Type 4 metallothionein genes are involved in ... - Wiley Online Library

Plant, Cell and Environment (2012) 35, 770–789

doi: 10.1111/j.1365-3040.2011.02450.x

Type 4 metallothionein genes are involved in regulating Zn ion accumulation in late embryo and in controlling early seedling growth in Arabidopsis pce_2450

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YUJUN REN, YANG LIU, HONGYU CHEN, GANG LI, XUELIAN ZHANG & JIE ZHAO

State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan 430072, China

ABSTRACT Type 4 metallothionein (MT) genes are recognized for their specific expression in higher plant seeds, but their functions are still unclear. In this study, the functions of two Arabidopsis metallothionein genes, AtMT4a and AtMT4b, are investigated in seed development, germination and early seedling growth. Transcriptional analysis showed that these two genes are specifically expressed in late embryos. Subcellular localization displayed that both AtMT4a and AtMT4b are widespread distributed in cytoplasm, nucleus and membrane. Co-silencing RNAi of AtMT4a and AtMT4b reduced seed weight and influenced the early seedling growth after germination, whereas overexpression of these two genes caused the opposite results. Detailed analysis showed clearly the correlation of AtMT4a and AtMT4b to the accumulation of some important metal ions in late embryos, especially to Zn ion storing in seeds, which then serves as part of early Zn ion resources for postgerminated seedling growth. Furthermore, phytohormone abscisic acid (ABA) and gibberellic acid (GA) may play roles in regulating the expression and function of AtMT4a and AtMT4b during seed development; and this may influence Zn accumulation in seeds and Zn ion nutrient supplementation in the early seedling growth after germination. Key-words: ABA; AtMT4a; AtMT4b; GA.

INTRODUCTION Metallothioneins (MTs) are a class of low-molecular-weight (4–14 kDa), cysteine (Cys)-rich proteins that have a high binding affinity to metals via the thiol groups of their Cys residues (Hamer 1986). Normally, metals binding to the thiol groups account for about 10% of the molecular weight of MTs and usually play important roles in defining the physiological and biochemical functions of MTs (Kagi 1991). MTs in higher plants are classified according to the Correspondence: J. Zhao. E-mail: [email protected] This research was supported by the Major State Basic Research Program of China (2012CB944801), the Key Grant Project of Chinese Ministry of Education (311026), the Chinese 111 project (B06018) and the National Natural Science Foundation of China (30970277). 770

arrangement of Cys residues in amino acid sequences into the following four types: MT1, MT2, MT3 and MT4 (Cobbett & Goldsbrough 2002). Genes encoding all four types of MTs are found in Arabidopsis, rice (Zhou et al. 2006), tomato (Giritch et al. 1998) and sugarcane (Figueira, Kido & Almeida 2001), indicating a complex evolutionary relationship of the MT gene family before the divergence of monocots and dicots. In Arabidopsis, seven active MT genes (MT1a, MT1c, MT2a, MT2b, MT3, MT4a and MT4b) and a pseudogene (MT1b) are identified (Zhou & Goldsbrough 1995). The regulation and characteristics of plant MTs are poorly understood. A major problem encountered when studying plant MTs is their high susceptibility to proteolytic digestion and oxidation during purification (Murphy et al. 1997). Many MT genes are expressed at high levels in plant organs such as root, leaf and fruit (Matsumura, Nirasawa & Terauchi 1999; Bausher et al. 2003; Moyle et al. 2005; White et al. 2006), which suggests that MTs may have fundamental roles in plant. However, there is little evidence for specific MT functions in plants. Nevertheless, plant MTs are implicated in a variety of physiological processes and stress responses, including specific roles in the phloem (Guo, Bundithya & Goldsbrough 2003; Vilaine et al. 2003; Chatthai et al. 2004); trichome (Foley & Singh 1994; Guo et al. 2003); seeds (Kawashima et al. 1992; White & Rivin 1995; Dong & Dunstan 1996; Chyan et al. 2005); senescence (Hsieh, Liu & Huang 1995; Guo et al. 2003); response to plant hormones such as abscisic acid (ABA), gibberellic acid (GA) and kinetin (KT) (Reynolds & Crawford 1996; Charbonnelcampaa, Lauge & Combes 2000; Thomas et al. 2005; Yuan et al. 2008); and in free radical scavenging and signalling (Wong et al. 2004; Xue et al. 2009). The majority of research on plant MTs focuses on their possible roles in metal absorption/excretion or tolerance and homeostasis (Chyan et al. 2005; An et al. 2006; Yang et al. 2009). MTs expressed in plant vegetative tissues have a higher binding affinity for Cu ions than for Cd or Zn ions (Evans et al. 1992; Murphy et al. 1997); MTs are also functional Cu chelators when expressed in yeast (Ma et al. 2003; Roosens et al. 2004). However, their specific relationships with respect to metal ions in plant have not been elucidated. The expression of type 4 MTs is normally restricted to developing seeds, which is distinct from other MTs © 2011 Blackwell Publishing Ltd

Functions of type 4 MT in Arabidopsis (Guo et al. 2003; Chyan et al. 2005). The first isolated plant MT4 is the wheat EC protein (Lane, Kajioka & Kennedy 1987), then the maize Ec (White & Rivin 1995), Arabidopsis MT4a and MT4b (Guo et al. 2003), sesame MT (Chyan et al. 2005) and other MT4 genes in Douglas fir (Chatthai et al. 1997) and white spruce (Dong & Dunstan 1996) were also isolated specifically from developing seeds. Until now, at least 17 MT4 genes or protein sequences derived from 13 plant species are deposited in NCBI, but most of them have unknown functions. The best-studied MT4 is the wheat EC; it can be induced by ABA, but not by Zn2+ (Kawashima et al. 1992) even if it was initially isolated as a Zn-binding protein in embryos (Lane et al. 1987). Recent research showed that the wheat Ec predominantly binds six Zn ions in an unprecedented cluster composition; interestingly, the affinity of Ec for Zn is significantly lower than that of other MTs (Lane et al. 1987; Kawashima et al. 1992; Freisinger 2008). Micronutrients are involved in all metabolic and cellular functions. Plants have developed finely tuned mechanisms to efficiently acquire and balance the levels of essential metal ions such as Fe, Zn, Cu, Mn and Co (Puig & Peñarrubia 2009). Several of these elements such as Cu, Zn and Fe are redox active, making them essential as catalytic active cofactors in enzymes; others like Mn and Co have enzyme-activating functions, and yet others like Fe and Zn also play a structural role in stabilizing proteins (Hänsch & Mendel 2009). Zn is an essential catalytic component of enzymes for protein metabolism, gene expression, energy production, and also maintains the structural and functional integrity of biomembranes (Grotz et al. 1998; Hänsch & Mendel 2009). It was found that Zn plays a role on the lateral root development in Arabidopsis (Richard et al. 2011). Until now, more than 1200 proteins are presumed to contain, bind and transport Zn ion; these include a large number of zinc-finger proteins and transcription factors, oxidoreductases, and hydrolytic enzymes (Krämer & Clemens 2005; Hänsch & Mendel 2009). Zn deficiency is one of the most widespread micronutrient deficiencies in plants, and it causes severe reduction in crop production. In Zn-deficient plants, many physiological impairments cause inhibition of their growth, differentiation and development (Cakmak 2000). Zn also plays an important role in seed development, and Zn-deficient plants show delayed maturity (Krämer & Clemens 2005). In Arabidopsis, the AtMT4a and AtMT4b genes were previously reported as being specifically expressed in seeds (Cobbett & Goldsbrough 2002); however, the mechanisms underlying their functions remain unknown. To understand the possible roles of these two genes in Arabidopsis, we investigated their expression and distribution at the tissue and subcellular level, and observed the influence of overexpression (OE) and double-RNA interference (RNAi) lines of these two genes on the phenotypes of transgenic plants. Then we found that these two proteins may play a role in storing and distributing Zn ion in seed; the Zn stored in the seeds is subsequently used as a nutrient in the growth of the early germinated seedling, and also discovered possible

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roles of ABA and GA in regulating the expression and functions of AtMT4a and AtMT4b during seed development and early seedling growth.

