Plant Cell Illumina, Inc.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

First published online July 2, 2009; 10.1105/tpc.109.067884

The Plant Cell 21:2133-2142 (2009)
© 2009 American Society of Plant Biologists

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow PPT slides of all figures
Right arrow Supplemental Data
Right arrow All Versions of this Article:
21/7/2133    most recent
tpc.109.067884v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via CrossRef
Right arrow Citing Articles via Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mitani, N.
Right arrow Articles by Ma, J. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mitani, N.
Right arrow Articles by Ma, J. F.
Agricola
Right arrow Articles by Mitani, N.
Right arrow Articles by Ma, J. F.

Identification and Characterization of Maize and Barley Lsi2-Like Silicon Efflux Transporters Reveals a Distinct Silicon Uptake System from That in Rice[W]

Namiki Mitani1, Yukako Chiba1,2, Naoki Yamaji and Jian Feng Ma3

Research Institute for Bioresources, Okayama University, Kurashiki 710-0046, Japan

3 Address correspondence to maj{at}rib.okayama-u.ac.jp.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 METHODS
 REFERENCES
 
Silicon (Si) uptake has been extensively examined in rice (Oryza sativa), but it is poorly understood in other gramineous crops. We identified Low Silicon Rice 2 (Lsi2)-like Si efflux transporters from two important gramineous crops: maize (Zea mays) and barley (Hordeum vulgare). Both maize and barley Lsi2 expressed in Xenopus laevis oocytes showed Si efflux transport activity. Furthermore, barley Lsi2 was able to recover Si uptake in a rice mutant defective in Si efflux. Maize and barley Lsi2 were only expressed in the roots. Expression of maize and barley Lsi2 was downregulated in response to exogenously applied Si. Moreover, there was a significant positive correlation between the ability of roots to absorb Si and the expression levels of Lsi2 in eight barley cultivars, suggesting that Lsi2 is a key Si transporter in barley. Immunostaining showed that maize and barley Lsi2 localized only at the endodermis, with no polarity. Protein gel blot analysis indicated that maize and barley Lsi2 localized on the plasma membrane. The unique features of maize and barley Si influx and efflux transporters, including their cell-type specificity and the lack of polarity of their localization in Lsi2, indicate that these crops have a different Si uptake system from that in rice.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 METHODS
 REFERENCES
 
Silicon (Si) is a beneficial element for plant growth. Several benefits of Si have been reported, such as enhancement of pest and pathogen resistance, drought and heavy metal tolerance, and improvement of agricultural crop quality and yield, in a wide variety of plant species (Richmond and Sussman, 2003Go; Fauteux et al., 2005Go; Ma and Yamaji, 2006Go, 2008Go; Liang et al., 2007Go). These beneficial effects are mostly attributed to the high accumulation of Si in plant shoots (Ma, 2004Go). However, the level of Si accumulation in the shoots differs greatly among plant species (Ma and Takahashi, 2002Go; Hodson et al., 2005Go). Physiological studies have shown that the differences in Si accumulation result from the capacity of the roots to absorb Si (Takahashi et al., 1990Go), but little is known about the molecular mechanisms underlying Si uptake in plants other than rice (Oryza sativa).

In rice, great progress has been made in the identification of Si transporters involved in the uptake and distribution of Si. Rice is a typical Si-accumulating plant and is able to accumulate Si to >10% of the shoot dry weight (Ma and Takahashi, 2002Go). This high accumulation in the shoots has been attributed to the presence of an influx Si transporter (Low Silicon Rice 1 [Lsi1]) and efflux transporter (Lsi2) found in rice roots. Both Lsi1 and Lsi2 of rice are localized at the plasma membrane of both exodermal and endodermal cells of the roots (Ma et al., 2006Go, 2007aGo). Furthermore, both show polar localization. Lsi1 is localized at the distal end of the cells, while Lsi2 is localized at the proximal end. Knockout of either Lsi1 or Lsi2 resulted in a significant reduction of Si uptake by rice plants (Ma et al., 2006Go, 2007aGo; Tamai and Ma, 2008Go). Therefore, the concerted functioning of these two transporters produces an effective flow of Si in the exodermis and endodermis to override the barrier of Casparian strips (Ma et al., 2006Go, 2007aGo).

More recently, Lsi6, the only homolog of Lsi1 in the rice genome, has been identified in rice (Yamaji et al., 2008Go). Unlike Lsi1, Lsi6 is mainly localized at the xylem parenchyma cells of the leaf and is responsible for unloading Si from the xylem. Knockout of Lsi6 affected the distribution of Si in the shoots (Yamaji et al., 2008Go).

In contrast with the situation in rice, little is known about the mechanisms of Si uptake in other gramineous crops. Gramineae plants have various Si uptake capacities and root structures (Tamai and Ma, 2003Go); therefore, it is possible that they have different Si uptake systems for Si. This possibility has been supported by the identification of homologs of rice Lsi1 in maize (Zea mays; Zm Lsi1) and barley (Hordeum vulgare; Hv Lsi1) (Chiba et al., 2009Go; Mitani et al., 2009Go). Both barley and maize Lsi1 show Si influx transport activity like rice Lsi1, but their cell-type specificities of localization and expression patterns are different from those of rice Lsi1. Barley and maize Lsi1 are localized at the epidermal, hypodermal, and cortical cells (Chiba et al., 2009Go; Mitani et al., 2009Go). Furthermore, the expression of rice Lsi1 is downregulated by supplementation with Si, whereas the expression levels of both barley and maize Lsi1 are unaffected by Si (Ma et al., 2006Go; Yamaji and Ma, 2007Go; Chiba et al., 2009Go; Mitani et al., 2009Go). Therefore, further elucidation of Si transporters in other plant species will help to better understand the various Si uptake systems in different gramineous plants.

