Arabidopsis HY1 Confers Cadmium Tolerance by Decreasing Nitric Oxide Production and Improving Iron Homeostasis

Molecular Plant Advance Access published September 20, 2013 Molecular Plant

RESEARCH ARTICLE

Arabidopsis HY1 Confers Cadmium Tolerance by Decreasing Nitric Oxide Production and Improving Iron Homeostasis Bin Hana, Zheng Yanga, Yanjie Xiea, Li Niea, Jin Cuib and Wenbiao Shena,1 a College of Life Sciences, Nanjing Agricultural University, Nanjing, 210095, China b Laboratory Center of Life Sciences, Nanjing Agricultural University, Nanjing, 210095, China

Key words:  Arabidopsis; cadmium tolerance; heme oxygenase 1; iron homeostasis; nitric oxide; primary root elongation.

Introduction Cadmium (Cd2+) is a non-essential and toxic element that accumulates in plant cells as a consequence of improper human activities. Besides inhibiting seed germination, Cd2+ reduces plant growth and photosynthesis, and impairs nutrient distribution (Das et  al., 1997). As a divalent ion, Cd2+ is taken up by the roots through low-specificity Fe2+, Zn2+, and Ca2+ transporters or channels, and is then translocated to the shoots. Therefore, Cd2+ toxicity can at least partly be attributed to competition of the essential metals uptake, especially iron (Clemens, 2006). Iron (Fe2+ and Fe3+) is an essential nutrient for plants. All plant species, except grasses (e.g. barley, maize, and rice), mobilize Fe via the Strategy I  pathway (Marschner and Römheld, 1994). After soil acidification, Fe3+ is reduced to Fe2+ by a plasma membrane-bound ferric chelate reductase, named Ferric Reduction Oxidase 2 (FRO2), which is one of eight members of the Arabidopsis FRO family (Robinson et al., 1999). Thereafter, Fe2+ is transported into the roots by Iron-Regulated Transporter 1 (IRT1), a member of the ZIP family of metal transporters, which is also responsible for Cd2+ uptake from soil into root cells (Vert et al., 2002). To maintain

the metal balance and detoxify heavy metals in plant cells, several members of the P1B-ATPase (Heavy metal ATPase, HMA) family, such as HMA2, HMA3, and HMA4, can be activated under Fe deficiency or Cd2+ stress (Eren and Argüello, 2004; Argüello et al., 2007; Courbot et al., 2007). The important role of glutathione (GSH) in Cd2+ detoxification was also elucidated (Noctor et al., 2012). However, the signaling mechanisms involved in Cd2+ toxicity and related Fe deficiency are still unclear. Nitric oxide (NO) is a free radical gas that has been shown to have multiple signaling functions in various species (Moreau et  al., 2010). In animals, the synthesis of NO from L-arginine is catalyzed by the heme-containing enzyme nitric oxide synthase (NOS). In plants, NO can be produced

1 To whom correspondence should be addressed. E-mail wbshenh@njau. edu.cn, tel. +86-25-8439-9032, fax +86-25-8439-6542.

© The Author 2013. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS. doi:10.1093/mp/sst122 Received 23 June 2013; accepted 13 August 2013

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ABSTRACT  Up-regulation of the gene that encodes intracellular heme oxygenase 1 (HO1) benefits plants under cadmium (Cd2+) stress; however, the molecular mechanisms remain unclear. Here, we elucidate the role of Arabidopsis HY1 (AtHO1) in Cd2+ tolerance by using genetic and molecular approaches. Analysis of two HY1 null mutants, three HY1 overexpression lines, HO double or triple mutants, as well as phyA and phyB mutants revealed the specific hypersensitivity of hy1 to Cd2+ stress. Supplementation with two enzymatic by-products of HY1, carbon monoxide (CO) and iron (Fe, especially), rescued the Cd2+ -induced inhibition of primary root (PR) elongation in hy1-100. The mutation of HY1, which exhibited lower glutathione content than Col-0 in root tissues, was able to induce nitric oxide (NO) overproduction, Cd2+ accumulation, and severe Fe deficiency in root tissues. However, the contrasting responses appeared in 35S:HY1-4. Additionally, reduced levels of Ferric Reduction Oxidase 2 (FRO2) and Iron-Regulated Transporter 1 (IRT1) transcripts, and increased levels of Heavy Metal ATPase 2/4 (HMA2/4) transcripts bolster the notion that HY1 up-regulation ameliorates Fe deficiency, and might increase Cd2+ exclusion. Taken together, these results showed that HY1 plays a common link in Cd2+ tolerance by decreasing NO production and improving Fe homeostasis in Arabidopsis root tissues.

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  Han et al.  •  HY1, NO, and Iron in Cadmium Tolerance

phenotypes. Meanwhile, lower GSH content was detected in root tissues of hy1-100 than that of Col-0, regardless of the treatment of Cd2+. Similarly to previous results (BessonBard et al., 2009; Ye et al., 2013), further results revealed that up-regulation of HY1 decreases Cd2+-induced NO production, resulting in enhanced Cd2+ tolerance in Arabidopsis roots. The expression of genes related to Fe uptake (FRO2, IRT1) and P1B-ATPase genes (HMA2, HMA4) strengthened the proposed roles of HY1 in reducing Cd2+ accumulation and ensuring Fe homeostasis. Our results therefore point to HY1 as a potential contributor of Cd2+ tolerance.

RESULTS Contribution of HOs to Cd2+ Hypersensitivity Pharmacological studies have shown that HO1 and CO contribute to Cd2+ tolerance in soybean (Noriega et  al., 2004) and alfalfa (Han et  al., 2008; Fu et  al., 2011). To generate corresponding genetic evidence, we compared Cd2+-induced phenotypic changes in hy1-100, ho2, ho3, and ho4 individual Arabidopsis mutants. Compared with Col-0, the relative fresh weight accumulation and germination frequency in these four individual ho mutants decreased to approximately similar extents after growing on Cd2+ plates (Supplemental Figures 1A and 2). Although hy1-100 exhibited a slight growth reduction compared to Col-0 under control conditions, the PR inhibition was more pronounced in hy1-100 than any other ho mutants (Supplemental Figures 1 and 3). Using transplanting tests, the inhibition of relative PR elongation in hy1-100 was 79.2 ± 4.7% after 5 d of Cd2+ stress, in comparison with 58.6  ±  6.1%, 58.7 ± 5.4%, 53.8 ± 7.5%, and 58.3 ± 11.5% in Col-0, ho2, ho3, and ho4, respectively (Figure 1A and 1B). These observations verify that hy1-100 is hypersensitive to Cd2+ on PR elongation. To exclude the involvement of HO2, HO3, and HO4 in Cd2+induced PR inhibition, redundancy tests were performed. In contrast to the inhibition responses of hy1-100/ho3 and hy1-100/ho4, neither ho2/ho3, ho2/ho4, and ho3/ho4 double mutants nor ho2/ho3/ho4 triple mutants were hypersensitive to Cd2+ in terms of relative PR elongation, when compared with Col-0 (Figure 1C). Further real-time RT–PCR showed that the transcript levels of HY1 were higher than those of HO2, HO3, and HO4 in Arabidopsis roots upon 50 μM Cd2+ stress (Figure 1D). HY1 transcripts were induced by Cd2+ stress, with the first strong peak at 6 h and the second peak at 24 h.

