Inoculation with abscisic acid (ABA)-catabolizing bacteria can improve phytoextraction of heavy metal in contaminated soil

Journal Pre-proof Inoculation with abscisic acid (ABA)-catabolizing bacteria can improve phytoextraction of heavy metal in contaminated soil Qi Lu, Yineng Weng, Yue You, Qianru Xu, Haiyue Li, Yuan Li, Huijun Liu, Shaoting Du PII:

S0269-7491(19)34207-1

DOI:

https://doi.org/10.1016/j.envpol.2019.113497

Reference:

ENPO 113497

To appear in:

Environmental Pollution

Received Date: 1 August 2019 Revised Date:

18 September 2019

Accepted Date: 25 October 2019

Please cite this article as: Lu, Q., Weng, Y., You, Y., Xu, Q., Li, H., Li, Y., Liu, H., Du, S., Inoculation with abscisic acid (ABA)-catabolizing bacteria can improve phytoextraction of heavy metal in contaminated soil, Environmental Pollution (2019), doi: https://doi.org/10.1016/j.envpol.2019.113497. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

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Inoculation with abscisic acid (ABA)-catabolizing bacteria can improve

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phytoextraction of heavy metal in contaminated soil

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Qi Lu, Yineng Weng, Yue You, Qianru Xu, Haiyue Li, Yuan Li, Huijun Liu, Shaoting Du*

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* Corresponding Author.

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College of Environmental Science and Engineering, Zhejiang Gongshang University,

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Hangzhou 310018, China

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E-mail [email protected]

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Fax: +86-571-88832369

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10

Abstract

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Promotion of plant capacity for accumulation of heavy metals (HMs) is one of the key

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strategies in enhancing phytoremediation in contaminated soils. Here we report that,

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Rhodococcus qingshengii, an abscisic acid (ABA)-catabolizing bacteria, clearly boosts

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levels of Cd, Zn, and Ni in wild-type Arabidopsis by 47, 24, and 30%, respectively, but

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no increase in Cu was noted, when compared with non-inoculated Arabidopsis plants in

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contaminated growth substrate. Furthermore, when compared with wild-type plants,

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R.qingshengii-induced increases in Cd, Zn, and Ni concentrations were more pronounced

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in abi1/hab1/abi2 (ABA-sensitive mutant) strains of Arabidopsis, whereas little effect

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was observed in snrk2.2/2.3 (ABA insensitive mutant). This demonstrates that

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metabolizing ABA might be indispensable for R. qingshengii to improve metal

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accumulation in plants. Bacterial inoculation significantly elevated the expression of Cd,

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Zn, and Ni-related transporters; whereas the transcript levels of Cu transporters remained

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unchanged. This result may be a reasonable explanation for why the uptake of Cd, Zn,

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and Ni in plants was stimulated by bacterial inoculation, while no effect was observed on

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Cu levels. From our results, we clearly demonstrate that R. qingshengii can increase the

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accumulation

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transporters-associated

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ABA-catabolizing bacterial inoculation might be an alternative strategy to improve

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phytoremediation efficiency in HMs contaminated soil.

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Keywords: ABA-catabolizing bacteria, heavy metal, phytoextraction, metal transporter

of

Cd,

Zn,

and

mechanism.

Ni

in

plants

Metabolizing

via

an

ABA

ABA-mediated in

the

plants

HM by

2

31

32 33

The main finding of the work Inoculation with R. qingshengii could increase the accumulation of Cd, Zn, and Ni in plants via an ABA-mediated HM transporter-associated (mostly of IRT1) mechanism

3

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1. Introduction

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Heavy metal contamination of soils resulting from rapid industrialization and

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widespread application of agrochemicals through agricultural activities has received

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considerable attention (He et al., 2019). Owing to their persistence and toxicity, heavy

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metals (HMs) in soils pose a severe threat to soil function, agricultural production, and the

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human food chain (Zhang et al., 2015; Sills et al., 2018). Therefore, there is a growing

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need for cost-effective strategies for soil decontamination. However, conventional

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physical and chemical remediation techniques have a high cost, are generally harmful to

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soil properties, or even produce susceptibility to secondary contamination (Liu et al.,

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2018a). In recent years, phytoremediation, an environment-friendly remediation

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technology, that repairs soil through accumulator or hyper accumulator plants, has become

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increasingly popular (Pidatala et al., 2016). However, large-scale application of

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phytoremediators is hampered by their low efficiency (Yang et al., 2019). As such,

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alternative strategies that can enhance the efficiency of the heavy metal phytoremediation

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process are still urgently needed. Thus, bacteria-associated phytoextraction may be a

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promising strategy (Lebeau et al., 2008; Sessitsch et al., 2013).

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A large number of studies revealed that abscisic acid (ABA), an endogenous

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phytohormone in plants, plays an important role in regulating the accumulation of metals

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in plants (Lauro et al., 2017). For example, exogenous ABA supply could significantly

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decrease cadmium (Cd) levels in Cd exposed-plants such as Arabidopsis (Fan et al., 2014),

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Solanum photeinocarpum (Wang et al., 2016) and Populus euphratica (Han et al., 2016);

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as well as contribute to the reduction of zinc (Zn) concentrations in Populus × canescens 4

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exposed to excessive Zn (Shi et al., 2015); and exogenous ABA could alleviate Ni uptake

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in Ni stressed-rice plants (Oryza sativa L.) (Rubio et al., 1994). Accordingly, exogenous

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ABA application was considered as a potential technique to minimize heavy metal

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accumulation in plants.

