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PubMed2001

Flue gas desulfurization by-products additions to acid soil: alfalfa productivity and environmental quality.

Flue gas desulfurization (FGD) by-products are created when coal is burned and SO2 is removed from the flue gases. These FGD by-products are often alkaline and contain many plant nutrients. Land application of FGD by-products is encouraged but little information is available related to plant responses and environmental impacts concerning such use. Agricultural lime (ag-lime) and several new types of FGD by-products which contain either vermiculite or perlite were applied at 0, 0.5, 1.0, and 2.0 times the soil's lime requirement (LR) rate to an acidic soil (Wooster silt loam). The highest FGD by-products application rate was equivalent to 75.2 Mg ha(-1). Growth of alfalfa (Medicago sativa L.) was significantly increased compared to the untreated control in the second year after treatment with yields for the 1 x LR rate of FGD approximately 7-8 times greater compared to the untreated control and 30% greater than for the commercial ag-lime. Concentrations of Mo in alfalfa were significantly increased by FGD by-products application, compared to the untreated control, while compared to the ag-lime treatment, concentrations of B increased and Ba decreased. No soil contamination problems were observed, even at the 2xLR rate, indicating these materials can be safely applied to agricultural soils.

Agriculture↗

PubMed2002

Effect of mixed cadmium, copper, nickel and zinc at different pHs upon alfalfa growth and heavy metal uptake.

Alfalfa plants were grown in soil-pots contaminated with a mixture of Cd(II), Cu(II), Ni(II), and Zn(II), (at 50 mg/kg each) at pHs of 4.5, 5.8, and 7.1. The plants were fertilized using a nutrient solution, which was adjusted appropriately to the same pH. Plants in the control treatment were grown in the absence of the heavy metals mixture. The growth of the control plants was the same at the three pHs studied and the heavy metal stressed plants also showed similar behavior at each pHs. There were statistically significant differences (P<0.05) between the shoot length of the control treatment plants and the length of plants grown in the presence of the heavy metal mixture. Under the effects of the heavy metal mixture, nickel was the most accumulated element in the shoot tissue, with 437, 333, and 308 ppm at pH 7.1, 5.8, and 4.5, respectively. Cadmium was found to be second in accumulated concentrations with 202 ppm, 124 ppm, and 132 ppm at pH 7.1, 5.8, and 4.5, respectively, while zinc was third, followed by copper. The maximum relative uptakes (element in plant/element in soil-water-solution) were found to be 26 times for nickel, 23 times for cadmium, 12 times for zinc. and 6 times for copper. We considered these relations as indicative of the ability of alfalfa plants to take up elements from a soil matrix contaminated with a mixture of cadmium, copper, nickel, and zinc.

Cadmium↗

PubMed1997

An alfalfa rubisco small subunit homologue shares cis-acting elements with the regulatory sequences of the RbcS-3A gene from pea.

A genomic clone of RbcS was isolated from an alfalfa (Medicago sativa L. cv. Apica) genomic library and characterized. Although this clone has structural features similar to a functional gene, the second exon is interrupted by a stop codon and thus is not fully translatable in the plant. Sequence analysis of the 5' and 3' noncoding regions of RbcSK-1A showed a high sequence homology to the flanking sequences of the RbcS-3A gene from pea. The regions of homology contain many important cis-regulatory elements shown to be essential for regulation of the RbcS-3A gene in pea. The promoter of this alfalfa rubisco clone was used in a translational fusion to test its ability to control the expression of the GUS reporter gene in an homologous nuclear background. High levels of GUS enzyme activity were recorded. These strong levels are comparable to some exceptionally high levels produced in other studies following the use of photosynthesis gene promoters in fusions with the GUS reporter gene.

Base Sequence↗

PubMed2001

Enzyme assisted ensiling of alfalfa with enzymes by solid substrate fermentation.

A crude enzyme preparation, obtained by solid substrate fermentation (SSF) with a Gliocladium spp. and added at the 5% level to wilted or non-wilted alfalfa, improved the fermentation characteristics and stability of alfalfa silages as effectively as commercial preparations, Novo-Nordisk Celluclast 1.5 L and Viscozyme 120 L, applied at the 0.025% level. The effective dose of the crude enzyme costs about one-fourth of the cost of the commercial enzymes.

Carbohydrate Metabolism↗

PubMed2000

Inhibition of cysteine and aspartyl proteinases in the alfalfa weevil midgut with biochemical and plant-derived proteinase inhibitors.

