Besse M , Knipfer T , Miller AJ , Verdeil JL , Jahn TP , Fricke W .

BBS/E/J/000C0651 Biotechnology and Biological Sciences Research Council , BBS/E/C/00004166 Biotechnology and Biological Sciences Research Council

	Fig. 1. Leaf regions analysed in the present study. Leaf samples were taken from up to three developmental zones along the elongating leaf three of barley, the elongation zone (EZ), the non-elongation zone (NEZ) and the emerged blade (EmBL). In addition, a leaf sample was taken from the mature blade and from midway along the sheath of leaf two. Leaf samples consisted of 2-cm-long segments which were taken from the centre region of the respective zone. For qPCR analyses of the elongation zone of leaf three, the younger leaf four which was wrapped within this region was removed.
	Fig. 2. Bayesian phylogenetic analysis of barley major intrinsic proteins (MIPs). Barley MIP protein sequences were aligned with MIPs of Arabidopsis, maize, and rice using ClustalW. Barley MIPs analysed are highlighted. Annotations and protein sequences are given in Supplementary File S2 at JXB online. Concerning HvNIP2;1, Fig. 2 refers to the gene annotated first (accession number BA166444) and not to the gene referred to by Schnurbusch et al. (2010) as a boron transporter (HvNIP2;1). The latter gene is identical in sequence to the silicon transporter (HvLsi1, shown) reported by Chiba et al. (2009). HvSIP2;1 is not annotated; only a partial sequence is known, which shows the highest homology to AtSIP2;1 among Arabidopsis SIPs. The distance scale represents the evolutionary distance, expressed as the number of substitutions per amino acid. The figures displayed on the main nodes reflect the posterior probability.
	Fig. 3. Real-time (qPCR) expression analyses of major intrinsic proteins in leaf regions of barley. The elongation zone (EZ), adjacent non-elongation zone (NEZ), and emerged-blade portion (EmBL) of the growing leaf three were analysed, together with the blade and sheath of the mature leaf two (L2 and Sh, respectively). (A, B, C, D) Values of relative expression show how many times higher or lower the expression of a candidate gene was compared with that of the reference genes (ubiquitin, H+-ATPase, HvSIP2;1), using the 2–(ΔCt) method. (E) Total expression of the PIP1 and PIP2 subfamily, together with (F) ratio of expression. Results are averages ±SD (error bars) of three experiments. Statistical significance of difference in expression between leaf regions is summarised in Supplementary File S4 at JXB online.
	Fig. 4. The percentage contribution of individual family members to total expression of PIP1s, PIP2s, and TIPs in leaf regions of barley. Data were calculated from the expression values shown in Fig. 3. The TIPs analysed should represent the bulk of expression of TIPs (Alexandersson et al., 2005; Sakurai et al., 2005). HvTIP2;1, HvTIP2;2, HvTIP3;1, and HvTIP5;1 together accounted for less than 1% of the expression of TIPs and are not included in the pie charts. Numbers give the mean percentage contribution calculated from three experiments. Standard deviations are given in Supplementary File S4 at JXB online.
	Fig. 5. Test of water channel function of selected barley MIPs through expression in yeast and subsequent swelling assays of isolated spheroplasts. (A) Swelling kinetics of a representative batch of spheroplasts. Each curve is the average of 10–20 swelling kinetics, fitted with two-exponential equations. Control spheroplasts were isolated from yeast which had been transformed with the empty vector used for transformation (pYeDP60u, negative control) or with an Arabidopsis MIP (AtTIP1;2) known to show water channel activity (positive control). (B) Rate constants of water flow in spheroplasts. Results are averages ±SD (error bars) of the analysis of (n) kinetics as shown in (A): HvPIP2;5, (4); HvTIP1;1, (8); HvTIP2;3, (8), HvPIP2;2, (8); HvPIP2;7, (2, no error bar shown, values of 4.5 and 3.6 s−1); HvPIP1;2, (3); HvTIP1;2 (4); negative control, (3); and positive control (4). Rate constants of swelling of barley-MIP expressing spheroplasts were significantly higher (P <0.001) than that of (negative) control spheroplasts containing the empty vector used for transformation.
	Fig. 6. Test of water channel function of HvPIP2;2 and HvPIP2;7 through transient (3 d) expression in Xenopus laevis oocytes. Results are means ±SD (error bars) of six (control) and eight (HvPIP2;2, HvPIP2;7) oocytes, analysed from one representative batch. Osmotic water permeability of oocytes expressing HvPIP2;2 or HvPIP2;7 was significantly higher (P <0.001) than that of water-injected (control) oocytes, as was the difference in water permeability between HvPIP2;2 and HvPIP2;7-expressing oocytes (P <0.001).
