Introduction

Metal nutrients, such as iron (Fe), manganese (Mn), zinc (Zn), and copper (Cu), are essential for normal plant growth. Fe can be readily reduced or oxidized in biochemical reactions, making it well suited for its role in redox active proteins involved in respiration, photosynthesis, and nitrogen fixation (Hell and Stephan 2003). Fe is also vital for completion of the citric acid cycle, assimilation of sulfur and nitrogen, and chlorophyll biosynthesis. Zn is an important structural component of protein domains such as the Zn-fingers found in many DNA-binding proteins, as well as enzymes such as alcohol dehydrogenase. During seed germination, drastic biological changes occur (Hoshikawa 1973). During rice seed germination, the nutrients stored in the seed are used for germination. The rice seed is composed mainly of embryo and endosperm and the metal nutrients are stored in both structures. An adequate flow of metal elements during seed germination is important for normal growth, but our understanding about the flow dynamics of these metal nutrients during rice seed germination is very limited.

Extensive regulatory cross talk is to be expected between the transition metal homeostasis network and the homeostatic systems of other nutrients (Huang et al. 2000). Maintaining availability and controlling the distribution of metal elements in a plant requires tight control of their transport across membranes and binding to organic and inorganic compounds (Finney and O’Halloran 2003). These plant metal transporter families and their biological roles have been reviewed (Pittman 2005; Williams and Mills 2005; Clemens 2006; Colangelo and Guerinot 2006; Grotz and Guerinot 2006; Hydon and Cobbett 2007; Krämer et al. 2007; Puig et al. 2007).

Synchrotron-based X-ray microfluorescenece (μ-XRE) is a technique well suited to the localization of essential elements in cells and tissues. Most inorganic element biochemistry studies rely to some extent on bulk analysis of the elements of interest. Direct chemical element imaging is often more reliable than bulk analysis because it is not affected by sample preparation, which may alter metal distribution. In addition, chemical element imaging enables us to correlate tissue distribution with biochemical functions, or alteration of these functions. Trace element analysis requires the use of a technique that has detection limits as low as a few micrograms per gram. Direct chemical element imaging has unprecedented detection limits compared to more conventional chemical imaging using electron microscopy (>100 μg/g). In addition, direct chemical element imaging can detect all elements, which is not possible using radioisotopes.

To examine metal flow during rice seed germination, we performed microarray analysis using mRNA extracted from germinating rice seeds, and found that many kinds of transporter genes involved in metal transport were strongly expressed and that their expression levels changed during seed germination. Furthermore, we examined the localization of the endogenous elements (Fe, Zn, Mn, and Cu) in rice seeds during germination using synchrotron-based X-ray microfluorescence (μ-XRF) at the Super Photon ring-8GeV (SPring-8) facility. The localization of Fe, Zn, Mn, and Cu was different from each other, and changed during seed germination. Using promoter–GUS analysis, we showed that the expression patterns of OsZIP4, a Zn transporter (Ishimaru et al. 2006), were similar to Zn localization in germinating rice seeds. Our data suggested that metal elements are dynamically mobilized to regions of embryonic growth during rice seed germination.

Material and methods

RNA extraction

Fifty ungerminated seeds and 50 seeds sown on Murashige and Skoog (MS) medium were collected and homogenized separately using MULTI-BEADS Shocker (Yasui-kikai, Osaka, Japan). RNA was isolated from fully mature and germinating seeds as described by Takaiwa et al. (1987). The RNA was purified using RNeasy mini columns (Qiagen, Tokyo, Japan) following the manufacturer’s instructions and used for microarray analysis.

