HYPERSENSITIVE TO HIGH LIGHT1 Interacts with LOW QUANTUM YIELD OF PHOTOSYSTEM II1 and Functions in Protection of Photosystem II from Photodamage in Arabidopsis

Under high-irradiance conditions, plants must efficiently protect photosystem II (PSII) from damage. In this study, we demonstrate that the chloroplast protein HYPERSENSITIVE TO HIGH LIGHT1 (HHL1) is expressed in response to high light and functions in protecting PSII against photodamage. Arabidopsis thaliana hhl1 mutants show hypersensitivity to high light, drastically decreased PSII photosynthetic activity, higher nonphotochemical quenching activity, a faster xanthophyll cycle, and increased accumulation of reactive oxygen species following high-light exposure. Moreover, HHL1 deficiency accelerated the degradation of PSII core subunits under high light, decreasing the accumulation of PSII core subunits and PSII–light-harvesting complex II supercomplex. HHL1 primarily localizes in the stroma-exposed thylakoid membranes and associates with the PSII core monomer complex through direct interaction with PSII core proteins CP43 and CP47. Interestingly, HHL1 also directly interacts, in vivo and in vitro, with LOW QUANTUM YIELD OF PHOTOSYSTEM II1 (LQY1), which functions in the repair and reassembly of PSII. Furthermore, the hhl1 lqy1 double mutants show increased photosensitivity compared with single mutants. Taken together, these results suggest that HHL1 forms a complex with LQY1 and participates in photodamage repair of PSII under high light.     

Nanodomains of Cytochrome b6f and Photosystem II Complexes in Spinach Grana Thylakoid Membranes

The cytochrome b₆f (cytb₆f) complex plays a central role in photosynthesis, coupling electron transport between photosystem II (PSII) and photosystem I to the generation of a transmembrane proton gradient used for the biosynthesis of ATP. Photosynthesis relies on rapid shuttling of electrons by plastoquinone (PQ) molecules between PSII and cytb₆f complexes in the lipid phase of the thylakoid membrane. Thus, the relative membrane location of these complexes is crucial, yet remains unknown. Here, we exploit the selective binding of the electron transfer protein plastocyanin (Pc) to the lumenal membrane surface of the cytb₆f complex using a Pc-functionalized atomic force microscope (AFM) probe to identify the position of cytb₆f complexes in grana thylakoid membranes from spinach (Spinacia oleracea). This affinity-mapping AFM method directly correlates membrane surface topography with Pc-cytb₆f interactions, allowing us to construct a map of the grana thylakoid membrane that reveals nanodomains of colocalized PSII and cytb₆f complexes. We suggest that the close proximity between PSII and cytb₆f complexes integrates solar energy conversion and electron transfer by fostering short-range diffusion of PQ in the protein-crowded thylakoid membrane, thereby optimizing photosynthetic efficiency.     

PsbN Is Required for Assembly of the Photosystem II Reaction Center in Nicotiana tabacum

The chloroplast-encoded low molecular weight protein PsbN is annotated as a photosystem II (PSII) subunit. To elucidate the localization and function of PsbN, encoded on the opposite strand to the psbB gene cluster, we raised antibodies and inserted a resistance cassette into PsbN in both directions. Both homoplastomic tobacco (Nicotiana tabacum) mutants ∆psbN-F and ∆psbN-R show essentially the same PSII deficiencies. The mutants are extremely light sensitive and failed to recover from photoinhibition. Although synthesis of PSII proteins was not altered significantly, both mutants accumulated only ∼25% of PSII proteins compared with the wild type. Assembly of PSII precomplexes occurred at normal rates, but heterodimeric PSII reaction centers (RCs) and higher order PSII assemblies were not formed efficiently in the mutants. The ∆psbN-R mutant was complemented by allotopic expression of the PsbN gene fused to the sequence of a chloroplast transit peptide in the nuclear genome. PsbN represents a bitopic trans-membrane peptide localized in stroma lamellae with its highly conserved C terminus exposed to the stroma. Significant amounts of PsbN were already present in dark-grown seedling. Our data prove that PsbN is not a constituent subunit of PSII but is required for repair from photoinhibition and efficient assembly of the PSII RC.     

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.     

