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Nucleotide sequence and structural determinants of specific binding of coat protein or coat protein peptides to the 3' untranslated region of alfalfa mosaic virus RNA 4.

The specific binding of alfalfa mosaic virus coat protein to viral RNA requires determinants in the 3' untranslated region (UTR). Coat protein and peptide binding sites in the 3' UTR of alfalfa mosaic virus RNA 4 have been analyzed by hydroxyl radical footprinting, deletion mapping, and site-directed mutagenesis experiments. The 3' UTR has several stable hairpins that are flanked by single-stranded (A/U)UGC sequences. Hydroxyl radical footprinting data show that five sites in the 3' UTR of alfalfa mosaic virus RNA 4 are protected by coat protein, and four of the five protected regions contain AUGC or UUGC. Electrophoretic mobility band shift results suggest four coat protein binding sites in the 3' UTR. A 3'-terminal 39-nucleotide RNA fragment containing four AUGC repeats bound coat protein and coat protein peptides with high affinity; however, coat protein bound poorly to antisense 3' UTR transcripts and poly(AUGC)10. Site-directed mutagenesis of AUGC865-868 resulted in a loss of coat protein binding and peptide binding by the RNA fragment. Alignment of alfalfa mosaic RNA sequences with those from several closely related ilarviruses demonstrates that AUGC865-868 is perfectly conserved; moreover, the RNAs are predicted to form similar 3'-terminal secondary structures. The data strongly suggest that alfalfa mosaic virus coat protein and ilavirus coat proteins recognize invariant AUGC sequences in the context of conserved structural elements.

Alfalfa mosaic virus↗

Multinational outbreak of Salmonella enterica serotype Newport infections due to contaminated alfalfa sprouts.

CONTEXT: In December 1995, reported Salmonella enterica serotype Newport (SN) infections increased sharply in Oregon and British Columbia but not elsewhere in North America. Similar unexplained increases had been noted in 6 other states in the fall of 1995. OBJECTIVE: To determine the source of the outbreak(s). DESIGN: Case-control studies, environmental investigations, bacterial subtyping, and surveillance information review. SETTINGS: Oregon and British Columbia communities (winter 1995-1996) and Georgia, Oklahoma, Pennsylvania, Vermont, Virginia, and West Virginia (fall 1995). PARTICIPANTS: Oregon and British Columbia residents with culture-confirmed SN infections and onset from December 1, 1995, through February 29, 1996, and healthy community controls. MAIN OUTCOME MEASURES: Odds ratio (OR) of illness associated with exposures; distribution patterns and culture of alfalfa seeds and sprouts; subtyping of SN isolates. RESULTS: We identified 133 cases in Oregon and British Columbia; 124 (93%) occurred in patients older than 18 years; 87 (65%) were female. Case patients were more likely than community control subjects to report having eaten alfalfa sprouts in the 5 days preceding illness (41% [17/41] vs 4% [3/75]; OR, 17.0; 95% confidence interval, 4.3-96.0). Case isolates shared a distinctive pulsed-field gel electrophoresis (PFGE) pattern. The SN was grown from seeds and alfalfa sprouts. The distribution of 1 seed lot to multiple growers corresponded to the distribution of cases. Distribution of a second seed lot from the same European wholesaler corresponded to the location of the fall outbreak, which was characterized by a similar demographic profile. The PFGE pattern of fall outbreak isolates and confiscated sprouts and seeds was indistinguishable from the Oregon and British Columbia outbreak and differed from background isolates. CONCLUSIONS: The SN-contaminated alfalfa seeds were distributed to multiple growers across North America in 1995 and resulted in a protracted international outbreak scattered over many months. Current sprouting methods are inadequate to protect consumers from such events. Alfalfa sprouts may be an elusive but important vehicle for salmonellosis and other enteric infections.

Case-Control Studies↗

Cloned and expressed fungal phyA gene in alfalfa produces a stable phytase.

