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. 2019 Nov 6;182(1):204–214. doi: 10.1104/pp.19.00710

A Natural Variant of miR397 Mediates a Feedback Loop in Circadian Rhythm1,[OPEN]

Yan-Zhao Feng a,2, Yang Yu a,2,3,✉, Yan-Fei Zhou a, Yu-Wei Yang a,✉, Meng-Qi Lei a, Jian-Ping Lian a, Huang He a, Yu-Chan Zhang a, Wei Huang b, Yue-Qin Chen a,3,4
PMCID: PMC6945863  PMID: 31694901

Flowering time in Arabidopsis is affected by a circadian clock-regulating feedback loop involving miR397b and its target gene CASEIN KINASE II SUBUNIT B3.

Abstract

MicroRNAs (miRNAs) are small noncoding RNAs of ∼21 nt in length, which have regulatory roles in many biological processes. In animals, proper functioning of the circadian clock, which is closely linked to the fitness of almost all living organisms, is regulated by miRNAs. However, to date, there have been no reports of the roles of miRNA in regulation of the plant circadian rhythm. Here, we report a natural variant of miR397 that lengthens the circadian period and controls flowering time in Arabidopsis (Arabidopsis thaliana). Highly conserved among angiosperms, the miRNA miR397 has two members in Arabidopsis: miR397a and miR397b. However, only miR397b significantly delayed flowering. Our results suggest that miR397b controls flowering by targeting CASEIN KINASE II SUBUNIT BETA3 (CKB3), in turn modulating the circadian period of CIRCADIAN CLOCK ASSOCIATED1 (CCA1). We further demonstrated that CCA1 directly bound to the promoter of MIR397B and suppressed its expression, forming a miR397b-CKB3-CCA1 circadian regulation feedback circuit. Evolutionary analysis revealed that miR397b is a newly evolved genetic variant in Arabidopsis, and the miR397b targeting mode may have a role in enhancing plant fitness. Our results provide evidence for miRNA-mediated circadian regulation in plants and suggest the existence of a feedback loop to manipulate plant flowering through the regulation of circadian rhythm.

Living on a rotating planet, organisms have evolved to form an endogenous clock that oscillates with a period of ∼24 h, i.e. the circadian clock. In plants, the circadian clock controls multiple physiological processes, including light perception and leaf movement, stomatal opening, hypocotyl elongation, and the timing of flowering (Barak et al., 2000; Li et al., 2011). The molecular mechanisms of circadian clock have been widely studied in Arabidopsis (Arabidopsis thaliana). Three major parts of the circadian system (input pathways, central oscillators, output pathways) interlock with each other, forming sophisticated transcriptional feedback loops to ensure that life processes are synchronized to ambient light and temperature (Harmer, 2009; Cui et al., 2013).

Plant circadian regulation network consists of a variety of proteins, such as CIRCADIAN CLOCK ASSOCIATED1 (CCA1), TIMING OF CAB EXPRESSION1 (TOC1), and other pseudoresponse regulators (PRRs) like PRR5, PRR7, and PRR9 (Harmer, 2009). Among them, CCA1 is an important component of central oscillator. Overexpression of CCA1 causes arrhythmic and severely delays flowering (Wang and Tobin, 1998). Negative regulators of CCA1 include the TOC1, PRR5, PRR7, and PRR9 (Nakamichi et al., 2005). Casein kinase II (CK2) modulates CCA1 activity by phosphorylation, thereby regulating the circadian clock (Daniel et al., 2004; Portolés and Mas, 2010; Lu et al., 2011). Overexpression of either the CK2 β-subunits CKB3 or CKB4 in Arabidopsis shortens the circadian period and leads to early flowering due to the disruption of balance between phosphorylated CCA1 and the nonphosphorylated CCA1 (Sugano et al., 1999; Portolés and Mas, 2010). In a recent model, CK2 was classified as a component of the morning loop in the regulatory network of the circadian clock (Chen, 2013).

In addition to protein coding genes, noncoding RNAs also involve in the circadian regulation. For instance, Arabidopsis CYCLING DOF FACTOR 5 (CDF5) and its natural antisense long noncoding RNA FLORE affect photoperiodic flowering under circadian control (Henriques et al., 2017). Small noncoding RNAs such as miRNAs and small interfering RNAs are also crucial regulators and widely reported to regulate the developmental processes in plants (Xu et al., 2018; Yu et al., 2018). MiRNAs are endogenous small RNAs of ∼21 nt in length, with a regulatory function in gene expression based on mRNA degradation or translational inhibition (Chen, 2004; Bagga et al., 2005). MiRNAs are proved to participate in circadian clock networks in animals (Kadener et al., 2009; Nagel et al., 2009; Szweykowska-Kulinska and Jarmolowski, 2018). In Arabidopsis, it has been reported that miR167, miR168, miR171, and miR398 presented diurnal oscillations but not under circadian control (Siré et al., 2009). Whether and how small noncoding RNAs could affect plant circadian clock remains to be declared.

