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. 2015 Apr 28;14(1):377–386. doi: 10.1111/pbi.12391

CLUSTERED PRIMARY BRANCH 1, a new allele of DWARF11, controls panicle architecture and seed size in rice

Yongzhen Wu 1, Yongcai Fu 1, Shuangshuang Zhao 1, Ping Gu 1, Zuofeng Zhu 1, Chuanqing Sun 1,2, Lubin Tan 1,✉
PMCID: PMC11388900  PMID: 25923523

Summary

Panicle architecture and seed size are important agronomic traits that directly determine grain yield in rice (Oryza sativa L.). Although a number of key genes controlling panicle architecture and seed size have been cloned and characterized in recent years, their genetic and molecular mechanisms remain unclear. In this study, we identified a mutant that produced panicles with fascicled primary branching and reduced seeds in size. We isolated the underlying CLUSTERED PRIMARY BRANCH 1 ( CPB1) gene, a new allele of DWARF11 (D11) encoding a cytochrome P450 protein involved in brassinosteroid (BR) biosynthesis pathway. Genetic transformation experiments confirmed that a His360Leu amino acid substitution residing in the highly conserved region of CPB1/D11 was responsible for the panicle architecture and seed size changes in the cpb1 mutants. Overexpression of CPB1/D11 under the background of cpb1 mutant not only rescued normal panicle architecture and plant height, but also had a larger leaf angle and seed size than the controls. Furthermore, the CPB1/D11 transgenic plants driven by panicle‐specific promoters can enlarge seed size and enhance grain yield without affecting other favourable agronomic traits. These results demonstrated that the specific mutation in CPB1/D11 influenced development of panicle architecture and seed size, and manipulation of CPB1/D11 expression using the panicle‐specific promoter could be used to increase seed size, leading to grain yield improvement in rice.

Keywords: Oryza sativa, brassinosteroid, panicle architecture, seed size, transgenic plants

Introduction

Rice (Oryza sativa L.) is a very important staple food crop which feeds more than half of the world's population. With an ever‐increasing global population, food security is becoming a serious problem. Accordingly, improving crop productivity is a major challenge for rice breeding programmes worldwide (Sakamoto and Matsuoka, 2004; Wang and Li, 2008). Both panicle architecture and seed size are important agronomic traits directly affecting grain yield, which have been a major purpose in crop improvement programmes. In recent years, several important genes, including LP, DEP1, OsLG1, GL3.1, TGW6, qSW5, qGW8 etc., have been identified to control the development of panicle morphology and seed size in rice (Huang et al., 2009; Ishimaru et al., 2013; Li et al., 2011; Qi et al., 2012; Shomura et al., 2008; Wang et al., 2012; Zhu et al., 2013). However, the molecular mechanisms underlying the development of panicle architecture and grain size remain unclear.

Brassinosteroids (BRs), a very important family of phytohormone, play critical roles in plant development, such as plant height, leaf angle, panicle morphology and seed size (Vriet et al., 2012). Multiple pieces of evidence indicated that genetic manipulation of BR biosynthesis or signalling pathways could significantly improve seed yield in plant. For instance, the overexpression of DWARF4 in Arabidopsis promoted seed yield by about 60% per plant with an increased number of branches and siliques (Choe et al., 2001). Likewise, an elevated silique number and seed yield were observed in transgenic Arabidopsis plants overexpressing the HSD1 gene that encodes a putative enzyme involved in BR synthesis (Li et al., 2007). In rice, the os‐dwarf4‐1 mutant increased grain yields when the plant density was relatively higher or where no extra fertilizer was added (Sakamoto et al., 2006). Driving the expression of DWARF4 homologous genes from rice, maize and Arabidopsis in rice, using a specific promoter, brought about 15%–44% increases in grain yield per plant compared to the wild type plants due to greater seed production and bigger seeds both in greenhouse and in field trials (Wu et al., 2008). In contrast, the expression of OsDWARF4 driven by a medium constitutive promoter in rice resulted in a severe depression in plant development leading to reduced seed production (Reuzeau et al., 2005). Therefore, an optimized expression of BR‐related genes using a tissue‐specific promoter will be helpful for improving grain size and yield in cereal crops.