MATERIALS AND METHODS Plant materials and growth condition Arabidopsis plants (Columbia ecotype) were used as test material and were grown under 16 h:8 h/light:dark period at 22–24 °C. For expression analysis of Arabidopsis MT gene family, materials including root, stem and leaf of 45-day-old plants, 15-day-old seedlings, siliques at 2 days after pollination (DAP), 4.5 DAP, 6 DAP and 9 DAP, 9 DAP silique pericarp, dry seeds, floral buds, flowers exposing petals, opening flowers and 12 DAP ovules were collected for further RNA extraction. For analysing AtMT4a and AtMT4b expressions in ABA and GA biosynthesis or catabolism-related gene mutants, we chose the ABA biosynthesis mutants of key genes ABA1 (AT5G67030; ABRC mutant CS3100; Rock & Zeevaart 1991), ABA2 (AT1G52340; ABRC mutant CS156; González-Guzmán et al. 2002), NCED6 (AT3G24220; mutant CS853988; Lefebvre et al. 2006) and AAO3 (AT2G27150; ABRC mutant CS5736; Salk_072361C; Seo et al. 2004), and ABA catabolism mutants of CYP707A1 (AT4G19230; mutant Salk_069127; Okamoto et al. 2006) and CYP707A2 (AT2G29090; mutant Salk_072410; Kushiro et al. 2004). For GA biosynthesis and catabolism mutants, the key gene GA3ox1 (AT1G15550; mutant CS6943; Mitchum et al. 2006) and GA2ox2 (AT1G30040; mutant Salk_051749C; Yamauchi et al. 2007) were selected, respectively.

Exogenous treatment For treatment with various exogenous factors, the 12 DAP siliques of wild type (WT) plants were collected and dissected to expose the ovules. The dissected siliques were washed with 1/2 Murashige and Skoog (MS) medium and submerged into the fresh medium supplemented with 200 mm ZnCl2, 100 mm CuCl2, 100 mm Fe-EDTA, 100 mm CoCl2, 100 mm CdCl2, 10 mm HgCl2, 10 mm ABA, 10 mm zeatin (ZT), 10 mm indoleacetic acid (IAA), 5 mm GA, 5 mm methyl jasmonic acid (MeJA), 100 mm NaCl, 0.1% H2O2 or 300 mm mannitol (Man) in a continuous illumination at 24 °C for 24 h. For cold or heat treatments, the dissected siliques were kept in 4 or 42 °C incubator for 24 h.

Semi- and real-time quantitative PCR analysis Total RNA was isolated from different kinds of Arabidopsis organs (root, stem, leaf, dry seed, various developmental stages of seedlings, siliques and flowers) or the above treated siliques using the TRIzol (Cat no. 15596-026) or Plant RNA Extraction Reagent (Cat no. 12322-012) guided by the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). For reverse transcription, total RNA was digested with DNase I (MBI, Fermentas, Vilnius, Lithuania)

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to remove the genome contamination, then subjected to the first stand cDNA synthesis using the ReverTra Ace-a-™ first strand cDNA synthesis kit (Cat no. FSK-100, TOYOBO). Semi-quantitative PCR analysis was adopted to examine the expression of the seven active MT genes in Arabidopsis as well as AtMT4a and AtMT4b in the OE or RNAi transgenic plants. The ubiquitously expressed AtGAPC (Glyceraldhyde-3-phosphate dehydrogenase C subunit gene, AT3G04120) was used as an internal control. The PCR procedure contained an initial 3 min denaturation step at 94 °C, followed by 30 cycles consisting of 94 °C for 20 s, 56 °C for 30 s and 72 °C for 30 s. Real-time quantitative PCR analysis was performed to examine the expression of AtMT4a and AtMT4b in the siliques of WT, transgenic plants, the above exogenous factors-treated, and ABA and GA biosynthesis and catabolism-related gene mutant plants by SYBR-green fluorescence using the Rotor-Gene Q real-time PCR machine (QIAGEN, Hilden, Genmany). The expression of ABA and GA biosynthesis or catabolism-related genes or other genes related to zinc ion binding or zinc finger transcription factor activities in the siliques and seedlings of AtMT4a and AtMT4b transgenic plants were also detected by using this method. The PCR procedure contained an initial 4 min denaturation step at 94 °C, followed by 40 cycles consisting of 94 °C for 18 s, 56 °C for 30 s and 72 °C for 30 s. The specificity of PCR amplification was checked with a heat dissociation curve (65–95 °C) after the final cycle of PCR. The primer pairs were either designed with the online primer designing tool ‘Primer-BLAST’ (http:// www.ncbi.nlm.nih.gov/tools/primer-blast/) or the Primer Premier Software (Premier Biosoft International, Palo Alto, CA, USA) to find primers specific for the respective genes in Arabidopsis.The primer pairs are listed in Supporting Information Table S1.

Construction of promoter fusion and subcellular localization vectors and microscopy analysis To obtain the promoters of AtMT4a and AtMT4b, genomic DNA fragments (~1600 and ~1400 bp) were amplified using the F/R-AtMT4aPF and F/R-AtMT4bPF primer pairs, respectively (Supporting Information Table S2). The reverse primers (R-AtMT4aPF or R-AtMT4bPF) were created for the translational fusion of the promoter with the GUS coding region in pCAMBIA1381Xb vector. After transformation of the vectors in Arabidopsis, the transgenic offsprings were selected in hygromycin B antibiotic medium, and the T3 homozygous generations were used for assay of GUS activity. The procedure for histochemical GUS staining was described by Ren & Zhao (2009). For observing AtMT4a and AtMT4b subcellular localization, the stop code-mutated (TAG→TCC) open reading frame (ORF) regions of AtMT4a and AtMT4b were cloned by using the primer pairs F/R-AtMT4aGF and F/R-

AtMT4bGF (Supporting Information Table S2), and inserted into the intermediate vector pPK100 to fuse with the eGFP expression cassette in the Nco I enzyme site. After sequencing to keep the right insertion of the fragments, the vectors were digested with EcoR I and Hind III to release the CaMV35S::AtMT4a-eGFP::NosT or CaMV35S::AtMT4b-eGFP::NosT cassette. These two cassettes were then inserted to pCAMBIA1300 (CAMBIA) to produce the final version of the subcellular localization vectors (Supporting Information Fig. S1).The existence of AtMT4a-eGFP and AtMT4b-eGFP in plant cells were verified by Western blot described by Zhang, Ren & Zhao (2008). The subcellular localization of AtMT4a-eGFP, AtMT4beGFP and the control eGFP proteins were observed in the hypocotyl cells of 7-day-old Arabidopsis seedlings by using confocal microscopy. In order to verify fluorescence distribution in the cell wall, plasmolysed hypocotyl cells were prepared in 5% NaCl. Transient experiment was performed by onion epidermal cells expression, with the method described by Kong et al. (2011).

