Exogenous 2-(3,4-Dichlorophenoxy) triethylamine ameliorates the soil drought effect on nitrogen metabolism in maize during the pre-female inflorescence emergence stage

Background Nitrogen (N) metabolism plays an important role in plant drought tolerance. 2-(3,4-Dichlorophenoxy) triethylamine (DCPTA) regulates many aspects of plant development; however, the effects of DCPTA on soil drought tolerance are poorly understood, and the possible role of DCPTA on nitrogen metabolism has not yet been explored. Results In the present study, the effects of DCPTA on N metabolism in maize (Zea mays L.) under soil drought and rewatering conditions during the pre-female inflorescence emergence stage were investigated in 2016 and 2017. The results demonstrated that the foliar application of DCPTA (25 mg/L) significantly alleviated drought-induced decreases in maize yield, shoot and root relative growth rate (RGR), leaf relative water content (RWC), net photosynthetic rate (Pn), stomatal conductance (Gs) and transpiration rate (Tr), and nitrate (NO3−), nitrite (NO2−), soluble protein contents, and nitrate reductase (NR), nitrite reductase (NiR), isocitrate dehydrogenase (ICDH), alanine aminotransferase (AlaAT) and aspartate aminotransferase (AspAT) activities. In addition, the foliar application of DCPTA suppressed the increases of intercellular CO2 concentration (Ci), ammonium (NH4+) and free amino acid contents, and the glutamate dehydrogenase (GDH) and protease activities of the maize. Simultaneously, under drought conditions, the DCPTA application improved the spatial and temporal distribution of roots, increased the root hydraulic conductivity (Lp), flow rate of root-bleeding sap and NO3− delivery rates of the maize. Moreover, the DCPTA application protected the chloroplast structure from drought injury. Conclusions The data show, exogenous DCPTA mitigates the repressive effects of drought on N metabolism by maintained a stabilized supply of 2-oxoglutarate (2-OG) and reducing equivalents provided by photosynthesis via favorable leaf water status and chloroplast structure, and NO3− uptake and long-distance transportation from the roots to the leaves via the production of excess roots, as a result, DCPTA application enhances drought tolerance during the pre-female inflorescence emergence stage of maize. Electronic supplementary material The online version of this article (10.1186/s12870-019-1710-5) contains supplementary material, which is available to authorized users.


Background
Crops are frequently exposed to drought during the growth period because of limited and erratic rainfall patterns due to global climate change, which leads to restrictions on agricultural productivity worldwide [1]. Maize (Zea mays L.), an essential component of global food security, is widely cultivated around the word. The majority of the cultivated area of maize is almost wholly rain-fed and experiences sporadic drought and rewetting cycles [2]. However, maize is considered to be a drought-sensitive crop and loses approximately 1/4 potential yield annually due to drought [3]. By 2050, the world population will reach 9 billion people, resulting in a high demand for maize (projected to double); furthermore, at that time, drought will severely restrict crop growth for more than 50% of the cultivated land [4].
To stabilize and increase global crop production to satisfy the demand of the globally burgeoning population, it is imperative to design agronomic research to improve maize performance under drought stress [5]. The application of plant growth regulators has been considered an effective way to enhance crop drought resistance [6]. Multiple investigations have indicated that a tertiary amine bioregulator known as 2-(3,4-dichlorophenoxy) triethylamine (DCPTA) regulates many aspects of plant development; for example, DCPTA promotes plant growth [7,8], enlarges chloroplast volume [9], enhances photosynthetic enzyme activity [10], accelerates CO 2 fixation [11], and stimulates carotenoid biosynthesis [12]. As far as we know, very few studies of DCPTA have focused on crops, and the effect of DCPTA on crops exposed to soil drought are still unclear.
Nitrogen (N) metabolism is a fundamental process in determining the growth and productivity of plants [13]. After being taken up by root systems, nitrate (NO 3 − ) is converted to nitrite (NO 2 − ) by nitrate reductase (NR), the first step of N uptake and utilization. Subsequently, nitrite (NO 2 − ) is converted to NH 4 + by nitrite reductase (NiR) with reduction-ferredoxin (Fd red ) as an electron donor [14]. Afterward, the ammonium (NH 4 + ), derived from NO 3 − reduction, photorespiration and/or other metabolic processes is assimilated into glutamine by the glutamine synthase/glutamine oxoglutarate aminotransferase (GS/GOGAT) cycle or the alternative glutamate dehydrogenase (GDH) pathway with 2-oxoglutarate (2-OG) and reducing equivalents provided by photosynthesis [15]. Subsequently, glutamate serving as a donor of the amino group is used for the synthesis of other amino acids, which are used for the synthesis of various organic molecules such as chlorophyll, proteins and nucleic acids. The reactions are catalysed by aminotransferases such as alanine aminotransferase (AlaAT) and aspartate aminotransferase (AspAT) [16].
Drought disrupts N metabolism mainly via inhibiting the uptake and/or long-distance transportation of NO 3 − [17], altering the activities of enzymes involved in N metabolism [18], inhibiting amino acid synthesis, and promoting protein hydrolysis [19]. At present, the study of plant growth regulators mainly concentrates on the improvement of photosynthesis and antioxidant systems, and there have been only a limited number of publications related to N metabolism.
Our previous hydroponic trial found that exogenous DCPTA drastically promoted growth under non-stress conditions and mitigated the PEG-simulated drought-induced growth inhibition of maize at the seedling stage by improving photosynthetic capacity [20] and modulating antioxidant system [21]. The present study was conducted to explore whether DCPTA can alleviate soil drought injuries to maize and whether the effects are associated with the modulation of N metabolism.

