Effect of root interaction on nodulation characteristics and isoflavonoid mechanism of alfalfa in the simulated alfalfa/oat in pots

: Rational intercropping is capable of promoting nodulation and facilitating the fixation and utilization of nitrogen (N) of legumes. Isoflavonoid is critical to form root nodules of legumes, whereas the regulation of isoflavonoid over nodulation and N fixation in legume/cereal intercropping remains unclear. In the present study, nutrient solution of different root partitions (e.g., no barrier (A-O), nylon mesh barrier (NA-O), plastic barrier (PA-O) and sole alfalfa (SA)) and nitrogen levels (e.g., 21 and 210 mg L -1 N) were used to delve into the variations of nodulation, N fixation, isoflavonoid content, isoflavonoid synthase ( IFS ) and nodulation-signaling pathway ( NOD ) genes in alfalfa. As suggested from the results, the parameters of nodulation and N fixation ability with A-O were all evidently higher than those with PA-O and SA. Formononetin and genistein with A-O had noticeably higher contents than those with PA-T and SA. Daidzein and luteolin with A-O had remarkably higher contents than those with NA-O in the root. IFR 1, IFR 4 , NOD 1 and NOD 2 with A-O and NA-O had noticeably higher relative expressions than those with SA and PA-O. IFR 2 and IFR 3 with A-T and NA-T had significantly lower relative expressions than those with PA-O and SA. The nodulation and isoflavonoids met significant positive associations. IFR and NOD genes and TNN and ENN followed significant positive associations. NOD genes and formononetin exhibited extremely significant positive associations. The conclusion was drawn that the closer root interaction between alfalfa and oat, the more effective the nodulation, N fixation ability and isoflavoids content of alfalfa will be. Isoflavonoids primarily affected nodulation and N fixation. The mechanism of isoflavonoids on nodulation and N fixation in alfalfa/oat intercropping was explained that root interaction results in the reduction of environmental N, thereby regulating the relative expressions of IFR genes, elevating the isoflavonoids contents, and up-regulating the relative expressions of NOD genes; as a result, the nodulation and N fixation ability in alfalfa are facilitated.


