08SG2/OsBAK1 regulates grain size and number, and functions differently in Indica and Japonica backgrounds in rice
- Hua Yuan†1, 2,
- Shijun Fan†1, 2,
- Juan Huang1, 3,
- Shijie Zhan1, 2,
- Shifu Wang1, 2,
- Peng Gao1, 2,
- Weilan Chen1, 2,
- Bin Tu1, 2,
- Bingtian Ma1, 2,
- Yuping Wang1, 2,
- Peng Qin1, 2Email author and
- Shigui Li1, 2Email authorView ORCID ID profile
© The Author(s). 2017
Received: 13 April 2017
Accepted: 18 May 2017
Published: 25 May 2017
Both grain size and grain number are significant for rice yield. In the past decade, a number of genes related to grain size and grain number have been documented, however, the regulatory mechanisms underlying them remains ambiguous.
We identified a rice small grain (sg2) mutant in an EMS mutant library generated from an indica variety, Shuhui498. Using the MutMap gene mapping strategy, we identified two linkage regions on chromosome 7 and 8, respectively, consistent with the segregation ratios in the F2 population. We focused on the linkage region on chromosome 8, and named this locus as 08sg2. One of three SNPs identified in the linkage region was located in an exon of OsBAK1, leading to a nonsynonymous mutation in the kinase domain. The plant harboring the mutant version 08sg2 locus exhibited a decreased grain size, grain number and plant height. Cytological analysis indicated that 08SG2 regulated spikelet hull development by affecting cell proliferation. The grain size and number of knockout mutants of OsBAK1 in the japonica background were significantly decreased, but less so than in 08sg2, supporting the idea that the SNP in OsBAK1 was responsible for the 08sg2 phenotype, but that 08SG2/OsBAK1 function differently in indica and japonica backgrounds. 08sg2 was insensitive to 24-epiBL, and the expression of BR-related genes was obviously altered in 08sg2. The proportionally decreased grain length when 08sg2 and GS3 were combined indicate that 08SG2 and GS3 regulate grain length independently.
Our work shows that 08SG2/OsBAK1 is important for rice yield in both indica and japonica backgrounds, by regulating grain size and grain number, and the function of 08SG2/OsBAK1 is obviously affected by genetic background. The amino acid substituted in 08sg2 is highly conserved among different species, supporting the idea that it is important for the molecular function of 08SG2/OsBAK1. Together, our work is helpful for fully understanding the function of 08SG2/OsBAK1.
Grain size and grain number are the most important factors determining grain yield in rice. So far, a lot of genes related to grain size and number have been documented. Regarding grain size, multiple signaling pathways that influence grain size have been identified in rice (Li and Li 2014, 2016; Zheng et al. 2015; Zuo and Li 2014), including the ubiquitination-mediated proteasomal degradation pathway, G-protein signaling pathway, the mitogen-activated protein kinase signaling pathway, transcriptional regulatory factors and phytohormone pathways. The ubiquitination-mediated proteasomal degradation pathway includes GW2 (Song et al. 2007) and GW5/qSW5 (Shomura et al. 2008; Weng et al. 2008), and the G-protein signaling pathway includes GS3 and DEP1. GS3 is a homolog of AGG3, the G protein γ-subunit of Arabidopsis (Li et al. 2012), which encodes a putative transmembrane protein and functions as a negative regulator for grain size (Fan et al. 2006; Mao et al. 2010). DEP1 encodes a plant-specific G protein γ subunit and mutants in it have short grains (Huang et al. 2009). The mitogen-activated protein kinase (MAPK) signaling pathway includes OsMKK4 and OsMAPK6. OsMAPK6 interacts strongly with OsMKK4, indicating that the OsMKK4-OsMAPK6 cascade influences grain size in rice (Duan et al. 2014; Liu et al. 2015). The transcriptional regulatory factors pathway includes GW8, GLW7 and GS2/GL2/GLW2/OsGRF4. GW8 and GLW7 encode the plant-specific transcription factors OsSPL16 and OsSPL13 (Si et al. 2016; Wang et al. 2012), respectively. GS2/GL2/GLW2 encodes a transcription factor Growth-Regulating Factor 4 (OsGRF4), which regulates grain length and width mainly by promoting cell expansion (Che et al. 2015; Duan