Botanical Studies (2010) 51: 17-26.
molecular biology
Arabidopsis floral regulators FVE and AGL24 are phloem-mobile RNAs
Horng-Woei YANG1,2 and Tien-Shin YU2 *
1 Graduate Institute of Life Science, National Defense Medical Center, Taipei, Taiwan
2Institute of Plant and Microbial Biology, Academia Sinica, Taipei 11529, Taiwan
(Received May 12, 2009; Accepted July 3, 2009)
ABSTRACT. Plants take advantage of the vascular system to operate environmental stimulates for fine-tuning their developmental programs. Recent evidence shows that the FLOWERING LOCUS T (FT) protein is the long-sought-after florigen that integrates the photoperiod variation perceived in the leaves. However, evidence also supports that other yet-to-be identified systemic regulators participate in floral induction. To this end, we investigated phloem exudates from excised broccoli (Brassica oleracea) inflorescences. Microar-ray and RT-PCR analyses revealed that at least two RNAs of floral regulators, FVE and AGAMOUS-LIKE 24 (AGL24), are present in the phloem sap. Enzymatic analysis demonstrated that the phloem-sap RNAs contain a 5' cap and a polyadenlylation tail, which suggests that phloem sap contains typical mRNAs. Arabidopsis grafting experiments were used to test whether these RNAs move long distance along the phloem transloca-tion stream. Consistent with previous reports, Arabidopsis transformants expressing FVE and AGL24 displayed an early flowering phenotype. When wild-type scions were grafted onto P35S-FVE or Ps5s_AGL24 transformant stocks, the RNAs of transgenic FVE and AGL24 were detected from the wild-type scions. Thus, both FVE and AGL24 RNAs can move long distance across the graft union. Our data support the notion that multiple sys­temic floral regulators may participate in floral regulation.
Keywords: Arabidopsis; Brassica oleracea; Flowering; Grafting; Phloem sap; RNA long-distance movement.
introduction
As sessile organisms, plants frequently face daily challenges from various environmental stresses. To com­municate between the tissues that perceive signals and those where growth takes place, plants have evolved many elegant mechanisms, including an RNA-based system, to incorporate the response to environmental signals into their developmental programs. This plant-unique systemic RNA regulatory network participates in gene silencing, pathogen defense, leaf development, tuber formation, phosphate homeostasis and many other physiological pro­cesses (Lucas et al., 1995; Palauqui et al., 1997; Kim et al" 2001; Haywood et al" 2005; Banerjee et al., 2006; Chiou et al., 2006; Buhtz et al., 2008; Pant et al., 2008; Turgeon and Wolf, 2009). It has been demonstrated that the non-cell autonomous RNA molecules are trafficking through the phloem translocation stream (Haywood et al., 2005), probably by forming an RNA-protein complex to allow the stable translocation of RNA molecules (Lucas et al., 2001).
The RNA complexity within the phloem translocation stream has been extensively characterized. Direct sequenc­ing of cDNA libraries constructed from collected phloem
*Corresponding author: E-mail: tienshin@gate.sinica.edu.tw; Tel: 886-2-27871159; Fax: 886-2-27827954.
sap (Ruiz-Medrano et al., 1999; Yoo et al., 2004; Doering-
Saad et al., 2006; Omid et al., 2007; Buhtz et al., 2008);
laser-captured phloem tissues (Asano et al., 2002; Nakazo-no et al., 2003); GFP-marked companion cells (Ivashikina et al., 2003); or peeled vasculature (Vilaine et al., 2003; Pommerrenig et al., 2006) provide evidence that thousands of RNA species are present in phloem sap. These phloem-sap RNAs encode proteins involved in many physiological processes, which is consistent with the notion that the sys­temic RNA regulatory network may function to fine-tune plant development (Long and Lucas, 2006).
Floral induction is orchestrated by leaf-derived systemic floral regulators, or florigen (Zeevaart, 1976). Recent evi­dence has shown that the FT protein may function as a flo-rigen (Corbesier et al., 2007). However, other molecules, probably FT RNA and yet-to-be-identified molecules, also function as systemic floral regulators. When FT RNA is fused with movement-defective virus, the chimeric virus RNA moves systemically, which suggests that FT RNA can mediate long-distance trafficking of heterologous RNA (Li et al., 2009). In addition, ectopic expression of FLC in companion cells revealed that FLC represses at least two systemic signals produced in the leaves, one of which is FT independent (Searle et al., 2006). Thus, floral initia­tion may be regulated by multiple florigens. However, the molecular basis of these systemic floral regulators remains unknown.
