JXB Advance Access originally published online on May 13, 2003
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Journal of Experimental Botany, Vol. 54, No. 388, pp. 1789-1791,
July 1, 2003
© 2003 Oxford University Press
A sucrose transporter, LjSUT4, is up-regulated during Lotus japonicus nodule development*
Received 27 January 2003; Accepted 26 March 2003
,1
1 Agricultural University of Athens, Department of Agricultural Biotechnology, Iera Odos 75, 118 55 Athens, Greece
2 Max Plank Institute for Molecular Plant Physiology, Am Mühlenberg, D-14476 Golm, Germany
* The nucleotide sequence appeared in the DDBJ/EMBL/GenBank database with the accession number AJ538041. To whom correspondence should be addressed. Fax: +30 210 5294314. E-mail: bmbi2kap{at}aua.gr
Abbreviations: dpi, days post-infection.
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Abstract |
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LjSUT4, encoding a putative sucrose transporter, was identified in a Lotus japonicus nodule cDNA library. The deduced amino acid sequence showed a high degree of identity with sucrose transporters from other plants. Semi-quantitative RT-PCR analysis demonstrated that the L. japonicus SUT4 gene was expressed at high levels in both roots and nodules. In situ hybridization revealed that, in young nodules, SUT4 mRNA transcripts are present in vascular bundles, inner cortex and both infected and uninfected cells while, in mature nodules, accumulation of transcripts was restricted only in vascular bundles and the inner cortex. The results indicated that LjSUT4 codes for a putative sucrose transporter, and its expression pattern suggests a possible shift in the mechanism of sugar transport during nodule development. The role of this polypeptide in sucrose transport and metabolism is discussed.
Key words: In situ hybridization, Lotus japonicus, root nodules, sucrose transporter, symbiosis.
Effective nitrogen fixation involves the complex interaction of legume plants with soil bacteria, Rhizobium, Bradyrhizobium, Sinorhizobium, Mesorhizobium, and Azorhizobium. A new organ is formed from this interaction, the root nodule. Within the nodule, the bacteria reduce atmospheric nitrogen to ammonia, which the plant assimilates in glutamine and glutamate. In turn, the plant provides the bacteria with carbon for growth and energy production. The carbon cost for this process is high, rendering the nodule a strong sink organ. Sucrose produced in photosynthetic tissues is the main carbohydrate translocated through the phloem to the nodules where it is rapidly metabolized (Vance et al., 1997).
In sink tissues sucrose transport is mediated by specific transporters, a family of highly hydrophobic proteins consisting of 12 transmembrane domains, which function as sucrose/H+ co-transporters (Delrot et al., 2001). Sink loading by sucrose transporters have been characterized in Plantago major, Vicia faba, Pisum sativum, and barley (Gahrtz et al., 1996; Lemoine, 2000; Weschke et al., 2000). In addition, enhanced expression of Arabidopsis sucrose transporters AtSUC2 and AtSUT4 was observed in various sink tissues including roots, flower, green fruit, sink leaves, and ovaries (Weise et al., 2000). Furthermore, a pollen-specific sucrose transporter-like protein (NtSUT3) has also been identified in tobacco (Lemoine et al., 1999). Moreover, gene expression analyses revealed that leaf sucrose transporters are also expressed in sink tissues (Lemoine, 2000).
In the present study, the spatial and temporal expression of a Lotus japonicus putative sucrose transporter gene whose expression is highly enhanced in nodules was investigated.
Recently, large numbers of expressed sequences tags (ESTs) from L. japonicus nodules have been deposited in public databases and analysed by DNA arrays for transcriptome analysis (Colebatch et al., 2002). Further analysis by digital northern revealed the presence of ESTs coding for polypeptides involved in monosaccharide and disaccharide transport. The complete nucleotide sequence of an EST clone showing high homology to previously characterized plant sucrose transporters was determined and designated as LjSUT4. The deduced amino acids of the LjSUT4 sequence revealed the presence of an open reading frame of 511 amino acids. The multiple amino acid sequence alignment of LjSUT4 with other known plant sucrose transporters expressed in sink tissues, revealed that the LjSUT4 exhibits 70%, 67.1%, 65.7%, and 49.7% similarity to Lycopersicon esculentum SUT4, Arabidopsis thaliana SUT4, Daucus carota SUT1, and Vicia faba SUT, respectively (Fig. 1). In silico analysis of the hydrophobic regions in LjSUT4 revealed the presence of 12 putative transmembrane domains, a characteristic of this family of membrane transporters (data not shown).
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The accumulation of LjSUT4 transcripts in different L. japonicus tissues (nodules, roots, leaves, stems, flowers, seedpods, cotyledons, hypocotyls, and apical meristems) was examined using a semi-quantitative reverse-transcription (RT)-PCR approach (Fig. 2A). The highest levels of LjSUT4 transcripts were observed in sink tissues such as roots and nodules. Relatively lower levels were found in green seedpods and hypocotyls, whereas, no detectable expression was found in leaves, stems, flowers, cotyledons, and apical meristems. Accumulation of LjSUT4 transcripts was also examined during nodule development. LjSUT4 transcripts were detectable at relatively low levels in emerging nodules 10 d post-infection (dpi), showed a maximum accumulation in young nodules (14 dpi), while at consequent developmental stages the transcript levels decreased (Fig. 2B).
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The spatial localization of LjSUT4 gene transcripts during nodule development was examined using an in situ hybridization approach. Sections of L. japonicus nodules at various stages of development were hybridized with 11-digoxigenin-rUTP-labelled RNA probes transcribed from LjSUT4 cDNA clone. Both antisense and sense labelled RNA transcripts were used as probes. At 14 dpi with Mesorhizobium loti, high levels of LjSUT4 transcripts were observed in the nodule parenchymatous cells, vascular bundles and in the infected and uninfected cells of the central tissue (Fig. 3A). In mature nodules at 28 dpi a strong signal was present mainly in the vascular bundles and nodule parenchymatous cells, while no hybridization signal could be detected in the cells of the central tissue (Fig. 3B). As a negative control, sections of L. japonicus nodules at 14 dpi with M. loti, were hybridized to sense digoxigenin-labelled RNA probes transcribed from a LjSUT4 clone (Fig. 3C). In this case no significant hybridization signal was detected.
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The results indicate that LjSUT4 codes for a putative sucrose transporter, which accumulates in various sink tissues of L. japonicus including root nodules. The differences of LjSUT4 spatial expression patterns during nodule development suggest that there is a possible shift in the transport and consequent metabolism of sugars associated with nodule maturation. This shift in the transport mechanism remains to be elucidated, especially with respect to the characterization and localization of additional transporters involved in sucrose transport. A developmentally similar spatial expression pattern of sucrose synthase transcripts was observed in soybean nodules (Kavroulakis et al., 2000). These data taken together suggest that sucrose may not be the immediate carbon source for cells located in the central tissue of mature nodules, but phosphorylated derivatives of sucrose catabolism (trioses or hexoses) are translocated from the inner cortex.
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Acknowledgement |
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This work was supported by an EU programme (HPRN-CT-2000-00086).
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