Downregulating the Sucrose Transporter Vpsut1 in Verbascum Phoeniceum Does Not Inhibit Phloem Loading

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 plasmodesmata 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

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B Fig. 2. Autoradiographs of Nicotiana tabacum (A–C) and Verbascum phoeniceum (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 normally (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). Transgenic Plants. RNAi constructs were designed for the down- The second mechanism, constituting approximately 28% of total regulation of SUT1 in V. phoeniceum and N. tabacum. Over 100 uptake in wild-type N. tabacum and V. phoeniceum,isnot independent transgenic lines of each species were generated. In involved in phloem loading (see Discussion). The results re- tobacco, visible chlorosis developed on the leaves of approxi- ported here are for the PCMBS-sensitive phase only (see Ma- mately 90% of the transgenic plants in a manner similar to that terials and Methods). described when NtSUT1 is downregulated by antisense technol- Leaf tissue from phenotypic transgenic tobacco lines took up ogy (4). The severity of the phenotype differed considerably in less [14C]sucrose than leaf tissue from wild-type plants (Fig. 3A). the various lines. In the weak phenotype tobacco line NtT1.27.2, In the 20 independent tobacco lines tested, inhibition ranged one area approximately 2 cm in diameter between the third and from 23–93% and was most severe in transgenic plants that fourth major vein on each mature leaf became chlorotic, while displayed the most severe chlorotic symptoms and retarded the rest of the leaf was healthy. The heights of the wild-type and growth. Downregulation had no statistically-significant effect on transgenic plants with weak phenotypes were indistinguishable. PCMBS-insensitive uptake. Downregulation of SUT1 expression However, transgenic tobacco plants with a more severe pheno- ranged from 4- to 7-fold in tobacco lines with moderate chlorosis type were noticeably stunted and the leaves were more chlorotic to approximately 23-fold in plants with severe chlorosis (Fig. 1A). Chlorosis developed in a basipetal pattern that (Fig. 4A). paralleled the sink-source transition and became progressively Veins were visible in autoradiographs of wild-type (Fig. 2A)

18850 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0904189106 Zhang and Turgeon Downloaded by guest on September 28, 2021 14 Fig. 3. PCMBS-sensitive [ C]sucrose uptake by leaf discs of (A) wild-type Fig. 5. Soluble sugars in mature leaves of Nicotiana tabacum (A) and (NtWT) and transgenic Nicotiana tabacum and (B) wild-type (VpWT) and Verbascum phoeniceum (B). Results are from individual transgenics and from transgenic Verbascum phoeniceum lines. Results are from individual trans- either two (A) or three (B) wild-type (WT) plants. Error bars, SE (n ϭ 6 for NtWT, genics and from either two (A) or three (B) wild-type plants. PCMBS-sensitive n ϭ 9 for VpWT, n ϭ 3 for transgenics) except N. tabacum line NtT1.27.2. Only (transporter driven) uptake values were obtained by subtracting uptake in the one sample was analyzed for this line due to the limited amount of plant presence of PCMBS from uptake without PCMBS. Error bars, SE (n ϭ 6 for material. NtWT, n ϭ 9 for VpWT, n ϭ 3 for transgenics). Uptake rates for discs from wild-type plants were 135 nmol cmϪ2 hϪ1 and 229 nmol cmϪ2 hϪ1 for tobacco

and V. phoeniceum, respectively. lation in most of the lines with moderate reduction of [14C]su- PLANT BIOLOGY crose uptake ranged from 6- to 10-fold while in lines VpT2.27.4 and VpT2.27.2, with the most severe repression of [14C]sucrose and most transgenic tobacco lines, although the intensity of the uptake, SUT1 was downregulated 17- and 24-fold, respectively vein images declined with the degree of downregulation (Fig. (Fig. 4B). It is possible that there are other sucrose transporters, 2B). No veins were visible in the autoradiographs from the most in addition to SUT1, in the V. phoeniceum leaf. However, if this severely downregulated lines (Fig. 2C). is so, they must not play an appreciable role in sucrose uptake Since transgenic V. phoeniceum plants did not exhibit chlorotic from the apoplast since downregulation of SUT1 alone results in symptoms, all of the more than 100 independently derived lines almost complete inhibition of [14C]sucrose uptake. As with were screened for [14C]sucrose uptake capacity. Inhibition of tobacco, SUT1 downregulation had no statistically-significant PCMBS-sensitive [14C]sucrose uptake ranged from 0–93% in the effect on PCMBS-insensitive uptake. transgenics, compared to wild-type plants (Fig. 3B). Quantita- Veins were visible in autoradiographs of leaf discs from tive RT-PCR on RNA of leaf tissue from representative plants 14 wild-type V. phoeniceum plants (Fig. 2D). Unlike tobacco, the indicated that, as in tobacco, plants with diminished [ C]sucrose intensity of vein images was not noticeably diminished in mod- uptake exhibited significant SUT1 downregulation. Downregu- erately downregulated lines (Fig. 2E). In the most severely downregulated line (VpT2.27.2), veins were apparent in autoradiographs of approximately 20% of the discs; no veins were apparent in the remaining 80% (Fig. 2F)

