Inoculation with Bacillus subtilis and Azospirillum brasilense produces

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Food Safety and Toxicology

Inoculation with Bacillus subtilis and Azospirillum brasilense produces abscisic acid that reduces IRT1-mediated cadmium uptake of roots Qianru Xu, Wei Pan, Ranran Zhang, Qi Lu, Wanlei Xue, Cainan Wu, Bixiu Song, and Shaoting Du J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00598 • Publication Date (Web): 08 May 2018 Downloaded from http://pubs.acs.org on May 8, 2018

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Journal of Agricultural and Food Chemistry

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Inoculation with Bacillus subtilis and Azospirillum brasilense

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produces abscisic acid that reduces IRT1-mediated cadmium uptake

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of roots

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Qianru Xu1, Wei Pan1, Ranran Zhang1, Qi Lu, Wanlei Xue, Cainan Wu, Bixiu Song,

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Shaoting Du*

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College of Environmental Science and Engineering, Zhejiang Gongshang University,

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Hangzhou 310018, China

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To whom correspondence should be addressed. E-mail [email protected]

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1

These authors contributed equally to this work.

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*Author to whom correspondence may be addressed.

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Phone: +86-571-28008209; Fax: +86-571-88832369

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E-mail: [email protected]

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Notes: The authors declare no competing financial interest.

1

ACS Paragon Plus Environment


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Abstract

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Cadmium (Cd) contamination of agricultural soils represents a serious risk to crop

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safety. A new strategy using abscisic acid (ABA)-generating bacteria, Bacillus subtilis

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or Azospirillum brasilense, was developed to reduce the Cd accumulation in plants

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grown in Cd-contaminated soil. Inoculation with either bacterium resulted in a

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pronounced increase in the ABA level in wild-type Arabidopsis Col-0 plants,

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accompanied by a decrease in Cd levels in plant tissues, which mitigated the Cd

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toxicity. As a consequence, the growth of plants exposed to Cd was improved.

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Nevertheless, B. subtilis and A. brasilense inoculation had little effect on Cd levels

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and toxicity in the ABA-insensitive mutant snrk 2.2/2.3, indicating that the action of

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ABA is required for these bacteria to reduce Cd accumulation in plants. Furthermore,

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inoculation with either B. subtilis or A. brasilense down-regulated the expression of

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IRT1 (IRON-REGULATED TRANSPORTER 1) in the roots of wild-type plants and

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had little effect on Cd levels in the IRT1-knockout mutants irt1-1 and irt1-2. In

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summary, we conclude that B. subtilis and A. brasilense can reduce Cd levels in

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plants via an IRT1-dependent ABA-mediated mechanism.

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Keywords: ABA-generating bacteria; Yield; Oxidative stress; Photosynthesis; Cd

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accumulation; Cd uptake transporter

2


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Introduction

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Cadmium (Cd) is recognized as a serious pollutant in agricultural soils owing to its

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high ecotoxicity and rate of bioaccumulation in organisms.1 As a consequence of

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increases in the mining and refinement of metal ores, and the application of

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Cd-containing phosphate fertilizers, sewage sludge, and municipal composts to

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agricultural soils,2 Cd contamination of agricultural soils has become a worldwide

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environmental issue in recent years.3,4 However, owing to the continual high demand

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for food, most of these Cd-contaminated lands are still being used for crop

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production.5,6 Soil-borne Cd can readily accumulate in the edible parts of crops, and

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subsequently enters the food chain, thereby posing a significant risk to human

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health.7,8 Many methods have been developed for lowering the pollution risk of Cd in

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crops, including (1) physical or chemical measures (such as encapsulation of the

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contaminated soil and chemical immobilization of Cd) and (2) phytoremediation by

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hyperaccumulating plants.9 However, physical and chemical strategies are expensive,

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and phytoremediation requires several growing seasons to be effective, rendering it

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impractical in regions where farmland is limited. Thus, alternative strategies that are

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cost-effective and interfere less with crop production are still urgently needed.

