Enterobacter asburiae reduces cadmium toxicity in maize plants by

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Enterobacter asburiae reduces cadmium toxicity in maize plants by repressing iron uptake-associated pathways Cheng Zhou, Ninggao Ge, Jiansheng Guo, Lin Zhu, Zhongyou Ma, Shiyong Chen, and Jianfei Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03293 • Publication Date (Web): 21 Aug 2019 Downloaded from pubs.acs.org on August 22, 2019

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Journal of Agricultural and Food Chemistry

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Enterobacter asburiae reduces cadmium toxicity in maize plants by repressing iron

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uptake-associated pathways

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Cheng Zhou1,2, Ninggao Ge1, Jiansheng Guo3, Lin Zhu1, Zhongyou Ma1, Shiyong Cheng1*,

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Jianfei Wang1*

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1Key

Lab of Bio-Organic Fertilizer Creation, Ministry of Agriculture, Anhui Science

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and Technology University, Bengbu 233100, China

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2Jiangsu

Provincial Key Lab of Solid Organic Waste Utilization, Jiangsu Collaborative

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Innovation Center of Solid Organic Wastes, Educational Ministry Engineering Center

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of Resource-Saving Fertilizers, Nanjing Agricultural University, Nanjing 210095, China

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3School

of Medicine, Zhejiang University, Hangzhou 310058, China

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Cheng Zhou and Ninggao Ge contributed equally to this work.

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*Corresponding

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Shiyong Cheng, [email protected];

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Jianfei Wang, [email protected]

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Phone: +86-0550-6733024; Fax: +86-0550-6733024

authors:

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ACS Paragon Plus Environment


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Abstract

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Soil microbes have recently been utilized to improve cadmium (Cd) tolerance and

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lower its accumulation in plants. Nevertheless, whether rhizobacteria can prevent Cd

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uptake by graminaceous plants and the underlying mechanisms remain elusive. In this

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study, inoculation with Enterobacter asburiae NC16 reduced transpiration rates and

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the expression of some iron (Fe) uptake-related genes including ZmFer, ZmYS1, ZmZIP

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and ZmNAS2 in maize (Zea mays) plants, which contributed to mitigation of Cd toxicity.

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However, the inoculation with NC16 failed to suppress the transpiration rates and

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transcription of these Fe uptake-related genes in plants treated with fluridon, an

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abscisic acid (ABA) biosynthetic inhibitor, indicating that the impacts of NC16-

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inoculation observed were dependent on the actions of ABA. We found that NC16

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increased host ABA levels by mediating the metabolism of ABA rather than its

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synthesis. Moreover, the capacity of NC16 to inhibit plant uptake of Cd was greatly

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weakened in plants overexpressing ZmZIP, encoding a zinc/iron transporter.

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Collectively, our findings indicated that E. asburiae NC16 reduced Cd toxicity in maize

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plants at least partially by hampering the Fe uptake-associated pathways.

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Key words

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Abscisic acid metabolism, Cadmium stress, Iron uptake transporter, Rhizobacteria

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Introduction

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Cadmium (Cd) is the most mobile heavy metal found in soils that exhibits high

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toxicity to almost all living organisms.1 Due to huge demands for foods, Cd is

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transferred considerably from Cd-contaminated soils into edible tissues of crops and

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thus enters into food chains, posing risks to animal or human health.2,3 Although the

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immobilization of heavy metals by physical adsorption and chemical treatments have

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so far been employed to lower Cd availability in soils, these methods are costly and

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instable, rendering it impractical for wide application in farmlands.4 Hence, it is urgent

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to develop efficient tactics to reduce Cd levels in crops grown in Cd-polluted

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agricultural soils.

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In plants, Cd stress often causes diverse cytotoxic effects such as inhibition of

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photosynthesis, mineral and water absorption, involving the competition of metal-

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binding molecules with other essential metals, especially iron (Fe).5 Cd can enter into

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plant cells via several transporters such as NRAMPs, IRT1 and Ca channels.6 However,

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nicotianamine (NA), a critical chelator involved in the Fe translocation, can reduce Cd

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transport from roots to shoots.7 It has well been documented that Cd stress often

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provokes Fe deficiency responses by activating the expression of Fe acquisition-

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related genes such IRT1 and FRO2.8 In Strategy I plants (dicots and non-

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graminaceous monocots), Cd stress can trigger the transcription of IRT1, thereby

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enhancing the IRT1-mediated Cd uptake.9,10 In Strategy II (graminaceous) plants, Cd-

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induced the release of phytosiderophores (PS) promotes the formation of Fe3+-PS

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complex, which can be taken up by the YS1/YSL proteins.11

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Root-secreted PS to chelate Cd has previously been considered as an important

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strategy for ameliorating Cd toxicity in maize plants, although the formed Cd2+-PS

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complex is relatively weak as compared to Fe3+ or Zn2+.12 In fact, the PS-alleviated Cd

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toxicity in plants is mainly attributable to improve Fe nutritional status rather than to

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lower Cd availability.12 Consistently, Strategy I plants such as Arabidopsis

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overexpressing ZmYS1 exhibit stronger Cd tolerance, which contributes to improved

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Fe acquisition via bypassing Cd inhibition of IRT1-mediated Fe uptake.12 Interestingly,

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more recently, a strategy I-like Fe uptake system has been found in Strategy II plants

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such as rice and maize.13,14 Fe-regulated transporters such as OsIRT1, OsIRT2, ZmIRT1

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and ZmZIP3 are identified as functional Fe and Zn transporters, indicating that Strategy

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II plants can utilize both the strategy I and II mechanisms to mine Fe.13,14 Considering

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that, the interdiction of strategy I-like Fe acquisition pathways in Strategy II plants may

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repress the entry of Cd into agricultural foods.