Introduction
Facilitative root interactions in mixed cropping systems are critical since they nutritionally improve crops grown in nutrient-poor soils and low-input agro-ecosystems 1 . Among the interactions, legume/cereal intercropping system has been extensively adopted for its ability of nitrogen (N) fixation by legume 2 . The legume may partially transfer symbiotically fixed N to the intercropped cereal 3 , while the cereal is likely to stimulate the N fixation activity of legume 4 . It has been widely evidenced that intercropping can stimulate nodulation growth and N fixation of legume, covering faba bean (Vicia faba L.)/maize (Zea mays L.) intercropping 2 , pea (Pisum sativum L.)/maize intercropping 5 and cowpea (Vigna unguiculata L.)/cotton (Gossypium spp.) intercropping 6 , etc.
Interspecific competitive and depletion N in legume/cereal intercropping system could down-regulate legume "N suppression" effects and facilitate N fixation 7 . This finding was because that the N content in the soil decreases; as a result, the diffusion of O2 is promoted in the nodule cortex, the respiration rate of nodule is accelerated, and the nitrogenase activity is enhanced. Moreover, the roots can be stimulated to secrete flavonoids (e.g., isoflavonoids) to further form nodules 8 . Isoflavonoids are critical to various plant-microbe interactions. The first effect is exerted in the rhizosphere, in which plants secrete (iso) flavonoids when N in the soil is scarce 9 . Perception of these compounds by rhizobia could activate the transcription of bacterial nodulation genes, thereby leading to the formation of nodulation factors 10 . The second proposed role of (iso) flavonoids during nodulation also took place inside the root, and it was associated with the notable capacity of (iso) flavonoids to inhibit auxin transport 11 . Kobayashi 12 also considered that daidzein and genistein are the major metabolites of soybean roots, and they could induce soybean nodulation. Several studies clearly suggested that the (ios) flavonoids are crucial for nodulation if the bacteria enter the plant, and exogenous (ios) flavonoids are no longer available 13,14 . Numerous existing studies have been conducted on the relationship between isoflavonoids secreted by roots and nodulation and N fixation in legume/cereal intercropping. However, how isoflavonoids in legume crops have the effect on nodules remains unclear. Isoflavonoid synthase (IFS) is a vital enzyme of isoflavone decomposition pathway, critically regulating the content and composition of isoflavonoids. Accordingly, to ascertain the variations of isoflavonoids contents and IFR and nodulation-signaling pathway (NOD) related genes in legume/cereal intercropping can further explore the regulation of isoflavones on nodulation and N fixation.
Alfalfa (Medicago sativa L.) refers to the crucial perennial legume forage; it can be used to achieve symbiotic N2 fixation 15 . Alfalfa exhibits the high-yield, rapidly regenerating many new stems after it is harvested; it can also be harvested multiple times in the growing season 16 . For this reason, it is one of the most commonly used species in arid and semi-arid regions. Oat (Avena sativa L.) is considered the most extensively used annual cool-season forage worldwide, which is an important source of nutrition for ruminant livestock 17 . It is applied in animal nutrition since it is a fast growing, palatable, succulent and nutritious fodder crop. Alfalfa/oat intercropping system is capable of enhancing N use efficiency, light use efficiency and land productivity; thus, it has become a major planting pattern in northern China. Nevertheless, the effects of root interaction on nodulation characteristics and isoflavonoids variations of alfalfa in this model remain unclear. Thus, in the present study, the root interaction intensity in alfalfa/oat intercropping was simulated, and sand culture nutrient solution was used to ascertain the nodulation, N fixation capacity, isoflavonoid content, IFR and NOD genes related expression of alfalfa with the use of different N treatment and planting systems. This study primarily aimed to elucidate how alfalfa/oat intercropping impacts the variation of isoflavonoids contents, as well as the potential role of isoflavonoids on nodulation in alfalfa, as attempt to create a novel perspective for facilitative root interactions and developing sustainable agriculture.

Nodulation and N fixation of alfalfa
TNN with A-T was significant higher than that with SA and NA-O (P < 0.05). ENN, PNW and SNW with A-O were significant higher than those with SA, NA-O and PA-O (P < 0.05) (Fig. 1). ENN and PNW with NA-O were significant higher than that with PA-O under N210. SNW with NA-O were significant higher than that with SA and PA-O (P < 0.05). NA with SA and NA-O was significant lower than that with A-O, but NA with SA and NA-O was significant higher than that with PA-O (P < 0.05). PNF with A-O was significant higher than that with NA-O; PNF with NA-O was significant higher than that with SA and PA-O (P < 0.05).   Table 1, the contents of daidzein and luteolin in stem and leaf, and those with SA and PA-O in root were not detected. The content of daidzein and luteolin with A-O was significant higher than that with NA-O in the root (P < 0.05). The content of formononetin with A-O was significantly higher than that with SA and NA-O, and the content of formononetin with NA-O was significantly higher than that with PA-O in stem and leaf (P < 0.05). The content of formononetin with A-O was significantly higher than that with PA-O in root (P < 0.05). The content of genistein with A-O was significantly higher than that with PA-O (P < 0.05).

