Light-Harvesting Complex Protein LHCBM9 Is Critical for Photosystem II Activity and Hydrogen Production in Chlamydomonas reinhardtii

Photosynthetic organisms developed multiple strategies for balancing light-harvesting versus intracellular energy utilization to survive ever-changing environmental conditions. The light-harvesting complex (LHC) protein family is of paramount importance for this function and can form light-harvesting pigment protein complexes. In this work, we describe detailed analyses of the photosystem II (PSII) LHC protein LHCBM9 of the microalga Chlamydomonas reinhardtii in terms of expression kinetics, localization, and function. In contrast to most LHC members described before, LHCBM9 expression was determined to be very low during standard cell cultivation but strongly increased as a response to specific stress conditions, e.g., when nutrient availability was limited. LHCBM9 was localized as part of PSII supercomplexes but was not found in association with photosystem I complexes. Knockdown cell lines with 50 to 70% reduced amounts of LHCBM9 showed reduced photosynthetic activity upon illumination and severe perturbation of hydrogen production activity. Functional analysis, performed on isolated PSII supercomplexes and recombinant LHCBM9 proteins, demonstrated that presence of LHCBM9 resulted in faster chlorophyll fluorescence decay and reduced production of singlet oxygen, indicating upgraded photoprotection. We conclude that LHCBM9 has a special role within the family of LHCII proteins and serves an important protective function during stress conditions by promoting efficient light energy dissipation and stabilizing PSII supercomplexes.     

MET1 Is a Thylakoid-Associated TPR Protein Involved in Photosystem II Supercomplex Formation and Repair in Arabidopsis

Photosystem II (PSII) requires constant disassembly and reassembly to accommodate replacement of the D1 protein. Here, we characterize Arabidopsis thaliana MET1, a PSII assembly factor with PDZ and TPR domains. The maize (Zea mays) MET1 homolog is enriched in mesophyll chloroplasts compared with bundle sheath chloroplasts, and MET1 mRNA and protein levels increase during leaf development concomitant with the thylakoid machinery. MET1 is conserved in C3 and C4 plants and green algae but is not found in prokaryotes. Arabidopsis MET1 is a peripheral thylakoid protein enriched in stroma lamellae and is also present in grana. Split-ubiquitin assays and coimmunoprecipitations showed interaction of MET1 with stromal loops of PSII core components CP43 and CP47. From native gels, we inferred that MET1 associates with PSII subcomplexes formed during the PSII repair cycle. When grown under fluctuating light intensities, the Arabidopsis MET1 null mutant (met1) showed conditional reduced growth, near complete blockage in PSII supercomplex formation, and concomitant increase of unassembled CP43. Growth of met1 in high light resulted in loss of PSII supercomplexes and accelerated D1 degradation. We propose that MET1 functions as a CP43/CP47 chaperone on the stromal side of the membrane during PSII assembly and repair. This function is consistent with the observed differential MET1 accumulation across dimorphic maize chloroplasts.     

Non-Recognition-of-BTH4, an Arabidopsis Mediator Subunit Homolog,Is Necessary for Development and Response to Salicylic Acid

Salicylic acid (SA) signaling acts in defense and plant development. The only gene demonstrated to be required for the response to SA is Arabidopsis thaliana NON-EXPRESSER OF PATHOGENESIS-RELATED GENE 1 (NPR1), and npr1 mutants are insensitive to SA. By focusing on the effect of analogs of SA on plant development, we identified mutants in additional genes acting in the SA response. In this work, we describe a gene necessary for the SA Non-Recognition-of-BTH4 (NRB4). Three nrb4 alleles recovered from the screen cause phenotypes similar to the wild type in the tested conditions, except for SA-related phenotypes. Plants with NRB4 null alleles express profound insensitivity to SA, even more than npr1. NRB4 null mutants are also sterile and their growth is compromised. Plants carrying weaker nrb4 alleles are also insensitive to SA, with some quantitative differences in some phenotypes, like systemic acquired resistance or pathogen growth restriction. When weak alleles are used, NPR1 and NRB4 mutations produce an additive phenotype, but we did not find evidence of a genetic interaction in F1 nor biochemical interaction in yeast or in planta. NRB4 is predicted to be a subunit of Mediator, the ortholog of MED15 in Arabidopsis. Mechanistically, NRB4 functions downstream of NPR1 to regulate the SA response.     

Cytochrome P450 CYP89A9 Is Involved in the Formation of Major Chlorophyll Catabolites during Leaf Senescence in Arabidopsis

Nonfluorescent chlorophyll catabolites (NCCs) were described as products of chlorophyll breakdown in Arabidopsis thaliana. NCCs are formyloxobilin-type catabolites derived from chlorophyll by oxygenolytic opening of the chlorin macrocycle. These linear tetrapyrroles are generated from their fluorescent chlorophyll catabolite (FCC) precursors by a nonenzymatic isomerization inside the vacuole of senescing cells. Here, we identified a group of distinct dioxobilin-type chlorophyll catabolites (DCCs) as the major breakdown products in wild-type Arabidopsis, representing more than 90% of the chlorophyll of green leaves. The molecular constitution of the most abundant nonfluorescent DCC (NDCC), At-NDCC-1, was determined. We further identified cytochrome P450 monooxygenase CYP89A9 as being responsible for NDCC accumulation in wild-type Arabidopsis; cyp89a9 mutants that are deficient in CYP89A9 function were devoid of NDCCs but accumulated proportionally higher amounts of NCCs. CYP89A9 localized outside the chloroplasts, implying that FCCs occurring in the cytosol might be its natural substrate. Using recombinant CYP89A9, we confirm FCC specificity and show that fluorescent DCCs are the products of the CYP89A9 reaction. Fluorescent DCCs, formed by this enzyme, isomerize to the respective NDCCs in weakly acidic medium, as found in vacuoles. We conclude that CYP89A9 is involved in the formation of dioxobilin-type catabolites of chlorophyll in Arabidopsis.     

