Monitoring Phloem Unloading and Post-Phloem Transport by Microperfusion of Attached Wheat Grains

Abstract

Phloem unloading and post-phloem transport in developing wheat (Trificum aestivum 1.) grains were investigated by perfusing the endosperm cavities of attached grains. Relative unloading ratio (RUR) and the rate of sucrose release into the endosperm cavity (SRR) were calculated, respectively, from 14C import and from sucrose washout from the cavity. RUR and SRR continued at or near in vivo rates over a wide range of cavity sap osmolality (90 to approximately 500 milliosmolal) and sucrose concentration (14-430 mM) and for long times (29 h). These are much greater ranges than have been observed for the endosperm cavity in vivo (230-300 milliosmolal, and 40-120 mM, respectively), indicating that neither the cavity sap osmolality nor sucrose concentration are controlling factors for the rate of assimilate import into the cavity. The maintenance of in vivo transport rates over a wide range of conditions strongly implicates the role of transport processes within the maternal tissues of the wheat grain, rather than activities of the embryo or endosperm, in determining the rate of assimilate import into the grain. RUR was decreased by high concentrations of sucrose and sorbitol, but not of mannitol. By plasmolyzing some chalazal cells, sorbitol appeared to block symplastic transport across the crease tissues, but neither sucrose nor mannitol caused plasmolysis in maternal tissues of attached grains. The inhibition of RUR by KCN and carbonyl cyanide m-chlorophenyl (CCCP) and the continued import of sucrose into grains against its concentration gradient suggest that solute movement into the endosperm cavity might occur by active membrane transport. However, the evidence is weak, since KCN and CCCP appeared to act primarily on some aspect of symplastic (i.e. nonmembrane) transport. Also, sucrose could move from the endosperm cavity into the maternal tissues (i.e. opposite to the normal direction of sucrose movement), suggesting that transmembrane movement in the nucellus may be a reversible process. Pressure-driven flow into the grain could account for movement against a concentration gradient.

Sucrose Release into the Endosperm Cavity of Wheat Grains Apparently Occurs by Facilitated Diffusion across the Nucellar Cell Membranes

“…There was no evident effect of pH on the SRR except at pH 9.5 (Table I), at which the SRR was about 60% higher than expected for intact grains (220 nmol h-' grain-'; Fisher, 1990). However, when making comparisons of import, we regard the RUR as a more reliable indicator, because the SRR can be biased upward by leakage of Suc from the endosperm (Wang and Fisher, 1994a). Quite likely, this explains the higher SRR at pH 9.5.…”

“…In recent work with wheat grains, we found that solute release from the maternal tissues into the endosperm cavity was inhibited by CN and by CCCP (Wang and Fisher, 1994a). However, because inhibition of import was accompanied by a reduction of the maternal Suc pool, phloem transport or SE/CC unloading, and not membrane transport, appeared to be the likely site of inhibition.…”

“…With the exception of a faster perfusion rate (12 pL h-' instead of 6 pL h-I), perfusion conditions and the measurements made to estimate import rates were as described previously (Wang and Fisher, 1994a). In brief, endosperm cavities of four grains on each ear were perfused with a test solution and, to measure the rate of assimilate import, the flag leaf was pulse labeled with I4CO,.…”

“…Two measures of assimilate import were used (Wang and Fisher, 1994a). The RUR is the ratio of the I4C import by perfused grains compared to nonperfused grains.…”

“…To clarify some of the effects of earlier mannitol and sorbitol perfusion treatments (Wang and Fisher, 1994a), we also addressed the possibility that these sugars might be absorbed by the matemal tissues from the endosperm cavity. Our results indicate that solute efflux from the maternal tissues of wheat grains differs in important respects from the corresponding step in legume seeds.…”