The phyA gene from Aspergillus ficuum that codes for a 441-amino-acid full-length phosphomonoesterase (phytase) was cloned and expressed in Medicago sativa (alfalfa) leaves. The expressed enzyme from alfalfa leaves was purified to homogeneity and biochemically characterized, and its catalytic properties were elucidated. The expressed phytase in alfalfa leaves retained all the biochemical properties of the benchmark A. ficuum phytase. Although the characteristic bi-hump pH optima were retained in the cloned phytase, the optimal pH shifted downward from 5.5 to 5.0. Also, the recombinant phytase was inhibited by the pseudo-substrate myo-inositol hexasulfate and also by antibody raised against a 20-mer peptide belonging to fungal phytase. The expressed phytase in alfalfa could also be modified by phenylglyoxal. Taken together, the results indicate that fungal phytase when cloned and expressed in alfalfa leaves produces stable and catalytically active phytase while retaining all the properties of the benchmark phytase. This affirms our view that "molecular biofarming" could be an alternative means of producing stable hydrolytic enzymes such as phytase.

6-Phytase↗

Alfalfa heat shock genes are differentially expressed during somatic embryogenesis.

We have isolated two cDNA clones (Mshsp18-1; Mshsp18-2) from alfalfa (Medicago sativa L.) which encode for small heat shock proteins (HSPs) belonging to the hsp17 subfamily. The predicted amino acid sequences of the two alfalfa proteins are 92% identical and a similar degree of homology (90%) can be detected between Mshsp18-2 and the pea hsp17. In comparison to various members of small HSPs from soybean amino acid sequence similarities of 80-86% were identified. The alfalfa HSPs share a homologous stretch of amino acids in the carboxy terminal region with hsp22, 23, 26 from Drosophila. This region contains the GVLTV motif which is characteristic of several members of small HSPs. At room temperature alfalfa hsp18 mRNAs were not detectable in root and leaf tissues but northern analysis showed a low level of expression in microcallus suspension (MCS). The transcription of Mshsp18 genes is induced by elevated temperature, CdCl2 treatment and osmotic shock in cultured cells. In alfalfa somatic embryos derived from MCS a considerable amount of hsp18 mRNA can be detected during the early embryogenic stages under normal culture conditions. The differential expression of these genes during embryo development suggests a specific functional role for HSPs in plant cells at the time of the developmental switch in vitro.

Amino Acid Sequence↗

Stress responses in alfalfa (Medicago sativa L.) 12. Sequence analysis of phenylalanine ammonia-lyase (PAL) cDNA clones and appearance of PAL transcripts in elicitor-treated cell cultures and developing plants.

An expression library containing cDNAs derived from transcripts from fungal elicitor-treated alfalfa cell suspension cultures was screened with an antiserum raised against phenylalanine ammonia-lyase (PAL) from alfalfa. A single immunoreactive clone was isolated which encoded a full-length PAL cDNA (APAL1) consisting of a 2175 bp open reading frame, 96 bp 5'-untranslated leader and 128 bp 3'-non-coding region. The deduced amino acid sequence was 86.5% similar to that of the PAL2 gene of bean, and encoded a polypeptide of Mr 78,865. A second PAL cDNA species was isolated, whose 3'-untranslated region was 86% identical to that of APAL1. Southern blot analysis indicated that PAL is encoded by a small multigene family in alfalfa. PAL transcript levels were rapidly and massively induced, and preceded increased PAL extractable activity, on exposure of alfalfa suspension cells to elicitor from baker's yeast. PAL transcripts were most abundant in roots, stems and petioles during growth and development of alfalfa seedlings. These studies provide the basis for an examination of the developmental and environmental control of a key enzyme of phenylpropanoid synthesis in a plant species which is readily amenable to stable genetic transformation.

