The Plant Journal (2009)

doi: 10.1111/j.1365-313X.2009.03918.x

The sequences of Arabidopsis GA-INSENSITIVE RNA constitute the motifs that are necessary and sufcient for RNA long-distance trafcking
1

Nien-Chen Huang1,2,3 and Tien-Shin Yu1,* Institute of Plant and Microbial Biology, Academia Sinica, Taipei, 11529, Taiwan, 2 Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, Chung-Hsing University and Academia Sinica, Taipei, 11529, Taiwan, and 3 Graduate Institute of Biotechnology, National Chung-Hsing University, Taichung, 402, Taiwan
Received 10 February 2009; revised 13 April 2009; accepted 5 May 2009. * For correspondence (fax +886 2 27827954; e-mail tienshin@gate.sinica.edu.tw).

SUMMARY In higher plants a number of physiological processes are regulated by systemic RNA signaling molecules. This phloem-mediated remote-control system provides specic and efcient regulation to ne-tune many plant developmental programs. However, the molecular mechanism underlying long-distance movement of RNA remains to be elucidated. To this end, we examined the long-distance movement of GA-insensitive (GAI) RNA by Arabidopsis inorescence grafting and RT-PCR analysis. Our results demonstrated that long-distance movement of RNA only occurred in specic transcripts. In addition, the sequences of GAI RNA are necessary and sufcient to target GREEN FLUORESCENT PROTEIN (GFP) RNA for long-distance movement, which indicates that the trafcking of GAI RNA is mediated by specic RNA motifs. Further analyses revealed that the motifs at coding sequences and 3 untranslated regions of GAI RNA play important roles during RNA movement. In addition, the structure of the RNA rather than its specic sequence may also be important in GAI RNA trafcking. However, the secondary structure of GAI RNA is not the only factor to target RNA for longdistance movement, because recovery of the secondary structure of movement-defective GAI RNA only partially rescued RNA movement. Taken together, our results show that long-distance movement of non-cell autonomous RNA operates by specic RNA mobile elements. Keywords: RNA long-distance trafcking, RNA mobile element, GA-insensitive, grafting, linker scanning, Arabidopsis.

INTRODUCTION As sessile organisms, plants recruit many mobile molecules, including RNA, as cell-to-cell and long-distance signals to orchestrate their developmental programs. These non-cell autonomous RNAs function as plant-unique remote-control systems in gene silencing, pathogen defense, leaf development, tuber formation, phosphate homeostasis and many other physiological processes (Lucas et al., 1995; Kuhn et al., 1997; Palauqui et al., 1997; Kim et al., 2001; Haywood et al., 2002, 2005; Banerjee et al., 2006; Chiou et al., 2006; Lough and Lucas, 2006; Buhtz et al., 2008; Lin et al., 2008; Pant et al., 2008; Turgeon and Wolf, 2009). Currently, it is well accepted that thousands of mRNAs and small RNAs trafc along the phloem translocation stream (Ruiz-Medrano et al., 1999; Yoo et al., 2004; Lough and Lucas, 2006; Kehr
2009 The Authors Journal compilation 2009 Blackwell Publishing Ltd

and Buhtz, 2008). Interestingly, the transport of these endogenous phloem-mobile RNAs does not simply follow the phloem mass ow translocation but rather is highly regulated by a yet-to be identied mechanism (Ruiz-Medrano et al., 1999; Haywood et al., 2005; Lough and Lucas, 2006). By application of the RNA delivery system that has been established in the literature, a hypothetical model has been proposed to elucidate the molecular mechanism underlying long-distance movement of RNA in plants (Lucas et al., 2001). In this model, the cis-acting sequence elements that are located on non-cell autonomous RNA, termed zip codes, may interact with putative companion cell-specic zip code-binding proteins to form an RNAprotein complex.
1

