Gibberellic Acid-Insensitive mRNA Transport in Pyrus

Wen-Na Zhang & Lei Gong & Chao Ma & Hai-Yan Xu &

Jian-Fang Hu & Takeo Harada & Tian-Zhong Li

Published online: 21 October 2011# Springer-Verlag 2011

Abstract Grafting is a common method for clonal propa-gation of fruit trees. Moreover, it serves as a mean to dealwith abiotic stress, adjust tree growth vigor, increase yield,and improve other fruit quality traits. Investigations ofrootstock and scion graft relationships have originallyfocused on anatomical and cellular development, nutrienttransport, and hormonal movement across graft union.Discovery of long distance transport of mRNA and smallRNAs in phloem tissues of rootstock and scion hasprovided new opportunities for investigation. In this study,we report on the endogenous transport of Gibberellic acidinsensitive (GAI) across graft union of a traditional localChinese pear cultivar, Pyrus bretschneideri cv. Yali (scion),and a wild Pyrus betulaefolia cv. Bunge (rootstock).Cleaved amplified polymorphic sequence analysis RT-PCR indicated that Pyrus-GAI can be transported within 4and 10 days after micro-grafting, and it can also betransported to a 10–50-cm tall scion of a 2-year-old graftingtree. To further investigate the transport capacity of Pyrus-GAI transcript, a 35S:pear (P. betulaefolia)-GAI transgenictobacco (Nicotiana tabacum L. cv. Samson.) was prepared

and grafted to wild-type tobacco. RT-PCR indicated thatsustained transmission of GAI mRNA through the graftunion occurred from the 15th day after grafting. The resultshave laid a foundation for improving rootstock andregulating the properties of scion in fruit trees by transgenictechnology.

Keywords Pyrus .GAI mRNA . Grafting . RNA transport .

Phloem

Introduction

Although major crops, such as rice, normally undergoseminal propagation, asexual propagation, such as grafting,rooting and layering, is important for perennial fruit trees(Westwood 1993). When scions are grafted onto rootstocks,not only do rootstocks confer support and adaptability forscion cultivars in different soil and climatic environments, butcertain rootstocks also endow advantageous properties onscions, such as dwarfing, desired physiological character-istics, early fertility, improved fruit quality, increased eco-nomic benefits, and so on (Soumelidou et al. 1994; Kambojet al. 1999a, b; Jensen et al. 2003, 2010; Khan et al. 2011; Suiet al. 2011). Therefore, it is important to study the relationshipbetween rootstock and scion of woody fruit trees.

The mechanism of physiological interactions between arootstock and a scion in fruit trees has been studied forsome time, but originally focused only on anatomicalinvestigations, nutrient transport, and hormonal move-ments. It remains largely unknown how rootstocks bringabout their effects on scions. Recent studies have suggestedthat certain mRNAs and small RNAs (miRNA and siRNA)can transport in plant vascular system in the form of RNA–protein complexes based on the Non-cell autonomous

Electronic supplementary material The online version of this article(doi:10.1007/s11105-011-0365-7) contains supplementary material,which is available to authorized users.

W.-N. Zhang : L. Gong : C. Ma : J.-F. Hu : T.-Z. Li (*)Laboratory of Fruit Cell and Molecular Breeding,China Agricultural University,Beijing 100193, People’s Republic of Chinae-mail: litianzhong1535@163.com

W.-N. Zhange-mail: wennafhxy@163.com

H.-Y. Xu : T. HaradaLaboratory of Plant Breeding and Genetics,Faculty of Agriculture and Life Science, Hirosaki University,Hirosaki 036-8561, Japan

Plant Mol Biol Rep (2012) 30:614–623DOI 10.1007/s11105-011-0365-7

pathway mechanism (Lucas et al. 1995; Ham et al. 2009).For example, transcripts of GAI (Haywood et al. 2005;Huang and Yu 2009), CmGAIP (Ruiz-Medrano et al.1999),PEP-Let6 (Kim et al. 2001; Kudo and Harada 2007), BEL-5(Banerjee et al. 2006) and CmPP16 (Xoconostle-Cazares etal. 1999) have been found transporting between rootstockand scion. Their transportation in grafting plants phloemaffects gene expression in target tissues and modulates plantdevelopment and morphogenesis (Doyle and Doyle 1990;Lucas et al. 1993; Lucas 1995; Goodenough et al. 1996;Gurdon et al. 1998; Clark 2001; Kim et al. 2001; Kragler etal. 2001; Haywood et al. 2005; Banerjee et al. 2006, 2009).So far, the only studies of long distance transport of mRNAin woody fruit trees that we are aware of are those byKanehira et al. (2009) and Xu et al. (2010), which reportedthat AUX/IAA14 and Gibberellic acid insensitive (GAI)transcripts in apple could transport between rootstock andscion via graft union, respectively.