In this study, we identified a mutant, clustered primary branch 1 (cpb1), showing phenotypes of increased BR‐sensitivity, clustered primary branch of panicle and smaller seed compared to wild type. Our genetic mapping experiments revealed that the underlying CPB1, a new allele of DWARF11 (D11), encodes a cytochrome P450 protein involved in BR biosynthesis pathway. Our studies also showed that a specific modification in the CPB1/D11 protein sequence altered the development of panicle architecture, plant height and seed size simultaneously. Furthermore, we found that an optimized expression of CPB1/D11 could lead to increased seed size and improved grain yields in rice plants.

Results

Characterization of cpb1

We identified a mutant, clustered primary branch 1 (cpb1) in an EMS‐mutagenized population of an indica line CP78. The panicle architecture of cpb1 mutants was dramatically altered compared to that of wild type, such as clustered primary branching and longer basal rachis internodes (Figure 1a, Table S1). Morphological analysis revealed that the internode of the primary branch was abnormally elongated in the cpb1 plants (Figure 1b), which might cause fascicled primary branches. The cpb1 appeared semidwarf phenotype (Figure 1c), featured with shorter culm internodes (Figure S1a,h), primarily caused by the shorter cell length in the cpb1 first culm internode revealed by a microscopic examination test (Figure S1a,b,c,h). Furthermore, the thicker stalk of cpb1 was observed that was mainly caused by increases in total number of cells and vascular bundles (Figure S1d–g,i). The cpb1 leaf angle was smaller compared to the wild type (Figure 1e). Histological examination revealed that the smaller leaf angle of the cpb1 plants was mainly due to shortened cell length in the lamina joint adaxial epidermis (Figure 1f,g). In addition, the cpb1 mutants had produced various unfavourable traits, such as delayed heading date, smaller grains, reduced seed setting ratio and lower thousand seed weight (Figure 1c,d, Table S1).

Figure 1.

Figure 1

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Phenotype comparison between the wild type and cpb1 mutant. (a) Panicle comparison of wild type (WT) and cpb1 plants at the grain filling stage. Brackets indicate the basal rachis internodes. Arrows indicate the formation of several primary branches into a cluster. (b) Micrographs of the clusters at the WT and cpb1 panicle internodes magnified from the boxed region in (a), respectively. (c) Gross plant morphology of the WT and cpb1 plants. (d) Comparison of the grains from the WT and cpb1 plants. (e) Comparison of lamina joint bending of the second leaf on the WT and cpb1 plants. (f and g): Microscopic observation of longitudinal sections of the adaxial epidermis of a lamina joint. (h) Skotomorphogenesis of WT and cpb1 plants. The arrows indicate nodes. (i) Lamina inclination after epiBL treatment in WT and cpb1 plants. (j) Measurements of lamina inclination in response to epiBL. Scale bars (a) 5 cm, (b) 1 mm, (c) 20 cm, (d) 2 mm, (e) 1 cm, (f, g) 200 μm and (h) 2 cm. Values in (j) are means ± SD (n = 20). Two‐tailed Student's t‐tests were performed between WT and cpb1 (*P < 0.05, **P < 0.01).

The observed multiple morphological defects of cpb1 mutants showed similar characteristics in appearance to the BR‐related mutants, such as d2, d11 and dlt (Hong et al., 2003; Tanabe et al., 2005; Tong et al., 2009). To test whether CPB1 was involved in BR‐mediated developmental processes, we measured the growth and morphological responses of the cpb1 mutants by observing internode elongation in the dark and inclination of the lamina joints after exposure to exogenous epiBL treatment. In the dark, BR‐defective rice mutants failed to elongate low internodes. Skotomorphogenesis was examined in cpb1 mutant seedlings. The results showed that etiolated seedlings of cpb1 mutant plants had shorter internodes than those of the wild type (Figure 1h). Lamina inclination assays indicated that the wild type plants had an epiBL dosage‐dependent lamina inclination. In contrast, cpb1 plants showed more obvious inclination of lamina joints relative to the wild type after treatment with 10–1000 ng/μL epiBL (Figure 1i,j). These results collectively indicated that the cpb1 was a BR‐related mutant and was sensitive to epiBL treatment.

