013; Mumm et al., 2013). Hereafter, we will follow this nomenclature. Deficiency of a functional AtQUAC1 has been documented to lead to changes in stomatal closure in response to high levels of CO₂, dark, and ABA (Meyer et al., 2010). Taken together, these studies have clearly demonstrated that both S- and R-type anion channels are key modulators of stomatal movements in response to several environmental factors.Despite a vast number of studies involving the above-mentioned anion channels, little information concerning the metabolic changes caused by their impairment is currently available. Such information is important to understand stomatal movements, mainly considering that organic acids, especially the levels of malate in apoplastic/mesophyll cells, have been highlighted as of key importance in leaf metabolism (Fernie and Martinoia, 2009; Araújo et al., 2011a, 2011b; Lawson et al., 2014; Medeiros et al., 2015). Here, we demonstrate that a disruption in the expression of AtQUAC1, which leads to impaired stomatal closure (Meyer et al., 2010), was accompanied by increases in mesophyll conductance (g_m), which is defined as the conductance for the transfer of CO₂ from the intercellular airspaces (C_i) to the sites of carboxylation in the chloroplastic stroma (C_c). By further characterization of atquac1 knockout plants, we demonstrated that reduced diffusive limitations resulted in higher photosynthetic rates and altered respiration that, in turn, led to enhanced biomass accumulation. Overall, the results obtained are discussed both in terms of the importance of organic acid transport in plant cell metabolism and with regard to the contribution that it plays in the regulation of both stomatal function and growth.     

A Host RNA Helicase-Like Protein,AtRH8, Interacts with the Potyviral Genome-Linked Protein,VPg, Associates with the Virus Accumulation Complex,and Is Essential for Infection

The viral genome-linked protein, VPg, of potyviruses is a multifunctional protein involved in viral genome translation and replication. Previous studies have shown that both eukaryotic translation initiation factor 4E (eIF4E) and eIF4G or their respective isoforms from the eIF4F complex, which modulates the initiation of protein translation, selectively interact with VPg and are required for potyvirus infection. Here, we report the identification of two DEAD-box RNA helicase-like proteins, PpDDXL and AtRH8 from peach (Prunus persica) and Arabidopsis (Arabidopsis thaliana), respectively, both interacting with VPg. We show that AtRH8 is dispensable for plant growth and development but necessary for potyvirus infection. In potyvirus-infected Nicotiana benthamiana leaf tissues, AtRH8 colocalizes with the chloroplast-bound virus accumulation vesicles, suggesting a possible role of AtRH8 in viral genome translation and replication. Deletion analyses of AtRH8 have identified the VPg-binding region. Comparison of this region and the corresponding region of PpDDXL suggests that they are highly conserved and share the same secondary structure. Moreover, overexpression of the VPg-binding region from either AtRH8 or PpDDXL suppresses potyvirus accumulation in infected N. benthamiana leaf tissues. Taken together, these data demonstrate that AtRH8, interacting with VPg, is a host factor required for the potyvirus infection process and that both AtRH8 and PpDDXL may be manipulated for the development of genetic resistance against potyvirus infections.Plant viruses are obligate intracellular parasites that infect many agriculturally important crops and cause severe losses each year. One of the common characteristics of plant viruses is their relatively small genome that encodes a limited number of viral proteins, making them dependent on host factors to fulfill their infection cycles (Maule et al., 2002; Whitham and Wang, 2004; Nelson and Citovsky, 2005; Decroocq et al., 2006). In order to establish a successful infection, the invading virus must recruit an array of host proteins (host factors) to translate and replicate its genome and to move locally from cell to cell via the plasmodesmata and systemically via the vascular system. It has been suggested that down-regulation or mutation of some of the required host factors may result in recessively inherited resistance to viruses (Kang et al., 2005b).Potyviruses, belonging to the genus Potyvirus in the family Potyviradae, constitute the largest group of plant viruses (Rajamäki et al., 2004). Potyviruses have a single positive-strand RNA genome approximately 10 kb in length, with a viral genome-linked protein (VPg) covalently attached to the 5′ end and a poly(A) tail at the 3′ end (Urcuqui-Inchima et al., 2001; Rajamäki et al., 2004). The viral genome contains a single open reading frame (ORF) that translates into a polypeptide with a molecular mass of approximately 350 kD, which is cleaved into 10 mature proteins by viral proteases (Urcuqui-Inchima et al., 2001). Recently, a novel viral protein resulting from a frameshift in the P3 cistron has been reported (Chung et al., 2008). Of