Effects of Intercropping Patterns on Cadmium Tolerance of Perilla Frutescens and Soybean

doi:10.21203/rs.3.rs-851176/v1

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Effects of Intercropping Patterns on Cadmium Tolerance of Perilla Frutescens and Soybean

https://doi.org/10.21203/rs.3.rs-851176/v1

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In this study, we intercropped hyperaccumulator Perilla frutescens with soybean to explore the effects of intercropping under cadmium stress based on the physiological growth indexes of plant, cadmium absorption capacity, and soil cadmium forms Experiments showed that compared with mono-cropping, intercropping soybean’s biomass, catalase (CAT) activity, peroxidase (POD) activity, total chlorophyll and net photosynthetic rate were significantly increased by 1042%~10658%, 1578%~7381%, 1345%~2337%, 376%~1500% and 7121%~31278%, while P frutescens’s significantly decreased by 9387%~9507%, 1562%~2572%, 12056%~15331%, 715%~756% and 3056%~5076% Under 5 and 10 mg kg^-1 Cd treatments, the Cd content in roots, stems, leaves, pods and grains of intercropping soybean decreased significantly by 1072%~3174%, 1488%~2168%, 1995%~3413%, 2222%~5270% and 791%~1786% along with enrichment coefficient decreasing by 1486%~2787%, while those of P frutescens significantly increased by 8700%~2141%, 948%~2155%, 2305%~3766% and 1818%~9412% along with enrichment coefficient significantly increasing by 2000%~7775% Additionally, citric acid, succinic acid, oxalic acid and tartaric acid worked importantly in heavy metal detoxification in plants Strikingly, the content of soil exchangeable Cd (EXC) under intercropping was the highest, even higher than that of mono-cropping soybean and P frutescens, which accounted for 37%~42% under the same stress condition Therefore, intercropping Perilla frutescens with soybean provide a potential strategy for Cd phytoremediation

Environmental Chemistry

Toxicology

heavy metal pollution

intercropping

hyperaccumulator

Perilla frutescens

Soybean

At present, serious cadmium (Cd)pollution in farmland directly leads to excessive Cd content in crops, which is harmful to human health(Kim et al, 2018)Therefore, how to reduce Cd pollution and ensure the safe production of food crops has become a current research hotspot in the field of soil and environment(Bashir et al, 2018; Saqib et al, 2018)To solve this problem, plant intercropping remediation technology is regarded as one of the most economical and effective technologies(Huang et al, 2018)In particular, the use of intercropping of hyperaccumulators with crops are currently the most effective treatment of heavy metals in soil The effective intercropping restoration mode can not only reduce the absorption of heavy metals by crops, but also achieve the purpose of production at the same time(Zou et al, 2016; Chen et al, 2019)For different absorption capacities of heavy metals between hyperaccumulators and crops, less heavy metals absorbed by crops can be achieved

The intercropping of hyperaccumulators and crops is firstly reflected in the physiological and biochemical effects of intercropping on the two plantsAccording to reports, the photosynthetic pigment content of tomato leaves increased by 1113%~3558% during the intercropping of Solanum and tomato, and the antioxidant enzyme activity increased by 050%~4706% compared with mono-cropping(Hu et al, 2019)Wang et al (2019) found that the photosynthetic pigment content of lettuce leaves increased by 144%~1440% after two ecological types of Solanumphoteinocarpum and its hybrid progeny were intercropped with lettuce, and antioxidant enzyme activity is significantly increased by 7330%~28095% In terms of the intercropping mechanism involving hyperaccumulators and crops, it is generally believed that the intercropping will change the type of plant root exudates Root exudates can change the rhizosphere microbial groups, soil enzyme activities and soil pH, leading to changes in the form of soil heavy metalsEventually,changes in the form of heavy metals in the soil willaffect the absorption of heavy metals by plantsRoot exudates of plants include organic acids, amino acids, soluble sugars and polysaccharidesOrganic acids asa ligand of heavy metal elements, participating in the absorption, transportation, and accumulation process of heavy metalsOrganic acids can also combine with heavy metals entering plantsto protect the activities of important proteins and enzymes in cells, thereby reducing the toxicity of heavy metals Therefore, organic acids play an important role in the detoxification mechanism of hyperaccumulation plants that are resistant to heavy metal poisoning(Zhang et al, 1999)Sha et al (2019)found that under the intercropping of Paspalumscrobiculatum and Macleaya cordatasoil enzyme activity was enhanced, and the proportion of tolerant bacteria in the soil also increasedThe changes in these intercropping soils eventually increased the amount of heavy metals enriched by hyperaccumulators Soil pH affects the bioavailability of heavy metals through adsorption and precipitation Under normal circumstances, the content of exchangeable heavy metals in the soil will decrease with the increase of pH(Liu et al, 2011), and there is a significant negative correlationIn the intercropping system of Oryza sativa and Thalia dealbata, the increasing of pH in rice rhizosphere soil can reduce the absorption of heavy metals by crops(Wang et al, 2020)Intercropping can change the form of heavy metals in the soil and enhance the bioavailability of heavy metals in the soil(Lu and Yan, 2007) For example, under the intercropping of Dipsacus chrysanthemum with broad bean, the Cd exchangeable content in the mature stage was significantly lower than that in the seedling stage by 1748% The possible reason is that under the intercropping mode, the roots secrete organic acids to complex with Cd to form a soluble complex, thereby increasing the activation efficiency of soil Cd, and then being absorbed by Dipsacanthus(Tan et al, 2016)

