Alternative splicing and nonsense-mediated decay of circadian clock genes under environmental stress conditions in Arabidopsis
© Kwon et al.; licensee BioMed Central Ltd. 2014
Received: 5 February 2014
Accepted: 14 May 2014
Published: 19 May 2014
The circadian clock enables living organisms to anticipate recurring daily and seasonal fluctuations in their growth habitats and synchronize their biology to the environmental cycle. The plant circadian clock consists of multiple transcription-translation feedback loops that are entrained by environmental signals, such as light and temperature. In recent years, alternative splicing emerges as an important molecular mechanism that modulates the clock function in plants. Several clock genes are known to undergo alternative splicing in response to changes in environmental conditions, suggesting that the clock function is intimately associated with environmental responses via the alternative splicing of the clock genes. However, the alternative splicing events of the clock genes have not been studied at the molecular level.
We systematically examined whether major clock genes undergo alternative splicing under various environmental conditions in Arabidopsis. We also investigated the fates of the RNA splice variants of the clock genes. It was found that the clock genes, including EARLY FLOWERING 3 (ELF3) and ZEITLUPE (ZTL) that have not been studied in terms of alternative splicing, undergo extensive alternative splicing through diverse modes of splicing events, such as intron retention, exon skipping, and selection of alternative 5′ splice site. Their alternative splicing patterns were differentially influenced by changes in photoperiod, temperature extremes, and salt stress. Notably, the RNA splice variants of TIMING OF CAB EXPRESSION 1 (TOC1) and ELF3 were degraded through the nonsense-mediated decay (NMD) pathway, whereas those of other clock genes were insensitive to NMD.
Taken together, our observations demonstrate that the major clock genes examined undergo extensive alternative splicing under various environmental conditions, suggesting that alternative splicing is a molecular scheme that underlies the linkage between the clock and environmental stress adaptation in plants. It is also envisioned that alternative splicing of the clock genes plays more complex roles than previously expected.
The circadian clock is an endogenous time-keeping system that coordinates the physiology and behavior of a living organism to its environment . In plants, the clock modulates rhythmic leaf movement, elongation rate of hypocotyls, roots, and stems, stomata aperture, stem circumnutation, and flower opening [1, 2].
Three major interlocked feedback loops constitute the plant circadian clock: the central loop, the morning loop, and the evening loop [3–5]. The central loop is mediated by the reciprocal repression between the morning-phased MYB transcription factors, CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), and the evening-phased pseudo-response regulator TIMING OF CAB EXPRESSION 1 (TOC1) [6, 7]. In the morning loop, CCA1 and LHY promote the transcription of PSEUDO-RESPONSE REGULATOR 9 (PRR9) and PRR7 genes [8, 9]. Closing the loop, the PRR members inhibit the transcription of CCA1 and LHY genes by sequentially binding to the gene promoters from early morning (PRR9) through mid-day (PRR7) to evening (PRR5) [10, 11]. The evening loop is illustrated by TOC1 and a hypothetical component Y, the expression of which is repressed by TOC1 and, in turn, activates TOC1 expression . Recent studies have shown that three evening-phased factors, EARLY FLOWERING 3 (ELF3), ELF4, and LUX ARRHYTHMO (LUX), form the EVENING COMPLEX (EC), which represses PRR9 gene and LUX gene itself [13, 14], indicating that the auto-inhibition of EC replaces the component Y in the evening loop .
The circadian system is substantially influenced by external cues. Phytochrome- and cryptochrome-mediated light signals mediate the induction of CCA1, LHY, and PRR9 genes [8, 16, 17]. Temperatures also affect the amplitudes and rhythms of the clock gene expression . In addition, growth hormones and abiotic stresses modulate the clock function. It has been observed that accumulation of CCA1, TOC1, and GIGANTEA (GI) gene transcripts is differentially regulated by abscisic acid, brassinosteroid, and auxin . High light stress induces CCA1 gene , linking the clock with plant stress adaptation.
