Pollen-Expressed Transcription Factor 2 Encodes a Novel Plant-Specific TFIIB-Related Protein that Is Required for Pollen Germination and Embryogenesis in Arabidopsis

Molecular Plant  •  Volume 6  •  Number 4  •  Pages 1091–1108  •  July 2013

RESEARCH ARTICLE

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 Niua, Yan Lianga, Jing-Jing Zhoua, Xiao-Ying Doua, Shu-Chen Gaoa, Li-Qun Chena, Xue-Qin Zhanga and De Yea,b,1 a State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China b National Center for Plant Gene Research (Beijing), Beijing 100101, China

ABSTRACT  Pollen germination and embryogenesis are important to sexual plant reproduction. The processes require a large number of genes to be expressed. Transcription of eukaryotic nuclear genes is accomplished by three conserved RNA polymerases acting in association with a set of auxiliary general transcription factors (GTFs), including B-type GTFs. The roles of B-type GTFs in plant reproduction remain poorly understood. Here we report functional characterization of a novel plant-specific TFIIB-related gene PTF2 in Arabidopsis. Mutation in PTF2 caused failure of pollen germination. Pollen-rescue revealed that the mutation also disrupted embryogenesis and resulted in seed abortion. PTF2 is expressed prolifically in developing pollen and the other tissues with active cell division and differentiation, including embryo and shoot apical meristem. The PTF2 protein shares a lower amino acid sequence similarity with other known TFIIB and TFIIB-related proteins in Arabidopsis. It can interact with TATA-box binding protein 2 (TBP2) and bind to the doublestranded DNA (dsDNA) as the other known TFIIB and TFIIB-related proteins do. In addition, PTF2 can form a homodimer and interact with the subunits of RNA polymerases (RNAPs), implying that it may be involved in the RNAPs transcription. These results suggest that PTF2 plays crucial roles in pollen germination and embryogenesis in Arabidopsis, possibly by regulating gene expression through interaction with TBP2 and the subunits of RNAPs. Key words: PTF2; TFIIB-related protein; transcription factor; pollen; embryogenesis; Arabidopsis.

Introduction Pollen germination and embryogenesis are two important steps of sexual plant reproduction. Pollen germination generates a pollen tube that delivers the male gametes into an embryo sac for double fertilization, while embryogenesis enables the zygote to develop into an embryo. The processes require a large number of nuclear genes to be accurately expressed (Tzafrir et  al., 2004; Wang et  al., 2008). In human and yeast, transcription of nuclear genes is accomplished by three conserved RNA polymerases (RNAPs), namely RNAP I, RNAP II, and RNAP III. In particular, RNAP I  is responsible for the transcription of ribosomal RNA (rRNA) genes; RNAP II primarily transcribes the protein-coding genes (mRNA genes); and RNAP III synthesizes 5S rRNAs, transfer RNAs (tRNAs), and small nuclear RNAs (snRNAs). In plants, except for the three conserved RNAPs, there are two additional nuclear RNAPs, namely RNAP IV and RNAP V, that play non-redundant roles in siRNA-directed DNA methylation and gene silencing

(Herr et  al., 2005; Kanno et  al., 2005; Onodera et  al., 2005; Wierzbicki et  al., 2008). RNAPs act in association with a set of auxiliary general transcription factors (GTFs), including B-type GTFs which are different in the three conserved RNAPs (Vannini and Cramer, 2012). In mammals and yeast, the TFIIB associates with RNAP II (Hahn, 2004; Liu et al., 2010), while the TFIIIB works with RNAP III (Kassavetis et  al., 1997; Schramm and Hernandez, 2002). TFIIB has a typical structure containing a zinc-ribbon domain, an adjacent B-finger domain, and a core domain composed of two imperfect direct repeats which are structurally related to cyclin-like fold (Buratowski and Zhou, 1 To whom correspondence should be addressed. E-mail [email protected], tel. +86-10-62734839, fax +86-10-62734839.

© The Author 2013. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS. doi:10.1093/mp/sst083, Advance Access publication 27 May 2013 Received 24 January 2013; accepted 5 May 2013

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1993; Chen and Hahn, 2003), while TFIIIB is composed of three subunits: TATA-box binding protein (TBP), TFIIB-related factor (Brf), and B double prime (Bdp). In addition to the domains similar to those in TFIIB, Brf1 also has other three conserved domains in its C-terminus (Khoo et al., 1994). Recent studies show that HsTAF1B and ScRrn7 are structurally related to TFIIB protein and associated with RNAP I  transcription (Knutson and Hahn, 2011; Naidu et al., 2011). The roles of these B-type GTFs in human and yeast have been well characterized (Kassavetis et  al., 1998; Lagrange et  al., 1998; Andrau et  al., 1999; Ferri et  al., 2000; Zhao et  al., 2003; Chen and Hahn, 2004; Knutson and Hahn, 2011; Naidu et  al., 2011). They function in recruitment of RNAPs to form the transcription pre-initiation complex (PIC), promoter opening, and selection of the transcription start site (Schramm and Hernandez, 2002; Woychik and Hampsey, 2002; Deng and Roberts, 2007; Kostrewa et al., 2009; Knutson and Hahn, 2011). In contrast, little has been known about the roles of B-type GTFs in plants. Based on their sequence characteristic, more than 10 TFIIB and TFIIB-related proteins/factors could be identified in the Arabidopsis genome (www.Arabidopsis.org). The plant-specific TFIIB-related protein (pBrp) is involved in RNAP I transcription (Lagrange et  al., 2003; Imamura et  al., 2008). The plant-specific TFIIB-related Protein 2 (pBRP2) was demonstrated playing a role in Arabidopsis endosperm proliferation (Cavel et  al., 2011). The Arabidopsis TFIIB1 (AtTFIIB1) plays important roles in pollen tube growth and seed formation (Zhou et al., 2013). In addition, Central Cell Guidance (CCG), which encodes a nuclear protein with a conserved N-terminal C2C2-type zinc-ribbon motif typical for TFIIB family, is essential for micropylar pollen tube guidance in Arabidopsis (Chen et al., 2007). All these data indicate that B-type GTFs play crucial roles in different plant development processes. However, only a few of them have been characterized. As the first step to study gene regulation in pollen development and pollen tube growth, we performed a global search of the Arabidopsis database (www.Arabidopsis.org) for the Pollenexpressed Transcription Factor genes (PTFs). Here, we report functional characterization of PTF2. PTF2 encodes a TFIIB-related protein, which shares a low amino acid sequence similarity with other known TFIIB and TFIIB-related proteins in Arabidopsis. Mutation in PTF2 led to failure of pollen germination and disrupted embryogenesis. The PTF2 protein can interact with TATAbox binding protein 2 (TBP2) and the subunits of RNAPs, bind the double-stranded DNA (dsDNA), and form a homodimer. Our results show that PTF2 is a novel plant-specific TFIIB-related protein and plays crucial roles in pollen germination and embryogenesis through regulation of gene transcription.

