Legume plants may facilitate Zanthoxylum bungeanum tolerance to extreme rainfall

A complete randomized design was implemented with two watering regimes (extreme rainfall and control) and three different plant combinations (Zanthoxylum bungeanum, Z. bungeanum + Capsicum annum, Z. bungeanum + Glycine max) in order to assess the morphological and physio-biochemical responses of focal and neighbor plants. The results indicated that, extreme rainfall had significantly negative impacts on Z. bungeanum in three intercropping systems. However, intercropping with G. max improved the transpiration rate (Tr) and stomatal conductance (Gs), raised leaf relative water content (LRWC), increased chlorophyll a (Chl a) and carotenoid (Car) content, and enhanced the superoxide dismutase activity (SOD) of Z. bungeanum. After recovery, the Z. bungeanum + G. max mixed culture significantly increased soil NO3−-N, improved the intercellular carbon dioxide concentration (Ci) and Tr, upregulated soluble sugar and proline, and enhanced hydrogen peroxidase activity (CAT). Moreover, the higher root biomass of G. max provided much more nitrogen for Z. bungeanum via the return of organic matter. However, intercropping with C. annum significantly increased active oxygen (ROS). Compared with neighboring species, in intercropping systems, G. max could have improved the tolerance of the focal species Z. bungeanum in response to extreme rainfall and its recovery after extreme rainfall.

As plants provide organic matter to decomposing microbes, the aboveground matter is closely linked with the belowground components. As such, the decomposer subsystem indirectly regulates plant growth and community composition by determining the supply of available soil nutrients 22 . To alleviate the negative effect of climate change, the characteristics of subordinate species are more important for focal species than their plant diversity [23][24][25] . Therefore, we assumed here that, under extreme rainfall, plant species composition might be crucial in promoting the absorption of nourishment and the physiological processes of focal species.
Z. bungeanum belongs to the family Rutaceae and is grown widely in India, North America, Australia and southwestern China 17 . The fruits of Z. bungeanum are medicinal raw materials used for the treatments of toothache and rheumatism and are an important flavoring in Chinese food 26 . It is drought resistant and grows quickly. Due to its important functions, researches into Z. bungeanum has gained increased attention from scientists 27 .
Most crops grow in soils with low nitrogen, which impacts plant nutrient uptake 28,29 . Tree intercropping systems can enhance environmental and economic benefits, for example, increasing land use efficiency and crop yield 30 and, conserving biodiversity 31 and carbon storage 32 . The use of nitrogen-fixing crops can be a strategy to enhance soil fertility 33 . As the third largest crop grown worldwide 34 , soybean is the main source of protein for both humans and animals 35 . Soybeans have the very important feature of being able to establish symbiotic relations with N 2 -fixing soil bacteria 36 . Moreover, soybean has been a well-studied crop with respect to environmental change 37 . For this reason, soybean is the best choice to explain the response of a target plant to environmental change. Hot pepper (Capsicum annuum L.) is a vital cash crop and is culinarily and medicinally important. It is also a non-nitrogen fixing plant that is sensitive to moisture 38 . Furthermore, hot pepper requires low organic fertilizer inputs when compared to soybean.
However, Z. bungeanum is vulnerable to extreme rainfall, which results in declines in yield, quality of fruit and even death 17 . A previous study has indicated that, under extreme rainfall conditions, the leaf nitrogen content of Z. bungeanum in a mixed legume culture was highest compared to other nonlegume mixed cultures 17 . This is because a significant increase in the root biomass of the soybeans enhanced microbial resistance and the N mineralization rate and promoted the nitrogen absorption of the neighboring crop-Z. bungeanum 17 . However, there remains a lack of understanding about the physio-biochemical response of Z. bungeanum to extreme rainfall stress, when grown together in combination with leguminous and nonleguminous plants.
The purpose of this study was to 1) Explore the growth, physiological and biochemical responses of Z. bungeanum in different intercropping systems under extreme rainfall; 2) Evaluate whether legume species can alleviate the negative impact of extreme rainfall on Z. bungeanum. To achieve these goals, we studied the plant growth index, photosynthetic traits, pigment content, free radical and antioxidant enzymes in different systems subjected to extreme rainfall. The present study would benefit the management of sustainable agriculture through the development of a reasonable ecosystem with higher resistance to extreme rainfall.

Result
Soil properties. After the recovery treatment, under previous normal rainfall conditions, the soil NH 4 + -N was higher in the Z-C mixed culture than in the Z monoculture and Z-G mixed culture (P < 0.05). Under the previous rainfall treatment, soil NO 3 − -N was higher in the Z-G mixed culture than in the Z monoculture and Z-C mixed culture (P < 0.05) ( Table 1). Planting systems, previous extreme rainfall, and the interactions of the planting system and previous extreme rainfall significantly affected both soil NH 4 + -N and NO 3 − -N (Table 1).
