Abstract
The plant circadian clock coordinates environmental signals with internal processes. We characterized the genomic and transcriptomic structure of the Petunia hybrida W115 clock in leaves and petals. We found three levels of evolutionary differences. First, PSEUDO-RESPONSE REGULATORS PhPRR5a, PhPRR5b, PhPRR7a, PhPRR7b, and GIGANTEA PhGI1 and PhGI2, differed in gene structure including exon number and deletions including the CCT domain of the PRR family. Second, leaves showed preferential day expression while petals tended to display night expression. Under continuous dark, most genes were delayed in leaves and petals. Importantly, photoperiod sensitivity of gene expression was tissue specific as TIMING OF CAB EXPRESSION PhNTOC1 was affected in leaves but not in petals, and PhPRR5b, PhPRR7b and the ZEITLUPE ortholog CHANEL, PhCHL, were modified in petals but not leaves. Third, we identified a strong transcriptional noise at different times of the day, and high robustness at dawn in leaves and dusk in petals, coinciding with the coordination of photosynthesis and scent emission. Our results indicate multilayered evolution of the Petunia clock including gene structure, number of genes and transcription patterns. The major transcriptional reprogramming of the clock in petals, with night expression may be involved in controlling scent emission in the dark.
Highlight The petunia leaf circadian clock shows maxima during the day while petal clock does it during the night. Reaction to dark is organ specific.
Introduction
Organisms, from bacteria to human beings, are subjected to periodic oscillations in the environment due the planet rotation around its axis. Circadian clocks are a complex set of genes allowing organisms to anticipate and adapt to daily environmental variations. In plants, the circadian clock is a network of interlocked loops comprising transcriptional, translational and posttranslational coordination (Harmer, 2009). Circadian processes have been studied in plants for a long period of time (see McClung for a historical overview, (McClung CR, 2006)). Most molecular studies have been done in Arabidopsis thaliana. The Arabidopsis core clock is formed by several genes. Two MYB transcription factors CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), LATE ELONGATED HYPOCOTYL (LHY) and the PSEUDO RESPONSE REGULATOR TIMING OF CAB EXPRESSION (TOC1) form the so-called core clock. Later studies found other clock components including the PSEUDO-RESPONSE REGULATOR gene family (PRR), out of which PRR3, PRR5, PRR7 and PRR9 are clock genes, and the Evening Complex (EC), which is formed by the EARLY FLOWERING 3 (ELF3), EARLY FLOWERING 4 (ELF4) and LUX ARRHYTMO (LUX) proteins. In addition, other genes playing a key role and considered part of the clock include the protein with blue light reception capacity ZEITLUPE (ZTL) and the single copy gene GIGANTEA (GI). The various models developed are based on mutually repressing genes and a set of activating genes coded by the REVEILLE MYB transcription factors (Hsu et al., 2013). Every new discover has added a level of complexity and new interpretation of the circadian clock model (Hernando et al., 2017).
Two aspects emerge from comparative genomics with lower organisms and within higher plants. First the core clock components identified in the picoeukaryote Ostreococcus comprise a MYB gene homolog to LHY and a PRR gene similar to TOC1 (Corellou et al., 2009). There is an additional blue-light receptor component with histidine kinase activity and circadian clock effects (Djouani-Tahri et al., 2011). So, basic clocks maybe found with two or maybe three components that function via transcriptional control. A second aspect is that the fine tuning of the different clock modules is based to a large extent on protein-protein interactions. As protein complexes require certain stoichiometries to maintain their function they are target of genetic constraints in terms of gene dosages and are especially sensitive to gene duplications. Duplicated genes follow four paths including gene loss, maintenance of redundancy, subfunctionalization or neofunctionalization (Airoldi and Davies, 2012). Plant genomes have been subject to genome duplications and, in some cases, followed by non-random elimination of duplicated genes (Adams and Wendel, 2005; Wendel et al., 2016). In Brassica, polyploidization events have involved subsequent gene loss but with a preferential retention of circadian clock genes as compared to house-keeping genes, supporting a gene dosage sensitivity model (Lou et al., 2012).
The genomes of the garden petunia and its ancestors Petunia axillaris and P. integrifolia have been recently sequenced (Bombarely et al., 2016). Petunia forms an early branching in the Solanaceae clade departing from Solanum lycopersicon, S. tuberosum, Nicotiana spp. and Capsicum spp. that have a chromosome number of n=12. Petunia has n=7 and this, together with a high activity of transposition, may have shaped a somewhat different genome evolution. Petunia shares a paleohexaplodization specific to the Solanaceae. A comprehensive analysis of the circadian clock genes found in the Petunia genomes shows that there is a set of genes that has remained as single copy. These include the petunia orthologs for PRR9, PRR3, TOC1 and LHY. In contrast, other genes are present in two to four copies, PRR7, PRR5, GI, ELF3 or ELF4 (Bombarely et al., 2016). Altogether these data indicate a possible departure of the circadian clock network from the one known in Arabidopsis, and suggests the evolution of the clock at different levels including gene structure, expression pattern and genetic functions.
