Article title
Postmeiotic development of pollen surface layers requires two Arabidopsis ABCG-type transporters
Journal name
Plant Cell Reports
Author name
Sojeong Yim1, Deepa Khare1, Joohyun Kang1, Jae-Ung Hwang1, Wanqi Liang3, Enrico Martinoia4, Dabing Zhang3, Byungho Kang5, Youngsook Lee1,2
Affiliation
1Department of Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang, 37673, Korea
2Division of Integrative Biosciences and Biotechnology, POSTECH, Pohang, 37673, Korea
3 Joint International Research Laboratory of Metabolic and Developmental Sciences, Shanghai Jiao Tong University-University of Adelaide Joint Center for Agriculture and Health, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
4Department of Plant and Microbial Biology, University Zurich, 8008 Zurich, Switzerland
5School of Life Sciences, Centre for Cell and Developmental Biology and State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Hong Kong, China
E-mail address of corresponding author
Supplemental Data Figure. 1. Isolation of ABCG1 and ABCG16 knockout mutants.
(a) Positions of T-DNA insertions in ABCG1 and ABCG16 are flagged. ABCG1 and ABCG16 do not possess introns. Arrows indicate the direction of inserted T-DNA.
(b) Genomic DNA PCR analysis of ABCG1 and ABCG16. PCR products with gene-specific primer pairs (Gene; upper) and with a gene-specific primer and a T-DNA-specific primer (T-ins; middle, lower). DNA samples were from wild-type (lanes 1 and 6), abcg1-1 (lane 2), abcg1-2 (lane 3), abcg16-1 (lane 7), abcg16-2 (lane 8), abcg1-1 abcg16-1 (lanes 4 and 9), and abcg1-2 abcg16-1 (lanes 5 and 10) plants.
(c) Expression levels of ABCG1 and ABCG16 in abcg1 abcg16 mutants quantified using qRT-PCR. The expression level of ABCG1 was reduced to 20-40% of the wild-type level in abcg1-1 abcg16-1 (dko-1) and abcg1-2 abcg16-1 (dko-2). ABCG16 transcript was not detected in dko-1 or dko-2.
Supplemental Data Figure. 2. Negative control experiment of AGP deposition.
Immunolocalization analysis of AGPs was done exactly as in Figure 3 except that the primary antibody, JIM13 was omitted. Images were taken under UV and FITC channels, respectively, and a merged image is presented. No FITC signal was observed in both the wild-type and abcg1 abcg16 samples. Blue fluorescence is from calcofluor white stain under UV light. Bar = 20 μm.
Supplemental Data Figure 3. Putative AMS-binding sites in the ABCG1 and ABCG16 promoters. Eight and five AMS-target E-box sequences (CANNTG, Xu et al., 2010) exist within the 1.6-kb promoter regions of ABCG1 and ABCG16, respectively. Numbers are in bp.
Supplemental Data Figure 4. ABCG1 and ABCG16 are required for normal pollen development in Arabidopsis.
(a) The siliques of double knockout mutants of ABCG1 and ABCG16 (dko-1, dko-2) failed to elongate (arrowheads), whereas those of wild type (Col-0) and single abcg1 and abcg16 knockout mutants (g1-1, g1-2, g16-1, and g16-2) elongated normally. Bar = 1 cm.
(b) Morphology of mature pollen grains. Light microscopy analysis revealed no morphological differences in the mature pollen grains of the wild type, abcg1, and abcg16, but showed that abcg1 abcg16 double knockout (dko-1 and dko-2) pollen grains were shriveled and stuck together. It was hard to separate pollen grains from abcg1 abcg16 anthers, since they were stuck to the anther surface. Bar = 20 μm.
Supplemental Data Figure 5. ABCG1 and ABCG16 have high levels of amino acid sequence similarity and identity. The amino acid sequences of ABCG1 and ABCG16 share 82.4% sequence identity and 89.3% sequence similarity as determined by a pairwise sequence alignment tool (http://www.ebi.ac.uk/Tools/psa/emboss_needle).
Supplemental Data Table 1. Primers used in this study