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Our recent study showed that exogenous abscisic acid (ABA) can clearly suppress

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root Cd uptake by inhibiting the activity of IRT1 (IRON-REGULATED

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TRANSPORTER 1), a Fe2+ transporter that also functions as a Cd transporter,2,10

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thereby indicating that we can develop techniques based on ABA application to

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minimize Cd accumulation in crop plants.11 However, due to its chemical instability 3



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and rapid catabolism, ABA is readily degraded in soils, and thus tends to have few

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applications in agriculture practice.12,13 Interestingly, however, in recent decades,

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several microorganisms that produce ABA have been identified.14-17 Theoretically,

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under favorable conditions, these microorganisms can sustainably produce ABA in

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soils, and in doing so counteract the degradation of ABA. Therefore, application of

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ABA-generating microbial inocula may represent a promising alternative strategy for

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reducing Cd accumulation in crops.

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In this study, we investigated the effect of two ABA-generating bacteria

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(Bacillus subtilis and Azospirillum brasilense) on Cd accumulation in plants by using

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wild-type Arabidopsis thaliana (ecotype Col-0) plants and ABA-insensitive mutants.

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We found that root inoculation with both ABA-generating bacteria inhibited the root

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expression of IRT1 in an ABA-dependent manner, thus decreasing IRT1-mediated Cd

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uptake. Application of the aforementioned bacteria to minimize Cd accumulation in

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crops is the subject of Chinese Patent No. ZL201510590310.8. Our findings also

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show that developing techniques based on the application of ABA-generating bacteria

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to Cd-contaminated soils may be an effective strategy for counteracting Cd

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contamination in crops.

71 72

Materials and methods

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Plants and soils

74

The Col-0 ecotype of Arabidopsis (wt) and mutants generated in its background

75

were used in this study, including an ABA-insensitive double mutant, snrk 2.2/2.3, 4


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and two IRT1 T-DNA insertion IRT1-null mutants, irt1-1 (SALK_054554C) and

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irt1-2 (SALK_024525). Seeds of snrk 2.2/2.3, in which the SnRK2.2- and

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SnRK2.3-mediated ABA signaling pathways have been lost, were obtained from Dr.

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Chongwei Jin (College of Natural Resources and Environmental Science, Zhejiang

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University, China). Further information on the snrk 2.2/2.3 double mutant can be

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found in the paper by Fujii et al. 18 Seeds of the two IRT1-null mutants were kindly

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provided by Dr. Takafumi Mizuno (Mie University, Japan).19

83

The soils used for pot experiments in the present study comprised a 6:3:1 (v/v/v)

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mixture of nutrient soil (Klasmann-Deilmann Gmbh), vermiculite, and perlite. The

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main physicochemical properties of the soils are listed in Table SI-1. The soil samples

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were sterilized using an autoclave (Panasonic) and then artificially contaminated with

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an aqueous solution of CdCl2 to achieve final concentrations of 0 and 3 mg Cd kg-1

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soil.

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Bacterial strains and culture conditions

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Two ABA-generating bacteria, B. subtilis (CGMCC1.4255) and A. brasilense

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(CGMCC1.10379), obtained from the China General Microbiological Culture

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Collection Center (CGMCC), were used in the present study. These bacterial strains

93

were grown in liquid medium for 24 h at 28°C with continuous shaking at 200 rpm.

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The compositions of the bacterial growth media were based on those recommended

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by the CGMCC. The B. subtilis liquid medium contained the following (g L-1): 10

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soya peptone, 3 beef extract, and 5 NaCl. The A. brasilense liquid medium contained 5



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the following (g L-1): 0.5 yeast extract, 20 mannitol, 0.8 K2HPO4, 0.2 KH2PO4, 0.2

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MgSO4·7H2O, 0.1 CaSO4·2H2O, 0.005 FeCl3·6H2O, and 0.001 NaMoO4·2H2O, pH

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adjusted to 7.2 with 0.1 M HCl or 1 M NaOH. Following incubation, the bacterial

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cells were collected by centrifugation at 5000 rpm for 10 min, washed three times

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with sterile normal saline, and then recentrifuged. The pelleted cells of B. subtilis and

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A. brasilense were diluted (10-1–10-8) and spread on nutrient agar plates (nutrient

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broth and 1.5% agar). The plates were incubated at 30°C for 24 h, and thereafter

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bacterial colonies were counted and the numbers of colony-forming units per milliliter

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(CFU mL-1) were calculated. Bacterial inocula were prepared by resuspending

106

pelleted cells in sterile normal saline to obtain an inoculum density of 108 CFU mL−1.

