phloem long-distance transport of cmnacpmrna: implications ... · the evolution of the phloem and...

INTRODUCTION

The evolution of the phloem and xylem, the plant′s long-distance translocation systems, played a critical role in thedevelopment of an effective whole-plant circulatory andcommunication network. Over the past several decades, muchattention has been focused on elucidating the mechanismsunderlying the operation of these two translocation systems.Although a considerable body of evidence exists concerningthe delivery of nutrients to the various plant organs (essentialminerals and water via the xylem and sugars and amino acidsvia the phloem; Zimmermann and Milburn, 1975; Turgeon,1996), only limited information is available concerning theroles played by the phloem and the xylem in the delivery ofinformation molecules (Zimmermann and Milburn, 1975;Schulz, 1998). However, it is generally accepted that bothlong-distance transport systems function in coordinatingphysiological processes occurring in specific organs withdevelopmental events taking place in meristematic tissues (e.g.,roots, shoots and flowers). As phytohormones have beendetected in the phloem and xylem sap, these small moleculeshave been implicated in signaling between, for example, rootsand shoots (Zimmermann and Milburn, 1975; Gowing et al.,1993; Munns and Sharp, 1993; Jackson, 1997). However, theseobservations do not exclude the possible involvement of novel

information macromolecules in the establishment of aneffective whole-plant, or systemic, communication network.

A role for small peptides in systemic signaling is supportedby studies conducted on systemin; this peptide is involved inthe establishment of systemic acquired resistance to pathogens(Pearce et al., 1991; Enyedi et al., 1992; Bergey et al., 1996).Systemin is first synthesized within the infected leaf andsubsequently translocated towards the vegetative apex via thephloem (Narváez-Vásquez et al., 1995). The involvement ofmacromolecules (proteins and nucleic acids) in this whole-plant signaling network is suggested by a number of recentfindings. Most cells within the body of the plant areinterconnected by an highly specialized intercellular organelle,termed the plasmodesmata (Robards and Lucas, 1990; Lucaset al., 1993a; Lucas, 1995). A range of experimentalapproaches has now provided a body of evidence consistentwith the hypothesis that plasmodesmata mediate the cell-to-cell trafficking of proteins and protein-nucleic acid complexes(Fujiwara et al., 1993; Noueiry et al., 1994; Ding et al., 1995;Balachandran et al., 1997; Ghoshroy et al., 1997; Itaya et al.,1997; Rojas et al., 1997; Ishiwatari et al., 1998; Lough et al.,1998). Given that plasmodesmata allow the trafficking oftranscription factors, such as KNOTTED1 (Lucas et al., 1995),and other homeotic proteins (Mezitt and Lucas, 1996; Perbalet al., 1996), this unique organelle appears to potentiate local

4405Development 126, 4405-4419 (1999)Printed in Great Britain © The Company of Biologists Limited 1999DEV0231

Direct support for the concept that RNA moleculescirculate throughout the plant, via the phloem, is providedthrough the characterisation of mRNA from phloem sap ofmature pumpkin (Cucurbita maxima) leaves and stems.One of these mRNAs, CmNACP, is a member of the NACdomain gene family, some of whose members have beenshown to be involved in apical meristem development. Insitu RT-PCR analysis revealed the presence of CmNACPRNA in the companion cell-sieve element complex of leaf,stem and root phloem. Longitudinal and transversesections showed continuity of transcript distributionbetween meristems and sieve elements of the protophloem,suggesting CmNACP mRNA transport over long distancesand accumulation in vegetative, root and floral meristems.In situ hybridization studies conducted on CmNACPconfirmed the results obtained using in situ RT-PCR.

Phloem transport of CmNACP mRNA was proved directlyby heterograft studies between pumpkin and cucumberplants, in which CmNACP transcripts were shown toaccumulate in cucumber scion phloem and apical tissues.Similar experiments were conducted with 7 additionalphloem-related transcripts. Collectively, these studiesestablished the existence of a system for the delivery ofspecific mRNA transcripts from the body of the plant to theshoot apex. These findings provide insight into the presenceof a novel mechanism likely used by higher plants tointegrate developmental and physiological processes on awhole-plant basis.

Key words: CmNACP, Cucurbita, Phloem-specific mRNA,Plasmodesmata, Shoot-to-meristem signaling

SUMMARY

Phloem long-distance transport of CmNACP mRNA: implications for

supracellular regulation in plants

Roberto Ruiz-Medrano, Beatriz Xoconostle-Cázares and William J. Lucas*

Section of Plant Biology, Division of Biological Sciences, University of California, One Shields Avenue, Davis, CA95616, USA*Author for correspondence (e-mail: [email protected])

Accepted 14 July; published on WWW 27 September 1999

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supracellular control over developmental events (Lucas et al.,1993a; Mezitt and Lucas, 1996; Hake and Char, 1997; McLeanet al., 1997; Jackson and Hake, 1997).

As plasmodesmata interconnect the functional, enucleate,sieve elements of the phloem to their neighboring companioncells (Esau, 1969; Lucas et al., 1993a; Turgeon, 1996), thispathway would allow the selective entry of informationmacromolecules into the phloem translocation stream.Experimental support for the involvement of proteins in sucha long-distance signaling system is provided by the observationthat the phloem sap contains a wide variety of proteins (Fisheret al., 1992; Nakamura et al., 1993; Sakuth et al., 1993;Tiedemann and Carstens-Behrens, 1994; Ishiwatari et al.,1995; Schobert et al., 1995, 1998). Furthermore, recentmicroinjection experiments provided direct proof that many ofthese phloem proteins have the capacity to mediate their owntransport through plasmodesmata (Balachandran et al., 1997;Ishiwatari et al., 1998; Xoconostle-Cázares et al., 1999).

A number of experimental observations provide support forthe notion that RNA also may be translocated within thephloem. Certain plant RNA viruses that lack a functionalcapsid protein can still systemically infect their hosts, thusindicating that infectious RNA can be transported, via thephloem, from the inoculated leaf to developing tissues(Gilbertson and Lucas, 1996; Carrington et al., 1996). Thatendogenous RNA can move from the companion cell (site oftranscription) through the connecting plasmodesmata into thesieve element gains support from SUT1 in situ localizationexperiments (Kühn et al., 1997). Additionally, a number ofexperimental systems exhibiting systemic acquired genesilencing (Lindbo et al., 1993; Elmayan and Vaucheret, 1996;Palauqui et al., 1996; Covey et al., 1997; English et al., 1997;Ratcliff et al., 1997; Voinnet and Baulcombe, 1997;Wassenegger and Pélissier, 1998) have now provided strongcircumstantial evidence that transmission of the cosuppressedstate may involve RNA transport within the phloem (Palauquiet al., 1997; Smyth, 1997; Jorgensen et al., 1998).

The recent characterization of a protein, CmPP16, presentwithin the phloem of Cucurbita maxima (pumpkin), whosefunctional properties are consistent with its involvement in thenon-sequence-specific transport of RNA between companioncells and functional sieve elements (Xoconostle-Cázares et al.,1999), provides additional support for the hypothesis that RNAmolecules move over long distances, via the phloem. Finally,molecular analysis of the phloem sap collected from Cucumissativus (cucumber) scions grafted onto pumpkin stockprovided direct experimental evidence for the translocation ofCmPP16 mRNA through the heterograft union (Xoconostle-Cázares et al., 1999).

In the present study, we document the existence of apopulation of RNA molecules within the phloem sap ofpumpkin. Further, using the pumpkin/cucumber heterograft asa model experimental system, in conjunction with in situ RT-PCR analysis, we demonstrate that a number of thesetranscripts move, via the phloem, from the pumpkin stock allthe way into meristematic tissues of the cucumber scion. Incontrast, other transcripts, although present within the phloemtranslocation stream, were not detected within scion meristemtissues. These experiments establish the operation of a systemfor the delivery of specific mRNAs from the body of the plantto the shoot apex. These findings are discussed in terms of a

novel mechanism likely used by plants to integratedevelopmental and physiological processes on a whole-plantbasis.

