Isolation and characterization of three maize

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Isolation and characterization of three maize aquaporin genes, ZmNIP2;1, ZmNIP2;4 and ZmTIP4;4 involved in urea transport Riliang Gu#, Xiaoling Chen#, Yuling Zhou & Lixing Yuan* Department of Plant Nutrition, Key Laboratory of Plant-Soil Interactions, MOE, College of Environmental and Resources Sciences, China Agricultural University, Beijing 100193, China

Urea-based nitrogen fertilizer was widely utilized in maize production, but transporters involved in urea uptake, translocation and cellular homeostasis have not been identified. Here, we isolated three maize aquapoin genes, ZmNIP2;1, ZmNIP2;4 and ZmTIP4;4, from a cDNA library by heterogous complementation of a urea uptake-defective yeast. ZmNIP2;1 and ZmNIP2;4 belonged to the nodulin 26-like intrinsic proteins (NIPs) localized at plasma membrane, and ZmTIP4;4 belonged to the tonoplast intrinsic protein (TIPs) at vacuolar membrane. Quantitative RT-PCR revealed that ZmNIP2;1 was expressed constitutively in various organs while ZmNIP2;4 and ZmTIP4;4 transcripts were abundant in reproductive organs and roots. Expression of ZmTIP4;4 was significantly increased in roots and expanded leaves under nitrogen starvation, while those of ZmNIP2;1 and ZmNIP2;4 remained unaffected. Functions of maize aquapoin genes in urea transport together with their distinct expression manners suggested that they might play diverse roles on urea uptake and translocation, or equilibrating urea concentration across tonoplast. [BMB reports 2012; 45(2): 96-101]

INTRODUCTION Urea is a ubiquitous nitrogen (N) source in soils and also the most widespread form of N fertilizer used in agricultural crop production (1). It was believed for a long time that plants absorb most urea-derived N in the form of ammonium because of the rapid degradation of urea by enzyme urease (2, 3). However, several physiological evidences for direct intake of urea by plants have been suggested (4-6). For example, the accumulation of urea into leaves was observed after urea foliar *Corresponding author. Tel: +86-10-62734424; Fax: +86-10-6273 1016; E-mail: [email protected] # These authors have contributed equally to this paper. http://dx.doi.org/10.5483/BMBRep.2012.45.2.96 Received 12 October 2011, Revised 18 October Accepted 21 October 2011 Keywords: Aquaporin, Maize, Membrane transport, Nitrogen nutrition, Urea 96 BMB reports

14 application in the presence of urease inhibitor (6). C-labeled urea short-term influx analysis also revealed urea transport across the plasma membrane of alga and Arabidopsis suspension cell (7, 8). Besides being taken up, urea is an important N metabolite within plant cell produced by both arginine degradation and ureide catabolism pathways. Arginase-derived urea is produced in mitochondria, and then exported to cytoplasm for hydrolyzing into ammonium by urease. Vacuolar loading can be also beneficial to transiently store excess amount of urea, and vacuolar unloading can mobilize urea into cytosol if N is limited (9). In many physiological processes, therefore, urea transport across different cellular membranes is essential, and requires different types of urea transporters. 　Membrane protein AtDUR3 for transporting urea has been successfully isolated from Arabidopsis cDNA library by growth complementation of a urea uptake-defective yeast mutant YNVW1 (8). Function expression in X. laevis oocytes further + revealed that AtDUR3 encoded a high-affinity urea/H symporter. AtDUR3 protein localized at the plasma membrane of root rhizodermis cells under N deficiency and atdur3 T-DNA insertion lines showed impaired growth on urea as the sole N source, suggesting its important function in high-affinity urea uptake in roots (1). 　Aquaporins have been classified into four subfamilies referred to as plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin 26-like intrinsic proteins (NIPs) and small and basic intrinsic proteins (SIPs) (10, 11). The PIP member (NtAQP1), NIP member (CpNIP1) and TIP members (AtTIP1;2, 2;1, 4;1) were also found to transport urea when they expressed in yeast or oocytes (12-14). The aquaporins facilitated urea transport in a pH-independent manner and with liner concentration dependency (14). NIPs and PIPs localized at plasma membrane, suggesting their roles in moving urea between the apoplast and symplast of cells in planta (15-17). In contrast, AtTIPs were mainly targeted to tonoplast or other endomembranes, and might play roles in equilibrating urea concentration between different cellular compartments (14). In particularly, AtTIP5;1 was localized in pollen mitochondria, probably involved in N remobilization via transport of mitochondrial urea to the cytoplasm (18). 　Maize (Zea mays L.) is one of the most widely cultivated crop plants providing food for human beings, feed for animals

