Karyotypic evolution of the Medicago complex: sativa-caerulea-falcata inferred from comparative cytogenetic analysis.

BACKGROUND: Polyploidy plays an important role in the adaptation and speciation of plants. The alteration of karyotype is a significant event during polyploidy formation. The Medicago sativa complex includes both diploid (2n = 2× = 16) and tetraploid (2n = 2× = 32) subspecies. The tetraploid M. ssp. sativa was regarded as having a simple autopolyploid origin from diploid ssp. caerulea, whereas the autopolyploid origin of tetraploid ssp. falcata from diploid form ssp. falcata is still in doubt. In this study, detailed comparative cytogenetic analysis between diploid to tetraploid species, as well as genomic affinity across different species in the M. sativa complex, were conducted based on comparative mapping of 11 repeated DNA sequences and two rDNA sequences by a fluorescence in situ hybridization (FISH) technique. RESULTS: FISH patterns of the repeats in diploid subspecies caerulea were highly similar to those in tetraploid subspecies sativa. Distinctly different FISH patterns were first observed in diploid ssp. falcata, with only centromeric hybridizations using centromeric and multiple region repeats and a few subtelomeric hybridizations using subtelomeric repeats. Tetraploid subspecies falcata was unexpectedly found to possess a highly variable karyotype, which agreed with neither diploid ssp. falcata nor ssp. sativa. Reconstruction of chromosome-doubling process of diploid ssp. caerulea showed that chromosome changes have occurred during polyploidization process. CONCLUSIONS: The comparative cytogenetic results provide reliable evidence that diploid subspecies caerulea is the direct progenitor of tetraploid subspecies sativa. And autotetraploid ssp. sativa has been suggested to undergo a partial diploidization by the progressive accumulation of chromosome structural rearrangements during evolution. However, the tetraploid subspecies falcata is far from a simple autopolyploid from diploid subspecies falcata although no obvious morphological change was observed between these two subspecies.

Detection of Changes in the Medicago sativa Retinoblastoma-Related Protein (MsRBR1) Phosphorylation During Cell Cycle Progression in Synchronized Cell Suspension Culture.

Deepening our knowledge on the regulation of the plant cell division cycle depends on techniques that allow for the enrichment of cell populations in defined cell cycle phases. Synchronization of cell division can be achieved using different plant tissues; however, well-established cell suspension cultures provide large amount of biological sample for further analyses. Here, we describe the methodology of the establishment, propagation, and analysis of a Medicago sativa suspension culture that can be used for efficient synchronization of the cell division. A novel 5-ethynyl-2'-deoxyuridine (EdU)-based method is used for the estimation of cell fraction that enters DNA synthesis phase of the cell cycle and we also demonstrate the changes in the phosphorylation level of Medicago sativa retinoblastoma-related protein (MsRBR1) during cell cycle progression.

The thiol compounds glutathione and homoglutathione differentially affect cell development in alfalfa (Medicago sativa L.).

Glutathione (GSH) is an important scavenger of Reactive Oxygen Species (ROS), precursor of metal chelating phytochelatins, xenobiotic defence compound and regulator of cell proliferation. Homoglutathione (hGSH) is a GSH homologue that is present in several taxa in the family of Fabaceae. It is thought that hGSH performs many of the stress-defence roles typically ascribed to GSH, yet little is known about the potential involvement of hGSH in controlling cell proliferation. Here we show that hGSH/GSH ratios vary across organs and cells and that these changes in hGSH/GSH ratio occur during dedifferentiation and/or cell cycle activation events. The use of a GSH/hGSH biosynthesis inhibitor resulted in impaired cytokinesis in isolated protoplasts, showing the critical importance of these thiol-compounds for cell division. However, exposure of isolated protoplasts to exogenous GSH accelerated cytokinesis, while exogenous hGSH was found to inhibit the same process. We conclude that GSH and hGSH have distinct functional roles in cell cycle regulation in Medicago sativa L. GSH is associated with meristemic cells, and promotes cell cycle activation and induction of somatic embryogenesis, while hGSH is associated with differentiated cells and embryo proliferation.

CdSe/ZnS quantum dots trigger DNA repair and antioxidant enzyme systems in Medicago sativa cells in suspension culture.

