J Plant Res (2017) 130:909–927
DOI 10.1007/s10265-017-0950-4
REGULAR PAPER
Enhanced photosynthetic capacity increases nitrogen metabolism
through the coordinated regulation of carbon and nitrogen
assimilation in Arabidopsis thaliana
Kumi Otori1,2 · Noriaki Tanabe1,2 · Toshiki Maruyama1 · Shigeru Sato3 ·
Shuichi Yanagisawa2,3 · Masahiro Tamoi1,2 · Shigeru Shigeoka1,2 
Received: 19 January 2017 / Accepted: 12 April 2017 / Published online: 3 May 2017
© The Botanical Society of Japan and Springer Japan 2017
Abstract  Plant growth and productivity depend on inter-
actions between the metabolism of carbon and nitrogen.
The sensing ability of internal carbon and nitrogen metabo-
lites (the C/N balance) enables plants to regulate metabo-
lism and development. In order to investigate the effects
of an enhanced photosynthetic capacity on the metabolism
of carbon and nitrogen in photosynthetically active tissus
(source leaves), we herein generated transgenic Arabidop-
sis thaliana plants (ApFS) that expressed cyanobacterial
fructose-1,6-/sedoheptulose-1,7-bisphosphatase in their
chloroplasts. The phenotype of ApFS plants was indistin-
guishable from that of wild-type plants at the immature
stage. However, as plants matured, the growth of ApFS
plants was superior to that of wild-type plants. Starch lev-
els were higher in ApFS plants than in wild-type plants at
2 and 5  weeks. Sucrose levels were also higher in ApFS
plants than in wild-type plants, but only at 5 weeks. On the
other hand, the contents of various free amino acids were
lower in ApFS plants than in wild-type plants at 2 weeks,
Kumi Otori and Noriaki Tanabe contributed equally to this work.
Electronic supplementary material  The online version of this
article (doi:10.1007/s10265-017-0950-4) contains supplementary
material, which is available to authorized users.
* Masahiro Tamoi
tamoi@nara.kindai.ac.jp
1 ductivity, the ratio between C and N must be tightly coor-
Department of Advanced Bioscience, Faculty of Agriculture,
Kindai University, Nakamachi, Nara 631‑8505, Japan
2 Bush 2001).
Core Research for Evolutional Science and Technology
(CREST), Japan Science and Technology Agency,
Kawaguchi 332‑0012, Japan
3
Biotechnology Research Center, The University of Tokyo,
Yayoi 1‑1‑1, Bunkyo‑ku, Tokyo 113‑8657, Japan
but were similar at 5  weeks. The total C/N ratio was the
same in ApFS plants and wild-type plants, whereas nitrite
levels increased in parallel with elevations in nitrate reduc-
tase activity at 5 weeks in ApFS plants. These results sug-
gest that increases in the contents of photosynthetic inter-
mediates at the early growth stage caused a temporary
imbalance in the free-C/free-N ratio and, thus, the feedback
inhibition of the expression of genes involved in the Cal-
vin cycle and induction of the expression of those involved
in nitrogen metabolism due to supply deficient free amino
acids for maintenance of the C/N balance in source leaves
of ApFS plants.
Keywords  Biomass · Calvin cycle · C/N balance ·
Fructose-1,6-bisphosphatase · Photosynthesis ·
Sedoheptulose-1,7-bisphosphatase
Introduction
Plants use energy from light to convert carbon dioxide
into carbon (C) metabolites, such as starch and sugars, and
take up nitrogen (N) by their roots in either of two forms,
­NH4+ or ­NO3−. These C and N metabolites are required for
the biosynthesis of macromolecules, including proteins,
nucleic acids, fatty acids, and a large number of secondary
metabolites. Since interactions between the metabolism of
C and N in plants is important for optimal growth and pro-
dinated according to the cellular C-N status (Coruzzi and
Therefore, plants possess a regulatory system to coor-
dinate the uptake and distribution of these nutrients in
response to metabolic and environmental cues (Coruzzi and
Zhou 2001). In Arabidopsis thaliana plants, the addition of
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glucose and/or nitrate to a medium has been shown to alter
the levels of genes involved in the metabolism of C and N
(Price 2004; Wang et  al. 2000, 2003). Recent studies on
C:N sensing and signaling in plants, which involved molec-
ular-genetic, genomic, and cell biological approaches, have
begun to uncover the regulation mechanisms between C
and N (Coruzzi and Zhou 2001; Gutiérrez et al. 2007; Kang
and Turano 2003; Maruta et  al. 2010; Tamoi et  al. 2010;
Yanagisawa et al. 2004; Zheng 2009). Furthermore, a plant
nuclear gene (GLB1), which encodes a PII-like protein in
Arabidopsis, plays a role in the coordinated regulation of
C/N metabolism and is also thought to function in C/N
signaling in plants (Hsieh et al. 1998). Sato and co-workers
identified a C/N response mutant that survived in medium
containing excess glucose under limited N conditions. The
response gene encoding a Ring-H2 type ubiquitin ligase
(ATL31) also plays a key role in regulating the response to
C/N conditions during the early post-germinative growth in
Arabidopsis (Sato et  al. 2009). Glutamate:glyoxylate ami-
notransferase (GGT) catalyzes the reaction of glutamate
and glyoxylate to 2-oxoglutarate and glycine (Igarashi et al.
2003). ggt1 mutants showed low N assimilation, leading
to a decreased Rubisco content; however, the C/N balance
was maintained in these mutants (Dellero et al. 2015). The
glutamate receptor (AtGLR1.1) functions as a C/N regula-
tor that regulates C/N metabolism and distinct physiologi-
cal processes such as germination through the control of
abscisic acid (ABA) biosynthesis (Ferrario-Mery et  al.
2005; Kang and Turano 2003). Additionally, ABA-insen-
sitive mutant 1 (abi1) exhibited more sensitive responses
to high C/low N stress, while the overexpression of ABI1
resulted in successful post-germination under these condi-
tions. The transcript levels of ABA-signaling markers, such
as RD29b, LEA3-4, and TSPO, were suppressed in ABI1-
overexpressing plants by high C/low N stress, suggesting
cross-talk between C/N and ABA signaling pathways under
the control of ABI1 (Lu et al. 2015).
We previously reported that transgenic tobacco and
lettuce plants that overexpressed fructose-1,6-/sedohep-
tulose-1,7-bisphosphatase (FBP/SBPase) of Synechococ-
cus elongatus PCC7942 (S.7942) in their chloroplasts had
an enhanced C ­ O2 assimilation rate and increased biomass
production (Ichikawa et  al. 2010; Miyagawa et  al. 2001;
Yabuta et  al. 2008). Furthermore, single or combinational
overexpression of sedoheptulose-1,7-bisphosphatase,
fructose-1,6-bisphosphate aldolase (FBPald) and cyano-
bacterial putative-inorganic carbon transporter B (ictB)
in tobacco plants also caused increased photosynthesis
and biomass production (Lefebvre et  al. 2005; Rosenthal
et  al. 2011; Simkin et  al. 2015). These transgenic plants
did not show the N-deficient phenotypes at mature stage.
These findings suggested that an essential N component for
plant growth, such as an amino acid and protein, should be
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J Plant Res (2017) 130:909–927
increased in photosynthesis-elevated transgenic plants at
a certain point. Based on these findings, we assumed that
an enhanced photosynthetic capacity at the immature stage
in transgenic plants affected cell differentiation, assimilate
partitioning, carbohydrate compartmentation, and various
metabolic pathways including N metabolism. Therefore,
these photosynthesis-elevated transgenic plants may be an