Ethanolamide Oxylipins of Linolenic Acid Can Negatively Regulate Arabidopsis Seedling Development

N-Acylethanolamines (NAEs) are fatty-acid derivatives with potent biological activities in a wide range of eukaryotic organisms. Polyunsaturated NAEs are among the most abundant NAE types in seeds of Arabidopsis thaliana, and they can be metabolized by either fatty acid amide hydrolase (FAAH) or by lipoxygenase (LOX) to low levels during seedling establishment. Here, we identify and quantify endogenous oxylipin metabolites of N-linolenoylethanolamine (NAE 18:3) in Arabidopsis seedlings and show that their levels were higher in faah knockout seedlings. Quantification of oxylipin metabolites in lox mutants demonstrated altered partitioning of NAE 18:3 into 9- or 13-LOX pathways, and this was especially exaggerated when exogenous NAE was added to seedlings. When maintained at micromolar concentrations, NAE 18:3 specifically induced cotyledon bleaching of light-grown seedlings within a restricted stage of development. Comprehensive oxylipin profiling together with genetic and pharmacological interference with LOX activity suggested that both 9-hydroxy and 13-hydroxy linolenoylethanolamides, but not corresponding free fatty-acid metabolites, contributed to the reversible disruption of thylakoid membranes in chloroplasts of seedling cotyledons. We suggest that NAE oxylipins of linolenic acid represent a newly identified, endogenous set of bioactive compounds that may act in opposition to progression of normal seedling development and must be depleted for successful establishment.     

Light-Harvesting Complex Stress-Related Proteins Catalyze Excess Energy Dissipation in Both Photosystems of Physcomitrella patens

Two LHC-like proteins, Photosystem II Subunit S (PSBS) and Light-Harvesting Complex Stress-Related (LHCSR), are essential for triggering excess energy dissipation in chloroplasts of vascular plants and green algae, respectively. The mechanism of quenching was studied in Physcomitrella patens, an early divergent streptophyta (including green algae and land plants) in which both proteins are active. PSBS was localized in grana together with photosystem II (PSII), but LHCSR was located mainly in stroma-exposed membranes together with photosystem I (PSI), and its distribution did not change upon high-light treatment. The quenched conformation can be preserved by rapidly freezing the high-light-treated tissues in liquid nitrogen. When using green fluorescent protein as an internal standard, 77K fluorescence emission spectra on isolated chloroplasts allowed for independent assessment of PSI and PSII fluorescence yield. Results showed that both photosystems underwent quenching upon high-light treatment in the wild type in contrast to mutants depleted of LHCSR, which lacked PSI quenching. Due to the contribution of LHCII, P. patens had a PSI antenna size twice as large with respect to higher plants. Thus, LHCII, which is highly abundant in stroma membranes, appears to be the target of quenching by LHCSR.     

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.     

Activation of the Stt7/STN7 Kinase through Dynamic Interactions with the Cytochrome b6f Complex

