Plant Physiology®, August 2018, Vol. 177, pp. 1691–1703, www.plantphysiol.org © 2018 American Society of Plant Biologists. All Rights Reserved. 1691

Cadmium (Cd) is a nonessential heavy metal harm-ful to human health. It is widely released into soil, air, and water mainly through industrial and mining ef-fluents and phosphate fertilization (Pinto et al., 2004). The toxic effect of Cd has been a worldwide concern since the outbreak of Itai-Itai disease in the mid-20th century in Japan caused by the daily consumption of Cd-contaminated rice (Horiguchi et al., 1994). For the general population, the major route of Cd exposure is food. Rice grains contaminated with Cd represent a major risk to the health of more than half of the world’s population that depends on rice as a basic staple.

Plants have evolved various molecular mechanisms to respond to heavy metal stress, including microR-NA (miRNA)-guided gene regulation at the posttran-scriptional level (Ding et al., 2016, 2017; Gielen et al., 2016). miRNAs are a class of endogenous noncoding small RNAs that can base pair their target mRNAs to induce their degradation or repress their translation in eukaryotic organisms. In plants, miRNAs have vital regulatory roles in plant growth, development, and re-sponses to biotic and abiotic stresses, including heavy metal stress (Bartel, 2004; Sunkar et al., 2006; Yang and Chen, 2013; Gu et al., 2017; Noman et al., 2017).

miR166 is a well-conserved miRNA present in all land plants (Floyd and Bowman, 2004; Axtell and Bowman, 2008). miR166 targets the transcripts of the class-III homeodomain-Leu zipper (HD-Zip) transcrip-tion factors in plants, including rice (Itoh et al., 2008). miR166-mediated cleavage of HD-Zip III mRNAs has been confirmed by 5′ RACE and degradome sequenc-ing analysis (Liu et al., 2009; Li et al., 2010). miR166 acts as a master regulator of plant development (Schwab et al., 2005). In Arabidopsis (Arabidopsis thaliana), miR166/165 and its targets regulate diverse aspects of plant developmental processes, including shoot apical and lateral meristem formation, leaf polarity, floral development, and vascular patterning of shoot and root (Jung and Park, 2007; Carlsbecker et al., 2010; Chen, 2012; Singh et al., 2014). Recent studies indicate

MicroRNA166 Modulates Cadmium Tolerance and Accumulation in Rice1

Yanfei Ding,a Shaohua Gong,a Yi Wang,a Feijuan Wang,a Hexigeduleng Bao,a Junwei Sun,a Chong Cai,a Keke Yi,b Zhixiang Chen,a,c and Cheng Zhua,2

aKey Laboratory of Marine, Food Quality and Hazard Controlling Technology of Zhejiang Province, College of Life Sciences, China Jiliang University, Hangzhou 310018, People’s Republic of ChinabKey Laboratory of Plant Nutrition and Fertilizer, Ministry of Agriculture, Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Science, Beijing 100081, ChinacDepartment of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana 47907-2054ORCID IDs: 0000‑0002‑3460‑1344 (Y.D.); 0000‑0002‑3232‑2675 (J.S.); 0000‑0002‑3916‑477X (K.Y.); 0000‑0002‑8056‑4943 (C.Z.)

MicroRNAs (miRNAs) are 20- to 24-nucleotide small noncoding RNAs that regulate gene expression in eukaryotic organisms. Several plant miRNAs, such as miR166, have vital roles in plant growth, development and responses to environmental stresses. One such environmental stress encountered by crop plants is exposure to cadmium (Cd), an element highly toxic to most organisms, including humans and plants. In this study, we analyzed the role of miR166 in Cd accumulation and tolerance in rice (Oryza sativa). The expression levels of miR166 in both root and leaf tissues were significantly higher in the reproductive stage than in the seedling stage in rice. The expression of miR166 in the roots of rice seedlings was reduced after Cd treatment. Overexpression of miR166 in rice improved Cd tolerance, a result associated with the reduction of Cd-induced oxidative stress in transgenic rice plants. Furthermore, overexpression of miR166 reduced both Cd translocation from roots to shoots and Cd accumulation in the grains. miR166 targets genes encoding the class-III homeodomain-Leu zipper (HD-Zip) family proteins in plants. In rice, HOMEODOMAIN CONTAINING PROTEIN4 (OsHB4) gene (Os03g43930), which encodes an HD-Zip protein, was up-regulated by Cd treatment but down-regulated by overexpression of miR166 in transgenic rice plants. Overexpression of OsHB4 increased Cd sensitivity and Cd accumulation in the leaves and grains of transgenic rice plants. By contrast, silencing OsHB4 by RNA interference enhanced Cd tolerance in transgenic rice plants. These results indicate a critical role for miR166 in Cd accumulation and tolerance through regulation of its target gene, OsHB4, in rice.

1This study was supported by the Natural Science Foundation of Zhejiang Province (LR17C130001), the National Natural Science Foundation of China (31771698), the Key Research and Development Project of Zhejiang Province (2015C03020-4), and the National Key Research and Development Program of China (2017YFD0801104).

2Address correspondence to pzhch@cjlu.edu.cn.The author responsible for distribution of materials integral to

the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Cheng Zhu (pzhch@cjlu.edu.cn).

