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PubMed2001

Early symbiotic responses induced by Sinorhizobium meliloti iIvC mutants in alfalfa.

A mutation in the ilvC gene of Sinorhizobium meliloti 1021 determines a symbiotically defective phenotype. ilvC mutants obtained from different S. meliloti wild-type strains are able to induce root hair deformation on alfalfa roots and show variable activation of the common nodulation genes nodABC. All of these mutants are noninfective. The presence of extra copies of nodD3-syrM in an IlvC- background does not promote nod expression but allows the detection of low levels of Nod factor production. The sulphation of the Nod factor metabolites, however, is not affected. Furthermore, IlvC- strains induce a specific pattern of starch accumulation on alfalfa roots as well as of early nodulin expression. Hence, the pleiotropic action of the ilvC gene in S. meliloti may reveal novel complexities involved in the symbiotic interaction.

Alcohol Oxidoreductases↗
PubMed1991

Acidic conditions permit effective nodulation of alfalfa by invasion-deficient Rhizobium meliloti exoD mutants.

Rhizobium meliloti exoD mutants are deficient in invasion of alfalfa nodules and, as a consequence, the nodules that exoD strains induce fail to fix nitrogen. These nodules appear to be arrested at the same stage as nodules induced by other exo mutants, which do not make an acidic exopolysaccharide called EPS I, or by ndv mutants, which do not produce a periplasmic cyclic beta(1,2) glucan. However previous genetic and biochemical evidence suggested that the nodule invasion defect of exoD mutants arose from a biochemical deficiency distinct from those of both EPS I-deficient exo mutants and ndv mutants. In this study, we characterize mutant phenotypes of exoD strains in both free-living and symbiotic states. Nodules induced by exoD mutants are generally small and empty of bacteria, and exhibit the same structural features as nodules induced by other invasion-deficient mutants. Putative incipient infection threads were visible in outer cortical cells of these nodules but not in the plant cells in the interior of the nodule. We show that exoD mutants are sensitive to alkaline conditions, ceasing to grow at elevated pH in liquid yeast extract cultures and exhibiting decreased viability in alkaline medium. Interestingly, we find that buffering the plant growth medium at slightly acidic pH (6.0-6.5) restores the ability of exoD mutants to invade alfalfa nodules. exoD mutants are thus alkali sensitive for both free-living and symbiotic phenotypes. This result implies that the nodule invasion defect of exoD mutants arises from their sensitivity to alkaline conditions and, furthermore, that alkaline conditions may obtain in the developing infection thread. The deduced amino acid sequence of ExoD is extremely hydrophobic, suggesting that the protein is membrane associated. We propose models whereby absence of a putative membrane protein might lead to sensitivity to alkaline conditions and consequent arrest of nodule invasion.

Amino Acid Sequence↗
PubMed2002

A unified framework for mapping quantitative trait loci in bivalent tetraploids using single-dose restriction fragments: a case study from alfalfa.

The development of statistical methodologies for quantitative trait locus (QTL) mapping in polyploids is complicated by complex polysomic inheritance. In this article, we propose a statistical method for mapping QTL in tetraploids undergoing bivalent formation at meiosis by using single-dose restriction fragments. Our method is based on a unified framework, one that uses chromosome bivalent pairing configuration and gametic recombination to discern different mechanisms of gamete formation. Our bivalent polyploid model can not only provide a simultaneous estimation of the linkage and chromosome pairing configuration-a cytological parameter of evolutionary and systematic interest-but also enhances the precision of estimating QTL effects and position by correctly characterizing gene segregation during polyploid meiosis. By using our method and a linkage map constructed in a previous study, we successfully identify several QTL affecting winter hardiness in bivalent tetraploid alfalfa. Moreover, our results reveal significant preferential chromosome pairing at meiosis in an F1 hybrid population, which indicates the importance of reassessing the traditional view of random chromosome segregation in alfalfa.

Chromosome Pairing↗
PubMed1993

Superoxide dismutase enhances tolerance of freezing stress in transgenic alfalfa (Medicago sativa L.).

