Arbuscular mycorrhizal fungi alleviate arsenic toxicity in Sophora viciifolia Hance . 1 by improving the growth , photosynthesis , reactive oxygen species and gene 2 expression of phytochelatin synthase

Arbuscular mycorrhizal fungi alleviate arsenic toxicity in Sophora viciifolia Hance. 1 by improving the growth, photosynthesis, reactive oxygen species and gene 2 expression of phytochelatin synthase 3 QiaoMing Zhang, Minggui Gong, Shanshan Xu, Angran Zhang, Jiangfeng Yuan, 4 Qingshan Chang 5 College of Forestry, Henan University of Science and Technology, Luoyang, Henan 471023, 6 China 7 College of Food and Bioengineering, Henan University of Science and Technology, Luoyang, 8 Henan 471023, China 9


INTRODUCTION 44
Arsenic (As) element is often defined as 'heavy metals' (HMs) due to its high density, and it 45 is one of widespread trace components in the earth's crust 1 . The normal concentration of As in 46 soil and water usually does not exceed 10 mg kg -1 2, 3 . In the past decades, As contamination in 47 the terrestrial ecosystem is getting worse due to excessive anthropogenic activities, such as 48 mining of As ores, smelting of metal, burning of fossil fuel, irrigating croplands with As-49 contaminated groundwater, and using As-based agrochemical and phosphate fertilizers 4-7 . In 50 consequence, the accumulation in agricultural products and water is continuing to rise beyond 51 the threshold values for the dietary recommendation, and pose significant problems for human 52 health through food chains 1 . As toxicity represents a threat to natural ecosystems, and As is a 53 potent carcinogenic substance for humans and other organisms 3 . As ions, which are present in  Therefore, the HM toxicity can be neutralized and alleviated, and this process are regarded as 74 a critical detoxification mechanism of HM in plant cells 5 . Some HM ions, such as Pb, Cd, Hg, 75 Cu, Cr and As, can induce to PCs synthesis. Howerver PCS genes in some higher plants are 76 isolated and described, and overexpression of Arabidopsis PCS genes from other plant species 77 directly led to the higher HM resistance 1 . 78 Higher plants, which are adapted to As-contaminated soils, are generally associated with 79 arbuscular mycorrhizal fungi (AMF) 6 . More than 80% of terrestrial higher plants can establish 80 symbiotic relationships with AMF 12,13 . AMF play an essential role as a "bridge" between plants 81 and rhizosphere soil, which acquire carbohydrate compounds and lipids from host plants, and 82 in return, transfer mineral nutrients (e.g., potassium (K), phosphorus (P), and nitrogen (N)) from 83 rhizosphere soil to host plants by their arbuscules and hyphal coils 14,15 . AMF enhance mineral 84 nutrient uptake of plant and soil quality, which improves the plant resistance to HM stress and 85 alleviates HM toxicity in host plants 12  symbiosis between AMF and woody legumes maybe represents a good strategy for the 127 restoration of vegetation and phytoremediation under As-contaminated soils.

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AM colonization rate. AMF colonization was readily detected in the roots of R. intraradices-130 inoculated S. viciifolia, but not found in the roots of non-inoculated plants ( Table 1). The 131 8 colonization rates of S. viciifolia by R. intraradices were 65.0%, 56%, and 43.5%, at 0, 50 and 132 100 mg kg −1 As levels. The addition of As to soils had a significantly negative influence on R. 133 intraradices colonization in S. viciifolia roots, which was decreased with the increase in As 134 concentration in soils.

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Plant growth. The two-way ANOVA revealed significant effects of As level and R. 136 intraradices inoculation on the growth of S. viciifolia seedlings (Table 1). Plants showed 137 symptoms of As toxicity, such as leaves wilting and yellowing, when exposed to high As stress.

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The shoot and root dry weight, plant height, and root length of S. viciifolia seedlings were 139 restrained with the increased As concentrations in soils (P < 0.01), except that the shoot dry 140 weight in S. viciifolia seedlings and root length in R. intraradices-inoculated seedlings had no 141 significant difference between 0 and 50 mg kg −1 As level.

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The shoot and root dry weight, plant height, and root length in S. viciifolia seedlings were 143 obviously benefitted by R. intraradices-inoculation (P < 0.05). R. intraradices-inoculated S. 144 viciifolia seedlings had greater shoot and root dry weight, plant height, and root length than non-145 inoculated S. viciifolia, irrespective of the As treatment. But the root length in 0 mg kg −1 As 146 level had no significant difference between R. intraradicesand non-inoculated seedlings.

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The As concentration in S. viciifolia shoots and roots enhanced with the increasing As level 148 in soils, irrespective of R. intraradices inoculation (P < 0.05). As concentration in S. viciifolia 149 shoots and roots was obviously decreased by R. intraradices inoculation at the same As level 150 in soils, except for that in 0 mg kg −1 As level. Significant interactive effects of As level × R. 151 intraradices inoculation on As concentrations in the shoots and roots were also found in this 152 study. Furthermore, As concentration in leaves had a very lower value, compared to roots at the 153 9 same As level in soils.

