Arabidopsis Transcription Factor Genes NF-YA1, 5, 6, and 9 Play Redundant Roles in Male Gametogenesis, Embryogenesis, and Seed Development  Jinye Mu, Helin Tan, Sulei Hong, Yan Liang, Jianru Zuo  Molecular Plant  Volume 6, Issue 1, Pages 188-201 (January 2013) DOI: 10.1093/mp/sss061 Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 1 The Expression Patterns of NF-YA Genes. (A) Phylogenetic analysis of NF-YA proteins from different plant species. The phylogenetic tree was constructed using the MEGA4 software after alignment with ClustalW (Tamura et al., 2007). Arabidopsis thaliana (At) NF-YA1 through NF-YA10 are given with their corresponding loci. The accession numbers for other proteins are as indicated. (B) Expression patterns of LEC1 and NF-YA genes at seed developmental stage 4 and stage 7 (seed developmental stages are defined according to Schmid et al. (2005)). The data presented were extracted from the Arabidopsis eFP Browser microarray database (http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi). (C) Expression of NF-YA1 analyzed by quantitative reverse transcription–PCR (qRT–PCR). The relative expression level of NF-YA1 was normalized with that of ACTIN7 (At5g09810). Error bars indicate SD of three independent experiments. (D) Expression of NF-YA9 analyzed by qRT–PCR. See (C) for technical details. Molecular Plant 2013 6, 188-201DOI: (10.1093/mp/sss061) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 2 Analysis of the Expression Pattern of the NF-YA1:GUS Reporter. The NF-YA1:GUS reporter construct was transformed into wild-type (Col-0) plants and the GUS expression was analyzed by histochemical staining. At least five independent transgenic lines were analyzed and similar results were obtained. (A) A 7-day-old seedling. (B) A 3-week-old seedling. (C) Inflorescences derived from a 6-week-old seedling. (D) A flower derived from a 6-week-old seedling. (E) Stamens derived from a 6-week-old seedling. (F) Floral organs derived from a 6-week-old seedling. (G) A matured silique derived from a 6-week-old seedling. (H) An immature seed at the early heart stage. (I) An immature seed at the early torpedo stage. (J) A matured seed. Molecular Plant 2013 6, 188-201DOI: (10.1093/mp/sss061) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 3 The nf-ya1-D Mutant Phenotype. (A) Schematic diagram of the T-DNA insertion site in the NF-YA1 locus. Exons are boxed and lines between boxes represent introns. (B) NF-YA1 expression in the wild-type and the NF-YA1/nf-ya1-D mutant analyzed by quantitative reverse transcription–PCR (qRT–PCR). RNA prepared from 8-week-old wild-type (Col-0) and NF-YA1/nf-ya1-D seedlings was used for the assay. See Figure 1C for technical details. (C) Ten-day-old wild-type (Col-0) and NF-YA1/nf-ya1-D seedlings. Bar = 2 mm. (D) Pollen grains collected from anthers of wild-type and NF-YA1/nf-ya1-D mutant plants stained with the Alexander solution. Normal and defective pollen grains are stained as brown and green, respectively. Defective pollen grains are denoted by arrows. (E) Analysis of microspore development in NF-YA1/nf-ya1-D plants. Microspores collected from anthers at different stages as indicated and stained with 4’6-diamidine-2’-phenylindole dihydrochloride (DAPI) are shown. Defective microspores are denoted by arrows. Bar = 20 µm. (F) Embryo development in siliques derived from NF-YA1/nf-ya1-D plants. Samples were collected from the same silique, cleared as described in the ‘Methods’ section, and analyzed under a microscope equipped with Nomarski optics. (G) Seeds in developing siliques of wild-type and NF-YA1/nf-ya1-D plants. Aborted seeds are denoted by arrows. (H) Seven-day-old nf-ya1-D/– seedlings rescued from immature seeds. Bar = 2 mm. (I) Seven-day-old nf-ya1-D/– seedlings rescued from immature seeds. Trichomes on the cotyledon margins are denoted by arrows. Bar = 2 mm. Molecular Plant 2013 6, 188-201DOI: (10.1093/mp/sss061) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 4 Defective Reproductive Development of the 35S:NF-YA Transgenic Plants. (A) Expression of NF-YA genes analyzed by reverse transcription–PCR (RT–PCR) in wild-type (Col-0) and 35S:NF-YA transgenic plants. At least six independent transgenic lines for each transgene were analyzed and results of two representative lines are shown. Transgenic line numbers are indicated on the top of the panel. Ubiquitin 5 (UBQ5; At3g62250) was used as an internal control. UBQ5 and NF-YA genes were amplified for 24 and 28 cycles, respectively. Primers used in PCR are the same pairs as for the cloning experiment (see Supplemental Table 2). (B) NF-YA1 expression in wild-type, 35S:NF-YA1 transgenic plants, and the NF-YA1/nf-ya1-D seedlings analyzed by quantitative reverse transcription–PCR (qRT–PCR). RNA prepared from 3-week-old wild-type (Col-0), 35S:NF-YA1 transgenic plants and NF-YA1/nf-ya1-D seedlings was used for the assay. See Figure 1C for technical details. (C) Pollen grains collected from anthers of wild-type, 35S:NF-YA1, and 35S:NF-YA9 plants stained with the Alexander solution. Normal and abnormal pollen grains are stained as brown and green, respectively. (D) Embryo development in the 35S:NF-YA transgenic plants. (E) Seeds in developing siliques of wild-type, 35S:NF-YA1, 35S:NF-YA5, and 35S:NF-YA9 plants. (F) The seeds of the 35S: NF-YA transgenic plants. Bar = 1 mm. Molecular Plant 2013 6, 188-201DOI: (10.1093/mp/sss061) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 5 The Germination Phenotype of 35S:NF-YA Transgenic Seeds. (A) The germination phenotype of the homozygous 35S:NF-YA1 plants. Seedlings were germinated and grown on MS medium for 5 d. (B) The germination phenotype of the homozygous 35S:NF-YA9 plants. Seedlings were germinated and grown on MS medium for 5 d. (C) The germination phenotype of the heterozygous 35S:NF-YA plants. Seedlings were germinated and grown on MS medium and MS medium containing 0.1 µM ABA for 5 d. Molecular Plant 2013 6, 188-201DOI: (10.1093/mp/sss061) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 6 Characterization of 35S:NF-YA Transgenic Plants. (A) Seven-day-old wild-type (Col-0) and NF-YA1 and NF-YA9 transgenic seedlings germinated and grown on ½ MS medium. Bar = 1 mm. (B) Seven-day-old wild-type (Col-0) and NF-YA5 and NF-YA6 transgenic seedlings germinated and grown on ½ MS medium. Bar = 1 mm. (C) Two-week-old NF-YA1-OX and NF-YA9-OX plants stained with the Lugol’s solution. The accumulation of starches is stained as dark brown. Bar = 1 mm. (D) Two-week-old NF-YA1-OX and NF-YA9-OX plants stained with Fat Red 7B to visualize lipid accumulation. Bar = 1 mm. (E) Five-week-old wild-type (Col-0) and NF-YA transgenic plants grown in soil. (F) The flowering phenotype of the 35S:NF-YA9 transgenic plants. The flowering time of wild-type and 35S:NF-YA9 transgenic plants grown under continuous white light was measured by counting rosette leaf number at bolting. The experiment was repeated three times and the average values are shown. Error bars represent SD (n > 20). Asterisks indicate statistically significant differences compared with wild-type plants (P < 0.01). Molecular Plant 2013 6, 188-201DOI: (10.1093/mp/sss061) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 7 Overexpression of NF-YA Genes Promotes the Vegetative-to-Embryonic Transition. (A) Expression of NF-YA genes analyzed by reverse transcription PCR (RT–PCR) in wild-type (Col-0) and NF-YA-OXi plants. See Figure 4A for technical details. (B) Seven-day-old wild-type (Col-0) and NF-YA transgenic seedlings germinated and grown on ½ MS medium containing 10 µM estradiol. (C) The formation of somatic embryos on the cotyledons or root tips of NF-YA-OXi seedlings. Images were taken from seedlings grown on ½ MS medium containing 10 µM estradiol for 4 weeks. Bar = 2 mm. (D) Fat Red 7B staining of seedlings shown in (B). Bar = 2 mm. (E) The accumulation of eicosenoic acid (C20:1; a fatty acid maker for the formation of TAG) in NF-YA-OXi transgenic seedlings germinated and grown in the presence of 10 µM estradiol for 10 d. Four independent transgenic lines (line numbers are given below the graph) were analyzed and results of two representative lines are shown. FW, fresh weight. (F) Analysis of expression of the seed-specific genes in the NF-YA1-OXi and NF-YA9-OXi transgenic seedlings by reverse transcription PCR (RT–PCR). RNA samples were prepared from seedlings germinated and grown in the presence of 10 µM estradiol for 7 d. Ubiquitin 5 (UBQ5; At3g62250) was used as an internal control. UBQ5 and seed-specific genes were amplified for 24 and 30 cycles, respectively. Primers used in PCR experiment listed in Supplemental Table 2. Molecular Plant 2013 6, 188-201DOI: (10.1093/mp/sss061) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions