RLB (RICE LATERAL BRANCH) recruits PRC2-mediated H3K27 tri-methylation on OsCKX4 to regulate lateral branching

Abstract

Lateral branches such as shoot and panicle are determining factors and target traits for rice (Oryza sativa L.) yield improvement. Cytokinin promotes rice lateral branching; however, the mechanism underlying the fine-tuning of cytokinin homeostasis in rice branching remains largely unknown. Here, we report the map-based cloning of RICE LATERAL BRANCH (RLB) encoding a nuclear-localized, KNOX-type homeobox protein from a rice cytokinin-deficient mutant showing more tillers, sparser panicles, defected floret morphology as well as attenuated shoot regeneration from callus. RLB directly binds to the promoter and represses the transcription of OsCKX4, a cytokinin oxidase gene with high abundance in panicle branch meristem. OsCKX4 over-expression lines phenocopied rlb, which showed upregulated OsCKX4 levels. Meanwhile, RLB physically binds to Polycomb repressive complex 2 (PRC2) components OsEMF2b and co-localized with H3K27me3, a suppressing histone modification mediated by PRC2, in the OsCKX4 promoter. We proposed that RLB recruits PRC2 to the OsCKX4 promoter to epigenetically repress its transcription, which suppresses the catabolism of cytokinin, thereby promoting rice lateral branching. Moreover, antisense inhibition of OsCKX4 under the LOG promoter successfully increased panicle size and spikelet number per plant without affecting other major agronomic traits. This study provides insight into cytokinin homeostasis, lateral branching in plants, and also promising target genes for rice genetic improvement.

Figure 1

Phenotypes of ZH11 and rlb. A, Plants morphology of ZH11 and rlb after heading, bar = 10 cm. B, Tillers from three plants of ZH11 and rlb at booting stage, bar = 5 cm. C, Main panicle of ZH11 and rlb, red arrows indicate the neck-panicle node, bar = 5 cm. D, Primary branch of ZH11 and rlb, red arrows indicate the degenerate floret points from the secondary branch of rlb, bar = 2.5 cm. E and F, A single primary branch of ZH11 and rlb, bar = 100 µm. G and H, Paraffin section of ZH11 and rlb at panicle initiation stage, bar = 500 µm. I and J, SEM analysis of ZH11 and rlb panicle at early differentiation stage, the red triangles indicate floret meristems, bar = 500 µm. K and L, Morphology of florets and seeds of ZH11 and rlb, red arrows indicate the sterile lemma, bar = 1 cm. M–O, Callus regeneration of ZH11 and rlb. M, basic medium was used. N, enlargement of the red frame from M. O, basic medium with the addition of 2.0 mg L⁻¹ zeatin was used. P, Regeneration ratios of ZH11 and rlb on the basic medium with or without the addition of zeatin, 50 independent calluses were used for each treatment. Different characters represent statistical difference at P < 0.05 by Student’s t test. Q and R, Endogenous hormone levels of ZH11 and rlb. TZR, trans-zeatin-riboside. IPA, isopentenyl adenosine. IP, N6-(2-Isopentenyl) adenosine. Data are shown as means ± sd (n = 3). **P ≤ 0.01 by the Student’s t test.

Figure 2

Map-based cloning and functional verification of RLB. A, RLB was preliminarily mapped between markers RM14584 and RM5711 on chromosome 7 (Chr.7) using 24 F₂ individuals. The region narrowed down to about 3.6 CM between RM3831 and RM5490 using 48 F₂ mutant individuals. Further fine-mapping analysis with a total of 882 F₂ segregants restricted the locus within a 44.2-kb region between Ind8 and Ind14. Seven putative ORFs in the mapped region were sequenced. A mutation was found in ORF1 (indicated in red). All F₂ mutant type plants were from the cross of rlb/TN1. B, Schematic presentation of RLB gene structure and the mutation site. Black rectangles represent exons, white rectangles represent untranslated region (UTR), and black arrows indicate the location of primers. C, RLB protein structure and deduced mutation in the amino acid sequence. The 237th to 244th residues were deleted in rlb. D, qRT-PCR analysis of RLB expression level in ZH11 and rlb using various primer pairs. E and F, Genetic complementation test of RLB. Plant morphology (E) and main panicles (F) morphology of ZH11, rlb and the genetic complementation lines (rlbcom-1 and rlbcom-3). Bar = 10 cm in (E) and bar = 5 cm in (F). Seed from rlbcom lines was shown with normal sterile lemma as indicated by the red arrows in (F). G, Plant morphology of Nipponbare and RLB over-expression lines (oxRLB-6 and oxRLB-9). Bar = 10 cm. H, qRT-PCR analysis of RLB expression level in OxRLB lines. I and J, Morphology of the mutated florets from RLB over-expression lines. Bar = 0.5 cm. K, Main panicles morphology of RLB over-expression lines. Bar = 5 cm. For D and H, data are shown as means ± sd (n = 3). **P ≤ 0.01 by Student’s t test.

Figure 3

Expression pattern of RLB. A and B, qRT-PCR analysis of the relative expression levels of RLB in different tissues (A) and panicle developmental stages (B). Flag-l-s, flag leaf sheath. Flag-l, flag leaf. B, Relative expression level of RLB in eight panicle development stage. For A and B, data are shown as means ± sd of three biological replicates. C, Subcellular localization of RLB. pro35s::RLB-eGFP, pro35s::RLB(M)-eGFP and control vector p35s::eGFP were transformed into rice protoplasts respectively, and observed using a confocal laser-scanning microscope. Green fluorescence shows eGFP, red fluorescence shows the chloroplast auto-fluorescence, Bars = 5 μm.

