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.     

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.     

Loss of Ceramide Kinase in Arabidopsis Impairs Defenses and Promotes Ceramide Accumulation and Mitochondrial H2O2 Bursts

Arabidopsis thaliana plants that lack ceramide kinase, encoded by ACCELERATED CELL DEATH5 (ACD5), display spontaneous programmed cell death late in development and accumulate substrates of ACD5. Here, we compared ceramide accumulation kinetics, defense responses, ultrastructural features, and sites of reactive oxygen species (ROS) production in wild-type and acd5 plants during development and/or Botrytis cinerea infection. Quantitative sphingolipid profiling indicated that ceramide accumulation in acd5 paralleled the appearance of spontaneous cell death, and it was accompanied by autophagy and mitochondrial ROS accumulation. Plants lacking ACD5 differed significantly from the wild type in their responses to B. cinerea, showing earlier and higher increases in ceramides, greater disease, smaller cell wall appositions (papillae), reduced callose deposition and apoplastic ROS, and increased mitochondrial ROS. Together, these data show that ceramide kinase greatly affects sphingolipid metabolism and the site of ROS accumulation during development and infection, which likely explains the developmental and infection-related cell death phenotypes. The acd5 plants also showed an early defect in restricting B. cinerea germination and growth, which occurred prior to the onset of cell death. This early defect in B. cinerea restriction in acd5 points to a role for ceramide phosphate and/or the balance of ceramides in mediating early antifungal responses that are independent of cell death.     

Peroxisomal ATP-Binding Cassette Transporter COMATOSE and the Multifunctional Protein ABNORMAL INFLORESCENCE MERISTEM Are Required for the Production of Benzoylated Metabolites in Arabidopsis Seeds

Secondary metabolites derived from benzoic acid (BA) are of central importance in the interactions of plants with pests, pathogens, and symbionts and are potentially important in plant development. Peroxisomal β-oxidation has recently been shown to contribute to BA biosynthesis in plants, but not all of the enzymes involved have been defined. In this report, we demonstrate that the peroxisomal ATP-binding cassette transporter COMATOSE is required for the accumulation of benzoylated secondary metabolites in Arabidopsis (Arabidopsis thaliana) seeds, including benzoylated glucosinolates and substituted hydroxybenzoylcholines. The ABNORMAL INFLORESCENCE MERISTEM protein, one of two multifunctional proteins encoded by Arabidopsis, is essential for the accumulation of these compounds, and MULTIFUNCTIONAL PROTEIN2 contributes to the synthesis of substituted hydroxybenzoylcholines. Of the two major 3-ketoacyl coenzyme A thiolases, KAT2 plays the primary role in BA synthesis. Thus, BA biosynthesis in Arabidopsis employs the same core set of β-oxidation enzymes as in the synthesis of indole-3-acetic acid from indole-3-butyric acid.Many important secondary metabolites in plants are derived from, or incorporate, benzoic acid (BA). These include compounds found in root exudates, inflorescences, stems, and flower volatiles (D’Auria and Gershenzon, 2005). BA is also potentially a precursor for the plant hormone salicylic acid (SA; Wildermuth et al., 2001). In Arabidopsis (Arabidopsis thaliana), benzoylated glucosinolates (BGs) accumulate in seeds, presumably as a deterrent against animal feeding. Thus, BA metabolites are believed to play key roles in the interactions of plants with microbial and animal pests as well as in beneficial relationships such as pollination systems (Boatright et al., 2004). Understanding the pathways and control of BA synthesis in plants, therefore, is very important.Three different pathways for the synthesis of BA have been proposed for plants (Boatright et al., 2004; Wildermuth, 2006). These begin with the first committed step of the phenylpropanoid pathway, the deamination of Phe by Phe ammonia lyase to produce trans-cinnamic acid (CA). CA can then be oxidized by CoA-independent reactions in the cytosol, or it may be activated with CoA and proceed through one cycle of peroxisomal β-oxidation. Alternatively, BA synthesis may proceed via a third, CoA-dependent but β-oxidation-independent, pathway that combines elements of the first two pathways (Wildermuth, 2006). Recent studies in Petunia hybrida have highlighted the importance of the peroxisomal β-oxidation pathway in the production of BA for incorporation into floral volatile benzenoids. Enzymes identified in this pathway to date are a cinnamate:CoA ligase (PhCNL/PhAAE [for acyl-activating enzyme]) that activates CA (Colquhoun et al., 2012; Klempien et al., 2012), a multifunctional protein (PhMFP) that hydrates and oxidizes the trans-cinnamoyl-CoA (Qualley et al., 2012), and a 3-ketoacyl CoA thiolase (PhKAT1) that cleaves the resultant β-keto thioester (Van Moerkercke et al., 2009).Seeds of Arabidopsis accumulate appreciable amounts of BGs (Reichelt et al., 2002; Kliebenstein et al., 2007). Thus, while free BA is not detected in Arabidopsis seeds (Ibdah and Pichersky, 2009), the accumulation of BGs and other BA-containing secondary metabolites in Arabidopsis seeds provides a powerful experimental system with which to determine the pathway and potential control of BA synthesis in plants. For example, a peroxisomal acyl-CoA ligase (BZO1, for benzoyloxy glucosinolate) has been identified in Arabidopsis that is closely related to PhCNL1 and is required for BG production in seeds (Kliebenstein et al., 2007). BZO1 has recently been shown to be an AAE with cinnamate:CoA ligase activity (Lee et al., 2012).To further investigate the requirement for peroxisomal β-oxidation in BA synthesis, and to identify key enzymes involved in Arabidopsis, we analyzed BA-containing secondary metabolites (BGs and substituted hydroxybenzoylated choline esters) of seeds from a suite of β-oxidation mutants covering the key steps of β-oxidation, including substrate import, activation, oxidation, and thiolysis. This work identifies specific isozymes in Arabidopsis that mediate these steps, defines a new role for ABNORMAL INFLORESCENCE MERISTEM (AIM1), and determines a route for the entry of CA into peroxisomes.     

