Plant Growth Regul (2012) 66:271–284 DOI 10.1007/s10725-011-9651-5

ORIGINAL PAPER

Maize transcription factor Zmdof1 involves in the regulation of Zm401 gene Xiyang Chen • Dongxue Wang • Chen Liu Meizhen Wang • Tao Wang • Qian Zhao • Jingjuan Yu

•

Received: 26 September 2011 / Accepted: 22 December 2011 / Published online: 25 January 2012 Ó Springer Science+Business Media B.V. 2012

Abstract Zmdof1 is a member of the maize Dof transcription factor family genes and participates in the regulation and control of the PEPC gene. The Zm401 gene, which contains short open reading frames (ORFs), has been cloned from maize, and its promoter contains several Zmdof1 recognition sites (DOFCORE, AAAG). Zm401 has an important role in anther development, and the protein encoded by the longest ORF, Zm401p10, localizes in the nucleus and is essential for maize anther development. In this study, we cloned Zmdof1, and expression pattern assay suggested that Zmdof1 has a role not only in nutrition organ development but also in maize pollen maturation. Transient expression of a Zmdof1::GFP fusion protein in onion epidermal cells showed a nuclear localization. 50 deletion analysis of the Zm401 promoter showed that the region of -670 to -510 is important for promoter activity.

Electronic supplementary material The online version of this article (doi:10.1007/s10725-011-9651-5) contains supplementary material, which is available to authorized users. X. Chen  D. Wang  C. Liu  M. Wang  T. Wang  Q. Zhao  J. Yu (&) State Key Laboratory for Agro-Biotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, People’s Republic of China e-mail: [email protected] Present Address: D. Wang Department of Biology, Stanford University, 385 Serra Mall, Stanford, CA 94305-5020, USA Present Address: T. Wang Plant Biotechnology Research Center, College of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China

Trans-activation assays in the yeast one-hybrid system confirmed that Zmdof1 had a strong interaction with AAAG elements in the Zm401 promoter. Co-transformation of a PZm401::Gus construct with UBI::Zmdof1 resulted in an approximately 40% decrease in GUS expression. Decrease of pollen viability resulting from ectopic expression of Zm401 controlled by its native promoter was recovered when Zmdof1 was transformed. Quantitative RT-PCR analysis showed that in PZm401::Zm401/UBI:: Zmdof1 transgenic tobacco, the expression of Zm401 decreased significantly, coupled with an increase of Zmdof1 expression. The results indicated that Zmdof1 interacts with the Zm401 promoter in vitro and downregulates Zm401 in transgenic tobacco pollen. A probable regulatory mechanism of Zmdof1 to Zm401 in pollen was proposed. Keywords Zea mays L.  Zm401  Zmdof1  Pollen  Dof  Transcription factor

Introduction Dof (DNA-binding with One Finger) domain proteins are plant-specific transcription factors with a highly conserved DNA-binding domain and are found in many monocotyledonous and dicotyledonous plants. The first Dof domain protein gene was isolated from maize by Yanagisawa and Izui (1993). Subsequently, many Dof domain protein genes have been cloned from many species. Investigation of these genes suggested that they have crucial roles in many physiological processes, including photosynthesis (Yanagisawa 2000; Yanagisawa and Sheen 1998), phytohormone response (Baumann et al. 1999; Kang et al. 2003; Kang and Singh 2000; Kisu et al. 1997, 1998; Mena 2002; Washio

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2001, 2003), seed storage protein expression (Diaz et al. 2005; Kawakatsu et al. 2009; Marza´bal et al. 2008; Mena et al. 1998; Vicente-Carbajosa et al. 1997; Yamamoto et al. 2006), regulation of stomata guard cell-specific genes (Plesch et al. 2001), establishment of leaf adaxial-abaxial polarity (Kim et al. 2010), phytochrome signal conduction (Park et al. 2003; Ward et al. 2005), stress response (Chen et al. 1996), seed germination (Diaz et al. 2005; Dong et al. 2007; Isabel-LaMoneda et al. 2003; Marza´bal et al. 2008; Mena 2002; Papi et al. 2000; Washio 2001), light-mediated circadian clock (Imaizumi and Kay 2006; Imaizumi et al. 2005; Iwamoto et al. 2009; Yang et al. 2010), and secondary metabolism (Skirycz et al. 2006). The conserved Dof DNA-binding domain is located in the N-termini of Dof proteins. The Dof domain, also called the N-terminal domain of Dof proteins, binds to DNA through a CX2CX21CX2C motif, which forms a single zinc finger when coordinating with a Zn2? ion (Yanagisawa 2002). This motif is essential for DNA recognition and named DNA-binding with One Finger (Dof). Any mutation of the four cysteine residues causes loss of function (Yanagisawa 2001). The core recognition site of Dof proteins (DOFCORE) is AAAG (or its reversibly complimentary sequence, CTTT), except in pumpkin (Kisu et al. 1998). The flanking sequences of DOFCORE also affect the binding of the protein to DNA, although they are not critical (Yanagisawa and Schmidt 1999). Almost all Dof proteins recognize the AAAG motif; this was confirmed by EMSA in vitro (Umemura et al. 2004; Yanagisawa 2002). The C-terminus of Dof proteins, unlike the N-terminus, varies among different Dof proteins, and it may interact with different regulatory proteins or signals to activate or repress gene expression. This may be the molecular basis of the different functions of Dof domain proteins (Yanagisawa 2001). Zmdof1 is the first isolated Dof domain protein. As a transcription factor, Zmdof1 protein is localized in nuclei and binds to the promoter region of the phosphoenolpyruvate carboxylase (PEPC) gene, which is involved in C4 photosynthesis (Yanagisawa 2000). Zmdof1 expressed in leaves, stems, and roots, and the activity of Zmdof1 is regulated by light in leaves (Yanagisawa 2000). Recent research has revealed that it also improves nitrogen assimilation and growth under low-nitrogen conditions (Yanagisawa 2004b). Previously, we identified several putative pollen-specific genes in maize by differential screening of a mature pollen cDNA library. We cloned the full-length cDNA of one of these genes, Zm401, using 50 and 30 RACE (Dai et al. 2004). Southern blot analysis showed that Zm401 is present at a low copy number in the maize genome. RT-PCR and in situ hybridization demonstrated that this gene specifically expressed in tapetum and pollen, from the floret-forming

