A total of 100 sorghum inbred lines were used in this study. The plant names are listed in Supplementary Table S1 . All sorghum plants were grown under hydroponic conditions at Shenyang Agricultural University, Shenyang, Liaoning, China.

Hydroponic experiments were conducted in a laboratory climate chamber. The plants were grown in a hydroponic box (0.4 × 0.25 × 0.2 m). Sorghum seeds of the same size and full grain were selected for sterilization with 10% NaClO solution for 5–10 min, washed with distilled water, placed in Petri dishes covered with wet filter paper, and cultured in an incubator under the following conditions: day/night temperatures of 28°C/25°C and light/dark periods of 12 h. After 3 days, seedlings were selected and transferred to 1/2 Hoagland nutrient solution, which (as control, normal N [NN]) consisted of 3 mM KNO₃, 0.5 mM NH₄H₂PO₄, 1 mM MgSO₄·7H₂O, 2 mM Ca(NO₃)₂·4H₂O, 0.05 M Fe-EDTA, 5 × 10^-3 mM MnCl₂·4H₂O, 2.5 × 10^-4 mM H₃BO₃, 0.5 × 10^-3 mM ZnSO₄·7H₂O, 0.2 × 10^-3 mM CuSO₄·5H₂O, and 0.1 × 10^-3 mM (NH₄)₆Mo₇O₂₄·4H₂O. After the third leaf stage, the seedlings were treated with NN or low-N (LN: 0.05 mmol·L^-1 N). The composition of the other plant nutrients remained unchanged during low-N treatment. The low-N treatment was performed for 10 days, after which the seedlings were harvested (four biological replicates). Shoot and root tissues were separated, and samples from each group were mixed in replicates. The leaves and roots were sampled for physiological analysis, frozen in liquid N, and stored at −80°C.

Five sorghum seedlings were selected from each experimental treatment. The absolute distance from the base of the stem to the longest leaf in the upper part of the stem (plant height) and that from the base of the plant to the lowest part of the root (root length) were measured. For each replicate, three randomly selected seedling leaves and roots were analyzed.

The plants were separated into roots and shoots and dried in an oven at 70°C until they reached a constant weight. The dry weights of shoots and roots were determined using an electronic balance with a precision of 0.001 g.

Leaf and root tip microstructures were prepared in accordance with the methodology described by Qi et al. (2014) to measure leaf and root cell structures. In brief, 15–20-mm leaf and 3-μm root tip transections were prepared and fixed in a fixing solution. The fixed leaves and roots were dehydrated, embedded, sliced, and stained for 2 h. Subsequently, they were decolorized with different alcohol gradients, dyed with solid green, and sealed with transparent xylene and neutral gum. The slices were scanned and processed using a scanner (Pannoramic DESK, P-MIDI, P250, Hungary) and scanning software (Pannoramic Scanner) to count the number of cortex cells in the meristematic zone and calculate the length of cells in the elongation zone of root tips.

A total of 12 sorghum seedling leaves were used in each treatment to quantify the leaf area, with the latter determined using length and width measurements. For each replicate, four sorghum seedling leaves were randomly selected and analyzed three times. A total of 12 sorghum seedling leaves were randomly selected from each treatment and used to measure the leaf chlorophyll content. Sorghum seedling leaves were sampled using a hole punch (diameter, 1.2 mm). After sampling, the leaves were placed in an opaque reagent bottle containing 10 mL of 80% acetone solution and kept in the dark for 24 h to extract the total chlorophyll content. The total chlorophyll content was determined to be 645 nm as described by Liu et al. (2022a).

SPAD values were determined using a portable SPAD-502 (Konica Minolta, Inc., Tokyo, Japan). Measurements were conducted five times for each leaf, and the mean value was calculated as the SPAD value for a given leaf. At least four readings were obtained for each treatment in each plot.

The leaf net photosynthetic rate (Pn), stomatal conductivity (Gs), transpiration rate (Tr), and intercellular CO₂ concentration (Ci) of the first fully expanded sorghum leaf were determined using Li-6400 (LI-COR, USA), and photosynthesis-related indices were determined four times for each treatment. The parameters were set as follows: light intensity, 1,200 μmol m^-2 s^-1; CO₂ concentration, 500 ± 5 μmol mol^-1.

