Comprehensive screening of low nitrogen tolerant maize based on multiple traits at the seedling stage

Plant material, growth and treatment conditions

The six maize, GEMS42-I, Ji846, SY998, CML223, CML114 and GEMS42-II, with a significant difference in grain yield and nitrogen tolerance were used in the present study. The surface-sterilized seeds germinated on wet sand in the culture room. Then, the 4-day-old seedlings were transferred into the nutrient solution for continuing growth. The complete basal nutrient solution contained 0.24 g/L NH₄NO₃, 0.50 g/L MgSO₄, 0.15 g/L KCl, 0.36 g/L CaCl₂, 0.05 mM EDTA-Fe and a microelement solution (Hoagland & Arnon, 1950). The nutrient solution containing 1/10 N of the complete nutrient solution was used for low nitrogen treatment (-N), and the seedlings growing under the complete nutrient were used as control (+N). Keep the culture room parameter as follow: 16 h of light (300–320 µmol m⁻² s⁻¹) at 24 °C and 8 h of darkness at 22 °C photoperiods, and relative humidity of 65–80%. Roots and leaves of all six maize were harvested separately after growing under low nitrogen conditions for 20 days. Each treatment was replicated three times.

Biomass and phenotypic characteristics of the root system

Root was floated in the water and scanned using the scanner (Epson Expression 11000XL) to get the image. The root total length, root volume, root surface area and root average diameter were calculated with Tennant’s statistical method in WinRHIZO Pro software (Version 2.0, 2005; Regent Instrument Inc., Quebec, Canada) as previous study (Altaf et al., 2022). The seedlings were washed with distilled water, and the fresh weight (FW) was measured after drying with bibulous paper.

Measurement the NO ${}_3^{-}$ and NH ${}_4^{+}$ content of the seedlings

For nitrates (NO₃⁻) determination, roots and shoots (approximately 0.5 g FW) were cut into pieces and suspended in 5 mL boiling water for 10 min (Tang et al., 2013). Then the supernatant was diluted to 25 mL. The assay mixture containing 0.1 mL samples and 0.4 mL 5% salicylic acid-sulfuric acid, was incubated at 20 °C for 20 min, then mixed with 9.5 mL 8% NaOH (w/v). Its absorbance was measured at 410 nm wavelength.

The ammonium (NH₄⁺) of root and shoot were extracted by homogenizing in 0.3 mM H₂SO₄ (pH 3.5). After centrifugation at 3,900 g for 10 min, the supernatant was collected using for the determination of ammonium (NH₄⁺) content as previously described (Lin & Kao, 1996). After NO₃⁻ the and NH₄⁺ determination, the root nitrogen accumulation, shoot nitrogen accumulation and total plant nitrogen accumulation were calculated.

Enzyme activity assays

Approximately 0.5 g of fresh roots were homogenized with 10 mM Tris–HCl buffer (pH 7.6) containing 1 mM MgCl₂, 1 mM EDTA and 1 mM β-mercaptoethanol in a chilled pestle and mortar. After centrifugation at 15,000 g for 30 min (4 °C), the supernatant was used as an enzyme extract (Ren et al., 2017).

The whole extraction procedure was carried out at 4 ° C.

For GS (EC6.3.1.2) activity assayed, a 1.0 mL reaction mixture (pH 8.0) contained 80 µmol Tris–HCl buffer, 40 µmol L-glutamic acid, 8.0 µmol ATP, 24 µmol MgSO₄, and 16 µmol NH₂OH and enzyme extract. The enzyme extract was added to initiate the reaction. After incubation for 30 min at 30 °C, the reaction was stopped by adding two mL 2.5% (w/v) FeCl₃ and 5% (w/v) trichloroacetic acid in 1.5 M HCl. After centrifugation at 3,000 g for 10 min, the absorbance of the supernatant was measured at 540 nm. GS activity was expressed as 1.0 µM L-glutamate γ-monohydroxamate (GHA) formed g⁻¹ FW h⁻¹, with µmol GHA g⁻¹ FW h⁻¹.

