Role of Glycine max in improving drought tolerance in Zanthoxylum bungeanum

Study site description

The study site, characterized by the Udic Luvisol soils, was located in Mao County (31°41′N, 103°53′E, 1,686 m a.s.l.), in southwestern China, where mean annual temperature is 8.9 °C, lowest mean annual temperature is –11.6 °C, and highest mean annual temperature is 32.3 °C; mean annual precipitation is 920 mm that mainly occurs during the period May–September (data collected at Mao County Ecological Station of Chinese Academy of Science). Extreme drought conditions in this region have increased significantly in recent years (Xia et al., 2018). Permission to conduct the study and collect samples at the field site was obtained from the farmer, Mr. Li Xingfu.

Experimental design

The experiment was conducted from 1−30 August 2015 as a randomized block experiment, comprising three replicates of six treatments applied to 2.6 × 2.6-m plots. The experimental plots were covered with a rainproof shield constructed as a 2 m high steel frame with a transparent plastic roof (Borken et al., 2006); there was a 1-m wide buffer area between each plot and plots were surrounded by plastic partitions to prevent flow of surface and soil water.

Treatments comprised application of average rainfall (control: CK) or extreme drought conditions to (i) Z. bungeanum monoculture (Z); (ii) intercropping of Z. bungeanum and G. max (Z-G) and (iii) intercropping of Z. bungeanum and C. annuum (Z-C). Z. bungeanum seedlings with the same basal stem and plant height were selected as experimental materials and planted in the center of the plots in April 2013, and G. max and C. annuum were evenly planted at a density of 0.27 m⁻² (25 plants) per plot in April 2015; plots were kept weed-free. Extreme drought is defined as continuous precipitation of <0.1 mm day⁻¹ in August (Zhiyuan Wang et al., 2015); thus, the extreme drought treatment comprised no watering. The CK was the average rainfall in August (1983–2013) at the study site of 3.0 mm day⁻¹ (Ng et al., 2015); CK plots were evenly irrigated twice a day (07:00–09:00 and 18:00–20:00, 1–30 August 2015) to allow for water loss through transpiration and evaporation.

On 30 August 2015, height (cm) of the experimental plants was recorded and crown size was measured as the average of two perpendicular transverse widths (Sánchez-González, ICRD & GM, 2007). Fully expanded leaves were sampled from the experimental plants, quickly placed in liquid N₂, and returned to laboratory for storage at −80 ° C prior to analysis.

Leaf relative water content

We measured leaf relative water content (LRWC) using the method described by Galle, Haldimann & Feller (2007) and calculated as LRWC = [(FW-DW)/(TW-DW)] ×100%, where FW is fresh weight of a single, fully expanded leaf per plant and DW is dry weight. After FW of the fully expanded leaves had been recorded, the samples were immediately dipped in distilled water, in the dark, at 4 °C. After 4 h, leaves were weighed to obtain turgor weight (TW), and then dried in an oven for 24 h at 70°; to determine DW.

Pigment and photosynthetic parameters

Pigment was measured from 0.2 g of fresh leaves that were placed in five mL of 100% acetone for 36 h at room temperature in the dark. Then, reflectance of the extracting solution was determined at 662, 645, and 470 nm using a spectrophotometer and calculation of pigment content was based on the method reported by Liu et al. (2014).

Net CO₂ assimilation rate (P_n), stomatal conductance (G_s), intercellular CO₂concentration (C_i), and transpiration rate (T_r) were measured on fully expanded leaves at the end of the experiment between 09:00 and 11:00 hrs using a portable photosynthetic instrument (LI-6400, LI-COR Inc., USA). Photon flux density, CO₂ concentration, and air relative humidity were maintained at 800 µmol m⁻² s⁻¹, 400 µmol mol⁻¹, and 70–80% respectively. Intrinsic water use efficiency (WUE_intr) was determined as WUE_intr = P_n/T_r, and L_s was calculated as L_s = 100% × (C_a–C_i)/C_a.

Soluble sugars, soluble protein, and proline

A supernatant was created from 0.2 g of DW leaves that were extracted three times using 6 ml of 80% ethanol at 80 °C for 30 min. Then, content of soluble sugars was separated with linear gradient from 50 to 150 Mm and determined following the method reported by Quan et al. (2004) and soluble protein content was determined using Bradford G-250 reagent. Proline was extracted from DW leaves using two mL of 10% acetic acid and five mL of 3% sulfosalicylic acid; proline content of the final supernatant was determined using the method described by Liu et al. (2014).