Construction of AtMT4a and AtMT4b OE and RNAi constructs In this study, we generated the OE and RNAi transgenic plants of these two genes in Arabidopsis.When constructing the OE vectors, AtMT4a and AtMT4b ORFs were amplified with the gene-specific primer pairs F/R-AtMT4aOE and F/R-AtMT4bOE, respectively (Supporting Information Table S2), and inserted into pCBIm vector after Xba I and Sac I digestion to form the CaMV35S::AtMT4a::NosT or CaMV35S::AtMT4b::NosT cassette in the final OE vectors (Supporting Information Fig. S1). For constructing RNAi vectors, an intermediate vector pCBIm-GusIn was created by inserting a 220 bp GUS intron fragment (Supporting Information Table S2) from the vector pCAMBIA1301 (CAMBIA) into pCBIm at the enzyme site BamH I and Sal I. The AtMT4a and AtMT4b RNAi vectors were then constructed by inserting two full-length AtMT4a or AtMT4b cDNA (the ATG start code was removed; Supporting Information Table S2) into the pCBIm-GusIn vector in an inverse orientation and separated by the GUS intron linker under the control of CaMV35S promoter (Supporting Information Fig. S1).

Phenotypic analysis of transgenic plants For determination of seed germination frequencies, the freshly harvested and fully dried seeds were sterilized and sowed on 1/2MS agar medium, kept at 4 °C in dark for 48 h, and then put in a growth chamber for 48 h at 24 °C under a cycle of 16 h light and 8 h darkness. After germination, the seedling growth statuses were confirmed by detecting the length of root, hypocotyl and cotyledon, and dry seedling weight at different germination time.


Functions of type 4 MT in Arabidopsis

Prokaryotic expression of AtMT4a and AtMT4b in Escherichia coli BL21 and measurement of metal accumulation A PCR fragment containing the entire AtMT4a or AtMT4b ORF region was cloned into the Sal I/EcoR I (for AtMT4a) or Sal I/BamH I (for AtMT4b) enzyme site of the E. coli expression vector pGEX-5X-1 (Pharmacia, Fairfield, CT, USA) to produce GST-AtMT4a or GST-AtMT4b (Supporting Information Table S2; Supporting Information Fig. S1). To express the proteins and the GST control, the plasmids were transformed into E. coli BL21 (DE3). The transformed cells were diluted 1:50 for overnight culture. The cells grew into OD600 0.8 at 37 °C before expression of the recombinant proteins were induced by addition of 0.5– 1.5 mm IPTG, followed by growth at 28 °C for 2–4.5 h. The cells were harvested and lysed by sonication, as described previously (Wong et al. 2004). The proteins (GST, GSTAtMT4a and GST-AtMT4b) in the recovered supernatant were purified by batch affinity chromatography with glutathione-Sepharose 4B (Pharmacia) according to the manufacturer’s instruction. To detect the metal accumulation of GST, GST-AtMT4a and GST-AtMT4b, 2 mm CuCl2 or 1.3 mm ZnCl2 was added into medium. After 7 h of culture at 28 °C, the cells were collected and washed thoroughly with deionized water, dried at 95 °C for 12 h and then digested in 100 mL concentrated nitric acid for 12 h at 115 °C. The digested samples were diluted to 4.5 mL with deionized water, and metal contents were measured by atomic absorption spectrometry (PEAA800, Thermo, Waltham, MA, USA).

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115 °C, when nitric acid was dried out, redissolve the tissue ash to 10 mL with deionized water, and analysed by atomic absorption spectrometry (PEAA800, Thermo) (Guo, Metha & Goldsbrough 2008).

In situ Zn localization in mature seeds For Zn localization, fresh mature seeds of WT and transgenic plants were quickly frozen in liquid nitrogen and sectioned at -20 °C using a cryomicrotome. The sections were collected and dried at air condition and fixed with cold acetone for 10 min followed by washing three times with phosphate-buffered saline (PBS). After that, the sections were incubated with 0.05 mm zinquin solution for 2 h in the dark on a gyratory shaker at room temperature and then washed three times with PBS. The samples were observed under microscope using a standard UV2-A set of filters.

In vitro metal ion addition tests Seeds were germinated for 2, 4.5 and 6 d, and the seedlings were transferred to the fresh medium supplemented with 1, 4 and 9 times ZnSO4, CuSO4, their equal amount combinations, and the mixture of various metal ions (Fe, Cu, Mn, Zn, Co and Mo) compared with the concentration of the metal ions in 1/2MS medium which are 4.3 mg L-1 ZnSO4 and 0.0125 mg L-1 CuSO4. The seedlings were turned into vertical position and further cultivated for 7 d, and then the dry weight is calculated.

RESULTS Resistance of AtMT4a and AtMT4b transgenic seedlings to excessive exogenous metal ions

Temporal and spatial expression of AtMT4a and AtMT4b genes

Seeds of transgenic T3 homozygous and WT plants were germinated on horizontally placed 1/2MS agar medium for 4 d. Then the seedlings were transferred into fresh 1/2MS plates supplemented with 400 mm ZnCl2, 30 mm CuCl2, 200 mm Fe-EDTA, 50 mm CoCl2, 40 mm CdCl2 or 10 mm HgCl2 after a resistance and toxicity examination using different concentrations of the metal ions (Supporting Information Table S4). The seedlings were turned into vertical position and further cultivated for 6 d, and the root lengths were calculated. For each treatment, 30 seedlings were used for calculation, and the treatments were repeated for at least three times.

To clarify the relations of AtMT4a and AtMT4b with respect to other active AtMTs in Arabidopsis, we analysed their expressions in different tissues and organs by semiquantitative PCR (Fig. 1a). The expressions of AtMTs of types 1 to 3 were observed in almost all organs, except for weaker or no expression in dry seeds (DS). The expression of AtMT4a and AtMT4b occurred in the siliques at the torpedo to the curled embryo transition stage (ST), and was highly expressed in the dry seeds, but not in the other organs including the silique pericarps (SP). By using quantitative real-time PCR, the transcription level of AtMT4b at 12 DAP in siliques was significantly higher than that of AtMT4a (Fig. 1b). When the seeds germinated, both AtMT4a and AtMT4b expression was gradually decreased in the imbibed seeds (0.5 and 12 h), and became nearly undetectable in the germinated and growing seedlings [1–10 day after germination (DAG)] (Fig. 1c). By using the promoter-GUS reporter system (Supporting Information Fig. S1), we observed that the expression of both AtMT4a and AtMT4b was gradually enhanced from the torpedo to the curled transition embryos (6 DAP) and achieved to the highest level in the mature embryos. But there were also difference between these two gene

Determination of Zn and Cu contents in plant tissues To measure Zn and Cu contents in WT and transgenic plants, we collected the 14 DAP siliques, a vegetative tissue mix sample including root, stem and leaf, and dry seeds. The samples were washed in deionized water for 5 min three times, dried at 95 °C for 24 h, and then the dry weight of each sample was determined. Then the tissues were thoroughly digested in 1 mL concentrated nitric acid for 24 h at


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Figure 1. Expression profiles of AtMT4a and AtMT4b in Arabidopsis by semi- and real-time quantitative PCR. (a) Expression of AtMT gene family in different tissues and organs. The ubiquitously expressed gene AtGAPC (AT3G04120) was used as an internal standardized control. R, root; S, stem; and L, leaf from a 45-day-old plant; SL, 15-day-old seedling; SG, silique at globular embryo stage (2 DAP); SH, silique at heart embryo stage (4.5 DAP); ST, silique at the transition of torpedo to curled embryo stage (6 DAP); SC, silique at bent cotyledon embryo stage (9 DAP); SP, silique pericarp (9 DAP); DS, dry seed; FB, floral bud; FE, flower exposing petal; OF, opening flower. (b) Relative transcription level of AtMT4a and AtMT4b in 12 DAP siliques compared with AtGAPC. The test was replicated in three samples with at least three technological replicates. (c) Expression of AtMT4a and AtMT4b in 0.5, 12 and 24 h seed germination and 4, 6 and 10 d seedling growth. DAP, days after pollination.

expressions. AtMT4a was highly expressed in vascular tissues, while AtMT4b was predominant in the whole embryo cells (Fig. 2). No GUS activity was found in the integuments and pericarps or other tissues (mature root, stem, leaf and flower) except for 1–6 d germinated seedlings (picture not shown).

