JXB Advance Access originally published online on March 12, 2004
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Journal of Experimental Botany, Vol. 55, No. 398, pp. 957-959, April 1, 2004
© 2004 Oxford University Press
GENE NOTE |
Novel expression patterns of phosphatidylinositol 3-hydroxy kinase in nodulated Medicago spp. plants
Received 12 Novenber 2003; Accepted 20 January 2004
1 Laboratory of Molecular Biology, Department of Plant Sciences, Wageningen Agricultural University, Dreijenlaan 3, 6703 HA Wageningen,The Netherlands
2 John Innes Centre, Norwich Research Park, Colney Lane, Norwich NR4 7UH, UK
* Present address and to whom correspondence should be sent: Laboratorio de Fisiología Vegetal, Departamento de Biología, Universidad Autónoma de Madrid, E-28049 Madrid, Spain. Fax: +34 913 978 191. E-mail: luise.hernandez{at}uam.es
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Abstract |
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A cDNA clone encoding a phosphatidylinositol 3-kinase (PtdIns 3-kinase) has been characterized from Medicago truncatula, Mtpi3k, that is highly homologous to their counterparts from soybean (over 84%). The results suggest the presence of at least two genes coding PtdIns 3-kinases in M. truncatula. Mtpi3k transcript levels increased in nodules, compared with non-infected roots. Strikingly, Mtpi3k mRNA accumulated in young elongating stems at higher levels than that observed in other organs. Enhanced transcription of genes coding PtdIns 3-kinases might occur in tissues experiencing a high degree of vesicle trafficking and cell elongation.
Key words: Elongating stems, Medicago truncatula, nodules, phosphatidylinositol 3-kinase, transcripts.
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The phosphatidylinositol (PtdIns) 3-kinases are responsible for the phosphorylation in the D-3 position of the inositol ring of PtdIns (Drøbak and Heras, 2002). Substantial evidence suggests that 3-phosphorylated phosphoinositides are involved in processes such as cell growth, vesicle trafficking, and secretion (Odorizzi et al., 2000). In plants, there is only evidence for the PtdIns 3-kinase homologous to the yeast protein VPS34P, which is apparently able to phosphorylate PtdIns exclusively in the D-3 position (Stack and Emr, 1994). Only three cDNAs with high homology to vps34 have been cloned in higher plants: one from Arabidopsis thaliana (Atvps34, accession number U10669 [GenBank] ; Welters et al., 1994) and two from Glycine max (Gmpi3k-1 and Gmpi3k-5, accession numbers L29770 [GenBank] and L27265 [GenBank] ; Hong and Verma, 1994). The importance of PtdIns 3-kinase activity for normal cell development in plants has been demonstrated using transgenic A. thaliana plants expressing antisense Atvps34, where the growth of plants was severely impaired (Welters et al., 1994). Furthermore, accumulation of Gmpi3k-l transcripts was observed in young soybean nodules (Hong and Verma, 1994), a developmental stage associated with a high rate of vesicle production and membrane biogenesis as the host cells accommodate thousands of bacteroids within symbiosomes (Brewin, 1991). Recent evidence suggests that PtdIns 3-kinases in plants might also be key mediators of other cellular events: they might play a role in nuclear function (Bunney et al., 2000; Drøbak and Heras, 2002) and during stomatal movements (Jung et al., 2002).
However, relatively limited direct information is available about the role of PtdIns 3-kinase in plants, and only a few genes encoding for this enzyme in plants have been cloned. To inspect the involvement of PtdIns 3-kinases during the establishment of the Rhizobium–legume symbiosis, the expression of a gene encoding PtdIns 3-kinase (Mtpi3k) in nodulated Medicago truncatula was analysed. This species has become established as a model plant for the study of molecular genetics in the Rhizobium–legume symbiosis and several databases are available with Expressed Sequence Tag (EST) cDNA libraries through the Medicago truncatula consortium. After a survey at http://bio-srI8.stanford.edu, the clone 00356 (accession number AA660470 [GenBank] ) was identified. Its complete sequence (accession number AJ517208 [GenBank] ) was compared in multialignment and pairwise analyses to their soybean and A. thaliana counterparts, showing an identity with Gmpi3k-1 and Gmpi3k-5 up to 84.7% and 85.3%, respectively, and up to 73.7% compared with Atpi3k. Interestingly, the open reading frame sequence is extremely conserved, where homology increases up to 90% relative to the soybean genes, being the largest differences confined to both 5'- and 3'-end non-coding regions.
Due to the existence of two highly homologous genes in soybean, the presence of more than one gene coding PtdIns 3-kinase in M. truncatula has been analysed by Southern blot hybridization. Genomic DNA was extracted from leaves of M. truncatula plants grown for 4 weeks on a commercial nursery substrate. Several aliquots were digested with different restriction endonucleases, separated by electrophoresis in 1% agarose gels, and blotted onto Hybond N+ membranes (Amersham Biosciences, UK), following standard protocols (Sambrook et al., 1989). The DNA probes were radiolabelled with 32P-dCTP using Klenow (Feinberg and Vogelstein, 1983) and membranes were hybridized under stringent conditions (0.1x SSC at 65 °C), before being exposed to a phosphorimager plate (Storm Molecular Dynamics, USA). To discriminate between the cloned cDNA and other putative homologous genes present in the genome of M. truncatula, two distinct probes were generated. The first one, comprising the full-length coding sequence, was prepared by digestion of the EST clone with XhoI/BamHI (approximately 3150 bp). The smaller probe was synthesized by PCR from the Mtpi3k 3'-end using the EST clone as the template (forward primer 5'-GCGTGAGTGCATTGTTCC-3' and reverse primer 5'-GGATTGTGAGTATTGAAAAGTGCG-3'). The amplified DNA fragment had a 517 bp size, spanning from position 2456 to 2973, and comprised the last 100 bp of the ORF region and almost the full-length 3'-end.