RESULTS Identification of PTF2 Gene and ptf2 Mutants PTF2 (At4g35540) was identified by its expression in developing pollen grains in a search of the Arabidopsis database

(www.Arabidopsis.org) for PTFs. It encoded a protein that shared a lower sequence similarity with the general transcription factor TFIIB and TFIIB-related proteins in Arabidopsis (Figure  1A). In comparison to the known TFIIB and TFIIBrelated proteins, PTF2 had a relatively conserved N-terminal zinc-ribbon domain and two regions with lower similarities to the B-finger and core domains of the TFIIB and TFIIBrelated proteins (Supplemental Figure 1). In addition, it also had an uncharacterized sequence in its C-terminal region (Supplemental Figure 1). Further BLASTP search of the nonredundant protein sequences database at NCBI (www.ncbi. nlm.nih.gov) using the full-length PTF2 amino acid sequence as a query showed that the proteins which shared the identity with PTF2 ranging from 33% to 47% were found only in plant genomes (Figure  1B). All these proteins remained uncharacterized. The ptf2-1 (CS_849168) and ptf2-2 (CS_808904) mutants were identified by search of the Arabidopsis Biological Resource Center (ABRC) database (www.Arabidopsis.org). The T-DNA in ptf2-1 was inserted at 74 bp upstream of the ATG codon or 30 bp downstream of the transcription start site of PTF2 as confirmed by PCR using the gene-specific primer P1 and T-DNA left border-specific primer P745 (Figure 2A and 2B). The T-DNA insertion site in ptf2-2 was located at 40 bp upstream of the transcription start site of PTF2 as confirmed by PCR using the gene-specific primer P2 and T-DNA left border-specific primer LB3 (Figure  2A and 2C). These data showed that the T-DNA insertion sites in the both mutants are in the promoter region of At4g35530 and PTF2, which faces each other with a 658-bp gap between the two ATG codons of these two genes (Figure  2A). Only ptf2-1 heterozygous mutant plants (ptf2-1/+) were identified (Figure  2B). In contrast, the ptf2-2 homozygous mutant plants (ptf2-2/–) were easily obtained (Figure  2C). The quantitative RT–PCR assays indicated that expression levels of At4g35530 were not reduced but increased by 1.2- and 1.9-fold in the ptf2-1/+ and ptf2-2/– seedlings, respectively, compared to those in wildtype (Figure 2D). However, the expression level of PTF2 in the ptf2-1/+ seedlings was greatly reduced to 53% of wild-type seedlings, and that in the ptf2-2/– seedlings was almost the same (1.1-fold) as that in wild-type (Figure 2D). Thus, taken together, ptf2-1 mutation drastically reduced the expression of PTF2 and had no significant impact on the expression of At4g35530, while ptf2-2 mutation did not reduce the expression of both At4g35530 and PTF2 genes. The T-DNA insertion in ptf2-1 created a genetic tag of Basta (Bas) resistance for the mutant plants. The progeny seedlings from the self-pollinated ptf2-1/+ plant segregated in a ratio of 0.90 (1608) Bas-resistant (BasR) to 1 (1779) Bassensitive (BasS) (Table  1), and no ptf2-1 homozygous plant was obtained. When ptf2-1/+ plants were used as recipients in crosses with wild-type pollen grains, 46.7% (1716/3672) of the resulting F1 progeny were resistant to Basta (Table 1). In contrast, when wild-type plants were used as recipients in crosses

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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).

with the ptf2-1/+ mutant plants, merely 1.1% (128/11195) of the F1 progeny was resistant to Basta (Table 1). These results indicated that transmission of ptf2-1 mutation was drastically reduced through the male gametophyte and that ptf2-1 mutation only had a slight impact on female gametophytic function. Genetic and phenotypic analyses showed that ptf2-2 mutant behaved as normal as wild-type plants (Table 1), indicating that ptf2-2 mutant did not have any visible phenotype. Therefore, the ptf2-1/+ plants were used for further study.

The ptf2-1 Mutant Is Defective in Pollen Germination To investigate how ptf2-1 mutation affected male gametophytic function, it was introgressed into a quartet1 (qrt1) mutant background. In the qrt1 homozygous mutant (qrt1/qrt1), the four microspores from a microsporocyte failed

to separate after meiosis, but their functions were virtually unaffected (Preuss et  al., 1994). A  quartet from the ptf21/+;qrt1/qrt1 plant had two mutant pollen grains (ptf2-1 qrt1) and two qrt1 pollen grains (presenting wild-type). First, we examined the pollen grains from ptf2-1/+ plants and the quartets from ptf2-1/+;qrt1/qrt1 plants using scanning electron microscopy (SEM), Alexander staining, and 4’,6-diamidino-2phenylindole (DAPI) staining, compared to those from wildtype and qrt1/qrt1 plants, respectively. No obvious defect in pollen morphology, pollen viability, and nuclear division was observed (Supplemental Figure 2), indicating that ptf2-1 mutation did not affect pollen formation. Then, we examined in vitro germination of the pollen grains from ptf2-1/+ and ptf2-1/+;qrt1/qrt1 plants. As shown in Figure  3A–3C, 43.7% (n  =  1292) of the pollen grains from

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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.

