Michel Matringe

Engineering Plant Shikimate Pathway for Production of Tocotrienol and Improving Herbicide Resistance

Tocochromanols (tocopherols and tocotrienols), collectively known as vitamin E, are essential antioxidant components of both human and animal diets. Because of their potential health benefits, there is a considerable interest in plants with increased or customized vitamin E content. Here, we have explored a new strategy to reach this goal. In plants, phenylalanine is the precursor of a myriad of secondary compounds termed phenylpropanoids. In contrast, much less carbon is incorporated into tyrosine that provides p-hydroxyphenylpyruvate and homogentisate, the aromatic precursors of vitamin E. Therefore, we intended to increase the flux of these two compounds by deriving their synthesis directly at the level of prephenate. This was achieved by the expression of the yeast (Saccharomyces cerevisiae) prephenate dehydrogenase gene in tobacco (Nicotiana tabacum) plants that already overexpress the Arabidopsis p-hydroxyphenylpyruvate dioxygenase coding sequence. A massive accumulation of tocotrienols was observed in leaves. These molecules, which were undetectable in wild-type leaves, became the major forms of vitamin E in the leaves of the transgenic lines. An increased resistance of the transgenic plants toward the herbicidal p-hydroxyphenylpyruvate dioxygenase inhibitor diketonitril was also observed. This work demonstrates that the synthesis of p-hydroxyphenylpyruvate is a limiting step for the accumulation of vitamin E in plants.

Tyrosine and Phenylalanine Are Synthesized within the Plastids in Arabidopsis

While the presence of a complete shikimate pathway within plant plastids is definitively established, the existence of a cytosolic postchorismate portion of the pathway is still debated. This question is alimented by the presence of a chorismate mutase (CM) within the cytosol. Until now, the only known destiny of prephenate, the product of CM, is incorporation into tyrosine (Tyr) and/or phenylalanine (Phe). Therefore, the presence of a cytosolic CM suggests that enzymes involved downstream of CM in Tyr or Phe biosynthesis could be present within the cytosol of plant cells. It was thus of particular interest to clarify the subcellular localization of arogenate dehydrogenases (TYRAs) and arogenate dehydratases (ADTs), which catalyze the ultimate steps in Tyr and Phe biosynthesis, respectively. The aim of this study was to address this question in Arabidopsis (Arabidopsis thaliana) by analysis of the subcellular localization of the two TYRAAts and the six AtADTs. This article excludes the occurrence of a spliced TYRAAt1 transcript encoding a cytosolic TYRA protein. Transient expression analyses of TYRA- and ADT-green fluorescent protein fusions reveal that the two Arabidopsis TYRA proteins and the six ADT proteins are all targeted within the plastid. Accordingly, TYRA and ADT proteins were both immunodetected in the chloroplast soluble protein fraction (stroma) of Arabidopsis. No TYRA or ADT proteins were immunodetected in the cytosol of Arabidopsis cells. Taken together, all our data exclude the possibility of Tyr and/or Phe synthesis within the cytosol, at least in green leaves and Arabidopsis cultured cells.

Protoporphyrinogen oxidase inhibition by three peroxidizing herbicides: Oxadiazon, LS 82-556 and M&B 39279

Three chemically unrelated peroxidizing molecules, namely oxadiazon [5‐(t‐butyl)‐3‐(2,4‐dichloro‐5‐isopropoxyphenyl)‐1,3,4‐oxadiazol‐2‐one], LS 82‐556 [(S)3‐N‐(methylbenzyl)carbamoyl‐5‐propionyl‐2,6‐lutidine] and M&B 39279 [5‐amino‐4‐cyano‐1‐(2,6‐dichloro‐4‐trifluoromethylphenyl)pyrazol], are potent inhibitors of plant, yeast and mouse protoporphyrinogen oxidase.