Effects of DCPTA on yield under soil drought and rewatering conditions
From the time point of maize at the nine-leaf stage, irrigation was stopped for 20 days (during the pre-female inflorescence emergence stage) to form the drought conditions, and then rehydration. Drought stress significantly inhibited the maize yield ( Fig. 1a and b). Compared with the well-watered treatment (plants were irrigated continuously), in the drought treatment, the grain number decreased by 29.23% in 2016 and 33.24% in 2017, and the grain yield decreased by 34.06% in 2016 and by 38.22% in 2017. However, the decrease in maize yield was partially recovered by DCPTA. Compared with the well-watered treatment, in the drought+DCPTA treatment, the grain number decreased by 14.01% in 2016 and by 16.55% in 2017, and the grain yield decreased by 17.98% in 2016 and by 20.54% in 2017. Moreover, the application of DCPTA improved maize yield under well-watered conditions. Compared with the well-watered treatment, in the DCPTA treatment, the grain number increased by 5.97% in 2016, and the grain yield increased by 7.31% in 2016 and by 8.02% in 2017.
Effects of DCPTA on relative growth rate (RGR) of shoot and root under soil drought and rewatering conditions During the drought period, the shoot RGR decreased, the root RGR firstly increased and then decreased, and the RGR of shoot and root recovered during rehydration ( Fig. 2a and   The root hydraulic conductivity, flow rate of root-bleeding sap and NO 3 − concentrations in the root-bleeding sap declined continuously during the drought period and recovered during rehydration ( Fig. 4a- Under well-watered conditions, DCPTA significantly increased root hydraulic conductivity on the 15th, 20th, 25th and 30th days in 2016 and on the 20th and 25th days in 2017; DCPTA significantly increased the flow rate of root-bleeding sap on the 10th, 15th, 20th and 25th days in both 2016 and 2017; DCPTA significantly increased the NO 3 − concentration in root-bleeding sap on the 10th, 15th, 20th, 25th and 30th days in 2016 and on the 10th, 15th, 20th and 25th days in 2017.

