Coupling photosynthetic physiology and C₄ enzyme regulation enhances grain yield of intercropped maize under no-tillage and moderate irrigation in oasis regions

Coupling photosynthetic physiology and C₄ enzyme regulation enhances grain yield of intercropped maize under no-tillage and moderate irrigation in oasis regions | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Coupling photosynthetic physiology and C₄ enzyme regulation enhances grain yield of intercropped maize under no-tillage and moderate irrigation in oasis regions Congcong Guo, Yan Wang, Xiaoyuan Bao, Hong Fan, Yali Sun, Wei He, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7635468/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background The Hexi Oasis irrigation area is endowed with abundant light and heat resources, making it suitable for intercropped maize systems. However, after the traditional “high-water-consumption–high-yield” pathway under conventional tillage and full irrigation was disrupted, studies on how photosynthetic physiology and the expression of key C₄ enzymes synergistically compensate for yield losses under limited water supply remain scarce. Methods To address this gap, a field experiment was conducted in 2024 to systematically evaluate the coupled effects of tillage (no-tillage, NT; conventional tillage, CT), cropping pattern (intercropping, IM; monocropping, SM), and three irrigation regimes (low, I1; medium, I2; high, I3) on maize grain yield, photosynthetic physiology, and key enzyme regulation. Results The results showed that the NT × IM × I2 combination achieved a grain yield of 12,400 kg·hm⁻² at 14% moisture in 7 m² yield plots, representing significant increases of 10.5% and 27.2% compared with CTIMI2 and NTSMI2, respectively, while reducing irrigation by 8.3% relative to I3. During the silking–grain filling stage, this treatment maintained the highest SPAD, Pn, Gs, and Y(II), along with the lowest Ci and Y(NO). Enzyme activities of phosphoenolpyruvate carboxylase, ribulose-1,5-bisphosphate carboxylase/oxygenase, and pyruvate phosphate dikinase increased by 6–11%, 8–10%, and 9–14%, respectively, with corresponding gene expression upregulated by 31–80%. Structural equation modeling indicated that the standardized path coefficient and explained variance of the photosynthesis–enzyme coupling on yield reached 0.977 and 94.8%, respectively. Conclusion In summary, no-tillage combined with moderate irrigation enhanced intercropped maize yield stability under limited water supply through a dual mechanism of “photosynthetic performance maintenance + C₄ enzyme activity/transcription enhancement.” Intercropped maize Photosynthetic physiology C₄ enzymes Yield No-tillage Irrigation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Background The contradiction between the rapidly growing population and the shrinking arable land area poses a serious threat to food security. Maximizing crop yields through the efficient use of limited resources in agricultural production practices is a crucial pathway to achieving sustainable agriculture and safeguarding national food security [ 1 ]. Intercropping systems can optimize the utilization of light, heat, water, and nutrients by exploiting niche separation mechanisms, thereby allowing two crops to complement each other spatially and temporally [ 2 ]. In the Hexi Oasis irrigation region, where light and heat resources are abundant, intercropping maize is particularly suitable. However, the region faces severe water scarcity [ 3 ], which restricts the sustainable development of the maize industry. Therefore, investigating intercropping techniques for stable maize production under limited irrigation and clarifying the theoretical basis of water-saving efficiency has become an urgent issue to address. Intercropping and no-tillage are regarded as core technologies for water-saving and yield stability due to their respective advantages in resource-use efficiency and soil moisture conservation. Studies have shown that intercropping allows for rational crop combinations, where differences in root architecture between species promote more effective utilization of nutrients and water, while reducing interspecific resource competition, thereby enhancing photosynthetic physiological activity [ 4 , 5 ]. Intercropping can also maintain a larger photosynthetic source and a longer functional period after flowering, facilitating the translocation and allocation of assimilates to grains, ultimately leading to higher grain yield [ 6 ]. No-tillage, on the other hand, helps maintain a strong photosynthetic source during the middle and late growth stages, slows down the decline in chlorophyll content, extends the photosynthetic duration, and promotes assimilate translocation to grains, thereby increasing yield [ 7 ]. Water availability is a key factor regulating crop growth, and some studies have indicated that limited irrigation encourages root penetration into deeper soil layers, improving water-use efficiency [ 8 – 10 ]. Under adequate soil moisture, crop leaves tend to open their stomata to enhance transpiration, which increases stomatal conductance, transpiration rate, and net photosynthetic rate [ 11 ]. Moreover, mild water stress has been shown to stimulate the activities of phosphoenolpyruvate carboxylase (PEPC), NADP-malate dehydrogenase (NADP-MDH), and NADP-malic enzyme (NADP-ME), thereby improving CO₂ fixation capacity and strengthening carbon assimilation, which collectively enhance photosynthetic efficiency. Additionally, limited irrigation can prevent the adverse effects of excessive water supply, help maintain leaf health, and sustain photosynthetic function, thereby promoting assimilate accumulation and grain yield formation [ 12 ]. However, most existing studies have focused on monocropped maize under conventional tillage or full irrigation, lacking systematic evidence for the combined effects of the “no-tillage × intercropping × limited irrigation” triad. More critically, whether such agronomic combinations can synergistically regulate photosynthetic machinery at the physiological and molecular levels to compensate for the potential biomass loss caused by reduced irrigation remains unclear. Photosynthetic performance represents the primary physiological bottleneck determining maize yield formation. More than 60% of dry matter accumulation during the grain-filling stage originates from post-anthesis photosynthesis, which coincides with the water-sensitive period in the Hexi irrigation region. Under limited irrigation, no-tillage can effectively improve the photosynthetic performance of monocropped maize by regulating soil physicochemical properties and enhancing soil water and nutrient availability, thereby sustaining relatively high grain yields [ 13 ]. As a C₄ crop, maize exhibits photosynthetic efficiency that is highly dependent on the activity and gene expression of PEPC, pyruvate phosphate dikinase (PPDK), and Rubisco. Meanwhile, chlorophyll fluorescence parameters (Fv/Fm, Y(II), Y(NPQ), Y(NO)) can dynamically capture changes in PSII photochemical efficiency and photoprotective capacity. Theoretically, the combined effects of no-tillage improving rhizosphere water status, intercropping optimizing canopy light distribution, and moderate irrigation inducing mild reversible stress may jointly activate a cascade pathway of “upregulated C₄ enzyme gene expression → enhanced enzyme activity → increased Pn and Y(II),” thus achieving photosynthetic compensation under limited water supply. However, such mechanisms remain to be validated through field experiments. Maize ( Zea mays L.), as one of the world’s most important cereal crops, serves not only as a staple for direct human consumption and livestock feed but also as a vital industrial raw material, playing an irreplaceable role in ensuring global food security and driving agricultural economic development. With the growth of the global population and the continuous upgrading of dietary structures, higher demands have been placed on maize yield and quality. However, in recent years, the rapid turnover of maize varieties has led to diminishing returns in yield and quality improvements achieved solely through genetic enhancement, with the marginal effects of traditional breeding approaches becoming increasingly limited. Consequently, optimizing agronomic practices to unlock maize’s production potential has emerged as a key direction in contemporary agricultural research. In the arid regions of Northwest China, the Hexi Oasis irrigation area, endowed with abundant light and heat resources, is particularly suitable for developing intercropping systems of maize, providing unique natural advantages for assimilate accumulation and grain filling [ 14 ]. However, the region faces severe resource-based water scarcity, which constrains the sustainable development of the maize industry. Therefore, under limited irrigation, identifying agronomic optimizations that enhance photosynthetic efficiency in intercropped maize to ensure yield stability is a key challenge for advancing water-saving, high-efficiency agriculture in this area. To this end, we systematically investigated: (1) how the coupling of no-tillage and intercropping regulates maize gas-exchange, chlorophyll fluorescence, and SPAD dynamics under limited irrigation; (2) whether these physiological responses are coordinated with changes in the activities of PEPC, PPDK, and Rubisco and the transcription of their corresponding genes ( pepc , ppdk , rbcL ); and (3) the relative contributions and path coefficients linking the photosynthesis–enzyme activity–molecular regulation network to grain yield and its components. The results aim to elucidate the photosynthetic physiological and molecular mechanisms by which the “no-tillage + moderate irrigation + intercropping” system achieves water-saving yield stability, providing quantifiable theoretical thresholds and practical pathways for maize production in the arid Northwest. Results Effects of moderate irrigation and no-tillage on grain yield and yield components of intercropped maize Moderate irrigation combined with no-tillage exerted a significant effect on grain yield in intercropped maize, with a significant two-way interaction but a non-significant three-way interaction (Fig. 1). Across tillage practices, no-tillage (NT) increased grain yield by 7.8% compared with conventional tillage (CT). At the same land area, intercropped maize (IM) produced 29.5% higher yield than monocropped maize (SM). Across irrigation regimes, grain yield declined with reduced irrigation, with low irrigation (I1) producing 18.9% less yield than high irrigation (I3), while no significant difference was observed between moderate (I2) and high irrigation (I3). Under intercropping, NT with moderate irrigation (NTIMI2) significantly outperformed CT with moderate irrigation (CTIMI2) by 10.5%, and NTIMI2 yielded 27.2% more than NT with monocropping under moderate irrigation (NTSMI2). Although NTIMI1 produced 14.5% less yield than NTIMI3, the difference between NTIMI2 and NTIMI3 was not significant. Overall, moderate irrigation under no-tillage ensured yield stability in intercropped maize. The yield advantages of NT and IM were mainly attributable to increases in ear number and 1000-grain weight (Table 1). Compared with CT, NT increased ear number and grain weight by 6.0% and 5.8%, respectively, while IM increased these components by 5.8% and 6.2% compared with SM. In contrast, tillage and planting patterns had no significant effect on kernel number per ear. Irrigation exerted a strong influence on yield components: relative to I3, I1 reduced both ear number and grain weight, whereas I2 had effects similar to I3. Notably, NTIMI2 significantly increased ear number (+8.0%) and grain weight (+6.3%) compared with CTIMI2. Effects of moderate irrigation and no-tillage on photosynthesis and chlorophyll fluorescence of intercropped maize Compared with the control, NT reduced Pn at the jointing stage but increased Pn by 7–11% from silking to maturity. Before silking, IM exhibited a lower net photosynthetic rate than SM, but during the reproductive stage, Pn in IM was 6–10% higher than in SM, indicating a delayed yet pronounced intercropping advantage. Irrigation had a significant effect on Pn. Under low irrigation (I1), Pn decreased continuously relative to I3 during both the vegetative stage (–7 to –13%) and the reproductive stage (–6 to –13%), whereas I2 maintained values comparable to I3. Notably, under moderate irrigation, NT (NTIMI2) significantly enhanced Pn during the reproductive stage compared with CTIMI2 (+9–20%) and NTSMI2 (+7–14%). In contrast, NTIMI1 showed a marked decline relative to NTIMI3, while NTIMI2 and NTIMI3 performed similarly (Fig. 2). Similar to Pn and Gs, Tr peaked at silking (Fig. 3). Compared with CT, NT reduced Tr before tasseling but increased it by 8–12% afterward. Relative to SM, IM exhibited lower Tr values before silking, but during the reproductive stage, Tr was 7–11% higher, reflecting an intercropping advantage. From silking to maturity, Tr under low irrigation (I1) was 9–13% lower than under I3, while I2 maintained values comparable to I3. Among treatment combinations, NTIMI2 showed a clear advantage, sustaining significantly higher Tr than CTIMI2 (+14–19%) and NTSMI2 (+9–13%) during the reproductive stage. In contrast, NTIMI1 exhibited a marked reduction compared with NTIMI2 and NTIMI3, whereas NTIMI2 and NTIMI3 did not differ significantly. Compared with the control, NT reduced Gs at the jointing stage but increased it by 9–13% from silking to maturity. Similarly, at the early growth stage, Gs in IM was lower than in SM, but from silking onward, Gs was 8–13% higher in IM, highlighting the delayed advantage of intercropping. Irrigation exerted a strong influence during the reproductive stage: Gs under I1 was 14–21% lower than under I3, while I2 remained comparable to I3. At the treatment-combination level, NTIMI2 maintained higher Gs than CTIMI2 (+9–18%) and NTSMI2 (+9–13%) from silking to maturity, whereas NTIMI1 was significantly lower than NTIMI3 (Fig. 4). At the jointing stage, NT increased Ci relative to CT, but from silking to maturity it consistently reduced Ci by 6–8%, suggesting improved CO₂ assimilation capacity. Similarly, IM showed higher Ci than SM before tasseling, but 7–11% lower values during reproductive stages, indicating enhanced CO₂ utilization efficiency under intercropping. Irrigation effects were pronounced during the reproductive period: both I2 and I3 significantly reduced Ci compared with I1, while no difference was observed between I2 and I3. Among treatment combinations, NTIMI2 reduced Ci by 7–10% compared with CTIMI2 and NTSMI2, and by 6–13% compared with NTIMI1, whereas no difference was observed between NTIMI2 and NTIMI3 (Fig. 5). Compared with CT, NT reduced Fv/Fm during early vegetative stages but increased it by 1–4% from silking to maturity. Similarly, IM exhibited slightly lower Fv/Fm than SM before tasseling but 1–3% higher values during reproductive stages. Irrigation exerted a strong influence: low irrigation (I1) significantly decreased Fv/Fm compared with I2 and I3, whereas no difference was detected between I2 and I3. At the treatment combination level, NTIMI2 maintained higher Fv/Fm than both CTIMI2 and NTSMI2 during reproductive stages, while NTIMI1 consistently showed lower values than NTIMI2 and NTIMI3 (Fig. 6). Compared with the control, NT reduced SPAD by 6.4% at the jointing stage but significantly increased it by 5.0–8.0% from silking to the dough stage. Similarly, before silking, SPAD values in IM were lower than in SM, whereas after silking, IM showed 5–7% higher SPAD values than SM. The irrigation effect was more pronounced: SPAD under I1 consistently remained lower than under I2 and I3, while no significant difference was observed between I2 and I3 (Fig. 7). Effects of moderate irrigation and no-tillage on the activities of key photosynthetic enzymes in intercropped maize leaves NT reduced PEPC activity at the jointing stage but increased it by 6–9% from silking to grain filling compared with CT. Similarly, IM exhibited slightly lower activity than SM during