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JXB Advance Access originally published online on December 12, 2003
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Journal of Experimental Botany, Vol. 55, No. 396, pp. 539-541, February 1, 2004
© 2004 Oxford University Press

GENE NOTE

Characterization in maize of ZmTIP2-3, a root-specific tonoplast intrinsic protein exhibiting aquaporin activity

Received 11 August 2003; Accepted 3 November 2003

Félicie Lopez*, Agnès Bousser, Igor Sissoëff, Jackson Hoarau and Aline Mahé{dagger}

Institut de Biotechnologie des Plantes, Bât. 630, CNRS/UMR 8618, Université Paris-Sud, F-91405 Orsay Cedex, France

* Present address: Université d’Avignon, Faculté des Sciences UMR A408, 33 rue Louis Pasteur, F-84000 Avignon. France.
{dagger} To whom correspondence should be addressed. Fax: +33 1 69 15 34 24. E-mail: mahe{at}ibp.u-psud.fr


   Abstract
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 Abstract
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The characterization of the tonoplast intrinsic protein ZmTIP2-3 cDNA isolated from maize roots is reported. ZmTIP2-3 belongs to the TIP2 group according to the present nomenclature. The aquaporin function of ZmTIP2-3 protein was demonstrated using expression in X. laevis oocytes. Northern blot analyses revealed that ZmTIP2-3 was specifically expressed in roots. Salt and water stresses induced the accumulation of ZmTIP2-3 transcripts. By contrast, no effect of ABA was observed. An oscillation of ZmTIP2-3 transcript amount during the day–night cycle was observed with some typical features of genes regulated by a circadian mechanism.

Key words: Aquaporin, root, tonoplast intrinsic protein (TIP), Zea mays.

The activity of plant water channels (aquaporins) localized in vacuolar or in plasma membranes increases membrane water permeability (Ohshima et al., 2001). RNA expression profiles of some water channels localized in the plasma membrane (named PIP) and in the tonoplast (named TIP) are modulated in response to water stress (Sarda et al., 1999; Smart et al., 2001; Suga et al., 2002). Thus, aquaporins could contribute to drought tolerance by facilitating water transport between different organs and at the cellular level by maintaining water homeostasis (Kjellbom et al., 1999; Tyerman et al., 2002).

In this paper, the characterization of one member of the maize TIP family, ZmTIP2-3, is reported.

ZmTIP2-3 cDNA was obtained by RT-PCR technology using a poly dT adapter primer (5'-GGCCACGCGTCGACTAGTAC(T)17-3') for the RT reaction and a primer located in the ATG region (5'-AAGATGGTGAAGCTCGCATTTG-3') and a primer corresponding to the restriction site of poly dT adapter primer (5'-GGCCACGCGTCGACTAGTAC-3') for the PCR. The RT reaction was performed on RNA extracted from roots of maize plants (F2 inbred line INRA) grown hydroponically under an osmotic stress generated with 8% PEG.

The nucleotide sequence of the PCR fragment obtained (968 pb) contains an open reading frame of 747 pb and a 3' non-coding region of 218 pb. This cDNA sequence has 98.76% identity with ZmTIP2-3 cDNA isolated by Chaumont et al. (2001). The divergence is essentially located in the 5' region (95.9% identity) and corresponds to the deletion of three nucleotides, the addition of one nucleotide, and to five substitutions. The open reading frame differs by three nucleotides over 747. The deduced protein is nearly identical, only amino acid –229 is changed (leucine is replaced by a proline). The ZmTIP2-3 cDNA has been isolated from the F2 maize inbred line whereas Chaumont et al. (2001) studied the B73 maize inbred line which could explain the polymorphism observed between the two cDNAs. The two sequences are probably allelic forms of one gene.

ZmTIP2-3 belongs to the TIP2 subgroup. In maize, only ZmTIP1-1 has been characterized so far (Barrieu et al., 1998). No report has appeared on the characterization of other maize TIPs, in particular for the ZmTIP2 group.