Photosynthetic organisms have the ability to adapt to changes in light quality by readjusting the cross sections of the light-harvesting systems of photosystem II (PSII) and photosystem I (PSI). This process, called state transitions, maintains the redox poise of the photosynthetic electron transfer chain and ensures a high photosynthetic yield when light is limiting. It is mediated by the Stt7/STN7 protein kinase, which is activated through the cytochrome b₆f complex upon reduction of the plastoquinone pool. Its probable major substrate, the light-harvesting complex of PSII, once phosphorylated, dissociates from PSII and docks to PSI, thereby restoring the balance of absorbed light excitation energy between the two photosystems. Although the kinase is known to be inactivated under high-light intensities, the molecular mechanisms governing its regulation remain unknown. In this study we monitored the redox state of a conserved and essential Cys pair of the Stt7/STN7 kinase and show that it forms a disulfide bridge. We could not detect any change in the redox state of these Cys during state transitions and high-light treatment. It is only after prolonged anaerobiosis that this disulfide bridge is reduced. It is likely to be mainly intramolecular, although kinase activation may involve a transient covalently linked kinase dimer with two intermolecular disulfide bonds. Using the yeast two-hybrid system, we have mapped one interaction site of the kinase on the Rieske protein of the cytochrome b₆f complex.Photosynthetic organisms are subjected to constant changes in light quality and quantity and need to adapt to these changes in order to optimize, on the one hand, their photosynthetic yield, and to minimize photo-oxidative damage on the other. The photosynthetic electron transfer chain consists of photosystem II (PSII), the plastoquinone (PQ) pool, the cytochrome b₆f complex (Cyt b₆f), plastocyanin, and photosystem I (PSI). All of these complexes and components are integrated or closely associated with the thylakoid membrane. The two antenna systems of PSII and PSI capture and direct the light excitation energy to the corresponding reaction centers in which a chlorophyll dimer is oxidized and charge separation occurs across the thylakoid membrane. These processes lead to the onset of electron flow from water on the donor side of PSII to ferredoxin on the acceptor side of PSI coupled with proton translocation across the thylakoid membrane. In order to sustain optimal electron flow along this electron transfer chain, the redox poise needs to be maintained under changing environmental conditions. Several mechanisms have evolved for the maintenance of this redox balance. In the case of over-reduction of the acceptor side of PSI, excess electrons can reduce molecular oxygen through the Mehler reaction to superoxide, which is then converted to hydrogen peroxide by a plastid superoxide dismutase and ultimately to water by a peroxidase (Asada, 2000). Over-reduction of the PQ pool can be alleviated by PTOX, the plastid terminal oxidase responsible for oxidizing PQH₂ to form hydrogen peroxide, which is subsequently converted to water (Carol et al., 1999; Cournac et al., 2000; Wu et al., 1999).In addition to these electron sinks that prevent the over-reduction of the electron transfer chain, the photosynthetic apparatus is able to maintain the redox poise of the PQ pool by readjusting the relative cross sections of the light harvesting systems of PSII and PSI upon unequal excitation of the two photosystems. This readjustment can occur both in the short term through state transitions and in the long term by changing the stoichiometry between PSII and PSI (Bonaventura and Myers, 1969; Murata, 1969; Pfannschmidt, 2003). State transitions occur because of perturbations of the redox state of the PQ pool due to unequal excitation of PSII and PSI, limitations in electron acceptors downstream of PSI, and/or in CO₂ availability. Excess excitation of PSII relative to PSI leads to reduction of the PQ pool and thus favors the docking of PQH₂ to the Qo site of the Cyt b₆f complex. This process activates the Stt7/STN7 protein kinase (Vener et al., 1997; Zito et al., 1999), which is closely associated with this complex and leads to the phosphorylation of some LHCII proteins and to their detachment from PSII and binding to PSI (Depège et al., 2003; Lemeille et al., 2009). Although both Lhcb1 and Lhcb2 are phosphorylated, only the phosphorylated form of Lhcb2 is associated with PSI whereas phosphorylated Lhcb1 is excluded from this complex (Longoni et al., 2015). This state corresponds to state 2. In this way the change in the relative antenna sizes of the two photosystems restores the redox poise of the PQ pool. The process is reversible as over-excitation of PSI relative to PSII leads to the oxidation of the PQ pool and to the inactivation of the kinase. Under these conditions, phosphorylated LHCII associated with PSI is dephosphorylated by the PPH1/TAP38 phosphatase (Pribil et al., 2010; Shapiguzov et al., 2010) and returns to PSII (state 1). It should be noted, however, that a strict causal link between LHCII phosphorylation and its migration from PSII to PSI has been questioned recently by the finding that some phosphorylated LHCII remains associated with PSII supercomplexes and that LHCII serves as antenna for both photosystems under most natural light conditions (Drop et al., 2014; Wientjes et al., 2013).State transitions are important at low light but do not occur under high light because the LHCII kinase is inactivated under these conditions (Schuster et al., 1986). It was proposed that inactivation of the kinase is mediated by the ferredoxin-thioredoxin system and that a disulfide bond in the kinase rather than in the substrate may be the target site of thioredoxin (Rintamäki et al., 1997, 2000). Analysis of the Stt7/STN7 protein sequences indeed reveals the presence of two conserved Cys residues close to the N-terminal end of this kinase, which are conserved in all species examined and both are essential for kinase activity although they are located outside of the kinase catalytic domain (Fig. 1) (Depège et al., 2003; Lemeille et al., 2009). Based on protease protection studies, this model of the Stt7/STN7 kinase proposes that the N-terminal end of the kinase is on the lumen side of the thylakoid membrane separated from the catalytic domain on the stromal side by an unusual transmembrane domain containing several Pro residues (Lemeille et al., 2009). This configuration of the kinase allows its catalytic domain to act on the substrate sites of the LHCII proteins, which are exposed to the stroma. Although in this model the conserved Cys residues in the lumen are on the opposite side from the stromal thioredoxins, it is possible that thiol-reducing equivalents are transferred across the thylakoid membrane through the CcdA and Hcf164 proteins, which have been shown to operate in this way during heme and Cyt b₆f assembly (Lennartz et al., 2001; Page et al., 2004) or through the LTO1 protein (Du et al., 2015; Karamoko et al., 2011).Figure 1.Conserved Cys in the Stt7/STN7 kinase. Alignment of the sequences of the Stt7/STN protein kinase from Selaginella moelendorffii (Sm), Physcomitrella patens (Pp), Oryza sativa (Os), Populus trichocarpa (Pt), Arabidopsis thaliana (At), Chlamydomonas reinhardtii ...Here we have examined the redox state of the Stt7/STN7 kinase during state transitions and after illumination with high light to test the proposed model. We find that the Stt7/STN7 kinase contains a disulfide bridge that appears to be intramolecular and maintained not only during state transitions but also in high light when the kinase is inactive. Although these results suggest at first sight that the disulfide bridge of Stt7/STN7 is maintained during its activation and inactivation, we propose that a transient opening of this bridge occurs during the activation process followed by the formation of an intermolecular disulfide bridge and the appearance of a short-lived, covalently linked kinase dimer.     