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Urea transport by maize aquaporins Riliang Gu, et al.

and materials for industries. Urea is a preferred N source for maize production because of its lower unit cost (19). However, the molecular basis of urea transport in maize plants is not well understood. In this present study, we aimed to isolate urea transporter genes from maize plants, and three aquaporins (ZmNIP2;1, ZmNIP2;4 and ZmTIP4;4) were obtained. We also examined their organ- and nitrogen-dependent expression patterns. These findings suggested important roles of aquaporins in urea transport throughout maize plants.

RESULTS Isolation and characterization of three maize aquaporins (ZmNIP2;1, ZmNIP2;4 and ZmTIP4;4) mediating urea transport in yeast

A cDNA library constructed from maize root tissue was transformed into the yeast mutant YNVW1 which is unable to grow on the medium containing ＜5 mM urea as sole N source (8). Of approx. 100,000 independent transformants, 9 independent clones were isolated by conferring growth complementation to yeast strain YNVW1 on 2 mM urea, and represented three genes with 6, 2 and 1 clones for ZmNIP2;1, ZmNIP2;4 and ZmTIP4;4, respectively. By retransformation to YNVW1, in contrast to empty vector, the transformates carrying all three isolated genes were able to grown on 2 mM urea,

indicating that ZmNIP2;1, ZmNIP2;4 and ZmTIP4;4 mediated urea transport in yeast (Fig. 1). We also conducted yeast growth complementation assay in the medium supplied with various external urea concentration (0.5 to 10 mM) under different pH levels (pH 4.5 to 6.5). It showed that ZmNIP2;1-, ZmNIP2;4- and ZmTIP4;4-expressing yeast could grow till on 0.5 mM urea, and their growth were hardly affected by raising external pH, indicating that these aquaporins mediated urea transport in yeast in pH-independent manners (Supplementary Fig. S1). 　ZmNIP2;1 has been shown to transport silicon (Si) in ooctyes as described by Mitani et al. (20). To investigate whether ZmNIP2;4 and ZmTIP4;4 could transport Si, the transformed yeast strains were grown in medium supplied with 2 mM Germanium dioxide (GeO2) in present of 1 mM arginin as N sources. Germanium (Ge) is an analogue of Si and toxic to yeast when it is taken up (21). ZmNIP2;1-expressing yeast showed growth sensitive to Ge (Fig. 1), confirming Si transport activity of ZmNIP2;1. ZmNIP2;4-expressing yeast also exhibited growth sensitive to Ge, suggesting that, similar to ZmNIP2;1, ZmNIP2;4 could transport Si in yeast. By contrast, ZmTIP4;4 seemed not to transport Ge and Si (Fig. 1). 　As shown in Supplementary Fig. 2, a phylogenic tree was constructed based on the alignment of all Arabidopsis aquaporin members, the rice and maize members within TIP4 and NIP2 subfamilies, and CpNIP2;1 and NaTIPa which have been identified as urea transporters in the previous studies (12, 13). ZmTIP4;4 was assigned into the TIP4 clade containing AtTIP4;1 and NaTIPa, and ZmNIP2;1 and ZmNIP2;4 into the NIP2 clade containing CpNIP2;1 (Supplementary Fig. 2). This phylogenic relationship supported the similar properties of urea transport for those members within TIP4 and NIP2 subfamilies in planta. In addition, ZmNIP2;1 and ZmNIP2;4 showed 94% identity at peptide level, and comparative genome analysis further mapped both genes to the maize chromosome regions micro-colinearized to OsNIP2;1 region (Supplemental Table S1). This indicated that ZmNIP2;1 and ZmNIP2;4 were the segmental duplicates arising by the specific whole genome duplication of maize genome (commonly named allotetraploidization) after the maize-rice split.

