Incorporation of radioactively labeled amino acids into protein in leaves does not require prior surface abrasion

Research interests in our laboratory require examination of the pattern of polypeptide synthesis in leaf tissue throughout the life of the plant. We find it desirable to label the leaf tissue of the intact plant since previous work in our laboratory (J. G. Boothe et al., MNL 56:113, 1982) suggested that injury to the leaf can induce a change in the pattern of polypeptide synthesis. Other workers have described a method for incorporating labeled amino acids into leaves of intact plants following abrasion of the upper leaf surface (P. Cooper et al., Plant Physiol. 75:431, 1984).

Various protocols for incorporation of 35S-Methionine into proteins of leaf tissue were conducted last summer. We compared the incorporation of 35S-Methionine into TCA-precipitable material of leaf tissue with and without abrasion immediately before delivery of the label. Abrasion was achieved by lightly rubbing a 4 cm x 2 cm area of the upper leaf surface with fine sandpaper (No. 400). 35S-Methionine (10 µCi) was applied to this area. Labeling time was 1 h; during the period of labeling, temperature was monitored using a thermo-couple and recorded every 10 min using a digital data logger (DL-2020, ECD). The average temperature over this time period was 20 C. Proteins were extracted, TCA precipitated and liquid scintillation counted as described previously (C. L. Baszczynski et al., Can. J. Biochem. 60:569, 1982). Representative data are:
Leaf Sample Treatment cpm/µl/2 cm2 sample
1 Abrasion 15,873
2 Abrasion 13,918
3 None 13,987
4 None 9,925

We conclude that abrasion is not required to achieve incorporation of 35S-Methionine into proteins of maize leaf tissue. We have omitted the use of an abrasive from our procedure since the injury response may alter the pattern of peptides synthesized.

C. A. Rees, T. G. Crowe and D. B. Walden

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