Jean-François Chollet

Salicylic Acid, an Ambimobile Molecule Exhibiting a High Ability to Accumulate in the Phloem

The ability of exogenous salicylic acid (SA) to accumulate in castor bean (Ricinus communis) phloem was evaluated by HPLC and liquid scintillation spectrometry analyses of phloem sap collected from the severed apical part of seedlings. Time-course experiments indicated that SA was transported to the root system via the phloem and redistributed upward in small amounts via the xylem. This helps to explain the peculiarities of SA distribution within the plant in response to biotic stress and exogenous SA application. Phloem loading of SA at 1, 10, or 100 μm was dependent on the pH of the cotyledon incubating solution, and accumulation in the phloem sap was the highest (about 10-fold) at the most acidic pH values tested (pH 4.6 and 5.0). As in animal cells, SA uptake still occurred at pH values close to neutrality (i.e. when SA is only in its dissociated form according to the calculations made by ACD LogD suite software). The analog 3,5-dichlorosalicylic acid, which is predicted to be nonmobile according to the models of Bromilow and Kleier, also moved in the sieve tubes. These discrepancies and other data may give rise to the hypothesis of a possible involvement of a pH-dependent carrier system translocating aromatic monocarboxylic acids in addition to the ion-trap mechanism.

Salicylic Acid Transport in Ricinus communis Involves a pH-Dependent Carrier System in Addition to Diffusion

Despite its important functions in plant physiology and defense, the membrane transport mechanism of salicylic acid (SA) is poorly documented due to the general assumption that SA is taken up by plant cells via the ion trap mechanism. Using Ricinus communis seedlings and modeling tools (ACD LogD and Vega ZZ softwares), we show that phloem accumulation of SA and hydroxylated analogs is completely uncorrelated with the physicochemical parameters suitable for diffusion (number of hydrogen bond donors, polar surface area, and, especially, LogD values at apoplastic pHs and Δ LogD between apoplast and phloem sap pH values). These and other data (such as accumulation in phloem sap of the poorly permeant dissociated form of monohalogen derivatives from apoplast and inhibition of SA transport by the thiol reagent p-chloromercuribenzenesulfonic acid [pCMBS]) lead to the following conclusions. As in intestinal cells, SA transport in Ricinus involves a pH-dependent carrier system sensitive to pCMBS; this carrier can translocate monohalogen analogs in the anionic form; the efficiency of phloem transport of hydroxylated benzoic acid derivatives is tightly dependent on the position of the hydroxyl group on the aromatic ring (SA corresponds to the optimal position) but moderately affected by halogen addition in position 5, which is known to increase plant defense. Furthermore, combining time-course experiments and pCMBS used as a tool, we give information about the localization of the SA carrier. SA uptake by epidermal cells (i.e. the step preceding the symplastic transport to veins) insensitive to pCMBS occurs via the ion-trap mechanism, whereas apoplastic vein loading involves a carrier-mediated mechanism (which is targeted by pCMBS) in addition to diffusion.

Proton-Peptide Co-Transport in Broad Bean leaf Tissues

The transport of [14C]glycyl-glycine (Gly-Gly) has been characterized in leaf discs from mature exporting leaves of broad bean (Vicia faba L.). In terms of glycine (Gly) equivalents, the rate of transport of Gly-Gly was similar to that of Gly uptake. Uptake of Gly-Gly was localized mainly in the mesophyll cells, with little accumulation in the veins. It was optimal at pH 6.0, sensitive to thiol reagents and metabolic inhibitors, and exhibited a single saturable phase with an apparent Michaelis constant of 16 mM. Gly-Gly did not inhibit the uptake of labeled Gly. Addition of Gly-Gly induced a concentration-dependent pH rise in the medium, showing that peptide uptake is mediated with proton co-transport. Gly-Gly also induced a concentration-dependent transmembrane depolarization of mesophyll cells with an apparent Michaelis constant of 15 mM. This depolarization was followed by a transient hyperpolarization. When present at a 10-fold excess, various peptides and tripeptides were able to inhibit Gly-Gly uptake with the following decreasing order of efficiency: Gly-Gly-Gly = leucine-Gly > Gly-tyrosine > Gly-glutamine = Gly-glutamic acid > Gly-phenylalanine > Gly-threonine > Gly-aspartic acid = Gly-asparagine = aspartic acid-Gly. Gly inhibited the uptake of Gly-Gly only slightly, whereas tetraGly and the tripeptide glutathione were not inhibitory. The dipeptides inhibiting Gly-Gly uptake also induced changes in the transmembrane potential difference of mesophyll cells and were able to affect in a complex way the response normally induced by Gly-Gly. Altogether, the data demonstrate the existence of a low-affinity, broad-specificity H+/peptide co-transporter at the plasma membrane of mesophyll cells. The physiological importance of this transporter for the exchange of nitrogenous compounds in mature leaves remains to be determined, as do the details of the electrophysiological events induced by the dipeptides.