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stage, increasing in concentration up to mature pollen, in vitro and in vivo. Open reading frame (ORF) prediction from the Zm401 cDNA sequence showed that no long ORFs were present in the 1,149-bp-length gene (Dai et al. 2004). We initially proposed that Zm401 functions as a short-open reading frame mRNA or noncoding RNA (Ma et al. 2008). A subsequent study confirmed that the longest ORF produced a functional protein, Zm401p10 (Wang et al. 2009). GFP fusion protein analysis demonstrated that Zm401p10 accumulated preferentially in the nucleus (Wang et al. 2009). Zm401p10 is also essential for tapetum and microspore development and can regulate floret formation in maize (Wang et al. 2009). Controlled by the Zm13 promoter, a maize pollen-specific promoter, ectopic expression of Zm401 in tobacco plants caused many abnormalities, including delayed degradation of the tapetum and asynchronous meiosis of microsporocyte, leading to different levels of pollen sterility in transgenic tobacco plants (Ma et al. 2005). Overexpression of Zm401 in maize showed similar features to those of transgenic tobacco pollen (Dai et al. 2007; Ma et al. 2008). Transcriptome analysis suggested that 278 genes were downregulated and 150 genes were upregulated in anthers when Zm401p10 was overexpressed (Wang et al. 2009). Genes actively involved in photosynthesis and transcriptional regulation were more numerous in the downregulated genes group, whereas those involved in translation/ribosomal structure/ biogenesis were relatively more numerous in the upregulated group (Wang et al. 2009). Investigation of the mechanism of pollen development gives us a better understanding of flower development in higher plants. Comparing reports on gene cloning and functional research shows that there is limited research focus on the transcriptional control of pollen development. More research has been carried out on the identification of gene function, and little attention has been paid to gene regulation. In the present study, we analyzed the regulation of pollen-specific gene Zm401 by a Dof domain protein. The maize Dof domain gene, Zmdof1, was cloned and characterized. In vivo and in vitro results showed that Zmdof1 could interact with Zm401 and downregulate Zm401 expression in transgenic tobacco pollen. These results provide additional insights into the molecular mechanism of maize pollen development.

Materials and methods Cloning of Zmdof1 gene Genomic DNA was extracted from maize (Zong31 inbred line) leaves by a revised SDS method (Sambrook et al. 1989). Zmdof1 is an intron-less gene; therefore, the PCR

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was carried out using 0.5 lg of genomic DNA to clone the full-length Zmdof1 gene. Primers were designed according to the sequence of Zmdof1 published on NCBI by Yanagisawa and Izwi (1993) [966076.1, or gi: 517257], and PCR primers Zmdof1 F and Zmdof1 R (Table 1) were synthesized by Invitrogen (Shanghai, China). The PCR amplification parameters were as follows: an initial denaturing step at 95°C for 5 min; 30 cycles at 94°C for 1 min, 61°C for 30 s, and 72°C for 30 s; and a final extension for 5 min at 72°C. Reactions were carried out with High Fidelity Taq enzyme (TaKaRa LA TaqTM with GC buffer) in a Biometra Thermo cycler (TG-96; Biometra, Germany). The PCR products were analyzed on 0.8% agarose gel, purified, ligated into vector pMD-19T (TaKaRa, Dalian, China), and sequenced twice with two different clones in different PCRs (Invitrogen, Shanghai, China). Transient expression of Zmdof1::GFP in onion epidermal cells The 35S::Zmdof1::GFP (Fig. 3b) and 35S::GFP chimeric constructs were used in this experiment, and 35S::GFP was used as a control. Fresh onion peels were placed on MS Table 1 Primers used in plasmid construction and molecular analysis (the underlined sequences in the primer sequences are enzyme digest sites)

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(Murashige-Skoog) culture medium (Murashige and Skoog 1962, 2% sucrose, 10% agar, pH 5.8) at 28°C in complete darkness 10 h before bombardment. Bombardment was carried out with a PDS-1000/He gene gun system (BioRad, America), and 5 lg of both plasmids were bombarded four times. Each bombardment was carried out at 1,100 psi of helium pressure at a distance of 7 cm. The onion cells were observed under an OlympusBX51 fluorescence microscope (Olympus Optical, Tokyo, Japan) 16 h after bombardment. At least three independent experiments were performed with each construct.