Proteolytic activities in alfalfa weevil (Hypera postica) larval midguts have been characterized. Effects of pH, thiol activators, low-molecular weight inhibitors, and proteinase inhibitors (PIs) on general substrate hydrolysis by midgut extracts were determined. Hemoglobinolytic activity was highest in the acidic to mildly acidic pH range, but was maximal at pH 3.5. Addition of thiol-activators dithiothreitol (DTT), 2-mercaptoethanol (2-ME), or L-cysteine had little effect on hemoglobin hydrolysis at pH 3.5, but enhanced azocaseinolytic activity two to three-fold at pH 5.0. The broad cysteine PI E-64 reduced azocaseinolytic activity by 64% or 42% at pH 5 in the presence or absence of 5 mM L-cysteine, respectively. Inhibition by diazomethyl ketones, Z-Phe-Phe-CHN(2) and Z-Phe-Ala-CHN(2), suggest that cathepsins L and B are present and comprise approximately 70% and 30% of the cysteine proteolytic activity, respectively. An aspartyl proteinase component was identified using pepstatin A, which inhibited 32% (pH 3.5, hemoglobin) and 50% (pH 5, azocasein) of total proteolytic activity. This activity was completely inhibited by an aspartyl proteinase inhibitor from potato (API), and is consistent with the action of a cathepsin D-like enzyme. Hence, genes encoding PIs with specificity toward cathepsins L, B and D could potentially be effective for control of alfalfa weevil using transgenic plants.

Animals↗

PubMed1985

Wound-induced trypsin inhibitor in alfalfa leaves: identity as a member of the Bowman-Birk inhibitor family.

The primary structure of the wound-inducible trypsin inhibitor from alfalfa (ATI) establishes it as a member of the Bowman-Birk proteinase inhibitor family. The time course of induction of ATI in alfalfa following wounding is similar to the induction of the nonhomologous proteinase inhibitors I and II in tomato and potato leaves, and, like inhibitors I and II, ATI is induced to accumulate in excised leaves supplied with the proteinase inhibitor inducing factor from tomato leaves. The similarity of the wound induction of ATI to that of inhibitors I and II indicates that wound-regulated systems are present in Solanaceae and Leguminosae plant families that possess a common fundamental recognition system regulating synthesis of proteinase inhibitors in response to pest attacks. ATI is the first Bowman-Birk inhibitor that has been found in leaves and is the only member of this family known to be regulated by wounding.

Amino Acid Sequence↗

PubMed1978

Coat protein binds to the 3'-terminal part of RNA 4 of alfalfa mosaic virus.

All four RNAs of alfalfa mosaic virus contain a limited number of sites with a high affinity for coat protein [Van Boxsel, J. A. M. (1976), Ph.D. Thesis, University of Leiden]. In order to localize these sites in the viral RNAs, RNA 4 Tthe subgenomic messenger for coat protein) was subjected to a very mild digestion with ribonucleast T1. The ten major fragments, apparently resulting from five preferential hits, were separated and tested for messenger activity in a wheat germ cell-free system, as well as for the capacity to withdraw coat protein from intact particles. Fragments which stimulated amino acid incorporation were assumed to contain the 5 terminus. Strong evidence was obtained for the location of sites with a high affinity for coat protein near the 3' terminus. The smallest fragment which has the 3'-terminal cytosine comprises only 10% of the length of intact RNA 4 but still possesses these sites. Evidence is presented that the complete coat protein cistron is in the complementing 90% fragment. Possibly, the high-affinity sites are entirely located in the 3'-terminal extracistronic part of RNA 4. They will have the same position in RNA 3 and, possibly, also in the other parts of the genome of alfalfa mosaic virus. The need of this genome for coat protein in order to become infectious may therefore find its explanation in the fact that a conformational change at the 3' ends of the genome parts brought about by the coat protein is required for recognition by the viral replicase.

Medicago sativa↗

PubMed2001

Alfalfa (Medicago sativa L.) flavonoids. 2. Tricin and chrysoeriol glycosides from aerial parts.