Carbohydrate Accumulation and Photosynthesis. Soluble carbohydrates accumulated in the mature leaves of tobacco transgenics, as expected when phloem transport is inhibited (4–9). In transgenics grown in the relatively low light of the growth chamber (350 ␮mol of photons mϪ2 sϪ1), the sum of sucrose, glucose and fructose levels ranged from 5-fold to 28-fold in excess of those found in wild-type plants (Fig. 5A). To determine whether transgenic plants are able to export carbohydrate stored as starch, plants were kept in the dark for periods of 12–72 h. Although the leaves of wild-type tobacco plants lost their starch within 12 h, starch was readily apparent in the mesophyll cells of transgenic tobacco plants with mild to severe downregulation of SUT1, even after 72 h, as demonstrated by I/KI staining. In plants with mild or moderate downregulation of SUT1, starch accumulated in some areas of the lamina but not in others. Chlorosis was also patchy in these leaves, whereas in leaves with severe downregulation chlorosis developed more evenly throughout the Fig. 4. Real-time PCR expression patterns of SUT1 in Nicotiana tabacum (A) and Verbascum phoeniceum (B) leaves. Results are from individual transgenics lamina. As previously described (4) downregulation of NtSUT1 and from either two (A) or three (B) wild-type (WT) plants. Expression levels led to pronounced inhibition of photosynthesis (Fig. 6A). This are normalized to wild-type. Error bars, SE (n ϭ 6 for NtWT, n ϭ 9 for VpWT, was the case in all tested tobacco transgenic lines, including the n ϭ 3 for transgenics). plant with the weakest phenotype (NtT1.27.2) (Fig. 6A).