Table 1.  Genetic Analysis of ptf2 Mutants. Crosses (female × male)

BasR or With insertion (W)

BasS or Without insertion (Wo)

W:Wo

TEF

TEM

ptf2-1/+ selfed

1608

1779

0.90:1

NA

NA

ptf2-1/+ × WT

1716

1956

0.88:1

88%

NA

WT × ptf2-1/+

128

11 067

0.01:1

NA

1%

ptf2-2/+ selfed

122

37

3.30:1

NA

NA

ptf2-2/+ × WT

75

85

0.90:1

90%

NA

WT × ptf2-2/+

57

51

1.12:1

NA

100%

BasR, Basta-resistant; BasS, Basta-sensitive; NA, not applicable; TE, transmission efficiency = (W/Wo) × 100%; TEF, female transmission efficiency; TEM, male transmission efficiency; WT, wild-type.

ptf2-1/+ plants were able to germinate, compared to that 86.9% (n = 1076) of wild-type pollen grains could germinate under the same conditions. Similarly, approximately 44.6% (n  =  233) of quartets from qrt1/qrt1 plants had three or four pollen grains germinated, while only 4.4% (n  =  272) of the quartets from ptf2-1/+;qrt1/qrt1 plants had three pollen grains germinated. No quartet from ptf2-1/+;qrt1/qrt1 plants had four pollen grains germinated and 80.1% of the quartets germinated one or two pollen tubes (Figure 3D–3F). These results showed that ptf2-1 mutation inhibited pollen germination in vitro. Time course examination of pollen germination was performed to further investigate in vivo germination of ptf21 pollen. Fewer than five quartets from the qrt1/qrt1 and

ptf2-1/+;qrt1/qrt1 plants were pollinated to a pre-emasculated wild-type stigma, respectively. The pollinated pistils were collected and stained with aniline blue to visualize the pollen tubes at 6, 12, and 24 h after pollination (hap). At 6 hap, 52.8% qrt1/qrt1 quartets had three or four pollen tubes (Figure 3G and 3H, and Table  2); only 3.2% ptf2-1/+;qrt1/qrt1 quartets had three pollen tubes (Figure  3I and 3J, and Table  2); the other ptf2-1/+;qrt1/qrt1 quartets produced one or two pollen tubes and no ptf2-1/+;qrt1/qrt1 quartet had four pollen tubes (Figure  3K and 3L, and Table  2). This result was consistent with the result achieved from the in vitro germination assay. At 12 and 24 hap, the rates of the qrt1/qrt1 and ptf2-1/+;qrt1/ qrt1 quartets with different numbers of pollen tubes were

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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).

almost the same as those observed at 6 hap. These results indicated that the ptf2-1 mutation disrupted germination of pollen in vivo and in vitro.

The ptf2-1 Mutation Disrupts Embryogenesis Genetic data showed that ptf2-1 mutation still could be transmitted at a low transmission efficiency through the male

gametes. To investigate why no ptf2-1 homozygous plant could be obtained in the progeny from self-pollinated ptf2-1/+ plants, we examined seed development in the siliques from ptf2-1/+ plants 10 d after natural self-pollination; 3.1% of seeds were found aborted in contrast to that no seed abortion occurred in the self-pollinated wild-type plants (Supplemental Figure 3A and 3B, and Table 3). When the pollen grains from

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Table 2.  Statistics of the In Vivo Germination Rates of ptf2-1/+;qrt1/qrt1 Pollen by Comparison with qrt1/qrt1. Times

Plants

Rates of the quartets that had different numbers of pollen grains germinated 4 (%)

6h 12 h 24 h

6.7 ± 3.2

qrt1/qrt1 ptf2-1/+;qrt1/qrt1

0

qrt1/qrt1

10.2 ± 3.6

ptf2-1/+;qrt1/qrt1

0

qrt1/qrt1

11.1 ± 2.8

ptf2-1/+;qrt1/qrt1

0

Total no.

3 (%)

2 (%)

1 (%)

46.1 ± 3.6

38.7 ± 6.3

8.4 ± 2.3

125

3.2 ± 2.5

45.7 ± 7.5

51.1 ± 5.8

168

45.1 ± 5.0

35.3 ± 6.4

9.4 ± 3.1

106

4.1 ± 1.8

43.8 ± 6.1

52.1 ± 4.6

145

35.0 ± 2.2

45.2 ± 4.8

8.7 ± 2.8

126

3.2 ± 3.2

45.6 ± 6.4

51.2 ± 6.0

117

Times, incubation times.

ptf2-1/+ plants were pollinated to wild-type plants, the seed setting was as normal as that in the self-pollinated wild-type siliques (Supplemental Figure 3C and Table 3). When the ptf21/+ plants were pollinated with wild-type pollen grains, no aborted seed was observed (Supplemental Figure  3D and Table  3). When the pre-emasculated ptf2-1/+ plants were manually pollinated with their own pollen grains, the results were similar to those achieved from natural self-pollination. Approximately 1.6% (19/1182) of the resulting seeds was aborted (Supplemental Figure  3E and Table  3). These data indicated that ptf2-1 mutation also led to seed abortion. To generate more ptf2-1 homozygous mutant seed materials, the coding sequence (CDS) of PTF2 (PTF2CDS) was fused to the promoter of the pollen-specific gene PPME1 (Louvet et  al., 2006). The resulting construct PPME1:PTF2CDS was introduced into ptf2-1/+ plants. Forty independent transgenic ptf2-1/+ plants were obtained. Genetic analysis indicated that 31 out of the 40 pollen-rescued ptf2-1/+ lines had restored the genetic transmission of ptf2-1 through male gametophyte (Supplemental Table  1). The pollen-rescued ptf2-1/+ plants with homozygous transgenic insertion (PRptf2-1/+) were generated in T2 progeny. They were cataloged into two phenotypic groups by statistics of their seed setting efficiency. The transgenic plants (eight out of the randomly selected 14 transgenic lines) in the first group had 10.7–22.6% seeds aborted (Figure 4A–4C), while the transgenic plants (six out of 14 lines) in the second group had almost 100% seed set as wild-type (data not shown). As shown in Figure 4A and 4B, the abnormal seeds had a lighter color than the normal seeds and most of them died finally in the self-pollinated PRptf2-1/+ siliques from the first

