The Arabidopsis Ethylene/Jasmonic Acid-NRT Signaling Module Coordinates Nitrate Reallocation and the Trade-Off between Growth and Environmental Adaptation

Stresses decouple nitrate assimilation and photosynthesis through stress-initiated nitrate allocation to roots (SINAR), which is mediated by the nitrate transporters NRT1.8 and NRT1.5 and functions to promote stress tolerance. However, how SINAR communicates with the environment remains unknown. Here, we present biochemical and genetic evidence demonstrating that in Arabidopsis thaliana, ethylene (ET) and jasmonic acid (JA) affect the crosstalk between SINAR and the environment. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that ethylene response factors (ERFs), including OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2/ERF59, bind to the GCC boxes in the NRT1.8 promoter region, while ETHYLENE INSENSITIVE3 (EIN3) binds to the EIN3 binding site motifs in the NRT1.5 promoter. Genetic assays showed that cadmium and sodium stresses initiated ET/JA signaling, which converged at EIN3/EIN3-Like1 (EIL1) to modulate ERF expression and hence to upregulate NRT1.8. By contrast, ET and JA signaling mediated the downregulation of NRT1.5 via EIN3/EIL1 and other, unknown component(s). SINAR enhanced stress tolerance and decreased plant growth under nonstressed conditions through the ET/JA-NRT1.5/NRT1.8 signaling module. Interestingly, when nitrate reductase was impaired, SINAR failed to affect either stress tolerance or plant growth. These data suggest that SINAR responds to environmental conditions through the ET/JA-NRT signaling module, which further modulates stress tolerance and plant growth in a nitrate reductase-dependent manner.     

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).     

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.     

Degradation of the Endoplasmic Reticulum by Autophagy during Endoplasmic Reticulum Stress in Arabidopsis

In this article, we show that the endoplasmic reticulum (ER) in Arabidopsis thaliana undergoes morphological changes in structure during ER stress that can be attributed to autophagy. ER stress agents trigger autophagy as demonstrated by increased production of autophagosomes. In response to ER stress, a soluble ER marker localizes to autophagosomes and accumulates in the vacuole upon inhibition of vacuolar proteases. Membrane lamellae decorated with ribosomes were observed inside autophagic bodies, demonstrating that portions of the ER are delivered to the vacuole by autophagy during ER stress. In addition, an ER stress sensor, INOSITOL-REQUIRING ENZYME-1b (IRE1b), was found to be required for ER stress–induced autophagy. However, the IRE1b splicing target, bZIP60, did not seem to be involved, suggesting the existence of an undiscovered signaling pathway to regulate ER stress–induced autophagy in plants. Together, these results suggest that autophagy serves as a pathway for the turnover of ER membrane and its contents in response to ER stress in plants.     

Arabidopsis DELLA and JAZ Proteins Bind the WD-Repeat/bHLH/MYB Complex to Modulate Gibberellin and Jasmonate Signaling Synergy

Integration of diverse environmental and endogenous signals to coordinately regulate growth, development, and defense is essential for plants to survive in their natural habitat. The hormonal signals gibberellin (GA) and jasmonate (JA) antagonistically and synergistically regulate diverse aspects of plant growth, development, and defense. GA and JA synergistically induce initiation of trichomes, which assist seed dispersal and act as barriers to protect plants against insect attack, pathogen infection, excessive water loss, and UV irradiation. However, the molecular mechanism underlying such synergism between GA and JA signaling remains unclear. In this study, we revealed a mechanism for GA and JA signaling synergy and identified a signaling complex of the GA pathway in regulation of trichome initiation. Molecular, biochemical, and genetic evidence showed that the WD-repeat/bHLH/MYB complex acts as a direct target of DELLAs in the GA pathway and that both DELLAs and JAZs interacted with the WD-repeat/bHLH/MYB complex to mediate synergism between GA and JA signaling in regulating trichome development. GA and JA induce degradation of DELLAs and JASMONATE ZIM-domain proteins to coordinately activate the WD-repeat/bHLH/MYB complex and synergistically and mutually dependently induce trichome initiation. This study provides deep insights into the molecular mechanisms for integration of different hormonal signals to synergistically regulate plant development.     