The bulk of work on plant circadian rhythms has been done in Arabidopsis using leaf tissue and seedlings. Like in animals, there is important evidence that the circadian clock expression network differs between different organs. The current view is that the shoot apical meristem may work as a center of coordination (Takahashi et al., 2015), and leaves and roots differ in the regulatory network, as a result of differences in light inputs (James et al., 2008; Bordage et al., 2016).
Petal development starts with the activation of the so-called B function genes in both gymnosperms and angiosperms (Theissen and Becker, 2004). The initial transcriptional activation is followed at early stages by an autoregulatory positive regulation of the MADS-box genes controlling petal morphogenesis in Antirrhinum, Arabidopsis and petunia (Schwarz-Sommer et al., 1992; Goto and Meyerowitz, 1994; Jack et al., 1994; Zachgo et al., 1995; Samach et al., 1997; Vandenbussche et al., 2004). Once organ identity is established and right after anthesis, there is a transcriptional reprogramming (Manchado-Rojo et al., 2012). Furthermore, in sympetalous flowers with petals forming a tube and a limb, both parts of the flower appear to have different functions and transcriptional control (Delgado-Benarroch et al., 2009; Manchado-Rojo et al., 2014). The petal function after anthesis includes concealing the sexual organs and attracting pollinators. The lifespan of a flower is relatively short with most flowers surviving two to five days after anthesis. After anthesis, metabolism and scent emission changes rapidly (Muhlemann et al., 2012; Weiss et al., 2016). Flowers enter rapid senescence upon pollination as a result of ethylene release (Shaw et al., 2002; van Doorn and Woltering, 2008; Liu et al., 2011).
Floral scent release depends on petal development in a quantitative way (Manchado-Rojo et al., 2012), and is circadian regulated in monocots and dicots such as Antirrhinum, Narcissus, rose or petunia (Helsper et al., 1998; Kolosova et al., 2001; Verdonk et al., 2003; Hoballah et al., 2005; Ruíz-Ramón et al., 2014). Most flowers analyzed emit scent preferentially during the day or during the night. The LHY and ZTL orthologs control scent emission in Petunia and Nicotiana attenuata (Fenske et al., 2015; Yon et al., 2015; Terry et al., 2019). Both emit higher quantities during the night, indicating an identity and circadian component controlling this trait.
In the current work, we have addressed the structure of the petunia circadian clock from three different perspectives. The gene structure diverges as PRR paralogs have different intron numbers and PhGI1 and PhGI2 vary in the coding region. The transcriptional structure showed maximum expression during the day in leaves and during the dark in petals. This maximum tended to delay in both tissues under constant darkness conditions. We further identified opposite levels of transcriptional noise at dawn in leaves and dusk in petals. Our results reflect the evolution of the plant circadian clock at different overlapping levels and indicate an organ specific transcriptional structure of the plant circadian clock.
Materials and Methods
Plant materials and experiment design
We used the Petunia hybrida W115 Mitchell for all the analysis. Plants were grown in the greenhouse under natural conditions. Experiments under controlled conditions in growth chambers were performed as described (Mallona et al., 2011a), with the following modifications. For the control experiment, plants were adapted to light:dark growth chamber conditions for at least 1 week. Day:night (12LD) conditions were matched with thermoperiods of 23 °C:18 °C during the light and dark periods. Zeitgeber time (ZT) was defined as ZT0 for light on and ZT12 for light off. In the second experiment, plants were transferred from 12LD cycle to a continuous dark cycle (12DD) with the same temperature regimes.
Flowers were marked before opening, and samples were taken at day 2-3 after anthesis. We used the petal limbs for all experimental procedures. We used young leaves with a length of 1.5-2.5 cm for all the experiments. Sampling of petal limbs and leaves was made every three hours, starting at ZT0 and tissues were immediately frozen in liquid nitrogen. In the case of 12DD experiment, sampling also started at ZT0, during the first 24h under continuous dark.
Phylogeny and bioinformatics
Gene models of Solanaceae were obtained from (https://solgenomics.net/), Antirrhinum from (http://bioinfo.sibs.ac.cn/Am/) (Li et al., 2019b), TAIR (https://www.arabidopsis.org/), Phytozome (https://phytozome.jgi.doe.gov/pz/portal.html) and NCBI (https://www.ncbi.nlm.nih.gov/). We used the corresponding predicted proteins to identify the intron-exon boundaries using Genewise (Birney et al., 2004). The corresponding exon-intron boundaries were plotted using the exon-intron graphic maker (http://wormweb.org/exonintron). Protein alignment was performed with CLUSTALX (Larkin et al., 2007). Phylogenetic analysis was performed with the R libraries “ape” and “phangorn” (Paradis et al., 2004; Schliep, 2011) (R version 3.5.1), using the Maximum Likelihood as statistical method, JTT (Jones, Taylor and Thornton, (Jones et al., 1992)) as model of amino acid substitution and 500 bootstrap replicates. Trees were visualized and annotated with “ggtree” (Yu et al., 2017) using R. Protein domains were predicted using the web-based tool PROSITE (Hulo et al., 2006), schematic proteins were plotted with the R package “drawProteins” (Brennan, 2018). The protein sequences used in the phylogenetic reconstruction are listed in the Supplementary Table S1 and Supplementary Table S2.