Ten flavone glycosides have been isolated and identified in aerial parts of alfalfa. These included six tricin, one 3'-O-methyltricetin, and three chrysoeriol glycosides. Most of these compounds were acylated with ferulic, coumaric, or sinapic acids, and acylation occurred on the terminal glucuronic acid. Eight of these compounds, including 7-O-beta-D-glucuronopyranosyl-3'-O-methyltricetin, 7-O-beta-D-glucuronopyranosyl-4'-O-beta-D-glucuronopyranosidechrysoeriol, 7-O-[2'-O-feruloyl-beta-D-glucuronopyranosyl(1-->2)-O-beta-D-glucuronopyranoside]chrysoeriol, 7-O-[2'-O-feruloyl-[beta-D-glucuronopyranosyl(1-->3)]-O-beta-D-glucuronopyranosyl(1-->2)-O-beta-D-glucuronopyranoside]chrysoeriol, 7-O-[2'-O-sinapoyl-beta-D-glucuronopyranosyl(1-->2)-O-beta-D-glucuronopyranoside]tricin, 7-O-[2'-O- feruloyl-beta-D-glucuronopyranosyl(1-->2)-O-beta-D-glucuronopyranoside]tricin, 7-O-[2'-O-p-coumaroyl-beta-D-glucuronopyranosyl(1-->2)-O-beta-D-glucuronopyranoside]tricin, and 7-O-[2'-O-feruloyl-[beta-D-glucuronopyranosyl(1-->3)]-O-beta-D-glucuronopyranosyl(1-->2)-O-beta-D-glucuronopyranoside]tricin, have not been reported previously in the plant kingdom. Two previously identified alfalfa flavones, 7-O-beta-D-glucuronopyranosidetricin and 7-O-[beta-D-glucuronopyranosyl(1-->2)-O-beta-D-glucuronopyranoside]tricin, were also isolated.

Carbohydrate Conformation↗

PubMed1998

Alfalfa Saponins and Their Implication in Animal Nutrition.

Alfalfa (Medicago sativa L.) is of immense importance as livestock fodder for both developed and lesser developed countries as it contains a high amount of protein, and yield of dry matter is also very high. The main antinutritional components present in this plant are saponins, and their unfavorable effects on animal performance have restricted the optimum use of this high-protein plant as an animal feed. The occurrence, chemistry, analysis, and consequences of intake of alfalfa saponins are reviewed. The information synthesized may lead to planning of more detailed studies on isolation and characterization of saponins and sapogenins to gain a better understanding of the biological activities of the aglycon and carbohydrate moieties of saponins.

Journal Article↗

PubMed1997

Isolation of pathogen/stress-inducible cDNAs from alfalfa by mRNA differential display.

Differential display of mRNA was used to isolate a full-length (SRG1) and a partial (SRG2) alfalfa cDNA induced during infection with the fungal pathogen Colletotrichum trifolii. The deduced amino acid sequences are similar to each other and resemble plant defense-related proteins and tree pollen allergens. SRG1 is a member of a gene family in alfalfa, which may also include the putative defense-related gene PR10. Unlike many defense-related genes described in similar systems, expression of SRG1-like genes does not correlate with resistance to C. trifolii. We speculate SRG1 is induced in response to plant stress.

Amino Acid Sequence↗

PubMed1997

Expression of a novel-type small proline-rich protein gene of alfalfa is induced by 2,4-dichlorophenoxiacetic acid in dedifferentiated callus cells.

Differential screening of a cDNA library of 2,4-dichlorophenoxiacetic acid (2,4-D)-treated alfalfa (Medicago sativa) callus tissues resulted in the isolation of a 571 bp cDNA clone (MsPRP5) encoding for a proline-rich protein (84 amino acids) with a specific repeat unit of TPVLPPRK/RGRPPPVPP. In addition, a characteristic amino acid block (PPVYK) previously found in other proline-rich proteins also occurs in the C-terminal region of MsPRP5. At the N-terminal, a signal peptide similar to leader sequences of extracellular proteins can be predicted. According to the northern analysis, the corresponding gene is not expressed or is weakly expressed in differentiated vegetative organs and somatic embryos. However the accumulation of MsPRP5 mRNA is auxin concentration-dependent in dedifferentiated callus tissue. An increase in the amount of steady-state mRNA was detected already 20 min after auxin shock (100 microM 2,4-D). Maximum expression was observed at 24-48 h in the presence of 2,4-D. Elevated expression was also found in cells recovering after heat shock and wounding stress. In synchronized alfalfa cells, the transcript level of MsPRP5 gene fluctuated during cell cycle progression with peaks in G1/S phase cells. Considering the structural features and expression properties of MsPRP5, this clone may represents a new type of proline-rich protein gene which responds to hormonal shock and some other stresses as well.

2,4-Dichlorophenoxyacetic Acid↗

PubMed1999

MsJ1, an alfalfa DnaJ-like gene, is tissue-specific and transcriptionally regulated during cell cycle.