Here, we report a connection of miRNA in plant circadian regulation. Overexpression of a conserved miRNA miR397b in Arabidopsis delayed flowering. Knock-out of CKB3, the target of miR397b, also delayed flowering time in Arabidopsis. We further demonstrated that the delayed flowering time was accompanied by prolonged circadian period and hypophosphorylation of CCA1. Intriguingly, CCA1 could in turn physically bind to the promoter of miR397b and suppress its expression, forming a miR397b-CKB3-CCA1 feedback circuit in circadian regulation. We also performed comparative analysis of miR397 and CKB3 in an evolutionary view and found this targeting mode is recently evolved and intraspecifically existed in Arabidopsis. Our results provide evidence of miRNA-mediated circadian regulation in plants, which further expand the regulatory role of miRNAs in plant development.

RESULTS

Overexpression of miR397b Delays Flowering in Arabidopsis

We previously found that miR397 regulates seed size and grain yield in both the monocotyledon rice (Oryza sativa) and the dicotyledon Arabidopsis, through down-regulating its target mRNAs OsLAC and AtLAC4, respectively (Zhang et al., 2013; Wang et al., 2014). Improvement of grain yield is usually correlated with altered flowering time in agricultural practice. In rice, we have showed that OsmiR397-overexpresing rice plants exhibited early flowering phenotype. MiR397 is a highly conserved miRNA that includes two members in Arabidopsis, miR397a and miR397b (Abdel-Ghany and Pilon, 2008). We asked whether miR397 could also promote flowering time in Arabidopsis. We subsequently overexpressed miR397a and miR397b in Arabidopsis and detected their effects. Unexpectedly, we found that overexpression of miR397b resulted in a delayed-flowering phenotype when compared with the wild-type Arabidopsis, which is opposite to the early-flowering phenotype caused by OsmiR397 expression in rice (Zhang et al., 2013). As shown in Figure 1A, OXmiR397b#7 and OXmiR397b#14 produced 51.6 ± 4.9 (n = 8, P < 0.01**) and 47.5 ± 4.3 (n = 8, P < 0.01**) leaves at bolting, respectively, whereas the wild-type Col-0 only produced 40.6 ± 4.9 leaves (n = 28; Fig. 1, A and B). Reverse transcription quantitative PCR (RT-qPCR) analysis indicated that the transcript level of the flowering-promoting gene FLOWERING LOCUS T (FT) in OXmiR397b plants was lower than that in wild-type plants (Fig. 1C), which is consistent with the late-flowering phenotype in OXmiR397b. Intriguingly, although miR397a has a highly similar mature sequence to miR397b, we did not observe significant late flowering in the miR397a-overexpressing line OXmiR397a#1 (43.5 ± 4.8, n = 10, P = 0.11) and OXmiR397a#2 (42.2 ± 4.5, n = 11, P = 0.35; Fig. 1, A and B). These findings suggest that miR397b may regulate flowering through a potentially distinct pathway in Arabidopsis.

Figure 1.

Figure 1.

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MiR397b delays Arabidopsis flowering time. A, Phenotypes of OXmiR397a and OXmiR397b lines versus wild-type (WT) plants (Col-0). Scale bar = 10 cm. B, Total leaf number at bolting for each line in (A), n ≥ 8. C, Diurnal expression of FT in OXmiR397b plants. Data were shown as mean ± sd of three replicates. UBQ10 was used as an endogenous control in RT-qPCR. Asterisks indicate statistically significant differences compared with wild type by Student's t test (*P < 0.05; **P < 0.01). Seedlings were grown under a 12-h light and 12-h dark photoperiod.

MiR397b Regulates Flowering Time by Suppressing CKB3 Expression, Not LAC2/LAC4/LAC17

MiRNAs function through negatively regulating their downstream target genes (Chen, 2004; Bagga et al., 2005). MiR397 has been shown to directly target LAC2, LAC4, and LAC17 in Arabidopsis (Wang et al., 2014). We thus examined the effects of these target genes on flowering time. The three mutant plants of miR397 targets, including lac2 (SALK_025690), lac4 (SALK_144432), and lac17 (SALK_016748; Cai et al., 2006; Berthet et al., 2011; Cesarino et al., 2013; Zhao et al., 2013; Wang et al., 2014), were applied to investigate whether disruption of these genes are responsible for late flowering in Arabidopsis. Unexpectedly, none of these mutants presented late flowering phenotype (Supplemental Figs. S1, A and B; Supplemental Table S1). We also detected the expression levels of LAC2, LAC4, and LAC17 in miR397a-overexpressing lines, and found that miR397a could dramatically and simultaneously suppress the levels of LAC2, LAC4, and LAC17 as that of miR397b overexpressing lines (Supplemental Fig. S1C; Wang et al., 2014). However, overexpression of miR397a did not delay flowering time (Fig. 1, A and B). The difference between OXmiR397a and OXmiR397b prompted us that suppression of these laccases genes could not explain the delayed flowering in OXmiR397b plants and other downstream targets need to be discovered.