Subcellular distribution of AtMT4a and AtMT4b To investigate the subcellular distribution of AtMT4a and AtMT4b, these two genes were fused with eGFP to

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generate AtMT4a-eGFP and AtMT4b-eGFP (Supporting Information Fig. S1), and then the fusion constructs were transformed into Arabidopsis plants. To verify the existence of fusion proteins, Western blot analysis was performed using anti-GFP. The result shows that the control eGFP is about 27 kDa, while both the fusion AtMT4a- and AtMT4b-eGFP are about 35 kDa (Supporting Information Fig. S2). By confocal laser scanning microscopy (CLSM), we observed that the control eGFP was located in the cytoplasm, nucleus and membrane of transgenic hypocotyl cells (Fig. 3a–d). Interestingly, the localizations of AtMT4a- and

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Figure 2. Distribution of AtMT4a and AtMT4b in transgenic plants harbouring the promoter-GUS reporter gene (a–l). GUS expression was observed from 6 DAP embryos to the mature seeds and ovules. DAP, day after pollination. Bar is 0.5 mm in a–l. © 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 770–789


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Figure 3. Subcellular localization of AtMT4a::eGFP and AtMT4b::eGFP in hypocotyl cells of transgenic Arabidopsis. (a)–(d) eGFP control. (e)–(h) AtMT4a::eGFP. (i)–(l) AtMT4b::eGFP. (a), (c), (e), (g), (i) and (k) Fluorescence images. (b), (d), (f), (h), (j), and (l) Overlay of fluorescence image with bright-field image. (a), (b), (e), (f), (i) and (j) Unplasmolyzed cells. (c), (d), (g), (h), (k) and (l) Plasmolyzed cells. Arrowheads in (a), (e) and (i) represent nuclei; arrows in (c), (g) and (k) represent cell wall. Bar is 40 mm in all pictures.

AtMT4b-eGFP were similar to that of the eGFP control (Fig. 3e–l). We also used transient expression method in onion epidermal cell and the result was consistent with their subcellular localization in hypocotyl cells of transgenic Arabidopsis (Supporting Information Fig. S3).

Expression of AtMT4a and AtMT4b in OE and RNAi transgenic plants Due to the difficulty in obtaining mutants for AtMT4a and AtMT4b, we overexpressed or knocked down the two genes to obtain OE or RNAi transgenic Arabidopsis plants. In mature leaves and 6 DAP siliques of OE transgenic lines, both AtMT4a and AtMT4b showed extremely high expression compared with the WT control (Fig. 4a,b). The homozygous lines, AtMT4a-OE16-4, 23-2 and AtMT4bOE3-2, 11-5, were selected for further phenotypic analysis.

As AtMT4a and AtMT4b are highly similar in mRNA region, their full-length mRNA sequences were directly used in the construction of RNAi vectors for obtaining the co-silencing RNAi transgenic plants (Supporting Information Fig. S1). The expression of both AtMT4a and AtMT4b dropped sharply in RNAi plants compared with WT plants, and they displayed severe inhibition to each other in 12 DAP siliques and 12 DAP ovules (Fig. 4c). Quantitative real-time PCR in AtMT4a RNAi plants demonstrated that the expression of AtMT4a and AtMT4b are severely suppressed (Fig. 4d), and a similar result was gotten in AtMT4b RNAi plants (Fig. 4e). To confirm the expressions, we took the ovules from the siliques in 12 DAP for real-time PCR, and obtained the similar results (Fig. 4f). This result indicates that all of them are co-silencing RNAi transgenic lines. Although AtMT4 genes show less homology with other MT genes (Supporting Information Fig. S4), we still want to confirm specificity of the silencing construct in the


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Figure 4. Overexpression (OE) and RNAi of AtMT4a and AtMT4b in transgenic Arabidopsis plants. (a)–(b) Verification of AtMT4a and AtMT4b OE by semi-quantitative PCR in mature leaves (a) and 6 days after pollination (DAP) siliques (b) of wild-type (WT) and OE transgenic lines. (c) Verification of AtMT4a and AtMT4b RNAi (RN) and their suppressive effect to each other by semi-quantitative PCR in 12 DAP siliques of WT and RNAi transgenic lines. The AtGAPC (AT3G04120) gene was used as an internal standardized control. (d)–(f) Verification of AtMT4a/b co-silencing RNAi in 12 DAP siliques and ovules of the selected RNAi transgenic lines compared with the WT plants by real-time quantitative PCR. The tests were replicated in three samples with at least three technological replicates. Each number over the lanes represents one line of the transgenic plants.

AtMT4a and AtMT4b RNAi plants. RT-PCR was used to detect the expression level of other AtMT genes, including AtMT1a, AtMT1c, AtMT2a, AtMT2b and AtMT3. Compared with WT plants, all the types 1 to 3 AtMT genes have

invisible expression alteration in 12 DAP ovules and 15-day-old seedlings in the AtMT4a and AtMT4b RNAi plants (Supporting Information Fig. S5). For this reason, the homozygous lines AtMT4b-RN4-25 and 30-14 were chosen



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Figure 5. Phenotype of AtMT4a and AtMT4b overexpression and co-silencing RNAi transgenic seeds in Arabidopsis. (a) Seed shape and size in wild-type (WT) and transgenic plants. A line on a seed represents the measuring direction of a seed length. Bar is 650 mm in all pictures. (b) and (c) Comparison of seed weight and length in WT and transgenic plants under fresh harvested, dry and 12 h imbibed condition. The transgenic lines used for analysis are marked at the bottom of the figures. The number of seeds for each analysis is more than 300; the value is represented as an average of at least three replicates.

mainly for phenotypic analysis and were named AtMT4b/ a-RN4-25 and 30-14; the AtMT4a-RN15-6 and 32-7 were also chosen and renamed to AtMT4a/b-RN15-6 and 32-7.

Phenotypes of the AtMT4a and AtMT4b OE lines and co-silencing RNAi transgenic plants The AtMT4a and AtMT4b OE as well as the co-silencing RNAi yielded no observable effects on mature seed shape (Fig. 5a), but influenced the weight and length of the seeds which were increased in the OE plants and reduced in co-silencing RNAi plants (Fig. 5b,c). Both the AtMT4a and AtMT4b OE lines and co-silencing RNAi had no obvious impact on the seed germination ratio (data not shown). The root growth length was significantly higher in the OE seedlings than in the WT seedlings at 3–6 DAG seedlings, although it was much lower in the AtMT4a/b co-silencing RNAi seedlings (Fig. 6a,b). In addition, both AtMT4a, AtMT4b OE and co-silencing RNAi influenced the overall size of cotyledons, the length of the hypocotyls and the weight of the seedlings (Fig. 6b,c; Supporting Information Fig. S6). AtMT4a and AtMT4b OE apparently promoted these parameters, while co-silencing RNAi resulted in the opposite effects. The 2 DAG seedlings showed almost no weight increase compared with the dry

seeds (0 DAG), suggesting that no nutrient absorption of seedlings occurred from the culture medium at that time. However, their weight of 4.5 and 6 DAG seedlings increased more than twofold than that of the dry seeds (Fig. 6c), implying that nutrient transport and accumulation was occurring in the seedlings from the culture medium.