Oligo DNA microarray analysis

A rice 22 K custom oligo DNA microarray kit (Agilent Technologies, Palo Alto, CA, USA), which contains approximately 22,000 oligonucleotides synthesized based on sequence data from the rice full-length cDNA project (http://cdna01.dna.affrc.go.jp/cDNA/), was used. The oligonucleotides were designed using the 3′-noncoding region of each full-length rice cDNA to detect gene-specific expression (www.agilent.com). The RNA yield and purity of the seeds and Fe-sufficient and -deficient shoots and roots were determined spectrophotometrically, and the integrity of the RNA was checked using an Agilent 2100 Bioanalyzer (Agilent Technologies). Hybridization was performed according to the manufacturer’s instructions. cDNAs were synthesized from total RNA (1 μg) and labeled with the fluorescent dye Cy3 or Cy5 using an Agilent Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies). The fluorescently-labeled targets were then hybridized to the Agilent rice 22 K oligo DNA microarrays. The hybridized microarrays were scanned using an Agilent Microarray Scanner, and extraction software (Feature Extraction version 7.1; Agilent Technologies) was used for image analysis and data extraction. For the microarray analysis, two hybridizations with reciprocally exchanged labeling dyes were performed with two independent biological samples. To detect genes whose expression levels changed during seed germination, the signal intensities from the labeled targets derived from germinating seeds 1–3 days after sowing were compared with those of fully mature seeds (0 days). The microarray results were filtered to select candidate clones with P-value log ratios of less than 0.001. A twofold expression cutoff was applied, and cases in which all four replications passed this cutoff were scored as differential expression.

Synchrotron-based X-ray microfluorescence (μ-XRE)

Oryza sativa L. cv. Nipponbare was used for synchrotron-based X-ray microfluorescence. Plants were grown on agar plates without nutrients, and harvested at 12 h, 24 h, and 36 h after sowing. The rice seeds were cut with a vertical slicer into about 100-μm sections, freeze-dried, and used for synchrotron-based X-ray microfluorescence. The in vivo μ-XRE imaging was carried out at BL37XU of the Super Photon ring-8 GeV (SPring-8) facility (Hyogo, Japan; Hokura et al. 2006). Elemental maps were obtained by scanning the samples with a 10.0-keV monochromatic beam. The X-ray beam was focused with a Fresnel Zone Plate (FZP) to a beam size of 0.8 μm (V) × 1.4 μm (H) full-width at half-maximum (FWHM), while recording the X-ray fluorescence with a Si (Li) solid-state detector. The FZP was produced by the sputtered-slice manufacturing method (Kamijo et al. 2003). The step size was set to 50 μm (for whole seed) or 10 μm (for embryo) and provided some oversampling, ensuring that no part of the target was missed. The integrated XRE intensity of each line was calculated from the spectrum and normalized to that of the incident bean, which was measured by an ionization chamber, and then the elemental map of the measured area was calculated.

Rice transformation

Genomic sequences containing the putative promoter regions of OsZIP4 (–3,000 bp to –1 bp from the translation initiation codon) were amplified from genomic DNA by the polymerase chain reaction (PCR) (Ishimaru et al. 2006). The DNA fragments of the entire promoter region were then fused upstream of the open reading frame of the uidA gene, which encodes GUS, in the pIG121Hm vector (Hiei et al. 1994). Agrobacterium tumefaciens (C58) carrying a binary vector was used to transform rice following the method of Higuchi et al. (2001).

Histochemical analysis

Histochemical assays for GUS activity were conducted according to Jefferson et al. (1987), with some modifications. First, the transgenic seeds were cut in half with a scalpel prior to incubation in the substrate solution. The plant material was fixed in 90% (v/v) acetone for 5 min and rinsed in a buffer containing 50 mM NaPO4 (pH 7.2), 0.5 mM K3Fe(CN)6, and 0.5 mM K4Fe(CN)6. GUS staining was performed using 4 mM 5-bromo-4-chloro-3-indolyl-β-glucuronide (GUS) in staining buffer [50 mM NaPO4 (pH 7.2), 0.5 mM K3Fe(CN)6, 0.5 mM K4Fe(CN)6, and 20% methanol] with vacuum infiltration for 30 min on ice, followed by incubation at 37°C in darkness for 24 h. GUS staining was observed under a substance microscope (B061; Olympus, Tokyo, Japan). Each assay was performed at least three times using more than three plant lines.