The clock components are also regulated at the posttranscriptional and protein levels. It has been shown that the stability of CCA1 mRNA and the translation of LHY mRNA are influenced by light [21, 22]. In addition, the F-box protein ZEITLUPE (ZTL) is responsible for the dark-induced degradation of TOC1 protein . Furthermore, temperature-dependent phosphorylation of CCA1 modulates its binding to target gene promoters . Most recently, chromatin remodeling and alternative splicing of the clock genes have been described as fundamental processes in the regulation of the clock function .
Some of the clock genes have been shown to undergo alternative splicing in response to abiotic stresses in plants [26, 27], among which temperature regulation of CCA1 alternative splicing is best characterized. CCA1 alternative splicing produces two protein isoforms, the full-size CCA1α form and the truncated CCA1β form that lacks the MYB DNA-binding motif . CCA1β competitively inhibits CCA1α activity by forming nonfunctional heterodimers that are excluded from DNA binding. CCA1 alternative splicing is suppressed by low temperatures. Under low temperature conditions, CCA1β production is reduced, and thus CCA1α activity is elevated, leading to the stimulation of freezing tolerance , linking the clock with temperature response.
Recently, it has been reported that alternatively spliced RNA isoforms of some clock genes are degraded through the nonsense-mediated decay (NMD) pathway [28–33], unlike the productive alternative splicing of CCA1 gene. NMD has evolved as an mRNA quality control mechanism that degrades mRNA molecules harboring premature termination codons (PTCs), which generate truncated proteins that are harmful to cellular energy metabolism, and those having aberrantly long 3′ untranslated regions (3′-UTRs) [32, 33]. It is thus possible that alternative splicing serves as a precise mechanism for controlling the mRNA levels of the clock genes, depending on endogenous and external conditions.
In this study, we systematically investigated the alternative splicing patterns of major clock genes under various environmental conditions. We also examined the fates of the RNA splice variants. Our study shows that alternative splicing of the clock genes is differentially influenced by photoperiod and a variety of abiotic stresses. The results of our study show that although RNA splice variants of some clock genes are predicted to encode truncated versions of the authentic proteins, those of other clock genes do not appear to encode specific proteins and, instead, are degraded through the NMD pathway. It is envisioned that alternative splicing plays more complex roles in the clock function than previously expected.
Major clock genes undergo extensive alternative splicing
On the basis of the prevalence of alternative splicing events in the plant circadian clock genes in the literature [20, 26, 27, 34, 35], we anticipated that alternative splicing of the core clock genes constitutes a critical component of the clock function. Previous reports have shown that alternative splicing of CCA1 is suppressed by low temperatures [20, 27, 35]. The alternative protein isoform (CCA1β), which lacks the protein domain required for DNA binding, acts as a dominant negative regulator of the authentic CCA1 transcription factor (CCA1α), thus providing a self-regulatory circuit that links the clock with temperature stress response.
To extend our understanding of the functional relationship between the clock genes and environmental stress responses, we selected a group of major clock genes that constitutes the plant circadian clock and investigated whether these undergo alternative splicing and their alternative splicing patterns are altered under environmental stress conditions.
CCA1 alternative splicing is mediated by the retention of intron 4 and introduces a PTC into CCA1β transcript (Figure 1A). PRR7 alternative splicing is somewhat complicated. It is mostly mediated by the retention of intron 3, resulting in PRR7β transcript (Figure 1B and Additional file 1). A PTC is introduced into the PRR7β transcript. Notably, it is also mediated by the skipping of exon 4 and the retention of introns 2 and 3 [26, 34, 35]. PRR9 alternative splicing is unique, among others, in that the major alternatively spliced variant (PRR9β) is produced by selection of alternative 5′ splice site in intron 2 (Figure 1C and Additional file 2). The presence of two additional RNA splice variants has also been recently reported [26, 34, 35].
A single TOC1 cDNA sequence was identified in the TAIR database. However, it has been shown that an alternative splicing event occurs by the retention of intron 4 [26, 34], introducing a PTC into TOC1β transcript (Figure 1D and Additional file 3). It has been reported that RNA splice variants of ELF3 gene are hardly detected in wild-type plants, but several RNA splice variants are detected in the skip-1 mutant, which is defective in its splicing machinery , possibly due to the retention of intron 2 or 3 (Figure 1E). We found that the ELF3 gene undergoes alternative splicing in wild-type plants (Additional file 4). In addition, it was found that the ELF3β transcript is derived from the inclusion of a new alternative exon and a PTC is introduced into the splice variant.