Detection of rhythmic gene expression was performed using the non-parametric statistical algorithm JTK_CYCLE (Hughes et al., 2010) implemented in the R package “MetaCycle” (Wu et al., 2016). We analyzed leaves and petals, under two light conditions, 12h light/12h dark (12LD) and constant darkness (12DD). Differences between two time series, were tested using an harmonic ANOVA (HANOVA) implemented in the R package “DODR” (Thaben and Westermark, 2016). We plotted the graphics with “ggplot2” (Wickman, 2017).
Gene expression analysis by qPCR
RNA was extracted from three biological replicates per time point of leaves and corollas using acid phenol (Box et al., 2011). Concentrations were measured using NanoDrop (Thermo-Fisher). Equal amounts of total RNA were used to obtain cDNA using Maxima kits (Thermo-Fisher).
PCR analysis was performed as described before (Mallona et al., 2010), the following protocol was used for 40 cycles: 95 °C for 5 s, 60 °C for 20 s and 72 °C for 15 s (Clontech SYBR Green Master Mix and Mx3000P qPCR Systems, Agilent Technologies). Primers for circadian clock genes were designed using pcrEfficiency (Mallona et al., 2011b) (Supplementary Table S3) and the following protocol was used for 40 cycles: 95 °C for 5 s, 60 °C for 20 s (55 °C for PhGI1 and PhGI2) and 72 °C for 15 s. Samples were run in duplicate. Primer combinations were tested with genomic DNA from Mitchell and we found that all of them gave a single copy DNA on agarose gels. The endpoint PCR was further verified by melting point analysis where all primer combinations gave a single peak of melting (Supplementary Fig. S1). Normalized expression was calculated as described (Schmittgen and Livak, 2008) and PhACT was the internal control gene, a stable gene in circadian studies in petunia leaves and petals (Terry et al., 2019).
Results
The duplicated PRR5, PRR7 and GI diverge in intron number and coding sequence
We used the laboratory line Petunia hybrida W115, also known as Mitchell, which contains the circadian clock genes corresponding to P. axillaris (Bombarely et al., 2016) for a detailed analysis of the structure of the PRR and GI paralogs. Several genes forming the morning and evening loops of the circadian clock in petunia have undergone gene duplication. The genome of petunia has seven PRR genes as PRR7 and PRR5 are duplicated both in P. axillaris and P. integrifolia while Arabidopsis has the canonical set of five genes, PRR1 or TOC1, PRR3, PRR5, PRR7 and PRR9 involved in circadian regulation (Bombarely et al., 2016). We reconstructed a phylogenetic tree of PRR genes of Solanaceae and Arabidopsis (Supplementary Table S1) in order to deduce the evolutionary relationships of the duplicated genes. As found previously for other Angiosperms, the PRR genes of Solanaceae form three major clades: the TOC1/PRR1 clade, the PRR7/3 clade and the PRR9/5 clade (Fig. 1) (Takata et al., 2010). The PRR5a genes of P. axillaris, P. integrifolia are closer to the Arabidopsis AtPRR5 while the rest of the PRR genes of Solanaceae, including the PRR5b, form an additional subclade. This topology indicates that the PRRa paralogs may be an ancestral form and the PRRb may have been formed later and retained, in some cases as single copy genes. The PRR7 genes also showed a similar topology where PaxiNPRR7a and PinfS6PRR7a are closer to the Arabidopsis gene than the single copy genes of the rest of the Solanaceae, and the PRR7b paralogs. This topology is also seen in petunia PRR9, PRR3 and TOC1 that are somewhat between the Arabidopsis gene and the rest of the Solanaceae, according to the early departure of Petunia from the rest of the family (Bombarely et al., 2016).
We found that the gene models for PhPRR5a and PhPRR5b differ in the number of exons comprising the coding region as PhPRR5a has seven and PhPRR5b eight exons (Supplementary Fig. S2). The gene model in Arabidopsis comprises 6 exons in AT5G24470 (AtPRR5), indicating that changes in intron-exon structure has occurred in the evolution of the PRR family. The number of exons also differed between PhPRR7a with eight exons while PhPRR7b had seven exons. The Arabidopsis AT5G02810 AtPRR7 has nine exons out of which eight correspond to coding region, thus coinciding with the phylogenetically closer PhPRR7a.
