AtLHT1 transporter can facilitate the uptake and translocation of a

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AtLHT1 transporter can facilitate the uptake and translocation of a glycinergic-chlorantraniliprole conjugate in Arabidopsis thaliana Yan Chen, Ying Yan, Zhan-Fu Ren, Ulrika Ganeteg, Guang-Kai Yao, ZiLin Li, Tian Huang, Jia-Hui Li, Yong-Qing Tian, Fei Lin, and Han-hong Xu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03591 • Publication Date (Web): 07 Nov 2018 Downloaded from http://pubs.acs.org on November 9, 2018

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Journal of Agricultural and Food Chemistry

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AtLHT1 transporter can facilitate the uptake and translocation of a glycinergic-

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chlorantraniliprole conjugate in Arabidopsis thaliana

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Yan Chen1, Ying Yan1, Zhan-Fu Ren1, Ulrika Ganeteg2, Guang-Kai Yao1, Zi-Lin Li1,

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Tian Huang1, Jia-Hui Li1, Yong-Qing Tian1, Fei Lin1*, Han-Hong Xu1*

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1State

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South China Agricultural University, Guangzhou, 510642, Guangdong, China; Key

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Laboratory of Natural Pesticide and Chemical Biology, Ministry of Education, South

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Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources,

China Agricultural University, Guangzhou, 510642, Guangdong, China

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2Umeå

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Swedish University of Agricultural Sciences (SLU), SE-901 83 Umeå, Sweden

Plant Science Centre, Department of Forest Genetics and Plant Physiology,

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Corresponding Author

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*Han-Hong

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[email protected]

Xu and Fei Lin; Tel: +86-20-85285127. Email: [email protected] and

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Author contributions

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HHX and FL conceived and designed the research. YC, YY, ZFR, ZLL, TH and JHL

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conducted the experiments and analyzed the data. GKY prepared the necessary

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compounds. UG and YQT participated in the data analysis and helped with writing of the

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manuscript. FL and HHX wrote the paper. All the authors have read and approved the

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manuscript.

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Abstract: Understanding of the transporters involved in the uptake and translocation of

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agrochemicals in plants could provide an opportunity to guide pesticide to the site of

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insect feeding. The product of Arabidopsis thaliana gene AtLHT1 makes a major

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contribution to the uptake into the roots of free amino acids and some of their derivatives.

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Here, a chlorantraniliprole-glycine conjugate (CAP-Gly-1) was tested for its affinity to

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AtLHT1 both in planta and in vitro. Seedlings deficient in AtLHT1 exhibited a reduction

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with respect to both the uptake and root-to -shoot transfer of CAP-Gly-1; plants in which

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AtLHT1 was constitutively expressed were more effective than wild type in term of their

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root uptake of CAP-Gly-1. Protoplast patch clamping showed that the presence in the

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external medium of CAP-Gly-1 was able to induce AtLHT1 genotypes dependent inward

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currents. An electrophysiology-based experiment carried out in Xenopus laevis oocytes

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expressing AtLHT1 showed that AtLHT1 had a high in vitro affinity for CAP-Gly-1. The

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observations supported the possibility of exploiting AtLHT1 as a critical component of a

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novel delivery system for amino acid-based pesticide conjugates.

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Keywords: Glycinergic-chlorantraniliprole conjugate, amino acid transporter, uptake,

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translocation, patch clamping

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INTRODUCTION

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As many of the bioactive compounds used for crop protection, especially pesticides,

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are toxic, efforts to reduce their usage are necessary, in the interests of both safeguarding

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human health and minimizing collateral damage to the environment1. Major losses are

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incurred by spray drift, run-off and photodegradation, and it has been estimated that less

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than 0.1% of the bioactive material applied actually reaches its target2,3.

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An attractive strategy to improve pesticide efficacy is to modify bioactive

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compounds in order to encourage their ability to cross the plasma membrane and be

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subsequently translocated through the plant's vascular system to reach the site of

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pest/pathogen damage2. The addition of either amino acids, amino acid esters or sugar

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moieties to a pesticide structures has been found to measurably improve mobility in

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plants, especially through the phloem, presumably through the involvement of the plant's

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native transporter machinery4-9. These conclusions have been primarily based on

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physiological experiments, for example, concentration-dependent kinetics, competition

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and inhibit studies. With the acquisition of extensive DNA sequence, it is increasingly

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possible to identify the carriers responsible for the movement of pesticide conjugates. The

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in silico identification of Ricinus communis sugar transporter genes and the monitoring of

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their transcriptional behavior has led to the identification of the GTF (glucose-fipronil

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conjugate) induced gene RcSTP1. Functional analysis based on the expression of this

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gene in Xenopus laevis oocytes has demonstrated that its product has a high affinity for

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GTF. Long-distance root-to-shoot transport of GTF was found to be enhanced by

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introducing a transgene which constitutively expressed RcSTP110. More recently studies

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were able to show that the distinctive tissue distribution of a glycine/fipronil conjugate

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(GlyF) is governed by the activity of four amino acid transporters, and one of them,

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RcANT15, has the ability to significantly increase the uptake of serine-fipronil conjugate

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in Xenopus oocytes significantly11,12.

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The Arabidopsis thaliana genome harbors a minimum of 67 genes encoding proteins

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likely involved in amino acid transport; several of these have been have been functionally

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characterized13. While AtLHT1 was initially held to be a high-affinity transporter for both

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lysine and histidine, in the meantime it has been shown to have much broader

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specificity14-16. Thus AtLHT1 appears not only responsible for the uptake of amino acids

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into the root, but also in their cycling within mesophyll cells15,16. AtLHT1 was also

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reported to negatively regulate aspects of the plant's pathogen defense response by

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altering the availability of glutamine17. Analysis of are2 mutant has revealed that AtLHT1

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is the gene responsible, indicating that AtLHT1 is involved in the transport of ACC (1-

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aminocylopropane carboxylic acid)18, and implying that AtLHT1 has a broad substrate-

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specificity. Given that AtLHT1 makes a major contribution to the uptake of amino acids

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into roots, as well as that of some amino acid derivatives, it was therefore of interest to

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test whether its functionality could be exploited to actively take up a pesticide/amino acid

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conjugate.