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The ability of the two bacteria to colonize soils was studied as previously described.20

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As shown in Fig. SI-1, there were no significant differences in the numbers of bacteria

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in the presence of Cd. During a 2-week period, the populations of B. subtilis and A.

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brasilense initially increased (1.3–2.4 × 107 CFU g-1) and then decreased (4–5 × 106

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CFU g-1). On the basis of the above results, bacteria were re-inoculated into the soils

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every 2 weeks to maintain sufficient bacterial numbers over the entire treatment

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period.

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Pot experiment

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Arabidopsis seeds were surface-sterilized with a mixture of ethanol and 30%

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H2O2 (1:1) for 20 min, washed extensively with sterile water, and then germinated on

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an agar-solidified nutrient medium in Petri dishes. The basal agar medium had the 6


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following nutrient composition: 750 µM NaH2PO4, 500 µM MgSO4, 1000 µM K2SO4,

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2.25 mM KNO3, 1000 µM (NH4)2SO4, 1 mM CaCl2, 10 µM H3BO3, 0.5 µM MnSO4,

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0.5 µM ZnSO4, 0.1 µM CuSO4, 0.1 µM (NH4)6Mo7O24, and 25 µM Fe-EDTA, pH 5.8.

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On day 7, the seedlings were transplanted to four independent aseptic pots per

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treatment. During the 4th week after transplantation, 2 mL of dead (autoclaved at

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121°C for 30 min) or live bacterial suspensions (108 cells mL-1) were inoculated onto

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the soil surface. As a control treatment, soil was inoculated with 2 mL sterile water.

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During the 6th and 8th weeks after transplantation, bacteria were re-inoculated into the

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soils to maintain sufficient bacterial activity. During plant growth, soil moisture was

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maintained at 60% of the water-holding capacity by watering with sterile deionized

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water. The average temperature of the greenhouse ranged from 24.0 ± 4.6°C (day) to

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18.3 ± 3.2°C (night), relative humidity in the leaf chamber remained at 70%, and the

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daily average photoperiod was 14 h. The daytime light intensity was 50 µmol photons

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m-2 s-1. Nine-week-old plants were harvested for further growth, Cd concentration,

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oxidative stress, and chlorophyll fluorescence analyses. The levels of available Cd in

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soils after plantation were also measured.

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Measurement of biomass and Cd concentration

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In the 9th week after transplantation, the plants were harvested and photographed,

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and then dried at 80°C for 24 h. Oven-dried shoot samples were ground into a fine

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powder for the analysis of Cd concentration. The dried shoot samples were digested in

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concentrated HNO3 at 120°C until no brown NO gas was emitted, and then further 7



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digested with HNO3/HClO4 at 180°C until the solution became transparent.21

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Digestates were diluted in ultrapure water and the concentration of Cd in the

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digestates was analyzed by flame atomic absorption spectrometry (iCE 3300; Thermo

142

Scientific).

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Chlorophyll fluorescence

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Nine-week-old Arabidopsis plants exposed to the different treatments were used to

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examine chlorophyll fluorescence using a pulse-amplitude-modulated fluorometer

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(IMAG-MAX/L, Germany). Whole plants were initially dark-adapted for 20 min prior

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to performing the exposure procedure. The first completely expanded leaves were

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then removed and arranged neatly on the fluorometer for measurements.

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Measurements were taken from each leaf at three areas of interest in the intercostal

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regions close to the main vein. Chlorophyll fluorescence parameters, including the

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maximum fluorescence (Fm), the yield of photochemical quantum [Y(II)], maximum

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effective quantum

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non-photochemical quenching (NPQ), and electron transport rate (ETR), were

154

measured.22

155

Determination of malondialdehyde (MDA) and reactive oxygen species (H2O2 and

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O2• -) concentrations

yield

of

PSII (Fv/Fm),

photochemical quenching (qP),

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The level of lipid peroxidation was measured by estimating the concentration of

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MDA, a decomposition product of the peroxidized polyunsaturated fatty acid

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component of membrane lipids, using thiobarbituric acid (TBA) as the reactive 8


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material following a previously described method.23 Leaf samples (0.1 g) from

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9-week-old plants were homogenized in 3 mL 5% (w/v) trichloroacetic acid (TCA)

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and the homogenate was centrifuged at 11,500 × g for 10 min. One milliliter of the

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resultant supernatant was mixed with 4 mL of TBA reagent (0.5% [w/v] of TBA in