In this study, in order to transform the transportablesignificant mRNA into rootstock of fruit tree and laterimprove rootstock and scion for fruit tree production, wehave investigated the endogenous Pyrus-GAI transcripttransportation in P. bretschneideri Yali–P. betulaefoliaBunge grafts, and the P. betulaefolia–GAI transcript as anexogenous RNA transportation in transgenic tobacco grafts.These results will lay a foundation for improving therootstock and regulating the targeted properties of the scionin fruit trees by fixed genes transformation.

Materials and Methods

Plant Materials

Pears (P. betulaefolia and P. bretschneideri) were subcul-tured on an MS medium (Murashige and Skoog 1962)containing 0.5 mg/L benzylaminopurine (BA) and 0.2 mg/Lindole acetic acid (IAA), whereas tobaccos (N. tabacum)were subcultured on an MS medium without any hormoneunder the following conditions: 25°C (day) and 23°C(night), under 14 and 10 h photoperiod, respectively, with85% relative humidity (RH) and 50 μmol m−2 s−1 photo-synthetically active radiation provided by cool whitefluorescent tubes. MS medium was 3.0% (w/v) sucrose,0.8% (w/v) agar (Difco Bacto, KS, USA), adjusted to pH5.9 with 1 M KOH, and sterilized by autoclaving.

Grafting

Micro-grafting with subcultured shoots of P. betulaefoliaand P. bretschneideri and their samplings was conductedaccording to Xu et al. (2010). Tissue samples werecollected every day from 1 to 10 days after grafting

(Fig. 1a). Ten individual samples were mixed for analysis.The controls were ungrafted P. betulaefolia, ungrafted P.bretschneideri and the mixture of P. betulaefolia and P.bretschneideri GAI cDNA.

Tissue from the phloem of a 50-cm tall, 2-year-old tree(P. bretschneideri as scion and P. betulaefolia as rootstock)(Fig. 3) was sampled every 10 cm along the scion stemfrom graft union site (Fig. 3a).

The tobacco micro-grafting experiment was performedexactly as described by Mallory et al. (2001). Briefly,tobaccos used for rootstocks were beheaded, removing allthe leaves, and a horizontal transverse was cut in the top ofthe stem. Scions were prepared by cutting the top 1.5 cm ofthe plant, removing all the leaves, and trimming the bottomof the stem into a wedge-shape. The plants used for bothrootstocks and scions were about 4 weeks old (Fig. 1b).The graft unions were wrapped with aluminum foil (10-mmwide×7-mm high), and the grafted tobaccos were grown inMS medium with 2.0 mg/L of indole butyric acid (IBA). Atleast three graft lines with ten grafts per line were preparedand sampled on a daily basis. New leaves appeared 5 daysafter grafting in the scion apex; therefore, tissue sampleswere collected on the 5th day, 15th day, 25th day, and 35thday after grafting. Grafting combinations, including 35 s:pBI 121/wild type (WT) and WT/35 s:pBI 121, and controlgrafts (including 35 s:pBI 121/35 s:pBI 121, WT/WT and35 s:P. betulaefolia (DL)]-GAI/35 s:P. betulaefolia (DL)-GAI) were also made. Due to slim micro-grafting tobaccolines, it was not possible to separate phloem from xylem;therefore, we sampled the whole stem of scion androotstock, as well as new leaves of the scions for analysis.

Cleaved Amplified Polymorphic Sequence Analysis

Total RNA was extracted from tissues of grafts using thecetyltrimethylammonium bromide method as described inChang et al. (1993) and then treated with RNase-freeDNaseI(Takara, Dalian, China). cDNA synthesis wasperformed with M-MLV (Promega, Madison, WI, USA)as described in Zambounis et al. (2011). RT-PCR productsamplified with the primers GAIha1f and GAIha2r (Table 1)were digested with HapII (Takara, Dalian, China) andseparated on a 3% agarose gel.

In situ hybridization

A 317-base pair (bp) fragment containing a specific sequenceregion was amplified using PCR and the primers GAIprobefand GAIprobef (Table 1) under the following conditions:initial denaturation at 94°C for 1 min followed by 35 cyclesof 94°C for 30 s, 55°C for 30 s, 72°C for 1 min, and a finalextension at 72°C for 7 min. Digoxigenin-labeled antisenseand sense RNA probes were made following the manufac-

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turer’s instructions of Digoxygenin RNA labeling kit (RocheApplied Science, Penzberg, Germany). Hybridization andimmunological detection of the hybridized probes wereperformed as described by Ma et al. (2011). The hybridiza-tion signals were observed and recorded by light microscopy(BX51 Olympus, Japan).

The segment between the apex and the base of P.betulaefolia stem (∼5-cm long) was used for in situhybridization (ISH). Segments were fixed in 4% parafor-maldehyde for 15 h at 4°C and then, dehydrated using

graded ethanol series containing 30%, 50%, 70%, 85% and95% ethanol. The materials were embedded in ParaplastPlus (Sherwood Medical, St. Louis, MO, USA). Ten-micrometer slices were cut with a microtome and mountedon glass slides.

