In leaves of C 3 plants, the stroma of the chloroplast is the site of the reductive pentose pathway (i.e.,
the Calvin cycle) and CO 2 fixation [35]. The primary inorganicsubstrate of ribulose-1,5-bisphosphate
carboxylase-oxygenase (Rubisco) is CO 2. The primary organicsubstrate is ribulose 1,5-bisphosphate
(RuBP). Although the Calvin cycle fixes inorganicsubstrate CO 2 , it also regenerates the acceptor RuBP.
The regeneration of RuBP provides a feedback mechanism for control of the rate of photosynthesis. How-
ever, metabolism and recycling of reduced C and N occur in subcellular compartments other than the
chloroplast and even outside the cell that may have been the primary site of C reduction. During pho-
torespiration, for example, the C in the phosphoglycolate molecule that is generated by the oxygenase re-
action is not recycled conservatively within the chloroplast [36,37] or in the cell [32]. Similarly, the su-
crose that is the major phloem mobile leaf product is not synthesized in the chloroplast but in the cytosol
[11,27,38]. In fact, phloem mobile sugars need not be synthesized in the same cell that initially fixed the
CO 2. It suffices here to note that the auxiliary phloem sugars such as raffinoses are made in phloem cells
distant from the site of CO 2 fixation [39]. It also suffices here to note that intercellular exchanges of as-
similates preceding export from the leaf are more complex in C 4 and C 3 -C 4 intermediate types than in the
leaves of C 3 species and that sucrose synthesis and CO 2 fixation occur in separate cells and tissues
[38,40,41]. Clearly, we are dealing with a complex set of processes when we try to understand how net
photosynthesis, which is measured as substrate utilization (e.g., mol of CO 2 fixed per second per m^2 ),
is affected by sink demand and product removal (mol of CO 2 exported per second per m^2 ). Subcellular,
cellular, and tissue level interactions are all involved. Is there any unifying mechanism that truly explains
how photosynthesis in the chloroplast of a chlorenchyma cell and movement in the phloem sieve cell are
linked?
The literature suggests that source photosynthesis and sink demand for assimilates are linked mech-
anistically, in part, by the operation of specific site exchanges in the light such as that mediated by oper-
ation of the phosphate translocator at the chloroplast membrane [11,25,27,38]. It is known that reserves
of sugars and starch buffer sink demand and affect growth and development [4,5]. Can we hope to corre-
late primary leaf parameters such as photosynthetic efficiency and leaf export capacity with sink demand?
We have developed a working hypothesis which states that in most plants the immediate export rates from
leaves during photosynthesis best predict RGR. If this hypothesis is valid, then in many cases the RGR of
a plant should be tightly correlated with the capacity of the leaves to export C in the light as the C is be-
ing fixed (i.e., immediately). How can one measure or estimate immediate C flux through a complex or-
gan such as the leaf?
We have defined immediate export as the direct or instantaneous flux of C from CO 2 to assimilates
to the phloem [18]. This operational definition of immediate export excludes for practical purposes ex-
port arising from storage reserves either during the same photoperiod or during subsequent night periods
[5]. Again, we emphasize that it is important to export total leaf assimilate supply at different times in the
day. The specific question that is being addressed is, how important to the maximum operation of the C
and N reduction pathways in the leaf tissue is the immediate rate of assimilate removal via the transloca-
tion stream in the light? The Vmaxof a single enzyme step in a complex reaction pathway can provide use-
ful information about the kinetics and the importance of that enzyme step. To obtain Vmaxthe enzyme rate
needs to be measured quantitatively. Similarly, the importance of immediate export rate from the leaf dur-
ing photosynthesis needs to be quantified experimentally.
II. METHODOLOGIES USED TO ESTIMATE IMMEDIATE C EXPORT
In this chapter we describe how steady-state^14 CO 2 labeling of leaf tissue is achieved and why data ob-
tained when^14 C isotopic equilibrium exists between photosynthesis and export provide useful estimates
of the immediate mass efflux of C. To appreciate the advantages and disadvantages of steady-state^14 CO 2
labeling, one needs to consider other methodologies that have been used to quantify C export [6–10].
A. Gas Exchange and Differential Dry Weight Analysis
Perhaps the least costly method to estimate mass rates of export from source leaves is to measure the net
CO 2 exchange rate with an infrared gas analyzer (IRGA) and the changes in the dry weight of the leaf
over time [3,42–46]. The IRGA quantifies the net amount of C fixed and thus the total dry weight that
should have been retained in the leaf in the absence of export. Photorespiratory CO 2 losses are accounted
408 LEONARDOS AND GRODZINSKI