In this study, we identified Lsi2-like efflux transporters of Si from two important gramineous crops: maize and barley. Both maize and barley accumulate Si, although to a lesser extent than does rice (Ma and Takahashi, 2002Go). Physiological studies have shown that maize and barley actively absorb Si (Ma and Takahashi, 2002Go). For example, Si uptake by maize and barley seedlings was inhibited by the presence of metabolic inhibitors and low temperature (Liang et al., 2006Go; Nikolic et al., 2007Go). The concentration of Si in the xylem sap is much higher than that in the external solution. However, barley and maize Lsi1 are thought to be passive Si transporters, which could drive Si from the external solution to the root cells, following the Si concentration gradient. Therefore, an active Si transporter is required to achieve the higher accumulation of Si in the xylem. Our results show that the Lsi2-like efflux transporters are responsible for the active transport of Si in maize and barley. Furthermore, we found that barley and maize Lsi2 have a different cell-type specificity of localization from rice Lsi2, revealing that a different uptake system exists for Si between rice and maize/barley.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 METHODS
 REFERENCES
 
Cloning of Maize and Barley Lsi2
Based on a database search using the Maize Genomic and Genetic Database (http://www.maizegdb.org/), we obtained the closest homolog of the rice efflux transporter Lsi2 in maize and designated it Zm Lsi2. By contrast, a partial fragment of barley Lsi2 was identified by genomic-PCR with a primer set designed from the conserved sequence of Os Lsi2 and Zm Lsi2. The full-length Hv Lsi2 cDNA was determined by a rapid amplification of cDNA ends method using total RNA isolated from barley root.

The predicted amino acid sequences indicated that maize and barley Lsi2 are membrane proteins consisting of 477 and 474 amino acids, respectively (accession numbers of AB495341 [Zm Lsi2] and AB447483 [Hv Lsi2]). Both shared 86% identity with rice Lsi2 at the amino acid level (BLAST search and ClustalW analysis at the DNA Data Bank of Japan) (Figures 1A and 1B ; see Supplemental Data Set 1 online). Similar to rice Lsi2, maize and barley Lsi2 are also putative anion transporters with 11 predicted transmembrane domains (SOSUI, http://bp.nuap.nagoya-u.ac.jp/sosui/).


Figure 1
View larger version (61K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 1. Alignment and Phylogenetic Tree of Maize, Barley, and Rice Lsi2 Homologs.

(A) Amino acid sequence similarity between maize (Zm), barley (Hv), and rice (Os) Lsi2. The peptide sequences in maize and barley Lsi2 against which the anti-ZmLsi2 and anti-HvLsi2 antibodies, respectively, were produced, are boxed.

(B) Phylogenetic tree of maize, barley, and rice Lsi2, and Lsi2-like proteins in rice. The amino acid sequences are presented in Supplemental Data Set 1 online. Phylogenetic analysis was performed using MEGA version 4 (Tamura et al., 2007Go).

 
Functional Analysis of Barley and Maize Lsi2 in a Heterologous Expression System
Considering the amino acid sequence similarity between maize, barley, and rice Lsi2, Zm Lsi2 and Hv Lsi2 are likely to function as Si efflux transporters. To evaluate whether maize and barley Lsi2 are able to transport silicic acid, we performed an analysis using Xenopus laevis oocytes. We monitored the radioactivity released from oocytes expressing Zm Lsi2, Hv Lsi2, or no additional transcript that had been injected with 68Ge-labeled silicic acid. We observed higher efflux transport activity of silicic acid in the oocytes expressing maize and barley Lsi2 compared with the negative control (i.e., oocytes injected with water instead of transcript) (Figures 2A and 2B ). This result indicates that maize and barley Lsi2 have Si efflux transport activity.


Figure 2
View larger version (14K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 2. Si Efflux Transport Activity in Heterologous Expression Systems.

(A) and (B) Assay in Xenopus oocytes. Si (1 mM) labeled with 68Ge was injected into ooctyes expressing Zm Lsi2 (A), Hv Lsi2 (B), or no additional gene (water). After 4 h of incubation, the level of released Si was determined. The amount of released Si relative to the total amount of injected Si was shown as efflux transport activity. Vertical bars represent the SD of four biological replicates for maize Lsi2 and three biological replicates for barley Lsi2.

(C) Complementation test of barley Lsi2 in the rice lsi2 mutant. A Si uptake experiment was performed by exposing two independent transgenic lines harboring Lsi2P-HvLsi2 and vector control lines to a nutrient solution containing 0.5 mM Si as silicic acid for 6 h. Vertical bars represent SD of four biological replicates. Different letters indicate significant difference at P < 0.05 (Tukey's test).

 
To further examine the transport activity of barley Lsi2 in planta, we transformed a rice mutant (lsi2) defective in Si uptake with a construct carrying the barley Lsi2 cDNA under the control of rice Lsi2 promoter (Lsi2P-HvLsi2). Uptake experiments using two independent transgenic lines showed that Si uptake was significantly increased in plants carrying Lsi2P-HvLsi2 compared with those transformed with the vector control (Figure 2C).

Expression of Maize and Barley Lsi2 mRNA
To determine the mRNA accumulation of maize and barley Lsi2, total RNAs were extracted from the roots and shoots of each species. RT-PCR analysis showed that maize and barley Lsi2 were both expressed only in the roots (Figures 3A and 3D ). Furthermore, spatial analysis showed that the expression was higher in the basal root regions than in the root tip region in both maize and barley (Figures 3B and 3E).


Figure 3
View larger version (29K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 3. Expression Pattern of Maize and Barley Lsi2 and Response to Exogenously Applied Si.