Expression of isoflavonoid and N-fixed related genes in alfalfa
To unravel the underlying molecular mechanism of the stimulatory effects of different alfalfa and oat cropping systems on alfalfa nodulation, we analyzed expression patterns of a series of key genes regulating isoflavonoids (Fig. 3) and nodulation ( Fig. 4) in alfalfa. The relative expressions of IFR 1 and IFR 4 with A-O were significantly higher than those with NA-O, and the relative expressions of IFR 1 and IFR 4 with NA-O were significantly higher than those with SA and PA-O (P < 0.05). The relative expression of IFR 1 with SA was significantly higher than that with PA-O under N210 (P < 0.05). The relative expression of IFR 4 with PA-O was significantly higher than that with SA in root under N21 (P < 0.05).The relative expressions of IFR 2 and IFR 3 with SA were significantly higher than those with PA-O, and the relative expressions of IFR 2 and IFR 3 with PA-O were significantly higher than those with NA-O and A-O (P < 0.05). The relative expressions of IFR 2 under N210 and N21 and IFR 3 under N210 with NA-O were significantly higher than those with SA (P < 0.05). The relative expressions of NOD 1 and NOD 2 with A-O and NA-O were significantly higher than those with SA and PA-O (P < 0.05). The relative expression of NOD 2 with A-O was significantly higher than that with NA-O except that in root under N21 (P < 0.05).

Correlation analysis of nodulation, isoflavonoids and genes
The parameters of nodulation (TNN, ENN, PNW, SNW, NA and PNF) and the parameters of isoflavonoids (daidzein, luteolin, formononetin and genistein) had extremely significant positive correlation (P < 0.01) except genistein in stem and leaf ( Table 2). The correlation coefficient between formononetin in stem and leaf and ENN was the highest (0.904).
IFR 2 or IFR 3 had negative correlation with the parameters of nodulation ( Table  3). And other parameters were positive correlation. The related genes except IFR 2 in root and TNN, the all related genes and ENN, the related genes except IFR 2 and NOD 2 in stem and leaf and NA or PNF were all extremely significant correlation (P < 0.01). The correlation coefficient between IFR 4 in stem and leaf and ENN, NOD 1 in root and PNF were the highest (0.83).