Effects of DCPTA on leaf relative water content (RWC) under soil drought and rewatering conditions
The RWC declined continuously over the drought period and recovered during rehydration (Fig. 5). In the drought treatment compared with the control, RWC decreased by 30.88% on day 20 and by 13.08% on day 30 in 2016 and decreased by 37.48% on day 20 and by 21.89% on day 30 in 2017. However, the DCPTA application partially reversed the decline in RWC caused by drought and resulted in a faster recovery of the foliar RWC contents after rehydration. In the drought+DCPTA treatment compared with the control, the RWC decreased by 11.75% on day 20 and by 3. In 2016 and 2017, Ci showed the same variation tendency during the drought period (Fig. 6d). In the drought treatment, Ci declined over days 0-10, subsequently increased over days 10-20, and then decreased after rehydration. In the drought+DCPTA treatment, Ci Effects of DCPTA on chloroplast ultrastructure under soil drought and rewatering conditions Regardless of whether DCPTA was applied, the photosynthetic mesophyll cells of the non-stressed seedlings included a delimited cell wall containing chloroplasts ( Fig. 7a, b, e and f ). These chloroplasts had intact membranes and a regular arrangement of granal and stromal thylakoids, which were attached to the cell wall and exhibited typical ellipsoidal shapes. However, in the stressed seedlings, the cell wall structure was incomplete and exhibited indistinct gradation, a lower density, and loose edges ( Fig. 7c and h). Plasmolysis and degradation were also evident in part of the cell membrane. Moreover, the chloroplasts, which separated from the plasma membrane, were nearly round and swelled asymmetrically, the thylakoids were overly disorganized, and the thylakoid membranes were loose and showed an increased number of plastoglobules. In the PEG-6000 + DCPTA treatment, the complete membrane structures of the chloroplasts were present, and the shapes of the chloroplasts changed slightly from elongated ellipses to ellipses close to the cell walls ( Fig. 7d and i). A well-aligned internal lamellar system and fewer plastoglobules were observed in the leaves of the PEG-6000 + DCPTA treatment compared with the leaves of the PEG-6000 treatment.
Effects of DCPTA on ICDH activity under soil drought and rewatering conditions ICDH activity declined continuously over the drought period and recovered during rehydration (Fig. 8 The foliar NO 3 − and NO 2 − contents declined continuously during the drought period and recovered during rehydration ( Fig. 9a and b) Under well-watered conditions, DCPTA significantly increased the foliar NO 3 − content on the 20th and 25th days in 2016 and on the 15th, 20th, 25th and 30th days in 2017. However, the DCPTA application had no significant effect on the foliar contents of NO 2 − and NH 4 + .

Effects of DCPTA on activities of NR and NiR under soil drought and rewatering conditions
The activities of foliar NR and NiR declined continuously during the drought period and recovered during rehydration ( Fig. 10a and b). In the drought treatment

Effects of DCPTA on activities of GS, GOGAT and GDH under soil drought and rewatering conditions
The foliar GS activity first increased and then decreased, and the foliar GOGAT activity decreased continuely during the drought period and recovered during rehydration ( Fig. 11a and b). In the drought treatment compared with the control, the activities of foliar GS and GOGAT decreased by 40  In contrast, drought led to marked increases in the activities of foliar NAD-GDH and NADH-GDH ( Fig. 11c and d). In the drought treatment compared with the control, the activities of foliar NAD-GDH and NADH-GDH increased by 87. 16

Effects of DCPTA on activities of AlaAT and AspAT under soil drought and rewatering conditions
The activities of foliar AlaAT and AspAT first increased and then continuously decreased during the drought period and recovered during rehydration ( Fig. 12a

Effects of DCPTA on protease activity, and contents of proteins and free amino acids under soil drought and rewatering conditions
The protease activity and free amino acid contents increased continuously during the drought period and decreased during rehydration (Fig. 13a and c). In the drought treatment compared with the control, the protease activity and free amino acid contents increased by 122.48 and 88.92%, respectively, on day 20 and by 55.03 and 34.51%, respectively, on day 30 in 2016 and increased by 145.15 and 78.58%, respectively, on day 20 and by 57.08 and 43.29%, respectively, on day 30 in 2017. However, the DCPTA application partially reversed the increases in the protease activity and free amino acid contents caused by drought. In the drought +DCPTA treatment compared with the control, the protease activity and free amino acid contents increased by 78.47 and 24.77%, respectively, on day 20 and by 16.46 and 6.62%, respectively, on day 30 in 2016 and increased In contrast, drought led to a marked decrease in the foliar protein content (Fig. 13b). In the drought treatment compared with the control, the foliar protein content decreased by 35.51% on day 20 and by 18.32% on day 30 in 2016 and decreased by 44.81% on day 20 and by 22.50% on day 30 in 2017. The DCPTA application suppressed the decrease in the foliar protein content induced by drought. In the drought+DCPTA treatment compared with the control, the foliar protein content decreased by 19.54% on day 20 and by 7.25% on day 30 in 2016 and decreased by 25.46% on day 20 and by 10.30% on day 30 in 2017. Significant differences between the foliar protein contents of the well-watered treatment and well-watered+DCPTA treatment were observed on day 10 in 2017 and on day 15 in 2017, and significant differences between the free amino acid contents of these treatments were observed on day 10 in 2017.