Tobacco transformation and selection The constructs for stable expression were introduced into tobacco plants (Nicotiana tabacum L. NC89) by Agrobacterium tumefaciens (LBA4404)-mediated leaf disk transformation (Gallois and Marinho 1995). After regeneration on kanamycin selective medium, transformed tobacco lines were checked for the presence of the transgene by PCR and Southern blot, and then, the transformants were transferred to a greenhouse.

Oligo names

Primer sequences

PZm401 (1791)F

50 -TTAGTGGCGGATTGGTCG-30

PZm401 (1791)R

50 -TTCACTTGATTTCCCACTAATTTT-30

Zmdof1F

50 -TCGCCTTCCATCTTTCTCCCTC-30

Zmdof1R

50 -ATAGCTGAAGAATCAAAGCGCTCCAT-30

Zmdof1(GFP)F

50 -GCTCTAGAATGCAGGAGGCGTC-30

Zmdof1(GFP)R

50 -CCGGATATCCGGGAGGTTGAGGAAGATG-30

PZm401 (YOH)F

50 -TCCCCCGGGCTGCAGACATAGCTCTC-30

PZm401 (YOH)R 60 bp(M)F

50 -CCCTCGAGTTTACTTATC AGGTTAAG-30 50 -CCCAAGCTTAAAAGGTTTTGTTTTG-30

60 bp(M)R

50 -CCCTCGAGCTTTTGTTTGTAAAAATTG-30

60 bp(DM)F

50 -CCCAAGCTTAAAAcGTTTTGTTTTGAAAAAC-30

60 bp(DM)R

50 -CCCTCGAGGTTTTGTTTGTAAAAATTG-30

B42AD-Zmdof1F

50 -GGAATTCATGCAGGAGGCGTC-30

B42AD-Zmdof1R

50 -CCGCTCGAGCGGGAGGTTGAGGAAG-30

GusF

50 -CTGCGACGCTCACACCGATACC-30

GusR

50 -TCACCGAAGTTCATGCCAGTCCAG-30

Zmdof1 (09.3.13)F

50 -GCGAGATCACCACGGAGACT-30

Zmdof1 (09.3.13)R

50 -TCACGGGAGGTTGAGGAAGA-30

ActinF

50 -GAACCAGAAGGATGCATATGTTG-30

ActinR

50 -GGTAATCAGTAAGGTCACGTCCA-30

qtactinF

50 -AAGGGATGCGAGGATGGA-30

qtactinR

50 -CAAGGAAATCACCGCTTTGG-30

qZm401p10F qZm401p10R

50 -ATCTTTTCACTCCTGCGGTAATATC-30 50 -TGGTAAATGAGCTATTCAAAATGAAC-30

qZmdof1F

50 -TCCTCCTCCTCGTCGTCGTCC-30

qZmdof1R

50 -GTCGTCCAGCACCGCCTTCC-30

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Analysis of pollen viability Mature pollen grains were collected from untransformed and transgenic tobacco plants and stained with I2–KI solution containing 0.5% (W/V) I2 and 3% (W/V) KI (Nelson 1968). The samples were then covered with a coverslip and viewed using an Olympus BX51 microscope (Olympus Optical, Tokyo). Images were captured using an Olympus digital microscope camera DT70 (Olympus Optical, Tokyo) and processed with the same software. The accumulation of starch in pollen grains was observed, and pollen viability (%) was expressed as the ratio of the number of stained pollen grains to the total number of pollen grains (Tsuchiya et al. 1995). The observation of pollen viability was performed at least three times per flower, for at least two flowers per plant, and at least three independent plants were observed. Quantitative analysis of GUS activity Quantitative analysis of GUS activity was determined according to Jefferson et al. (1987). One hundred milligrams of mature pollen collected from untransformed and transgenic tobacco plants were ground in liquid nitrogen, and 39 volume of GUS extract buffer (50 mM pH7.0 sodium phosphate buffer, 10 mM EDTA, 0.1% Triton X-100, 0.1% SDS, 10 mM b-mercaptoethanol) was then added and grinding continued for 2 min. The samples were transferred to 1.5-ml eppendorf centrifuge tubes, shaken for 5 min, and then centrifuged with 12,000 rpm for 10 min at 4°C, and the supernatant were collected and stored at 4°C. The protein concentration was determined as described by Bradford (1976). GUS activity was calculated as nmol 4-Methylumbelliferone (4-MU) per minute per gram protein. For each construct, six independent assays were performed and the average was calculated. Yeast one-hybrid assays Three Zm401 promoter fragments were inserted to pLACZi, producing PZm401::LacZ, 60 bp(M)::LacZ, and 60 bp(DM)::LacZ, respectively. 60 bp (M) contained three AAAG elements located in the -635 to -574 region of PZm401with a mutation of AGAA to ACAA in the -593 and -580 site to avoid self-activation by yeast and resulted in an AAAG mutated to AAAC in this region. 60 bp (DM) was the same sequence as 60 bp (M) except that all the AAAG motifs were mutated to AAAC, this construct was used as a negative control (Fig. 4a). The three plasmids (PZm401::LacZ, 60 bp(M)::LacZ, and 60 bp(DM)::LacZ) were constructed as bait vectors (BD), and 60 bp(DM)::LacZ was set as the negative control. PGAL1:: AD::Zmdof1, created by inserting Zmdof1 ORF (open

