Multiple Sequence Motifs in the Rubisco Small Subunit Transit Peptide Independently Contribute to Toc159-Dependent Import of Proteins into Chloroplasts

A large number of plastid proteins encoded by the nuclear genome are posttranslationally imported into plastids by at least two distinct mechanisms: the Toc159-dependent and Toc132/Toc120-dependent pathways. Light-induced photosynthetic proteins are imported through the Toc159-dependent pathway, whereas constitutive housekeeping plastid proteins are imported into plastids through the Toc132/Toc120 pathway. However, it remains unknown which features of the plastid protein transit peptide (TP) determine the import pathway. We have discovered sequence elements of the Rubisco small subunit TP (RbcS-tp) that play a role in determining import through the Toc159-dependent pathway in vivo. We generated multiple hybrid mutants using the RbcS-tp and the E1α-subunit of pyruvate dehydrogenase TP (E1α-tp) as representative peptides mediating import through the Toc159-dependent and Toc159-independent pathways, respectively. Import experiments using these hybrid mutants in wild-type and ppi2 mutant protoplasts revealed that multiple sequence motifs in the RbcS-tp independently contribute to Toc159-dependent protein import into chloroplasts. One of these motifs is the group of serine residues located in the N-terminal 12-amino acid segment and the other is the C-terminal T5 region of the RbcS-tp ranging from amino acid positions 41 to 49. Based on these findings, we propose that multiple sequence elements in the RbcS-tp contribute independently to Toc159-dependent import of proteins into chloroplasts.The plastid is a crucial organelle in plant cells. It plays a role in critical cellular processes such as photosynthesis, ATP generation, amino acid metabolism, and synthesis of fatty acids and lipid components. Accordingly, a large number of proteins are required for all these activities in plastids. Some of these proteins are encoded by the chloroplast genetic system and are translated in the plastids. However, most plastid proteins (over 90%) are encoded by the nuclear genome and are imported into plastids from the cytosol posttranslationally (Kessler and Schnell, 2006; Jarvis, 2008).Most plastid interior proteins that undergo posttranslational import from the cytosol contain a cleavable N-terminal targeting signal, a transit peptide (TP), of 50 to 70 amino acid residues (Jarvis, 2008; Lee et al., 2008). However, recently, some plastid interior proteins have been identified that do not have the N-terminal canonical TP (Miras et al., 2002, 2007; Nada and Soll, 2004). The long TP consists of multiple domains or motifs that encode information for preprotein import into plastids (von Heijne et al., 1989; Pilon et al., 1995; Rensink et al., 2000; Lee et al., 2006, 2008). The preproteins transit through the cytosol as unfolded protein. During passage through the cytosol, they may form a complex with heat shock proteins, such as Hsp70 and Hsp90, and guidance factors such as 14-3-3 (May and Soll, 2000; Qbadou et al., 2006). However, 14-3-3 may not be essential for the targeting of these proteins to chloroplasts (Lee et al., 2002, 2006; Nakrieko et al., 2004). To cross the two envelope membranes, the TP interacts with components of the Toc and Tic complexes located at the outer and inner envelopes of chloroplasts, respectively (Jarvis, 2008). These include members of the Toc159 family, Toc33/Toc34, Toc75, and Tic20. At the late stage or after translocation, the TP is recognized and cleaved off by stromal processing peptidases (Richter and Lamppa, 1999; Chen and Li, 2007).Despite extensive study of the TPs, it is not fully understood how the information encoded in these peptides is decoded by the plastid protein import machinery. TPs display some degree of similarity in their amino acid composition, including a higher content of Ala, Gly, and the hydroxylated amino acids Ser and Thr, and a lack of acidic amino acids (von Heijne et al., 1989; Bruce, 2001; Zhang and Glaser, 2002). However, it is clear that the entire family of TPs, termed the transit peptidome, cannot be represented by a single consensus sequence. Growing evidence has pointed to a functional classification of TPs. The first indication is that the transit peptidome may be classified into two groups: Toc159-dependent and Toc159-independent TPs (Ivanova et al., 2004; Kubis et al., 2004; Smith et al., 2004). The TPs that confer Toc159 dependence in protein import are typically used by light-induced photosynthetic proteins, whereas Toc159-independent TPs are used by nonphotosynthetic and housekeeping proteins (Kessler and Schnell, 2006). This was clearly demonstrated in the ppi2 mutant that has a T-DNA insertion in atTOC159 (Smith et al., 2004). In accord with this observation, the expression of atTOC159 is high in young and photosynthetic tissues whereas atTOC132 and atTOC120 are expressed uniformly in all plant tissues at low levels (Kubis et al., 2004). In addition, in nonphotosynthetic tissues, such as roots, the mRNA level of atTOC132 or atTOC120 is much higher than that of atTOC159. These results are consistent with the hypothesis that TPs may contain sequence motifs that determine the targeting pathway. However, the sequence information that confers Toc159 dependence or Toc132/120 dependence on these proteins during protein import remains unknown. In addition, Lee et al. (2008) recently demonstrated that the transit peptidome may be divided into several groups based on critical sequence motifs present in the TP. However, the role of the sequence motifs embedded in the TPs is not entirely clear yet with respect to translocation through the envelope membranes and also to the molecular machinery that recognizes these sequence motifs. Furthermore, the sequence information that confers Toc159 dependence or Toc132/120 dependence in protein import on these proteins remains unknown.The Rubisco small subunit (RbcS) and E1α TPs (RbcS-tp and E1α-tp) confer Toc159 dependence and Toc159 independence in protein import into chloroplasts, respectively (Smith et al., 2004). In this study, using these two TPs, we have determined the RbcS-tp sequence motifs that confer Toc159 dependence. Here, we have demonstrated that Toc159-dependent protein import is mediated independently by multiple sequence motifs: one of them is the group of Ser residues located in the N-terminal 12-amino acid segment and the other is in the C-terminal region ranging from amino acid positions 41 to 49.     

