Differential expression patterns of 13 OsMBD family genes in rice tissues
A bioinformatics analysis has identified 17 putative OsMBD proteins in rice . Considering the annotation updates of the rice genome over the past decades, in this study, we verified the predicted OsMBD family genes by searching the National Center for Biotechnology Information (NCBI) and MSU Rice Genome Annotation Project (RGAP) databases. Our search revealed 15 genes in both NCBI and RGAP databases matching with previously predicted OsMBD701, OsMBD703, OsMBD704, OsMBD705, OsMBD706, OsMBD707, OsMBD708, OsMBD709, OsMBD710, OsMBD711, OsMBD713, OsMBD714, OsMBD715, OsMBD717, and OsMBD718, respectively (Additional file 1: Table S1). However, no NCBI RAP locus or MSU RGAP locus has been identified that match with OsMBD712 or OsMBD716 (Additional file 1: Table S1). In addition, a RAP locus Os04g0192775 and a MSU RGAP locus LOC_Os04g11510 were retrieved as genes putatively encoding MBD-containing proteins (Additional file 1: Table S1).
Quantitative RT-PCR (qRT-PCR) was performed to explore the expression patterns of the OsMBD family genes in the roots, stems, leaves, spikelets, seeds, and panicle axes of rice plants. Among the 17 putative OsMBD-encoding genes retrieved from NCBI and/or MSU RGAP databases, no transcripts of the predicted OsMBD703/Os06g0702100/LOC_Os06g48870, OsMBD713/Os04g0193900/LOC_Os04g11730, Os04g0192775, or LOC_Os04g11510 were detected in any tested tissues (Additional file 1: Table S1). While OsMBD704 and OsMBD714 were detected to be preferentially expressed in the seeds, OsMBD701, OsMBD705, OsMBD706, OsMBD707, OsMBD708, OsMBD709, OsMBD710, OsMBD711, OsMBD715, OsMBD717, and OsMBD718 were observed to be differentially expressed in various tissues (Fig. 1).
OsMBD707 is constitutively expressed and localized in the nucleus
Rice OsMBDs could be divided into six classes . In the present study, we focused on functional analysis of OsMBD707, which belongs to class Ⅰ . Phylogenetic analysis of OsMBD707 and closely related MBDs in various plant species showed that OsMBD707 was clustered together with ObMBD11-like in Oryza brachyantha, hypothetical protein TVU48968.1 in Eragrostis curvula, SiMBD10 and SiMBD11 in Setaria italica, ZmMBD105 and ZmMBD106 in Zea mays, BdMBD11 in Brachypodium distachyon, and SbMBD10 and SbxP2 in Sorghum bicolor (Fig. 2). qRT-PCR analysis showed that the mRNA of OsMBD707 was expressed in various tissues, although the transcript level in the spikelets was relatively low (Fig. 1). A 1931-bp promoter fragment upstream of the translational start of OsMBD707 was cloned and fused with the GUS reporter gene. Histochemical staining of rice plants transformed with the OsMBD707 promoter-GUS fusion construct showed that GUS was expressed throughout the tested tissues, including the roots, stems, leaves, and spikelets, although the expression level was weaker than that of 35S promoter-GUS transgenic plants (Fig. 3a). Overall, these results indicated that OsMBD707 is constitutively expressed in various rice tissues.
OsMBD707 was predicted to generate two alternative transcripts, XM_015764399.1/LOC_Os12g42550.1, and XM_015764400.2/LOC_Os12g42550.2 (Additional file 2: Figure S1A, B). We performed RT-PCR to clone the cDNA fragment of OsMBD707, but obtained only XM_015764399.1/LOC_Os12g42550.1. An RT-PCR using primers (Additional file 2: Figure S1A) designed to distinguish the two predicted alternative transcripts was further performed. Consistently, only XM_015764399.1/LOC_Os12g42550.1 was detected in all tested tissues, including the roots, stems, leaves, spikelets, seeds, and panicle axes (Additional file 2: Figure S1C, D), suggesting that there is only one isoform, LOC_Os12g42550.1, of OsMBD707. To explore the subcellular localization of OsMBD707, we performed transient expression of a GFP-OsMBD707 fusion construct in rice protoplasts. Microscopy revealed that the fluorescence signal of GFP-OsMBD707 was inside the nucleus region (Fig. 3b), demonstrating that OsMBD707 is a nuclear-localized protein which is consistent with a function as a methyl-CpG-binding protein.
Overexpression of OsMBD707 causes larger tiller angles and reduced photoperiod sensitivity in rice
To explore the function of OsMBD707 in rice, we generated overexpression and RNAi knockdown transgenic plants. For each type, more than 40 independent transgenic T0 plants were generated, and five independent plants were chosen for initial analysis. qRT-PCR analysis showed that the transcription levels of OsMBD707 were significantly higher in plants transformed with OsMBD707-overexpression construct (about 12- to 43- fold) and lower in plants transformed with OsMBD707-RNAi construct (about 11–27%), as compared to wild-type plants (Fig. 4a, b), confirming the overexpression and knock-down of OsMBD707, respectively, in the transgenic plants. In addition, we generated CRISPR/Cas9 knockout plants of OsMBD707. Genotyping of the CRISPR/Cas9 transgenic plants identified nine independent T0 plants with homozygous mutations in at least one of the single guide RNA (sgRNA) targeting sites of OsMBD707 (Fig. 4c). Initial phenotypic observation showed that the OsMBD707-overexpression plants (referred to as OX707) displayed a larger tiller angle after tillering stage compared to wild-type. In contrast, no obvious morphological differences were observed among wild-type, the OsMBD707-RNAi plants (referred to as 707i) and the CRISPR/Cas9 knockout plants (referred to as mbd707).
