Abstract
Phosphate (Pi) is essential to plant growth and crop yield. However, it remains unknown how Pi homeostasis is maintained during cereal grain filling. Here, we identified a rice grain-filling-controlling PHO1-type Pi transporter, OsPHO1;2, through map-based cloning. Pi efflux activity and its localization to the plasma membrane of seed tissues implicated a specific role for OsPHO1;2 in Pi reallocation during grain filling. Indeed, Pi over-accumulated in developing seeds of the Ospho1;2 mutant, which inhibited the activity of ADP-glucose pyrophosphorylase (AGPase), important for starch synthesis, and the grain-filling defect was alleviated by overexpression of AGPase in Ospho1;2-mutant plants. A conserved function was recognized for the maize transporter ZmPHO1;2. Importantly, ectopic overexpression of OsPHO1;2 enhanced grain yield, especially under low-Pi conditions. Collectively, we discovered a mechanism underlying Pi transport, grain filling and P-use efficiency, providing an efficient strategy for improving grain yield with minimal P-fertilizer input in cereals.
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Data availability
Entire RNA-seq datasets were deposited in the NCBI Gene Expression Omnibus under accession number GSE149101. Source data are provided with this paper.
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Acknowledgements
We thank J. F. Ma for useful discussions, G. Xu for providing the yeast mutant line, expression vector and advice about Pi application, J. Wan for providing OsAGPL2- and OsAGPS2b-specific antibodies, W. Zhang for maize transformation, Q. Shu for rice materials, J. Gong and D. Chao for assistance with P measurements, X. Gao and Z. Zhang for sample preparation and electron microscopic observations. This work was supported by the National Key Research and Development Program of China (2016YFD0100600), the Chinese Academy of Sciences (XDB27040201) and the National Key Laboratory of Plant Molecular Genetics. This work was also supported, in part, by a grant from the National Science Foundation (MCB-1714795 to S.L.) and the National Natural Science Foundation of China (31871217).
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Z.H., S.L. and W.T. conceived and designed the research. B.M., L.Z., Q.G., J.W., X.L., H.W., Y.L., J.L., X.W., Q.L. and Y.D. performed experiments. B.M., L.Z., Q.G., J.W. and Z.H. analyzed data and oversaw the entire study. B.M., L.Z., Q.G., W.T., S.L. and Z.H. wrote the manuscript.
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Extended data
Extended Data Fig. 1 Map-based cloning of Ospho1;2 for grain incomplete filling phenotype.
Primary mapping region a and fine mapping b of the locus to a ~5 kb region of the NIP genome with 3600 individuals from NILs population, denoted by double black headed arrowheads. This fine mapping region includes only one gene LOC_Os02g56510 that encodes OsPHO1;2. Eight recombinants delimiting the mapping region for detailed progeny analysis is presented by bars. Grey bars refer to the heterozygous allele, white and black bars refer to NIL-OsPHO1;2 and NIL-Ospho1;2 homozygous alleles, respectively. Phenotypic statistics of 100-grain weight from F3 segregation of the 8 recombinants were shown on the right. Data are means ±s.d. (n = 8 plants). *** indicates significant difference at P < 0.001 according to two-tailed Student’s t-test. 1-30-1: P = 2.88 × 10−15; 1-177-3: P = 7.10 × 10−15; 2-125-6: P = 3.31 × 10−18.
Extended Data Fig. 2 Agronomic traits of nearly isogenic lines of OsPHO1;2 and Ospho1;2.
(a, b) Morphology of mature plants a and panicles b of NIL-OsPHO1;2 and NIL-Ospho1;2. Scale bars, 20 cm a and 5 cm b, respectively. (c-i) Comparison of grain thickness (c), 1,000-grain weight (d), plant height (e), grain number per panicle (f), seed setting rate (g), tiller number (h) and grain yield per plant (i) between NIL-OsPHO1;2 and NIL-Ospho1;2. (j) Lesions and lesion length of the representative NIL plants inoculated with Xanthomonas oryzae pv. oryzae (Xoo) (strain PXO99A) for 2 weeks, indicating that Ospho1;2 mutant plants significantly increased Xoo resistance. Scale bars, 2 cm. n = 12 plants for c-g, n = 16 plants for h, n = 24 plants for i and j, P-values were indicated according to two-tailed Student’s t-test (c-j). For the box-and-whisker plots, the central line, box and whiskers indicate the median, IQR and 1.5 times the IQR, respectively (c-j).
Extended Data Fig. 3 Mutant sites and agronomic traits of CRISPR-Cas9 knockout alleles.
a, Gene structure and mutation sites in OsPHO1;2, including nucleotide substitutions and deletions. b, Different mutant alleles (Ospho1;2-ko1 to Ospho1;2-ko8) generated by CRISPR-Cas9 in Nip background, and the mutant target was indicated in (a) with blue star. (c-i) Trait comparison of grain thickness (c), 1,000-grain weight (d), grain yield per plant (e), plant height (f), grain number per panicle (g), seed setting rate (h) and tiller number (i) between wild type and various mutant alleles. n = 12 plants for c-j, P-values were indicated according to two-tailed Student’s t-test. For the box-and-whisker plots, the central line, box and whiskers indicate the median, IQR and 1.5 times the IQR, respectively (c-i).
