Expression of an alfalfa (Medicago sativa L.) peroxidase gene in transgenic Arabidopsis thaliana enhances resistance to NaCl and H2O2.

Peroxidases (PODs) are enzymes that play important roles in catalyzing the reduction of H2O2 and the oxidation of various substrates. They function in many different and important biological processes, such as defense mechanisms, immune responses, and pathogeny. The POD genes have been cloned and identified in many plants, but their function in alfalfa (Medicago sativa L.) is not known, to date. Based on the POD gene sequence (GenBank accession No. L36157.1), we cloned the POD gene in alfalfa, which was named MsPOD. MsPOD expression increased with increasing H2O2. The gene was expressed in all of the tissues, including the roots, stems, leaves, and flowers, particularly in stems and leaves under light/dark conditions. A subcellular analysis showed that MsPOD was localized outside the cells. Transgenic Arabidopsis with MsPOD exhibited increased resistance to H2O2 and NaCl. Moreover, POD activity in the transgenic plants was significantly higher than that in wild-type Arabidopsis. These results show that MsPOD plays an important role in resistance to H2O2 and NaCl.

Activity of chalcones derived from 2,4,5-trimethoxybenzaldehyde against Meloidogyne exigua and in silico interaction of one chalcone with a putative caffeic acid 3-O-methyltransferase from Meloidogyne incognita.

Meloidogyne exigua is a parasitic nematode of plants that causes great losses to coffee farmers. In an effort to develop parasitic controls, 154 chalcones were synthesized and screened for activity against this nematode. The best results were obtained with (2E)-1-(4'-nitrophenyl)-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-one (6) with a 50% lethal concentration (LC50) of 171 µg/ml against M. exigua second-stage juveniles, in comparison to the commercially-available nematicide carbofuran which had an LC50 of 260 µg/ml under the same conditions. When coffee plants were used, 6 reduced the nematode population to ~50% of that observed in control plants. To investigate the mechanism of action of 6, an in silico study was carried out, which indicated that 6 may act against M. exigua through inhibition of a putative caffeic acid 3-O-methyltransferase homodimer, the amino acid sequence of which was determined by examining the genome of Meloidogyne incognita.

Determination of thiodicarb using a biosensor based on alfalfa sprout peroxidase immobilized in self-assembled monolayers.

A biosensor based on alfalfa sprout (Medicago sativa) homogenate as a source of peroxidase is proposed for the determination of thiodicarb by square-wave voltammetry. This enzyme was immobilized in self-assembled monolayers of l-cysteine on a gold electrode. Several parameters were investigated to evaluate the optimum conditions for operation of the biosensor. The analytical curve was linear for thiodicarb concentrations of 2.27 x 10(-6) to 4.40 x 10(-5) mol L(-1) with a detection limit of 5.75 x 10(-7) mol L(-1). The lifetime of the Au-alfalfa sprout-SAMs was 20 days (at least 220 determinations). The average recovery of thiodicarb from samples of vegetable extracts ranged from 99.02 to 101.04%. The results obtained for thiodicarb in vegetable extracts using the proposed method are in close agreement with those using a high performance liquid chromatography procedure at the 95% confidence level.

Identification of Fructose-1,6-bisphosphate aldolase cytosolic class I as an NMH7 MADS domain associated protein.

We are interested in identifying proteins that interact with the MADS domain protein NMH7 of Medicago sativa. We use an affinity column with a synthetic peptide derived from the MADS domain of NMH7 which has been reported to mediate protein-protein interaction with non-MADS domain interacting proteins. We identified approximately 40 and approximately 80kDa specifically bound proteins as the monomeric and dimeric forms of Fructose-1,6-bisphosphate aldolase cytosolic class I. NiNTA pull down assays revealed that K- and C-terminus regions of NMH7 are not required for the interaction with aldolase. Aldolase enzymatic activity is not required for the interaction with NMH7. NMH7 and aldolase were coimmunoprecipitated from non-inoculated seed and seedlings extracts. Colocalization studies using confocal microscopy showed that aldolase and NMH7 are localized in the cytoplasm and the nucleus of the cortical cells. These data together show that M. sativa aldolase is a novel MADS domain binding protein, and suggest a broader functional repertory for this enzyme, as has been proposed for other glycolytic enzymes.

A CDPK isoform participates in the regulation of nodule number in Medicago truncatula.

Medicago spp. are able to develop root nodules via symbiotic interaction with Sinorhizobium meliloti. Calcium-dependent protein kinases (CDPKs) are involved in various signalling pathways in plants, and we found that expression of MtCPK3, a CDPK isoform present in roots of the model legume Medicago truncatula, is regulated during the nodulation process. Early inductions were detected 15 min and 3-4 days post-inoculation (dpi). The very early induction of CPK3 messengers was also present in inoculated M. truncatula dmi mutants and in wild-type roots subjected to salt stress, indicating that this rapid response is probably stress-related. In contrast, the later response was concomitant with cortical cell division and the formation of nodule primordia, and was not observed in wild-type roots inoculated with nod (-) strains. This late induction correlated with a change in the subcellular distribution of CDPK activities. Accordingly, an anti-MtCPK3 antibody detected two bands in soluble root extracts and one in the particulate fraction. CPK3::GFP fusions are targeted to the plasma membrane in epidermal onion cells, a localization that depends on myristoylation and palmitoylation sites of the protein, suggesting a dual subcellular localization. MtCPK3 mRNA and protein were also up-regulated by cytokinin treatment, a hormone linked to the regulation of cortical cell division and other nodulation-related responses. An RNAi-CDPK construction was used to silence CPK3 in Agrobacterium rhizogenes-transformed roots. Although no major phenotype was detected in these roots, when infected with rhizobia, the total number of nodules was, on average, twofold higher than in controls. This correlates with the lack of MtCPK3 induction in the inoculated super-nodulator sunn mutant. Our results suggest that CPK3 participates in the regulation of the symbiotic interaction.

Transcription of the Azospirillum brasilense nifH gene is positively regulated by NifA and NtrA and is negatively controlled by the cellular nitrogen status.

The expression of a translational Azospirillum brasilense nifH-uidA fusion was studied in A. brasilense and in Rhizobium meliloti strains with mutations in nifA, ntrA and ntrC. Induction of the fusion was observed in the R. meliloti wild-type and NtrC- strains on incubation under microaerobic conditions but not in the NifA- and NtrA- strains, showing the absolute requirement of both sigma 54 and NifA for activation of the nifH promoter. Histochemical analysis of the root nodules elicited by R. meliloti wild-type showed expression of the fusion in the late symbiotic zone but not in the meristematic and the early symbiotic zones. No induction of the nifH-uidA fusion was observed in the R. meliloti wild-type or NifA- strains incubated aerobically in nitrogen-free medium, indicating that, in contrast to R. meliloti nifH, A. brasilense nifH cannot be activated directly by NtrC. Expression of the nifH gene in A. brasilense only occurs under nitrogen-limiting, microaerobic conditions, suggesting the presence of a nitrogen-dependent control system for nif gene expression.

Adenylate cyclase activity in a higher plant, alfalfa (Medicago sativa).

An adenylate cyclase activity in Medicago sativa L. (alfalfa) roots was partially characterized. The enzyme activity remains in the supernatant fluid after centrifugation at 105,000 g and shows in crude extracts an apparent Mr of about 84,000. The enzyme is active with Mg2+ and Ca2+ as bivalent cations, and is inhibited by EGTA and by chlorpromazine. Calmodulin from bovine brain or spinach leaves activates this adenylate cyclase.