In fact, the selection of hyperaccumulators is the key to the restoration of heavy metal-contaminated soil by intercropping with hyperaccumulators and cropsAt present, there are still deficiencies in hyperaccumulators Field restoration experiments have shown that many kinds of hyperaccumulators, such as Solanum nigrum, Sedum alfredii andBidenspilosahave slow growth, small biomass, and unsatisfactory restoration effects(Cao et al, 2014; Chen et al, 2014b; Lin et al, 2020)Choosing hyperaccumulators with suitable growth habits and higher biomass for better extraction of heavy metals, which is essential for the intercropping systemPerilla frutescens is a Cd hyperaccumulator with the advantages of large biomass, short growth cycle and strong adaptability to the growth environment,which is an ideal Cd accumulating plant for intercropping research(Xiao et al, 2020)Currently, the post-treatment problem of hyperaccumulators has always limited the development of phytoremediation technology In fact, P frutescensis still a kind of aromatic plant, rich in volatile oil(Zhong et al, 2021)Therefore, the cadmium-rich P frutescenscan be separated by steam distillation to separate the volatile oil without Cd pollution In addition,P frutescensrich in heavy metal Cd can also be prepared into high-performance activated carbon, which shows a good removal effect on Pb²⁺ and Cd²⁺ in water, and effectively inactivates Cd in biomass(Zheng et al, 2019)These characteristics ofP frutescensgreatly improve the economic benefits of repairing hyperenrichment plants Soybean is an important oil crop and economic crop in china, and it is sensitive to Cd and can easily absorb heavy metals(Finger-Teixeira et al, 2010)According to reports, under the condition of intercropping between legumes and hyperaccumulators, legumes can provide nitrogen to hyperaccumulators through nitrogen fixation, thus promoting the accumulation of hyperaccumulators(Li et al, 2007)Studies have found that Cd stress seriously affects the growth and development of soybean, which in turn affects soybean yields(Huang et al, 2006)Therefore, research to improve the situation of soybean growth under Cd contamination is of great significanceAt present, our lab has conducted preliminary explorations on the Cd enrichment capacity of the hyperaccumulatorP frutescens(Xie et al, 2011),and explained the Cd tolerance of P frutescensfrom the perspective of organic acids(Chen et al, 2018) and plant chelating peptides(Xiao et al, 2020)Nevertheless, whether the intercropping of P frutescensand crops can reduce the Cd content in the crops while repairing the soil, and the mechanism of Cd tolerance in plants has not been studied yet

In this paper, the effects of intercropping patterns on Cd tolerance of P frutescens and soybean will be explored from the aspects of plant growth indicators, physiological levels, the correlation between plant organic acids and Cd content, and soil Cd forms This study aims to provide a reference for the study of the mechanism of Cd tolerance in P frutescens and soybean

21Plant materials

In this experiment, the soil was collected in the westernmost glass room (26°5′22″ N, 119°13′49″ E) of the College of Life Sciences of Fujian Agriculture and Forestry University on March 16, 2019 Firstly,the soil was placed in the laboratory to be air-dried naturally, and the stones and other debris in the soil were removed before being crushed Secondly, the ground soil of the same weight was then weighed and placed in the culture basins (62 cm×35 cm×26 cm, length × width × height), with or without partitions Finally, the chemical reagent CdCl₂·25H₂O was used to prepare the soil treated with 0, 5 and 10 mg kg^-1 Cd separately,and the soil was mixed thoroughly and equilibrated for 14 dThe pH of the collected soil was 572, organic matter was 4312%, total nitrogen was 3090 mg kg^-1, total phosphorus was 37490 mg kg^-1, total potassium was 45878 mg kg^-1, and available phosphorus was 12015 mg kg^-1, quick-acting potassium was 14986mg kg^-1