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Our recent studies demonstrated that ABA-generating bacteria could increase

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endogenous ABA levels in plants and thereby minimize Cd accumulation in contaminated

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Arabidopsis (Xu et al., 2018) and pakchoi (Pan et al., 2019). Thus, ABA-generating

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bacterial application in contaminated soil was considered as a feasible strategy to

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counteract Cd contamination in agricultural produce. However, the question of whether

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catabolizing the endogenous ABA in plants via a bacterial pathway could promote metal

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accumulation in plants had not been identified. Interestingly, several plant growth

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promoting bacteria (PGPB) that have the ability to sustainably catabolize ABA have been

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identified (Kang et al, 2014; Belimov et al, 2014). In addition, our previous study found

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that inoculation of ABA-generating bacteria could significantly down-regulate the

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expression of Fe-regulated transporter 1 (IRT1, a metal transporter with a broad substrate

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range) in the Cd stressed-plants, which dominantly contribute to the ABA induced-

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inhibition of Cd uptake by the roots (Xu et al., 2018). Accordingly, it is reasonable to

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suppose that inoculation of ABA-catabolizing bacteria into metal contaminated soil

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might promote Cd phytoextraction by enhancing the expression of IRT1. Another

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question arising is whether ABA-catabolizing bacteria could affect the expression of

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other HMs (such as Zn, Ni, and Cu) transporters in roots of stressed-plants, e.g. ZIP

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(iron-regulated transporter (IRT)-like proteins) (Milner et al., 2013), NRAMP (natural 5

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resistance associated macrophage protein) (Pottier et al., 2015; Castaings et al., 2016),

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HMA (A P-type ATPase) (Park and Ahn , 2017), and COPT (copper transporter) families

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(Sanz et al., 2019), among others. If so, the question arises of whether the plants

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inoculated with ABA-catabolizing bacteria were also more likely to accumulate Zn, Ni,

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and Cu but information on this aspect is scarce.

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To answer the above questions, we studied the effects of Rhodococcus qingshengii

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(an ABA-catabolizing bacteria) on the accumulation of HMs in Cd, Zn, Ni, and Cu

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stressed-plants using wild-type Arabidopsis, an ABA sensitive mutant, and an

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ABA-insensitive mutant. Because oxidative stress is a mechanism which inhibits plant

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biomass production during heavy metal stress (Jin et al., 2013) and photosynthetic

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inhibition of plants has also been implicated as a toxicity response to HM stress (Rai et

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al., 2016), these growth vigor indices were also analyzed. In order to further verify the

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pathways in which ABA-catabolizing bacteria promote the accumulation of heavy metals

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in plants, the expression of metal transporter genes in IRT1, ZIP, NRAMP, HMA, and

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COPT families related to Cd, Zn, Ni, and Cu uptake were also determined. IRT1 has broad

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specificity for divalent heavy metals, mediating the transport of Cd, Zn, and Ni (Martos

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et al., 2016; He et al., 2017; Nishida et al., 2011; Zhu et al., 2019); ZIP-transporters 1 and

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4 confer Cd and Zn uptake activity (Upadhyaya et al., 2016; Zeng et al., 2017;

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Shanmugam et al., 2013); HMA3, member of the HMA (A P1B type ATPase) family, are

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responsible for the uptakes of Cd and Zn in roots (Gravot et al., 2004; Moreira et al.,

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2018). In addition, Cd transporters also include NRAMP1, NRAMP3, and NRAMP4

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(Thomine et al., 2000; Pottier et al., 2015), and NRAMP4 is also a Ni transporter 6

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(Mizuno et al., 2005). The copper transporters COPT1 and COPT2 are involved in the

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transportation of Cu (Sanz et al., 2019). Thus, the effects of R. qingshengii on the

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expression of the abovementioned transporters were analyzed. And lastly, IRT1-null

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mutants were applied to further demonstrate the critical role of IRT1 in the promotion of

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metal phytoextraction in plants by ABA-catabolizing bacteria.

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2 Materials and methods

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2.1 Plants, growth substrates, and bacterial strains

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The Col-0 ecotype of Arabidopsis (wt), ABA-associated mutants (ABA-sensitive triple

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mutant abi1/hab1/abi2 and ABA-insensitive double mutant snrk 2.2/2.3), and IRT1

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knockout mutants (irt1-1 and irt1-2) were used in this study. Growth substrates for pot

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experiments in this study comprised Klasmann-Deilmann Gmbh nutrient soil, perlite, and

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vermiculite at 6:1:3 ratio (v/v/v) (Table SI-1). The sterilized growth substrates were

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mixed thoroughly with HMs to final levels of 1.25 mg Cd, 125 mg Zn, 50 mg Ni, and 50

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mg Cu kg-1 growth substrates. After aging for more than four months, the contaminated

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growth substrates were used in the following pot experiment.

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The ABA-catabolizing bacteria, R. qingshengii (BNCC203056), obtained from the

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Bena Culture Collection (BNCC), were used in this study. R. qingshengii was cultured in

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Luria-Bertani's (LB) liquid medium (10 g/L soya peptone, 5 g/L beef extract, and 10 g/L

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NaCl, pH 7.2) and shaken in flasks for 3 d at 30°C at 150 rpm. Then the bacteria cells

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were harvested by centrifugation at 3000 ×g for 10 min. After washing with sterile 0.9% 7

121

NaCl solution, the bacteria cells were resuspended in sterile saline to obtain a bacterial

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suspension with 108 CFU mL-1.

