OsbHLH069 Negatively Regulates Rice Panicle Development by Competitively Interfering with the LAX1-LAX2 Complex

doi:10.13560/j.cnki.biotech.bull.1985.2025-1282

Abstract

Abstract:

Objective The LAX1-LAX2 complex is a key module regulating axillary meristem development in the rice panicle. The bHLH (basic helix-loop-helix) transcription factor OsbHLH069 interacts with LAX1, and its gain-of-function mutant nsp1-D (no spikelet 1-dominant) exhibits a sparse panicle phenotype. This study aimed to elucidate the molecular interaction model among these three proteins and clarify the mechanism by which they regulate panicle branching development. Methods The nuclear localization signal and transcriptional activation domain of OsbHLH069 were analyzed using rice protoplasts and yeast systems. The interaction between OsbHLH069 and LAX2 was investigated through yeast two-hybrid assays, bimolecular fluorescence complementation, and in vitro pull-down assays. The lax2-4 nsp1-D double mutant was generated by field crossing, and genetic interactions were analyzed by quantitative real-time PCR. The interaction regions among the three proteins were mapped using truncation mutants and yeast two-hybrid assays. The interaction model was further explored via luciferase complementation assays. Results OsbHLH069 is a typical nuclear-localized transcriptional activator, with its nuclear localization signal located at amino acids 266–285 and its transcriptional activation domain at the N-terminal region (amino acids 1–120). OsbHLH069 directly interacted with LAX2 both in vivo and in vitro. OsbHLH069 interacted with LAX1 through its bHLH domain and with LAX2 through its C-terminal region; these interaction regions overlapped with the LAX1-LAX2 interaction interface. OsbHLH069 competitively inhibited the stability of the LAX1-LAX2 complex. The lax2-4 nsp1-D double mutant showed more severe defects in branch number and spikelets per panicle than either single mutant, resembling the previously reported phenotype of the lax1 nsp1-D double mutant. Conclusion A phased regulatory model is proposed: during early panicle axillary meristem development, the LAX1-LAX2 complex promotes axillary meristem initiation; as development progresses, OsbHLH069 achieves fine-tuned negative regulation of the panicle axillary meristem by competitively disassembling the LAX1-LAX2 complex.

Key words: rice, panicle development, OsbHLH069, LAX1, LAX2, protein interaction

YANG Qun, LI Jing, FU De-bao, XU Ting-ting. OsbHLH069 Negatively Regulates Rice Panicle Development by Competitively Interfering with the LAX1-LAX2 Complex[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2025-1282.

Figures/Tables 5

Fig. 1 Analysis of the nuclear localization signal peptide of OsbHLH06935S:OsAPI5-RFP served as a nuclear localization protein marker. Scale bar=10 μm

Fig. 2 Transcriptional activity analysis of OsbHLH069 truncated proteinA: Analysis of the transcriptional activity of truncated OsbHLH069 proteins in yeast cells. B: Schematic diagram of the vectors. C: Analysis of the transcriptional activity of truncated OsbHLH069 proteins in rice protoplasts. ***: A significant difference (P<0.001). ns: No significant difference (P>0.05)

Fig. 3 Validation of the interaction between OsbHLH069 and LAX2 in vivo and in vitroA: Yeast two-hybrid assay. B: Bimolecular fluorescence complementation assay. 35S:OsAPI5-YFP serves as a nuclear marker. Scale bar = 10 µm. C: In vitro pull-down assay

Fig. 4 Phenotypic analysis of lax2-4 nsp1-D double mutantsA‒D: Plant architecture, bars=11 cm. E: Panicle morphology, bar=6 cm. F: Tiller number. G: Primary branch number. H: Secondary branch number. I: Spikelet number per primary branch. J: Spikelets number per secondary branch. K: Total spikelet number per panicle. L: Quantitative real-time PCR detection of LAX2 expression. M: Quantitative real-time PCR detection of OsbHLH069 expression. Different lowercase letters indicate significant differences between groups (P<0.05); ns refers to no significant difference (P>0.05)

Fig. 5 Study on the interaction modes of OsbHLH069, LAX1, and LAX2A: Schematic diagram of amino acid truncation for LAX1, LAX2, and OsbHLH069. B‒F: Yeast two-hybrid analysis. Double Dropout Medium (DDO) refers to SD/-Leu/-Trp. Quadruple Dropout Medium (QDO) refers to SD/-Leu/-Trp/-His/-Ade. G: Luciferase complementation assay