Activated oxygen or oxygen free radicals have been implicated in a number of physiological disorders in plants including freezing injury. Superoxide dismutase (SOD) catalyzes the dismutation of superoxide into O2 and H2O2 and thereby reduces the titer of activated oxygen molecules in the cell. To further examine the relationship between oxidative and freezing stresses, the expression of SOD was modified in transgenic alfalfa (Medicago sativa L.). The Mn-SOD cDNA from Nicotiana plumbaginifolia under the control of the cauliflower mosaic virus 35S promoter was introduced into alfalfa using Agrobacterium tumefaciens-mediated transformation. Two plasmid vectors, pMitSOD and pChlSOD, contained a chimeric Mn-SOD construct with a transit peptide for targeting to the mitochondria or one for targeting to the chloroplast, respectively. The putatively transgenic plants were selected for resistance to kanamycin and screened for neomycin phosphotransferase activity and the presence of an additional Mn-SOD isozyme. Detailed analysis of a set of four selected transformants indicated that some had enhanced SOD activity, increased tolerance to the diphenyl ether herbicide, acifluorfen, and increased regrowth after freezing stress. The F1 progeny of one line, RA3-ChlSOD-30, were analyzed by SOD isozyme activity, by polymerase chain reaction for the Mn-SOD gene, and by polymerase chain reaction for the neo gene. RA3-ChlSOD-30 had three sites of insertion of pChlSOD, but only one gave a functional Mn-SOD isozyme; the other two were apparently partial insertions. The progeny with a functional Mn-SOD transgene had more rapid regrowth following freezing stress than those progeny lacking the functional Mn-SOD transgene, suggesting that Mn-SOD serves a protective role by minimizing oxygen free radical production after freezing stress.

Base Sequence↗
PubMed2005

Aphid infestation causes different changes in carbon and nitrogen allocation in alfalfa stems as well as different inhibitions of longitudinal and radial expansion.

Alfalfa (Medicago sativa) stem elongation is strongly reduced by a pea aphid (Acyrthosiphon pisum Harris) infestation. As pea aphid is a phloem feeder that does not transmit virus or toxins, assimilate withdrawal is generally considered as the main mechanism responsible for growth reduction. Using a kinematic analysis, we investigated the spatial distributions of relative elemental growth rates of control and infested alfalfa stems. The water, carbon, and nitrogen contents per unit stem length were measured along the growth zone. Deposition rates and growth-sustaining fluxes were estimated from these patterns. Severe short-term aphid infestation (200 young adults over a 24-h period) induced a strong and synchronized reduction in rates of elongation and of water and carbon deposition. Reduced nitrogen content and associated negative nitrogen deposition rates were observed in some parts of the infested stems, especially in the apex. This suggested a mobilization of nitrogen from the apical part of the growth zone, converted from a sink tissue into a source tissue by aphids. Calculation of radial growth rates suggested that aphid infestation led to a smaller reduction in radial expansion than in elongation. Together with earlier observations of long-lasting effects of a short-term infestation, this supports the hypothesis that in addition to nutrient withdrawal, a thigmomorphogenesis-like mechanism is involved in the effect of aphid infestation on stem growth.

Animals↗
PubMed1994

A Vesicular Arbuscular Mycorrhizal Fungus (Glomus intraradix) Induces a Defense Response in Alfalfa Roots.

Flavonoid accumulation and activities of phenylalanine ammonia-lyase (PAL), chalcone isomerase (CHI), and chitinase were followed during early colonization of alfalfa roots (Medicago sativa L. cv Gilboa) by vesicular arbuscular (VA) fungi (Glomus intraradix). Formononetin was the only flavonoid detected that showed a consistent increase in the inoculated roots. This increase depended only on the presence of the fungus in the plant rhizosphere; no colonization of the root tissue was required. CHI and chitinase activities increased in inoculated roots prior to colonization, whereas the increase in PAL activity coincided with colonization. After reaching a maximum, activities of all enzymes declined to below those of uninoculated roots. PAL inactivation was not caused by a soluble inhibitor. Our results indicate that VA fungi initiate a host defense response in alfalfa roots, which is subsequently suppressed.

