Mulched Drip Fertigation with Growth Inhibitors Reduces Bundle-Sheath Cell Leakage and Improves Photosynthesis Capacity and Barley Production in Semi-Arid Regions

A better understanding of the factors that reduce bundle-sheath cell leakage to CO2 (Փ), enhance 13C carbon isotope discrimination, and enhance the photosynthetic capacity of barley leaves will be useful to develop a nutrient- and water-saving strategy for dry-land farming systems. Therefore, barley plants were exposed to a novel nitrification inhibitor (NI) (3,4-dimethyl-1H-pyrazol-1-yl succinic acid) (DMPSA) and a urease inhibitor (UI) (N-butyl thiophosphorictriamide (NBPT)) with mulched drip fertigation treatments, which included HF (high-drip fertigation (370 mm) under a ridge furrow system), MF (75% of HF, moderate-drip fertigation under a ridge furrow system), LF (50% of HF, low-drip fertigation under a ridge furrow system), and TP (traditional planting with no inhibitors or drip fertigation strategies). The results indicated that the nitrification inhibitor combined with mulched drip fertigation significantly reduced bundle-sheath cell leakage to CO2 (Փ) as a result of increased soil water content; this was demonstrated by the light and CO2 response curves of the photosynthesis capacity (An), the apparent quantum efficiency (α), and the 13C-photosynthate distribution. In the inhibitor-based strategy, the use of the urease and nitrification inhibitors reduced Փ by 35% and 39% compared with TP. In the NI-HF strategy, it was found that barley could retain the maximum photosynthesis capacity by increasing the leaf area index (LAI), An, rubisco content, soluble protein, dry matter per plant, and productivity. The CO2 and light response curves were considerably improved in the NI-HF and NI-MF treatments due to a higher 13C carbon isotope (Δ‰), respiration rate (Rd), and Ci/Ca, therefore obtaining the minimum Փ value. With both inhibitors, there was a significant difference between HF and LF drip fertigation. The NI-MF treatment significantly increased the grain yield, total chlorophyll content, WUE, and NUE by 52%, 47%, 57%, and 45%, respectively. Collectively, the results suggest that the new nitrification inhibitor (DMPSA) with HF or MF mulched drip fertigation could be promoted in semi-arid regions in order to mitigate bundle-sheath cell leakage to CO2 (Փ), without negatively affecting barley production and leading to the nutrient and water use efficiency of barley.


Introduction
The shortage of irrigation water constrains crop production worldwide, and, with the projected climate change, its impacts will become more significant in the near future [1].To cope with the water shortage, it is necessary to develop water-saving agriculture countermeasures, thereby producing more crops per drop.Rainwater is the main factor influencing barley production in the rain-fed areas of Northwest China [2].Water shortages in semi-arid regions are also a severe ecological issue [3].The shortage of irrigation water is the main constraint on worldwide crop productivity.Mulched drip fertigation with inhibitors is the best nutrient-and water-saving farming approach, widely used in dry-land farming systems [1].Plastic film mulching is a technique that reduces soil water loss via evaporation, with the objectives of reducing water stress and improving crop production [4].As the global population continues to grow, the demand for food is increasing, and the use of fertilizers is also increasing in farming systems [5].Due to the high demand for water and fertilizers, especially nitrogen fertilizers, barley cultivation may result in significant nitrogen losses due to NH 3 evaporation and nitrate leaching [6][7][8].Finding the best management practices that lead to lower N losses while maintaining yields is therefore crucial in barley cropping areas to ensure both the economic and environmental sustainability of these agro-ecosystems.
Many physiological processes have an effect on crop yields, in which photosynthesis is the foundation of dry matter accumulation.Drought stress during the flowering stage has a deleterious effect on the photosynthetic capacity due to leaf senescence and photosynthesis reduction [9].The senescence of leaves speeds up the degradation of stored dry matter, resulting in decreased ∆ 13 C and negatively affecting the net photosynthetic rates of crops [10,11].Understanding the effect of mulched drip fertigation with two novel nitrification and urease inhibitors on yields through photosynthesis will help to increase crop production [2].Nitrification and urease inhibitors [12,13], water-saving drip fertigation approaches [14], and distributed nitrogen fertilization have been used to improve the management of the nitrogen supply and barley production [15], resulting in increased yields, reduced water consumption, and reduced agricultural costs [8].Drip irrigation is often used for plant production in water-poor areas [14].Mulched drip fertigation is combined with inhibitors to achieve the integrated fertigation of water and fertilizers, save irrigation water, reduce the evapotranspiration (ET) rate, and improve production and resource use efficiency [16,17].Therefore, it is necessary to study the effects of nitrification and urease inhibitors on photosynthetic capacity and water use efficiency and the impact of drip irrigation on the ∆ 13 C and barley yield.
Basic photosynthesis parameters are associated with leaf chlorophyll content and the bundle-sheath cell leakiness of CO 2 (Φ).There is a considerable relationship between the leaf photosynthesis capacity and chlorophyll content [18].However, it remains unclear how various mulched drip fertigation treatments with nitrification and urease inhibitors affect this relationship.Moreover, the carbon assimilation rates of barley leaves may be considerably improved by slight improvements in the soil water content [19].The use of urease inhibitors is an effective strategy to mitigate Φ [20].Nitrification inhibitors have been defined as suitable tools to improve the nitrogen utilization efficiency, thereby reducing nitrogen loss [21,22], which can also improve photosynthesis, the chlorophyl content, and the barley yield [13].In addition, in C 4 crops, the ratio of the carboxylation rate of phosphoenolpyruvate (PEP) to the decarboxylation acid rate of C 4 to the bundle-sheath leakage of the CO 2 rate is in equilibrium, maximizing the C 4 photosynthesis capacity [23,24], indicating a negative correlation between PEP and photosynthesis [25,26].Research on C 4 grasses and crops has shown that Φ may be considerably influenced by environmental stress, such as rainwater stress, thus considerably increasing Φ [27,28].However, so far, it is not clear whether it is influenced by nutrition and water-saving management strategies, such as mulched drip irrigation using nitrification and urease inhibitors [29].
In view of these challenges, there is limited research on the synergistic influence of urease (NBPT) and nitrification (DMPSA) inhibitors in mulched drip fertigation strategies.Mulched drip fertigation is a modern and innovative irrigation method that uses a drip fertigation system to apply fertilizer and irrigation water together, with the aim of improving the photosynthetic CO 2 and light response curves, carbon isotope discrimination, nitrogen use efficiency (NUE), and water use efficiency (WUE); reducing CO 2 sheath cell leakage (Φ) and surface evaporation; and increasing barley production.Still, it is unknown whether ∆ 13 C is affected by water-saving agricultural strategies, such as mulched drip fertigation with nitrification and urease inhibitors, where the barley crop is exposed to moderate water stress.The specific aims of this study were to reveal the effect of mulched drip fertigation on physiological processes, CO 2 sheath cell leakage (Φ), and production and to improve the photosynthesis capacity of barley using two novel nitrification and urease inhibitors.These mechanisms are examined in semi-arid areas where the photosynthesis capacity of barley leaves and Φ are affected by various nutrient-and water-saving agricultural strategies.