Reprogramming the Phenylpropanoid Metabolism in Seeds of Oilseed Rape by Suppressing the Orthologs of REDUCED EPIDERMAL FLUORESCENCE1

As a result of the phenylpropanoid pathway, many Brassicaceae produce considerable amounts of soluble hydroxycinnamate conjugates, mainly sinapate esters. From oilseed rape (Brassica napus), we cloned two orthologs of the Arabidopsis (Arabidopsis thaliana) gene REDUCED EPIDERMAL FLUORESCENCE1 (REF1) encoding a coniferaldehyde/sinapaldehyde dehydrogenase. The enzyme is involved in the formation of ferulate and sinapate from the corresponding aldehydes, thereby linking lignin and hydroxycinnamate biosynthesis as a potential branch-point enzyme. We used RNA interference to silence REF1 genes in seeds of oilseed rape. Nontargeted metabolite profiling showed that BnREF1-suppressing seeds produced a novel chemotype characterized by reduced levels of sinapate esters, the appearance of conjugated monolignols, dilignols, and trilignols, altered accumulation patterns of kaempferol glycosides, and changes in minor conjugates of caffeate, ferulate, and 5-hydroxyferulate. BnREF1 suppression affected the level of minor sinapate conjugates more severely than that of the major component sinapine. Mapping of the changed metabolites onto the phenylpropanoid metabolic network revealed partial redirection of metabolic sequences as a major impact of BnREF1 suppression.Phenylpropanoid metabolism provides plants with a vast array of phenolic compounds that contribute to nearly all aspects of plant life (Vogt, 2010). In species of the Brassicaceae family, soluble hydroxycinnamate conjugates, mainly sinapate esters, constitute an abundant metabolite fraction produced from a branch of the phenylpropanoid pathway (Fraser and Chapple, 2011). As major compounds, sinapoylmalate accumulates in leaves (Hause et al., 2002) and sinapoylcholine (sinapine) in seeds (Bouchereau et al., 1991; Fig. 1).Open in a separate windowFigure 1.Biosynthesis of sinapate esters emphasizing the crucial role of the bifunctional hydroxycinnamaldehyde dehydrogenase CALDH/SALDH encoded by the gene REF1. Dashed arrows symbolize multistep biosyntheses. Abbreviations for enzymes are as follows: CAD, (hydroxy)cinnamyl alcohol dehydrogenase; SAD, sinapyl alcohol dehydrogenase; SCT, 1-O-sinapoylglucose:choline sinapoyltransferase; SGT, UDP-Glc:sinapate glucosyltransferase; SMT, 1-O-sinapoylglucose:malate sinapoyltransferase.In Arabidopsis (Arabidopsis thaliana), the disturbed accumulation of sinapoylmalate caused the mutant phenotype reduced epidermal fluorescence (ref; Ruegger and Chapple, 2001). Molecular characterization of the ref1 mutant led to the identification of the gene At3g24503 (REF1) encoding coniferaldehyde dehydrogenase/sinapaldehyde dehydrogenase (CALDH/SALDH; EC 1.2.1.68; Nair et al., 2004). The bifunctional enzyme CALDH/SALDH was shown to catalyze the NADP⁺-dependent oxidation of coniferaldehyde and sinapaldehyde to yield the corresponding hydroxycinnamates ferulate and sinapate. As a potential branching enzyme, the enzymatic activity of the REF1-encoded CALDH/SALDH might be crucial for the partition ratio of metabolites between lignin and hydroxycinnamate biosynthesis (Fig. 1). Therefore, manipulation of REF1 expression coupled to a comprehensive metabolite analysis of transgenic plants appeared as an interesting strategy to gain a deeper understanding of the plant phenylpropanoid metabolic network. Moreover, in crop plants like oilseed rape (Brassica napus), where high levels of sinapate esters contribute to antinutritive features, the suppression of REF1 orthologs might cause increased quality.This work describes the isolation of REF1 orthologous genes from oilseed rape (BnREF1). By RNA interference, transgenic lines of oilseed rape were generated that suppress BnREF1 gene expression during seed development. Homozygous transgenic progeny were developed and used in a comprehensive liquid chromatography-mass spectrometry (LC/MS)-based metabolite profiling to investigate the impact of BnREF1 silencing in seeds. Mapping of changed metabolite patterns onto the phenylpropanoid metabolic network revealed insight into the molecular mechanisms by which silencing of BnREF1 produced a novel seed chemotype in oilseed rape.     