Arabidopsis SEIPIN Proteins Modulate Triacylglycerol Accumulation and Influence Lipid Droplet Proliferation

The lipodystrophy protein SEIPIN is important for lipid droplet (LD) biogenesis in human and yeast cells. In contrast with the single SEIPIN genes in humans and yeast, there are three SEIPIN homologs in Arabidopsis thaliana, designated SEIPIN1, SEIPIN2, and SEIPIN3. Essentially nothing is known about the functions of SEIPIN homologs in plants. Here, a yeast (Saccharomyces cerevisiae) SEIPIN deletion mutant strain and a plant (Nicotiana benthamiana) transient expression system were used to test the ability of Arabidopsis SEIPINs to influence LD morphology. In both species, expression of SEIPIN1 promoted accumulation of large-sized lipid droplets, while expression of SEIPIN2 and especially SEIPIN3 promoted small LDs. Arabidopsis SEIPINs increased triacylglycerol levels and altered composition. In tobacco, endoplasmic reticulum (ER)-localized SEIPINs reorganized the normal, reticulated ER structure into discrete ER domains that colocalized with LDs. N-terminal deletions and swapping experiments of SEIPIN1 and 3 revealed that this region of SEIPIN determines LD size. Ectopic overexpression of SEIPIN1 in Arabidopsis resulted in increased numbers of large LDs in leaves, as well as in seeds, and increased seed oil content by up to 10% over wild-type seeds. By contrast, RNAi suppression of SEIPIN1 resulted in smaller seeds and, as a consequence, a reduction in the amount of oil per seed compared with the wild type. Overall, our results indicate that Arabidopsis SEIPINs are part of a conserved LD biogenesis machinery in eukaryotes and that in plants these proteins may have evolved specialized roles in the storage of neutral lipids by differentially modulating the number and sizes of lipid droplets.     

A Wheat SIMILAR TO RCD-ONE Gene Enhances Seedling Growth and Abiotic Stress Resistance by Modulating Redox Homeostasis and Maintaining Genomic Integrity

Plant growth inhibition is a common response to salinity. Under saline conditions, Shanrong No. 3 (SR3), a bread wheat (Triticum aestivum) introgression line, performs better than its parent wheat variety Jinan 177 (JN177) with respect to both seedling growth and abiotic stress tolerance. Furthermore, the endogenous reactive oxygen species (ROS) was also elevated in SR3 relative to JN177. The SR3 allele of sro1, a gene encoding a poly(ADP ribose) polymerase (PARP) domain protein, was identified to be crucial for both aspects of its superior performance. Unlike RADICAL-INDUCED CELL DEATH1 and other Arabidopsis thaliana SIMILAR TO RCD-ONE (SRO) proteins, sro1 has PARP activity. Both the overexpression of Ta-sro1 in wheat and its heterologous expression in Arabidopsis promote the accumulation of ROS, mainly by enhancing the activity of NADPH oxidase and the expression of NAD(P)H dehydrogenase, in conjunction with the suppression of alternative oxidase expression. Moreover, it promotes the activity of ascorbate-GSH cycle enzymes and GSH peroxidase cycle enzymes, which regulate ROS content and cellular redox homeostasis. sro1 is also found to be involved in the maintenance of genomic integrity. We show here that the wheat SRO has PARP activity; such activity could be manipulated to improve the growth of seedlings exposed to salinity stress by modulating redox homeostasis and maintaining genomic stability.     

Arabidopsis Small Rubber Particle Protein Homolog SRPs Play Dual Roles as Positive Factors for Tissue Growth and Development and in Drought Stress Responses