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A previous study has detailed the designed and synthesized a series of ester-capped

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amino acid-conjugated chlorantraniliproles (CAPs), as well as their acidified forms. Most

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of these conjugates were shown to both accumulate readily in Ricinus communis and to

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move freely through the phloem and xylem. Both model predictions and experimental

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data support the notion that the phloem transport of these conjugates requires an active

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carrier19. Here, the potential of amino acid transporter AtLHT1 to facilitate the

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translocation of the conjugates in planta has been investigated. The findings suggest

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opportunities for exploiting the activity of amino acid transporters to promote the uptake

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of pesticide conjugates, thereby representing a potential means to manipulate the

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distribution of a pesticide within a plant.

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MATERIALS AND METHODS

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Plant materials. Seeds of the A. thaliana AtLHT1-5 mutant AtLHT1-KO16; and of a

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line over-expressing AtLHT1 (35S-AtLHT11)20 were obtained from the Department of

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Forest Genetics and Plant Physiology, Umeå Plant Science Centre, Swedish University of

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Agricultural Sciences, and the stocks was multiplied in house. For uptake experiments

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and the isolation of root protoplasts, seeds were surface-sterilized by immersion in 70%

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(v/v) ethanol, then transferred for 5 min into 2% (v/v) NaClO. After rinsing three times in

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sterile water, the seeds were imbibed for three days at 4ºC, and sown on dishes containing

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solidified 0.65% (w/v) Phytagel (DuchefaBiochemie, Haarlem, The Netherlands), half-

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strength Murashige and Skoog (1962) medium supplemented with 0.5% (w/v) sucrose

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and adjusted to pH5.8 with KOH. The material was held in a growth chamber delivering

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a 12 h photoperiod provided by lamps emitting 125-130 μmol photons m-2s-1, a relative

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humidity of 50% and a constant temperature of 21ºC.

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Conjugate structures. The structure of the chlorantraniliprole conjugates, involving

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glycine (CAP-Gly-1 and CAP-Gly-2), alanine (CAP-Ala-1 and CAP-Ala-2) and serine

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(CAP-Ser-1 and CAP-Ser-2) are presented as Fig. 1. The conjugates were prepared

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following the methods given by previous study19.

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Uptake of CAP conjugates by A. thaliana roots. After growing for 14-18 days, sets

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of 30 seedlings of each of AtLHT1-KO, 35S-AtLHT1 and the wild type ecotype Col-0

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(WT), were removed from the medium and their roots immersed for 1 h in individual

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wells of a 96-well Costar 3599 micotiter plate (Corning Inc., New York City, NY, USA)

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with one plant per well, which contained 0.5 mM CaCl2. The solution in each well was

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replaced by 30 μl uptake solution (0.5 mM CaCl2 plus 30 μM of the CAP conjugate). In

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competition and inhibition experiments, either 50 μM CCCP or 1mM pCMBS or 30 μM

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or 100 μM glycine was added to each well. As a control, a set of wells filled with the 30

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μM CAP conjugate was set up in parallel. After 6 h of treatment, the seedlings were

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rinsed three times in 0.5 mM CaCl2 and separated into root and shoot tissue, which was

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snap-frozen in liquid nitrogen, macerated in 200 μL methanol and ultrasonicated for 30

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min. The resulting suspensions were centrifuged (14,000 g, 10 min). A 100 μL aliquot of

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the supernatant was subjected to HPLC to quantify the content of each compound, using

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an Agilent 1100 device, attached to a C18 reverse-phase column (Agilent, Santa Clara,

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CA. USA). The solvent was a 1:1 (v/v) mixture of acetonitrile and aqueous 0.1% (v/v)

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trifluoroacetic acid. The injection volume was 10 μL, and the flow rate was 1 mL per min.

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A. thaliana root and leaf protoplasts isolation. Root and leaf protoplasts were

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isolated from 14 day old and 6 week old seedlings, following the method described by

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Demidchik et al. with minor modifications22. Cellulase onozula RS, cellulysin and

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pectolyase were replaced by 2% (w/v) cellulase C-1794 (Yakult Honsha, Tokyo, Japan)

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and 0.4% (w/v) macerozyme (Yakult Honsha) for the preparation of root protoplasts,

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while W5 (5 mM KCl, 125 mM CaCl2, 154 mM NaCl, 5 mM glucose, 0.02 mM MES,

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pH 5.7 adjusted with Tris, π0=290~300 mOsMol·kg-1 adjusted with mannitol) was used

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instead of the holding solution described above. Root digests were shaken gently (at 60

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rpm) at 28°C for 60 min, while the leaf digests were shaken gently (40 rpm) at 28°C for

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180 min. Protoplasts were isolated by filtering through a nylon mesh with 70 μm

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diameter pores, and the residue remaining on the mesh was rinsed with W5 solution. The

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resulting preparation of protoplasts was collected by imposing a 3 min centrifugation at

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150 g, after which the pellet was rinsed 2-3 times in W5, followed by a second

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centrifugation (150 g, 3 min). The method using for isolating leaf protoplasts was the

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same as the root materials, except that that the enzymes concentrations were halved.

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Finally, the protoplasts were suspended in 1 mL W5, representing a titer of 1× 105 per

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mL, and held on ice for at most 3 h before being used for the patch clamp experiments.

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Leaf protoplasts were counted using a haemocytometer and used for uptake experiments.

