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First published online November 3, 2003
doi: 10.1242/10.1242/dev.00814

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Regulation of storage protein gene expression in Arabidopsis

Thomas Kroj*, Gil Savino, Christiane Valon, Jérôme Giraudat and François Parcy{dagger}

Institut des Sciences du Végétal, UPR2355 Centre National de la Recherche Scientifique, Avenue de la Terrasse, 91190 Gif-sur-Yvette, France



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  		Fig. 1. FUS3 and LEC2 activate the At2S3 promoter. (A) Comparison of nucleotide sequences of cis-elements present on At2S3 (upper sequence) and Brassica napus napA (bottom sequence) promoters. The B-box element contains DistB and ProxB, the RY-G-box complex contains a G-box surrounded by 2 RY motifs. (B) FUS3 and LEC2 bind to the RY-G-box complex in the At2S3 promoter. Yeast reporter strains B::HIS3 and RY-G::HIS3 carrying either the control plasmid pCV70 or expressing FUS3 or LEC2 were streaked (as depicted on the right) on medium containing histidine (+His) or restrictive medium (–His, + 1 mM 3-AT). (C) FUS3 protein forms a gel retardation complex (arrow) with both RY elements. FUS3 protein was incubated with a radiolabelled fragment of the At2S3 promoter (–98 to –48 relative to transcription start) containing both RY elements and the G-box. The reactions contain in vitro transcribed and translated control plasmids (lane 1) or FUS3 expression plasmid (lanes 2-17). Non-labelled competitor DNA (see Materials and methods) was added in 5-fold molar excess (lanes 3, 6, 9, 12, 15), 10-fold molar excess (lanes 4, 7, 10, 13, 16) and 20-fold (lanes 5, 8, 11, 14, 17). Competitor DNA used was wild type (lanes 3-5), mutant RY1 (lanes 6-8), mutant RY2 (lanes 9-11), mutant RYs (lanes 12-14) and mutant G-box (lanes 15-17).

 


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  		Fig. 2. abi3, fus3 and lec2 mutations affect At2S3 expression in seeds. (A) Northern blot analysis of At2S3 and At2S3::GFP expression. All plants analysed are homozygous for the At2S3::GFP transgene introduced in wild-type Col-0 (lanes 1-3), abi3 (lanes 4-6), lec2 (lanes 7-9) and fus3 (lanes 10-12). Total RNA was extracted from 11 DAP siliques (lanes 1, 4, 7, 10), 12 DAP siliques (lanes 2, 5, 8, 11) and 13 DAP siliques (lanes 3, 6, 9, 12). (B) The At2S3 expression pattern is altered in abi3, fus3 and lec2 mutants. Non-radioactive in situ hybridisation with an At2S3 probe was performed on 11-13 DAP seeds of the same genotypes as in A: Col-0 (panels 1 and 7), fus3 (panels 2, 3, 8), abi3 (panels 4 and 9), and lec2 (panels 5, 6, 10) homozygous for the At2S3::GFP transgene. In wild type, expression was detected in both cotyledons (c), the embryo axis (a) and endosperm cell layer (e). Arrows in panels 5 and 6 indicate sectors of lec2 cotyledons with reduced At2S3 expression. Scale bars: 50 µm (1 to 6) and 25 µm (7-10).

 


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  		Fig. 3. At23S::GFP expression pattern in abi3, fus3 and lec2 mutants. Seeds homozygous for the At2S3::GFP transgene were used to visualise the At2S3 promoter activity in wild-type Col-0 (A,E,I,S), abi3 (B,F,J,K,T), fus3 (D,H,L,M,V), lec2 (C,G,N-R,U). (A-D) Seeds harvested just before complete desiccation (yellow siliques, 15-16 DAP) visualised under white light. (E-H) Green fluorescence of the seeds shown in A-D. (I-R) Green fluorescence of isolated embryos extracted from seed coats at 11-13 DAP. All embryo images were taken with the same exposure except K,which is identical to picture J but exposed 10 times longer to visualise the low At2S3::GFP fluorescence present in abi3. (S-V) Green fluorescence signal present in the seed envelope at 16 DAP and arising from the endosperm layer. Scale bars: 500 µm (A-H) and 50 µm (I-W).

 


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  		Fig. 4. Storage protein gene expression is dramatically reduced in lec2 fus3 double mutant. (A,B) At2S3::GFP seeds in Col-0 background (top) and lec2 fus3 At2S3::GFP seeds (bottom) at 16 DAP visualised under white light (A) or blue light (B). (C-E) Green fluorescence in lec2 fus3 At2S3::GFP (D) embryos as compared to At2S3::GFP Col-0 (C) at 14-16 DAP. (E) Same embryo as in D but under white light to show the presence of green and purple pigments in cotyledons. (F) Quantification of storage protein gene expression by northern blot analysis of seed RNA from wild-type, abi3, fus3, lec2 and lec2 fus3 seeds in the At2S3::GFP background. Specific probes used were At2S1 (lanes 1), At2S2 (lanes 2), At2S3 (lanes 3), At2S4 (lanes 4), CRUCIFERIN A (lanes a), CRUCIFERIN B (lanes b), CRUCIFERIN C (lanes c). (G) Analysis of ABI3 expression by western blot of total protein extracts from wild-type, abi3, fus3, lec2 and lec2 fus3 seeds.

 


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  		Fig. 5. FUS3 and LEC2 promoter activities in embryos and endosperm. LEC2::GUS (A-F) and FUS3::GUS (G-K) activities were assayed in whole seeds (A,B,H,I: globular stages), isolated embryos (C,D, torpedo stages; E, early mature embryos around 12 DAP; J, heart stage; K, mature embryo before desiccation) or sections of seed envelopes (F,G). FUS3::GUS (M,O,Q) expression pattern and At2S3::GFP green fluorescence (L,N,P) were compared in individual lec2 mutant embryos just before desiccation (at 15-16 DAP). Scale bars: 50 µm. (R) Time course of FUS3 and LEC2 mRNA expression during silique development analysed by RT-PCR. The developmental stage of samples is indicated in DAP. EF1{alpha} was used as a control.

 


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  		Fig. 6. Models for SSP gene regulation. (A) ABI3, FUS3 and LEC2 positively regulate At2S3 expression. We propose that FUS3 and LEC2 act directly in a similar and partially redundant manner. ABI3 is also crucial for At2S3 expression but functions differently from FUS3 and LEC2. In addition, LEC2 is needed to ensure FUS3 uniform expression in wild-type embryos. (B) Speculative model of how FUS3, LEC2 and ABI3 proteins might be acting according to our study and data presented by Lara et al. (Lara et al., 2003Go).

 

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© The Company of Biologists Ltd 2003