Results

Expression patterns of transporter genes involved in metal transport

Using the 22 K oligo array, expression profiles of transporter genes were analyzed during rice seed germination (Fig. 1). mRNA extracted from mature rice seeds or seeds 1, 2, and 3, days after sowing were used for oligo array analysis. We examined the expression patterns of the metal transporters, which were previously reported (reviewed in Pittman 2005; Williams and Mills 2005; Clemens 2006; Colangelo and Guerinot 2006; Grotz and Guerinot 2006; Hydon and Cobbett 2007; Krämer et al. 2007; Puig et al. 2007). First, the genes whose signal intensities during seed germination were greater than 1,000 were labeled as having a high level during this stage (Fig. 1, yellow). Second, the transcriptional levels of seeds 1 day, 2 days, and 3 days after sowing were compared with those of fully mature seeds (0 days) (Fig. 1, red, upregulated; blue, downregulated). These data made it possible to calculate the level of gene expression at each stage relative to that in fully ripened seeds, and therefore to understand the temporal changes in the mRNA levels.

Fig. 1
figure 1

Expression patterns of metal transporter genes during seed germination. Red indicates upregulation, blue indicates downregulation, and yellow indicates signal intensities greater than 1,000

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The expression levels of the zinc-regulated transporter and the iron-regulated transporter protein (ZIP) family (Eide et al. 1996; Korshunova et al. 1999; Vert et al. 2002; Ishimaru et al. 2006) tended to decrease upon germination (also observed by Nozoye et al. 2007). A member of the cation diffusion facilitator (CDF) family (Williams et al. 2000), the P1B-type ATPase family (Mills et al. 2003), the oligopeptide transporter (OPT) superfamily (Curie et al. 2001), the iron-regulated protein (IREG) family (McKie et al. 2000; Schaaf et al. 2006), IDI7, an Fe deficiency-induced ABC transporter identified in barley (Yamagichi et al. 2002), PIC1, an Fe transporter in the chloroplast (Duy et al. 2007), and the copper transporter (COPT) family (Kampfenkel et al. 1995) were strongly expressed and their expression levels tended to be upregulated during germination (Fig. 1). Rice homologs of the Ca2+-sensitive cross complementer 1 (CCC1) family (OsVIT1: Kim et al. 2006), RAN1 (AtHMA7) belonging to the P-type ATPase subfamily (Hirayama et al. 1999; Woeste and Kieber 2000), Ferric Reductase Defective 3 (FRD3), a citrate transporter of the large multidrug and toxin efflux (MATE) family (Durrett et al. 2007), PAA1 (AtHMA6), the P-type ATPase involved in Cu transfer across the chloroplast envelope (Shikanai et al. 2003), and ECA1, a member of the P-type ATPase subfamily transporting Mn2+ (Axelaen and Palmgren 2001) were not strongly expressed and showed no pronounced changes in their expression levels during germination.

To clarify what was happening during rice seed germination, we also compiled a list of the genes whose expression levels (signal intensities) were the highest in mature seeds or seeds 1–3 days after sowing (Table 1); those with the highest ratios in seeds 1 day, 2 days, and 3 days after sowing were compared to mature seeds (Table 2). A gene encoding a seed storage protein was included in the list with the highest signal intensities in the mature seeds and in seeds 1 day after sowing (Table 1). It also included the gene annotated as a Zn-induced protein. Expression of these genes decreased dramatically during seed germination. By 2–3 days after sowing, the signal intensities for several genes increased during seed germination. Metallothionein was included among these genes. Among the genes with the highest expression ratios 1 day after sowing compared to fully mature seeds were genes identified as encoding amylase as well as enzymes involved in reduction (Table 2). Three days after sowing, the genes involved in respiration and photosynthesis such as ferredoxin and chlorophyll-binding protein were identified among the genes with the highest expression ratios.