attractive tool for analyzing the relationship between the
metabolism of C and N at the immature and mature stages.
In the present study, we generated a transgenic Arabi-
dopsis plant (ApFS) that expressed FBP/SBPase in its chlo-
roplasts in order to clarify the effects of alterations in pho-
tosynthetic capacity on various metabolic pathways and the
expression of genes involved in the metabolism of C and N.
The photosynthetic C ­ O2 fixation rate was higher in ApFS
plants than in wild-type plants under saturating irradiance
conditions, resulting in a larger total dry weight in ApFS
plants than in wild-type plants. We analyzed the expression
of various genes and metabolites involved in the metabo-
lism of C and N in ApFS plants at the immature (2 weeks)
and mature (5 weeks) stages in order to clarify the effects
of an enhanced photosynthetic capacity on nitrogen metab-
olism in plants at various growth stages.
Materials and methods
Plant materials and growth conditions
The seeds of Arabidopsis thaliana (L.) Heynh. wild-
type (ecotype Columbia-0) and transgenic plant (ApFS)
lines were sown on a 1:1 perlite/soil mix or Murashige
and Skoog (MS) medium after stratification at 4 °C for
at least 48  h. Sterilized seeds of A. thaliana were placed
on Murashige and Skoog (MS) sucrose-free gellan gum
medium or a 1:1 perlite/soil mix in 6-cm pots. Tsuchi-
taro (Sumirin agro-products co., Ltd) which contains N
8.4  g  l−1, P 136  g  l−1, K 4.9  g  l−1 was used as a soil for
cultivation. Two-week-old plants grown on pots were trans-
ferred to 10-cm pots. To avoid the effects of insufficient
of nutrients on nitrogen metabolism and gene expression,
all plants grown in pots were given Hoagland modified
solution, which contained 10  mM (­NH4)H2PO4, 60  mM
­KNO3, 20  mM ­MgSO4, 40  mM Ca(NO3)24H2O, 0.25%
Fe(III)EDTA, and micronutrients, 3 ml plant−1 day−1 until
2  weeks and 20  ml  plant−1  day−1 after that. Since FBP/
SBPase was continuously expressed by the CaMV35S pro-
moter in transgenic plants, all plants were cultured under
the following conditions to avoid secondary effects by the
futile cycle and circadian responses to gene expression
(Velez-Ramirez et  al. 2011): 24-h continuous white light
(ca. 100–150 µmol photons m ­ −2 s−1), temperature of 23 °C,
and air humidity of 60%.
J Plant Res (2017) 130:909–927 911
Generation of transgenic plants
In order to generate Arabidopsis plants overexpressing
cyanobacterial FBP/SBPase under the control of the cauli-
flower mosaic virus 35S promoter, a cDNA clone contain-
ing the complete cyanobacterial FBP/SBPase open read-
ing frame was fused with the transit peptide of a tomato
rbcS3C gene and cloned into pBI101 (Fig. S1a). The result-
ant plasmid was introduced into wild-type Arabidopsis
by Agrobacterium tumefaciens (C58)-mediated transfor-
mation utilizing the floral dip method (Clough and Bent
1998). Transgenic plants were selected on 0.8% (w/v) agar
plates containing MS medium and 50 µg ml−1 kanamycin.
Homozygous T3 generation plants harboring the transgene
were used in further analyses.
Measurement of enzymatic activities
Rosette leaves (60–100 mg FW) were harvested and ground
to a fine powder in liquid ­N2 using a mortar and pestle.
Activities of FBPase, NADPH-dependent glyceralde-
hyde-3-phosphate dehydrogenase (­NADP+-GAPDH) and
phosphoribulokinase (PRK) and Rubisco were assayed as
previously described (Miyagawa et  al. 2001; Tamoi et  al.
1998). Activites of nitrate reductase (NR) were assayed as
described in Konishi and Yanagisawa (2011). Glutamine
synthetase (GS) activity was assayed as described in Schei-
ble et al. (1997).
Isolation of stromal proteins and immunoblotting
The extraction of stromal protein was carried out according
to Aronsson and Jarvis (2002) with some modifications.
All procedures were carried out at 4 °C. Intact chloroplasts
were isolated from laws of wild-type and ApFS plants at
2  weeks old. Plants were homogenized with a small rotor
in 100  ml isolation buffer (0.3  M sorbitol, 5  mM ­MgCl2,
5 mM EDTA, 20 mM HEPES-KOH, pH8.0). The homoge-
nate was filtered through a double layer of Miracloth
(Merck Millipore, Darmstadt, Germany). The homogen-
ate was centrifuged at 1000g for 5 min and the pellet was
resuspended in 3 ml isolation buffer. The resuspended chlo-
roplasts were loaded onto a two-step Percoll gradient. Two-
step gradients consisted of a bottom layer comprising 75%
Percol solution (w/v) and a top layer comprising 30% Per-
coll solution (w/v). Two-step gradients were centrifuged in
a swing-out rotor at 1500g for 10 min. Isolated chloroplasts
were ruptured by hypotonic shock in buffer (10 mM M ­ gCl2
and 20  mM HEPES, pH 7.6) on ice for 10  min. Stroma
proteins were separated from thylakoid membranes by cen-
trifugation (20,000g for 10  min at 4 °C). Stromal proteins
were concentrated using Amicon Ultra Centrifugal Filters
3  K (Merc Millipore, Darmstadt, Germany), and protein
concentrations were determined using Bradford protein
assay.
Supernatants containing solubilized proteins were sepa-
rated by SDS–PAGE on 12% polyacrylamide gels. Sepa-
rated proteins were electrophoretically transferred to a
Hybond-P PVDF membrane (GE Healthcare, UK). The
proteins of cyanobacterial FBP/SBPase and ascorbate per-
oxidase (sAPX and tAPX: chloroplastic isoform, cAPX:
sytosolic isoform) were detected using a specific monoclo-
nal antibody as previously described by Miyagawa et  al.
(2001) and Ishikawa et al. (1996), respectively. The primary
antibody was detected using a horseradish peroxidase-
linked, antimouse, goat secondary antibody (Bio-Rad).
Measurement of photosynthesis
The rates of photosynthesis were measured using a LI-
6400XT portable infrared gas analyzer (Li-Cor, Lin-
coln, NE, USA) under a fixed blue-red light-emitting
diode (LED) light source, 360  ppm ­CO2, temperature of
25 °C, and relative humidity of 65%. Measurements were
taken from a leaf area of 0.785 cm2 and at actinic light of
0–1000 µmol photons m−2 s−1 using the instrument’s auto
program function.
Metabolite analysis
Rosette leaves (100  mg FW) were immediately placed
into liquid ­N2, ground in liquid N
­ 2 using a pestle and mor-
tar with 1  ml of 6% perchloric acid, and centrifuged at
15,000×g for 5 min. Pellets were used for starch measure-
ments and supernatants were enzymatically examined for
sugars (glucose, fructose, and sucrose) as described by Gal-
tier et al. (1995).
Nitrate and nitrite levels were analyzed enzymatically
and spectrophotometrically as described by Konishi and
Yanagisawa (2011) and measured using calibration curves
obtained with standard mixtures. Glutamate levels were
analyzed spectrophotometrically with a l-glutamic acid
measuring kit (Roche Applied Science) according to the
manufacturer’s instructions. Glutamine was catalyzed by
glutaminase (Daiwa Fine Chemicals, Japan) and formed
glutamic acid then analyzed spectrophotometrically.
Ammonia was extracted from approximately 100  mg of
leaves and analyzed spectrophotometrically by indophe-
nol blue absorptiometry using an ammonia measuring kit
(Wako Pure Chemical Industries, Japan) according to the
manufacturer’s instructions.
The extraction and analysis of various amino acids by
capillary electrophoresis-mass spectrometry (CE-MS) were
performed according to previous studies (Hachiya et  al.
2014; Sato et al. 2004; Sato and Yanagisawa 2010).
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912 J Plant Res (2017) 130:909–927