ROS and membrane lipid peroxidation

Superoxide anion (O₂⁻) production rate in leaves was determined from oxidation of hydroxylamine (Zhou et al., 2004) that was extracted from 0.2 g of DW leaves using two mL of 65 mM phosphate buffer (pH 7.8) followed by centrifugation at 5,000 g for 10 min. Then, 0.1 mL of supernatant was incubated at 25 °C for 20 min in 0.9 mL of 65 Mm phosphate buffer (pH 7.8), 0.1 mL of 10 mM hydroxylammonium chloride; next, 17 mM sulphanilamide and 7 mM α-naphthylamine were added to the mixture and incubated again at 25 °C for 20 min. Ethyl ether at the same volume was added to the mixture that was then centrifuged at 1,500 g for 5 min, and absorbance was read at 530 nm using a spectrophotometer (UV 330, Perkin Elmer, USA).

Content of hydrogen peroxide (H₂O₂) was determined by measuring absorbance of the titanium–peroxide complex at 410 nm (Zhou et al., 2004). Fresh leaves (0.2 g) were ground with five mL of acetone and centrifuged at 3,000 g for 10 min; then, 0.1 mL of titanium reagent (50 µL of 20% titanium tetrachloride in concentrated HCl), 0.2 mL of ammonia, and one mL of supernatant were added and the mixture was centrifuged at 3,000 g for 10 min. The resulting precipitate was washed five times with acetone and centrifuged at 10,000 g for 5 min, before it was dissolved in three mL of 1 M H₂SO₄; finally absorbance was measured at 410 nm using a spectrophotometer.

We used the thiobarbituric acid (TBA) method (Zhou, Lam & Zhang, 2007) to determine malondialdehyde (MDA) content. We ground 0.2 g of fresh leaves in two mL of 50 mM phosphate buffer (pH 7.8) that was then centrifuged at 12,000 g for 20 min. Next, one mL of the supernatant was mixed with three mL of 20% trichloroacetic acid solution that contained 2% TBA, and the reaction mixture was incubated in water at 95 °C for 30 min before it was cooled quickly in an ice bath and centrifuged at 15,000 g for 10 min. Absorption at 532 and 600 nm was determined using a spectrophotometer, and MDA content was calculated according to an extinction coefficient of 155 mM⁻¹ cm⁻¹.

Antioxidant enzyme activity

The NBT method (Fu & Huang, 2001) was used to determine superoxide dismutase (SOD) activity, where one unit of SOD activity was defined as the amount of enzyme required for 50% inhibition of NBT reduction at 560 nm. Activity of catalase (CAT) was measured using the method described by Fu & Huang (2001), by determining H₂O₂ decomposition per 1 min at 240 nm absorbance, where one unit of CAT activity was defined as change in absorbance of 0.01 units per min.

Statistical analyses

We used two-way ANOVA to test for effects of cropping system and extreme drought on physio-biochemical parameters, and one-way ANOVA to test effects of all treatments on physio-biochemical parameters. Data were checked for normality and homogeneity of variances prior to ANOVA, and means were compared using Duncan’s test. Statistical analyses were conducted in SPSS v.17.0 (SPSS Inc., Chicago, IL). Presented data are means ± SE.

Morphological traits

Overall, there was a strong reduction in the morphological traits in extreme drought stressed plants compared with the control across the three cropping systems (Table 1). Extreme drought reduced the crown diameter of Z. bungeanum across the three cropping systems (decreases of 60.27, 32.12, and 44.33% in Z, Z-C, and Z-G, respectively), but there was no effect on tree height. Although height and crown diameter of C. annuum and G. max decreased slightly after 30 d of extreme drought stress, there were no significant effects of drought (Table 2).

Leaf relative water content, photosynthetic pigments, and gas exchange

Z. bungeanum LRWC was slightly reduced by extreme drought across the three cropping systems. We found that the LRWC content in Z. bungeanum was significantly lower in the cropping system, regardless of drought conditions, than in the other two systems (Table 1) and LRWC in C. annuum was greater than in G. max (Table 2). There was no effect of extreme drought stress on chlorophyll b (Chl b) or carotenoid (Car) content, but it decreased Chl a content in Z. bungeanum (Table 1). Irrespective of extreme drought, content of Chl a, Chl b, and Car in G. max was greater than in C. annuum (Table 2). Extreme drought led to a decrease in Car in G. max, but there were not effects on Chl a or Chl b (Table 1). In general, extreme drought decreased photosynthesis and increased L_s in Z. bungeanum across the three cropping systems. There were no effects of extreme drought on C_ior G_s in Z. bungeanum across the three cropping systems; however, there were decreases in T_r in Z. bungeanum in Z and Z-C. WUE_intr of Z. bungeanum in Z was greater under extreme drought conditions. There were no effects of extreme drought on photosynthetic parameters in C. annuum or G. max across the three cropping systems (Table 2).