Cell Growth Defect Factor1/CHAPERONE-LIKE PROTEIN OF POR1 Plays a Role in Stabilization of Light-Dependent Protochlorophyllide Oxidoreductase in Nicotiana benthamiana and Arabidopsis

Angiosperms require light for chlorophyll biosynthesis because one reaction in the pathway, the reduction of protochlorophyllide (Pchlide) to chlorophyllide, is catalyzed by the light-dependent protochlorophyllide oxidoreductase (POR). Here, we report that Cell growth defect factor1 (Cdf1), renamed here as CHAPERONE-LIKE PROTEIN OF POR1 (CPP1), an essential protein for chloroplast development, plays a role in the regulation of POR stability and function. Cdf1/CPP1 contains a J-like domain and three transmembrane domains, is localized in the thylakoid and envelope membranes, and interacts with POR isoforms in chloroplasts. CPP1 can stabilize POR proteins with its holdase chaperone activity. CPP1 deficiency results in diminished POR protein accumulation and defective chlorophyll synthesis, leading to photobleaching and growth inhibition of plants under light conditions. CPP1 depletion also causes reduced POR accumulation in etioplasts of dark-grown plants and as a result impairs the formation of prolamellar bodies, which subsequently affects chloroplast biogenesis upon illumination. Furthermore, in cyanobacteria, the CPP1 homolog critically regulates POR accumulation and chlorophyll synthesis under high-light conditions, in which the dark-operative Pchlide oxidoreductase is repressed by its oxygen sensitivity. These findings and the ubiquitous presence of CPP1 in oxygenic photosynthetic organisms suggest the conserved nature of CPP1 function in the regulation of POR.     

Phosphatidylinositol 4-Phosphate Negatively Regulates Chloroplast Division in Arabidopsis

Chloroplast division is performed by the constriction of envelope membranes at the division site. Although constriction of a ring-like protein complex has been shown to be involved in chloroplast division, it remains unknown how membrane lipids participate in the process. Here, we show that phosphoinositides with unknown function in envelope membranes are involved in the regulation of chloroplast division in Arabidopsis thaliana. PLASTID DIVISION1 (PDV1) and PDV2 proteins interacted specifically with phosphatidylinositol 4-phosphate (PI4P). Inhibition of phosphatidylinositol 4-kinase (PI4K) decreased the level of PI4P in chloroplasts and accelerated chloroplast division. Knockout of PI4Kβ2 expression or downregulation of PI4Kα1 expression resulted in decreased levels of PI4P in chloroplasts and increased chloroplast numbers. PI4Kα1 is the main contributor to PI4P synthesis in chloroplasts, and the effect of PI4K inhibition was largely abolished in the pdv1 mutant. Overexpression of DYNAMIN-RELATED PROTEIN5B (DRP5B), another component of the chloroplast division machinery, which is recruited to chloroplasts by PDV1 and PDV2, enhanced the effect of PI4K inhibition, whereas overexpression of PDV1 and PDV2 had additive effects. The amount of DRP5B that associated with chloroplasts increased upon PI4K inhibition. These findings suggest that PI4P is a regulator of chloroplast division in a PDV1- and DRP5B-dependent manner.     

Knocking Down of Isoprene Emission Modifies the Lipid Matrix of Thylakoid Membranes and Influences the Chloroplast Ultrastructure in Poplar