Large-Scale Proteomics of the Cassava Storage Root and Identification of a Target Gene to Reduce Postharvest Deterioration

Cassava (Manihot esculenta) is the most important root crop in the tropics, but rapid postharvest physiological deterioration (PPD) of the root is a major constraint to commercial cassava production. We established a reliable method for image-based PPD symptom quantification and used label-free quantitative proteomics to generate an extensive cassava root and PPD proteome. Over 2600 unique proteins were identified in the cassava root, and nearly 300 proteins showed significant abundance regulation during PPD. We identified protein abundance modulation in pathways associated with oxidative stress, phenylpropanoid biosynthesis (including scopoletin), the glutathione cycle, fatty acid α-oxidation, folate transformation, and the sulfate reduction II pathway. Increasing protein abundances and enzymatic activities of glutathione-associated enzymes, including glutathione reductases, glutaredoxins, and glutathione S-transferases, indicated a key role for ascorbate/glutathione cycles. Based on combined proteomics data, enzymatic activities, and lipid peroxidation assays, we identified glutathione peroxidase as a candidate for reducing PPD. Transgenic cassava overexpressing a cytosolic glutathione peroxidase in storage roots showed delayed PPD and reduced lipid peroxidation as well as decreased H₂O₂ accumulation. Quantitative proteomics data from ethene and phenylpropanoid pathways indicate additional gene candidates to further delay PPD. Cassava root proteomics data are available at www.pep2pro.ethz.ch for easy access and comparison with other proteomics data.     

Pipecolic Acid,an Endogenous Mediator of Defense Amplification and Priming,Is a Critical Regulator of Inducible Plant Immunity

Metabolic signals orchestrate plant defenses against microbial pathogen invasion. Here, we report the identification of the non-protein amino acid pipecolic acid (Pip), a common Lys catabolite in plants and animals, as a critical regulator of inducible plant immunity. Following pathogen recognition, Pip accumulates in inoculated Arabidopsis thaliana leaves, in leaves distal from the site of inoculation, and, most specifically, in petiole exudates from inoculated leaves. Defects of mutants in AGD2-LIKE DEFENSE RESPONSE PROTEIN1 (ALD1) in systemic acquired resistance (SAR) and in basal, specific, and β-aminobutyric acid–induced resistance to bacterial infection are associated with a lack of Pip production. Exogenous Pip complements these resistance defects and increases pathogen resistance of wild-type plants. We conclude that Pip accumulation is critical for SAR and local resistance to bacterial pathogens. Our data indicate that biologically induced SAR conditions plants to more effectively synthesize the phytoalexin camalexin, Pip, and salicylic acid and primes plants for early defense gene expression. Biological priming is absent in the pipecolate-deficient ald1 mutants. Exogenous pipecolate induces SAR-related defense priming and partly restores priming responses in ald1. We conclude that Pip orchestrates defense amplification, positive regulation of salicylic acid biosynthesis, and priming to guarantee effective local resistance induction and the establishment of SAR.     

