A two-year field experiment was carried out at Huihe village, Chengdu plain, Sichuan province (102°54-104°53 E, 30°05-31°26 N) from 2020-22. It is a subtropical region with an average temperature of 16.1°C, annual total precipitation of 1780 mm, and a total sunshine duration of 1050 h (Sichuan Province Agro-meteorological Center, China). The basic soil fertility of soil includes organic matter (20.3 g/kg), total nitrogen (1.3 g/kg), available phosphorus (0.015 mg/kg), available potassium (0.118 mg/kg), and pH (6.7) in the topsoil layer (0-20cm). The monthly annual temperature and rainfall of the rapeseed growing season are demonstrated in Figure 1 .

The two rapeseed genotypes (Chuannong and Zhongyouza) were involved in the two-year field trial. These rapeseed genotypes are abundantly cultivated in Sichuan province, especially in the higher reaches of the Yangtze river basin. The experiment employed a two-factor split-plot design. Three shading treatments were established at various growth stages of rapeseed; S0 = control (ambient light), S1 = shade from GS5 to GS6 and S2 = shade from GS7 to GS8 ( Figure 2 ). The plants were enclosed by a layer of black polyethylene nets, which blocked approximately 35% of solar radiation. Two cultivars were assigned to the main plot and subplots received shading treatment. All treatments were carried out three times, yielding 24 plots with a 12 m² plot size. The prior harvested crop was rice, and the soil fertility was medium. The field was rotated before sowing, and a rope was manually pulled on-line while maintaining a row-row distance of 33 cm and a plant-plant gap of 20 cm. One seedling was left in each hole after emergence, and the baseline planting density was 150,000 plants/ha. Phosphorus and potassium fertilizers were applied at a rate of 90 kg/ha as base fertilizer. Nitrogen fertilizer was used at a 90 kg/ha rate in split dosages of 50% as base fertilizer + 50% topdressing at seedling stage. Local measurements were practiced to control the pests and weeds.

The data was recorded and sorted out by Microsoft Excel 2019. SPSS 19.0 (SPSS, Chicago, IL, USA) software was used to statistically analyze all the data. To estimate the differences among treatments, ANOVA with three-way analysis of variance followed by least significant difference (LSD) at p<0.05 significance level was performed. Pearson correlation coefficient were calculated to determine the relationship between different parameters. All the tables and figures were shaped by Excel 2019 and Origin 2021 software (OriginLab Co., Northampton, MA, USA).

Different shading treatments significantly altered the yield variables of both investigated rapeseed genotypes. In contrast to S0, the Chuannong genotype showed a decreased number of pods by 7.40 and 9.23% and Zhongyouza genotype experienced the 7.16 and 8.25% after S1 and S2 treatments as compared to S0, respectively. While the number of seeds per pod was lowered by 5.91 and 39.60% in Chuannong and 7.58 and 33.85% in Zhongyouza after the respective shading treatments. Under S1 and S2, the Chuannong exhibited 2.78 and 19.73% reduction and Zhongyouza showed 4.42 and 12.04% decline in 1000-seed weight following S1 and S2, respectively. In case of yield, the S1 and S2 declined the yield of Chuannong genotype by 13.31 and 50.03% and this reduction was 11.06 and 37.01% in Zhongyouza, respectively. Under various shading treatments, the aforementioned yield characteristics in both years demonstrated a similar trend, while 2020-21 year significantly exhibited higher yield in both genotypes ( Figure 3 ). Additionally, S2 had a significant impact on all yield parameters. Taken altogether, it was observed that the Chuannong genotype was more shade-sensitive and showed lower yield than Zhongyouza under shade treatment.

Shade stress significantly reduced the dry matter of both rapeseed genotypes. According to two-year average data, the DM was reduced by 7.28 and 33.32% in Chuannong and 7.16 and 31.91% in Zhongyouza following S1 and S2 treatment as compared to S0, respectively. Shading at both growth stages disrupted the accumulation and distribution of DM in the organs of rapeseed. Under S2, a significant drop of DM was detected in the rapeseed organs. The seed weight was more affected by shading than stem and pod shells at the organ level under S2.

