Long-distance transport of Gibberellic Acid Insensitive mRNA in Nicotiana benthamiana
© Xu et al.; licensee BioMed Central Ltd. 2013
Received: 3 November 2012
Accepted: 10 October 2013
Published: 21 October 2013
The Gibberellic Acid (GA) signal is governed by the GAI (Gibberellic Acid Insensitive) repressor, which is characterized by a highly conserved N-terminal DELLA domain. Deletion of the DELLA domain results in constitutive suppression of GA signaling. As the GAI transcript is transportable in phloem elements, a Δ-DELLA GAI (gai) transgenic stock plant can reduce the stature of a scion through transport of gai mRNA from the stock. However, little is known about the characteristics of a scion on a gai stock.
Arabidopsis Δ-DELLA GAI (gai) was fused with a T7 epitope tag and expressed under the control of a companion cell-specific expression promoter, Commelina yellow mottle virus promoter (CoYMVp), to enhance transport in the phloem. The CoYMVp:Atgai-T7 (CgT) transgenic Nicotiana benthamiana exhibited a dwarf phenotype and lower sensitivity to GA enhancement of shoot stature. A wild-type (WT) scion on a CgT stock contained both Atgai-T7 mRNA and the translated product. Microarray analysis to clarify the effect of the CgT stock on the gene expression pattern in the scion clearly revealed that the WT scions on CgT stocks had fewer genes whose expression was altered in response to GA treatment. An apple rootstock variety, Malus prunifolia, integrating CoYMVp:Atgai moderately reduced the tree height of the apple cultivar scion.
Our results demonstrate that Atgai mRNA can move from companion cells to sieve tubes and that the translated product remains at the sites to which it is transported, resulting in attenuation of GA responses by reducing the expression of many genes. The induction of semi-dwarfism in an apple cultivar on root stock harbouring Atgai suggests that long-distance transport of mRNA from grafts would be applicable to horticulture crops.
KeywordsGAI mRNA Transport Grafting Dwarf Phloem
The importance of gibberellins (GAs) to angiosperm growth regulation has been demonstrated by the phenotype of GA-deficient mutants. The GA-deficient Arabidopsis thaliana ga1-3 mutant that lacks ent-kaurene synthetase A, an enzyme in the GA biosynthesis pathway, exhibits a characteristic severe dwarf phenotype . Mutants such as ga1-3 are GA-sensitive dwarf mutants that have been observed in a number of different plant species and typically carry recessive mutations that reduce the activity of GA biosynthesis enzymes . Further molecular characterization of various GA response mutants led to the discovery of the GID1 (GIBBERELLIC INSENSITIVE DWARF1) and DELLA proteins, which are key components of the molecular GA-GID1-DELLA mechanism that enables plants to respond to GA . Genetic and molecular studies have identified the GA receptors and several positive and negative components of the GA signaling cascade [4, 5]. Among them, the three major players are the GA receptors, the DELLA repressor proteins, and the F-box proteins that control the stability of DELLA proteins. Ueguchi-Tanaka et al.  demonstrated that GID1 is a soluble GA receptor in rice (Oryza sativa). Discovery of the molecular identity of the endogenous plant GA-opposable growth-inhibitory factor resulted from molecular cloning of the genes encoding what are now known as the DELLA proteins.
The Arabidopsis gai mutation confers dominant, GA-insensitive dwarfism [7, 8]. An insertional mutagenesis approach has facilitated the molecular cloning of gai via isolation of a Ds transposon-inactivated allele . The gai open reading frame carries a small in-frame deletion mutation and thus encodes an altered product, a mutant gai protein that lacks a 17-amino-acid segment, now known as the DELLA domain, named after its first five amino acids. Molecular genetic analysis of GA-insensitive dwarf mutants has also identified an F-box protein (SLY1) that is part of a DELLA-interacting E3 ubiquitin ligase that interacts with a C-terminal region of the DELLA protein [10–12] and targets DELLAs for breakdown by the proteasome. DELLA proteins are thought to repress plant growth, and gibberellins promote growth by overcoming the repressive effects of these proteins. The Arabidopsis gai-1 mutant has a 51-bp deletion that encodes part of the conserved DELLA domain. As mentioned above, the Δ-DELLA form of GAI acts as a gain-of-function mutant that can inhibit some components of the GA signaling pathway . Expression of Arabidopsis gai in rice yields a dwarf phenotype, suggesting that GAI is sufficiently conserved between plant families to allow it to function .