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Patch clamp recordings. Experiments were performed using the whole-cell current

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clamp mode. The standard extracellular solution contained 10 mM potassium glutamate,

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1 mM CaCl2, 5 mM MES, 4 mM MgCl2 (pH5.8, 300 mOsM·kg-1, adjusted with

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mannitol). The electrical resistance of the pipettes when filled with ‘pipette solution’ (140

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mM potassium glutamate, 0.1 mM CaCl2, 10 mM EDTA, 2 mM MgCl2, 10 mM Hepes, 2

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mM ATP-Mg (pH7.2, 300-320 mOsM·kg-1 adjusted with mannitol) was in the range 10-

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15 MΩ. After gigaohm seals were formed, whole cell configurations were achieved by

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gentle suction, and the membrane was immediately clamped to a holding potential of -70

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mV. The resting membrane potential and spontaneous membrane potential activity of

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protoplasts were recorded in the current clamp mode (I = 0). After the protoplasts had

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been perfused for 5 min, the various compounds were added and the changes of

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membrane potential recorded. All recording experiments were carried out at room

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temperature (22-24ºC). A BX51WI positive fixed stage microscope (Olympus, Shinjuku-

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ku, Tokyo, Japan) and a Multiclamp 700B amplifier, Digital 1440A D-A converter

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(Molecular Devices, Foster City, CA, USA) were used as recording instruments. Data

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were collected at 5 kHz and filtered at 1 kHz and analyzed using the pClamp 10 software

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package (Molecular Devices) and Origin 8.6 software (OriginLab, Northampton, MA,

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USA)

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Uptake of CAP conjugates by A. thaliana leaf protoplasts. A 1 ml aliquot of leaf

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protoplasts was combined with 1 mL W5 for 15-180 min in individual wells of a 12-well

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Costar 3599 micotiter plate (Corning Inc., New York City, NY, USA). Thereafter, the

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suspensions were pelleted by a 3 min centrifugation at 150 g and then rinsed 2-3 times in

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W5. Finally, the protoplasts were counted using hemocytometer and then re-centrifuged

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for 3 min at 150 g, macerated in 200 μL methanol and ultrasonicated for 30 min. The

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resulting suspensions were centrifuged (14,000 g, 10 min). A 100 μL aliquot of the

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supernatant was subjected to HPLC to quantify the content of each target compound.

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Functional expression of AtLHT1 in Xenopus oocytes. Detailed protocols for the

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preparation and injection of the oocytes and the associated electrophysiology have been

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described elsewhere10,23. The 1.71 kbp AtLHT1 opening reading frame was amplified

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using

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GATCTGATATCACTAGTGCCACCATGGTAGCTCAAGCTCCTCA/5’-CGCGG

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CCGCCTCGAGGCATGCTTAGTGGAATTACATAGCGTTTA.

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plasmid was linearized by SpeI/SphI restriction, then subjected to recombination with the

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AtLHT1 fragment using an In-Fusion Cloning Kit (Clontech, Mountain view, CA, USA).

a

gene-specific

the

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primer

pair

The

5’-

pT7TSHA

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The resulting plasmid was linearized by restriction with SmaI, after which the capped

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mRNA was reverse transcribed in vitro using an mMessage mMachine kit

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(www.thermofisher.com/cn/zh/home/brands/ambion.html). Stage V and VI X. laevis

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oocytes were isolated and each injected with 27.6 nL (1 ng per nL) AtLHT1 cRNA and

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incubated for 2-4 days at 18ºC in Barth’s medium supplemented with 10 µg per mL

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gentamycin. The oocytes were subsequently bathed in modified sodium Ringer solution

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(96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 5 mM MgCl2, 5 mM HEPES, pH 5.5) with

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continuous perfusion at 3 mL min-1. Recording pipettes were filled with 3 M KCl,

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delivering an electrical resistance between 0.5 to 1 MΩ. Currents were measured using a

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Model OC-725C oocyte clamp amplifier (Warner Instruments, Hamden, CT, USA),

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filtered at 200 Hz and digitized at 2,000 Hz. Holding potential was -50 mV, and voltage

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pulses from -150 mV to +50 mV were applied for 100 ms. Substrate-dependent currents

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were obtained by subtracting an average of background currents recorded before and after

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CAP exposure. Base line currents at pH 7.6 were monitored throughout the recording.

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The oocytes were equilibrated in the test solution for about 2 min before being exposed to

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the test compounds. After each exposure, they were rinsed in amino acid- or CAP

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conjugate-free solution at pH 7.6 until the currents had returned to base line. Data were

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acquired and analysed with the help of Digidata 1440A and pClamp10.0 software (Axon

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Instruments Inc., Union City, CA, USA). OriginPro8.0 software (www.originlab.com/)

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was utilized for Km fitting. All experiments were performed at room temperature (22-

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24ºC).

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HPLC determination. The extracts were analyzed using a Waters 1100 device,

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attached to a C18 reverse-phase column (Waters, Milford, MA. USA). The solvent was a

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1:1 (v/v) mixture of acetonitrile and aqueous 0.1% (v/v) trifluoroacetic acid. The

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injection volume was 10 μL, and the flow rate was 1 mL per min.

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Statistical analyses. Statistical analyses were conducted using IBM SPSS Statistics

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20 for Windows (https://www.ibm.com/analytics/us/en/technology/spss/). The data are

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presented in the form mean ± SEM. For the CAP conjugate uptake into A.thaliana roots

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data and the two electrode voltage clamp experiment in X. laevis oocytes, a one way

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analysis of variance (ANOVA), followed by the LSD test was carried out to compare the

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differences among treatments (P < 0.05). For the uptake inhibition experiments,

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differences within each ATLHT1 genotype were determined by one way ANOVA,

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followed by Dunnett’s test (*: P < 0.05, **: P < 0.001). Measurements of the

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depolarization magnitude in A. thaliana root protoplasts in response to the various

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treatments were analyzed by a two way ANOVA, followed by Scheffe’s test (P < 0.05).