Nitrogen content of plant leaves. Irrespective of extreme rainfall, when compared with the Z-G mixed culture, the NH 4 + -N content of the Z. bungeanum leaves decreased significantly in the Z monoculture and Z-C mixed culture. Extreme rainfall significantly decreased the NO 3 − -N content of G. max. The two-way ANOVA showed that planting systems, extreme rainfall, and their interaction significantly affected the NH 4 + -N content of the Z. bungeanum leaves ( Table 2).
Focal plant growth and neighbor species biomass. Under normal water conditions, the LRWC of Z. bungeanum decreased significantly in the Z-C culture when compared with the monoculture and Z-G mixed culture (P < 0.05) ( Table 3). Extreme rainfall reduced LRWC and the height of Z. bungeanum in all cultures, and it significantly decreased the aboveground weight of G. max and C. annum (P < 0.01). A two-way ANOVA showed that the planting system significantly affected the LRWC of Z. bungeanum (P < 0.001) and that the extreme rainfall significantly affected the LRWC and height of Z. bungeanum (P < 0.05) ( Table 3).  Table 1. Soil physical and chemical properties after recovery. "Z-G" denotes Z. bungeanum intercropping with G. max, "Z-C" denotes Z. bungeanum intercropping with C. annuum, and "Z" denotes the Z. bungeanum monoculture. Different uppercase letters indicate significant differences among all treatments. ANOVA was used to assess the effects of extreme rainfall on soil properties. *P < 0.05, **P < 0.01, ***P < 0.001, ns nonsignificant (P > 0.05). After recovery treatments, the LRWC of Z. bungeanum in the Z-G mixed culture was higher than that in the Z-C mixed culture and Z monoculture (P < 0.05). The root biomass of G. max previously treated with extreme rainfall was significantly larger than that of the control after recovery (P < 0.05). The previous extreme rainfall significantly reduced the yield of C. annuum and G. max (P < 0.01). A two-way ANOVA showed that after one month of the recovery treatment, the planting system did not have significant effects on the growth of Z. bungeanum (Table 3).

Photosynthetic parameters.
Under the normal rainfall treatment, the C i of Z. bungeanum in Z-C mixed culture decreased significantly in comparison with the Z-G mixed culture and Z monoculture. The extreme rainfall treatment significantly reduced the P n of Z. bungeanum in the Z monoculture, Z-C and Z-G mixed cultures by 12.83%, 25.71% and 22.96%, respectively (P < 0.05). The T r of Z. bungeanum in the Z-C mixed culture was significantly smaller than that in the Z-G mixed culture and Z monoculture under extreme rainfall (P < 0.05). The extreme rainfall treatment significantly decreased the T r of Z. bungeanum in the Z-C mixed culture by 30.27% (P < 0.05). The planting systems significantly affected the P n , G s , C i and T r ; the extreme rainfall significantly affected the P n and T r (P < 0.001). There was a significant interaction effect between the planting system and extreme rainfall on T r ( Table 4). The order of the effects on the photosynthetic parameters of Z. bungeanum is planting system > extreme rainfall > interaction of planting system and extreme rainfall.
After recovery with the previous normal rainfall treatment, the P n of Z. bungeanum of the Z-C and Z-G mixed cultures increased significantly (P < 0.05), while the T r of Z. bungeanum in the Z-C and Z-G mixed cultures decreased significantly in comparison with the Z monoculture (P < 0.05). After recovery with the previously extreme rainfall, the C i of the Z. bungeanum in the Z-C mixed culture was significantly lower than that in the Z-G mixed culture (P < 0.05). Irrespective of whether the previous treatments were of normal or extreme rainfall, the T r of Z. bungeanum in the Z monoculture was significantly higher than that in the Z-C and Z-G mixed cultures (P < 0.05). The planting system significantly affected the P n , C i and T r ; the extreme rainfall significantly affected the  Table 2. Leaf nitrogen content of Z. bungeanum and neighbor crops. "Z-G" denotes Z. bungeanum intercropping with G. max, "Z-C" denotes Z. bungeanum intercropping with C. annuum, and "Z" denotes the Z. bungeanum monoculture. Different uppercase letters indicate significant differences among all treatments. ANOVA was used to assess the effects of extreme rainfall on leaf nitrogen content. *P < 0.05, **P < 0.01, ***P < 0.001, ns non-significant (P > 0.05).  Table 3. Effects on the growth parameters of Z. bungeanum and neighbor crops after treatment and recovery. "Z-G" denotes Z. bungeanum intercropping with G. max, "Z-C" denotes Z. bungeanum intercropping with C. annuum, and "Z" denotes the Z. bungeanum monoculture. "LRWC" denotes leaf relative water content. Different uppercase letters indicate significant differences among all treatments. ANOVA was used to assess the effects of extreme rainfall on plant growth. *P < 0.05, **P < 0.01, ***P < 0.001, ns non-significant (P > 0.05).