early growth, but 9–10% higher activity during reproductive stages. Irrigation exerted a pronounced effect: I2 consistently maintained the highest PEPC activity, whereas I1 reduced it by 7–16% compared with I2 and I3. Treatment combinations confirmed this trend: NTIMI2 showed significantly higher PEPC activity during silking to grain filling compared with CTIMI2 (+6–11%) and NTSMI2 (+7–10%), while NTIMI1 reduced activity by 12–16% relative to NTIMI2 (Fig. 8). Rubisco activity was lowest at the jointing stage and peaked at silking. Compared with CT, NT significantly increased Rubisco activity by 10–11% from silking to grain filling, although it was slightly lower at the jointing stage. Likewise, IM enhanced Rubisco activity by 10–13% compared with SM during reproductive stages. Irrigation markedly affected Rubisco activity: I1 reduced activity by 18–19% compared with I3, while no significant difference was observed between I2 and I3. At the treatment combination level, NTIMI2 consistently exhibited higher Rubisco activity during reproductive stages, exceeding CTIMI2 (+10%) and NTSMI2 (+8–10%), while NTIMI1 showed significant reductions relative to NTIMI3 (Fig. 9). Compared with CT, NT decreased PPDK activity at the jointing stage but increased it by ~9–10% from silking to grain filling. Similarly, IM significantly reduced PPDK activity during early growth but enhanced it by 9–12% during reproductive stages relative to SM. Irrigation strongly regulated PPDK activity. I1 consistently reduced activity by 13–20% compared with I3, while I2 maintained values comparable to I3. Treatment combinations confirmed that NTIMI2 promoted the highest PPDK activity during reproductive stages, increasing it by 9–14% relative to CTIMI2 and NTSMI2, whereas NTIMI1 significantly reduced activity compared with NTIMI3 (Fig. 10). Effects of moderate irrigation and no-tillage on the relative expression of key photosynthetic enzyme genes in intercropped maize At the jointing stage, pepc expression in NTIMI2 leaves was downregulated by 22.0% compared with CTIMI2, but at the grain-filling stage, it was upregulated by 39.4%. Relative to NTSMI2, pepc expression in NTIMI2 leaves was 10.6% lower at the jointing stage but 31.0% higher at the grain-filling stage. From jointing to grain filling, pepc expression in NTIMI1 leaves was markedly downregulated by 55.3–77.2% and 54.2–67.9% compared with NTIMI2 and NTIMI3, respectively. While no significant difference was observed between NTIMI2 and NTIMI3 at the jointing stage, pepc expression in NTIMI2 was 41.2% higher than in NTIMI3 at the grain-filling stage (Fig. 11A). At the jointing stage, rbc expression in NTIMI2 leaves was lower than in CTIMI2, whereas at the grain-filling stage, NTIMI2 was 63.5% higher than CTIMI2. Compared with NTSMI2, rbc expression in NTIMI2 leaves was reduced by 11.0% at the jointing stage but increased by 80.3% at the grain-filling stage. From jointing to grain filling, rbc expression in NTIMI1 leaves was 63.8–75.8% lower than in NTIMI3. At the jointing stage, NTIMI2 expression was 9.4% lower than NTIMI3, while at the grain-filling stage, CTIMI2 was 13.1% lower than CTIMI3. No significant difference was observed between NTIMI2 and NTIMI3 at the grain-filling stage, but CTIMI2 showed a 38.7% reduction compared with CTIMI3 (Fig. 11B). Compared with CTIMI2, the relative expression of the ppdk gene in NTIMI2 maize leaves was downregulated by 14.2% at the jointing stage but upregulated by 53.6% at the grain-filling stage. Relative to NTSMI2, ppdk expression in NTIMI2 was 16.0% lower at the jointing stage but 61.1% higher at the grain-filling stage. At the jointing stage, ppdk expression in NTIMI1 leaves was reduced by 59.4% and 64.8% compared with NTIMI2 and NTIMI3, respectively, while NTIMI2 was 13.4% lower than NTIMI3 and CTIMI2 was 8.0% lower than CTIMI3. At the grain-filling stage, ppdk expression in NTIMI1 leaves was downregulated by 73.4% and 74.1% compared with NTIMI2 and NTIMI3, respectively. No significant difference was observed between NTIMI2 and NTIMI3, whereas CTIMI2 was 30.0% lower than CTIMI3 (Fig. 11C). Correlation between grain yield and photosynthetic physiological parameters and structural equation modeling analysis Principal component analysis (PCA) was conducted on the relationships between grain yield and photosynthetic physiological parameters of intercropped maize under moderate irrigation and no-tillage (Fig. 12). The cumulative contribution rate of PC1 and PC2 reached 94.8%, indicating strong representativeness. Correlation analysis among maize grain yield, yield components, and photosynthetic physiological parameters (Fig. 13-A) revealed that grain yield, ear number, kernel number per ear, and 1000-grain weight were all significantly and positively correlated with SPAD values, net photosynthetic rate (Pn), stomatal conductance (Gs), and transpiration rate (Tr), while being significantly and negatively correlated with intercellular CO₂ concentration (Ci). Thus, enhancing SPAD, Pn, Gs, and Tr while reducing Ci can optimize photosynthetic performance, thereby synergistically improving ear number, kernel number per ear, and 1000-grain weight, ultimately sustaining higher grain yield. Furthermore, the structural equation model (Fig. 13-B) demonstrated that pepc and rbc exerted significant positive effects on PEPC and Rubisco, respectively, across different tillage, planting, and irrigation treatments. Both PEPC and Rubisco had significant positive effects on Pn and Y(II). In addition, Y(II) positively affected Pn, while Pn regulated grain yield (GY) indirectly by influencing LAI and LAD. Among these pathways, LAI had the most pronounced effect on GY, with a standardized path coefficient as high as 0.977***. Therefore, under moderate irrigation, no-tillage can effectively enhance the activities of key photosynthetic enzymes and the relative expression of their genes, as well as leaf actual photochemical efficiency and net photosynthetic rate during the middle and late growth stages of intercropped maize. This maintains a high level of photosynthetic performance, optimizes photosynthetic efficiency, and ultimately secures higher grain yield. Discussion Effects of moderate irrigation and no-tillage on grain yield of intercropped maize Optimized agronomic practices are essential for achieving stable and increased crop yields. Among them, appropriate tillage methods, mulching techniques, planting patterns, and irrigation regimes play key roles in improving resource-use efficiency and ensuring food security [ 15 ]. Previous studies have demonstrated that intercropping can significantly enhance land-use efficiency and farmland productivity by improving canopy structure and light distribution, extending the functional duration of border-row leaves, and facilitating nutrient complementarity between crops [ 16 , 17 ]. The present study similarly showed that intercropped maize achieved significantly higher grain yield than monocropped maize, confirming the advantages of intercropping in promoting photosynthetic efficiency and nutrient utilization. However, under the constrained water resources of the Northwest Oasis irrigation area, intercropping combined with conventional tillage alone cannot ensure yield stability. Our findings revealed that integrating no-tillage with moderate irrigation markedly increased intercropped maize yield, producing 10.5% higher yield than intercropping under conventional tillage, while maintaining a grain yield comparable to high irrigation but with reduced water consumption. This highlights that the combination of no-tillage and moderate irrigation is a critical agronomic strategy for achieving “water-saving and yield stability” in oasis maize production. The yield-enhancing mechanism is primarily reflected in two aspects: (1) Under moderate irrigation, no-tillage significantly increased SPAD values and the peak leaf area index (LAI) at the silking stage, while mitigating their decline during grain filling. This delayed leaf senescence, enhanced photosynthetic capacity, and strengthened grain-filling intensity, thereby providing more assimilates for kernel development [ 18 , 19 ]. (2) No-tillage combined with moderate irrigation significantly improved the activities of PEPC and Rubisco in maize leaves from silking to grain filling, enhancing CO₂ fixation capacity, net photosynthetic rate (Pn), and transpiration rate (Tr). This facilitated the accumulation and translocation of assimilates to grains [ 20 , 21 ]. Together, these effects ensured steady improvements in maize yield under limited irrigation conditions. Effects of moderate irrigation and no-tillage on photosynthetic physiological characteristics of intercropped maize leaves Grain yield largely depends on the accumulation of assimilates from post-anthesis photosynthesis; thus, improving leaf photosynthetic performance is a key pathway to achieving stable and increased yields [ 22 , 23 ]. Compared with monocropping, intercropped maize exhibited higher SPAD values and net photosynthetic rate (Pn) from silking to grain filling, indicating that intercropping can delay leaf senescence, extend the stay-green period, and enhance photosynthetic efficiency. This finding is consistent with previous studies, which demonstrated that cereal–legume intercropping improves photosynthetic performance by optimizing the light environment and promoting nutrient complementarity. The underlying mechanism may be that, after pea harvest, maize benefits from more space and resources, utilizes residual organic matter and the nitrogen-fixing effect of legumes, thereby enhancing nutrient supply and SPAD levels. Additionally, intercropping forms a more rational canopy structure, improving light conditions and creating a favorable environment for photosynthesis. Furthermore, our study found that under moderate irrigation, combining no-tillage with intercropping significantly increased SPAD, Pn, and stomatal conductance (Gs) from silking to grain filling. This aligns with earlier findings, suggesting that the integration of no-tillage with intercropping enhances post-anthesis photosynthetic potential [ 18 ]. The underlying mechanisms may involve two aspects: (1) No-tillage reduces soil temperature and energy consumption in the early stages, leading to a marked compensatory effect after silking that delays leaf senescence and maintains higher SPAD levels and photosynthetic activity [ 24 ]. (2) During the fallow period, no-tillage fields retain more soil moisture, which provides sufficient water for crops in the middle and late growth stages. This improves root activity and leaf water potential, promotes stomatal opening and water–vapor exchange, thereby increasing Gs and Tr, and ultimately enhances CO₂ assimilation capacity [ 25 ]. In summary, the combination of no-tillage and moderate irrigation effectively improves the photosynthetic physiological state of maize during the late growth stages in intercropping systems, sustaining high photosynthetic efficiency and assimilate accumulation rates, and thus providing a physiological foundation for stable yield improvement. Chlorophyll fluorescence parameters sensitively reflect the efficiency of light absorption and utilization, as well as the response of the photosynthetic apparatus to stress, and are thus important indicators for evaluating crop photosynthetic performance [ 26 ]. Fv/Fm represents the maximum quantum efficiency of PSII, while Y(II) reflects the actual photochemical efficiency of PSII under illumination; together, they measure potential photosynthetic capacity and actual photosynthetic activity, respectively. Y(NPQ) and Y(NO), in turn, indicate photoprotective capacity and the degree of photodamage [ 27 , 28 ]. In this study, Fv/Fm and Y(II) significantly declined with decreasing irrigation, whereas under moderate irrigation, no-tillage maintained higher Fv/Fm and Y(II) values during the later growth stages. This suggests that no-tillage combined with moderate irrigation can mitigate the inhibitory effects of water deficit on PSII energy conversion, thereby sustaining strong photosynthetic activity. The underlying reason may lie in the early suppression of photosynthetic performance due to lower soil temperature and intensified resource competition under no-tillage and intercropping conditions ; however, during the middle and late growth stages, improved soil conditions and the independent growth of intercropped maize allow greater acquisition of light, water, and nutrients, thereby enhancing PSII energy conversion efficiency [ 29 ]. At the same time, our results showed that no-tillage with moderate irrigation significantly increased Y(NPQ) while reducing Y(NO), indicating that this treatment enhanced leaf photoprotective capacity and reduced the risk of photodamage. These findings are consistent with previous studies showing that no-tillage and intercropping improve canopy light environments and optimize soil hydrothermal conditions, which promote root development and maintain photosynthetic apparatus homeostasis, thereby strengthening plant adaptability to light stress [ 18 ]. In summary, no-tillage combined with moderate irrigation not only improved PSII energy conversion efficiency and photoprotective capacity in intercropped maize but also effectively reduced non-regulated energy dissipation, alleviating the decline in photosynthetic efficiency during the late growth stages. This, in turn, provided a physiological foundation for assimilate accumulation during grain filling and yield formation. Effects of moderate irrigation and no-tillage on the activities of key photosynthetic enzymes and the relative expression of their genes in intercropped maize leaves PEPC, Rubisco, and PPDK are among the most critical enzymes in crop photosynthesis, and their activities directly determine leaf photosynthetic efficiency. PEPC initiates the C₄ pathway by catalyzing the fixation of CO₂ with pyruvate to form oxaloacetate, which subsequently enters the C₄ cycle; Rubisco is the principal carbon-fixing enzyme in both C₃ and C₄ plants; and PPDK converts oxaloacetate into phosphoenolpyruvate (PEP), sustaining the C₄ pathway [ 30 ]. Previous studies have shown that the decline in leaf photosynthetic rate is largely attributable to reductions in photosynthetic enzyme activities and the relocation of enzymes. Slowing down the reduction in enzyme activity and limiting enzyme relocation are conducive to maintaining high photosynthetic efficiency, thereby facilitating assimilate accumulation and translocation [ 31 ]. In this study, we found that under moderate irrigation, applying no-tillage to intercropping systems reduced photosynthetic enzyme activity at the jointing stage but significantly enhanced it from silking to grain filling. This could be explained by the relatively lower soil temperature under no-tillage intercropping during the jointing stage, where cold stress suppressed enzyme activity. However, as crop development progressed, soil temperatures increased, and tillage practices had little effect on soil thermal conditions. From silking to grain filling, soil moisture became the dominant factor influencing photosynthetic enzyme activity; no-tillage effectively reduced soil water loss and evaporation [159] , thereby ensuring sufficient water supply for vegetative growth, promoting stomatal opening, lowering CO₂ diffusion resistance, and ultimately increasing photosynthetic enzyme activity [ 18 ]. Other studies have also reported that mild water stress can enhance photosynthetic enzyme activity and gene expression, accelerating metabolic processes and improving photosynthetic capacity. This mechanism enables crops to maintain strong growth and yield potential even under water deficit conditions [ 20 ]. Our findings are consistent with this: under no-tillage intercropping, moderate irrigation significantly upregulated pepc gene expression and increased PEPC activity from the jointing to grain-filling stages, whereas under low irrigation, the activities of all three enzymes and the relative expression of their associated genes were lowest. The likely explanation is that mild water stress enhances PEPC activity and its gene expression, while