Journal of Experimental Botany, Vol. 53, No. 369, pp. 631-637,
April 1, 2002
© 2002 Oxford University Press
Original Papers |
Use of aphid stylectomy and RT-PCR for the detection of transporter mRNAs in sieve elements
School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
Received 16 July 2001; Accepted 20 November 2001
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Abstract |
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Unmodified samples of barley (Hordeum vulgare) sieve tube sap have been obtained by severing the stylets (stylectomy) of feeding aphids and collecting the exuding liquid. Primers were designed to direct the amplification of a series of specific cDNAs encoding barley proteins selected because of their significance in sieve tube function. mRNA encoding the H+/sucrose co-transporter SUT1, a putative aquaporin and the H+/ATPase PPA1 were detected in sieve tube sap. These mRNA species appear to be present at very low concentrations. mRNA encoding the potassium transporter HAK1 could not be detected. The results strongly suggest that some mRNA species are imported into sieve elements, which are enucleate, from neighbouring companion cells.
Key words: Aphid, barley, phloem, RT-PCR, stylectomy, transporter.
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Introduction |
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Translocation through the phloem provides arguably the most important transport conduit of the plant. One of the difficulties of studying the mechanism and control of translocation is the problem of access to phloem cell contents. Some studies have made use of the fact that sieve element exudate can be collected from cut stems. While such techniques allow the collection of ‘phloem-enriched’ samples, there is an obvious potential problem of contamination with the contents of other cells that are damaged by cutting. This is a particular problem when one is analysing mRNA populations; the concentration of mRNAs in sieve elements is many orders of magnitude lower than that of the cytoplasm of nearby parenchyma cells. The effects of contamination of ‘sieve element exudate’ samples by other cell types has been demonstrated by Ruiz-Medrano et al., who showed the presence of Rubisco mRNA after 30 cycles of RT-PCR (Ruiz-Medrano et al., 1999
). Phloem-feeding insects, such as aphids, offer the opportunity of specifically sampling the contents of sieve elements. Using aphid stylectomy, one can allow the insects to initiate feeding and then sever the stylets. Pure sieve element sap can be collected as it exudes from the stump of stylets; this material has only been exposed to the inert inner surfaces of the stylets and hence is not altered by the insect. Stylectomy has previously been used to characterize soluble proteins in wheat sieve elements (Fisher et al., 1992) and to detect protein kinase activity in rice phloem sap (Nakamura et al., 1995). Sasaki et al. were able to demonstrate the presence of mRNA for thioredoxin, oryzacystain-I and actin in pure rice phloem sap using stylectomy (Sasaki et al., 1998). Their target mRNA species were chosen on the basis of the demonstrated presence of abundant, specific proteins in the sap. Other studies have indirectly indicated the presence of mRNA in the sieve elements using in situ RT-PCR approaches. The mRNAs included those for proteins CmPP16 and CmNACP in cucumber (Xoconostle-Cazares et al., 1999; Ruiz-Medrano et al., 1999) and, significantly, the mRNA for the H+/sucrose co-transporter SUT1 in sieve elements of potato and tomato (Kühn et al., 1999).
In this paper, the detection of mRNA species selected for their relevance to phloem function in pure sieve element sap is described. Aphid stylectomy has been used to collect barley sieve element sap samples followed by RT-PCR. A number of membrane transporters are involved in accumulating solute and facilitating bulk flow. The mRNA H+/sucrose co-transporter SUT1 was selected as a target because sucrose is the major solute in phloem sap. SUT1 has already been convincingly localized in source sieve elements of Solanum species using in situ hybridization (Kühn et al., 1997). H+/sucrose symporters are energized by outwardly directed H+/ATPases that acidify the apoplast (Morsomme and Boutry, 2000) and for this reason barley H+/ATPase PPA1 was also selected as a target. In Vica faba (Bouche-Pillon et al., 1994) and Arabidopsis thaliana (DeWitt and Sussman, 1995), H+/ATPase transporters have been convincingly located in the companion cells.
Potassium is a major inorganic solute found in the sieve elements and its transport is facilitated by a range of ion channels (Schachtman, 2000). A cDNA for the potassium channel HAK1 has recently been isolated from the roots of barley (Santa-Maria et al., 1997) and HAK1 mRNA was selected as the third target. Finally, the bulk flow that drives translocation requires the influx of water into sieve elements. This is facilitated by the water channels aquaporins (Chrispeels et al., 1997). While none of these transporter proteins have yet been localized to the sieve element–companion cell complex, their presence in either, or both, of the sieve elements or companion cells is consistent with current theories of phloem function. A major intrinsic protein (MIP) family, with an expression pattern consistent with a role in this process has previously been identified in barley leaves (Hollenbach and Dietz, 1995). Since this protein is a candidate molecule for controlling flow through aquaporins within the phloem, the mRNA for this MIP protein was selected as the fourth target in the study. As far as is known, this is the first demonstration of the presence of specific transporter mRNAs in sieve element sap using aphid stylectomy.