BACKGROUND: Nanoparticles appear to be promising devices for application in the agriculture and food industries, but information regarding the response of plants to contact with nano-devices is scarce. Toxic effects may be imposed depending on the type and concentration of nanoparticle as well as time of exposure. A number of mechanisms may underlie the ability of nanoparticles to cause genotoxicity, besides the activation of ROS scavenging mechanisms. In a previous study, we showed that plant cells accumulate 3-Mercaptopropanoic acid-CdSe/ZnS quantum dots (MPA-CdSe/ZnS QD) in their cytosol and nucleus and increased production of ROS in a dose dependent manner when exposed to QD and that a concentration of 10 nM should be cyto-compatible. RESULTS: When Medicago sativa cells were exposed to 10, 50 and 100 nM MPA-CdSe/ZnS QD a correspondent increase in the activity of Superoxide dismutase, Catalase and Glutathione reductase was registered. Different versions of the COMET assay were used to assess the genotoxicity of MPA-CdSe/ZnS QD. The number of DNA single and double strand breaks increased with increasing concentrations of MPA-CdSe/ZnS QD. At the highest concentrations, tested purine bases were more oxidized than the pyrimidine ones. The transcription of the DNA repair enzymes Formamidopyrimidine DNA glycosylase, Tyrosyl-DNA phosphodiesterase I and DNA Topoisomerase I was up-regulated in the presence of increasing concentrations of MPA-CdSe/ZnS QD. CONCLUSIONS: Concentrations as low as 10 nM MPA-CdSe/ZnS Quantum Dots are cytotoxic and genotoxic to plant cells, although not lethal. This sets a limit for the concentrations to be used when practical applications using nanodevices of this type on plants are being considered. This work describes for the first time the genotoxic effect of Quantum Dots in plant cells and demonstrates that both the DNA repair genes (Tdp1ß, Top1ß and Fpg) and the ROS scavenging mechanisms are activated when MPA-CdSe/ZnS QD contact M. sativa cells.

The Sinorhizobium meliloti MsbA2 protein is essential for the legume symbiosis.

Sinorhizobium meliloti is a beneficial legume symbiont, closely related to Brucella species, which are chronic mammalian pathogens. We discovered that the S. meliloti MsbA2 protein is essential to ensure the symbiotic interaction with the host plant, alfalfa. S. meliloti invades plant cells via plant-derived structures known as infection threads. However, in the absence of MsbA2, S. meliloti remains trapped within abnormally thickened infection threads and induces a heightened plant defence response, characterized by a substantial thickening of the nodule endodermis layer and the accumulation of polyphenolic compounds. The S. meliloti MsbA2 protein is homologous to the Escherichia coli lipopolysaccharide/phospholipid trafficking protein MsbA. However, MsbA2 was not essential for the membrane transport of either lipopolysaccharide or phospholipids in S. meliloti. We determined that the msbA2 gene is transcribed in free-living S. meliloti and that in the absence of MsbA2 the polysaccharide content of S. meliloti is altered. Consequently, we propose a model whereby the altered polysaccharide content of the S. meliloti msbA2 mutant could be responsible for its symbiotic defect by inducing an inappropriate host response.

Excretion of polyamines in alfalfa and tobacco suspension-cultured cells and its possible role in maintenance of intracellular polyamine contents.

Changes in polyamines (PAs) in cells and cultivation media of alfalfa (Medicago sativa L.) and tobacco bright yellow 2 (BY-2) (Nicotiana tabacum L.) cell suspension cultures were studied over their growth cycles. The total content of PAs (both free and conjugated forms) was nearly 10 times higher in alfalfa, with high level of free putrescine (Put) (in exponential growth phase it represented about 65-73% of the intracellular Put pool). In contrast, the high content of soluble Put conjugates was found in tobacco cells (in exponential phase about 70% of the intracellular Put). Marked differences occurred in the amount of PAs excreted into the cultivation medium: alfalfa cells excreted at the first day after inoculation 2117.0, 230.5, 29.0 and 88.0 nmol g(-1) of cell fresh weight (FW) of Put, spermidine (Spd), spermine (Spm) and cadaverine (Cad), respectively, while at the same time tobacco cells excreted only small amount of Put and Spd (12.7 and 2.4 nmol g(-1) FW, respectively). On day 1 the amounts of Put, Spd, Spm and Cad excreted by alfalfa cells represented 21, 38, 12 and 15% of the total pool (intra- plus extra-cellular contents) of Put, Spd, Spm and Cad, respectively. In the course of lag-phase and the beginning of exponential phase the relative contents of extracellular PAs continually decreased (with the exception of Cad). On day 10, the extracellular Put, Spd, Spm and Cad still represented 11.3, 10.9, 2.1 and 27% of their total pools. The extracellular PAs in tobacco cells represented from day 3 only 0.1% from their total pools. The possible role of PA excretion into the cultivation medium in maintenance of intracellular PA contents in the cells of the two cell culture systems, differing markedly in growth rate and PA metabolism is discussed.