DnaJ-like proteins are molecular chaperones that regulate Hsp70 ATPase activity both in protein folding, assembly and disassembly of protein complexes. Here we report the isolation of MsJ1, an alfalfa gene encoding a protein homologous to cytosolic DnaJ-like proteins. MsJ1 was induced under heat-shock treatment in both leaves and stems of adult plants. In the absence of heat shock MsJ1 expression was tissue-specific with the highest levels of mRNA in roots and in embryonal structures. High levels of transcript were also detected in cotyledons where active degradation of storage protein occurs. In synchronized alfalfa suspension-cultured cells the MsJ1 transcript was actively expressed and showed a phase-specific modulation during cell cycle with a 2-fold induction in G2/M. These findings suggest that DnaJ-like proteins play an active role in regulating normal cellular events like protein degradation, morphogenesis and cell cycle progression.

Amino Acid Sequence↗

PubMed2000

Enhanced levels of free and protein-bound threonine in transgenic alfalfa (Medicago sativa L.) expressing a bacterial feedback-insensitive aspartate kinase gene.

Threonine, lysine, methionine, and tryptophan are essential amino acids for humans and monogastric animals. Many of the commonly used diet formulations, particularly for pigs and poultry, contain limiting amounts of these amino acids. One approach for raising the level of essential amino acids is based on altering the regulation of their biosynthetic pathways in transgenic plants. Here we describe the first production of a transgenic forage plant, alfalfa (Medicago sativa L.) with modified regulation of the aspartate-family amino acid biosynthetic pathway. This was achieved by over-expressing the Escherichia coli feedback-insensitive aspartate kinase (AK) in transgenic plants. These plants showed enhanced levels of both free and protein-bound threonine. In many transgenic plants the rise in free threonine was accompanied by a significant reduction both in aspartate and in glutamate. Our data suggest that in alfalfa, AK might not be the only limiting factor for threonine biosynthesis, and that the free threonine pool in this plant limits its incorporation into plant proteins.

Aspartate Kinase↗

PubMedDate not supplied

Degradation of benzo[a]pyrene in soil with arbuscular mycorrhizal alfalfa.

Mycorrhizal and non-mycorrhizal alfalfa (Medicago sativa) was grown in pots containing soil artificially contaminated with various levels of benzo[a]pyrene (B[a]P) (0, 1, 10 and 100 mg kg(-1)). Soil and plants were sampled after 30, 40, 50, 60 and 90 days and compared with unlanted pots. The percentage of mycorrhizal root length colonized by Glomus caledoniun was not significantly affected by the addition of B[a]P up to 10 mg kg(-1) but was significantly lower at 100 mg kg(-1)B[a]P compared with low concentrations (p < 0.05). There was no difference in soil polyphenol oxidase and dehydrogenase activity among the controls and applications of 1 and 10 mg kg(-1) of B[a]P. However, enzyme activities were significantly higher at 100 mg kg(-1) B[a]P compared with the other three treatments, and there was no mycorrhizal effect. Over a period of 90 days the concentration of B[a]P in soil in which alfalfa was grown was significantly lower than in unplanted soil (p < 0.05). Degradation rates of B[a]P added at 1, 10 and 100 mg kg(-1) without G. caledonium were 76, 78 and 53%, and with mycorrhizal inoculation were 86, 87 and 57%. The degradation rate in unplanted soil was significantly lower than in planted soil, and was significantly higher in medium- and low-B[a]P treatments than in the high B[a]P concentration tested. There is a possibility of enhancement phytoremediation of PAHs in rhizosphere soil with arbuscular mycorrhizal fungi.

Benzo(a)pyrene↗

PubMed1997

Analyses of phosphoenolpyruvate carboxylase gene structure and expression in alfalfa nodules.

Phosphoenolpyruvate carboxylase (PEPC) plays a crucial role in the assimilation of CO2 during symbiotic N2 fixation in legume root nodules. In this study, an alfalfa PEPC gene (PEPC-7), whose transcripts are found at elevated levels in nodules relative to either leaves or roots, has been isolated and characterized. The intron/exon structure of this gene is identical to that of most other plant PEPC genes except for the presence of an additional intron in the 5' untranslated region. In situ RNA hybridization studies showed that PEPC transcripts were present in the nodule meristem, the infection zone, the nitrogen-fixing zone, and the parenchyma. PEPC transcripts were also found in vascular tissue of roots and nodules and in the pulvinus of petioles. In transgenic alfalfa, a chimeric reporter gene was expressed in these same regions except that little expression was found in the nodule meristem. Analyses of promoter deletions suggest that the region between -634 and -536 is of particular importance in directing transcriptional activity to the infected zone of nodules. Within this region is a mirror repeat sequence that is potentially capable of forming an H-DNA structure. These results indicate that PEPC-7 has a central role in nitrogen-fixing nodules and that regulation of transcription is an important determinant of its activity.

Base Sequence↗

PubMed2001

Substrate preferences of O-methyltransferases in alfalfa suggest new pathways for 3-O-methylation of monolignols.