MATERIALS AND METHODS

Isolation and cloning of RNA from phloem sapPhloem sap poly(A)+ RNA was isolated from 4-week-old,greenhouse-grown, pumpkin (Cucurbita maxima cv. Big Max) plantsand cDNA synthesized. Phloem sap was collected as follows: stemsor petioles were excised from the plant and blotted, twice, for severalseconds onto sterile filter paper (#3 MM; Whatman, Maidstone, UK).Phloem sap exuded thereafter was collected using sterile micropipettetips (200 µl) and immediately mixed with an equal volume of 8 Mguanidinium buffer (Logemann et al., 1987). Proteins in this sap werethen extracted, twice, with a 25:24:1 phenol:chloroform:isoamylalcohol mixture. The remaining RNA was then precipitated with 0.7volumes of absolute ethanol and 0.2 volumes of 1 M acetic acid,centrifuged at 4°C for 45 minutes, and then resuspended in sterilizeddeionized water. Typically, 400-500 ng of phloem sap RNA werecollected from each plant. Poly(A)+ RNA was then isolated, from totalRNA, using an oligotex mRNA extraction kit (Qiagen, Santa Clarita,CA) and first-strand cDNA synthesized with Superscript ReverseTranscriptase (Gibco, Bethesda, MD). Stem RNA was also obtainedfrom these same pumpkin plants, and poly(A)+ RNA isolation andcDNA synthesis was performed as described for phloem sap.

PCR analysis Phloem-sap-derived and stem cDNAs were used as templates forLong-Distance-PCR (LD-PCR) according to the manufacturer′srecommendations (Clontech, Palo Alto, CA) using a Robocycler PCRsystem (Stratagene, La Jolla, CA). Stem total RNA and phloem sapRNA were used at 30 ng/µl for poly(A)+ RNA isolation, cDNAsynthesis and LD-PCR. For these assays, template cDNA wasnormalized after this amplification. Primers used to amplify PP2,CmrbcS, Cmthioredoxin-h, Cmimportin α CmNACP, CmRINGP,CmRABP, CmGAIP, CmWRKYP, CmSTMP and CmCYCLINP, forsemiquantitative PCR analysis are presented in Table 1. PCRs wereperformed under the following standard conditions: 94°C for 60seconds (1 cycle); 94°C for 30 seconds; 60°C for 30 seconds; 72°Cfor 60 seconds (20 cycles). In order to identify the PCR products, theDNA was run on an agarose gel, blotted to a Hybond N-plus nylonmembrane (Amersham Pharmacia Biotech, Arlington Heights, IL)and hybridized, at 65°C, with probes labeled with [α-32P]dCTP usinga Random Priming labeling Kit (NEN Life Science, Boston, MA)(hybridization buffer: 0.5 M Na2HPO4, pH 7.2, 7% SDS, 1% BSAand 1.0 mM EDTA). Blots were washed with a high stringencysolution (15 mM NaCl plus 1.5 mM sodium citrate, pH 8), at 65°C,and then exposed, at −80°C, to Biomax X-ray film (Kodak, Rochester,NY).

To determine the relative amounts of cDNA from stem and phloemsap used in these assays, five stem and three phloem sap samples wererun in agarose gels, blotted to nylon membrane, and then hybridizedwith phloem sap cDNA labeled with [α-32P]dCTP, and washed underhigh stringency conditions. These same cDNA samples were thensubjected to LD-PCR and the resultant products were used for furtheramplification of specific transcripts (Table 1). The PCR conditionswere analyzed to ensure amplification was being carried out over thelinear range for all genes analyzed within these independent stem andphloem sap samples.

For detection of pumpkin transcripts in apical tissues of control andheterografted cucumber scions, extracted RNA was reversetranscribed (as above), and the cDNAs amplified by PCR (two rounds;a 1 µl aliquot of the original reaction was used as template for thesecond round) under the following conditions: 94°C, 60 seconds (1

R. Ruiz-Medrano, B. Xoconostle-Cázares and W. J. Lucas

4407Long distance delivery of CmNACP mRNA

cycle); 94°C for 30 seconds; 60°C for 30 seconds, 72°C for 60seconds (35 cycles). The resultant products were run on agarose gels,blotted and, for a more specific detection, hybridized with [α-32P]dCTP-labeled internal probes (generated by PCR using the nestedprimers presented in Table 1), and then washed under stringentconditions (see above). For CmSTMP, the following degenerateprimers were used to clone the gene from phloem sap cDNA: forward,5′-GWIRTIGARGCIHTIAARATHATH-3′, corresponding to theamino acid sequence DVEAIKAKII; and reverse, 5′-CATYTCIS-WIGTIGGYTTCCARTGICKYTT-3′, corresponding to the aminoacid sequence KRHWKPTDEM.

RNase protection assayPetiole and stem phloem sap was collected from 18-day-old pumpkinplants as described by Balachandran et al. (1997). Stem tissue wasexcised from these same plants, washed twice with DEPC-water, andhomogenized using RNase-free materials. The homogenate wascentrifuged, at 22°C, for 20 minutes at 2,000 g and the supernatantcollected and used in RNA stability assays. Aliquots (0.2 µg) oftobacco mosaic virus (TMV) RNA (Boehringer Mannheim,Indianapolis, IN) were mixed with phloem sap (1.5 µg phloemprotein), stem extract (1.4 µg total protein) or DEPC-treated water,and then either incubated for 5 and 10 minutes, or immediatelycombined with formaldehyde-loading buffer (Ambion, Austin, TX).Ethidium bromide was added to these RNA samples prior to beingrun in formaldehyde-agarose gels.

In situ RT-PCR protocolIn situ RT-PCR experiments were performed by adapting the protocolof Chen and Fuggle (1997), in that the use of rTth polymerase (PEApplied Biosystems, Foster City, CA) was incorporated in the RT-PCR protocol. A reverse transcriptase cocktail [containing, at 0.2 mM,dATP, dCTP, dGTP, and dTTP (Boehringer Mannheim), plus 2.5 mMmanganese acetate, reverse primer at 75 µM and 5 units of rTth

polymerase] was prepared immediately prior to use. Fresh 150-200µm-thick sections were obtained using a model TC-2 Sorvallmicrotome (DuPont, Wilmington, DE). Sections were placed onto aglass slide, covered by a 25 µl aliquot of the above cocktail and thensealed with amplicover discs and clips (PE Applied Biosystems). Thereverse transcription step was performed at 60°C for 20 minutes. Forthe PCR step, the cocktail was replaced with a solution having thesame composition, except that the forward primer was added, dTTPwas reduced to 10 µM, and Oregon Green-labeled dUTP (20 µM;Molecular Probes, Eugene, OR) was added. The amplificationprotocol consisted of 10 cycles; 30 seconds at 94°C, 30 seconds at60°C and 60 seconds at 72°C. A Perkin-Elmer GenAmp In SituThermal PCR System-1000 was used for these experiments (PEApplied Biosystems). After this reaction series, sections wereincubated (1 minute) in absolute ethanol, followed by rinsing in 1 mMEDTA, and then overnight washing (16 hours), at 22°C, in this EDTAsolution. Primers used for in situ RT-PCR are presented in Table 1.

Tissue sections were visualized using a Leica TCS-4D confocallaser scanning microscope (CLSM; Leica Lasertechnik, Heidelberg,Germany). Fluorescence associated with Oregon Green-labeledCmNACP transcripts was detected using a krypton/argon laser and thefollowing filter settings: 488 nm excitation and 525 nm emission.CLSM images were recorded and processed as described byXoconostle-Cázares et al. (1999). After image collection, the tissuewas irrigated with 2 mM fluorescein isothiocyanate (MolecularProbes) and structural images subsequently collected and recordedusing the same filter settings as for Oregon Green. Images wererecorded with a PC workstation and were processed in AdobePhotoshop (version 5.0). Images presented are representative of 10replicate experiments in which a minimum of 3 tissue sections wereanalyzed.

To overcome the problem of lignin autofluorescence associatedwith soil-grown pumpkin roots, plants were grown using hydroponictechniques (Lucas et al., 1993b). Seedlings were grown for 10 days

Table 1. Primers employed for transcript analysis within Cucurbita maxima, Cucumis sativus and Petunia hybrida tissues Gene Forward primer Reverse primer

PP2 5′-GCAATGGACAACAAAGAGAAGGAAGCC-3′ 5′-CGATCATGCGCAACCACATCCCTTTGC-3′CmrbcS 5′-ATGGCTTCCATCGTCTCATCCGCC-3′ 5′-TTGTCGAAGCCAATGACTCTGATGAA-3′Cmthioredoxin-h 5′-TCCAGCTTGGAGACTGCTGGCCCGACT-3′ 5′-ATCAATGGCGAGAACCAAATCATCAAA-3′Cmimportin α 5′-GGACTATATCTAATATAACAGCCGGGAACA-3′ 5′-AACTGGGACTGGAATAAGCATGGCGAA-3′CmNACP 5′-GTCATGCATGAATTTCGACTCGAACCC-3′ 5′-GCATCGCCATTGTTCGATCATAACATC-3′

