References

Dupuy et al. (1992) Appl. Environ. Microbiol. 58, 2415-2419 Normand et al. (1996) Mol. Microbial Ecol. Manual 3.4.5, 1-12 Ponsonnet, Nesme (1994) Arch. Microbiol. 161, 300-309 Willems et al. (2001) Int. J. Syst. Microbiol. 51, 623-632

CONSTITUTIVE EXPRESSION OF A NODULE ENHANCED MALATE DEHYDROGENASE GENE IN ALFALFA ALTERS ORGANIC ACID SYNTHESIS AND IMPROVES GROWTH IN ALUMINUM-CONTAINING ACID CULTURE

M. Tesfaye1, D.L. Allan2, C.P. Vance3.4, D.A. Samac1-4

'Dcpt Plant Pathology; 2Soil, Water and Climate

3Agronomy and Plant Genetics; 4Plant Science Research Unit, USDA-ARS Univ. of Minnesota, St. Paul, MN 55108, USA

1. Introduction

Alfalfa is very sensitive to aluminum (Al) toxicity in acid soils, which make up about 40% of the world's arable land. Addition of organic acids to nutrient solutions alleviates phytotoxic Al effects. Malate dehydrogenase catalyzes the reversible reduction of oxaloacetate to malate. A unique form of malate dehydrogenase (neMDH), expressed 5-15-fold higher in alfalfa nodules, was identified previously. This non-photo synthetic isoform plays a crucial role in providing malate to bacteroids to fix N2 and by the plant for assimilation of N2. This research aimed to constitutively over-express neMDH enzyme in alfalfa, to test whether such transgenic plants had enhanced organic acid synthesis and excretion, and to evaluate if selected transgenic plants had increased Al tolerance.

2. Procedures and Results

Leaf pieces from a highly regenerable alfalfa cultivar Regen-SY were transformed with A. tumefaciens that contains a chimeric gene consisting of a full-length neMDH eDNA under the CaMV 35S promoter. Transgene integration and expression were evaluated by DNA-blot, PCR, RNA-blot, RT-PCR, Western blot and specific enzyme analysis. PCR analysis and DNA blot hybridization confirmed that at least one copy of the transgene was present in the transgenic lines. Transgenic lines showed up to 1.6-fold increase in specific MDH enzyme activity. Gel-blot analysis of total RNA clearly showed increased levels of neMDH transcripts in both root-tips and leaf samples of transgenic lines, compared to untransformed RegenSY plants. Amounts of neMDH polypeptides were also considerably higher in root-tip samples of selected neMDH transgenic lines than in root-tips of untransformed plants. Transgenic alfalfa showed reduced biomass accumulation during the first year of growth in field plots of neutral soil at pH 7.3. When plants were grown in 50 I^M Al-containing acid culture at pH 4.3 however, transgenic plants had at least 3.4-fold greater root elongation than untransformed plants, indicating the set plants may be suited to cultivation in Al-containing acid soils. Although we have no direct evidence for the mechanism of Al tolerance by transgenic alfalfa, the enhanced Al tolerance by transgenic lines and the poor growth of untransformed Regen-SY plants in Al-containing hydroponic solution or soil culture coincided with the organic acid synthesis and exudation patterns of these plants. Transgenic plants showed up to 5-fold higher leaf and root concentrations and 7-fold root exudation of oxalate, citrate, succinate, malate and acetate than Regen SY plants. One transgenic alfalfa over-expressing organic acids was crossed with GA-AT, an acid soil tolerant alfalfa previously selected in Georgia, USA. Cosegregation of the neMDH transgene and enhanced neMDH mRNA expression was observed in F1 crosses. Transgenic alfalfa will be evaluated for nodulation and biological N-fixation ability in acid soils.

3. References

Kochian LV (1995) Annu. Rev. Plant. Physiol. Plant. Mol. Biol. 46, 237-260 Miller et al. (1998) Plant J. 15, 173-184

COMMON MECHANISMS FOR THE INTRACELLULAR SURVIVAL OF BACTERIA WITHIN PLANTS AND ANIMALS

L. Barra1'2, G.P. Ferguson1, K. LeVier1, G.R. Campbell1, C. Blanco2, G.C. Walker1

'Dept of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA 2Lab UMR CNRS-6026, Université de Rennes-1, 35042 Rennes Cedex, France

1. Introduction

Sinorhizobium meliloti can establish a symbiotic relationship with alfalfa plants. Once inside the plant cells, S. meliloti differentiate into bacteroids and persist in a membrane-bound acidic compartment. The bacteria need to adapt to a variety of stresses during this association. The symbiosis can be disrupted at the stage of the initial root hair colonization, infection thread formation, release into the plant cell, and finally differentiation of the bacteria into nitrogen-fixing bacteroids.

S. meliloti mutants have been isolated that are affected at different stages in the alfalfa symbiosis (Glazebrook et al. 1993). Some of these mutants were released into the plant cell but were impaired in the differentiation into bacteroids. One of these mutants lacks BacA activity, a 420 amino acid, putative inner membrane protein, which is apparently essential for S. meliloti bacteroid development and/or intracellular survival. A homolog of the S. meliloti BacA protein was found to be essential for the intracellular survival of the mammalian pathogen, Brucella abortus, which is phylogenetically close to S. meliloti. Both bacA mutants exhibit increased resistance to bleomycin, suggesting that BacA could take part in the uptake of bleomycin (Ichige et al. 1997; Glazebrook et al. 1993).

2. Results and Discussion

New bacA phenotypes have been highlighted by recent physiological studies (Ferguson, unpublished) and genetic analysis (LeVier 2000), suggesting that BacA has the ability to carry out more than one function. BacA could be involved in host signal, or compound importer or exporter. These signal or compound transporters essential to the survival within the cell could not be transduced to their target in a BacA- background, leading to the lysis or early senescence of the bacteria. Recent data, using membrane destabilizing agents, suggest that BacA could also be involved in the cell envelope homeostasis. We are currently investigating, by physiological and genetic analysis, the mechanisms and pathways by which BacA could modulate the intracellular survival.

3. References

Glazebrook JIA et al. (1993) Genes Dev. 7, 1485-97 Ichige A et al. (1997) J. Bacteriol. 179, 209-16 LeVier K et al. (2000) Science 287, 2492-3

4. Acknowledgements

This work was supported by Public Health Services Grant GM31030 from the National Institute of Health (NIH) to GCW. LB acknowledges the receipt of a doctoral fellowship from the "Region Bretagne - Conseil Regional de Bretagne - France".

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