ZmSOC1, an MADS-Box Transcription Factor from Zea mays, Promotes Flowering in Arabidopsis

Zea mays is an economically important crop, but its molecular mechanism of flowering remains largely uncharacterized. The gene, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), integrates multiple flowering signals to regulate floral transition in Arabidopsis. In this study, ZmSOC1 was isolated from Zea mays. Sequence alignment and phylogenetic analysis demonstrated that the ZmSOC1 protein contained a highly conserved MADS domain and a typical SOC1 motif. ZmSOC1 protein was localized in the nucleus in protoplasts and showed no transcriptional activation activity in yeast cells. ZmSOC1 was highly expressed in maize reproductive organs, including filaments, ear and endosperm, but expression was very low in embryos; on the other hand, the abiotic stresses could repress ZmSOC1 expression. Overexpression of ZmSOC1 resulted in early flowering in Arabidopsis through increasing the expression of AtLFY and AtAP1. Overall, these results suggest that ZmSOC1 is a flowering promoter in Arabidopsis.

The MADS-box family of transcription factors in plants is known for its role in developmental processes [8]. Most of the members of this family are involved in flowering regulation, vegetative development, meristem identity regulation, floral organ development and the formation of fruit and seed [9][10][11][12][13]. The MADS-box family contains a DNA binding domain of about 58 amino acids that binds DNA sequences known as the CArG box (CC(A/T)6GG) [14,15].
Maize is an economically important crop, and a comprehensive understanding of flowering behaviors is required to increase production and improve seed quality. However, the molecular mechanisms of flowering control in maize remain poorly understood. The ZmMADS1/ZMM5, which is a SOC1/TM3-like gene in Zea mays, is expressed in vegetative organs at a low level, increased in reproductive tissues and parallels the expression pattern in the development of OsSOC1 and AtSOC1 [7,28,[33][34][35][36]. ZmMADS1/ZMM5 not only contains the typical Mikc domain, but also has a highly conserved SOC1 motif at the C-terminal, which uniquely exists in the SOC1/TM3-like gene [36,37]. Based on the similarity between ZmMADS1/ZMM5 and SOC1, we renamed ZmMADS1/ZMM5 as ZmSOC1 [38].
In the present study, we isolated the ZmSOC1 gene from Zea mays (B73). The protein sequence information, subcellular localization, transcriptional activation activity and expression pattern of ZmSOC1 in maize were investigated. Our results show that ZmSOC1 promotes flowering in Arabidopsis and suggest that the gene may play a similar role in maize.

Isolation and Characterization of ZmSOC1
The ORF of the ZmSOC1 gene was 696 base pairs (bp) in length and identical to ZmMADS1 (GenBank ID: NM_001111682.1), encoding a protein of 232 amino acids with an estimated molecular mass of 26.4 kDa. A BLAST search of GenBank revealed that the ZmSOC1 protein was similar to SbSOC1 (87.50% identity), OsSOC1 (75.11% identity), AtSOC1 (53.99% identity), HvSOC1 (65.09% identity), VvSOC1 (54.17% identity), PtSOC1 (52.05% identity) and VuSOC1 (50.48% identity), respectively ( Figure 1A). Multiple sequence alignment indicated that ZmSOC1 contained a well-conserved MADS domain and a less-conserved K domain ( Figure 1A). However, there are significant variations appearing in their C-terminals. In addition, a nuclear localization signal was predicted ( Figure 1A). To investigate the relationship between ZmSOC1 and other SOC1-like genes, a phylogenetic tree was constructed based on the amino acid sequences in their MADS-box domain ( Figure 1B). The phylogenetic tree showed that all members could be divided into dicot and monocot clades, and the ZmSOC1 protein belonged to the monocot clade together with other SOC1 homologs from the Gramineae ( Figure 1B).

