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ARTICLE

Plasma membrane H+-ATPase overexpressio

increases rice yield via simultaneous enhancement
of nutrient uptake and photosynthesis
Maoxing Zhang1,2,13, Yin Wang2,3,13, Xi Chen4,13, Feiyun Xu1,13, Ming Ding1, Wenxiu Ye 2,5, Yuya Kawai6,

Yosuke Toda 2,7, Yuki Hayashi6, Takamasa Suzuki 8, Houqing Zeng9, Liang Xiao1, Xin Xiao10, Jin Xu11,

Shiwei Guo1, Feng Yan12, Qirong Shen 1, Guohua Xu 1, Toshinori Kinoshita 2,6✉ & Yiyong Zhu 1✉

Nitrogen (N) and carbon (C) are essential elements for plant growth and crop yield. Thus,

improved N and C utilisation contributes to agricultural productivity and reduces the need for

fertilisation. In the present study, we find that overexpression of a single rice gene, Oryz

sativa plasma membrane (PM) H+-ATPase 1 (OSA1), facilitates ammonium absorption an

assimilation in roots and enhanced light-induced stomatal opening with higher photosynth-

esis rate in leaves. As a result, OSA1 overexpression in rice plants causes a 33% increase in

grain yield and a 46% increase in N use efficiency overall. As PM H+-ATPase is highly

conserved in plants, these findings indicate that the manipulation of PM H+-ATPase could

cooperatively improve N and C utilisation, potentially providing a vital tool for food security

and sustainable agriculture.

https://doi.org/10.1038/s41467-021-20964-4 OP

1 Jiangsu Collaborative Innovation Center for Solid Organic Waste Resource Utilization, College of Resources and Environment Sciences, Nanjing Agricultura

University, Nanjing, China. 2 Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Nagoya, Japan. 3 Institute of Ecology, College o

Urban and Environmental Sciences and Key Laboratory for Earth Surface Processes of Ministry of Education, Peking University, Beijing, China. 4 College of Lif

Sciences, Nanjing Agricultural University, Nanjing, China. 5 School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, China. 6 Graduate
School of Science, Nagoya University, Nagoya, Japan. 7 Japan Science and Technology Agency (JST), PRESTO, Kawaguchi, Japan. 8 Department of Biological
Chemistry, College of Bioscience and Biotechnology, Chubu University, Kasugai, Aichi, Japan. 9 College of Life and Environmental Sciences, Hangzhou Normal
University, Hangzhou, China. 10 College of Resources and Environment, Anhui Science and Technology University, Fengyang, China. 11 College of Horticulture,
Shanxi Agricultural University, Taigu, China. 12 Institute of Agronomy and Plant Breeding, Justus Liebig University, Giessen, Germany. 13These authors
contributed equally: Maoxing Zhang, Yin Wang, Xi Chen, Feiyun Xu. ✉email: kinoshita@bio.nagoya-u.ac.jp; yiyong1973@njau.edu.cn

NATURE COMMUNICATIONS | (2021) 12:735 | https://doi.org/10.1038/s41467-021-20964-4 | www.nature.com/naturecommunications

6
7
8
9

(

)

:,;

http://crossmark.crossref.org/dialog/?doi=10.1038/s41467-021-20964-4&domain=pdf

http://orcid.org/0000-0003-3464-507X

http://orcid.org/0000-0003-2421-4743

http://orcid.org/0000-0002-1977-05

http://orcid.org/0000-0002-1977-0510

http://orcid.org/0000-0002-5662-96

http://orcid.org/0000-0002-5662-9620

http://orcid.org/0000-0002-3283-2392

http://orcid.org/0000-0001-7621-1259

http://orcid.org/0000-0003-2695-8199

mailto:kinoshita@bio.nagoya-u.ac.jp

mailto:yiyong1973@njau.edu.cn

www.nature.com/naturecommunications

N
itrogen (N) and carbon (C) are indispensable elements for
plant growth and are required in large quantities for crop
production1. Crops largely obtain N from the soil as

NH4+

and/or NO3− and C from the atmosphere as CO2. Syn-
thetic N fertilisers are also applied in large amounts, with annual
rates of >120 million tons worldwide2. Crops have a limited
ability to utilise N3; thus, excess N is continuously lost fro

agricultural systems, which pollutes the environment4. In addi-
tion, the productivity of C3 plants such as rice and wheat is
limited by inefficient CO2 fixation by RuBisCO during photo-
synthesis, due to low CO2 concentrations within the mesophyll
cells of leaves. Plant biomass and crop production can be
improved by the enhancement of intercellular CO2 concentration,
which creates an effect similar to CO2 fertilisation5, but also emits
excess CO2 into the atmosphere6. Thus, it is critically important
to determine how best to enhance N and CO2 uptake by plants to
improve crop production and environmental performance.

Plasma membrane (PM) H+-ATPase, a subfamily of P-type
ATPases, generates a membrane potential and H+ gradient across
the PM, energising multiple ion channels and various H+-cou-
pled transporters for diverse physiological processes7,8. In pre-
vious studies, we demonstrated that the PM H+-ATPase mediates
light-induced stomatal opening9,10. Overexpression of the PM
H+-ATPase in guard cells significantly enhances stomatal open-
ing, photosynthesis and, subsequently, growth in Arabidopsis
thaliana, a model plant11. It remains unknown if this manip-
ulation would be efficient in crops, such as rice, which is the
staple food for three billion people worldwide12.

Unlike most terrestrial plants, paddy rice grows in flooded soils
where ammonium (NH4+) ions constitute the dominant N source
for root uptake13. To use NH4+ as an N source, rice roots require
efficient uptake ability and corresponding assimilation capacity for
NH4+. Conversely, high tissue accumulation of unassimilated
NH4+ is usually negatively correlated with plant growth14,15. The
assimilation of NH4+ in root cells requires a C skeleton as the
substrate for the synthesis of amino acids through the glutamine
synthetase (GS)/glutamate synthase (GOGAT) cycle. The assim-
ilation of one molecule of NH4+ generates two molecules of H+ in
the cytoplasm16. PM H+-ATPase facilitates the transport of var-
ious nutrients, such as nitrate, phosphate and potassium (K+)17,18,
and maintains cytosolic H+ homeostasis by pumping H+ outside
the cells19. In our previous study, NH4+ nutrition was found to
induce upregulation of PM H+-ATPase activity in rice roots20.
Recently, we determined that enhanced PM H+-ATPase activity
in rice roots ensures rice growth at high NH4+ concentrations21.
Therefore, we hypothesised that PM H+-ATPase may be involved
in NH4+ metabolism in rice plants.

In this study, we examined the involvement of PM H+-ATPase
in NH4+ uptake by rice roots and stomatal opening for CO2
uptake and photosynthesis in rice leaves, with the aim of devel-
oping a new strategy to improve rice yield and N use efficiency
(NUE) via the overexpression of a single gene, Oryza sativa PM
H+-ATPase 1 (OSA1).

Results
PM H+-ATPase mediates NH4+ absorption. We first investi-
gated the relationship between PM H+-ATPase and NH4+ uptake
by rice roots. We treated rice roots with the fungal toxin fusi-
coccin (FC), a stimulator for PM H+-ATPase activity22, and
found that the rate of 15NH4+ absorption increased by 17% in
darkness and by 11% under illumination, compared to the cor-
responding controls (mock) (Fig. 1a). These results clearly indi-
cate that PM H+-ATPase is involved in NH4+ uptake in rice
roots. Under illumination, we also observed an additional
increase in the 15NH4+ absorption rate (Fig. 1a) and induction of

leaf stomatal opening for transpiration (Fig. 1b). These results
suggest that enhanced transpiration in leaves also contributes to
NH4+ uptake by roots. Therefore, we inferred that the over-
expression of PM H+-ATPase in rice roots and/or stomatal guard
cells would efficiently improve NH4+ absorption.

Phenotype of OSA1 overexpression and mutation rice lines. To
evaluate the effects of PM H+-ATPase on NH4+ and CO2 uptake
in rice, we focused on a typical PM H+-ATPase isoform, OSA1,
and investigated the phenotypes of OSA1-overexpressing lines
(OSA1-oxs, driven by the CaMV-35S promoter23, OSA1#1 to
OSA1#3) (Fig. 2a), and osa1 knockout mutants (osa1-1 to

osa1-3

,
TOS17 insertional mutants) (Fig. 3a and Supplementary Fig. 1).
Compared to wild-type (WT) plants, OSA1 expression was
7.4–8.6-fold higher in roots and 3.5–5.3-fold higher in leaves of
OSA1-oxs (Fig. 2c), without affecting the expression levels of
other PM H+-ATPase isoforms (Supplementary Table 1). OSA1-
ox plants exhibited ~40% higher H+-ATPase protein levels and
~30% higher PM H+-ATPase activity than did WT plants
(Fig. 2d, e), whereas these values were reduced in osa1 mutants
(Fig. 3c–e). We confirmed higher H+ extrusion from roots in
OSA1-oxs (Supplementary Fig. 2) and proper localisation of
overexpressed PM H+-ATPase in roots (Supplementary Fig. 3).
When grown under hydroponic conditions, 4-week-old OSA1-ox
lines exhibited enhanced plant growth, with 18–33% greater
dry weight compared to the WT (Fig. 2a, b). By contrast, osa1
mutants exhibited 33–52% lower dry weight compared to
WT plants (Fig. 3a, b). These results indicate that OSA1 (PM
H+-ATPase) is a key factor regulating growth in rice. We
observed no significant phenotype changes related to growth,
relative OSA1 gene expression, PM H+-ATPase protein levels,
stomatal opening (stomatal conductance) and photosynthesis rate
in the empty vector-transformed rice under hydroponic condi-
tions (Supplementary Fig. 4).

Overexpression of PM H+-ATPase enhanced NH4+ uptake. To
understand the effects of PM H+-ATPase overexpression on N
uptake, we compared the isotopic 15N (15NH4+) absorption rate
between WT and OSA1-oxs (or osa1 mutants), and determined
the absorption rate of 15NH4+ within 5 min by roots. 15

NH4+

concentrations ranging from 0.5 to 8 mM were used to test
15NH4+ uptake via different NH4+ transport systems in rice
roots. Interestingly, the 15NH4+ absorption rate in all OSA1-oxs
was significantly higher than that in the WT, under both low (≤1
mM) and high (≥1 mM) NH4+ concentration conditions invol-
ving the high- and low-affinity transport systems, respectively24

(Fig. 2f). By contrast, all osa1 mutants exhibited lower 15NH4+

absorption rates under all NH4+ concentration conditions
(Fig. 3f). We also examined the 15NH4+ absorption rate within
30 min under 2 mM 15NH4+. The rate of 15NH4+ absorption was
20–30% higher in OSA1-oxs than in the WT, but was markedly
lower in osa1 mutants (Supplementary Fig. 5). In all rice lines,
15NH4+ absorption rates were significantly repressed by treat-
ment with 0.35 µM vanadate, an inhibitor for PM H+-ATPase
(Supplementary Fig. 5). These results confirmed that PM
H+-ATPase modification in rice roots regulated NH4+ absorp-
tion. Consequently, under laboratory hydroponic conditions,
total N accumulation was found to be 16–57% higher in OSA1-
oxs (Fig. 2g) but lower in osa1 mutants (Fig. 3g) compared to the
WT. In addition, the contents of other nutrients such as K, P, Ca,
S, Fe, and Zn were also increased in OSA1-oxs and decreased in
osa1 mutants compared to the WT (Supplementary Fig. 6a–c, f, j).
Interestingly, total C accumulation was 21–47% higher in OSA1-
oxs but lower in osa1 mutants compared to the WT (Figs. 2h and
3h). Because C is not taken up by plant roots, these results suggest

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that OSA1 modification influenced CO2 uptake and/or fixation in
rice leaves.

PM H+-ATPase overexpression enhanced stomatal con-
ductance and photosynthetic activity. Stomata are crucial for gas
exchange, particularly for CO2 diffusion into the leaf12. Light, the
most effective environmental signal for stomatal opening, then
activates PM H+-ATPase10,11,25–28. PM H+-ATPase-induced
hyperpolarisation in the PM of guard cells enables K+ uptake
through inward-rectifying K+ channels. The accumulation of K+

and its counter ions in guard cells prompts guard-cell swellin

and stomatal opening29. Therefore, we investigated stomatal
phenotypes in OSA1-oxs. Representative closed and open stomata
in a WT rice leaf are shown in Fig. 4a. In darkness, the level of
stomatal closure in OSA1-oxs was similar to that in the WT,
whereas under light, the ratio of open to closed stomata was
significantly higher in OSA1-oxs (Fig. 4b). Conversely, in osa1
mutants, the ratio of open to closed stomata was significantly
lower than in the WT under light treatment (Supplementary
Fig. 7a). In all rice lines, stomatal opening was suppressed by the
plant hormone abscisic acid (ABA) (Fig. 4b and Supplementary
Fig. 7a), suggesting that ABA action was unaffected in guard cells
of both OSA1-ox and osa1 mutant plants. Stomatal density, size,
and shape in OSA1-oxs and osa1 mutants were comparable to
those of the WT (Supplementary Fig. 8), suggesting that over-
expression or mutation of PM H+-ATPase in rice had no effect
on stomatal morphology or development; these results were
similar to our observations in Arabidopsis thaliana12.