HY1 Confers Cd2+ Tolerance Subsequent functional analysis of HY1 was performed by using transgenic plant lines 35S:HY1-3, 35S:HY1-4, and 35S:HY1-5, which had been validated by Western blotting, and real-time RT–PCR (Figure  2A and 2B) by using two internal control genes F-box and SAND (Supplemental Figure 4). We further observed that Cd2+-induced inhibition of PR elongation was significantly alleviated in 35S:HY1-3, 35S:HY1-4, and 35S:HY1-5

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by nitrate reductase (NR), a well-documented route of reductive reactions. However, the mechanisms underlying oxidative routes, such as NOS-like biochemical pathway and polyamine-mediated NO production, remain elusive (Gupta et  al., 2011). Meanwhile, NO has been indicated as a cellular messenger in numerous plant physiological processes (Delledonne, 2005), owing in part to its high affinity and reactivity towards transition metals, especially Fe (Graziano and Lamattina, 2005). Besides mediating responses to Fe deprivation (Chen et al., 2010), NO is able to protect plants against the oxidative damage triggered by Cd2+ (Kopyra and Gwóźdź, 2003). By contrast, Besson-Bard et al. (2009) showed that NO contributes to Cd2+ toxicity by initiating a Cd2+ induced Fe deficiency pathway in Arabidopsis. The aggressive effects of NO were also suggested by Ye et  al. (2013), who demonstrated the accumulation of NO in Cd2+ -induced programmed cell death. In view of these conflicting data, further research and other functional elements are required to clarify cell signaling networks of NO in Cd2+ tolerance as well as an interference of Cd2+ with Fe homeostasis. The heme–heme oxygenase (HOs; EC 1.14.99.3) system has been recently recognized as another signaling system in animal physiological processes (Wagener et al., 2003). Heme is an important nutritional source of Fe in both animal and plant cells (Graziano and Lamattina, 2005; Yanatori et  al., 2010). Normally, HO catabolizes heme into three products: Fe2+, carbon monoxide (CO), and biliverdin (BV), which is rapidly converted to bilirubin (BR) (Shekhawat and Verma, 2010). In Arabidopsis, four HO genes, including HY1, HO2, HO3, and HO4, have been characterized (Gisk et  al., 2010). Among these, HY1, an inducible isoform of HO, is the most highly expressed. Meanwhile, expression levels of HO2, HO3, and HO4 are extremely low (Matsumoto et al., 2004). Interestingly, the expression of HO1 and/or its catalytic product CO were suggested to be induced by Cd2+ in alfalfa (Han et  al., 2008; Cui et  al., 2011; Fu et  al., 2011) and soybean (Noriega et al., 2012). The up-regulation of HO1 could protect against Cd2+ -induced oxidative cell damage by the regulation of GSH metabolism (Noriega et  al., 2004; Han et  al., 2008). Previous results also revealed that HO may interact with NO in modulating cucumber adventitious root formation (Xuan et  al., 2008) and Cd2+ -induced oxidative damage in soybean leaves (Noriega et  al., 2007). However, the detailed mechanisms of HOs in Cd2+ tolerance are still elusive. The aim of this work was to elucidate the interaction among Cd2+ tolerance, Fe homeostasis, and NO signaling with emphasis on the possible roles of HY1 in this process. Genetic and molecular analysis demonstrated that hy1-null mutants are more sensitive to Cd2+ stress than Col-0. This was demonstrated by comparing relative primary root (PR) elongation in Col-0, four individual ho mutants (hy1-100, ho2, ho3, and ho4), and several double and triple ho mutants. The second allele of hy1 mutant (hy1-1), as well as phyA and phyB mutants also confirmed that the Cd2+ hypersensitivity is hy1-specific. By contrast, lines that overexpress HY1 displayed tolerant

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seedlings (Figure  2C). Consistently with these results, different degrees of damage in root cells of hy1-100 and 35S:HY1-4 were observed (Supplemental Figure 5). For example, after 2 d of 50 μM Cd2+ treatments, hy1-100 exhibited enlarged and unusually shaped root cells, in contrast with the small cells containing dense cytoplasm which divide and expand in Col-0. The delayed phenomenon was observed in 35S:HY1-4. To further confirm the phenotypes of hy1-100, a second line of hy1 mutant hy1-1 with the ecotype Ler as background was performed. After 5 d of 50 μM Cd2+ treatments, the inhibition of PR elongation in hy1-1 was 75.6 ± 8.2%, in comparison with 51.4 ± 5.2% in Ler-0 (Figure  2D). Taken together, these genetic data confirmed that the loss of HY1 function increases sensitivity to Cd2+ stress. We then used hy1-100 and 35S:HY1-4 plants for subsequent experiments.

Alteration of Cd2+ Toxicity by HY1 The experiments described above evaluated the contribution of HY1 to Cd2+ tolerance, and thus raised the question of how HY1 modulates Cd2+ tolerance in Arabidopsis. To answer this question, we first assessed the role of HY1 by the addition of hemin (a HY1 inducer) to the media.

Pharmacological tests showed that, under the normal conditions, hemin significantly enhanced PR elongation in Col-0 and 35S:HY1-4 (in particular) (Figure 3A). By contrast, a significant inhibition of PR elongation was observed in hy1100 treated with hemin alone. Likewise, hemin significantly alleviated Cd2+-induced PR inhibition in Col-0 and 35S:HY1-4, but not in hy1-100. To investigate which enzymatic by-product(s) of HY1 was involved in this process, we tested the effects of BR, CORM-2 (a CO donor), and Fe-EDTA on Cd2+ tolerance. It showed that Cd2+-induced PR inhibition in Col-0 and 35S:HY1-4 could be differentially rescued by CORM-2 or Fe-EDTA exposure. However, no significant differences were observed when 50  μM BR was supplied (Figure  3A). Unlike hemin, both CORM-2 and Fe-EDTA partially alleviated Cd2+-induced inhibition of PR elongation in hy1-100, with more effects observed in Fe-EDTA than CORM-2. Thus, Fe and CO may be potential candidates for HY1-triggered Cd2+ tolerance.

NO Production under Cd2+ Stress Similarly to a previous report (Besson-Bard et al., 2009), 50 μM Cd2+ triggered a slight but not significant increase in DAF-FM

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Figure 1. Cd2+-Sensitivity and Redundancy Analysis of Four Individual HO Mutants, and Relative Expression of Four HO Genes in Col-0 Roots upon Cd2+ Stress. Morphology and fresh weight (A), and relative PR elongation (B, C) of 5-day-old seedlings transferred to liquid medium with or without 50 μM CdSO4 for another 5 d. Arrows indicate the PR position of 5-day-old seedlings of each genotype. Relative PR elongation was calculated from arrows to root tips. The grid scale of the plate is 15 mm. Meanwhile, time course of four HO genes expression in Col-0 roots was analyzed by real-time RT–PCR (D). Expression levels were relative to that of F-box and SAND. Mean values with different letters denote significant differences according to multiple comparisons (P < 0.05). Asterisks indicate significant differences compared with HY1 transcript of 0 h at P < 0.05 or 0.01 levels according to t-test.