The water channel activity of ZmTIP2-3 was assayed in Xenopus laevis oocytes as previously described by Gaspar et al. (2003). Oocytes injected with ZmTIP2-3 cRNA have a water permeability (Pf) approximately 7-fold higher than the control (Table 1).


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  		Table 1 . Assays for ZmTIP2-3 water transport activity in Xenopus oocytes The water permeability (Pf) was calculated by measuring the swelling of cRNA-injected oocytes submitted to hypotonic shock as previously described by Gaspar et al. (2003). Oocytes were injected with ZmTIP2-3 cRNA, Hv-{gamma}TIP cRNA or with water. The {gamma}TIP from Hordeum vulgare was used as a positive control. The Pf values are given as the mean ±SE.
 
The accumulation of ZmTIP2-3 transcripts in different vegetative tissues (leaves, roots), in reproductive tissues (anthers, ear) and in developing seeds was examined (Fig. 1). ZmTIP2-3 mRNA was detected only in root tissues whereas no detectable signal was found in other maize organs. To ensure that mRNA in various organ samples was intact, the blot was hybridized with the 18S rRNA probe. These data were consistent with the fact that ESTs corresponding to ZmTIP2-3 were found only in root tissue library (Chaumont et al., 2001).



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  		Fig. 1. Expression pattern of ZmTIP2-3 mRNA in various organs of maize. Total RNA was isolated from roots (R), stem (St), juvenile and mature leaves (JL, ML), juvenile and mature silk (JS, MS), young ear (YE), anther (A), and seeds harvested 10 d and 30 d after pollination (S10, S30). Total RNA (10 µg) was subjected to northern analysis as previously described by Gaspar et al. (2003). The membrane was hybridized with the 32P-labelled specific probes corresponding to the 3' end cDNA of ZmTIP2-3. The 3' UTR of ZmTIP2-3 cDNA was obtained by PCR using the following primers: 5'-GCCAGCAGGAGTACCCATGA-3' and 5'-GAAACGCCGAACTGTGCA-3'. Each membrane was hybridized with a constitutively expressed gene encoding 18S rRNA to check RNA loading. The membrane was exposed for 24 h (root extractions) and then for 3 weeks to detect the ZmTIP2-3 signal in the other organs.

 
The effects on the expression of ZmTIP2-3 mRNA of salt and water stresses (37 mM NaCl and 8% polyethylene glycol) and abscisic acid (10 µM), which could act as a signal in response to a water deficit, were then investigated. Stresses were imposed on 11-d-old seedlings grown in hydroponic medium under a 16/8 h light/dark photoperiod. ZmTIP2-3 transcript amounts clearly increased in PEG-stressed roots and in NaCl-treated roots compared with the control after 8 h of treatment (Fig. 2). This increase was maintained after 16 h of treatment. In addition, the induction seems to be stronger for the NaCl treatment. ZmTIP2-3 mRNA remained undetectable in leaves of stressed maize (data not shown). When ABA (10 µM) was added to the culture medium, the amount of ZmTIP2-3 mRNA remained unchanged. ABA action was checked by observing stomata closure. These results suggested that regulation of ZmTIP2-3 gene expression in response to osmotic stress is not mediated by ABA.



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  		Fig. 2. Effect of osmotic and ABA treatments on ZmTIP2-3 mRNA levels. 37 mM NaCl, 8% PEG or 10 µM ABA were added to hydroponic medium of 10-d-old maize root grown in under 16/8 h light/dark photoperiod condition as described by Gaspar et al. (2003). The different stresses were applied 2 h before midday and roots were harvested 8 h or 16 h after treatment. Northern analysis was performed as described in Fig. 1.

 
The expression pattern of ZmTIP2-3 was followed during two consecutive day/night cycles (Fig. 3). The ZmTIP2-3 mRNA abundance revealed a diurnal rhythm during the consecutive day-night cycles. The amount of transcripts increased shortly before the light period, reached a maximum of expression after 4 h light and declined to a low level during the end of the light period and the beginning of the dark period (Fig. 3A). When photoperiod length was inverted (8/16 h light/dark) or when continuous darkness was imposed, the diurnal oscillations were maintained (Fig. 3B, C). These results suggest that ZmTIP2-3 gene expression is regulated diurnally at the transcriptional level and that this regulation could be controlled by an internal clock.