Growth of Transplastomic Cells Expressing d-Amino Acid Oxidase in Chloroplasts Is Tolerant to d-Alanine and Inhibited by d-Valine

Dual-conditional positive/negative selection markers are versatile genetic tools for manipulating genomes. Plastid genomes are relatively small and conserved DNA molecules that can be manipulated precisely by homologous recombination. High-yield expression of recombinant products and maternal inheritance of plastid-encoded traits make plastids attractive sites for modification. Here, we describe the cloning and expression of a dao gene encoding d-amino acid oxidase from Schizosaccharomyces pombe in tobacco (Nicotiana tabacum) plastids. The results provide genetic evidence for the uptake of d-amino acids into plastids, which contain a target that is inhibited by d-alanine. Importantly, this nonantibiotic-based selection system allows the use of cheap and widely available d-amino acids, which are relatively nontoxic to animals and microbes, to either select against (d-valine) or for (d-alanine) cells containing transgenic plastids. Positive/negative selection with d-amino acids was effective in vitro and against transplastomic seedlings grown in soil. The dual functionality of dao is highly suited to the polyploid plastid compartment, where it can be used to provide tolerance against potential d-alanine-based herbicides, control the timing of recombination events such as marker excision, influence the segregation of transgenic plastid genomes, identify loci affecting dao function in mutant screens, and develop d-valine-based methods to manage the spread of transgenic plastids tagged with dao.Selectable marker genes provide powerful genetic tools for manipulating plant genomes (Miki and McHugh, 2004; Day and Goldschmidt-Clermont, 2011). High and stable accumulation of high-value products in transgenic plastids (Daniell et al., 2009; Maliga and Bock, 2011), combined with restricted dissemination of plastid transgenes in pollen (Daniell et al., 1998; Ruf et al., 2007; Svab and Maliga, 2007), make plastid genomes an attractive target for modification. Reverse genetics allows site-directed changes to be introduced into the important set of genes present in plastids to study and ultimately improve their function (Whitney et al., 2011; Day, 2012). Positive conditional selectable marker genes allow transgenic cells to divide in the presence of chemicals such as antibiotics or herbicides that inhibit wild-type untransformed cells and underpin the development of herbicide-resistant transplastomic crops (Daniell et al., 1998, 2009; Iamtham and Day, 2000; Lutz et al., 2001; Ye et al., 2001; Dufourmantel et al., 2007; Shimizu et al., 2008). Negative conditional selectable marker genes inhibit the proliferation of transgenic cells when exposed to compounds that have a limited impact on the viability of wild-type cells (Miki and McHugh, 2004). Most commonly, the enzyme product of a negative selection gene converts an exogenous chemical substrate into a toxic product. Negative selectable markers allow genetic restriction technologies to manage the spread of transgenic crops (Daniell, 2002; Hills et al., 2007) and are valuable components of genome manipulation technologies involving recombination (Hardy et al., 2010). Negative selection can be used to promote genome changes by, for example, linking recombination events to the excision of a negative selection marker gene. Dual positive/negative selectable marker genes are particularly versatile tools for genome engineering. An example is the uracil3 (URA3) gene marker in Saccharomyces cerevisiae, where selection for or against URA3 encoding orotidine 5''-phosphate decarboxylase has been used to influence the timing of recombination events to manipulate chromosomes and introduce site-directed mutations into S. cerevisiae genes (Boeke et al., 1987).Homologous recombination is an effective tool for genome engineering in bacteria (Link et al., 1997) and S. cerevisiae (Boeke et al., 1987) and is the predominant recombination pathway operating in plastids (Day and Madesis, 2007). Plastid genomes are relatively small, sequenced in over 200 species (Jansen and Ruhlman, 2012), polyploid, and are highly suitable targets for genome engineering (O’Neill et al., 2012). Establishing a dual-marker system in plastids would allow control over the timing and efficiency of homologous recombination events, such as marker excision between direct repeats (Iamtham and Day, 2000). New positive selection markers not involving antibiotics are candidates for developing herbicide-tolerant transplastomic crops, contained by maternal inheritance of plastids (Daniell et al., 1998). Genetic containment methods require the insertion of a conditional negative selectable marker into the plastid genome and may be desirable in transplastomic “pharma” crops and cells expressing products for medicine (Daniell et al., 2009; Oey et al., 2009a, 2009b; Ruhlman et al., 2010; Gisby et al., 2011). Negative markers inserted into the nucleus would be ineffective for preventing the spread of transgenic plastids because nuclear and chloroplast genes are inherited independently and by different mechanisms.Nuclear expression of the Rhodotorula gracilis dao gene encoding d-amino acid oxidase (DAAO; EC 1.4.3.3) allows positive or negative selection of transgenic plant cells (Erikson et al., 2004). Transgenic nuclear dao plants were tolerant to d-Ala and d-Ser but sensitive to d-Val and d-Ile. A reversal of the tolerance pattern was observed in wild-type plants, which were sensitive to d-Ala and d-Ser but tolerant to d-Val and d-Ile. The toxic ketoacids 3-methyl-2-oxobutanoate and 3-methyl-2-oxopentanoate are produced by DAAO-catalyzed deamination of d-Val and d-Ile, respectively (Erikson et al., 2004). Originally developed in Arabidopsis (Arabidopsis thaliana), the system has been applied to crops (Lai et al., 2007). Importantly, d-amino acids are relatively cheap and nontoxic (Gullino et al., 1956; Friedman, 1999), thereby facilitating their use outside the laboratory. Cost and safety are important factors for applications as positive selection herbicides to control weeds in transgenic crops and negative selection agents to control the spread of transgenic crops. A dual selectable marker gene has not previously been described in plastids. The bacterial codA gene, encoding cytosine deaminase (Mullen et al., 1992), is the sole negative selection marker shown to work in plastids (Serino and Maliga, 1997). Transplastomic cells expressing codA are sensitive to the antibiotic 5-fluorocytosine (Serino and Maliga, 1997), which is used to treat fungal infections in humans, with possible side effects including hepatotoxicity (Steer et al., 1972). The relatively high cost of 5-fluorocytosine and its potential toxicity on nontarget organisms would hinder its use as a negative selection agent to manage the spread of transgenic crops expressing codA.Plastids are important centers for amino acid synthesis and metabolism (Lancien et al., 2006), but the impact of d-amino acids on plastids had not been reported previously. Here, we have developed a plastid marker based on the Schizosaccharomyces pombe dao gene (Wood et al., 2002) that confers either positive (d-Ala) or negative (d-Val) selection on transplastomic cells. Our results are consistent with d-amino acids being transported into plastids, where d-Ala inhibits one or more plastid functions and d-Val is converted into a toxic product by DAAO. The plastid dao gene provides a new versatile marker for plastid genetics, with applications for developing d-Ala-tolerant transplastomic plants, d-Val-based containment of transgenic plastids, and controlling the timing of recombination events in plastid genomes.     