Isoprene is a small lipophilic molecule with important functions in plant protection against abiotic stresses. Here, we studied the lipid composition of thylakoid membranes and chloroplast ultrastructure in isoprene-emitting (IE) and nonisoprene-emitting (NE) poplar (Populus × canescens). We demonstrated that the total amount of monogalactosyldiacylglycerols, digalactosyldiacylglycerols, phospholipids, and fatty acids is reduced in chloroplasts when isoprene biosynthesis is blocked. A significantly lower amount of unsaturated fatty acids, particularly linolenic acid in NE chloroplasts, was associated with the reduced fluidity of thylakoid membranes, which in turn negatively affects photosystem II photochemical efficiency. The low photosystem II photochemical efficiency in NE plants was negatively correlated with nonphotochemical quenching and the energy-dependent component of nonphotochemical quenching. Transmission electron microscopy revealed alterations in the chloroplast ultrastructure in NE compared with IE plants. NE chloroplasts were more rounded and contained fewer grana stacks and longer stroma thylakoids, more plastoglobules, and larger associative zones between chloroplasts and mitochondria. These results strongly support the idea that in IE species, the function of this molecule is closely associated with the structural organization and functioning of plastidic membranes.Isoprene is globally the most abundant biogenic hydrocarbon constitutively emitted from many plant species (Guenther et al., 2012). It has been proposed that leaf isoprene emission is an important adaptation for plants, conferring tolerance to different environmental constraints (Vickers et al., 2009; Loreto and Schnitzler, 2010; Loreto and Fineschi, 2014). However, biogenic isoprene emission represents a nontrivial carbon loss in plants, particularly under stress conditions (Fang et al., 1996; Brilli et al., 2007; Teuber et al., 2008; Ghirardo et al., 2014), and the reason(s) why plants emit isoprene are still ambiguous, and the true role of isoprene emission remains elusive. Different approaches and techniques have been used to determine whether and how the cost of this expensive carbon emission is matched by the accomplishment of the physiological function in planta. It has been shown that isoprene might quench and/or regulate reactive oxygen and nitrogen species formation (Behnke et al., 2010a; Velikova et al., 2012), thereby indirectly providing a general antioxidant action (for review, see Vickers et al., 2009; Loreto and Schnitzler, 2010) and stabilizing thylakoid membrane structures due to the lipophilic properties of this molecule (Sharkey et al., 2001; Velikova et al., 2011).Protein and pigment-protein complexes are assembled and embedded in a lipid matrix, which has a unique lipid composition. The thylakoid lipid bilayer of chloroplasts is characterized by a high proportion of galactolipids with one (monogalactosyldiacylglycerol [MGDG]) or two (digalactosyldiacylglycerol [DGDG]) Gal molecules (Joyard et al., 2010). MGDGs are the primary constituents (approximately 50%) of thylakoid membrane glycerolipids, followed by DGDGs (approximately 30%), sulfoquinovosyldiacylglycerol (approximately 5%–12%), and phosphatidylglycerol (approximately 5%–12%; Kirchhoff et al., 2002). Galactolipids contain a large proportion of polyunsaturated fatty acids, and consequently, the thylakoid membrane is a relatively fluid system (Gounaris and Barber, 1983) compared with other biological membranes. The fluidity of the thylakoid membrane is essential for photosynthetic processes.The thylakoid membranes are highly organized internal membrane chloroplast systems that conduct the light reactions of photosynthesis. These membranes comprise pigments and proteins organized in complexes. Thylakoid membranes are arranged into stacked and unstacked regions called grana and stroma thylakoids, respectively, differentially enriched in PSI and PSII complexes (Mustárdy et al., 2008). The spatial separation of the PSI and PSII complexes in the stacked and unstacked membrane regions and the macromolecular organization of PSII in stacked grana thylakoids are self-organizing processes and important features to maintain the functional integrity of the photosynthetic apparatus (Kirchhoff et al., 2007).It is not known how changes in the lipid matrix affect lipid-protein interactions and vice versa, and how membrane macroorganization ensures the efficient diffusion of protein complexes associated with plant adaptation to the changing environment remains elusive. The isoprene impact on thylakoid intactness and functionality has been assessed using different biophysical techniques (Velikova et al., 2011). Thermoluminescence data demonstrated that the position of the main peak (Qb peak; Sane, 2004) was upshifted approximately 10° in isoprene-emitting (IE) plants, suggesting modifications in the lipid environment due to the presence of isoprene in heterologous Arabidopsis (Arabidopsis thaliana) plants expressing the isoprene synthase gene from poplar (Populus spp.). It was also shown that isoprene improves the stability of PSII light-harvesting complex II (LHCII) through the modification of pigment-protein complex organization in thylakoid membranes (Velikova et al., 2011). Moreover, we recently showed that knocking down isoprene emission in poplar remodels the chloroplast proteome (Velikova et al., 2014). The lack of isoprene resulted in the down-regulation of proteins associated with the light reactions of photosynthesis, redox regulation, and oxidative stress defenses and several proteins responsible for lipid metabolism (Velikova et al., 2014).In this study, we focused on the lipid composition of thylakoid membranes in IE and nonisoprene-emitting (NE) poplar (Populus × canescens) leaves. Specifically, we determined whether the translational suppression of isoprene synthase in NE leaves influences the lipid matrix of thylakoids and how this phenomenon affects membrane structure and function. Here, we provide evidence that the suppression of isoprene biosynthesis in poplar (1) reduced total galactolipids, phospholipids (PLs), and linolenic fatty acid (18:3), (2) altered the chloroplast ultrastructure, and (3) stimulated photoprotective mechanisms.     