SCIEntIfIC RePoRTS | (2018) 8:15996 | DOI:10.1038/s41598-018-34449-w P n , G s and T r . There was a significant interaction effect between the planting system and extreme rainfall on P n and T r (Table 4). Either at the stage of extreme rainfall or at the recovery phase, the T r was the most affected parameter.
Photosynthetic pigments. Under normal rainfall conditions for all cultures, no significant difference was found in the Car content of the Z. bungeanum leaves. Irrespective of normal or extreme rainfall treatments, the Chl a/Chl b of the Z. bungeanum leaves of the Z-C mixed culture and Z monoculture was significantly lower when compared with Z-G (P < 0.05). Under extreme rainfall, in comparison with the Z monoculture, the content of Chl a, Chl b and Car, and Chl a/Chl b of Z. bungeanum in the Z-G mixed culture significantly increased by 52.74%, 24.30%, 31.35% and 23.47%, respectively (P < 0.05). There were no significant differences in the Chl a, Chl b and Car contents, and the Chl a/Chl b of Z. bungeanum between the Z-C mixed culture and Z monoculture.
The planting system significantly affected the Chl a and Car contents and the Chl a/Chl b; the extreme rainfall significantly affected the content of Car, while the planting system and extreme rainfall interactively impacted the content of Chl a and Chl b (P < 0.01). In the extreme rainfall phase, the influence of extreme rainfall and that of the interaction of the planting system and extreme rainfall were more obvious that of the planting system alone ( Table 5). After recovery, the previously extreme rainfall treatment significantly increased the contents of Chl a, Chl b and Car of the Z. bungeanum leaves in the Z monoculture (P < 0.05). There were no significant differences in the Chl a, Chl b, and Car content and Chl a/Chl b of Z. bungeanum in the Z-G between the previously extreme rainfall treatment and control. The Chl a and Chl b contents of Z. bungeanum in the Z-C mixed culture with the previously extreme rainfall treatment increased significantly (P < 0.01), while the Chl a/Chl b significantly decreased. The planting systems significantly affected the Car content (P < 0.001), and the previously extreme rainfall significantly affected the Chl a, Chl b and Car contents, while the interaction of the planting system and extreme rainfall significantly affected the Chl b content and Chl a/Chl b (P < 0.01).

ROS production and lipid peroxidation.
Under normal rainfall, the O 2 −· . production rate of the Z. bungeanum leaves of the Z-G mixed culture decreased significantly (P < 0.05), while the H 2 O 2 content increased significantly compared with the Z-C mixed culture and Z monoculture (P < 0.05) (Fig. 1A,C). The extreme rainfall significantly increased the O 2 −· . production rate of the Z. bungeanum leaves by 9.21%, 12.33% and 16.42% (P < 0.05); raised the H 2 O 2 content by 89.69%, 20.94% and 4.73%; and increased the malondialdehyde (MDA) content by 11.49%, 4.09% and 4.00% in the Z monoculture, Z-C and Z-G mixed cultures, respectively. The two-way ANOVA showed that the planting system and extreme rainfall both significantly affected the O 2 −· . production rate and H 2 O 2 content. The interaction effects of planting systems and extreme rainfall significantly affected H 2 O 2 content (P < 0.001) (Fig. 1A,C).
After recovery with the previously normal rainfall treatment, the O 2 −· . production rate and MDA content of the Z. bungeanum leaves of the Z-C mixed culture increased significantly while the H 2 O 2 content decreased significantly compared with the Z monoculture and Z-G mixed cultures (P < 0.05) (Fig. 1B,D,F). However, after recovery with the previously extreme rainfall treatment, the O 2 −· . production rate of Z. bungeanum increased significantly while, compared with the Z monoculture and Z-G mixed cultures, the H 2 O 2 content decreased significantly in the Z-C mixed culture (P < 0.05). The planting system significantly affected the O 2 −· . production rate and H 2 O 2 content (P < 0.001) (Fig. 1B-D).
