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PubMed2006

Effect of low and high-saponin lines of alfalfa on pea aphid.

Pea aphid fed on a high-saponin line of alfalfa showed reduction of reproduction and survival, and disturbance in development of its population. This line negatively influenced aphid probing behaviour, particularly prolonging the non-probing period and probing of the peripheral tissues (epidermis and mesophyll) and shortening the period of phloem sap ingestion. The high-saponin line of alfalfa differed from the low-saponin one by the presence of zanhic acid tridesmoside and a higher level of 3-GlcA,28-AraRhaXyl medicagenic acid glycoside. The saponins incorporated into sucrose-agarose gels significantly reduced number of the aphid probes into the gels and extended their duration in comparison to the control gels (without tested compounds). Role of zanhic acid tridesmoside and 3-GlcA,28-AraRhaXyl medicagenic acid glycoside as potential factors for partial resistance of alfalfa towards the pea aphid is discussed.

Animals↗

PubMed2000

Nuclear localization of a hypoxia-inducible novel non-symbiotic hemoglobin in cultured alfalfa cells.

We have isolated a 483-bp-long full-length cDNA clone encoding a non-symbiotic hemoglobin called Mhb1, the first one found in alfalfa. This non-symbiotic hemoglobin is a single copy gene localized in linkage group 4 in diploid Medicago genome. The Mhb1 mRNA was found only in the roots of alfalfa plants. The Mhb1 gene was inducible by hypoxia and showed no induction by cold stress treatment. The Mhb1 transcript level increased at the G2/M boundary in a synchronized alfalfa cell suspension culture. The majority of Mhb1 protein was shown to be localized in the nucleus and smaller amounts were detected in the cytoplasm. A potential link to the nitric oxide signalling pathway is also discussed.

Amino Acid Sequence↗

PubMed1999

Alfalfa contains substantial 9-hydroperoxide lyase activity and a 3Z:2E-enal isomerase.

Fatty acid hydroperoxides formed by lipoxygenase can be cleaved by hydroperoxide lyase resulting in the formation of short-chain aldehydes and omega-oxo acids. Plant hydroperoxide lyases use 13- or 9-hydroperoxy linoleic and linolenic acid as substrates. Alfalfa (Medicago sativa L.) has been reported to contain a hydroperoxide lyase specific for 13-hydroperoxy linoleic and linolenic acid only. However, in addition to 13-hydroperoxide lyase activity we found substantial 9-hydroperoxide lyase activity in alfalfa seedlings as well. The specific activity for 9-hydroperoxy fatty acids was about 50% of the activity for the 13-isomers. Furthermore, alfalfa seedlings contain a 3Z:2E-enal isomerase that converts the 3Z-enal products to their 2E-enal isoforms.

Aldehyde-Lyases↗

PubMed2002

Monolignol biosynthesis in microsomal preparations from lignifying stems of alfalfa (Medicago sativa L.).

Microsomal preparations from lignifying stems of alfalfa (Medicago sativa L.) contained coniferaldehyde 5-hydroxylase activity and immunodetectable caffeic acid 3-O-methyltransferase (COMT), and catalyzed the S-adenosyl L-methionine (SAM) dependent methylation of caffeic acid, caffeyl aldehyde and caffeyl alcohol. When supplied with NADPH and SAM, the microsomes converted caffeyl aldehyde to coniferaldehyde, 5-hydroxyconiferaldehyde, and traces of sinapaldehyde. Coniferaldehyde was a better precursor of sinapaldehyde than was 5-hydroxyconiferaldehyde. The alfalfa microsomes could not metabolize 4-coumaric acid, 4-coumaraldehyde, 4-coumaroyl CoA, or ferulic acid. No metabolism of monolignol precursors was observed in microsomal preparations from transgenic alfalfa down-regulated in COMT expression. In most microsomal preparations, the level of the metabolic conversions was independent of added recombinant COMT. Taken together, the data provide only limited support for the concept of metabolic channeling in the biosynthesis of S monolignols via coniferaldehyde.