Hybridization of DNA digested with PstI, NcoI, HindIII, and BamHI with the full-length probe revealed the presence of more than one band (Fig. 1A), but when hybridized with the 3'-end specific probe there was only one band (Fig. 1B). This is in agreement with the restriction mapping of Mtpi3k, where PstI, NcoI, and BamHI did not cut the sequence, although their putative presence at intron non-coding regions cannot be excluded. Digestion with EcoRI, an enzyme that has one restriction site at position 1906, resulted in two bands when hybridized with the 3'-end probe (Fig. 1B), but it increased to five bands when hybridized with the full-length probe (Fig. 1A). Thus, it is conceivable that at least another gene homologous to Mtpi3k is present in the M. truncatula genome. To analyse this possibility further, a survey was made at the non-redundant ESTs databases and five putative ESTs of M. truncatula were identified (accession numbers AW688500 [GenBank] , BG448131 [GenBank] , AW690678 [GenBank] , BQ124464 [GenBank] , and BG453095 [GenBank] ). After multialignment analysis, two (AW688500 [GenBank] and AW690678 [GenBank] ) of the three clones overlapping at the 5'-end region expanded upstream between 19– 34 bp compared with Mtpi3k. In this region, comprising from 45 to at least 11 bp, the nucleotide sequence is completely mismatched to Mtpi3k. This region is expected to be highly variable between different genes, although no other significant mismatches were observed downstream. It is predicted that these ESTs might be representative of another PtdIns 3-kinase gene but, due to the extremely conserved sequence, this will only be confirmed when their 3'-end sequences are available.
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Accessible information about the transcription pattern of PtdIns 3-kinase genes in plant tissues is extremely scarce, as only the study by Hong and Verma (1994) has addressed this issue. Therefore, the differential gene expression of Mtpi3k in M. truncatula and the closely related species M. sativa, inoculated with Rhizobium meliloti (Van de Wiel et al., 1990) were studied. Plants were collected after 3–4 weeks post-inoculation; shoot meristems, leaves, young elongating stems, non-infected roots, and nodules were sampled and RNA extracted (Greissen and Mullineaux, 1995). Total RNA was separated in 1% (w/v) denaturation formaldehyde agarose gels and transferred onto Hybond N+ membranes (Sambrook et al., 1989). Phosphorimager images after hybridization with the previously described radiolabelled probes were analysed (Kodak 1D 3.5, USA), and the signals were normalized against the rRNA 18S band (data not shown).
The accumulation of transcripts varied significantly between tissues, with higher levels in stems and nodules. Blots containing RNA from M. truncatula hybridized with the 3'-end specific probe had higher expression in nodules than in uninfected roots (Fig. 2a). A similar pattern was observed when the full-length probe was used, although differences were smaller (Fig. 2b). Therefore, Mtpi3k might be up-regulated in nodules, in agreement with early results in soybean, where Gmpi3k-1 accumulated in nodules (Hong and Verma, 1994). Considering the extremely high homology of the legume PtdIns 3-kinase cDNAs (over 84%), it is difficult to establish clearly their phylogenetic relationship based on the expression pattern.
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Striking results arose when the mRNA abundance of Mtpi3k was analysed in shoot organs, as hybridization with both probes showed a high level in young stems compared with that found in shoot apical meristems and expanded leaves. To verify these results, the pattern of Mtpi3k transcript accumulation was also analysed in organs of M. sativa (Fig. 2d), that was hybridized with the heterologous full-length probe in medium-stringent conditions (0.5x SSC, 65 °C). Similar pattern was observed, with a putative up-regulation of the Mtpi3k homologues of M. sativa in stems and nodules. The young developing stems used for RNA extraction contain tissues undergoing cell elongation, where high rates of vacuolization are observed, and proper sorting of vacuolar vesicles is essential for normal shoot development (Morita et al., 2002). The importance of PtdIns 3-kinases in normal shoot development was highlighted in transgenic Arabidopsis plants expressing antisense Atpi3k. These plants showed phenotypes with petioles that had not elongated and extremely short stems, although the growth of callus was not severely affected (Welters et al., 1994); trait that might be related to the relatively low level of Mtpi3k expression in tissues experiencing cell division (Fig. 2). Additional evidence was found using the Electronic Northern facility at http://medicago.toulouse.inra.fr, where the frequencies of identified EST clones homologous to Mtpi3k were compared between libraries produced from several plant tissues. This analysis showed relevant ratios in immature seeds, developing stems, roots under elongation, and roots at early stages of Rhizobium infection (data not shown).
In conclusion, the observed putative up-regulation of Mtpi3k in nodules and in stems might indicate a role in tissues experiencing high rates of vesicle trafficking and vacuolar differentiation. It is envisaged that the development and architecture of several plant organs and tissues might depend on PtdIns 3-kinase function, prompting further research in this respect.
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Acknowledgements |
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LEH and NJB gratefully acknowledge financial support from the EU HCM Programme: Signals in Symbiosis (CHRX-CR94–0699). We are grateful to Professor SR Long (Stanford, USA) for the donation of the clone EST00356
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