Differential expression of the two cytosolic glutamine synthetase genes in various organs of Medicago truncatula.

In order to clarify the physiological roles of the cytosolic forms of glutamine synthetase (GS) in Medicago truncatula, we have performed a detailed analysis of the expression of the two functional cytosolic GS genes, MtGSa and MtGSb in several organs of the plant. Transcriptional fusions were made between the 2.6 or 3.1 kbp 5' upstream regions of MtGSa or MtGSb, respectively, and the reporter gene gusA encoding beta-glucuronidase and introduced into the homologous transgenic system. MtGSa and MtGSb were found to be differentially expressed in most of the organs, both temporally and spatially. The presence of GS proteins at the sites where the promoters were active was confirmed by immunocytochemistry, providing the means to correlate gene expression with the protein products. These studies have shown that the putative MtGSa and MtGSb promoter fragments were sufficient to drive GUS expression in all the tissues and cell types where cytosolic GS proteins were located. This result indicates that the cis acting regulatory elements responsible for conferring the contrasting expression patterns are located within the region upstream of the coding sequences. MtGSa was preferentially expressed in the vascular tissues of almost all the organs examined, whereas MtGSb was preferentially expressed in the root cortex and in leaf pulvini. The location and high abundance of GS in the vascular tissues of almost all the organs analysed suggest that the enzyme encoded by MtGSa plays an important role in the production of nitrogen transport compounds. The enzyme synthesised by MtGSb appears to have more ubiquitous functions for ammonium assimilation and detoxification in a variety of organs.

Cellular expression and regulation of the Medicago truncatula cytosolic glutamine synthetase genes in root nodules.

In this paper we have studied the localisation of expression of the two functional cytosolic glutamine synthetase (GS) genes, MtGSa and MtGSb, in root nodules of the model legume Medicago truncatula. We have used a combination of different techniques, including immunocytochemistry, in situ hybridisation and promoter beta-glucuronidase (GUS) fusions in transgenic plants, to provide the means of correlating gene expression with protein localisation. These studies revealed that transcriptional regulation (mRNA synthesis) plays an important part in controlling GS protein levels in nodules of M. truncatula. The major locations of cytosolic GS mRNA and protein are the central tissue, the parenchyma and the pericycle of the vascular bundles. These findings indicate that in nodules, GS might be involved in other physiological processes in addition to the primary assimilation of ammonia released by the bacterial nitrogenase. The two genes show different but overlapping patterns of expression with MtGSa being the major gene expressed in the infected cells of the nodule. Promoter fragments of 2.6 kb and 3.1 kb of MtGSa and MtGSb, respectively, have been sequenced and primer extension revealed that the MtGSb promoter is expressed in nodules from an additional start site that is not used in roots. Generally these fragments in the homologous transgenic system were sufficient to drive GUS expression in almost all the tissues and cell types where GS proteins and transcripts are located except that the MtGSa promoter fragment did not express GUS highly in the nodule infected cells. These results indicate that the cis-acting regulatory elements responsible for infected-cell expression are missing from the MtGSa promoter fragment.

Plant interstitial telomere motifs participate in the control of gene expression in root meristems.

The promoters of Arabidopsis eEF1A genes contain a telomere motif, the telo-box, associated with an activating sequence, the tef-box. Database searches indicated the presence of telo-boxes in the 5' region of numerous genes encoding components of the translational apparatus. By using several promoter constructs we demonstrate that the telo-box is required for the expression of a beta-glucoronidase gene in root primordia of transgenic Arabidopsis. This effect was observed when a telo-box was inserted upstream or downstream from the transcription initiation site, and occurred in synergy with the tef-box. These results clearly indicate that interstitial telomere motifs in plants are involved in control of gene expression. South-western screening of a lambdaZAP library with a double-stranded Arabidopsis telomere motif resulted in characterization of a protein related to the conserved animal protein Puralpha. The possibility of a regulation process similar to that achieved by the Rap1p in Saccharomyces cerevisiae is discussed.

The Medicago truncatula MtAnn1 gene encoding an annexin is induced by Nod factors and during the symbiotic interaction with Rhizobium meliloti.

Here we report the characterization of a new Nod factor-induced gene from Medicago truncatula identified by mRNA differential display. This gene, designated MtAnn1, encodes a protein homologous to the annexin family of calcium- and phospholipid-binding proteins. We further show that the MtAnn1 gene is also induced during symbiotic associations with Rhizobium meliloti, both at early stages in bacterial-inoculated roots and in nodule structures. By in situ hybridization, we demonstrate that MtAnn1 expression in nodules is mainly associated with the distal region of invasion zone II not containing infection threads, revealing MtAnn1 as a new marker gene of the pre-infection zone. Moreover, analyses of MtAnn1 expression in response to bacterial symbiotic mutants suggest that the expression of MtAnn1 during nodulation requires biologically active Nod factors and is independent of the infection process.

Use of a subtractive hybridization approach to identify new Medicago truncatula genes induced during root nodule development.

We report the identification of new molecular markers associated with different stages of Rhizobium-induced nodule development in the legume Medicago truncatula. A cDNA library was constructed from pre-nitrogen-fixing M. Truncatula nodules, and differentially screened with a polymerase chain reaction-amplified subtracted probe. Twenty-nine new families of nodulin cDNA clones, designated MtN1 to MtN29, were thus identified in addition to clones for several known nodulins. All MtN genes were shown by Northern (RNA) hybridization analysis to be induced during nodulation, some of them well before nodule emergence. The MtN genes were classified into three groups depending on their expression kinetics. The expression of three MtN genes showed a limited induction by Nod factors purified from Rhizobium meliloti. Homologies with a variety of proteins were found for the deduced amino acid sequences of 10 of the MtN genes.

Assaying synthetic ribozymes in plants: high-level expression of a functional hammerhead structure fails to inhibit target gene activity in transiently transformed protoplasts.

A hammerhead ribozyme designed against the mRNA coding for the Escherichia coli beta-glucuronidase (GUS) reporter enzyme was constructed. The synthetic ribozyme appeared able to correctly cleave in vitro the target RNA. This catalytic molecule was then assayed for in vivo activity in plant protoplasts. Plasmids coding either for the ribozyme or for the GUS target gene were cotransfected into the cells by the PEG-calcium procedure and GUS gene expression monitored following transient expression by measuring the intracellular GUS enzymatic activity. Expression of the ribozyme to high molar excess over the GUS transcript did not lead to any significant decrease of GUS activity in the transfected protoplasts. Insertion of the ribozyme sequence in the 3'-untranslated region of the GUS mRNA also had no detectable effect on GUS reporter gene expression whereas the corresponding RNA appeared able to self-cleave in vitro. These results indicate that the ability of ribozymes to perform catalytic cleavage of their substrate mRNA in vitro is essential but clearly not sufficient to ensure that efficient inhibition of the corresponding target gene will occur upon endogenous expression of this catalytic RNA in the plant cell.

Dinoflagellate luminescence: purification of a NAD(P)H-dependent reductase and of its substrate.

The soluble enzymatic luminescent system of the dinoflagellate Pyrocystis lunula (luciferase-luciferin) is coupled with an enzymatic NAD(P)H-dependent reaction. The enzyme is a soluble reductase (Mr 47,000) which catalyzes, in the presence of NAD(P)H, the reduction of a molecule called P630. Reduced P630 has the same spectral characteristics as the purified luciferin. The luciferase can oxidize this reduced molecule with a light emission at 480 nm. These observations suggest that reduced P630 is a luciferin molecule. The oxidized form seems, in these conditions, to be the precursor of luciferin.