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  		Fig. 3. Day–night oscillations of ZmTIP2-3 transcript levels in maize roots grown under different photoperiods. Roots from 8-d-old maize grown in various photoperiod conditions were harvested every 2 h for 48 h. Northern analysis were performed on total RNA (10 µg) extracted from roots of plants grown under a 16/8 h day/night photoperiod (A), a 8/16 h light/dark cycle (B) or under a 16/8 h day/night photoperiod and maintained in darkness for the last cycles (C). RNA hybridization was performed as described in Fig. 1. White and grey boxes indicate day and night periods, respectively.

 
To summarize, a cDNA encoding ZmTIP2-3, a root-specific vacuolar aquaporin corresponding to the TIP2 subgroup has been isolated and characterized. The results of this study demonstrate that ZmTIP2-3 gene expression is modulated by osmotic stress and is insensitive to ABA. In addition, a circadian regulation of ZmTIP2-3 mRNA accumulation was observed. The fact that ZmTIP2-3 is expressed early in maize roots subjected to osmotic stress strengthens the idea that ZmTIP2-3 might have a role in the regulation of water transfer across the tonoplast in order to maintain homeostasis of the cell.


   Acknowledgements
 
We are grateful to J Grisvard for providing us with an 18S probe, PH Schunmann for the Hv-{gamma}TIP clone and JP Muller for the gift of oocytes. P Lopez was financially supported by a grant from Biogemma. We thank Professor G Noctor for his critical reading of the manuscript.


   References
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 Abstract
 References
 
Barrieu F, Chaumont F, Chrispeels MJ. 1998. High expression of the tonoplast aquaporin ZmTIP1 in epidermal and conducting tissues of maize. Plant Physiology 117, 1153–1163.[Abstract/Free Full Text]

Chaumont F, Barrieu F, Wojcik E, Chrispeels MJ, Jung R. 2001. Aquaporins constitute a large and highly divergent protein family in maize. Plant Physiology 125, 1206–1215.[Abstract/Free Full Text]

Gaspar M, Sissoëff I, Bousser A, Roche O, Hoarau J, Mahé A. 2003. Cloning and characterization of ZmPIP1-5, an aquaporin transporting water and urea. Plant Science 165, 21–31.[CrossRef]

Kjellbom P, Larsson C, Johansson II, Karlsson M, Johanson U. 1999. Aquaporins and water homeostasis in plants. Trends in Plant Science 4, 308–314.[CrossRef][Web of Science][Medline]

Ohshima Y, Iwasaki I, Suga S, Murakami M, Inoue K, Maeshima M. 2001. Low aquaporin content and low osmotic water permeability of the plasma and vacuolar membranes of a CAM plant Graptopetalum paraguayense: comparison with radish. Plant and Cell Physiology 42, 1119–1129.[Abstract/Free Full Text]

Sarda X, Tousch D, Ferrare K, Cellier F, Alcon C, Dupuis JM, Casse F, Lamaze T. 1999. Characterization of closely related delta-TIP genes encoding aquaporins which are differentially expressed in sunflower roots upon water deprivation through exposure to air. Plant Molecular Biology 40, 179–191.[CrossRef][Web of Science][Medline]

Smart LB, Moskal WA, Cameron KD, Bennett AB. 2001. MIP genes are down-regulated under drought stress in Nicotiana glauca. Plant and Cell Physiology 42, 686–693.[Abstract/Free Full Text]

Suga S, Komatsu S, Maeshima M. 2002. Aquaporin isoforms responsive to salt and water stresses and phytohormones in radish seedlings. Plant and Cell Physiology 43, 1229–1237.[Abstract/Free Full Text]

Tyerman SD, Niemietz CM, Bramley H. 2002. Plant aquaporins: multifunctional water and solute channels with expanding roles. Plant, Cell and Environment 25, 173–194.[CrossRef][Medline]


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This Article
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