Expression Profiles of ZmSOC1 in Maize
To explore whether ZmSOC1 expression was regulated by abiotic stresses, maize seedlings were treated with NaCl, PEG6000, Abscisic Acid (ABA), Salicylic acid (SA), low temperature (4 °C) and water without nutrients. At 4 °C, ZmSOC1 was slightly downregulated after 6 h; for NaCl treatment, ZmSOC1 was significantly repressed after 3 h; for PEG6000 treatment, ZmSOC1 was repressed quickly; for ABA treatment, ZmSOC1 increased within 3 h and subsequently decreased after 6 h; for SA treatment, ZmSOC1 expression decreased (but not significantly before 6 h), with a 40% reduction after 12 h, but the expression level of ZmSOC1 was lightly changed in the water without nutrients ( Figure 2A). Taken together, these results indicate that ZmSOC1 expression was downregulated by the abiotic stresses.
To profile expression of ZmSOC1 in different plant tissues and different developmental stages, we analyzed its expression using a GeneChip. ZmSOC1 maintained expression at a moderate level with relatively stable expression in roots, stems, leaves and the shoot apex ( Figure 2B). The expression level increased two-to three-fold in the filaments and ears, demonstrating that ZmSOC1 plays an important role in maize floral organ development. Interestingly, its expression remained high for 10-25 days in endosperms, but in the embryos, the expression levels decreased to almost undetectable levels, indicating that ZmSOC1 may be an essential factor for endosperm development ( Figure 2B).
MADS-box gene-specific effects are closely related to internal and external environmental factors. Zhang and Forde [39] found NO3 − as a signaling molecule stimulating lateral root elongation in Arabidopsis and is dependent of the expression of the MADS-box gene, ANR1. Wang [40] found that the RM1 (MADS-box) gene in rice callus tissue regulates plant cell dedifferentiation and re-differentiation processes and acts on target genes. After the target genes are activated, the cells transform into a distinct morphology. Overall, ZmSOC1 as a MADS-box gene may play an important role in plant growth and developmental processes.   ZmSOC1  159  AtSOC1  158  SbSOC1  158  HvSOC1  158  OsSOC1  160  VvSOC1  159  VuSOC1 159 PtSOC1 ZmSOC1  214  AtSOC1  232  SbSOC1  212  HvSOC1  229  OsSOC1  218  VvSOC1  211  VuSOC1 220 PtSOC1

Subcellular Localization and Yeast Transcriptional Activation of ZmSOC1
ZmSOC1 amino acid sequence analysis revealed a nuclear localization signal in the MADS-box domain ( Figure 1A). To validate the subcellular localization of ZmSOC1 protein, ZmSOC1:GFP was transformed into Arabidopsis mesophyll protoplasts. The fluorescence of ZmSOC1:GFP was localized in the nucleus as a blue spot according to Hoechst staining ( Figure 3A). These data demonstrate that ZmSOC1 entered the nucleus as a transcription factor and was involved in transcriptional regulation. The transcriptional activation activity of ZmSOC1 protein was analyzed using a yeast two-hybrid system. The recombinant plasmid pAS2-1:ZmSOC1, together with the positive control pGBK-GAL4-SV40-T53 and the negative control pAS2-1 plasmids, respectively, were transformed into the yeast strain AH109 containing the reporter gene HIS. All transformants grew well on the SD (Synthetic Dropout)/-Ade/-Trp media ( Figure 3B), whereas on SD/-Ade/-Trp/-His media, only the positive control pGBK-GAL4-SV40-T53 transformants survived. These results demonstrate that ZmSOC1 could not activate the expression of HIS genes in yeast cells, suggesting that ZmSOC1 protein may have no transcriptional activation activity in yeast.
Transcription factors containing sequence-specific DNA-binding motifs are key molecular switches for nuclear localization, DNA binding and dimerization [24,25,41]. In the present study, a predicted nuclear localization signal was present in the MADS domain of ZmSOC1 ( Figure 1). As expected, the ZmSOC1 protein mainly localized to the Arabidopsis nucleus ( Figure 3). However, MADS-box transcriptional activation has not been widely investigated. The C-terminus in some MADS-box proteins functions as a core transcriptional activation domain [37,42]. In the present study, we demonstrated that the ZmSOC1 protein had no transcriptional activation activity in yeast cells (Figure 3), similar to other MADS-box proteins, such as AGAMOUS (AG), APETALA3 (AP3) and PISTILLATA (PI) [43]. Because the C-terminal domain is the most divergent region among the MADS-box proteins, it is well established that some MADS-box proteins have transcriptional activity, while others do not [43,44].

