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Study of vacuole glycerate transporter NPF8.4 reveals a new role of photorespiration in C/N balance

Nature Plants volume 9, pages 803–816 (2023)Cite this article

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Abstract

The photorespiratory intermediate glycerate is known to be shuttled between the peroxisome and chloroplast. Here, localization of NPF8.4 in the tonoplast, together with the reduced vacuolar glycerate content displayed by an npf8.4 mutant and the glycerate efflux activity detected in an oocyte expression system, identifies NPF8.4 as a tonoplast glycerate influx transporter. Our study shows that expression of NPF8.4 and most photorespiration-associated genes, as well as the photorespiration rate, is upregulated in response to short-term nitrogen (N) depletion. We report growth retardation and early senescence phenotypes for npf8.4 mutants specifically upon N depletion, suggesting that the NPF8.4-mediated regulatory pathway for sequestering the photorespiratory carbon intermediate glycerate in vacuoles is important to alleviate the impact of an increased C/N ratio under N deficiency. Thus, our study of NPF8.4 reveals a novel role for photorespiration in N flux to cope with short-term N depletion.

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Fig. 1: Induction of three NPF8.4 transcripts upon N depletion.
Fig. 2: NPF8.4b encoded by the major transcript of NPF8.4 localizes in the tonoplast.
Fig. 3: NPF8.4b is expressed in mesophyll cells near the midrib.
Fig. 4: npf8.4-1 and npf8.4-2 mutants, but not npf8.4-3, exhibit growth retardation and early senescence phenotypes under N depletion conditions.
Fig. 5: NPF8.4 is responsible for glycerate storage in the vacuole in response to N depletion.
Fig. 6: NPF8.4a and NPF8.4b exhibit glycerate efflux activity.
Fig. 7: The photorespiration pathway is stimulated in response to 2-day N depletion.
Fig. 8: Schematic model of how NPF8.4 sequesters the carbon intermediate glycerate into vacuoles when the photorespiration pathway is stimulated in response to short-term N depletion.

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Data availability

All data supporting the findings of this study are available within the article, Extended Data Figs. 1–8 and the Supplementary Information, or from the corresponding author upon reasonable request. The RNA-seq raw data are deposited in the NCBI Gene Expression Omnibus under GEO accession number GSE224214. TAIR 10 Genome (www.arabidopsis.org) and Araport11 (www.araport.org) were used as references to identify potential transcripts. Source data are provided with this paper.

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Acknowledgements

We thank S.-H. Lin in our lab for assistance with the 15N analysis; S.-P. Li and S.-M. Huang from our Confocal Microscope Facility for assistance with the acquisition of confocal microscopy images; S.-Y. Tung and C.-I. Yu from our Genomics Core facility and H.-N. Lin from our Bioinformatics Core facility for help with the RNA-seq experiment and analysis; and J. O’Brien for English editing. We thank C.-Y. Lin, C.-Y. Ting and T.-H. Chang from the Metabolomics Core Facility of the Agricultural Biotechnology Research Center for GC/MS and UPLC–MS/MS parameter optimization and the mass spectrometry analysis and the Plant Tech Core Facility in the Agricultural Biotechnology Research Center for generating the CRISPR constructs. This work was supported by grants from National Science and Technology Council (MOST 108-2311-B-001-006-MY3; MOST 111-2326-B-001-010-), an investigation award from Academia Sinica and a grant from the Institute of Molecular Biology, Academia Sinica, Taiwan.

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    Yi-Chen Lin & Yi-Fang Tsay

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Y.-C.L. and Y.-F.T. conceived and designed the project. Y.-C.L. performed the experiments and analysed the data. Y.-C.L. and Y.-F.T. wrote the manuscript.

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Correspondence to Yi-Fang Tsay.

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Extended data

Extended Data Fig. 1 Diurnal expression pattern of NPF8.4 under normal light or elevated light conditions.

Transcript levels of NPF8.4a (a), NPF8.4b (b), and NPF8.4c (c) were determined by RT-qPCR amplicons against the specific region marked in Fig. 1a. Plants were grown hydroponically with 5 mM KNO3 for 15 days and then shifted to N-depleted medium for 2 days under normal light (100 μmol m–2 s–1) or elevated light (350 μmol m–2 s–1), with a 16-h-light/8-h dark cycle. Relative transcript levels on the second day of N depletion were determined by RT-qPCR using ACT2 as an internal control. Values represent mean ± SD for three biological repeats, with each repeat comprising five plants. Different letters represent significant differences based on one-way ANOVA (p < 0.0001) with Tukey’s HSD post hoc test (p ≤ 0.05) in a-c. Two-way ANOVA results are also shown in the top right corner (*p = 0.0019; **p = 0.0001; ***p < 0.0001 in (a), ***p < 0.0001 in (b), and **p = 0.0001; ***p < 0.0001 in (c).

