Embryonic Photosynthesis Affects Post-Germination Plant Growth.

Photosynthesis is the fundamental process fueling plant vegetative growth and development. The progeny of plants relies on maternal photosynthesis, via food reserves in the seed, to supply the necessary energy for seed germination and early seedling establishment. Intriguingly, before seed maturation, Arabidopsis (Arabidopsis thaliana) embryos are also photosynthetically active, the biological significance of which remains poorly understood. Investigating this system is genetically challenging because mutations perturbing photosynthesis are expected to affect both embryonic and vegetative tissues. Here, we isolated a temperature-sensitive mutation affecting CPN60α2, which encodes a subunit of the chloroplast chaperonin complex CPN60. When exposed to cold temperatures, cpn60α2 mutants accumulate less chlorophyll in newly produced tissues, thus allowing the specific disturbance of embryonic photosynthesis. Analyses of cpn60α2 mutants were combined with independent genetic and pharmacological approaches to show that embryonic photosynthetic activity is necessary for normal skoto- and photomorphogenesis in juvenile seedlings as well as long-term adult plant development. Our results reveal the importance of embryonic photosynthetic activity for normal adult plant growth, development, and health.

Modification of sperm fatty acid composition during epididymal maturation in bats.

Biochemical properties of polyunsaturated fatty acids (PUFAs) are fundamental to sperm movements. Amongst all adjustments operated during epididymal maturation, sperm membrane lipid composition is remodelled. Specifically, the proportion of PUFAs usually increases from the caput towards the cauda epididymidis. In mammals, PUFAs are predominantly acquired through the diet, which can consequently impact male fertility. We aimed at analysing to what extent n-6 and n-3 PUFAs are incorporated into sperm in the Seba's short-tailed bat (Carollia perspicillata), and at demonstrating the effect of the sperm fatty acid composition on sperm mobility. We therefore provided food varying in fatty acid composition to males of C. perspicillata and measured the fatty acid composition and mobility traits in spermatozoa collected from the caput and cauda epididymides. We found that n-6 and n-3 PUFAs and saturated fatty acids were significantly related to sperm velocity but not to the proportion of progressive sperm (i.e. motility). Concomitant to an increase in sperm velocity, the level of fatty acid saturation increased from the caput to the cauda epididymidis, while the proportion of PUFAs remained similar along the epididymis. A reduction in n-6 PUFAs counterbalanced an increase in n-3 PUFAs. The food treatments did not affect the sperm fatty acid composition. Our results suggest that a precise endogenous control rather than dietary effects determines sperm fatty acid composition in C. perspicillata.

The pathogen-related yeast protein Pry1, a member of the CAP protein superfamily, is a fatty acid-binding protein.

Members of the CAP superfamily (cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins), also known as SCP superfamily (sperm-coating proteins), have been implicated in many physiological processes, including immune defenses, venom toxicity, and sperm maturation. Their mode of action, however, remains poorly understood. Three proteins of the CAP superfamily, Pry1, -2, and -3 (pathogen related in yeast), are encoded in the Saccharomyces cerevisiae genome. We have shown previously that Pry1 binds cholesterol in vitro and that Pry function is required for sterol secretion in yeast cells, indicating that members of this superfamily may generally bind sterols or related small hydrophobic compounds. On the other hand, tablysin-15, a CAP protein from the horsefly Tabanus yao, has been shown to bind leukotrienes and free fatty acids in vitro Therefore, here we assessed whether the yeast Pry1 protein binds fatty acids. Computational modeling and site-directed mutagenesis indicated that the mode of fatty acid binding is conserved between tablysin-15 and Pry1. Pry1 bound fatty acids with micromolar affinity in vitro, and its function was essential for fatty acid export in cells lacking the acyl-CoA synthetases Faa1 and Faa4. Fatty acid binding of Pry1 is independent of its capacity to bind sterols, and the two sterol- and fatty acid-binding sites are nonoverlapping. These results indicate that some CAP family members, such as Pry1, can bind different lipids, particularly sterols and fatty acids, at distinct binding sites, suggesting that the CAP domain may serve as a stable, secreted protein domain that can accommodate multiple ligand-binding sites.

