Rice axillary meristems (AMs) are essential to the formation of tillers and panicle branches in rice, and therefore play a determining role in rice yield. However, the regulation of inflorescence AM development in rice remains elusive. In this study, we identified no spikelet 1-Dominant (nsp1-D), a sparse spikelet mutant, with obvious reduction of panicle branches and spikelets. Inflorescence AM deficiency in nsp1-D could be ascribed to the overexpression of OsbHLH069. OsbHLH069 functions redundantly with OsbHLH067 and OsbHLH068 in panicle AM formation. The Osbhlh067 Osbhlh068 Osbhlh069 triple mutant had smaller panicles and fewer branches and spikelets. OsbHLH067, OsbHLH068, and OsbHLH069 were preferentially expressed in the developing inflorescence AMs and their proteins could physically interact with LAX1. Both nsp1-D and lax1 showed sparse panicles. Transcriptomic data indicated that OsbHLH067/068/069 may be involved in the metabolic pathway during panicle AM formation. Quantitative RT-PCR results demonstrated that the expression of genes involved in meristem development and starch/sucrose metabolism was down-regulated in the triple mutant. Collectively, our study demonstrates that OsbHLH067, OsbHLH068, and OsbHLH069 have redundant functions in regulating the formation of inflorescence AMs during panicle development in rice.
Axillary meristems (AMs) generate branches and determine the inflorescence pattern, and further define the overall architecture of plants. In addition, they have great impacts on the tiller number and panicle size, and therefore significantly influence the seed number and yield of crops. Hence, understanding the molecular mechanism for AM development is of both scientific and application significance. Although some genes involved in panicle development of rice have been reported to date, the underlying mechanism remains largely unknown in rice. In this study, we reported that OsbHLH067, OsbHLH068, and OsbHLH069 redundantly regulate the formation of inflorescence AMs in rice. OsbHLH067, OsbHLH068, and OsbHLH069 were preferentially expressed in developing inflorescence AMs. Overexpression of OsbHLH069 resulted in sparse panicles. The Osbhlh067 Osbhlh068 Osbhlh069 triple mutant exhibited small panicles with fewer branches and spikelets. OsbHLH067/068/069 were found to interact with LAX1, which might be involved in the metabolism pathway and influence the gene expression related to panicle development.
Citation: Xu T, Fu D, Xiong X, Zhu J, Feng Z, Liu X, et al. (2023) OsbHLH067, OsbHLH068, and OsbHLH069 redundantly regulate inflorescence axillary meristem formation in rice. PLoS Genet 19(4): e1010698. https://doi.org/10.1371/journal.pgen.1010698
Editor: Yuling Jiao, Peking University, CHINA
Received: June 20, 2022; Accepted: March 8, 2023; Published: April 13, 2023
Copyright: © 2023 Xu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: C. W. received the fundings from the National Natural Science Foundation of China (U20A2023, 31630054 and 31821005), the National Key Research and Development Program of Hubei Province (2022BBA54), the Natural Science Foundation of Hubei Province (2022CFA024), and the Foundation of Hubei Hongshan Laboratory (2021hszd010). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Flowering plants can undergo reiterative growth and continuous organogenesis during their lifespan. Axillary meristems (AMs) play a central role at both the vegetative and reproductive growth stages to determine rice plant architecture. At the vegetative growth stage, AMs are initiated from the boundary between the shoot apical meristem and leaf primordium, and then develop into rice tillers. At the reproductive growth stage, after the shoot apical meristem is transformed into the inflorescence meristem (IM), inflorescence AMs hierarchically transformed into branch meristems (BMs) and spikelet meristems (SMs) and then finally develop into a rice panicle . In the inflorescence architecture, the primary branch meristem initiates at the boundary between the IM and bract primordia, and then generates primary branches (PBs). Secondary branch and spikelet meristems are generated at the boundary between the elongated primary branch and bract primordia, thereby differentiating into secondary branches (SBs) and spikelets, respectively. Ultimately, the number of tillers, branches, and spikelets derived from AMs together determine the yield of rice .
Multiple transcriptional factors involved in inflorescence AM formation have been identified in rice. For instance, LAX1 encodes a bHLH transcription factor and regulates AM formation during inflorescence development [3,4]. LAX2 encodes a nuclear protein and physically interacts with LAX1 to mediate the process of AM formation . MOC1 is a transcriptional regulator of the GRAS family and mainly regulates the formation of vegetative and reproductive AMs in rice . A genetic analysis has revealed that LAX1, LAX2, and MOC1 have overlapping functions involved in distinct pathways that regulate AM formation during vegetative and reproductive development . TAB1/OsWUS is another transcription factor identified for inflorescence AM formation in rice . It seems that all the transcriptional factors identified in AM-defective rice mutants are conserved in various plant species. LAX1 and LAX2 are orthologues of the maize genes BA1 and BA2 respectively [8,9]; and rice MOC1 is homologous to tomato LS and Arabidopsis LAS [6,10]. Although some of these genes are only essential for the formation of vegetative AMs but not for that of reproductive AMs, they play some conserved roles in initiating AMs in various plant species [4–6,8–11].
Auxin biosynthesis, transport, and signaling have been demonstrated to be required for inflorescence AM formation and lateral organ initiation . In Arabidopsis, AM formation involves auxin synthesis genes YUC1, 4, and 6 ; auxin polar transporter genes PIN1, and AUX1 [14,15]; auxin polar transport regulator gene PID [16,17]; and the auxin signal transduction gene MONOPTEROS (MP) . Notably, certain homologous genes in maize and rice also participate in inflorescence AM development. For example, mutation of the auxin biosynthesis/signaling pathway genes, including SPI1, VT2, BIF2, ZMAUX1, BIF1, and BIF4, impaired inflorescence in maize [19–23]. In rice, OsPIN1c/d and OsPID are required for AM formation during inflorescence development [24,25]. In Arabidopsis and maize, some transcriptional factors are associated with auxin signaling pathway to regulate inflorescence AM development. For instance, BIF1 and BIF4 are integral for auxin signaling modules that dynamically regulate the expression of BA1 . However, transcriptional factors involved in the influence of phytohormone and metabolic pathway on rice AM development remain largely unknown.
The basic/helix-loop-helix (bHLH) proteins form one of the largest transcription factor families. The bHLH domain, which is composed of about 60 amino acids, enables the formation of the homodimeric or heterodimeric complex through the HLH region and determines the ability to bind downstream genes through the basic region . LAX1 encodes a bHLH protein and is a key factor determining the formation of AM in rice . LAX1 mRNA accumulates in 2–3 layers of cells in the boundary region between initiating AM and the shoot apical meristem [3,27]. LAX1 protein accumulates transiently in initiating AMs and is subsequently trafficked to the AM in a stage- and direction-specific manner for the establishment of new AMs . Mutation of LAX1 was found to severely suppress the initiation of lateral spikelets and affect both vegetative and reproductive branching [3,27]. Ectopic expression of LAX1 also causes pleiotropic effects, including dwarfing, reduced branching, and severe sterility , indicating that fine regulation of LAX1 expression is essential for normal AM formation. LAX2 is a novel nuclear protein acting synergistically with LAX1 in rice to regulate the process of AM formation . SPL protein has been reported to possibly regulate LAX1 expression directly at the transcription level . Recent studies have suggested that the LAX1 haplotype contributes to the number of panicle branches and grain weight, thereby affecting the rice yield [29,30].
In the present study, we identified a no spikelet 1-Dominant rice mutant (nsp1-D) with fewer branches and spikelets. Genetic analysis suggested that the overexpression of OsbHLH069 resulted in the nsp1-D morphology. OsbHLH069 belongs to subfamily F of the bHLH transcription factor family in rice , and is functionally redundant with its homologs, OsbHLH067 and OsbHLH068, in regulating panicle AM formation. In situ hybridization results indicated that OsbHLH067, OsbHLH068, and OsbHLH069 are preferentially expressed in the inflorescence AM, and can physically interact with LAX1 individually. In addition, the Osbhlh067 Osbhlh068 Osbhlh069 triple mutant showed significant variations in the expression of AM formation genes such as RCN4, OsSPL14, NL1 and PLA1. Our findings suggest that the OsbHLH067/068/069-LAX1 module might act through metabolism pathways such as starch and sucrose metabolism to regulate inflorescence AM development.
Identification of a mutant with sparse panicles
A sparse panicle mutant (03Z11CH32) with few or no spikelets was identified from our T-DNA insertional mutant library [32,33]. The heterozygous mutant exhibited a semi-dominant mutation segregation with a single Mendelian locus in the progeny (sparse: normal = 210: 72, χ2 = 0.08 < 3.84 for 3:1). Therefore, the mutant was designated as no spikelet 1-Dominant (nsp1-D). Compared with WT, heterozygotes (nsp1-D/+) showed a weaker phenotype of sparse panicles with reduction of grain setting, and homozygous mutants nsp1-D displayed seriously sparse panicles (Fig 1A and 1B).
(A) and (B) Architecture of plant (A) and panicle (B) in wild type (WT), nsp1-D/+, and nsp1-D during reproductive growth. Bars = 20 cm in (A) and 5 cm in (B). (C) to (G) Statistical analysis of the number of primary branches (PBs) (C), secondary branches (SBs) (D), spikelets in PBs (SPBs) (E), spikelets in SBs (SSBs) (F), and total spikelets (G) per panicle among WT, nsp1-D/+, and nsp1-D plants. Values shown are the means ± SEM from 10 replicates. Letters denote significant differences as ranked by the Dunnett’s test (one-way analysis of variance, P < 0.05).
