Ethylene stimulation of latex yield depends on the expression of a
sucrose transporter (
HbSUT1B) in rubber tree (Hevea brasiliensis)
ANAÏS DUSOTOIT-COUCAUD,
1,2PANIDA KONGSAWADWORAKUL,
3LAURENCE MAUROUSSET,
4UNSHIRA VIBOONJUN,
3NICOLE BRUNEL,
1,2VALÉRIE PUJADE-RENAUD,
1,5HERVÉ CHRESTIN
3,6and SOULAÏMAN SAKR
1,7,81 Clermont Université, Université Blaise Pascal, UMRA 547 PIAF, F-63177 Aubière, France 2 INRA, UMRA 547 PIAF, F-63100 Clermont-Ferrand, France
3 Department of Plant Science, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand 4 Free 3091 PHYMOTS, Université de Poitiers, 40 avenue du Recteur Pineau, Poitiers cedex, France
5 Centre International de Recherche et d’Aide au Développement, UMR DAP, 24 avenue des Landais, 63177 Aubière cedex, France
6 Institut de Recherche pour le Développement (IRD), UR 060 CLIFA/CEFE-CNRS, 1919 route de Mende, F34293, Montpellier cedex 5, France 7 Agrocampus Ouest, Centre d’Angers, UMR SAGAH, IFR QUASAV 149, 2 rue Le Nôtre, 49045 Angers cedex, France
8 Corresponding author (soulaiman.sakr@agrocampus-ouest.fr)
Received February 20, 2010; accepted September 5, 2010; published online October 26, 2010; handling Editor Ron Sederoff
Summary Hevea brasiliensis is an important industrial crop for natural rubber production. Latex biosynthesis occurs in the cytoplasm of highly specialized latex cells and requires sucrose as the unique precursor. Ethylene stimulation of latex production results in high sugar flow from the surrounding cells of inner bark towards the latex cells. The aim of this work was to understand the role of seven sucrose transporters (HbSUTs) and one hexose transporter (HbHXT1) in this process. Two Hevea clones were used: PB217 and PB260, respectively described as high and low yielding clones. The expression pattern of these sugar transporters (HbSUTs and HbHXT1) was monitored under different physiological con-ditions and found to be maximal in latex cells. HbSUT1, one of the most abundant isoforms, displayed the greatest response to ethylene treatment. In clone PB217, ethylene treatment led to a higher accumulation of HbSUT1B in latex cells than in the inner bark tissues. Conversely, stronger expression of HbSUT1B was observed in inner bark tissues than in latex cells of PB260. A positive correlation with HbSUT1B transcript accumulation and increased latex pro-duction was further supported by its lower expression in latex cells of the virgin clone PB217.
Keywords: ethylene, latex yield, laticifers, sugar transporters.
Introduction
Hevea brasiliensis is a tree of the Euphorbiaceae family, originating in the Amazonian basin and now cultivated in
numerous tropical countries. This tree produces natural rubber (cis-polyisoprene), used in many industries. Natural rubber biosynthesis occurs in the latex, the cytoplasm of highly specialized latex cells (laticifers). These cells are rhythmically differentiated as single cells from the cambium and arranged in concentric layers (mantels). Upon matu-ration, the laticifers anastomose within each mantel, build-ing laticifer networks, referred to as a para-circulatory system (Hébant and de Faÿ 1980,de Faÿ and Jacob 1989). Their cytoplasm contains numerous rubber particles in suspension, accounting for as much as 90% of the latex dry weight. Bark treatment with Ethrel® (ethephon i.e., 2-chloroethylphosphonic acid, an ethylene releaser) stimu-lates latex regeneration (d’Auzac and Ribaillier 1969) and such treatment is now widely used in rubber estates to increase yield.
Polyisoprene (rubber) biosynthesis may follow two differ-ent pathways: (i) the mevalonate pathway, which that has been well characterized in the latex (Bandurski and Teas 1957, Hepper and Audley 1969) and (ii) the DEX/MEP (1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol)
pathway, which was proposed by Ko et al. (2003), but has been called into question (Sando et al. 2008). Whatever the pathway, sucrose is the unique precursor of natural rubber (d’Auzac 1964,Chow et al. 2007), and its transport into latex cells may be key to latex generation.
Latex cells are heterotrophic and need to be supplied with photoassimilates to meet their high carbon and energy demands (Tupy 1973,Eschbach et al. 1986,d’Auzac et al. 1989, Silpi et al. 2007). Data from cytological studies reporting that the laticifers are devoid of plasmodesmata
Tree Physiology30, 1586–1598 doi:10.1093/treephys/tpq088
© The Author 2010. Published by Oxford University Press. All rights reserved.
(Hébant 1981,de Faÿ et al. 1989), and electrophysiological investigations (Bouteau et al. 1991, 1992, 1999) have suggested the presence of active sugar/H+symporters on the latex cell plasma membrane. In support of this hypothesis, seven putative sucrose transporters (HbSUTs) were cloned from a latex cDNA library, and were classified into three groups: group II for HbSUT1A and HbSUT1B, group III for HBSUT2A, HbSUT2B and HbSUT2C and group IV for HbSUT4 and HbSUT5 (Dusotoit-Coucaud et al. 2009). All these putative transporters were expressed in source and sink organs, including latex cells. In the virgin (unexploited) tree, ethylene-stimulated latex production was only associ-ated with high expression of two putative sucrose trans-porters, i.e., HbSUT1A and HbSUT2A. In addition, this response was specific to latex cells and was not found in other living cells in the inner bark (Dusotoit-Coucaud et al. 2009). No information is available on the role of these genes in ethylene-enhanced latex production of exploited trees.
In sink organs, sugar absorption can operate in two dis-tinct ways (Lemoine 2000,Lalonde et al. 2004,Rolland et al. 2006, Sauer 2007, Braun and Slewinski 2009). In Hevea trees, sucrose is known to be the main sugar trans-ported into sink latex cells, where it is used for latex gen-eration, including rubber synthesis (90% of total metabolism), as well as in osmoregulation (Jacob et al. 1989). As in the case of sucrose, electrophysiological data showed that exogenous glucose induced plasma membrane depolarization of isolated latex cells or protoplasts, suggesting the presence of a putative glucose/H+ sympor-ter in these cells (Lacrotte 1991, Bouteau et al. 1992,
1999). In this case, glucose would directly enter glycoly-sis to provide pyruvate and then acetyl-CoA, the precursor of cis-polyisoprene biosynthesis. No hexose transporter has been isolated to date.
To acquire an initial insight into the role of sugar (sucrose and hexose) transporters in ethylene-stimulated latex production in industrially exploited trees, this work has been conducted on two widely cultivated H. brasiliensis clones: PB217 and PB260. These clones differ in their metabolism and their response to ethylene (Jacob et al. 1995,Gohet 1996,Gohet et al. 2003). Clone PB260 has an active metabolism and an intermediate sugar loading capacity. This clone expresses its high yield, without stimu-lation, but responds poorly to Ethrel®. Clone PB217 has lower metabolic activity, but probably high sugar loading capacity. Clone PB217 is low yielding when not stimulated, but high yielding when it is stimulated with Ethrel®.
