The Fate of 14C-Labelled Hurnic Substances in Rice Cells in Culture

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Summary Peat and aquatic humic acids covering a molecular mass range from approximately 40,000 to 8,000 were found to stimulate the growth of rice calli and rice cells in suspension culture. Growth stimulation was investigated as a function of humic acid concentration up to 100 ppm and growth time up to 60 days. The humic acids increase calli weight by approximately 80% and cell numbers in culture approximately 160 × after 42 days. The humic acids had no effect on the protein and DNA contents of rice cells; however, by using the radioactive 14 C N-methylamide derivatives of the humic acids it has been found that both rice cell protein and DNA become radiolabelled during culture. The incorporation of 14 C into DNA is approximately 10× that into protein. Extraction of cell DNA followed by hydrolysis with DNAase I and HPLC analysis of the resulting deoxynucleotides has demonstrated that approximately 70% of the 14 C activity is incorporated into the purine deoxyribonucleotides dAMP and dGMP. Analysis of the humic acids in the culture medium during growth showed that while a major proportion of the humic molecules remained, there was some breakdown to lower molecular mass species and possibly some release of the radiolabelled methylamine. However, the asymmetric incorporation of the major proportion of the 14 C into the purine-containing deoxynucleotides did not arise from the putative release of the label from the humic acids, since 14 C-methylamine itself incorporated 14 C evenly into the four deoxynucleotides. It is suggested that aromatic moeties derived from the humic acids could result in phenylalanine and tyrosine entering the metabolic pathways to glycine and aspartate, which are used in the de novo synthesis of the deoxynucleotide bases. The incorporation of 14 C from 14 C-glycine and 14 C-aspartate into rice DNA deoxynucleotides was confirmed in control experiments. The results suggest that rice cells can use humic breakdown products in the synthesis of proteins and DNA.