Zhang and Turgeon PNAS ͉ November 3, 2009 ͉ vol. 106 ͉ no. 44 ͉ 18851 Downloaded by guest on September 28, 2021 conclusion that NtSUT1 is primarily responsible for phloem loading in tobacco leaves. To determine if downregulating NtSUT1 inhibits sucrose uptake from the apoplast, we measured uptake of [14C]sucrose into leaf discs from wild-type and transgenic tobacco plants. The leaf disc technique has been widely used to characterize the in vivo properties of mono- and disaccharide transporters (28–33) and to correlate rates of phloem loading with photosynthetic capacity (34). Sucrose uptake into leaf discs is biphasic (35). A saturable, PCMBS-sensitive phase displaying Michaelis-Menten kinetics is superimposed on a linear phase that is insensitive to PCMBS. The linear phase does not drive phloem loading (36, 37). The PCMBS-sensitive portion of total uptake represents sucrose-transporter-mediated uptake activity (25–27). In transgenic tobacco lines, PCMBS-sensitive uptake into leaf discs declined with increasing downregulation of NtSUT1 expression. It is apparent that if all three members of the NtSUT1 family are active, they were all downregulated by the partial sequence from CAA57727 because the sucrose uptake ability in severe pheno- Fig. 6. Net photosynthesis in wild-type (WT) and transgenic lines of Nicoti- type lines almost disappeared. Autoradiographic analysis indi- ana tabacum (A) and Verbascum phoeniceum (B). Results are from individual cated that exogenous [14C]sucrose was primarily taken up by the transgenics and from either two (A) or three (B) wild-type plants. Error bars veins, consistent with SUT1 localization in the phloem (23), and ϭ ϭ ϭ indicate SE (n 6 for NtWT, n 9 for VpWT, n 3 for transgenics). that the intensity of the vein images declined with downregulation of SUT1. The leaves of most tested V. phoeniceum transgenic plants with The phenotype of transgenic tobacco plants in this study is essentially the same as that described for plants in which SUT1 significant down regulation of SUT1 did not accumulate soluble is downregulated by antisense technology. We noted, as did carbohydrates in excess of amounts in wild-type plants (Fig. 5B). Ku¨hn et al. (38), that some areas of affected leaves accumulate Line VpT2.27.2, with the most severe downregulation of RNA starch while others do not. Therefore, quantitative measurement and sucrose transport activity, accumulated 2.8-fold more solu- of starch in whole transgenic leaves may not accurately reflect ble carbohydrate than wild-type (Fig. 5B). processes occurring within localized affected regions. The amount of soluble carbohydrate accumulation in trans- The same experimental design used on tobacco was used to genic V. phoeniceum leaves was negligible compared to the test the hypothesis that phloem loading is driven by sucrose amount exported. Given the rate of net photosynthesis, the fresh Ϫ transporter activity in V. phoeniceum. When VpSUT1 was down- weight of mature leaves (32.4 mg cm 2), and an assumed export regulated in V. phoeniceum, PCMBS-sensitive [14C]sucrose up- rate of 80% of carbon fixed daily (which is typical for herbaceous take also declined, indicating that, as in tobacco, SUT1 activity Ϫ plants), the leaves exported approximately 154 mg carbon g 1 is primarily, if not entirely, responsible for energized uptake of fresh weight from the time they reached full size to harvest, sucrose from the apoplast. We cannot exclude the possibility that approximately 8 days later. Since the leaves of line VpT2.27.2. other sucrose transporters are expressed in V. phoeniceum accumulated only 2 mg carbon gϪ1 fresh weight more than leaves, but if they are, they must share enough sequence simi- wild-type leaves during this period, this constituted less than 2% larity with the identified VpSUT1 to be downregulated simulta- of the amount exported. neously, or these sucrose transporters do not account for an Transgenic V. phoeniceum plants exported carbon from starch appreciable amount of sucrose transporter activity, since se- normally. Even in line VpT2.27.2, starch was no longer visible verely downregulating VpSUT1 essentially eliminated all PC- after the plants had been kept in the dark for 12 h. Rates of net MBS-sensitive sucrose uptake into the leaf discs (Fig. 3B). It photosynthesis were indistinguishable from wild-type plants should be noted that, although SUT1 downregulation appears to (Fig. 6B). be more uniformly severe in transgenic V. phoeniceum in com- parison to tobacco in Figs. 3 and 4, this is due to the conscious Discussion choice of lines with these properties, not due to inherent Downregulating sucrose transporters is the most compelling differences in susceptibility. From autoradiographs of leaf discs it is clear that exogenous approach found to date to test models of phloem loading. 14 Downregulation of SUT1 by antisense technology or DNA [ C]sucrose accumulates in the veins of wild-type V. phoeniceum leaves, as in tobacco. However, downregulation of SUT1 did not insertion results in severe inhibition of phloem loading as appreciably modify the V. phoeniceum vein images, except in the demonstrated by accumulation of soluble carbohydrates in most severely downregulated line, and then not consistently. leaves, reduced photosynthesis, stunted growth and leaf chlorosis These results are similar to those in which sucrose transporter (4–6, 8, 9). According to these results, SUT1 appears to be the activity in RFO-transporting plants is inhibited with PCMBS primary, if not the only, sucrose transporter responsible for (19). Apparently, when sucrose transporter activity is blocked, apoplastic phloem loading. [14C]sucrose enters the tissue by the non-saturable, PCMBS- In our experiments, a 393-bp fragment based on tobacco SUT1 insensitive uptake mechanism (36, 37) and is then sequestered in cDNA sequence CAA57727 was amplified to make an RNAi the veins by diffusion through plasmodesmata and polymer construct. The amplified partial sequence shared 98 and 95% trapping in the intermediary cells (34). However, the genetic similarity at the nucleic acid level with CAQ58422 and technique used here circumvents several objections raised FM164638, two identified NtSUT1 genes, respectively (24). against the pharmacological approach to blocking sucrose trans- Considering that downregulation of NtSUT1 resulted in virtually porter activity with PCMBS, including lack of specificity and identical symptoms of loading inhibition described for NtSUT1 difficulties with tissue infiltration (39). antisense plants (4), and did so in approximately 90% of the The major difference between the results on V. phoeniceum independently-derived transgenic lines, the results support the and tobacco is that downregulating SUT1 and reducing sucrose

