Involvement of miR528 in the Regulation of Arsenite Tolerance in Rice

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Involvement of miR528 in the regulation of arsenite tolerance in rice (Oryza sativa L.) Qingpo Liu, Haichao Hu, Leyi Zhu, Ruochen Li, Ying Feng, Liqing Zhang, Yuyan Yang, Xingquan Liu, and Heng-Mu Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b04191 • Publication Date (Web): 25 Sep 2015 Downloaded from http://pubs.acs.org on September 29, 2015

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Journal of Agricultural and Food Chemistry

Title: Involvement of miR528 in the regulation of arsenite tolerance in rice (Oryza sativa L.)

Authors: Qingpo Liu,*,†,‡ Haichao Hu,†,# Leyi Zhu,†,# Ruochen Li,† Ying Feng,§ Liqing Zhang,† Yuyan Yang,† Xingquan Liu,‡ Hengmu Zhang,*, £

Institutions: †Department

of Agronomy, and ‡The Key Laboratory for Quality Improvement of Agricultural Products of Zhejiang

Province, College of Agriculture and Food Science, Zhejiang A & F University, Lin’an, Hangzhou 311300, PR China §College

of Environmental and Resources Science, Zhejiang University, Hangzhou 310058, PR China

£Institute of

Virology and Biotechnology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, PR

China

*Corresponding authors #H.H

and L.Z. contributed equally to this work

Tel: 86-0571-63742087 Fax: 86-0571-63741276 E-mail address: [email protected] (Q.L.) [email protected] (H.Z.)

1

ACS Paragon Plus Environment


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Abstract

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Tens of miRNAs were previously established as being arsenic (As) stress responsive in rice. However, their functional role in

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As tolerance remains unclear. Here, we demonstrated that transgenic plants overexpressing miR528 (Ubi::MIR528) were

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more sensitive to arsenite [As(III)] compared with wild-type (WT) rice. Under normal and stress conditions, miR528-5p and

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-3p were highly up-regulated in both the roots and leaves of transgenic plants, which exhibited a negative correlation with

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the expression of seven target genes. Compared with WT plants, Ubi::MIR528 plants showed excessive oxidative stress

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generation and remarkable amino acid content changes in the roots and leaves upon As(III) exposure. Notably, the

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expression profiles of diverse functional genes were clearly different between WT and transgenic plants. Thus, the observed

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As(III) sensitivity of Ubi::MIR528 plants was likely due to the strong alteration of antioxidant enzyme activity and amino acid

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profiles and the impairment of the As(III) uptake, translocation, and tolerance systems of rice.

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Keywords: Rice; miR528; overexpression; arsenite tolerance; gene expression pattern

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INTRODUCTION

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Arsenic (As) is a class of environmental metalloid that is ubiquitous and abundant in nature and is highly toxic to

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living organisms.1 In nature, As can exist in both inorganic and organic forms, with the former being more toxic than

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methylated As species.1 A growing body of evidence shows that chronic exposure to As, either by drinking

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As-contaminated water or through the food chain, causes severe health problems in humans.2,3 In South and

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Southeast Asia, dietary consumption of rice is considered to be a major risk for As exposure,4,5 given that rice, which is

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a staple food for nearly half of the world’s population, is more efficient in As uptake, translocation and accumulation

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than land-planted crops, such as wheat and barley.6,7 Accordingly, the issue of As contamination in rice has raised

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much concern worldwide.

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Improving plant tolerance to As may be considered as an effective way to mitigate As contamination in rice.

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However, only a few protein-coding genes have been identified as playing a possible role in As tolerance in rice thus

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far. As early as 2004, Dasgupta et al.8 identified and mapped an As tolerance-associated quantitative trait locus (QTL)

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to chromosome 6 of rice using a recombinant inbred line (RIL) population. Since then, no great progress has been

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made in the identification of genes functioning in the control and regulation of As sensitivity in rice. Recently, using

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heterologous expression systems, three independent research groups demonstrated that transgenic Arabidopsis

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plants that separately carried overexpression vectors for the rice genes PIP2;4, PIP2;6, PIP2;7, NRAMP1, and HIR1

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showed remarkable tolerance to As compared with wild-type (WT) plants.9-11 However, no direct evidence has been

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provided for the role of these genes in As tolerance in rice.

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It has been widely reported that regulatory miRNA molecules, by interacting with their target genes, play extensive

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and critical roles in the response to environmental stresses in plants.12 miRNAs are a class of endogenous non-coding

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small RNAs, 20-24 nt in length, that efficiently regulate the expression of target genes at the post-transcriptional level.

