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At least 325 records · Page 18Linked to original sources

PubMed1993

Identification of a RAPD marker associated with somatic embryogenesis in alfalfa.

The current study was conducted to identify random amplified polymorphic DNA (RAPD) markers linked to genes controlling somatic embryogenesis in alfalfa. Segregation analyses of the somatic embryogenesis trait and the RAPD markers in an F1 population of 83 plants, derived from a cross between embryogenic A70-34 and non-embryogenic Arrow36 alfalfa plants, identified a polymorphic band that is associated with somatic embryogenesis. Based on the assumptions that somatic embryogenesis in alfalfa is controlled by two dominant genes with complementary effects and that the genotypes of A70-34 and Arrow36 are AAaaBbbb and aaaabbbb, respectively, the segregation data for the marker and the somatic embryogenesis trait in the F1s indicate that the marker is linked to the A locus. The maximum recombination fraction estimated for the linkage between the marker and the gene is 36.3%.

Base Sequence↗

PubMed1995

Post-transcriptional regulation of a salt-inducible alfalfa gene encoding a putative chimeric proline-rich cell wall protein.

A cDNA previously shown to identify a salt-inducible root-specific transcript in Medicago sativa was used to screen an alfalfa library for the corresponding genomic sequence. One positive clone was recovered. The nucleotide sequence of a subclone contained a 329 bp 5' region upstream of the first ATG codon, a 1143 bp coding segment, and a 447 bp 3'-untranslated region interrupted by a single 475 bp intron. Translation of the coding segment, which was designated MsPRP2, suggested it encodes a chimeric 40,569 Da cell wall protein with an amino-terminal signal sequence, a repetitive proline-rich sequence, and a cysteine-rich carboxyl-terminal sequence homologous to nonspecific lipid transfer proteins. The 3'-untranslated region of MsPRP2 contained a sequence similar to one found to destabilize mRNAs transcribed from the elicitor-regulated proline-rich protein gene PvPRP1. Transcription run-on experiments using nuclei from salt-sensitive and salt-tolerant alfalfa callus suggested that the accumulation of MsPRP2 transcripts in salt-tolerant alfalfa cells grown in the presence of salt is due primarily to increased mRNA stability. The MsPRP2 gene thus may be a useful model for studying post-transcriptional salt-regulated expression of cell wall proteins.

Amino Acid Sequence↗

PubMed1992

Molecular characterization and expression of an isocitrate dehydrogenase from alfalfa (Medicago sativa L).

A putative isocitrate dehydrogenase (IDH) cDNA from alfalfa has been cloned and sequenced. The derived amino acid sequence of 433 residues contains the isocitrate and isopropylmalate dehydrogenase signatures, is 63% identical to yeast mitochondrial NADP-IDH and shares high sequence identity with peptides of pig heart NADP-IDH. The sequence contains a potential N-terminal leader with similarities to a thylakoid transit peptide. IDH transcripts and NADP-IDH activity were detected in all alfalfa tissues examined, their levels depending upon the tissue type and its developmental stage. Transcripts and enzymatic activity were not induced on exposure of cell suspension cultures to a fungal elicitor. IDH is encoded by a small gene family in alfalfa.

3-Isopropylmalate Dehydrogenase↗

PubMed1992

Differential expression of a chimeric CaMV-tomato proteinase Inhibitor I gene in leaves of transformed nightshade, tobacco and alfalfa plants.

