Open Access Research article

Arabidopsis mutant sk156 reveals complex regulation of SPL15 in a miR156-controlled gene network

Shu Wei12, Margaret Y Gruber2*, Bianyun Yu23, Ming-Jun Gao2, George G Khachatourians4, Dwayne D Hegedus2, Isobel AP Parkin2 and Abdelali Hannoufa5*

Author Affiliations

1 College of Tea & Food Science and Technology, Anhui Agricultural University, 130 Changjiang Blvd West, Hefei, 230036, China

2 Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK, S7N 0X2, Canada

3 Current address: Plant Biotechnology Institute, National Research Council of Canada, 110 Gymnasium Place, Saskatoon, SK, S7N 0W9, Canada

4 Department of Food and Bioproduct Sciences, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK, S7N 5A8, Canada

5 Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, N5V 5T3, Canada

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BMC Plant Biology 2012, 12:169  doi:10.1186/1471-2229-12-169

Published: 18 September 2012

Additional files

Additional file 1:

Schematic diagram of disrupted carotenoid cleavage dioxygenase genes CCD7 and CCD8 in the max3-9 and max4-1 mutants used in this study. Boxes represent exons and lines represent introns. Triangles show the T-DNA insertion sites. The max mutants were previously reported by (Booker et al. 2004).

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Additional file 2:

SALK and FLAG T-DNA insertion lines for miR156-targeted SPL genes.

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Additional file 3:

Reduced lengths of cauline stem basal internodes in three spl15 mutants compared with WT Arabidopsis. Length of the cauline stem basal inter-node was measured from the rosette core up to the first visible basal node for WT, three spl mutants and sk156 plants grown for 6 weeks.

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Additional file 4:

Confirmation by PCR of transgene presence in sk156 lines transformed with a 35S:SPL15m gene. The miR156 insensitive SPL15m contained 11 mutated nucleotides as described in Materials and Methods. Primer sequences are listed in Additional file 5. (A) Activation-tag from pSKI015 T-DNA detected in transgenic sk156 plants carrying 35S:SPL15m cassette (lanes 0-11) and in the sk156 background alone (lanes 12 and 13). Primers SK2222-F (430bp upstream) and SK2222-R (1830bp downstream) flanking the T-DNA insertion site and primer pSKI015-GW-LB2 (439bp to the T-DNA left border) were used to detect the insert. In WT lane, no T-DNA insert was detected and only a fragment close to 1.9kb was present due to genomic DNA amplified with the primers flanking the T-DNA insertion site. In homozygous sk156 plants which did not carry 35S:SPL15m, a single T-DNA fragment (869bp) was generated. 1kb, 1-kb Plus DNA ladder (Invitrogen); WT, Col-4; pSKI015, plasmid containing the activation tag present in sk156. (B) Transgene SPL15m detected in the transgenic sk156 lines carrying 35S:SPL15m (lanes 0-14), but not in sk156 alone using primers 35SF3 and SPL15R.

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Additional file 5:

Primers used in this study.

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Additional file 6:

PCR confirmation of transgene presence in four different miR156-sensitive or miR156-insensitive transgenic Arabidopsis populations used in this manuscript. (A) Lanes 2-8, PCR product (818bp) for 7 transgenic plants carrying a 35S:AT4G30975 cassette in a WT background using primers 35S-F3 and p795-3R. P, binary plasmid pBI121 containing 35S:AT4G30975 as a positive control. WT, Col-4. (B), (C) and (D) Transgene PCR product (636bp) carrying AS1:SPL15m, AS1:SPL15n and SPL:SPL15m cassettes in a sk156 background, respectively, using primers SPL15-871F and NosTer-R6. P, plasmid containing 35S:SPL15 as a positive control. Black arrows points to the DNA marker. Primer sequences are listed in Additional file 5. 1kb, 1-kb Plus DNA ladder (Invitrogen).

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