JXB Advance Access originally published online on July 28, 2003
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Journal of Experimental Botany, Vol. 54, No. 390, pp. 2193-2194,
September 1, 2003
© 2003 Oxford University Press
Putative PIP1 genes isolated from apple: expression analyses during fruit development and under osmotic stress
Received 13 March 2003; Accepted 15 June 2003
1 National Institute of Fruit Tree Science, Tsukuba, Ibaraki 305-8605, Japan
2 Huazhong Agricultural University, Wuhan, Hubei 430070, China
* To whom correspondence should be addressed. Fax: +81 29 838 6437. E-mail: takaya{at}affrc.go.jp
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Abstract |
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To gain insight into the function of plasma membrane intrinsic protein (PIP) genes in apple, two genes, MdPIP1a and MdPIP1b, were isolated. MdPIP1 expression was in accordance with the volume increase during fruit development, which is a loading process of water and solutes. In addition, the expression of MdPIP1 was up-regulated in the stems by osmotic stress. These results indicate that MdPIP1 may play important roles not only in fruit expansion, but also in maintaining water homeostasis under stress conditions.
Key words: Apple, fruit expansion, gene expression, osmotic stress, plasma membrane intrinsic protein (PIP).
Most fruits contain 80–95% water on a fresh weight basis. Rapid expansion of fruit is achieved by accumulating a large amount of water during the developmental processes (Coombe, 1976). Water deficiency makes fruits reduce in size and even fail to expand so as to drop, while excessive water supply decreases sugar content and results in poor taste. A dramatic change in water condition also leads to unfavourable fruit quality. For example, irrigation or rain following a long time of drought usually incurs fruit splitting. All these factors will greatly reduce the market value of fruits and bring economic loss to farmers. In addition, the plant stem dedicates itself to fruit yield and quality by transporting water and nutrients. Water homeostasis in this organ is important for the proper maintenance of this function. Fruit trees as perennial plants have to survive in the located environment for a long time once they have been planted. Therefore, many environmental factors can affect the water status. Recently, it has been documented that aquaporins are crucial to controlling water homeostasis in plants (Chrispeels and Agree, 1994). Investigations on the role of plasma membrane intrinsic protein (PIP) genes in regulating the accumulation of water during fruit development and under stress conditions should be valuable.
To isolate a partial PIP cDNA, RT-PCR was performed. The primer pairs 5'-GAC TAC AA(G/A) GA(G/A) CCA CCA CC-3' (forward) and 5'-AGT GCA GCT CCA ATG AAG G-3' (reverse) were designed for first PCR and the primer pairs 5'-ATC TC(T/C) GGT GGI CAC (A/G)TI AAC CC-3' (forward) and 5'-G(T/G)C C(G/A/C)(G/A) CCC A(G/A)(T/A) AIA (C/T)CC AGT GG-3' (reverse) for nested PCR. The PCR product was subcloned into the pCR®2.1 vector. After confirming the homology by searching the DNA databank, the partial PIP fragment was used as a probe to screen the cDNA library from apple shoots. As a result, two full-length cDNAs (MdPIP1a, AB100869 [GenBank] and MdPIP1b, AB100870 [GenBank] ) were isolated and identified as PIP1 members. Due to the high identity of MdPIP1a and MdPIP1b, it was difficult to make specific probes to distinguish them. Hence DIG-labelled MdPIP1a was used as a probe for expression analysis. Northern blotting using total RNA from different apple tissues detected two different mRNA bands in all tissues tested, which might arise from the expression of a homologous clone such as MdPIP1b or additional RNA species as yet unknown (Fig. 1A). Among non-fruit tissues, MdPIP1 showed the highest expression level in shoots and the second-highest level in young leaves, while the hybridization signal was just a trace in mature leaves (Fig. 1A). Among fruit tissues, young fruits at 19 d after full bloom (DAF) showed MdPIP1 expression at a low level, while it was expressed strongly in apple fruits at later stages, from 61–174 DAF (Fig. 1A). Thus MdPIP1 expression levels were in accordance with the patterns of fruit volume increase (Fig. 1B).