Responses of AtMT4a and AtMT4b to various exogenous factors In higher plants, the expression of MT genes are usually induced by excess of metal ions (Cobbett & Goldsbrough 2002); we therefore analysed the expression level of AtMT4a and AtMT4b under the treatments of ZnCl2, CuCl2, Fe-EDTA, CoCl2, CdCl2 and HgCl2, respectively. The results show that none of the genes display a significant response to metals, except for weak responses to Cd, Fe and Co ions (Fig. 7a,b). The responses of AtMT4a and AtMT4b to various phytohormones in vitro were also examined (Fig. 7a,b). ABA and MeJA strongly enhanced the expression of AtMT4a and AtMT4b, but GA and ZT resulted in an antagonistic effect and reduce their expression compared with the control. In addition, IAA decreased the expression of AtMT4b, but not AtMT4a. These results suggest that the



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Figure 6. Phenotype of AtMT4a and AtMT4b overexpression (OE) and co-silencing RNAi transgenic seedlings in Arabidopsis. (a) Morphological changes of seed germination and early seedling growth in WT and AtMT4b transgenic plants. DAG, day after germination. (b) Length of cotyledon, hypocotyl and root in 6 DAG seedlings of AtMT4b OE and AtMT4b/a co-silencing RNAi (RN) transgenic plants. (c) Weight of 0–6 DAG seeds or seedlings in AtMT4b OE and AtMT4b/a co-silencing RN transgenic plants. Significant difference between WT and transgenic plants were made by software Origin 7.5 (one-way anova test). * represents P < 0.05; ** represents P < 0.01. The values are the mean ⫾ SD from at least 200 seedlings and three independent tests.

expression of AtMT4a and AtMT4b may be regulated by hormones, especially by ABA and GA. Under some environmental stresses in vitro, Man and NaCl significantly enhanced the expression of AtMT4a and

AtMT4b, but cold decreased their expression (Fig. 7a,b). Heat and H2O2 did not influence AtMT4a expression; however, heat decreased the expression of AtMT4b, while H2O2 caused the reverse effect. These results indicate that



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HgCl2 for resistance and toxicity examination (Supporting Information Table S3). Under whatever the conditions tested, the bacteria expressing GST-AtMT4a and GSTAtMT4b showed no significant enhanced metal tolerance than the GST control (data not shown). We also detected the metal contents of E. coli strains expressing GST-AtMT4a and GST-AtMT4b in the bacterial heterologous expression system; the corresponding host bacteria were respectively grown in medium added with ZnCl2 or CuCl2, and then the metal contents were measured by atomic absorption spectrometry. It was found that host bacteria containing these two proteins showed significantly enhanced contents of Zn2+ or Cu2+ compared with the single GST protein, and metal contents in these two strains were different (Fig. 8a,b).

Changes in the Zn and Cu contents in siliques and seeds of AtMT4a and AtMT4b OE and co-silencing RNAi transgenic plants

Figure 7. Real-time quantitative PCR analysis of AtMT4a and AtMT4b expression after treatment with various exogenous factors and stresses in 12 days after pollination (DAP) siliques of Arabidopsis in vitro. (a) Response of AtMT4a to the treatments of various factors and stresses. (b) Response of AtMT4b to the treatments of various factors and stresses. The methods for treatments are described in Materials and Methods. The result listed in the chart is an average from three independent biological samples with at least three technological replicates. The values are the mean ⫾ SD. * and **: significant difference between the control and the treatment (one-way anova test proceeded by software Origin 7.5; * represents P < 0.05; ** represents P < 0.01). ABA, abscisic acid; GA, gibberellic acid; ZT, zeatin; JA, jasmonic acid; IAA, indoleacetic acid; Man, mannitol.

AtMT4a and AtMT4b may respond to some stresses during seed development and maturation.

E. coli BL21 accumulated more metal ions when AtMT4a and AtMT4b proteins are expressed As AtMT4a and AtMT4b have no significantly induced expression to metals (Fig. 7a,b), we investigated their influence on metal accumulation in the bacterial heterologous expression system. By expressing the fusion proteins in E. coli BL21, both GST-AtMT4a and GST-AtMT4b were easily recognized (picture not shown). To detect the metal tolerance of E. coli containing fusion proteins of GST-AtMT4a and GST-AtMT4b in the bacterial heterologous expression system, the corresponding host bacteria expressing these proteins were dropped on Luria Broth (LB) solid plates supplemented with different concentration of ZnCl2, CuCl2, Fe-EDTA, CoCl2, CdCl2 and

In the study, we observed the AtMT4a and AtMT4b OE and co-silencing RNAi plant growth under treatment with the excess of different metal ions, including ZnCl2, CuCl2, Fe-EDTA, CoCl2, CdCl2 and HgCl2. However, the results showed no significant growth changes compared with the untreated control (data not shown) As AtMT4a and AtMT4b have no relation to metal tolerance in Arabidopsis, we determined whether they are involved in storing metal ions in vivo. In WT plants, the Zn content in siliques and seeds was higher than in the vegetative tissues (Fig. 9a), while the Cu content in the vegetative tissues was higher than in siliques and seeds (Fig. 9b). This indicates that Zn is preferentially accumulated in siliques and seeds, whereas Cu accumulates more in vegetative tissues. In the AtMT4a and AtMT4b OE transgenic lines, both the Zn and Cu contents were significantly increased in siliques and seeds, but only the Zn ion content decreased significantly in the same tissues of AtMT4a/b co-silencing RNAi lines (Fig. 9a,b). However, in vegetative tissues, only Zn was significantly increased in the OE plants (Fig. 9a); the Cu content, however, showed no obvious changes compared with the WT control (Fig. 9b). By using the Zn-specific fluorescent probe zinquin (Coyle et al. 1994; Hendrickson et al. 2003), the Zn fluorescence was observed as much stronger in AtMT4b OE embryos but lower in AtMT4a/b co-silencing RNAi embryos than in WT ones (Fig. 9c). These results indicate that AtMT4a and AtMT4b may be involved in the Zn-storing mechanism in the embryos.

Addition of Zn improves the early AtMT4a/b co-silencing RNAi seedling growth As AtMT4a and AtMT4b are likely involved in storing Zn and Cu in Arabidopsis siliques and seeds, we questioned whether their transgenic phenotypes are related to these metal ions. To this effect, we performed the metal addition


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Figure 8. Binding affinities of AtMT4a and AtMT4b to various metal ions in prokaryotic expression. (a),(b) Binding affinities of GST, GST-AtMT4a and GST-AtMT4b to Zn2+ (a) and Cu2+ (b) when expressed in Escherichia coli BL21. The treated concentration of each metal ion in LB medium is listed in Materials and Methods. **: significant difference of metal binding affinity between GST and GST-AtMT4a or GST-AtMT4b (one-way anova test proceeded by software Origin 7.5; ** represents P < 0.01). All the examinations were done in three independent samples with three technological replicates; the results displayed an average of these tests with a standard deviation (⫾SD).

tests of different folds (1, 4 and 9 times) Zn or/and Cu or a mixture of metal microelements (Fe, Cu, Mn, Zn, Co and Mo) to assess transgenic seedling growth. In 4.5 and 6 DAG of culture, the growth of all transgenic seedlings showed no significant discrepancy. However, in 2 d of culture supplemented with either onefold of Zn, Zn and Cu or metal ion mix, the seedling growth of AtMT4a/b co-silencing RNAi lines was significantly improved (Fig. 10a,b,d), while there was no restoration of their growth under the addition of Cu ion (Fig. 10c), indicating that Zn plays a pivotal role in restoring the growth of AtMT4a/b co-silencing RNAi seedlings.