C.Z., K.Y., and Y.D. conceived the original research plan and su-pervised the experiments; S.G., Y.D., and Y.W. performed most of the experiments; F.W., H.B., J.S., and C.C. performed some of the exper-iments; C.Z., Y.D., and Z.C. analyzed the data and wrote the article.

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that miR166 also regulates plant responses to abiotic stresses, including drought in Triticum dicoccoides and rice (Oryza sativa; Kantar et al., 2011; Zhang et al., 2018) and mercury treatment in Medicago truncatula (Zhou et al., 2012). Our previous work from microarray assays showed that miR166 was strongly down-regulated in rice by Cd stress (Ding et al., 2011). However, the roles of miR166 and its target genes in Cd accumulation and tolerance in rice have not been established.

In the current study, we performed a comprehensive functional analysis of rice miR166 and its target genes in Cd accumulation and tolerance using both gene expression and transgenic approaches. Our results showed that Cd stress down-regulated the expression of miR166 but up-regulated the expression of its tar-get gene HOMEODOMAIN CONTAINING PROTEIN4 (OsHB4) in rice. In addition, overexpression of miR166 down-regulated OsHB4 expression, decreased Cd- induced oxidative stress, and reduced Cd accumulation in rice grains. By contrast, overexpression of OsHB4 in-creased both Cd sensitivity and Cd accumulation in the leaves and grains of transgenic rice plants, whereas si-lencing OsHB4 by RNA interference (RNAi) enhanced Cd tolerance in transgenic rice plants. Based on these results, we conclude that miR166 plays a critical role in Cd accumulation and tolerance through regulation of its HD-Zip target genes in rice.

RESULTS

Cd‑Responsive Expression of Rice miR166

We previously reported our investigation of rice miRNA expression patterns under Cd treatment, us-ing a microarray assay with probes complementary to the miRNA sequences of rice from miRBase Release 11.0 (http://microrna.sanger.ac.uk/; Ding et al., 2011). Rice seedlings were treated with 60 μm CdCl2 for 6 h, and small RNAs were isolated from roots and used to screen for differentially expressed miRNAs by mi-croarray analysis. The microarray results showed that the expression of miR166 was substantially decreased under Cd stress (Ding et al., 2011).

To further analyze miR166, we monitored the ex-pression profile of miR166 in various rice tissues at different growth stages by quantitative real-time PCR (RT-qPCR) analysis. As shown in Figure 1, miR166 was expressed at high levels in flag leaves and leaves during the grain-ripening stage but at relatively low levels in shoots and roots in the vegetative growth stage. These results suggested a role of miR166 in the ripening stage.

RT-PCR analysis also confirmed findings of the pre-viously reported microarray analysis on the reduction in expression of miR166 after Cd treatment (Fig. 2A). RT-qPCR analysis was then performed to examine the time course in the changes of miR166 expression in response to Cd. In rice roots, miR166 expression was reduced substantially during the first 6 h of Cd

treatment (Fig. 2B). We also examined the response of rice class III HD-Zip genes, the targets of miR166, to Cd treatment. There are five closely related class III HD-Zip genes (OsHB1 to OsHB5) in rice that are predicted to be targets of miR166 based on sequence analysis. However, our expression analysis showed that among these, only OsHB4 (Os03g43930) was responsive to Cd. OsHB4 was induced after exposure to Cd (Fig. 2B). Thus, both miR166 and OsHB4 were responsive to Cd stress. Their opposite expression patterns in response to Cd were consistent with the HD-Zip gene being a target of miR166 in plants.

To further analyze miR166, we overexpressed its pre-cursor in rice to determine the effects of miR166 on the expression of class III HD-Zip genes (Fig. 2C). We placed the miR166 precursor gene behind the cauliflower mosaic virus (CaMV) 35S promoter (35S:MIR166) and transformed it into rice plants. In transgenic rice lines, the transcript levels of mature miR166 increased by 2- to 6-fold over those in wild-type plants (Fig. 2C). We also examined the expression of the class III HD-Zip genes in these 35S:MIR166 lines and found that the transcript levels of only OsHB4 were substantial-ly reduced relative to the levels in wild-type plants. Furthermore, those lines with relatively high levels

Figure 1. Expression profiles of miR166 in rice. cDNAs were synthe-sized from total RNA extracted from various tissues of rice grown in a greenhouse, and the mRNA levels were quantified by RT-qPCR. The data were normalized to β-ACTIN and were shown relative to the veg-etative root sample. Error bars represent the se for three independent experiments.

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of miR166 transcripts (lines #15-1, #10-4, and #12-2) displayed relatively strong reduction in the transcript levels of OsHB4 (Fig. 2C). On the other hand, line #11-1 had a lower level of the miR166 transcripts and, at the same time, a higher level of OsHB4 transcripts than other transgenic lines (Fig. 2C). This observation supports that OsHB4 is a major target gene of miR166 and as a result, transcripts of OsHB4 are subjected to miR166-mediated degradation.