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reading frame) to the MCSs (Multiple Cloning Sites) of pB42AD plasmid (Fig. 4b), was used as AD vector in this system. Colony lift filter assay was applied to monitor the expression of the LacZ reporter gene using X-Gal as a substrate. All procedures were conducted strictly according to the manufacturer’s protocol (Clontech, Yeast Protocols Handbook PT3024-1). Semiquantitative and quantitative RT-PCR Total RNA was isolated from mature pollen of wild-type and transgenic tobacco plants using the procedure developed by Logemann et al. (1987). After treatment with RNase-free DNase I (Promega), the total RNA (5 lg) was reverse-transcribed and used as a template. The PCR was carried out with the Zmdof1-specific primers Zmdof1 (09.3.13F, R) and Gus (F, R) (Table 1), which were designed according to the 30 end of the Zmdof1 gene and the GUS gene (GenBank no. AF234297) by PRIMER PREMIER 5. Actin (using primers ActinF and ActinR) was used as a loading control (Table 1). The products were separated on a 1.0% agarose gel. Quantitative RT-PCR was carried out using Applied Biosystems 7900HT Real-Time PCR system and SYBR Premix EX TaqTM (Perfect real time, DRR041A, TaKaRa). The tobacco actin gene was used as a reference gene. The primers qActinF and qActinR were used for the tobacco actin gene, qZmdof1F and qZmdof1R for the Zmdof1 gene, and qZm401p10F and qZm401p10R for the Zm401 gene; all designed using the Primer3 program (http:// frodo.wi.mit.edu/primer3/) to produce PCR products of *150 bp in length. At least three repetitions per sample were conducted. The gene expression changes were calculated according to the following formula: ratio = 2^ (Dct target (sample - control) - Dct ref (sample - control) (Pfaffl 2001). In the formula, ‘target’ refers to the Zm401 gene and Zmdof1 gene, ‘ref’ refers to the actin gene, ‘sample’ refers to the transgenic samples, and ‘control’ refers to wild-type samples.

Results 50 Deletion analysis of Zm401 promoter The Zm401 promoter has been cloned and analyzed previously (Li et al. 2001; Wang et al. 2009). To identify functional elements in this promoter, we carried out 50 deletion analyses. Seven different lengths of the promoter (1,791, 1,470, 1,370, 1,070, 970, 670, and 510 bp) were fused to the Gus gene, respectively (Fig. 1a), and were transformed into tobacco. After characterization by PCR and RT-PCR, more than six transgenic tobacco plants for

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275

a

b GUS GUS GUS GUS GUS GUS GUS CaMV 35S

GUS

Fig. 1 Loss-of-function analysis of Zm401 promoter. a Zm401 promoter deletion fragments and CaMV35S fused to the gus reporter gene were used for a loss-of-function analysis of the promoter. DOFCOREs are represented by dotted line, b quantitative analysis of GUS activity in mature pollen with different promoter fragments and

Gus fusions. CaMV35S fused with Gus was used as a control. Data represent mean values, the error bars represent standard deviations of the means, and more than six transgenic plants were selected for analysis in each experiment

one copy of construct were selected for further analysis. Quantitative analysis of GUS activity in mature pollen of all seven constructs of transgenic tobacco plants was performed when the flower bud reached 25 mm in length. The results showed that the GUS activity from 1,791 to 670 was low except a little high in -1,370 to -1,070 region and fluctuated little among these constructs. However, when the promoter was deleted to 510, GUS activity in the mature pollen was greatly increased, reaching a maximum level, suggesting the region of -670 to -510 may have some negative elements (Fig. 1b). Online analysis using software PlantCARE and PLACE (Higo et al. 1999; Lescot et al. 2002) showed that there are five DOFCORE elements in the region of -670 to -510 (Fig. S1).

in nucleus and act as a transcription factor. To test this hypothesis, onion epidermal cells were bombarded with 35S::Zmdof1::GFP and a control plasmid 35S::GFP (Fig. 3b). In the case of control plasmid, GFP was detected in both the cytoplasm and the nucleus, whereas the 35S::Zmdof1::GFP fusion protein accumulated only in the nucleus of transfected cells (Fig. 3a). Previously, results indicated that Zmdof1 is expressed in almost all vegetative tissues, including green leaves, etiolated leaves, stems, and roots (Yanagisawa 1996; Yanagisawa and Izui 1993; Yanagisawa and Sheen 1998). To analyze whether Zmdof1 is expressed in maize pollen grains, RT-PCR was carried out. The results shown in Fig. 3c indicated that Zmdof1 is expressed in pollen, as well as in stems and green leaves.

