Fertilization controls tiller numbers via transcriptional regulation of a MAX1-like gene in rice cultivation

LC-MS/MS analysis of endogenous 4DO, CL, CLA and MeCLA

Rice plants were grown as previously described68, with minor modifications. Eleven-day-old seedlings grown on 0.6% agar media of

hydroponic nutrients under a 16 h light/8 h dark photoperiod were

transferred to glass vials containing 50 mL hydroponic nutrient

solution without inorganic phosphate to activate SL biosynthesis25.

The shoot and root of the 32-day-old seedlings were harvested and

homogenized in 12.5 mL of acetone containing stable isotopelabeled internal standards [[6’-D1]−4DO11, [1-13CH3]-(S)-CL69,

[1-13CH3]-rac-CLA22, [10-D1]-rac-MeCLA22 using POLYTRON PT3100D

(Kinematica).

The filtrates were evaporated under nitrogen gas until acetone

was almost removed. Then, 1 mL of distilled water and 2 mL of

Nature Communications | (2023)14:3191

1.

2.

3.

4.

5.

6.

Wang, B., Smith, S. M. & Li, J. Genetic regulation of shoot architecture. Annu. Rev. plant Biol. 69, 437–468 (2018).

Evenson, R. E. & Gollin, D. Assessing the impact of the green

revolution, 1960 to 2000. science 300, 758–762 (2003).

Hedden, P. The genes of the green revolution. TRENDS Genet. 19,

5–9 (2003).

Khush, G. S. Green revolution: preparing for the 21st century.

Genome 42, 646–655 (1999).

Pingali, P. L. Green revolution: impacts, limits, and the path ahead.

Proc. Natl Acad. Sci. USA 109, 12302–12308 (2012).

Wu, K. et al. Enhanced sustainable green revolution yield via

nitrogen-responsive chromatin modulation in rice. Science 367,

eaaz2046 (2020).

11

Article

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

Marzec, M., Muszynska, A. & Gruszka, D. The role of strigolactones in

nutrient-stress responses in plants. Int. J. Mol. Sci. 14,

9286–9304 (2013).

Sun, H. et al. Strigolactones are involved in phosphate-and nitratedeficiency-induced root development and auxin transport in rice. J.

Exp. Bot. 65, 6735–6746 (2014).

Booker, J. et al. MAX1 encodes a cytochrome P450 family member

that acts downstream of MAX3/4 to produce a carotenoid-derived

branch-inhibiting hormone. Dev. Cell 8, 443–449 (2005).

Gomez-Roldan, V. et al. Strigolactone inhibition of shoot branching.

Nature 455, 189–194 (2008).

Umehara, M. et al. Inhibition of shoot branching by new terpenoid

plant hormones. Nature 455, 195–200 (2008).

Al-Babili, S. & Bouwmeester, H. J. Strigolactones, a novel

carotenoid-derived plant hormone. Annu. Rev. Plant Biol. 66,

161–186 (2015).

Péret, B., Clément, M., Nussaume, L. & Desnos, T. Root developmental adaptation to phosphate starvation: better safe than sorry.

Trends Plant Sci. 16, 442–450 (2011).

Nussaume, L. et al. Phosphate import in plants: focus on the PHT1

transporters. Front. Plant Sci. 2, 83 (2011).

Smith, S. E., Smith, F. A. & Jakobsen, I. Mycorrhizal fungi can

dominate phosphate supply to plants irrespective of growth

responses. Plant Physiol. 133, 16–20 (2003).

Shi, J. et al. A phosphate starvation response-centered network regulates mycorrhizal symbiosis. Cell 184, 5527–5540. e5518 (2021).

Akiyama, K., Matsuzaki, K.-i & Hayashi, H. Plant sesquiterpenes

induce hyphal branching in arbuscular mycorrhizal fungi. Nature

435, 824–827 (2005).

Yoneyama, K. et al. Conversion of carlactone to carlactonoic acid is

a conserved function of MAX 1 homologs in strigolactone biosynthesis. N. Phytologist 218, 1522–1533 (2018).

Zhang, Y. et al. Rice cytochrome P450 MAX1 homologs catalyze

distinct steps in strigolactone biosynthesis. Nat. Chem. Biol. 10,

1028–1033 (2014).