Overexpression of miR166 Improved Rice Tolerance to Cd Stress

The Cd-responsive expression of both miR166 and its target gene OsHB4 shows that miR166 likely plays a role in the accumulation and tolerance of the heavy metal in plants. To test this possibility, we compared responses to Cd in wild-type plants with those in transgenic 35S:MIR166 lines (#15-1, #10-4, and #12-2) with relatively high miR166 expression levels. The wild-type and transgenic 35S:MIR166 plants were grown for 8 weeks in Yoshida’s culture solution and then treated with 2 μm CdCl2 for 14 d. Although Cd treatment inhibited the growth of both wild-type and transgenic miR166 plants, the extent of growth inhi-bition in transgenic 35S:MIR166 plants was slightly lower than that in wild-type plants (Fig. 3A). The chlo-rophyll content in wild-type and transgenic plants

was also evaluated. Prior to Cd treatment, wild-type and transgenic 35S:MIR166 lines had similar levels of chlorophyll (Fig. 3B). After Cd treatment, however, the chlorophyll levels in the 35S:MIR166 plants were slightly higher than those in wild-type plants (Fig. 3B). On the other hand, the levels of oxidative stress in the transgenic 35S:MIR166 lines were substantial-ly lower than those in wild-type plants, based on the levels of malonyldialdehyde (MDA), an indicator of oxidized membrane lipids under oxidative stress (Fig. 3C). Under normal growth conditions, there was no significant difference in MDA content between wild-type and transgenic 35S:MIR166 plants (Fig. 3C). Cd treatment increased MDA levels by almost 100% in wild-type plants but by only 20 to 30% in the transgen-ic 35S:MIR166 lines (Fig. 3C). Thus, the levels of MDA in transgenic 35S:MIR166 lines #15-1, #12-2, and #10-4 were 47.4, 39.5, and 42.1% lower than that in wild-type plants, respectively (Fig. 3C). These results indicated that overexpression of miR166 decreased membrane oxidation under Cd stress.

Stresses usually cause damage in plants via oxida-tive stress from increased accumulation of reactive oxygen species, such as hydrogen peroxide (H2O2). Therefore, the H2O2 content in wild-type and trans-genic 35S:MIR166 plants was examined. As shown in Figure 3D, under normal conditions, there was no significant difference in the H2O2 content between

Figure 2. Expression levels of miR166 and its target gene OsHB4. A, RT-PCR of miR166 precursor from roots under Cd stress for 6 h. B, Detection of miR166 and OsHB4 transcript levels in roots of 1-week-old rice seedlings treated for the indicated times with 60 μm CdCl2 by RT-qPCR. C, Detection of miR166 and OsHB4 gene transcripts in 35S:MIR166 transgenic plant lines by RT-qPCR. Quan-tifications were normalized to the expres-sion of β-ACTIN. Error bars represent se for three independent experiments.

Overexpression of miR166 Increases Cd Tolerance

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wild-type and transgenic 35S:MIR166 lines, and after Cd treatment, the H2O2 content increased by 2.3-fold in leaves of wild-type plants but only about 2.0-fold in leaves of 35S:MIR166 lines. As a result, the levels of H2O2 in Cd-treated transgenic 35S:MIR166 lines were significantly lower than those in the Cd-treated wild-type plants (Fig. 3D). Thus, the increased Cd tolerance of miR166-overexpressing rice lines was associated with reduced accumulation of reactive oxygen species and reduced oxidative stress under Cd stress.

Overexpression of miR166 Reduced Cd Translocation in Rice

To examine the role of miR166 in rice Cd accumu-lation, we measured Cd levels in the roots and shoots of rice plants grown hydroponically for 14 d in the presence of 2 μm CdCl2. The Cd content in the roots of transgenic 35S:MIR166 plants was 10%–20% higher than that in the roots of wild-type plants (Fig. 4A). On the other hand, the shoots of 35S:MIR166 plants con-tained about 15%–30% less Cd than those of wild-type plants (Fig. 4B). Based on the Cd contents in both roots

and shoots, we further calculated the Cd translocation factor (the ratio of Cd amount in shoots to that in roots) to examine Cd translocation from roots to above-ground parts. The calculated ratios indicated that the translocation factor in 35S:MIR166 plants was about 40% lower than that in wild-type plants (Fig. 4C), sug-gesting that overexpression of miR166 led to reduced Cd translocation from roots to shoots in hydroponically grown rice plants.

To further analyze the effect of miR166 on the root-to-shoot translocation of Cd, we estimated Cd accumula-tion in the xylem and phloem sap after Cd treatment in hydroponically grown plants. As shown in Figure 4D, the Cd concentrations in the xylem sap were more than 10 times higher than those in the phloem sap in both wild-type and transgenic 35S:MIR166 plants, indicat-ing that Cd root-to-shoot translocation was primarily through xylem. Furthermore, the Cd concentrations in the xylem sap in the 35S:MIR166 plants were 1.5 times lower than those in wild-type plants (Fig. 4D). These results indicate that miR166 plays a negative role in Cd loading to the xylem for root-to-shoot translocation in rice.

Figure 3. Performance of wild-type and 35S:MIR166 transgenic plants under Cd stress. A, Phenotypic comparison of wild-type (WT) and 35S:MIR166 transgenic rice lines (#10-4, #12-2, and #15-1) under Cd stress. Top: Plants grown for 8 weeks under normal conditions before treatment. Bottom, Eight-week-old plants treated with 2 μm CdCl2 for 14 d. B, Effect of Cd stress on chlorophyll levels. C, Effect of Cd stress on MDA levels. D, Effect of Cd stress on H2O2 content. Asterisks indicate significant differences between wild-type and transgenic plants at *P ≤ 0.05 and **P ≤ 0.01.