Antioxidant stress components. Irrespective of the normal or extreme rainfall treatments, no significant difference of SOD activity in Z. bungeanum leaves was found between the Z monoculture, Z-C and Z-G mixed cultures (Fig. 2A). The extreme rainfall treatment significantly raised the SOD activity of the Z. bungeanum leaves Treatment  Table 4. Effects on the photosynthetic parameters of Z. bungeanum after treatment and recovery. "Z-G" denotes Z. bungeanum intercropping with G. max, "Z-C" denotes Z. bungeanum intercropping with C. annuum, and "Z" denotes Z. bungeanum monoculture. "P n " denotes net photosynthetic, "G s " denotes stomatal conductance, "C i " denotes intercellular carbon dioxide concentration, "T r " denotes transpiration rate. Different uppercase letters indicate significant differences between the control (normal rainfall) treatments; different lowercase letters indicate significant differences between the extreme rainfall treatments. ANOVA was used to assess the effects of extreme rainfall on photosynthetic parameters. *P < 0.05, **P < 0.01, ***P < 0.001, ns non-significant (P > 0.05). in the Z-G mixed culture by 33.21% (P < 0.05). Compared to the Z monoculture, and Z-C under normal rainfall and extreme rainfall treatments, the CAT activity of the Z. bungeanum leaves increased significantly in the Z-G mixed culture (Fig. 2C). Extreme rainfall significantly affected the SOD activity of the Z. bungeanum leaves (P < 0.05), the planting system significantly affected the CAT activity of the Z. bungeanum leaves (P < 0.001) ( Fig. 2A-C).
After recovery under the previously extreme rainfall treatment, the SOD activity of the Z. bungeanum leaves of Z-C was significantly higher than that of Z-G (Fig. 2B). Under the previously normal rainfall condition, the Z monoculture had higher SOD activity compared to the Z-C and Z-G mixed cultures. Irrespective of previously normal or extreme rainfall treatments, the Z-C and Z-G mixed cultures had higher CAT activity than the Z monoculture (Fig. 2D). In the Z-G mixed culture, the previously extreme rainfall treatment significantly increased the CAT activity by 64.71%. The interaction effects of planting systems and extreme rainfall significantly affected the SOD and CAT activity of the Z. bungeanum leaves (Fig. B,D).

Biochemical parameters.
Under normal rainfall, compared to the Z monoculture, the soluble sugar content of the Z. bungeanum leaves increased significantly and the proline content decreased significantly in the Z-C and Z-G mixed cultures (P < 0.05) (Fig. 3A,E). Compared with the Z and Z-G cultures, the soluble protein content of the Z. bungeanum leaves increased significantly in the Z-C culture (Fig. 3C). The extreme rainfall significantly decreased the soluble sugar content of Z. bungeanum in the Z-G mixed cultures. In the extreme rainfall treatment, the proline content of the Z. bungeanum leaves of the Z-C culture increased significantly (P < 0.05), while the soluble protein content of decreased significantly, compared to the control (P < 0.01). The interaction effects of planting systems and extreme rainfall significantly affected the proline content of the Z. bungeanum leaves (P < 0.01) (Fig. 3E).
After recovery with the previously normal rainfall treatment, the soluble sugar content of the Z. bungeanum leaves decreased significantly in the Z-C and Z-G mixed cultures (P < 0.05), and the soluble protein content increased significantly increased in the Z-G mixed culture, compared to the Z monoculture. The proline content increased significantly in the Z-C and Z-G mixed cultures than in the Z monoculture (P < 0.05). After recovery, compared to the control, the previously extreme rainfall treatment significantly increased the soluble sugar and proline content of the Z. bungeanum leaves in the Z-G mixed culture (P < 0.01) (Fig. 3B,F). The planting system significantly affected the soluble sugar, soluble protein and proline content of the Z. bungeanum leaves (Fig. 3B,D,F).

Discussion
Extreme rainfall has been increasing globally and seriously affecting plant growth and yield 39 . The present study indicated that irrespective of the planting system, the LRWC and height of Z. bungeanum decreased under extreme rainfall compared to the control (Table 3). This was due to excessive water causing anoxia in the rhizosphere and hindering nutrient uptake by plants 40 . Many previous studies have shown that the presence of a legume has a positive effect on the growth of neighboring species [41][42][43] . Our results indicated that extreme rainfall did not significantly decreased the LRWC and height of Z. bungeanum in a legume mixed culture (G. max.), and it reduced the height of Z. bungeanum in the nonleguminous mixed culture (C. annuum) ( Table 3). This could be attributed to the higher soil NH 4 + -N and NO 3 − -N in the legume mixed culture (as shown in our previous experiment 17 ). In the Z-G mixed culture, G. max could maintain adequate physiological functioning in wet soils. Its moisture resistance property is associated with the formation of aeration tissue 44-46 . Our study found that the root biomass of G. max increased significantly under extreme rainfall (P < 0.05) ( Table 3). The increase in root biomass provides the basis for the aeration of tissue and the nitrogen-fixing activity of leguminous crop 17,47 . Moreover, under extreme rainfall, the NH 4 + -N of Z. bungeanum leaves was higher in the Z-G mixed culture than in the Z monoculture and Z-C mixed cultures (P < 0.05) (  Table 5. Effects on pigment parameters of Z. bungeanum after treatment and recovery. "Z-G" denotes Z. bungeanum intercropping with G. max, "Z-C" denotes Z. bungeanum intercropping with C. annuum, and "Z" denotes the Z. bungeanum monoculture. "Chl a" denotes chlorophyll a, "Chl b" denotes chlorophyll b, and "Car" denotes carotenoid. Different uppercase letters indicate significant differences among all treatments. ANOVA was used to assess the effects of extreme rainfall on leaf pigment content. *P < 0.05, **P < 0.01, ***P < 0.001, ns non-significant (P > 0.05).
