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OsNF-YB1, a rice endosperm-specific gene, is essential for cell proliferation in endosperm development
Gene 551 (2014) 214–221
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OsNF-YB1, a rice endosperm-specific gene, is essential for cell proliferation in endosperm development Xiaocong Sun a, Sheng Ling a, Zhanhua Lu b, Yi-dan Ouyang a, Shasha Liu a, Jialing Yao a,⁎ a b
College of Life Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China
a r t i c l e
i n f o
Article history: Received 25 May 2014 Received in revised form 19 August 2014 Accepted 28 August 2014 Available online 29 August 2014 Keywords: Endosperm-specific expression NF-YB Cell cycle regulator Rice
a b s t r a c t Cell cycle regulators are crucial for normal endosperm development and seed size determination. However, how the cell cycle related genes regulate endosperm development remains unclear. In this study, we reported a rice Nuclear Factor Y (NF-Y) gene OsNF-YB1, which was also identified as an endosperm-specific gene. Transcriptional profiling and promoter analysis revealed that OsNF-YB1 was highly expressed at the early stages of rice endosperm development (5–7 DAP, days after pollination). Repression of OsNF-YB1 resulted in differential expression of the genes in cell cycle pathway, which caused abnormal seeds with defected embryo and endosperm. Basic cytological analysis demonstrated that the reduced endosperm cell numbers disintegrated with the development of those abnormal seeds in OsNF-YB1 RNAi plants. Taken together, these results suggested that the endospermspecific gene OsNF-YB1 might be a cell cycle regulator and played a role in maintaining the endosperm cell proliferation. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Seed is critical for plant reproduction and crop breeding. In cereal crop, endosperm occupies the major part of the inner seed space and stored most of the seed nutrients. Thus, rice endosperm is of importance for energy providing, seed germination, and seeding development. Besides, it serves as an important food source for humans. The endosperm results from the fertilization of two polar nuclei in the central cell of the embryo sac by one sperm cell nucleus, which generates a triploid nucleus, whereas the diploid embryo originates from fertilization of the egg cell by the other sperm cell (Sabelli and Larkins, 2009). Endosperm development of rice involves several different types of coordinated cell cycle programs: early in development, after double fertilization, endosperm undergoes syncytium formation (0–2 DAP) and cellularization (3–4 DAP). At 4–10 DAP, endosperm cells undergo mitotic division rapidly that coincides with the differentiation of cell types such as aleurone, basal endosperm transfer cells, embryo-surrounding cells and starchy endosperm. At 8–10 DAP, the endosperm endoreduplicates and becomes polyploid. Finally, at 14–20 DAP, the endosperm cells begin to undergo programmed cell death (Agarwal et al., 2011; Olsen, 2001; Young and Gallie, 2000). During the early stages of seed development,
Abbreviations: NF-Y, Nuclear Factor Y; DAP, days after pollination; RBR, retinoblastomarelated; CDK, Cyclin Dependent Kinase; QRT-PCR, quantitative real-time polymerase chain reaction; RNAi, RNA interference; GFP, green fluorescent protein. ⁎ Corresponding author. E-mail address: [email protected] (J. Yao).
http://dx.doi.org/10.1016/j.gene.2014.08.059 0378-1119/© 2014 Elsevier B.V. All rights reserved.
the cell cycle regulation of endosperm is important in determining final seed size and accumulation of storages. Three different types of cell cycles are included during endosperm development: one is acytokinetic mitosis which results in a syncytium; the second is normal mitosis that produces most cells comprising the mature endosperm; and the third is endoreduplication, which entails reiterated rounds of DNA replication without chromatin condensation, sister chromatid segregation, or cytokinesis, and resulting in endopolyploid cells (Sabelli and Larkins, 2009). Generally, retinoblastoma-related (RBR) or RB protein acts as a restrictor of cell cycle progression by preventing G1 to S phase transition through inhibiting gene expression programs of E2F-dependent transcription factors involved in DNA replication, cell-cycle progression and chromatin dynamics (Sabelli et al., 2005a). Conversely, RBR/RB can be inhibited by the phosphorylation activity of Cyclin Dependent Kinase (CDK) during G1/S transition, stimulating the expression of E2F-dependent transcription factors, and complete cell cycle entry (Boniotti and Gutierrez, 2001; Cheng et al., 2013; Nakagami et al., 2002). Several genes regulating cell cycle have been identified and are crucial for normal endosperm development. Recently, maize RBR homologue of RBR1 has been demonstrated to play roles in endosperm cell proliferation, endoreduplication, as well as cell death in maize endosperm (Sabelli et al., 2005b, 2013). In rice, Orysa;KRP1 is reported as a cyclin-dependent kinase (CDK) inhibitor and plays an important role in the exit from the mitotic cell cycle during rice grain formation. Orysa;KRP1 overexpression reduced seed filling and suppressed endoreduplication by disturbing the cell expansion and/or cell division during endosperm formation (Barroco et al., 2006). Another gene Orysa;
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CycB1;1 encodes a B type cyclin, and suppression of its expression results in abnormal endosperm cellularization and produces seeds containing only an enlarged embryo at maturity (Guo et al., 2010). Rice cell cycle switch 52A (OsCCS52A) is an APC activator that is involved in maintaining normal seed size formation by mediating the exit from mitotic cell division to enter the endoreduplication cycles in rice endosperm (Su'udi et al., 2012). Multiple approaches have been adopted to investigate the genetic and epigenetic controls in rice endosperm development, including traditional genetic studies, transcriptome and proteomics analysis, and high-throughput sequencing (RNA-seq, ChIP-seq) (Gao et al., 2013; Huh et al., 2008; Xue et al., 2012). Recently, the genome-wide analysis of rice indicated that 191 transcription factor (TF)-encoding genes were predominantly expressed in seed and 59 TFs were regulated during seed development, some of which are homologues of seed-specific TFs (Xue et al., 2012). Newly, 151 endosperm-specific genes (OsEnS) were identified and classified into 12 groups by a genomic survey in rice (Nie et al., 2013). These various omics data provide informative clues for understanding the complex transcriptional networks of developing rice seeds. However, it is still unclear that how the transcription factors regulate seed development, especially in endosperm cell division cycle. Nuclear Factor Y (NF-Y) transcription factors (also known as Hemeassociated proteins [HAPs] and CCAAT box binding factors [CBFs]) are present in all higher eukaryotes. Studies in animals have revealed that NF-Ys are important regulators for cell cycle regulation and function as heterotrimeric complexes composed of single subunits from each of three protein families: NF-YA, NF-YB, and NF-YC. In animal systems, NF-Y complex has been extensively studied: NF-YA is the subunit that makes sequence-specific contact with CCAAT boxes (Xing et al., 1993); NF-YB and NF-YC possess histone-fold motifs that allow them to form a determined tight dimer (Romier et al., 2003). In mammals, NF-Y controls the activation of mitotic cyclins, and is required to activate cell cycle regulated genes (Di Agostino et al., 2006; Hu et al., 2006). Compared with the understanding of NF-Y in animals, the function research about NF-Y in plants is lagging some way behind. Unlike animals and fungi, plants have significantly expanded the number of genes encoding NF-Y subunits (Petroni et al., 2012). In rice, 28 NF-Y genes including 10 NFYA, 11 NF-YB, and 7 NF-YC, have been identified (Thirumurugan et al., 2008). And during the course of evolution, the plant NF-Ys seem to have diversified into at least two main groups. The first group has more general expression patterns and/or functions, whereas the second group has acquired more specific expression patterns and/or functions and could play key roles in specific pathways (Laloum et al., 2013). In Arabidopsis, LEAFY COTYLEDON 1 (LEC1) and the related LEC1like gene (L1L) are NF-YB genes that play essential yet complementary roles during embryo development (Braybrook and Harada, 2008; Kwong et al., 2003). In rice, silencing the expression of OsNF-YB2, OsNF-YB3, and OsNF-YB4 with RNAi method resulted in reduced accumulation of nuclear-encoded photosynthesis transcripts (Miyoshi et al., 2003). In our previous analysis, five NF-Y transcription factors have been identified in endosperm-specific genes (OsEnS) of rice (Nie et al., 2013). Compared with the identified 28 NF-Y genes (Thirumurugan et al., 2008), OsEnS-41 (LOC_Os02g49410) has been reported and named as OsNF-YB1 previously (Masiero et al., 2002; Thirumurugan et al., 2008). In this study, we further confirmed the expression pattern of OsNF-YB1 using transcriptional profiling and promoter analysis. RNAi strategy was carried out to reduce the expression of OsNFYB1, which leads to defects of seed development in transgenic plants. Further studies showed that down-regulation of OsNF-YB1 resulted in reducing endosperm cell numbers compared to WT, and suppressed expression of OsNF-YB1 can result in the differential expression of cell cycle related genes. These results implied that OsNF-YB1 might be a potential regulator for controlling the cell cycle of endosperm cell division in rice.