Effects of acifluorfen-methyl on cucumber cotyledons: Porphyrin accumulation☆

Abstract The nitrodiphenyl ether herbicide acifluorfen-methyl and the pyridine derivative LS 82-556 induce porphyrin accumulation in green cucumber cotyledons. When experiments are done with intact plants absorbing the herbicide through the roots, that accumulation is light-dependent. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU) which prevents cellular damages under these conditions (M. Matringe and R. Scalla, Pestic. Biochem. Physiol. 26 , 150 (1986), also inhibits porphyrin accumulation. In contrast, when detached cotyledons are cut into pieces and floated on herbicide solutions, porphyrins accumulate in the dark. Accordingly, DCMU does not inhibit porphyrin accumulation or protect the tissues against herbicidal effects. Furthermore, 4,6-dioxoheptanoic acid, and inhibitor of tetrapyrrole biosynthesis, prevents both porphyrin accumulation and phytotoxic effects. The same results can be obtained with norflurazon, an inhibitor of carotenoid biosynthesis; in this case, inhibition of porphyrin biosynthesis is probably a secondary consequence of abnormal development of chloroplasts. These results indicate that the herbicidal activity of diphenyl ether-type herbicides probably results from their ability to interfere with the metabolism of tetrapyrroles.

Tocotrienols, the Unsaturated Forms of Vitamin E, Can Function as Antioxidants and Lipid Protectors in Tobacco Leaves

Vitamin E is a generic term for a group of lipid-soluble antioxidant compounds, the tocopherols and tocotrienols. While tocotrienols are considered as important vitamin E components in humans, with functions in health and disease, the protective functions of tocotrienols have never been investigated in plants, contrary to tocopherols. We took advantage of the strong accumulation of tocotrienols in leaves of double transgenic tobacco (Nicotiana tabacum) plants that coexpressed the yeast (Saccharomyces cerevisiae) prephenate dehydrogenase gene (PDH) and the Arabidopsis (Arabidopsis thaliana) hydroxyphenylpyruvate dioxygenase gene (HPPD) to study the antioxidant function of those compounds in vivo. In young leaves of wild-type and transgenic tobacco plants, the majority of vitamin E was stored in thylakoid membranes, while plastoglobules contained mainly δ-tocopherol, a very minor component of vitamin E in tobacco. However, the vitamin E composition of plastoglobules was observed to change substantially during leaf aging, with α-tocopherol becoming the major form. Tocotrienol accumulation in young transgenic HPPD-PDH leaves occurred without any significant perturbation of photosynthetic electron transport. Tocotrienols noticeably reinforced the tolerance of HPPD-PDH leaves to high light stress at chilling temperature, with photosystem II photoinhibition and lipid peroxidation being maintained at low levels relative to wild-type leaves. Very young leaves of wild-type tobacco plants turned yellow during chilling stress, because of the strongly reduced levels of chlorophylls and carotenoids, and this phenomenon was attenuated in transgenic HPPD-PDH plants. While sugars accumulated similarly in young wild-type and HPPD-PDH leaves exposed to chilling stress in high light, a substantial decrease in tocotrienols was observed in the transgenic leaves only, suggesting vitamin E consumption during oxygen radical scavenging. Our results demonstrate that tocotrienols can function in vivo as efficient antioxidants protecting membrane lipids from peroxidation.

The Biosynthetic Capacities of the Plastids and Integration Between Cytoplasmic and Chloroplast Processes

Plastids are semiautonomous organelles derived from cyanobacterial ancestors. Following endosymbiosis, plastids have evolved to optimize their functions, thereby limiting metabolic redundancy with other cell compartments. Contemporary plastids have also recruited proteins produced by the nuclear genome of the host cell. In addition, many genes acquired from the cyanobacterial ancestor evolved to code for proteins that are targeted to cell compartments other than the plastid. Consequently, metabolic pathways are now a patchwork of enzymes of diverse origins, located in various cell compartments. Because of this, a wide range of metabolites and ions traffic between the plastids and other cell compartments. In this review, we provide a comprehensive analysis of the well-known, and of the as yet uncharacterized, chloroplast/cytosol exchange processes, which can be deduced from what is currently known about compartmentation of plant-cell metabolism.