Given that stomatal aperture is a limiting factor for
photosynthesis12,30, we examined the photosynthetic properties
of OSA1-ox plants. Under saturated white light (WL) conditions,
stomatal conductance in OSA1-oxs was almost double that in the
WT (Fig. 4c and

Supplementary Table 2

), and photosyntheti

rates in OSA1-oxs were 26–28% higher than in the WT (Fig. 4d
and Supplementary Table 2), indicating that enhanced light-
induced stomatal opening in OSA1-oxs conferred higher photo-
synthesis rates. By contrast, osa1 mutants exhibited 22–37% lower
stomatal conductance and 27–35% lower photosynthetic rates
(Supplementary Fig. 7b, c). Next, we examined photosynthetic
light response curves in detail. Along with increased stomatal

conductance (Fig. 4e), the photosynthetic rates of OSA1-ox plants
were 15–34% higher than those of the WT (Fig. 4f), particularly
under high-intensity light (500–1500 µmol m−2 s−1). Photosyn-
thetic CO2 response curves (A–Ci curves) were also higher for
OSA1-oxs than for the WT (Fig. 4g), indicating a higher
photosynthetic capacity among OSA1-ox plants. The water use
efficiency of OSA1-oxs was 13–21% lower than that of the

(Supplementary Table 2).

Genome-wide effect of OSA1 on gene expression. To identify
differentially expressed genes (DEGs) and associated pathways
that may provide a molecular basis for the described OSA1-ox
and osa1 mutant phenotypes, we analysed the comprehensive
gene expression profiles in the leaves and roots of 4-week-old
WT, OSA1-ox (OSA1#2) and osa1-2 mutant plants using RNA-
sequencing (RNA-seq) analysis. Among the DEGs, 1373 and 1124
transcripts were upregulated in the leaves and roots of the OSA1-
ox line, and 347 and 3295 transcripts were downregulated in the
leaves and roots of the osa1-2 mutant, respectively (Fig. 5a). By
contrast, 1895 and 1304 transcripts were downregulated in the
leaves and roots of the OSA1-ox line, and 1859 and 2913 tran-
scripts were upregulated in the leaves and roots of the

osa1-2

mutant, respectively (Supplementary Fig. 9a–c). Consistent wit

OSA1 expression levels, we detected 59 and 82 genes in the leaves
and roots, respectively, that were upregulated in the OSA1-ox line
but downregulated in the osa1-2 mutant (Fig. 5a).

We then performed Gene Ontology (GO) term enrichment
analysis of the DEGs upregulated in the OSA1-ox line and
downregulated in the osa1-2 mutant to investigate the molecular
mechanisms underlying OSA1-mediated biological processes
(Supplementary Fig. 9d, e and Supplementary Data 1). The
results indicated that 12 biological processes were significantly
enriched, including photosynthesis, NH4+ assimilation, gluta-
mate biosynthesis, amino acid metabolism, carbohydrate trans-
membrane transport, various ion transport, and N utilisation
(Supplementary Fig. 9d, e and Supplementary Data 1). Genes
associated with transmembrane transporter activity, ion trans-
port, substrate-specific transmembrane transporter activity,
cation transmembrane transporter activity, carbohydrate trans-
membrane transporter activity, and PM part were also

Fig. 1 Plasma membrane (PM) H+-ATPase regulates ammonium (NH4+) uptake in rice. a 15NH4+ absorption rate in wild-type (WT) rice. To determine
15NH4+ absorption rates, rice seedlings were incubated in 2 mM 15NH4+ solution with 5 μM fusicoccin (FC) for 30 min under dark or illuminated
conditions. b Average transpiration rates of rice leaves under dark and illuminated conditions over 30 min. Small circles in a, b represent data points for
individual experiments; three biological replicates were analysed for each treatment. Columns and error bars in a, b represent the means ± standard errors
(SEs; n = 3). Differences were evaluated using the two-tailed Student’s t test (

P < 0.05; ***P < 0.005; n.s., not significant). The exact P values are 0.0018 (mock in dark vs. FC in dark), 0.0025 (mock in dark vs. mock in light), 0.0143 (mock in light vs. FC in light) and 0.0016 (FC in dark vs. FC in light) for (a); 0.8194 (mock in dark vs. FC in dark), 0.0006 (mock in dark vs. mock in light), 0.0851 (mock in light vs. FC in light), and 4.27 × 10−5 (FC in dark vs. FC in light) for (b). n.s. Not significant.

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significantly enriched in overlapping genes that were upregulated
in OSA1-ox roots and leaves, but downregulated in the osa1-2
mutant (Supplementary Data 2). In addition, we compared leaf
and root transcriptomes between the WT, OSA1-ox line, and
osa1-2 mutant, and found that genes associated with nucleic acid
binding transcription factor activity, response to chitin, response
to organonitrogen compound, regulation of N compound
metabolic process, regulation of nucleobase-containing com-
pound metabolic process, and RNA biosynthetic process were
significantly enriched in the overlapping genes downregulated in
OSA1-ox leaves and upregulated in osa1 mutant leaves (false
discovery rate [FDR] < 0.05) (Supplementary Data 3). However, no GO terms were found to be significantly enriched in overlapping genes downregulated in OSA1-ox roots and upregu- lated in osa1-2 mutant roots (Supplementary Data 3).

A set of genes were enriched in “membrane transport”
category. Six NH4+ transporter genes were enriched in the
membrane transport category: AMT3;3, AMT3;1, AMT2;3,
AMT2;1, AMT1;2 and AMT1.1 (Fig. 5b). These genes encode
both high- and low-affinity NH4+ transporters and were
significantly upregulated in the OSA1-ox line and downregulated

in the osa1 mutant in both leaves and roots (Fig. 5b). Transporter
genes encoding other cation transporters (e.g. HAK1, CAX1a and
CAT1), electroneutral substance transporters (e.g. PIP1;3) or
anion transporters (e.g. PT8) were also affected by the modifica-
tion of OSA1 (Fig. 5b). These results suggest a potential role for
OSA1 in modulating ion and solute transport in plants.

Genes involved in NH4+ assimilation such as GS (GS2 and
GS1;2) and glutamate synthase (NADH-GOGAT2, Fd-GOGAT2
and NADH-GOGAT1) were also strongly affected by the OSA1
modification (Fig. 5b). Genes associated with photosynthesis were
induced by OSA1 overexpression and repressed by OSA1
knockout in leaves; these included Psb28, PsbQ, PsaH, PFPA2,
PsbR1, GLO4 and RbcS (Fig. 5b).

We examined the expression levels of NH4+-responsive genes,
including AMT1;1, GS1;2, NADH-GOGAT1,

NADH-GOGAT2

and GS2 using quantitative reverse-transcription polymerase
chain reaction (PCR) (Fig. 5d–h). The expression of all
investigated genes increased significantly in the OSA1-ox lines.
Notably, GRF4, a key transcription factor in N metabolism and C
fixation in rice31, was highly expressed in response to OSA1
overexpression (Fig. 5c).

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Fig. 2 OSA1 overexpression promotes nitrogen (N) and carbon (C) uptake in rice. a Phenotypes of 4-week-old WT and OSA1-overexpressing (ox) plants.
b Dry weights of WT and OSA1-ox plants. c Relative OSA1 expression levels in WT and OSA1-ox plants. d Relative PM H+-ATPase protein levels in WT and
OSA1-ox plants. e Hydrolytic activity of PM H+-ATPase in WT and OSA1-ox plants. f 15NH4+ absorption rates in the roots of WT and OSA1-ox plants under
different NH4+ concentrations. To determine 15NH4+ absorption rates, seedlings were incubated with 0.5–8 mM 15NH4+ for 5 min to reflect the net uptake
rate. g, h Total N and C levels in WT and OSA1-ox plants. Plants were grown hydroponically in a greenhouse for 4 weeks. Small circles in b–h represent data
points for individual experiments; three biological replicates were analysed for each treatment. Values in b–h are presented as the means ± SEs (n = 3).
Differences were evaluated using the two-tailed Student’s t test (*P < 0.05; **P < 0.01). The exact P values are provided in the Source Data file.

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Overexpression of PM H+-ATPase promoted field production.
To verify the effects of OSA1 overexpression on rice yield under
field conditions, we conducted trials over two growing seasons at
three different locations in the middle of China (Nanjing-S in
2016, and Nanjing-N and Fengyang in 2017). We applied urea as
an N fertiliser at four different levels: 0 kg ha−1 (no N [N–N]),
100 kg ha−1 (low N [L–N]), 200 kg ha−1 (moderate or normal N
[M–N]) and 300 kg ha−1 (high N [H–N]). Rice seedlings were
planted at a spacing of 25 cm between rows and 20 cm between
hills, with a total area of 26 m2 per transect. Stomatal conductance
and photosynthetic rates during the vegetative stage exhibited
similar trends in the field and laboratory (Supplementary Fig. 10).
Representative plants at the reproductive stage under M–N
conditions at Nanjing-N are shown in Fig. 6a, b. At all three
locations, grain yield of the OSA1-ox lines was 27–39% (mean,
33%) higher than that of the WT (Fig. 6e and Supplementary
Tables 3–5). Conversely, in osa1 mutants, grain yield was sig-
nificantly lower than that of the WT at all three locations (Sup-
plementary Tables 3–5). In OSA1-oxs, the higher yield was
correlated with higher panicle weight (18–42%) (Fig. 6f), which

was attributed to increased numbers of panicles per hill (15–20%)
(Fig. 6c, g) and spikelets per panicle (8–16%) (Fig. 6d, h). Plant
height, panicle length, filled grain rate and 1000-grain weight
were nearly identical between OSA1-ox, osa1 mutant and WT
plants (Supplementary Tables 3–5). Similar patterns were
observed across fertilisation levels (Fig. 6j, Supplementary Fig. 11
and Supplementary Tables 3–5). Notably, under N–N conditions,
grain yield was 12–20% higher in OSA1-oxs than in the WT at all
test locations (Fig. 6j and Supplementary Tables 3–5). The NUE
of OSA1-oxs was ~46% higher than that of the WT at all N
fertilisation levels (Fig. 6i). Even when treated with only half the
amount of N fertiliser (L–N, 100 kg ha−1), the grain yield of the
OSA1-ox lines was significantly higher than that of the WT grown
under M–N conditions (200 kg ha−1) (Fig. 6j). Thus, the same
grain yield was attained using only half the amount of N fertiliser
when the WT was replaced with OSA1-oxs.

To further verify the practical outcome of OSA1 overexpression
in rice, we conducted an independent field trial in Hainan,
southern China, which has a tropical climate and short-day
conditions, and therefore produces lower yield than the

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Fig. 3 osa1 mutant plants exhibit lower N uptake and decreased C content. a Phenotypes of WT and osa1 plants (scale = 10 cm). b Root and shoot dry
weights of WT plants and osa1 mutants. c Relative expression levels of OSA1 in the roots and leaves of WT plants and osa1 mutants. d Relative PM H+-
ATPase protein levels in the roots and leaves of WT plants and osa1 mutants. e ATP hydrolytic activity of PM H+-ATPase in WT plants and osa1 mutants.
f 15NH4+ absorption rates in the roots of WT plants and osa1 mutants under different NH4+ concentrations. The 15NH4+ absorption rate was determined
after seedlings were incubated with 0.5–8 mM 15NH4+ for 5 min. g, h Total N and C levels in roots and leaves of WT plants and osa1 mutants. Plants were
grown hydroponically in a greenhouse for 4 weeks; small circles in b–h represent data points for individual experiments; three biological replicates
were analysed for each treatment. Values in b–h are presented as the means ± SEs (n = 3). Differences were evaluated using the two-tailed Student’s t test
(*P < 0.05; **P < 0.01). The exact P values are provided in the Source Data file.

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subtropical areas of central China. Under these conditions, the
OSA1-ox lines also produced significantly higher grain yield than
the WT (Supplementary Table 6).

Discussion
Increasing crop yield by improving NUE and C fixation is
important for sustainable agriculture and environment perfor-
mance. In this study, we demonstrated the critical role of the PM
H+-ATPase gene OSA1 in controlling both NUE and photo-
synthesis in paddy rice production. Overexpression of OSA1 in
rice plants increased the activity of PM H+-ATPase (Fig. 2e),
promoted NH4+ uptake and assimilation in roots (Fig. 2f, g) and
enhanced light-induced stomatal opening and stomatal con-
ductance and photosynthetic rate under saturated WL in leaves
(Fig. 4b–d and Supplementary Table 2), leading to higher NUE
and grain yield (Fig. 6). Our results demonstrate the cooperative

enhancement of NH4+ metabolism, photosynthesis rate and
grain yield through the expression modulation of a single PM
H+-ATPase gene in rice plants.

PM H+-ATPase was found to regulate NH4+ uptake in rice
(Fig. 1 and Supplementary Fig. 5). Furthermore, genetic evidence
based on OSA1 overexpression/knockout showed that OSA1
modulation regulated the rate of NH4+ absorption by rice roots
across a wide range of rhizosphere NH4+ concentrations (Figs. 2f
and 3f). RNA-seq analyses revealed the upregulation of at least six
NH4+ transporter genes (AMT3;3, AMT3;1, AMT2;3, AMT2;1,
AMT1;2 and AMT1.1) that encode both high- and low-affinity
NH4+ transporters in OSA1-overexpressing lines (OSA1-oxs),
and the downregulation of these genes in the osa1 mutant (Fig. 5).
These results indicate that there is a close relationship between
PM H+-ATPase and NH4+ transporters in rice root cells. These
coordinated expression pattern of different genes is also the
fundamental mechanisms that enable OSA1-oxs rice roots to

Fig. 4 Stomatal and photosynthetic properties of OSA1-ox plants. a Representative stomata in the epidermis of WT plants. Experiments were repeated
three occasions with similar results. b Percentage of open stomata observed after 3 h of darkness (DK), red light plus blue light (RL + BL) or RL + BL in the
presence of 20 μΜ abscisic acid (ABA) in WT and OSA1-ox plants (for the details see the “Methods” section). c, d Stomatal conductance (c) and CO2
assimilation rate under (d) DK (30 min), white light (WL; 2 h) and a second DK treatment (30 min) in WT and OSA1-ox plants. e, f Stomatal conductance
(e) and CO2 assimilation rate (f) in response to light in WT and OSA1-ox plants. g Relationship between CO2 assimilation rate and intercellular CO2
concentration in WT and OSA1-ox plants. Small circles in b–d represent data points for individual experiments; three biological replicates were analysed for
each treatment. Values in b–d are presented as the means ± SEs (n = 3) and those in e–g are the means ± SDs (n = 3). Differences were evaluated using
the two-tailed Student’s t test (*P < 0.05; **P < 0.01). The exact P values are provided in the Source Data file.