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2.2 Pot experiment

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Arabidopsis seeds were treated with 75% ethanol for 15 min and rinsed with sterilized

125

water at least three times. The sterilized seeds were germinated on an 80 mesh nylon net

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floating in nutrient solution under dark conditions at 4°C for 2 d. The nutrient solution

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had the following composition (µM): 1500 KNO3, 750 NaH2PO4, 375 K2SO4, 1000 CaCl2,

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500 MgSO4, 25 Fe-EDTA, 0.1 CuSO4, 0.5 MnSO4, 10 H3BO3, 0.5 ZnSO4, and 0.1

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(NH4)6Mo7O24, pH 5.8 (Guan et al., 2018). 7-d-old seedlings were then transplanted into

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the contaminated growth substrates. After 3 weeks, the growth substrates surface was

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inoculated with 2 mL of 108 CFU mL-1 bacterial suspension once per week, except for the

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un-inoculated controls. Seedlings were cultured in a growth room under controlled

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conditions of (temperatures: 25°C day/20°C night; photoperiod: 12 h day/12 h night;

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relative humidity: 60% day/70% night). During plant growth, the growth substrate was

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watered with sterile deionized water to maintain the appropriate water-holding capacity.

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The pot experiments were conducted with five replicates and each pot contained 9

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seedlings. After 5 weeks of the inoculation treatment, plants were harvested for further

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analysis.

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2.3 Harvest and metal concentration determinations

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Three replicate pots were harvested when the plants were in the 9th wk. The

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aboveground part of the plants was carefully separated, photographed, weighed, and rinsed 8

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with deionized water. The oven-dried tissues were digested in 5:1(v/v) HNO3/HClO4 at

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180°C till for approximately 4-5 h (Jin et al., 2009). The concentrations of Cd, Zn, and Cu

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in the digests were estimated by a Thermo Scientific iCE 3300 flame atomic absorption

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spectrometry after dilution of the digestate with ultrapure water and filtration using a filter

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membrane (0.22 µm). The concentration of Ni was analyzed using the Agilent

147

Technologies 4210 microwave plasma atomic emission spectrometer (MP-AES).

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2.4 Chlorophyll content and non-photochemical quenching test

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The chlorophyll concentrations of plant leaves were determined with Konica Minolta

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SPAD-502 meter. For each plant, measurements were taken of all fully expanded leaves

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in the same rosette of a 9-week-old plant and the SPAD value was recorded (Zhang et al.,

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2016). Furthermore, the chlorophyll fluorescence parameter (NPQ), a sensitive early

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indicator reflecting the excited state of thylakoid membranes (Krause and Jahns, 2014;

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Fan et al., 2019) was measured. Briefly, the NPQ of 9-week-old plants was measured

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with a Walz IMAG-MAX/L pulse-amplitude-modulated (PAM) fluorometer (Liu et al.,

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2018b). The leaves from the same position on each plant were selected for NPQ detection.

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2.5 Determination of malondialdehyde (MDA) concentrations

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According to Du et al. (2016), plant samples (0.5 g) were homogenized in 5 mL of

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ice-cold 10% trichloroacetic acid (TCA) solution, and then centrifuged at 12000 ×g for 10

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min. The resulting supernatant was mixed with 2 mL of thiobarbituric acid (TBA) reagent

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and the reaction was initiated by boiling the mixture in a water-bath. After 30 min, the

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reaction was terminated by cooling on ice. After centrifugation at 10000 ×g for 10 min, 9

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the formation of thiobarbituric acid substances was monitored at 450, 532, and 600 nm

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using a spectrophotometer (Puxi TU1901, China).

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2.6 Measurements of ABA catabolic capacity of bacteria

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In order to explore the ability of R.qingshengii in metabolizing ABA, the bacterial

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strain was added to an ABA-supplemented (75 ng/mL) LB medium at 30°C on a rotatory

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shaker at 150 rpm. After incubation for 48 h, the ABA concentration in the resulting

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supernatant was determined by an enzyme-linked immunosorbent assay (ELISA) kit

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(Laierbio) using SpectraMAX 190 microplate spectrophotometer (Molecular Devices) at

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450 nm.

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Furthermore, 3 days after the first inoculation with R.qingshengii, we also analyzed the

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endogenous levels of ABA in the rosette leaves and roots (Xu et al., 2018). Briefly, 0.1 g

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tissues were homogenized in 1 mL pH 7.2 phosphate buffer. After centrifugation, a

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Laierbio ELISA kit was used to analyze the ABA levels in the supernatant as described

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by the manufacturer’s directions. The PGP attributes of the strain used in this study were

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also estimated (Fig. SI-1), including indole acetic acid (IAA) and siderophore production,

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and aminiocyclopropane-1-carboxiylate (ACC) deaminase activity.

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2.7 Measurements of metal bioavailability in growth substrates

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The bioavailabilities of Cd, Zn, Ni, and Cu in growth substrates were assessed by

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monitoring the extractable HMs contents released by growth substrates as described by

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Bao (2000). Dried growth substrate sample (10.0 g) passed through a 1 mm sieve was

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placed in a 100 mL hard glass flask. Extractable Zn, Ni, and Cu in growth substrates were 10

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extracted with 0.1 mol L-1 HCl solution, and the extractable Cd in growth substrates was

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extracted by diethylenetriaminep entaacetic acid (DTPA) solution (0.005 mol/L DTPA,

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0.01mol/L CaCl2, and 0.1 mol/L triethanolamine). Multiple metals (Cd, Zn, and Cu) in the

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extracted solution were analyzed by Thermo Scientific iCE 3300 atomic absorption

188

spectrometer, the concentration of Ni was measured by MP-AES (4210; Agilent

189

Technologies).