Synthesis and structure-activity relationships of some pesticides with an α-amino acid function

Abstract The synthesis of several derivatives of a xenobiotic (phenoxyalkanecarboxylic acid) and an α -amino acid ( l -lysine) is described. Various substituents were introduced in the aromatic ring of the phenoxyalkanecarboxylic acid and the side-chain was modified. They were tested for their effect on the transport of a neutral ( l -threonine), an acidic ( l -glutamic acid) and a basic ( l -lysine) amino acid, and a sugar (sucrose). All compounds markedly inhibited threonine uptake by leaf tissues of Vicia faba L. – and more specifically phloem loading – with two exceptions, when the aromatic ring was substituted in the 4-position by a primary amino group or when d -lysine was used instead of l -lysine. By contrast, the addition of a chlorine atom in the 4-position of the aromatic ring enhanced the inhibitory activity. Similar results were obtained for inhibition of glutamate uptake and, to a lesser extent, for lysine uptake. pH dependence of the inhibitory activity as well as electrophysiological data indicate that permeases mediating active transport of amino acids are the target of these conjugates. These, in addition to other data obtained with other xenobiotics, show that the amino acid carrier system is capable of recognizing a wide range of conjugates of various sizes, structures and octanol/water partition coefficients.

Transport of Salicylic Acid and Related Compounds

Various stresses promote SA accumulation. SA is in part conjugated in the cytoplasm to inactive compounds such as salicylic acid O-β-glucoside (SAG) or modified to active compounds such as methylsalicylate (MeSA). SAG is sequestered in the vacuole by an ATP-binding cassette transporter mechanism or an H+-antiporter mechanism. Free SA is mobile and can be transported within the plant, mainly via the phloem. SA molecules found in the phloem sap may come from the synthesis area via the symplastic route in symplastic loaders or may be taken up from the phloem apoplast (apoplastic loaders). In this latter case, SA must cross the plasma membrane of the companion cell-sieve cell complex. Similarly, synthetic derivatives or analogs applied to the foliage to enhance plant defence must cross at least once the plasma membrane before reaching the sieve tubes. The ability of molecules to diffuse through the plasma membrane is dependent on their chemical properties (size of the molecule, Log D, polar surface area, number of hydrogen bond donors). On these bases, the discrepancies between the computed predictions of phloem mobility of SA and various analogs and the actual results, as well as the effect of pCMBS on uptake suggest that SA transport involves a pH-dependent carrier system in addition to the ion trap mechanism, at least in the cotyledons of Ricinus communis. Although SA levels increase in both the phloem and systemic leaves after mature leaf infection, this salicylate is clearly not the primary systemic signal which contributes to SAR. Several data strongly suggest that MeSA as well as azelaic acid and small lipids are earlier signals. As MeSA is predicted to be very poorly phloem mobile, the mechanism of long distance transport of this volatile compound remains to be elucidated.

Vectorization of agrochemicals: amino acid carriers are more efficient than sugar carriers to translocate phenylpyrrole conjugates in the Ricinus system

Producing quality food in sufficient quantity while using less agrochemical inputs will be one of the great challenges of the twenty-first century. One way of achieving this goal is to greatly reduce the doses of plant protection compounds by improving the targeting of pests to eradicate. Therefore, we developed a vectorization strategy to confer phloem mobility to fenpiclonil, a contact fungicide from the phenylpyrrole family used as a model molecule. It consists in coupling the antifungal compound to an amino acid or a sugar, so that the resulting conjugates are handled by active nutrient transport systems. The method of click chemistry was used to synthesize three conjugates combining fenpiclonil to glucose or glutamic acid with a spacer containing a triazole ring. Systemicity tests with the Ricinus model have shown that the amino acid promoiety was clearly more favorable to phloem mobility than that of glucose. In addition, the transport of the amino acid conjugate is carrier mediated since the derivative of the L series was about five times more concentrated in the phloem sap than its counterpart of the D series. The systemicity of the L-derivative is pH dependent and almost completely inhibited by the protonophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP). These data suggest that the phloem transport of the L-derivative is governed by a stereospecific amino acid carrier system energized by the proton motive force.