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20% TCA). The reaction mixture was heated at 95°C for 30 min in a water bath and

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then rapidly cooled in an ice bath and centrifuged at 11,500 × g for 15 min. The

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absorbance of the colored supernatant was monitored at 450, 532, and 600 nm. The

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concentration of MDA was calculated according to the following equation:

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Concentration (µmol l-1) = 6.45 × (OD532–OD600) – 0.56 × OD450,

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where OD is the optical density. The concentration of MDA in leaves was expressed

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as µmol of MDA g-1 fresh weight. H2O2 in leaf tissue was precisely determined

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spectrophotometrically (410 nm) using the titanium method described by Hossain et

172

al.24 Briefly, H2O2 was extracted by homogenizing 0.1 g of leaf samples with 3 mL of

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50 mM potassium phosphate buffer pH (6.5) at 4°C. The homogenate was centrifuged

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at 11,500 × g for 15 min. Three milliliters of the resultant supernatant was mixed with

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1 mL of 0.1% TiCl4 in 20% H2SO4 (v/v), and the mixture was then centrifuged at

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11,500 × g for 12 min. The optical absorption of the supernatant was measured

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spectrophotometrically at 410 nm and expressed as µmol g-1 fresh weight. The rate of

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O2• - production in leaf tissue was assayed by adding sulfanilic acid and

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α-naphthylamine according to the method described by He et al.25 Briefly, 0.1 g of

180

leaf samples was homogenized with 2 ml of 50 mM potassium phosphate buffer (pH 9



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7.8) at 4°C, and the mixture was then centrifuged at 10,000 × g for 10 min at 4°C.

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One milliliter of the supernatant was mixed with 0.9 mL of 50 mM potassium

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phosphate buffer (pH 7.8) and 0.1 mL of 10 mM hydroxylamine hydrochloride.

184

Subsequently, the reaction mixture was incubated at 25°C for 20 min before adding 1

185

mL of 17 mM p-aminobenzene sulfonic acid and 1 mL of 7 mM α-naphthylamine.

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After further incubation for 20 min, the optical density of the supernatant was

187

measured spectrophotometrically at 530 nm and expressed as nmol min-1 g-1 fresh

188

weight.

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Measurement of Cd bioavailability in soils

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Cd bioavailability was assessed by monitoring 0.01 M CaCl2 (1:10, m/v)

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extractable Cd concentrations released by soils after shaking for 3 h.26 Cd in the

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extracted solution was analyzed by flame atomic absorption spectrometry (iCE 3300;

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Thermo Scientific). The results provided information regarding the effects of bacterial

194

inoculation on the available Cd levels in soils. As shown in Fig. SI-2, there were no

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obvious differences in Cd availability in Cd-contaminated soils after inoculation with

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B. subtilis and A. brasilense.

197

Measurement of ABA levels

198

The effects of the two bacterial species on total endogenous levels of ABA in the

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roots and shoots of plants were determined 7 d after the first inoculation treatment

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using an enzyme-linked immunosorbent assay (ELISA) kit via the one-step

201

double-antibody sandwich method.27 Briefly, having carefully extracted plants from 10


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the pots, their roots were rinsed in distilled water to remove all loosely adhering soil

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particles, and then blotted dry with a paper towel. Roots or rosette leaf samples (0.1 g)

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were ground in 5 mL 80% (v/v) methanol extraction medium in an ice-cooled mortar,

205

incubated at 4°C for 4 h, and then centrifuged at 4,000 rpm for 15 min at 4°C. The

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resulting supernatant was analyzed using a Phytodtek ELISA kit according the

207

manufacturer’s instructions. ABA concentrations were determined by measuring the

208

optical density at 450 nm using a microplate reader (SpectraMAX 190; Molecular

209

Devices). Values were expressed as the means of four replicates.