Organ- and nitrogen-dependent expressions of ZmNIP2;1, ZmNIP2;4 and ZmTIP4;4 genes in maize plants

Fig. 1. Yeast growth of the urea uptake-defective strain YNVW1 by expressing ZmNIP2;1, ZmNIP2;4 or ZmTIP4;4. The yeast mutant YNVW1 was transformed with the empty vector pDR, and pDR carrying ZmNIP2;1, ZmNIP2;4 or ZmTIP4;4 cDNA. Transformants were precultured on YNB medium supplemented with 1 mM arginine. A 1 ml aliquot of a saturated culture was harvested, and resuspended in 1 ml of water. One to 5-fold dilutions of a 5 ml aliquot of yeast cell suspension were spotted on YNB medium supplemented with 1 mM arginine (Arg, as control), 2 mM Urea or 1 mM Arg + 2 mM GeO2 at pH 5.2 for 5 days. http://bmbreports.org

The expressions of ZmNIP2;1, ZmNIP2;4 and ZmTIP4;4 were investigated in various maize organs at different developmental stages: seedling stage, silking stage and 15 days after pollination (Fig. 2). The three investigated genes were highly expressed in maize roots, which was coincident with their isolation from root cDNA library (Fig. 1). ZmNIP2;1 expressed constitutively in almost all tested organs, while ZmNIP2;4 and ZmTIP4;4 expressed in tissue-specific manners (Fig. 2). Besides root tissue, ZmNIP2;4 and ZmTIP4;4 transcripts were abundant in tissue of axis, and the latter was also abundant in premature ear (Fig. 2). BMB reports

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Fig. 2. Expressions of ZmNIP2;1, ZmNIP2;4 and ZmTIP4;4 transcripts in various maize organs. Maize plants were grown in field and the corresponding tissues at three developmental stages (seedling stage, silking stage and stage of 15 days after pollination) were collected for qPCR analysis of ZmNIP2;1, ZmNIP2;4 and ZmTIP4;4 expressions. Expression level was the relative level to that of control gene ZmGAPDH. Bars indicate means ± SD (n = 3).

　 The expressions of ZmNIP2;1, ZmNIP2;4 and ZmTIP4;4 were also investigated in maize plants under different N nutrition status. ZmTIP4;4 transcript was significantly up-regulated in roots by removing N from the solution, while expressions of ZmNIP2;1 and ZmNIP2;4 were not affected (Fig. 3A). In shoots, ZmTIP4;4 showed distinct expression patterns in between expanded and unexpanded leaves (Fig. 3B). In contrast to that of unexpanded leaves, ZmTIP4;4 transcript in expanded leaves was significantly increased by approx. 8-folds under N deficiency. However, expressions of ZmNIP2;1 and ZmNIP2;4 remained unaffected in either expanded or unexpanded leaves, similar to those in roots (Fig. 3). These results revealed a nitrogen-dependent expression manner of ZmTIP4;4 in roots and expanded leaves, suggesting a potential physiological link of ZmTIP4;4-mediated urea transport to plant N nutritional status. 98 BMB reports

Fig. 3. Expressions of ZmNIP2;1, ZmNIP2;4 and ZmTIP4;4 transcripts in maize root (A) and leaves (B) under N deficiency. Tendays-old maize plants were precultured hydroponically with continuous supply of 2 mM NH4NO3, and then subjected to N-free nutrient solution for 0, 1 and 3 days. Roots, unexpanded and expanded leaves were collected for qPCR analysis of ZmNIP2;1, ZmNIP2;4 and ZmTIP4;4 expressions. Expression levels were the relative levels to that of control gene Alpha tubulin4. The data were gene-wise normalized to the control plant (grown under N sufficient conditions), in which expression level was fixed to 1. Bars indicate means ± SD (n = 3), and significant differences at P ＜ 0.05 are indicated by different letters.