(A) and (D) Tissue-specific expression of maize Lsi2 (A) and barley Lsi2 (D). Roots and shoots were harvested from maize and barley. Relative maize or barley Lsi2 expression levels were determined by RT-PCR. Actin mRNA level was used as an internal control.

(B) and (E) Spatial expression of maize (B) and barley (E) Lsi2 in different root regions. Cyclophilin (CyP) or Tubulin mRNA level was used as an internal control.

(C) and (F) Effect of Si on the expression of maize Lsi2 (C) and barley (F) Lsi2. Seedlings were exposed to the nutrient solution with or without 1 mM Si for 7 d. The roots were sampled at different days, and the expression level was determined with real-time RT-PCR. Vertical bars represent the SD of three biological replicates. Different letters indicate significant difference at P < 0.05 (Tukey's test).

 
The effect of Si supply on the expression of maize and barley Lsi2 was also investigated. The expression of both maize and barley Lsi2 was downregulated in response to the Si supply, and the expression level was decreased to 50 and 20% that of the original level, respectively, at the seventh day (Figures 3C and 3F).

Correlation between Barley Lsi2 Expression and Si Uptake Levels
There is a genotypic variation in Si uptake among barley cultivars (Chiba et al., 2009Go). To understand the molecular basis of this variation, the Hv Lsi2 expression level was investigated in eight cultivars differing in Si uptake. Interestingly, clear variation was observed in the Hv Lsi2 expression level. Statistical analysis indicated a significant positive correlation between Si uptake and the Hv Lsi2 expression level, with a correlation coefficient of r = 0.87 (P < 0.05) (Figure 4 ).


Figure 4
View larger version (7K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 4. Correlation between Si Uptake Ability and the Expression of Lsi2 in Eight Barley Cultivars.

Si uptake ability in eight barley cultivars was determined by exposing seedlings (14 d old) to 0.5 mM Si for 24 h. The expression of Lsi2 in the roots of different barley cultivars was determined by quantitative real-time RT-PCR. Pearson's correlation coefficient (r) at P < 0.05 was indicated. Dashed line shows approximate curve. Vertical and horizontal bars represent SD of three biological replicates.

 
Cell-Type Specificity of Zm Lsi2 and Hv Lsi2 Localization
To investigate the cell-type specificity of maize and barley Lsi2 localization, immunostaining with rabbit anti-Zm Lsi2 and anti-Hv Lsi2 antibody, respectively, was performed. Microscopy observations indicated that both maize and barley Lsi2 were localized at the endodermis of the basal region of both the seminal and lateral roots in maize and barley (Figures 5A to 5C and 6A to 6C ). Localization of maize Lsi2 in the crown roots was not observed (Figure 5D). Interestingly, neither maize nor barley Lsi2 exhibited polar localization (Figures 5 and 6).


Figure 5
View larger version (102K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 5. Cell-Type Specificity of Localization of Lsi2 in Maize Roots.

Immunostaining with anti-ZmLsi2 antibody was performed using the basal region of seminal root ([A] and [B]), lateral root (C), and crown root (D). Bars = 50 µm.

 

Figure 6
View larger version (31K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 6. Cell-Type Specificity of Localization of Lsi2 in Barley Roots.

Immunostaining with anti-HvLsi2 antibody was performed using the basal region of seminal roots ([A] and [B]) and lateral root (C). Bars = 50 µm.

 
To further examine the effect of aerenchyma formation on the localization of maize and barley Lsi2, both barley and maize seedlings were grown in a nutrient solution with or without aeration. Under nonaerated conditions, aerenchyma was observed in the basal root region of both barley and maize (see Supplemental Figure 1 online). However, aerenchyma formation did not affect the cell-type specificity of maize and barley Lsi2 localization (see Supplemental Figure 1 online).

Subcellular Localization of Maize and Barley Lsi2
The subcellular localization of maize and barley Lsi2 was investigated by protein gel blot analysis of microsomes isolated from maize or barley roots. Anti-ZmLsi2 or anti-HvLsi2 antibody resulted in detection of a single band at approximately the expected size in the microsome fraction (Figures 7A and 7C ), supporting the specificity of these antibodies to maize Lsi2 or barley Lsi2, respectively. Fractionation of the root microsomal membranes with sucrose density gradients showed that maize and barley Lsi2 were detected in the heavy density fractions (mainly at the 50/60% sucrose boundary for Zm Lsi2 and the 40/50% for Hv Lsi2), as is the case for Hv PIP2;1, a plasma membrane protein (Katsuhara et al., 2002Go) (Figures 7B and 7D), whereas tonoplast intrinsic protein ({gamma}-TIP) was detected in the light density fractions (Maeshima, 1992Go). These results indicate that maize and barley Lsi2 proteins are localized at the plasma membrane and not at the tonoplast.


Figure 7
View larger version (48K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 7. Subcellular Localization Analysis of Maize and Barley Lsi2 by Protein Gel Blots.

(A) and (C) Specificity of anti-ZmLsi2 (A) and anti-HvLsi2 (C) antibody. The microsomal fraction was isolated from maize or barley roots. SDS-PAGE and protein gel blot analyses were conducted using anti-ZmLsi2 or anti-HvLsi2 antibody.