Zmdof1 cloning and characterization Zmdof1 is an intron-less and high GC content (73.5%) gene; therefore, we cloned the Zmdof1 gene from maize TM leaves genomic DNA by PCR using TaKaRa LA Taq with GC buffer. Sequence analysis showed that the nucleotide sequences similarity between our cloned Zmdof1 and published on NCBI by Yanagisawa and Izui (1993) was more than 98%, and the deduced amino acid sequences similarity was about 95%. However, from the nucleotide sequence alignment, we found that there were three obvious gaps in the aligned sequences, and Zmdof1 has 12 extra nucleotides compare to that published by Yanagisawa and Izui (1993, Fig. 2a). In the amino acid sequence alignment, there were six differences, all in the N-terminus. The Dof domain and the speculated NLS (Nuclear localization signal, Yanagisawa and Sheen 1998; Yanagisawa 2001), however, had no changes (Fig. 2b). This implies that the Zmdof1 we cloned should be located

Zmdof1 could interact with the promoter of the Zm401 gene in yeast To analyze whether Zmdof1 could interact with the Dof core recognition site in the promoter of Zm401, we constructed three vectors for yeast one-hybrid system. These comprised the full length of promoter of Zm401 (PZm401), one truncated fragments [60 bp (M)], and one fragment with all DOFCOREs of 60 bp (M) mutated from AAAG to AAAC, which was used as a negative control [60 bp (DM)] (Fig. 4a). The 60 bp (M) and 60 bp (DM) were located in the region of -670 to -510. We used 60 bp first, but the fragment showed self-activation; therefore, we mutated this element and used 60 bp (M) to replace 60 bp. The three BD vectors were transformed into yeast host YM4271 after linearization. PCR analysis was conducted to confirm that the three fragments were successfully integrated in the yeast genomes. b-Galactosidase assays showed that there

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Fig. 2 Sequences alignment of the cloned Zmdof1 gene and the published Zmdof1 gene (966076.1, or gi: 517257 Yanagisawa and Izui 1993). a DNA sequence alignment; b deduced amino acid sequences alignment. The four cysteine residues are indicated by arrows, Dof

domain (CPRCASRDTKFCYYNNYNTSQPRHFCKGCRRYWTKGG TLRNVPVGGGTRK) is indicated by thick line under the amino acid sequences, and the NLS (sequences are PKKKPASKKRRVVAPAP) is underlined (also highlighted by bidirection arrows)

was no blue color in the reaction filters (see the left of Fig. 4c), which indicated that there were no other activators in yeast. PGAL1::AD::Zmdof1, the fusion construct of AD::Zmdof1 under the control of the GAL1 promoter (Fig. 4b), was

transformed to the yeast containing the BD vectors. b-Galactosidase assays showed that the intensity of blue color of Zmdof1 with PZm401 and Zmdof1 with 60 bp (M) was almost equal and moderate (see upper two pictures in the right of Fig. 4c), and the color was absolutely

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a

Fluorescence

277

Merge

Light

b Zmdof1

35S::Zmdof1::GFP 35S::Zmdof1 ::GFP 35S::GFP CaMV 35s Pro

GFP

NOS-ter

c Stem

Green leaf

Pollen

Zmdof1

35S::

Actin

GFP

Fig. 3 Characterization of Zmdof1. a Subcellular localization. The chimeric 35S::GFP and 35S::Zmdof1::GFP plasmids were transiently expressed in onion epidermal cells and detected under excitation light (fluorescence, left), visible light (light, middle), or both lights (merged, right). Bar = 30 lm; b schematic diagrams of 35S::Zmdof1::GFP. CaMV 35S pro, cauliflower mosaic virus 35S promoter; Zmdof1: Zmdof1 ORF; GFP green fluorescence protein

gene; Nos-ter: nopaline synthase gene terminator. c RT-PCR analyses of Zmdof1 expression. Total RNAs were extracted from stem, green leaf, and pollen of maize, respectively. The primers Zmdof1 (09.3.13 F, R) were used in the RT-PCR analysis. The PCR products of Zmdof1 (upper) and Actin (lower: control) were separated on agarose gel

undetected in 60 bp (DM) (bottom two pictures of Fig. 4c). The result indicated that Zmdof1 could interact with the promoter of Zm401 gene in yeast.

Almost no GUS activity was detected in the pollen of the wild-type tobacco and transgenic tobacco harboring Zmdof1 only (Fig. 6a). The feature was preserved in T1 generation, and the ratio of GUS activity between transgenic tobacco pollen harboring PZm401::Gus and transgenic tobacco pollen harboring PZm401::Gus together with Zmdof1 was 1.52 (Fig. 6b). Taking together, these results suggest that Zmdof1 is a transcriptional repressor of the Zm401 promoter.

Zmdof1 downregulates the Zm401 gene in tobacco For further analysis of the interaction of Zmdof1 with Zm401 in vivo, we generated transgenic tobacco plants harboring PZm401::Gus (Fig. 5b), PZm401::Gus/UBI::Zmdof1 by transforming PZm401::Gus into wild-type and UBI::Zmdof1 (Fig. 5a) tobacco plants. More than six independent transgenic lines were chosen for analysis, and we also kept a track for their progenies (T1). Mature pollen was collected, and quantitative analysis of GUS activity was carried out in T0 and T1 generations of these transgenic tobacco plants. The GUS activity of transgenic tobacco pollens harboring PZm401::Gus was 164.04 nmol 4-MU min-1 mg-1 protein, which about 1.72-fold of that harboring PZm401::Gus together with Zmdof1 in the T0 generation.