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To explore possible mechanisms by which miR166 regulates Cd accumulation and translocation, we com-pared the expression of Cd transporter genes in wild-type and 35S:MIR166 roots using RT-qPCR (Fig. 4E). OsHMA3 is a tonoplast-localized Cd transporter in rice roots that belongs to the heavy metal ATPase (HMA) family. OsHMA3 is mainly expressed in roots, and its overexpression enhances Cd accumulation in roots (Sasaki et al., 2014). Our RT-qPCR analysis revealed that miR166 positively regulated OsHMA3, based on increased levels of OsHMA3 transcripts in 35S:MIR166 roots (Fig. 4E). The increased expression of OsHMA3 in the miR166-overexpressing lines was consistent with increased Cd accumulation in the transgenic rice roots. OsHMA2 is a major Zn/Cd transporter in rice roots (Takahashi et al., 2012). It has been reported that mutations in OsHMA2 restricted Cd translocation (Satoh- Nagasawa et al., 2012). In the 35S:MIR166 plants, in which Cd translocation was also reduced, OsHMA2 expression was decreased (Fig. 4E), which was consis-tent with the positive role of OsHMA2 in Cd transloca-tion in rice (Satoh-Nagasawa et al., 2012).

We also analyzed the agronomic traits of and Cd accumulation in soil-grown wild-type and transgenic 35S:MIR166 lines under field conditions in 2013. The

experiments were conducted in a restricted experi-mental field where the soil was artificially contami-nated with Cd (average of 3 μg/g). Plants were grown until grain ripening, and both the agronomical traits and Cd content in wild-type and miR166-overexpress-ing lines were compared. The transgenic 35S:MIR166 plants had normal growth and yield, with no signifi-cant difference in plant size, leaf dry weight, and grain weight from wild-type plants (Fig. 5). However, the transgenic plants accumulated about 50% less Cd in the stems and leaves than wild-type plants (Fig. 5, C and D). Moreover, Cd content in the grains of 35S: MIR166 plants was 57% lower than that in wild-type (Fig. 5E). These results indicate that overexpression of miR166 can be explored as an effective way to reduce grain Cd accumulation.

These measurements showed that in the shoots of soil-grown rice, Cd accumulated primarily in the stem tissues, while the Cd levels in the leaves were gener-ally very low (Fig. 5). In fact, Cd levels were lower in leaves than in grains in both wild-type and transgenic miR166-overexpressing lines (Fig. 5). It appears that a large proportion of the Cd content in the shoots stays in or near the vascular tissues of the stem, where its translocation takes place. Previous studies have also

Figure 4. Cd content in wild-type and 35S:MIR166 transgenic rice lines (#10-4, #12-2, and #15-1) after exposure to 2 μm CdCl2 for 14 d. A, Cd content in roots. B, Cd content in shoots. C, Ratio of Cd content in shoots to that in roots. D, Cd content in the xylem and phloem sap. E, Relative expression levels of metal transporter genes in the roots of wild-type (WT) and 35S:MIR166 transgenic plants under Cd stress. Vertical bars represent sd of the mean (n = 3). Asterisks indicate mean values are significantly different between wild-type and transgenic plants at *P ≤ 0.05 and **P ≤ 0.01.

Overexpression of miR166 Increases Cd Tolerance

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shown that the levels of Cd in the leaves are similar to or even lower than those in the grains in soil-grown rice plants (Shimo et al., 2011; Siebers et al., 2013).

Overexpression of OsHB4 Increased Cd Sensitivity

To determine the role of OsHB4, a target gene of miR166, in Cd accumulation and tolerance, we also generated an OsHB4-overexpression construct driv-en by the CaMV 35S promoter and introduced it into rice plants using Agrobacterium-mediated transforma-tion. Three transgenic lines (#5, #6, and #11), which contained OsHB4 transcripts at least 25 times higher than in wild-type plants (Fig. 6A), were chosen for fur-ther analysis. To evaluate the effect of OsHB4 overex-pression, we again treated 8-week-old wild-type and 35S:OsHB4 plants with 2 μm CdCl2 in Yoshida nutri-ent solution for 14 d. As shown in Figure 6B, more ex-tensive wilting occurred in the transgenic 35S:OsHB4 lines than in wild-type plants after 14 d of Cd treat-ment (Fig. 6B). In addition, under Cd stress, both the H2O2 and MDA levels were significantly higher in the 35S:OsHB4 lines than in wild-type plants (Fig. 6, C and D). These results indicated that overexpression of OsHB4 reduced plant Cd tolerance.

We then analyzed the effect of OsHB4 overexpres-sion on Cd content in hydroponically grown and soil-grown plants (Fig. 7). The 35S:OsHB4 lines had significantly higher Cd levels in the shoots but lower Cd levels in the roots than wild-type plants (Fig. 7). As a result, the Cd translocation factors were signifi-cantly higher in the 35S:OsHB4 plants than in wild-type plants (Fig. 7C). Furthermore, Cd concentrations in the xylem sap were 30 to 40% higher in 35S:OsHB4 plants than in wild-type plants (Fig. 7D). These results indicate that OsHB4 plays a positive role in Cd load-ing to the xylem to promote the root-to-shoot translo-cation in rice. When grown under field conditions in soil artificially contaminated with Cd, the transgenic 35S:OsHB4 lines grew and developed quite normally (Fig. 8). However, these transgenic plants accumulated more Cd in shoots and grains than wild-type plants (Fig. 8). These results suggest that OsHB4 plays a role opposite to that of miR166 in promoting Cd transloca-tion, thereby increasing Cd accumulation in both rice shoots and grains.