OsMBD707-overexpression, -knockdown, and -knockout lines were generated up to T3 to T4 generations, and two independent overexpression lines, one knockdown line, and one knockout line were chosen for further analysis. Consistent with initial phenotypic observation, the two OsMBD707-overexpression lines OX707-#20 and OX707-#21 displayed larger tiller angles, compared to wild-type, the knockdown line 707i-#30, and the knockout line mbd707-#15 (Fig. 5a). In addition, we observed significant delays in flowering of the two overexpression lines grown under short day (SD) condition (Fig. 5a). We further investigated the heading dates of the OsMBD707-overexpression, -knockdown, and -knockout lines in growth chambers under SD and long day (LD) conditions. As showed in Fig. 5b, under SD, the flowering times of OX707-#20 and OX707-#21 were significantly delayed (about 15–17 days) compared with that of wild-type, 707i-#30 or mbd707-#15 (Fig. 5b). In contrast, under LD, the flowering times of OX707-#20 and OX707-#21 were significantly earlier (about 10.5–12.5 days) compared with that of wild-type, 707i-#30 or mbd707-#15 (Fig. 5c), indicating that overexpression of OsMBD707 caused reduced photoperiod sensitivity in transgenic rice plants.
Global transcriptome analysis reveals transcriptional changes in key flowering regulator genes induced by overexpression of MBD707
RNA-seq-based transcriptome analysis was performed to investigate global transcript changes in the OsMBD707-overexpression transgenic line OX707-#21 under both SD and LD conditions. Under SD, about 1,026 genes were identified that were differentially expressed between OX707-#21 and wild-type, and of these, 616 genes were up-regulated, whereas 410 genes were down-regulated in OX707-#21 (Additional file 3: Figure S2A, Additional file 4: Table S2). Under LD, about 1,653 differentially expressed genes (DEGs) were identified between OX707-#21 and wild-type, including 997 up-regulated genes and 656 down-regulated genes in OX707-#21 (Additional file 3: Figure S2A, Additional file 5: Table S3). In total, about 2,353 DEGs were identified under SD and/or LD (Additional file 3: Figure S2B).
Gene Ontology (GO) analysis of these 2,353 DEGs showed that the most common biological process categories were associated with translation, peptide biosynthetic, peptide metabolic, amide biosynthetic, and cellular amide metabolic processes; the most common cellular component categories were ribosome, ribonucleoprotein complex, non-membrane-bounded organelle, intracellular non-membrane-bounded organelle, and cytoplasmic part; and the top three common molecular function categories were structural molecule activity, structural constituent of ribosome, and transferase activity/transferring glycosyl groups (Additional file 6: Figure S3, Additional file 7: Table S4). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the identified DEGs were significantly enriched in ribosome and phenylpropanoid biosynthesis pathways (Additional file 8: Figure S4, Additional file 9: Table S5).
We further surveyed the DEGs with known or putative functions involved in tiller angle or flowering time regulation. The phytochromeinteracting factorlike protein gene OsPIL15 (Os01g0286100) that negatively regulates tiller angle  was significantly down-regulated in OX707-#21 under both SD and LD (Additional file 4: Table S2, Additional file 5: Table S3). Notably, a number of genes with functions in controlling flowering time were identified among the DEGs, including FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (OsFKF1) , Early heading date 1 (Ehd1) [31, 32], Days to heading on chromosome 2 (DTH2) , Heading date 3a (Hd3a) and RICE FLOWERING LOCUS T1 (RFT1) [34, 35], OsMADs14 and OsMADS15 , and Flowering Locus T gene homologs FT-L7, FT-L8 and FT-L12 that promote flowering, and Grain number, plant height, and heading date 2 (GHd2)  that inhibits flowering. Under SD, Hd3a, RFT1, FT-L7, OsMADs14, and OsMADS15 were down- regulated in OX707-#21. In contrast, OsFKF1, Ehd1, Hd3a, RFT1, FT-L8, FT-L12, and OsMADs14 were up-regulated, whereas Ghd2 was down-regulated in OX707-#21 under LD (Fig. 6a, b). The transcriptional changes of these flowering regulator genes in OX707-#21 were consistent with the delayed flowering and early flowering phenotypes of the MBD707-overexpression line under SD and LD, respectively, except that the minor-effect heading promoting gene DTH2 was paradoxically down-regulated under LD (Fig. 6a, b). The transcriptional profiles of five key flowering regulator genes, Ehd1, Hd3a, RFT1, OsMADS14, and OsMADS15 were verified by qRT-PCR, and the results were consistent with the RNA-seq data (Fig. 6c).