Overexpression of ZmSOC1 Promotes Flowering in Arabidopsis
To examine the role of ZmSOC1 at flowering time, transgenic ZmSOC1 Arabidopsis plants driven by the Ubi promoter were generated. The transgenic plants were analyzed by qRT-PCR to confirm the expression level of ZmSOC1, and the transgenic lines all showed high ZmSOC1 expression levels compared to wild-type ( Figure 4A). Three independent T3 lines were selected for flowering time analysis under LD (Long-day) conditions. Compared to the wild-type, overexpression of ZmSCOC1 significantly promoted flowering in Arabidopsis. The number of rosette leaves showing bolting ranged from 7.2 to 9.3 in ZmSOC1-overexpressing plants and was 14.5 for wild-type plants. In addition, the transgenic lines showed no morphological changes (excluding the leaves becoming slightly smaller). However, overexpression of another nine maize MADS-box genes was not observed to promote flowering in transgenic Arabidopsis. These results indicate that ZmSOC1 functions as a flowering activator in Arabidopsis.  To examine the molecular mechanisms by which ZmSOC1 promotes flowering, the expression patterns of AtAGL24, AtSOC1, AtLFY and AtAP1 were analyzed using qRT-PCR in wild-type and transgenic seedlings under LD conditions. AtSOC1 expression levels in transgenic plants were similar to the wild-type. AtAGL24 expression levels increased slightly, demonstrating that ZmSOC1 overexpression did not influence the SOC1 upstream gene in the flowering pathway. The downstream gene, AtLFY, was obviously upregulated in transgenic plants ( Figure 5), suggesting that upregulation of AtLFY was the result of ZmSOC1. The expression of AP1 (downstream of AtLFY) was significantly upregulated in transgenic compared to wild-type plants ( Figure 5). These results demonstrate that the high expression of AtLFY and AtAP1, which are upregulated by the overexpression of ZmSOC1, should be involved in the promotion of flowering time in transgenic plants.
The overexpression of ZmSOC1 in Arabidopsis suggests that ZmSOC1 plays an evolutionarily conserved role in the promotion of flowering. First, it caused early flowering (Figure 4), in agreement with previous studies [45][46][47]. Second, it upregulated the expression of AtLFY and AtAP1 (Figure 5), which were directly or indirectly upregulated by SOC1 during the floral transition [7,25,34]. Taken together, these results demonstrate that ZmSOC1 may be a flowering promoter in maize. Because heterodimerization of AtSOC1 and AtAGL24 is a key mechanism activating AtLFY expression, we predicted that the interaction between ZmSOC1 and AtAGL24 would result in early flowering in transgenic Arabidopsis by activating AtLFY expression [7,25,34,48,49].

Plant Material and Growth Conditions
Arabidopsis thaliana Columbia plants were grown in a greenhouse with a 14/10 h light/dark cycle at 22 °C. Seeds were sterilized for 10 min in 75% ethanol with 0.1% Triton X-100, for 5 min in 95% ethanol and finally washed with absolute ethanol on filter paper for drying. Seeds were germinated on half-strength Murashige and Skoog selective plates with 50 mg/L kanamycin. After 10 days of incubation in a growth chamber (14/10 h light/dark, 22 °C), resistant plants were transferred to soil.