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other biotic17 or abiotic stresses have been identified in the rice genome. Thus, elucidation of the biological functions

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of these differentially expressed miRNAs in the regulation of plant adaptation is an intriguing topic. Among the

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identified differentially expressed miRNAs, miR528 was found to be significantly differentially regulated by distinct

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abiotic stresses. In addition, miR528 is strongly up-regulated by As(III) in the roots of Minghui 86, a rice cultivar that is

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highly sensitive to arsenite.16 Furthermore, a search for homologous sequences showed that miR528 is expected to

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be monocot species-specific.18 The targets of miR528 were predicted to include some multi-copper oxidase

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domain-containing proteins, laccases, and plastocyanin-like domain-containing proteins that are important in the

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regulation of copper homeostasis, lignin biosynthesis, and other biological processes in plant cells.19,20 However, the

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biological function of miR528 in the adaptive response to environmental factors in rice remains unclear.

In recent years, an abundance of miRNAs that are specifically responsive to Cd,14 H2O2,15 arsenite [As(III)],16 and

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Thus, in the present study, a construct carrying the overexpressed miR528 precursor was subsequently transferred

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to the japonica rice Nipponbare cultivar, which is more tolerant to As(III) than Minghui 86. As expected, the transgenic

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rice plants became more sensitive to As(III) compared with WT plants. On this basis, the physiological and molecular

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mechanisms of miR528-mediated As(III) sensitivity were preliminarily investigated.

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MATERIALS AND METHODS

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Rice cultivation and treatments

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The japonica rice cv. Nipponbare was used in all experiments. Rice seeds were surface sterilized with 0.5% NaOCl

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for 15 min, then rinsed thoroughly and soaked in deionized water overnight. Next, the seeds were placed on filter

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paper and germinated for 5 days by supplying a 0.5 mM CaCl2 solution. Subsequently, the rice seedlings were

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transferred to 10-L containers filled with half-strength Kimura solution21 for an additional 21 days of hydroponic

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cultivation in a growth chamber under a 16-h-light (30˚ C) and 8-h dark (20˚ C) photoperiod. Then, the seedlings were

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treated with 25 µM sodium arsenite (NaAsO2) for 6 days. During rice cultivation and treatment, the nutrient solution

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was renewed every 3 days. At different time points in the As(III) treatment (6 h, 24 h, 72 h, and 144 h), rice roots,

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shoots and leaves were sampled separately for further analyses. At harvest, the As(III)-treated seedling roots were

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first immersed in an ice-cold desorption solution for 10 min to remove apoplastic As. The desorption solution was

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composed of 1 mM K2HPO4, 0.5 mM Ca(NO3)2, and 5 mM MES (pH 5.5).21

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Cloning of miR528 and rice transformation

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A 400-bp fragment harboring the miR528 precursor sequence was amplified from the genomic DNA of cv.

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Nipponbare using the primers 5’-AAA GAG CTC CCA CCC TTC ACC AAT GGA TGC ATC-3’ (forward) and 5’-AAA

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GGA TCC GAA ATG GAT ATG AAT TCA GAC CTG G-3’ (reverse). The amplified PCR product was cloned into the

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Ubiquitin promoter cassette of pCAMBIA1300 between the Sac I and BamH I restriction sites, and this construct was

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subsequently transformed into Escherichia coli strain DH5α for proliferation. After confirmation through sequencing,

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the plasmid carrying pCAMBIA1300-MIR528 was electroporated into Agrobacterium tumefaciens EHA105, which was

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then transformed into rice to generate transgenic plants. T3 homozygous lines were used for all experiments.

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Real-time quantitative RT-PCR (qPCR)

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Total RNA and small RNAs from different tissues were extracted using the RNAiso plus and RNAiso for small RNA

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Kits (TaKaRa, Shiga, Japan), respectively. For real-time RT-PCR analysis, a universal reverse primer and two pairs of

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specific primers were designed for miR528-5p and -3p, respectively (Supplementary Table 1). The real-time qPCR mix

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included 5 µL of cDNA, 5 µL of primers, and 10 µL of 2× SYBR Green Mix, in a final volume of 20 µL. Real-time

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qPCR was carried out in a Mastercycler ep realplex system (Eppendorf, Westbury, USA) using the SYBR PrimeScript

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miRNA RT-PCR Kit (TaKaRa, Shiga, Japan) under conditions of 95˚ C for 1 min, followed by 40 cycles of 95˚ C for 15

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s and 60˚ C for 30 s.

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The expression analyses of the target genes and six functional genes were also performed using real-time qPCR.

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In these analyses, the RNA was first treated with DNase I and then reverse transcribed using an oligo(dT) primer. The

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PrimeScript RT Reagent Kit (TaKaRa, Shiga, Japan) was used to generate cDNA, at 50˚ C for 30-60 min. The primers

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employed for PCR amplification are listed in Supplementary Table 1. The Mastercycler ep realplex system (Eppendorf,

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Westbury, USA) was also used to carry out real-time qPCR with the SYBR Premix Ex TaqTM Kit (TaKaRa, Shiga,

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Japan).