Journal Article↗
PubMed1994

Alfalfa Enod12 genes are differentially regulated during nodule development by Nod factors and Rhizobium invasion.

MsEnod12A and MsEnod12B are two early nodulin genes from alfalfa (Medicago sativa). Differential expression of these genes was demonstrated using a reverse transcription-polymerase chain reaction approach. MsEnod12A RNA was detected only in nodules and not in other plant tissues. In contrast, MsEnod12B transcripts were found in nodules and also at low levels in roots, flowers, stems, and leaves. MsEnod12B expression was enhanced in the root early after inoculation with the microsymbiont Rhizobium meliloti and after treatment with purified Nod factors, whereas MsEnod12A induction was detected only when developing nodules were visible. In situ hybridization showed that in nodules, MsEnod12 expression occurred in the infection zone. In empty Fix- nodules the MsEnod12A transcript level was much reduced, and in spontaneous nodules it was not detectable. These data indicate that MsEnod12B expression in roots is related to the action of Nod factors, whereas MsEnod12A expression is associated with the invasion process in nodules. Therefore, alfalfa possesses different mechanisms regulating MsEnod12A and MsEnod12B expression.

Amino Acid Sequence↗
PubMed1995

Suppression of an Isoflavonoid Phytoalexin Defense Response in Mycorrhizal Alfalfa Roots.

Isoflavonoids and steady-state mRNA levels of phenylalanine ammonia-lyase, chalcone isomerase, and isoflavone reductase were followed during a rapid, nearly synchronous infection of alfalfa (Medicago sativa L.) roots by the vesicular arbuscular fungus Glomus intraradices (Schenck & Smith) to test whether previously indicated suppression of the host defense response is regulated by changes in the steady-state mRNA level. Relative amounts of steady-state phenylalanine ammonia-lyase mRNA in the mycorrhizal roots doubled between d 14 and 18 and then immediately declined by 75% to reach and maintain a value lower than the control roots through d 21. Relative levels of chalcone isomerase mRNA in the inoculated roots increased 6-fold between d 14 and 17 and then decreased rapidly to the control level. Isoflavone reductase mRNA was not induced by mycorrhizal colonization. High-performance liquid chromatography, proton-nuclear magnetic resonance, and fast atom bombardment-mass spectrometry analyses showed consistent increases in formononetin levels and transient increases in medicarpin-3-O-glycoside and formononetin conjugates in the inoculated roots when colonization began. As colonization increased, levels of formononetin conjugates declined in mycorrhizal roots below those in uncolonized controls. Medicarpin aglycone, an alfalfa phytoalexin normally associated with pathogenic infections, was not detected at any stage. These findings supply detailed evidence that, during early colonization of plant roots by symbiotic Glomus, defense transcripts are induced and then subsequently suppressed.

Journal Article↗
PubMed1996

Water Deficit-Induced Changes in Concentrations in Proline and Some Other Amino Acids in the Phloem Sap of Alfalfa.

Changes in amino acid composition of alfalfa (Medicago sativa L.) phloem sap were studies in response to a water deficit. Sap was collected by stylectomy. As the leaf water potential ([psi]) decreased from -0.4 to -2.0 MPa, there was significant increase of the total amino acid concentration, due to that of some amino acids: proline, valine, isoleucine, leucine, glutamic acid, aspartic acid, and threonine. Asparagine concentration, which is the main amino acid assayed in the phloem sap of alfalfa (it accounts for 70% of the total content), did not vary with the plant water status. The other amino acid concentrations remained stable as [psi] varied; in particular, [gamma]-amino butyric acid concentration remained unchanged, whereas it varied in response to wounding. The more striking change in the sieve tubes was the accumulation of proline, which was observed below a [psi] threshold value of about -0.9 MPa (concentration x60 for a decrease of [psi] from -0.9 to -2.0 MPa). The role of such changes in phloem sap amino acid concentration in osmotic adjustment of growing tissues is discussed.