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efficiently take up NH4+ in the field soils with frequently fluc-
tuated NH4+ concentration. Our results also indicate that OSA1
overexpression may enhance NH4+ assimilation capacity. Genes
responsible for NH4+ assimilation such as glutamine synthetase
(GS1;2 and GS2) and glutamate synthase (NADH-GOGAT1,
NADH-GOGAT2 and Fd-GOGAT) were upregulated in OSA1-oxs
(Fig. 5b, e–h). Because NH4+ uptake and assimilation are closely

synchronised in plant roots32, enhanced GS and

GOGAT

activity can transfer root-absorbed NH4+ to amino acids for the
synthesis of various N-containing compounds during plant
growth and development, which in turn prevent NH4+ over-
loading in the root cytoplasm due to the acceleration of NH4+

uptake in OSA1-oxs (Fig. 2f). However, the process of NH4+

assimilation also generates H+, which is toxic if excessively

Up-regulated genes
from OSA1-ox roots

Up-regulated genes
from OSA1-ox leaves

Down-regulated genes
from osa1-2 leaves

Down-regulated genes
from osa1-2 roots

NADH-GOGAT2

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Fig. 5 Differentially expressed genes (DEGs) enrichment caused by the modification of OSA1 in rice. a Venn diagram representing overlapping
upregulated genes in OSA1-ox and downregulated genes in the osa1-2 mutant (osa1) in roots and leaves (false discovery rate [FDR] < 0.05). b Heat map of the DEGs. Significant genes in response to N, C metabolism and ion transport are listed (FDR < 0.05). c–h Relative expression of N metabolism-related genes in WT and OSA1-ox roots (c–f) and leaves (c, g, h). Plants were grown hydroponically in a greenhouse for 4 weeks. Small circles in c–h represent data points for individual experiments; three biological replicates were analysed for each treatment. Columns and error bars in c–h represent the means ± SEs (n = 3). Differences were evaluated using the two-tailed Student’s t test (*P < 0.05; **P < 0.01). The exact P values are provided in the Source Data file.

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accumulated in the cytoplasm33,34. Notably, the overexpression of
only NH4+ transporter genes (AMT1;1 and AMT1;3)35,36 or
glutamine synthetase (GS1;1 and GS1;2)37 alone led to higher
NH4+ uptake or assimilation rates, but caused poor growth
and yields in paddy rice. Considering the important role of
PM H+-ATPase in maintaining intracellular pH, enhanced PM

H+-ATPase activity through OSA1 overexpression (Fig. 2c–e)
pumped excessive H+ out of root cells during NH4+ assimilation
(Supplementary Fig. 12). Through this feedback, OSA1-ox
rice absorbs and uses NH4+ more efficiently than do WT
plants (Figs. 2f–g and 3f–g), which is important for NUE
improvement.

WT OSA1#1 OSA1#2 OSA1#3

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We also observed that OSA1 is involved in C fixation through
the regulation of stomatal opening (Supplementary Fig. 12).
Stomatal conductance and photosynthetic rates were enhanced in
OSA1-oxs due to increased stomatal aperture opening (Fig. 3),
compared with rates in the WT and osa1 mutants (Supplemen-
tary Fig. 7). This result is consistent with the finding that genes
related to photosynthesis were upregulated by OSA1 over-
expression and downregulated by OSA1 knockout (Fig. 5b), for
example, Psb28, PsbQ, PsaH, PFPA2, PsbR1, GLO4 and RbcS38–40.
Enhanced photosynthesis in OSA1-oxs might also have con-
tributed to the uptake and assimilation of NH4+ in rice roots by
providing more C skeletons and energy for NH4+ metabolism
processes41. Therefore, N acquisition and photosynthetic activity
are intrinsically linked through overall N and C status in rice
plants42, resulting in a globally coordinated increase in C and N
accumulation in rice plants through OSA1 overexpression (Sup-
plementary Fig. 13). Together, these results show that OSA1
overexpression promotes both C and N uptake and assimilation,
which further regulate the expression of genes involved in C and
N metabolism and contribute to plant growth and grain yield.

PM H+-ATPase is also involved in the uptake of various
nutrients from plant roots by providing proton motive
force17,18,43. In this study, the stronger acidification in OSA1-ox
rice roots (Supplementary Fig. 2) could provide higher proton
motive force in the rhizosphere for the uptake of nutrients. This is
consistent with the enhanced NH4+ uptake rate in OSA1-oxs as
compared with WT plants (Fig. 2f–g and Supplementary Fig. 6),
and also consistent with upregulating the expression of various
nutrient transporter genes, such as ammonium transporters
AMT1;1/1;2/2;1/3;1/3;3, phosphate transporter PT1/PT8/PHO1.1
and potassium transporter HAK1 in roots of OSA1-oxs (Fig. 5b).
These results coincided with the increased contents of N, P and K
in OSA1-oxs as compared with WT plants (Fig. 2g and Supple-
mentary Fig. 6). Recently, GRF4, a transcription factor in rice,
was reported to integrate N assimilation, C fixation and plant
growth; multiple N metabolism genes, such as AMT1;1, GS1;2,
GS2 and NADH-GOGAT2, are positively regulated by GRF431.
Here, GRF4 was found to be upregulated by OSA1 overexpression
(Fig. 5b, c). It is possible that some master regulators of nutrient
uptake and metabolism, such as GRF4, could be activated by
OSA1 overexpression. The enhanced C fixation and N metabo-
lism could also have a feedback on the expression of nutrient
transporter genes in order to ensure sufficient supply of nutrients
for the promotion of the plant growth. Further study is deserved
to investigate the underlying molecular mechanisms responsible
for the signal transduction initiated by OSA1 overexpression.

In contrast to CO2, which is taken up from the atmosphere, N
is derived from fertilisers for most non-legume crops. Thus,
cultivars with improved NUE are in urgent demand for the
sustainable development of agriculture. The green revolution has
boosted crop yields; however, the resulting cereal varieties are
associated with reduced NUE44. Even precision crop manage-
ment has led to only a slight improvement in NUE3. In this study,

OSA1-ox rice exhibited both higher yield and higher NUE
than the WT under a wide range of N fertilisation rates, from 0 to
300 kg N ha−1 (Fig. 6i, j and Supplementary Tables 3–5). Higher
NUE in OSA1-ox rice leads to a lower demand for N fertilisers to
produce similar yields of rice grain. To obtain the same yield as
WT rice, OSA1-ox rice requires only half the amount of N fer-
tiliser (Fig. 6j). This benefit will drastically reduce the cost of rice
production as well as the environmental load produced by excess
N accumulation due to rice production.

Given that the molecular mechanisms of nutrient uptake17,43

and light-induced stomatal opening27 are conserved in most plant
species, this manipulation strategy could be applicable to many
valuable crops. Therefore, we suggest designating plants over-
expressing PM H+-ATPase as promotion and upregulation of
plasma membrane proton-ATPase (PUMP) plants. If PM
H+-ATPase overexpression can be realised using non-transgenic
methods such as genome editing, these crops could have great
potential for commercial use, conferring greater yields and
potentially critical environmental benefits.

Methods
Plant cultivation. Seeds of WT rice (Oryza sativa L. ssp. japonica cv. Nipponbare),
overexpression lines OSA1#1, OSA1#2 and OSA1#3 and mutant lines

osa1-1

(TOS17 line ND3017), osa1-2 (TOS17 line ND3025), osa1-3 (TOS17 line ND3033)
and CaMV-35S empty vector were surface sterilised in 10% (v:v) H2O2 for 30 min
and preincubated in aerated 0.5 mM CaSO4 solution. After 2 days, all seeds were
germinated on plastic support nets (mesh size, 2 mm2) floating on 1 mM CaSO4
solution for ~1 week, followed by application of IRRI (International Rice Research
Institute) nutrient solution (2 mM NH4Cl, 0.5 mM K2SO4, 0.3 mM KH2PO4, 1 mM
CaCl2, 1 mM MgSO4, 0.5 mM Na2SiO3·9H2O, 9 μM MnCl2, 0.39 μM Na2MoO4,
20 μM H3BO4, 0.77 μM ZnSO4, 0.32 μM CuSO4, 20 μM EDTA-Fe) at pH 5.5. For
the gas-exchange experiment, seedlings of the WT, OSA1-overexpressing lines and
osa1 mutants were grown in ½ IRRI nutrient solution for 1 week, followed by 5
more weeks of growth in modified IRRI nutrient solution (pH 5.5) containing
2 mM NH4Cl. The solution in the containers was replaced every 3 days. Plants for
most of the laboratory experiments were grown in a greenhouse at 30 °C/24 °C
(day/night) and 60–80% relative humidity. Plants for stomatal aperture and gas-
exchange measurements (Fig. 4 and Supplementary Figs. 4 and 7) were grown in a
growth chamber (NC-410HC, Nippon Medical & Chemical Instruments Co.,
Ltd, Osaka, Japan) under ~150 μmol m−2 s−1 fluorescent light at 30 °C/24 °C
(12 h/12 h) and 60–80% relative humidity. The rice seeds for these experiments
were of the same age.

For field experiments, plants were grown in the summer of 2016 and 2017 at
four well-controlled biological experimental stations in Hainan in 2016 (N18°67′,
E108°76′), southern Nanjing in 2016 (N32°01′, E118°51′), northern Nanjing in
2017 (N32°11′, E118°46′) and Fengyang in 2017 (N32°52′, E117°33′). Hainan is in a
tropical monsoon zone with sandy soil, whereas the experimental sites in Nanjing
and Fengyang are in a subtropical monsoon climate zone with yellow-brown soil.
For each field experiment (Fig. 6, Supplementary Fig. 11 and Supplementary
Tables 3–6), four levels of N (urea) fertiliser were applied: 0, 100, 200 and 300 kg N
ha−1 (N–N, L–N, M–N and H–N). Seeds were germinated and seedlings were
grown in a greenhouse for ~1 month at the beginning of May. The rice seedlings
were hand-transplanted in a flooded field with regular hill spacing. Each
fertilisation treatment was performed in one plot (6.5 m × 4 m). Rice seedlings were
planted in 14 rows with 20 hills per row, for a spacing of 25 and 20 cm, respectively.
Each OSA1-ox, osa1 mutant and WT line was planted in three rows (excluding
border hills). At the edge of each plot, the same rice line of inside neighbour was
also planted as the border hills (red box) to avoid the margin effects on the rice
growth inside the plot. Each plot contained 480 hills, for a total of 1920 hills. Each
field experiment consisted of four plots with different N fertilisation levels. Prior to

Fig. 6 Overexpression of OSA1 increases grain yield and N use efficiency (NUE) in the field. a–d Photographs of 100-day-old WT and OSA1-ox plants
in the field (a), in pots in the field (b) and harvested panicles (c) and spikelets (d) in 2017 in northern Nanjing under 200 kg N ha−1 (M–N) fertilisation.
e Grain yield, f panicle weight per plant, g panicles per hill and h spikelets per panicle of WT and OSA1-ox plants in field tests at three locations
(n ≥ 6). i Relative agronomic NUE in WT and OSA1-ox plants in field tests under low (L–N; 100 kg N ha−1), moderate (M–N; 200 kg N ha−1) or high (H–N;
300 kg N ha−1) levels of N fertilisation. Columns and error bars represent the means ± SEs (n = 3). j Grain yield of WT and OSA1-ox plants in field tests
under different N conditions. Black asterisks represent significant differences between WT and OSA1-ox plants under the same N fertilisation level; small
circles in e–h, j represent data points of collected samples in individual experiments (n = 6 in 2017 Nanjing-N and n = 8 in 2016 Nanjing-S and 2017
Fengyang). Centre line indicates the median, upper and lower bounds represent the 75th and the 25th percentile, respectively. Whiskers indicate the
minimum and the maximum in the box plots (e–h, j). Differences were evaluated using the two-tailed Student’s t test (*P < 0.05; **P < 0.01; n.s., not significant). The exact P values are provided in the Source Data file.

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seedling transplantation, the paddy field was fertilised with 80 kg P ha−1 as Ca
(H2PO4)2 and 110 kg K ha−1 as K2SO4. The first N fertilisation was carried out at
2 days before transplantation using 33.3% of the total amount of N fertiliser, which
was mixed into the soil. At the tillering stage (~1 week after transplanting), the
second N fertilisation (33.3% of the total N) was carried out. The final N
application (33.3% of the total N) was conducted 4 weeks later. The plant growth
period (transplantation to harvest) differed among rice lines and N levels, as
follows. At 0 or 100 kg N ha−1, the growth period was 109 ± 3 days for the WT and
osa1 mutant and 102 ± 2 days for the overexpression lines; at 200 or 300 kg N ha−1,
the growth period was 119 ± 2 days for the WT and osa1 mutant and 112 ± 2 days
for the overexpression lines.