severe water stress suppresses the activities of PEPC, Rubisco, and PPDK. Therefore, under moderate irrigation, no-tillage promotes the enhancement of key photosynthetic enzyme activities and their gene expression in intercropped maize leaves during the middle and late growth stages. This maintains a high carboxylation capacity for CO₂, strengthens carbon assimilation, improves Pn and related gas-exchange parameters, and synergistically enhances photosynthetic efficiency. Conclusion From a holistic “water–light–enzyme–yield” perspective, this study is the first to demonstrate under limited water supply in oasis irrigation regions that coupling no-tillage with intercropping, supplemented by moderate irrigation, can trigger a three-tiered “photosynthesis–enzyme activity–gene expression” cascade response in maize, thereby achieving the breakthrough goal of “reduced irrigation without yield loss” or even “yield gain.” Specifically, this model translates physiological advantages into yield advantages through three key mechanisms: (i) no-tillage conserves soil water and improves soil moisture, while intercropping optimizes light distribution, jointly delaying leaf senescence and sustaining strong post-anthesis photosynthetic sources; (ii) moderate water stress acts as a “mild elicitor,” upregulating pepc , ppdk , and rbcL expression, enhancing PEPC, PPDK, and Rubisco activities, thereby improving CO₂ carboxylation capacity and photochemical efficiency; and (iii) increasing Y(II) and Y(NPQ) while decreasing Y(NO), thus maintaining efficient PSII function under water-saving conditions and reducing non-regulated energy dissipation. Structural equation modeling further revealed that the coupled physiological–molecular effects generate a standardized path coefficient of 0.977 along the Pn → LAI → yield pathway, explaining 94.8% of the yield variation and providing direct evidence for the quantitative relationship between photosynthesis, enzyme activity, and yield. Beyond maize, the “tillage–planting–irrigation” triadic paradigm established in this study can be extended to other C₄ crops (e.g., sorghum, sugarcane) and cereal–legume intercropping systems. This offers a replicable and scalable theoretical template for developing new cropping systems characterized by “high yield and efficiency, water conservation, and low carbon emissions” in arid regions worldwide. Materials and methods Plant materials The maize cultivar used in the experiment was Xianyu 335 ( Zea mays L.), and the pea cultivar was Longwan No. 1 ( Pisum sativum L.). Seeds of maize (cv. Xianyu 335) and pea (Longwan No. 1) were purchased from local commercial suppliers in Lanzhou and Wuwei, Gansu, China. Xianyu 335 is a nationally approved hybrid in China (approval Nos. Guoshenyu 2004017 and Guoshenyu 2006026), and Longwan No. 1 is nationally registered as a non-major crop variety (registration No. GPD-Pea(2018)620005). No wild materials were collected and no permits were required. Experimental site description The experiment was conducted in 2024 at the “Oasis Agricultural Research and Teaching Base of Gansu Agricultural University” in Huangyang Town, Liangzhou District, Wuwei City, Gansu Province, China (37°30′N, 103°5′E). The site is located in a cold temperate arid climate zone, with an average annual precipitation of approximately 156 mm, annual evaporation of about 2400 mm, total sunshine duration of 2969.2 h, mean annual temperature of 7.2°C, and a frost-free period of 156 days. The region has abundant solar radiation, making it well suited for maize intercropping systems. Local agricultural production is predominantly based on conventional tillage with plowing, and plastic film mulching is commonly applied. The soil type at the experimental site was irrigated desert soil. In 2024, the nutrient contents of the plow layer were as follows: total nitrogen, 0.89 g kg⁻¹; available phosphorus, 24.98 mg kg⁻¹; available potassium, 138.44 mg kg⁻¹; organic matter, 14.53 g kg⁻¹; and bulk density, 1.24 g cm⁻³. Variations in temperature and precipitation at the site throughout the 2024 growing season are shown in Fig. S1 . Based on a long-term experiment initiated in 2015, this study adopted a split-split plot design with three factors. The main plots included two tillage practices: no-tillage (NT, where maize was directly sown the following year onto the residual film after the previous maize harvest without tillage) and conventional tillage (CT, where the field was deep-plowed after the previous maize harvest, and new plastic film was applied after land preparation before sowing the following year). The subplots consisted of two planting patterns: intercropped maize (IM) and monocropped maize (SM). The sub-subplots were assigned three irrigation levels: low irrigation (I1, 4500 m³ ha⁻²), moderate irrigation (I2, 4950 m³ ha⁻²), and high irrigation (I3, 5400 m³ ha⁻²), with the high irrigation treatment representing the local conventional irrigation level. In total, 12 treatment combinations were established, each replicated three times, resulting in 36 plots. Each plot had an area of 63 m² (7 m × 9 m). The treatment codes are provided in Table S1 . The planting density of monocropped maize was 90,000 plants ha⁻¹, with a row spacing of 40 cm and a plant spacing of 27 cm. In the maize–pea intercropping system, a 3:4 planting ratio (three rows of maize to four rows of pea) was adopted, with a spacing of 25 cm between maize and pea rows. The planting density of intercropped maize was 52,000 plants ha⁻¹, while that of pea was 760,000 plants ha⁻¹, with an inter-row ratio of 8:11 between pea and maize strips. Maize and pea were sown on April 18 and April 7, 2024, respectively, and harvested on September 27 and July 6, 2024, respectively. After harvest, all crop residues were removed from the field. All irrigation treatments were applied using drip irrigation under plastic film, with water volume monitored by flow meters. Specific irrigation quotas at each growth stage are provided in Table S2. The maize fertilization regime followed local conventional management, with a total nitrogen application of 360 kg ha⁻¹, split at a ratio of 3:5:2 across the basal stage, the big-tassel stage, and the grain-filling stage. Phosphorus fertilizer was applied at an N:P ratio of 2:1, equivalent to 180 kg ha⁻¹, incorporated entirely as a basal application. For pea, total nitrogen and phosphorus application rates were 90 kg ha⁻¹ and 45 kg ha⁻¹, respectively, both applied entirely as basal fertilizer across all treatments. The chemical nitrogen fertilizers applied included urea and diammonium phosphate. White agricultural plastic film was used for mulching, with a width of 120 cm and a thickness of 0.01 mm. Given the high native potassium content of the soil in this region, no potassium fertilizer was applied. Pest, disease, and weed management practices were carried out according to local conventional tillage practices. Sampling and measurements The yield and yield components At maturity, a 5 m row length in the center of each plot (one film width of 1.4 m, totaling 7 m²) with continuous plants and no gaps was selected as the sampling area for yield measurement. Ears were harvested manually, shelled on site, and cleaned of impurities. Fresh grain weight was measured using an electronic balance with an accuracy of 0.01 kg. A 500 g grain subsample was randomly collected, inactivated at 105°C for 30 min, and then oven-dried at 80°C to a constant weight to determine actual moisture content. Grain weight was converted to a standard moisture content of 14% according to national maize yield determination standards and further converted to yield per hectare (kg ha⁻¹). Leaves relative chlorophyll content At the jointing, big-tassel, silking, grain-filling, and dough stages, the relative chlorophyll content (SPAD value) of maize leaves was measured on sunny mornings between 9:30 and 11:30. SPAD values were determined using a SPAD-502 Plus chlorophyll meter. For each measurement, three representative maize plants of uniform growth were randomly selected, and three readings were taken per plant; the average value was recorded as the observation, avoiding interference from leaf veins. At the jointing stage, measurements were taken on the second fully expanded leaf from the top of the plant. At the big-tassel, silking, grain-filling, and dough stages, measurements were taken on the ear leaf, with the reading location at the middle portion of the leaf, avoiding veins and leaf margins. Leaves gas exchange parameters At the jointing, big-tassel, silking, grain-filling, and dough stages, the net photosynthetic rate (Pn), transpiration rate (Tr), intercellular CO₂ concentration (Ci), and stomatal conductance (Gs) of maize leaves were measured between 9:30 and 11:30 a.m. on sunny days using a LI–6800XT portable photosynthesis system. Three representative maize plants of uniform growth were randomly selected for measurement, with care taken to avoid leaf veins. During measurement, the leaf chamber area was set to 2 cm², with an airflow rate of 500 µmol s⁻¹. The photosynthetic photon flux density was set at 1200 µmol m⁻² s⁻¹. Leaf temperature was maintained equal to air temperature, and relative humidity was controlled at 55%. At the jointing stage, the second fully expanded leaf from the top was selected, while at the big-tassel, silking, grain-filling, and dough stages, the ear leaf was measured at the middle portion, avoiding veins and leaf margins. Leaves chlorophyll fluorescence parameters At the jointing, big-tassel, silking, grain-filling, and dough stages, chlorophyll fluorescence parameters of maize leaves were measured between 9:30 and 11:30 a.m. on sunny days using an FMS-II pulse-modulated fluorometer (PAM-2500). Three representative maize plants of uniform growth were randomly selected, and each was measured three times; the average value was recorded as the observation, avoiding interference from leaf veins. At the jointing stage, the second fully expanded leaf from the top was selected, while at the big-tassel, silking, grain-filling, and dough stages, the ear leaf was measured at the middle portion of the leaf, avoiding veins and margins. Prior to measurement, target leaves were dark-adapted for 30 min using dark adaptation clips. Thereafter, a measuring light (< 0.05 µmol m⁻² s⁻¹) followed by a saturating pulse light (6000 µmol m⁻² s⁻¹) was applied to obtain the minimum fluorescence (Fo) and maximum fluorescence (Fm) under dark-adapted conditions. The maximum photochemical efficiency was then calculated as: Fv/Fm = (Fm – Fo) / Fm Subsequently, actinic light (1000 µmol m⁻² s⁻¹) was applied for 3 min. Once the fluorescence signal stabilized, the steady-state fluorescence (Fs) was recorded. A saturating pulse light (6000 µmol m⁻² s⁻¹) was then applied to determine the maximum fluorescence under light (Fm′) and the minimum fluorescence under light (Fo′). The following parameters were calculated: Actual photochemical efficiency of PSII: Y(II)=(Fm′-Fs)/Fm Quantum yield of regulated energy dissipation: Y(NPQ) = F/Fm′-F/Fm Quantum yield of non-regulated energy dissipation: Y(NO) = F/Fm Relative electron transport rate: ETR = PAR×Y(II)×0.84×0.5 where PAR represents photosynthetically active radiation (1000 µmol m⁻² s⁻¹), 0.84 is the leaf absorption coefficient, and 0.5 assumes equal partitioning of excitation energy between PSⅠ and PSⅡ. The activity of key photosynthetic enzymes At the jointing stage of maize, the second fully expanded leaf from the top was sampled, while at the silking and grain-filling stages, fresh samples were collected from the middle section of ear leaves for the determination of key photosynthetic enzymes. Three replicates were taken for each treatment, with 0.2 g of tissue per replicate. Samples were immediately frozen in liquid nitrogen and then stored in an ultra-low temperature freezer at − 80℃ to preserve tissue structure and biological activity. The activities of PEPC, PPDK, and Rubisco were measured using assay kits supplied by Suzhou Comin Biotechnology Co., Ltd., following the manufacturer’s instructions. qRT-PCR验证 To analyze the expression dynamics of key photosynthetic enzyme genes in maize leaves under different tillage practices, planting patterns, and irrigation regimes, samples were collected at three growth stages: jointing, silking, and grain filling. For each treatment, three maize plants of uniform growth were randomly selected. At the jointing stage, the second fully expanded leaf from the top was sampled; at the silking and grain-filling stages, the middle portion of the ear leaf was collected. After removing the veins, 0.2 g of fresh tissue was quickly weighed, flash-frozen in liquid nitrogen, and stored at − 80°C. Total RNA was extracted from maize leaves using the HiScript® II Q RT SuperMix for qPCR (+ gDNA wiper) kit (Nanjing). RNA quality was assessed with an Agilent 2100 Bioanalyzer and verified by RNase-free agarose gel electrophoresis. RNA was then reverse-transcribed into cDNA using the FastPure® Plant Total RNA Isolation Kit (Nanjing), following the instructions provided with the PrimeScript reverse transcription kit. The cDNA synthesis reaction system (20 µL) contained 4 µL of 5× HiScript II qRT SuperMix II, 4 µL of 4× gDNA Wiper Mix, 12 µL of RNase-free H₂O, and RNA (1000 ng). Reaction conditions were 50°C for 15 min and 85°C for 5 s. The cDNA products were stored at − 20°C until use. Subsequently, quantitative real-time PCR (qRT-PCR) was performed. The maize coding sequences were obtained from the MaizeGDB database ( http://www.maizegdb.org/ ), and primers were designed using Primer Premier 5.0 (Canada) and synthesized by Shanghai OE Biotech Co., Ltd. (primer sequences are provided in Table 1 ). The reactions were carried out using the Taq Pro Universal SYBR qPCR Master Mix kit on a LightCycler® 480 Ⅱ real-time PCR system (Switzerland). The reaction mixture (20 µL) consisted of 10 µL of 2× Taq Pro Universal SYBR qPCR Master Mix, 0.4 µL of 10 µM Primer 1, 0.4 µL of 10 µM Primer 2, 1 µL of cDNA, and 8.5 µL of nuclease-free H₂O. The PCR program was set as follows: 95°C for 30 s; followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. Gene expression levels were calculated using the 2⁻(ΔΔCt) method, with GhActin serving as the internal reference gene. Each treatment included three biological replicates. The specific primer sequences are listed in Table S3. Statistical analysis Data were recorded and organized using Microsoft Excel 2019. Duncan’s multiple range tests were performed in SPSS 21.0. Figures and correlation analyses were generated with Origin 2019b and GraphPad Prism 9.0. Results are expressed as mean ± standard error (mean ± SE). One-way ANOVA followed by the least significant difference (LSD) test was used to determine significance at the 95% or 99% confidence level. Image assembly was conducted in Adobe Illustrator 2020. Structural equation modeling (SEM) was performed using AMOS 22.0. Declarations Clinical trial number Not applicable. Authors’ contributions C.C.G., and Y.W., Writing –original draft, Conceptualization, Data curation, Visualization, Writing – original draft. H.F., and Y.L.S., Methodology, Conceptualization, Methodology, Software. W.H., and C.S.B., Writing – review & editing. Data curation, Supervision. F.Y.C., C.X.B., and X.Y.L., Formal analysis, Visualization. C.Z., and X.Y.B., Conceptualization, , Writing – review & editing, Project administration, Supervision, Writing – review& editing Funding We would like to thank to the Major Science and Technology Projects of Gansu Province (No. 22ZD6NA009, No. 23ZDNA008), the Youth Science and Technology Fund of Gansu Province (25JRRA371), the Joint Fund of Gansu Agricultural University (GSAU-DKZY-2025-001) and the Key Project of Natural Science Foundation of Gansu Province (No. 21JR7RA802). Data Availability Data will be made available on request. Ethics approval and consent to participate I have permission to collect Plant Material. All experimental research on plants complied with institutional, national, and international guidelines. Field studies were conducted in accordance with Chinese national legislation on crop variety release and agricultural experimentation. No wild plants were collected in this study, and therefore no specific collection permits were required. The commercial cultivars used in this study—Xianyu 335 (national approvals Guoshenyu 2004017; Guoshenyu 2006026) and Longwan No. 1 (national variety registration GPD-Pea(2018)620005)—are available for purchase from local suppliers in [City, Province, Country] upon request. Consent for publication Not applicable. Competing interests The authors declare no competing interests Author details 1 State Key Laboratory of Aridland Crop Science, College of Agronomy, Gansu Agricultural University, Lanzhou 730070, China References Rudel TK, Schneider L, Uriarte M, Turner BL, DeFries R, Lawrence D, et al. Agricultural intensification and changes in cultivated areas, 1970–2005. Proc. Natl. Acad. Sci. U. S. A. 2009;106:20675–80. Raza MA, Yasin HS, Gul H, Qin R, Mohi Ud Din A, Khalid MHB, et al. Maize/soybean strip intercropping produces higher crop yields and saves water under semi-arid conditions. Front. Plant Sci. 2022;13:1006720. Pan X, Zhang H, Deng H, Yu S, Zhou C, Li F. 