Plant Physiology and Biochemistry 185 (2022) 35–44

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Plant Physiology and Biochemistry

journal homepage: www.elsevier.com/locate/plaphy

Foxtail millet [Setaria italica (L.) Beauv.] over-accumulates ammonium

under low nitrogen supply
Faisal Nadeem a, b, Rashid Mahmood b, Muhammad Sabir c, Waqas-ud-Din Khan d,
Muhammad Saleem Haider e, Ruifeng Wang a, Yanting Zhong f, Muhammad Ishfaq a,
Xuexian Li a, *
a
MOE Key Laboratory of Plant-Soil Interactions, Department of Plant Nutrition, China Agricultural University, Beijing, 100193, China
b
Department of Soil Science, University of the Punjab, Lahore, 54590, Pakistan
c
Institute of Soil and Environmental Sciences, University of Agriculture, Faisalabad, 38040, Pakistan
d
Sustainable Development Study Centre, Government College University, Lahore, 54000, Pakistan
e
Department of Plant Pathology, University of the Punjab, Lahore, 54590, Pakistan
f
Department of Vegetable Sciences, China Agricultural University, Beijing, 100193, China

A R T I C L E I N F O A B S T R A C T

Keywords: Nitrogen (N) deficiency is a primary limiting factor for crop production worldwide. Previously, we reported root
Foxtail millet system architectural modifications of hydroponically cultured foxtail millet [Setaria italica (L.) Beauv.] to
Nitrogen limitation (LN) facilitate N translocation under N limitation. Here, we investigated foxtail millet for its shoot adaptation to low N
NH+4 over-accumulation
in terms of internal N regulation under hydroponic culture. The results of this study revealed that the shoot N and
Glutamate synthase (GOGAT)
nitrate (NO−3 ) concentrations significantly declined as compared to control (CK); however, the shoot over-
Glutamine synthetase (GS)
Photosynthesis accumulated ammonium (NH+ 4 ) under low N (LN). N shortage resulted in down-regulation of expressions of
Gene expression SiPetA, SiccsA, SipsbA, SirpoB, SipsaA, SiatpA, Sirps16, and SiPEPC which, undermined chloroplast functioning and
CO2 assimilation for the provision of carbon skeleton. Carbon deficiency and lower activities of GS decelerated
ammonia assimilation and led to over-accumulation of NH+ 4 in the LN-shoot, as indicated by lower concentra­
tions of total amino acids. Thus, enhanced GOGAT activity was to assimilate NH+ 4 while, those of catalase (CAT),
superoxide dismutase (SOD) and peroxidase (POD) were to scavenge reactive oxygen species (ROS) of NH+ 4
toxicity framework. The weakened chloroplast factory eventually minimized photosynthesis and reduced dry
mass of the LN shoot. Such regulation of N by the shoot, perhaps, resurrected physiological functions which
maintained internal mineral status under nitrogen limitation in foxtail millet.

1. Introduction Europe (Li and Wu, 1996; Yang et al., 2012). The availability of refer­
ence genome sequence (Bennetzen et al., 2012; Zhang et al., 2012) has
Millets (C4 panicoid grasses) are known for their features of climate made it an interesting crop for C4 photosynthesis, stress biology and
resilience through a wide range of ecological adaptations such as less bioenergy related studies (Muthamilarasan and Prasad, 2015). Among
irrigational and nutrient input requirements and withstanding envi­ monocotyledons, foxtail millet is ideal to study plant stress tolerance
ronmental stresses (Kole et al., 2015). Their nutritional richness in di­ mechanisms (Doust et al., 2009).
etary fibers, resistant starches, vitamins, essential amino acids, proteins Nitrogen (N) is an essential primary macronutrient as well as yield
and other bioactive compounds make them superior to other major ce­ limiting factor for plants in most terrestrial ecosystems (Wang et al.,
reals (Amadou et al., 2013). Among millets, foxtail millet [Setaria italica 2012; Xu et al., 2012). Many physiological processes, like chlorophyll
(L.) Beauv.] is an important fodder and cereal crop of arid and semi-arid biosynthesis and photosynthetic CO2 assimilation capacity of plant
regions of Asia and ranks second in total millet production worldwide leaves, are changed upon N limitation (Jin et al., 2015; Rascher et al.,
(Wang et al., 2016). It is one of the oldest cereal crops which provides six 2000; Shrestha et al., 2012). With the aim to meet the food demands of
million tons of grains to the people throughout Asia and southern world’s growing population, agriculture has become intensive and thus

* Corresponding author.
E-mail address: [email protected] (X. Li).