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.     

Recruitment of PLANT U-BOX13 and the PI4Kβ1/β2 Phosphatidylinositol-4 Kinases by the Small GTPase RabA4B Plays Important Roles during Salicylic Acid-Mediated Plant Defense Signaling in Arabidopsis

Protection against microbial pathogens involves the activation of cellular immune responses in eukaryotes, and this cellular immunity likely involves changes in subcellular membrane trafficking. In eukaryotes, members of the Rab GTPase family of small monomeric regulatory GTPases play prominent roles in the regulation of membrane trafficking. We previously showed that RabA4B is recruited to vesicles that emerge from trans-Golgi network (TGN) compartments and regulates polarized membrane trafficking in plant cells. As part of this regulation, RabA4B recruits the closely related phosphatidylinositol 4-kinase (PI4K) PI4Kβ1 and PI4Kβ2 lipid kinases. Here, we identify a second Arabidopsis thaliana RabA4B-interacting protein, PLANT U-BOX13 (PUB13), which has recently been identified to play important roles in salicylic acid (SA)-mediated defense signaling. We show that PUB13 interacts with RabA4B through N-terminal domains and with phosphatidylinositol 4-phosphate (PI-4P) through a C-terminal armadillo domain. Furthermore, we demonstrate that a functional fluorescent PUB13 fusion protein (YFP-PUB13) localizes to TGN and Golgi compartments and that PUB13, PI4Kβ1, and PI4Kβ2 are negative regulators of SA-mediated induction of pathogenesis-related gene expression. Taken together, these results highlight a role for RabA4B and PI-4P in SA-dependent defense responses.     

Retromer Contributes to Immunity-Associated Cell Death in Arabidopsis

Membrane trafficking is required during plant immune responses, but its contribution to the hypersensitive response (HR), a form of programmed cell death (PCD) associated with effector-triggered immunity, is not well understood. HR is induced by nucleotide binding-leucine-rich repeat (NB-LRR) immune receptors and can involve vacuole-mediated processes, including autophagy. We previously isolated lazarus (laz) suppressors of autoimmunity-triggered PCD in the Arabidopsis thaliana mutant accelerated cell death11 (acd11) and demonstrated that the cell death phenotype is due to ectopic activation of the LAZ5 NB-LRR. We report here that laz4 is mutated in one of three VACUOLAR PROTEIN SORTING35 (VPS35) genes. We verify that LAZ4/VPS35B is part of the retromer complex, which functions in endosomal protein sorting and vacuolar trafficking. We show that VPS35B acts in an endosomal trafficking pathway and plays a role in LAZ5-dependent acd11 cell death. Furthermore, we find that VPS35 homologs contribute to certain forms of NB-LRR protein-mediated autoimmunity as well as pathogen-triggered HR. Finally, we demonstrate that retromer deficiency causes defects in late endocytic/lytic compartments and impairs autophagy-associated vacuolar processes. Our findings indicate important roles of retromer-mediated trafficking during the HR; these may include endosomal sorting of immune components and targeting of vacuolar cargo.     

Novel Plant Immune-Priming Compounds Identified via High-Throughput Chemical Screening Target Salicylic Acid Glucosyltransferases in Arabidopsis

Plant activators are compounds, such as analogs of the defense hormone salicylic acid (SA), that protect plants from pathogens by activating the plant immune system. Although some plant activators have been widely used in agriculture, the molecular mechanisms of immune induction are largely unknown. Using a newly established high-throughput screening procedure that screens for compounds that specifically potentiate pathogen-activated cell death in Arabidopsis thaliana cultured suspension cells, we identified five compounds that prime the immune response. These compounds enhanced disease resistance against pathogenic Pseudomonas bacteria in Arabidopsis plants. Pretreatments increased the accumulation of endogenous SA, but reduced its metabolite, SA-O-β-d-glucoside. Inducing compounds inhibited two SA glucosyltransferases (SAGTs) in vitro. Double knockout plants that lack both SAGTs consistently exhibited enhanced disease resistance. Our results demonstrate that manipulation of the active free SA pool via SA-inactivating enzymes can be a useful strategy for fortifying plant disease resistance and may identify useful crop protectants.