Journal Article↗
PubMed1996

Stress Responses in Alfalfa (XXI. Activation of Caffeic Acid 3-O-Methyltransferase and Caffeoyl Coenzyme A 3-O-Methyltransferase Genes Does Not Contribute to Changes in Metabolite Accumulation in Elicitor-Treated Cell-Suspension Cultures).

Transcription of genes encoding L-phenylalanine ammonia-lyase (PAL), the first enzyme of the phenylpropanoid pathway, and caffeic acid 3-O-methyltransferase (COMT) and caffeoyl CoA 3-O-methyltransferase (CCOMT), enzymes involved in the synthesis of lignin and wall-esterified phenolic compounds, was strongly activated in elicitor-treated cell-suspension cultures of alfalfa (Medicago sativa L.). However, consequent changes in the extractable activities of COMT and CCOMT were small to nonexistent compared with a 15- to 16-fold increase in PAL activity. Only low levels of COMT and CCOMT transcripts were reflected in the total and polysomal RNA fractions compared with PAL transcripts. Elicited cell cultures did not accumulate lignin or the products of COMT and CCOMT in the soluble and wall-esterified phenolic fractions. In one alfalfa cell line in which elicitation resulted in very high PAL activity and increased deposition of methoxyl groups in the insoluble wall fraction, there was still no change in COMT and CCOMT activities. Overall, these results indicate that the initial gene transcription events in elicited cells may be less selective than the subsequent metabolic changes, highlighting the importance of posttranscriptional events in the control of phenylpropanoid biosynthesis.

Journal Article↗
PubMed1998

Studying early nodulin gene ENOD40 expression and induction by nodulation factor and cytokinin in transgenic alfalfa.

ENOD40, an early nodulin gene, is expressed following inoculation with Rhizobium meliloti or by adding R. meliloti-produced nodulation (Nod) factors or the plant hormone cytokinin to uninoculated roots. We isolated two MsENOD40 clones, designated MsENOD40-1 and MsENOD40-2, with distinct promoters from an alfalfa (Medicago sativa cv Chief) genomic library. The promoters were fused to the reporter gene uidA (gus), and the constructs were introduced into alfalfa. We observed that the MsENOD40-1 construct was expressed almost exclusively under symbiotic conditions. The MsENOD40-2 construct was transcribed under both symbiotic and nonsymbiotic conditions and in nonnodular and nodular tissues. Both MsENOD40 promoter-gus constructs were similarly expressed as nodules developed, and both were expressed in roots treated with 6-benzylaminopurine or purified Nod factor. However, no blue color was detected in nodule-like structures induced by the auxin transport inhibitor N-1-(naphthyl)phthalamic acid on roots of plants containing the MsENOD40-1 promoter construct, whereas pseudonodules from plants containing the MsENOD40-2 promoter construct stained blue. A 616-bp region at the distal 5' end of the promoter is important for proper spatial expression of MsENOD40 in nodules and also for Nod-factor and cytokinin-induced expression.

Bacterial Proteins↗
PubMed1998

Respiratory Elicitors from Rhizobium meliloti Affect Intact Alfalfa Roots

Molecules produced by Rhizobium meliloti increase respiration of alfalfa (Medicago sativa L.) roots. Maximum respiratory increases, measured either as CO2 evolution or as O2 uptake, were elicited in roots of 3-d-old seedlings by 16 h of exposure to living or dead R. meliloti cells at densities of 10(7) bacteria/mL. Excising roots after exposure to bacteria and separating them into root-tip- and root-hair-containing segments showed that respiratory increases occurred only in the root-hair region. In such assays, CO2 production by segments with root hairs increased by as much as 100% in the presence of bacteria. Two partially purified compounds from R. meliloti 1021 increased root respiration at very low, possibly picomolar, concentrations. One factor, peak B, resembled known pathogenic elicitors because it produced a rapid (15-min), transitory increase in respiration. A second factor, peak D, was quite different because root respiration increased slowly for 8 h and was maintained at the higher level. These molecules differ from lipo-chitin oligosaccharides active in root nodulation for the following reasons: (a) they do not curl alfalfa root hairs, (b) they are synthesized by bacteria in the absence of known plant inducer molecules, and (c) they are produced by a mutant R. meliloti that does not synthesize known lipo-chitin oligosaccharides. The peak-D compound(s) may benefit both symbionts by increasing CO2, which is required for growth of R. meliloti, and possibly by increasing the energy that is available in the plant to form root nodules.