Grain yield was determined at harvest in October. At maturity, 6–8 hills of
plants from each rice line were randomly selected at the centre of the plot from
among a 22 × 18 array of hills (excluding the border hills) and harvested. Yield and
its components were determined45,46 with minor modifications. The samples were
divided into grain and straw for nutrient content analysis. Agronomic NUE was
defined as the yield increase per kg N fertiliser in the field experiment. Relative
agronomic NUE (Fig. 6i) was calculated as the ratio to WT rice under L–N
treatment in each field trial. Statistical analyses were performed using two-tailed
Student’s t tests and one-way analysis of variance followed by Tukey’s test.

Construction of the overexpression vector and transgenic plants. The open-
reading frame of OSA1 was amplified using gene-specific primers (Supplementary
Table 7). The fragment was treated with restriction enzymes, inserted into vectors
and sequenced before transformation. Embryonic rice (O. sativa) calli were
transformed via Agrobacterium-mediated transformation47. Three independent
homozygous T2 or T3 lines (OSA1#1, OSA1#2 and OSA1#3) were used for all
phenotypic analyses.

Quantitative reverse-transcription PCR. Total RNA was isolated from the roots
of WT and transgenic plants using TRIzol reagent according to the manufacturer’s
instructions48 (Invitrogen Life Technologies, Carlsbad, CA, USA). Quantitative
PCR was performed using an SYBR Premix Ex Taq II (Perfect Real Time) Kit
(TaKaRa Biotechnology, Dalian, China) on a Step One Plus Real-Time PCR System
(Applied Biosystems, Bio-Rad, CA, USA), and the data were analysed using the
2−ΔΔCT method. The OsActin and OsGAPDH genes were used as internal refer-
ences to normalise the test gene expression data. All analyses were repeated at
least three times. PCR primer sets for gene amplification are listed in Supple-
mentary Table 7.

Immunodetection. Leaf and root samples were harvested separately. The samples
were immediately homogenised in liquid N and then in ice-cold homogenisation buffer
with a mortar and pestle49. The membrane proteins were collected by centrifugation
and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and
immunoblot analysis; PM H+-ATPase was detected using anti-H+-ATPase antibody50.
Actin was used as an internal control protein and was detected using anti-actin anti-
bodies (1:3000 dilution, Sigma-Aldrich, St. Louis, MO, USA, Cat#057M4548). Relative
PM H+-ATPase levels (Figs. 2d and 3d and Supplementary Fig. 4c) were estimated
from the ratio of the signal intensity of PM H+-ATPase to that of actin from the same
sample. WT and OSA1-ox seedlings were grown in IRRI nutrient solution containing
2 mM NH4+ for 3 weeks in a growth chamber. Immunohistochemical detection of PM
H+-ATPase was performed9 (Supplementary Fig. 3). Roots of 3-week-old seedlings
were harvested separately and placed in fixation buffer (4% paraformaldehyde, 60 mM
sucrose and 50 mM cacodylic acid; pH 7.4) for 2 h at room temperature. Fixed samples
were washed five times with phosphate-buffered saline (PBS) and embedded in 5% agar
dissolved in PBS. Sections of 100-mm thickness were prepared using a vibratome
(ZERO1; Dosaka EM, Kyoto, Japan) and placed on a glass slide. Samples were treated
with enzyme solution (0.1% pectolyase and 0.3% Triton X-100 in PBS) for 2 h, washed
five times with PBS, washed once with blocking solution (5% bovine serum albumin)
for 10 min and incubated with primary antibody (anti-H+-ATPase) diluted 1000-fold
in PBS overnight. On the second day, samples were washed five times with PBS, washed
in blocking solution for 10 min and incubated with secondary antibody (Alexa 546,
diluted 1000-fold in PBS) for 2 h. Finally, the samples were observed under confocal
laser scanning microscopy (FV-10i; Olympus, Tokyo, Japan).

Measurement of PM H+-ATPase activity. We determined ATP hydrolytic
activity of PM H+-ATPase51. Root and leaf tissues were ground in ice-cold
homogenisation buffer to isolate the PM in a two-phase partitioning method51. PM
H+-ATPase hydrolysis activity was determined as the difference between assay
results with and without the addition of 0.1 mM Na3VO4 to the reaction solution
(Figs. 2e and 3e). The assay was performed in 0.5 mL of reaction solution con-
taining 30 mM BTP/MES, 5 mM MgSO4, 50 mM KCl, 50 mM KNO3, 1 mM
Na2MoO4, 1 mM NaN3, 0.02% (w/v) Brij 58, and 5 mM disodium-ATP (substrate
for PM H+ ATPase). The reaction was initiated by adding 30 µL of a membrane
vesicle suspension containing 1–2 µg total protein and proceeded for 30 min at
30 °C; thus, inorganic phosphate was liberated after the hydrolysis of ATP. The
reaction was stopped by adding 1 mL reagent (2% [v/v] concentrated H2SO4, 5%
[w/v] sodium dodecyl sulfate and 0.7% [w/v] (NH4)2MoO4), followed by 50 µL 10%
(w/v) ascorbic acid. After 10 min, 1.45 mL arsenite-citrate reagent (2% [w/v]

sodium citrate, 2% [w/v] sodium arsenite and 2% [w/v] glacial acetic acid) was
added52. Colour development was completed after 30 min and measured spectro-
photometrically at 720 nm. In each test, H+-ATPase activity was calculated as the
amount of phosphate liberated within 30 min mg−1 membrane protein in excess of
the boiled-membrane protein control.

15N absorption rates in roots of WT, OSA1-overexpressing and osa1 plants.
WT, OSA1-ox and osa1 mutant seedlings were grown in IRRI nutrient solution
containing 2 mM NH4+ for 4 weeks in a growth chamber (NC-410HC, Nippon
Medical & Chemical Instruments Co., Ltd.) under ~150 μmol m−2 s−1 fluorescent
light at 30 °C/24 °C (12 h/12 h) and 60–80% relative humidity. To determine
15NH4+ absorption rates within 30 min, seedlings were rinsed in 0.1 mM CaSO4
for 1 min, transferred to modified IRRI nutrient solution containing 2 mM
(15NH4)2SO4 (atom% 15N: 98%) incubated with mock 5 μM FC (Fig. 1a) or 350 μM
vanadate (Supplementary Fig. 5) for 30 min53 and rinsed again with 0.1 mM CaSO4
for 1 min48. To determine 15NH4+ absorption rates within 5 min in roots of WT,
OSA1-ox and osa1 mutant plants under different NH4+ concentrations, seedlings
were incubated with 0.5, 1, 2, 4 and 8 mM 15NH4+ for 5 min (Figs. 2f and 3f).

Roots and shoots were separated for weighing, and then immediately frozen in
liquid N2. After grinding, an aliquot of the powder was dried to a constant weight
at 70 °C, and 10 mg of each sample was analysed using the MAT253-Flash EA1112-
MS system (Thermo Fisher Scientific, Inc., USA). Each experiment was performed
with three independent biological replicates, and statistical analyses were
performed using two-tailed Student’s t tests.

Nutrient element analysis of plant samples. Roots and leaves were harvested
from 6-week-old plants, washed three times with tap water and rinsed twice (5 min
each) with deionised water to remove any adhering nutrients. The leaves and roots
were dried in a forced-air oven at 70 °C for ~48 h to a constant weight for dry
weight measurements (Figs. 2b and 3b and Supplementary Fig. 4a). The dried
samples were ground and passed through a 1.0-mm screen. Total N/C contents
(Figs. 2g, h and 3g, h) were determined via the dry combustion method using an
Element Analyser (vario EL, Elementar, Langenselbold, Germany). For the analysis
of mineral elements, the dry biomass was digested in H2SO4 or HClO4. P con-
centrations were determined using the molybdate yellow method and K con-
centrations were determined by flame emission photometry (Supplementary
Fig. 6)54,55. The other nutrient elements were measured by ICP (Agilent 710 ICP-
OES). At least three plants per treatment were harvested, and three independent
biological replicates were analysed for each treatment.

Stomatal observation. Stomatal observation and quantification were performed10.
Briefly, epidermal fragments were obtained by homogenising 1-week-old rice
seedlings that had been grown in ½ Murashige and Skoog agar medium using a
Waring blender. After passing the tissue through a 58-µm nylon mesh, the material
was incubated in observation buffer containing 50 mM KCl, 0.1 mM CaCl2 and
5 mM MES-BTP with pH 6.5. After ~3-h incubation in darkness or light (150 µmol
photon m−2 s−1 red light [LED-R; EYELA] plus 50 µmol photon m−2 s−1 blue
light [Stick-B-32]) in 20 µM ABA, epidermal fragments were collected for micro-
scopic observation. The percentage of open stomata (Fig. 4b and Supplementary
Figs. 7a and 8c) was quantified as the number of open stomata per total stomata
observed. At least 100 stomata were observed per treatment; three biological
replicates were analysed for each treatment, and statistical analysis was conducted
using two-tailed Student’s t tests.

Gas-exchange measurements. Gas-exchange measurements were performed
using the LI-6400 System (Li-Cor) with a standard chamber. Light and CO2
response curves were constructed based on data obtained using measurement
processes and light sources12,56. The flow rate, leaf temperature and relative
humidity were kept constant at 500 μmol s−1, 24 °C and 60–75% (Pa/Pa), respec-
tively. Under each light/CO2 condition, photosynthetic rate and stomatal con-
ductance data were collected after these values reached a steady state (15–30 min).
Fully expanded leaves from 6-week-old plants were used in these experiments
(Figs. 1b and 4 and Supplementary Figs. 7b, c and 4d, e). WL was provided by a
fibre optic illuminator with a halogen projector lamp (15 V/150 W, Moritex, San
Jose, CA, USA) as a light source powered by an MHAB-150W (Moritex) power
supply. For CO2 response curves, leaves were measured at saturating WL condi-
tions (~1500 μmol m−2 s−1) (Fig. 4g and Supplementary Fig. 7b, c). To obtain the
stomatal conductance and CO2 assimilation rate data shown in Fig. 4c, d and
Supplementary Table 2, leaves were measured under saturating WL conditions
(~1000 μmol m−2 s−1).

For field gas-exchange measurements (Supplementary Fig. 10), the flow rate of
the Li-6400 system was kept constant at 500 μmol s−1 at a leaf temperature and
relative humidity of 28 °C and 40–50% (Pa/Pa), respectively. All measurements
were performed before the heading stage. At least three plants were selected for
measurement, and three biological replicates were analysed for each treatment.

Stomatal density and size. Three to four fully expanded leaves of 6-week-old rice
plants were selected. At least five microphotographs were randomly taken of the

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adaxial or abaxial surface of the leaf lamina. The average stomatal density and size
(long axis of each stoma) were calculated57 (Supplementary Fig. 8a, b).

High-throughput RNA-seq analysis. For RNA-seq analysis, total RNA was
extracted from leaves and roots collected from 4-week-old WT, OSA1-ox (OSA1#2)
and osa1 (osa1-2) mutant rice lines using a TRIzol Plus RNA Purification Kit
(Thermo Fisher Scientific, Waltham, MA, USA). Complementary DNA libraries
were constructed using a TruSeq RNA Sample Prep Kit v. 2 (Illumina, San Diego,
CA, USA) and sequenced using a NextSeq 500 system (Illumina). Base-calling of
sequence reads was performed using the NextSeq 500 pipeline software. Only high-
quality sequence reads (50 continuous nucleotides with quality values >25) were
used for mapping (Fig. 5a, b and Supplementary Fig. 9). Reads were mapped to O.
sativa (IRGSP v. 1.0 2019.8.29) transcripts using the Bowtie software58. Experiments
were repeated three times separately. We obtained 10.1–13.6 million sequence reads
per experiment. Gene expression values were reported in RPM (reads per million
mapped reads) units. Normalisation of read counts and statistical analyses were
performed using the EdgeR software package59,60 and the Degust Ver. 3.1.0 web tool
(http://degust.erc.monash.edu). Obtained RPM values were further analysed using
MS Excel software. Only genes with log2 fold change ≥1 or ≤−1, and an FDR < 0.05 were considered to be significant DEGs. GO term enrichment was conducted using GO Term Enrichment tool in the Plant Transcriptional Regulatory Map (Plan- tRegMap) website61 (http://plantregmap.gao-lab.org/go.php). GO category (http:// geneontology.org/) FDR ≤ 0.05 was regarded as significantly enriched.

Detection of rhizosphere acidification in roots. Rhizosphere acidification in WT
and OSA1-oxs roots was determined51. The roots of 7-day-old plants were thor-
oughly washed with deionised water and spread on an agar sheet containing 0.7%
(w/v) agar, 0.02% (w/v) bromocresol purple, 2 mM NH4Cl and 1 mM CaSO4 at pH
5.6. The roots were carefully pressed into the agar to avoid damage. For visuali-
sation of rhizosphere acidification, incubation was conducted in a growth chamber
in the dark for 12 h. The relative area of rhizosphere acidification (Supplementary
Fig. 2b) was estimated as a ratio to the WT area (yellow area on agar sheet;
Supplementary Fig. 2a). At least three plants per treatment were harvested, and
three independent biological replicates were analysed for each treatment.