Map‐based cloning and functional analysis of CPB1

To analyse the genetic factor for the altered cpb1 phenotypes, we produced a cross between mutant cpb1 and wild type CP78. All resultant F1 plants showed phenotype similar to the wild type, and the phenotypic segregation ratio of wild type (WT) to cpb1 mutant plants in the F2 population in a mendelian model of 3 : 1 (155 WT plants/45 cpb1 plants; χ2 C < χ2 0.05,1 = 3.84). These results indicated that the clustered primary branch in cpb1 mutants was controlled by a single recessive nuclear gene.

To map CPB1, we next generated a F2 population derived from a cross between cpb1 and a japonica cultivar C418. CPB1 was roughly mapped on the long arm of chromosome 4 between InDel (insertion‐deletion) markers S1 and S2 (Figure 2a). To fine‐map the CPB1 gene, we used 168 recessive individuals from the same F2 population and finally narrowed down the CPB1 locus to a 176‐kb region between markers C1 and C2 within the overlapped bacterial artificial chromosome (BAC) clones AL606588 and AL606441 (Figure 2b). A total of 20 putative genes were localized in the 176‐kb region according to the genome annotation of Nipponbare (http://rice.plantbiology.msu.edu/). On comparison of the mapped genomic region between WT and cpb1, we only detected one single nucleotide polymorphism (SNP) at the sixth exon of LOC_Os04 g39430, from A (WT) to T (cpb1) (Figure 2c), causing a His360Leu amino acid substitution (Figure 2d).

Figure 2.

Figure 2

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Map‐based cloning of CPB1. (a) CPB1 was mapped primarily to the long arm of chromosome 4 between markers S1 and S2. (b) Fine‐mapping of CPB1. The numerals indicate the number of recombinants. (c) The gene structure and the mutation sites in cpb1. (d) The cytochrome P450 superfamily domain was predicted in the CPB1/D11 protein. Solid lines show the position of the amino acid transition. (e) Constructs for complementary analyses; pOE‐ CPB1 contains the CPB1/D11 ORF used for overexpression; pRNAi‐ CPB1 denotes the RNA interference construct; UBI is a maize ubiquitin promoter. (f) Conservation analysis of the amino acid substitution region and the frequency of the 100 homologous genes. The red‐boxed region indicates the position of the amino acid transition.

The DWARF11 (D11) gene is annotated as the LOC_Os04 g39430 gene model, encoding a cytochrome P450 superfamily protein, CYP724B1, involved in the BR biosynthesis pathway (Tanabe et al., 2005). To verify the function of D11 in rescuing the cpb1 phenotype, we introduced an overexpressed transgenic construct of CPB1 allele (OE‐CPB1) and reduced transcript transgenic construct by a RNAi strategy (RNAi‐CPB1) (Figure 2e), respectively, into the cpb1 mutant and japonica cultivar Zhonghua 17 (ZH17) using Agrobacterium‐mediated transformation. The results showed that all 15 independent OE‐CPB1 lines rescued normal panicle architecture and plant height of the cpb1 plants (Figure 3a,b). Furthermore, quantitative RT‐PCR (qRT‐PCR) analysis showed that the CPB1/D11 transcript levels in positive transgenic plants were heavily elevated relative to these of the control (cpb1) plants (Figure 3c). In contrast, all 12 independent RNAi‐CPB1 transgenic plants showed semidwarf in stature, later in heading date and smaller in grain size (Figure 3e, Figure S2), with significantly down‐regulated CPB1/D11 transcripts compared to the control (ZH17) plants (Figure 3f). However, no clustered primary branch was observed in the RNAi‐CPB1 transgenic plants (Figure 3d). We speculated that the fascicled primary branch may be associated with a specific variation in the protein sequence, but not with a reduction in transcript level. These genetic evidences and transformation results confirmed that CPB1 was a new D11 allele which influenced the development of panicle architecture and other traits related to BR defects.

Figure 3.

