Plant Gene Transcription Research Articles

Shedding light on clock controlled cab gene transcription in higher plants

Research from several laboratories has revealed that the circadian clock regulates the transcription of many higher plant genes. Amongst the most well characterized of these genes are those that encode the chlorophyll a/b binding (Cab) proteins of the light harvesting complexes of photosystem II. Analysis of cab gene expression in both etiolated and green plants reveals a complex interaction between phytochrome(s) and the circadian clock, in which phytochrome appears to regulate the level of cab transcription and the circadian clock regulates the phase. The development of a non-invasive assay for cab transcription based on firefly luciferase expression in transgenic plants has not only made it possible to analyze clock regulated transcription in living plants, but also provides a means to screen for clock mutants in Arabidopsis.

The expression of barley ?-amylase genes inXenopus laevis oocytes

The synthesis and secretion of α-amylase (EC 3.2.1.1) fromXenopus laevis oocytes injected with plasmids containing barley α-amylase complementary DNA (cDNA), genomic DNA, or synthetic α-amylase mRNA were studied. α-Amylase accumulated within the oocytes beginning 12 h after injection of DNA and in the medium 12 h later as a result of secretion. S1 mapping showed that the transcription of genomic DNA was initiated at the same site in oocytes as in barley aleurone, but that the transcription of cDNAs was less precise than that of genomic DNA. The α-amylase secreted by oocytes injected with either RNA or DNA had a molecular mass (Mr) of 44000 daltons (Da) and was indistinguishable from native barley α-amylase in size, isoelectric point, antigenicity and enzymatic activity. Isoelectric focussing showed that two enzymatically active isoforms of α-amylase were synthesized and secreted from oocytes injected with synthetic RNA or DNA. The results permit us to assign specific electrophoretic bands to specific cDNA clones. We conclude that theXenopus oocyte is a promising surrogate system for the study of transcription and translation of plant genes.

Characterization of the Arabidopsis Adh G-box binding factor.

Protein-DNA interaction at an inverted repeat of the sequence 5'-GTGG-3' (G-box) has been associated with the transcription of several plant genes [Giuliano, G., et al. (1988). Proc. Natl. Acad. Sci. USA 85, 7089-7093; Ferl, R.J., and Laughner, B.H. (1989). Plant Mol. Biol. 12, 357-366; Schulze-Lefert, P., et al. (1989). EMBO J. 8, 651-656]. We characterized the binding of the Arabidopsis G-box binding factor (GBF) from whole-cell extracts and fractionated extracts to the G-box of alcohol dehydrogenase (Adh) using gel mobility shift assays. DNase I footprinting localized the region of GBF/G-box interaction to two sites, one apparent high-affinity binding site (-227 to -201) and a possible low-affinity binding site (-193 to -182). DNA-protein cross-linking demonstrated that the G-box is bound by proteins of two sizes, 31 kilodaltons and 18 kilodaltons. In addition, we found that in vitro the interaction of GBF from Arabidopsis suspension cultures or leaves with the Adh G-box is indistinguishable, and that there is evidence of multiple protein-protein interactions.

The expression of barley ?-amylase genes in Xenopus laevis oocytes

The synthesis and secretion of α-amylase (EC 3.2.1.1) from Xenopus laevis oocytes injected with plasmids containing barley α-amylase complementary DNA (cDNA), genomic DNA, or synthetic α-amylase mRNA were studied. α-Amylase accumulated within the oocytes beginning 12 h after injection of DNA and in the medium 12 h later as a result of secretion. S1 mapping showed that the transcription of genomic DNA was initiated at the same site in oocytes as in barley aleurone, but that the transcription of cDNAs was less precise than that of genomic DNA. The α-amylase secreted by oocytes injected with either RNA or DNA had a molecular mass (Mr) of 44000 daltons (Da) and was indistinguishable from native barley α-amylase in size, isoelectric point, antigenicity and enzymatic activity. Isoelectric focussing showed that two enzymatically active isoforms of α-amylase were synthesized and secreted from oocytes injected with synthetic RNA or DNA. The results permit us to assign specific electrophoretic bands to specific cDNA clones. We conclude that the Xenopus oocyte is a promising surrogate system for the study of transcription and translation of plant genes.

A light-entrained circadian clock controls transcription of several plant genes.

Diurnal oscillations in steady-state mRNA levels and transcription rates were measured for seven transcripts (five of which encode chloroplast-localized proteins) in tomato seedlings: photosystem I and photosystem II chlorophyll a/b binding proteins (CAB/I and CAB/II), small subunit of RuBisCO (RBCS), actin, subunit II of the photosystem I reaction center (PSAD), subunit I of the photosystem II oxygen-evolving enzyme (OEE1), and a biotin-binding protein of unknown function. CAB/II mRNA levels were found to oscillate greater than 20-fold, showing a peak at noon, while only marginal diurnal oscillations are seen in RBCS transcripts. The oscillations are at least partially controlled at the transcriptional level. Transcription rates of both CAB/II and RBCS, measured by nuclear run-on experiments, were found to oscillate, with a peak around 8 a.m. Transcription rates of the 'biotin' clone also oscillated, with a peak around noon. Transfer of plants to constant darkness or constant light conditions alters the amplitude of the transcriptional oscillation, but does not abolish it, suggesting that it is at least partially controlled by a circadian clock. The oscillations are still visible after three days in complete darkness, and have a period very close to 24 h. The oscillator phase can be reset by out-of-phase light treatment.

Induction of Responsiveness to Octopine and Lysopine in Crown-Gall Tumors

Bean leaf tumors induced by Agrobacterium tumefaciens strain 181 show enhanced growth in response to octopine or lysopine only if one of these compounds is present during the period of tumor induction. Either compound applied during this period results in tumors which subsequently respond to both. The combined action of the bacterium plus octopine or lysopine at induction is proposed to induce transcription of plant genes coding for enzymes involved in the degradation and/or biosynthesis of octopine and lysopine.