Nested 5′-CTTCTCCCACCTCTAATAGATCCCAC-3′ 5′-CGACGATGATATCTCCGGTGTCACACC-3′CmRINGP 5′-TCATTTGTTGTAATGGAAGAGCAAGAG-3′ 5′-CTTTTCCCCCCTTTTGCAATCAGAATT-3′

Nested 5′-TCCCGTGCTTATGTGGAACAGGTGAAC-3′ 5′-GACTTGGAGGATGAAACTTCTGTGCCA-3′CmRABP 5′-CATTTTTGTTCTGTCAACGCCTGTTCT-3′ 5′-CATTCTGCAGACATCTTAAGAGTAGAA-3′

Nested 5′-GTGCTGGGAAGTCGAGTCTAGTGTTGC-3′ 5′-ACGAGCAACACGATGTGCTTGGAACCC-3′CmGAIP 5′-GTGTCGAATAGCTTGGCGGATCTGGAC-3′ 5′-GAGCATGCTTGCTTGCTTGAATGCATT-3′

Nested 5′-GAAATGTACCTTGGGAAACAAATCTCCC-3′ 5′-CCCACCAAACTAAGACGAAGGGCGGGA-3′CmWRKYP 5′-GAAACAGCTGAGGCCATTGATGCCTCATCC-3′ 5′-CCGAGCAGCAGGAACATCGTGATTGTGCTT-3′

Nested 5′-GTTCCTGCTGCTCGCAACAGCAGCCAC-3′ 5′-CCTAGTGAACAAATATTGGGGGGAAAAGG-3′CmSTMP 5′-GTGAATTGTCAGAAGGTGGGTGCACCG-3′ 5′-CTTCCTTTGGTTAATGAACCAATTATT-3′

Nested 5′-CAGGCTTGTACGGTGGCTGCTGGGAAT-3′ 5′-CAAGCAGCTGCTGCCGAGCTTCTTTAGG-3′CmCYCLINP 5′-ATGGCTGCTAATTTCTGGACTTCATCGCAC-3′ 5′-ATGGCTTCATAGACAGCTTGGTTCAGAGCCG-3′

Nested 5′-GGCCAATTATATACTGAAGTTGGCTC-3′ 5′-TCTCAATAGAGATATTCTTGACCACGT-3′CmPP16 5′-GTGGTAAAGGACTTCAAGCCCACGACC-3′ 5′-ATGGGTTTGAAGAAGCCAAGCCACTTA-3′

Nested 5′-CAAGCCCACGACCCTCTTAATAAACC-3′ 5′-CGCCGCCTTGCAGCTTGAAGGACACTC-3′CmSUTP1 5′-ATIGCIGCIGGIGTICARTTYGGITGGGCIYTICA-3′ 5′-CCYTGIARCATRTTRTTIGCIARCTCIARIATCCAT-3′

Nested 5′-TTGCAGCTCTCTCTTCTTACTCCTTAT-3′ 5′-GTTGGCGACATCGAGGATCCAAAATC-3′CsNACP 5′-CCIGGITTYYGICAYCCIACIGAYGARGAR-3′ 5′-RTCIDYICCIGTIGCYTTCCARAICC-3′

Specific 5′-CCGGGGTTTTGGCATCCGACGGACGAG-3′ 5′-GTCGGTGCCGGTGGCCTTCCAGTAGCCAT-3′PhNAM 5′-CCTACTGGGCTAAGGACTAACAGAGCTACT-3′ 5′-GAGTTGAAATAGTGGAGAAACAGGACACGT-3′

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in soil in a controlled environmental chamber (model MTR30;Conviron, Winnipeg, Canada; 650 µmol/m2/secondphotosynthetically active radiation [PAR], 16-hour photoperiod, 70%relative humidity, 26°C). Roots were then washed free of soil and theplants transferred to one-liter glass jars containing aerated nutrientsolution (Lucas et al., 1993b). Seven to ten days later, root tissue wasexcised and processed for CmNACP in situ RT-PCR analysis.

In situ hybridization and immunolocalization methodsPumpkin petiole, stem and meristematic tissues (vegetative and floral)were excised from 4-week-old plants and fixed, dehydrated andparaffin-embedded according to the method described by Smith et al.(1992) and Jackson et al. (1994). Generation of riboprobes was carriedout by in vitro transcription (Ambion) of sense and antisenseCmNACP RNA, using digoxigenin (Dig)-labeled UTP (BoehringerMannheim), with cDNA for CmNACP cloned in pCR II TOPO(Invitrogen, Carlsbad, CA), digested with either BamHI (sense) orXbaI (antisense), as template. Paraffin-embedded tissues weresectioned (10 µm; model HM340E microtome, Microm, Walldorf,Germany) and rehydrated for further incubation with sense andantisense riboprobes. Following hybridization and high-stringencywashes, Dig-labeled RNA was detected using anti-Dig antibody(Boehringer Mannheim) conjugated to alkaline phosphatase (AP). APactivity was measured using NBT (Nitro Blue Tetrazolium) and BCIP(bromo-chloro-indolyl phosphate), and the chromogen produced wasimaged by phase contrast light microscopy.

Recombinant CmNACP was expressed in and purified fromEscherichia coli as a His-tagged fusion protein. An oligopeptide, H-KEWYFFCQRDRKYPTGMR-(MAP)8, representing an antigenicregion present within CmNACP, was synthesized (Research Genetics,Huntsville, AL) and used, in combination with recombinantCmNACP, to produce polyclonal antibodies in rabbits. (Prescreenedrabbits having serum-negative response to CmNACP were usedto raise antibodies). After the immunological scheme, IgG waspurified using a Protein A column and used for western andimmunolocalization assays. Detection of CmNACP was carried outon fixed tissue using the methods described by Smith et al. (1992) andJackson et al. (1994). Anti-CmNACP antibodies were used in adilution of 1:2000 and were detected by goat anti-rabbit APantibodies; AP was assayed using NBT and BCIP and imaged asabove.

Northern analysisTotal RNA was extracted from 5 g of plant tissue (leaf, stem, roots,flowers and vegetative apices) as described by Logemann et al. (1987).For each tissue, 10 µg of total RNA was run in a formaldehyde gel,blotted to a nylon membrane and then hybridized as described aboveto a [α32P]dCTP-labeled full-length CmNACP cDNA cloned in thepCRII TOPO vector (Invitrogen). Filters were then washed under highstringency conditions (see above) and exposed to X-ray film.

Identification of functional sieve elementsCarboxyfluorescein diacetate loading experiments were performed asdescribed by Grignon et al. (1989). Four hours after introducingcarboxyfluorescein diacetate into the apoplasm of fully expandedleaves (4-week-old pumpkin plants), petioles were excised at the base.Thick (~2 mm) sections of petiole were then cut and examinedimmediately, using CLSM, to visualize the presence ofcarboxyfluorescein (CF) within mature, functional sieve elements.Transverse sections of this thickness were necessary as, in unfixed 200µm-thick sections, CF was found to rapidly diffuse across the cut CC-SE surface.

Grafting protocolsThe side-grafting technique of Tiedemann (1989) was employed, withsome modifications, to generate heterografts between scions cut from4-week-old cucumber (Cucumis sativus cv. Straight Eight) plants

(vegetative apex to the second expanded leaf) and stocks provided byequivalent-aged pumpkin plants. Each excised cucumber scion(approx. 5-10 cm in length) was carefully inserted into an incisionmade in a pumpkin (or cucumber for autografts) stem at a location 10cm back from the vegetative apex. The graft site was fastened andsealed with parafilm and the plant was then covered with a clearplastic bag. Grafted plants were grown in a controlled environmentchamber for 17 days under low light (100 µmol/m2/second PAR) andhigh humidity (95%) to optimize conditions for the formation offunctional graft unions. Plants were then grown under higher lightintensity (650 µmol/m2/second PAR; 70% relative humidity, 26°C)until used in experiments.

Plants were employed for phloem sap analysis 3-4 weeks afterbeing grafted. Phloem sap was collected from the grafted scions ofcucumber as described for pumpkin. Typically, 5-15 µl of phloem sapwas collected, per scion, and was immediately processed to synthesizecDNA, which was amplified using CmNACP-specific primersaccording to the following procedure: 60 seconds at 94°C (1 cycle);30 seconds at 94°C; 30 seconds at 61°C; 60 seconds at 72°C (35cycles, two rounds). Preliminary RT-PCR experiments conducted onpumpkin and cucumber confirmed the specificity of the primersdesigned to amplify CmNACP. The amplified products were run onan agarose gel, blotted to a Hybond plus nylon membrane (AmershamPharmacia Biotech) and hybridized under stringent conditions to a[α32P]dCTP-labeled CmNACP probe, washed under stringentconditions and then exposed, at −80°C, to X-ray film.