Dual Role for Phospholipid:Diacylglycerol Acyltransferase: Enhancing Fatty Acid Synthesis and Diverting Fatty Acids from Membrane Lipids to Triacylglycerol in Arabidopsis Leaves

There is growing interest in engineering green biomass to expand the production of plant oils as feed and biofuels. Here, we show that PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE1 (PDAT1) is a critical enzyme involved in triacylglycerol (TAG) synthesis in leaves. Overexpression of PDAT1 increases leaf TAG accumulation, leading to oil droplet overexpansion through fusion. Ectopic expression of oleosin promotes the clustering of small oil droplets. Coexpression of PDAT1 with oleosin boosts leaf TAG content by up to 6.4% of the dry weight without affecting membrane lipid composition and plant growth. PDAT1 overexpression stimulates fatty acid synthesis (FAS) and increases fatty acid flux toward the prokaryotic glycerolipid pathway. In the trigalactosyldiacylglycerol1-1 mutant, which is defective in eukaryotic thylakoid lipid synthesis, the combined overexpression of PDAT1 with oleosin increases leaf TAG content to 8.6% of the dry weight and total leaf lipid by fourfold. In the plastidic glycerol-3-phosphate acyltransferase1 mutant, which is defective in the prokaryotic glycerolipid pathway, PDAT1 overexpression enhances TAG content at the expense of thylakoid membrane lipids, leading to defects in chloroplast division and thylakoid biogenesis. Collectively, these results reveal a dual role for PDAT1 in enhancing fatty acid and TAG synthesis in leaves and suggest that increasing FAS is the key to engineering high levels of TAG accumulation in green biomass.     

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.     

Lack of Phosphatidylglycerol Inhibits Chlorophyll Biosynthesis at Multiple Sites and Limits Chlorophyllide Reutilization in Synechocystis sp. Strain PCC 6803