Lipid droplets (LDs) act as repositories for fatty acids and sterols, which are used for various cellular processes such as energy production and membrane and hormone synthesis. LD-associated proteins play important roles in seed development and germination, but their functions in postgermination growth are not well understood. Arabidopsis (Arabidopsis thaliana) contains three SRP homologs (SRP1, SRP2, and SRP3) that share sequence identities with small rubber particle proteins of the rubber tree (Hevea brasiliensis). In this report, the possible cellular roles of SRPs in postgermination growth and the drought tolerance response were investigated. Arabidopsis SRPs appeared to be LD-associated proteins and displayed polymerization properties in vivo and in vitro. SRP-overexpressing transgenic Arabidopsis plants (35S:SRP1, 35S:SRP2, and 35S:SRP3) exhibited higher vegetative and reproductive growth and markedly better tolerance to drought stress than wild-type Arabidopsis. In addition, constitutive over-expression of SRPs resulted in increased numbers of large LDs in postgermination seedlings. In contrast, single (srp1, 35S:SRP2-RNAi, and srp3) and triple (35S:SRP2-RNAi/srp1srp3) loss-of-function mutant lines exhibited the opposite phenotypes. Our results suggest that Arabidopsis SRPs play dual roles as positive factors in postgermination growth and the drought stress tolerance response. The possible relationships between LD-associated proteins and the drought stress response are discussed.Environmental stresses, including drought, high salinity, oxidative stress, and unfavorable temperatures, profoundly affect the growth and development of higher plants. Because of their sessile life cycle, plants have developed self-protective mechanisms to increase their tolerance to short- and long-term stresses by triggering diverse sets of signal transduction pathways and activating stress-responsive genes. The genetic and cellular mechanisms in response to abiotic stress have been widely documented in higher plants (Shinozaki and Yamaguchi-Shinozaki, 1996; Bray, 1997; Ishitani et al., 1997; Zhu, 2002; Bohnert et al., 2006; Shinozaki and Yamaguchi-Shinozaki, 2007; Vij and Tyagi, 2007).Lipid droplets (LDs) are dynamic subcellular organelles enclosed by a monolayer of phospholipid. LDs act as repositories for fatty acids and sterols, which are used for energy production and membrane and hormone synthesis. LDs are also involved in various cellular processes, including intracellular protein storage, stress responses, and lipid signaling (Bartz et al., 2007; Zehmer et al., 2009; Carman, 2012; Herker and Ott, 2012; Murphy, 2012; Sun et al., 2013; Kory et al., 2015). LDs bud from the endoplasmic reticulum (ER), where they become enriched with triacylglycerols and subsequently enlarged, until they pinch off to form an LD (Chapman et al., 2012; Chapman and Ohlrogge, 2012; Jacquier et al., 2013). Several reports suggest that LD-associated proteins, such as fat-specific protein 27 (FSP27), SEIPIN, and PERILIPIN1 (Plin1), are key regulators of LD formation in mammals, Drosophila, and yeasts (Farese and Walther, 2009; Xu et al., 2012; Yang et al., 2012). After budding from the ER, LDs fuse with each other and expand. In adipocytes, Plin1 functions as an enhancer of FSP27-mediated lipid transfer and LD growth, indicating that Plin1 and FSP27 participate in LD formation and fusion (Sun et al., 2013). Enlarged LDs provide surfaces to allow the attachment of numerous LD-associated proteins, which are later displaced during shrinkage of LDs by lipolysis (Kory et al., 2015).Because LDs are mainly present in seeds, studies on LD-associated proteins in higher plants have focused on seed development and germination (Chapman et al., 2013; Gidda et al., 2013; Horn et al., 2013; Szymanski et al., 2014). For example, oleosins regulate LD size in Arabidopsis (Arabidopsis thaliana) seed development (Siloto et al., 2006). Arabidopsis SEIPINs modulate LD proliferation and neutral lipid accumulation in developing seeds (Cai et al., 2015). On the other hand, the cellular roles of LD-associated proteins in postgermination growth remain largely unraveled.CaSRP1 (Capsicum annuum stress-related protein 1) was previously identified as a hot pepper small rubber particle protein (SRPPs) homolog (Hong and Kim, 2005). CaSRP1 was induced in response to water stress in hot pepper plants. Constitutive over-expression of CaSRP1 in transgenic Arabidopsis plants resulted in elevated growth and increased drought tolerance relative to wild-type Arabidopsis (Kim et al., 2010). CaSRP1 is evolutionarily related to SRPPs in rubber-producing plants (Wititsuwannakul et al., 2008). Rubber particles are single-membrane organelles that store rubber (cis-1,4-polyisoprene). Although rubber particles and LDs have different lipid compositions, their basic architectures are similar (Cornish et al., 1999). Thus, SRPP homologs may have common properties in the formation and biogenesis of rubber particles and/or LDs in rubber-producing and non-rubber-producing plants.In this report, we identified and characterized three SRPP homologs, SRP1, SRP2, and SRP3, in Arabidopsis. The SRP genes were differentially expressed in various tissues and induced by abscisic acid (ABA) and a broad spectrum of abiotic stress, including drought, high salinity, and low temperature. SRP-overexpressing transgenic Arabidopsis plants (35S:SRP1, 35S:SRP2, and 35S:SRP3) exhibited higher vegetative and reproductive growth and markedly better tolerance to drought stress than wild-type Arabidopsis plants. In addition, ectopic expression of SRPs resulted in increased numbers of large LDs in postgermination seedlings. In contrast, single (srp1, 35S:SRP2-RNAi, and srp3) and triple (35S:SRP2-RNAi/srp1srp3) loss-of-function mutant lines showed the opposite phenotypes. Arabidopsis SRPs appeared to be LD-associated proteins and displayed polymerization properties in vivo and in vitro. These results are discussed in light of the suggestion that Arabidopsis SRPs play dual roles as positive factors in postgermination growth and drought stress response. The possible relationships between LD-associated proteins and stress tolerance response are also discussed.     

Rhamnolipids Elicit Defense Responses and Induce Disease Resistance against Biotrophic, Hemibiotrophic, and Necrotrophic Pathogens That Require Different Signaling Pathways in Arabidopsis and Highlight a Central Role for Salicylic Acid

