for because net CO 2 exchange is measured. The difference in dry weight between samples in the light and
the theoretical amount of C that should have been retained can only be due to export. Whereas the IRGA
measurements are nondestructive, the differential dry weight estimations of leaf tissue are destructive,
and many small samples need to be taken from several leaves. Subsampling reduces disturbance of a sin-
gle leaf and eliminates the large variability in dry weight within the same leaf and heterogeneity among
different leaves. A disadvantage of this protocol is that it is difficult to evaluate short-term changes in ex-
port. Also, measurable changes in leaf dry weight do not occur rapidly, usually taking several hours. Nev-
ertheless, destructive sampling does provide the tissue necessary for biochemical characterization of the
leaf intermediates and, coupled with labeling procedures, can provide important information regarding
the specific pools of assimilates contributing to leaf metabolism and export [18].
B. Isotopes of C
In most instances 95% of the photoassimilates being transported via the phloem are carbohydrates such
as sucrose. The mass (e.g.,^13 C) and radioactive (e.g.,^11 C,^14 C) isotopes of C introduced as labeled CO 2
are excellent tools for directly tracing the movement of photoassimilates in plants. Two of the advantages
of using radioactive and mass isotopes are that information about metabolism can be obtained and a less
invasive method of measuring C export can be achieved. However, sufficient time is required for proper
labeling of the pools of the primary phloem mobile assimilates, and care must be exercised in calculating
for instrument sensitivity and isotope discrimination.
- The Mass Isotope^13 C
The mass isotope^13 C has been used extensively in studies of isotopic discrimination as a tool to dis-
tinguish photosynthetic pathways in C 4 , C 3 , and C 3 -C 4 intermediate plants [47–49]. Mass isotopes such
as^13 C and^15 N are currently not used as extensively as they should be to study export patterns (Ref. 50
and references therein). The problem at the moment is the lack of an inexpensive detection method for
the mass isotopes that does not require destructive sampling [51,52]. In most studies, a destructive sam-
pling step is required to determine the enrichment level of the assimilates in samples that are often pre-
pared for analysis by mass spectrophotometry coupled to gas or liquid chromatography. However, it is
quite clear that the use of mass isotopes to probe assimilate translocation can and should be coupled
with improving technologies such as nuclear magnetic resonance (NMR) imaging [19,53,54]. NMR
imaging in medicine has revolutionized remote sensing of tissues [55]. With steady-state labeling of
leaves with mass isotopes such as^13 CO 2 we should be able to replace other methods of quantifying ex-
port as well as monitor partitioning and allocation patterns. It is theoretically possible to analyze export
of labeled assimilates from leaves, movement within individual bundles of phloem cells, and sink
metabolism noninvasively.
- The Radioisotope^11 C
The short-lived radioisotope^11 C has been used extensively as a noninvasive probe of translocation pro-
cesses [17,56–63]. However, because of the short half-life of^11 C (i.e., 20.4 min), experiments are re-
stricted in time and must be performed in proximity to a particle accelerator. The main advantage of^11 C
is that the isotope emits particles of much higher energy than the particles that are emitted from
(^14) C, and thus, translocation of the (^11) C-labeled intermediates is easier to monitor remotely using Geiger-
Müller (GM) detectors. However, heavy shielding of the detectors is required [64], which limits an anal-
ysis of partitioning of label within different tissues in the same organ [54]. Nevertheless,^11 C has been
used to study directional movements and translocation speed in stems [56,63,65]. In other studies, trans-
fer function and compartmental analysis have been used to quantify^11 C-photoassimilate export from
source leaves [62,66,67]. Given the cost, time, and effort of setting up labeling experiments, the overall
value of using short-lived isotopes must be considered carefully. Although the procedure is noninvasive
and the short half-life of^11 C and^13 N permits repetition of tests using the same leaf [58,60,64], a signifi-
cant disadvantage of these isotopes is their short half-life, which makes analysis of labeled assimilates by
current biochemical techniques very difficult. Sap samples and tissue extracts must be purified and ana-
lyzed immediately. We have been able to use^13 N (half-life 10 min) fed as^13 NH 3 to probe leaf photores-
piration directly [58]. In the first hour,^13 N-labeled glutamate, alanine, serine, and glycine were detected
(Grodzinski and Lapointe, unpublished) and export of these photorespiratory intermediates (using^14 C)
QUANTIFYING IMMEDIATE C EXPORT FROM LEAVES 409