The negatively charged lipid phosphatidylglycerol (PG) constitutes up to 10% of total lipids in photosynthetic membranes, and its deprivation in cyanobacteria is accompanied by chlorophyll (Chl) depletion. Indeed, radioactive labeling of the PG-depleted ΔpgsA mutant of Synechocystis sp. strain PCC 6803, which is not able to synthesize PG, proved the inhibition of Chl biosynthesis caused by restriction on the formation of 5-aminolevulinic acid and protochlorophyllide. Although the mutant accumulated chlorophyllide, the last Chl precursor, we showed that it originated from dephytylation of existing Chl and not from the block in the Chl biosynthesis. The lack of de novo-produced Chl under PG depletion was accompanied by a significantly weakened biosynthesis of both monomeric and trimeric photosystem I (PSI) complexes, although the decrease in cellular content was manifested only for the trimeric form. However, our analysis of ΔpgsA mutant, which lacked trimeric PSI because of the absence of the PsaL subunit, suggested that the virtual stability of monomeric PSI is a result of disintegration of PSI trimers. Interestingly, the loss of trimeric PSI was accompanied by accumulation of monomeric PSI associated with the newly synthesized CP43 subunit of photosystem II. We conclude that the absence of PG results in the inhibition of Chl biosynthetic pathway, which impairs synthesis of PSI, despite the accumulation of chlorophyllide released from the degraded Chl proteins. Based on the knowledge about the role of PG in prokaryotes, we hypothesize that the synthesis of Chl and PSI complexes are colocated in a membrane microdomain requiring PG for integrity.Photosynthetic membrane of oxygenic phototrophs has a unique lipid composition that has been conserved during billions of years of evolution from cyanobacteria and algae to modern higher plants. With no known exception, this membrane system always contains the uncharged glycolipids monogalactosyldiacylglycerol and digalactosyldiacylglycerol (DGDG) as well as the negatively charged lipids sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG; Murata and Siegenthaler, 1998). Interestingly, it seems that PG is the only lipid completely essential for the oxygenic photosynthesis. The loss of DGDG has only a mild impact on the cyanobacterial cell (Awai et al., 2007), and as shown recently in the cyanobacterium Synechocystis sp. strain PCC 6803, both galactolipids can be in fact replaced by glucolipids (Awai et al., 2014). SQDG and PG are only minor lipid components, each accounting for 5% to 12% of total lipids (Murata and Siegenthaler, 1998). SQDG is dispensable, although its lack results in various defects (Yu et al., 2002; Aoki et al., 2004), but PG plays an essential role in both cyanobacterial cells and plant chloroplasts (Hagio et al., 2000; Babiychuk et al., 2003).The critical role of PG has been mostly connected to the function of PSII. In both cyanobacteria and plants, lack of PG impairs the stability of PSII complexes and the electron transport between primary and secondary quinone acceptors inside the PSII reaction center. As shown in Synechocystis sp., PG molecules stabilize PSII dimers and facilitate the binding of inner antenna protein CP43 within the PSII core (Laczkó-Dobos et al., 2008). Indeed, according to the PSII crystal structure, two PG molecules are located at the interface between CP43 and the D1-D2 heterodimer (Guskov et al., 2009). As a consequence, the PG depletion inhibits and destabilizes PSII complexes and also, impairs assembly of new PSII complexes, although all PSII subunits are still synthesized in the cell (Laczkó-Dobos et al., 2008).Despite the fact that the vital link between PG and PSII is now well established, the phenotypic traits of PG-depleted cells signal that there are other sites in the photosynthetic membrane requiring strictly PG molecules. In Synechocystis sp., lack of PG triggers rapid loss of trimeric PSI complexes (Domonkos et al., 2004; Sato et al., 2004), and because PSI complexes bind more than 80% of chlorophyll (Chl) in the Synechocystis sp. cell, the PG depletion is accompanied by a characteristic Chl bleaching (Domonkos et al., 2004). However, the reasons for this symptom are still unclear. Chl metabolism is tightly coordinated with synthesis, assembly, and degradation of photosystem complexes (for review, see Komenda et al., 2012b; Sobotka, 2014), and we have shown recently that the PSI complexes are the main sink for de novo Chl produced in cyanobacteria (Kopečná et al., 2012). Given the drastic decrease in PSI content in the PG-depleted cells, Chl biosynthesis must be directly or indirectly affected after the PG concentration in membranes drops below a critical value. Although it was recently suggested that galactolipid and Chl biosyntheses are coregulated during chloroplast biogenesis (Kobayashi et al., 2014), a response of the Chl biosynthetic pathway to the altered lipid content has not been examined.To investigate Chl metabolism during PG starvation, we used the Synechocystis sp. ΔpgsA mutant, which is unable to synthesize PG (Hagio et al., 2000). The advantage of using the ΔpgsA strain is in its ability to utilize exogenous PG from growth medium, which allows monitoring of phenotypic changes from a wild type-like situation to completely PG-depleted cells. Chl biosynthesis shares the same metabolic pathway with heme and other tetrapyrroles. At the beginning of tetrapyrrole biosynthesis, the initial precursor, 5-aminolevulinic acid (ALA), is made from Glu through glutamyl-tRNA and subsequently converted in several steps to protoporphyrin IX. The pathway branches at the point where protoporphyrin IX is chelated by magnesium to produce Mg-protoporphyrin IX, the first intermediate on the Chl branch. This step is catalyzed by Mg-chelatase, a multisubunit enzyme that associates relatively weakly with the membrane; however, all following enzymes downward in the pathway are almost exclusively bound to membranes (Masuda and Fujita, 2008; Kopečná et al., 2012). The last enzyme of the Chl pathway, Chl synthase, is an integral membrane protein that attaches a phytyl chain to the last intermediate chlorophyllide (Chlide) to finalize Chl formation (Oster et al., 1997; Addlesee et al., 2000). According to current views, Chl synthase should also be involved in reutilization of Chl molecules from degraded Chl-binding proteins, which includes dephytylation and phytylation of Chl molecules with Chlide as an intermediate (Vavilin and Vermaas, 2007).In this study, we show a complex impact of PG deficiency on Chl metabolism. The lack of PG inhibited Chl biosynthesis at the two different steps: first, it drastically reduced formation of the initial precursor ALA, and second, it impaired the Mg-protoporphyrin methyl ester IX (MgPME) cyclase enzyme catalyzing synthesis of protochlorophyllide (Pchlide). The diminished rate of Chl formation was accompanied by impaired synthesis of both trimeric and monomeric PSI complexes and accumulation of a PSI monomer associated with the CP43 subunit of PSII. We also showed that the PG-depleted cells accumulated Chlide, originating from dephytylation of existing Chl, which suggests an inability to reutilize Chl for the PSI synthesis. We discuss a scenario that the Chl biosynthesis and synthesis of core PSI subunits are colocated in PG-enriched membrane microdomains.     

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.     

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.     

Chloroplast Activity and 3′phosphadenosine 5′phosphate Signaling Regulate Programmed Cell Death in Arabidopsis