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 J Plant Res (2017) 130:909–927 913
◂Fig. 1  Phenotype of wild-type and ApFS plants. Plants were grown
for 2  weeks (a) or 5  weeks (d) on soil. b Number of rosette leaves
in wild-type and ApFS plants grown for 2 weeks on soil. Error bars
indicate SE (n = 40–60 plants). There was no statistically significant
difference between wild-type and ApFS plants. c Shoot dry weights
of wild-type and ApFS plants grown for 2 weeks on soil. Error bars
indicate SE (n = 4–5 plants). There was no statistically significant dif-
ference between wild-type and ApFS plants. e Number of primary
rosette branches of wild-type and ApFS plants grown for 5 weeks on
soil. Error bars indicate SE (n = 8–12 plants). There was no statis-
tically significant difference between wild-type and ApFS plants. f
Number of lateral branches of wild-type and ApFS plants grown for
5 weeks on soil. Error bars indicate SE (n = 8–12 plants). g Number
of rosette leaves of wild-type and ApFS plants grown for 5 weeks on
soil. Error bars indicate SE (n = 8–12 plants). h Shoot dry weights
of wild-type and ApFS plants grown for 5 weeks on soil. Error bars
indicate SE (n = 8–12  plants). Asterisks indicate that mean values
were significantly different from those in wild-type plants by a one-
way ANOVA with Dunnett’s post hoc test (*P < 0.05, **P < 0.01).
The entire experiment was replicated three times with similar results
Analyses of total C and total N were performed with
whole plants using a Thermo Fisher Scientific Model Flash
EA1112 (SI SIENCE, Japan) and CHN analyzer vario EL
(Sumika Chemical Analysis Service, Osaka, Japan).
Quantitative real‑time PCR experiments
Quantitative real-time PCR (q-PCR) was performed with
an Applied Biosystems 7300 Real Time PCR System and
Roche LightCycler 96 system, using the SYBR Premix Ex
Taq (Takara) and FastStart Universal SYBR Green Master
(ROX). Primer pairs for q-PCR are shown in Supplemental
Table S1. Actin2 mRNA was used as an internal standard in
all experiments.
Statistical analysis
Statistical analyses were performed using EZR soft-
ware (Saitama Medical Center, Jichi Medical University,
Saitama, Japan), which is a graphical user interface for R
(The R Foundation for Statistical Computing, Vienna, Aus-
tria) (Kanda 2013). More precisely, it is a modified version
of R commander designed to add statistical functions fre-
quently used in biostatistics.
Results
Generation of transgenic Arabidopsis having FBP/
SBPase
We generated transgenic Arabidopsis plants that
expressed cyanobacterial FBP/SBPase in their chloro-
plasts and selected three T3-generation lines (ApFS-11,
ApFS-19, and ApFS-9) displaying different FBP/SBPase
overexpression patterns (Fig. S1b). The total activities of
FBPase derived from endogenous FBPase (plastidic and
cytosolic) and cyanobacterial FBP/SBPase were 1.4- to
2.6-fold higher in ApFS plants than in wild-type plants.
No significant differences were observed in the total activ-
ities of ­NADP+-GAPDH and PRK between ApFS plants
and wild-type plants (Fig. S1c). The activation state of
Rubisco was 1.2-fold higher in ApFS plants than in wild-
type plants, whereas no significant differences were noted
in the total activities of Rubisco, indicating an increase
in the in vivo activation state of Rubisco in ApFS plants
(Fig. S1c). Two weeks after planting, the growth of ApFS
plants was indistinguishable from that of wild-type plants
(Fig. 1a). There was no significant difference in the num-
ber of rosette leaves or shoot dry weights between wild-
type and ApFS plants at 2  weeks (Fig.  1b, c). Although
no significant differences were observed in the number
of primary rosette branches between wild-type and ApFS
plants, the numbers of lateral branches and rosette leaves
were significantly higher in ApFS plants than in wild-
type plants 5  weeks after planting (Fig.  1d, f, g). ApFS
plants also had higher shoot dry weights associated with
larger numbers of lateral branches and rosette leaves than
wild-type plants 5 weeks after planting (Fig. 1h). Photo-
synthetic activities in 5-week-old plants were measured
under atmospheric conditions (360 ppm ­CO2) and various
light intensities (50–1000 μmol photons m−2 s−1). At irra-
diances less than 100  μmol  photons  m−2  s−1, photosyn-
thetic activities were not significantly different between
wild-type and ApFS plants. However, at irradiances
greater than 250  μmol  photons  m−2  s−1, photosynthetic
activities were significantly stronger in ApFS plants than
in wild-type plants (Fig.  2a). No significant difference
was observed in the content of chlorophyll or anthocya-
nin between wild-type and ApFS plants (Fig. 2b).
Modulation of various sugars in transgenic Arabidopsis
plants
In order to investigate the effects of an enhanced photo-
synthetic capacity on C metabolism, we measured vari-
ous sugar levels in rosette leaves at immature (2-week-
old) and mature (5-week-old) stages. Starch levels at
immature and mature leaves were higher in ApFS plants
than in wild-type plants (Fig.  3a). At immature stage,
no significant difference was observed in sucrose levels
between wild-type and ApFS plants. However, sucrose
levels were higher in ApFS plants than in wild-type
plants at mature stage (Fig.  3b). On the other hand, no
significant difference was observed in glucose or fructose
levels between wild-type and ApFS plants at both imma-
ture and mature stage (Fig. 3c, d).
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914 J Plant Res (2017) 130:909–927