18852 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0904189106 Zhang and Turgeon Downloaded by guest on September 28, 2021 uptake capacity in V. phoeniceum did not have an appreciable AbSUT1 (14), StSUT1 (23), PmSUC2 (22), and AtSUC1 and AtSUC2 (45). Primers effect on phloem loading in any of the more than 100 indepen- used for the amplification of the V. phoeniceum SUT1 fragment were 5Ј- dently derived transgenic lines grown either in low (growth GGSGTGCARTTCGGNTGGGCNYT-3 (forward) and 5Ј-GTAMACCTCYTTNC- Ј chamber) or high (greenhouse) light. It could be argued that V. CCATCCARTC-3 (reverse). RNA was isolated from mature leaves of V. phoeniceum using the Plant RNA Mini Kit (Omega Bio-Tek). cDNA was synthesized phoeniceum does not display the same symptoms as tobacco using oligo(dT) primer and SuperScript RT (Invitrogen). The 816-bp fragment when loading is inhibited, but this is clearly not the case since amplified from V. phoeniceum was used as a 32P labeled probe to screen a downregulating RFO synthesis in the same cultivar of this cDNA library from mature leaves with different stringencies (18). Clones were species, using RNAi technology as in the present study, results sequenced by Cornell Bioresource Center and the homology analysis of de- in the classic symptoms of transport inhibition: excessive accu- rived sequences was conducted with BLASTx. mulation of soluble carbohydrates, inability to transport starch during an extended dark period, stunted growth, and leaf RNAi Vector Construction and Plant Transformation. For tobacco, based on chlorosis (18). GenBank sequence CAA57727, a 393-bp sense fragment, with XhoI and EcoRI What is the function of VpSUT1, if not in phloem loading? at the two ends, was amplified by forward primer 5Ј-AATGCCTCGAGGGCG- Ј Ј Results from several types of experimentation indicate that sucrose GAAAAGCGAGGATGA-3 and reverse primer 5 AATGCGAATTCCGGAAAC- CTCGCAATCCAG-3Ј. A 393-bp anti-sense fragment, with XbaI and HindIII at leaks continuously from phloem cells into the apoplast and must be the two ends, was amplified by forward primer 5Ј-AATGCTCTAGAGGCG- retrieved in order for long-distance transport to operate efficiently GAAAAGCGAGGATGA-3Ј and reverse primer 5Ј-AATGCAAGCTTCGGAAAC- (13, 40–43). This may be the reason that sucrose transporters CTCGCAATCCAG-3Ј. For V. phoeniceum, a 352-bp sense fragment, with XhoI localize to the phloem in the petiole and stem, in addition to the and KpnI at the two ends, was amplified by forward primer 5Ј-AATGCCTC- minor veins, in many species. In A. meridionalis, SUT1 localizes to GAGCGATTGGATGGGTCGGGA-3Ј and reverse primer 5Ј-AATGCGGTACCTGC- the ‘ordinary’ CCs that are present in the minor veins in addition CGCGAGAGGGATACC-3Ј. A 352-bp anti-sense fragment, with XbaI and ClaIat to intermediary cells. A. meridionalis is in the same family as V. the two ends, was amplified by forward primer 5Ј-AATGCTCTAGACGATTG- phoeniceum and its minor vein anatomy and transport sugars are GATGGGTCGGGA-3Ј and reverse primer 5Ј-AATGCATCGATTGCCGCGAGAGG- Ј essentially identical (13, 44). GATACC-3 . The two hairpin cassettes were released from pHANNIBAL by restriction digestion and subcloned into the NotI site of binary vector pART27 The retrieval function could also explain why leaf discs of for Agrobacterium (GV3101)-mediate transformation in the two species. wild-type V. phoeniceum take up more exogenous sucrose than Transformation of tobacco followed standard methods. Transformation of V. discs from wild-type N. tabacum (Fig. 3). If retrieval along the phoeniceum followed the procedure previously described (18), but with re- path phloem requires more transporter activity than loading visions as detailed in SI Text.