The PRR family of Arabidopsis has two conserved domains: REG (Response Regulatory Domain) and a CCT (CONSTANS, CONSTANS-like, and TIMING OF CAB EXPRESSION 1 [TOC1/PRR1]) (Liu et al., 2016) (Supplementary Fig. S3A). We used Arabidopsis as model and we compared it with petunia sequences. We found that all the PRR members of P. axillaris and P. inflata shared the REG domain (Supplementary Fig. S3A). The CCT domain was found in all the coding genes except for PaxiNPRR7b, PinfPRR7a and PinfPRR7b. The presence of the CCT domain in PaxiNPRR7a and absence from the rest of the gene group in petunia was surprising, thus we analyzed other Solanaceae, member of the Convolvulaceae (Cuscuta australis and Ipomea nil) and Plantaginaceae (Antirrhinum majus). We found that the CCT domain was absent in the Solanaceae analyzed (Capsicum annuum, C.baccatum, Nicotiana benthamiana, N.sylvestris, N.tabacum, N.tomentosiformis, Petunia axillaris, P. inflata, Solanum lycopersicum, S.melongena, S.pennellii, S.pimpinellifolium, S.tuberosum) (Supplementary Fig. S3B). However, the CCT domain could be found in the rest of the species analyzed. This indicates an early change in the PRR7 family in Solanaceae with possible implications in clock functioning.
GIGANTEA is a single copy gene in the Arabidopsis genome (Fowler et al., 1999) and it is found in one to three copies in the Solanaceae genomes (Bombarely et al., 2016). The genes PaxiNGI1 and PaxiNGI2 are present in the genome of P. hybrida Mitchell. PhGI1, PinfS6GI1 and PinfS6GI1 share an N-terminus conserved with AtGI that was absent in PhGI2 (Fig. 2, Supplementary Fig. S4, Supplementary Table S2). Furthermore, PhGI2 has a 41 amino acid insertion that was not conserved in PinfS6GI2 or other GI genes. The PinfS6GI3 is much shorter that the other paralogs, a feature conserved in N. benthamiana GI3 (Fig. 2). The PinfSGI1 had an additional C-terminal fragment of 105 aminoacids absent from the rest of the GI genes analyzed (Fig. 2, Supplementary Fig. S4).
We can conclude that the structural evolution of core circadian clock genes has occurred at several levels including changes in the number of retained paralogs, gene structure and coding region.
The leaf clock has its maximum during the day while the petal clock shifts towards the night
The current model of the plant circadian clock defines three loops called morning, central and evening loop. These describe the time of the day when certain genes are preferentially expressed (Pokhilko et al., 2012). We established the expression patterns of the different clock genes in leaves and petals. As the genes contained in P.hybrida cv Mitchell correspond to P.axillaris, we further describe them as Ph genes. These included the morning loop genes PhPRR9, PhPRR7a, PhPRR7b, PhPRR5a, PhPRR5b and PhPRR3. The core loop was represented by PhTOC1 and PhLHY. Finally, the evening genes analyzed included PhGI1, PhGI2, PhELF4, PhCHL and PhFKF. This analysis was performed in petunia that was acclimated to light:dark conditions of 12 hour light and 12 dark (12LD) or continuous dark (12DD) conditions.
We compared three parameters between leaves and petals at 12 hours light/12 hours dark: rhythmicity of expression (oscillation), time point with maximum expression (phase) as well as amplitude, defined as is the difference between the peak or trough (maximum or minimum) and the mean value of a wave (Supplementary Table S4). Concerning the rhythmicity, most genes showed a rhythmic oscillation pattern except PhELF4 and PhCHL in leaves, and PhCHL and PhPRR7 in petals (Supplementary Table S4).
Concerning the time of peak expression, most genes had their maximum expression during the light phase in leaves, except PhELF4 and PhLHY at ZT15 and ZT21 respectively. The light phased genes peaked either during the morning at ZT4.5 (PhPRR5a and PhCHL), during midday at ZT 7.5 (PhPRR5b, PhPRR7a, PhPRR9 and PhTOC1), towards the afternoon at ZT9 (PhGI1, PhGI2, PhPRR3 and PhPRR7b) or at dask at ZT 10.5 (PhFKF). In contrast, most of these genes shifted their expression maximum to the dark period in petals (Fig. 3) with the exception of PhCHL, PhPRR9 and PhPRR7a. Among those genes that maintained their expression peak during the day or night, PhPRR9, PhCHL and PhELF4 showed a delay and PhPRR7a an advance of 1.5 hours compared to leaves. The genes that reached their maximum during the dark period in petals could be divided in those with a peak expression early at night at ZT12 (PhGI2 and PhPRR7b), a peak towards the middle of the night at ZT 13.5 and ZT15 (PhGI1, PhPRR3, PhPRR5a and PhPRR5b, PhFKF) and those with a maximum expression at the end of the night at ZT21 (PaxiELF4 and PhTOC1) (Table 1). The only gene showing a maintained expression maximum in leaves and petals was PhLHY.