https://doi.org/10.1016/j.plaphy.2022.05.031
Received 25 January 2022; Received in revised form 22 May 2022; Accepted 24 May 2022
Available online 27 May 2022
0981-9428/© 2022 Elsevier Masson SAS. All rights reserved.
F. Nadeem et al. Plant Physiology and Biochemistry 185 (2022) 35–44

high amounts of N fertilizers are applied to sustain crop productivity. was 2 mM NH4NO3, 0.25 mM KH2PO4, 0.75 mM K2SO4, 0.1 mM KCl, 2
This excessive use of N negatively impacts not only the environment, mM CaCl2, 0.65 mM MgSO4, 0.2 mM Fe-EDTA, 1 × 10− 3 mM MnSO4, 1
through eutrophication, algal blooms and hypoxia (Diaz and Rosenberg, × 10− 3 mM ZnSO4, 1 × 10− 4 mM CuSO4, 5 × 10− 6 mM (NH4)6Mo7O24
2008; Sharma and Bali, 2018), but also the farm economy (Ray et al., and 1 × 10− 3 mM H3BO3. For the imposition of N limitation (LN treat­
2013; Tilman et al., 2002, 2011). It is, hence, imperative to manage and ment) NH4NO3 was reduced to 0.02 mM in the nutrient solution keeping
optimize plant N utilization, by exploring nitrogen use efficient plant other nutrients unchanged in concentration. The nutrient solution was
species, for the sustainability of agricultural production as well as changed every 2 days and the pH of both CK and LN nutrient solutions
environment. There exists a strongly positive correlation between N was kept at 6.0. Each treatment had six biological replicates and the
contents of leaves and the photosynthetic capacity (Makino et al., 2003; experiment was reproduced three times. The temperature of standard
Takashima et al., 2004; Tazoe et al., 2006). Thus, there is an inevitable growth chamber was 28/22 ◦ C (day/night), relative humidity 65%,
dependence of plant growth, development and yield formation on N illumination 300 mmol photons m− 2 s− 1 and photoperiod 14/10 h
status. An optimal distribution of N determines efficient photosynthesis (day/night). On 21st day, after transfer to the nutrient solution, the
in plants. An efficient photosynthesis needs the exploration of chloro­ shoot samples were harvested, frozen immediately in liquid N2, and
plast development associated genes viz. SiPetA, SiccsA, SipsbA, SirpoB, stored at − 80 ◦ C for RNA isolation and determination of soluble pro­
SipsaA, SiatpA, and Sirps16 encoding cytochrome f, heme attachment teins, total amino acids and enzyme activities. Shoot dry weight and
protein in cytochrome c, photosystem-II protein D1, RNA polymerase other physiological measurements were carried out after the shoot
beta subunit, PS-I apoprotein A1, ATP synthase CF1 alpha subunit and samples were harvested and washed three times, oven-dried at 105 ◦ C
cytochrome c heme attachment protein, respectively (Zhang et al., for 30 min and then dried at 70 ◦ C until constant weight. Number of
2018). Furthermore, the role of GS-GOGAT (Glutamine leaves was counted manually and the plant height was determined as the
synthetase-Glutamate synthase) cycle is vital in the N metabolism sum of the internode space using a centimeter scale.
(Masclaux-Daubresse et al., 2006). Maize reduces N allocation to chlo­
rophyll and light harvesting proteins as a strategy to control excess 2.1. SPAD value, chlorophyll a, b and total carotenoids
electron production in low nitrogen provision. Soluble proteins pro­
duction also decreases in a view to reduce nitrogen storage reservoirs Chlorophyll Meter (SPAD-502, Konica Minolta Sensing Inc., Japan)
(Mu et al., 2016). was used to measure SPAD values at three different points of the
Nitrate (NO−3 ) and ammonium (NH+ 4 ) are the inorganic forms of N youngest fully expanded 4th leaf on the day of harvest at 8:00–9:00 a.m.
mostly taken up by plants from the soil (Cui et al., 2017; Hobbie and The average of three SPAD reads from the same leaf was taken as the
Högberg, 2012; Tho et al., 2017). The ability of plants to preferentially SPAD value for that leaf. This process was extended to all four plants of
take up NO−3 or NH+ 4 varies differentially with species and environ­ every pot. Thus every pot gave four averages of SPAD value which were
mental conditions (Hawkins and Robbins, 2010; Kronzucker et al., considered as technical replicates. There were six biological replicates of