et al. 2015; Hu et al. 2015; Li et al. 2016). Another important signaling pathway controlling grain size is phytohormone pathway, which has been proven to play a crucial role in rice development, especially in the course of grain growth. For example, GS5, a putative serine carboxypeptidase, functions as a positive regulator of grain size (Li et al. 2011), and inhibits the interaction between OsBAK1-7 and OsMSBP1, suggesting that GS5 might regulate grain size through the brassinosteroids (BR) signaling pathway (Xu et al. 2015). GL3.1/qGL3 encodes a putative protein phosphatase with a Kelch-like repeat domain (OsPPKL1), and may function as a negative regulator through BR signaling in rice (Hu et al. 2012; Qi et al. 2012; Zhang et al. 2012). Regarding grain number, many QTLs and genes have been cloned, such as Gn1a (Ashikari et al. 2005), DEP1 (Yan et al. 2007), DEP3 (Qiao et al. 2011), GNP1 (Wu et al. 2016b) and GAD1 (Jin et al. 2016). However, as complex agronomic traits, the regulatory mechanism of grain size and number remains largely unknown.
OsBAK1, as a BRI1-associated receptor kinase (BAK1), has been proven to participate in BR signal transduction and regulate plant architecture in rice (Li et al. 2009; Shimin et al. 2014), but no mutants have been identified so far, and there was no comprehensive investigation of OsBAK1 function on yield-related traits. In this study, we identified a mutant of OsBAK1 in the indica background, named 08sg2, which exhibited a decreased plant height, grain size, grain number and panicle length. Loss of function of OsBAK1 in the japonica background exhibited significantly decreased grain size and number, but less so than in the indica background. Our results show that the reduced grain length of 08sg2 was due to decreased cell proliferation. 08sg2 was insensitive to BR, and 08SG2 affected the expression of BR-related genes. Pyramiding analysis indicated that 08SG2/OsBAK1 regulate grain length independently of GS3.
Isolation the 08sg2 mutant from a small grain (sg2) mutant
We identified a small grain (sg2) mutant from an EMS mutant library generated from an indica variety Shuhui498 (R498), which is an excellent restorer of hybrid rice. The sg2 mutant showed a significant change of plant height, grain size and panicle architecture (Additional file 1: Figure S1, Additional file 1: Table S1). To identify the gene responsible for the sg2 phenotype, we generated a F2 population from a cross between sg2 mutant and the parental line R498. Only 28 plants, about one-sixteenth of the plants (χ2 (15:1) = 1.45 < (χ2 (0.05,1) = 3.84) showed short grain length (Additional file 1: Figure S2A), suggesting that two recessive loci were responsible for the sg2 phenotype. Subsequently, we applied the MutMap strategy for gene mapping (Abe et al. 2012). Consistent with the segregation ratio, we identified two linkage regions on chromosome 7 and 8, respectively (Additional file 1: Figure S2B). These results suggest that the sg2 mutant phenotype is due to two recessive mutations, simultaneously. In this study, we focused on the linkage region on chromosome 8, and named this locus 08sg2.
Three linkage SNPs (SNP index = 1) on chromosome 8 were identified (Additional file 1: Table S2), which were located in LOC_Os08g05980, LOC_Os08g07760 and LOC_Os08g08000, respectively. RNA-sequencing showed that only LOC_Os08g07760 and LOC_Os08g08000 were expressed in young panicles, furthermore, only the SNP2 in LOC_Os08g07760 led to a nonsynonymous mutation of a glycine (GGT) to an aspartic acid (GAT) (Additional file 1: Table S2). Therefore, we considered LOC_Os08g07760 as the best candidate gene for 08SG2, which was identified as a BRI1-associated receptor kinase (BAK1) that participates in BR signal transduction (Li et al. 2009). Then, we obtained plants harboring only the 08sg2 locus using markers based on the SNP in 08SG2/OsBAK1, and generated a F2 segregation population from a cross between 08sg2 and R498. The SNP in LOC_Os08g07760 was co-segregated with the plants with extremely short grain from F2 population (Additional file 1: Figure S3), confirming LOC_Os08g07760 as the best candidate gene for 08SG2.