Focus on Ethylene: Role of Ethylene and Its Cross Talk with Other Signaling Molecules in Plant Responses to Heavy Metal Stress

Excessive heavy metals (HMs) in agricultural lands cause toxicities to plants, resulting in declines in crop productivity. Recent advances in ethylene biology research have established that ethylene is not only responsible for many important physiological activities in plants but also plays a pivotal role in HM stress tolerance. The manipulation of ethylene in plants to cope with HM stress through various approaches targeting either ethylene biosynthesis or the ethylene signaling pathway has brought promising outcomes. This review covers ethylene production and signal transduction in plant responses to HM stress, cross talk between ethylene and other signaling molecules under adverse HM stress conditions, and approaches to modify ethylene action to improve HM tolerance. From our current understanding about ethylene and its regulatory activities, it is believed that the optimization of endogenous ethylene levels in plants under HM stress would pave the way for developing transgenic crops with improved HM tolerance.In addition to common abiotic stresses seen in agricultural production, such as drought, submerging, and extreme temperatures (Thao and Tran, 2012; Xia et al., 2015), heavy metal (HM) stress has arisen as a new pervasive threat (Srivastava et al., 2014; Ahmad et al., 2015). This is mainly due to the unrestricted industrialization and urbanization carried out during the past few decades, which have led to the increase of HMs in soils. Plants naturally require more than 15 different types of HM as nutrients serving for biological activities in cells (Sharma and Chakraverty, 2013). However, when the nutritional/nonnutritional HMs are present in excess, plants have to either suffer or take these up from the soil in an unwilling manner (Nies, 1999; Sharma and Chakraverty, 2013). Upon HM stress exposure, plants induce oxidative stress due to the excessive production of reactive oxygen species (ROS) and methylglyoxal (Sharma and Chakraverty, 2013). High levels of these compounds have been shown to negatively affect cellular structure maintenance (e.g. induction of lipid peroxidation in the membrane, biological macromolecule deterioration, ion leakage, and DNA strand cleavage; Gill and Tuteja, 2010; Nagajyoti et al., 2010) as well as many other biochemical and physiological processes (Dugardeyn and Van Der Straeten, 2008). As a result, plant growth is retarded and, ultimately, economic yield is decreased (Yadav, 2010; Anjum et al., 2012; Hossain et al., 2012; Asgher et al., 2015). Moreover, the accumulation of metal residues in the major food chain has been shown to cause serious ecological and health problems (Malik, 2004; Verstraeten et al., 2008).Plants employ different strategies to detoxify the unwanted HMs. Among the common responses of plants to HM stress are increases in ethylene production due to the enhanced expression of ethylene-related biosynthetic genes (Asgher et al., 2014; Khan and Khan, 2014; Khan et al., 2015b) and/or changes in the expression of ethylene-responsive genes (Maksymiec, 2007). Conventionally, this hormone has been established to modulate a number of important plant physiological activities, including seed germination, root hair and root nodule formation, and maturation (fruit ripening in particular; Dugardeyn and Van Der Straeten, 2008). On the other hand, although ethylene has also been suggested to be a stress-related hormone responding to a number of biotic and abiotic triggers, little is known about the exact role of elevated HM stress-related ethylene in plants (Zapata et al., 2003). Enhanced production of ethylene in plants subjected to toxic levels of cadmium (Cd), copper (Cu), iron (Fe), nickel (Ni), and zinc (Zn) has been shown (Maksymiec, 2007). As an example, Cd- and Cu-mediated stimulation of ethylene synthesis has been reported as a result of the increase of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) activity, one of the enzymes involved in the ethylene synthesis pathway (Schlagnhaufer and Arteca, 1997; Khan et al., 2015b).Plants tend to adjust or induce adaptation or tolerance mechanisms to overcome stress conditions. To develop stress tolerance, plants trigger a network of hormonal cross talk and signaling, among which ethylene production and signaling are prominently involved in stress-induced symptoms in acclimation processes (Gazzarrini and McCourt, 2003). Therefore, the necessity of controlling ethylene homeostasis and signal transduction using biochemical and molecular tools remains open to combat stress situations. Stress-induced ethylene acts to trigger stress-related effects on plants because of the autocatalytic ethylene synthesis. Autocatalytic stress-related ethylene production is controlled by mitogen-activated protein kinase (MAPK) phosphorylation cascades (Takahashi et al., 2007) and through stabilizing ACS2/6 (Li et al., 2012). Strong lines of evidence have shown the multiple facets of ethylene in plant responses to different abiotic stresses, including excessive HM, depending upon endogenous ethylene concentration and ethylene sensitivities that differ in developmental stage, plant species, and culture systems (Pierik et al., 2006; Kim et al., 2008; Khan and Khan, 2014). Under HM stress conditions, plants show a rapid increase in ethylene production and reduced plant growth and development, suggesting a negative regulatory role of ethylene in plant responses to HM stress (Schellingen et al., 2014; Khan et al., 2015b). On the other hand, a potential involvement of ETHYLENE INSENSITIVE2 (EIN2), a central component of the ethylene signaling pathway, as a positive regulator in lead (Pb) resistance in Arabidopsis (Arabidopsis thaliana) has also been demonstrated (Cao et al., 2009). More recently, Khan and Khan (2014) showed that ethylene-regulated antioxidant metabolism maintained a higher level of reduced glutathione (GSH) and alleviated photosynthetic inhibition in mustard (Brassica juncea) plants exposed to Ni, Zn, or Cd through the optimization of ethylene homeostasis (Masood et al., 2012). Taken together, the purpose of this review is to update the research community with our current understanding of the roles of ethylene and its signaling in plant responses to HM stress. Moreover, the cross talk of ethylene with other phytohormones and signaling molecules upon HM stress will also be discussed.     

