JXB Advance Access originally published online on March 3, 2003
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Journal of Experimental Botany, Vol. 54, No. 385, pp. 1193-1204,
April 1, 2003
© 2003 Oxford University Press
Expression of a putative grapevine hexose transporter in tobacco alters morphogenesis and assimilate partitioning
Received 2 August 2002; Accepted 16 December 2002
UMR CNRS 6161, Transport des Assimilats, Laboratoire de Physiologie, Biochimie et Biologie Moléculaire Végétales, Bâtiment Botanique, UFR Sciences, 40 Avenue du Recteur Pineau, F-86022 Poitiers Cédex, France
1 To whom correspondence should be addressed. Fax: +33 5 49 45 41 86. E-mail: serge.delrot{at}univ-poitiers.fr
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Abstract |
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Tobacco plants were transformed by leaf disc regeneration with the VvHT1 (Vitis vinifera hexose transporter 1) cDNA under the control of the constitutive CaMV 35S promoter in a sense or antisense orientation. Among the 20 sense plants and 10 antisense plants obtained, two sense plants showed a mutant phenotype when grown in vitro, with stunted growth and an increase in the (leaves+stem)/roots dry weight ratio. The rate of [3H]-glucose uptake in leaf discs from these plants was decreased to 25% of the value measured in control plants. The amount of VvHT1 transgene and of host monosaccharide transporter MST transcripts in the leaves were studied by RNA gel blot analysis. The VvHT1 transcripts were usually present, but the amount of MST transcripts was the lowest in the plants that exhibited the most marked phenotype. Although the phenotype was lost when the plants were transferred from in vitro to greenhouse conditions, it was found again in vitro in the progeny obtained by self-pollination or by back-cross. The data show that VvHT1 sense expression resulted in unidirectional post-transcriptional gene inactivation of MST in some of the transformants, with dramatic effects on growth. They provide the first example of plants modified for hexose transport by post-transcriptional gene silencing. Some of the antisense plants also showed reduced expression of MST, and decreased growth. These results indicate that, like the sucrose transporters, hexose transporters play an important role in assimilate transport and in morphogenesis.
Key words: Assimilate transport, gene silencing, grapevine, monosaccharide transport, sink/source relationship, tobacco.
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Introduction |
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The plasma membrane transporters mediating the transport of sucrose (Ward et al., 1998), reducing monosaccharides (Büttner and Sauer, 2000) and polyols (Noiraud et al., 2001) control the loading and the unloading of the conducting complex, as well as the accumulation of sugars in sink cells. In this way, they play a major role in morphogenesis and plant productivity, and may also interfere with the expression of various genes through sugar-sensing mechanisms (Koch, 1996; Lalonde et al., 1999; Gibson, 2000; Smeekens, 2000).
The cloning of the sucrose transporter in spinach (Riesmeier et al., 1992) and of the monosaccharide transporter in Chlorella (Sauer and Tanner, 1989) has opened wide avenues related to the molecular characterization of sugar transporters. Many clones belonging to these gene families have now been isolated from various species and organs (for reviews, see Büttner and Sauer, 2000; Lemoine, 2000). The achievement of Arabidopsis genome sequencing has shown that among the numerous genes encoding membrane transporters, about 100 sequences were related to sugar transporters with, respectively, nine sucrose transporters and 14 monosaccharide-transporters. Relatively few of the putative sugar transporters isolated have been successfully expressed in yeasts or Xenopus. The potential problems linked to heterologous expression include the codon bias (Sauer and Tanner, 1993), the role of the 5' and 3' untranslated region (Stadler et al., 1995), the lipid environment required for full activity of the transporter (Robl et al., 2000), mistargeting, and toxicity. The exact physiological function of many of these clones is, therefore, still unknown.