(B) and (D) Subcellular localization of maize (B) and barley (D) Lsi2 protein. Membrane fractions were separated by sucrose gradient centrifugation as indicated, and each fraction was subjected to SDS-PAGE and protein gel blot analysis. Anti-HvPIP2;1 and anti-{gamma}-TIP antibodies were used as markers of the plasma membrane and tonoplast, respectively.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
METHODS
 REFERENCES
 
In this study, we identified two Si efflux transporters (Zm Lsi2 and Hv Lsi2) from two gramineous plants: maize and barley (Figure 1). Heterologous expression of maize or barley Lsi2 in Xenopus oocytes showed that these proteins had efflux transport activity for Si (Figures 2A and 2B). Furthermore, when barley Lsi2 was introduced into the rice lsi2 mutant, which has a dysfunctional Si efflux transporter due to a single nucleotide substitution in the open reading frame (ORF) (Ma et al., 2007aGo), Si uptake was recovered (Figure 2C). These results indicate that maize and barley Lsi2 function as efflux transporters of Si, similar to rice Lsi2. However, Os Lsi2 and Zm Lsi2/Hv Lsi2 exhibited different features in cell-type specificity and in the polarity of their localizations. Rice Lsi2 is localized both in the exodermis and endodermis (Ma et al., 2007aGo), whereas maize and barley Lsi2 are only present in the endodermis (Figures 5 and 6). Furthermore, rice Lsi2 is characterized by polar localization at the proximal side of both the exodermis and endodermis of rice roots (Ma et al., 2007aGo). However, neither maize nor barley Lsi2 showed such polar localization (Figures 5 and 6). Although the mechanism that regulates polar localization has yet to be elucidated, it will be interesting to compare Os Lsi2 and Zm Lsi2/Hv Lsi2 in terms of polar localization mechanisms in the future.

Spatial expression analysis showed that maize and barley Lsi2 were mainly expressed in the basal root region (Figures 3B and 3E). Considering that Si influx transporters in maize and barley are also expressed in the same region (Chiba et al., 2009Go), these influx and efflux transporters are likely to work together to transfer Si from the external soil to the stele. However, the response of maize and barley Lsi2 expression to Si supply was different from that of Lsi1. The expression of barley and maize Lsi2 was downregulated in response to exogenously applied Si (Figures 3C and 3F), whereas no response was observed in the expression of maize and barley Lsi1 (Chiba et al., 2009Go; Mitani et al., 2009Go), indicating that these influx and efflux transporters in maize and barley might be regulated in a distinct way.

There is a genotypic variation in Si uptake among barley cultivars (Ma et al., 2003Go; Chiba et al., 2009Go). Previously, only a weak correlation between Lsi1 expression level and Si uptake ability was observed in eight barley cultivars (Chiba et al., 2009Go). In this study, we found a positive correlation between barley Lsi2 expression level and Si uptake (Figure 4). These results indicate that the expression level of Lsi2 is a key factor in determining Si uptake in barley, which is different from in rice. In the case of rice, expression of both Lsi1 and Lsi2 is responsible for genotypic differences in Si uptake (Ma et al., 2007bGo). The Lsi2-dependent Si uptake system in barley is consistent with the fact that only Lsi2 expression is affected by Si, whereas both Lsi1 and Lsi2 are downregulated by Si in rice (Ma et al., 2006Go, 2007aGo).

Maize is characterized by the formation of crown roots. Maize Lsi1 and Lsi6 are expressed in the crown roots (Mitani et al., 2009Go), but the expression of maize Lsi2 was not observed (Figure 5D). Crown roots form the major part of the adult rootstock and are the basis for lodging resistance of the plants (Hochholdinger et al., 2004Go). Si taken up by the crown roots through maize Lsi1 and Lsi6 may increase their mechanical resistance (Mitani et al., 2009Go). Since maize Lsi2 is mainly responsible for the transport of Si into the stele in seminal and lateral roots, it seems that Lsi2 is not necessary in the crown roots of maize.

Identification of maize and barley Lsi2 in this study together with Si influx transporters identified previously (Chiba et al., 2009Go; Mitani et al., 2009Go) reveals that the uptake system for Si differs between rice and maize/barley. This difference results from the different localization patterns of Si influx and efflux transporters at different cells. In rice, both influx and efflux transporters of Si are polarly localized on the same cells of the exodermis and endodermis (Ma and Yamaji, 2008Go). By contrast, maize and barley influx transporters (Zm Lsi1/Hv Lsi1) and efflux transporters (Zm Lsi2/Hv Lsi2) are located at distinct cells (Figure 8 ); maize and barley influx transporters are polarly localized on the epidermal, hypodermal, and cortical cells, and efflux transporters are nonpolarly localized on the endodermis only (Figure 8). Therefore, in maize and barley, Si can be taken up from external solution (soil solution) by Lsi1 at distinct cells, such as epidermal, hypodermal, and cortical cells (Figure 8). By contrast, in rice, Si is only taken up at the exodermal cells by Lsi1. After being taken up into maize and barley root cells, Si is transported to the endodermis by the symplastic pathway and then released to the stele by Zm Lsi2/Hv Lsi2 in (Figure 8). By contrast, in rice, Si taken up by Lsi1 at the exodermal cells is released by Lsi2 to the apoplast and then transported into the stele by both Lsi1 and Lsi2 again at the endodermal cells.


Figure 8
View larger version (21K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 8. Schematic Model of the Si Transport System in Maize/Barley and Rice.

In maize and barley, Si as silicic acid [Si(OH)4] is taken up from the external solution by the influx transporter (Zm Lsi1/Hv Lsi1) localized on the distal side of cells in the epidermis and cortex layer and then transferred to the endodermis through the symplastic pathway (blue arrow). At the endodermis, the Si is released by an active Si efflux transporter (Zm Lsi2/Hv Lsi2) to the stele. In rice, Si is taken up from the external solution by Lsi1 at the distal side and released to the apoplast of aerenchyma by Lsi2 at the proximal side of the exodermal cells. Si is then transported to the stele by Lsi1 and Lsi2 at the endodermal cells.

