Changes in the gene expression profile of Arabidopsis thaliana under chromium stress

Ecotoxicology and Environmental Safety 193 (2020) 110302

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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv

Changes in the gene expression profile of Arabidopsis thaliana under chromium stress

T

Jianxia Liua,1, Guotao Dingb,1, Zikuan Gaia, Wei Zhangc, Yonghong Hanb, Weihao Lib,∗ a

Affiliated Hospital of Hebei Engineering University, Hebei, Handan, China Handan Municipal Center for Disease Control and Prevention, Hebei, Handan, China c College of Life Sciences Agricultural University of Hebei, Baoding, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Chromium Cr(III) Cr(VI) Arabidopsis thaliana Gene expression profile

Based on previous studies and preliminary test results, 200 μM was used as the test concentration of chromium (Cr), and changes in the gene expression profile of Arabidopsis thaliana in response to 24-h treatments of Cr(III) and Cr(VI) were analyzed using the Arabidopsis ATH1 Genome Array. The results were as follows. There were 238 upregulated genes and 858 downregulated genes in response to treatments with Cr(III) and Cr(VI). For Cr (III) and Cr(VI) treatments, there were 185 and 587 specifically upregulated genes as well as 220 and 956 specifically downregulated genes, respectively. Among the common differentially expressed genes (DEGs), the expression levels of genes involved in redox, secondary metabolism, and energy metabolism processes were significantly downregulated, while those of genes related to the stress response, photosynthesis, and sulfur metabolism were significantly upregulated. These findings indicated that Cr seriously affected the normal activities of A. thaliana cells. Some genes associated with stress and regulation were upregulated to adapt to the stress caused by Cr. Among the unique DEGs, the expression levels of genes involved in indole-3-acetic acid (IAA) regulatory pathway were significantly increased in response to Cr(III) treatment; the expression levels of genes involved in the abscisic acid (ABA) regulation pathway and carotenoid synthesis were significantly increased following Cr(VI) treatment. These results revealed some differences in response to Cr(III) and Cr(VI) in A. thaliana.

1. Introduction Heavy metal pollution is a major environmental hazard factor worldwide (Abou-Elwafa et al., 2019). Heavy metals in water, air, and soil can be enriched progressively through the food chain and pose a hazard to the health of plants and humans (Bertucci et al., 2018). Chromium (Cr) is the seventh most abundant element on earth, and it is widely used in leather tanning, printing and dyeing, and electroplating, among other processes (Lunk, 2015; Singh et al., 2013; Zayed and Terry, 2003). Due to its widespread industrial use, a large amount of Cr waste is discharged into the environment in the form of liquids, solids, and gases, eventually causing serious harm to the ecological environment. Many studies have shown that Cr is toxic to the roots, stems and leaves of plants (Lopez-Bucio et al., 2014). Cr generally exists in the form of trivalent Cr(III) and hexavalent Cr (VI) in nature. Cr(III) is an essential trace element for glucose metabolism in mammals (Evert et al., 2013). In contrast, Cr(VI) is

carcinogenic and is classified as a type A carcinogen. Although it has been reported that a low concentration of Cr(III) can stimulate plant growth, high concentrations of Cr(III) and Cr(VI) can severely inhibit plant growth. Generally, Cr(VI) is more toxic than Cr(III) in plants; however, in some plants, the latter has been found to exhibit more toxic effects than the former. In recent years, many studies have been conducted to examine the phytotoxicity of Cr. However, most of the reported data were obtained based on the total Cr concentration, which rarely distinguishes differences in Cr toxicity in different valence states. Some studies were about the changes of ultrastructural induced by Cr(VI) in Roots of Arabidopsis thaliana (Eleftheriou et al., 2015). Studies have found that plant defense mechanisms are not passive when subjected to stresses; instead, they take proactive measures to cope with the stressors. These processes include adaptive morphological, physiological, and biochemical changes, with changes at the molecular level playing a decisive role. Therefore, only at the molecular level can the complex biological

∗

Corresponding author. Handan Municipal Center for Disease Control and Prevention, Hebei, China. Tel.: +86 0310 8168766; fax: +86 0310 8168129. E-mail address: [email protected] (W. Li). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.ecoenv.2020.110302 Received 3 October 2019; Received in revised form 26 December 2019; Accepted 4 February 2020 0147-6513/ © 2020 Elsevier Inc. All rights reserved.

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mechanism of plant stress finally be revealed. To the best of our knowledge, there are currently no data regarding the influences of Cr (III) and Cr(VI) on the gene expression profile of Arabidopsis thaliana. The purpose of this study was to investigate the molecular regulatory network of the Cr stress response by using gene chip technology at the transcriptional level, to discover important genes related to stress, and to provide supportive data and theoretical bases for Cr stress at the molecular level.

0.5 h). The absorbance of the total RNA was measured under 260/280nm UV light to calculate the purity of the total RNA. Agilent's microarray hybridization and hybridization microarray scanning experiments were performed by Shanghai OE Biotech Co., Ltd.

2.3. Data analysis of the expression profile microarray Standardization: All the microarray samples in this study were considered by the standardization method, which is a full data algorithm (it differs from the baseline algorithm, which selects one benchmark in all microarray samples, all other microarray data are converted using this benchmark to obtain the same mean density.) The purpose of this algorithm is to maintain consistency of the probe intensity distribution on the chip for all the current samples. If the two N-dimensional data vectors X and Y have the same data distribution, then the quantile-quantile figure based on these two vectors should be a straight line parallel to the diagonal line. Based on this statistical idea, the data vector can be projected onto the diagonal to check whether the same data distribution is obtained. The analysis was performed using GeneSpring software, and the quantile standardization method was used. Probe filtering:

2. Materials and methods 2.1. Plant cultivation Murashige and Skoog (MS) basal medium were purchased from Biotech (Shanghai, China). The Cr(NO)3 and K2Cr2O7 were purchased from Sinopharm Chemical Reagention corporation (Beijing China). The A. thaliana ecotype Columbia (Col-0) was selected. After sterilization, the seeds of A. thaliana were sown in Murashige and Skoog (MS) basal medium. Seeds were placed at 4 °C for 3 d and then moved to light and germinated. The culture temperature of the plant growth chamber was 22 °C, the light source was fluorescent, the light and dark cycle was 8/16 h, the light intensity was approximately 90 μM m−2 s−1, and the relative humidity was approximately 70%. After 10 days of growth, the plants were transferred to MS medium and then, at 14 d, to MS medium containing 200 μM of Cr(III) or Cr(VI) for 24 h. Consistent with the previous culture conditions, the cultivation temperature of the plant growth chamber is 22 °C, the light and dark cycle is 8/16h, the light intensity is about 90 μMm−2 s-1, and the relative humidity is about 70%. The tissues were then collected for later use. Simultaneously, some plants were transferred from MS medium to new MS medium without Cr(III) or Cr(VI) and used as the control group. Cr (III) and Cr(VI) were provided by Cr(NO)3 and K2Cr2O7, respectively. We used the Arabidopsis ATH1 Genome Array chip from Affymetrix, USA, which contains 22,500 probe sets representing approximately 24,000 genes. There were 2 test samples for each of the Cr(III) and Cr (VI) treatments, and each sample consisted of 3 biological replicates. For 1 blank control sample, there were 2 replicates.