Crop protection: new strategies for sustainable development

GFP 2012 was the 42nd congress organized by the French Group of Pesticide research (“Groupe Francais des Pesticides”, GFP), an association created in 1977 by a group of scientists from several French universities and research institutes. Initially, this conference was held twice a year, but nowadays, the adopted rhythm is once a year over a period of 3 days, usually in May. All scientific and technical aspects of the synthesis, reactivity, mode of action, toxicology, ecotoxicology, and environmental fate of pesticides are discussed. In this special issue, ESPR publishes 17 articles that are based on oral presentations at GFP 2012 in Poitiers. It covers the latest advances in the field of pesticide research with a special focus on innovative strategies for plant protection. This key theme was chosen by the organizing team according to the main research conducted in the location hosting the conference, namely, original strategies to confer systemicity to nonmobile compounds. The purpose of this research is to improve the bioavailability of active compounds, thereby reducing the quantity of pesticides used and pollution. It also aims at managing parasites that remain uncontrolled because of their location, e.g., fungi responsible for grapevine trunk diseases. In 2008, France, which is the fourth largest market, behind the USA, Brazil, and Japan, has implemented an action plan called “Ecophyto”. The Ecophyto plan embodies the commitment given by the authorities, industry professionals, and representatives of civil society to cut the nationwide use of pesticides by 50 % within 10 years, if possible (Ministere de l’Agriculture et de la Peche 2008). The most notable goal of Ecophyto is to reduce the dependency of farms on plant protection products, without affecting agricultural yields and maintaining high levels both in terms of quality and quantity. In this context, the aim of the conference was to provide some answers to current societal challenges, i.e., producing larger amounts of food for an exponentially growing population while reducing agronomic inputs for pest and weed control, thereby reducing pollution as well as potential toxicity to the environment or human health. In order to achieve these various objectives, GFP 2012 was organized in four sessions as follows:

A Convenient Method of Protection and Mild Deprotection of α-Aminoacids

Abstract Functionalisation of β or γ carboxyl group of aspartic and glutamic acids with labile substituents was performed via the use of N-trichloroethoxycarbonyl5-oxazolidinone as protective group.

Vectorization of agrochemicals via amino acid carriers: influence of the spacer arm structure on the phloem mobility of phenylpyrrole conjugates in the Ricinus system

BACKGROUND Excessive agrochemical use poses significant threats to environmental safety and human health. Reducing pesticide use without reducing yield is necessary for sustainable agriculture. Therefore, we developed a vectorisation strategy to enhance agrochemical delivery through plant amino acid carriers. RESULTS In addition to a fenpiclonil conjugate recently described, three new amino acid conjugates were synthesised by coupling fenpiclonil to an l-α-amino acid. Phloem mobility of these conjugates, which exhibit different structures of the spacer arm introduced between fenpiclonil and the α-amino acid function, was studied using the Ricinus model. Conjugate L-14, which contains a triazole ring with the shortest amino acid chain, showed the best phloem systemicity among the four conjugates. By contrast, removing the triazole ring in the spacer arm did not improve systemicity. L-14 exhibited phloem systemicity at all reported pH values (pH values from 5.0 to 6.5) of the foliar apoplast, while acidic derivatives of fenpiclonil were translocated only at pH values near 5.0. CONCLUSION The conjugates were recognised by a pH-dependent transporter system and translocated at distance in the phloem. They exhibited a broader phloem systemicity than fenpiclonil acidic derivatives within the pH value range of the foliar apoplast.

Modifications of the chemical structure of phenolics differentially affect physiological activities in pulvinar cells of Mimosa pudica L. II. Influence of various molecular properties in relation to membrane transport

Early prediction of compound absorption by cells is of considerable importance in the building of an integrated scheme describing the impact of a compound on intracellular biological processes. In this scope, we study the structure-activity relationships of several benzoic acid-related phenolics which are involved in many plant biological phenomena (growth, flowering, allelopathy, defense processes). Using the partial least squares (PLS) regression method, the impact of molecular descriptors that have been shown to play an important role concerning the uptake of pharmacologically active compounds by animal cells was analyzed in terms of the modification of membrane potential, variations in proton flux, and inhibition of the osmocontractile reaction of pulvinar cells of Mimosa pudica leaves. The hydrogen bond donors (HBD) and hydrogen bond acceptors (HBA), polar surface area (PSA), halogen ratio (Hal ratio), number of rotatable bonds (FRB), molar volume (MV), molecular weight (MW), and molar refractivity (MR) were considered in addition to two physicochemical properties (logD and the amount of non-dissociated form in relation to pKa). HBD + HBA and PSA predominantly impacted the three biological processes compared to the other descriptors. The coefficient of determination in the quantitative structure-activity relationship (QSAR) models indicated that a major part of the observed seismonasty inhibition and proton flux modification can be explained by the impact of these descriptors, whereas this was not the case for membrane potential variations. These results indicate that the transmembrane transport of the compounds is a predominant component. An increasing number of implicated descriptors as the biological processes become more complex may reflect their impacts on an increasing number of sites in the cell. The determination of the most efficient effectors may lead to a practical use to improve drugs in the control of microbial attacks on plants.