Downregulating the sucrose transporter VpSUT1 in Verbascum phoeniceum does not inhibit phloem loading Cankui Zhang and Robert Turgeon1 Department of Plant Biology, Cornell University, Ithaca, NY 14853 Edited by Maarten J. Chrispeels, University of California San Diego, La Jolla, CA, and approved September 16, 2009 (received for review April 15, 2009) Sucrose is loaded into the phloem in the minor veins of leaves raffinose and stachyose. The presence of these abundant plas- before export. Two active, species-specific loading mechanisms modesmata raises the possibility that, in RFO plants, sucrose have been proposed. One involves transporter-mediated sucrose stays within the symplast all the way from the mesophyll cells to transfer from the apoplast into the sieve element-companion cell the SEs, so-called symplastic loading. complex, so-called apoplastic loading. In the putative second mech- The concept of apoplastic loading, mediated by transporters, anism, sucrose follows an entirely symplastic pathway, and the is intuitively attractive because it employs the proton motive solute concentration is elevated by the synthesis of raffinose and force to transfer sucrose into the phloem against a thermody- stachyose in the phloem, not by transporter activity. Several sucrose- namic gradient. Symplastic loading, on the other hand, has been transporting plants have been shown to be apoplastic loaders by viewed with more caution because symplastic flux, through the downregulating sucrose transporter 1 (SUT1), leading to accumula- cytoplasmic sleeve of plasmodesmata, is passive; there is no tion of sugars and leaf chlorosis. In this study we compared the effect obvious direct mechanism for establishing an uphill concentra- of downregulating SUT1 in Nicotiana tabacum, a sucrose transporter, tion gradient (13–16). Yet, in RFO-transporting plants, the and Verbascum phoeniceum, a species that transports raffinose and concentration of sugar in the phloem sap is comparable to that stachyose. To test the effectiveness of RNAi downregulation, we of apoplastic loaders and considerably higher than in bundle .measured SUT1 mRNA levels and sucrose-H؉ symport in leaf discs PLANT BIOLOGY Mild NtSUT1 downregulation in N. tabacum resulted in the pro- sheath and mesophyll cells (16, 17). This is a phenomenon that nounced phenotype associated with loading inhibition. In contrast, requires explanation: two adjacent plant cell types—bundle no such phenotype developed when VpSUT1 was downregulated in sheath and companion cell—linked by abundant plasmodes- V. phoeniceum, leaving minimal sucrose transport activity. Only those mata, yet with highly different solute concentrations and hydro- plants with the most severe VpSUT1 downregulation accumulated static pressures. more carbohydrate than usual and these plants were normal by other A ‘‘polymer trap’’ mechanism (18–20) has been proposed to criteria: growth rate, photosynthesis, and ability to clear starch during explain how sucrose is loaded through plasmodesmata in RFO the night. The results provide direct evidence that the mechanism of plants. According to this model, sucrose diffuses through plas- phloem loading in V. phoeniceum does not require active sucrose modesmata from mesophyll cells to intermediary cells, where it uptake from the apoplast and strongly supports the conclusion that is converted into RFOs. These larger sugars cannot diffuse back the loading pathway is symplastic in this species. again due to their size. If this hypothesis is correct, sucrose transporters do not play a direct role in loading and their plasmodesmata ͉ raffinose ͉ stachyose downregulation should have little, if any, effect on translocation. Although the polymer trap hypothesis provides a theoretical hloem loading provides the driving force for long-distance framework for symplastic loading, it has been suggested that Ptransport from leaves to sink organs in many plants by loading in RFO plants is nonetheless apoplastic and transporter elevating the carbohydrate content and hydrostatic pressure in driven (13, 14, 21). Indeed, sucrose transporters are found in the sieve elements (SE) and companion cells (CC) of minor veins the leaves of plants with abundant plasmodesmata, and SUT1 (1–3). In a large number of species, phloem loading is mediated has been localized to the minor veins of an RFO-transporting by transporters on the plasma membranes of the SEs and CCs. species (13). These transporters drive sucrose, and in some cases sugar Until recently it has not been possible to test the concept of alcohol, from the apoplast into the SE-CC complex. The most symplastic loading directly by downregulating SUT1, due to the direct proof of apoplastic loading has come from studies in which lack of a readily transformable model plant. However, Verbas- sucrose transporter 1 (SUT1/SUC2) is downregulated by antisense cum phoeniceum, an RFO-transporting species, has been found technology or DNA insertion (4–9). Such downregulation in- to transform with ease (18). Earlier experiments demonstrated hibits phloem loading and results in accumulation of sugars and that