The 08sg2 mutant exhibits a decreased grain size and grain number
08SG2 regulates the development of spikelet hull by affecting cell proliferation
Furthermore, we analyzed the expression level of several genes related to grain size. The expression of TGW6, which negatively controls grain length and thousand-grain weight (Ishimaru et al. 2013), was increased 1.8 times in 08sg2 (Additional file 1: Figure S7), consistent with the 08sg2 small grain phenotype. Therefore, 08SG2 is likely to regulate grain length through TGW6.
08sg2 is a loss of function mutant of OsBAK1
Comparisons of the reduction percentage of 08SG2/OsBAK1 mutation in indica and japonica backgrounds
Number of grains per panicle
Grain weight per panicle
08sg2 vs R498
−10.2 ± 0.2
−2.7 ± 0.7
−8 ± 1.1
−11.9 ± 2.8
−9.7 ± 2.1
−23.3 ± 3.1
KO vs Nipponbare
−6.3 ± 0.6**
−3.4 ± 1.0
−3.8 ± 0.5**
−7.1 ± 1.7*
−6.0 ± 1.0*
−18.1 ± 3.5*
OsBAK1 was expressed ubiquitously in all organs examined, with a relatively higher expression in developing young inflorescences (Additional file 1: Figure S10), supporting the biological roles of 08SG2/OsBAK1 in regulating grain size and grain number. The sequence alignment and phylogenetic analysis of the homologs of OsBAK1 showed that OsBAK1 was conserved in different species (Additional file 1: Figure S11; Additional file 1: Figure S12), notably, the amino acid substitution in 08sg2 (G310D), which was located in the kinase domain of OsBAK1 (Fig. 3a), was highly conserved among different species (Additional file 1: Figure S11), suggesting that this amino acid may be important for 08SG2/OsBAK1 function.
The 08sg2 mutant is insensitive to 24-epiBL
08sg2 affects the expression of BR-related genes
08SG2/OsBAK1 was involved in BR signal transduction (Li et al. 2009) and the 08sg2 mutant appeared insensitive to BR (Fig. 4a-h). We therefore analyzed the expression level of BR signaling-related genes, including OsBRI1, OsMDP1, D1, OsBZR1, OsLIC1, DLT, ILI1, BU1, RAVL1, GSK2 and TUD1 (Bai et al. 2007; Duan et al. 2006; Hu et al. 2013; Je et al. 2010; Tanaka et al. 2009; Tong et al. 2009; Tong et al. 2012; Wang et al. 2008; Wang et al. 2006; Yamamuro et al. 2000; Zhang et al. 2009). Only the expression of BU1, which was reported to be a positive regulator of BR signaling, was down-regulated in 08sg2 (Fig. 4i).
In addition, many studies had reported that BR mutants usually had feedback regulation of BR biosynthetic genes (Duan et al. 2014; Fang et al. 2016; Hong et al. 2003; Liu et al. 2015; Yamamuro et al. 2000). We therefore analyzed the expression of BR biosynthetic genes in R498 and 08sg2 in young panicles, including OsDWARF, D2, D11, OsDWARF4 (Hong et al. 2003; Mori et al. 2002; Sakamoto et al. 2006; Tanabe et al. 2005). The expression levels of OsDWARF, D2 and OsDWARF4 were significantly up-regulated in 08sg2 (Fig. 4i). These results suggest that mutation in 08SG2/OsBAK1 results in the feedback regulation of BR biosynthesis in rice.