Haywood et al.  have reported the long-distance delivery of RNA for the Arabidopsis ΔDELLA-gai (Atgai) genes. In grafting experiments, they demonstrated that the gai transcript specifically entered functional sieve elements and induced a highly reproducible change of leaf phenotype in tomato when Atgai mRNA was transported into the tomato shoot apex . Long-distance transport of the GAI transcript in woody plants (Malus and Pyrus) has also been demonstrated [15, 16]. Ham et al.  reported that the polypyrimidine tract binding motif within the GAI mRNA is involved in the formation of a mobile ribonucleoprotein complex, and proposed the presence of motifs that are necessary and sufficient for long-distance trafficking of the GAI transcript. Furthermore, Huang and Yu  reported that the trafficking of GAI RNA is mediated by specific RNA motifs existing among coding sequences and the 3′-untranslated regions.
Experiments using a grafting system have provided long-distance transport of several transcripts across a graft union, such as CmPP16 (encoding a 16-kDa Cucurbita maxima phloem protein) , CmNACP (Cucurbita maxima non-cell-autonomous protein) , PFP (pyrophosphate-dependent phosphofructokinase) -LeT6[21, 22], StBEL5 (Solanum tuberosum BEL1-like transcription factor) [23, 24] and AUX/IAA14[25, 26]. However, no details of the molecular mechanism involved, especially the physiological function of the GAI mRNA transport system, have been clarified. In the present study, we characterized GAI mRNA transport through phloem using Atgai transgenic tobacco as the experimental material. The results proved that a WT scion on Atgai rootstock contained the Atgai protein and that its growth reflected attenuation of the expression of many GA response genes.
Atgai transgenic tobacco exhibits dwarfism and lower sensitive to GA3
CgTrootstock affects WT scion growth
Transport of AtgaimRNA through the graft union
Detection of Atgai-T7 protein in the scion of the WT/CgTcombination
Attenuation of the GA response of the WT scion on CgTstock revealed by microarray
Numbers of genes exhibiting different degrees of GA response
Class (degree of change)
Numbers of genes
*WT/WT(GA) to *WT/WT
*WT/ CgT(GA) to *WT/WT
A (<20 ~ ≤100)
B (<10 ~ ≤20)
C (<5 ~ ≤10)
D (<2 ~ ≤5)
E (<0.5 ~ ≤2)
F (>0.5 ~ ≥0.4)
G (>0.4 ~ ≥0.3)
H (>0.3 ~ ≥0.2)
I (>0.2 ~ ≥0.01)
Atgaiapple rootstock reduces the stature of the scion cultivar
Lack of the DELLA domain of GAI results in constitutive activation of a mutant growth inhibitor whose genetically dominant action can no longer be opposed by GA. Since gai is a semi-dominant and gain-of-function mutation , integration of gai into WT results in a semi-dwarf phenotype, and this has contributed to the Green Revolution . The stature of several crops, including rice , Chrysanthemum, tobacco , and apple , has been reduced by transformation with ΔDELLA-GAI (gai), as is the case for the Arabidopsis gai-1 allele. In all cases, ΔDELLA-GAI (gai) was expressed under the control of CaMV 35S (Cauliflower Mosaic Virus promoter 35S).