The open reading frame and terminator region of a wound-inducible tomato Inhibitor I gene, regulated by the CaMV 35S promoter, was stably integrated into the genomes of nightshade (Solanum nigrum), tobacco (Nicotiana tabacum), and alfalfa (Medicago sativa), using an Agrobacterium-mediated transformation system. The expression of the foreign Inhibitor I gene in leaves of each species was studied at the mRNA and protein levels. The levels of Inhibitor I protein present in leaves of each species correlated with the levels of mRNA. The average levels of both mRNA and Inhibitor I protein were highest in leaves of transgenic nightshade plants (over 125 micrograms of Inhibitor I per g tissue), less in tobacco plants (about 75 micrograms/g tissue), and lowest in leaves of transgenic alfalfa plants (below 20 micrograms/g tissue). Inhibitor I protein was observed in all tissues throughout transgenic plant species, but inhibitor concentration per gram of tissue was 2-3 times higher in young developing leaf tissues and floral organs. The differences in the expression of the CaMV-tomato Inhibitor I gene among the different plant genera suggests that either the rate of transcription of the foreign gene or the rate of degradation of the nascent Inhibitor I mRNA varies among genera. Using electron microscopy techniques, the newly synthesized pre-pro-Inhibitor I protein was shown to be correctly processed and stored as a mature Inhibitor I protein within the central vacuoles of leaves of transgenic nightshade and alfalfa. The results of these experiments suggest that maximal expression of foreign proteinase inhibitor genes, and perhaps other foreign defense genes, may require gene constructs that are fashioned with promoters and terminators that allow maximum expression in the selected plant species.

Chymotrypsin↗

PubMed1992

Alfalfa root nodule phosphoenolpyruvate carboxylase: characterization of the cDNA and expression in effective and plant-controlled ineffective nodules.

Phosphoenolpyruvate carboxylase (PEPC) plays a key role in N2 fixation and ammonia assimilation in legume root nodules. The enzyme can comprise up to 2% of the soluble protein in root nodules. We report here the isolation and characterization of a cDNA encoding the nodule-enhanced form of PEPC. Initially, a 2945 bp partial-length cDNA was selected by screening an effective alfalfa nodule cDNA library with antibodies prepared against root nodule PEPC. The nucleotide sequence encoding the N-terminal region of the protein was obtained by primer-extension cDNA synthesis and PCR amplification. The complete amino acid sequence of alfalfa PEPC was deduced from these cDNA sequences and shown to bear striking similarity to other plant PEPCs. Southern blots of alfalfa genomic DNA indicate that nodule PEPC is a member of a small gene family. During the development of effective root nodules, nodule PEPC activity increases to a level that is 10- to 15-fold greater than that in root and leaf tissue. This increase appears to be the result of increases in amount of enzyme protein and PEPC mRNA. Ineffective nodules have substantially less PEPC mRNA, enzyme protein and activity than do effective nodules. Maximum expression of root nodule PEPC appears to be related to two signals. The first signal is associated with nodule initiation while the second signal is associated with nodule effectiveness. Regulation of root nodule PEPC activity may also involve post-translational processes affecting enzyme activity and/or degradation.

Amino Acid Sequence↗

PubMed1995

Stress responses in alfalfa (Medicago sativa L.) XIX. Transcriptional activation of oxidative pentose phosphate pathway genes at the onset of the isoflavonoid phytoalexin response.

We have isolated cDNA clones encoding the pentose phosphate pathway enzymes 6-phosphogluconate dehydrogenase (6PGDH, EC 1.1.1.44) and glucose 6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) from alfalfa (Medicago sativa L.). These exhibit extensive nucleotide and amino acid sequence similarity to the corresponding genes from bacteria, Drosophila and mammals. Transcripts encoding both enzymes are expressed at high levels in roots and nodules. Exposure of alfalfa suspension cells to an elicitor from yeast cell walls results in co-ordinated increases in transcription rates for both genes, followed by increased steady state transcript levels but only slightly increased extractable enzyme activities, at the onset of accumulation of isoflavonoid phytoalexins. Levels of NADPH and NADP remain relatively constant in alfalfa cells following elicitation. The rapid transcriptional activation of 6PGDH and G6PDH does not therefore appear to be a response to altered pyridine nucleotide redox state. These genes appear to respond to early events in elicitor-mediated signalling rather than to subsequent elicitor-induced changes in secondary metabolism. Hydrogen peroxide, a potential signal for elicitation of anti-oxidative genes in biologically stressed plant cells, did not induce 6PGDH or G6PDH transcripts or enzymatic activity.

Amino Acid Sequence↗

PubMed1994

Isolation and characterization of phosphoprotein phosphatase 1 from alfalfa.