08SG2/OsBAK1 and GS3 regulate grain length in independent pathways
08SG2/OsBAK1 plays a crucial role in the regulation of grain size and number
08SG2/OsBAK1, also called OsSERK1, belongs to the Somatic Embryogenesis Receptor Kinases (SERKs) family in rice (Hu et al. 2005; Ito et al. 2005). As a SERK family gene, OsBAK1 was proven to play a basic role in mediating somatic embryogenesis in rice, and might play wider roles in other organs (Hu et al. 2005; Ito et al. 2005). Overexpression and knockdown of OsBAK1 showed that OsBAK1 controlled plant architecture and was involved in BR signaling (Li et al. 2009; Shimin et al. 2014). But there was no comprehensive investigation of OsBAK1 function on yield-related traits, such as grain size and number, except that overexpression and knockdown of OsBAK1 showed decreased grain length and increased grain width, respectively, in the ZH11 background (Li et al. 2009). However, another study showed that OsBAK1 didn’t affect grain size in the Kitaake background (Shimin et al. 2014). In this study, we used loss of function mutants of 08SG2/OsBAK1 in the indica and japonica backgrounds to investigate its genetic effect on grain size and number, and also measured other agronomic traits such as plant height, panicle length and grain weight (Fig. 1). Our results showed that the grain length and width were decreased in both indica (Shuhui498) and japonica (Nipponbare) background (Fig. 1f, g; Fig. 3f, g). These findings, different with previous reports, might be explained by ZH11 (japonica) and Kitaake (japonica) backgrounds, or because expression of OsBAK1 was not completely silenced by RNAi. Also, the decreased grain number in 08sg2/Osbak1 suggested that 08SG2/OsBAK1 is essential for rice grain number in both the indica and japonica backgrounds (Fig. 1m; Fig. 3k), which may be due to the up-regulated expression level of two negative regulators of panicle branching OsCLV1 and OsCLV2 (Additional file 1: Figure S6).
Many studies had indicated that BR-related genes regulated grain size through altering cell expansion or cell proliferation, for example: D2 (Fang et al. 2016) and D11 (Wu et al. 2016a) regulated grain size by promoting cell expansion; XIAO (Jiang et al. 2012), SMG1 (Duan et al. 2014) and DSG1 (Liu et al. 2015) regulated grain size by affecting cell proliferation through cell cycle genes. Our results indicated that 08SG2/OsBAK1 positively regulates the development of spikelet hull by affecting cell proliferation (Fig. 2h, j), consistent with this notion, the expression levels of some cell-cycle related genes were down-regulated in the 08sg2 mutant (Fig. 2k), and this result is consistent with the cytological mechanism in internodes in the OsBAK1 overexpression plants (Li et al. 2009). Our results provide a new evidence that BR may control grain size via altering cell proliferation through cell cycle genes.
Even the loss of function of 08SG2/OsBAK1 showed obvious reduction on grain size and grain number in both indica (Shuhui498) and japonica (Nipponbare) backgrounds, the differences of effect on grain size and grain number were still observed at different background, for example: loss of function of 08sg2/Osbak1 led to a 10.2% or 6.3% decrease in grain length, and to 9.7% or 6.0% decrease in grain number (Table 1) in indica (Shuhui498) and japonica (Nipponbare) backgrounds, respectively. This indicates that the function of 08SG2/OsBAK1 on grain size and number is affected by genetic background.
08SG2/OsBAK1 regulates grain length independently with GS3
08SG2/OsBAK1 positively regulated grain length in rice. However, GS3 is a negative regulator of grain length (Fan et al. 2006). The SNP substitution in 08sg2/Osbak1 led to similar reductions in the grain length in the gs3 and GS3 background (reduced 10.20% or 12.57%, respectively), and the mutation of GS3 also led to a comparable increase in the grain length in the 08sg2/Osbak1 and 08SG2/OsBAK1 backgrounds (increased 16.09% or 13.03%, respectively) (Fig. 5). In addition, the expression levels of O8SG2/OsBAK1 and GS3 were not affected by each other, suggesting that 08SG2/OsBAK1 and GS3 regulate grain length in independent pathways, with no genetic interaction.