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  Han et al.  •  HY1, NO, and Iron in Cadmium Tolerance

fluorescence in Col-0 roots during the first 6 h of Cd2+ treatment. Thereafter, the fluorescence increased progressively (Figure  3B). This increase of fluorescence can specifically be attributed to NO, because it could be significantly suppressed in the presence of potassium salt of 2-(4-carboxyphenyl)4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), which is considered a NO scavenger (Chen et  al., 2010) (Figure  4A and 4B). Further comparison showed that the mutation of HY1 increased NO accumulation in Cd2+-treated root tissues. By contrast, Cd2+-induced NO production was significantly lower in 35S:HY1-4 than in either Col-0 or hy1-100, with a larger difference seen between 35S:HY1-4 and hy1-100 than 35S:HY1-4 and Col-0 (Figure 3B). In order to further confirm that the changes in DAF-FM fluorescence were caused by NO, the highly specific electron paramagnetic resonance (EPR) analysis was performed. Similarly, Cd2+-induced EPR signal in hy1-100 was higher than those of Col-0 and 35S:HY1-4 (in particular). Although no significant NO production was observed in both Fe2+(DETC)2 and ethyl acetate, the mutation of HY1 was able to induce NO accumulation even under control conditions (Figure 3C). These results supported the idea that the induction of HY1 plays a role in decreasing NO overproduction.

NO scavenger. As a positive control, exogenous application of fresh sodium nitroprusside (SNP; a NO-releasing compound) alone obviously increased NO content in Col-0, hy1-100, and 35S:HY1-4 (Figure  4A and 4B). This effect was not observed in seedlings treated with old SNP solution, which contains no NO, but nitrate, nitrite, and ferrocyanide (Tossi et al., 2009). The significant inhibition of PR elongation in Col-0, hy1-100, and 35S:HY1-4 suggested the aggravating effects of fresh SNP under control (in particular) and Cd2+-stressed conditions (Figure  4C). By contrast, no such significant inhibition was observed when the same seedlings were treated with old SNP. These results demonstrate that Cd2+-induced NO accumulation may trigger PR inhibition. This conclusion was further supported by the demonstration that cPTIO was able to prevent Cd2+-induced NO production and differentially abolish the subsequent inhibition of PR elongation. Comparatively, in the presence of Cd2+, hy1-100 showed less PR elongation than Col-0, regardless of SNP or cPTIO treatment (Figure 4C). Except for Cd2+ plus cPTIO treatment, the opposite phenotypes were observed in 35S:HY1-4. The above-mentioned results implied that HY1 is able to affect PR elongation in response to Cd2+ exposure by modulating NO production.

The HY1-Induced Decrease in NO Production Is Related to Cd2+ Tolerance

The Origin of NO Production in HY1-Modulated Cd2+ Tolerance

To evaluate the contribution of NO, we compared the effects of Cd2+ on NO production and PR elongation in hy1-100 and 35S:HY1-4 in the presence of a NO-releasing compound and a

To characterize the source of NO under our experimental conditions, we studied the effects of NG-nitro-L-arginine methyl ester hydrochloride (L-NAME) on NO production, as well as

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Figure 2.  Western Blot, Transcript Expression, and Cd2+-Hypersensitive Phenotypic Analysis. (A) 10-day-old seedlings were used for Western blot analysis. The number below the band indicates the relative abundance of the corresponding HY1 protein compared with that of Col-0. (B) The transcript levels of HY1 in 5-day-old seedlings were analyzed by real-time RT–PCR. The expression levels were relative to that of F-box and SAND. (C, D) 5-day-old seedlings were transferred to liquid medium with or without 50  μM CdSO4 for another 5 d; relative PR elongation was then recorded. Mean values with different letters denote significant differences according to multiple comparisons (P < 0.05).

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PR elongation in the absence or presence of Cd2+. The mammalian NOS inhibitor L-NAME was used to partially inhibit NO production in Arabidopsis and green algae (Foresi et al., 2010; Van Ree et al., 2011). As shown in Figure 4A and 4B, L-NAME differentially decreased endogenous NO production in Col-0, hy1-100, and 35S:HY1-4 regardless of Cd2+ stress. Cd2+-induced PR inhibition in hy1-100 was significantly blocked by L-NAME. Compared to the samples treated with Cd2+ alone, however, an obvious increase or a slight but not significant decrease in relative PR elongation was observed in Col-0 and 35S:HY1-4 co-treated by L-NAME. To confirm the effects of L-NAME, we tested the origin of Cd2+-induced NO production in nia1/nia2 or atnoa1 mutant, which exhibit defects in NR or indirectly reduce NO production, respectively (Lozano-Juste and León, 2010; Moreau et al., 2010). As shown in Figure 5A–5C, with respect to Col-0 upon Cd2+ stress, atnoa1 produced less NO and exhibited significant alleviation of relative PR inhibition. Moreover, the mutation of AtNOA1 could not fully block the increase of NO production upon Cd2+ treatment. Interestingly, the addition of hemin resulted in similar levels of PR elongation in Col0, nia1/nia2, and atnoa1 seedlings upon Cd2+ stress. In view of the fact that hemin brought about the similar decreasing tendencies in NO production in Col-0, nia1/nia2, and atnoa1,

compared to Cd2+-stressed alone samples, we suggested that there may be other NO synthetic pathway(s) in hemininduced Cd2+ tolerance. Given that Cd2+ and polyamines (PAs) treatments induce NO formation in plant seedling roots which, in turn, is involved in root growth inhibition (Tun et al., 2006; Groppa et  al., 2008a; Arasimowicz-Jelonek et  al., 2011), the contribution of PAs on NO production and PR elongation was investigated. First, we observed that NO production was significantly increased in the roots of both Cd2+-stressed and PAs-treated plants, especially in those exposed to Cd2+ and spermine (Spm; Supplemental Figure 6), in comparison with the chemical-free control samples. As expected (Groppa et al., 2007; Yang et al., 2010), Cd2+ increased putrescine (Put) content in all of three different Arabidopsis genotypes, whereas spermidine (Spd) content remained unaltered (Figure 6B and 6C). Interestingly, compared with Col-0 in the control conditions, the Spm content was 61.9 ± 2.1% and 16.8 ± 1.3% lower in roots of hy1-100 and 35S:HY1-4, respectively (Figure  6D). Cd2+ treatment significantly reduced Spm content with 66.8 ± 1.7%, 76.6 ± 0.3%, and 53.6 ± 1.5% reductions in Col-0, hy1-100, and 35S:HY1-4 seedlings, respectively. We also found that 35S:HY1-4 contained a slight but not significant increase in total PA content (Put+Spd+Spm) than Col-0 under

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Figure 3.  Effects of Hemin, BR, CORM-2, Fe-EDTA on PR Elongation and the NO Production under Cd2+ Stress. (A) 5-day-old seedlings were transferred to liquid medium containing 50 μM hemin, 50 μM BR, 100 nM CORM-2, or 50 μM Fe-EDTA alone or in combination with 50 μM CdSO4 for another 5 d; relative PR elongation was then measured. (B) 5-day-old seedlings were treated with or without 50 μM CdSO4. After treatments at the indicated time points, root tissues were loaded with 5 μM DAF-FM DA for 30 min. NO production was then detected by confocal laser scanning microscopy. (C) 5-day-old seedlings were treated with or without 50 μM CdSO4 for 3 d. Whole seedlings were used for NO detection by EPR analysis. Mean values with different letters denote significant differences according to multiple comparisons (P < 0.05). Asterisks indicate significant differences between Col-0 and hy1-100 or Col-0 and 35S:HY1-4 upon Cd2+ stress at the indicated time points according to t-test (P < 0.05).

