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See detailUnveiling two new trichome-specific promoters of interest for metabolic engineering in Nicotiana tabacum
Pottier, Mathieu ULiege; Laterre, Raphaëlle; Van Wessem, Astrid et al

E-print/Working paper (2019)

Nicotiana tabacum has emerged as a suitable host for metabolic engineering of terpenoids and derivatives in tall glandular trichomes, which actively synthesize and secrete specialized metabolites. However ... [more ▼]

Nicotiana tabacum has emerged as a suitable host for metabolic engineering of terpenoids and derivatives in tall glandular trichomes, which actively synthesize and secrete specialized metabolites. However, implementation of an entire biosynthetic pathway in glandular trichomes requires the identification of trichome-specific promoters to appropriately drive the expression of the transgenes needed to set up the desired pathway. In this context, RT-qPCR analysis was carried out on wild-type N. tabacum plants to compare the expression pattern and level of NtRbS-T1 and NtMALD1, two newly identified genes expressed in glandular trichomes, with those of NtCYP71D16, NtCBTS2α, NtCPS2, NtLTP1, which were reported in the literature to be specifically expressed in glandular trichomes. The latter were previously investigated separately, preventing any accurate comparison of their expression level. We show that NtRbcS-T1 and NtMALD1 are specifically expressed in glandular trichomes just like NtCYP71D16, NtCBTS2α, NtCPS2, while NtLTP1 was also expressed in other leaf tissues as well as in the stem. Transcriptional fusions of all six promoters to the GUS-VENUS reporter gene were introduced in N. tabacum by Agrobacterium-mediated transformation. Most transgenic lines displayed GUS activity in tall glandular trichomes. In some transgenic lines, except for pNtLTP1:GUS-VENUS, this expression was specific. In other transgenic lines, GUS expression was extended to other tissues, probably resulting from a position effect during transgene integration. We discuss alternatives to overcome this lack of tissue specificity in some transgenic lines, should some of these promoters be used in the context of metabolic engineering in N. tabacum. [less ▲]

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See detailAutophagy is essential for optimal translocation for iron to seeds in Arabidopsis
Pottier, Mathieu ULiege; Dumont, Jean; Masclaux-Daubresse, Céline et al

in Journal of Experimental Botany (2019), 70(3), 859-869

Micronutrient deficiencies affect a large part of the world population. They are mostly due to the consumption of grains with insufficient content of Fe or Zn. It is therefore important to improve our ... [more ▼]

Micronutrient deficiencies affect a large part of the world population. They are mostly due to the consumption of grains with insufficient content of Fe or Zn. It is therefore important to improve our knowledge of the mechanisms of micronutrient loading to seeds. Nutrient loaded in seeds originate either from de novo uptake by roots or recycling from leaves. Autophagy is a conserved mechanism for nutrient recycling in eukaryotes and was shown to be involved in nitrogen remobilization to seeds. Measuring the distribution of metal nutrients at the end of the life in Arabidopsis thaliana plants impaired in autophagy, we have investigated the role of autophagy in metal micronutrient translocation to seeds. We found that several Arabidopsis genotypes impaired in autophagy display defects in nutrient remobilization to seeds. In atg5-1, which is completely defective in autophagy, the efficiency of Fe translocation from vegetative organs to seeds was severely decreased even when Fe was provided during seed formation. Combining atg5-1 with sid2 mutation that counteracts premature senescence associated to autophagy deficiency and using 57Fe pulse labelling, we could propose a two step mechanism in which iron taken up de novo during seed formation is first accumulated in vegetative organs and subsequently remobilized to seeds. Finally, we showed that translocations of zinc and manganese to seeds are also dependent on autophagy. Our results highlight the importance of autophagy for optimal micronutrient remobilization to seeds. Fine tuning autophagy during seed formation opens new possibilities to improve this trait. [less ▲]

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See detailThe Nicotiana tabacum ABC transporter NtPDR3 secretes O-methylated coumarins in response to iron deficiency
Lefèvre, François; Fourmeau, Justine; Pottier, Mathieu ULiege et al

in Journal of Experimental Botany (2018), 69(18), 4419-4431

Although iron is present in large amounts in the soil, its poor solubility means that plants have to use various strategies to facilitate its uptake. In this study, we show that expression of NtPDR3 ... [more ▼]

Although iron is present in large amounts in the soil, its poor solubility means that plants have to use various strategies to facilitate its uptake. In this study, we show that expression of NtPDR3/NtABCG3, a Nicotiana tabacum plasma-membrane ABC transporter in the pleiotropic drug resistance (PDR) subfamily, is strongly induced in the root epidermis under iron deficiency conditions. Prevention of NtPDR3 expression resulted in N. tabacum plants that were less tolerant to iron-deficient conditions, displaying stronger chlorosis and slower growth than those of the wildtype when not supplied with iron. Metabolic profiling of roots and root exudates revealed that, upon iron deficiency, secretion of catechol-bearing O-methylated coumarins such as fraxetin, hydroxyfraxetin, and methoxyfraxetin to the rhizosphere was compromised in NtPDR3-silenced plants. However, exudation of flavins such as riboflavin was not markedly affected by NtPDR3-silencing. Expression of NtPDR3 in N. tabacum Bright Yellow-2 (BY-2) cells resulted in altered intra- and extracellular coumarin pools, supporting coumarin transport by this transporter. The results demonstrate that N. tabacum secretes both coumarins and flavins in response to iron deficiency and that NtPDR3 plays an essential role in the plant response to iron deficiency by mediating secretion of O-methylated coumarins to the rhizosphere. [less ▲]