 
These differences in the uptake system of these plants may be attributed to the root structures observed in rice versus maize and barley. In rice roots, there are two Casparian strips: one at the exodermis and one at the endodermis (Ma et al., 2006Go; Yamaji and Ma, 2007Go), whereas in both maize and barley, one Casparian strip usually develops at the endodermis under nonstressed conditions. Moreover, mature rice roots have a distinct structure, with a highly developed aerenchyma, and hardly have cortical cells between the exodermis and endodermis (Kawai et al., 1998Go). Therefore, Si transported into rice exodermal cells by the influx transporter, Lsi1, has to be released by the efflux transporter, Lsi2, into the apoplast of a spoke-like structure across the aerenchyma. However, in maize and barley roots, although aerenchyma is also formed under certain growth conditions (Evans, 2003), it occurs in mature root regions. Furthermore, at the root regions where aerenchyma formation was observed in maize and barley, the localization of Lsi2 was not changed compared with the localization in the root without aerenchyma formation (see Supplemental Figure 1 online). These observations suggest that the localization of Si efflux transporters is inherently programmed and is not affected by growth conditions.

These differences in the localization of Si transporters and polarity between rice and maize/barley may be one of the reasons for the different Si uptake capacity among species, although further studies are needed to address this issue. Radical transport of a mineral includes both the apoplastic pathway through cell walls and intercellular spaces and symplastic pathway from cell to cell (Marschner, 1995Go). Our results provide a molecular basis for these pathways in different plant species.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 METHODS
REFERENCES
 
Plant Materials and Growth Conditions
Maize (Zea mays cv B73) seeds were soaked in deionized water for 2 h and then placed on a sheet of moist filter paper in a Petri dish incubated overnight at 4°C. Germinated seeds were transferred to a floating nylon mesh on 0.5 mM CaCl2 solution in a growth cabinet at 25°C in the dark. Seedlings were transferred to 3-liter plastic pots containing continuously aerated one-fifth Hoagland solution [1 mM KNO3, 1 mM Ca(NO3)2, 0.4 mM MgSO4, 0.2 mM KH2PO4, 20 µM Fe-EDTA, 3 µM H3BO3, 1 µM (NH4)6Mo7O24, 0.5 µM MnCl2, 0.4 µM ZnSO4, and 0.2 µM CuSO4, pH 6.0]. The plants were grown in a greenhouse with natural light.

Barley seeds (Hordeum vulgare cv Haruna-Nijo) were soaked in deionized water for 2 h and then incubated overnight on a sheet of moist filter paper in the dark at 20°C. Germinated seeds were transferred to a floating nylon mesh in a continuously aerated solution containing 0.5 mM CaCl2 in the dark. Seedlings were then transferred into the aerated one-fifth Hoagland solution as described above in a growth chamber at 20°C.

Cloning of Maize and Barley Lsi2 cDNA
For cloning of Zm Lsi2, RNA was extracted from maize roots (14 d old) using an RNeasy plant mini kit (Qiagen). Total RNA (1.5 µg) was reverse-transcribed into cDNA by the SuperScript first-strand synthesis system (Invitrogen). Based on the homology search in Maize Genomics and Genetics Database (http://www.maizegdb.org/), the ORF of Zm Lsi2 with a flanking BamHI site was amplified with the following primers from the cDNA samples: forward 5'- ATGGATCCAT CATGGCTCTT GCTTCTGTGG-3' and reverse 5'-TTGGATCCTA CGATCGAGTTCAGATGTTGA-3. These amplified ORFs were inserted into TA cloning vector pTA2 (TOYOBO), according to the protocol of the manufacturer. The sequence of the cloned gene was confirmed by an ABI PRISM 310 genetic analyzer using a BigDye Terminators v3.1 cycle sequencing kit (Applied Biosystems).

For cloning of Hv Lsi2, partial fragment was obtained by PCR using primers (5'- CCTACATGGTGGGGCTCA-3' and 5'-GAAGAAGACGAGCAGCGAGT-3') designed from the conserved sequence of Os Lsi2 and Zm Lsi2. Barley genomic DNA was used as a template. The full-length Hv Lsi2 cDNA was generated by the rapid amplification of cDNA ends method from total RNA prepared from barley roots (SMART RACE cDNA amplification kit; Clontech) with primers corresponding to the partial fragment of Hv Lsi2. The entire Hv Lsi2 cDNA was subcloned into the pGEM-T Easy vector (Promega). The sequences of cloned genes were confirmed as described above.

Phylogenetic Analysis
Alignments were performed with Clustal W using default setting, and phylogenetic trees were constructed using MEGA version 4 (Tamura et al., 2007Go) with 1000 bootstrap trials.

Si Efflux Transport Activity Assay
Oocytes were isolated from Xenopus laevis frogs. Procedures for defolliculation, culture condition, and selection were the same as described previously (Mitani et al., 2008Go). For synthesis of capped RNA, the inserts of maize Lsi2 were digested from the pTA2 vector with BamHI and ligated into pXβG-ev1 (Preston et al., 1992Go). For barley Lsi2 cDNA, the ORF was amplified by RT-PCR with the following primers: 5'-GAAAGATCTATGGCGCTCGCGTCTCTCCCCAAGG-3' and 5'-GAAAGATCTCTACATCTTTCCGATGAGGGGGATG-3'. The fragment containing the ORF was inserted into the BglII site of a Xenopus oocytes expression vector, pXβG-ev1.

