https://orcid.org/0000-0002-3262-0859
Figures
Abstract
Plants are prone to genome duplications and tend to preserve multiple gene copies. This is also the case for the genes encoding the Sm proteins of Arabidopsis thaliana (L). The Sm proteins are best known for their roles in RNA processing such as pre-mRNA splicing and nonsense-mediated mRNA decay. In this study, we have taken a closer look at the phylogeny and differential regulation of the SmE-coding genes found in A. thaliana, PCP/SmE1, best known for its cold-sensitive phenotype, and its paralog, PCPL/SmE2. The phylogeny of the PCP homologs in the green lineage shows that SmE duplications happened multiple times independently in different plant clades and that the duplication that gave rise to PCP and PCPL occurred only in the Brassicaceae family. Our analysis revealed that A. thaliana PCP and PCPL proteins, which only differ in two amino acids, exhibit a very high level of functional conservation and can perform the same function in the cell. However, our results indicate that PCP is the prevailing copy of the two SmE genes in A. thaliana as it is more highly expressed and that the main difference between PCP and PCPL resides in their transcriptional regulation, which is strongly linked to intronic sequences. Our results provide insight into the complex mechanisms that underlie the differentiation of the paralogous gene expression as an adaptation to stress.
Citation: Dikaya V, Rojas-Murcia N, Benstein RM, Eiserhardt WL, Schmid M (2025) The Arabidopsis thaliana core splicing factor PORCUPINE/SmE1 requires intron-mediated expression. PLoS ONE 20(3): e0318163. https://doi.org/10.1371/journal.pone.0318163
Editor: Ayaz Ahmad, Abdul Wali Khan University Mardan, PAKISTAN
Received: August 14, 2024; Accepted: January 12, 2025; Published: March 26, 2025
Copyright: © 2025 Dikaya et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files. Excel spreadsheets containing the RT-qPCR Ct values and calculations, the root length measurements, and the ovule abortion counts have been deposited on FigShare: https://figshare.com/projects/PCP_regulation/229497
Funding: This work was supported by grants from the Knut och Alice Wallenbergs Stiftelse (KAW 2018.0202) and Svenska Forskningsrådet Formas (2023-01077) to M.S. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Rooted in place, plants cannot escape changes in their environment. Instead, they constantly monitor their surroundings and adjust their growth and physiology to ensure survival. Daily and seasonal temperature fluctuations in particular are important environmental cues that, together with other external signals, inform plants about their surroundings. The actual responses differ depending on the temperature the plant experiences. For example, moderately elevated temperature triggers a reaction known as thermomorphogenesis whereas severe heat stress can stop plant growth altogether and ultimately result in the death of the plant [1,2]. Similarly, cold is usually divided into low permissive temperatures that only mildly affect plant development, chilling that induces a cold and general stress response, and temperatures below 0°C, or freezing, that might cause ice formation and severe damage inside the plant cell. Importantly, exposure to chilling conditions can prime and prepare plants for a further temperature drop [3,4]. Depending on their habitat, plants may face either only chilling, freezing, or both.
Perception of temperature in plants is mediated by a combination of multiple signaling pathways rather than a single dedicated sensor. Initially, temperature modulates the physical properties of cellular components such as the rigidity of membranes, enzymatic activity, conformational changes of protein and nucleic acid structures, (dis-) assembly of the cytoskeleton, and changes in the viscosity of solutions [5–8]. These primary physical responses initiate signals via multiple pathways that work simultaneously: MAPK cascades, Ca2 + influx into the cell, and rearrangement of transcription and translation [9]. Many players of cold signaling, e.g., phytochromes, abscisic acid, and reactive oxygen species, are not specific to temperature responses but also participate in other signal pathways such as light, osmotic stress, drought, and general stress response [10–12]. The ultimate result of the signal transmission is an adjustment of the gene regulation, metabolism, overall developmental program of the plant, and its acclimatization to the current growth conditions. Not surprisingly given the scope and importance of temperature regulation, multiple mechanisms, including regulation at the epigenetic level, transcription, translation, and post-translational modifications, contribute to this process [11].