Discussions
Legume/cereal intercropping can enhance N fertilizer utilization efficiency, which is characterized by high yield and low consumption 18 . In legume/cereal intercropping systems, the reliance on symbiotic N2 fixation is enhanced as a result of a strong competition for soil mineral N by the intercropped cereal. The N competition in intercropping is primarily achieved by root interaction.
The root separation method was adopted here to clarify the effect of root interaction on nodulation and N fixation of alfalfa and the regulation of isoflavones on nodulation and N fixation. In this study, the nodulation (TNN, ENN, PNW and SNW) and N fixation (NA and PNF) under normal N level were higher than those under low N level of alfalfa. It was therefore suggested that low N level could promote the nodule and N fixation of alfalfa. It has also been confirmed in other studies that the nodulation and N fixation of legumes were largely affected by the N concentration, namely, the lower N concentration, the higher the nodulation and N fixation ability will be 19 . Compared with NA-O, nodulation and N fixation of alfalfa increased significantly with A-O. When the roots of the two crops with A-O, they can directly compete and exchange environment N through connection of Rhizobia or interception of root surface area. However, the competition and exchange of N with NA-O were achieved primarily through mass flow and diffusion 20 . Accordingly, the N compete ability with A-O was more robust than that with NA-O of oat, and the nodulation and N fixation with A-O was stronger than that with NA-O of alfalfa. Meantime, the nodulation and N fixation with NA-O of alfalfa were more obvious than those with PA-O and SA, since PA-O and SA had no root interaction, and N exchange could not take place. It was therefore indicated that the nodulation and N fixation of alfalfa increased with the rise in root interaction. Some scholars also consider that legume/cereal intercropping cannot up-regulate the amount of N fixation 21 , whereas the majority of scholars consider that legume/cereal intercropping can facilitate the nodulation and N fixation of legume 2,22 . Furthermore, this promotion ability is determined by the combination of crops and cultivation methods 23 .
Iosflavonoids refer to the major signal components of legume root extracts 24 . In the present study, in the roots, daidzein and luteolin contents with A-O were noticeably larger than those with NA-O, and those with no PA-O and SA detected. Formononetin and genistein contents with A-O were obviously larger than those with PA-O and SA in different parts. All of these findings revealed that alfalfa/oat intercropping gave rise to the differences of isoflavonoids contents in alfalfa; the closer the root interaction, the higher the isoflavonoids contents will be. Meantime, daidzein, luteolin, formononetin and genistein all displayed positive correlations with nodulation and N fixation, suggesting that isoflavonoids contents evidently impacted root nodules in alfalfa. This result was achieved because isoflavonoids participate in the signaling exchange between roots of legumes and N-fixing rhizobia, and they can promote division of cortical cells during nodule formation by hindering auxin transport. Ferguson 25 also reported that genistein application could enhance nitrogenase activity, thus, protein content enhancement could be attributed to hastened N fixation. As revealed from the results of Dolatabadian's experiment, the genistein used elevated nodule number and nodule weight, as well as nitrogenase activity 26 . In brief, root interaction considerably up-regulated the isoflavonoids contents, and isoflavonoids are indispensable for nodulation in alfalfa.
The existing research primarily focuses on the necessity and evidence of isoflavonoids in legume nodulation, whereas isoflavonoids exert less effect in legume, especially in the field of gene research 13 . To delve into the nodulation and N fixation mechanism in alfalfa/oat intercropping, the IFR and NOD related genes were detected. As suggested from the results, the relative expressions of IFR 1, IFR 4, NOD 1 and NOD 2 with A-T were evidently higher than those with PA-T and SA, while the relative expressions of IFR 2 and IFR 3 exhibited opposite performance. It is therefore suggested that root interaction exerted noticeable regulatory effect on IFR and NOD genes, thereby affecting the isoflavonoids contents and nodulation characteristics in alfalfa. The correlation revealed that IFR genes were closely correlated with TNN and ENN, IFR 1, IFR 3 and IFR 4 were tightly associated with NA and PNF. It can also be demonstrated that IFR can significantly regulate the nodules formation of alfalfa, and IFR 1, IFR 3 and IFR 4 can significantly regulate alfalfa N fixation ability. Subramanian 27 also considered that during soybean nodulation, isoflavonoids are necessary as inducers of nodulation genes in roots, and they are critical to determine the degree of nodulation. The relative expressions of NOD genes with A-T were obviously higher than those with PA-T and SA. This finding complies with the nodulation, N fixation and isoflavonoids contents, because the N competition in intercropping system is more intense under nutrient stress. To fix more N, the relative expression of NOD genes as the signal material of nodulation was up-regulated 28 . Moreover, NOD genes in root were positively correlated with nodulation and N fixation, while NOD genes displayed positive associations with daidzein, luteolin and formononetin. It is therefore demonstrated that the isoflavonoids in alfalfa are the main inducers of NOD genes expression. Subramanian et al. 13 also reported that isoflavonoids are critical to endogenous NOD gene induction in the determinate legume. For this reason, the essential effect exerted by isoflavonoids in alfalfa nodulation is associated with their effects in NOD gene induction.

Conclusions
In alfalfa/oat intercropping, the closer root interaction between alfalfa and oat, the more effective the nodulation, N fixation ability and isoflavoids content of alfalfa will be. Isoflavonoids primarily affected nodulation and N fixation. The mechanism of isoflavonoids on nodulation and N fixation in alfalfa/oat intercropping was explained that root interaction results in the reduction of environmental N, thereby regulating the relative expressions of IFR genes, elevating the isoflavonoids contents, and up-regulating the relative expressions of NOD genes; as a result, the nodulation and N fixation ability in alfalfa are facilitated.