Amino Acid Sequence↗

Use of NIRS technology with a remote reflectance fibre-optic probe for predicting mineral composition (Ca, K, P, Fe, Mn, Na, Zn), protein and moisture in alfalfa.

In the present work we study the use of near-infrared spectroscopy (NIRS) technology together with a remote reflectance fibre-optic probe for the analysis of major (Ca, K, P) and minor (Fe, Mn, Na, Zn) elements, protein and moisture in alfalfa. The method allows immediate analysis of the alfalfa without prior sample treatment or destruction through direct application of the fibre-optic probe on ground samples in the case of the mineral composition and on-ground and compacted (baled) samples in the case of protein and humidity. The regression method employed was modified partial least-squares (MPLS). The calibration results obtained using samples of alfalfa allowed the determination of Ca, K, P, Fe, Mn, Na and Zn, with a standard error of prediction (SEP(C)) and a correlation coefficient (RSQ) expressed in mg/kg of alfalfa of 1.37x10(3) and 0.878 for Ca, 1.10x10(3) and 0.899 for K, 227 and 0.909 for P, 103 and 0.948 for Fe, 5.1 and 0.843 for Mn, 86.2 and 0.979 for Na, and of 1.9 and 0.853 for Zn, respectively. The SEP(C) and RSQ values (in %) for protein and moisture in ground samples were 0.548 and 0.871 and 0.150 and 0.981, respectively; while in the compacted samples they were 0.564 and 0.826 and 0.262 and 0.935, respectively. The prediction capacity of the model and the robustness of the method were checked in the external validation in alfalfa samples of unknown composition, and the results confirmed the suitability of the method.

Calibration↗

Molecular cloning and expression of alfalfa (Medicago sativa L.) vestitone reductase, the penultimate enzyme in medicarpin biosynthesis.

Medicarpin, the major phytoalexin in alfalfa, is synthesized by way of the isoflavonoid branch of phenylpropanoid metabolism. One of the final steps of medicarpin biosynthesis, from vestitone to 7,2'-dihydroxy-4'-methoxyisoflavanol, is catalyzed by vestitone reductase. A 1245-bp cDNA clone which encodes vestitone reductase was identified utilizing internal amino acid sequence of purified vestitone reductase. When expressed in Escherichia coli, the cloned enzyme exhibits strict substrate stereospecificity for (3R)-vestitone, as was observed for vestitone reductase purified from alfalfa. The calculated molecular weight of the protein (35,918) is similar to that of purified vestitone reductase from alfalfa (38 kDa by SDS-PAGE). The levels of vestitone reductase transcript (1.35 kb) greatly increase within 2 h of elicitor addition to alfalfa cell suspension cultures, preceding the rapid increases in vestitione reductase enzyme activity and medicarpin biosynthesis. In healthy alfalfa plants, the highest levels of transcripts were detected in roots and root nodules, consistent with the synthesis of medicarpin and its conjugate in these tissues. The cloning of the vestitone reductase gene provides a specific tool for the study and manipulation of pterocarpan biosynthesis in legumes.

Amino Acid Sequence↗

Alfalfa seeds lower low density lipoprotein cholesterol and apolipoprotein B concentrations in patients with type II hyperlipoproteinemia.

Fifteen patients with hyperlipoproteinemia (HLP), types IIA (n = 8), IIB (n = 3) and IV (n = 4) were given 40 g of heat prepared alfalfa seeds 3 times daily at mealtimes for 8 weeks with otherwise unchanged diet. In patients with type II HLP alfalfa treatment caused after 8 weeks a maximal lowering of pretreatment median values of total plasma cholesterol from 9.58 to 8.00 mmol/l (P less than 0.001) and low density lipoprotein (LDL) cholesterol from 7.69 to 6.33 mmol/l (P less than 0.01), which corresponds to decreases of 17% and 18%, respectively. Maximal decrease was 26% in total cholesterol and 30% in LDL cholesterol. In two patients with hypercholesterolemia the LDL cholesterol decreased less than 5%. Apolipoprotein B decreased in the same period from 2.17 to 1.43 g/l (P less than 0.05) in type II HLP, corresponding to 34% decrease, whereas apolipoprotein A-I did not change. Body weight increased slightly during the first 4 weeks of alfalfa treatment (P less than 0.001) probably because of the caloric content in the alfalfa seeds. After cessation of treatment, all lipoprotein concentrations returned to pretreatment levels. We conclude that alfalfa seeds can be added to the diet to help normalize serum cholesterol concentrations in patients with type II HLP.