On the other hand, GAI mRNA is able to move through phloem, and is considered to function at the site(s) to which it has been transported [14, 18]. As plasmodesmata connect the functional enucleate sieve elements of the phloem to their neighboring companion cells , this pathway is considered to allow the selective entry of information macromolecules into the phloem translocation stream. In this study, the Atgai transgene was delivered via the promoter of a plant virus, CoYMV, which is expressed strongly only in companion cells . Intriguingly, the CgT transgenic tobacco plants also exhibited semi-dwarf phenotypes, as was the case when the 35S promoter was used, indicating that expression of Atgai only in companion cells can induce a definite dwarf phenotype. Therefore, it is suggested that the GAI transcript in companion cells acts non-cell-autonomously to regulate the growth of plants. Furthermore, the less sensitive GA response of the WT scion on CgT rootstock strongly indicated the non-cell-autonomous effect of the GAI transcript. In higher plants the system of GAI mRNA transport through the phloem might function to integrate growth modalities among plant organs . In our experiments, not only the integrated Atgai but also the extant N. benthamiana GAI were considered to be transported by the same molecular mechanism. Detailed analysis of the transport of both gene transcripts via the graft union would be expected to clarify the physiological role of GAI mRNA long-distance movement.
Approximately one-fourth of the grafts we examined did not show the RT-PCR product derived from the gai mRNA transported from the stock. Long-distance transport of RNA in sieve tubes appears to be mediated by RNA-binding proteins. Ham et al.  identified RNA-binding proteins involved in mRNA transport, and proposed a model in which a ribonucleoprotein complex moves in the phloem. It is clear that RNA also binds to chaperone proteins for stability and delivery to target tissues . As a matter of course, such large complexes must pass through the graft union, where vascular bundles are developed in the callus (Additional file 6). De novo passage through the sieve tube tends to be unorthodox, showing features such as a winding path, thus disrupting the passage of large ribonucleoprotein complexes, and in extreme cases such complexes would become clogged. Since the conductance of a vessel is proportional to the fourth power of the vessel radius (Hagen-Poiseulle law), a slightly reduced diameter would pose an obstacle to passage. On the other hand, grafting of many horticultural crops is a well-developed technology, and this phenomenon in such plants might be less problematic, suggesting that they can establish near-complete connection of the sieve tube at the graft union as the growth progressed.
No other report has identified the translated protein derived from the transported gai mRNA in grafting experiments. In the WT scion on the CgT stock, the gai protein fused with the T7-tag peptide was clearly detectable. Its amount was considered to be approximately one-third of that in the CgT stock, suggesting effective translation of the gai mRNA in the scion. However, there is a possibility that the protein might have been translated in companion cells and then moved through the graft union. For clarification of this issue, a high-accuracy experiment to detect the GAI protein in sieve elements would be necessary.
Our present microarray experiment was also the first of its kind to investigate the long-distant distance transport of mRNA in grafted plants. The overall results clearly revealed that the WT scions on CgT stocks had fewer genes whose expression was altered in response to GA treatment than in the WT scions on WT stocks. It is known that GA has pronounced effects on overall reproductive growth from flowering to fruiting . Therefore, although we succeeded in growing a dwarf apple tree using the Atgai rootstock, the effect of this manipulation on subsequent fruit development will require careful investigation. However, semi-dwarf cultivation of many crops might be feasible using Atgai-expressing rootstock. Furthermore, the application of not only GAI but also other phloem-transportable mRNAs [21, 23, 26] might become possible by means of grafting. Finally, the present results imply that a non-genetically modified scion is capable of improvement by a genetically modified rootstock, and consequently, the fruits of the plant would not contain the inserted DNA sequence. Grafting using gene-modified rootstock is expected to become a focus of interest as an innovative approach to agriculture .
This study has demonstrated for the first time that transgenic stock expressing a ΔDELLA-gai gene by the companion cells specific-promoter is able to transfer the gai-mRNA to a wild-type scion. Furthermore, this study provides the first evidence that the translated product of the gai mRNA is present in the scion. In addition, microarray data clearly indicated that many GA-responsive genes in the wild-type scion on ΔDELLA-gai stock show attenuated responses.
