Roger E. Wyse

Adaptation of the enzyme-linked immunosorbent assay (ELISA) to the quantitative analysis of abscisic acid

Abstract We have developed an enzyme-linked immunosorbent assay (ELISA) for the quantitative analysis of abscisic acid (ABA). The method is based on competitive binding between free and enzyme-labeled antigen with antibodies. Anti-ABA antibodies were produced in rabbits against a conjugate of human serum albumin and ABA. The antibodies were adsorbed on polystyrene microtiter plates. Free and alkaline-phosphatase-labeled ABA competed for binding sites on the antibody. Bound enzyme activity was colorimetrically determined after addition of the p-nitrophenyl phosphate substrate. Sample ABA concentrations are calculated based on an inverse relationship between enzyme activity and ABA concentration. Absorbance was linear between 25 pg and 250 ng ABA/assay. The sensitivity of the assay, 25 pg, is comparable to the radioimmunoassay and other established chromatographic methods for ABA detection. Partially purified plant extracts showed parallelism with ABA standards. The technique is sensitive, rapid, and inexpensive. However, the need for partial sample purification is still a hindrance to the speed of the method.

The regulation of turgor pressure during sucrose mobilisation and salt accumulation by excised storage-root tissue of red beet

The changes in turgor pressure that accompany the mobilisation of sucrose and accumulation of salts by excised disks of storage-root tissue of red beet (Beta vulgaris L.) have been investigated. Disks were washed in solutions containing mannitol until all of their sucrose had disappeared and then were transferred to solutions containing 5 mol·m-3 KCl+5 mol·m-3 NaCl in addition to the mannitol. Changes in solute contents, osmotic pressure and turgor pressure (measured with a pressure probe) were followed. As sucrose disappeared from the tissue, reducing sugars were accumulated. For disks in 200 mol·m-3 mannitol, the final reducing-sugar concentration equalled the initial sucrose concentration so there was no change in osmotic pressure or turgor pressure. At lower mannitol concentrations, there was a decrease in tissue osmotic pressure which was caused by a turgor-driven leakage of solutes. At concentrations of mannitol greater than 200 mol·m-3, osmotic pressure and turgor pressure increased because reducing-sugar accumulation exceeded the initial sucrose concentration. When salts were provided they were absorbed by the tissue and reducing-sugar concentrations fell. This indicated that salts were replacing sugars in the vacuole and releasing them for metabolism. The changes in salf and sugar concentrations were not equal because there was an increase in osmotic pressure and turgor pressure. The amount of salt absorbed was not affected by the external mannitol concentration, indicating that turgor pressure did not affect this process. The implications of the results for the control of turgor pressure during the mobilisation of vacuolar sucrose are discussed.

In vitro and in vivo modification of sugar transport and translocation in celery by phytohormones

Abstract In an attempt to address the controversy in the literature as to whether phytohormones have any direct effect on phloem loading of sucrose, we investigated the effect of gibberellic acid (GA 3 ) and indoleacetic acid (IAA) on sugar transport and translocation in celery ( Apium graveolens L. cv. Utah 5270). Both hormones enhanced sucrose uptake into isolated vascular bundles and phloem tissue of celery and enhanced the export of 14 C assimilates from leaves of intact plants in vivo. The hormone-induced increase of uptake into isolated vascular bundles or phloem was specific for sucrose and mannitol which are translocated in phloem. Furthermore, the hormone-induced increase in translocation was not due to an increase in sink demand, since neither glucose nor sucrose uptake rates were affected in the storage parenchyma tissue discs (sink region) in the presence of GA 3 or IAA. The evidence suggests that phytohormones may have a direct effect on phloem loading of sucrose. The possibility of short-term GA 3 and IAA effects on processes resulting in membrane transport of sugars in celery is discussed.