Figure 3

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CPB1/D11 functional verification. (a): Panicle morphology comparison between control (cpb1) and OE‐ CPB1. (b): Plant phenotypic comparison between control (cpb1) and OE‐ CPB1. (c): Real‐time PCR analysis of CPB1/D11 expression in OE‐ CPB1 transgenic lines. (d): Panicle morphology comparison between control (ZH17) and RNAi‐ CPB1. (e): Plant phenotypic comparison among control (ZH17), RNAi‐ CPB1‐1 and RNAi‐ CPB1‐2. (f): Real‐time PCR analysis of CPB1/D11 expression in RNAi‐ CPB1 transgenic lines. Scale bars (a, b) 5 cm and (c, d) 20 cm. Values in (e‐f) are means ± SD of three independent experiments. The expression levels have been normalized to those of rice Ubiquitin.

Phylogenetic analysis of CPB1/D11 homologues

To identify the conservation of specific modification of CPB1/D11 in this study, we retrieved protein sequences by BLASTP using the full‐length protein sequence of CPB1/D11 as a query against the nonredundant protein database (http://www.ncbi.nlm.nih.gov/BLAST/) and identified 99 proteins showing high sequence similarity with CPB1/D11 in higher plants. A total of 32 putative homologues (more than 60% identity with CPB1/D11) from these 99 proteins were selected for phylogenetic analysis. The results showed that CPB1/D11 is closely related to genes found in other monocots, such as Oryza brachyantha, Hordeum vulgare, Zea mays and Sorghum bicolor (Figure S3). Notably, multiple sequence alignment and motif analysis showed that the amino acid substitution site in the cpb1 mutant was a highly conserved region in higher plants (Figure 2f), which indicated that this site played a very important role in maintaining the function of CPB1/D11 protein.

CPB1/D11 expression pattern

We performed quantitative RT‐PCR analysis to examine the temporal and spatial CPB1/D11 expression pattern in different tissues from both wild type and cpb1. The results showed that CPB1/D11 was highly expressed in young roots, tiller bases, leaf sheaths, leaf blades and particularly in young panicles (Figure 4a). In comparison with the WT, the expression level was significantly increased in the nodes, leaf pulvinus and young panicles of the cpb1 mutant (Figure 4a), which is consistent with previous reports that D11 mRNA accumulated at a higher level in d11 mutants (Tanabe et al., 2005).

Figure 4.

Figure 4

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CPB1 / D11 expression pattern and feedback regulation of BR‐related genes. (a) CPB1/D11 expression in different WT and cpb1 mutant tissues. YR, young roots; TB, tiller bases; N, nodes; CU, culms; LS, leaf sheaths; LP, leaf pulvini; LB, leaf blades; YP, young panicles; PB, panicle branches; HU, hulls. (b) Relative expression levels of the genes related to BR biosynthesis in the young WT and cpb1 panicles. (c): Relative expression levels of the genes related to BR signalling in the young WT and cpb1 panicles. Values in (a–c) are means ± SD of three independent experiments. The expression levels have been normalized to those of rice ubiquitin.

Expression analysis of BR, panicle architecture and grain shape‐related gene between WT and cpb1

To investigate the feedback regulations of BR‐related genes in young panicles, we analysed the expression levels of 13 BR‐related genes in WT and cpb1 young panicles by qRT‐PCR, including four BR biosynthesis genes and nine BR‐signalling genes (Table S2). The results showed that the expression levels of most BR biosynthesis genes in the cpb1 mutant were significantly higher than in WT except for BRD2 (Figure 4b) involving in the sterol biosynthetic pathway (Hong et al., 2005). However, no significant difference of expression level was observed for the BR‐signalling genes between the WT and cpb1 mutant, except for BU1 participating in two BR‐signalling pathways through OsBRI1 and D1 (Tanaka et al., 2009). These results indicated that the feedback mechanism within young panicles in WT and cpb1 mutant plants mainly occurs in the biosynthesis process, instead of the signalling pathways of BR.

To identify the panicle architecture regulatory pathway associated with the CPB1/D11 gene, we measured the expression levels of five panicle architecture‐related genes (Table S2) in young panicles. The results showed that the expression levels of all five known panicle architecture‐related genes in cpb1 were not significantly different from the WT (Figure S4a), which indicated that the CPB1/D11 gene might be involved in a specific pathway that regulated the development of panicle architecture. Furthermore, we analysed the expression levels of 10 grain shape‐related genes (Table S2). The expressions of both qSW5 (Shomura et al., 2008) and TGW6 (Ishimaru et al., 2013) significantly increased in the cpb1 compared to the WT (Figure S4b). This result suggested that the expression levels of qSW5 and TGW6 might be affected in the cpb1 plants and that this led to the cpb1 small grain phenotype.