Genetic Interactions between PEROXIN12 and Other Peroxisome-Associated Ubiquitination Components

Most eukaryotic cells require peroxisomes, organelles housing fatty acid β-oxidation and other critical metabolic reactions. Peroxisomal matrix proteins carry peroxisome-targeting signals that are recognized by one of two receptors, PEX5 or PEX7, in the cytosol. After delivering the matrix proteins to the organelle, these receptors are removed from the peroxisomal membrane or matrix. Receptor retrotranslocation not only facilitates further rounds of matrix protein import but also prevents deleterious PEX5 retention in the membrane. Three peroxisome-associated ubiquitin-protein ligases in the Really Interesting New Gene (RING) family, PEX2, PEX10, and PEX12, facilitate PEX5 retrotranslocation. However, the detailed mechanism of receptor retrotranslocation remains unclear in plants. We identified an Arabidopsis (Arabidopsis thaliana) pex12 Glu-to-Lys missense allele that conferred severe peroxisomal defects, including impaired β-oxidation, inefficient matrix protein import, and decreased growth. We compared this pex12-1 mutant to other peroxisome-associated ubiquitination-related mutants and found that RING peroxin mutants displayed elevated PEX5 and PEX7 levels, supporting the involvement of RING peroxins in receptor ubiquitination in Arabidopsis. Also, we observed that disruption of any Arabidopsis RING peroxin led to decreased PEX10 levels, as seen in yeast and mammals. Peroxisomal defects were exacerbated in RING peroxin double mutants, suggesting distinct roles of individual RING peroxins. Finally, reducing function of the peroxisome-associated ubiquitin-conjugating enzyme PEX4 restored PEX10 levels and partially ameliorated the other molecular and physiological defects of the pex12-1 mutant. Future biochemical analyses will be needed to determine whether destabilization of the RING peroxin complex observed in pex12-1 stems from PEX4-dependent ubiquitination on the pex12-1 ectopic Lys residue.Oilseed plants obtain energy for germination and early development by utilizing stored fatty acids (Graham, 2008). This β-oxidation of fatty acids to acetyl-CoA occurs in peroxisomes, organelles that also house other important metabolic reactions, including the glyoxylate cycle, several steps in photorespiration, and phytohormone production (Hu et al., 2012). For example, indole-3-butyric acid (IBA) is β-oxidized into the active auxin indole-3-acetic acid (IAA) in peroxisomes (Zolman et al., 2000, 2007, 2008; Strader et al., 2010; Strader and Bartel, 2011). Many peroxisomal metabolic pathways generate reactive oxygen species (Inestrosa et al., 1979; Hu et al., 2012), and peroxisomes also house antioxidative enzymes, like catalase and ascorbate peroxidase, to detoxify hydrogen peroxide (Wang et al., 1999; Mhamdi et al., 2012).Peroxisomes can divide by fission or be synthesized de novo from the endoplasmic reticulum (ER). Preperoxisomes with peroxisomal membrane proteins bud from the ER and fuse, allowing matrix proteins to be imported to form mature peroxisomes (van der Zand et al., 2012; Mayerhofer, 2016). Peroxin (PEX) proteins facilitate peroxisome biogenesis and matrix protein import. Most peroxins are involved in importing proteins destined for the peroxisome matrix, which are imported after recognition of a type 1 or type 2 peroxisome-targeting signal (PTS). The PTS1 is a tripeptide located at the C terminus of most peroxisome-bound proteins (Gould et al., 1989; Chowdhary et al., 2012). The less common PTS2 is a nonapeptide usually located near the N terminus (Swinkels et al., 1991; Reumann, 2004). PTS1 proteins are recognized by PEX5 (van der Leij et al., 1993; Zolman et al., 2000), PTS2 proteins are recognized by PEX7 (Marzioch et al., 1994; Braverman et al., 1997; Woodward and Bartel, 2005), and PEX7 binds to PEX5 to allow matrix protein delivery in plants and mammals (Otera et al., 1998; Hayashi et al., 2005; Woodward and Bartel, 2005). The cargo-receptor complex docks with the membrane peroxins PEX13 and PEX14 (Urquhart et al., 2000; Otera et al., 2002; Woodward et al., 2014), and PEX5 assists cargo translocation into the peroxisomal matrix (Meinecke et al., 2010) before dissociating from its cargo (Freitas et al., 2011).After cargo delivery, PEX5 is recycled to enable further rounds of cargo recruitment (Thoms and Erdmann, 2006). This process requires a set of peroxins that is implicated in ubiquitinating PEX5 so that it can be retrotranslocated back to the cytosol. PEX5 ubiquitination is best understood in yeast. In Saccharomyces cerevisiae, Pex5 is monoubiquitinated through the action of the peroxisome-tethered ubiquitin-conjugating enzyme Pex4 and the peroxisomal ubiquitin-protein ligase Pex12 (Platta et al., 2009) and returned to the cytosol with the assistance of a peroxisome-tethered ATPase complex containing Pex1 and Pex6 (Grimm et al., 2012). S. cerevisiae Pex5 also can be polyubiquitinated and targeted for