the 11 viral proteins, VPg is a multifunctional protein and the only other viral protein present in the viral particles (virions) besides the coat protein and the cylindrical inclusion protein (CI; Oruetxebarria et al., 2001; Puustinen et al., 2002; Gabrenaite-Verkhovskaya et al., 2008). The nonstructural protein is linked to the viral RNA by a phosphodiester bond between the 5′ terminal uridine residue of the RNA and the O⁴-hydroxyl group of amino acid Tyr (Murphy et al., 1996; Oruetxebarria et al., 2001; Puustinen et al., 2002). Mutation of the Tyr residue that links VPg to the viral RNA abolishes virus infectivity completely (Murphy et al., 1996). In infected cells, VPg and its precursor NIa are present in the nucleus and in the membrane-associated virus replication vesicles in the cytoplasm (Carrington et al., 1993; Rajamäki and Valkonen, 2003; Cotton et al., 2009). As a component of the replication complex, VPg may serve as a primer for viral RNA replication (Puustinen and Mäkinen, 2004) and as an analog of the m⁷G cap of mRNAs for the viral genome to recruit the translation complex for translation (Michon et al., 2006; Beauchemin et al., 2007; Khan et al., 2008). Furthermore, VPg has been suggested to be an avirulence factor for recessive resistance genes in diverse plant species (Moury et al., 2004; Kang et al., 2005b; Bruun-Rasmussen et al., 2007). Thus, VPg plays a pivotal role in the virus infection process. The molecular identification of VPg-interacting host proteins and the subsequent functional characterization of such interactions may advance knowledge of the intricate virus replication mechanisms and help develop novel antiviral strategies.Previous studies have shown that VPg and its precursor NIa interact with several host proteins, including three essential components of the host protein translation apparatus (Thivierge et al., 2008). The first protein is the cellular translation initiation factor eIF4E or its isoform eIF(iso)4E, identified through a yeast two-hybrid screen using VPg as a bait (Wittmann et al., 1997; Schaad et al., 2000). The protein complex of VPg and eIF4E is an essential component for virus infectivity (Robaglia and Caranta, 2006). Mutations and knockout of eIF4E or eIF(iso)4E confer resistance to infection (Lellis et al., 2002; Ruffel et al., 2002; Nicaise et al., 2003; Gao et al., 2004; Kang et al., 2005a; Ruffel et al., 2005; Decroocq et al., 2006; Bruun-Rasmussen et al., 2007). It is well known that potyviruses recruit selectively one of the eIF4E isoforms, depending on specific virus-host combinations (German-Retana et al., 2008). For instance, in Arabidopsis (Arabidopsis thaliana), eIF(iso)4E is required for infection by Turnip mosaic virus (TuMV), Plum pox virus (PPV), and Lettuce mosaic virus, while eIF4E is indispensable for infection by Clover yellow vein virus (Duprat et al., 2002; Lellis et al., 2002; Sato et al., 2005; Decroocq et al., 2006). The second cellular protein interacting with VPg is another translation initiation factor, eIF4G. Analysis of Arabidopsis knockout mutants for eIF4G or its isomers eIF(iso)4G1 and eIF(iso)4G2 has yielded results supporting the idea that the recruitment of eIF4G for potyvirus infection is also isoform dependent (Nicaise et al., 2007). Recently, poly(A)-binding protein (PABP), the translation initiation factor that bridges the 5′ and 3′ termini of the mRNA into proximity, has been proposed to be essential for efficient multiplication of TuMV (Dufresne et al., 2008). PABP was previously documented to interact with NIa, a VPg precursor containing both VPg and the proteinase NIa-Pro (Léonard et al., 2004). As the translation factors eIF(iso)4E and PABP have been found to be internalized in virus-induced vesicles, it has been suggested that the interactions between VPg and these translation factors are crucial for viral RNA translation and/or replication (Beauchemin and Laliberté, 2007; Beauchemin et al., 2007; Cotton et al., 2009). Besides these three translation factors, a Cys-rich plant protein, potyvirus VPg-interaction protein, was also found to associate with VPg (Dunoyer et al., 2004). This plant-specific VPg-interacting host protein contains a PHD finger domain and acts as an ancillary factor to support potyvirus infection and movement (Dunoyer et al., 2004).In this study, we describe the identification of an Arabidopsis DEAD-box RNA helicase (DDX), AtRH8, and a peach (Prunus persica) DDX-like protein, PpDDXL, both interacting with the potyviral VPg protein. Using the atrh8 mutant, we demonstrate that AtRH8 is not required for plant growth and development in Arabidopsis but is necessary for infection by two plant potyviruses, PPV and TuMV. Furthermore, we present evidence that AtRH8 colocalizes with the virus accumulation complex in potyvirus-infected leaf tissues, which reveals a possible role of AtRH8 in virus infection. Finally, we have identified the VPg-binding region (VPg-BR) of AtRH8 and PpDDX and show that overexpression of the VPg-BR either from AtRH8 or PpDDXL suppresses virus accumulation.     