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RESULTS AND DISCUSSION

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Root uptake of the CAP conjugates. Since some CAP conjugates exhibit a

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comparably higher level of insecticidal activity in vivo against beet armyworm when

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compared with their parent compound CAP19, three conjugates CAP-Gly-1, CAP-Ala-1

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and CAP-Ser-1 were selected for this study, along with their corresponding esterified

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forms CAP-Gly-2; CAP-Ala-2 and CAP-Ser-2. The conjugates were all tested for their

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capacity to be taken up by A. thaliana roots over a 6 h exposure period (Fig. 1 and Fig. 2).

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The root uptake of CAP-Gly-2 reached a level of 200.18 nmol/g after exposure, making it

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the most effectively taken up compound, followed by the CAP-Ala-2; the other four

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conjugates were all less effectively taken up than their parent compound CAP (Fig. 2).

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However, the concentration of CAP-Ala-2 was higher than that of CAP-Gly-2 in both

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phloem and xylem fluids when taken up by R. communis 19, suggesting that mobility of

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compounds may differ in plant species.

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Although CAP-Gly-2 was the most effectively taken up compound and has a

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comparable insecticidal activity to CAP in plants, the carboxymethyl groups present in it

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underwent total de-esterification to become CAP-Gly-1 during the uptake process. Only

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CAP-Gly-1 could be detected in plants when the seedlings were treated with CAP-Gly-2.

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The retention time (RT) of CAP-Gly-1 and CAP-Gly-2 standard sample was 5.950 min

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and 10.234 min, respectively. Chromatogram of samples extracted from the seedlings that

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were exposed to the CAP-Gly-2 totally overlapped with that of the CAP-Gly-1 (Fig. S1).

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These results suggested that CAP-Gly-1 is the actual compound being transported and

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plays insecticidal role in planta. Therefore, the focus was placed on identification of the

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transporters responsible for the uptake and transport of the of CAP-Gly-1 in A. thaliana.

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The root uptake of CAP-Gly-1 is influenced by AtLHT1 activity. As the transport

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of CAP-Gly-1 through the phloem likely involves an active carrier, and since AtLHT1

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makes an important contribution to root uptake, the behavior of a set of plants carrying

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modified alleles of AtLHT1 was tested to investigate whether AtLHT1 was responsible

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for the uptake of CAP-Gly-1. The uptake of CAP-Gly-1 into the roots was greater for the

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35S-AtLHT1 line than for WT, which in turn was much higher than that was achieved by

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AtLHT1-KO (Fig. 3b). The effect of AtLHT1 genotype on the accumulation of CAP-Gly-

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1 was similar to that shown by the whole plant (Fig. 3a), indicating the AtLHT1 play

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important role in the root uptake of CAP-Gly-1.

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AtLHT1 is believed to provide one of the more important facilitators of the uptake of

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free amino acid from the soil15,16. The knocking out or over-expression of AtLHT1 has a

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major effect on the uptake kinetics of a variety of amino acids24. Plants lacking a

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functional copy of AtLHT1 exhibit a reduced uptake of L-Ser, D-Ala and Gly with rates

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of 100%, 92% and 84%, respectively15,16. AtLHT1 contributes more than 75% for the

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root uptake of CAP-Gly-1 (Fig. 3a), indicating that AtLHT1 functions in the uptake of

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CAP-Gly-1. AtLHT1 also participates in the transport of ACC, thereby influencing ACC-

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induced ethylene responses18. Although there is still a little evidence to support the notion

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that AtLHT1 is involved in the transport of amino acid derivatives, its broad substrate

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specificity probably enables it to recognize amino acid-related compounds.

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Plants carrying a non-functional version of AtLHT1 gene led to a reduced

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accumulation of CAP-Gly-1 in the shoot, whereas the over-expression of the gene had no

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effect on this ability (Fig. 3c). The implication is that the root-to-shoot translocation of

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CAP-Gly-1 is also fulfill by AtLHT1. However, simply increase AtLHT1 expression

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could not improve transport ability of CAP-Gly-1 in the plants significantly. Nevertheless,

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at least 45% of the CAP-Gly-1 transport to shoot relied on other transport mechanisms

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(Fig. 3d). Transport of xenobiotics can be via the symplast, following their entry into the

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root epidermis, root hair and root cap cells25. In addition, some transport can take place

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via the apoplast, at least until it reaches the endodermis Casparian strip. Thereafter, the

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xenobiotics are transferred to xylem and hence enter the transpiration stream to the shoot.

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Other, as yet not characterized transporters are probably involved in these processes.

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Given that AtLHT1 interacts with most neutral and alkaline amino acids, a wide range of

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modification targets should be available for conjugation with a protective compound in

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order to enhance its in planta mobility.

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The uptake of CAP-Gly-1 is inhibited by CCCP, pCMBS and glycine. CCCP,

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widely used to study the transmembrane transport of pesticides, is an uncoupling agent

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for oxidative phosphorylation, as it removes the proton driving force in the

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transmembrane process4,5; pCMBS is a transmembrane transport inhibitor which inhibits

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the transport of protease activity on the plasma membrane26. A series of effector

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inhibition and substrate competition experiments were performed in order to clarify the

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mechanistic basis of CAP-Gly-1 uptake. In the absence of AtLHT1 activity (KO-

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AtLHT1), the addition of neither of these two compounds had any no effect on CAP-Gly-

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1 uptake, while the uptake of CAP-Gly-1 into the roots of both WT and 35S-AtLHT1 was

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significantly inhibited. In both WT and 35S-AtLHT1, the inhibition imposed by both

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supplements exceeded 50% (Fig. 4a), meaning that both CCCP and pCMBS were able to

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inhibit AtLHT1 activity. These results suggested that the CAP-Gly-1 process is energy-

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consuming and is carrier-mediated.

