JXB Advance Access originally published online on May 16, 2005
Journal of Experimental Botany 2005 56(417):1805-1819; doi:10.1093/jxb/eri169
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
RESEARCH PAPER |
Phytosulphokine gene regulation during maize (Zea mays L.) reproduction*
1Biozentrum Klein-Flottbek und Botanischer Garten, Universität Hamburg, Ohnhorststrasse 18, D-22609 Hamburg, Germany
2Botanisches Institut der Christian-Albrechts-Universität zu Kiel, Olshausenstrasse. 40, D-24098 Kiel, Germany
To whom correspondence should be addressed. Fax: +49 431 880 4222. E-mail: msauter{at}bot.uni-kiel.de
Received 10 December 2004; Accepted 14 March 2005
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The sulphated pentapeptide phytosulphokine (PSK) was identified as a substance that promotes cell division in low-density suspension cultures and has been implicated in various aspects of tissue differentiation in plants. The peptide is derived from PSK precursor proteins that are encoded by small gene families. The physiological roles of PSK are still not clearly defined and little is known about expression of members of the PSK precursor gene family in any plant species. In this study, highly regulated tissue and cell type-specific expression are described for four PSK genes from maize (Zea mays L.) in female and male gametophytes, and during seed development. ZmPSK1 and ZmPSK3 were specifically and differentially expressed in cells of female and male gametophytes and in female and male gametes. In anthers ZmPSK1 or ZmPSK3 transcripts were found, for example, at high levels in secretory tapetal cells which support developing microspores. ZmPSK1 mRNA was abundant in mature pollen including sperm cells. ZmPSK1 and ZmPSK3 transcripts were also detected in egg and central cells of the female gametophyte and ZmPSK1 mRNA was present in synergids, indicating that the PSK peptide probably plays a role during gametogenesis, pollen germination, and fertilization. In developing maize kernels all four ZmPSK genes were expressed, albeit with striking differences in their expression patterns. It is proposed here that PSK is required for numerous but defined processes during gametophyte and early sporophyte development. In general, PSK availability appears to be controlled through transcriptional regulation in a tissue and cell type-specific and development-dependent manner.
Key words: Anther, corn development, embryo sac, gametophyte, maize, peptide signalling, phytosulphokine, pollen, reproduction, Zea mays L
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Maize (Zea mays L.) is an important crop plant and a long-standing model plant for genetic studies. Understanding floral and gametophytic development, fertilization, and embryogenesis is one particular research interest that is being studied in maize. Male and female gametophytes are essential structures for angiosperm reproduction. The female gametophyte produces a number of specific cell types such as the egg and central cell which give rise to the embryo and endosperm, respectively. It is involved in pollen tube guidance, fertilization, induction of seed development, and perhaps also in the maternal control of embryo development (reviewed in Drews et al., 1998
; Drews and Yadegari, 2002). The male gametophyte or pollen grain must achieve stigma recognition, pollen tube growth, and fertilization. Seed development requires co-ordinated expression of embryo and endosperm and is controlled by sporophytic, male gametophytic, and female gametophytic genes. Much has been learned about these processes at the cytological level not only in maize but also in the model plant Arabidopsis and many genes with a possible function in reproduction have been described in Arabidopsis (da Costa-Nunes and Grossniklaus, 2004; Hennig et al., 2004). A detailed understanding of how gametophytic differentiation and seed development are regulated is, however, still being awaited.
Phytosulphokine (PSK) was identified as a plant-specific sulphated pentapeptide that can induce cell division in suspension cells which are kept at low density and which normally would not divide (Matsubayashi and Sakagami, 1996; Yang et al., 2000). PSK genes were identified and the preproprotein that is processed to release PSK was described (Yang et al., 1999, 2001; Lorbiecke and Sauter, 2002). A leucine-rich repeat receptor kinase was identified as a putative PSK receptor that binds PSK and enhances PSK-dependent growth upon over-expression in carrot suspension cells (Matsubayashi et al., 1997, 2002). While much progress has been made in identifying PSK precursor and PSK receptor genes, the physiological roles for the PSK peptide are still not clearly defined. Studies performed with in vitro-cultured cells implied a role for PSK in cell proliferation (Matsubayashi et al., 1996, 1999a; Chen et al., 2000) and also in cell differentiation (Matsubayashi et al., 1999b). PSK was shown to be involved in density-dependent in vitro germination of tobacco pollen (Chen et al., 2000). Density-dependent pollen germination and tube growth is known as a pollen population effect (Brewbaker and Majumder, 1961; Pasonen and Kapyla, 1998; Chen et al., 2000). Chen and co-workers (2000) suggested that PSK is produced in the pollen and should be released into the germination medium within less than 2 h. They concluded that PSK precursor mRNAs should be present in mature pollen grains. However, PSK transcription during pollen development and/or pollen germination was not determined.
PSK effects observed in planta indicate that PSK may not simply promote cell proliferation but appears to play a role in diverse aspects of plant development and reproduction. In Cryptomeria japonica and in carrot, PSK had a stimulatory effect on the formation of somatic embryos (Kobayashi et al., 1999; Igasaki et al., 2003). When hypocotyl segments of Asparagus were treated with PSK, a promotive effect on the formation of adventitious roots was observed (Yamakawa et al., 1998). In Arabidopsis, a protective effect of PSK against high night temperature was described (Yamakawa et al., 1999).
It is conceivable that PSK acts as a developmental factor that influences proliferation, differentiation, and survival of cells. All three mechanisms play important roles during gametophyte and seed development. Maize is well suited to study PSK function during these processes: maize kernels are large in size and many events relating to seed development have been described in detail (Kiesselbach, 1949; Lur and Setter, 1993; Grafi and Larkins, 1995; Larkins et al., 2001; Olsen, 2001). Maize pollen can be harvested in large amounts and can be triggered to germinate in vitro. Furthermore, single cells can be isolated from the female and male gametophytes and used to study gene expression even at low transcript levels (Sauter et al., 1998).