proteasomal degradation (Kiel et al., 2005). The cytosolic ubiquitin-conjugating enzyme Ubc4 cooperates with the peroxisomal ubiquitin-protein ligase Pex2 to polyubiquitinate Pex5 (Platta et al., 2009). Pex10 has ubiquitin-protein ligase activity (Williams et al., 2008; Platta et al., 2009; El Magraoui et al., 2012), but whether Pex10 directly ubiquitinates Pex5 is controversial. Pex10 promotes Ubc4-dependent Pex5 polyubiquitination when Pex4 is absent (Williams et al., 2008); however, Pex10 is not essential for Pex5 mono- or polyubiquitination (Platta et al., 2009), but rather enhances both Pex4/Pex12- and Ubc4/Pex2-mediated ubiquitination (El Magraoui et al., 2012). Recycling of the PTS2 receptor PEX7 is less understood, although the Pex5 recycling pathways are implicated in shuttling and degrading Pex7 in Pichia pastoris (Hagstrom et al., 2014).Although PEX5 ubiquitination has not been directly demonstrated in plants, the implicated peroxins are conserved in Arabidopsis, and several have been connected to PEX5 retrotranslocation. The PEX4 ubiquitin-conjugating enzyme binds to PEX22, which is predicted to be a peroxisomal membrane protein based on ability to restore peroxisome function to yeast mutants (Zolman et al., 2005). The pex4-1 mutant displays increased membrane-associated PEX5 (Ratzel et al., 2011; Kao and Bartel, 2015), suggesting that ubiquitin supplied by PEX4 promotes PEX5 retrotranslocation. PEX1 and PEX6 are members of the ATPases associated with diverse cellular activities (AAA) family and are tethered to peroxisomes by the peroxisomal membrane protein PEX26 (Goto et al., 2011; Li et al., 2014). The pex6-1 mutant displays PTS1 import defects and decreased PEX5 levels (Zolman and Bartel, 2004), suggesting that impaired PEX5 recycling can lead to increased PEX5 degradation. Indeed, pex4-1 restores PEX5 levels in the pex6-1 mutant (Ratzel et al., 2011), suggesting that Arabidopsis PEX4 also is involved in PEX5 ubiquitination and degradation when retrotranslocation is impeded.In addition to allowing for further rounds of PTS1 cargo import, several lines of evidence suggest that in the absence of efficient retrotranslocation, PEX5 retention in the peroxisomal membrane impairs peroxisome function. Slightly reducing levels of the PEX13 docking peroxin ameliorates the physiological defects of pex4-1 without restoring matrix protein import (Ratzel et al., 2011), presumably because decreasing PEX5 docking reduces its accumulation in the peroxisomal membrane. In addition, overexpressing PEX5 exacerbates rather than ameliorates the peroxisomal defects of pex4-1 (Kao and Bartel, 2015), suggesting that pex4-1 defects are linked to excessive PEX5 lingering in the peroxisome membrane rather than a lack of PEX5 available for import.The three Really Interesting New Gene (RING) peroxins (PEX2, PEX10, and PEX12) from Arabidopsis each possesses in vitro ubiquitin-protein ligase activity (Kaur et al., 2013). Null mutations in the RING peroxin genes confer embryo lethality in Arabidopsis (Hu et al., 2002; Schumann et al., 2003; Sparkes et al., 2003; Fan et al., 2005; Prestele et al., 2010), necessitating other approaches to study the in vivo functions of these peroxins. Expressing RING peroxins with mutations in the C-terminal zinc-binding RING domains (ΔZn) confers matrix protein import defects for PEX2-ΔZn and photorespiration defects for PEX10-ΔZn but no apparent defects for PEX12-ΔZn (Prestele et al., 2010). Targeting individual RING peroxins using RNAi confers β-oxidation deficiencies and impairs PTS1 cargo import (Fan et al., 2005; Nito et al., 2007). A screen for delayed matrix protein degradation (Burkhart et al., 2013) uncovered a missense pex2-1 mutant and a splicing pex10-2 mutant that both display PTS1 import defects (Burkhart et al., 2014), suggesting roles in regulating the PTS1 receptor, PEX5. A missense pex12 mutant (aberrant peroxisome morphology 4, apm4) has defects in β-oxidation and PTS1 import and increased membrane-associated PEX5 (Mano et al., 2006). These findings highlight the essential roles of the RING peroxins in Arabidopsis development and peroxisomal functions, but the RING peroxin interactions and the individual roles of the RING peroxins in PEX5 retrotranslocation remain incompletely understood.In this study, we describe a missense pex12-1 mutant recovered from a forward genetic screen for β-oxidation deficient mutants. The pex12-1 mutant displayed severe peroxisomal defects, including reduced growth, β-oxidation deficiencies, matrix protein import defects, and inefficient processing of PTS2 proteins. Comparing single and double mutants with impaired RING peroxins revealed that each RING peroxin contributes to complex stability and influences PEX5 accumulation. Furthermore, decreasing PEX4 function ameliorated pex12-1 defects, suggesting that the Glu-to-Lys substitution in pex12-1 lures ubiquitination, perhaps by pex12-1 itself, leading to PEX4-dependent degradation of the mutant protein.     

