2-Hydroxy-phytanoyl-CoA lyase (AtHPCL) is involved in phytol metabolism in Arabidopsis.

During chlorophyll degradation, large amounts of the isoprenoid alcohol phytol are released. The pathway of phytol catabolism has been studied in humans, because chlorophyll is part of the human diet, but little is known for plants. In humans, phytanoyl-CoA derived from phytol is degraded via α-oxidation by phytanoyl-CoA hydroxylase (PAHX) and 2-hydroxy-phytanoyl-CoA lyase (HPCL). Arabidopsis contains two sequences homologous to the human proteins AtPAHX and AtHPCL. Insertional mutants of Arabidopsis (pahx, hpcl) were grown under N deprivation to stimulate chlorophyll breakdown or supplemented with phytol to increase the endogenous amount of phytol. During N deprivation, chlorophyll, phytol, phytenal, upstream metabolites of phytol breakdown, and tocopherol and fatty acid phytyl esters, alternative phytol-derived lipids, accumulated in pahx and hpcl mutants, in line with the scenario that the mutations interfere with phytol degradation. AtHPCL was localized to the peroxisomes. Expression analysis of the AtHPCL sequence in the yeast Δpxp1 or Δmpo1 mutants followed by supplementation with 2-hydroxy-palmitic acid and enzyme assays of peroxisomal proteins from Col-0 and hpcl plants with 2-hydroxy-stearoyl-CoA revealed that AtHPCL harbors 2-hydroxy-acyl-CoA lyase activity. The α-dioxygenases αDOX1 and αDOX2 are involved in α-oxidation of fatty acids and could be involved in an alternative pathway of phytol degradation. However, phytol-related lipids in the αdox1, αdox2, or αdox1 αdox2 mutants were not altered compared with Col-0, indicating that αDOX1 and αDOX2 are not involved in phytol degradation. These results demonstrate that phytol degradation in Arabidopsis involves α-oxidation by AtPAHX and AtHPCL, but that it is independent of αDOX1/αDOX2.

The Bacterial Gq Signal Transduction Inhibitor FR900359 Impairs Soil-Associated Nematodes.

The cyclic depsipeptide FR900359 (FR) is derived from the soil bacterium Chromobacterium vaccinii and known to bind Gq proteins of mammals and insects, thereby abolishing the signal transduction of their Gq protein-coupled receptors, a process that leads to severe physiological consequences. Due to their highly conserved structure, Gq family of proteins are a superior ecological target for FR producing organisms, resulting in a defense towards a broad range of harmful organisms. Here, we focus on the question whether bacteria like C. vaccinii are important factors in soil in that their secondary metabolites impair, e.g., plant harming organisms like nematodes. We prove that the Gq inhibitor FR is produced under soil-like conditions. Furthermore, FR inhibits heterologously expressed Gαq proteins of the nematodes Caenorhabditis elegans and Heterodera schachtii in the micromolar range. Additionally, in vivo experiments with C. elegans and the plant parasitic cyst nematode H. schachtii demonstrated that FR reduces locomotion of C. elegans and H. schachtii. Finally, egg-laying of C. elegans and hatching of juvenile stage 2 of H. schachtii from its cysts is inhibited by FR, suggesting that FR might reduce nematode dispersion and proliferation. This study supports the idea that C. vaccinii and its excreted metabolome in the soil might contribute to an ecological equilibrium, maintaining and establishing the successful growth of plants.

Phytol derived from chlorophyll hydrolysis in plants is metabolized via phytenal.

Phytol is the isoprenoid alcohol bound in ester linkage to chlorophyll, the most abundant photosynthetic pigment in plants. During leaf senescence, large amounts of phytol are released by chlorophyll degradation. However, the pathway of phytol catabolism in plants is unknown. We hypothesized that phytol degradation in plants might involve its oxidation into the long-chain aldehyde phytenal. Using GC-MS for aldehyde quantification after derivatization with methylhydroxylamine, phytenal was identified in leaves, whereas other long-chain aldehydes (phytanal and pristanal) were barely detectable. We found that phytenal accumulates during chlorotic stresses, for example, salt stress, dark-induced senescence, and nitrogen deprivation. The increase in the phytenal content is mediated at least in part independently of enzyme activities, and it is independent of light. Characterization of phytenal accumulation in the pao1 mutant affected in chlorophyll degradation revealed that phytenal is an authentic phytol metabolite derived from chlorophyll breakdown. The increase in phytenal was even stronger in mutants affected in the production of other phytol metabolites including vte5-2 (tocopherol deficient) and pes1 pes2 (fatty acid phytyl ester deficient). Therefore, phytenal accumulation is controlled by competing, alternative pathways of phosphorylation (leading to tocopherol production) or esterification (fatty acid phytyl ester production). As a consequence, the content of phytenal is maintained at low levels, presumably to minimize its toxic effects caused by its highly reactive aldehyde group that can form covalent bonds with and inactivate the amino groups of proteins.

