Competition for in vitro [h]gibberellin a(4) binding in cucumber by substituted phthalimides: comparison with in vivo gibberellin-like activity.

Certain N-substituted phthalimides (NSPs) have gibberellin (GA)-like activity in a number of GA bioassays. The interaction between representative NSPs and a protein fraction from cucumber (Cucumis sativus L.) hypocotyls that has GA-binding characteristics consistent with those expected of GA receptors was studied. Analysis of in vitro equilibrium saturation data indicated the presence of only one class of high affinity [(3)H]GA(4) binding sites (K(d) approximately 30 nanomolar, n = 0.25 picomole per milligram of protein). In the presence of 6 or 60 micromolar 1-[3-chlorophthalimido]-cyclohexanecarboximide (AC-94,377), the K(d) for [(3)H]GA(4) increased, whereas the maximum number of saturable [(3)H]GA(4) binding sites did not change significantly. The dissociation of [(3)H]GA(4) from its binding sites was complex and was best described by a bi-exponential equation. AC-94,377 did not affect the rates of [(3)H]GA(4) dissociation from its binding sites. These results implied that AC-94,377 and [(3)H]GA(4) compete for binding to the same sites. A correlation was observed between the activity of over 20 NSPs in the cucumber hypocotyl bioassay and their in vitro affinity for the GA binding sites. Our observations lend further support to the notion that certain GA binding proteins in cucumber cytosol are GA receptors and also provide a molecular explanation for the GA-like in vivo activity of some NSPs.

RNA synthesis in germinating embryonic axes of soybean and wheat.

The rate of synthesis of RNA during early germination of wheat and soybean embryos was investigated by ascertaining the incorporation of radioactive uridine into RNA. In wheat embryos, where the lag period preceding rapid growth is 5.5 hours, there is a 2-fold increase in RNA synthesis between 1.5 and 5.5 hours, with half of the increase occurring by 3.5 hours. In soybean axes, where the lag period is 9.5 hours, the increased rate of RNA synthesis is 5.5-fold between 1.5 and 9.5 hours, with three fourths of this increase occurring after 4 hours.Analysis of the ratio of radioactivity incorporated into the 18S and 26S rRNAs of the germinating embryos provided a further measure of the increased rates of RNA synthesis. With wheat embryos, the 26S/18S ratio increased from 1.0 at 1.5 hours to 1.5 at 3.5 hours, while with the soybean axes, distinct ribosomal patterns were obtained only after 4 hours and the 26S/18S rRNA ratio increased from 0.4 at 4 hours to 1.0 at 9 hours. The extent of methylation of the rRNA synthesized at 4 and 9 hours in the soybean axes was similar, indicating that the methylating capacity of the axes is probably not rate limiting to rRNA synthesis. In both seed embryo systems the level of UTP increased 2 to 3-fold during the lag phase of germination. With wheat embryos, the time course of the increase in UTP correlated approximately with the change in the rate of RNA synthesis. With the soybean axes, however, the increase in the rate of RNA synthesis occurred predominantly after the rise in the level of UTP.

No Effect of 5-Fluorouracil on the Properties of Purified alpha-Amylase from Barley Half-seeds.

alpha-Amylase has been purified from de-embryonated seeds of barley (Hordeum vulgare L. cv. Betzes) which have been incubated on 10(-6)m gibberellic acid (GA(3)) following 3 days of imbibition in buffer. Incubation of the half-seeds in up to 10(-2)m 5-fluorouracil (5-FU) during the entire incubation period, including imbibition, had no effect on any of the following characteristics of purified alpha-amylase: thermal stability in the absence of calcium, molecular weight of the enzyme, isozyme composition, specific activity, or the amount of alpha-amylase synthesized by the aleurone tissue. The synthesis of rRNA and tRNA was strongly inhibited by 5-FU, indicating that the analog had entered the aleurone cells. These results are not in agreement with those of Carlson (Nature New Biology 237: 39-41 [1972]) who found that treatment of barley aleurone with 10(-4)m 5-FU prior to the addition of GA(3) resulted in decreased thermal stability of GA(3)-induced alpha-amylase and who interpreted this as evidence that the mRNA for alpha-amylase was synthesized during the imbibition of the aleurone tissue and independently of gibberellin action. Results of the present experiments indicate that the thermal stability of highly purified alpha-amylase is not altered by treatment of barley half-seeds with 5-FU, and that 5-FU cannot be used as a probe to examine the timing of alpha-amylase mRNA synthesis.

Control and Kinetics of Branch Root Formation in Cultured Root Segments of Haplopappus ravenii.

Branch root formation required only the presence of minerals, sucrose as a carbon source, and an auxin. The number of primordia formed was a function of auxin concentration. With naphthaleneacetic acid at 0.1 mg/l, up to 60 or more branches were formed per centimeter of Haplopappus ravenii root segment. Under our conditions, pea root segments formed only five or six branches per centimeter, but tomato and radish, like H. ravenii, formed large numbers of branches. Cytokinin inhibited branch formation, while gibberellic acid was without effect. Vitamins were not required for branch formation, although they enhanced elongation. Up to 5 days were required for the maximum number of stable branch primordia to form under the influence of naphthaleneacetic acid. If naphthaleneacetic acid was withdrawn earlier, fewer branch primordia developed. The requirement for a lengthy exposure to naphthaleneacetic acid, the kinetics of the response, and the ease with which naphthaleneacetic acid could be rinsed out of the tissue with consequent cessation of branch root formation, were similar to other hormone-regulated developmental systems. Anatomical and cytological studies were made of segments exposed for various times to auxin. The segments were mostly diarch, and branches formed obliquely to protoxylem poles. While primarily only pericycle-endodermis cells divided, both these and cortex cells responded in the first 24 hours exposure to naphthaleneacetic acid with enlarged nuclei and nucleoli, and a few cortical cells divided. Maximum nucleus and nucleolus size was reached approximately 9 hours after exposure to naphthaleneacetic acid. Branches rarely elongated more than 5 cm before their meristems died. The H. ravenii culture is maintained only by the frequent formation of new naphthaleneacetic acid-induced branches.