Protein phosphatases are central regulatory components of diverse processes in eukaryotes and are among the most highly conserved proteins known. In this paper, we report the cloning and sequencing of a type 1 protein phosphatase (pp1Ms) cDNA from alfalfa. Southern analysis indicates the presence of a gene family of PP1 proteins in alfalfa. The pp1Ms open reading frame is very similar to one of five predicted Arabidopsis type 1 protein phosphatases, indicating that different subtypes are individually conserved. Expression of the alfalfa pp1Ms in a temperature-sensitive Schizosaccharomyces pombe PP1 mutant, dis2-11, revealed no complementation, suggesting that PP1Ms is not involved in mitotic regulation. In different plant organs, different pp1Ms transcript levels were observed; in contrast, mRNA levels remained constant in all phases of the cell cycle and in logarithmically growing cells. However, when cells entered stationary phase pp1Ms transcript levels decreased considerably.

Amino Acid Sequence↗

PubMed1991

Isolation and analysis of a cDNA clone that encodes an alfalfa (Medicago sativa) aspartate aminotransferase.

We have isolated an alfalfa leaf cDNA clone that encodes aspartate aminotransferase (AAT, EC 2.6.1.1) by direct complementation of an Escherichia coli aspartate auxotroph with a plasmid cDNA library. DNA sequence analysis of the recombinant plasmid, pMU1, revealed that a 1514 bp cDNA was inserted in the correct orientation and in-frame with the start of the lacZ coding sequence in the vector, pUC18. The resulting fusion protein is predicted to be 424 amino acids in length with a molecular weight of 46387 Daltons. The cDNA-encoded protein has a characteristic pyridoxal phosphate attachment site motif and has substantial amino acid sequence homology to both animal and bacterial AATs. Plasmid pMU1 encodes an AAT with a Km for aspartate of 3.3 mM, a Km for 2-oxoglutarate of 0.28 mM, and a pH optimum between 8.0 and 8.5. Several lines of evidence including Western blot analysis, the isoelectric point of the encoded protein, and the effect of pH on the activity of the fusion protein, suggest that the cDNA encodes the isozyme AAT-1 rather than AAT-2. Northern blot analysis showed that the aat-1 clone hybridized to a 1.6 kb transcript present in alfalfa leaves, roots and nodules. The relative concentrations of aat-1 mRNA in these tissues were 1:2:5, respectively. Thus, transcription of aat-1 appears to be induced during nodule development. Southern blot analysis suggested that AAT-1 in alfalfa is encoded by either a single-copy gene or a small, multigene family.

Amino Acid Sequence↗

PubMed1995

MMK2, a novel alfalfa MAP kinase, specifically complements the yeast MPK1 function.

Mitogen-activated protein (MAP) kinases are serine/threonine protein kinases that are activated in response to a variety of stimuli. Here we report the isolation of an alfalfa cDNA encoding a functional MAP kinase, termed MMK2. The predicted amino acid sequence of MMK2 shares 65% identity with a previously identified alfalfa MAP kinase, termed MMK1. Both alfalfa cDNA clones encode functional kinases when expressed in bacteria, undergoing autophosphorylation and activation to phosphorylate myelin basic protein in vitro. However, only MMK2 was able to phosphorylate a 39 kDa protein from the detergent-resistant cytoskeleton of carrot cells. The distinctiveness of MMK2 was further shown by complementation analysis of three different MAP kinase-dependent yeast pathways; this revealed a highly specific replacement of the yeast MPK1(SLT2) kinase by MMK2, which was found to be dependent on activation by the upstream regulators of the pathway. These results establish the existence of MAP kinases with different characteristics in higher plants, suggesting the possibility that they could mediate different cellular responses.

Amino Acid Sequence↗

PubMed2002

Isolation from alfalfa of resistance gene analogues containing nucleotide binding sites.

Cloned resistance (R) genes from a broad range of plant species are known to share similarities in DNA sequence and structural motifs. Degenerate oligonucleotide primers designed from conserved regions of the nucleotide binding site (NBS), common to many R genes, were used to amplify the NBS regions from genomic DNA from alfalfa ( Medicago sativa L). Sequence comparisons of the amplified fragments indicated that at least 18 families of NBS-containing R genes are present in alfalfa. Comparisons to R genes from other species suggested a polyphyletic origin of these gene families. Using the same degenerate primers, PCR analysis of cDNA prepared from a plant not challenged with a pest or pathogen revealed that many of the NBS-containing gene families were transcribed actively. Amplification of NBS regions from other Medicago species showed the presence of some NBS-containing genes not present in alfalfa. These results indicate that the NBS-containing R genes comprise a large gene family in Medicago, at least some of which are transcribed in healthy plants, and that different Medicago species carry unique NBS genes.