References 47

[1]	Xing YZ, Zhang QF. Genetic and molecular bases of rice yield [J]. Annu Rev Plant Biol, 2010, 61: 421-442.
[2]	刘园园, 陈析丰, 钱前, 等. 水稻穗发育调控的分子机制研究进展 [J]. 生物技术通报, 2025, 41(5): 1-13.
	Liu YY, Chen XF, Qian Q, et al. Advances in molecular mechanisms regulating panicle development in rice [J]. Biotechnol Bull, 2025, 41(5): 1-13.
[3]	Komatsu K, Maekawa M, Ujiie S, et al. LAX and SPA: Major regulators of shoot branching in rice [J]. Proc Natl Acad Sci USA, 2003, 100(20): 11765-11770.
[4]	Tabuchi H, Zhang Y, Hattori S, et al. LAX PANICLE2 of rice encodes a novel nuclear protein and regulates the formation of axillary meristems [J]. Plant Cell, 2011, 23(9): 3276-3287.
[5]	Ritter MK, Padilla CM, Schmidt RJ. The maize mutant barren stalk1 is defective in axillary meristem development [J]. American J Botany, 2002, 89(2): 203-210.
[6]	Yao H, Skirpan A, Wardell B, et al. The barren stalk2 gene is required for axillary meristem development in maize [J]. Mol Plant, 2019, 12(3): 374-389.
[7]	Greb T, Clarenz O, Schäfer E, et al. Molecular analysis of the LATERAL SUPPRESSOR gene in Arabidopsis reveals a conserved control mechanism for axillary meristem formation [J]. Genes Dev, 2003, 17(9): 1175-1187.
[8]	Gälweiler L, Guan CH, Müller A, et al. Regulation of polar auxin transport by AtPIN1 in Arabidopsis vascular tissue [J]. Science, 1998, 282(5397): 2226-2230.
[9]	Bennett SRM, Alvarez J, Bossinger G, et al. Morphogenesis in pinoid mutants of Arabidopsis thaliana [J]. Plant J, 1995, 8(4): 505-520.
[10]	Li Y, Zhu JS, Wu LL, et al. Functional divergence of PIN1 paralogous genes in rice [J]. Plant Cell Physiol, 2019, 60(12): 2720-2732.
[11]	He YB, Yan L, Ge CN, et al. PINOID is required for formation of the stigma and style in rice [J]. Plant Physiol, 2019, 180(2): 926-936.
[12]	McSteen P, Hake S. barren inflorescence2 regulates axillary meristem development in the maize inflorescence [J]. Development, 2001, 128(15): 2881-2891.
[13]	McSteen P, Malcomber S, Skirpan A, 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 [J]. Plant Physiol, 2007, 144(2): 1000-1011.
[14]	Skirpan A, Culler AH, Gallavotti A, et al. BARREN INFLORESCENCE2 interaction with ZmPIN1a suggests a role in auxin transport during maize inflorescence development [J]. Plant Cell Physiol, 2009, 50(3): 652-657.
[15]	Massari ME, Murre C. Helix-loop-helix proteins: regulators of transcription in eucaryotic organisms [J]. Mol Cell Biol, 2000, 20(2): 429-440.
[16]	Poirier BC, Feldman MJ, Lange BM. bHLH093/NFL and bHLH061 are required for apical meristem function in Arabidopsis thaliana [J]. Plant Signal Behav, 2018, 13(7): e1486146.
[17]	Jung KH, Han MJ, Lee YS, et al. Rice Undeveloped Tapetum1 is a major regulator of early tapetum development [J]. Plant Cell, 2005, 17(10): 2705-2722.
[18]	Gallavotti A, Zhao Q, Kyozuka J, et al. The role of barren stalk1 in the architecture of maize [J]. Nature, 2004, 432(7017): 630-635.
[19]	Dong HJ, Zhao H, Li SL, 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) [J]. PLoS Genet, 2018, 14(4): e1007323.
[20]	Gonzalez A, Zhao MZ, Leavitt JM, et al. Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings [J]. Plant J, 2008, 53(5): 814-827.
[21]	Friedrichsen DM, Nemhauser J, Muramitsu T, et al. Three redundant brassinosteroid early response genes encode putative bHLH transcription factors required for normal growth [J]. Genetics, 2002, 162(3): 1445-1456.
[22]	Uji Y, Taniguchi S, Tamaoki D, et al. Overexpression of OsMYC in the up-regulation of early JA-rresponsive genes and bacterial blight resistance in rice [J]. Plant Cell Physiol, 2016, 57(9): 1814-1827.
[23]	Wang YJ, Zhang ZG, He XJ, et al. A rice transcription factor OsbHLH1 is involved in cold stress response [J]. Theor Appl Genet, 2003, 107(8): 1402-1409.
[24]	Yi KK, Wu ZC, Zhou J, et al. OsPTF1, a novel transcription factor involved in tolerance to phosphate starvation in rice [J]. Plant Physiol, 2005, 138(4): 2087-2096.
[25]	Liu WW, Tai HH, Li SS, et al. bHLH122 is important for drought and osmotic stress resistance in Arabidopsis and in the repression of ABA catabolism [J]. New Phytol, 2014, 201(4): 1192-1204.