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See detailThe Hidden Face of Rubisco
Pottier, Mathieu ULiege; Gilis, Dimitri; Boutry, Marc

in Trends in Plant Science (2018), 23(5), 382-392

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) fixes atmospheric CO2 into organic compounds and is composed of eight copies each of a large subunit (RbcL) and a small subunit (RbcS). Recent ... [more ▼]

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) fixes atmospheric CO2 into organic compounds and is composed of eight copies each of a large subunit (RbcL) and a small subunit (RbcS). Recent reports have revealed unusual RbcS, which are expressed in particular tissues and confer higher catalytic rate, lesser affinity for CO2, and a more acidic profile of the activity versus pH. The resulting Rubisco was proposed to be adapted to a high CO2 environment and recycle CO2 generated by the metabolism. These RbcS belong to a cluster named T (for trichome), phylogenetically distant from cluster M, which gathers well-characterized RbcS expressed in mesophyll or bundle-sheath tissues. Cluster T is largely represented in different plant phyla, including pteridophytes and bryophytes, indicating an ancient origin. [less ▲]

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See detailPhotosynthetic Trichomes Contain a Specific Rubisco with a Modified pH-Dependent Activity.
Laterre, Raphaelle; Pottier, Mathieu ULiege; Remacle, Claire ULiege et al

in Plant Physiology (2017), 173(4), 2110-2120

Ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) is the most abundant enzyme in plants and is responsible for CO2 fixation during photosynthesis. This enzyme is assembled from eight large subunits ... [more ▼]

Ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) is the most abundant enzyme in plants and is responsible for CO2 fixation during photosynthesis. This enzyme is assembled from eight large subunits (RbcL) encoded by a single chloroplast gene and eight small subunits (RbcS) encoded by a nuclear gene family. Rubisco is primarily found in the chloroplasts of mesophyll (C3 plants), bundle-sheath (C4 plants), and guard cells. In certain species, photosynthesis also takes place in the secretory cells of glandular trichomes, which are epidermal outgrowths (hairs) involved in the secretion of specialized metabolites. However, photosynthesis and, in particular, Rubisco have not been characterized in trichomes. Here, we show that tobacco (Nicotiana tabacum) trichomes contain a specific Rubisco small subunit, NtRbcS-T, which belongs to an uncharacterized phylogenetic cluster (T). This cluster contains RbcS from at least 33 species, including monocots, many of which are known to possess glandular trichomes. Cluster T is distinct from the cluster M, which includes the abundant, functionally characterized RbcS isoforms expressed in mesophyll or bundle-sheath cells. Expression of NtRbcS-T in Chlamydomonas reinhardtii and purification of the full Rubisco complex showed that this isoform conferred higher Vmax and Km values as well as higher acidic pH-dependent activity than NtRbcS-M, an isoform expressed in the mesophyll. This observation was confirmed with trichome extracts. These data show that an ancient divergence allowed for the emergence of a so-far-uncharacterized RbcS cluster. We propose that secretory trichomes have a particular Rubisco uniquely adapted to secretory cells where CO2 is released by the active specialized metabolism. [less ▲]

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See detailTransient production of artemisinin in Nicotiana benthamiana is boosted by a specific lipid transfer protein from A. annua.
Wang, Bo; Kashkooli, Arman Beyraghdar; Sallets, Adrienne et al

in Metabolic Engineering (2016), 38

Our lack of full understanding of transport and sequestration of the heterologous products currently limit metabolic engineering in plants for the production of high value terpenes. For instance, although ... [more ▼]

Our lack of full understanding of transport and sequestration of the heterologous products currently limit metabolic engineering in plants for the production of high value terpenes. For instance, although all genes of the artemisinin/arteannuin B (AN/AB) biosynthesis pathway (AN-PW) from Artemisia annua have been identified, ectopic expression of these genes in Nicotiana benthamiana yielded mostly glycosylated pathway intermediates and only very little free (dihydro)artemisinic acid [(DH)AA]. Here we demonstrate that Lipid Transfer Protein 3 (AaLTP3) and the transporter Pleiotropic Drug Resistance 2 (AaPDR2) from A. annua enhance accumulation of (DH)AA in the apoplast of N. benthamiana leaves. Analysis of apoplast and cell content and apoplast exclusion assays show that AaLTP3 and AaPDR2 prevent reflux of (DH)AA from the apoplast back into the cells and enhances overall flux through the pathway. Moreover, AaLTP3 is stabilized in the presence of AN-PW activity and co-expression of AN-PW+AaLTP3+AaPDR2 genes yielded AN and AB in necrotic N. benthamiana leaves at 13 days post-agroinfiltration. This newly discovered function of LTPs opens up new possibilities for the engineering of biosynthesis pathways of high value terpenes in heterologous expression systems. [less ▲]