group. Subsequently, we observed these two types of seeds using differential interference contrast (DIC) microscope. The results showed that the aborted seeds had abnormal embryos compared to that in the normal seeds (Figure 4D–4M). When the normal embryos had developed to the globular stage (Figure  4D), the abnormal embryos were still at the 8-cell or 16-cell stages and the cell arrangement in such defective embryos was abnormal (Figure  4E) compared to that in the normal embryos. When the normal embryos proceeded to the heart stage (Figure  4F), the defective embryos were at globular stage with an irregular cell arrangement (Figure 4G). When the normal embryos were at the torpedo stage (Figure  4H), the mutant embryos were still at the globular stage and had an expanded aberrant shape with a characteristic similar to that of the embryos from transition to heart stage in wild-type (Figure 4I). In the older siliques, the normal embryos were at the walking-stick stage (Figure 4J), yet the defective embryos were still at the globular stage and continued to divide, resulting in a defective globular embryo that failed to develop into a heart embryo (Figure  4K). All the images showed that the defective embryos exhibited an irregular cell division and obviously altered cell arrangement. Such defective embryos did not proceed to the heart stage and started to degrade before they matured, resulting in seed abortion (Figure  4B). In a few cases, there were the abnormal seeds with irregular embryo which could survive to mature (Figure 4L and 4M). Such survive mutant seeds could germinate and produce abnormal seedlings, which had an unusual number of cotyledons and defective morphology in some tissues, especially in roots (Figure 5A–5D) compared to the wild-type seedlings (Figure 5E). As shown in Table 4, there

Table 3.  Statistics of Seed Setting in ptf2-1/+ Siliques Compared with Wild-Type. Crosses (female × male)

Normal seeds (%)

Aborted seeds (%)

Unfertilized ovules (%)

Total no.

WT selfed

99.5 ± 0.0

0

0.5 ± 0.0

3166

ptf2-1/+ selfed

91.4 ± 2.3

3.1 ± 1.7

5.5 ± 1.5

3107

WT × ptf2-1/+

98.5 ± 0.2

0

1.5 ± 0.2

1579

ptf2-1/+ × WT

95.9 ± 1.4

0

4.1 ± 1.4

565

ptf2-1/+ ×ptf2-1/+

94.0 ± 1.2

1.6 ± 0.1

4.4 ± 1.0

1182

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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).

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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.

Table 4.  Segregation of the Seedlings from Different Self-Pollinated PRptf2-1/+ Lines. Lines

BasR seedlings

BasS seedlings

Total

Rates

13.1%

Normal

Defective

PRptf2-1/+ (3)

252

56

120

428

PRptf2-1/+ (4)

276

16

151

443

3.6%

PRptf2-1/+ (12)

534

6

340

880

0.7%

PRptf2-1/+ (21)

400

35

215

650

5.4%

Rates = (Defective/Total) × 100%.

were 0.7–13.1% seedlings with defective morphology from the progeny of the four independent transgenic lines. Some of the abnormal PRptf2-1 seedlings could grow up in soil. The plants had more branches compared to wild-type plants (Figure  5F). PCR assay using the DNAs from the abnormal seedlings indicated that they were homozygous for ptf2-1 mutation (Figure  5G). Quantitative RT–PCR assay using the

RNAs extracted from the PRptf2-1 seedlings of two pollenrescued lines showed that the expression levels of PTF2 were dramatically reduced to 4.2% and 9.5% of that in wild-type seedlings (Figure  5H). The expression levels of At4g35530 were not reduced (1.3- and 1.4-fold of that in wild-type, respectively) in these PRptf2-1 seedlings, suggesting that the phenotypes of PRptf2-1 are related to PTF2 instead of

Niu et al.  •  PTF2 for Pollen Germination and Embryogenesis   

At4g35530. In contrast, the plants in wild-type background, which carried the same insertion of PPME1:PTF2CDS construct as the PRptf2-1 plants, exhibited normal embryogenesis and growth pattern as wild-type plants (Supplemental Figure 4A and 4B). These results indicated that ptf2-1 mutation also affected embryogenesis and plant growth pattern.

The Phenotype of ptf2-1 Mutant Is Complemented by PTF2 Considering that PTF2 faces the neighbor gene At4g35530 with only a 658-bp gap between the two ATG codons (Figure  2A), the different length genomic DNA fragments (2914 and 2318 bp) of PTF2 were used in mutant complementation assays, which comprised the 1150- and 554-bp sequences upstream of the PTF2 transcription start site, respectively. The DNA fragments also contained the 5’-UTR, coding region, and 3’-UTR of PTF2. In particular, the 2914-bp fragments included part of the At4g35530 coding sequence, while the 2318-bp fragment did not contain any At4g35530 coding sequence. The two fragments were amplified and subcloned into the Ti-derived vector pBI121 (CloneTech). The resulting constructs were introduced into the ptf2-1/+ plants. In total, 25 and 24 independent transformants were obtained, respectively, in a screen using Basta and Kanamycin double selections. In T2 generation, most of the transgenic lines (22 of 25 and 22 of 24) showed a segregation ratio of approximately 2 BasR to 1 BasS compared to the original segregation ratio of 0.90 BasR to 1 BasS (Supplemental Tables 2 and 3), suggesting that the DNA fragments are sufficient to complement the mutant. Then, the plants Comp(1150) and Comp(554) homozygous for ptf2-1 mutation were obtained from the transgenic ptf2-1/+ progeny. The in vitro germination rates of the pollen grains from the two independent complemented lines randomly selected were significantly recovered and close to that of wild-type pollen grains (Figure 6A–6D). Moreover, the complemented plants had normal seed setting as wild-type (Figure 6E–6G), and also had a normal growth pattern (data not shown). All these results demonstrated that the defective phenotype observed in ptf2-1 mutant plants was resulted from the mutation in PTF2 gene.