[26]	Ikeda M, Fujiwara S, Mitsuda N, et al. A triantagonistic basic helix-loop-helix system regulates cell elongation in Arabidopsis [J]. Plant Cell, 2012, 24(11): 4483-4497.
[27]	Górska AM, Gouveia P, Borba AR, et al. ZmbHLH80 and ZmbHLH90 transcription factors act antagonistically and contribute to regulate PEPC1 cell-specific gene expression in maize [J]. Plant J, 2019, 99(2): 270-285.
[28]	Cui J, You CJ, Zhu EG, et al. Feedback regulation of DYT1 by interactions with downstream bHLH factors promotes DYT1 nuclear localization and anther development [J]. Plant Cell, 2016, 28(5): 1078-1093.
[29]	Ko SS, Li MJ, Sun-Ben Ku M, et al. The bHLH142 transcription factor coordinates with TDR1 to modulate the expression of EAT1 and regulate pollen development in rice [J]. Plant Cell, 2014, 26(6): 2486-2504.
[30]	Dubos C, Le Gourrierec J, Baudry A, et al. MYBL2 is a new regulator of flavonoid biosynthesis in Arabidopsis thaliana [J]. Plant J, 2008, 55(6): 940-953.
[31]	Bello BK, Hou YX, Zhao J, et al. NF-YB1-YC12-bHLH144 complex directly activates W_x to regulate grain quality in rice (Oryza sativa L.) [J]. Plant Biotechnol J, 2019, 17(7): 1222-1235.
[32]	Wen CK, Chang CR. Arabidopsis RGL1 encodes a negative regulator of gibberellin responses [J]. Plant Cell, 2002, 14(1): 87-100.
[33]	Abbai R, Singh VK, Nachimuthu VV, 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 [J]. Plant Biotechnol J, 2019, 17(8): 1612-1622.
[34]	Huang XH, Yang SH, Gong JY, et al. Genomic architecture of heterosis for yield traits in rice [J]. Nature, 2016, 537(7622): 629-633.
[35]	Oikawa T, Kyozuka J. Two-step regulation of LAX PANICLE1 protein accumulation in axillary meristem formation in rice [J]. Plant Cell, 2009, 21(4): 1095-1108.
[36]	Lv YP, Zhang XF, Hu YJ, et al. BOS1 is a basic helix-loop-helix transcription factor involved in regulating panicle development in rice [J]. Front Plant Sci, 2023, 14: 1162828.
[37]	Zheng Y, Fu DB, Yang ZN. OsDPE2 regulates rice panicle morphogenesis by modulating the content of starch [J]. Rice, 2023, 16: 5.
[38]	Gallavotti A, Malcomber S, Gaines C, et al. BARREN STALK FASTIGIATE1 is an AT-hook protein required for the formation of maize ears [J]. Plant Cell, 2011, 23(5): 1756-1771.
[39]	Dai DQ, Zhang HL, He L, et al. Panicle apical abortion 7 regulates panicle development in rice (Oryza sativa L.) [J]. Int J Mol Sci, 2022, 23(16): 9487.
[40]	Zhang ZY, Li JJ, Tang ZS, et al. Gnp4/LAX2, a RAWUL protein, interferes with the OsIAA3-OsARF25 interaction to regulate grain length via the auxin signaling pathway in rice [J]. J Exp Bot, 2018, 69(20): 4723-4737.
[41]	Xu TT, Fu DB, Xiong XH, et al. OsbHLH067, OsbHLH068, and OsbHLH069 redundantly regulate inflorescence axillary meristem formation in rice [J]. PLoS Genet, 2023, 19(4): e1010698.
[42]	冯志赟, 符德保, 张亚芳, 等. 一个控制水稻侧生小穗形成基因的遗传定位分析 [J]. 分子植物育种, 2015, 13(3): 483-490.
	Feng ZY, Fu DB, Zhang YF, et al. Genetic mapping of a gene that determines the formation of lateral spikelets in rice [J]. Mol Plant Breed, 2015, 13(3): 483-490.
[43]	Li XW, Gao XQ, Wei Y, et al. Rice APOPTOSIS INHIBITOR5 coupled with two DEAD-box adenosine 5′-triphosphate-dependent RNA helicases regulates tapetum degeneration [J]. Plant Cell, 2011, 23(4): 1416-1434.
[44]	Bart R, Chern M, Park CJ, et al. A novel system for gene silencing using siRNAs in rice leaf and stem-derived protoplasts [J]. Plant Meth, 2006, 2: 13.
[45]	张彩霞. 水稻株型相关基因的定位及LAX互作蛋白的筛选 [D]. 武汉: 华中农业大学, 2014.
	Zhang CX. Gentic mapping of the rice plant architecture genes and screening proteins interacting with LAX [D]. Wuhan: Huazhong Agricultural University, 2014.
[46]	Pufall MA, Graves BJ. Autoinhibitory domains: modular effectors of cellular regulation [J]. Annu Rev Cell Dev Biol, 2002, 18: 421-462.
[47]	Eastwood K, Yin C, Bandyopadhyay M, et al. New insights into the orange domain of E(spl)-M8, and the roles of the C-terminal domain in autoinhibition and Groucho recruitment [J]. Mol Cell Biochem, 2011, 356(1/2): 217-225.