HvLsi1 is a silicon influx transporter in barley

Authors


*(fax +81 86 434 1209; e-mail maj@rib.okayama-u.ac.jp).

Summary

Most plants accumulate silicon in their bodies, and this is thought to be important for resistance against biotic and abiotic stresses; however, the molecular mechanisms for Si uptake and accumulation are poorly understood. Here, we describe an Si influx transporter, HvLsi1, in barley. This protein is homologous to rice influx transporter OsLsi1 with 81% identity, and belongs to a Nod26-like major intrinsic protein sub-family of aquaporins. Heterologous expression in both Xenopus laevis oocytes and a rice mutant defective in Si uptake showed that HvLsi1 has transport activity for silicic acid. Expression of HvLsi1 was detected specifically in the basal root, and the expression level was not affected by Si supply. There was a weak correlation between Si uptake and the expression level of HvLsi1 in eight cultivars tested. In the seminal roots, HvLsi1 is localized on the plasma membrane on the distal side of epidermal and cortical cells. HvLsi1 is also located in lateral roots on the plasma membrane of hypodermal cells. These cell-type specificity of localization and expression patterns of HvLsi1 are different from those of OsLsi1. These observations indicate that HvLsi1 is a silicon influx transporter that is involved in radial transport of Si through the epidermal and cortical layers of the basal roots of barley.

Introduction

Silicon (Si) is the second most abundant element after oxygen in the earth’s crust. As silicon dioxide constitutes 50–70% of the soil mass, all plants grown in soil will contain some Si in their tissues. Silicon exerts beneficial effects on plant growth and production by alleviating both biotic and abiotic stresses, including infections, pests, lodging, drought and nutrient imbalance, in a wide variety of plant species (Fauteux et al., 2005; Ma and Yamaji, 2006, 2008; Richmond and Sussman, 2003). Recently, it has also been reported that boosting silica levels in wheat leaves reduces feeding damage by rabbits, the major vertebrate pest of cereal crops in the UK (Cotterill et al., 2007). These beneficial effects are mostly associated with the level of Si accumulation in shoots (Ma, 2004). However, among plants grown under similar conditions, the shoot Si concentration varied from 0.02 to 10.6% (Ma and Takahashi, 2002). This variation is mainly attributed to differences in Si uptake ability by the roots of various species. Three possible modes of Si uptake, namely active, passive and rejective modes, have been proposed for higher plants. Plants that actively take up Si tend to have higher Si concentrations in the xylem than found in the growth medium, those that passively take up Si have similar Si concentrations in the xylem sap and growth medium, and those that reject Si have a lower Si xylem sap concentration than that in the growth medium (Takahashi et al., 1990). However, the molecular mechanisms responsible for the various uptake modes are poorly understood.

Plant species of the Gramineae show high Si accumulation, but the accumulation extent differs between plant species (Hodson et al., 2005; Ma and Takahashi, 2002). Rice is an Si hyper-accumulator with an active mode of Si uptake. Recently, two transporters responsible for the high Si uptake capacity have been identified using rice mutants (lsi1 and lsi2) defective in Si uptake. Lsi1 is localized on the distal side of the exo- and endodermal cells and functions as an influx transporter (Ma et al., 2006). Lsi2 is located on the proximal side of the exo- and endodermal cells and functions as an efflux transporter (Ma et al., 2007a). Combination of Lsi1 and Lsi2 enables rice to transfer Si efficiently from the external solution to the xylem in the roots while overcoming the obstacle caused by Casparian bands in the exodermis and endodermis (Ma et al., 2006, 2007a). More recently, an Si transporter (Lsi6) responsible for xylem unloading was identified (Yamaji et al., 2008). This transporter is localized at the xylem parenchyma cells of leaf blades and sheaths.

Little is known about the molecular mechanism of Si uptake in plants other than rice. In the present study, we identified a gene encoding an Si transporter, HvLsi1, in barley. This transporter was characterized in terms of its expression patterns, transport activity, and intracellular localization and cell-type specificity. We found that, although both HvLsi1 and OsLsi1 function as Si influx transporters, they have different cell-type specificity of localization and expression patterns.

