Journal of Experimental Botany, Vol. 53, No. 369, pp. 639-649,
April 1, 2002
© 2002 Oxford University Press
Original Papers |
High expression of putative aquaporin genes in cells with transporting and nutritive functions during seed development in Norway spruce (Picea abies)
1 Department of Biology and Environmental Science, Kalmar University, S-391 82 Kalmar, Sweden
2 Department of Botany, Stockholm University, S-106 91 Stockholm, Sweden
Received 23 May 2001; Accepted 20 November 2001
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Abstract |
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Aquaporins mediate the bidirectional passage of water over membranes and are present in tonoplasts (TIPs) and in plasma membranes (PIPs) of plant cells. Knowing their expression in different tissues is valuable when assessing their contribution to plant water relations. A TIP-gene has been cloned from developing female gametophytes of Picea abies, a conifer displaying an embryology different from the angiosperms. Probes were made from conserved regions of the TIP gene and used for in situ hybridization to examine the gene expression pattern in developing female reproductive structures. Early during development high transcript expression was found in the spongy tissue encasing the developing female gametophyte, in cells of the future seed coat of young ovules and in vascular tissue of the ovuliferous scale. At later stages a strong signal was seen in archegonia jacket cells surrounding egg cells and, still later, at the time of storage protein accumulation, in storage parenchyma cells of the gametophyte as well. These aquaporin-homologues probably participate in regulating water balance in the cells although they could also be permeable to other molecules than water.
Key words: Aquaporin, conifer, in situ hybridization, seed, tonoplast intrinsic protein (TIP).
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Introduction |
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With a few exceptions, a major point of difference between the embryogenesis of gymnosperms and angiosperms is that the former generally show a free-nuclear phase, while in the angiosperms, the first and subsequent divisions of the zygote are invariably followed by wall formation (Raghavan and Sharma, 1995
). Other major differences reside in the size and structure of the female gametophyte, the origin and formation of the nutritive tissue of the seed, and whether the ovules are naked or enclosed in an ovary (Biswas and Johri, 1997). In all the Archegoniate (including the majority of gymnosperms) fertilization and the first critical phases of embryogeny occur within the shelter provided by the archegonium and the adjacent gametophytic tissue (Gifford and Foster, 1987). Thus in gymnosperms, the female gametophyte serves the dual function of bearing gametes and nourishing the embryo. However, in its early stages of development the embryo is nourished by the egg cytoplasm through the suspensor system and it is only later that it begins to draw upon cells of the female gametophyte which, as the embryo begins to differentiate, starts to accumulate enormous reserves of starch, fats and proteins (Raghavan and Sharma, 1995).
The maturation phase is central for viable seed production in conifers and, hence, storage nutrient synthesis is essential in that process. In a previous study, the accumulation of storage proteins and protein storage vacuole (PSV) formation in developing seeds of Picea abies was followed (Hakman, 1993a, b). An abundant intrinsic protein of PSV membranes from mature seeds was also identified as 
-TIP (tonoplast intrinsic protein) (Oliviusson and Hakman, 1995). Many plants contain in their PSV membranes a large quantity of TIPs (Johnson et al., 1989), proteins that belong to the major intrinsic protein (MIP) class of membrane proteins. There is currently an intense interest in the MIP family of proteins as many of them have been shown to function as water channels (aquaporins) by facilitating an osmotically driven permeation of water across membranes (Preston et al., 1992; Chrispeels and Maurel, 1994; Agre et al., 1995; Maurel, 1997). However, more recent research also indicates that at least some plant aquaporins may have a dual function in transporting both water and small solutes (Biela et al., 1999; Gerbeau et al., 1999).
All members of the MIP family, although not all of them are aquaporins, are characterized by the presence of six transmembrane helices and the NPA (Asn-Pro-Ala) signature motif. In plants, MIPs have been localized both in the plasma membrane (PIPs) and in the vacuolar or vacuolar-derived membranes (TIPs). Several isoforms can be found in one plant, conferring developmental or tissue specificity. In Arabidopsis thaliana, for example, the two isoforms -TIP and 
-TIP are expressed in seed tissues and vegetative tissues, respectively (Höfte et al., 1992). In the genomic sequence of Arabidopsis, 35 different MIP-encoding genes were identified and found to form four subfamilies (Johanson et al., 2001), similar to the result from Zea mays based on comparison of amino acid sequences and phylogenetic analysis of 31 full-length cDNAs (Chaumont et al., 2001). There is also a growing use of different TIP isoforms as functional markers for specific types of vacuolar membranes (Jauh et al., 1999; Vitale and Galili, 2001).