Journal Article↗
PubMed1999

Winter survival of transgenic alfalfa overexpressing superoxide dismutase

To test the hypothesis that enhanced tolerance of oxidative stress would improve winter survival, two clones of alfalfa (Medicago sativa) were transformed with a Mn-superoxide dismutase (Mn-SOD) targeted to the mitochondria or to the chloroplast. Although Mn-SOD activity increased in most primary transgenic plants, both cytosolic and chloroplastic forms of Cu/Zn-SOD had lower activity in the chloroplast SOD transgenic plants than in the nontransgenic plants. In a field trial at Elora, Ontario, Canada, the survival and yield of 33 primary transgenic and control plants were compared. After one winter most transgenic plants had higher survival rates than control plants, with some at 100%. Similarly, some independent transgenic plants had twice the herbage yield of the control plants. Prescreening the transgenic plants for SOD activity, vigor, or freezing tolerance in the greenhouse was not effective in identifying individual transgenic plants with improved field performance. Freezing injury to leaf blades and fibrous roots, measured by electrolyte leakage from greenhouse-grown acclimated plants, indicated that the most tolerant were only 1 degrees C more freezing-tolerant than alfalfa clone N4. There were no differences among transgenic and control plants for tetrazolium staining of field-grown plants at any freezing temperature. Therefore, although many of the transgenic plants had higher winter survival rates and herbage yield, there was no apparent difference in primary freezing injury, and therefore, the trait is not associated with a change in the primary site of freezing injury.

Journal Article↗
PubMed2000

Iron-superoxide dismutase expression in transgenic alfalfa increases winter survival without a detectable increase in photosynthetic oxidative stress tolerance.

To determine whether overexpression of Fe-superoxide (SOD) dismutase would increase superoxide-scavenging capacity and thereby improve the winter survival of transgenic alfalfa (Medicago sativa L.) plants, two genotypes were transformed with the vector pEXSOD10, which contains a cDNA for Arabidopsis Fe-SOD with a chloroplast transit peptide and cauliflower mosaic virus 35S promoter. A novel Fe-SOD was detected by native PAGE in both greenhouse- and field-grown transgenic plants, but activity varied among independent transgenic plants. The increased Fe-SOD activity was associated with increased winter survival over 2 years in field trials, but not with oxidative stress tolerance as measured by resistance of leaves to methyl viologen, a superoxide generator. Total shoot dry matter production over 2 harvest years was not associated with Fe-SOD activity. There was no detectable difference in the pattern of primary freezing injury, as shown by vital staining, nor was there additional accumulation of carbohydrates in field-acclimated roots of the transgenic alfalfa plants. We did not detect any difference in growth of one transgenic plant with high Fe-SOD activity compared with a non-transgenic control. Therefore, the improvement in winter survival did not appear to be a consequence of improved oxidative stress tolerance associated with photosynthesis, nor was it a consequence of a change in primary freezing injury. We suggest that Fe-SOD overexpression reduced secondary injury symptoms and thereby enhanced recovery from stresses experienced during winter.

Adaptation, Physiological↗
PubMed2000

Oxygen regulation of a nodule-located carbonic anhydrase in alfalfa.

Control of the permeability to oxygen is critical for the function of symbiotic nitrogen fixation in legume nodules. The inner cortex (IC) seems to be a primary site for this regulation. In alfalfa (Medicago sativa) nodules, expression of the Msca1 gene encoding a carbonic anhydrase (CA) was previously found to be restricted to the IC. We have now raised antibodies against recombinant Msca1 protein and used them, together with antibodies raised against potato leaf CA, to demonstrate the presence of two forms of CA in mature nodules. Each antibody recognizes a different CA isoform in nodule tissues. Immunolocalization revealed that leaf-related CAs were localized primarily in the nitrogen-fixing zone, whereas the Msca1 protein was restricted exclusively to the IC region, in indeterminate and determinate nodules. In alfalfa nodules grown at various O(2) concentrations, an inverse correlation was observed between the external oxygen pressure and Msca1 protein content in the IC, the site of the putative diffusion barrier. Thus Msca1 is a molecular target of physiological processes occurring in the IC cells involved in gas exchange in the nodule.