Constitutively Active Mitogen-Activated Protein Kinase Versions Reveal Functions of Arabidopsis MPK4 in Pathogen Defense Signaling

Plant mitogen-activated protein kinases (MAPKs) are involved in important processes, including stress signaling and development. In a functional yeast screen, we identified mutations that render Arabidopsis thaliana MAPKs constitutively active (CA). Importantly, CA-MAPKs maintain their specificity toward known activators and substrates. As a proof-of-concept, Arabidopsis MAPK4 (MPK4) function in plant immunity was investigated. In agreement with the phenotype of mpk4 mutants, CA-MPK4 plants were compromised in pathogen-induced salicylic acid accumulation and disease resistance. MPK4 activity was found to negatively regulate pathogen-associated molecular pattern-induced reactive oxygen species production but had no impact on callose deposition, indicating that CA-MPK4 allows discriminating between processes regulated by MPK4 activity from processes indirectly affected by mpk4 mutation. Finally, MPK4 activity was also found to compromise effector-triggered immunity conditioned by the Toll Interleukin-1 Receptor–nucleotide binding (NB)–Leu-rich repeat (LRR) receptors RPS4 and RPP4 but not by the coiled coil–NB-LRR receptors RPM1 and RPS2. Overall, these data reveal important insights on how MPK4 regulates plant defenses and establishes that CA-MAPKs offer a powerful tool to analyze the function of plant MAPK pathways.     