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  Han et al.  •  HY1, NO, and Iron in Cadmium Tolerance

Cd2+-stressed conditions (Figure  6E). By contrast, the lowest levels of PAs were observed in hy1-100. Comparatively, the total content of PAs in three genotypes were respectively decreased in Cd2+-stressed conditions with respect to the corresponding control plants. Responses of PR elongation to PAs in these mutants with or without Cd2+ stress were also analyzed (Figure  6A). We observed that, under the control conditions, PAs treatments caused decreases in root elongation, with Spm (100 μM) giving the greatest level of inhibition (61.9 ± 7.1%, 62.1 ± 4.1%, and 49.3 ± 5.2% reduction in Col-0, hy1-100, and 35S:HY1-4,

respectively). These decreasing effects were significantly enhanced in the presence of Cd2+. However, a high dose of Spm (1 mM) alone resulted in even more phytotoxic effects than those of 50 μM Cd2+, which led to bleach and death (data not shown). Among these genotypes, we observed that relative PR elongation in 35S:HY1-4 was less sensitive to PAs and Cd2+ than those in Col-0 and hy1-100. Compared to the samples treated with Cd2+ alone, a significant alleviation in PR inhibition was found in hy1-100 co-treated with Put or Spd (in particular). We also noticed that the addition of Spm aggravated Cd2+ toxicity in three genotypes.

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Figure 4.  Involvement of NO in Cd2+-Triggered Inhibition of PR Elongation Mediated by HY1. 5-day-old seedlings were transferred to liquid medium containing 50 μM sodium nitroprusside (SNP) or old SNP, 250 μM cPTIO, 100 μM NG-nitro-Larginine methyl ester hydrochloride (L-NAME) alone or in combination with 50 μM CdSO4. (A) After 3-d treatments, root tissues were loaded with 5 μM DAF-FM DA for 30 min. NO production was then detected by confocal laser scanning microscopy. Scale bar = 150 μm. Meanwhile, corresponding fluorescence intensity of NO (B) and relative PR elongation after 5-d treatments (C) were also provided. Mean values with different letters denote significant differences according to multiple comparisons (P < 0.05).

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NO biosynthesis was previously suggested as a result of catabolism of PA by copper-binding amine oxidases (CuAO, also called as DAO; Gil-Amado and Gomez-Jimenez, 2012) and FAD-binding polyamine oxidases (PAO) (Wimalasekera et al., 2011a, 2011b). Subsequent experiments revealed that, with respect to the control samples, DAO and PAO activities in Col-0, hy1-100, and 35S:HY1-4 seedling roots were significantly reduced by Cd2+ treatment (Figure 6F and 6G). However, NO

production was induced (Figure 4A and 4B). To further confirm these results, β-hydroxyethylhydrazine (β-HEH), an inhibitor of DAO/PAO, was applied (Su et  al., 2006). Surprisingly, 100 μM β-HEH significantly increased DAF-FM fluorescence in the roots of Arabidopsis (Supplemental Figure 6). Moreover, β-HEH caused a significant decrease in root elongation under the control or Cd2+-stressed conditions (Figure  6A). Together, the above results clearly excluded the possibility

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Figure 5.  The Role of HY1 in Cd2+-Induced NO Production and Glutathione Reduction. (A–C) 5-day-old seedlings were transferred to liquid medium containing 50 μM hemin or 50 μM CdSO4 individually or simultaneously. After 3-d treatments, root tissues were loaded with 5 μM DAF-FM DA for 30 min. NO production was then detected by confocal laser scanning microscopy (A). Scale bar = 150 μm. Meanwhile, corresponding fluorescence intensity of NO (B) and relative PR elongation after 5 d treatments (C) were also provided. (D, E) 5-day-old seedlings were transferred to liquid medium containing 0 (Control) or 50  μM CdSO4 (Cd2+) for 3 d; root tissues were loaded with 50 μM monochlorobimane (MCB) for 20 min. Glutathione content was then detected by a fluorescence microscope (D). Scale bar = 200 μm. Meanwhile, corresponding intensity of MCB fluorescence (E) was also provided. AU, arbitrary units. Mean values with different letters denote significant differences according to multiple comparisons (P < 0.05).

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  Han et al.  •  HY1, NO, and Iron in Cadmium Tolerance

of PAs-mediated NO biosynthesis in HY1-modulated Cd2+ tolerance.

Opposite Effects of HY1 Ablation and HY1 Overexpression Mutant on Cd2+ Accumulation

Relationship among HY1-Modulated NO Production, PR Elongation, and Cd2+ -Induced Iron Deficiency Previous studies demonstrated that Cd2+ competes with Fe2+ uptake by up-regulating the expression of IRT1 gene and initiates an Fe-deprivation signaling pathway (Vert et  al., 2002). Both Fe deficiency and Cd2+ stress promote NO production (Graziano and Lamattina, 2007; BessonBard et  al., 2009). Thus, besides increasing NO production, HY1 ablation may amplify Cd2+ -induced Fe deficiency. To test this hypothesis, we analyzed the Fe content in three different genotypes. As expected, the concentration of Fe was differentially reduced in all of the tested genotypes upon Cd2+ treatment (Table 1). In contrast with a reduction to 74.5 ± 6.3% or 74.0 ± 2.4% of Fe levels seen in roots of hy1-100 with or without Cd2+ treatment, the Fe content in 35S:HY1-4 was 17.0 ± 7.7% or 13.4 ± 4.5% higher than that of Col-0. Similar tendencies were apparent in shoot tissues of these three different genotypes. Subsequent experiments were performed to assess a functional link between HY1-modulated NO production and Fe deficiency. We illustrated that NO production was significantly enhanced in Col-0, hy1-100, and 35S:HY1-4 under Fe deficiency, in comparison with those grown in Fe-sufficient media (Figure  7A and 7B). These effects were significantly enhanced in the presence of Cd2+. Among these genotypes, hy1-100 exhibited maximal NO production in root tissues. Given that the production of NO in roots of tomato and Arabidopsis is an early and sustained response to Fe deprivation and Cd2+ exposure (Graziano and Lamattina, 2007; BessonBard et  al., 2009), we wondered about the effects of these changes in metal levels on other phenotypic responses. As expected, relative PR elongation in hy1-100 was more sensitive

Expression of Genes Responsible for Heavy Metal Homeostasis To further establish the relationship between HY1 and the genes related to Fe uptake or heavy metal detoxification under Cd2+ treatment, we analyzed the expression profiles of FRO2, IRT1, HMA2, and HMA4. As shown in Figure 8, Cd2+ treatment increased the levels of FRO2 and IRT1 transcripts in Col-0. Additionally, Cd2+-induced expression of FRO2 and IRT1 genes was weaker in 35S:HY1-4 than in Col-0. By contrast, hy1-100 exhibited the strongest levels of FRO2 and IRT1 expression. Subsequent results showed that HMA2 and HMA4 transcripts were increased in 35S:HY1-4 upon Cd2+ exposure, while no significant differences were observed in either Col-0 or hy1-100 with or without Cd2+ stress. Together, the abovementioned experiments partially support previous results obtained using ICP–OES (Table  1), and reinforce the close link among HY1, Cd2+-induced Fe deficiency and heavy metal homeostasis.

