was observed [31,68]. It is impossible to follow the fate of the intermediates using short-lived isotopes
during pulse-chase experiments that extend into a normal day-night cycle.
- The Radioisotope^14 C
The most widely used radioisotope in the study of leaf photosynthesis and phloem translocation is^14 C.
The main advantage of^14 C is its commercial availability in different forms (e.g.,^14 CO 2 ,^14 C-sucrose) and
its long half-life (5730 years). It can be used in both short- and long-term studies of export and partition-
ing. Although^14 C emits (negatron) of low energy, translocation of^14 C-labeled assimilates can be
monitored in a noninvasive manner with GM detectors and products can be analyzed following sampling
[18,69]. Unfortunately, GM detectors cannot measure^14 C in tissues farther than 1 mm from the plant sur-
face and a correction must be made for each leaf [18,69]. The manner in which^14 CO 2 is introduced and
export of^14 C assimilates is monitored varies. Both pulse-chase and steady-state labeling protocols have
been used to obtain useful data regarding early export of photoassimilates.
Pulse-chase labeling with^14 CO 2 is the most common procedure used to study translocation of^14 C-
photoassimilates [31,70–75]. Typically, the label is fed to a source leaf as^14 CO 2 and either its disappear-
ance from the leaf or appearance in sink tissue is analyzed. Sizes and rates of turnover of sugar pools (e.g.,
transport and vacuolar sucrose pools) in the light have been estimated by monitoring the translocatory ef-
flux of^14 C from leaves pulse labeled with^14 CO 2 and employing compartmental models [72,73,76,77].
However, pulse-chase experiments may not provide precise measurements of the mass transfer rate of C
during photosynthesis because the specific activity of^14 C in the pools changes dramatically during chase
periods, especially in leaves subjected to different environmental conditions [69,78]. We have used pulse-
chase experiments primarily to follow the export and respiration of reserves during a dark period, when
no label can be incorporated into the transport products directly via photosynthesis [79,80]. Steady-state
labeling has been the method used to quantify the mass transfer rate of immediate export during
photosynthesis.
During steady-state labeling, transport pools of sugars achieve isotopic equilibrium with the^14 CO 2 ,
which is supplied continuously at a constant specific activity [18,69]. There are different protocols for es-
tablishing steady-state labeling with^14 CO 2 [7,18,34,69,81]. By calculating export fluxes only when iso-
topic equilibrium has been achieved, errors associated with determining immediate export rates using
non-steady-state labeling and pulse-chase experiments are significantly reduced [18,78].
III. STEADY-STATE^14 CO 2 LABELING AND MEASUREMENT OF
IMMEDIATE C EXPORT RATES DURING PHOTOSYNTHESISA. Open-Flow Gas Analysis System
We have used open-flow gas analysis systems similar to that in Figure 1 to establish steady-state^14 CO 2
labeling conditions [33,34,63,65]. During the leaf gas exchange analysis and labeling experiments,
plants were held in a growth chamber in which irradiance, temperature, and humidity were controlled.
The middle portion of a leaf was enclosed in a brass leaf chamber that had been chrome plated to re-
duce problems associated with water exchange [82]. The leaf chamber consisted of a top part (16 cm^2
exposing area through a glass window) and a bottom part in which was mounted a GM detector (model
EWGM, window area 6.8 cm^2 , Bicron Corp., Newbury, OH). Both upper and lower sections of the leaf
chamber were designed as water circulating jackets for leaf temperature control. Leaf and gas stream
temperatures were measured with two thermistors (YSI 44003 A, YSI Inc., Yellow Spring, OH) inside
the leaf chamber. Photosynthetic photon flux density (PPFD) (400–700 nm) was provided by three
1,000 W metal halide lamps (Sylvania, GTE, Toronto, ON, Canada) and measured with a Li-Cor quan-
tum sensor (model LI-189, Li-Cor Inc., Lincoln, NE) positioned at the surface level of the leaf. The de-
sired CO 2 concentration (35 or 90 Pa) was obtained by mixing CO 2 –free air that had been passed
through soda lime with pure CO 2 using two mass flow controllers (Side-Trak, Sierra Instruments, Inc.,
Monterey, CA). The CO 2 concentration in the gas stream entering and exiting the leaf chamber was
measured with an IRGA (model 6262, Li-Cor Inc.). Humidity in the gas entering the leaf chamber was
controlled by first passing the gas stream through a gas bubbler placed in a temperature-controlled wa-
ter bath (model RTE-9, Neslab Instruments Inc., Portmouth, NH). The dew point in the gas stream en-
tering and exiting the leaf chamber was monitored with a digital humidity analyzer (Dew point meter;
410 LEONARDOS AND GRODZINSKI