Transgenic Construct

Experiment with transgenic construct was performedexactly as described by Hynes et al. (2003). The plantexpression vector pBI 121 (GenBank ID, AF485783)containing 35S CaMV promoter, resistant tab neomycinphosphotransferaseII (NPTII) and a nopaline synthase(NOS) 3’ transcriptional terminator sequence, was used forexpression studies in transgenic tobaccos. P. betulaefolia(DL)-GAI cDNA sequences were obtained by RT-PCRusing the primers XBAGAIF and SACGAIR listed inTable 1 under the following conditions: initial denaturationat 94°C for 1 min followed by 35 cycles at 94°C for 30 s,60°C for 30 s and 72°C for 2 min, then a final extension at72°C for 10 min. For DL-GAI mRNA plasmid constructs,the sense primer XBAGAIF contained XbaIsite at 5’ end,whereas the antisense primer SACGAIR contained SacIsiteat 3’ end. Amplified DL-GAI cDNA sequences werepurified and subcloned into PMD18-T simple vector(Takara, Dalian, China). T vector containing DL-GAIcDNA was digested with XbaI and SacI, and then insertedinto the same sites of pBI 121 vector without β-glucuronidase

Table 1 Primer sequences used in the current study

Primers Sequence (5’-3’)

GAIFW -TTGATTTCCGAGCCCTACCC

GAIRV -AACTCGGTCATCGCTCACTGA

GAIha1f -AACTCGGTCATCGCTCACTGA

GAIha2r -CAGTGGCCCGCTCTGATGCA

ActinF1 -CATACATGGCAGGCACATTG

ActinR1 -ATTGGAATGGAAGCTGCTGG

XBAGAIF -GCTCTAGATTGATTTCCGAGCCCTA

SACGAIR -GTCATCGCTCACTGAGAGCTCG

NPTIIF -CGGCTATGACTGGGCACAACAGACAAT

NPTIIR -AGCGGCGATACCGTAAAGCACGAGGAA

GAIprobef -TTAAAGCTTAAGCCGTCCAGCAGA

GAIprobeR -GCTCTAGAATACCCGTGAGCCGA

Fig. 1 Heterografting of Pyrusand tobacco. a P. bretschneideriscion on P. betulaefolia root-stock and sampling position. (1)Leaf of scion, (2) shoot tip ofscion, (3) phloem tissue fromscion stem, (4) graft-union, (5)phloem tissue from stock stem,(6) xylem tissue from stockstem. b Tobacco micro-grafting.c Separation of inner tissue(xylem) and outer tissue(phloem) of the scion stem.Scale bars=1 cm

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(GUS) gene (Fig. S4). The positive control construct waspBI 121 without β-glucuronidase (GUS).

Tobacco Transformation

Tobacco was transformed by the leaf disk method asdescribed in Mallory et al. (2003) and Viswanathan et al.(2011). Disks of plant tissue were cut from tobacco leavesand cultured at 25°C for 2 days (light) before soaked in asuspension of transgenic-carrying Agrobacterium bacterialstrain EHA105 cells. The leaf disks were incubated at 25°Cfor 2 days (dark) on the feeder plates and transferred to MSmedium with 500 mg/L cefalotin, 0.5 mg/L indoleaceticacid (IAA), 2.0 mg/L zeatin (ZT) which are used forinhibiting the growth of Agrobacterium agent. After 7–10 days of light culturing, the leaf disks were transferred toMS medium with 500 mg/L cefalotin and 100 mg/LKanamycin which act as selective agent. Calli formed fromthe leaf disks were separated. Shoots that grew from thecalli were transplanted to MS medium with 2.0 mg/L indolebutyric acid (IBA) to encourage root development.

Results

GAI Genes in P. bretschneideri and P. Betulaefoliaand Cleaved Amplified Polymorphic Sequence Analysis

According to DL-GAI (GenBank ID, JF304102) and YL-GAI (GenBank ID, JF304103) by National Center forBiotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/), GAI genes were RT-PCR amplified from cDNAof P. bretschneideri and P. betulaefolia using the specificprimers, GAIFW and GAIRV. Both genes encode polypep-tides of 634 amino acid residues within the N-terminal ofDELLA domain. They share 98.92% sequence identity with12 single nucleotide polymorphisms (SNPs) (Fig. S1).

A cleaved amplified polymorphisms sequence (CAPS)marker was developed in order to discriminate DL-GAI andYL-GAI transportation in P. bretschneideri and P. Betulaefoliagrafts using the restriction enzyme HapII. A 361-bp RT-PCRproduct amplified from DL-GAI cDNA (P. betulaefolia) wascleaved into three fragments of 24, 45 and 292 bp, whereasthat of YL-GAI cDNA (P. bretschneideri) was cleaved intofour fragments of 24, 44, 45 and 248 bp (Fig. 2; Fig. S2). RT–PCR restriction fragment length polymorphism (RFLP) ofrootstock and scion showed that all six tissues including thescion apex (Fig. 2, lane 2), new leaves of the scion (Fig. 2,lane 1), the phloem of P. bretschneideri (Fig. 2, lane 3), thegraft union (Fig. 2, lane 4) and the xylem of P. betulaefolia(Fig. 2, lane 6) exhibited unique restriction band patterns until4th day after grafting. However, the patterns of all tissuesexcept for the xylem of P. betulaefolia (Fig. 2, lane 6)

displayed mixed 24, 44, 45, 248 and 292 bp from 4th day to10th day after grafting. The result suggested that eitherrootstock or scion has incorporated fragments from its graftingpartner (Fig. 2), though the band of 24 bp was too small todisplay, and the bands of 44 and 45 bp were too close todistinguish on 3% agarose gel. It indicated that Pyrus-GAIgene, as endogenous mRNA, can simultaneously transportback and forth from the tissue cultured P. betulaefolia to P.bretschneideri within the 4th to 10th day after grafting.