Contrary to S0, the Chuannong genotype showed the 8.96 and 58.34% decline in seed weights after S1 and S2 treatments as compared to S0, respectively, while Zhongyouza exhibited 22.9 and 49.63% inhibition after the respective shading treatments. The stem, pod shell, and seed weights of both genotypes under shading followed a similar decreasing trend: S2<S1<S0 ( Figure 4 ). The DM of all organs followed the same reducing tendency in both years. but 2020-21 displayed higher dry matter than 2021-22 year. Aside from that, shade during the pod stage (S2) substantially impacted both cultivars’ dry matter.

The differences in nitrogen accumulation and distribution were found under shading stress at distinct growth stages. The values in Tables 1 and 2 represent the mean value for two-year experiment. The total nitrogen (TN) of both genotypes was detected in the following increasing order: S0>S1>S2 at maturity stage. In contrast to S0, S1 and S2 treatments reduced the TN distribution of Chuannong by 17.84 and 73.29%, respectively, however this reduction was 8.47 and 40.27% in Zhongyouza, respectively ( Table 1 ). Shading had an impact on rapeseed organs of both genotypes. For instance, S1 had lower nitrogen values for leaves and stems, whereas pod shells and seeds showed lower nitrogen values under S2. Regarding genotypes, a higher TN was observed in Zhongyouza. Moreover, shading treatments affected both genotypes’ N contents of the leaves, stems, and pods ( Table 1 ).

The lower value of N translocation (NT), N translocation efficiency (NTE) and N contribution proportion (NCP) was perceived in S1, whereas higher values were examined in S2 treatment. The NTE was 5.30 and 36.78% lower in Chuannong and 23.08 and 37.08% in Zhongyouza genotype under S1 as compared to S0 and S2, respectively. The N harvest index (NHI) and N assimilation (NA) values of both genotypes were lowest in S2 treatment ( Table 2 ).

Shading decreased the distribution of ¹⁵N isotopes in different organs of rapeseed ( Figure 5 ). Compared to S0, the stem ¹⁵N accumulation was declined in Chuannong genotype by 69.31 and 12.85% under S1 and S2, respectively, however, this change was 54.14 and 5.12% in Zhongyouza genotype. In Chuannong, the reduction of 7.17 and 72.35% in seeds ¹⁵N accumulation was observed following S1and S2 relative to S0, respectively. While this inhibition was 15.31 and 86.38% for Zhongyouza. Leaf and stem ¹⁵N accumulation of both genotypes displayed a increasing trend; S0>S2>S1. While pod shell and seeds exhibited a increasing trend; S0>S1>S2. The ¹⁵N accumulation in the entire plant decreased under both shade tratements, compared to S0. In general, both rapeseed genotypes exhibited the following ¹⁵N accumulation trend; S0>S1>S2. Taken altogether, it was noticed that the Zhongyouza displayed a higher accumulation of ¹⁵N than Chuannong ( Figure 5 ).

The shading stress at both growth stages considerably influenced the enzymatic activities in the leaves and pod shells of both the studied genotypes. Relative to S0, the S1 reduced the NR, NiR, GS and GOGAT activities by 20.65, 8.60, 33.74 and 9.24% in leaves of Chuannong, respectively. Whereas the Zhongyouza experienced a 28.31, 12.96, 21.47 and 14.05% reduction following S1 as compared to S0, respectively ( Figure 6 ).

In case of pod shell, the NR activity of Chuannong was reduced by 6.37 and 28.33% after S1 and S2 relative to S0, respectively, while this decline was 11.51 and 30% for Zhongyouza genotype. A decline of 7.75 and 11.47% was detected in NiR activity of Chuannong and 4.98 and 10.56% Zhongyouza genotypes under S1 and S2 when compared with S0, respectively. The S1 and S2 treatments also declined the pod shell GS activity of Chuannong (8.91 and 25.45%) and Zhongyouza (9.31 and 27.74%), respectively. Similarly, the pod shell GOGAT activity showed 15.05 and 24.67% decline in Chuannong and 6.50 and 16.46% in Zhongyouza genotype following S1 and S2, respectively ( Figure 6 ). Comparing both genotypes, our findings unveiled that the Chuannong cultivar showed higher NR and GOGAT activity while Zhongyouza showed more NiR and GS enzymatic activities under S1 and S2 treatments. Furthermore, comparing S1 and S2, S2 significantly lowered all the enzymatic activities in both genotypes.