Adult↗

Ozone effects on growth and assimilate partitioning in alfalfa, Medicago sativa L.

Alfalfa (Medicago sativa L.) were exposed to O(3) concentrations varying between 118 x 10(-6) microg cm(-3) (0.06 ppm) and 157 x 10(-6) microg cm(-3) (0.08 ppm) for 6 h per day 5 days per week for several weeks. Typical plants were sacrificed weekly, and growth parameters were measured. O(3) reduced overall growth, relative growth rates and unit leaf rates in alfalfa before it was cut, indicating that O(3) had reduced photosynthesis. However, after the alfalfa was cut, these same parameters indicated that in some cases, O(3)-stressed plants had greater photosynthetic rates than controls during regrowth. O(3) also altered dry matter partitioning. Roots were most affected, followed by leaves and stems, respectively. In general, O(3) reduced photosynthate production and reduced the proportion of photosynthate partitioned to roots relative to leaves and stems. This could reduce starch reserves in alfalfa, and be detrimental to stand longevity. However, the post-cutting study indicated that at least some alfalfa cultivars may be able to acclimate to O(3)-stress, though plants did not fully recover from pre-cutting differences.

Journal Article↗

The de novo designed nutritive protein MB-1Trp does not resist proteolytic degradation in alfalfa leaves.

We previously reported on a de novo designed protein "milk bundle-1Trp" (MB-1Trp) as a source of selected essential amino acids (EAA) for ruminant feeding. Here, we attempt to express this de novo designed protein in alfalfa. The microbial version of the gene encoding the protein was modified in order to achieve two expression strategies in transgenic alfalfa plants. Chimeric MB-1Trp genes alone or fused to a signal peptide and an endoplasmic reticulum retention sequence were introduced into alfalfa via Agrobacterium-mediated transformation. Polymerase chain reaction and reverse transcriptase polymerase chain reaction analysis performed on individual transgenic lines demonstrated that the MB-1Trp gene was correctly integrated and transcribed into mRNA. However, under our conditions, it was impossible to detect MB-1Trp protein expression in any of the transgenic plants analyzed. In order to assess MB-1Trp stability in alfalfa, Escherichia coli-derived MB-1Trp was incubated with proteins extracted from leaves of a non-transgenic plant. This study revealed a high susceptibility of mature MB-1Trp to alfalfa proteases, which may have contributed to its lack of accumulation.

Amino Acids, Essential↗

Removal of azinphos methyl by alfalfa plants (Medicago sativa L.) in a soil-free system.

The objective of this study was to investigate the removal of azinphos methyl assisted by alfalfa plants, with special emphasis on the effects of this compound on some plant's physiological parameters. Hydroponic cultures of alfalfa (Medicago sativa L., var Romagnola) were employed as a model system. These cultures were exposed to a nutrient medium containing 10 mg/l of azinphos methyl. A first-order kinetic approach was used to describe the removal of azinphos methyl from the solution. After 20 days of culture, the initial amount of azinphos methyl was reduced to non-detectable levels in the presence of plants. In the absence of plants, 20% of azinphos methyl remained in the solution after 30 days of treatment. The half-life of the pesticide was reduced from 10.8 to 3.4 days in the presence of plants. The growth index of alfalfa plants exposed to azinphos methyl was negatively affected. Chlorophyll contents were reduced after 24 h of treatment and thereafter the levels were comparable to that of control plants. The peroxidase activity of alfalfa roots was not affected by the presence of azinphos methyl. In conclusion, alfalfa plants were able to survive when exposed to an effective concentration of 10 mg/l of azinphos methyl in the root zone, with some alterations on their physiological parameters.