Quantification of H+ extrusion rate. The H+ efflux rates from the WT and
OSA1-ox rice roots were measured using the scanning ion-selective electrode
technique (SIET System BIO-003A, Younger USA Science and Technology Corp.,
Applicable Electronics Inc., Science Wares Inc., Falmouth, MA, USA) (Supple-
mentary Fig. 2f)15,21. Briefly, seedlings were placed in 50 mL of growth solution
with 2 mM NH4+ for 12 h. Then, the rice roots of 7-day-old plants were washed
with deionised water and equilibrated in the measuring solution for 10 min. The
equilibrated roots were then transferred to a measuring chamber, which contained
3 mL of a solution comprising 0.2 mM CaCl2, 0.1 mM KCl, 0.1 mM NaNO3 and
0.5 g L−1 MES (2-morpholinoethanesulfonic acid sodium salt) (pH 5.7). At least
three plants per treatment were analysed, and three independent biological repli-
cates were performed.

Reporting summary. Further information on research design is available in the Nature
Research Reporting Summary linked to this article.

Data availability
The authors declare that the data supporting the findings of this study are available
within the paper and the Supplementary information. The RNA-seq data that support
the findings of this study have been deposited in the DNA Data Bank of Japan (DDBJ)
with the accession number DRA011260. Source data are provided with this paper.

Received: 1 October 2019; Accepted: 4 January 2021;

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Acknowledgements
This work was supported by grants from the National Key Basic Research and Devel-
opment Program (2017YFD0200200/0200206 to Y.Z.), the Natural Science Foundation
of China (NSFC 31471937 to Y.Z.), Technology of Japan and the Advanced Low Carbon
Technology Research and Development Program from the Japan Science and Technol-
ogy Agency (JPMJAL1011 to T.K.) and the Natural Science Foundation of Anhui Pro-
vince, China (1608085MC59 to X.X.), as well as Grants-in-Aid for Scientific Research on
Innovative Areas (15H05956, 20H05687 and 20H05910 to T.K.).

Author contributions
Y.W., T.K. and Y.Z. conceived the research project and designed the experiments. M.Z.,
Y.W., F.X., M.D., Y.K., Y.T., Y.H., T.S., H.Z., L.X., X.X., S.G., T.K. and Y.Z. conducted
experiments and performed data analyses. Y.W., X.C., W.Y., J.X., F.Y., Q.S., G.X., T.K.
and Y.Z. oversaw the entire study and wrote 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/s41467-021-20964-4.

Correspondence and requests for materials should be addressed to T.K. or Y.Z.

Peer review information Nature Communications thanks Xiangdong Fu, Brent Kaiser,
Beom-Gi Kin, and the other, anonymous, reviewer(s) for their contribution to the peer
review of this work. Peer review reports are available.

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(osa1-1) (osa1-2) (osa1-3)

a
b

26 cyc

30 cyc

ND3017 ND3025 NF3033

Supplementary Figure 1

Supplementary Figure 1 TOS17 insertion site and RT-PCR of OSA1 in osa1 mutants. a
Schematic diagram of the genome structure of OSA1. Lines represent UTRs and introns. Exons
are represented by white boxes. Positions of the TOS17 insertion are indicated by small triangles.
b OSA1 expression in the roots of WT and osa1 mutants by RT-PCR. Experiments were repeated
three occasions with similar results.

WT osa1-1 osa1-2 osa1-3

OSActin

OSA1

700 bp
500 bp

1K bp

700 bp
500 bp
1K bp

maker

Supplementary Figure 2

WT OSA1#1 OSA1#2 OSA1#3
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em
in
al
ro
ot

n
um

be
re

**
n.s. n.s.

n.s.

n.s.
n.s.

n.s.
n.s.
n.s.
n.s.

Time（min）

H
+

ef
flu

x
ra

te
s（

m
ol

·c
m

2 ·s

-1
）

0
10
20

40
50
60
70
80

0 2 4 6 8 10

OSA1#1

OSA1#2

OSA1#3

f
*

Supplementary Figure 2

Supplementary Figure 2 Rhizosphere acidification and H+ efflux properties of WT and OSA1-oxs rice.
a Monitoring of rhizosphere acidification around WT and OSA1-oxs rice roots. Plants were grown in nutrient
solution for 7 days. After washing with deionized water, roots were carefully spread onto solid medium
containing 0.02% (w/v) bromocresol purple and 0.7% (w/v) agar adjusted to pH 5.6. After incubation for 12 hr
in the dark, the plates were photographed. b Relative area of rhizosphere acidification (yellow area) on the
surface of agar plates. c-e Surface area (c), length(d) and number (e) of seminal roots in WT and OSA1-oxs.
Small circles represent the data points of individual experiments were performed. Values are mean ± SEs (n
= 5). f Quantification of H+ efflux rates from WT and OSA1-oxs roots. Intact roots (3-5 cm in length from the
root tips) were equilibrated in the measuring solution for 10 min. Values are mean ± SEs (n = 6). Differences
were evaluated using the two-tailed Student’s t-test (*P < 0.05; **P < 0.01, n.s., not significant). The exact p values are provided in the Supplementary Data 5.

OSA1#1
WT
OSA1#2
OSA1#3

EX
EP

i EN

jhgf

EPn EN

PH XY

omlk

EPs
EN

PH
XY

trqp

PH
XY

Supplementary Figure 3 Localization of PM H+ ATPase in rice roots. Immunohistochemical staining of 3-weeks-old
seedlings in WT and OSA1-oxs, with polyclonal antibodies recognizing rice PM H+-ATPase (anti-H+-ATPase ) was
performed in rice root. Red color shows the signal of anti-H+-ATPase (b, j), blue color shows autofluorescence of cell wall
stained by DAPI(a, f), and the merged image (c-e and h-j). EP, epidermis; EX, exodermis; EN, endodermis; PH, phloem;
XY, xylem. Bars = 150 µm (a-c, f-h, k-m and p-r) ,20 µm (d, i, n and s) and 60 µm (e, j, o and t). Experiments were
repeated three occasions (a-t ) with similar results.

Auto
fluorescence Anti-H+-ATPase Merged

Supplementary Figure 3

a b c d

Cortex

Cortex
Cortex
Cortex

D
ry

w
ei

gh
t (

g/
pl

an
t)

Root Shoot

n.s.
n.s.

Irradiance (μmol m-2 s-1)

C
O

2
as

si
m

ila
tio

n
ra

te
(μ

m
ol

m
-2

1 )

0
5
10

0 500 1000 1500 2000 2500

+CaMV 35S

Irradiance (μmol m-2 s-1)
0

0.2

0.4

0.6

0.8

1
0 500 1000 1500 2000 2500
WT

S
to

m
at

al
c

on
du

ct
an

ce
(m

ol
m

-2
s

-1
）
+CaMV 35S
a

d e

R
el
at
iv

e
pr

ot
ei

n
le

ve
l

R
el
at
iv

e
ex

pr
es

si
on

o
f

O
S
A
1

Roots Leaves Roots Leaves

Supplementary Figure 4

Supplementary Figure 4 Plant growth and gas-exchange properties in CaMV 35S empty
vector transformed rice. a Dry weights of root and shoot in WT and CaMV 35S empty vector
transformed plants. b Relative expression level of OSA1 in the roots and leaves of WT and
CaMV 35S empty vector transformed plants. c Relative PM H+-ATPase protein levels in the
roots and leaves WT and CaMV 35S empty vector transformed plants. d, e Stomatal
conductance (d) and CO2 assimilation rate (e) in response to light in WT and CaMV 35S empty
vector transformed plants. Small circles in (a-c) represent the data points for individual
experiments and three biological replicates were performed. Values in (a–c) are presented as
the means ± SEs (n = 3) and those in (d, e) are the means ± standard deviations (n = 3).
Differences in (a–c) were evaluated using the two-tailed Student’s t-test (*P < 0.05; ** P < 0.01).

n.s. n.s. n.s.
n.s.

Supplementary Figure 5 15N absorption rate by WT and OSA1-oxs, and osa1 mutants. Rice
plants were grown hydroponically in a greenhouse for 4 weeks. To determine 15NH4+ absorption
rates, seedlings were incubated in 2 mM 15NH4+ solution for 30 min under darkness with the
treatment of 350 μM vanadate (VA) for rice roots. Values are the mean ± SEs (n = 3). Differences
were evaluated using the two-tailed Student’s t-test (*P < 0.05; ** P < 0.01). The exact p values are provided in the Supplementary Data 5.

Supplementary Figure 5

W
T

os
a1
-1

os
a1
-2

os
a1
-3

OS
A1
#1

OS
A1
#2

OS
A1
#3

**
* *

* *

15
N

H
4+

ab
so

rp
tio

n
ra
te
(μ
m
ol

h
-1

g-
1
D

W
)
**

Mock
VA

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

16.0

a b

Supplementary Figure 6

To
ta

l a
m

ou
nt

o
f P

in
p

la
nt

g/
pl

t）

Roots Leaves Roots Leaves
To
ta
l a
m
ou
nt

o
f C

a
in

p
la

nt
(m

g/
pl

an
t）

To
ta
l a
m
ou
nt

o
f S

i i
n

pl
an

t
(m

g/
pl
an
t）
*
Roots Leaves Roots Leaves
To
ta
l a
m
ou
nt

o
f M

g
in

p
la
nt
(m
g/
pl
an
t）
*
To
ta
l a
m
ou
nt
o
f S
in
p
la
nt

(m
g/

pl
an
t）
*
**
Roots Leaves
c

d e f
Roots Leaves

To
ta
l a
m
ou
nt

o
f K

in
p
la
nt
(m
g/
pl
an
t）
* ****
******
* ***
**
** **

**
*

**** **
* *
*
**
**** **
**
*
*
**
*
** *
*
*
**
**
*
*
**
**
**
*
* *
** **
****

**
**** **

* *
* *
Roots Leaves
To
ta
l a
m
ou
nt

o
f F

e
in

p
la
nt
(m
g/
pl
an
t）
*
Roots Leaves
To
ta
l a
m
ou
nt

o
f B

in
p
la
nt

(m
g/
pl
an
t）
*
To
ta
l a
m
ou
nt
o
f M

n
in

p
la
nt
(m
g/
pl
an
t）
*
**
Roots Leaves
To
ta
l a
m
ou
nt
o
f C

u
in

p
la
nt
(m
g/
pl
an
t）
***
Roots Leaves
To
ta
l a
m
ou
nt

o
f Z

n
in
p
la
nt
(m
g/
pl
an
t）
***
Roots Leaves

Supplementary Figure 6 OSA1 overexpression promotes nutrition elements uptake in
rice. a-k Total amount of K (a) , P (b) ,Ca (c) , Si (d) ,Mg (e) , S (f) ,Fe (j) , Mn (h) ,B (i) , Zn
(j) and Cu (k) in WT and OSA1-ox and osa1 mutant plants. Plants were grown hydroponically
in a greenhouse for 3 weeks. Small circles in (a-k) represent the data points for individual
experiments and three biological replicates were performed. Values in (a–k) are presented as
the means ± SEs (n = 3). Differences were evaluated using the two-tailed Student’s t-test
(*P < 0.05; ** P < 0.01). The exact p values are provided in the Supplementary Data 5.

ih
g

*
**
*
*
**
*
** **
**
*
*
* **
****

****
**

* ****

St
om

at
al

c
on

du
ct

an
ce

（

m
ol
m
-2

s
-1
）

DK WL 2nd DK

b c

a
C
O
2
as
si
m
ila
tio
n
ra

(μ
m

ol
m

-2
s-

1 )
DK WL 2nd DK
**
*
** **
*

Pe
rc

en
ta

ge
o

f o
pe

n
st

om
at

a
(%

)

DK ABARL+BL

**
** **
*

Supplementary Figure 7

Supplementary Figure 7 Gas-exchange properties of osa1 mutants. a Percentage of
open stomata observed after 3 h of DK, RL+BL, or RL+BL in the presence of 20 μΜ ABA in
WT. Differences were assessed using the Student’s t-test. Error bars represent the SE (n = 3;
at least 100 stomata observed under each condition). b, c Stomatal conductance (b) and CO2
assimilation rate (c) in the DK (30 min), WL (2 h), and a 2nd DK treatment (30 min) in WT and
osa1 mutants, where light intensity was 1,000 μmol m-2 s-1. Small circles in (a) to (c) represent
the data points for individual experiments and three biological replicates were
performed.Values in (a–c) are presented as the means ± SEs (n = 3). Differences in (a–c)
were evaluated using the two-tailed Student’s t-test (*P < 0.05; ** P< 0.01). The exact p values are provided in the Supplementary Data 5.

adaxial abaxial

S
to
m
at

al
s

iz
e

(μ
m
)
S
to
m
at

al
d

en
si

ty
(/

m
m

2 )

adaxial abaxial

Supplementary Figure 8

b
OSA1#3
OSA1#1
WT

Bar = 8 μm

OSA1#2
c
osa1-1
osa1-2
osa1-3

Supplementary Figure 8

Supplementary Figure 8 Stomatal density, size, and shape in WT, OSA1-oxs and osa1
mutant plants. a Stomatal size in WT, OSA1-oxs, and osa1 mutants (in each experiment, 60
stomata were examined). b Stomatal density in WT, OSA1-oxs, and osa1 mutants (in each
experiment, 5 microphotographs were examined). c Randomly selected stomata observed
after 3 h of red light and blue light (RL+BL) in WT, OSA1-oxs, and osa1 mutants. Experiments
were repeated three occasions with similar results. Small circles in (a) and (b) represent the
data points for individual experiments and three biological replicates were performed. Values
in (a, b) are presented as the means ± SEs (n = 3). Differences in (a, b) were evaluated
using the two-tailed Student’s t-test, and no significant difference was found.