Fig. 2  Photosynthetic param-
eters of wild-type and ApFS
plants grown for 5 weeks on
soil. a Effects of increasing irra-
diance on ­CO2 assimilation at
360 ppm ­CO2, 25 °C, and 60%
relative humidity. Error bars
indicate SE (n = 5–6 plants). b
Total chlorophyll (left panel)
and anthocyanin (light panel).
Error bars indicate SE (n = 4–6
plants). Asterisks indicate that
mean values were signifi-
cantly different from those in
wild-type plants by a one-way
ANOVA with Dunnett’s post
hoc test (*P < 0.05, **P < 0.01).
The entire experiment was repli-
cated three times with similar
results

Effects of an enhanced photosynthetic capacity
on the expression of genes involved in the Calvin cycle
and starch and sucrose biosynthesis
The high assimilation of C in transgenic plants may affect
the expression of genes involved in C metabolism. In order
to address this possibility, we analyzed the transcript levels
of genes encoding chloroplastic FBPase, SBPase, RBCS1A,
and PRK at immature and mature stages using q-PCR. At
both immature and mature stage, the transcript levels of

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J Plant Res (2017) 130:909–927 915
Fig. 3  Contents of starch (a), sucrose (b), glucose (c), and fructose
(d) in rosette leaves of wild-type and ApFS plants grown on soil at
2- (left panel) and 5-week-old stage (right panel). Error bars indicate
SE (n = 5–10 plants). Asterisks indicate that mean values were signifi-
these genes were lower in ApFS plants than in wild-type
plants (Fig. S2). Since an enhanced photosynthetic capacity
increased starch and sucrose levels in ApFS plants grown
for 5 weeks (Fig. 3a, b), we examined the transcript levels
of starch synthase (SS1-4), ADP-glucose pyrophosphory-
lase large subunit (APL1-4), ADP-glucose pyrophosphory-
lase small subunit (APS1), Beta-amylase (BAM3), cytosolic
FBPase (cFBPase), sucrose-phosphate synthase (SPSA and
SPSC), and sucrose synthetases (SUS1, SUS2, SUS3, SUS4,
and SUS5) (Fig.  4). The transcript levels of SS1, SS2,
SPSC, and SUS3 were consistently higher in ApFS plants
than in wild-type plants. The transcript levels of BAM3
and APL2 were higher in ApFS plants than in wild-type
plants at immature stage, whereas these levels were lower
in ApFS plants than in wild-type plants at mature stage.
The transcript levels of SS3 and SS4 were similar in ApFS
and wild-type plants at immature stage, but were signifi-
cantly lower in mature ApFS plants than those of wild-type
plants. The transcript levels of APL1 and APL3 were sig-
nificantly higher in ApFS plants than in wild-type plants at
immature stage, whereas these levels were similar in ApFS
and wild-type plants at mature stage. The transcript levels
of cFBPase and SUS5 were similar in ApFS and wild-type
plants at immature stage, but were significantly higher in
ApFS plants than in wild-type plants at mature stage. The
transcript levels of SUS1 were significantly lower in ApFS
plants than in wild-type plants at immature stage, whereas
this level was similar in ApFS and wild-type plants at
mature stage. No significant differences were observed in
the transcript levels of APS1, SPSA, and SUS4 in wild-type
and ApFS plants at both immature and mature stage. The
transcript levels of SUS2 and SUS6 were below or near the
detection limit at both immature and mature stage.
cantly different from those in wild-type plants by a one-way ANOVA
with Dunnett’s post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001).
The entire experiment was replicated three times with similar results
Effects of an enhanced photosynthetic capacity
on PEPC
Phosphoenolpyruvate carboxylase (PEPC) 1 and 2 dou-
ble knockout mutants showed prominent changes in C/N
metabolism and plant growth in Arabidopsis, suggesting
that PEPC in leaves plays a crucial role in modulating the
balance of C and N metabolism (Shi et al. 2015). The high
assimilation of C in ApFS plants may affect the expres-
sion levels of PEPC and its activity. The transcript lev-
els of PEPC1 tended to be higher in ApFS plants than in
wild-type plants at immature stage, but were lower in ApFS
plants than in wild-type plants at mature stage (Fig. 5a). No
significant changes were observed in the transcript levels of
PEPC2 between wild-type and ApFS plants at both imma-
ture and mature stage. However, the transcript levels of
PEPC2 were lower in ApFS plants than in wild-type plants
at mature stage (Fig. 5a). Since the relative expression lev-
els of PEPC3 and PEPC4 in leaves were lower than those
of PEPC1 and PEPC2 (Shi et al. 2015), the expression lev-
els of PEPC3 and PEPC4 were below or near the detec-
tion limit at both immature and mature stage. Despite the
differences observed in the transcript levels of PEPC1 and
PEPC2 between wild-type and ApFS plants, no significant
difference was noted in the total activities of PEPC between
wild-type and ApFS plants at both immature and mature
stage (Fig. 5b).
Effects of an enhanced photosynthetic capacity on N
metabolism
In order to establish whether an enhanced photosynthetic
capacity contributed to maintaining the C/N balance, we
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916
Fig. 4  Expression of genes involved in sucrose and starch synthe-
sis in rosette leaves of wild-type and ApFS plants grown on soil at
2- (left panel) and 5-week-old stage (right panel). The transcript lev-
els of these genes were measured by a q-PCR analysis. Error bars
indicate SE (n = 6–9  plants). Asterisks indicate that mean values
13
J Plant Res (2017) 130:909–927
were significantly different from those in wild-type plants by a one-
way ANOVA with Dunnett’s post hoc test (*P < 0.05, **P < 0.01,
***P < 0.001). The entire experiment was replicated three times with
similar results
J Plant Res (2017) 130:909–927 917
Fig. 5  Expression of PEPC genes (a) and PEPC activity (b) in
rosette leaves of wild-type and ApFS plants grown on soil at 2- (left
panel) and 5-week-old stage (right panel). The transcript levels of
these genes were measured by a q-PCR analysis. Error bars indi-
cate SE [n = 6–9  plants (a), n = 4–6  plants (b)]. Asterisks indicate
measured the total contents of C and N in leaves from wild-
type and ApFS plants. No significant difference was noted
in the total contents of C and N or in the C/N ratio between
wild-type and ApFS plants grown for 5  weeks (Table  1).
Furthermore, no significant difference was observed in
the contents of total soluble protein between wild-type
and ApFS plants at both immature and mature stage (Fig.
S3). No significant difference was observed in the levels of
nitrate between wild-type and ApFS plants at both imma-
ture and mature stage (Fig.  6a). At immature stage, there
was no significant difference in the levels of nitrite between
wild-type and ApFS plants. At mature stage, nitrite lev-
els were higher in ApFS plants than in wild-type plants
(Fig.  6b). Although there was no significant difference in
the total activity of NR between wild-type and ApFS plants
at immature stage, it was higher in ApFS plants than those
of wild-type plants at mature stage (Fig.  6f), suggesting
that the uptake capacity of nitrate increased as ApFS plants
matured.
We measured the concentration of N-containing metab-
olites (free amino acids) in wild-type and ApFS plants
grown for 2 or 5  weeks. The level of the ammonium ion
was not altered between wild-type and ApFS plants at
2  weeks, but was lower in ApFS plants than in wild-type
plants at 5 weeks (Fig. 6c). The content of glutamine was
lower in ApFS plants than in wild-type plants at 2 weeks,
and was higher in ApFS plants than in wild-type plants at
5  weeks (Fig.  6d). No significant difference was observed
in the levels of glutamate or total activities of GS between
ApFS and wild-type plants at 2 and 5  weeks (Fig.  6e, g).
We also measured C and N metabolite contents in plants
grown for 2 weeks on MS medium (Table S2). Glycine and
FBP levels were significantly higher in ApFS19 and ApFS9
plants, and RuBP levels were higher in ApFS19 plants than
that mean values were significantly different from those in wild-type
plants by a one-way ANOVA with Dunnett’s post hoc test (*P < 0.05,
**P < 0.01, ***P < 0.001). The entire experiment was replicated three
times with similar results
in wild-type plants (Table  S2). Similar to plants grown
on MS medium, the amounts of a large number of amino
acids, fumarate, succinate, and N­ AD+ were lower in ApFS
plants than in wild-type plants at 2 weeks.
We next examined the transcript levels of genes involved
in N metabolism at immature and mature stage using
q-PCR (Fig.  7). The transcript levels of asparagine syn-
thetase 1 (ASN1) were consistently higher in ApFS plants
than in wild-type plants. At immature stage, the transcript
levels of these genes, except for those of ASN1 in ApFS
plants, were similar or lower than those in wild-type plants.
However, the transcript levels of nitrate reductase 1 (NR1)
and nitrate reductase 2 (NR2) and glutamine synthase 1
(GLN1;1) were higher in ApFS plants than in wild-type
plants at mature stage. The transcript levels of asparagine
synthetase 2 (ASN2) were lower in ApFS plants than in
wild-type plants at mature stage. The transcript levels of
ferredoxin-glutamate synthase1 (Fd-GOGAT1) and glu-
tamine synthase2 (GLN2) were lower in ApFS plants than
in wild-type plants at immature stage. However, no sig-
nificant difference was observed in the transcript levels of
nitrite reductase1 (NIR1), asparagine synthetase 3 (ASN3),
GLN1;2, GLN1;3, Fd-GOGAT2 or NADH-dependent glu-
tamate synthase (NADH-GOGAT1) between wild-type and
ApFS plants at both immature and mature stage (Fig. 7).
Effects of an enhanced photosynthetic capacity
on the expression of genes involved in nitrate transport
We examined the transcript levels of nitrate transporters
(NRT1. 1, NRT1.2, NRT1.3, NRT1.4, NRT1.5, NRT1.7,
NRT1.8, NRT1.9, NRT 1.11, NRT1.12, NRT2.1, NRT2.3,
NRT2.5, NRT2.7, and NRT3.1) between wild-type and
ApFS plants at immature and mature stage (Fig. 8). The
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918
Fig. 6  Contents of the nitrate ion (a), nitrite ion (b), free ammonium
ion (c), free glutamine (d), and free glutamate (e), and activities of
NR (f) and GS (g) in rosette leaves of wild-type and ApFS plants
grown on soil at 2- (left panel) and 5-week-old stage (right panel).
Error bars indicate SE (n = 4–5 plants). Asterisks indicate that mean
transcript levels of NRT1.4 and NRT1.11 were consist-
ently higher in ApFS plants than in wild-type plants. In
contrast, the transcript levels of NRT2.3 were consist-
ently lower in ApFS plants than in wild-type plants. The
transcript levels of NRT1.8 and NRT1.12 were higher in
ApFS plants than in wild-type plants at immature stage,
but were lower in ApFS plants than in wild-type plants at
mature stage. The transcript levels of NRT1.5 and NRT2.5
were higher in ApFS plants than in wild-type plants at
immature stage, but were similar in wild-type and ApFS
plants at mature stage. Conversely, the transcript levels
13
J Plant Res (2017) 130:909–927
values were significantly different from those in wild-type plants by a
one-way ANOVA with Dunnett’s post hoc test (*P < 0.05, **P < 0.01,
***P < 0.001). The entire experiment was replicated three times with
similar results
of NRT1.1 and NRT2.7 were lower in ApFS plants than
in wild-type plants at immature stage, but were similar
in ApFS and wild-type plants at mature stage. The tran-
script levels of NRT2.1 and NRT3.1 were similar in wild-
type and ApFS plants at immature stage, but were higher
or lower, respectively, in ApFS plants than those of wild-
type plants at mature stage. No significant differences
were observed in the transcript levels of NRT1.2, NRT1.3,
NRT1.7, or NRT1.9 between ApFS and wild-type plants
at both immature and mature stage.
J Plant Res (2017) 130:909–927 919