Results

Isolation of HvLsi1 cDNA

A partial sequence of HvLsi1 was obtained from the barley EST clone (BM372236; Gene Index Databases, http://biocomp.dfci.harvard.edu/tgi/tgipage.html) that has the highest amino acid sequence similarity to the rice influx Si transporter, OsLsi1. The full-length cDNA sequence for HvLsi1 was obtained by RACE using total RNA isolated from barley root. The cDNA is 1344 bp long and is deduced to encode a polypeptide consisting of 295 amino acids (accession number AB447482). blast search and clustal w analysis (DNA Data Bank of Japan) indicated that HvLsi1 belongs to a Nod26-like major intrinsic protein sub-family of aquaporins. The predicted amino acid sequence indicated that HvLsi1 is a membrane protein that is most similar to OsLsi1, with 81.8% identity (Figure 1). HvLsi1 has six transmembrane domains and two conserved Asn-Pro-Ala (NPA) motifs. Furthermore, four residues forming an aromatic/arginine (ar/R) selectivity filter that are thought to be important for Si transport were also conserved (Figure 1).

Figure 1.

 Alignment of HvLsi1 and OsLsi1 amino acid sequences.
Predicted transmembrane domains in HvLsi1 are underlined, and the conserved NPA motifs (NPA1 and NPA2) and the ar/R selectivity filter (H2, H5, LE1 and LE2) are boxed. Asterisks indicate the peptide sequence to which the anti-HvLsi1 antibody was raised.

Functional analysis of HvLsi1 in the heterologous system

To demonstrate whether HvLsi1 shows transport activity for silicic acid, heterologous analysis was performed in Xenopus oocytes. Injection of OsLsi1 cRNA was used as a positive control and injection of water as a negative control. The level of 68Ge-labelled silicic acid transferred into oocytes was monitored. When HvLsi1 cRNA prepared by in vitro transcription was micro-injected into the oocytes, a high transport activity for Si similar to that for OsLSi1 was observed in the oocytes (Figure 2). This result indicates that HvLsi1 has Si influx transport activity.

Figure 2.

 Transport activity for Si in Xenopus oocytes.
HvLsi1 cRNA, OsLsi1 cRNA or water were injected into Xenopus oocytes. The oocytes were exposed to 1 mm silicic acid labelled with 68Ge. After 30 min incubation, radioactivity in the oocytes was determined. Values are means ± SD of three replicates.

To further analyse the function of HvLsi1, the gene was transformed into rice mutants (lsi1-3) using a construct carrying the HvLsi1 cDNA under the control of the rice Lsi1 promoter (Lsi1P–HvLsi1). lsi1-3 is a newly isolated mutant from M3 seeds of rice (cv. Nipponbare) irradiated with γ-rays. This mutant is allelic to the lsi1 mutant reported previously (Ma et al., 2001), and has a single nucleotide deletion 202 bases from the ATG codon (Figure S1), resulting in a frame shift in OsLsi1. The lsi1-3 mutant was used for this experiment because its background (cv. Nipponbare) gives a better yield in transformation experiments.

First, the localization of HvLsi1 in the transgenic plant roots was investigated by immunostaining using both anti-HvLsi1 and anti-OsLsi1 antibodies. HvLsi1 protein was localized at the exodermis and endodermis of transgenic plants carrying an Lsi1P–HvLsi1 construct, but not in those transformed with an empty vector (control) (Figure 3a,b). Furthermore, HvLsi1 showed polar localization at the distal side of both exodermis and endodermis cells. This localization pattern is the same as that of OsLsi1 in wild-type rice (Ma et al., 2006). When the roots of transgenic plants were stained with anti-OsLsi1 antibody, no signal was observed (Figure 3c,d). This result indicates that there is no cross-reaction between HvLsi1 and OsLsi1 antibodies.

Figure 3.

 Cell-type specificity of localization of HvLsi1 in the rice mutant lsi1-3.
A construct comprising HvLsi1 cDNA controlled by the OsLsi1 promoter (Lsi1P–HvLsi1) or an empty vector (EV) were individually introduced into the rice mutant lsi1-3 that is defective in Si uptake. Immunostaining by anti-HvLsi1 antibody (a, b) and anti-OsLsi1 antibody (c, d) was performed using these transgenic rice plants. Scale bars = 50 μm.