For a better understanding of the role of sugar transpor-ters in ethylene stimulation of latex production in H. brasiliensis, a programme aimed at the characterization of various membrane transporters in latex cells is being developed. This work characterizes seven sucrose transpor-ters and one hexose transporter in latex cells and their surrounding cells of inner bark, in these two widely used clones PB217 and PB260.
Materials and methods Plant material
Field experiments were carried out at the Bongo/SAPH plantation (Côte d’Ivoire). Latex and bark samples were col-lected from the trunks of mature PB217 and PB260 rubber trees (10 years old), industrially harvested for 2 years. These trees had been regularly tapped (three times per week) without Ethrel® stimulation for 3 months and left untapped for 1 week before Ethrel®treatment and the first sample collection. The latex samples were collected as described byPujade-Renaud et al. (1994). Briefly, after dis-carding the first 20 drops, the latex samples were collected from three trees and pooled in the same tube, as a mixture of 2 ml per sample in an equal volume of 2× fixation buffer. The bark samples were collected 5–15 cm below the tapping cut in the same tube. These latex and bark samples were immediately deep-frozen in liquid nitrogen and stored at−80 °C.
Back-cut trees (cut 6 months earlier) grown in a controlled environment chamber (CPN, Michelin Group, Clermont-Ferrand, France) were used to analyse hexose transporter gene expression in different organs (mature leaves, bark and xylem) and for immunolocalization of HbSUT1B in stems. The culture conditions of these young Hevea trees were described inDusotoit-Coucaud et al. (2009).
Field experiment
Seven groups of three trees, tapped for 2 years, of clones PB217 and PB260, selected for their growth and yield hom-ogeneity, as well as seven batches of mature virgin trees selected for their growth homogeneity, were sampled in a similar way, as described by Pujade-Renaud et al. (1994)
and Sookmark et al. (2002). Two remained unstimulated (control) and five were treated with 1 g of 2% Ethephon/ palm oil mixture on a 1-cm wide, slightly scraped bark band, just beneath the half spiral (S/2) tapping cut. Trees were treated for 4, 8, 16 and 24 h, respectively, before sim-ultaneous sampling. Each treatment was carried out on three independent trees. The control trees were also treated the same way, but only with 1 g of palm oil without ethephon. The latex and bark samples were collected on the same day and at the same hour.
RNA extraction
Total RNA extraction from latex was performed according to Pujade-Renaud et al. (1994,1997) and Sookmark et al. (2002). Total inner bark RNA extraction from mature trees was performed using a caesium chloride cushion (Sambrook et al. 1989), with 2 g of inner bark ground in liquid nitrogen. Total RNA extraction from leaves, bark and xylem of young trees was carried out as described by Dusotoit-Coucaud et al. (2009).
Molecular cloning of hexose transporter (HXT) full-length cDNA
One latex hexose transporter probe was obtained by poly-merase chain reaction (PCR) directly from latex cDNA libraries. Degenerate primers were designed from conserved regions of plant hexose transporter cDNAs (EMBL data library): [ primer HXT1F (50-TTBCARCANBTNACNGG NATNAA-30) and primer HXT1R (50-GGNCCCCANG ACCANGCAAA-30)]. An amplified fragment of expected size 345 bp was cloned and sequenced, and then used as an HXT homologous probe to screen the latex cDNA libraries under low stringency. Two clones were obtained and proved to correspond to one isoform, referred to as HbHXT1 (H. brasiliensis HeXose Transporter 1), registered in the EMBL/GenBank/DDBJ database under the accession number FM244737.
Specific primer for real-time RT–PCR
Previously described SUT-specific primers for HbSUTs were used to carry out real-time RT–PCR ( Dusotoit-Coucaud et al. 2009). For HbHXT1, a specific primer pair was designed [ primer HXT1F (50-GGGCTTTTCCTCTTC TTTGC-30) and primer HXT1R (50-ACACAGTCTTGGGA CCTTGG-30)] and its specificity was tested by carrying out semi-quantitative and real-time RT–PCR on each full-length cDNA cloned in the pGEM-T easy vector (Promega, Madison, WI, USA).
Quantification of sugar transporter transcripts by real-time RT–PCR
After treatment with DNase I, 2 µg of total RNA were used as template for first-strand cDNA synthesis (SuperScript-III, Invitrogen, Carlsbad, CA, USA). Real-time PCR was carried out using the cDNA as template, the specific primers, a fluorescent dye (SYBR-Green) and an iCycler (Bio-Rad, Hercules, CA, USA). Rubber tree cDNA encod-ing actin was used as an internal control. Polymerase chain reactions were performed as follows: 5 min at 95 °C for denaturation, 20 s at 95 °C, 35 cycles of 20 s at 58 °C, 20 s at 72 °C for amplification and 10 min at 72 °C for final extension. Relative PCR efficiencies were determined using a dilution series of RT–PCR products as standards. All reac-tions were performed in triplicate, and ΔCt was calculated as described inDusotoit-Coucaud et al. (2009). To compare the abundance of sugar transporter transcripts in both clones, the expression levels were calculated from the formula: Expression = E(Ct(actin)−Ct(sugar transporter)). Real-time RT–PCR data are the means of three replicates. The error bars are technical standard deviation.
Preparation of anti-HbSUT1B antibody
Anti-HbSUT1B antiserum was developed in rabbits (Proteogenix, Oberhausbergen, France) against a synthetic peptide (PDAPSAKTSRAVTAAFH) coupled to a carrier
protein (KLH). Its sequence was derived from the specific C-terminal region of the HbSUT1B protein.