Since attempts to detect the PSK peptide in planta by way of immunocytology have not been successful to date, expression of PSK precursor genes as an approximation to PSK abundance was studied. PSK precursor proteins are encoded by small gene families. Little is known about the regulation of the different family members in any plant species. In maize, four PSK genes were previously identified based on available EST sequences (Lorbiecke and Sauter, 2002). In this study, the PSK gene family was classified phylogenetically and the expression of the ZmPSK genes during male and female gametogenesis and during seed development was analysed. Based on this study's findings it was concluded that maize gametophytic and early sporophytic development may depend on PSK availability which is tightly controlled through differential expression of PSK genes in a cell type-specific manner.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Plant material, treatments and tissue preparation
The Zea mays L. lines used in this study were the inbred lines LC (a colour converted W22 line; Wienand et al., 1986
) and A188, the flavonole-deficient conditionally male-sterile line ‘white pollen’ (genotype: whp/whp, c2/c2; Coe et al., 1981) and its corresponding fertile wild-type line (whp/whp, C2/C2 or whp/whp, C2/c2). Plants were grown in a greenhouse with 16 h of light at 25 °C and a relative humidity of 55–95%. Controlled pollination was achieved by the use of standard breeding procedures. For RNA isolation, all harvested tissues were frozen immediately in liquid nitrogen and stored at –70 °C until use. Unfertilized pistillate spikelets were harvested at the time of silking. Silks and glumes were removed from the pistils (ovules) for separate analyses. For RT-PCR, pistils were dissected into three parts. Developing kernels were harvested between 6 d and 20 d after pollination (dap). In addition, embryos were dissected from 20 dap kernels and both tissues were harvested separately. Tassels were harvested immediately before the pollen-shedding stage. Staminate spikelets were harvested during tassel development up to the time point of anthesis. At anthesis, days before anthesis (dba) were calculated for all previously collected samples. Mature pollen was harvested at anthesis. Pollen was germinated in vitro in liquid medium (Schreiber and Dresselhaus, 2003) for 10 min and harvested by centrifugation. Coleoptiles and primary roots were harvested from shoots grown on moist filter paper in Petri dishes for 3 d in the dark at 25 °C. In additional experiments seedlings were grown for 7 d in paper rolls as described by Hetz et al. (1996) in the greenhouse and were dissected afterwards into roots, kernel, and leaves. Seedlings were grown for 16 d in soil in the greenhouse until harvest of the above-ground parts. Mature plants were raised in the greenhouse. Third or fourth leaves and stem sections of the youngest internode were excised at the time of pollen shedding.
Cloning of ZmPSK sequences
A 214 bp fragment corresponding to the 3'-region of ZmPSK1 was amplified from a 
-ZAPII cDNA library constructed from mRNA of LC kernels harvested between 13 d and 28 d after pollination. The primer pair ZmPSK1-F2 (5'-GAGGGCGCCAACGACGAGGAC-3') and ZmPSK1-R2 (5'-CAGAAGCAGCACGCGCTGGCG-3') was used for PCR amplification. The primer sequences were derived from the EST-based ZmPSK1 sequence of the maize line OH43 (BK000133
[GenBank]
, Lorbiecke and Sauter, 2002). The amplified fragment was digoxigenin-labelled and used for plaque screening of the same cDNA library according to standard procedures. pBluescript clones carrying cDNA inserts were excised in vivo from selected positive -ZAPII clones by the ExAssistTM Interference-Resistant Helper phage with SOLR strain (Stratagene, Heidelberg, Germany). Two identical clones carrying the ZmPSK1 cDNA (572 bp, Accession number AY842490) were identified.
A 251 bp fragment of the 3'-UTR of ZmPSK2 was amplified from LC genomic DNA using two primers deduced from a ZmPSK2 EST of line OH43 (BK000134 [GenBank] , Lorbiecke and Sauter, 2000): ZmPSK2-F2 (5'-GGCAAGCCATGAGGAGGAGGTAG-3') and ZmPSK2-R2 (5'-GATGATGGATCTCGGACGATAC-3'). One SNP was found in the PCR product when compared with the OH43 ZmPSK2 (data not shown).
For ZmPSK3, a 3'-UTR-specific 324 bp fragment was amplified by RT-PCR from total RNA of suspension-cultured maize cells of the hybrid line A188xB73. Reverse transcription was performed with the primer ZmPSK3-R2 (5'-CCATGATATCTATATGGATAG-3') in standard conditions. PCR was performed with primers ZmPSK3-R2 and ZmPSK3-F2 (5'-CAACTAGCTAGTCACTGCTCA-3').
A 203 bp fragment corresponding to the 5'-region of ZmPSK4 was amplified from LC genomic DNA using primers deduced from an EST-derived ZmPSK4 sequence of line OH43 (BK000136 [GenBank] ) with primers ZmPSK4-F2 (5'-CCAACACTGTTCCAGACTTCCAG-3') and ZmPSK4-R2 (5'-CGAGCAGCTGCCGCGCAGTTG-3'). ZmPSK4 sequences from LC and OH43 turned out to be identical in the amplified regions.
Sequence analysis
cDNA clones and PCR fragments were sequenced with the ABI PRISMTM Big-Dye Terminator Cycle Sequencing Kit with Ampli-Taq® DNA Polymerase FS (Applied Biosystems, Weiterstadt, Germany). Samples were run on a 377A sequencing device (Applied Biosystems, Weiterstadt, Germany). Data were analysed using programs provided by the ExPASy proteomics server (http://www.expasy.ch/). Alignment was calculated with the Clustal X 1.83 program (Thomson et al., 1997) and manually edited using GeneDoc 2.6 (Nicholas and Nicholas, 1997). Phylogenetic relationships were displayed using TreeView 1.6.6.
Identification of PSK gene loci on genome linkage maps were identified using online services provided by the Maize Genetics and Genomics Database (http://www.maizegdb.org/), the Rice Genome Research Program (http://rgp.dna.affrc.go.jp/), US Rice Genome Sequencing (http://www.usricegenome.org/), and WebChrom (http://www.genome.clemson.edu/projects/rice/fpc/WebChrom/).
Northern blot analysis
Total RNA from developing maize kernels and pollen was isolated as described by Kluth et al. (2002). Total RNA from other tissues was isolated with TRIfast reagent (PeqLab, Erlangen, Germany) as described by Lorbiecke and Sauter (1998). Twenty to 30 µg RNA each were loaded on gels, separated, and blotted as described by Lorbiecke and Sauter (1998). 32P-labelled probes were generated using random primers (Sambrook et al., 1989) and hybridized successively to the same blot (Sauter, 1997). As a control for equal loading, RNAs were hybridized with a cDNA fragment of ZmJR27 that encodes a proteasome subunit. ZmJR27 was previously found to be ubiquitously expressed in maize (R Lorbiecke, unpublished data).