(1) Statistical analysis of the probe filtering: Probes filtering steps were performed in the following order: 1) assessment of whether the background and signal could be clearly distinguished; 2) assessment of whether the probe signal was detected; 3) assessment of whether the probe signal was effectively greater than the negative control; 4) examination of the probe signal strength; 5) confirmation that for the two sets of data used for comparison, at least 75% of one data set was labeled as ‘Detected’. (2) IsNOTWellAboveBG: probe was filtered using the limWellAbove function. (3) Processed data: After filtration, in the expression results of probes used for gene expression analysis, repeated data were first combined, and then complete data were preprocessed, to obtain nonrepetitive probes.

2.2. Agilent expression profile microarray experiments Gene probe annotation: The gene annotation was based on the Agilent commercialization chip annotation and completed using GeneSpring software.

Total RNA was extracted using the RNeasy Mini Kit (QIAGEN). The procedure was as follows:The total plants under 200 mM Cr stress were ground in liquid nitrogen, followed by the addition of 100 mg of reagent in a 1.5-mL centrifuge tube. Immediately after volatilization of the liquid nitrogen, 450 μL of radio link control (RLC) buffer (with 1% β-mercaptoethanol added) was added, mixed thoroughly, and placed in a warm bath (56 °C) for 1–3 min. The mixture was aspirated into a QIAshredder centrifuge column and centrifuged at 13,000 rpm for 2 min at room temperature. The supernatant was transferred to a new 1.5-mL centrifuge tube. Anhydrous ethanol at half the volume of the supernatant was added to the centrifuge tube to generate a precipitate, which was then pipetted back and forth using a pipette. The samples were immediately transferred to an RNAeasy mini centrifuge column and centrifuged at 10,000 rpm for 30 s. The bottom layer filtrate was discarded, and 700 μL of Buffer RW 1 was added and centrifuged at 10,000 rpm for 15 s at room temperature. The bottom layer filtrate was discarded, and 500 μL of Buffer RPE was added was centrifuged at 10,000 rpm for 15 s at room temperature. Repeat this step one time. The bottom layer filtrate was discarded, and the empty centrifuge column was centrifuged at 13,000 rpm for 1 min at room temperature. A total of 30 μL RNase-free water was added and centrifuged at 10,000 rpm for 1 min at room temperature (a new 1.5-mL centrifuge tube was used to collect the RNA solution after centrifugation). Total RNA was purified according to the instruction supplied with the QIAGEN's RNeasy mini kit. The total RNA integrity was assessed by measuring the ratio of 28S to 18S by agarose gel electrophoresis (180 V,

2.4. Statistical analysis Significant differences were analyzed using the unpaired t-test, and the screening criterion was that the absolute fold change (FC(abs)) was greater than or equal to 2 (i.e., the threshold of the Log2Fold change was set to −1, 1) and p ≤ 0.05. If two samples were labeled as T and N, then T was the treatment group and N was the control group. A negative FC value indicated that the expression of T was lower than N, and a positive value denoted relatively higher expression, and whereas FC < −1 indicated significantly low expression, FC > 1 indicated significantly high expression. Clustering and functional enrichment analysis: A heatmap was generated using MeV software, and the parameters were “Distance Metrics: Euclidean Distance; Linkage Method: Complete linkage. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed using the online analysis software FunNet, and the results and bar charts generated using the web version are provided.

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2.5. Verification of genes with altered expression by fluorescence quantitative reverse transcription-polymerase chain reaction (qRT-PCR) using SYBR green I

The microarray results showed that 1844 DEGs (probes) accounted for approximately 8.6% of the total number of probes. Following the Cr (III) treatment, there were 1501 DEGs, including 423 upregulated and 1078 downregulated genes; upon Cr(VI) treatment, there were 2639 DEGs, including 825 upregulated and 1814 downregulated genes. There were 238 upregulated common genes and 858 downregulated common genes in response to Cr(III) and Cr(VI) treatments. There were 185 specifically upregulated genes and 220 specifically downregulated genes following Cr(III) treatment, and 587 specifically upregulated genes and 956 specifically downregulated genes upon Cr(VI) treatment (Supplementary Fig. 1).

According to the gene expression microarray results, 10 upregulated or downregulated genes with various fold changes were selected, and qRT-PCR primers were designed based on the messenger RNA (mRNA) sequences in the GenBank database to verify the microarray results by qRT-PCR. PrimerQuest software was used to design the primers, and the primers were synthesized by Shanghai Biological Engineering Co., Ltd. The primer sequences are shown in Supplementary Table 1. Fluorescence qRT-PCR was performed according to the instruction manual supplied with the SYBR® Premix Ex Taq™ reagent kit, and the reaction was performed on the ABI 7500 fluorescence qRT-PCR instrument. The housekeeping gene ACTIN2 was used as a control for standardization of data, and the fold change in gene expression was calculated using the 2−ΔΔCT method. To ensure consistency of the Ct value, each sample including the control gene was tested three times. The reaction system consisted of the following:

3.2. Validation of DEGs by fluorescence qRT-PCR Fluorescence qRT-PCR is a reliable method to verify microarray results. In this study, 10 significant DEG sequences were selected for primer design for fluorescence qRT-PCR analysis to verify the accuracy and reliability of the gene chip analysis. The results are shown in Supplementary Table 2. Six upregulated genes were confirmed by fluorescence qRT-PCR, and the expression levels of 4 downregulated genes were inhibited. These results indicated that the microarray data were biologically reproducible, and the gene chip analysis results were reliable. The relative expression levels of the same gene detected by the two methods were not consistent, and the fold change in expression of the same gene differed between the gene chip and fluorescence qRTPCR results. For GSTU4, PAD3, CYP87A2, and RBOHB4, the results obtained using the two methods were very similar, but the fold changes of the other 6 upregulated genes were very different between the two methods. These differences may have been due to the different sensitivity and reaction kinetics of the two methods. In general, upregulated gene expression detected by microarray was slightly lower than that detected by fluorescence qRT-PCR, especially for those genes that were significantly overexpressed. This phenomenon is mainly a consequence of the higher sensitivity of fluorescence qRT-PCR than microarray, as well as the fluorescence signal saturation phenomenon in gene microarray hybridization.