Functional homology within this family is also exemplified by the cross-reactivity of antibodies raised against one MIP protein to similar proteins from different species; this indicates binding to conserved epitopes within the proteins, as discussed previously (Chaumont et al., 1998).
Aquaporin expression has been shown to be up-regulated during cell elongation (Ludevid et al., 1992; Schünmann and Ougham, 1996; Smart et al., 1998), and during stress by desiccation, increased ABA levels (Mariaux et al., 1998) and salt (Uno et al., 1998); it is also down-regulated by salt stress (Yamada et al., 1995). Apart from expression levels, the aquaporin activity is thought to be regulated by phosphorylation as was shown for the tonoplast aquaporin -TIP from Phaseolus vulgaris (Johnson and Chrispeels, 1992; Maurel et al., 1995) and the plasma membrane aquaporin PM28A from Spinacia oleracea (Johansson et al., 1996, 1998). Many of the gene-expression (mRNA abundance) studies imply that aquaporins are highly expressed in vascular tissues, in meristems and in tissues with high water or metabolite flux (Chrispeels et al., 1999) and could thus account for transcellular water flow and osmoregulation of cells such as during the rapid physical/structural changes that occur during seed development and germination (Maurel et al., 1997a; Barrieu et al., 1998).
Recently, the expression pattern of aquaporin homologues was investigated in seedlings, mature roots and needles of P. abies and a general strong signal associated to the vascular tissue was found (Oliviusson et al., 2001). In this work, the investigation on P. abies aquaporin homologues was extended to developing seeds with the hypothesis that a conifer, with its marked differences in embryology and anatomy from angiosperms, would add valuable knowledge of the possible function(s) of the proteins. In an earlier investigation it was found that extracts from developing seeds gave cross-reactive bands at 26–27 kDa on blots probed with antiserum against P. vulgaris -TIP. The antibodies also labelled a protein in the PSV membrane of mature seeds of P. abies (Oliviusson and Hakman, 1995). This protein was purified from isolated PSVs and a partial amino acid sequence derived from it showed high identity to other aquaporin-related plant proteins. This information was used to produce primers that allowed a full-length sequence of the gene to be obtained. In addition to this PSV-protein present in seed parenchyma cells heavily engaged in storage protein synthesis and accumulation, the -TIP antiserum also recognized a protein with a slightly different mobility on immunoblots from extracts of female gametophytes of an earlier stage, i.e. before the formation of PSVs (see Fig. 1 in Oliviusson and Hakman, 1995). The reason for the change seen in the protein profile could be due either to post-translational modifications of a protein or from dissimilar proteins being synthesized during female gametophyte development. If different proteins were synthesized as these observations may indicate, it was reasoned that an early isoform could be associated with archegonia formation as the band is strongest in the gametophyte extract of this stage. Each gametophyte contains many archegonia with a layer of jacket cells surrounding a central cell that increases rapidly in size before dividing into a ventral canal cell and a giant egg cell, which are characteristic features of conifers. A MIP isoform could possibly be present in cells engaged in this size increment or in the vacuolar inclusions (‘proteid vacuoles’) contained in the egg cells. In order to test these ideas in situ hybridization experiments of developing ovules and seeds have been performed with probes made within the conserved regions of the cloned gene. The results indicate that both the tapetal-like cells of the spongy tissue surrounding the growing female gametophyte as well as the jacket cells around the central cell/egg cell have an increased gene activity. Both types of cells/tissues are believed to have transport functions to the structures that they surround.