In situ hybridization
Maize anthers were harvested at different developmental stages and were fixed in phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4) with 4% (w/v) paraformaldehyde. Fixed anthers were frozen and cut into 25 µm thick sections. The sections were mounted on polylysine-coated slides (Super-Frost® Plus, Menzel, Braunschweig, Germany) and dried for 1 h at 60 °C. Subsequently the sections were washed first in PBS and then in 0.2 M HCl for 10 min each by dipping slides into the solution. Sections were then treated with 10 µg ml–1 proteinase K in PBS (Roche, Mannheim, Germany) for 20 min at 37 °C followed by washing once with 2 mg ml–1 glycine in PBS for 1 min, once in PBS for 1 min, once in 4% paraformaldehyde in PBS for 10 min, twice in PBS for 3 min each, once in 50 mM HCl, 5.4 mM acetic anhydride, and 13 µl ml–1 triethanolamine for 10 min, and once in H2O for 2 min prior to prehybridization. Prehybridization was carried out for 1 h at 42 °C in a buffer containing 500 µl ml–1 formamide, 100 µl ml–1 10x PBS, 20 µg ml–1 NaCl, 10 µl ml–1 Tween 20, 50 µg ml–1 heparin, and 100 µg ml–1 fishsperm DNA. RNA probes were generated from the 3'-UTR of ZmPSK1 and ZmPSK3. Antisense and sense RNA probes were transcribed and DIG-labelled (Roche, Mannheim, Germany) in vitro using T3 or T7 RNA polymerase (MBI Fermentas, St Leon-Rot, Germany). For hybridization, 10 µl slide–1 containing 100 ng RNA probe, 50 µg PolyA RNA, and 15 µg tRNA were denatured at 99 °C for 5 min and then mixed with 90 µl of prehybridization solution. Each slide was covered with 100 µl of RNA mix in prehybridization solution as described and incubated at 42 °C for 16 h in a moist chamber. After hybridization sections were washed four times with 5x SSC (0.75 M NaCl, 75 mM sodium citrate) for 15 min each at 42 °C, once in 10 mM TRIS–HCl, pH 7.5, 2 mM EDTA, 500 mM NaCl (buffer A) for 5 min at 37 °C followed by treatment for 30 min at 37 °C with 20 µg ml–1 RNase A (Sigma, Taufkirchen, Germany) in buffer A. After RNase treatment slides were washed four times for 10 min each at 37 °C in buffer A and once in PBS for 5 min. Unspecific antigens were blocked by incubation in blocking solution (Roche) for 1 h. RNA–RNA hybrids were detected after incubation for 1 h with an anti-digoxigenin monoclonal antibody (Roche, Mannheim, Germany) diluted 1:1000 in blocking solution and incubation overnight with an NBT-BCIP-solution (Roche, Mannheim, Germany) in accordance with manufacturer's instructions. All treatments were carried out at room temperature unless stated otherwise.
Single cell and tissue isolation
Single central cells, egg cells, synergids, and sperm cells were isolated and selected from Zea mays L. line A188 as described by Kranz et al. (1991). Antipodal cells were isolated in groups of approximately 15 cells. To isolate nucellus tissue, the chalazal part of ovules without the embryo sac was dissected: by separating the outer and inner integuments, nucellus tissue was released. Part of the inner integument remained attached to the nucellus tissue. After washing in 650 mOsm mannitol solution, cells or tissues were transferred into tubes, quick frozen in liquid nitrogen, and stored until use.
cDNA synthesis
Isolation of mRNA from 25 synergids, 25 eggs, 25 central cells, 4 clusters of approximately 15 antipodal cells, 400 sperm cells, or 5 nucellus tissue pieces were carried out with oligo dT(25) magnetic beads (Dynal, Hamburg, Germany). Nucellus tissue was homogenized in liquid nitrogen before mRNA isolation. Two times concentrated lysis/binding buffer (200 mM TRIS–HCl, pH 7.5, 1 M LiCl, 20 mM EDTA, pH 8.0, 10 mM DTT, and 2% lithium dodecyl sulphate) was added to the frozen cells or tissues in mannitol solution. The volume was adjusted with deionized H2O to give a final concentration of 1x lysis/binding buffer in a total volume of 20–40 µl. For each cell or tissue type 15 µl oligo dT(25) magnetic beads were washed once with lysis/binding buffer, the lysed cells or tissue were added and incubated for 15 min under continuous rotation to allow annealing of the mRNA to oligo dT(25). Washes were carried out twice each, first with 50 µl washing buffer (10 mM TRIS–HCl, pH 7.5, 0.15 M LiCl, and 1 mM EDTA) and then with 50 µl first strand buffer (50 mM TRIS–HCl, pH 8.3, 75 mM KCl, and 3 mM MgCl2). Magnetic beads with mRNA were resuspended in 3 µl deionized H2O and used for first strand cDNA synthesis applying the template switch mechanism at the 5' end and long-distance PCR (SMART-LDPCR; BD Bioscience, Clontech) following the manufacturer's instructions. For each cell or tissue type the optimal PCR cycle number was determined empirically to generate cDNAs with a size distribution between 0.5 kb and 3 kb. The PCR conditions led to similar cDNA concentrations of approximately 20 ng µl–1 for all cell types and nucellus tissue.
PCR amplification of cDNAs
PCR analysis was conducted in 50 µl reactions with 1 µl cell or tissue type-specific cDNA or 500 ng genomic DNA as template.
The following primers were used: for ZmPSK1: ZmPSK1-F2 and ZmPSK1-R2; for ZmPSK2: ZmPSK2-F1 (5'-CACTCGCTCGCTCACTACG-3') and ZmPSK2-R1 (5'-CTAGCATGCACAAGGTACTAC-3'); for ZmPSK3: ZmPSK3-F1 (5'-CAAGCAGCTGAAGGCTAGTC-3') and ZmPSK3-R1 (5'-CTAGGCTAGCTCGTGAACC-3'); for ZmPSK4: ZmPSK4-F1 (5'-TTCCACAGCGCAAAGCTCCG-3') and ZmPSK4-R1 (5'-CTCGTCCTTGTCGTCGTTCC-3') and for ZmWee1: ZmWee1-F (5'-CACCCCGCTTCCCGAGTCT-3') and ZmWee1-R (5'-CAGCAGCCACAGCCTTTAGTCCTT-3').
PCR was performed for 35 cycles with cDNA or genomic DNA as templates. The thermal cycling parameters were as follows: denaturation at 94 °C for 1 min, annealing at 65 °C for 30 s in the case of ZmPSK1, or at 56 °C for 30 s in the case of ZmPSK2, ZmPSK3, and ZmPSK4; strand elongation at 72 °C for 45 s. Twenty-five µl of each PCR reaction were loaded on an agarose gel for separation of PCR products.