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Table Table 1 Three factors of maize yield under different treatments Tillage initiatives Planting pattern Irrigation amounts Ear numbers (ear m -2 ) Kernel number per spilk 1000-kernel weight (g) NT IM I1 8.1±0.12 d 562.0±18.6 c 436.0±7.5 d I2 9.5±0.21 a 643.5±21.2 ab 489.1±18.6 a I3 9.6±0.06 a 652.2±17.0 a 194.6±5.0 a SM I1 7.4±0.15 e 528.3±6.9 d 410.7±13.2 f I2 9.0±0.10 bc 586.9±14.9 c 461.9±16.5 bc I3 9.1±0.20 b 619.7±19.3 b 463.0±21.6 b CT IM I1 7.5±0.15 e 523.5±24.4 d 413.9±8.6 ef I2 8.8±0.12 c 620.8±33.0 b 460.2±12.7 bc I3 9.1±0.17 b 631.5±21.6 ab 465.5±18.8 b SM I1 7.0±0.10 f 492.1±20.7 e 388.5±7.0 g I2 8.3±0.26 d 583.4±16.4 c 343.4±13.5 de I3 9.0±0.10 bc 617.9±9.0 b 440.6±10.0 cd Variance analysis Tillage initiatives (T) ** NS ** Planting pattern (P) ** NS * Irrigation amounts (I) ** * * T×P * NS ** T×I * NS * P×I NS * T×P×I NS NS NS Note: NT and CT represent no-tillage and conventional tillage, respectively. IM and SM were intercrop and monocrop maize, respectively. I1, I2, and I3 represent low, medium, and high irrigation amount, respectively. Differences between treatments are denoted by different lowercase letters, with ** and * indicating significant effects of the experimental factors on the parameter at the 0.01 and 0.05 significance levels, respectively, while NS denotes no significant effect of the experimental factors on the parameter. Additional Declarations No competing interests reported. Supplementary Files Supplementary9.15.doc Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7635468","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":524906144,"identity":"ae3fe78e-36c2-4ff5-bff8-e338b736235f","order_by":0,"name":"Congcong Guo","email":"","orcid":"","institution":"Gansu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Congcong","middleName":"","lastName":"Guo","suffix":""},{"id":524906146,"identity":"599e9114-fd02-4f69-86ca-af4a45417052","order_by":1,"name":"Yan Wang","email":"","orcid":"","institution":"Gansu Agricultural 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1","display":"","copyAsset":false,"role":"figure","size":68373,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of moderate irrigation under no-tillage on grain yield of intercropped maize.Note: NT and CT represent no-tillage and conventional tillage, respectively. IM and SM were intercrop and monocrop maize, respectively. I1, I2, and I3 represent low, medium, and high irrigation amount, respectively. Differences between treatments are denoted by different lowercase letters, with a significance level of 0.05, and the same applies hereafter.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7635468/v1/9590992a2ad46ec38f8a0c3d.png"},{"id":92962564,"identity":"36ba481d-3df9-4a55-bfcb-1b93debdc289","added_by":"auto","created_at":"2025-10-07 15:11:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":75680,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of moderate irrigation under no-tillage on the net photosynthetic rate (Pn) of intercropped maize at different growth stages. Note: NT and CT represent no-tillage and conventional tillage, respectively. IM and SM were intercrop and monocrop maize, respectively. I1, I2, and I3 represent low, medium, and high irrigation amount, respectively. V6, R1 and R2 respectively represent the jointing stage, the filament stage and the grouting stage.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7635468/v1/e4464c06c7104f6391086fd6.png"},{"id":92963327,"identity":"c9354925-41f1-4dca-af6d-598d661ad862","added_by":"auto","created_at":"2025-10-07 15:19:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":76949,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of moderate irrigation under no-tillage on the transpiration rate (Tr) of intercropped maize at different growth stages. Note: NT and CT represent no-tillage and conventional tillage, respectively. IM and SM were intercrop and monocrop maize, respectively. I1, I2, and I3 represent low, medium, and high irrigation amount, respectively. V6, R1 and R2 respectively represent the jointing stage, the filament stage and the grouting stage.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7635468/v1/8877fe99ca54c47d6e47ba73.png"},{"id":92962566,"identity":"b64f2b8e-4361-42a7-a84f-2b02ab13bf92","added_by":"auto","created_at":"2025-10-07 15:11:40","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":82526,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of moderate irrigation under no-tillage on stomatal conductance (Gs) of intercropped maize at different growth stages. Note: NT and CT represent no-tillage and conventional tillage, respectively. IM and SM were intercrop and monocrop maize, respectively. I1, I2, and I3 represent low, medium, and high irrigation amount, respectively. V6, R1 and R2 respectively represent the jointing stage, the filament stage and the grouting stage.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7635468/v1/3e9a034a2e2680d7fddac075.png"},{"id":92963328,"identity":"f77473ba-db84-4bd6-8053-da7d81c9e1ca","added_by":"auto","created_at":"2025-10-07 15:19:40","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":78979,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of moderate irrigation under no-tillage on intercellular CO₂ concentration (Ci) of intercropped maize at different growth stages. Note: NT and CT represent no-tillage and conventional tillage, respectively. IM and SM were intercrop and monocrop maize, respectively. I1, I2, and I3 represent low, medium, and high irrigation amount, respectively. V6, R1 and R2 respectively represent the jointing stage, the filament stage and the grouting stage.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7635468/v1/95fba6cd314ae645e21e8158.png"},{"id":92962569,"identity":"76e7221e-5cca-4ee8-80f1-08ad5d142122","added_by":"auto","created_at":"2025-10-07 15:11:40","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":78200,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of moderate irrigation under no-tillage on the maximum photochemical efficiency of intercropped maize at different growth stages. Note: NT and CT represent no-tillage and conventional tillage, respectively. IM and SM were intercrop and monocrop maize, respectively. I1, I2, and I3 represent low, medium, and high irrigation amount, respectively. V6, R1 and R2 respectively represent the jointing stage, the filament stage and the grouting stage.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7635468/v1/cc734c42d42a96d62ba6c78b.png"},{"id":92962587,"identity":"0bac7958-c9db-4b51-9ff0-d0642e03ad3e","added_by":"auto","created_at":"2025-10-07 15:11:40","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":75935,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of moderate irrigation under no-tillage on SPAD values of intercropped maize at different growth stages. Note: NT and CT represent no-tillage and conventional tillage, respectively. IM and SM were intercrop and monocrop maize, respectively. I1, I2, and I3 represent low, medium, and high irrigation amount, respectively. V6, R1 and R2 respectively represent the jointing stage, the filament stage and the grouting stage.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7635468/v1/e166d4b657268e7efefe10bc.png"},{"id":92963345,"identity":"7769b29e-4373-4d2d-b7bd-fde5fcd57051","added_by":"auto","created_at":"2025-10-07 15:19:41","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":65172,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of moderate irrigation under no-tillage on leaf PEPC activity of intercropped maize at different growth stages. Note: NT and CT represent no-tillage and conventional tillage, respectively. IM and SM were intercrop and monocrop maize, respectively. I1, I2, and I3 represent low, medium, and high irrigation amount, respectively. V6, R1 and R2 respectively represent the jointing stage, the filament stage and the grouting stage.Different lowercase letters in the same year mean significant difference at the 0.05 probability level among treatments，and the same applies hereafter.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7635468/v1/00352cc3ca0e8e585de55057.png"},{"id":92963329,"identity":"7d95552e-80a7-425c-a479-96f6ea7453ce","added_by":"auto","created_at":"2025-10-07 15:19:40","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":65805,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of moderate irrigation under no-tillage on leaf Rubisco activity of intercropped maize at different growth stages. Note: NT and CT represent no-tillage and conventional tillage, respectively. IM and SM were intercrop and monocrop maize, respectively. I1, I2, and I3 represent low, medium, and high irrigation amount, respectively. V6, R1 and R2 respectively represent the jointing stage, the filament stage and the grouting stage.Different lowercase letters in the same year mean significant difference at the 0.05 probability level among treatments，and the same applies hereafter.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-7635468/v1/56140991b7ddbb6b31bf1b77.png"},{"id":92962576,"identity":"998a6324-5bfc-4d42-b2f4-bbb97ce88311","added_by":"auto","created_at":"2025-10-07 15:11:40","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":60667,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of moderate irrigation under no-tillage on leaf PPDK activity of intercropped maize at different growth stages. Note: NT and CT represent no-tillage and conventional tillage, respectively. IM and SM were intercrop and monocrop maize, respectively. I1, I2, and I3 represent low, medium, and high irrigation amount, respectively. V6, R1 and R2 respectively represent the jointing stage, the filament stage and the grouting stage.Different lowercase letters in the same year mean significant difference at the 0.05 probability level among treatments，and the same applies hereafter.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-7635468/v1/90860664eadbb4e2694965b5.png"},{"id":92963330,"identity":"634f3827-daa1-4f41-b53a-350807463f6f","added_by":"auto","created_at":"2025-10-07 15:19:40","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":82139,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of moderate irrigation under no-tillage on the relative expression levels of \u003cem\u003epepc\u003c/em\u003e, \u003cem\u003erbc\u003c/em\u003e, and \u003cem\u003eppdk\u003c/em\u003e genes in intercropped maize leaves at different growth stages. Note: NT and CT represent no-tillage and conventional tillage, respectively. IM and SM were intercrop and monocrop maize, respectively. I1, I2, and I3 represent low, medium, and high irrigation amount, respectively. V6 and R2 respectively represent the jointing stage and the grouting stage.Different lowercase letters in the same year mean significant difference at the 0.05 probability level among treatments，and the same applies hereafter.\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-7635468/v1/26b6e524a6a84a218f07a83f.png"},{"id":92964412,"identity":"9ddf95b2-e6f9-4497-8a7d-396d02807cc1","added_by":"auto","created_at":"2025-10-07 15:27:40","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":52206,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal component analysis of grain yield and photosynthetic physiological parameters of intercropped maize under moderate irrigation with no-tillage.\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-7635468/v1/2f0cada76028683834ca08ac.png"},{"id":92962590,"identity":"736ca0cf-35ab-48f5-a384-d7d073c07ed3","added_by":"auto","created_at":"2025-10-07 15:11:40","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":147777,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation and structural equation model analysis between grain yield and photosynthetic physiological parameters of maize. Note: GY, Grian yield; EN, Ear numbers; KNS, Kernel number per spike; TKW, 1000-grain weight; \u003cem\u003eP\u003c/em\u003e\u003csub\u003en,\u003c/sub\u003e\u003csub\u003e\u003cem\u003e \u003c/em\u003e\u003c/sub\u003eNet photosynthetic rate; \u003cem\u003eT\u003c/em\u003e\u003csub\u003er,\u003c/sub\u003e\u003csub\u003e\u003cem\u003e \u003c/em\u003e\u003c/sub\u003eTranspiration rate; \u003cem\u003eG\u003c/em\u003e\u003csub\u003es,\u003c/sub\u003e\u003csub\u003e\u003cem\u003e \u003c/em\u003e\u003c/sub\u003eStomatal conductance; \u003cem\u003eC\u003c/em\u003e\u003csub\u003ei, \u003c/sub\u003eIntercellular CO\u003csub\u003e2\u003c/sub\u003e concentration; SPAD, Chlorophyll relative content; PEPC, Phosphoenolpyruvate carboxylase; Y(II), Actual photochemical efficiency; LAI, Leaf area index; LAD, Leaf area duration.\u003c/p\u003e","description":"","filename":"13.png","url":"https://assets-eu.researchsquare.com/files/rs-7635468/v1/62588e2ae2ba1f52839f2a6c.png"},{"id":95654860,"identity":"89fe0894-e96e-4c5a-b6c0-4be8e444e6e6","added_by":"auto","created_at":"2025-11-11 16:13:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2086411,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7635468/v1/7519fda8-c5b4-4656-91dc-038516bcd03b.pdf"},{"id":92963326,"identity":"1fce5adb-df99-4c6b-92ff-4fe5089f9cc4","added_by":"auto","created_at":"2025-10-07 15:19:40","extension":"doc","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":93184,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary9.15.doc","url":"https://assets-eu.researchsquare.com/files/rs-7635468/v1/b3b748e14d3a2fd4b0b429fd.doc"}],"financialInterests":"No competing interests reported.","formattedTitle":"Coupling photosynthetic physiology and C₄ enzyme regulation enhances grain yield of intercropped maize under no-tillage and moderate irrigation in oasis regions","fulltext":[{"header":"Background","content":"\u003cp\u003eThe contradiction between the rapidly growing population and the shrinking arable land area poses a serious threat to food security. Maximizing crop yields through the efficient use of limited resources in agricultural production practices is a crucial pathway to achieving sustainable agriculture and safeguarding national food security [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Intercropping systems can optimize the utilization of light, heat, water, and nutrients by exploiting niche separation mechanisms, thereby allowing two crops to complement each other spatially and temporally [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In the Hexi Oasis irrigation region, where light and heat resources are abundant, intercropping maize is particularly suitable. However, the region faces severe water scarcity [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], which restricts the sustainable development of the maize industry. Therefore, investigating intercropping techniques for stable maize production under limited irrigation and clarifying the theoretical basis of water-saving efficiency has become an urgent issue to address.