2 Nien-Chen Huang and Tien-Shin Yu This RNAprotein complex is subsequently transported into sieve elements through plasmodesmata (Lucas et al., 2001). Consistent with this hypothesis, mutations altering the stemloop structure of a viroid specically disrupted entry of the viroid from the bundle sheath into mesophyll cells (Qi et al., 2004; Zhong et al., 2007 & Zhong et al., 2008). This evidence supports the idea that RNA structural motifs directly mediate the trafcking of viroid RNA. However, whether this structural motif is sufcient to target other cellautonomous RNAs for long-distance movement has yet to be investigated. In Arabidopsis and tomato, grafting experiments performed with transformant stocks of PCaMV35SGAI and PCaMV35SGFP showed that GAI but not GFP RNA can move long distances, which suggests that the trafcking of GAI RNA may be mediated by a motif that is absent from GFP RNA (Haywood et al., 2005). In contrast, efforts with a potato non-cell autonomous RNA, SUCROSE TRANSPORTER 1 (SUT1), failed to identify the cis-acting elements involved in SUT1 RNA movement (Kuhn et al., 1997; Lalonde et al., 2003). Thus, whether the long-distance movement of plant endogenous, non-cell autonomous RNA is mediated by a specic RNA motif remains to be determined. GAI encodes a nuclear protein that belongs to the DELLA subfamily of GRAS transcription factors (Pysh et al., 1999). Arabidopsis has ve DELLA proteins: GAI, RGA, RGL1, RGL2 and RGL3. The function of these proteins is partially redundant in negative regulation of GA responses (Peng et al., 1997; Silverstone et al., 1998; Dill and Sun, 2001; Wen and Chang, 2002; Tyler et al., 2004). The application of GA stimulates the interaction between DELLA proteins and the GA receptor GIBBERELLIN INSENSITIVE DWARF1 (GID1), which leads to proteolysis or inactivation of the DELLA proteins and consequently relieves various GA-dependent responses (Olszewski et al., 2002; Schwechheimer, 2008). GAI may act in a non-cell autonomous manner. GAI RNA is detected in pumpkin phloem sap, and heterografting experiments have demonstrated that GAI RNA moves from the stock to the scion apex (Ruiz-Medrano et al., 1999; Haywood et al., 2005). In tomato grafting, the long-distance movement of DELLA-deleted GAI RNA is associated with phenotypic alterations in scion leaves, which suggests that GAI RNA is a systemic signaling molecule (Haywood et al., 2005). In this study, we investigated whether specic RNA motifs are involved in long-distance trafcking of plant non-cell autonomous RNA. We found that the GAI RNA does indeed contain the specic RNA motifs that are necessary and sufcient to target GFP RNA, a cell-autonomous RNA, for long-distance movement. Deletion and linker-scanning analyses demonstrated that the motifs required for GAI RNA movement are located in the 3 sequences of GAI RNA. Taken together, our data show that the RNA mobile elements in GAI RNA play important roles in RNA movement. RESULTS Long-distance movement of GAI RNA is impaired when ectopically expressed in mesophyll cells To understand the molecular mechanism underlying GAI RNA long-distance trafcking, we rst tested whether the movement of GAI RNA occurs only in specic cell types. The full-length GAI RNA, which contains 2146 nucleotides (nt) including 5 (208 nt) and 3 (340 nt) untranslated regions (UTRs), was amplied and driven by an Arabidopsis mesophyll-specic promoter, RbcS-2b (At5g38420; Kim et al., 2003), or a companion cell-specic promoter, SUC2 (Truernit and Sauer, 1995). To distinguish the transgene from the endogenous RNA, the nopaline synthase (NOS) terminator was included after the 3 UTR of GAI, because it has been shown that this RNA fragment does not interfere the long-distance movement of GAI RNA (Haywood et al., 2005). For each construct, 10 independent transformants were selected as stocks and grafted with wild-type inorescences. Two weeks after grafting, the scion apices were harvested for RNA extraction. In addition, the mature leaves of stocks were used as controls (Figure 1a). Reverse transcription-polymerase chain reaction (RT-PCR) was conducted to examine whether GAI RNA was transported from stocks to the scion apices. Total RNA of scion apices or stock leaves was used for RT reactions, and an aliquot of the rst-strand cDNA was subjected to PCR with primers against GAI and NOS terminator (see Experimental Procedures). The PCR was conducted with 35 cycles, and the

(a)

(b)

(c)

Figure 1. Long-distance movement of GAI RNA is impaired when it is ectopically expressed in mesophyll cells. (a) Image of Arabidopsis inorescence grafting. (b) The RT-PCR analyses of RNA extracted from apices of wild-type scions (SC) and mature leaves of PSUC2GAI transformant stocks (ST). The RNA was extracted from wild-type scions grafted onto 10 individual PSUC2GAI transformants (lanes 110); PCaMV35SGAI (lane 11, positive control); or PCaMV35S GFP stocks (lane 12, negative control). (c) The RT-PCR analyses of RNA extracted from wild-type scions grafted onto 10 independent PRbcS2bGAI transformant stocks (lanes 110). The RT-PCR was performed with specic primers GAI-RT-For and the NOS terminator (primer sequences are listed in Table S2) to distinguish the transgene from endogenous GAI RNA. The primers GFPFor and GFPRev were used as a negative control. The gene IMPORTIN-a (IMP-a) was used as a loading control.

2009 The Authors Journal compilation 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), doi: 10.1111/j.1365-313X.2009.03918.x