Perilla frutescens (L) Britt was a Cd hyperaccumulator screened in the laboratory early, which was purchased from a plant farm in Huichang county,Jiangxi province Soybean (Glycine max (L)Merrill) is a dicotyledonous herb of the genus legume and is cultivated worldwide The soybean variety "Ruidou No 1" was adopted in this experiment, purchased from Zhencheng Agricultural Materials Management Department in Muyang county, Jiangsu province

22 Intercropping experiment

The experiments were set with Cd treatment conditions at concentrations of 0, 5 and 10 mg kg^-1and three planting methods of P frutescensmonocropping, soybean monocropping and P frutescenssoybean intercropping in potted soil culture experimentsAfter the soil was aged, the soybean and P frutescensseeds were soaked firstlyand then put directly in the Cd-treated potting soil The planting method of 5 cm×5 cm intervals was used in each treatment, and 30 P frutescensand 30 soybeans were planted in each potIntercropping planting mode were set as 1:1 By externally adding with Hoagland nutrient solution, same nutrient supply and soil moisture content of soybeans and P frutescensof were ensued during cultivationSamples of soybeans and P frutescensof each treatment were collected during the soybean bulging periodThe fresh plant samples were cleaned with distilled water, then quickly frozen with liquid nitrogen, and kept in a refrigerator at -20°C for the determination of physiological and biochemical indexSome plant samples were weighed on the electronic balance as fresh weight, then dried in the oven at 105℃to constant weight at 72℃ for the measurement of the dry weight of plants and the determination of Cd contentAfter the plants were collected, the soil samples were collected immediately, then were mixed evenly, air-dried, and passed through a 30-mesh sieve for use

23Experiment methods for plant samples

The biomass of plant samples was measured by tape and balanceThe determination of net photosynthetic rate: taking three function leaves from plants for measurement by the portable meter plant photosynthesis (LI-COR6400,LI-COR), and select 3 strains of each processingDetermination of chlorophyll content: referring to the method of Wang et al (2006)The activity of antioxidant enzymes was determined by ultraviolet spectrophotometry:0500 g fresh sample of plant leaves was taken and ground into a powder with liquid nitrogen Plant sample powder were collcted from the previous step into a 10 ml centrifuge tube, and added 8 ml of 50 mmol L^-1 phosphate buffer (pH 78) containing 1% PVP After mixing, the obtained samples were centrifugated at 10000 r min^-1 and 4℃ for 20 min in a high-speed cryogenic centrifuge Malondialdehyde (MDA) content was determined by the thiobarbituric acid method, catalase (CAT) activity was determined by the ultraviolet absorption method, and peroxidase (POD) activity was determined by the guaiacol method (Wang et al, 2006)

The determination of organic acid content was slightly modified according to Chen et al (2018) The plant roots, stems and leaves are dried at 60℃ to a constant mass, then were crushed and passed through a 60-mesh sieve (05~12 mm), and stored under dry conditions for later useAccurately, 01 g of the plant sample were weighed, then 10 mL of 01% potassium dihydrogen phosphate solution were added to dissolve it, and were soak for 4 hours, ultrasonically extracted for 30 minutes, filtered and centrifuged for 10 minutes (3500 r min^-1)The supernatant was filtered through a 045 μm organic filter membrane and sampled for analysis

Determination of Cd content in plant samples: The Cd determination method in the national food safety standard (GB500915-2014) was adopted (National Health Commission, 2015)The digested plant samples’ Cd content was determined by atomic absorption spectrometry (WFX-130A, Beijing Beifen-Ruili) The determination of different Cd forms in soil was based on Bi et al (2006)

24 Data analysis

The ANOVA in Microsoft Office and IBM SPSS Statistics 210 software was used for data processing, analysis and mapping, and Duncan's new complex range method was used for significant difference test at a level of P< 005 All data in the table are mean ± standard deviation (n=3)

Bioconcentration factor (BCF) = total Plant Cd content/soil Cd content

Transfer coefficient (TF) = Aboveground Cd content/underground Cd content of plants

31 P frutescensand soybean biomass under intercropping

As shown in Table S1, under the three Cd treatments, the biomass of intercropped soybean increased by 1042%~10658% compared with mono-cropped soybean The results showed that with the increase of Cd concentration, the biomass of mono-cropped soybeans first decreased and then increased, the biomass of intercropped soybeans first increased and then decreased, and the biomass of intercropped soybeans was significantly higher than that of mono-cropped soybeans With the increase of Cd concentration, the biomass of P frutescens decreased Interestingly, the biomass of intercropping P frutescens was significantly lower than monocropping

