Glutamine synthetase (GS), the key enzyme in plant nitrogen assimilation, is strictly regulated at multiple levels, but the most relevant reports focus on the mRNA level. Using specific antibodies as probes, the effects of nitrogen on the expression and localization of individual wheat GS (TaGS) isoforms were studied. In addition to TaGS2, TaGS1;1 with high affinity to substrate and TaGS1;3 with high catalytic activity were also localized in mesophyll, and may participate in cytoplasmic assimilation of ammonium (NH₄⁺) released from photorespiration or absorbed by roots; TaGS1;2 was localized in xylem of leaves. In roots, although there were hundreds of times more TaGS1;1 than TaGS1;2 transcripts, the amount of TaGS1;1 subunit was not higher than that of TaGS1;2; NH₄⁺ inhibited TaGS1;1 expression but stimulated TaGS1;3 expression. In root tips, nitrate stimulated TaGS1;1, TaGS1;3, and TaGS2 expression in meristem, while NH₄⁺ promoted tissue differentiation and TaGS1;2 expression in endodermis and vascular tissue. Only TaGS1;2 was located in vascular tissue of leaves and roots, and was activated by glutamine, suggesting a role in nitrogen transport. TaGS1;3 was induced by NH₄⁺ in root endodermis and mesophyll, suggesting a function in relieving NH₄⁺ toxicity. Thus, TaGS isoforms play distinct roles in nitrogen assimilation for their different kinetic properties, tissue locations, and response to nitrogen regimes.

Glutamine synthetase (GS), a key enzyme in plant nitrogen metabolism, is closely related to nitrogen remobilization. However, how GS isoforms participate in nitrogen remobilization remains unclear. Here, the spatiotemporal expression of the TaGS gene family after anthesis was investigated, and the results showed that TaGS1;1 was mainly encoded by TaGS1;1-6A, while the other isozymes were mainly encoded by TaGS localized on the A and D subgenomes. TaGS1;2-4A/4D had the highest expression level, especially in rachis and peduncle. Furthermore, immunofluorescence showed TaGS1;2 was located in the phloem of rachis and peduncle. GUS (β-glucuronidase) staining confirmed that ProTaGS1;2-4A/4D::GUS activity was mainly present in the vascular system of leaves, roots, and petal of Arabidopsis. Ureides, an important transport form of nitrogen, were mainly synthesized in flag leaves and transported to grains through the phloem of peduncle and rachis during grain filling. TaAAH, which encodes the enzyme that degrades ureides to release NH₄⁺, had a higher expression in rachis and peduncle and was synchronized with the increase in NH₄⁺ concentration in phloem, indicating that NH₄⁺ in phloem is from ureide degradation. Taking the above into account, TaGS1;2, which is highly expressed in the phloem of peduncle and rachis, may participate in N remobilization by assimilating NH₄⁺ released from ureide degradation.

Studies of traits related to nitrogen (N)-use efficiency (NUE) in wheat cultivars are important for breeding N-efficient cultivars. Canopy structure has a major effect on NUE, as it determines the distribution of light and N. However, the mechanism by which canopy structure affects the distribution of light and N within the canopy remains unclear. The N-efficient winter wheat varieties YM49 and ZM27 and N-inefficient winter wheat varieties XN509 and AK58 were grown in the field under two N levels. Light transmittance was enhanced, and the leaf area index and photosynthetically active radiation were lower in the N-efficient cultivar population, which was characterized by moderately sized flag leaves, a low frequency of canopy leaf curling, a low light attenuation coefficient (K_L), and high plant compactness. Reductions in the amount of shade increased the distribution of light and N resources to the middle and lower layers. The photosynthetic rate, transpiration rate, instant water-use efficiency, and canopy photosynthetic NUE were higher, N remobilization of the upper and middle canopy leaves was reduced, and the leaf N content was high in the N-efficient cultivars. A higher ratio of the N extinction coefficient (K_N) to K_L reflects the assimilation ability of the N-efficient winter wheat cultivars, resulting in improved canopy structure and distribution of light and N, higher 1000-grain weight and grain yield, and significantly increased light and NUE. An improved match between gradients of light and N in the leaf canopy promotes balanced C and N metabolism and reduces energy and nutrient losses. This should be a goal when breeding N-efficient wheat cultivars and implementing tillage regimes.