SCIEntIfIC RePoRTS | (2018) 8:15996 | DOI:10.1038/s41598-018-34449-w legumes facilitate focal species nutrient uptake via increasing N 2 fixation 48 . After recovery, the LRWC of the focal species in three mixed cultures decreased compared to before the recovery. This may be due to the large diurnal temperature differences of the experiment site between different periods. Additionally, Z. bungeanum passes into a different growth phase, causing the lower LRWC. Altered precipitation regimes influence plant eco-physiology by enhancing plant photosynthesis, transpiration and the leaf respiration rate 49,50 . Plant photosynthesis is a fundamental biological process and is greatly dependent on precipitation 51 . Previous studies have shown that increased precipitation could enhance plant photosynthesis and ecosystem carbon uptake 52 . An increase in precipitation can also indirectly affect focal species via its influence on the surrounding community 53 . The present study found that extreme rainfall significantly decreased the P n of the focal species in three mixed cultures. Previous studies have revealed that the changes in P n are mainly induced by the stimulation of stomatal conductance under increased precipitation 54 . Our results show that extreme rainfall significantly decreased G s and T r , thereby resulting in the decreased P n of focal species in the Z-C mixed culture. However, the T r of focal species increased significantly in the Z-G mixed culture, which might be due to the formation of root aeration tissue of G. max 44,45,55,56 . After one month of rain recovery treatments, the P n of focal species decreased compared with before recovery. This result may be due to the growth stage changes in Z. bungeanum. Moreover, when compared to August, the photosynthetic radiation in September declined. The P n of the focal species in Z-C was lower compared with its control (P < 0.05). This clearly indicated that the Figure 1. Effects of free radical and MDA content on focal species after extreme rainfall and recovery. The left column represents 30 days of rainfall, the right column represents recovery for 30 days; "Z-G" denotes Z. bungeanum intercropping with G. max, "Z-C" denotes Z. bungeanum intercropping with C. annuum, and "Z" denotes the Z. bungeanum monoculture. Vertical bars show ± SE of the mean (n± SE). Different uppercase letters indicate significant differences between the control (normal rainfall) treatments; different lowercase letters indicate significant differences between the extreme rainfall treatments. "r" denotes extreme rainfall; "s" denotes planting system; "r × s" denotes the interaction of extreme rainfall and planting system; Proportion of explained variance by extreme rainfall and planting system effects and by their interactions (two-way ANOVA). Significant levels: *P < 0.05, **P < 0.01, ***P < 0.001, "ns" no significant. photosynthetic resilience of focal species in Z-C mixed culture was not strong enough. This shows that legumes can alleviate the decline of photosynthetic function in Z. bungeanum. The content of chlorophyll pigments in leaves is an indicator of plant physiological status 57 . Due to degradation of the chloroplast pigment in the Z monoculture and Z-C mixed cultures, extreme rainfall caused significant damage to the focal species. It has been previously demonstrated that, in the absence of any disturbance, pigment value also declines 58 . Nitrogen is critical for the growth and development of crop plants. The literature data shows that, in mixed stands, depending on the legume species and cultivar, from forage legumes to companion grasses, the rates of nitrogen transfer range from 0 to 73% 59 . Nitrogen fertilizer can increase chlorophyll and carotenoid content in the leaves of plants 60 ; Sun et al. 17 found that the soil nitrogen content in the Z-G mixed culture was significantly higher than in the Z monoculture. They also found that the leaf NH 4 + -N and NO 3 − -N content in the focal species of the Z-G mixed culture was significantly higher than that of the Z monoculture and Z-C mixed culture. Moreover, the leaf NH 4 + -N and NO 3 − -N content of G. max significantly decreased 17 . This proved that G. max could promote the nitrogen uptake of focal species. The present study shows that the Chl a, Chl b and Car content of the focal species in the Z-G mixed culture increased significantly under extreme rainfall. After recovery, the pigment content of the leaves of the focal species changed significantly in the Z monoculture and the Z-C mixed culture. This may be because, compared to the Z-G mixed culture, the pigment content of Z. bungeanum in the Z monoculture and Z-C mixed culture did not reach a steady state. This further proved that there was a significantly different impact on P n in Z. bungeanum in the Z-C mixed culture (Table 4). However, in the Z-G mixed culture, there was no significant difference in the Chl a, Chl b, Car and Chl a/Chl b content of the focal species between the normal and extreme rainfall recovery treatments. These results indicate that the intercropping of leguminous crops could stabilize the pigmentation resilience of focal species.