Down‑Regulation of OsHB4 Improves Cd Tolerance

To further investigate the function of OsHB4 in Cd response, we attempted to down-regulate its expression

Figure 5. Agronomic traits and Cd content in miR166-overexpressing lines grown in Cd-containing soil. A, Shoot length. B, Dry weight (DW) of 100 grains. C, Stem Cd concentration. D, Leaf Cd concentration. E, Grain Cd concentration in wild-type (WT) and 35S:MIR166 plants (n = 6). Asterisks indicate significant differences between wild-type and transgenic plants at *P ≤ 0.05 and **P ≤ 0.01.

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through gene silencing. An RNAi construct for OsHB4 was generated and transformed into rice. Three inde-pendent transgenic OsHB4 RNAi lines (#1, #2, and #3) with substantial reduction in the level of OsHB4 tran-scripts were identified by RT-qPCR analysis (Fig. 9B). Homozygous progeny of the independent RNAi trans-genic lines were further analyzed for Cd tolerance.

Similar to the transgenic 35S:MIR166 plants, the transgenic OsHB4 RNAi plants displayed improved Cd tolerance. After exposure to 2 μm CdCl2 for 14 d, the MDA and H2O2 levels in the OsHB4 RNAi lines were substantially lower than those in Cd-stressed wild-type seedlings (Fig. 9, C and D). In addition, the OsHB4 RNAi plants accumulated less Cd in shoots but more Cd in roots than wild-type plants (Fig. 10, A and B). Furthermore, Cd concentration in the xy-lem sap in the OsHB4 RNAi plants was significantly lower than that in wild-type plants (Fig. 10C). When grown in Cd-contaminated soil, OsHB4 RNAi plants accumulated less Cd in the grains than wild-type plants (Fig. 10D). These results indicated that in ad-dition to overexpression of miR166, down-regulation of its target gene OsHB4 could also reduce Cd trans-location to grains.

DISCUSSION

Heavy metals such as Cd are highly toxic to plants, and plants have evolved a variety of adaptive strate-gies to reduce Cd toxicity. Over the past several years, a substantial number of studies have revealed that miRNAs play critical roles in the posttranscriptional regulation of plant genes in response to heavy metals. Microarray analysis in Glycine max led to the identifi-cation of 26 Cd-responsive miRNAs (Fang et al., 2013). Solexa sequencing in radish (Raphanus sativus) iden-tified 54 known and 16 novel miRNAs that were dif-ferentially expressed under Cr stress (Liu et al., 2015). Deep sequencing in the Cd hyperaccumulator Sedum alfredii identified 356 miRNAs, of which 79 miRNAs were differentially expressed under Cd stress (Han et al., 2016). Although an increasing number of heavy metal-responsive miRNAs has been identified, further functional analysis is necessary to establish their roles in heavy metal accumulation and tolerance in plants. Overexpression, mutations, and silencing of miRNAs and their targets have proved useful for analyzing the function of stress-responsive miRNAs and their targeted transcripts in plant stress responses. These molecular

Figure 6. Performance of wild-type and 35S:OsHB4 transgenic plants treated with 2 μm CdCl2 for 14 d. A, Detection of OsHB4 mRNA in 35S:OsHB4 transgenic rice. Real-time PCR quantifications were normalized to the expression of β-ACTIN. Error bars represent se (n = 3). B, Phenotypic comparison of wild-type and 35S:OsHB4 transgenic rice lines (#5, #6, and #11) under Cd stress. A representative picture is shown. C, MDA content in plants treated with 2 μm CdCl2 for 14 d. D, H2O2 content in plants treated with 2 μm CdCl2 for 14 d. Asterisks indicate significant differences between wild-type and transgenic plants at *P ≤ 0.05 and **P ≤ 0.01.

Overexpression of miR166 Increases Cd Tolerance

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Figure 7. Cd accumulation in wild-type and 35S:OsHB4 transgenic rice lines (#5, #6, and #11) after exposure to 2 μm CdCl2 for 14 d. A, Cd content in roots. B, Cd content in shoots. C, Ratio of Cd content in shoots to that in roots. D, Cd content in the xylem and phloem sap. Vertical bars represent sd of the mean (n = 3). Asterisks indicate mean values are significantly different between wild-type (WT) and transgenic plants at *P ≤ 0.05 and **P ≤ 0.01.

Figure 8. Agronomic traits and Cd content in OsHB4-overexpressing lines grown in Cd-containing soil. A, Shoot length. B, Dry weight (DW) of 100 grains. C, Leaf Cd concentration. D, Grain Cd concentration in wild-type (WT) and 35S:OsHB4 plants. n = 6; asterisks indicate significant differences between wild-type and transgenic plants at *P ≤ 0.05 and **P ≤ 0.01.

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genetic approaches have also been used to improve plant tolerance to heavy metals. Zhang et al. (2013) reported that overexpression of miR395 in rapeseed (Brassica napus) reduced Cd-induced oxidative stress and increased the biomass and sulfur levels under Cd exposure in transgenic plants. On the other hand, we have recently reported that overexpression of miR390 in transgenic rice plants increased Cd accumulation and reduced Cd tolerance, indicating that miR390 is a negative regulator of plant adaptive responses to Cd in rice (Ding et al., 2016).