SYBR® Premix Ex Taq™ (2 × ) 10.0 μL PCR Forward Primer (10 μM) 0.4 μL PCR Reverse Primer (10 μM) 0.4 μL ROX Reference Dye II (50 × ) 0.4 μL DNA template 2.0 μL ddH2O 6.8 μL Total volume 20.0 μL The reaction conditions were as follows: 95.0 °C for 30 s; 95.0 °C for 5 s; 60.0 °C for 34 s; absorbance measurement; proceed to line 2 for 40 cycles; 95.0 °C for 15 s; 60.0 °C for 60 s; 95.0 °C for 15 s; completion.

3. Results and discussion 3.1. Quantitative analysis of differentially expressed genes in A. thaliana in response to Cr treatments In this experiment, the Arabidopsis ATH1 Genome Array was used to study the gene expression patterns in A. thaliana seedlings subjected to Cr(III) and Cr(VI) treatments. In comparison to the untreated control group, genes with changes in expression intensity by two or more times (FC ≥ 2 and P < 0.05) in the treatment group were defined as differentially expressed genes (DEGs) (Fig. 1).

3.3. GO enrichment analysis of DEGs in response to Cr(III) and Cr(VI) treatments GO consists of three parts, namely, biological process, molecular function, and cellular component. By means of ID matching or sequence annotation, each protein or gene can be matched with its corresponding

Fig. 1. Volcano plot of DEGs in response to Cr(III) and Cr(VI) treatments. 3

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nonbiological damage (Chen et al., 2010). It is speculated that this transcription factor is important in the response to Cr stress. MYB transcription factors represent one of the most abundant and functionally diverse transcription factor families in plants, with approximately 130 members identified in A. thaliana. These transcription factors play an important regulatory role in plant response processes and are widely involved in the regulation of plant secondary metabolism and responses to hormones and environmental factors. They also play a regulatory role in cell differentiation, organ formation, plant leaf morphogenesis, and disease resistance. The MYB domain is a peptide segment with approximately 51–52 amino acids, containing 3 tryptophan residues that are separated by 18–19 amino acid residues; it forms a hydrophobic region containing conserved amino acid residues and spacer sequences, folding the MYB domain into a helix-turn-helix structure. MYB is widely involved in various physiological activities of plants, such as secondary metabolism, cell morphogenesis, signal transduction in plant growth, biotic and abiotic stress responses, and ABA sensitivity. Analysis of the promoter regions of high-salt-, drought-, and ABA-induced genes in A. thaliana has shown that the core sequence of the MYB binding site is TAACTG. These transcription factors are present in ABA-dependent high-salt, drought, low-temperature, and damage response pathways, bind to the core sequence TAACTG of the promoter of downstream target genes, and can induce the expression of stress-resistant genes in plants. Following Cr treatment, MYB45, MYB62, and MYB79 were significantly upregulated, among which MYB62 has been reported to be correlated with the phosphate-starvation response and to regulate the biosynthesis of gibberellic acid. NAC transcription factors are a class of transcription factors specific to higher plants. The N-terminus of this family contains a conserved NAC domain (approximately 150 amino acids), and the C-terminus is a highly variable transcriptional activation region, which plays a very important role in plant direction and stress responses. Huang (Huang et al., 2012) reported that overexpression of rice SNACl (stress-responsive NaCl) can enhance the salt tolerance of transgenic rice. Hu (Hu et al., 2009) also found that overexpression of different NAC genes in A. thaliana can improve their drought resistance. NAC042 in A. thaliana was significantly upregulated under Cr treatment; this gene is induced by H2O2. It is speculated that oxidative damage on A. thaliana could lead to the accumulation of H2O2, thereby inducing NAC042 upregulation (Wu et al., 2012). The AP2/EREBP transcription factor family is a class of transcription factors unique to plants. The common feature of this family of transcription factors is that their DNA binding domain is a conserved AP2/REBP domain. According to the number of conserved domains, this family is divided into two subfamilies, AP2 and EREBP. The AP2 subfamily transcription factors are related to the regulation of plant growth and development. The EREBP subfamily transcription factors respond to plant abiotic stress. ERF069 (AT1G22985), RWP-RK domaincontaining protein (AT2G43500), and dehydration-responsive elementbinding protein 2E (AT2G38340) were significantly upregulated in response to Cr treatment, suggesting that AP2/EREBP transcription factors might be involved in the response to Cr stress. Zinc finger family proteins make up the most abundant transcription factor family in eukaryotes. These proteins can bind to zinc ions to form a stable finger-like structure and play a broad role in regulating cellular differentiation and embryonic development through the process of gene expression. Under biotic and abiotic stresses, the expression of a variety of transcription factors in zinc finger protein family factors was induced. In this experiment, BHLH038, BHLH100, and BHLH101 were upregulated, whereas the upregulation of BHLH100 was particularly significant, with fold changes of 17.83 and 41.13 in response to Cr(III) and Cr(VI) treatments, respectively. The 3 transcription factors belonged to the BHLH 1b subgroup, and studies have shown that they respond to iron, nickel, zinc, and copper (Wu et al., 2012). It is speculated that this phenomenon is associated with Cr stress.