Blotting, Western↗
PubMed2000

How alfalfa root hairs discriminate between Nod factors and oligochitin elicitors.

Using ion-selective microelectrodes, the problem of how signals coming from symbiotic partners or from potential microbial intruders are distinguished was investigated on root hairs of alfalfa (Medicago sativa). The Nod factor, NodRm-IV(C16:2,S), was used to trigger the symbiotic signal and (GlcNAc)(8) was selected from (GlcNAc)(4-8), to elicit defense-related reactions. To both compounds, root hairs responded with initial transient depolarizations and alkalinizations, which were followed by a hyperpolarization and external acidification in the presence of (GlcNAc)(8). We propose that alfalfa recognizes tetrameric Nod factors and N-acetylchitooligosaccharides (n = 4-8) with separate perception sites: (a) (GlcNAc)(4) and (GlcNAc)(6) reduced the depolarization response to (GlcNAc)(8), but not to NodRm-IV(C16:2, S); and (b) depolarization and external alkalization were enhanced when NodRm-IV(C16:2,S) and (GlcNAc)(8) were added jointly without preincubation. We suggest further that changes in cytosolic pH and Ca(2+) are key events in the transduction, as well as in the discrimination of signals leading to symbiotic responses or defense-related reactions. To (GlcNAc)(8), cells responded with a cytosolic acidification, and they responded to NodRm-IV(C16:2,S) with a sustained alkalinization. When both agents were added jointly, the cytosol first alkalized and then acidified. (GlcNAc)(8) and NodRm-IV(C16:2,S) transiently increased cytosolic Ca(2+) activity, whereby the response to (GlcNAc)(8) exceeded the one to NodRm-IV(C16:2,S) by at least a factor of two.

Calcium↗
PubMed2001

Constitutive overexpression of cytosolic glutamine synthetase (GS1) gene in transgenic alfalfa demonstrates that GS1 may be regulated at the level of RNA stability and protein turnover.

Glutamine synthetase (GS) catalyzes the ATP-dependent condensation of NH4+ with glutanate to yield glutamine. Gene constructs consisting of the cauliflower mosaic virus (CaMV) 35S promoter driving a cytosolic isoform of GS (GS1) gene have been introduced into alfalfa (Medicago sativa). Although transcripts for the transgene were shown to accumulate to high levels in the leaves, they were undetectable in the nodules. However, significant amounts of beta-glucuronidase activity could be detected in nodules of plants containing the CaMV 35S promoter-beta-glucuronidase gene construct, suggesting that the transcript for the GS1 transgene is not stable in the root nodules. Leaves of alfalfa plants with the CaMV 35S promoter-GS1 gene showed high levels of accumulation of the transcript for the transgene when grown under low-nitrogen conditions and showed a significant drop in the level of GS1 transcripts when fed with high levels of NO3-. However, no increase in GS activity or polypeptide level was detected in the leaves of transgenic plants. The results suggest that GS1 is regulated at the level of RNA stability and protein turnover.

Cytosol↗
PubMed1967

Soluble proteins in alfalfa roots as related to cold hardiness.

Soluble proteins extracted from alfalfa roots of hardy and nonhardy varieties were studied in relation to cold hardiness with polyacrylamide gel electrophoresis and quantitative enzyme analysis. Soluble protein content of alfalfa roots increased during hardening in all varieties. Two new isoenzymes with peroxidase activities were found in the fully hardened samples but no large shifts in the electrophoretic pattern were detected with polyacrylamide gel electrophoresis. Peroxidase and catalase activities increased during hardening in all varieties, but only small differences among hardy and nonhardy varieties were detectable. The studies indicated that protein metabolism was altered during the hardening process.

Journal Article↗