We further characterized the panicle traits of nsp1-D and nsp1-D/+ plants (Fig 1C–1G). Compared with WT, these plants showed no obvious defects in the number of PBs (Fig 1C). The number of SBs was slightly reduced in nsp1-D/+ and remarkably decreased in nsp1-D plants (Fig 1D). In both nsp1-D and nsp1-D/+ plants, the number of spikelets in PBs (SPBs) and spikelets in SBs (SSBs) significantly decreased, ultimately resulting in a severe reduction of the number of spikelets per panicle (Fig 1E–1G). These observations suggested that defective panicle development occurred in a dose-dependent manner in nsp1-D.
Characterization of inflorescence development in nsp1-D
To further explore the development of nsp1-D panicles, we compared the panicle development between WT and nsp1-D by histological section analysis and scanning electron microscopy (SEM). Histological section analysis revealed no remarkable morphological differences in apices between WT and nsp1-D (Fig 2A and 2E). At the reproductive stage, WT showed normal generation of PBs (Fig 2B), but PB primordia in nsp1-D were largely suppressed (Fig 2F). In addition, the SB primordia (Fig 2G) and spikelet primordia (Fig 2H) were significantly reduced in nsp1-D compared with in WT (Fig 2C and 2D). SEM results further demonstrated that nearly no SB primordia were formed, and there were only elongated PB primordia in nsp1-D at the spikelet primordia formation stage (Fig 2I–2P). These results suggested that inflorescence AM formation was compromised in nsp1-D.
(A) to (H) Longitudinal section of a developing inflorescence in WT (A to D) and nsp1-D plants (E to H) during formation of the first bract primordium (A, E), the primary branch primordia (B, F), the secondary branch primordia or spikelet primordia (C, G), and the floret primordia (D, H). Red arrows (in G, H) indicate the empty bracts; Yellow arrowheads (in G) indicate the positions where the axillary meristems should initiate. Purple stars (in H) indicate the bract-like knobs. Bars = 200 μm. (I) to (P) Scanning electron microscopy images showing the inflorescence development for WT (I to L) and nsp1-D plants (M to P) during formation of the first bract primordium (I, M), the primary branch primordia (J, N), the secondary branch primordia or spikelet primordia (K, O), and the floret primordia (L, P). White stars indicate inflorescence meristem; green and red asterisks denote primary and secondary branch primordia, respectively; the red bracket outlines the region where secondary branch primordia failed to initiate; the yellow, green, and red arrows denote spikelet primordia, floret primordia, and elongated primary branch primordia, respectively. Bars = 100 μm. (Q) to (T) In situ localization of OSH1 in WT (Q, R) and nsp1-D (S, T) inflorescences at the stage of primary branch meristem (Q, S) and secondary branch meristem (R, T) differentiation. Bars = 100 μm.
OSH1 is considered as a marker gene of meristematic cells in rice . We therefore examined the expression pattern of OSH1 in nsp1-D by in situ hybridization. OSH1 signals could be detected in PB and SB meristems in WT plants (Fig 2Q and 2R). However, in nsp1-D, these signals were greatly reduced in PB meristem, and even not detected in the undeveloped meristem of SB and spikelets (Fig 2S and 2T). Taken together, it could be speculated that the corresponding gene in nsp1-D may be involved in the inflorescence AM formation in rice.
Identification of the NSP1 gene
Thermal asymmetric interlaced PCR (Tail-PCR) was performed to identify the T-DNA insertion site in nsp1-D . The flanking sequence of the T-DNA insertion site indicated the presence of a truncated T-DNA at –6138 bp in the promoter of the LOC_Os01g57580 gene in nsp1-D (Fig 3A). PCR amplification results suggested that the insertion was well co-segregated with the panicle morphology in the progenies (n = 20) of the nsp1-D/+ plant (Fig 3B). All the T-DNA insertion homozygotes showed severely sparse panicles, and heterozygotes exhibited a weaker phenotype of sparse panicles. Quantitative RT-PCR (qRT-PCR) analysis revealed a notable increase in the expression of LOC_Os01g57580, and normal expression of other genes surrounding the T-DNA insertion site in the 100 kb region of nsp1-D relative to that in WT (Fig 3C). Therefore, LOC_Os01g57580 might be the gene responsible for the sparse panicle phenotype of nsp1-D.
(A) Structure of the NSP1 genome and the T-DNA insertion site. Black boxes represent exons; lines between the boxes represent introns; the inverted triangle indicates T-DNA. Primers L and R on the NSP1 genome and primer TL14 at the T-DNA left border used for genotype analysis are marked with arrows. (B) Co-segregation analysis of nsp1-D/+. W, H, and M indicate wild type (WT), heterozygous, and homozygous for T-DNA insertion, respectively. (C) Quantitative RT-PCR analysis of genes flanking the T-DNA insertion site in young panicles (< 5 mm) of WT and nsp1-D. The internal rice Ubiquitin (UBQ) gene was used to normalize gene expression. Data are the means ± SEM from nine replicates. (D) to (F) Plant morphology (D) and panicle morphology (E) of 35S-pOsbHLH069::OsbHLH069 transgenic plants (OE-3 and OE-12). (F) Closeup view of one primary branch in (E). (G) Expression analysis of OsbHLH069 in the leaves of the 35S-pOsbHLH069::OsbHLH069 transgenic plants (OE-3 and OE-12). Gene expression was normalized to the rice UBQ gene. Values shown indicate the means ± SEM from three replicates. Different letters denote significant differences ranked by the Dunnett’s test (one-way analysis of variance, P < 0.05). Bars = 20 cm in (D) and 4 cm in (E, F).
LOC_Os01g57580 encodes a typical bHLH transcription factor, and is designated as OsbHLH069 . We then overexpressed OsbHLH069 driven by the CaMV35S (35S-pOsbHLH069::OsbHLH069) in rice. Among the 45 putative transgenic plants, 13 positive transgenic plants exhibited obvious sparse panicle phenotype. We selected two independent transgenic lines (OE-3 and OE-12, heterozygous and homozygous of 35S-pOsbHLH069::OsbHLH069, respectively) for further examination. Compared with negative transgenic plants, the OE-3 plant showed a mild phenotype with a few branches and spikelets, and the OE-12 plant displayed a severe phenotype without SBs and spikelets (Fig 3D–3F). qRT-PCR analysis demonstrated that OE-3 and OE-12 plants had significantly higher OsbHLH069 transcript levels than WT plants (Fig 3G). Based on these results, it could be concluded that OsbHLH069 is NSP1, and its overexpression would result in sparse panicles in nsp1-D plants.
OsbHLH067, OsbHLH068, and OsbHLH069 are preferentially expressed in inflorescence AM
It has been reported that the rice genome contains 167 bHLH genes, which can be subdivided into 22 subfamilies named as A–V . OsbHLH069 belongs to subfamily F (14 bHLH proteins), and OsbHLH067, OsbHLH068, and OsbHLH070 are closely homologous to OsbHLH069 (S1A Fig).
Alignment analysis revealed that OsbHLH067, OsbHLH068, and OsbHLH069 contain a conserved bHLH domain (S1B Fig), but OsbHLH070 contains an atypical bHLH domain that does not bind DNA (S1C Fig) . qRT-PCR results demonstrated that OsbHLH067, OsbHLH068, and OsbHLH069 are constitutively expressed in various organs, including the root, culm, leaf, leaf sheath, and young panicles of less than 5 mm (S2A–S2C Fig). We subsequently carried out in situ hybridization for more precise examination of the spatial expression patterns of OsbHLH067, OsbHLH068, and OsbHLH069 in young panicles. Just like OSH1, OsbHLH069 (Fig 4A–4D) and OsbHLH067 (Fig 4E–4H) were expressed in all reproductive meristems, such as IM, PB meristem, SB meristem, and SM (Fig 4M–4P) . OsbHLH068 mRNA showed similar expression patterns to OsbHLH069 and OsbHLH067 except for in the IM (Fig 4I–4L). Moreover, LAX1 mRNA was preferentially accumulated in the boundary of initiating AM (Fig 4Q–4T) . Movement of the LAX1 protein towards the future AM has been reported to be required for maintaining the AM development . Our results suggested that OsbHLH067/068/069 are preferentially expressed in inflorescence AM, and may act together with LAX1 for the AM development.
In situ localization of OsbHLH069 (A to D), OsbHLH067 (E to H), OsbHLH068 (I to L), OSH1 (M to P), and LAX1 (Q to T) in developing inflorescences. (D), (H), (L), (P) and (T) The sense probes of OsbHLH069 (D), OsbHLH067 (H), OsbHLH068 (L), OSH1 (P), and LAX1 (T) served as controls. (A), (E), (I), (M) and (Q) A developing inflorescence at the primary branch meristem (PBM, green triangle) differentiation stage. (B), (F), (J), (N) and (R) A developing inflorescence at the secondary branch meristem (SBM, red triangle) differentiation stage. (C), (G), (K), (O) and (S) A developing inflorescence at the spikelet meristem (SM, yellow triangle) initiation stage. IM, inflorescence meristem. Bars = 100 μm.
