Expression in anthers of two genes encoding Brassica ... - Springer Link

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Plant Molecular Biology 34: 163–168, 1997. c 1997 Kluwer Academic Publishers. Printed in Belgium.

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Expression in anthers of two genes encoding Brassica oleracea transmembrane channel proteins Ren´e K. Ruiter1 , Gerben J. van Eldik, Marinus M.A. van Herpen, Jan A.M. Schrauwen and George J. Wullems Department of Experimental Botany, University of Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, Netherlands ( author for correspondence); 1 Present address: Department of Brassica and Oilseeds Research, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK Received 24 June 1996; accepted in revised form 31 January 1997

Key words: Brassica oleracea, transmembrane channel proteins, ...

Abstract Screening of an anther cDNA expression library resulted in the isolation of two almost identical cDNA clones, termed mipA and mipB, showing homology with sequences encoding transmembrane channel proteins from the MIP family. Both clones were expressed in several tissues, but not in pollen. MipA was preferentially expressed in the surrounding sporophytic tissues of stamens. Anthers subjected to drought were induced to accumulate even more mip transcripts, which was entirely due to higher mipA gene expression. On basis of isolation procedures, sequence homology and drought inducibility of mipA we conclude that the encoded proteins probably are constituents of the pollen coat and are aquaporins. During early pollen development, a flow of water occurs from the tapetum to the growing pollen grains. This water is supplied via the vascular system in the filament, and, within the anther, transported towards the various tissues. At shedding, pollen is strongly dehydrated, which may be seen as a condition essential to survive water stress during the period of pollen transfer [30]. Water is lost from the grains, but also from the locular space, prior to shedding, at the end of pollen development [18]. Moreover, specific water relocation in endothecial and epidermal cells is a prerequisite to anther dehiscence [4, 18, 29]. After landing of the pollen grains on the stigma, pollen hydrates by absorption of stigmatic water, and then germinates [10, 27]. Thus, transport of water is an essential element for successful anther and pollen development, anther dehiscence and pollen hydration.

The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X95639 (mipA) and X95640 (mipB).

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Membranes are barriers to water flow [5], which can be overcome by aquaporin-like proteins that form water-selective transmembrane channels [2, 32]. Aquaporins occur both in the plasma membrane [7, 26] and the tonoplast [32]. These proteins are assumed to be involved in symplastic and transcellular transport of water. Aquaporins are members of the MIP family of passive transmembrane transporters, a family named after the major integral membrane protein (MIP) of the bovine lens fibre cell membrane [17]. These MIPs are specific to a single type of membrane, such as the plasma membrane (PIP), the tonoplast (TIP) or the peribacteroid membrane of root nodules [7, 12, 19, 22, 25, 26]. The occurrence of individual MIP proteins is often restricted to single-cell types. Expression of the corresponding genes in tissue-specific, as is described for genes encoding -TIPs in seeds, proteins located in the tonoplast of protein storage vacuoles [19, 20, 21, 22], and for the root-specific mip genes [34, 42, 44]. In Brassica oleracea, the pollen grains are surrounded by a lipidic pollen coat, also containing proteins. Screening of an anther cDNA expression library with