A novel aldose/aldehyde reductase protects transgenic plants against lipid peroxidation under chemical and drought stresses.

Rapid accumulation of toxic products from reactions of reactive oxygen species (ROS) with lipids and proteins significantly contributes to the damage of crop plants under biotic and abiotic stresses. Here we have identified a stress-activated alfalfa gene encoding a novel plant NADPH-dependent aldose/aldehyde reductase that also exhibited characteristics of the homologous human enzyme. The recombinant alfalfa enzyme is active on 4-hydroxynon-2-enal, a known cytotoxic lipid peroxide degradation product. Ectopic synthesis of this enzyme in transgenic tobacco plants provided considerable tolerance against oxidative damage caused by paraquat and heavy metal treatment. These transformants could also resist a long period of water deficiency and exhibited improved recovery after rehydration. We found a reduced production of lipid peroxidation-derived reactive aldehydes in these transformed plants under different stresses. These studies reveal a new and efficient detoxification pathway in plants.

Cell cycle regulation in the course of nodule organogenesis in Medicago.

The molecular mechanisms of de novo meristem formation, cell differentiation and the integration of the cell cycle machinery into appropriate stages of the developmental programmes are still largely unknown in plants. Legume root nodules, which house nitrogen-fixing rhizobia, are unique plant organs and their development may serve as a model for organogenetic processes in plants. Nodules form and are essential for the plant only under limitation of combined nitrogen in the soil. Moreover, their development is triggered by external mitogenic signals produced by their symbiotic partners, the rhizobia. These signals, the lipochitooligosaccharide Nod factors, act as host-specific morphogens and induce the re-entry of root cortical cells into mitotic cycles. Maintenance of cell division activity leads to the formation of a persistent nodule meristem from which cells exit continuously and enter the nodule differentiation programme, involving multiple cycles of endoreduplication and enlargement of nuclear and cell volumes. While the small diploid 2C cells remain uninfected, the large polyploid cells can be invaded and, after completing the differentiation programme, host the nitrogen-fixing bacteroids. This review summarizes the present knowledge on cell cycle reactivation and meristem formation in response to Nod factors and reports on a novel plant cell cycle regulator that can switch mitotic cycles to differentiation programmes.

Cell cycle function of a Medicago sativa A2-type cyclin interacting with a PSTAIRE-type cyclin-dependent kinase and a retinoblastoma protein.

In plants multiple A-type cyclins with distinct expression patterns have been isolated and classified into three subgroups (A1-A3), while in animal somatic cells a single type of cyclin A is required for cell-cycle regulation from the S to M phases. We studied the function of an A2-type cyclin from Medicago sativa (Medsa;cycA2) which, in contrast to animal and most plant A-type cyclins, was expressed in all phases of the cell cycle. Using synchronized alfalfa cell cultures and anti-Medsa;CycA2 polyclonal antibodies, we showed that while the mRNA level increased steadily from the late G1 to the G2-M phase, the protein level after a rapid increase in S-phase reached a plateau during the G2 phase. In the yeast two-hybrid system, the Medsa;CycA2 protein interacted with the PSTAIRE-motif-containing cyclin-dependent kinase Cdc2MsA and with the maize retinoblastoma protein. Unexpectedly, the CycA2-associated kinase activity was biphasic: a first activity peak occurred in the S phase while the major one occurred during the G2/M transition, with no apparent dependence upon the actual levels of the Medsa;CycA2 and Cdc2MsA proteins. Immunohistological localization of the cyclin A2 protein by immunofluorescence and immunogold labelling revealed the presence of Medsa;CycA2 in the nucleus of the interphase and prophase cells, while it was undetectable thereafter during mitosis. Together these data suggest that Medsa;CycA2 plays a role both in the S phase and at the G2/M transition.

Inheritance and mapping of 2n-egg production in diploid alfalfa.