Table 1 Genes whose signal intensities were highest in mature seeds or in seeds 1–3 days after sowing
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Table 2 Genes whose expression ratios were highest in mature seeds or in seeds 1–3 days after sowing
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Fe, Zn, Mn, and Cu localization changes during rice seed germination

To examine the localization of the metal ions (Fe, Zn, Mn, and Cu) during germination, we performed μ-XRE analysis in germinating seeds prior to radicle protrusion (i.e., the germination stage). Rice seeds were removed from agar plates without nutrients and sliced 12 h, 24 h, and 36 h after sowing. The samples were freeze-dried and used for X-ray imaging. The elemental maps of each element are presented for the half-sliced rice seeds (Fig. 2) and the enlarged images of the embryo (Fig. 3). Twelve hours after sowing, Fe accumulated in the dorsal vascular bundle, aleurone layer, and the endosperm (Fig. 2). In the embryo, Fe accumulated in the scutellum facing the endosperm near the ventral vascular bundle and the vascular bundle of the scutellum 12 h after sowing (Fig. 3). Twenty-four hours after sowing, Fe was still detectable in the dorsal vascular bundle. In the embryo, Fe distribution was dispersed in the scutellum, and had accumulated in the coleoptile. Fe accumulation in the epithelium and endosperm near the scutellum was also observed. Thirty-six hours after sowing, Fe was detected in the root tips. In the embryo, Fe was observed not only in epithelium, scutellum, and coleoptile, but also in the leaf primordium and radicle.

Fig. 2
figure 2

The elemental maps of Fe, Zn, Mn, and Cu in half-sliced germinating rice seeds. The normalized X-ray fluorescence intensities are scaled from red (maximum) to blue (minimum). Each image indicates the relative distribution of the specific element, and thus the concentration scale varies for each image. sc, scutellum; en, endosperm; lp, leaf primordium; rp, root tip; ep, epithelium

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Fig. 3
figure 3

The elemental maps of Fe, Zn, Mn, and Cu in the embryo of the germinating rice seeds. The normalized X-ray fluorescence intensities are scaled from red (maximum) to blue (minimum). Each image shows the relative distribution of the specific element, and thus the concentration scale varies for each image. sc, scutellum; en, endosperm; lp, leaf primordium; ep, epithelium

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Zn was most abundant in the embryo. Zn was also distributed in the endosperm and was most abundant in the aleurone layer (Fig. 2). After sowing, Zn in the endosperm decreased compared to Zn in the embryo. In the embryo, high levels of Zn accumulated in the radicle and leaf primordium (Fig. 3). Twenty-four hours after sowing, Zn accumulation increased in the scutellum and the vascular bundle of the scutellum. In the scutellum, Zn accumulated in the endosperm similarly to Fe. After 36 h, Zn was distributed in the leaf primordium and the root tip. Zn was also detected in a specific area that was assumed to be the junction between the embryo and the dorsal vascular bundle.

Mn was accumulated in the endosperm and embryo (Fig. 2). In the embryo, Mn accumulation in the scutellum decreased after sowing, whereas accumulation in the coleoptile increased (Fig. 3). Thirty-six hours after sowing, Mn was also observed in the root tip.

Cu is also an important element for plants, but its concentration in the plant body is extremely low. Using the SPring-8 facilities, we succeeded in detecting Cu in the rice seed. Cu was detected not only in the embryo but also in the endosperm (Figs. 2 and 3). After sowing, Cu in the scutellum decreased and accumulation in the coleoptile and root were observed.

To examine the localization of the transporter gene involved in the mobilization of stored Zn during germination, promoter-GUS fusion activity was analyzed in germinating seeds. We monitored the promoter activity of OsZIP4, which encodes a Zn transporter (Fig. 4). OsZIP4 expression was observed in fully mature seeds in the bud scale, coleorhizae, vascular bundle of the scutellum, and leaf primordium (Fig. 4a). Upon germination, OsZIP4 expression was induced in the dorsal vascular bundle (Fig. 4b). In the embryo, OsZIP4 expression was strongly induced in the vascular bundle of the scutellum (Fig. 4c, d). Three days after sowing, OsZIP4 expression was storongly observed in the scutellum, the vascular bundle of the scutellum, coleoptile, and radicle (Fig. 4d).