Experimental Design
In order to prevent the effect of precipitation on the N concentration in the experiment, it was carried out in the growth chamber (light at 28℃/14 h and darkness at 20 ℃ /10 h, light intensity was 260~350 mol m -2 s -1 , and relative humidity was 60%~70%) at Gansu Agricultural University, Lanzhou, China. In order to provide an accurate and stable N environment, and reduce root nodule abscission and RNA degradation though rapid and complete root sampling, nutrient solution sand culture method was used. Nutrient solution sand culture method was conducted with sand culture (25 kg pot -1 sand washed clean by distilled water) with 5 cropping systems in plastic pots (47 cm diameter, 40 cm height) under 2 N treatments. The 2 N treatments were (1) the low N level, 21 mg N L -1 as mixed N source of Ca(NO3)2 and (NH4)2SO4 (N21), (2) the medium N level (appropriate nitrogen levels of alfalfa), 210 mg N L -1 as mixed N source of Ca(NO3)2 and (NH4)2SO4 (N210). Hoagland-Arnon solution as the basic nutrient solution was used in the N21 and N210 nutrient solutions, and the ration of NO3 --N: NH4 + -N was 1:1. The 5 cropping systems were (1) alfalfa as a monoculture (SA), (2) oat as a monoculture (ST), (3) alfalfa/oat intercropping with no barrier (A-O), (4) alfalfa/oat intercropping with nylon mesh barrier (NA-O), (5) and alfalfa/oat intercropping with plastic barrier (PA-O). Plastic pots were cut in the middle, separated into two compartments and then placed nylon mesh (diameter of 0.2 mm) or plastic film in the middle, and then reconstructed 29 . The A-O planting pattern had no artificial physical barrier between alfalfa and oat, allowing water and nutrients to exchange and possible root interaction between alfalfa and oat. NA-O planting pattern obstructed overlapping of alfalfa roots with oat roots but allowed water and nutrients to exchange through the nylon meshes. The PA-O planting pattern preventing water and nutrients exchanged between alfalfa and oat and with no overlapping of alfalfa roots with oat roots. There were 3 replications in each treatment.
The seeds of alfalfa and oat were chosen and disinfected, and then were planted in separated one side of plastic pots filled with sand. When the alfalfa and oat grew to 3 cm in height, healthy seedlings were kept in each plastic pot with the rest removed by hand. Sole alfalfa and sole triticale kept 20 seedlings, no barrier, nylon mesh barrier and plastic barrier of alfalfa and triticale kept 10 seedlings, respectively. Seven days after alfalfa seedlings emergence, two levels of N nutrient solution (1000 mL per pot) were added to the pots, and then rhizobium (Sinorhizobium meliloti, 12531, provided by College of Pratacultural Science, GAU) liquid was inoculated (50 mL pot-1, OD600 between 0.63 to 0.64). Sand was rinsed with distilled water and nutrient solution was replaced once a week. Distilled water (1 L) was slowly added in each pot (prevent salt ion accumulation) in each time. Then pots with plants were rinsed with distilled water for 12 h, and then added nutrient solution (2 L pot -1 ). Distilled water was supplemented every day to the location of the nutrient solution for the first time.
Sowing date for alfalfa was 1 March 2018; and sowing date for oat was 23 March 2018; and harvest date for alfalfa and oat was 13 July 2018 (flowering stage of alfalfa). the original source of method descriptions should be clearly acknowledged and cited. The method descriptions were cited from Zhao's research which is our team's preliminary research 30 .

Plant dry matter weight and N accumulate
During the sampling period, the whole 5 plants of alfalfa and oat were selected from each pot for whole plant dry matter. 3 pots were taken for each treatment. Whole plant dry weight (PDW, mg plant -1 ): put fresh samples in 105℃ oven for 15 min and then put them in 60～70℃ oven until a constant weight.
Plant nitrogen content (PNC) was determined by Kjeldahl procedure after digestion in a mixture of concentrated H2SO4 and H2O2 31 . Powdered sample was digested in the Kjeldahl digestion flask by boiling with H2SO4-H2O2 until the mixture became clear. The digested liquid was filtered and volume. Ammonia was steam distilled from the digest to which NaOH solution was added. The distillate was collected in a conical flask containing HCl and red methyl indicator. The ammonia that was distilled into the receiving conical flask reacted with the acid and the excess acid in the flask was estimated by back titration against NaOH with color change from red to yellow (end point). Determinations were made on all reagents alone (blank determinations). N (%) was calculated as [(ml standard acid × N of acid) -(ml blank × N of base)] × (ml std base × N of base) × 1.4007/ Weight of sample in grams × 100%. Plant N accumulation (PNA, mg plant -1 ) = PNC × PDW.