Extended Data Fig. 4 Tissue specific expression pattern of OsPHO1;2.
a, mRNA levels of OsPHO1;2 in different tissues by qRT-PCR, normalized to the rice Actin gene. IM, inflorescence meristem; YP, young panicle; hull, and filling seeds. Values are means ± s.d. (n = 3 technical replicates). b, Western blot detection of OsPHO1;2 in various tissues using anti-OsPHO1;2, and Actin was immunodetected as a loading control. c, qRT-PCR analysis of relative mRNA levels of OsPHO1;2 in different developing seed tissues, including embryo, endosperm, pericarp and nucellar epidermis, normalized to the rice Actin gene. Data are means ± s.d. (n = 3 technical replicates). d, Western blot analysis of OsPHO1;2 in developing seed tissues using anti-OsPHO1;2, and anti-Actin was immunodetected as a loading control. e, Tissue specificity of OsPHO1;2 expression in early-milking seeds. Immunostaining with an anti-GUS antibody was performed in milky grains (5 DAF) of transgenic lines with pOsPHO1;2-GUS fusion reporter driven by the native promoter. The image is a representative of three independent lines. Scale bars, 200 μm. f, Blue box area in (c) was magnified. Scale bars, 50 μm. The white arrowheads indicate the nucellar epidermis (NE, e, f). Independent experiments were repeated at least 2 times with similar results (a-f).
Extended Data Fig. 5 Immunoelectron microscopy analysis shows that OsPHO1;2 localizes at the plasma membrane.
(a-c) Immunoelectron microscopy analysis in the node vascular bundles cells from NIP plants. Samples were immunodetected with the antibody of OsPHO1;2 (b, c) or 1% BSA (a, control). (c) was magnified from blue box area in (b). Scale bars, 500 nm. (d-f) Immunoelectron microscopy analysis in seed nucellar epidermis cells from wild type seeds, and samples were immunodetected with the antibody of OsPHO1;2 (e, f) or 1% BSA (d, control). Scale bars, 200 nm. Black arrowheads denote the immunogold particles in plasma membranes (PM), and positions of cell wall (CW) and cytosol (Cy) are also marked. Images are representatives of at least three independent cells.
Extended Data Fig. 6 Transporter activity assay of OsPHO1s under different pH in Xenopus oocytes.
a, Average current-voltage curves for phosphate conductance by OsPHO1;2 and Ospho1;2 in Xenopus oocytes. b, A detailed exhibition of the current signals at +60 mV and -180 mV from (a). Similar analyses were conducted for OsPHO1;1 (c and d) and OsPHO1;3 (e and f). The exact Xenopus oocytes number (n) was indicated in each graph. Error bars depict means ± s.e.m (a, c, e). Significant difference was analyzed according to two-tailed Student’s t-test (a-f). P-values were calculated through the comparison with Mock pH 5.5. * P < 0.05, ** P < 0.01, *** P < 0.001. OsPHO1;2 pH 5.5 at +60 mV: P = 1.60 × 10−7; OsPHO1;2 pH 6.5 at +60 mV: P = 2.52 × 10−4; OsPHO1;2 pH 7.5 at +60 mV: P = 1.96 × 10−5; OsPHO1;2 pH 5.5 at -180 mV: P = 6.65 × 10−6; OsPHO1;2 pH 6.5 at -180 mV: P = 0.012; OsPHO1;2 pH 7.5 at -180 mV: P = 4.23 × 10−3; OsPHO1;3 pH 5.5 at -180 mV: P = 1.32 × 10−8; OsPHO1;3 pH 6.5 at -180 mV: P = 2.35 × 10−7; OsPHO1;3 pH 7.5 at -180 mV: P = 1.40 × 10−9. For the box-and-whisker plots, the central line, box and whiskers represent the median, IQR and 1.5 times the IQR, respectively (b, d, f).
Extended Data Fig. 7 OsPHO1;2 functions in Pi transfer from root to shoot and reallocation.
(a, b) Pi contents were measured in root (a) and shoot (b) of seedlings (n = 4 plants). Germinating seeds were grown for a week in the presence of 1 mM Pi, and then were transferred to Pi-deficient media for the desired period (0-21 d). After 21 days of Pi starvation, plants were transferred to Pi-sufficient conditions for 3 days (21 + 3 d). Control plants were grown for 25 d in the presence of 1 mM Pi. c, Pairwise comparison of inorganic phosphate (Pi) distribution in different tissues, brown rice, husk, rachis, node I, stem I and flag leaf in NILs and WT/Ospho1;2-ko1 (n = 6 plants). For the box-and-whisker plots, the central line, box and whiskers represent the median, IQR and 1.5 times the IQR, respectively (a–c). d, Pi distribution in the panicle and leaf tissues of NILs and WT/Ospho1;2-ko1. Values are means ± s.d. (n = 3 plants). e, Measurement of Pi contents in different parts of developing seeds (15 DAF). Tissues of pericarp, endosperm and nucellar epidermis were pairwise compared in NIL-OsPHO1;2 and NIL-Ospho1;2. Data are means ± s.d. (n = 3 biologically independent samples). P-values were indicated according to two-tailed Student’s t-test (a-e).
Extended Data Fig. 8 Pi accumulation inhibits AGPase production and activity.
a, b, Relative mRNA levels of OsAGPL2 (a) and OsAGPS2b (b) in suspension cells of NILs under various Pi treatments. Values are means ± s.d. (n = 3 biologically independent samples). P = 1.56 × 10−8 (a) and P = 1.10 × 10−5 (b) by one-way ANOVA. Different letters denote significant differences (P < 0.05) from Tukey’s HSD test. c, Measurement of AGPase activity in the WT and Ospho1;2-ko1 grains from 5 DAF to 30 DAF, values are means ± s.d. (n = 3 biologically independent samples). 5 DAF: P = 2.72 × 10−3; 7 DAF: P = 1.41 × 10−4; 10 DAF: P = 7.56 × 10−4; 15 DAF: P = 2.33 × 10−4; 20 DAF: P = 0.4068; 25 DAF: P = 0.0617; 30 DAF: P = 0.1132; **P < 0.01, ***P < 0.001, ns. not significant, by two-tailed Student’s t-test.
Extended Data Fig. 9 Evolutionary analysis of OsPHO1s and its homologs.
a, Phylogenic analysis of PHO1 family proteins in rice, Arabidopsis and homologs of OsPHO1;2 in other cereal crops including Zea mays, Triticum aestivum, Sorghum bicolor and Setaria italica. The tree was built by Mega 5.0. Scale bars, 0.05. b, Mutation sites of Zmpho1;2a (top) and Zmpho1;2b (bottom) generated by CRISPR-Cas9.
Extended Data Fig. 10 Ectopic overexpression of OsPHO1;2 significantly enhances grain yield and Pi reallocation.
a, Western blot analysis of OsPHO1;2 protein levels in grains (15 DAF) of the ectopic overexpression lines and WT. Actin was detected as a loading control. Independent experiments were repeated at least 2 times with similar results. b, c, Plant morphology (b) and seeds phenotype (c) of WT and OsPHO1;2-OE lines. Scale bars, 20 cm (b) and 2 cm (c). (d–f) Comparison of 1,000-grain weight (d), grain length (e) and seed setting rate (f) between WT and OsPHO1;2-OE lines (n = 28 plants). (g) Inorganic phosphate (Pi) distribution in different tissues (n = 6 plants). Pi was measured in husk, rachis, node I, stem I and flag leaf in WT and OsPHO1;2-OE lines (left), and Pi contents in pericarp, endosperm and nucellar epidermis of developing seeds (15 DAF) were also detected (right). P = 3.42 × 10−4 (d), P = 0.170 (e), P = 0.476 (f), for P-values in (g): P = 0.481 (Husk), P = 0.011 (Rachis), P = 2.07 × 10−4 (Flag leaf), P = 0.803 (Stem I), P = 0.010 (Node I), P = 2.40 × 10−4 (Endosperm), P = 0.150 (Nucellar Epidermis), P = 0.167 (Pericarp), by one-way ANOVA. The different letters denote significant differences (P < 0.05) from Tukey’s HSD test. For the box-and-whisker plots, the central line, box and whiskers represent the median, IQR and 1.5 times the IQR, respectively (d–g).
Supplementary information
Supplementary Information
Supplementary Notes 1 and 2 and Figs. 1–11
Supplementary Tables
Supplementary Tables 1–4. Primers used in this paper; recombinant constructs and plasmids used in this paper; antibodies used in this paper; strains used in this paper.
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Unprocessed western blots for Fig. 2b.
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Unprocessed western blots for Fig. 4b,e.
Source Data Fig. 5
Unprocessed western blots for Fig. 5g.
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Unprocessed western blots for Fig. 6c.
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Unprocessed western blots for Extended Data Fig. 4b,d.
Source Data Extended Data Fig. 10
Unprocessed western blots for Extended Data Fig. 10a.
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Ma, B., Zhang, L., Gao, Q. et al. A plasma membrane transporter coordinates phosphate reallocation and grain filling in cereals. Nat Genet 53, 906–915 (2021). https://doi.org/10.1038/s41588-021-00855-6
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DOI: https://doi.org/10.1038/s41588-021-00855-6
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