RNA splicing [13,14], the process during which non-coding introns are removed from the pre-mRNA and exons are joined, has emerged as a key regulatory mechanism that enables plants to respond to and adjust their physiology, growth, and development to their surroundings [15–18]. Conceptually, RNA splicing is often divided into constitutive splicing, during which a prevalent RNA isoform is formed, and alternative splicing (AS), which enables the organism to produce multiple distinct mature mRNAs from one pre-mRNA [19,20]. In Arabidopsis thaliana, about 60% of genes undergo AS [21–23].
AS has been shown to play a role in establishing organ identity, regulating transitions between developmental stages, and contributing to the response of plants to various environmental stimuli [24–26]. For example, it has been shown that the number and type of AS events in the cell change significantly in response to cold and approximately 27% of the cold-responsive genes showed differential AS in A. thaliana [27]. However, even though multiple observations demonstrate the importance of RNA splicing in coordinating a plant’s response and acclimation to temperature [28–30], the contribution of individual splicing factors and their connection to canonical temperature signaling pathways is largely unknown [31].
A gene that has recently been shown to be important for the proper growth and development of A. thaliana, particularly under cool ambient temperatures and in cold conditions, is PORCUPINE (PCP; SmE1; At2g18740) [32–34]. PCP encodes an E-subunit of the evolutionary conserved Sm-ring that together with U-rich small nuclear RNAs forms the core of the U1, U2, U4, and U5 small nuclear ribonucleoproteins (snRNPs) [35,36]. All members of the Sm-ring are highly conserved in their structure and possess an N-terminal α-helix followed by a five-stranded antiparallel β-sheet which forms characteristic Sm1 and Sm2 motifs [35,37,38]. Structural analyses of the human Sm-ring revealed that the ring-like structure is formed by the interaction between the β4 strand of one Sm-protein and the β5 strand of its counterclockwise neighbor [39]. The Sm-site that binds snRNA inside the central opening of the Sm-ring is formed by residues that are positioned in the loops L3 (between strands β2 and β3) and L5 (between β4 and β5) and belong to the Sm1 and Sm2 motif, respectively [39]. Together with other proteins, snRNPs form the core of the spliceosome, the molecular machinery that catalyzes the splicing reaction [36]. An intriguing feature of A. thaliana plants deficient in PCP is that these mutants appear macroscopically like wild-type plants at 23°C but display severe developmental phenotypes at moderately cool temperatures of 16°C including significant aberrations in shoot- and root apical meristem development, delayed flowering time, and male sterility [32]. More recently, it has been shown, however, that the pcp-1 mutant exhibits subtle phenotypes at the cellular level, for example, in the root meristem, when grown at a temperature of 23 °C [40]. These findings demonstrate that the PCP gene is important for normal development across the entire temperature range, but that mutant phenotypes primarily manifest at cooler temperatures.
Sm genes have been duplicated in plants, and often two or more copies of each subunit are found in a given species [41,42]. This is also the case in A. thaliana where each of the seven subunits that form the Sm-ring has been duplicated [43]. The coding sequences of A. thaliana PCP (SmE1) and its paralog PCP-LIKE (PCPL; SmE2) are highly similar and the two proteins differ in only two amino acids [32]. The biological function of PCPL has, however, not yet been investigated in detail.
Here we have conducted a detailed phylogenetic analysis of SmE proteins in green plants (Viridiplantae). It revealed that duplication of SmE likely occurred multiple times and independently in different clades of the Viridiplantae. A comparison of the predicted structure of the two A. thaliana SmE proteins suggested that two amino acid differences that distinguish PCP and PCPL are unlikely to affect their function. In contrast to mutations in PCP, pcpl mutant plants did not display any phenotypes under any of the temperatures tested, even though PCPL gene expression is modulated by temperature. Functional analyses indicate that the two A. thaliana SmE proteins can, in principle, fulfill the same function, and differences reside mostly in their transcriptional regulation. Importantly, the expression of PCP was found to be strongly linked to its introns. Among them, the 1st intron might be of particular importance and its deletion can have a strong effect on PCP expression. However, other introns also seem to contribute to the regulation of PCP expression. These findings suggest that cis-regulatory elements distributed across multiple introns or complex interactions between non-coding and coding sequences during transcription and RNA processing ensure timely and correct expression of PCP in response to temperature.