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To understand whether and how the MdPIP1 gene in the stem is expressed in response to various conditions of water supply, shoot cuts were treated by dipping in PEG solutions. The expression of MdPIP1 in osmotic treatment by –2 and –4 MPa PEG solutions was up-regulated in 6 h compared with that in the reference solution. A similar trend was observed with a prolonged treatment time (Fig. 2). MdPIP1 gene expression in the reference solution showed almost the same levels for 12 h, but its level increased at 24 h.
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Cell expansion in fruits occurs mainly by water uptake accompanied by the transport of sugars and other solutes into vacuoles (Coombe, 1976). Therefore, water channels on the vacuole membrane are considered to be important for this phenomenon. Tonoplast intrinsic protein (TIP) genes were isolated and assayed during fruit development in pear (Shiratake et al., 2001) and peach (Sugaya et al., 2001). However,
TIP mRNA accumulation failed to be detected at the cell-expanding stage in both fruits. In this study, up-regulated expression of MdPIP1 coincided with the fruit cell-expanding stage, suggesting the involvement of MdPIP1 in water absorption during fruit expansion. The regulation of PIP upon osmotic stress varies with plant organs and species. Under water stress, the expression of PIP is down-regulated in rice roots and leaves (Li et al., 2000) and in the common ice plant (Yamada et al., 1995), but up-regulated in rice stems (Malz and Sauter, 1999) and in rape seed (Gao et al., 1999) as in the present result in apple stems. Despite the controversial results on PIP that expression may be, in part, due to the complex functions of this gene family in different cell types and at different development stages (Yamada et al., 1995), the present results still indicate that the expression of MdPIP1 is regulated in response to various conditions of water supply through intracellular water transport in apple stems.
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Acknowledgements |
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We thank Dr K Shiratake (Nagoya University) for providing information on PIP primers. We also thank Dr H Yakushiji (National Institute of Fruit Tree Science) for comments on the manuscript.
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References |
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TopAbstractReferences |
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Chrispeels MJ, Agree P. 1994. Aquaporins: water channel proteins of plant and animal cells. Trends in Biochemical Sciences 19, 421–425.[CrossRef][Web of Science][Medline]
Coombe BG. 1976. The development of fleshy fruits. Annual Review of Plant Physiology 27, 207–228.[Web of Science]
Gao Y-P, Young L, Bonham-Smith P, Gusta LV. 1999. Characterization and expression of plasma membrane aquaporins in primed seed of Brassica napus during germination under stress conditions. Plant Molecular Biology 40, 635–644.[CrossRef][Web of Science][Medline]
Li L, Li S, Tao Y, Kitagawa Y. 2000. Molecular cloning of a novel water channel from rice: its products expression in Xenopus oocytes and involvement in chilling tolerance. Plant Science 154, 43–51.
Malz S, Sauter M. 1999. Expression of two PIP genes in rapidly growing internodes of rice is not primarily controlled by meristem activity or cell expansion. Plant Molecular Biology 40, 985–995.[CrossRef][Web of Science][Medline]
Michel BE, Kaufmann MR. 1973. The osmotic potential of polyethylene glycol 6000. Plant Physiology 51, 914–916.
Shiratake K, Kobae Y, Suzuki Y, Nakaune M, Tanase K, Yamaki S. 2001. Molecular cloning of a cDNA encoding tonoplast water channel of pear fruit and its expression during development. Journal of the Japanese Society for Horticultural Science 70, 281–286.
Sugaya S, Gemma H, Iwahori S. 2001. Isolation and expression analysis of a gene encoding a vacuolar-type water channel protein in peach fruit. Journal of the Japanese Society for Horticultural Science 70, 716–718.
Yamada S, Katasuhara M, Kelly WB, Michalowski CB, Bohnert HJ. 1995. A family of transcripts encoding water channel proteins: tissue-specific expression in the common ice plant. The Plant Cell 7, 1129–1142.[Abstract]
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