The changing trend of carbohydrates content of the both rapeseed genotypes was the same in both years. The shading treatment considerably reduced the sucrose, reducing sugar and soluble sugar contents of the stem and pod shell of both tested genotypes. The sucrose content of stem was declined by 14.08 and 41.28% in Chuannong and 6.87 and 35.36% in Zhongyouza, while pod shell showed a reduction of 16.45 and 35.18% in Chuannong and 14.19 and 37.99% in Zhongyouza following S1 and S2 treatments as compared to S0, respectively (average value based on two years). Generally, it was noticed that Zhongyouza genotype showed higher sucrose content than Chuannong genotype under all treatments.

Under various shading treatments, the reducing sugar content of Chuannong and Zhongyouza showed the following trend: S0>S1>S2 in both years. Compared with S0, the stem reducing sugar contents of Chuannong genotype experienced a decline by 15.21 and 76.66%, while this reduction for Zhongyouza genotype 10.71 and 51.21% after S1 and S2 treatments, respectively. In addition, the S1 and S2 decreased the reducing sugar of pod shell by 25.53 and 84.37% in Chuannong genotype and 15.52 and 55.81% in Zhongyouza genotype, respectively.

The soluble sugar content of Zhongyouza genotype was higher than that of Chuannong under all the treatments. Contrary to control, the stem soluble sugar of Chuannong genotype was inhibited by 10.52 and 46.72% and Zhongyouza genotype was reduced by 10 and 44.26% following S1 and S2 treatments, respectively. However, pod shell soluble sugar content showed a decline of 8.56 and 36.24% in Chuannong and 8.21 and 34.39% in Zhongyouza genotypes after the respective shading treatments. Moreover, the following inclination of carbohydrates was observed in both cultivars; S0>S1>S2 ( Table 3 ). Furthermore, Zhongyouza showed significantly higher carbohydrates content in stem and pod shell and 2020-21 year showed higher values of carbohydrates as compared to 2021-22. Collectively, it was seen that the shade at the pod development stage (S2) significantly affected the carbohydrates content in both years.

The current study’s correlation analysis demonstrated that shade stress was substantially connected to yield metrics, nitrogen absorption and carbohydrates transportation. All the enzyme activities were significantly positive correlated with N transportation to different organs but a non-significant correlation of enzymatic activities with yield was observed. A negative correlation of NT, NTE, NCP and NA with carbohydrates were examined but carbohydrates exhibited positive correlation with yield parameters. Moreover, total dry matter and ¹⁵N displayed a significantly positive correlation with seed yield ( Figure 7 ).

Light is a critical environmental component impacting the growth and development of crops (Guoping et al., 2008; Zhong et al., 2014). Numerous studies have documented a decrease in yield due to shading stress (Cantagallo et al., 2004; Zhang et al., 2006; Acreche et al., 2009; Mu et al., 2010). In two-year studies, there were no significant declines in pods per plant but a significant fall in pod filling and grain weight (Wang et al., 2015). Previous researches have demonstrated that the drop in grain production was due to a reduction in grain number and weight (Acreche et al., 2009; Mu et al., 2010; Polthanee et al., 2011). Variations in ovule fertility and seed number per pod were impacted by changes in growth circumstances such as N availability, light and temperature (Bouttier and Morgan, 1992). Grain yield and spikelet filling had significant positive linear associations, while grain yield and grain weight showed a positive relationship (Wang et al., 2015). Shading reduced grain dry weight during grain filling, lowering grain yield (Ishibashi et al., 2014). To pinpoint crucial growth stage, most other reported field experiments have not used sufficiently defined durations of shading. For instance, (Habekotté, 1993) and (Iglesias and Miralles, 2014) both used shade (60% and 50%, respectively) for entire anthesis stage and resulting in yield losses of 50% and 15%, respectively, but no particular growth stage could be determined. Thus, to our knowledge, our study is among the fewer studies of rapeseed that has identified a relatively critical growth stage which affected by shading.