There are two ZTL-specific cDNA sequences (ZTLα and ZTLβ) in the public database. Sequence comparison and direct sequencing of RT-PCR products revealed that the ZTL alternative splicing is mediated by the retention of intron 2 (Figure 1F and Additional file 5). The ZTLβ-encoded protein has been considered as an authentic ZTL enzyme in the literature , which is probably because the abundance of the ZTLβ transcript is much higher than that of the ZTLα transcript (see below).
The modes of splicing events are diverse in the clock genes
The modes of alternative splicing are diverse in the clock genes (Figure 2B). Retention of specific introns mediates the alternative splicing of CCA1, PRR7, TOC1, ZTL, and ELF3 genes. Exon skipping is involved in PRR7 alternative splicing. Meanwhile, alternative 5′ splice site contributes to PRR9 alternative splicing. Alternative splicing of ELF3 gene was the most complicated. Retention of intron 2 or 3 has been implicated in the ELF3 alternative splicing . However, direct sequencing of PCR products revealed that an additional RNA splice variant (ELF3β), which is probably most abundant among the splice variants, was produced by the inclusion of an alternative exon.
Some RNA splice variants of the clock genes are degraded by NMD
Alternatively spliced RNA variants containing a PTC enter either the productive or unproductive pathway. In the productive pathway, the mRNA is translated into a protein that is structurally distinct from the authentic protein. One example is the alternative splicing of CCA1, in which the CCA1β protein isoform possesses protein domains required for dimer formation and transcriptional activation but lacks the MYB DNA-binding domain . In contrast, in the unproductive pathway, the transcript is degraded via the NMD-mediated degradation pathway .
We next examined the β transcript levels of each clock gene in the upf1-5 and upf3-1 Arabidopsis mutants, in which the NMD pathway is impaired [40, 41]. The levels of PRR7β, PRR9β, and ZTLβ transcripts in the mutants were comparable to those in wild-type plants (Figure 4A, right panels). In contrast, those of TOC1β and ELF3β transcripts were higher by approximately two-fold in the mutants than in wild-type plants (Figure 4B, right panels). We also examined the levels of TOC1β and ELF3β transcripts in the upf1-5 and upf3-1 mutants under heat conditions. The β transcript levels were even higher in the mutants than in wild type plants when grown at 37°C (Additional file 6). The more prominent differences in the TOC1β and ELF3β transcript levels at 37°C is due to the heat-induced alternative splicing of TOC1 and ELF3 genes (see below). Based on these observations, it was concluded that whereas the PRR7β, PRR9β, and ZTLβ transcripts are likely to encode specific proteins, like the CCA1β transcript , the TOC1β and ELF3β transcripts are probably targeted by NMD. The sensitivity of the TOC1β and ELF3β transcripts to NMD is also consistent with the notion that the steady-state levels of NMD target mRNAs were relatively low in many cases [30, 42, 43].
Since the identities of ZTLα and ZTLβ transcripts are currently unclear (, this work), we also examined the effects of CHX and upf1-5 and upf3-1 mutations on the accumulation of ZTLα transcript. We found that the ZTLα transcript level was not affected by CHX treatments (Additional file 7). It was also unaltered in the upf1-5 and upf3-1 mutants, like that of ZTLβ transcript under identical assay conditions. It is therefore likely that the ZTLα transcript is not targeted by NMD and, instead, encodes a distinct protein, like the ZTLβ transcript.
Protein isoforms of the clock components are defective in different functional domains
Some RNA splice variants, such as PRR7β, PRR9β, and ZTLβ transcripts that are insensitive to NMD, were predicted to encode truncated proteins that harbor altered protein structural organizations, as has been demonstrated with CCA1β protein isoform . In many cases, these structural alterations in the truncated forms include deletions, insertions, or substitutions of certain protein domains .
We analyzed the structural organization of the predicted protein isoforms of CCA1, PRR7, PRR9, and ZTL using the analysis tools in the SMART and Pfam databases (http://smart.embl-heidelberg.de/ and http://pfam.sanger.ac.uk/, respectively). The amino acid sequences of the protein isoforms were obtained either from the TAIR database or deduced from the nucleotide sequences of RT-PCR products.