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Gene expression analysis

211

Root tissues were collected 24 h after the first inoculation with bacteria and

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immediately frozen in liquid nitrogen prior to total RNA extraction. First-strand

213

cDNA was synthesized from the total RNA using a PrimeScript reverse transcription

214

(RT) reagent kit (TaKaRa).28 The levels of IRT1 mRNA were determined using a

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SYBR Green RT-PCR kit (TaKaRa) with the following gene-specific primers: fw,

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AAGCTTTGATCACGGTTGG; rev, TTAGGTCCCATGAACTCCG. RT-PCR analysis was

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performed using the Option 2 Real-Time PCR System (MJ Research) under the

218

following cycling conditions: 30 s at 95°C, and then 40 cycles of 95°C for 5 s, 55°C

219

for 30 s, and 72°C for 30 s. Primers for the housekeeping gene UBQ10 used as a

220

control in the PCR were as follows: fw, ACCCTAACGGGAAAGACGA; rev,

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GGAGCCTGAGAACAAGATGAA. Amplification of the PCR products was monitored

222

via intercalation of SYBR-Green and relative expression of the target genes was 11



223

calculated according to a previously described equation.21

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Statistical analysis

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The values presented in this manuscript represent the means of four or five

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replicated experiments. Statistical analyses were performed using the SPSS program.

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Data were subjected to analysis of variance (ANOVA), and the statistical significance

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of differences (P < 0.05) between means was determined using the Duncan test.

229

230

Results

231

B. subtilis and A. brasilense decreased Cd levels in plants

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Although the amount of available Cd in soil amended with CdCl2 was only

233

approximately 0.3 mg kg-1 (Fig. SI-2), the Col-0 plants grown in this soil accumulated

234

a higher amount of Cd (more than 15 mg kg-1) in their aboveground biomass (Fig. 1),

235

which is consistent with the results of previous studies.29,30 Interestingly, when either

236

B. subtilis or A. brasilense was inoculated into the Cd-contaminated soil, there was a

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significant decrease of approximately 40% in Cd levels in the shoots compared with

238

the treatment without bacterial inoculation (ControlBacteria-free) (Fig. 1). However,

239

compared with the ControlBacteria-free treatment, the addition of dead bacteria did not

240

affect Cd levels in the aboveground biomass of Col-0 plants. These results indicate

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that the putative preventative effect of B. subtilis or A. brasilense on Cd accumulation

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in plants grown in Cd-contaminated soil could be attributed to the bio-activity of these 12


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bacteria.

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B. subtilis and A. brasilense mitigated Cd toxicity in plants

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Next, we investigated the effect of B. subtilis or A. brasilense inoculation on plant

247

growth. In the control soil with no Cd amendment, inoculation with either bacteria

248

(and the addition of dead bacteria) had little effect on the aboveground biomass of

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Col-0 plants (Fig. 2a and b). However, in the Cd-contaminated soil, the aboveground

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biomass markedly decreased by approximately 35%, indicating that 0.3 mg kg-1 of

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available Cd was sufficient to cause stress. As expected, the addition of dead B.

252

subtilis or A. brasilense did not affect plant growth in Cd-contaminated soil. However,

253

in the same Cd-contaminated soil, inoculation with either live bacterium reversed the

254

Cd stress-induced growth suppression, resulting in an approximately one-fold increase

255

in aboveground biomass. These results indicate that Cd-induced damage to plant

256

growth could be mitigated by the bio-activity of B. subtilis and A. brasilense.

257

Oxidative stress has been recognized as a mechanism by which plant biomass

258

production is inhibited during Cd stress.31, 32 Here, we found that the levels of MDA,

259

H2O2, and O2•- in Col-0 plants were elevated by Cd in the soil. These Cd-induced

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oxidative stress indices were clearly lowered by the inoculation of live B. subtilis or A.

261

brasilense, but not by the addition of dead bacteria (Fig. 2e-g). These results indicate

262

that the bio-activities of B. subtilis and A. brasilense tend to alleviate Cd-induced

263

oxidative stress in plants.

264

An increase in oxidative load due to Cd stress could increase the inhibition of 13



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photosynthesis.33 As photosynthesis is the main source of plant biomass, the effects of

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B. subtilis and A. brasilense inoculation on photosynthesis were further studied by

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measuring parameters of chlorophyll fluorescence. We found that bacterial

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inoculation resulted in blue imaging of the maximum fluorescence yield (Fm) in

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leaves when the plants were grown in Cd-contaminated soil, similar to the phenotypes

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observed in control soil, but different from those for the ControlBacteria-free and dead

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bacteria treatments under Cd-contaminated conditions (Fig. 2c). Furthermore,

272

significant increases in the efficiency of Y(II) were observed with live bacteria

273

treatments

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Cd-contaminated soil (Fig. 2d). These results indicate that B. subtilis and A. brasilense

275

inoculation facilitates the maintenance of high PSII activity in plant leaves under Cd

276

stress.

under Cd

stress

compared

with

dead

bacteria

treatments

in

277 278

Alleviating effects of B. subtilis and A. brasilense on Cd Stress in plants are

279

associated with ABA

280

As mentioned previously, the plant hormone ABA may be involved in decreasing

281

Cd exposure-induced plant stress in the B. subtilis and A. brasilense treatments. We

282

therefore analyzed ABA levels after inoculation with each bacterial culture.

