Volume 6, Issue 4, Pages 1091-1108 (July 2013) Pollen-Expressed Transcription Factor 2 Encodes a Novel Plant-Specific TFIIB-Related Protein that Is Required for Pollen Germination and Embryogenesis in Arabidopsis  Qian-Kun Niu, Yan Liang, Jing-Jing Zhou, Xiao-Ying Dou, Shu-Chen Gao, Li-Qun Chen, Xue-Qin Zhang, De Ye  Molecular Plant  Volume 6, Issue 4, Pages 1091-1108 (July 2013) DOI: 10.1093/mp/sst083 Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 1 Characterization of PTF2 Gene. (A) A phylogenetic tree of the TFIIB and TFIIB-related proteins from Arabidopsis. The sequence identities between any two clusters or two proteins can be determined by aligning the vertical lines of the tree with the percentage scale bar. (B) Phylogenetic analysis of PTF2 and the homologous proteins with the highest similarity from seven plant species (www.ncbi.nlm.nih.gov) using DNAMAN software (www.lynnon.com). Molecular Plant 2013 6, 1091-1108DOI: (10.1093/mp/sst083) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 2 Identification of the ptf2 Mutants. (A) Schematic diagram of PTF2 gene structure, showing the T-DNA insertion sites in the ptf2 mutants. The black and white boxes indicate translated and untranslated regions, respectively. The arrow heads indicate the positions of the primers used for PCR assays. (B, C) Confirmation of the T-DNA insertion in ptf2-1/+ (B) and ptf2-2/– (C) plants by PCR. (D) Expression levels of PTF2 and At4g35530 in the ptf2-1/+ and ptf2-2/– seedlings compared to that in wild-type seedlings. Molecular Plant 2013 6, 1091-1108DOI: (10.1093/mp/sst083) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 3 The ptf2-1 Mutation Caused Failure of Pollen Germination. (A, B) The in vitro germination of the pollen grains from wild-type (A) and ptf2-1/+ (B) plants. (C) Statistics of in vitro pollen germination rates of the wild-type (WT) and ptf2-1/+ plants. (D, E) The in vitro germination of the pollen grains from qrt1/qrt1 (D) and ptf2/+;qrt1/qrt1 (E) plants. (F) Statistics of in vitro pollen germination rates of the qrt1/qrt1 and ptf2-1/+;qrt1/qrt1 plants. The numbers under x-axis present number of germinated pollen grains per tetrad. (G, H) The bright field (G) and fluorescent (H) images showing that a qrt1/qrt1 quartet germinated four pollen tubes in vivo. (I–L) The bright field (I, K) and fluorescent (J, L) images showing that the ptf2-1/+;qrt1/qrt1 quartets germinated three (I, J) or two pollen tubes (K, L) in vivo. The in vitro pollen germination rates were calculated after incubated at 22°C for 6 h. The white arrows indicate the ungerminated pollen grains. Pt, pollen tube. ** P < 0.01, by Student’s t-test. Bars = 100 μm in (A, B); 20 μm in (D, E) and (G–L). Molecular Plant 2013 6, 1091-1108DOI: (10.1093/mp/sst083) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 4 The ptf2-1 Mutation Caused Embryonic Defect Revealed by Pollen-Rescue. (A) Seed setting in the siliques from the wild-type (WT) and pollen-rescued ptf2-1/+ (PRptf2-1/+) plants 7 d after self-pollination. The red arrow heads indicate the aborted seeds. (B) Seed setting in the mature siliques from the WT and pollen-rescued ptf2-1/+ (PRptf2-1/+) plants. The black arrow heads indicate the aborted seeds. (C) Statistical analysis of the seed setting in the PRptf2-1/+ siliques by comparison with WT siliques. The seeds were counted in the siliques from WT or PRptf2-1/+ plants seven d after fertilization. * P < 0.05; ** P < 0.01, by Student’s t-test. (D, E) DIC images of the embryos at the globular stage from a PRptf2-1/+ silique, showing the normal embryo (D) and abnormal embryo (E). (F, G) DIC images of the embryos at the heart stage from a PRptf2-1/+ silique, showing a normal embryo (F) and an abnormal embryo with irregular cell organization (G). (H, I) DIC images of the embryos at the torpedo stage from a PRptf2-1/+ silique, showing a normal embryo (H) and an abnormal embryo (I). (J, K) DIC images of embryos at the walking-stick stage from a PRptf2-1/+ silique, showing the normal embryo (J) and the abnormal embryo (K). (L, M) DIC images of embryos at the matured stage from a PRptf2-1/+ silique, showing a normal embryo (L) and an abnormal embryo with an irregular structural pattern (M). Bars = 200 μm in (A, B); 50 μm in (D–M). Molecular Plant 2013 6, 1091-1108DOI: (10.1093/mp/sst083) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 