For GOGAT (EC1.4.7.1) activity assayed, a three mL reaction solution was prepared with 25 mM Tris–HCl buffer (pH 7.6), which contained 0.5 mL enzyme extract, 0.05 mL 0.1 M 2-oxoglutarate, 0.1 mL 10 mM KCl, 0.2 mL 3 mM NADH and 0.4 mL 20 mM L-glutamine. The reaction was initiated by adding L-glutamine immediately following the enzyme preparation. The decrease in absorbance was recorded for 3 min at 340 nm. The GOGAT activity was expressed as µmol NADH g⁻¹ FW h⁻¹.

NR (EC1.7.1.1) activity was determined according to Wojciechowska et al. with minor modifications (Wojciechowska et al., 2016). The NR activity was expressed as µg NO₂⁻ g⁻¹ FW h⁻¹.

Quantitative RT-PCR analysis

Total RNA was isolated using TRIzol reagent (Invitrogen, CA, USA) and then first-strand cDNA was synthesized using the M-MLV Reverse Transcriptase (Promega, WI, USA) according to the manufacturer’s instructions. For the quantitative real-time PCR (qRT-PCR) experiment, 20 µL reaction components were prepared according to the manufacturer’s protocol for SYBR Green Real Master Mix (TIANGEN, Beijing, China). Using GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as the endogenous control. Real-time PCR was conducted on the CFX96™ Real-Time PCR Detection System (Bio-Rad, CA, USA), and the primer pairs used for quantitative RT-PCR were shown in supplemental Table S1.

Data analysis and D value calculation

The standard deviation (SD) was used to express the sample variability in the present study. All analyses of significance were conducted at the p < 0.05 level. Considering that the biological differences among the different maize genotypes, evaluation of the low nitrogen tolerance of maize by NTC may be more reasonable. The NTC was calculated as $\mathrm{NTC}=\frac{\text{low nitrogen trait}}{\text{high nitrogen trait}}$. Then principal component analysis was conducted based on the NTC in SPSS (Statistical Product and Service Solutions) software (version 18.0). The principal component (PCi, i = 1, 2 …n) with eigenvalue (λ_i, i = 1, 2 …n) >1 was selected as new index. PCi is the i-the principal component. λ_i is the eigenvalue of the i-the principal component. The eigenvalue (λ_i, i = 1, 2 …n) and factor score (FACi, i = 1, 2 …n) were present in the results of the principal component analysis. The principal component value Xi was calculated as Xi =FAC $i\times \sqrt[2]{{\lambda }_i}$ (i = 1, 2 …n). Then the subordinate function value was calculated as $U\left(Xi\right)=\frac{Xi-Xmin}{Xmax-Xmin}$ (i = 1, 2 …n). Xmax and Xmin represent the maximum and minimum value of the ith principal component, respectively. The weight coefficient was calculated as $W\left(i\right)=\frac{\mathrm{P}i}{{\sum }_{i=1}^n\mathrm{P}i}$ (i = 1, 2 …n). Pi represents the proportion of variance explained by the i-the principal component. Finally, the D value was calculated as $D={\sum }_{i=1}^n\left[\mathrm{U}\left(Xi\right)\times \mathrm{W}\left(i\right)\right]$ (i = 1, 2 …n).

Statistical analysis

The standard deviation was used to express the sample variability in the present study. The significance of differences was conducted using SAS 9.2 (SAS Institute, Cary, NC, USA). Data were subjected to ANOVA using PROC LSD (p <  0.05) in SAS.

Plant physiological changes in response to nitrogen stress

Low nitrogen (ca. 0.3 mM NH₄NO₃) significantly inhibited the growth of Ji846 but not of the other five genotypes of maize (Fig. 1), and even significantly increased the root biomass of SY998 and GEMS42-II by 46% and 66%, respectively (Fig. 1C). Consider that root is the primary organ for water and nutrients capturing, the morphology of root is investigated by WinRHIZO Pro software. All of the root total length, root surface and root volume of Ji846 were significantly decreased in response to low nitrogen stress, which decreased by 56%, 65% and 74%, respectively (Fig. 2). Root diameter of the six maize were decreased in response to low nitrogen stress (Fig. 2B). In contrast, the root total length, root surface and root volume of SY998 and GEMS42-II were prominently increased under low nitrogen (Figs. 2B, 2D, 2E). Low nitrogen not affected the root total length, root surface and root volume of GEMS42-I, CML223 and CML114 (Figs. 2B, 2D, 2E). These results indicated that Ji846 was sensitive to low nitrogen stress.