Overexpression of CPB1/D11 using the maize Ubiquitin promoter increased grain length and thousand grain weight

We compared the yield‐related traits among the control (cpb1), OE‐CPB1 transgenic and wild type plants in order to investigate its influence to yield‐related traits by overexpressing the CPB1/D11 gene. Notably, OE‐CPB1 plants produced dramatically larger seeds than the control and WT plants (Figure 5c). We found that OE‐CPB1 transgenic plants showed a substantial increase in grain length compared to the WT (+19.1%, Figure 5g), but there were slight decreases in grain width (−2.4%, Figure 5h) and thickness (−2.6%, Figure 5i). We also detected a significant increase in thousand grain weight (+11.5%, Figure 5j) in OE‐CPB1 transgenic plants. Scanning electron microscopy showed that the outer epidermal cell size of the lemma in OE‐CPB1 grains became significantly larger than it was in the control and wild type grains (Figure 5d–f). However, the grain yield per plant of OE‐CPB1 transgenic plants showed no significant increase (Figure 5k) compared to the wild type, because the OE‐CPB1 transgenic plants showed profound changes in plant architecture, for instance larger leaf angles and narrower leaves (Figure 5a,b).

Figure 5.

Figure 5

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Phenotypes of the control (cpb1), OE‐ CPB1 transgenic and wild type plants. (a) Angle of the second leaf comparison among the control, OE‐ CPB1 and WT plants. (b) Second leaf comparison among the control, OE‐ CPB1 and WT plants. (c) Grain size comparison among the control, OE‐ CPB1 and WT seeds. (d–f) Scanning electron microscopy photographs of lemma outer epidermis cells from the control, OE‐ CPB1 and WT plants. (g) Grain lengths of the control, OE‐ CPB1 and WT seeds. (h): Grain widths of the control, OE‐ CPB1 and WT seeds. (i) Grain thicknesses of the control, OE‐ CPB1 and WT seeds. (j) 1000‐grain weights of the control, OE‐ CPB1 and WT seeds. (k) Grain yields per plant for the control, OE‐ CPB1 and WT plants. Scale bars (a) 2 cm, (b) 1 cm, (c) 2 mm and (d–f) 100 μm. Values in (g–k) are means ± SD (n = 20). Two‐tailed Student's t‐tests were performed between the control and OE‐ CPB1, and OE‐ CPB1 and WT, respectively (*P < 0.05, **P < 0.01, NS, not significant).

Optimized expression of CPB1/D11 using panicle‐specific promoters improved grain size and yield per plant

To explore the potential improvement in grain yield through genetic transformation of the CPB1/D11 gene in rice, we developed two constructs, pTH1::CPB1 and pDEP1::CPB1, using two rice young panicle‐specific promoters TH1 (Li et al., 2012) and DEP1 (Huang et al., 2009), driving CPB1/D11 gene expression, and transformed them into japonica cv. ZH17 using Agrobacterium tumefaciens. Although expression levels of CPB1/D11 in both the leaf blades and young panicles of transgenic plants (TH1::CPB1 and DEP1::CPB1) were higher than those in the control plants (Figure 6c,d), the plant architecture did not exhibit obvious difference, but the grain shape dramatically changed between the transgenic (TH1::CPB1 and DEP1::CPB1) and control plants (Figure 6a,b). Both TH1::CPB1 and DEP1::CPB1 plants showed a substantial increase in grain length (+10.9% and +15.7%, respectively, Figure 6e) compared to the controls, a slight increase in grain width (+3.1% and +1.1%, respectively, Figure 6f), and a significant decrease in grain thickness (−8.9% and −9.7%, respectively, Figure 6g), with no effects on grain number per panicle (Figure 6h). These phenotypic changes ultimately resulted in significant increase in thousand grain weight (+5.8% and +6.1%, respectively, Figure 6i) and grain yield per plant (+5.1% and +5.6%, respectively, Figure 6j) in the TH1::CPB1 and DEP1::CPB1 transgenic plants. These results demonstrated that optimizing CPB1/D11 expression using the appropriate promoters could enlarge seed size and enhance grain yield without affecting plant architecture and that CPB1/D11 could be useful in molecular rice breeding programmes.