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See detailIdentification of mutations allowing Natural Resistance Associated Macrophage Proteins (NRAMP) to discriminate against cadmium.
Pottier, Mathieu ULiege; Oomen, Ronald; Picco, Cristiana et al

in the plant journal (2015), 83(4), 625-37

Each essential transition metal plays a specific role in metabolic processes and has to be selectively transported. Living organisms need to discriminate between essential and non-essential metals such as ... [more ▼]

Each essential transition metal plays a specific role in metabolic processes and has to be selectively transported. Living organisms need to discriminate between essential and non-essential metals such as cadmium (Cd(2+) ), which is highly toxic. However, transporters of the natural resistance-associated macrophage protein (NRAMP) family, which are involved in metal uptake and homeostasis, generally display poor selectivity towards divalent metal cations. In the present study we used a unique combination of yeast-based selection, electrophysiology on Xenopus oocytes and plant phenotyping to identify and characterize mutations that allow plant and mammalian NRAMP transporters to discriminate between their metal substrates. We took advantage of the increased Cd(2+) sensitivity of yeast expressing AtNRAMP4 to select mutations that decrease Cd(2+) sensitivity while maintaining the ability of AtNRAMP4 to transport Fe(2+) in a population of randomly mutagenized AtNRAMP4 cDNAs. The selection identified mutations in three residues. Among the selected mutations, several affect Zn(2+) transport, whereas only one, E401K, impairs Mn(2+) transport by AtNRAMP4. Introduction of the mutation F413I, located in a highly conserved domain, into the mammalian DMT1 transporter indicated that the importance of this residue in metal selectivity is conserved among NRAMP transporters from plant and animal kingdoms. Analyses of overexpressing plants showed that AtNRAMP4 affects the accumulation of metals in roots. Interestingly, the mutations selectively modify Cd(2+) and Zn(2+) accumulation without affecting Fe transport mediated by NRAMP4 in planta. This knowledge may be applicable for limiting Cd(2+) transport by other NRAMP transporters from animals or plants. [less ▲]

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See detailGenotypic variations in the dynamics of metal concentrations in poplar leaves: a field study with a perspective on phytoremediation.
Pottier, Mathieu ULiege; Garcia de la Torre, Vanesa S.; Victor, Cindy et al

in Environmental Pollution (2015), 199

Poplar is commonly used for phytoremediation of metal polluted soils. However, the high concentrations of trace elements present in leaves may return to soil upon leaf abscission. To investigate the ... [more ▼]

Poplar is commonly used for phytoremediation of metal polluted soils. However, the high concentrations of trace elements present in leaves may return to soil upon leaf abscission. To investigate the mechanisms controlling leaf metal content, metal concentrations and expression levels of genes involved in metal transport were monitored at different developmental stages on leaves from different poplar genotypes growing on a contaminated field. Large differences in leaf metal concentrations were observed among genotypes. Whereas Mg was remobilized during senescence, Zn and Cd accumulation continued until leaf abscission in all genotypes. A positive correlation between Natural Resistance Associated Macrophage Protein 1 (NRAMP1) expression levels and Zn bio-concentration factors was observed. Principal component analyses of metal concentrations and gene expression levels clearly discriminated poplar genotypes. This study highlights a general absence of trace element remobilization from poplar leaves despite genotype specificities in the control of leaf metal homeostasis. [less ▲]

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See detailAutophagy as a possible mechanism for micronutrient remobilization from leaves to seeds.
Pottier, Mathieu ULiege; Masclaux-Daubresse, Celine; Yoshimoto, Kohki et al

in Frontiers in Plant Science (2014), 5

Seed formation is an important step of plant development which depends on nutrient allocation. Uptake from soil is an obvious source of nutrients which mainly occurs during vegetative stage. Because seed ... [more ▼]

Seed formation is an important step of plant development which depends on nutrient allocation. Uptake from soil is an obvious source of nutrients which mainly occurs during vegetative stage. Because seed filling and leaf senescence are synchronized, subsequent mobilization of nutrients from vegetative organs also play an essential role in nutrient use efficiency, providing source-sink relationships. However, nutrient accumulation during the formation of seeds may be limited by their availability in source tissues. While several mechanisms contributing to make leaf macronutrients available were already described, little is known regarding micronutrients such as metals. Autophagy, which is involved in nutrient recycling, was already shown to play a critical role in nitrogen remobilization to seeds during leaf senescence. Because it is a non-specific mechanism, it could also control remobilization of metals. This article reviews actors and processes involved in metal remobilization with emphasis on autophagy and methodology to study metal fluxes inside the plant. A better understanding of metal remobilization is needed to improve metal use efficiency in the context of biofortification. [less ▲]

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