WRINKLED1 and ACYL-COA:DIACYLGLYCEROL ACYLTRANSFERASE1 regulate tocochromanol metabolism in Arabidopsis.

Photosynthetic organisms such as plants, algae and some cyanobacteria synthesize tocochromanols, a group of compounds that encompasses tocopherols and tocotrienols and that exhibits vitamin E activity in animals. While most vitamin E biosynthetic genes have been identified in plant genomes, regulatory genes controlling tocopherol accumulation are currently unknown. We isolated by forward genetics Arabidopsis enhanced vitamin E (eve) mutants that overaccumulate the classic tocopherols and plastochromanol-8, and a tocochromanol unknown in this species. We mapped eve1 and eve4, and identified the unknown Arabidopsis tocochromanol by using a combination of analytical tools. In addition, we determined its biosynthetic pathway with a series of tocochromanol biosynthetic mutants and transgenic lines. eve1 and eve4 are two seed lipid mutants affecting the WRINKLED1 (WRI1) and ACYL-COA:DIACYLGLYCEROL ACYLTRANSFERASE1 (DGAT1) genes, respectively. The unknown tocochromanol is 11'-12' γ-tocomonoenol, whose biosynthesis is VITAMIN E 1 (VTE1) - and VTE2-dependent and is initiated by the condensation of homogentisate (HGA) and tetrahydrogeranylgeranyl pyrophosphate. This study identifies the first two regulatory genes, WRI1 and DGAT1, that control the synthesis of all tocochromanol forms in seeds, and shows the existence of a metabolic trade-off between lipid and tocochromanol metabolisms. Moreover, it shows that Arabidopsis possesses a tocomonoenol biosynthetic pathway that competes with tocopherol synthesis.

An Endosperm-Associated Cuticle Is Required for Arabidopsis Seed Viability, Dormancy and Early Control of Germination.

Cuticular layers and seeds are prominent plant adaptations to terrestrial life that appeared early and late during plant evolution, respectively. The cuticle is a waterproof film covering plant aerial organs preventing excessive water loss and protecting against biotic and abiotic stresses. Cutin, consisting of crosslinked fatty acid monomers, is the most abundant and studied cuticular component. Seeds are dry, metabolically inert structures promoting plant dispersal by keeping the plant embryo in an arrested protected state. In Arabidopsis thaliana seeds, the embryo is surrounded by a single cell endosperm layer itself surrounded by a seed coat layer, the testa. Whole genome analyses lead us to identify cutin biosynthesis genes as regulatory targets of the phytohormones gibberellins (GA) and abscisic acid (ABA) signaling pathways that control seed germination. Cutin-containing layers are present in seed coats of numerous species, including Arabidopsis, where they regulate permeability to outer compounds. However, the role of cutin in mature seed physiology and germination remains poorly understood. Here we identify in mature seeds a thick cuticular film covering the entire outer surface of the endosperm. This seed cuticle is defective in cutin-deficient bodyguard1 seeds, which is associated with alterations in endospermic permeability. Furthermore, mutants affected in cutin biosynthesis display low seed dormancy and viability levels, which correlates with higher levels of seed lipid oxidative stress. Upon seed imbibition cutin biosynthesis genes are essential to prevent endosperm cellular expansion and testa rupture in response to low GA synthesis. Taken together, our findings suggest that in the course of land plant evolution cuticular structures were co-opted to achieve key physiological seed properties.

Induced jasmonate signaling leads to contrasting effects on root damage and herbivore performance.