Dry Matter Partitioning and 13 C-Photosynthates in Different Organs
The synergistic influence of the urease (NBPT) and nitrification (DMPSA) inhibitors under the mulched drip fertigation strategies considerably affected dry matter partitioning to various organs under different growth stages (Table 1).At the maturity stage, the highest dry matter partitioning plant −1 was noted under the NI-HF treatment.There were significant effects of the mulched drip fertigation strategies on the amount of dry matter (g plant −1 ) at various growth stages of barley.On average, the data showed that the dry matter partitioning (g plant −1 ) was higher for NI-HF, and the lowest was noted with the TP planting.Compared with UI-HF, the dry matter (g plant −1 ) was considerably improved by 25.4% in the NI-HF treatment and 23.2% in NI-MF, whereas, in NI-HF, it was enhanced by 71.6%, compared with TP planting at the maturity stage.The application of urease (NBPT) and nitrification (DMPSA) inhibitors under mulched drip irrigation changed the distribution of 13 C photosynthetic patterns among several plant organs (Table 2).During physiological maturity, the enrichment rate of 13 C is relatively high in the stems, grains, spikes, and leaves of barley crops.The average data show that under NI-HF treatment, the distribution of 13 C photosynthesis in grains was the highest, followed by UI-HF treatment, and the distribution was the smallest under TP treatment.Compared with the UI-HF treatment, the distribution of 13 C photosynthesis in the grains of NI-HF treatment and NI-MF treatment significantly increased by 10.1% and 4.2%, respectively, while that in NI-HF treatment increased by 30.0%compared to TP treatment.In addition, traditional planting and no drip irrigation and fertilization significantly reduced the photosynthetic distribution pattern of 13 C in the leaves and stems, Plants 2024, 13, 239 4 of 18 while mulched drip irrigation and fertilization under urease (NBPT) and nitrification (DMPSA) inhibitors considerably enhanced the 13 C in the stems and spikes.Under both urease (NBPT) and nitrification (DMPSA) inhibitors with mulched drip fertigation, the C i /C a concentration ratio improved considerably (Figure 1).In the NI-HF treatment, the C i /C a was considerably improved by 14.8% compared to TP treatment.The mean of the two years' data indicated that the C i /C a of the NI-MF and UI-HF treatments was enhanced by 7.7% and 11.1% compared to TP treatment.The Ci/Ca of the NI-LF treatment was improved considerably by 7.2%, and that in UI-MF was improved by 6.5%, compared with the TP treatment.Compared with the TP treatment, in the NI-HF treatment, (∆ 13 C) carbon isotope discrimination was considerably enhanced by 29.1%.Compared with TP treatment, the NI-HF, NI-MF, NI-LF, UI-HF, UI-MF, and UI-LF treatments significantly increased ∆ 13 C by 29.1%, 20.4%, 18.2%, 22.5%, 18.9%, and 12.7%, respectively.Under the TP treatment, the leakage of bundle-sheath cells to CO 2 (Φ) was 35

Respiration Rate and Apparent Quantum Efficiency (α)
For the apparent quantum efficiency (α), in the research years of 2020-2021 and 2021-2022, there were considerable changes under mulched drip irrigation combined with urease (NBPT) and nitrification (DMPSA) inhibitor strategies (Table 3).Compared to TP treatment, in the NI-HF and UI-HF treatments, the α increased by 17.1% and 12.6%, respectively, while that in the NI-MF treatment increased by 9.9%.Compared to TP treatment, at the grain filling stage, the average α values of the NI-HF and UI-HF treatments increased by 12.8% and 8.8%, respectively.The two-year average data show that under the NI-HF and TP treatments at different growth stages, the α also decreased from 0.056 to 0.044 and from 0.046 to 0.039, respectively.Compared with TP treatment, the NI-HF treatment increased the average respiratory rate (Rd) by 33.6%, 24.0%, and

Respiration Rate and Apparent Quantum Efficiency (α)
For the apparent quantum efficiency (α), in the research years of 2020-2021 and 2021-2022, there were considerable changes under mulched drip irrigation combined with urease (NBPT) and nitrification (DMPSA) inhibitor strategies (Table 3).Compared to TP treatment, in the NI-HF and UI-HF treatments, the α increased by 17.1% and 12.6%, respectively, while that in the NI-MF treatment increased by 9.9%.Compared to TP treatment, at the grain filling stage, the average α values of the NI-HF and UI-HF treatments increased by 12.8% and 8.8%, respectively.The two-year average data show that under the NI-HF and TP treatments at different growth stages, the α also decreased from 0.056 to 0.044 and from 0.046 to 0.039, respectively.Compared with TP treatment, the NI-HF treatment increased the average respiratory rate (Rd) by 33.6%, 24.0%, and 20.5%.Compared with TP treatment, the mean Rd of the NI-HF and UI-HF treatments in the grain filling stage increased by 42.0% and 31.6%.