We found that shade in the beginning of the pod’s development limited the assimilates transfer and decreased the weight of the pod shell and the number of seeds per pod (Tayo and Morgan, 1979). In the current study, the S1 demonstrated a relatively high yield when the supply of pods and seeds resumes to normal levels after the shade has been removed, although a shortage of assimilates on flowers under shade is also damaging and diminishes the potential for compensatory growth. As previously discussed, I canola appears more vulnerable to severe temperatures and water deficits during late blooming and early pod set, aligning its sensitive periods with those of pulses rather than cereals (Sadras and Dreccer, 2015). Thus, they function similarly to the shade treatments applied in this study. In general, the observed correlations between the time of shade treatments and their effects on yield components and their relationships at maturity are similar to previously described physiological consequences of reduced assimilate supply (Tayo and Morgan, 1979; Keiller and Morgan, 1988; Diepenbrock, 2000). Dry matter production and accumulation are the primary determinants of crop yield, which are also limited by different environmental factors (CH, 1995). Shading stress greatly changed the physiology and morphology of the plant and eventually decreased the dry matter accumulation and distribution, resulting in decreased grain yield (Acreche et al., 2009; Li et al., 2010; Mauro et al., 2011). Dry matter accumulation in high-yield maize accounts for more than 60% of the total dry matter. Grain yield is influenced by the development and distribution of dry matter in vegetative organs such as the stem, leaf, and sheath (Huang et al., 2007). Our results demonstrated that S2 treatment considerably reduced the pod shell and seeds dry weight, which caused the yield drop in both rapeseed genotypes. We can conclude that shade at pod development stage (S2) is crucial to cause reduction in dry matter and yield.

Increasing biological yield is the foundation for increasing output; nutrient intake and distribution are key prerequisites for biological yield (Hirel et al., 2007). This study examined the changes in N accumulation and transportation under shade at various growth stages and deduced a portion of the mechanism underlying the grain yield response to N use. The remobilization of nitrogen in vegetative organs and the uptake of additional nitrogen throughout the grain-filling cycle provide grain N (Mueller and Vyn, 2016). Furthermore, nitrogen remobilization in the stem and leaf accounts for 69 to 80% of grain N (Subedi and Ma, 2005; Chen et al., 2014b). As a result, N accumulation and distribution in vegetative and reproductive organs play a pivotal role in dry matter weight at maturity and influence grain yield. According to our findings, shade declined the total N accumulation of rapeseed in the following order: S0>S1>S2>. The total N of the pod shell and seeds were significantly reduced by shade at pod development (S2) compared to the flowering stage (S1). Furthermore, N buildup of S1 raised after the light was restored, but it did not return to normal levels ( Table 1 ; Figure 5 ). The S2 inhibited the amount of N translocation towards pod shell and seeds compared to other treatments ( Table 1 ), resulting in poorer yields (Chen et al., 2015b). When the accumulated N at pod development is smaller than the grain requirements, nitrogen transport rises (Chen et al., 2015b), as evidenced by our findings. Late-season shade (S2) reduced the N translocation towards economic organs ( Table 1 ). As a result, we found that shading reduced N uptake distribution in all organs, resulting in a decrease in seed yield. In conclusion, shade reduced N buildup and impeded N transfer from vegetative organs to grain, such as leaves, stems, and pod shells. This study found that shading at pod developmental stage (S2) had a greater detrimental impact on N uptake than at flowering stage (S1), consistent with root shape and root physiology changes during shading ( Figure 8 ) (Gao et al., 2017a). Shading altered the root structure, reducing root dry weight, absorption area, and active absorption area. Weather, climate, and air pollution contribute to shade, which is a challenging problem to solve in the manufacturing process. Changing sowing times is an excellent way to deal with low-light prone areas at later growth stage of crop, but it can be effected by temperature, soil moisture, and crop rotation as well (Gao et al., 2017a; Zhao et al., 2018).