1997). However, NH+ 4 causes toxicity at elevated concentrations in plant each treatment (i.e. six pots). To determine chlorophyll a, b and total
tissues (Britto and Kronzucker, 2002). Many studies on uptake, alloca­ carotenoids, 0.5 g of fresh leave samples was homogenized and extrac­
tion, assimilation, and signaling of NO−3 and NH+ 4 and their interactions ted using 80% acetone. The absorbance readings were taken using a
in various plant species (reviewed in (Hachiya and Sakakibara, 2016, spectrophotometer (Shah et al., 2017; Wellburn, 1994) and calculations
2017), along with their root system architecture alterations under N were made according to the following formulae:
limitations, are available (Engineer and Kranz, 2007; Guo et al., 2014;
Gao et al., 2015). However, the studies reporting NH+ 4 accumulation in Chl a = 12.25A663.2 − 2.79A646.8 ×
ml of acetone
plants, foxtail millet in particular, under low nitrogen supply are limited. weight of sample
In our previous study, we reported foxtail millet as a nitrogen utilization
ml of acetone
efficient crop which increases its specific root length, root diameter and Chl b = 21.5A646.8 − 5.1A663.2 ×
Weight of Sample
biomass accumulation under N limitation. The mechanism of root sys­
tem architectural modification of foxtail millet in response to N limita­ ml of acetone
tion is different from other cereals like maize and wheat (Nadeem et al., Total Carotenoids = 1000A470 − 2.13Chl a − 85.02Chl b ×
Weight of Sample
2018). In current study, the shoot of foxtail millet seedlings
over-accumulated ammonium (NH+ 4 ) under low nitrogen (LN) as a result
of hampered photosynthesis and insufficient provision of carbon 2.2. Analysis of the N and total mineral concentrations
skeleton.
The dried shoot samples were ground into fine powder. 0.3 g, of
2. Materials and methods these samples, was digested by H2SO4–H2O2 and analyzed using a
modified Kjeldahl distillation method by collecting the ammonia liber­
This experiment was conducted at China Agricultural University, ated as ammonium borate, during the alkalinization with NaOH, in boric
Beijing, China in a standard growth chamber using Yugu 1 (a standard acid solution (4%) followed by titration with 0.02 N HCl (Baker and
variety of foxtail millet) (Cheng and Dong, 2010). Seeds of Yugu 1 were Thompson, 1992) to determine total N. Nitrate (NO−3 ) was determined
washed with deionized water, sterilized with 10% H2O2 for 30 min, by extracting 0.1 g of dried and finely ground shoot sample using 50 ml
imbibed in saturated CaSO4 solution with continuous aeration for 5 h of 2% acetic acid. The extract was diluted to contain 0–10 ppm NO−3 in
and germinated on moist filter paper in the growth chamber. Consistent test tubes having 10 ml final volume of every dilution. To every dilution,
seedlings with 2 cm embryonic roots were first enfolded cylindrically, 0.5 g of finely ground powder mixture (37 g citric acid, 5 g manganese
using two layers of deionized water saturated filter paper, then placed sulphate monohydrate, 2 g sulphanilamide, 1 g N-(1-Naphthyl)ethyl­
vertically in a growth holder containing deionized water, to ensure enediamine dihydrochloride and 1 g zinc) was added. The extract
continuous supply of deionized water to the seedlings, and finally mixture was shaken and centrifuged for 10 min to decant a pinkish
covered with black plastic until leaf emergence. The uniform and purple colored supernatant. The intensity of color was measured
consistent seedlings, at the stage of two fully expanded leaves, were through spectrophotometer at 540 nm (Singh et al., 1988). Ammonium
transferred into a pot containing 3.4-L of 25%-strength nutrient solution (NH+ 4 ) was determined by homogenizing, approximately, 0.5 g of shoot
for 3 days, 50% for 4 days, and 100% for 7 days (14 days in total). tissue in liquid N2 using a mortar and pestle followed by the extraction
During the next 7 days (i.e. day 15–21), these seedlings were subjected with 6 ml of 10 mM formic acid (Husted et al., 2000). The homogenate
to low nitrogen (LN). The recipe of nutrient solution used as control (CK) (1 ml) was centrifuged for 10 min at 2.53 g and 2 ◦ C. The supernatant

