Light regulatory sequences are located within the 5'' portion of the Fed-1 message sequence.

We have previously shown that element(s) mediating a light-induced increase in the abundance of Fed-1 mRNA in the leaves of transgenic tobacco plants are located within the transcribed portion of the gene. As part of an effort to define the mechanism of this effect, we report here that cis-acting elements capable of mediating a 5-fold light-induced increase in the abundance of this mRNA are located within a region comprising the 5' leader and first third of the Fed-1 coding sequence. No activity was detected in the 3' untranslated region of the gene. In a gain-of-function assay, the 5' region was found to be capable of conferring light responsiveness on three different reporter sequences, although experiments with the gusA reporter were complicated by an apparent negative light effect on the stability of this mRNA. Deletion experiments show that at least one essential light regulatory element is located in the 5' untranslated region of Fed-1 between nucleotides +19 and +57. Additional Fed-1 sequences, including a portion of the protein coding region, are required to confer positive responsiveness on the gusA reporter. These additional sequences may include specific light regulatory elements or simply provide an environment in which the leader element can function normally.     

The 5' end of the pea ferredoxin-1 mRNA mediates rapid and reversible light-directed changes in translation in tobacco

Ferredoxin-1 (Fed-1) mRNA contains an internal light response element (iLRE) that destabilizes mRNA when light-grown plants are placed in darkness. mRNAs containing this element dissociate from polyribosomes in the leaves of transgenic tobacco (Nicotiana tabacum) plants transferred to the dark for 2 d. Here, we report in vivo labeling experiments with a chloramphenicol acetyl transferase mRNA fused to the Fed-1 iLRE. Our data indicate that the Fed-1 iLRE mediates a rapid decline in translational efficiency and that iLRE-containing mRNAs dissociate from polyribosomes within 20 min after plants are transferred to darkness. Both events occur before the decline in mRNA abundance, and polyribosome association is rapidly reversible if plants are re-illuminated. These observations support a model in which Fed-1 mRNA in illuminated leaves is stabilized by its association with polyribosomes, and/or by translation. In darkness a large portion of the mRNA dissociates from polyribosomes and is subsequently degraded. We also show that a significant portion of total tobacco leaf mRNA is shifted from polyribosomal to non-polyribosomal fractions after 20 min in the dark, indicating that translation of other mRNAs is also rapidly down-regulated in response to darkness. This class includes some, but not all, cytoplasmic mRNAs encoding proteins involved in photosynthesis.     

Premature termination codons destabilize ferredoxin-1 mRNA when ferredoxin-1 is translated

Ferredoxin-1 (Fed-1) mRNA is poorly translated in dark-treated tobacco (Nicotiana tabacum) leaves, resulting in destabilization of Fed-1 mRNA and a differential light/dark accumulation of the mRNA. Insertion of nonsense codons within the Fed-1 coding sequence disrupts the light regulation of Fed-1 mRNA abundance. Here we show that the nonsense codon effect results primarily from lowering the Fed-1 mRNA stability in light-treated leaf tissue and in rapidly growing tobacco cell cultures, but not in dark-treated leaf tissue. These results suggest that nonsense codons trigger a decay pathway distinct from that seen for Fed-1 mRNA in the dark. We propose that nonsense-mediated decay of nonsense-containing Fed-1 mRNA occurs in light-treated leaves and in non-photosynthetic tobacco culture cells where Fed-1 mRNA is being actively translated.     

Analysis of regulatory elements involved in the induction of two tobacco genes by salicylate treatment and virus infection.

Tobacco genes encoding the PR-1a protein and a glycine-rich protein are expressed after treatment of plants with salicylate or infection with tobacco mosaic virus. Upstream sequences of these genes were fused to reporter genes, and these constructs were used to transform tobacco. Upstream sequences of the PR-1a gene of 689 base pairs or longer were sufficient for induction of the reporter gene in tobacco mosaic virus-inoculated leaves, systemically induced leaves from infected plants, and leaves treated with salicylate. No such induction was found with upstream sequences of 643 base pairs or shorter of the PR-1a gene. When the PR-1a upstream sequence from nucleotides -625 to -902 was fused to the cauliflower mosaic virus 35S core promoter, a construct was obtained that conferred tobacco mosaic virus and salicylate inducibility to the reporter gene in transgenic plants. This confirmed the localization of tobacco mosaic virus- and salicylate-responsive elements between positions -643 and -689 in the PR-1a promoter. With the glycine-rich protein gene, an upstream sequence of 645 base pairs was sufficient for tobacco mosaic virus and salicylate inducibility of the reporter gene, whereas constructs containing 400 base pairs or fewer of the glycine-rich protein promoter were largely inactive.