RNA motifs required for GAI RNA long-distance trafcking 3 PCR products were visualized in agarose gels. For those samples that did not produce detectable signals in 35 cycles, the number of PCR cycles was increased to 40 cycles. The non-detectable sample was dened as no visualized PCR products in both 35 and 40 cycles. The PCR was conducted at least twice for each sample to make sure the data are representative. When wild-type scions were grafted onto PSUC2GAI transformants, the transgenic GAI RNA was detected in 7 out of 10 wild-type scions (Figure 1b; represented as 70% of the scion detection rate), which suggests that the GAI RNA readily moved long distances when expressed in companion cells. In contrast, GAI RNA was detected in only 1 out of 10 scions grafted onto PRbcS2bGAI transformant stocks (10% scion detection rate), which suggests that the movement of GAI RNA was limited when expressed in the mesophyll cells (Figure 1c). In control experiments, the detection rate of GAI RNA from the scions was 92% (46 out of 50 grafts) when wild-type scions were grafted onto PCaMV35SGAI transformant stocks, or 0% (0 out of 40 grafts) when PCaMV35SGFP transformants were used as stocks (Figure 1b; Haywood et al., 2005). As it is possible that low-level accumulation of transgenic GAI RNA in PRbcS2bGAI stocks may have accounted for the lack of detected GAI RNA in scions, RT-PCR was used to examine the level of transgenic GAI RNA in PRbcS2bGAI and PSUC2GAI transformants. The level of transgenic GAI RNA in stocks was not correlated with the detection of translocated GAI RNA in scions (Figure S1), which suggests that a high accumulation of GAI RNA in the stock is not sufcient to trigger RNA movement. Thus, the machinery required for GAI RNA trafcking might be mainly located in companion cells. RNA long-distance movement occurs only with specic transcripts In Arabidopsis, sequence comparison of the ve DELLA proteins, GAI, RGA, RGL1, RGL2 and RGL3, revealed that the coding regions of GAI paralogs are conserved, but the UTRs are relatively diverse (Figure S2). To test whether the sequence variations among GAI paralogs may affect RNA trafcking, we amplied the full-length cDNA (containing 5 and 3 UTRs) of the ve GAI paralogs and driven by a CaMV35S promoter. Again, Arabidopsis inorescence grafting and RT-PCR analysis were performed to examine the long-distance trafcking of transgenic RNA. The detection rate of the GAI RNA from wild-type scions grafted onto PCaMV35SGAI stocks was 90% (Table 1). In contrast, the detection rate of RGA, RGL1, RGL2 or RGL3 RNA was greatly reduced from the scions that had been grafted onto PCaMV35SRGA, RGL1, RGL2 or RGL3 transformant stocks (Table 1). Therefore, although the ve GAI paralogs share high sequence similarity, only GAI RNA efciently moves long distances.
Table 1 Arabidopsis grafting performed with wild-type scions grafted onto individual GAI paralog transformants Scion detection (%) 90 7 0 4 14

Stocks PCaMV35SGAI PCaMV35SRGA PCaMV35SRGL1 PCaMV35SRGL2 PCaMV35SRGL3

No. of grafts 45 70 43 145 105

No. of lines 5 5 15 7 6

(a)

(b)

Figure 2. GAI RNA is sufcient to target GFP RNA for long-distance movement. (a) Reverse transcription-PCR was used to analyze the samples collected from an Arabidopsis wild-type plant (WT), PCaMV35SgaiGFP, PCaMV35SGFP-gai or PCaMV35SGFP transformant stock (ST) or wild-type scion (SC). The PCR was conducted with 35 cycles with the same gene-specic primers GFPFor and GFPRev. Note that the GFP RNA was not detected from wild-type scions even though the PCR was conducted with 40 cycles. IMPORTIN-a (IMP-a) was used as a loading control. The insertion point of GFP is illustrated in the diagram. 5, 5 UTR; 3, 3 UTR. (b) Analysis of RNA movement in Arabidopsis grafting experiments. No. lines represents the number of independent transformants that were used as stocks.

GAI RNA is sufcient to target GFP RNA for long-distance trafcking If GAI RNA contains the motif required for RNA long-distance movement, this motif may be able to target other cellautonomous RNAs to move over long distances. To test this possibility, Arabidopsis transformants carrying PCaMV35S GAIGFP or PCaMV35SGFPGAI transgenes were used as stocks and grafted with wild-type scions. In control experiments, GFP RNA was not detected in wild-type scions with PCaMV35SGFP used as a stock (Figure 2; Haywood et al., 2005). However, when PCaMV35SGAIGFP or PCaMV35SGFP GAI transformants were used as stocks, the RNA of GFPGAI and GAIGFP was detected in wild-type scions (Figure 2a). In our 80 grafting samples, detection of translocated RNA was highly reproducible (Figure 2b). Thus, the information contained within GAI RNA is sufcient to target cell-autonomous RNA for long-distance delivery.

2009 The Authors Journal compilation 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), doi: 10.1111/j.1365-313X.2009.03918.x

4 Nien-Chen Huang and Tien-Shin Yu

(a)

(b)

(c)

(d)

Figure 3. Deletion analysis to identify motifs required for movement of GAI RNA. (a, b) Reverse transcription-PCR analyses using RNA extracted from wild-type scions grafted onto (a) PCaMV35S-GAI D5 UTR or PCaMV35SGAI D3 UTR, or (b) PCaMV35SGFP-GAI18082146 or PCaMV35S-GFP-GAI995-2146 transformant stocks. The PCR was conducted with 35 cycles and the primers GAIRTFor and NOS term Rev (a); or GFP-For and GFP-Rev (b). The gene IMPORTIN-a (IMP-a) was used as a loading control. (c) Statistical data from Arabidopsis grafting experiments performed with wild-type scions and transformant stocks carrying different UTR-truncated GAIs. For each construct, at least four independent transformants were used as stocks. The PCR was conducted with 35 cycles and the non-detection samples were further conrmed with 40 cycles. (d) Arabidopsis grafting experiments performed with transformant stocks carrying different GFPGAI transgenes. The RNA of GAI9952146 is sufcient to target GFP for long-distance movement.