Heterodimeric Capping Protein from Arabidopsis Is a Membrane-Associated,Actin-Binding Protein

The actin cytoskeleton is a major regulator of cell morphogenesis and responses to biotic and abiotic stimuli. The organization and activities of the cytoskeleton are choreographed by hundreds of accessory proteins. Many actin-binding proteins are thought to be stimulus-response regulators that bind to signaling phospholipids and change their activity upon lipid binding. Whether these proteins associate with and/or are regulated by signaling lipids in plant cells remains poorly understood. Heterodimeric capping protein (CP) is a conserved and ubiquitous regulator of actin dynamics. It binds to the barbed end of filaments with high affinity and modulates filament assembly and disassembly reactions in vitro. Direct interaction of CP with phospholipids, including phosphatidic acid, results in uncapping of filament ends in vitro. Live-cell imaging and reverse-genetic analyses of cp mutants in Arabidopsis (Arabidopsis thaliana) recently provided compelling support for a model in which CP activity is negatively regulated by phosphatidic acid in vivo. Here, we used complementary biochemical, subcellular fractionation, and immunofluorescence microscopy approaches to elucidate CP-membrane association. We found that CP is moderately abundant in Arabidopsis tissues and present in a microsomal membrane fraction. Sucrose density gradient separation and immunoblotting with known compartment markers were used to demonstrate that CP is enriched on membrane-bound organelles such as the endoplasmic reticulum and Golgi. This association could facilitate cross talk between the actin cytoskeleton and a wide spectrum of essential cellular functions such as organelle motility and signal transduction.The cellular levels of membrane-associated lipids undergo dynamic changes in response to developmental and environmental stimuli. Different species of phospholipids target specific proteins and this often affects the activity and/or subcellular localization of these lipid-binding proteins. One such membrane lipid, phosphatidic acid (PA), serves as a second messenger and regulates multiple developmental processes in plants, including seedling development, root hair growth and pattern formation, pollen tube growth, leaf senescence, and fruit ripening. PA levels also change during various stress responses, including high salinity and dehydration, pathogen attack, and cold tolerance (Testerink and Munnik, 2005, 2011; Wang, 2005; Li et al., 2009). In mammalian cells, PA is critical for vesicle trafficking events, such as vesicle budding from the Golgi apparatus, vesicle transport, exocytosis, endocytosis, and vesicle fusion (Liscovitch et al., 2000; Freyberg et al., 2003; Jenkins and Frohman, 2005).The actin cytoskeleton and a plethora of actin-binding proteins (ABPs) are well-known targets and transducers of lipid signaling (Drøbak et al., 2004; Saarikangas et al., 2010; Pleskot et al., 2013). For example, several ABPs have the ability to bind phosphoinositide lipids, such as phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂]. The severing or actin filament depolymerizing proteins such as villin, cofilin, and profilin are inhibited when bound to PtdIns(4,5)P₂. One ABP appears to be strongly regulated by another phospholipid; human gelsolin binds to lysophosphatidic acid and its filament severing and barbed-end capping activities are inhibited by this biologically active lipid (Meerschaert et al., 1998). Gelsolin is not, however, regulated by PA (Meerschaert et al., 1998), nor are profilin (Lassing and Lindberg, 1985), α-actinin (Fraley et al., 2003), or chicken CapZ (Schafer et al., 1996).The heterodimeric capping protein (CP) from Arabidopsis (Arabidopsis thaliana) also binds to and its activity is inhibited by phospholipids, including both PtdIns(4,5)P₂ and PA (Huang et al., 2003, 2006). PA and phospholipase D activity have been implicated in the actin-dependent tip growth of root hairs and pollen tubes (Ohashi et al., 2003; Potocký et al., 2003; Samaj et al., 2004; Monteiro et al., 2005a; Pleskot et al., 2010). Exogenous application of PA causes an elevation of actin filament levels in suspension cells, pollen, and Arabidopsis epidermal cells (Lee et al., 2003; Potocký et al., 2003; Huang et al., 2006; Li et al., 2012; Pleskot et al., 2013). Capping protein (CP) binds to the barbed end of actin filaments with high (nanomolar) affinity, dissociates quite slowly, and prevents the addition of actin subunits at this end (Huang et al., 2003, 2006; Kim et al., 2007). In the presence of phospholipids, AtCP is not able to bind to the barbed end of actin filaments (Huang et al., 2003, 2006). Furthermore, capped filament ends are uncapped by the addition of PA, allowing actin assembly from a pool of profilin-actin (Huang et al., 2006). Collectively, these data lead to a simple model whereby CP, working in concert with profilin-actin, serves to maintain tight regulation of actin assembly at filament barbed ends (Huang et al., 2006; Blanchoin et al., 2010; Henty-Ridilla