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F. Nadeem et al. Plant Physiology and Biochemistry 185 (2022) 35–44

Fig. 1. Foxtail millet seedlings grown under CK and LN conditions (A), plant height (B), number of leaves (C), shoot dry weight (D), chlorophyll a (Chl a), chlorophyll
b (Chl b) and total carotenoid concentrations (E), and SPAD value of leaves (F). Standard error of six biological replicates was indicated by error bars and the
significant changes under LN in comparison to CK were represented by asterisks (*P < 0.05; **P < 0.01).

was transferred to polypropylene tubes (2 ml) having nylon filters (0.45 acid) were added to detect NO−2 calorimetrically and then optical density
mm) (Costar, Corning Inc., Lowell, MA, USA) and centrifuged for 5 min of solution was measured at 540 nm after 30 min (Majláth et al., 2016).
at 53000 g and 2 ◦ C. The supernatant, from this step, was analyzed by GOGAT (Glutamate synthase) activity was determined, following
the o-phthalaldehyde (OPA) method for the determination of total tissue slightly modified procedures (Zhang et al., 1997; Zhong et al., 2017),
NH+ 4 content using spectrophotometer (Goyal et al., 1988). A 10 ml of using a reaction mixture containing hydroxylamine hydrochloride
tissue extract was added in 3 ml of OPA reagent and incubated in dark buffer (pH 7.4). The reaction was terminated following the incubation at
for 30 min at room temperature (25 ◦ C) for color development. The 37 ◦ C by adding acidic FeCl3. After centrifugation at 8000 rpm for 10
absorbance was measured at 410 nm using spectrophotometer. ICP-MS min, the absorbance was measured at 540 nm using spectrophotometer.
was used to determine the concentrations of P, K, Ca, Mg, Fe, Zn, B, Approximately, 100 mg of fresh shoot tissue was ground and incubated
Mn, Cu, Al, and Na (Masson et al., 2010). with 1.5 ml of 5% Trichloroacetic acid (TCA) for 1 h at room tempera­
ture for amino acid profiling using Waters e2695 HPLC (Waters Corp,
15 15 Milford, MA, USA). Total soluble proteins were analyzed using a stan­
2.3. Transient NH+
4 and NO−3 uptake
dard kit (Coomassie Protein assay reagent; Bio-Rad, Hercules, CA,
United States) having bovine serum albumin as a reference. Total sol­
For the analysis of 15NH+ 4 and
15
NO−3 uptake in shoot, foxtail millet
uble sugars in the shoot were determined by using a commercially
seedlings were rinsed for 1 min in a 1 mM CaSO4 solution before
available kit (Boehringer Mannheim, Germany). Approximately, 50 g of
transferring to uptake solution for 10 min. The uptake solution
finely ground shoot sample was loaded into Elementar Vario Macro CN
comprised of 2 mM/200 μM 15(NH4)2SO4 (99.12 atom% 15N) or 4 mM/
analyzer (Elementar Technologies, Hanau, Germany) to analyze the
400 μM K15NO3 (99.29 atom% 15N). To balance K+ in the uptake solu­
carbon to nitrogen (C/N) ratio.
tion, 0.6 mM K2SO4 was added where 400 μM K15NO3 whereas no K+
was added where 4 mM K15NO3 was applied. Before the harvest, roots
were again rinsed for 1 min in 1 mM CaSO4 solution and then dried at
2.5. Determination of catalase (CAT), superoxide dismutase (SOD) and
70 ◦ C for 48 h. Isotope ratio mass spectrometry (DELTAplus XP, Thermo
peroxidase (POD) activities
Finnigan) was used to analyze 15N accumulation in the shoot.
The shoot tissue (~0.1 g) was added in 4 ml of 50 mM potassium
2.4. Enzyme activity, total amino acids, total soluble proteins and sugars phosphate buffer of pH 7.0, homogenized and centrifuged for 20 min at
determination 15000 rpm at 4 ◦ C. The supernatant was obtained and used to analyze
enzyme activities. Superoxide dismutase (SOD) activity was determined
For the determination of nitrate reductase (NR) activity, 0.2 g of by measuring the inhibition of photochemical reduction of nitroblue
fresh leaf sample was cut and incubated in 1 ml mixture of 100 mmol L− 1 tetrazolium (NBT) (Giannopolitis and Ries, 1977). Color development
Na-phosphate buffer (pH 7.5) and 200 mmol L− 1 KNO3 at 37 ◦ C for 1 h in was done by illuminating 0.2 ml of 195 mM methionine, 2.4 ml of 50
dark. The conversion of NO−3 into NO−2 was stopped by adding 0.4 ml of mM potassium phosphate buffer solution (pH 7.8), 0.1 ml of 0.3 mM
30% trichloroacetic acid (m/v). To the reaction mixture, 2 ml of 0.2% 1- ethylenediaminetetraacetic acid (EDTA), 0.2 ml of 1.125 mM NBT, 0.2
naphthylamine and 2 ml of 1% sulphanilamide (dissolved in 30% acetic ml of 60 μM riboflavin and 50 μl enzyme extract for 15 min at light

37
F. Nadeem et al. Plant Physiology and Biochemistry 185 (2022) 35–44

15
Fig. 2. Foxtail millet shoot nitrogen (N) concentration (A), shoot nitrate (NO−3 ) concentration (B), shoot ammonium (NH+ 4 ) concentration (C) and shoot N influx
(D) under LN in comparison to CK. Standard error of six biological replicates was indicated by error bars and the significant changes under LN in comparison to CK
were represented by asterisks (*P < 0.05; **P < 0.01).