As in Arabidopsis (see above), the monosaccharide transporters are encoded by large gene families in Ricinus communis (Weig et al., 1994) and Chenopodium rubrum (Roitsch and Tanner, 1994). Most monosaccharide transporters described so far are expressed exclusively or mostly in various sinks: AtSTP2 (Truernit et al., 1999) and Petunia PMT1 (Ylstra et al., 1998) in the pollen, AtSTP3 in the sepals and stigma, AtSTP4 in the anthers and root tips (Caspari et al., 1994; Truernit et al., 1996), MST1 in tobacco roots and young leaves (Sauer and Stadler, 1993), and MtST1 in Medicago truncatula roots (Harrison, 1996). It has therefore been inferred that their main physiological function is to take up monosaccharides from the free space into various sink cells (Sauer and Tanner, 1993). However, source tissues are also able to take up hexoses from the external medium (Delrot, 1981, 1989), and some monosaccharide transporters are also expressed in source leaves, either at a low (MST1, Sauer and Stadler, 1993; AtSTP4, Truernit et al., 1996) or high (AtSTP1, Sauer et al., 1990) level. Thus, monosaccharide transporters, as well as sucrose transporters, may play a physiological role both in source and in sink tissues.
In addition to heterologous expression, one of the ways available to elucidate the function and physiological role of the sugar transporters is to prepare plants modified for these transporters by sense overexpression, antisense inhibition or gene disruption. These approaches have still been quite limited for sugar transporters. The existence of several homologues in multigenic families may lead to compensation phenomena and a lack of apparent phenotype in the case of gene disruption. In spite of these limitations, Sherson et al. (2000) recently isolated and described a knock-out Arabidopsis mutant for the monosacharide transporter AtSTP1. The mutant plants grow and develop normally, but the uptake of glucose in seedlings is strongly reduced, suggesting that AtSTP1 is involved in the uptake of extracellular monosaccharides by the embryo and in the seedlings.
Although the effects of antisense expression of the sucrose transporter have been documented in detail in potato and tobacco (Riesmeier et al., 1992; Kühn et al., 1996; Bürkle et al., 1998), no report of overexpression or antisense inhibition of monosaccharide transporter expression has yet been described. Potato plants transformed with the antisense construct of the sucrose transporter StSUT1 under the control of the CaMV 35S constitutive promoter accumulate a high level of sucrose in the leaves, whereas root development and tuber yield are strongly reduced (Riesmeier et al., 1992). These plants, as well as plants transformed by the same construct under the control of the companion cell specific promoter rolC, exhibit the same altered leaf phenotype (Kühn et al., 1996) and contain a high number of modified starch grains and oleosomes (Schulz et al., 1998). The level of the SUT1 transcripts, SUT1 protein and the sucrose uptake capacity of plasma membrane vesicles from leaves are strongly reduced in CaMV 35S-StSUT1 antisense plants, but not in rolC-StSUT1 antisense plants (Kühn et al., 1996; Lemoine et al., 1996). These data indicate that inhibition of the sucrose transporter in the companion cell is sufficient to impair phloem loading, but also that this transporter is expressed at low levels in the strong majority of non-phloem cells contained in the leaf.
The present study reports on the characterization of transgenic tobacco plants, where sense or antisense expression of the putative hexose transporter VvHT1 from grapevine altered the expression of the endogenous MST monosaccharide transporter. This epigenetic down-regulation of the host gene resulted in changes of growth phenotype, of transport properties of the tissues and of the shoot/root relationships.
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Materials and methods |
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Chimeric constructs and plant transformation
The binary vector pFB8 used in this work carried three cassettes between the T-DNA borders (Atanassova et al., 1995): (i) the npt-II gene conferring kanamycin resistance of the transformants; (ii) the VvHT1 cDNA in sense and antisense orientation, controlled by the 35S promoter and terminator sequences; (iii) the uidA gene, driven by the same 35S 5' region, as the internal control of transgene expression.
The sequence and the orientation of the inserts were determined by sequencing and restriction digestion of plasmids recovered from transformed Escherichia coli DH5
. The binary vectors were transferred to Agrobacterium tumefaciens LBA 4404 and leaf discs were infected as previously described (Atanassova et al., 1995).
The number of transgene insertions in the tobacco genome was checked by segregation of an average of 500–600 seeds per clone, obtained by selfing, and germinated on culture medium supplemented with 350 µg ml–1 kanamycin.
Nucleic acid purification and blot hybridization
Total RNA from frozen plant material, corresponding to different organs of 6-week-old transformed tobacco plantlets, was obtained as described by Howell and Hull (1978), and submitted to selective precipitation with 2 M LiCl (Verwoerd et al., 1989). Purified RNA samples, 20 µg each, were separated by formaldehyde-agarose gel electrophoresis, and transferred to Hybond N+ membranes (Amersham Life Science).