Table 1  Total C, N and C/N ratio of wild-type and ApFS plants lateral branches at 5  weeks increased, and, thus, shoot
Total-C (%) Total-N (%) C/N ratio
dry weight was higher in ApFS plants than in wild-type
plants at 5  weeks (Fig.  1). Photosynthetic activity was
2 weeks greater in ApFS plants than in wild-type plants (Fig.  2),
 Wild type 37.8 ± 0.7 6.9 ± 0.2 5.5 ± 0.1 indicating that the increment observed in ApFS plant
 ApFS-11 37.0 ± 1.5 6.2 ± 0.7 6.1 ± 0.7 growth was due to increased photosynthetic capacity.
 ApFS-19 38.1 ± 0.8 6.9 ± 0.3 5.5 ± 0.1 Transgenic tobacco plants with increased SBPase
 ApFS-9 38.6 ± 0.4 6.7 ± 0.4 5.8 ± 0.4 activity were previously shown to exhibit a higher pho-
5 weeks tosynthetic capacity and biomass production than those
 Wild type 35.5 ± 1.5 5.6 ± 0.4 6.4 ± 0.6 of wild-type plants (Lefebvre et  al. 2005; Rosenthal et  al.
 ApFS-11 38.4 ± 1.4 4.6 ± 1.0 9.1 ± 1.8 2011). Moreover, when plants grew under field condi-
 ApFS-19 35.7 ± 0.9 5.0 ± 0.4 7.3 ± 0.4 tions at elevated ­CO2, leaf N contents were significantly
 ApFS-9 35.9 ± 1.6 5.5 ± 0.5 6.6 ± 1.0 decreased in transgenic plants (Rosenthal et al. 2011). Fur-
Values are indicated as the mean ± SE for three individual experi- ther enhancement of photosynthesis and biomass produc-
ments tion was observed when SBPase, FBPald and ictB were
There was no statistically significant difference between wild-type simultaneously expressed in tobacco plants (Simkin et  al.
and ApFS plants (P > 0.05, by a one-way ANOVA) 2015). Furthermore, the levels of various amino acids were
found to change in tobacco plants under different C and N
statuses in order to correct for the imbalance between C
Effects of an enhanced photosynthetic capacity and N in leaves (Fritz et al. 2006). In ApFS plants, the level
on the expression of genes involved in C/N coordination of starch increased at 2 and 5  weeks due to an enhanced
photosynthesis capacity (Fig.  3a). However, the C/N ratio
We examined the transcript levels of transcription factors was similar to that in wild-type plants at 5 weeks (Table 1).
involved in C and/or N metabolism between wild-type and Therefore, we assumed that the expression levels of genes
ApFS plants at immature and mature stage (Fig. 9). No sig- involved in nitrate metabolism are regulated in order to
nificant difference was observed in the transcript levels of supply deficient free amino acids and modulate growth
NIN-like proteins (NLP6 and NLP7) between ApFS and characteristics at the immature stage of ApFS plants, but
wild-type plants at immature stage, whereas those of NLP6 not the mature stage (Fig. 10).
and NLP7 in ApFS plants were higher than those in wild- The transcript levels of the genes involved in the Cal-
type plants at mature stage. The transcript levels of PII-like vin cycle are known to be regulated by negative feedback
protein (GLB1) were consistently lower in ApFS plants control with the accumulation of photosynthates in leaves
than in wild-type plants at both immature and mature stage. (Sheen 1990). The transcript levels of genes encoding chlo-
No significant difference was observed in the transcript roplastic FBPase, SBPase, RBCS1A, and PRK were consist-
levels of Snf1-related protein kinases (SnRK1.1/Akin10) ently lower in ApFS plants than those of wild-type plants at
between ApFS and wild-type plants at both immature and both immature and mature stage (Fig. S3). This decrease in
mature stage. On the other hand, the transcript levels of the transcript levels of genes involved in the Calvin cycle
SnRK1,2/Akin11 were lower in ApFS plants than in wild- was attributed to an intracellular unbalanced C/N ratio such
type plants at immature stage, but were similar in ApFS as high C and low N (Rideout et  al. 1992). Conversely,
and wild-type plants at mature stage. At immature stage, transgenic tobacco and Arabidopsis plants that had reduced
the transcript levels of Abscisic acid insensitive 1 (ABI1) SBPase levels showed decreased photosynthetic activ-
were lower in ApFS plants than in wild-type plants, but ity and dwarfing phenotypes in parallel with a decrease in
were higher in ApFS plants than those of wild-type plants starch content (Harrison et  al. 2001; Liu et  al. 2012). No
at mature stage. significant difference was observed in the total activities of
­NADP+-GAPDH, PRK, or Rubisco between wild-type and
ApFS plants at 5  weeks. Meanwhile, the Rubisco activity
Discussion ratio was higher in ApFS plants than in wild-type plants
(Fig. S1). These results were consistent with previous find-
In the present study, we generated Arabidopsis plants ings obtained in FBP/SBPase-overexpressing transgenic
overexpressing FBP/SBPase in order to evaluate the tobacco plants indicating that an enhanced photosynthetic
effects of an enhanced photosynthetic capacity on capacity in ApFS plants temporarily inhibited the tran-
C/N metabolism. Although the phenotype of ApFS script levels of the genes involved in the Calvin cycle due
plants was indistinguishable from that of wild-type to increased levels of photosynthates, but had no influence
plants at 2  weeks, the numbers of rosette leaves and on these enzymatic activities (Miyagawa et al. 2001).