intensity of 5000 lux and absorbance was measured at 560 nm. One unit method (Livak and Schmittgen, 2001). There were four biological rep­
of SOD was defined as the amount of enzyme causing half-maximal in­ licates and three technical replicates for each treatment.
hibition of the NBT reduction under the assay condition. Catalse (CAT)
activity was determined by the consumption of hydrogen peroxide 2.7. Statistical analysis
(H2O2) at 240 nm for 1 min (Aebi, 1984). Enzyme extract (50 μl) was
added in a 3-ml tube containing 100 mM potassium phosphate buffer One-way PROC ANOVA in SAS (Sas Institute Inc., 2004) was used to
(pH 7.0) and 15 mM H2O2. For the calculation of CAT activity extinction analyze data. Means of control (CK) and low nitrogen (LN) treatments
coefficient (39.4 mM− 1 cm− 1) was used. Peroxidase (POD) activity was were compared through least significant difference (LSD) test at a 0.05
determined by milling 1 g of shoot tissue with 5 ml of assay buffer (pH or 0.01 level of probability.
6.8) containing phosphate buffer (125 μM), pyrogallol (50 μM), H2O2
(50 mM) and enzyme extract (20 times diluted). After incubating the 3. Results
reaction mixture at 25 ◦ C for 5 min, the reaction was stopped by adding
0.5 ml of 5% (v/v) H2SO4. The absorbance of purpurogallin was 3.1. Photosynthesis credentials and dry mass accumulation
measured at 420 nm (Kar and Mishra, 1976).
The leaves of foxtail millet plants, provisioned with low nitrogen
2.6. RNA isolation and relative gene expression (LN), turned light green and produced 34.43% lower SPAD values cor­
responding to 58.05%, 39.52% and 34.59% reductions in chlorophyll a
Trizol reagent (Invitrogen protocol) was used to extract total RNA (Chl a), chlorophyll b (Chl b) and total carotenoids concentrations,
from shoot samples of foxtail millet. DNase 1 was used to digest total respectively, in comparison to control (CK) (Fig. 1A, E, F). The plant
RNA (4–5 g) to remove any potential DNA (Takara Biomedicals, Kyoto, height and the number of leaves per plant were also decreased (22.61%
Japan). M-MLV reverse transcriptase (Invitrogen) was used for the and 5.26%, respectively) in LN plants. These reductions in plant height
reverse transcription of RNA samples into cDNA. Genes of interest were and number of leaves cumulatively decreased biomass accumulation
obtained as reported (Zhang et al., 2018) and the specific primer of and produced a significantly lower shoot dry weight (37.35%) in LN
elongation factor 1-α (EF-1α) (as internal control or reference gene) was plants as compared to CK (Fig. 1B, C, D).
constructed using Primer Premier 5.0 (Table S1). Relative gene
expression was analyzed through Quantitative real-time PCR (qRT-PCR) 3.2. Nitrogen (N), nitrate (NO−3 ) and ammonium (NH+
4 ) evolution in the
in a Bio-Rad iCycler iQ5 system (Bio-Rad, Hercules, CA, United States) shoot
using SYBR® Premix Ex TaqTM (Takara). The program was set at
pre-incubation for 10 min at 95 ◦ C, 40 cycles of determination at 95 ◦ C There was a significant drop (68.02%) in the shoot nitrogen (N)
for 15 s, annealing at 60 ◦ C for 30 s, and, finally, extension at 72 ◦ C for concentration under LN (Fig. 2A). Interestingly, the LN shoot accumu­
30 s. Quantification was performed by the standard comparative CT lated 95.66% less nitrate (NO−3 ) with respect to CK (Fig. 2B). In contrast,

38
F. Nadeem et al. Plant Physiology and Biochemistry 185 (2022) 35–44

Fig. 3. Total amino acids (A), total soluble proteins (B), the carbon contents (C) and the nitrogen contents (D) in foxtail millet shoot under LN. Standard error of six
biological replicates was indicated by error bars and the significant changes under LN in comparison to CK were represented by asterisks (*P < 0.05; **P < 0.01).