GO number, which then corresponds to the term, namely, the functional category or cellular localization. At the biological process level, for upregulated genes in response to both Cr(III) and Cr(VI) treatments, significant enrichment was found in transcriptional regulation and defense response; for the downregulated genes in response to both Cr(III) and Cr(VI) treatments, significant enrichment was found in redox processes and chitin responses. As an upregulated gene under both Cr(III) and Cr(VI) treatments, auxin stimulation was significantly enriched under Cr(III) treatment and relatively enriched under Cr(VI) treatment; as a downregulated gene, transcription regulation was significantly enriched only under Cr(VI) treatment (Supplementary Fig. 2). These results indicate that under chromium stress, A. thaliana was damaged by oxidation and the redox process was blocked, resulting in transcriptional regulation. At the molecular function level, for both up and downregulated genes in response to both Cr(III) and Cr(VI) treatments, transcription factor activity was significantly enriched; for the upregulated genes in response to both Cr(III) and Cr(VI) treatments, protein kinase activity was significantly enriched; for downregulated genes in response to both Cr(III) and Cr(VI) treatments, catalytic activity and heme-binding were significantly enriched (Supplementary Fig. 3). These results indicate that A. thaliana can regulate the activity of transcription factors through upregulation of kinase expression, thus responding to Cr stress. At the cellular component level, for both up and downregulated genes in response to both Cr(III) and Cr(VI) treatments, the intimal system, apoplast, and cell wall were significantly enriched. In addition, for upregulated genes in response to Cr(VI) treatment, the chloroplast thylakoid membrane was significantly enriched (Supplementary Fig. 4). 3.4. Common DEGs in response to Cr(III) and Cr(VI) treatments 3.4.1. Transcription factor genes Transcription factors play an important role in transmitting signals and regulating the expression of downstream functional genes (Guo and Ecker, 2003; Kim and Kim, 2006; Nakano et al., 2006). In this experiment, the expression levels of 63 transcription factor genes were significantly altered under Cr(III) and Cr(VI) stress, including 20 upregulated and 43 downregulated genes. Upregulated transcription factor genes mainly included several families, such as WRKY, MYB, NAC, AP2/EREBP, and zinc finger protein genes. The upregulated transcription factor genes are shown in Supplementary Table 3. Transcription factors play important roles in signal transduction pathways. They are DNA-binding proteins that can specifically interact with cis-acting elements in the promoter regions of eukaryotic genes. Through their interactions with other related proteins, transcription factors activate or suppress gene transcription. The interactions with the cis-acting elements activate or inhibit transcription initiation or shut down the processes of related genes. When plants sense and transmit external stress signals, they can induce changes in expression levels of specific functional genes, causing physiological and biological responses in plants. WRKY-like transcription factors are plant-specific zinc finger type transcription regulatory factors. In A. thaliana, 74 members of the WRKY family are involved in the physiological regulation of A. thaliana growth and development, aging, biological and abiotic stress responses, and defense responses, among others. Nearly all WRKY proteins bind to the W-box (TGAC(C/T)). W-box and WRKY transcription factors regulate the expression levels of downstream genes of W-box to play a protective and defensive role. The WRKY gene is not constitutively expressed in plants, and it is induced by the external environment. The expression of WRKY genes can be induced under biological stresses, such as pathogen defense, response signaling molecules, and environmental stresses, such as drought, low temperature, trauma, and heavy metals. In the present study, WRKY8 (AT5G46350) was significantly upregulated. This transcription factor is induced by abscisic acid (ABA) and H2O2 and is a positive regulatory factor against biological and 4

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extrusion (MATE) efflux proteins. The ABC family is a membrane protein family with strong transport functions, which are mainly located in the tonoplast membrane. All ABC vectors contain 4-6 transmembrane hydrophobic regions, ATP binding regions, and nucleotide-binding regions. Most ABC transporters have transport activity in organisms and depend on the energy generated by ATP hydrolysis to achieve the transmembrane transport of substrates inside and outside the cell. The substrates include polysaccharides, peptides, heavy metal chelate complexes, alkaloids, and drugs. Plants have a greater number of ABC transporter proteins than animals or microorganisms. For example, there are 128 varieties in rice and 129 varieties in A. thaliana, most of which require further study. The known plant ABC transporter proteins are associated with stress resistance and heavy metal detoxification. Bovet (Bovet et al., 2003) found that expression of the AtMRP3 gene in A. thaliana was upregulated as the Cd2+ concentration increased, and it was speculated to be a detoxification protein. Lee (Lee et al., 2005) found that A. thaliana plants expressing the ABC vector family member AtPDR12 exhibited high tolerance to Pb2+. Plants carrying a deletion of the AtPDR12 gene showed reduced growth compared with normal wild-type plants but had a higher concentration of Pb2+; in contrast, plants with overexpression of AtPDR12 showed improved growth and less Pb content, suggesting that AtPDR12 could serve as an ion pump to eliminate Pb2+ or buffer Pb2+ toxicity. In the present study, MRP3 (AT3G13080), MRP7 (AT3G13100), and ABC transporter-like protein (AT3G21080) were significantly upregulated, which may have been related to the detoxification of Cr. The MATE family is a new family of transporter proteins that are extensively found in prokaryotes and eukaryotes. MATE transport proteins consist of 400–700 amino acids, most of which contain 12 transmembrane helices, and all MATE proteins exhibit approximately 40% sequence similarity. In 2003, Hvorup (Hvorup et al., 2003) found 58 MATE transporters in A. thaliana, which was a significantly greater number compared with the previously identified MATE transporters in humans and bacteria. Plant MATE transporter proteins are associated with endogenous and exogenous detoxification mechanisms of plant secondary metabolites. Upon Cr(III) and Cr(VI) treatments, the expression levels of two MATE protein genes (AT2G04070 and AT1G71140) were significantly upregulated, which may have been associated with Cr efflux.

Table 1 DEGs related to the stress response in response to Cr(III) and Cr(VI) treatments. AGI code

Gene name or description

AT5G14130 AT2G18980 AT2G39040 AT5G64100 AT1G14540 AT5G64120 AT3G01190 AT2G38390 AT2G38380 AT5G15180 AT3G32980 AT4G37520 AT4G30170 AT2G30750

peroxidase 55 peroxidase 16 peroxidase 24 peroxidase 69 peroxidase 4 peroxidase 71 peroxidase 27 peroxidase 23 peroxidase 22 peroxidase 56 peroxidase 32 peroxidase 50 peroxidase 45 CYP71A12

AT1G53540

HSP20-like chaperone HSP23.6-MITO HSP17.6A MRP3 MRP7 AT3G21080 MATE efflux family protein MATE efflux family protein

AT4G25200 AT5G12030 AT3G13080 AT3G13100 AT3G21080 AT2G04070 AT1G71140

Category

Peroxidase

Cytochrome P450 monooxygenase Heat shock proteins

ABC transporter proteins

MATE efflux proteins

Fold-change Cr(III)

Cr(VI)

−2.31 −3.08 −6.27 −2.28 −3.52 −4.70 −2.75 −2.59 −2.33 −8.40 −3.28 −2.91 −2.95 12.23

−4.20 −9.64 −7.32 −3.22 −5.91 −8.64 −5.86 −4.62 −2.78 −4.29 −3.44 3.00 −5.61 21.78