Deletion analyses were conducted to locate the potential motifs required for GAI RNA movement. To test whether the 5 or 3 UTR is required for GAI RNA trafcking, GAI with different truncated UTRs was generated, and driven by a CaMV35S promoter. The NOS-terminator was again used to distinguish the transgene from the endogenous GAI. Arabidopsis transformants carrying GAI transgenes were rst analyzed by RT-PCR with specic primers against the transgene, and the transformants with similar RNA accumulation level of transgene were used as stocks (data not shown). Our grafting results indicated that the 5 UTR of GAI RNA was dispensable for RNA trafcking (Figure 3a,c). In contrast, deletion of the GAI 3 UTR greatly reduced the rate of detection of GAI RNA in the scions, which suggests that the 3 UTR is required for long-distance movement of GAI RNA (Figure 3a,c). To examine whether the 3 UTR of GAI RNA is sufcient to target GFP RNA movement, GFP was fused with the GAI 3 UTR and driven by a CaMV35S promoter. Chimeric GFP RNA was not detected in the wildtype scions grafted onto transformants carrying GFP GAI18082146 (3 UTR), GFPGAI17522146 (50 nt of coding

sequence +3 UTR) or GFPGAI17002146 (100 nt of coding sequence +3 UTR) transgene, which indicates that the 3 UTR of GAI RNA is not sufcient for RNA movement (Figure 3b,d). However, chimeric RNA was detected in wild-type scions grafted onto transformants carrying PCaMV35SGFPGAI9952146 transgene, which indicates that the RNA of GAI9952146 is sufcient to target GFP RNA for long-distance movement (Figure 3b,d). Thus, the GAI9952146 RNA fragment is necessary and sufcient for long-distance trafcking of RNA. In addition, our data also suggest that at least two RNA mobile elements, one located in the GAI coding sequence and the other in the 3 UTR, are required for movement of GAI RNA. Alternatively, the RNA structure created by folding of the GAI coding sequence and the 3 UTR may determine the long-distance trafcking of GAI RNA. Linker-scanning analysis of the long-distance movement of GAI9952146 RNA Linker-scanning analysis was employed to further characterize the RNA mobile elements within GAI9952146 RNA.

2009 The Authors Journal compilation 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), doi: 10.1111/j.1365-313X.2009.03918.x

RNA motifs required for GAI RNA long-distance trafcking 5 Site-directed mutagenesis was used to create 26 sequencereplaced mutants (M1M26) across the 9952146 nt of GAI RNA (Figure S3). Arabidopsis transformants expressing mutant GAI RNA were used as stocks and grafted with wildtype scions. The scion detection rate of the GAI9952146 RNA was 67% when wild-type scions were grafted onto PCaMV35S GAI9952146 transformant stocks (Figure 4a,c; Table S1). However, the scion detection rate of mutated GAI RNA was reduced to 30% or less when M1, M3, M4, M5, M7, M11, M13, M14, M17 and M18 transformants were used as stocks, which suggests that these regions are important during GAI RNA movement (Figure 4c, Table S1). When grafting used M6 and M9 stocks, the scion detection rate of the GAI RNA was reduced to 44 and 50%, respectively. In contrast, the scion detection rate of mutated GAI RNA was similar to or slightly better than that of GAI9952146 when M2, M8, M10, M13, M15, M16 and M19M26 transformants were used as stocks (Figure 4c, Table S1). The mutations leading to defective trafcking of GAI RNA were mainly located in three regions (motifs A, B and C), which suggest that multiple motifs are required for movement of GAI RNA (Figure 4c). In addition, because two-thirds of the mutations occurring in the fragment of GAI9952146 strongly or partially disrupted RNA movement, the RNA structure may play a more important role in targeting GAI RNA for long-distance movement in RNA trafcking. In yeast and animal cells, it has been shown that a specic stemloop structure is required to target RNA for asymmetric localization (Chartrand et al., 1999; Gonzalez et al., 1999; Kloc et al., 2002). To test whether the stemloop structure of

(a)

(b)

(c)

Figure 4. Linker-scanning analysis of the long-distance movement of GAI9952146 RNA. (a, b) The RT-PCR analyses using RNA extracted from wild-type scions grafted onto (a) PCaMV35S-GAI9952146 or PCaMV35SM1 and (b) PCaMV35SM4 or PCaMV35SM4-rev transformant stocks. The gene IMPORTIN-a (IMP-a) was used as a loading control. (c) Schematic illustration of the RT-PCR analyses performed with wild-type scions grafted onto PCaMV35SGAI9952146 or 27 linker-scanning mutant transformants. The scion detection rate (%) is shown above each bar. For each construct, ve independent transformants were used as the stocks and grafted with wild-type scions. The scion detection rate was calculated based on RT-PCR analysis of at least 50 successful grafts. The three potential motifs (A, B and C) involved in movement of GAI RNA and their relative positions on the fragment of GAI9952146 are illustrated.

2009 The Authors Journal compilation 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), doi: 10.1111/j.1365-313X.2009.03918.x