The leaf N content and enzyme activities are closely associated with each other (Sinclair et al., 2000). We observed that, shade inhibited the activity of NR, NiR, GS, and GOGAT, as was shown by the earlier research (Wang et al., 2020). GS and GOGAT are two essential enzymes involved in the N metabolism (Nigro et al., 2017). Due to shade, GS and GOGAT activities reduced gradually in the present study. This observation in grains was the same as in leaves (Wang et al., 2020). Wheat responds as a sensitive to ammonium nutrition at low light intensities, and its low GS activity is insufficient for ammonium assimilation. This occurrence apparently arose as a result of the significantly decreased light intensity in southern China, where plants face weak light stress during grain filling, which is relatable to our findings (Setién et al., 2013; Gao et al., 2017b). When plants were subjected to shade, nitrate delivery to the tops dropped considerably. The drop in NR activity resulted from the reduction in nitrate concentration (Udayakumar et al., 1981). Reduced NR activities in shade-adapted plants make it easier for the plants to coordinate their N and carbon uptake across a variety of light conditions (Fredeen et al., 1991). The 50.3, 24 and 30.4% inhibition was also observed in NR, GS and GOGAT enzymatic activities following shade treatment (Yu et al., 2011). We concluded that leaves and pods’ enzymatic activities are greatly reduced by shade. Among the shading treatments, the S2 treatment considerably declined the enzyme activities in the pods, which restricted the nitrogen transport towards seeds that led to low grain yield in both the investigated rapeseed genotypes.

Shading limited the transformation of photosynthetic products. It accelerates the consumption of assimilates in leaves and stems and reduces grain yield (Li et al., 2013). Studies on different crops showed that the carbohydrate accumulation in leaves, stems, and roots decreased significantly under shading (Chen et al., 2014a; Hussain et al., 2021). As one of the main photosynthetic products, sucrose is significantly affected by light intensity and light cycle (Emerson, 1958). The decrease in sucrose content is closely related to light intensity. Shade reduce the output of leaves, the primary organ responsible for the formation of photosynthetic products, and eventually results in a decrease in sucrose content (Wu et al., 2017). The deleterious effect of whole-plant shade during grain filling on grain yield has been ascribed to photo-assimilate deficiency (Singh and Jenner, 1984; Okawa et al., 2003). However, there were differences in the accumulation and transport of carbohydrates under shading stress at different growth stages. This study showed that shading at pod stage (S2) had a more serious impact than shading at flowering stage (S1), which directly led to the reduction in grain yield. This could be because of leaf senescence, reducing photosynthetic potential, carbon fixation, and assimilates at pod development stage (Brouwer et al., 2012), which resulted in insufficient transportation of photosynthetic products. It was discovered that shaded wheat reduced grain output by speeding up the consumption of assimilates in the leaves and stems (Li et al., 2013). The authors further found that the carbohydrate of pod photosynthesis is mostly transported to the grain. In maize plants, the post-anthesis shading weakened the ability of nitrogen accumulation and stimulated the obvious re mobilization of carbohydrate reserves from stem to grain, but the decrease of grain filling rate eventually led to the decrease of grain yield (Reed et al., 1988). In this study, shading at flowering stage still reduced grain yield. The retardation can be attributed to the loss of non-structural carbohydrate transport to the kernel as a result of light deprivation (Mu et al., 2009) and kernel filling rate (Jichao and Zhiyong, 2005), which decreased the endosperm cell number and volume (Jia et al., 2011) and kernel set as a result of accelerated senescence. Starch deposition was decreased by shading, particularly under high shading. Additionally, ear shading decreased the kernel starch (Cui et al., 2012). Based on our findings, we can conclude that S2 treatment significantly reduced the carbohydrates translocation towards economic organs, leading to lower yields in both rapeseed genotypes.