Journal Article↗

PubMed2005

An expressed sequence tag SSR map of tetraploid alfalfa (Medicago sativa L.).

A genetic map constructed from a population segregating for a trait of interest is required for QTL identification. The goal of this study was to construct a molecular map of tetraploid alfalfa (Medicago sativa.) using simple sequence repeat (SSR) markers derived primarily from expressed sequence tags (ESTs) and bacterial artificial chromosome (BAC) inserts of M. truncatula. This map will be used for the identification of drought tolerance QTL in alfalfa. Two first generation backcross populations were constructed from a cross between a water-use efficient, M. sativa subsp. falcata genotype and a low water-use efficient M. sativa subsp. sativa genotype. The two parents and their F(1) were screened with 1,680 primer pairs designed to amplify SSRs, and 605 single dose alleles (SDAs) were amplified. In the F(1), 351 SDAs from 256 loci were mapped to 41 linkage groups. SDAs not inherited by the F(1), but transmitted through the recurrent parents and segregating in the backcross populations, were mapped to 43 linkage groups, and 44 of these loci were incorporated into the composite maps. Homologous linkage groups were joined to form eight composite linkage groups representing the eight chromosomes of M. sativa. The composite maps consist of eight composite linkage groups with 243 SDAs from M. truncatula EST sequences, 38 SDAs from M. truncatula BAC clone sequences, and five SDAs from alfalfa genomic SSRs. The total composite map length is 624 cM, with average marker density per composite linkage group ranging from 1.5 to 4.4 cM, and an overall average density of 2.2 cM. Segregation distortion was 10%, and distorted loci tended to cluster on individual homologues of several linkage groups.

Chromosome Mapping↗

PubMed2005

Use of real time PCR to determine population profiles of individual species of lactic acid bacteria in alfalfa silage and stored corn stover.

Real-time polymerase chain reaction (RT-PCR) was used to quantify seven species of lactic acid bacteria (LAB) in alfalfa silage prepared in the presence or absence of four commercial inoculants and in uninoculated corn stover harvested and stored under a variety of field conditions. Species-specific PCR primers were designed based on recA gene sequences. Commercial inoculants improved the quality of alfalfa silage, but species corresponding to those in the inoculants displayed variations in persistence over the next 96 h. Lactobacillus brevis was the most abundant LAB (12 to 32% of total sample DNA) in all of the alfalfa silages by 96 h. Modest populations (up to 10%) of Lactobacillus plantarum were also observed in inoculated silages. Pediococcus pentosaceus populations increased over time but did not exceed 2% of the total. Small populations (0.1 to 1%) of Lactobacillus buchneri and Lactococcus lactis were observed in all silages, while Lactobacillus pentosus and Enterococcus faecium were near or below detection limits. Corn stover generally displayed higher populations of L. plantarum and L. brevis and lower populations of other LAB species. The data illustrate the utility of RT-PCR for quantifying individual species of LAB in conserved forages prepared under a wide variety of conditions.

DNA, Bacterial↗

PubMed2025

Whole genome duplication drives transcriptome reprogramming in response to drought in alfalfa.