Isolation of the microsomal fraction, sodium dodecyl sulphate–polyacrylamide gel electrophoresis and western blotting
Isolation of bark microsomal fractions was performed accord-ing toDecourteix et al. (2006). Protein samples (30 mg) were subjected to sodium dodecyl sulphate (SDS)–polyacrylamide gel electrophoresis (PAGE) according to Laemmli (1970). The gel system consisted of a 5% stacking gel and a 10% resolving gel. After gel electrophoresis, polypeptides were electroblotted onto a nitrocellulose membrane (Trans-Blot®, Bio-Rad). The transfer buffer contained 25 mM Tris, 193 mM glycine, 0.1% (w/v) SDS and 20% (v/v) methanol. The trans-fer was performed at a constant current of 400 mA for 2 h at room temperature. Tris-buffered saline (TBS) was used as the buffer for immunoblotting. Tween 20 [0.1% (v/v)] and 10% (w/v) dry milk were used to block nitrocellulose filters. Immunodetection was achieved with the HbSUT1B rabbit antiserum diluted 1/2000 as primary antibody, and antirabbit IgG (H + L) (P.A.R.I.S., Compiègne, France) labelled with peroxidase as a secondary antibody, diluted 1/10,000. The protein–antibody complex was detected with Western LightningTMPlus-ECL (Perkin Elmer, Shelton, CT, USA). HbSUT1B immunolocalization in young stems
Young stem pieces from back-cut trees were fixed in FAA (3.7% (v/v) formaldehyde, 50% (v/v) ethanol, 5% (v/v) acetic acid) at 4 °C overnight. Fixed tissues were dehydrated through a graded series of ethanol and the samples were gradually infiltrated with medium grade L.R. White resin (Sigma-Aldrich, St Louis, MO, USA) with solutions of 25:75, 50:50 and 75:25 (v/v) L.R. White: 100% ethanol for 1 h each. Infiltration was completed with two incubations in 100% L.R. White at room temperature for 1 h and overnight, respectively. The samples were placed in gelatin embedding capsules, filled with fresh resin and heat poly-merized at 55 °C for at least 15 h. Samples were cut into 2–3 µm sections with a microtome. Sections were rinsed in phosphate-buffered saline (PBS), pH 7.2, and held for 15 min in PBS, 0.1% (v/v) Triton X-100. Sections were then placed for 15 min in PBS supplemented with 0.1% (v/v) Triton X-100, 0.2% (w/v) glycine ( pH 7.2). After washing in PBS–Triton (0.1%) medium, non-specific sites were satu-rated for 45 min by 5% (v/v) normal goat serum in PBS, pH 7.2, 0.1% (v/v) Triton X-100, 0.2% (v/v) Tween and 0.1% (w/v) bovine serum albumin (BSA), and incubated overnight at room temperature with 1/2000 diluted anti-serum against HbSUT1B. After washing in PBS, pH 7.2, and 0.1% (v/v) Triton X-100 (2 × 20 min) and then in TBS, pH 8.2, 0.2% (v/v) Triton X-100 (2 × 15 min), the sections were placed for 45 min in blocking solution: TBS, pH 8.2, 0.2% (v/v) Triton, 0.2% (v/v) Tween 20, 1% (w/v) BSA and 5% (v/v) normal goat serum. Sections were then
incubated at room temperature for 2 h 30 min with goat anti-rabbit antibody conjugated to alkaline phosphatase (Sigma), diluted 1/40 in blocking solution. Tissue sections were washed with TBS, 0.2% Triton X-100 (3 × 20 min) and then incubated, at room temperature, with chromogenic substrates nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) (Bio-Rad). Colour development was carried out at room temperature and was stopped by washing in H2O. Sections were then mounted
onto microscope slides, air-dried and covered with coverslips for microscopy using Eukitt (Kindler GmbH & Co., Freiburg, Germany) mounting medium.
Statistical analysis
Because empirical errors on Qr increased with Qr values,
consistent with the above exponential formulation, statistical procedures were performed on log-transformed data. By using Student’s t-test, we considered only the results statisti-cally different with P < 0.01.
Results
Cloning cDNA of a putative hexose transporter from latex A latex-derived cDNA library was screened using a PCR-amplified HXT-homologous probe. Only one full-length cDNA encoding a putative hexose transporter (accession number FM244737) was isolated, even under low stringency screening conditions. Sequence identity shared by HbHXT1 and the previously cloned hexose trans-porters from other plant species ranged between 89 and 57% (Figure1a). The highest percentage identity was found for a Populus tremuloides monosaccharide transporter (PtMT). Finally, HbHXT1 has the characteristics of the published plant hexose transporters, such as membrane topology with 12 predicted membrane spanning domains (Büttner and Sauer 2000,Büttner 2007).
The expression of HbHXT1 was monitored by real-time RT–PCR in different organs: mature leaves, bark and xylem of young PB217 and PB260 trees (Figure1b). HbHXT1 was more abundant in PB217 than in PB260. In PB217, HbHXT1 was very highly (120-fold over actin) and moderately (35-fold) expressed in leaves and bark, respectively, and almost not detectable in xylem. In PB260, HbHXT1 also exhibited the highest relative abundance in leaves (38-fold).
Figure 1. Characterization of HbHXT1. (a) Phylogenetic tree of plant hexose transporters exhibiting sequence similarity with HbHXT1, was constructed from public databases (EMBL/ GenBank/DDBJ). Deduced protein sequences were aligned using Multiple Sequence Comparison by Log-Expectation (http://www. ebi.ac.uk/Tools/muscle/). The tree was constructed using the program PhyML (http://www.phylogeny.fr/version2_cgi/one_task. cgi?task_type=phyml) and Treeview. The branch numbers rep-resent the bootstrap values (100 bootstraps). The percentages of identity with HbHXT1 were obtained from the Blast software (http ://blast.ncbi.nlm.nih.gov/Blast.cgi). Accession numbers of presented sucrose transporter sequences are: AtGlc1 (A. thaliana; CAA39037.1), AtGlcT2 (A. thaliana; AAM67326.1), AtSTP1 (A. thaliana; NM_100998.3), AtSTP4 (A. thaliana; NM_112883.4), AtSTP9 (A. thaliana; NP_175449.1), AtSTP11 (A. thaliana; NP_197718.1), AtSTP13 (A. thaliana; NP_198006), DgMS (Datisca glomerata; CAD30830.1), GmMT (Glycine max; AJ563365.1), HbHXT1 (H. brasiliensis; AM745116.1), JrHXT1 (Juglans regia; DQ026508.1), JrHXT2 (Juglans regia; DQ026509.1), NtMT (Nicotiana tabacum; X66856.1), OeMT2 (Olea europaea; ABJ98314.1), OsMT4 (Oryza sativa, AAQ24871.1), OsMT6 (O. sativa; AY342322), PhMT1 (Petunia × hybrida; AAC61852.1), PtMT (Populus tremula; CAG27609.1), RcST (R. communis, XM_002524644.1), SlHT (Solanum lycoper-sicum, CAB52689.1), VfMT (Vitis vinifera; Z93775.1), VvHT (V. vinifera; Y09590.1), VvHT2 (V. vinifera; AAT77693.2), VvHT7
(V. vinifera; AAX47308.1), ZmMT1 (Zea mays; NP_001105681.1), ZmSCA (Z. mays; ACG38482.1), ZmScC (Z. mays, NP_001148007.1). (b) Transcript abundance of HbHXT1 in different organs and tissues of young trees. cDNA from leaves, bark and xylem of young rejuvenated trees (back-cut 6 months earlier) were used for real-time RT–PCR analysis. Bars represent the technical standard deviation. Results were statistically evaluated using Student’s t-test.
Basal expression of sugar transporters in latex
First, the basal expression of the eight isoforms of sugar transporters previously cloned from H. brasiliensis was ana-lysed in the latex of both PB217 and PB260 (Figure2a). In the PB217 clone, HbSUT1B was the most highly expressed isoform in latex (50-fold over actin) and was at least sixfold more abundant than the other SUT isoforms. The isoforms of the SUT2 group (HbSUT2A, HbSUT2B and HbSUT2C), the SUT4 group (HbSUT4 and HbSUT5) and the SUT1 group (HbSUT1A) were weakly expressed in PB217 latex, compared with the housekeeping gene. In the PB260 clone, HbSUT1B was also one of the most highly expressed isoforms in latex (50-fold). It was 10-fold higher than the minor isoforms, HbSUT1A, HbSUT2A, HbSUT4 and HBSUT5. Two other isoforms were also found to be rela-tively highly expressed in PB260 latex: HbSUT2B and HbSUT2C (5- and 10-fold more expressed than the minor isoforms). Finally, the two isoforms of the SUT4 group (HbSUT4 and HbSUT5) were also weakly expressed in PB217 (Figure2a). The putative hexose transporter HbHXT1 was expressed in both PB260 and PB217 latex (Figure2a).