Discussion Genetic Evidence Supports that Arabidopsis HY1 Attenuates Cd2+ Toxicity Through physiological and pharmacological approaches, it has been documented that HO1 participates in plant stress responses following Cd2+ exposure (Noriega et  al., 2004; Fu et al., 2011; Noriega et al., 2012). In this report, besides photomorphogenesis (Muramoto et  al., 1999; Shekhawat and Verma, 2010), genetic and molecular evidence demonstrated that Arabidopsis HY1 (AtHO1) also confers Cd2+ tolerance. This conclusion is based on several pieces of evidence. First, although the mutation of HY1 causes a slight growth reduction compared to Col-0 under the control conditions, the relative PR elongation of hy1-100 was more sensitive to Cd2+ stress than Col-0 and the three other ho mutants (Figure 1A and 1B, and Supplemental Figures 1 and 3). It was suggested that phytochromes might participate in the regulation of plant stress tolerance (reviewed by Carvalho et al., 2011). Interestingly, Sung et al. (2007) showed that the mutation of PHYA exhibits an enhanced thiol synthesis mechanism and increased tolerance to arsenate. Furthermore, arsenic tolerance assays with phyB-9 and phot1/phot2 mutants showed that these photoreceptor mutants do not exhibit phyA-like arsenic tolerance. As HY1 was originally identified as being involved in the pathway of phytochrome chromophore

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The next question concerns whether HY1 decreases Cd2+ accumulation. Given that Cd2+ can be taken up through Fe2+, Zn2+, and Ca2+ transporters or channels (Clemens, 2006), we used inductively coupled plasma–optical emission spectrometry (ICP–OES) to measure the levels of Cd and Fe in roots and shoots of Arabidopsis seedlings. The results shown in Table 1 illustrate that HY1 affected Cd2+ content. For example, compared with Col-0 roots, the Cd2+ concentration was 36.6 ± 2.3% higher in roots of hy1-100 and 25.6 ± 1.5% lower in 35S:HY1-4. However, Cd2+ concentrations were approximately similar in shoots of three different genotypes. These results clearly indicated that HY1 ablation can increase Cd2+ accumulation and sensitivity in root tissues. Additionally, GSH content was decreased under Cd2+ exposure (Figure  5D and 5E). Interestingly, a lower level of GSH content was detected in root tissues of hy1-100 than that of Col-0, regardless of Cd2+ stress.

to Cd2+ stress than those in Col-0 and 35S:HY1-4 (Figure 7C and 7D). The different levels of inhibition of PR elongation in different genotypes became more evident when Fe deficiency was combined with Cd2+ treatment. In comparison with Col-0 and hy1-100, 35S:HY1-4 was relatively insensitive to Fe deficiency- and Cd2+-induced inhibition of PR elongation. Thus, we further deduced that HY1 is able to modulate root development during Cd2+ exposure and Fe deficiency.

Han et al.  •  HY1, NO, and Iron in Cadmium Tolerance   

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biosynthesis (Muramoto et  al., 1999), the possible interrelationship between phytochromes and Cd2+ tolerance was investigated. The relative expression values of five phytochrome genes in Arabidopsis root tissues (Supplemental Figure  7A) showed that the expression levels of PHYA and PHYB are much higher than that of other phytochrome genes. Subsequently, two phytochrome mutants phyA and phyB were individually used. Although phyA and phyB displayed shorter roots (data not shown), no significant difference in Cd2+ sensitivity was observed among phyA and phyB mutants, and Ler-0 (Supplemental Figure 7B). Therefore, we concluded that phytochromes, at least PHYA and PHYB individually, are not involved in Cd2+ tolerance. However, understanding how

HY1 and phytochromes separately or dependently modulate specific stress responses might be a central question in plant biology. It was also noticed that HY1-mediated Cd2+hypersensitive phenotype manifested primarily as reduced PR elongation rather than decreased germination frequency and fresh weight accumulation (Figure  1A and 1B, and Supplemental Figures 1 and 2). Second, in contrast with the increased sensitivity observed in the two hy1-null mutants, three lines that overexpress HY1 were more tolerant to Cd2+ stress than Col-0 (Figures 1A, 1B, and 2). The contrasting effects of ablating and enhancing HY1 expression may be attributed to the accelerated root cell damage in hy1-100 and the delayed effects

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Figure 6.  Effects of Polyamines on HY1-Mediated Cd2+ Tolerance. (A) 5-day-old seedlings were transferred to liquid medium containing 100  μM putrescine (Put), spermidine (Spd), spermine (Spm), and β-hydroxyethylhydrazine (β-HEH) alone or in combination with 50 μM CdSO4 for another 5 d; relative PR elongation was then measured. (B–G) Seedlings were transferred to liquid medium containing 0 (Control) or 50 μM CdSO4 (Cd2+) for 3 d. Free Put (B), Spd (C), Spm (D), and total polyamines (E) in roots were quantified by UPLC. Meanwhile, activities of diamine oxidase (DAO; (F)) and polyamine oxidase (PAO; (G)) were also measured. Mean values with different letters denote significant differences according to multiple comparisons (P < 0.05).

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  Han et al.  •  HY1, NO, and Iron in Cadmium Tolerance

in 35S:HY1-4 (Supplemental Figure  5). Consistently with this proposal, Cd2+ levels were lower in 35S:HY1-4 than in Col-0, whereas hy1-100 accumulated more Cd2+, especially in root tissues (Table  1). Functional redundancy analysis of hy1-100/ho3, hy1-100/ho4, ho2/ho3, ho2/ho4, ho3/ho4, and ho2/ho3/ho4 double or triple mutants supports the conclusion that Cd2+ hypersensitivity is hy1-specific (Figure  1C). Molecular evidence further illustrated that Cd2+ -induced

HY1 ­transcripts were higher than those of HO2, HO3, or HO4 transcripts, each of which was expressed at extremely low levels (Figure 1D). Previous results suggested that the cytoprotective roles of HO1 may be attributed to the catalytic products of its enzymatic reactions (Wagener et al., 2003; Zhang et al., 2012). We showed that co-treatment with the CO donor CORM-2 could partially rescue the inhibition of PR elongation observed

Table 1.  Metal Content of Col-0, hy1-100, and 35S:HY1-4 upon Cd2+ Stress. Ion (mg g–1 DW)

Shoots

Cd2+

Col-0

hy1-100

35S:HY1-4

Col-0

hy1-100

35S:HY1-4

Cadmium

ND

ND

ND

0.82 ± 0.13b

1.12 ± 0.19a

0.61 ± 0.12c

Iron

3.35 ± 0.13B

2.48 ± 0.08D

3.80 ± 0.15A

2.71 ± 0.18C

2.02 ± 0.17E

3.17 ± 0.21B

Cadmium

ND

ND

ND

0.47 ± 0.03AB

0.52 ± 0.07A

0.41 ± 0.08B

Iron

0.46 ± 0.07bc

0.34 ± 0.02cd

0.71 ± 0.05a

0.38 ± 0.08c

0.21 ± 0.04e

0.53 ± 0.09b

Five-day-old seedlings were transferred to liquid medium containing 0 (Control) or 50 μM CdSO4 (Cd2+) for another 5 d. In the specific metal content of root or shoot tissues, mean values with different letters denote significant differences according to multiple comparisons (P < 0.05). DW, dry weight; ND, none detected.