\u003c/p\u003e\u003cp\u003eIntercropping and no-tillage are regarded as core technologies for water-saving and yield stability due to their respective advantages in resource-use efficiency and soil moisture conservation. Studies have shown that intercropping allows for rational crop combinations, where differences in root architecture between species promote more effective utilization of nutrients and water, while reducing interspecific resource competition, thereby enhancing photosynthetic physiological activity [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Intercropping can also maintain a larger photosynthetic source and a longer functional period after flowering, facilitating the translocation and allocation of assimilates to grains, ultimately leading to higher grain yield [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. No-tillage, on the other hand, helps maintain a strong photosynthetic source during the middle and late growth stages, slows down the decline in chlorophyll content, extends the photosynthetic duration, and promotes assimilate translocation to grains, thereby increasing yield [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Water availability is a key factor regulating crop growth, and some studies have indicated that limited irrigation encourages root penetration into deeper soil layers, improving water-use efficiency [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Under adequate soil moisture, crop leaves tend to open their stomata to enhance transpiration, which increases stomatal conductance, transpiration rate, and net photosynthetic rate [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Moreover, mild water stress has been shown to stimulate the activities of phosphoenolpyruvate carboxylase (PEPC), NADP-malate dehydrogenase (NADP-MDH), and NADP-malic enzyme (NADP-ME), thereby improving CO₂ fixation capacity and strengthening carbon assimilation, which collectively enhance photosynthetic efficiency. Additionally, limited irrigation can prevent the adverse effects of excessive water supply, help maintain leaf health, and sustain photosynthetic function, thereby promoting assimilate accumulation and grain yield formation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, most existing studies have focused on monocropped maize under conventional tillage or full irrigation, lacking systematic evidence for the combined effects of the \u0026ldquo;no-tillage \u0026times; intercropping \u0026times; limited irrigation\u0026rdquo; triad. More critically, whether such agronomic combinations can synergistically regulate photosynthetic machinery at the physiological and molecular levels to compensate for the potential biomass loss caused by reduced irrigation remains unclear.\u003c/p\u003e\u003cp\u003ePhotosynthetic performance represents the primary physiological bottleneck determining maize yield formation. More than 60% of dry matter accumulation during the grain-filling stage originates from post-anthesis photosynthesis, which coincides with the water-sensitive period in the Hexi irrigation region. Under limited irrigation, no-tillage can effectively improve the photosynthetic performance of monocropped maize by regulating soil physicochemical properties and enhancing soil water and nutrient availability, thereby sustaining relatively high grain yields [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. As a C₄ crop, maize exhibits photosynthetic efficiency that is highly dependent on the activity and gene expression of PEPC, pyruvate phosphate dikinase (PPDK), and Rubisco. Meanwhile, chlorophyll fluorescence parameters (Fv/Fm, Y(II), Y(NPQ), Y(NO)) can dynamically capture changes in PSII photochemical efficiency and photoprotective capacity. Theoretically, the combined effects of no-tillage improving rhizosphere water status, intercropping optimizing canopy light distribution, and moderate irrigation inducing mild reversible stress may jointly activate a cascade pathway of \u0026ldquo;upregulated C₄ enzyme gene expression \u0026rarr; enhanced enzyme activity \u0026rarr; increased Pn and Y(II),\u0026rdquo; thus achieving photosynthetic compensation under limited water supply. However, such mechanisms remain to be validated through field experiments.\u003c/p\u003e\u003cp\u003eMaize (\u003cem\u003eZea mays\u003c/em\u003e L.), as one of the world\u0026rsquo;s most important cereal crops, serves not only as a staple for direct human consumption and livestock feed but also as a vital industrial raw material, playing an irreplaceable role in ensuring global food security and driving agricultural economic development. With the growth of the global population and the continuous upgrading of dietary structures, higher demands have been placed on maize yield and quality. However, in recent years, the rapid turnover of maize varieties has led to diminishing returns in yield and quality improvements achieved solely through genetic enhancement, with the marginal effects of traditional breeding approaches becoming increasingly limited. Consequently, optimizing agronomic practices to unlock maize\u0026rsquo;s production potential has emerged as a key direction in contemporary agricultural research. In the arid regions of Northwest China, the Hexi Oasis irrigation area, endowed with abundant light and heat resources, is particularly suitable for developing intercropping systems of maize, providing unique natural advantages for assimilate accumulation and grain filling [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHowever, the region faces severe resource-based water scarcity, which constrains the sustainable development of the maize industry. Therefore, under limited irrigation, identifying agronomic optimizations that enhance photosynthetic efficiency in intercropped maize to ensure yield stability is a key challenge for advancing water-saving, high-efficiency agriculture in this area. To this end, we systematically investigated: (1) how the coupling of no-tillage and intercropping regulates maize gas-exchange, chlorophyll fluorescence, and SPAD dynamics under limited irrigation; (2) whether these physiological responses are coordinated with changes in the activities of PEPC, PPDK, and Rubisco and the transcription of their corresponding genes (\u003cem\u003epepc\u003c/em\u003e, \u003cem\u003eppdk\u003c/em\u003e, \u003cem\u003erbcL\u003c/em\u003e); and (3) the relative contributions and path coefficients linking the photosynthesis\u0026ndash;enzyme activity\u0026ndash;molecular regulation network to grain yield and its components. The results aim to elucidate the photosynthetic physiological and molecular mechanisms by which the \u0026ldquo;no-tillage\u0026thinsp;+\u0026thinsp;moderate irrigation\u0026thinsp;+\u0026thinsp;intercropping\u0026rdquo; system achieves water-saving yield stability, providing quantifiable theoretical thresholds and practical pathways for maize production in the arid Northwest.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eEffects of moderate irrigation and no-tillage on grain yield and yield components of intercropped maize\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eModerate irrigation combined with no-tillage exerted a significant effect on grain yield in intercropped maize, with a significant two-way interaction but a non-significant three-way interaction (Fig. 1). Across tillage practices, no-tillage (NT) increased grain yield by 7.8% compared with conventional tillage (CT). At the same land area, intercropped maize (IM) produced 29.5% higher yield than monocropped maize (SM). Across irrigation regimes, grain yield declined with reduced irrigation, with low irrigation (I1) producing 18.9% less yield than high irrigation (I3), while no significant difference was observed between moderate (I2) and high irrigation (I3). Under intercropping, NT with moderate irrigation (NTIMI2) significantly outperformed CT with moderate irrigation (CTIMI2) by 10.5%, and NTIMI2 yielded 27.2% more than NT with monocropping under moderate irrigation (NTSMI2). Although NTIMI1 produced 14.5% less yield than NTIMI3, the difference between NTIMI2 and NTIMI3 was not significant. Overall, moderate irrigation under no-tillage ensured yield stability in intercropped maize.\u003c/p\u003e\n\u003cp\u003eThe yield advantages of NT and IM were mainly attributable to increases in ear number and 1000-grain weight (Table 1). Compared with CT, NT increased ear number and grain weight by 6.0% and 5.8%, respectively, while IM increased these components by 5.8% and 6.2% compared with SM. In contrast, tillage and planting patterns had no significant effect on kernel number per ear. Irrigation exerted a strong influence on yield components: relative to I3, I1 reduced both ear number and grain weight, whereas I2 had effects similar to I3. Notably, NTIMI2 significantly increased ear number (+8.0%) and grain weight (+6.3%) compared with CTIMI2.\u003c/p\u003e\n\u003cp id=\"_Toc198845261\"\u003e\u003cstrong\u003eEffects of moderate irrigation and no-tillage on photosynthesis and chlorophyll fluorescence of intercropped maize\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompared with the control, NT reduced Pn at the jointing stage but increased Pn by 7–11% from silking to maturity. Before silking, IM exhibited a lower net photosynthetic rate than SM, but during the reproductive stage, Pn in IM was 6–10% higher than in SM, indicating a delayed yet pronounced intercropping advantage. Irrigation had a significant effect on Pn. Under low irrigation (I1), Pn decreased continuously relative to I3 during both the vegetative stage (–7 to –13%) and the reproductive stage (–6 to –13%), whereas I2 maintained values comparable to I3. Notably, under moderate irrigation, NT (NTIMI2) significantly enhanced Pn during the reproductive stage compared with CTIMI2 (+9–20%) and NTSMI2 (+7–14%). In contrast, NTIMI1 showed a marked decline relative to NTIMI3, while NTIMI2 and NTIMI3 performed similarly (Fig. 2).\u003c/p\u003e\n\u003cp\u003eSimilar to Pn and Gs, Tr peaked at silking (Fig. 3). Compared with CT, NT reduced Tr before tasseling but increased it by 8–12% afterward. Relative to SM, IM exhibited lower Tr values before silking, but during the reproductive stage, Tr was 7–11% higher, reflecting an intercropping advantage. From silking to maturity, Tr under low irrigation (I1) was 9–13% lower than under I3, while I2 maintained values comparable to I3. Among treatment combinations, NTIMI2 showed a clear advantage, sustaining significantly higher Tr than CTIMI2 (+14–19%) and NTSMI2 (+9–13%) during the reproductive stage. In contrast, NTIMI1 exhibited a marked reduction compared with NTIMI2 and NTIMI3, whereas NTIMI2 and NTIMI3 did not differ significantly.\u003c/p\u003e\n\u003cp\u003eCompared with the control, NT reduced Gs at the jointing stage but increased it by 9–13% from silking to maturity. Similarly, at the early growth stage, Gs in IM was lower than in SM, but from silking onward, Gs was 8–13% higher in IM, highlighting the delayed advantage of intercropping. Irrigation exerted a strong influence during the reproductive stage: Gs under I1 was 14–21% lower than under I3, while I2 remained comparable to I3. At the treatment-combination level, NTIMI2 maintained higher Gs than CTIMI2 (+9–18%) and NTSMI2 (+9–13%) from silking to maturity, whereas NTIMI1 was significantly lower than NTIMI3 (Fig. 4).\u003c/p\u003e\n\u003cp\u003eAt the jointing stage, NT increased Ci relative to CT, but from silking to maturity it consistently reduced Ci by 6–8%, suggesting improved CO₂ assimilation capacity. \u0026nbsp;Similarly, IM showed higher Ci than SM before tasseling, but 7–11% lower values during reproductive stages, indicating enhanced CO₂ utilization efficiency under intercropping. Irrigation effects were pronounced during the reproductive period: both I2 and I3 significantly reduced Ci compared with I1, while no difference was observed between I2 and I3. \u0026nbsp;Among treatment combinations, NTIMI2 reduced Ci by 7–10% compared with CTIMI2 and NTSMI2, and by 6–13% compared with NTIMI1, whereas no difference was observed between NTIMI2 and NTIMI3 (Fig. 5).\u003c/p\u003e\n\u003cp\u003eCompared with CT, NT reduced Fv/Fm during early vegetative stages but increased it by 1–4% from silking to maturity. Similarly, IM exhibited slightly lower Fv/Fm than SM before tasseling but 1–3% higher values during reproductive stages. Irrigation exerted a strong influence: low irrigation (I1) significantly decreased Fv/Fm compared with I2 and I3, whereas no difference was detected between I2 and I3. At the treatment combination level, NTIMI2 maintained higher Fv/Fm than both CTIMI2 and NTSMI2 during reproductive stages, while NTIMI1 consistently showed lower values than NTIMI2 and NTIMI3 (Fig. 6).\u003c/p\u003e\n\u003cp\u003eCompared with the control, NT reduced SPAD by 6.4% at the jointing stage but significantly increased it by 5.0–8.0% from silking to the dough stage. Similarly, before silking, SPAD values in IM were lower than in SM, whereas after silking, IM showed 5–7% higher SPAD values than SM. The irrigation effect was more pronounced: SPAD under I1 consistently remained lower than under I2 and I3, while no significant difference was observed between I2 and I3 (Fig. 7).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of moderate irrigation and no-tillage on the activities of key photosynthetic enzymes in intercropped maize leaves\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNT reduced PEPC activity at the jointing stage but increased it by 6–9% from silking to grain filling compared with CT. Similarly, IM exhibited slightly lower activity than SM during early growth, but 9–10% higher activity during reproductive stages. Irrigation exerted a pronounced effect: I2 consistently maintained the highest PEPC activity, whereas I1 reduced it by 7–16% compared with I2 and I3. Treatment combinations confirmed this trend: NTIMI2 showed significantly higher PEPC activity during silking to grain filling compared with CTIMI2 (+6–11%) and NTSMI2 (+7–10%), while NTIMI1 reduced activity by 12–16% relative to NTIMI2 (Fig. 8).\u003c/p\u003e\n\u003cp\u003eRubisco activity was lowest at the jointing stage and peaked at silking. Compared with CT, NT significantly increased Rubisco activity by 10–11% from silking to grain filling, although it was slightly lower at the jointing stage. Likewise, IM enhanced Rubisco activity by 10–13% compared with SM during reproductive stages. Irrigation markedly affected Rubisco activity: I1 reduced activity by 18–19% compared with I3, while no significant difference was observed between I2 and I3. At the treatment combination level, NTIMI2 consistently exhibited higher Rubisco activity during reproductive stages, exceeding CTIMI2 (+10%) and NTSMI2 (+8–10%), while NTIMI1 showed significant reductions relative to NTIMI3 (Fig. 9).\u003c/p\u003e\n\u003cp\u003eCompared with CT, NT decreased PPDK activity at the jointing stage but increased it by ~9–10% from silking to grain filling. Similarly, IM significantly reduced PPDK activity during early growth but enhanced it by 9–12% during reproductive stages relative to SM. Irrigation strongly regulated PPDK activity. I1 consistently reduced activity by 13–20% compared with I3, while I2 maintained values comparable to I3. Treatment combinations confirmed that NTIMI2 promoted the highest PPDK activity during reproductive stages, increasing it by 9–14% relative to CTIMI2 and NTSMI2, whereas NTIMI1 significantly reduced activity compared with NTIMI3 (Fig. 10).\u003c/p\u003e\n\u003cp id=\"_Toc198845267\"\u003e\u003cstrong\u003eEffects of moderate irrigation and no-tillage on the relative expression of key photosynthetic enzyme genes in intercropped maize\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt the jointing stage, \u003cem\u003epepc\u003c/em\u003e expression in NTIMI2 leaves was downregulated by 22.0% compared with CTIMI2, but at the grain-filling stage, it was upregulated by 39.4%. Relative to NTSMI2, \u003cem\u003epepc\u003c/em\u003e expression in NTIMI2 leaves was 10.6% lower at the jointing stage but 31.0% higher at the grain-filling stage. From jointing to grain filling, \u003cem\u003epepc\u003c/em\u003e expression in NTIMI1 leaves was markedly downregulated by 55.3–77.2% and 54.2–67.9% compared with NTIMI2 and NTIMI3, respectively. While no significant difference was observed between NTIMI2 and NTIMI3 at the jointing stage, \u003cem\u003epepc\u0026nbsp;\u003c/em\u003eexpression in NTIMI2 was 41.2% higher than in NTIMI3 at the grain-filling stage (Fig. 11A).