itself, this could mask differences in the latter activity. Also, it PLANT BIOLOGY should be kept in mind that rates of uptake from exogenous Quantitative RT-PCR Expression Analysis. To prepare template for quantitative solutions in different species can differ due to dissimilarities in RT-PCR, 0.5 ␮g total RNA was used to synthesize cDNA using the iScript cDNA tissue thickness and geometry that affect diffusion rates. synthesis kit (Bio-Rad). Quantitative RT-PCR was performed using SYBR Green Inefficient retrieval may be the reason that soluble carbohy- I technology on a Bio-Rad iQ5 system (Bio-Rad Laboratories). Primer sequences Ј Ј Ј drates accumulate to a limited extent in the most severely were forward 5 -GGGCTCTAAAGTTCTCTTAATGGGC-3 and reverse 5 - CTCTAACACACCCTTAGCATTTCC-3Ј for V. phoeniceum, forward 5Ј-TACAGT- downregulated V. phoeniceum transgenics. However, even in GACAATTGCTCCTCCCGT-3Ј and reverse 5Ј-GGCATGTCCAAGATCAGCAG- these severely affected plants, with almost no residual active CAAA-3Ј for tobacco, and forward 5Ј-CGCGGAAGTTTGAGGCAATAA-3Ј and sucrose uptake capacity, photosynthetic rates are normal and reverse 5Ј-TCGGCCAAGGCTATAGACTCGT-3Ј for 18S rRNA. The 50-␮L quanti- starch is cleared from mesophyll cells during the night period, tative RT-PCRs contained 1ϫ iQ SYBR Green PCR supermix (Bio-Rad Labora- indicating that there is little effect on the overall capacity of tories), 300 nM of each primer, and 1 ␮L template cDNA. The amplification leaves to produce, load and transport soluble carbohydrates. protocol consisted of an initial cycle of 95 °C for 10 min, followed by 40 cycles These data provide molecular genetic evidence that, in species of 95 °C for 15 s and 55 °C for 1 min. Primer specificity was evaluated by with abundant minor vein plasmodesmata, photosynthates are melting curve analysis. All samples were amplified in triplicate and averaged. able to migrate from mesophyll cells to the phloem along an Data analysis was performed using the relative standard curve method. entirely symplastic pathway, and that differences in sugar con- Sucrose Uptake Assay. Leaf discs (1.6-mm diameter) from the first fully ex- centration and hydrostatic pressure between mesophyll cells and panded mature leaf were collected with a belt punch (C. S. Osborne) at 14:00 the phloem, sufficient to drive long-distance transport, can be to 15:00 and incubated in Mes [2-(N-Morpholino) ethanesulfonic acid] buffer established and maintained without the need for active sucrose pH 5.5, containing 9.9 Bq [14C]sucrose and 1 mM unlabeled sucrose, with or uptake from the apoplast. Since phloem unloading is often without PCMBS (5 mM) for 1 h, and washed three times with ice-cold buffer symplastic as well (3), it seems likely that, in many species, for a total of 1 h. Sugar extraction and scintillation counting procedures were photoassimilate travels from the cytosol of mesophyll cells to as described (18). The PCMBS-insensitive portion of uptake was subtracted sink cells without crossing a single plasma membrane, a finding from total uptake to obtain PCMBS-sensitive values. Thirty leaf discs were used with potentially important implications in the study of carbon per replicate and three replicates were conducted per experiment. For auto- partitioning. radiography, leaf discs were flash frozen, lyophilized, pressed thin, and ap- plied to BioMax MR film (Kodak) (32). Materials and Methods Photosynthesis and Carbohydrate Content. A LI-COR 6400 (LI-COR Biotechnol- Plant Material. Seeds of V. phoeniceum L. cv. ‘‘Flush of White’’ (Garden ogy) was used to analyze CO uptake in the first three fully expanded leaves Makers) and N. tabacum ‘‘Petite Havana SRI’’ were surface-sterilized and 2 of each plant at 600 ␮mol mϪ2 sϪ1 with a CO concentration of 360 ␮mol mϪ2 germinated as described (18). Plants were kept in a growth chamber with 14-h 2 sϪ1. Sugars were analyzed by HPLC as described (18, 46), using xylitol as day/10-h night cycles at approximately 100 ␮mol photons mϪ2 sϪ1 for plant internal standard. transformation. Transgenic plants of both species were grown either in a growth chamber on a 14-h day/10-h night cycle with 350 ␮mol photons mϪ2 ACKNOWLEDGMENTS. We thank L. Cheng, P. Li, W. Miller, and R. Harmon for sϪ1, or in a greenhouse with 600–800 ␮mol photons mϪ2 sϪ1. the use of equipment; Bertrand Lasseur, Edwin Reidel, and Jingying Liu for assistance; and Andre Jagendorf for critical reading of the manuscript. This Gene Cloning. Degenerate primers for sucrose transporter 1 (SUT1) were study was supported by National Science Foundation Grant IOB 0444119 designed based on the amino acid sequence alignment of AmSUT1 (13), (to R.T.).

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