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2. Materials and methods 2.1. Plant materials and tissue preparation Oryza sativa L. japonica cultivar Zhong hua 11 (ZH11) was used for genetic analysis. Both wild type (WT) and transgenic rice plants were grown in the field at the Agricultural Experiment Station of Huazhong Agricultural University under normal cultural conditions. The tissues (root, stem, leaf, and seeds of different developmental stages) used for RNA isolation were frozen in liquid nitrogen when collected and stored at −80 °C for further use.
2.2. Structural and sequence analysis of OsNF-YB1 Genomic sequence, protein sequence and promoter sequences (− 2000 bp) of OsNF-YB1 were obtained from MSU (http://rice. plantbiology.msu.edu/) or RAP (http://rapdb.dna.affrc.go.jp/tools/ dump). Analyses of genomic sequence, protein sequence and promoter sequence of OsNF-YB1 were carried out in GSDS (http://gsds.cbi.pku. edu.cn/index.php), Pfam (http://pfam.sanger.ac.uk/search) and PLACE (http://www.dna.affrc.go.jp/PLACE/) respectively. Homologous genes of OsNF-YB1 were collected from NCBI database (http://www.ncbi. nlm.nih.gov/) using pblast. Protein sequences of these homologous genes were analyzed in ClustalX (version 2.0) and were used to construct a neighbor-joining tree in MEGA4.
2.3. RNA isolation and QRT-PCR Total RNAs were extracted from various ZH11 tissues [root (booting stages), stem (booting stages), flag leaf (1 day before heading), seeds (1 and 3 DAP), endosperms (5, 7, 10, 14 and 21 DAP)] or OsNF-YB1 RNAi seeds (7 DAP) using TRIzol reagent (TransGen) according to the manufacturer's instructions. RNAs were simultaneously reversetranscribed to first-strand cDNAs using M-MLV reverse transcriptase (Promega). The QRT-PCR was performed using 2 × SYBR Premix EX Taq (Takara) on ABI StepOne™. Rice Ubiguitin (Ubi) gene was amplified and used as an internal standard to normalize the expression of tested genes. Each experiment was replicated independently for three times under identical conditions. Primer sequences used in this study were listed in Supplemental Table 1.
2.4. Vector construction and rice transformation To construct an RNAi vector for OsNF-YB1, a 477 bp cDNA fragment of OsNF-YB1 was amplified using primers RNAi-F (5′-GGGACTA GTGGTACCAGCATGGCAGG GAACAAA-3′ with a SpeI site, bold letters, and a KpnI site, underlined integrated) and RNAi-R (5′-GGGGAGC TCGGATCCGATCACCGACCGGAGAAA-3′ with a SacI site and a BamHI site). PCR products were digested with KpnI/BamHI and SacI/SpeI, and were inserted into pDS1301 (Supplemental Fig. 1A) (Chu et al., 2006). We extracted genomic DNA from the leaves of ZH11 by the CTAB method (Murray and Thompson, 1980). According to the available DNA sequence, primers pM-F (5′-CGCGGATCCCCCCTTTTGAAAACAGTA TGCAGG-3′ with a BamHI site, underlined integrated) and pM-R (5′CCCAAGCTTCGCTCTATGCTTGGGCAGTATTTA-3′ with a HindIII site) were designed to isolate the promoter sequence (1974 bp) of OsNFYB1 by PCR amplification. PCR products were digested with BamHI and HindIII restriction enzymes, and inserted into pDX2181 (Supplemental Fig. 1B) at the BamHI/HindIII sites (Ye et al., 2012). Both the two constructed vectors were transformed into rice ZH11 by using the high efficient Agrobacterium-mediated transformation system (Wu et al., 2003).
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2.5. Phenotypic analysis in rice transgenic plants Transgenic rice plants were confirmed by PCR analysis using Hptspecific primers (Hpt-F: TCCATACAAGCCAACCAC; Hpt-R: TGAAAAAG CCTGAACTCAC). PCR products were detected in transgenic rice lines, using the wild type (WT) as a negative control. Seed setting rate was defined as the ratio of seed-contained grain (abnormal ones included) numbers to the total grain numbers, with 30 individual plants measured for each line. Percentage of abnormal seeds was defined as the ratio of abnormal seed numbers to the total seed numbers. Significant differences of seed setting rate and thousand seeds weight between the treatment groups (WT and RNAi) were carried out using t test in Microsoft Office Excel 2007 and the significant levels were indicated by *(P b 0.05) and **(P b 0.01).
of GFP fluorescence was carried out in transgenic plants and also nontransgenic controls. 2.7. Cytological research Rice seeds of different developmental stages were fixed in FAA containing 5% glacial acetic acid, 5% formaldehyde, 63% ethanol, and 27% water at room temperature for 24 h. After dehydration and infiltration, the samples were embedded in paraffin and cut into 10 μm thick sections. The paraffin sections were stained with ammonium methylbenzene blue. The sections were observed and captured using a BX53 microscope (Olympus, Japan). 3. Results
2.6. Detection of GFP fluorescence
3.1. Endosperm-specific gene OsNF-YB1 is a presumptive histone-like transcription factor
The independent promoter:gfp transgenic plants of T0 generation were obtained for fluorescence analysis. Rice plant roots, stems, leaves, flowers and seeds of different developing stages were photographed using a SZX16 Zoom Stereo Microscope (Olympus, Japan) equipped with an attachment for fluorescence observations. Images were captured using an Olympus E-330 camera (Olympus, Japan). The detection
Based on the genome-wide analysis of endosperm-specific genes in rice (Nie et al., 2013), OsNF-YB1 gene is regarded as an endospermspecific gene and located on chromosome 2 of the rice genome, with a 561 bp coding sequence. Two exons and one intron are included in OsNF-YB1 by analyzing its full-length genomic sequence (Fig. 1A).
Fig. 1. Structural and sequence analysis of OsNF-YB1. (A) Full-length genomic sequence of OsNF-YB1. Two exons and one intron are included. (B) Neighbor-joining tree of OsNF-YB1 in different species. Thirty genes/proteins were used in this diagram. Abbreviations are as follows: at, Arabidopsis thaliana; cs, Cucumis sativus; gm, Glycine max; os, Oryza sativa; pt, Populus trichocarpa; sb, Sorghum bicolor; si, Setaria italic; sl, Solanum lycopersicum; sm, Selaginella moellendorffii; ss, Selaginella sinensis; tu, Triticum urartu; vv, Vitis vinifera; zm, Zea mays. (C) Protein sequence analysis of OsNF-YB1 and its three homologous genes in different species.
Fig. 2. Spatial and temporal expression of OsNF-YB1. (A) Expression profiling of OsNF-YB1 in ZH11. Total RNAs were extracted from root (booting stages), stem (booting stages), leaf (1 day before heading), developing seeds (1 and 3 DAP; S1 and S3) and endosperms (5, 7, 10, 14, and 21 DAP; En5, En7, En10, En14, and En21). (B) Promoter activity in developing seeds of the pOsNF-YB1:gfp transgenic plants. Transgenic plants harboring the OsNF-YB1 promoter were analysis in comparison with WT. Developing seeds in 3, 5, 7, 10, and 14DAP were selected for photographing. Light and GFP indicate images captured using light and florescence microscopes respectively. en, endosperm; em, embryo. Bars = 1 mm.
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Table 1 The putative cis-regulatory elements related to endosperm-specific expression present in OsNF-YB1 promoter. Element name
Motif sequence
Numbers
Positions
ACGTATERD1 E-Box CAATBOX1 GATA BOX LAT52
ACGT CANNTG CAAT GATA AGAAA
7 8 14 6 3
−94, −351, −738, −1584, −1598, −1715, −1803 −77, −108, −397, −996, −1320, −1621, −1861, −1922 −143, −524, −562, −600, −1039, −1076, −1086, −1279, −1304, −1426, −1471, −1488, −1674, −1751 −544, −912, −991, −1182, −1461, −1917 −304, −1440, −1834
Protein sequence analysis revealed that there was a CBFD_NFYB_HMF domain (pfam 00808) which is described as a histone-like transcription factor (CBF/NF-Y) and archaeal histone located from 32 to 92 sites in its full 187 amino acids. Homologous genes of OsNF-YB1 in thirteen different plant species were collected to find their evolutionary relationship by drawing a neighbor-joining tree (Fig. 1B). Gramineous plants including Triticum urartu (TRIUR3_32790), Sorghum bicolor (Sb04g029340) and Setaria italica (Si018339) are found to contain the homologous genes closely related to OsNF-YB1. The same as OsNF-YB1, all these proteins are confirmed to contain a CBFD_NFYB_HMF domain in their protein sequence (Fig. 1C). So far, no related functional reports can be found about these genes.