NO${}_3^{-}$ and NH ${}_4^{+}$ content in maize

Nitrate ion (NO₃⁻) and ammonium ion (NH₄⁺) are the main form of nitrogen for plant absorption. Maize can uptake both nitrate and ammonium. Both the NO₃⁻ and NH₄⁺ contents were significantly decreased in all maize genotypes under low nitrogen stress (Fig. 3). However, only the root NO₃⁻ content of SY998 was significantly higher than the other genotype under low nitrogen (Fig. 3A). Under low nitrogen stress, Ji846 has the lowest NH₄⁺ content both in root and shoot, while the GEMS42-I and SY998 have the highest NH₄⁺ content both in root and shoot (Figs. 3C and 3D). The lowest nitrogen accumulation was observed in Ji846 (8.78 µg N plant⁻¹) under nitrogen limitation, no matter in the root, shoot, or total plant (Table 1). While the highest nitrogen accumulation was observed in SY998 (153.87 µg N plant⁻¹) under nitrogen limitation (Table 1). Interestingly, the NO₃⁻ content was higher than the NH₄ ⁺ content of all six maize, irrespective of the nitrogen nutritional status of the plants. In the high nitrogen condition, the NO₃⁻ of root and shoot was 12.3 and 10.4 times the NH₄⁺ in Ji846, respectively. While under the low nitrogen condition, the NO₃⁻ of root and shoot was 7.1 and 6.6 times the NH₄⁺ in Ji846, respectively (Fig. 3).

Expression of the nitrate and ammonium transporter genes

The expression of ZmNRT2;1 and ZmNRT2;2 in Ji846 and SY998 were significantly increased under low nitrogen conditions (Figs. 4A and 4B). The expression of ZmNRT2;1 under low nitrogen was nine and three times the high nitrogen condition in Ji846 and SY998, respectively. The expression of ZmNRT2;2 under low nitrogen was 41 and 11 times the high nitrogen condition in Ji846 and SY998, respectively (Figs. 4A and 4B). In addition, the expression of ZmNRT2;2 in CML223 was also significantly increased (5 times) under nitrogen limitation (Fig. 4B). For the ammonium transporters genes, the expression of ZmAMT1;1a was significantly increased under nitrogen limitation in GEMS42-I, Ji846, SY998 and GEMS42-II, which increased 16, 10, 20 and 3 times, respectively (Fig. 4C). The expression of ZmAMT1;3 was significantly increased under nitrogen limitation in Ji846, CML223, CML114 and GEMS42-II, which varied from 1.4 to 4.3 times (Fig. 4).

Nitrogen metabolism-related key enzymes activity assay

Low nitrogen significantly decreased the activities of key nitrogen metabolism enzymes in some maize. The greatest reduction in the activities of NR (approximately 82%) in Ji846 (Fig. 5A), GS (approximately 88%) in Ji846 (Fig. 5B), (GOGAT approximately 56%) in CML223 (Fig. 5C). The NR activities of GEMS42-I, CML223 and CML114 were decreased 60.3%, 68.6% and 48.6%, respectively (Fig. 5A). In addition, the lower activities of NR and GS were observed in SY998, CML223 and GEMS42-II, irrespective of the nitrogen condition (Figs. 5A and 5B). Nitrogen limitation affected the activity of GOGAT less than that of NR and GS. The GOGAT activities only decreased in CML223 and GEMS42-II in response to nitrogen limitation (Fig. 5C).

Principal component analysis based on the nitrogen tolerance coefficient (NTC)

The first four principal components jointly explain the major part of the total variance (96.8%), being PC1 responsible for 47.4%, PC2 for 21.6%, PC3 for 15.5% and PC4 for 12.3% of the total variance, respectively (Table 2). The eigenvalue of PC1, PC2, PC3 and PC4 were 8.054, 3.676, 2.627 and 2.091, respectively (Table 2). The factor score of the four principal components of each maize was directly extracted from the principal component analysis results (Table 3). Then, the principal component value and subordinate function value were calculated. Finally, each maize has a D value (Table 3). The SY998, GEMS42-I and GEMS42-II have the higher D value that can define as low nitrogen tolerant maize, while the Ji846 with a lower D value was defined as low nitrogen sensitive maize (Table 3).