6 Nien-Chen Huang and Tien-Shin Yu deletion analyses, we have also shown that the GAI9952146 RNA fragment is necessary and sufcient to target other cell autonomous RNAs for long-distance trafcking (Figure 3). In addition, our linker-scanning analysis revealed that at least three motifs are required for long-distance movement of GAI RNA (Figure 4a). Because the successful trafcking of GAI RNA from stock to scion apex requires multiple translocation steps, which include vascular entry, transport along the phloem and exit (Lucas et al., 2001), it is possible that each step may require a specic motif to interact with putative translocation machinery. Consistent with this notion, the results of our grafting experiments showed that the RNA movement is not associated with over-accumulation of GAI RNA in the stocks (Figures 1 and S1). Thus, it is possible that an active transport system, which involves RNA-binding proteins or other yet to be identied proteins, participates in the selection of specic transcripts for long-distance trafcking. Interestingly, the long-distance movement of GAI RNA was signicantly higher when GAI was expressed in the companion cells rather than in the mesophyll cells (Table 1). This suggests that the machinery required for GAI RNA movement is mainly located in companion cells. However, because the cellular boundary may serve as a regulatory point for viroid RNA trafcking (Qi et al., 2004), it is possible that GAI RNA moves efciently from cell to cell in mesophyll cells but in a limited fashion across the boundary into companion cells. Our linker-scanning analysis showed that over two-thirds of mutations occurring at GAI9952146 fragment affect GAI RNA movement, which suggests that the structure rather than specic sequences is important for recognition by RNA movement machinery. The recovery of the stemloop structure in movement-defective GAI RNA only resulted in partial rescue of RNA movement (Figure 4b,c). Therefore, the stemloop structure may not be the only factor for the recognition of RNA movement machinery. More detailed analyses are required to identify the nature of the RNA mobile elements. In viroids, it has been shown that the tertiary structure of the RNA motif plays an important role during the vascular entry of viroid RNA (Zhong et al., 2007). It is possible that the tertiary structure of GAI RNA or other factors may also contribute to the interaction with transacting factors. Interestingly, RNA tertiary structures are also important for asymmetric localization of yeast ASH1 mRNA (Chartrand et al., 1999; Gonzalez et al., 1999). This may raise the intriguing question of whether the mechanism of RNA long-distance movement and RNA asymmetrical localization shares the same evolutionary origin. The scion detection rate of mutant GAI RNA was strongly affected when mutations occurred in motif C (M17 and M18), indicating that motif C is important for RNA movement (Figures 4 and S3). However, the insertion of GFP into the junction between the coding sequence and the 3 UTR did not disrupt the movement of chimeric GAIGFP RNA

Figure 5. Computer-predicted secondary structure of GAI9952146 RNA and the location of 26 linker-scanning mutations. The secondary structure of GAI9952146 RNA, as predicted by the computer program MFOLD. The mutation sites of each linker scanning mutation are depicted with black lines (almost no defect in RNA movement), blue lines (mild defects in RNA movement) or red lines (strong defects in RNA movement).

GAI RNA is involved in the movement of RNA, we predicted the secondary structure of GAI9952146 RNA using the computer program MFOLD (Zuker, 2003). We selected the M4 mutation for further analysis, because the sequence of M4 was located on a separate stemloop arm and mutations occurring on this arm (M4 and M5) strongly reduced RNA movement to 20 or 10% (Figure 5). We speculated that if a specic stemloop structure participates in GAI RNA movement, the recovery of this structure might rescue RNA movement to the wild-type level. To this end, the nucleotides that complemented the M4 sequence were mutated to recover a stemloop structure similar to that of the wild type (Figure S4). Computer prediction showed that the entire structure of M4-rev RNA was the same as GAI9952146 RNA (data not shown). In 50 grafting samples, the scion detection rate of M4-rev RNA was increased to 43% (Figure 4b,c), which indicates that the recovery of the stemloop structure only partially rescued the long-distance movement of GAI RNA. Thus, the stemloop structure of GAI RNA is not the only factor determining RNA movement. DISCUSSION In this study we have shown that the long-distance trafcking of GAI RNA is mediated by specic RNA motifs. Based on

2009 The Authors Journal compilation 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), doi: 10.1111/j.1365-313X.2009.03918.x

RNA motifs required for GAI RNA long-distance trafcking 7 (Figure 2), suggesting that motif C may act separately from other motifs during GAI RNA trafcking. Alternatively, it is possible that specic sequences, or RNA structures of individual motifs, may interact with putative RNA movement machinery and assemble into a complicated RNAprotein complex. In yeast, it has been demonstrated that the interaction between RNA structure and a protein complex is important for RNA asymmetric localization (Chartrand et al., 1999, 2001). Whether a similar model can be applied to GAI RNA long-distance movement needs further investigation. The primary sequence comparison among ve GAI paralogs indicated that RGA is more similar to GAI (Figure S2). However, our grafting analyses showed that the movement efciency of RGL3 is slightly better than RGA, RGL1 or RGL2 (Table 1). This result is consistent with the notion that the structure rather than the primary sequences of GAI RNA participates in RNA movement. Whether the structure of RGL3 is more accessible to RNA movement machinery remains to be investigated. When GAI 3 UTRs were deleted, the detection of translocated GAI RNA from the scions was signicantly reduced (Figure 3a). The truncated or mutant forms of GAI RNA may have been rapidly degraded before they could interact with the RNA trafcking machinery. However, our RT-PCR analyses showed that accumulation of the UTR-truncated or mutated GAI RNA in most of the GAI transformants was not signicantly reduced (data not shown). Therefore, RNA stability was unlikely to be a major factor affecting the movement of GAI RNA in our experiments. In higher plants, several thousand RNAs have been identied from analyses of the phloem sap RNA composition (Sasaki et al., 1998; Ruiz-Medrano et al., 1999; Lough and Lucas, 2006; Kehr and Buhtz, 2008). Because the sieve elements are enucleate cells, these phloem sap RNAs might be synthesized in the companion cells (or other cell types) and then transported into the sieve element. This nding raises the possibility that most, if not all, of the phloem sap RNA may have the ability to move from companion cells into sieve elements. Currently, it has been shown that at least four mRNAs (BEL5, LeT6, KNOTTED1 and GAI) are able to move for long distances or cell-to-cell in Arabidopsis or tomato (Lucas et al., 1995; Kim et al., 2001, 2005; Haywood et al., 2005; Banerjee et al., 2006). However, the sequence comparison or secondary structure prediction between GAI and BEL5 or other phloem sap RNAs failed to identify the conserved domain among these genes (data not shown). It is possible that more than one RNA trafcking machine participates in the movement of individual non-cell autonomous RNA. Consistent with this hypothesis, proteomic or mass spectrometric analysis of pumpkin or melon phloem sap proteins has identied a set of putative RNA-binding proteins, including a polypyrimidine tract-binding protein, RBP50 (Gomez et al., 2005; Lin et al., 2009; Ham et al., 2009). These proteins may be involved in the translocation of phloem-mobile RNA. EXPERIMENTAL PROCEDURES Plant material
Arabidopsis thaliana plants were obtained from the Arabidopsis Biological Resource Center (ABRC, Columbus, OH, USA) and grown in growth chambers under 16-h/8-h, 22C/20C day/night cycle, under white uorescent light at an intensity of 100 lmol m)2 sec)1.