Plant materials and growth conditions
Nicotiana benthamiana was grown on MS  agar (Wako Pure Chemical Industries Ltd., Osaka, Japan) plates in a Petri dish or a glass bottle. Grafted tobacco plants were cultivated on soil in a pod. Hydroponic culture was also performed using a styrofoam plate floating on hydroponic solution (Otsuka House Nos. 1 and 2, Otsuka Chemical Co., Osaka, Japan). The cultures were incubated at 24°C with a 16:8-h photoperiod under 100 μmol m-2 s-1 provided by cool-white fluorescent tubes. Wild apple rootstock (Malus prunifolia Borkh. var. ringo Asami Mo 84-A) was used for transformation. The grafts of Malus plants were transplanted on a pod and grown in a greenhouse .
Construction of the binary vector
Arabidopsis seed (gai-1, CS63) was provided by the Arabidopsis Biological Resource Center (Ohio State University). The CS63 plantlets were used to extract the RNA fraction to obtain the full cDNA of the Atgai gene harboring a 51-bp deletion from the region encoding the conserved DELLA domain . XbaI and SacI restriction sites were added to the 5′- and 3′- sites of the Atgai fragment by PCR using the primers AtgaiXba and AtgaiSac (Additional file 7). The GUS fragment was deleted from pBI121 with XbaI and SacI , and then replaced with the new Atgai fragment. The 35S promoter was deleted from the vector by SalI and double-digested by with SpeI and XbaI. The Commelina yellow mottle virus promoter (CoYMVp) of pCOI  (from Prof. Neil Olszewski, University of Minnesota, St. Paul, MN, USA), which is expressed specifically in companion cells , was amplified by the primers CoYMVproFPSal and CoYMVproRPSpe, and then used to replaced the 35S promoter sequence with the SalI and SpeI sites (Additional file 7). A T7-epitope tag sequence (MASMTGGQQMG, Invitrogen, USA) was inserted into the 3′- site of CoYMVp: Atgai by PCR. The forward primer was CoYMVproFl, and the primer T7tag R including the T7-epitope tag sequence was used as the reverse primer. The new vector was sequenced to ensure correct insertion. The CoYMVp:Atgai-T7 (CgT) fused gene was cloned in pBI121 (Figure 1) carrying the nos-kanamycin resistance cassette. Transgenic lines were identified by PCR with the primers CoYMproFP1 and AtgaiR1 to detect the Δ-DELLA –gai. kihyufg (Additional file 7).
Micrografting was performed according to the method described by Bai et al. . Briefly, as shown in Figure 2, plantlets 10 days after germination were grafted under a stereomicroscope on a clean bench. The plant was cut horizontally approximately 3 mm below the cotyledon. Then the scion (tissue with the cotyledon) and rootstock (tissue at the bottom with root) were fastened together with a silicone tube (ф 0.4 mm × 0.1 mm, 3 mm length; TechJam, Osaka, Japan). The grafted plants were each propped against an agar block on MS agar medium. At 7 days after grafting, the silicone tube was cut off. The grafts were then cultivated in soil or using a standard nutrient solution (Otsuka House Nos. 1 and 2, Otsuka Chemical Co., Osaka, Japan) until phenotype observation or sampling. In the case of Malus plants, cleft grafting between subcultured shoots was performed.
Grafted plants that had been grown for 7 days in a Petri dish were transferred to pots with nursery soil. After 1 week, they were sprayed with 0.1 mM GA3 solution (Nakarai Tesque, Inc. Kyoto, Japan) containing 0.02% Tween 20 once every two days for three weeks. For RT-PCR, protein extraction, microarray and seedling stature measurement, plants were cultured hydroponically for 9 days, then sprayed with 0.1 mM GA3 solution.