PTF2 Is Expressed Prolifically in the Tissues with Active Cell Division and Differentiation The quantitative RT–PCR assays showed that PTF2 had a ubiquitous expression pattern with higher transcription levels in flowers and siliques (Figure 7A). To further investigate PTF2 expression in more detail, two constructs for expressing PTF2– GUS and PTF2–GFP fusion proteins under the control of the 1150-bp PTF2 promoter were introduced into ptf2-1/+ plants, respectively. The constructs could complement the defective phenotype of ptf2-1 mutant plants (Supplemental Tables 4 and 5), indicating that the fusion proteins were expressed in the right places. In the T2 PTF2–GUS transgenic plants, GUS stains were observed in many tissues including inflorescences,

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developing pollen grains, and embryos (Figure 7B–7F), where the phenotype appeared in ptf2-1 mutant. Interestingly, relatively stronger GUS activity was detected in shoot apical meristems, root tips, and primordia of lateral roots (Figure 7G and 7H), but no GUS stain was observed in leaves (Figure 7I). In the roots of PTF2–GFP transgenic plants, GFP signal was colocalized in the nuclei of root cells with the propidium iodide (PI) stain (Supplemental Figure  5). In the developing pollen grains from the PTF2–GFP transgenic plants, GFP signal was first observed in the vegetative nuclei of the pollen grains at the early binucleate stage (Figure 8A–8H). The signal was persistent during the second pollen mitosis (Figure 8I and 8M) and then significantly decreased at the late trinucleate developmental stage (Figure 8J–8K and 8N–8O). No GFP signal was detected in the mature pollen grains after being released from the anthers (Figure  8L and 8P), germinating pollen grains, and pollen tubes (Figure  8Q–8T). In summary, PTF2 was expressed prolifically in developing pollen grains and the other tissues with active cell division and differentiation.

PTF2 Interacts with TBP2 and Binds to dsDNA To test whether PTF2 could interact with TBP2, the recombinant proteins GST-PTF2 and His-TBP2 were expressed and purified from the prokaryotic expression system and used for pull-down assay. As shown in Figure 9A, the recombinant GST-PTF2 protein was able to pull down the His-TBP2 protein, while the control GST did not, indicating that PTF2 could physically interact with TBP2 in vitro. To determine whether PTF2 could bind to dsDNA, the recombinant GST-PTF2 proteins were incubated with DNAcellulose resin for 2 h at 4°C. The matrix was eluted after washing five times with washing buffer and boiled in SDS sample buffer. Then the eluted samples were subjected to SDS–PAGE. As shown in Figure 9B, GST proteins could not be detected from the eluted samples, while GST-PTF2 proteins could (Figure 9C). After the dsDNAs were removed from the cellulose resin by DNase I  treatment, GST-PTF2 disappeared from the eluted sample (Figure  9C). These results indicated that PTF2 could bind the dsDNAs that were covalently linked to cellulose resin in the assay system.

PTF2 Can Form a Homodimer When the purified recombinant GST-PTF2 proteins were fractionated in the native PAGE electrophoresis, the proteins appeared as a band of approximately 180 kD (Supplemental Figure  6), compared to the related band of 95 kD appearing in SDS–PAGE electrophoresis (Figure 9C). These data indicated that the GST-PTF2 proteins could form a homodimer in vitro. To verify this observation, the PTF2 protein was used as the bait (BD–PTF2) and prey (AD–PTF2) to perform yeast two-hybrid (Y2H) assays. The result showed that the transformed yeast cells with BD–PTF2 plus AD–PTF2 could grow well on SD/-Leu-Trp-His-Ade medium and exhibited positive X-Gal stain. In contrast, the cells transformed with the

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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(1150 bp) (F) and Comp(554 bp) (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).

negative control combination sets of BD–PTF2 plus AD, AD– PTF2 plus BD, and BD plus AD did not grow on the selective medium (Figure 9D). All the four group cells could grow well on the SD/-Leu-Trp medium (Figure 9D). To further verify its homodimerization in plant cells, we performed a bimolecular fluorescence complementation (BiFC) assay. The PTF2 CDS was subcloned into the vectors pSPYNE–35S and pSPYCE–35S to generate the fusion protein-expressing constructs PTF2– YFPN and PTF2–YFPC . The plasmid mixture containing PTF2– YFPN and PTF2–YFPC was bombarded into onion epidermal cells. The YFP fluorescence was accumulated exclusively in the nuclei of the onion epidermal cells. In contrast, no YFP

fluorescence was accumulated in the two negative controls (Figure  9E). These results suggested that PTF2 could form a homodimer in plant cells.

PTF2 Interacts with the Predicted Subunits of RNAPs from Arabidopsis in Y2H Assays To investigate the interaction between PTF2 and the subunits of RNAPs, we searched the Arabidopsis database (www. Arabidopsis.org) and indentified 30 genes that were predicted to encode the subunits of RNAPs, among which some were predicted to be specific for one RNAP, and the others were general for two or more RNAPs (Supplemental Table 6).

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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).