Plasmid construction
The full-length GAI cDNA was described previously (Haywood et al., 2005). The Arabidopsis full-length RGA, RGL1, RGL2 and RGL3 cDNAs were amplied by RT-PCR with RNA extracted from Landsberg erecta (Ler) seedlings and gene-specic primers (sequences of the primers are in Table S2). For generation of GAI, RGA, RGL1, RGL2 and RGL3 DDELLA lines, the full-length cDNA served as a template to generate a 51-bp deletion at the DELLA domain, and GAI DDELLA was used to generate all the other GAI deletion lines by PCR amplication. To generate GFPGAI and GAIGFP clones, the GFP fragment was directly cloned into GAI full-length cDNA, which introduced a BamHI site by site-directed mutagenesis at positions 209 nt or 1807 nt of GAI, respectively. Polymerase chain reaction with suitable primers (Table S2) was used to create the varieties of GAI deletion clones. All the constructs were conrmed by sequencing and subcloned to binary vectors containing a CaMV35S or a SUC2 promoter. Arabidopsis was transformed by oral dip transformation (Clough and Bent, 1998). The transformants were selected on MS medium containing 40 lg ml)1 hygromycin. The antibiotic-resistant plants were transferred to soil for further analysis.

Arabidopsis inorescence grafting

Arabidopsis grafting experiments were performed using the following procedures: Arabidopsis plants (Ler ecotype) were grown for 45 weeks until bolting. The inorescence (about 4 cm long, containing two to three lateral oral buds) from scions was cut into a V-shape under microscopy and inserted into the stock, which had a vertical cut in the middle of the primary inorescence and a piece of polyethylene tubing was inserted over the base of the cut bolt. The grafting junction was secured with the polyethylene tubing and sealed with Paralm. Grafted plants were kept at high humidity for 7 days then returned to normal growth conditions for another week. Scion RNA samples were extracted 14 days after grafting.

RNA extraction and RT-PCR analysis

Total RNA from plant tissues was extracted using TRIZOL reagent (Invitrogen, http://www.invitrogen.com/). For RT-PCR analysis, 5 lg of total RNA was used in RT reactions performed with oligo(dT)20 and SuperScript III reverse transcriptase (Invitrogen). One microliter of cDNA was used for the PCR reaction with the following conditions: 1 min at 94C for 1 cycle; 30 sec at 94C, 30 sec at 60C, 1 min at 68C for 35 cycles and 10 min at 68C for 1 cycle. Specic primers against the transgene and NOS terminator were used for PCR reactions to distinguish transgenes from endogenous genes (primer sequences are in Table S3). To minimize the bias during PCR, the same primers were used for each set of experiments. The PCR was conducted at least twice for each sample to make sure the data were representative. An aliquot (5 ll) of PCR products was separated on 1.5% agarose gels.

2009 The Authors Journal compilation 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), doi: 10.1111/j.1365-313X.2009.03918.x