To determine the mechanism of late flowering in OXmiR397b plants and the difference between miR397a and miR397b, we compared the mature sequences of the two members of miR397. As shown in Figure 2A, only the 13th nucleotide of the 21 nt miR397a and miR397b were found to be different. The 13th nucleotide in miR397b is U, whereas it is G in miR397a. We further compared the target genes of miR397a and miR397b by using psRNATarget (Dai et al., 2018). We noted that, in addition to LAC2, LAC4, and LAC17, a gene encoding one of the β-subunits of protein kinase CK2 (CKB3) showed a higher expected complementarity score with miR397b than miR397a (Supplemental Table S2). CKB3 is involved in circadian rhythms and affects flowering time (Sugano et al., 1999). Overexpression of CKB3 promotes flowering (Sugano et al., 1999), which is opposite to the effect of OXmiR397b. Based on these facts, we speculated that CKB3 could be the target of miR397b and is responsible for late flowering in OXmiR397b plants. In contrast, due to the mismatch base existing near the putative slicing site (10th/11th; Fig. 2A), miR397a may not target CKB3 mRNA.

Figure 2.

Figure 2.

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MiR397b delays flowering by down-regulating CKB3. A, Sequence comparison of miR397a and miR397b, and 5′RACE product and the sequencing results are also indicated. B and C, Relative expression of CKB3 and miR397b in OXmiR397b plants (B) and miR397a in OXmiR397a plants (C). Data were shown as mean ± sd of three replicates. U6 and UBQ10 were used as endogenous control in RT-qPCR for miRNA and mRNA, respectively. Asterisks indicate statistically significant differences compared with wild type (WT) by Student's t test (*P < 0.05; **P < 0.01). D and E, Phenotypes of OXCKB3, OXmiR397b×OXCKB3, and ckb3 CRISPR (#guide1-8) plants. Scale bar = 5 cm (D) and 10 cm (E). F and G, Total leaf number of ckb3 CRISPR lines (n ≥ 12) and wild type, OXmiR397b#7, OXCKB3, and OXmiR397#7×OXCKB3 plants (n ≥ 8).

To verify this speculation, we performed 5′ RNA ligase mediated-rapid amplification of cDNA ends (RLM-RACE) experiment to confirm whether CKB3 mRNA is exactly cleaved in the putative cleavage site. By sequencing the PCR products following 5′ RLM-RACE experiments, we showed that the cleavage site was between the 10th and 11th nucleotides of the predicted miR397-CKB3 matching sequence (Fig. 2A), indicating that there is a miRNA-directed CKB3 mRNA cleavage pattern. Next, we measured CKB3 expression levels in OXmiR397b and OXmiR397a plants using qPCR. The results showed that CKB3 was down-regulated by ∼40% in OXmiR397b plants, whereas it was not significantly altered in OXmiR397a plants (Fig. 2, B and C), suggesting that CKB3 is a target of miR397b but not miR397a. To further verify whether it is the 13th nucleotide abolishing the slicing ability of miR397a, we performed 5′ RLM -RACE in rice protoplasts, which do not have the background expression of AtmiR397b variant. We coexpressed OXCKB3 plasmid together with a point-mutated G13U in AtmiR397a backbone (AtmiR397aG13U) as well as the wild-type AtmiR397a and AtmiR397b (Supplemental Fig. S2A). The results showed that both AtmiR397aG13U and AtmiR397b could cleave CKB3 mRNA in rice protoplasts, whereas AtmiR397a is failed to mediate the cleavage (Supplemental Fig. S2B). This result further confirmed that the CKB3 mRNA cannot be cleaved by miR397a for its 13th nucleotide mismatching.

To further confirm that late flowering was caused by CKB3 suppression, we developed CKB3 knock-out plants by using CRISPR-Cas9. Transgenic plants were screened and examined for genome editing effects (Supplemental Fig. S3). The ckb3 knock-out plants showed enlarged rosette size and delayed flowering (Fig. 2, D–F), as the leaf number at bolting was 49.2 ± 4.4 for ckb3#guide1-8 (n = 16, P < 0.01**), 46.7 ± 5.7 for ckb3#guide2-2 (n = 15, P < 0.05*), 45.6 ± 3.5 for ckb3#guide2-5 (n = 13, P < 0.01**), and 46.5 ± 3.9 for ckb3#guide2-11 (n = 12, P < 0.01**) compared with 39.9 ± 3.8 (n = 12) in wild type.

To investigate whether the late flowering in OXmiR397b plants could be restored by CKB3 expression, we crossed OXCKB3 plants with OXmiR397b. As expected, when CKB3 was introduced into OXmiR397b plants, the enlarged rosette size and the late flowering were abolished (Fig. 2G), with the CKB3 expression level being compromised to some extent (Supplemental Fig. S4). Altogether, these results demonstrated that miR397b delayed flowering via down-regulation of CKB3 rather than LAC2/LAC4/LAC17.