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Plant materials
Female cones of Norway spruce (Picea abies [L.] Karst.) were collected during 1993, 1995, 1996, 1998, and 2000 from trees growing in a seed orchard in Uppsala or in the surroundings of Stockholm. Collections were made from late May until early September, although in some years the collection had to stop earlier due to a lack of material. Developing ovules or female gametophytes were isolated and processed for microscopy or were immediately frozen in liquid nitrogen and stored at -80 °C. Mature dry seeds were imbibed for 18 h at 4 °C prior to RNA extraction.
RNA extraction
The method by Chang et al. was used for extraction of total RNA from tissues (Chang et al., 1993). The RNA concentration was measured spectrophotometrically and the RNA integrity determined by EtBr-staining of RNA separated in formaldehyde-containing agarose gels. Poly(A)+ RNA was isolated from total RNA on oligo-d(T) columns (according to Sambrook et al., 1989) or by oligo-d(T) coupled magnetic beads following instructions from the manufacturer (Dynal, Oslo, Norway). Isolated RNA was used in cDNA library constructions, RT-PCR, RACE and for Northern analysis.
RT-PCR
A 361 bp mip homologous cDNA resulting from RT-PCR with degenerative primers (sense [F3]: 5'-T(A/C/T)T(A/T)(C/T)TACTGGAT(C/T)GC(C/T)CA(A/G)CTTC-3' and antisense [B3]: 5'-AGTGG(A/T)CC(A/C)ACCCAG(A/T)AGA(C/T)CC-3') and poly(A)+ RNA from maturing female gametophytes, using the Titan RT-PCR System (Boehringer Mannheim, Mannheim, Germany), was cloned into a pCRII vector (TA cloning kit; Invitrogen, Leek, The Netherlands) according to the manufacturer's instructions. Sequence information from this fragment was used to construct specific primers for PCR. The primers (sense [F1]: 5'-ACTGGATTGCTCAGCTTCTTGG-3' and antisense [B4]: 5'-AAACCAGCCCGAACGTCATCAC-3'), were chosen within a conserved region of plant mip genes.
cRNA/cDNA probes
The insert orientation of the 361 bp mip homologous fragment ligated into the pCRII vector was checked by sequencing and restriction enzyme digestion. Sense and antisense cRNA probes were made by in vitro transcription and labelled with digoxigenin (DIG) using Sp6 and T7 RNA polymerases, respectively, with the DIG RNA Labeling Mix (Boehringer Mannheim, Mannheim, Germany), after 5'-overhang linearization of the plasmid with XhoI and BamHI, respectively.
DIG-cDNA probes were made from 612 bp 5' RACE inserts excised from the pCRII vector by EcoRI and were separated by gel electrophoresis and gel-purified. The cDNA was denatured and both strands individually labelled using the DIG DNA Labeling Kit from the same supplier.
cDNA library construction and screening
Two µg poly(A)+ RNA from female gametophytes collected 26 June and 18 July 1995, respectively, were reverse-transcribed and an EcoRI-linker was ligated using the Universal RiboClone cDNA Synthesis System (Promega, Madison, WI, USA). The cDNA was ligated into the lambda ZAPII/EcoRI vector (Stratagene, La Jolla, CA, USA) and further packaged and amplified using the GigapackIII Gold Packaging Extract (Stratagene) according to the manufacturer's instruction. Approximately 500000 plaque forming units of the respective library were screened with the DIG-labelled 361 bp cRNA (100 ng ml-1) or cDNA (25 ng ml-1), described above. Positive clones were rescreened with the same probe, amplified and sequenced. However no full-length cDNA was found and the remaining part of the 5' end of the cDNA was identified using the 5' RACE procedure.
5'- and 3'-RACE
The RACE-procedure (Frohman et al., 1988) was used to obtain a full-length cDNA from poly(A)+ RNA isolated from female gametophytes collected 11 June 1998, 26 June and 18 July 1995, respectively. The 5'-AmpliFINDERTM RACE KIT (Clontech, Palo Alto, CA, USA) was used according to the manufacturer's instructions, first in a single-stranded cDNA synthesis reaction with 2 µg poly(A)+ RNA and a custom designed cDNA synthesis anchor (5'-GACTCGAGTCGACATCGA(T)17–3'). The 5'-RACE anchor supplied with the kit was then ligated to the 5'-end of the cDNA. The gene-specific primer B4 and the supplied primer complementary to the 5'-anchor were used in a PCR reaction (94 °C, 45 s; 60 °C, 45 s; 72 °C, 2 min; 35 cycles with a final extension step at 72 °C for 7 min). 3'-RACE was performed with the same single-stranded cDNA described above, in a PCR reaction (94 °C, 40 s; 59 °C, 60 s; 72 °C, 1 min 30 s with 5 s increment per cycle, 35 cycles with a final elongation step at 72 °C for 15 min) with the gene-specific primer F1 and a primer (5'-GACTCGAGTCGACATCG-3') complementary to the cDNA synthesis anchor at the 3'-end of the cDNA.