5 Phenotypic Characterization of PRptf2-1 Seedlings and Plants. (A–D) Showing the abnormal mutant seedlings with three cotyledons (A), morphology-defective cotyledons (B–D), and shorter roots (A–D). (E) A normal wild-type (WT) seedling. (F) Showing a branchy pollen-rescued mutant plant compared to a WT plant. (G) Molecular confirmation of the pollen-rescued ptf2-1 homozygous mutant plants (PRptf2-1) by PCR, showing that the PRptf2-1 samples had the transgenic PTF2 gene and lacked the PCR fragment related to T-DNA insertion, compared to the control WT and ptf2-1/+ plants. (H) Quantitative RT–PCR assay, showing a significant reduction of PTF2 expression levels and a slight increase of At4g35530 expression levels in the PRptf2-1 seedlings, compared to that in WT. Bars = 500 μm. Molecular Plant 2013 6, 1091-1108DOI: (10.1093/mp/sst083) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 6 Complementation of ptf2-1 Mutant. (A–C) The in vitro germination of pollen grains from wild-type (WT) plants (A) and the complemented ptf2-1 homozygous plants Comp(1150) (B) and Comp(554) (C). (D) Statistics of the in vitro pollen germination rates of the complemented plants Comp(1150) and Comp(554) by comparison with that of WT plants. (E–G) Showing the siliques with full seed sets from WT plant (E) and the complemented plant Comp(1150bp) (F) and Comp(554bp) (G). The in vitro pollen germination rates were calculated after incubation at 22°C for 6 h. Bars = 50 μm in (A–C), 200 μm in (E–G). Molecular Plant 2013 6, 1091-1108DOI: (10.1093/mp/sst083) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 7 The Expression Pattern of PTF2. (A) Relative expression levels of PTF2 in various tissues, revealed by quantitative RT–PCR. (B–I) Showing that GUS stains were observed in inflorescence (B), flower (C), the pollen grains before released from anther (D), developing embryos (E, F), seedling (G), root tips and primordia of lateral root (H) of the transgenic ptf2-1 plants carrying the PTF2(1150bp):cDNA–GUS construct, but was absent from the mature leaf (I). Bars = 50 μm in (B, H); 200 μm in (C, G); 20 μm in (D–F); 1000 μm in (I). Molecular Plant 2013 6, 1091-1108DOI: (10.1093/mp/sst083) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 8 PTF2–GFP Was Localized in Vegetative Nuclei of Developing Pollen Grains. (A–D) The DAPI-stained pollen grains at the uninucleate stage (A, B) and the binucleate stage (C, D). (E–H) Showing that the GFP signal started to appear in the vegetative nucleus of the pollen grain at the binucleate stage. (I–L) The DAPI-stained pollen grains at the trinucleate stage (I–K) and mature pollen grain (L). (M–P) The GFP images of (I–L), showing that the GFP signal persisted in the vegetative nucleus of the pollen grain at the early trinucleate stage (M), then was significantly reduced at the late trinucleate stage (N, O) and disappeared in the mature pollen grain (P). (Q, R) The DAPI-stained germinating pollen grain (Q) and growing pollen tube (R). (S, T) The GFP images of (Q, R), showing that no GFP signal was observed in the germinating pollen grain (S) and growing pollen tube (T). GN, generative nucleus; Pg, pollen grain; Pt, pollen tube; SN, sperm nuclei; VN, vegetative nucleus. Bars = 5 μm. Molecular Plant 2013 6, 1091-1108DOI: (10.1093/mp/sst083) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 9 PTF2 Interacted with TBP2, Bound to dsDNA, and Formed a Homodimer. (A) Interaction of PTF2 with TBP2 protein in vitro revealed by protein pull-down assay. (B) SDS–PAGE electrophoresis, showing that GST did not bind to the dsDNA-cellulose resin as a negative control. (C) SDS–PAGE electrophoresis showed that GST–PTF2 bound to dsDNA. (D) Homodimerization of PTF2 protein revealed by Y2H assay. (E) Homodimerization of PTF2 protein revealed by BiFC assay. FT, flow through; Bars = 20 μm. Molecular Plant 2013 6, 1091-1108DOI: (10.1093/mp/sst083) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 10 PTF2 and AtTFIIB1 Interacted with the Subunits of RANPs. (A) The co-transformed yeast cells grown on SD/-Leu-Trp for 3 d, showing that the cells from all the combination groups could grow well. (B, C) The co-transformed yeast cells grown on SD/-Leu-Trp-His-Ade for 3 d (B) and 7 d (C). Molecular Plant 2013 6, 1091-1108DOI: (10.1093/mp/sst083) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions