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Visualizing plant intracellular inorganic orthophosphate distribution

Nature Plants volume 10, pages 315–326 (2024)Cite this article

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Abstract

Intracellular inorganic orthophosphate (Pi) distribution and homeostasis profoundly affect plant growth and development. However, its distribution patterns remain elusive owing to the lack of efficient cellular Pi imaging methods. Here we develop a rapid colorimetric Pi imaging method, inorganic orthophosphate staining assay (IOSA), that can semi-quantitatively image intracellular Pi with high resolution. We used IOSA to reveal the alteration of cellular Pi distribution caused by Pi starvation or mutations that alter Pi homeostasis in two model plants, rice and Arabidopsis, and found that xylem parenchyma cells and basal node sieve tube element cells play a critical role in Pi homeostasis in rice. We also used IOSA to screen for mutants altered in cellular Pi homeostasis. From this, we have identified a novel cellular Pi distribution regulator, HPA1/PHO1;1, specifically expressed in the companion and xylem parenchyma cells regulating phloem Pi translocation from the leaf tip to the leaf base in rice. Taken together, IOSA provides a powerful method for visualizing cellular Pi distribution and facilitates the analysis of Pi signalling and homeostasis from the level of the cell to the whole plant.

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Fig. 1: Development of the IOSA method for Pi visualization at the organ level in plants.
Fig. 2: Cellular Pi distribution patterns in rice and Arabidopsis.
Fig. 3: Pi master regulators control cellular Pi distribution in plants.
Fig. 4: Screening and cloning of novel cellular Pi distribution regulators of leaf using the IOSA method.

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Acknowledgements

This work was funded by grants from the National Natural Science Foundation of China (32130096, 32222078, 32272810 and 31972493) and the National Key Research and Development Program of China (number 2021YFF1000400). K.Y. and W.R. were supported by the Innovation Program of the Chinese Academy of Agricultural Sciences.

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  1. These authors contributed equally: Meina Guo, Wenyuan Ruan, Ruili Li, Lei Xu.

Authors and Affiliations

  1. State Key Laboratory of Efficient Utilization of Arid and Semi-arid Arable Land in Northern China/Key Laboratory of Plant Nutrition and Fertilizer, Ministry of Agriculture, Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing, China

    Meina Guo, Wenyuan Ruan, Ruili Li, Lei Xu, Qianqian Zhang, Jianhao Ren & Keke Yi

  2. State Key Laboratory of Efficient Production of Forest Resources/ National Engineering Research Center of Tree Breeding and Ecological Restoration, College of Biological Sciences and Technology, Beijing Forestry University, Beijing, People’s Republic of China

    Meina Guo

  3. EBMP (Environnement, Bioénergies, Microalgues et Plantes), Aix Marseille Univ, CEA, CNRS, UMR7265, BIAM, Saint-Paul lez Durance, France

    Sahar Hani, Pascale David & Laurent Nussaume

  4. State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University, Hangzhou, China

    Bingsong Zheng

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Contributions

W.R., L.N. and K.Y. designed this research. M.G., W.R., R.L. and S.H. performed most of the described experiments. Q.Z., M.G. and S.H. measured the Pi concentration. W.R., M.G., R.L., S.H., J.R., L.X., P.D., B.Z., L.N. and K.Y. analysed the data. W.R., M.G., L.N. and K.Y. wrote the paper. All authors reviewed the paper.

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Correspondence to Wenyuan Ruan or Keke Yi.

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Extended data

Extended Data Fig. 1 IOSA detection buffer (IDB) is specific to the detection of Pi.

a, Reaction analysis of IOSA detection buffer with P-contained molecules. The phosphate (Pi, KH2PO4, 0.05 mM), phosphite (Phi, KH2PO3, 5 mM), adenosine triphosphate (ATP, 5 mM), deoxyribonucleic acid (DNA, 1 μg/ml), ribonucleic acid (RNA, 1 μg/ml), bovine serum albumin (BSA, 1 mg/ml), phytic acid (IP6, 5 mM), ployphosphate (ploy-Pi, 1 μg/ml), hydrolyzed ployphosphate (PloyP-hyd), and the extraction of rice root grown under +P ( + PE) and –P (–PE) conditions, were used to react with IOSA detection buffer (IDB) for 20 min. b, Absorbance measurement of the reaction solution in (a) at the wavelength of 860 nm. Values represent the means ± SD of four replicates. Different letters above the bars indicate significant differences between groups. Statistics, one-way ANOVA with post-hoc Tukey’s test (P < 0.05). nd, indicates no signal was detected. c. Linear range analysis of reaction between IDB and different Pi concentrations.

Source data

Extended Data Fig. 2 IDB can be used directly for Pi staining in plants.

The germinated wild type (Nip) seeds were directly sown on +P (200 μM Pi) cultural solution for 7 days before the Pi-staining assay. Total roots were directly stained with IOSA detecting buffer (+IDB) under vacuum conditions for 20 min. The reaction buffers of H2O (without IDB: –IDB), +AM (only plus ammonium molybdate), and IDB(–AM) (IDB buffer without ammonium molybdate) were used as negative control. Scale bar, 1 cm.

