*2.1. Physiological Mechanisms of Crop N*

Focusing on crop physiological mechanisms, N is closely linked to Chl. N is an important component in the formation of chloroplasts and Rubisco enzymes. Increasing leaf N content will significantly increase leaf chlorophyll content (LCC) and Rubisco enzymes, ultimately leading to a significant increase in crop photosynthetic rate and consequent changes in the external morphology and internal structure of crop leaves [9,29]. Chl can respond to N uptake by crops, and the strength of the relationship directly affects the accuracy of N estimation. However, N is redistributed and reused in the crop during growth. Early in crop development, N is initially concentrated in nutrient bodies such as leaves, and as nutritional and reproductive growth coexist, N starts to be distributed to nutrient bodies and reproductive organs; at the crop maturity stage, N in nutrient bodies is transferred to reproductive organs. This resulted in varying degrees of N and Chl content reduction in the canopy leaves, which changed the correlations between crop N and Chl at various growth stages [25]. In addition to Chl, other mineral deficits, diseases, frost damage, and water stress may also produce leaf yellowing, and using Chl content as a proxy for N is misleading, which limits the ability to estimate N directly from Chl [28]. It has been recommended to utilize leaf protein as a substitute for leaf N content in several studies [30,31], because in contrast to Chl, protein is also a major nitrogenous component in crops and contributes to the varied distribution of N in crop plants. The mechanism of protein–N interactions is under investigation.

#### *2.2. Spectral Response Properties of Canopy N*

N affects the spectral reflectance of crops by influencing the Chl content of green crops (Figure 2). Healthy crops' VIS reflectance spectrum is determined by the Chl's absorption effect, which forms a prominent reflectance peak near 550 nm. Multiple reflections in the NIR combine to generate a red edge region of reflectance in the range of 700–780 nm, and the rising slope of the curve reflects the Chl content per unit area to some extent. NIR (780–1350 nm) is closely related to leaf structure and is instructive for exploring whether N is influenced by leaf structure. Under N stress, both the canopy spectral reflectance and the vertical distribution of N will alter.

**Figure 2.** Spectral reflectance properties of the wheat canopy: (**A**) spectral reflectance under N stress; (**B**) spectral reflectance at different growth stages under normal N; and (**C**) correlation between leaf nitrogen concentration (LNC) and spectral reflectance at different growth stages.

During N deficiency, the VIS reflectance of the crop canopy spectrum increased, while the NIR reflectance and red-edge position (REP) decreased; in excess of N, the VIS reflectance decreased, while the NIR reflectance and REP increased (Figure 2) [14,32–34]. As the growth stages proceed, the response of canopy spectral reflectance to crop N status reduced, and the VIS regions also displayed "red shift" and "blue shift" with the development [33,35,36]. It can be found that the analysis of crop N abundance and deficiency using spectral techniques can be specific to a band interval [7,37]. Hyperspectral techniques can even be precise to a specific band [38,39], which provides the possibility of effective identification of crop N deficiency.

The top leaves of the plant under N stress will use the N transferred from the bottom leaves, causing the bottom leaves to yellow and decline prematurely, while the top leaves color changes are not obvious because of being less stressed by N [40,41]. As influenced by the level of soil N supply, the spectral reflectance of leaves at different leaf positions differed erratically in VIS and SWIR, while showing a clear gradient in NIR [42]. Duan et al. [43] suggested that N concentration at different leaf positions decreases from top to bottom at the jointing stage, flowering stages, and filling stage, while the flag leaf stage shows an increasing and then decreasing trend. The vertical distribution of N in the plant is not constant and varies between N conditions, planting densities and growth stages [41,43,44]. Exploring the spectral response properties of the different leaf positions can serve as a foundation for precise N quantification.