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Materials and methods |
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Plant growth
Barley seedlings were germinated on wet filter paper in the dark at 20 °C for 48 h. Germinated seeds were transferred to plastic mesh suspended over aerated 0.5 mol m-3 CaCl2 and kept in the dark for a further 24 h. After this period plants were transferred to aerated Long Ashton nutrient solution at 20 °C and subjected to a 16/8 h light period (
200 µmol m-2 s-1). Plants were used 6–7 d from germination.
Aphid stylectomy
The method used was similar to previously published approaches (Pritchard, 1996). Plants were secured into specially constructed chambers which allowed the roots to be bathed in hydroponic medium while the basal 20% of the leaf projected into a second leaf chamber. The remainder of the leaf was uncovered. Aphids (5–10 per plant) were restricted to the leaf chamber by means of a tight-fitting lid. Aphids (Rhopalosiphum padi L.) were allowed to feed overnight, after which the lid was removed and the tungsten needle of a microcautery unit (Synthech CA-50, Hilversum, Netherlands) introduced while observing under the microscope. The stylets of aphids in the correct orientation were cut and examined for any exudation. Once stylets had been cut and were exuding, the chamber was flooded with water-saturated paraffin oil. The whole procedure, from removing the aphid-chamber lid to filling with oil, took less than 5 min (Pritchard, 1996).
Sieve element sap collection and handling
The objective of this section was to minimize contamination of mRNA by external sources and rapidly to inhibit any phloem RNase. Typically 300–600 nl of sap were collected from each severed stylet tube at a rate of approximately 60–120 nl h-1. Stylets were left to exude under paraffin oil to prevent evaporation and to protect the exudate from environmental RNases. Accumulating sap was aspirated off the stylets before the droplet touched the leaf epidermis and the sap was periodically collected into a glass capillary backloaded with RNase inhibitor buffer (0.5 unit µl-1 ANTI-RNase (Ambion), 1 unit µl-1 RNAsin (Promega) in 10 mM Tris, pH 8.0)
Relative volumes of sap and buffer were monitored (using an eyepiece graticule and assuming sap droplets were spherical) throughout the collection and additional buffer was taken up at appropriate points to ensure good mixing and to maintain an approximate 1:2 ratio of sap:buffer. Paraffin oil was sucked up after the sap/buffer mixture and samples stored at -20 °C in the collection capillary for up to 6 weeks prior to use. Thawed sap/buffer mixture was expelled into a microfuge tube immediately prior to RT-PCR experimentation. No attempt was made to exclude the paraffin oil which preliminary experiments showed did not interfere with subsequent RT-PCRs.
RT-PCR
The small quantities of phloem sap and the extremely low copy number necessitated extreme care during processing of the samples to prevent contamination. This included a separate area for pre-PCR manipulations, dedicated air-displacement pipettes, barrier tips, and aliquoted stocks. Negative controls included water samples subjected to PCR (which would allow detection of amplification of the target gene due to contamination with genomic DNA) or to both PCR and RT-PCR. Positive controls, containing approximately ten copies of each single-copy target gene, were included for each RT-PCR. Samples of sieve element sap (ranging from 0.42 to 0.63 µl volume) were reverse transcribed using the Sensiscript Reverse Transcription Kit (Qiagen) in the presence of the primer ENNT24 (5'-TATAGAATTCGCGGCCGCTCGCGA (T)24) in a 10 µl reaction. Aliquots (0.1–1.0 µl) of the reverse transcription reaction were subjected to PCR for 50 cycles (94 °C for 30 s, 60 °C for 30 s, 72 °C for 60 s, with an initial 95 °C for 10 min to activate the HotStarTaq enzyme (Qiagen)) on a Hybaid OnmiGene cycler. The PCR reaction contained HotStarTaq bufferx1, including 1.5 mM MgCl2, 0.2 mM each dNTP, 0.2 µM of each forward and reverse gene-specific primer and 4 units HotStarTaq per 100 µl reaction. PCR volumes were kept small (15 µl) to allow multiple amplifications from each sample of reverse transcribed phloem sap mRNA. The primers used are given in Table 1. Products were visualized by agarose gel electrophoresis. Bands obtained were stabbed with a hypodermic needle and the resulting agarose plug re-amplified for no more than 25 cycles using the original primer pair. Resulting products were purified using the Qiaquick PCR purification Kit (Qiagen) and sequenced (Lark Biotechnologies, Saffron Walden, UK) to verify their identity.