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Beneficial bacteria colonized in plant rhizosphere have so far received great

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attentions.15-17 Diverse rhizobacteria establish mutual relationships with various plant

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species to benefit two parties.18,19 These beneficial bacteria are collectively considered

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as plant growth-promoting rhizobacteria (PGPR) that can assist plants in antagonizing

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phytopathogens, promoting root growth and surviving under adverse stresses.15-20

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Plant-microbes interactions have been shown to improve the efficiency of

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phytoremediation and lower Cd accumulation in plants by various mechanisms, such

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as microbial adsorption and auxin-mediated signaling pathways.17,21 Some abscisic

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acid (ABA)-generating bacteria have recently been demonstrated to inhibit IRT1-

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mediated Cd uptake and thus reduce Cd toxicity in plants.3,22 Nevertheless, ABA can

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be readily degraded in soils because of its chemical instability and the occurrence of

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ABA-metabolizing bacteria, which may result in the instability of bacteria-derived ABA

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in the soils.23-25

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In the present study, inoculation of maize plants with Cd-tolerant bacterium

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Enterobacter asburiae NC16 reduced Cd toxicity. Transcriptome analyses revealed

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that the expression of some Fe uptake-related genes including ZmFer, ZmYS1, ZmZIP

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and ZmNAS2 was greatly suppressed in the Cd-exposed roots by NC16. Moreover,

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NC16-mediated inhibition of host ABA metabolism resulted in increases of ABA levels,

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which was responsible for reducing the absorption of Cd by host plants. Our results

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further suggested that the inoculation of plants with NC16 relieved Cd toxicity

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primarily by interrupting the Fe uptake-related pathways.

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Materials and methods

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Plant materials, bacterial isolation and growth conditions

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Maize (Zea mays L. inbred line A32) seeds were treated with 0.1% HgCl2 for 10

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min and rinsed with sterile water at least three times. The sterilized seeds were

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germinated under dark and high humidity conditions at 30°C for 3 days (d). Soil

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samples were air-dried, ground and sieved through a 3-mm sieve for pot experiments.

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An aqueous solution of CdCl2 was used to treat the soils and keep it at 100% of water-

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holding capacity while ensuring an enrichment of 50 mg kg-1 Cd. The physicochemical

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properties of the soils were shown in Supplementary Table S1. 10-d-old seedlings were

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then transplanted into the Cd-polluted soils and placed in a growth chamber at 28°C

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light (16 h)/25°C dark (8 h) and 70% relative humidity. After three months,

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rhizospheric soils were used to isolate Cd-tolerant bacteria based on the methods

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described by Lin et al.26 One Cd-tolerant bacterial strain was purified and identified as

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E. asburiae NC16 by 16s rRNA sequencing (Genbank No. MK968154). This strain was

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stored in LB medium (10 g L-1 tryptone, 5 g L-1 yeast extract and 10 g L-1 NaCl)

108

containing 20% glycerol at −80°C for further experiments.

109

Assays of pot and split-root systems

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For pot experiments, maize seedlings were grown in Cd-polluted soils with the

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content of 50 mg kg-1 Cd. Pot experiments were conducted three times and each

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replicate contained 15 pots. The isolated bacteria were cultured in liquid LB medium

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at 30°C in a shaker at 200 rpm for 18 h. The culture was centrifuged at 8000 × g for 10

114

min, and the precipitate was then washed three times with sterile normal saline.

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Bacterial suspensions were prepared in sterile water to obtain an inoculum density of

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2 × 108 CFU mL-1. The bacterial suspensions were poured into the soil at the final

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density of 5 × 107 CFU g-1. To further assess the colonization of E. asburiae NC16 in

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plant rhizosphere, the plasmid pPROBE was firstly transferred into this strain. Serial

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dilutions from rhizopsheric soils were spread on LB agar medium supplemented with

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50 μg mL-1 of kanamycin, and incubated at 30°C to enumerate bacterial colonies.

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In a split-root system, a two-compartment plastic chamber was designed, and

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maize roots were put into two side chambers of the root box. To perform the split-

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root assays, 10-d-old maize seedlings grown in 1/2 Hoagland medium were

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transferred into split-root systems for 5 weeks. Bacterial strains were collected and

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washed three time with sterile normal saline, and then resuspended in 1/2 Hoagland

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medium. Bacterial suspensions were adjusted to an optical density of 5 × 107 CFU mL-1.

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The left chamber of split-root systems was inoculated with the bacterial suspensions

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containing 0 or 0.3 mM CdCl2, and the other chamber was not inoculated. The growth

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medium was replaced every 3 d during the whole plant growth.

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Transcriptome analyses and quantitative real time PCR

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In a split-root system, 10-d-old maize seedlings were treated with or without

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bacterial suspensions, and were subjected to 0 or 0.3 mM CdCl2. After 48 h of

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treatments, root samples were separated for extracting total RNA using Trizol reagent

134

(Invitrogen, USA). The quality and quantity of RNA was examined by the Agilent 2100

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Bioanalyzer (Agilent, USA). The RNA samples were used to construct cDNA libraries

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using the Illumina Hiseq 2500 platform (Illumina, USA). Raw reads were filtered by

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removing the low-quality reads and adaptor sequences, and were deposited in the

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NCBI SRA database (accession No. SRR9306628, SRR9306629, SRR9306630 and

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SRR9306631). Log2(Fold-change) > 1.0 and FDR adjusted p-value < 0.05 was set as the

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cutoff to screen differentially expressed genes (DEGs) among different treatments.

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Gene Ontology (GO) enrichment analysis was conducted as reported by Wang et al.27

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To perform quantitative real time PCR (qRT-PCR), RNA samples were reversely

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transcribed into cDNA using M-MLV reverse transcriptase (TaKaRa, Japan) according

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to the manufacturer’s instructions. The synthesized cDNA samples were used as the

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templates of qRT-PCR reactions that were carried out in an Applied Biosystems (ABI)

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7500 PCR machine as described by Zhou et al.20 The maize Actin (ZmActin) was used

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to normalize the expression levels of targeted genes. The primers used in this study

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were listed in Supplementary Table S2.

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Generation of transgenic maize plants

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To generate 35Spro::ZmZIP (ZIP-ox) plants, the coding sequence of ZmZIP was

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amplified by PCR. The PCR conditions were as follows: 95°C (5 min), 35 cycles of 95°C

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(30 s), 58°C (30 s), 72°C (90 s), and 72°C (5 min). More, PCR-amplified products were

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inserted into the cloning vector pUC18 for sequencing. The PCR products of ZmZIP

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were then digested and ligated into the binary vector pBI121 using Xbal and Kpnl.

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Transgenic plants were obtained by the Agrobacterium-mediated transformation of

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maize shoot tips.28 Briefly, Agrobacterium tumefaciens carrying the recombinant

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plasmids pBI121-ZmZIP at OD600 = 0.8 was collected and resuspended in 1/2 MS

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liquid medium containing 100 µM acetosyringone. Injured shoot tips were incubated

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in the bacterial suspension for 15 min, and then cultured on MS agar medium for 72 h

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in the dark at 25 °C. Subsequently, transformed plantlets were transferred into pots,

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and the seedlings were screened by spraying with 350 μg mL-1 of kanamycin after 2

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weeks of growth. Additionally, PCR assays were also conducted on these transgenic

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lines. T3 generation lines displaying the highest transcription of ZmZIP were used in

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next experiments. The primers used in this experiment were listed in Supplementary

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Table S2.