PRR7β and PRR9β protein isoforms possess the CONSTANS, CONSTANS-LIKE, and TOC1 (CCT) domains but lack the N-terminal PR domain (Figure 5), which mediates interactions with other proteins, such as PRR5 [23, 45–47]. The overall structures of the predicted ZTLα and ZTLβ protein isoforms were similar to each other except for the short C-terminal sequence. The ZTLβ isoform is slightly smaller than the ZTLα isoform by 17 residues (Figure 5A). We were unable to identify any distinct protein motifs in the C-terminal region of the ZTL proteins, and thus it is currently unclear whether the two ZTL protein isoforms are functionally distinct or not.
Our data showed that TOC1β and ELF3β transcripts were targeted by NMD and were not expected to encode any proteins (Figure 5B).
Short days suppress the alternative splicing of TOC1 and ELF3genes
Plants use the circadian clock to monitor daylength changes in inducing seasonal developmental responses [48, 49]. We therefore hypothesized that photoperiod influences the alternative splicing patterns of the clock genes.
We observed that the levels of TOC1α and ELF3α transcripts were higher under SDs than under LDs, evidently during the night (Figure 6D and E, respectively). In contrast, the levels of TOC1β transcript were lower during the night under SDs, and those of ELF3β transcript were not altered under identical conditions compared with LDs. Notably, the peak level of the TOC1β transcript shifted from ZT12 under LDs to ZT8 under SDs. Together, these observations indicate that SDs suppress the alternative splicing of the TOC1 and ELF3 genes. There were no discernible effects of SDs on the alternative splicing of ZTL gene (Figure 6F).
Low temperatures suppress CCA1 and ELF3 alternative splicing but induce TOC1alternative splicing
Low temperatures dampen the cyclic expression of the clock genes, resulting in the repression of the clock function . Similarly, low temperatures suppress the alternative splicing of CCA1, and the imbalance between CCA1α and CCA1β protein isoforms leads to disturbed circadian rhythms and induction of freezing tolerance . It was therefore suspected that low temperatures would also affect the alternative splicing of other clock genes.
The levels of both PRR7α and PRR7β transcripts were lower during the subjective day and higher during the subjective night compared to those at 23°C (Figure 7B), indicating that low temperatures do not affect PRR7 alternative splicing but diminish its rhythmic expression. PRR9 is functionally redundant with PRR7 . However, the effects of low temperatures on PRR9 expression were distinct from those on PRR7 expression. The levels of both PRR9α and PRR9β transcripts were markedly elevated at low temperatures throughout the time course, and the rhythmic expression was enhanced (Figure 7C), indicating that low temperatures do not affect its alternative splicing.
The effects of low temperatures on the alternative splicing of the evening-phased genes were quite diverse. While the levels of TOC1α transcripts remained unchanged, those of TOC1β transcripts were markedly higher at low temperatures (Figure 7D), indicating that low temperatures induce TOC1 alternative splicing. The levels of ELF3α transcripts were largely unaffected but loosed rhythmicity at low temperatures (Figure 7E). In contrast, the levels of ELF3β transcripts were drastically reduced, showing that ELF3 alternative splicing is suppressed at low temperatures. ZTL expression was suppressed at low temperatures, but its alternative splicing remained unaltered (Figure 7F).
Heat induces the alternative splicing of CCA1, PRR7, TOC1, and ELF3genes
Heat stress has become an important issue in the field because of recent global warming that extensively affects plant growth and distribution . Because the clock is entrained at least in part by temperature, heat would certainly influence the clock function. However, little is known about the relationship between heat stress and the clock. We therefore examined the effects of heat on the alternative splicing of the clock genes. The heat assays were performed under continuous light conditions to eliminate the effects of light–dark transitions.
High salinity suppresses ELF3alternative splicing
Salt stress influences plant growth and developmental processes, such as flowering time, which is closely associated with the clock function [51–53]. We therefore examined the effects of high salinity on the alternative splicing of the clock genes.