3.10

41.88

16.10 4.43 2.19 2.01 3.15 5.89

40.37 41.01 2.87 2.50 2.45 8.16

2.33

2.82

3.4.2. Stress response-related genes When external environmental conditions change, organisms must rapidly regulate their own gene expression programs and initiate protection mechanisms to adapt to various changes. Microarray results showed that following Cr treatment, the expression fold change of some genes related to stress response changed significantly in A. thaliana (Table 1). The above DEGs were subjected to cluster analysis, and the results are shown in Fig. 2. The figure shows that the expression patterns of these genes following Cr(III) and Cr(VI) treatments were significantly different from those in the control group. Peroxidase. In this experiment, a large number of peroxidases were downregulated, which may have been related to the thylakoid peroxidation caused by oxidative damage of Cr to A. thaliana. Heat shock proteins (HSP). HSPs make up a highly conserved family of molecular chaperones that participate in protein folding, unfolding, assembly, transport, and degradation. HSPs can enhance the tolerance of cells to damage and maintain the normal metabolic function of cells. For example, HSP150 protein can alleviate the damage caused by Al virus in Saccharomyces cerevisiae (Ezaki et al., 1998). In the present study, HSP20-like chaperones (T1G53540), HSP17.6A (T5G12030), and HSP23.6-MITO (AT4G25200) were significantly upregulated, whereas HSP17.6A and HSP23.6-MITO have been confirmed to be associated with hyperosmotic stress and Cd stress (Nishizawa et al., 2006; Sun et al., 2001). Cytochrome P450 monooxygenase. Cytochrome P450 monooxygenase plays an important role in lignin, defense proteins, hormones, fatty acid signal transduction or catalytic degradation of endogenous and exogenous toxins in the detoxification pathway (Lopes et al., 2012; van den Bout-van den Beukel et al., 2008; Waller et al., 2012). Here, we found that CYP71A12 (AT2G30750), which encodes a cytochrome P450 monooxygenase, was upregulated 12.2-fold and 21.8-fold in response to Cr(III) and Cr(VI) treatment, respectively, potentially in relation to the candidate gene for Cr tolerance in A. thaliana. ABC transporter proteins and multidrug and toxic compound

3.4.3. Photosynthesis-related DEGs Photosynthesis is the basis of the life activities of plants, and capturing solar energy by antenna pigments is the first step of photosynthesis. Among the DEGs related to Cr stress were four coding genes for the light-harvesting pigment-protein complexes, AT2G05070, AT3G27690, AT5G54270, and AT3G08940, the genes in photosystem II of the light-harvesting light pigment complexes Lhcb2.2, Lhcb2.3, Lhcb3, and Lhcb4.2 (Supplementary Fig. 5), respectively, and their expression levels were significantly increased. Cr treatment damaged the membrane system of the plant, causing the degradation of cytochrome and inhibiting photosynthesis. We speculated that at that time, pigment-protein mRNA was expressed in large quantities to synthesize related proteins to maintain normal photosynthesis. 3.4.4. Electron transport chain-related DEGs Cytochrome c oxidase is an oxidoreductase, a large transmembrane protein complex that is present in the mitochondrial inner membrane. Because it is the fourth central enzyme complex in the respiratory electron transport chain, it is also called complex IV. It can accept four electrons from four cytochrome C proteins and transfer them to an oxygen molecule to convert oxygen into two water molecules. During this process, it combines the four matrix protons to produce water molecules while transporting four protons across the membrane, helping to form the transmembrane proton potential difference. This potential energy difference can be used by adenosine triphosphate 5

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Fig. 2. Cluster chromatography of DEGs related to stress response in response to Cr(III) and Cr(VI) treatments.

synthase generating ATP, the most basic energy molecule in organisms (Jobson et al., 2004; Rodriguez-Roldan et al., 2006; Wishkerman et al., 2011). Among DEGs related to Cr stress, two genes (AT2G14270 and AT1G29620) encoding the subunit VIB of cytochrome C oxidase were significantly downregulated, indicating that Cr had an inhibitory effect on the electron transport chains and affected energy synthesis.

Table 2 Sulfur metabolism-related DEGs in response to Cr(III) and Cr(VI) treatments. AGI code

AT4G08620 AT1G78000 AT3G51895 AT4G02700 AT2G17640 AT4G31870 AT2G29480 AT2G29470 AT2G29460 AT1G69930 AT1G69920 AT1G17180 AT1G02930 AT1G02920 AT5G02780

3.4.5. Sulfur metabolism Sulfur is an essential mineral nutrient in the growth and development of plants. It is mainly absorbed by plant roots in the form of SO42− and enters into the organic skeleton after a series of reduction and assimilation reactions to generate Cys. Plants use Cys as a precursor to synthesize numerous metabolic products with important biological functions, such as GSH, PCs, and MTs, which are directly related to plant stress tolerance. The microarray results showed that the change fold in expression levels of some genes related to sulfur metabolism changed significantly in A. thaliana after Cr treatment (Table 2). During the process of sulfur absorption, a variety of sulfur transport proteins play synergistic roles, with 12 sulfate transporter family members in A. thaliana. According to the coding sequence homology, the difference in tissue or cell localization, and the kinetic characteristics, they can be divided into four subfamilies: AtSultr1 (Sultr1;1, Sultr1;2, Sultr1;3), AtSultr2 (Sultr2;1, Sultr2;2), AtSultr3 (Sultr3;1, Sultr3;2, Sultr3;3, Sultr3;4, Sultr3;5), and AtSultr4 (Sultr4;1, Sultr4;2). AtSultr1-4 are responsible for the transport of sulfates in different tissues and cells. Members of the AtSultr1 subfamily are high-affinity sulfur transporters that are mainly but not specifically expressed in roots (Maruyama-Nakashita et al., 2004; Yoshimoto et al., 2003). When the sulfur content in the environment decreases, the members of this subfamily are regulated at the transcriptional level. Sultr1;1 and

Gene

SULTR1;1 SULTR1;2 SULTR3;1 SULTR3;2 SERAT3;1 GPX7 GSTU2 GSTU3 GSTU4 GSTU11 GSTU12 GSTU25 GSTF6 GSTF7 GSTL1

Category

sulfur transport proteins

Cys synthetase Glutathione peroxidase Glutathione-S-transferase

Fold-change Cr(III)

Cr(VI)