To determine whether Pyrus-GAI mRNA can betransported within a 50-cm tall adult tree as well as thedistance, samples were collected from a 2-year-old tree (P.bretschneideri as scion and P. betulaefolia as rootstock)(Fig. 3a) for the same experiment (Fig. 3b). Pyrus-GAItranscripts were PCR amplified and analyzed with CAPSmarker to discriminate SNPs. RT-PCR cDNA-RFLP.Results showed that DL-GAI mRNA of a 50-cm tall treecan transport to the phloem of scion in 10–40 cm awayfrom the graft union, and YL-GAI mRNA can transport tothe rootstock (Fig. 3b).

To confirm that the cDNAs were not contaminated bygenomic DNA (gDNA), actin genes of Pyrus (Pyrus ESTaccession no. ab190176) were amplified with ActinF1 andActinR1. The result showed that gDNA product was longerthan that of cDNA because of the intron, indicating that theprepared DL-GAI and YL-GAI cDNA was not contaminatedwith gDNA (Fig. S3).

In Situ hybridization of DL-GAI

In order to identify the location of DL-GAI transcripts, ISHwas conducted on cross-sectioned P. betulaefolia. From thecell size and array pattern, it was possible to identifyphloem, which was located near xylem (Fig. 4a, b). DL-GAI mRNA was detected in phloem by an antisense probe(Fig. 4c, d); however, no signal was recorded either fromxylem or when utilizing a sense probe (Fig. 4a, b). Thepositive signal sizes are derived from phloem sieve tube–companion cells (SE-CC).

Analysis of DL-GAI mRNA Transport in TransgenicTobaccos

To evaluate possible application of exogenous GAI mRNAon transgenic rootstocks of fruit trees later, 35S:DL-GAIcDNA was initially transformed into tobaccos. Six lines ofthe transgenic tobacco were identified by RT-PCR withprimers GAIF/GAIR and NPTIIF/NPTIIR, respectively(Fig. 5d). The phenotypes and growth indicators of 35 s:DL-GAI cDNA transgenic tobaccos were shown obviouslyshorter internode and dwarfish comparing to wild types(WT) (Fig.5a, b, e), hence it was confirmed that 35S:DL-GAI cDNA had been transformed into tobaccos.

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In order to detect DL-GAI mRNA transmission fromrootstock to scion in transgenic tobaccos, grafting wasconducted using 35S:DL-GAI cDNA transgenic line as therootstock and WT as the scion. NPTII gene and GAI cDNAwere amplified from scions by specific primer pairsNPTIIF/NPTIIR and GAIFW/GAIRV, respectively. It sug-gested that DL-GAI mRNA and NPTII gene are all able to

simultaneously transport between rootstock and scion15 days after grafting. Then when the pBI 121(withoutGUS gene) transgenic tobacco line was grafted with WT,NPTII gene was observed only in the rootstock, but not inthe scion (Fig. 6a). Therefore, it is confirmed that neitherpBI 121 vector nor NPTII gene is able to transport upwardon their own.

Fig. 2 CAPS analysis on micro-grafting Pyrus. Scion (P. bretschneideri),(1) Leaf of scion, (2) Shoot tip of scion, (3) Phloem tissue fromscion stem, (4) graft union; rootstock (P. betulaefolia), (5) phloemtissue from stock stem, (6) xylem tissue from stock stem. CK

control, D ungrafted P. betulaefolia, M’ ungrafted mixture cDNA ofP. bretschneideri and P. betulaefolia, Y ungrafted P. bretschneideri,M DNA ladder 2000, 1–10 d 1–10 days after grafting

Fig. 3 GAI mRNA transport in a2-year-old grafted tree of P.bretschneideri scion on P. betu-laefolia. a A 50-cm tall 2-year-oldgrafted tree of P. bretschneideriscion on P. betulaefolia wassampled from every 10 cm apartfrom the graft union (SCP, 1–5),the graft union (J) and therootstock phloem (STP). Blackarrowhead shows sampledpositions. b Cleaved amplifiedpolymorphic sequence (CAPS)analysis on RT-PCR products ofthe scion and the rootstock. MDNA Ladder 2000, CK control, Dungrafted P. betulaefolia, M’ungrafted mixture cDNA of P.bretschneideri and P. betulaefolia,Y ungrafted P. bretschneideri. cSeparation of inner tissue (xylem)and outer tissue (phloem, SCP) ofthe stem, the phloem (SCP)stripped from the samples wasused as the materials for theRT-PCR experiments.Scale bars=1.5 cm