GR: 201001894, Pips nr. 134105 BIO2KAP plan3591.tex; 14/04/1997; 7:24; v.7; p.1

164 antibodies raised against a mixture of pollen coat proteins [37] led to the isolation of two sequences, mipA and mipB, that encode proteins of 286 amino acids (Fig. 1) with a predicted molecular mass of 31 kDa. Amino acid sequence comparison identified only 4 amino acid differences between the two sequences (resulting from 51 nucleic acid differences), and revealed a strong resemblance with the MIP family of transmembrane channel proteins (Fig. 1). Most homologous to MIPA and MIPB (94–99% identity) is a set of aquaporins present in the Arabidopsis thaliana plasma membrane [24, 26], PIP1a, PIP1b and PIP1c. Identity with other MIPs is present in the six membrane-spanning domains, as well as in the hydrophilic regions between spanner 2 and 3, and spanner 5 and 6 [36]. A set of 22 amino acids present in almost all members of the MIP family [36] is also present in the deduced MIPA and MIPB protein sequences (Fig. 1). The spatial expression patterns of mipA and mipB were studied by northern blot analysis of RNA isolated from several tissues [40] (Fig. 2). To discriminate between mipA and mipB gene expression, we used the 30 -untranslated region (30 -UTR) of the two cDNA clones. MipB transcripts were present in stamens, sepals, petals, carpels and roots, but not in mature pollen or in seeds. In leaves, a clear signal could only be observed after longer exposure times (data not shown). MipA was expressed in the same tissues, but preferentially in stamens. Both 30 -UTR probes detected a 1200 nt transcript (Fig. 2). To determine whether the genes are expressed in pollen at earlier stages during development, RNA was isolated from bicellular pollen grains [39], and compared with RNA from whole anthers at the same developmental stage. No mip transcripts could be detected in bicellular pollen grains (Fig. 2), in contrast to the entire anthers, which strongly indicates that the genes are expressed in a sporophytic portion of the anther. Hybridisation of genomic DNA digested with EcoRI or HindIII with the mipA sequence revealed at least three strongly hybridizing bands, and some weaker hybridizing bands (Fig. 3). Hybridization with the complete mipB sequence revealed the same pattern, whereas both 30 -UTR probes only detected the strongly hybridizing bands (data not shown). The results demonstrate the existence of a set of mip-like sequences within the B. oleracea genome. The sequence similarity of MIPA and MIPB to A. thaliana aquaporins (Fig. 1) led to the question of whether the corresponding genes are inducible by drought. Therefore, we quantified mip transcript levels

in anthers from 5–6 mm flower buds (anthers at the bicellular pollen stage) exposed to drought (Fig. 4). Drought increased the level of mipA transcripts in anthers and rehydration partially reverted this effect. In contrast, the mipB transcript level was not affected by drought. In petals, sepals, carpels and seedlings the total mip transcript level remained constant over a period of 24 h of drought (data not shown). During anther development, mip transcripts were detected from the unicellular microspore stage up to anthesis, with increased transcript levels in anthers at the tricellular stage (Fig. 5). So, mip gene expression preceded anther dehydration, which in B. oleracea anthers starts in 9 mm flower buds [8], but is further increased upon dehydration itself. Similar results were obtained with both the whole sequences, and with the 30 -UTR probes (data not shown). Using the entire cDNAs as probes, a second 1400 nt transcript was detected (Fig. 5). It was only present in immature anthers and not detected by the 30 -UTR probes (Fig. 2), the latter indicating that it probably represents a transcript from another gene. From our results it can be concluded that mip expression in anthers is regulated in different ways. First, expression is drought-inducible, as shown for anthers from 5–6 mm flower buds (Fig. 4). However, drought treatment does not lead to overall loss of water from anthers of 5–6 mm buds [38]. So, undetectably small, local changes in water content, or signal transduction from other flower organs may induce the observed increase in expression. Drought inducibility has also been found for many other mip genes [12, 14, 16, 23, 31, 42, 43]. Second, mipA and mipB expression is linked to anther development, as transcripts accumulate from the unicellular microspore stage on (Fig. 5). So, expression precedes anther dehydration, enabling the plant to organise the water transporting machinery before dehydration is occurring. Whether local dehydration, undetectable at the level of anther fresh weight, induces mip expression early during anther development, is unclear [38]. Third, expression increases during dehydration in anthers from flower buds of 9 mm and larger. As mip gene expression increases upon imposed drought stress, also the programmed form of dehydration at the end of anther development may have resulted in the increased expression in these anthers. Mip gene expression is similar to the described expression of bopc15 and bopc34 [38]. The latter two genes are also expressed in anthers prior to dehydration, and by artificially imposed drought.

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Figure 1. Alignment of the deduced amino acid sequences of the Brassica oleracea cDNA clones mipA and mipB, and three Arabidopsis thaliana aquaporin encoding sequences. The consensus line shows residues conserved in all 5 sequences. Underlined, bold face letters in the consensus line represent 22 amino acids conserved among most transmembrane channel proteins [36]. Amino acid stretches forming membrane spanning domains are shaded. The lengths of the sequences are indicated at the top. Sequences used in the alignment with the B. oleracea (BO) sequences MIPA and MIPB (accession numbers X95639 and X95640) are the Arabidopsis thaliana (AT) sequences PIP1a, PIP1b, PIP1c (accession numbers X75881, X68293 and X75882 [26]).

Figure 2. Spatial expression of mipA and mipB. Northern blot analysis of total RNA isolated from several Brassica oleracea tissues. Each lane contained 10 g RNA. A northern blot was consecutively hybridized with the 30 -UTRs of the two cDNA clones (indicated as 30 -UTR mipA and 30 mipB), and a 25S rRNA probe as a control. In the right panel, total RNA isolated from bicellular pollen was compared with total RNA isolated from anthers at the bicellular pollen stage. The lengths of the detected mip transcripts (in nucleotides) are shown at the left.