Extreme rainfall causes a reduction in oxygen supply in the soil that further leads the over-production of ROS in plants 5,61 . The dramatic increase in the ROS level triggers protein degradation, lipid peroxidation, and deoxyribonucleic acid (DNA) fragmentation and causes cell death 62 . Our study found that, under extreme rainfall, when compared with their controls, the rates of O 2 −· . production were significantly higher in all cultures. The H 2 O 2 content was significantly higher only in the Z monoculture, and no significant differences were observed in the Z-C and Z-G mixed cultures. Therefore, under extreme rainfall, the MDA content of the focal species increased Figure 2. Effects of leaf SOD and CAT activity on focal species after extreme rainfall and recovery. The left column represents 30 days of rainfall, the right column represents recovery for 30 days; "Z-G" denotes Z. bungeanum intercropping with G. max, "Z-C" denotes Z. bungeanum intercropping with C. annuum, and "Z" denotes the Z. bungeanum monoculture. Vertical bars show ± SE of the mean (n± SE). Different uppercase letters indicate significant differences between the control (normal rainfall) treatments; different lowercase letters indicate significant differences between the extreme rainfall treatments. "r" denotes extreme rainfall; "s" denotes planting system; "r × s" denotes the interaction of extreme rainfall and planting system; Proportion of explained variance by extreme rainfall and planting system effects and by their interactions (two-way ANOVA). Significant levels: *P < 0.05, **P < 0.01, ***P < 0.001, "ns" no significant. . The SOD activity of Z. bungeanum was significantly enhanced in the Z-G mixed culture compared with its control. Moreover, the CAT activity of Z. bungeanum was the highest in the Z-G mixed culture in comparison with the Z-C mixed and Z monoculture cultures. This proved that species specificity plays a very important role in enhancing antioxidant enzyme activities to improve tolerance of focal species. The present findings are in line with a previous study that showed that facilitation should be more common when plants are subject to high abiotic conditions 65 . After recovery, the MDA content of focal species recovered to control levels for all cultures. Moreover, in extreme rainfall recovery treatments, the CAT activity of Z. bungeanum was significantly higher in Z-G compared with its control. That means that the damage from extreme rainfall on Z. bungeanum in the Z monoculture, Z-C and Z-G mixed cultures could be recovered from in 30 days. The present finding gives an indication of the strong tolerance to extreme rainfall of focal species in a legume mixed culture. The left column represents 30 days of rainfall, the right column represents recovery for 30 days; "Z-G" denotes Z. bungeanum intercropping with G. max, "Z-C" denotes Z. bungeanum intercropping with C. annuum, and "Z" denotes the Z. bungeanum monoculture. Vertical bars show ± SE of the mean (n± SE). Different uppercase letters indicate significant differences between the control (normal rainfall) treatments; different lowercase letters indicate significant differences between the extreme rainfall treatments. "r" denotes extreme rainfall; "s" denotes planting system; "r × s" denotes the interaction of extreme rainfall and planting system; Proportion of explained variance by extreme rainfall and planting system effects and by their interactions (two-way ANOVA). Significant levels: *P < 0.05, **P < 0.01, ***P < 0.001, "ns" no significant. Osmoregulation is an important adaptation strategy to external stress 66 . Different patterns of carbohydrate accumulation in the leaves of flooded and control plants suggest that the decrease in water soluble carbohydrates in control plants is caused by the translocation from leaves to other plant organs 67 . Our results show that, under extreme rainfall, the soluble sugar content of focal species in the Z-G mixed culture was higher than that in the Z monoculture and Z-C mixed culture. This may suggest either a higher rate of soluble sugar or a high molecular weight fructan 68 . In addition, proline has several roles in osmotic adjustment, the elimination of ROS, and the maintance of cell redox status under stress 69 . Our study found that, under extreme rainfall the proline content of the focal species increased in the Z-C mixed cultures compared with their controls. This showed that proline accumulation in focal species might have a scavenging function 70 and act as an osmolytes 71 . In recovery treatments, the soluble sugar and proline contents of focal species leaves in the Z monoculture and Z-G mixed culture were significantly increased compared with the control. The accumulation of these osmolytes represents an important adaptive response during the recovery period 72 .