\u003c/p\u003e\n\u003cp\u003eAt the jointing stage, \u003cem\u003erbc\u003c/em\u003e expression in NTIMI2 leaves was lower than in CTIMI2, whereas at the grain-filling stage, NTIMI2 was 63.5% higher than CTIMI2. Compared with NTSMI2, \u003cem\u003erbc\u003c/em\u003e expression in NTIMI2 leaves was reduced by 11.0% at the jointing stage but increased by 80.3% at the grain-filling stage. From jointing to grain filling, \u003cem\u003erbc\u0026nbsp;\u003c/em\u003eexpression in NTIMI1 leaves was 63.8–75.8% lower than in NTIMI3. At the jointing stage, NTIMI2 expression was 9.4% lower than NTIMI3, while at the grain-filling stage, CTIMI2 was 13.1% lower than CTIMI3. No significant difference was observed between NTIMI2 and NTIMI3 at the grain-filling stage, but CTIMI2 showed a 38.7% reduction compared with CTIMI3 (Fig. 11B).\u003c/p\u003e\n\u003cp\u003eCompared with CTIMI2, the relative expression of the \u003cem\u003eppdk\u0026nbsp;\u003c/em\u003egene in NTIMI2 maize leaves was downregulated by 14.2% at the jointing stage but upregulated by 53.6% at the grain-filling stage. Relative to NTSMI2,\u003cem\u003e\u0026nbsp;ppdk\u0026nbsp;\u003c/em\u003eexpression in NTIMI2 was 16.0% lower at the jointing stage but 61.1% higher at the grain-filling stage. At the jointing stage, \u003cem\u003eppdk\u003c/em\u003e expression in NTIMI1 leaves was reduced by 59.4% and 64.8% compared with NTIMI2 and NTIMI3, respectively, while NTIMI2 was 13.4% lower than NTIMI3 and CTIMI2 was 8.0% lower than CTIMI3. At the grain-filling stage, \u003cem\u003eppdk\u003c/em\u003e expression in NTIMI1 leaves was downregulated by 73.4% and 74.1% compared with NTIMI2 and NTIMI3, respectively. No significant difference was observed between NTIMI2 and NTIMI3, whereas CTIMI2 was 30.0% lower than CTIMI3 (Fig. 11C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrelation between grain yield and photosynthetic physiological parameters and structural equation modeling analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrincipal component analysis (PCA) was conducted on the relationships between grain yield and photosynthetic physiological parameters of intercropped maize under moderate irrigation and no-tillage (Fig. 12). The cumulative contribution rate of PC1 and PC2 reached 94.8%, indicating strong representativeness. Correlation analysis among maize grain yield, yield components, and photosynthetic physiological parameters (Fig. 13-A) revealed that grain yield, ear number, kernel number per ear, and 1000-grain weight were all significantly and positively correlated with SPAD values, net photosynthetic rate (Pn), stomatal conductance (Gs), and transpiration rate (Tr), while being significantly and negatively correlated with intercellular CO₂ concentration (Ci). Thus, enhancing SPAD, Pn, Gs, and Tr while reducing Ci can optimize photosynthetic performance, thereby synergistically improving ear number, kernel number per ear, and 1000-grain weight, ultimately sustaining higher grain yield.\u003c/p\u003e\n\u003cp\u003eFurthermore, the structural equation model (Fig. 13-B) demonstrated that \u003cem\u003epepc\u0026nbsp;\u003c/em\u003eand \u003cem\u003erbc\u003c/em\u003e exerted significant positive effects on PEPC and Rubisco, respectively, across different tillage, planting, and irrigation treatments. Both PEPC and Rubisco had significant positive effects on Pn and Y(II). In addition, Y(II) positively affected Pn, while Pn regulated grain yield (GY) indirectly by influencing LAI and LAD. Among these pathways, LAI had the most pronounced effect on GY, with a standardized path coefficient as high as 0.977***. Therefore, under moderate irrigation, no-tillage can effectively enhance the activities of key photosynthetic enzymes and the relative expression of their genes, as well as leaf actual photochemical efficiency and net photosynthetic rate during the middle and late growth stages of intercropped maize. This maintains a high level of photosynthetic performance, optimizes photosynthetic efficiency, and ultimately secures higher grain yield.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eEffects of moderate irrigation and no-tillage on grain yield of intercropped maize\u003c/h2\u003e\u003cp\u003eOptimized agronomic practices are essential for achieving stable and increased crop yields. Among them, appropriate tillage methods, mulching techniques, planting patterns, and irrigation regimes play key roles in improving resource-use efficiency and ensuring food security [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Previous studies have demonstrated that intercropping can significantly enhance land-use efficiency and farmland productivity by improving canopy structure and light distribution, extending the functional duration of border-row leaves, and facilitating nutrient complementarity between crops [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The present study similarly showed that intercropped maize achieved significantly higher grain yield than monocropped maize, confirming the advantages of intercropping in promoting photosynthetic efficiency and nutrient utilization.\u003c/p\u003e\u003cp\u003eHowever, under the constrained water resources of the Northwest Oasis irrigation area, intercropping combined with conventional tillage alone cannot ensure yield stability. Our findings revealed that integrating no-tillage with moderate irrigation markedly increased intercropped maize yield, producing 10.5% higher yield than intercropping under conventional tillage, while maintaining a grain yield comparable to high irrigation but with reduced water consumption. This highlights that the combination of no-tillage and moderate irrigation is a critical agronomic strategy for achieving \u0026ldquo;water-saving and yield stability\u0026rdquo; in oasis maize production.\u003c/p\u003e\u003cp\u003eThe yield-enhancing mechanism is primarily reflected in two aspects: (1) Under moderate irrigation, no-tillage significantly increased SPAD values and the peak leaf area index (LAI) at the silking stage, while mitigating their decline during grain filling. This delayed leaf senescence, enhanced photosynthetic capacity, and strengthened grain-filling intensity, thereby providing more assimilates for kernel development [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. (2) No-tillage combined with moderate irrigation significantly improved the activities of PEPC and Rubisco in maize leaves from silking to grain filling, enhancing CO₂ fixation capacity, net photosynthetic rate (Pn), and transpiration rate (Tr). This facilitated the accumulation and translocation of assimilates to grains [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Together, these effects ensured steady improvements in maize yield under limited irrigation conditions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eEffects of moderate irrigation and no-tillage on photosynthetic physiological characteristics of intercropped maize leaves\u003c/h2\u003e\u003cp\u003eGrain yield largely depends on the accumulation of assimilates from post-anthesis photosynthesis; thus, improving leaf photosynthetic performance is a key pathway to achieving stable and increased yields [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Compared with monocropping, intercropped maize exhibited higher SPAD values and net photosynthetic rate (Pn) from silking to grain filling, indicating that intercropping can delay leaf senescence, extend the stay-green period, and enhance photosynthetic efficiency. This finding is consistent with previous studies, which demonstrated that cereal\u0026ndash;legume intercropping improves photosynthetic performance by optimizing the light environment and promoting nutrient complementarity. The underlying mechanism may be that, after pea harvest, maize benefits from more space and resources, utilizes residual organic matter and the nitrogen-fixing effect of legumes, thereby enhancing nutrient supply and SPAD levels. Additionally, intercropping forms a more rational canopy structure, improving light conditions and creating a favorable environment for photosynthesis. Furthermore, our study found that under moderate irrigation, combining no-tillage with intercropping significantly increased SPAD, Pn, and stomatal conductance (Gs) from silking to grain filling. This aligns with earlier findings, suggesting that the integration of no-tillage with intercropping enhances post-anthesis photosynthetic potential [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The underlying mechanisms may involve two aspects: (1) No-tillage reduces soil temperature and energy consumption in the early stages, leading to a marked compensatory effect after silking that delays leaf senescence and maintains higher SPAD levels and photosynthetic activity [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. (2) During the fallow period, no-tillage fields retain more soil moisture, which provides sufficient water for crops in the middle and late growth stages. This improves root activity and leaf water potential, promotes stomatal opening and water\u0026ndash;vapor exchange, thereby increasing Gs and Tr, and ultimately enhances CO₂ assimilation capacity [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In summary, the combination of no-tillage and moderate irrigation effectively improves the photosynthetic physiological state of maize during the late growth stages in intercropping systems, sustaining high photosynthetic efficiency and assimilate accumulation rates, and thus providing a physiological foundation for stable yield improvement.\u003c/p\u003e\u003cp\u003eChlorophyll fluorescence parameters sensitively reflect the efficiency of light absorption and utilization, as well as the response of the photosynthetic apparatus to stress, and are thus important indicators for evaluating crop photosynthetic performance [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Fv/Fm represents the maximum quantum efficiency of PSII, while Y(II) reflects the actual photochemical efficiency of PSII under illumination; together, they measure potential photosynthetic capacity and actual photosynthetic activity, respectively. Y(NPQ) and Y(NO), in turn, indicate photoprotective capacity and the degree of photodamage [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In this study, Fv/Fm and Y(II) significantly declined with decreasing irrigation, whereas under moderate irrigation, no-tillage maintained higher Fv/Fm and Y(II) values during the later growth stages. This suggests that no-tillage combined with moderate irrigation can mitigate the inhibitory effects of water deficit on PSII energy conversion, thereby sustaining strong photosynthetic activity. The underlying reason may lie in the early suppression of photosynthetic performance due to lower soil temperature and intensified resource competition under no-tillage and intercropping conditions ; however, during the middle and late growth stages, improved soil conditions and the independent growth of intercropped maize allow greater acquisition of light, water, and nutrients, thereby enhancing PSII energy conversion efficiency [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. At the same time, our results showed that no-tillage with moderate irrigation significantly increased Y(NPQ) while reducing Y(NO), indicating that this treatment enhanced leaf photoprotective capacity and reduced the risk of photodamage. These findings are consistent with previous studies showing that no-tillage and intercropping improve canopy light environments and optimize soil hydrothermal conditions, which promote root development and maintain photosynthetic apparatus homeostasis, thereby strengthening plant adaptability to light stress [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In summary, no-tillage combined with moderate irrigation not only improved PSII energy conversion efficiency and photoprotective capacity in intercropped maize but also effectively reduced non-regulated energy dissipation, alleviating the decline in photosynthetic efficiency during the late growth stages. This, in turn, provided a physiological foundation for assimilate accumulation during grain filling and yield formation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffects of moderate irrigation and no-tillage on the activities of key photosynthetic enzymes and the relative expression of their genes in intercropped maize leaves\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePEPC, Rubisco, and PPDK are among the most critical enzymes in crop photosynthesis, and their activities directly determine leaf photosynthetic efficiency. PEPC initiates the C₄ pathway by catalyzing the fixation of CO₂ with pyruvate to form oxaloacetate, which subsequently enters the C₄ cycle; Rubisco is the principal carbon-fixing enzyme in both C₃ and C₄ plants; and PPDK converts oxaloacetate into phosphoenolpyruvate (PEP), sustaining the C₄ pathway [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Previous studies have shown that the decline in leaf photosynthetic rate is largely attributable to reductions in photosynthetic enzyme activities and the relocation of enzymes. Slowing down the reduction in enzyme activity and limiting enzyme relocation are conducive to maintaining high photosynthetic efficiency, thereby facilitating assimilate accumulation and translocation [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In this study, we found that under moderate irrigation, applying no-tillage to intercropping systems reduced photosynthetic enzyme activity at the jointing stage but significantly enhanced it from silking to grain filling. This could be explained by the relatively lower soil temperature under no-tillage intercropping during the jointing stage, where cold stress suppressed enzyme activity. However, as crop development progressed, soil temperatures increased, and tillage practices had little effect on soil thermal conditions.\u003c/p\u003e\u003cp\u003eFrom silking to grain filling, soil moisture became the dominant factor influencing photosynthetic enzyme activity; no-tillage effectively reduced soil water loss and evaporation \u003csup\u003e[159]\u003c/sup\u003e, thereby ensuring sufficient water supply for vegetative growth, promoting stomatal opening, lowering CO₂ diffusion resistance, and ultimately increasing photosynthetic enzyme activity [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Other studies have also reported that mild water stress can enhance photosynthetic enzyme activity and gene expression, accelerating metabolic processes and improving photosynthetic capacity. This mechanism enables crops to maintain strong growth and yield potential even under water deficit conditions [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Our findings are consistent with this: under no-tillage intercropping, moderate irrigation significantly upregulated \u003cem\u003epepc\u003c/em\u003e gene expression and increased PEPC activity from the jointing to grain-filling stages, whereas under low irrigation, the activities of all three enzymes and the relative expression of their associated genes were lowest. The likely explanation is that mild water stress enhances PEPC activity and its gene expression, while severe water stress suppresses the activities of PEPC, Rubisco, and PPDK.\u003c/p\u003e\u003cp\u003eTherefore, under moderate irrigation, no-tillage promotes the enhancement of key photosynthetic enzyme activities and their gene expression in intercropped maize leaves during the middle and late growth stages. This maintains a high carboxylation capacity for CO₂, strengthens carbon assimilation, improves Pn and related gas-exchange parameters, and synergistically enhances photosynthetic efficiency.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eFrom a holistic \u0026ldquo;water\u0026ndash;light\u0026ndash;enzyme\u0026ndash;yield\u0026rdquo; perspective, this study is the first to demonstrate under limited water supply in oasis irrigation regions that coupling no-tillage with intercropping, supplemented by moderate irrigation, can trigger a three-tiered \u0026ldquo;photosynthesis\u0026ndash;enzyme activity\u0026ndash;gene expression\u0026rdquo; cascade response in maize, thereby achieving the breakthrough goal of \u0026ldquo;reduced irrigation without yield loss\u0026rdquo; or even \u0026ldquo;yield gain.