32 Effects of intercropping on Cd content and Cd absorption capacity of soybean and P frutescens organs under Cd stress

321Cd content and absorption capacity of soybean

The Cd content of soybean organs is shown in Fig 1 Under 0 mg kg^-1 Cd treatment, the Cd content of intercropped soybean roots was significantly lower than that of monocropping by 7027% In addition, no Cd was detected in other soybean organsUnder 5 and 10 mg kg^-1 Cd treatments, compared with monocropping, the Cd content of intercropping soybean roots, stems, leaves, pods and grains decreased significantly by 1072%~3174%, 1488%~2168%, 1995%~3413%, 2222%~5270% and 791%~1786% The BCF of intercropping soybean was significantly decreased by 1486%~2787% Under 5 mg kg^-1 Cd treatment, the TF of intercropped soybean was significantly reduced by 1500%, while under 10 mg kg^-1Cd treatment, it was significantly increased by 1200% (Table S2)These results confirmed that intercropping significantly reduced the Cd content and Cd absorption capacity of soybean

322 Cd content and absorption capacity of P frutescens treated

The content of Cd in all organs of P frutescensis shown in Fig 1 Under 0 mg kg^-1 Cd treatment, the Cd content in the roots, stems and leaves of the intercropping P frutescenswas significantly increased by 11898%, 3092% and 3689%, respectively There was no Cd detected in the spicas of the intercropping P frutescens Under 5 and 10 mg kg^-1 Cd treatments, in intercropping P frutescens,Cd content of roots, stems, leaves and spicas were significantly increased by 8700%~2141%, 948%~2155%, 2305%~3766% and 1818%~9412% Under 0, 5 and 10 mg kg^-1 Cd treatments, the BCF of intercropping P frutescenssignificantly increased by 2000%~7775% The TF of intercropped P frutescensdecreased by 4043% and 2941% under 0 and 5 mg·kg^-1Cd treatments, while there was no significant change under 10 mg·kg^-1Cd treatment (Table 2)These results showed that intercropping significantly increased the Cd content and Cd absorption capacity of P frutescens

33 Effect of intercropping on physiological characteristics of soybean and P frutescens

331 Effect of intercropping on chlorophyll of soybean and P frutescens

The Chlorophyll A (Chla) andTotal chlorophyll (ChlT) content of intercropped soybean leaves were significantly increased by 209% and 376%, and there was no significant difference in Chlorophyll B (Chlb) content under 0 mg kg^-1 Cd treatmentUnder 5 and 10 mgkg^-1 Cd treatments, the content of Chla, Chlb and ChlTof intercropped soybean leaves were obviously increased by 370%~904%, 1446%~3529%, and 546%~1500% (Fig 2) The content of Chla, Chlb and ChlTof intercropping P frutescenswas significantly increased by 6214%, 7048% and 6217% under 0 mg·kg^-1 Cd treatmentHowever, under 5 and 10 mgkg^-1 Cd treatments, the content of Chla, Chlb and ChlT in intercropping P frutescenswas reduced by 785%~833%, 487%~1771% and 715%~756% The net photosynthetic rate (Pn) of intercropped soybean leaves was significantly increased by 7121%, 19429% and 31278%under different Cd treatmentsInterestingly, the Pn of intercropping P frutescensleaves significantly decreased by 3056%, 5076% and 4490% This study found that under the same Cd stress, intercropping significantly increased soybean chlorophyll content and Pn, while significantly reducing P frutescens’s(Fig 2)

332 Effects of intercropping on malondialdehyde content and antioxidant enzyme activities of soybean and P frutescensleaves

Under 0, 5, and 10 mg kg^-1 Cd treatments, themalondialdehyde (MDA) contents of intercropped soybean leaves not only caused a certain degree of reduction by 1692%~1152%, but also the activities of POD and CAT were significantly increased by 1345%~2337% and 1578%~7381% Similarly, P frutescens appeared the same results The MDA content of P frutescensleaves was significantly reduced by 5765%~7278%, and the activities of POD and CAT were increased by 1562%~2572% and 12056%~15331% under intercropping In summary, intercropping reduced the MDA content, and increased the CAT and POD activity of soybeanand P frutescens leaves(Table S3)

34 Effect of intercropping on the organic acid content of soybean and P frutescens

341 Determination of organic acid content

Under the set chromatographic conditions, the mixed standards containing eight organic acids are completely separated within fourteen minutes The standard chromatogram measured by HPLC is shown in Fig 3, and the linear regression equation is shown in Table 4Chromatographic conditions: Chromatographic column is ZORBAX SB-C18 (250 mm×46 mm, 5 μm); mobile phase: methanol (A); 01% potassium dihydrogen phosphate solution (B); Isocratic elution: 5%A~95%B; flow rate 08mL min^-1; injection volume 10 μL; detection wavelength 210 nm; column temperature 30℃

342 Correlation between Cd content and organic acid in various organs of soybean and P frutescens under different planting methods

