Many experimental procedures require the manipulation of tissue in order that the particular aspects of interest be made more readily available for study. When such procedures are employed it is important to determine whether or not they will affect the results obtained and if so, take this into account when evaluations are being made. Recent evidence has shown that even relatively minor manipulation of some tissues may result in readily observable changes in the patterns of protein synthesis (Currie and White, 1981).

We report some preliminary results with maize protoplasts which indicate that alterations in the patterns of newly synthesized polypeptides are associated with the preparation of the protoplasts from maize leaf tissue. The plant material employed was a commercial hybrid (PX-11) grown under standard greenhouse conditions. Protoplasts were obtained from 25-33 day old plants utilizing a modified version of a method developed earlier in our laboratory (Meadows and Walden, MGCNL, 1978). Young, folded leaf tissue was excised, macerated and placed in a freshly prepared osmoticum of 0.4 M mannitol. This tissue was subsequently transferred to an enzyme solution consisting of 0.4 M mannitol, 5 mM CaCl₂*2H₂O and 0.5% cellulase pH 5.7, in which it was allowed to incubate in the dark at 27 C, with shaking over a 3-1/2 hr period with two changes of enzyme solution. Following enzymatic digestion, the crude protoplast fractions were combined, centrifuged at low speed and the pellet resuspended in a wash medium of 0.4 M mannitol and 5 mM CaCl₂*2H₂O.

The protein synthetic capabilities of freshly isolated protoplasts were examined through the addition of various concentrations of ¹⁴C-leucine for different lengths of time. Following labelling, the protoplasts were rinsed in wash medium, pelleted and protein extracted through homogenization in plant extraction buffer (PEB - Hughes, Baszczsynski and Ketola-Pirie, MGCNL, 1981) using a glass homogenizer with Teflon pestle. Incorporation of the radioactive precursor was determined through scintillation counting of TCA precipitated samples. The results revealed an almost complete absence of protein synthesis in these cells. In contrast to this, intact leaf tissue showed a high degree of incorporation when labelled under similar conditions in glass distilled water and homogenized as described above, after previously grinding the tissue with a ceramic mortar and pestle. A similar analysis performed with tissues taken at various stages in the course of protoplast preparation revealed a steady decrease in the protein synthetic capabilities associated with an increasing degree of tissue manipulation.

Separation of polypeptides extracted from intact, macerated and enzyme treated leaf tissue was achieved through SDS polyacrylamide gel electrophoresis of protein samples on 10-20% linear gradient gels (U. K. Laemmli, 1970). Examination of the stained gel patterns showed no significant differences between tissues of the various treatments. However, certain changes in the patterns of newly synthesized polypeptides were observed between the different treatments when fluorographic analysis was undertaken. These alterations were most evident in a few higher molecular weight bands and appear to represent either changes in the amounts of certain polypeptides synthesized and/or novel protein synthesis.

It is possible that some of these differences observed may in fact reflect the synthesis of repair proteins initiated as a result of cell injury. If this is the case, this phenomenon may be viewed as a stress type response. Investigations are currently underway to further characterize this response and also to compare it with those elicited by various other forms of stress.

J. G. Boothe, D. B. Walden and B. G. Atkinson

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