8 Nien-Chen Huang and Tien-Shin Yu Site-directed mutagenesis

To generate the linker-scanning mutations on GAI RNA, the plasmid containing the GAI9952146 fragment served as a template for site-directed mutagenesis. These primers are listed in Table S4. Site-directed mutagenesis was performed under the following conditions: 1 min at 95C for 1 cycle; 30 sec at 95C, 1 min at 55C, 8 min 30 sec at 68C for 18 cycles and 1 min at 94C, 1 min at 55C, 10 min at 72C for 1 cycle. The PCR product was digested with DpnI at 37C for 1 h to remove the template DNA. To generate M4-rev, the M4-containing plasmid was used as a template for site-directed mutagenesis. All of the mutant clones were conrmed by sequencing analysis. The mutant constructs were driven by a CaMV35S promoter. For grafting experiments, ve independent transformant plants from each mutant construct were used as the stocks and were grafted with wild-type scions.
Chiou, T.J., Aung, K., Lin, S.I., Wu, C.C., Chiang, S.F. and Su, C.L. (2006) Regulation of phosphate homeostasis by microRNA in Arabidopsis. Plant Cell, 18, 412421. Clough, S.J. and Bent, A.F. (1998) Floral dip: a simple method for Agrabacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735 743. Dill, A. and Sun, T.-P. (2001) Synergistic derepression of gibberellin signaling by removing RGA and GAI function in Arabidopsis thaliana. Genetics, 159, 777785. Gomez, G., Torres, H. and Pallas, V. (2005) Identication of translocatable RNA-binding phloem proteins from melon, potential components of the long-distance RNA transport system. Plant J. 41, 107116. Gonzalez, I., Buonomo, S.B.C., Nasmyth, K. and von Ahsen, U. (1999) ASH1 mRNA localization in yeast involves multiple secondary structure elements and Ash1 protein translation. Curr. Biol. 9, 337340. Ham, B.-K., Brandom, J.L., Xoconostle-Cazares, B., Ringgold, V., Long, T.J. and Lucas, W.J. (2009) A polypyrimidine tract binding protein, pumpkin RBP50, forms the basis of a phloem-mobile ribonucleoprotein complex. Plant Cell, 21, 197215. Haywood, V., Kragler, F. and Lucas, W.J. (2002) Plasmodesmata: pathways for protein and ribonucleoprotein signaling. Plant Cell, 14, S303S325. Haywood, V., Yu, T.-S., Huang, N.-C. and Lucas, W.J. (2005) Phloem longdistance trafcking of GIBBERELLIC ACID INSENSITIVE RNA regulates leaf development. Plant J. 42, 4968. Kehr, J. and Buhtz, A. (2008) Long distance transport and movement of RNA through the phloem. J. Exp. Bot. 59, 8592. Kim, M., Canio, W., Kessler, S. and Sinha, N. (2001) Developmental changes due to long-distance movement of a homeobox fusion transcript in tomato. Science, 293, 287289. Kim, J.-Y., Yuan, Z. and Jackson, D. (2003) Developmental regulation and signicance of KNOX protein trafcking in Arabidopsis. Development, 130, 43514362. Kim, J.-Y., Rim, Y., Wang, J. and Jackson, D. (2005) A novel cell-to-cell trafcking assay indicates that the KNOX homeodomain is necessary and sufcient for intercellular protein and mRNA trafcking. Genes Dev. 19, 788793. Kloc, M., Zearfoss, N.R. and Etkin, L.D. (2002) Mechanism of subcellular mRNA localization. Cell, 108, 533544. Kuhn, C., Franceschi, V.R., Schulz, A., Lemoine, R. and Frommer, W.B. (1997) macromolecular trafcking indicated by localization and turnover of sucrose transporters in enucleate sieve elements. Science, 275, 12981300. Lalonde, S., Weise, A., Walsh, R.P., Ward, J.M. and Frommer, W.B. (2003) Fusion to GFP blocks intercellular trafcking of the sucrose transporter SUT1 leading to accumulation in companion cells. BMC Plant Biol. 3, 8. Lin, S.-I., Chiang, S.-F., Lin, W.-Y., Chen, J.-W., Tseng, C.-Y., Wu, P.-C. and Chiou, T.-J. (2008) Regulatory network of microRNA399 and Pho2 by systemic signaling. Plant Physiol. 147, 732746. Lin, M.-K., Lee, Y.-J., Lough, T.J., Phinney, B.S. and Lucas, W.J. (2009) Analysis of the pumpkin phloem proteome provides insights into angiosperm sieve tube function. Mol. Cell Proteomics, 8, 343356. Lough, T.J. and Lucas, W.J. (2006) Integrative plant biology: role of phloem long-distance macromolecular trafcking. Annu. Rev. Plant Biol. 57, 203 232. Lucas, W.J., Bouche-Pillon, S., Jackson, D.P., Nguyen, L., Baker, L., Ding, B. and Hake, S. (1995) Selective trafcking of KNOTTED1 homeodomain protein and its mRNA through plasmodesmata. Science, 270, 1980 1983. Lucas, W.J., Yoo, B.-C. and Kragler, F. (2001) RNA as a long-distance information macromolecule in plant. Nature Rev. Mol. Cell Biol. 2, 849857. Olszewski, N., Sun, T.-p. and Gubler, F. (2002) Gibberellin signaling: biosynthesis, catabolism, and response pathways. Plant Cell, 14, S61S80. Palauqui, J.C., Elmayan, T., Pollien, J.-M. and Vaucheret, H. (1997) Systemic acquired silencing: transgene-specic post-transcriptional silencing is transmitted by grafting from silenced stocks to non-silenced scions. EMBO J. 16, 47384745. Pant, B.D., Buhtz, A., Kehr, J. and Scheible, W.-R. (2008) MicroRNA399 is a long-distance signal for the regulation of plant phosphate homeostasis. Plant J. 53, 731738. Peng, J., Carol, P., Richard, D.E., King, K.E., Cowling, R.J., Murphy, G.P. and Harberd, N.P. (1997) The Arabidopsis GAI gene denes a signaling pathway that negative regulates gibberellin responses. Genes Dev. 11, 31943205.

