Ascertaing metabolic pathway sites in orobanche aegyptiaca that can be inhibited using anti-metabolites in vitro
Mgutu, Allan Jalemba
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Some parasitic angiosperms cause considerable damage to food and fodder plants resulting in very low yield or none at all. The pernicious root parasites Striga and Orobanche are among the most devastating weeds and are not effectively controlled due to various reasons. The principal interest of this study was the biochemical understanding of the growth and development processes of the weeds. In this study, biochemical pathways of Orobanche aegyptiaca that can be inhibited with anti-metabolites were investigated using in vitro experimental systems. In vitro cultures of Orobanche aegyptiaca were established both on solid and liquid media with potential use in bioassays and biochemical studies. A modified MS medium composition was adopted as most suitable and consisted of a 10-fold reduction in ammonium nitrate. The medium was further supplemented with 2 mg/L 2,4-dichlorophenoxy acetic acid and 0.1 mg/L kinetin as plant growth regulators and standard MS vitamins. The effect of anti metabolites inhibiting amino acid, fatty acid, carotenoid, cellulose and folate biosyntheses, photosynthesis and tubulin polymerization were determined by measuring the growth of the in vitro tissues and elongating radicles. Branched chain amino acid biosynthesis was the most sensitive pathway to inhibition among all the investigated biosynthesis pathways. Glutamine biosynthesis and/or ammonium assimilation was equally sensitive to inhibition in the in vitro tissues. The significance of amino acid biosyntheses in Orobanche aegyptiaca was shown when the activities of the specific enzymes were also found to be quite sensitive to inhibition by the same anti-metabolites. Acetolactate synthase in branched chain amino acid biosynthesis was effectively inhibited by all the ALS targeting anti-metabolites used i.e. chlorsulfuron (IC5o 1.0 nM), imazapyr (IC5o 0.88µM) and imazaquin (IC5o 1.97 µM). Despite the common inhibitory activity, imazaquin was completely ineffective on the radicles. Glutamine synthetase was sensitive to glufosinate when partially purified (IC5o 9.1 µM). Cellulose biosynthesis was sensitive to inhibition by dichlobenil in callus tissues (IC5o0.1 nM) and elongating radicles (IC5o 10 nM) only. Cell suspensions seemed to have developed the ability to grow without real cell walls. Inhibitors of microtubulin polymerization were only effective in inhibiting growth of callus tissues. Folate biosynthesis was mildly sensitive to inhibition by sulfadiazine and asulam possibly due to a competent folate recycling system and/or sufficient stored reserves. Inhibition of fatty acid, carotenoid and plastoquinone/tocopherol biosyntheses, and photosynthesis, had little effect on growth of the tissues.