Effects of environmental conditions on the alternative splicing of the clock genes
Rhythmic expression of stress response genes and distinct phenotypes of clock mutants under abiotic stress conditions underscore the close connection between the circadian clock and environmental stress response in plants. One of the best-understood mechanisms is the clock control of C-REPEAT BINDING FACTOR (CBF) genes that play a pivotal role in cold stress response [18, 54]. The central oscillators CCA1 and LHY regulate the expression of the CBF genes by binding directly to their gene promoters . The CBF genes are also directly regulated by PRR9, PRR7, and PRR5 [56, 57]. In addition, the transcription of CBF target genes, such as COLD REGULATED 15 A (COR15A) and RESPONSIVE TO DISSECATION 29 A (RD29A) [58, 59], is clock-controlled .
Altered stress responses of various clock mutants further support the clock control of abiotic stress adaptation. The prr9 prr7 prr5 triple mutants exhibit enhanced resistance to drought and cold stresses . TOC1-deficient mutants display drought-tolerant phenotypes . In addition, it has been shown that Arabidopsis plants that are defective in CCA1, LHY, and GI genes are susceptible to freezing temperatures [55, 62].
Although the linkage between the clock and environmental stress responses has been widely explored, molecular mechanisms and underlying signaling schemes have not been studied at the molecular level in most cases. It has been reported that low temperatures reduce the amplitude of the clock gene expression . Meanwhile, it is known that the clock genes are regulated by extensive alternative splicing, which is influenced by low temperatures. It is therefore evident that alternative splicing of the clock genes should be taken into the interpretation of the expression analysis data under abiotic stress conditions.
This study shows that a group of major clock genes undergoes alternative splicing through a variety of splicing modes, such as intron retention, exon skipping, and selection of alternative 5′ splice sites, resulting in two or more RNA splice variants for each clock gene. It was also found that photoperiod and abiotic stresses, such as temperature extremes and high salinity, broadly affect the alternative splicing of the clock genes. On the basis of the effects of CHX on the relative levels of RNA splice variants and expression assays in NMD-defective mutants, we propose that the alternative splicing of CCA1, PRR7, PRR9, and ZTL genes is productive with RNA splice variants encoding distinct proteins. In contrast, the RNA splice variants of TOC1 and ELF3 genes are predicted to be degraded through the NMD-mediated degradation pathway.
Collectively, our data strongly support the notion that the major clock genes are also regulated at the posttranscriptional level through alternative splicing in addition to the transcriptional control under both normal and environmental stress conditions. Alternative splicing-mediated control of the clock genes would serve as a molecular scheme that incorporates environmental stress signals into the clock, as has been verified with CCA1 alternative splicing that links low temperature signals with the clock .
In this work, we focused on two major RNA splice variants of each clock gene, although additional RNA splice variants have been identified or predicted for some of the clock genes examined (Figure 1). More works on the additional RNA splice variants are required to further extend our understanding on the linkage between alternative splicing events of the clock genes and environmental stress responses. Searching for a full set of RNA splice variants of each clock gene, as has been performed by RNA sequencing method , will also be helpful for the elucidation of the clock function in abiotic stress adaptation. We are currently working on plants that are impaired in the alternative splicing of each clock gene and those expressing a specific RNA splice variant to investigate the physiological roles of the alternative splicing of the clock genes.
Function of alternative protein isoforms
Recent studies have shown that protein isoforms that lack specific functional domains, which are produced by the alternative splicing of transcription factor genes, act as competitive inhibitors of the authentic transcription factors by forming nonfunctional heterodimers [27, 63]. The best-characterized mechanism is the dominant negative regulation of the CCA1 transcription factor (CCA1α) by the protein isoform CCA1β. While the CCA1β isoform possesses protein domains required for dimmer formation and transcriptional activation, it lacks the MYB domain necessary for DNA binding . Therefore, CCA1β is capable of interacting with CCA1α, forming CCA1α-CCA1β heterodimers that are excluded from DNA binding.