108.35% higher accumulation of ammonium (NH+ 4 ) occurred in the expression of SiPEPC (a gene involved in CO2 fixation during photo­
shoot under LN than CK (Fig. 2C). However, to our surprise, transient synthesis) was also down-regulated in the shoot under LN (Fig. 4A).
15 15
NH+4 and NO−3 uptake assay showed significant decreases in 15NH+4 Besides, carbon (C%) and nitrogen (N%) contents of the LN shoot also
uptake both under 2 mM and 200 μM 15(NH4)2SO4 supplies whereas, decreased in comparison to CK (Fig. 3C and D).
15
NO−3 uptake remained non-significant under 4 mM as well as 400 μM
K15NO3 supplies (Fig. 2D). 3.5. N assimilation enzymes and antioxidants activities

Over-accumulation of NH+ 4 in the LN shoot made it worth to inves­

3.3. Total amino acids profile and total soluble proteins
tigate activities of N assimilation enzymes viz. Glutamate synthase
(Glutamate oxoglutarate aminotransferase; GOGAT) and Glutamine
Foxtail millet shoot responded to LN provision, physiologically, by
synthetase (GS). There was a reciprocal response whereby, the activity
reducing the concentrations of total amino acids such as aspartate (Asp),
of GOGAT enhanced by 135.72% and that of GS decreased by 32.44% in
glutamate (Glu), glutamine (Gln), serine (Ser), histidine (His), glycine
LN shoot (Fig. 5C and D). Along N assimilation, NO−3 and NO−2 reduction
(Gly), threonine (Thr), arginine (Arg), alanine (Ala), valine (Val),
activities through nitrate (NR) and nitrite reductase (NiR) enzymes,
phenylalanine (Phe), isoleucine (Ile) leucine (Leu) and lysine (Lys)
respectively, were also analyzed. A co-reduction (81.65 and 15.98%,
(Fig. 3A). Moreover, the lowering of total amino acids in the shoot was
respectively) in the activities of both enzymes was observed (Fig. 5A and
accompanied by a decrease (50.03%) in the concentration of total sol­
B) synergistic to lower NO−3 concentrations (Fig. 3B) in the LN shoot.
uble proteins under LN (Fig. 3B).
Closely related to countering NH+ 4 -toxicity was the behavior of antiox­
idants such as catalase (CAT), superoxide dismutase (SOD) and peroxi­
3.4. Expression of chloroplast degradation and carbon skeleton related dase (POD) (ROS-scavenging enzymes). The activities of CAT, SOD and
genes POD were induced by 86.98%, 30.55% and 43.85%, respectively, in LN
shoot (Fig. 6A, B, C). Lastly, the total mineral content of LN shoot
The decreases in total soluble proteins and total amino acids called remained unchanged overall, with the exception of magnesium (Mg)
for the assessment of chloroplast functioning through investigations of which decreased by 22.06% (Table 1).
expressions of chloroplast related genes. The expressions of SiPetA,
SiccsA, SipsbA, SirpoB, SipsaA, SiatpA, and Sirps16 were down-regulated
in foxtail millet shoot under LN (Fig. 4B–H). Furthermore, the

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F. Nadeem et al. Plant Physiology and Biochemistry 185 (2022) 35–44

Fig. 4. Relative expression of SiPEPC (A), SiPetA (B), SiccsA (C), SipsbA (D), SirpoB (E), SipsaA (F), SiatpA (G) and Sirps16 (H) in foxtail millet shoot under LN.
Standard error of six biological replicates was indicated by error bars and the significant changes under LN in comparison to CK were represented by asterisks (*P <
0.05; **P < 0.01).