Recovery of the viability of PZm401::Zm401 transgenic tobacco pollen by Zmdof1 expression PZm401::Zm401 (Fig. 5b) and UBI::Zmdof1 (Fig. 5a) were separately transformed into tobacco. After PCR and RT-PCR confirmation, transgenic plants were obtained. Furthermore, PZm401::Zm401 was transformed into UBI::Zmdof1 transgenic tobacco, generating transgenic plants with PZm401::Zm401/UBI::Zmdof1. Pollen grains from PZm401::Zm401, UBI::Zmdof1, and PZm401::Zm401/

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a -1

PZm401 -2000

60bp(M) -635

-574

60bp(DM) -635

-574

pLacZi PCYC1

Ampr

lacZ

b pB42AD-Zmdof1

URA3

Zmdof1

pB42AD Ampr

PGAL1

AD

TADH1

TRP1

c PZm401

Zmdof1+ PZm401

60bp(M)

Zmdof1+ 60bp(M)

60bp(DM)

Zmdof1+ 60bp(DM)

Fig. 4 The transactivation activities of Zmdof1 to the promoter of Zm401 in yeast. a Schematic diagrams of the reporter (BD) vectors used in one yeast hybridization. PZm401: whole length promoter of Zm401; 60 bp (M): -635 to -574 of Zm401 promoter, with two mutations (AGAA to ACAA), and resulted in an AAAG mutated to AAAC in this region; 60 bp (DM): -635 to -574 of Zm401 promoter, with all four DOFCORE mutations (AAAG to AAAC). The vertical line represents AAAG (DOFCORE), and the oblique line represents AAAG mutated to AAAC. pLacZi: a plasmid used for constructing BD with a reporter gene URA3 for selection.

b Schematic diagrams of the effectors (AD) plasmids used in the transactivation assays. pB42AD: a plasmid used for constructing AD with a reporter gene TRP1 for selection. c The transformants were streaked on SD(Ura-) or SD(Ura-? Trp-) medium, and the bgalactosidase activity was examined. Yeast harboring PZm401, 60 bp (M), and 60(DM) (left side) only have BD and were used as controls. Yeast harboring Zmdof1 ? PZm401, Zmdof1 ? 60 bp (M), and Zmdof1 ? 60(DM) (right side) have both AD and BD. 60 bp (DM) and Zmdof1 ? 60(DM) was set as negative controls. M mutation

UBI::Zmdof1 transgenic plants were stained with I2/KI solution to test viability. All pollen from wild-type and UBI::Zmdof1 transgenic plants were perfectly normal round shape and stained deeply (Fig. 7a), as expected of pollen with normal viability (Fig. 7b). Pollen grains from PZm401::Zm401 transgenic plants were misshapen and poorly stained, whereas most pollen grains from PZm401::Zm401/UBI::Zmdof1 transgenic plants were normal (Fig. 7a). The average viability of the pollen from PZm401::Zm401 was 13.8%, which was reduced significantly compared with the wild type, and the viability of pollen from PZm401::Zm401/UBI::Zmdof1 was

51.3% in average, significantly higher than that from PZm401::Zm401 (Fig. 7b). Total RNA was extracted from the mature pollen of PZm401::Zm401 and PZm401::Zm401/UBI::Zmdof1 transgenic tobacco plants. Quantitative RT-PCR was conducted to analyze the expression levels of Zm401 and Zmdof1. The results showed that when the level of Zmdof1 expression increased to 4.7-fold, Zm401 expression levels decreased more than 40% in PZm401::Zm401/UBI::Zmdof1 transgenic plants (Fig. 7c). These results demonstrate that Zmdof1 downregulates Zm401 transcription in transgenic tobacco pollen.

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279

a Zmdof1

UBI::Zmdof1

LB

RB

Ubi Pro

NPT

pCOU CaMV 35S Pro

LB

Zm401

PZm401

b

NOS-ter

NOS-ter

PZm401::Zm401 NOS-ter RB

LB

GUS

CaMV 35S Pro

NPT

pBI121 NOS-Pro

NOS-ter

NOS-ter

LB

GUS

PZm401 PZm401::Gus

NOS-ter

Fig. 5 Constructions of expression vectors for tobacco transformation. a The ORF of Zmdof1 was inserted to the MCS of pCOU, producing UBI::Zmdof1. b The binary vector pBI121 was used as basic backbone. PZm401::Gus: the CaMV 35S promoter of the plasmid pBI121 was replaced by PZm401; PZm401::Zm401: the CaMV 35S promoter and Gus gene of the plasmid pBI121 were

GUS activity (nmol 4-MU/mg/min)

a

replaced by PZm401 and Zm401, respectively. CaMV 35S, cauliflower mosaic virus 35S promoter; nptII, gene-encoding neomycin phosphotransferase II; NOS-ter: nopaline synthase gene terminator; Ubi Pro: ubiquitin promoter; Zmdof1: Zmdof1 ORF; PZm401: promoter of Zm401; Zm401: Zm401 ORF; LB left border; RB right border

300

***

250 200 150

***

100 50

***:P<0.001

0 WT

GUS activity (nmol 4-MU/mg/min)

b

160 140 120 100 80 60 40 20 0

Zmdof1

PZm401

PZm401/Zmdof1

** **

Zmdof1

PZm401

PZm401/Zmdof1

***:P<0.01

Fig. 6 Quantitative analysis of GUS activity. Mature pollen collected from untransformed and transgenic tobacco plants. 4-MUG was used as substrate in this quantitative analysis of GUS activity, which was calculated as nmol 4-Methylumbelliferone (4-MU) per minute per gram of protein. Chart showing comparative GUS activity of transgenic tobacco harboring PZm401::Gus and PZm401::Gus/