Expression of AtMT4a and AtMT4b in ABA and GA biosynthesis and catabolism-related mutants Our exogenous hormone treatment experiments showed that AtMT4a and AtMT4b respond to ABA and GA (Fig. 7). To determine whether they respond to ABA and GA in vivo, the expression of the two genes was examined in mutants of ABA and GA biosynthesis or catabolismrelated genes. In the ABA biosynthesis pathway, the genes encoding zeaxanthin epoxidase (ABA1/ZEP), shortchain dehydrogenase/reductase (ABA2/SDR1), 9-cisepoxycarotenoid dioxygenase (NCED) and abscisic aldehyde oxidase (AAO) are essential (Seo et al. 2000a,b; Audran et al. 2001; Iuchi et al. 2001; Cheng et al. 2002; González-Guzmán et al. 2002; Lefebvre et al. 2006). The ABA1/ZEP expression was ubiquitous in Arabidopsis seed tissues (Audran et al. 2001), and that of ABA2/SDR1 was strong in seed funicules and at the junction of pedicels and young siliques (Cheng et al. 2002). NCED and AAO are multigene families in Arabidopsis (Seo & Koshiba 2002); NCED6 and NCED9 are required for ABA biosynthesis

during seed development (Lefebvre et al. 2006), while among the four AAOs, AAO3 plays a major role in ABA biosynthesis in seeds (Seo et al. 2004). Mutations of these ABA biosynthesis genes lowered the ABA level in seeds (Seo et al. 2004). In this study, the AtMT4a and AtMT4b expression was significantly decreased in the 12 DAP siliques of the aba1, aba2, nced6 and aao3 mutants (Fig. 11a,c), but significantly increased in two key mutants of ABA catabolism-related genes, CYP707A1 and CYP707A2. The latter genes are highly expressed in dry Arabidopsis seeds (Kushiro et al. 2004; Okamoto et al. 2006). These results imply that the endogenous seed ABA level influences the expression of AtMT4a and AtMT4b. GAs usually play a role antagonistic to that of ABA in seed development, and are involved in maturation and germination (Seo et al. 2006; Gutierrez et al. 2007; SantosMendoza et al. 2008). In Arabidopsis, the GA level in seeds is presumably regulated by the GA biosynthesis genes GA3ox1 and GA3ox2, and a deactivation gene GA2ox2 (Yamaguchi et al. 1998; Oh et al. 2006; Seo et al. 2006; Yamauchi et al. 2007). We detected AtMT4a and AtMT4b expression in the mutant ga3ox1 and ga2ox2, as shown in Fig. 11b,d; AtMT4a and AtMT4b expression was increased in the ga3ox1 mutant but decreased in the ga2ox2 mutant. Therefore, the endogenous seed GA level influences the expression of AtMT4a and AtMT4b, but the influence is opposite to that of ABA.

Changes in Zn accumulation in seeds of ABA and GA biosynthetic and catabolism-related mutants As the endogenous ABA and GA levels influence AtMT4a and AtMT4b expression during seed development, we assessed whether they further influence the accumulation of Zn in seed. We examined the Zn content in seeds of ABA



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Figure 9. Zn and Cu accumulation in vegetative tissues, siliques, and seeds of WT, AtMT4a and AtMT4b overexpression and co-silencing RNAi transgenic Arabidopsis plants. (a)–(b) Accumulation of Zn (a) and Cu (b) in mixed mature vegetative tissues (root, stem and leaf), siliques and seeds of wild type (WT), AtMT4a and AtMT4b overexpression and co-silencing RNAi transgenic lines. All of the examinations were done in three independent samples with three technological repeats; the results display an average of these tests with a standard deviation (⫾SD). **: significant difference between WT and transgenic plants (one-way anova test proceeded by software Origin 7.5; ** represents P < 0.01). (c) In situ Zn localization in mature seeds of WT, AtMT4b overexpression and AtMT4a/b co-silencing RNAi transgenic plants. The larger pictures are the labelled Zn fluorescence images; the smaller ones are the corresponding bright-field images, respectively. The detection was done at least in 10 independent seeds of WT and transgenic plants. Bar is 200 mm in all pictures of Fig. 9.

and GA biosynthesis or catabolism-related mutants, as shown in Fig. 11e, and observed that the Zn content in the ABA biosynthetic mutants aba1, aba2, nced6, aao3 and the GA catabolic mutant ga2ox2 was lower than in the WT plants, but it was higher than in the AtMT4a/b doubleRNAi plants (AtMT4b/a-RN30-14); in the ABA catabolic mutants cyp707a1, cyp707a2 and the GA biosynthetic mutant ga3ox1, the Zn content was increased compared with the WT, but it was still lower than in the AtMT4b OE plants (AtMT4b-OE3-2).

Expression of some zinc finger transcription factor- and zinc ion-binding protein-related genes in the AtMT4a and AtMT4b transgenic plants We tracked the possible influence of Zn accumulation in AtMT4a and AtMT4b OE or co-silencing RNAi transgenic

seeds to subsequent seedling growth by examining the expression of some zinc finger transcription factor- and zinc ion-binding protein-related genes in 2-day-germinated seedlings. These genes were obtained by searching the TAIR keyword search program (http://www.arabidopsis. org/servlets/Search ? action=new _ search&type=keyword) using the keyword ‘seedling’; in the ‘growth and development stages’ category, 891 gene loci were analysed and at last 10 genes that showed relationships to Zn were selected for further expression analysis (Fig. 12). All of the selected genes showed expression changes in AtMT4a and AtMT4b OE or co-silencing RNAi seedlings. The genes U2AF35a and U2AF35b (AT1G27650 and AT5G42820, U2 small nuclear ribonucleoprotein auxiliary factor subunits a and b;Wang & Brendel 2006), GATA21 (AT5G56860, GATA transcription factor 21; Bi et al. 2005), WIP4 (AT3G20880, C2H2 type zinc finger protein; Nawy et al. 2005), CA1 and CA2 (AT3G01500 and AT5G14740, carbonic anhydrase 1 and 2; Tanz et al. 2009), and MPPB (AT3G02090,



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Figure 10. Effects of metal ion addition starting from 2 day after germination (DAG) to the restoration growth of wild type (WT), AtMT4b overexpression and AtMT4a/b co-silencing RNAi transgenic Arabidopsis seedlings. The changes of seedling growth are displayed by weight variations. The results are calculated at the seventh day after addition. (a) Effects of Zn2+ addition to the restoration of seedling growth. (b) Effects of Cu2+ addition to the restoration of seedling growth. (c) Effects of Zn2+ and Cu2+ combinational addition to the restoration of seedling growth. (d) Effects of metal ion mixture (Fe2+, Cu2+, Mn2+, Zn2+ and Co2+) addition to the restoration of seedling growth. The concentration for each metal ions is based on the original concentrations in 1/2MS medium. 1¥, the control 1/2MS medium which contains the original concentration of each metal ions; 2¥, 5¥ and 10¥, folds of metal ions supplemented to the control 1/2MS medium based on the original concentration of these metal ions in the medium. All of the experiments are repeated three times; the results display an average of these experiments with a standard deviation (⫾SD). **: significant difference between the controls (1¥) and the addition tests (one-way anova test proceeded by software Origin 7.5; ** represents P < 0.01).

metalloendopeptidase; Hajduch et al. 2010) showed significantly enhanced expression in the OE seedlings, but were decreased in expression in the co-silencing RNAi lines. In contrast, the genes OBE1 (AT3G07780, plant homeodomain finger protein; Saiga et al. 2008), VAR2 (AT2G30950, metalloprotease that functions in thylakoid membrane biogenesis; Sakamoto et al. 2003) and ZBD (AT1G23740, zinc-binding dehydrogenase family protein; Giacomelli, Rudella & van Wijk 2006) showed significantly decreased expression in the OE seedlings, but were increased in the co-silencing RNAi transgenic line.

