CCA1 alternative splicing as a way of linking the circadian clock to temperature response in Arabidopsis

Abstract

Most living organisms on the earth have the circadian clock to synchronize their biochemical processes and physiological activities with environmental changes to optimize their propagation and survival. CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) is one of the core clock components in Arabidopsis. Notably, it is also associated with cold acclimation. However, it is largely unknown how CCA1 activity is modulated by low temperatures. We found that the CCA1 activity is self-regulated by a splice variant CCA1β and the CCA1β production is modulated by low temperatures, linking the circadian clock with cold acclimation. CCA1β competitively inhibits the activities of functional CCA1α and LATE ELONGATED HYPOCOTYL (LHY) transcription factors by forming nonfunctional CCA1α-CCA1β and LHY-CCA1β heterodimers. Consequently, CCA1β-overexpressing plants (35S:CCA1β) exhibit shortened circadian periods as observed in cca1 lhy double mutants. In addition, elongated hypocotyls and petioles and delayed flowering of CCA1α-overexpressing plants (35S:CCA1α) were rescued by coexpression of CCA1β. Interestingly, low temperatures suppress CCA1 alternative splicing and thus derepress the CCA1α activity in inducing cold tolerance. These observations indicate that a cold-responsive self-regulatory circuit of CCA1 plays a role in plant responses to low temperatures.

The earth’s rotation on its axis causes rhythmic environmental changes. Therefore, most living organisms possess the circadian clock to anticipate upcoming environmental changes and synchronize many biological processes, such as stress responses, hormone signaling, metabolism, growth, and development, with the surroundings.^1,2

Light and temperature are two major determinants that affect the circadian clock. The effect of ambient temperature changes in the circadian clock has been examined in both animals and plants, such as Neurospora, Drosophila, and Arabidopsis.^1,3-6 An additional temperature-related characteristics of the clock is temperature compensation, which refers to the maintenance of circadian periods within the physiological range of changes in ambient temperatures.^1,7,8

It has been shown that circadian clock-defective mutants show alterations in freezing tolerance, further supporting the significance of the clock in plant responses to cold temperatures.^9-14 GIGANTEA (GI), which constitutes the clock output pathway, is involved in a cold response pathway that is associated with sugar accumulation in a C-repeat (CRT)/dehydration responsive element-binding factor (CBF)-independent manner.^11,12 Clock central oscillators also regulate the CBF-COLD-REGULATED (COR) regulon, a major cold response module. For example, prr7 prr9 prr5 triple mutants exhibit enhanced cold resistance with elevated CBF and COR expression and accumulation of raffinose and proline.¹³ In addition, CCA1 and LHY transcription factors regulate the expression of CBF genes by binding directly to the gene promoters.¹⁴ Together, these observations suggest that the clock is closely associated with cold response in plants.

The activities of transcription factors are regulated at various steps, such as transcriptional and posttranscriptional regulation, posttranslational modifications, dynamic protein-protein interactions, and protein turnover.¹⁵ Dynamic dimer formation modulates many aspects of transcription factor activities, for instance, DNA-binding affinity, transcriptional regulation activity, or subcellular localization. Recently, it has been shown that a group of small proteins that possess protein domains required for dimer formation but lack DNA-binding domains, including splice variants of transcription factors lacking functional protein domains, form nonfunctional heterodimers with transcription factors to attenuate their activities.^16-18

We recently found that CCA1 alternative splicing produces two isoforms, the full-size CCA1α and the truncated CCA1β. The CCA1β isoform has the protein-protein interaction domain that mediates dimer formation. However, it lacks the N-terminal MYB DNA-binding domain, unlike the CCA1α isoform.¹⁹ CCA1β competitively inhibits CCA1α activity by forming nonfunctional heterodimers CCA1α-CCA1β and LHY-CCA1β, which have reduced DNA-binding affinities. The similar disruptions of circadian rhythmic patterns in 35S:CCA1β transgenic plants and cca1 lhy double mutants and the attenuation of 35S:CCA1α transgenic plants by CCA1β coexpression further support the dominant negative regulation of CCA1α by CCA1β.

Of particular interest is the fact that alternative splicing of CCA1 is suppressed by low temperatures, resulting in upregulation of CCA1α activity.¹⁹ Under cold conditions, CCA1α overcomes the repressible effects of CCA1β and turns out to be fully active in inducing freezing tolerance. Consistent with this, 35S:CCA1α transgenic plants exhibit enhanced freezing tolerance, whereas 35S:CCA1β transgenic plants are susceptible to freezing.¹⁹ Thus, it is concluded that the CCA1α activity is modulated by CCA1β in response to low temperatures (Fig. 1).

Figure 1. Schematic model depicting the linkage between the clock and cold response. The CCA1 gene is alternatively spliced to produce two splice variants, CCA1α and CCA1β. Balanced CCA1 alternative splicing is physiologically important for the clock function under normal conditions. Under cold conditions, the CCA1 alternative splicing is suppressed, resulting in the reduction of CCA1β. As a result, the clock is suppressed, although underlying molecular mechanisms are not fully understood, and freezing tolerance is enhanced because of the elevation of CCA1α activity that induces expression of CBF genes. Therefore, the self-regulatory circuit of CCA1 underlies the signaling linkage between the clock function and cold response, which contributes to plant survival at low temperatures.

Recently, it has been reported that alternative splicing modulates the role of LHY in cold responses by producing nonfunctional transcripts.²⁰ Moreover, other clock components, such as TIMING OF CAB EXPRESSION1 (TOC1), PSEUDO RESPONSE REGULATOR3 (PRR3), PRR5, PRR7 and PRR9, are also alternatively spliced.²⁰ Circadian rhythms were disrupted in plants having mutations in PROTEIN ARGININE METHYLTRANSFERASE 5 (PRMT5) that catalyzes arginine methylation in the splicing factor responsible for PRR5 alternative splicing.²⁰ In Drosophila, a PRMT5 homolog also regulates alternative splicing of clock genes.²² In addition, in Neurospora, alternative splicing of FREQUENCY (FRQ) mediates clock control of ambient temperature responses.²³ It therefore appears that alternative splicing of circadian clock genes is a widespread molecular mechanism that mediates clock function in eukaryotes.

Alternative splicing of pre-mRNAs occurs in splicesome consisting of five small nuclear ribonuleoproteins (snRNPs) and many non-snRNP proteins.²⁴ In Arabidopsis, approximately 70 snRNP genes and 400 genes encoding splicesomal and splicesome-associated proteins have been identified.²⁵ Recent reports indicate that genes encoding splicing regulators, such as serine/arginine-rich (SR) proteins, are also alternatively spliced in response to abiotic stresses, including cold and heat, which in turn alter the splicing of other pre-mRNAs.²⁴ Considering the alternative splicing of clock components and its relevance in regulating clock function in a temperature-dependent manner, studies on splicing factors or splicesomes will give more insights into understanding the link between the circadian clock and temperature responses.

Seo PJ, Park MJ, Lim MH, Kim SG, Lee M, Baldwin IT, et al. A self-regulatory circuit of CIRCADIAN CLOCK-ASSOCIATED1 underlies the circadian clock regulation of temperature responses in Arabidopsis. Plant Cell. 2012;24:2427–42. doi: 10.1105/tpc.112.098723.