Soil Water Content (SWC) and Chlorophyll Content
The mulched drip fertigation strategies had significant effects on rainfall collection and enhanced SWC at the 0-200 cm layers compared to traditional planting with no inhibitors and drip fertigation (Figure 2).The SWC at 0-200 cm under the NI-HF treatment was significantly different at various growth stages.At the flowering and grain filling stages, the SWC at the 0-200 cm depth improved considerably under both urease (NBPT) and nitrification (DMPSA) inhibitors with mulched drip fertigation, while, under the UI-LF and TP treatments, the SWC gradually decreased as compared to the rest of the treatments.In addition, the nitrification (DMPSA) inhibitor with high drip mulched fertigation increased the SWC.Furthermore, at the flowering stage, the SWC under NI-HF and UI-HF was considerably improved by 55.1% and 49.4% compared to the TP treatment.The mulched drip fertigation in combination with urease (NBPT) and nitrification (DMPSA) inhibitor strategies considerably improved the Chl ab content and chlorophyll stability index (CSI) of barley flag leaves.In both urease (NBPT) and nitrification (DMPSA) inhibitor with mulched drip fertigation strategies, the Chl ab content and CSI of barley improved with increasing mulched drip fertigation, while the CSI was considerably reduced with increasing mulched drip fertigation with both urease and nitrification inhibitors (Table 4).However, when mulched drip fertigation increased from moderate to high, there was no significant variance noted in the Chl ab content and CSI under both urease and nitrification inhibitors.The mean data from both years revealed that the NI-HF, NI-MF, NI-LF, UI-HF, UI-MF, and UI-LF treatments considerably improved with increasing mulched drip fertigation with both urease and nitrification inhibitors (Table 4).However, when mulched drip fertigation increased from moderate to high, there was no significant variance noted in the Chl ab content and CSI under both urease and nitrification inhibitors.The mean data from both years revealed that the NI-HF, NI-MF, NI-LF, UI-HF, UI-MF, and UI-LF treatments considerably improved the Chl ab content by 48.6%, 46.8%, 32.9%, 42.5%, 39.7%, and 20.3% compared to the TP treatment.

Soluble Protein, Rubisco Content, and Leaf Area Index (LAI)
In the two-year study, the soluble protein content in the leaves increased from 0 DAF to 40 DAF under the NI-HF and UI-HF treatments.The difference between the NI-HF and UI-HF treatments was significant from 0 to 32 DAF, while the change between NI-MF and TP was considerable between 0 and 40 DAF (Figure 3).In addition, from 0 to 40 DAF, the NI-HF and NI-MF treatments led to higher rubisco content in the leaves compared to all other treatments, and the NI-HF treatment showed the greatest improvement during the two-year study period (Figure 4).These results showed that, after flowering, mulching drip irrigation with urease (NBPT) and nitrification (DMPSA) inhibitors increased the content of soluble protein and rubisco in leaf photosynthesis, which was helpful for the accumulation of photosynthesis products and improved the photosynthetic capacity.
Under different strategies of mulched drip irrigation combined with urease (NBPT) and nitrification (DMPSA) inhibitors, the LAI from 2020 to 2022 was significantly higher (Figure 5).Under NI-HF, there were significant variations in the LAI of barley crops at various growth stages.The change in LAI started at 60DAP and reached its maximum at 100 DAP; it then gradually decreased at 100-140 DAP due to leaf senescence, indicating that early leaf senescence occurs due to inadequate rainwater.Under the action of urease and nitrification inhibitors, except for 30 DAP, different mulching drip irrigation fertilization strategies had a significant impact on the LAI.For example, the NI-HF treatment increased the LAI on different days after planting.However, the difference between the NI-MF and UI-HF treatments was not significant.from 0 to 40 DAF, the NI-HF and NI-MF treatments led to higher rubisco content in the leaves compared to all other treatments, and the NI-HF treatment showed the greatest improvement during the two-year study period (Figure 4).These results showed that, after flowering, mulching drip irrigation with urease (NBPT) and nitrification (DMPSA) inhibitors increased the content of soluble protein and rubisco in leaf photosynthesis, which was helpful for the accumulation of photosynthesis products and improved the photosynthetic capacity.Under different strategies of mulched drip irrigation combined with urease (NBPT) and nitrification (DMPSA) inhibitors, the LAI from 2020 to 2022 was significantly higher (Figure 5).Under NI-HF, there were significant variations in the LAI of barley crops at various growth stages.The change in LAI started at 60DAP and reached its maximum at 100 DAP; it then gradually decreased at 100-140 DAP due to leaf senescence, indicating that early leaf senescence occurs due to inadequate rainwater.Under the action of urease and nitrification inhibitors, except for 30 DAP, different mulching drip irrigation fertilization strategies had a significant impact on the LAI.For example, the NI-HF treatment increased the LAI on different days after planting.However, the difference between the NI-MF and UI-HF treatments was not significant.Under different strategies of mulched drip irrigation combined with urease (NBP and nitrification (DMPSA) inhibitors, the LAI from 2020 to 2022 was significantly high (Figure 5).Under NI-HF, there were significant variations in the LAI of barley crops various growth stages.The change in LAI started at 60DAP and reached its maximum 100 DAP; it then gradually decreased at 100-140 DAP due to leaf senescence, indicatin that early leaf senescence occurs due to inadequate rainwater.Under the action of urea and nitrification inhibitors, except for 30 DAP, different mulching drip irrigation ferti zation strategies had a significant impact on the LAI.For example, the NI-HF treatme increased the LAI on different days after planting.However, the difference between th NI-MF and UI-HF treatments was not significant.