PLASTID MOVEMENT IMPAIRED1 and PLASTID MOVEMENT IMPAIRED1-RELATED1 Mediate Photorelocation Movements of Both Chloroplasts and Nuclei

Organelle movement and positioning play important roles in fundamental cellular activities and adaptive responses to environmental stress in plants. To optimize photosynthetic light utilization, chloroplasts move toward weak blue light (the accumulation response) and escape from strong blue light (the avoidance response). Nuclei also move in response to strong blue light by utilizing the light-induced movement of attached plastids in leaf cells. Blue light receptor phototropins and several factors for chloroplast photorelocation movement have been identified through molecular genetic analysis of Arabidopsis (Arabidopsis thaliana). PLASTID MOVEMENT IMPAIRED1 (PMI1) is a plant-specific C2-domain protein that is required for efficient chloroplast photorelocation movement. There are two PLASTID MOVEMENT IMPAIRED1-RELATED (PMIR) genes, PMIR1 and PMIR2, in the Arabidopsis genome. However, the mechanism in which PMI1 regulates chloroplast and nuclear photorelocation movements and the involvement of PMIR1 and PMIR2 in these organelle movements remained unknown. Here, we analyzed chloroplast and nuclear photorelocation movements in mutant lines of PMI1, PMIR1, and PMIR2. In mesophyll cells, the pmi1 single mutant showed severe defects in both chloroplast and nuclear photorelocation movements resulting from the impaired regulation of chloroplast-actin filaments. In pavement cells, pmi1 mutant plants were partially defective in both plastid and nuclear photorelocation movements, but pmi1pmir1 and pmi1pmir1pmir2 mutant lines lacked the blue light-induced movement responses of plastids and nuclei completely. These results indicated that PMI1 is essential for chloroplast and nuclear photorelocation movements in mesophyll cells and that both PMI1 and PMIR1 are indispensable for photorelocation movements of plastids and thus, nuclei in pavement cells.In plants, organelles move within the cell and become appropriately positioned to accomplish their functions and adapt to the environment (for review, see Wada and Suetsugu, 2004). Light-induced chloroplast movement (chloroplast photorelocation movement) is one of the best characterized organelle movements in plants (Suetsugu and Wada, 2012). Under weak light conditions, chloroplasts move toward light to capture light efficiently (the accumulation response; Zurzycki, 1955). Under strong light conditions, chloroplasts escape from light to avoid photodamage (the avoidance response; Kasahara et al., 2002; Sztatelman et al., 2010; Davis and Hangarter, 2012; Cazzaniga et al., 2013). In most green plant species, these responses are induced primarily by the blue light receptor phototropin (phot) in response to a range of wavelengths from UVA to blue light (approximately 320–500 nm; for review, see Suetsugu and Wada, 2012; Wada and Suetsugu, 2013; Kong and Wada, 2014). Phot-mediated chloroplast movement has been shown in land plants, such as Arabidopsis (Arabidopsis thaliana; Jarillo et al., 2001; Kagawa et al., 2001; Sakai et al., 2001), the fern Adiantum capillus-veneris (Kagawa et al., 2004), the moss Physcomitrella patens (Kasahara et al., 2004), and the liverwort Marchantia polymorpha (Komatsu et al., 2014). Two phots in Arabidopsis, phot1 and phot2, redundantly mediate the accumulation response (Sakai et al., 2001), whereas phot2 primarily regulates the avoidance response (Jarillo et al., 2001; Kagawa et al., 2001; Luesse et al., 2010). M. polymorpha has only one phot that mediates both the accumulation and avoidance responses (Komatsu et al., 2014), although two or more phots mediate chloroplast photorelocation movement in A. capillus-veneris (Kagawa et al., 2004) and P. patens (Kasahara et al., 2004). Thus, duplication and functional diversification of PHOT genes have occurred during land plant evolution, and plants have gained a sophisticated light sensing system for chloroplast photorelocation movement.In general, movements of plant organelles, including chloroplasts, are dependent on actin filaments (for review, see Wada and Suetsugu, 2004). Most organelles common in eukaryotes, such as mitochondria, peroxisomes, and Golgi bodies, use the myosin motor for their movements, but there is no clear evidence that chloroplast movement is myosin dependent (for review, see Suetsugu et al., 2010a). Land plants have innovated a novel actin-based motility system that is specialized for chloroplast movement as well as a photoreceptor system (for review, see Suetsugu et al., 2010a; Wada and Suetsugu, 2013; Kong and Wada, 2014). Chloroplast-actin (cp-actin) filaments, which were first found in Arabidopsis, are short actin filaments specifically localized around the chloroplast periphery at the interface between the chloroplast and the plasma membrane (Kadota et al., 2009). Strong blue light induces the rapid disappearance of cp-actin filaments and then, their subsequent reappearance preferentially at the front region of the moving chloroplasts. This asymmetric distribution of cp-actin filaments is essential for directional chloroplast movement (Kadota et al., 2009; Kong et al., 2013a). The greater the difference in the amount of cp-actin filaments between the front and rear regions of chloroplasts becomes, the faster the chloroplasts move, in which the magnitude of the difference is determined by fluence rate (Kagawa and Wada, 2004; Kadota et al., 2009; Kong et al., 2013a). Strong blue light-induced disappearance of cp-actin filaments is regulated in a phot2-dependent manner before the intensive polymerization of cp-actin filaments at the front region occurs (Kadota et al., 2009; Ichikawa et al., 2011; Kong et al., 2013a). This phot2-dependent response contributes to the greater difference in the amount of cp-actin filaments between the front and rear regions of chloroplasts. Similar behavior of cp-actin filaments has also been observed in A. capillus-veneris (Tsuboi and Wada, 2012) and P. patens (Yamashita et al., 2011).Like chloroplasts, nuclei also show light-mediated movement and positioning (nuclear photorelocation movement) in land plants (for review, see Higa et al., 2014b). In gametophytic cells of A. capillus-veneris, weak light induced the accumulation responses of both chloroplasts and nuclei, whereas strong light induced avoidance responses (Kagawa and Wada, 1993, 1995; Tsuboi et al., 2007). However, in mesophyll cells of Arabidopsis, strong blue light induced both chloroplast and nuclear avoidance responses, but weak blue light induced only the chloroplast accumulation response (Iwabuchi et al., 2007, 2010; Higa et al., 2014a). In Arabidopsis pavement cells, small numbers of tiny plastids were found and showed autofluorescence under the confocal laser-scanning microscopy (Iwabuchi et al., 2010; Higa et al., 2014a). Hereafter, the plastid in the pavement cells is called the pavement cell plastid. Strong blue light-induced avoidance responses of