was determined by liquid scintillation counting (model LS-6800, Beckman Instruments Inc, San Ramon,
CA). The C export rate was calculated as the difference between the C fixation rate measured continu-
ously by the IRGA and the C retention rate estimated by the GM trace and corrected for the efficiency of
the GM tube.
C. GM Detector Counting Efficiency
Because of the difference in morphological, anatomical, and biochemical leaf characteristics (e.g., leaf
thickness, venation pattern, partitioning) among species that we have examined, the counting efficiency
of the GM detectors varied between 0.1 and 1.0%. The counting efficiencies of the GM detectors for a
representative number of different photosynthetic types of Panicumspecies [30,34] are shown in Figure
- In spite of the low counting efficiency (i.e., 0.5–1.0%), for each species there was a high linear corre-
lation between the radioactivity determined by destructive analysis and that counted by the GM detector.
The coefficient of determination (r^2 ) varied from 0.79 to 0.98. These data support the view that the GM
detectors can be used to monitor^14 C in the leaf nondestructively.
D. Calculation of Concurrent Export During Steady-State^14 CO 2
FeedingThe theories behind determining mass fluxes of C during photosynthesis using either the method of dif-
ferential weight analysis or^14 CO 2 steady-state labeling coupled with net gas exchange are very similar.
Figure 3 shows the net CO 2 assimilation calculated from the photosynthetic rate obtained from the IRGA
for representative C 3 , C 3 -C 4 intermediate, and C 4 species that transport sucrose (Figure 3A, B, and C, re-
spectively) and a C 3 species that translocates auxiliary sugars as well as sucrose (Figure 3D). The IRGA
was used to estimate the rate of assimilation (dashed line) throughout the experiment. In each case, the
photosynthetic rate was constant before^14 CO 2 was supplied at a constant specific activity. The retention
of^14 C was measured nondestructively with the GM detector and was corrected with measurements of ra-
dioactivity made by destructive sampling at the end of the feeding period (solid line in Figure 3A–D). Im-
mediate export of^14 C-assimilates (dotted line) was calculated as the difference between fixation and re-
tention rate given by the data derived from the IRGA and the GM detector during an appropriate period
(shaded area in Figure 3A–D).
In order to determine the appropriate period, a series of destructive experiments were designed to ap-
proximate the time required for transport pools to reach isotopic equilibrium. When the leaf was sampled
during a typical 2-hr feeding period, the pattern of^14 C partitioning in the transport sugars indicated that
isotopic equilibrium between the^14 CO 2 in the air stream and the major^14 C-translocates was generally not
achieved in the first hour (Figure 3M–P). A period of 60 to 90 min was usually required before the spe-
cific activity of the major sugar (sucrose) reached a steady level. The sugar pools in the C 4 species gen-
erally reached isotopic equilibrium earlier than in the C 3 and C 3 -C 4 intermediates species. Normally, the
data between 90 and 120 min were used to calculate values for photosynthesis and the corresponding con-
current export rate (shaded area in Figure 3A–D). During this period the^14 C-sucrose pool was in isotopic
equilibrium with the^14 CO 2 being assimilated. Similarly, in species such as C. sativusthe^14 C-stachyose
pool (auxiliary phloem mobile sugar that was used as a marker of transport) was in isotopic equilibrium.
Figure 3I, J, and K show the accumulation of sucrose, which is the main form of assimilates being ex-
ported in the Panicumspecies. In C. sativusauxiliary sugars accumulated as well as sucrose (Figure 3L).
However, in some species there was a large pool of labeled hexoses [e.g., in the C 4 speciesP. miliaceum
and in the C 3 speciesC. sativus(data not shown)]. In all species^14 C accumulated in sugar and starch (Fig-
ure 3E–H) that sustain metabolic requirements within the leaf and export during subsequent periods of
light or darkness [30,63,79,83]. The fate of these pools and their contribution to export and respiration
could be determined in pulse-chase experiments.
The rates of photosynthesis and immediate export obtained when isotopic equilibrium was first es-
tablished (e.g., 90–120 min) were the data sets we used to evaluate changes in immediate export rates in
leaves challenged with environmental stresses [18,33,34,63], or diseases [78,80]. These data also provide
comparisons of immediate export capacity among leaves with naturally different CO 2 fixation pathways
(i.e., C 3 , C 3 -C 4 intermediate, and C 4 ) [30,33,34] or transgenics with specifically altered C metabolism
[83].
412 LEONARDOS AND GRODZINSKI