Photosynthesis Light and CO2 Response Curve
The light response curve of barley leaves under the combined action of vario mulched drip irrigation fertilization and urease (NBPT) and nitrification (DMPSA) i hibitor strategies is shown in Figure 6.The photosynthetic response curve indicates th

Photosynthesis Light and CO 2 Response Curve
The light response curve of barley leaves under the combined action of various mulched drip irrigation fertilization and urease (NBPT) and nitrification (DMPSA) inhibitor strategies is shown in Figure 6.The photosynthetic response curve indicates the relationships between important photosynthetic parameters, such as A n .The light response curve shows that, due to the different mulched drip irrigation conditions with different combinations of urease and nitrification inhibitors, the dynamic changes in the number of standard stems of A n during barley flowering increased (Figure 7).Under different urease and nitrification inhibitor strategies, the A n of the NI-HF and UI-HF treatments was significantly higher as compared with the rest of the treatments.Compared with the UI-HF and UI-MF treatments, a higher A n was recorded under the NI-HF and NI-MF treatments.Similarly, when the number of standard rods exceeded 1600 µmol m −2 s −1 , the Pn curve became constant.In descending order, the A n at various PAR levels in the different treatments was as follows: NI-HF > UI-HF > NI-MF > UI-MF > NI-LF > UI-LF and TP treatments.We used the C4 photosynthetic model to simulate the CO2 response curve of photosynthesis at the flowering stage of barley flag leaves.The An CO2 response curve indicates that, at the flowering period of barley, with an increase in Ci, drip irrigation fertilization combined with urease and nitrification inhibitor strategies under various mulches, the An rate was significantly increased (Figure 7).When observed under the nitrification inhibitor strategy, the An content in flag leaves treated with NI-HF was the maximum as compared with the rest of the treatments.Compared with the TP treatment, NI-HF and UI-HF indicated the maximum An.Likewise, when Ci exceeded 600 μmol mol −1 , the An curve became constant and saturated.

Resource Use Efficiency and Barley Production
The field study showed that the grain yield, WUE, and NUE of barley were significantly affected by the various mulched drip fertigation strategies in combination with urease (NBPT) and nitrification (DMPSA) inhibitors (Table 4).As the mulched drip fer- We used the C 4 photosynthetic model to simulate the CO 2 response curve of photosynthesis at the flowering stage of barley flag leaves.The An CO 2 response curve indicates that, at the flowering period of barley, with an increase in C i , drip irrigation fertilization combined with urease and nitrification inhibitor strategies under various mulches, the An rate was significantly increased (Figure 7).When observed under the nitrification inhibitor strategy, the A n content in flag leaves treated with NI-HF was the maximum as compared with the rest of the treatments.Compared with the TP treatment, NI-HF and UI-HF indicated the maximum A n .Likewise, when C i exceeded 600 µmol mol −1 , the A n curve became constant and saturated.

Resource Use Efficiency and Barley Production
The field study showed that the grain yield, WUE, and NUE of barley were significantly affected by the various mulched drip fertigation strategies in combination with urease (NBPT) and nitrification (DMPSA) inhibitors (Table 4).As the mulched drip fertigation increased, the grain yield considerably improved, but the differences were non-significant when the irrigation exceeded moderate mulched drip fertigation under both urease (NBPT) and nitrification (DMPSA) inhibitors.In addition, mulched drip fertigation irrigation combined with DMPSA considerably enhanced the NUE.Compared with UI-LF, the NUE of the NI-HF, NI-MF, NI-LF, UI-HF, and UI-MF treatments increased by 51.3%, 45.0%, 28.6%, 39.8%, and 27.0%.Meanwhile, the WUE in the NI-HF, NI-MF, NI-LF, UI-HF, UI-MF, and UI-LF treatments was increased by 59.3%, 56.9%, 44.0%, 48.4%, 44.8%, and 27.7% when compared with TP treatment.The application of the DMPSA inhibitor had the maximum effect on the WUE and NUE of barley.The grain yield in the NI-HF, NI-MF, NI-LF, UI-HF, UI-MF, and UI-LF treatments was increased by 55.8%, 51.5%, 34.5%, 48.7%, 42.1%, and 21.9% compared with TP treatment.
Financial profit is one of the most valuable estimation indexes for the management productivity of barley.The total costs of the different treatments had clear variations due to the use of plastic films, growth inhibitors, and irrigation (Table 5).The main output value for the barley crop was the grain yield.The output value under NI-HF was significantly higher than that of the rest of the treatments.The net incomes with the NI-HF, NI-MF, NI-LF, UI-HF, UI-MF, UI-LF, and TP treatments were 16,897, 14,895, 9295, 13,537, 11,295, 6655, and 4965 (Yuan ha −1 ).Among the various treatments, the net profit of NI-HF was the highest (16897 CNY ha −1 ).