The expression of HvLsi1 and OsLsi1 and the Si uptake by the roots were compared among three independent transgenic plants carrying an Lsi1P–HvLsi1 construct and control plants with an empty vector. Wild-type rice and the lsi1-3 mutant were used as positive and negative controls, respectively. Expression of HvLsi1 was only observed in the Lsi1P–HvLsi1 plants (Figure 4a). The expression level of HvLsi1 in the transgenic plants varied to some extent, probably due to the positional effect of the trans-gene (Figure 3a). The lsi1-3 mutant showed low Si uptake, but the Si uptake was significantly increased in plants carrying Lsi1P–HvLsi1 (Figure 4b). Furthermore, the Si uptake by transgenic plants was similar to that by wild-type rice (Figure 4b). These results clearly indicate that HvLsi1 is able to complement OsLsi1 as an Si influx transporter. Interestingly, no correlation was observed between the HvLsi1 expression level and the Si uptake capacity in the transgenic lines (Figure 4a,b).

Figure 4.

 Complementation test of HvLsi1 in the rice mutant lsi1-3.
(a) Relative expression levels of HvLsi1 were determined by real-time RT-PCR in three independent transgenic lines and vector control lines. Wild-type (WT; cv. Nipponbare) and the lsi1-3 mutant were used as controls. The HvLsi1 levels were normalized to that in transgenic plant Lsi1P–HvLsi1 19.
(b) Si uptake experiment performed in the nutrient solution containing 0.5 mm Si for 24 h. Values are means ± SD of three replicates.

Expression pattern of HvLsi1 and Si uptake

The expression pattern of the HvLsi1 transcript was investigated in barley roots and shoots using real-time RT-PCR. HvLsi1 mRNA was expressed specifically in the roots (Figure 5a). Furthermore, higher HvLsi1 expression was observed in the basal zone (15–30 mm) than the root tip zone (0–15 mm) (Figure 5b). To link the spatial expression of HvLsi1 and Si uptake, we compared Si uptake in two root zones using a compartment transport box (Ma et al., 2001). In contrast to the expression pattern (Figure 5a), uptake was higher in the root tip zones (0–15 mm) than in the basal zone (15–30 mm) (Figure 5c). The effect of Si supply on expression of root HvLsi1 was also examined. Continuous Si supply for up to 7 days did not affect the expression of HvLsi1 (Figure 6).

Figure 5.

 Expression pattern of HvLsi1 and spatial uptake of Si.
(a) Roots and shoots were harvested from 2-week-old seedlings of barley. Relative HvLsi1 expression levels were determined by real-time RT-PCR. The Actin mRNA level was used as an internal control.
(b) Relative HvLsi1 mRNA levels in two root zones of 5-day-old seedlings were measured by real-time RT-PCR. The Cyclophilin (CyP) mRNA level was used as an internal control.
(c) Si uptake by the two root zones. Either the root tip (0–15 mm) or the basal root (15–30 mm) was exposed to nutrient solution containing 2 mm silicic acid in the multi-compartment transport box. The Si concentration in the fourth compartment that included the cut end of the root was measured over the time course as indicated. Values are means ± SD of three replicates.

Figure 6.

 Effect of Si on the expression of HvLsi1.
Seedlings were exposed to nutrient solution with or without 1 mm Si. On various days, the roots were sampled and the expression level was determined using real-time RT-PCR. Values are means ± SD of three replicates.

Genotypic variation in HvLsi1 expression and Si uptake

To investigate the relationship between the HvLsi1 expression and Si uptake by the roots, eight barley cultivars were tested. The Si uptake differed between cultivars (Figure 7a). The expression level of HvLsi1 in the roots also varied among these cultivars (Figure 7b). However, there was a weak correlation between the HvLsi1 expression level and the Si uptake (correlation coefficient = 0.696, < 0.05, Figure 7c).

Figure 7.