DNA sequencing
The clones resulting from the screening of the cDNA libraries and the products of the RT-PCR and 5'- and 3'-RACE reactions were sequenced on both strands manually on a 5% polyacrylamide gel after a reaction with the Thermo Sequenase Cycle Sequencing Kit (Amersham, Little Chalfont, UK), or automatically on ABI PRISM 7700 Sequence Detection System (Perkin-Elmer/Roche Molecular Systems, Branchburg, New Jersey, USA) following a reaction with the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit from the same manufacturer. A full-length cDNA sequence (mipfg) was obtained by aligning sequences from the cDNA library clones with 5'-RACE clones as well as with the 3'-RACE clones resulting from several PCR reactions with the same cDNA. One mip homologous gene transcript was identified and it was identical in all of the developmental stages investigated.
Multiple sequence analysis of the deduced P. abies MIPFG with the best scoring results from similarity searches of sequence databanks, was made using Clustal W (Thompson et al., 1994) at the European Bioinformatics Institute.
Northern analysis
Poly(A)+ RNA was isolated from developing ovules of 11 June, female gametophytes of 25 June, 6 July and 18 July 1998, from mature dry seeds and mature seeds that were imbibed for 18 h at 4 °C. RNA was separated on formaldehyde agarose gels (Sambrook et al., 1989) and capillary transferred to positively charged membranes (ZetaProbe GT Genomic; Bio-Rad, Hercules, California, USA) in 20xSSC for 5 h at room temperature (RT) or for 16 h at 4 °C. Membranes were dried in vacuum for 2 h at 80 °C and prehybridized (50% formamide (v/v), 5xSSC, 0.02% SDS (w/v), 2% Blocking Reagent [Boehringer Mannheim] (w/v), 0.1% lauroyl-sarcosine (w/v)) for 1 h and incubated with a similar solution containing antisense DIG-cRNA probe (100 ng ml-1) at 68 °C for 18 h prior to high stringency washes (2x5 min in 2xSSC/0.1% SDS (w/v) at RT, 2x15 min in 0.1xSSC/0.1% SDS (w/v) at 68 °C). Alternatively, a cDNA probe (25 ng ml-1) was used with a high SDS hybridization solution (50% formamide (v/v), 0.12 M Na2HPO4, 0.25 M NaCl, 7% SDS (w/v), 2% Blocking Reagent [Boehringer Mannheim] (w/v), 5xSSC, 50 µg ml-1 yeast tRNA, 0.1% lauroyl-sarcosine (w/v)) at 50 °C for 20 h, and then washed (2x5 min in 2xSSC/0.1% SDS (w/v) at RT, 1x15 min in 0.1xSSC/0.1% SDS (w/v) at RT and 1x15 min in 0.1xSSC/0.1% SDS (w/v) at 68 °C). Detection was made by colometry using the DIG Detection Kit and BCIP/NBT as substrate according to the manufacturer's instructions.
Light and electron microscopy
Female gametophytes collected around the time of fertilization, during maturation and from mature dry seeds were embedded in EPON and LR White and processed for light and electron microscopy as described earlier (Hakman, 1993a, b). Ultrathin sections were examined in a Zeiss 906 transmission electron microscope (Oberkochen, Germany).