Genomic DNA was isolated from the supernatant of the LiCl RNA precipitation by addition of 2 vols of ethanol followed by a 15 min centrifugation at 5000 g. The DNA pellet was resuspended in water and treated with RNase (1 g l–1) and proteinase K20 (50 mg l–1) for 60 min at 37 °C. After a phenol–chloroform–isoamyl alcohol (25/24/1, by vol.) extraction, the DNA was precipitated by the addition of isopropanol. The pellet was washed in 70% ethanol and dissolved in water. The DNA (7–10 µg) was digested with either HindIII or EcoRI for 16 h. The DNA fragments were separated on a 0.8% agarose gel overnight at 30 V and blotted onto Hybond N+ membranes (Amersham Life Science) with alkali fixation as described by the manufacturer.
DNA and RNA blots were hybridized with randomly primed [32P] specific probes for uidA, MST1, NPTII or VvHT1 genes. Washings were made at high stringency (65 °C, 0.1x SSC (sodium saline chloride), 0.1% SDS). For rehybridization, the RNA and DNA filters were washed in boiling 0.1% and 0.5% SDS, respectively.
Plant material
Uptake experiments were performed on 6-week-old tobacco (Nicotiana tabacum cv. Paraguay Bell) plants. The plants were grown in vitro at a constant temperature of 22 °C with a 16/8 h day/night regime (150 µmol m–2 s–1). The culture medium was an agar MS modified medium (Duchefa, the Netherlands) containing 0.85 g l–1 NH4+NO3 and 15 g l–1 sucrose except when stated otherwise.
Uptake activity
Leaf discs (0.28 cm2 surface) were excised with a cork borer from young fully expanded leaves. The lower epidermis was not been stripped off because plants grown in vitro have a very thin epidermis which allows sufficient exchanges with the incubation medium. The discs were floated on a preincubation medium (mannitol 175 mM, CaCl2 0.5 mM, K2SO4 0.5 mM, MES 20 mM, pH 5.8, 198 mOsmol) for a 30 min period. Then the preincubation medium was replaced with the same buffered medium containing 1 mM glucose and 10 MBq ml–1glucose-[3H] (NEN, Saclay, France). This glucose concentration is slighly higher than the Km usually determined for plant monosaccharide transporters in plant plasma membrane vesicles (0.3 mM, Tubbe and Buckhout, 1992) and is expected to saturate the transporter without a strong contribution of a diffusional component. At the end of incubation, leaf discs were rinsed (three times for 3 min each) on the preincubation medium. Throughout the experiment, the discs were placed on a reciprocal shaker at room temperature. After sampling, each disc was digested in a solution of perchloric acid (65% [w/w]; 100 µl) and hydrogen peroxide (33% [w/w]; 200 µl) at 55 °C for about 16 h. Radioactivity was counted by liquid scintillation spectrometry (Packard Tricarb 1900TR, Packard instruments, Rungis, France).
Sugar measurements
About 100 mg of plant tissue material were ground in liquid nitrogen and the powder was suspended in 800 µl of extraction buffer (HEPES 50 mM, MgCl2 5 mM, EDTA-Na2 1 mM, pH 7.5) and 800 µl H20. After 20 min incubation on ice, the slurry was centrifuged for 3 min at 12 000 g and soluble sugars were determined in the supernatant. This supernatant was incubated for 3 min at 95–100 °C to denature proteins and to avoid sucrose hydrolysis. Sugar content was determined enzymatically according to Jones et al. (1977). Twenty-five µl of each sample, diluted 50-fold with water, were mixed with 30 µl (16 U) of invertase (dissolved in 320 mM citrate buffer, pH 4.6) and 145 µl of water. After 30 min incubation at 55 °C, the samples were spectrophotometrically assayed in 1 ml of buffer (0.1 M HEPES, pH 7.6, 0.4 mM NADP, 1 mM ATP, 5 mM MgCl2, 0.5 mM dithioerythritol), at 340 nm. The contents of sucrose, glucose and fructose were determined under the same conditions after the addition of hexokinase (0.5 U), phosphoglucose isomerase (2 U) and glucose-6-phosphate dehydrogenase (2.5 U) with an incubation time of 30 min at 28 °C. To obtain the values of fructose and glucose, invertase was deliberately omitted. To determine the glucose content only, both invertase and phosphoglucose isomerase were omitted. All enzymes were purchased from Sigma.