et al., 2013; Pleskot et al., 2013). Furthermore, the availability of CP for filament ends can be modulated by fluxes in signaling lipids. Genetic evidence for this model was recently obtained by analyzing the dynamic behavior of actin filament ends in living Arabidopsis epidermal cells after treatment with exogenous PA (Li et al., 2012). Specifically, changes in the architecture of cortical actin arrays and dynamics of individual actin filaments that are induced by PA treatment were found to be attenuated in cp mutant cells (Li et al., 2012; Pleskot et al., 2013).Structural characterization of chicken CapZ demonstrates that the α- and β-subunits of the heterodimer form a compact structure resembling a mushroom with pseudo-two-fold rotational symmetry (Yamashita et al., 2003). Actin- and phospholipid-binding sites are conserved on the C-terminal regions, sometimes referred to as tentacles, which comprise amphipathic α-helices (Cooper and Sept, 2008; Pleskot et al., 2012). Coarse-grained molecular dynamics (CG-MD) simulations recently revealed the mechanism of chicken and AtCP association with membranes (Pleskot et al., 2012). AtCP interacts specifically with lipid bilayers through interactions between PA and the amphipathic helix of the α-subunit tentacle. Extensive polar contacts between lipid headgroups and basic residues on CP (including K278, which is unique to plant CP), as well as partial embedding of nonpolar groups into the lipid bilayer, are observed (Pleskot et al., 2012). Moreover, a glutathione S-transferase fusion protein containing the C-terminal 38 amino acids from capping protein α subunit (CPA) is sufficient to bind PA-containing liposomes in vitro (Pleskot et al., 2012). Collectively, these findings lead us to predict that AtCP will behave like a membrane-associated protein in plant cells.Additional evidence from animal and microbial cells supports the association of CP with biological membranes. In Acanthamoeba castellanii, CP is localized primarily to the hyaline ectoplasm in a region of the cytoplasm just under the plasma membrane that contains a high concentration of actin filaments (Cooper et al., 1984). Localization of CP with regions rich in actin filaments and with membranes was supported by subcellular fractionation experiments, in which CP was associated with a crude membrane fraction that included plasma membrane (Cooper et al., 1984). Further evidence demonstrates that CP localizes to cortical actin patches at sites of new cell wall growth in budding yeast (Saccharomyces cerevisiae), including the site of bud emergence. By contrast, CP did not colocalize with actin cables in S. cerevisiae (Amatruda and Cooper, 1992). CP may localize to these sites by direct interactions with membrane lipids, through binding the ends of actin filaments, or by association with another protein different from actin. In support of this hypothesis, GFP-CP fusion proteins demonstrate that sites of actin assembling in living cells contain both CP and the actin-related protein2/3 (Arp2/3) complex, and CP is located in two types of structures: (1) motile regions of the cell periphery, which reflect movement of the edge of the lamella during extension and ruffling; and (2) dynamic spots within the lamella (Schafer et al., 1998). CP has been colocalized to the F-actin patches in fission yeast (Schizosaccharomyces pombe; Kovar et al., 2005), which promotes Arp2/3-dependent nucleation and branching and limits the extent of filament elongation (Akin and Mullins, 2008). These findings lend additional support for a model whereby CP cooperates with the Arp2/3 complex to regulate actin dynamics (Nakano and Mabuchi, 2006). Activities and localization of other plant ABPs are linked to membranes. Membrane association has been linked to the assembly status of the ARP2/3 complex, an actin filament nucleator, in Arabidopsis (Kotchoni et al., 2009). SPIKE1 (SPK1), a Rho of plants (Rop)-guanine nucleotide exchange factor (GEF) and peripheral membrane protein, maintains the homeostasis of the early secretory pathway and signal integration during morphogenesis through specialized domains in the endoplasmic reticulum (ER; Zhang et al., 2010). Furthermore, Nck-associated protein1 (NAP1), a component of the suppressor of cAMP receptor/WASP-family verprolin homology protein (SCAR/WAVE) complex, strongly associates with membranes and is particularly enriched in ER membranes (Zhang et al., 2013a). Finally, a superfamily of plant ABPs, called NETWORKED proteins, was recently discovered; these link the actin cytoskeleton to various cellular membranes (Deeks et al., 2012; Hawkins et al., 2014; Wang et al., 2014).In this work, we demonstrate that CP is a membrane-associated protein in Arabidopsis. To our knowledge, this is the first direct evidence for CP-membrane association in plants. This interaction likely targets CP to cellular compartments such as the ER and Golgi. This unique location may allow CP to remodel the actin cytoskeleton in the vicinity of endomembrane compartments and/or to respond rapidly to fluxes in signaling lipids.     