RT-PCR
First strand synthesis was performed for 75 min at 42 °C using 0.25 µM oligo-dT primer, 25 U RevertAidTM H Minus M-MuLV Reverse Transcriptase (MBI Fermentas, Germany), 50 mM TRIS–HCl, pH 8.3, 50 mM KCl, 4 mM MgCl2, 20 mM DTT, 40 U Ribonuclease inhibitor (MBI Fermentas, Germany), and 100 ng total RNA from developing kernels at 0–20 dap in a total volume of 100 µl. To amplify specific cDNAs, 1 µM cDNA-specific primer pair, 0.75 mM MgCl2, 0.1 mM dNTP, 1% DMSO, 10 mM TRIS–HCl, pH 8.8, 50 mM KCl, 0.8% Nonidet P40, and 0.5 U Taq polymerase (MBI Fermentas, Germany) were added to 10 µl of the RT reaction (5 µl in the case of ZmCycB2;1) in a total volume of 50 µl. The thermal cycling parameters were 94 °C for 3 min, 94 °C for 1 min, 56 °C for 1 min in the case of ZmPSK3, 55 °C for 1 min in the case of all other cDNAs, 72 °C for 1 min, 30 cycles in the case of ZmWee1, ZmJR27, and ZmCycB2;1 and 35 cycles for all other cDNAs; final extension at 72 °C for 10 min. Cycle numbers were determined empirically for each cDNA such that amplifications did not exceed the exponential phase. Primer pairs were: ZmPSK1-F2 and ZmPSK1-R2, ZmWee1-F and ZmWee1-R; ZmPSK2-F2 and ZmPSK2-R2, ZmPSK3-F1 and ZmPSK3-R1, ZmPSK4-F2 and ZmPSK4-R2, JR27-F21 (5'-CTTCGGATGCTGGCTATCAGGC-3') and JR27-R15 (5'-AAGACAACCGTGTCGAGCATGGTTCC-3'), ZmCycB2;1-MS10 and ZmCycB2;1-MS11 (Sauter et al., 1998). Twenty µl of each amplification product were subjected to electrophoresis. Abundance of PCR fragments was calculated using the PCBAS 2.09 program (Raytest, Straubenhardt, Germany).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Phylogenetic relationship of the PSK gene family from maize
PSK precursor genes exist in small gene families in higher plants. It is assumed that they all encode the same PSK growth factor. The overall amino acid sequence similarity between PSK precursor proteins is relatively low, both within a PSK family and between PSK families of different plant species. However, the secondary structure of PSK precursor proteins was predicted to be highly conserved (Lorbiecke and Sauter, 2002
).
In maize, the four known putative PSK precursor homologues were predicted based on publicly available EST data (Lorbiecke and Sauter, 2002). In this study, the full-length sequence of ZmPSK1 was cloned from a -cDNA library of immature kernels from the maize line LC. Four insertion/deletions (indels) and seven single nucleotide polymorphisms (SNPs) were detected when the cloned sequence was compared with the ZmPSK1 cDNA from line OH43. They were ascribed to line-specific variations. These differences resulted in two changes in the deduced amino acid sequence, V16
L16, K25T25, in the region of the putative signal peptide and in the region of its predicted cleavage site, respectively (Fig. 1A). Sequence identities between the deduced ZmPSK precursor proteins were below 43% and similarities were below 52% and were mostly restricted to the region of the PSK domain (Yang et al., 2001) and the PSK signature motif (Lorbiecke and Sauter, 2002). Additional residues were conserved between particular ZmPSK family members (Fig. 1A). Calculation of protein similarity revealed a closer relationship between ZmPSK1 and ZmPSK3 (identity: 28%) and between ZmPSK2 and ZmPSK4 (43%) (data not shown). In accordance with this finding, phylogenetic analysis of PSK precursor proteins from different plant species revealed two large clusters, designated cluster I and cluster II (Fig. 1B). PSK precursor proteins from dicotyledonous and monocotyledonous species grouped separately within each cluster. This finding suggested that despite of an unusually low sequence similarity among PSK precursor proteins a certain degree of phylogenetic conservation was retained during evolution (Fig. 1B).
|
ZmPSK1 was found to be 50% identical to OsPSK4 and ZmPSK3 was found to be 51% identical to OsPSK6, respectively (data not shown). Both maize PSKs are therefore more similar to these rice PSK precursor proteins than to any of the known PSK precursors from maize (Fig. 1B). Using publicly available maize linkage data maps the ZmPSK3 gene was mapped at
260 cM/bin9.06 on the long arm of chromosome 9 (data not shown). Based on genome synteny analyses of the macrocolinearity between maize and rice chromosomes (Salse et al., 2004) a close evolutionary linkage was found between this region of maize chromosome 9 and a region of rice chromosome 3. In fact, the rice OsPSK6 gene was found to be localized at the corresponding position on the rice genome that is on chromosome 3 at 34.8 cM (data not shown) indicating that it was likely that both genes originated from the same ancestor gene during grass evolution.
Mapping of ZmPSK1 was carried out as part of the Maize Assembled Genomic Island (MAGI) project (Emrich et al., 2004) by using sequence data of a genomic ZmPSK1 clone (MAGI_80480). The ZmPSK1 gene was mapped on chromosome 7 at 56 cM/bin7.00 between markers umc1694 and bnlg2132 (P. Schnable, Iowa State University, USA; personal communication). This chromosome region is linked by macrocolinearity to the top of chromosome 7 from rice. Accordingly, OsPSK4 was on this corresponding region at 5.8 cM (data not shown). Thus, ZmPSK1 and OsPSK4 are also linked by synteny like ZmPSK3 and OsPSK6.
The ZmPSK2 gene mapped at 300 cM/bin3.05 on the long arm of chromosome 3. The position of ZmPSK4 is unknown. So far, no phenotypic mutations have been mapped to any region of the known ZmPSK gene loci. However, the available mapping data of ZmPSK genes suggested a widely spread distribution of the PSK gene family over the maize genome as was also found for PSK genes in the Arabidopsis (Lorbiecke and Sauter, 2002) and rice genomes (R Lorbiecke, unpublished results).
Although PSK gene families are little conserved at the amino acid level, phylogenetic data indicated the presence of two PSK subfamilies in both monocotyledonous and in dicotyledonous plants. Synteny data of maize and rice genomes indicated that at least two PSK genes found in each plant probably originated from the same two ancestor genes. It might be hypothesized that these genes may not only have retained a similar genomic context but also a similar function during evolution.