Then these genes were cloned into pGADT7 vector to generate AD fusion protein, and co-transformed into yeast AH109 cells with BD–PTF2 and BD–AtTFIIB1, respectively. The cotransformation with BD was used as a negative control. When the AD fusion protein constructs were co-transformed with BD– PTF2, only the four combination groups AD–RPC4/BD–PTF2, AD–RPC34/BD–PTF2, AD–RPAC42/BD–PTF2, and AD–RPD7/ BD–PTF2 could grow on both SD/-Trp-Leu and SD/-Trp-LeuHis-Ade media (Figure 10A and 10B). In contrast, the four corresponding negative control groups AD–RPC4/BD, AD–RPC34/ BD, AD–RPAC42/BD and AD–RPD7/BD could not grow on SD/Trp-Leu-His-Ade medium, although they grew well on SD/Trp-Leu medium (Figure 10A and 10B). These results indicated that PTF2 had interaction with RPC4, RPC34, RPAC42, and

RPD7 in the yeast assay system. When the AD fusion protein constructs were co-transformed with BD–AtTFIIB1, only the four combination groups AD–RPC4/BD–AtTFIIB1, AD–RPC34/ BD–AtTFIIB1, AD–RPAC42/BD–AtTFIIB1, and AD–RPD7/BD– AtTFIIB1 grew well on SD/-Trp-Leu-His-Ade medium, showing that AtTFIIB1 also had interaction with RPC4, RPC34, RPAC42, and RPD7. Therefore, RPC4, RPC34, RPAC42, and RPD7 could interact with both PTF2 and AtTFIIB1. In addition, when the incubation times were extended from 3 to 7 d, more cotransformation combination groups could grow up slowly in contrast to those in the 3-d incubation assay. As shown in Figure 10C, PTF2 weakly interacted with RPAC43 and AtTFIIB1 weakly interacted with RPAC43, RPB5C, and RPB7NT. Taken together, two subunits predicted general to RNAP I and RNAP

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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.

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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.

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).

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III (RPAC42 and RPAC43), two subunits predicted specific for RNAP III (RPC4 and RPC34), and one subunit predicted specific for RNAP IV (RPD7) had interaction with PTF2 and AtTFIIB1, while the two subunits predicted specific for RNAP II (RPB5C and RPB7NT) had interaction with only AtTFIIB1 in Y2H assays.

Discussion In this study, we have identified and characterized a novel pollen-expressed transcription factor PTF2 in Arabidopsis. Mutation in PTF2 led to failure of pollen germination, indicating that it is important to pollen germination. Our data showed that PTF2 was expressed from the early binucleate stage to trinucleate stage of developing pollen grains but absent from mature pollen grains and pollen tubes. PTF2 was located in the vegetative nucleus, but not in the generative nucleus and sperm cells. Therefore, it likely influences pollen germination by controlling the gene expression in the vegetative cell of pollen grains during pollen development. The ptf2-1 pollen grains did not exhibit any observable defect in nucleate division, morphology, and viability, indicating that the mutation did not affect pollen formation. Therefore, failure of pollen germination in ptf2-1 was unlikely caused by developmental defect of the pollen grains. Studies have shown that many of the genes required for pollen germination and pollen tube growth are expressed during the late stages of pollen development. The RNAs are stored in the mature pollen grains and translated during pollen germination and pollen tube growth (Mascarenhas, 1989; Guyon et  al., 2000; Honys and Twell, 2004; Ishimizu et  al., 2010). Therefore, we propose that PTF2 may be involved in expression of those genes whose RNAs are expressed and stored to be used for pollen germination during pollen maturation. PTF2 is also expressed in the vegetative tissues with active cell division and differentiation, such as developing embryos, root tips, and shoot apical meristems. These data implied that PTF2 might be also involved in embryogenesis and vegetative development. We addressed this question by pollen-rescue. In the pollen-rescued ptf2-1 (PRptf2-1) mutant seeds, the defects occurred in embryo, different from the observation of the mutations in the other two general transcription factor genes AtTFIIB1 (Zhou et al., 2013) and PBRP2 (Cavel et al., 2011) in Arabidopsis. The mutation in AtTFIIB1 causes retarded proliferation of endosperm nuclei, resulting in embryonic defect and seed abortion. The pbrp2 mutant exhibits defect in endosperm development, but did not affect seed formation. Moreover, the PRptf2-1 plants had more lateral shoots compared to wild-type plants, indicating that PTF2 also plays important roles in plant growth. PTF2 encodes a protein that shares a lower sequence similarity with a class of B-type GTFs found in plants. The typical B-type GTFs, such as the Arabidopsis AtTFIIB1 and human TFIIB, have a typical structure containing three conserved domains: a zinc-ribbon domain, an adjacent B-finger domain,

and a core domain composed of two imperfect direct repeats. The sequence comparison showed that PTF2 does not have the characteristic significance typical to that of the typical TFIIB factors. However, it has a highly conserved zinc-ribbon structure at the N-terminal and shares a relatively lower similarity of amino acid sequence in the regions related to the B-finger and core domains of other TFIIB proteins (Supplemental Figure 1). In human and yeast, the C-terminal core domain of TFIIB is responsible for interacting with TBP and promoter DNA (Barberis et  al., 1993; Nikolov et  al., 1995); the zinc-ribbon interacts with the subunit of RNAP II to recruit RNAP II to the target promoter and functions as a bridge between the promoter and RNAP II complex (Buratowski and Zhou, 1993); the B-finger projects into the RNA catalytic center of RNAP II and plays roles in DNA opening and selection of transcription start site after PIC formation (Kostrewa et  al., 2009). TFIIB also can have an intramolecular interaction between the N- and C-terminal domains, which involves direct binding of the B-finger and the second repeat of core domain (Roberts and Green, 1994). Such an intramolecular interaction of TFIIB leads to formation of ‘closed’ form conformation (Roberts and Green, 1994; Wu and Hampsey, 1999). Thus, the typical TFIIB can interact with TBP, double-stranded DNA, and the subunits of RNAP II, and has an intramolecular interaction. Our data showed that PTF2 could interact with the Arabidopsis TBP2, bind to double-stranded DNA, and interact with the subunits predicted for RNAP I, III, and IV complexes as demonstrated by Y2H and pull-down assays. We have not obtained any datum to show whether PTF2 could have an intramolecular interaction, but our data show that it could form a homodimer in the in vitro and in vivo assays. Therefore, we propose that PTF2 is functionally similar to B-type GTFs. The subunits of RNAP I, RNAP II, and RNAP III complexes have been identified in human and yeast (Vannini and Cramer, 2012). However, most of the analogs in plants still remain unknown. In this study, four subunits predicted for RNAP complexes, namely RPC4, RPC34, RPAC42, and RPD7, were indentified to interact with PTF2 and AtTFIIB1 in Y2H assays. These subunits do not exhibit any significant sequence similarity with each other. However, they share a sequence similarity with the subunits of RNAPs in human and yeast. In particular, RPC34 shares 25% identity and 41% similarity with hsRPC6 that belongs to the RNAP III complex; and RPAC42 shares 45% and 40% identity with hsRPAC1 and ScRPAC40 sequences, respectively, which all exist in both RNAP I  and RNAP III complexes (Schramm and Hernandez, 2002). In addition, the Arabidopsis RPC4, hsRPC4, and ScRPC53 all belong to the RPC4 super family, the special subunits in RNAP III (Schramm and Hernandez, 2002). RPD7 has been proposed to be a subunit of RNAP IV (Ream et  al., 2009). Therefore, PTF2 and AtTFIIB1 may be associated with RNAP I, RNAP III, and RNAP IV transcription processes. In addition, as a typical