LAX1 has been reported to be predominantly localized in the nucleus . We fused LAX1 with RFP as a nuclear marker, and then investigated the subcellular localization of OsbHLH067, OsbHLH068, and OsbHLH069 by individually expressing their fusion proteins with GFP driven by a CaMV35S promoter in rice protoplasts. Compared with the empty GFP protein evenly distributed in the cytoplasm and nucleus, the GFP-fused proteins were co-localized with RFP-fused LAX1 in the nucleus (S2D Fig), suggesting that OsbHLH067, OsbHLH068, and OsbHLH069 may function in the nucleus.
OsbHLH067/068/069 redundantly regulate inflorescence AM formation
Considering that OsbHLH067, OsbHLH068, and OsbHLH069 are homologous genes with similar expression patterns during inflorescence development in rice, we speculated that they might be involved in panicle AM development. Hence, CRISPR/Cas9 system was used to generate single, double, and triple mutants for them (S3 Fig). Compared with WT plants, the single and double mutants showed no noticeable change in morphology (Fig 5A–5H, S4 Fig). However, the triple mutant displayed severe defects, including dwarf stature, single culm, and small panicle with few branches and spikelets (Fig 5A–5H). Histological analysis revealed that the triple mutant showed significant reduction of branch primordia, indicating that inflorescence AM formation is compromised in the absence of OsbHLH067, OsbHLH068, and OsbHLH069 (Fig 5I). In addition, in situ hybridization of OSH1 mRNA suggested that AM formation was arrested in the panicles of the triple mutant (Fig 5J). Taken together, our results suggest that OsbHLH067, OsbHLH068, and OsbHLH069 are functionally redundant for inflorescence AM formation.
(A) and (B) Phenotype comparisons of the plant (A) and panicle (B) for wild type (WT), Osbhlh067 Osbhlh069, Osbhlh068 Osbhlh069, and Osbhlh067 Osbhlh068 double mutants, and the Osbhlh067 Osbhlh068 Osbhlh069 triple mutant. Bars = 20 cm in (A) and 4 cm in (B). (C) to (H) Quantification of the number of (C) tillers, (D) primary branches (PBs), (E) secondary branches (SBs), (F) spikelets in PBs (SPBs), (G) spikelets in SBs (SSBs) and (H) total spikelets per panicle in WT, Osbhlh067 Osbhlh069, Osbhlh068 Osbhlh069, and Osbhlh067 Osbhlh068 double mutants, and the Osbhlh067 Osbhlh068 Osbhlh069 triple mutant. Data are the means ± SEM from 12 replicates. Different letters denote significant differences ranked by the Dunnett’s test (one-way analysis of variance, P < 0.05). (I) Paraffin sections of inflorescences with SB primordia from WT and the Osbhlh067 Osbhlh068 Osbhlh069 triple mutant. Bars = 100 μm. (J) In situ localization of OSH1 in WT and Osbhlh067 Osbhlh068 Osbhlh069 triple mutant panicles at the secondary branch primordia differentiation stage. Bars = 100 μm.
Genetic interaction between OsbHLH069 and LAX1
LAX1 acts as a major regulator on AM formation in rice [3,27]. In our T-DNA insertion mutant library, an identified allelic mutant of lax1 showed reduction of branches and spikelets (S5A–S5G Fig). To examine the genetic interaction between OsbHLH069 and LAX1, we attempted to generate a double mutant of nsp1-D/+ and lax1 (Fig 6). Given the close physical locations of OsbHLH069 and LAX1 on chromosome 1, we failed to obtain the nsp1-D lax1 double mutant. As described above, compared with WT plants, nsp1-D and nsp1-D/+ showed reduction of SPBs and SSBs in panicle (Figs 1A–1G and 6A–6I). The defects in lateral branching of the panicle became more severe when lax1 or lax1/+ was combined with nsp1-D and nsp1-D/+ (Fig 6), indicating that OsbHLH069 and LAX might have a synergistic effect on panicle AM formation.
(A) to (C) Architectures of (A) the plant, (B) panicle, and (C) primary branch (PB) in the progeny. (D) to (I) Quantitative statistics of the number of (D) tillers, (E) PBs, (F) secondary branches (SBs), (G) spikelets of PBs (SPBs), (H) spikelets of SBs (SSBs) and (I) total spikelets in the progeny. Data are the means ± SEM from 10 replicates. Different letters denote significant differences ranked by the Dunnett’s test (one-way analysis of variance, P < 0.05). WH, NSP1 lax1/+; HW, nsp1-D/+ LAX1; MW, nsp1-D LAX1; WM, NSP1 lax1; HH, nsp1-D/+ lax1/+; HM, nsp1-D/+ lax1; MH, nsp1-D lax1/+. Bars = 20 cm in (A) and 3 cm in (B, C).
We then investigated the possibility of transcriptional regulation among OsbHLH067, OsbHLH068, OsbHLH069, and LAX1. qRT-PCR analysis showed that the expression of OsbHLH067, OsbHLH068, and OsbHLH069 was not affected in lax1 plants (S5H Fig). In the Osbhlh067 Osbhlh068 Osbhlh069 triple mutant plants, the transcription level of LAX1 was slightly increased as indicated by qRT-PCR (S5I Fig). In situ hybridization analysis revealed that the accumulation of LAX1 mRNA was not affected in the few AMs of the triple mutant (S5J–S5L Fig). These results indicated that OsbHLH067/068/069 and LAX1 have no significant mutual effect at the transcriptional level.
OsbHLH067/068/069 physically interact with LAX1
Considering that LAX1 moves directionally to the newly formed AM , where it might have overlapping functions with OsbHLH067/068/069, it is possible that OsbHLH067/068/069 physically interact with LAX1 in the developing AM. To test this hypothesis, we carried out a yeast two-hybrid assay. Because OsbHLH067, OsbHLH068, OsbHLH069, and LAX1 all showed self-activation activities in yeast cells (Fig 7A), we co-transformed a truncated LAX1 fragment (1–159 aa) without self-activation activity as a bait in yeast cell, with the preys OsbHLH067/068/069 (Fig 7B). The resultant transformants all grew on the diluted selective medium, except for the control transformant of pGAD-T7 or pGAD-OsbHLH003 (Fig 7B), suggesting that OsbHLH067/068/069 interact with LAX1 in yeast cells. We further used the truncated LAX1 (31–99 aa) containing the bHLH domain (41–89 aa) as a bait for the interaction assay. The results showed that OsbHLH067/068/069 could interact with the bHLH domain of LAX1, but not with the control of pGAD-OsbHLH003 (Fig 7B).
(A) Transcription activity assay of OsbHLH067, OsbHLH068, OsbHLH069, and LAX1 in yeast cells. The coding sequences (CDS) of LAX1, OsbHLH067, OsbHLH068, and OsbHLH069 were introduced into the pGBK-T7 vector, respectively. The empty vector pGBK-T7 served as a negative control. Both vectors were transformed into AH109, respectively. Cultures were diluted (1:10 successive dilution series) and spotted onto the control medium without Trp and selective medium without Trp and Ade. (B) In yeast two-hybrid assays, OsbHLH067/068/069 interacts with LAX1 through the bHLH domain of LAX1. The pGBK-LAX1(1–159)/pGBK-LAX1(31–99) and pGAD-OsbHLH003 combinations were separately used as negative controls. (C) Bimolecular fluorescence complementation (BiFC) analysis shows the interaction between OsbHLH067/068/069 and LAX1 in rice protoplasts. API5-YFP served as a nuclear marker. Bars = 10 μm. (D) and (E) In vitro pull-down assay showing the direct interaction between OsbHLH067/068/069 and LAX1. MBP-LAX1 was pulled down (PD) by GST-OsbHLH069 immobilized on glutathione-conjugated agarose beads and by His-OsbHLH067/068 immobilized on Ni sepharose beads, and analyzed by immunoblotting (IB) using an anti-MBP antibody. Each input (IN) lane was immunoblotted using an anti-His, anti-GST, or anti-MBP antibody.
We then carried out bimolecular fluorescence complementation (BiFC) assays to confirm the interaction between OsbHLH067/068/069 and LAX1 in rice protoplasts. As anticipated, reconstructed CFP fluorescence was detected in the nuclei of rice protoplasts when co-transforming cCFP-LAX1 with OsbHLH067-nCFP, OsbHLH068-nCFP, or OsbHLH069-nCFP (Fig 7C). We further confirmed the interaction between OsbHLH69/067/068 and LAX1 by pull down assays, respectively (Fig 7D and 7E). Taken together, our results suggested that OsbHLH067/068/069 physically interact with LAX1 and might act synergistically with it to regulate panicle AM formation in rice.