	Fig. 7. Tissue-specific expression of barley MIPs in leaf regions. (A) Scheme and micrograph of a cross-section of a mature blade, identifying tissues. (B) In situ hybridization data. Expression is shown as blue colour, and was detected using antisense probes of genes of interest. An antisense designed against 18S ribosomal RNA was used as positive control. A sense probe was tested for all genes as negative control and is shown representatively for ribosomal RNA. PBS, parenchymatous bundle sheath; L3, leaf three; Sh L2, sheath of leaf two; scale bar=50 μm.
	Fig. 8. Subcellular localization of barley MIPs as studied through transient (2 d) expression in onion epidermis. Expression was viewed in epidermal peels using an Olympus FV1000 confocal laser scanning microscope. (A) Candidates genes fused to the enhanced yellow fluorescent protein (EYFP); (B) Cytoplasm marker pSAT6-DsRed2-N1 (DsRED, red fluorescence) and pBIN20-plasma membrane-mCherry marker (pm mCherry, red fluorescence); (C) overlay of (A) and (B); (D) magnified parts of cells shown in (C); (E) transmitted light micrographs of cells shown in (A) to (D). Scale bar (A, B, C, E)=50 μm, Scale bar (D)=25 μm.
	Fig. 9. Casparian bands in the mestome sheath in different developmental regions of barley leaves. Cross-sections were stained with berberin-hemisulphate/toluidine blue and viewed under UV-light (Brundrett et al., 1988). Occurrence of Casparian bands (yellow fluorescence) is indicated by arrows. Xylem vessels showed autofluorescence. MX, metaxylem; PX, protoxylem; MSH, mestome sheath; PH, phloem. Scale bar (A, C, E, G)=50 μm, scale bar (B, D, F, H)=25 μm.

Alexandersson, Whole gene family expression and drought stress regulation of aquaporins. 2005, Pubmed

Alexandersson, Whole gene family expression and drought stress regulation of aquaporins. 2005, Pubmed
Balk, Rapid stalk elongation in tulip (Tulipa gesneriana L. cv. Apeldoorn) and the combined action of cold-induced invertase and the water-channel protein gammaTIP. 1999, Pubmed
Barrieu, Tonoplast intrinsic proteins from cauliflower (Brassica oleracea L. var. botrytis): immunological analysis, cDNA cloning and evidence for expression in meristematic tissues. 1998, Pubmed
Bienert, Specific aquaporins facilitate the diffusion of hydrogen peroxide across membranes. 2007, Pubmed
Boscari, Potassium channels in barley: cloning, functional characterization and expression analyses in relation to leaf growth and development. 2009, Pubmed , Xenbase
Boursiac, Stimulus-induced downregulation of root water transport involves reactive oxygen species-activated cell signalling and plasma membrane intrinsic protein internalization. 2008, Pubmed , Xenbase
Breitling, Rank products: a simple, yet powerful, new method to detect differentially regulated genes in replicated microarray experiments. 2004, Pubmed
Breitling, Rank-based methods as a non-parametric alternative of the T-statistic for the analysis of biological microarray data. 2005, Pubmed
Calamita, Water permeability of rat liver mitochondria: A biophysical study. 2006, Pubmed
Chaumont, Characterization of a maize tonoplast aquaporin expressed in zones of cell division and elongation. 1998, Pubmed , Xenbase
Chiba, HvLsi1 is a silicon influx transporter in barley. 2009, Pubmed , Xenbase
Close, A new resource for cereal genomics: 22K barley GeneChip comes of age. 2004, Pubmed
Danielson, Unexpected complexity of the aquaporin gene family in the moss Physcomitrella patens. 2008, Pubmed
Drummond, BEAST: Bayesian evolutionary analysis by sampling trees. 2007, Pubmed
Fetter, Interactions between plasma membrane aquaporins modulate their water channel activity. 2004, Pubmed , Xenbase
Flexas, Tobacco aquaporin NtAQP1 is involved in mesophyll conductance to CO2 in vivo. 2006, Pubmed , Xenbase
Frangne, Expression and distribution of a vaculoar aquaporin in young and mature leaf tissues of Brassica napus in relation to water fluxes. 2001, Pubmed
Fricke, Biophysical limitation of cell elongation in cereal leaves. 2002, Pubmed
Hachez, The expression pattern of plasma membrane aquaporins in maize leaf highlights their role in hydraulic regulation. 2008, Pubmed
Hachez, Localization and quantification of plasma membrane aquaporin expression in maize primary root: a clue to understanding their role as cellular plumbers. 2006, Pubmed
Hachez, Modulating the expression of aquaporin genes in planta: A key to understand their physiological functions? 2006, Pubmed
Hamann, Improved cloning and expression of cytochrome P450s and cytochrome P450 reductase in yeast. 2007, Pubmed