It can be seen from Table 1 that more organic acids were significantly positively correlated with Cd content in mono-cropped soybeans than in intercropped soybeans, and the organic acids with the largest correlation coefficient were citric acid, succinic acid, tartaric acid and oxalic acid There was no significant difference between the organic acids of mono-cropped P frutescensand intercropped P frutescens The largest correlation coefficients were citric acid, tartaric acid and oxalic acid of intercropped P frutescens

The correlation coefficient of organic acids and Cd in the roots of intercropped soybean was higher than that of mono-cropped soybean except fumaric acid and tartaric acid, and the greatest correlation was citric acidThe number of species with a positive correlation between organic acid and Cd content in intercropped soybean stems was less than that in mono-cropped soybean Among them, the greatest correlation was oxalic acid and tartaric acid The number of species with a positive correlation between organic acid and Cd content in intercropped soybean leaves was less than that in mono-cropped soybean, and the greatest correlation was citric acid Except that, the correlation coefficient of fumaric acid and Cd in stems of intercropped P frutescenswas higher than mono-cropped The correlation coefficient of organic acid and Cd in other organs of intercropped P frutescenswas lower than mono-cropped

In general, intercropping changed the correlation between Cd content and free organic acids in P frutescensand soybean Citric acid, oxalic acid and tartaric acid were the most important in soybean, meantime, tartaric acid, citric acid and succinic acid was the most important in P frutescens

Table1 Correlation between Cd content and organic acids in roots, stems and leaves of soybean and P frutescens

Plant	Organs	Treatment	Malic acid	Citric acid	Succinic acid	Fumaric acid	Oxalic acid	Tartaric acid	Total organic acid
Soybean	Root	M	-0984**	0982**	-0887**	0933**	-0521	0721*	0982**
	Root	I	0346	0984**	-0416	0514	0275	0347	0986**
	Stem	M	0578	0998**	0964**	0348	0339	0176	0998**
	Stem	I	-0805**	-0854**	0198	-0963**	0950**	0905**	-0819**
	Leaf	M	0485	0023	0990**	0675*	-0963**	-0428	0769*
	Leaf	I	-0752*	0075	-0085	-0926**	-0347	-0045	-0259
Perilla frutescen	Root	M	-0118	0886**	-0016	-0249	0271	0955**	0861**
	Root	I	-0403	0127	-0421	-0506	-0798**	0912**	006
	Stem	M	-0678*	0963**	-0940**	0677*	0821**	a	0965**
	Stem	I	-0931**	0881**	-0675*	0880**	-0288	a	0868**
	Leaf	M	0727*	0921**	0565	-0857**	0424	a	0858**
	Leaf	I	0438	-0092	0560	-0905**	-0084	a	-0199

Note: "*" indicates significant correlation at 005 level; "**" indicates a significant correlation at 001 level "a" At least one variable is a constant, so it cannot be calculated

35 Effect of P frutescens and soybean intercropping on Cd forms in cadmium-contaminated soil

There are five forms of Cd in soil, namely, exchangeable form (EXC-Cd), carbonate binding form (Crab-Cd), iron and manganese oxidation form (FeMnO₂-Cd), organic form (OM-Cd) and residual form (RES-Cd) As shown in Fig 4, in the soil by monocropping soybean and monocropping P frutescens, the content of FeMnO₂-Cd was the largest, accounting for 34% and 36% under the condition of 0 mg kg^-1 Cd treatmentHowever, under the conditions of 5 and 10 mg kg^-1 Cd treatments, the EXC-Cd content was the highest, accounting for 37% and 35% in monocropping soybean soil, and 35% and 32% in monocropping P frutescens soil Remarkably, EXC-Cd content in the soil of the intercropping planting mode is the largest, even higher than that of the monocropping soybean and P frutescens, accounting for 37% to 42% (Fig 4)

In conclusion, in intercropping mode, the proportion of EXC-Cd increased with increasing Cd concentration, and EXC-Cd content was significantly higher than that in monocropping mode, and it accounted for the largest proportion of chemical forms of Cd in soil