Genome doubling did not enhance drought tolerance in alfalfa, but may set the stage for long-term adaptation to drought through a novel transcriptional landscape. Whole genome duplication (WGD) has been shown to enhance stress tolerance in plants. Cultivated alfalfa is autotetraploid, but diploid wild relatives are important sources of genetic variation for breeding. Investigating how WGD affects gene expression in stress conditions could provide better understanding for use of diploid genetic resources. In this work, we compared the drought response of neotetraploid plants obtained by bilateral sexual polyploidization with diploid full sibs, by measuring physiological and biochemical traits and RNA-seq. Without drought, 4x plants had lower photosynthetic potential than 2x plants per unit leaf area, but larger leaves allowed them to outperform the per leaf photosynthetic potential of 2x plants. Physiological and biochemical traits were significantly affected by drought in both 2x and 4x plants, but the differences between ploidies were small and nonsignificant. Proline levels were higher in 4x than 2x plants, both in control and drought conditions, indicating that larger cells with higher volume-to-surface ratio of 4x  plants require a higher osmolyte concentration. RNA-seq and gene network analyses showed that more genes were affected by drought at 4x than at 2x level, with downregulation of hundreds of genes involved in photosynthesis and stomatal movement at 4x level, suggesting that WGD made the 4x plants more responsive to drought. Genes involved in proline, phytormone and cell wall functions were also transcriptionally affected by drought in 4x plants. We conclude that WGD did not immediately enhance drought tolerance in alfalfa, but may set the stage for long-term adaptation to drought through a novel transcriptional landscape.

Medicago sativa↗

PubMed2004

Sinorhizobium meliloti RpoH1 is required for effective nitrogen-fixing symbiosis with alfalfa.

Sinorhizobium meliloti is a root-nodulating, nitrogen-fixing bacterium. An S. meliloti strain that is mutant for the rpoH(1) gene, which encodes a sigma(32)-like protein, elicits the formation of ineffective nodules on the host plant alfalfa. We characterized the rpoH(1) mutant for phenotypes related to symbiosis. Alfalfa nodules formed by the rpoH(1) mutant exhibited greatly reduced levels of acetylene reduction activity compared to the wild-type nodules. Whereas intracellular colonization by rhizobia was observed in a zone just below the apical meristem, we found ultrastructural abnormalities and signs of degeneration of bacteroids within many host cells in the proximally adjacent zone. In the proximal part of the nodule, only a few nodule cells contained bacteroids. In contrast, the rpoH(1) mutant showed normal induction of nitrogen fixation gene expression in microaerobic culture. These results suggest that the rpoH(1) mutation causes early senescence of bacteroids during the endosymbiotic process, but does not affect the invasion process or the synthesis of the nitrogenase machinery. The rpoH(1) mutant exhibited increased sensitivity to various agents and to acid pH, suggesting that RpoH(1) is required to protect the bacterial cell against environmental stresses encountered within the host. Since RpoH(1) was previously reported to be required for the synthesis of some heat shock proteins (Hsps), we examined the transcription of several genes for Hsp homologs. We found that transcription of groESL(5), lon, and clpB after heat shock was RpoH(1)-dependent, and conserved nucleotide sequences were found in the -35 and -10 regions upstream of the transcription start sites of these genes. Although groESL(5) expression is almost completely dependent on RpoH(1), we found that a groESL(5) mutant strain is still capable of normal symbiotic nitrogen fixation on alfalfa.

Base Sequence↗

PubMed1978

Effect of alfalfa meal on shrinkage (regression) of atherosclerotic plaques during cholesterol feeding in monkeys.

A semipurified diet containing 1.2 mg of cholesterol/Cal was fed to cynomolgus monkeys (Macaca fascicularis). At the end of 6 months, a group of 18 animals was killed for evaluation of atherosclerosis in the aorta and the coronary arteries. The remaining monkeys were assigned to three groups of 18 animals each and fed, during the following 18 months, semipurified diets containing 0.34 mg of cholesterol/Cal with or without alfalfa meal, or a diet consisting entirely of Monkey Chow. a decrease in cholesterolemia and plasma phospholipid levels, normalization in the distribution of plasma lipoproteins, and reduction in the extent of aortic and coronary atherosclerosis were observed in monkeys fed the semipurified diet containing alfalfa, although the intake of cholesterol remained as high as in the usual American diet. These changes, also observed in monkeys fed a chow diet almost devoid of cholesterol, suggest that alfalfa counteracts the atherogenic effect of dietary cholesterol.

Animals↗

PubMed1989

Ovarian cyclicity and serum progesterone and luteinizing hormone in fine-wool ewes supplemented with alfalfa or pinto beans.