Apical tissues were also dissected from non-grafted (control) andheterografted cucumber scions and RNA extracted for RT-PCRanalysis. A NAC-related partial cDNA from cucumber phloem sapwas obtained by PCR using the degenerate primers presented in Table1. The product was cloned, sequenced, and specific primers (Table 1)used to assay for the corresponding product in cucumber andpumpkin. Poly(A)+ RNA was obtained from control and heterograftedcucumber scion apical tissues, reverse-transcribed and subjected toLD-PCR. The product was used as template to assay for the presenceof pumpkin transcripts, by PCR, using the primers presented in Table1. The standard conditions for the PCR (two rounds) were 60 secondsat 94°C (1 cycle); 30 seconds at 94°C; 30 seconds at 61°C; 60 secondsat 72°C (35 cycles). Products were blotted to a nylon membrane andhybridized, under high stringency conditions, with the corresponding[α32P]dCTP-labeled probes generated by PCR using nested primers(Table 1) to confirm the identity of the amplified products.

RESULTS

Detection of RNA transcripts in the functionalphloemThe present studies were performed on pumpkin because: (i)analytical quantities of phloem sap could be readily collectedfrom excised petioles and stems (Sabnis and Hart, 1976), and(ii) previous studies had established that specific phloemproteins can be transported through a heterograft union(Tiedemann and Carstens-Behrens, 1994). Initial experimentsestablished the presence of RNA within the phloem sapcollected from excised petiole and stem tissues. Poly(A)+ RNAcontained within the pumpkin phloem sap was first reverse-transcribed to produce cDNA which was then amplified andcloned into the phage vector, λgt11, yielding 1×105

independent recombinants. An aliquot of the phloem-sap-derived cDNA was cloned into pCR II TOPO vector and 100clones were chosen, at random, for sequencing and subsequentanalysis.

Although many of these phloem transcripts appeared toencode unique sequences (not yet present in any database),



Table 2 presents some of the clones for which homology toknown genes could be established. Other clones showedhomology to genes whose functions have yet to be elucidated.Interestingly, some of these phloem transcripts appeared tocontain factors putatively involved in signal transduction andtranscriptional regulation, a finding that has significance giventhe enucleate condition of the functional sieve elements. Onesuch clone, CmNACP (a transcript containing a conserved NACdomain; Aida et al., 1997), was selected for furthercharacterization, since some members of this large gene familyappear to play a pivotal role in meristem (leaf and floral organ)development. CmNACP displays a high level of homology(Table 2) to the NAC domain of other members of this family(Fig. 1). In petunia and Arabidopsis, NAM (Souer et al., 1996),CUC2 (Aida et al., 1997) and NAP (Sablowski andMeyerowitz, 1998) transcripts were reported predominantly invegetative and floral apices. Hence, the presence of CmNACPmRNA in the phloem sap within a region of the plant distantfrom vegetative buds or floral tissues, suggested to us that thesetranscripts may be translocated to these sites through the

phloem. Alternatively, CmNACP could function in otherpumpkin tissues, perhaps associated with the vascularcambium (meristem), or in the control over senescence, asseems to be the case for SenU5 in leaves of tomato (John etal., 1997).

This phloem sap cDNA library was screened with CmNACP,CmRINGP and CmPP16 cDNAs in order to determine thefrequency at which such transcripts were present. Out of 5×103

pfu plated, approximately 1% gave a positive signal for eachgene tested. Thus, these transcripts appear to be enriched inthis subset of phloem-derived transcripts.

CmNACP and other phloem-related transcripts arenot contaminants from non phloem tissuesExcision-induced release of pressure within functional sieveelements has long been considered to result in contaminationof the exuded phloem sap by entry of proteins and RNA fromneighboring parenchyma or companion cells (Kollmann et al.,1970). In view of such a possibility, a series of controlexperiments was performed in order to verify whether

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Fig. 1. Multiple alignments of CmNACP-related amino acid sequences. The predicted sequence for CmNACP is compared with ATAF1,ATAF2, CUC2 and NAP of Arabidopsis (GenBank accession numbers X74755, X74756, AB002560 and AJ222713, respectively), NAM ofpetunia (X92205) (Souer et al., 1996) and senU5 of tomato (Z75524) (John et al., 1997). The N-terminal half of CmNACP displays clearhomology with the NAC domain (Aida et al., 1997); conserved NAC domain motifs are identified by rectangular blocks. The reverse primer forCmNACP corresponded to the 3′ untranslated region for higher specificity of detection (see Fig. 7). Sequences were aligned using the Clustal WProgram (version 1.7) (Thompson et al., 1994).

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CmNACP mRNA and other phloem-related transcripts arebona fide constituents of the pumpkin phloem sap. To this end,poly(A)+ RNA was isolated from stem phloem sap collectedfrom five different plants (all of the same age) grown underidentical conditions. Total poly(A)+ RNA was also isolatedfrom stem tissue excised from each site where phloem sap hadbeen collected. These individual mRNA samples were reverse-transcribed to cDNA and then employed as templates for semi-quantitative PCR analysis.

As transcripts of pumpkin phloem protein 2 (PP2) werepreviously shown to be both confined to companion cells

(Bostwick et al., 1992) and not translocated across aheterograft union (Golecki et al., 1999), initial controlexperiments were conducted with this gene. Although CmPP2transcripts were readily detected in the cDNA prepared fromstem tissue, reflecting the high expression level of this gene incompanion cells (Bostwick et al., 1992), they were not detectedwithin the cDNA obtained from the phloem sap, even after 20cycles of amplification (Fig. 2A). This experiment establishedthat RNA contamination, originating from tissues outside ofthe SE, must have been present at a low level.

This conclusion was further supported by amplificationof nuclear-encoded RUBISCO small subunit (CmrbcS),Cmimportin α (a protein involved in nuclear transport; Gorlichet al., 1995; Loeb et al., 1995; Hicks et al., 1996) and


Table 2. Representative transcripts identified within Cucurbita maxima phloem sapGene Similarity (%)@ Possible function Reference

CmNACP NAM (99)* , Meristem maintenance Souer et al. (1996)CUC2 (97)*NAP (97)* Floral development Aida et al. (1997)

Sablowski and Meyerowitz (1998)SenU5 (95)* Leaf senescence John et al. (1997)

CmRINGP RING (41)** Transcriptional regulation von Arnim and Deng (1993)CmRABP RAB (84) Intracellular vesicular trafficking Haizel et al. (1995)CmGAIP GAI (78) Regulator of gibberellin response Silverstone et al. (1998)CmWRKYP WRKY (70) Defense response Rushton et al. (1996)CmSTMP STM (51) Cell fate in meristems Long et al. (1996)CmCYCLINP G1-S cyclin (72) Cell cycle Sauter (1997)CmPP16 - RNA transport Xoconostle-Cázares et al. (1999)CmSUTP1 SUT1 (77) SE sucrose transport Riesmeier et al. (1992)CmPDHPP PDHP (38) Regulation of glycolysis Lawson et al. (1993)

@Similarities determined by the methods of Gish et al. (1993) and Altschul et al. (1990, 1997).*Similarity to the conserved NAC domain.**Identity to the zinc-finger domain of nodulation-associated RING-finger protein from Lotus japonicus (accession number S49446).

Fig. 2. Phloem sap of pumpkin contains an enriched subset oftranscripts. (A) Gene-specific primers were employed to amplifyCmPP2, Cmthioredoxin-h, CmrbcS, Cmimportin α, CmNACP andCmRINGP (expected size of products being 620, 382, 480, 610,610 and 696 bp, respectively) from whole-stem tissue (first lane)and phloem sap cDNAs (remaining five lanes) prepared frompoly(A)+ RNA collected from five different 4-week-old pumpkinplants, all grown under the same conditions. The cDNA wassubjected to LD-PCR and the products used as template for theamplification of specific transcripts. The PCR products were thenblotted to a nylon membrane and hybridized, under stringentconditions, to the corresponding probes. (B) Relative abundanceof transcripts in stem and phloem sap cDNA and absence ofcontaminating DNA. Upper panel shows autoradiographicanalysis of individual stem and phloem sap cDNA sampleshybridized against 32P-labeled phloem sap cDNA, to establish theamount of cDNA present in each assay. Lower panel showsspecific primers used to amplify CmPP16 and CmRINGP fromthese individual cDNA samples. Arrowheads indicate the locationof predicted bands, being 490 and 700 bp for CmPP16 andCmRINGP, respectively; a band at 800 bp corresponding toCmPP16 genomic clone (upper arrowhead) was not detected,indicating the absence of contaminating DNA. (C) Phloem sapproteins protect RNA. TMV RNA (0.2 µg) was incubated withphloem sap (1.5 µg), stem extract (1.4 µg), or DEPC-treatedwater, for various times prior to being run on an agarosedenaturing gel. Incubation times were as follows; lanes 1, 4 and 7,immediate loading; lane 2, 5 and 8, 5 minutes; lanes 3, 6 and 9, 10minutes. Lanes 1-3; phloem sap; lanes 4-6, stem extract; lanes 7-9, DEPC-treated water.