41 Effects of the intercropping system on biomass and photosynthesis of P frutescens and soybean

Different plants possess different uses of soil nutrients, water, light, and heat, thereby brings differences to the growth, which depends on photosynthesis and antioxidant systems (Mnk et al, 2020) of plants in intercropping system Chlorophyll plays a very keystone role in the process of photosynthesis (Paul et al, 2017)Intercropping can change the utilization of light energy by varying the chlorophyll content of plants，so the photosynthetic capacity and dry matter accumulation of leaves changed (Li et al, 2019)In this study, the biomass, chlorophyll content and net photosynthetic rate of intercropped soybean was significantly higher than that of mono-cropped, while those of intercropped P frutescenswere significantly lower than mono-cropped P frutescens,which indicated that intercropping patterns could significantly promote the growth of soybean and inhibited the growth of P frutescens For one thing, it is speculated that soybean and P frutescens had the same needs or uses for soil resources, so interspecific competition was gradually formed during the growth process As more nitrogen, phosphorus and organic matter in soil utilized by soybean, a disadvantageous position of P frutescenshad been resulted The other assumption suggested that P frutescenshave a strong ability to absorb heavy metals in the soil, so the absorption of heavy metals by soybean reducedCertainly, excessive Cd would bring necrosis and withering of plant leaves, photosynthesis efficiency decline, biomass reduction and even death (Xie et al, 2011; Yang et al, 2020)Tang et al (2020) found that compared with monoculture, the contents of chlorophyll A, total chlorophyll and carotenoids were significantly increased in intercropped spinach, and this result is consistent with our experimentAfter intercropping of hyperaccumulator Tagetespatulawith Brassica chinenesis, the biomass of Tagetespatula increased significantly while the biomass and net photosynthetic rate of Brassica chinenesisdecreased significantly (Yan et al, 2020), which is contrary to the results of this experimentIt is suggested thatthe competitive ability of Tagetespatula for soil nutrients and other resources was significantly stronger than that of Brassica chinenesisTherefore, intercropping with suitable hyperaccumulators and crops can restore soil and maintain crop yield

42 Effects of the intercropping system on Cd absorption capacity of P frutescens and soybean

Plant absorption of heavy metal Cd in soil is mainly related to the form of Cd in soil Tessier et al (1979)divided forms of heavy metals in sediment or soil into five forms: exchangeable form (EXC-Cd), carbonate binding form (Crab-Cd), iron and manganese oxidation form (FeMnO₂-Cd), organic form (OM-Cd) and residual form (RES-Cd)Easy being transferred and transformed, these five forms can change with the environmental conditions (Giasson et al, 2005; Shang et al, 2016)They also have very different bioavailability Heavy metals in Crab and EXC forms have high bioavailability, which can be easily absorbed by plants, while the FeMnO₂ form and OM form are not easy to be absorbed directly, but can be transformed into EXC form and Crab form under certain redox conditions Lastly, the RES form is generally not bioavailable (Fei et al, 2012; Zu et al, 2014)In this work, 0 mg kg^-1 Cd intercropping significantly increased the EXC-Cd content in soil, accounting for 3716% of the total, and significantly decreased the Crab-Cd, OM-Cd and RES-Cd contentUnder 5 and 10 mg kg^-1Cd treatments, the intercropping mode significantly increased EXC-Cd in soil, separately at a proportion of 3963% and 4206%, and reduced FeMnO₂-Cd and OM-Cd content It is clear that intercropping could significantly increase the content of bioavailable Cd in soil under Cd stressThese results indicated that soil EXC-Cd was converted from Crab-Cd, FeMnO₂-Cd and OM-Cd under intercropping conditionsThere are two possible reasons for this phenomenonFirstly, low-molecular-weight organic acids, such as oxalic, and citric et al could form soluble complexes with Cd, which would be released from the soil matrix then (Krishnamurti et al, 1997) In this study, the bioavailability of Cd in the soil enhanced may be resulted in the root exudates, mainly organic acids, released by soybean and P frutescens roots As is known to all, leguminous plants can release a large amount of H⁺ into the soil in the process of biological nitrogen fixation to acidify the soil (Chang et al, 2006) So the second reason is that the low pH value of the soil will increase the dissolution and release of heavy metals such as carbonate and Fe-Mn combination (Sappin-Didier et al, 2005; Yang et al, 2005)Furthermore, the Cd enrichment amount and enrichment coefficient of intercropped P frutescens were significantly higher than those of mono-cropped, confirming that most EXC-Cd released from soil was absorbed by P frutescensHowever, the hyperaccumulatorP frutescensmay have a similar "foraging" for heavy metals in the soil environment (Guo et al, 2021), which inhibits the absorption of heavy metals in soybeansInterestingly, Tan et al (2016)found that EXC-Cd in soil was significantly lower at the maturity stage than seedling stage of crops, which was inconsistent with the results of this study It was indicated that the intercropped P reticulum grew faster, having greater biomass, and absorbed more Cd from the soil, which led to the decrease of exchangeable Cd content in the soil during the mature stage Different from the P reticulum, the biomass of P frutescenssignificantly decreased in intercropping conditions, which could not completely absorb the EXC-Cd in the soil, increasing exchangeable Cd content in the soilThese results suggest that the accumulation of heavy metals in soil by plants depends on the bioavailability of heavy metals in soil and which species are under intercropping treatment In this study, it was found that intercropping increased the bioavailability of Cd, and more EXC-Cd was absorbed by P frutescens