Chromatography, High Pressure Liquid↗

PubMed2003

Structural and compositional modifications in lignin of transgenic alfalfa down-regulated in caffeic acid 3-O-methyltransferase and caffeoyl coenzyme A 3-O-methyltransferase.

Isolated lignins from alfalfa deficient in caffeic acid 3-O-methyltransferase contained benzodioxanes resulting from the incorporation of the novel monomer, 5-hydroxyconiferyl alcohol. Due to the high level incorporated into the soluble lignin fraction and the use of sensitive NMR instrumentation, unique structural features were revealed. A new type of end-unit, the 5-hydroxyguaiacyl glycerol unit, was identified. It was possible to establish that coniferyl alcohol, sinapyl alcohol, and the novel 5-hydroxyconiferyl alcohol can cross-couple with the 5-hydroxyguaiacyl units that are formed in the lignin, the latter giving rise to extended chains of benzodioxane units. There is also evidence that 5-hydroxyconiferyl alcohol couples with normal (guaiacyl or syringyl) lignin units. Lignin in the alfalfa deficient in caffeoyl CoA 3-O-methyltransferase was structurally similar to the control lignin but the transgenic exhibited a dramatic decrease in lignin content (approximately 20%) and modest increase in cellulose (approximately 10%) reflecting a 30% increase in cellulose:lignin ratio. The compositional changes in both transgenics potentially allow enhanced utilization of alfalfa as a major forage crop by increasing the digestibility of its stem fraction.

Down-Regulation↗

PubMed2003

Profiling phenolic metabolites in transgenic alfalfa modified in lignin biosynthesis.

Soluble phenolics, wall-bound phenolics and soluble and core lignin were analyzed in transgenic alfalfa with genetically down-regulated O-methyltransferase genes involved in lignin biosynthesis. High performance liquid chromatography and principal component analysis were used to distinguish metabolic phenotypes of different transgenic alfalfa genotypes growing under standard greenhouse conditions. Principal component analysis of HPLC chromatograms did not resolve differences in leaf metabolite profiles between wild-type and transgenic plants of the same genetic background, although stem phenolic profiles were clearly different between wild-type and transgenic plants. However, the analytical methods clearly differentiated two non-transgenic alfalfa cultivars based on either leaf or stem profiles. Metabolic profiling provides a useful approach to monitoring the broader biochemical phenotypes of transgenic plants with altered expression of lignin pathway enzymes.

Cell Wall↗

PubMed2000

Efficacy of novel organic acid and hypochlorite treatments for eliminating Escherichia coli O157:H7 from alfalfa seeds prior to sprouting.

This study investigated novel two-step organic acid/hypochlorite treatments as alternatives to 20000 ppm active chlorine (from calcium hypochlorite) for eliminating Escherichia coli O157:H7 from alfalfa seeds prior to sprouting. Commercially available alfalfa seeds were inoculated with a five-strain E. coli O157:H7 mixture and dried to attain ca. 10(6) CFU/g of seeds. Seeds then underwent one of several soak treatments including: (1) 5% (v/v) lactic acid for 10 min at 42 degrees C; (2) 5% acetic acid (v/v) for 10 min at 42 degrees C; (3) 2.5% lactic acid for 10 min at 42 degrees C followed by 2000 ppm active chlorine (from calcium hypochlorite) for 15 min at 25 degrees C; (4) 5% lactic acid for 10 min at 42 degrees C followed by 2000 ppm active chlorine for 15 min at 25 degrees C; or (5) 20000 ppm active chlorine for 15 min at 25 degrees C. Each treatment reduced numbers of inoculum cells by about 6.0 log10 CFU/g as determined by plating on Sorbitol MacConkey agar (SMac). Plating on non-selective brain heart infusion agar (BHI) showed that treatments 1-4 reduced counts by 2.3-4.1 log10 CFU/g, thus indicating a large proportion of injured cells. Successive lactic acid and hypochlorite treatments (3 and 4) were more lethal than either organic acid alone (1 and 2). No surviving cells were detected on SMac or BHI following treatment with 20000 ppm active chlorine (treatment 5). Regardless of the previous treatment, E. coli O157:H7 counts increased to 10(7)-10(8) CFU/g during sprouting. Germination of seeds was not adversely affected by any of the treatments (germination > 90%). Results of this study show that: (a) non-lethal cell injury must be considered when evaluating intervention treatments against E. coli O157:H7 on alfalfa seeds; (b) reductions of 2-4 log10 CFU/g can be attained without using 20000 ppm active chlorine; (c) successive lactic acid and hypochlorite treatments have greater lethality than organic acid treatments alone; and (d) none of the treatments tested can prevent regrowth of surviving E. coli O157:H7 during sprouting.