In this study, we discovered that expression of miR166 was significantly reduced under Cd expo-sure (Fig. 2). Transgenic rice plants overexpressing miR166 displayed improved growth under Cd stress, unlike wild-type (Fig. 3). Increased growth of the miR166-overexpressing rice plants was associated with significant increase in the chlorophyll content and attenuation in Cd-induced oxidative damage in rice (Fig. 3). Furthermore, overexpression of miR166 re-duced root-to-shoot Cd translocation rate (Fig. 4). The 35S:MIR166 plants had substantially lower Cd concen-trations in the xylem sap than the wild-type plants (Fig. 4D). It has been well established that root-to-shoot Cd translocation via the xylem is the major and common physiological process determining Cd accumulation in

shoots and grains of rice (Uraguchi et al., 2009). These results suggest that miR166 plays a role in Cd loading to the xylem and regulates root-to-shoot translocation in rice. Indeed, overexpression of miR166 changed the expression pattern of OsHMA metal transporter genes with important roles in the transport and homeosta-sis of metals in plants (Sasaki et al., 2014). The previ-ous finding that mutations in OsHMA2 restrict Zn/Cd translocation (Satoh-Nagasawa et al., 2012) show that OsHMA2 encodes a major Zn/Cd transporter that promotes Zn/Cd root-to-shoot transport. Our discov-ery that overexpression of miR166 reduces both the expression of OsHMA2 and root-to-shoot translocation in the transgenic rice plants (Fig. 4) is consistent with this role of OsHMA2. OsHMA3, on the other hand, is a tonoplast-localized Cd transporter that promotes vac-uolar compartmentalization of Cd, thereby restricting its root-to-shoot translocation (Uraguchi et al., 2009; Sasaki et al., 2014). Overexpression of OsHMA3 also increased Cd tolerance in rice, likely due to increased vacuolar sequestration of Cd in the roots (Sasaki et al., 2014). Likewise, in 35S:MIR166 plants, we observed increased expression of OsHMA3 (Fig. 4E). Thus, the increased Cd tolerance of and decreased Cd transloca-tion in the 35S:MIR166 plants could be attributable at

Figure 9. Molecular characterization and Cd tolerance analysis of OsHB4 RNAi rice plants. A, Phenotypic comparison of wild-type (WT) and RNA interference (RNAi) transgenic rice lines (-1, -2 and -3) under Cd stress. A, Representative picture taken after Cd treatment for 14 d. B, Reverse0transcription quantitative PCR analysis of the expression of OsHB4 intransgenic rice relative to that of β-ACTIN. Data represent means and se of three replicates. C, MDA content in plants treated with 2 μm CdCl2 for 14 d. D, H2O2 content in plants treated with 2 μm CdCl2 for 14 d. Asterisks indicate significant differences between wild-type and transgenic plants at *P ≤ 0.05 and **P ≤ 0.01.

Overexpression of miR166 Increases Cd Tolerance

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least in part to the opposite effects on the expression of OsHMA2 and OsHMA3.

A key finding of this study is the substantial reduc-tion of leaf and grain Cd contents in the transgenic miR166-overexpressing rice lines grown in soil con-taining a low Cd concentration (Fig. 5), making miR166 a potential candidate for genetic engineering to reduce Cd content in rice grains. The reduced Cd content in the leaves and grains of transgenic miR166-overex-pressing lines likely resulted from the reduction in the root-to-shoot translocation of the heavy metal. Expres-sion of miR166 in leaves and flag leaves during the grain-ripening stage was substantially higher than that in leaves during the vegetative growth stage (Fig. 1). Since the grain-ripening stage is a critical period for grain Cd accumulation (Arao et al., 2009), the increased expression of miR166 in leaves and flag leaves during the grain-ripening stage may help reduce Cd transport into rice grains. Intriguingly, miR166 expression was reduced by Cd treatment (Fig. 2). The physiological significance of Cd-responsive reduction in miR166 ex-pression is unclear but may be related to the other roles of this miRNA in plant growth and development un-der stress conditions.

The biological functions of miRNAs are mediated through negative regulation of their specific targets. In rice, miR166 targets genes encoding the class III HD-Zip, as shown by sequencing analysis and confirmation by 5′

RACE and degradome sequencing (Liu et al., 2009; Li et al., 2010). In this study, we found that miR166 and OsHB4, one of the HD-Zip genes, exhibited oppo-site expression patterns in rice roots after treatment with Cd (Fig. 2B). OsHB4 and miR166 also showed negatively correlated expression patterns in miR166- overexpression lines (Fig. 2C). These results confirmed that OsHB4 was targeted by miR166. In rice and Ara-bidopsis, about 50% of all homeobox proteins belong to the HD-Zip superfamily (Chan et al., 1998; Jain et al., 2008). HD-Zip transcription factors regulate the enlargement of shoot apical meristem and vascular development (Prigge et al., 2005; Williams et al., 2005). There are also HD-Zip family genes that are respon-sive to various stress conditions, including drought, salt, and abscisic acid (Agalou et al., 2008; Zhang et al., 2012). Shin et al. (2004) reported that Athb-12, an HD-Zip protein from Arabidopsis, increased salt tol-erance in yeast by promoting sodium exclusion. Al-though some members of the HD-Zip gene family have been implicated in the regulation of development and stress responses, the functions of most HD-Zip genes are still unknown. Based on our comprehensive analy-sis, we have provided strong evidence for a critical role of a rice HD-Zip gene, OsHB4, as an important target of miR166 in the regulation of Cd accumulation and tolerance in rice. Expression of OsHB4 in Cd-treated rice seedlings was negatively regulated by miR166

Figure 10. Cd accumulation in wild-type and OsHB4 RNAi transgenic rice after exposure to 2 μm CdCl2 for 14 d. A, Cd content in roots. B, Cd content in shoots. C, Cd content in the xylem and phloem sap. D, Grain Cd concentration in wild-type (WT) and OsHB4 RNAi plants grown in Cd-containing soil. Asterisks indicate significant differences between wild-type and transgenic plants at *P ≤ 0.05 and **P ≤ 0.01.

