Selection of soybean plant leaves which yield mesophyll cell isolates with maximal rates of CO₂ and NO ^sup−_inf2 photoassimilation

Abstract

A problem often encountered when assaying mesophyll cell isolates prepared from mature soybean leaves, was that of poor reproducibility in rates of net ¹⁴CO₂ photoassimilation and NO₂ ⁻ photoreduction. It was known that soybean source leaves repeatedly displayed their most active net CO₂ photoassimilation in the period from attainment of maximal leaf area to approximately two to five days subsequent to that point. Advantage was taken of the fact that when soybean leaflets of each leaf reach their maximal area they also have reached their maximal leaf length from base to tip. This facilitates a more rapid determination of the point in time in which leaflet areas had reached A_max. Soybean plants (Glycine max cv. Williams) were propagated in the growth chamber with a 12 h light-12 h dark cycle, 25δC, 65% RH, and 700 microeinsteins per meter squared per second. At 24 d post-emergence, the third leaf (numbered acropetally from the unifoliates) of each plant had just attained maximum leaflet areas (≈110 cm²) and lengths (≈13 cm). For this study, leaf mesophyll cells were enzymatically isolated, using commercially prepared pectinase, from leaflet sets of leaves selected from each of the second, third, and fourth leaf positions. Maximal rates of net ¹⁴CO₂ photoassimilation (with 5 mM HCO₃ ⁻) for the second, third and fourth leaf (leaflet) isolates were, respectively, 27.0, 57.0, and 41.7 μmol ¹⁴CO₂ assimilated per milligram chlorophyll per hour; simultaneously maximal rates of NO ^sup−_inf2 photoreduction (1 mM NO ^sup−_inf2 ) were, respectively, 4.4, 8.1, and 0.0 μmol NO ^sup−_inf2 reduced per milligram chlorophyll per hour. These studies made it clear that in order repeatedly to attain reproducible maximal rates of leaf cell isolate net ¹⁴CO₂ photoassimilation and NO ^sup−_inf2 photoreduction, it always was necessary to select the newest, fully expanded leaves (e.g. leaf number 3) for cell isolation. Leaves from several plants only were pooled if they were excised from identically the same node on each of the plants.