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Glycine is a specific substrate for AtLHT1, so would be expected to compete with

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CAP-Gly-1. The inclusion of either 30 or 100 μM glycine in the uptake medium had no

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inhibitory effect on CAP-Gly-1 uptake in AtLHT1-KO (Fig. 4b), but it did significantly

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reduce its uptake in both WT and 35S-AtLHT1 (Fig. 4b). In WT, the uptake fell by

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62.8% in the presence of 30 μM glycine and by 70.5% in the presence of 100 μM glycine;

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the respective reductions in uptake in 35S-AtLHT1 were 52.9% and 44.8% (Fig. 4b).

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Overall, the uptake of CAP-Gly-1 in both WT and AtLHT1-overexpressing plants could

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be significantly inhibited by glycine, indicting the uptake process shared the transport

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strategies as A. thaliana uptake glycine.

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The interaction of AtLHT1 with CAP conjugates in A. thaliana root protoplasts.

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Patch clamping can be used to characterize both the net ion flux across the entire surface

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area of a cell and ion currents flowing through a single open channel in cells. AtLHT1 is

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a plasmalemma-localied amino acid transporter, expressed in the rhizodermis and leaf

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mesophyll cells15. Plant cells are encased a cell wall which can insulate the cell's surface

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from physical contact with a xenobiotics. As a result, in order to explore the ability of

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AtLHT1 to transport CAP-Gly-1, the whole cell patch clamp approach was adopted using

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A. thaliana root protoplasts. The membrane potential response of 35S-AtLHT1 root

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protoplasts to the addition of the various substrates is shown in Fig. 5a-e. There was little

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response to the presence of either DMSO or CAP, in contrast to the major effect of both

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glycine and glutamic acid. The observation that CAP-induced little membrane potential

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changes were minor in protoplast further supported the notion that mobility of CAP in

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planta is mediated in a passive way. In contrast, the responses to CAP-Gly-1 was above

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the background level, but was less pronounced than those to either glycine or glutamic

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acid, suggesting that the addition of an amino acid moiety to CAP increases the affinity

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between CAP-Gly-1 and AtLHT1, in spite of the nonnegligible effect of CAP moiety.

316

The normalized depolarization amplitudes of the membrane potential for the various

317

substrates are illustrated in Fig. 5f. There were significant differences between the

318

substrates for each of the three AtLHT1 genotypes. The depolarization amplitudes

319

induced by CAP-Gly-1 was not differ from those induced by the positive controls glycine

320

and glutamic acid, but was significantly greater than those induced by the negative

321

controls (CAP and DMSO). Of the three AtLHT1 genotypes, the 35S-AtLHT1 protoplasts

322

appeared to respond most strongly to CAP-Gly-1. The overall conclusion was that CAP-

323

Gly-1 was able to stimulate AtLHT1 activity in protoplasts.

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The uptake of CAP conjugates by A. thaliana leaf protoplasts. As AtLHT1 also

325

functions in the uptake of amino acid into the leaf mesophyll, a time course experiment

326

was conducted to characterize the uptake of CAP conjugates into A. thaliana leaf

327

mesophyll protoplasts. Between 15 mins to 180 mins, the protoplast isolated from plants

328

carrying various alleles of AtLHT1, absorbed CAP-Gly-1 at a steadily increasing rate (Fig.

329

6). With respect to the effect of AtLHT1, the uptake of CAP-Gly-1 into the protoplasts

330

was greater for the 35S-AtLHT1 line than for WT, which in turn was much higher than

331

was achieved by AtLHT1-KO (Fig. 6). This observation implied that AtLHT1 indeed

332

mediates the uptake of CAP-Gly-1 into plant cells.

333

The interaction of AtLHT1 with CAP-Gly-1 in X. laevis oocytes. The

334

identification of AtLHT1's involvement in the uptake of CAP-Gly-1 prompted an effort

335

to study the interaction between AtLHT1 and the conjugates in X. laevis oocytes, a

336

classical heterologous expression model used for electrophysiological investigations of

337

ion channels. AtLHT1 was expressed in X. laevis oocytes by injecting its cRNA, and the

338

two microelectrodes voltage clamp technique was employed to monitor the transport of

339

the CAP conjugate by measuring the inward currents thereby induced (Fig. 7). There was

340

no observed change in the size of the current induced by the presence of either the control

341

or the test substrates CAP-Gly-1 in the negative control (oocytes injected with water) (Fig.

342

7a). However, detectable inward currents were induced by adding CAP-Gly-1 to the

343

AtLHT1-expressing X. laevis oocytes. When the substrates were removed from the

344

medium, the currents returned to the base line (Fig. 7b). CAP induced detectable inward

345

current, might be caused by the unwanted effects of endogenous ion channels, but the

346

current induced was significantly less than that induced by CAP-Gly-1 (Fig. 7b and 7c).

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The implication was that the proton-coupled transporter activity of AtLHT1 could

348

potentially be used to transport CAP-Gly-1.

349

The affinity of AtLHT1 for CAP-Gly-1 was then explored by performing a kinetic

350

analysis. A non-linear regression analysis was applied to the concentration–activity plots

351

to calculate the relevant K0.5 values. Those for glutamic acid and CAP-Gly-1 were

352

comparable to one another, while the value for glycine was lower. The value for CAP

353

was much higher than that of either CAP-Gly-1 (Fig. 8a-e), indicating that the addition of

354

an amino acid moiety to CAP significantly improved its affinity with AtLHT1.

355

In conclusion, is has been shown that A. thaliana roots are able to take up the

356

conjugate CAP-Gly-1 via the activity of the lysine/histidine transporter AtLHT1.

357

Characterizing the influence of AtLHT1 on the transport of a wider range of amino

358

acid/CAP conjugates should provide more information on the potential of AtLHT1 to

359

deliver xenobiotics in plants.