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To detect DL-GAI mRNA transmission from scion torootstock in transgenic tobaccos, 35S:pBI 121(without GUSgene) and DL-GAI cDNA transgenic tobaccos as the scionswere both grafted with WT. As shown in Fig. 6b, NPTIIgene and DL-GAI mRNA was observed to be existed onlyin the scions, but not in the rootstocks, which indicates thatboth pBI 121 vector and NPTII gene also cannot transportdownward on their own. Nonetheless, within DL-GAIcDNA transgenic tobacco as the scion grafts, NPTII geneand DL-GAI can simultaneously transport between scionand rootstock on 15 days after grafting (Fig. 6b) like theprevious case.

To avoid the calli of graft union blocking the macro-molecules transportation in plant vascular system, and toavoid amplifying tobacco GAI cDNA by the specific primerpairs GAIFW/GAIRV in Pyrus, self-grafting was conductedwith WT, 35S:pBI 121 (without GUS gene) and DL-GAIcDNA transgenic tobaccos, to be used as controls.Transmission of NPTII and GAI cDNA can be bothdetected in scion’s new leaves (Fig. 6c).

In addition, the grafted wild types/35 s: DL-GAI tobaccowas dwarfish compared to the ungrafted wild type 50 daysafter grafting (Fig. 5c, e), thus it was confirmed that 35S:

DL-GAI cDNA had transported from transgenic rootstocksinto WT scions.

All these results led to a conclusion that Pyrus-GAIas an exogenous mRNA can transport within a transgenicgraft system from rootstock to scion, and vice versa,simultaneously.

Discussion

Endogenous GAI mRNA Transport Between the Rootstockand the Scion in Phloem of Pyrus

Grafting is a cultivation method in fruit tree production, whichwas used for improving varieties and acquiring excellentcharacteristics, such as dwarfism and resistance, so as toincrease fruit yield. The phenotype, growth habit and fruitquality of fruit trees will be changed when the same scion isgrafted onto different rootstocks (Samanci and Uslu 1995;Jensen et al. 2003; Smaka et al. 2010). Therefore, it is ofgreat interest to study the complicated interactions betweenrootstock and scion of fruit trees for strengthening resistance,increasing fruit yield and quality (Li et al. 2011).

Fig. 4 In situ hybridization oftransverse sections of P.betulaefolia stem. Sections werehybridized with a digoxigenin-labeled 317-base mRNA copyof a sequence of DL-GAI. a, bSense probe. c, d Antisenseprobe. The boxed area in a andc is enlarged in b and d,respectively. The arrows in dindicate DL-GAI mRNA signals.P phloem, X xylem.Scale bars=50 μm

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Due to the abundance in phloem sap, simple phloemseparation procedure and well-established transformationplatform, the transportation of mRNA has already beenreported in Arabidopsis (Deeken et al. 2008), pumpkin(Gómez et al. 2005), cucumber (Ruiz-Medrano et al. 1999),tomato (Kim et al. 2001; Haywood et al. 2005), potato(Banerjee et al. 2006; Kudo and Harada 2007; Schmitt et al.2008; Banerjee et al. 2009). However, woody fruit trees havelittle or less phloem eluate, elementary transformation andregeneration technology; therefore, it is difficult to study fruittree propagation by grafting (Soria-Guerra et al. 2011). Ourcurrent study aims at improving rootstock of fruit tree bymRNA transportation from rootstock to scion, which will thenlead to the regulation and control of various scion characters.

In this research, tissue cultured P. bretschneideri Yaliand P. betulaefolia Bunge were used for simulating fruit

tree growth in the field. Pyrus-GAI mRNA can betransported between the rootstock and the scion within 4–10 days after grafting in dual directions (Fig. 2). Phloem,especially SE-CC is the primary tunnel for nutritiontransportation in plants. Sink-to-source transportation inphloem occurs from the tip down the leaf axis toward thepetiole. In situ hybridization showed accumulation ofPyrus-GAI mRNA in the phloem of woody fruit tree(Fig. 4). Moreover, Pyrus-GAI mRNA can be transportedfrom source to distant sink tissues in phloem of scionwithin an adult tree, except for the scion apex (Fig. 3b, lane5). The character of GAI mRNA transportation was similarto Cmgaip mRNA in transgenic tomatoes, which waspredominantly transported and accumulated in strong andupper sink tissues (Haywood et al. 2005). It suggested thatsource-to-sink transportation of mRNA in phloem possibly