The amino acid substitution in the kinase domain of 08SG2/OsBAK1 lead to loss function
The amino acid substitution (G310D) in 08sg2 is located in the kinase domain of OsBAK1 and this glycine is highly conserved in other species (Fig. 3a, Additional file 1: Figure S11). This amino acid substitution leads a phenotype similar to the loss of function mutant (Fig. 1; Fig. 3). OsBAK1 encodes a receptor-like protein kinase (RLK) (Ito et al. 2005). Both OsBAK1 and its homolog BAK1 in Arabidopsis are functional protein kinases capable of auto-phosphorylation, and a mutation at a conserved site (K329E, K330E, respectively) in the kinase domain led to loss of kinase activity (Li et al. 2002; Shimin et al. 2014). Several other studies in Arabidopsis showed that the amino acid substitution at a conserved site in the kinase domain of RLKs abolished kinase activity (Braun et al. 1997; Horn and Walker 1994), but the amino acid change in 08sg2 is different. Therefore, this substituted amino acid might play a role in protein function of 08SG2/OsBAK1, and it is worthy to investigate whether this amino acid substitution affects protein kinase activity, or other molecular functions of 08SG2/OsBAK1.
08SG2/OsBAK1 plays an essential role in the regulation of grain size, grain number and plant height in both the indica and japonica backgrounds, and is important for rice yield. The function of 08SG2/OsBAK1 on grain size and number is affected by genetic background. 08SG2/OsBAK1 regulates grain size by participating in cell proliferation, and is independent of GS3. The glycine substituted in 08sg2 is located in the kinase domain, is highly conserved among different species, and is presumably important for the molecular function of 08SG2/OsBAK1. Together, our work is helpful for unveiling the molecular function of 08SG2/OsBAK1, and indicating that 08SG2/OsBAK1 is a potential target to manipulate for increasing rice yield.
Plant materials and growth conditions
The small grain mutant (sg2) was initially identified from an ethyl methanesulfonate (EMS) mutant population of an indica variety Shuhui498 (R498). 08sg2 was isolated from sg2 in the segregation population by linkage marker and backcrossed with R498 three times. All plants were grown in paddies at the Rice Research Institute of Sichuan Agricultural University (Chengdu, China) and Hainan (Lingshui, China) under normal cultivation conditions.
Cytological analysis by scanning electron microscopy (SEM)
Spikelet hulls from 08sg2 and R498 plants were collected before fertilization and fixed in 2.5% glutaraldehyde, which was prepared as described (Hu et al. 2015). The fixed samples were dehydrated in a graded ethanol series (from 30% to 100%, 100%), followed by substitution using isopentyl acetate. The critical-point Dryer (Quorum K850, England) was used to dry samples. The samples were sputter-coated with platinum using a magnetron sputtering apparatus (JS-1600, Beijing, China). The inner and outer epidermal cells of lemma of the spikelet hulls were observed by SEM (JSM-7500 F, JEOL, Japan). Cell lengths were measured using Image J software.
MutMap (Abe et al. 2012) was applied for gene mapping. Briefly, we generated an F2 population of a cross between the sg2 mutant and the wild type (R498). DNA of 25 F2 progeny with small grains as sg2 was extracted and bulked in an equal ratio, then this bulked DNA was subjected to whole-genome sequencing. DNA from R498 was re-sequenced as a control. The SNPs/INDELs indexes were calculated as previously described (Abe et al. 2012). We simultaneously measured allele segregation using Euclidean distance (ED) (Additional file 1: Figure S2B) as previously described (Hill et al. 2013).