UBI::Zmdof1 in T0 generation (a) and T1 generation (b). Wild-type plants and plants transformed with Zmdof1 only were used as controls. Data represent mean values, the error bars represent standard deviations of the means, and more than six transgenic plants were selected for analysis in each experiment. Significance was determined by Student’s t test with **P \ 0.01 and ***P \ 0.001

Discussion

processes (Shaw et al. 2009; Yanagisawa 2001). Dof genes are predominately expressed in vegetative organs, whereas some expressed in flower organs, concerning with flowering. CDF1 in Arabidopsis, Rdd1 in rice, and JcDof1 in

Dof protein is a plant-specific transcription factor, involved in the regulation of many important plant-specific

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Plant Growth Regul (2012) 66:271–284

a

wt

UBI::Zmdof1

c

PZm401::Zm401 PZm401::Zm401/UBI::Zmdof1

5.3 4.8

change fold(%)

4.3 3.8 3.3 2.8 2.3 1.8 1.3 0.8 0.3

PZm401::Zm401

pollen viability(%)

b

PZm401::Zm401/ UBI:: Zmdof1

Zm401

Zmdof1

120 100 80

***

60 40

***

20 0

wt

UBI::Zmdof1

PZm401::Zm401

***:P<0.001 PZm401::Zm401/UBI::Zmdof1

Fig. 7 a Pollen grains collected from wild-type and transgenic tobacco plants, respectively, were stained with I2–KI. Bar = 100 lm. b Comparison of the pollen viability of PZm401::Zm401 and PZm401::Zm401/UBI:: Zmdof1 transgenic plants. Wild-type plants (wt) and plants transformed with Zmdof1 only were used as control. Data represent mean values, the error bars represent standard deviations of the means, and more than six transgenic plants were selected for analysis in each experiment. Significance was determined

by Student’s t test with ***P \ 0.001. c Quantitative RT-PCR analyses of the expression of Zm401 and Zmdof1 in pollens of transgenic tobacco harboring PZm401::Zm401 and PZm401::Zm401/ UBI::Zmdof1. The expressions of Zm401 and Zmdof1 in the transgenic tobacco plants of PZm401::Zm401 were set as 1 artificially. Data represent mean values, the error bars represent standard deviations of the means, and more than six transgenic plants were selected for analysis in each experiment

manioca were reported to participate in the process of lightmediated flowering (Imaizumi et al. 2005; Iwamoto et al. 2009; Yang et al. 2010). OBP2 in Arabidopsis is involved in secondary metabolism expressed in flower stalks and petals (Skirycz et al. 2006). Recently, Shaw et al. (2009) found that there are some wheat Dof genes expressed in mature anthers. In this paper, we reported that Zmdof1 expressed in pollen (Fig. 3c) and regulated the pollenspecific gene expression, giving another clue to that Dof protein participates in plants reproductive growth. The 50 deletion results and online software analysis showed that there was one cluster of Dof protein recognition sites (DOFCORE) in the Zm401 promoter. To analyze the regulation of Zm401 expression, we cloned a maize Dof

transcription factor gene, Zmdof1, and analyzed it. Using the sequence of Zmdof1 published by Yanagisawa and Izui (1993), a highly homologous gene was cloned by PCR. Sequence analysis of the encoded protein showed that six changes had occurred in N-terminus, but the four cysteine residues, which are essential for DNA recognition in Dof domain, were unchanged, and no change had occurred in the C-terminus. The results of a yeast one-hybridization assay indicated that the changes in the N-terminus did not affect the function of the Dof domain. In a review, Yanagisawa stated that the N-terminus of the Dof domain protein is conserved, and the variability of the C-terminus reflects the different functions of distinct Dof domain proteins (Yanagisawa 2002). The different Dof domain

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proteins were classified by their C-termini. In addition to Zmdof1, three other Dof genes have been found in maize, Zmdof2, Zmdof3, and ZmPBF (Vicente-Carbajosa et al. 1997; Yanagisawa 1995). We compared our cloned gene with the ORFs of these three genes and found that the lengths of the four ORFs were almost different, and there was little similarity except in the Dof domain (Fig. S2). The results indicated that the cloned gene is more likely to be Zmdof1 than other Dof family members. Transient expression of Zmdof1 in onion epidermal cells indicated that this protein localizes in the nucleus (Fig. 3a). This suggests that the speculated NLS sequence in the Zmdof1 protein is functional. From the results, we confirmed that we had cloned Zmdof1 and the sequence differences were due to the different maize strain used. Many works have been done on analyzing the function of Zm401gene on pollen development previously (Dai et al. 2004, 2007; Li et al. 2001; Ma et al. 2005, 2008; Wang et al. 2009). In this work, we intend to elucidate the regulatory mechanism of Zm401. 50 deletion analysis of the Zm401 promoter showed that there may be some negative elements in the -670 to -510 region (Fig. 1). Interestingly, this region contains tandem DORCOREZM elements (Fig. S1). To confirm whether these elements interact with Zmdof1, a yeast one-hybridization assay was conducted using these two regions. b-Galactosidase assays showed that blue color developed in the reaction filter. By contrast, no blue color was detected when 60 bp (DM), with all the AAAG (DOFCORE) motifs mutated, was transformed together with PGAL1::AD::Zmdof1 into yeast (Fig. 4). Yeast one-hybrid system was widely used to confirm the interaction of transcription factors and DNA sequence. Using this system, Pruneda-Paz et al. (2009)confirmed that CHE binds to the TBS (TCP-binding site) in CCA1 promoter not in LHY promoter; Mak et al. (1996) confirmed that Gal4–MRF4 fusion protein activates expression of an E-box HIS3 reporter gene in yeast. In addition, a yeast one-hybridization assay results demonstrated that FOG interacts specifically with the N-f but not the C-f of GATA-1 to form a ternary complex on DNA in yeast cells (Tsang et al. 1997). The one yeast hybridization result shown in this report indicated that Zmdof1 interacts with the promoter of Zm401 by binding to the AAAG elements of -670 to -510 region. The expression of Zmdof1 (Fig. 3c) and Zm401 (Dai et al. 2004) in pollen suggested the possibility that Zmdof1 interacts with the promoter of Zm401 in vivo. The results shown in this report verified this suggestion. In transgenic tobacco harboring PZm401::Gus, the GUS activity was high; however, following transformation with a construct expressing Zmdof1, the GUS activity under the control of Zm401 promoter in PZm401::Gus/UBI::Zmdof1 transgenic plants decreased significantly (Fig. 6). Ma (2005) reported