We also found differences in amplitude between tissues. In general, amplitude of the clock genes was higher in leaves than in petals including PhGI1, PhGI2, PhFKF and the PRR genes PhPRR9, PhPRR7b and PhTOC1. The only gene showing larger amplitude in petals was PhELF4 (Fig. 4, Supplementary Table S4). From all our observations we can conclude that the clock transcriptional structure differs in several ways between leaves and petals. First a robust rhythmic pattern was observed for all genes tested except PhCHL that was arrhythmic, PhELF4 in leaves and PhPRR7a in petals. Most genes showed day phase in leaves and night phase in petals. Finally, the petal clock was somewhat dampened compared to leaves.
The clock shows higher oscillation in petals than leaves under continuous dark
In order to study the entrainment of the petunia circadian clock to the light:dark cycle, petunia plants were transferred from light:dark (12LD) conditions to continuous darkness (12DD). Under constant darkness the genes PhLHY and PhPRR7a lost their significant oscillations in leaves (Table1). Interestingly, the gene PhELF4 that was not rhythmic under LD conditions (Table 1) but displayed a robust oscillation in leaves under 12DD conditions. Finally, PhPRR9 was not rhythmically expressed under a 12DD cycle in petals (Table 1). The rest of the genes analyzed maintained a rhythmic expression except for PhCHL that lacked a rhythm in any of the tissues or conditions analyzed, and PhPRR7a that was not rhythmic in petals.
We compared the expression between 12LD and 12DD in leaves (Fig. 4). We classified the clock genes in three groups either showing a delay in maximum expression between 1.5 and 7.5 hours (PhPRR9, PhPRR5a, PhPRR5b, PhTOC1, PhGI1, PhGI2, PhFKF and PhCHL) an advance: PhPRR7b, PhPRR3 (1.5 hours) and PhLHY (18 hours) or a maintained maximum expression regardless of photoperiod (Table 1) (PhPRR7a and PhELF4).
In petals, PhPRR9, PhPRR7b, PhPRR5b, PhGI1, PhGI2 and PhCHL delayed their maximum expression between 1.5 and 10.5 hours. PhPRR7a and PhLHY, peaked 1.5 and 19.5 hours earlier, respectively. The last group included those genes that did not show differences in phase under 12LD or 12DD conditions: PhPRR5a, PhPRR3, PhTOC1, PhELF4 and PhFKF (Table 1).
Altogether, PhLHY showed advanced expression under DD conditions while PhGI1, PhGI2, PhPRR5b, PhPRR9 and PhCHL were delayed in both leaves and petals. The only gene that remained robust was PhELF4. Thus, these genes were homogenously affected by photoperiod. In contrast, PhFKF, PhPRR5a, PhPRR7a, PhPRR7b, PhPRR3 and PhTOC1 showed an organ specific change in phase in response to free running conditions (Table 1).
We compared the amplitude of clock genes in petunia leaves and petals under 12LD and 12DD. In leaves, we found that all genes showed a lower amplitude in continuous darkness except PhELF4 displaying higher amplitude under 12DD (Supplementary Table S4). In petals the rhythmic expression dampened in PhPRR9, PhPRR7a, PhPRR7b, PhPRR3, PhTOC1, PhGI1, PhGI2, PhFKF, PhCHL and PhLHY. In contrast, the rhythm of PhPRR5a, PhPRR5b and PhELF4 had higher amplitudes (Supplementary Table S4).
Rhythmicity and photoperiod-sensitivity are tissue specific
An important paradigm in the analysis of circadian clock gene expression is the effect of free running conditions on the genes thought to have a circadian control (Somers et al., 1998). We analyzed several parameters of circadian clock genes including phase, noise or amplitude in two tissues and light conditions using Harmonic ANOVA (Thaben and Westermark, 2016). These parameters resulted in a specific gene expression pattern that was compared in both tissues under LD and DD cycles (Table 2). We found that PhELF4, PhLHY, PhPRR5a, PhPRR7a and PhPRR9 were stable regardless of the tissue or photoperiod (p > 0.05). In contrast, PhFKF, PhGI1, PhPRR3 and PhTOC1 showed a different expression pattern between leaves and petals under a 12LD cycle (p < 0.05). In contrast to LD conditions, under 12DD PhGI1, PhPRR5b and PhPRR7b were differentially expressed in leaf versus petal. When we compared leaves at 12LD versus 12DD, PhGI1, PhGI2 and PhTOC1 showed significant changes whereas in petals this group included PhGI1, PhGI2, PhPRR5b, PhPRR7b and PhCHL (Table 2).
These results indicate that there are two sets of genes with different rhythms in leaves and petals and a group of stable genes comprising PhELF4, PhLHY, PhPRR5a, PhPRR7a and PhPRR9. Furthermore, the effect of photoperiod appeared to be organ-specific for those genes that showed significant changes.