Induced defenses play a key role in plant resistance against leaf feeders. However, very little is known about the signals that are involved in defending plants against root feeders and how they are influenced by abiotic factors. We investigated these aspects for the interaction between rice (Oryza sativa) and two root-feeding insects: the generalist cucumber beetle (Diabrotica balteata) and the more specialized rice water weevil (Lissorhoptrus oryzophilus). Rice plants responded to root attack by increasing the production of jasmonic acid (JA) and abscisic acid, whereas in contrast to in herbivore-attacked leaves, salicylic acid and ethylene levels remained unchanged. The JA response was decoupled from flooding and remained constant over different soil moisture levels. Exogenous application of methyl JA to the roots markedly decreased the performance of both root herbivores, whereas abscisic acid and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid did not have any effect. JA-deficient antisense 13-lipoxygenase (asLOX) and mutant allene oxide cyclase hebiba plants lost more root biomass under attack from both root herbivores. Surprisingly, herbivore weight gain was decreased markedly in asLOX but not hebiba mutant plants, despite the higher root biomass removal. This effect was correlated with a herbivore-induced reduction of sucrose pools in asLOX roots. Taken together, our experiments show that jasmonates are induced signals that protect rice roots from herbivores under varying abiotic conditions and that boosting jasmonate responses can strongly enhance rice resistance against root pests. Furthermore, we show that a rice 13-lipoxygenase regulates root primary metabolites and specifically improves root herbivore growth.

Yeast Integral Membrane Proteins Apq12, Brl1, and Brr6 Form a Complex Important for Regulation of Membrane Homeostasis and Nuclear Pore Complex Biogenesis.

Proper functioning of intracellular membranes is critical for many cellular processes. A key feature of membranes is their ability to adapt to changes in environmental conditions by adjusting their composition so as to maintain constant biophysical properties, including fluidity and flexibility. Similar changes in the biophysical properties of membranes likely occur when intracellular processes, such as vesicle formation and fusion, require dramatic changes in membrane curvature. Similar modifications must also be made when nuclear pore complexes (NPCs) are constructed within the existing nuclear membrane, as occurs during interphase in all eukaryotes. Here we report on the role of the essential nuclear envelope/endoplasmic reticulum (NE/ER) protein Brl1 in regulating the membrane composition of the NE/ER. We show that Brl1 and two other proteins characterized previously-Brr6, which is closely related to Brl1, and Apq12-function together and are required for lipid homeostasis. All three transmembrane proteins are localized to the NE and can be coprecipitated. As has been shown for mutations affecting Brr6 and Apq12, mutations in Brl1 lead to defects in lipid metabolism, increased sensitivity to drugs that inhibit enzymes involved in lipid synthesis, and strong genetic interactions with mutations affecting lipid metabolism. Mutations affecting Brl1 or Brr6 or the absence of Apq12 leads to hyperfluid membranes, because mutant cells are hypersensitive to agents that increase membrane fluidity. We suggest that the defects in nuclear pore complex biogenesis and mRNA export seen in these mutants are consequences of defects in maintaining the biophysical properties of the NE.

Plastochromanol-8 and tocopherols are essential lipid-soluble antioxidants during seed desiccation and quiescence in Arabidopsis.

Given their essential role as vitamin E, tocopherols and tocotrienols have been studied extensively in animals and plants. In contrast, our understanding of the function of plastochromanol-8 (PC-8), a third type of tocochromanol with a longer side chain, is very limited despite the wide distribution of PC-8 in the plant kingdom, including species consumed by humans. To investigate PC-8 function in vivo, we combined the Arabidopsis vte1 mutation that eliminates tocopherols and PC-8 and causes the accumulation of 2,3-dimethyl-6-phytyl-1,4-benzoquinol (DMPBQ), a redox-active tocopherol precursor, and the vte2 mutation that eliminates tocopherols without affecting PC-8. The vte2 vte1 double mutant lacks tocopherols, PC-8, and DMPBQ, and exhibits the most severe physiological and biochemical phenotypes of any tocochromanol-affected genotype isolated to date, most notably a severe seedling developmental phenotype associated with massive lipid oxidation initiated during seed desiccation and amplified during seed quiescence. In contrast, the presence of PC-8 in vte2 suppresses or attenuates all of the developmental and biochemical phenotypes observed in vte2 vte1, demonstrating that PC-8 is a lipid antioxidant in vivo. Finally, the low relative fitness of vte2 vte1 demonstrates that tocopherols and PC-8 are in vivo lipid antioxidants essential for seed plant survival.