DISCUSSION To understand the molecular basis for urea transport in plants, an approach on growth complementation of urea-transport-deficient yeast strain has been successfully used to isolated plant genes encoding urea transport proteins (13, 14). Using the similar approach in this study, three maize aquaporin genes, ZmNIP2;1, ZmNIP2;4 and ZmTIP4;4, were obtained (Fig. 1). Although the yeast mutant (Δdur3) was defective in urea transporter at the plasma membrane, not only expression of plasma membrane proteins (ZmNIP2;1, ZmNIP2;4) but also vacuolar proteins (ZmTIP4;4) were able to complement their growth under low urea concentration. This was in agreement with the previous studies on isolation of both CpNIP1 from zucchini and several AtTIPs from Arabidopsis (13, 14). Although no any PIP member for transporting urea was screened from the cDNA library of maize, Arabidopsis and zucchini plants (Fig. 1) (13), one of maize PIP member, ZmPIP1-5b, was characterized to transport urea when expressed in oocytes (22). At least in maize cells, therefore, aquaprions of NIPs and PIPs could mediate the moving of urea across plasma membrane, http://bmbreports.org


and TIPs for transporting urea across tonoplast. 　ZmNIP2;1 (also named ZmLsi1) has been characterized as a Si transporter (20), similar to its close homolog OsNIP2;1 (also named OsLsi1) (16). They all belonged to the NIP III subgroup of NIP aquaporins, including another rice homolog OsNIP2;2 (also named OsLsi6) (23) and other three maize homologs ZmNIP2;2 (also named ZmLsi6) (20), ZmNIP2;3 and ZmNIP2;4 (Supplementary Fig. 2). Heterologous expression in oocytes showed that OsNIP2;1, OsNIP2;2 and ZmNIP2;1 were permeable to Si (16, 20, 23). Heterologous expression in yeast also showed that ZmNIP2;1 and ZmNIP2;4 could transport Si because the growth of transformed yeast were more sensitive to Ge, an toxic analog of Si (Fig. 1). Although NIP III subgroup members were suggested to be unique for Si transport (16), OsNIP2;1 was also permeable to other solutes, such as urea and boric acid (24), arsenite (25) and selenite (26) when it was expressed in oocytes. The mechanisms controlling the selectivity of transport substrates among NIPs were probably dependent on the NPA and aromatic/arginine regions of proteins (27). NIP III subgroup members normally had a larger, more open pore by compare to other subgroup members, resulting in a wider channel selectivity filter for different substrates (28). Considering the plasma membrane localization of ZmNIP2;1 in maize root epidermal cells (20), ZmNIP2;1 (and ZmNIP2;4) might function in maize roots on uptake of urea, as well as silicon, arsentie, selentie and probably other solutes. In maize roots ZmNIP2;1 and ZmNIP2;4 expressions were unaffected by N deficiency (Fig. 3) and ZmNIP2;1 and ZmNIP2;2 expressions were also unaffected by Si supplement (20), further supporting that they might not only transport certain substrate. 　ZmNIP2;1 and ZmNIP2;4 were segmental duplicates arising by maize genome allotetroplidization (Supplementary Table S1). Although both transporters shared similar transport activity to urea or Si, their expression patterns were distinct in various maize organs (Fig. 2). Expression of ZmNIP2;4 was more specific in roots and axis in contrast to the constitutive expression manner of ZmNIP2;1. Expression pattern shifts of the duplicated paralogous genes could reflect the divergence hypotheses that a duplicate gene pair might be involved in: nonfunctionalization, subfunctionalization and neofunctionalization (29). The specific organ-dependent expression of ZmNIP 2;4 suggested a neofunctionlization fate of this gene after it duplicated from the constitutive expressed ZmNIP2;1. Thus, besides urea uptake in root, ZmNIPs might also be involved in urea uptake and translocation in leaves and/or reproductive organs. 　Heterologous expression in yeast showed that ZmTIP4;4 was permeable to urea, neither to Si (Fig. 1) nor to ammonia (data not shown), similar to its homolog AtTIP4;1 and NtTIPa (12, 14). ZmTIP4;4 also seemed to be targeted to tonoplast in planta and might mediate urea loading and unloading through the vacuole membrane in plant cells (14). Like AtTIP4;1 in Arabidopsis roots (14), ZmTIP4;4 expression in maize roots was up-regulated by N starvation (Fig. 3A). This suggested that, http://bmbreports.org