Figure 6.

Figure 6

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Phenotypes of the control (ZH17), TH1:: CPB1 and DEP1:: CPB1 transgenic plants. (a) Plant architecture comparison of the control, TH1:: CPB1 and DEP1:: CPB1 plants. (b) Grain size comparison of the control, TH1:: CPB1 and DEP1:: CPB1 seeds. (c–d) Real‐time PCR analysis of CPB1/D11 expression in the leaf blades and young panicles of the control, TH1:: CPB1 and DEP1:: CPB1 plants. (e) Grain length comparison of the control, TH1:: CPB1 and DEP1:: CPB1 seeds. (f) Grain width comparison of the control, TH1:: CPB1 and DEP1:: CPB1 seeds. (g) Grain thickness comparison of the control, TH1:: CPB1 and DEP1:: CPB1 seeds. (h): Grain number per panicle among the control, TH1:: CPB1 and DEP1:: CPB1 plants. (i): 1000‐grain weight comparison of the seeds from the control, TH1:: CPB1 and DEP1:: CPB1. (j): Grain yield per plant among the control, TH1:: CPB1 and DEP1:: CPB1 plants. Scale bars (a) 20 cm and (b) 2 mm. Values in (e–h) are means ± SD (n = 20). Two‐tailed Student's t‐tests were performed between the control and TH1:: CPB1, and control and DEP1:: CPB1, respectively (*P < 0.05, **P < 0.01, NS, not significant).

Discussion

In this study, we characterized a mutant cpb1 showing fascicled primary branch and smaller seeds compared to WT. Through map‐based cloning and genetic transformation, CPB1 was identified to be a new allele of DWARF11 (D11), belonging to the cytochrome P450 superfamily, functioning in the BR biosynthesis pathway (Tanabe et al., 2005). Interestingly, in the lamina joint inclination test, we found that the cpb1 became more sensitive to epiBL treatment relative to the wild type (Figure 1i,j), which is consistent with previous reports in BR‐deficient mutants, such as d2 and d11 (Hong et al., 2003; Tanabe et al., 2005). We speculated that the higher sensitivity of BR‐deficient mutants in the lamina joint to BL may be due to the specific changes of BR‐signalling pathway through the feedback regulation of BR‐biosynthetic genes.

Overexpression of CPB1/D11 under the background of cpb1 mutant could rescue normal panicle architecture and plant height (Figure 3a,b). However, a RNAi‐mediated knockdown of CPB1/D11 assay did not show clustered primary branch phenotype in the positive transgenic plants, but only cpb1‐like phenotypes (Figure 3d,e, Figure S2). Previously four allelic D11 mutations have been identified, among which d11‐1, d11‐4 and d11‐2 generated a premature stop codon in exon 2, exon 4 and exon 7, respectively, whilst d11‐3 led to an amino acid substitution in exon 4 (Tanabe et al., 2005). Nevertheless, none of these mutants had a clustered primary branch. This suggested that the amino acid substitution site in exon 6 in CPB1/D11 was a specific variation controlling the development inflorescence of panicle architecture. Furthermore, expression analysis of the genes involved in the panicle architecture of the wild type and cpb1 mutant young panicles showed that there were no significant expression changes between WT and cpb1 (Figure S4a). This result suggested that CPB1/D11 might be involved in other regulatory pathways influencing rice panicle formation.

