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The family of Ricinus communis monosaccharide transporters and the RcSTP1 in promoting the uptake of a glucose-fipronil conjugate Gen-Lin Mao, Ying Yan, Yan Chen, Bing-Feng Wang, Fei-Fei Xu, Zhi-xiang Zhang, Fei Lin, and Han-Hong Xu J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 10 Jul 2017 Downloaded from http://pubs.acs.org on July 10, 2017

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Journal of Agricultural and Food Chemistry

1

The family of Ricinus communis monosaccharide transporters and the RcSTP1

2

in promoting the uptake of a glucose-fipronil conjugate

3

Gen-Lin Mao ,Yin Yan , Yan Chen, Bing-Feng Wang, Fei-Fei Xu, Zhi-Xiang Zhang,

4

Fei Lin*, Han-Hong Xu*

§

§

5 6 7 8 9

State

Key

Laboratory

for

Conservation

and

Utilization

of

Subtropical

10

Agro-bioresources, and Key Laboratory of Natural Pesticide and Chemical Biology,

11

Ministry of Education, South China Agricultural University, Guangzhou, Guangdong

12

510642, Guangdong, People’s Republic of China.

13 14

§

These authors contributed equally to this work

15

*To whom correspondence should be addressed. Tel: +86-20-85285127. Fax:

16

+86-20-38604926. Email: [email protected] and [email protected]

17 18 19 20 21 22

ACS Paragon Plus Environment


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ABSTRACT: Enhancing the systemic distribution of a bioactive compound by

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exploiting the plant's vascular transport system presents a means of reducing both the

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volume and frequency of pesticide/fungicide application. The foliar uptake of the

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glucose-fipronil

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N-[3-cyano-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(trifluoromethyl)sulfinyl]-1

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H-pyrazol-5-yl]-1-(β-D-glucopyranosyl)-1H-1,2,3-triazole-4-methanamine

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achieved in castor bean (Ricinus communis), and its transport via the phloem is known

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to be mediated by monosaccharide transporter(s) (MSTs), although neither the

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identity of the key MST(s) involved, nor the mechanistic basis of its movement have

32

yet to be described. Based on homology with Arabidopsis thaliana sugar transporters,

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the castor bean genome was concluded to harbour 53 genes encoding a sugar

34

transporter, falling into the eight previously defined sub-families INT, PMT, VGT,

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STP, ERD6, pGlucT, TMT and SUT. Transcriptional profiling identified the product

36

of RcSTP1 as a candidate for mediating GTF uptake, since this gene was induced by

37

exposure of the plant to GTF. By transiently expressing RcSTP1 in onion epidermis

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cells, the site of RcSTP1 deposition was shown to be the plasma membrane. A

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functional analysis based on RcSTP1 expression in Xenopus laevis oocytes

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demonstrated that its product has a high affinity for GTF. The long distance

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root-to-shoot transport of GTF was enhanced in a transgenic soybean chimera

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constitutively expressing RcSTP1.

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KEYWORDS: monosaccharide transporter, Ricinus communis, glucose-fipronil,

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transgenic soybean composite plants, Xenopus oocyte

conjugate


(GTF)

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Certain pesticidal compounds are taken up by plants and then distributed systemically

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via both phloem and xylem. This characteristic is highly desirable, as it allows full

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protection to be achieved following either a foliar application or a simple irrigation of

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the roots1,2. As yet only a small number of insecticides are known to have this

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property: one of these is spirotetramat, a relatively new product derived from tetramic

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acid, which has been shown to be transported to both the shoot and the roots via the

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phloem and xylem, thereby providing a good level of control against aphids,

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whiteflies and psyllids3.

INTRODUCTION

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It has been suggested that systemic pesticides could be engineered by various

55

relatively simple chemical modifications of the active molecule4-6. One possibility is

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to confer phloem mobility by exploiting the activity of native transporters. An

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example is the modified form of E-2,4-dichlorophenoxyacetyl (2,4-D), which when

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conjugated with an α-amino acid, is readily translocated via the phloem in Vicia faba

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via an carrier system5. Similar results have also been achieved for the herbicide

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glyphosate and 2,4-D, which are also mediated by phloem mobility and may be

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loaded by a phosphate transporter and an Aromatic and Neutral Amino acid

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transporter 1 (ANT1), respectively7,8.