3.2. OsNF-YB1 is highly expressed at the early stages in rice seed development The expression level of OsNF-YB1 in different developmental stages was investigated. The results showed that OsNF-YB1 was highly expressed in the developing rice endosperm, with a maximum expression levels at around 5–7 DAP, whereas its expression levels in root, stem, and leaf were much lower (Fig. 2A). This result suggested that OsNF-YB1 expressed highly in rice endosperm, especially at the early stages. Promoter sequence analysis revealed that several confirmed endosperm-specific cis-regulatory elements existed in OsNF-YB1 promoter sequence (Table 1). We further analyzed the promoter by the
Fig. 3. Reduction of OsNF-YB1 expression influenced the seeds morphology. A. The detection of interferential efficiency to the OsNF-YB1 RNAi plants in T0 generation. B. Comparison of seeds morphology between WT and OsNF-YB1 RNAi plants. Developing seeds in 3, 5, 7, 10, and 14 DAP and mature stage were photographed. Bars = 1 mm.
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Table 2 Statistical analysis to the three independent OsNF-YB1 RNAi transgenic rice seeds in T2 generation. Transgenic plants
Abnormal seed rate (%)
Seed setting rate (%)
Thousand seed weight (g)
WT L2 L6 L8
0 28.61 ± 2.57 31.65 ± 2.96 33.53 ± 1.88
88.52 66.03 58.93 61.77
24.05 21.22 20.38 20.64
± ± ± ±
2.04 3.05** 3.30** 2.43**
± ± ± ±
0.45 1.00** 0.90** 1.29**
Data are presented as mean ± SE. Significant differences from WT were carried out using t test in Microsoft Office Excel 2007 and indicated by *(P b 0.05) and **(P b 0.01) (n = 30).
gfp reported gene. Clear signals of GFP fluorescence were detected in different stages of pOsNF-YB1:gfp transgenic rice endosperm. However, no signal was observed in the roots, stems, leaves, flowers, and even rice embryo (Fig. 2B, Supplemental Fig. 2). These results thus indicated that the OsNF-YB1 promoter was sufficient to drive the endosperm-specific expression of the GFP protein in the transgenic rice plants. 3.3. Reduction of OsNF-YB1 expression results in seed developmental defect in rice In order to investigate the function of OsNF-YB1 during the seed development, RNAi strategy was performed to down-regulate the expression level of OsNF-YB1 in transgenic plants. After obtaining 25 positive transgenic rice plants, we detected the expression of OsNF-YB1 in their seeds (7 DAP) by using OsNF-YB1 specific primers (Fig. 3A).
The interferential efficiency of these single lines showed a great difference in T0 generation, and three ones with much lower expression levels compared to WT were selected for going down to the next generation (s2, s6, and s8 in Fig. 3A). Finally, the OsNF-YB1 RNAi transgenic plants in T2 generation were chosen for further analysis. The phenotype of three OsNF-YB1 RNAi lines in T2 generation were analyzed and compared to the WT. No obvious differences were observed in the vegetative growth and developmental phase between OsNF-YB1 RNAi plants and WT. However, the OsNF-YB1 RNAi plants showed a significantly reduction in both seed setting rate and thousand seed weight (t-test, P b 0.01) (Table 2). Observation in developmental seeds further detected some abnormal seeds with unfilled and shrunken phenotype compared to WT. These abnormal seeds appeared at around 5 DAP and persisted until mature stage (Fig. 3B). Statistical analysis in the mature seeds revealed that these abnormal seeds took a proportion of approximately 30% in all seeds in each line (Table 2). Paraffin section of these abnormal seeds in different developmental stages was carried out to detect the variation in the internal seeds. In WT seeds, the endosperm cells proliferated continuously with the embryo enlarged gradually from 3 DAP to 7 DAP. In contrast, the endosperm of those abnormal seeds had reduced cell numbers with much looser and more swollen morphology of cells from 3 DAP to 7 DAP (Fig. 4 RNAi type I). And the endosperm cells appeared a phenomenon of disintegrating at 5 and 7 DAP (Fig. 4 RNAi type I). The embryos developed more slowly and had an irregular shape compared with WT. In the severely defective seeds, both the endosperm and the embryos stopped
Fig. 4. Paraffin section to the abnormal seeds in different developmental stages (3, 5, and 7DAP) (Bars = 1 mm). The squared regions are enlarged in its right (Bars = 50 μm). en, endosperm; em, embryo.
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Fig. 5. QRT-PCR analysis confirmed the altered expression of different genes in independent OsNF-YB1 RNAi transgenic rice seeds (A2, A6, A8). Seeds at 7 DAP were analyzed. (A) Early endosperm development genes in rice. (B) Cell cycle-related genes in rice. (C) Starch synthesis-related genes in rice.
developing and disappeared in the end (Fig. 4 RNAi type II). All these results indicated that the OsNF-YB1 could be a critical regulator for seed formation by controlling endosperm development.
3.4. Interference of OsNF-YB1 down-regulate the expression of other early endosperm development genes in rice As described above, OsNF-YB1 is highly expressed at the early stages of seed development, and suppressed its expression can result in the seed developmental defect in rice. We thus speculated that other early endosperm development genes might be influenced in the OsNF-YB1 RNAi plants. Four early endosperm development genes in rice (MADS29, ROS1a, OsFIE1, and Orysa;CycB1;1) have been reported and their transcript levels were detected in OsNF-YB1 RNAi plants. The results showed that all the four genes were down-regulated with the suppressed expression of OsNF-YB1 (Fig. 5A). In mammals, NF-Y functions as a central regulator of cell proliferation and participate in the cell cycle regulation (Bhattacharya et al., 2003; Hu et al., 2006). Therefore, the expression level of ten genes that related to cell cycle regulation were examined in OsNF-YB1 RNAi plants. The results revealed that the expression of E2F1, E2F2, ORC1, and ORC2 were down-regulated, while RBR1 and RBR2 were up-regulated in
OsNF-YB1 RNAi plants. But the expression levels of other four genes in transgenic plants were not changed compared to WT (Fig. 5B). In crop plants, starch granule formed and deposited in endosperm since approximately 4 days after fertilization (Burrell, 2003). As thousand seeds weight of OsNF-YB1 RNAi plants were significantly reduced compared with those of WT, we further detected the expression of five starch synthesis-related genes (AGPL2, Glucan, GBSSI, BEI, and AP2/EREBP) to validate whether the process of starch synthesis was interrupted in those OsNF-YB1 RNAi plants. However, no obvious alternation was found compared to the WT (Fig. 5C). Thus in all, we speculate that interference of OsNF-YB1 results in differential expression of cell cycle-related genes in early endosperm development, which might result in abnormal endosperm cell proliferation in OsNF-YB1 RNAi transgenic rice plants. 4. Discussion Systematic transcriptional profiles have provided us informative data in understanding the tissue-specific genes, which might be involved in developmental process (Fujita et al., 2010; Sharma et al., 2012; Wang et al., 2010). Here, OsNF-YB1, an endosperm-specific gene in rice (Nie et al., 2013), was further confirmed to express in the early stages of seed development (5–7 DAP) by QRT-PCR analysis. The