Transcriptional noise is gene and tissue specific
Although gene expression quantities were determined for the same set of mRNA extractions, the degree of significance in terms of gene expression levels was not always as expected based on average expressions. This indicated that some genes had robust expression levels while others appeared to be very variable. In order to quantify the dispersion of data, we plotted the normalized Ct values for all genes, dividing the Ct of the clock gene by the Ct of the reference gene PhACT (Fig. 5, Fig. 6) and calculated the coefficient of variation (CV) for all time points (Supplementary Table S5). We found that the data dispersion was very different between genes, tissues and light conditions. The gene with the maximum transcriptional noise was PhLHY in petals at ZT0 and 12LD (CV 24.81) while PhPRR7a in leaves showed the lowest at ZT0 and 12LD (CV 0.56) (Supplementary Table S5). In addition, transcriptional noise seemed to change during the day. In leaves under a light:dark cycle, the highest noise was found at ZT9 (average CV 9.19) and the lowest, at ZT18 (average CV 4.35). In contrast, in petals, the maximum noise was at ZT0 (average CV 10.33) and the minimum, at ZT12 (average CV 3.44) (Fig. 5, Supplemental Table S5). Under constant darkness, this pattern varied. Leaves, displayed the highest CV at ZT12 (average CV 7.89) and the lowest, at ZT0 (average CV 4.34). Petals showed the maximum transcriptional noise at ZT9 (average CV 9.41) and the minimum at ZT12 (average CV 3.31) (Fig. 6, Supplementary Table S5).
We can conclude that subjective time ZT0 i.e. when lights are turned on, displayed the lowest transcriptional noise in leaves and the highest in petals. When day advanced, noise increased in leaves that showed its maximum at ZT9 with opposite behavior in petals that had its lowest level of noise at ZT12 i.e. when lights were turned off. Under free running conditions, the same pattern was found as the lowest and highest noise for leaves coincided with early and late day respectively, while in petals transcriptional noise was low in the subjective night and higher noise was found at subjective time ZT9. This indicates that an endogenous component governs transcriptional noise of the clock genes, which also differs in leaves and petals.
Discussion
The petunia clock gene show structural evolutionary changes
The evolution of the plant circadian clock is considered an important driver of adaptation in a variety of plants including tomato, Opuntia ficus-indica or barley (Mallona et al., 2011a; Zakhrabekova et al., 2012; Müller et al., 2016; Müller et al., 2018). The plant clock is an important coordinator of primary and secondary metabolism in plants. It defines the timing of floral scent emission in a variety of plants including Petunia or Nicotiana attenuatta (Fenske et al., 2015; Yon et al., 2015; Terry et al., 2019). The plant circadian clock appears to have a specific transcriptional structure in different tissues such as leaves, pods, seeds, or roots (Thain et al., 2002; James et al., 2008; Bordage et al., 2016; Weiss et al., 2018). As the transcriptional structure of the clock in petal is currently unknown, we used Petunia hybrida to perform a detailed analysis. We have characterized the structural changes in PhPRR5a, PhPRR5b, PhPRR7a, PhPRR7b, PhGI1 and PhGI2 and the transcriptional structure of the petunia circadian clock in petals and leaves, using standard growth and free running conditions of continuous darkness.
The complete genome paleohexaploidization of petunia, found in the Solanaceae group (Bombarely et al., 2016) is reflected in the retaining of several clock genes as duplications that are found as single copy genes in Arabidopsis and other species. These include PhPRR5a, PhPRR5b, PhPRR7a, PhPRR7b, PhGI1 and PhGI2. Other genes that are found as single copy include PhLHY, PhPRR9, PhPRR3, PhTOC1, PhFKF and PhCHL. Interestingly genes found as single copy in petunia such as PhTOC1, PhPRR9 and PhPRR3 are found as single copy in most Solanaceae except for N. benthamiana that appears to have two copies of each gene (Fig 1). Two of the petunia paralogs PhPRR7a, PinfS6PRR7a and PhPRR5a and PinfS6PRR5a cluster between Arabidopsis and the rest of the Solanaceae genes. In contrast the single copy genes TOC1, PRR3 and PRR9 are found as a subclade for all the Solanaceae together including Petunia. This indicates that there has been a loss of PRR5 and PRR7 paralogs in the Solanaceae that have a single copy gene, while Petunia has retained the older copy closer to the Arabidopsis, Vitis vinifera and Amborella trichopoda genes. The additional changes observed in the number of exons indicate a specific evolution of one paralog. Indeed, AtPRR5 has six exons whereas AtPRR7 presents nine exons (AT5G24470.1 and AT5G02810, consulted in TAIR database) while PhPRR5a and PhPRR7b present 7 exons whereas PhPRR5b and PhPRR7a have 8 exons, indicating possible sub or neofunctionalization of these paralogs (see below).
We found two domains, REG and CCT in all analyzed TOC1, PRR3, PRR5 and PRR9 sequences. In contrast, the CCT domain was absent in most PRR7 paralogs in Capsicum spp., Petunia spp., Solanum spp. and Nicotiana spp. Interestingly, we only found the CCT domain in PhPRR7a, which shared more similarities in the amino acids sequence with AtPRR7. The lack of CCT domains in Solanaceae but not in the related Convolvulaceae family suggests that this event occurred in the early history of Solanaceae. In addition, this alteration, which has been has been described in PRR orthologs in crops such as rice and soybean, can modify growth and flowering time (Lenser and Theißen, 2013; Li et al., 2019a). This may result in a specific clock in the Solanaceae family.