Capped RNA was then synthesized from linearized pXβG-ev1 plasmids by in vitro transcription with an mMASSAGE mMACHINE high yield capped RNA transcription kit (Ambion), according to the manufacturer's instructions. A volume of 50 nL (1 ng nL–1) in vitro cRNA transcripts was injected into the selected oocytes using a Nanoject II automatic injector (Drummond Scientific). As a negative control, 50 nL of RNase-free water was also injected. After incubation in MBS at 18°C for 1 d, the oocytes were used for the transport activity assay as described previously (Ma et al., 2007aGo; Mitani et al., 2008Go). Briefly, 1 mM silicic acid labeled with 68Ge (Perkin-Elmer; 2 GBq mmol–1) was injected into oocytes. After 4 h of incubation, the external solutions were collected and the remaining oocytes were homogenized with 0.1 N HNO3. Radioactivities of the external solution and homogenized oocytes were determined by a liquid scintillation analyzer (Perkin-Elmer).

Complementation Analysis of the Rice Mutant lsi2
The promoter region of Os Lsi2 (2.0 kb) was amplified from rice (Oryza sativa cv Nipponbare) genomic DNA using the following primers: 5'-AAAGCTTGTTATTAACGCTGAGACTGAA-3' and 5'-TTCTAGATTATAGCTAGCTTAGCTACTTG-3'. The ORF of Hv Lsi2 and the terminator region of the Noparine synthase gene were amplified from pGFP-HvLsi2 by PCR with the following primers: 5'-GCTCTAGAATGGCGCTCGCGTCTCTCCCCAA-3' and 5'-GCTCTAGAGATCTAGTAACATAGATGACACC-3'. These two fragments were inserted into a binary vector (pPZP2H-lac), and the final construct was designated as pLsi2P-HvLsi2. The plasmid was transformed into Agrobacterium tumefaciens strain EHA101 and then transferred into calli derived from the rice mutant lsi2 using an Agrobacterium-mediated transformation system.

The Si uptake experiment and gene expression analysis were performed with two independent transgenic lines and vector control lines. The uptake experiment was conducted in the half-strength Kimura B solution, pH 5.6, containing 0.5 mM Si as silicic acid for 6 h (Ma et al., 2006Go).

Expression Analyses
Roots and shoots of maize or barley were sampled and subjected to RNA extraction as described below, and the expression of maize or barley Lsi2 in the tissues was examined by RT-PCR. To investigate the effect of Si supply on Zm Lsi2 and Hv Lsi2 gene expression, seedlings (20 d old for maize and 14 d old for barley) were cultured in one-fifth Hoagland solution containing 0 or 1 mM silicic acid. At different time points, the roots were sampled and subjected to RNA extraction. The RNA was used for quantitative analysis of Zm Lsi2 or Hv Lsi2 gene expression as described below. The expression level in different root regions (0 to 10 mm, 10 to 20 mm, and 20 to 30 mm from the maize apex; 0 to 15 mm and 15 to 30 mm from the barley apex) was also examined similarly. All experiments were biologically replicated three times. Actin, {alpha}-tubulin, or cyclophilin was used as an internal standard with primers described below.

Genotypic Variation in Si Uptake
Eight cultivars, including Haruna-Nijo, Golden promise, SV183, BCUS126, SV75, SV119, BCUS93, and SV92, were used for Si uptake analysis. 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, 2005Go). The roots from each cultivar were also sampled for RNA extraction and used for quantitative analysis of Hv Lsi2 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) and then converted to cDNA followed by DNase I treatment using SuperScript II (Invitrogen), according to the manufacturer's instructions. Specific cDNAs were amplified by SYBR premix Ex Taq (Takara) and real-time RT-PCR (ABI Prism 7500; Applied Biosystems), with the following primer sets: 5'-GTCCTAGCCTCCGTTTTCGT-3' and 5'-CCATCTCCCTCTCCCTCTCTT-3' for Hv Lsi2; 5'-GACTCTGGTCATGGTGTCAGC-3' and 5'-GGCTGGAAGAGGACCTCAGG-3' for actin; 5'-CCTGTCGTGTCGTCGGTCTAAA-3' and 5'- ACGCAGATCCAGCAGCCTAAAG-3' for cyclophilin; 5'-ACGTGCCAAC AGGTGCTTCT TATG-3' and 5'-TACGATCGAGGCATACAATTATG-3' for Zm Lsi2; and 5'-ATGGCATCCAGGCTGATGGT-3' and 5'-TATGGCTCAACTACCGAAGT-3' for {alpha}-tubulin.

Immunological Staining
The synthetic peptide C-SGSGMDLDGKRMEA (positions 210 to 223 of Zm Lsi2) or C-GVAPDADGKQMA (positions 209 to 220 of Hv Lsi2) was used to immunize rabbits to obtain antibodies against maize and barley Lsi2, respectively. The obtained antiserum was purified through a peptide affinity column before use. The seminal, crown, and lateral roots of maize or the seminal and lateral roots of barley were used for immunostaining of Zm Lsi2 or Hv Lsi2 protein with a 1:300 diluted anti-ZmLsi2 or anti-HvLsi2 as described previously (Yamaji and Ma, 2007Go). Fluorescence of secondary antibody (Alexa Fluor 555 goat anti-rabbit IgG; Molecular Probes) was observed with a fluorescence microscope (Axio Imager with Apotome; Carl Zeiss).

To investigate the effect of growth conditions on the localization of Zm Lsi2 and Hv Lsi2, maize and barley were grown in the nutrient solution with or without aeration for 14 d. The roots were then used for immunostaining as described above.