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The classic grafting experiments revealed that the puta­tive floral regulators may traffic along the phloem trans­location stream (Zeevaart, 1976). In addition, FT RNA is capable to move cell-to-cell (Li e al., 2009), which sug­gests that the non-cell autonomous RNA may systemically regulate floral initiation. We therefore focused on phloem-sap RNAs. By using microaray and RT-PCR analysis, we have identified at least two RNA of floral regulators (FVE and AGL24) from broccoli (Brassica oleracea) phloem sap. Enzymatic analysis showed that the phloem-sap RNAs contained a 5' cap and a polyadenylation tail, which suggests that these RNAs are typical mRNAs. Arabidopsis grafting experiments demonstrated that the RNAs of FVE and AGL24 move long distance, indicating that FVE and AGL24 RNAs are phloem-mobile RNA. We proposed that FVE and AGL24 may function as systemic floral regula­tors.
materials and methods
Plant materials and growth conditions
Broccoli (Brassica oleracea var. italica) seeds were obtained from a local company (cultivar Green-Huei; Known-You Seed Co, Taiwan). Broccoli plants were grown in the field or in a growth chamber under 16-h day/ 8-h night and 25°C /20°C day/night condition under white fluorescent light, with light intensity 200 fimol m-2s-1. Arabidopsis seeds were obtained from the Arabidopsis Biological Resource Center (Ohio, USA) and grown in
a growth chamber under 16-h day/8-h night, 22°C /20°C
day/night conditions under white fluorescent light, with light intensity 100 fimol m-2s-1.
Broccoli phloem sap collection and RNA extraction
The young inflorescences of 2-month-old broccoli were excised by use of a sharp blade and blotted with 3MM filter paper twice. The first drop of exudates was discarded to minimize contamination by surrounding tissues. To extract phloem-sap RNA, the exudates were immediately mixed wi th an equal volume of TRIzol reagent (Invitrogene, CA). The phloem-sap RNA was further purified by use of the RNeasy plant RNA mini kit (Qiagen) according to the manufacturer’s recommendations.
DNS analysis
DNS method was used to measure the sugar content of broccoli phloem sap (Bernfeld, 1995). The phloem sap was diluted in 20 mM Tris-HCl pH 7.0 and treated with invertase (5U/mg, Fluka) at 37°C for 30 min to convert sucrose to glucose and fructose. After treatment, the phloem sap was mixed with DNS solution (43 mM 3, 5, Di-NitroSalicylic acid, 400 mM NaOH, 1 M potassium sodium-tartrate tetrahydrate) and boiled for 5 min. The solution was diluted and OD540 was read. The concentration of sucrose was calculated by comparison with the glucose standard.
Microarray analysis
To synthesize the probe of broccoli phloem-sap RNA, the first-strand and second-strand cDNA was synthesized from total phloem-sap RNA by reverse transcriptase and DNA polymerase, respectively. The biotin-labeled probe was synthesized by T7 RNA polymerase with double-strand cDNA template. The probe was fragmented and hybridized with Arabidopsis ATH1 GeneChip at 16oC for 16 hr. The GeneChip was washed and scanned by afftmetrix scanner 3000. The data were analyzed by use of the Genespring software (ver 7.3, Agilent Technologies). To improve the sensitivity of microarray applied to heterologous species, we used an open-source software (http://affymetrix.arabidopsis.info/xspecies/) to mask poorly hybridized probe sets. We used the intensity threshold 400 as recommended (Hammond et al., 2005) to calculate the RNA accumulation level. The hybridization intensity value was calculated by Genespring software and defined as arbitrary unit (AU).
RNA de-capping assay
The broccoli phloem-sap RNA was pre-treated with DNase I (Invitrogen, CA) to remove potential DNA contamination. For mRNA de-capping assay, the DNase I-treated phloem-sap RNA was incubated with tobacco acid pyrophosphatase (TAP, Invitrogen, CA) to remove the 5'-cap. The purified TAP-treated RNA was subsequently treated with terminator 5'-phosphate-dependent exonucle-ase (TerExo, Epicentre). For control experiments, the DN-ase I-treated phloem-sap RNA was directly incubated with TerExo. After treatment, the phloem-sap RNA was puri­fied and underwent RT-PCR with SuperScipt III reverse transcriptase (Invitrogen, CA) and oligo (dT)20 primer ac­cording to the manufacturer's recommendations. The PCR program was as follows: 94°C for 5 min (1 cycle); 94°C for 30 sec, 60°C for 30 sec, 68°C for 30 sec (30 cycles); and 68°C for 7 min (1 cycle). Gene specific primers are listed in Table 1.