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promoter of OsNF-YB1 was active in rice endosperm, which can drive endosperm-specific expression of the gfp gene in its promoter:gfp transgenic rice plants. Our results are consistent with previous report which showed that OsNF-YB1 transcripts can only be detected in developing seed tissues (Masiero et al., 2002). Several endosperm-specific cisregulatory elements were found in OsNF-YB1 promoter region, including seven ACGT-core elements (Schindler et al., 1992), eight E-Box motifs (CANNTG) (Stalberg et al., 1996), fourteen CAAT BOX consensus sequence (Shirsat et al., 1989), six GATA BOX consensus sequence (Gidoni et al., 1989; Lam et al., 1989) and three LAT52 motifs (AGAAA) (Filichkin et al., 2004). These results provide an explanation to the endospermspecific expression pattern of OsNF-YB1 gene. It has been reported that rice NF-Ys proteins contain a conserved domain in their middle and variable sequences in their N- and C-terminus regions (Thirumurugan et al., 2008). Here, based on the protein sequence analysis, a conserved CBFD_NFYB_HMF domain presented in OsNF-YB1 and its homologous proteins (Fig. 1C). In addition, OsNFYB1 has been identified as a partner of OsMADS18 (Masiero et al., 2002). Functional analysis indicated that OsMADS18 is involved in the control of the flowering and fruit development (Immink et al., 1999). In this study, our results showed that suppressed expression of OsNFYB1 could result in unfilled and shrunken seeds, and reducing of both seed setting rate and thousand seed weight. It is likely that OsNF-YB1 functions in rice seed development via forming a complex with MADSbox proteins to control the expression of its target genes. Moreover, expression levels of OsMADS29, OsROS1a, OsFIE, and Orysa;CycB1;1, which were involved in early endosperm development, were changed in OsNF-YB1 RNAi plants. MADS29, a transcription factor that preferentially expressed in nucellus and nucellar projection, can promote the degradation of maternal tissues by directly regulating the expression of genes encoding a cysteine protease and NBS-LRR proteins, and affects the dry matter accumulation and seed morphology (Yin and Xue, 2012). ROS1a encodes a putative bi-functional DNA demethylase, and the maternal ros1a allele can cause failure of early-stage endosperm development (Ono et al., 2012; Zemach et al., 2010). The expression and phenotype were similar between OsNF-YB1 and these genes, indicating that OsNF-YB1 might be integrated into the regulating network of seed early development in rice. Furthermore, we found that the abnormal seeds showed reduced endosperm cell numbers and even disintegrated at later developmental stage. Moreover, the expression level of six cell cycle related genes showed obviously alternation in OsNF-YB1 RNAi plants, with downregulation of E2F1, E2F2, ORC1, and ORC2, which have been proved to be positive regulators in the cell cycle (Diaz-Trivino et al., 2005; Ren et al., 2002), and up-regulation of RBR1 and RBR2, which has been reported as negative regulators (Sabelli et al., 2005a). E2F controls the transcription of a wide range of genes including genes involved in cellcycle progression and DNA synthesis, replication and repair, and plays a crucial role in the regulation of G1-to-S phase transition (Menges et al., 2002; Ren et al., 2002; Shen, 2002). RBRs belong to a conserved protein family that primarily prevent cells from entering S phase by inhibiting E2F transcription factors, the activity of which is required for the expression of many S-phase genes (Sabelli et al., 2005a, 2013). In Arabidopsis, ORC genes contain the binding sites for the E2F family transcription factors and function as the E2F targets (Diaz-Trivino et al., 2005). Thus, it is assumed that OsNF-YB1 functions as a cell cycle regulator and plays a crucial role in regulating cell cycle-related genes in rice endosperm. In addition, five starch synthesis genes did not change their expression when the expression of OsNF-YB1 was suppressed. This indicates that the reduction of thousand seeds weight in mutant is not because of the starch accumulation, but owning to the reducing of endosperm cell numbers. The abnormal embryos in OsNF-YB1 RNAi seeds was speculated to ascribe failure endosperm development. It is well-known that endosperm is critical for development of young embryo and the size of endosperm and embryo limit each other physically (Zhou et al., 2013). Mature seed of rice endospermless mutant en1-1 and en1-2 has no
endosperm but an extremely large embryo (Hong et al., 1996). Knockdown of Orysa;CycB1;1 results in abnormal seeds with absent endosperm and enlarged embryo (Guo et al., 2010). Whereas in our present research, interference of OsNF-YB1 can result in the missing of both endosperm and embryo. Therefore, it is possible that the early abortion of endosperm would result in embryo development ceasing, while the late disintegration of endosperm may produce a large embryo. In conclusion, the results of this study display that the OsNF-YB1, a rice endosperm-specific gene, is essential for cell proliferation in endosperm development. It is useful to understand the function and mechanism of plant NF-YB proteins which has acquired more specific expression patterns and could play key roles in specific pathways. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2014.08.059. Conflict of interest We declare that there is no conflict of interest. Acknowledgments We thank Dr. Chungen Hu for his helpful suggestions and discussion. This research was supported by grants from the National Natural Science Foundation of China (Grant No. 30971551 and Project J1103510), and the Fundamental Research Funds for the Central Universities (Grant No. 2012MBDX012). References Agarwal, P., Kapoor, S., Tyagi, A.K., 2011. Transcription factors regulating the progression of monocot and dicot seed development. Bioessays 33, 189–202. Barroco, R.M., Peres, A., Droual, A.M., De Veylder, L., Nguyen le, S.L., De Wolf, J., Mironov, V., Peerbolte, R., Beemster, G.T., Inze, D., Broekaert, W.F., Frankard, V., 2006. The cyclindependent kinase inhibitor Orysa;KRP1 plays an important role in seed development of rice. Plant Physiol. 142, 1053–1064. Bhattacharya, A., Deng, J.M., Zhang, Z., Behringer, R., de Crombrugghe, B., Maity, S.N., 2003. The B subunit of the CCAAT box binding transcription factor complex (CBF/NF-Y) is essential for early mouse development and cell proliferation. Cancer Res. 63, 8167–8172. Boniotti, M.B., Gutierrez, C., 2001. A cell-cycle-regulated kinase activity phosphorylates plant retinoblastoma protein and contains, in Arabidopsis, a CDKA/cyclin D complex. Plant J. 28, 341–350. Braybrook, S.A., Harada, J.J., 2008. LECs go crazy in embryo development. Trends Plant Sci. 13, 624–630. Burrell, M.M., 2003. Starch: the need for improved quality or quantity: an overview. J. Exp. Bot. 54, 451–456. Cheng, Y., Cao, L., Wang, S., Li, Y., Shi, X., Liu, H., Li, L., Zhang, Z., Fowke, L.C., Wang, H., Zhou, Y., 2013. Downregulation of multiple CDK inhibitor ICK/KRP genes upregulates the E2F pathway and increases cell proliferation, and organ and seed sizes in Arabidopsis. Plant J. 75, 642–655. Chu, Z., Yuan, M., Yao, L., Ge, X., Yuan, B., Xu, C., Li, X., Fu, B., Li, Z., Bennetzen, J.L., Zhang, Q., Wang, S., 2006. Promoter mutations of an essential gene for pollen development result in disease resistance in rice. Genes Dev. 20, 1250–1255. Di Agostino, S., Strano, S., Emiliozzi, V., Zerbini, V., Mottolese, M., Sacchi, A., Blandino, G., Piaggio, G., 2006. Gain of function of mutant p53: the mutant p53/NF-Y protein complex reveals an aberrant transcriptional mechanism of cell cycle regulation. Cancer Cell 10, 191–202. Diaz-Trivino, S., del Mar Castellano, M., de la Paz Sanchez, M., Ramirez-Parra, E., Desvoyes, B., Gutierrez, C., 2005. The genes encoding Arabidopsis ORC subunits are E2F targets and the two ORC1 genes are differently expressed in proliferating and endoreplicating cells. Nucleic Acids Res. 33, 5404–5414. Filichkin, S.A., Leonard, J.M., Monteros, A., Liu, P.P., Nonogaki, H., 2004. A novel endo-betamannanase associated with anther and gene in tomato LeMAN5 is pollen development. Plant Physiol. 134, 1080–1087. Fujita, M., Horiuchi, Y., Ueda, Y., Mizuta, Y., Kubo, T., Yano, K., Yamaki, S., Tsuda, K., Nagata, T., Niihama, M., Kato, H., Kikuchi, S., Hamada, K., Mochizuki, T., Ishimizu, T., Iwai, H., Tsutsumi, N., Kurata, N., 2010. Rice expression atlas in reproductive development. Plant Cell Physiol. 51, 2060–2081. Gao, Y., Xu, H., Shen, Y., Wang, J., 2013. Transcriptomic analysis of rice (Oryza sativa) endosperm using the RNA-Seq technique. Plant Mol. Biol. 81, 363–378. Gidoni, D., Brosio, P., Bond-Nutter, D., Bedbrook, J., Dunsmuir, P., 1989. Novel cis-acting elements in Petunia Cab gene promoters. Mol. Gen. Genet. 215, 337–344. Guo, J., Wang, F., Song, J., Sun, W., Zhang, X.S., 2010. The expression of Orysa;CycB1;1 is essential for endosperm formation and causes embryo enlargement in rice. Planta 231, 293–303. Hong, S.K., Kitano, H., Satoh, H., Nagato, Y., 1996. How is embryo size genetically regulated in rice? Development 122, 2051–2058.
