Li R , Wang J , Li S , Zhang L , Qi C , Weeda S , Zhao B , Ren S , Guo YD .

	Figure 1. Heatmap of PIPs and TIPs expression in root, stem, flower, and root tissues of tomato plants. SlPIP2s transcript abundance in leaves, roots, stems and flowers was analyzed using qRT-PCR.The expression values were calculated using the 2–Δt method and the 18S housekeeping gene, and the average log2 values of three replicates were used to generate a heat map in cluster 3.0 software. Green represents low expression, and red denotes high expression. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).
	Figure 2. Phylogenetic relationship of SlPIP2;1, SlPIP2;7 and SlPIP2;5 with PIP proteins. The predicted amino acid sequences of the three SlPIP2s and their corresponding sequences from other species were aligned using the ClustalW2 sequence alignment program. The phylogenetic tree was constructed using MEGA6 software.
	Figure 3. Alignment of predicted amino acid sequence of tomato aquaporins (SlPIP2;1, SlPIP2;7 and SlPIP2;5) with other aquaporins (DNAMAN). The predicted amino acid sequences of SlPIP2;1, SlPIP2;7 and SlPIP2;5 were compared with aquaporins from different sources. Transmembrane domains are shown with a dashed line below the alignment; circles indicate the NPA selectivity filter.
	Figure 4. (a–p) Subcellular localizations of SlPIP2;1, SlPIP2;7 and SlPIP2;5 in onion epidermal cells. The fusion protein SlPIP2;1–GFP (a,e,i,m), SlPIP2;7–GFP (b,f,j,n), SlPIP2;5–GFP (c,g,k,o) and GFP alone (d,h,l,p) were transiently expressed under the control of the cauliflower mosaic virus 35S promoter in onion epidermal cells.Images are dark field for green fluorescence (a–d, i–l,); merged (e–h, m–p); plasmolysed the cells with 0.8 M mannitol (i–p). PM, plasma membrane; CW, cell wall. (r–u) Tissue localization of expression of aquaporins in roots of tomato. Expression was analyzed by in situ hybridization in root. Three candidate aquaporin genes SlPIP2;7 (t) and SlPIP2;1 (u) were studied, panel (r) is the positive control and panel (s) is the negative control. EP:epidermis, C: cortex, EN: endodermis, S: stele. Bar = 400 μm.
	Figure 5. Assay on functional expression of SlPIP2;1, SlPIP2;7 and SlPIP2;5 in Xenopus oocytes.(A) Increase in relative volume of oocytes injected with SlPIP2;1, SlPIP2;7 and SlPIP2;5 cDNA after transfer to hypoosmotic medium, using water as control. (B) Pf-values of oocytes injected with SlPIP2;1, SlPIP2;7 and SlPIP2;5 cRNA, using water as the control. Pf of oocytes expressing SlPIP2;1, SlPIP2;7 and SlPIP2;5 present significant differences from negative control. Significant differences were determined by Duncan’s multiple range test (P < 0.05). Error bars indicate SD (n = 3). (C) SlPIP2;1, SlPIP2;7 and SlPIP2;5 expressing oocytes were exposed to different external (pHe) or internal (pHi) pH conditions. Pf for oocytes expressing SlPIP2s exposed to internal acidification is statistically different from itscontrol (i.e. pHi = 7.5), while treatment with pHe = 6.0 does notresult in a significant inhibition when compared with itscontrol (pHe = 7.5). Significant differences were determined by Duncan’s multiple range test (P < 0.05). Error bars indicate SD (n = 3). (D) SlPIP2;1, SlPIP2;7 and SlPIP2;5 expressing oocytes were exposed to 100 mM HgCl2 condition. Pf for oocytes expressing SlPIP2s exposed toHg result in a significant inhibition when compared with itscontrol. Significant differences were determined by Duncan’s multiple range test (P < 0.05). Error bars indicate SD (n = 3).
	Figure 6. Root hydraulic conductivity of tomato plants. (A) The relation between P to Jv of water supplied in tomato root system. (B) Hydraulic conductivity of tomato roots under normal and drought stress. Theroot hydraulic conductance was down regulated under drought treatment. Significant differences were determined by Duncan’s multiple range test (P < 0.05). Error bars indicate SD (n = 3). Drought (C) and salt (D) regulation of SlPIP2;1, SlPIP2;7 and SlPIP2;5 expression in tomato roots. Significant differences were determined by Duncan’s multiple range test (P < 0.05). Error bars indicate SD (n = 3).