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The

glucose-fipronil

conjugate

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N-[3-cyano-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(trifluoromethyl)sulfinyl]-1

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H-pyrazol-5-yl]-1-(β-D-glucopyranosyl)-1H-1,2,3-triazole-4-methanamine (GTF) has

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been shown to enjoy a higher level of phloem mobility than that of fipronil itself4. The



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fluorescence-enabled visualization of the uptake and transport of GTF in the castor

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bean (Ricinus communis) seedling has shown that, after transiting the epidermis and

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mesophyll, the GTF is loaded into sieve tubes and then translocated from the

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cotyledon to the hypocotyl via the phloem9. Both phloridzin and carbonyl cyanide

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m-chlorophenylhydrazone markedly inhibit GTF uptake, while D-glucose is only a

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moderate competitor for GTF and sucrose is non-competitive. The implication has

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been drawn that GTF uptake, at least in the castor bean seedling, is partially mediated

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by

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monosaccharides across a hydrophobic membrane11.

monosaccharide

transporters

(MSTs)10,

proteins

which

shuttle

soluble

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The MST polypeptide family has been organized into a number of sub-groups,

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referred to as sugar transporter proteins (STPs), polyol/monosaccharide transporters

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(PMTs), inositol transporters (INTs), plastidial glucose transporters/suppressors of G

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protein (pGlucTs/SBGs), tonoplastic monosaccharide transporters (TMTs), early

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response to dehydration transporters (EDRs) and vacuolar glucose transporters

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(VGTs)12. The STPs, PMTs and INTs are all associated with the plasma membrane

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and facilitate the import of glucose, fructose, galactose, mannose, xylose, sorbitol,

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mannitol, xylitol and myo-inositol; the pGlucTs export glucose from the plastid, while

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the SGBs enable glucose to be imported into the Golgi apparatus; the TMTs and

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ERD-like transporters assist in the import/export of monosaccharides across the

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vacuolar membrane; finally, the VGTs are important during both germination and

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flowering13. The MSTs as a whole are able to transport a broad spectrum of

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monosaccharides, but individual members of the family show distinct substrate


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specificity11,14. The Arabidopsis thaliana genome harbors 53 distinct MST-encoding

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genes, but to date only two have been described in the castor bean15. The focus of the

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present study was to identify which MST(s) in castor bean is/are involved in the

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uptake and translocation of GTF.

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MATERIALS AND METHODS

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Plant Materials, Chemicals and Plasmid Vectors. R. communis seeds were

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obtained from the Agricultural Science Academy (Zibo, Shandong, China) and those

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of soybean cv. Yuechun 04-5 were obtained from the Root Biology Centre (South

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China Agricultural University, Guangzhou, China). GTF was prepared according to

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our previously described4. GTF and fipronil were dissolved in DMSO. And in all

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treatments, the final concentration of DMSO was below 1% (v/v). Agrobacterium

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rhizogenes strain K599 was also provided by the Root Biology Centre (South China

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Agricultural University). The vectors pCam-35S and pEGFP were obtained from the

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South China Agricultural University College of Life Sciences. The vector pT7TSHA

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was kindly provided by the Institute of Plant Protection (Chinese Academy of

105

Agricultural Sciences, Beijing, China). Six days after sowing, the cotyledons of

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uniformly-sized seedling, with residual endosperm being removed, were incubated in

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a solution containing 20 mM MES (pH 5.5), 0.25 mM MgCl2, and 0.5 mM CaCl2

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with 100 µM GTF or without 100 µM GTF. As solvent control, a solution of 1% (v/v)

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DMSO replaced the 100 µM GTF was also included in the treatment. The cotyledons

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were harvested at 3 h and 6 h post treatments, snap-frozen in liquid nitrogen and



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stored at −80°C until ready for RNA extraction.

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In silico Identification of R. communis Sugar Transporter Genes and Their

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Phylogeny. The R. communis genome sequence, downloaded from the Phytozome

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v9.1 database (www.phytozome.net/ricinus.php), was scanned using the HMM search

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program for the presence of sequences harboring motifs associated with “sugar (and

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other) transporters” (Pfam HMM profile PF00083) (hmmer.janelia.org/). The selected

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sequences were first validated by subjecting them to a BlastP search against the set of

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known A. thaliana sugar transporters, and finally, all available EST data related to

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these genes were assembled following a BlastN search against the R. communis NCBI

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dbEST dataset (http://www.ncbi.nlm.nih.gov). Predicted products of below 300

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residues in length were excluded, since these are thought unlikely to be functional16,17.

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The set of possible R. communis sugar transporters was aligned using the Clustal X2

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algorithm and a phylogenetic tree was constructed based on the neighbor-joining

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method, using routines implemented in MEGA6 software18. Sub-family identity was

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assigned on the basis of homology with A. thaliana sequences (www.arabidopsis.org).

