The experiment was conducted during 2020-2022 in the 5 villages in Ayikule Township, Zepu County, Xinjiang, China (38°18′N, 77°17′E). The altitude ranges from 1215 to 1490 m. The climate is warm temperate continental arid, and the daily average temperature and rainfall during the growth period of winter wheat are shown in Figure 1A . The daily solar radiation and total solar radiation for two growing seasons are shown in Figure 1B . The former crop was summer soybean, and the soil of the experimental site was sandy loam. The initial soil nutrients of the upper soil layer (0-20 cm) contained 1.5 g kg^-1 soil organic matter, 0.7 g kg^-1 total nitrogen, 38.4 mg kg^-1 alkaline nitrogen, 17.9 mg kg^-1 available phosphorus, and 102.6 mg kg^-1 available potassium.

Field experiment was conducted based on the mode of walnut intercropping with wheat, and a two-factor split-plot experimental design was used in the experiment. The degree of shade corresponding to different canopy areas of the walnut trees were the main factor, including below canopy area (BCA) and far canopy area (FCA) ( Figure 2 ); exogenous spraying as secondary factors, including exogenous 6-benzylaminopurine (6-BA) spraying (BCA+6-BA and FCA+6-BA, respectively) and water spraying as control. 6-BA was sprayed once at the booting stage and once at the jointing stage of wheat at a concentration of 60 mg L^-1, with a spray rate of 750 L ha^-1, and each treatment was repeated three times. The wheat variety Xindong 60 was sown on October 6 and 8 in 2020, 2021, and harvested on June 10 and 12 in 2021, 2022.The sowing rate was 300 kg ha^-1 and row spacing of row planting was 15 cm. The walnut tree was 8 years old and evenly spaced north-south with 8 m row spacing and 4 m plant spacing. The width of the walnut-wheat belt was 7.2 m and the minimum distance between the walnut trees and the wheat rows was 1.0 m. The FCA was the area between two corresponding tree trunks located 0.4-2.8 m from the trunk; the BCA referred to the area between two corresponding tree trunks located 2 m from the trunks, as shown in Figure 2 . The data in the BCA was the average data from two BCA. Before sowing, 150 kg ha^-1 of urea (containing 46% N) and 300 kg ha^-1 of diammonium phosphate (containing 46% P₂O₅, 18% N) were applied as basic fertilizers. Flood irrigation was applied six times during the whole growing season (overwintering, regreening, jointing, booting, flowering and filling) with a total irrigation volume of 4350-4950 m³ ha^-1. Other cultural practices remained constant in all treatments.

The data were subjected to analysis of variance (ANOVA) using SPSS Statistics 26.0 (Chicago, USA). The differences between means were compared using the least significant difference (LSD) test with a P value of < 0.05. The random forest model analysis to rank the contribution of physiological indices at flowering and filling stage to yield was performed using the “ randomForest “ and “ rfPermute “ packages in R. Graphs were generated using Origin 2024b and Microsoft PowerPoint 2016 (Microsoft, Redmond, USA).

In the walnut-wheat intercrop, shade stress significantly inhibited wheat growth ( Figure 3 ). Specifically, the dry matter weight of single wheat stems was significantly lower in the BCA treatment compared to FCA, especially at maturity ( Figure 3A ). Meanwhile, compared to the FCA treatment, the LAI of wheat in the BCA treatment was reduced by 10.4%-23.9% at the flowering and filling stages ( Figure 3B ). Interestingly, wheat dry matter accumulation and LAI (except for 2022) from flowering to filling were increased by exogenous 6-BA, both under BCA and FCA conditions. Correspondingly, affected by canopy shading of walnut trees, the number of effective spikes decreased by 10.81%-11.64%, number of grains per spikes decreased by 7.9%-10.1%, thousand grain weight decreased by 8.1%-10.7%, and grain yield of wheat decreased by 26.6%-27.0% in 2 years in BCA treatment compared with FCA. In the BCA and FCA treatments, the number of effective spikes increased by 1.39% to 3.44%, the number of grains per spike by 4.4% to 9.18%, the thousand grain weight by 2.3% to 5.5% and the yield by 10.4% to 16.5% when exogenous 6-BA was applied compared to spray water ( Table 1 ). There was a significant positive correlation between wheat dry matter weight at flowering and grain filling and yield at maturity, with R2 values of 0.78 and 0.81, respectively ( Figure 3C ).