Fig. 4
figure 4

Histochemical localization of GUS activity derived from OsZIP4 promoter-GUS transformants in fully mature seeds (a, 0 days) and seeds 1–3 days (b–d) after sowing. sc, scutellum; en, endosperm; lp, leaf primordium; rp, root tip; ep, epithelium

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Discussion

Metal transporters are involved in metal flow during rice seed germination

Using microarray analysis, we found that many transporter genes were strongly expressed and that their expression levels changed during rice seed germination (Fig. 1). These results suggested that localization of metal ions is regulated at the molecular level. Genes involved in seed protein storage and the gene annotated as the Zn-induced protein were included in the list of genes having the highest signal intensities in mature seeds and in seeds 1 day after sowing (Table 1). Note that this Zn-induced protein is also identified as the Zn-finger protein, RAMY, which binds to the α-amylase gene and is probably involved in its gibberellin-induced expression (Peng et al. 2004). Expression of these genes decreased dramatically during seed germination. This tendency suggested that genes important in the first phase of seed germination need to be downregulated during the latter stages of seed germination, and Zn may play an important role here. During the 2–3 days after sowing, the gene encoding metallothionein was upregulated. Metallothionein is reported to be involved in metal translocation (Fukuzawa et al. 2004; Zhou et al. 2006), suggesting that metal translocation is important during seed germination. One day after sowing, the genes for amylase and enzymes involved in reduction and were upregulated (Table 2). Starch degradation triggered by plant hormones was suggested to occur during this period. Since metal nutrients are important as cofactors for some enzymes, metal translocation might be important for these enzyme activities. Three days after sowing, genes involved in respiration and photosynthesis such as ferredoxin and the chlorophyll-binding protein were induced. These data suggest that proteins abundant in seeds decrease 1–2 days after sowing and biological functions such as respiration become active 3 days after sowing. Furthermore, metals, especially Fe and Zn, are suggested to be important for these activities. In our microarray results, many transporter genes thought to be involved in metal transport were expressed at high levels during rice seed germination (Fig. 1). Genes encoding ZIP family members putatively involved in Zn transport decreased during seed germination (Nozoye et al. 2007; Fig. 1). This trend was observed for the most abundant genes in mature seeds, which were downregulated during seed germination (Table 1). In the μ-XRE Zn imaging analysis, Zn accumulation in meristematic tissues was limited in the embryo (Figs. 2 and 3). A decrease in OsZIP family transcripts might be necessary for this type of partial localization of Zn. Similarly to other members of the rice ZIP family genes, OsZIP4 expression in whole seeds decreased in the 2–3 days after sowing. OsZIP4 promoter-GUS activity in the embryo, however, was strong and did not decrease during seed germination (Fig. 4). Therefore, expression in specific tissues and downregulation in other parts of the seed might be important for specific Zn translocation.

Free Fe ion is extremely toxic to plants, as it injures cells by catalyzing the generation of cellular free radicals. Therefore, small-molecule chelators have been speculated to be required for the utilization of the Fe. Mugineic acid family phytosiderophores (MAs) are natural Fe chelators that graminaceous plants secrete from their roots to solubilize Fe in the soil (Takagi 1976). As MAs have been identified in the xylem and phloem of rice and barley (Mori et al. 1991; Kawai et al. 2001), MAs may play an important role in the long-distance transport of Fe in graminaceous plants as well. Nicotianamine (NA), an intermediate in the MA biosynthetic pathway, is also thought to be involved in the long-distance transport of metal cations in the plant body (Stephan and Scholz 1993; Higuchi et al. 1996; Pich and Scholz 1996; Stephan et al. 1996; Ling et al. 1999; Takahashi et al. 2003). Moreover, NA has been suggested to play an essential role in metal translocation and accumulation in developing seeds, based on analysis of NA-deficient transgenic tobacco (Nicotiana tabacum) plants (Takahashi et al. 2003). Rice produces and secretes deoxymugineic acid (DMA), the initial compound synthesized in the MA biosynthetic pathway. We recently suggested that DMA and NA are involved in Fe transport during rice seed germination based on results from promoter-GUS and microarray analysis (Nozoye et al. 2007). We previously reported that genes not obviously induced under Fe-deficient conditions in vegetative tissues are strongly expressed during seed germination (Nozoye et al. 2007). Among the three nicotianamine synthase genes in rice (OsNAS1–3), OsNAS3, the gene least induced under Fe deficiency in roots (Inoue et al. 2003), was found to be most abundantly expressed during rice seed germination (Nozoye et al. 2007). Similar tendencies were observed for the expression of metal transporters during rice seed germination. For example, OsNramp2 and OsFRDL1 expression were strongly induced during germination, even though these genes were not induced under Fe-deficient conditions. Different regulatory mechanisms may be operating in expression during rice seed germination compared to that under nutrient-deficient conditions.