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Gene journal homepage: www.elsevier.com/locate/gene
OsNF-YB1, a rice endosperm-specific gene, is essential for cell proliferation in endosperm development Xiaocong Sun a, Sheng Ling a, Zhanhua Lu b, Yi-dan Ouyang a, Shasha Liu a, Jialing Yao a,⁎ a b
College of Life Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China
a r t i c l e
i n f o
Article history: Received 25 May 2014 Received in revised form 19 August 2014 Accepted 28 August 2014 Available online 29 August 2014 Keywords: Endosperm-specific expression NF-YB Cell cycle regulator Rice
a b s t r a c t Cell cycle regulators are crucial for normal endosperm development and seed size determination. However, how the cell cycle related genes regulate endosperm development remains unclear. In this study, we reported a rice Nuclear Factor Y (NF-Y) gene OsNF-YB1, which was also identified as an endosperm-specific gene. Transcriptional profiling and promoter analysis revealed that OsNF-YB1 was highly expressed at the early stages of rice endosperm development (5–7 DAP, days after pollination). Repression of OsNF-YB1 resulted in differential expression of the genes in cell cycle pathway, which caused abnormal seeds with defected embryo and endosperm. Basic cytological analysis demonstrated that the reduced endosperm cell numbers disintegrated with the development of those abnormal seeds in OsNF-YB1 RNAi plants. Taken together, these results suggested that the endospermspecific gene OsNF-YB1 might be a cell cycle regulator and played a role in maintaining the endosperm cell proliferation. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Seed is critical for plant reproduction and crop breeding. In cereal crop, endosperm occupies the major part of the inner seed space and stored most of the seed nutrients. Thus, rice endosperm is of importance for energy providing, seed germination, and seeding development. Besides, it serves as an important food source for humans. The endosperm results from the fertilization of two polar nuclei in the central cell of the embryo sac by one sperm cell nucleus, which generates a triploid nucleus, whereas the diploid embryo originates from fertilization of the egg cell by the other sperm cell (Sabelli and Larkins, 2009). Endosperm development of rice involves several different types of coordinated cell cycle programs: early in development, after double fertilization, endosperm undergoes syncytium formation (0–2 DAP) and cellularization (3–4 DAP). At 4–10 DAP, endosperm cells undergo mitotic division rapidly that coincides with the differentiation of cell types such as aleurone, basal endosperm transfer cells, embryo-surrounding cells and starchy endosperm. At 8–10 DAP, the endosperm endoreduplicates and becomes polyploid. Finally, at 14–20 DAP, the endosperm cells begin to undergo programmed cell death (Agarwal et al., 2011; Olsen, 2001; Young and Gallie, 2000). During the early stages of seed development,
Abbreviations: NF-Y, Nuclear Factor Y; DAP, days after pollination; RBR, retinoblastomarelated; CDK, Cyclin Dependent Kinase; QRT-PCR, quantitative real-time polymerase chain reaction; RNAi, RNA interference; GFP, green fluorescent protein. ⁎ Corresponding author. E-mail address: [email protected] (J. Yao).
http://dx.doi.org/10.1016/j.gene.2014.08.059 0378-1119/© 2014 Elsevier B.V. All rights reserved.
the cell cycle regulation of endosperm is important in determining final seed size and accumulation of storages. Three different types of cell cycles are included during endosperm development: one is acytokinetic mitosis which results in a syncytium; the second is normal mitosis that produces most cells comprising the mature endosperm; and the third is endoreduplication, which entails reiterated rounds of DNA replication without chromatin condensation, sister chromatid segregation, or cytokinesis, and resulting in endopolyploid cells (Sabelli and Larkins, 2009). Generally, retinoblastoma-related (RBR) or RB protein acts as a restrictor of cell cycle progression by preventing G1 to S phase transition through inhibiting gene expression programs of E2F-dependent transcription factors involved in DNA replication, cell-cycle progression and chromatin dynamics (Sabelli et al., 2005a). Conversely, RBR/RB can be inhibited by the phosphorylation activity of Cyclin Dependent Kinase (CDK) during G1/S transition, stimulating the expression of E2F-dependent transcription factors, and complete cell cycle entry (Boniotti and Gutierrez, 2001; Cheng et al., 2013; Nakagami et al., 2002). Several genes regulating cell cycle have been identified and are crucial for normal endosperm development. Recently, maize RBR homologue of RBR1 has been demonstrated to play roles in endosperm cell proliferation, endoreduplication, as well as cell death in maize endosperm (Sabelli et al., 2005b, 2013). In rice, Orysa;KRP1 is reported as a cyclin-dependent kinase (CDK) inhibitor and plays an important role in the exit from the mitotic cell cycle during rice grain formation. Orysa;KRP1 overexpression reduced seed filling and suppressed endoreduplication by disturbing the cell expansion and/or cell division during endosperm formation (Barroco et al., 2006). Another gene Orysa;
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CycB1;1 encodes a B type cyclin, and suppression of its expression results in abnormal endosperm cellularization and produces seeds containing only an enlarged embryo at maturity (Guo et al., 2010). Rice cell cycle switch 52A (OsCCS52A) is an APC activator that is involved in maintaining normal seed size formation by mediating the exit from mitotic cell division to enter the endoreduplication cycles in rice endosperm (Su'udi et al., 2012). Multiple approaches have been adopted to investigate the genetic and epigenetic controls in rice endosperm development, including traditional genetic studies, transcriptome and proteomics analysis, and high-throughput sequencing (RNA-seq, ChIP-seq) (Gao et al., 2013; Huh et al., 2008; Xue et al., 2012). Recently, the genome-wide analysis of rice indicated that 191 transcription factor (TF)-encoding genes were predominantly expressed in seed and 59 TFs were regulated during seed development, some of which are homologues of seed-specific TFs (Xue et al., 2012). Newly, 151 endosperm-specific genes (OsEnS) were identified and classified into 12 groups by a genomic survey in rice (Nie et al., 2013). These various omics data provide informative clues for understanding the complex transcriptional networks of developing rice seeds. However, it is still unclear that how the transcription factors regulate seed development, especially in endosperm cell division cycle. Nuclear Factor Y (NF-Y) transcription factors (also known as Hemeassociated proteins [HAPs] and CCAAT box binding factors [CBFs]) are present in all higher eukaryotes. Studies in animals have revealed that NF-Ys are important regulators for cell cycle regulation and function as heterotrimeric complexes composed of single subunits from each of three protein families: NF-YA, NF-YB, and NF-YC. In animal systems, NF-Y complex has been extensively studied: NF-YA is the subunit that makes sequence-specific contact with CCAAT boxes (Xing et al., 1993); NF-YB and NF-YC possess histone-fold motifs that allow them to form a determined tight dimer (Romier et al., 2003). In mammals, NF-Y controls the activation of mitotic cyclins, and is required to activate cell cycle regulated genes (Di Agostino et al., 2006; Hu et al., 2006). Compared with the understanding of NF-Y in animals, the function research about NF-Y in plants is lagging some way behind. Unlike animals and fungi, plants have significantly expanded the number of genes encoding NF-Y subunits (Petroni et al., 2012). In rice, 28 NF-Y genes including 10 NFYA, 11 NF-YB, and 7 NF-YC, have been identified (Thirumurugan et al., 2008). And during the course of evolution, the plant NF-Ys seem to have diversified into at least two main groups. The first group has more general expression patterns and/or functions, whereas the second group has acquired more specific expression patterns and/or functions and could play key roles in specific pathways (Laloum et al., 2013). In Arabidopsis, LEAFY COTYLEDON 1 (LEC1) and the related LEC1like gene (L1L) are NF-YB genes that play essential yet complementary roles during embryo development (Braybrook and Harada, 2008; Kwong et al., 2003). In rice, silencing the expression of OsNF-YB2, OsNF-YB3, and OsNF-YB4 with RNAi method resulted in reduced accumulation of nuclear-encoded photosynthesis transcripts (Miyoshi et al., 2003). In our previous analysis, five NF-Y transcription factors have been identified in endosperm-specific genes (OsEnS) of rice (Nie et al., 2013). Compared with the identified 28 NF-Y genes (Thirumurugan et al., 2008), OsEnS-41 (LOC_Os02g49410) has been reported and named as OsNF-YB1 previously (Masiero et al., 2002; Thirumurugan et al., 2008). In this study, we further confirmed the expression pattern of OsNF-YB1 using transcriptional profiling and promoter analysis. RNAi strategy was carried out to reduce the expression of OsNFYB1, which leads to defects of seed development in transgenic plants. Further studies showed that down-regulation of OsNF-YB1 resulted in reducing endosperm cell numbers compared to WT, and suppressed expression of OsNF-YB1 can result in the differential expression of cell cycle related genes. These results implied that OsNF-YB1 might be a potential regulator for controlling the cell cycle of endosperm cell division in rice.