ZmPSK1 is abundantly expressed in the male gametophyte
PSK gene regulation was studied during gametophyte and early sporophyte development to see if PSK might play a role in sexual reproduction. To that end, regulation of the four known members of the PSK gene family in maize was analysed. Northern blot analysis revealed high mRNA levels for ZmPSK1 in various reproductive tissues and low or undetectable transcript abundance for ZmPSK2, ZmPSK3, and ZmPSK4 (Fig. 2A, B; data not shown).
|
ZmPSK1 was predominantly expressed in pistillate staminate spikelets, in male inflorescences (in maize called ‘tassel’), and in kernels (Fig. 2A). ZmPSK1 mRNA was not detected in styles of the female flower (‘silk’; Fig. 2A). This result suggested a study in more detail of the expression of ZmPSK1 during male gametophyte and pollen development. For this analysis not only was wild-type maize used but also a maize line that is deficient in anthocyanin synthesis and therefore produces conditionally sterile pollen (‘white pollen’, whp; Coe et al., 1981
), to see if the loss of pollen fertility affected ZmPSK regulation. Expression analysis of developing male staminate spikelets showed that ZmPSK1 transcripts increased strongly between 9 d before anthesis and anthesis (Fig. 2B, –9 dba to 0 dba). Transcripts were also highly abundant in mature pollen (Fig. 2B). No changes in ZmPSK1 transcript levels were observed when pollen grains were germinated in vitro for 10 min (Fig. 2B). No difference in ZmPSK1 transcript abundance was observed between the pollen-sterile mutant whp and its corresponding wild-type line (WT) indicating that pollen sterility, due to the lack of flavonoid biosynthesis, did not affect ZmPSK1 expression.
ZmPSK1 transcript amounts were similar in spikelets at anthesis (Fig. 2B, 0 dba) and in isolated pollen of the same stage (Fig. 2B, pollen mature). In order to elucidate in which cells ZmPSK1 transcripts were localized during anther development, anthers at two different developmental stages were subjected to in situ hybridization (Fig. 3). The younger anther stage to be analysed was characterized by uninucleate microspores with small vacuoles and a nucleus that was centred and a living tapetal cell layer (data not shown; compare with Bedinger, 1992; Fig. 2D). At this stage ZmPSK1 transcripts were detected at high levels in tapetal cells of the sporangium wall (Fig. 3A, B). Neither the epidermal nor endothecial cells (Fig. 3A, B), nor the connective with the vascular bundle (Fig. 3A) nor the developing microspores (Fig. 3B) revealed specific staining. Control assays with sense RNA as the probe did not result in staining (Fig. 3C, D). The second stage analysed was characterized by a degenerated tapetum, uninucleate microspores with one large central vacuole, and a nucleus that was positioned opposite the aperture (data not shown, compare with Bedinger, 1992; Fig. 2E). At this later stage ZmPSK1 mRNA was detected exclusively and strongly in microspores (Fig. 3E–H). Tapetal cells were degenerated at this stage and staining in this cell layer was therefore no longer visible. RT-PCR analysis of sperm cells isolated from mature pollen showed that ZmPSK1 was expressed in male gametes (Fig. 4A, B). In situ hybridization data revealed that the maturing pollen cytoplasm was strongly stained (Fig. 3F). Both results taken together indicate that ZmPSK1 was expressed in generative cells and in the vegetative cell of the pollen. Out of the four ZmPSK genes ZmPSK1 was the only gene to be expressed in sperm cells.
|
|
For ZmPSK3, a weak hybridization signal in northern analysis was noted in tassels (Fig. 2A) and in developing staminate spikelets (Fig. 2B). In situ hybridization performed on developing anthers at the younger stage described above revealed ZmPSK3 transcripts in the epidermis, endothecium, and tapetal cell layer (Fig. 3I, J, L), in the connective tissue (Fig. 3I) including the vascular bundle (Fig. 3K), but not in microspores (Fig. 3I). During late anther development, expression of ZmPSK3 in epidermal and in tapetal cells ceased (Fig. 3Q, T), but staining was still observed in endothecial cells (Fig. 3Q, T). Cells at the stomium showed particularly strong staining (Fig. 3Q, T). In maize, the stomium is located at the meeting point of the two loculi that make up each half anther. It is the site where the anther wall opens to release pollen at anthesis. ZmPSK3 transcripts were still present in the vasculature of older stage anthers (Fig. 3Q, S) and ZmPSK3 transcripts were detected in microspores at this uninucleate, large vacuolar stage (Fig. 3R). Northern analysis indicated that mature pollen did not contain detectable transcript levels of ZmPSK3 (Fig. 2B). Using RT-PCR studies on mature pollen it was observed that ZmPSK3 transcripts were also not detected in sperm cell preparations (Fig. 4A, B) which confirmed northern data (Fig. 2B). In conclusion, it can be said that ZmPSK3 was transiently expressed in developing microspores. Expression began at the uninucleate, large vacuolar stage and ceased as pollen matured.
Transcripts of ZmPSK2 and ZmPSK4 were neither detected in male inflorescences by northern blot (Fig. 2A, B), nor by RT-PCR in sperm cells isolated from mature pollen (Fig. 4A, B). The results clearly indicate a role for ZmPSK1 and ZmPSK3 in male gametophyte development. ZmPSK1 appears to have a unique role because its transcripts are found in developing anthers and in the vegetative pollen cell. ZmPSK1 is the only PSK gene that is also expressed in male gametes of maize. PSK was reported to act on density-dependent pollen germination in tobacco (Chen et al., 2000). It is conceivable that ZmPSK1, ZmPSK3, or both provide active PSK for pollen germination in maize.
ZmPSK genes are differentially expressed in the female gametophyte
Northern blot analyses showed expression of ZmPSK1 in ovaries of female flowers and to some extent in glumes, albeit in both tissues at a much lower level than that found in male staminate spikelets or developing kernels (Fig. 2A). A more detailed RT-PCR analysis using three sections of the pistillate spikelet showed the highest transcript levels of ZmPSK1 in the upper part of the nucellus when compared with the upper ovary wall and the bottom region of the pistil (which consists of pistil axis, nucellar tissue, and embryo sac; Fig. 5A).
|
ZmPSK1 expression in nucellar tissue was verified by RT-PCR studies (Fig. 5A). RT-PCR analysis of single cells from the embryo sac (Fig. 4A) showed that ZmPSK1 was expressed in all cell types (Fig. 4B), except in antipodal cells (data not shown).
ZmPSK2 transcripts were not detected in pistillate spikelets by northern blot hybridization (data not shown). However, ZmPSK2 transcripts were amplified by RT-PCR from RNA obtained from the bottom pistil region and the ovary wall, respectively. Higher transcript amounts were detected in the bottom part of the pistil (Fig. 5A). ZmPSK2 was neither expressed in cells of the embryo sac nor in nucellar cells (Fig. 4B). This finding suggested that ZmPSK2 expression was restricted to tissues other than the nucellus and embryo sac in the female flower. These could be the pistil axis or the ovary wall.