NdhV Is a Subunit of NADPH Dehydrogenase Essential for Cyclic Electron Transport in Synechocystis sp. Strain PCC 6803

Two mutants sensitive to heat stress for growth and impaired in NADPH dehydrogenase (NDH-1)-dependent cyclic electron transport around photosystem I (NDH-CET) were isolated from the cyanobacterium Synechocystis sp. strain PCC 6803 transformed with a transposon-bearing library. Both mutants had a tag in the same sll0272 gene, encoding a protein highly homologous to NdhV identified in Arabidopsis (Arabidopsis thaliana). Deletion of the sll0272 gene (ndhV) did not influence the assembly of NDH-1 complexes and the activities of CO₂ uptake and respiration but reduced the activity of NDH-CET. NdhV interacted with NdhS, a ferredoxin-binding subunit of cyanobacterial NDH-1 complex. Deletion of NdhS completely abolished NdhV, but deletion of NdhV had no effect on the amount of NdhS. Reduction of NDH-CET activity was more significant in ΔndhS than in ΔndhV. We therefore propose that NdhV cooperates with NdhS to accept electrons from reduced ferredoxin.Cyanobacterial NADPH dehydrogenase (NDH-1) complexes are localized in the thylakoid membrane (Ohkawa et al., 2001, 2002; Zhang et al., 2004; Xu et al., 2008; Battchikova et al., 2011b) and participate in a variety of bioenergetic reactions, such as respiration, cyclic electron transport around photosystem I (NDH-CET), and CO₂ uptake (Ogawa, 1991; Mi et al., 1992; Ohkawa et al., 2000). Structurally, the cyanobacterial NDH-1 complexes closely resemble energy-converting complex I in eubacteria and the mitochondrial respiratory chain regardless of the absence of homologs of three subunits in cyanobacterial genomes that constitute the catalytically active core of complex I (Friedrich et al., 1995; Friedrich and Scheide, 2000; Arteni et al., 2006). Over the past decade, new subunits of NDH-1 complexes specific to oxygenic photosynthesis have been identified in several cyanobacterial strains. They are NdhM to NdhQ and NdhS (Prommeenate et al., 2004; Battchikova et al., 2005, 2011b; Nowaczyk et al., 2011; Wulfhorst et al., 2014; Zhang et al., 2014; Zhao et al., 2014b, 2015), in addition to NdhL first identified in the cyanobacterium Synechocystis sp. strain PCC 6803 (hereafter Synechocystis 6803) about 20 years ago (Ogawa, 1992). Among them, NdhS possesses a ferredoxin (Fd)-binding motif and was shown to bind Fd, which suggested that Fd is one of the electron donors to NDH-1 complexes (Mi et al., 1995; Battchikova et al., 2011b; Ma and Ogawa, 2015). Deletion of NdhS strongly reduced the activity of NDH-CET but had no effect on respiration and CO₂ uptake (Battchikova et al., 2011b; Ma and Ogawa, 2015). The NDH-CET plays an important role in coping with various environmental stresses regardless of its elusive mechanism. For example, this function can greatly alleviate heat-sensitive growth phenotypes (Wang et al., 2006a; Zhao et al., 2014a). Thus, heat treatment strategy can help in identifying the proteins essential to NDH-CET.Here, a new oxygenic photosynthesis-specific (OPS) subunit NdhV was identified in Synechocystis 6803 with the help of heat treatment strategy, and its deletion did not influence the assembly of NDH-1L and NDH-1MS complexes and the activities of CO₂ uptake and respiration but impaired the NDH-CET activity. We give evidence that NdhV interacts with NdhS and is another component of Fd-binding domain of cyanobacterial NDH-1 complex. A possible role of NdhV on the NDH-CET activity is discussed.     