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2.8 RT-PCR analysis

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Root tissue was harvested 3 days after the first inoculation of R.qingshengii, and then

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frozen in liquid nitrogen immediately before total RNA extraction. The total RNA was

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extracted using the TRIzol kit (Ambion, Life Technologies). Total RNA was reverse

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transcribed with PrimeScriptTM RT reagent kit (TaKaRa). The expression levels of IRT1,

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ZIP1, ZIP4, NRAMP1, NRAMP3, NRAMP4, HMA3, COPT1, and COPT2 were analyzed

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using SGExcel FastSYBR qPCR kit with MJ OptionTM 2 RT-PCR System (Fang et al.,

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2016). The thermal cycling conditions included 3 min at 95 °C; 5 s at 95 °C, 30 s at 55 °C,

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30 s at 72 °C (35 cycles); 15 s at 95 °C; 1 min at 60 °C; 15 s at 95 °C; 15 s at 60 °C. All of

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the gene-specific primers are listed in Table SI-2.

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2.9 Statistical analysis

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Bioconcentration factor (BCF) is calculated as the concentration ratio of plant shoot to

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soil. Statistical analysis was performed using SPSS for Windows. Means were compared

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either by a t test or ANOVA, as indicated in the figure legends. Different letters on the

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error bars denote the differences between control and inoculation treatments; where 11

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significant. * and n.s. denote a significant and non-significant difference between bacterial

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inoculation treatments or different Arabidopsis varieties.

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3. Results

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3.1 R.qingshengii increased Cd, Zn, and Ni levels in plants

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To investigate the effects of R.qingshengii on the metal phytoextraction, we first

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assessed metal accumulation in plants. As shown in Fig. 1A-C, inoculation with

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R.qingshengii significantly increased the concentrations of Cd, Zn, and Ni in shoots of wt

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plants, up to 47, 24, and 30%, respectively, when compared with bacteria
free plants.

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Considering that BCF was previously used to evaluate plants for phytoextraction purpose

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(Bizo et al., 1986), we also investigated the effects of bacteria on BCFs in the present

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study. Our results showed that R.qingshengii inoculation significantly increased Cd-, Zn-,

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and Ni-BCFs levels of plants grown in the HMs growth substrate (Fig. 1-I), indicating that

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R.qingshengii could promote the concentration coefficient for Cd, Zn, and Ni in plants.

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HMs accumulation is another common indicator for evaluating phytoextraction capacity

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of plants (Kumar et al., 1995). For this purpose, the accumulation of HMs in plants tissue

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after bacterial inoculation was also investigated. As shown in Fig. 1 E-G, the

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accumulation of Cd, Zn, and Ni in shoots per plant increased substantially after

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inoculation, by 72%, 46%, and 50%, respectively, when compared with the bacteria free

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control (controlbacteria-free). Interestingly, inoculation of R.qingshengii did not significantly

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alter the concentration and accumulation of Cu in wt plants, when compared with the 12

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controlbacteria-free (Fig. 1D and H). These results indicated that R.qingshengii inhabiting

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metalliferous growth substrates could increase the capacity of phytoremediation of Cd, Zn,

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and Ni, but not of Cu.

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3.2 R.qingshengii mitigated HMs toxicity in plants

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The presence of HMs in soil often leads to plant growth retardation. Increasing the

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biomass of plants is also an important means to enhance the efficiency of

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phytoremediation. Therefore, we first investigated the effect of R.qingshengii inoculation

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on plant growth vigor. Inoculation promoted the growth of Arabidopsis rosettes in both the

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non-contaminated and contaminated growth substrates (Fig. 2-A). The fresh and dry

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biomasses

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non-contaminated/contaminated soils (Fig. 2B-C). Interestingly, two-way ANOVA

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analysis showed that, although there was a significant increase in fresh weight with

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bacterial inoculation between non-contaminated and contaminated soils (Fig. 2-B), there

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was no significant difference in the dry weight (Fig. 2-C). The results suggested that the

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stimulating effect of R.qingshengii on dry biomass production was irrespective of the

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status of the heavy metal concentration of the growth substrate. These growth promoting

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attributes of R.qingshengii (secretion of IAA) might play an active role in promoting

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plant growth (Fig. SI-1).

of

plants

were

greatly

increased

after

inoculation

in

both

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Here, MDA was measured to reflect the oxidative status of plants. Inoculation with

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R.qingshengii significantly reduced MDA levels in the plants under HMs stress, as 13

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compared with controlbacteria
free (Fig. 2-F). This result indicates that R.qingshengii could

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alleviate HMs-induced oxidative stress. In addition, the effects of R.qingshengii on

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photosynthesis were further investigated by measuring chlorophyll level and the

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chlorophyll fluorescence parameter NPQ. Our data revealed that inoculated with

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R.qingshengii significantly enhanced the leaf SPAD values by 22% and 21% in the

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presence of non-spiked and spiked growth substrates, respectively, compared to the

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bacteria-free control plants (Fig. 2-E). Bacteria treatments also enhanced the NPQ in the

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leaves under HMs stress (Fig. 2-D). The above-mentioned results illustrate that

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R.qingshengii could alleviate HMs-induced oxidative stress and photosynthesis

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inhibition.