13

920 J Plant Res (2017) 130:909–927

Fig. 7  Expression of genes involved in N metabolism in rosette (n = 6–9  plants). Asterisks indicate that mean values were signifi-
leaves of wild-type and ApFS plants grown on soil at 2- (left panel) cantly different from those in wild-type plants by a one-way ANOVA
and 5-week-old stage (right panel). The transcript levels of these with Dunnett’s post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001).
genes were measured by a q-PCR analysis. Error bars indicate SE The entire experiment was replicated three times with similar results

13
J Plant Res (2017) 130:909–927 921

Fig. 8  Expression of NRT genes in rosette leaves of wild-type and indicate that mean values were significantly different from those in
ApFS plants grown on soil at 2- (left panel) and 5-week-old stage wild-type plants by a one-way ANOVA with Dunnett’s post hoc test
(right panel). The transcript levels of these genes were measured by (*P < 0.05, **P < 0.01, ***P < 0.001). The entire experiment was
a q-PCR analysis. Error bars indicate SE (n = 6–9  plants). Asterisks replicated three times with similar results

13

922
Fig. 9  Expression of genes involved in C/N sensing and coordina-
tion in rosette leaves of wild-type and ApFS plants grown on soil at
2- (left panel) and 5-week-old stage (right panel). The transcript lev-
els of these genes were measured by a q-PCR analysis. Error bars
indicate SE (n = 6–9  plants). Asterisks indicate that mean values
It has been reported that the disruption of chloroplastic
FBPase caused alteration of various metabolites including
starch and sucrose by a decreased photosynthetic capacity
(Rojas-González et  al. 2015). Also in ApFS plants, some
of the metabolite changes observed may be associated with
an enhanced photosynthetic capacity. Starch levels were
consistently higher in ApFS plants than in wild-type plants
(Fig.  3a). ADP-glucose pyrophosphorylase is an essential
enzyme for the regulation of starch synthesis and comprises
small and large subunits in plants (Crevillén et  al. 2003).
In Arabidopsis, four genes encode the large subunit of
ADP-glucose pyrophosphorylase. Among them, APL1 and
APL2 may be the catalytic isoforms, and APL1 is the main
L subunit in leaves, whereas other APLs are mainly pre-
sent in sink tissues (Crevillén et al. 2005; Ventriglia et al.
2008). The expression levels of APL1-3 were markedly
13
J Plant Res (2017) 130:909–927
were significantly different from those in wild-type plants by a one-
way ANOVA with Dunnett’s post hoc test (*P < 0.05, **P < 0.01,
***P < 0.001). The entire experiment was replicated three times with
similar results
higher in ApFS plants than in wild-type plants at immature
stage (Fig.  4). β-amylases, which generate maltose from
starch for export to the cytosol, are committed to a starch
degradation pathway (Fulton et  al. 2008; Stitt and Zee-
man 2012). In Arabidopsis, nine genes encode β-amylase
and β-amylase-like proteins. The importance of these
enzymes was analyzed using single knockout mutants, and
only bam3 was found to accumulate starch at mature stage
(Monroe et al. 2014). The expression levels of BAM3 were
higher at 2  weeks, but were lower in ApFS plants than in
wild-type plants at 5 weeks (Fig. 4). These data suggest that
the biosynthesis and degradation of starch are enhanced in
ApFS plants at immature stage. SS catalyzes the transfer of
the glucosyl moiety of ADP-glucose to a pre-existing α-(1,
4) glucan primer (Roldán et al. 2007). Mutants lacking SS3
and SS4 are completely starchless (Szydlowski et al. 2009).
J Plant Res (2017) 130:909–927 923