The OsbHLH067/068/069-LAX1 module is associated with starch and sucrose metabolism
To further understand the biological functions of OsbHLH067/068/069 required for AM development, we used young panicles (≤ 2 mm) from the Osbhlh067 Osbhlh068 Osbhlh069 triple mutant and WT plants to perform a comparative RNA-seq. Principal component (PC) analysis demonstrated that the RNA-seq data from three biological replicates were closely clustered (Fig 8A). Compared with WT, the triple mutant was found to contain 1533 down-regulated and 1526 up-regulated genes (Q value ≤ 0.05, fold change > 1.5; Fig 8B, S1 Table). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment results suggested that these down-regulated genes were significantly enriched in multiple biological processes, including secondary metabolite biosynthesis, fatty acid metabolism, alanine aspartate and glutamate metabolism, starch and sucrose metabolism, cell cycle, and phototransduction (Q value ≤ 0.05; Fig 8C, S2 Table). Up-regulated genes were significantly enriched only in “protein processing in endoplasmic reticulum” (Q value ≤ 0.05) (Fig 8D, S3 Table).
(A) Principal component analysis of transcriptome data with three biological replicates in the young panicles (< 2 mm) of Osbhlh067 Osbhlh068 Osbhlh069 triple mutant. Samples with high similarity will be clustered together. (B) Number of DEGs (Q value ≤ 0.05 and fold change >1.5) between WT and Osbhlh067 Osbhlh068 Osbhlh069 triple mutant. (C) and (D) Representative KEGG terms (Q value ≤ 0.05) from the down-regulated genes (C) and the up-regulated genes (D) in the Osbhlh067 Osbhlh068 Osbhlh069 triple mutant. (E) Quantitative RT-PCR analysis of genes involved in secondary metabolite biosynthesis, alanine aspartate and glutamate metabolism, fatty acid metabolism, cell cycle, and phototransduction in the Osbhlh067 Osbhlh068 Osbhlh069 triple mutant and lax1 panicles. (F) Quantitative RT-PCR analysis of genes involved in meristem formation and starch and sucrose metabolism for WT and the Osbhlh067 Osbhlh068 Osbhlh069 triple mutant. Data in (E) and (F) were normalized to the rice UBQ gene, and values represent the means ± SEM from three replicates. Significant difference (two-tailed Student’s t-test, *P< 0.05, **P< 0.01, ***P< 0.001); ns indicates not significant (two-tailed Student’s t-test; P > 0.05).
Because OsbHLH067, OsbHLH068, and OsbHLH069 are typical transcription factors with transcriptional activation activities (Fig 7A), we examined these down-regulated genes in the triple mutant by a comparison with WT by qRT-PCR analysis (S4 Table). As expected, the expression levels of the selected genes (sorted by Q value) were confirmed to be down-regulated in the triple mutant (Fig 8E and 8F). Interestingly, all the examined genes involved in sucrose metabolism were significantly down-regulated in lax1 (Fig 8F). For the selected genes enriched in the other biological processes, they showed no obvious change in expression in lax1 compared with WT (Fig 8E). Among the down-regulated genes in the triple mutant, some of them have been suggested to play important roles in regulating panicle development, such as RCN4, OsTB1, OsSPL14, NL1, and PLA1 [35–42]. Their expression levels were also significantly reduced in both the triple mutant and lax1 compared with WT (Fig 8F, S1 Table). Thus, we speculate OsbHLH067/068/069 function redundantly and might be involved in starch and sucrose metabolism process to modulate AM development by interacting with LAX1.
The function of OsbHLH067, OsbHLH068, and OsbHLH069 in plants
The bHLH protein transcription factors are ubiquitous transcriptional regulators that control many different developmental and physiological processes , which can be divided into 32 subfamilies . Usually, the bHLH genes in the same subfamily participate in the same biological process with partial or complete functional redundancy. For instance, all six members in the bHLH subfamily 16 have the conserved function of regulating flag leaf angle in rice . Arabidopsis PIF-family members PIF1, PIF3, PIF4, and PIF5 (belonging to subfamily 24) regulate seedling morphogenesis through differential expression-patterning of shared target genes . The bHLH010, bHLH089, and bHLH091 genes from bHLH subfamily 9 are redundantly required for Arabidopsis anther development . In our study, OsbHLH067, OsbHLH068, OsbHLH069, and OsbHLH070 belong to the subfamily F of the bHLH transcription factor in rice, although OsbHLH070 does not contain a typical bHLH domain . OsbHLH069 was demonstrated to function redundantly with OsbHLH067 and OsbHLH068, which are all involved in panicle AM development. Because we have not obtained the Osbhlh070, the role of OsbHLH070 in inflorescence AM development cannot be ruled out.
Notably, rice bHLH subfamily F and Arabidopsis bHLH subfamily X  are clustered into the phylogenetic clade 15 . A recent study reported that some members of subfamily X in Arabidopsis determine the competence of the pericycle for lateral root initiation . Overexpression of AtbHLH112 suppressed lateral root emergence, but the Atbhlh112 mutant exhibited no obvious defect . OsbHLH067, OsbHLH068, and OsbHLH069 are the close homologs to Arabidopsis AtbHLH112 . OsbHLH068 and AtbHLH112 showed a similar expression patterns in transgenic Arabidopsis and partially redundant functions in salt stress response and opposite functions in flowering transition . In this study, overexpression of OsbHLH069 resulted in an obvious defect in AM development (Figs 1, 2 and 3) and partially delayed flowering (Fig 3D). These observations can be explained by the fact that genes in the bHLH subfamily potentially have redundant but distinct functions in rice and Arabidopsis, presumably due to the evolutionary functional divergence of homolog-encoded proteins.
OsbHLH067/068/069 may participate in meristem development through metabolism-related pathways
In situ hybridization revealed that OsbHLH067/068/069 was predominantly expressed in both inflorescence AM and IM (Fig 4). We suspect that OsbHLH067/068/069 may be mainly involved in inflorescence meristem development. RNA-Seq data analysis demonstrated that the differentially expressed genes in the Osbhlh067 Osbhlh068 Osbhlh069 triple mutant were significantly enriched in multiple metabolic pathways such as amino acid metabolism, fatty acid metabolism, secondary metabolism, and cell cycle (Fig 8C). qRT-PCR analysis further confirmed the downregulation of those genes involved in starch and sucrose metabolic pathway in the triple mutant (Fig 8F). Although the functions of genes associated with AM development have not been reported in rice, some of their homologs involved in meristem development in other plants have been characterized. The maize genome encodes 14 trehalose-6-phosphate synthase (TPS) genes and 11 trehalose-6-phosphate phosphatase (TPP) genes. ZmTPP4 is the ortholog of OsTPP4 in maize ; ZmTPP4 is a complete paralogue of RA3; and loss of ZmTPP4 and RA3 would reduce meristem determinacy and increase inflorescence branching [53,54]. The level of trehalose-6-phosphate levels influences plant growth and development through perturbations of the glucose sensor HEXOKINASE 1 (HXK1). HXK1 over-expressing lines exhibited an increase in the number of primary rosette branches despite of no elevation of sugar levels in Arabidopsis [55,56]. In this study, the expression of OsTPP4 and OsHXK7 was significantly suppressed in both the triple mutant and lax1. Multiple investigations have demonstrated that bud outgrowth might be mediated by specific nutritional and hormonal signaling pathways . Thus, we conjecture that OsbHLH067/068/069 influence AM development partially through the starch and sucrose metabolism pathway.
Interactions between OsbHLH067/068/069 and LAX1 in regulating inflorescence AM development
In rice, AM development during the reproductive stage entails stem cell maintenance, AM initiation, AM differentiation, and branching outgrowth. Previous studies have demonstrated that LAX1 mRNA is restricted within a few layers of cells at the boundary region between the SAM and initiating AM  and that LAX1 protein accumulates transiently in initiating AM . Here, we found that LAX1 mRNA indeed accumulates in the boundary region where AM occurs (Fig 4Q–4S). Considering the movement of LAX1 protein, OsbHLH067/068/069 are proposed to interact with LAX1 protein, which coordinates AM development.
This study investigated the interaction between OsbHLH067/068/069 and LAX1 for AM formation. Like the mutation of LAX1 gene, the Osbhlh067 Osbhlh068 Osbhlh069 triple mutant plants showed significant decreases in SBs rather than lateral spikelets on PBs (Fig 5) . Thus, it can be deduced that the interaction of OsbHLH067/068/069 and LAX1 may have certain spatiotemporal characteristic, and determines their transcriptional activity or their ability to bind specific target genes. Indeed, our RNA-seq data and qRT-PCR analysis suggested that five unigenes annotated as contributors to inflorescence architecture in plants were down-regulated in the triple mutant and lax1 (S1 Table, Fig 8F). Among them, RCN4, a TFL1-like gene, acts as a direct downstream target of OsMADS5 and OsMADS34 and precisely regulates inflorescence and SM determinacy . OsTB1 was found to work downstream strigolactones to inhibit the outgrowth of axillary buds and increase the inflorescence size and spikelet number in rice [36,41]. An appropriate increase in the expression of OsSPL14 at the reproductive stage could promote panicle branching and higher grain yield in rice [35,37]. PLASTOCHRON1 (PLA1), a cytochrome P450 gene, is mainly expressed in bracts of the panicle and acts as a timekeeper of panicle development . A recent study has shown that bract suppression regulated by the miR156/529-SPLs-NL1-PLA1 module is required for the transition from vegetative to reproductive branching in rice . In addition, it has been reported that both OsTB1 and OsSPL14 control panicle development through strigolactone signaling and sugar sensing . RNA-seq data and qRT-PCR results demonstrated that some genes involved in the starch and sucrose metabolic pathway were also downregulated in the triple mutant and lax1 (Fig 8F). Therefore, we speculate that the interaction of OsbHLH067/068/069 with LAX1 is required to regulate AM development.