 Genotypic variation of Si uptake and expression of HvLsi1.
(a) Si uptake by eight barley cultivars (GP, Golden Promise; SV183; Haruna, Haruna-Nijo; BCUS126; SV75; SV119; BCUS93; SV92). Seedlings (14 days old) were exposed to 0.5 mm Si and allowed to take it up for 24 h.
(b) Expression of HvLsi1 in the roots of the various cultivars.
(c) Relationship between Si uptake by the roots and the expression of HvLsi1. The correlation coefficient (r) and P-value were calculated using a standard formula. Values are means ± SD of three replicates.

Intracellular localization and cell-type specificity of HvLsi1

To determine the intracellular localization of HvLsi1, an N-terminal GFP fusion of HvLsi1 (GFP–HvLsi1) was introduced into onion epidermal cells by particle bombardment. GFP fluorescence from fused GFP–HvLsi1 was observed on the outer layer of cells, but the signal from GFP alone was observed both in the cytoplasm and nucleus (Figure 8a,b). In order to distinguish between localization in the cell wall or the plasma membrane, plasmolysis was induced by adding 1 m mannitol, and the cell wall was stained with propidium iodide. GFP fluorescence of GFP–HvLsi1 in the plasmolysed cells clearly indicated that HvLsi1 is localized exclusively on the plasma membrane (Figure 8c).

Figure 8.

 Subcellular localization of HvLsi1.
A construct carrying GFP–HvLsi1 was introduced into onion epidermal cells by particle bombardment. GFP was used as a control. The upper panels show GFP fluorescence (a–c), and the lower panels show bright-field images (d–f). Images of the plasmolysed cell transformed with GFP–HvLsi1 are shown in (c) and (f). The red signal in (c) indicates cell walls counter-stained using propidium iodide. Scale bars = 50 μm. Arrowheads indicate nuclei.

In barley, immunostaining with a rabbit anti-HvLsi1 polyclonal antibody showed that HvLsi1 was localized in the epidermal and cortical cells in the basal part of the seminal roots (Figure 9a). The signal was barely observed in the root tip region (Figure 9b). This result is consistent with the expression data of HvLsi1 in two root regions (Figure 5b). In the lateral roots, HvLsi1 was only localized at the hypodermis (the outermost layer of the cortex cells) (Figure 9c). Staining with pre-immune serum did not show any signal (Figure 9d–f). Interestingly, in both seminal and lateral roots, HvLsi1 showed polar localization at the distal side of the cells (Figure 9a,c).

Figure 9.

 Cell-type specificity of localization of HvLsi1 in barley root.
(a–c) Immunostaining with anti-HvLsi1 antibody was performed using the basal and root tip regions of seminal roots and the lateral roots of barley (cv. Golden Promise).
(d–f) Pre-immune serum was used as a negative control (NC).
Scale bars = 50 μm.

Discussion

Previous results indicate that the Si uptake kinetics in barley follow a typical Michaelis–Menten curve, and uptake was inhibited by both 2,4-dinitrophenol and HgCl2, suggesting that barley actively takes up Si (Nikolic et al., 2007). However, the transporters involved in Si uptake had not been identified in barley. In the present study, we used sequence homology with a previously identified Si influx transporter in rice, OsLsi1, to identify HvLsi1 in barley (Figure 1). Heterologous expression in both Xenopus oocytes and a rice mutant showed that HvLsi1 functions as an Si influx transporter similar to OsLsi1 (Figures 2 and 3). However, the cell-type specificity of localization and expression response to Si differ between OsLsi1 and HvLsi1.

HvLsi1 is localized in epidermal cells and all cortical cells in the seminal roots and in hypodermal cells (corresponding to the exodermis in rice) in the lateral roots (Figure 9a,c). In contrast, OsLsi1 is localized at the exodermis and endodermis of all roots types, including seminal, lateral and crown roots (Ma et al., 2006; Yamaji and Ma, 2007). This difference may be attributed to the distinct root structures observed in rice and barley. Rice has Casparian Strips in the exodermis which usually obstruct a possible apoplastic pathway to the endodermis. Another structural difference is the development of aerenchyma, a tissue containing enlarged gas spaces in the cortex layer. Aerenchyma formation is required for rice due to the vital importance of internal aeration of roots in paddy fields (Lux et al., 2004). Therefore, in rice, Si is taken up by OsLsi1 at the exodermis first and is then released to the aerenchyma (apoplastic space). After that, Si has to be taken up again by OsLsi1 at the endodermis to reach the stele. In contrast, barley lacks both aerenchyma and Casparian strips on the exodermis; therefore, Si may be transported into the symplast from any cells in the epidermis and cortex layer via the Hvlsi1 transporter.