pavement cell plastids and nuclei were induced in a phot2-dependent manner, but the accumulation response was not detected for either organelle (Iwabuchi et al., 2007, 2010; Higa et al., 2014a). In both Arabidopsis and A. capillus-veneris, phots mediate nuclear photorelocation movement, and phot2 mediates the nuclear avoidance response (Iwabuchi et al., 2007, 2010; Tsuboi et al., 2007). The nuclear avoidance response is dependent on actin filaments in both mesophyll and pavement cells of Arabidopsis (Iwabuchi et al., 2010). Recently, it was shown that the nuclear avoidance response relies on cp-actin-dependent movement of pavement cell plastids, where nuclei are associated with pavement cell plastids of Arabidopsis (Higa et al., 2014a). In mesophyll cells, nuclear avoidance response is likely dependent on cp-actin filament-mediated chloroplast movement, because the mutants deficient in chloroplast movement were also defective in nuclear avoidance response (Higa et al., 2014a). Thus, phots mediate both chloroplast (and pavement cell plastid) and nuclear photorelocation movement by regulating cp-actin filaments.Molecular genetic analyses of Arabidopsis mutants deficient in chloroplast photorelocation movement have identified many molecular factors involved in signal transduction and/or motility systems as well as those involved in the photoreceptor system for chloroplast photorelocation movement (and thus, nuclear photorelocation movement; for review, see Suetsugu and Wada, 2012; Wada and Suetsugu, 2013; Kong and Wada, 2014). CHLOROPLAST UNUSUAL POSITIONING1 (CHUP1; Oikawa et al., 2003) and KINESIN-LIKE PROTEIN FOR ACTIN-BASED CHLOROPLAST MOVEMENT (KAC; Suetsugu et al., 2010b) are key factors for generating and/or maintaining cp-actin filaments. Both proteins are highly conserved in land plants and essential for the movement and attachment of chloroplasts to the plasma membrane in Arabidopsis (Oikawa et al., 2003, 2008; Suetsugu et al., 2010b), A. capillus-veneris (Suetsugu et al., 2012), and P. patens (Suetsugu et al., 2012; Usami et al., 2012). CHUP1 is localized on the chloroplast outer membrane and binds to globular and filamentous actins and profilin in vitro (Oikawa et al., 2003, 2008; Schmidt von Braun and Schleiff, 2008). Although KAC is a kinesin-like protein, it lacks microtubule-dependent motor activity but has filamentous actin binding activity (Suetsugu et al., 2010b). An actin-bundling protein THRUMIN1 (THRUM1) is required for efficient chloroplast photorelocation movement (Whippo et al., 2011) and interacts with cp-actin filaments (Kong et al., 2013a). chup1 and kac mutant plants were shown to lack detectable cp-actin filaments (Kadota et al., 2009; Suetsugu et al., 2010b; Ichikawa et al., 2011; Kong et al., 2013a). Similarly, cp-actin filaments were rarely detected in thrum1 mutant plants (Kong et al., 2013a), indicating that THRUM1 also plays an important role in maintaining cp-actin filaments.Other proteins J-DOMAIN PROTEIN REQUIRED FOR CHLOROPLAST ACCUMULATION RESPONSE1 (JAC1; Suetsugu et al., 2005), WEAK CHLOROPLAST MOVEMENT UNDER BLUE LIGHT1 (WEB1; Kodama et al., 2010), and PLASTID MOVEMENT IMPAIRED2 (PMI2; Luesse et al., 2006; Kodama et al., 2010) are involved in the light regulation of cp-actin filaments and chloroplast photorelocation movement. JAC1 is an auxilin-like J-domain protein that mediates the chloroplast accumulation response through its J-domain function (Suetsugu et al., 2005; Takano et al., 2010). WEB1 and PMI2 are coiled-coil proteins that interact with each other (Kodama et al., 2010). Although web1 and pmi2 were partially defective in the avoidance response, the jac1 mutation completely suppressed the phenotype of web1 and pmi2, suggesting that the WEB1/PMI2 complex suppresses JAC1 function (i.e. the accumulation response) under strong light conditions (Kodama et al., 2010). Both web1 and pmi2 showed impaired disappearance of cp-actin filaments in response to strong blue light (Kodama et al., 2010). However, the exact molecular functions of these proteins are unknown.In this study, we characterized mutant plants deficient in the PMI1 gene and two homologous genes PLASTID MOVEMENT IMPAIRED1-RELATED1 (PMIR1) and PMIR2. PMI1 was identified through molecular genetic analyses of pmi1 mutants that showed severe defects in chloroplast accumulation and avoidance responses (DeBlasio et al., 2005). PMI1 is a plant-specific C2-domain protein (DeBlasio et al., 2005; Zhang and Aravind, 2010), but its roles and those of PMIRs in cp-actin-mediated chloroplast and nuclear photorelocation movements remained unclear. Thus, we analyzed chloroplast and nuclear photorelocation movements in the single, double, and triple mutants of pmi1, pmir1, and pmir2.     

Quantitative Analysis of the Mitochondrial and Plastid Proteomes of the Moss Physcomitrella patens Reveals Protein Macrocompartmentation and Microcompartmentation

Extant eukaryotes are highly compartmentalized and have integrated endosymbionts as organelles, namely mitochondria and plastids in plants. During evolution, organellar proteomes are modified by gene gain and loss, by gene subfunctionalization and neofunctionalization, and by changes in protein targeting. To date, proteomics data for plastids and mitochondria are available for only a few plant model species, and evolutionary analyses of high-throughput data are scarce. We combined quantitative proteomics, cross-species comparative analysis of metabolic pathways, and localizations by fluorescent proteins in the model plant Physcomitrella patens in order to assess evolutionary changes in mitochondrial and plastid proteomes. This study implements data-mining methodology to classify and reliably reconstruct subcellular proteomes, to map metabolic pathways, and to study the effects of postendosymbiotic evolution on organellar pathway partitioning. Our results indicate that, although plant morphologies changed substantially during plant evolution, metabolic integration of organelles is largely conserved, with exceptions in amino acid and carbon metabolism. Retargeting or regulatory subfunctionalization are common in the studied nucleus-encoded gene families of organelle-targeted proteins. Moreover, complementing the proteomic analysis, fluorescent protein fusions revealed novel proteins at organelle interfaces such as plastid stromules (stroma-filled tubules) and highlight microcompartments as well as intercellular and intracellular heterogeneity of mitochondria and plastids. Thus, we establish a comprehensive data set for mitochondrial and plastid proteomes in moss, present a novel multilevel approach to organelle biology in plants, and place our findings into an evolutionary context.Endosymbiosis has enabled and shaped eukaryotic evolution. The engulfment of an ancestral α-proteobacterium by a presumably archaebacterial host cell stands at the origin of mitochondrial and eukaryotic evolution over 1.5 billion years ago (Dyall et al., 2004). In plants, the subsequent uptake of a photosynthetic