Acetic Acid↗

PubMed2001

Factors influencing the detection and enumeration of Escherichia coli O157:H7 on alfalfa seeds.

Isolating Escherichia coli O157:H7 from batches of alfalfa seeds used to produce sprouts implicated in human illness has been difficult, perhaps due to nonhomogenous and very low-level contamination and inaccessibility of the pathogen entrapped in protected areas of the seed coat. We evaluated the effectiveness of various treatments in releasing E. coli O157:H7 from seeds. The influence of homogenization (blending or stomaching for 1 or 2 min), rinsing method (shaking for 5 min), soaking time (0. 1, 3, 6, or 15 h), soaking temperature (4 or 21 degrees C), and the addition of surfactants (0.1%, 0.5%, or 1.0% Tween 80 or Span 20) to rinse water was determined. Blending or stomaching for 1 or 2 min, and soaking for 1 h or longer at 4 or 21 degrees C, respectively, resulted in maximum release of E. coli O157:H7 from seeds. Soaking seeds at 37 degrees C for 15 h increased cell populations of E. coli O157:H7 by approximately 3.6 log10 CFU/g, likely due to bacterial growth. The maximum number of cells released from seeds by rinse water containing 1.0% Span 20 was at 21 degrees C, whereas at 37 degrees C, 0.1% or 0.5% Tween 80 was more effective. Detection of E. coli O157:H7 on seeds stored at 37 degrees C for up to 13 weeks and on sprouts derived from these seeds was compared. E. coli O157:H7 inoculated on seeds at 2.0 log10 CFU/g was detected after storage of seeds for up to 8 weeks at 37 degrees C and in sprouts produced from the seeds. The pathogen was not detected on seeds stored for 13 weeks at 37 degrees C and was not isolated from sprouts produced from these seeds. Identifying seed treatment methods that enhance removal of E. coli O157:H7 from alfalfa seeds can aid the isolation and enumeration of the pathogen on seeds. With a combination of optimal conditions for detecting the pathogen, i.e. soaking seeds for 1 h and pummeling seeds for 1 min, an enrichment step in modified tryptic soy broth (TSB), and the use of immunomagnetic beads for separation of E. coli O157:H7 cells, E. coli O157:H7 was detected in alfalfa seeds incubated at 37 degrees C for up to 8 weeks as effectively as in sprouts produced from the seeds.

Colony Count, Microbial↗

PubMed2003

Efficacy of chlorine and calcinated calcium treatment of alfalfa seeds and sprouts to eliminate Salmonella.

The efficacy of a 20,000 ppm calcium hypochlorite treatment of alfalfa seeds artificially contaminated with Salmonella was studied. Salmonella populations reached >7.0 log on sprouts grown from seeds artificially contaminated with Salmonella and then treated with 20,000 ppm Ca(OCl)(2). The efficacy of spray application of chlorine (100 ppm) to eliminate Salmonella during germination and growth of alfalfa was assessed. Alfalfa seed artificially contaminated with Salmonella was treated at germination, on day 2 or day 4, or for the duration of the growth period. Spray application of 100 ppm chlorine at germination, day 2, or day 4 of growth was minimally effective resulting in approximately a 0.5-log decrease in population of Salmonella. Treatment on each of the 4 days of growth reduced populations of Salmonella by only 1.5 log. Combined treatment of seeds with 20,000 ppm Ca(OCl)(2) and followed by 100 ppm chlorine or calcinated calcium during germination and sprout growth did not eliminate Salmonella.