Identification of a plant gene encoding glutamate/aspartate-prephenate aminotransferase: the last homeless enzyme of aromatic amino acids biosynthesis.

In all organisms synthesising phenylalanine and/or tyrosine via arogenate, a prephenate aminotransferase is required for the transamination of prephenate into arogenate. The identity of the gene encoding this enzyme in the organisms where this activity occurs is still unknown. Glutamate/aspartate‐prephenate aminotransferase (PAT) is thus the last homeless enzyme in the aromatic amino acids pathway. We report on the purification, mass spectrometry identification and biochemical characterization of Arabidopsis thaliana prephenate aminotransferase. Our data revealed that this activity is housed by the prokaryotic‐type plastidic aspartate aminotransferase (At2g22250). This represents the first identification of a gene encoding PAT.

Competitive interaction of three peroxidizing herbicides with the binding of [3H]acifluorfen to corn etioplast membranes

The specific binding of the herbicide acifluorfen 5‐[2‐chloro‐4‐(trifluoromethyl)phenoxy]‐2‐nitrobenzoic acid to corn etioplast membranes is competitively inhibited by protoporphyrinogen IX, the substrate of protoporphyrinogen oxidase. Three other peroxidizing molecules, oxadiazon [5‐ter‐butyl‐3‐(2,4‐dichloro‐5‐isopropoxyphenyl)‐1,3,4‐oxadiazol‐2‐one], LS 82556 [(S)3‐N‐(methylbenzyl)carbamoyl‐5‐propionyl‐2,6‐lutidine], and M&B 39279 [5‐amino‐4‐cyano‐1‐(2,6‐dichloro‐4‐trifluoromethylphenyl)pyrazol], also compete with acifluorfen for its binding site. The four herbicides thus bind to the same site, or to closely located sites, on the enzyme protoporphyrinogen oxidase.

4-Hydroxyphenylpyruvate Dioxygenase Catalysis IDENTIFICATION OF CATALYTIC RESIDUES AND PRODUCTION OF A HYDROXYLATED INTERMEDIATE SHARED WITH A STRUCTURALLY UNRELATED ENZYME

4-Hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the conversion of 4-hydroxyphenylpyruvate (HPP) into homogentisate. HPPD is the molecular target of very effective synthetic herbicides. HPPD inhibitors may also be useful in treating life-threatening tyrosinemia type I and are currently in trials for treatment of Parkinson disease. The reaction mechanism of this key enzyme in both plants and animals has not yet been fully elucidated. In this study, using site-directed mutagenesis supported by quantum mechanical/molecular mechanical theoretical calculations, we investigated the role of catalytic residues potentially interacting with the substrate/intermediates. These results highlight the following: (i) the central role of Gln-272, Gln-286, and Gln-358 in HPP binding and the first nucleophilic attack; (ii) the important movement of the aromatic ring of HPP during the reaction, and (iii) the key role played by Asn-261 and Ser-246 in C1 hydroxylation and the final ortho-rearrangement steps (numbering according to the Arabidopsis HPPD crystal structure 1SQD). Furthermore, this study reveals that the last step of the catalytic reaction, the 1,2 shift of the acetate side chain, which was believed to be unique to the HPPD activity, is also catalyzed by a structurally unrelated enzyme.

Effects of peroxidizing herbicides on protoporphyrin IX levels in non-chlorophyllous soybean cell culture

Abstract The mode of action of 16 peroxidizing herbicides belonging to four different families (diphenyl ethers, oxadiazon, pyridine derivatives, and pyrazole derivatives) has been studied in nonchlorophyllous soybean cell cultures. Whenever possible, we have compared active and inactive compounds. Phytotoxic effects were estimated on the basis of growth inhibition, either in the dark or in the light. Protoporphyrin IX accumulations were estimated in dark-treated samples, using a simple methodology. In all cases, we have found a positive correlation between cellular damages and protoporphyrin IX accumulations. The results provide further evidences in favor of the light-dependent activity of peroxidizing herbicides being due to their capacity to induce protoporphyrin IX accumulation.