Fig. 5 Identification of transgenic tobaccos. a Transplanted N.tabacum wild type. b Transplanted transgenic N. tabacum of 35S:DL-GAI cDNA. c Wild-type tobacco (scion) and 35 s:DL-GAI cDNA(rootstock) grafts. The arrow indicated the graft union. Scale bars=50 cm. d RT-PCR identification of NPTII gene and DL-GAI cDNA in35 s:DL-GAI cDNA transgenic tobaccos. NPTII (NPTIIF/NPTIIR),NPTIIgenes were amplified by primer pairs NPTIIF/NPTIIR. DL-GAI(GAIFW/GAIRV), DL-GAI cDNA was amplified by primer pairsGAIFW/GAIRV. pBI 121, pBI 121(without GUS gene) transgenic

tobaccos were used as positive controls. WT wild-type tobaccos wereused as negative control. Lanes 1–6, 35 s:DL-GAI cDNA transgenictobacco lines. e The height (centimeters) and internode length(centimeters) of wild-type tobaccos, 35 s:DL-GAI transgenic tobaccosand wild-type/35 s:DL-GAI grafts tobacco at eight-leaf -stage on50 days after grafting. Results are shown as mean (error barsrepresent standard error) height (centimeters) of three lines in eachtobacco

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Fig. 6 RT-PCR of NPTII to identify DL-GAI mRNA transport intransforms grafts. a WT tobaccos were used as scions and thetransgenic 35 s:pBI 121 without GUS transgenic tobaccos asrootstocks (WT/pBI 121), and WT tobaccos were used as scions andthe DL-GAI cDNA transgenic tobaccos as rootstocks (WT/p 35 s:DL-GAI). b The opposite grafts combination to the a. WT tobaccos wereused as rootstocks and the transgenic 35 s:pBI 121 without GUStransgenic tobaccos as scions (pBI 121/WT), and WT tobaccos were

used as rootstocks and the DL-GAI cDNA transgenic tobaccos asscions (p 35 s:DL-GAI/WT). 1–3, grafted lines. L scion new leaves,SC scion stem, ST rootstock stem. c Control grafts. WT, the DL-GAIcDNA transgenic tobaccos (35 s:DL-GAI), and the transgenic 35 s:pBI121 without GUS gene (pBI 121) grafted by themselves respectively.NPTII and DL-GAI cDNA were amplified by primer pairs NPTIIF/NPTIIR and GAIFW/GAIRV, respectively on the 5th day (5 d), 15thday (15 d), 25th day (25 d) and 35th day (35 d) after grafting

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reflects a complex process. Due to root pressure andtranspiration pull, the macromolecules, such as mRNA,can be initially transported from the source to nearby sinkand then reached to the further locations in phloem flows.The number of signaling macromolecules also will bereduced from the stock. Similarly, the level of Pyrus-GAImRNA presented in the scion apex gradually diminishedduring its growth, so it was not suspected that GAI mRNAwas not detected in scion apex of an adult tree (Fig. 3b).

Exogenous DL-GAI mRNATransport in Transgenic Tobaccos

To evaluate exogenous Pyrus-GAI mRNA transportation intransgenic grafts and establish a theoretical foundation forthe future analysis on improving the rootstock of fruit treesby transgenic technology, the tobacco was used astransgenic model plant in our study.

NPTII gene in pBI 121 did not exhibit independent dualtransport when only a vector (pBI 121) is used; however, itcan be transported when DL-GAI is fused into pBI 12115 days after grafting (Fig. 6a, b). Figure 3 showed the dualtransport of Pyrus-GAI even without any vector, indicatingthat the presence of a vector is not a prerequisite conditionfor mRNA transport. Similarly, it has been reported that FTand Cmgaip in the rootstock do not move to scion part inapple (Tränkner et al. 2010) and tomato (Haywood et al.2005). Thus, the transport of mRNA may depend on somespecial condition, such as homodomain (HD) of the mRNAcoding protein (Kim et al. 2001) and the hairpin structure ofthe mRNA (Huang and Yu 2009). It is possible to concludethat DL-GAI mRNA can be transported like an “engine”,which can carry or drive other sequences such as NPTIIgene or pBI 121 vector transportation. The same conclusionhad been proven by FT mRNA in TCV viral vector (Li etal. 2009). Consequently, we will continue to study Pyrus-GAI mRNA transportable capacity of carrying other genesin future researches. These results confirmed the possibilityof sustained exogenous mRNA transport in transgenicgrafts in dual directions, proposing a new means ofregulating the properties of the plants (Fig. 5c, e).

Based on the current studies on pear stock regeneration,it is possible to apply transportable mRNA to fruit treegrafting. It is of great interest to make transgenic rootstocka transmissible mRNA with significant agronomic traits,such as self-compatibility, reduced juvenility, and antivirusand dwarf phenotypes (Rivera-Vega et al. 2011). Thiswould enable the scion to be improved through transpor-tation of mRNA from transgenic rootstock, which couldalso avoid the controversy on transgenic fruits. In terms ofPurus-GAI mRNA in this study, we could transform otherdual-direction transmissible mRNAs into the interstocks offruit trees, so as to modulate the agronomic traits andimprove the development and adaptability of scions and

rootstocks. Such a study will lay a foundation forapplication of RNA transport and innovating propertiesfor fruit tree.

Acknowledgments We would like to thank Song-ling Bai (HirosakiUniversity, Japan) and Ai-de Wang (Cornell University, USA) for thetechnical assistance. This work was supported by the DoctoralProgram Special Fund of the Ministry of Education in China(2010000811036), National Natural Science Foundation of China(30871697) and Beijing Natural Science Foundation (6102017).