A transit peptide-like sorting signal at the C terminus directs the Bienertia sinuspersici preprotein receptor Toc159 to the chloroplast outer membrane

Although Toc159 is known to be one of the key GTPase receptors for selective recognition of chloroplast preproteins, the mechanism for its targeting to the chloroplast surface remains unclear. To compare the targeting of these GTPase receptors, we identified two Toc159 isoforms and a Toc34 from Bienertia sinuspersici, a single-cell C₄ species with dimorphic chloroplasts in individual chlorenchyma cells. Fluorescent protein tagging and immunogold studies revealed that the localization patterns of Toc159 were distinctive from those of Toc34, suggesting different targeting pathways. Bioinformatics analyses indicated that the C-terminal tails (CTs) of Toc159 possess physicochemical and structural properties of chloroplast transit peptides (cTPs). These results were further confirmed by fluorescent protein tagging, which showed the targeting of CT fusion proteins to the chloroplast surface. The CT of Bs Toc159 in reverse orientation functioned as a cleavable cTP that guided the fluorescent protein to the stroma. Moreover, a Bs Toc34 mutant protein was retargeted to the chloroplast envelope using the CTs of Toc159 or reverse sequences of other cTPs, suggesting their conserved functions. Together, our data show that the C terminus and the central GTPase domain represent a novel dual domain–mediated sorting mechanism that might account for the partitioning of Toc159 between the cytosol and the chloroplast envelope for preprotein recognition.     

Probing Arabidopsis Chloroplast Diacylglycerol Pools by Selectively Targeting Bacterial Diacylglycerol Kinase to Suborganellar Membranes