Sorghum seedling leaves were digested with 1 mL of 6 M HCl at 110°C for 24 h. Next, 1 mL of the sample was centrifuged at 15,000 ×g for 10 min. The supernatant was neutralized using 2 M NaOH. The neutralized and diluted samples were subjected to ultra-performance liquid chromatography (UPLC) coupled to a Vanquish mass spectrometer (MS) equipped with a quadrupole electrospray ionization (QE) source. The analytical conditions were as follows: a C18 column (1.7 μm, 50 × 2.1 mm) was maintained at 55°C with a flow rate of 0.5 mL/min and injection volume of 1 μL. Gradient elution was then performed. Finally, the data were analyzed using LC–MS/MS on a Q Exactive hybrid Q–Orbitrap mass spectrometer equipped with a heated ESI source (Thermo Fisher Scientific) using the full MS acquisition method (Glauser et al., 2016).

The leaves and roots were dried, pulverized, and boiled in concentrated sulfuric acid. Total N content was determined using the Kjeldahl method. The nitrate and ammonium contents in the roots were determined using colorimetric and ninhydrin colorimetric methods, respectively, with some modifications (Wang et al., 2018a). Briefly, fresh root (0.1 g) was ground into a homogenate with distilled water, transferred to a centrifuge tube, and boiled for 10 min. Subsequently, the supernatant was centrifuged at 15,000 ×g for 10 min, and 0.1 mL of the supernatant was added to 0.4 mL of 5% salicylic-sulfuric acid solution and reacted at 20°C under light for 20 min. Furthermore, 9.5 mL of 8% NaOH solution was added slowly. Finally, the NO₃ ^- content was quantified at 410 nm using a spectrophotometer.

Similarly, roots (0.1 g) were placed in 10% acetate to extract NH₄ ⁺, mixed with distilled water, and filtered. The supernatant was combined with a solution of ninhydrin reagent (ninhydrin with propanol, butyl alcohol, glycol, and acetate, pH 5.4) and 1% ascorbic acid and boiled for 15 min. The concentration of NH₄ ⁺ was quantified at 580 nm.

Nitrate reductase (NR) and nitrite reductase (NiR) activities were determined using nitrate as described by Qu et al. (2023). Briefly, samples (200 mg) were ground in an extraction buffer containing potassium phosphate (pH 8.8), EDTA, cysteine, and 3% (w/v) bovine serum albumin (BSA). The absorbance values of the NR and NiR supernatants were measured at 540 nm.

Glutamine synthetase (GS) and glutamate synthase (GOGAT) activity levels were analyzed using methods described by Wang et al. (2018a) and Luo et al. (2013). Briefly, leaves or roots (200 mg) were extracted in buffer (Tris-HCl, pH 8.0; MgSO₄, DTT, and sucrose) to measure GS activity. In addition, the leaves and roots were extracted with Tris-HCl (pH 7.6), MgC1₂, EDTA, and β-mercaptoethanol at 4°C to measure GOGAT activity. After the homogenate was centrifuged at 15,000 × g for 30 min at 4°C, the absorbance of the GS and GOGAT supernatants was measured at 540 and 340 nm, respectively.

The soluble sugar, sucrose, and starch contents were determined by anthrone sulfate colorimetry (Ren et al., 2023). Fresh leaf and root samples (0.1 g) were extracted using 80% ethanol solution. The supernatant was collected, and its volume was fixed at 20 mL with 80% ethanol to determine the soluble sugar and sucrose contents. The starch content was determined by precipitation.

The leaves and roots were extracted in a buffer (Tris-HCl, β-mercaptoethanol, MgCl₂, ethylene glycol-bis-β-aminoethyl ether, ethylenediaminetetraacetic acid, and 15% glycerol). The mixed solutions were centrifuged for 15 min at 17,000 ×g (4°C). The supernatant was used to determine enzyme activity. The activities of sucrose phosphate synthase (SPS), sucrose synthase (SuSy), and invertase (INV) were measured as described by Shahid et al. (2019).

Data from at least three replicates are presented as mean ± SE. Analysis of variance (ANOVA) was performed using the Duncan test design method with SPSS software (version 19.0, IBM, USA) at probability levels of 5%, 1%, and 0.1% for each data point with at least three duplications. Graphics were created using GraphPad Prism 8.3.0 and Adobe Photoshop 2020.