Figure 7.  Effects of Iron Deficiency and Cd2+ on NO Production and Primary Root Elongation. 5-day-old seedlings were transferred to liquid medium with or without Fe supply and 50 μM Cd2+. (A) After 3-d treatments, root tissues were loaded with 5 μM DAF-FM DA for 30 min. NO production was then detected by confocal laser scanning microscopy. Scale bar = 150 μm. Meanwhile, corresponding fluorescence intensity of NO (B) was also provided. The dashed line denotes the relative fluorescence intensity of the normal (left) or 50 μM CdSO4 (right) conditions, respectively. After 5-d treatments, morphology and fresh weight (C), and relative PR elongation (D) were recorded. Arrows indicate the PR position of 5-day-old seedlings of each genotype. Relative PR elongation was calculated from arrows to root tips. The grid scale of the plate is 15 mm. Mean values with different letters denote significant differences according to multiple comparisons (P < 0.05).

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Roots

Control

Han et al.  •  HY1, NO, and Iron in Cadmium Tolerance   

Figure 8.  Expression Analyses of FRO2, IRT1, HMA2, and HMA4 Genes by Real-Time RT–PCR. 5-day-old Arabidopsis seedlings were treated with or without 50 μM Cd2+ for another 1 d. Total RNA from the different seedling roots were extracted. Expression levels are presented relative to that of F-box and SAND. Mean values with different letters denote significant differences (P < 0.05) according to multiple comparisons.

and suggested that Arabidopsis HY1 contributes to the alleviation of Cd2+ toxicity. In particular, our findings highlight that HY1 may serve as a component in the complex signaling networks in response to Cd2+ exposure, salt stress (Xie et al., 2011), and light stimuli (Muramoto et  al., 1999; Gisk et  al., 2010).

HY1 Links Improved Iron Homeostasis and Reduced NO Production under Cd2+ Stress The role of the HO1/CO system in ROS scavenging has been reported in alfalfa under Cd2+ stress (Han et  al., 2008; Cui et  al., 2011). However, the mechanisms of HO1 in reducing Cd2+ accumulation is not yet clear. It was suggested that HO1 might be required for Fe assimilation from heme in mammals, pathogenic bacteria, and plants (Graziano and Lamattina, 2005; Yanatori et  al., 2010). Under our experimental conditions, we noticed that, in comparison with Col-0, hy1-100 had lower Fe levels in both root and shoot tissues regardless of the presence of Cd2+ (Table 1). The opposite trend was observed in 35S:HY1-4. The above-mentioned results suggest a functional link between HY1 and Fe deficiency. Previous reports have shown that Fe deficiency, which can be induced by Cd2+ stress, induces the generation of NO (Chen et al., 2010). NO contributes to Cd2+ toxicity and increases of the expression of genes related to Fe uptake (Besson-Bard et al., 2009) and HO1 (Noriega et al., 2007). Moreover, Cd2+ tolerance can be enhanced by the up-regulation of HO1 expression (Noriega et al., 2004; Cui et al., 2011; Noriega et al., 2012). Clearly, HY1 is a common element that links these phenomena. Many reports have indicated that the phenotypes associated with Cd2+ toxicity and the molecular responses to Cd2+ stress are similar to those of Fe deficiency. Cd2+ toxicity can at least partly be attributed to disruption of the uptake and homeostasis of essential metals, especially Fe. For example, Wu et al. (2012) concluded that chlorosis of new Arabidopsis leaves and the arrested root growth caused by Cd2+ were alleviated by increasing levels of Fe in the growth medium. Here, we showed that additional supplementation of Fe-EDTA was able to partially rescue the Cd2+-induced inhibition of PR elongation, not only in Col-0, but also in hy1-100, while to a lesser extent in 35S:HY1-4 (Figure 3A). In addition, ICP–OES results supported that plants became Fe-deficient following exposure to Cd2+. Interestingly, hy1-100 accumulated less Fe and more Cd2+ than Col-0 in both roots and shoots. The opposite effects were observed in 35S:HY1-4 (Table 1). Combined with Cd2+ hypersensitivity tests (Figures 1 and 2), these results suggest that HY1 improves Fe status, thus reducing Cd2+ accumulation in root cells. Increased NO production is an early response of roots to Fe deprivation (Graziano and Lamattina, 2007). A  recent study also provided evidence of the involvement of NO in mediating Cd2+ toxicity (Besson-Bard et  al., 2009). In the present study, we confirmed that Cd2+ increased NO production in a time-dependent manner in Arabidopsis roots (Figure  3B).

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in hy1-100 subjected to Cd2+ stress (Figure  3A). This can be explained by the fact that CO plays a role in quenching reactive oxygen species (ROS) produced by Cd2+ (Han et al., 2008). However, no such response was observed following co-treatment with BR and Cd2+. In fact, BR or BV is regarded as an antioxidant in animals and plants (Shekhawat and Verma, 2010). Therefore, Cd2+ -induced lipid peroxidation alone may not contribute directly to the inhibition of PR elongation. This explanation is supported by the fact that application of butylated hydroxyanisole (BHA), an efficient lipophilic antioxidant, suppresses the Cd2+ -induced activation of sulfur assimilation genes (Jobe et al., 2012), and could not rescue the aluminum-induced inhibition of root elongation in pea plants (Yamamoto et  al., 2001). Given the fact that GSH depletion, depletion of upstream thiols together with an enhanced oxidative state are required to induce Cd2+ -induced responses (Jobe et  al., 2012), GSH profiles of Col-0 and hy1-100 were also investigated. Using monochlorobimane (MCB) fluorescence, it was confirmed that hy1-100 exhibited lower GSH content than Col-0 when subjected to Cd2+ stress (Figure  5D and 5E). Given the important role of GSH in Cd2+ detoxification (Noctor et  al., 2012), we suggested that the mutation of HY1-induced sensitivity to Cd2+ stress might be partially explained by the decreased content of GSH in seedling roots. However, detailed relationship between GSH and HY1 during Cd2+ stress should be further investigated in the near future. Taken together, our results support previous results (Noriega et  al., 2004; Cui et  al., 2011; Noriega et  al., 2012),

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  Han et al.  •  HY1, NO, and Iron in Cadmium Tolerance

reported extensively that the competition between Cd2+ and Fe2+ leads to a reduction in the levels of intracellular Fe and initiates a Fe-starvation pathway. Regarding this context, FRO2 and IRT1 play the important roles in Fe uptake in plants (Graziano and Lamattina, 2005). The P1B-ATPases (HMAs) are membrane proteins that transport heavy metal ions across biological membranes using ATP-hydrolysis, and therefore contribute to metal homeostasis in plant cells (Argüello et al., 2007). The HMA2 and HMA4 proteins are responsible for Cd2+ efflux from the cells (Eren and Argüello, 2004; Courbot et al., 2007). In the present study, we showed that HY1 ablation amplified the expression of FRO2 and IRT1 in Cd2+ -stressed plants, whereas overexpression of HY1 decreased the expression of FRO2 and IRT1 (Figure  8). Moreover, following Cd2+ stress, levels of HMA2 and HMA4 transcripts were increased obviously in 35S:HY1-4. Meanwhile, no significant differences were observed in hy1-100 in the presence or absence of Cd2+ stress. This observation corroborates the increased Cd2+ accumulation and Fe deficiency in hy1-100 as well as decreased Cd2+ accumulation and Fe deficiency in both root (especially) and shoot tissues of 35S:HY1-4 (Table  1). These data thus underscore the involvement of HY1 in reestablishing Fe homeostasis and reducing Cd2+ content under Cd2+ stress. Together, our findings are consistent with previous reports, and demonstrated that Cd2+ favors Fe deficiency and induces NO production before inhibition of PR elongation. To maintain metal balance and detoxify heavy metals in plant cells, HY1 might act as a common link in the improved Fe homeostasis and decreased NO production in Cd2+-stressed Arabidopsis, thus alleviating inhibition of PR elongation and Cd2+ accumulation (Figure 9).