RT-PCR analysis
The first-strand cDNA was synthesized from total RNA extracted from phloem sap or leaf tissues with use of Su­perScript III reverse transcriptase (Invitrogene, CA) and oligo (dT)20 primer according to the manufacturer's rec­ommendations. An aliquot of first-strand cDNA was used for PCR with the following conditions: 94°C for 5 min, 1
cycle; 94°C for 30 sec, 60°C for 30 sec, 68°C for 90 sec,
30 cycles; 68°C for 7 min, 1 cycle. The sequences of gene-specific primers used for RT-PCR are in Table 1.
Plasmid construction and Arabidopsis transformation
The full-length cDNA of Arabidopsis FVE and AGL24 (including the 5' and 3' untranslated regions [UTRs]) was amplified by PCR with gene-specific primers (Table 1). The PCR products were cloned and confirmed by sequencing. FVE and AGL24 were driven by a CaMV35S
YANG and YU ― RNA movement of FVE and AGL24 19
Table 1. Primers used in PCR analysis.
Primers Sequence
AtFVE
For:
5'
GGATCCGAAGAGAGAGAGATATAGAGACAC
Rev:
5'
GGGCCCACAGAGAAGGAATCATTAGGTTCA
AtAGL24
For:
5'
CCCGGGACAAACACAGTCACCATCTCTCTC
Rev:
5'
AGATCTGGTGCAAAATAGTTATAAAGACCA
AtCO
For:
5'
TCTAGAATTAGCCCCTTCTTTCAGATACCA
Rev:
5'
AGATCTAAAGAAGAATACTATAGTTTTAAT
AtFVE-RT
For:
5'
TTGAACATCTGGGATTATGAC
AtAGL24-RT
For:
5'
AGACAAAACCAAGCAGCTACG
AtCO-RT
For:
5'
CCTCAGGGACTCACTACAACGACAA
NOS term
Rev:
5'
GTGGTGGTGGTGGTGGCTAGCGTTAAC
AtIMP-a-RT
For:
5'
ATGTCACTGAGACCCAACGCTAAGACGGAG
Rev:
5'
GAATTAGTCGTTCAAGGGCGGGAAGAGCAGG
BoACX1-RT
For:
5'
GCCTGCCTTTGTTGATCTTC
Rev:
5'
TACCCATCTTCGTCCCGATA
BoAGL24-RT
For:
5'
GGGAAGGTATAATCTTCATGCAAGTAAC
Rev:
5'
TGTCATAGCTTGACACATTTGTGGTC
BoAP1-RT
For:
5'
ACTGGTCGATGGAGTATAACAGGCTT
Rev:
5'
AGCAGCCAAGGTTGCAGTTGTAAACG
BoCO-RT
For:
5'
AACGTGTTCCTATTCTCCCAATCTC
Rev:
5'
CTCCATATCCTGTGTTGAACATTATC
BoEBF2-RT
For:
5'
GCTCCTTGCGGTCTTTATCA
Rev:
5'
TTTATCCGTGATCCCAGAGC
BoEIN4-RT
For:
5'
GAGCCTCGACCAGAAGATCA
Rev:
5'
CGAGCGAATCATCAGACAAA
BoFAD3-RT
For:
5'
GGTCAATAATGTTGGCCACT
Rev:
5'
ACTTGCGACCAAACTCTCCA
BoFLC-RT
For:
5'
GAAAGCTCGTCAGCTTTCTGTTCTCT
Rev:
5'
CAAAGCCTGATTCTCTTCTTTCAGCA
BoFVE-RT
For:
5'
GCGCATGATGCTGATCTTCATTGTG
Rev:
5'
GGTCTGTAAATCAAGTCACTCATCC
BoLOX2-RT
For:
5'
ACTTTCCCGTCCCGTTCTTGG
Rev:
5'
GATTGTCGTGCCCGTGAATGC
BoRbcS-RT
For:
5'
TTCACCGGCTTGAAGTCATC
Rev:
5'
CCGGGTACTCCTTCTTGCAT
BoSEN1-RT
For:
5'
TTTAAACACAACCGCACGAA
Rev:
5'
AGACGGATGTCCGATACTGAA
BoThio-h-RT
For:
5'
CTCAAGGCAGCCAAAGAATC
Rev:
5'
ATGGCCTGAACATCGAACTC
BoWRKY40-RT
For:
5'
CCGAAGCTTCTGACACTACCC
Rev:
5'
GCTGCTAATGCTGCTGTGAA
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Botanical Studies, Vol. 51, 2010
promoter and introduced into the Agrobacterium tumefaciens strain AGL1. Arabidopsis was transformed by the floral dip transformation method (Clough and Bent, 1998). The transformants were selected on MS medium containing 40 fig/mL hygromycin.