Four characteristics, including grain weight, grain number per panicle, panicle number per plant and proportion of filled grains, are main factors influencing grain yield in rice (Sakamoto and Matsuoka, 2008). Most reported the BR‐related mutants showed altered seed size, such as brd1 and d2, which produced small grains that were shorter in both grain length and width relative to the wild type (Hong et al., 2002, 2003). However, the molecular mechanisms underlying BR regulation of seed size are still unclear. Previous studies suggested that BR can regulate grain filling by modulating the carbon flux in rice, which influences seed size (Wu et al., 2008). In this study, we analysed the expression patterns of 10 grain shape‐related genes and found that the small grains produced by cpb1 might have a negative correlation with qSW5 and TGW6 expression, which function as negative regulators of grain width and length, respectively, during young panicle development in rice (Ishimaru et al., 2013; Shomura et al., 2008). The scanning electron microscopy assays of the lemma outer epidermis cells of the control (cpb1), OE‐CPB1 and WT plants revealed that the changes in seed size were positively associated with changes to the size of the lemma outer epidermis cell. Furthermore, a recent study showed that BR regulates cell elongation by modulating GA metabolism through the regulation of GA metabolic gene expression in rice (Tong et al., 2014). These results suggested that the BR regulation of grain size might be due to cell elongation in the lemma outer epidermis through modulation of GA metabolism in rice.

As they have been discovered in 1979, BRs have been thought to have promising applications in agriculture and their economic value as yield‐promoting agents was predicted by the early 1990s (Khripach et al., 2000). Therefore, fine tuning the BR level by genetic manipulation of BR biosynthesis or signalling pathways may be an important way of improving yields in cereal crops. Previous report demonstrated that OsmiR397 could enhance brassinosteroid signalling by down‐regulating the OsLAC, which in turn increases grain size, panicle branching and grain yield in rice (Zhang et al., 2013). In this study, CPB1/D11, as an important BR biosynthesis gene, also possessed the potential to increase grain size and rice yield by regulating brassinosteroid accumulation. Compared with the wild type plants, the overexpressing CPB1/D11 transgenic plants showed larger seed size and profound changes in plant architecture, resulting in no significant increase in grain yield. We further used two rice panicle‐specific promoters of TH1 and DEP1, which are involved in driving CPB1/D11 expression. The transgenic plants not only increased seed size and grain yield per plant, but also retained other favourable agronomic traits, including grain number per panicle and plant architecture. Taken together, optimizing CPB1/D11 expression using tissue‐specific promoters could be a promising approach for increasing seed size and improving grain yield in rice.

Experimental procedures

Plant materials and growth conditions

The clustered primary branch 1 (cpb1) mutants were identified from the EMS‐mutagenized population of an indica line, CP78, according to Wu et al. (2005). The CPB1 mapping population was generated from the cross between cpb1 and japonica cv. C418. The mapping population and all the requisite cultivars, CP78, C418 and Zhonghua 17, were grown in a field at the China Agricultural University, Beijing Experimental Station.

Histological analysis

Tissues were fixed in FAA (70% ethanol, 5% glacial acetic acid, 3.7% formaldehyde) overnight. Following this, they underwent a series of dehydration and infiltration stages and were then embedded in paraffin. A Leica microtome was used to cut 8‐μm sections, which were then stained with 1% safranine and 1% fast green and observed under an Olympus BX51 microscope (Japan).

Scanning electron microscopy

The samples were fixed in 2.5% glutaraldehyde solution for more than 2 h and then dehydrated through an ethanol series. The samples were critical‐point‐dried, mounted, sputter‐coated with platinum, observed and photographed using a Hitachi S‐3400 scanning electron microscope (Japan).

Lamina inclination assay and skotomorphogenesis of cpb1

Sterilized seeds were grown for 5d in a chamber kept at 28 °C with a light/dark cycle of 16/8 h. One microlitre of ethanol containing 0, 1, 10, 100 or 1000 ng of epiBL (Sigma‐Aldrich, St. Louis) was spotted onto the tip of the lamina of 6‐d‐old seedlings (Fujioka et al., 1998). The treated seedlings were grown for three additional days and then the inclination of the lamina joint of the second leaf was measured. For the de‐etiolation phenotype analyses of the cpb1 mutants, sterilized seeds were grown on 1% agar medium containing half‐strength MS medium in complete darkness at 28 °C for 3 weeks.