Plant resistance to phytopathogenic microorganisms mainly relies on the activation of an innate immune response usually launched after recognition by the plant cells of microbe-associated molecular patterns. The plant hormones, salicylic acid (SA), jasmonic acid, and ethylene have emerged as key players in the signaling networks involved in plant immunity. Rhamnolipids (RLs) are glycolipids produced by bacteria and are involved in surface motility and biofilm development. Here we report that RLs trigger an immune response in Arabidopsis (Arabidopsis thaliana) characterized by signaling molecules accumulation and defense gene activation. This immune response participates to resistance against the hemibiotrophic bacterium Pseudomonas syringae pv tomato, the biotrophic oomycete Hyaloperonospora arabidopsidis, and the necrotrophic fungus Botrytis cinerea. We show that RL-mediated resistance involves different signaling pathways that depend on the type of pathogen. Ethylene is involved in RL-induced resistance to H. arabidopsidis and to P. syringae pv tomato whereas jasmonic acid is essential for the resistance to B. cinerea. SA participates to the restriction of all pathogens. We also show evidence that SA-dependent plant defenses are potentiated by RLs following challenge by B. cinerea or P. syringae pv tomato. These results highlight a central role for SA in RL-mediated resistance. In addition to the activation of plant defense responses, antimicrobial properties of RLs are thought to participate in the protection against the fungus and the oomycete. Our data highlight the intricate mechanisms involved in plant protection triggered by a new type of molecule that can be perceived by plant cells and that can also act directly onto pathogens.In their environment, plants are challenged by potentially pathogenic microorganisms. In response, they express a set of defense mechanisms including preformed structural and chemical barriers, as well as an innate immune response quickly activated after microorganism perception (Boller and Felix, 2009). Plant innate immunity is triggered after recognition by pattern recognition receptors of conserved pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs, respectively) or by plant endogenous molecules released by pathogen invasion and called danger-associated molecular patterns (Boller and Felix, 2009; Dodds and Rathjen, 2010). This first step of recognition leads to the activation of MAMP-triggered immunity (MTI). Successful pathogens can secrete effectors that interfere or suppress MTI, resulting in effector-triggered susceptibility. A second level of perception involves the direct or indirect recognition by specific receptors of pathogen effectors leading to effector-triggered immunity (ETI; Boller and Felix, 2009; Dodds and Rathjen, 2010). Whereas MTI and ETI are thought to involve common signaling network, ETI is usually quantitatively stronger than MTI and associated with more sustained and robust immune responses (Katagiri and Tsuda, 2010; Tsuda and Katagiri, 2010).The plant hormones, salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) have emerged as key players in the signaling networks involved in MTI and ETI (Robert-Seilaniantz et al., 2007; Tsuda et al., 2009; Katagiri and Tsuda, 2010; Mersmann et al., 2010; Tsuda and Katagiri, 2010; Robert-Seilaniantz et al., 2011). Interactions between these signal molecules allow the plant to activate and/or modulate an appropriate spectrum of responses, depending on the pathogen lifestyle, necrotroph or biotroph (Glazebrook, 2005; Koornneef and Pieterse, 2008). It is assumed that JA and ET signaling pathways are important for resistance to necrotrophic fungi including Botrytis cinerea and Alternaria brassicicola (Thomma et al., 2001; Ferrari et al., 2003; Glazebrook, 2005). Infection of Arabidopsis (Arabidopsis thaliana) with B. cinerea causes the induction of the JA/ET responsive gene PLANT DEFENSIN1.2 (PDF1.2; Penninckx et al., 1996; Zimmerli et al., 2001). Induction of PDF1.2 by B. cinerea is blocked in ethylene-insensitive2 (ein2) and coronatine-insensitive1 (coi1) mutants that are respectively defective in ET and JA signal transduction pathways. Moreover, ein2 and coi1 plants are highly susceptible to B. cinerea infection (Thomma et al., 1998; Thomma et al., 1999). JA/ET-dependent responses do not seem to be usually induced during resistance to biotrophs, but they can be effective if they are stimulated prior to pathogen challenge (Glazebrook, 2005). Plants impaired in SA signaling are highly susceptible to biotrophic and hemibiotrophic pathogens. Following pathogen infection, SA hydroxylase (NahG), enhanced disease susceptibility5 (eds5), or SA induction-deficient2 (sid2) plants are unable to accumulate high SA levels and they display heightened susceptibility to Pseudomonas syringae pv tomato (Pst), Hyaloperonospora arabidopsidis, or Erysiphe orontii (Delaney et al., 1994; Lawton et al., 1995; Wildermuth et al., 2001; Nawrath et al., 2002; Vlot et al., 2009). Mutants that are insensitive to SA, such as nonexpressor of PATHOGENESIS-RELATED (PR) genes1 (npr1), have enhanced susceptibility to these pathogens (Cao et al., 1994; Glazebrook et al., 1996; Shah et al., 1997; Dong, 2004). According to some reports, plant defense against necrotrophs also involves SA. Arabidopsis plants expressing the nahG gene and infected with B. cinerea show larger lesions compared with wild-type plants (Govrin and Levine, 2002). In tobacco (Nicotiana tabacum), acidic isoforms of PR3 and PR5 gene that are specifically induced by SA (Ménard et al., 2004) are up-regulated after challenge by B. cinerea (El Oirdi et al., 2010). Resistance to some necrotrophs like Fusarium graminearum involves both SA and JA signaling pathways (Makandar et al., 2010). It is assumed that SA and JA signaling can be antagonistic (Bostock, 2005; Koornneef and Pieterse, 2008; Pieterse et al., 2009; Thaler et al., 2012). In Arabidopsis, SA inhibits JA-dependent resistance against A. brassicicola or B. cinerea (Spoel et al., 2007; Koornneef et al., 2008). Recent studies demonstrated that ET modulates the NPR1-mediated antagonism between SA and JA (Leon-Reyes et al., 2009; Leon-Reyes et al., 2010a) and suppression by SA of JA-responsive gene expression is targeted at a position downstream of the JA biosynthesis pathway (Leon-Reyes et al., 2010b). Synergistic effects of SA- and JA-dependent signaling are also well documented (Schenk et al., 2000; van Wees et al., 2000; Mur et al., 2006) and induction of some defense responses after pathogen challenge requires intact JA, ET, and SA signaling pathways (Campbell et al., 2003).Isolated MAMPs trigger defense responses that also require the activation of SA, JA, and ET signaling pathways (Tsuda et al., 2009; Katagiri and Tsuda, 2010). For instance, treatment with the flagellin peptide flg22 induces many SA-related genes including SID2, EDS5, NPR1, and PR1 (Ferrari et al., 2007; Denoux et al., 2008), causes SA accumulation (Tsuda et al., 2008; Wang et al., 2009), and activates ET signaling (Bethke et al., 2009; Mersmann et al., 2010). Local application of lipopolysaccharides elevates the level of SA (Mishina and Zeier, 2007). The oomycete Pep13 peptide induces defense responses in potato (Solanum tuberosum) that require both SA and JA (Halim et al., 2009). Although signaling networks induced by isolated MAMPs are well documented, the contribution of SA, JA, and ET in MAMP- or PAMP-induced resistance to biotrophs and necrotrophs is poorly understood.Rhamnolipids (RLs) are glycolipids produced by various bacteria species including some Pseudomonas and Burkholderia species. They are essential for bacterial surface motility and biofilm development (Vatsa et al., 2010; Chrzanowski et al., 2012). RLs are potent stimulators of animal immunity (Vatsa et al., 2010). They have recently been shown to elicit plant defense responses and to induce resistance against B. cinerea in grapevine (Vitis vinifera; Varnier et al., 2009). They also participate to biocontrol activity of the plant beneficial bacteria Pseudomonas aeruginosa PNA1 against oomycetes (Perneel et al., 2008). However, the signaling pathways used by RLs to stimulate plant innate immunity are not known. To gain more insights into RL-induced MTI, we investigated RL-triggered defense responses and resistance to the necrotrophic fungus B. cinerea, the biotroph oomycete H. arabidopsidis, and the hemibiotroph bacterium Pst in Arabidopsis. Our results show that RLs trigger an innate immune response in Arabidopsis that protects the plant against these different lifestyle pathogens. We demonstrate that RL-mediated resistance involves separated signaling sectors that depend on the type of pathogen. In plants challenged by RLs, SA has a central role and participates to the restriction of the three pathogens. ET is fully involved in RL-induced resistance to the biotrophic oomycete and to the hemibiotrophic bacterium whereas JA is essential for the resistance to the necrotrophic fungus.     