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2. Materials and methods 2.1. Plant materials and tissue preparation Oryza sativa L. japonica cultivar Zhong hua 11 (ZH11) was used for genetic analysis. Both wild type (WT) and transgenic rice plants were grown in the field at the Agricultural Experiment Station of Huazhong Agricultural University under normal cultural conditions. The tissues (root, stem, leaf, and seeds of different developmental stages) used for RNA isolation were frozen in liquid nitrogen when collected and stored at −80 °C for further use.
2.2. Structural and sequence analysis of OsNF-YB1 Genomic sequence, protein sequence and promoter sequences (− 2000 bp) of OsNF-YB1 were obtained from MSU (http://rice. plantbiology.msu.edu/) or RAP (http://rapdb.dna.affrc.go.jp/tools/ dump). Analyses of genomic sequence, protein sequence and promoter sequence of OsNF-YB1 were carried out in GSDS (http://gsds.cbi.pku. edu.cn/index.php), Pfam (http://pfam.sanger.ac.uk/search) and PLACE (http://www.dna.affrc.go.jp/PLACE/) respectively. Homologous genes of OsNF-YB1 were collected from NCBI database (http://www.ncbi. nlm.nih.gov/) using pblast. Protein sequences of these homologous genes were analyzed in ClustalX (version 2.0) and were used to construct a neighbor-joining tree in MEGA4.
2.3. RNA isolation and QRT-PCR Total RNAs were extracted from various ZH11 tissues [root (booting stages), stem (booting stages), flag leaf (1 day before heading), seeds (1 and 3 DAP), endosperms (5, 7, 10, 14 and 21 DAP)] or OsNF-YB1 RNAi seeds (7 DAP) using TRIzol reagent (TransGen) according to the manufacturer's instructions. RNAs were simultaneously reversetranscribed to first-strand cDNAs using M-MLV reverse transcriptase (Promega). The QRT-PCR was performed using 2 × SYBR Premix EX Taq (Takara) on ABI StepOne™. Rice Ubiguitin (Ubi) gene was amplified and used as an internal standard to normalize the expression of tested genes. Each experiment was replicated independently for three times under identical conditions. Primer sequences used in this study were listed in Supplemental Table 1.
2.4. Vector construction and rice transformation To construct an RNAi vector for OsNF-YB1, a 477 bp cDNA fragment of OsNF-YB1 was amplified using primers RNAi-F (5′-GGGACTA GTGGTACCAGCATGGCAGG GAACAAA-3′ with a SpeI site, bold letters, and a KpnI site, underlined integrated) and RNAi-R (5′-GGGGAGC TCGGATCCGATCACCGACCGGAGAAA-3′ with a SacI site and a BamHI site). PCR products were digested with KpnI/BamHI and SacI/SpeI, and were inserted into pDS1301 (Supplemental Fig. 1A) (Chu et al., 2006). We extracted genomic DNA from the leaves of ZH11 by the CTAB method (Murray and Thompson, 1980). According to the available DNA sequence, primers pM-F (5′-CGCGGATCCCCCCTTTTGAAAACAGTA TGCAGG-3′ with a BamHI site, underlined integrated) and pM-R (5′CCCAAGCTTCGCTCTATGCTTGGGCAGTATTTA-3′ with a HindIII site) were designed to isolate the promoter sequence (1974 bp) of OsNFYB1 by PCR amplification. PCR products were digested with BamHI and HindIII restriction enzymes, and inserted into pDX2181 (Supplemental Fig. 1B) at the BamHI/HindIII sites (Ye et al., 2012). Both the two constructed vectors were transformed into rice ZH11 by using the high efficient Agrobacterium-mediated transformation system (Wu et al., 2003).
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2.5. Phenotypic analysis in rice transgenic plants Transgenic rice plants were confirmed by PCR analysis using Hptspecific primers (Hpt-F: TCCATACAAGCCAACCAC; Hpt-R: TGAAAAAG CCTGAACTCAC). PCR products were detected in transgenic rice lines, using the wild type (WT) as a negative control. Seed setting rate was defined as the ratio of seed-contained grain (abnormal ones included) numbers to the total grain numbers, with 30 individual plants measured for each line. Percentage of abnormal seeds was defined as the ratio of abnormal seed numbers to the total seed numbers. Significant differences of seed setting rate and thousand seeds weight between the treatment groups (WT and RNAi) were carried out using t test in Microsoft Office Excel 2007 and the significant levels were indicated by *(P b 0.05) and **(P b 0.01).
of GFP fluorescence was carried out in transgenic plants and also nontransgenic controls. 2.7. Cytological research Rice seeds of different developmental stages were fixed in FAA containing 5% glacial acetic acid, 5% formaldehyde, 63% ethanol, and 27% water at room temperature for 24 h. After dehydration and infiltration, the samples were embedded in paraffin and cut into 10 μm thick sections. The paraffin sections were stained with ammonium methylbenzene blue. The sections were observed and captured using a BX53 microscope (Olympus, Japan). 3. Results
2.6. Detection of GFP fluorescence
3.1. Endosperm-specific gene OsNF-YB1 is a presumptive histone-like transcription factor
The independent promoter:gfp transgenic plants of T0 generation were obtained for fluorescence analysis. Rice plant roots, stems, leaves, flowers and seeds of different developing stages were photographed using a SZX16 Zoom Stereo Microscope (Olympus, Japan) equipped with an attachment for fluorescence observations. Images were captured using an Olympus E-330 camera (Olympus, Japan). The detection
Based on the genome-wide analysis of endosperm-specific genes in rice (Nie et al., 2013), OsNF-YB1 gene is regarded as an endospermspecific gene and located on chromosome 2 of the rice genome, with a 561 bp coding sequence. Two exons and one intron are included in OsNF-YB1 by analyzing its full-length genomic sequence (Fig. 1A).
Fig. 1. Structural and sequence analysis of OsNF-YB1. (A) Full-length genomic sequence of OsNF-YB1. Two exons and one intron are included. (B) Neighbor-joining tree of OsNF-YB1 in different species. Thirty genes/proteins were used in this diagram. Abbreviations are as follows: at, Arabidopsis thaliana; cs, Cucumis sativus; gm, Glycine max; os, Oryza sativa; pt, Populus trichocarpa; sb, Sorghum bicolor; si, Setaria italic; sl, Solanum lycopersicum; sm, Selaginella moellendorffii; ss, Selaginella sinensis; tu, Triticum urartu; vv, Vitis vinifera; zm, Zea mays. (C) Protein sequence analysis of OsNF-YB1 and its three homologous genes in different species.
Fig. 2. Spatial and temporal expression of OsNF-YB1. (A) Expression profiling of OsNF-YB1 in ZH11. Total RNAs were extracted from root (booting stages), stem (booting stages), leaf (1 day before heading), developing seeds (1 and 3 DAP; S1 and S3) and endosperms (5, 7, 10, 14, and 21 DAP; En5, En7, En10, En14, and En21). (B) Promoter activity in developing seeds of the pOsNF-YB1:gfp transgenic plants. Transgenic plants harboring the OsNF-YB1 promoter were analysis in comparison with WT. Developing seeds in 3, 5, 7, 10, and 14DAP were selected for photographing. Light and GFP indicate images captured using light and florescence microscopes respectively. en, endosperm; em, embryo. Bars = 1 mm.
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Table 1 The putative cis-regulatory elements related to endosperm-specific expression present in OsNF-YB1 promoter. Element name
Motif sequence
Numbers
Positions
ACGTATERD1 E-Box CAATBOX1 GATA BOX LAT52
ACGT CANNTG CAAT GATA AGAAA
7 8 14 6 3
−94, −351, −738, −1584, −1598, −1715, −1803 −77, −108, −397, −996, −1320, −1621, −1861, −1922 −143, −524, −562, −600, −1039, −1076, −1086, −1279, −1304, −1426, −1471, −1488, −1674, −1751 −544, −912, −991, −1182, −1461, −1917 −304, −1440, −1834
Protein sequence analysis revealed that there was a CBFD_NFYB_HMF domain (pfam 00808) which is described as a histone-like transcription factor (CBF/NF-Y) and archaeal histone located from 32 to 92 sites in its full 187 amino acids. Homologous genes of OsNF-YB1 in thirteen different plant species were collected to find their evolutionary relationship by drawing a neighbor-joining tree (Fig. 1B). Gramineous plants including Triticum urartu (TRIUR3_32790), Sorghum bicolor (Sb04g029340) and Setaria italica (Si018339) are found to contain the homologous genes closely related to OsNF-YB1. The same as OsNF-YB1, all these proteins are confirmed to contain a CBFD_NFYB_HMF domain in their protein sequence (Fig. 1C). So far, no related functional reports can be found about these genes.