References

Banerjee AK, Chatterjee M, Yu Y, Suh SG, Wa M, Hannapel DJ(2006) Dynamics of a mobile RNA of potato involved in a longdistance signaling pathway. Plant Cell 18:3443–3457

Banerjee AK, Lin T, Hannapel DJ (2009) Untranslated regions of amobile transcript mediate RNA metabolism. Plant Physiol151:1831–1843

Chang S, Puryear J, Cairney J (1993) A simple and efficientmethod for isolating RNA from pine trees. Plant Mol BiolRep 11:113–116

Clark SE (2001) Cell signalling at the shoot meristem. NatureReviews Mol Cell Biol 2:276–284

Deeken R, Ache P, Kajahn I, Klinkerberg J, Bringmann G, Hedrich R(2008) Identification of Arabidopsis thaliana phloem RNAsprovides a search criterion for phloem-based transcripts hidden incomplex datasets of microarray experiments. Plant J 55:746–759

Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue.Focus 12:13–15

Gómez G, Torres H, Pallás V (2005) Identification of translocatableRNA-binding phloem proteins from melon, potential componentsof the long-distance RNA transport system. Plant J 41(1):107–16

Goodenough DA, Goliger JA, Paul DL (1996) Connexins, connexons,and intercellular communication. Annu Rev Biochem 65:475–502

Gurdon JB, Dyson S, Johnson D (1998) Cells’ perception of positionin a concentration gradient. Cell 95:159–162

Ham B-K, Jeri L, Brandom, Xoconostle-Cázares B, Ringgold V,Lough TJ (2009) A polypyrimidine tract binding protein,pumpkin RBP50, forms the basis of a phloem-mobile ribonu-cleoprotein complex. Plant Cell 21:197–215

Haywood V, Yu TS, Huang NC, Lucas WJ (2005) Phloem longdistance trafficking of Gibberellic Acid-Insensitive RNA regu-lates leaf development. Plant J 42:49–68

Huang NC, Yu TS (2009) The sequence of Arabidopsis GA insensitiveRNA constitute the motifs that are necessary and sufficient forRNA long-distance trafficking. Plant J 59:921–929

Hynes LW, Peng JR, Richards DE, Harberd NP (2003) Transgenicexpression of the Arabidopsis DELLA proteins GAI and gaiconfers altered gibberellin response in tobacco. Transgenic Res12:707–714

Jensen PJ, Rytter J, Detwiler EA, Travis JW, Mcnellis TW (2003)Rootstock effects on gene expression patterns in apple treescions. Plant Mol Biol 493:493–511

Jensen PJ, Makalowska I, Altman N, Fazio G, Praul C, Naximova SN,Crassweller RM, Travis JW, Mcnellis TW (2010) Rootstockregulated gene expression patterns in apple tree scion. Tree GenetGenomes 6:57–72

Kamboj JS, Blake PS, Quinlan JD, Baker DA (1999a) Identificationand quantitation of GC-MS of zeatin and zeatin riboside in xylemsap from rootstock and scion of grafted apple trees. Plant GrowthRegul 28:199–205

622 Plant Mol Biol Rep (2012) 30:614–623

Kamboj JS, Browning G, Blake PS, Quinlan JD, Baker DA (1999b)GC-MS-SIM analysis of abscisic acid and indole-3-acetic acid inshoot bark of apple rootstocks. Plant Growth Regul 28:21–27

Kanehira A, Yamada K, Iwaya T, Tsuwamoto R, Kasai A,Nakazono M, Harada T (2009) Apple phloem cells containsome mRNAs transported over long distances. Tree GenetGenomes 6:635–642

Khan MA, Zhao Y-F, Korban SS (2011) Molecular mechanisms ofpathogenesis and resistance to the bacterial pathogen Erwiniaamylovora, causal agent of fire blight disease in rosaceae. PlantMol Biol Rep. doi:10.1007/s11105-011-0334-1

Kim M, Canio W, Kessler S, Shinha N (2001) Developmental changesdue to long-distance movement of a homeobox fusion transcriptin tomato. Science 293:287–289

Kragler F, Yoo BC, Lucas WJ (2001) RNA as a long-distanceinformation macromolecule in plants. Mol Cell Biol 2:849–856

Kudo H, Harada T (2007) A graft-transmissible RNA from tomatorootstock changes leaf morphology of potato scion. HortSci42:225–226

Li C-Y, Zhang K, Zeng X-W, Jackson S, Zhou Y, and Hong Y-G(2009) A cis element within flowering locus T mRNA deter-mines its mobility and facilitates trafficking of heterologous viralRNA. J Virology (83):3540–3548

Li F, Lei H-J, Zhao X-J, Tian R-R, Li T-H (2011) Characterization ofthree sorbitol transporter genes in micropropagated apple plantsgrown under drought stress. Plant Mol Biol Rep. doi:10.1007/s11105-011-0323-4