4. Discussion requirement hence, compromising the photosynthesis and carbon

provision.
Cereal crops like maize, for instance, tend to reduce nitrogen allo­ The capability of C4 photosynthesis makes foxtail millet an important
cation to chlorophyll under low nitrogen availability (Mu et al., 2016) crop (Zhang et al., 2017). Photosynthesis is directly influenced or
whereas, cultivars of rice having higher nitrogen use efficiencies give regulated by the N contents through chlorophyll production, regulation
better agronomic response to partial nitrate nutrition (Duan et al., of size, number and composition of chloroplast and functionality of
2007). In the same context, plants alter biomass allocation under photosynthetic enzymes in plant tissues (Bassi et al., 2018; Zheng,
different N availabilities (Müller et al., 2000; Poorter et al., 2012; 2009). As far as photosynthesis is concerned, N shortage (LN)
Shipley and Meziane, 2002). Foxtail millet adapts low nitrogen condi­ down-regulated the expressions of chloroplast related genes, viz. SiPetA
tion through the enhancements of specific root length and root diameter (encoding cytochrome f), SiccsA (encoding heme attachment protein in
to facilitate efficient transport of photo-assimilates (Nadeem et al., cytochrome c), SipsbA (encoding photosystem-II protein D1), SirpoB
2018); however, how foxtail millet adjusts internal nitrogen reserves (encoding RNA polymerase beta subunit), SipsaA (encoding PS-I apo­
with respect to carbon skeleton provision and dry mass accumulation protein A1), SiatpA (encoding ATP synthase CF1 alpha subunit) and
under nitrogen limitation remains unknown. The root specific response Sirps16 (encoding cytochrome c heme attachment protein) (Zhang et al.,
of foxtail millet made it worth to study its aboveground adaptation to 2018), in N deprived foxtail millet shoot (Fig. 4B–H). The incapacitated
nitrogen limitation in terms of N accumulation, which has not been chloroplast factory reduced chlorophyll components i.e. Chl a, Chl b and
explored yet. As a matter of fact, the concentration of the shoot N total carotenoids which, further, lowered the SPAD value indicating the
decreased (Fig. 3A) which enforced the shoot of foxtail millet plants to minimal photosynthetic activities and carbon skeleton provision, as
turn light green or yellowish in color under LN condition, indicating the pronounced by the down regulation of expression of SiPEPC (a C4
N stress (Fig. 1A). photosynthesis gene) (Zhang et al., 2018) in the LN shoot (Fig. 1E and F
Nitrate (NO−3 ) and ammonium (NH+ 4 ) are the inorganic forms of N and 4A). There must exist a close coordination between cellular carbon
taken up by plant roots (Maathuis, 2009) with NO−3 being the preferred and nitrogen in plants. A balance between carbon to nitrogen (C/N) ratio
source (Hobbie and Högberg, 2012). To our surprise, the NO−3 concen­ in plants is critical for appropriate functioning of metabolic related ac­
tration decreased while the NH+ 4 concentration increased in LN shoot in tivities (Zheng, 2009). The process of photosynthesis quenches atmo­
this study. Foxtail millet seedlings were provisioned with 0.02 mM spheric CO2 and converts it into plant biomass in the presence of solar
NH4NO3 as the LN treatment which means almost 100% reduction as light depending upon the N availability (Kim et al., 2001). For instance,
compared to CK (2 mM NH4NO3). Therefore, the reduction in the NO−3 maize increases its leaf dry weight as the N level increases during the
concentration of foxtail millet shoot under LN was due to the low vegetative growth stages (Jin et al., 2015). The co-reductions in carbon
availability of external NO−3 and its subsequent low uptake through roots (C%) and nitrogen contents (N%) (Fig. 3C and D) reinforced the lack of
(Nadeem et al., 2018). On the other hand, the dramatically elevated sufficient carbon to facilitate N assimilation in the LN shoot. Alterna­
NH+ 4 concentration in LN shoot could be the result of diminishing carbon tively, NH+ 4 dominant conditions lead plants to generate N at the
skeleton provision due to compromised photosynthetic abilities (Fig. 2B expense of their internal carbon reserves (Hessini et al., 2009a). Alto­
and C and 1F) under nitrogen limitation. Once the plants sense nitrogen gether, nitrogen shortage triggered the decline in chlorophyll biosyn­
limitations, they tend to remobilize N (Park, 2005) to fulfill essential N thesis and corresponding photosynthetic CO2 assimilation capacity (Jin

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F. Nadeem et al. Plant Physiology and Biochemistry 185 (2022) 35–44

Fig. 5. Nitrate reductase; NR (A), Nitrite reductase; NiR (B), Glutamate oxoglutarate aminotransferase; GOGAT (C) and glutamate synthetase; GS (D) activities in
foxtail millet shoot under LN. Standard error of six biological replicates was indicated by error bars and the significant changes under LN in comparison to CK were
represented by asterisks (*P < 0.05; **P < 0.01).

Fig. 6. Catalase; CAT (A) superoxide dismutase; SOD (B) and peroxidase; POD (C) antioxidant activities in foxtail millet shoot under LN. Standard error of six
biological replicates was indicated by error bars and the significant changes under LN in comparison to CK were represented by asterisks (*P < 0.05).

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F. Nadeem et al. Plant Physiology and Biochemistry 185 (2022) 35–44

Table 1
Mineral Contents of Foxtail millet shoot under control (CK) and Low nitrogen (LN).
Mineral Contents (mg kg− 1)

P K Ca Mg Fe Zn B Mn Cu Al Na

CK 3580.38 ± 53706.21 ± 1746.62 ± 2008.85 ± 113.21 ± 17.03 ± 2.64 ± 19.20 ± 3.58 ± 34.13 ± 202.22 ±
156.91 1959.13 59.09 128.39 6.08 1.55 0.26 2.66 0.47 4.43 27.02
LN 2994.95 ± 56821.29 ± 1825.23 ± 1565.67 ± 99.72 ± 18.48 ± 1.58 ± 25.00 ± 3.78 ± 24.81 ± 195.92 ±
684.43 1240.59 81.55 32.08** 23.88 4.63 0.71 6.85 0.89 6.42 23.77

All the values indicated Means ± SE. ** represented significant changes under LN with respect to CK (P < 0.01).