Cmthioredoxin-h (the expression of which is confined tocompanion cells in rice; Ishiwatari et al., 1998) cDNAs. Forthese experiments, phloem sap poly(A)+ RNA was collectedfrom an unblotted cut, reverse-transcribed, amplified andcloned (R. R.-M., B. X.-C. and W. J. L., unpublished results).

Transcripts for these three genes were detected in stem tissue,but not in the phloem sap, after 20 cycles of amplification (Fig.2A). These results are consistent with the known ultrastructuralfeatures of the phloem, in that the functional sieve elementsare enucleate, and thus, would not contain the components

Fig. 3. CmNACP transcripts are presentwithin the companion cell-sieve elementcomplexes of the pumpkin vascularsystem. Fresh transverse sections ofpetiole or stem tissue were processed forin situ RT-PCR analysis. (A) Schematictransverse section of a pumpkinstem/petiole. Vascular bundles comprisean internal and external phloem (IP andEP, respectively), the internal andexternal cambium (CA), and the xylem(X). The outer region of the stem/petiolecontains supporting collenchyma tissue(CO) and connecting phloem strandsinter-link the vascular bundles.(B) CmrbcS-specific primers produced agreen fluorescent signal that wasassociated with chloroplast-containingcortical cells that surround the vascularbundles. A weaker CmrbcS signal wasdetected over immature phloem cells,whereas signal was absent from the areaoccupied by the functional phloem (FP).(C) Higher magnification of the EP in(B). Note the absence of any greenfluorescent signal over the functionalcompanion cell (CC) and sieve element(SE) complexes. (D) Cmimportin α-specific primers produced a greenfluorescent signal that was located overthe CC. Note the absence of signal fromthe associated SE. (E) CmNACP-specific primers produced a strong greenfluorescent signal that was confined toCC and SE located in a petiole vascularbundle. (F) Same transverse section asin E, after staining with fluoresceinisothiocyanate in order to revealanatomical details of the phloem.(G) Location of mature (enucleate),functional SE identified using thecarboxyfluorescein diacetate loadingtechnique. A green fluorescent signal,corresponding to the presence of CF,was detected only in SE positioneddistal to the CA; i.e., in the functionalphloem (FP). The absence of fluorescent signal from the associated CC reflects the speed with which CF diffused out of the sections into thebathing medium. (H) CmSUT1-specific primers produced a strong fluorescent signal that was confined to immature (lower) and functional(upper) CC-SE complexes. (I) Same section as in H, after staining with fluorescein isothiocyanate to reveal anatomical details of the phloem.Immature SE indicated by asterisk (∗ ). (J) Negative control, in which CmNACP primers were omitted from the reaction mixture. Only anextremely low background level of fluorescence was detected in such experiments. (K) Same section as shown in J, after staining with fluoresceinisothiocyanate to reveal anatomical details of the tissue. (L) Green CmNACP RNA fluorescent signal present in CC and SE of a connectingphloem strand. These simple strands consist of functional phloem CC-SE complexes (arrows), embedded in parenchyma tissue (red fluorescentsignal (arrowheads) associated with chloroplasts in these cells). (M) Image collected within the plane of a functional sieve plate (SP). SE and CC(end wall) outlined for orientation. Note that, as the SP was positioned at an oblique angle to the long axis of the SE, only a portion of the platewas in the focal plane. A strong green fluorescent signal was present within the individual pores of this SP. In this image plane the CC end wallwas devoid of signal. (N) Same as in M, except that this image was collected 20 µm beneath the SP depicted in M. (O,Q) Distribution ofCmNACP mRNA, within a petiole vascular bundle equivalent to that depicted in E, as detected by Dig-antisense riboprobe. (P,R) Equivalentsections as in O and Q, but hybridized with Dig-sense riboprobe. All bars, 50 µm, except for B and M and N, which are 100 and 25 µm,respectively.

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required for nuclear trafficking, and contain plastids rather thantrue chloroplasts (Schulz, 1998).

The reverse situation was observed when experiments wereconducted with CmNACP and CmRINGP. Here, low transcriptlevels were detected in stem tissue, whereas in the phloem sapthe levels ranged from highly abundant down to those detectedin stem tissue (Fig. 2A). It is important to note that, after10-15 more amplification cycles, CmPP2, CmrbcS andCmthioredoxin-h transcripts could be detected in phloem sapsamples. Thus, high transcript levels in the phloem sap andlower levels in stem tissue, such as was found for CmNACPand CmRINGP, suggest that these are bona fide constituents ofthe phloem sap. Conversely, low levels of a specific transcriptin the phloem sap do not necessarily establish that it representscontamination from surrounding tissues.

In an effort to normalize transcript detection by PCR, thetemplate was quantified as follows: poly(A)+ RNA was isolatedfrom five stem and three phloem sap samples collected fromindividual plants, cDNA synthesized, blotted and thenhybridized with [α-32P]dCTP-labeled phloem sap cDNA (Fig.2B, upper panel). Similar amounts of synthesized cDNA werethen subjected to LD-PCR, and the resultant products were thenused to amplify specific cDNAs. Under these conditions,CmPP16 transcript was readily detected in both stem andphloem sap samples (Fig. 2B). In contrast, with the exact samestarting template, and identical amplification conditions,CmRINGP was only detected in the three phloem sap samples.These experiments revealed the different relative abundance ofthe CmPP16 and CmRINGP transcripts in the phloem sap andstem tissue. To test for the possible presence of contaminatingDNA, amplification of CmPP16 was carried out as we hadearlier identified introns in this gene (Xoconostle-Cázares et al.,1999). The absence of an 800 bp band (Fig. 2B), which wouldbe the PCR product of the gene, indicated that neither DNA norunspliced RNA were present in the phloem sap samples.

Phloem sap has the capacity to complex and protectRNAThe stability of phloem sap RNA was next examined byconducting in vitro experiments in which TMV RNA wasmixed with phloem sap or stem extract. The formation of anRNA-protein complex was observed when TMV RNA wasincubated with phloem sap. These phloem sap proteinsappeared to stabilize this RNA, as only limited degradation wasdetected (Fig. 2C). In contrast, incubation with stem extractresulted in significant RNA degradation. Interestingly, theRNA-phloem sap complex was stable even under the denaturingconditions imposed by the presence of formaldehyde.

Localization of CmNACP and other phloem-relatedtranscripts to companion cell-sieve elementcomplexTo further confirm that CmNACP mRNA is present within thephloem, a modified version of in situ RT-PCR (Chen andFuggle, 1997) was carried out on fresh sections obtained fromstems and petioles. Primers specific for CmrbcS, Cmimportinα and CmNACP were employed and fluorescence associatedwith each transcript was visualized using a CLSM. A simplediagram of the vascular arrangement in pumpkin petiole/stemtissue is presented in Fig. 3A. The fluorescent signal associatedwith CmrbcS transcripts was detected in cortical cells and, in

particular, in the cells surrounding the vascular bundles (Fig.3B). No fluorescent signal was detected in association withfunctional companion cell-sieve element (CC-SE) complexes(Fig. 3C). In the case of Cmimportin α, a clear signal wasdetected over the CCs, but no fluorescence was observed in thefunctional SEs (Fig. 3D). This finding is consistent with theknown high transcriptional/translational activity of maturecompanion cells (Fisher et al., 1992).

In situ RT-PCR experiments conducted with CmNACPprimers revealed the presence of a strong fluorescent signalconfined to developing and functional CC-SE complexeslocated within the petiole (Fig. 3E,F); a similar pattern wasobserved in stem tissues (see Fig. 5I-L). Interestingly, closeinspection of the innermost region of the vascular cambium(involving more than 50 sections) indicated a complete absenceof signal, thus eliminating this meristematic tissue as apotential source of contaminating CmNACP mRNA.Furthermore, as the cambium is the site where the sieve mothercells are formed, this lack of signal precludes the possibilitythat CmNACP mRNA is synthesized in sieve mother cells andthen retained until the generation of mature CC-SE complexes.