In situ hybridization (ISH)
Fresh tissues were fixed on ice in a freshly prepared solution of 4% (w/v) paraformaldehyde and 0.25% (v/v) glutaraldehyde in phosphate buffer (0.1 M, pH 7.0) for 3–5 h, dehydrated through an ethanol series, infiltrated and embedded with paraffin (Paraplast Plus; Sherwood Medical Industries, St Louis, MI, USA). Sections about 6 µm thick were cut with a Microm HM 355 microtome (Heidelberg, Germany) and stretched on poly-L-lysine coated glass slides. In situ hybridization with DIG-labelled probes was performed as described earlier (Oliviusson et al., 2001). The slides were hybridized with either antisense or sense strands of the 361 bp P. abies mipfg cRNA probe or with the 612 bp cDNA probe. After colour development for up to 24 h, sections were observed with a Nikon Optiphot microscope and photographed. Digitized images were adjusted and mounted with Photoshop 4.0.
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Structural changes of the female gametophyte in developing seeds
Since climatic variations influence the rate of Picea abies seed development, in addition to the collection dates references will also be made to the development of certain important features in the material collected over the various years. The female gametophyte at the time of fertilization, which in Uppsala and Stockholm occurs in the later part of June, contains one to many large archegonia (Fig. 1A
). The size of the central cell/egg cell in comparison to the other cells of the gametophyte is striking. A layer of jacket cells surrounds each egg cell (Fig. 1B). Parenchyma cells of female gametophytes at this developmental stage are dominated by large central vacuoles (Fig. 1C) which, during the course of seed maturation, are replaced by smaller vacuoles and vesicles filled with storage proteins (Fig. 1D). In storage parenchyma of mature female gametophytes, the cytoplasm is densely packed with nutrients, lipids and storage proteins contained in oil bodies and in PSVs, respectively. In a previous investigation (Oliviusson and Hakman, 1995) the accumulation pattern of TIPs in relation to the storage proteins was followed in developing seeds by using antibodies against Phaseolus vulgaris -TIP. Immunoblots showed two bands. A lower molecular mass band that was most abundant in early-stage female gametophytes, prior to early embryo formation and the degeneration of the egg cell, that later was replaced by a higher molecular mass protein, the accumulation pattern of which followed that of the storage proteins. Both were in the range of 26–27 kDa. A similar accumulation pattern of MIPs, with the relative molecular masses and staining intensities of the bands remaining the same, has been seen in developing seeds from several collections made over many years. As a subcellular localization to the tonoplast has only been made for the protein in mature seeds (Oliviusson and Hakman, 1995) the proteins in general will be referred to as MIPs.
In this work, the general structure of the jacket cells and the formation of PSVs in storage parenchyma are of special interest, since accumulation of the MIP proteins is associated with them (Fig. 1A–D). In light microscopic sections the jacket cells are seen to have larger nuclei and more cytoplasmic staining than the general storage parenchyma cells of the gametophyte (Fig. 1B). The jacket cells enclosing the egg cell are assumed to have transport functions which are supported by their general fine structure characterized by a cytoplasm filled with many organelles, small vacuoles and vesicles, and numerous poorly differentiated plastids and mitochondria (Fig. 1E). The latter are often seen close to the cell wall abutting the egg cell. The cell wall in this region is particularly thick with many pits traversed by numerous plasmodesmata (Fig. 1F).
Characterization of Picea abies mipfg
A 361 bp mip cDNA probe resulting from RT-PCR with degenerative primers and poly(A)+ RNA from maturing female gametophytes was used to screen two cDNA libraries of developing female gametophytes isolated from cones of 26 June and 18 July 1995, respectively. Approximately 500000 plaque forming units were screened from each library. Several positive clones from both cDNA libraries were isolated, rescreened, sequenced, and shown to be of the same gene. However, no full-length clones were identified from the cDNA libraries and the remaining sequence at the 5' end of the transcript was revealed by 5' RACE using a gene-specific primer. Also from ovules collected on 11 June 1998, an identical sequence resulted after 5'- and 3' RACE with the same primers. A cDNA library from female gametophytes of that stage was not screened and therefore the presence cannot be excluded of other mip homologues not identified with the primer pairs used. The complete cDNA presented in Fig. 2 (mipfg; EMBL accession number AJ133748) contains an open reading frame that encodes a 253 amino acid protein with a calculated molecular mass of 26374 Da. This deduced protein sequence showed a close relationship with the TIP subclass of the MIP family of proteins in homology searches of sequence databanks. A multiple alignment of proteins that gave the highest scores in databank searches is shown in Fig. 3. Hydropathy plots (not shown) predicted six transmembrane regions (underlined in Fig. 2) of the deduced protein. The protein also displays the two conserved NPA-motifs and the signature sequence of the MIP family, [HNQA]-x-N-P-[STA]-[LIVMF]-[ST]-[LIVMF]-[GSTAFY] of the PROSITE database (Bairoch et al., 1996). Moreover, it contains the amino acid sequence (AEEATHPDSIR) identical to the tryptic fragment near the N-terminal of the TIP protein that earlier had been purified and sequenced from protein storage vacuolar membranes of mature seeds (Oliviusson and Hakman, 1995).