Azinphosmethyl↗

Enzymic O-methylation of isoliquiritigenin and licodione in alfalfa and licorice cultures.

S-Adenosyl-L-methionine (SAM): isoliquiritigenin (2',4,4'-trihydroxychalcone) 2'-O-methyltransferase (CHMT) of alfalfa (Medicago sativa) catalyses the formation of 4,4'-dihydroxy-2'-methoxychalcone, which is the most potent inducer of nodulation-genes of Rhizobium meliloti, the symbiont of alfalfa which forms nitrogen-fixing nodules. SAM: licodione 2'-O-methyltransferase (LMT) is involved in the biosynthesis of a retrochalcone in cultured licorice (Glycyrrhiza echinata) cells and has been shown to be induced as a defence response of the cells. Because licodione exists in an equilibrium mixture of tautomeric 2',4,4',beta-tetrahydroxychalcone (major) and 1-(2,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)-1,3-propanedione (minor), the apparent mode of action of both enzymes is very similar. In this study, cultured alfalfa cells were shown to exhibit rapid and transient increases in the extractable activities of both CHMT and LMT after treatment with yeast extract (YE). Treatment of solution-cultured alfalfa seedlings with YE also resulted in a similar induction of both CHMT and LMT activities in the roots, but no activity was detected in the shoots. These activities were attributed to a single gene product, the CHMT protein, as extracts of Escherichia coli transformed with the CHMT cDNA exhibited both CHMT and LMT activities. In contrast, in G. echinata cells, LMT was induced after YE treatment, but no CHMT activity was observed. It is concluded that alfalfa CHMT and licorice LMT are distinct enzymes, the former displaying the wider substrate specificity.

Chalcone↗

Stress responses in alfalfa (Medicago sativa L). XXII. cDNA cloning and characterization of an elicitor-inducible isoflavone 7-O-methyltransferase.

Medicarpin, the major phytoalexin in alfalfa, is synthesized via the isoflavonoid branch of phenylpropanoid metabolism. The methyl group at the 9 position of medicarpin is generally accepted to arise via the methylation of the 4' position (B-ring) of daidzein. Surprisingly, the isoflavone-O-methyltransferase (IOMT), which is induced along with other enzymes involved in medicarpin biosynthesis, methylates the A-ring 7-hydroxyl group of daidzein in vitro, a reaction that probably does not occur in vivo. Utilizing internal amino acid sequence information from purified alfalfa IOMT, we have isolated three full-length IOMT cDNA clones. A search of the protein databases revealed sequence similarities to O-methyltransferases from various sources. The highest match (50.5% identity) was found between IOMT8 and 6a-hydroxymaackiain 3-O-methyltransferase from Pisum sativum. The molecular weight of alfalfa IOMT expressed in Escherichia coli was similar to that of purified IOMT from alfalfa cell cultures (41 kDa by SDS-PAGE). The recombinant enzyme catalyzed the O-methylation of A-ring hydroxyl group(s) of isoflavones, and could also methylate the pterocarpan (+) 6a-hydroxymaackiain. Alfalfa contains multiple IOMT genes, and closely related sequences are present in the genomes of chickpea and cowpea, species that also produce B-ring methylated isoflavonoids in vivo. Northern blot analysis indicated that IOMT transcripts are rapidly induced following elicitation, prior to the increase in IOMT activity and medicarpin accumulation. The possible role of the isoflavone 7-OMT in the synthesis of formononetin in vivo is discussed.

Amino Acid Sequence↗

Down-regulation of specific members of the glutamine synthetase gene family in alfalfa by antisense RNA technology.