A parasitic nematode releases cytokinin that controls cell division and orchestrates feeding site formation in host plants.

Sedentary plant-parasitic cyst nematodes are biotrophs that cause significant losses in agriculture. Parasitism is based on modifications of host root cells that lead to the formation of a hypermetabolic feeding site (a syncytium) from which nematodes withdraw nutrients. The host cell cycle is activated in an initial cell selected by the nematode for feeding, followed by activation of neighboring cells and subsequent expansion of feeding site through fusion of hundreds of cells. It is generally assumed that nematodes manipulate production and signaling of the plant hormone cytokinin to activate cell division. In fact, nematodes have been shown to produce cytokinin in vitro; however, whether the hormone is secreted into host plants and plays a role in parasitism remained unknown. Here, we analyzed the spatiotemporal activation of cytokinin signaling during interaction between the cyst nematode, Heterodera schachtii, and Arabidopsis using cytokinin-responsive promoter:reporter lines. Our results showed that cytokinin signaling is activated not only in the syncytium but also in neighboring cells to be incorporated into syncytium. An analysis of nematode infection on mutants that are deficient in cytokinin or cytokinin signaling revealed a significant decrease in susceptibility of these plants to nematodes. Further, we identified a cytokinin-synthesizing isopentenyltransferase gene in H. schachtii and show that silencing of this gene in nematodes leads to a significant decrease in virulence due to a reduced expansion of feeding sites. Our findings demonstrate the ability of a plant-parasitic nematode to synthesize a functional plant hormone to manipulate the host system and establish a long-term parasitic interaction.

Unusual vitamin E profile in the oil of a wild African oil palm tree (Elaeis guineensis Jacq.) enhances oxidative stability of provitamin A.

Introduction: The African oil palm (Elaeis guineensis Jacq.) is the predominant oil crop in the world. In addition to triacylglycerols, crude palm oil (CPO) extracted from the mesocarp of the fruits, contains high amounts of provitamin A (carotenes) and vitamin E (tocochromanols). Because of their unsaturated nature, the carotenes are prone to oxidation and therefore are in part limiting for the shelf life of CPO. Methods: A tree with unusual toochromanol composition was identified by HPLC screening of the mesocarp of wild trees. Polymorphisms in a candidate gene were identified by DNA sequencing. The candidate protein was heterologously expressed in Escherichia coli coli and Arabidopsis thaliana to test for enzyme activity. Oxidative stability of the CPO was studied by following carotene degradation over time. Results: In the present study, a wild Oil Palm tree (C59) from Cameroon was identified that lacks α-tocopherol and α-tocotrienol and instead accumulates the respective γ forms, suggesting that the activity of γ-tocopherol methyltransferase (VTE4) was affected. Sequencing of the VTE4 locus in the genome of plant C59 identified a G/C polymorphism that causes the exchange of a highly conserved tryptophan at position 290 with serine. The W290S exchange renders the VTE4 enzyme inactive, as shown after expression in Escherichia coli and Arabidopsis thaliana. The oxidative stability of carotenes in the mesocarp of the wild palm C59 was enhanced compared with control accessions. Furthermore, supplementation of commercial palm oil with different tocochromanols showed that γ-tocotrienol exerts a stronger effect during the protection of carotenes against oxidation than α-tocotrienol. Discussion: Therefore, the introduction of the high γ-tocotrienol trait into elite breeding lines represents a potent strategy to protect carotenes against oxidation and extend the shelf life of CPO, hence allowing the development of a value added high-carotene CPO to be used to fight against vitamin A deficiency.

Lipid Analysis by Gas Chromatography and Gas Chromatography-Mass Spectrometry.

Gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) represent powerful tools for the quantitative and structural analysis of plant lipids. Here, we outline protocols for the isolation, separation, and derivatization of plant lipids for subsequent GC and GC-MS analysis. Plant lipids are extracted with organic solvents and separated according to their polarity by thin-layer chromatography or solid phase extraction. As most lipids are not volatile, the analytes are derivatized by transmethylation or trimethylsilylation to enable the transition of the molecules into the gas phase. After separation on the polymer matrix of the GC column, the analytes are detected by flame ionization or mass spectrometry. This chapter includes methods suitable for the analysis of lipid-bound or free fatty acids, long chain alcohols, and monoacylglycerols and for the determination of double bond positions in fatty acids.

