Identification and characterization of circadian clock genes in a native tobacco, Nicotiana attenuata

Sep 26, 2012BMC plant biology

Finding and describing daily rhythm genes in wild tobacco plants

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Abstract

Three core circadian clock genes, NaLHY, NaTOC1, and NaZTL, were identified in Nicotiana attenuata.

Orthologous genes of core circadian clock components were isolated based on protein similarity and transcript levels.
Transcript levels of NaLHY peaked at dawn, while NaTOC1 peaked at dusk under long day conditions.
Ectopic expression of NaLHY and NaZTL in Arabidopsis led to elongated hypocotyl and late-flowering traits.
Protein interactions between NaTOC1 and NaZTL were confirmed using yeast two-hybrid assays.
Silencing NaTOC1 in N. attenuata resulted in late-flowering phenotypes under long day conditions.

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BACKGROUND: A plant's endogenous clock (circadian clock) entrains physiological processes to light/dark and temperature cycles. Forward and reverse genetic approaches in Arabidopsis have revealed the mechanisms of the circadian clock and its components in the genome. Similar approaches have been used to characterize conserved clock elements in several plant species. A wild tobacco, Nicotiana attenuata has been studied extensively to understand responses to biotic or abiotic stress in the glasshouse and also in their native habitat. During two decades of field experiment, we observed several diurnal rhythmic traits of N. attenuata in nature. To expand our knowledge of circadian clock function into the entrainment of traits important for ecological processes, we here report three core clock components in N. attenuata.

RESULTS: Protein similarity and transcript accumulation allowed us to isolate orthologous genes of the core circadian clock components, LATE ELONGATED HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION 1/PSEUDO-RESPONSE REGULATOR 1 (TOC1/PRR1), and ZEITLUPE (ZTL). Transcript accumulation of NaLHY peaked at dawn and NaTOC1 peaked at dusk in plants grown under long day conditions. Ectopic expression of NaLHY and NaZTL in Arabidopsis resulted in elongated hypocotyl and late-flowering phenotypes. Protein interactions between NaTOC1 and NaZTL were confirmed by yeast two-hybrid assays. Finally, when NaTOC1 was silenced in N. attenuata, late-flowering phenotypes under long day conditions were clearly observed.

CONCLUSIONS: We identified three core circadian clock genes in N. attenuata and demonstrated the functional and biochemical conservation of NaLHY, NaTOC1, and NaZTL.

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Identification and characterization of circadian clock genes in a native tobacco, Nicotiana attenuata

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