Vector construction and rice transformation

To generate the RNAi vector for CPB1/D11, two copies of the 384‐bp fragment (from +421 to +804 bases) of CPB1/D11 cDNA at inverted repeats, driven by the maize Ubiquitin promoter, were introduced into the pJL1460 vector (Wang et al., 2004). To generate the overexpression vector for CPB1/D11, a 1443‐bp fragment containing the entire CPB1/D11 ORF was amplified using primer pair OE‐1F/1R from CP78 first‐strand cDNA and cloned into pCAMBIA1301, which was driven by the maize Ubiquitin promoter. The pOE‐CPB1 and pRNAi‐CPB1 were introduced into Agrobacterium tumefaciens strain EHA105, and the resulting strains were used to transform the cpb1 mutant and japonica cv. Zhonghua 17, respectively.

To generate the vectors for pTH1::CPB1 and pDEP1::CPB1, the entire CPB1/D11 ORF was amplified and cloned into pCAMBIA1300. Then, the 2274‐bp and 2050‐bp fragments harbouring the endogenous promoters of TH1 and DEP1 (Huang et al., 2009; Li et al., 2012), respectively, were introduced into the pCAMBIA1300‐CPB1 using amplification primers pTH1F/1R and pDEP1F/1R. Both pTH1::CPB1 and pDEP1::CPB1 were transformed into japonica cv. Zhonghua 17 independently by means of the method described above.

Evaluation of the yield‐related traits

A total of 20 plants each of the wild type and cpb1 mutant plants were used to measure the grain size (length, width and thickness), 1000‐grain weight, seed setting and grain yield per plant. Phenotypic measurements of the positive transgenic plants were undertaken using three independent lines (20 plants from each line).

Phylogenetic analysis

All the protein sequences were obtained from The National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST/) using the CPB1/D11 protein sequence as a query. All obtained protein sequences were aligned using ClustalX 1.83, and motif analyses were conducted using WebLogo (Crooks et al., 2004). The phylogenetic tree was constructed using MEGA 5.0 (Tamura et al., 2011) based on the neighbour‐joining method, and the bootstrap values were estimated with 1000 replicates.

Quantitative RT‐PCR analysis

Total RNA was extracted from various samples using an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). First‐strand cDNA synthesis was carried out using a Superscript III RT Kit (Invitrogen, Carlsbad). The expression levels were analysed using a CFX96 Real‐Time System (Bio‐Rad, Hercules) with rice Ubiquitin as the internal control. Each set of experiments was repeated three times, and the relative quantification method (DDCT) was used to evaluate quantitative variation.

Statistical analysis

The two‐tailed Student's t‐test was carried out on all the data using SPSS version 16 (SPSS Inc, Chicago). Significance was accepted at P < 0.05 and P < 0.01.

Supporting information

Figure S1 WT and cpb1 internode characteristics.

Figure S2 Phenotype comparison between the control (ZH17) and RNAi‐ CPB1 transgenic plants.

Figure S3 Phylogenetic tree for CPB1/D11 and its homologues.

Figure S4 Expression of panicle architecture and grain shape‐related genes.

PBI-14-377-s002.pdf (203.7KB, pdf)

Table S1 WT and cpb1 agronomic trait measurement data.

PBI-14-377-s001.xlsx (10.3KB, xlsx)

Table S2 Primers used in this study.

PBI-14-377-s003.xlsx (11.5KB, xlsx)

Acknowledgements

This work was supported by grants 31222040 and 91435103 from the National Natural Science Foundation of China, and Program for New Century Excellent Talents in University from Ministry of Education, China (NCET‐12‐0517). There is no conflict of interest.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1 WT and cpb1 internode characteristics.

Figure S2 Phenotype comparison between the control (ZH17) and RNAi‐ CPB1 transgenic plants.

Figure S3 Phylogenetic tree for CPB1/D11 and its homologues.

Figure S4 Expression of panicle architecture and grain shape‐related genes.

PBI-14-377-s002.pdf (203.7KB, pdf)

Table S1 WT and cpb1 agronomic trait measurement data.

PBI-14-377-s001.xlsx (10.3KB, xlsx)

Table S2 Primers used in this study.

PBI-14-377-s003.xlsx (11.5KB, xlsx)

Articles from Plant Biotechnology Journal are provided here courtesy of Society for Experimental Biology (SEB) and the Association of Applied Biologists (AAB) and John Wiley and Sons, Ltd

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