The production of eggs with the sporophytic chromosome number (2n eggs) in diploid alfalfa (Medicago spp.) is mainly associated with the absence of cytokinesis after restitutional meiosis. The formation of 2n eggs through diplosporic apomeiosis has also been documented in a diploid mutant of M. sativa subsp. falcata (L.) Arcang. (2n = 2x = 16), named PG-F9. Molecular tagging of 2n-egg formation appears to be an essential step towards marker-assisted breeding and map-based cloning strategies aimed at investigating and manipulating reproductive mutants of the M. sativa complex. We made controlled crosses between PG-F9 and three wild type plants of M. sativa subsp. coerulea (Less.) Schm. (2n = 2x = 16) and then hand-pollinated the F1 progenies with tetraploid plants of M. sativa subsp. sativa L. (2n = 4x = 32). As a triploid embryo block prevents the formation of 3x progenies in alfalfa because of endosperm imbalance, and owing to the negligible selfing rate, seed set in 2x-4x crosses was used to discriminate the genetic capacity for 2n-egg production. F1 plants that exhibited null or very low seed sets were classified as normal egg producers and plants with high seed sets as 2n-egg producers. A bulked segregant analysis (BSA) with RAPD (random amplified polymorphic DNA), ISSR (inter-simple sequence repeat), and AFLP (amplified fragment length polymorphism) markers was employed to identify a genetic linkage group related to the 2n-egg trait using one of the three F1 progenies. This approach enabled us to detect a paternal ISSR marker of 610 bp, generated by primer (CA)8-GC, located 9.8 cM from a putative gene (termed Tne1, two-n-eggs) that in its recessive form determines 2n eggs and a 30% recombination genomic window surrounding the target locus. Eight additional RAPD and AFLP markers, seven of maternal, and one of paternal origin, significantly co-segregated with the trait under investigation. The minimum number of quantitative trait loci (QTLs) controlling seed set in 2x-4x crosses was estimated by ANOVA and regression analysis. Four maternal and three paternal independent molecular markers significantly affected the trait. A paternal RAPD marker allele, mapped in the same linkage group of Tne1, explained 43% of the variation for seed set in 2x-4x crosses indicating the presence of a major QTL. A map of the PG-F9 chromosome regions carrying the minor genes that determine the expression level of 2n eggs was constructed using selected RAPD and AFLP markers. Two of these genes were linked to previously mapped RFLP loci belonging to groups 1 and 8. Molecular and genetic evidence support the involvement of at least five genes.

Elevation of the cytosolic free [Ca2+] is indispensable for the transduction of the Nod factor signal in alfalfa.

In root hairs of alfalfa (Medicago sativa), the requirement of Ca(2+) for Nod factor signaling has been investigated by means of ion-selective microelectrodes. Measured 50 to 100 microm behind the growing tip, 0.1 microM NodRm-IV(C16:2,S) increased the cytosolic free [Ca2+] by about 0.2 pCa, while the same concentration of chitotetraose, the nonactive glucosamine backbone, had no effect. We demonstrate that NodRm-IV(C16:2,S) still depolarized the plasma membrane at external Ca(2+) concentrations below cytosolic values if the free EGTA concentration remained low (

Cloning of a WD-repeat-containing gene from alfalfa (Medicago sativa): a role in hormone-mediated cell division?

Rhizobium meliloti can interact symbiotically with Medicago plants thereby inducing the formation of root nodules. Screening of a young nodule cDNA library led to the isolation of a cDNA from Medicago sativa, Msgbl, that comprises a new member of the RACK1 (Receptor of Activated C Kinase) subfamily of WD-repeat proteins. This subfamily shows homology to the beta-subunit of heterotrimeric G proteins. Besides RACK1, this subfamily contains several plant genes including the well characterized auxin-inducible ArcA of tobacco. The Msgbl gene is strongly expressed in young embryos and in leaves, and is induced upon cytokinin treatment of roots. Whereas northern analysis failed to reveal differences in expression between total RNA from roots and nodules, in situ hybridization demonstrated that the transcript was most abundant in dividing cells of nodule primordia and in the nodule meristem. Msgbl may be related to the signal transduction acting in response to hormone-mediated cell division.

Association of caffeoyl coenzyme A 3-O-methyltransferase expression with lignifying tissues in several dicot plants.