Lucas WJ (1995) Plasmodesmata: intercellular channels for macro-molecular transport in plants. Curr Opin Cell Biol 7:673–680

Lucas WJ, Ding B, Van der Schoot C (1993) Plasmodesmata and thesupracellular nature of plants. New Phytol 125:435–476

Lucas WJ, Bouché-Pillon S, Jackson DP, Nguyen L, Baker L, Ding B,Hake S (1995) Selective traffic king of KNOTTED1 homeo-domain protein and its mRNA through plasmodesmata. Science270:1980–1983

Ma J, He Y-H, Wu C-H, Liu H-P, Hu Z-Y, Sun G-M (2011) Cloningand molecular characterization of a SERK gene transcriptionallyinduced during somatic embryogenesis in Ananas comosus cv.Shenwan Plant Mol Biol Rep. doi:10.1007/s11105-011-0330-5

Mallory C, Ely L, Smith TH, Marathe R, Anandalakshmi R, FagardM, Vaucheret H, Pruss G, Bowman L, Vance VB (2001) HC-Prosuppression of transgene silencing eliminates the small RNAs butnot transgene methylation or the mobile signal. Plant Cell13:571–583

Mallory C, Mlotshwa S, Bowman LH, Vance VB (2003) The capacityof transgenic tobacco to send a systemic RNA silencing signaldepends on the nature of the inducing transgene locus. Plant J35:82–92

Murashige T, Skoog F (1962) A revised medium for rapid growth andbioassays with tobacco tissue cultures. Physiol Plant 15:473–497

Rivera-Vega L, Mamidala P, Koch JL, Mason ME, Mittapalli O (2011)Evaluation of reference genes for expression studies in ash

(Fraxinus spp.) Plant Mol Biol Rep doi. 10.1007/s11105-011-0340-3

Ruiz-Medrano R, Xoconostle-Cázares B, Lucas WJ (1999) Phloemand long-distance transport of CmNACP mRNA: implications forsupracellular regulation in plants. Development 126:4405–4419

Samanci H, Uslu I (1995) Rootstock adaptation and affinity studies onRazaki and Muskule grape varieties. ACHRI p 30

Schmitt B, Stadler R, Sauer N (2008) Immunolocalization ofsolanaceous SUT1 proteins in companion cells and xylemparenchyma: new perspectives for phloem loading and transport.Plant Physiol 148:187–199

Smaka A, Li X-Y, Heikelt C, Welander M, Zhu L-H (2010)Effects of transgenic rootstocks on growth and developmentof non-transgenic scion cultivars in apple. Transgenic Res 19(6):933–948

Soria-Guerra RE, Rosales-Mendoza S, Gasic K, Wisniewski ME,Band M, Korban SS (2011) Gene expression is highly regulatedin early developing fruit of apple. Plant Mol Biol Rep.doi:10.1007/s11105-011-0300-y

Soumelidou K, Morris DA, Battey NH, Barnett JR, John P (1994)Auxin transport capacity in relation to the dwarfing effect ofapple root-stocks. J Hort Sci 69:719–725

Sui J-M, Guo B-T, Wang J-S, Qiao L-X, Zhou Y, Zhang H-G, Gu M-H,Liang G-H (2011) A new GA-insensitive semidwarf mutant of rice(Oryza sativa L.) with a missense mutation in the SDG Gene. PlantMol Biol Rep doi. 10.1007/s11105-011-0321-6

Tränkner C, Lehmann S, Hoenicka H, Hanke M-V, Fladung M,Lenhardt D, Dunemann F, Gau A, Schlangen K, Malnoy M,Flachowsky H (2010) Overexpression of an FT-homologous geneof apple induces early flowering in annual and perennial plants.Planta 232:1309–1324

Viswanathan A, Kuriakose B, Bharadwaj S, Thomas G (2011)Expression of Aprotinin in anther causes male sterility in tobaccovar Petit havana. Plant Mol Biol Rep. doi:10.1007/s11105-011-0288-3

Westwood MN (1993) Hormones and growth regulators. In: WestwoodMN (ed) Temperate-zone pomology, physiology and culture, 3rd ed.Timber Press Inc, Portland, pp 364–381

Xoconostle-Cazares B, Xiang Y, Ruiz-Medrano R, Wang HL, MonzerJ, Yoo BC, McFarland KC, Franceschi VR, Lucas WJ (1999)Plant paralog to viral movement protein that potentiates transportof mRNA into the phloem. Science 283:94–98

Xu H-Y, Zhang W-N, Li M-F, Harada T, Han Z-H, Li T-Z (2010)Gibberellic acid insensitive mRNA transport in both directionsbetween stock and scion in Malus. Tree Genet Genomes 6:1013–1019

Zambounis AG, Kalamaki MS, Tani EE, Paplomatas EJ, Tsaftaris AS(2011) Expression analysis of defense-related genes in cotton(Gossypium hirsutum) after Fusarium oxysporum f. sp. vasinfec-tum infection and following chemical elicitation using a salicylicacid analog and methyl jasmonate. Plant Mol Biol Rep doi.10.1007/s11105-011-0335-0

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