Gene Cloning and Vector Construction
To clone the ZmSOC1 gene, the AtSOC1 protein (GenBank ID: NP_182090.1) was used for a BLAST search in the Maize Genetics and Genomics Database [50], and a protein (GRMZM2G171365) with high similarity to AtSOC1 was identified. Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) from the ear of maize (B73). Total RNA was treated with DNase I (Promega, Madison, WI, USA), and first-strand cDNA synthesis was performed using a First Strand cDNA Synthesis Kit following the manufacturer's instructions (Promega, Madison, WI, USA).

Bioinformatics Analysis
Genes homologous to ZmSOC1 were identified using BLAST searches against GenBank [53], and potential sequence motifs were identified using SMART [54]. Protein sequence similarity ratio analysis was performed using DNAMAN software. Based on the e-value and identity, the orthologous of ZmSOC1 were selected for the construction of the phylogenetic tree using DNAMAN software.

Yeast Transcriptional Activation Assay
The full-length coding sequence of ZmSOC1 was amplified with the primer pair 5'-ACCATGGTGCGGGGCAAGACGCAG-3' (NdeI site) and 5'-AGGATCCGCCTGACCTGACCG CCACT-3' (BamHI site). The PCR product was cloned into the pAS2-1 vector. For interaction studies, the plasmid was transformed into yeast strain AH109. Yeast cells were made competent for transformation by incubation in lithium acetate solution. Then transformation was performed by incubating the cells with transforming DNA, carrier DNA and PEG3350. The Yeast Transformation Kit (SIGMA, St. Louis, MO, USA) used the lithium acetate method. The transformed strains were cultured on SD/-Ade/-Trp and SD/-Ade/-His/-Trp synthetic complete drop-out media (SC drop-out) with 3-amino-1,2,4-triazole at 30 °C.

Stress Treatments in Maize
The maize inbred line Z31 was used in this study. Z31 seeds were washed with distilled water and set in a sprout machine at 28 °C for 4 days in the dark. Seeds with 2-3 cm germs were transferred to vermiculite to grow at 28 [57]. After 10 days, the maize contained two leaves and a core stop providing nutrient solution. Subsequently, maize seedlings were treated with NaCl (250 mM), PEG6000 (20%), ABA (100 μM), and SA (100 μM) at 4 °C, with H2O as a control, and sampled at 0, 0.5, 1, 3, 6, 12 and 24 h. The samples were immediately frozen in liquid nitrogen, with three seedlings for each replicate.

Material Collecting for Microarray
Maize plants of inbred line B73 have been sequenced and have a mature GeneChip, so this was used to harvest these tissues. We harvested vegetative tissues at the big trumpet stage, including the roots, stems, leaves and the shoot apex, as well as reproductive tissues, including endosperms and embryos at 10, 15, 20 and 25 days after pollination; filaments and ear were harvested before pollination [58]. The above material was divided in three to get total RNA, and we used the Affymetrix GeneChip of maize to analysis in triplicate.

Statistics of Transgenic Arabidopsis in Phenotypes and Expression
The T3 transgenic and wild-type Arabidopsis were directly planted in nutritive soil, and total RNA was extracted from leaves after four weeks. qRT-PCR was used to analyze ZmSOC1 expression levels in transgenic Arabidopsis. The number of rosette leaves was counted when these Arabidopsis began bolting, and 30 transgenic plants and wild-type Arabidopsis were counted, respectively.

Conclusions
ZmSOC1 was isolated from maize and characterized. It showed high identity to other SOC1 homologs and contained the well-conserved MADS domain and SOC1 motif. The protein was localized to the Arabidopsis nucleus and showed no transcriptional activation activity in yeast cells. Overexpression of ZmSOC1 promoted flowering through increasing the expression of AtLFY and AtAP1 in transgenic Arabidopsis. ZmSOC1 has a high expression level in floral organs, suggesting that ZmSOC1 should be a flowering promoter in maize. These findings increase our understanding of the mechanism of flowering control in maize.