ZmPSK3 transcripts were detected in the pistil, but not in glumes or in silks (Figs 2A, B,5A). Within the pistil, expression was highest in the lower part containing the embryo sac, nucellar tissue, and pistil axis (Fig. 5A). Detailed analysis of the embryo sac revealed highly cell-type specific expression in egg and central cells (Fig. 4B). Synergids and antipodals had no ZmPSK3 transcripts. Expression of ZmPSK3 was also verified for nucellar tissue (Fig. 4B). Interestingly, the cell-cycle regulatory gene ZmWee1 showed a similar expression pattern to ZmPSK3 in the embryo sac (Fig. 4B). Transcripts were present in egg and central cells, but not in antipodals or synergids. Surrounding nucellar tissue also expressed both ZmWee1 and ZmPSK3 possibly pointing to a link between fertilization-induced cell division activity and PSK function.
ZmPSK4 was expressed in the ovary of unfertilized pistillate spikelets as shown by northern blot where a signal in the size range of 1.3 kb was detected (Fig. 2A) and by RT-PCR analysis (Fig. 5A). However, in both cases, transcript levels appeared to be rather low. In single cells of the embryo sac and in nucellar cells, mRNA of ZmPSK4 was not present.
Since transcripts of ZmPSK2 and ZmPSK4 were detected in unfertilized pistillate spikelets by RT-PCR (Fig. 5A, B) it was concluded that both genes were expressed in tissues other than gametophytic and nucellar. In the female flower these might be the carpels (ovary wall), palets or axis. In accordance with this interpretation ZmPSK2 and ZmPSK4 mRNAs were detected in the ovary wall and in the bottom part of the female flower, but not in nucellar tissue (Figs 4B, 5A).
ZmPSK gene expression in the female gametophyte was highly cell-type specific and it is conceivable that transcript levels in single cells may be comparable to transcript levels found in pollen. Since PCR amplification with single cell types of the embryo sac were performed with standardized cDNA populations until saturation, these data (Fig. 4B) cannot be interpreted quantitatively. Nonetheless, they showed specific and differential expression of ZmPSK1 and ZmPSK3 genes in the embryo sac (Fig. 4B) indicating that these genes may be important for sexual plant reproduction. The egg cell and central cell, which both participate in double fertilization, expressed ZmPSK1 and ZmPSK3. Synergids, which aid fertilization, transcribed ZmPSK1 mRNA only. On the other hand, antipodals, which are not involved in fertilization, did not express any ZmPSK gene (data not shown).
By contrast with ZmPSK1 and ZmPSK3, ZmPSK2 and ZmPSK4 did not appear to play a role in female gametogenesis. Transcripts were neither found in cells of the embryo sac nor in nucellar tissue surrounding the embryo sac. ZmPSK2 and ZmPSK4 genes were also not active in male gametogenesis nor in sperm cells. It was therefore concluded that ZmPSK2 and ZmPSK4 do not play a role in sexual reproduction in maize.
PSK genes are differentially controlled during kernel development
ZmPSK genes were shown to be differentially expressed in female and male gametophytes and in female and male gametes. There is now interest in the regulation of gene expression following fertilization. Therefore, expression of ZmPSK genes during kernel development was examined by semi-quantitative RT-PCR (Fig. 5B). To verify the reliability of the RT-PCR method, analysis of ZmPSK1 and ZmJR27 was included in this experiment. For both genes, the data obtained by RT-PCR resembled results obtained by northern analysis (Figs 2A, 5B).
During kernel development, ZmPSK1 expression increased strongly between pollination (Fig. 2A, pistillate spikelet 0 dap) and 6 d after pollination (Fig. 2A, 6 dap). Transcript abundance remained high in the developing corn until 20 dap. In 20 dap kernels, ZmPSK1 transcripts were present in both the embryo and the remaining kernel tissues, with higher amounts in the kernel tissue (Fig. 2A), indicating a role for PSK in embryogenesis as well as in caryopsis development or possibly in endosperm development. The amount of ZmPSK1 mRNA in the kernel decreased drastically after germination (Fig. 2B, kernel germ.)
Compared with ZmPSK1, mRNA amounts of ZmPSK2, ZmPSK3, and ZmPSK4 were very low. Nonetheless, RT-PCR analysis revealed that all four genes were expressed in a highly regulated manner during different phases of corn development (Fig. 5B). For ZmPSK2, expression was at a low but constant level between 0 dap and 12 dap (Fig. 5B). Transcript levels decreased thereafter and were near zero at 20 dap. ZmPSK3 was also expressed prior to fertilization (Figs 2A, 5B) and decreased subsequently until at 12 dap a second, transient increase occurred. At this time of kernel development, rates of mitotic cell division and endoreduplication in the starchy endosperm declined (Fig. 6A, B). ZmPSK4 mRNA was expressed between 0 dap and 6 dap (Fig. 6B). Thereafter, mRNA levels decreased until 20 dap.
|
PSK was implicated in regulating cell division activity in suspension-cultured cells. Evidence for a role of PSK in regulating cell division activity in planta is, however, lacking. Maize kernel development relies on two modes of cell cycle progression. Kernel growth is supported by cell production through mitotic cell divisions. On the other hand, endosperm development involves endoreduplication, i.e. DNA replication without nuclear and cell divisions (Larkins et al., 2001
). To compare ZmPSK gene expression patterns to these cytological events during kernel and endosperm development, expression of the mitotic B2-type cyclin ZmCycB2;1 (Sauter et al., 1995; Renaudin et al., 1996) was analysed as a molecular marker for mitotic cell division activity. ZmWee1 expression was studied as molecular marker for endoreduplication. ZmWee1 kinase mRNA was previously shown to accumulate preferentially during DNA endoreduplication (Sun et al., 1999). The Wee1 kinase phosphorylates M- and/or S-phase-specific cyclin-dependent kinases and is most likely involved in the regulatory switch between the mitotic and the endoreduplication cell cycle in the developing endosperm of maize (Sun et al., 1999; Larkins et al., 2001).
ZmCycB2;1 transcripts increased steadily and strongly between 0 dap and 10 dap and decreased sharply between 10 dap and 12 dap (Figs 5B, 6B). ZmCycB2;1 expression thus indicated that mitotic cell division activity in the kernel was maximal at 10 dap and became very low at 12 dap, a finding that is in good agreement with previously published cytological data (Lur and Setter, 1993; Fig. 6).
ZmWee1 expression reached maximal levels at 10 dap (Figs 5B, 6B) and declined gradually thereafter. Molecular data obtained for ZmWee1 expression were in agreement with the suggested role of this kinase as a regulator of cell division and endoreduplication activity in maize kernel development (Sun et al., 1999) and also with the time curve of cytologically determined mitotic and endoreduplication rates (Lur and Setter; 1993; Fig. 6A).