CRISPR/Cas9 plasmid construction, plant transformation and mutation detection
To obtain loss of function mutants, we designed the target site at the third exon of 08SG2/OsBAK1 (Fig. 3a), then generated the CRISPR/Cas9 plasmid construct using the Biogle CRISPR/Cas kit (BGK03, http://www.biogle.cn/) according to instructions. The Oligo sequence pair (F:5′-TGTGTGCTTGGGAATGCTGCACTATC-3′, R:5′-AAACGATAGTGCAGCATTCCCAAGCA-3′) was generated using the online system (BGK03, http://www.biogle.cn/index/excrispr). The final CRISPR/Cas9 plasmid construction pBGK03-OsBAK1 was introduced into Agrobacterium tumefaciens strain EHA105, and transformation of Nipponbare was performed as previously described (Hiei et al. 1994). For mutation detection, genomic DNA was extracted from T0 transgenic plants and primer pairs flanking the designed target site were used for PCR amplification. PCR products were sequenced to obtain knockout plants. The sequencing primers are listed in Additional file 2: Table S3.
Brassinosteroid sensitivity assay
For lamina joint inclination analysis, after soaking and pregermination, the uniform germinated seeds of R498 and 08sg2 were sown in soil and cultured in the plant growth chamber at 28 °C until two-leaf stage. 1 μl of ethanol solution containing various concentrations (0, 5, 50, 500 μM) of 24-epiBL were dropped at the lamina joints of the second leaf of R498 and 08sg2. After cultured for 3 days, the angles between the lamina and its leaf sheath were photographed and measured. 30 plants were measured for each treatment with three replications.
For the mesocotyl elongation assay, after soaking and pre-germination, the uniform germinated seeds of R498 and 08sg2 were sown in soil and cultured in the plant growth chamber at 28 °C in complete darkness for about 12 days. The mesocotyls of R498 and 08sg2 were photographed and measured. 30 plants were measured for each treatment with three replications.
For the root elongation inhibition and coleoptile elongation assays, seeds of R498 and 08sg2 were dehusked, and sterilized by washing the seeds as follows: 75% ethanol for 1 min, sterilized water for five times, 2% NaClO for 30 min, then rinsed several times with sterilized water. The seeds were then germinated on half-strength Murashige and Skoog medium supplemented with 0, lμM 24-epiBL at 28 °C. About a week later, root and coleoptile were photographed and lengths measured. 30 plants were measured for each treatment with three replications.
RNA extraction, cDNA synthesis and quantitative real-time RT-PCR
Total RNA was extracted from young panicles of R498, 08sg2, NIL-GS3 R3551 using Plant RNA Kit according to the product manual (OMEGA). To obtain cDNA, about 500 ng of the total RNA was used for a genomic DNA elimination reaction and a reverse-transcription reaction using PrimeScriptTM RT Reagent Kit with gDNA Eraser according to the product manual (TaKaRa). Quantitative real-time RT-PCR analysis was performed on the CFX96TM Real-Time PCR system (Bio-Rad) with KAPA SYBR® FAST qPCR Kit (KAPA). Actin was used as an internal control in all analyses. Three biological replicates were performed for each gene. The primers used in quantitative real-time RT-PCR are listed in Additional file 2: Table S4.
We thank Hang He and Wei Yan at Peking University for MutMap analysis, and Chengzhi Liang and Qiang Gao at Institute of Genetics and Development Biology, CAS for RNA-seq analysis, and Dr. Sheila McCormick at UC Berkeley for editing.
This work was supported by grants from the National Natural Science Foundation of China (91535102 and 31471475), and the Breeding of New Varieties of Green Super Rice (2014AA10A604).
PQ and BT generated the ethyl methanesulfonate (EMS) population. PQ, SF and JH identified the sg2 mutant and generated the F2 population. HY and SF isolated the 08sg2 mutant and investigated the agronomic traits. SZ, YW and PG performed the cytological analysis and performed the brassinosteroid sensitivity test. SW and BM performed quantitative real-time RT-PCR. HY and WC performed the plasmid construction, plant transformation and mutation detection. HY and SF wrote the manuscript and equally contributed to this work. PQ and SL designed and guided this research. All authors approved the manuscript.
The authors declare that they have no competing interests.
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