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3.3 Enhancement of Cd, Zn, and Ni accumulation in R.qingshengii inoculated-plants

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was ABA-associated

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To explore whether the alterations of Cd, Zn, and Ni accumulation in plants by

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R.qingshengii inoculation were related to an ABA-associated process, we first analyzed

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the ability of R.qingshengii to metabolize ABA. Our study showed that R.qingshengii

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could significantly reduce ABA concentration in ABA-supplemented LB medium (Fig.

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3A). Meanwhile, inoculation of aforementioned bacteria could also reduce ABA

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concentrations in the shoots and roots of plants in contaminated growth substrate by 22

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and 28%, respectively, compared to the bacteria
free plants (Fig. 3B). The above results

266

indicated that the bacteria used in this study were capable of metabolizing ABA and

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decreasing ABA concentrations in plants.

14

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Next, in order to better understand the role of ABA underpinning the increases in

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HMs phytoextraction by R.qingshengii inoculation, an ABA-insensitive mutant and an

270

ABA-sensitive mutant were selected and analyzed for the differences caused by bacterial

271

inoculation treatment between the plant varieties. The levels of Cd, Zn, and Ni in the

272

shoots of abi1/hab1/abi2 after inoculation drastically increased by 113%, 32%, and 64%,

273

respectively, and the accumulation of the metals increased by 148%, 65%, and 79%,

274

respectively, more than those in the bacteria
free plants (Fig. 4). These results indicated

275

that ABA-sensitive mutants exhibited a stronger increase in the uptake of Cd, Zn, and Ni

276

after inoculation, than those in the wt plants. However, R.qingshengii did not affect the

277

levels of Cd, Zn, and Ni in the snrk2.2/2.3 plants. These findings suggested that the

278

incremental effects of R.qingshengii on the uptakes of Cd, Zn, and Ni in plants were

279

attributed to ABA-associated processes. It is worth mentioning that, consistent with that in

280

wt plants, inoculation of R.qingshengii did not cause changes in Cu concentrations in the

281

ABA-sensitive mutant nor in the ABA-insensitive mutant.

282

Additionally, treatments with R.qingshengii promoted the growth of plant rosettes

283

and clearly increased the SPAD levels in both ABA-sensitive and ABA-insensitive

284

mutants, compared with bacteria
free plants (Fig. SI-2A and 2B), indicating that the

285

effect of bacterial inoculation on plant growth was via an ABA independent mechanism.

286

Besides, the NPQ of the leaf edges in above two inoculated mutants under HMs stress

287

remained greenish-yellow instead of orange (Fig. SI-2C), and the changes in the levels of

288

MDA by bacterial inoculation between two mutants were also not statistically significant

289

(Fig. SI-2D). These results indicated that the beneficial impact on mitigation of oxidative 15

290

stress and photosynthetic inhibition by bacterial inoculation were not correlated with an

291

ABA-mediated mechanism.

292

293

3.4 Promotion of HMs transporters underlies the enhancement of phytoextraction

294

by R.qingshengii

295

First, we analyzed the effects of bacterial treatment on the expression of the major

296

transporters responsible for Cd, Zn, Ni, and Cu influx into plants (Fig. 5A). Compared

297

with the non-inoculated control, R.qingshengii significantly upregulated the relative

298

expression levels of IRT1, ZIP1, ZIP4, NRAMP1, NRAMP3, NRAMP4, and HAM3 in the

299

wt plant roots by 196%, 76%, 76% ,40%, 53%, 79%, and 50%, respectively, in

300

contaminated growth substrate, while no obvious changes were observed in COPT1 and

301

COPT2 (Fig. 5C). These results confirmed that inoculation with R.qingshengii could

302

up-regulate the transcription levels of Cd, Zn, and Ni uptake-related genes, whereas those

303

of Cu transporter-encoding genes remained unchanged. Moreover, from the radar chart in

304

Fig. 5-B, we can intuitively observe that the regulation of IRT1 by R.qingshengii

305

inoculation was most prominent.

306

Since IRT1 can act as Cd, Zn, and Ni transporter and the transcriptional level of IRT1

307

increased the most among all above transporters under bacterial inoculation, we

308

investigated whether the promotion of Cd, Zn, and Ni phytoextraction by R.qingshengii

309

were mostly attributed to increases in IRT1 activity, by employing two IRT1-null mutants

310

irt1-1 and irt1-2. Interestingly, the concentrations of Cd, Zn, and Ni in shoots of mutants 16

311

had slight but not significant increases after inoculation, in comparison with the

312

controlbacteria-free (Fig. 6). These results support the assumption that the increased heavy

313

metal (Cd, Zn, and Ni) accumulation by R.qingshengii treatment might be mainly

314

attributed to the promotion of IRT1 expression. Moreover, similar to wild-type

315

Arabidopsis, the biomass of irt1-1 and irt1-2 increased significantly after inoculation

316

when compared with the controlbacteria
free (Fig. SI-3), which further confirmed that

317

beneficial effects of bacterial inoculation on plant growth might be IRT1-independent.

318

319

4. Discussion

320

Developing strategies for improving the extraction efficiency of hyper accumulators

321

has become a hot topic in HMs phytoremediation (Yang et al., 2019). The ability of

322

PGPB to assist in increasing the efficiency of phytoremediation of HMs contaminated

323

soil is a promising possibility (Ma et al., 2016; Hrynkiewicz et al., 2018). In most cases,

324

the rhizospheric bacteria can alter metal accumulation capacity in plants by changing

325

HMs bioavailability in soil by excreting siderophores, organic acids, biosurfactants, and

326

other substances (Lebeau et al., 2008; Sessitsch et al., 2013; Ullah et al., 2015). Here, we

327

found that PGPB could also assisted phytoextraction as a stimulator of root HMs uptake

328

ability. This study proposed a way that ABA-catabolizing bacteria R.qingshengii could

329

facilitate Cd, Zn, and Ni uptake by host plants through stimulation of HMs uptake-related

330

transporters.