	Figure 7. Growth of the wild-type and transgenic plants under normal and drought growth conditions. (A) Germination rates of the wild-type and transgenic Arabidopsis under normal conditions. Error bars indicate SD (n = 3). (B) Germination rates of the wild-type and transgenic Arabidopsis under drought conditions. Error bars indicate SD (n = 3). (C) The wild-type and transgenic Arabidopsis plants were photographed 20 days after germination on soil before drought treatment. (D) Phenotypes of wild-type and transgenic Arabidopsis plants subjected to drought stress for 10 days. (E) The wild-type and transgenic tomato plants were photographed 30 d after germination on soil before drought treatment. (F) Phenotypes of wild-type and transgenic tomato plants subjected to drought stress for 15 days. L1: Transgenic line1; L2: Transgenic line2.Confirmation of the transgenic lines. RT- PCR or real time quantitative analyses of the expression of SlPIP2;4, SlPIP2;5 or SlPIP2;7 in the wild-type plants and independent transgenic lines (L-1and L-2) in (G) two week-old whole Arabidopsis plants grown in MS medium and (H) two-week-old tomato plants grown in MS medium.
	Figure 8. Analysis of RWC and MDA in transgenic lines under drought stress. Tomato leaves were sampled from WT and transgenic lines under drought stress for 15 d to detect RWC (A) and MDA (B). Significant differences were determined by Duncan’s multiple range test (P < 0.05). Error bars indicate SD (n = 3).

Aharon, Overexpression of a plasma membrane aquaporin in transgenic tobacco improves plant vigor under favorable growth conditions but not under drought or salt stress. 2003, Pubmed

Aharon, Overexpression of a plasma membrane aquaporin in transgenic tobacco improves plant vigor under favorable growth conditions but not under drought or salt stress. 2003, Pubmed
Alexandersson, Transcriptional regulation of aquaporins in accessions of Arabidopsis in response to drought stress. 2010, Pubmed
Alleva, Cloning, functional characterization, and co-expression studies of a novel aquaporin (FaPIP2;1) of strawberry fruit. 2010, Pubmed , Xenbase
Alleva, Plasma membrane of Beta vulgaris storage root shows high water channel activity regulated by cytoplasmic pH and a dual range of calcium concentrations. 2006, Pubmed
Aroca, Regulation of root water uptake under abiotic stress conditions. 2012, Pubmed
Ayadi, Identification and characterization of two plasma membrane aquaporins in durum wheat (Triticum turgidum L. subsp. durum) and their role in abiotic stress tolerance. 2011, Pubmed , Xenbase
Beebo, Life with and without AtTIP1;1, an Arabidopsis aquaporin preferentially localized in the apposing tonoplasts of adjacent vacuoles. 2009, Pubmed
Bellati, Intracellular pH sensing is altered by plasma membrane PIP aquaporin co-expression. 2010, Pubmed , Xenbase
Bienert, Solanaceae XIPs are plasma membrane aquaporins that facilitate the transport of many uncharged substrates. 2011, Pubmed , Xenbase
Bienert, A conserved cysteine residue is involved in disulfide bond formation between plant plasma membrane aquaporin monomers. 2012, Pubmed , Xenbase
Chrispeels, Aquaporins: the molecular basis of facilitated water movement through living plant cells? 1994, Pubmed
Daniels, The plasma membrane of Arabidopsis thaliana contains a mercury-insensitive aquaporin that is a homolog of the tonoplast water channel protein TIP. 1994, Pubmed , Xenbase
Fischer, On the pH regulation of plant aquaporins. 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
Heath, Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation. 1968, Pubmed
Heymann, Aquaporins: Phylogeny, Structure, and Physiology of Water Channels. 1999, Pubmed
Jang, An expression analysis of a gene family encoding plasma membrane aquaporins in response to abiotic stresses in Arabidopsis thaliana. 2004, Pubmed
Jang, Transgenic Arabidopsis and tobacco plants overexpressing an aquaporin respond differently to various abiotic stresses. 2007, Pubmed
Javot, Role of a single aquaporin isoform in root water uptake. 2003, 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