3.2. OsNF-YB1 is highly expressed at the early stages in rice seed development The expression level of OsNF-YB1 in different developmental stages was investigated. The results showed that OsNF-YB1 was highly expressed in the developing rice endosperm, with a maximum expression levels at around 5–7 DAP, whereas its expression levels in root, stem, and leaf were much lower (Fig. 2A). This result suggested that OsNF-YB1 expressed highly in rice endosperm, especially at the early stages. Promoter sequence analysis revealed that several confirmed endosperm-specific cis-regulatory elements existed in OsNF-YB1 promoter sequence (Table 1). We further analyzed the promoter by the
Fig. 3. Reduction of OsNF-YB1 expression influenced the seeds morphology. A. The detection of interferential efficiency to the OsNF-YB1 RNAi plants in T0 generation. B. Comparison of seeds morphology between WT and OsNF-YB1 RNAi plants. Developing seeds in 3, 5, 7, 10, and 14 DAP and mature stage were photographed. Bars = 1 mm.
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Table 2 Statistical analysis to the three independent OsNF-YB1 RNAi transgenic rice seeds in T2 generation. Transgenic plants
Abnormal seed rate (%)
Seed setting rate (%)
Thousand seed weight (g)
WT L2 L6 L8
0 28.61 ± 2.57 31.65 ± 2.96 33.53 ± 1.88
88.52 66.03 58.93 61.77
24.05 21.22 20.38 20.64
± ± ± ±
2.04 3.05** 3.30** 2.43**
± ± ± ±
0.45 1.00** 0.90** 1.29**
Data are presented as mean ± SE. Significant differences from WT were carried out using t test in Microsoft Office Excel 2007 and indicated by *(P b 0.05) and **(P b 0.01) (n = 30).
gfp reported gene. Clear signals of GFP fluorescence were detected in different stages of pOsNF-YB1:gfp transgenic rice endosperm. However, no signal was observed in the roots, stems, leaves, flowers, and even rice embryo (Fig. 2B, Supplemental Fig. 2). These results thus indicated that the OsNF-YB1 promoter was sufficient to drive the endosperm-specific expression of the GFP protein in the transgenic rice plants. 3.3. Reduction of OsNF-YB1 expression results in seed developmental defect in rice In order to investigate the function of OsNF-YB1 during the seed development, RNAi strategy was performed to down-regulate the expression level of OsNF-YB1 in transgenic plants. After obtaining 25 positive transgenic rice plants, we detected the expression of OsNF-YB1 in their seeds (7 DAP) by using OsNF-YB1 specific primers (Fig. 3A).
The interferential efficiency of these single lines showed a great difference in T0 generation, and three ones with much lower expression levels compared to WT were selected for going down to the next generation (s2, s6, and s8 in Fig. 3A). Finally, the OsNF-YB1 RNAi transgenic plants in T2 generation were chosen for further analysis. The phenotype of three OsNF-YB1 RNAi lines in T2 generation were analyzed and compared to the WT. No obvious differences were observed in the vegetative growth and developmental phase between OsNF-YB1 RNAi plants and WT. However, the OsNF-YB1 RNAi plants showed a significantly reduction in both seed setting rate and thousand seed weight (t-test, P b 0.01) (Table 2). Observation in developmental seeds further detected some abnormal seeds with unfilled and shrunken phenotype compared to WT. These abnormal seeds appeared at around 5 DAP and persisted until mature stage (Fig. 3B). Statistical analysis in the mature seeds revealed that these abnormal seeds took a proportion of approximately 30% in all seeds in each line (Table 2). Paraffin section of these abnormal seeds in different developmental stages was carried out to detect the variation in the internal seeds. In WT seeds, the endosperm cells proliferated continuously with the embryo enlarged gradually from 3 DAP to 7 DAP. In contrast, the endosperm of those abnormal seeds had reduced cell numbers with much looser and more swollen morphology of cells from 3 DAP to 7 DAP (Fig. 4 RNAi type I). And the endosperm cells appeared a phenomenon of disintegrating at 5 and 7 DAP (Fig. 4 RNAi type I). The embryos developed more slowly and had an irregular shape compared with WT. In the severely defective seeds, both the endosperm and the embryos stopped
Fig. 4. Paraffin section to the abnormal seeds in different developmental stages (3, 5, and 7DAP) (Bars = 1 mm). The squared regions are enlarged in its right (Bars = 50 μm). en, endosperm; em, embryo.
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Fig. 5. QRT-PCR analysis confirmed the altered expression of different genes in independent OsNF-YB1 RNAi transgenic rice seeds (A2, A6, A8). Seeds at 7 DAP were analyzed. (A) Early endosperm development genes in rice. (B) Cell cycle-related genes in rice. (C) Starch synthesis-related genes in rice.
developing and disappeared in the end (Fig. 4 RNAi type II). All these results indicated that the OsNF-YB1 could be a critical regulator for seed formation by controlling endosperm development.
3.4. Interference of OsNF-YB1 down-regulate the expression of other early endosperm development genes in rice As described above, OsNF-YB1 is highly expressed at the early stages of seed development, and suppressed its expression can result in the seed developmental defect in rice. We thus speculated that other early endosperm development genes might be influenced in the OsNF-YB1 RNAi plants. Four early endosperm development genes in rice (MADS29, ROS1a, OsFIE1, and Orysa;CycB1;1) have been reported and their transcript levels were detected in OsNF-YB1 RNAi plants. The results showed that all the four genes were down-regulated with the suppressed expression of OsNF-YB1 (Fig. 5A). In mammals, NF-Y functions as a central regulator of cell proliferation and participate in the cell cycle regulation (Bhattacharya et al., 2003; Hu et al., 2006). Therefore, the expression level of ten genes that related to cell cycle regulation were examined in OsNF-YB1 RNAi plants. The results revealed that the expression of E2F1, E2F2, ORC1, and ORC2 were down-regulated, while RBR1 and RBR2 were up-regulated in
OsNF-YB1 RNAi plants. But the expression levels of other four genes in transgenic plants were not changed compared to WT (Fig. 5B). In crop plants, starch granule formed and deposited in endosperm since approximately 4 days after fertilization (Burrell, 2003). As thousand seeds weight of OsNF-YB1 RNAi plants were significantly reduced compared with those of WT, we further detected the expression of five starch synthesis-related genes (AGPL2, Glucan, GBSSI, BEI, and AP2/EREBP) to validate whether the process of starch synthesis was interrupted in those OsNF-YB1 RNAi plants. However, no obvious alternation was found compared to the WT (Fig. 5C). Thus in all, we speculate that interference of OsNF-YB1 results in differential expression of cell cycle-related genes in early endosperm development, which might result in abnormal endosperm cell proliferation in OsNF-YB1 RNAi transgenic rice plants. 4. Discussion Systematic transcriptional profiles have provided us informative data in understanding the tissue-specific genes, which might be involved in developmental process (Fujita et al., 2010; Sharma et al., 2012; Wang et al., 2010). Here, OsNF-YB1, an endosperm-specific gene in rice (Nie et al., 2013), was further confirmed to express in the early stages of seed development (5–7 DAP) by QRT-PCR analysis. The