Antioxidant defense system

Extreme drought increased the O²⁻ production rate and H₂O₂ content in Z. bungeanum in the Z and Z-C treatments, but there was no effect in the Z-G treatment. There was no effect of extreme drought stress on MDA content in Z. bungeanum in the three cropping systems (Fig. 1); however, extreme drought stress led to increased SOD activity in Z. bungeanum in Z-C and Z-G and greater CAT activity in Z and Z-G (Fig. 2). Content of H₂O₂ in C. annuum and MDA in G. max were greater under extreme drought conditions, and there was no effect on SOD or CAT activity (Table 2).

Osmolytes and soluble proteins

Content of soluble sugar and proline in Z. bungeanum did not vary with cropping system under extreme drought stress conditions; however, content of soluble protein of Z. bungeanum was reduced in Z-C and Z-G (Table 1). There were no effects of extreme drought conditions on soluble sugar, soluble protein or proline content in C. annuum and G. max (Table 2).

Plant growth, LRWC, pigment content, and gas exchange

Drought is the principal abiotic stress factor that limits agricultural crop productivity, growth, and development by lowering plant nutrient uptake from arid soils due to reduced expansion of plant cells (Ihsan et al., 2016; Luo et al., 2010). As shown in Tables 1 and 2, short-term extreme drought had no effect on height of Z. bungeanum, C. annuum, or G. max, indicating that plant height is a long-term nutrient accumulation process (Table 2). However, we found that extreme drought reduced Z. bungeanum crown diameter in Z, Z-C and Z-G cropping systems (Table 1). Water deficit reduces soil nutrient availability and plant nutrient uptake, and has a negative effects on soil microbe activities (Hu & Schmidhalter, 2001). Our experiment showed that crown diameter in Z. bungeanum is sensitive to extreme drought conditions that will then likely affect levels of light radiation interception.

Leguminous crops may provide additional nutrients to nearby crops, and our previous study (Sun et al., 2019) showed that the Z-G treatment led to lower content of soil and leaf N content than the Z-C and Z treatment. This effect is probably due to the sensitivity of synergetic N fixation to drought during the reproductive stage that leads to reductions in N accumulation in leguminous crops (Serraj, Sinclair & Purcell, 1999), and the greater sensitivity of N₂ fixation to drought stress than N uptake and assimilation in soil (Purcell & Andyking, 1996).

Plant pigment is a receptor of light radiation, and its level of content directly affects intensity of photosynthesis and formation of dry matter (Ghosh et al., 2004). Declines in Chl content commonly occur under drought stress (Bijanzadeh & Emam, 2010; Din et al., 2011), indicating effects on the pigment synthesis pathway or pigment degradation (Batra, Sharma & Kumari, 2014). However, we found there were no effects of extreme drought conditions on pigment content in Z. bungeanum in any of the three cropping systems, indicating that short-term extreme drought conditions did not affect the synthesis of chlorophyll encoded by the Cab gene family (Din et al., 2011). Efficient N nutrition alleviates effects of extreme drought conditions, by maintaining normal crop metabolism, even under low tissue water condition (Abid et al., 2016), and we found that Car content in G. max decreased under extreme drought conditions (Table 2), possibly due to reductions in NH₄⁺-N and NO₃⁻-N absorption.