Translocation of metal nutrients (Fe, Zn, Mn, and Cu) during rice seed germination involves NA and DMA

Using μ-XRE analysis at the SPring-8 facility, we have for the first time succeeded in documenting the changes in distribution of Fe, Mn, Zn, and Cu during rice seed germination (Figs. 2 and 3). Changes in distribution showed distinct patterns between the elements. Several regulatory mechanisms were suggested to exist for metal homeostasis during rice seed germination.

Most of the Fe in fully mature rice seeds is associated with the embryo and the aleurone layer (Fig. 2); Fe distribution in the scutellum increased after sowing. Recently, we hypothesized that Fe stored in the endosperm was transported into the scutellum through the epithelium, collected into the vascular bundle of the scutellum, and then transported to the leaf primordium and seminal root based on promoter–GUS analysis of the genes involved in DMA synthesis and Fe transport (Nozoye et al. 2007). Fe localization visualized by SPring-8 in the present study strongly supports our hypothesis that DMA and NA are involved in Fe transport from the aleurone to the leaf primordium and seminal root. NA is thought to be produced because OsNAS2 is expressed in the endosperm (Nozoye et al. 2007). However, Fe concentrations were highest in the aleurone layer, but not throughout the endosperm (Fig. 2). DMA and NA also have the ability to chelate Zn, Mn, and Cu (von Wirén et al. 1999), suggesting their role in transporting these metals during rice seed germination.

Zn accumulated in the whole endosperm (Fig. 2). NA was suggested to have an important role in Zn mobilization in the endosperm. Fe is associated with ferritin or phytate in the endosperm. Zn accumulated in the endosperm is associated not only with phytate, but also with protein (Cakmak et al. 2004). The differences in metal distribution might suggest the differences of their storage form. Zn flow was quite dynamic compared to other metals analyzed. Zn stored in the endosperm was transported to the scutellum and collected in the vascular bundle of the scutellum and then transported to the seminal root and leaf primordium (Figs. 2 and 3). Zn accumulation in the junction between the embryo and the dorsal vascular bundle increased after sowing. The dorsal vascular bundle seemed to be the route of Zn transport to the embryo. Zn is the most critical micronutrient affecting protein synthesis in plants (Cakmak et al. 1989; Obata et al. 1999). In actively growing root and shoot meristematic tissues, Zn is most likely utilized in protein synthesis, membrane structure and function, gene expression, and oxidative stress tolerance (Cakmak 2000). High Zn concentrations in newly developed tissues were confirmed to be important during rice seed germination.

Mn distribution in the endosperm was similar to that of Fe (Fig. 2), and Cu distribution in the endosperm was similar to that of Zn (Fig. 2). This similarity might be related to the differences in storage forms. Following sowing, distribution of Mn and Cu had spread to the scutellum and increased in the seminal root and leaf primordium. In the scutellum, the accumulation of Mn and Cu decreased, and the accumulation in the coleoptile and root increased. The possibility exists that Mn and Cu in the embryo are actively used for growth of the coleoptile and radicle during seed germination, and flow from the endosperm to embryo was not as active compared to Fe and Zn.

In conclusion, we succeeded in visualizing the Fe, Zn, Cu, and Mn flow during rice seed germination for the first time. Fe, Zn, Cu, and Mn flow were different from each other, and NA and DMA were suggested to be involved in their flow during rice seed germination.