Abstract
One-step and two-step pathways are proposed to synthesize cytokinin in plants. The one-step pathway is mediated by LONELY GUY (LOG) proteins. However, the enzyme for the two-step pathway remains to be identified. Here, we show that quantitative trait locus GY3 may boost grain yield by more than 20% through manipulating a two-step pathway. Locus GY3 encodes a LOG protein that acts as a 5′-ribonucleotide phosphohydrolase by excessively consuming the cytokinin precursors, which contrasts with the activity of canonical LOG members as phosphoribohydrolases in a one-step pathway. The residue S41 of GY3 is crucial for the dephosphorylation of iPRMP to produce iPR. A solo-LTR insertion within the promoter of GY3 suppressed its expression and resulted in a higher content of active cytokinins in young panicles. Introgression of GY302428 increased grain yield per plot by 7.4% to 16.3% in all investigated indica backgrounds, which demonstrates the great value of GY302428 in indica rice production.
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Data availability
Sequence data from this study can be found in GenBank under accession number PRJEB6180, SRX502298–SRX502317 and SRX502162–SRX502255 for 3,010 diverse accessions, 20 African cultivated rice accessions and 94 accessions of O. barthii, respectively. The eChlp–seq sequence data and BED files were deposited in the NCBI Gene Expression Omnibus with accession number GSE231360. The reference genome of MH63RS2 and Nipponbare can be found in Rice Information GateWay (http://rice.hzau.edu.cn/rice_rs2/) and The Rice Annotation Project (https://rapdb.dna.affrc.go.jp/). All data are available in the main text or the Supplementary Information and Supplementary Data. Source data are provided with this paper.
Code availability
All software used in the study are publicly available as described in Methods and Reporting Summary.
References
Osugi, A. & Sakakibara, H. Q&A: how do plants respond to cytokinins and what is their importance? BMC Biol. 13, 102 (2015).
Ashikari, M. et al. Cytokinin oxidase regulates rice grain production. Science 309, 741–745 (2005).
Sakakibara, H. Cytokinins: activity, biosynthesis, and translocation. Annu. Rev. Plant Biol. 57, 431–449 (2006).
Kiba, T., Takei, K., Kojima, M. & Sakakibara, H. Side-chain modification of cytokinins controls shoot growth in Arabidopsis. Dev. Cell 27, 452–461 (2013).
Sakakibara, H. Cytokinin biosynthesis and transport for systemic nitrogen signaling. Plant J. 105, 421–430 (2021).
Osugi, A. et al. Systemic transport of trans-zeatin and its precursor have differing roles in Arabidopsis shoots. Nat. Plants 3, 17112 (2017).
Kurakawa, T. et al. Direct control of shoot meristem activity by a cytokinin-activating enzyme. Nature 445, 652–655 (2007).
Chen, C. M. & Kristopeit, S. M. Metabolism of cytokinin: deribosylation of cytokinin ribonucleoside by adenosine nucleosidase from wheat germ cells. Plant Physiol. 68, 1020–1023 (1981).
Chen, C. M. & Kristopeit, S. M. Metabolism of cytokinin: dephosphorylation of cytokinin ribonucleotide by 5′-nucleotidases from wheat germ cytosol. Plant Physiol. 67, 494–498 (1981).
Gu, B. et al. An-2 encodes a cytokinin synthesis enzyme that regulates awn length and grain production in rice. Mol. Plant 8, 1635–1650 (2015).
Hua, L. et al. LABA1, a domestication gene associated with long, barbed awns in wild rice. Plant Cell 27, 1875–1888 (2015).
Hinsch, J. et al. De novo biosynthesis of cytokinins in the biotrophic fungus Claviceps purpurea. Environ. Microbiol. 17, 2935–2951 (2015).
Radhika, V. et al. Methylated cytokinins from the phytopathogen Rhodococcus fascians mimic plant hormone activity. Plant Physiol. 169, 1118–1126 (2015).
Samanovic, M. I. et al. Proteasomal control of cytokinin synthesis protects Mycobacterium tuberculosis against nitric oxide. Mol. Cell 57, 984–994 (2015).
Kuroha, T. et al. Functional analyses of LONELY GUY cytokinin-activating enzymes reveal the importance of the direct activation pathway in Arabidopsis. Plant Cell 21, 3152–3169 (2009).
Seo, H. & Kim, K. J. Structural basis for a novel type of cytokinin-activating protein. Sci. Rep. 7, 45985 (2017).
Naseem, M., Bencurova, E. & Dandekar, T. The cytokinin-activating LOG-family proteins are not lysine decarboxylases. Trends Biochem. Sci. 43, 232–236 (2018).