Figure 4

RLB directly binds to the OsCKX4 promoter and suppresses its transcription. A, qRT-PCR analysis of the relative expression level of some reported development-related genes in ZH11, rlb, and OxRLB line. Data are shown as means ± sd of three biological replicates. B, ChIP-qPCR analysis of the RLB binding sites on OsCKX4. Positions of region P1–P10 are shown in (C). The enrichment values were normalized to IgG-immunoprecipitated DNA, actin primers was used as a CK. C, Schematic presentation of the OsCKX4 gene structures. Black boxes: coding region, blank box: untranslated region, line: promoter. +1: Transcription starting site, red dots: TGACA motif. Short black lines P1–P10 indicate the tested regions in ChIP-qPCR. D, EMSA assay showing RLB directly bind to the promoter of OsCKX4. The 5-, 10-, 50-, and 100-fold excess nonlabeled probes were used for competition. E, EMSA assay showing G-box mutation and RLB(M) weaken the bind between the protein and the promoter of OsCKX4. F, LUC transient transcriptional activity assay in rice protoplast. Reporter: proCKX4::19fLUC:tNOS; effectors: pro35S::RLB:tNOS and pro35S::RLB(M):tNOS. The fLUC/rLUC ratio represents the relative activity of 35S promoter. For A, B, and F data are shown as means ± sd of three biological replicates. **P < 0.01 by the Student’s t test, *P < 0.05 by the Student’s t test. For E different characters represent statistical difference at P < 0.05 by Student’s t test.

Figure 5

Identification of OsCKX4 gene. A–E, mRNA in-situ hybridization alaysis of OsCKX4 transcript in developing panicle. Longitudinal sections of ZH11 panicles in 0.1 cm (A), 0.3 cm (B), and 0.5 cm (C) were hybridized with digoxigenin-labeled antisense OsCKX4 RNA probes. D, enlargement of the red frame from C. E, Sense probe was used as a negative contronl. FM, floret meristem; St, stamen; Pl, palea; Le, lemma; Lo, lodicule; Sl, sterile lemma. For A–C and E, bars = 100 μm, for D, bar = 25 μm. F, Expression pattern of OsCKX4 in various tissues. Data are shown as means ± sd of three biological replicates. G, The relative expression level of OsCKX4 in ZH11 and the two representative OxCKX4 lines. For F and G, data are shown as means ± sd (n = 3). **P < 0.01 by the Student’s t test. H and I, Agronomic performance of the OxCKX4 lines and ZH11 lines. For H, bar = 10 cm. For I, bar = 5 cm.

Figure 6

RLB recruits PRC2 to CKX4 to deposite H3K27me3. A, Yeast two-hybrid assay revealing the interaction of RLB and PRC2 members. Y2H assays were performed using the matchmaker GAL4 two-hybrid system and selected on synthetic medium lacking Leucine, Tryptophan and Histidine with 100 ng mL⁻¹ Aureobasidin A applied. B, Pull-down assay of GST-RLB and His-EMF2b. 6XHis-EMF2B was incubated with GST or GST-RLB in GST beads and was pulled down from the RLB-GST conjugated GST beads. C, BiFC analysis of RLB and EMF2B in rice protoplasts. Representative cells are used for imaging by laser-scanning confocal microscopy at excitation and emission wavelength 514 nm and 527 nm, respectively. GFP signal image gain value is 600. Positive interaction of nYFP-RLB::EMF2B-cYFP is indicated by the green fluorescence signal. Vector nYFP-RLB::-cYFP and nYFP::EMF2B-cYFP were used as control. Bars = 50 μm. D–F, mRNA in-situ hybridization alaysis of EMF2b transcript in developing panicle. Longitudinal sections of ZH11 panicle in 0.5 cm were hybridized with digoxigenin-labeled EMF2b antisense RNA probes. E, enlargement of the red frame from D. F, Sense probe was used as a negative control. PBM, primary branch meristem. FM, floret meristem; St, stamen; Pl, palea; Le, lemma; Lo, lodicule; Sl, sterile lemma. For D and F, bars = 100 μm, for E, bar = 25 μm. G and H, Plant and panicles morphology of the wild-type (Dongjin) and emf2b mutant. Bar = 10 cm for plant and 5 cm for panicle, respectively. I, ChIP-qPCR analysis of H3K27me3 deposition on OsCKX4 in ZH11 and rlb. Positions of the tested regions are indicated in Figure  4C. All values are shown as means ± sd with biological triplicates. **P < 0.01 by the Student’s t test.

Figure 7

Performance of proLOG::aCKX4 lines. A and B, Plant and main panicles morphology of ZH11 and the two representative proLOG::aCKX4 lines. For A, bar = 10 cm. For B, bar = 5 cm. C and D, The relative expression level of OsCKX4 in panicle and leaf of ZH11 and proLOG::aCKX4 lines. E–K, Agronomic traits of ZH11 and proLOG::aCKX4 lines. E, Tiller number. F, Total spikelets per plant. G, Seed setting ratio. H, 1,000-grain weight. I, Seed length. J, Seed width. K, Yield per plant. All values are means ± sd, with biological triplicates for gene expression analysis and five biological repeats for agronomic traits analysis. For seed length and width analysis 100 full seeds were used. *P ≤ 0.05, **P ≤ 0.01 by the Student’s t test.

Figure 8

Working model for RLB in rice lateral branch development.