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First published online April 30, 2007
doi: 10.1242/10.1242/dev.001891

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Arabidopsis homologs of components of the SWR1 complex regulate flowering and plant development

¹ National Research Laboratory of Plant Developmental Genetics, Department of Biological Sciences, Seoul National University, Seoul, 151-742, Korea.
² Global Research Laboratory for Flowering at SNU and UW, Seoul, 151-742, Korea.
³ Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju 660-701, Korea.
⁴ Plant Metabolism Research Center, Kyung Hee University, Suwon 449-701, Korea.

View larger version (88K): [in this window] [in a new window]   Fig. 1. Mutations in AtSWC6 and SUF3 cause the same phenotype. (A) Genomic structure of AtSWC6 and T-DNA location in the atswc6 mutant (SAIL_1142/CS841940). Black and gray boxes indicate exons and untranslated regions, respectively; lines between these boxes indicate introns; white triangle indicates T-DNA insertion; arrowhead indicates transcription start site. (B) RT-PCR analysis of AtSWC6, FLC and SUF3 expression in wild type and atswc6 mutant. RNA was extracted from 30-day-old Arabidopsis plants grown in long-day conditions. TUBULIN (TUB) was used as an internal control. (C) Northern blot analysis of AtSWC6 and FLC expression. RNA was extracted from 10-day-old FRI, atswc6 FRI, suf3 FRI and Col. The AtSWC6 transcript was not detected in the atswc6 mutant, indicating that it is a null allele. (D) Flowering time of FRI, atswc6 FRI and suf3 FRI and of Col (fri), atswc6 fri and suf3 fri grown under long-day conditions. (E) Morphology of wild type (Col) and atswc6 grown for 20 days under long-day conditions. (F) Morphology of FRI, suf3 FRI, atswc6 FRI and suf3 atswc6 FRI grown for 30 days under long-day conditions. (G) Leaf shape of wild type (Col) and atswc6 mutant. (H) atswc6 flower with five petals. Scale bars: in E and G, 1 cm.

View larger version (40K): [in this window] [in a new window]   Fig. 2. Phenotype of AtSWC6 overexpression line and chromatin immunoprecipitation. (A) Col, atswc6 and 35S-myc:AtSWC6 atswc6 transgenic Arabidopsis plants grown for 35 days. (B) Col, atswc6 and 35S-AtSWC6:GFP atswc6 transgenic plant grown for 35 days. (C) Number of rosette leaves at flowering of Col, atswc6, 35S-myc:AtSWC6 atswc6 and 35S-AtSWC6:GFP atswc6. All plants in A-C were grown in long-day conditions. (D) Expression of FT, SOC1 and FLC in Col, suf3, atswc6, 35S-myc:AtSWC6 atswc6 and 35S-AtSWC6:GFP atswc6 grown in short-day conditions for 4 weeks. Early flowering of atswc6 and suf3 is caused by the increased expression of the floral integrators FT and SOC1. (E) Col and 35S-myc:SUF3 suf3 (for SUF3 protein) or 35S-myc:AtSWC6 atswc6 (for AtSWC6 protein) transgenic seedlings grown under long-day conditions were used for ChIP assay using anti-myc antibody. Above is a map of the FLC gene showing the location of the regions examined in the FLC promoter (FLC-A for -1288 to -1470, FLC-B for -360 to -497, FLC-C for -45 to -164, relative to the ATG codon) and first intron (FLC-V for +1469-+1607). TUB was used as an internal control (white bars). The x-axis indicates relative enrichment.

View larger version (82K): [in this window] [in a new window]   Fig. 3. Spatial and temporal expression of AtSWC6. (A) Northern blot analysis of AtSWC6 expression in different Arabidopsis tissues. RNA was extracted from 20-day-old Col grown in long-day conditions for whole plants (Wh), root (Rt), shoot apex (SA) and rosette leaves (RL). RNA for old leaves (OL), stem (St) and inflorescence with flowers (Inf) was extracted from 40-day-old plants. (B) Temporal expression of AtSWC6 assessed by RT-PCR. RNA was extracted from 5-, 8-, 11- and 14-day-old Col grown in long-day conditions. All the samples were harvested 4 hours after light on. (C) Diurnal rhythm of AtSWC6 expression assessed by RT-PCR. RNA was extracted from 10-day-old Col grown in long-day conditions. Northern blot analysis with the same RNA showed diurnal rhythm in SUF3 expression. (D) The effect of photoperiod and vernalization on AtSWC6 expression. FRI with (+) and without (-) vernalization, and Col grown under long-day (LD) and short-day (SD) conditions were used for northern blot and RT-PCR analyses. For RT-PCR analysis, TUB expression served as an internal control. (E) Spatial expression patterns of AtSWC6 were examined by GUS staining in the AtSWC6p-AtSWC6:GUS transgenic plant. Histochemical GUS staining was performed in whole (a) 3-, (b) 5- and (c) 10-day-old plants, in (d) inflorescence and flowers, (e) an axillary bud, (f,g) developing seeds and (g) siliques of different stages.