Fig. 10  Effects of FBP/SBPase
overexpression on gene expres-
sion and concentrations of
metabolites involved in C and N
metabolism. Boxed compounds
and italicized genes indicate
metabolites and genes measured
in this study. Solid lines indicate
several enzyme reactions,
and broken lines indicate an
abbreviation for the transport
of metabolites across the mem-
brane. The colors of compounds
and genes indicate the relative
abundance. The following color
codes are used: red lower tran-
script and metabolite levels in
ApFS plants than in wild-type
plants, blue higher transcript
and metabolite levels in ApFS
plants than in wild-type plants.
ADP-G ADP-glucose, Asn
asparagine, Asp aspartate, F6P
fructose 6-phosphate, FBP fruc-
tose 1,6-bisphosphate, G1P glu-
cose 1-phosphate, G6P glucose
6-phosphate, Gln glutamine,
Glu glutamate, OAA oxaloace-
tate, 2-OG 2-oxoglutarate, PEP
phosphoenolpyruvate, 3-PGA
3-phosphoglycerate, Ru5P
ribulose 5-phosphate, RuBP
ribulose 1,5-bisphosphate, S7P
sedoheptulose 7-phosphate, SBP
sedoheptulose 1,7-bisphosphate,
TP triosephosphate, UDP-G
UDP-glucose

Decreases in the expression levels of APL2, BAM3, SS3
and SS4 were observed in ApFS plants at 5 weeks, suggest-
ing that the pathway for starch degradation is promoted in
order to provide energy for growth at this stage.
Sucrose synthase (SUS) catalyzes the reversible reac-
tion of sucrose synthesis and breakdown, and an equi-
librium between these two reactions predominates in the
sucrose breakdown step to produce UDP-glucose, which
is a cell wall precursor (Baroja-Fernández et  al. 2012).
Among the six SUS genes (SUS1-6) in Arabidopsis,
SUS2 and SUS6 transcripts are lower than other isoforms
in leaves (Bieniawska et  al. 2007). A previous study
reported that the expression level of SUS1 was increased
in response to a sucrose treatment and osmotic stress,
while the transcript levels of SUS3 increased in response
to osmotic stress only (Baud et  al. 2004). Although no
significant differences were observed in the sucrose con-
tents of ApFS and wild-type plants, the transcript levels
of SUS1 were lower, while those of SUS3 were higher in
ApFS plants than in wild-type plants at 2 weeks (Fig. 4).
At mature stage, sucrose contents were higher than those
in WT plants; however, no significant differences were
noted in SUS1 levels between ApFS plants and wild-type
plants. On the other hand, SUS3 levels were still higher in
ApFS than in wild-type plants (Fig.  4). SUS1 and SUS4
are closely related, but exhibit distinct expression profiles
(Baud et al. 2004). In the present study, we showed that
the expression patterns of SUS1 and SUS4 were differ-
ent in ApFS plants. On the other hand, a previous study
reported that the transcript levels of SUS5 were regulated
by a sucrose or stress treatment (Baud et  al. 2004). In
ApFS plants, the transcript levels of SUS5 were higher
than that in wild-type plants at 5 weeks (Fig. 4). Previous
reports and our results suggest that the transcript levels of
SUS family members are regulated by osmotic conditions
with a balance between starch, sucrose, and hexose lev-
els; however, the underlying regulatory mechanisms have
not yet been elucidated in detail.