Tokunaga, H. et al. Arabidopsis LONELY GUY (LOG) multiple mutants reveal a central role of the LOG-dependent pathway in cytokinin activation. Plant J. 69, 355–365 (2012).
Wu, B., Mao, D. H., Liu, T. M., Li, Z. X. & Xing, Y. Z. Two quantitative trait loci for grain yield and plant height on chromosome 3 are tightly linked in coupling phase in rice. Mol. Breed. 35, 156 (2015).
Wu, Y. et al. The QTL GNP1 encodes GA20ox1, which increases grain number and yield by increasing cytokinin activity in rice panicle meristems. PLoS Genet. 12, e1006386 (2016).
Du, J. et al. Dual binding of chromomethylase domains to H3K9me2-containing nucleosomes directs DNA methylation in plants. Cell 151, 167–180 (2012).
Schaller, G. E., Street, I. H. & Kieber, J. J. Cytokinin and the cell cycle. Curr. Opin. Plant Biol. 21, 7–15 (2014).
Marzluff, W. F. & Duronio, R. J. Histone mRNA expression: multiple levels of cell cycle regulation and important developmental consequences. Curr. Opin. Cell Biol. 14, 692–699 (2002).
Chen, W. et al. Genome-wide association analyses provide genetic and biochemical insights into natural variation in rice metabolism. Nat. Genet. 46, 714–721 (2014).
Kumar, A. & Bennetzen, J. L. Plant retrotransposons. Annu. Rev. Genet. 33, 479–532 (1999).
Vitte, C. & Panaud, O. Formation of solo-LTRs through unequal homologous recombination counterbalances amplifications of LTR retrotransposons in rice Oryza sativa L. Mol. Biol. Evol. 20, 528–540 (2003).
Kim, J. M., Vanguri, S., Boeke, J. D., Gabriel, A. & Voytas, D. F. Transposable elements and genome organization: a comprehensive survey of retrotransposons revealed by the complete Saccharomyces cerevisiae genome sequence. Genome Res. 8, 464–478 (1998).
Goodchild, N. L., Wilkinson, D. A. & Mager, D. L. Recent evolutionary expansion of a subfamily of RTVL-H human endogenous retrovirus-like elements. Virology 196, 778–788 (1993).
SanMiguel, P. et al. Nested retrotransposons in the intergenic regions of the maize genome. Science 274, 765–768 (1996).
Vitte, C., Panaud, O. & Quesneville, H. LTR retrotransposons in rice (Oryza sativa, L.): recent burst amplifications followed by rapid DNA loss. BMC Genom. 8, 218 (2007).
Ma, J., Devos, K. M. & Bennetzen, J. L. Analyses of LTR-retrotransposon structures reveal recent and rapid genomic DNA loss in rice. Genome Res. 14, 860–869 (2004).
Wang, W. et al. Genomic variation in 3,010 diverse accessions of Asian cultivated rice. Nature 557, 43–49 (2018).
Ambavaram, M. M. et al. Coordinated regulation of photosynthesis in rice increases yield and tolerance to environmental stress. Nat. Commun. 5, 5302 (2014).
Stein, J. C. et al. Genomes of 13 domesticated and wild rice relatives highlight genetic conservation, turnover and innovation across the genus Oryza. Nat. Genet. 50, 285–296 (2018).
Choi, J. Y. et al. Nanopore sequencing-based genome assembly and evolutionary genomics of circum-basmati rice. Genome Biol. 21, 21 (2020).
Seo, H. & Kim, K. J. Structural insight into molecular mechanism of cytokinin activating protein from Pseudomonas aeruginosa PAO1. Environ. Microbiol. 20, 3214–3223 (2018).
Denning, G. L. & Mew, T. W. China and IRRI: Improving China’s Rice Productivity in the 21st Century (IRRI, 1997).
Xue, W. et al. Natural variation in Ghd7 is an important regulator of heading date and yield potential in rice. Nat. Genet. 40, 761–767 (2008).
Zhang, X. et al. TaCol-B5 modifies spike architecture and enhances grain yield in wheat. Science 376, 180–183 (2022).
Kahle, D. & Wickham, H. ggmap: spatial visualization with ggplot2. R J. 5, 144–161 (2013).
Haklay, M. & Weber, P. OpenStreetMap: user-generated street maps. IEEE Pervasive Comput. 7, 12–18 (2008).
Yu, H., Xie, W., Li, J., Zhou, F. & Zhang, Q. A whole-genome SNP array (RICE6K) for genomic breeding in rice. Plant Biotechnol. J. 12, 28–37 (2014).
Ma, X. et al. A Robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol. Plant 8, 1274–1284 (2015).
Takei, K., Sakakibara, H. & Sugiyama, T. Identification of genes encoding adenylate isopentenyltransferase, a cytokinin biosynthesis enzyme, in Arabidopsis thaliana. J. Biol. Chem. 276, 26405–26410 (2001).
Zhang, J. et al. Extensive sequence divergence between the reference genomes of two elite indica rice varieties Zhenshan 97 and Minghui 63. Proc. Natl Acad. Sci. USA 113, E5163–E5171 (2016).
International Rice Genome Sequencing, P. The map-based sequence of the rice genome. Nature 436, 793–800 (2005).
Krueger, F. & Andrews, S. R. Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications. Bioinformatics 27, 1571–1572 (2011).
Zhao, L. et al. Integrative analysis of reference epigenomes in 20 rice varieties. Nat. Commun. 11, 2658 (2020).
Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).
Li, H. et al. The sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).
Liu, Z., Wei, F. & Feng, Y. Q. Determination of cytokinins in plant samples by polymer monolith microextraction coupled with hydrophilic interaction chromatography-tandem mass spectrometry. Anal. Methods 2, 1676–1685 (2010).
Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).
Baek, M. et al. Accurate prediction of protein structures and interactions using a three-track neural network. Science 373, 871–876 (2021).
Forli, S. et al. Computational protein–ligand docking and virtual drug screening with the AutoDock suite. Nat. Protoc. 11, 905–919 (2016).
Wang, M. et al. The genome sequence of African rice (Oryza glaberrima) and evidence for independent domestication. Nat. Genet. 46, 982–988 (2014).
Li, W. et al. Draft genomes of two outcrossing wild rice, Oryza rufipogon and O. longistaminata, reveal genomic features associated with mating-system evolution. Plant Direct 4, e00232 (2020).
Chen, X. et al. Manta: rapid detection of structural variants and indels for germline and cancer sequencing applications. Bioinformatics 32, 1220–1222 (2016).
Martin, S. H., Davey, J. W. & Jiggins, C. D. Evaluating the use of ABBA-BABA statistics to locate introgressed loci. Mol. Biol. Evol. 32, 244–257 (2015).
Malinsky, M. et al. Genomic islands of speciation separate cichlid ecomorphs in an East African crater lake. Science 350, 1493–1498 (2015).
Browning, B. L., Zhou, Y. & Browning, S. R. A one-penny imputed genome from next-generation reference panels. Am. J. Hum. Genet. 103, 338–348 (2018).
Garrison, E., Kronenberg, Z. N., Dawson, E. T., Pedersen, B. S. & Prins, P. A spectrum of free software tools for processing the VCF variant call format: vcflib, bio-vcf, cyvcf2, hts-nim and slivar. PLoS Comput. Biol. 18, e1009123 (2022).
Ahlmann-Eltze, C. & Patil, I. ggsignif: R package for displaying significance brackets for “Ggplot2.” Preprint at https://psyarxiv.com/7awm6/ (2021).
De Mendiburu, F. Agricolae: Statistical Procedures For Agricultural Research. R package version 1.3–6 https://CRAN.R-project.org/package=agricolae (2023).
Acknowledgements
We thank B. Han for sharing the clone An-2, M. J. Han for assistance in analyzing the retrotransposon structure and J. B. Wang for his excellent fieldwork. The computations in this paper were performed on the bioinformatics computing platform of the National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University. This work was partially supported by funds from the National Natural Science Foundation of China (31821005 to Q.Z., U20A2031 to Y.X., 32061143042 to Y.X. and 31901519 to X.Z.), the Earmarked Fund for China Agriculture Research System (CARS-01 to Q.Z.) and the National Key Laboratory of Crop Genetic Improvement Self-research Program (ZW22B0204 to Y.X.).
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Y.X. conceived and supervised this study and reviewed and modified the manuscript. B.W. designed and performed the experiments, collected and analyzed the data and wrote the manuscript. J.M. collected young panicle samples. Hongbo Liu, H.Y. and W.C. measured the cytokinin contents. D.M. and A.S. developed the materials. Z.Z. conducted the eChlp–seq. X.Z. collected the indica backbone parents. B.Z. collected the phenotypic data of 533 accessions. Xianghua Li and J.X. organized the laboratory operations. Haiyang Liu, Xingwang Li, W.Y., W.X., P.Y., L.W. and Q.Z. contributed valuable suggestions for the manuscript. All the authors read and approved the manuscript.