The circadian clock Is altered in OXmiR397b by hypophosphorylation of CCA1

Previous studies reported that CK2 affects circadian rhythm by disrupting the balance between phosphorylated and the nonphosphorylated circadian regulators (Sugano et al., 1999; Portolés and Mas, 2010; Mulekar and Huq, 2014), and overexpressing CKB3 alters the circadian clock and shortens the period (Sugano et al., 1999). We asked if the delayed flowering phenotype in OXmiR397b plants was also caused by an altered circadian rhythm. To address this question, we first examined the expression pattern of CCA1, a component of the circadian oscillator, in wild-type and OXmiR397b plants grown in constant light (LL) at 4-h interval after 7 d entrainment in light/dark cycle using real-time quantitative PCR. As shown in Figure 3A, OXmiR397b#7 and OXmiR397b#14 displayed a lengthened period of the CCA1 expression, which were 23.48 ± 0.08 h and 23.1 ± 0.18 h, respectively, compared with 22.86 ± 0.3 h in wild type.

Figure 3.

Figure 3.

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MiR397b lengthens circadian period accompanied by hypophosphorylation of CCA1. A, reverse transcription quantitative PCR (RT-qPCR) of CCA1 expression pattern of OXmiR397b#7, OXmiR397b#14, and wild-type (WT). Data were shown as mean ± sd of three replicates. UBQ10 was used as an endogenous control in RT-qPCR. The shading represents subjective night in free running conditions. B, Bioluminescence assay of OXmiR397b and wild type under LL using CCA1::LUC as reporter. The dark gray represents night and light gray represents subjective night in free running conditions. C, Period lengths of OXmiR397b and wild type. D, Relative amplitude errors of OXmiR397b and wild type (n = 12). Similar results were acquired for three experiments. E, In vitro kinase assay of wild type, OXmiR397b, OXCKB3, and OXmiR397b × OXCKB3. Quantification of the grayscale was determined in three independent replicates, and asterisks indicate statistically significant differences compared with wild type by Student's t test (*P < 0.05; **P < 0.01).

We also performed a bioluminescence assay to detect the circadian oscillation in wild type and OXmiR397b plants at 2-h interval, which provided a higher resolution for dissecting CCA1 expression by using CCA1::LUC as the reporter. In wild-type plants, luciferase activity oscillated in a 24.69 ± 0.23 h (n = 12) period under the drive of the CCA1 promoter, peaking at the subjective dawn. However, when CCA1::LUC plants were crossed with OXmiR397b, their offspring displayed lengthened period of 25.32 ± 0.73 h (n = 12, P < 0.05*; Fig. 3, B–D). These results demonstrated that miR397b lengthens the circadian period and results in late flowering by targeting CKB3, in turn modulating the circadian period of CCA1.

To determine whether phosphorylated CCA1 was compromised when CKB3 was suppressed by miR397b, an in vitro kinase assay was used to measure kinase activity in different mutants. As shown in Figure 3E, when the same amount of His-CCA1 protein was added into the reaction, the radioactive phosphate signal was enhanced in OXCKB3, whereas a decreased signal was detected in OXmiR397b. These results indicated that CCA1 is hypophosphorylated in OXmiR397b and the balance between phosphorylated CCA1 and the nonphosphorylated CCA1 is disrupted.

CCA1 Physically Binds to the Promoter of MIR397B and Forms a Feedback Regulation Loop in the Circadian Clock

Because the essential part of circadian oscillation is feedback regulation (Hardin et al., 1990; Somers, 1999), we ask whether CCA1 could feed back to regulate miR397b. We applied luciferase assays to monitor the expression pattern of miR397b. Segments from −1531 to +16 bp of premiR397b were cloned and fused to the firefly luciferase gene (Fig. 4A). When plants were transferred to LL after entraining under LD conditions, transgenic plants of MIR397B::LUC also showed rhythmic expression (Fig. 4B). Interestingly, the phase of MIR397B peaked at subjective dusk, which is approximately opposite to that of CCA1, indicating a negative regulation of CCA1 and MIR397B. We then detected the expression level of miR397b by qPCR in CCA1-overexpressing and cca1-1/lhy-21 plants. The results showed that miR397b was down-regulated in OXCCA1 plants and was up-regulated in the null mutant (Fig. 4C). Taken together, these results suggest that CCA1 may act antagonistically on miR397b.

Figure 4.

Figure 4.

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MiR397b is regulated by the circadian clock. A, Schematic of miR397b::LUC construct. B, Expression of MIR397B::LUC under LL. C, Expression of miR397b in wild-type (WT), OXCCA1, and cca1-1/lhy-21. Data were shown as mean ± sd of three replicates. U6 was used as an endogenous control in RT-qPCR for miR397b. Asterisks indicate statistically significant differences compared with wild type by Student's t test (*P < 0.05; **P < 0.01).

