Quantitative Analysis of Photosynthate Unloading in Developing Seeds of Phaseolus vulgaris L.

Abstract

The pathway and kinetics of photosynthate unloading in developing seeds of bean (Phaseolus vulgaris L.) were investigated using steady-state labeling with '4C02. The continuous assimilation of 14CO2 at constant specific activity produced stable tracer fluxes that facilitated straighiforward analyses of photosynthate import and unloading in developing seeds. The kinetics of tracer equilibration within intact seeds were compatible with a symplastic route of photosynthate unloading in the seed coat. The import and partitioning of tracer within seeds were partially disrupted by the surgical excision of the distal halves of seeds as practiced during the preparation of "empty" seed coats for perfusion.The mechanisms that regulate photosynthate allocation within developing legume seeds have been the focus of many recent studies (for reviews see refs. 16, 25, 30). Photosynthates accumulated by the embryo must first be unloaded to the seed coat apoplast because the seed coat and embryo of legume seeds are not symplastically linked (25). The pathway of unloading to the seed coat apoplast has not been conclusively determined. Theoretically, photosynthates could unload directly from the phloem to the apoplast (apoplastic pathway) (29, 30), or they could move symplastically from the phloem throughout the tissues of the seed coat before unloading to the apoplast (symplastic pathway) (13,16,17,21). The symplastic unloading pathway has received the most support.The study of photosynthate unloading from legume seed coats has been advanced by the development of the "empty seed coat technique," in which the embryo of a developing seed is removed through an incision in the seed coat and a perfusion solution (or agar trap) is used to collect materials released from the seed coat for subsequent analysis (20,26,31). The use of the empty seed coat technique is complicated by the possibility that the surgical procedure, or the introduction of a solution into the seed coat "cup," may disturb phloem import into the seed coat and/or the process of unloading to the seed coat apoplast (15). Early studies using 'Supported by grant 87-CRCR-1-2397 from the U.S. Department of Agriculture (R.M.S.), grant DCB-8803837 from the National Science Foundation (R.T.), and funds provided by the Hatch Program.pulse-labeling of leaves (or petioles) indicated that the total amounts of labeled photosynthates imported to perfused and intact seeds were similar (20,26,31). However, quantitative comparisons of pulse-label accumulation by perfused versus intact seeds are unreliable when made over long time periods because the tracer fluxes produced by this technique are not sustained over time.Many problems inherent in pulse-labeling are circumvented by the use of steady-state labeling techniques. Steady-state labeling has been used for the quantitative analysis of various aspects of carbon partitioning in plants because the stable tracer fluxes produced by the continuous assimilation of "'CO2 at constant specific activity result in relatively simple trace...

Quantitative Analysis of Photosynthate Unloading in Developing Seeds of Phaseolus vulgaris L.

“…Plants were labeled in a forced-draft hood as described by Ellis et al (2). In summary, steady-state labeling was initiated between 8:00 and 9:30 AM by sealing the entire top leaf in an assimilation chamber about 9 min after beginning the light period.…”

“…Methods for the analysis of sugar and 14C in tissues have been described (2). Perfusate sugar and '4C were assayed directly by methods used previously for neutral column eluates (2).…”

“…Apparently there are sufficient plasmodesmata to accommodate symplastic transfer of photosynthate throughout the seed coat before it is unloaded into the apoplast in Phaseolus vulgaris L. (the symplastic unloading route hypothesis) (1 1). This hypothesis has also received support from empty seed coat experiments (6,14) and studies of tracer kinetics (2,15). An alternative to the symplastic unloading route hypothesis is that photosynthates are unloaded directly from the phloem into the apoplast, without passing through the seed coat symplast (the apoplastic unloading route hypothesis) (20).…”

“…Steady-state labeling with '4C02 will be used to simplify analysis of tracer fluxes within seed coats (2). Disruption of phloem import and unloading by the empty seed coat technique has recently been reassessed (2,10), and will be evaluated quantitatively in this study.…”

“…Steady-state labeling with '4C02 will be used to simplify analysis of tracer fluxes within seed coats (2). Disruption of phloem import and unloading by the empty seed coat technique has recently been reassessed (2,10), and will be evaluated quantitatively in this study. To clarify discussion of photosynthate movement into and out of perfused seed coats, we will use the following terms, as proposed by Grusak and Minchin (6); phloem import: the movement of photosynthates into the seed coat via the phloem; phloem unloading: the movement of assimilates from the phloem to the apoplast or the symplast of the seed coat; and seed coat unloading-the movement of photosynthates from the seed coat symplast to the apoplast.…”