bacterium between 1.5 and 1.2 billion years ago led to the formation of chloroplasts (Dyall et al., 2004). Plants thereby evolved by the integration of three distinct genetic compartments. After the establishment of endosymbiosis, genes were transferred to a great extent, mainly from mitochondria and plastids to the nucleus (Bock and Timmis, 2008), necessitating an orchestrated flux of information in the form of proteins and metabolites between the compartments of eukaryotic cells to ensure homeostasis, growth, and development. This communication between organelles is facilitated by physical interactions (Kornmann et al., 2009), control of protein import (Ling et al., 2012), and retrograde signaling (Nargund et al., 2012). During radiation and diversification, especially of land plants, nuclear genomes substantially changed due to endosymbiotic and horizontal (Yue et al., 2012) gene transfer, genome duplication, and gene gain and loss (Duarte et al., 2006; Lang et al., 2010; Martin, 2010), obtruding the question of whether these phenomena are linked to alterations in metabolic pathway partitioning between organelles. Retained paralogs can either introduce a new function (neofunctionalization) or reconstitute existing functions (subfunctionalization; Duarte et al., 2006), for example by distinct spatiotemporal expression profiles or distinct subcellular localizations, resulting in the modulation or introduction of metabolic functions in the respective cellular compartments. Moreover, proteins can localize to several subcellular compartments, a phenomenon called dual or multiple targeting (Yogev and Pines, 2011; Xu et al., 2013). Consequently, many eukaroytic metabolic pathways, as well as the plastid and mitochondrial proteomes, are constituted of a mosaic of proteins of diverse evolutionary origins (Szklarczyk and Huynen, 2010), and evolution has shaped variable organellar functionalities across taxa. To date, the evolution and variability of postendosymbiotic metabolic partitioning is largely not characterized on a high-throughput level. So far, large-scale mitochondrial proteome data sets are only available for the green alga Chlamydomonas reinhardtii (Atteia et al., 2009), rice (Oryza sativa; Huang et al., 2009), and the model flowering plant Arabidopsis (Arabidopsis thaliana; Millar et al., 2001; Heazlewood et al., 2004), whereas plastid proteomics in plants is on an advanced level and covers more species (Polyakov et al., 2010; van Wijk and Baginsky, 2011).While higher plants diversified relatively recently but massively, simple moss plants can be traced back 330 million years (Hubers and Kerp, 2012), identifying them as prime candidates for an evolutionary view of organellar proteomes and organelle biology at a genome-wide scale. In contrast to specialized flowering plants, mosses are generalists with few tissues, high metabolic variability, and ancestral features such as high abiotic stress tolerance (Frank et al., 2007) and few plastid types (Cove, 2005).By integrating quantitative proteomics, multivariate analysis, metabolic pathway maps, phylogenomics, and localization with fluorescent proteins, we reliably characterize subcellular proteomes and gene family diversification. Key characteristics of postendosymbiotic organellar proteome evolution are identified by cross-species comparative analysis. In support of our high-throughput analyses, we conduct single-protein analyses and identify proteins that mark microcompartments within organelles and localize to dynamic contact sites between organelles. These proteins may facilitate the exchange of proteins and metabolites, while others influence the dynamics of individual chloroplasts and mitochondria. This study characterizes the mitochondrial and plastid proteomes of moss and reveals the heterogeneity of organelles within a single cell.     

Photosystem II and Pigment Dynamics among Ecotypes of the Green Alga Ostreococcus

We investigated the photophysiological responses of three ecotypes of the picophytoplankter Ostreococcus and a larger prasinophyte Pyramimonas obovata to a sudden increase in light irradiance. The deepwater Ostreococcus sp. RCC809 showed very high susceptibility to primary photoinactivation, likely a consequence of high oxidative stress, which may relate to the recently noted plastid terminal oxidase activity in this strain. The three Ostreococcus ecotypes were all capable of deploying modulation of the photosystem II repair cycle in order to cope with the light increase, but the effective clearance of photoinactivated D1 protein appeared to be slower in the deepwater Ostreococcus sp. RCC809, suggesting that this step is rate limiting in the photosystem II repair cycle in this strain. Moreover, the deepwater Ostreococcus accumulated lutein and showed substantial use of the xanthophyll cycle under light stress, demonstrating its high sensitivity to light fluctuations. The sustained component of the nonphotochemical quenching of fluorescence correlated well with the xanthophyll deepoxidation activity. Comparisons with the larger prasinophyte P. obovata suggest that the photophysiology of Ostreococcus ecotypes requires high photosystem II repair rates to counter a high susceptibility to photoinactivation, consistent with low pigment package effects in their minute-sized cells.The prasinophytes are marine planktonic green algae with a phylogenetic position branching near the base of the green lineage (Baldauf, 2003; Turmel et al., 2009). They are widespread in temperate (Diez et al., 2001; Zhu et al., 2005) and polar (Lovejoy et al., 2007) marine habitats, in which they are often significant contributors to primary production (Not et al., 2004). The prasinophytes include the smallest known eukaryotic photoautotroph, Ostreococcus tauri (Courties et al., 1994; Chrétiennot-Dinet et al., 1995), whose particularly simple structure makes it an attractive model minimal chlorophyte, and indeed, minimal eukaryote (Derelle et al., 2006). Recently, genomic sequences for three Ostreococcus strains, isolated from different ecological niches, have become available (Derelle et al., 2006; Palenik et al., 2007), thus increasing the interest of these models for understanding acclimation processes in this deep-branching group of chlorophytes.Photoacclimation strategies differ in two Ostreococcus strains (Cardol et al., 2008; Six et al., 2008), which, although belonging to different phylogenetic clades, are nonetheless morphologically indistinguishable (Rodríguez et al., 2005). O. tauri, a eutrophilic lagoon species, modulates PSII content to enable acclimation and growth over a wide range of irradiances. In marked contrast, Ostreococcus sp. (O. sp.) RCC809, isolated at 105 m depth in the tropical Atlantic Ocean, modulates the size of its large PSII antenna in a strategy that accommodates a narrower range of light levels but that incurs lower nutrient costs compared with photoacclimation in O. tauri (Six et al., 2008). The evidence for different light