Dry weight determination
The transformed tobacco plants, 4–6 for each independent clone, were cultured in vitro for 6 weeks. Roots, leaves and stems of each plant were cut, and weighed separately after a 48 h incubation at 80 °C. A longer incubation period is not necessary as the weight did not change after 48 h of drying.
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cDNA reconstruction strategy and transgenic tobacco production
The VvHT1 cDNA, obtained after screening of a library prepared from grape (Vitis vinifera cv. Pinot Noir) berries at the veraison stage (Fillion et al., 1999), was cloned in the EcoRI site of the pSK II phagemid (Fig. 1A). This cDNA contained a 500 bp rRNA DNA fused to a fragment of the 5' untranslated region upstream of the translation initiation codon of VvHT1 cDNA. To remove the contaminating rRNA DNA sequence, a double digestion with KpnI and BamHI was made. This produced a VvHT1 coding region fragment (KpnI–BamHI) of 1700 bp (Fig. 1B), which lacks the ATG portion of 50 bp (KpnI being an internal restriction site in VvHT1 cDNA). The coding sequence lacking VvHT1 was produced as a PCR fragment, with a direct primer containing the cDNA sequence just upstream the ATG codon and introducing a BamHI restriction site, and a reverse primer corresponding to the VvHT1 sequence 150 pb downstream of the KpnI internal site (Fig. 1C). The PCR product was subcloned in the pGEM-T easy vector (Fig. 1D) and its sequence was checked. After double digestion by KpnI/PstI (Fig. 1E), the coding region lacking part of the VvHT1 (50 bp) was ligated to the original 1700 bp portion, thus producing the entire cDNA as a BamHI–BamHI fragment of 1750 bp (Fig. 1F). This cDNA was inserted into the BamHI site of the pKS II vector (Fig. 1G) and of the pFB8 binary vector (Fig. 1H). The chimeric constructs also contained the kanamycin resistance gene for selection and the GUS reporter gene as the internal control of transgene expression. These constructs were used to transform tobacco leaf discs via the Agrobacterium-mediated transformation method. Leaf discs were also used to regenerate non-transformed plants grown in vitro as controls. The phenotype and the growth of 30 transformants (20 sense and 10 antisense) grown in vitro was monitored. For convenience, throughout this report the plants transformed with the sense and antisense constructs will be referred to as S and AS plants, respectively.
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Phenotype of the transformants
Most of the plants transformed with the sense or antisense construct did not show any differences compared to controls. However, two sense transformants, S6 and S7, and three antisense transformants, AS36, AS40 and AS41, exhibited an apparent phenotype. This phenotype was most pronounced for the sense transformants S6 and S7. Compared to control plants, the modified phenotype was characterized by stunted growth, small leaves and short stems (Fig. 2). The root system of these transformants was also much less developed than that of sense transformants without apparent phenotype such as S24 or wild-type C22 plants (Fig. 2). The same phenotype appeared in vitro for plants grown either on a sucrose (Fig. 2) or a glucose (data not shown) medium. Although the mutant phenotype was lost when the plants were transferred to the greenhouse, it was consistently observed for all subsequent subcultures in vitro. Furthermore, the marked phenotype exhibited in vitro by these primary transformants reappeared in their progeny, obtained both by selfing or backcross, but only and always when cultured under the same in vitro conditions. These findings led to investigate in more detail the physiological and genetic properties of the transformants exhibiting this phenotype.
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The dry weight of different parts of the plants was measured 6 weeks after in vitro multiplication. Among the 20 sense transformants studied in detail, two (i.e. S6 and S7) exhibited a significantly smaller leaf dry weight than that of control plants (Fig. 3A). However, this decrease was more pronounced for the roots (Fig. 3C), and even more for the stems (Fig. 3B). Among the 10 antisense transformants tested, only AS40 was significantly affected in stem and root dry weight (Fig. 3A, B, C). To assess the partitioning of assimilates between the aerial parts of the plants and the roots, the dry weight ratio (leaves+stem)/root was also calculated. This ratio was significantly increased in S6 and S7 (Fig. 3D). Concerning the antisense plants, the (leaves+stem)/root ratio was also significantly increased in AS36, AS40, and AS41. The growth rate of the S6, S7, AS36, AS40, and AS41 transformants was strongly reduced compared to that of control plants (Fig. 3E).