Diacylglycerol (DAG) is an intermediate in metabolism of both triacylglycerols and membrane lipids. Probing the steady-state pools of DAG and understanding how they contribute to the synthesis of different lipids is important when designing plants with altered lipid metabolism. However, traditional methods of assaying DAG pools are difficult, because its abundance is low and because fractionation of subcellular membranes affects DAG pools. To manipulate and probe DAG pools in an in vivo context, we generated multiple stable transgenic lines of Arabidopsis (Arabidopsis thaliana) that target an Escherichia coli DAG kinase (DAGK) to each leaflet of each chloroplast envelope membrane. E. coli DAGK is small, self inserts into membranes, and has catalytic activity on only one side of each membrane. By comparing whole-tissue lipid profiles between our lines, we show that each line has an individual pattern of DAG, phosphatidic acid, phosphatidylcholine, and triacylglycerol steady-state levels, which supports an individual function of DAG in each membrane leaflet. Furthermore, conversion of DAG in the leaflets facing the chloroplast intermembrane space by DAGK impairs plant growth. As a result of DAGK presence in the outer leaflet of the outer envelope membrane, phosphatidic acid accumulation is not observed, likely because it is either converted into other lipids or removed to other membranes. Finally, we use the outer envelope-targeted DAGK line as a tool to probe the accessibility of DAG generated in response to osmotic stress.Diacylglycerol (DAG) is a central metabolite in plant lipid metabolism. Its glycerol backbone is modified with two acyl chains. If a third acyl chain is added, triacylglycerol (TAG) is formed, whereas if a head group is added, it is converted into polar lipids such as a galactolipid. In green tissues, the majority of DAG is used as an intermediate in galactolipid synthesis, because the extensive thylakoid membrane system consists of approximately 85% galactolipids (Block et al., 1983). Although under normal conditions the galactolipids are exclusively chloroplastic, in Arabidopsis (Arabidopsis thaliana), the DAG used to make galactolipids is derived from assembly pathways in both the chloroplast and the endoplasmic reticulum (ER; Benning, 2009). In both pathways, the bulk of the fatty acids are synthesized in the chloroplast stroma (Browse et al., 1986) in the following order of abundance: 18:1, 16:0, and 18:0 (Wallis and Browse, 2002).In the chloroplast pathway, these fatty acids are directly attached to a glycerol-3-P, generating first lyso-phosphatidic acid (l-PtdOH) and then phosphatidic acid (PtdOH) in the inner leaflet of the chloroplast inner envelope (Fig. 1; Frentzen et al., 1983). The acyltransferases involved are specific to the extent that the sn-2 position of the glycerol backbone predominantly receives a 16:0 fatty acid. PtdOH is then used directly for phosphatidylglycerol (PtdGro) synthesis (Babiychuk et al., 2003) or converted to DAG by a PtdOH phosphatase (Joyard and Douce, 1977). The PtdOH phosphatase activity is known to be associated with the inner envelope, though which leaflet is obscured by the fact that DAG can efficiently flip across membranes (Hamilton et al., 1991) and the actual enzyme has not been unambiguously identified and located (Nakamura et al., 2007). However, the leaflet associations of two other enzymes that use DAG in the inner envelope have been established. MGD1, which uses DAG to synthesize the most abundant galactolipid, monogalactosyldiacylglycerol (MGDG), is on the outer leaflet of the inner envelope membrane (Xu et al., 2005), while SQD2, which uses DAG to generate the less abundant sulfolipid, sulfoquinovosyldiacylglycerol (SQDG), is located on the inner leaflet of the inner envelope membrane (Tietje and Heinz, 1998). Also associated with the inner envelope membrane are a number of fatty acid desaturases, including FAD4, FAD5, FAD6, FAD7, and FAD8 (Joyard et al., 2010). Two of these are specific, generating lipids with signature desaturations: FAD4 desaturates only the 16:0 fatty acid of PtdGro, giving plastidic PtdGro a distinct 16:1 Δ3 trans moiety (Browse et al., 1985; Gao et al., 2009), and FAD5 desaturates primarily the 16:0 fatty acid of MGDG, producing 16:1 Δ7 cis (Kunst et al., 1989). The remaining desaturases are less specific, with little preference for head group or acyl tail. They further desaturate 16:1 or 18:1 in the cis conformation to 16:2 or 18:2 (FAD6; Browse et al., 1989) and on to 16:3 or 18:3 (FAD7 and FAD8; Wallis and Browse, 2002). The combined actions of these FADs result in the highly desaturated fatty acid profiles seen for most chloroplast lipids.Open in a separate windowFigure 1.Overview of DAG pools in the chloroplast envelope membranes. Processes that are known to have activity feeding into or withdrawing from DAG pools in the chloroplast envelope membranes are shown. Enzymes are indicated, and their substrates and products are connected with black arrows. However, for space reasons, not all reactants are shown. Membrane leaflets are indicated, and enzymes with known membrane topology are displayed correctly, while those without known topology are displayed in the center of the appropriate membrane. The acyl group preferred by each l-PtdOH acyltransferase is given in parentheses. Proposed processes transporting lipids from the ER to the chloroplast are shown with dashed arrows. Enzymes are as follows: 