One Divinyl Reductase Reduces the 8-Vinyl Groups in Various Intermediates of Chlorophyll Biosynthesis in a Given Higher Plant Species,But the Isozyme Differs between Species

Divinyl reductase (DVR) converts 8-vinyl groups on various chlorophyll intermediates to ethyl groups, which is indispensable for chlorophyll biosynthesis. To date, five DVR activities have been detected, but adequate evidence of enzymatic assays using purified or recombinant DVR proteins has not been demonstrated, and it is unclear whether one or multiple enzymes catalyze these activities. In this study, we systematically carried out enzymatic assays using four recombinant DVR proteins and five divinyl substrates and then investigated the in vivo accumulation of various chlorophyll intermediates in rice (Oryza sativa), maize (Zea mays), and cucumber (Cucumis sativus). The results demonstrated that both rice and maize DVR proteins can convert all of the five divinyl substrates to corresponding monovinyl compounds, while both cucumber and Arabidopsis (Arabidopsis thaliana) DVR proteins can convert three of them. Meanwhile, the OsDVR (Os03g22780)-inactivated 824ys mutant of rice exclusively accumulated divinyl chlorophylls in its various organs during different developmental stages. Collectively, we conclude that a single DVR with broad substrate specificity is responsible for reducing the 8-vinyl groups of various chlorophyll intermediates in higher plants, but DVR proteins from different species have diverse and differing substrate preferences, although they are homologous.Chlorophyll (Chl) molecules universally exist in photosynthetic organisms. As the main component of the photosynthetic pigments, Chl molecules perform essential processes of absorbing light and transferring the light energy in the reaction center of the photosystems (Fromme et al., 2003). Based on the number of vinyl side chains, Chls are classified into two groups, 3,8-divinyl (DV)-Chl and 3-monovinyl (MV)-Chl. The DV-Chl molecule contains two vinyl groups at positions 3 and 8 of the tetrapyrrole macrocycle, whereas the MV-Chl molecule contains a vinyl group at position 3 and an ethyl group at position 8 of the macrocycle. Almost all of the oxygenic photosynthetic organisms contain MV-Chls, with the exceptions of some marine picophytoplankton species that contain only DV-Chls as their primary photosynthetic pigments (Chisholm et al., 1992; Goericke and Repeta, 1992; Porra, 1997).The classical single-branched Chl biosynthetic pathway proposed by Granick (1950) and modified by Jones (1963) assumed the rapid reduction of the 8-vinyl group of DV-protochlorophyllide (Pchlide) catalyzed by a putative 8-vinyl reductase. Ellsworth and Aronoff (1969) found evidence for both MV and DV forms of several Chl biosynthetic intermediates between magnesium-protoporphyrin IX monomethyl ester (MPE) and Pchlide in Chlorella spp. mutants. Belanger and Rebeiz (1979, 1980) reported that the Pchlide pool of etiolated higher plants contains both MV- and DV-Pchlide. Afterward, following the further detection of MV- and DV-tetrapyrrole intermediates and their biosynthetic interconversion in tissues and extracts of different plants (Belanger and Rebeiz, 1982; Duggan and Rebeiz, 1982; Tripathy and Rebeiz, 1986, 1988; Parham and Rebeiz, 1992, 1995; Kim and Rebeiz, 1996), a multibranched Chl biosynthetic heterogeneity was proposed (Rebeiz et al., 1983, 1986, 1999; Whyte and Griffiths, 1993; Kolossov and Rebeiz, 2010).Biosynthetic heterogeneity refers to the biosynthesis of a particular metabolite by an organelle, tissue, or organism via multiple biosynthetic routes. Varieties of reports lead to the assumption that Chl biosynthetic heterogeneity originates mainly in parallel DV- and MV-Chl biosynthetic routes. These routes are interconnected by 8-vinyl reductases that convert DV-tetrapyrroles to MV-tetrapyrroles by conversion of the vinyl group at position 8 of ring B to the ethyl group (Parham and Rebeiz, 1995; Rebeiz et al., 2003). DV-MPE could be converted to MV-MPE in crude homogenates from etiolated wheat (Triticum aestivum) seedlings (Ellsworth and Hsing, 1974). Exogenous DV-Pchlide could be partially converted to MV-Pchlide in barley (Hordeum vulgare) plastids (Tripathy and Rebeiz, 1988). 8-Vinyl chlorophyllide (Chlide) a reductases in etioplast membranes isolated from etiolated cucumber (Cucumis sativus) cotyledons and barley and maize (Zea mays) leaves were found to be very active in the conversion of exogenous DV-Chlide a to MV-Chlide a (Parham and Rebeiz, 1992, 1995). Kim and Rebeiz (1996) suggested that Chl biosynthetic heterogeneity in higher plants may originate at the level of DV magnesium-protoporphyrin IX (Mg-Proto) and would be mediated by the activity of a putative 8-vinyl Mg-Proto reductase in barley etiochloroplasts and plastid membranes. However, since these reports did not use purified or recombinant enzyme, it is not clear whether the reductions of the 8-vinyl groups of various Chl intermediates are catalyzed by one enzyme of broad specificity or by multiple enzymes of narrow specificity, which actually has become one of the focus issues in Chl biosynthesis.Nagata et al. (2005) and Nakanishi et al. (2005) independently identified the AT5G18660 gene of Arabidopsis (Arabidopsis thaliana) as an 8-vinyl reductase, namely, divinyl reductase (DVR). Chew and Bryant (2007) identified the DVR BciA (CT1063) gene of the green sulfur bacterium Chlorobium tepidum, which is homologous to AT5G18660. An enzymatic assay using a recombinant Arabidopsis DVR (AtDVR) on five DV substrates revealed that the major substrate of AtDVR is DV-Chlide a, while the other four DV substrates could not be converted to corresponding MV compounds (Nagata et al., 2007). Nevertheless, a recombinant BciA is able to reduce the 8-vinyl group of DV-Pchlide to generate MV-Pchlide (Chew and Bryant, 2007). Recently, we identified the rice (Oryza sativa) DVR encoded by Os03g22780 that has sequence similarity with the Arabidopsis DVR gene AT5G18660. We also confirmed that the recombinant rice DVR (OsDVR) is able to not only convert DV-Chlide a to MV-Chlide a but also to convert DV-Chl a to MV-Chl a (Wang et al., 2010). Thus, it is possible that the reductions of the 8-vinyl groups of various Chl biosynthetic intermediates are catalyzed by one enzyme of broad specificity.In this report, we extended our studies to four DVR proteins and five DV substrates. First, ZmDVR and CsDVR genes were isolated from maize and cucumber genomes, respectively, using a homology-based cloning approach. Second, enzymatic assays were systematically carried out using recombinant OsDVR, ZmDVR, CsDVR, and AtDVR as representative DVR proteins and using DV-Chl a, DV-Chlide a, DV-Pchlide a, DV-MPE, and DV-Mg-Proto as DV substrates. Third, we examined the in vivo accumulations of various Chl intermediates in rice, maize, and cucumber. Finally, we systematically investigated the in vivo accumulations of Chl and its various intermediates in the OsDVR (Os03g22780)-inactivated 824ys mutant of rice (Wang et al., 2010). The results strongly suggested that a single DVR protein with broad substrate specificity is responsible for reducing the 8-vinyl groups of various intermediate molecules of Chl biosynthesis in higher plants, but DVR proteins from different species could have diverse and differing substrate preferences even though they are homologous.     