In this study, Cd in roots of soybean and roots, stems, and leaves of P frutescens were detected under 0 mg kg^-1Cd treatmentIt is showed that P frutescenscould still absorb Cd from soil and transport it to stems and leaves from roots when the background concentration of Cd in soil is low Apparently, P frutescenshas a stronger ability to deal with Cd stress problem than soybeanUnder the same Cd stress condition, the Cd content and its enrichment coefficient of soybean under intercropping conditions were significantly lower than those of mono-cropped Conversely, the Cd content and Cd enrichment coefficient of intercropped P frutescenswere significantly higher than those of mono-cropped The transfer coefficient of intercropped soybean first decreased and then increased with increasing Cd content while that of intercropped P frutescensdecreased The transfer coefficient of soybean in intercropping was significantly increased under 10 mg kg^-1 Cd treatment compared with that in monocropping It is likely that the biomass of soybean in intercropping was significantly increased, and the Cd content was higher In order to improve the Cd tolerance of soybean, the aboveground biomass provided more locations for heavy metals to accumulate This situation would increase the transfer coefficient so as to avoid the negative impact on plants caused by the excessive accumulation of heavy metals in the ground(Zhang et al, 2020)Transfer coefficient of intercropped P frutescensdecreased with the increase of Cd content The reason for this phenomenon is that the P frutescens above-ground biomass decreased and enabled to provide more heavy metal accumulation locations This change allows P frutescens to retain more of the heavy metal Cd in its underground parts, which is consistent with the study of Xieet al (2011)In conclusion, under intercropping patterns, Cd enrichment of soybean mainly occurred in the roots, and then gradually transferred to the stems and leaves with the increase of biomass On the contrary, P frutescens’suptake and tolerance of Cd is stronger, after the absorption of Cd, quickly from their roots transferred to the stems and leaves, whichled the biomass and transfer coefficient decreases

43 Resistance mechanism of antioxidant enzymeactivity system and organic acids to Cd

Heavy metal stress can cause plants to produce a large amount of active oxygen free radicalsExcessive accumulation of reactive oxygen species will break the balance and lead to protein degeneration, DNA damage, cell enzyme inactivation and membrane lipid peroxidation damage, and evendeath(Wang et al, 2014; Jiang et al, 2016)The antioxidant enzyme system is an important mechanism of plant tolerance under heavy metal stress and plays a keystone role in plant adaptation to stress(Zhang et al, 2014)The changes in antioxidant enzyme activities and MDA content in plants were closely related to Cd stress (Yi and Kao, 2007)POD can regulate the level of indole-acetic acid (IAA) in plants and avoid the toxic effect of H₂O₂ in the organism CAT converts H₂O₂ to H₂O and O₂ and participates in the glutathione-ascorbic acid cycle to remove H₂O₂ from cells, and the change of MDA content could reflect the damage degree of the plant cell membrane(Jamieson et al, 2012; Chen et al, 2014a; Guo et al, 2019)In this study, intercropping increased CAT activity and POD activity of soybean and P frutescens leaves under Cd stress, and decreased MDA content under Cd stress The finding of Tang et al (2017)also confirmed that the activity of antioxidant enzymes increased under the intercropping of Solanum nigrum and Solanum photeinocarpum, which was consistent with this studyThe resistance of antioxidant enzymes to heavy metal stress is a complex physiological process, which is affected by plant species, heavy metal concentration and propertiesFor example, the SOD activity of intercroppedSolanum nigrum and Lycopersiconesculentum had no significant difference compared with that of monoculture, but the POD and CAT activities were significantly increased(Hu et al, 2019)In this study, the changes in the antioxidant enzyme activity systembrought by inter-copping could reduce the oxidative stress response of cells to a certain extent and improve the heavy metal tolerance of P frutescensand soybeanHowever, the intercropping of hyperaccumulators and crops may produce opposite results in this study, such as the intercropping of hyperaccumulators Pteris vittata L and Broussonetiapapyrifera L, which decreased POD activity and increased MDA content in leaves of Broussonetiapapyrifera L(Peng et al, 2018)That`s because Pteris vittata L is an Ashyperaccumulator,with the increasing of root exudates, the absorption of As increasedOn the contrary,Broussonetiapapyrifera L is not a hyperaccumulator but inhibited by ASTherefore, only by selecting suitable plants for intercropping can the toxicity of reactive oxygen species to plants under heavy metal stress be alleviated to varying degrees by increasing antioxidant enzyme activity and reducing MDA content without damaging plant growth