The gene GI appeared in flowering plants and is absent in mosses or picoalgae (Linde et al., 2017). In the Solanaceae we found two to three copies, and in Petunia hybrida, there are significant differences in the coding region between PhGI1 and PhGI2 suggesting a diversification of functions. Furthermore, the amino acid differences between P. axillaris and P. inflata indicate species specific changes in this master regulator that maybe related to the differing environmental niches where both species grow.
We used the predicted protein sequences to infer the domain structure of GIGANTEA. Although a previous study describes that GI encodes a protein with six transmembrane domains (Park et al., 1999), the biochemical functions of GI are not understood. Yeast two hybrid experiments performed with the Arabidopsis GI protein show that the N-terminal domain interacts with FKF1 (Sawa et al., 2007), while the complete protein shows interactions with the CYCLING DOF FACTOR6 protein (Krahmer et al., 2019). As the differences in protein structure found between PhGI1 and PhGI2 do not match well known domains we cannot understand their functional differences. Nevertheless, the PinfS6GI3 does lack the N terminus required for interactions with FKF1 and ZTL in Arabidopsis.
Daily expression of petunia clock genes is tissue specific
The current transcriptional model of the plant circadian clock is largely based on the expression of genes in the Arabidopsis hypocotyls and leaves (Staiger et al., 2013). It includes the morning, midday or core and the evening loops. During the morning, the genes CCA1 and LHY repress the evening genes GI and TOC1 and activate PRR9 and PRR7. At the same time, TOC1 acts repressing GI and PRR9 but activating CCA1/LHY. On the other hand, GI stabilizes ZTL that is a TOC1 repressor (Pokhilko et al., 2010).
Previous studies have revealed that the circadian clock is tissue-specific (Thain et al., 2002; Endo et al., 2014; Bordage et al., 2016). Differential expression of clock genes has been reported in several tissues including seeds, roots, leaves, stems and flowers at several developmental stages in different plant species such as bamboo (Dutta et al., 2018), radish (Wang et al., 2017) or daisy (Fu et al., 2014). The present study has covered several clock genes, including GI and PRRs paralogs, in petunia leaves and petals and our results are consistent with the existence of organ-specific biological clocks in plants.
The expression of clock genes differs between paralogs
Changes in gene expression concerning timing, quantity and rhythm may hint at possible subfunctionalization or neofunctionalization of duplicated clock genes. We found that PhGI1, PhGI2, PhPRR7b and PhPRR5b had similar expression patterns to those previously described in other plants in leaves (Fowler et al., 1999; Matsushika et al., 2000; Marcolino-Gomes et al., 2014). In contrast, PhPRR5a and PhPRR7a that were the closest paralogs to the rest of the species, showed modified expression patterns. PhPRR5a and PhPRR7a showed an advanced phase, peaking before their respective paralogs, PhPRR5b and PhPRR7b. Interestingly, in petals, PhPRR7a displayed a profile similar to the canonical AtPRR7. Moreover, the paralogs PhGI1, PhGI2, PhPRR5a, PhPRR5b and PhPRR7b delayed their maxima to the dark period.
Leaves and petals have different clock coordination
In the present work we identified significant oscillations in gene expression using the JTK_CYCLE algorithm, a non-parametric method which also provided measures of phase and period (Hughes et al., 2010). As mentioned above, most analyzed genes displayed a robust rhythm. Second, we performed an HANOVA test and we found genes that displayed a differential expression pattern, comparing tissues and light conditions. The core clock genes LHY and TOC1 are found in basal picoeukaryotes, mosses, Marchantia polymorpha and all higher plants (Corellou et al., 2009; Holm et al., 2010; Linde et al., 2017). We found that PhLHY and PhPRR9 did not show any statistical differences regardless the tissue or light cycle. In contrast, PhTOC1 expression pattern differed between leaves and petals. This indicates a basal change in the clock coordination between both tissues. This scenario maybe further supported by the significant changes found for PhFKF, PhPRR3, and PhGI1 between tissues. Finally, PhGI1, a gene found only in flowering plants showed significant changes between tissues and photoperiods indicating that it may play a role in the coordination between development and environmental signals.
Photoperiod sensitivity is organ-specific
The effect of day length on biological clocks has been widely studied. For example, floral transition is controlled by CONSTANS (CO) and FLOWERING LOCUS T (FT) genes which are regulated by the circadian clock, including ELF3, ELF4, GI, LHY, PRRs and ZTL genes (Samach et al., 2000; Suárez-López et al., 2001; Valverde et al., 2004). These genes are capable to integrate environmental cues, mainly day length, but also temperature. Clock genes are therefore sensitive to ambient changes resulting in an adaptive advantage (Dodd et al., 2005). The present study revealed that a constant dark regime induced phase-shift even in the first 24h. Most analyzed genes tended to delay their maximum expression, especially in leaves. Only PhLHY advanced its phase both in leaves and petals. Interestingly PhLHY lost its rhythmic expression in leaves but it persisted in petals, similar to previous studies (Fenske et al., 2015). Other genes, PhPRR7a (in leaves) and PhPRR9 (in petals), did not retain their rhythmicity, suggesting that the integration of environmental cues and phototransduction varies depending on the tissue. This is consistent with previous studies, that have reported the effect of light on organ-specific circadian clocks and photoperiodic sensitivity (Shimizu et al., 2015; Bordage et al., 2016).