DISCUSSION AtMT4a and AtMT4b are embryo-specific genes and have different expression patterns in embryos Although the expression of AtMT4a and AtMT4b is restricted to developing seeds (Guo et al. 2003), their specific expression and distribution were not clear. In this study, by using semi- and real-time quantitative PCR, we observed that AtMT4a and AtMT4b start to express at the



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Figure 11. Expression analysis of AtMT4a and AtMT4b in ABA and GA biosynthesis and catabolism-related mutants, and Zn accumulation in seeds of these mutants in Arabidopsis. (a) and (b) Real-time quantitative PCR analysis of AtMT4a expression in the mutants of ABA (a) and GA (b) biosynthesis and catabolism-related genes. (c) and (d) Real-time quantitative PCR analysis of AtMT4b expression in the mutants of ABA (c) and GA (d) biosynthesis and catabolism-related genes. The experiments were done in 12 DAP siliques of these mutants. The results listed in the charts are average values from three independent biological samples with at least three technological replicates. (e) Zn accumulation in seeds of ABA and GA biosynthesis and catabolism-related gene mutants. All the examinations were repeated in three independent samples, the results display average values of these experiments with a standard deviation (⫾SD). The Zn content in AtMT4b overexpression and AtMT4a/b double-RNAi transgenic seeds are used as a positive control. **: significant difference between WT and the mutants (one-way anova test proceeded by software Origin 7.5; ** represents P < 0.01). ABA, abscisic acid; DAP, days after pollination; GA, gibberellic acid; WT, wild type.

torpedo to curled embryo transition stage, with their expression gradually rising until the highest level is observed in seeds of the late bend cotyledon stage; we noted that AtMT4b shows significantly higher expression than AtMT4a (Fig. 1b). By using the promoter-GUS

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reporter system, the tissue-specific expression of AtMT4a and AtMT4b in Arabidopsis embryos was further observed. AtMT4a was much more highly expressed in vascular tissues, while AtMT4b was predominant in the whole embryo cells (Fig. 2). This result may be the first showing of different expression patterns between two AtMT4 genes. Until now, very few plant species with two or more MT4 genes in the genome have been reported (Leszczyszyn, Schmid & Blindauer 2007); in most cases, only one MT4 gene is characterized, like in rice for instance (Zhou et al. 2006). In other words, whether the finding in our study also exists in other plant species that have two or more MT4s needs to be explored further. Regardless, in our results about AtMT4a and AtMT4b expression and distribution, analysis in Arabidopsis offered an example for the similar analysis of other type 4 MTs in other plants.

AtMT4a and AtMT4b influence metal accumulation in E. coli but do not correlate with metal tolerance Several reports had demonstrated that plant MTs can bind various metal ions with different affinities in vitro (Evans et al. 1992; Chyan et al. 2005; Guo et al. 2008). The wheat EC is an example of a type 4 MT, and it was proven to have metal binding affinity to Zn2+ (Lane et al. 1987; Kawashima et al. 1992). In sesame, a seed-specific MT4, SiMT, can bind Zn and Cu ions, but not Mg ions (Chyan et al. 2005). In the Cu-sensitive yeast mutant Dcup1 and the Zn-sensitive yeast mutants Dzrc1Dcot1, all four types of Arabidopsis MTs displayed similar levels of Cu accumulation in the Dcup1 mutant, while the type 4 MTs (AtMT4a and AtMT4b) showed higher Zn accumulation than the other MTs in the Dzrc1Dcot1 mutant (Guo et al. 2008). However, other than the metal affinity analysis of these MT4s, none of them were really referred in the context of the protein functions in plants before. In this study, we first dissected the relationships of Arabidopsis AtMT4a and AtMT4b with metal ions in the bacterial heterologous expression system and in vivo, by expressing them as GST-AtMT4a and GST-AtMT4b in E. coli BL21; both fusion protein strains displayed stronger metal accumulation (including Zn2+ and Cu2+) than the GST control (Fig. 8). This result was consistent with our observation of AtMT4a and AtMT4b accumulating Zn and Cu in the Zn- and Cu-sensitive yeast mutants (Guo et al. 2008). In our study, neither AtMT4a nor AtMT4b showed properties related to metal tolerance or sensitivity in OE or co-silencing RNAi transgenic Arabidopsis plants. A possible explanation for this phenomenon may be due to the metal binding properties and the metal-thiolate cluster formation of the type 4 MTs. As in wheat, the EC protein binds a total of six divalent metal ions in two separate metalthiolate clusters. One cluster consists of four metal ions and is made up by a part of the protein containing 11 Cys residues. The second cluster features two metal ions coordinated by six Cys residues (Peroza & Freisinger 2007). These typical separated metal-thiolate clusters make EC


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Figure 12. Real-time quantitative PCR analysis of some zinc finger transcription factor- and zinc ion-binding protein-genes in AtMT4a and AtMT4b overexpression and co-silencing RNAi transgenic Arabidopsis plants. The name and locus for each gene is marked above the corresponding chart (a–j). The PCR was done in 2 d of geminated seedlings. The results listed in the chart are average values from three independent biological samples with at least three technological replicates. * and **: significant difference between wild type (WT) and the transgenic plants (one-way anova test proceeded by software Origin 7.5; * represents P < 0.05, ** represents P < 0.01). © 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 770–789

Functions of type 4 MT in Arabidopsis much more sensitive to ionic strength and pH changes than the MTs in the mammalian, cyanobacterial and other types of plant MTs, and result in implications for Zn binding thermodynamics and kinetics. The apparent binding constant of EC that was derived is lower than that of rabbit liver MT-2 (Hasler et al. 2000), and the midpoint of titration curve for the pH-dependent zinc-bound amount to EC is higher than that of other MTs (Öz, Pountney & Armitage 1998; Blindauer et al. 2002). As physiological ionic strengths tend to be higher and the pH lower than the above tested conditions, the dramatically lower Zn affinity of EC at moderate ionic strength makes it difficult to assign the role of metal resistance (Leszczyszyn et al. 2007, 2010). As AtMT4a and AtMT4b in Arabidopsis have very similar composition patterns of 17 Cys and the two critical histidines (His) to the wheat EC, the protein metal binding properties and metalthiolate cluster formation of these two MT4s may also have characteristics similar to that of EC. This may explain why AtMT4a and AtMT4b control metal accumulation but do not confer metal tolerance in Arabidopsis.