Crops often exhibit morphological and physiological responses to environmental stress, with the seedling stage being the primary and most sensitive stage of development. Consequently, seedling growth parameters must be incorporated into low-N tolerance evaluations. Evaluating seedling growth in low-N environments is essential to elucidate the low-N tolerance of sorghum. However, there is currently a paucity of systematic studies on low-N tolerance in sorghum. Zhang et al. (2017) compared nine genotypes of Fagopyrum tararicum and identified relative plant height, stem diameter, root/shoot ratio, leaf area, and chlorophyll concentration as indicators of low-N tolerance. This study further demonstrated that these indicators can be used to evaluate the tolerance of various genotypes, revealing similar patterns of morphological changes under low-N stress, although the extent of these changes varied among genotypes. PCA, Pearson’s correlation analysis, and Y value comprehensive analysis were used to evaluate the tolerance of 100 sorghum genotypes under N stress. The identified sorghum indicators were classified into three factors, with the maximum eigenvector load corresponding to plant height, shoot dry weight, and root dry weight. Ultimately, this systematic approach allowed the selection of 100 sorghum genotypes for a detailed evaluation of low-N tolerance during the seedling stage. The genotypes were then categorized into four groups according to tolerance levels. Identification and validation of low-N tolerance indicators are vital for breeders and will help improve available genetic resources. Furthermore, future studies should focus on employing dimensionality reduction techniques, simplifying complex traits, and applying visualization methods to provide a more detailed reflection of low-N tolerance across various sorghum genotypes.

Plants can adapt to diverse environmental stimuli by modifying their fundamental root structures—for example, plants can adjust their root architecture to enhance N acquisition under N-deficient conditions, thereby adapting to an N-deficient environment. The “steep, cheap, and deep” ideotype for maize (Zea mays) exemplifies this concept, proposing that root phenotypes optimize N capture under limited N resource availability (Gao et al., 2015). The results of our study indicate that the root morphologies of the two sorghum genotypes exhibit distinct responses to N-deficient conditions. Specifically, genotype 398B had a longer root length and greater root dry weight than CS3541. This suggests that a plastic root system can enhance low-N tolerance in sorghum. Furthermore, faster root growth rates are conducive to plant growth (Yuan et al., 2022). Similar responses to N starvation were observed in terms of plant height and shoot dry weight in 398B and CS3541 plants under low-N conditions. However, 398B plants exhibited greater plant height and shoot dry weight, indicating higher N assimilation efficiency during growth.

The architecture of the root system, which is the physical configuration of the roots, governs their distribution within the soil over time and space, making it a primary determinant of N capture, especially in low-N environments. Anatomical phenotypes that reduce the carbon and nutrient requirements of root growth and maintenance should consequently improve the acquisition of soil N resource (Sun et al., 2018; Lynch et al., 2021). Lynch et al. (2023) indicated that an elongation of root cortical cells can ameliorate N capture by accelerating the root elongation rate, thereby improving the efficiency of N capture and utilization. This is corroborated by our study that N starvation triggered an increase in cortical cell length in the root elongation zone, which, in turn, altered the anatomical structure to support root elongation under low-N conditions. Root growth is characterized by its indeterminate nature, marked by the continual succession of cell division, regulated cell expansion, and differentiation within the meristem and adjacent root regions. Root morphogenesis is controlled by the interplay of cell division and expansion regulation (Strader et al., 2010). In this study, it was observed that the meristematic zone of the roots in both sorghum genotypes exhibited a denser packing of cells compared to those grown under N-sufficient conditions. It was particularly noteworthy that 398B had a significantly higher number of cells in the meristematic zone of the root tips compared to CS3541 under low-N stress. As soil hardness typically escalates with depth, hindering the capture of N from deep soil, longer roots that can penetrate hard soil may also improve the capture of N from deep regions. Both cell division and expansion were pivotal phenotypic traits that merit further investigation as an avenue to improve N capture and use efficiency in sorghum, especially in response to N deficiency.

The leaf blade is specialized to capture light and utilize the energy for photosynthetic carbon assimilation while also being capable of limiting N content, surface area, and resistance for gas exchange (Wakayama et al., 2006). The specialized anatomy requires the development of two distinct chloroplast-containing cell types within the leaf: the mesophyll cells, which constitute the majority of cells within the leaf and are predominantly located between the parallel vascular strands, and the bundle sheath cells, which encircle the vascular tissue, forming a column of cells that extend along the length of each vascular strand (Smillie et al., 2012). Under low-N stress, we observed a degradation of bulliform cells and garland structures in CS3541 leaves, which was not apparent in the leaves of 398B. Furthermore, there was no significant variation in leaf thickness and the cross-sectional area of vascular bundles in 398B. However, a greater leaf epidermal thickness and a larger cross-sectional area of vascular bundle were observed in 398B leaves than in CS3541 leaves under low-N stress. This suggests that 398B accumulates biomass in leaves and promotes robust leaf growth and development by maintaining a stable internal leaf structure. The photosynthetic parameters, including photosynthetic rate (Pn), transpiration rate (Tr), intercellular CO₂ concentration (Ci), and stomatal conductance (Gs), serve as internal markers of photosynthetic metabolism in plants (Liu et al., 2022b; Qin et al., 2021). Therefore, it can be postulated that improvements in these gas exchange parameters under stress conditions could bolster plant stress resistance. Zhao et al. (2005) found that N deficiency significantly reduced the leaf chlorophyll concentration, the rate of leaf net photosynthesis, and biomass production in sorghum. In this study, Pn, Tr, Ci, and Gs were significantly reduced in 398B and CS3541 plants under N stress. However, the decrease was more pronounced in CS3541 than in 398B, suggesting that the N-efficient genotype has a higher capacity for C assimilation than the N-inefficient genotype, as discussed further below.