Fatty acid isoprenoid alcohol ester synthesis in fruits of the African Oil Palm (Elaeis guineensis).

The African Oil Palm (Elaeis guineensis; family Arecaceae) represents the most important oil crop for food and feed production and for biotechnological applications. Two types of oil can be extracted from palm fruits, the mesocarp oil which is rich in palmitic acid and in carotenoids (provitamin A) and tocochromanols (vitamin E), and the kernel oil with high amounts of lauric and myristic acid. We identified fatty acid phytyl esters (FAPEs) in the mesocarp and kernel tissues of mature fruits, mostly esterified with oleic acid and very long chain fatty acids. In addition, fatty acid geranylgeranyl esters (FAGGEs) accumulated in mesocarp and kernels to even larger amounts. In contrast, FAPEs and FAGGEs amounts and fatty acid composition in leaves were very similar. Analysis of wild accessions of African Oil Palm from Cameroon revealed a considerable variation in the amounts and composition of FAPEs and FAGGEs in mesocarp and kernel tissues. Exogenous supplementation of phytol or geranylgeraniol to mesocarp slices resulted in the incorporation of these alcohols into FAPEs and FAGGEs, respectively, indicating that they are synthesized via enzymatic reactions. Three candidate genes of the esterase/lipase/thioesterase (ELT) family were identified in the Oil Palm genome. The genes are differentially expressed in mesocarp tissue with EgELT1 showing the highest expression. Geranylgeraniol from FAGGE might be recycled and used as a substrate for the synthesis of carotenoids and tocotrienols during fruit development. Thus, FAPEs and FAGGEs in the mesocarp and kernel of Oil Palm provide an additional metabolic source for fatty acids and phytol or geranylgeraniol, respectively.

Inhibition of acetyl-CoA carboxylase by spirotetramat causes growth arrest and lipid depletion in nematodes.

Plant-parasitic nematodes pose a significant threat to agriculture causing annual yield losses worth more than 100 billion US$. Nematode control often involves the use of nematicides, but many of them including non-selective fumigants have been phased out, particularly due to ecotoxicological concerns. Thus new control strategies are urgently needed. Spirotetramat (SPT) is used as phloem-mobile systemic insecticide targeting acetyl-CoA carboxylase (ACC) of pest insects and mites upon foliar application. However, in nematodes the mode of action of SPT and its effect on their development have not been studied so far. Our studies revealed that SPT known to be activated in planta to SPT-enol acts as a developmental inhibitor of the free-living nematode Caenorhabditis elegans and the plant-parasitic nematode Heterodera schachtii. Exposure to SPT-enol leads to larval arrest and disruption of the life cycle. Furthermore, SPT-enol inhibits nematode ACC activity, affects storage lipids and fatty acid composition. Silencing of H. schachtii ACC by RNAi induced similar phenotypes and thus mimics the effects of SPT-enol, supporting the conclusion that SPT-enol acts on nematodes by inhibiting ACC. Our studies demonstrated that the inhibition of de novo lipid biosynthesis by interfering with nematode ACC is a new nematicidal mode of action addressed by SPT, a well-known systemic insecticide for sucking pest control.

Parasitic worms stimulate host NADPH oxidases to produce reactive oxygen species that limit plant cell death and promote infection.

Plants and animals produce reactive oxygen species (ROS) in response to infection. In plants, ROS not only activate defense responses and promote cell death to limit the spread of pathogens but also restrict the amount of cell death in response to pathogen recognition. Plants also use hormones, such as salicylic acid, to mediate immune responses to infection. However, there are long-lasting biotrophic plant-pathogen interactions, such as the interaction between parasitic nematodes and plant roots during which defense responses are suppressed and root cells are reorganized to specific nurse cell systems. In plants, ROS are primarily generated by plasma membrane-localized NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidases, and loss of NADPH oxidase activity compromises immune responses and cell death. We found that infection of Arabidopsis thaliana by the parasitic nematode Heterodera schachtii activated the NADPH oxidases RbohD and RbohF to produce ROS, which was necessary to restrict infected plant cell death and promote nurse cell formation. RbohD- and RbohF-deficient plants exhibited larger regions of cell death in response to nematode infection, and nurse cell formation was greatly reduced. Genetic disruption of SID2, which is required for salicylic acid accumulation and immune activation in nematode-infected plants, led to the increased size of nematodes in RbohD- and RbohF-deficient plants, but did not decrease plant cell death. Thus, by stimulating NADPH oxidase-generated ROS, parasitic nematodes fine-tune the pattern of plant cell death during the destructive root invasion and may antagonize salicylic acid-induced defense responses during biotrophic life stages.