281

that transgenic tobacco plants containing the Zm401 gene driven by the promoter of Zm13 displayed varying degrees of aberrant flowers, and the average viability was only about 7.6%. In the present study, Zm401 was transformed into tobacco under the control of its own promoter, resulting in low pollen viability. The pollen viability recovered when Zmdof1 was transformed into PZm401::Zm401 plants (Fig. 7). RT-PCR analysis showed that the expression of Zm401 decreased by almost 40% when the expression of Zmdof1 increased about fourfold in PZm401::Zm401/UBI::Zmdof1 transgenic tobacco pollen (Fig. 7c). These results indicated that Zmdof1 expression downregulated the expression of Zm401 controlled by Zm401 promoter and resulted in the recovery of low pollen viability. Zmdof1 is a first isolated Dof domain protein in plants, works as a trancriptional activator to C4 photosynthetic PEPC in green leaves. Depending on the result that Zmdof1 enhanced transcription differently in greening and etiolated protoplasts, Yanagisawa suggested that Dof1 is associated with the light-regulated expression (Yanagisawa 2004a; Yanagisawa and Sheen 1998). In present report, Zmdof1 expresses in pollen and downregulates Zm401 in tobacco pollen (Figs. 6, 7). Based on the Dof1 enhances gene transcription in green leaves and represses in pollen, a regulatory mechanism for tissue-specific gene expression is proposed (Fig. 8).The Zmdof1 may have dual functions in photosynthesis and pollen development in maize. Zmdof1 works as a transcriptional activator in green leaves for photosynthesis (Yanagisawa 2004a; Yanagisawa and Sheen 1998) and as a transcriptional repressor in pollen for pollen development in maize. We propose two models for the function of Zmdof1 as a suppressor in pollen development. One model is that there may exist other Dof domain protein, which binds to the Zm401 promoter and activates the Zm401 gene expression. The Zmdof1 inhibits the expression of Zm401 through competitive binding to the DOFCORE in Zm401 promoter Plant protein(s)

component or mediator +

Zmdof1

+ component or mediator Light

Zm401

Pollen development

Pollen

PEPC

Photosynthesis

Green leaves

Fig. 8 Schematic model of Zmdof1 regulates Zm401 in pollen and green leaves

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(Fig. S3 a). The other model is the involvement of the mediator(s). Mediator was first found in yeast as a co-regulator required for transcriptional activation (Flanagan et al. 1991) and was recently identified within the plant kingdom in Arabidopsis thaliana (Ba¨ckstro¨m et al. 2007). The mediators have diverse functions concerning from plant development to flowering (Kidd et al. 2011). The mediator complex performs a crucial function in gene regulation by forming a link between gene-specific transcription factors and RNA polymerase II and works as a factor required for the general initiation factors to DNA-binding transactivators (Conaway and Conaway 2011b). That mediator is suggested to be important for both activation and repression of transcription. Most mediators were reported to work as activators (Ba¨ckstro¨m et al. 2007; Bjo¨rklund and Kim 1996; Borggrefe and Yue 2011; Conaway and Conaway 2009, 2011a, b; Goodrich et al. 1996; Kidd et al. 2011; Kornberg 2005; Wang et al. 2004), while the IXL, a subunit of the mammalian mediator complex, encoded by a human gene intersex like (IXL) and identified from an embryonic heart cDNA library, was suggested to function as a transcriptional suppressor (Wang et al. 2004). In maize pollen, Zmdof1 might interact with a (some) mediator(s), which mediates the repression of transcription and results in suppressing the Zm401 gene expression (Fig. S3 b). In this report, the results indicated that Zmdof1 contacts with the promoter of Zm401 in vitro and may involve in maize pollen development by downregulation of Zm401 in vivo. In addition, the possible function mechanism of Zmof1 in maize is proposed. All these may be very informative to understand the new function of Dof domain transcription factors and the molecular mechanism of maize pollen development. Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant No. 30971555 and 30671124), National Basic Research Program of China (Grant No.2012CB215300), National Transgenic Major Program of China (Grant Nos. 2008ZX003-002 and 2009ZX08009-093B).

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