Calcium↗

PubMed2000

Efficacy of allyl isothiocyanate in killing enterohemorrhagic Escherichia coli O157:H7 on alfalfa seeds.

Volatile compounds occurring in the essential oil of plants were tested for their efficacy in killing Escherichia coli O157:H7. Experiments using an agar disk assay revealed that exposure of the pathogen to 50 microl of eugenol, carvacrol, linalool, or methyl jasmonate in a 950-cc jar at 20, 37 or 47 degrees C for up to 48 h failed to inhibit colony formation. However, exposure to 8 microl of allyl isothiocyanate (AIT) (equivalent to 8.4 ppm in the air within the jar, if completely volatilized) resulted in more than a 7-log10 reduction in population of E. coli O157:H7 at 37 degrees C within 48 h; significant (P < or = 0.05) reduction in populations also occurred in the presence of 4 microl of AIT compared to 2 microl, which had no lethal affect. At 20 degrees C, the lethality of AIT was substantially less, although significant reduction occurred when disks were exposed to 8 or 10 microl of AIT compared to 4 or 6 microl and when exposed to 4 or 6 microl compared to 2 microl. Treatment with 10 microl of AIT for 5 h at 47 degrees C resulted in death of 6 log10 of E. coli O157:H7. The efficacy of AIT in killing E. coli O157:H7 on dry and wet alfalfa seeds was investigated. The pathogen, at an initial population of 2.7 log10 cfu/g of seed, was not recovered by direct plating (< 0.7 log10 cfu/g) or enrichment of wet seeds exposed to 50 microl of AIT/950-cc jar for 24 h at 37 or 47 degrees C. Exposure of dry seeds containing 2.9 log10 cfu of E. coli O157:H7 per g to an atmosphere containing 100 microl of AIT/950-cc jar (ca. 105 ppm AIT if completely volatilized) for 24 h at 47 degrees C did not eliminate viable E. coli O157:H7 cells. Unfortunately, the enhanced effectiveness of AIT in killing the pathogen on wet alfalfa seeds is offset by a dramatic reduction in seed viability. Nevertheless, the use of AIT as an alternative to chlorine for the purpose of killing E. coli O157:H7 and perhaps other pathogens on alfalfa seed holds promise. Factors that may influence conditions rendering increased sensitivity of E. coli O157:H7 to AIT without compromising seed viability should be investigated.

Escherichia coli O157↗

PubMed2000

Alfalfa (Medicago sativa) carbamoylphosphate synthetase gene structure records the deep lineage of plants.

Given the central role of carbamoylphosphate synthetases in pyrimidine and arginine metabolism in all living organisms, the absence of fundamental information regarding plant CPSase genes is a striking omission [Lawson et al., Mol. Biol. Evol. 13 (1996) 970-977; van den Hoff et al., J. Mol. Evol. 41 (1995) 813-832]. Whereas CPSase gene architecture and aa sequence have proven to be useful characters in establishing ancient and modern genetic affinities, phylogenetic analysis cannot be completed without the inclusion of plant CPSases. We describe the first isolation by molecular cloning of a plant CPSase gene (CPAII) derived from alfalfa (Medicago sativa). DNA sequence analysis reveals a proteobacterial architecture, namely closely linked carA and carB coding domains separated by a short intergenic region, and transcribed as a polycistronic mRNA. CPAII encodes the amino acid residues that typify a CPSase type II enzyme. In addition, an ancient internal duplication has been retained in the plant carB sequence. Partial nucleotide sequencing of additional clones reveals that the alfalfa genome contains multiple CPSase II gene copies which may be tissue-specific in their expression. It appears that with respect to CPSase genes, CPAII resembles the carAB gene of bacteria, and may have preserved much of this ancient gene structure in the alfalfa genome.