The Arabidopsis mature endosperm promotes seedling cuticle formation via release of sulfated peptides.

In Arabidopsis mature seeds, the onset of the embryo-to-seedling transition is nonautonomously controlled, being blocked by endospermic abscisic acid (ABA) release under unfavorable conditions. Whether the mature endosperm governs additional nonautonomous developmental processes during this transition is unknown. Mature embryos have a more permeable cuticle than seedlings, consistent with their endospermic ABA uptake capability. Seedlings acquire their well-sealing cuticles adapted to aerial lifestyle during germination. Endosperm removal prevents seedling cuticle formation, and seed reconstitution by endosperm grafting onto embryos shows that the endosperm promotes seedling cuticle development. Grafting different endosperm and embryo mutant combinations, together with biochemical, microscopy, and mass spectrometry approaches, reveal that the release of tyrosylprotein sulfotransferase (TPST)-sulfated CIF2 and PSY1 peptides from the endosperm promotes seedling cuticle development. Endosperm-deprived embryos produced nonviable seedlings bearing numerous developmental defects, not related to embryo malnutrition, all restored by exogenously provided endosperm. Hence, seedling establishment is nonautonomous, requiring the mature endosperm.

Contribution of Hydrogen Cyanide to the Antagonistic Activity of Pseudomonas Strains Against Phytophthora infestans.

Plants face many biotic and abiotic challenges in nature; one of them is attack by disease-causing microbes. Phytophthora infestans, the causal agent of late blight is one of the most prominent pathogens of the potato responsible for multi-billion-dollar losses every year. We have previously reported that potato-associated Pseudomonas strains inhibited P. infestans at various developmental stages. A comparative genomics approach identified several factors putatively involved in this anti-oomycete activity, among which was the production of hydrogen cyanide (HCN). Here, we report the relative contribution of HCN emission to the overall anti-Phytophthora activity of two cyanogenic Pseudomonas strains, P. putida R32 and P. chlororaphis R47. To quantify this contribution, we generated HCN-negative mutants (Δhcn) and compared their activities to those of their respective wild types in different experiments assessing P. infestans mycelial growth, zoospore germination, and infection of potato leaf disks. Using in vitro experiments allowing only volatile-mediated interactions, we observed that HCN accounted for most of the mycelial growth inhibition (57% in R47 and 80% in R32). However, when allowing both volatile and diffusible compound-mediated interactions, HCN only accounted for 1% (R47) and 18% (R32) of mycelial growth inhibition. Likewise, both mutants inhibited zoospore germination in a similar way as their respective wild types. More importantly, leaf disk experiments showed that both wild-type and Δhcn strains of R47 and R32 were able to limit P. infestans infection to a similar extent. Our results suggest that while HCN is a major contributor to the in vitro volatile-mediated restriction of P. infestans mycelial growth, it does not play a major role in the inhibition of other disease-related features such as zoospore germination or infection of plant tissues.

Efficiency of natural substances to protect Beauveria bassiana conidia from UV radiation.

BACKGROUND: Solar radiation is assumed to be a major factor limiting the efficacy of entomopathogenic fungi used as biocontrol agents in open field applications. We evaluated 12 natural UV-protective co-formulants for their effect on the survival of UV-exposed Beauveria bassiana spores on agar plates, colza leaf discs and in the field. RESULTS: Colony-forming unit (CFU) counts of unformulated conidia on agar plates and leaf discs dropped to ≤ 50% after exposure to UV radiation. The highest UV protection was achieved with humic acid, which provided > 90% protection of UV-B-exposed conidia in laboratory experiments. In the field, 10% humic acid increased spore persistence up to 87% at 7 days after application. Sesame and colza oil also provided high UV protection in both assays (> 73% and > 70%, respectively). CONCLUSIONS: This study shows that it is possible to increase the persistence of B. bassiana spores under exposure to UV radiation by formulation with natural UV-protective additives. UV protectants might, therefore, increase the efficacy of entomopathogenic fungi as biocontrol agents in open field applications. © 2018 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Valuable carcasses: postmortem preservation of fatty acid composition in heart tissue.

