A high zinc concentration of 520 M, approximately 100 times that used most often in standard plant tissue culture media, was found to be superior in liquid callus cultures of japonica rice, increasing growth to 146% compared with standard N6 medium. At the same time, the internal zinc concentration increased 40 times in fast growing cells; soluble protein doubled, and free amino acids decreased. Under zinc-free conditions the cultures slowed in growth, and several free amino acids such as aspartic acid, glutamic acid, asparagine, and glutamine accumulated. We suggest that zinc acts as a direct regulatory factor in inducing auxin activity, but not auxin levels, making high internal zinc accumulation mandatory if high auxin concentrations are required as in rice callus cultures.

In many developmentally and functionally important higher plant plastid genes, expression depends on a specific nuclear-encoded RNA polymerase (NEP). Molecular mechanisms for NEP-mediated gene expression are poorly understood. We have improved a transient expression assay based on biolistics and the dual-luciferase reporter technique, which facilitated investigations into the regulation of plastid genes in vivo. We scrutinized the 5'-flanking region and the 5'-untranslated region (5'UTR) of accD, a plastid gene encoding a subunit of the prokaryotic-type acetyl-CoA carboxylase which is transcribed exclusively by NEP. The results indicated that two AT-rich sequences, one of them containing two overlapping YRTA-like motifs, were essential for accD expression in vivo. The results also revealed that the length of the 5'UTR rather than a particular sequence element was a determinant for the level of accD expression. Because transcripts accumulated in proportion to reporter enzyme activity and protein levels, and transcript degradation rates were independent of the nature of the 5'UTR, it was unlikely that the 5'UTR acts as a translational enhancer or a stabilizer of the transcripts. Therefore, the length of 5'UTR might be a factor contributing to the efficiency of NEP-dependent transcription in plastids.

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