RFO synthesis is required for efficient phloem loading in V. starch, stunted growth, diminished photosynthesis, and leaf phoeniceum (18) but these experiments did not address the chlorosis. These experiments confirm the necessity of transmem- fundamental question of how sucrose enters the minor veins. The brane transport of sucrose in these species and the indispensable results presented here indicate that severe downregulation of role of SUT1 in apoplastic phloem loading. In species that load from the apoplast, relatively few plasmod- esmata connect the SE-CC complex to surrounding cells (10, 11). Author contributions: C.Z. and R.T. designed research; C.Z. performed research; C.Z. and This is unsurprising given that extensive symplastic continuity R.T. analyzed data; and C.Z. and R.T. wrote the paper. would create a futile cycle of active loading from the apoplast The authors declare no conflict of interest. and passive leakage back to mesophyll cells. However, in many This article is a PNAS Direct Submission. other species plasmodesmata in the minor veins are numerous, Data deposition: The sequence reported in this paper has been deposited in the GenBank sometimes extremely so (10–12). Plasmodesmata are especially database (accession no. FJ797307). numerous between bundle sheath cells and specialized compan- 1To whom correspondence should be addressed. E-mail: [email protected]. ion cells, known as intermediary cells, in the minor veins of This article contains supporting information online at www.pnas.org/cgi/content/full/ species that transport raffinose-family oligosaccharides (RFOs), 0904189106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0904189106 PNAS ͉ November 3, 2009 ͉ vol. 106 ͉ no. 44 ͉ 18849–18854 Downloaded by guest on September 28, 2021 A ABC DEF B Fig. 2. Autoradiographs of Nicotiana tabacum (A–C) and Verbascum phoe- niceum (D–F) leaf discs exposed to [14C]sucrose. A and D are from wild-type plants, B and E from plants with moderate SUT1 downregulation (NtT1.19.5 and VpT2.27.10, respectively), and C and F from plants with severe SUT1 downregulation (NtT1.19.6 and VpT2.27.2, respectively). Diameter of leaf discs, 1.6 mm. more acute with leaf age. This chlorosis was more evenly Fig. 1. Representative wild-type and transgenic Nicotiana tabacum (A) and distributed throughout the lamina than in plants with a weak Verbascum phoeniceum (B) plants. Left to right (A): wild-type, weak, moder- phenotype and the leaves were smaller than in wild-type plants. ate and severely downregulated NtSUT 1 RNAi lines; (B): wild-type (left) and three significantly downregulated VpSUT1 RNA lines. Note that the degree of Transgenic plants with very severe chlorosis grew to a height of downregulation of NtT1.19.7 and VpT2.27.2 is similar. (Scale bars, A ϭ 10 cm less than 15 cm and did not produce seeds, even after 12 months and B ϭ 4 cm.) in the greenhouse. Transgenic V. phoeniceum plants grew and developed nor- mally (Fig. 1B) in growth chambers and in a greenhouse with SUT1, leading to almost complete loss of sucrose transporter supplemental lighting (600–800 ␮mol photons mϪ2 sϪ1). With activity, has little effect on long-distance transport. the exception of a single individual, apparently suffering from an unrelated problem sometimes noted in wild-type plants, none of Results the more than 100 transgenic plants displayed evidence of SUT1 in V. phoeniceum. Degenerate primers were designed based chlorosis and there was no distinguishable difference in the size on the alignment of SUT1 sequences from five species, including of leaves of transgenics and wild-type plants. AmSUT1 from Alonsoa meridionalis, an RFO plant. An 816-bp fragment amplified from V. phoeniceum leaf cDNA with these Downregulation of Sucrose Uptake and SUT1 Expression. The capac- primers was used as a probe to screen a cDNA library of mature ity of leaf tissue to take up exogenous sucrose, a measure of V. phoeniceum leaves. Numerous screenings were carried out sucrose transporter activity (20), was measured by incubating with different stringencies to identify members of the SUT1 gene leaf discs in Mes-buffered [14C]sucrose for 1 h before washing family in V. phoeniceum. All identified clones encoded the same and scintillation counting. Preliminary experiments indicated sequence with an ORF of 511 amino acids. The deduced amino that uptake was linear with time for more than 2 h. Autoradio- acid sequence is highly homologous to SUT1 from A. meridi- graphs of leaf discs demonstrated that radiolabel was localized onalis (80%), Asarina barclaiana (79%), Nicotiana tabacum primarily in the veins of wild-type plants of both species (Fig. 2 (74%), Solanum tuberosum (73%), and Plantago major (75%) A and D). (13, 14, 22–24). The identified sucrose transporter gene from V. Quantitative uptake experiments were conducted with and phoeniceum was named VpSUT1 and the sequence deposited in without p-chloromercuribenzenesulfonic acid (PCMBS). PC- GenBank (accession FJ797307). MBS completely inhibits sucrose transporters but does not block uptake by a second, non-saturable transport mechanism (25–27).