Discussion
Due to water shortages, barley production can be limited by poor photosynthesis in rain-fed regions [30,31].Earlier research works revealed that carbon isotope discrimination (∆ 13 C) plays a vital role in increasing the photosynthesis capacity and reducing the cell leakage of a bundle sheath to CO 2 [23,32].Compared with UI-HF, the dry matter (g plant −1 ) was considerably improved by 25.4% in the NI-HF treatment and 23.2% in NI-MF, whereas the NI-HF treatment enhanced it by 71.6% compared with TP planting at the maturity stage.In addition, traditional planting and drip irrigation and fertilization significantly reduced the photosynthetic distribution pattern of 13 C in the leaves and stems, while mulched drip irrigation and fertilization under urease (NBPT) and nitrification (DMPSA) inhibitors considerably enhanced the 13 C distribution in the spikes and stems.Earlier research has also reported similar results [33][34][35].The timing of deficit irrigation can significantly improve the chlorophyll ab content and photosynthesis capacity and delay leaf senescence [10].Compared to TP treatment, in the NI-HF treatment, the (∆ 13 C) carbon isotope identification was significantly improved by 29.1%.Under TP treatment, bundle sheath cell leakage was sensitive to CO 2 (Φ) and was 35.2% higher than in the NI-HF treatment.The barley leaf carbon ∆ 13 C can reveal the ecological effects of the barley crop Pn [36,37].Ali et al. [1] reported that ∆ 13 C was changed with the influence of a greater ρ and was considerably enhanced under the CF rather than the RB planting technique.Under the NI-HF treatment, Ci/Ca was considerably enhanced by 14.8% compared to the TP treatment.The Ci/Ca of the NI-MF and UI-HF treatments increased by 7.7% and 11.1%, respectively, compared to the TP treatment.The study by Wang et al. [32] showed that the maximum Ci/Ca value can lead to a significant increase in the ∆ 13 C value.Thomas et al. [38] also found that, compared to DI treatment, PRI treatment had a significantly better ∆13C value.In the present research, the estimations of ∆ 13 C and C i /C a are similar to those of other C 4 crops [39,40].
The barley yield is related to the photosynthetic capacity [41] and decreases under rain-fed agricultural systems Φ [38].Compared to TP treatment, the average α of the NI-HF treatment and UI-HF treatment increased by 17.1% and 12.6%, respectively, while that of the NI-MF treatment increased by 9.9%.The two-year average data show that under the NI-HF and TP treatments at different growth stages, α decreased from 0.056 to 0.044 and from 0.046 to 0.039, respectively.Compared with TP treatment, the NI-HF treatment increased the mean Rd by 33.6%, 24.0%, and 20.5%, respectively.Mulching coverage leads to a rise in barley production, which is related to the A n , Rd, and α values [29].Greater P n and α values and a lower Φ are possible causes.Water scarcity decreases CO 2 availability and stomatal conductance, thus decreasing the A n and resource use efficiency [42,43].Numerous studies have confirmed that DMPSA inhibitors can improve the photosynthesis, Rd, and α values [44,45].In irrigation experiments conducted in the same experimental area, Ali et al. [1] found that the impact of NBPT on the soluble protein and rubisco content was largely influenced by irrigation management in the weeks after fertilization, and the impact was negligible at the highest irrigation frequency.In this two-year study, the soluble protein content in the leaves increased from 0 DAF to 40 DAF under the NI-HF and UI-HF treatments.The difference between the NI-HF and UI-HF treatments was significant from 0 to 32 DAF, while the change between NI-MF and TP was considerable between 0 and 40 DAF.The rubisco enzyme in the Calvin cycle accounts for 50% of the soluble protein of leaves [46,47], so it is the most obvious target to improve the photosynthesis of barley [48].These results showed that after flowering, the mulched drip irrigation with NBPT and DMPSA inhibitors increased the rubisco content under leaf photosynthesis, which was conducive to the accumulation of photosynthetic products and the improvement of photosynthesis.
Under the urease (NBPT) and nitrification (DMPSA) inhibitors and film drip irrigation strategies, the chlorophyll ab content and CSI in barley flag leaves increased with the increase in film drip irrigation fertilization, while the CSI decreased significantly with the increase in film irrigation under different urease and nitrification inhibitors.The amount and duration of irrigation considerably affects leaf senescence and increases the chlorophyll content and Pn [49,50].However, when the level of covered drip irrigation fertilization increased from moderate to high, there was insignificant variance in the Chl ab content and CSI under the action of the urease and nitrification inhibitors.Increasing photosynthesis is one of the essential strategies to increase grain yields and biomass [51,52].Previous studies have reported that RF systems play a crucial role in improving the water use efficiency in dry-land agricultural systems [39,49].In this study, in the flowering and filling stages, under the urease (NBPT) and nitrification (DMPSA) inhibitors and the mulched drip irrigation fertilization strategy, the SWC at a 0-200 cm depth significantly increased (p < 0.05), while, under UI-LF and TP, the SWC gradually decreased.The light response curve shows that under the combined action of urease and nitrification inhibitors, the An and PAR of barley during the flowering period increased under different mulching drip irrigation and fertilization conditions.Under different urease and nitrification inhibitor strategies, the A n of the NI-HF and UI-HF treatments was considerably greater.Compared with the UI-HF and UI-MF treatments, a higher A n was recorded under the NI-HF and NI-MF treatments.Similarly, when the PAR exceeded 1600 µmol m −2 s −1 , the A n curve became constant.We used the photosynthesis model developed by [53,54] to simulate the photosynthesis CO 2 response curve at the flowering stage for barley flag leaves.Compared with TP treatment, NI-HF and UI-HF yielded the maximum A n values.Likewise, when C i exceeded 600 µmol mol −1 , the A n curve became constant and saturated.When Ci was 400 µmol mol −1 , the CO 2 response curve of A n in the barley leaves was the maximum [50].Higher ∆ 13 C may also contribute to a higher P n value, barley yield, and photosynthesis with low Φ values [11,54].This is consistent with our research results.
With the increase in the fertilization amount under mulched drip irrigation, the grain yield significantly increases, but when the fertilization amount under drip irrigation exceeds the moderate level of NBPT and DMPSA inhibitors, the yield change is not significant.In addition, the combination of mulched drip irrigation and DMPSA irrigation significantly improves the NUE.Wang et al. [31] found that the slow release of a nitrogen fertilizer can promote the transfer of nitrogen to barley grains.The improvement in water and nutrient utilization efficiency makes drip irrigation fertilization a promising strategy to improve plant nutrition and reduce nutrient losses (such as N) [48,53,55].The WUE in the NI-HF, NI-MF, NI-LF, UI-HF, UI-MF, and UI-LF treatments was increased by 59.3%, 56.9%, 44.0%, 48.4%, 44.8%, and 27.7% when compared with TP treatment.The application of the DMPSA inhibitor had the maximum effect on the WUE and NUE of barley.The grain yield for the NI-HF, NI-MF, NI-LF, UI-HF, UI-MF, and UI-LF treatments was 55.8%, 51.5%, 34.5%, 48.7%, 42.1%, and 21.9% higher compared with TP treatment.The impact of nitrogen on barley yields is indirectly generated by growth effects [52,56].The interaction between deficit irrigation and inhibitors is a key factor affecting barley growth and yields [51, 54,57].Furthermore, urease inhibitors inhibit the hydrolysis rate of amide nitrogen, reduce the volatilization of NH 4 + , prolong nitrogen fertilizer utilization, and ensure a sufficient nutrient supply for yield formation in the later growth stage [1], ultimately improving the NUE, WUE, and barley yield.