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(Fig. 2). The transgenic plants overexpressing OsHB4 displayed phenotypes opposite to those of transgen-ic plants overexpressing miR166, including changes in Cd tolerance, Cd concentrations in the xylem sap, Cd root-to-shoot translocation, and Cd content in the grains. In contrast, silencing of OsHB4 improved Cd tolerance and reduced Cd translocation to grains (Figs. 9 and 10). These phenotypes of OsHB4-silenced rice plants were similar to those of 35S:MIR166 plants (Fig. 10). These results further support the notion that OsHB4 is a critical target of miR166 in the regulation of Cd translocation and tolerance in rice. As a transcrip-tion factor, OsHB4’s role in Cd transport and tolerance is likely through regulation of other genes, including OsHMA2 and OsHMA3, which affect various aspects of Cd uptake, sequestration, translocation, and detox-ification in rice. Future studies should identify the di-rect target genes of OsHB4 and associated molecular and biochemical processes in order to establish the miR166/OsHB4-mediated pathway of plant-adaptive responses to this toxic heavy metal.

MATERIALS AND METHODS

Plant Growth Conditions, Seedling Cultivation, and Stress Treatment

Seeds of rice cultivar Zhonghua 11 (Oryza sativa subsp. japonica) were sterilized with 10% sodium hypochlorite for 20 min, rinsed three times with distilled water, and soaked for 2 d at 37°C in the dark. The seeds were then transferred onto moist filter paper and cultured at 30°C in the dark for 1 d. The germinated seeds were then transferred to Yoshida nutrient solution under a 13 h light (29°C)/11 h dark (22°C) photoperiod. For Cd treatment, 1-week-old seedlings were transferred to plastic containers containing Yoshida nutrient solution with appropriate concentrations of CdCl2. Seedlings that were not treated with Cd were used as controls. After Cd treatment, rice tissues were collected separately and immediately frozen in liquid nitrogen for further analysis.

RT‑qPCR Assay

Total RNA collected from rice roots after exposure to 60 μm CdCl2 for 0, 1, 3, 6, 12, and 24 h was separately isolated using Trizol reagent (Invitrogen) and treated with RNase-free DNase I (TaKaRa) to remove contaminated DNA. Real-time PCRs were carried out using a Rotor-Gene Q machine (parameters, 95°C for 1 min, followed by 45 cycles of 95°C for 10 s, 58°C for 15 s, and 72°C for 15 s). All reactions were performed in triplicate. Quantification of gene expression was performed using the comparative Ct method. The β-ACTIN gene was chosen as the internal control. The primer pairs for RT-qPCR analysis of these genes were as follows: β-ACTIN forward, 5′-GCCGTCCTCTCTCTG-TATGC-3′; reverse, 5′-GGGGACAGTGTGGCTGAC-3′; miR166, stem-loop RT primer, 5′-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTG-GATACGACAGGGAA-3′; forward, 5′-GTCGGACCAGGCTTCA-3′; reverse, 5′-TGCGTGTCGTGGAGTC-3′; OsHB4 forward, 5′-ATGTCAAGAACCTC-CCCAG-3′; reverse, 5′-CACACGCAAAAATCACTCA-3′; OsHMA2 forward, 5′-TGGCACCACAAAAGGCTATT-3′; reverse, 5′-CACCGTCGATTGGAAT-GACT-3′; OsHMA3 forward, 5′-TGGGGATCTTCATCAAGGGT-3′; reverse, 5′-AACGAGTCGATGCTGAACTC-3′.

Vector Construction and Rice Transformation

For generation of 35S:MIR166 constructs, a 299-bp fragment surrounding the miRNA sequence that includes the foldback structure of miR166 was am-plified from rice genomic DNA using the following primers: miR166 forward 5′-CGGGGTACCCTTTCTCTGCTTTGGTGGTT-3′ and reverse 5′-AACTG-

CAGTTCCGATGGATTTCCTGGAC-3′. The amplified fragments were digest-ed and subcloned into KpnI and PstI sites in pCAMBIA1300-35S-NOS between the CaMV 35S promoter and the NOS terminator. The hygromycin phospho-transferase gene (hpt) was used as a selectable marker. To generate 35S:OsHB4 constructs, the OsHB4 (Os03g43930) open reading frame was amplified by RT-PCR with the forward primer 5′-GGGGTACCGGAGAAGGGTCGACGG-GGATG-3′ and reverse primer 5′-CGGGATCCGACGAATGACCAGTTGAC-GAAC-3′. The PCR products were first cloned into pMD-19T vector (TaKaRa) and verified by sequencing. Then, the OsHB4 open reading frame was released by digestion with KpnI and BamHI and subcloned into pCAMBIA1300. To generate the RNAi constructs, an OsHB4 gene fragment was PCR-amplified using gene-specific primers (5′-GGGGATCCGAGCTCAGGCAGGACAG-GTCGTCG-3′ and 5′-CGACTAGTGGTACCCCTTGCGCTGCTTCTCG-3′) and cloned into pCAMBIA1300-35SI-X by forward and reverse insertions.