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Measurement of metal ion in plants, and Cd availability in soils

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To measure metal ion content, shoot and root samples were firstly rinsed with 0.5

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mM CaCl2 and dried overnight at 65°C. 0.1 g of dried samples was digested by

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HNO3/HClO4 (4:1, v/v) according to the method described by Lei et al.29 Elemental

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analysis was performed by an inductively coupled plasma atomic emission

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spectroscopy (ICP-AES; Perkin Elmer, USA). In addition, the bioavailability of Cd in soils

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was assessed by monitoring the extractable Cd content released by soils as described

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by Houba et al.30

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Analyses of physiological parameters, chloroplast structure and phytosiderophore

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H2O2 content was determined as previously described by Brennan and Frenkel.31

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Malondialdehyde (MDA) levels were measured by using thiobarbituric acid-based

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colorimetric method described by Quan et al.32 Electrolyte leakage (EL) was assayed

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based on the formulae: EL (%) = Initial electrical conductance (C1)/Final electric

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conductance (C2) × 100% as described by Shou et al.33 Chlorophyll content was

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extracted and determined according to the method described by Porra.34 Chloroplast

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ultrastructure was examined by transmission electron microscopy (TEM) based on the

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method described by Zhou et al.20 In addition, to analyze the root-released

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phytosiderophore 2’-deoxymugineic acid (DMA), the 1/2 Hoagland medium in the

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non-inoculated chamber was replaced with ultrapure water for harvesting root

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exudates. Root dry weight was used to normalize the content of root exudates. The

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concentrations of DMA were quantified by HPLC.12 The maximal PSII photochemical

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efficiency (Fv/Fm) was measured on maize leaves after 0.5 h of dark adaptation by a

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chlorophyll fluorescence imaging systems FluorCam 7 (Photon Systems Instruments,

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Brno, Czech Republic).

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Detection of ABA content and ABA 8′-hydroxylase activity

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The content of ABA was quantified by an indirect enzyme-linked immunosorbent

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assay (ELISA) according to the method described by Yang et al.35 To assess the capacity

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of plants to metabolize the hormone ABA, 10-d-old maize seedling grown in the split

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root system were treated with or without cell suspensions of NC16 for 48 h. Then, the

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roots were incubated with the specific radioactivity of 3.0 × 104 Bq/mL 3H-ABA for 24

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h. The metabolism of ABA and phaseic acid (PA) was analyzed as previously described

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by Wang et al.36

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Statistical Analysis

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The data presented in this study indicated the means of at least three replicated

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experiments. Statistical analysis for the data of physiological parameters such as

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biomass and metal ion content, qRT-PCR, stomatal aperture, transpiration rate and

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ABA levels was conducted using one-way analysis of variance (ANOVA) and Duncan’s

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multiple range tests at p < 0.05 in the statistical package of IBM SPSS Statistics 21.0.

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Results

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Inoculation of maize plants with E. asburiae ameliorates Cd stress-induced toxicity

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To investigate the effects of E. asburiae NC16 on the resistance of maize plants to

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Cd stress, we assessed plant growth performance. When plants were grown in Cd-

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polluted soils for two weeks, Cd stress resulted in plant growth retardation with low

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biomass. However, inoculation of plants with NC16 obviously alleviated Cd toxicity,

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displaying higher biomass and chlorophyll levels (Fig. 1a-c). In addition, no phenotypic

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difference was observed between the control and inoculated plants under non-Cd

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stress (Fig. 1a). The colonization abilities of NC16 in the plant rhizosphere were further

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examined. As shown in Supplementary Fig. S1, the population of NC16 initially

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increased (1.7 × 107 CFU g-1) and then decreased (5.3 × 106 CFU g-1) in the Cd-polluted

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soils after two weeks of inoculation. However, the bacterial colonization was

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observably higher in the non-Cd-treated soils than in the Cd-polluted soils.

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In the split-root system (Fig. 2a), there was no significant difference in the plant

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growth between the control and inoculated plants under non-Cd stress (Fig. 2b). The

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control plants displayed more severe root growth inhibition than the inoculated plants

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after 5 weeks of Cd treatment (Fig. 2c). Shoot and root fresh weights were significantly

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higher in the inoculated plants than in the controls (Fig. 2d,e). The production of H2O2

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was greatly elevated in the Cd-treated leaves. By contrast, Cd stress-induced increases

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of H2O2 levels were remarkably decreased in the inoculated plants (Fig. 2f). EL and

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MDA, important indicators of stress injuries, were also determined in both the control

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and inoculated plants. As shown in Fig. 2g,h, the inoculated leaves had less the values

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of EL and MDA than the controls after Cd treatment. These data indicated that the

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inoculation of maize plants with NC16 could mitigate Cd stress.

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Effects of E. asburiae on plant photosynthesis and chloroplast structures

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Alleviation of leaf chlorosis by NC16 was in line with the measured chlorophyll

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levels (Fig. 3a). The value of Fv/Fm was no significant difference between the control

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and inoculated plants under non-Cd stress, although the value of Fv/Fm in the controls

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was significantly lower than that in the inoculated plants after Cd treatment (Fig. 3b).

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As shown in Fig. 3c, TEM observation of chloroplast structures of mesophyll and

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bundle sheath cells in the Cd-treated leaves showed more swollen chloroplasts and

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less number of thylakoids, grana stacking and lamellae. Compared with the controls,

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when the Cd-treated plants were inoculated with NC16, more thylakoids, grana

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stacking and lamellae were found in the chloroplasts, indicating that these harmful

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effects could be alleviated by NC16. Nevertheless, the chloroplast structures were no

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observable difference between the control and inoculated plants under non-Cd stress.

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Transcriptome analysis of E. asburiae-colonized maize roots

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To explore the mechanisms underlying the inoculation with NC16 enhanced the

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adaptation of maize plants to Cd stress, the whole genome expression profiles of

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maize roots were conducted by RNA-Sequencing to identify root-expressed genes in

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response to NC16 or Cd stress. For this purpose, 10-d-old maize seedlings were

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treated with cell suspensions of NC16 and/or 0.3 mM Cd2+ for 48 h. After different

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treatments, we examined differential gene expression by comparison of the control

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plants with Cd stress (+Cd), NC16 (+NC16), and Cd stress plus NC16 (+Cd+NC16) (Fig.