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Extended data
Extended Data Fig. 1 Plant architecture and yield performance of near-isogenic lines of GY3.
a–d, Comparison of whole plant (a), panicle (b), primary branch (c) and panicle architecture (d) between 02428 and 02428-GY3TQ at maturation stage. Bars, 5 cm. e–q, Performance of grain yield per plant (e), secondary branches per panicle (f), spikelets per panicle (g), grains per panicle (h), plant height (i), tillers per plant (j), heading date (k), primary branches per panicle (l), panicle length (m), 1000-grains weight (n), seed setting rate (o), biomass weight per plant (p) and harvest index (q) between 02428 and 02428-GY3TQ. r, Relative expression level of GY3 in leaves at seedling stage. Data are means ± SE (n = 30 for e–q, n = 3 for r). P values were calculated by two-sided paired Student’s t-test.
Extended Data Fig. 2 Improvement of the yield performance of TQ and its related hybrid by GY302428.
a–d, Performances of whole plant (a), panicle (b), branch (c) and panicle architecture (d) of TQ, TQ-GY302428 and their hybrids with G46B at maturation stage, Bars, 5 cm. e, Genetic constitution of TQ-GY302428 identified by 6 K SNP array. f, Relative expression level of GY3 in TQ, TQ-GY302428 and their hybrids with G46B in leaves at seedling stage. g, Grain yield per plot of TQ and TQ-GY302428. Data are mean ± SE (n = 3 for f, n = 6 for g); P values were calculated by two-sided paired Student’s t-test.
Extended Data Fig. 3 Natural variations and complementation test of GY3.
a, Gene structure and sequence variations between 02428 and TQ alleles. b, Schematic representation of the complementing (CP) and over-expressing (OE) constructs of GY3. c–n, Comparisons of yield-related traits including grain yield per plant (c), secondary branches per panicle (d), spikelets per panicle (e), grains per panicle (f), tillers per plant (g), primary branches per panicle (h),1000-grains weight (i), panicle length (j), seed setting rate (k), biomass per plant (l), harvest index (m) and plant height (n) between 02428 and 02428-CP lines. For box plots in c–n, the box limits indicate the 25th and 75th percentiles, whiskers further extend by ±1.5 times the interquartile range from the limits of each box, and the center line indicates the median, individual data points are plotted (n = 29, 29, 23 and 21 for 02428, 02428-CP_1, 02428-CP_2 and 02428-CP_3, respectively). P values were calculated by two-sided paired Student’s t-test.
Extended Data Fig. 4 Performances of the over-expressor of GY3.
a–d, Comparisons of whole plant (a), panicle (b), primary branch (c) and panicle architecture (d) between 02428 and 02428-OE plant. Bars, 5 cm. (e) Expression level of GY3 in the leaves of 02428 and 02428-OE plants at seedling stage. Data are means ± SE (n = 4). f–q, Performances of tillers per plant (f), primary branches per panicle (g), secondary branches per panicle (h), panicle length (i), spikelets per panicle (j), grains per panicle (k) 1000-grains per plant (l), seed setting rate (m), grain yield per plant (n), biomass per plant (o), harvest index (p) and plant height (q) of 02428 and 02428-OE plants. For box plots in f–q, the box limits indicate the 25th and 75th percentiles, whiskers further extend by ±1.5 times the interquartile range from the limits of each box, and the center line indicates the median, individual data points are plotted (n = 29, 30, 32 and 29 for 02428, 02428-OE_1, 02428-OE_2 and 02428-OE_3, respectively). P values were calculated by two-sided paired Student’s t-test.
Extended Data Fig. 5 Performances of loss-of-function of GY3 in Zhonghua 11 (ZH11) background.
a–d, Comparisons of whole plant (a), panicle (b), and panicle architecture (c) between ZH11 and ZH11GY3-KO plant. Bars, 5 cm. d–h, Performance of spikelets per panicle (d), primary branches per panicle (e), secondary branches per panicle (f), tillers per plant (g), panicle length (h) between ZH11 (n = 12) and ZH11GY3-KO (n = 13) T0 plants with varied insertions or deletions resulted GY3 loss-of-function. Data are means ± SE. (i), The cytokinin contents in young leaves of ZH11 and ZH11GY3-KO plants. Data are means ± SE (n = 3). P values were calculated by two-sided paired Student’s t-test. n.d. represents not detected.