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Detection of transgenes in the transformants
The number of transgene copies present in the transformants was checked by DNA gel blot analysis. DNA extracted from tobacco plants was digested with HindIII and hybridized with either GUS or NPTII, both markers which flank the VvHT1 sense/antisense cassette in the constructs used for transformation (Fig. 1). Hybridization with the GUS probe (Fig. 4A) indicated that one copy of the uidA coding sequence was present in S6 and AS41 genomes, two copies in S7 and S24, genomes, and multiple copies in AS 36. This was confirmed by the DNA blots hybridized with the NPTII probe (data not shown).
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The integration of the VvHT1 sequence in the transformants was revealed with the VvHT1 probe and also checked by PCR (Fig. 4B, D). Because of the presence of an internal asymetric HindIII site in the VvHT1 coding region (Fig. 1), hybridization with this probe produced two different, but constant, patterns in sense and antisense clones. However, Southern analysis never revealed any VvHT1 band in S24. For this reason, PCR assays were run using VvHT1 specific primers described in the reconstitution strategy (Fig. 1). This allowed the detection of an amplification product of the expected size (180 bp), corresponding to the 5' end of VvHT1 cDNA. The presence of the VvHT1 fragment was confirmed in all transformants studied without exception, even in S24, and in the cloning pKS II–VvHT1 vector, the positive control, whereas it was absent in the regenerated wild-type plant C22, the negative control. Hybridization with the MST1 probe (Fig. 4C) revealed the presence of four labelled bands in wild-type tobacco plants as well as in the transformants. Because Nicotiana tabacum results from crossing two parents N. sylvestrisxN. tomentosiformis and because of an internal HindIII site in the genomic sequence of MST1 (Sauer and Stadler, 1993), the four labelled bands observed are consistent with the presence of two fragments from each of both genes in the tobacco genome, each copy coming from one parent. Transformation did not affect the pattern of labelling, which shows that the MST1 gene was not disrupted by the transformation events.
Effects of transgenesis on gene expression
The effects of transformation were also assessed at the transcript level by RNA gel blot analysis. Total RNA extracted from leaves of various plants was hybridized with the MST1, VvHT1 and GUS probes. Because the tobacco genome probably contains several genes encoding monosaccharide transporters close to MST1, it is likely that the results of RNA gel blot hybridization with MST1 concern several monosaccharide transporters rather than MST1 alone. The amount of RNA loaded for each lane was constant, as assessed by the signals obtained with 25S rRNA probe (Fig. 5, bottom line). Hybridization with the MST1 probe showed that, compared to controls, the level of MST transcripts was significantly reduced in the S6 and S7 sense transformants, and in the AS36 and AS41 antisense plants. By contrast, no significant change was observed in the amount of MST messengers for S24 (Fig. 5, top line).
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The signals detected for the VvHT1 transcripts were strong in S6 and S7, and apparent in AS36 and AS41 transgenic tobaccos. This indicated that the transgene was not only present, but also transcribed in these transformants. No detectable labelling was found after hybridization with the VvHT1 probe in the S24 clone. Importantly, there was no signal detected in control plants, showing that the VvHT1 probe used did not cross-react with the endogenous MST transcripts and was, therefore, specific for the VvHT1 transgene products.
Hybridization with the GUS probe indicated that this transgene is strongly transcribed in S7 and S24 transformants, at a lower level in the S6 and AS36 clones, and at a just detectable level in AS41 one. As expected, no uidA transcripts were detected in control plants.
Effects of transgenesis on glucose uptake and sugar levels
The S6 and S7 primary transformants, which combined the most apparent modifications of the phenotype (stunted growth, total weight, aerial/root weight) and the strongest MST1 transcripts reduction were selected for further analysis. Uptake of [3H]-glucose by the leaf tissues was monitored. Glucose uptake was linear for at least 60 min for all plants tested, and the rates of uptake were significantly decreased in leaf discs from S6 and S7 transformants (Fig. 6). The inhibition of glucose uptake in leaf discs from these plants was about 75%, when compared to the controls C19 and C22 (regenerated non-transformed plants).