Protein Gel Blot Analysis
Membrane proteins were prepared from maize or barley roots basically according to the methods of Sugiyama et al. (2007)Go. Thirty grams of roots harvested from maize (14 d old) or barley (23 d old) seedlings were homogenized in 70 mL of ice-cold homogenizing buffer composed of 100 mM Tris-HCl, pH 8.0, 150 mM KCl, 0.5% (w/v) polyvinylpolypyrrolidone, 5 mM EDTA, 3.3 mM DTT, 1 mM phenylmethylsulfonyl fluoride, and 10% (v/v) glycerol (Sugiyama et al., 2007Go). After filtration, the homogenates were centrifuged at 8000g for 10 min to yield the supernatant and centrifuged again under the same conditions. The supernatants were then ultracentrifuged at 100,000g for 30 min. The pellets were resuspended in 1 mL of resuspension buffer containing 10 mM Tris-HCl, pH 7.6, 10% (v/v) glycerol, 1 mM EDTA, 1 mM DTT, and 1/100 volume of Protease Inhibitor Cocktail for plant cell and tissue extracts (Sigma-Aldrich). The suspended solution was then fractionated with discontinuous sucrose gradients (20 to 60% sucrose in 10 mM Tris-HCl, pH 7.6, 1 mM EDTA, and 1 mM DTT) by ultracentrifugation at 100,000g for 120 min. The fractionated membranes were recovered by ultracentrifugation at 100,000g for 30 min. Each pellet was resuspended in 100 µL resuspension buffer supplemented with 1/100 volume of Protease Inhibitor Cocktail for plant cell and tissue extracts (Sigma-Aldrich) and 1 mM DTT.

Equal amounts of each fraction were mixed with the same volume of sample buffer containing 250 mM Tris-HCl, pH 6.8, 8% (w/v) SDS, 40% (v/v) glycerol, 0.01% (w/v) bromophenol blue, and 200 mM β-mercaptoethanol. The mixture was incubated for 1 min at 95°C for Zm Lsi2, for 10 min at 37°C for Hv Lsi2, for 10 min at 95°C for {gamma}-TIP, and for 1 min at 100°C for HvPIP2;1. SDS-PAGE was then performed using 5 to 20% gradient polyacrylamide gels (ATTO). The transfer to polyvinylidene difluoride membrane was performed with a semidry blotting system, and the membrane was treated with the purified primary rabbit anti-ZmLsi2, HvLsi2, and HvPIP2;1 (100 times dilution) or {gamma}-TIP polyclonal antibody (1000 times dilution). ECL Peroxidase labeled anti-rabbit antibody (1:10,000 dilution; GE Healthcare) was used as a secondary antibody, and an ECL Plus western blotting detection system (GE Healthcare) was used for detection via chemiluminescence.

Statistic Analysis
Analysis of variance was performed on all data sets. Tukey's honestly significant difference test was used to compare treatment means. Pearson's correlation coefficient (r) was calculated using Excel.

Accession Numbers
Sequence data from this article can be found in the GenBank/EMBL/DDBJ databases under accession numbers AB495341 (Zm Lsi2) and AB447483 (Hv Lsi2).

Supplemental Data
The following materials are available in the online version of this article.

Supplemental Figure 1. Cellular Localization of Zm Lsi2 and Hv Lsi2 under Nonaerated Conditions.
Supplemental Data Set 1. Protein Sequences Used to Generate the Phylogenetic Tree in Figure 1B.


   Acknowledgments
 
This work was supported by the Program of Promotion of Basic Research Activities for Innovative Biosciences, a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (17078008 to J.F.M.), and a grant from the Ministry of Agriculture, Forestry, and Fisheries of Japan (Genomics for Agricultural Innovation IPG-006 to J.F.M.). The work was also supported by a Sunbor grant and the Ohara Foundation for Agricultural Science. We thank M. Maeshima for providing the anti-{gamma}-TIP antibody, M. Katsuhara for the HvPIP2;1 antibody, K. Sato for barley seeds, and Y. Mano for maize seeds. We also thank Jared Westbrooks for critical reading of this manuscript.


   Footnotes
 
1 These authors contributed equally to this work. Back

2 Current address: Creative Research Initiative Sousei, Hokkaido University, North-21, West-10, Kita-ku, Sapporo, 001-0021, Japan. Back

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Jian Feng Ma (maj{at}rib.okayama-u.ac.jp).

[W] Online version contains Web-only data. Back

www.plantcell.org/cgi/doi/10.1105/tpc.109.067884

Received April 13, 2009; Revision received June 10, 2009. accepted June 19, 2009.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 METHODS
 REFERENCES
 
Chiba, Y., Mitani, N., Yamaji, N., and Ma, J.F. (2009). HvLsi1 is a silicon influx transporter in barley. Plant J. 57: 810–818.[CrossRef][Web of Science][Medline]

Evans, E.D. (2004). Aerenchyma formation. New Phytol. 161: 35–49.[CrossRef][Web of Science]

Fauteux, F., Remus-Borel, W., Menzies, J.G., and Belanger, R.R. (2005). Silicon and plant disease resistance against pathogenic fungi. FEMS Microbiol. Lett. 249: 1–6.[Web of Science][Medline]

Hochholdinger, F., Woll, K., Sauer, M., and Dembinsky, D. (2004). Genetic dissection of root formation in maize (Zea mays) reveals root-type specific developmental programmes. Ann. Bot. (Lond.) 93: 359–368.[Abstract/Free Full Text]

Hodson, M.J., White, P.J., Mead, A., and Broadley, M.R. (2005). Phylogenetic variation in the silicon composition of plants. Ann. Bot. (Lond.) 96: 1027–1046.[Abstract/Free Full Text]

Katsuhara, M., Akiyama, Y., Koshio, K., Shibasaka, M., and Kasamo, K. (2002). Functional analysis of water channels in barley roots. Plant Cell Physiol. 43: 885–893.[Abstract/Free Full Text]

Kawai, M., Samarajeewa, P.K., Barrero, R.A., Nishiguchi, M., and Uchimiya, H. (1998). Cellular dissection of the degradation pattern of cortical cell death during aerenchyma formation of rice roots. Planta 204: 277–287.[CrossRef][Web of Science]

Liang, Y., Sun, W., Zhu, Y.-G., and Christie, P. (2007). Mechanisms of silicon-mediated alleviation of abiotic stresses in higher plants: A review. Environ. Pollut. 147: 422–428.[CrossRef][Medline]

Liang, Y.C., Hua, H.X., Zhu, Y.G., Zhang, J., Cheng, C.M., and Romheld, V. (2006). Importance of plant species and external silicon concentration to active silicon uptake and transport. New Phytol. 172: 63–72.[CrossRef][Web of Science][Medline]

Ma, J.F. (2004). Role of silicon in enhancing the resistance of plants to biotic and abiotic stresses. Soil Sci. Plant Nutr. 50: 11–18.