283

Inoculation with either bacterium promoted a significant increase in the ABA

284

concentrations in roots and shoots (Fig. 3). To gain additional insights into the role of

285

ABA in the mechanism underlying the decrease in Cd accumulation in plants, we

286

further analyzed the effects of these ABA-generating bacteria on Cd concentrations in 14


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ABA-insensitive mutants grown in Cd-contaminated soil. Neither the inoculation of B.

288

subtilis nor that of A. brasilense affected the levels of Cd in snrk 2.2/2.3 plants, in

289

which the SnRK2.2- and SnRK2.3-mediated ABA signaling pathways had been lost.18

290

(Fig. 4a). This result differs from the effect observed in the wt plants. The above

291

findings indicate that the effect of B. subtilis and A. brasilense on decreasing Cd

292

accumulation in wt plants is dependent on ABA-associated activity.

293

We accordingly further investigated whether the mitigation of Cd toxicity

294

following inoculation with B. subtilis or A. brasilense is associated with ABA. The

295

growth promotion detected in Cd-stressed wt plants after inoculation with either live

296

bacterium was not observed in the ABA-insensitive mutant snrk 2.2/2.3 (Fig. 4b). In

297

addition, live B. subtilis and A. brasilense treatments had minimal effect on MDA,

298

H2O2, and O2•- levels in the shoots of Cd-stressed snrk 2.2/2.3 mutants, which

299

contrasts with the effect observed in Cd-stressed wt plants (Fig. 4c, d, and e).

300

Furthermore, no obvious change was detected in PSII activity (indicated by Fv/Fm and

301

Y(II),

302

non-photochemical quenching [NPQ]) in the leaves of snrk 2.2/2.3 plants in the

303

presence of ABA-generating bacteria. This also differed from the results obtained for

304

wt plants (Table SI-2). On the basis of these observations, we conclude that

305

ABA-associated activity plays a paramount role in the mechanism(s) whereby B.

306

subtilis and A. brasilense contribute to alleviating Cd toxicity in plants.

electron

transport

rate

[ETR],

photochemical quenching [qP], and

307 308

Inhibition of IRT1 underlies the prevention of plant Cd accumulation by B. subtilis 15



309

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and A. brasilense

310

The ferrous iron transporter IRT1 has been shown to play an important role in Cd

311

uptake by plant roots.2,10 Therefore, we investigated whether the observed decrease in

312

Cd accumulation after B. subtilis and A. brasilense inoculation is associated with IRT1.

313

Inoculation of live B. subtilis and A. brasilense into a Cd-contaminated growth

314

medium resulted in decreases of 93% and 78%, respectively, in IRT1 transcript levels

315

in wt plant roots when compared with those observed in the un-inoculation treatment

316

(Fig. 5). This indicated a pronounced down-regulation of IRT1 following bacterial

317

inoculation. To verify whether B. subtilis and A. brasilense reduced Cd levels in

318

plants by inhibiting IRT1 activity in roots, we investigated the effect of these bacteria

319

using the IRT1-null mutants irt1-1 and irt1-2. Although inoculation with both bacteria

320

slightly decreased the Cd levels in both IRT1-null mutants, two-way ANOVA analysis

321

showed that the decreases were considerably lower than those observed in wt plants

322

(Fig. 6). Moreover, biomass production among the bacteria inoculation treatments

323

using irt-1 and irt1-2 mutants did not show any significant differences (Fig. SI-3).

324

These results indicate that IRT1 plays a key role in reducing Cd levels in the plants

325

subjected to B. subtilis and A. brasilense treatments.