We next analyzed the promoter sequence of MIR397B. We found an evening element (EE; AAAATATCT) located at −22 bp upstream of MIR397B (Fig. 5A). Coincidentally, previous studies reported that CCA1 bound to EE (Harmer and Kay, 2005; Andronis et al., 2008). Thus we speculated that the rhythmic expression of miR397b was controlled by CCA1. To determine whether CCA1 could bind MIR397B promoter in vitro, we first performed Electrophoresis Mobile Shift Assay (EMSA) to test their interaction. From the results, an obvious shifted band was observed when recombinant His-CCA1 was incubated with a synthetic biotin-labeled probe identical to −30 to −4 of miR397b locus, which could be reversed when competed with the unlabeled probe, rather than the unlabeled mutated probe (Fig. 5B). These results suggested that CCA1 could bind to MIR397B promoter in vitro. We also performed chromatin immunoprecipitation (ChIP) assay to evaluate the binding of CCA1 to MIR397B promoter. The ChIP and following PCR results showed that fragments of MIR397B promoter were significantly enriched. Real-time PCR revealed that there was an 8-fold enrichment compared with the negative control (Fig. 5, C and D). The above findings showed that CCA1 could directly bind to the promoter region of MIR397B and suppress its expression, forming a miR397b-CKB3-CCA1 feedback circuit in circadian regulation.

Figure 5.

Figure 5.

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CCA1 binds to the promoter of miR397b. A, Schematic of the miR397b promoter; the Evening Element (EE) and primers used for ChIP assay are also indicated. B, EMSA of CCA1 using the probe harboring EE or mutated EE. C and D, ChIP of FLAG-tagged CCA1 with an anti-FLAG antibody, determined by PCR (C) and RT-qPCR (D).

A Natural Genetic Variant of miR397 Acquired CKB3 as a New Target

MiR397 is a highly conserved miRNA [21-22]. To evaluate the prevalence of miR397 targeting CKB3 in plants and investigate its evolutionary significance, we comparatively analyzed the sequences of 241 CKB3 homologs in 64 plant species and found that the “A” of the ninth nucleotide, which matches to the 13th “U” in miR397b, is highly conserved in plants (Fig. 6A). We then analyzed the sequence identity of 80 mature miR397 sequences in 42 plant species. The result showed that miR397 was conserved among most of these plant species, whereas a natural variant of a “U” instead of a “G” appeared in the 13th nucleotide only in three plant species, that is, Arabidopsis (ath-miR397b), Capsella rubella (cru-miR397), and Cynara cardunculus (cca-miR397g; Fig. 6, B and C). Both cru-miR397 and cca-miR397g exhibit low complementarities to their CKB3 homologs (Supplemental Fig. S5), implying that miR397 failed to target CKB3 homologs in these two plants and the miR397-CKB3 targeting mode is specifically existed in Arabidopsis.

Figure 6.

Figure 6.

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The conservation of miR397 in plants. A, Sequence logo of CKB3 homologs in 64 species. The asterisk indicates that “A” is the common base among these species. B, Sequence logo of mature miR397 sequence among 44 species. The asterisk indicates the natural variant site. C, The dendrogram with evolutionary relationship of plants basing on Angiosperm Phylogeny Group IV. Circles in red show the three plant species that have a variant of “U” instead of “G” appeared in the 13th nucleotide of miR397. D, Sequences of miR397b in different Arabidopsis thaliana ecotypes. The “G” highlighted in purple in the 13th nucleotide is the same as that of miR397a in Col-0.

To further survey the intraspecific genome variation in Arabidopsis, we compared MIR397B sequences in different Arabidopsis accessions according to the data released in 1001 Genomes project (1001 Genomes Consortium, 2016). Among the 1014 sequenced genomes that covered the locus of miR397b, we found that the sequences of miR397b are identical to miR397a in most of the ecotypes, whereas it is evolved in 7 ecotypes (Fig. 6D). Geographically, most of the ecotypes are collected from Europe, and the longitude ranges from 4.11667 to 29.0000 (time zone ranging from UTC to UTC+3; Supplemental Table S3). We collected seeds of 3 of the seven ecotypes together with 2 accessions without the 13th G to U variant in miR397b from NASC (Nottingham Arabidopsis Stock Centre) to investigate their growth status. We observed a larger rosette size and delayed flowering time in three ecotypes with G to U variant when compared with the ecotypes without the variation (Supplemental Fig. S6).

These results implied that the miR397-CKB3 targeting mode could be recently evolved and specifically existed in Arabidopsis. Given the expression of miR397b delayed flowering and increased seed size and seed number in Arabidopsis (Wang et al., 2014), we speculated that the natural variation of miR397 would promote the fitness of plants through regulating circadian rhythm.