360 361

Supplementary data

362

Fig. S1. Detection of CAP-Gly-1 in different AtLHT1 genotypes plants by HPLC. The

363

red line is the chromatogram following HPLC of 40 μM CAP-Gly-1 standard sample.

364

The gray line is the chromatogram following HPLC of 30 μM CAP-Gly-2 standard

365

sample. The green line is the chromatogram following HPLC of the 35S-LHT1 shoot

366

extracts what been measured after a 6 h exposure to a 30 μM concentration of CAP-Gly-2.

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The blue line is the chromatogram following HPLC of the WT shoot extracts what been

368

measured after a 6 h exposure to a 30 μM concentration of CAP-Gly-2.

369

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Acknowledgements

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We acknowledge the guidance of Huaiyu Gu (Department of Anatomy and Neurobiology,

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Zhongshan School of Medicine, Sun Yat-sen University) in the use of the whole cell

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patch clamp approach, and of Xiurong Wang (Root Biology Centre, South China

375

Agricultural University) for providing the pT7TSHA plasmid and for advice regarding

376

the functional expression of AtLHT1 in Xenopus oocytes.

377 378 379

Funding

380

This research was supported by the National Key R&D Program of China

381

(2017YFD0200307 and 2018YFD020044), the Project of Science and Technology in

382

Guangdong province (grant No. 2014A050503056), the Scientific Project in Guangzhou

383

City (grant No. 201707020013 and 201704030027).

384 385

References

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(1) Massinon, M.; Lebeau, F. Comparison of spray retention on synthetic

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superhydrophobic surface with retention on outdoor grown wheat leaves. Ann. Appl. Biol.

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2011, 114, 261-268.

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(2) Wang, C. J.; Liu, Z. Q. Foliar uptake of pesticides—present status and future

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challenge. Pestic. Biochem. Physiol. 2007, 87, 1-8.

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(3) Damak, M.; Mahmoudi, S. R.; Hyder, M. N.; Varanasi, K. K. Enhancing droplet

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deposition through in-situ precipitation. Nat. Commun. 2016, 7, 12560.

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(4) Denis, M. H.; Delrot, S. Carrier-mediated uptake of glyphosate in broad bean (Vicia

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faba) via a phosphate transporter. Physiol. Plant. 1993, 87, 569-575.

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(5) Delétage-Grandon, C.; Chollet, J. F.; Faucher, M.; Rocher, F.; Komor, E.; Bonnemain,

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J. L. Carrier-mediated uptake and phloem systemy of a 350-Dalton chlorinated xenobiotic

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with an alpha-amino acid function. Plant Physiol. 2001, 125, 1620-1632.

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(6) Chollet, J. F.; Rocher, F.; Jousse, C.; Delétage-Grandon, C.; Bashiardes, G.;

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Bonnemain, J. L. Synthesis and phloem mobility of acidic derivatives of the fungicide

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fenpiclonil. Pest Manag. Sci. 2004, 60, 1063-1072.

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(7) Jiang, D. X.; Lu, X. L.; Hu, S.; Zhang, X. B.; Xu, H. H. A new derivative of fipronil:

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effect of adding a glycinyl group to the 5-amine of pyrazole on phloem mobility and

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insecticidal activity. Pestic. Biochem. Physiol. 2009, 95, 126-130.

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(8) Yang, W.; Wu, H. X.; Xu, H. H.; Hu, A. L.; Lu, M. L. Synthesis of glucose–gipronil

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gonjugate and its phloem mobility. J Agric. Food Chem. 2011, 59, 12534-12542.

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(9) Wu, H.; Marhadour, S.; Lei, Z. W.; Yang, W.; Marivingtmounir, C.; Bonnemain, J. L.;

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Chollet, J. F. Vectorization of agrochemicals: amino acid carriers are more efficient than

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sugar carriers to translocate phenylpyrrole conjugates in the Ricinus system. Environ. Sci.

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Pollut. Res. Int. 2018, 25, 14336-14349.

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(10) Mao, G. L.; Yan, Y.; Chen, Y.; Wang, B. F.; Xu, F. F.; Zhang, Z. X.; Lin, F.; Xu, H.

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H. Family of Ricinus communis monosaccharide transporters and RcSTP1 in promoting

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the uptake of a glucose-fipronil conjugate. J Agric. Food Chem. 2017, 65, 6169-6178.

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(11) Xie, Y.; Zhao, J. L.; Wang, C. W.; Yu, A. X.; Liu, N.; Chen, L.; Lin, F.; Xu, H. H.

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Glycinergic-fipronil uptake is mediated by an amino acid carrier system and induces the

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expression of amino acid transporter genes in Ricinus communis seedlings. J Agric. Food

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Chem. 2016, 64, 3810-3818.

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(12) Sheng, Q.; Liu, X.; Xie, Y.; Lin, F.; Zhang, Z.; Zhao, C.; Xu, H. H. Synthesis of

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novel amino acid-fipronil conjugates and study on their phloem loading mechanism.

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Molecules 2018, 23, 778; doi:10.3390/molecules23040778.

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(13) Tegeder, M. Transporters for amino acids in plant cells: some functions and many

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unknowns. Curr. Opin. Plant Biol. 2012, 15, 315-321.

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(14) Chen, L.; Bush, D. R. LHT1, a lysine- and histidine-specific amino acid transporter

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in Arabidopsis. Plant Physiol. 1997, 115, 1127-1134.

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(15) Himer, A.; Ladwig, F.; Stransky, H.; Okumoto, S.; Keinath, M.; Harms, A.;

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Frommer, W. B.; Koch, W. Arabidopsis LHT1 is a high-affinity transporter for cellular

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amino acid uptake in both root epidermis and leaf mesophyll. Plant Cell 2006, 18, 1931-

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1946.

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(16) Svennerstam, H.; Ganeteg, U.; Bellini, C.; Nasholm, T. Comprehensive screening of

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Arabidopsis mutants suggests the lysine histidine transporter 1 to be involved in plant

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uptake of amino acids. Plant Physiol. 2007, 143, 1853-1860.