Constant dark also had an effect on oscillations, which in general tended to decrease in most analyzed genes in leaves and petals. Similar results have been reported in other plants species: LHY/CCA1, ELF4, GI and TOC1 gene expression dampens under constant light or constant dark conditions in Arabidopsis (Wang and Tobin, 1998; Park et al., 1999; Liew et al., 2014; Fenske et al., 2015). Loss of circadian rhythmicity could be key and be involved in responses to environmental changes, such as seasonal dormancy during winter in Japanese cedar or chestnut (Ramos et al., 2005; Nose and Watanabe, 2014).
Transcriptional noise is tissue-specific and depends on the photoperiod
One of the main features of the transcriptional structure of circadian clocks is the capacity to integrate noisy environmental signals and internal transcriptional variation (Hogenesch and Ueda, 2011). The robustness of circadian oscillation is related to the number of mRNA molecules, interactions and complex formation, and it is stabilized by the entrainment to the light:dark cycle (Gonze et al., 2002).
In the present work we found that molecular noise differed in leaves and petals and it was influenced by the time of the day. While in leaves highest stability appeared at the beginning of the subjective day, petals displayed the lowest stability. This was also noticeable when plants were transferred to continuous darkness. Interestingly, the time point with the highest transcriptional noise shifted both in leaves and petals. The lowest stability advanced in petals, and delayed in leaves. Furthermore, the increased transcriptional robustness early in the day in leaves, and in the late day-early night in petals, coincide with the major functional changes in both tissues, initiation of photosynthesis and scent emission. As noise increases thereafter in both tissues, it could be that funneling transcriptional noise into robustness at certain times of the day may have biological implications to achieve consistent outputs. However, the molecular function, if any, is not understood as this is the first report of this phenomenon.
Taken together the differential transcriptional structure and response to light, we conclude that the circadian clock in leaves and petals show substantial differences, that may reflect the underlying function in controlling photosynthesis and secondary metabolism in both tissues. The functional differences between leaves and petals may rely in part on a circadian clock reprogramming during flower development.
Supporting information
Fig. S1. Melt or dissociation curve analysis of petunia genes.
Fig. S2. Exon-intron structure of Petunia axillaris (PaxiN) PRR5 and PRR7 genes.
Fig. S3. (A) Domain structure of PRRs proteins.
Fig. S4. Local alignment of GIGANTEA proteins.
Table S1. PSEUDO-RESPONSE REGULATORs (PRRs) protein accessions used in the phylogenetic reconstruction and for the annotation of protein sequences.
Table S2. GIGANTEA (GI) protein accessions used in the phylogenetic reconstruction.
Table S3. Primers used for qPCR.
Table S4. Rhythmic analysis of transcriptional data.
Table S5: Coefficient of variation, gene expressions.
Authors’ contributions
MIT, MCS, and MEC performed the experimental work; MIT, JW and MEC designed the research programme; JW and MEC secured funds; MIT, JW and MEC wrote the first draft of the manuscript and all authors commented and corrected the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Acknowledgements
This work was developed under projects Fundación Séneca 19398/PI/14, MICINN-FEDER BFU-2013-45148-R and BFU-2017-88300-C2-1-R.
Footnotes
Marta I. Terry - marta.terry{at}edu.upct.es, Marta Carrera-Alesina - martacalesina{at}gmail.com, Julia Weiss - julia.weiss{at}upct.es
Abbreviations
- CCT
- CONSTANS, CONSTANS-like, and TIMING OF CAB EXPRESSION 1 domain
- Ct
- Cycle threshold
- PaxiN
- Petunia axillaris
- PhACT
- ACTIN
- PhELF4
- EARLY FLOWERING 4
- PhFKF
- FLAVIN-BINDING KELCH REPEAT F-BOX
- PhGI1
- GIGANTEA 1
- PhGI2
- GIGANTEA 2
- PhLHY
- LATE ELONGATED HYPOCOTYL
- PhPRRs (PhPRR3, PhPRR5a, PhPRR5b, PhPRR7a, PhPRR7b and PhPRR9)
- PSEUDO-RESPONSE REGULATORS
- PhTOC1
- TIMING OF CAB EXPRESSION 1
- PhCHL
- CHANEL (ZEITLUPE)
- Ph
- Petunia hybrida
- PinfS6
- Petunia inflata
- REG
- Response regulatory domain
- ZT
- Zeitgeber time.