HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES1 is required for circadian periodicity through the promotion of nucleo-cytoplasmic mRNA export in Arabidopsis

Abstract

Cold acclimation has been shown to be attenuated by the degradation of the INDUCER OF CBF EXPRESSION1 protein by the E3 ubiquitin ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES1 (HOS1). However, recent work has suggested that HOS1 may have a wider range of roles in plants than previously appreciated. Here, we show that hos1 mutants are affected in circadian clock function, exhibiting a long-period phenotype in a wide range of temperature and light environments. We demonstrate that hos1 mutants accumulate polyadenylated mRNA in the nucleus and that the circadian defect in hos1 is shared by multiple mutants with aberrant mRNA export, but not in a mutant attenuated in nucleo-cytoplasmic transport of microRNAs. As revealed by RNA sequencing, hos1 exhibits gross changes to the transcriptome with genes in multiple functional categories being affected. In addition, we show that hos1 and other previously described mutants with altered mRNA export affect cold signaling in a similar manner. Our data support a model in which altered mRNA export is important for the manifestation of hos1 circadian clock defects and suggest that HOS1 may indirectly affect cold signaling through disruption of the circadian clock.

Figure 1.

Mutations to HOS1 Result in a Long-Period Circadian Phenotype across a Range of Physiological Temperatures and Light Conditions. (A) Schematic diagram showing positions of the hos1 mutations across the HOS1 locus. Exons are shown in black boxes and introns in black lines. The position of the hos1 mutations and allele designations are labeled. Triangles represent T-DNA insertions and the asterisk represents point mutations. The primer positions in exons 2 and 10 are marked to illustrate the positions of qPCR primers used to produce the data in Figure 1B. (B) qPCR data showing reduced levels of HOS1 mRNA expression in each of the hos1 mutants studied. Primers for exon 2 (dark gray bars) are located upstream of and exon 10 (white bars) are located downstream of all hos1 mutations studied (see [A]). Seedlings were grown for 10 d before being harvested at dawn. Data are averages of biological triplicate ± se normalized for transcript abundance to UBQ10. Stars indicate significant difference to the respective wild type as measured by Student’s t test where one star indicates P ≤ 0.05 and two stars P ≤ 0.01. (C) to (F) Delayed fluorescence assays comparing the wild type (Col) to the hos1 alleles. Seedlings were entrained for 7 d in 12L:12D white-light cycles at 22°C before transfer to experimental conditions of constant 12, 17, or 27°C and constant red light ([C] and [D]) or constant blue light ([E] and [F]). Delayed fluorescence was then measured in several hos1 mutants (white circles) compared with wild-type data (Col, black circles). (C) and (E) Plots showing period estimations produced via mathematical analysis plotted versus RAE for the wild type (Col) versus the hos1 mutants. Data are from individual wells containing 10 to 20 seedlings. (D) and (F) Plot illustrating the effect of temperature on the variable weighted mean of the period for the wild type (Col) versus hos1 mutants. Data are variable weighted mean ± se. Stars indicate significant difference to the respective wild type as measured by Student’s t test where one star indicates P ≤ 0.05 and two stars P ≤ 0.01.

Figure 2.

The Long-Period Phenotype in hos1 Does Not Require Selected Individual Clock Components. (A) Delayed fluorescence assays comparing the wild type (Col, black circles) to hos1 (gray circles) and the single (white circles) and double (red circles) mutants. Seedlings were entrained for 16 d in 12L:12D white-light cycles at 22°C before transfer to experimental conditions of constant 12, 17, or 27°C and constant red light. Plots show period estimations produced via mathematical analysis plotted versus RAE. Data are from individual wells containing 10 to 20 seedlings. (B) Plots illustrating the effect of temperature on the variable weighted mean of the period for the wild type (Col, black circles) to hos1 (gray circles) and the single (white circles) and double (red circles) mutants. Data are variable weighted mean ± se. Stars indicate significant difference to the respective wild type as measured by Student’s t test where one star indicates P ≤ 0.05 and two stars P ≤ 0.01.

Figure 3.

Under Constant Red Light, HOS1 Is Required for Correct Expression of Clock Genes. Plants were grown for 7 d under 12L:12D white-light cycles at 22°C followed by 2 d in 12L:12D red-light cycles at 17°C before being transferred to constant red light at 17°C for sampling in biological triplicate at the Zeitgeber time (ZT) times indicated. The cDNA were analyzed by qPCR three times for each biological replicate. Data presented are the average of three biological repeats ± se. Transcript abundance levels were normalized to ACT7. Blue diamonds with solid lines are data from the wild type (Col), whereas red squares with dotted lines are data from hos1-3.