According to the domain structures of the protein isoforms encoded by the NMD-insensitive RNA splice variants, the PRR7β and PRR9β protein isoforms are predicted to function in a way that is distinct from that of CCA1β. Unlike CCA1β that lacks the MYB DNA-binding domain, PRR7β and PRR9β have the CCT domain, which is responsible for DNA binding, but lack the PR domain that mediates protein-protein interactions [7, 23, 45–47]. A plausible working mechanism of the PRR7β and PRR9β protein isoforms would be that they compete with the authentic PRR7α and PRR9α transcription factors for binding to the promoters of target genes, as has been previously proposed . Further investigations are required to determine the functional modes of PRR7β and PRR9β.
Two RNA splice variants of ZTL gene are also insensitive to NMD, and two protein isoforms, ZTLα and ZTLβ, are expected to be produced. The ZTLα and ZTLβ isoforms are identical except for the far C-terminal sequences; the former is larger than the latter by 17 residues. The functional mode of ZTLβ thus might differ from the β protein isoforms of other clock components. The lack of the C-terminal extension might also influence the protein conformation of the ZTLβ isoform, which would affect its substrate specificity or enzymatic activity. The smaller ZTLβ isoform has been annotated as the authentic ZTL protein in the literature [23, 65]. We found that the level of ZTLβ transcript is much higher than that of ZTLα transcript, which is in contrast to the α/β ratios of other clock genes. It is currently unclear whether ZTLα or ZTLβ or both is an authentic enzyme. Phenotypic and physiological assays on transgenic plants that specifically express either ZTLα or ZTLβ cDNA would help clarify this uncertainty.
NMD-mediated control of the clock gene expression
Unlike the NMD-insensitive β transcripts of CCA1, PRR7, PRR9, and ZTL genes, TOC1β and ELF3β transcripts are apparently targeted by NMD. The TOC1β and ELF3β transcripts possess sequence features that are frequently observed in NMD substrates, in which they have splice junctions downstream of the PTC and very long 3′-UTRs .
Physiological roles of the NMD pathway are somewhat controversial. According to the “noise” hypothesis, NMD-sensitive RNA splice variants occur as a result of splicing error and are eventually removed through the NMD pathway . In contrast, in the “regulated unproductive splicing and translation (RUST)” hypothesis, alternative splicing is coupled with NMD as a regulatory mechanism for monitoring the abundance of full-size RNA splice variants . We believe that the RUST hypothesis fits well into the alternative splicing of the TOC1 and ELF3 genes, based on the following reasons. First, the RUST hypothesis depicts that alternative splicing occurs through distinct modes of splicing events [26, 34]. We found that the alternative splicing of the TOC1 and ELF3 genes is mediated by the retention of specific introns, supporting the notion that their alternative splicing is a regulated process rather than a simple splicing error. Second, their alternative splicing is regulated by environmental factors in a discrete manner. Production of the ELF3β transcript is suppressed by cold and high salinity conditions but induced under heat stress conditions. Third, whereas their alternative splicing is markedly influenced by abiotic stresses, the levels of TOC1α and ELF3α transcripts are less affected under identical conditions. However, it is still possible that the RNA splice variants may be at least in part translated into proteins. It has recently been reported that some NMD targets are stabilized and translated into proteins under certain conditions .
We investigated the alternative splicing events of major clock genes under various environmental conditions and the sensitivity of their RNA splice variants to NMD. Alternative splicing patterns of the clock genes were differently affected by changes in photoperiod and abiotic stresses, such as cold, heat, and high salinity. Based on the results of this study, we propose that alternative splicing of the clock genes, either by producing truncated isoforms that act as self-regulators or by regulating the abundance of full-size transcripts at the posttranscriptional level, contributes to the precise regulation of the clock function, particularly under fluctuating environmental conditions. It may also serve as a web that integrates environmental stress signals into the clock, providing an adaptation strategy in response to unpredictable environmental changes.
Gene sequences and their exon-intron structures were obtained from the Arabidopsis Information Resource (TAIR, http://www.arabidopsis.org/). Alternative splicing modes of PRR7 and TOC1 genes, which have not been annotated in TAIR, were predicted based on the sequence analysis and the previous reports describing the predicted types of alternative splicing [26, 34]. For ELF3 gene that has not been studied, the presence of alternatively spliced RNA variants was verified by direct sequencing of RT-PCR products. Protein domain structures were predicted using the SMART (http://smart.embl-heidelberg.de/) and Pfam (http://www.sanger.ac.uk/Software/Pfam/) databases.