2.91 1.52 2.12 2.41 2.07 1.52 1.55 9.21 1.65 1.54 2.52 3.33 2.81 2.61 2.90

1.5 2.02 3.03 1.12 2.24 2.45 2.33 9.54 2.55 3.06 4.27 4.14 1.83 3.16 3.62

Sultr1;2 have 72.6% homology and are responsible for the transport and absorption of sulfates from the environment by roots of A. thaliana. There are many unanswered questions concerning the third subfamily, and the specific functions of the transporters (Sultr3;1, Sultr3;2, Sultr3;3 and Sultr3;4) have not been reported. In this experiment, Sultr1;1, Sultr1;2, Sultr3;1, and Sultr3;2 were upregulated, which indicated that Cr could stimulate sulfur absorption in A. thaliana. Cys is the hub of sulfur nutrient metabolism and is the precursor of many sulfur-containing compounds with important functions. In addition, reduced Cys is also a sulfur donor for vitamins and coenzymes, 6

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downregulated genes mainly acted on the synthesis of S-lignin, Hlignin, and G-lignin (Fig. 3). These results indicated that Cr could inhibit lignin biosynthesis in A. thaliana.

such as thiamin, lipoic acid, biotin, and iron-sulfur clusters. Cys synthetase is a multimeric complex composed of SAT and OASTL. Five genes encode SAT in A. thaliana, Serat1;1(At5g56760), Serat2;1(At1g55920), Serat2;2(At3g13110), Serat3;1(At2g17640), and Serat3;2(At4g35640). Serat3;1 and Serat3;2 are located in the cytoplasm. Upon Cr treatment, Serat3;1 was upregulated, which could improve Cys synthesis to cope with stress. Glutathione peroxidase (GSH-Px) is an important enzyme that clears H2O2 and many organic hydroperoxides. Glutathione-S-transferase (GST) has dual functions of removing peroxides and detoxifying plants and bodies. The synergistic action of these two enzymes plays an important protective role in the plant antioxidant defense system, protecting cells from lipid oxides produced by oxidative stress and external toxic substances. The results showed that the expression levels of some GSH-Px and GST genes were upregulated in A. thaliana in response to Cr treatments to alleviate the oxidative damage caused by Cr in A. thaliana.

3.5. DEGs specific to Cr(III) and Cr(VI) In the Cr(III) and Cr(VI) groups, 185 and 587 genes were specifically upregulated, while 220 and 956 genes were specifically downregulated, respectively. The number of specific DEGs was greater in the Cr(VI) group than the Cr(III) group, indicating that Cr(VI) had a greater effect on A. thaliana than Cr(III). A comparison revealed a specificity of the expression levels of several important genes in response to the two Cr treatments. 3.5.1. Analysis of DEGs specific to Cr(III) The hormone is a trace substance synthesized by plants and plays an important regulatory role in plant growth and development. When plants receive external stimuli, the signal transduction pathways mediated by indole-3-acetic acid (IAA), ABA, salicylic acid (SA), jasmonic acid (JA), ethylene (ETH), and others begin to respond to external stimulation of the plant. Under Cr(III) stress, 9 genes in the IAAregulated pathway in A. thaliana were significantly upregulated (Supplementary Table 4). After enrichment analysis by KEGG, it was found that these genes encode GH3 and SAUR, two auxin-responsive proteins (shown in red in Fig. 4), which are used to stimulate cellular growth.

3.4.6. Phenylpropanoid metabolism The phenylpropanoid metabolic pathway is an important pathway in plant secondary biomass metabolism and is related to the synthesis of phenolic compounds, such as phenolic acids and flavonoids, as well as the synthesis of many secondary metabolites, such as lignin and hydroxycinnamate. All substances containing a phenylpropanoid skeleton are generated directly or indirectly through this metabolic pathway, and these substances exert various physiological functions in plants. Phenylpropanoid metabolism has important physiological significance in plants, which is mainly reflected by changes in its enzyme activity, intermediate metabolites, and their further transformation products (lignin, phytochemicals, phenolic acids, flavonoids, and other phenolics), cellular differentiation during plant growth and development, and a close relationship with physiological and abiotic stress resistance as well as physiological activities such as coloration in plants. Lignin is a structural product that is produced during the vascular plant evolution process. Lignin gradually infiltrates the cell wall during cell lignification and fills the cellulosic framework to increase the cell wall hardness, enhance the mechanical support and compressive strength of cells, promote the formation of mechanical tissues, and consolidate and support the plant body and water transport. In addition, the insoluble properties of lignin and chemical properties of polyphenols can also greatly enhance plant resistance to stress (Ali et al., 2006; Mukai et al., 2011). In the present study, a total of 16 genes in the phenylpropanoid metabolism were significantly downregulated (Table 3). After KEGG enrichment analysis, we found that these

3.5.2. Analysis of DEGs specific to Cr(VI) ABA is a sesquiterpenoid that is synthesized from xanthophyll and affects plant growth and development. ABA is increased in plants exposed to environmental stresses, such as drought, cold, and salinity. It plays an important role in regulating stomatal movement, stabilizing photosynthetic organs, increasing antioxidant enzyme activities, protecting photoinhibition, and regulating gene expression. Studies have shown that ABA augments plant resistance to environmental stress. Therefore, it is considered to be a stress hormone. The present study showed that expression levels of 16 genes in the ABA signal transduction pathway were upregulated in response to Cr(VI) treatment (Supplementary Table 5). Among them, AT2G40340 can upregulate the mRNA expression of ascorbic acid peroxidase (APX) and increase APX enzyme activity, and it plays an important role in the antioxidation function in A. thaliana (Lee et al., 2010). Carotenoids are a large group of pigment substances (red, orangered, and yellow) that are synthesized by isoprene. Excluding a few carotenoids (C30, C45 and C50) that are synthesized by nonphotosynthetic bacteria, carotenoids are mainly triterpenoids (C40) that are synthesized by the condensation of 8 isoprene units. Thus far, more than 600 kinds of carotenoids (C40) have been found in nature, mainly as important components of the photosynthetic membrane of higher plants, algae, and cyanobacteria. Carotenoids are mainly distributed in plant chloroplasts and chloroplast membranes, including carotene and xanthophylls. Plant carotenoids are important components of the lightharvesting complexes, mainly exist in the chloroplasts of plant leaves and chloroplast membranes of flowers and fruits, have the ability to absorb and transmit electrons, and play an important role in the elimination of chlorophyll triplets and singlets, superoxide anion, and other free radicals that are produced during photosynthesis (Auldridge et al., 2006; Caliandro et al., 2013; Wei et al., 2010). Carotenoids are also precursors of the biosynthesis of many physiologically active substances, such as plant hormones (ABA), defense compounds, and aromatic flavor compounds (Gunel et al., 2001; Oliveira et al., 2003; Taylor and Ramsay, 2005). Five genes were significantly upregulated in the carotenoid synthesis pathway in the present study (AT5G67030, AT5G17230, AT5G52570, AT2G32640, and AT4G19170), encoding zeaxanthin epoxidase, lycopene synthase, carotenoid B-cyclohydroxylase 2,