Results
SmE duplication is a reoccurring event throughout plant evolution
The Arabidopsis thaliana genome encodes two SmE proteins, AtPCP (SmE1; At2g18740) and AtPCPL (AtPCP-like; SmE2; At4g30330). To reconstruct the evolution of the SmE paralogs we retrieved SmE gene and protein sequences from 24 species of green plants. These represented major lineages such as green algae (Chlamydomonas reinhardtii), bryophytes (Physcomitrium patens), lycophytes (Selaginella moellendorffii), and ferns (Ceratopteris richardii). Most of the SmE genes and proteins, however, belong to the angiosperms, including Amborella trichopoda (the sister species to the rest of angiosperms), as well as several monocotyledonous species and representatives of two major Eudicot clades, namely the Asterids and Rosids. Five Brassicaceae species were added to the analysis to increase local resolution in proximity to A. thaliana. Out of these 24 species, two, Chlamydomonas reinhardtii and Ceratopteris richardii, possessed only one SmE gene while in the remaining species between 2 to 6 paralogs of SmE could be identified (S1 Table).
Initial alignment of the genomic DNA sequences showed very high conservation within the exon sequences only. The low sequence similarity of the introns as well as a big variability of their lengths prevented the construction of a reliable phylogenetic tree based on the genomic DNA alignment. Instead, we proceeded with reconstructing a SmE phylogeny using amino acid and cDNA sequence alignments. The phylogenetic tree overall mapped to the currently accepted evolutionary tree of angiosperms [44] (Fig 1). SmE genes did not split into two distinct groups, SmE1-like (including AtPCP) and SmE2-like (including AtPCP-like), rejecting our initial hypothesis that A. thaliana PCP and PCPL were the results of an ancient gene duplication in the green lineage. Instead, our results suggest that SmE duplications occurred multiple times independently in various branches of the green lineage and that the SmE duplication that led to AtPCP and AtPCPL appears to be specific to the Brassicaceae family. Interestingly, we did not observe SmE2-like genes in Brassica rapa and Brassica oleracea, which instead contained additional copies of SmE1, suggesting the secondary loss of PCPL-like genes and additional PCP duplication in the Brassica genus. Similar duplication events that were inherited by a whole clade of closely related species occurred in grasses (Poaceae) and presumably in the Solanaceae family. Duplications observed in the remaining examined species did not group inside clades above the species level.
A maximum likelihood tree based on the CDS alignment was constructed using IQ-TREE software. Nodes are colored according to the bigger clades they belong to: green algae (no color), Bryophytes (light blue), Lycophytes (dark blue), Ferns (grey), Monocots (yellow), Eudicots (orange for Asterids and magenta for Rosids). The Brassicaceae family is highlighted in a dashed box. Protein sequences are colored according to the ClustalX color scheme [45]. SmE copies are numbered for each species except Arabidopsis thaliana, where gene names PCP and PCPL are given instead and highlighted in bold. Corresponding gene IDs can be found in S1 Table. The schematic drawing above the protein alignment represents the secondary structure of the protein. The N-terminal alpha-helix is indicated in orange, and teal arrows mark beta-strands. Asterisks mark amino acid positions 65 and 86 that discriminate PCP (SmE1) and PCPL (SmE2).
Brassicaceae-specific amino acid substitutions are unlikely to affect SmE protein function
Our phylogenetic analysis indicated that A. thaliana PCP and PCPL are the result of a Brassicaceae-specific duplication. Specifically, the SmE1-like and SmE2-like proteins differ in two amino acids, an asparagine to lysine substitution in position 65 and a threonine to alanine substitution in position 86. The question arises if these amino acid substitutions affect the function of the proteins.