As an internal control for the in situ RT-PCR method,CmSUTP1 was detected using specific primers (Table 1).Previously, SUT1 mRNA was shown, by electron microscopy-based in situ hybridization, to be restricted to the CC-SEcomplexes of potato and tobacco (Kühn et al., 1997). In situRT-PCR experiments revealed that, as with CmNACP,CmSUTP1 transcripts were confined to developing andfunctional CC-SE complexes (Fig. 3H,I). The absence of signalwithin petiole and stem tissues in experiments in which one orboth primers were omitted from the reaction mixtureestablished the specificity of the protocol employed in thesestudies (Fig. 3J,K).

The location of mature, functional, sieve elements, withinthe vascular bundles, was confirmed both by electronmicroscopy and by performing carboxyfluorescein diacetateloading and phloem translocation experiments. Based oncomparative analyses of in situ RT-PCR and CF-labeledfluorescent images, performed on petioles from equivalentlyaged plants, it was possible to establish that CmNACP mRNAwas present in the same relative position as the functional CC-SE complexes (compare Fig. 3E and 3G). In addition,CmNACP transcripts were also detected in interconnectingphloem strands (Fig. 3L) that are embedded in theparenchymatous tissue of the cortex (see Fig. 3A), which wasfree of CmNACP mRNA. Finally, CmNACP transcripts weredetected in the region of functional sieve plates. Here a strongfluorescent signal was detected within the actual sieve platepores (Fig. 3M) and extended out into the lumen of theadjoining sieve elements (Fig. 3N).

In situ hybridization experiments, performed on pumpkinpetiole cross sections, revealed the presence of CmNACPmRNA within developing and mature CC-SE complexes (Fig.3O), with a strong signal being detected in mature SEs (Fig.3Q). Control experiments, using sense riboprobe establishedthe specificity of this hybridization (Fig. 3P,R). Interestingly, aweak signal was detected over the nuclei of both immature andmature CCs, likely reflecting the presence of mRNA, ratherthan the detection of chromosomal DNA, as no signal wasdetected in other cell types. Collectively, these experimentsprovided strong support for the conclusion that (i) CmNACP



mRNA is present within the phloem long-distancetranslocation stream of pumpkin, and (ii) the in situ RT-PCRmethod accurately reports the cellular site(s) of transcriptaccumulation.

The presence of CmPP16 transcripts within the CC-SEcomplexes of pumpkin petiole and stem tissues has alreadybeen established (Xoconostle-Cázares et al., 1999). To confirmthe presence and cellular distribution of CmRINGP, CmRABP,CmGAIP, CmWRKYP, CmSTMP and CmCYCLINP, in situ RT-PCR experiments were next performed with the appropriateprimer sets. CmRINGP, CmRABP and CmSTMP were found tobe confined to developing and mature CC-SE complexes.CmGAIP was detected in vascular tissues, including xylemparenchyma and the cambium, with the highest signalbeing detected in association with the CC-SE complexes.CmCYCLINP transcripts were detected in the cambium and inassociation with developing and mature CC-SE complexes.Lastly, CmWRKYP transcripts were detected both within thephloem and, at a lower level, in the surrounding cortex (datanot shown).

Tissue and cellular distribution of CmNACP mRNAand proteinThe tissue-specific distribution and abundance of CmNACPmRNA was assessed by northern analysis. These experimentsdemonstrated that transcripts were present in all plant organs;low levels were present in leaf, stem, floral and apical tissues,whereas in roots the level was higher (Fig. 4). The tissue andcellular distribution of CmNACP mRNA was next examined byin situ RT-PCR experiments (Fig. 5). Analysis of longitudinaland transverse sections established the continuity between thefluorescence detected within apical (Fig. 5B) and floral (Fig.5F) meristems and CmNACP mRNA within SEs of theprotophloem, which is located distal to these tissues (Fig. 5I,J). These in situ RT-PCR results further confirmed the presenceof CmNACP mRNA within the CC-SE complexes of the upperstem; fluorescence detected in equivalent cells located in thelower stems (Fig. 5K,L) and petioles of senescing leaves (datanot shown) was lower in intensity.

Parallel in situ hybridization experiments, performed onsimilar meristematic tissues as used for in situ RT-PCR

analysis, revealed an equivalent pattern for CmNACP mRNAdistribution in apical (Fig. 5C), axillary (Fig. 5E) and floral(Fig. 5G) meristems. Control sections hybridized with senseDig-labeled riboprobes were devoid of signal (Fig. 5D,H)which confirmed the specificity of this method of transcriptdetection.

In agreement with the high level of CmNACP transcriptdetected in root tissue by northern analysis (Fig. 4), a strongfluorescent signal was observed in transverse and longitudinalsections of pumpkin roots (Fig. 5M and N, and P, respectively).Similar to the situation found in petiole and stem tissues, onlybackground levels of fluorescence were detected in theappropriate control experiments where primers were omittedfrom the reaction mixture (Fig. 5O). Interestingly, transversesections of tissue near the root apex revealed that thefluorescent signal extended out beyond the phloem into corticaland epidermal cells (Fig. 5M). A strong fluorescent signal alsoextended from the terminal phloem through the primarymeristem to the root initials (Fig. 5P). However, the root capcells appeared to be devoid of signal. These results areconsistent with a role for CmNACP in both vegetative, floraland root meristematic tissues.

Polyclonal antibodies directed against CmNACP were usedfor immunolocalization studies. A strong CmNACP signal wasdetected within nuclei of mature CC and developing CC-SEcomplexes (Fig. 5Q). Signal was not detected in mature SEs,consistent with Western analysis performed on phloem sap(data not shown). Analysis of floral tissues revealed thepresence of CmNACP signal in the apical region of developingovaries (Fig. 5S). Here, the signal was present in both nucleiand cytoplasm (Fig. 5U). In vegetative axillary meristems,CmNACP exhibited a gradient in distribution, with the highestlevels being detected in the L1 layer (Fig. 5V).

Movement of CmNACP mRNA into scion meristemof cucumberHeterograft experiments were performed, using pumpkin as thestock and cucumber as the scion, to investigate whether theCmNACP mRNA, present in the pumpkin phloem translocationstream, can move into the meristem of the scion. Earlier studiesusing this heterograft system established that, once theconnections between vascular bundles of the scion and stockwere established, pumpkin phloem proteins can be translocatedthrough the graft union into the cucumber scion (Tiedemannand Carstens-Behrens, 1994; Golecki et al., 1998, 1999;Xoconostle-Cázares et al., 1999). Cucumber scions weregrafted onto the stems of 4-week-old pumpkin plants and, 3weeks later, scion phloem sap was collected for RT-PCRanalysis (Fig. 6A). Appropriate autograft controls were alsoperformed using cucumber as both stock and scion. To preventcross-contamination of scion phloem sap by the stock tissue,all scions were severed by a transverse cut made 3 cm from thegraft union (see Fig. 6A).

These grafting experiments demonstrated that CmNACPtranscripts could be detected in the phloem sap collected fromthe cucumber scion of heterografted plants, but wereundetectable in sap from the cucumber controls (Fig. 6B). Insome samples, an additional band was present in the scionphloem sap, but was never detected in cucumber controls. Thisband could represent a second CmNACP-related transcript. ThePCR products obtained from cucumber scions were cloned and

Fig. 4. Northern analyses indicate that CmNACP mRNA is present inall plant tissues. Total RNA (10 µg) from the indicated tissues waselectrophoresed and blotted and RNA was then probed with[α32P]dCTP-labeled CmNACP cDNA under stringent conditions.Lower panel presents the corresponding agarose gel, stained withethidium bromide, showing the position of the 28S and 18Sribosomal RNA.

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sequenced, and confirmedto be CmNACP sequence.Parallel in situ RT-PCRexperiments also confirmedthe presence of CmNACP andCmPP16 transcripts in the SEof the scion cucumber stem.Strong fluorescent signal forboth transcripts was detectedin peripheral sieve elements(Fig. 6C and D). Controlsperformed on non-graftedcucumber stems (with bothsets of primers) indicated anabsence of signal within theSEs (Fig. 6E). The presenceof CmRINGP, CmRABP,CmGAIP, CmWRKYP,CmSTMP and CmCYCLINPtranscripts was also analyzedin cucumber grafted scions.In all cases, fluorescent signalwas detected in the functionalSEs (data not shown).

The tissue-specificdistribution of CmNACPtranscripts within cucumberscions was established by insitu RT-PCR experiments.Analysis of heterograftedcucumber scion tissuesrevealed the presence ofCmNACP transcripts invegetative meristems (Fig.6F, G) and developing floralorgans (Fig. 6H,J). Incontrast, in equivalent tissuesof autografted or controlcucumber plants no signal, oronly a very weak fluorescentsignal, could be detected(Fig. 6I,K).