a
b
c

Supplementary Figure 9 The DEGs and GO enrichment caused by modification of OSA1.
Plants were grown hydroponically in a greenhouse for 4 weeks. a DEGs of OSA1-ox plants in
leaves. b DEGs of OSA1-ox plants in roots. Small pink circles in (a-b) represent the up-regulation
of individual gene, blue circles represent the down-regulation gene, grey circles represent the not
significantly regulated gene. c Venn diagram representing the overlaps of the down-regulated
genes in OSA1-ox and the up-regulated genes in osa1-2 mutant (osa1) from roots and leaves
(FDR <0.05). d-e The significant GO analysis of genes in leaves (d) and roots (e), which were up regulated by OSA1 overexpression, and down regulated by mutation of OSA1 (FDR< 0.05).

log2 (FC)

-lo
g2

(F
D

R
)

log2 (FC)
-lo
g2
(F
D
R
)
d
e

Supplementary Figure 9

Down-regulated genes
from OSA1-ox leaves

Up-regulated genes
from osa1-2 roots

Down-regulated genes
from OSA1-ox roots

Up-regulated genes
from osa1-2 leaves

S
to
m
at

al
co

nd
uc

ta
nc

e
(m

ol
m
-2
s

-1
)

N-N

L-N

M-N H-N
a
b
N-N L-N
M-N H-N
C
O
2
as
si
m
ila
tio
n
ra
te
(μ
m
ol
m
-2

s-
1 )

* *
* *
*
*
* *
*
* *
**

* * *

*
*
* *
*
* *
**
*
** **
**
**
**
**
**
**
* *

Supplementary Figure 10

Supplementary Figure 10 Gas-exchange properties of WT, OSA1-oxs, and mutant plants in
the field. Stomatal conductance (a) and net photosynthetic rate (b) in WT, OSA1-oxs, and osa1
mutants. Plants were grown in the summer of 2017 at Nanjing-N under N-N (0 kg N/ha), L-N (100
kg N/ha), M-N (200 kg N/ha), or H-N (300 kg N/ha) fertilisation. Measurements were conducted at
ambient [CO2] (~400 μL L-1) at the flowering stage. Leaf temperature and relative humidity in the
leaf chamber were maintained at 28℃ and 40–50% (Pa/Pa), respectively. Small circles in (a) and
(b) represent the data points for individual experiments and three replicates were performed.
Values in (a, b) are presented as the means ± SEs (n = 3). Differences were evaluated using the
two-tailed Student’s t-test (*P < 0.05; ** P < 0.01). The exact p values are provided in the Supplementary Data 5.

OsA1#3

OsA1#3OsA1#3

a b
c d
H-N
L-N
N-N
H-N
L-N
N-N
WT OSA1#1 OSA1#2 OSA1#3
WT OSA1#1 OSA1#2 OSA1#3
WT OSA1#1 OSA1#2 OSA1#3
WT OSA1#1 OSA1#2 OSA1#3
WT OSA1#1 OSA1#2 OSA1#3
WT OSA1#1 OSA1#2 OSA1#3
WT OSA1#1 OSA1#2 OSA1#3
WT OSA1#1 OSA1#2 OSA1#3
WT OSA1#1 OSA1#2 OSA1#3
WT OSA1#1 OSA1#2 OSA1#3
WT OSA1#1 OSA1#2 OSA1#3
WT OSA1#1 OSA1#2 OSA1#3

30
c

m
30

c
m
30
c

m
8

8
cm

8
cm
8
cm
8
cm

Supplementary Figure 11

Supplementary Figure 11 Overexpression of OSA1 increases grain yield in the field. a-d
Photographs of 100-day-old WT and OSA1-oxs plants under H-N (300 kg N/ha), L-N (100 kg N/ha),
or N-N (0 kg N/ha) fertilisation in the field (a) and in pots (b) in the summer of 2017 at Nanjing-N. c,
d panicles (c) and spikelets (d) under different N fertilisation levels.

Supplementary Figure 12 Schematic diagram of the model for the effect of overexpression of
OSA1 on the interaction between C and N uptake and metabolism in paddy rice.
Overexpression of OSA1 in roots facilitates NH4+ uptake and also pumps excessive H+ outside the root
cells to guarantee the assimilation of NH4+ in cytoplasm. As the result, efficient utilization of NH4+ in rice
roots provide amino acids and protein for the plant growth and photosynthesis. On the other side,
overexpression of OSA1 in guard cells polarized the membrane potential and trigger the K+ influx for the
opening of stomatal under light, which enhanced the uptake of CO2 and improve the photosynthesis, which
can provide carbon skeleton (2-OG) for the assimilation of NH4+ in roots. In addition, the enhanced
transpiration by stomatal opening also improves the nutrients uptake due to the accelerated water transport
in plants.
AMT: ammonium transporter, GS: glutamine synthetase, Gln: Glutamine, GOGAT: glutamate synthase,
Glu: Glutamate, 2-OG: 2 oxoglutatate, TCA: Tricarboxylic acids, PGA: Phosphoglycerate, G3P:
Glyceraldehyde 3-phosphate, Rubisco: Rubisco ribulose-1,5-bishosphate carboxylase.

Supplementary Figure 12

C
CO2

NH4+

2-OG

CO2
Transpiration

NH4+
assimilation

↑

ATP

H+

ADP
OSA

Gln

Glu

AMT GS

Root Cell

↑
Glu

Sugar

TCA
cycle

GOGAT
H+

Ammino acid

H
2 O

O
2

3-PGA

G3P

ATP

NADPH

NADP
+

ADP+Pi
Rubisco

Leaf Cell
Photosynthesis↑

ATPADP

H+ H+
H+

OS
A

K+ K+
K+

+ + + + +

H2O

Guard Cell

NH4+

a
b

Culm nitrogen content （mg/plant）

C
ul

m
c

ar
bo

n
co

nt
en

t (
m

g/
pl
an
t）

Leaf nitrogen content （mg/plant）

Le
af

c
ar

bo
n

co
nt

en
t(

m
g/

pl
an
t）

Grain nitrogen content （mg/plant）

G
ra

in
c

ar
bo
n
co
nt
en
t (
m
g/
pl
an
t）
c
0

1800

3600

5400

7200

0 200 400 600 800

WT
OSA1#1
OSA1#2
OSA1#3
osa1-1
osa1-2
osa1-3

4000

8000

12000

16000

0 300 600 900 1200

WT
OSA1#1
OSA1#2
OSA1#3
osa1-1
osa1-2
osa1-3
0

5000

10000

15000

20000

0 2000 4000 6000 8000

WT
OSA1#1
OSA1#2
OSA1#3
osa1-1
osa1-2
osa1-3

Supplementary Figure 13

Supplementary Figure 13 Correlations of N and C content in WT, OSA1-oxs, and osa1
mutant plants under various fertilisation levels in the field. Correlations between N content
and C content in leaves (a), culms (b), and grain (c) of all rice genotypes harvested in the summer
of 2017 at Nanjing-N.

y = 2.0615x + 2637.4
R² = 0.9383

y = 8.7141x – 176.37
R² = 0.9962

y = 12.032x + 101.22
R² = 0.9909

Supplementary Table 1 Relative expression of OSA (OSA2, OSA3, OSA4, OSA5, OSA6, OSA7, OSA8,
OSA9, OSA10) genes in rice leaves and roots of WT, OSA1-oxs, and osa1 mutant lines. There were no
significant difference among OSA isoforms. Differences were evaluated using the two-tailed Student’s t-
test. n.d. represents the gene expression that cannot be detected.

Supplementary Table 1

Root OSA2 OSA3 OSA4 OSA5 OSA6 OSA7 OSA8 OSA9 OSA10

WT 1.00±0.08 1.00±0.26 n.d. 1.00±0.09 n.d. 1.00±0.18 1.06±0.08 1.27±0.16 1.10±0.19

OSA1#1 1.18±0.22 1.52±0.20 n.d. 0.93±0.11 n.d. 1.33±0.28 1.19±0.21 1.68±0.14 1.29±0.24

OSA1#2 1.23±0.25 1.31±0.18 n.d. 0.87±0.04 n.d. 1.22±0.11 1.16±0.10 1.71±0.36 1.23±0.13

OSA1#3 1.04±0.13 1.60±0.40 n.d. 1.18±0.20 n.d. 1.38±0.23 1.18±0.14 1.43±0.13 1.16±0.26

osa1-1 1.23±0.13 1.17±0.08 n.d. 0.98±0.16 n.d. 1.21±0.11 0.96±0.07 0.90±0.24 1.03±0.15

osa1-2 1.13±0.20 1.13±0.24 n.d. 0.92±0.23 n.d. 1.25±0.07 1.10±0.16 1.01±0.24 1.04±0.20

osa1-3 1.34±0.28 1.36±0.16 n.d. 1.05±0.25 n.d. 1.13±0.15 0.93±0.18 0.97±0.20 1.05±0.16
Leaf OSA2 OSA3 OSA4 OSA5 OSA6 OSA7 OSA8 OSA9 OSA10

WT 1.00±0.06 1.10±0.17 n.d. 1.33±0.40 n.d. 1.14±0.10 1.16±0.07 n.d. n.d.

OSA1#1 0.87±0.04 1.25±0.14 n.d. 1.64±0.44 n.d. 1.23±0.08 1.32±0.29 n.d. n.d.

OSA1#2 1.09±0.05 1.44±0.21 n.d. 1.41±0.39 n.d. 1.33±0.16 1.15±0.16 n.d. n.d.

OSA1#3 0.87±0.09 1.35±0.18 n.d. 1.32±0.39 n.d. 1.32±0.23 1.24±0.11 n.d. n.d.

osa1-1 0.70±0.11 0.67±0.08 n.d. 1.64±0.34 n.d. 1.11±0.19 1.12±0.22 n.d. n.d.

osa1-2 0.82±0.11 0.73±0.07 n.d. 1.18±0.47 n.d. 1.05±0.21 1.11±0.22 n.d. n.d.

osa1-3 0.68±0.20 0.68±0.08 n.d. 1.55±0.73 n.d. 1.14±0.20 1.35±0.29 n.d. n.d.

Supplementary Table 2 Gas-exchange properties of WT, OSA1-oxs, and mutant lines of
hydroponically grown rice plants. Measurements were conducted at 400 μL L–1 CO2. Light intensity was
1,000 μmol m–2 s–1. Leaf temperature and relative humidity of leaf chamber were maintained at 24°C
and 60–75% (Pa/Pa), respectively. Water use efficiency was calculated as the ratio between the CO2
assimilation rate and transpiration rate. Differences were evaluated using the two-tailed Student’s t-test
(*P < 0.05; ** P < 0.01) ±SEs (n = 3).

WT OSA1#1 OSA1#2 OSA1#3

CO2 assimilation rate
(μmol m−2 s−1) 11.61±0.53 14.62±0.87* (P=0.023) 14.88±1.39 (P=0.055) 14.59±0.78* (P=0.018)

Stomatal conductance
(mol m−2 s−1) 0.41±0.05 0.73±0.02** (P=0.002) 0.88±0.08** (P=0.003) 0.83±0.07** (P=0.003)

Ci (μL L−1) 336.67±8.46 349.05±3.62 (P=0.175) 354.34±4.80 (P=0.090) 353.53±1.16 (P=0.073)

Transpiration rate
(mmol m−2 s−1) 3.48±0.37 4.96±0.18* (P=0.012) 5.61±0.12** (P=0.003) 5.06±0.42* (P=0.027)

Water use
efficiency 3.38±0.23 2.94±0.20 (P=0.150) 2.66±0.29 (P=0.075) 2.90±0.14 (P=0.089)

Supplementary Table 2

Supplementary Table 3 Agronomic traits and grain yields of WT, OSA1-oxs, and osa1 mutant lines in
the field (2016 Nanjing-S). Plant height, tiller number, panicle numbers, panicle length, spikelets, 1,000
grain weight, filled grains, and grain yield for plants grown in 2016 at Nanjing-S under 0 kg N/ha (N-N),
100 kg N/ha (L-N), 200 kg N/ha (M-N) and 300 kg N/ha (H-N) fertilisation. Differences were evaluated
using one-way ANOVA. Values are the mean ±SEs (n ≥ 3). Letters indicates significant differences at
P < 0.05 and the order starts with OSA1-oxs.