Expression analysis pointed to a major role for ZmPSK1 and a minor role for the remaining ZmPSK genes in maize endosperm development. Even though for ZmPSK2, ZmPSK3, and ZmPSK4 overall mRNA levels appeared to be low, all genes were differentially regulated. The fact that ZmPSK genes were expressed at specific stages during kernel development may indicate that they were subject to regulation by defined signals that are released as embryo and corn development progress. Taking into account that PSK gene expressions seem to be highly tissue-specific and that the relative amount of each tissue changes during kernel development, it may be that expression patterns reflect tissue-specific expression of each PSK gene in, for example, starchy endosperm, aleurone, embryo, or the basal transfer layer. Overall, ZmPSK genes were expressed throughout kernel development.
PSK gene expression in seedlings and plants
Whereas ZmPSK1 showed high expression in gametophytes and developing kernels (Fig. 2A) transcript levels in seedlings and in mature plants were low (Fig. 2A, B) indicating that ZmPSK1 may have a predominant role in generative but not in vegetative plant development. By contrast, ZmPSK3 transcripts were more abundant in leaves from seedlings and mature plants than in generative tissues (Fig. 2A, B). Expression of ZmPSK3 was also higher in seedling leaves than in roots (Fig. 2B).
For ZmPSK2 and ZmPSK4 transcript abundance in seedlings or mature plant organs was below the detection limit of the northern blot (Fig. 2; data not shown) even though several sequences of ZmPSK2 were found in a database search in EST libraries derived from developing green tissue (data not shown) indicating that it is a transcribed gene.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
In this study, the PSK gene family from maize has been described. It has been shown that gene expression of each of its family members is tissue-specific and is regulated during plant development and tissue differentiation. Expression of ZmPSK1 was very strong compared with other PSK genes and was predominantly linked to reproductive organs. ZmPSK1 and ZmPSK3 were found to be differentially expressed in cells of the female gametophyte. Both genes were expressed in egg cells, central cells, and nucellar tissue. By contrast with ZmPSK1, ZmPSK3 was not expressed in synergids. In antipodal cells ZmPSK genes appeared not to be active. These findings suggested that ZmPSK3 expression was limited to gametophytic cells that retain the potential for proliferation. Expression of the cell cycle regulatory kinase ZmWee1 was also detected only in cells that are able to proliferate. Igasaki et al. (2003)
showed that PSK has a strong stimulatory effect on in vitro formation of somatic embryos of Cryptomeria japonica. These data support the notion that PSK might also be necessary for female gametophyte and/or embryo development in planta.
ZmPSK1 and ZmPSK3 were the only PSK genes expressed in the male gametophyte, albeit expression patterns differed between both genes. In anthers that contained a functional tapetum and microspores with one nucleus and multiple vacuoles, transcripts of both ZmPSK1 and ZmPSK3 were detected at high levels in tapetal cells. Maize possesses a secretory tapetum that secretes nutrients to the microspore and precursors for the biosynthesis of the outer pollen wall called exine (Bedinger, 1992). Tapetal cells are thus well equipped to provide large amounts of PSK to the developing microspores. It would be interesting to test, through genetic means, if, and if so at which stage, microspore development is hampered when PSK expression is suppressed.
At this early stage of microspore development ZmPSK3 was transcribed not only in tapetal cells but throughout the anther, i.e. in the epidermis, the endothecium, and the connective tissue. Upon anther maturation when uninucleate microspores aquired one large vacuole and tapetal cells degenerated, expression of ZmPSK3 in epidermal and tapetal cells ceased. Expression persisted in endothecial cells and was especially strong in endothecial cells proximal to the stomium. Cells at the stomium are thought to undergo programmed cell death to allow opening of the anther wall and pollen release during dehiscence (Keijzer et al., 1996; Wu and Cheung, 2000). Light and electron microscopic studies of the dehiscence process in tobacco anthers implied that a cascade of unique gene expression events throughout anther development is required for the dehiscence programme and that differentiation of the stomium and of other cell-types in the interlocular region involves cell signalling processes (Sanders et al., 2005).
Tapetal cells in which PSK transcripts were highly abundant in the early stages also undergo programmed cell death. Moreover, progressive ethylene-mediated programmed cell death is a hallmark of endosperm development in maize kernels (Young and Gallie, 2000). During endosperm development all four PSK genes were expressed, with a specific pattern found for each gene. Taking these coincident data into consideration it may be worthwhile to look into a possible role of PSK in cell death-related processes in plants.
In uninucleate microspores with one large vacuole, high mRNA levels of both ZmPSK1 and ZmPSK3 were found. These microspores subsequently undergo cell division that results in formation of an immature pollen grain with a vegetative and a generative cell (McCormick, 1993). ZmPSK3 expression appeared shortly before this microspore mitosis I and ceased upon pollen maturation. One possible function of ZmPSK3 could be to ensure co-ordinated mitotic divisions among all microspores contained in one loculus or co-ordinated pollen wall synthesis or both. Induction of mitotic cell division by PSK was shown previously in low density suspension cells which would not proliferate in the absence of a trigger (Matsubayashi et al., 1996). Since pollen are released at the same time their maturation must be completed essentially at the same time. A diffusible signalling molecule like PSK could help synchronize this process.
Transcripts of ZmPSK1 remained abundant in mature pollen and expression was shown to continue until after pollen germination. Previous analyses demonstrated that the PSK peptide stimulates germination of tobacco pollen in vitro and predicted that PSK mRNA be present in mature pollen (Chen et al., 2000). This assumption was now confirmed and the gene responsible for PSK expression in germinating pollen was identified. If PSK transcript abundance reflects PSK peptide expression in maize, the ZmPSK1 gene is a likely candidate to be involved in regulatory mechanisms related to both pollen development and pollen germination in maize. In addition, ZmPSK1 was the only PSK gene expressed in sperm cells and in synergids. Hence, ZmPSK1 gene function may be required for cell–cell communication between the pollen tube and the synergid that initiates fertilization.
Interestingly, both gametophytically expressed ZmPSK genes showed closest relationship to each other at the amino acid level and clustered to subgroup II of the phylogenetic tree. By contrast, ZmPSK2 and ZmPSK4, the two genes that were not expressed in gametophytic cells, clustered to subgroup I. A similar clustering into two groups was found for the PSK family members from other plants. Moreover, based on their genomic locations, ZmPSK1 and ZmPSK3 seem to be linked evolutionary to their closest homologues from rice, OsPSK6 and OsPSK4. Thus, phylogenetic relationship of PSK genes might reflect at least to some extent similar expression patterns and hence similar functions. Since detailed PSK gene expression data are largely missing from other plant species, future experiments will be necessary to assess this conclusion. Expression data from rice showed that OsPSK genes were differentially expressed in various sporophytic tissues (Lorbiecke and Sauter, 2002). By contrast, expression patterns of PSK genes from Arabidopsis were more or less similar in the tissues examined (Yang et al., 2001). However, only a rather crude analysis on stems, leaves, and roots was performed in that study.