METHODS Plant Materials and Growth Conditions Arabidopsis (Arabidopsis thaliana) seeds of hy1-100 (CS236), hy1-1 (CS67), ho2 (SALK_025840), ho3 (SALK_034321), ho4 (SALK_044934), nia1/nia2 (CS2356), atnoa1 (CS6511), phyA (CS6219), and phyB (CS6211) were obtained from the Arabidopsis Biological Resource Center (ABRC). Except for hy1-1, phyA, and phyB with the ecotype Landsberg erecta (Ler) as background, all mutants were isolated from the ecotype Columbia (Col-0). Using specific primers (Supplemental Table  1), the homozygous lines were verified. Meanwhile, the binary vector pCAMBIA 1302 (AF234298) was used for Arabidopsis HY1 overexpression (Supplemental Figure 8). The homogenous HY1 overexpression lines were then used and generated according to Xie et al. (2011). Seeds were surface-sterilized and rinsed three times with sterile water, then cultured in 0.5 Murashige and Skoog (MS, pH 5.8) solid medium containing 1% (w/v) agar and 1% (w/v) sucrose. Plates containing seeds were kept at 4°C for 2 d, and then transferred into a growth chamber with 16/8-h (day/

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Whereas hy1-100 produced more NO, the opposite response was observed in 35S:HY1-4. The above results were further confirmed by EPR analysis (Figure 3C). Furthermore, the fresh NO-releasing compound SNP strongly enhanced NO production and Cd2+ toxicity in roots of all of the tested Arabidopsis lines (Figure  4). In contrast, no significant differences were observed following treatment with old SNP, except for an increase in NO level in Col-0 under Cd2+ stress. The NO scavenger cPTIO and the mammalian NOS inhibitor L-NAME differentially counteracted the Cd2+-induced NO production and subsequent PR inhibition, with more dramatic effects seen following application of cPTIO than L-NAME. Previous reports showed that Cd2+ induces NO synthesis in both roots and leaves, and neither AtNOA1 nor NR catalyzes this production (Besson-Bard et  al., 2009). However, in our system, Cd2+-induced NO production was significantly lower in atnoa1 than in Col-0 (Figure 5A and 5B). This discrepancy is likely due to the different incubation times of loading probe for the determination of NO content or various Arabidopsis tissues tested. Subsequent results (Figure  5A–5C) further support the idea that there may be other NO synthetic pathway(s) in hemin-induced Cd2+ tolerance. However, our previous results revealed that the majority of NO production in Arabidopsis is attributed to NIA/NR/NOA1 upon salinity stress. Compensatory and synergistic modes of NIA/NR/NOA1dependent NO production and HY1 expression were found in the modulation of salt tolerance (Xie et al., 2013). Recent results also showed that oleic acid (18:1) levels could regulate NO synthesis, and thereby NO-mediated signaling, by regulating NOA1 levels in Arabidopsis (Mandal et al., 2012). Therefore, the different origins of NO production caused by different stressed conditions cannot be eliminated. It was reported that DAO- and/or PAO-mediated NO biosynthesis could be one hypothesis to explain many findings in PA-mediated stress responses (Wimalasekera et al., 2011a, 2011b). Therefore, the contribution of polyamine-mediated NO production were tested in this study. Although 100  μM PAs could promote NO production (Supplemental Figure  6) and inhibit PR elongation (Figure 6A) under the control conditions, Cd2+-induced reduction of PAO and DAO activities, and total PAs content (Figure 6B–6G) suggested that polyamine oxidases may be not the enzymatic source of Cd2+-induced NO production. These results could be supported by several previous reports (Groppa et al., 2007, 2008b), both of which illustrated that DAO activity is reduced under cadmium or copper stress. The above results were also consistent with previous reports (Groppa et al., 2008a; Arasimowicz-Jelonek et al., 2011), revealing that NO contributes to Cd2+ toxicity, at least under our experimental conditions. Certainly, the functional links between HY1 and NOA1 or other NO synthetic pathway(s) should be further investigated in the future. The NO overproduction and Cd2+ -hypersensitivity phenotypes observed in hy1-100 following Cd2+ exposure were very sensitive to Fe deficiency, whereas the same two parameters in 35S:HY1-4 were relatively insensitive (Figure 7). It has been

Han et al.  •  HY1, NO, and Iron in Cadmium Tolerance   

night) regimes at 22°C and 120 μmol m–2 s–1 irradiation for the indicated time points.

Chemicals All chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA) unless stated otherwise. The old SNP solution was obtained as a negative control by maintaining a 50-μM SNP solution for at least 5 d in the light in an open tube to eliminate NO (Tossi et al., 2009).

Phenotypic Analysis

Real-Time RT–PCR Analysis Real-time PCR experiments were performed using a Mastercycler® ep realplex real-time PCR system (Eppendorf, Hamburg, Germany) with a SYBR® Premix Ex Taq™ (TaKaRa). Using specific primers (Supplemental Table 2), the expression levels of corresponding genes were calculated relative to the maximum abundance in the different samples. According to the geNorm algorithm, all values were normalized against those of two internal control genes F-box (NM_121575) and SAND (NM_128399) under the identical conditions (Supplemental Figure 4).

Western Blot Analysis Fifty micrograms of protein from homogenates were subjected to SDS–PAGE using a 12% acrylamide resolving gel (Mini Protean II System, Bio-Rad, Hertz, UK). Rabbit polyclonal antibody raised against the AtHY1 was used as primary antibody. The monoclonal anti-β-Tubulin (T5201; SigmaAldrich, USA) was presented as a loading control. The films were scanned and analyzed using Quantity One v4.4.0 software (Bio-Rad, USA).

Confocal Determination of Endogenous NO Production The endogenous NO level was detected using a TCS-SP2 confocal laser scanning microscope (Leica Lasertechnik GmbH, Heidelberg, Germany; excitation 488  nm, emission 500– 530 nm). Roots were collected at the indicated times and loaded with 5  μM 4-amino-5-methylamino-2’7’-difluorofluorescein diacetate (DAF-FM DA) (Ye et  al., 2013) in 20 mM HEPES/NaOH buffer (pH 7.5) for 30 min, then washed three times and analyzed microscopically. Results are from four representative experiments. Fluorescence was expressed as relative fluorescence units using Leica Confocal Software 2.5.