Extended Data Fig. 2 Genomic PCR and RT-PCR analyses of npf8.4-1.

a. Genomic PCR. b. RT-PCR. Total RNA was extracted from the shoots and NPF8.4 expression was analyzed using primers F and R (see Supplementary Table 4). UBQ10, internal RT-PCR control. Positions of the primers F, R, and LB are shown in Fig. 4a. Similar results were observed for three independent experiments. Uncropped scans of gels are provided as Source Data Fig. 1.

Extended Data Fig. 3 The growth phenotypes of 15-day-old wild-type and the npf8.4-1 mutant shifted to N depletion for the indicated number of days.

a, Photos of wild-type and npf8.4-1 mutant in response to N depletion. b. Shoot dry weight of Col-0 and the npf8.4-1 mutant in response to N depletion. Values represent mean ± SD for three biological repeats. Plants were grown hydroponically with 5 mM KNO3 for 15 days and then transferred to N-depleted medium for the number of days indicated. Different letters represent significant differences based on one-way ANOVA (p < 0.0001) with Tukey’s HSD post hoc test (p ≤ 0.05). Two-way ANOVA results are also shown in the top right corner. ‘***’, p < 0.0001. Similar results were obtained from three independent experiments.

Extended Data Fig. 4 Protein expression levels in X. laevis oocytes.

Protein levels of NPF8.4a and NPF8.4b in water-, NPF8.4a- and NPF8.4b-injected oocytes were analyzed by hybridizing to anti-NPF8.4 antibodies. Similar results were observed for three independent experiments.

Extended Data Fig. 5 Metabolomics and RNAseq analysis reveal increased photorespiration flux in response to 2-day N depletion.

For ease of comparison, the gene expression levels shown in boxes have been normalized to the same gene in wild-type at day 0 (representing 1). Metabolite content shown in circles is normalized to the same metabolite in wild-type at day 0 (representing 100). Transcriptome fold-change and magnitude of metabolite accumulation are illustrated according to the color key at bottom. Thick red lines indicate reactions upregulated in response to 2-day N depletion. Glycerate is highlighted in yellow, and NH4+ is highlighted in cyan. ‘*’ represents a significant difference between wild-type and mutants in the metabolomics analysis (p ≤ 0.05; Student’s t-test). Boxes with a thick frame reflect significant differences in gene expression or metabolite content before and after N depletion (p ≤ 0.05; Student’s t-test). Plant growth conditions are the same as for those detailed in Supplementary Table 2. Data represent the mean values of three biological replicates, with each replicate consisting of 15 plants.

Extended Data Fig. 6 The growth retardation phenotype of the npf8.4 mutants under N deficiency can be rescued by elevated CO2 conditions when the photorespiration pathway is attenuated.

a, c, Photos of 16-day-old plants in response to N deficiency (a) or N sufficiency (c) under ambient CO2 (400 ppm) or high CO2 (900 ppm). b, d, Shoot dry weight of Col-0 and npf8.4 mutants in response to N deficiency (b) or N sufficiency (d) under ambient CO2 (400 ppm) or high CO2 (900 ppm). Plants were grown hydroponically with 5 mM KNO3 for 8 days and then kept under the same condition (5 KN) or shifted to N depletion (-N) for a further 8 days under ambient CO2 (400 ppm) or high CO2 (900 ppm), respectively. Values represent mean ± SD for four biological repeats. Different letters represent significant differences based on one-way ANOVA (p < 0.0001 in (b); p = 0.0001 in (d)) with Tukey’s HSD post hoc test (p ≤ 0.05). Two-way ANOVA results are also shown in the top right corner. ‘***’, p < 0.0001; ‘ns’, no significance. Similar results were obtained from two independent experiments.

Extended Data Fig. 7 Scatterplot of the C/N atom ratio v.s. fold-change in amino acids in response to 2-day N depletion.

Fold-changes in amino acids after 2 days of N depletion (D2) were calculated by normalizing to the same amino acid at day 0 (D0, representing 100%). Statistical significance of the linear relationship between the C/N atom ratio and fold-change in amino acids was calculated using the Pearson correlation coefficient (r = 0.76, p = 0.007).

Extended Data Fig. 8 Homo-oligomerization of NPF8.4b in a yeast-based split ubiquitin system.

a, An X-Gal filter assay and b, quantitative β-galactosidase measurements were performed using NPF8.4b-Cub-PLV as bait and NubG, NubI, or NubG-NPF8.4b as prey. The N-terminal part of wild-type ubiquitin (NubI) or of a mutant (NubG) that does not interact with the C-terminal part of ubiquitin was used as the positive and negative control, respectively. The data in (b) represent the mean ± SD from three biological repeats. Similar results were observed for three independent experiments.

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Unprocessed gels of Extended Data Fig. 2.

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Lin, YC., Tsay, YF. Study of vacuole glycerate transporter NPF8.4 reveals a new role of photorespiration in C/N balance. Nat. Plants 9, 803–816 (2023). https://doi.org/10.1038/s41477-023-01392-2

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