DISCUSSION

The results reported here reveal a role for miR397b in regulating circadian clock and subsequently modulating flowering time in Arabidopsis. Circadian clock with approximate 24-h rhythm is generated by organisms to better acclimate the daily changing environment on earth, which controls nearly 50% of genes at transcriptional level in mammals (Zhang et al., 2014), and nearly 40% in plants (Hernando et al., 2017), suggesting broad and profound effects across kingdoms. Although proteins of the circadian clock do not share similarity in amino acid composition, their regulatory mechanisms are similar. Besides protein coding genes, noncoding RNAs especially miRNAs further expand the regulatory manner at posttranscriptional level. In humans (Homo sapiens), miRNA-192/194 cluster shortens the period by directly repressing the core component, Per gene (Nagel et al., 2009). In Drosophila (Drosophila melanogaster), the microRNA bantam was also identified to regulate the translation of the core clock components using a tilling array (Kadener et al., 2009).

In plants, only a few studies concerning the relationship of miRNAs and circadian have been reported. Siré et al. reported Arabidopsis miR171, miR398, miR168a, and miR167a cycle in diurnal conditions, whereas the oscillations were not driven by circadian clock (Siré et al., 2009). A member of MIR167 family, miR167d, did show rhythmicity under circadian control together with miR160b, miR158a, and miR157a (Hazen et al., 2009). However, no clear clues about how does the circadian rhythmicity generate for these miRNAs were shown. The clock-controlled miR164 modulates leaf senescence via ORE1, serving as a feed-forward pathway of PRR9 in Arabidopsis (Kim et al., 2018). In wheat, miR408 was reported to target TaTOC1, the key component of circadian clock, and thus promoting heading date (Zhao et al., 2016). Still, the studies in plants lack information on whether and how miRNAs affect the circadian clock.

In this study, we showed that miR397b lengthened the period of CCA1::LUC under constant light, by targeting CKB3 to reduce the phosphorylation status of CCA1, which in turn binding to the promoter of miR397b and inhibiting its transcription, providing evidence of miRNA regulating circadian clock in plants. With this effect, a negative feedback loop was formed among miR397b-CKB3-CCA1, suggesting noncoding RNA can also participate in feed-back circadian regulation as protein coding genes do (Fig. 7). Moreover, this study also provides evidence that miRNAs can alter the circadian period in plants, which was found in Drosophila and human previously (Kadener et al., 2009; Nagel et al., 2009). It is interesting to explore whether other types of noncoding RNAs such as phased small-interfering RNAs and long noncoding RNAs will involve in circadian network.

Figure 7.

Figure 7.

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Model of interactions between miR397b and the circadian clock. MiR397b targets CKB3 at posttranscriptional level, which further reduced the phosphorylation status of CCA1, a component of the core oscillator in circadian clock, leading to flowering time alteration.

Unlike animals, it is believed that mRNA cleavage is the prevalent mode of function due to the perfect or near-perfect complementation in plants (Iwakawa and Tomari, 2015). Indeed, many miRNAs target mRNAs in a canonical cleavage manner (Vaucheret et al., 2004; Millar and Gubler, 2005; Zhang et al., 2017). Mismatches near the cleavage site could lower the slicer activity of Argonaute (Iwakawa and Tomari, 2015). Hence, it is important to design artificial miRNAs that pair to miRNA positions at 2 to 12 as suggested by Schwab et al. (Schwab et al., 2005). It is reported that the single mismatch at miRNA position 13 gave rise to a reduction in cleavage rate (Lutz et al., 2017). In this study, we found that AtmiR397a distinguishes AtmiR397b only at the 13th nucleotide; as a result, CKB3 was not cleaved by miR397a (Fig. 2, B and C). This result features the importance of perfect base pairing near the cleavage site in plants (Schwab et al., 2005), and suggests that more strict rules to choose candidate amiRNAs are necessary.

MiR397 is highly conserved across plant species. However, the effect of miR397 expression to flowering time differs between Arabidopsis and the monocot model plant rice. In rice, overexpression of miR397 could promote the heading date, which is thought to be a result of brassinosteroid signaling manipulation (Zhang et al., 2013). In this study, we present an opposite delayed-flowering phenotype when overexpressing miR397b in Arabidopsis, implying the complicated network of controlling flowering time in plants. The functional diversity of miR397 regulation to flowering time in different species largely depends on whether the miRNA sequence matches to target genes related to flowering time pathway, as a consequence of evolution. Our results suggest the miR397b-CKB3 regulation mode is newly acquired by a natural genetic variation in Arabidopsis thaliana. We also try to understand the significance of this natural variation in an evolutionary view. The mutation of G to U in MIR397 may improve the ability of Arabidopsis to survive and reproduce, which is known as fitness. Plant fitness is associated with total seed production (Baker et al., 1994). Our previous work demonstrated that expression of miR397b in Arabidopsis increased inflorescence shoots number, seed size, and seed number (Wang et al., 2014), indicating the potential impact of miR397b in enhancing plant fitness. The enlarged rossette size and delayed flowering time prolonged vegetative stage and facilitated nutrient accumulation, which ensures the increased seed production in OXmiR397b. The recently evolved highly specifically manipulation of CKB3 expression by miR397b highlights the miRNA-mediated fine-tuning of circadian clock in plant fitness, indicating the coevolution between plant miRNAs and circadian rhythm.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