In order to effectively conserve species, we must understand the structure and function of integral mechanisms at all levels of organismal organisation, from intracellular biochemistry to whole animal ecophysiology. The accuracy of biochemical analyses depend on the quality and integrity of the samples analysed. It is believed that tissue samples collected immediately postmortem provide the most reliable depiction of the living animal. Yet, euthanasia of threatened or protected species for the collection of tissue presents a number of ethical complications. Polyunsaturated fatty acids (PUFA) are essential to the cardiovascular system of all animals and the structure of PUFA can be degraded by peroxidation, potentially modifying the fatty acid composition of the tissue over postmortem time. Here, we assessed the composition of PUFA in cardiac tissue of bats (Carollia perspicillata) over the course of 12-h postmortem. We show that PUFA are resistant to naturally occurring postmortem degradation in heart tissue, with no difference in the overall composition of fatty acids across all time classes (0, 3, 6 or 12-h postmortem). Our results suggest that carcasses that would otherwise be discarded may actually be viable for the assessment of fatty acid composition in a number of tissues. We hope to spur further investigations into the viability of carcasses for other biochemical analyses as they may be an untapped resource available to biologists.

A sulfur-containing volatile emitted by potato-associated bacteria confers protection against late blight through direct anti-oomycete activity.

Plant diseases are a major cause for yield losses and new strategies to control them without harming the environment are urgently needed. Plant-associated bacteria contribute to their host's health in diverse ways, among which the emission of disease-inhibiting volatile organic compounds (VOCs). We have previously reported that VOCs emitted by potato-associated bacteria caused strong in vitro growth inhibition of the late blight causing agent Phytophthora infestans. This work focuses on sulfur-containing VOCs (sVOCs) and demonstrates the high in planta protective potential of S-methyl methane thiosulfonate (MMTS), which fully prevented late blight disease in potato leaves and plantlets without phytotoxic effects, in contrast to other sVOCs. Short exposure times were sufficient to protect plants against infection. We further showed that MMTS's protective activity was not mediated by the plant immune system but lied in its anti-oomycete activity. Using quantitative proteomics, we determined that different sVOCs caused specific proteome changes in P. infestans, indicating perturbations in sulfur metabolism, protein translation and redox balance. This work brings new perspectives for plant protection against the devastating Irish Famine pathogen, while opening new research avenues on the role of sVOCs in the interaction between plants and their microbiome.

Vitamin E Biosynthesis and Its Regulation in Plants.

Vitamin E is one of the 13 vitamins that are essential to animals that do not produce them. To date, six natural organic compounds belonging to the chemical family of tocochromanols-four tocopherols and two tocotrienols-have been demonstrated as exhibiting vitamin E activity in animals. Edible plant-derived products, notably seed oils, are the main sources of vitamin E in the human diet. Although this vitamin is readily available, independent nutritional surveys have shown that human populations do not consume enough vitamin E, and suffer from mild to severe deficiency. Tocochromanols are mostly produced by plants, algae, and some cyanobacteria. Tocochromanol metabolism has been mainly studied in higher plants that produce tocopherols, tocotrienols, plastochromanol-8, and tocomonoenols. In contrast to the tocochromanol biosynthetic pathways that are well characterized, our understanding of the physiological and molecular mechanisms regulating tocochromanol biosynthesis is in its infancy. Although it is known that tocochromanol biosynthesis is strongly conditioned by the availability in homogentisate and polyprenyl pyrophosphate, its polar and lipophilic biosynthetic precursors, respectively, the mechanisms regulating their biosyntheses are barely known. This review summarizes our current knowledge of tocochromanol biosynthesis in plants, and highlights future challenges regarding the understanding of its regulation.

Current strategies for vitamin E biofortification of crops.