Cultivars of japonica rice Zhonghua 11 were used for transformation. Trans-genic lines were generated by cocultivation of rice calli with Agrobacterium tumefaciens strain EHA105, which contained the constructs (Hiei et al., 1994). Transgenic rice seedlings were selected using 50 mg/L hygromycin. All the putative T0 transgenic plants were screened using PCR analysis with genomic DNA from their leaves. The PCR was performed with hpt-specific primers in a 20-µL reaction system. Expression levels of miR166 and OsHB4 in transgen-ic rice plants were examined using RT-qPCR. The transformation vector also contains a GUS reporter gene, and GUS staining was also used to confirm the transgenic plants (Jefferson, 1987).

Effect of Cd Stress on Rice Seedling Growth

To determine the role of miR166 on rice response to Cd stress, wild-type and 35S:MIR166 seedlings were cultured with Yoshida nutrient solution for 8 weeks. After exposure to 2 μm CdCl2 for 14 d, phenotypes of the plants were examined by photographing plants, and the chlorophyll, MDA, H2O2, and Cd contents were also determined. All experiments were performed in triplicate. Data points were presented as the mean ± sd of three replications. Significant differences among the wild-type and transgenic lines were analyzed using Student’s t test.

The chlorophyll content was determined according to the method of Knud-son et al. (1977). In brief, 0.1 g fresh rice leaf was extracted in 2 mL 95% ethanol for 36 h in the dark, and the extracted solution was analyzed spectrophoto-metrically. The H2O2 content was measured using a Hydrogen Peroxide Assay Kit (Nanjing Jiancheng). Leaf tissues (0.5 g) were ground into powder using liquid nitrogen and homogenized in 0.9 mL of precooled 50 mm phosphate buffer (pH 7.0). The homogenate was centrifuged at 10,000g for 10 min, and the supernatants were used for the H2O2 assay. Absorbance of the supernatant was recorded at 405 nm.

The MDA content was measured as described by Heath and Packer (1968), with slight modification. In brief, rice shoots of 0.1 g were ground in 1 mL of 10% (w/v) trichloroacetic acid. After centrifugation at 12,000g for 10 min at 4°C, the supernatants were collected, and 2 mL of the supernatant fraction was mixed with 2 mL 0.6% TBA solution. The mixtures were heated at 95°C for 30 min and then cooled quickly in an ice bath. The resulting mixtures were centrifuged at 10,000g for 10 min, and the absorbance of the supernatants was measured at 450, 532, and 600 nm.

Determination of Cd Content

After treatment with 2 μm CdCl2 for 14 d, roots of rice seedlings were im-mersed in 20 mm disodium ethylenediamine tetra-acetic acid for 20 min and then rinsed three times with deionized water. The roots and shoots were dried at 105°C for 2 h and then at 70°C to achieve a constant weight. In total, 200 mg of dry plant material was treated with 6 mL HNO3 at 120°C with a Mul-tiwave until they were completely digested. Concentrations of Cd were then determined using an atomic absorption spectrometer (AA-7000; Shimadzu). Xylem sap exuded from the cut surface was collected by trapping it into a 1.5-mL plastic vial filled with a small piece of cotton for 1 h after cutting the shoots at 3 cm above the roots. The amount of collected sap was weighed. Cd concentration in the sap was quantified using atomic absorption spectrometry.

Soil Growing of Rice Plants and Analysis of Cd Content

Soil for the experiments was collected from the surface layer (0–20 cm) of an unpolluted paddy field in Hangzhou, China. The soil was sieved using a 2-mm grid sieve and air dried. Fertilizers were added to the soil and incubated

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1702 Plant Physiol. Vol. 177, 2018

for 2 weeks before Cd treatment, which was added by manually mixing in a CdCl2 solution. To ensure Cd homogeneity, the soil was mixed every 5 d for 30 d. Before planting, the Cd level was determined to be about 3 μg per gram of soil.

For analysis of Cd content in soil-grown rice plants, wild-type, miR166, and OsHB4 transgenic rice seeds were surface-sterilized and germinated in deion-ized water for 3 d. Two-week-old rice seedlings were transplanted to a plastic rectangular pot filled with Cd-containing soil (∼3 μg/g) in May, 2013 at China Jiliang University in Hangzhou, China. The pots were set up outdoors, with a glass shelter to protect from rainfall and pests. Intermittent flooding was introduced throughout cultivation to enhance Cd availability in the soil. Plants were harvested in September and washed thoroughly with tap water and then distilled water after grain ripening. The plant samples were then divided into stems, leaves, and grains. After harvesting, the fresh weights were recorded, and the plants were dried at 70°C. Then, the dry weights were recorded and the plant materials were ground using a stainless steel grinder. For grain Cd analysis, dried grain samples were hulled and milled into powder. Brown rice (0.3 g) was treated with 6 mL HNO3 at 120°C with a Multiwave until they were completely digested. The Cd content of the grains was quantified by an atomic absorption spectrometer (AA-7000; Shimadzu).

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number AK103284.Received April 24, 2018; accepted June 4, 2018; published June 20, 2018.

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