OsbHLH067, OsbHLH068, and OsbHLH069 redundantly regulate inflorescence axillary meristem formation in rice

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.


Introduction
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 [1]. 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 [2].
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 [5]. MOC1 is a transcriptional regulator of the GRAS family and mainly regulates the formation of vegetative and reproductive AMs in rice [6]. 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 [5]. TAB1/OsWUS is another transcription factor identified for inflorescence AM formation in rice [7]. 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][5][6][8][9][10][11].
Auxin biosynthesis, transport, and signaling have been demonstrated to be required for inflorescence AM formation and lateral organ initiation [12]. In Arabidopsis, AM formation involves auxin synthesis genes YUC1, 4, and 6 [13]; 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) [18]. 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][20][21][22][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 [20]. 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 [26]. LAX1 encodes a bHLH protein and is a key factor determining the formation of AM in rice [3]. 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 [27]. 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 [3], 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 [5]. SPL protein has been reported to possibly regulate LAX1 expression directly at the transcription level [28]. 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 [31], 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, χ 2 = 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).
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.
OSH1 is considered as a marker gene of meristematic cells in rice [34]. 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 [33]. 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

PLOS GENETICS
OsbHLH067/068/069 regulates rice panicle development 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. 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. LOC_Os01g57580 encodes a typical bHLH transcription factor, and is designated as OsbHLH069 [31]. 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 [31]. 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) [26]. 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) [3]. 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) [3]. Movement of the LAX1 protein towards the future AM has been reported to be required for maintaining the AM development [27]. Our results suggested that OsbHLH067/068/069 are preferentially expressed in inflorescence AM, and may act together with LAX1 for the AM development.
LAX1 has been reported to be predominantly localized in the nucleus [27]. 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 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.

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.
We then investigated the possibility of transcriptional regulation among OsbHLH067, OsbHLH068, OsbHLH069, and LAX1. qRT-PCR analysis showed that the expression of

OsbHLH067/068/069 physically interact with LAX1
Considering that LAX1 moves directionally to the newly formed AM [27], 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).
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 downregulated 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).
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

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 [43], which can be divided into 32 subfamilies [44]. 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 [45]. Arabidopsis PIF-family members PIF1, PIF3, PIF4, and PIF5 (belonging to subfamily 24) regulate seedling morphogenesis through differential expression-patterning of shared target genes [46]. The bHLH010, bHLH089, and bHLH091 genes from bHLH subfamily 9 are redundantly required for Arabidopsis anther development [47]. 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 [26]. 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 [48] are clustered into the phylogenetic clade 15 [44]. A recent study reported that some members of subfamily X in Arabidopsis determine the competence of the pericycle for lateral root initiation [49]. Overexpression of AtbHLH112 suppressed lateral root emergence, but the Atbhlh112 mutant exhibited no obvious defect [50]. OsbHLH067, OsbHLH068, and OsbHLH069 are the close homologs to Arabidopsis AtbHLH112 [31]. 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 [51]. 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 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. https://doi.org/10.1371/journal.pgen.1010698.g007

PLOS GENETICS
OsbHLH067/068/069 regulates rice panicle development ZmTPP4 is the ortholog of OsTPP4 in maize [52]; 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 [55]. 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 [3] and that LAX1 protein accumulates transiently in initiating AM [27]. 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) [3]. 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 [42]. 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 [38]. 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 [40]. In addition, it has been reported that both OsTB1 and OsSPL14 control panicle development through strigolactone signaling and sugar sensing [39]. 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 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). https://doi.org/10.1371/journal.pgen.1010698.g008

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 [57]. 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 [20]. 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 [57]. Therefore, the comprehensive genetic networks associated with inflorescence architecture must be precisely regulated. In rice, LAX1 might be directly regulated by SPL14 [59]. 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 [59]. 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 [3]. 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 [60]. TAWAWA1 is a unique regulator of SM phase transition in rice [61]. OsMADS1 is first expressed in SMs and required for floral meristem identity [62]. 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 [20]; at the inflorescence AM development stage, OsbHLH067/068/069 interact with LAX1 and mainly participate in metabolism pathways, finetuning 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.

Plant materials and growth conditions
The nsp1-D (03Z11CH32) and lax1 mutants (03Z11JS33) were identified from our T-DNA insertion mutant library [32]. 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 [33]. 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 pCAM-BIA2301 (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 [32].

Generation of CRISPR lines
OsbHLH067, OsbHLH068, and OsbHLH069 were mutated using the CRISPR/Cas9 technique [63]. 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 [59]. 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 [64].

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 pGA-D-OsbHLH003 was used as a negative control [65]. 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 Nterminal 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) [66]. The tested constructs were transformed into rice protoplasts as previously reported [67]. 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.
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.

RNA-seq analysis
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.

Statistical analysis
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 oneway analysis of variance (ANOVA) in the IBM SPSS Statistics software application (version 25.0: IBM, Armonk, NY, USA).

Primers
All primers used in this study are listed in S5 Table.

Accession numbers
Sequence data in this study can be found in the Rice Genome Annotation Project (http://rice. . 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). (TIF) S1