Nodulation
Roots were rinsed with distilled water, and absorbed residual water with absorbent paper, then removed the nodules quickly. All fresh nodules were detached from the roots to collect, and then counted and weighted. Nodulation, including total nodule number (TNN), effective nodule number (ENN), fresh nodule weight per plant (PNW), single fresh nodule weight (SNW) of alfalfa, was measured. At the time of sampling, some nodules are pink; they can effectively fixing N, so they are called "effective nodules". In addition, some nodules are white or brown, which may be immature or aging, so they are called "non-effective nodules". The TNN is the sum of ENN and ineffective nodule number. The ratio of ENN to TNN was calculated.

Nitrogenase activity
The nitrogenase activity (NA) was measured by the acetylene reduction assay 32 . Weigh fresh nodules about 0.2 g into a 7 ml glass bottle, seal with a rubber stopper. 10% volume of air was removed and replaced with an equal volume of acetylene. After 30 min at room temperature, duplicate 25 μL gas samples were removed and analyzed by gas chromatography for peak of ethylene and acetylene. The standard curve of ethylene was determined and measured under standard conditions using standard ethylene to calculate the nitrogenase activity of the nodule sample. The instrument was the GC-7890F gas chromatograph with column temperature of 180°C , sampler of 150°C, and FID detector of 170°C. Gas pressure: N2 is 0.3 mPa, H2 is 0.08 mPa, and air is 0.15 mPa. C2H4 level (μmol g -1 h -1 ) = hx (sample peak area) × C (standard C2H4 level, μmol mL -1 ) / hs (standard C2H4 peak area) × 24.9 × t (C2H2 reaction time, h) ×m (tumor weight, g). N fixation capacity (umol h -1 ) =NA × SNW.

Content of isoflavonoids
1g of hay sample was accurately weighed (having been powdered and screened for 1mm). Subsequently, 20 ml of methanol solution was added and soaked for 6 h, extracted by ultrasound for 30 min, and then filtered through filter paper. The filter residue was collected again, and 20ml of methanol solution was added. The mixture solution was ultrasonically treated for 20min and filtered with filter paper. The two filter solutions were mixed and spin-dried in vacuum at 30 ℃. The crude extract was dissolved in methanol to 10ml and filtered with 0.22 μ m PTFE membrane for HPLC analysis.
Preparation of standard curve: the quality of standard products (daidzein, luteolin, formononetin, genistein) was accurately weighted, and then the products were dissolved in methanol to prepare standard solution at a certain concentration. The chromatographic peak area of each component was ascertained using the automatic injector. The injection quality of the standard and the corresponding peak area acted as the standard curve.

Quantitative Real-time PCR Analysis of isoflavonoids
For quantitative analysis, 3 independent biological replicates of stem and leaf and root samples of alfalfa from three different pots for each treatment were harvested. The each provided sample obtained from the different alfalfa plants in each pot, frozen in liquid N, and kept at −80°C for further analyses. Total RNA extraction was isolated using TransZOL method. The concentration of the total RNA was determined by ultra-micro UV spectrophotometer (Quawell-Q5000, USA). Five grams of total RNA were used to synthesize cDNA by reverse transcriptase powerscript™ (Hiscript II Q Sellect RT SuperMix for qPCR (+ gDNA wiper) test kit; Vazyme; China) following the manufacturer's protocol. The cDNA samples were used as template to quantify the target gene expression levels (quantitative real-time PCR) by ChamQ SYBR Color qPCR Master Mix Test Kit (Vazyme, China) following the manufacturer's protocol.