Temporal-spatial expression of OsbHLH067/068/069 is essential for regulating panicle branching
It has been demonstrated that inflorescence AM formation are mediated by phytohormones . In maize, BIF1 and BIF4, two AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) proteins, are involved in the regulation of the BA1 orthologous of LAX1, suggesting that auxin signaling modules are directly responsible for AM formation . Loss-of-function or overexpression of MONOPTEROS (MP)/ARF5 led to strongly suppressed reproductive AM initiation, resulting in "pin"-like inflorescence in Arabidopsis [18,58]. These findings suggest that auxin plays an essential role in ensuring the proper inflorescence architecture of plants. Considering that rice LAX1 is the ortholog of BA1, we hypothesized that LAX1 acts a conserved mechanism in boundary domains for AM formation by the auxin pathway. qRT-PCR analysis suggested some auxin-related genes were consistently suppressed in lax1 plants (S6A Fig). In contrast, most of them have normal expression levels in the triple mutant Osbhlh067 Osbhlh068 Osbhlh069 triple mutant (S6B Fig). These results indicated that LAX1 might participate in auxin pathway alone, but not through the interaction with OsbHLH067/068/069.
The inflorescence architecture is determined by meristem size, bud initiation and outgrowth, and controlled by endogenous and external factors. After transition from vegetative to reproductive development, rice panicle development is set to undergo an interned process, that is IM initiates indeterminate BMs, which in turn produce a series of SMs . Therefore, the comprehensive genetic networks associated with inflorescence architecture must be precisely regulated. In rice, LAX1 might be directly regulated by SPL14 . Both RNAi and overexpression lines of SPL genes showed remarkably reduced panicle branches, indicating that the expression of LAX1 must be fine-tuned for reproductive branching . Genetic analysis has revealed that mutation in LAX1 severely suppresses the initiation of lateral spikelets and affects both vegetative and reproductive branching [3,4]. Overexpression of LAX1 also causes reduction of branching . Our results suggest that OsbHLH067/068/069 physically interact with LAX1 and may tightly regulate LAX1 activity to control panicle AM formation (Fig 7B–7E). Both the OsbHLH069-overexpressing plants and the Osbhlh067 Osbhlh068 Osbhlh069 triple mutant showed some defects in reproductive AM development, resulting in reduction of panicle branches and spikelets (Figs 1, 3 and 5). OsbHLH067/068/069 have broad expression pattern including IM, PB meristem, SB meristem, and SM (Fig 4A–4P), which suggest that they may have multiple functions in regulating reproductive branching. FRIZY PANICLE (FZP) is required to establish SM by inhibiting the formation of BMs . TAWAWA1 is a unique regulator of SM phase transition in rice . OsMADS1 is first expressed in SMs and required for floral meristem identity . To elucidate the function of OsbHLH067/068/069 on SM development, the expression patterns of TAWAWA1, OsMADS1 and FZP, would be examined in the future.
In this study, we examined that the OsbHLH067/068/069-LAX1 module is essential for the regulation of inflorescence AM development in rice. With the onset of inflorescence AM development, the boundary expressed LAX1 might be required for auxin signal transduction to promote inflorescence AM initiation ; at the inflorescence AM development stage, OsbHLH067/068/069 interact with LAX1 and mainly participate in metabolism pathways, fine-tuning the hormone and nutritional signaling to maintain inflorescence AM development. In addition, both OsbHLH067/068/069 and LAX1 are bHLH transcription factors, and thus their self-regulation of molecular networks in meristem development requires further examination.
Materials and methods
Plant materials and growth conditions
The nsp1-D (03Z11CH32) and lax1 mutants (03Z11JS33) were identified from our T-DNA insertion mutant library . All rice plants used in the study were derived from Oryza sativa Japonica variety Zhonghua 11 (ZH11), which was designed as wild type (WT). Rice plants were cultivated during the normal growing season in the experimental field of Huazhong Agricultural University in Wuhan, China (latitude 30.5°N, 15m above sea level; average daily temperature approximately 28°C).
Isolation of the flanking sequences of T-DNA in nsp1-D
Thermal asymmetric interleaved polymerase chain reaction (PCR) was adopted to isolate the flanking sequence of T-DNA in nsp1-D . In the 3-step PCR reactions, one end primer was on the T-DNA left border, namely IN-R, TL14, and LBT2 in turn, and the other end primer was always random primer AD8. The 3rd-round PCR product was sequenced and aligned.
Plasmid construction for generation of OsbHLH069 overexpressing plants
The Cauliflower mosaic virus (CaMV) 35S enhancer sequence was amplified from pCAMBIA2301 (pC2301) and cloned to pC2301 to construct the 35S-pC2301 vector. To construct the 35S-pOsbHLH069::OsbHLH069 vector, two fragments were obtained. One fragment was an approximately 3 kb promoter region of OsbHLH069 amplified from the ZH11 genome using the primer pair 35S-pC2301-OsbHLH069-L1/35S-pC2301-OsbHLH069-R1. Another fragment was composed of approximately 2 kb of the OsbHLH069 genome and a 1 kb region behind the termination codon amplified from the ZH11 genome using the primer pair 35S-pC2301-OsbHLH069-L2/35S-pC2301-OsbHLH069-R2. Using Gibson Assembly Master Mix (E2611S: New England BioLabs, Ipswich, MA, USA), the two fragments were cloned to the linearized vector 35S-pC2301 digested by EcoRI. The constructed plasmid was introduced into Agrobacterium tumefaciens EHA105, and finally transformed into rice callus to generate transgenic plants .
Generation of CRISPR lines
OsbHLH067, OsbHLH068, and OsbHLH069 were mutated using the CRISPR/Cas9 technique . Two gRNA targets for each gene were selected. Two sgRNA sites shared by OsbHLH067 and OsbHLH069 were chosen to generate the double mutant Osbhlh067 Osbhlh069. Homozygotes of Osbhlh068 and Osbhlh069 were identified to develop the double mutant Osbhlh068 Osbhlh069. The CRISPR/Cas9 vector of OsbHLH068 was transformed into Osbhlh067 callus to obtain the double mutant Osbhlh067 Osbhlh068 and into Osbhlh067 Osbhlh069 callus to develop the triple mutant Osbhlh067 Osbhlh068 Osbhlh069. These resultant transgenic plants were identified by 2.5% agarose gel electrophoresis and sequencing.
Scanning electron microscopy (SEM) analysis
For scanning electron microscopy (SEM), young panicles from wild type and nsp1-D mutants at typical development period were carefully dissected to remove bract hairs and keeping tissue integrity. The samples were fixed in 2.5% glutaraldehyde (2.5% GA in a 50mM phosphate buffer, pH 7.0) at 4°C overnight, dehydrated with an ethanol series of from 25% to 100%, and dried. Then, the tissues were coated by using an E-100 ion sputter, and observed under a scanning electron microscope (S570, Hitachi, Tokyo, Japan).
Histology and in situ hybridization
Young panicles from wild type and nsp1-D as well as Osbhlh067 Osbhlh068 Osbhlh069 triple mutant at different developmental stages were fixed with 50% FAA solution containing 50% ethanol, 3.7% formaldehyde, and 5% acetic acid at 4°C overnight. The samples were dehydrated by gradient ethanol and made transparent by xylene, followed by embedding in paraffin, and then sliced into 8 μm sections for 0.5% toluidine blue staining and in situ hybridization.
The templates for the OsbHLH067, OsbHLH068, OsbHLH069, LAX1, and OSH1 probes were amplified from ZH11 cDNA using gene-specific primers joined with T7 or SP6 promoters as previously reported . Probes were synthesized using a digoxigenin (DIG)-labeling kit (Millipore Sigma, Burlington, MA, USA). Hybridizations were conducted at 50°C overnight for total probes as previously described .
Yeast two-hybrid assay
The Matchmaker Gold yeast two-hybrid system (Clontech Laboratories, Mountain View, CA, USA) was used. The CDS of OsbHLH067, OsbHLH068, OsbHLH069, and LAX1 was amplified from ZH11 cDNA and then cloned to pGBK-T7 and pGAD-T7, respectively. The pGAD-OsbHLH003 was used as a negative control . Truncated fragments of LAX1 (1–159) and LAX1 (31–99) were obtained by PCR amplification and then cloned to pGBK-T7. Combined constructs were transformed into AH109 strains in an AD-BK-coupled manner.
Subcellular localization and bimolecular fluorescence complementation (BiFC) assay
To test the subcellular localization of OsbHLH067, OsbHLH068 and OsbHLH069, their coding sequences (CDS) without the termination codon were separately amplified from ZH11 cDNA and then ligated in-frame to PM999-GFP containing a CaMV35S promoter at the N-terminal end and a GFP coding sequence at the C-terminal end. To obtain the BiFC constructs, the CDS of OsbHLH067, OsbHLH068, OsbHLH069, and LAX1 was amplified and cloned into pSCYNE (nCFP) and pSCYCE (cCFP) . The tested constructs were transformed into rice protoplasts as previously reported . After incubation overnight at 23°C, fluorescence in the transformants was observed using the Olympus FV1000.