Site Description
This field experiment research was conducted in Huangyang Town, Liangzhou District, Wuwei City, Agricultural Science, Gansu Province, in 2020-2021 and 2021-2022, located at a latitude of 37 • 30 ′ N and longitude of 103 • 50 ′ E. The annual sunshine duration was 2360-2920 h, the height above mean sea level was 1766 m, and the frost-free period was 135-150 days.The rainfall in 2020-2021 was 230 mm, and that in 2021-2022 was 200 mm.The monthly rainfall and temperature in 2020-2021 and 2021-2022, as well as the monthly average over 40 years (1980-2020), are shown in Figure 8. Table 6 provides the soil characteristics at a depth of 0-20 cm in farmland over the past two years.

Experimental Design
The on-site study was carried out using a random complete block design (RCBD) with four replicates, each covering an area of 60 square meters (20 × 3 m).The study consisted of seven treatments: NI-HF-nitrification inhibitor (3,4-dimethyl-1H-pyrazole-1-yl succinic acid) (DMPSA) (Zhongsheng Agricultural Science (Shandong) Fertilizer Co., Ltd., Liaocheng City, China) with (370 mm) high drip fertigation irrigation under a ridge furrow system; NI-MF-nitrification inhibitor DMPSA with 75% of H, moderate drip fertigation irrigation under a ridge furrow system; NI-LF-nitrification inhibitor DMPSA with 50% of H, low drip fertigation irrigation under a ridge furrow system; UI-HF-urease inhibitor (N-butyl thiophosphorictriamide) (NBPT) with (370 mm) high drip fertigation irrigation under a ridge furrow system; UI-MF-urease inhibitor NBPT with 75% of H, moderate drip fertigation irrigation under a ridge furrow system; UI-LF-urease inhibitor NBPT with 50% of H, low drip fertigation irrigation under a ridge furrow system; TP-traditional planting with no inhibitor and drip irrigation.Drip irrigation under a plastic film cover was used for fertigation and irrigation.Before sowing, a drip irrigation belt was laid and then the ridge furrow area was covered with plastic film mulching.Each plot was equipped with high-precision water meters (LXS-25, Ningbo, China), pressure gauges, and control valves to ensure accurate discharge and a stable pressure.All experimental plots were irrigated at 50 mm after sowing to achieve uniform germination.Throughout the growing season, single water applications (64, 45, and 27 mm) were applied for the H, M, and L treatments, totaling 5 times.The AZ34 barley variety was sown with a population of 2.25 × 10 6 seeds ha −1 .The seeds were sown on 5 October 2020 and 9 October 2021, with row spacing of 20 cm.Barley was harvested on 19 May 2021 and 21 May 2022.A large space of 0.8 m was reserved between the bordering plots to prevent the leakage of water and nutrients.In these two years, 40 kg N ha −1 , 200 kg P 2 O 5 ha −1 , and 27.5 kg K ha −1 were applied before planting, and 160 and 40 kg ha −1 of N were applied through drip irrigation fertilization during the jointing and flowering stages.

Soil Water Content
The soil moisture content at different growth stages was measured.A TDR machine (Time-Domain Reflectometry, Trase System, Soil Moisture Equipment Corp., Germany) was used to record the soil depth at 0-200 cm, with intervals of 20 cm.

δ 13 C Isotope and Chlorophyll Content
In the mature stage, the biomass of leaves was used for δ 13 C measurement.A fine powder was prepared from dry leaf samples using the Isoprime 100 instrument (Isoprime 100%, Cheadle, UK) for 13 C analysis.
The 13 C composition (δ 13 C) was calculated as where R standard and R sample are the Pee Dee Belemnite (PDB) standard and 13 C/ 12 C ratios of the dry leaf sample.
The carbon isotope ∆ 13 C was calculated according to Farquhar et al. [28].
In addition, five plant samples were obtained from the center of each plot at the maturity stage and subsequently divided into the ear leaf, stem (including sheath), other leaves, cob, ear bracts, tassel, and grain.All separated components were oven-dried at 80 • C to a constant weight.A sub-sample of 4 mg was used to determine the isotopic abundance.The total chlorophyll ab content of barley flag leaves was calculated based on the program and formula explained by von Camemmer and Furbank [29].
where A n is the net photosynthetic rate and x is C i .The light response curves were measured during the same days on which the CO 2 response curves were measured.The different PPFD levels were measured using the LI-Cor LI-6400XT portable photosynthesis system at a CO 2 concentration of 400 µL L −1 .PPFD levels were taken at 1800, 1600, 1400, 1000, 600, 400, 200, 100, 80, 60, 40, 20, and 0 µmol m −2 s −1 .The light response curve was modeled by a non-rectangular hyperbolic model.Photosynthetic parameters derived from the light response curves were determined according to the method described by Ye [30].
Among them, A n is photosynthesis; Q is PPFD; A max is the irradiance saturation rate of total photosynthesis; R d is the dark breathing rate; a is the maximum apparent quantum yield of CO 2 ; J is a dimensionless convexity term (0 < j < 1).The slope of this line is the apparent quantum efficiency (α), and the absolute value of the intercept is the respiratory rate (R d ).