Electron Paramagnetic Resonance (EPR) Imaging of NO After different treatments, about 0.1 g Arabidopsis seedlings were crushed with a mortar and pestle, then incubated

in 0.3 ml of buffer solution (50 mM HEPES, 1 mM dithiothreitol, 1 mM MgCl2, pH 7.6) at room temperature for 2 min. The mixture was added to 0.3 ml of freshly made Fe2+(DETC)2 solution (2 M Na2S2O4 , 3.3 mM DETC, 3.3 mM FeSO4 , 3.3 g l–1 bovine serum albumin) in dark or low-light conditions (Sun et  al., 2012). After incubation at room temperature for 2 min, 0.2 ml of ethyl acetate was added to the mixture, shaken for 3 min and centrifuged at 4°C and 12  000  g for 5 min (Xu et al., 2004). The organic solvent layer was used to determine NO on a Bruker A300 spectrometer (Bruker Instrument, Germany) under the following conditions: room temperature; microwave frequency, 9.85 GHz; microwave power, 63.49 mW; modulation frequency, 100.00 kHz.

Determination of Metal Concentrations by ICP–OES Experiments Fresh samples were washed four times with 5 mM CaSO4 and 10 mM EDTA-Na2 solution and rinsed briefly in de-ionized water after treatments (Graziano and Lamattina, 2007). Afterwards, roots and shoots were cut into smaller pieces and oven-dried at 60°C separately, then digested with HNO3 using a Digital Block Sample Digestion System (LabTech ED54 DigiBlock). The metal content was determined using an Inductively Coupled Plasma-Optical Emission Spectrometer (ICP–OES, Perkin Elmer Optima 2100DV).

Ultra-High-Performance Liquid Chromatography Analysis of Polyamines Endogenous free polyamine content was determined according to the methods previously reported (Su et al., 2006; Yang et  al., 2010) with minor modification. Arabidopsis root tissues (approximately 0.06 g) were homogenized with 1 ml 5% (v/v) perchloric acid. After extraction for 1 h in an ice bath, the homogenate were centrifuged at 10  000 g for 30 min. Afterwards, the supernatant (about 750 μl) was extracted and mixed with 1.5 ml 2  M NaOH and 15  μl bezoylchloride. The mixture was vortexed and incubated for 20 min at 37°C, and then 3 ml saturated sodium chloride solution and 4.5 ml diethyl ether were added. After centrifugation (1500 g for 5 min), 2 ml ether phase was collected. Samples were evaporated to dryness and re-dissolved in 200  μl methanol (high-performance liquid chromatography grade). Standards were treated in the same way with 10 μM each polyamine in the reaction mixture. The benzoyl derivatives were separated and analyzed by an UHPLC system (Agilent 1290 Infinity, USA) equipped with an UV detector under the following conditions: 2.1 × 100 mm ZORBAX Eclipse plus C18 reverse-phase column (Agilent, USA); particle size, 1.8 μm; column temperature, 30°C; mobile phase, 60% (v/v) methanol; flow rate, 0.4 ml min–1; detected wavelength, 254 nm. The results were expressed as nmol g–1 FW.

Assays of PAO and DAO Activities Arabidopsis root tissues (approximately 0.06 g) were ground with a mortar and pestle at 4°C in 2.0 ml 0.1 M sodium phosphate buffer (pH 6.5). Homogenates were centrifuged at

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Five-day-old seedlings were transferred to 0.5 MS liquid medium containing 1% (w/v) sucrose with or without the indicated chemicals under the corresponding conditions. After treatments, seedlings were transferred to agar plates and imaged using a Canon IXUS 130 camera. Relative PR elongation (%) and fresh weight (FW; mg seedling–1) were then recorded.

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  Han et al.  •  HY1, NO, and Iron in Cadmium Tolerance

At1g69720 (HO3), At1g58300 (HO4), At1g01580 (FRO2), At4g19690 (IRT1), At4g30110 (HMA2), At2g19110 (HMA4), At3g47450 (NOA1), At1g77760 (NIA1), At1g37130 (NIA2), At5g15710 (F-box), At2g28390 (SAND), At5g08290 (YLS8), At4g05320 (UBQ10), At5g60390 (EF-1α), and At5g09810 (ACT7), At1g09570 (PHYA), At2g18790 (PHYB).

SUPPLEMENTARY DATA

10 000 g for 20 min at 4°C. Supernatants were used for PAO and DAO activities measurement. PAO and DAO activities were determined according to the method of Su et al. (2006) with minor modification. Reaction solutions (3.0 ml) contained 1.8 ml 0.1  M sodium phosphate buffer (pH 6.5), 0.9 ml crude enzyme extracts, 0.1 ml peroxidase (250 U ml–1), 0.2 ml 4-aminoantipyrine (0.1 g l–1) and N,N’dimethylaniline (0.025%). The reaction was initiated by the addition of 15 μl Spm (20 mM) for the determination of PAO activity, or 15  μl Put (20 mM) for DAO activity. 0.001∆OD550 min–1 was regarded as one enzyme activity unit (U). The results were expressed as U g–1 FW.

Determination of Glutathione by Fluorescence Microscopy The endogenous glutathione level was detected by a fluorescent microscope (Axio Imager A1, Carl Zeiss, Germany; excitation 365 nm). Roots were collected at the indicated times and loaded with 50  μM monochlorobimane (MCB; Müller et  al., 2002) in a phosphate buffer (pH 7.2) for 20 min, then washed three times and analyzed microscopically. Images of the fluorescent signal were captured using a digital camera. Results are from four representative experiments.

Statistical Analysis All data presented were the mean values of at least three independent experiments. Each value was expressed as means ± SE. Statistical analysis was performed using SPSS 16.0 software. Differences among treatments were analyzed by oneway ANOVA, taking P < 0.05 and 0.01 as significant according to multiple comparisons or t-test.

Accession Numbers The AGI locus identifier for the Arabidopsis genes described in this work are: At2g26670 (HY1), At2g26550 (HO2),

Supplementary Data are available at Molecular Plant Online.

FUNDING This work was supported by the Fundamental Research Funds for the Central Universities (KYZ201316), Scientific Innovation Research of College Graduate in Jiangsu Province (CXZZ12_0268), the National Natural Science Foundation of China (30971711), and the Priority Academic Program Development of Jiangsu Higher Education Institutions.

Acknowledgments We thank the ABRC for providing seed of SALK T-DNA insertion lines and Dr. Chuanyou Li (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, China) for providing seed of Ler-0 wild-type. No conflict of interest declared.

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Figure  9. Schematic Representation of the Proposed HY1-Mediated Cd2+ Tolerance Signaling Pathway. Cd2+ favors Fe deficiency and induces nitric oxide (NO) synthesis, thereafter contributing to primary root (PR) inhibition and Cd2+ accumulation. To keep the metal balance and detoxify heavy metals in cells, Fe and NO homeostasis is reestablished by HY1, which in turn increases glutathione (GSH) content, alleviates PR inhibition, and decreases Cd2+ content in roots. The dashed lines denote incompletely characterized pathways. T bars: inhibition.

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tolerance via increased cadmium sequestration in roots and improved iron homeostasis of shoots. Plant Physiol. 158, 790–800.

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SUMMARY  Heme oxygenase 1 (HO1) is a potential contributor of Cd2+ tolerance; however, the molecular mechanisms are largely unclear. Using genetic and molecular approaches, we revealed that Arabidopsis HY1 (AtHO1) contributes to the alleviation of Cd2+ toxicity by decreasing NO production and improving Fe homeostasis in root tissues.

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