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4a). The data of RNA-Sequencing showed that 962 genes, corresponding to 544 up-

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regulated and 418 down-regulated genes, exhibited significantly differential

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expression in the Comparison I (+Cd vs –Cd; Supplementary Table S3). However, more

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total number of DEGs was regulated by NC16 in the Comparison II (+NC16-Cd vs –Cd;

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Supplementary Table S4) or combination of NC16 and Cd stress in the Comparison III

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(+NC16+Cd vs +Cd) than was regulated by Cd stress alone (Fig. 4b; Supplementary

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Table S5).

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As shown in Fig. 4c, 810 up-regulated and 693 down-regulated DEGs were

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commonly expressed in both the Comparison II and III, respectively. It was observed

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that up-regulated DEGs in the Comparison I shared 46 DEGs with the down-regulated

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DEGs of both the Comparison II and III (Supplementary Table S6). Among these DEGs,

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the expression of several Fe uptake-related genes including ZmFer, ZmYS1, ZmZIP and

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ZmNAS2 was up-regulated in the Comparison I, but the inoculation with NC16 notably

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repressed their transcripts in both Comparison II and III (Fig. 4d), indicating that NC16

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negatively regulated plant’s Fe acquisition systems. Moreover, the DEGs in the

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Comparison III were functionally classified by the GO enrichment analysis, and were

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categorized into biological process (BP), cellular component (CC) and molecular

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function (MF) (Supplementary Fig. S2). In the BP category, most DEGs were involved

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in the processes such as oxidation reduction, carbohydrate metabolism, transport and

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photosynthesis. In the CC category, the majority of the DEGs were associated with

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membrane, cell wall, apoplast and photosystem. In the MF category, the DEGs were

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mainly related to oxidoreductase activity, transporter activity, heme binding and iron

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ion binding. Additionally, the results of qRT-PCR analyses indicated that the

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transcriptional patterns of some randomly selected genes were in accordance with

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the RNA-Seq data (Supplementary Fig. S3).

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Inoculation of maize plants with E. asburiae reduces Cd accumulation and its

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translocation from roots to shoots

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The content of Cd was firstly quantified in maize plants. Less Cd levels were found

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in the NC16-inoculated plants compared with the controls (Fig. 5a). After 1 week of Cd

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treatment, the inoculated shoots had 52% lower Cd content than the control shoots.

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In roots, the Cd content of the control plants was about 30% higher than that of the

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inoculated plants (Fig. 5a). The Cd translocation ratio to shoots (shoot Cd content/total

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Cd amount) was significantly lower in inoculated plants than the controls

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(Supplementary Fig. S4). Similarly, the inoculated plants had less Cd content (Fig. 5a)

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and lower Cd translocation ratio (Supplementary Fig. S4) compared with the controls

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after 5 weeks of Cd treatment. Furthermore, histochemical staining of Cd localization

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showed that the inoculated with NC16 reduced reddish precipitates in the roots

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compared with the controls after 1 week of Cd treatment (Fig. 5b).

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In this study, Cd exposure led to up-regulation of ZmYS1 transcription and more

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the release of the phytosiderophore DMA by maize roots, but these effects were

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dramatically inhibited by NC16 (Fig. 5c,d). A similar trend was also observed for the

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release of DMA under non-Cd stress. As shown in Fig. 5e, the content of Fe was

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markedly higher in the Cd-treated roots than the non-Cd-treated roots, and the

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inoculation of plants with NC16 reduced root Fe content under Cd stress. However, a

292

converse trend was observed for the shoots (Fig. 5f). There was no significant

293

difference in the shoot and root Fe levels between the control and inoculated plants

294

under non-Cd stress (Fig. 5e,f). Thus, the NC16-alleviated Cd toxicity in plants was not

295

involved in the strategy II responses including the release of DMA and ZmYS1 gene

296

expression but the strategy I-like Fe uptake pathways that likely impacted the entry

297

route of Cd.

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E. asburiae increases host ABA levels via repression of its metabolism

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As shown in Fig. 6a,b, the NC16-inoculated plants had more root ABA levels than

300

the controls under non-Cd stress after 48 h of inoculation. Similarly, upon exposure to

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Cd stress, the inoculated plants displayed 26% and 32% more shoot and root ABA

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levels than the controls, respectively. However, NC16 was not able to synthesize ABA.

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And the transcription of several ABA biosynthetic genes including ZmNCED, ZmABA1,

304

ZmAAO3 and ZmLOS5 in the plants was not notably induced by NC16 after 48 h of

305

treatments (Supplementary Fig. S5). Hence, the NC16-induced increases of host ABA

306

levels were neither attributable to bacterial secretion nor promotion of host ABA

307

synthesis.

308

We further tested whether regulation of ABA 8′-hydroxylase activity by NC16

309

mediated ABA levels in maize plants. For this reason, a split-root system was designed

310

as described above, in which the left chamber was inoculated into NC16 or not

311

inoculated as the controls. After 48 h of treatments, the roots were incubated with

312

3H-ABA.

313

of 3H-ABA compared with the controls (Fig. 6c). Consistently, the production of 3H-PA

314

was distinctly decreased in the inoculated plants (Fig. 6d). These data indicated that

315

the inoculation of plants with NC16 remarkably increased host ABA levels by inhibiting

316

its metabolism.

317

Inhibited host uptake of Fe by E. asburiae confers increased Cd resistance

After 24 h of incubation, the inoculation with NC16 reduced the metabolism

318

To test whether ABA was involved in the NC16-alleviated Cd toxicity in plants, we

319

examined the effects of NC16 on Cd levels in plants exposed to 10 μM fluridon (FLU),

320

an ABA biosynthetic inhibitor. Alleviation of Cd toxicity in the Cd-treated plants after

321

NC16 exposure was not found in the FLU-treated plants, as evidenced by stronger

322

inhibition of root growth and lower biomass (Fig. 7a-c). FLU exposure greatly 15



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weakened the NC16-indcued decreases of Cd levels in plants (Fig. 7d). Stomatal

324

apertures of the inoculated plants were reduced by 30% compared with the controls

325

after 48 h of Cd treatment, while that of the inoculated plants were not significantly

326

differed from the controls under non-Cd stress (Fig. 7e). In accordance to the changes

327

of stomatal apertures, the inoculation with NC16 increased leaf surface temperature

328

and reduced transpiration rates compared with the controls, although that was

329

markedly attenuated in the Cd-treated plants after FLU exposure (Fig. 7f,g).