Leaf Area Index (LAI) and Resource Use Efficiency
The LAI was calculated as LAI = GLA × N/S (6) where GLA is the green leaf area, N is the number of plants within a unit area of land, and S is the unit area of land.The nitrogen use efficiency (NUE) was determined as below [33].NUE = (NF − NC)/NA × 100 (7) where NF is the N content in the fertilization plot, NC is the N content in the control plot, and NA is the nitrogen amount.
The soluble protein content in leaves was measured according to Bradford's [34] improved method.As mentioned earlier, the rubisco content in leaves was measured using SDS-PAGE [35].Four rows from each plot were manually harvested to determine the grain yield.

Economic Analysis
The economic benefit for each plot was determined using the following equations:

Statistical Analysis
The data were analyzed for each sampled event separately using the SPSS 22.0 software (SPSS Inc., Chicago, IL, USA) and plotted with Origin Pro 8.5 (Origin Lab Corporation, Northampton, MA, USA).Means and standard errors were calculated for individual measurements taken at each sampling date.To identify significant treatment effects, multiple comparisons were performed with the least significant difference (LSD) test, and the significance level was set at the 0.05 probability level.

Conclusions
Mulched drip fertigation with a new nitrification inhibitor (DMPSA) had a significant impact on the soil water content and bundle-sheath cell leakage to CO 2 (Φ) and enhanced the 13 C-photosynthate distribution in different organs, the photosynthetic capacity, the apparent quantum efficiency (α), and barley production.Under the inhibitor-based strategy,

Figure 1 .
Figure 1.Effects of urease and nitrification inhibitor application with drip irrigation fertigation on the intercellular to ambient CO2 concentration ratio (Ci/Ca), bundle-sheath leakiness to CO2 (Փ), and carbon isotope discrimination (Δ 13 C) during the flowering stage.Vertical bars represent the (means ± SR) (n = 3), while lowercase letters represent LSD values.

Figure 1 .
Figure 1.Effects of urease and nitrification inhibitor application with drip irrigation fertigation on the intercellular to ambient CO 2 concentration ratio (Ci/Ca), bundle-sheath leakiness to CO 2 (Φ), and carbon isotope discrimination (∆ 13 C) during the flowering stage.Vertical bars represent the (means ± SR) (n = 3), while lowercase letters represent LSD values.

Figure 2 .
Figure 2. Dynamic changes in the soil water content (%) at different barley growth stages with 0-200 cm depth under different treatments during 2020-2022.Vertical bars represent the means ± SR (n = 3).

Figure 2 .
Figure 2. Dynamic changes in the soil water content (%) at different barley growth stages with 0-200 cm depth under different treatments during 2020-2022.Vertical bars represent the means ± SR (n = 3).The mulched drip fertigation in combination with urease (NBPT) and nitrification (DMPSA) inhibitor strategies considerably improved the Chl ab content and chlorophyll stability index (CSI) of barley flag leaves.In both urease (NBPT) and nitrification (DMPSA) inhibitor with mulched drip fertigation strategies, the Chl ab content and CSI of barley improved with increasing mulched drip fertigation, while the CSI was considerably reduced

Figure 3 .
Figure 3. Effects of urease and nitrification inhibitor application with drip irrigation fertigation on the soluble protein content at different days after flowering.Vertical bars represent the means ± SR (n = 3).

Figure 3 .
Figure 3. Effects of urease and nitrification inhibitor application with drip irrigation fertigation on the soluble protein content at different days after flowering.Vertical bars represent the means ± SR (n = 3).Plants 2024, 13, x FOR PEER REVIEW 9 of 20

Figure 4 .
Figure 4. Effects of urease and nitrification inhibitor application with drip irrigation fertigation on the rubisco content at different days after flowering.Vertical bars represent the means ± SR (n = 3).

Figure 4 .Figure 4 .
Figure 4. Effects of urease and nitrification inhibitor application with drip irrigation fertigation on the rubisco content at different days after flowering.Vertical bars represent the means ± SR (n = 3).

Figure 5 .
Figure 5. Dynamic changes in the leaf area index (LAI) at different barley growth stages und different treatments during 2020-2022.Vertical bars represent the means ± SR (n = 3).

Figure 5 .
Figure 5. Dynamic changes in the leaf area index (LAI) at different barley growth stages under different treatments during 2020-2022.Vertical bars represent the means ± SR (n = 3).

Plants 2024 ,
13,  x FOR PEER REVIEW 10 of 20 sponse curve shows that, due to the different mulched drip irrigation conditions with different combinations of urease and nitrification inhibitors, the dynamic changes in the number of standard stems of An during barley flowering increased (Figure7).Under different urease and nitrification inhibitor strategies, the An of the NI-HF and UI-HF treatments was significantly higher as compared with the rest of the treatments.Compared with the UI-HF and UI-MF treatments, a higher An was recorded under the NI-HF and NI-MF treatments.Similarly, when the number of standard rods exceeded 1600 μmol m −2 s −1 , the Pn curve became constant.In descending order, the An at various PAR levels in the different treatments was as follows: NI-HF > UI-HF > NI-MF > UI-MF > NI-LF > UI-LF and TP treatments.