Both OsLsi1 and HvLsi1 are mainly expressed in roots (Figure 5a) (Ma et al., 2006). However, the expression of OsLsi1 was decreased by Si supply, whereas that of HvLsi1 was not (Figure 6). This suggests that the mechanism regulating expression of Si influx transporter genes differs between rice and barley. Rice accumulates more Si than barley, but it is not clear whether the accumulation extent is related to regulation of expression of the Si transporter gene.

Spatial expression analysis in the roots showed that HvLsi1 is expressed more in the basal region than in the root tip region (Figure 5b). Immunostaining showed the same pattern (Figure 9a,b). This expression pattern of HvLsi1 is similar to that of OsLsi1 in rice (Yamaji and Ma, 2007). However, the contribution of HvLsi1 to the Si uptake differs between barley and rice. In rice, Si was taken up more in the basal region of the roots than in the root tip region, in accordance with the expression pattern of OsLsi1 (Yamaji and Ma, 2007). In contrast, in barley, higher uptake was observed in the root tip region (Figure 5c). This result suggests that other unidentified transporters are involved in Si uptake in the root tip region in barley. Identification of these transporters will facilitate understanding of the molecular mechanism of Si uptake in barley and the differences in Si uptake mechanisms between barley and rice.

Polar localization at the distal side of epidermal and cortical cells of both seminal and lateral roots indicates that HvLsi1 functions as an influx transporter (Figure 9a,c), similar to OsLsi1 (Ma et al., 2006). Interestingly, when HvLsi1 was expressed in a rice mutant, it also showed polar localization in rice roots (Figure 3a). This suggests that the mechanism controlling polar localization is similar between rice and barley, although the mechanisms involved remains to be determined.

The level of HvLsi1 expression in the rice mutant was not correlated with the level of Si uptake, suggesting that the minimum expression level observed in the Lsi1P–HvLsi1 plants was more than sufficient to maintain maximum Si uptake in rice (Figure 4a,b). In fact, the line with the lowest HvLsi1 expression level showed similar Si uptake to the wild-type rice (Figure 4b).

It has been reported that there is genotypic variation in Si accumulation in the barley grain. The Si concentration in the grain ranged from 0 to 3800 mg kg−1, depending on the cultivar. This genotypic variation may be associated with many factors, but the major one is the Si uptake capacity of the roots (Ma, 2003). In rice, cultivars with lower Si uptake tended to have lower expression of OsLsi1 (Ma et al., 2007b). In the present study, we examined the relationship between Si uptake and the expression of HvLsi1. Genotypic variation in Si uptake by the roots was observed, but there is only a weak correlation between the Si uptake and the expression of HvLsi1 (Figure 7). These results suggest that HvLsi1 expression is differently regulated in response to Si application, and/or the function of HvLsi1 differs among the cultivars. Alternatively, other transporters might be responsible for the genotypic variation in Si uptake. In fact, another transporter of Si (Lsi2) has been identified in rice. This transporter is responsible for actively releasing Si from of the cells to the xylem. The expression level of this gene is also correlated with Si uptake in rice (Ma et al., 2007b). We have identified an EST homologous to OsLsi2 in barley. It will be interesting to examine whether expression of this gene is responsible for the genotypic variation in Si uptake of barley cultivars.

In conclusion, our results clearly demonstrate that HvLsi1 functions as an influx transporter in barley roots that is responsible for transport of Si from the external solution to the root cells. Although its function in transporting Si is similar to that of OsLsi1 in rice, the cell-type specificity of localization and expression pattern differ between HvLsi1 and OsLsi1. Identification and characterization of additional transporter(s) involved in Si accumulation in barley will enable a general understanding of the Si uptake mechanisms in plants, as well as development of a genetic modification strategy to enhance Si accumulation in various species.