Glutamine synthetase (GS) catalyzes the ATP-dependent condensation of NH3 with glutamate to produce glutamine. In plants GS is an octameric enzyme and is located either in the cytoplasm (GS1) or in the chloroplast (GS2). Two distinct classes of GS1 genes with unique 3'-untranslated region (3'UTR) have been identified in alfalfa. We have demonstrated that the two classes exhibit differential expression pattern in the different plant organs suggesting different functional roles for the different isozymes. To determine the functional significance of the two classes of GS1 genes in alfalfa, we have utilized antisense gene constructs aimed specifically at the 3'UTR of the two GS1 genes and introduced them individually into alfalfa. Our data show that the gene constructs are effective in lowering the corresponding transcript level very effectively though there were organ-specific differences in the level of reduction. No transcript corresponding to the antisense gene construct was detected in any of the alfalfa transformants though they accumulated to significant levels in transgenic tobacco containing the same construct. This suggests that the antisense transcript was not stable in the presence of the homologous target sequence. Transgenic alfalfa with up to 80% reduction in the transcript level corresponding to each gene class, however, showed no reduction in GS activity or GS1 polypeptide level. The results suggest that GS1 mRNA levels are not rate-limiting for GS1 polypeptide synthesis and that GS levels are controlled both at the transcriptional and translational/post-translational level.

DNA, Recombinant↗

Combined effects of elevated CO2 and herbivore damage on alfalfa and cotton.

We examined herbivore-induced responses of alfalfa (Medicago sativa) and cotton (Gossypium hirsutum) under different CO2 conditions. Plants were grown under ambient (350 ppm) or elevated (700 ppm) CO2 levels, and were either damaged or undamaged by Spodoptera littoralis larvae. At harvest, growth of undamaged (control) plants was determined, and foliar chemical composition of both undamaged and damaged plants was analyzed. Cotton grew faster overall and showed a greater increase in growth in response to CO2 enrichment than did alfalfa. Elevated CO2 levels increased starch and decreased nitrogen levels in damaged alfalfa and undamaged cotton plants. Alfalfa saponin levels were significantly increased by elevated CO2 and damage. Regarding specific saponins, medicagenic acid bidesmoside (3GlcA,28AraRhaXyl medicagenate) concentrations were reduced by high CO2, whereas zanhic acid tridesmoside (3GlcGlcGlc,23Ara,28AraRhaXylApi Za) levels were unaffected by the treatments. Soyasaponin I (3GlcAGalRha soyasapogenol B) was only detected in minute amounts. Alfalfa flavonoid analyses showed that total flavonoid levels were similar between treatments, although free apigenin increased and apigenin glucoside (7-O-[2-O-feruloyl-beta-D-glucuronopyranozyl (1-->2)-O-8-D-glucuronopyranozyl]-4'-O-beta-D-glucuronopyranozide apigenin) decreased in CO2-enriched plants. In cotton, herbivore damage increased levels of total terpenoid aldehydes, gossypol, hemigossypolone, the heliocides H1 and H4, but not H2 and H3, whereas CO2 enrichment had no effect. These results demonstrate that combined effects of CO2 and herbivore damage vary between plant species, which has implications for the competitive balance within plant communities.

Animals↗

A comparison of constitutive promoters for expression of transgenes in alfalfa (Medicago sativa).