Basal expression of putative sugar transporters in bark tissues
Overall, HbSUTs presented similar basal expression patterns in the inner bark of both clones (Figure 2b). Judging by their basal expression level, the sucrose transporters could be classified into three groups in the bark of PB217. HbSUT1A, HbSUT1B and HbSUT2B were the most highly expressed isoforms, with relative expression levels of 30-, 50- and 10-fold, respectively, over actin. HbSUT4 was moderately expressed (threefold) and HbSUT2A, HbSUT2C and HbSUT5 were the isoforms displaying the lowest expression. These three groups were also found in PB260 bark (Figure2b). HbSUT1A, HbSUT1B and HbSUT2B were again the most highly expressed isoforms, with expression levels of 200-, 180- and 110-fold, respectively. HbSUT4 and HbSUT5 isoforms were moderately expressed (15-fold), and HbSUT2A and HbSUT2C were the most weakly expressed isoforms (Figure 2b). HbHXT1 was also expressed in the bark of PB260 and PB217 (Figure2b). Increase in latex production after Ethrel®stimulation of PB217 and PB260 clones
Bark Ethrel®treatment beneath the tapping cut provokes an increase in latex production in both PB217 and PB260 clones within the first 24 h (Figure 3). The increase in latex yield, expressed as a percentage of the unstimulated control, was generally higher in PB217 than in PB260. Regarding PB217, a significant increase in latex yield was seen as soon as 8 h after stimulation (35% over the control) and reached its maximum at 16 h after ethylene treatment (in average 80% over the control). PB260 exhibited an earlier yield response to Ethrel®stimulation (+30% within thefirst 4 h) and reached its maximum (+50%) 16 h after ethylene treatment.
Effect of Ethrel®treatment on the transcript levels of putative sugar transporters in the latex
Ethylene treatment enhanced transcript accumulation of HbSUT1B, HbSUT2A and HbSUT5, in the latex of PB217
Figure 2. Basal expression of HbSUTs and HbHXT1 in latex and bark of exploited trees of the PB217 and PB260 clones. Transcript levels of the seven HbSUTs and of the HbHXT1 isoforms were monitored by real-time RT–PCR, carried out on latex (a) and bark tissues (b) of clones PB217 and PB260, regularly tapped for 2 years. To compare the abundance of sugar transporter transcripts in each clone, relative transcripts levels of sugar transporters were determined using real-time PCR and normalized using actin as the reference gene. Expression is represented on a logarithmic scale. Bars represent the technical standard errors of three independent replicates. Results were statistically evaluated using Student’s t-test (P < 0.01).
Figure 3. Latex yield of exploited PB217 and PB260 trees after ethylene stimulation. Batches of three homogeneous mature trees were treated with 2% ethephon, 4, 8, 16, 24 and 40 h before simultaneous tapping. Latex production is expressed according to the following formula: production increase (%) = [( production of stimulated tree– production of unstimulated tree)/production of unstimulated tree] × 100.
(Figure 4). HbSUT1B showed a very early (within the first 4 h) and high (>8-fold) transcript accumulation which lasted 24 h at least after ethylene treatment (Figure 4a). Under the same experimental conditions, transcript levels of HbSUT2A and HbSUT5 were transiently increased within the first 4 h of treatment (seven- and fivefold, respectively) but progressively decreased thereafter (Figure4b and c).
In PB260, no ethylene-induced overexpression of HbSUT1B was observed over 24 h after ethylene treatment. Similar data were obtained for HbSUT1A, HbSUT4 and HbSUT5 (Figure 4a and g). Conversely, HbSUT2A and HbSUT2C exhibited slight and gradual ethylene up-regulation during this period (from 3.5- to 5-fold).
HbHXT1 exhibited clone-dependent stimulation of its expression in response to the ethylene treatment (Figure5d and h). In PB217, its transcript level was early and signi fi-cantly (eightfold) increased during the first 16 h, but decreased after 24 h of treatment (Figure 4d). Despite its high basal level in PB260, no regulation of HBHXT1 tran-script level in response to ethylene was seen in the latex of this Hevea clone.
Effect of Ethrel®treatment on the transcript levels of putative sugar transporters in the inner bark
In PB217 bark, the transcript levels of three SUT isoforms (HbSUT1B, HbSUT2A and HbSUT2B) were significantly
Figure 4. Transcript accumulation of sugar transporters after ethylene stimulation in the latex of mature exploited PB217 and PB260 trees. Transcript levels of sucrose transporters of the SUT1 group (a and e), SUT2 group (b and f ), SUT4 group (c and g) and hexose transporter (d and h) were monitored after ethylene stimulation in the latex of exploited trees of PB217 (a–d) and PB260 (e–h) clones. Samples were collected 4, 8, 16, 24 and 40 h after the treatment (Tap1) and after 3 days rest (Tap2). Relative transcripts levels of these sugar transporters were determined using real-time PCR and normalized using actin as the reference gene. Bars represent the technical standard deviation of three independent replicates. Results were statistically evaluated with Student’s t-test (P < 0.01).
regulated by ethylene (Figure 5a–c). The transcript level of HbSUT1B was up-regulated by ethylene for 24 h after treat-ment, with a maximum accumulation (sixfold) at 8 h after the treatment (Figure 5a). HbSUT2A and HbSUT2B dis-played a slight increase in their respective transcript levels during the time-course of treatment, with a maximum at 8 h for HbSUT2A (fivefold) and at 24 h for HbSUT2B (eight-fold; Figure 5b). The isoforms HbSUT1A, HbSUT2C, HbSUT4 and HbSUT5 did not show any response to ethyl-ene treatment (Figure5a–c).
In PB260 bark, HbSUT1B also displayed ethylene-induced up-regulation. Its transcript level was strongly increased after 8 h and reached a maximum of 14-fold at 24 h of treatment (Figure 5e). The transcript levels of
HbSUT4 and HbSUT5 were also up-regulated by ethylene. They were up-regulated during thefirst 4 h of the treatment (12-fold) and then returned to their initial level after 24 h of ethylene treatment (Figure5g).
HbHXT1 transcript level did not exhibit any significant ethylene-induced regulation in bark tissues of either PB260 or PB217 clone (Figure5d and h).
Immunolocalization of HbSUT1B in young Hevea stems HbSUT1B appears to be a potentially important candidate gene for ethylene stimulation of latex production and for clonal differentiation. Additional data on the localization of its corresponding putative protein would support the role
Figure 5. Transcript accumulation of sugar transporters after ethylene stimulation in bark of mature exploited PB217 and PB260 trees. Transcript levels of sucrose transporters of the SUT1 group (a and e), SUT2 group (b and f ), SUT4 group (c and g) and hexose transporters (d and h) were monitored after ethylene stimulation in the bark tissues of exploited trees of PB217 (a–d) and PB260 (e–h) clones. Samples were collected 4, 8, 16 and 24 h after ethylene treatment. Qr was obtained using the E−ΔΔCtmethod with the unstimulated controls as refer-ence and with actin as a standard. Bars represent the technical standard deviation. Results were statistically evaluated with Student’s t-test (P < 0.01).
of HbSUT1B in the functioning of latex cells. To this aim, an HbSUT1B antibody was raised and its specificity was tested on bark microsomal proteins extracted from young Hevea stem sections (Figure6a). Pre-immune serum (PS) was used as a negative control, and no immunological recognition could be detected (Figure 6a). With the antiserum, a specific band of the expected apparent molecular weight (56 kDa), as deduced from the HbSUT1B sequence, was observed.