Discussion
Similar to previous reports for tomato [22] and wheat [19], drought significantly diminished the NO 3 − content in maize leaves in both the DCPTA-treated and non-treated leaves (Fig. 9a). This decrease may be explained by drought-induced inhibitions in nitrate uptake from the roots and/or nitrate transport. However, in this study, the reduction in the non-treated leaves was greater than that in the DCPTA-treated leaves. In this study, there was a significant difference between the RLD and RSD of the well-watered treatment and DCPTA treatment in 0-20 cm and 0-30 cm in 2016 and 2017 on day 30 ( Fig. 3b and d). This result suggests that DCPTA promoted root development in maize under well-watered conditions. Interestingly, under drought conditions, the DCPTA application significantly increased RLD, RSD and the root RGR (Fig. 3a-d). These results indicate that the DCPTA application also promoted maize root growth and improved the spatial and temporal distribution of roots, which was beneficial to NO 3 − uptake under drought conditions. The xylem sap transports water and nutrients from the roots throughout the plant and depends on transpiration intensity and root pressure. The increased root hydraulic conductivity and flow rate of root-bleeding sap induced by the DCPTA application may due to the enhanced root pressure, which depend on physiological activity of the whole root system (Fig. 4a and b) [23,24]. In addition, the NO 3 − delivery rate in the presence of DCPTA was significantly higher than that without DCPTA under drought conditions, which may partly result from the improved NO 3 − absorption and enhanced root pressure induced by DCPTA (Fig. 4c). The stable RWC in the drought+DCPTA treatment suggests an abundant supply water to the aboveground parts, and balanced transpirational loss and water uptake under drought conditions; as a result, the DCPTA-treated plants maintained Gs, which reduced the leaf epidermal resistance and promoted the mass flow of water to the leaf surface and the transportation of the NO 3 − required for N metabolism in leaves (Figs. 5 and 6b). Under drought conditions, increases in the foliar NO 3 − were observed in the DCPTA-treated plants (Fig. 9b).
Whether stomatal or non-stomatal factors are the main cause of a reduced Pn may be determined by changes in Gs and Ci [25]. During the early period of drought, the change of Ci were accompanied by continuously declined Gs, then Gs decreases, but Ci shows an increase ( Fig. 6b and d). Thus, the decrease of the Pn in drought-treated plants was mainly attributed to stomatal limitations firstly, and then, non-stomatal limitations induced by the damage of photochemical mechanism, partly reflected by damaged chloroplast (Figs. 6a and 7). However, DCPTA application maintains relatively high Gs, ensuring the availability of CO 2 for the carbon reduction cycle. Simultaneously, the DCPTA application delayed the increase in Ci and protected the chloroplast ultrastructure against drought-induced oxidative damage, which suggests that DCPTA can protect the photochemical mechanism and, as a result, ensures a more efficient photosynthesis process after rehydration. Moreover, similar to previous studies on spruce [7], sugar beets [9] and guayule [10], DCPTA application also promoted photosynthesis under well-watered conditions.
In most plants, nitrate reduction occurs in leaves. NO 3 − , after being taken up into the leaf cell, is converted to NH 4 + by two successive steps catalysed by NR and NiR. NR, the rate-limiting enzyme of nitrogen assimilation, is highly sensitive to stress [26]. Similar to previous studies on wheat and barley, the NR activity continuously declined in response to drought (Fig. 10a) [18,19]. As a typical nitrate-induced enzyme, NR activity is primarily regulated by the NO 3 − concentration in the leaves [27]. The up-regulation of foliar NR activity in the drought+DCPTA treatment may result from the increase in the foliar NO 3 − content (Fig. 9a). Moreover, the reduction in the foliar NiR activity under drought conditions was significantly reversed by the application of DCPTA, which may be because the DCPTA-stabilized photosynthesis resulted in a sufficient supply of Fd red (Figs. 6a and 10b), thus promoting the conversion of NO 2 − to NH 4 + . The present results indicate that DCPTA treatment could maintain a high NO 3 − assimilation ability in maize under drought conditions.
Although the foliar NR and NiR activities declined during the drought period, the foliar NH 4 + content exhibited an increasing tendency in our experiment (Fig. 9c). This increase may be associated with the glycine oxidation in activated photorespiration, which is induced by decreases in Ci levels under drought conditions [28]. The increased Gs induced by DCPTA was beneficial to the increase in CO 2 in the cellular spaces of the leaf, implying that photorespiration was partly alleviated (Fig. 6b).