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Figure 4. Quantification of mipA and mipB transcript levels after artificial drought and subsequent rehydration. Brassica oleracea anthers were harvested from flower buds of 5–6 mm after drought periods of up to 18 h, and rehydration for 3 or 6 h after 18 h of drought (indicated as 18/3 and 18/6). Northern blots with 10 g total RNA isolated from these anthers were probed with a full-length mip cDNA clone (indicated as mip) or the 30 -UTRs of both cDNA clones (indicated as 30 -UTR mipA and 30 -UTR mipB). The amounts of radioactivity measured (in Bq) are represented by the vertical bars.

Figure 3. Southern blot analysis of Brassica oleracea genomic DNA. Genomic DNA was digested with EcoRI, BamHI and HindIII as shown at the top. The Southern blot was probed with the fulllength mipA cDNA clone. A DNA molecular weight marker (in kb) is shown at the right.

MIPs are located in the membranes of the cells in which they are synthesised, forming transmembrane channels [5, 7]. B. oleracea mipA and mipB were isolated as sequences encoding pollen surface proteins. Substances at the outside of the pollen exine probably originate from the tapetum [8]. So, most likely, the MIPs are synthesised in the tapetal cells and find their way to the pollen coat when incorporated in the tapetal membranes. Membrane lipids have been detected in the pollen coat [11]. In pollen, the MIPs could be located in the membranous coating superficial layer (CSL) [9], or the exinic outer layer (EOL) [14], two membranelike layers observed outside the exine. Unfortunately, antisera raised against synthetic peptides of other MIPs failed to detect any similarity to MIPA or MIPB from B. oleracea. MIPs are known to be involved in specific transport of ions, glycerol, sugars or water [1, 32, 33, 41]. It may be assumed from the high protein sequence identity with the A. thaliana PIP1 sequences, that MIPA and MIPB are plasma membrane-located aquaporins, proteins specifically involved in transmembrane water

Figure 5. Mip gene expression during Brassica oleracea anther development. Northern blot analysis of 10 g total RNA isolated from anthers at different developmental stages and probed with the full-length mipA cDNA clone. The lengths of the detected transcripts (in nucleotides) are shown at the left. The relation between flower bud length (in mm) and pollen developmental stage is shown below the northern blot (uni, unicellular microspore stage; bi, bicellular pollen stage; tri, tricellular pollen stage).

transport [3]. Such a function is further indicated by the anther-specific inducibility of the corresponding genes by drought stress, and by the elevated expression in anthers from the onset of anther dehydration. Increased mip gene expression may serve to increase the amount of MIP protein in the membranes, thereby accelerating transmembrane water transport [6]. Whether the proteins fulfil this function only in the tapetum or whether

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167 they are also functional in the pollen coat, channelling water into the pollen or pollen coat, remains to be established. Indication for such a function can be obtained by injection of mipA and mipB transcripts into Xenopus oocytes may elucidate whether these sequences encode functional aquaporins [35].

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Acknowledgements The authors are very grateful to Dr A.F. Croes for critical and helpful discussion when preparing this manuscript. We are grateful to Gerard van der Weerden and Wim van den Brink for growing our plant material. This research was supported by funds of the BRIDGE programme of the Commission of the European Communities (Contract BIOT 900172).