\u0026rdquo; Specifically, this model translates physiological advantages into yield advantages through three key mechanisms: (i) no-tillage conserves soil water and improves soil moisture, while intercropping optimizes light distribution, jointly delaying leaf senescence and sustaining strong post-anthesis photosynthetic sources; (ii) moderate water stress acts as a \u0026ldquo;mild elicitor,\u0026rdquo; upregulating \u003cem\u003epepc\u003c/em\u003e, \u003cem\u003eppdk\u003c/em\u003e, and \u003cem\u003erbcL\u003c/em\u003e expression, enhancing PEPC, PPDK, and Rubisco activities, thereby improving CO₂ carboxylation capacity and photochemical efficiency; and (iii) increasing Y(II) and Y(NPQ) while decreasing Y(NO), thus maintaining efficient PSII function under water-saving conditions and reducing non-regulated energy dissipation. Structural equation modeling further revealed that the coupled physiological\u0026ndash;molecular effects generate a standardized path coefficient of 0.977 along the Pn \u0026rarr; LAI \u0026rarr; yield pathway, explaining 94.8% of the yield variation and providing direct evidence for the quantitative relationship between photosynthesis, enzyme activity, and yield. Beyond maize, the \u0026ldquo;tillage\u0026ndash;planting\u0026ndash;irrigation\u0026rdquo; triadic paradigm established in this study can be extended to other C₄ crops (e.g., sorghum, sugarcane) and cereal\u0026ndash;legume intercropping systems. This offers a replicable and scalable theoretical template for developing new cropping systems characterized by \u0026ldquo;high yield and efficiency, water conservation, and low carbon emissions\u0026rdquo; in arid regions worldwide.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003ePlant materials\u003c/h2\u003e\n \u003cp\u003eThe maize cultivar used in the experiment was Xianyu 335 (\u003cem\u003eZea mays\u003c/em\u003e L.), and the pea cultivar was Longwan No. 1 (\u003cem\u003ePisum sativum\u003c/em\u003e L.). Seeds of maize (cv. Xianyu 335) and pea (Longwan No. 1) were purchased from local commercial suppliers in Lanzhou and Wuwei, Gansu, China. Xianyu 335 is a nationally approved hybrid in China (approval Nos. Guoshenyu 2004017 and Guoshenyu 2006026), and Longwan No. 1 is nationally registered as a non-major crop variety (registration No. GPD-Pea(2018)620005). No wild materials were collected and no permits were required.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eExperimental site description\u003c/h2\u003e\n \u003cp\u003eThe experiment was conducted in 2024 at the \u0026ldquo;Oasis Agricultural Research and Teaching Base of Gansu Agricultural University\u0026rdquo; in Huangyang Town, Liangzhou District, Wuwei City, Gansu Province, China (37\u0026deg;30\u0026prime;N, 103\u0026deg;5\u0026prime;E). The site is located in a cold temperate arid climate zone, with an average annual precipitation of approximately 156 mm, annual evaporation of about 2400 mm, total sunshine duration of 2969.2 h, mean annual temperature of 7.2\u0026deg;C, and a frost-free period of 156 days. The region has abundant solar radiation, making it well suited for maize intercropping systems. Local agricultural production is predominantly based on conventional tillage with plowing, and plastic film mulching is commonly applied.\u003c/p\u003e\n \u003cp\u003eThe soil type at the experimental site was irrigated desert soil. In 2024, the nutrient contents of the plow layer were as follows: total nitrogen, 0.89 g kg⁻\u0026sup1;; available phosphorus, 24.98 mg kg⁻\u0026sup1;; available potassium, 138.44 mg kg⁻\u0026sup1;; organic matter, 14.53 g kg⁻\u0026sup1;; and bulk density, 1.24 g cm⁻\u0026sup3;. Variations in temperature and precipitation at the site throughout the 2024 growing season are shown in Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\n \u003cp\u003eBased on a long-term experiment initiated in 2015, this study adopted a split-split plot design with three factors. The main plots included two tillage practices: no-tillage (NT, where maize was directly sown the following year onto the residual film after the previous maize harvest without tillage) and conventional tillage (CT, where the field was deep-plowed after the previous maize harvest, and new plastic film was applied after land preparation before sowing the following year). The subplots consisted of two planting patterns: intercropped maize (IM) and monocropped maize (SM). The sub-subplots were assigned three irrigation levels: low irrigation (I1, 4500 m\u0026sup3; ha⁻\u0026sup2;), moderate irrigation (I2, 4950 m\u0026sup3; ha⁻\u0026sup2;), and high irrigation (I3, 5400 m\u0026sup3; ha⁻\u0026sup2;), with the high irrigation treatment representing the local conventional irrigation level. In total, 12 treatment combinations were established, each replicated three times, resulting in 36 plots. Each plot had an area of 63 m\u0026sup2; (7 m \u0026times; 9 m). The treatment codes are provided in Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\n \u003cp\u003eThe planting density of monocropped maize was 90,000 plants ha⁻\u0026sup1;, with a row spacing of 40 cm and a plant spacing of 27 cm. In the maize\u0026ndash;pea intercropping system, a 3:4 planting ratio (three rows of maize to four rows of pea) was adopted, with a spacing of 25 cm between maize and pea rows. The planting density of intercropped maize was 52,000 plants ha⁻\u0026sup1;, while that of pea was 760,000 plants ha⁻\u0026sup1;, with an inter-row ratio of 8:11 between pea and maize strips. Maize and pea were sown on April 18 and April 7, 2024, respectively, and harvested on September 27 and July 6, 2024, respectively. After harvest, all crop residues were removed from the field.\u003c/p\u003e\n \u003cp\u003eAll irrigation treatments were applied using drip irrigation under plastic film, with water volume monitored by flow meters. Specific irrigation quotas at each growth stage are provided in Table S2. The maize fertilization regime followed local conventional management, with a total nitrogen application of 360 kg ha⁻\u0026sup1;, split at a ratio of 3:5:2 across the basal stage, the big-tassel stage, and the grain-filling stage. Phosphorus fertilizer was applied at an N:P ratio of 2:1, equivalent to 180 kg ha⁻\u0026sup1;, incorporated entirely as a basal application. For pea, total nitrogen and phosphorus application rates were 90 kg ha⁻\u0026sup1; and 45 kg ha⁻\u0026sup1;, respectively, both applied entirely as basal fertilizer across all treatments. The chemical nitrogen fertilizers applied included urea and diammonium phosphate. White agricultural plastic film was used for mulching, with a width of 120 cm and a thickness of 0.01 mm. Given the high native potassium content of the soil in this region, no potassium fertilizer was applied. Pest, disease, and weed management practices were carried out according to local conventional tillage practices.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eSampling and measurements\u003c/h2\u003e\n \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\n \u003ch2\u003eThe yield and yield components\u003c/h2\u003e\n \u003cp\u003eAt maturity, a 5 m row length in the center of each plot (one film width of 1.4 m, totaling 7 m\u0026sup2;) with continuous plants and no gaps was selected as the sampling area for yield measurement. Ears were harvested manually, shelled on site, and cleaned of impurities. Fresh grain weight was measured using an electronic balance with an accuracy of 0.01 kg. A 500 g grain subsample was randomly collected, inactivated at 105\u0026deg;C for 30 min, and then oven-dried at 80\u0026deg;C to a constant weight to determine actual moisture content. Grain weight was converted to a standard moisture content of 14% according to national maize yield determination standards and further converted to yield per hectare (kg ha⁻\u0026sup1;).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eLeaves relative chlorophyll content\u003c/h2\u003e\n \u003cp\u003eAt the jointing, big-tassel, silking, grain-filling, and dough stages, the relative chlorophyll content (SPAD value) of maize leaves was measured on sunny mornings between 9:30 and 11:30. SPAD values were determined using a SPAD-502 Plus chlorophyll meter. For each measurement, three representative maize plants of uniform growth were randomly selected, and three readings were taken per plant; the average value was recorded as the observation, avoiding interference from leaf veins. At the jointing stage, measurements were taken on the second fully expanded leaf from the top of the plant. At the big-tassel, silking, grain-filling, and dough stages, measurements were taken on the ear leaf, with the reading location at the middle portion of the leaf, avoiding veins and leaf margins.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eLeaves gas exchange parameters\u003c/h2\u003e\n \u003cp\u003eAt the jointing, big-tassel, silking, grain-filling, and dough stages, the net photosynthetic rate (Pn), transpiration rate (Tr), intercellular CO₂ concentration (Ci), and stomatal conductance (Gs) of maize leaves were measured between 9:30 and 11:30 a.m. on sunny days using a LI\u0026ndash;6800XT portable photosynthesis system. Three representative maize plants of uniform growth were randomly selected for measurement, with care taken to avoid leaf veins. During measurement, the leaf chamber area was set to 2 cm\u0026sup2;, with an airflow rate of 500 \u0026micro;mol s⁻\u0026sup1;. The photosynthetic photon flux density was set at 1200 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1;. Leaf temperature was maintained equal to air temperature, and relative humidity was controlled at 55%. At the jointing stage, the second fully expanded leaf from the top was selected, while at the big-tassel, silking, grain-filling, and dough stages, the ear leaf was measured at the middle portion, avoiding veins and leaf margins.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eLeaves chlorophyll fluorescence parameters\u003c/h2\u003e\n \u003cp\u003eAt the jointing, big-tassel, silking, grain-filling, and dough stages, chlorophyll fluorescence parameters of maize leaves were measured between 9:30 and 11:30 a.m. on sunny days using an FMS-II pulse-modulated fluorometer (PAM-2500). Three representative maize plants of uniform growth were randomly selected, and each was measured three times; the average value was recorded as the observation, avoiding interference from leaf veins. At the jointing stage, the second fully expanded leaf from the top was selected, while at the big-tassel, silking, grain-filling, and dough stages, the ear leaf was measured at the middle portion of the leaf, avoiding veins and margins.\u003c/p\u003e\n \u003cp\u003ePrior to measurement, target leaves were dark-adapted for 30 min using dark adaptation clips. Thereafter, a measuring light (\u0026lt;\u0026thinsp;0.05 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1;) followed by a saturating pulse light (6000 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1;) was applied to obtain the minimum fluorescence (Fo) and maximum fluorescence (Fm) under dark-adapted conditions. The maximum photochemical efficiency was then calculated as: Fv/Fm = (Fm \u0026ndash; Fo) / Fm\u003c/p\u003e\n \u003cp\u003eSubsequently, actinic light (1000 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1;) was applied for 3 min. Once the fluorescence signal stabilized, the steady-state fluorescence (Fs) was recorded. A saturating pulse light (6000 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1;) was then applied to determine the maximum fluorescence under light (Fm\u0026prime;) and the minimum fluorescence under light (Fo\u0026prime;). The following parameters were calculated:\u003c/p\u003e\n \u003cp\u003eActual photochemical efficiency of PSII: Y(II)=(Fm\u0026prime;-Fs)/Fm\u003c/p\u003e\n \u003cp\u003eQuantum yield of regulated energy dissipation: Y(NPQ)\u0026thinsp;=\u0026thinsp;F/Fm\u0026prime;-F/Fm\u003c/p\u003e\n \u003cp\u003eQuantum yield of non-regulated energy dissipation: Y(NO)\u0026thinsp;=\u0026thinsp;F/Fm\u003c/p\u003e\n \u003cp\u003eRelative electron transport rate: ETR\u0026thinsp;=\u0026thinsp;PAR\u0026times;Y(II)\u0026times;0.84\u0026times;0.5\u003c/p\u003e\n \u003cp\u003ewhere PAR represents photosynthetically active radiation (1000 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1;), 0.84 is the leaf absorption coefficient, and 0.5 assumes equal partitioning of excitation energy between PSⅠ and PSⅡ.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eThe activity of key photosynthetic enzymes\u003c/h2\u003e\n \u003cp\u003eAt the jointing stage of maize, the second fully expanded leaf from the top was sampled, while at the silking and grain-filling stages, fresh samples were collected from the middle section of ear leaves for the determination of key photosynthetic enzymes. Three replicates were taken for each treatment, with 0.2 g of tissue per replicate. Samples were immediately frozen in liquid nitrogen and then stored in an ultra-low temperature freezer at \u0026minus;\u0026thinsp;80℃ to preserve tissue structure and biological activity.\u003c/p\u003e\n \u003cp\u003eThe activities of PEPC, PPDK, and Rubisco were measured using assay kits supplied by Suzhou Comin Biotechnology Co., Ltd., following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003eqRT-PCR验证\u003c/h2\u003e\n \u003cp\u003eTo analyze the expression dynamics of key photosynthetic enzyme genes in maize leaves under different tillage practices, planting patterns, and irrigation regimes, samples were collected at three growth stages: jointing, silking, and grain filling. For each treatment, three maize plants of uniform growth were randomly selected. At the jointing stage, the second fully expanded leaf from the top was sampled; at the silking and grain-filling stages, the middle portion of the ear leaf was collected. After removing the veins, 0.2 g of fresh tissue was quickly weighed, flash-frozen in liquid nitrogen, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C. Total RNA was extracted from maize leaves using the HiScript\u0026reg; II Q RT SuperMix for qPCR (+\u0026thinsp;gDNA wiper) kit (Nanjing). RNA quality was assessed with an Agilent 2100 Bioanalyzer and verified by RNase-free agarose gel electrophoresis. RNA was then reverse-transcribed into cDNA using the FastPure\u0026reg; Plant Total RNA Isolation Kit (Nanjing), following the instructions provided with the PrimeScript reverse transcription kit. The cDNA synthesis reaction system (20 \u0026micro;L) contained 4 \u0026micro;L of 5\u0026times; HiScript II qRT SuperMix II, 4 \u0026micro;L of 4\u0026times; gDNA Wiper Mix, 12 \u0026micro;L of RNase-free H₂O, and RNA (1000 ng). Reaction conditions were 50\u0026deg;C for 15 min and 85\u0026deg;C for 5 s. The cDNA products were stored at \u0026minus;\u0026thinsp;20\u0026deg;C until use.\u003c/p\u003e\n \u003cp\u003eSubsequently, quantitative real-time PCR (qRT-PCR) was performed. The maize coding sequences were obtained from the MaizeGDB database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.maizegdb.org/\u003c/span\u003e\u003c/span\u003e), and primers were designed using Primer Premier 5.0 (Canada) and synthesized by Shanghai OE Biotech Co., Ltd. (primer sequences are provided in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The reactions were carried out using the Taq Pro Universal SYBR qPCR Master Mix kit on a LightCycler\u0026reg; 480 Ⅱ real-time PCR system (Switzerland). The reaction mixture (20 \u0026micro;L) consisted of 10 \u0026micro;L of 2\u0026times; Taq Pro Universal SYBR qPCR Master Mix, 0.4 \u0026micro;L of 10 \u0026micro;M Primer 1, 0.4 \u0026micro;L of 10 \u0026micro;M Primer 2, 1 \u0026micro;L of cDNA, and 8.5 \u0026micro;L of nuclease-free H₂O. The PCR program was set as follows: 95\u0026deg;C for 30 s; followed by 40 cycles of 95\u0026deg;C for 5 s and 60\u0026deg;C for 30 s. Gene expression levels were calculated using the 2⁻(\u0026Delta;\u0026Delta;Ct) method, with GhActin serving as the internal reference gene. Each treatment included three biological replicates. The specific primer sequences are listed in Table S3.