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Regulation of MIPs by phosphorylation of serine residues has been shown. Nine of the serines in MIPFG (two facing the cytoplasm) were predicted as possible phosphorylation sites using the PROSITE database (indicated in Fig. 2
). However, it is not known whether they are phosphorylated in vivo.
Temporal accumulation of mip transcripts in ovules and female gametophytes
Northern blot analyses were performed using poly(A)+ RNA isolated from ovules and female gametophytes collected at different developmental stages during 1998 and from mature dry and imbibed seeds, and the blots were probed with a DIG-labelled 361 bp mipfg cRNA (Fig. 4). The highest accumulation of transcripts was seen in ovules collected 11 June (lane 1) and in isolated female gametophytes from 18 July (lane 4). The female gametophyte collected on 11 June, 1998 was too small for isolation and therefore the complete ovules were included in the Northern blots from that collection. As there are no corresponding data of the protein profile from ovules of that date nothing can be said about the gene product(s). The transcripts detected 18 July (Fig. 4, lane 4) coincided with the accumulation of the higher molecular mass protein seen on immunoblots. A low level of transcripts was also present in the female gametophytes collected on 25 June and 6 July (Fig. 4, lanes 2 and 3, respectively) that coincided with the transition phase from the lower to the higher molecular mass bands on the immunoblots. No MIP transcripts could be detected in the mature dry or imbibed seeds (Fig. 4, lanes 5 and 6). All transcripts on the Northern blots gave a rather broad band of the same size class and the small variation seen in the protein molecular masses on immunoblots can not be deduced from it. Similar expression patterns were also seen when the membranes were reprobed with the longer (612 bp) DIG-labelled 5' RACE mipfg cDNA clone (not shown).
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In situ hybridization
Ovuliferous scales of 2 June 1998, bore at their base two ovules containing the female gametophyte (Fig. 5A–C). A nucellar dome has been formed in the massive nucellar cap and the integument has overgrown the nucellus and formed a wide-open micropyle. The female gametophyte is surrounded by a prominent megaspore membrane (wall) and a distinct layer of spongy tissue that stains intensively after in situ hybridization, both with the cDNA probe (Fig. 5A, D) and with the cRNA antisense probe (Fig. 5B, E). The RNA sense probe only gave a very weak staining reaction (Fig. 5C, F) while the control, incubated only with antibodies against DIG, did not stain at all (not shown). A terminal part of a vascular strand present just below the ovule also gave a strong staining reaction after in situ hybridization. In addition, the region of the ovule that will later differentiate into the seed coat gave a positive signal as did vascular bundles in the ovuliferous scale (Fig. 5G). Since the cDNA probe gave similar but more lucid staining patterns compared to the antisense cRNA probe, some of the following sections are only shown with the cDNA probe. Using cDNA as probes for in situ expression studies has been shown, for example, for rTip1 in rice roots (Samarajeewa et al., 1999).
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On 12 June 1998, the female gametophytes had increased slightly in size but the staining pattern of ovules was otherwise the same as on 2 June (Fig. 5A
–F), and is therefore not shown. This stage however, corresponded to the ovules giving a high transcript signal on the Northern blot (Fig. 4, lane 1), which suggests that the spongy tissue and/or the integument, also constituting a large part of the ovule, are responsible for that high signal.