330

Furthermore, treatment with ABA alone could obviously mitigate Cd toxicity in plants

331

(Fig. 7a-d). We thus concluded that the roles of NC16 in reducing Cd toxicity in plants

332

were positively associated with the actions of ABA.

333

As aforementioned, the data of RNA-Seq analyses showed that the transcription

334

of some Fe uptake-related genes in plants was strikingly lower in the inoculated plants

335

than the controls under Cd stress, indicating that reduced uptake of Cd by host plants

336

may be tightly related to the Fe uptake-associated genes. To verify this assumption,

337

we thus probed the impacts of NC16 on the expression of Fe uptake-related genes

338

including ZmFer, ZmYS1, ZmZIP and ZmNAS2 in the Cd-treated plants. The inoculation

339

with NC16 remarkably reduced the transcription of these Fe uptake-associated genes

340

in the Cd-treated roots compared with the controls. Nevertheless, FLU treatments

341

abrogated the inhibition of NC16 on these Fe uptake-related genes (Supplementary

342

Fig. S6). Furthermore, the capacity of NC16 to reduce Cd toxicity was largely

343

attenuated in ZmZIP-overexpressing (ZIP-ox) plants. Upon exposure to Cd stress, the

344

ZIP-ox plants had lower biomass and more Cd levels than the wild-type plants in the

16


Page 17 of 43


345

presence of NC16 (Fig. 7c,d). These results strongly indicated that the NC16-mediated

346

repression of the zinc/iron transporter contributed to lower Cd accumulation, thus

347

reducing Cd toxicity in maize plants.

348

Discussion

349

Considerable efforts have recently been focused on ameliorating Cd toxicity in

350

crop plants by manipulation of soil Cd-tolerant bacteria.3,10,22,26 However, it remains

351

largely unknown whether rhizobacteria can directly regulate host hormone levels and

352

that the molecular mechanisms underlying the rhizobacteria prevent Cd uptake by

353

host plants. In this study, inoculation of maize plants with E. asburiae NC16

354

substantially reduced Cd levels. NC16-mediated suppression of host ABA metabolism

355

conferred increased cellular ABA levels, thereby impeding Cd absorption through

356

modulation of Fe uptake-related pathways.

357

Cd stress often leads to inhibition of plant growth, reduction of chlorophyll

358

content, and destruction of the intracellular structures of chloroplasts, manifesting by

359

reduction of chloroplast number, size and grana stacking.37,38 In accordance to

360

previous studies on the toxic effects of Cd stress, total chlorophyll levels were

361

obviously decreased in the Cd-treated leaves. At the ultrastructural level, Cd stress

362

seriously affects the photosynthetic apparatus with abnormal chloroplast structures

363

such as chloroplast shape and swollen thylakoids. The impact of abiotic stress on the

364

photosynthesis is severed as a key indicator of plant adaption to adverse stresses.3,17,22

365

Compared with the controls, the inoculation of maize plants with NC16 had higher

366

chlorophyll levels, Fv/Fm, and better chloroplast development under Cd stress,

17



Page 18 of 43

367

indicating that this strain could improve the adaptation of plants to Cd stress. It has

368

well been shown that Cd toxicity is mainly resulted from the dysfunction of essential

369

nutrient uptake, especially Fe nutritional status since the chlorotic symptoms and

370

molecular responses imposed by Cd stress share the similarity to a certain extent with

371

those induced by Fe deficiency.8 Accordantly, it was observed here that the expression

372

levels of Fe acquisition-related genes were evidently increased in the Cd-treated roots.

373

Cd stress markedly decreased shoot Fe levels, but increased in roots, which was in

374

concert with the earlier study reported by Wang et al.39

375

Improved Fe acquisition has recently been shown to enhance the resistance of

376

plants to Cd stress.17,40 Arabidopsis plants co-overexpressing of FIT with bHLH38 or

377

bHLH39 exhibit promoted Fe assimilation and its root-to-shoot translocation, and

378

enhanced Cd sequestration in roots, thus conferring the strong tolerance of plants to

379

Cd stress.40 Increased Fe supply or source can also enhance Cd tolerance in plants by

380

reducing Cd absorption.40-42 In this study, higher shoot and less root Fe content was

381

found in the NC16-inoculated plants than the controls under Cd stress, while shoot

382

and root Cd levels were notably lower in the inoculated plants. It has previously been

383

demonstrated that NA plays a key role in protecting plants from Cd toxicity.40,43

384

Increased NA levels can promote the Fe translocation from roots to shoots, and

385

augment the Fe levels in shoots.40,43,44 However, the inoculation with NC16 lowered

386

the transcription of ZmNAS2, encoding a putative NA synthase, in maize plants

387

compared with the controls. We further observed that the inoculation of maize plants

388

with NC16 did not affect the Fe uptake, but increased shoot Fe accumulation under

18


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389

Cd stress. Thus, the effects of NC16-inoculation observed here were not attributable

390

to the ZmNAS2-mediated Fe uptake and translocation of Cd-treated plants.

391

The bioavailability of Cd in soils is a pivotal factor influencing plant uptake of Cd.45

392

However, the inoculation with NC16 did not impact the Cd availability in the soils

393

(Supplementary Fig. S7). These findings were similar to that reported by a recent study,

394

in which soil inoculation with beneficial microbes such as B. subtilis and A. brasilense

395

did not result in a significant increase in the availability of Cd.22 Hence, the NC16-

396

alleviated Cd toxicity in plants was likely resulted from the prevention of Cd entry into

397

the roots. Mounting evidence indicates that the increased ABA levels can improve Cd

398

tolerance in Arabidopsis and rice.10,46 We thus probed whether the NC16-induced Cd

399

tolerance of plants was tightly related to the changes of cellular ABA levels. As

400

expected, the content of ABA was much higher in the inoculated plants than the

401

controls after Cd treatment. Furthermore, the ABA biosynthetic inhibitor FLU

402

abrogated the effects of NC16 on mitigating Cd toxicity in plants. In view of the results,

403

the ABA-dependent pathways was essential for the NC16-induced inhibition of Cd

404

uptake by host plants, which was further supported by recent studies, in which

405

elevation of plant ABA levels strikingly lowed Cd levels by application of exogenous

406

ABA or ABA-generating bacteria.3,10,22 However, we found that NC16 was not able to

407

secrete and synthesize ABA. Thus, it was possible that the biosynthetic and metabolic

408

pathways of ABA in host plants were mediated by NC16. However, we found that the

409

transcription of ABA biosynthetic genes was not significantly induced by NC16. It has

410

previously been indicated that the ABA 8′-hydroxylase is essential for the metabolism

19



Page 20 of 43

411

of ABA in plants, thereby fine tuning cellular ABA levels.47,48 We thus examined

412

whether regulation of host ABA levels was attributable to the NC16-induced inhibition

413

of ABA metabolism. Our data showed that the converting ability of ABA into PA was

414

much lower in the inoculated plants than in the non-inoculated plants, implying that

415

the activities of ABA 8′-hydroxylase were markedly repressed in the inoculated plants.