Amino Acid Sequence↗

PubMed2005

Temporally distinct Escherichia coli 0157 outbreaks associated with alfalfa sprouts linked to a common seed source--Colorado and Minnesota, 2003.

Escherichia coli O157 outbreaks were identified in Minnesota in February 2003 involving seven persons and in Colorado in July 2003 involving 13 persons. Case isolates from the two states had matching pulsed-field gel electrophoresis (PFGE) patterns. Independent case-control studies linked infections in each outbreak with eating alfalfa sprouts that were traced to the same seed distributor. The Colorado sprouter reportedly complied with the Food and Drug Administration (FDA) sprout guidance, whereas the Minnesota sprouter did not. These investigations revealed that increased compliance with existing FDA guidance is needed and that additional research is needed to improve the alfalfa seed decontamination process. This reaffirms the FDA recommendation that raw alfalfa sprouts should be considered potentially contaminated and avoided by persons at high-risk such as the elderly, young children, and immunocompromised persons. PFGE played an essential role in linking these two temporally and geographically distinct E. coli O157 outbreaks.

Adult↗

PubMed1992

Identification of five sites of acetylation in alfalfa histone H4.

Radioactive acetylation in vivo of plant histone H4 of alfalfa, Arabidopsis, tobacco, and carrot revealed five distinct forms of radioactive, acetylated histone. In histone H4 of eukaryotes ranging from fungi to man, acetylation is restricted to four lysines (residues 5, 8, 12, and 16) possibly caused by a quantitative methylation of lysine-20. Chemical and proteolytic fragmentation of the amino terminally blocked alfalfa H4 protein, dynamically acetylated by radioactive acetate in vivo, allowed protein sequencing and identification of selected peptides. Peptide identification was facilitated by analyzing fully characterized calf histone H4 in parallel. Acetylation in vivo of alfalfa histone H4 was restricted to the lysines in the amino-terminal domain of the protein, residues 1-23. Lysine-20 was shown to be free of methylation, as in pea histone H4. This apparently makes lysine-20 accessible as a novel target for histone acetylation. The in vivo pattern of lysine acetylation (16 greater than 12 greater than 8 greater than or equal to 5 = 20) revealed a preference for lysines -16 and -12 without an apparent strict sequential specificity of acetylation.

Acetylation↗

PubMed1984

Isolation and characterization of a wound-induced trypsin inhibitor from alfalfa leaves.

A trypsin inhibitor from leaves of field-grown alfalfa plants has been purified and shown to be the same trypsin inhibitor that is wound induced in leaves of young growth chamber grown plants. This inhibitor accounts for the major trypsin inhibitory activity found in both field-grown and wound-induced plants. The inhibitor exhibits a molecular weight of about 7500 and is specific for trypsin with a Ki of 1 X 10(-10) M. Analysis of the purified inhibitor by cation-exchange high-performance liquid chromatography revealed the presence of four isoinhibitor species that have identical immunological and inhibitory properties. The amino acid analysis of the four species indicates small but significant differences. Immunological double diffusion comparisons of the alfalfa inhibitor with the Bowman-Birk and Kunitz soybean inhibitors did not reveal any cross-reactivity although the amino acid content of the alfalfa inhibitor resembles those of Bowman-Birk family members.

Amino Acids↗

PubMed1980

Complexes of alfalfa mosaic virus RNA 4 with one and three coat protein dimers.