when internal N sources is limited, ZmTIP4;4 might be involved in unloading urea from vacuolar storage to cytoplasm, which subsequently degraded into ammonium for further N assimilation. In particular, ZmTIP4;4 expression was significantly increased in expanded leaves under N deficiency, rather than that in unexpanded leaves (Fig. 3B). In contrast to unexpanded leaves, the expanded leaves serve as source of N for remobilzation under N deficiency. Thus, this result suggested that ZmTIP4;4-mediated urea transport was important for unloading vacuolar urea across tonoplast under N deficient conditions. 　Taken together, in the present study we isolated three maize aquaporin genes encoded urea-transporting proteins in yeast. Their organ- and nitrogen-dependent expression manners may suggest the diverse roles of these aquaporins on urea uptake and translocation through plasma membrane, or urea homeostasis across tonoplast. However, the physiological roles of these urea transporters in maize plants still remain to be elucidated by gain or loss of function analysis in the further studies.

MATERIALS AND METHODS Yeast complementation assay

A cDNA library was constructed from root tissue of maize inbred line B73. Total RNA was extracted using Trizol Reagent, + + and poly(A ) mRNA was enriched using poly(A ) RNA Tract kit (Invitrogen, Catalog No. K1520-02). Maize cDNA library TM was constructed into pDONR222 vector using CloneMiner cDNA Library Construction Kit as described by the manufactory manual (Invitrogen, Catalog No. 18249-029). By gateway LR recombination, this library was then transferred into a yeast expression vector pDR-GW vector (30). This cDNA library was transformed into the urea uptake-defective yeast strain YNVW1 (8). Transformants were plated and selected on solid yeast nitrogen base (YNB) medium supplemented with 2 mM urea as sole N source, buffered at pH 5.2 by MES-Tris. The plasmids were extracted from the obtained yeast clones and the inserted cDNA were sequenced. 　A 1 ml saturated cultures of yeast transformates carrying the corresponding constructs were harvested. The pellets were resuspended in 1 ml of water, and then spotted in 10-fold dilution on solid YNB medium supplement with 1 mM arginine, or additionally with 2 mM GeO2, or different concentration of urea as sole N source buffered at different pH by MES-Tris.

Expression analysis of maize genes

Maize plants (inbred line B73) were grown in field and the corresponding tissues were collected for gene expression analysis. Roots and leaves of maize seedlings were harvested rd at five-leaf stage. At silking stage, young leaves (the 3 leaf up rd the ear leaf), old leaves (the 3 leaf down the ear leaf), ear leaves, tassels and immature ears were collected. Axis and seeds were sampled 15 days after pollination (DAP). For N nutrition treatment, maize seedlings were grown in nutrient solBMB reports

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ution with 2 mM NH4NO3. Five-leaf stage plants were transferred to fresh solution without N for 1, or 3 days. Roots, unexpanded leaves (the first visible leaf) and expanded leaves (the middle matured leaf) were collected. 　The expression profiles of maize genes were examined using quantitative RT-PCR method (qPCR). The PCR reactions were performed using SYBR Green dye, and data were analyzed using 7500 SDS software 1.3 (Applied Biosystems). Expression of the ZmGAPDH gene (NM_001111943.1) was served as an internal control for organs specific expression, and Alpha tubulin4 gene (ZmTUB4, AJ420856.1) for N dependent expression (31). Three biological replicates of qPCR were performed for each sample. The primers for the aimed or control genes were: NIP2;1-F: 5'-GTGTGATTCATGCTCCATC GATC-3', NIP2;1-R: 5'-GGATCGAGAGAAGAGCGACACA-3'; NIP2;4-F: 5'-CACTCGCATTGTGTGTCCGGTTG-3', NIP2;4-R: 5'-GTACGAACGCTTGCTTGGCACG-3'; TIP4;4-F: 5'-ACACGAACCGCTTCCCAGGG-3', TIP4;4-R: 5'-CATTCCAT TCGAATCGAAACCG-3'; GAPDH-F: 5'-CTGGTTTCTACCGAC TTCCTTG-3', GAPDH-R: 5'-CGGCATACACAAGCAGCAAC-3'; TUB4-F: 5'-GCTATCCTGTGATCTGCCCTGA-3', TUB4-R: 5'-C GCCAAACTTAATAACCCAGTA-3'.