326 327

Discussion

328

Although several strategies have been proposed to remediate Cd-contaminated soils

329

with the aim of minimizing Cd accumulation in edible crops grown in affected soils,9

330

these strategies tend to be expensive in terms of labor and cost, or are time-consuming 16


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to implement. Therefore, strategies that can effectively prevent Cd uptake by crop

332

roots need to be developed.11,

333

Cd-contaminated soil with B. subtilis or A. brasilense can effectively decrease Cd

334

levels in Arabidopsis plants. Furthermore, it is noteworthy that in a lightly polluted

335

soil, root inoculation with either of the aforementioned bacteria, also lowered Cd in

336

the edible parts of pakchoi cabbage, one of the most popular vegetables in China,

337

from 0.27 to approximately 0.13 mg kg-1 FW, which is below the permissible limit for

338

Cd concentrations in leafy vegetables in China (0.2 mg kg-1 FW) (unpublished work).

339

Therefore, our findings may provide a new effective strategy for counteracting Cd

340

contamination in crops.

34-36

In this study, we found that inoculation of

341

In this study, we showed that the addition of dead B. subtilis or A. brasilense did

342

not affect the Cd level in plants (Fig. 1), which indicates that these bacteria prevent

343

Cd entry into plants via their bio-activity. A change in Cd availability in the soil is a

344

key factor affecting Cd uptake by plant roots.37 However, inoculation of live B.

345

subtilis or A. brasilense had little effect on the availability of Cd in the soil (Fig. SI-2).

346

Thus, the decreases in Cd levels in plants attributable to B. subtilis or A. brasilense are

347

assumed to be induced by inhibition of Cd entry into the root cells. Since B. subtilis

348

and A. brasilense have previously been identified as ABA-generating bacteria,14-16 we

349

aimed to investigate whether ABA plays a role in the prevention of Cd uptake by plant

350

roots. We found that both B. subtilis and A. brasilense clearly elevated ABA levels

351

(Fig. 3). Furthermore, both bacteria had a negligible effect on Cd levels in the snrk

352

2.2/2.3 mutants, in which the SnRK2.2- and SnRK2.3-mediated ABA signaling 17



Page 18 of 37

353

pathways had been lost.18 Considering these results, we conclude that an

354

ABA-dependent pathway is required for B. subtilis or A. brasilense to reduce Cd

355

levels in plants. This conclusion is also supported by the results of Fan et al.,11 who

356

showed that exogenous ABA application in a Cd-amended hydroponic culture system

357

clearly decreased Cd accumulation in plants. Inhibition of growth, oxidative stress,

358

and suppression of photosynthesis are typical symptoms associated with the excess

359

accumulation of Cd in plants,35, 38-40 and were also observed in the current study (Fig.

360

2). The inoculation of Cd-contaminated soil with B. subtilis or A. brasilense Cd

361

clearly alleviated growth inhibition, oxidative stress, and photosynthesis suppression

362

in wt Arabidopsis plants induced by Cd excess accumulation, whereas these

363

alleviation effects were not observed in the ABA-insensitive mutant snrk 2.2/2.3.

364

These results provide further evidence that an ABA-associated process is required for

365

B. subtilis or A. brasilense to minimize Cd accumulation and its toxicity in plants.

366

Next, we sought to examine the mechanism whereby B. subtilis and A. brasilense

367

reduce Cd accumulation in plants. IRT1 plays an important role in the process of Cd2+

368

absorption by roots.2, 10, 41, 42 Further, Seguela et al.43 showed that exogenous ABA

369

application in an Fe-limited growth medium inhibited the root expression of IRT1.

370

Under conditions of Cd contamination, we observed that inoculation with the

371

ABA-generating bacteria B. subtilis and A. brasilense can substantially reduce the

372

IRT1 transcript level in roots (Fig. 5). Interestingly, the effect of bacterial inoculation

373

on reducing plant Cd levels was almost non-existent in the IRT1-knockout mutants

374

irt1-1 and irt1-2 (Fig. 6). These results provided direct evidence that IRT1 plays a key 18


Page 19 of 37


375

role in the mechanism(s) whereby B. subtilis and A. brasilense reduce plant Cd levels.

376

Nevertheless, we observed that bacterial inoculation also led to a slight decline in the

377

Cd concentrations of IRT1 mutants, thereby indicating that other pathways might be

378

involved in the reduction of plant Cd levels following inoculation with

379

ABA-generating bacteria. In addition to IRT1, other transporters such as

380

zinc-regulated transporter/IRT-like protein (ZIP) and natural resistance-associated

381

macrophage protein family transporter (NRAMP) families have been proposed to be

382

involved in the Cd uptake by plants roots.44-49 Future studies need to clarify whether

383

these Cd uptake-associated transporters play a role in the reduction of Cd

384

accumulation in plants promoted by ABA-generating bacteria.