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RT-PCR and Quantitative Real Time PCR (qRT-PCR) Analysis. The set of

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RcMSTs,

obtained

from

the

128

(www.phytozome.net/ricinus.php), was targeted for amplification using primer pairs

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listed in Table S1. RNA was extracted from frozen cotyledons using a Plant RNA kit

130

(OMEGA, Guangzhou, China). An 1 µg aliquot used as the template for synthesizing

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the cDNA first strand, using the iScript™ Reverse Transcription system (Bio-Rad,

132

Philadelphia, PA, USA) after treating with DNase I (NEB, Ipswich, MA, England)

R.

communis

gene


annotation

database

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was used to remove the contaminating genomic DNA. An RT-PCR assay, based on a

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bulked sample of cDNA template, was performed to confirm the presence of MST

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transcript, with R. communis genomic DNA included as a positive control. The

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resulting amplicons were visualized by electrophoretic separation through a 2% (w/v)

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agarose gel. Genes displaying a detectable level of transcription were monitored via a

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quantitative real time PCR (qRT-PCR) assay of the response of cotyledon explants to

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a 6 h exposure to GTF. The qRT-PCRs were performed as described elsewhere19,

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Relative transcript abundances were calculated from the abundance of RcActin

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transcript (GenBank accession: NM_001323740.1, for primer sequence please see

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Table S1), using the ∆∆Ct method20. Difference between treatment means value were

143

tested

144

(https://spss.en.softonic.com/)

for

significance

using

routines

implemented

in

SPSS

software

145

Subcellular Localization of Gene Expression. The open reading frame of

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RcSTP1, previously known as RcHEX315, was inserted in frame in front of the

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enhanced green fluorescent protein (EGFP) coding sequence within a pEGFP vector

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using a two-step Ω-PCR procedure21. Specifically, in the first reaction, the chimeric

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primer pair pGFP-RcSTP1F and pGFP-RcSTP1R (see Table S1) was used to amplify

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the RcSTP1 sequence, and a 2-3 µL aliquot of this reaction was then used as the

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primer for the second reaction with pEGPF as template. The final product was

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incubated at 37℃ for 30 min in the presence 5-10 U DpnI to degrade the plasmid

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sequence, and the product was inserted into E. coli (DH5α) competent cells. Positive

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clones were validated by sequencing. The fusion vector pEGFP-RcSTP1 was



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transiently transformed by bombardment (PDS/1000 device, Bio-Rad) into onion

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epidermal strips supported on an agar plate. The bombardment parameters were:

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bombardment pressure 1,100 psi, gold particle diameter 1.0 mm, separation distance 9

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cm, and decompression vacuum 1,000 psi. After a 16 h incubation, fluorescence was

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captured by a laser scanning confocal microscope (Zeiss LSM780, Germany), using a

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wavelength of 488 nm for excitation and 515-545 nm and 610 nm filters for detection.

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Functional Analysis in Xenopus laevis Oocytes. Transporter activity was

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determined using a two electrode voltage clamp technique22-24. The 1.6 kb opening

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reading frame of RcSTP1 gene was amplified by primer pair pT7-RcSTP1F and

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pT7-RcSTP1R (Table S1). The plasmid pT7TSHA was linearized by cutting at its Spe

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℃ and Sph ℃ sites and then recombined with the RcSTP1 amplicon using an In-Fusion

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Cloning Kit (Clontech, Mountain view, CA, USA). The resulting plasmid was

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linearized by restriction with Sma I, then a transcribed the Capped mRNA in vitro

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using

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http://www.thermofisher.com/cn/zh/home/brands/ ambion.html).

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laevis oocytes were isolated and injected with 27.6 nL (1 ng/nL) RcSTP1 cRNA and

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

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gentamycin. The oocytes were then bathed in modified sodium Ringer solution (96

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mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 5 mM HEPES, 5 mM MgCl2, pH 5.5 adjusted

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with NaOH) with continuous perfusion at a rate of 3 mL/min. The recording pipettes

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were filled with 3M KCl, delivering an electrical resistance of 1-5 MΩ. Currents were

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measured using a oocyte clamp amplifier (Model OC-725C, Warner Instruments,

mMessage

Mmachine

kits


(Thermo

Fisher,

Stage ℃ and ℃ X.

Page 9 of 40


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Hamden, CT, USA), filtered at 200 Hz and digitized at 2,000 Hz. Holding potential

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was -90 mV, and voltage pulses from -150 mV to +50 mV were applied for 100 ms.