Nitrogen is one of the essential elements for wheat growth and development, and NO3--N is the main form of nitrogen that wheat obtains from soil. NRT/NPF genes family encode membrane transporters, which are mainly involved in NO3--N absorption, transport, and allocation in plants. In order to understand the relationship between NRT/NPF family and nitrogen utilization in wheat, the relative expression characteristic of TaNRT/TaNPF family in flag leaves of N-efficient wheat cultivars Zhoumai 27 (ZM27) and N-inefficient wheat cultivars Aikang 58 (AK58) at flowering stage were studied with the second-generation sequencing technology. The results showed that 386 genes of TaNRT/TaNPF family were identified in the second generation transcriptome database. Compared with AK58, there were 27, 16, and 23 differentially expressed genes in ZM27 in reducing (N120), normal (N225), and excessive (N330) nitrogen treatments. There were 16 (59.26%), 12 (75%), and 19 (82.61%) up-regulated genes in ZM27, respectively. Seven genes were down-regulated in ZM27 in reducing nitrogen treatment. The relative expression level of TaNPF8.1 was the highest and significantly up-regulated by 1.5 times in nitrogen excessive condition. In conclusion, the relative expression of TaNRT/TaNPF family genes was regulated by nitrogen application rate and cultivar. Wheat network database showed that the relative expression of TaNRT/TaNPF family had tissue specificity and chromosomal preference. The highest expression level of TaNPF8.1 in flag leaf was located on chromosome 3A, and the root specific expression TaNRT2.2 and TaNRT3.1 were mainly distributed on chromosome 6. The stem specific expression of TaNPF4.5 was mainly distributed on chromosome 2. The qRT-PCR of TaNRT/TaNPF genes were consistent with the results of the second-generation transcriptome and network data. Interaction analysis of TaNPF8.1, TaNPF4.5, and TaNRT3.1 revealed that NO3--N transport may also require the collaborative participation of transcription factor MYB, chlorophyll A-B binding protein, and chaperone protein. These findings laid a foundation for further studies on the relationship between TaNRT/TaNPF family expression and nitrogen uptake and utilization.

Canopy ammonia (NH₃) exchange is a major contributor to agricultural NH₃ emissions and is closely linked to nitrogen-use efficiency. Glutamine synthetase (GS) mediates plant NH₃ assimilation, yet the specific roles of different GS isoenzymes in regulating wheat canopy NH₃ exchange remain unclear. This study aimed to clarify the functional differences of wheat TaGS isoenzymes in modulating canopy–atmosphere NH₃ exchange dynamics using two wheat cultivars (Yumai 49-198 and Xinong 509) under two nitrogen application levels (120 and 225 kg N ha⁻¹). Field experiments combined with FTIR-based NH₃ flux measurement, biochemical assays, and molecular analyses were conducted at anthesis and 16, 24, and 30 days after anthesis (DAA). Results showed that the leaf NH₃ compensation point, determined by apoplastic NH₄⁺ concentration, is a key factor influencing canopy NH₃ exchange. Leaf NH₃ sources exhibited distinct temporal specificity: photorespiration and nitrate reduction dominated at anthesis to 16 DAA, whereas nitrogenous compound degradation prevailed at 24–30 DAA. This temporal partitioning was highly coordinated with TaGS isoenzyme expression: TaGS2 was highest in early grain filling, potentially supporting assimilate NH₃ from photorespiration/nitrate reduction, while TaGS1;1 expression increased progressively, aligning with the scavenging of NH₃ from organic nitrogen degradation. These coordinated patterns suggest that the TaGS isoenzymes play differentiated roles in influencing wheat canopy NH₃ exchange. This study thus provides correlative insights that point to potential molecular targets for breeding nitrogen-efficient wheat cultivars and mitigating agricultural NH₃ emissions sustainably.

Nitrous oxide (N₂O) is a potent greenhouse gas and a major ozone-depleting substance of the nitrogen cycle. While global N₂O budgets focus on microbial soil sources, evidence suggests that living plants can also contribute to N₂O emissions. However, the origin of plant-associated N₂O fluxes is contested: measured emissions may arise from endogenous plant metabolism or merely from transport of soil-derived N₂O. In this review, we clarify these pathways and synthesize current findings from laboratory and field studies. Experimental evidence from ¹⁵N-tracer and axenic-culture studies supports N₂O formation linked to nitrate (NO₃⁻) and nitrite (NO₂⁻) in photosynthetic organisms, although mechanistic resolution varies among taxa. In angiosperms, proposed plastid/chloroplast and hypoxia-associated mitochondrial routes are linked to NO₃⁻/NO₂⁻ metabolism and NO formation, but the terminal NO-to-N₂O step remains unresolved and differs from the flavodiiron-dependent mechanism demonstrated in algae. We discuss key environmental and physiological controls (substrate availability, light, O₂ status, reductant supply) on these processes and highlight methodological challenges in source attribution (chamber artefacts, destructive sampling, isotope analysis). Our analysis suggests that plant-associated N₂O fluxes are real, but highly variable and context-dependent. Rather than assuming generic “plant emission factors”, we emphasize source-specific approaches that distinguish endogenous formation from soil-derived transport and microbial production. This framework can improve N₂O budgets and guide mitigation by indicating whether management should target soil N availability and microbial processes or physiological conditions favouring plant-associated N₂O formation.