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Results |
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Determination of the relative amount of RNases in phloem sap
It was determined whether sieve element sap contains significant levels of RNases by comparison with RNase A standards. 250 ng samples of rRNA were incubated with a range of concentrations of Rnase A, or with samples of barley leaf extract or barley sieve element sap (obtained by stylectomy), and checked for RNA degradation by agarose gel electrophoresis. The lowest level of RNase detectable by this assay was 1 ng RNAse A ml-1. The results showed that the barley leaf extract contained the equivalent of at least 300 ng RNaseA activity per g leaf extract (1 g leaf=1 ml extract). Purified sieve element sap contained no detectable RNase activity. However, since the activity could have been below the limits of detection of this technique, sieve element sap was subsequently collected into buffer containing a cocktail of RNase inhibitors as a precautionary measure.
Demonstrating the presence of transport specific mRNAs in sieve element sap
Reverse transcription was carried out using an oligo-dT primer which allowed subsequent detection of multiple species of cDNAs from the same sample of sieve element sap. Small aliquots (0.1–1 µl) of the RT-reaction were amplified using gene-specific primers specific to four Hordeum vulgare genes.
Sucrose/proton symporter SUT1
Primers were designed using the known sequence of the Hordeum vulgare H+/sucrose symporter sut1 (Weschke et al., 2000; accession no. AJ272309). These primers directed the amplification of a single product when used in PCRs with barley total leaf cDNA template. Sequencing of the 0.8 kb product confirmed it to be barley SUT1 cDNA; the corresponding amplification product using genomic DNA template was 3.5 kb. Next, a total of 13 PCRs were performed using these primers in combination with template cDNA produced by RT-PCR using stylectomy-purified barley sieve element sap. cDNA present in the equivalent of 42–63 nl of sieve element sap was amplified in 15 µl PCRs, 5 µl of which were analysed by gel electrophoresis. Three PCRs resulted in bands of approximately 0.8 kb (Fig. 1) and sequencing these confirmed them to represent cDNA of the barley sucrose transporter SUT1 (Fig. 5a). An additional band of approximately 1.3 kb re-amplified, using internal primers specific to sut1, and was confirmed as being H. vulgare SUT1 cDNA by sequencing the resultant PCR fragment. Performing more than 50 cycles of PCR on sieve element sap did not result in the production of any additional products.
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ATPase
Reverse PCR primers were designed to a known barley plasmamembrane H+/ATPase (Hess et al., 1998; accession no. AJ222786) previously shown to be expressed in leaves. As this sequence was too short to design suitable forward primers, several degenerate forward primers were designed against conserved regions of plasma membrane H+/ATPases of various monocotyledonous species. Using these primers in combination with barley leaf cDNA resulted in the amplification of several fragments all of which were sequenced. A database search showed that one of them had very high homology (87% identity) to maize H+/ATPase mRNA; this novel sequence was deposited in the EMBL database as Hordeum vulgare PPA1 mRNA (accession no. AJ295612). Using this sequence information, a new set of gene-specific primers was designed against barley ppa1, which amplified single fragments of 0.8 kb and 0.5 kb using barley genomic DNA and cDNA as templates, respectively. Sequencing of both products confirmed their identities. A total of 12 PCRs were performed using sieve element sap cDNA and the barley gene-specific primers. cDNA present in the equivalent of 13–42 nl of sieve element sap was amplified in 15 µl PCRs. Single fragments of approximately 0.5 kb were obtained on two out of 12 occasions (Fig. 2). Sequencing these confirmed them to represent the barley H+/ATPase PPA1 (Fig. 5b
). An additional two faint bands initially sized to 1.5 kb were amplified from sieve element sap cDNA; these re-amplified using the original primers to the correct size of 0.5 kb. Sequencing confirmed both to be the barley H+-ATPase PPA1 cDNA.
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Aquaporin
Degenerate primers were designed against the conserved region of plasma membrane aquaporins (PIPs) from various monocotyledonous species. Using the barley leaf cDNA template, these degenerate primers resulted in a product which, when sequenced, showed high homology with a previously described PIP from barley root (98% identity to EMBL accession no. AB009309) and leaf (100% identity EMBL accession no. X76911; Hollenbach and Dietz, 1995). A second generation of gene-specific primers was designed against this barley aquaporin sequence, which successfully directed amplification of products of 1.3 kb and 0.7 kb using barley genomic DNA and leaf cDNA templates, respectively. A total of 15 PCRs were performed using sieve element sap cDNA with the barley aquaporin-specific primers. cDNA present in an equivalent of 5–63 nl of sieve element sap was amplified in 15 µl PCRs. Initially, bands of approximately 0.6–0.7 kb (Fig. 3) were obtained in two samples and sequencing these confirmed one of them to represent the Hordeum aquaporin cDNA (Fig. 5c). Interestingly, the second band showed homology to the C-terminal region of the structural polyprotein of a single-stranded RNA virus of the aphid Rhopalosiphum padi (EMBL AF022937). Performing an additional 20 cycles of PCR on the previously negative PCRs resulted in two additional bands of approximately 0.6–0.7 kb (Fig. 4). Sequencing these revealed one of them to represent the Hordeum aquaporin as expected. The second fragment, however, again showed homology to the same R. padi virus structural protein gene as previously (Fig. 5d). The two PCRs producing the viral fragments originated from the same sample of sieve element sap collected using the same aphid.