STATE TRANSITION7-Dependent Phosphorylation Is Modulated by Changing Environmental Conditions,and Its Absence Triggers Remodeling of Photosynthetic Protein Complexes

In plants and algae, the serine/threonine kinase STN7/STT7, orthologous protein kinases in Chlamydomonas reinhardtii and Arabidopsis (Arabidopsis thaliana), respectively, is an important regulator in acclimation to changing light environments. In this work, we assessed STT7-dependent protein phosphorylation under high light in C. reinhardtii, known to fully induce the expression of LIGHT-HARVESTING COMPLEX STRESS-RELATED PROTEIN3 (LHCSR3) and a nonphotochemical quenching mechanism, in relationship to anoxia where the activity of cyclic electron flow is stimulated. Our quantitative proteomics data revealed numerous unique STT7 protein substrates and STT7-dependent protein phosphorylation variations that were reliant on the environmental condition. These results indicate that STT7-dependent phosphorylation is modulated by the environment and point to an intricate chloroplast phosphorylation network responding in a highly sensitive and dynamic manner to environmental cues and alterations in kinase function. Functionally, the absence of the STT7 kinase triggered changes in protein expression and photoinhibition of photosystem I (PSI) and resulted in the remodeling of photosynthetic complexes. This remodeling initiated a pronounced association of LHCSR3 with PSI-LIGHT HARVESTING COMPLEX I (LHCI)-ferredoxin-NADPH oxidoreductase supercomplexes. Lack of STT7 kinase strongly diminished PSII-LHCII supercomplexes, while PSII core complex phosphorylation and accumulation were significantly enhanced. In conclusion, our study provides strong evidence that the regulation of protein phosphorylation is critical for driving successful acclimation to high light and anoxic growth environments and gives new insights into acclimation strategies to these environmental conditions.Oxygenic photosynthesis converts solar energy into chemical energy. This energy is utilized for carbon dioxide assimilation, allowing the formation of complex organic material. Plant photosynthesis is performed by a series of reactions in and at the thylakoid membrane, resulting in light-dependent water oxidation, NADP reduction, and ATP formation (Whatley et al., 1963). These light reactions are catalyzed by two photosystems (PSI and PSII). A third multiprotein complex, also embedded in the thylakoid membrane, is the cytochrome b₆f (cyt b₆f) complex that links photosynthetic electron transfer processes between the two photosystems and functions in proton translocation. The ATP synthase takes advantage of the proton-motive force that is generated by the light reactions (Mitchell, 1961) to produce ATP. ATP and NADPH, generated through linear electron flow from PSII to PSI, drive the Calvin-Benson-Bassham cycle (Bassham et al., 1950) to fix CO₂. Alternatively, cyclic electron flow (CEF) between PSI and the cyt b₆f complex solely produces ATP (Arnon, 1959).Under normal growth conditions, CEF provides additionally required ATP for CO₂ fixation (Lucker and Kramer, 2013), counteracts overreduction of the PSI acceptor side under stressful environmental cues, and readjusts the ATP poise, leading to increased lumen acidification important for photoprotection (Alric, 2010; Peltier et al., 2010; Leister and Shikanai, 2013; Shikanai, 2014). In microalgae and vascular plants, CEF relies on the NAD(P)H dehydrogenase-dependent and/or PROTON GRADIENT REGULATION5 (PGR5)-related pathways (Munekage et al., 2002, 2004; Petroutsos et al., 2009; Tolleter et al., 2011; Johnson et al., 2014). For both pathways, supercomplexes consisting of PSI-LIGHT HARVESTING COMPLEX I (LHCI) and components of the respective electron transfer routes have been identified. In Arabidopsis (Arabidopsis thaliana), a unique NAD(P)H dehydrogenase-PSI supercomplex with a molecular mass of more than 1,000 kD was discovered (Peng et al., 2008). From Chlamydomonas reinhardtii, Iwai et al. (2010) isolated a protein supercomplex composed of PSI-LHCI, LHCII, the cyt b₆/f complex, ferredoxin-NADPH oxidoreductase (FNR), and PROTON GRADIENT REGULATION-LIKE1 (PGRL1).PGRL1 and PGR5 interact physically in Arabidopsis and associate with PSI to allow the operation of CEF (DalCorso et al., 2008). Functional data suggest that PGRL1 might operate as a ferredoxin-plastoquinone reductase (Hertle et al., 2013). The PGRL1-containing CEF supercomplex isolated from C. reinhardtii is capable of CEF under in vitro conditions in the presence of exogenously added soluble plastocyanin and ferredoxin (Iwai et al., 2010). Terashima et al. (2012) isolated a CEF supercomplex of