Ma, J.F., Higashitani, A., Sato, K., and Takeda, K. (2003). Genotypic variation in silicon concentration of barley grain. Plant Soil 249: 383–387.[CrossRef][Web of Science]

Ma, J.F., and Takahashi, E. (2002). Soil, Fertilizer, and Plant Silicon Research in Japan. (Amsterdam: Elsevier).

Ma, J.F., Tamai, K., Yamaji, N., Mitani, N., Konishi, S., Katsuhara, M., Ishiguro, M., Murata, Y., and Yano, M. (2006). A silicon transporter in rice. Nature 440: 688–691.[CrossRef][Medline]

Ma, J.F., and Yamaji, N. (2006). Silicon uptake and accumulation in higher plants. Trends Plant Sci. 11: 392–397.[CrossRef][Web of Science][Medline]

Ma, J.F., and Yamaji, N. (2008). Functions and transport of silicon in plants. Cell. Mol. Life Sci. 65: 3049–3057.[CrossRef][Web of Science][Medline]

Ma, J.F., Yamaji, N., Mitani, N., Tamai, K., Konishi, S., Fujiwara, T., Katsuhara, M., and Yano, M. (2007a). An efflux transporter of silicon in rice. Nature 448: 209–212.[CrossRef][Medline]

Ma, J.F., Yamaji, N., Tamai, K., and Mitani, N. (2007b). Genotypic difference in Si uptake and expression of Si transporter genes in rice. Plant Physiol. 145: 919–924.[Abstract/Free Full Text]

Maeshima, M. (1992). Characterization of the major integral protein of vacuolar membrane. Plant Physiol. 98: 1248–1254.[Abstract/Free Full Text]

Marschner, H. (1995). Mineral Nutrition of Higher Plants. (San Diego, CA: Academic Press).

Mitani, N., and Ma, J.F. (2005). Uptake system of silicon in different plant species. J. Exp. Bot. 56: 1255–1261.[Abstract/Free Full Text]

Mitani, N., Yamaji, N., and Ma, J.F. (2008). Characterization of substrate specificity of a rice silicon transporter, Lsi1. Pflugers Arch. 456: 679–686.[CrossRef][Web of Science][Medline]

Mitani, N., Yamaji, N., and Ma, J.F. (2009). Identification of maize silicon influx transporters. Plant Cell Physiol. 50: 5–12.[Abstract/Free Full Text]

Nikolic, M., Nikolic, N., Liang, Y., Kirkby, E.A., and Romheld, V. (2007). Germanium-68 as an adequate tracer for silicon transport in plants. Characterization of silicon uptake in different crop species. Plant Physiol. 143: 495–503.[Abstract/Free Full Text]

Preston, G.M., Carroll, T.P., Guggino, W.B., and Agre, P. (1992). Appearance of water channels in Xenopus oocytes expressing red cell CHIP28 protein. Science 256: 385–387.[Abstract/Free Full Text]

Richmond, K.E., and Sussman, M. (2003). Got silicon? The non-essential beneficial plant nutrient. Curr. Opin. Plant Biol. 6: 268–272.[CrossRef][Web of Science][Medline]

Sugiyama, A., Shitan, N., and Yazaki, K. (2007). Involvement of a soybean ATP-binding cassette-type transporter in the secretion of genistein, asignal flavonoid in legume-Rhizobium symbiosis. Plant Physiol. 144: 2000–2008.[Abstract/Free Full Text]

Takahashi, E., Ma, J.F., and Miyake, Y. (1990). The possibility of silicon as an essential element for higher plants. Comments Agric. Food Chem. 2: 99–122.

Tamai, K., and Ma, J.F. (2003). Characterization of silicon uptake by rice roots. New Phytol. 158: 431–436.[CrossRef][Web of Science]

Tamura, K., Dudleym, J., Nei, M., and Kumar, S. (2007). MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24: 1596–1599.[Abstract/Free Full Text]

Tamai, K., and Ma, J.F. (2008). Reexamination of silicon effects on rice growth and production under field conditions using a low silicon mutant. Plant Soil 307: 21–27.[CrossRef][Web of Science]

Yamaji, N., and Ma, J.F. (2007). Spatial distribution and temporal variation of the rice silicon transporter Lsi1. Plant Physiol. 143: 1306–1313.[Abstract/Free Full Text]

Yamaji, N., Mitatni, N., and Ma, J.F. (2008). A transporter regulating silicon distribution in rice shoots. Plant Cell 20: 1381–1389.[Abstract/Free Full Text]





This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow PPT slides of all figures
Right arrow Supplemental Data
Right arrow All Versions of this Article:
21/7/2133    most recent
tpc.109.067884v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via CrossRef
Right arrow Citing Articles via Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mitani, N.
Right arrow Articles by Ma, J. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mitani, N.
Right arrow Articles by Ma, J. F.
Agricola
Right arrow Articles by Mitani, N.
Right arrow Articles by Ma, J. F.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
ASPB Publications THE PLANT CELL PLANT PHYSIOLOGY
Copyright © 2009 by the American Society of Plant Biologists