Selectivity of mRNAdelivered to the apicaltissuesIn a final series ofexperiments, the questionwas addressed as to whetherother transcripts, presentwithin the phloemtranslocation stream, are alsodelivered, via the phloem, to apical tissues. The presenceof CmNACP, CmRINGP, CmRABP, CmGAIP, CmWRKYP,CmSTMP, CmCYCLINP and CmPP16 was investigated byPCR analysis using specific primers (Table 1). As expectedfrom our earlier studies, CmNACP was detected inheterografted but not in control cucumber tissues (Fig. 7). Inaddition, CmGAIP and CmPP16 were also amplified from theheterografted scion tissues, but products were not amplifiedfor CmRINGP, CmRABP, CmWRKYP, CmSTMP and

CmCYCLINP. As the tissue used for these RT-PCRexperiments contained functional phloem, detection ofCmGAIP and CmPP16 transcripts could reflect their presencein SE; movement beyond the protophloem will require adetailed in situ RT-PCR analysis, as performed for CmNACP.Furthermore, failure to detect CmRINGP, CmRABP,CmWRKYP, CmSTMP and CmCYCLINP could be due to theirpresence, in the scion apex, at very low levels, rather than totheir total absence. In any event, these results implicate



Fig. 6. Translocation of CmNACP mRNA through aheterograft union into apical tissues of cucumber scions.(A) Diagrammatic representation of the morphology ofa pumpkin plant on which is indicated the site of aheterograft union (gu) between pumpkin, acting as thestock, and cucumber as the scion: abbreviations as inFig. 5; CAM, cucumber axillary meristem. Asteriskindicates the site at which apical tissues were excisedfor RT-PCR analysis (see Fig. 7). (B) Detection ofCmNACP in cucumber scion phloem sap collected fromlocation indicated by the double asterisk. Lane 1,phloem sap cDNA prepared from the scion of acucumber autograft union hybridized to a 32P-labeledCmNACP probe. (Phloem sap cDNA prepared fromcontrol (autografted and ungrafted) cucumber plantsgave a similar negative result.) Lanes 2-4, phloem sapcDNA from three cucumber scions of heterograftedplants. In situ RT-PCR detection of CmNACPtranscripts (C) and CmPP16 transcripts (D) in crosssections of cucumber scion stem vascular tissue. Thescion was first excised, and cross sections cut prior tocollection of phloem sap. (E) Equivalent cross sectiontaken from a cucumber control and subjected to RT-PCR using CmNACP primers. Longitudinal sections ofan axillary meristem (F) and an apical meristem (G)taken from cucumber scions (see A for location). Notethat in F the CmNACP RNA signal extended from thephloem strands (P) in the petiole (Pe) all the way to thedome of the meristem. The underlying stem (St) tissuewas lost from this section. In G, the base of the AM isensheathed in developing trichomes that also emit agreen autofluorescent signal. The yellow signal presentwithin the AM and LP represents the combination of the green CmNACP RNA signal and the red autofluorescence; note that the strength of theCmNACP RNA signal in this tissue was approx. 20% of that detected in the pumpkin AM. Longitudinal sections through developing floralorgans from cucumber heterografted (H and J) or autografted (I and K) scions. A background green autofluorescent signal was at times detectedin developing floral tissues, particularly in petals (see K). CmNACP transcripts were readily detectable in the ovary (Ov), petals (P), sepals (S)and floral meristems (Fm) of the heterografted, but were absent from equivalent tissues within autografted cucumber scions and control plants.All bars are 100 µm.

Fig. 5. Localization of CmNACP mRNA and CmNACP in shoot, floral and root meristem tissues of pumpkin. (A) Diagrammatic representationof the morphology of a pumpkin: AM, apical meristem; LM, lateral meristem (includes floral meristems); LP, leaf primordia. Direction ofphloem translocation is indicated by red arrows. Arrowheads identify the positions where sections were obtained for protein and mRNAlocalization. (B) Longitudinal section of pumpkin vegetative apex showing a strong CmNACP-associated signal (green fluorescence) in theapical meristem; less intense signals were detectable in the adjacent organ primordia (developing glandular trichomes often exhibited greenautofluorescence (arrowheads) that was also detected in controls (not shown); red autofluorescence signal was employed to display generaltissue outline). (C) Longitudinal section of vegetative apex hybridized with CmNACP Dig-labeled antisense riboprobe. Arrow indicates strongsignal associated with the provascular tissues. (D) Equivalent section hybridized with CmNACP Dig-labeled sense riboprobe. (E) Longitudinalsection through an axillary meristem hybridized with antisense riboprobe. (F,G) Longitudinal sections of developing flower where CmNACPtranscripts were detected by in situ RT-PCR (F) and in situ hybridization (G). Petals (P) and sepals (S). Note that CmNACP mRNA was presentover the region of the protophloem (lower arrows) and extended beyond the functional phloem to the base of the ovary. (H) Control for floralmeristem, as in D. (I) Transverse stem section taken from the base of the longitudinal section displayed in B. CmNACP transcripts were presentin both immature (proximal to the cambium; CA) and functional (outer) CC-SE complexes. (K) Transverse section of a vascular bundle, locatedin the region of the main stem, demonstrating the continuity of CmNACP RNA signal within the phloem long-distance translocation system.(J) Same section as shown in I, after staining with fluorescein isothiocyanate to reveal anatomical details of the phloem. (L) Anatomical detailsof the section displayed in K, imaged as in J. (M) Transverse section of a pumpkin root demonstrating the presence of CmNACP transcriptswithin the phloem (P) and extending out in an irregular pattern into the cortex (C) and epidermis (E). (N) Anatomical details of the pumpkinroot (M), imaged as in J. (O) Negative control, performed on a transverse root section taken from the same region as that used in M, in whichCmNACP primers were omitted from the reaction mixture. Only background levels of fluorescence were detected in such experiments. Locationof tetrarch phloem tissue indicated by arrows. (P) Longitudinal section of a pumpkin root apex, illustrating the cellular distribution of CmNACPtranscripts within the primary meristem (PM) and extending back into the vascular tissue (phloem; P) and epidermis (E). Fluorescent signal wasnot detected in the root cap (RC) cells (arrows). (Q,R) Immunolocalization of CmNACP detected in the outer phloem of a petiole cross sectionusing immune and preimmune sera, respectively. (S,T) Immunolocalization of CmNACP detected in a longitudinal section of a developingflower, using immune and preimmune sera, respectively. Ov, ovary; arrow identifies signal associated with the protophloem. (U) Highermagnification of the ovary shown in S illustrating CmNACP signal in the distal region of the ovules. (V) CmNACP detected in developingaxillary meristem. Bars are 100 µm, except for U and V, which are 50 µm.

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specificity in terms of the mRNA molecules delivered to thescion apex.

DISCUSSION

Specific population of mRNA molecules moves inthe phloemIn the present study, we provide experimental evidence thathigher plants have evolved a mechanism that allows theselective translocation of specific mRNA molecules, by thephloem, for delivery to distant organs of the plant. Althoughearlier studies identified nucleic acids within the phloem sap(see Zimmermann and Milburn, 1975), these findings wereconsidered to reflect contamination resulting from a pressuresurge generated during tissue excision (Kollmann, 1970).However, we here present several lines of evidence which offerstrong support for the concept that many of the mRNAmolecules present within the pumpkin phloem sap are bonafide constituents of the translocation stream.

Semi-quantitative RT-PCR experiments, performed on stemtissues and phloem sap, established that transcripts known toaccumulate in cells surrounding the SE, including the CC, wereeither not detected, or were present at low levels within thephloem sap (Fig. 2). It is important to note that our results withCmPP2 and Cmthioredoxin-h are consistent with earlierreports on the characterization of these phloem-related genes(Bostwick et al., 1992; Golecki et al., 1998, 1999; Ishiwatariet al., 1998). However, in a recent study conducted on rice,thioredoxin-h mRNA was detected in the phloem sap (Sasakiet al., 1998). In the absence of internal controls for the ricestudy, this inconsistency likely reflects the non-quantitative RT-PCR approach used by Sasaki et al. (1998). It is important tonote that the presence of rare transcripts that are translocated,via the phloem, may be difficult to characterize, and thus will

require a combination of heterografting and in situ RT-PCRstudies.