Immunolocalization experiments were then carried out on young Hevea stem sections. With the PS, non-specific
colouration was observed in xylem (Figure 6b); conse-quently, the same colour with antiserum was not considered as true immunological recognition in this tissue (Figure6c). With the antiserum, specific colour was detected in the schlerenchyma cells and especially in the latex cells (Figure 6d and e), confirming the presence of the HbSUT1B protein in these tissues. The very young latex cells were identified by their irregular shape and dense granular cytoplasm, and by the presence of some nascent anastomoses (arrows in Figure6e)
Figure 6. Western blot of bark microsomal proteins and immunolocalization of HbSUT1B in the PB217 clone. HbSUT1B antiserum was produced in rabbits (Proteogenix, Oberhausbergen, France). (a) Western blot with the antiserum (AS) carried out on 30 µg of protein from a bark microsomal fraction. Pre-immune serum was used as a control. (b) and (c) Immunolocalization experiments were conducted on young stem sections from rejuvenated PB217 tree. Pre-immune serum (b) and anti-HbSUT1B serum (c) were used as negative and positive controls, respectively. The arrows show some young latex cells.
Transcript accumulation of HbSUT1 and HBHXT1 in virgin and exploited tree of the PB217 clone
Tapping represents a serious abiotic wounding stress for exploited trees (under regular tapping). It requires latex cells to completely regenerate their cytoplasm after latex expulsion. In this context, to check whether the repeated tappings affect the transcript level of sugar transporters, the same ethylene treatments were carried out both on virgin trees (at bark opening) and on trees exploited for 2 years. This comparison was only conducted for HbSUT1B and HbHXT1, which were found to be up-regulated by ethylene in the latex of commercial PB217 trees.
Ethylene treatment induced an increased level of HbSUT1B in the latex of exploited trees, whereas HbSUT1A was decreased in virgin trees (Figure 7a). Similarly, the HbHXT1 transcript level was markedly up-regulated by ethylene in the latex of exploited trees but almost not up-regulated in virgin trees (Figure 7c). This means that HbSUT1B and HbHXT1 are more respon-sive to ethylene in exploited trees (under regular tapping) than in virgin trees. In bark (Figure 7b and d), a similar pattern was observed, especially for HbSUT1. The HbSUT1B level was significantly up-regulated by ethylene in exploited trees, whereas it was barely increased in virgin trees (Figure 7b). HbHXT1 did not appear to be ethylene regulated in the bark of either virgin or exploited trees (Figure 7d).
Discussion
Laticiferous cells harbour hexose and sucrose transporters
One full-length cDNA encoding a putative hexose transporter was cloned from a latex-specific cDNA library (HbHXT1, accession number FM244737). HbHXT1 shares high sequence identity with previously published plant hexose transporters (Caspari et al. 1994, Büttner and Sauer 2000,
Büttner 2007). The presence of such a hexose tranporterfits well with the glucose-induced plasma membrane depolariz-ation of the latex cells (Lacrotte 1991,Bouteau et al. 1992,
1999). The cloning of only one putative hexose transporter contrasts with the presence of seven putative sucrose transpor-ters in the same cells (Dusotoit-Coucaud et al. 2009). Plasma membrane-localized hexose transporters are encoded by a multigenic family in some species (Caspari et al. 1994,
Büttner 2007). For example, 14 isoforms were identified in Arabidopsis thaliana (Büttner and Sauer 2000), three isoforms in Solanum lycopersicon (Gear et al. 2000), eight isoforms in Ricinus communis (Weig et al. 1994), but only one isoform in walnut tree (Decourteix et al. 2008). The existence of other isoforms of hexose transporter in latex is not excluded, but they may be very poorly expressed in the latex cells.
Sucrose and hexose transporters are expressed in most of the sink organs, indicating that sucrose released into the extracellular space can be taken up directly via sucrose transporters and/or via hexose transporters after being split
Figure 7. Transcript accumulation of HbSUT1B and HbHXT1 in latex and bark of virgin and exploited trees. Transcript levels of sucrose transporters HbSUT1B (a and b) and hexose transporter HbHXT1 (c and d) were monitored by real-time RT–PCR on RNA from latex (a and c) and bark tissues (b and d) of PB217 virgin or 2-year exploited trees, after ethylene stimulation. Qr was obtained using the E−ΔΔCtmethod with the unstimulated controls as reference and with actin as the reference gene. Bars represent the technical standard deviation of three inde-pendent replicates. Results were statistically evaluated using Student’s t-test (P < 0.01).
by a cell wall invertase (Braun and Slewinski 2009). The isolation of HbHXT1 and seven HbSUTs from latex reflects the ability of the latex cells to import both sucrose and hexoses. However, the high diversity of sucrose transporters in the latex cells, coupled with the central role of sucrose in rubber synthesis, suggests a preference by these cells for the importation of sucrose rather than hexoses.
The basal level of sugar transporter transcripts is clone dependent
In accordance with previous data (Gohet et al. 2003), clone PB217 produced less latex (220 g tap−1tree−1) than clone PB260 (350 g tap−1tree−1) in the absence of ethylene treat-ment. This difference could coincide with higher basal expression of sugar transporter in PB260 than in PB217 (Figure 2) and, incidentally, with the ability of cells to import sugar. Correlation between transcript accumulation of sugar transporters and their transport activity has been reported (Sakr et al. 1997, Lemoine et al. 1999, Rosche et al. 2002;Decourteix et al. 2006,2008,Hayes et al. 2007,
Zhou et al. 2009). For example, the accumulation of Juglans regia sucrose transporter 1 (JrSUT1) in xylem par-enchyma cells during winter led to higher active sucrose uptake by the same cells (Decourteix et al. 2006). The above elevated basal latex production (without Ethrel® treat-ment) of clone PB260 could result from a concomitant augmentation of metabolic activity (Gohet et al. 2003) and sucrose importation (Figure 2b), causing low sucrose con-centration in latex cells (Jacob et al. 1995, Gohet et al. 2003). In contrast, the low basal level of latex production in PB217 can be related to weaker metabolic activity (Gohet et al. 2003) and to relatively low basal abundance of sucrose transporters in its latex cells (Figure 2a). Because intracellular sucrose negatively regulates the expression of its own transporter (Chiou and Bush 1998, Aoki et al. 1999, Matsukura et al. 2000, Vaughn et al. 2002, Ransom-Hodgkins et al. 2003, Zhou et al. 2009), we suggest that this low basal level of HbSUTs is associated with the high sucrose concentration of its latex cells (Gohet et al. 2003).
HbSUT1B could be a crucial player for the sustained sucrose importation required for high ethylene-stimulated latex production in PB217
Ethrel® treatment of bark induced a higher stimulation of latex production in clone PB217 than in clone PB260 (an increase of, respectively, 80 and 50% after 24 h of ethylene treatment, Figure3). Ethylene increases latex production by enhancing the sink strength of latex cells, through stimu-lation of latex cytosolic invertase activity (Tupy and Primot 1982), latex sucrose concentration (Tupy and Primot 1976,
Low and Gomez 1982) and both sucrose influx and H+/ ATPase activity (d’Auzac et al. 1982,Tupy 1984,Lacrotte et al. 1985,Lacrotte 1991). Considering the significance of
sugar transporters in meeting the carbon demand of different sink organs (Lemoine 2000, Sauer 2007, Braun and Slewinski 2009,Dusotoit-Coucaud et al. 2009,Zhou et al. 2009), the strong ethylene stimulation of latex production in PB217-exploited trees may involve up-regulation of at least one of the identified sugar transporters.