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Acknowledgements

This work received financial supports from the National Natural Science Foundation of China (30971863 and 31121062), the Ministry of Agriculture of China (2009ZX08009-131B and 2011ZX08003-005), and the Ministry of Education of China (NCET-08-0528).

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REFERENCES 1. Kojima, S., Bohner, A., Gassert, B., Yuan, L. and von Wiren, N. (2007) AtDUR3 represents the major transporter for high-affinity urea transport across the plasma membrane of nitrogen-deficient Arabidopsis roots. Plant. J. 52, 30-40. 2. Watson, C. J., Miller, H., Poland, P., Kilpatrick, D. J., Allen, M. D. B., Garrett, M. K. and Christianson, C. B. (1994) Soil properties and the ability of the urease inhibitor N-(n-BUTYL) thiophosphoric triamide (nBTPT) to reduce ammonia volatilization from surface-applied urea. Soil Biol. Biochem. 26, 1165-1171. 3. Marschner, H. (1995) Mineral Nutrition of Higher Plants. Academic Press, London, UK. 4. Hine, J. C. and Sprent, J. I. (1988) Growth of phaseolus vulgaris on various nitrogen sources: the importance of urease. J. Exp. Bot. 39, 1505-1512. 5. Gerendas, J., Zhu, Z. and Sattelmacher, B. (1998) Influence of N and Ni supply on nitrogen metabolism and urease activity in rice (Oryza sativa L.). J. Exp. Bot. 49, 1545-1554. 6. Krogmeier, M. J., McCarty, G. W. and Bremner, J. M. (1989) Phytotoxicity of foliar-applied urea. Proc. Natl. Acad. Sci. U.S.A. 86, 8189-8191. 7. Wilson, M. R., O'Donohue, S. I. and Walker, N. A. (1988) 100 BMB reports

16. 17.

18.

19.

20. 21.

The transport and metabolism of urea in Chara australis. J. Exp. Bot. 39, 763-774. Liu, L. H., Ludewig, U., Frommer, W. B. and von Wiren, N. (2003) AtDUR3 encodes a new type of high-affinity urea/H+ symporter in Arabidopsis. Plant Cell. 15, 790-800. Kojima, S., Bohner, A. and von Wiren, N. (2006) Molecular mechanisms of urea transport in plants. J. Membrane Biol. 212, 83-91. Johanson, U., Karlsson, M., Johansson, I., Gustavsson, S., Sjovall, S., Fraysse, L., Weig, A. R. and Kjellbom, P. (2001) The complete set of genes encoding major intrinsic proteins in arabidopsis provides a framework for a new nomenclature for major intrinsic proteins in plants. Plant Physiol. 126, 1358-1369. Chaumont, F., Barrieu, F., Wojcik, E., Chrispeels, M. J. and Jung, R. (2001) Aquaporins constitute a large and highly divergent protein family in maize. Plant. Physiol. 125, 1206-1215. Gerbeau, P., Guclu, J., Ripoche, P. and Maurel, C. (1999) Aquaporin Nt-TIPa can account for the high permeability of tobacco cell vacuolar membrane to small neutral solutes. Plant J. 18, 577-587. Klebl, F., Wolf, M. and Sauer, N. (2003) A defect in the yeast plasma membrane urea transporter Dur3p is complemented by CpNIP1, a Nod26-like protein from zucchini (Cucurbita pepo L.), and by Arabidopsis thaliana [delta]-TIP or [gamma]-TIP. FEBS Letters. 547, 69-74. Liu, L. H., Ludewig, U., Gassert, B., Frommer, W. B. and von Wiren, N. (2003) Urea transport by nitrogen-regulated tonoplast intrinsic proteins in Arabidopsis. Plant Physiol. 133, 1220-1228. Chaumont, F., Barrieu, F., Jung, R. and Chrispeels, M. J. (2000) Plasma membrane intrinsic proteins from maize cluster in two sequence subgroups with differential aquaporin activity. Plant Physiol. 122, 1025-1034. 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. Gao, Z., He, X., Zhao, B., Zhou, C., Liang, Y., Ge, R., Shen, Y. and Huang, Z. (2010) Overexpressing a putative aquaporin gene from wheat, TaNIP, enhances salt tolerance in transgenic Arabidopsis. Plant Cell Physiol. 51, 767-775. Soto, G., Fox, R., Ayub, N., Alleva, K., Guaimas, F., Erijman, E. J., Mazzella, A., Amodeo, G. and Muschietti, J. (2010) TIP5;1 is an aquaporin specifically targeted to pollen mitochondria and is probably involved in nitrogen remobilization in Arabidopsis thaliana. Plant J. 64, 1038-1047. Denning, G., Kabambe, P., Sanchez, P., Malik, A., Flor, R., Harawa, R., Nkhoma, P., Zamba, C., Banda, C., Magombo, C., Keating, M., Wangila, J. and Sachs, J. (2009) Input subsidies to improve smallholder maize productivity in Malawi: toward an african green revolution. PLoS Biol. 7, e23 Mitani, N., Yamaji, N. and Ma, J. F. (2009) Identification of maize silicon influx transporters. Plant Cell Physiol. 50, 5-12 Tallberg, P., Koski-Vahala, J. and Hartikainen, H. (2002) Germanium-68 as a tracer for silicon fluxes in freshwater http://bmbreports.org