Extended Data Fig. 6 Cytokinin content in young panicles and enzyme-catalysed reactions.
a, b, Comparisons of cytokinin contents including DHZ (a) and DHZR (b) in young panicles of 02428, 02428-GY3TQ, 02428-CP, 02428-OE, and 02428LTR-KO. Data are means ± SE (three biological and three technical replicates). Different letters upon the bars indicated significant difference at P < 0.01 via Duncan test (a and b). c, Detection of the enzyme-catalysed reaction products of GY3 (red) and An-2 (blue) to iP and iPR, respectively. Std, standards of iPRMP, iP and iPR. d, Enzymatic product molar mass ratio of iP to iPR in An-2 (I), GY3 (IX) and mixtures with a molar mass ratio of An-2 to GY3 from 27:1 to 1:27 in 3-fold increments (from II to VIII with 27:1, 9:1, 3:1, 1:1, 1:3, 1:9, 1:27, respectively). e, Detection the enzyme-catalysed reaction products of newly recombinant enzymes (A-B-III, A-II-C, I-II-C, I-B-C, I-B-III). Std, standards of iPRMP, iP and iPR.
Extended Data Fig. 7 Expression profiles of GY3 in young panicle.
a, The expression patterns of GY3 in 02428 and 02428-GY3TQ plants. Data are means ± SE (n = 3). P values were calculated by two-sided paired Student’s t-test. b–g, RNA in situ hybridization of GY3 by anti-sense probes in inflorescence meristems at primary branch initiating stage (b and e), secondary branching formation stage (c and f) and floral meristem developing stage (d and g) between 02428 (b, c and d) and 02428-GY3TQ (e, f and g). (h), the signals of sense probe of GY3 in 02428 were used as the negative control. Bars, 20 μm.
Extended Data Fig. 8 Improvements of the yield performances of MH63 and its related hybrids by GY302428.
a–d, Performances of whole plant (a), panicle (b), branch (c) and panicle architecture (d) of MH63, MH63-GY302428 and their hybrids crossed with G46B at maturation stage. Bars, 5 cm. e, Genetic components of MH63-GY302428 identified by the 6 K SNP array. f, Relative expression level of GY3 in leaves at seedling stage. g, Comparison of grain yield per plot (each plot contained 40 plants) of MH63, MH63-GY302428 and their hybrids crossed with G46B. Data are mean ± SE (n = 3). P values were calculated by two-sided paired Student’s t-test.
Extended Data Fig. 9 Improvements of the yield performances of SH527 and its related hybrids by GY302428.
a–d, Performances of whole plant (a), panicle (b), branch (c) and panicle architecture (d) of SH527, SH527-GY302428 and their hybrids crossed with G46B at maturation stage. Bars, 5 cm. e, Genetic components of SH527-GY302428 identified by the 6 K SNP array. f, Relative expression level of GY3 in leaves at seedling stage. g, Comparisons of grain yield per plot (each plot contained 40 plants) of SH527, SH527-GY302428 and their hybrids crossed with G46B. Data are mean ± SE (n = 3); P values were calculated by two-sided paired Student’s t-test.
Extended Data Fig. 10 Structure analysis of An-2 and GY3.
Illustration of the substrate-binding cavities of An-2 (a) and GY3 (d) with docking its substrate iPRMP, the structure of An-2 and GY3 are predicted by AlphaFold. (b and e) show the hydrogen bond and its lengths between riboside of iPRMP and its interaction amino acid residues in An-2 (b) and GY3 (e). (c and f) show the hydrogen bond and its lengths between the phosphate of iPRMP and its interaction amino acid residues in An-2 (c) and GY3 (f). Residues involved in enzyme catalysis, AMP-binding residues show as red and green sticks, respectively. The yellow dash lines represent the hydrogen bond and the number adjacent to each bond represent hydrogen bond lengths. The red dash lines (e and f) represent lengths between atoms in GY3 catalysis residue for the corresponding hydrogen bond formation in An-2.
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Supplementary Data 2: Solo-LTR genotype list of 533 Oryza sativa accessions based PCR products. 3: Solo-LTR genotype list of AA genome Oryza species based on WGS. 4: Solo-LTR genotype list of African cultivated rice based on WGS. 5: Solo-LTR genotype list of 3,010 rice population based on WGS. 6: Solo-LTR genotype list of backbone lines for hybrids in China based on PCR products. 7: Primer used in this study.
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Wu, B., Meng, J., Liu, H. et al. Suppressing a phosphohydrolase of cytokinin nucleotide enhances grain yield in rice. Nat Genet 55, 1381–1389 (2023). https://doi.org/10.1038/s41588-023-01454-3
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DOI: https://doi.org/10.1038/s41588-023-01454-3