Plant materials and growth conditions
Arabidopsis thaliana ecotype Columbia-0 (Col-0) was used, unless otherwise specified. The upf1-5 and upf3-1 mutants, which have been previously described [26, 34], were kindly provided by Dr. Jeong Sheop Shin (Korea University, Seoul, Korea) and Dr. Hee-Jeong Jeong (Kyung Hee University, Yongin, Korea). Plants were grown on ½ X Murashige & Skoog media containing 0.6% (w/v) agar (hereafter referred to as MS-agar plates) in a growth chamber set at 23°C with relative humidity of 60% under either long day conditions (LDs, 16-h light and 8-h dark) or short day conditions (SDs, 8-h light and 16-h dark) with white light illumination (120 μM photons m-2 s-1) provided by fluorescent FLR40D/A tubes (Osram, Seoul, Korea).
Analysis of gene transcript levels
Extraction of total RNA samples from appropriate plant materials and RT-PCR conditions have been described previously . Total RNA samples were extensively pretreated with an RNase-free DNase to eliminate contaminating genomic DNA prior to analysis.
Quantitative real-time RT-PCR (qRT-PCR) was employed to determine the levels of gene transcripts. RNA sample preparation, reverse transcription, and quantitative PCR were conducted according to the rules that have been proposed to ensure reproducible and accurate measurements .
qRT-PCR reactions were performed in 96-well blocks using an Applied Biosystems 7500 Real-Time PCR System (Foster City, CA) using the SYBR Green I master mix in a volume of 20 μl. The PCR primers were designed using the Primer Express Software installed in the system and listed in Additional file 8. The two-step thermal cycling profile used was 15 s at 94°C and 1 min at 68°C. The eIF4A gene (At3g13920) was included in the reactions as internal control to normalize the variations in the amounts of cDNA used . All qRT-PCR reactions were performed in biological triplicates using RNA samples extracted from three independent plant materials grown under identical conditions. The comparative ΔΔCT method was used to evaluate the relative quantities of each amplified product in the samples. The threshold cycle (CT) was automatically determined for each reaction using the default parameters of the system. The specificity of PCR reactions was determined by melt curve analysis of the amplified products using the standard methods installed in the system.
Absolute quantification of gene transcripts
Absolute quantification of gene transcripts was conducted as previously described . The cDNAs of alternatively spliced RNA variants were subcloned into a pGADT7 vector (Clontech, Mountain View, CA), and the absolute standard curve of each transcript was obtained by a series of 10-fold dilutions covering from 10-19 to 10-23 mol/μl, as described elsewhere [36, 37]. Quantitative RT-PCR was conducted using a SYBR Green I master mix (Applied Biosystems) with splice variant-specific primers listed in Additional file 8.
Abiotic stress treatments
Arabidopsis plants grown for 10 days on MS-agar plates under LDs were used for abiotic stress treatments. For cold and heat treatments, plants were incubated at 4°C or at 37°C under continuous light conditions for appropriate time durations before harvesting plant materials. To examine the effects of high salinity on the alternative splicing of the clock genes, plants were transferred to MS liquid medium containing 200 mM NaCl under continuous light conditions for appropriate time durations.
Cycloheximide (CHX) treatments
The CHX treatments were performed as described elsewhere [29, 30]. Ten-day-old plants grown on MS-agar plates were transferred to MS liquid medium containing 20 μM CHX. Following vacuum infiltration for 10 min, the plants were incubated for 5 h at 23°C under normal growth conditions before harvesting plant materials for total RNA extraction.
We thank Drs. Jeong Sheop Shin and Hee-Jeong Jeong for kindly providing the upf1 and upf3 mutants. This work was supported by the International Exchange Program for University Researchers through the National Research Foundation of Korea (013-2011-1-C00048) and the Next-Generation BioGreen 21 Program (Plant Molecular Breeding Center No. 201203013055290010200) provided by the Rural Development Administration, Korea. It was also supported in part by the Human Frontier Science Program (RGP0002/2012).
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