Vitamin E refers to four tocopherols and four tocotrienols that are exclusively synthesized by photosynthetic organisms. While α-tocopherol is the most potent vitamin E compound, it is not the main form consumed since the composition of most major crops is dominated by γ-tocopherol. Nutritional studies show that populations of developed countries do not consume enough vitamin E and that a large proportion of individuals exhibit plasma α-tocopherol deficiency. Following the identification of vitamin E biosynthetic genes, several strategies including metabolic engineering, classic breeding and mutation breeding, have been undertaken to improve the vitamin E content of crops. In addition to providing crops in which vitamin E content is enhanced, these studies are revealing the bottlenecks limiting its biosynthesis.

Metabolic Origins and Transport of Vitamin E Biosynthetic Precursors.

Tocochromanols are organic compounds mostly produced by photosynthetic organisms that exhibit vitamin E activity in animals. They result from the condensation of homogentisate with four different polyprenyl side chains derived all from geranylgeranyl pyrophosphate. The core tocochromanol biosynthesis has been investigated in several photosynthetic organisms and is now well-characterized. In contrast, our current knowledge of the biosynthesis and transport of tocochromanol biosynthetic precursors is much more limited. While tocochromanol synthesis occurs in plastids, converging genetic data in Arabidopsis and soybean demonstrate that the synthesis of the polar precursor homogentisate is located in the cytoplasm. These data implies that tocochromanol synthesis involves several plastidic membrane transporter(s) that remain to be identified. In addition, the metabolic origin of the lipophilic isoprenoid precursor is not fully elucidated. While some genetic data exclusively attribute the synthesis of the prenyl component of tocochromanols to the plastidic methyl erythritol phosphate pathway, multiple lines of evidence provided by feeding experiments and metabolic engineering studies indicate that it might partially originate from the cytoplasmic mevalonate pathway. Although this question is still open, these data demonstrate the existence of membrane transporter(s) capable of importing cytosolic polyprenyl pyrophosphate such as farnesyl pyrophosphate into plastids. Since the availability of both homogentisate and polyprenyl pyrophosphates are currently accepted as major mechanisms controlling the type and amount of tocochromanols produced in plant tissues, we summarized our current knowledge and research gaps concerning the biosynthesis, metabolic origins and transport of tocochromanol biosynthetic precursors in plant cells.

Biosynthesis, regulation and functions of tocochromanols in plants.

Tocopherols and tocotrienols have been originally identified as essential nutrients in mammals based on their vitamin E activity. These lipid-soluble compounds are potent antioxidants that protect polyunsaturated fatty acids from lipid peroxidation. The biosynthesis of tocopherols and tocotrienols occurs exclusively in photosynthetic organisms. The biosynthetic precursors and the different pathway intermediates have been identified by biochemical studies and the different vitamin E biosynthetic genes (VTE genes) have been isolated in several plants and cyanobacteria. The characterization of transgenic plants overexpressing one or multiple VTE genes combined with the study of vitamin E deficient mutants allows from now on understanding the regulation and the function of tocopherols and tocotrienols in plants.

Nonenzymatic oxidation of trienoic fatty acids contributes to reactive oxygen species management in Arabidopsis.

In higher plants such as Arabidopsis thaliana, omega-3 trienoic fatty acids (TFAs), represented mainly by alpha-linolenic acid, serve as precursors of jasmonic acid (JA), a potent lipid signal molecule essential for defense. The JA-independent roles of TFAs were investigated by comparing the TFA- and JA-deficient fatty acid desaturase triple mutant (fad3-2 fad7-2 fad8 (fad3 fad7 fad8)) with the aos (allene oxide synthase) mutant that contains TFAs but is JA-deficient. When challenged with the fungus Botrytis, resistance of the fad3 fad7 fad8 mutant was reduced when compared with the aos mutant, suggesting that TFAs play a role in cell survival independently of being the precursors of JA. An independent genetic approach using the lesion mimic mutant accelerated cell death2 (acd2-2) confirmed the importance of TFAs in containing lesion spread, which was increased in the lines in which the fad3 fad7 fad8 and acd2-2 mutations were combined when compared with the aos acd2-2 lines. Malondialdehyde, found to result from oxidative TFA fragmentation during lesion formation, was measured by gas chromatography-mass spectrometry. Its levels correlated with the survival of the tissue. Furthermore, plants lacking TFAs overproduced salicylic acid (SA), hydrogen peroxide, and transcripts encoding several SA-regulated and SA biosynthetic proteins. The data suggest a physiological role for TFAs as sinks for reactive oxygen species.