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(17) Liu, G.; Ji, Y.; Bhuiyan, N. H.; Pilot, G.; Selvaraj, G.; Zou, J.; Wei, Y. Amino acid

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homeostasis modulates salicylic acid-associated redox status and defense responses in

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Arabidopsis. Plant Cell 2010, 22, 3845-3863.

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(18) Shin, K.; Lee, S.; Song, W. Y.; Lee, R.; Lee, I.; Ha, K.; Koo, J. C.; Park, S. K.; Nam,

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H. G.; Lee, Y. Genetic identification of ACC-resistance 2 reveals involvement of Lysine

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Histidine Transporter 1 in the uptake of 1-aminocyclopropane-1-carboxylic acid in

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Arabidopsis thaliana. Plant Cell Physiol. 2015, 56, 572-582.

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(19) Yao, G.; Wen, Y.; Chen, Z.; Xu, H. Novel amino acid ester–chlorantraniliprole

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conjugates: design, synthesis, phloem accumulation and bioactivity. Pest Manag. Sci.

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2017, 73, 2131-2137.

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(20) Foster, J.; Lee, Y. H.; Tegeder, M. Distinct expression of members of the LHT

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amino acid transporter family in flowers indicates specific roles in plant reproduction. Sex.

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Plant Reprod. 2008, 21, 143-152.

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(21) Murashige, T.; Skoog, F. A revised medium for rapid growth bioassays with tobacco

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tissues cultures. Physiol. Plant. 1962, 15, 473-497.

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(22) Demidchik, V.; Essah, P. A.; Tester, M. Glutamate activates cation currents in the

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plasma membrane of Arabidopsis root cells. Planta 2004, 219, 167-175.

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(23) Lübbert, H.; Hoffman, B. J.; Snutch, T. P.; Dyke, T. V.; Levine, A. J.; Hartig, P. R.;

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Lester, H. A.; Davidson, N. cDNA cloning of a serotonin 5-HT1C receptor by

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electrophysiological assays of mRNA-injected Xenopus Oocytes. Proc. Natl. Acad. Sci. U.

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S. A. 1987, 84, 4332-4336.

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(24) Svennerstam, H.; Jämtgård, S.; Ahmad, I.; Huss Danell, K.; Näsholm, T.; Ganeteg,

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U. Transporters in Arabidopsis roots mediating uptake of amino acids at naturally

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occurring concentrations. New Phytol. 2011, 191, 459–467.

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(25) Lee, Y.; Foster, J.; Chen, J.; Voll, L. M.; Weber, A. P. M.; Tegeder, M. AAP1

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transports uncharged amino acids into roots of Arabidopsis. Plant J. 2007, 50, 305-319.

457

(26) M'Batchi, B.; Ayadi, R. E.; Delrot, S.; Bonnemain, J. L. Direct versus indirect

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effects of p-chloromercuribenzenesulphonic acid on sucrose uptake by plant tissues: The

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electrophysiological evidence. Physiol. Plant. 1986, 68, 391-395.

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Figure legends

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Fig. 1. The structure of the amino acid chlorantraniliprole (CAP) conjugates used in the

464

study. (a) CAP amino acid ester conjugates and (b) CAP amino acid conjugates Numbers

465

in parentheses corresponds to the code referred in previous study19.

466 467

Fig. 2. The uptake of CAP conjugates in wild type A. thaliana. The concentration of the

468

conjugates within the plant was measured after a 6 h exposure to a 30 μM concentration

469

of each compound. Uptake is expressed as nmol (whole plant) / root FW (fresh weight).

470

Bars represent mean values ± SEM (n = 3). Means differing significantly from the control

471

on the basis of Dunnett’s test are indicated by asterisks (*: P < 0.05, **: P < 0.01).

472 473

Fig. 3. The CAP-Gly-1 in planta content is dependent on the AtLHT1 genotype. The

474

content of CAP-Gly-1 (nmolg-1 fresh weight) after a 6 h exposure in (a) the whole plant,

475

(b) the root, (c) the shoot. (d) The root-to-shoot translocation of CAP-Gly-1 content. Each

476

bar represents a mean ± SEM (n = 3). Means differing significantly (P < 0.05) from one

477

another are headed by a different letter.

478 479

Fig. 4. The effects of inhibitors and competitors on the amount of CAP-Gly-1 in plants

480

carrying variant alleles of AtLHT1 (KO-AtLHT1, WT and 35S-AtLHT1). (a) the effect of

481

known inhibitors of amino acid transporters (50 µM CCCP or 1 mM pCMBS) and (b) the

482

effect of the competitor glycine (30 µM or 100 µM). The data shown represent the mean

483

of batches of 30 plants ± SEM (n = 3). Dunnett’s test was applied to test for differences

484

between means in comparison with the control group. ***: means differed significantly

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(P < 0.001).

486 487

Fig. 5. The activation of membrane potential across the plasma membrane of patch-

488

clamped protoplasts isolated from a line over-expressing AtLHT1 (35S-AtLHT1). The

489

traces show the response to the presence in the external medium of (a) CAP-Gly-1, (b)

490

glycine, (c) glutamic acid, (d) DMSO, (e) CAP; (f)The amplitude of depolarization

491

induced by various substrates (glycine, glutamic acid, CAP-Gly-1 and CAP) in the

492

plasma membrane of protoplasts isolated from the roots of 35S-AtLTH1, WT and 35S-

493

AtLHT1. The variation was analyzed by a two way ANOVA, followed by the application

494

of Scheffe’s test to identify significant differences between means. Different lower case

495

letters indicate differences between genotypic means within each substrate, and different

496

upper case letters indicate differences between treatments within each genotype.