Arabidopsis inflorescence grafting
Arabidopsis transformant stocks were grown for 4-5 weeks until bolting. The primary inflorescences of stocks were cut vertically 3-4 cm above the base. The scions were obtained from wild-type inflorescences with 2 to 3 lateral buds and cut into a V shape under microscopy. Both scion and stock were merged together with a piece of polyethylene tubing for physical support. The junction of the scion and stock was sealed with Parafilm and kept under high humidity for 7 days. The RNA samples were extracted at 14 days after grafting.
results
Phloem sap collection from broccoli (Brassica oleracea)
To investigate whether the RNAs of floral regulators are present in phloem sap, we chose broccoli (Brassica oleracea) as a system for phloem sap collection. Excised broccoli inflorescences showed high sucrose content (134.8±15.0 mg/mL) but low glucose content (5.7±0.1 mg/ mL) in exudates, which suggests that the exudates were largely derived from phloem. To test whether the exudates were contaminated by ruptured surrounding tissues during phloem sap collection, RT-PCR with primers of Rubisco small subunit (RbcS), a mesophyll-expressed gene, or Thioredoxin-h (Thio-h), a confirmed phloem-sap RNA (Giavalisco et al., 2006; Sasaki et al., 1998), were used. RT-PCR with Thio-h primers obtained signals from both phloem-sap and leaves (Figure 1). In contrast, when RbcS primers were used for RT-PCR with 30 cycles, the PCR products were detected only in RNA extracted from leaves but not phloem sap (Figure 1). However, when PCR was conducted with 35 cycles, a weak signal was detected from phloem sap sample (data not shown, Figure 2).
Figure 2. RT-PCR analysis of phloem-sap RNA. Total RNA was extracted from broccoli phloem sap and mature leaf (A) or flower apex (B).
Thus, although the phloem sap was contaminated by the surrounding tissues, the contamination was relatively low abundant.
Detection of floral regulators RNA in phloem sap
To analyze whether the RNA of floral regulators are present in the phloem sap, the broccoli phloem-sap RNA was hybridized with Arabidopsis ATH1 GeneChip. As shown in Table 2, the hybridization intensity (represented as arbitrary unit, the definition of the arbitrary unit was described in the materials and methods) of FVE and AGL24 in the phloem sap was significantly higher than that of other floral regulators, which suggests the presence
of FVE and AGL24 RNAs in the phloem sap.
To further verify whether the RNAs of FVE and AGL24 were present in the phloem sap, RT-PCR was conducted. When PCR was performed with 35 cycles, the signals
Figure 1. RT-PCR analysis of RNA extracted from broccoli leaf and phloem sap with use of gene-specific primers for Rubisco small subunit (RbcS), or Thioredoxin-h (Thio-h).
YANG and YU ― RNA movement of FVE and AGL24
21
of FVE and AGL24 were detected in both phloem sap and leaves (Figure 2). In contrast, the RT-PCR products of FLOWERING LOCUS C (FLC), CONSTANS (CO), SENESCNECE 1 (SEN1), APETALA1 (AP1) and RbcS were detected in leaves but only at a background level in phloem sap, which suggests that the RNAs of FLC, CO, SEN1 , and AP1 were not present in phloem sap, or low abundant in the phloem sap.