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Steady-state currents are presented as the mean current between 50 and 100 ms

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following the onset of voltage pulses and were obtained by subtracting an average of

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the background current recorded before and after substrate application. The data were

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acquired and analyzed with the help of Digidata 1440A and pClamp10.0 software

183

(Axon Instruments Inc., Union City, CA, USA). Dose-response data were analyzed

184

using GraphPad Prism 5 (http://www.graphpad.com/scientific-software/prism/).

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Root Uptake and Root-shoot Translocation of GTF and Fipronil in Soybean

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Seedlings. Due to the current lack of efficient transformation technology for R.

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communis, a soybean model was used to explore the function of RcSTP1 with respect

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to the uptake of GTF and its translocation to the root and shoot. Four day-old soybean

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seedlings were cultured hydroponically in half-strength Hoagland’s solution for one

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week under controlled conditions (16 h photoperiod; light period temperature 28 ±

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2°C, dark period temperature 22 ± 2°C; relative humidity 70%). The roots were

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bathed for either 6 h or 12 h in half strength Hoagland’s solution containing 0.1%

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(v/v) Tween-80 and 50 µM GTF or 50 µM fipronil, after which the plants were

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separated into their aerial part and their roots, macerated and extracted with acetone.

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The extractant was purified using an AccuBond C18 SPE solid-phase extraction kit

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(Agilent Technologies, Santa Clara, CA, USA), following a previously published

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protocol4. The content of the resulting solutions was analyzed using an Agilent

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Technologies 1100 HPLC system equipped with a vacuum degasser, a quaternary



199

pump, an autosampler, a UV-visible detector and an Agilent C18 reverse-phase

200

column (5 µm, 250 mm × 4.6 mm i.d.) held at 30°C. The flow rate was 1 mL/min, and

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the injection volume was 10 µL. The mobile phase consisted of a 1:1 mixture of

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acetonitrile and water and the absorbance wavelength was 210 nm. A series of

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standard solutions of GTF or fipronil (0.5, 1, 5, 10, 25, and 50 µM) was prepared in

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methanol for calibration purposes. In order to measure the content of GTF and

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fipronil, standard curves were made. The linear equation of GTF was y = 20.31x -

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0.9439 (r = 0.9993) and that for fipronil was y = 20.26x + 0.3991 (r = 0.9999).

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Recovery studies were conducted at three spiking levels: 0.1 mg/Kg, 0.5 mg/Kg and 1

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mg/Kg.

209

were all >80%.

The individual mean recovery rates for GTF and fipronil in the plant tissue

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The Heterologous Expression of RcSTP1 in Soybean Hairy Roots. The

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RcSTP1 cDNA sequence was PCR-amplified using the primer pair p35S-STP1F and

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p35S-STP1R (Table S1). The amplified fragment was then transferred into the

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pCam-35S vector using the appropriate restriction enzyme sites. The resulting

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construct, comprising the RcSTP1 opening reading frame driven by a CaMV 35S

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promoter, was transformed into Agrobacterium rhizogenes strain K599 by

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electroporation. Transgenic chimeric soybean plants, consisting of a wild type shoot

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and transgenic hairy roots, were treated to induce the expression of the transgene

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using the hypocotyl injection method described by25,26. Briefly, seeds were

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surface-sterilized by immersion for 1 min in 3% (v/v) H2O2, rinsed with sterile water

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and germinated in sand medium. The hypocotyls of five day-old seedlings (cotyledons


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unfolded) were infected with A. rhizogenes strain K599 carrying the transgene and

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empty vector, and the plants were kept under high humidity. When newly developed

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hairy roots had reached a length of ~10 cm, the wild type roots were excised and the

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plants were cultured in half-strength Hoagland's solution for 1~2 weeks to allow for

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further growth of the hairy roots. Hairy roots harbouring an empty vector were stained

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for GUS activity following a published method27. DNA was extracted from hairy root

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segments, and subjected to a PCR assay targeting RcSTP1 to identify those which had

228

been successfully transformed. The roots of confirmed transformants were incubated

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in half strength Hoagland’s solution containing 0.1% (v/v) Tween-80 and 50 µM GTF,

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and harvested after 2 h, 6 h or 12 h. The plants were separated into upper and lower

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parts and roots (Fig. S1). GTF extraction and sample analysis were performed as

232

described above.