sediment. Water Res. 36, 956-962. 22. Gaspar, M., Bousser, A., Sissoeff, I., Roche, O., Hoarau, J. and Mahe, A. (2003) Cloning and characterization of ZmPIP1-5b, an aquaporin transporting water and urea. Plant Sci. 165, 21-31. 23. Yamaji, N., Mitatni, N. and Ma, J. F. (2008) A transporter regulating silicon distribution in rice shoots. Plant Cell 20, 1381-1389. 24. Mitani, N., Yamaji, N. and Ma, J. F. (2008) Characterization of substrate specificity of a rice silicon transporter, Lsi1. Pflugers Archiv. Eur. J. Physiol. 456, 679-686. 25. Zhao, F.-J., Ago, Y., Mitani, N., Li, R.-Y., Su, Y.-H., Yamaji, N., McGrath, S. P. and Ma, J. F. (2010) The role of the rice aquaporin Lsi1 in arsenite efflux from roots. New Phytol. 186, 392-399. 26. Zhao, X. Q., Mitani, N., Yamaji, N., Shen, R. F. and Ma, J. F. (2010) Involvement of Silicon Influx Transporter OsNIP2;1 in Selenite Uptake in Rice. Plant Physiol. 153, 1871-1877. 27. Mitani, N., Yamaji, N., Zhao, F. J. and Ma, J. F. (2011) The aromatic/arginine selectivity filter of NIP aquaporins

http://bmbreports.org

28.

29.

30.

31.

plays a critical role in substrate selectivity for silicon, boron, and arsenic. J. Exp. Bot. 62, 4391-4398. Schnurbusch, T., Hayes, J., Hrmova, M., Baumann, U., Ramesh, S. A., Tyerman, S. D., Langridge, P. and Sutton, T. (2010) Boron toxicity tolerance in barley through reduced expression of the multifunctional aquaporin HvNIP2;1. Plant Physiol. 153, 1706-1715. Duarte, J. M., Cui, L., Wall, P. K., Zhang, Q., Zhang, X., Leebens-Mack, J., Ma, H., Altman, N. and dePamphilis, C. W. (2006) Expression pattern shifts following duplication indicative of subfunctionalization and neofunctionalization in regulatory genes of Arabidopsis. Mol. Biol. Evol. 23, 469-478. Loque, D., Lalonde, S., Looger, L. L., von Wiren, N. and Frommer, W. B. (2007) A cytosolic trans-activation domain essential for ammonium uptake. Nature 446, 195-198. Czechowski, T., Stitt, M., Altmann, T., Udvardi, M. K. and Scheible, W. R. (2005) Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol. 139, 5-17.

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