Effects of season and supplementation on the incidence and rate of ovulation and hormone profiles in multiparous, nonlactating ewes were investigated under range and drylot conditions during anestrus (February through August). Ninety ewes received one of six nutritional treatments: 1) range forage, 2) range forage plus 0.33 kg.hd(-1).d(-1) pinto beans, 3) 0.45 kg.hd(-1).d(-1) alfalfa pellets, 4) dry lot plus 1.33 kg.hd(-1).d(-1) prairie hay only, 5) pinto beans or 6) alfalfa pellets at rates used on the range and prairie hay. Supplemented ewes were heavier (P < 0.05) than ewes fed range forage or prairie hay during most of the study. Seasonality of ovulation, in terms of incidence and rate, was not affected (P > 0.40) by supplementation in range ewes. The ovulation rate tended to be highest in prairie hay plus pinto bean drylot ewes in February (P = 0.21) and in prairie hay plus alfalfa pellet and prairie hay plus pinto bean ewes in March (P = 0.13) compared with prairie hay ewes. Generally, seasonality of ovulation was not influenced by supplementation (P > 0.40). The incidence of ovulation approached zero for drylot ewes in April, May, June and July, while it was 43, 27, 35 and 21% for range ewes, in those same months. Supplementation did not affect serum progesterone during the estrous cycle. Luteinizing hormone (LH) concentrations were similar (P > 0.50) among drylot treatment groups before a 50-ug gonadotropin releasing hormone (GnRH) challenge. Pinto bean supplementation enhanced serum LH response to GnRH (P < 0.10).

Journal Article↗

PubMed1990

Dynamic histone acetylation in alfalfa cells. Butyrate interference with acetate labeling.

Dynamic histone acetylation of alfalfa (Medicago sativa) was studied in suspension cultures by short-term labeling with radioactive acetate. The relative labeling rates for the acetylated histones were in order of decreasing incorporation; H3.2 greater than H3.1 greater than H4 greater than H2B.1 greater than H2A.3. Histone H3 showed at least seven sites of acetylation, histone H2B.1 had six sites and histone H4 had five sites. Low numbers of acetylation sites were observed for histone H2B.2 and all histone H2A variants. The mass ratio, steady state acetylation and dynamic acetylation between major variant H3.1 and minor variant H3.2 were approx. 2:1, 1:2 and 2:5, respectively. Treatment of alfalfa cells with 50 mM n-butyrate did not lead to histone hyperacetylation, but instead interfered with histone acetylation labeling by acetate. The extent of apparent inhibition increased with time and concentration of butyrate. It is likely that the conversion of butyrate to acetylCoA results in dilution of the specific radioactivity of [3H]acetate in the acetylCoA pool thereby inhibiting the labeling reaction. This interpretation is supported by 14C-labeling of alfalfa acetylated histones by [1-14C]butyrate.

Acetates↗

PubMed2006

Compression of fractionated sun-cured and dehydrated alfalfa chops into cubes--specific energy models.

The objective of this study was to determine the specific energy requirements for the compression of fractionated sun-cured and dehydrated alfalfa chops, when subjected to different pressures and holding times. The compression behavior of fractionated sun-cured and dehydrated alfalfa chops was studied using a single cubing unit capable of making one cube in a single stroke of the plunger. The cube die dimensions were 30 mm x 30 mm in cross-section and an effective depth of compression of 0.38 m. The initial moisture content of dehydrated and sun-cured chops were 6% and 7% (wb), respectively. A stack of two sieves (instead of five) was used along with a pan to achieve leaf and stem separation. The nominal opening sizes of two sieves with square holes were 3.96 and 1.17 mm, respectively. Leaf and stem fractions were combined later to obtain five different samples each for sun-cured and dehydrated alfalfa with leaf content ranging from 0% to 100% by mass in increments of 25%. The chop moisture content and preheat temperature before compaction was 10% (wb) and 75 degrees C, respectively. The cube die temperature was maintained at 90+/-5 degrees C. The mass of chops used for making each cube was 23+/-02 g. A hydraulic press was used to apply 9.0, 12.0 and 14.0 MPa of pressures through a plunger. After compression, the plunger was held in place for 10 and 30s, before the compacted forage was extracted. Empirical equations were fitted to the data relating specific energy for cube making to pressure, residence time, and leaf content.

Biomass↗