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OsbHLH067, OsbHLH068, and OsbHLH069 redundantly regulate inflorescence axillary meristem formation in rice [1]

['Tingting Xu', 'National Key Laboratory Of Crop Genetic Improvement', 'National Center Of Plant Gene Research', 'Wuhan', 'Hubei Hongshan Laboratory', 'Huazhong Agricultural University', 'Debao Fu', 'Xiaohu Xiong', 'Junkai Zhu', 'Jiangsu Kingearth Seed Co.']

Date: 2023-07

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.

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.

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.

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.

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 ].

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 – 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.

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 – 6 , 8 – 11 ].

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 ].

Results

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 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. PPT PowerPoint slide

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TIFF original image Download: Fig 3. Identification of No Spikelet 1 (NSP1). (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). https://doi.org/10.1371/journal.pgen.1010698.g003 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.

OsbHLH067/068/069 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. PPT PowerPoint slide

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TIFF original image Download: Fig 5. Characterization of double and triple mutants of OsbHLH067, OsbHLH068, and OsbHLH069. (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. https://doi.org/10.1371/journal.pgen.1010698.g005

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. PPT PowerPoint slide

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TIFF original image Download: Fig 6. Phenotype analysis of plants in the progenies of nsp1-D/+ lax1/+. (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). https://doi.org/10.1371/journal.pgen.1010698.g006 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.

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[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1010698

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