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References 1. Aelst LVS, Hohmann FK, Zimmermann FK, Jans AWH, Thevelein JM: A yeast homologue of the bovine lens fibre MIP gene family complements the growth defect of a Saccharomyces cerevisiae mutant on fermentable sugars but not its defect in glucose-induced RAS-mediated cAMP signalling. EMBO J 10: 2095–2104 (1991). 2. Agre P, Sasaki S, Chrispeels MJ: Aquaporins: a family of water channel proteins. Am J Physiol 265: F461 (1993). 3. Agre P, Preston GM, Smith BL, Jung JS, Raina S, Moon C, Guggino WB, Nielsen S: Aquaporin CHIP: the archetypal molecular water channel. Am J Physiol 265: 463–476 (1993). 4. Bonner LL, Dickinson HG: Anther dehiscence in Lycopersicon esculentum. I. Structural aspects. New Phytol 113: 97–115 (1989). 5. Chrispeels MJ, Agre P: Aquaporins: water channel proteins of plant and animal cells. Trends Biochem Sc 19: 421–425 (1994). 6. Chrispeels MJ, Maurel C: Aquaporins: the molecular basis of facilitated water movement through living plant cells. Plant Physiol 105: 9–13 (1994). 7. Daniels MJ, Mirkov TE, Chrispeels MJ: The plasma membrane of Arabidopsis thaliana contains a mercuryinsensitive aquaporin that is a homolog of the tonoplast water channel protein TIP. Plant Physiol 106: 1325–1333 (1994). 8. Dickinson HG: The role of plastids in the formation of pollen grain coatings. Cytobios 8: 25–40 (1973). 9. Dickinson HG, Elleman CJ: Structural changes in the pollen grain of Brassica oleracea during dehydration in the anther and development on the stigma as revealed by anhydrous fixation techniques. Micron Microsc Acta 16: 255–270 (1985). 10. Dickinson HG: Dry stigmas, water and self-incompatibility in Brassica. Sex Plant Reprod 8: 1–10 (1995). 11. Evans DE, Rothnie NE, Sang JP, Palmer MV, Mulcahy DL, Singh MB, Knox RB: Correlations between gametophytic (pollen) and sporphytic (seed) generations of polyunsaturated fatty acids in oilseed rape Brassica napus L. Theor Appl Genet 76: 411–419 (1988).

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

Fortin MG, Morrison NA, Verma DPS: Nodulin-26, a peribacteroid membrane nodulin, is expressed independently of the development of the peribacteroid compartment. Nucl Acids Res 15: 813–824 (1987). Fray RG, Wallace A, Grierson D, Lycett GW: Nucleotide sequence and expression of a ripening and water stress-related cDNA from tomato with homology to the MIP class of membrane channel proteins. Plant Mol Biol 24: 539–543 (1994). Gaude T, Dumas C: A membrane-like structure on the pollen wall surface in Brassica. Ann Bot 54: 821–825 (1984). Guerrero FD, Jones TJ, Mullet JE: Turgor-responsive gene transcription and RNA levels increase rapidly when pea shoots are wilted. Sequence and expression of three inducible genes. Plant Mol Biol 15: 11–26 (1990). Guerrero FD, Crossland L: Tissue-specific expression of a turgor-responsive gene with amino acid sequence homology to transport-facilitating proteins. Plant Mol Biol 21: 929–935 (1993). Gorin MB, Yancey SB, Cline J, Revel J-P, Horwitz J: The major intrinsic protein (MIP) of the bovine lens fibre membrane: characterization and structure based on cDNA cloning. Cell 39: 49–59 (1984). Heslop-Harrison J: An interpretation of the hydrodynamics of pollen. Am J Bot 66: 737–745 (1979). H¨ofte H, Hubbard L, Reizer J, Ludevid D, Herman EM, Chrispeels MJ: Vegetative and seed-specific forms of tonoplast intrinsic protein in the vacuolar membrane of Arabidopsis thaliana. Plant Physiol 99: 561–570 (1992). Inoue K, Takeuchi Y, Nishimura M, Hara-Nishimura I: Characterization of two integral membrane proteins located in the protein bodies of pumpkin seeds. Plant Mol Biol 28: 1089– 1101 (1995). Johnson KD, Herman EM, Chrispeels MJ: An abundant, highly conserved tonoplast protein in seeds. Plant Physiol 91: 1006– 1013 (1989). Johnson KD, H¨ofte H, Chrispeels MJ: An intrinsic tonoplast protein of protein storage vacuoles in seeds in structurally related to a bacterial solute transporter (GLpF). Plant Cell 2: 525–532 (1990). Jones JT, Mullet JE: Developmental expression of a turgorresponsive gene that encodes an intrinsic membrane protein. Plant Mol Biol 28: 983–996 (1995). Kaldenhoff R, K¨olling A, Richter G: A novel blue light- and abscisic acid-inducible gene of Arabidopsis thaliana encoding an intrinsic membrane protein. Plant Mol Biol 23: 1187–1198 (1993). Kaldenhoff R, K¨olling A, Meyers J, Karmann U, Ruppel G, Richter G: The blue light-responsive AthH2 gene of Arabidopsis thaliana is primarily expressed in expanding as well as in differentiating cells and encodes a putative channel protein of the plasmalemma. Plant J 7: 87–95 (1995). Kammerloher W, Fisher U, Piechottka GP, Sch¨affner AR: Water channels from the plasma membrane cloned by immunoselection from a mammalian expression system. Plant J 6: 187–199 (1994). Kandasamy MK, Nasrallah JB, Nasrallah ME: Pollen-pistil interactions and developmental regulation of pollen tube growth in Arabidopsis. Development 120: 3405–3418 (1995). Keijzer CJ: Hydration changes during anther development. In: Mulcahy DL, Ottaviano E (eds) Pollen: Biology and Implications for Plant Breeding, pp. 197–202. Elsevier, Amsterdam (1983).