331

In the present study, higher shoot Cd, Zn, and Ni concentrations and increased Cd-, 17

332

Zn-, and Ni-BCFs were found in the R.qingshengii-inoculated plants than in

333

controlbacteria-free under HMs stress (Fig. 1). These data shown that R. qingshengii could

334

promote phytoextraction of Cd, Zn, and Ni. We believe that ABA appears to be involved

335

in above regulatory process. This assumption was based on the following evidence: First,

336

the bioavailability of HMs in soil is the key to phytoextraction (Petruzzelli et al., 2014).

337

Interestingly, inoculation with R.qingshengi did not change the availability of HMs in

338

growth substrate to any extent (Fig. SI-4). In this context, the enhancement of HMs

339

phytoextraction by R.qingshengii might be unrelated to bioavailability of HMs. Secondly,

340

R.qingshengii was capable of catabolizing ABA, and ABA levels in R.qingshengii

341

inoculated plants were greatly decreased (Fig. 3), which was consistent with the findings

342

of an earlier study reported by Belimov et al. (2014). Thirdly, the promotional effects of

343

R.qingshengii on Cd, Zn, and Ni phytoextraction were more significant in ABA-sensitive

344

mutant when compared with wt plants, whereas no effects were clearly produced in the

345

ABA-insensitive mutant (Fig. 4), further confirming that the stimulating effects of

346

R.qingshengii on Cd, Zn, and Ni uptake in plants are associated with an ABA-associated

347

pathway.

348

A question arises as to how ABA-catabolizing bacteria promote Cd, Zn, and Ni

349

uptake in roots of plants. Ample evidence has revealed that modification of HMs uptake

350

in roots by external treatments is associated with expression of HMs uptake-related genes

351

(Fan et al., 2014; He et al., 2017). For example, overexpression of AtHMA3 could

352

improve Cd, Pb, and Zn accumulation in transgenic plants (Morel et al., 2008);

353

promotion of LeIRT1 expression may be a crucial mechanism for nitrate-facilitated Cd 18

354

uptake in tomato plants (Luo et al., 2012). A hydroponic experiment also reported that

355

inoculation of S.alfredii with an endophytic bacterium SaMR12 could increase Cd

356

accumulation in the plant, accompanied with elevated expression of SaZIP3, SaNarmp1,

357

SaNarmp3, and SaNarmp6 (Pan et al., 2017). The transcription analyses in this present

358

study showed that inoculated plants had much higher expression of Cd, Zn, and Ni

359

uptake-related genes compared with the control plants under HMs stress, while no

360

obvious changes were detected in the expression of Cu transporter-encoding genes (Fig.

361

5C). These findings are compatible with the fact that R.qingshengii inoculation increased

362

phytoextraction of Cd, Zn, and Ni while had no impact on that of Cu. Previous studies

363

reported that upregulation of Cu uptake-related genes (Yruela, 2009) or an increase of Cu

364

mobility in soils (Cornu et al., 2014; Huguenot et al., 2015; Cornu et al., 2017) could

365

improve Cu phytoextraction. However, neither the expression of Cu transporter genes nor

366

Cu availability in soil was improved by R. qingshengii (Fig. 5 and Fig. SI-4), which may

367

explain why the Cu level in plants was barely affected by R. qingshengii. Taken together,

368

these results confirmed that the effects of R. qingshengii on the facilitation of HMs

369

phytoextraction might be attributed to an increase in expression of HMs associated

370

transporters. Notably, R. qingshengii can also produce IAA (Fig. SI-1), which is another

371

phytohormone that has been shown to upregulate the expression of transporter genes

372

related to HM uptake, such as IRT1, ZIP4, and Nramp6 (Chen et al., 2017). Therefore,

373

IAA may also play a role in the elevation of HM phytoextraction by R. qingshengii.

374

However, in our present study, inoculation with R. qingshengii did not significantly

375

increase the HM concentration in an ABA-insensitive mutant (Fig. 4). This suggests that 19

376

the process by which R. qingshengii increases HM phytoextraction should be mainly

377

attributed to the production of ABA, but not or only minorly attributed to the secretion of

378

IAA. Furthermore, we also quantitatively observed that the up-regulation of IRT1 by R.

379

qingshengii was the most pronounced, by approximately 2.5-fold when compared with

380

ZIP1, ZIP4, and NRAMP4 (Fig. 5C). Moreover, R.qingshengii inoculation led to a slight

381

but not significant increase in Cd, Zn, and Ni levels of IRT1-knockout mutants (Fig. 6),

382

indicating that the increase in IRT1 transcript level in plants caused by R.qingshengii

383

could be crucial in promoting HMs uptake in plants. On the other hand, since R.

384

qingshengii also slightly increased HMs concentrations in plants when IRT1 was knocked

385

out, other transporters including ZIPs, NRAMPs, and HMAs were possibly involved in

386

the above-mentioned processes.