Experimental procedures

Plant materials and growth conditions

Barley (Hordeum vulgare L.) cultivars Haruna-Nijo and Golden Promise were used for all experiments. Seeds were soaked in de-ionized water for 2 h, and then incubated overnight on a moist paper in the dark at 20°C. Germinated seeds were transferred to a nylon mesh floating on a continuously aerated solution containing 0.5 mm CaCl2 (pH 5.6) in the dark. Seedlings were then transferred into aerated one-fifth Hoagland’s solution (pH 6.0) containing 1 mm KNO3, 1 mm Ca(NO3)2, 1.4 mm MgSO4 and 0.2 mm KH2PO4, and micronutrients comprising 20 μm Fe-EDTA, 3 μm H3BO3, 1.0 μm (NH4)6Mo7O24, 0.5 μm MmCl2, 0.4 μm ZnSO4 and 0.2 μm CuSO4. All plants were grown in a growth chamber at 20°C.

Molecular cloning of HvLsi1

The full-length HvLsi1 cDNA was generated by the RACE method from total RNA prepared from barley root (SMART™ RACE cDNA amplification kit; Clontech, http://www.clontech.com/) using primers designed according to the EST information in the Gene Index Databases. The entire HvLsi1 cDNA was subcloned into the pGEM-T Easy vector (Promega, http://www.promega.com/) and the sequence was determined using a Big-Dye sequencing kit (Applied Biosystems, http://www.appliedbiosystems.com/) with universal and gene-specific primers on an ABI PRISM 310 Genetic Analyzer (Applied Biosystems).

Si influx transport activity assay

The ORF of HvLsi1 cDNA was amplified by RT-PCR using primers 5′-GAAAGATCTATGGCCAGCAACTCGAGATCGAAC-3′ and 5′-GAAAGATCTTCAGACGGGGATGTGGTCGAGCTC-3′. The fragment containing the ORF was inserted into the BglII site of a Xenopus oocyte expression vector, pXbG-ev1 (Preston et al., 1992). cRNA preparation, micro-injection into oocytes and Si influx transport activity assay were performed as described previously (Mitani et al., 2008).

Transformation of HvLsi1 into the rice mutant

The promoter region of OsLsi1 (1.6 kb) was amplified from rice genomic DNA using primers 5′-ATAGATCTTCAGATGATATGTCGATTTCTG-3′ and 5′-TTTCTAGAGCTACTTGTGCAGCACGCG-3′. The ORF of HvLsi1 and the nopaline synthase terminator region was amplified from pGFP-HvLsi1 described below by PCR using primers 5′-GCTCTAGAATGGCCAGCAACTCGAGATCGAAC-3′ and 5′-GCTCTAGAGATCTAGTAACATAGATGACACC-3′. These two clones were inserted into a vector (pPZP2H-lac) to produce pLsi1P-HvLsi1. The resulting plasmid was transformed into Agrobacterium tumefaciens (strain EHA101), and then transferred into calluses derived from the rice mutant lsi1-3 using an Agrobacterium-mediated transformation system. lsi1-3 was isolated from M3 seeds of the rice cultivar Nipponbare irradiated with γ-rays as described previously (Ma et al., 2001). The Si uptake experiment and gene expression analysis were performed with three independent transgenic lines, vector control lines, wild-type rice and the mutant. The uptake experiment was conducted in a nutrient solution containing 0.5 mm Si as silicic acid for 24 h. Immunostaining was also performed using both anti-HvLsi1 and anti-OsLsi1 antibodies as described below.

Expression pattern

Barley roots and shoots were sampled and subjected to RNA extraction as described below. Expression of HvLsi1 in various tissues was examined by RT-PCR. To investigate the effect of Si supply on gene expression, seedlings (14 days old) were cultured in one-fifth Hoagland’s solution containing 0 or 1 mm silicic acid. At days 0, 1, 4 and 7, the roots were sampled and subjected to RNA extraction and quantitative analysis of gene expression as described below. The expression level in two root regions (0–15 and 15–30 mm) was also examined similarly. All experiments were replicated three times.