Genetic removal of tri-unsaturated fatty acids suppresses developmental and molecular phenotypes of an Arabidopsis tocopherol-deficient mutant. Whole-body mapping of malondialdehyde pools in a complex eukaryote.

Malondialdehyde (MDA) is a small, ubiquitous, and potentially toxic aldehyde that is produced in vivo by lipid oxidation and that is able to affect gene expression. Tocopherol deficiency in the vitamin E2 mutant vte2-1 of Arabidopsis thaliana leads to massive lipid oxidation and MDA accumulation shortly after germination. MDA accumulation correlates with a strong visual phenotype (growth reduction, cotyledon bleaching) and aberrant GST1 (glutathione S-transferase 1) expression. We suppressed MDA accumulation in the vte2-1 background by genetically removing tri-unsaturated fatty acids. The resulting quadruple mutant, fad3-2 fad7-2 fad8 vte2-1, did not display the visual phenotype or the aberrant GST1 expression observed in vte2-1. Moreover, cotyledon bleaching in vte2-1 was chemically phenocopied by treatment of wild-type plants with MDA. These data suggest that products of tri-unsaturated fatty acid oxidation underlie the vte2-1 seedling phenotype, including cellular toxicity and gene regulation properties. Generation of the quadruple mutant facilitated the development of an in situ fluorescence assay based on the formation of adducts of MDA with 2-thiobarbituric acid at 37 degrees C. Specificity was verified by measuring pentafluorophenylhydrazine derivatives of MDA and by liquid chromatography analysis of MDA-2-thiobarbituric acid adducts. Potentially applicable to other organisms, this method allowed the localization of MDA pools throughout the body of Arabidopsis and revealed an undiscovered pool of the compound unlikely to be derived from trienoic fatty acids in the vicinity of the root tip quiescent center.

Characterization of a divinyl ether biosynthetic pathway specifically associated with pathogenesis in tobacco.

In tobacco (Nicotiana tabacum), an elicitor- and pathogen-induced 9-lipoxygenase (LOX) gene, NtLOX1, is essential for full resistance to pathogens, notably to an incompatible race of Phytophthora parasitica var. nicotianae (Ppn race 0). In this work, we aimed to identify those oxylipins induced during attempted infection by Ppn race 0 and down-regulated in NtLOX1 antisense plants. Here we show that colneleic and colnelenic acids, which significantly inhibit germination of Ppn zoospores, are produced in roots of wild-type plants inoculated with Ppn, but are down-regulated in NtLOX1 antisense plants. A search for a tobacco gene encoding the enzyme involved in the formation of these divinyl ether (DVE) fatty acids resulted in the cloning and characterization of a DVE synthase (DES) clone (NtDES1). NtDES1 is a 9-DES, specifically converting fatty acid 9-hydroperoxides into DVE fatty acids. NtDES1 has the potential to act in combination with NtLOX1 because, in the presence of the two enzymes, linoleic and linolenic acids were converted in vitro into colneleic and colnelenic acids, respectively. In addition, the pattern of NtDES1 gene expression was quite similar to that of NtLOX1. Their transcripts were undetected in healthy tissues from different plant organs, and accumulated locally and transiently after elicitation and fungal infection, but not after wounding. Visualization of NtDES1-yellow fluorescent protein and NtLOX1-cyan fluorescent protein fusion proteins in tobacco leaves indicated that both localize in the cytosol and are excluded from plastids, consistent with the presumed location of the 9-LOX pathway in plants and the lack of transit peptides for NtLOX1 and NtDES1, respectively. Our data suggest that, in tobacco, NtDES1 and NtLOX1 act together and form DVEs in response to pathogen attack and that this class of oxylipins modulates in vivo the outcome of the tobacco-Ppn race 0 interaction.