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The relative expression level was calculated using the comparative CT method. The results were normalized to the

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expression of U6 or β-tubulin. Each experiment was replicated at least three times.

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Quantification of total As and Copper (Cu)

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After 6 days of As(III) treatment, the contents of total As and Cu in the samples were quantified via inductively

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coupled plasma mass spectrometry (ICP-MS, Agilent 7500a; Agilent Technologies, USA). Briefly, the roots and shoots

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of the rice seedlings were separately sampled and oven dried, after apoplastic As in the roots was removed by

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treatment with desorption solution for 10 min. The samples were then powdered using a ball mill (Retsch, MM301,

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Germany), and 0.3 g of the ground powder from each sample was digested with 0.5 mL of H2O2 and 2 mL of nitric acid

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at 120˚ C for 24 hours. When the digestion solution had cooled, deionized water was added to a final volume of 20 mL

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for the quantification of total As and Cu through ICP-MS.

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Antioxidant enzyme assays

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After 3 and 6 days of As(III) treatment, fresh root and leaf samples from each treatment were used to assess the

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activity of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase

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(APX). The procedures and methods applied for the enzymatic activity analyses were described as previously.22

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Amino acid analysis

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Fresh root and leaf samples from each treatment were milled to a fine powder for amino acid analysis. Briefly, 0.3 g

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of the ground samples was digested with 10 mL of HCl (6 mol L-1) at 110˚ C for 24 hours. After cooling, the digestion

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solution was diluted by adding 15 mL of deionized water to a final volume of 25 mL. Then, 1 mL of the diluted digestion

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solution was transferred to a glass tube and dried in a vacuum oven at 50˚ C. After being dried completely, the sample

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was re-dried once more after adding 1 mL of deionized water. After re-drying, 1 mL of deionized water, 0.5 mL of

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triethylamine hexanenitrile (14%), 0.5 mL of phenyl isothiocyanate and 2 mL of n-hexane were successively added to

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the glass tube. The mixture was rocked intensely for 2 minutes and was then placed on a table for a static duration of

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1 hour. Finally, the obtained subnatant was filtered and injected into a Waters HPLC system to quantify the contents of

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different amino acids.

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Statistical analysis

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All of the data are presented as the mean value ± SE (standard error) of three or four replicates. The data were

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analyzed via two-way analysis of variance to confirm the variability and validity of the results and using the least

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significant difference (LSD) test to examine the significant differences between treatments in SPSS 19.0.

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RESULTS

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Analysis of the expression pattern of miR528 at different developmental stages in transgenic plants

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To better understand the relationship between plant growth and miR528 expression, we investigated the expression

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patterns of miR528-5p and -3p at four different stages of development in WT and transgenic plants under normal field

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conditions. As clearly shown in Figure 1, both miR528-5p and -3p were highly expressed in transgenic seedlings

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compared with WT plants. With the time of plant growth, the expression levels of the two miR528s decreased

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significantly (Figure 1). Both miR528-5p and -3p reached their minimum expression level at the booting stage and then

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rebounded at the heading stage. Notably, the expression levels of both miR528s were over 5-fold higher in the

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transgenic plants than in their WT counterparts at the booting stage. These observations indicated that miR528 was

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indeed highly expressed throughout all stages of development in Ubi::MIR528 transgenic plants.

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Comparison of the phenotypes of WT and Ubi::MIR528 transgenic plants under arsenite stress

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Under normal growth conditions, the transgenic plants showed no apparent phenotypic changes compared with WT

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plants, although miR528 was highly expressed throughout all developmental stages in the transgenic plants (Figure 1).

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However, when challenged with 25 µM As(III) for 3 days, almost the entire Ubi::MIR528 seedling exhibited the typical

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As-poisoning phenotype, with chlorosis being observed in the leaves and shrinking or necrosis in aerial parts, in

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contrast to WT (Figure 2). The observed sensitivity of Ubi::MIR528 plants to As(III) revealed that miR528 is directly

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involved in the process of As tolerance in rice.

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Moreover, compared with WT plants, Ubi::MIR528 plants accumulated a significantly lower total As level in the roots

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(485.2 ± 21.8 vs. 1153.7 ± 69.2 mg kg-1) but showed a higher concentration in the shoots (39.8 ± 1.63 vs. 25.7 ± 1.15

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mg kg-1). Total As was decreased by 137.8% in the roots, but was increased by 54.9% in the shoots of transgenic

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plants. Accordingly, transgenic plants overexpressing MIR528 exhibited significantly weaker As uptake and

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accumulation (588.0 ± 29.4 vs. 1213.2 ± 60.7 mg kg-1; p

Involvement of miR528 in the Regulation of Arsenite Tolerance in Rice

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