Sections made on 21 June 1998, showed female gametophytes with well-developed archegonia (Fig. 5H). At this stage an intense staining was seen particularly in the jacket cells surrounding the giant egg cells while the rest of the female gametophyte did not stain (Fig. 5H, I). This stage corresponded to the stages of female gametophytes giving the lower molecular band on the immunoblot, supporting the view of a different MIP homologue functioning during archegonia formation. On sections including the integument as well, the region of the ovule which later will differentiate into the seed coat was noticeable. Resin cavities lined by secretory cells had developed in the inner tissue (the future endotesta) of the seed coat in the vicinity of the female gametophyte. With phase contrast microscopy the cells in the middle tissue (the future thick-walled sclerotesta) were seen to have slightly sculptured cell walls while the outer cells of the future sarcotesta were thin-walled. At this developmental stage the cells of the future sclerotesta stained intensively with both the cDNA probe (not shown) and cRNA antisense probe (Fig. 5J) while the cRNA sense probe only gave a very weak staining reaction (Fig. 5K). The seed coat now starts to harden, as noticed during tissue isolation.
At the time of embryo formation and the onset of storage protein accumulation, a strong staining reaction was seen all over the storage parenchyma of the female gametophyte (Fig. 5L, M). The staining signal decreased slightly later on as the embryo differentiated further and storage materials were built up in the cells, which possibly concealed the signal (Fig. 5N, O). These two stages corresponded to the time when the higher molecular mass MIP accumulated on immunoblots. The latter was also identical to the stage giving a strong signal on the Northern blot (Fig. 4, lane 4).
Mature dry seeds, however, did not give any staining signal in the in situ experiments (not shown), which also is in accordance with the results obtained with Northern analysis (Fig. 4, lanes 5 and 6).
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Discussion |
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Aquaporin homologues (MIPs) of Picea abies show a changing protein profile during seed development. In mature seeds the protein is known to reside in the PSV membrane of parenchyma cells (Oliviusson and Hakman, 1995
). In the present work, an aim was also to investigate which were the cells/tissues that expressed the gene(s) during the earlier stages. After cloning a gene from maturing seeds probes were made within regions known to be conserved among different aquaporin homologues (Fig. 2) with the prospect that such probes would also detect related genes in other tissues. A developmental analysis showed that jacket cells of the archegonium (Fig. 5H, I) in addition to the seed parenchyma at the onset of protein storage vacuolar formation (Fig. 5L–O) had a high gene activity. This suggests that the transition previously seen on the protein profile of developing female gametophytes (Oliviusson and Hakman, 1995) is the result of these gene activities. However, the identity of the lower molecular mass protein is still not known despite much effort to isolate it, and, consequently, neither if it belongs to the PIP or TIP subclass although its size suggests the latter. On immunoblots it has a size that is similar to an abundant MIP present in roots that was recently cloned and found to belong to the -TIP subgroup (MIPR, EMBL accession number AJ005078; Oliviusson et al., 2001).
A high expression of transcripts was also seen in the tapetal-like cells of the spongy tissue around the enlarging female gametophytes (Fig. 5A–F). Thus, the high activity of aquaporin homologues in these cells implies an important water/solute transporting function during the enlargement growth of the structures that they enclose. These results agree with the finding of high MIP transcripts of the PIP subclass in Brassica oleracea anthers with a probable location in the tapetum, as these cells share similar functions (Ruiter et al., 1997), although a precise in situ localization was not done. Interestingly, in a recent investigation of the in situ expression pattern of the tonoplast aquaporin ZmTIP1, Barrieu et al. found high transcript levels of the gene encoding a tonoplast intrinsic protein around the nucellus and in basal endosperm transfer cells of developing maize caryopses, cells with a symplastic transport of assimilates (Barrieu et al., 1998). They proposed that cells experiencing such cellular activities need a capacity for adjusting water potential of the cytoplasm (Barrieu et al., 1998).
Although it was not possible to isolate more than one gene from the developing female gametophytes, there are probably others expressed during the earlier stages. The RT-PCR and RACE products obtained with degenerate and specific primer pairs with female gametophytes from 11 June were identical to the gene cloned from tissues of 26 June and 18 July, which showed high identity to other plant TIPs. This product is, however, not limited to the female gametophyte, as identical PCR products were also obtained from roots, hypocotyls and cotyledons of 2-week-old seedlings by using the same primer pairs (Oliviusson et al., 2001).