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eData were recorded and organized using Microsoft Excel 2019. Duncan\u0026rsquo;s multiple range tests were performed in SPSS 21.0. Figures and correlation analyses were generated with Origin 2019b and GraphPad Prism 9.0. Results are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE). One-way ANOVA followed by the least significant difference (LSD) test was used to determine significance at the 95% or 99% confidence level. Image assembly was conducted in Adobe Illustrator 2020. Structural equation modeling (SEM) was performed using AMOS 22.0.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003cbr\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC.C.G., and Y.W., Writing \u0026ndash;original draft, Conceptualization, Data curation, Visualization, Writing \u0026ndash; original draft. H.F., and Y.L.S., Methodology, \u0026nbsp; Conceptualization, Methodology, Software. W.H., and C.S.B., Writing \u0026ndash; review \u0026amp; editing. Data curation, Supervision. F.Y.C., C.X.B., and X.Y.L., Formal analysis, \u0026nbsp;Visualization. C.Z., and X.Y.B., Conceptualization, , Writing \u0026ndash; review \u0026amp; editing, Project administration, Supervision, Writing \u0026ndash; review\u0026amp; editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank to the Major Science and Technology Projects of Gansu Province (No. 22ZD6NA009, No. 23ZDNA008), the Youth Science and Technology Fund of Gansu Province (25JRRA371), the Joint Fund of Gansu Agricultural University (GSAU-DKZY-2025-001) and the Key Project of Natural Science Foundation of Gansu Province (No. 21JR7RA802).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;I have permission to collect Plant Material. All experimental research on plants complied with institutional, national, and international guidelines. Field studies were conducted in accordance with Chinese national legislation on crop variety release and agricultural experimentation. No wild plants were collected in this study, and therefore no specific collection permits were required. The commercial cultivars used in this study\u0026mdash;Xianyu 335 (national approvals Guoshenyu 2004017; Guoshenyu 2006026) and Longwan No. 1 (national variety registration GPD-Pea(2018)620005)\u0026mdash;are available for purchase from local suppliers in [City, Province, Country] upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;Not applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eState Key Laboratory of Aridland Crop Science, College of Agronomy, Gansu Agricultural University, Lanzhou 730070, China\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eRudel TK, Schneider L, Uriarte M, Turner BL, DeFries R, Lawrence D, et al. Agricultural intensification and changes in cultivated areas, 1970\u0026ndash;2005. Proc. Natl. Acad. Sci. U. S. A. 2009;106:20675\u0026ndash;80.\u003c/li\u003e\n \u003cli\u003eRaza MA, Yasin HS, Gul H, Qin R, Mohi Ud Din A, Khalid MHB, et al. Maize/soybean strip intercropping produces higher crop yields and saves water under semi-arid conditions. Front. Plant Sci. 2022;13:1006720.\u003c/li\u003e\n \u003cli\u003ePan X, Zhang H, Deng H, Yu S, Zhou C, Li F. Selecting reasonable soil moisture-maintaining measures to improve the soil physicochemical properties and achieve high yield and quality of purple garlic in the China Hexi Corridor oasis agricultural area. Front. Plant Sci. 2024;15:1447469.\u003c/li\u003e\n \u003cli\u003eKhashi U Rahman M, Saati-Santamar\u0026iacute;a Z, Garc\u0026iacute;a-Fraile P. Intercropping of non-leguminous crops improves soil biochemistry and crop productivity: a meta-analysis. 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Field Crops Res. 2021;271:108249.\u003c/li\u003e\n \u003cli\u003eMart\u0026iacute;nez-Go\u0026ntilde;i XS, Miranda-Apodaca J, P\u0026eacute;rez-L\u0026oacute;pez U. Could buckwheat and spelt be alternatives to wheat under future environmental conditions? Study of their physiological response to drought. Agric. Water Manag. 2023;278:108176.\u003c/li\u003e\n \u003cli\u003eJha PK, Araya A, Stewart ZP, Faye A, Traore H, Middendorf BJ, et al. Projecting potential impact of COVID-19 on major cereal crops in Senegal and Burkina Faso using crop simulation models. Agric. Syst. 2021;190:103107.\u003c/li\u003e\n \u003cli\u003eZhang M, Gao Y, Zhang Y, Fischer T, Zhao Z, Zhou X, et al. The contribution of spike photosynthesis to wheat yield needs to be considered in process-based crop models. Field Crops Res. 2020;257:107931.\u003c/li\u003e\n \u003cli\u003eHu F, Tan Y, Yu A, Zhao C, Coulter JA, Fan Z, et al. 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Plant Cell Environ. 2020;43:1259\u0026ndash;72.\u003c/li\u003e\n \u003cli\u003eScafaro AP, Fan Y, Posch BC, Garcia A, Coast O, Atkin OK. Responses of leaf respiration to heatwaves. Plant Cell Environ. 2021;44:2090\u0026ndash;101.\u003c/li\u003e\n \u003cli\u003eHosseini SH, Rahemi Karizaki A, Nakhzari Moghadam A, Biabani A, Taliei F. Light interception and radiation use efficiency in quinoa (\u003cem\u003eChenopodium quinoa Willd.\u003c/em\u003e). Crop Sci. 2023;63:2438\u0026ndash;49.\u003c/li\u003e\n \u003cli\u003eRalph J. Tricin and tricin-lignins in Medicago versus in monocots. New Phytol. 2020;228:11\u0026ndash;4.\u003c/li\u003e\n \u003cli\u003eLiu X, Rahman T, Song C, Yang F, Yang W. Relationships among light distribution, radiation use efficiency and land equivalent ratio in maize-soybean strip intercropping. Field Crops Res. 2018;224:91\u0026ndash;101.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003e\u003cstrong\u003eTable 1\u0026nbsp;\u003c/strong\u003eThree factors of maize yield under different treatments\u003c/p\u003e\n\u003cdiv align=\"Left\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"627\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTillage initiatives\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePlanting pattern\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIrrigation amounts\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eEar numbers\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(ear m\u003csup\u003e-2\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 133px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eKernel number per spilk\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1000-kernel weight (g)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"6\" style=\"width: 83px;\"\u003e\n \u003cp\u003eNT\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 75px;\"\u003e\n \u003cp\u003eIM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003eI1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e8.1\u0026plusmn;0.12 d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 133px;\"\u003e\n \u003cp\u003e562.0\u0026plusmn;18.6 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e436.0\u0026plusmn;7.5 d\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003eI2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e9.5\u0026plusmn;0.21 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 133px;\"\u003e\n \u003cp\u003e643.5\u0026plusmn;21.2 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e489.1\u0026plusmn;18.6 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003eI3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e9.6\u0026plusmn;0.06 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 133px;\"\u003e\n \u003cp\u003e652.2\u0026plusmn;17.0 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e194.6\u0026plusmn;5.0 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 75px;\"\u003e\n \u003cp\u003eSM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003eI1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e7.4\u0026plusmn;0.15 e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 133px;\"\u003e\n \u003cp\u003e528.3\u0026plusmn;6.9 d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e410.7\u0026plusmn;13.2 f\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003eI2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e9.0\u0026plusmn;0.10 bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 133px;\"\u003e\n \u003cp\u003e586.9\u0026plusmn;14.9 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e461.9\u0026plusmn;16.5 bc\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003eI3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e9.1\u0026plusmn;0.20 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 133px;\"\u003e\n \u003cp\u003e619.7\u0026plusmn;19.3 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e463.0\u0026plusmn;21.6 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"6\" style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eCT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 75px;\"\u003e\n \u003cp\u003eIM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003eI1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e7.5\u0026plusmn;0.15 e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 133px;\"\u003e\n \u003cp\u003e523.5\u0026plusmn;24.4 d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e413.9\u0026plusmn;8.6 ef\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003eI2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e8.8\u0026plusmn;0.12 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 133px;\"\u003e\n \u003cp\u003e620.8\u0026plusmn;33.0 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e460.2\u0026plusmn;12.7 bc\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003eI3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e9.1\u0026plusmn;0.17 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 133px;\"\u003e\n \u003cp\u003e631.5\u0026plusmn;21.6 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e465.5\u0026plusmn;18.8 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 75px;\"\u003e\n \u003cp\u003eSM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003eI1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e7.0\u0026plusmn;0.10 f\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 133px;\"\u003e\n \u003cp\u003e492.1\u0026plusmn;20.7 e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e388.5\u0026plusmn;7.0 g\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003eI2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e8.3\u0026plusmn;0.26 d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 133px;\"\u003e\n \u003cp\u003e583.4\u0026plusmn;16.4 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e343.4\u0026plusmn;13.5 de\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003eI3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e9.0\u0026plusmn;0.10 bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 133px;\"\u003e\n \u003cp\u003e617.9\u0026plusmn;9.0 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e440.6\u0026plusmn;10.0 cd\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\" style=\"width: 127px;\"\u003e\n \u003cp\u003eVariance analysis\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" style=\"width: 105px;\"\u003e\n \u003cp\u003eTillage initiatives (T)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 133px;\"\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" style=\"width: 105px;\"\u003e\n \u003cp\u003ePlanting pattern (P)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 133px;\"\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" style=\"width: 105px;\"\u003e\n \u003cp\u003eIrrigation amounts (I)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 133px;\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" style=\"width: 105px;\"\u003e\n \u003cp\u003eT\u0026times;P\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 133px;\"\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" style=\"width: 105px;\"\u003e\n \u003cp\u003eT\u0026times;I\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 133px;\"\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" style=\"width: 105px;\"\u003e\n \u003cp\u003eP\u0026times;I\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 133px;\"\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" style=\"width: 105px;\"\u003e\n \u003cp\u003eT\u0026times;P\u0026times;I\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 133px;\"\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eNote: NT and CT represent no-tillage and conventional tillage, respectively. IM and SM were intercrop and monocrop maize, respectively. I1, I2, and I3 represent low, medium, and high irrigation amount, respectively. Differences between treatments are denoted by different lowercase letters, with ** and * indicating significant effects of the experimental factors on the parameter at the 0.01 and 0.05 significance levels, respectively, while NS denotes no significant effect of the experimental factors on the parameter.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Intercropped maize, Photosynthetic physiology, C₄ enzymes, Yield, No-tillage, Irrigation","lastPublishedDoi":"10.21203/rs.3.rs-7635468/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7635468/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eThe Hexi Oasis irrigation area is endowed with abundant light and heat resources, making it suitable for intercropped maize systems. However, after the traditional \u0026ldquo;high-water-consumption\u0026ndash;high-yield\u0026rdquo; pathway under conventional tillage and full irrigation was disrupted, studies on how photosynthetic physiology and the expression of key C₄ enzymes synergistically compensate for yield losses under limited water supply remain scarce.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eTo address this gap, a field experiment was conducted in 2024 to systematically evaluate the coupled effects of tillage (no-tillage, NT; conventional tillage, CT), cropping pattern (intercropping, IM; monocropping, SM), and three irrigation regimes (low, I1; medium, I2; high, I3) on maize grain yield, photosynthetic physiology, and key enzyme regulation.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eThe results showed that the NT \u0026times; IM \u0026times; I2 combination achieved a grain yield of 12,400 kg\u0026middot;hm⁻\u0026sup2; at 14% moisture in 7 m\u0026sup2; yield plots, representing significant increases of 10.5% and 27.2% compared with CTIMI2 and NTSMI2, respectively, while reducing irrigation by 8.3% relative to I3. During the silking\u0026ndash;grain filling stage, this treatment maintained the highest SPAD, Pn, Gs, and Y(II), along with the lowest Ci and Y(NO). Enzyme activities of phosphoenolpyruvate carboxylase, ribulose-1,5-bisphosphate carboxylase/oxygenase, and pyruvate phosphate dikinase increased by 6\u0026ndash;11%, 8\u0026ndash;10%, and 9\u0026ndash;14%, respectively, with corresponding gene expression upregulated by 31\u0026ndash;80%. Structural equation modeling indicated that the standardized path coefficient and explained variance of the photosynthesis\u0026ndash;enzyme coupling on yield reached 0.977 and 94.8%, respectively.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eIn summary, no-tillage combined with moderate irrigation enhanced intercropped maize yield stability under limited water supply through a dual mechanism of \u0026ldquo;photosynthetic performance maintenance\u0026thinsp;+\u0026thinsp;C₄ enzyme activity/transcription enhancement.\u0026rdquo;\u003c/p\u003e","manuscriptTitle":"Coupling photosynthetic physiology and C₄ enzyme regulation enhances grain yield of intercropped maize under no-tillage and moderate irrigation in oasis regions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-07 15:11:35","doi":"10.21203/rs.3.rs-7635468/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e8665640-f04c-4823-aecc-97fefe4eb39a","owner":[],"postedDate":"October 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-10T13:53:52+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-07 15:11:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7635468","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7635468","identity":"rs-7635468","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}