Arabidopsis (Arabidopsis thaliana) plants of the Columbia (Col-0) ecotype were grown on soil at 22°C under a 12-h:12-h light:dark cycle following stratification at 4°C for 2 d. Flowering time and total leaf number were calculated when the floral buds were first seen. OXCKB3 seeds were kindly provided by Elaine M. Tobin (University of California; Sugano et al., 1999), and CCA1::LUC was provided by C.  Robertson McClung (Dartmouth College; Salomé and McClung, 2005). For relative expression level determination of FT, 10-d-old plants were grown on Murashige and Skoog medium with 1.5% (w/v) Suc and 0.4% (w/v) Phytagel after stratification at 4°C for 2 d at 12-h:12-h light:dark cycle. For expression pattern of CCA1, plants were grown in 12-h:12-h light:dark cycle for 7 d and then transferred to constant light, and are sampled at 4-h interval starting from ZT0.

Real-time Quantitative PCR

Total RNAs were isolated using RNAiso Plus (Takara, Cat. 9108) with chloroform and isopropanol, washed with ethanol, and dissolved in RNase-free distilled water. RNAs were reverse-transcribed by PrimeScript RT reagent Kit (Perfect Real Time; Takara Cat. RR037A). Stem-loop RT primers were designed for miRNA reverse transcription, whose 3′ end were specifically reverse complement with the 3′ end of their corresponding miRNA by a 9-nucleotide overhang. Real-time qPCR was performed with SYBR Premix Ex Taq II (code No. RR820A). qPCR for miRNAs forward primers for miRNAs were designed into an oligo with 6-nucleotide overhang at 5′ end to increase the Tm. Reverse primer was a universal primer reverse complemented to the “loop” region of the stem-loop RT primers. Expression levels were normalized to U6 (for miRNAs) and UBQ10 (for protein-coding genes) by the 2−ΔΔCT method. Primers were listed in Supplemental Table S4.

5′RLM-RACE Assay

Total RNAs were isolated from OXmiR397b plants, and 5′ RLM-RACE was done using a Takara 5′ Full RACE kit (D315) following the manufacturer’s instructions but without calf intestinal alkaline phosphatase and tobacco acid pyrophosphatase treatments. Briefly, 2 μg total RNA was ligated to 15 pmol 5′ RACE adaptor at 16°C for 1 h, purified with pheno/chloroform/isoamylol, and then precipitated with isopropanol. The ligated RNA was reverse-transcribed in 1× Moloney Murine Leukemia Virus reverse transcriptase buffer with 2.5 μm Random 9 mers, 1 μM dNTPs 10U RNase inhibitor, and 50U Moloney Murine Leukemia Virus reverse transcriptase in a total volume of 10 μL, and the complementary DNA was amplified by nested PCR using Takara LA Taq followed by the procedure: 94°C for 3 min, and then 20 cycles of denaturing at 94°C for 30 s, annealing at 55°C for 30 s, and then extension at 72°C for 1 min, and finally, finished by 72°C for 5 min. complementary DNA (2 μL) was used in a total volume of 50 μL in outer PCR, and 1 μL of the outer PCR product was used in a total volume of 50 μL for inner PCR. PCR products were separated in 1% agarose gel and ligated to pEASY- Blunt simple vector for Sanger Sequencing.

Vector Construction and Plant Transformation

For miR397b::LUC lines, the coding sequence of the firefly luciferase gene was amplified and cloned into the modified pCAMBIA1390 vector, and the tobacco mosaic virus 35S promoter was removed and replaced by the −1531 to +16 region of the miR397b locus. For FLAG-tagged CCA1 expression lines, the coding sequence of CCA1 was amplified and fused to a 3×FLAG tag, and the cassette was inserted into the modified pCAMBIA 1390 vector. To generate CKB3 CRISPR plants, guide RNAs were designed to target the Exon 1 (#guide 1, see CKB3-T1F/R in Supplemental Table S4) and Exon 2 (#guide 2, see CKB3-T2F/R in Supplemental Table S4) of CKB3, respectively. Oligonucleotides for the same target site were annealed and ligated to BsaI-digested AtU6-guide RNA-SK, and the ligated vector was digested with NheI and SpeI, followed by ligation to SpeI-digested pCAMBIA-pYao::Cas9 (Yan et al., 2015). The constructs were introduced into wild-type plants using the floral dip method.