The photosynthetic characteristics of wheat flag leaves were significant differences during the flowering and grain filling stages in different canopy regions. In particular, the SPAD value of wheat flag leaf was significantly higher in the BCA than in the FCA ( Figure 4 ). The SPAD value of flag leaves was significantly increased by exogenous 6-BA under BCA and FCA conditions. Meanwhile, Pn, Gs and Tr of winter wheat flag leaves were significantly reduced in BCA compared with FCA, while Ci was significantly increased ( Figure 4 ). The Pn, Tr and Gs of wheat flag leaves during the flowering and grain filling stages were significantly increased under exogenous 6-BA, while the Ci of flag leaves was reduced. Further analysis of photosynthetic physiological parameters revealed that the activity of RuBPCase and PEPCase in wheat flag leaves gradually decreased from flowering to grain filling stage ( Figure 4 ). The RuBPCase and PEPCase activities of wheat flag leaves in BCA were higher than those in FCA, and the change pattern of 2-year experimental results between treatments was consistent; the RuBPCase and PEPCase activities of wheat flag leaves in BCA and FCA under the intercropping mode of walnut wheat were increased to different degrees by spraying exogenous 6-BA. During the two years, the Fv/Fm and φ II of wheat flag leaves in the flowering period showed similar trends ( Figure 4 ).

During the flowering and filling stages, the activities of SOD, POD, CAT and APX in wheat flag leaves were significantly lower in BCA than in FCA ( Figure 5 ). The activity of GR in the flowering period was higher in BCA than in FCA, but during the grain filling period was opposite. Compare with spraying water, the activities of SOD, POD, CAT and APX in wheat leaves were significantly increased after spraying 6-BA under intercropping mode, especially under FCA. Moreover, the activity of GR was significantly increased by exogenous 6-BA under FCA. Therefore, exogenous 6-BA enhanced the antioxidant capacity of wheat flag leaf under shade stress caused by walnut-wheat intercrop. There was also a highly significant positive correlation between APX and GR activity and Pn, Tr and Gs ( Figure 6 ).

Shade stress and exogenous 6-BA treatments regulated endogenous hormone homeostasis in wheat flag leaves during both the flowering and grain filling stages. Specifically, IAA and GA contents in wheat flag leaves under BCA were significantly higher than those under FCA, while the ABA and ZT contents were much lower than those under FCA. Further analysis shows that the endogenous IAA, GA and ZT contents in wheat leaves were significantly increased by spraying 6-BA under BCA and FCA compared to not spraying 6-BA, with the most pronounced increase at maturity, while the ABA content of wheat flag leaves was significantly reduced ( Figure 7 ).

The results of the principal component analysis between the photosynthetic parameters, antioxidant enzymes, hormone content and dry matter accumulation in wheat during the flowering and grain filling stages showed that PC1 and PC2 together explained 57% of the changes in dry matter weight ( Figure 8A ). The main factors influencing dry matter accumulation in wheat were Pn, Tr, Gs, ABA content and APX activity in flag leaves. Furthermore, the random forest model quantified the contributions of photosynthetic and physiological indicators of wheat to mature yield ( Figure 8B ) and the results showed that the activities of RuBPCase and PEPCase, the value of Gs, Tr, Pn and Ci, the content of ABA and ZT of flag leaves during flowering significantly contributed to yield, when the activity of RuBPCase, PEPCase and APX, the value of Gs, Pn, Ci and Tr, the content of GA, ZT, ABA of flag leaves during grain filling stage significantly contributed to yield of maturity.