In vitro pull-down assays
To examine the interaction between OsbHLH069 and LAX1, the coding sequences of OsbHLH069 and LAX1 were separately cloned into the pGEX-4T-1 and pMAL-c2X vector to generate GST-OsbHLH069 and MBP-LAX1, respectively. For the interaction between OsbHLH067/OsbHLH068 and LAX1, the His-fused proteins of His-OsbHLH067 and His-OsbHLH068 were produced in the pET-32a vector.
For in vitro LAX1 and OsbHLH067/068/069 interaction, bacterial lysates containing ~15 mg MBP-LAX1 fusion protein was mixed with lysates containing ~30 mg GST-OsbHLH069 or His-OsbHLH067 or His-OsbHLH068 fusion proteins, respectively. Glutathione sepharose (30 μL; GE Life Sciences) was added to MBP-LAX1 and GST-OsbHLH069 combined solution or Ni Sepharose (30 μL; GE Life Sciences) to MBP-LAX1 and His-OsbHLH067/068 combined solution with rocking at 4°C for 60 min. Beads were washed four times with the TGH buffer (50 mM HEPES, pH 7.5, 1.5 mM MgCl2, 150 mM NaCl, 1 mM EGTA, pH 8.0, 1% Triton X-100, 10% glycerol, 1 mM PMSF, and 1x Complete protease inhibitor cocktail [Roche]), and the isolated proteins were further separated on a 12% SDS-PAGE gels.
Then, the isolated proteins were detected by immunoblot analysis with anti-GST antibody (Abmart) and anti-MBP antibody (NEB) for LAX1-OsbHLH069 interaction, respectively. For LAX1-OsbHLH067 and LAX1-OsbHLH068 interactions, the isolated proteins were detected by immunoblot analysis with anti-His antibody (Abmart) and anti-MBP antibody (NEB), respectively. The immunoblot bands were visualized on a chemiluminescent imaging system (Tanon-5200, Tanon Science and Technology).
RNA extraction and quantitative RT-PCR
Total RNA was extracted from various tissues of plants by using the TRIzol reagent (Invitrogen) according to the manufacturer’s instructions, and first-strand cDNA was synthesized from 2.5 μg of total RNA with SuperScript III Reverse Transcriptase (Invitrogen) and oligo (dT)18 primer (Takara). By using the Applied Biosystems 7500 real-Time PCR system, qRT-PCR experiments were performed with SYBR Green Master Mix (Roche) in a total 10 μL reaction system according to the manufacturer’s instructions, and the resultant data were normalized by the internal rice ubiquitin (UBQ) gene and analyzed by using the relative quantification method (2(-Delta Delta CT)). S5 Table lists the primers used in the qRT-PCR assays.
Total mRNA from young panicles (<2 mm) of wild type and the Osbhlh067 Osbhlh068 Osbhlh069 triple mutant with three biological replicates was isolated using Tri Reagent (Sigma-Aldrich, St. Louis, MO, USA) and purified using oligo(dT)-attached magnetic beads. The quality was checked using the NanoDrop 8000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). The mRNA libraries were constructed and sequenced using the DNBSeq platform at BGI Genomics (Shenzhen, PRC). High-quality reads were filtered with SOAPnuke (version 1.5.2) and then mapped to the rice reference genome IRGSP-1.0 using HISAT2 (version 2.0.4). Gene expression level was calculated with RSEM (version 1.2.12) after alignment with Bowtie2 (version 2.2.5). Differential expression analysis was conducted using the DESeq2 (version 1.4.5). Genes whose expression had a Q value ≤ 0.05 and fold change > 1.5 were chosen for further analysis. A Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of annotated DEGs was conducted by Phyper (https://en.wikipedia.org/wiki/Hypergeometric_distribution) based on the Hypergeometric test.
The two-tailed Student’s t-test in Microsoft Excel (Redmond WA, USA) was used for comparing means between two samples. A Dunnett’s test was used for multiple comparisons by one-way analysis of variance (ANOVA) in the IBM SPSS Statistics software application (version 25.0: IBM, Armonk, NY, USA).
All primers used in this study are listed in S5 Table.
Sequence data in this study can be found in the Rice Genome Annotation Project (http://rice.uga.edu/) under the following accession numbers: NSP1 (OsbHLH069, LOC_Os01g57580), LAX1 (LOC_Os01g61480), OSH1 (LOC_Os03g51690), OsbHLH065 (LOC_Os04g41570), OsbHLH066 (LOC_Os03g55220), OsbHLH067 (LOC_Os05g42180), OsbHLH068 (LOC_Os04g53990), and OsbHLH070 (LOC_Os08g08160).
S1 Fig. Phylogenetic analysis of OsbHLH069.
(A) Phylogenetic analysis of putative OsbHLH069 homologs in rice using MEGA5.1 with neighbor-joining method and the following parameters: Poisson correction, pairwise deletion, and bootstrap (1,000 replicates; random seed). (B) Alignment of OsbHLH067, OsbHLH068, OsbHLH069, and OsbHLH070. Alignment was conducted with ClustalW from a MEGA5.1 program and then mapped using GeneDOC software. Red, orange, and yellow shading represent residues conserved in 100%, 80%, and 60% of the sequences, respectively. (C) Comparison of the bHLH domain of OsbHLH067, OsbHLH068, OsbHLH069, and OsbHLH070.
S2 Fig. Expression profiles and subcellular localization of OsbHLH067/068/069.
(A) to (C) Expression levels of OsbHLH069 (A), OsbHLH067 (B), and OsbHLH068 (C) in various organs, including R (root), C (culm), L (leaf), LS (leaf sheath), and P (panicle < 5 mm). Rice UBQ gene acted as a control. Values represent means ± SEM from nine replicates. (D) Subcellular localization of OsbHLH069, OsbHLH067, and OsbHLH068 in rice protoplasts. The LAX1-RFP vector was used as a nuclear marker. The 35S-GFP vector served as a control. Bars = 10 μm.
S3 Fig. Mutation site analysis of single, double, and triple mutants of OsbHLH067, OsbHLH068, and OsbHLH069.
(A) Schematic diagram of sgRNA sites of OsbHLH069, OsbHLH067, and OsbHLH068 by CRISPR/Cas9 system, respectively. Boxes denote exons, and lines between the boxes indicate introns. (B) to (H) Analysis of the mutation sites in single mutants Osbhlh069 (B), Osbhlh067 (C), Osbhlh068 (D), double mutants of Osbhlh067 Osbhlh069 (E), Osbhlh068 Osbhlh069 (F), Osbhlh067 Osbhlh068 (G), and triple mutant of Osbhlh067 Osbhlh068 Osbhlh069 (H). The red ellipsis represents the missing base; the double slash represents the omitted base; the bases underlined correspond to the target sequences; the bases in blue represent PAM.
S4 Fig. Characterization of single mutant of Osbhlh067, Osbhlh068, and Osbhlh069.
(A) and (B) Comparison of the gross plant (A) and panicle (B) among WT, Osbhlh067, Osbhlh068, and Osbhlh069 during reproductive growth. Bars in (A) and (B) = 20 cm and 4 cm, respectively. (C) to (H) Quantification of the number of tillers (C), PBs (D), SBs (E), SPBs (F), SSBs (G), and total spikelets (H) among WT, Osbhlh067, Osbhlh068, and Osbhlh069. Values in (C) to (H) are shown as means ± SEM from 12 replicates. Different letters denote significant differences ranked by the Dunnett’s test (one-way analysis of variance, P < 0.05).
S5 Fig. Expression relationship between OsbHLH067/068/069 and LAX1.
(A) and (B) Gross morphology of WT (A) and lax1 (B) during reproductive growth. Bars = 20 cm. (C) to (E) Panicle morphology of WT (C), lax1/+ (D), and lax1 (E). Bars = 4 cm. (F) The T-DNA insertion site in lax1. Box represents the LAX1 genome, and the triangle indicates T-DNA. The primers L1, R1, and URB4 used for genotype analysis are marked with arrows. (G) The co-segregation analysis of lax1. W, H, and M indicate WT, heterozygous, and homozygous for T-DNA insertion, respectively. (H) Expression analysis of OsbHLH069, OsbHLH067, OsbHLH068, and LAX1 in the young panicles (< 2 mm) of lax1. (I) Quantitative RT-PCR analysis of LAX1 expression in the young panicles (< 2 mm) of WT and triple mutant Osbhlh067 Osbhlh068 Osbhlh069. (J) to (L) In situ hybridization with a LAX1 probe on WT (J) and Osbhlh067 Osbhlh068 Osbhlh069 triple mutant (K, L) inflorescences at the differentiation stage of secondary branch meristem. Bars = 100 μm. The rice UBQ gene was used to normalize gene expression. The values shown in (H) and (I) are means ± SEM from nine replicates. Significant difference (two-tailed Student’s t-test, ***P < 0.001); ns indicates not significant (two-tailed Student’s t-test; P > 0.05).
Quantitative RT-PCR analysis of eight auxin-related genes in the young panicles (< 2 mm) of lax1 (A) and Osbhlh067 Osbhlh068 Osbhlh069 triple mutants (B). Data were normalized to the rice UBQ gene, and values represent means ± SEM from three replicates. Significant difference (two-tailed Student’s t-test, **P < 0.01, ***P < 0.001); ns indicates not significant (two-tailed Student’s t-test; P > 0.05).