233 234

RESULTS

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The Members of the R. communis Sugar Transporter Family. A total of 99

236

protein sequences were identified as harboring a potential “sugar (and other)

237

transporters” domain was recovered. Of these, 63 shared at least 45% (and up to 84%)

238

similarity to an A. thaliana sugar transporter; all but ten of the 63 specified a product

239

longer than the 300 residues required to act as a functional transporter. Their encoding

240

genes were distributed across 33 scaffolds of the R. communis genome (Table 1). The

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final set of 53 sugar transporters contained three sucrose transporters (SUTs) and 50

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MSTs. MSTs were classified into seven sub-families: RcINTs (six proteins), RcPMTs



243

(seven), RcVGTs (two), RcSTPs (ninteen), RcERD6s (nine), RcpGlucTs (four),

244

RcTMTs (three) (Table 1). Three of the genes (RcSTP1, RcSTP9 and RcSUC2) are

245

synonyms of the genes RcHEX3, RcHEX6 and RcScr1, respectively15,28.A

246

phylogenetic analysis, based on the proteins' full length sequences with the

247

neighbor-joining method and 1000 bootstrap replicates, showed the 53 genes belong

248

to eight established sub-groups (Fig. 1).

249

Transcriptional Response of the RcMSTs to GTF Treatment. Based on

250

RT-PCR analysis of cotyledons subjected to a 6 h exposure to GTF, nine of the

251

RcMSTs showed no trace of transcription, four showed spliceosome transcripts and

252

nine were weakly transcribed (Fig. 2A, table 1). A more detailed analysis of 35 of the

253

RcMSTs (Table 1) was conducted using qRT-PCR: this showed that after a 6 h

254

exposure to GTF, 26 of them were up-regulated (seven by more than two fold) (Fig.

255

2B) and eleven were down-regulated (the only gene by more than two fold was

256

RcTMT3) (Fig. 2C). The behavior of the RcSTPs was also monitored following a 3 h

257

exposure to GTF; all of these, with the sole exception of RcSTP1, were

258

down-regulated by the treatment (data not shown), with RcSTP1 transcript abundance

259

being reduced by around five fold when compared with the negative control (Fig. 3).

260

At the 6 h time point, the abundance of RcSTP1 transcript induced by GTF was

261

significantly (p 0.05). 223x167mm (300 x 300 DPI)



Figure 4 The sub-cellular localization of RcSTP1 in transiently transformed onion epidermal cells. The transgenes were (A, C) p35S::GFP and (B, D) p35S::RcSTP1cDNA-GFP. (A, B) Z-projections of optical sections. (C, D) individual optical sections from the center of the cells show in (A) and (B), respectively. RcSTP1 localizes to the plasma membrane and lacks cytoplasmic strands (arrowed in (C)) as introduced by the p35S::GFP transgene. 190x190mm (150 x 150 DPI)


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Figure 5 Inward currents in X. laevis oocytes in response to the treatments with glucose and GTF. The oocytes were tested using voltage clamped at -90 mV. Non-injected oocytes are shown in the upper trace and those injected with RcSTP1 cRNA are shown in the lower trace. Oocytes were perfused with 200 µM glucose or GTF at the indicated times points. 77x38mm (300 x 300 DPI)



Figure 6 Kinetic analysis of glucose- and GTF-induced currents. Oocytes harboring RcSTP1 were perfused at a holding potential of -50mV. The data have been fitted to the Michaelis-Menten equation. The error bars show the SE (n = 5 oocytes). (A) K0.5 value for glucose (0.059 ± 0.023 mM ). (B) K0.5 value for GTF (0.042 ± 0.003 mM). 146x50mm (220 x 220 DPI)


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Figure 7 The distribution of fipronil and GTF in wild type soybean seedlings. The detection is carried out following a 6 h or a 12 h incubation of the roots in (A) fipronil, or (B) GTF. Data were shown in the form mean ± SE (n = 3) with a column topped by different letters are significantly different from each other as determined by Duncan’s multiple-range test (P > 0.05). 191x74mm (150 x 150 DPI)



Figure 8 PCR-based validation of transgenicity of hairy root cultures transformed with pCaMV35S::RcSTP1. M: DL5000 size marker; lanes 1-4: template extracted from the root of putative transgenic lines. Con: hairy roots harboring an empty vector, N: non-transgenic roots. 813x437mm (150 x 150 DPI)


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Figure 9 The distribution of GTF in transgenic soybean seedings following the incubation of hairy roots in 100 µM GTF. Plants harboring either an empty vector or pCaMV35S::RcSTP1. Samples were taken after (A) 1 h, (B) 2 h, (C) 6 h, (D) 12 h. Data shown in the form mean ± SE (n = 3). Differences being statistical significant (p < 0.05) were determined by the Students' t test, and indicated by asterisks. 201x141mm (150 x 150 DPI)



537x218mm (150 x 150 DPI)


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