Expression conservation within the circadian clock of a monocot: natural variation at barley Ppd-H1 affects circadian expression of flowering time genes, but not clock orthologs

Abstract

Background: The circadian clock is an endogenous mechanism that coordinates biological processes with daily changes in the environment. In plants, circadian rhythms contribute to both agricultural productivity and evolutionary fitness. In barley, the photoperiod response regulator and flowering-time gene Ppd-H1 is orthologous to the Arabidopsis core-clock gene PRR7. However, relatively little is known about the role of Ppd-H1 and other components of the circadian clock in temperate crop species. In this study, we identified barley clock orthologs and tested the effects of natural genetic variation at Ppd-H1 on diurnal and circadian expression of clock and output genes from the photoperiod-response pathway.

Results: Barley clock orthologs HvCCA1, HvGI, HvPRR1, HvPRR37 (Ppd-H1), HvPRR73, HvPRR59 and HvPRR95 showed a high level of sequence similarity and conservation of diurnal and circadian expression patterns, when compared to Arabidopsis. The natural mutation at Ppd-H1 did not affect diurnal or circadian cycling of barley clock genes. However, the Ppd-H1 mutant was found to be arrhythmic under free-running conditions for the photoperiod-response genes HvCO1, HvCO2, and the MADS-box transcription factor and vernalization responsive gene Vrn-H1.

Conclusion: We suggest that the described eudicot clock is largely conserved in the monocot barley. However, genetic differentiation within gene families and differences in the function of Ppd-H1 suggest evolutionary modification in the angiosperm clock. Our data indicates that natural variation at Ppd-H1 does not affect the expression level of clock genes, but controls photoperiodic output genes. Circadian control of Vrn-H1 in barley suggests that this vernalization responsive gene is also controlled by the photoperiod-response pathway. Structural and functional characterization of the barley circadian clock will set the basis for future studies of the adaptive significance of the circadian clock in Triticeae species.

Figure 1

Phylogenetic relationships of circadian clock related proteins. Neighbor-joining phylogenetic trees were built from the protein alignments of the indicated sequences. a) PRR gene family, b) CCA1/LHY gene family, c) GI genes. Accession numbers are given in Table 1. Bootstrap values were calculated from 10,000 permutations.

Figure 2

Diurnal and circadian expression of PRR genes in the spring cultivar Scarlett (ppd-H1 ) and the introgression line S42 IL-107 (Ppd-H1 ) under long day and free running conditions. Scarlett (solid line) and S42-IL107 (dashed line) were grown under long day conditions (16 hours light - left panel) for two weeks and then released in continuous light (right panel). Transcript accumulation was measured at two-hour intervals (one-hour interval at the end of the day and beginning of the night/subjective nights) by qRT-PCR analysis of specific genes and normalized to HvActin. Values represent the average of two technical replicates ± standard error. Black and grey bars indicate objective and subjective nights.

Figure 3

Diurnal and circadian expression of HvCCA1 and HVGI in the spring cultivar Scarlett (ppd-H1 ) and the introgression line S42-IL107 (Ppd-H1) under long day and constant light. Scarlett (solid line) and S42-IL107 (dashed line) were grown under long day conditions (16 hours light - left panel) for two weeks and then released in continuous light (right panel). Transcript accumulation was measured at two hour intervals (one-hour interval at the end of the day and beginning of the night/subjective nights) by qRT-PCR analysis of specific genes and normalized to HvActin. Values represent the average of two technical replicates ± standard error. Black and grey bars indicate objective and subjective nights.

Figure 4

Diurnal and circadian expression of flowering genes in the spring cultivar Scarlett (ppd-H1 ) and the introgression line S42 IL-107 (Ppd-H1 ) under long day and free running conditions. Scarlett (solid line) and S42-IL107 (dashed line) were grown under long day conditions (16 hours light - left panel) for two weeks and then released in continuous light (right panel). Transcript accumulation was measured at two hour intervals (one-hour interval at the end of the day and beginning of the night/subjective nights) by qRT-PCR analysis of specific genes and normalized to HvActin. Values represent the average of two technical replicates ± standard error. Black and grey bars indicate objective and subjective nights.

Figure 5

Diurnal and circadian expression of clock output genes HvCABII and HvGRP7 in the spring cultivar Scarlett (ppd-H1) and the introgression line S42 IL-107 (Ppd-H1 ) under long day and free running conditions. Scarlett (solid line) and S42-IL107 (dashed line) were grown under long day conditions (16 hours light - left panel) for two weeks and then released in continuous light (right panel). Transcript accumulation was measured at two hour intervals (one-hour interval at the end of the day and beginning of the night/subjective nights) by qRT-PCR analysis of specific genes and normalized to HvActin. Values represent the average of two technical replicates ± standard error. Black and grey bars indicate objective and subjective nights.