Kaldenhoff, Significance of plasmalemma aquaporins for water-transport in Arabidopsis thaliana. 1998, Pubmed , Xenbase
Katsuhara, Over-expression of a barley aquaporin increased the shoot/root ratio and raised salt sensitivity in transgenic rice plants. 2003, Pubmed
Knepper, The aquaporin family of molecular water channels. 1994, Pubmed
Li, Cotton plasma membrane intrinsic protein 2s (PIP2s) selectively interact to regulate their water channel activities and are required for fibre development. 2013, Pubmed
Li, Expressions of three cotton genes encoding the PIP proteins are regulated in root development and in response to stresses. 2009, Pubmed
Lian, The role of aquaporin RWC3 in drought avoidance in rice. 2004, Pubmed , Xenbase
Liu, Proline accumulation and salt-stress-induced gene expression in a salt-hypersensitive mutant of Arabidopsis. 1997, Pubmed
Ma, Loss of TIP1;1 aquaporin in Arabidopsis leads to cell and plant death. 2004, Pubmed
Maurel, AQUAPORINS AND WATER PERMEABILITY OF PLANT MEMBRANES. 1997, Pubmed , Xenbase
Maurel, The vacuolar membrane protein gamma-TIP creates water specific channels in Xenopus oocytes. 1993, Pubmed , Xenbase
Maurel, Plant aquaporins: membrane channels with multiple integrated functions. 2008, Pubmed
McLean, Root hydraulic conductance and aquaporin abundance respond rapidly to partial root-zone drying events in a riparian Melaleuca species. 2011, Pubmed
Nguyen, Genome-wide expression analysis of rice aquaporin genes and development of a functional gene network mediated by aquaporin expression in roots. 2013, Pubmed
Postaire, A PIP1 aquaporin contributes to hydrostatic pressure-induced water transport in both the root and rosette of Arabidopsis. 2010, Pubmed
Regan, Accurate and high resolution in situ hybridization analysis of gene expression in secondary stem tissues. 1999, Pubmed
Reuscher, Genome-wide identification and expression analysis of aquaporins in tomato. 2013, Pubmed
Sade, Improving plant stress tolerance and yield production: is the tonoplast aquaporin SlTIP2;2 a key to isohydric to anisohydric conversion? 2009, Pubmed
Schüssler, The effects of the loss of TIP1;1 and TIP1;2 aquaporins in Arabidopsis thaliana. 2008, Pubmed
Siemens, Changes in root water flow properties of solution culture-grown trembling aspen (Populus tremuloides) seedlings under different intensities of water-deficit stress. 2004, Pubmed
Smart, MIP genes are down-regulated under drought stress in Nicotiana glauca. 2001, Pubmed
Sutka, Natural variation of root hydraulics in Arabidopsis grown in normal and salt-stressed conditions. 2011, Pubmed
Toenniessen, Advances in plant biotechnology and its adoption in developing countries. 2003, Pubmed
Tournaire-Roux, Cytosolic pH regulates root water transport during anoxic stress through gating of aquaporins. 2003, Pubmed , Xenbase
Uehlein, The tobacco aquaporin NtAQP1 is a membrane CO2 pore with physiological functions. 2003, Pubmed , Xenbase
Verslues, Methods and concepts in quantifying resistance to drought, salt and freezing, abiotic stresses that affect plant water status. 2006, Pubmed
Xu, A banana aquaporin gene, MaPIP1;1, is involved in tolerance to drought and salt stresses. 2014, Pubmed
Yue, Molecular cloning and expression analysis of tea plant aquaporin (AQP) gene family. 2014, Pubmed
Zhang, A cotton gene encoding a plasma membrane aquaporin is involved in seedling development and in response to drought stress. 2013, Pubmed
Zhang, Water and urea permeability properties of Xenopus oocytes: expression of mRNA from toad urinary bladder. 1991, Pubmed , Xenbase
Zhou, Constitutive overexpression of soybean plasma membrane intrinsic protein GmPIP1;6 confers salt tolerance. 2014, Pubmed
Zhou, Overexpression of the wheat aquaporin gene, TaAQP7, enhances drought tolerance in transgenic tobacco. 2012, Pubmed , Xenbase
Šurbanovski, Expression of Fragaria vesca PIP aquaporins in response to drought stress: PIP down-regulation correlates with the decline in substrate moisture content. 2013, Pubmed