416

Thus, NC16 could mediate host ABA metabolism rather than regulate its synthesis.

417

Indeed, the biosynthesis of ABA is involved in a series of ABA biosynthetic genes such

418

as NCED, ABA1, AAO3, and LOS5, and upregulation of the single gene cannot

419

considerably increase ABA levels.49,50 These findings provided important evidence that

420

rhizobacteria regulated the metabolic and physiological processes of host plants

421

preferentially by operating more critical pathways during plant-microbe interactions.

422

We deeply explored the potential mechanisms underlying the inoculation with

423

NC16 reduced Cd toxicity in maize plants. It has previously been shown that

424

exogenous application of ABA in rice enhances Cd tolerance with decreased Cd

425

content by inhibiting transpiration rates.46 Cd translocation from roots to shoots can

426

be driven by leaf transpiration. Reduced transpiration rates is thus conducive to

427

repressing the root-to-shoot transportation of Cd, which has been suggested to be a

428

crucial mechanism of Cd tolerance in plants.46 In this study, the NC16-inoculated

429

plants had smaller stomatal apertures than the controls under Cd stress, which

430

resulted in higher leaf temperature and lower transpiration rates. Moreover, the

431

effects of FLU treatments on the NC16-inoculated plants indicated that the

432

transpiration rates and Cd levels were substantially increased. These results indicated

20


Page 21 of 43


433

that higher ABA levels in the inoculated plants contributed to the decrease in

434

transpiration rates and Cd levels, thereby increasing the resistance of plants to Cd

435

stress.

436

Besides reduction of the transpiration rates, the NC16-induced increases of ABA

437

levels in maize may provoke an alternative complement pathway for lowering Cd

438

sensitivity. In Strategy I plants, the Fe2+ transporter IRT1 has been recognized as a key

439

transport route for Cd uptake in Fe-deficient plants.10 It is well documented that ABA

440

plays a negative role in regulating the Arabidopsis IRT1, thereby conferring the strong

441

resistance of plants to Cd stress.10 Recently, the identification of IRT orthologs in

442

Strategy II plants, rice (OsIRT1 and OsIRT2) and maize (ZmIRT1 and ZmZIP3) has also

443

been shown to function as Fe2+ transporters,13,14 which raise a question whether these

444

transporters lead to Cd sensitivity in graminaceous species. In fact, heterologous

445

expression of root-specific OsIRT1 and OsIRT2 markedly enhances Cd uptake in

446

yeast.51 To alleviate Cd stress-induced Fe deficiency, graminaceous plants have

447

adopted an alternative mechanism of root-secreted PS to chelate Fe.11 Transgenic

448

Arabidopsis expressing ZmYS1 exhibits greater growth performance under Cd stress.12

449

However, decreased Cd toxicity in the NC16-inoculated plants appeared not to be

450

associated with the function of ZmYS1. Because the inoculation with NC16 did not

451

upregulate the expression of ZmYS1, on the contrary, lowered its transcripts in the Cd-

452

treated roots. Likely, the inoculated plants experienced less Cd-induced Fe deficiency,

453

thus mildly activating the expression of ZmYS1 for chelating Fe, as evidenced by higher

454

shoot Fe levels in the inoculated plants. Intriguingly, the transcription of some strategy

21



Page 22 of 43

455

I-like Fe uptake-related genes including ZmFer and ZmZIP was markedly repressed in

456

the Cd-treated roots exposed to NC16. So this problem was whether there existed a

457

conserved mechanism in maize plants that ABA blocked plant uptake of Cd via

458

repression of the zinc/iron-regulated transporters ZIPs or IRTs. To verify this

459

hypothesis, we examined the effects of NC16-inoculation on plants overexpressing

460

ZmZIP under Cd stress. We found that the inoculation with NC16 failed to reduction

461

of Cd accumulation in the transgenic plants. Connolly et al. (2002) have previously

462

reported that Arabidopsis plants overexpressing IRT1 display greater sensitivity to Cd

463

stress than the wild-type plants, due to higher accumulation of Cd in the tissue.52

464

Therefore, our findings provided key evidence that the zinc/iron transporter play an

465

essential role in the mechanisms whereby NC16 inhibited the absorption of Cd by

466

plants.

467

In conclusion, although previous studies have explored the applications of soil

468

microbe-induced Cd resistance in crop plants, the underlying mechanisms are rarely

469

clear. In this study, a model is proposed for the NC16-alleviated Cd toxicity in maize

470

plants (Fig. 8), in which Cd stress stimulates the expression of Fe uptake-related genes

471

and thus promotes Cd accumulation in plants. However, the inoculation with NC16

472

can repress host ABA metabolism, and in turn increase ABA levels, contributing to

473

reduction of transpiration rates. We also unravel other key mechanisms whereby the

474

interdiction of host Cd uptake by NC16 is dependent on the ABA-mediated inhibition

475

of Fe uptake transporters such as ZmZIP. Therefore, application of the Cd-tolerant

476

bacteria E. asburiae will be a feasible way to lower Cd accumulation in crop plants.

22


Page 23 of 43

477


Acknowledgments

478

This work was supported by the National Natural Science Foundation of China

479

(31600210), China Postdoctoral Science Foundation (2017M620214), the Key

480

Research Project of the Anhui Science and Technology Committee (16030701102) and

481

the Natural Science Foundation of Education Department of Anhui province

482

(KJ2018ZD051).

483

Supporting Information Available: [Fig. S1 The colonization abilities of E. asburiae

484

NC16 in the rhizosphere of maize plants; Fig. S2 GO enrichment analysis of

485

differentially expressed genes (DEGs) in the Comparison III (+NC16+Cd vs +Cd); Fig. S3

486

qRT-PCR analyses of nine randomly selective genes from the DEGs among different

487

treatments; Fig. S4 Effects of E. asburiae NC16 on the translocation ratio of Cd to

488

shoots; Fig. S5 Effects of E. asburiae NC16 on the transcription of the ABA biosynthetic

489

genes in maize plants; Fig. S6 Effects of FLU treatments on the expression of Fe uptake-

490

related genes in NC16-inoculated maize plants under Cd stress; Fig. S7 Effects of E.