Materials and methods
Study site. The experimental site was located in Mao county eastern Qinghai-Tibet Plateau (31°41′N, 103°53′E, elevation 1686 m). According to meteorological monitoring data from the Mao County Ecological Station of Chinese Academy of Science, the mean annual precipitation in the area is 920 mm, mean annual temperature is 8.9 °C and extreme minimum and maximum temperatures recorded are −11.6 °C, and 32.2 °C, respectively. The total precipitation in August is approximately 90 mm, and it is considered as the month with the most rainfall. According to a previous study, extreme rainfall is expected to increase in this area 73  annuum were planted in April 2015. One Z. bungeanum was grown in the center of each plot, while species of G. max and C. annuum were planted at the same density (0.27 m 2 /individual) in all plots. No additional fertilization was applied after the experiment commenced, and the weeds in each plot were completely removed by hand each week. Z. bungeanum and intercrops were grown under natural rainfall before simulating extreme precipitation. In August 2015, we exposed our plots, to the precipitation treatment at random, in which triplicate plots per system received either normal (control) or extreme rainfall. To avoid external rainfall effects, all plots were kept under rainout shelters during the experimental period (from 1 st August to 30 th September 2015) to control soil moisture. To minimize greenhouse effects, the rainout shelters for each plot were situated 2 m aboveground 75 . Tap water was used to mimic extreme rainfall events, and a watering pot was used to compensate for rain.
Rainfall regimes were designated 76 , based on the average rainfall in the area during August of 3 mm/day (based on the average rainfall data during 1983-2013 from the Mao County Ecological Station of Chinese Academy of Science). This was designed as the control rain regime, while extreme rainfall was designated according to the abnormally high rainfall in August of 9.5 mm/day. Each planting system was first divided into two groups of different treatments: (1) Extreme rainfall (9.5 mm/day) and (2) Mean rainfall (Control, 3.0 mm/day). During the two-month-long experimental period, all the plots were watered in the morning (7-9 am) and evening (6-8 pm). After one month of extreme rainfall and control treatments, the systems were subsequently subjected to one month of recovery with rainfall of 3.0 mm/day. Around all plots, thick PVC panels were inserted to a depth of 0.5 m into the soil to prevent the lateral water movement between the plots and prevent interactions with roots from neighboring plots.

Plant leaf collection.
At the end of each stage, the youngest fully expanded Z. bungeanum, G. max, and C.
annum leaves at the same developmental stage among plots were collected and placed in a liquid nitrogen container. The samples were taken back to the laboratory and stored at −80 °C to determine their physiological and biochemical parameters.
Soil properties analysis. Soil sampling was taken randomly at 50 cm distance from the Z. bungeanum (in 0-10, 10-20, and 20-30 cm depth) in each plot 77 . The estimation of soil moisture content was performed gravimetrically by oven drying (105 °C for 24 h) 20 g of the soil sample. Soil NH 4 + -N and NO 3 − -N were determined with the help of a flow injection auto analyzer (AA3, Bran + Luebbe, Germany).
Plant leaves nitrogen analysis. Approximately 0.1 g fresh leaves was ground and extracted in 1 mL of distilled water for 2.5 h. The NO 3 − -N content of the solution was determined with 10% (w/v) salicylic acid in 96% sulfuric acid. The values were quantified after generating a standard curve 78 . The NH 4 + -N content was determined with the colorimetric assay described by Krom 79 . Analysis of growth and biomass. Plant height (cm) was measured with the help of a measuring tape. The roots, shoots and leaves of neighboring plants were separated after digging the plants out from the soil, weighting, and subjecting them to oven drying at 70 °C for 24 h to measure dry weight. Determination of yield by weighing method.
Determination of leaf relative water content. Leaf relative water content (LRWC) 80  The expanded leaves were collected from each pot as samples and weighed to obtain their fresh weight (FW). Then, the samples were immediately dipped into deionized water, in dark conditions at room temperature. After 12 h, the leaves were weighted to obtain turgor weight (TW) and then subjected to oven drying at 70 °C for 24 h to determine the dry weight (DW). Pigment and Photosynthetic parameter. Chl a, Chl b, and Car were determined using 0.2 g of leaf sample and 5 mL of 100% acetone as a solvent. The samples were placed in dark conditions for 36 h at room temperature, and the extracting solution was determined spectrophotometrically at 662, 645, and 470 nm 81 . The value of Chl a/Chl b was calculated by dividing Chl a by Chl b. The P n , G s , C i and T r were measured for fully expanded leaves at similar development stages with a portable open-flow gas exchange system (LI-6400, LI-COR Inc., USA) during the late morning (9:00-11:00 h). The air relative humidity, CO 2 concentration and photon flux density were maintained at 70-80%, 400 μmol mol −1 and 800 µmol m −2 s −1 respectively in all cases.