To rule out the possibility that the RNAs detected in phloem sap could be the result associated with RNAs
highly accumulated in the leaves, we selected the genes with different RNA accumulated level in phloem sap and tested the RNA accumulation level of these RNA in the leaves. Based on our result of phloem-sap microarray analysis, we randomly chose 20 genes with different level of RNA accumulation in phloem sap and subjected to RT-PCR analysis. The results showed that the level of RNA accumulation in phloem sap was not related to that in leaves (Figure 3, 6 out of 20 genes was shown in the figure), which suggests that the RNAs we detected in the sap were most likely actively transported into the sap, or alternatively, were more stable in the sap.
Phloem sap RNA has a 5' cap and is polyadenylated
Result of RT-PCR analysis of phloem-sap RNA by use of cDNA synthesized with an oligo (dT) primer suggested that the phloem-sap RNA contains a poly A tail (Figure
1B, 2A, and 3). The RT-PCR performed with oligo d(T)
failed to amplify signal by using in vitro synthesized RNA, further confirmed that the phloem-sap RNA is polyadenylated (data not shown). To examine whether the phloem-sap RNA also contains a 5' cap similar to a typical mRNA, the de-capping assay was performed. The 5' cap protects mRNA from degradation by terminator exonuclease (TerExo, Wierzbicki et al., 2008). Tobacco acid pyrophosphatase (TAP) removes the 5' cap structure and allows RNA to be completely degraded by TerExo (Figure 4A). In our control experiments, after in vitro synthesized RNA (IVT) was incubated with TerExo, RT-PCR failed to produce positive signals, which suggests that the un-capped RNA was completely degraded when being incubated with TerExo (Figure 4B). In contrast, when phloem-sap RNA was incubated with TerExo, the
Figure 3. RT-PCR analysis of RNA accumulation level in phloem sap and leaves.
A B
Figure 4. Enzymatic analysis of phloem-sap RNA. (A) Illustration of mRNA de-capping assay. Typical mRNA contains 5' cap (m7G), which is resistant to degradation by terminator exonuclease (TerExo). Tobacco acid pyrophosphatase (TAP) removes the 5' cap and allows the RNA to be degraded by TerExo. (B) RT-PCR analysis of Thio-h, FVE and AGL24 in TAP- and TerExo-treated phloem-sap RNA. For control experiments, the in vitro synthesized RNA (IVT) was used.
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Botanical Studies, Vol. 51, 2010
RNA of Thio-h, FVE and AGL24 can be detected by RT-PCR, suggesting that a population of the phloem sap RNA was TerExo resistant (Figure 4B). However, when phloem-
sap RNA was treated with TAP and TerExo, RT-PCR did
not produce detectable signals, suggesting that the TerExo resistant RNA was 5' capped (Figure 4B). Taken together, our results suggest that the phloem-sap RNAs contain a population of typical mRNAs that have a 5' cap and a polyadenlyation tail.
Arabidopsis FVE and AGL24 mRNA move long distance from stock to scion
To test whether the RNAs detected in the phloem sap move long distance, Arabidopsis grafting experiments were conducted. The full-length cDNAs of FVE and AGL24 (containing 5' and 3' UTRs) were PCR amplified and dri ven by a CaMV35S promoter. For control experiments, the full-length cDNA of CO was amplified, because CO RNA acts in a cell-autonomous manner (An et al., 2004; Ayre et al., 2004). Similar to previous results, Arabidopsis transformants carrying P35S-AGL24, P35S-FVE or P35S-CO transgenes displayed an early flowering phenotype, which indicates the functions of these genes are to promote flowering (Figure 5A). Consistent with the early flowering phenotype, RT-PCR analysis of transformants showed a high expression of the transgenes (Figure 5B). These transformants were used as stocks and grafted to wild-type scions. Two weeks after grafting, the scion apices and stock leaves were harvested for RNA extraction and subjected to RT-PCR analyses. The PCR products were amplified from stocks and scions when FVE or AGL24 gene-specific primers were used (Figure 5B). In contrast, the PCR products were obtained from stock but not wild-type scion when CO gene-specific primers were used (Figure 5B). Therefore, the mRNAs of FVE and AGL24 but not the mRNA of CO move long distance in grafted Arabidopsis.