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Genetic Analysis

medium (Murashige and Skoog, 1962) containing 25  mg  L–1 kanamycin and 10  mg  L–1 Basta (Sigma, 45520, USA), confirmed by PCR using the primer pair PTF2-CON-F/pBI121-CONR (Supplemental Table 7) and used for further analyses. For the complementation experiments, the 2914- and 2318bp genomic DNA fragments were amplified by PCR with the primer pairs PTF2-COMP1150-F(XhoI)/PTF2-COMP-R(SacI) and PTF2-COMP554-F(XhoI)/PTF2-COMP-R(SacI) (Supplemental Table  7). After sequence validation, the fragments were cloned into vector pBI121 and introduced into ptf2-1/+ plants. The transformed plants were screened on the selective MS medium and further verified by PCR assays. The transformants homozygous for ptf2-1 mutation were selected in T2 generations and used for further analyses. For expression pattern assays, the PTF2 cDNA fragment without the stop codon was fused with GFP and GUS, respectively, under the control of native PTF2 promoter (1150 bp). The two fragments were amplified by PCR with primer pairs PTF2-G-F(SpeI)/PTF2-G-R(BamHI) and PTF2-COMP1150F(XhoI)/PTF2-P-R(SpeI) (Supplemental Table  7). Then they were cloned into vector pBI121 to create the C-terminal fusion protein-expressing constructs PTF2:cDNA–GFP and PTF2:cDNA–GUS. The constructs were then transformed into ptf2-1/+ plants, respectively. The complemented ptf2-1 plants were selected for expression pattern assays in T2 generation.

All genetic analyses were performed as described by Yang et al. (2003) and Jiang et al. (2005).

Quantitative RT–PCR

TFIIB, AtTFIIB1 also interacted with the predicted RNAP II subunits RPB5C and RPB7NT, implying that AtTFIIB1 may be also involved in RNAP II transcription. However, whether PTF2 is needed for RNAP II transcription remains unclear. More studies are still required to address this question.

METHODS Phylogenetic Analysis The sequences of PTF2-homologous proteins from different species were obtained from the National Center for Biotechnology Information by BLASTP search (www.ncbi. nlm.nih.gov). Multiple sequence alignment and phylogenetic analyses were performed using the DNAMAN software package (www.lynnon.com).

Plant Materials and Growth Conditions The wild-type and mutant plants were from Arabidopsis thaliana Colombia background (Col) and obtained from the ABRC (www.Arabidopsis.org). The mutant plants were backcrossed with wild-type for three generations to purify the ptf2 mutations. All the plants were grown in soil at 22°C with a 16-h light/8-h dark cycle.

Phenotypic Characterization Morphology of pollen grains was observed using a SEM (Hitachi, S-3400N) as described by Hulskamp et al. (1995). For the Alexander staining assay, the mature pollen grains were collected in the 20% Alexander staining solution prepared according to Alexander (1969). DAPI-staining assay was performed as described previously (Kang et  al., 2003). The in vitro and in vivo pollen germination assays were performed as described by Li et al. (1999) and Jiang et al. (2005), respectively. To examine their developmental patterns, the seeds were clarified in Hoyer’s solution (Liu and Meinke, 1998) for several minutes to 3 h and observed with a Leica DM2500 microscope equipped with a DIC system (Leica, Wetzlar, Germany).

Vector Construction and Mutant Complementation For the pollen-rescue experiments, the promoter of PPME1 (Louvet et al., 2006) and the coding sequence (CDS) of PTF2 were amplified by PCR using the primer pairs PPME1-P-F(SalI)/ PPME1-P-R(SpeI) and PTF2-PR-F(SpeI)/PTF2-PR-R(SacI), respectively (Supplemental Table  7). The resulting DNA fragments were first cloned in the T-vector pMD18 (Takara, D101A, China) for sequence validation. Then they were subcloned into Ti-derived vector pBI121 (CloneTech). The constructs were introduced into the ptf2-1/+ plants using the Agrobacterium (strain GV3101)-mediated infiltration method (Bechtold and Pelletier, 1998). The transformed plants were selected on MS

The total RNAs were isolated using a total RNAs extract kit (Bioteke, China) from different plant tissues and treated with DNase I  (Sigma, AMPD1, USA) at 25°C for 15  min. The first-strand cDNA was synthesized using a SuperScript II kit (Invitrogen, 18064–014, USA). Quantitative RT–PCR and statistical analysis were performed according to Liu et al. (2007). The assays were run using Power SYBR Green PCR Master Mix and the gene-specific primers (Supplemental Table  7) on an ABI 7500 real-time instrument (Applied Biosystems, www.appliedbiosystems.com). The PCR program was 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The gene expression levels in three biological replicates were normalized to that of ACTIN 2/8 in the same cDNA samples used. The relative expression levels were calculated with a ΔCt (threshold cycle) method.