To assign a function of the MIPFG protein that has been cloned and identified as a TIP of PSVs, as a transporter of water is reasonable. Several of the proteins with high homology to the deduced MIPFG sequence have selective water transport capacity. Recent reports also support the idea that functional residues or motifs in a protein sequence can be predicted to a given transport function, as more MIP sequences with known functions are gathered in databanks (Froger et al., 1998; Kuwahara et al., 1998; Lagrée et al., 1998; 1999). Froger et al. (1998) proposed a signature pattern for water transport and by comparing that sequence with that of the P. abies MIPFG, defines it as a water channel. The presence of water channels in the PSVs of seeds would allow maintenance of a cellular osmotic balance during the dynamic phases of protein loading into the PSVs formed during seed maturation, followed by the breakdown of the reserve products and the vacuolar redistribution that occur during germination and seedling growth, as discussed previously (Maurel et al., 1997). In P. abies the storage parenchyma of the female gametophytes is the major sources of storage proteins in the seeds, and is consumed during seedling growth. In the embryos, on the other hand, which also contain TIP proteins in their PSV membranes, this protein is replaced by another isoform in the seedlings (see Fig. 4 in Oliviusson et al., 2001; MIPR, EMBL accession number AJ005078).
On Northern blots (Fig. 4) and after in situ hybridization (Fig. 5) an elevated accumulation of transcripts was seen at the time when PSVs were formed and storage proteins started to accumulate in them (Fig. 1), which also correlates well with the protein accumulation profile seen earlier on immunoblots of developing seeds (Oliviusson and Hakman, 1995). Turgor regulation is known to occur in Phaseolus vulgaris seeds (Zhang et al., 1996) and may be important during seed maturation and germination. However, this process might be more efficient in the seed coat than in the cotyledons known to contain large amounts of -TIP (Maurel et al., 1997). The analysis of turgor-dependent efflux of assimilates from P. vulgaris seed coat (Zhang et al., 1996) indicates that the layer of ground parenchyma cells of the coat have an important regulatory function in the turgor-homeostat mechanism. It is interesting to note that high transcript levels were found in the sclerotesta of the future seed coat during development of P. abies seeds, even if there are no protein data validating the presence of aquaporin-related proteins in that tissue. Also, it is not known if the seed coat of P. abies has a similar function in assimilate partitioning, although it is most likely, considering its association with vascular tissue at the ovular base (Fig. 5A).
Both the archegonia jacket cells and the spongy cells have characteristics of cells with high metabolite fluxes and share possibly a similar requirement of osmoregulation. Considering the recent observations that both plant TIPs (Gerbeau et al., 1999) and PIPs (Biela et al., 1999) can have dual functions in transporting both water and small solutes, extensive studies of substrate selectivity of aquaporins are needed before their functional significance can be properly understood (Eckert et al., 1999; Tyerman et al., 1999).
It is interesting to note that in a textbook about plant embryogenesis (Wardlaw, 1955) the following sentences, that is a citation from Chamberlain of 1935, describing a gymnosperm can be found: ‘The turgidity of the female gametophyte (in the cycas), and later, the turgidity of the central cell, and still later, the egg cell is extreme’, followed by, ‘that if small cuts are made in the prothallus near the egg membrane, the liquid contents of the egg will spurt out, sometimes to a distance of 20 cm’, which indeed is a remarkable physiological situation probably not met by many other cells!
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We thank Dr Jan Salaj, Institute of Plant Genetics, Slovak Academy of Sciences, Nitra, Slovak Republic, for advice concerning in situ hybridization. We gratefully acknowledge financial support from the Carl Tryggers Foundation to I.H.
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3 Present address: Heidelberger Institut für Pflanzenwissenschaften, Zellbiologie, Im Neuenheimer Feld 230, D-69120, Heidelberg, Germany.
4 To whom correspondence should be addressed. Fax: +46(0)480446244. E-mail: inger.hakman{at}hik.se
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