Improvements in plant growth in the external environment are often correlated with C and N metabolism (Woo et al., 2004; Meng et al., 2018). In particular, the longer roots in 398B exhibited significantly higher total N and NO₃ ^- content under low-N conditions than the CS3541 roots. This increase is likely associated with NRT function (Hawkins and Robbins, 2010). NR is a pivotal enzyme that regulates the rate-limiting step of the NO₃ ^- assimilation pathway. Subsequently, nitrate is converted to ammonium, which serves as a precursor for the synthesis of various amino acids, including glutamate, arginine, proline, and 4-hydroxyproline, via the GS/GOGAT cycle (Xu et al., 2012). The 398B plants were found to have higher NR, NiR, GS, and GOGAT activity levels in their leaves and roots than the CS3541 plants during N starvation. This indicates that 398B may have a greater capacity for amino acid biosynthesis under low-N conditions. This was further supported by the higher concentrations of most amino acids in 398B than in CS3541 under low-N conditions. Consequently, greater N assimilation capacities may enhance low-N tolerance in sorghum.

Glutamic acid (Glu), the primary amino acid produced by the GS/GOGAT cycle, serves as an amino group donor for the synthesis of numerous other amino acids (Forde and Lea, 2007). This study observed a significant reduction in the concentration of glutamic acid (Glu) in sorghum leaves under low-N conditions. Interestingly, no difference in the Glu concentration was observed between the two genotypes under low-N conditions, indicating that the biosynthesis of various amino acids may also be influenced by the scarcity of amino group donors. However, a considerable number of essential amino acids, including histidine, glycine, threonine, cysteine, tyrosine, isoleucine, leucine, and phenylalanine, were found to be significantly higher in 398B plants than in CS3541 plants under low-N stress. This robust individual response to low-N conditions, particularly for leucine, has also been observed in tomato leaves (Urbanczyk-Wochniak and Fernie, 2005). In the face of nutrient limitation, amino acids in leaves are recycled and allocated to young leaves or sink tissues for the synthesis of specific proteins, and this process occurs frequently (Watanabe et al., 2013; Hildebrandt et al., 2015)—for instance, the concentrations of threonine, isoleucine, and leucine were markedly diminished under conditions of low-N availability, which is likely due to the limited availability of C for N assimilation. Furthermore, the levels of glycolysis-derived phenylalanine and tyrosine also significantly decreased under low-N conditions, consistent with the findings of Schlüter et al. (2012). This evidence underscores that N deficiency is accompanied by significant alterations in different amino acids, indicating that amino acids play an important role in the adaptation of source-leaf metabolism to N deprivation and the export of N resources to sink organs.

Photosynthesis and C metabolism are well recognized as primary sources of energy, and the C skeleton is essential for plant growth and N assimilation (Du et al., 2023). Our findings indicate that sucrose, soluble sugar, and total sugar levels were significantly lower in 398B leaves than in CS3541 leaves regardless of N supply. Conversely, the roots exhibit the opposite trend. Consequently, it is postulated that C assimilation in 398B leaves is more efficient than that in CS3541 leaves. Furthermore, under low-N conditions, there was a significant increase in the activities of SS and SPS, whereas INV activity was induced in leaves. It is conceivable that C metabolism may be reprogrammed to favor C storage as starch or sucrose rather than exporting it to achieve a balance between C and N (Lu et al., 2020). In contrast, the roots of both 398B and CS3541 showed lower soluble sugar and total sugar contents but higher sucrose and starch contents under low-N levels. It was speculated that root growth retardation may be due to changes in sink demand, allowing C accumulation under N stress. This occurred despite a significant increase in the activities of SS, SPS, and INV in the roots. Notably, the sucrose content and activities of SS and SPS were greater in 398B roots than in CS3541 roots regardless of N supply. This is consistent with previous studies (Gelli et al., 2014; Bollam et al., 2021). These results indicate that the C/N balance, which is crucial to maintain optimal plant growth and respond to N limitations, is modulated differently in the two sorghum genotypes with contrasting tolerances to low-N. Understanding these genotype-specific responses is vital to develop strategies to enhance crop productivity in N-deficient environments.