Cardoso, C. et al. Natural variation of rice strigolactone biosynthesis

is associated with the deletion of two MAX1 orthologs. Proc. Natl

Acad. Sci. USA 111, 2379–2384 (2014).

Mashiguchi, K., Seto, Y. & Yamaguchi, S. Strigolactone biosynthesis,

transport and perception. Plant J. 105, 335–350 (2021).

Abe, S. et al. Carlactone is converted to carlactonoic acid by MAX1

in Arabidopsis and its methyl ester can directly interact with AtD14

in vitro. Proc. Natl Acad. Sci. USA 111, 18084–18089 (2014).

Zhang, Y. et al. The tomato MAX1 homolog, SlMAX1, is involved in

the biosynthesis of tomato strigolactones from carlactone. N. Phytologist 219, 297–309 (2018).

Ito, S. et al. Canonical strigolactones are not the major determinant

of tillering but important rhizospheric signals in rice. Sci. Adv. 8,

eadd1278 (2022).

Umehara, M., Hanada, A., Magome, H., Takeda-Kamiya, N. &

Yamaguchi, S. Contribution of strigolactones to the inhibition of

tiller bud outgrowth under phosphate deficiency in rice. Plant Cell

Physiol. 51, 1118–1126 (2010).

Challis, R. J., Hepworth, J., Mouchel, C., Waites, R. & Leyser, O. A

role for more axillary growth1 (MAX1) in evolutionary diversity in

strigolactone signaling upstream of MAX2. Plant Physiol. 161,

1885–1902 (2013).

McSteen, P. Hormonal regulation of branching in grasses. Plant

Physiol. 149, 46–55 (2009).

de Jong, M. et al. Auxin and strigolactone signaling are required for

modulation of Arabidopsis shoot branching by nitrogen supply.

Plant Physiol. 166, 384–395 (2014).

Qin, P. et al. Pan-genome analysis of 33 genetically diverse rice

accessions reveals hidden genomic variations. Cell 184,

3542–3558. e3516 (2021).

Nature Communications | (2023)14:3191

https://doi.org/10.1038/s41467-023-38670-8

30. Xie, X., Yoneyama, K. & Yoneyama, K. The strigolactone story. Annu.

Rev. Phytopathol. 48, 93–117 (2010).

31. Arite, T. et al. d14, a strigolactone-insensitive mutant of rice, shows

an accelerated outgrowth of tillers. Plant Cell Physiol. 50,

1416–1424 (2009).

32. Minakuchi, K. et al. FINE CULM1 (FC1) works downstream of strigolactones to inhibit the outgrowth of axillary buds in rice. Plant Cell

Physiol. 51, 1127–1135 (2010).

33. Zhang, J. et al. Strigolactones inhibit auxin feedback on PINdependent auxin transport canalization. Nat. Commun. 11,

1–10 (2020).

34. Domagalska, M. A. & Leyser, O. Signal integration in the control of

shoot branching. Nat. Rev. Mol. Cell Biol. 12, 211–221 (2011).

35. Luo, L. et al. Developmental analysis of the early steps in strigolactone‐mediated axillary bud dormancy in rice. Plant J. 97,

1006–1021 (2019).

36. Khan, W., Prithiviraj, B. & Smith, D. L. Photosynthetic responses of

corn and soybean to foliar application of salicylates. J. Plant Physiol.

160, 485–492 (2003).

37. Mashiguchi, K. et al. A carlactonoic acid methyltransferase that

contributes to the inhibition of shoot branching in Arabidopsis.

Proc. Natl Acad. Sci. USA 119, e2111565119 (2022).

38. Wakabayashi, T. et al. Direct conversion of carlactonoic acid to

orobanchol by cytochrome P450 CYP722C in strigolactone biosynthesis. Sci. Adv. 5, eaax9067 (2019).

39. Tsuji, H. et al. Hd3a promotes lateral branching in rice. Plant J. 82,

256–266 (2015).

40. Böhlenius, H. et al. CO/FT regulatory module controls timing of

flowering and seasonal growth cessation in trees. Science 312,

1040–1043 (2006).

41. Hsu, C.-Y., Liu, Y., Luthe, D. S. & Yuceer, C. Poplar FT2 shortens the

juvenile phase and promotes seasonal flowering. Plant Cell 18,

1846–1861 (2006).