Trafficking of Vacuolar Proteins: The Crucial Role of Arabidopsis Vacuolar Protein Sorting 29 in Recycling Vacuolar Sorting Receptor

The retromer is involved in recycling lysosomal sorting receptors in mammals. A component of the retromer complex in Arabidopsis thaliana, vacuolar protein sorting 29 (VPS29), plays a crucial role in trafficking storage proteins to protein storage vacuoles. However, it is not known whether or how vacuolar sorting receptors (VSRs) are recycled from the prevacuolar compartment (PVC) to the trans-Golgi network (TGN) during trafficking to the lytic vacuole (LV). Here, we report that VPS29 plays an essential role in the trafficking of soluble proteins to the LV from the TGN to the PVC. maigo1-1 (mag1-1) mutants, which harbor a knockdown mutation in VPS29, were defective in trafficking of two soluble proteins, Arabidopsis aleurain-like protein (AALP):green fluorescent protein (GFP) and sporamin:GFP, to the LV but not in trafficking membrane proteins to the LV or plasma membrane or via the secretory pathway. AALP:GFP and sporamin:GFP in mag1-1 protoplasts accumulated in the TGN but were also secreted into the medium. In mag1-1 mutants, VSR1 failed to recycle from the PVC to the TGN; rather, a significant proportion was transported to the LV; VSR1 overexpression rescued this defect. Moreover, endogenous VSRs were expressed at higher levels in mag1-1 plants. Based on these results, we propose that VPS29 plays a crucial role in recycling VSRs from the PVC to the TGN during the trafficking of soluble proteins to the LV.     

Comparative Analysis of Light-Harvesting Antennae and State Transition in chlorina and cpSRP Mutants