385

A further mechanism proposed to explain the observed decreases in plant Cd

386

concentrations is the biomass dilution effect due to promotion of plant growth.50 In

387

this regard, it is worth noting that, because the inoculation of Cd-contaminated soil

388

with B. subtilis and A. brasilense clearly promoted plant growth, the decrease in Cd

389

level in plants we observed might also be due to a biomass dilution effect. However,

390

we believe this assumption to be unlikely based on the following two findings: (1)

391

bacterial inoculation promoted the growth of wt plants only in Cd-contaminated soil

392

and not in uncontaminated soil (Fig. 2b); (2) bacterial inoculation resulted in a marked

393

decrease in the Cd levels of wt plants, whereas this effect was minimized or

394

eliminated in ABA-insensitive and IRT1-knockout mutants (Fig. 4a and 6). Therefore,

395

growth promotion under Cd-contaminated conditions does not appear to be the

396

mechanism by which B. subtilis and A. brasilense reduce the Cd levels in plants. 19



Page 20 of 37

397

Instead, we suspect that plant growth promotion probably resulted from a decrease in

398

Cd levels, which alleviated the toxicity in plants.

399

In conclusion, our results revealed that inoculating the rhizosphere with

400

ABA-generating bacteria (B. subtilis and A. brasilense) can reduce Cd levels and

401

improve plant growth by alleviating Cd-induced oxidative stress and photosynthetic

402

inhibition via ABA-mediated and IRT1-dependent mechanisms. On the basis of our

403

results, we suggest that developing techniques based on applications of

404

ABA-generating bacteria may represent a promising approach for reducing Cd

405

accumulation in edible plant organs, thus improving food safety in Cd-contaminated

406

areas.

407 408

Supplementary material

409

Table SI-1: Physicochemical properties of experimental soils

410

Table SI-2: Effects of Bacillus subtilis and Azospirillum brasilense inoculation on the

411

photosynthetic parameters of wt and snrk 2.2/2.3 plants under Cd stress

412

Figure SI-1: The colonization abilities of Bacillus subtilis and Azospirillum brasilense

413

in Cd-contaminated soils.

414

Figure SI-2: Effects of inoculation with ABA-generating bacteria on the available Cd

415

contents in Cd-contaminated soils.

416

Figure SI-3: Effects of inoculation with ABA-generating bacteria on the growth of wt

417

Arabidopsis and irt1-1 and irt1-2 mutants.

418 20


Page 21 of 37


419

Acknowledgments

420

This work was financially supported by the Science and Technology Department

421

Commonwealth Technology Applied Research Project of Zhejiang Province

422

(2017C32001), the Zhejiang Province Natural Science Foundation (LY14C130001),

423

College

424

2016r408057, 2017R408056), and the Student’s Science and Technology Project of

425

Zhejiang Gongshang University (14020000341, CX201723005).

Students’ Science-Technology

Innovation

Program

(201710353033,

426 427

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Figure Captions

588

Fig. 1. Effects of inoculating roots with abscisic acid (ABA)-generating bacteria on

589

Cd concentrations in the shoots of wt Arabidopsis plants under Cd stress. Bacterial

590

suspensions were inoculated onto the soil surface around the rhizosphere during the

591

4th, 6th, and 8th weeks after transplantation, and plants were harvested during the 9th

592

week. Values represent the means ± standard errors (n= 4). Different letters denote

593

significant differences among bacteria inoculation treatments at P < 0.05.

594

Fig. 2. Effects of inoculating roots with abscisic acid (ABA)-generating bacteria on

595

the growth of wt Arabidopsis plants under Cd stress. (a) Image of the plants; (b) fresh

596

weight of shoots; (c) image of the maximum fluorescence (Fm); (d) yield of

597

photochemical quantum Y(II); (e) concentration of malondialdehyde (MDA); (f)

598

concentration of H2O2; (g) rate of O2• - production. Treatments were the same as those

599

described in Fig. 1. Values represent the means ± standard errors (n = 4). Different

600

letters denote significant differences among bacteria inoculation treatments at P

Inoculation with Bacillus subtilis and Azospirillum brasilense produces

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