Figure 6 .
Figure 6.Dynamic photosynthetic light response curves of barley leaves exposed to urease and nitrification inhibitor application with drip irrigation fertigation during flowering stage (the measurements were made at a CO2 concentration of 400 μL L −1 ).Vertical bars represent the means ± SR (n = 3).

Figure 6 . 20 Figure 7 .
Figure 6.Dynamic photosynthetic light response curves of barley leaves exposed to urease and nitrification inhibitor application with drip irrigation fertigation during flowering stage (the measurements were made at a CO 2 concentration of 400 µL L −1 ).Vertical bars represent the means ± SR (n = 3).Plants 2024, 13, x FOR PEER REVIEW 11 of 20

Figure 7 .
Figure 7. Photosynthetic CO 2 response curves of barley leaves exposed to urease and nitrification inhibitor application with drip irrigation fertigation during flowering stage (measurements were made at a PPFD of 1500 µmol•m −2 •s −1 ).Vertical bars represent the means ± SR (n = 3).
located at a latitude of 37°30′ N and longitude of 103°50′ E. The annual sunshine duration was 2360-2920 h, the height above mean sea level was 1766 m, and the frost-free period was 135-150 days.The rainfall in 2020-2021 was 230 mm, and that in 2021-2022 was 200 mm.The monthly rainfall and temperature in 2020-2021 and 2021-2022, as well as the monthly average over 40 years, are shown in Figure8.Table6provides the soil characteristics at a depth of 0-20 cm in farmland over the past two years.

Figure 8 .
Figure 8. Monthly rainfall and temperature distribution during the barley-growing seasons.

Figure 8 .
Figure 8. Monthly rainfall and temperature distribution during the barley-growing seasons.

Table 1 .
Effects of different treatments on dry matter partitioning (g plant −1 ) in different organs (%) at physiological maturity of barley during 2020-2022.
NI-HF: nitrification inhibitor DMPSA with 370 mm high drip fertigation irrigation under ridge furrow system; NI-MF: nitrification inhibitor DMPSA with 75% of H, moderate drip fertigation irrigation under ridge furrow system; NI-LF: nitrification inhibitor DMPSA with 50% of H, low drip fertigation irrigation under ridge furrow system; UI-HF: urease inhibitor NBPT with 370 mm high drip fertigation irrigation under ridge furrow system; UI-MF: urease inhibitor NBPT with 75% of H, moderate drip fertigation irrigation under ridge furrow system; UI-LF: urease inhibitor NBPT with 50% of H, low drip fertigation irrigation under ridge furrow system; TP: traditional planting with no inhibitor and drip irrigation.TS: tillering stage; RWS: re-wintering stage; JS: jointing stage; FS: flowering stage; MS: maturity stage.Values are given as means, and different lowercase letters indicate significant differences at p ≤ 0.05 level in the same line (LSD test) (n = 3).

Table 2 .
Effects of different treatments on 13C-photosynthates distribution in different organs (%) at physiological maturity of barley during 2020-2022.

Table 3 .
Cont. -HF: nitrification inhibitor DMPSA with 370 mm high drip fertigation irrigation under ridge furrow system; NI-MF: nitrification inhibitor DMPSA with 75% of H, moderate drip fertigation irrigation under ridge furrow system; NI-LF: nitrification inhibitor DMPSA with 50% of H, low drip fertigation irrigation under ridge furrow system; UI-HF: urease inhibitor NBPT with 370 mm high drip fertigation irrigation under ridge furrow system; UI-MF: urease inhibitor NBPT with 75% of H, moderate drip fertigation irrigation under ridge furrow system; UI-LF: urease inhibitor NBPT with 50% of H, low drip fertigation irrigation under ridge furrow system; NITP: traditional planting with no inhibitor and drip irrigation.FS: flowering stage; GFS: grain filling stage.Values are given as means, and different lowercase letters indicate significant differences at p ≤ 0.05 level in the same line (LSD test) (n = 3).

Table 4 .
Effects of different treatments on grain yield (t ha −1 ), total chlorophyll ab (mg g −1 ), and chlorophyll stability index (CSI, %) at average of three different growth stages (jointing, flowering, and grain filling stage), as well as nitrogen use efficiency and water use efficiency (WUE kg mm −1 ) of barley during 2020-2022.
NI-HF: nitrification inhibitor DMPSA with 370 mm high drip fertigation irrigation under ridge furrow system; NI-MF: nitrification inhibitor DMPSA with 75% of H, moderate drip fertigation irrigation under ridge furrow system; NI-LF: nitrification inhibitor DMPSA with 50% of H, low drip fertigation irrigation under ridge furrow system; UI-HF: urease inhibitor NBPT with 370 mm high drip fertigation irrigation under ridge furrow system; UI-MF: urease inhibitor NBPT with 75% of H, moderate drip fertigation irrigation under ridge furrow system; UI-LF: urease inhibitor NBPT with 50% of H, low drip fertigation irrigation under ridge furrow system; TP: traditional planting with no inhibitor and drip irrigation.Values are given as means, and different lowercase letters indicate significant differences at p ≤ 0.05 level in the same line (LSD test) (n = 3).

Table 5 .
Average economic benefits in Chinese yuan (CNY ha −1 ) under different treatments.

Table 6 .
The chemical properties of the soil layers at the experimental site (0-20 cm).

Table 6 .
The chemical properties of the soil layers at the experimental site (0-20 cm).