Medicago truncatula contains a second gene encoding a plastid located glutamine synthetase exclusively expressed in developing seeds

Abstract

Background: Nitrogen is a crucial nutrient that is both essential and rate limiting for plant growth and seed production. Glutamine synthetase (GS), occupies a central position in nitrogen assimilation and recycling, justifying the extensive number of studies that have been dedicated to this enzyme from several plant sources. All plants species studied to date have been reported as containing a single, nuclear gene encoding a plastid located GS isoenzyme per haploid genome. This study reports the existence of a second nuclear gene encoding a plastid located GS in Medicago truncatula.

Results: This study characterizes a new, second gene encoding a plastid located glutamine synthetase (GS2) in M. truncatula. The gene encodes a functional GS isoenzyme with unique kinetic properties, which is exclusively expressed in developing seeds. Based on molecular data and the assumption of a molecular clock, it is estimated that the gene arose from a duplication event that occurred about 10 My ago, after legume speciation and that duplicated sequences are also present in closely related species of the Vicioide subclade. Expression analysis by RT-PCR and western blot indicate that the gene is exclusively expressed in developing seeds and its expression is related to seed filling, suggesting a specific function of the enzyme associated to legume seed metabolism. Interestingly, the gene was found to be subjected to alternative splicing over the first intron, leading to the formation of two transcripts with similar open reading frames but varying 5' UTR lengths, due to retention of the first intron. To our knowledge, this is the first report of alternative splicing on a plant GS gene.

Conclusions: This study shows that Medicago truncatula contains an additional GS gene encoding a plastid located isoenzyme, which is functional and exclusively expressed during seed development. Legumes produce protein-rich seeds requiring high amounts of nitrogen, we postulate that this gene duplication represents a functional innovation of plastid located GS related to storage protein accumulation exclusive to legume seed metabolism.

Figure 1

Southern blot analysis of GS2 genes in Medicago truncatula. A. Schematic representation of BAC mth2-53e90 indicating the position of MtGS2a (AC148968-43) and MtGS2b (AC 1448968-42) and the restriction sites relevant for the southern analysis. The position of the probe used for the Southern analysis is also indicated. B. Southern hybridization of M. truncatula J5 genomic DNA and BAC mth2-53e90 DNA. 20 μg of genomic DNA and 5 μg of BAC DNA were digested with BgLII (1), NcoI (2) and EcoRV (3) and probed with a 260 bp DNA fragment corresponding to part of the 5'UTR and coding sequence of GS2a cDNA.

Figure 2

Exon/intron organization of the two Medicago truncatula GS2 genes. Exons are numbered and represented by dark gray vertical boxes. The two MtGS2b alternatively spliced transcripts are shown, highlighting the retention of the first intron in the MtGS2b-α transcript. The start (ATG) and the stop codon (TAA) are indicated.

Figure 3

Phylogenetic relationships of GS2 genes in relevant legume species. Bayesian unconstrained tree. In brackets are GenBank accession numbers. Numbers represent posterior probability values.

Figure 4

Semi-quantitative RT-PCR analysis of GS2 trancripts in different organs of the plant. Total RNA was extracted from roots of plants growing on ammonium nitrate (1), roots of plants growing in symbiosis with S. meliloti (2), 14 day old nodules (3), leaves (4), stems (5), cotyledons from light grown seedlings (6), cotyledons from dark grown seedlings (7), flowers (8), pods (9) and developing seeds collected at 20 DAP (10). 50 ng of cDNA was used in each assay. Transcript levels for MtGS2a, MtGS2b-α, MtGS2b-β and the elongation factor 1-a (elf1-a), as internal reference gene are shown. Values correspond to quantified band intensities of GS2 relative to the housekeeping gene elf1-α.

Figure 5

Semi-quantitative RT-PCR analysis of GS2 transcripts during seed development, seed imbibition and in major seed tissues. Total RNA was extracted from 3 DAP seeds (1), 6 DAP (2), 10 DAP (3), 14 DAP (4), 20 DAP (5), 24 DAP (6) and 36 DAP (7), dry seeds (after-ripenning) (8), dry seeds 1,5 h after imbibition (9) and dry seeds 24 h after imbibition (10), seed coat (11), endosperm (12) and whole embryo (13). 50 ng of cDNA was used in each assay. Transcript levels for MtGS2a, MtGS2b-α, MtGS2b-β and the elongation factor 1-a (elf1-α), as internal reference gene are shown. Values correspond to quantified band intensities of each GS2 transcript relative to the housekeeping gene elf1-α.

Figure 6

Western blot analysis of GS2 polypeptides during seed development and in major seed tissues. Proteins were extracted from seeds collected at different times following pollination (as indicated) and in seed tissues dissected from 20 DAP seeds: seed coat (SC), endosperm (ES) and embryo (E). Protein extracts (10 μg per lane) were subjected to SDS-PAGE followed by western-blot analysis using a specific anti-GS2 antibody.

Figure 7

Clustal X alignment of GS2a and GS2b deduced amino acid sequences. Residues fully conserved (*), highly conserved (:), poorly conserved (.) and not conserved (). The transit peptide is represented in gray and was predicted by analogy with the GS2 from pea [9]. Black boxes outline potential modified residues affecting GS2b activity. The black dots indicate two cysteine residues which are conserved in all plastid located isoenzymes [69]. I-IV-identifies the conserved segments between GS proteins defined by Eisenberg et al. [49].

Figure 8

Analysis of the catalytic functionality of GS2b. A. Complementation analysis of an E. coli glnA mutant expressing either GS2b or GS2a plant cDNAs. The plant cDNA corresponding to the coding sequences, without the predicted transit peptide were introduced into pTrc99A. Controls were transformed with the empty plasmid. Bacteria were grown on Minimal Medium (M9) agar plates with (+GLN) and without (-GLN) a glutamine supplement (0.25 mg mL-1). B. Western blot analysis of E. coli glnA extracts expressing recombinant GS2a or GS2b mature proteins probed with anti-GS antibody. C. GS activity assays of E.coli glnA- extracts expressing either GS2a or GS2b mature proteins. Results represent the mean of three independent experiments.