In the fruit-grain intercropping mode of land use optimization, grain crops are disadvantaged in growth, especially for cereal crops such as wheat (Charbonnier et al., 2017; Liu et al., 2020). Similar to the results of Nasielski et al. (2015), who found that soybean growth was significantly inhibited in agroforestry intercropping areas, the present study found a significant decrease in dry matter accumulation and yield of wheat in BCA compared to FCA in a walnut-wheat intercropping pattern in Xinjiang, China ( Figure 3A ; Table 1 ). A highly significant positive correlation was found between dry matter weight and yield at flowering and filling stages ( Figure 2C ). Photosynthesis provides the material basis for yield formation, with more than 90% of wheat yield derived from photosynthesis (Parry et al., 2011), and flag leaf photosynthesis contributing about 40% of yield (Carmo-Silva et al., 2017). The efficiency and regulation of Rubisco and PEPCase are reasonable strategies to increase photosynthesis and yield (Carmo-Silva et al., 2015). However, despite the increase in RuBPCase and PEPCase activities induced by shade stress, Pn, Tr and Gs were significantly suppressed in wheat flag leaves under BCA ( Figure 4 ; Figure 6 ), which may be related to the insufficient CO₂ supply. Interestingly, this study also found a significant positive correlation between APX and CAT activities and photosynthetic parameters ( Figure 6 ). Hussain et al. (2020) showed that shade increased antioxidant enzyme activities in soybean and that the magnitude of the effect was greater in the early than in the late stages of shading. However, in the present study, enzymatic antioxidant systems such as SOD, POD, APX were significantly weakened in wheat flag leaves in the near-canopy area ( Figure 5 ), which is connected with the plant adaptation mechanism to shade. Hormones are key physiological indicators that balance plant growth and stress tolerance (Stamm and Kumar, 2010; Xu et al., 2021). Previous studies have shown that post-anthesis shade reduces IAA levels but increases ABA levels in maize kernels (Wang et al., 2020). The study on Rumex palustris showed that flooding and shading not only induced GA production but also enhanced sensitivity to GA (Benschop, 2004). In the present study, shading significantly increased IAA and GA contents and decreased ABA and ZT contents in wheat flag leaves ( Figure 7 ). The contribution of ZT and ABA contents to yield was significant in wheat flag leaves at the flowering and filling stages ( Figure 8B ). Thus, walnut tree shading significantly altered flag leaf photosynthesis, antioxidant enzyme and hormone contents, which in turn reduced dry matter accumulation and yield of winter wheat.

Exogenous hormones alleviate plant stress and promote plant growth under stress conditions by improving plant physiological metabolism (Panigrahy et al., 2019; Mao et al., 2020; Li et al., 2020, 2023). A series of studies have shown that 6-BA plays an important role in plant resistance to drought, waterlogging, high temperature, salinity and other stresses (Wu et al., 2012; Ghaleh et al., 2020; Hu et al., 2022). In maize, 6-BA application improved light energy uptake and dissipation, carbon assimilation and metabolism, and chloroplast ultrastructure, thereby increasing photosynthetic rate in flooded summer maize (Hu et al., 2022). In this study, exogenous 6-BA significantly increased RuBPCase activity in wheat flag leaves ( Figure 4 ), thereby promoting photosystem II efficiency and net photosynthesis in wheat ( Figure 3 ). Similar to the results of Pan et al. (2013) in hybrid rice, exogenous 6-BA significantly increased the activities of SOD, POD, CAT and APX in flag leaves at anthesis and filling stages, especially at FCA ( Figure 5 ), which also indicates that 6-BA application effectively enhanced the scavenging capacity of leaves under shade for superoxide anion, hydrogen peroxide and hydroxyl radical (Li et al., 2020). The most important steps in photosynthesis and the synthesis of hormones for the defensive classes of plants both take place in the chloroplasts (Lu and Yao, 2018). During the flowering and filling stages, exogenous 6-BA increased the content of IAA, ZT, and GA in wheat flag leaves, while reducing the content of ABA ( Figure 7 ). Cytokinin is an essential phytohormone involved in the regulation of photosynthesis (Hudecek et al., 2022). Compared to water spraying, 6-BA application decreased growth hormone content, increased cytokinin content and young spike dry matter, and transported more soluble sugars and sucrose from non-spike organs to young spikes (Li et al., 2019). The main factors influencing dry matter accumulation in wheat were Pn, Tr, Gs, ABA content and APX activity in flag leaves ( Figure 8A ). Importantly, exogenous 6-BA also significantly increased wheat dry matter accumulation in BCA and FCA ( Figure 3 ). The flowering to grain-filling stage is a critical period for wheat grain development and filling (Djanaguiraman et al., 2020). Increasing photosynthetic capacity by exogenous 6-BA significantly increases wheat thousand kernel weight, which in turn increases yield ( Table 1 ). Overall, exogenous 6-BA facilitated the enhancement of physiological activity and photosynthesis in wheat, which in turn partially mitigated the detrimental effects of shading on wheat yield development. 6-BA is also a potentially valuable chemical control regulator that is expected to be beneficial in intensive agriculture.