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Phenotypic and functional analysis of self-pollinated transgenic tobacco progeny
The primary tobacco transformants (T0) grown from the transformed leaf discs expressing VvHT1 cDNA, were selfed, giving the T1 generation. The growth phenotype observed in the primary transformants for leaves, stems and roots was also clearly apparent in this T1 progeny. The mutant phenotype is shown for T1S6, T1S7, T1AS40, and T1AS41 compared with the control plant T1C22 (Fig. 7). The number of transgene insertions in the genome of the different tobacco clones was checked by segregation of T1 selfed seeds on a kanamycin medium. It revealed the presence of one insertion locus for T1S6, T1S7, T1AS40, and T1AS41, and two loci for T1S24. The transgene insertion number obtained from segregation analysis of T1 progeny corresponded to transgene copy number revealed by Southern blot in primary transformants. The only exception concerns T1S7, in which the two present copies must be integrated at a single locus.
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The T0 transformants were also back-crossed with wild-type plants and all the T1 transformants issued from phenotypically affected T0 transformants exhibited the same phenotype, indicating that the heterozygous state is sufficient for the expression of this phenotype (data not shown).
The decrease of glucose absorption measured in the leaves of primary transformants was also confirmed in leaf tissues of T1S6, T1S7 and T1S41, when compared to the progeny of control T1C22 (Fig. 8A). The concentrations of glucose, fructose and sucrose were also determined in leaves of T1C19 and T1C22 control plants and of selfed transformants T1S6 and T1AS41. No significant difference in soluble sugar concentrations was observed between the clones tested (Fig. 8B).
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Discussion |
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Given that sugars may act as signalling molecules in addition to their roles as metabolic resources and structural constituents of cells, it is interesting to study the effects of a general or selective repression of monosaccharide transport. The effects of repressing sucrose transport in plants by the introduction of antisense constructs have been described in detail (see Introduction). The only work available so far on a modification of monosaccharide transport is a recent report by Sherson et al. (2000) concerning a knock-out mutant for AtSTP1.
In the present work, transformed tobacco plants with modified monosaccharide transport were produced and characterized. These plants were obtained by RNA sense and antisense expression of the grapevine putative monosaccharide transporter VvHT1. VvHT1 encodes a protein highly homologous with several monosaccharide transporters, including MST1, a tobacco monosaccharide transporter mainly expressed in sink tissues (roots, flowers and young leaves; Sauer and Stadler, 1993). VvHT1 is expressed in sink organs as young grapevine leaves and grape berries (Fillion et al., 1999). Attempts to detect monosaccharide transport after expression of the corresponding cDNA in yeasts or oocyte have failed so far, possibly due to problems resulting from heterologous expression. Embryogenic grapevine cultures transformed with sense and antisense VvHT1 constructs have been obtained (P Coutos-Thévenot and S Delrot, unpublished results), but will require several years of growth and development before the physiological effects on the plants can be studied. Therefore, in the present work, the effects of VvHT1 sense and antisense expression were studied in tobacco plants. This model was chosen because of the high similarity between VvHT1 and MST1, and because tobacco can easily be transformed and grows rapidly. Whatever the exact function of VvHT1 in grapevine, its similarity to MST1 resulted in changes in the expression of the endogenous monosaccharide transporters from tobacco and allowed the consequences of these changes to be studied. Because both MST1 and VvHT1 are mainly expressed in sink tissues, transcript analysis and sugar uptake measurements on transgenic tobaccos were conducted with young leaves from in vitro-grown plants, which are expected to be mainly heterotrophic.
The phenotypically modified plants, whose dry weight and/or growth curves were affected (Fig. 3) also contained lower amounts of the host MST transcripts (Fig. 5) in their leaves. This holds true for the sense transformants S6 and S7, and for the antisense transformants AS36 and AS41. In the S24 clone, where the decrease in MST transcripts is less apparent, no marked effect on growth was observed.