In plants cells, excessive levels of NH 4 + are destructive, and the major NH 4 + assimilation pathway is the GS/ GOGAT cycle in higher plants. When the GS/GOGAT cycle is suppressed and the NH 4 + content rises continuously under stress, NH 4 + could serve as a substrate to form glutamate via the reversible amination of 2-OG, the process is catalyzed by GDH, although the enzyme has a lower affinity for NH 4 + [29]. In general, drought inhibited NH 4 + assimilation [30]. During the early period of drought, GDH activity increased sharply, GS activity increased slightly (Fig. 11a-d). These results suggest that accelerated NH 4 + assimilation in maize may be an adaptive mechanism to produce more glutamate and eliminate the accumulation of excess foliar NH 4 + . Subsequently, the GS and GOGAT activities decreased, which may have resulted from an inadequate supply of energy and 2-OG because of photosynthetic inhibition and decreased ICDH activity (Fig. 8).
The DCPTA application altered the major NH 4 + assimilation pathway, maintained the GOGAT/GS cycle and suppressed the GDH pathway, which may have contributed to maintaining the conversion of NH 4 + to glutamine and the subsequent formation of glutamate from glutamine. This result may occur because the photosynthetic stability and ICDH activity induced by the DCPTA application promoted 2-OG synthesis and the reducing power (i.e., NADPH, ATP, or Fd red ) in plants during the drought period, thus providing the GS/GOGAT cycle with relatively sufficient substrates and energy and favouring the enhancement of foliar GOGAT and GS activities [31]. As a result, with the application of DCPTA, drought had less of an effect on the activities of GS and GOGAT.
Although DCPTA promoted NO 3 − assimilation, as expressed by the increased NR and NiR activities ( Fig. 10a and b), this treatment compared to the drought treatment caused significant decreases in the NH 4 + content, which means that exogenous DCPTA resulted in the integration of NH 4 + into the structure of organic compounds, thereby contributing to the reduction in the NH 4 + content. Therefore, the DCPTA application effectively modulated the activities of ICDH, GS, GOGAT and GDH and accelerated the conversion of NH 4 + to glutamate, which is the precursor of other amino acids.
Transamination is a key step in the biosynthesis of various amino acids from glutamate, with the availability of C skeletons from the Krebs cycle [32]. In our studies, both the aminotransferases studied, AlaAT and AspAT, showed increased activities in maize during the early drought period (Fig. 12a and b). Such increases in aminotransferases activities under drought conditions might help in the synthesis of increased amounts of amino acids that act as compatible cytoplasmic solutes and protect cell organelles and biomolecules, thus reducing the adverse effects of drought on maize [33]. Subsequently, the AlaAT and AspAT activities decreased, which may be attributable to the weakened GS/ GOGAT pathway ( Fig. 11a and b) [34]. Moreover, stable aminotransferase activities were observed in DCPTA-treated plants. This finding may be associated with increased GS/GOGAT activities, which can generate more glutamate to serve as a substrate for transamination reactions in maize treated with DCPTA under drought conditions.
Most soluble proteins are enzymes that participate in various metabolic pathways in plants; therefore, the soluble protein content is considered one of the most important indices reflecting the overall metabolic level in plants. Protein synthesis in plants is very sensitive to abiotic stresses and is positively correlated with stress tolerance [19]. Free amino acids are the building blocks of proteins. Drought increased the free amino acid contents, which may mainly be attributed to the increased AlaAT and AspAT activities in the early drought period and the promotion of protein degradation (Figs. 12a, b,  13a, b, c) [35]. However, DCPTA-treated seedlings maintained higher soluble protein levels and lower free amino acid levels than did non-DCPTA-treated seedlings in response to drought. This result may occur because DCPTA inhibited protein degradation by stable protease activities and maintained protein stability, ensuring the series of physiological and biochemical processes that occur normally under stress conditions. Additionally, the DCPTA application increased the amino acid contents under well-watered conditions, which may be attributable to the promoted biosynthesis and accumulation of amino acids, which ultimately improved plant growth and development [36].