AtMT4a and AtMT4b may play cooperative roles in controlling Zn ion accumulation in seeds When using promoter-GUS fusion constructs to assay the tissue-specific expressions of AtMT4a and AtMT4b, both genes have high activities in embryos at the late stage of seed development (Fig. 2) at that time nutrients are stored in large quantities in embryos. The expression of AtMT4a in vascular tissues of embryos indicates that it may act as a transporter or as a ligand that participates in the transport of some substances. In contrast, the expression of AtMT4b in whole embryo cells indicates that it may be involved in the storage of nutrients. As AtMT4b expression in embryos is earlier and higher than that of AtMT4a (Figs 1 & 2), it is possible that AtMT4b first stores nutrient in the embryo cells, then AtMT4a distributes them into the whole embryo cells through the vascular tissues. This speculation is based on two aspects of the results observed in this study. The first is attributed to the subcellular localization characteristics of AtMT4a and AtMT4b. As both proteins localize in nearly all the organelles and cytoplasm (Fig. 3), it is possible that the embryo cells offer an adequate environment for AtMT4a and AtMT4b to store substances in the organelles and cytoplasm. The second aspect is based on the metal-binding capacities of AtMT4a and AtMT4b. As they have metal-binding affinities when expressed in E. coli BL21 (Fig. 8) and yeast cells (Guo et al. 2008), it is possible that AtMT4a and AtMT4b cooperatively bind and store metal ions in late embryos; then these ions are subsequently provided as mineral nutrients for the early seedling growth in a very short stage. The most likely metal ion that AtMT4a and AtMT4b engage in seed storage is Zn ion; this could be concluded from their Zn ion binding capacity in E. coli (Fig. 8a), and from the effects of AtMT4a and AtMT4b overexpression and co-silencing on seed Zn accumulation in transgenic plants. In AtMT4a and AtMT4b co-silencing

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RNAi seeds, Zn content drops in a significant level could visibly reduce seedling growth at the early stage in which time seeds use their own stored Zn ions to support the fast nutrient consumption shortly after seed germination if this part of Zn accumulation was deprived during seed maturation (Fig. 6). Therefore, the portion of Zn ions which is bound to AtMT4a and AtMT4b in seeds could be mainly implicated in the quick Zn supply and simulate early seedling growth. The other portion of seed Zn ion accumulation may have relationship with the ZIP proteins which define a family of metal ion transporters that are conservatively identified in plants, protozoa, fungi, invertebrates and vertebrates (Grotz et al. 1998).This type of proteins mainly function in metal ion accumulation and homeostasis in diverse organisms (Grotz et al. 1998). But whether this is true, the functional deprivation of both AtMT4a/b and the ZIP gene family in seeds needs to be studied in the future. Our result shows that AtMT4a and AtMT4b also have some affinity to Cu ions, exemplified by the binding constants (Fig. 8b) and Cu content increase in OE plants (Fig. 9b). Cu storage in embryos may play a minor role regarding the functions of AtMT4a and AtMT4b in Arabidopsis; it is because AtMT4a/b double-RNAi transgenic plants showed no significant decrease in Cu content. Also, the in vitro Cu ion addition did not improve the growth of AtMT4a/b co-silencing seedlings. Taken together, the studies on AtMT4a and AtMT4b functions indicate that they may play cooperative roles in regulating Zn accumulation in the embryos during seed development and maturation, and provide a mechanism for storing the Zn that is required for early seedling growth after germination.

ABA and GA may play roles in regulating the expression of the AtMT4a and AtMT4b genes which function in Zn accumulation during seed development Seed development and germination are crucial events in the life cycle of higher plants. In the late stage of seed development, seed maturation, storage substance accumulation and seed dormancy are induced. After imbibition, the seeds are induced to germinate (Goldberg, de Paiva & Yadegari 1994; Wobus & Webber 1999). The plant hormones ABA and GA play opposite roles in regulating these processes (Seo et al. 2006; Gutierrez et al. 2007; Santos-Mendoza et al. 2008). In developing seeds, ABA is necessary for inducing the synthesis of storage proteins and lipids for the onset of seed dormancy and for the acquisition of desiccation tolerance; it is also a negative regulator of seed germination (Finkelstein, Gampala & Rock 2002; Nambara & Marion-Poll 2003; Gutierrez et al. 2007). The endogenous ABA level reaches its peak during seed maturation and primary dormancy (Kucera, Cohn & Leubner-Metzger 2005). Endogenous GA inhibits dormancy, promotes germination and counteracts the effects of ABA (Finkelstein et al. 2002; Kucera et al. 2005). In this study, the expression of AtMT4a and AtMT4b starts from the late stage of embryos and reaches a peak value in the mature and dormancy embryos;


786 Y. Ren et al. this is concomitant with the changes of the endogenous ABA/GA ratio in seeds. In 12 DAP WT siliques treated with ABA and GA (Fig. 7a,b), both AtMT4a and AtMT4b expressions were positively up-regulated by ABA, but negatively down-regulated by GA. By further studying their expression in the 12 DAP siliques of ABA and GA biosynthesis and catabolism-related gene mutants in vivo (Fig. 11a–d), both genes were also up-regulated in the ABA catabolism- and GA biosynthesis-related gene mutants, but were down-regulated in the ABA biosynthesis- and GA catabolism-related gene mutants. However, the critical ABA and GA biosynthesis- and catabolism-related genes show no expression changes in the AtMT4a and AtMT4b overexpressors or in the co-silencing RNAi transgenic plants compared with the WT control (data not shown). These findings indicate that ABA and GA play important roles in regulating AtMT4a and AtMT4b expression in Arabidopsis. Furthermore, as the changes of AtMT4a and AtMT4b expression are consistent with the changes of ABA/GA ratio at different stages of seed development, the two gene functions may be regulated by ABA and GA. This was verified by detecting the Zn content in the mature seeds of deficient ABA and GA biosynthesis and catabolic mutants (Fig. 11e). Because the Zn content changes were consistent with the AtMT4a and AtMT4b expression changes in these mutants, this may prove that the endogenous ABA/GA ratio influenced Zn storage in seeds by regulating the expression of AtMT4a and AtMT4b. As the moderate excess or lack of Zn content in the seeds of AtMt4a and AtMT4b OE and co-silencing RNAi influence the expression of some zinc finger transcription factor- and zinc-binding protein-related genes in the early germinated seedlings (Fig. 12), AtMT4a and AtMT4b may regulate seedling growth by controlling Zn supplementation at the early germination stage. Taken together, these results suggest that ABA and GA play important roles in regulating the expression of AtMT4a and AtMT4b in Arabidopsis; the expressed genes influence the storage of Zn in seeds and provide Zn as a nutrient supplement for early seedling growth after germination.

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SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article:


Functions of type 4 MT in Arabidopsis Figure S1. Schematic diagrams of the vectors used for expression and function analysis of AtMT4a and AtMT4b in Arabidopsis. Figure S2. Western blot of GFP, AtMT4a-GFP and AtMT4b-GFP in transgenic Arabidopsis plants. Figure S3. Subcellular localization of AtMT4a-eGFP and AtMT4b-eGFP in onion epidermal cells by particle bombardment. Figure S4. Similarity of the Arabidopsis MT gene’s open reading frame. Figure S5. Expressions of AtMT1a, AtMT1c, AtMT2a, AtMT2b, AtMT3 genes in 12 d ovules and 15 d seedlings in WT and the AtMT4a/b double-RNAi transgenic plants. Figure S6. Growth status of AtMT4a overexpression and AtMT4a/b co-silencing RNAi transgenic Arabidopsis seedlings after seed germination. Table S1. Primer sequences used for semi-quantitative or real-time quantitative PCR in Arabidopsis.

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Table S2. Primer sequences used for amplification of the respective gene regions of AtMT4a and AtMT4b related to construction of the transgenic vectors in Arabidopsis. Table S3. Concentration areas of different metal ions used for detecting resistance and toxicity reactions of Escherichia coli BL21 cells expressing GST, GST-AtMT4a and GST-AtMT4b. Table S4. Concentration areas of different metal ions used for detecting resistance and toxicity reactions of AtMT4a and AtMT4b transgenic plants in Arabidopsis. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.