State transitions in photosynthesis provide for the dynamic allocation of a mobile fraction of light-harvesting complex II (LHCII) to photosystem II (PSII) in state I and to photosystem I (PSI) in state II. In the state I-to-state II transition, LHCII is phosphorylated by STN7 and associates with PSI to favor absorption cross-section of PSI. Here, we used Arabidopsis (Arabidopsis thaliana) mutants with defects in chlorophyll (Chl) b biosynthesis or in the chloroplast signal recognition particle (cpSRP) machinery to study the flexible formation of PS-LHC supercomplexes. Intriguingly, we found that impaired Chl b biosynthesis in chlorina1-2 (ch1-2) led to preferentially stabilized LHCI rather than LHCII, while the contents of both LHCI and LHCII were equally depressed in the cpSRP43-deficient mutant (chaos). In view of recent findings on the modified state transitions in LHCI-deficient mutants (Benson et al., 2015), the ch1-2 and chaos mutants were used to assess the influence of varying LHCI/LHCII antenna size on state transitions. Under state II conditions, LHCII-PSI supercomplexes were not formed in both ch1-2 and chaos plants. LHCII phosphorylation was drastically reduced in ch1-2, and the inactivation of STN7 correlates with the lack of state transitions. In contrast, phosphorylated LHCII in chaos was observed to be exclusively associated with PSII complexes, indicating a lack of mobile LHCII in chaos. Thus, the comparative analysis of ch1-2 and chaos mutants provides new evidence for the flexible organization of LHCs and enhances our understanding of the reversible allocation of LHCII to the two photosystems.In oxygenic photosynthesis, PSII and PSI function in series to convert light energy into the chemical energy that fuels multiple metabolic processes. Most of this light energy is captured by the chlorophyll (Chl) and carotenoid pigments in the light-harvesting antenna complexes (LHCs) that are peripherally associated with the core complexes of both photosystems (Wobbe et al., 2016). However, since the two photosystems exhibit different absorption spectra (Nelson and Yocum, 2006; Nield and Barber, 2006; Qin et al., 2015), PSI or PSII is preferentially excited under naturally fluctuating light intensities and qualities. To optimize photosynthetic electron transfer, the excitation state of the two photosystems must be rebalanced in response to changes in lighting conditions. To achieve this, higher plants and green algae require rapid and precise acclimatory mechanisms to adjust the relative absorption cross-sections of the two photosystems.To date, the phenomenon of state transitions is one of the well-documented short-term acclimatory mechanisms. It allows a mobile portion of the light-harvesting antenna complex II (LHCII) to be allocated to either photosystem, depending on the spectral composition and intensity of the ambient light (Allen and Forsberg, 2001; Rochaix, 2011; Goldschmidt-Clermont and Bassi, 2015; Gollan et al., 2015). State transitions are driven by the redox state of the plastoquinone (PQ) pool (Vener et al., 1997; Zito et al., 1999). When PSI is preferentially excited (by far-red light), the PQ pool is oxidized and all the LHCII is associated with PSII. This allocation of antenna complexes is defined as state I. When light conditions (blue/red light or low light) favor exciton trapping of PSII, the transition from state I to state II occurs. The over-reduced PQ pool triggers the activation of the membrane-localized Ser-Thr kinase STN7, which phosphorylates an N-terminal Thr on each of two major LHCII proteins, LHCB1 and LHCB2 (Allen, 1992; Bellafiore et al., 2005; Shapiguzov et al., 2016). Phosphorylation of LHCII results in the dissociation of LHCII from PSII and triggers its reversible relocation to PSI (Allen, 1992; Rochaix, 2011). Conversely, when the PQ pool is reoxidized, STN7 is inactivated and the constitutively active, thylakoid-associated phosphatase TAP38/PPH1 dephosphorylates LHCII, which then reassociates with PSII (Pribil et al., 2010; Shapiguzov et al., 2010). The physiological significance of state transitions has been demonstrated by the reduction in growth rate seen in the stn7 knock-out mutant under fluctuating light conditions (Bellafiore et al., 2005; Tikkanen et al., 2010).The canonical state transitions model implies spatial and temporal regulation of the allocation of LHC between the two spatially segregated photosystems (Dekker and Boekema, 2005). PSII-LHCII supercomplexes are organized in a tightly packed form in the stacked grana regions of thylakoid membranes, while PSI-LHCI supercomplexes are mainly localized in the nonstacked stromal lamellae and grana margin regions (Dekker and Boekema, 2005; Haferkamp et al., 2010). It has been proposed that, in the grana margin regions, which harbor LHCII and both photosystems, LHCII can migrate rapidly between them (Albertsson et al., 1990; Albertsson, 2001). This idea is supported by the recent discovery of mega complexes containing both photosystems in the grana margin regions (Yokono et al., 2015). Furthermore, phosphorylation of LHCII was found to increase not only the amount of PSI found in the grana margin region of thylakoid membranes (Tikkanen et al., 2008a), but also to modulate the pattern of PSI-PSII megacomplexes under changing light conditions (Suorsa et al., 2015). Nonetheless, open questions remain in relation to the physiological significance of the detection of phosphorylated LHCII in all thylakoid regions, even under the constant light conditions (Grieco et al., 2012; Leoni et al., 2013; Wientjes et al., 2013), although LHCII phosphorylation has been shown to modify the stacking of thylakoid membranes (Chuartzman et al., 2008; Pietrzykowska et al., 2014).State I-to-state II transition is featured by the formation of LHCII-PSI-LHCI supercomplexes, in which LHCII favors the light-harvesting capacity of PSI. Recently, LHCII-PSI-LHCI supercomplexes have been successfully isolated and purified using various detergents (Galka et al., 2012; Drop et al., 2014; Crepin and Caffarri, 2015) or a styrene-maleic acid copolymer (Bell et al., 2015). These findings yielded further insights into the reorganization of supercomplexes associated with state transitions, and it was suggested that phosphorylation of LHCB2 rather than LHCB1 is the essential trigger for the formation of state transition supercomplexes (Leoni et al., 2013; Pietrzykowska et al., 2014; Crepin and Caffarri, 2015; Longoni et al., 2015). Furthermore, characterization of mutants deficient in individual PSI core subunits indicates that PsaH, L, and I are required for docking of LHCII at PSI (Lunde et al., 2000; Zhang and Scheller, 2004; Kouril et al., 2005; Plöchinger et al., 2016).Recently, the state transition capacity has been characterized in the Arabidopsis (Arabidopsis thaliana) mutants with missing LHCI components. Although the Arabidopsis knock-out mutants lacking one of the four LHCI proteins (LHCA1-4) showed enhanced accumulation of LHCII-PSI complexes, the absorption cross-section of PSI under state II conditions was still compromised in the lhca1-4 mutants, and it is suggested that LHCI mediates the detergent-sensitive interaction between ‘extra LHCII’ and PSI (Benson et al., 2015; Grieco et al., 2015). Furthermore, the Arabidopsis mutant ΔLhca lacking all LHCA1-4 proteins was shown to be compensated for the deficiency of LHCI by binding LHCII under state II conditions (Bressan et al., 2016). In spite of this finding, the significant reduction in the absorption cross-section of PSI was still observed in the ΔLhca mutant, suggesting a substantial role of LHCI in light absorption under canopy conditions (Bressan et al., 2016). However, these findings emphasize the acclimatory function of state transitions in balancing light absorption capacity between the two photosystems by modifying their relative antenna size and imply the dynamic and variable organization of PS-LHC supercomplexes.LHC proteins are encoded by the nuclear Lhc superfamily (Jansson, 1994). The biogenesis of LHCs includes the cytoplasmic synthesis of the LHC precursor proteins, their translocation into chloroplasts via the TOC/TIC complex, and their posttranslational targeting and integration into the thylakoid membranes by means of the chloroplast signal recognition particle (cpSRP) machinery (Jarvis and Lopez-Juez, 2013). The posttranslational cpSRP-dependent pathway for the final translocation of LHC proteins into the thylakoid membrane includes interaction of cpSRP43 with LHC apo-proteins and recruitment of cpSRP54 to form a transit complex. Then binding of this tripartite cpSRP transit complex to the SRP receptor cpFtsY follows, which supports docking of the transit complex to thylakoid membranes and its association with the LHC translocase ALB3. Ultimately, ALB3 inserts LHC apo-proteins into the thylakoid membrane (Richter et al., 2010). Importantly, stoichiometric amounts of newly synthesized Chl a and Chl b as well as carotenoid are inserted into the LHC apo-proteins by unknown mechanisms to form the functional LHCs that associate with the core complexes of both photosystems in the thylakoid membranes (Dall’Osto et al., 2015; Wang and Grimm, 2015).The first committed steps in Chl synthesis occur in the Mg branch of the tetrapyrrole biosynthesis pathway. 5-Aminolevulinic acid synthesis provides the precursor for the formation of protoporphyrin IX, which is directed into the Mg branch (Tanaka and Tanaka, 2007; Brzezowski et al., 2015). Chl synthesis ends with the conversion of Chl a to Chl b catalyzed by Chl a oxygenase (CAO; Tanaka et al., 1998; Tomitani et al., 1999). It has been hypothesized that coordination between Chl synthesis and the posttranslational cpSRP pathway is a prerequisite for the efficient integration of Chls into LHC apo-proteins.In this study, we intend to characterize the assembly of LHCs when the availability of Chl molecules or the integration of LHC apo-proteins into thylakoid membranes is limiting. To this end, we compared the assembly of LHCs and the organization of PS-LHC complexes in two different sets of Arabidopsis mutants. Firstly, we used the chlorina1-2 (ch1-2) mutant, which is defective in the CAO gene. The members of the second set of mutants carry knock-out mutations in genes involved in the chloroplast SRP pathway (Richter et al., 2010).Our studies revealed distinct accumulation of PS-LHC supercomplexes between the two sets of mutant relative to wild-type plants. In spite of the defect in synthesis of Chl b, ch1-2 retains predominantly intact PSI-LHCI supercomplexes but has strongly reduced amounts of LHCII. In contrast, the chaos (cpSRP43) mutant exhibits synchronously reduced contents of both LHCI and LHCII, which results in the accumulation of PS core complexes without accompanying LHCs. Thus, the distribution of LHCs in the thylakoid membranes of the two mutants, ch1-2 and chaos, were explored under varying light conditions with the aim of elucidating the influence of modified LHCI/LHCII antenna size on state transitions. Our results contribute to an expanding view on the variety of photosynthetic complexes, which can be observed in Arabidopsis plants with specified mutations in LHC biogenesis.     

