Use of leaf discs to monitor protein synthesis under field conditions

In recent Newsletters, we have reported a series of studies in which a physical, chemical or biological 'stress-inducing' factor has been examined, usually in a 5 day old plumule and/or primary radicle, grown under stringent laboratory conditions. Altered or enhanced protein synthesis as revealed by fluorography has been our main criterion for the assignment to a factor of the capacity to induce stress. We have shown also that the laboratory techniques can be adapted to the study of certain other tissues of maize to address problems of development, etc.

This report outlines attempts to extend our studies to field grown material, i.e., to monitor the growth of a tissue or organ during the growing season. During the fall of 1984, in our nursery we created two plots (each approx. 15 x 20 m), designating one a minimum productivity plot and the other a maximum productivity plot. A third plot was selected as a control and left unaltered (normal nursery maintenance). In the minimum productivity plot the top soil was mixed with an equal amount of bank-run gravel; in the maximum plot, the top soil was enhanced with liberal applications of manure and premium top soil, to provide the best texture, etc. we could produce. Management practices (fertilization, irrigation, etc.) in 1985 were consistent with the intent of the plot, i.e. maximum or minimum (stressed) growth.

A selection of inbreds and some of their reciprocal hybrids were sown at two planting dates and thinned to a density of 60,000 plants per hectare in each plot. Measurements (height, number of leaves, etc.) were recorded several times through the growing season to provide a 'base-line' of growth. Fortunately we had a reasonable growing season such that the expected differences were obtained among plots, planting dates and cultivars.

Two cm (diameter) leaf discs, exposed to 35S-methionine for 1.5 h in situ, provided leaf material to yield TCA precipitable protein in sufficient quantity to monitor uptake and to run two dimensional gels followed by appropriate fluorography. One to several leaves were sampled from representative plants of each cultivar in each planting every two weeks from mid-June until mid-September. The field techniques (MGCNL 59:75, 1985) and the laboratory techniques (Baszczynski et al., Can. J. Biochem. 60:569, 1982) required little modification.

This study will require at least the 1986 growing season before convincing data analysis can be offered. We are particularly interested in examining proteins that may be unique to cultivars, those that may have a role in heterosis, and those that are of developmental significance. We do not know yet whether this system (protein isolation - electrophoretic separation - fluorographic analysis) is sensitive enough to address any of the three questions. Our preliminary data from the 1985 fluorograms do demonstrate qualitative differences among cultivars in the three performance plots, including some inbred-hybrid comparisons. Interestingly, no qualitative differences have been found between planting dates of the same cultivar/plot when the leaf is of the same age, say 60 days. If such fidelity is the rule, differences among genotypes should be easier to detect.

D. B. Walden and T. G. Crowe
 
 


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