Caffeoyl coenzyme A 3-O-methyltransferase (CCoAOMT) was previously shown to be associated with lignification in both in vitro tracheary elements (TEs) and organs of zinnia (Zinnia elegans). However, it is not known whether this is a general pattern in dicot plants. To address this question, polyclonal antibodies against zinnia recombinant CCoAOMT fusion protein were raiseed and used for immunolocalization in several dicot plants. The antibodies predominantly recognized a protein band with a molecular mass of 28 kD on western analysis of tissue extracts from zinnia, forsythia (Forsythia suspensa), tobacco (Nicotiana tabacum), alfalfa (Medicago sativa), and soybean (Glycine max). Western analyses showed that the accumulation of CCoAOMT protein was closely correlated with lignification in in vitro TEs of zinnia. Immunolocalization results showed that CCoAOMT was localized in developing TEs of young zinnia stems and in TEs, xylem fibers, and phloem fibers of old stems. CCoAOMT was also found to be specifically associated with all lignifying tissues, including TEs, xylem fibers, and phloem fibers in stems of forsythia, tobacco, alfalfa, soybean, and tomato (Lycopersicon esculentum). The presence of CCoAOMT was evident in xylem ray parenchyma cells of forsythia, tobacco, and tomato. In forsythia and alfalfa, pith parenchyma cells next to the vascular cylinder were lignified. Accordingly, marked accumulation of CCoAOMT in these cells was observed. Taken together, these results showed a close association of CCoAOMT expression with lignification in dicot plants. This supports the hypothesis that the CCoAOMT-mediated methylation branch is a general one in lignin biosynthesis during normal growth and development in dicot plants.

The D-type alfalfa cyclin gene cycMs4 complements G1 cyclin-deficient yeast and is induced in the G1 phase of the cell cycle.

Cyclins are key regulators of the cell cycle in all eukaryotes. In alfalfa, we have previously isolated three B-type cyclins. The closely related cycMs1 and cycMs2 genes are expressed primarily during the G2 and M phases and are most likely mitotic cyclins; expression of the cycMs3 gene is induced in the G0-to-G1 transition, when cells reenter the cell cycle. By complementation of G1 cyclin-deficient yeast cells, a novel alfalfa cyclin, designated cycMs4, was isolated. The predicted amino acid sequence of the cycMs4 gene is most similar to that of the Arabidopsis cyclin delta 3 gene. CycMs4 and cyclin delta 3 belong to the class of D-type cyclins and contain PEST-rich regions and a retinoblastoma binding motif. When comparing expression levels in different organs, cycMs4 transcripts were present predominantly in roots. Whereas expression of the cycMs4 gene was cell cycle-regulated in suspension-cultured cells, transcription in roots was observed to depend also on the positional context of the cell. When differentiated G0-arrested leaf cells were induced to resume cell division by treatment with plant hormones, cycMs4 transcription was induced before the onset of DNA synthesis. Whereas this induction was preceded by that of the cycMs3 gene, cycMs2 expression occurred later and at the same time as mitotic activity. These data suggest that cycMs4 plays a role in the G1-to-S transition and provide a model to investigate the plant cell cycle at the molecular level.

cDNA sequence, expression, and transcript stability of a cold acclimation-specific gene, cas18, of alfalfa (Medicago falcata) cells.

The nucleotide sequence of a full-length cDNA, the deduced amino acid sequence, and the regulation of expression of a cold acclimation-specific gene, cas18, in cell suspension cultures of a freezing-tolerant cultivar of alfalfa (Medicago falcata cv Anik) have been determined. The deduced polypeptide, CAS18, is relatively small (17.6 kD), is highly hydrophilic, is rich in glycine and threonine, and contains two distinctive repeat elements. It exhibits homology with members of the LEA/RAB/dehydrin family of proteins, which accumulate in response to abscisic acid (ABA) or water stress. It is intriguing that cas18 is induced by neither ABA nor water stress. The cas18 cDNA hybridizes to three transcripts of 1.6, 1.4, and 1.0 kb, and the cDNA characterized here corresponds to the 1.0-kb transcript. The expression of this gene is about 30-fold greater in cold-acclimated cells than in nonacclimated cells. Although the accumulation of transcripts during cold acclimation is relatively slow, their disappearance during deacclimation is dramatically rapid, becoming undetectable in less than 5 h. Studies of nuclear run-on transcription show that cold acclimation enhances the transcription of this gene nearly 9-fold. The stability of cas18-detectable transcripts during deacclimation is considerably increased if transcription is inhibited with cordycepin. It therefore appears that low temperature regulates the expression of cas18 at both the transcriptional and posttranscriptional levels.