Table 3 DEGs associated with phenylpropanoid metabolism in response to Cr(III) and Cr (VI) treatments. AGI code

AT5G64120 UGT72E3 AT2G38380 BGLU46 AT4G37520 AT2G37130 AT5G64100 AT2G38390 AT4G30170 AT3G01190 AT2G41480 AT2G39040 AT2G18980 AT5G14130 AT1G14540 AT5G15180

Annotation

peroxidase 71 coniferyl-alcohol glucosyltransferase peroxidase 22 beta glucosidase 46 peroxidase 50 peroxidase peroxidase 69 peroxidase 23 peroxidase 45 peroxidase 27 putative peroxidase peroxidase 24 peroxidase 16 peroxidase 55 peroxidase 4 peroxidase 56

Fold-change Cr(III)

Cr(VI)

−4.70 −2.43 −2.33 −4.79 −2.91 −2.59 −2.28 −2.59 −2.95 −2.75 −3.47 −6.27 −3.08 −2.31 −3.52 −8.40

−8.64 −3.12 −2.78 −4.09 −3.00 −4.62 −3.22 −4.62 −5.61 −5.86 −3.19 −7.32 −9.64 −4.20 −5.91 −4.29

7

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Fig. 3. DEGs associated with the phenylpropanoid metabolic pathway in response to Cr(III) and Cr(VI) treatments. The red regions are DEG sites. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Two genes encoding the subunit VIB of cytochrome C oxidase were significantly downregulated, which indicating that Cr had an inhibitory effect on the electron transport chains. Under Cr(III) stress, 9 genes in the IAA-regulated pathway in A. thaliana were significantly upregulated, which are used to stimulate cellular growth. Low concentrations caused a statistically significant increase in dry biomass as compared to the control. We attribute this phenomenon to the hormesis (Calabrese and Baldwin, 2003). Similar findings have been found in many studies (Dell'Amico et al., 2008; Jiang et al., 2008; Zeid, 2001). It is speculated that the concept of hormesis is the reason for the increased expression of IAA under Cr(III) treatment and the increased expression of ABA genes following Cr(VI) treatment.

lycopene B-cyclase, and carotenoid cleavage dioxygenase, respectively. It is speculated that the synthesis of carotenoids in A. thaliana was upregulated under Cr(VI) treatment in response to the oxidative damage of Cr(VI) to chloroplasts. In this study, we have discussed 6 aspects of genes, namely, transcription factor genes, stress response-related genes, photosynthesisrelated DEGs, electron transport chain-related DEGs, sulfur metabolism and phenylpropanoid metabolism. These altered genes are involved in a variety of plant responses including biological and nonbiological damage, phosphate-starvation, oxidative damage, and hyperosmotic stress (Angkawijaya et al., 2019; Bouain et al., 2019). In the present study, 3 genes of ABC transporter proteins were significantly upregulated, which may have been related to the detoxification of Cr (Navazas et al., 2019). Four coding genes for the light-harvesting pigment-protein complexes, were expressed in large quantities to maintain normal photosynthesis.

Fig. 4. Action sites of DEGs in the auxin regulatory pathway in response to Cr(III) treatment. 8

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4. Conclusion

Ezaki, B., et al., 1998. Protective roles of two aluminum (Al)-induced genes, HSP150 and SED1 of Saccharomyces cerevisiae, in Al and oxidative stresses. FEMS Microbiol. Lett. 159, 99–105. Gunel, T., et al., 2001. Expression of plant carotenoid genes in Schizsaccharomyces pombe. Yeast 18 S302-S302. Guo, H.W., Ecker, J.R., 2003. Plant responses to ethylene gas are mediated by SCF (EBF1/ EBF2)-dependent proteolysis of EIN3 transcription factor. Cell 115, 667–677. Hu, Y., et al., 2009. Differential expression of candidate genes for lignin biosynthesis under drought stress in maize leaves. J. Appl. Genet. 50, 213–223. Huang, H., et al., 2012. Transcriptome-wide survey and expression analysis of stressresponsive NAC genes in Chrysanthemum lavandulifolium. Plant Sci. 193, 18–27. Hvorup, R.N., et al., 2003. The multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) exporter superfamily. Eur. J. Biochem. 270, 799–813. Jiang, C.Y., et al., 2008. Isolation and characterization of a heavy metal-resistant Burkholderia sp from heavy metal-contaminated paddy field soil and its potential in promoting plant growth and heavy metal accumulation in metal-polluted soil. Chemosphere 72, 157–164. Jobson, R.W., et al., 2004. Adaptive evolution of cytochrome c oxidase: infrastructure for a carnivorous plant radiation. Proc. Natl. Acad. Sci. U. S. A 101, 18064–18068. Kim, J., Kim, H.-Y., 2006. Functional analysis of a calcium-binding transcription factor involved in plant salt stress signaling. FEBS Lett. 580, 5251–5256. Lee, M., et al., 2005. AtPDR12 contributes to lead resistance in arabidopsis. Plant Physiol. 138, 827–836. Lee, S.-j., et al., 2010. DREB2C interacts with ABF2, a bZIP protein regulating abscisic acid-responsive gene expression, and its overexpression affects abscisic acid sensitivity. Plant Physiol. 153, 716–727. Lopes, B., et al., 2012. Responses of CYP450 dependent system to aliphatic and aromatic hydrocarbons body burden in transplanted mussels from South coast of Portugal. Ecotoxicology 21, 730–749. Lopez-Bucio, J., et al., 2014. Phosphate relieves chromium toxicity in Arabidopsis thaliana plants by interfering with chromate uptake. Biometals 27, 363–370. Lunk, H.J., 2015. Discovery, properties and applications of chromium and its compounds. Chemtexts 1. Maruyama-Nakashita, A., et al., 2004. Regulation of high-affinity sulphate transporters in plants: towards systematic analysis of sulphur signalling and regulation. J. Exp. Bot. 55, 1843–1849. Mukai, Y., et al., 2011. Effect of lignin-derived lignophenols on vascular oxidative stress and inflammation in streptozotocin-induced diabetic rats. Mol. Cell. Biochem. 348, 117–124. Nakano, T., et al., 2006. Identification of genes of the plant-specific transcription-factor families cooperatively regulated by ethylene and jasmonate in Arabidopsis thaliana. J. Plant Res. 119, 407–413. Navazas, A., et al., 2019. Integrative response of arsenic uptake, speciation and detoxification by Salix atrocinerea. Sci. Total Environ. 689, 422–433. Nishizawa, A., et al., 2006. Arabidopsis heat shock transcription factor A2 as a key regulator in response to several types of environmental stress. Plant J. 48, 535–547. Oliveira, C., et al., 2003. Carotenoid compounds in grapes and their relationship to plant water status. J. Agric. Food Chem. 51, 5967–5971. Rodriguez-Roldan, V., et al., 2006. A comparative kinetic analysis of the reactivity of plant, horse, and human respiratory cytochrome c towards cytochrome c oxidase. Biochem. Biophys. Res. Commun. 346, 1108–1113. Singh, H.P., et al., 2013. Chromium toxicity and tolerance in plants. Environ. Chem. Lett. 11, 229–254. Sun, W.N., et al., 2001. At-HSP17.6A, encoding a small heat-shock protein in Arabidopsis, can enhance osmotolerance upon overexpression. Plant J. 27, 407–415. Taylor, M., Ramsay, G., 2005. Carotenoid biosynthesis in plant storage organs: recent advances and prospects for improving plant food quality. Physiol. Plantarum 124, 143–151. van den Bout-van den Beukel, C.J.P., et al., 2008. Evaluation of cytotoxic, genotoxic and CYP450 enzymatic competition effects of tanzanian plant extracts traditionally used for treatment of fungal infections. Basic Clin. Pharmacol. Toxicol. 102, 515–526. Waller, D., et al., 2012. Geographic variability of CYP450-mediated metabolism in cynomolgus macaques (Macaca fascicularis). JAALAS 51, 689–690. Wei, J., et al., 2010. The role of carotenoid isomerase in maintenance of photosynthetic oxygen evolution in rice plant. Acta Biochim. Biophys. Sin. 42, 457–463. Wishkerman, A., et al., 2011. Enhanced formation of methane in plant cell cultures by inhibition of cytochrome c oxidase. Plant Cell Environ. 34, 457–464. Wu, A., et al., 2012. JUNGBRUNNEN1, a reactive oxygen species-responsive NAC transcription factor, regulates longevity in arabidopsis. Plant Cell 24, 482–506. Yoshimoto, N., et al., 2003. Phloem-localizing sulfate transporter, Sultr1;3, mediates redistribution of sulfur from source to sink organs in Arabidopsis. Plant Physiol. 131, 1511–1517. Zayed, A.M., Terry, N., 2003. Chromium in the environment: factors affecting biological remediation. Plant Soil 249, 139–156. Zeid, I.M., 2001. Responses of Phaseolus vulgaris to chromium and cobalt treatments. Biol. Plantarum 44, 111–115.