Conclusions
The present study suggested that DCPTA treatment increased NO 3 − uptake and the long-distance transportation of NO 3 − from the roots to the leaves via the production of excess roots and maintained a stabilized transpiration rate. The increased foliar NO 3 − content up-regulated NR activity and maintained a high N assimilation ability that was restrained by drought. Exogenous DCPTA effectively regulated the ICDH, GS, GOGAT and GDH activities to speed up the conversion of NH 4 + to Glu, reduced the toxicity of excess NH 4 + to the plant, and accelerated the synthesis of proteins and amino acids. Moreover, DCPTA treatment maintained increased the photosynthetic capacity, supply nitrogen metabolism of energy and carbon skeleton thus alleviating the inhibition of growth by drought in maize.

Plant material, growth conditions, design and sampling
Seeds of the maize cultivar ZhengDan 958 and DCPTA were obtained from the Henan Academy of Agricultural Sciences in China and the China Zhengzhou Zhengshi Chemical Limited Company, respectively.
These experiments were performed in 2016 and 2017 at the Experimental Station of Northeast Agricultural University, Harbin (126°73′E, 45°73′N), Heilongjiang province, China. The research field area has a temperate continental monsoon climate. The rainfall and mean temperature data during the study period (2016 and 2017, May-October) are listed in Additional file 2. Pits (inner length, 10 m; width, 7 m; and height, 1.2 m) in the field were used as experiment containers (Additional file 3). Plastic sheets were used to cover the inner sides of the pits, and a rain-proof shed was used to ensure the crops were solely dependent on soil moisture and irrigation over the course of the experiment to maintain the soil water conditions. The soil used was Chernozem and was sieved (pore size, 1 cm) and diluted with vermiculite (particle diameter, 4-8 mm; soil to vermiculite, v/v, 2:1). Before planting, soil chemical analysis was conducted according to Cottenie et al. (1982) [37], and the results are presented in Table 1. Fertilization was carried out by adding ammonium nitrate (33.5% N), calcium superphosphate (15.5% P 2 O 5 ), and potassium sulfate (48% K 2 O) at the rates of 8.0, 8.0 and 20 kg pit − 1 , respectively, before planting. No fertilizer was applied after planting. All containers were watered to 85% before planting. The seeds were manually sown on 2nd May 2016 and 4th May 2017 and were harvested on 7th October 2016 and 3rd October 2017, respectively. Three seeds were sowed per hole to ensure germination, and only the healthiest seedling within 20 days was kept at each site. Each container consisted of 10 rows, and the plant-to-plant and row-to-row distances were 20 cm and 65 cm, respectively. In addition, the ground around the containers was manually sown with the same plant-to-plant and row-to-row distances. The control of plant diseases and insect pests was conducted by managers.
The maize at the nine-leaf stage (during the pre-female inflorescence emergence stage) were treated as follows: (1) plants were irrigated continuously and sprayed with either 10 mL water (well-watered) or DCPTA (well-watered+DCPTA) per plant; (2) irrigation was stopped to form the drought conditions and sprayed with 10 mL of either water (drought) or DCPTA (drought +DCPTA) per plant; plants were rehydrated after 20 days of drought treatment.
The concentrations of DCPTA (25 mg/L) were based on the results of previous screening experiments, and Tween-20 (0.03%) was added as a surfactant to the solution for spraying. Each treatment had five replicates, and experiments were performed in a completely randomized design. The dynamic changes in soil water contents during the experimental stage are exhibited in Additional file 4. Random plants from each treatment were sampled on days 0, 5, 10, 15, 20, 25 and 30. For leaf sampling, the middle part of the 9th leaf (numbered basipetally) was sampled for analysis of leaf gas exchange, and the same part of the leaf was stored at − 80°C after immersion in liquid nitrogen for 30 min for determination of physiological parameters. For root sampling, a hand-held soil auger (inner diameter of 20 cm) was used to obtain soil cores from 0 to100 cm depth of the soil profile at 10 cm increments. The soil cores were soaked in a plastic container overnight, and roots were stirred and sieved through a mesh (400 holes cm − 2 ). The soil cores were the carefully washed by swirling water through the cores. The soil material and old dead roots debris were manually separated from the live roots.
No permissions or licenses were needed to obtain our plant sample.