The activity of constitutive promoters was compared in transgenic alfalfa plants using two marker genes. Three promoters, the 35S promoter from cauliflower mosaic virus (CaMV), the cassava vein mosaic virus (CsVMV) promoter, and the sugarcane bacilliform badnavirus (ScBV) promoter were each fused to the beta-glucuronidase (gusA) gene. The highest GUS enzyme activity was obtained using the CsVMV promoter and all alfalfa cells assayed by in situ staining had high levels of enzyme activity. The 35S promoter was expressed in leaves, roots, and stems at moderate levels, but the promoter was not active in stem pith cells, root cortical cells, or in the symbiotic zones of nodules. The ScBV promoter was active primarily in vascular tissues throughout the plant. In leaves, GUS activity driven by the CsVMV promoter was approximately 24-fold greater than the activity from the 35S promoter and 38-fold greater than the activity from the ScBV promoter. Five promoters, the double 35S promoter, figwort mosaic virus (FMV) promoter, CsVMV promoter, ScBV promoter, and alfalfa small subunit Rubisco (RbcS) promoter were used to control expression of a cDNA from Trichoderma atroviride encoding an endochitinase (ech42). Highest chitinase activity in leaves, roots, and root nodules was obtained in plants containing the CsVMV:ech42 transgene. Plants expressing the endochitinase were challenged with Phoma medicaginis var. medicaginis, the causal agent of spring black stem and leaf spot of alfalfa. Although endochitinase activity in leaves of transgenic plants was 50- to 2650-fold greater than activity in control plants, none of the transgenic plants showed a consistent increase in disease resistance compared to controls. The high constitutive levels of both GUS and endochitinase activity obtained demonstrate that the CsVMV promoter is useful for high-level transgene expression in alfalfa.

Badnavirus↗

The development of virus-resistant alfalfa, Medicago sativa L.

We have generated more than 100 transgenic alfalfa plants, via Agrobacterium-mediated gene transfer, from genotypes selected from five alfalfa cultivars. These plants express the genes for kanamycin resistance and for the coat protein of alfalfa mosaic virus (AMV). The strongest expressers accumulated nearly 500 ng coat protein per mg soluble leaf protein. AMV inoculation of protoplasts from these strong expressers indicated that they were resistant to infection by AMV, while protoplasts from plants containing about a hundred-fold less coat protein and from control untransformed plants were not. Transgenic alfalfa plants containing large amounts of coat protein were, likewise, resistant to AMV. These plants did not develop systemic infections following inoculation with up to 50 micrograms/ml AMV, while inoculated control plants developed systemic infections following inoculation with as little as 10 micrograms/ml AMV. These results demonstrate that expression of the AMV coat protein gene confers resistance to AMV infection in transgenic alfalfa plants.

Capsid↗

Extractable copper, arsenic and antimony by EDTA solution from agricultural Chilean soils and its transfer to alfalfa plants (Medicago sativa L.).

Following our research on copper, arsenic and antimony in Chilean ecosystems, a study to understand the mobility and transport of these elements from soil to plants was carried out. So, the aim of this study, which follows on from the previous work, was to demonstrate if the total concentrations of these elements or their fractions extracted by 0.05 M EDTA pH 7 from different Chilean soils correlate with the respective total concentrations in the edible tissue of alfalfa plants collected simultaneously from 20 different sites affected or unaffected by mining activities. The highest copper fractions extracted by EDTA solutions were obtained in contaminated soils from the central region (41-69%); however the northern soils presented the highest extractable fractions of arsenic (9-34%). The antimony fraction was low in all soils (0.4-8.0%). Alfalfa plants from all contaminated sites presented high copper, arsenic and antimony concentrations (19-126 mg kg(-1), 5.7-16.3 mg kg(-1) and 0.16-1.7 mg kg(-1), respectively). Statistically significant correlations were obtained between the total contents of copper and arsenic and their respective extractable fractions in soils. Good correlations were found between elements in alfalfa plants. Correlations were also obtained between the total concentrations of three elements in soils and in alfalfa plants. However, excepting for antimony in the northern samples, higher correlation coefficients were evaluated when the extractable fractions were considered. Samples from the north region presented the highest copper transfer factor and the lowest for arsenic, in spite of the high concentration of this metalloid extracted by EDTA solution in these soils. There was not a clear trend on the transfer factor for antimony, probably due to the low content of this element in alfalfa plants and/or the low recovery obtained for this element by EDTA.

Agriculture↗