497 498

Fig. 6. The content of CAP-Gly-1 in leaf mesophyll protoplasts is associated with the

499

AtLHT1 genotypes. The content of CAP-Gly-1 in protoplasts was measured after the

500

lapse of 15, 30, 60, 120, and 180 mins. Different letters indicate differed significantly

501

between genotypic means at each time point (P < 0.001).

502 503

Fig. 7. Substrates inducing an inward current in AtLHT1-expressing Xenopus laevis

504

oocytes. (a) Oocytes injected with water, (b) oocyte injected with AtLHT1 cRNA.

505

Downward deflections indicate inward currents induced by the treatment. The horizontal

506

black bars indicate the 30 s exposure period for each substrate. (c) Comparison of inward

507

currents induce by different substrates. Gly and Glu set up as positive control, while CAP

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and DMSO as negative control. The data shown represent the mean of batches of 3

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oocytes ± SEM (n = 5). Dunnett’s test was applied to test for differences between means

510

in comparison with the control group. ***: means differed significantly (P < 0.001).

511 512

Fig. 8. Kinetic analysis of the various substrates and the associated current–voltage

513

relationships for substrate-induced currents. (a) glycine, (b) glutamic acid, (c) CAP-Gly-1,

514

(d) CAP. Substrate-induced currents (background subtracted) were measured at –150 mV.

515

Values displayed in the form mean ± SEM (n = 4). Currents were normalized to Vmax. (e)

516

Current-to-voltage curves. Oocytes were stepped through +50 to -150mV for 300 ms in

517

20 mV decrements. The currents shown represent the difference between those flowing at

518

+300 ms in the presence or absence of the substrates within a single oocyte.

519 520 521 522 523 524 525 526 527 528 529 530

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TOC

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The structure of the amino acid chlorantraniliprole (CAP) conjugates used in the study. (a) CAP amino acid ester conjugates and (b) CAP amino acid conjugates Numbers in parentheses corresponds to the code referred in previous study19. 258x130mm (300 x 300 DPI)

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Fig. 2. The uptake of CAP conjugates in wild type A. thaliana. The concentration of the conjugates within the plant was measured after a 6 h exposure to a 30 μM concentration of each compound. Uptake is expressed as nmol (whole plant) / root FW (fresh weight). Bars represent mean values ± SEM (n = 3). Means differing significantly from the control on the basis of Dunnett’s test are indicated by asterisks (*: P < 0.05, **: P < 0.01). 149x87mm (300 x 300 DPI)

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Fig. 3. The CAP-Gly-1 in planta content is dependent on the AtLHT1 genotype. The content of CAP-Gly-1 (nmolg-1 fresh weight) after a 6 h exposure in (a) the whole plant, (b) the root, (c) the shoot. (d) The rootto-shoot translocation of CAP-Gly-1 content. Each bar represents a mean ± SEM (n = 3). Means differing significantly (P < 0.05) from one another are headed by a different letter. 174x186mm (300 x 300 DPI)

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Fig. 4. The effects of inhibitors and competitors on the amount of CAP-Gly-1 in plants carrying variant alleles of AtLHT1 (KO-AtLHT1, WT and 35S-AtLHT1). (a) the effect of known inhibitors of amino acid transporters (50 µM CCCP or 1 mM pCMBS) and (b) the effect of the competitor glycine (30 µM or 100 µM). The data shown represent the mean of batches of 30 plants ± SEM (n = 3). Dunnett’s test was applied to test for differences between means in comparison with the control group. ***: means differed significantly (P < 0.001). 267x419mm (300 x 300 DPI)

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Figure 5

Fig. 5. The activation of membrane potential across the plasma membrane of patchclamped protoplasts isolated from a line over-expressing AtLHT1 (35S-AtLHT1). The traces show the response to the presence in the external medium of (a) CAP-Gly-1, (b) glycine, (c) glutamic acid, (d) DMSO, (e) CAP; (f)The amplitude of depolarization induced by various substrates (glycine, glutamic acid, CAP-Gly-1 and CAP) in the plasma membrane of protoplasts isolated from the roots of 35S-AtLTH1, WT and 35SAtLHT1. The variation was analyzed by a two way ANOVA, followed by the application of Scheffe’s test to identify significant differences between means. Different lower case letters indicate differences between genotypic means within each substrate, and different upper case letters indicate differences between treatments within each genotype.

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Fig. 6. The content of CAP-Gly-1 in leaf mesophyll protoplasts is associated with the AtLHT1 genotypes. The content of CAP-Gly-1 in protoplasts was measured after the lapse of 15, 30, 60, 120, and 180 mins. Different letters indicate differed significantly between genotypic means at each time point (P < 0.001). 180x149mm (300 x 300 DPI)

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Figure 7

Fig. 7. Substrates inducing an inward current in AtLHT1-expressing Xenopus laevis oocytes. (a) Oocytes injected with water, (b) oocyte injected with AtLHT1 cRNA. Downward deflections indicate inward currents induced by the treatment. The horizontal black bars indicate the 30 s exposure period for each substrate. (c) Comparison of inward currents induce by different substrates. Gly and Glu set up as positive control, while CAP and DMSO as negative control. The data shown represent the mean of batches of 3 oocytes ± SEM (n = 5). Dunnett’s test was applied to test for differences between means in comparison with the control group. ***: means differed significantly (P < 0.001).

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Fig. 8. Kinetic analysis of the various substrates and the associated current–voltage relationships for substrate-induced currents. (a) glycine, (b) glutamic acid, (c) CAP-Gly-1, (d) CAP. Substrate-induced currents (background subtracted) were measured at –150 mV. Values displayed in the form mean ± SEM (n = 4). Currents were normalized to Vmax. (e) Current-to-voltage curves. Oocytes were stepped through +50 to -150mV for 300 ms in 20 mV decrements. The currents shown represent the difference between those flowing at +300 ms in the presence or absence of the substrates within a single oocyte. 277x383mm (300 x 300 DPI)

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