42. Danilevskaya, O. N., Meng, X., Hou, Z., Ananiev, E. V. & Simmons,

C. R. A genomic and expression compendium of the

expanded PEBP gene family from maize. Plant Physiol. 146,

250–264 (2008).

43. Rodríguez-Leal, D., Lemmon, Z. H., Man, J., Bartlett, M. E. &

Lippman, Z. B. Engineering quantitative trait variation for crop

improvement by genome editing. Cell 171, 470–480. e478 (2017).

44. Hendelman, A. et al. Conserved pleiotropy of an ancient plant

homeobox gene uncovered by cis-regulatory dissection. Cell 184,

1724–1739. e1716 (2021).

45. Meyer, R. S. & Purugganan, M. D. Evolution of crop species: genetics

of domestication and diversification. Nat. Rev. Genet. 14, 840–852

(2013).

46. Ishikawa, S. et al. Low-cesium rice: mutation in OsSOS2 reduces

radiocesium in rice grains. Sci. Rep. 7, 1–15 (2017).

47. Workman, C. et al. A new non-linear normalization method for

reducing variability in DNA microarray experiments. Genome Biol.

3, 1–16 (2002).

48. Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120

(2014).

49. Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).

50. Li, B. & Dewey, C. N. RSEM: accurate transcript quantification from

RNA-Seq data with or without a reference genome. BMC Bioinforma. 12, 1–16 (2011).

51. Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital

gene expression data. Bioinformatics 26, 139–140 (2010).

52. Duan, J. et al. Strigolactone promotes cytokinin degradation through

transcriptional activation of CYTOKININ OXIDASE/DEHYDROGENASE

9 in rice. Proc. Natl Acad. Sci. USA 116, 14319–14324 (2019).

12

Article

53. Itoh, H. & Izawa, T. A study of phytohormone biosynthetic gene

expression using a circadian clock-related mutant in rice. Plant

Signal. Behav. 6, 1932–1936 (2011).

54. Rong, C. et al. Cytokinin oxidase/dehydrogenase family genes exhibit

functional divergence and overlap in rice growth and development,

especially in control of tillering. J. Exp. Bot. 73, 3552–3568 (2022).

55. Hirano, K. et al. Comprehensive transcriptome analysis of phytohormone biosynthesis and signaling genes in microspore/pollen

and tapetum of rice. Plant Cell Physiol. 49, 1429–1450 (2008).

56. Liu, X. et al. ζ-Carotene isomerase suppresses tillering in rice

through the coordinated biosynthesis of strigolactone and abscisic

acid. Mol. Plant 13, 1784–1801 (2020).

57. Lefevere, H., Bauters, L. & Gheysen, G. Salicylic acid biosynthesis in

plants. Front Plant Sci. 11, 338 (2020).

58. Lu, Z. et al. Genome-wide binding analysis of the transcription

activator IDEAL PLANT ARCHITECTURE1 reveals a complex network

regulating rice plant architecture. Plant Cell 25, 3743–3759 (2013).

59. Meng, F. et al. A bHLH transcription activator regulates defense

signaling by nucleo-cytosolic trafficking in rice. J. Integr. Plant Biol.

62, 1552–1573 (2020).

60. Seo, J. S. et al. OsbHLH148, a basic helix-loop-helix protein, interacts with OsJAZ proteins in a jasmonate signaling pathway leading

to drought tolerance in rice. Plant J. 65, 907–921 (2011).

61. Sun, H. et al. SPL14/17 act downstream of strigolactone signalling to

modulate rice root elongation in response to nitrate supply. Plant J.

106, 649–660 (2021).

62. Yamamoto, T. et al. Expression of RSOsPR10 in rice roots is antagonistically regulated by jasmonate/ethylene and salicylic acid via

the activator OsERF87 and the repressor OsWRKY76, respectively.

Plant Direct 2, e00049 (2018).

63. Tamiru, M. et al. The tillering phenotype of the rice plastid terminal

oxidase (PTOX) loss-of-function mutant is associated with strigolactone deficiency. N. Phytologist 202, 116–131 (2014).