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promoter of OsNF-YB1 was active in rice endosperm, which can drive endosperm-specific expression of the gfp gene in its promoter:gfp transgenic rice plants. Our results are consistent with previous report which showed that OsNF-YB1 transcripts can only be detected in developing seed tissues (Masiero et al., 2002). Several endosperm-specific cisregulatory elements were found in OsNF-YB1 promoter region, including seven ACGT-core elements (Schindler et al., 1992), eight E-Box motifs (CANNTG) (Stalberg et al., 1996), fourteen CAAT BOX consensus sequence (Shirsat et al., 1989), six GATA BOX consensus sequence (Gidoni et al., 1989; Lam et al., 1989) and three LAT52 motifs (AGAAA) (Filichkin et al., 2004). These results provide an explanation to the endospermspecific expression pattern of OsNF-YB1 gene. It has been reported that rice NF-Ys proteins contain a conserved domain in their middle and variable sequences in their N- and C-terminus regions (Thirumurugan et al., 2008). Here, based on the protein sequence analysis, a conserved CBFD_NFYB_HMF domain presented in OsNF-YB1 and its homologous proteins (Fig. 1C). In addition, OsNFYB1 has been identified as a partner of OsMADS18 (Masiero et al., 2002). Functional analysis indicated that OsMADS18 is involved in the control of the flowering and fruit development (Immink et al., 1999). In this study, our results showed that suppressed expression of OsNFYB1 could result in unfilled and shrunken seeds, and reducing of both seed setting rate and thousand seed weight. It is likely that OsNF-YB1 functions in rice seed development via forming a complex with MADSbox proteins to control the expression of its target genes. Moreover, expression levels of OsMADS29, OsROS1a, OsFIE, and Orysa;CycB1;1, which were involved in early endosperm development, were changed in OsNF-YB1 RNAi plants. MADS29, a transcription factor that preferentially expressed in nucellus and nucellar projection, can promote the degradation of maternal tissues by directly regulating the expression of genes encoding a cysteine protease and NBS-LRR proteins, and affects the dry matter accumulation and seed morphology (Yin and Xue, 2012). ROS1a encodes a putative bi-functional DNA demethylase, and the maternal ros1a allele can cause failure of early-stage endosperm development (Ono et al., 2012; Zemach et al., 2010). The expression and phenotype were similar between OsNF-YB1 and these genes, indicating that OsNF-YB1 might be integrated into the regulating network of seed early development in rice. Furthermore, we found that the abnormal seeds showed reduced endosperm cell numbers and even disintegrated at later developmental stage. Moreover, the expression level of six cell cycle related genes showed obviously alternation in OsNF-YB1 RNAi plants, with downregulation of E2F1, E2F2, ORC1, and ORC2, which have been proved to be positive regulators in the cell cycle (Diaz-Trivino et al., 2005; Ren et al., 2002), and up-regulation of RBR1 and RBR2, which has been reported as negative regulators (Sabelli et al., 2005a). E2F controls the transcription of a wide range of genes including genes involved in cellcycle progression and DNA synthesis, replication and repair, and plays a crucial role in the regulation of G1-to-S phase transition (Menges et al., 2002; Ren et al., 2002; Shen, 2002). RBRs belong to a conserved protein family that primarily prevent cells from entering S phase by inhibiting E2F transcription factors, the activity of which is required for the expression of many S-phase genes (Sabelli et al., 2005a, 2013). In Arabidopsis, ORC genes contain the binding sites for the E2F family transcription factors and function as the E2F targets (Diaz-Trivino et al., 2005). Thus, it is assumed that OsNF-YB1 functions as a cell cycle regulator and plays a crucial role in regulating cell cycle-related genes in rice endosperm. In addition, five starch synthesis genes did not change their expression when the expression of OsNF-YB1 was suppressed. This indicates that the reduction of thousand seeds weight in mutant is not because of the starch accumulation, but owning to the reducing of endosperm cell numbers. The abnormal embryos in OsNF-YB1 RNAi seeds was speculated to ascribe failure endosperm development. It is well-known that endosperm is critical for development of young embryo and the size of endosperm and embryo limit each other physically (Zhou et al., 2013). Mature seed of rice endospermless mutant en1-1 and en1-2 has no
endosperm but an extremely large embryo (Hong et al., 1996). Knockdown of Orysa;CycB1;1 results in abnormal seeds with absent endosperm and enlarged embryo (Guo et al., 2010). Whereas in our present research, interference of OsNF-YB1 can result in the missing of both endosperm and embryo. Therefore, it is possible that the early abortion of endosperm would result in embryo development ceasing, while the late disintegration of endosperm may produce a large embryo. In conclusion, the results of this study display that the OsNF-YB1, a rice endosperm-specific gene, is essential for cell proliferation in endosperm development. It is useful to understand the function and mechanism of plant NF-YB proteins which has acquired more specific expression patterns and could play key roles in specific pathways. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2014.08.059. Conflict of interest We declare that there is no conflict of interest. Acknowledgments We thank Dr. Chungen Hu for his helpful suggestions and discussion. This research was supported by grants from the National Natural Science Foundation of China (Grant No. 30971551 and Project J1103510), and the Fundamental Research Funds for the Central Universities (Grant No. 2012MBDX012). References Agarwal, P., Kapoor, S., Tyagi, A.K., 2011. Transcription factors regulating the progression of monocot and dicot seed development. Bioessays 33, 189–202. Barroco, R.M., Peres, A., Droual, A.M., De Veylder, L., Nguyen le, S.L., De Wolf, J., Mironov, V., Peerbolte, R., Beemster, G.T., Inze, D., Broekaert, W.F., Frankard, V., 2006. The cyclindependent kinase inhibitor Orysa;KRP1 plays an important role in seed development of rice. Plant Physiol. 142, 1053–1064. Bhattacharya, A., Deng, J.M., Zhang, Z., Behringer, R., de Crombrugghe, B., Maity, S.N., 2003. The B subunit of the CCAAT box binding transcription factor complex (CBF/NF-Y) is essential for early mouse development and cell proliferation. Cancer Res. 63, 8167–8172. Boniotti, M.B., Gutierrez, C., 2001. A cell-cycle-regulated kinase activity phosphorylates plant retinoblastoma protein and contains, in Arabidopsis, a CDKA/cyclin D complex. Plant J. 28, 341–350. Braybrook, S.A., Harada, J.J., 2008. LECs go crazy in embryo development. Trends Plant Sci. 13, 624–630. Burrell, M.M., 2003. Starch: the need for improved quality or quantity: an overview. J. Exp. Bot. 54, 451–456. Cheng, Y., Cao, L., Wang, S., Li, Y., Shi, X., Liu, H., Li, L., Zhang, Z., Fowke, L.C., Wang, H., Zhou, Y., 2013. Downregulation of multiple CDK inhibitor ICK/KRP genes upregulates the E2F pathway and increases cell proliferation, and organ and seed sizes in Arabidopsis. Plant J. 75, 642–655. Chu, Z., Yuan, M., Yao, L., Ge, X., Yuan, B., Xu, C., Li, X., Fu, B., Li, Z., Bennetzen, J.L., Zhang, Q., Wang, S., 2006. Promoter mutations of an essential gene for pollen development result in disease resistance in rice. Genes Dev. 20, 1250–1255. Di Agostino, S., Strano, S., Emiliozzi, V., Zerbini, V., Mottolese, M., Sacchi, A., Blandino, G., Piaggio, G., 2006. Gain of function of mutant p53: the mutant p53/NF-Y protein complex reveals an aberrant transcriptional mechanism of cell cycle regulation. Cancer Cell 10, 191–202. Diaz-Trivino, S., del Mar Castellano, M., de la Paz Sanchez, M., Ramirez-Parra, E., Desvoyes, B., Gutierrez, C., 2005. The genes encoding Arabidopsis ORC subunits are E2F targets and the two ORC1 genes are differently expressed in proliferating and endoreplicating cells. Nucleic Acids Res. 33, 5404–5414. Filichkin, S.A., Leonard, J.M., Monteros, A., Liu, P.P., Nonogaki, H., 2004. A novel endo-betamannanase associated with anther and gene in tomato LeMAN5 is pollen development. Plant Physiol. 134, 1080–1087. Fujita, M., Horiuchi, Y., Ueda, Y., Mizuta, Y., Kubo, T., Yano, K., Yamaki, S., Tsuda, K., Nagata, T., Niihama, M., Kato, H., Kikuchi, S., Hamada, K., Mochizuki, T., Ishimizu, T., Iwai, H., Tsutsumi, N., Kurata, N., 2010. Rice expression atlas in reproductive development. Plant Cell Physiol. 51, 2060–2081. Gao, Y., Xu, H., Shen, Y., Wang, J., 2013. Transcriptomic analysis of rice (Oryza sativa) endosperm using the RNA-Seq technique. Plant Mol. Biol. 81, 363–378. Gidoni, D., Brosio, P., Bond-Nutter, D., Bedbrook, J., Dunsmuir, P., 1989. Novel cis-acting elements in Petunia Cab gene promoters. Mol. Gen. Genet. 215, 337–344. Guo, J., Wang, F., Song, J., Sun, W., Zhang, X.S., 2010. The expression of Orysa;CycB1;1 is essential for endosperm formation and causes embryo enlargement in rice. Planta 231, 293–303. Hong, S.K., Kitano, H., Satoh, H., Nagato, Y., 1996. How is embryo size genetically regulated in rice? Development 122, 2051–2058.
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