discussion
In current studies, we have identified at least two floral regulators (FVE and AGL24) from broccoli phloem sap (Table 2 and Figure 2). Our Arabidopsis grafting experiments showed that the RNAs of FVE and AGL24 but not the RNA of CO move long distance across the graft union (Figure 5), which suggests that FVE and AGL24 are phloem-mobile RNAs. The trafficking of FVE and AGL24 RNAs may function as a systemic signal to communicate the environmental inputs to the apices and regulate floral initiation. Arabidopsis FVE encodes a retinoblastoma-associated protein that represses FLC transcription through a histone deacetylation mechanism (Ausin et al., 2004; Kim et al., 2004). AGL24 encodes a MADS-domam protein that functions as a floral activator (Yu et al., 2002). Both FVE and AGL24 participate in floral regulation by the cold response (Ausin et al., 2004; Kim et al., 2004; Michaels et al., 2003). Interestingly, classic physiological
Figure 5. Arabidopsis grafting experiments with wild-type scions and transformant stocks. (A) Flowering time of Arabidopsis transformants harboring P35S-FVE, P35s-AGL24, and P35S-CO. (B) RT-PCR analysis of RNA from Arabidopsis wild-type plants (Col), wild-type scions (SC) grafted onto transformant stock, or transformant stocks (ST) harboring P35S-FVE, P35s-AGL24, or Ps^CO transgene. IMPORTIN-a (IMP-a) was used as a loading control.
experiments have demonstrated that the regulation of flowering by the cold response appears to act at the whole plant level (Zeevaart, 1976). In addition, the ectopic expression of FLC in the companion cells showed that FLC represses at least two systemic signals produced in the leaves, one of the signals being FT independent (Searle et al., 2006). We proposed that the trafficking of the FVE and AGL24 RNA may incorporate the temperature variation that was perceived in the leaves to the apex and regulate floral initiation. Whether FVE and AGL24 are required for the cold signal received in the leaves remains to be investigated.
Similar to typical mRNAs present in cells, our de-capping assay showed that the mRNAs in broccoli phloem sap contains a 5' cap and a polyadenylation tail (Figure 4). Although significant amount of the phloem-sap RNA were degraded when phloem sap was incubated with TerExo, it remains a population phloem sap RNA to be TerExo resistant. It is possible that most of the phloem-sap RNAs were truncated during RNA extraction, or alternatively, only part of the phloem sap RNAs contain 5' cap structure. Because the RNA in the phloem sap seems to be identical
YANG and YU ― RNA movement of FVE and AGL24
23
Table 2. Floral regulatory genes in phloem sap and accession numbers.
Gene
AGI
AUa
Description
FVE
At2g19520
1091.9
WD-40 repeat family protein, AtMSI4
AGL24
At4g24540
765.1
MADS-box protein, AGAMOUS-LIKE 24
GI
At1g22770
70.8
GIGANTEA
UFO
At1g30950
65.2
Ubiquitin-protein ligase, Unusual Floral Organ
ESD4
At4g15880
56.6
Cysteine-type peptidase, Early in Short Days 4
FKF1
At1g68050
30.9
Ubiquitin-protein ligase, Flavin-binding Kelch domain F-box protein 1
FRI
At4g00650
28.8
FRIGIDA
TOC1
At5g61380
27.1
Transcription regulator, Timing of CAB expression 1
ZTL
At5g57360
17.6
Ubiquitin-protein ligase, ZEITLUPE
FLC
At5g10140
16.1
MADS-box protein, FLOWERING LOCUS C
FCA
At4g16280
15.5
RNA binding
ELF4
At2g40080
14.8
EARLY FLOWERING 4
VIP4
At5g61150
14.4
VERNALIZATION INDEPENDENCE 4
LFY
At5g61850
14.3
LEAFY
CO
At5g15840
13.9
Zinc finger protein, CONSTANS
VRN2
At4g16845
13.7
Polycomb group protein, REDUCED VERNALIZATION RESPONSE 2
SVP
At2g22550
13.6
MADS-box protein, SHORT VEGETATIVE PHASE
FWA
At4g25530
13.4
Homeo-box protein
AP1
At1g69120
13.3
Transcription factor, APETALA1
TFL1
At5g03840
13.2
TERMINAL FLOWER 1
ELF3
At2g25930
12.4
Transcription factor, EARLY FLOWERING 3
VRN1
At3g18990
11.6
REDUCED VERNALIZATION RESPONSE 1
FY
At5g13480
9.4
WD-40 repeat family protein
FD
At4g35900
9.2
DNA-binding protein-related, ATBZIP14
VIN5
At3g24440
9.2
VIN3-LIKE 1
FLD
At3g10390
8.3
FLOWERING LOCUS D
LHY1
At1g01060
8.1
MYB protein, LATE ELONGATED HYPOCOTYL 1
EMF1
At5g11530
7.2
EMBRYONIC FLOWER 1
CCA1
At2g46830
6.1
MYB protein, CIRCADIAN CLOCK ASSOCIATED 1
LD
At4g02560
5.9
Homeo-box protein, LUMINIDEPENDENS
AGL19
At4g22950
5.7
AGAMOUS-LIKE 19
SOC1
At2g45660
5.4
SUPPRESSOR OF OVEREXPRESSION OF CO 1
aAU: Arbitrary Unit.