Supplementary Table 3

2016
Nanjing-S

Material

Plant Tiller Panicles Panicles spikelet 1000 grain Filled Yield

height number number length number weight grains (kg/ha)

(cm) per hill per hill (cm) per panicle (g) rate (%)

N-N
(0 kg N/ha)

WT 81.3±1.1a 26.6±1.1b 24.9±0.8ab 17.2±0.5a 63.8±1.3b 26.1±0.5a 84.0±0.6a 6931±214b

OSA1#1 83.0±1.4a 30.3±1.7a 27.4±1.4a 16.5±0.6a 69.1±1.4a 25.1±0.7a 86.7±1.3a 8191±423a

OSA1#2 83.3±1.1a 33.8±1.3a 26.4±1.0a 16.8±0.2a 68.9±3.2a 25.4±0.6a 88.2±1.4a 8091±297a

OSA1#3 81.7±0.8a 32.8±2.3a 26.3±2.3a 16.8±0.2a 69.5±2.1a 26.1±0.3a 86.1±2.4a 8157±721a

osa1-1 77.0±3.1ab 25.1±0.6ab 22.5±0.7b 16.8±0.7a 58.4±1.5c 25.5±0.6a 84.6±0.8a 5644±176c

osa1-2 78.0±3.1ab 25.3±0.7b 22.5±0.8b 16.5±0.6a 58.0±1.5c 25.0±0.5a 85.4±1.2a 5554±187c

osa1-3 74.3±4.1b 24.6±0.7b 22.1±0.6b 16.3±0.5a 58.1±1.6c 25.6±1.0a 83.0±4.1a 5448±144c

L-N
(100 kg N/ha)

WT 87.0±2.1a 29.8±1.2b 25.9±0.8b 19.5±0.9a 79.1±0.8b 25.8±0.4a 84.0±2.9a 8836±262b
OSA1#1 91.7±5.7a 37.3±1.4a 30.8±1.1a 21.0±1.5a 83.5±2.0a 26.3±0.9a 87.4±0.4a 11757±414a
OSA1#2 86.7±0.8a 38.0±0.9a 31.1±1.9a 18.9±0.6a 84.3±1.8a 26.1±0.4a 88.1±0.8a 12018±729a
OSA1#3 85.3±0.8a 39.1±1.6a 32.3±1.7a 19.5±0.4a 85.0±1.9a 25.3±0.3a 87.4±2.2a 12088±632a
osa1-1 84.7±3.6a 26.5±0.6c 22.9±0.7b 18.4±1.0a 74.1±0.9c 25.4±0.8a 84.7±1.2a 7268±217c

osa1-2 84.0±2.5a 26.3±0.4c 22.8±0.5b 18.5±0.9a 74.8±0.7c 24.6±1.3a 83.7±1.8a 6987±149c

osa1-3 83.3±2.5a 26.3±0.7c 22.9±0.4b 18.4±1.2a 75.5±1.0bc 25.6±0.6a 84.8±1.0a 7463±122c

M-N
(200 kg N/ha)

WT 95.7±3.3a 33.5±2.0b 27.8±1.7b 20.0±1.3a 82.3±1.5b 25.1±1.3a 84.6±2.5a 9672±588b
OSA1#1 96.7±1.5a 40.6±2.2a 33.3±1.2a 20.0±0.7a 88.6±1.0a 25.6±0.5a 86.9±3.4a 13060±454a
OSA1#2 95.0±4.9a 41.6±2.0a 32.8±1.0a 19.7±1.5a 89.8±1.0a 25.9±0.3a 87.0±1.9a 13211±414a

OSA1#3 96.0±2.4a 41.5±1.4a 32.6±0.9a 19.3±0.4a 90.4±2.2a 26.6±0.3a 85.8±2.6a 13393±370a

osa1-1 92.3±4.3a 29.5±0.9c 25.1±0.9b 18.1±1.2a 76.5±1.5c 25.2±0.8a 83.9±2.3a 8097±294c

osa1-2 91.7±3.3a 30.1±0.8c 25.8±0.9b 18.5±0.7a 76.0±1.5c 25.2±0.9a 83.8±2.2a 8243±272c

osa1-3 92.3±2.2a 30.5±0.7c 25.9±0.6b 18.7±1.1a 75.5±1.6c 25.8±0.3a 83.4±3.1a 8370±190c

H-N
(300 kg N/ha)

WT 95.3±2.0b 35.5±1.4b 30.0±0.8b 20.0±1.0a 84.5±1.9b 26.1±0.3a 83.7±1.0ab 11044±298b
OSA1#1 104.0±3.2a 42.1±1.3a 34.9±2.2a 21.4±0.6a 92.5±1.7a 25.7±0.9a 85.6±1.8a 14120±883a
OSA1#2 104.7±0.8a 43.8±2.2a 35.9±1.2a 21.5±1.1a 93.4±1.9a 25.7±0.6a 85.7±0.5a 14684±478a

OSA1#3 102.7±0.4a 41.5±1.4a 36.0±1.6a 21.2±0.5a 93.1±1.3a 26.4±0.1a 86.4±0.7a 15205±677a

osa1-1 95.7±1.6b 30.9±0.8c 26.8±0.6c 20.5±0.9a 78.6±1.8c 26.1±0.3a 83.2±1.0ab 9091±214c

osa1-2 94.3±2.3b 30.6±1.0c 26.5±0.7c 20.1±0.2a 78.9±1.6c 26.1±0.1a 83.0±0.5ab 9008±228c

osa1-3 96.0±1.2b 29.4±0.7c 25.5±0.5c 19.8±0.2a 78.5±1.3c 25.3±0.7a 81.6±2.7b 8232±146c

Supplementary Table 4 Agronomic traits and grain yields of WT, OSA1-oxs, and osa1 mutant lines
in the field (2017 Nanjing-N). Plant height, tiller number, panicle numbers, panicle length, spikelets,
1,000 grain weight, filled grains, and grain yield for plants grown in 2017 at Nanjing-N under 0 kg N/ha
(N-N), 100 kg N/ha (L-N), 200 kg N/ha (M-N) and 300 kg N/ha (H-N) fertilisation. Differences were
evaluated using one-way ANOVA. Values are the mean ± SEs (n ≥ 3). Letters indicates significant
differences at P < 0.05 and the order starts with OSA1-oxs.

Supplementary Table 4

2017
Material

Plant Tiller Panicles Panicles spikelet 1000 grain Filled grains

Yield (kg/ha)Nanjing-N height number number per hill length
number per

panicle weight rate (%)

(cm) per hill (cm) (g)

N-N
(0

kgN/ha)

WT 81.7±1.5ab 25.0±1.2b 22.8±1.0bc 18.0±0.6a 64.7±2.9b 26.5±0.8a 81.3±1.5a 6340±265b

OSA1#1 83.3±1.1a 30.4±1.9a 25.8±0.3a 17.3±1.2a 68.7±0.5ab 26.3±0.4a 80.8±5.0a 7517±98a

OSA1#2 83.7±1.1a 34.4±2.1a 23.8±0.8ab 17.6±0.7a 71.7±1.8a 26.4±0.6a 82.6±0.9a 7422±240a

OSA1#3 82.0±0.7ab 32.1±2.1a 24.5±1.6ab 17.6±0.4a 72.5±3.0a 26.6±0.7a 78.0±4.7a 7347±493a

osa1-1 79.0±2.1ab 23.0±0.9b 20.2±1.1cd 17.5±0.6a 64.0±0.8b 26.5±0.8a 81.3±1.3a 5539±315b

osa1-2 78.7±3.3ab 23.1±0.8b 20.3±0.9cd 17.1±0.7a 63.7±0.6b 26.4±1.0a 84.0±0.8a 5717±247b

osa1-3 76.7±3.6b 22.8±0.6b 19.8±0.7d 17.3±0.2a 64.7±0.6b 26.4±0.4a 83.5±3.2a 5633±187b

L-N
(100

kgN/ha)

WT 90.7±1.1a 29.5±1.6b 25.2±1.0b 19.2±2.1a 74.5±0.8b 25.9±0.4a 83.1±3.1a 8034±305b
OSA1#1 95.7±3.2a 35.0±2.7a 27.8±0.9ab 19.4±1.9a 82.7±2.0a 26.6±0.7a 82.5±4.7a 10067±314a
OSA1#2 90.7±3.6a 38.5±2.8a 28.5±1.3a 18.5±1.7a 84.0±2.2a 25.9±0.8a 85.4±3.7a 10533±499a
OSA1#3 89.3±4.3a 36.8±2.2a 29.0±2.1a 19.2±1.8a 85.0±3.6a 25.6±0.7a 81.0±6.0a 10176±743a
osa1-1 88.3±2.9a 24.9±0.6b 22.0±1.0c 19.2±0.9a 68.8±1.1c 26.1±0.8a 85.7±1.3a 6747±300c

osa1-2 88.0±2.5a 24.8±0.8b 21.8±0.4c 18.5±1.1a 69.0±1.0c 26.6±0.4a 84.8±0.4a 6762±136c

osa1-3 88.3±2.5a 24.9±0.7b 21.7±0.6c 18.9±0.8a 69.2±0.9c 26.4±1.3a 84.9±2.4a 6687±189c

M-N
(200

kgN/ha)

WT 92.7±5.1a 30.3±1.2c 26.2±1.6b 19.9±2.7a 79.2±2.1b 25.6±0.7a 86.7±0.8a 9177±556b
OSA1#1 93.7±1.8a 35.9±1.4b 31.3±1.8a 20.0±1.9a 87.5±1.6a 26.1±0.7a 81.4±5.5a 11615±660a
OSA1#2 92.0±4.4a 39.1±1.8a 31.5±2.1a 19.5±2.7a 90.2±1.9a 26.0±0.7a 80.2±4.8a 11785±797a

OSA1#3 93.0±3.5a 38.5±1.1ab 30.8±1.1a 19.7±1.5a 90.0±1.4a 25.8±1.0a 82.8±5.3a 11834±426a

osa1-1 91.7±2.7a 26.9±0.7d 23.2±1.3b 19.6±0.5a 73.3±2.0c 24.9±0.7a 84.6±1.0a 7139±413c

osa1-2 89.0±2.8a 26.3±0.6d 22.7±0.7b 19.1±1.3a 73.5±2.0c 26.1±1.1a 84.1±2.7a 7298±235c

osa1-3 90.7±3.3a 26.9±0.7d 23.7±0.6b 19.6±1.0a 74.2±1.8c 26.9±.0.8a 81.2±8.5a 7648±197c

H-N
(300

kgN/ha)

WT 97.3±6.7a 31.6±0.8b 30.0±0.9b 20.2±1.5a 82.2±2.8b 25.4±0.7b 82.0±1.4ab 10230±305b
OSA1#1 106.0±5.8a 38.1±2.7a 32.5±1.8ab 21.6±2.0a 92.7±2.3a 25.4±0.7b 86.2±1.7ab 13119±729a
OSA1#2 106.7±4.6a 40.0±1.2a 34.7±1.0a 21.7±1.5a 95.3±2.8a 25.8±0.4ab 78.1±6.8b 13273±370a

OSA1#3 104.7±5.2a 40.1±0.8a 34.2±0.8a 21.4±1.7a 96.8±1.5a 25.9±0.7ab 82.9±0.6ab 14162±319a

osa1-1 97.7±6.4a 29.5±0.6b 26.5±0.8c 20.6±1.2a 78.2±1.3b 26.9±0.6ab 83.9±0.9ab 9315±277b

osa1-2 96.0±4.4a 29.4±0.4b 26.5±0.4c 20.4±1.8a 78.0±1.6b 26.4±0.6ab 87.0±3.7a 9450±133b

osa1-3 98.0±6.3a 28.9±0.6b 27.0±0.7c 20.0±1.6a 77.8±1.3b 27.7±1.2a 82.3±0.4ab 9558±245b

Supplementary Table 5 Agronomic traits and grain yields of WT , OSA1-oxs, and osa1 mutant lines
in the field (2017 Fengyang). Plant height, tiller number, panicle numbers, panicle length, spikelets,
1,000 grain weight, filled grains, and grain yield for plants grown in 2017 at Fengyang under 0 kg N/ha
(N-N), 100 kg N/ha (L-N), 200 kg N/ha (M-N) and 300 kg N/ha (H-N) fertilisation. Differences were
evaluated using one-way ANOVA. Values are the mean ± SEs (n ≥ 3). Letters indicates significant
differences at P < 0.05 and the order starts with OSA1-oxs.

Supplementary Table 5

2017
Material

Plant Tiller Panicles Panicles spikelet 1000 grain Filled

Yield (kg/ha)Fengyang height number number per hill length
number per

panicle weight grains

(cm) per hill (cm) (g) rate (%)

N-N
(0 kgN/ha)

WT 72.0±1.4a 24.5±1.5b 23.5±0.9a 16.8±0.9a 63.0±1.3b 25.8±0.4a 86.5±2.1a 6588±247b

OSA1#1 73.7±1.1a 28.7±1.4a 25.5±1.2a 16.2±1.7a 69.0±1.6a 25.8±0.8a 86.1±5.9a 7798±381a

OSA1#2 73.7±1.8a 29.8±1.6a 24.5±0.5a 16.5±1.1a 69.4±1.0a 26.3±0.2a 83.3±1.5a 7404±137a

OSA1#3 72.3±0.4a 30.0±1.7a 24.9±0.8a 16.5±1.3a 71.5±1.2a 25.7±1.3a 87.1±6.3a 7935±269a

osa1-1 69.7±1.8a 22.8±1.1b 20.5±0.9b 16.4±0.8a 58.5±1.3c 25.9±0.5a 84.3±2.9a 5212±235c

osa1-2 70.0±1.4a 22.3±0.6b 20.5±0.7b 15.9±0.5a 57.5±1.5c 26.1±0.6a 84.3±0.2a 5162±183c

osa1-3 69.7±2.2a 22.7±1.0b 20.3±0.6b 16.0±1.4a 57.5±1.7c 26.8±0.7a 85.0±1.9a 5292±165c

L-N
(100
kgN/ha)

WT 87.0±1.9a 28.2±0.9b 23.8±1.2b 18.7±1.2a 72.1±1.3c 25.7±0.3a 86.0±3.0ab 7545±378b
OSA1#1 91.7±3.5a 39.2±2.3a 27.9±1.8a 19.7±0.8a 80.4±1.5ab 26.2±0.7a 87.1±4.2a 10177±645a
OSA1#2 87.0±4.2a 39.0±4.2a 28.8±0.9a 18.6±1.2a 79.9±1.3b 27.2±1.3a 79.4±1.7b 9884±301a
OSA1#3 85.7±5.0a 39.5±2.7a 28.1±1.1a 18.7±1.5a 84.0±1.4a 26.4±0.9a 81.8±1.9ab 10144±382a
osa1-1 84.7±2.7a 25.0±1.3b 21.1±0.7b 17.3±1.5a 67.0±1.5d 25.4±0.9a 83.2±0.3ab 5955±193c

osa1-2 84.3±3.3a 25.3±0.5b 21.3±0.6b 17.4±1.3a 66.0±1.3d 26.2±0.9a 79.5±2.2b 5809±163c

osa1-3 84.7±3.6a 25.3±0.8b 21.4±0.5b 17.6±1.4a 68.0±1.6d 26.1±0.7a 84.5±2.2ab 6386±147c