Figure 4.

hos1 Mutants Exhibit Long Period but Robust Protein and Promoter Rhythms Despite Poor mRNA Rhythm Robustness. (A) The expression of endogenous CAB2, CCR2, and LHY in the wild type (Col) and hos1-3 in constant red light (see Methods). Data are the average of the three biological repeats ± se. Transcript abundance levels were normalized to ACT7. Samples were taken at the Zeitgeber time (ZT) indicated. Blue diamonds are data from Col, whereas red squares show data from hos1-3. (B) Plots to show the normalized mean CAB2:LUC, CCR2:LUC, and LHY:LUC luminescence profiles for the wild type and hos1-3 mutant. Data are presented as means ± se. Transgenic seedlings containing the CAB2:LUC or CCR2:LUC reporter were entrained for 7 d in 12L:12D white-light cycles at 22°C before transferred to experimental conditions of constant 17°C and 2 d of 12L:12D red-light cycles followed by 6 d of constant red light. Transgenic seedlings containing the LHY:LUC reporter were entrained for 6 d in 12L:12D white-light cycles at 22°C before transferred to experimental conditions of constant white light at room temperature. hos1-3 (gray filled circles) is compared with wild-type data (black filled circles). (C) Plots to show the period estimations for CAB2:LUC, CCR2:LUC, and LHY:LUC versus RAE for the wild type versus the hos1-3 mutant. (D) Immunoblot analysis showing endogenous LHY levels in the wild type (Col) compared with hos1-3, with LHY-OX and lhy-20 lines as positive and negative controls, respectively. The blots were reprobed using an antibody against UGPase for the loading control. Plants were grown for 7 d under 12L:12D white-light cycles at 22°C followed by 2 d in 12L:12D red-light cycles at 17°C before being transferred to constant red light at 17°C for sampling at the Zeitgeber time (ZT) times indicated. These images are representative images from technical duplicates.

Figure 5.

HOS1 Is Required for Nucleo-Cytoplasmic mRNA Export. (A) In situ hybridization with a fluorescein-labeled oligo(dT) probe in wild-type, hos1, los4, ztl (long period), and toc1 prr9 prr7 prr5 (t975) quadruple mutant (arrhythmic) roots. Plants were fixed at dawn (unless otherwise indicated) after 2 weeks of growth under 12L:12D white-light cycles. Bars = 50 µm. (B) In situ hybridization with a fluorescein-labeled oligo(dT) in wild-type, hos1, and sar1 roots counterstained with DAPI. Bars = 10 µm.

Figure 6.

Mutants Resulting in Altered mRNA Export Exhibit Long-Period Circadian Phenotypes and Alterations to Clock Gene Expression Similar to hos1. (A) Delayed fluorescence was performed on a range of nuclear pore–associated mutants. Seedlings were grown in 12L:12D white-light cycles for 16 d prior to transferring to constant 17°C and constant red light for analysis. Each plot shows period estimations plotted versus RAE comparing the wild type (WT; black circles) and mutant (gray circles). (B) and (C) RNA analysis comparing the expression of clock genes in hos1 alleles, sar1-4 and los4-1, to their appropriate wild types. Plants were grown for 2 weeks in 12L:12D white-light cycles and harvested in triplicate at dawn CT0 (B) or dusk CT12 (C). Data are shown relative to the wild-type values for comparison. Data are averages of biological triplicate ± se normalized for transcript abundance to TUB9 (B) or UBQ10 (C). Bar colors are as follows: hos1-3, blue; sar1-4, red; hos1-1, green; and los4-1, purple.

Figure 7.

Expression of Cold Signaling Genes Analyzed in Parallel Indicates Similar Perturbations in hos1 as in sar1-4 and los4-1 and This Perturbation Does Not Require LHY. Expression analysis of CBF3 ([A], [C], and [E]) and RD29A ([B], [D], and [F]) in the wild type (Col, dark-blue diamonds; C24, light-blue diamonds), hos1 alleles (hos1-3, red squares; hos1-1, pink squares), mRNA export mutants (sar1-4, dark-green triangles; los4-1, light-green triangles), lhy (lhy-20, purple circles), and hos1-3 lhy-20 double mutants (hos1lhy, orange Xs). Plants were grown for 2 weeks under 12L:12D white-light cycles and harvested at dawn (CT0), 8 h after dawn (CT8), or at dusk (CT12). Data are averages of biological triplicate ± se normalized for transcript abundance to UBQ10.