et al., 2015; Rascher et al., 2000; Shrestha et al., 2012), resulting in NH+ 4 alanine (Ala), valine (Val), phenylalanine (Phe), isoleucine(Ile) and
over-accumulation in the shoot of foxtail millet under LN (Fig. 2C). leucine (Leu), which could not sustain protein synthesis in the LN shoot
During the primary nitrogen assimilation pathway, N is taken up by (Fig. 3A and B). Similarly, OsGS1;1 knockout mutants reduced amino
plants and incorporated into amino acids which are the building blocks acid accumulation in rice (Tabuchi et al., 2005). Chloroplast contains
of nitrogenous compounds (Kruse et al., 2002), like proteins. Given the about 80% of N primarily in the form of proteins as an important N sink
toxic nature of NH+ 4 , its assimilation to organic compounds is necessary and functions to provide carbon skeleton supporting plant growth and
(Bittsánszky et al., 2015; Xu et al., 2012). The tolerance of a plant for crop production (Adam et al., 2001). Thus, malfunctioning of chloro­
NH+ 4 depends on its capacity to assimilate it (Cruz et al., 2006). The plast and decreased biosynthesis of total amino acids and total soluble
GS-GOGAT (Glutamine synthetase-Glutamate synthase) cycle is proteins decreased plant height, number of leaves and shoot dry weight
involved in NH+ 4 -assimilation (Masclaux-Daubresse et al., 2006). The of foxtail millet under LN (Fig. 1B–D). Considerable decreases observed
shoot of foxtail millet under LN tackled over-accumulation of NH+ 4 in NR and NiR activities were due to lower concentrations of NO−3 in the
through higher GOGAT activities and lower GS activities. These results LN shoot (Fig. 5A and B).
meant that foxtail millet went to the NH+ 4 -assimilation mode by accel­ A smaller foxtail millet plant under LN (Fig. 1A) represented a
erating the GOGAT activity but could not complete the cycle due to smaller sink for other nutrients, so it absorbed and accumulated ele­
lower GS activities which, tactically, highlighted the lack of sufficient ments accordingly; therefore, the concentrations of other mineral con­
carbon skeleton to favor effective ammonia assimilation (Fig. 5C and D). tents, largely, remained unchanged except the decrease in magnesium
This argument is also supported by a well-established fact that the (Mg) (Table 1) which could also have contributed to decreased chloro­
proper functioning of GS-GOGAT cycle and NH+ 4 assimilation/tolerance phyll biosynthesis. The incapacitation of photosynthesis framework
require the regulation of carbon skeleton through proper carbon man­ tends to accumulate NH+ 4 which could either be toxic through the gen­
agement in plants (Roosta and Schjoerring, 2008; Vega-Mas et al., eration of reactive oxygen species (ROS) (Hessini et al., 2009b) or impart
2015). On the other hand, lower GS-activity could also be a component priming effect in plant species (Hessini, 2022) through the activities of
favoring NH+ 4 tolerance in foxtail millet as is the case in Spartina alter­ CAT, SOD and POD (ROS-scavenging enzymes). We speculated that the
niflora (Hessini et al., 2021). Interestingly, the OsGS1;1 knockout mu­ activation of SOD could have countered the NH+ 4 -generated superoxide
tants in rice show over-accumulation of NH+ 4 (Tabuchi et al., 2005) radicals while the inductions of CAT and POD (Fig. 6A–C) were to
proposing, hypothetically, that the lowered GS activity in shoot of fox­ convert NH+ 4 -generated H2O2 to H2O (Patterson et al., 2010; Xie et al.,
tail millet could have also been a consequence of downregulation of GS 2015) as a part of physiological resurrections involved in internal N
associated transcription factors under LN, which needs to be verified in regulation and NH+ 4 -stress alleviation strategies of foxtail millet under
future studies. Furthermore, the lack in NH+ 4 -assimilation, through the LN (Fig. 7).
dysfunctional GS-GOGAT cycle, lowered concentrations of total amino
acids, such as aspartate (Asp), glutamate (Glu), glutamine (Gln), serine
(Ser), histidine (His), glycine (Gly), threonine (Thr), arginine (Arg),

Fig. 7. A flow chart of systematic adaptation of foxtail millet shoot to low nitrogen (LN). P; Phosphorous, K; Potassium, Ca; Calcium, Fe; Iron, Zn; Zinc, Mn;
Manganese, Cu; Copper, Al; Alluminium, B; Boron, Na; Sodium, Mg; Magnesium, NH+ 4 ; Ammonium, GOGAT; Glutamate synthase, GS; Glutamine synthetase, CAT;
Catalase, SOD; Superoxide dismutase, POD; Peroxidase.

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