Multi-compartment transport box experiment

Regional differences in Si uptake were examined using a multi-compartment transport box as described previously (Ma et al., 2001) with several modifications. The first and second compartments were separated by 20 mm width, and the third and fourth were separated by 10 mm width. Ten excised roots from 5-day-old seedlings were placed in a box such that the root tip (approximately 0–15 mm) is placed in the first compartment or the basal root (approximately 15–30 mm) in a second compartment. Nutrient solution containing 2 mm silicic acid was applied to the first or second compartment, and the amount of Si exuded from the cutting end in the fourth compartment was determined as described previously (Ma et al., 2001).

Genotypic variation in Si uptake

Eight cultivars including Golden Promise, SV183, Haruna-Nijo, BCUS126, SV75, SV119, BCUS93 and SV92 were used for the Si uptake experiment. Seedlings prepared as described above were exposed to a nutrient solution containing 0.5 mm Si as silicic acid with three replicates. After 24 h, the Si uptake was determined as described previously (Mitani and Ma, 2005). The roots for each cultivar were also sampled for RNA extraction and quantitative analysis of gene expression.

RNA extraction and quantitative real-time RT-PCR

Total RNA was extracted from frozen plant samples using an RNeasy plant mini kit (Qiagen, http://www.qiagen.com/), and converted to cRNA followed by DNase I treatment with SuperScript II (Invitrogen, http://www.invitrogen.com/) as described by the manufacturer. Specific cDNAs were amplified using SYBR Premix EX Taq™ (Takara, http://www.takara-bio.co.jp) and real-time RT-PCR (ABI Prism 7500; Applied Biosystems) with the following primer pairs: 5′-TTATGCGTGTGCGTGTGTGT-3′ and 5′-TGAACAGAGCGAGAGAGAGCA-3′ for HvLsi1, 5′-GACTCTGGTCATGGTGTCAGC-3′ and 5′-GGCTGGAAGAGGACCTCAGG-3′ for Actin, 5′-CCTGTCGTGTCGTCGGTCTAAA-3′ and 5′- ACGCAGATCCAGCAGCCTAAAG-3′ for Cyclophilin, and 5′-CGGTGGATGTGATCGGAACCA-3′ and 5′-CGTCGAACTTGTTGCTCGCCA-3′ for OsLsi1.

Construction and transient expression analysis of a GFP–HvLsi1 fusion

The ORF of the HvLSi1 cDNA fragment was amplified using primers 5′-GATGTACAAGATGGCCAGCAACTCGAGATCGAAC-3′ and 5′-GATGTACAGTCAGACGGGGATGTGGTCGAGCTC-3′. The HvLsi1 fragment was ligated to the 3′ end of GFP and placed under the control of the CaMV 35S promoter in pBluescript SK- (Stratagene, http://www.stratagene.com). The resulting plasmid was designated pGFP-HvLsi1. Gold particles with a diameter of 1 μm, coated with pGFP-HvLsi1 or GFP control, were introduced into onion epidermal cells using particle bombardment (PDS-1000/He particle delivery system, Bio-Rad, http://www.bio-rad.com/) using 1100 psi pressure disks. To induce plasmolysis, cells were incubated with 1 mm mannitol for 10 min. GFP fluorescence was observed using an Axio imager with Apotome (Carl Zeiss, http://www.zeiss.com/) with appropriate filters.

Immunohistological staining

Antibody against HvLsi1 was prepared by immunizing rabbits with synthetic peptide C-SVAADDDELDHIPV (positions 282–295 of HvLsi1). Antibody against OsLsi1 has been described previously (Yamaji and Ma, 2007). HvLsi1 immunostaining was performed in both seminal and lateral roots as described previously (Murata et al., 2006) with minor modifications. The sliced section was 100 μm thick, and an Axio imager with Apotome (Carl Zeiss) was used for observation.

Acknowledgements

This work was mainly supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (BRAIN). The work was also supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (number 18380052 to J.F.M.) and by a Sunbor grant and the Ohara Foundation for Agricultural Science. We thank Kazuhiro Sato for providing barley seeds and Jared Westbrooks (The University of Florida) for critical reading.

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