Hanba, Overexpression of the barley aquaporin HvPIP2;1 increases internal CO(2) conductance and CO(2) assimilation in the leaves of transgenic rice plants. 2004, Pubmed
Heinen, Role of aquaporins in leaf physiology. 2009, Pubmed
Hukin, Sensitivity of cell hydraulic conductivity to mercury is coincident with symplasmic isolation and expression of plasmalemma aquaporin genes in growing maize roots. 2002, Pubmed
Jabnoune, Diversity in expression patterns and functional properties in the rice HKT transporter family. 2009, Pubmed , Xenbase
Jang, An expression analysis of a gene family encoding plasma membrane aquaporins in response to abiotic stresses in Arabidopsis thaliana. 2004, Pubmed
Johanson, The complete set of genes encoding major intrinsic proteins in Arabidopsis provides a framework for a new nomenclature for major intrinsic proteins in plants. 2001, Pubmed
Johansson, The role of aquaporins in cellular and whole plant water balance. 2000, Pubmed , Xenbase
Kaldenhoff, Functional aquaporin diversity in plants. 2006, Pubmed
Katsuhara, Barley plasma membrane intrinsic proteins (PIP Aquaporins) as water and CO2 transporters. 2008, Pubmed
Katsuhara, Functional analysis of water channels in barley roots. 2002, Pubmed , Xenbase
Katsuhara, Over-expression of a barley aquaporin increased the shoot/root ratio and raised salt sensitivity in transgenic rice plants. 2003, Pubmed
Knipfer, Root pressure and a solute reflection coefficient close to unity exclude a purely apoplastic pathway of radial water transport in barley (Hordeum vulgare). 2010, Pubmed
Knipfer, Aquaporin-facilitated water uptake in barley (Hordeum vulgare L.) roots. 2011, Pubmed
Li, Characterization of OsPIP2;7, a water channel protein in rice. 2008, Pubmed , Xenbase
Liu, Purification and functional characterization of aquaporin-8. 2006, Pubmed
Ma, A silicon transporter in rice. 2006, Pubmed
Marini, A family of ammonium transporters in Saccharomyces cerevisiae. 1997, Pubmed
Maurel, Plant aquaporins: membrane channels with multiple integrated functions. 2008, Pubmed
Maurel, The vacuolar membrane protein gamma-TIP creates water specific channels in Xenopus oocytes. 1993, Pubmed , Xenbase
Maurel, Plant aquaporins: novel functions and regulation properties. 2007, Pubmed
Nelson, A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. 2007, Pubmed
Nour-Eldin, Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments. 2006, Pubmed
Pata, Molecular cloning and characterization of OsCDase, a ceramidase enzyme from rice. 2008, Pubmed
Pfaffl, A new mathematical model for relative quantification in real-time RT-PCR. 2001, Pubmed
Reisen, New insights into the tonoplast architecture of plant vacuoles and vacuolar dynamics during osmotic stress. 2005, Pubmed
Richardson, Cuticular permeance in relation to wax and cutin development along the growing barley (Hordeum vulgare) leaf. 2007, Pubmed
Richardson, Cloning and expression analysis of candidate genes involved in wax deposition along the growing barley (Hordeum vulgare) leaf. 2007, Pubmed
Sakurai, Identification of 33 rice aquaporin genes and analysis of their expression and function. 2005, Pubmed
Schnurbusch, Boron toxicity tolerance in barley through reduced expression of the multifunctional aquaporin HvNIP2;1. 2010, Pubmed , Xenbase
Schüssler, The effects of the loss of TIP1;1 and TIP1;2 aquaporins in Arabidopsis thaliana. 2008, Pubmed
Shen, BarleyBase--an expression profiling database for plant genomics. 2005, Pubmed
Takano, The Arabidopsis major intrinsic protein NIP5;1 is essential for efficient boron uptake and plant development under boron limitation. 2006, Pubmed , Xenbase
Tournaire-Roux, Cytosolic pH regulates root water transport during anoxic stress through gating of aquaporins. 2003, Pubmed , Xenbase
Volkov, Water permeability differs between growing and non-growing barley leaf tissues. 2007, Pubmed
Wei, HvPIP1;6, a barley (Hordeum vulgare L.) plasma membrane water channel particularly expressed in growing compared with non-growing leaf tissues. 2007, Pubmed , Xenbase
Wu, Casparian strips in needles are more solute permeable than endodermal transport barriers in roots of Pinus bungeana. 2005, Pubmed
Ye, Cell-to-cell pathway dominates xylem-epidermis hydraulic connection in Tradescantia fluminensis (Vell. Conc.) leaves. 2008, Pubmed
Zelazny, FRET imaging in living maize cells reveals that plasma membrane aquaporins interact to regulate their subcellular localization. 2007, Pubmed , Xenbase