References

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Carlier et al. (1997) Antimicrob. Agent Chemotherapy, 1495-1499

Kaiser et al. (1996) Physiol. Plant. 98, 833-837

Soberon et al. (2001) Molec. Plant-Microbe. Int. 14, 572-576

EFFECT OF OVEREXPRESSION OF A SOYBEAN CYTOSOLIC

GLUT AMINE SYNTHETASE GENE ON NODULATION AND GROWTH OF

TRANSGENIC PEA GROWN AT DIFFERENT LEVELS OF NITRATE

H. Fei1, J.K. Vessey1, B. Luit1, S. Chaillou2, B. Hirel2, E. Carrayol2, J.D. Mahon3,

P.L. Polowick3

^ept of Plant Science, U. Manitoba, Winnipeg, MB, Canada

2Laboratoire du M├ętabolisme et de la Nutrition des Plantes, INRA, Route de St-Cyr, Versailles Cedex, France

3Plant Biotechnology Institute, Saskatoon, SK, Canada

The impact of overexpression of a soybean cytosolic glutamine synthetase gene (GS15) on nodulation and growth of pea (Pisum sativum L. cv. Green feast) was studied. Three constructs of the GS15 fused with a constitutive 35S CaMV promoter, a nodule-specific promoter (LBC3) and a root-specific promoter (rolD), respectively, were introduced into pea plants via Agrobacterium rhizogenes. The transgenic lines homozygous for GS15 were isolated from 16 plants in each line by PCR analysis, and analyzed by Southern blot hybridization. Four lines with only one copy of GS15, i.e. 35SGS15-DB917, LBC3GS15-PLP225, rolDGS15-DB681 and rolDGS15-DB779, were selected for Western blots and hydroponic culture.

Western blot analysis revealed that the 35SGS15 construct containing a constitutive promoter was overexpressed in the leaves and roots. The rolDGS15 with a root-specific promoter was strongly overexpressed in roots and nodules, but also was detectable in leaves. The LBC3GSI5 protein was relatively abundant in the nodules and roots compared to control.

The hydroponic experiment showed that the overexpression of 35SGS15 and LBC3GSI5 constructs did not affect nodulation in the N free treatment, while rolDGSIS suppressed nodulation in the rolDGS 15-DB681 line, but not in rolDGS15-DB779 line. In the treatment with 0.1 mM N03", nodulation was inhibited in the 35SGS15-DB917 and rolDGS15-DB779 lines. Although nodule numbers for transgenic lines tended to be higher at the 1.0 mM NO3" level, these values were not significantly different (P = 0.05). Overexpression of rolDGS15 also suppressed nodulation in DB681 line in the 10.0 mM N03" treatment.

Overexpression of the 35SGS15 did not influence biomass accumulation in all NO3" treatments, but the LBC3GS15 tended to enhance biomass production, particularly in the treatments with 0.1 and 10.0 mM N03" (biomass increased by about 20% in both treatments). Overexpression of the rolDGS15 significantly inhibited biomass in DB681 line in all NO3" treatments, but stimulated biomass accumulation in DB779 line at 0.1 mM NO3" level and did not affect biomass in other treatments.

These results showed that the constructs of soybean cytosolic GS15 fused with different promoters were mainly overexpressed in specific tissues. The impact of overexpression of the GS15 on biomass accumulation of pea at different N03" levels was different from that on nodulation, implying that overexpression of GS15 may affect biomass accumulation by means other than nodulation. We are currently analyzing GS activity and nitrogen fixation rates to further characterize the impact of overexpression of GS15 on biomass production.

WHOLE GENOME STUDY OF FixLJ TARGETS IN SINORHIZOBIUM MELILOTI

Laboratoire de Biologie Mol├ęculaire des Relations Plantes-Microorganismes INRA/CNRS, BP 27, 31326 Castanet-Tolosan Cedex, France

In Sinorhizobium meliloti, expression of nitrogen fixation genes is controlled by a hierarchically organized regulatory cascade (David et al. 1988). The two component FixLJ system operates at the head of this cascade, which suggests that it plays a global regulatory role in response to the microaerobic conditions found in alfalfa root nodules. However, up to now only two genes were known to be directly activated by the FixJ transcriptional activator - nifA and fixK.

In order to identify potential new FixJ targets in the genome of S. meliloti we have developed an in vitro cyclic selection (SELEX) procedure using a fusion protein between GST and the DNA binding domain of FixJ. In a first phase, the procedure was applied to synthetic oligonucleotides randomized over 20 bp in order to select DNA fragments with good affinity for FixJ. Protein/DNA complexes were adsorbed on glutathione-sepharose beads, bound DNA was eluted, amplified and protein/DNA complexes were selected again. After six rounds of such a selection the randomized DNA appeared highly enriched in FixJ binding fragments. These fragments were cloned, sequenced and their complexes with phosphorylated FixJ (FixJ~P) were characterized by gel retardation and DNase I foot-printing experiments. This analysis led to the definition of two recognition patterns for FixJ~P. The first pattern CTAAGTAGTTTCCC (14 bp) is similar to the high affinity FixJ~P binding site mapped in the flxK promoter (Galinier et al. 1994). The second pattern CTACGTAG (8 bp) lies in the middle of a 40 bp region protected by FixJ~P against DNase I attack.

In a second phase, the SELEX procedure was applied to a pool of DNA fragments covering the entire S. meliloti genome, generated by random priming as described by Singer et al. (1997). This resulted in the identification of 20 new FixJ binding sites in addition to the known fixK and jixK ' promoters. These sites appear unequally distributed over the three replicons constituting the S. meliloti genome (Galibert et al. 2001); while the chromosome and the pSymA megaplasmid contain 9 and 10 sites respectively, only 3 sites are located on pSymB. Two of the new FixJ targets appear to result from a duplication of thcfixK promoter, evident from the presence of a downstream truncated fixK ORF. This duplication of the fixK promoter confers FixJ-dependent microaerobic induction to the downstream gene, as evidenced by RT-PCR and reporter gene experiments. Similar promoter duplications, including a truncated nifH ORF, were previously observed for the nifH promoter and were found to confer nifA regulation (Better et al. 1983; Murphy et al. 1993). Such a 'promoter hijacking' may therefore be a more common phenomenon in the S. meliloti genome than originally thought, allowing for the recruitment of new genes under particular physiological conditions.

References

Better et al. (1983) Cell 35, 479-485

Galibert et al. (2001) Science 293, 668-672

Galinier et al. (1994) J. Biol. Chem. 269, 23784-23789

Murphy P et al. (1993) J. Bacteriol. 175, 5193-5204

Singer B et al. (1997) Nucleic Acids Res. 25, 781-786

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