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924
PEPC is a crucial enzyme that functions in primary
metabolism by irreversibly catalyzing the conversion of
phosphoenolpyruvate (PEP) and ­ HCO3− to oxaloacetate
and inorganic phosphate, and plays an important role in
modulating the balance between C and N metabolism
in Arabidopsis leaves (Shi et  al. 2015). Although no sig-
nificant differences were observed in the total activities of
PEPC between ApFS and wild-type plants, the transcript
levels of PEPC1 and PEPC2 were more varied in ApFS
plants than in wild-type plants (Fig.  5). The differences
observed in the expression levels of PEPC1 and PEPC2
in ApFS plants may be due to changes in C and nitrate
metabolites.
The content of Gln was lower in ApFS plants at
2  weeks, but higher at 5  weeks than that in wild-type
plants (Fig.  6d). Although the amounts of a large number
of amino acids were decreased in ApFS plants, the level
of Gly was higher than that in wild-type plants grown on
MS medium at 2 weeks (Table S2). The increase detected
in the Gly content in ApFS plants at 2 weeks was attributed
to the temporary accumulation of a sugar pool derived from
an enhanced photosynthetic capacity and the photorespira-
tion by increased Rubisco activity. Nitrite contents and NR
activity were higher in ApFS plants than in wild-type plants
at mature stage, but were the same between wild-type and
ApFS plants at immature stage (Fig. 6b, f). Previous studies
reported that the transcript levels of many genes involved in
N metabolism were induced by light and/or photosynthetic
metabolites (Foyer et al. 1994; Koch 1996; Lillo 2008). In
the present study, the transcript levels of NR1 (NIA1), NR2
(NIA2), ASN1, and GLN1;1s were higher in ApFS plants
than in wild-type plants at mature stage (Fig. 7).
The supply of exogenous C was previously shown to
repress the level of ASN1 in Arabidopsis seedlings, and this
effect was relieved by supplementation with amino acids,
such as Asn, Gln, and Glu (Lam et  al. 1994, 1998). The
contents of sucrose and Gln were higher in ApFS plants
than in wild-type plants at 5 weeks (Figs. 3b, 6e). Since the
accumulation of ASN1 is regulated by the C/N ratio (Lam
et  al. 1994), ASN1 levels may be higher in ApFS plants
with constitutively enhanced C metabolism than in their
wild-type counterparts at 2 and 5 weeks in order to accel-
erate the metabolism of N (Fig. 7). On the other hand, the
expression of ASN2 is the strongest in the vegetative leaves
of A. thaliana under darkness and also strongly correlated
with the content of Asp (Gaufichon et al. 2013). In ApFS
plants, lower expression levels of ASN2 may have contrib-
uted to increases in the content of Gln at 5 weeks.
Taken together, an enhanced photosynthetic capacity in
ApFS plants accelerated C metabolism and consumption
of the amino acid pool for protein biosynthesis at 2 weeks.
A decrease in amino acid contents may have triggered an
increase in the transcript levels of genes involved in amino
13
J Plant Res (2017) 130:909–927
acid metabolism in order to maintain the C/N balance. At
5  weeks, the accelerated metabolism of C in ApFS plants
led to an increase in N metabolism in parallel with the
induced transcript levels of genes involved in nitrate metab-
olism. The resulting effect mediated an increase in nitrite
and Gln contents in ApFS plants at 5  weeks (Figs.  6, 7).
These results suggest that the transcript levels of the genes
involved in N metabolism are regulated by the overall inter-
nal status of organic C and N.
The transcript levels of NRT1.1 in ApFS plants were
lower than in wild-type plants at immature stage, but were
still higher than those in wild-type plants at mature stage
(Fig.  8). A previous study reported that NRT1.1 has been
identified as a nitrate sensor and NRT1.11 and NRT1.12
were plasma membrane transporters expressed in the com-
panion cells of the major vein and were involved in xylem-
to-phloem transfer for redistributing nitrate into developing
leaves (Ho et al. 2009, Hsu and Tsay 2013). The transcript
levels of NRT1.11 and NRT1.12 in ApFS plants were
higher than in wild-type plants at immature stage, but var-
ied in ApFS plants at mature stage (Fig. 8). In ApFS plants,
the higher expression levels of NRT1.11 at both immature
and mature stage (Fig.  8) suggested that the translocation
of N for redistributing nitrate into developing leaves was
activated. The transcript levels of NRT2.1 were previously
shown to be induced and those of NRT2.5 suppressed by
supplying exogenous nitrate to Arabidopsis plants (Oka-
moto et al. 2003). Although the transcript levels of NRT2.1
remained unchanged in ApFS plants, NRT2.5 transcript lev-
els in ApFS plants were higher than in wild-type plants at
immature stage (Fig. 8). The transcript levels of NRT2.1 in
ApFS plants were higher than in wild-type plants at mature
stage, whereas no significant difference was observed in
the transcript levels of NRT2.5 between ApFS and wild-
type plants at mature stage. The altered expression of NRTs
observed at both immature and mature stage, which were
grown under a constant supply of N, implied that C/N sen-
sor factors may regulate nitrate uptake in response to the
contents of internal N metabolites.
The mechanisms underlying metabolite sensing have
been elucidated in bacteria; a protein called PII regulates
the activities and transcription of enzymes involved in the
metabolism of N (Stadtman 2001). Hsieh et al. (1998) were
the first to identify the plant PII homolog (GLB1) from
Arabidopsis, and reported that the expression of GLB1 was
induced by sucrose and amino acid treatments. Further-
more, Ferrario-Méry et al. (2005) reported that the T-DNA
insertion mutants of AtGLB1 showed greater sensitivity to
nitrite ­(NO2−) than wild-type plants and had slightly higher
levels of carbohydrate and amino acids in response to a
­NH4+ supply. These findings suggest that the PII protein
plays an important role in the sensing and regulation of C
and N statuses in higher plants. GLB1 transcript levels in
J Plant Res (2017) 130:909–927 925
ApFS plants were lower than in wild-type plants at both
immature and mature stage (Fig.  9), indicating that GLB1
is also involved in the optimization of the C/N imbalance
caused by an enhanced photosynthetic capacity. Previous
studies reported that NIN-like transcription factors (NLPs)
regulated the transcript levels of NRTs, such as NRT1.1 and
2.1, in response to N availability (Konishi and Yanagisawa
2014; Krapp et  al. 2014). Marchive et  al. (2013) reported
that NLP7 regulated many steps of the primary N assimila-
tion and nitrate signaling pathways and coordinated regu-
lation by NLP7 which was ensured rapid adaptation to N
availability to maintain plant nitrate homeostasis. Actually,
in ApFS plants, NR1, NR2, and NRT2,1 increased with
NLP7. On the other hand, the transcript levels of NIR1
which was regulated by NLP7 did not increase in ApFS
plants. Huang et al. (2015) reported that the transcript lev-
els of NIR1 were directly regulated by HY5 in Arabidopsis
seedlings. These reports support that the transcript levels of
NIR1 did not increase in contrast to those of NR1, NR2, and
NRT2,1 in ApFS plants. Accordingly, the higher transcript
levels of NLP6 and NLP7 in ApFS plants than in wild-
type plants correlated with the higher transcript levels of
NR1, NR2, and NRT2,1 in ApFS plants due to a probable
increase in the uptake of nitrate ions and increase in nitrite
ions at mature stage (Figs. 6, 7, 8, 9).
Sucrose non-fermenting related kinase 1 (SnRK1) pro-
teins have been linked to the regulation of energy and
stress signaling in eukaryotes, and plants possess a small
SnRK1 gene family. Transgenic Arabidopsis overexpress-
ing SnRK1.2 showed a larger leaf size and rosette diameter
during the early development stage and early flowering,
while transgenic Arabidopsis expressing SnRK1.1 showed
the opposite phenotypes (Williams et al. 2014). Moreover,
SnRK1.1/Akin10 control the timing of flowering via the
INDETERMINATE DOMAIN (IDD)-containing transcrip-
tion factors IDD8 and SUS4 depending on sugar metabo-
lism in Arabidopsis (Jeong et al. 2015). Although the con-
tents of sucrose and starch were higher in ApFS plants than
in wild-type plants at mature stage, the transcript levels of
SnRK1.1 and SUS4 were not altered, suggesting that sugar
contents do not directly regulate various metabolic path-
ways via SnRK1.1 and SUS4 (Figs.  4, 9). On the other
hand, transcript levels of SnRK1.2 were lower in ApFS
plants than in wild-type plants at immature stage. How-
ever, ApFS plants did not exhibit any discernible flowering
phenotypes. Lu et al. (2015) reported that ABI1 regulated
post-germination growth in response to C/N balance. ABI1
could directly target SnRK1s, suggesting that importance
of the SnRK1s in C/N signal mediation under ABI1 con-
trol. In ApFS plants, however, the transcript levels of ABI1
and SnRK1s were not correlated with each other at both
immature and mature stages. These results suggest that
ABI1 and SnRK1,2 function as C/N sensors and regulate N
uptake through NRTs at both immature and mature stages
in ApFS plants.
In summary, the increase generated in photosynthetic
intermediates by accelerated photosynthesis in the imma-
ture leaves of ApFS transgenic plants was used in the bio-
synthesis of amino acids and proteins as well as carbohy-
drates. Therefore, an enhanced photosynthetic capacity
provisionally results in temporary free-amino acids deficits,
particularly in immature plants without an increased N sup-
ply. The temporary imbalance in the free-C/free-N ratio in
ApFS plants acted as a signal to regulate the expression
levels of NLPs, GLB1, SnRK1,2, and ABI1, suppressed the
expression of the genes involved in the Calvin cycle, and
also regulated the expression of the genes involved in N
metabolism and amino acid biosynthesis. These changes
facilitated nitrate metabolism and regulated the C/N bal-
ance as plants matured.
Acknowledgements  This work was supported by JST CREST Grant
Number JPMJCR12B3 (S.S.) and JPMJCR15O5 (S.Y.), Japan.
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