Plant measurement and analysis
Relative growth rate (RGR) and plant productivity The shoots and roots of maize were oven dried at 105°C for 45 min and then held at 80°C for 48 h; the shoot and root dry weights plant − 1 were determined soon afterwards. The RGR was determined as follows: RGR (fresh weight) = [ln (final dry weight)ln (initial dry weight)]/ (duration of treatment days) [38]. A leaf area metre (Li-COR 3100; Li-COR, Lincoln, NE, USA) was used to estimate the leaf area; number of grains plant − 1 (GN) and grain yield plant − 1 (GY) were recorded at the maize physiological maturity stage.
Leaf relative water content (RWC) and soil water content (SWC) The RWC and SWC were determined according to the methods of Machado and Paulsen (2001) [39] and Turner (1981) [40], respectively.
The RWC was determined on fresh leaf disks (2 × 2 cm) from the middle part of the 8th leaves (numbered basipetally). After they were weighed (FW), the disks were immersed in distilled water at 25°C overnight to obtain the turgid weight (TW). The leaves were dried at 80°C for 48 h and then weighed a third time (DW). RWC was calculated as follows: SWC was determined in the soil from the internal area of each container. After being weighed (FW), the soil portion was dried at 85°C for 96 h and then weighed (DW). SWC was calculated as follows:

Transmission electron microscopy of chloroplasts
Observations were performed according to the description of Hu et al. (2014) [41], and the chloroplast ultrastructure was observed under a H-7650 transmission electron microscope (manufacture: Hitachi, Japan).
Root morphological traits, root hydraulic conductivity, and the collection of root-bleeding sap Roots from each soil core were scanned using a digital scanner (Epson V700, Indonesia). The root images were analysed using the WinRHIZO Image Analysis system (Version 2013e) (Regent Instruments Inc., Canada). The root length density (RLD, cm root cm − 3 soil) and root square area density (RSD, cm 2 root cm − 3 soil) were calculated according to the method described by Mosaddeghi et al. (2009) [42]. The hydrostatic root hydraulic conductivity (Lp) was measured with a Scholander pressure chamber according to the method described by López-Pérez et al. (2007) [43].
The tools used for collection of root-bleeding sap are exhibited in Additional file 5. The plants were cut by scissors at 10-12 cm above the soil surface at 18:00-19:00. Centrifuge tubes (inner diameter 40 mm) with cotton were placed on the upper end of the stalks, and the stalk joints and centrifuge tubes were wrapped by plastic film to keep impurities and insects out. The bleeding sap was collected for 12 h; then, the cotton was extracted from each centrifuge tube and placed into a glass syringe (100 ml), and the root-bleeding sap was squeezed out for volume measurement. The NO 3 − content in the root-bleeding sap was determined by AA3 Continuous Flow Analytical System (Seal, Germany) according to Guan et al. (2014) [44]. The flow rate of the root-bleeding sap and the NO 3 − delivery rate were expressed as ml h − 1 root − 1 and μg h − 1 root − 1 , respectively. The foliar NO 3 − content determination by the reduction of NO 3 − to NO 2 − followed the salicylic acid methods of Cataldo et al. (1975) [45], the absorbance was monitored at 410 nm. The foliar NO 2 − content was determined