To varying degrees, ethylene treatment of the outer bark of the PB217 clone led to a higher level of sucrose transporter (HbSUT1B, HbSUT2A, HbSUT4) and hexose transporter (HbHXT) transcripts (Figure4). Yet, many lines of evidence support a significant contribution of HbSUT1B to the sustained sucrose importation required for the high stimulation by ethylene of latex production in PB217. HbSUT1B is by far the most abundant sucrose transporter isoform in the latex cells of exploited PB217 trees (Figure 2a). The presence of its corresponding protein in these cells was confirmed by immunolocalization (Figure 6). HbSUT1B expression was more sensitive to ethylene treatment than that of any other sugar transporters. In this respect, HbSUT1B accumulation increased very early (from as short a period as 4 h) and very considerably (up to 10-fold) during the time-course of ethylene treatment (Figure 4a). Interestingly, no up-regulation of HbSUT1B was seen in the latex cells of clone PB260 (Figure 4e), known for its low response to ethylene in terms of latex production (Figure 3; Gohet et al. 2003). In this Hevea clone, ethylene treatment rather induced the stimulation of HbSUT2A and HbSUT2C expression (Figure 4f ), whose basal expression levels in PB260 latex were low (Figure2). On the other hand, these two clones exhibited a contrasting pattern of HbSUT1B expression in the inner bark of exploited trees. The inner bark consists of many concentric layers of latex cells that are surrounded by heterotrophic living cells ( phloem, cortical parenchyma cells and trans-verse vascular rays) and constitute no more than 5% of all the inner bark living cells (d’Auzac et al. 1989). In clone PB217, the expression of HbSUTs was globally weaker in the bark tissues than in latex cells after ethylene treatment (6- and 10-fold, respectively, Figures4a and5a). This is not the case for clone PB260, for which HbSUT1B and, to a lesser extent, HbSUT4 and HbSUT5 were more expressed in the inner bark than in the latex (Figures 4e and 5e). Collectively, these findings lend support to the hypothesis that HbSUT1B regulation contributes to ethylene-induced stimulation of latex production in the exploited Hevea tree, and accounts for the differential response of PB217 and PB260 to ethylene treatment. For the former, ethylene treat-ment triggered stronger HbSUT1B expression in the latex cells, in comparison with the inner bark. The opposite effect was found in PB260, in which ethylene treatment induced elevated expression of HbSUT1B in the inner bark cells but not in the latex cells. This situation enables bark cells of PB260 to divert phloem-delivered sucrose to the detriment of latex cells, preventing them from efficiently importing the sucrose required for a high yield in ethylene-stimulated latex production.
Latex cells may modulate their sugar transport in relation to carbon demand
Expression patterns of HbSUTs and HbHXT1 were not the same in virgin (untapped trees, Dusotoit-Coucaud et al. 2009) and in exploited trees (Figure 7). In PB217, the ethylene-treated tree produced about 235 g tap−1tree−1 after 24 h of treatment, which seemingly correlates well with higher levels of HbSUT2A and HbSUT1A transcripts (Dusotoit-Coucaud et al. 2009). In exploited trees, this sti-mulating effect of ethylene was even higher and reached 440 g tap−1tree−1 during the first 24 h of treatment, and could be related to an increased level of HbSUT1 in the latex cells (Figures4and 5). The same adjustment of sugar transporters was documented in many active sink organs (Weber et al. 1997,Truernit et al. 1999,Schneidereit et al. 2003,2005,Scholz-Starke et al. 2003,Hayes et al. 2007). For example, 5 out of 14 hexose transporters are expressed at very specific stages of pollen development, depending on the carbon demand. AtSTP2 is expressed only during the early storage stages, whereas it is absent before germination (Truernit et al. 1999). The expression of AtSTP6 begins in pollen grains, also during storage (Scholz-Starke et al. 2003), AtSTP9 is expressed from Stage 10 of the flower until pollen germination (Schneidereit et al. 2003) and, finally, ATSTP11 is expressed during germination, corre-sponding to a high carbon demand stage (Schneidereit et al. 2005). In our Hevea model, sucrose demand of latex cells could be much higher in exploited trees than in unexploited ones. Indeed, the regularly exploited rubber tree produces more latex than the virgin ones (450 and 220 g tap−1tree−1, respectively) and these exploited trees are subjected to repeated severe wounding, combined with constitutive high metabolic activity required for latex regeneration between consecutive tappings (from every 2 to 5 days, depending on the plantations). The ethylene-mediated differential regu-lation of HbSUT1B may explain such difference between exploited and unexploited trees. HbSUT1B is by far the most highly expressed sucrose transporter isoform in the latex of virgin (Dusotoit-Coucaud et al. 2009) and exploited trees of PB217 (Figure 2a). Its transcript amount was considerably increased by ethylene in the latex cells of exploited trees (10-fold) while it decreased in virgin trees (Dusotoit-Coucaud et al. 2009). In parallel, the transcripts of HbSUT1A and HbSUT2A, whose basal levels were low in the latex cells, were enhanced by ethylene treatment in virgin trees (Dusotoit-Coucaud et al. 2009).
In conclusion, we report on the potential role of sugar trans-porters in the ethylene-stimulated production of latex in industrially exploited Hevea trees. We have studied eight puta-tive sugar transporters in two Hevea clones, differing in their metabolic activity and their response to ethylene stimulation. Of the eight putative sugar transporters previously isolated from latex cells (Dusotoit-Coucaud et al. 2009), HbSUT1B could be important in meeting the sustained sucrose demand of the latex cells necessary for ethylene-stimulated
latex production in PB217. In addition, ethylene-mediated HbSUT1B regulation may account for the differential response of latex production to ethylene application between virgin and exploited trees, as well as between clones PB217 and PB260. These data provide a cornerstone for the role of sucrose transporters in latex production by exploited trees and deserve to be extended to other Hevea clones. An understanding of the molecular basis of HbSUT1B regulation and its co-localization with production QTLs would enable us to learn more about the function of this sucrose transporter in such an economically significant process.
Acknowledgments
We are grateful to the staff of the SIPH Headquarters and of the Bongo Rubber Plantation in Côte d’Ivoire (West Africa) for their logistical and technical help in field experiments, as well as for allowing access to all their plant materials and laboratory facilities.
Funding
We are grateful to the Michelin group, which provided funding for this work.