View larger version (56K): [in this window] [in a new window]   Fig. 4. Interaction of atswc6 with other flowering-time mutants. Comparison of Arabidopsis flowering phenotype in (A) ld-1 and ld-1 atswc6,(B) fca-9 and fca-9 atswc6, (C) flc-3 and flc-3 atswc6, (D) ft-1 and ft-1 atswc6. Photographs were taken after 40 days in A and B, 20 days in C and 35 days in D. All plants were grown in long-day conditions. (E) Northern blot analysis of FLC and SOC1 in single and double mutants. The transcript level of FLC was reduced by approximately half in double mutants (fca-9 atswc6, ld-1 atswc6) as compared with each single mutant. (F) RT-PCR analysis of FLC and SOC1 in ft-1 and ft-1 atswc6. TUB was used as an internal control. In E and F, RNA was extracted from 10-day-old plants grown in long day conditions.

View larger version (36K): [in this window] [in a new window]   Fig. 5. AtSWC6 protein is colocalized with SUF3 in nucleus. Upper panels, Arabidopsis protoplast expressing AtSWC6-GFP and AtSWC6-RFP simultaneously. Lower panels, protoplast expressing SUF3-GFP and AtSWC6-RFP simultaneously. The right-most panels are merged images. The left-most panels show bright-field images of protoplasts.

View larger version (101K): [in this window] [in a new window]   Fig. 6. Arabidopsis protein interactions analyzed by yeast two-hybrid and coimmunoprecipitation assays. (A) Interactions between SUF3, AtSWC6 and AtSWC2 (SWC2). (B) Interactions between SUF3, AtSWC6, AtSWC2 and HTA8, HTA9 and HTA11. (C) The same interaction analysis as B but with baits and preys changed. (D) Interaction between regions of PIE1 and SUF3, AtSWC6, AtSWC2, HTA8, HTA9 and HTA11. PIE1-N, PIE1-M and PIE1-C indicate PIE1 regions comprising amino acids 1-520, 520-1220 and 1220-2055, respectively. (E) The same interaction analysis as D but with baits and preys changed. (F) Protein gel blots showing interaction between AtSWC6 and SUF3. IP indicates immunoprecipitation of protein extracts from tobacco leaves transiently co-expressing epitope-tagged proteins, as listed above the blot, with anti-GFP (-GFP) or anti-Flag (-Flag) antibodies, as indicated below the blot. About 10% of the total sample used in each IP was loaded as an input control. Mock indicates IP with anti-Flag antibody. The immunoprecipitates and protein extracts were separated by 9% SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with anti-myc antibody. (G) Protein gel blots showing interaction between AtSWC6, AtSWC2 and HTA11. Tobacco leaves co-expressing myc:SUF4 and AtSWC6:GFP were used as a negative control of IP. The protein blots were probed with anti-GFP antibody. (H) Protein gel blots showing interaction between PIE1 and SUF3 and AtSWC6. Mock indicates IP with no antibody. The protein blots were probed with anti-Flag (left) or anti-myc (right) antibodies.

View larger version (55K): [in this window] [in a new window]   Fig. 7. Knockdown of H2AZ causes a similar phenotype to atswc6. (A) The RNAi construct targeting all three Arabidopsis H2AZ genes - HTA8, HTA9 and HTA11 (AtH2AZs). The tandemly fused HTA8, HTA9 and HTA11 cDNAs were arranged as inverted repeats which were separated by a spliceable intron. The construct was inserted between the CaMV 35S promoter and the OCS terminator. (B) FRI and AtH2AZs RNAi transgenic plants grown for 45 days under long-day conditions. (C) Distribution of flowering time in AtH2AZs RNAi T1 transformants. Plants were grown in long-day conditions. The number of plants in each category is given above each bar. (D) Expression of HTA8, HTA9, HTA11 and FLC in three AtH2AZs RNAi lines that flowered with 35, 30 and 30 leaves. Homozygous lines were selected from the T2 generation of the three transgenic plants and RNA was extracted from 10-day-old plants grown in long-day conditions. All three AtH2AZs RNAi lines showed reduced expression of HTA8, HTA9 and HTA11. The transcript level of FLC was also reduced. (E) The relative levels of HTA8, HTA9 and HTA11 transcripts in the three AtH2AZs RNAi lines as compared with FRI wild type. (F) FRI, atswc6 FRI, amiR-HTA8, amiR-HTA9, amiR-HTA11 and amiR-HTA9&11 T1 plants grown for 40 days after germination in long-day conditions. (G) RT-PCR analysis of expression of HTA8, HTA9, HTA11 and FLC in FRI, atswc6 FRI, amiR-HTA8, amiR-HTA9, amiR-HTA11 and amiR-HTA9&11 T1 plants. RNA was extracted from leaves of T1 plants. TUB was used as an internal control.