491

asburiae NC16 on the bioavailability of Cd in Cd-polluted soils; Table S1

492

Physicochemical properties of soil samples; Table S2 Primers used in this study; Table

493

S3 Up- and down-regulated DEGs in the Comparison I (+Cd vs –Cd); Table S4 Up- and

494

down-regulated DEGs in the Comparison II (+NC16-Cd vs –Cd); Table S5 Up- and down-

495

regulated DEGs in the Comparison III (+NC16+Cd vs +Cd); Table S6 Analyses of shared

496

DEGs between up-regulated DEGs of the Comparison I and down-regulated DEGs of

497

both the Comparison II and III].

498

23



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499

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Figure captions

663

Fig. 1 Effects of inoculating maize roots with E. asburiae NC16 on plant growth. Maize

664

seedlings were grown in Cd-polluted soils and were inoculated with cell suspensions

665

of NC16. After 2 weeks of Cd treatment, plant growth was monitored, including (a)

666

plant phenotypes, (b) dry weights, and (c) chlorophyll content. -Cd, non-Cd treatment;

667

+Cd, Cd treatment; -NC16, no inoculation with NC16; +NC16, inoculation with NC16.

668

Error bars represent ± SD (n = 3), and different letters show significant difference

669

at p < 0.05.

670

Fig. 2 Effects of E. asburiae NC16 on maize adaptation to Cd stress. In the split-root

671

system, the left chamber was poured with cell suspensions of NC16 (+NC16), and that

672

without bacterial inoculation was used as the controls (-NC16). (a) 10-d-old maize

673

seedlings were transplanted to the split-root system with or without bacterial

674

suspensions, and were treated with 0 (-Cd) or 0.3 mM Cd2+ (+Cd). After 5 weeks of

675

treatments, plant growth performance were assessed, including (b) plant phenotypes,

676

(c) root growth status, (d) shoot and (e) root dry weights, (f) H2O2 content, (g) EL and

677

(h) MDA values. Error bars represent ± SD (n = 3), and different letters show significant

678

difference at p < 0.05.

679

Fig. 3 Effects of E. asburiae NC16 on plant photosynthesis under Cd stress. 10-d-old

680

maize seedlings were transplanted to the split-root system with or without cell

681

suspensions of NC16, and were subjected to 0 (-Cd) or 0.3 mM Cd2+ (+Cd). (a)

682

Chlorophyll content and (b) Fv/Fm were measured after 5 weeks of treatments. In

683

addition, (c) TEM observation of chloroplasts in both the mesophyll and bundle sheath

32


Page 33 of 43


684

cells was conducted. Error bars represent ± SD (n = 3), and different letters show

685

significant difference at p < 0.05.

686

Fig. 4 Transcriptome analyses of E. asburiae NC16-inoculated roots exposed to non-

687

Cd or Cd stress. 10-d-old maize seedlings were transplanted to the split-root system

688

with or without bacterial suspensions (-NC16 or +NC16), and were suffered from 0 or

689

0.3 mM Cd2+ (+Cd) treatments. (a) After 48 h of treatments, root tissues were

690

harvested for comparative transcriptome analyses, including Comparison I (+Cd vs -

691

Cd), II (+NC16-Cd vs -Cd), and III (+NC16+Cd vs +Cd). (b) Statistics of up- and down-

692

regulated DEGs. (c) Venn diagram indicating shared and specific DEGs among up- and

693

down-regulated DEGs. (d) Transcription profiles of some Fe uptake-related genes

694

(ZmFer, ZmYS1, ZmZIP and ZmNAS2) in different treatments.

695

Fig. 5 Effects of E. asburiae NC16 on the absorption of Cd and Fe in maize plants. (a)

696

After 1 week and 5 weeks of Cd treatment, the content of Cd in both non-inoculated

697

and NC16-inoculated plants grown in the split-root system was measured. (b) After 1

698

week of Cd treatment, in vivo localization of Cd in the roots was imaged. (c) After 12

699

and 24 h of Cd treatment, the expression levels of ZmYS1 in the roots were quantified

700

in the plants by qRT-PCR. (d) Rates of DMA release within 24 h from the roots grown

701

in the split-root system. After 5 weeks of Cd treatment, (e) shoot and (f) root Fe

702

contents in plants grown in the split-root system was quantified. Error bars represent

703

± SD (n = 3), and different letters show significant difference at p < 0.05.

704

Fig. 6 Effects of E. asburiae NC16 on the ABA content and its metabolism in maize

705

plants. After 48 h of inoculation, the content of ABA in the (a) shoots and (b) roots of

33



Page 34 of 43

706

plants grown in the split-root system was determined under non-Cd or Cd stress. The

707

metabolism of ABA in plants subjected to different treatments was further examined.

708

Changes of (c) 3H-ABA catabolized to (d) PA in different treatments were compared

709

after 24 h of incubation. Error bars represent ± SD (n = 3), and different letters show

710

significant difference at p < 0.05.

711

Fig. 7 Effects of ABA on the E. asburiae NC16-mediated Cd stress responses in maize

712

plants. In the split-root system, 10-d-old wild-type or ZmZIP-overexpressing (ZIP-ox)

713

plants were treated with or without 5 μM ABA, 10 μM FLU and NC16 at the final

714

concentration of 5 X 107 CFU mL-1 upon exposure to 0.3 mM Cd2+ (+Cd). After 5 weeks

715

of Cd treatment, plant growth was evaluated, including (a) plant phenotypes, (b) root

716

growth, and (c) plant dry weights. The content of Cd was quantified in the plants after

717

1 week and 5 weeks of Cd treatment, respectively (d). After 48 h of treatments, (e)

718

stomatal apertures, (f) transpiration rates and (g) leaf surface temperature were

719

examined in the plants treated with NC16 and/or FLU under Cd stress. Error bars

720

represent ± SD (n = 3), and different letters show significant difference at p < 0.05.

721

Fig. 8 A proposed model unraveling the mechanisms of E. asburiae NC16-mitigated Cd

722

toxicity in maize plants. Inoculation of plants with NC16 represses host ABA

723

metabolism and thus increases its levels, thereby reducing transpiration rates. The

724

increased ABA levels can inhibit the transcription of Fe uptake-related genes such as

725

ZmZIP encoding a putative zinc/iron transporter. The impacts of NC16-inoculation

726

observed further contribute to ameliorated Cd toxicity in plants.