RNA 4, the subgenomic coat protein messenger of alfalfa mosaic virus, was loaded with small amounts of coat protein in a reaction in which complete virions were the protein donor. In such a reaction the protein subunits attach to the high-affinity binding sites near the 3' end of RNA 4 [Houwing, C. J., & Jaspars, E. M. J. (1978) Biochemistry 17, 2927-2933]. At a ratio of up to 13 coat protein subunits to 1 mol of RNA 4, complexes with one and three protein dimers, designated complex I and complex III, respectively, were formed. These complexes were isolated by preparative electrophoresis in 4% polyacrylamide gel. At a large excess of the protein donor (280 protein subunit/mol of complex), both complexes I and III were converted into uniform complexes with 10 protein dimers. There were no indications for stable intermediate complexes. A model is suggested for the structure of the complexes which is based on the model proposed for the protein coat of alfalfa mosaic virus [Mellema, J. E., & Van Den Berg, H. J. N. (1974) J. Supramol. Struct. 2, 17-31]. The complexes possibly serve as successive stages in virion assembly. More intriguingly, the complexes could be of regulatory significance. Since the four RNA species of alfalfa mosaic virus have an extensive 3'-terminal homology, and since 3'-terminal interaction with coat protein subunits is thought to be a process leading to recognition of the viral genome by the viral replicase and thus to infectivity, complexes analogous to complexes I and III could represent the infectious forms of the genome RNAs.

Binding Sites↗

PubMed1980

Preferential binding of 3'-terminal fragments of alfalfa mosaic virus RNA 4 to virions.

3'-Terminal fragments present in a partial ribonuclease T1 digest of RNA 4 of alfalfa mosaic virus were selectively bound to virions of the same virus by incubation in 10 mM sodium phosphate and 1 mM EDTA, pH 7.0. The virions with the associated fragments were separated from the remainder of the digest by velocity gradient centrifugation. When the incubation was performed in 27.7 mM Na2HPO4 and 6.2 mM citric acid, pH 6.8, plus 10% glycerol, much more, but less specific, binding was obtained. However, in this case the terminal fragments present in unbound material of the digest were almost exclusively 5' termini. The four RNA species species of alfalfa mosaic virus have an extensive 3'-terminal homology. Therefore, it is expected that specific virion binding can be used to obtain 3'-terminal fragments of different lengths of the three genome RNA species (RNAs 1, 2, and 3) of alfalfa mosaic virus and, possibly, of related viruses. Such fragments are of great importance for the study of the specific 3'-terminal interaction with coat protein subunits which is thought to be responsible for the activation of the viral genome.

Binding Sites↗

PubMed2002

Changes in the cell wall network during the thermal dehydration of alfalfa stems.

The effects of heat treatments used to dry alfalfa stems were investigated. Heating at 70 or 100 degrees C caused no major change in the cell wall composition, but xylanase had lower activity on the cell wall of heated material and the amount of xylose released varied with the temperature used. Chemical fractionation of cell wall carbohydrates showed that the main changes occurring during stem dehydration concerned pectic polymers and probably hemicelluloses. There was less material soluble in ammonium oxalate from alfalfa heated at 100 degrees C than from fresh alfalfa. The results suggest that heat processing causes some changes in the cell wall network. Environmental scanning electron microscopy was used to examine fully hydrated tissues at high resolution. There was cell distortion without disruption of cell walls as water was lost.

Carbohydrates↗

PubMed2002

Chemical composition and enzymatic degradability of xylem and nonxylem walls isolated from alfalfa internodes.

During plant maturation, degradability of alfalfa (Medicago sativa L.) stems declines due to accumulation of highly lignified xylary tissue. Xylem and nonxylem tissues dissected from lower alfalfa internodes were analyzed for cell wall constituents and degradability. Cell walls comprised 740 mg g(-1) of xylem and 533 mg g(-1) of nonxylem tissues. Xylem tissues contributed about 60% of the cell wall mass in internodes. Xylem walls contained 28% lignin, 4% pectin, 29% hemicellulose, and 39% cellulose as compared to 15% lignin, 25% pectin, 30% hemicellulose, and 30% cellulose in nonxylem walls. Fungal enzymes hydrolyzed 22 and 73% of the structural carbohydrates in xylem and nonxylem walls, respectively. In both cell wall fractions, the release of xylose was 56-90% lower than that of other sugars, indicating that lignin preferentially restricted xylan degradation in secondary walls and xyloglucan degradation in primary walls. Elucidation of lignin-xylose interactions may reveal strategies for improving fiber degradability of alfalfa.

Cell Wall↗