References
Aoki, N., T. Hirose, S. Takahashi, K. Ono, K. Ishimaru and R. Ohsugi. 1999. Molecular cloning and expression analysis of a gene for a sucrose transporter in maize (Zea mays L.). Plant Cell Physiol. 40:1072–1078.
d’Auzac, J. 1964. Mise en evidence de la glycolyse et de ses relations avec la biosynthèse du caoutchouc au sein du latex d’ Hevea brasiliensis. Rev. Gén. Caoutch. Plast. 41:1831–1834. d’Auzac, J. and D. Ribaillier. 1969. L’ethylène, un nouveau
stimu-lant de la production de latex chez l’Hevea brasiliensis. C. R. Acad. Sci. D. 268:3046–3049.
d’Auzac, J., H. Crétin, B. Martin and C. Lioret. 1982. A plant vacuolar system: the lutoïds from Hevea brasiliensis latex. Physiol. Vég. 20:311–331.
d’Auzac, J., J.-L. Jacob and H. Chrestin. 1989. Physiology of rubber tree latex. The laticiferous cell and latex – a model of cytoplasm. In Physiology of Rubber Tree Latex. Eds. J. d’Auzac, J.L. Jacob and H. Chrestin. CRC Press Inc., Boca Raton, FL, USA, pp 470–482.
Bandurski, R.S. and H.J. Teas. 1957. Rubber biosynthesis in latex of Hevea brasiliensis. Plant Physiol. 32:643–648.
Bouteau, F., R. Lacrotte, D. Cornel, M. Monestiez, U. Bousquet, A. Pennarun and J. Rona. 1991. Electrogenic active proton pumps in Hevea brasiliensis laticiferous cells: its role in activat-ing sucrose/H+ and glucose/H+ symports at the plasma mem-brane. Bioelectrochem. Bioenerg. 26:223–236.
Bouteau, F., U. Bousquet, R. Lacrotte, D. Cornel, M. Monestiez and J. Rona. 1992. Sucrose/H+ and glucose/H+ symports at the plasma membrane of laticiferous cells and protoplasts of Hevea brasiliensis. Indian J. Nat. Rubber Res. 5:25–37.
Bouteau, F., O. Dellis and J.P. Rona. 1999. Transient outward K+ currents across the plasma membrane of laticifer from Hevea brasiliensis. FEBS Lett. 458:185–187.
Braun, D. and T. Slewinski. 2009. Genetic control of carbon parti-tioning in grasses: roles of sucrose transporters and tie-dyed loci in phloem loading. Plant Physiol. 149:71–81.
Büttner, M. 2007. The monosaccharide transporter(-like) gene family in Arabidopsis. FEBS Lett. 581:2318–2324.
Büttner, M. and N. Sauer. 2000. Monosaccharide transporters in plants: structure, function and physiology. Biochim. Biophys. Acta 1465:263–274.
Caspari, T., R. Stadler, N. Sauer and W. Tanner. 1994. Structure/ function relationship of the Chlorella glucose/H+ symporter. J. Biol. Chem. 4:3498–3502.
Chiou, T.J. and D.R. Bush. 1998. Sucrose is a signal molecule in assimilate partitioning. Proc. Natl Acad. Sci. USA 95:4784–4788.
Chow, K.S., K.L. Wan, M.N.M. Isa, A. Bahari, S.H. Tan, K. Harikrishna and H.Y. Yeang. 2007. Insights into rubber bio-synthesis from transcriptome analysis of Hevea brasiliensis latex. J. Exp. Bot. 58:2429–2440.
Decourteix, M., G. Alves, N. Brunel, T. Ameglio, A. Guilliot, R. Lemoine, G. Petel and S. Sakr. 2006. JrSUT1, a putative xylem sucrose transporter, could mediate sucrose influx into xylem par-enchyma cells and be up-regulated by freeze–thaw cycles over the autumn–winter period in walnut tree (Juglans regia L.). Plant Cell Environ. 29:36–47.
Decourteix, M., G. Alves, M. Bonhomme et al. 2008. Sucrose (JrSUT1) and hexose (JrHT1 and JrHT2) transporters in walnut xylem parenchyma cells: their potential role in early events of growth resumption. Tree Physiol. 28:214–224.
Dusotoit-Coucaud, A., N. Brunel, P. Kongsawadworakul, U. Viboonjun, A. Lacointe, J.-L. Julien, H. Chrestin and S. Sakr. 2009. Sucrose importation into laticifers of Hevea brasiliensis, in relation to ethylene stimulation of latex production. Ann. Bot. 104:635–647.
Eschbach, J.-M., J. Tupy and R. Lacrotte. 1986. Photosynthate allo-cation and productivity of latex vessels in Hevea brasiliensis. Biol. Plant. 28:321–328.
de Faÿ, E. and J.-L. Jacob. 1989. Anatomical organization of the laticiferous system in the bark. In Physiology of Rubber Tree Latex. Eds. J. d’Auzac, J.L. Jacob and H. Chrestin. CRC Press Inc., Boca Raton, FL, USA, pp 3–14.
de Faÿ, E., C. Hébant and J.-L. Jacob. 1989. Cytology and cyto-chemistry of the laticiferous system. In Physiology of Rubber Tree Latex. Eds. J. d’Auzac, J.L. Jacob and H. Chrestin. CRC Press Inc., Boca Raton, FL, USA, pp 15–29.
Gear, M.L., M.L. McPhillips, J.W. Patrick and D.W. McCurdy. 2000. Hexose transporters of tomato: molecular cloning, expression analysis and functional characterization. Plant Mol. Biol. 44:687–697.
Gohet, E. 1996. La production de latex par Hevea brasiliensis, relations avec la croissance. Influence de différents fac-teurs: origine clonale, stimulation hormonale, réserves hydrocarbonées. Thesis, Université Montpellier II, Montpellier, 343 p.
Gohet, E., P. Chantuma, R. Lacote, S. Obouayeba, K. Ian, A. Clément-demange, D. Kurnia and J.M. Eschbach. 2003. Latex clonal typology of Hevea brasiliensis. Physiological modelling of yield potential and clonal response to Ethephon stimulation. In IRRDB Workshop on Exploitation Technology, Kottayam, India.
Hayes, M.A., C. Davies and I.B. Dry. 2007. Isolation, functional characterization, and expression analysis of grapevine (Vitis vini-fera L.) hexose transporters: differential roles in sink and source tissues. J. Exp. Bot. 58:1985–1997.
Hébant, C. 1981. Ontogénie des laticifères du système primaire de l’Hevea brasiliensis, une etude ultra structurale et cytochimique. Can. J. Bot. 59:974–985.
Hébant, C. and E.D. Faÿ. 1980. Functional organization of the bark of Hevea brasiliensis (rubber tree): a structural and histoen-zymological study. Pflanzenphysiology. 97:391–398.
Hepper, C.M. and B.G. Audley. 1969. The biosynthesis of rubber from hydroxy–methyl–glutaryl coenzyme A in Hevea brasilien-sis latex. Biochem. J. 114:379.
Jacob, J., J. Prévôt, D. Roussel, R. Lacrotte, E. Serres, J. d’Auzac, J. Eschbach and H. Omont. 1989. Hevea brasiliensis yield-limiting factors, latex physiological parameters, latex diag-nosis and clonal typology. In Physiology of Rubber Tree Latex. Eds. J.J. d’Auzac, J.L. Jacob and H. Chrestin. CRC Press, Boca Raton, FL, USA, pp 345–382.
Jacob, J.L., J.C. Prevot, R. Lacrotte and J.M. Eschbach. 1995. Latex diagnosis. Plant Rech Dév. 2:33–37.
Ko, J., K. Chow and K. Han. 2003. Transcriptome analysis reveals novel features of the molecular events occurring in the laticifers of Hevea brasiliensis ( para rubber tree). Plant Mol. Biol. 53:479–492.