S1 Table. Differentially expressed genes in the Osbhlh067 Osbhlh068 Osbhlh069 triple mutant.
S2 Table. KEGG terms from the down-regulated genes in the Osbhlh067 Osbhlh068 Osbhlh069 triple mutant.
S3 Table. KEGG terms from the up-regulated genes in the Osbhlh067 Osbhlh068 Osbhlh069 triple mutant.
S4 Table. Down-regulated genes involved in various biological processes in the Osbhlh067 Osbhlh068 Osbhlh069 triple mutant.
S5 Table. Primers used in the study.
- 1. McSteen P, Leyser O. Shoot branching. Annu Rev Plant Biol. 2005; 56:353–74. pmid:15862100
- 2. Xing Y, Zhang Q. Genetic and molecular bases of rice yield. Annu Rev Plant Biol. 2010; 61:421–42. pmid:20192739
- 3. Komatsu K, Maekawa M, Ujiie S, Satake Y, Furutani I, Okamoto H, et al. LAX and SPA: major regulators of shoot branching in rice. Proc Natl Acad Sci U S A. 2003; 100(20):11765–70. pmid:13130077
- 4. Komatsu M, Maekawa M, Shimamoto K, Kyozuka J. The LAX1 and FRIZZY PANICLE 2 genes determine the inflorescence architecture of rice by controlling rachis-branch and spikelet development. Dev Biol. 2001; 231(2):364–73. pmid:11237465
- 5. Tabuchi H, Zhang Y, Hattori S, Omae M, Shimizu-Sato S, Oikawa T, et al. LAX PANICLE2 of rice encodes a novel nuclear protein and regulates the formation of axillary meristems. Plant Cell. 2011; 23(9):3276–87. pmid:21963665
- 6. Li XY, Qian Q, Fu ZM, Wang YH, Xiong GS, Zeng DL, et al. Control of tillering in rice. Nature. 2003; 422(6932):618–21. pmid:12687001
- 7. Tanaka W, Ohmori Y, Ushijima T, Matsusaka H, Matsushita T, Kumamaru T, et al. Axillary meristem formation in rice requires the WUSCHEL ortholog TILLERS ABSENT1. Plant Cell. 2015; 27(4):1173–84. pmid:25841039
- 8. Gallavotti A, Zhao Q, Kyozuka J, Meeley RB, Ritter MK, Doebley JF, et al. The role of barren stalk1 in the architecture of maize. Nature. 2004; 432(7017):630–5. pmid:15577912
- 9. Yao H, Skirpan A, Wardell B, Matthes MS, Best NB, McCubbin T, et al. The barren stalk2 gene is required for axillary meristem development in maize. Mol Plant. 2019; 12(3):374–89. pmid:30690173
- 10. Greb T, Clarenz O, Schafer E, Muller D, Herrero R, Schmitz G, et al. Molecular analysis of the LATERAL SUPPRESSOR gene in Arabidopsis reveals a conserved control mechanism for axillary meristem formation. Gene Dev. 2003; 17(9):1175–87. pmid:12730136
- 11. Schumacher K, Schmitt T, Rossberg M, Schmitz G, Theres K. The Lateral suppressor (Ls) gene of tomato encodes a new member of the VHIID protein family. Proc Natl Acad Sci U S A. 1999; 96(1):290–5. pmid:9874811
- 12. Matthes MS, Best NB, Robil JM, Malcomber S, Gallavotti A, McSteen P. Auxin evodevo: conservation and diversification of genes regulating auxin biosynthesis, transport, and signaling. Mol Plant. 2019; 12(3):298–320. pmid:30590136
- 13. Cheng Y, Dai X, Zhao Y. Auxin biosynthesis by the YUCCA flavin monooxygenases controls the formation of floral organs and vascular tissues in Arabidopsis. Genes Develop. 2006; 20(13):1790–9. pmid:16818609
- 14. Gälweiler L, Guan CH, Muller A, Wisman E, Mendgen K, Yephremov A, et al. Regulation of polar auxin transport by AtPIN1 in Arabidopsis vascular tissue. Science. 1998; 282(5397):2226–30. pmid:9856939
- 15. Wang Q, Kohlen W, Rossmann S, Vernoux T, Theres K. Auxin depletion from the leaf axil conditions competence for axillary meristem formation in Arabidopsis and tomato. Plant Cell. 2014; 26(5):2068–79. pmid:24850851
- 16. Benjamins R, Quint A, Weijers D, Hooykaas P, Offringa R. The PINOID protein kinase regulates organ development in Arabidopsis by enhancing polar auxin transport. Development. 2001; 128(20):4057–67. pmid:11641228
- 17. Friml J, Yang X, Michniewicz M, Weijers D, Quint A, Tietz O, et al. A PINOID-dependent binary switch in apical-basal PIN polar targeting directs auxin efflux. Science. 2004; 306(5697):862–5. pmid:15514156
- 18. Hardtke CS, Ckurshumova W, Vidaurre DP, Singh SA, Stamatiou G, Tiwari SB, et al. Overlapping and non-redundant functions of the Arabidopsis auxin response factors MONOPTEROS and NONPHOTOTROPIC HYPOCOTYL 4. Development. 2004; 131(5):1089–100. pmid:14973283
- 19. Gallavotti A, Barazesh S, Malcomber S, Hall D, Jackson D, Schmidt RJ, et al. sparse inflorescence1 encodes a monocot-specific YUCCA-like gene required for vegetative and reproductive development in maize. Proc Natl Acad Sci U S A. 2008; 105(39):15196–201. pmid:18799737
- 20. Galli M, Liu QJ, Moss BL, Malcomber S, Li W, Gaines C, et al. Auxin signaling modules regulate maize inflorescence architecture. Proc Natl Acad Sci U S A. 2015; 112(43):13372–7. pmid:26464512
- 21. Huang P, Jiang H, Zhu C, Barry K, Jenkins J, Sandor L, et al. Sparse panicle1 is required for inflorescence development in Setaria viridis and maize. Nat Plants. 2017; 3:17054. pmid:28418381
- 22. McSteen P, Malcomber S, Skirpan A, Lunde C, Wu X, Kellogg E, et al. barren inflorescence2 encodes a co-ortholog of the PINOID serine/threonine kinase and is required for organogenesis during inflorescence and vegetative development in maize. Plant Physiol. 2007; 144(2):1000–11. pmid:17449648
- 23. Phillips KA, Skirpan AL, Liu X, Christensen A, Slewinski TL, Hudson C, et al. vanishing tassel2 encodes a grass-specific tryptophan aminotransferase required for vegetative and reproductive development in maize. Plant Cell. 2011; 23(2):550–66. pmid:21335375
- 24. He Y, Yan L, Ge C, Yao XF, Han X, Wang R, et al. PINOID is required for formation of the stigma and style in rice. Plant Physiol. 2019; 180(2):926–36. pmid:30918083
- 25. Li Y, Zhu J, Wu L, Shao Y, Wu Y, Mao C. Functional divergence of PIN1 paralogous genes in rice. Plant Cell Physiol. 2019; 60(12):2720–32. pmid:31410483
- 26. Massari ME, Murre C. Helix-loop-helix proteins: Regulators of transcription in eucaryotic organisms. Mol Cell Biol. 2000; 20(2):429–40. pmid:10611221
- 27. Oikawa T, Kyozuka J. Two-step regulation of LAX PANICLE1 protein accumulation in axillary meristem formation in rice. Plant Cell. 2009; 21(4):1095–108. pmid:19346465
- 28. Yang X, Wang J, Dai Z, Zhao X, Miao X, Shi Z. miR156f integrates panicle architecture through genetic modulation of branch number and pedicel length pathways. Rice. 2019; 12(1):40. pmid:31147794
- 29. Abbai R, Singh VK, Nachimuthu VV, Sinha P, Selvaraj R, Vipparla AK, et al. Haplotype analysis of key genes governing grain yield and quality traits across 3K RG panel reveals scope for the development of tailor-made rice with enhanced genetic gains. Plant Biotechnol J. 2019; 17(8):1612–22. pmid:30701663
- 30. Huang X, Yang S, Gong J, Zhao Q, Feng Q, Zhan Q, et al. Genomic architecture of heterosis for yield traits in rice. Nature. 2016; 537(7622):629–33. pmid:27602511
- 31. Li X, Duan X, Jiang H, Sun Y, Tang Y, Yuan Z, et al. Genome-wide analysis of basic/helix-loop-helix transcription factor family in rice and Arabidopsis. Plant Physiol. 2006; 141(4):1167–84. pmid:16896230
- 32. Wu CY, Li XJ, Yuan WY, Chen GX, Kilian A, Li J, et al. Development of enhancer trap lines for functional analysis of the rice genome. Plant J. 2003; 35(3):418–27. pmid:12887592
- 33. Zhang J, Guo D, Chang Y, You C, Li X, Dai X, et al. Non-random distribution of T-DNA insertions at various levels of the genome hierarchy as revealed by analyzing 13 804 T-DNA flanking sequences from an enhancer-trap mutant library. Plant J. 2007; 49(5):947–59. pmid:17253985
- 34. Matsuoka M, Ichikawa H, Saito A, Tada Y, Fujimura T, Kanomurakami Y. Expression of a rice homeobox gene causes altered morphology of transgenic plants. Plant Cell. 1993; 5(9):1039–48. pmid:8104574