M-N
(200
kgN/ha)

WT 87.7±4.5a 30.3±1.8b 25.4±0.8b 19.0±1.4ab 76.1±1.1c 26.4±0.9ab 82.6±2.2a 8400±250b
OSA1#1 88.7±1.6a 40.3±1.7a 29.8±1.3a 19.7±0.6ab 84.6±1.2b 24.8±0.4b 88.9±2.9a 11044±490a
OSA1#2 87.0±3.7a 42.0±1.7a 30.4±0.7a 20.1±0.7a 84.1±0.9b 25.3±1.0ab 85.3±3.9a 10995±242a

OSA1#3 88.0±3.2a 40.2±2.3a 29.3±0.7a 19.3±0.5ab 88.6±1.8a 25.8±0.8ab 86.3±6.2a 11494±272a

osa1-1 83.0±4.6a 27.3±1.5b 22.3±0.5c 17.7±1.0b 71.0±1.0d 26.9±0.6a 80.7±1.5a 6822±161c

osa1-2 85.3±2.9a 27.0±0.8b 22.0±0.5c 17.8±0.2ab 71.4±0.8d 25.1±0.4ab 84.4±0.7a 6615±149c

osa1-3 83.3±3.9a 28.0±0.7b 22.8±0.4c 18.5±0.9ab 71.8±0.7d 25.6±0.8ab 83.0±2.4a 6909±119c

H-N
(300
kgN/ha)

WT 93.0±3.9ab 32.0±1.1b 28.0±0.5b 19.4±1.2a 79.0±1.8b 25.1±0.6b 83.9±2.3a 9267±150b
OSA1#1 101.3±4.5a 42.2±1.4a 31.3±1.5a 21.4±2.6a 88.9±2.0a 26.0±1.4ab 84.3±2.5a 12117±569a
OSA1#2 101.7±2.5a 42.0±1.9a 31.6±0.9a 20.9±1.3a 89.6±1.2a 24.9±0.5b 86.9±9.0a 12233±349a

OSA1#3 99.7±2.9ab 42.3±1.9a 31.6±1.3a 20.9±1.7a 93.3±2.5a 27.1±0.4a 79.9±4.6a 12742±533a

osa1-1 93.3±3.6ab 30.8±0.8b 24.9±0.4c 19.7±1.3a 73.4±1.8c 25.8±0.4ab 88.1±0.5a 8263±141c

osa1-2 91.7±1.6b 30.8±0.7b 24.8±0.6c 20.1±1.9a 73.5±1.9c 25.1±0.1b 82.2±0.9a 7477±170c

osa1-3 93.7±3.9ab 31.3±0.8b 24.5±0.5c 19.6±1.9a 73.8±1.4c 25.1±0.8b 84.8±2.3a 7659±141c

Supplementary Table 6 Agronomic traits and grain yields of WT , OSA1-oxs, and osa1 mutant lines in
the field (2016 Hainan ). Plant height, tiller number, panicle numbers, panicle length, spikelets, 1,000
grain weight, filled grains, and grain yield for plants grown in 2016 at Hainan under 0 kg N/ha (N-N), 100
kg N/ha (L-N), 200 kg N/ha (M-N) and 300 kg N/ha (H-N) fertilisation. Differences were evaluated using
one-way ANOVA. Values are the mean ± SEs (n ≥ 3). Letters indicates significant differences at P < 0.05 and the order starts with OSA1-oxs.

Supplementary Table 6

2016
Material
Plant Tiller Panicles Panicles spikelet 1000 grain Filled grains

Yield (kg/ha)Hainan
Height number number per hill length

number per
panicle weight rate (%)

(cm) per hill (cm) (g)
N-N
(0 kgN/ha)

WT 67.2±2.1ab 16.8±0.7b 14.5±0.5c 16.4±0.4a 60.5±2.6bc 25.1±0.2a 78.6±2.5a 3446±127c

OSA1#1 70.8±1.6a 18.6±0.8ab 16.0±0.6abc 15.9±0.7a 65.5±2.2ab 24.5±1.3a 80.6±2.4a 4112±164b

OSA1#2 70.2±1.1ab 20.2±1.0ab 17.3±0.4a 16.8±0.8a 67.7±2.4a 24.3±0.9a 81.7±1.0a 4607±104a

OSA1#3 71.2±2.4a 20.0±2.1ab 16.8±0.9ab 17.1±0.8a 67.5±1.6a 25.5±0.7a 81.4±1.8a 4663±237a

osa1-1 65.6±2.9ab 17.8±1.2ab 14.8±0.8c 16.8±1.4a 51.0±1.4d 24.7±0.7a 79.6±2.4a 2948±155d

osa1-2 65.4±2.3ab 20.0±0.8ab 15.1±0.6bc 16.7±0.8a 57.5±3.1cd 24.4±0.9a 78.4±3.1a 3313±121cd

osa1-3 64.0±3.0b 20.6±1.0a 15.0±0.6bc 16.4±1.0a 56.5±2.9cd 24.4±0.8a 79.0±3.7a 3248±138cd

L-N
(100
kgN/ha)

WT 77.2±3.0a 31.8±1.7b 21.9±0.9b 19.5±1.6a 69.0±4.0bc 25.5±0.3a 80.3±2.4a 6160±251b
OSA1#1 80.4±1.4a 39.0±0.6a 25.0±0.4a 21.1±0.8a 77.2±3.7ab 25.7±0.5a 81.4±3.0a 8032±130a
OSA1#2 82.2±2.0a 38.0±2.3a 25.4±0.6a 19.2±0.7a 76.5±2.9ab 26.2±1.0a 82.1±1.8a 8309±209a
OSA1#3 82.0±2.7a 40.8±0.9a 25.0±0.3a 19.9±1.1a 79.2±3.1a 25.5±0.7a 82.5±0.8a 8298±95a
osa1-1 74.2±4.6a 30.6±2.1b 20.1±0.9bc 16.3±0.3b 64.0±2.5c 24.4±0.8a 80.7±5.0a 5058±224c

osa1-2 75.8±3.9a 29.4±1.8b 19.3±0.7c 16.3±1.5b 61.3±3.3c 24.4±1.2a 79.3±1.5a 4547±164c

osa1-3 75.4±3.7a 29.8±1.0b 17.4±0.6d 16.3±0.4b 65.8±2.0c 25.1±0.5a 82.8±1.6a 4736±165c

M-N
(200
kgN/ha)

WT 83.8±2.3a 33.8±1.4b 24.3±0.8b 19.1±1.2a 73.8±3.1bc 24.7±0.5a 81.0±2.6a 7119±228c
OSA1#1 86.2±2.4a 42.6±1.9a 27.6±0.7a 20.1±1.3a 80.3±2.7ab 25.1±0.5a 83.5±2.4a 9286±235ab
OSA1#2 85.6±1.9a 42.4±2.0a 28.3±0.6a 19.4±1.2a 80.5±2.8ab 24.2±0.5a 82.9±2.1a 9087±203b

OSA1#3 85.0±2.1a 43.4±1.2a 27.4±0.5a 19.1±0.9a 85.5±3.6a 24.5±0.6a 84.4±2.5a 9662±174a

osa1-1 83.4±3.0a 34.8±0.8b 20.6±0.6c 18.7±0.7a 65.7±2.9d 24.1±1.8a 83.5±1.0a 5431±168de

osa1-2 80.0±4.9a 31.6±1.0bc 21.1±0.9c 18.3±0.6a 67.5±2.8cd 25.0±0.2a 81.8±1.3a 5814±245d

osa1-3 81.6±3.4a 29.2±2.2c 20.0±0.6c 18.6±1.8a 64.5±2.2d 24.6±0.7a 80.2±1.7a 5074±162e

H-N
(300
kgN/ha)

WT 89.0±2.4ab 33.8±3.0b 26.8±0.5b 20.1±1.2a 77.3±2.6bc 24.4±.0.8a 84.1±2.1a 8469±153c
OSA1#1 91.0±2.4a 40.0±1.5a 29.1±0.6a 21.1±0.4a 83.3±2.2ab 25.2±0.3a 81.6±5.4a 9933±200b
OSA1#2 92.2±2.1a 40.8±1.0a 29.9±0.5a 21.5±0.8a 85.2±2.0a 25.5±0.7a 83.1±1.5a 10723±184a

OSA1#3 91.2±1.6a 44.0±1.7a 29.3±1.0a 21.1±1.4a 85.7±1.9a 24.7±0.6a 84.0±0.9a 10361±341ab

osa1-1 82.0±1.4b 34.0±1.3b 23.5±0.7c 20.4±1.3a 71.5±1.6c 25.4±1.4a 82.5±0.9a 7017±200de

osa1-2 85.2±3.5ab 34.6±1.6b 23.9±0.7c 20.2±0.6a 72.7±2.5c 26.0±0.3a 82.1±0.9a 7368±229d

osa1-3 85.6±4.5ab 33.8±0.8b 23.4±0.7c 20.4±0.6a 73.2±3.8c 24.3±0.6a 80.8±2.1a 6684±200e

Supplementary Table 7 List of primers used in this study.

Supplementary Table 7

Gene name
Forward (F)/
Reverse (R) Sequences (5’–3’) Application

OSA1
F AGGTCGACTCTAGAGGATCCTAGGGTCAGCATAGCAGT

TransformationR CTCAGATCTACCATGGTACCGGAGGCGACCTCCCACACCT

OsActin
F GGAACTGGTATGGTCAAGGC

RT-PCRR AGTCTCATGGATAACCGCAG

OSA1
F TGGCTGGCATGGATGTTCTT

RT-PCRR TTCCTAGACGACGCCCTGTT

OsActin
F TTATGGTTGGGATGGGACA

quantitative RT-PCRR AGCACGGCTTGAATAGCG

OsGAPDH
F TCAAATGCTAGCTGCACCAC

quantitative RT-PCRR GCAGTGATGGCATGAACAGT

OSA1
F GTGTTTGGGTTTATGCTGCT

quantitative RT-PCRR GTATCCACCCAGCACAACTC

OSA2
F ACTGAGCCAGGCCTTAGTGT

quantitative RT-PCRR TCATTGAGGATGGCAATGAT

OSA3
F GAGGAGAGGGAGCTCAAGTG

quantitative RT-PCRR CACAACCGATTCTACATGCC

OSA4
F TCAGCATCGTCACCTTCTTC

quantitative RT-PCRR CTCTTCCCGTAGTCCAGCTC

OSA5
F TCTGGCTCTACAGCATCGTC

quantitative RT-PCRR TCCCGTAATCCTTCTTGCTC

OSA6
F TCAGCGTGGTGACCTACTTC

quantitative RT-PCRR CCCGTAGTCGTTCTTGTTCA

OSA7
F GGGCTGGGCTGGCGTTATCT

quantitative RT-PCRR TTGAAGAGCGTGTTGGAGGC

OSA8
F TCAACCAAATGGCTGAAGAG

quantitative RT-PCRR CCACAGATTCCACCTTTCCT

OSA9
F GTCCTTCCTCGAGAGACCTG

quantitative RT-PCRR GCGTAGAACACCAGGCTGTA

OSA10
F GCGGATGAAGAACTACACCA

quantitative RT-PCRR CTTGGAGATGGTCATGATGG

OsGS1.2
F TGTTTCTCCTCATCCCTGC

quantitative RT-PCRR TCACAGTCCTCGCTTTGC

OsGS2
F GGAGAGGTCATGCCTGGTCAGT

quantitative RT-PCRR ACTACACCAGCCTGCTCCGTTA

OsNADH-GOGAT1
F GTGCAGCCTGTTGCAGCATAAA

quantitative RT-PCRR CGGCATTTCACCATGCAAATC

OsNADH-GOGAT2
F CCTGTCGAAGGATGATGAAGGTGAAACC

quantitative RT-PCRR TGCATGGCCCTACTATCTTCGCATCA

OsAMT1.1
F GGTTTCTCTCCCTCTCCGAT

quantitative RT-PCRR CCACCTTCACACCACACATT

OsGRF4
F GAAAGCCTGTGGAAACGCA

quantitative RT-PCRR CAACGCCGAGCCAAATGAG

s41467-021-20964-4-1

Plasma membrane H+-ATPase overexpression increases rice yield via simultaneous enhancement of nutrient uptake and photosynthesis
Results
PM H+-ATPase mediates NH4+ absorption
Phenotype of OSA1 overexpression and mutation rice lines
Overexpression of PM H+-ATPase enhanced NH4+ uptake
PM H+-ATPase overexpression enhanced stomatal conductance and photosynthetic activity
Genome-wide effect of OSA1 on gene expression
Overexpression of PM H+-ATPase promoted field production
Discussion
Methods
Plant cultivation
Construction of the overexpression vector and transgenic plants
Quantitative reverse-transcription PCR
Immunodetection
Measurement of PM H+-ATPase activity
15N absorption rates in roots of WT, OSA1-overexpressing and osa1 plants
Nutrient element analysis of plant samples
Stomatal observation
Gas-exchange measurements
Stomatal density and size
High-throughput RNA-seq analysis
Detection of rhizosphere acidification in roots
Quantification of H+ extrusion rate
Reporting summary
Data availability
References
Acknowledgements
Author contributions
Competing interests
Additional information

41467_2021_20964_MOESM1_ESM

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