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Gas exchange and chlorophyll fluorescence. As stress had a noticeable effect on the 155 parameters of gas exchange in S. viciifolia plants (P< 0.01), which depressed the Pn, gs, and E, 156 and improved the Ci (Fig 1). But there were no significant differences in the Pn between 0 and 157 50 mg kg −1 As levels in R. intraradices-inoculated seedlings. Inoculation with R. intraradices 158 in the roots of S. viciifolia remarkably increased the Pn and gs and decreased the Ci (P < 0.01).

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But it had no obvious effect on the E, irrespective of R. intraradices-inoculation. The results of 160 two-way ANOVA indicated the Pn, Ci and E exhibited highly significant differences with the 161 interaction between As level in soils and R. intraradices-inoculation (P<0.01).

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The Fv/Fm, PSII, and qP in S. viciifolia leaves were obviously decreased, and the NPQ was 163 significantly increased with enhancing As level in soils (P< 0.01) (Fig 2). Except that the PSII 164 in R. intraradices-inoculated seedlings has an apparent difference between 0 and 50 mg kg−1 165 As levels. Inoculation with R. intraradices significantly increased the Fv/Fm, PSII, and NPQ, 166 and decreased qP, compared with non-inoculated plants in the same As level (P< 0.01). As treatments. Still, the H2O2 concentration was the highest at 100 mg kg −1 As, under both R.  were increased with the enhance of As level (P < 0.01), and they were highest at 100 mg kg −1 185 As (Fig 4). Inoculation with R. intraradices obviously increased the SOD, POD, and CAT were found in this study (P < 0.01). Furthermore, the expression level of SvPCS1 gene in the 203 roots were significantly higher than those in the leaves at the same As level and R. intraradices-204 inoculated treatment (P < 0.01).

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The colonization rate of R. intraradices in S. viciifolia roots decreased with the increase of 207 the As concentrations in our study. This result was in accordance with other pot-based studies 208 using Glycine max L. 17 , Lolium perenne L. 24 , and Helianthus annuus L. 25 under As stress. In 209 contrast, some studies reported no decrease in AM colonization rate 21 , and also an increase 26 , 210 when the As solution was artificially added into soils. Despite this difference of AM 211 colonization rate to As stress, each AMF symbiosis conferred the benefits to host plants 6 .

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The plant performance against HM stress and the symbiosis efficiency of AMF-inoculation 213 could be visually reflected by plant biomass 27 . High levels of As in soils often jeopardized 214 normal plant growth with toxicity symptoms like biomass decrease, stagnation in plant growth, 215 wilting, and necrosis of leaf blades 28 . In our study, the growth parameters of S. viciifolia 216 seedlings were restrained with the increasing the As concentration in soils. The lower AM 217 colonization rate in As contaminated soils did not mean that the symbiosis was not effective, 218 and R. intraradices-inoculated S. viciifolia grew better than non-inoculated seedlings at all As 219 12 levels, which suggests that the R. intraradices symbiosis played a positive role in mitigated As 220 stress in S. viciifolia seedlings. AMF hyphae colonized inside root cells and formed vesicle and 221 arbuscular structures, they enhanced As binding in roots, and restrict its further translocation to 222 shoots, thus inhibiting As toxicity 12 . Compared with the non-inoculated controls, inoculation 223 with Glomus mosseae decreases As accumulation in Trifolium repens and Lolium perenne 224 seedlings and this inoculation resulted in greater plant biomass and more As tolerance of host 225 plants 24 . Enhanced parameters of plant growth under As stress were closed due to improved 226 mineral nutrition through AM extraradical hyphal networks 16,29 . In the present study, As 227 concentrations in the shoots and roots of S. viciifolia seedlings increased proportionately with 228 the As level added into the soils, however, As concentrations in the roots and shoots of R. 229 intraradices-inoculated S. viciifolia were obviously lower than those of non-inoculated 230 seedlings, which indicated that R. intraradices symbiosis decreased the As accumulation in S. HM toxicity, which was also closely related to improving antioxidant defence for host plants 30 .

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When higher plants were exposed to As stress, higher production of O 2·-, OH-, perhydroxyl 235 (HO 2-), singlet oxygen (1O 2-), and H2O2 resulted from the transform of As(V) to As(III) 6,31 . The up-regulated the gene expression of SvPCS1in S. viciifolia seedlings, which showed that R. 325 intraradices alleviated the As toxicity and decreased As accumulation in S. viciifolia seedlings. 326 Thus, this investigation demonstrated the significant role of AMF in the resistance of host plants 327 to As toxicity. AMF inoculation was recommend as a potential contributor to alleviate As stress 328 in leguminous species. Further studies would focus on the cooperative mechanism of PCS gene 329 17 expression in both AMF and host plants under As toxicity stress.

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Experimental design. This experiment consisted of six treatments and was set up in a complete 332 randomized block design with two factors: (1) AMF treatments, i.e., R. intraradices and a non-333 AM inoculated control; (2) three As levels in soils, i.e., 0, 50, and 100 mg As/kg dry soil.  were respectively added into per 1 kg dry soil mixture, which was stirred fully with the blender.

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Eventually, three As concentrations in soils (0, 50, and 100 mg As per kg dry soil) were prepared.

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For this potted experiment, the soil mixture was autoclaved for 2 h at 121 C and 0.11 MPa 345 prior to use.