As the ligand of heavy metal elements, organic acids have multiple functions, including participating in the absorption, transportation, and accumulation of heavy metals These functions can not only promote the accumulation of metals in plants, but also improve the tolerance of plants to heavy metals (Henry et al, 2007)When plants are stressed by heavy metals, many plants can actively respond to organic acid metabolism in order to improve their resistance Organic acids in plants combine with heavy metal ions to form complexes, which are deposited in cell walls or vacuoles, reducing their free concentration in plants and thus the toxicity of heavy metals decreased(Polle and Schützendübel, 2003)As we all known, the molecular weight of each organic acid is different, and the coordination ability with Cd ion is also different Therefore, it is of little significance to compare the absolute amount of each organic acid to measure the detoxification ability of organic acid in P frutescensand soybean with the change of Cd concentration For this reason, in intercropping system of P frutescens and soybean, there were two explanationsin regulation and detoxification mechanism of Cd in these two plantsIt can be seen from Table 5 thatin the intercropping soybean’s roots and leaves, citric acid has the largest correlation coefficient with the Cd content Meanwhile，in the intercropping soybean stems, oxalic acid has the largest correlation coefficient with the Cd contentThis showed that under the intercropping system, the citric acid and oxalic acid in soybeans played a key role in detoxification of heavy metalsP frutescens in the intercropping mode, the tartaric acid in the roots, citric acid in the stems, and succinic acid in the leaves have the largest correlation coefficientAccording to the correlation coefficient, tartaric acid, citric acid and succinic acid in P frutescensplays a greater role in the detoxification processDifferent kinds of organic acids have different complexing and detoxification abilities with heavy metals in plants For example, the stability coefficient of citric acid complexing with Cd (10³¹⁵⁾ is significantly higher than that of malic acid (10¹³⁴)Thus, the low-toxic or non-toxic complex formed by citric acid and Cd in plants is more stable and plays a certain role in detoxification(Shi et al, 2003) At the same time, studies have shown that the extraction rate of succinic acid for heavy metals is similar to the extraction rate of citric acid(Wu et al, 2011),which indicates that succinic acid can also form non-toxic or low-toxic complex complexes with CdOxalic acid plays an important biological role in plants and participates in the detoxification of metals(Klug and Horst, 2010)Oxalic acid is mainly involved in the transport of Cd in soybean stems, transporting heavy metals to various organs of soybeanTartaric acid has an obvious detoxification effect on Cd and can reduce the toxicity of plants(Chen et al, 2000)Cd content in plants is closely related to the synthesis and accumulation of organic acids in plant species and organs of the same plant Studies on exogenous addition of organic acids have proved that organic acids can reduce the toxicity of heavy metals to plants(Guo and Zhuang, 2021)Therefore, organic acids can complex with heavy metals in plants, reducing the chance for heavy metals to bind to important proteins and enzymes in cells, thus reducing the toxicity of heavy metals in plants

Thus, antioxidant enzymes can alleviate the toxicity of ROS to plants under heavy metal stress in varying degrees Organic acids can also participate in the detoxification process of heavy metals, which also explained why P frutescenscould still grow after accumulated a large amount of heavy metals

In this study, the intercropping of P frutescensand soybean could not only enhance the tolerance ofhyperaccumulatorP frutescensto Cd and improve its ability to absorb heavy metals, which is a "win-win" model The intercropping system can strengthen the efficiency of phytoremediation, but not damage the economic benefits of farmers, and reduce the content of heavy metals in the soil, so the intercropping system of P frutescensand soybean has important practical significance

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Availability of data and materials

All data generated or analyzed during this study are included in this published article [and its supplementary information files].

Competing interest

The authors declare that they have no known competing interests.

Funding

This research work was supported by the Natural Science Foundation of Fujian Province, China (Grant No.2019J01383) in the design of the study and collection, and Fujian Agriculture and Forestry University Science and Technology Innovation Special Fund (Grant. No. CXZX2018053) in the analysis, interpretation of data and in writing the manuscript.

Author contributions

All authors contributed to the study conception and design. Bolun Han: Conceptualization, Software, Writing-original draft; Wan Zhan: Resources, Investigstion, Data curation; Rongrong Fan: Data curation, Software, Writing-review & editing; Rui Jing: Writing-review & editing; Ruiyu Lin: Writing-review & editing; Haibin He: Writing-review & editing; Xinyu Zheng: Methodology, Writing-review & editing, Supervision, Data curation. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Acknowledgements

I would like to express my gratitude to all authors and the Natural Science Foundation of Fujian Province, China (Grant No.2019J01383), and Fujian Agriculture and Forestry University Science and Technology Innovation Special Fund (Grant. No. CXZX2018053).

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Effects of Intercropping Patterns on Cadmium Tolerance of Perilla Frutescens and Soybean

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