Programmed cell death (PCD) is a crucial process both for plant development and responses to biotic and abiotic stress. There is accumulating evidence that chloroplasts may play a central role during plant PCD as for mitochondria in animal cells, but it is still unclear whether they participate in PCD onset, execution, or both. To tackle this question, we have analyzed the contribution of chloroplast function to the cell death phenotype of the myoinositol phosphate synthase1 (mips1) mutant that forms spontaneous lesions in a light-dependent manner. We show that photosynthetically active chloroplasts are required for PCD to occur in mips1, but this process is independent of the redox state of the chloroplast. Systematic genetic analyses with retrograde signaling mutants reveal that 3′-phosphoadenosine 5′-phosphate, a chloroplast retrograde signal that modulates nuclear gene expression in response to stress, can inhibit cell death and compromises plant innate immunity via inhibition of the RNA-processing 5′-3′ exoribonucleases. Our results provide evidence for the role of chloroplast-derived signal and RNA metabolism in the control of cell death and biotic stress response.Programmed cell death (PCD) is a universal process in multicellular organisms, contributing to the controlled and active degradation of the cell. In plants, PCD is required for processes as diverse as development, self-incompatibility, and stress response. One well-documented example is the induction of PCD upon pathogen attack, allowing the confinement of the infection, and resistance of the plant. The signaling events leading to the onset of PCD have been extensively studied: pathogen recognition triggers activation of mitogen-activated protein kinase cascades, as well as production of reactive oxygen species (ROS) and salicylic acid (SA), which lead to a hypersensitive response (Coll et al., 2011).From a cellular point of view, several classes of plant PCD have been described and compared with the ones found in animal cells (van Doorn, 2011). PCD is thought to have evolved independently in plants and animals, and genes underlying these mechanisms are therefore poorly conserved between the two kingdoms. However, most cellular features are conserved between plant and animal PCD that are both characterized by cell shrinkage, chromatin condensation, DNA laddering, mitochondria permeabilization, and depolarization (Dickman and Fluhr, 2013). In animal cells, mitochondria play a central role in the regulation of apoptosis (Czabotar et al., 2014; Mariño et al., 2014), and this role is likely shared between the two kingdoms (Lord and Gunawardena, 2012). That said, additional mitochondria-independent PCD pathways have clearly evolved in plants.Genetic approaches have greatly contributed to our understanding of cellular pathways governing PCD in plants. For example, the isolation of lesion mimic mutants (LMMs), in which cell death occurs spontaneously, has allowed the identification of several negative regulators of cell death (for review, see Bruggeman et al., 2015b). Interestingly, lesion formation is light dependent in several of these mutants, which include one of the best characterized LMMs—lesions simulating disease1 (lsd1; Dietrich et al., 1994). The LSD1 protein is required for plant acclimation to excess excitation energy (Mateo et al., 2004): when plants are exposed to excessive amounts of light, the redox status of the plastoquinone pool in the chloroplastic electron transfer chain is thought to influence LSD1-dependent signaling to modulate cell death (Mühlenbock et al., 2008). Additionally, we have previously identified the myoinositol phosphate synthase1 (mips1) mutant as a LMM, in which lesion formation is also light dependent (Meng et al., 2009). This mutant is deficient in the myoinositol (MI) phosphate synthase that catalyzes the first committed step of MI biosynthesis and displays pleiotropic defects such as reduced root growth, abnormal vein development, and spontaneous cell death on leaves, together with severe growth reduction after lesions begin to develop (Meng et al., 2009; Donahue et al., 2010). The light-dependent PCD in the mips1 mutant, as observed for lsd1, suggests that chloroplasts may play a role in the MI-dependent cell death regulation. Accumulating evidence suggests that chloroplasts may play a central role in PCD regulation like mitochondria in animal cells (Wang and Bayles, 2013). First, as described in the case of lsd1, excess light energy received by the chloroplast can function as a trigger for PCD. Furthermore, singlet oxygen (¹O₂), a ROS, can activate the EXECUTER1 (EX1) and EX2 proteins in the chloroplasts to initiate PCD (Lee et al., 2007). Likewise, ROS generated by chloroplasts play a major role for PCD onset during nonhost interaction between tobacco (Nicotiana tabacum) and Xanthomonas campestris (Zurbriggen et al., 2009). Finally, functional chloroplasts have also been shown to be required for PCD in cell suspensions (Gutierrez et al., 2014) and in a number of LMMs (Mateo et al., 2004; Meng et al., 2009; Bruggeman et al., 2015b). Thus, chloroplasts are now recognized as important components of plant defense response against pathogens (Stael et al., 2015) and are proposed to function with mitochondria in the execution of PCD (Van Aken and Van Breusegem, 2015). However, the exact signaling and metabolic contribution of chloroplasts to PCD remain to be elucidated. Furthermore, cross talk between chloroplasts and mitochondria does occur, such as during photorespiration (Sunil et al., 2013), but whether such communication functions sequentially or in parallel in the control of PCD remains to be determined (Van Aken and Van Breusegem, 2015).To further investigate how chloroplasts contribute to the regulation of cell death, we performed both forward and reverse genetics on the mips1 mutant. An extragenic secondary mutation in divinyl protochlorophyllide 8-vinyl reductase involved in chlorophyll biosynthesis leads to chlorophyll deficiency that abolishes the mips1 cell death phenotype, as do changes in CO₂ availability. These findings provide evidence for a link between photosynthetic activity and PCD induction in mips1. Additionally, we investigated the contribution of several retrograde signaling pathways (Chan et al., 2015) to the control of PCD in mips1. This process was independent of GENOMES UNCOUPLED (GUN) and EX signaling pathways, but we found that the SAL1-PAP_XRN retrograde signaling pathway inhibits cell death as well as basal defense reactions in Arabidopsis (Arabidopsis thaliana).