similar composition from anaerobic growth conditions that was active in vitro and contained proteins such as the chloroplast-localized Ca²⁺ sensor CAS and ANAEROBIC RESPONSE1 (ANR1), which were also shown to be functionally important for efficient CEF in the alga. Notably, it was suggested that the onset of CEF in C. reinhardtii is redox controlled (Takahashi et al., 2013).It has been demonstrated that efficient CEF is crucial for successful acclimation to excess light (Munekage et al., 2004; Dang et al., 2014; Johnson et al., 2014; Kukuczka et al., 2014). The most rapid response to excess light, however, relies on a mechanism called nonphotochemical quenching (NPQ). The fastest constituent of NPQ is energy-dependent (qE) quenching, which operates at a time scale of seconds to minutes and regulates the thermal dissipation of excess absorbed light energy, thereby providing effective photoprotection. In vascular plants, the PSII protein PSII SUBUNIT S is essential for qE (Li et al., 2000), whereas qE induction in the green alga C. reinhardtii is mediated by LIGHT-HARVESTING COMPLEX STRESS-RELATED PROTEIN3 (LHCSR3), an ancient light-harvesting protein that is missing in vascular plants (Peers et al., 2009). CEF and qE are complementary for acclimation to excess light, as double mutants deficient in both mechanisms possess additive phenotypes and are highly sensitive to light (Kukuczka et al., 2014). Another constituent of NPQ is the quenching by state transitions. State transitions are important to balance the excitation energy between PSI and PSII (Bonaventura and Myers, 1969; Murata, 1969). Under light conditions where PSII is preferentially excited, both PSII core and LHCII proteins become phosphorylated (Lemeille and Rochaix, 2010). As a consequence, phosphorylated LHCII proteins detach from PSII and partly connect to PSI (state 2). Under conditions where PSI excitation is predominant, this process is reversed. LHCII proteins are dephosphorylated and associate with PSII (state 1). The extent of state transition between vascular plants such as Arabidopsis and C. reinhardtii differs significantly. The proportion of mobile LHCII antenna is about 80% in the alga, whereas in Arabidopsis, only 15% to 20% of LHCII is transferred to PSI under state 2 conditions (Lemeille and Rochaix, 2010). However, the large increase in PSI antenna size in C. reinhardtii has recently been challenged (Nagy et al., 2014; Ünlü et al., 2014): while 70% to 80% of mobile LHCII detached from PSII in response to transition to state 2 conditions, only a fraction of about 20% functionally attached to PSI.Phosphorylation of LHC proteins requires the function of the STT7 kinase or its ortholog STN7 in C. reinhardtii or Arabidopsis, respectively. In the absence of the STT7/STN7 kinase, the initiation of state transitions is blocked (Depège et al., 2003; Bellafiore et al., 2005). The mobile LHCII fraction of C. reinhardtii includes the two monomeric minor LHCII antenna proteins, CP26 and CP29 (encoded by lhcb5 and lhcb4 genes), and the major chlorophyll a/b binding protein of LHCII, LHCBM5 (Takahashi et al., 2006), but also the LHCSR3 protein was suggested to migrate during state transitions (Allorent et al., 2013). Takahashi et al. (2014) suggested that only CP29 and LHCBM5 directly associate with PSI to form the PSI-LHCI-LHCII supercomplex, while the binding of CP26 could occur indirectly or via the other two proteins. However, it is not yet known whether STT7 directly phosphorylates the LHCII proteins or if this takes place as part of a kinase cascade (Rochaix, 2007). Nevertheless, the direct interaction between STT7 and the LHCII proteins is quite likely, since none of the other chloroplast kinases was found to be specifically required for LHCII phosphorylation (Rochaix, 2014). The activity of the STT7 kinase is mainly determined by the redox status of the plastoquinone pool (Vener et al., 1997; Zito et al., 1999). The identification of a PROTEIN PHOSPHATASE 2C (PP2C)-type phosphatase responsible for the dephosphorylation of the LHCII proteins in Arabidopsis has been described by two studies in parallel pointing to the fact that this enzyme, called PROTEIN PHOSPHATASE1/THYLAKOID-ASSOCIATED PHOSPHATASE38, acts directly on phosphorylated LHCII proteins, in particular when they are associated with the PSI-LHCI supercomplex (Pribil et al., 2010; Shapiguzov et al., 2010). Moreover, it is not known whether these phosphatases are constitutively active or if they are regulated by other means, for example through the redox state of the plastoquinone pool. Nonetheless, both enzymes are conserved in land plants and exhibit orthologous proteins in C. reinhardtii (Rochaix et al., 2012).Another kinase related to STN7/STT7 is encoded in the Arabidopsis and C. reinhardtii genomes and named STN8 and STATE TRANSITION-LIKE1 (STL1), respectively. STN8 is involved in PSII core subunit phosphorylation and influences the repair of PSII after photodamage (Bonardi et al., 2005; Vainonen et al., 2005). Remarkably, the disassembly of the PSII holocomplex is inhibited in STN7/STN8 double mutants (Tikkanen et al., 2008; Fristedt et al., 2009; Dietzel et al., 2011; Nath et al., 2013), suggesting that the phosphorylation of core subunits is required for PSII disassembly. It was further suggested that STN8 controls the transition between linear electron flow and CEF by the phosphorylation of PGRL1 in Arabidopsis (Reiland et al., 2011). As described for STN7, the activity of STN8 is probably regulated via the redox state of the plastoquinone pool (Bennett, 1991; Fristedt et al., 2009). Notably, the action of STN8 is counteracted by a chloroplast PP2C phosphatase (Samol et al., 2012), allowing for the fast reversibility of STN8-mediated acclimation responses. Thus, it appears that an intricate regulatory network of chloroplast protein kinases and phosphatases evolved in vascular plants and algae that drives the acclimation response to various environmental cues, including excess and changing light settings (Rochaix et al., 2012). As STN7/STT7 and STN8/STL1 kinase activities appear to be controlled by the redox poise of the plastoquinone pool, the plastoquinone pool would be a central player in these acclimation responses. On the other hand, the kinases themselves are subjected to phosphorylation (Reiland et al., 2009, 2011; Lemeille et al., 2010; Wang et al., 2013). However, the functional consequences of this phosphorylation are unknown.Recent comparative analyses revealed the presence of at least 15 distinct chloroplast protein kinases, suggesting an intricate kinase phosphorylation network in the chloroplast (Bayer et al., 2012). Generally, the phosphorylation of proteins is one of the most abundant posttranslational modifications. In complex eukaryotic systems, protein phosphorylation occurs most frequently on Ser followed by Thr residues, whereas protein phosphorylation of Tyr residues (1,800:200:1) is comparatively rare (Hunter, 1998; Mann et al., 2002). Protein phosphorylation is a general phenomenon in vivo; it is assumed that about one-third of all proteins are phosphorylated at a given time (Cohen, 2000; Ahn and Resing, 2001; Venter et al., 2001; Manning et al., 2002; Knight et al., 2003). A recent large-scale quantitative evaluation of human proteomic data strengthened the importance of protein phosphorylation for cellular function and human biology (Wilhelm et al., 2014). The C. reinhardtii and Arabidopsis genomes encode large kinase families (Arabidopsis Genome Initiative, 2000; Kerk et al., 2002; Merchant et al., 2007), supporting the view that protein phosphorylation also plays an important role in a plant’s life cycle. It is thus evident that the understanding of protein phosphorylation, including the specificity of residues phosphorylated or dephosphorylated in response to cellular as well as environmental factors, is one key to understanding the complex functional biological networks at the whole-system level. Likewise, it is crucial to design experimental setups allowing the linkage between phosphorylation events and particular physiological consequences to be elucidated.In this regard, we designed experiments to investigate STT7 kinase-dependent phosphorylation dynamics in C. reinhardtii in response to high light and anoxia, employing quantitative proteomics in conjunction with in-depth physiological characterization. These conditions are particularly interesting, as high light conditions are known to fully induce LHCSR3 protein expression and qE, while anoxia promotes CEF activity. Recently, it was demonstrated that qE and CEF are complementary and crucial in acclimation to these environmental cues (Kukuczka et al., 2014). Notably, LHCSR3 phosphorylation was suggested to depend on STT7 function (Bonente et al., 2011), while CEF supercomplex formation was found to be independent of STT7 kinase function (Takahashi et al., 2013), indicating that STT7 function might impact the acclimation to high light and anoxia in different ways. However, our quantitative proteomics and physiological data reveal that STT7-dependent variations in protein phosphorylation profiles have similar dramatic phenotypic consequences in both conditions, strongly suggesting that the regulation of protein phosphorylation is critical for driving successful acclimation to high light and anoxic growth environments.