acclimation strategies between these two Ostreococcus ecotypes raises the question of the underlying physiological processes for niche adaptation in these closely related organisms. Cardol et al. (2008) recently analyzed the coastal O. tauri and the deepwater O. sp. RCC809 grown under low to moderate light and found exciting evidence for a plastid terminal oxidase electron flow path in O. sp. RCC809 from PSII back to oxygen, short-circuiting the usual Z scheme in a mechanism to generate a transthylakoidal pH gradient without net generation of reductant. Like all oxygenic photoautotrophs, the prasinophytes suffer photoinactivation of PSII (Aro et al., 1993; Tyystjarvi, 2008; Guskov et al., 2009) at a rate approximately proportional to the incident irradiance (Nagy et al., 1995; Hakala et al., 2005). To counter this photoinactivation, a PSII repair cycle proteolytically removes the photoinactivated D1 protein (Silva et al., 2003) and replaces it through de novo synthesis and reassembly with the remaining subunits (Aro et al., 1993). If photoinactivation outruns the rate of repair, the PSII pool suffers net photoinhibition (Aro et al., 2005; Nishiyama et al., 2005, 2006; Murata et al., 2007), leading to a decrease in photosynthetic capacity and potentially to a decrease in growth. To limit photoinhibition, photosynthetic cells use physiological processes that dissipate excess light energy into heat, thereby preempting the generation of toxic reactive oxygen species (Baroli et al., 2004; Holt et al., 2004) that can inhibit metabolism, notably including the PSII repair processes (Nishiyama et al., 2006; Murata et al., 2007). These excitation dissipation mechanisms manifest as a drop in PSII fluorescence yield termed nonphotochemical quenching of fluorescence (NPQ). In land plants and characterized chlorophytes, NPQ is notably associated with changes in light-harvesting complex conformation along with pigmentation changes (Demmig-Adams and Adams, 1992; Baroli et al., 2004; Holt et al., 2004; Li et al., 2004).The specialization of Ostreococcus ecotypes to contrasting environments suggests that they may have evolved distinct capacities to cope with rapid fluctuations in light. Here, we investigate this question by subjecting three different Ostreococcus ecotypes to short-term increases in light irradiance to uncover their capacities for PSII repair and susceptibilities to photoinactivation. We use a target theory approach (Nagy et al., 1995; Sinclair et al., 1996) to parameterize their susceptibility to primary photoinactivation in a form useful for predicting and modeling responses to changes in irradiance. We moreover compare the Ostreococcus strains to a much larger prasinophyte derived from temperate surface waters, Pyramimonas obovata, to explore how cell size can influence photophysiology in the prasinophytes.     

Hunting for Plant Nitric Oxide Synthase Provides New Evidence of a Central Role for Plastids in Nitric Oxide Metabolism

Nitric oxide (NO) has emerged as a central signaling molecule in plants and animals. However, the long search for a plant NO synthase (NOS) enzyme has only encountered false leads. The first works describing a pathogen-induced NOS-like plant protein were soon retracted. New hope came from the identification of NOS1, an Arabidopsis thaliana protein with an atypical NOS activity that was found to be targeted to mitochondria in roots. Although concerns about the NO-producing activity of this protein were raised (causing the renaming of the protein to NO-associated 1), compelling data on its biological role were missing until recently. Strong evidence is now available that this protein functions as a GTPase that is actually targeted to plastids, where it might be required for ribosome function. These and other results support the argument that the defective NO production in loss-of-function mutants is an indirect effect of interfering with normal plastid functions and that plastids play an important role in regulating NO levels in plant cells.A major revolution in biology took place by the early 1990s after the discovery that nitric oxide (NO), a free radical, was not a toxic by-product of oxidative metabolism but had a fundamental role as a signaling molecule regulating normal physiological processes in animal cells (Culotta and Koshland, 1992). A role of this volatile molecule in plant defense responses was subsequently reported, and it is now well established that NO is also a key player in the regulation of different plant developmental processes, including germination, root growth, vascular differentiation, stomatal closure, and flowering (Lamattina et al., 2003; Wendehenne et al., 2004; Crawford and Guo, 2005). Animal cells synthesize NO primarily by the activity of NO synthase (NOS) enzymes. There are several NOS isoforms, but all of them catalyze the same basic reaction: a NADPH-dependent oxidation of l-Arg to NO and l-citrulline. By contrast, the synthesis of NO in plant cells remains a matter of debate. The first reported mechanism to make NO in plants was the reduction of nitrite to NO catalyzed (with low efficiency) by nitrate reductase (NR), a cytosolic enzyme that normally reduces nitrate to nitrite (Yamasaki et al., 1999). But the contribution of NR to NO synthesis is still controversial.The analysis of the Arabidopsis thaliana nia1 nia2 double mutant, which shows substantially reduced NR activity levels, has shown that such activity is required for NO synthesis during flowering (Seligman et al., 2008), auxin-induced lateral root development (Kolbert et al., 2008), and abscisic acid (ABA)-induced stomatal closure (Desikan et al., 2002; Bright et al., 2006) but not during infection (Zhang et al., 2003), salicylic acid treatment (Zottini et al., 2007), or mechanical stress (Garces et al., 2001). Furthermore, foliar extracts of the mutant show the same capacity to produce NO as wild-type plants when nitrite is exogenously supplied (Modolo et al., 2005). These results indicate that additional mechanisms to reduce nitrite into NO exist in plant cells and that the decreased capability for NO synthesis of mutant plants with defective NR activity might result from their reduced nitrite levels (Modolo et al., 2005). Other enzymatic sources for nitrite-dependent NO synthesis exist in the plasma membrane (Stohr et al., 2001) and mitochondria (Planchet et al., 2005), whereas nonenzymatic production of NO from nitrite has been shown to occur in acidic and reducing environments, such as the apoplasm (Bethke et al., 2004) and plastids (Cooney et al., 1994). The highly reduced levels of l-Arg in the nia1 nia2 mutant (Modolo et al., 2006) might also compromise its ability to produce NO. This amino acid is a substrate for the production of polyamines, compounds that have been proposed to participate in NO synthesis (Tun et al., 2006). Additionally, plants have been found to synthesize NO by an Arg-dependent NOS activity similar to that present in animal cells, as detailed in the next section.