All four ZmPSK genes analysed were expressed in developing maize kernels, albeit with striking differences in their expression patterns. Expression of the mitotic cyclin ZmCycB2;1 and of the cell cycle kinase ZmWee1 served as molecular markers to correlate PSK gene expression with cytological events related to kernel development (Fig. 6; Lur and Setter, 1993). These time-courses agreed well with previously published data on histone H1 kinase activity indicative of CDK activity (Grafi and Larkins, 1995), ZmWee1 expression (Sun et al., 1999), mitotic index and endoreduplication activtiy during corn development (Schweizer et al. 1995; Larkins et al., 2001). The strong increase in ZmPSK1 transcripts paralleled the increase in kernel fresh weight as well as the increase in endosperm cell division rate pointing to a role of ZmPSK1 in endosperm development. By contrast, ZmPSK4 transcripts decreased during the phases of most rapid cell division and growth of the endosperm. ZmPSK2 transcripts dropped significantly when mitotic and endoreduplication activity in the endosperm slowed down in favour of storage protein accumulation, suggesting that expression of ZmPSK2 and ZmPSK4 was not directly linked to production of endosperm cells or to endosperm development.
Cytokinins and auxin are known to regulate cell division and auxin has also been implicated in endosperm endoreduplication and zein expression. Lur and Setter (1993) showed that auxin concentration abruptly increased and the ratio of cytokinin to auxin declined at the time cell division decreased and endoreduplication increased (Fig. 6; zeatin riboside, IAA, Lur and Setter, 1993). ZmPSK3 transcript levels increased transiently when endoreduplication activity declined. ZmPSK3 transcript abundance also paralleled to a certain degree changes in auxin to cytokinin concentration. This may suggest a functional link between ZmPSK3 expression and hormone homeostasis. One possibility would be that ZmPSK3 participates in the molecular switch from endoreduplication to storage protein synthesis. In suspension-cultured mesophyll cells of asparagus Matsubayashi et al. (1999a) demonstrated that the production of active PSK-
was closely correlated with the signal transduction pathway mediated by auxin and cytokinin.
As a consequence of the tremendous increase in starchy endosperm, the proportional composition of tissue types within the maize kernel is altered significantly during development (Larkins et al., 2001). Hence, the varied ZmPSK expression patterns could either reflect developmentally dependent regulation of ZmPSK genes in the same tissue or tissue-type dependent expression. It is also possible that both expression modes occur during kernel development as was observed in gametophytes where cell-type specific expression as well as expression of two ZmPSK genes in the same cell type occurred.
In this context it has to be noted that all putative PSK precursor proteins are assumed to generate the same bioactive pentapeptide. Nevertheless, the expression data presented here indicate that different PSK precursors seem to be important in different cell types and at different developmental stages. It is conceivable that regulatory steps beyond gene expression are involved in the release of the bioactive PSK peptide. One additional level of regulation might be through differentially expressed proteases that are specific for each precursor type.
In plants, a number of peptides have been identified which are believed to act as signalling molecules, for example, CLAVATA3 (CLV3), ENOD40, SCR, RALF, systemins and a 4 kDa peptide from legumes (Franssen and Bisseling, 2001; reviewed in Ryan et al., 2002; Yamazaki et al., 2003). All of these peptides are thought to be secreted into the extracellular space. In Arabidopsis, for instance, the ligand-receptor pair CLV3 and CLV1 is assumed to play important roles in the regulation of shoot meristem fate (Clark, 2001; reviewed in Traas and Vernoux, 2002). CLV1 is a leucine-rich repeat receptor-like kinase (RLK) and CLV3 is a small secreted peptide that has been proposed to travel through the extracellular space to its receptor (Clark, 2001). Both proteins are involved in meristem maintenance (Traas and Vernoux, 2002). In carrot, a putative PSK receptor was found to be a leucine-rich repeat RLK (Matsubayashi et al., 2002). Experimental evidence (Matsubayashi and Sakagami, 2000) and sequence similarity analysis (R Lorbiecke, unpublished data) indicated that at least in rice more than one PSK receptor might exist. The biochemical similarities between the CLV1/CLV3 and the PSK/LRR RLK peptide-receptor pairs may be indicative of a similar mechanism through which PSK, CLV1 or peptide ligands in general act in plant cells.
PSK activity might depend on tightly modulated and tissue-specific PSK gradients. Hence, a specific concentration of PSK peptide in the extracellular space in relation to available PSK binding sites could be a key to PSK signal transduction. Such gradients might be established as a combination of cell- and tissue-type specific expression of different PSK genes, specific cleavage of the various PSK precursors, and diffusion and/or targeted transport of the PSK peptide. In Arabidopsis, not only di- and tripeptide transporters but also tetra- and pentapeptide transporters have been identified (Stacey et al., 2002). Due to the high abundance of peptide transporters in Arabidopsis, diverse and important roles are expected for these transporters during plant development. PSK was proposed to be one of their putative substrates (Stacey et al., 2002). Assuming PSK uptake into the cell, the existence of intracellular PSK receptors must be envisioned.
Currently, it is still unknown why more than one PSK peptide precursor gene is expressed in some cells, as in the case of egg and central cells. One explanation might be that the interplay between differently regulated PSK genes is necessary to fine-tune PKS abundance in a certain cell type, tissue, or organ. The existence of differentially regulated ZmPSK promoters might also facilitate the regulation of PSK expression by a larger spectrum of molecular signals.
|
Acknowledgements |
|---|
We are indebted to Dr Patrick S Schnable, Dr Tsui-Jung Wen, Debbie Che, and Scott Emrich (Iowa State University, USA) for mapping the ZmPSK1 gene locus. We thank Dr MM Herrmann (Universität Hamburg, Germany) for providing the northern blot of the whp mutant line and its corresponding wild type, and Katja Müller (Universität Hamburg, Germany) for her excellent technical assistance. We also thank Dr M Mulisch (Zentrale Mikroskopie, Biologiezentrum, Universität Kiel) for support with in situ studies. Part of this work was supported through grant SA495/8-1 from the Deutsche Forschungsgemeinschaft to MS.
|
Footnotes |
|---|
* Accession numbers of sequences that have been deposited in GenBank: ZmPSK1, complete mRNA (LC)=AY842490.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Bedinger P. 1992. The remarkable biology of pollen. The Plant Cell 4, 879–887.
Brewbaker JL, Majumder SK. 1961. Cultural studies of the pollen population effect and the self-incompatibility inhibition. American Journal of Botany 48, 457–464.[CrossRef][ISI]
Chen YF, Matsubayashi Y, Sakagami Y. 2000. Peptide growth factor phytosulphokine-alpha contributes to the pollen population effect. Planta 211, 752–755.[CrossRef]