Measurement of relative O-methyltransferase activities against all potential substrates in the monolignol pathway in developing alfalfa stem extracts revealed activities in the order: caffeoyl CoA > caffeoyl alcohol > 5-hydroxyferulic acid > caffeoyl aldehyde > 5-hydroxyconiferyl alcohol > 5-hydroxyferuloyl CoA > 5-hydroxyconiferaldehyde > caffeic acid. Maxima for all activities occurred in the seventh internode. In stem extracts from transgenic alfalfa with antisense downregulated caffeoyl CoA O-methyltransferase (CCoAOMT), activities with all substrates except for the two coenzyme A esters were unaffected. In contrast, downregulation of caffeic acid O-methyltransferase (COMT) reduced activities against the non-esterifed substrates in the order: 5-hydroxyconiferyl alcohol > 5-hydroxyferulic acid and caffeoyl alcohol > caffeoyl aldehyde > caffeic acid > 5-hydroxyconiferaldehyde. Recombinant COMT expressed in Escherichia coli exhibited the highest V(max)/K(m) values with 5-hydroxyconiferaldehyde and caffeoyl aldehyde, and the lowest with caffeic acid. These results indicate that COMT is unlikely to methylate caffeic acid during lignin biosynthesis in vivo, and provide enzymatic evidence for an alternative pathway to monolignols involving methylation of caffeoyl aldehyde and/or caffeoyl alcohol by COMT. The concept of independent pathways to guaiacyl and syringyl monolignols is discussed.

Catechol O-Methyltransferase↗

PubMed2002

Genotype-dependent transcriptional activation of novel repetitive elements during cold acclimation of alfalfa (Medicago sativa).

In a search for cold-regulated genes that are differentially expressed in alfalfa genotypes of contrasting freezing tolerance, we screened 1036 arrayed cDNA clones. The screening resulted in isolation of cDNA clones, which demonstrated dramatic differences in expression between hardy and un-hardy alfalfa varieties. Detailed analysis revealed that these cDNAs represent parts of novel non-coding repetitive elements carrying long-terminal repeats (LTR) and other retroelement-like features. Despite strong expression under low temperatures, DNA templates remained highly methylated, and a drug-induced decrease in methylation did not activate transcription under normal temperatures. We identified that these repetitive elements represent a large family and could insert into, or be adjacent to, the unrelated polyprotein sequences of putative retrotransposons. These retrotransposons also showed low temperature-induced transcriptional activation; however, this activation was not genotype-dependent. The retroelements described in this study are the first retroelement characterized in the Medicago genus. Furthermore, they represent the only known example of genotype-specific cold-induced transcriptional activation of multiple copies of a repetitive element whose expression is associated with a genotype difference in cold acclimation.

Acclimatization↗

PubMed2003

Antisense inhibition of NADH glutamate synthase impairs carbon/nitrogen assimilation in nodules of alfalfa (Medicago sativa L.).

Legumes acquire significant amounts of nitrogen for growth from symbiotic nitrogen fixation. The glutamine synthetase (GS)/NADH-dependent glutamate synthase (NADH-GOGAT) cycle catalyzes initial nitrogen assimilation. This report describes the impact of specifically reducing nodule NADH-GOGAT activity on symbiotic performance of alfalfa (Medicago sativa L.). Four independent transgenic alfalfa lines, designated GA89, GA87, GA88, and GA82 (for GOGATantisense), containing an antisense NADH-GOGAT cDNA fragment under the control of the soybean leghemoglobin (lbc3) promoter were evaluated. The GA plants were fertile and showed normal growth in non-symbiotic conditions. The NADH-GOGAT antisense transgene was heritable and the T1 plants showed phenotypic alterations - similar to primary transformants. Clonally propagated plants were inoculated with Sinorhizobium meliloti after rooting and the symbiotic phenotype was analyzed 21 days post-inoculation. Nodules of each GA line had reduced NADH-GOGAT activity, ranging from 33 to 87% of control plants, that was accompanied by comparable decreases in RNA and protein. Plants from the GA89 line, with the lowest NADH-GOGAT activity (c. 30%), presented a strikingly altered symbiotic phenotype: concomitantly activities of key enzyme for carbon and nitrogen assimilation decreased; nodule amino acids and amides were reduced while sucrose accumulated. Antisense GOGAT plants were chlorotic, reduced in fresh weight, and had a lower N content than control plants. Photosynthesis was also impaired in antisense plants. Specifically, reducing NADH-GOGAT in nodules resulted in plants having impaired nitrogen assimilation and altered carbon/nitrogen metabolic flux.

Amino Acid Oxidoreductases↗