387

Taken together, our findings provide key evidence that R.qingshengii increased

388

accumulation of Cd, Zn, and Ni in plants through a mechanism involving ABA-mediated

389

HMs transporters-associated gene expression (mostly of IRT1). Mostly, HMs in

390

contaminated soils exist in a multiple or combined form rather than the component with a

391

single metal (Wei and Yang, 2010). In this context, the R. qingshengii-improved

392

phytoextraction for multi-metals may provide a promising strategy for remediation of

393

multiple or combined HM-contaminated soils. However, notably, the enhancement of the

394

phytoextraction efficiency for Cd was more pronounced than Zn and Ni. Therefore, the

395

use of R. qingshengii in Cd-contaminated soil should be a priority. Besides, since IRT1 is

396

also involved in transportation of other metals, e.g. lead (Pb), manganese (Mn), and

397

cobalt (Co) (Korshunova et al., 1999; Jamil et al., 2018), whether inoculation with 20

398

R.qingshengii would have a similar promotional effect on the phytoextraction of these

399

metals in plants could be elucidated in future studies. It is also worth noting that the

400

R.qingshengii used in the present study could promote plant growth and alleviate

401

HMs-induced oxidative stress and photosynthesis inhibition in all plant varieties

402

including wt plants, ABA-insensitive mutants, and ABA-sensitive mutants (Fig. 2 and

403

Fig. SI-2). Therefore, the beneficial impact on plant vigor after R.qingshengii inoculation

404

might be independent of ABA, and is more likely be related to PGP activities of

405

R.qingshengii (Fig. SI-1). Since plant biomass is also a key factor for phytoextraction

406

(Sarwar et al., 2017), enabling vigorous growth of phytoextracting plants might also

407

favor phytoremediation efficiency.

408

409

5. Conclusions

410

We conclude that R.qingshengii can promote Cd, Zn, and Ni uptake via an

411

ABA-mediated HM transporter-associated mechanism. We suggest that application of

412

ABA-catabolizing bacteria in HMs contaminated soils might be an alternative strategy to

413

improve the phytoremediation potential of plants.

414

415

416

417

Acknowledgments This work was funded by grants from National Natural Science Foundation of China

21

418

(41977145), and Science and Technology Innovation Activity Program (201710353033,

419

2019R408055).

420

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Figure Legends

594

Fig. 1. Effects of R.qingshengii on concentrations (A-D) and accumulation (E-H) of Cd, Zn, Ni, and

595

Cu in the shoots of wt Arabidopsis plants grown in contaminated soils. (I) Bioconcentration factors

596

(BCF) by wt Arabidopsis plants grown in HMs contaminated growth substrates with and without

597

R.qingshengii inoculation (t test, P < 0.05, n = 5).

598

Fig. 2. Effects of R.qingshengii on the growth of wt plants in contaminated growth substrates (P <

599

0.05, n = 5, two-way ANOVA). (A) Photo, (B) fresh weight, (C) dry weight, (D) NPQ, (E) SPAD,

600

and (F) MDA.

601

Fig. 3. Effects of R.qingshengii on ABA concentrations in the LB liquid medium (A) and in the

602

shoots and roots of wt plants in contaminated growth substrates (B) (t test, P < 0.05, n = 5).

603

Fig. 4. Effects of R.qingshengii on concentrations (A-D) and accumulation (E-H) of Cd, Zn, Ni, and

604

Cu in shoots of snrk 2.2/2.3, wt, and abi1/hab1/abi2 Arabidopsis plants grown in contaminated

605

growth substrates (t test, P < 0.05, n = 5).

606

Fig. 5. (A) Metal-transporters involved in Cd, Zn, Ni, and Cu uptake. (B-C) Effects of

607

R.qingshengii on related gene expression in HMs-stressed wt plants (t test, P < 0.05, n = 5).

608

Fig. 6. Effects of R.qingshengii on the Cd, Zn, and Ni concentrations in IRT1 mutants in

609

contaminated growth substrates (t test, P < 0.05, n = 5).

31

6

400

200

0

Bacteria-free Bacteria inoculation

b 0.2 0.1

Bacteria-free Bacteria inoculation

Shoot Zn accumulation (µg plant-1 DW)

Shoot Cd accumulation (µg plant-1 DW)

0.3

10 5

12

a b

8

4

0

a

1.0 b

0.6 0.4 0.2 0.0

Bacteria-free Bacteria inoculation

a

a

9 6 3

Bacteria-free Bacteria inoculation 0.25

(G)

0.8

(D) 12

0

Bacteria-free Bacteria inoculation

1.2 (F)

a

15

0

16

0.4

b

Bacteria-free Bacteria inoculation

(E)

0.0

b

15

Cu concentration in shoots (mg kg-1 DW)

12

600

a

20

(H) Shoot Ni accumulation (µg plant-1 DW)

b

a

Cu 25 (C)

Ni concentration in shoots （ mg kg-1 DW）

18

0.5

Accumulation

Ni

800 (B)

a

0

(I)

Zn (A)

Shoot Ni accumulation (µg plant-1 DW)

24

Zn concentration in shoots (mg kg-1 DW)

Cd Cd concentration in shoots (mg kg-1 DW)

Concentration

0.20

a

a

0.15 0.10 0.05 0.00

Bacteria-free Bacteria inoculation

Bacteria-free Bacteria inoculation

BCF

Treatment Cd

Zn

Ni

Cu

Bacteria-free

10.081±1.449b

4.030±0.229b

0.299±0.010b

0.191±0.017a

Bacteria inoculation

14.828±0.388a

5.014±0.335a

0.389±0.001a

0.196±0.025a

610 611

Fig. 1

32

Bacteria inoculation 0.6

(A) Non-contaminated soil

0.07 (B)

* a

0.4

a

0.3

b

b

0.2 0.1 0.0

Non-contaminated soil

Non-contaminated soil

0.5 Contaminated soil

Contaminated soil

a

0.03

a b

0.02

b

0.00 Non-contaminated soil

Contaminated soil

n.s.