- 35. Jiao Y, Wang Y, Xue D, Wang J, Yan M, Liu G, et al. Regulation of OsSPL14 by OsmiR156 defines ideal plant architecture in rice. Nature genetics. 2010; 42(6):541–4. pmid:20495565
- 36. Minakuchi K, Kameoka H, Yasuno N, Umehara M, Luo L, Kobayashi K, et al. FINE CULM1 (FC1) works downstream of strigolactones to inhibit the outgrowth of axillary buds in rice. Plant Cell Physiol. 2010; 51(7):1127–35. pmid:20547591
- 37. Miura K, Ikeda M, Matsubara A, Song XJ, Ito M, Asano K, et al. OsSPL14 promotes panicle branching and higher grain productivity in rice. Nature genetics. 2010; 42(6):545–9. pmid:20495564
- 38. Miyoshi K, Ahn BO, Kawakatsu T, Ito Y, Itoh J, Nagato Y, et al. PLASTOCHRON1, a timekeeper of leaf initiation in rice, encodes cytochrome P450. Proc Natl Acad Sci U S A. 2004; 101(3):875–80. pmid:14711998
- 39. Wang F, Han T, Song Q, Ye W, Song X, Chu J, et al. The rice circadian clock regulates tiller growth and panicle development through strigolactone signaling and sugar sensing. Plant Cell. 2020; 32(10):3124–38. pmid:32796126
- 40. Wang L, Ming L, Liao K, Xia C, Sun S, Chang Y, et al. Bract suppression regulated by the miR156/529-SPLs-NL1-PLA1 module is required for the transition from vegetative to reproductive branching in rice. Mol Plant. 2021; 14(7):1168–84. pmid:33933648
- 41. Yano K, Ookawa T, Aya K, Ochiai Y, Hirasawa T, Ebitani T, et al. Isolation of a novel lodging resistance QTL gene involved in strigolactone signaling and its pyramiding with a QTL gene involved in another mechanism. Mol Plant. 2015; 8(2):303–14. pmid:25616386
- 42. Zhu W, Yang L, Wu D, Meng Q, Deng X, Huang G, et al. Rice SEPALLATA genes OsMADS5 and OsMADS34 cooperate to limit inflorescence branching by repressing the TERMINAL FLOWER1-like gene RCN4. New Phytol. 2022; 233(4):1682–700. pmid:34767634
- 43. Feller A, Machemer K, Braun EL, Grotewold E. Evolutionary and comparative analysis of MYB and bHLH plant transcription factors. Plant J. 2011; 66(1):94–116. pmid:21443626
- 44. Carretero-Paulet L, Galstyan A, Roig-Villanova I, Martinez-Garcia JF, Bilbao-Castro JR, Robertson DL. Genome-wide classification and evolutionary analysis of the bHLH family of transcription factors in Arabidopsis, poplar, rice, moss, and algae. Plant Physiol. 2010; 153(3):1398–412. pmid:20472752
- 45. Dong H, Zhao H, Li S, Han Z, Hu G, Liu C, et al. Genome-wide association studies reveal that members of bHLH subfamily 16 share a conserved function in regulating flag leaf angle in rice (Oryza sativa). PLoS Genet. 2018; 14(4):e1007323. pmid:29617374
- 46. Zhang Y, Mayba O, Pfeiffer A, Shi H, Tepperman JM, Speed TP, et al. A quartet of PIF bHLH factors provides a transcriptionally centered signaling hub that regulates seedling morphogenesis through differential expression-patterning of shared target genes in Arabidopsis. PLoS Genet. 2013; 9(1):e1003244. pmid:23382695
- 47. Zhu E, You C, Wang S, Cui J, Niu B, Wang Y, et al. The DYT1-interacting proteins bHLH010, bHLH089 and bHLH091 are redundantly required for Arabidopsis anther development and transcriptome. Plant J. 2015; 83(6):976–90. pmid:26216374
- 48. Pires N, Dolan L. Origin and diversification of basic-helix-loop-helix proteins in plants. Mol Biol Evol. 2010; 27(4):862–74. pmid:19942615
- 49. Zhang Y, Mitsuda N, Yoshizumi T, Horii Y, Oshima Y, Ohme-Takagi M, et al. Two types of bHLH transcription factor determine the competence of the pericycle for lateral root initiation. Nat Plants. 2021; 7(5):633–43. pmid:34007039
- 50. Wang WS, Zhu J, Lu YT. Overexpression of AtbHLH112 suppresses lateral root emergence in Arabidopsis. Funct Plant Biol. 2014; 41(4):342–52. pmid:32480995
- 51. Chen HC, Hsieh-Feng V, Liao PC, Cheng WH, Liu LY, Yang YW, et al. The function of OsbHLH068 is partially redundant with its homolog, AtbHLH112, in the regulation of the salt stress response but has opposite functions to control flowering in Arabidopsis. Plant Mol Biol. 2017; 94(4–5):531–48. pmid:28631168
- 52. Henry C, Bledsoe SW, Siekman A, Kollman A, Waters BM, Feil R, et al. The trehalose pathway in maize: conservation and gene regulation in response to the diurnal cycle and extended darkness. J Exp Bot. 2014; 65(20):5959–73. pmid:25271261
- 53. Claeys H, Vi SL, Xu X, Satoh-Nagasawa N, Eveland AL, Goldshmidt A, et al. Control of meristem determinacy by trehalose 6-phosphate phosphatases is uncoupled from enzymatic activity. Nat Plants. 2019; 5(4):352–7. pmid:30936436
- 54. Satoh-Nagasawa N, Nagasawa N, Malcomber S, Sakai H, Jackson D. A trehalose metabolic enzyme controls inflorescence architecture in maize. Nature. 2006; 441(7090):227–30. pmid:16688177
- 55. Barbier FF, Lunn JE, Beveridge CA. Ready, steady, go! A sugar hit starts the race to shoot branching. Curr Opin Plant Biol. 2015; 25:39–45. pmid:25938609
- 56. Kelly G, David-Schwartz R, Sade N, Moshelion M, Levi A, Alchanatis V, et al. The pitfalls of transgenic selection and new roles of AtHXK1: a high level of AtHXK1 expression uncouples hexokinase1-dependent sugar signaling from exogenous sugar. Plant Physiol. 2012; 159(1):47–51. pmid:22451715
- 57. Du Y, Wu B, Xing Y, Zhang Z. Conservation and divergence: Regulatory networks underlying reproductive branching in rice and maize. J Adv Res. 2022; 41:179–90. pmid:36328747
- 58. Przemeck GK, Mattsson J, Hardtke CS, Sung ZR, Berleth T. Studies on the role of the Arabidopsis gene MONOPTEROS in vascular development and plant cell axialization. Planta. 1996; 200(2):229–37. pmid:8904808
- 59. Wang L, Sun SY, Jin JY, Fu DB, Yang XF, Weng XY, et al. Coordinated regulation of vegetative and reproductive branching in rice. Proc Natl Acad Sci U S A. 2015;1 12(50):15504–9. pmid:26631749
- 60. Komatsu M, Chujo A, Nagato Y, Shimamoto K, Kyozuka J. FRIZZY PANICLE is required to prevent the formation of axillary meristems and to establish floral meristem identity in rice spikelets. Development. 2003; 130(16):3841–50. pmid:12835399
- 61. Yoshida A, Sasao M, Yasuno N, Takagi K, Daimon Y, Chen R, et al. TAWAWA1, a regulator of rice inflorescence architecture, functions through the suppression of meristem phase transition. Proc Natl Acad Sci U S A. 2013; 110(2):767–72. pmid:23267064
- 62. Prasad K, Sriram P, Kumar CS, Kushalappa K, Vijayraghavan U. Ectopic expression of rice OsMADS1 reveals a role in specifying the lemma and palea, grass floral organs analogous to sepals. Dev Genes Evol. 2001; 211(6):281–90. pmid:11466523
- 63. Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, et al. A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant. 2015; 8(8):1274–84. pmid:25917172
- 64. Li X, Gao X, Wei Y, Deng L, Ouyang Y, Chen G, et al. Rice APOPTOSIS INHIBITOR5 coupled with two DEAD-box adenosine 5’-triphosphate-dependent RNA helicases regulates tapetum degeneration. Plant Cell. 2011; 23(4):1416–34. pmid:21467577
- 65. Kim SH, Oikawa T, Kyozuka J, Wong HL, Umemura K, Kishi-Kaboshi M, et al. The bHLH Rac immunity1 (RAI1) is activated by OsRac1 via OsMAPK3 and OsMAPK6 in rice immunity. Plant Cell Physiol. 2012; 53(4):740–54. pmid:22437844
- 66. Waadt R, Schmidt LK, Lohse M, Hashimoto K, Bock R, Kudla J. Multicolor bimolecular fluorescence complementation reveals simultaneous formation of alternative CBL/CIPK complexes in planta. Plant J. 2008; 56(3):505–16. pmid:18643980
- 67. Bart R, Chern M, Park CJ, Bartley L, Ronald PC. A novel system for gene silencing using siRNAs in rice leaf and stem-derived protoplasts. Plant Methods. 2006; 2:13. pmid:16808845