Leaf growth reflects the physiological state of the plant when there is a water deficit (Parida et al., 2007), and LRWC directly reflects leaf water status. We found that LRWC of Z. bungeanum was lower in the Z monoculture than in the two intercropping systems (Table 1); however, the slight decreases in Z. bungeanum LRWC in Z-C and Z-G indicate that increasing diversity of crops may increase the water-holding capacity of the main crop species. Water-holding capacity directly affects plant metabolism, and leaf water status is closely related to photosynthetic activity (Ramachandra, Chaitanya & Vivekanandan, 2004). Our findings revealed that P_n in Z. bungeanum decreased in the Z monoculture and Z-C intercropping system under extreme drought stress conditions (Table 2), indicating that intercropping with G. max not only enhanced the water holding capacity of Z. bungeanum, but also promoted its photosynthetic capacity under extreme drought stress conditions. A key driver of decline in photosynthetic capacity is stomatal closure (Cornic, 2000); however, it is unclear whether drought inhibits photosynthesis due to stomatal closure or other metabolic impairments (Tezara et al., 1999). We found that extreme drought stress conditions led to increases in L_s in the three cropping systems and reduced levels of C_i in all three species, indicated that the decrease in photosynthetic capacity was related to water holding capacity and stomatal closure. Intercropping with G. max facilitated the maintenance of levels of P_n and G_s in Z. bungeanum under extreme drought conditions, indicating that stable LRWC allows normal photosynthetic activity to continue. Intercrops tend to have high water-use efficiency compared with single cropping (Morris & Garrity, 1993); however, our experiment showed there were high levels of WUE_intr in Z. bungeanum in the Z monoculture _,probably in response to drought. The lack of effect of extreme drought on levels of P_n in Z. bungeanum in the Z-G mixed culture showed that intercropping with G. max may alleviate declines in Z. bungeanum photosynthetic activity under extreme drought stress conditions. We also found that G_s in G. max decreased under extreme drought conditions, indicating that water absorption was blocked that may have reduced competition for soil water between G. max and Z. bungeanum. Supporting our findings, previous work has shown that levels of T_r in G. max decrease under drought stress (He et al., 2017). Extreme drought conditions led to decreases in P_n in Z. bungeanum, but no changes in WUE_intr or G_s in the Z-C intercropping system. However, we found that compared with Z-G, levels of T_r in Z. bungeanum were greater in the Z-C intercropping system, indicating greater water demand in C. annuum and competition for water between C. annuum and Z. bungeanum that likely affected levels of water absorption in Z. bungeanum.

ROS production and antioxidant enzymes

Plants that suffer drought stress accumulate free radicals, including superoxide anion, and hydrogen peroxide (Munné-Bosch & Penuelas, 2003), and sudden rises in ROS production induce DNA breakage, lipid peroxidation, protein degradation, and ultimately, cell death (Apel & Hirt, 2004). Responses of Z. bungeanum to drought stress conditions include increases in MDA content (Duling & Liu, 2010), and in this experiment, we found that Z. bungeanum ROS (O²⁻ and H₂O₂) production and MDA content were greater under extreme drought stress conditions in the Z-C intercropping system than in the control (Fig. 1). Similarly, SOD activity was greater in the Z-C intercropping system, supporting previous findings that elevated levels of SOD activity improve tolerance of oxidative stress (Anikó Mátai & Éva, 2019; Oliveira et al., 2014; Pan, Wu & Yu, 2006) and demonstrating antioxidant defense mechanisms are active in Z. bungeanum under drought conditions. We found that H₂O₂ and MDA content were also greater in C. annuum under extreme drought conditions. In the Z-G intercropping systems, there were no effects of extreme drought on ROS or MDA content in Z. bungeanum; however, SOD activity in Z. bungeanum (Fig. 2) and MDA content in G. max were greater, indicating that G. max facilitates greater drought tolerance in Z. bungeanum than when Z. bungeanum is grown in a monoculture or with C. annuum.

Soluble sugars, proline and soluble proteins

Under normal moisture conditions, content of soluble sugars and soluble protein in Z. bungeanum were found to be greater in the Z-C intercropping system than in the Z monoculture or Z-G intercropping system, while proline content was greater in the Z monoculture than in the Z-G intercropping system. In contrast, under extreme drought conditions, soluble sugars and soluble protein content in Z. bungeanum was greater in the Z monoculture and Z-C intercropping system than in the Z-G treatment. We found that extreme drought conditions had no effect on content of soluble sugars, soluble protein, or proline in C. annuum and G. max.

When osmotic regulation reduces osmotic potential, plants accumulate a variety of osmolytes that allow the absorption of water by cells that is essential during prolonged drought for the maintenance of physiological activity (Farooq et al., 2009; Ramanjulu & Bartels, 2002). Short periods of drought stress increase soluble sugar content in leaves (Haiyan Wang et al., 2015), and our results showed that, under normal moisture conditions, soluble sugar and soluble protein content in Z. bungeanum was greatest in the Z-G intercropping system than the Z monoculture or Z-C treatment, but under extreme drought conditions, they were lower in the Z-G treatment. These results may reflect the drought-mediated limitation of normal utilization and transport of osmotic substances or subsequent hydrolysis of starch (Shubhra Dayal, Goswami & Munjal, 2004). Our results show that osmoregulation in Z. bungeanum under extreme drought conditions was lowest in the Z-C intercropping system.