Determination of biochemical parameters. The leaves (0.2 DW) were extracted three times with 6 ml of 80% ethanol at 80 °C for 30 min. The resulting supernatant was analyzed for soluble sugar by the modified anthrone method 82 . Proline was extracted with 2 mL of 10% acetic acid and 5 mL of 3% sulfosalicylic acid. The resulting supernatants were analyzed according to a previously described method 83 . Soluble proteins were determined using the Bradford G-250 reagent.
Determination of ROS and lipid peroxidation. The production rate of O 2 −· . was measured by monitoring the nitrite formation from hydroxylamine in the presence of O 2 − 84 . Fresh leaves (0.2 g) were homogenized with 2 mL of 65 mM phosphate buffer (pH 7.8) and centrifuged at 5000 g for 10 min. The incubation mixture contained 0.9 mL of 65 Mm phosphate buffer (pH 7.8), 0.1 mL of 10 mM hydroxylammonium chloride and 1 mL of supernatant. After incubation at 25 °C for 20 min, 17 mM sulphanilamide and 7 mM α-naphthylamine were added to the incubation mixture, and kept at 25 °C for 20 min. The same volume of ethyl ether was added and centrifuged at 1500 g for 5 min. The absorbance wavelength of the aqueous solution was 530 nm. H 2 O 2 was determined by monitoring the absorbance of the titanium-peroxide complex 85 . Fresh leaf tissue (0.2 g) was homogenized with 5 mL of acetone and centrifuged at 3000 g for 10 min. The reaction mixture contained 0.1 mL of titanium reagent (50 μL of 20% titanium tetrachloride in concentrated HCl), 0.2 mL of ammonia and 1 mL of supernatant, and was then centrifuged at 3000 g for 10 min. The resulting precipitate was washed five times with acetone and centrifuged at 10,000 g for 5 min. The precipitate was solubilized in 3 mL of 1 M sulfuric acid (H 2 SO 4 ) and the absorbance was read at 410 nm. The MDA content was determined according to the thiobarbituric acid (TBA) method 86 . Fresh leaf tissue (0.2 g) was homogenized with 2 mL of 50 mM phosphate buffer (pH 7.8) and centrifuged at 12,000 g for 20 min. One milliliter of supernatant was mixed with 3 mL of 20% trichloroacetic acid (TCA) solution containing 2% TBA. The reaction mixture was incubated in a water bath at 95 °C for 30 min, rapidly cooled in an ice bath and then centrifuged at 15,000 g for 10 min. The absorbance was read at 532 and 600 nm. The amount of MDA was calculated using an extinction coefficient of 155 mM −1 cm −1 .
Determination of antioxidant enzyme activities. The SOD activity was determined by the nitroblue tetrazolium (NBT) method 87 . One unit of SOD activity was defined as the quantity of enzyme required for 50% inhibition of NBT reduction at 560 nm. The CAT was determined using the methods of Fu 87 . For CAT, the decomposition of H 2 O 2 was measured by the reduction in absorbance at 240 nm for 1 min. One unit of CAT activity was defined as an absorbance change of 0.01 units per min. Statistical analysis. Statistical analysis was performed using SPSS v.17.0 (SPSS Inc., Chicago, IL). One-way and two-way ANOVA were used to test the effects of planting systems and extreme rainfall on the soil properties and physio-biochemical parameters. Before the ANOVA, the data were checked for normality and the homogeneity of variances. Origin pro 8.5 was used for graphical presentation; error bars represent standard errors, and all data in the figures represent the mean ± SE.

Conclusion
Extreme rainfall significantly impacted the growth and metabolism of Z. bungeanum in the three intercropping systems, especially for the Z monoculture. In Z. bungeanum, a significant increase in ROS (O 2 −· . and H 2 O 2 ) and MDA content was found, while there were no significantly changes between the antioxidative (SOD and CAT) activities and the accumulation of osmoprotectants (proline, soluble sugars and soluble proteins) confirming its weak resistance potential in response to extreme rainfall. However, under extreme rainfall, intercropping with G. max had a significantly positive effect on the antioxidative activities, pigmentation and photosynthesis of Z. bungeanum. Intercropping with C. annum inhibited the growth, had a negative effect on T r , and decreased the CAT activity and soluble protein content of Z. bungeanum. After recovery, the soil NO 3 − -N significantly decreased in the Z-C mixed culture, resulting in a significant decrease in the P n of Z. bungeanum. By increasing its root biomass, G. max enhanced soil NO 3 − -N in the Z-G mixed culture and improved the T r and CAT activity of Z. bungeanum. Legumes could enhance the tolerance of Z. bungeanum to extreme rainfall. The present findings provide baseline information about the effects of neighbor species (leguminous and nonleguminous) on focal species under extreme rainfall stress. However, more detailed studies are required to explore the interactive mechanisms of different agroforestry plant species, which could lead to a better understanding of the possible physiological responses in coping with future climate change.