Our in situ RT-PCR experiments also provided strongsupport for the hypothesis that mRNA molecules are presentwithin the functional sieve tube system. In all cases examined,the transcripts cloned from the pumpkin phloem sap werepresent in CC-SE complexes of the petiole and stems.Furthermore, the pattern of CmSUTP-1 mRNA withinpumpkin CC-SE was the same as that reported for SUT1 inpotato, tomato and tobacco (Kühn et al., 1997). In contrast,transcripts for CmrbcS and Cmimportin α that were notdetected by semi-quantitative RT-PCR analysis were confinedto CCs and/or the surrounding tissues. Compelling evidence insupport of the phloem mRNA translocation hypothesis wasprovided by the combination of grafting, in situ localizationand in vitro RT-PCR techniques. With these experimentalapproaches, we were able to establish that specific mRNAmolecules are translocated through the pumpkin stock into thecucumber scion, via the phloem. Moreover, three of the eighttranscripts investigated in detail were detected within the apicaltissues of the heterografted cucumber scion.

Specificity of CC-SE protein and mRNA exchangeThe selectivity associated with protein exchange through theCC-SE plasmodesmata is likely imparted by proteins localizedto the CC (Lee et al., 1999). This concept is supported by therecent findings of Golecki et al. (1999). In this study, it wasdemonstrated that both C. maxima phloem protein 1 and PP2were transported into scion SEs, where they could then movethrough the interconnecting SE-CC plasmodesmata, but wereclearly unable to traffic into neighboring phloem parenchymacells. Earlier studies on SUT1 (Kühn et al., 1997) and CmPP16(Xoconostle-Cázares et al., 1999), in conjunction with the dataobtained in the present study, established that a selectiveprocess also operates within the CC to deliver a uniquepopulation of mRNA molecules into the sieve tube system.Although it has been demonstrated that CmPP16 can mediatethe cell-to-cell transport of mRNA, in a non-sequence-specificmanner, the CC proteins responsible for the targeting ofspecific proteins and ribonucleoprotein complexes to the CC-SE plasmodesmata remain to be elucidated.

Experiments carried out on grafted plants established thatCmNACP, CmGAIP and CmPP16 transcripts are beingdelivered to cucumber apical tissues, via the phloem. Basedon the results obtained with CmNACP, these transcriptsappear to be able to move from the terminal protophloem intovegetative and/or floral meristems. As many plant virusesappear to be excluded from moving along this route(Gilbertson and Lucas, 1996), the present findings indicatethat, of the total subset of mRNA molecules present withinthe phloem sap, specific transcripts must be recognized by themolecular machinery underlying this selective delivery ofmacromolecules, from the phloem to meristematic tissues.Evidence consistent with such a pathway for the transport ofmacromolecules, from the phloem, into developing organs(leaves and meristems), was recently provided by studies withtransgenic plants expressing the green fluorescent proteinunder the SUT1 promoter (Imlau et al., 1999). It would seemreasonable to deduce that CmNACP, CmGAIP and CmPP16transcripts are similarly being delivered to the meristems inpumpkin plants.


Fig. 7. Selective delivery of specific pumpkin phloem transcripts intodeveloping apical tissues of heterografted cucumber scions. cDNAobtained from control and heterografted cucumber tissues was usedas template to amplify the indicated transcripts with primerspresented in Table 1. Products were blotted and hybridized with thecorresponding internal probes produced by nested PCR, understringent conditions. (Expected sizes as in Fig. 3, except forCmGAIP, which is 640 bp.) Lane 1, cucumber apical tissue cDNA;lane 2, heterograft scion apical tissue cDNA. CsNACP: Detection ofCucumis sativus NACP in cucumber (lane 1) but not in pumpkin(lane 2) apical tissues (expected size of CsNACP, 250 bp).


Possible functions for translocated mRNAEvidence consistent with the long-distance translocation ofRNA transcripts has recently been provided by studies on themolecular determinants associated with the induction ofsystemic acquired silencing of viral and endogenous genes.Transgenic plants expressing high levels of a specific viralgene, or a fragment thereof, develop resistance whenchallenged with the corresponding virus (Lindbo et al., 1993;Covey et al., 1997; English et al., 1997; Ratcliff et al., 1997;Voinnet and Baulcombe, 1997; Tanzer et al., 1997; Ruiz et al.,1998). Similarly, transgenic plants expressing endogenousgenes at high levels have been found to display phenotypesconsistent with post-transcriptional silencing of these genes(Jorgensen, 1995; Elmayan and Vaucheret, 1996; Palauqui etal., 1996). In this regard, grafting experiments provided strongevidence that the transmission of the cosuppression signal,from the stock to the scion, moved through the phloem and washighly gene-specific in nature (Palauqui et al., 1997; Smyth,1997). A mechanism that could account for the long-distancetransmission of such a cosuppression signal has recently beenadvanced (Jorgensen et al., 1998). In this model, protein-mediated movement of specific complementary RNAmolecules (full-length or fragments) was proposed to occurinto and out of the phloem, via plasmodesmata.

The transcripts characterized in the present study (Table 2)were selected, at random, from a pumpkin phloem sap cDNAlibrary. The nature of these cDNAs likely reflects the uniquenature of the RNA population within the phloem sap.Moreover, the cDNAs for which a clear homology could beestablished indicate a likely role in the regulation of geneexpression or signal transduction. The cDNAs to which afunction could not be assigned may well reflect components ofa novel set of genes involved in phloem function, includingboth the maintenance of the enucleate sieve tube system andcommunication between distant organs.

Presently, no information is available on the role played byCmNACP in pumpkin. However, its RNA localization invegetative and floral meristems is similar to that reported forpetunia (Souer et al., 1996) and Arabidopsis (Aida et al., 1997;Sablowski and Meyerowitz, 1998). This pattern of RNAdistribution is consistent with a role for CmNACP in meristemdevelopment. The presence of full-length, polyadenylatedCmNACP mRNA in the pumpkin phloem sap, in conjunctionwith the detection of transcripts in both immature andfunctional CC-SE complexes, greatly extends the tissues inwhich this gene likely functions. However, as the in situ RT-PCR technique only allows the identification of the cells inwhich these transcripts accumulate, currently it is not possibleto discriminate between the site of transcription and RNAaccumulation. This question will be addressed in future studiesby conducting nuclear run-on experiments.

The database for Arabidopsis indicates that the NAC-domainencoding genes constitute a large gene family. A similarsituation is likely to exist in pumpkin and, thus, CmNACP maybe a member of a large gene family in which related genes maydisplay diverse or common functions, with different oroverlapping patterns of gene expression. In this regard,northern and in situ RT-PCR analyses revealed that root tipsaccumulate high levels of CmNACP mRNA, establishing anovel pattern for this member of the NAC-domain family (cf.Aida et al., 1997).

Our results demonstrate that CmNACP may act both at thelevel of RNA and protein. Immunolocalization studiesestablish that CmNACP is synthesized within the petiole andstem phloem (Fig. 5Q). Furthermore, detection of CmNACPwithin the nuclei of CC and meristematic cells suggests a rolein gene expression. The presence of CmNACP mRNA, and theabsence of CmNACP, in the phloem translocation stream, isconsistent with these transcripts having a role in long-distancesignaling. Following its delivery into distant, target cells, theCmNACP mRNA may be translated, or act as a signal toregulate the transcription of related genes, as appears to be thecase for CmPP16 in the heterografted cucumber scion(Xoconostle-Cázares et al., 1999). Heterografting of mutantscions onto wild-type plants would have provided a powerfulexperimental system to test whether CmNACP mRNA actsnon-cell-autonomously to rescue the mutant phenotype.Unfortunately, NAC-domain mutants in petunia andArabidopsis lack an apical meristem, thus making graftingexperiments impractical.

Information superhighway may coordinate meristemdevelopmentPhloem sap-specific transcripts, or their encoded proteins, mayplay a general role in the integration of physiologicalprocesses, occurring within leaves, and developmental eventstaking place in the various meristems (Mezitt and Lucas,1996). In this regard, it has long been recognized that floweringsignals are translocated to the vegetative apex, via the phloem(Zeevaart, 1962; Lang, 1965; Kinet et al., 1994; Colasanti etal., 1998). Interestingly, it was recently reported that thesymplasmic pathway through which such flower signalingmolecules may well traffic, appears to be modulated after thevegetative meristem is committed to flowering (Gisel et al.,1999). Although the nature of this signal(s) remains to beelucidated, it is possible that it moves from the leaves to theapex as a specific RNA-protein complex. The selective natureof the delivery and exit of phloem-specific transcripts, to themeristem of the cucumber scion (Fig. 6 and Fig. 7), providesadditional experimental foundation for this concept.

Thanks are due to Richard Jorgensen, John Bowman and ouranonymous reviewers for their insightful comments on themanuscript. Invaluable assistance in preparing the figures forpublication was provided by K. C. McFarland and Samantha Barling-Silva. This work was supported by DOE Biosciences grant DE-FG03-94ER20134 and NSF grant IBN-94-06974 (to W. J. L.).

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