RNA extraction, RT-PCR and qRT-PCR analysis
Total RNA was extracted from leaves with TRIzol reagent (Invitrogen, Tokyo, Japan), and genomic DNA was eliminated with a TURBO DNA-free Kit (Ambion Inc., Austin, USA). Reverse-transcribed cDNAs were prepared using a SuperScript® VILO™ cDNA Synthesis Kit (Invitrogen, USA). The cDNA corresponding to 50 ng of total RNA was used in 10-μl reactions with an S1000 Thermal Cycler (Bio-Rad, USA). To amplify Atgai mRNA in the WT scion, primers (AtgaiF2 and AtgaiR2) and the nested primers (AtgaiF3 and AtgaiR3) were prepared. The amplification condition were as follows: initial denaturing at 94°C for 4 min, 25 cycles at 94°C for 30 s, 58°C for 30 s, and 72°C for 1 min; and extension at 72°C for 3 min. For the nested PCR, 1 μl of the first PCR product was used as the template and subjected to 30 cycles. For qRT-PCR, 1 μl of the cDNA corresponding to 50 ng of total RNA was used in 20-μl reactions with iQ SYBR Green Supermix (Bio-Rad, USA). Triplicate reactions for each sample were amplified along with non-template controls on a Chromo 4 real-time PCR detector (Bio-Rad, USA). NbUbi (Accession No.: AY912494) was used to normalize the expression levels of Atgai. Primers (Atgai QF/Atgai QR and Ubi QF/ Ubi QR) specific to the Atgai and NbUbi genes were used in this experiment (Additional file 7).
Protein extraction and immunoblotting
About 3.0 g of scion shoot tissue (n = 5) was sampled after mRNA transport had been positive detected, and then ground in liquid nitrogen using a pre-cooled mortar and pestle. Protein was extracted according to the method described previously . The concentration of protein was measured by DC Protein Assay (Bio-Rad, USA). Total proteins (25 μg) were mixed with a SDS loading buffer, and heated at 95°C for 5 minutes for denaturation, then fractionated by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). SDS-PAGE was performed in 12.5% polyacrylamide gels using Bio-Rad Mini-PROTEAN 4 equipment at 200V for 1 h. Proteins were transferred to Immun-Blot™ PVDF membranes (Bio-Rad, USA). Then, each membrane was blocked with BSA (bovine serum albumin, Sigma, Germany) at room temperature for 1 h (in the blocking buffer, BSA was dissolved in 1 × TBS- 0.1% Tween to a final concentration 0.02 g/mL). For analysis of the immunoblots, the membranes were incubated with 0.1 μg/mL anti-T7-peptide monoclonal antibody (Novagen, USA) at 4°C overnight, and then washed 4 times for 1 h at room temperature. The membranes were then incubated with a 2,000-fold dilution of anti-mouse IgG, Goat Poly HRP (Cosmo Bio Co., Ltd., Tokyo, Japan) for 1 h, and washed 3 times for 1 h at room temperature. The signals were detected using an Amersham ECL Plus Western Blot Detection System (GE Healthcare, UK). A duplicate gel was run at the same time and then stained with Coomassie Brilliant Blue R250 as a loading control.
Five WT scions in which the Atgai mRNA transported from the CgT stock had been detected by RT-PCR were combined, and total RNA was prepared from the sample. Five scions for the WT/WT combination with and without GA3 treatment were also combined and used as samples for RNA extraction. The purification, labeling of cRNA, hybridization to 44K Tobacco DNA microarray (Agilent Techologies), signal scanning, and processing were performed by Hokkaido System Science Co., Ltd. (Japan) with a Low Input Quick Amp Labeling Kit using an Agilent Technologies Microarray Scanner (Agilent Techologies, USA). A total of 18,588 unique genes that passed the stringent quality control were used for inspection.
Gibberellic acid insensitive
Murashige and Skoog medium
T7 (bacteriophage) -epitope tag
Commelina yellow mottle virus.
This research was funded by the Adaptable and Seamless Technology Transfer Program through target-derived R&D of the Japan Science and Technology Agency, and by a Grant for Institutional Research at Hirosaki University.
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