Lacrotte, R. 1991. Relationship between the sucrose content of the rubber latex and the production: approach of the mechanisms involved during the saccharose loading of the laticifers of Hevea brasiliensis. Thesis, Université de Montpellier 2, Montpellier, 226 p.
Lacrotte, R., H. Van de Sype and H. Chrestin. 1985. Influence de l’éthylène sur l’utilisation du saccharose exogène par les laticifères d’Hevea brasiliensis. Proposition d’un mécanisme d’action. Physiol. Vég. 23:187–198.
Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. Lalonde, S., D. Wipf and W. Frommer. 2004. Transport
mechan-isms for organic forms of carbon and nitrogen between source and sink. Annu. Rev. Plant Biol. 55:341–372.
Lemoine, R. 2000. Sucrose transporters in plants: update on func-tion and structure. Biochim. Biophys. Acta Biomembr. 1465:246–262.
Lemoine, R., L. Burkle, L. Barker, S. Sakr, C. Kuhn, M. Regnacq, C. Gaillard, S. Delrot and W.B. Frommer. 1999. Identification of a pollen-specific sucrose transporter-like protein NtSUT3 from tobacco. FEBS Lett. 454:325–330.
Low, F.C. and J.B. Gomez. 1982. Carbohydrate status of exploited Hevea. I. the effect of different exploitation systems on the con-centration of the major soluble carbohydrates in latex. J. Rubb. Res. Inst. Malaysia 30:1–18.
Matsukura, C., T. Saitoh, T. Hirose, R. Ohsugi, P. Perata and J. Yamaguchi. 2000. Sugar uptake and transport in rice embryo. Expression of companion cell-specific sucrose transporter (OsSUT1) induced by sugar and light. Plant Physiol. 124:85–93. Pujade-Renaud, V., A. Clement, C. Perrot-Rechenmann, J.C. Prevot, H. Chrestin, J.L. Jacob and J. Guern. 1994. Ethylene-induced increase in glutamine synthetase activity and mRNA levels in Hevea brasiliensis latex cells. Plant Physiol. 105:127–132.
Pujade-Renaud, V., C. Perrot-Rechenmann, H. Chrestin, R. Lacrotte and J. Guern. 1997. Characterization of a full-length cDNA clone encoding glutamine synthetase from rubber tree latex. Plant Physiol. Biochem. (Paris) 35:85–93.
Ransom-Hodgkins, W., V. Vaughn and D. Bush. 2003. Protein phosphorylation plays a key role in sucrose-mediated transcrip-tional regulation of phloem-specific proton–sucrose symporter. Planta 217:483–489.
Rolland, F., E. Baena-Gonzalez and J. Sheen. 2006. Sugar sensing and signaling in plants: conserved and novel mechanisms. Annu. Rev. Plant Biol. 57:675–709.
Rosche, E., D. Blackmore, M. Tegeder, T. Richardson, H. Schroeder, T.J.V. Higgins, W.B. Frommer, C.E. Offler and J.W. Patrick. 2002. Seed-specific overexpression of a potato sucrose transporter increases sucrose uptake and growth rates of develop-ing pea cotyledons. Plant J. 30:165–175.
Sakr, S., M. Noubahni, A. Bourbouloux, J. Riesmeier, W. Frommer, N. Sauer and S. Delrot. 1997. Cutting, ageing and expression of plant membrane transporters. Biochim. Biophys. Acta 1330:207–216.
Sambrook, J., E.F. Fritsch and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Sando, T., S. Taken, N. Watanabe, H. Okumoto, T. Kuzuyama, A. Yamashita, M. Hattori, N. Ogasawara, E. Fukusaki and A. Kobayashi. 2008. Cloning and characterization of the 2-C-Methyl-D-erythritol 4-phosphate (MEP) pathway genes of a natural rubber producing plant, Hevea brasiliensis. Biosci. Biotechnol. Biochem. 72:2903–2917.
Sauer, N. 2007. Molecular physiology of higher plant sucrose transporters. FEBS Lett. 581:2309–2317.
Schneidereit, A., J. Scholz-starke and M. Buttner. 2003. Functional characterization and expression analysis of the glucose-specific AtSTP9 monosaccharide transporter in pollen of Arabidopsis. Plant Physiol. 133:182–190.
Schneidereit, A., J. Scholz-Starke and N. Sauer. 2005. AtSTP11, a pollen tube-specific monosaccharide transporter in Arabidopsis. Planta 221:48–55.
Scholz-Starke, J., M. Büttner and N. Sauer. 2003. AtSTP6, a new pollen-specific H+-monosaccharide symporter from Arabidopsis. Plant Physiol. 131:70–77.
Silpi, U., A. Lacointe, P. Kasempsap, S. Thanysawanyangkura, P. Chantuma, E. Gohet, N. Musigamart, A. Clement, T. Ameglio and P. Thaler. 2007. Carbohydrate reserves as a competing sink: evidence from tapping rubber trees. Tree Physiol. 27:881–889.
Sookmark, U., V. Pujade-Renaud, H. Chrestin, R. Lacote, C. Naiyanetr, M. Seguin, P. Romruensukharom and J. Narangajavana. 2002. Characterization of polypeptides accumu-lated in the latex cytosol of rubber trees affected by the tapping panel dryness syndrome. Plant Cell Physiol. 43:1323–1333. Truernit, E., R. Stadler, K. Baier and N. Sauer. 1999. A male
gametophyte-specific monosaccharide transporter in Arabidopsis. Plant J. 17:191–201.
Tupy, J. 1973. The level and distribution pattern of latex sucrose along the trunk of Hevea brasiliensis Mull.Arg. as affected by the sink region induced by latex tapping. Physiol. Vèg. 11:1–11. Tupy, J. 1984. Translocation, utilization and availability of sucrose
for latex production in Hevea. In Proceedings of the Symposium ‘Exploitation, Physiology and Improvement of Hevea’. Irca-Gerdat, Paris, France.
Tupy, J. and L. Primot. 1976. Control of carbohydrate metabolism by ethylene in latex vessels of Hevea brasiliensis Müll. Arg. in relation to rubber production. Biol. Plant. 18:373–383.
Tupy, J. and L. Primot. 1982. Sucrose synthetase in the latex of Hevea brasiliensis Müll. Arg. J. Exp. Bot. 33:988–995. Vaughn, M.W., G.N. Harrington and D.R. Bush. 2002.
Sucrose-mediated transcriptional regulation of sucrose symporter activity in the phloem. Proc. Natl Acad. Sci. USA 99:10876–10880. Weber, H., I. Borisjuk, N. Sauer and U. Wobus. 1997. A role for
sugar transporters during seed development: molecular character-isation of a hexose and a sucrose carrier in fava bean seeds. Plant Cell 9:895–908.
Weig, A., J. Franz, N. Sauer and E. Komor. 1994. Isolation of a family of cDNA clones from Ricinus communis L. with close hom-ology to the hexose carriers. Plant Genet. Breed. 143:178–183. Zhou, Y., K. Chan, T.L. Wang, C.L. Hedley, C.E. Offler and J.W.
Patrick. 2009. Intracellular sucrose communicates metabolic demand to sucrose transporters in developing pea cotyledons. J. Exp Bot. 60:71–85.