Bioluminescence Assay

Plants were entrained at 22°C under a 12-h:12-h light:dark cycle on Murashige and Skoog medium for 7 d, then transferred to constant light (120 μmol/m2/s), and luciferin was added to a final concentration of 2.5 mm. Bioluminescence was determined with an LB-960 (Berthold Technologies), every 2 h with the exposure time of 2 s. The bioluminescence intensity of each plant was normalized according to their minimums. The averages of the same line were plotted on the graph, and the period length and relative amplitude were analyzed by Biodare2 (https://biodare2.ed.ac.uk/welcome) using FFT NLLS method.

In Vitro Kinase Assay

The in vitro kinase assay was based on Lu et al. (2011)’s method. In short, His-CCA1 was expressed in BL21(DE3) bacterial strain with pEASY–Blunt E1 vector (Transgen). The bacteria were lysed in the suspension buffer (20 mM NaH2SO4/Na2HSO4, 0.5 m NaCl, 10 mm imidazole) and sonicated. The inclusion bodies were further lysed in lysis buffer (20 mm NaH2SO4/Na2HSO4, 0.5 m NaCl, 10 mm imidazole, 8 m urea, and 0.077% dithiothreitol) and purified by Ni Sepharose 6 Fast Flow (GE healthcare). The protein were renatured by dialyzing to phosphate-buffered saline to eliminate the urea and bound to Beaverbeads IDA-Nickel. Plant extracts containing 10 μg total proteins in the CK2 buffer (50 mm Tris-HCl, 10 mm MgCl2, 25 mm KCl, 1 mm phenylmethylsulfonyl fluoride, 1× protease inhibitor cocktail) were incubated with His-CCA1-bound magnetic beads in the reaction buffer (1×CK2 buffer, 5 mm EGTA, 0.1 mm NaVO3, 20 μm GTP, 5 μCi [γ-32P]ATP) at 30°C for 10 min. The reaction was stopped by adding 2 μL 0.5 m EDTA and analyzed by 10% SDS-PAGE and exposure to a phosphor screen following by scanning with Typhoon Image System. The quantification of the grayscale was determined by ImageJ.

EMSA

The assay was performed using Chemiluminescent EMSA Kit (GS009, Beyotime) following the manufacturer’s protocol with slight modifications. In brief, the probe was labeled with biotin and annealed. Escherichia coli (2 μg) expressed His-CCA1 was incubated with 40 nM biotin-labeled probes (or with 1 μm unlabeled probe or mutated probe) in 10 μL of 1×EMSA/Gel-Shift binding buffer at room temperature for 20 min. The samples were then added 1 μL of EMSA/Gel-Shift loading buffer (without bromophenol blue) and were separated on 6% PAGE gel. The samples on the gel were then transferred to positive charged Nylon membrane. The membrane was incubated with Streptavidin-HRP Conjugate followed by luminizing with ECL.

Chromatin Immunoprecipitation Assay

One gram of 9-d-old seedlings was collected and crosslinked with 1% formaldehyde. Crosslinking was stopped with Gly at the final concentration of 0.125 m. The tissues were then ground to fine powder in liquid nitrogen, and ChIP experiments were done using the EpiQuik Plant ChIP Kit (Cat. P2014). Briefly, the cell lysates were sonicated for DNA shearing and incubated with the antibody-bound (anti-FLAG M2 antibody, F1804, Sigma; anti-IgG, provided by the kit) assay plate for immunoprecipitation. The protein/DNA complex was cleaned and reverse-crosslinked. Following protease K digestion, DNA was purified by the column. Primers were designed to amplify the DNA region containing EE.

Evolutionary Analysis

The 241 CKB3 sequences of the homologous genes were collected from phytozyme (v12.1.6). The mature miR397 sequences and stem-loop hairpins were downloaded from MIRBASE or could be identified by National Center for Biotechnology Information BLAST. The miR397 sequences of different Arabidopsis thaliana accessions are downloaded from 1001 Genomes project (1001 Genomes Consortium, 2016). Data used for evolutionary analysis in this study can be found in Supplemental Tables S5 and 6. The evolutionary relationship of plants was performed by TBtools software (Chen et al., 2018).

Accession Numbers

Sequence data from this article can be found in The Arabidopsis Information Resource (TAIR) under the following accession numbers: At4g05105 (AtMIR397a), At4g13555 (AtMIR397b), At3g60250 (AtCKB3), At2g46830 (AtCCA1), At2g29130 (AtLAC2), At2g38080 (AtLAC4), and At5g60020 (AtLAC17).

Supplemental Data

The following supplemental materials are available.

Acknowledgments

We highly appreciate the help in providing circadian-related seeds by Dr. Elaine M Tobin, Paloma Mas, Steve A. Kay, and Alex A. R. Webb. We are also grateful to Qi Xie for providing CRISPR vectors.

Footnotes

1

This work was supported by the National Natural Science Foundation of China (NSFC) (U190120069, 91640202, 91940301, 31970606, and 31801082), the National Science Foundation of Guangdong Province (2016A030308015), and the National Science Foundation of Guangzhou (201707020018).

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