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Potassium transporter (HAK1)
Degenerate primers were produced using conserved regions common to all barley and rice potassium transporters. Use of these primers with barley genomic DNA led to the cloning of a variant of the hak1 gene (98% identity to EMBL AF025292), which was submitted to EMBL (accession AJ297886). The corresponding barley leaf cDNA template was detected by PCR and its sequence deposited with EMBL (accession AJ297888). 14 PCRs were performed using sieve element sap cDNA and barley hak1-specific primers. cDNA present in the equivalent of 11–43 nl of sieve element sap was used in 15 µl PCRs. None of these reactions resulted in amplification of a product (not shown). HAK1 cDNA originating from barley leaf was successfully amplified, demonstrating that the gene is expressed in leaf tissue and could be amplified using our primers.
A summary of the PCR analysis for the four transporters is shown in Table 1. All mRNA species except the potassium transporter HAK1 could be detected in phloem sap.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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This study has demonstrated that it is possible to detect mRNA for specific plasmamembrane transporters in as little as 10 nl of sieve element sap collected by aphid stylectomy using RT-PCR. The message for the SUT1 H+/sucrose transporter was detected in the sieve element sap, although the absolute level of the message was low. In tobacco, in situ hybridization has previously located SUT1 mRNA in both the companion cell and the sieve element, in particular, in the orifices of the plasmodesmata connecting these two cell types (Kühn et al., 1997
). It remains to be seen whether this finding is the result of directed export or simple leakage of the SUT1 mRNA from the companion cell into the sieve element. Similarly, the message for the PPA H+/ATPase and a putative aquaporin (a member of the MIP family) was detected in sieve element sap, again at a low absolute abundance of both transcripts. At present the mechanism by which these mRNAs entered the sieve element cannot be determined. By contrast, it was not possible to detect the message for the HAK1 potassium transporter in sieve element sap.
It is clear that the RT-PCR approach could be extended to develop a catalogue of mRNA species that are detectable in the sieve element. However, care must be taken in the interpretation of results obtained following the use of stylectomy. For instance, it is possible that the mRNA encoding viral polyprotein detected in the sieve element sap sample was present within the saliva of the aphid. It was not possible to define the exact concentration of mRNAs in this study's sieve element sap samples, but since sap is actually being collected from a file of sieve element cells, the level of message per cell appears extremely low in comparison to other studies on single cells (Karrer et al. 1995). An indication of target mRNA abundance was gained through the use of appropriate negative and positive controls. The positive control routinely used was barley genomic DNA (50 pg estimated to contain about ten copies of a single-copy gene) and this template always supported the amplification of the target product, visible as a strong band, after 50 cycles of PCR. However, only about 20% of PCRs containing sieve element sap cDNA treated in this way resulted in amplification of the target sequence. The low proportion of positive amplifications indicates that the concentrations of the target mRNAs in sieve element sap are very low. Other workers have also used the proportion of successful amplifications as a measure of mRNA abundance in low volume samples (Karrer et al., 1995). With regard to this study's failure to detect HAK1 transcript in sieve element sap, there appear to be three possible explanations: (a) HAK1 mRNA is completely absent from the companion cells; if the HAK1 transporter protein is not present in the sieve element–companion cell complex, its function must be performed by a different potassium transporter such as AKT2/3 (Deeken et al., 2000): (b) the low abundance of HAK1 mRNA present in sieve element sap is below this study's detection limit: (c) the HAK1 message is present in the companion cells, but the plasmodesmata exert a degree of specificity that does not allow the HAK1 message to move into the sieve element sap. In the latter case, the HAK1 message could be translated in the companion cell and the protein moved through plasmodesmata into the sieve element. The explicit assumption in the present work was that detection of the mRNA in the sieve element is evidence for translation in the companion cell. However, non-detection of a specific message in the sieve element clearly does not exclude the possibility of companion cell-based translation. The validity of these assumptions can be tested when there is a greater understanding of the selectivity to mRNA and proteins of plasmodesmata. Recent reviews have suggested that this is potentially complex (Thompson and Schulz, 1999; Oparka and Cruz, 2000) and is therefore a critical question for further research.
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Acknowledgments |
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This work was funded by a BBSRC ROPA award.
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Notes |
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1 To whom correspondence should be addressed. Fax: +44(0)1214145925. E-mail: h.j.newbury{at}bham.ac.uk
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