Source data

Extended Data Fig. 3 Pi staining assay in Arabidopsis roots grown under different Pi culture conditions.

The 10-d-old Arabidopsis primary roots grown under different Pi-containing medium (0, 50, 100, 250, 500 μM Pi) were used to perform IOSA. Different root regions were imaged. Scale bar, 1 mm. Three times each experiment was repeated independently with similar results.

Source data

Extended Data Fig. 4 Pi staining assay in Arabidopsis roots grown under 0 and 50 μM Pi conditions.

a, Phenotypic performance of 14-d-old Arabidopsis grown under 0 and 50 μM Pi conditions. Scale bar, 1 cm. b, Pi measurement of Arabidopsis roots in (a). c, Pi-staining assay of Arabidopsis roots in (a). Values represent the means ± SD of three independent bulking samples. Statistics, Student t-test, two-sided. Scale bar, 1 mm. Three times each experiment was repeated independently with similar results.

Extended Data Fig. 5 Pi-staining assay of young lateral root tip grown under long term Pi-deficient stress.

The germinated wild type (Nip) seeds were directly sown on –P (0 μM Pi) cultural solution for 15 days before Pi-staining assay. The whole roots were stained directly with IOSA detection buffer. The emerged young lateral roots were photographed by microscope. Bar = 100 μm. Three times each experiment was repeated independently with similar results.

Extended Data Fig. 6 Development of the IOSA method to visualize Pi at the cellular level in plants.

a, Pi staining steps of plant samples. b-c, The optimization of section thickness for roots and leaves. *, indicates the broken mesophyll cells. d, Optimization of Pi staining steps for leaves and roots. The numerical order indicates the Pi staining assay steps in (a). e, IOSA for the samples by free hand sectioning. f, IOSA procedures for cellular Pi-distribution analysis in plant tissues. Scale bar, 50 μm. Three times each experiment was repeated independently with similar results.

Extended Data Fig. 7 Comparison of the distribution of total P determined by LA-ICP-MS and Pi stained by IOSA in rice.

The images in the right panel were copied from the paper of Yamaji and Ma (2019, the Plant Journal). Their leaf and root P distributions were analyzed by LA-ICP-MS technique. Left panel images were performed by IOSA. Three times each experiment was repeated independently with similar results.

Extended Data Fig. 8 Cellular Pi distribution assay in node side longitudinal section.

a, Node I was longitudinally sectioned from the position of L1. b, Different parts of the longitudinal section image were zoomed into b1-b9 areas, respectively. The different cell types were indicated: evb, enlarged vascular bundle; dvb, diffusion vascular bundle; vbsc, vascular bundle sheath cells; tra, tracheids; pc, parenchyma cells; ave, annular vessels. Bar = 50 μm. Three times each experiment was repeated independently with similar results.

Extended Data Fig. 9 Cellular Pi distribution assay in node longitudinal section at center position.

a, Node I was sectioned longitudinally from the position of L1. The slides were stained with IDB. b, Different parts of the longitudinal section image were zoomed into b1-b12 areas, respectively. c, Cellular Pi-distribution in the nodal vascular anastomosis (nva). Different cell types were indicated: ph, phloem; xy, xylem; cc, companion cells; stec, sieve tube element cells; bar = 50 μm. Three times each experiment was repeated independently with similar results.

Extended Data Fig. 10 IOSA can discriminate difference in the Pi distribution in wild-type, Os-pho2, OsPHR2-OE-1, OsPHF1-OE-1, Os-pt8-1, Os-phf1-1, and Os-phr1 phr2 plants.

a, Phenotypic performance of 21-d-old wild-type (NIP), Os-pho2, OsPHR2-OE-1, OsPHF1-OE-1, Os-pt8-1, Os-phf1-1, and Os-phr1phr2 plants grown under 200 μM Pi cultural solution. Bar = 10 cm. b, Pi-concentration measurement of plants in (a). Third leaves were used for Pi measurement. Values represent the means ± SD of four independent plants. Different letters above the bars indicate significant differences between groups. Statistics, one-way ANOVA with post-hoc Tukey’s test (P < 0.05). c, Pi-staining assay of the third leaves of plants in (a). The middle area of the third leaf was sectioned transversely to 40 μm and stained by IOSA method. d, Pi-distribution assay of vascular bundle cells in plants of wild-type (NIP), Os-pho2, OsPHR2-OE-1, OsPHF1-OE-1, OsPT8-OE-1, Os-pt8-1, Os-phr1phr2, and Os-phf1-1 in rice. The third leaf middle area of 14-d-old plants were transversely sectioned and stained by IOSA method. Different cell types are indicated: msc, mestome sheath cell; cc, companion cells; mx, metaxylem; px, protoxylem; stec, sieve tube element cells; xpc, xylem parenchyma cells; vbsc, vascular bundle sheath cell; fi, fibers; mec, mesophyll cells. Scale bar, 50 μm. Four times each experiment was repeated independently with similar results.

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Guo, M., Ruan, W., Li, R. et al. Visualizing plant intracellular inorganic orthophosphate distribution. Nat. Plants 10, 315–326 (2024). https://doi.org/10.1038/s41477-023-01612-9

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