GUS Assay GUS activity assays were performed as described previously by Yang et al. (1999) with a small modification. The tissues were incubated in GUS staining solution (1 mg ml–1 bromochloroindoyl-b-glucuronide (X-Gluc), 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferricyanide, 10 mM EDTA, and 0.1% Triton X-100 in 100 mM sodium phosphate buffer, pH 7.0) for 3–6 h at 37°C after an initial 45-min vacuum. The stained samples were clarified in 25% acetic acid/75% ethanol solution, and observed under Leica MZ10F and Leica DM2500 microscopes (Leica, Wetzlar, Germany).

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Yeast Two-Hybrid Assays The yeast two-hybrid (Y2H) assays were performed using the Gal4 vector system (Clontech, www.clontech.com/). The CDS of PTF2 was cloned into both pGBKT7 vector and pGADT7 vector, respectively. The CDS of AtTFIIB1 was cloned into vector pGBKT7. The CDSs of the RNAPs subunits were cloned into pGADT7 vector to generate AD fusion proteins. The constructs were co-transformed into yeast strain AH109, according to Causier and Davies (2002). The transformed cells were adjusted to OD600  =  0.3–0.4 and grown on SD/Trp-Leu plates and SD/-Trp-Leu-His-Ade plates for 3 or 7 d at 30°C. The primers used in this experiment are listed in Supplemental Table 8.

BiFC Assay The CDS without the stop codon of PTF2 was amplified by PCR using the primer pair PTF2-BiFC-F(BamHI)/PTF2-BiFC-R(XhoI) (Supplemental Table 7) and cloned into vectors pSPYNE–35S and pSPYCE–35S (Walter et al., 2004) to generate PTF2–YFPN and PTF2–YFPC expression constructs, respectively. The constructs were introduced into onion epidermal cells using a biolistic PDS-1000/He gene gun system (Bio-Rad, www.biorad. com/). The fluorescence signal was examined using confocal laser scanning microscope (LSM510, Carl Zeiss, www.zeiss. com/) at 16–36 h after transformation.

Protein Production and Purification The CDS of Arabidopsis PTF2 was amplified by PCR using primers PTF2-GST-F(BamHI)/PTF2-GST-R(XhoI) listed in Supplemental Table  7 and cloned into vector pGEX4T-1 to generate a fusion protein GST–PTF2 expression construct. The CDS of Arabidopsis TBP2 was amplified by PCR using primers TBP2-His-F(BamHI)/TBP2-His-R(XhoI) (Supplemental Table  7) and cloned into vector pET30a to generate His–TBP2 fusion protein expression construct. The constructs were introduced into Escherichia coli strain BL21 (DE3). The recombinant proteins were purified using glutathione Sepharose 4B (GE healthcare, 17–0756–01, Sweden) and Ni Sepharose 6 Fast Flow (GE healthcare, 17–5318–01, Sweden), respectively, according to the manufacturers’ instructions.

Pull-Down Assays For the protein pull-down assays, 0.1 mg GST–PTF2 or GST protein was bound to glutathione Sepharose 4B beads (GE healthcare, Sweden) at 4°C for 2 h, and subsequently His-TBP2 recombinant protein (0.02 mg) was added after washing the beads five times with the solution containing 25 mM Tris–HCl (pH 7.3), 100 mM NaCl, and 0.1% Nonidet P-40. The pulldown mixtures contained 25 mM Tris–HCl (pH 7.3), 100 mM NaCl, 0.5 mM DTT, and 0.1% Nonidet P-40. The mixtures were rotated at 4°C for 3 h and washed at least eight times with pull-down buffer. The beads were re-suspended and boiled in the sample buffer (Sigma, S3401, USA) for 10 min, and the eluted fractions were subject to sodium dodecyl sulfate

polyacrylamide gel electrophoresis (SDS–PAGE). The in-put GST and GST–PTF2 proteins were examined by Western blotting with the anti-GST antibody (Sigma, A5838, Germany). The His-TBP2 was detected by Western blotting with the antiHis antibody (Sigma, A5588, Germany).

Protein–DNA Binding In vitro protein–DNA binding assays were performed as described by Jurvansuu and Goldman (2011) with a small modification. The purified recombinant proteins were incubated in a 10 mM Tris (pH 7.4) buffer containing 0.1 M NaCl with 1/100 vol of dsDNA-cellulose resin (Sigma, D8515, USA) and rotated at 4°C for 2 h. Then the matrix was washed five times with the buffer containing 10 mM Tris (pH 7.4) and 0.2  M NaCl and the proteins were eluted with 10 mM Tris (pH 7.4), 2 M NaCl buffer. The eluted proteins were boiled in the sample buffer (Sigma, S3401, USA) for 10 min and subject to SDS–PAGE. The DNase I treatment of the matrix was performed as described by Jurvansuu and Goldman (2011).

Accession Numbers Sequence data for the genes presented in this study can be found in the GenBank data libraries under the following accession numbers: PTF2 (At4g35540), AtTFIIB1 (At2g41630), AtTFIIB2 (At3g10330), pBrp (At4g36650), pBRP2 (At3g29380), TBP2 (At1g55520), Ricinus communis (XP_002517218), Populus trichoca (XP_002305222), Vitis vinifera (XP_002266602), Oryza sativa (BAD11651), Sorghum bicolor (XP_002445808), Zea mays (GRMZM2G398135_P01).

SUPPLEMENTARY DATA Supplementary Data are available at Molecular Plant Online.

FUNDING This work was supported by the research grants from the Ministry of Sciences and Technology (project number: 2013CB1945100), the Natural Science Foundation of China (NSFC, project numbers: 31130007 and 31273106), and the Ministry of Education (111 project numbered B06003).

Acknowledgments We thank Dr Shuhua Yang for the vectors pBI121, pSPYNE– 35S, and pSPYCE–35S. No conflict of interest declared.

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