34


Page 35 of 43


Fig. 1

(a)

- NC16

+ NC16

(b)

Shoots

- Cd

Dry weight (mg plant -1 )

200

+ Cd

Chlorophyll content (mg g-1 FW)

(c)

160

Roots

a

a

b

120

c

80

d

d

40

e

f

0

3

a

a

2.5

b

2 1.5 1 0.5 0


c


Fig. 2 (a)

(b)

- NC16

+ NC16

- Cd

- NC16

(g)

+ Cd

0.4

b

0.2

(h)

60

0

a

a

2 b

1

c

0

a

30 20

b c

10

c

0

a

45 EL (%)

c

0

15

3

+ Cd

40

a

H2O2 content (μmol g-1 FW)

a

0.6

30

- NC16

(f)

b c

c

MDA content (μmol g-1 FW)

Root dry weight (g plant-1)

0.8

+ NC16

- Cd

(d) Shoot dry weight (g plant-1)

+ NC16

(e)

- NC16

+ NC16

- NC16

(c)

+ NC16

Page 36 of 43

a

50 40

b

30 20 10 0


c

c

Page 37 of 43


Fig. 3

4 3

a

1 a b

2

a

0.8 c

1

a

b c

0.6 0.4 0.2

0

0

- Cd

+ Cd + NC16

+ Cd

Chloroplast

- Cd + NC16

1 μm

500 nm

500 nm

500 nm

1 μm

1 μm

500 nm

Chloroplast

Grana stacking

1 μm

1 μm

Grana lamella

Bundle sheath

Mesophyll cell

(c)

(b)

Fv/Fm

Chlorophyll content (mg g-1 FW)

(a)

500 nm

1 μm

500 nm

1 μm

1 μm

500 nm


500 nm


Fig. 4

Page 38 of 43

(a)

Comparative transcriptome analyses

+ NC16

- NC16

Groups

+/- Cd

(c)

Number of DEGs

Up Down

1500 1000

I

+Cd vs -Cd

II

+NC16-Cd vs -Cd

III

+NC16+Cd vs +Cd

+ NC16 +/- Cd

(b) 2000

Comparision

1729

1473

1699

1527

+Cd vs -Cd Up

+NC16-Cd vs -Cd Up

+Cd vs -Cd Down

+NC16-Cd vs -Cd Down

+NC16+Cd vs +Cd Up +NC16+Cd vs +Cd Down 695

544

500

418 630 0

0 1

779

752

132 31

2

3

4

5

6

0

0 0

675

146 191 2 0 0 0 0 143 0 0 0 46 18 68

38112 2 0 0 0 0 11 0 0

0 0

0

0 0 1 0

0

0 0 0

0 0

0 0

4 0 0

0 0 0

0

21 0 643

1

0

0

(d)

ID

Annota�on

+Cd vs –Cd

-Cd+NC16 vs –Cd +Cd+NC16 vs +Cd

[Log2(Fold change)] [Log2(Fold change)] [Log2(Fold change)]

Zm00001d004007 Fer-like Fe deﬁciency-induced transcrip�on factor

1.0

-1.2

-1.5

Zm00001d017429 Fe-phytosiderophore transporter yellow stripe 1

1.4

-2.0

-2.0

Zm00001d019228 ZIP zinc/iron transport family protein

1.5

-2.3

-3.1

Zm00001d028887 Nico�anamine synthase 2

3.0

-2.2

-3.1


Page 39 of 43


Fig. 5

(b)

2500

-NC16 +NC16

2000

- Cd

1000

a

c

d

c

d

b

0

+ Cd 1 week

Relative expression level of ZmYS1

(c)

5 weeks (d)

8

a

10

6 b

4 2

b c

d

0 0h

12 h

24 h

12 h

- NC16

a

8 6

b

4 2

c

0

d

24 h

+ NC16

(e)

(f) 1500 900 600 300 0

100

a

1200 c

b c

Shoot Fe content (mg kg -1 DW)

Root Fe content (mg kg -1 DW)

+ NC16

b

1500

500

- NC16

a

DMA release (μM g -1 DW d -1)

Cd content (μg g-1 DW)

(a)

80

a

a b

60 40 20 0


c


Fig. 6

400

a

300 200 100

b c

c

a

500 400

b

300 200 100

d

c

0

0

(c)

(d) 50

a

40 30

b

20 10 0

c d

PA (% of

Radioactivity (%)

Root ABA content (ng g -1 DW)

(b)

3H-ABA)

Shoot ABA content (ng g -1 DW)

(a)

Page 40 of 43

100 80 60 40 20 0


a

b

c d

Page 41 of 43


Fig. 7

Wild-type plants +NC16 ( ZIP -ox)

+ABA

+NC16

(d)

Dry weight (mg plant -1)

(c)

1.2 a

1 0.8

+NC16(ZIP-ox)

a

+ABA +NC16

c

+NC16+FLU

d

0.6

0.4 0.2

c

a

b

c

0 Shoots

(e) 0.12 Stomatal aperture (width/length)

(b)

+NC16+FLU

Cd content (μg g-1 DW)

(a)

3000 2000 1000

f gg

e

a

c d

a

a b

b e f f

c

d

0

Roots 1 week

a

a

+NC16(ZIP-ox ) +ABA +NC16 +NC16+FLU

5 weeks

b

0.09

(g)

c

0.06

+Cd

+NC16

+NC16+FLU

28° C

0.03 0

0h

(f) Transpiration rate (mmol m-2 S -1)

1.6

+Cd

+NC16

a a a

1.2

24° C

24 h

b c

0.8

+NC16+FLU

d

c

d e

0.4 48 h

0 0h

24 h

48 h


Fig. 8


Page 42 of 43

Reduction Transpira�on rate

Stomatal aperture

ABA ABA metabolism Microbes

Cd stress

Fe2+ PS-Fe3+

ZmYS1

Cd2+ ZmZIP

Fe3+


Increase

Page 43 of 43


For Table of Contents Only

Cd

ABA Transpiration rate

Cd

ABA Cd

Cd

Protoplast ABA metabolism Cd

？

ABA Cd

Cd

Cd

ZmZIP

Cd

ABA

ABA

Cd

mRNA Membrane

Cd Cd

Cd

Cd-tolerant bacteria


Cd

Apoplast

Enterobacter asburiae reduces cadmium toxicity in maize plants by

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