(F) 10

a a b

b

18 9

1.0

0.04

12

36 27

0.05

n.s.

(E) 0.0 SPAD readlngs

(D)

Bacteria-free Bacteria inoculation n.s.

0.01

45

NPQ

Shoot dry weight (g plant-1 DW)

Shoot fresh weight (g plant-1 FW)

Contaminated soil

(C)

0.06

0.5

MDA concentration (nmol g-1 FW)

Bacteria-free

a 8

b

a 6

b

4 2 0

0 Non-contaminated soil

Contaminated soil

Non-contaminated soil Contaminated soil

612 613

Fig. 2

33

250 (A)

(B)

a

75

70

b

65

ABA concentration in plant (ng g-1 FW)

ABA concentration in the LB liquid medium (ng mL-1)

80

Bacteria-free Bacteria inoculation

a

200 b

150 100

a

50

b

0

60 Bacteria-free Bacteria inoculation

Root

Shoot

614 615

Fig.3

34

47% a 18

a

a

b

9

snrk 2.2/2.3

Cd accumulation in shoots by per plant (µg plant-1)

b

1.0

(E)

wt

a 148%

0.6 0.4

a

a

72% a

b

b 0.2 0.0 snrk 2.2/2.3

wt

abi1/hab1/abi2

a

32% b

0

(F)

wt

Bacteria-free Bacteria inoculation

36

a 64%

27 a

a

18

30%

b a

b

9

abi1/hab1/abi2

Bacteria-free Bacteria inoculation

18 15

Cu (C)

0 snrk 2.2/2.3

21

45

a

300

abi1/hab1/abi2

Bacteria-free Bacteria inoculation

0.8

600

Bacteria-free Bacteria inoculation 24% a a b

Cu concentration in shoots (mg kg-1 DW)

27

Ni (B)

a

a

12

46% b

a

a 65% b

9 6 3 0 snrk 2.2/2.3

wt

abi1/hab1/abi2

3.0

(G)

wt

(D)

15

a

Bacteria-free Bacteria inoculation

a 12

a

a

6 3

a 79%

2.0

b

a a

50% a

1.0 b 0.5 0.0 snrk 2.2/2.3

wt

abi1/hab1/abi2

snrk 2.2/2.3

0.5

abi1/hab1/abi2

Bacteria-free Bacteria inoculation

(H) 0.4 0.3

wt

a

a a a

0.2

a

a

0.1 0.0 snrk 2.2/2.3

wt

abi1/hab1/abi2

616 617

a

a

9

abi1/hab1/abi2

Bacteria-free Bacteria inoculation

2.5

1.5

18

0 snrk 2.2/2.3

Cu cumulation by per weight of shoots (µg plant-1)

113%

900

Ni cumulation by per weight of shoots (µg plant-1)

36

0

Accumulation

a

Bacteria-free Bacteria inoculation

Ni concentration in shoots (mg kg-1 DW)

Zn (A)

Zn concentration in shoots (mg kg-1 DW)

45

Zn cumulation by per weight of shoots (µg plant-1)

Cd Cd concentration in shoots (mg kg-1 DW)

Concentration

Fig.4

35

(B)

(A) Transporters

HMs

IRT1 ZIP1 ZIP4 NRAMP1 NRAMP3 NRAMP4 HMA3 COPT1 COPT2

+ + +

Cd Zn Ni

+ +

+ +

+

+

+

+ +

+

Cu

+

Relative expression (% of the control)

4

+

(C) a

Bacteria-free Bacteria inoculation

3 a a

2

a

a

a

b

b

b

IRT 1

ZIP 1

ZIP 4

b

b

a b

b

a a

1

a

a

0 NR NR NR AM AM AM P3 P4 P1

HM A3

CO PT 1

CO PT 2

618 619

Fig.5

36

500

4 a

a

3 2 1 0

400

a

200 100 0

a 6

4

2

0

400

18 9

Bacteria-free Bacteria inoculation 25 (F)

a a

300 200 100 0

Bacteria-free Bacteria inoculation

27

(E) Zn concentration in shoots (mg kg-1 DW)

a

a

0

500 (D)

a

36

Bacteria-free Bacteria inoculation

8 Cd concentration in shoots (mg kg-1 DW)

(C)

a

300

Bacteria-free Bacteria inoculation

irt1-2

45 (B)

Zn concentration in shoots (mg kg-1 DW)

Cd concentration in shoots (mg kg-1 DW)

(A)

Ni concentration in shoots (mg kg-1 DW)

5

Bacteria-free Bacteria inoculation

Ni concentration in shoots (mg kg-1 DW)

irt1-1

20

a

a

15 10 5 0

Bacteria-free Bacteria inoculation

620 621

Fig.6

37

Highlights:




R. qingshengii improved the phytoextraction efficacy of Cd, Zn, and Ni, but not Cu.




R. qingshengii improved plant uptake of Cd, Zn, and Ni by an ABA-mediated process.




R. qingshengii upregulated expression of Cd, Zn, and Ni associated transporters.




R. qingshengii did not significantly affect Cu transporter transcription levels.




R.qingshengii barely affected Cd, Zn, and Ni uptake in the irt1 deletion mutants.

Conflict of interest statement The authors declared that they have no conflicts of interest to this work. We declared that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.