The Coronatine Toxin of Pseudomonas syringae Is a Multifunctional Suppressor of Arabidopsis Defense

The phytotoxin coronatine (COR) promotes various aspects of Pseudomonas syringae virulence, including invasion through stomata, growth in the apoplast, and induction of disease symptoms. COR is a structural mimic of active jasmonic acid (JA) conjugates. Known activities of COR are mediated through its binding to the F-box–containing JA coreceptor CORONATINE INSENSITIVE1. By analyzing the interaction of P. syringae mutants with Arabidopsis thaliana mutants, we demonstrate that, in the apoplastic space of Arabidopsis, COR is a multifunctional defense suppressor. COR and the critical P. syringae type III effector HopM1 target distinct signaling steps to suppress callose deposition. In addition to its well-documented ability to suppress salicylic acid (SA) signaling, COR suppresses an SA-independent pathway contributing to callose deposition by reducing accumulation of an indole glucosinolate upstream of the activity of the PEN2 myrosinase. COR also suppresses callose deposition and promotes bacterial growth in coi1 mutant plants, indicating that COR may have multiple targets inside plant cells.     

The DUF59 Family Gene AE7 Acts in the Cytosolic Iron-Sulfur Cluster Assembly Pathway to Maintain Nuclear Genome Integrity in Arabidopsis

Eukaryotic organisms have evolved a set of strategies to safeguard genome integrity, but the underlying mechanisms remain poorly understood. Here, we report that ASYMMETRIC LEAVES1/2 ENHANCER7 (AE7), an Arabidopsis thaliana gene encoding a protein in the evolutionarily conserved Domain of Unknown Function 59 family, participates in the cytosolic iron-sulfur (Fe-S) cluster assembly (CIA) pathway to maintain genome integrity. The severe ae7-2 allele is embryo lethal, whereas plants with the weak ae7 (ae7-1) allele are viable but exhibit highly accumulated DNA damage that activates the DNA damage response to arrest the cell cycle. AE7 is part of a protein complex with CIA1, NAR1, and MET18, which are highly conserved in eukaryotes and are involved in the biogenesis of cytosolic and nuclear Fe-S proteins. ae7-1 plants have lower activities of the cytosolic [4Fe-4S] enzyme aconitase and the nuclear [4Fe-4S] enzyme DNA glycosylase ROS1. Additionally, mutations in the gene encoding the mitochondrial ATP binding cassette transporter ATM3/ABCB25, which is required for the activity of cytosolic Fe-S enzymes in Arabidopsis, also result in defective genome integrity similar to that of ae7-1. These results indicate that AE7 is a central member of the CIA pathway, linking plant mitochondria to nuclear genome integrity through assembly of Fe-S proteins.