RNA secondary structure prediction

The secondary structure of GAI RNA was predicted with the computer program MFOLD (Zuker, 2003; http://mobyle.pasteur.fr/cgi-bin/ portal.py?form=mfold).

ACKNOWLEDGEMENTS
We thank the ABRC for providing Arabidopsis seeds, and Drs Hsoumin Li and Shu-Hsing Wu and our lab members for critically reading the manuscript. This work was supported by grants NSC 95-2311-B001-060 and NSC 96-2311-B-001-021-MY3 from the National Science Council, Taiwan.

SUPPORTING INFORMATION
Additional Supporting Information may be found in the online version of this article: Figure S1. Semi-quantitative RT-PCR analysis showed that longdistance RNA movement is not correlated with the level of accumulation of transgenic GAI RNA. Figure S2. Sequence comparison of ve Arabidopsis GAI paralogs. Figure S3. Site-directed mutagenesis to create 26 mutations of the GAI9952146 fragment. Figure S4. Secondary structure of the stemloop arm containing the M4 and M4-rev mutations. Table S1. Linker-scanning analysis of long-distance movement of the 3 half of GAI RNA. Table S2. Primers used in the construction of GAI paralogs. Table S3. Primers used in RT-PCR analysis. Table S4. Primers used in site-directed mutagenesis. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

REFERENCES
Banerjee, A.K., Chatterjee, M., Yu, Y., Suh, S.G., Miller, W.A. and Hannapel, D.J. (2006) Dynamics of a mobile RNA of potato involved in a long-distance signaling pathway. Plant Cell, 18, 34433457. Buhtz, A., Springer, F., Chappell, L., Baulcombe, D.C. and Kehr, J. (2008) Identication and characterization of small RNAs from the phloem of Brassica napus. Plant J. 53, 739749. Chartrand, P., Meng, X.-H., Singer, R.H. and Long, R.M. (1999) Structure elements required for the localization of ASH1 mRNA and of a green uorescent protein reporter particle in vivo. Curr. Biol. 9, 333 336. Chartrand, P., Singer, R.H. and Long, R.M. (2001) RNP localization and transport in yeast. Annu. Rev. Cell Dev. Biol. 17, 297310.

2009 The Authors Journal compilation 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), doi: 10.1111/j.1365-313X.2009.03918.x

RNA motifs required for GAI RNA long-distance trafcking 9

Pysh, L.D., Wysocka-Diller, J.W., Camilleri, C., Bouchez, D. and Benfey, P.N. (1999) The GRAS gene family in Arabidopsis: sequence characterization and basic expression analysis of the SCARECROW-LIKE genes. Plant J. 18, 111119. Qi, Y., Pelissier, T., Itaya, A., Hunt, E., Wassenegger, M. and Ding, B. (2004) Direct role of a viroid RNA motif in mediating directional RNA trafcking across a specic cellular boundary. Plant Cell, 16, 1741 1752. Ruiz-Medrano, R., Xoconostle-Cazares, B. and Lucas, W.J. (1999) Phloem long-distance transport of CmNACP mRNA: implications for supracellular regulation in plants. Development, 126, 44054419. Sasaki, T., Chino, M., Hayashi, H. and Fujiwara, T. (1998) Detection of several mRNA species in rice phloem sap. Plant Cell Physiol. 39, 895897. Schwechheimer, C. (2008) Understanding gibberellic acid signaling-are we there yet? Curr. Opin. Plant Biol. 11, 915. Silverstone, A.L., Ciampaglio, C.N. and Sun, T.-P. (1998) The Arabidopsis RGA gene encodes a transcriptional regulator repressing the gibberellin signal transduction pathway. Plant Cell, 10, 155169. Truernit, E. and Sauer, N. (1995) The promoter of the Arabidopsis thaliana SUC2 sucrose-H+ symporter gene directs expression of b-glucuronidase to the phloem: evidence for phloem loading and unloading by SUC2. Planta,, 196, 564570. Turgeon, R. and Wolf, S. (2009) Phloem transport: cellular pathways and molecular trafcking. Annu. Rev. Plant Biol. 60, 207221. Tyler, L., Thomas, S.G., Hu, J., Dill, A., Alonso, J.M., Ecker, J.R. and Sun, T.-P. (2004) DELLA proteins and gibberellin-regulated seed germination and oral development in Arabidopsis. Plant Physiol. 135, 10081019. Wen, C.-K. and Chang, C. (2002) Arabidopsis RGL1 encodes a negative regulator of gibberellin responses. Plant Cell, 14, 87100. Yoo, B.-C., Kragler, F., Varkonyi-Gasic, E., Haywood, V., Archer-Evans, S., Lee, Y.M., Lough, T.J. and Lucas, W.J. (2004) A systemic small RNA signaling system in plants. Plant Cell, 16, 19792000. Zhong, X., Tao, X., Stombaugh, J., Leontis, N. and Ding, B. (2007) Tertiary structure and function of an RNA motif required for plant vascular entry to initiate systemic trafcking. EMBO J. 26, 38363846. Zhong, X., Archual, A.J., Amin, A.A. and Ding, B. (2008) A genomic map of viroid RNA motifs critical for replication and systemic trafcking. Plant Cell, 20, 3547. Zuker, M. (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 110.

2009 The Authors Journal compilation 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), doi: 10.1111/j.1365-313X.2009.03918.x