Gene expression profile analysis revealed both common and specific DEGs in response to Cr(III) and Cr(VI) treatments. Among the common DEGs, the expression levels of related genes involved in redox, secondary metabolism and energy metabolism processes were significantly downregulated, while those of genes related to the stress response, photosynthesis, and sulfur metabolism were significantly upregulated, indicating that Cr seriously affected the normal activities of A. thaliana cells and induced the upregulation of some genes related to stress and regulation to adapt to the Cr stress. Among the specific DEGs, genes related to IAA regulatory pathways were significantly upregulated upon Cr(III) treatment; genes related to ABA regulatory pathways and genes involved in carotenoid synthesis were significantly upregulated upon Cr (VI) treatment, revealing some differences in response to Cr(III) and Cr (VI) in A. thaliana. CRediT authorship contribution statement Jianxia Liu: Data curation, Writing - original draft. Guotao Ding: Methodology, Software. Zikuan Gai: Supervision. Wei Zhang: Writing - review & editing. Yonghong Han: Visualization, Investigation. Weihao Li: Conceptualization. Declaration of competing interests The authors declare no conflict of interests. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ecoenv.2020.110302. References Abou-Elwafa, S.F., et al., 2019. Genetic mapping and transcriptional profiling of phytoremediation and heavy metals responsive genes in sorghum. Ecotoxicol. Environ. Saf. 173, 366–372. Ali, M.B., et al., 2006. Phenolics metabolism and lignin synthesis in root suspension cultures of Panax ginseng in response to copper stress. Plant Sci. 171, 147–154. Angkawijaya, A.E., et al., 2019. Expression profiles of 2 phosphate starvation-inducible phosphocholine/phosphoethanolamine phosphatases, PECP1 and PS2, in arabidopsis. Front. Plant Sci. 10. Auldridge, M.E., et al., 2006. Plant carotenoid cleavage oxygenases and their apocarotenoid products. Curr. Opin. Plant Biol. 9, 315–321. Bertucci, A., et al., 2018. Whole-transcriptome response to wastewater treatment plant and stormwater effluents in the Asian clam, Corbicula fluminea. Ecotoxicol. Environ. Saf. 165, 96–106. Bouain, N., et al., 2019. Systems genomics approaches provide new insights into Arabidopsis thaliana root growth regulation under combinatorial mineral nutrient limitation. PLoS Genet. 15. Bovet, L., et al., 2003. Transcript levels of AtMRPs after cadmium treatment: induction of AtMRP3. Plant Cell Environ. 26, 371–381. Calabrese, E.J., Baldwin, L.A., 2003. Hormesis: the dose-response revolution. Annu. Rev. Pharmacol. Toxicol. 43, 175–197. Caliandro, R., et al., 2013. Effects of altered alpha- and beta-branch carotenoid biosynthesis on photoprotection and whole-plant acclimation of Arabidopsis to photooxidative stress. Plant Cell Environ. 36, 438–453. Chen, L., et al., 2010. Wounding-induced WRKY8 is involved in basal defense in arabidopsis. Mol. Plant Microbe Interact. 23, 558–565. Dell'Amico, E., et al., 2008. Improvement of Brassica napus growth under cadmium stress by cadmium-resistant rhizobacteria. Soil Biol. Biochem. 40, 74–84. Eleftheriou, E.P., et al., 2015. Chromium-induced ultrastructural changes and oxidative stress in roots of Arabidopsis thaliana. Int. J. Mol. Sci. 16, 15852–15871. Evert, A.B., et al., 2013. Nutrition therapy recommendations for the management of adults with diabetes. Diabetes Care 36, 3821–3842.

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