1, ATS1; 2, ATS2; 3, lipid phosphate phosphatase γ; 4, MGD1; 5, SQD2; 6, cytosolic phospholipases; 7, MGD2 or MGD3; 8, SFR2; 9, acyl-CoA:glycerol-3-P acyltransferase; 10, l-PtdOH acyltransferase; 11, PtdOH phosphatase; 12, cytidine diphosphate-choline:DAG cholinephosphotransferase; 13, TGD4; and 14, TGD1, TGD 2, TGD3 lipid transport complex. OE, Chloroplast outer envelope membrane; IE, chloroplast inner envelope membrane; ACP, acyl carrier protein. [See online article for color version of this figure.]In unstressed plants, DAG seems to be used primarily in the inner chloroplast envelope. However, several conditions are known to cause extensive DAG use in the chloroplast outer envelope. During phosphate deprivation, MGD2 and MDG3 synthesize MGDG from DAG on the outer leaflet of the outer envelope membrane (Kobayashi et al., 2009). The DAG backbones are probably supplied from the phosphatidylcholine (PtdCho) pool by phospholipase activity, which was shown to be simultaneously up-regulated (Andersson et al., 2004; Nakamura et al., 2005). DAG is also generated during freezing stress by a galactolipid:galactolipid galactosyltransferase named Sensitive to FReezing2 (SFR2). This enzyme transfers the galactosyl head group of MGDG onto another MGDG, giving rise to digalactosyldiacylglycerol (DGDG) and DAG (Moellering et al., 2010). The DAG is subsequently sequestered into a lipid droplet by formation of TAG by an as yet unidentified enzyme.In the ER pathway, fatty acids synthesized in the chloroplast stroma are exported through a still poorly defined mechanism to the ER and activated to acyl-CoAs. Acyltransferases sequentially catalyze formation of l-PtdOH and PtdOH from glycerol-3-P and acyl-CoAs. Again, the acyltransferase working on the sn-2 position of the glycerol backbone is specific, but unlike the chloroplast isoform, it prefers an 18:1 carbon fatty acid (Frentzen et al., 1983). Newly generated PtdOH can be converted to PtdGro or phosphatidyl inositol (PtdIns) (Collin et al., 1999) or hydrolyzed to DAG (Shimojima et al., 2009). DAG can then be further metabolized to TAG and PtdCho. PtdCho acyl groups (18:1/18:1 and 18:1/16:0) are desaturated sequentially by desaturases FAD2 (Okuley et al., 1994) and FAD3 (Browse et al., 1993). These desaturases prefer PtdCho as substrate. The acyl chains modified on PtdCho are transferred to other ER lipids, including DAG, as a result of continual acyl editing of the PtdCho pool (Bates et al., 2012). Furthermore, PtdOH and many of the other extraplastidic phospholipids can be converted to DAG by action of phospholipases (Shimojima et al., 2009). These have as yet partially defined roles in response to stress or recycling of membrane lipids (Testerink and Munnik, 2005).Glycerolipid precursors generated by de novo synthesis, acyl editing, and possibly stress conditions in the ER are transported to the chloroplast by a mechanism that is likely to involve at least two putative lipid transporters: trigalactosyldiacylglycerol4 (TGD4) in the chloroplast outer envelope membrane and the TGD1, TGD2, and TGD3 complex in the inner envelope membrane (Wang and Benning, 2012). The actual lipid species transported remains unclear, but PtdCho, lyso-phosphatidylcholine, PtdOH, and DAG have been discussed in the literature (Andersson and Dörmann, 2009). The DAG moieties are then fully incorporated into all plastidic lipids except PtdGro, presumably using the same pathways that metabolize plastidic DAG, described above. Because of the preference of chloroplast and ER sn-2 acyltransferases for 16 or 18 carbon fatty acids, respectively, the origin of the DAG moieties can be distinguished by positional analysis of the acyl groups on the glycerol backbone (Roughan and Slack, 1982). In Arabidopsis, the chloroplast and ER lipid synthesis pathways contribute nearly equally to mature chloroplast lipids (Browse et al., 1986; Mongrand et al., 1998). Thus, the DAG pools described so far in the chloroplast inner and outer envelope membranes are each of dual origin.A challenge for the analysis of the different DAG pools is that this compound is not a bilayer-forming lipid and thus does not accumulate stably to high levels. Furthermore, during any lengthy fractionation procedure, its levels can be expected to alter, as DAG-modifying enzymes exist in multiple membranes. Moreover, because DAG is quickly metabolized and may have efficient transport systems (Dong et al., 2012), it is difficult to confirm whether metabolizing enzymes are accessing the same or separate DAG pools.To probe different DAG pools of chloroplast membranes in vivo, we have generated a series of stable transgenic Arabidopsis lines in which we target an Escherichia coli DAG kinase (DAGK) to each leaflet of the chloroplast envelope membranes. The basic utility of this approach was previously shown by targeting a DAGK to the chloroplast in tobacco (Nicotiana tabacum) using a single construct fusing the bacterial protein to the Rubisco small subunit N-terminal peptide (Fritz et al., 2007). Here, we present a full phenotypic analysis of these lines, determining which chloroplast membranes show steady-state alterations of DAG and PtdOH levels predicted by ectopic DAGK activity. We further determine the accessibility of DAG pools generated on the outer leaflet of the chloroplast outer envelope membrane during osmotic stress. Having this system established in Arabidopsis will allow characterization of DAG pools in multiple lipid mutant lines.