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to that within the cell, thus, what is the molecular basis for the determination of RNA long-distance trafficking? It has been proposed that the phloem-mobile RNAs may contain the putative motif required for long-distance movement (Lucas et al., 2001). Given that thousands of RNAs may be traveling through the phloem translocation stream (Long and Lucas, 2006), the possibility of only a conserved motif present in all of the phloem-mobile RNAs is unlikely. Thus, multiple RNA transport mechanisms and RNA mobile motifs may be involved in transporting these non-cell-autonomous RNAs. Recently, we showed that the RNA of GA-INSENSITIVE (GAI), a phloem-mobile RNA, contains RNA motifs required for RNA movement (Huang and Yu, 2009). The primary sequences of the putative RNA mobile motifs failed to reveal conserved motifs from other phloem-sap RNA, which is consistent with the notion that different RNA mobile motifs may participate in RNA movement. However, we cannot exclude that the structure of the RNA motifs is responsible for RNA long-distance trafficking. Therefore, more detailed analysis is required to understand the molecular nature of RNA mobile motifs.
Our Arabidopsis grafting experiments showed that the capacity for RNA long-distance movement is associated with the presence of the RNAs in the phloem sap (Figure 5). Thus, a large number of the phloem-sap RNAs may be able to move long distance. In addition to detecting the floral regulators, we also detected many other RNAs, which involved in diverse processes, in broccoli phloem sap. Thus, our system provides a genome-wide approach for investigating the non-cell-autonomous RNA.
Acknowledgments. We thank Hsueh-Huei Chen for helping collect broccoli phloem sap. Min-Yan Kuo and Jin-Yao Lai from the Affymetrix Gene Expression Service Lab for GeneChip hybridization and data output. Shu-Jen Chou and Pei-Chen Yu for suggestions on microarray analysis, and all the lab members for critical comment on the manuscript. This work was supported by grants NSC
95-2311-B-001-060 and NSC 96-2311-B-001-021-MY3
from the National Science Council, Taiwan.
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Botanical Studies, Vol. 51, 2010
阿拉伯芥開花調控基因FVE與AGI24為篩管中之
移動性RNA
楊鴻偉12 余天心2
1國防醫學院生命科學研究所
2中央研究院植物暨微生物學研究所
植物運用維管束系統來面對環境中多樣的刺激，以調節自身的生長發育。近來的研究證實，植物在
葉部整合光週期的變化並利用維管束系統長距離輸送 FLOWERING LOCUS T (FT) 至頂芽，促進植物的
開花。因此，FT被認為是追尋已久的開花素（Florigen)。然而，影響植物開花的因素除了光週期外，
證據顯示尚有其他基因在植物開花的路徑中扮演系統性的調控因子。我們分析青花菜收取之篩管液，
企圖找出潛在的系統性開花調控因子。利用基因微陣列（Microarray)及反轉錄聚合酶連鎖反應 (RT-PCR)
的分析方法，從青花菜篩管液中鑑定出 FVE 與 AGAMOUS-LItKE 24 (AGL24) 兩個與開花調控有關的
RNA 。酵素學上的分析證明 FVE 與 AGL24的RNA 帶有5'-cap結構及polyadenylation tail ，為典型之訊
息RNA (mRNA)。文獻資料顯示，大量表現FVE與AGL24可促進植物提早開花。我們以野生型阿拉伯
芥為接穗嫁接至FVE或AGL24轉殖株系之砧木上。利用RT-PCR的方式，可由野生型接穗偵測出FVE
或AGL24轉殖基因之RNA 。顯示FVE與AGL24RNA可經由維管束系統進行長距離運送。我們的實驗
結果顯示，除了 FT ，尚有多個基因對於植物開花具有系統性的調控。
關鍵詞：阿拉伯芥；青花菜；開花；嫁接；篩管液；RNA長距離移動。