It is likely that down-regulation of MST in sense plants expressing VvHT1 at a high level (S6, S7) results from post-transcriptional gene silencing, due to the strong sequence homology between the host MST1 gene and the VvHT1 transgene. This homology reaches 75%, and the phenomenon of unidirectional or bi-directional (i.e. co-suppression) post-transcriptional gene silencing has been observed when sequence homology is as low as 55% (Mol et al., 1994). The decrease in transcript level due to RNA degradation may affect only the host gene, only the transgene or both, and the extent of this decrease is not necessarily related to the level of transgene expression (Vaucheret et al., 1998; Fagard and Vaucheret, 2000). In S6 and S7, the host gene was markedly repressed and VvHT1 transcripts were present at high levels in both plants. The effects observed in S6 and S7 may, therefore, be explained by a unidirectional gene silencing, a particular case of post-transcriptional gene silencing where the host gene is the target, whereas the transgene is only the source of this epigenetic phenomenon (Vaucheret et al., 1998). In the case of S24, since the PCR amplification proved the presence of VvHT1 and hybridization with the GUS probe confirmed the transgene expression, the roles of both orthologues in post-transcriptional gene inactivation are different. In this case, silencing affects the transgene, while the host gene remains not down-regulated.
In AS36, AS40 and AS41 plants, the shoot/root ratio was also significantly increased. However, the weight of the individual organs was much less affected than in the sense transformants S6 and S7. Although the physiological consequences of the down-regulation of MST expression through sense or antisense expression of VvHT1 are similar, they appear stronger in the sense transformants, which suggests that the sense-induced epigenetic inactivation is more efficient than the antisense one.
The data provide evidence for MST playing a key role in the development and carbohydrate exchanges in tobacco plant. This is further confirmed by a close examination of dry weight results, which show that not all organs are affected in the same way by the decrease in MST expression (Fig. 3). The stems and roots are more affected than the leaves in S6 and S7. This results in a significant increase of the shoot/root ratio. Because glucose uptake is reduced in leaf discs (Fig. 6), the alteration of growth rate and sugar partitioning observed in sense transformants may be at least partly due to changes of sugar transport in the leaf.
The fact that the phenotype was lost when the plants were transferred from in vitro conditions to the greenhouse may result mainly from more efficient photosynthesis in the latter environment and, consequently, better conditions for normal leaves/roots exchanges and relationships. Successful in vitro restoration of the mutant phenotype in the T0 generation as well in the selfed (Fig. 7) or back-crossed transgenic tobaccos, confirms the importance of these culture conditions for the appearance of post-transcriptional gene silencing. These results suggest that the phenotype only appears under conditions where photosynthesis may be limiting, as is probably the case in the present studies. Under conditions of limiting photosynthesis, the supply of sugar in the growth medium increases the growth of tomato plantlets grown in vitro, whereas it is detrimental to growth under high photosynthetic photon flux (Le et al., 2001).
In addition, this mutant phenotype also correlates again with a significant inhibition of glucose absorption activity in the T1 progeny (Fig. 8A). These data confirm the functional relationship between stunted growth and modified glucose transport activity. No direct comparison can be made between the effects observed here for plants affected on hexose transport and grown in vitro, and the effects resulting from repression of sucrose transport observed in tobacco or potato plants grown in the greenhouse (Kühn et al., 1996; Bürkle et al., 1998; Schulz et al., 1998). It is likely that in tobacco as in Arabidopsis (Büttner and Sauer, 2000), Ricinus (Weig et al., 1994) and Chenopodium (Roitsch and Tanner, 1994), the hexose transporters are encoded by multigenic families. Therefore, VvHT1-related impact on MST1 gene expression may also concern MST1 paralogues, i.e. other members of the same gene family, thus causing the observed mutant phenotype. Interestingly, for the only knock-out mutant affected on a monosaccharide transporter (AtSTP1) known so far (Sherson et al., 2000), the plants grow and develop normally. This implies that phenotypic changes may require the repression of more than one member of multigenic families. In this context, the classical antisense and sense silencing remain successful approaches to modulate simultaneously the expression of several paralogues of plant transporters.
The fact that the expression of a presumably functional VvHT1 protein in the sense plants does not compensate for the suppression of MST1 and its paralogues is intriguing. Two hypothesis may be envisaged: (a) the overexpression of VvHT1 also induces a translational control that prevents the activity of the translated transporter and (b) although highly homologous to a functional hexose transporter, VvHT1 does not actually encode a functional hexose transporter. The latter hypothesis is at least indirectly supported by attempts to express VvHT1 in yeasts or Xenopus oocytes, which so far do not allow a firm conclusion about any glucose uptake activity (L Laquitaine, R Atanassova, unpublished data).
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Acknowledgements |
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Marina Leterrier was supported by a grant from the Conseil Régional Poitou-Charentes. The authors are indebted to Bruno Faure for taking care of the plants in the greenhouse.
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References |
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