Leaf protoplasts isolated by enzymatic digestion [3 percent Cellulysin in osmoticum (OS-0.6M sorbitol, 5 mM CaSO4 and 5 M MgCl2)] from seven-day old seedlings of a maize hybrid and its inbred parents (N28 and Mo17) were utilized for a study of RNA synthesis as a measure of heterosis. The influence of the growth hormone IAA and of ethanol (EtOH) at a concentration of 10-7 molar upon tritiated uridine (3H-U) incorporation were measured over a seven hour period.
Incorporation of 3H-leucine into protein over a 6 hour period was used as a measure of cell viability It was found that the kinetics of 3H-leucine incorporation paralleled those of 3H-U (Figure 1) and that cells were retaining their viability over the time of the experiment.
No statistically significant differences were found in the quantity of RNA isolated from N28, Mo17 and their hybrid when their protoplasts were incubated in OS (Table 1). Treatment of N28, Mo17 and of the hybrid with EtOH produced an increase of 63.4, 23.5 and 79.8 percent respectively in the content of total RNA. Treatment of the same strains by 10-7M IAA produced increases of 82.8, 42.1 and 109.7 percent respectively for N28, Mo17 and the hybrid. The IAA effect was, however, confounded because small quantities of EtOH were utilized to dissolve this hydrophobic compound into OS. Experiments are now in progress to measure the effect of IAA alone.
Oligo (dt)- columns were used to separate poly(A) + mRNA from the non-poly(A) species (Table 2). The quantity of poly(A) + RNA did not differ significantly between the strains when protoplasts were incubated in OS. Treatment with 10-7M IAA and EtOH increased the synthesis of poly(A) + mRNA species with the exception of Mo17, which showed a decrease of 12 percent when exposed to EtOH only. N28 and the hybrid showed a 9 percent increase each. The effect of EtOH may be related to alterations in membrane permeability which permitted greater entry of 3H-U into the protoplasts. This "leaky" response may not have been a characteristic of the Mo17 plasmalemma.
The hybrid manifested the greatest increase in poly(A) + RNA with IAA. Although this result is in general agreement with Nebiolo et al. (Plant Sci. Lett. 28:195-206, 1982/83) it was less than that observed with application of 10-7M gibberellic acid (GA3) to maize coleoptile cells. This variation may be related in part to differences in the 260OD/280OD ratio between the two studies and to the fact that auxins are known to specifically increase (100-250%) rRNA content but induce a smaller increase (25-50%) of poly(A) + RNA (Ann. Rev. Plant Physiol. 28:537-564, 1977). GA3 is also a specific inducer of poly (A) + RNA in immature plant tissue. The difference may therefore result from the specific action of each hormone. A significant difference also exists between the EtOH and the IAA treatment. The IAA treated protoplasts for N28, Mo17 and the hybrid have 19.2, 40.2 and 19.1 percent more poly (A) + RNA respectively than do the EtOH treated protoplasts. The increased synthesis may have resulted from enhanced activity and/or quantity of DNA-dependent RNA polymerase II produced by the IAA treatment or increased stability of the poly(A) + RNA. Part of the increase may also result from the availability of increased 3H-U pools. However, regardless of treatment the hybrid manifested the greatest potential for 3H-U incorporation.
Table 1. RNA extracted from 10-7M 1AA treated and untreated corn mesophyll protoplasts.
Valentin Ulrich and Patrick Smith
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