plan3591.tex; 14/04/1997; 7:24; v.7; p.5

168 29. Keijzer CJ: The process of anther dehiscence and pollen dispersal. I. The opening mechanism of longitudinally dehiscing anthers. New Phytol 105: 487–498 (1987). 30. Kerhoas C, Dumas C: Nuclear Magnetic Resonance and pollen quality. In: Linskens HF, Jackson JF (eds), Nuclear Magnetic Resonance. Modern Methods of Plant Analysis, vol 2, pp. 169– 190. Springer-Verlag, New York (1986). 31. Liu Q, Umeda M, Uchimiya H: Isolation and expression of two rice genes encoding the major intrinsic protein. Plant Mol Biol 26: 2003–2007 (1994). 32. Maurel C, Reizer J, Schroeder JI, Chrispeels MJ: The vacuolar membrane protein -TIP creates water specific channels in Xenopus oocytes. EMBO J 12: 2241–2247 (1993). 33. Maurel C, Reizer J, Schroeder JI, Chrispeels MJ, Saier HS: Functional characterization of the Escherichia coli glycerol facilitator, GLpF, in Xenopus oocytes. J Biol Chem 269: 11 869–11 872 (1994). 34. Opperman CH, Taylor CG, Conkling MA: Root-knot nematode-directed expression of a plant root-specific gene. Science 263: 221–223 (1994). 35. Preston GM, Carroll TP, Guggino WB, Agre P: Appearance of water channels in Xenopus oocytes expressing red cell CHIP28 protein. Science 256: 385–387 (1992). 36. Reizer J, Reizer A, Saier MH: The MIP family of integral membrane proteins: sequence comparisons, evolutionary relationships, reconstructed pathway of evolution, and proposed functional differentiation of the two repeated halves of the protein. Crit Rev Biochem Mol Biol 28: 235–257 (1993). 37. Ruiter RK, Mettenmeyer T, van Laarhoven DMC, van Eldik GJ, Doughty J, Van Herpen MMA, Schrauwen JAM, Dickinson HG, Wullems GJ: Proteins of the pollen coat of Brassica oleracea. J Plant Physiol, in press (1996).

38.

39.

40.

41.

42.

43.

44.

Ruiter RK, van Laarhoven DMC, Meyer H, van Eldik GJ, van Herpen MMA, Schrauwen JAM, Wullems GJ: Expression patterns in anthers of two Brassica oleracea genes encoding water stress-inducible proteins. Submitted (1996). Schrauwen JAM, de Groot PFM, van Herpen MMA, van der Lee T, Reijnen WH, Weterings KAP, Wullems GJ: Stagerelated expression of mRNAs during pollen development in lily and tobacco. Planta 182: 298–304 (1990). van Eldik GJ, Vriezen WH, Wingens M, Ruiter RK, van Herpen MMA, Schrauwen JAM, Wullems GJ: A pistil-specific gene of Solanum tuberosum is predominantly expressed in the stylar cortex. Sex Plant Reprod 8: 173–179 (1995). Weaver CD, Shomers NH, Louis CF, Roberts DM: Nodulin 26, a nodule-specific symbiosome membrane protein from soybean, is an ion channel. J Biol Chem 269: 17 858–17 862 (1994). Yamada S, Katsuhara M, Kelly WB, Michalowski CB, Bohnert HJ: A family of transcripts encoding water channel proteins: tissue-specific expression in the common ice plant. Plant Cell 7: 1129–1142 (1995). Yamaguchi-Shinozaki K, Koizumi M, Urao S, Shinozaki K: Molecular cloning and characterization of 9 cDNAs for genes that are responsive to desiccation in Arabidopsis thaliana: sequence analysis of one cDNA clone that encodes a putative transmembrane channel protein. Plant Cell Physiol 33: 217– 224 (1992). Yamamoto YT, Cheng C-L, Conkling MA: Root-specific genes from tobacco and Arabidopsis homologous to an evolutionarily conserved gene family of membrane channel proteins. Nucl Acids Res 18: 7449 (1990).

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