64. Yang, D. L. et al. Plant hormone jasmonate prioritizes defense over

growth by interfering with gibberellin signaling cascade. Proc. Natl

Acad. Sci. USA 109, E1192–E1200 (2012).

65. Wickham, H. ggplot2: Elegant Graphics for Data Analysis (SpringerVerlag New York, 2016).

66. Itoh, T. et al. Foreign DNA detection by high-throughput sequencing

to regulate genome-edited agricultural products. Sci. Rep. 10,

1–10 (2020).

67. Kouchi, H. & Hata, S. Isolation and characterization of novel nodulin

cDNAs representing genes expressed at early stages of soybean

nodule development. Mol. Gen. Genet. 238, 106–119 (1993).

68. Umehara, M. et al. Structural requirements of strigolactones for

shoot branching inhibition in rice and Arabidopsis. Plant Cell Physiol. 56, 1059–1072 (2015).

69. Seto, Y. et al. Carlactone is an endogenous biosynthetic precursor

for strigolactones. Proc. Natl Acad. Sci. USA 111, 1640–1645 (2014).

Acknowledgements

We would like to thank Drs. Masaki Endo and Seiichi Toki (National

Agriculture and Food Research Organization) for providing CRISPR/

Cas9 vectors. We also thank Dr. Kenichiro Hibara (currently, Kibi International University) for teaching some key protocols to J. Cui at the

beginning in this study and Dr. Mingchao Wang (Dresden University of

Technology) for proofreading some languages. We are grateful to the

System for Development and Assessment of Sustainable Humanosphere (RISH, Kyoto University) for providing a greenhouse to KM and

SY. This work was supported by JSPS KAKENHI under grant number

Nature Communications | (2023)14:3191

https://doi.org/10.1038/s41467-023-38670-8

JP17H06246 (to T.I.), JP22H05180 (to T.I.), JP22H0517222 (to T.I.),

JP22H00367 (to T.I.), JP19H02892 (to K.M.), JP17H06474 (to S.Y.), by the

Human Frontier Science Program Organization under grant number

RGP0011/2019 (to T.I.), and by Cabinet Office, Government of Japan,

Cross-ministerial Moonshot Agriculture, Forestry and Fisheries Research

and Development Program, “Technologies for Smart Bio-industry and

Agriculture”(funding agency: Bio-oriented Technology Research

Advancement Institution) (JPJ009237 to T.I.). the Collaborative Research

Program of Institute for Chemical Research, Kyoto University (grant #

2020-92) (to T.I., K.M., and S.Y.)

Author contributions

T.I. conceived the original idea on this work, organized all the experiments and mainly revised the manuscript. T.I. and N.N. performed the

field transcriptome analysis. With substantial support by K.K. and K.S.,

N.N. made the os1900 & os5100 and os900 & os1400 double mutants

and check the tiller number phenotypes firstly. K.M. and S.Y. performed

SL analysis and GR24 application assay. M.M. and J.I. performed in situ

hybridization analysis and RNA-seq analysis. J.C. performed all of the

rest experiments including related to genotyping and phenotyping of all

mutants, the entire experiments on the promoter deletion mutant analysis and wrote the original manuscript. All the authors revised the

manuscript.

Competing interests

The authors declare no competing interests.

Additional information

Supplementary information The online version contains

supplementary material available at

https://doi.org/10.1038/s41467-023-38670-8.

Correspondence and requests for materials should be addressed to

Takeshi Izawa.

Peer review information Nature Communications thanks Bing Wang and

the other, anonymous, reviewer(s) for their contribution to the peer

review of this work. A peer review file is available.

Reprints and permissions information is available at

http://www.nature.com/reprints

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Open Access This article is licensed under a Creative Commons

Attribution 4.0 International License, which permits use, sharing,

adaptation, distribution and reproduction in any medium or format, as

long as you give appropriate credit to the original author(s) and the

source, provide a link to the Creative Commons license, and indicate if

changes were made. The images or other third party material in this

article are included in the article’s Creative Commons license, unless

indicated otherwise in a credit line to the material. If material is not

included in the article’s Creative Commons license and your intended

use is not permitted by statutory regulation or exceeds the permitted

use, you will need to obtain permission directly from the copyright

holder. To view a copy of this license, visit http://creativecommons.org/

licenses/by/4.0/.

© The Author(s) 2023

13

...