Both amino acids map to regions that are unlikely to play crucial roles in protein folding and affect its function. Position 65 falls into the loop region between two beta-strands or the very beginning of the beta-strand, and position 86 lies in the unstructured C-terminal region of the protein after the final predicted beta-strand. Furthermore, substitutions in position 65 are quite diverse in their physical properties and occurred independently several times in the green lineage (Fig 1). In contrast, substitutions in position 86 are not as widespread, but the substitution of threonine (T) in PCP by alanine (A) in PCPL could in principle affect post-translational protein modifications, in particular phosphorylation. Nevertheless, considering the position and nature of the two amino acid substitutions they seem unlikely to affect protein function. This notion is supported by the predicted structures of PCP and PCPL, which are largely congruent (S1 Fig). In particular, the substitutions in positions 65 and 86 do not appear to affect the protein backbone and structure (S1 Fig).
SmE proteins are essential for plant development
Loss of PCP (SmE1) in A. thaliana has been reported to result in temperature-sensitive phenotypes. pcp-1 was initially described as a mutant sensitive to cold (4°C) and cool ambient temperature of 16°C [32,33]. In agreement with these previous reports, we observed an intermediate phenotype with drastic shortening of the root and anthocyanin accumulation in the cotyledons in pcp-1 grown at 10°C (S3 Fig). We also observed a mild delay in the development of 10-day-old pcp-1 at 23°C. Mutants had smaller leaf rosettes and shorter roots with less abundant and developed lateral roots in comparison to Col-0 (S4 Fig). This delay in development appeared to be transient, and pcp-1 plants eventually caught up and developed similarly to WT at 23°C [32]. Confirming its cold temperature-sensitive nature, 7-day-old pcp-1 seedlings grown at 27°C did not exhibit significant alterations in shoot and root development compared to Col-0 plants growing in the same conditions (S3 Fig).
Consistent with the cold-sensitivity of the pcp-1 mutants, PCP expression in the WT was induced two-fold at 16°C and almost 3.5-fold at 10°C in comparison to 23°C (Fig 2A). We also observed a mild induction of PCP in response to elevated temperature. Interestingly, our qPCR data showed that PCPL in WT displayed the same overall expression profile as PCP (Fig 2B) but the expression changes in response to cool and warm temperatures were much weaker. For example, whereas PCP expression was induced more than 3-fold at 10°C when compared to 23°C, PCPL expression increased only 1.5-fold. These findings suggested that PCP might be the more important of the two SmE genes in response to temperature.
PCP (SmE1) (A) and PCPL (SmE2) (B) expression was determined in the Col-0, pcp-1, and pcpl-1 grown at 10°C, 16°C, 23°C and 27°C by RT-qPCR. Bars show the mean expression based on 3 biological replicates with 3 technical replicates each. Error bars indicate the standard deviation (SD).
To test this hypothesis, we generated two pcpl mutant alleles using CRISPR/Cas9 mutagenesis. Interestingly, we found that pcpl-1 and pcpl-2 mutants were indistinguishable from wild-type across a wide range of temperatures (S3 and S4 Figs), which is remarkably different from the temperature-sensitive pcp-1 (S3 and S4 Figs). Possibly, the expression of PCPL is sufficient to compensate for the loss of PCP in pcp-1 at 23°C and higher temperatures, but PCPL is not enough to fulfill the splicing needs of the cell at lower temperatures. Alternatively, there might be differences in the processing of the transcripts or even in the proteins themselves.
However, plants clearly require at least one functional copy of SmE as demonstrated by the finding that 25% of the developing seeds of self-pollinated double-heterozygous pcp pcpl plants were aborted (Fig 3), indicating gametophytic lethality. The same experiment also revealed a moderately increased seed abortion rate in the pcp-1 single mutant. In contrast, the seed set was essentially unaffected in the pcpl single mutants (Fig 3), again highlighting phenotypic differences between the mutants in these paralogous genes.
The fraction of aborted ovules relative to the total number of ovules in the siliques (n = 20) of the six different genotypes was calculated and plotted as a box-with-whiskers plot. The box spans over the 25th to 75th percentiles, the line inside the box represents the median, whiskers reach out to the minimal and maximal values in each dataset. A one-way ANOVA test with Tukey correction was performed after the removal of outliers using the ROUT method (Q = 1%). All statistical tests were run using GraphPad Prism. Letters represent significantly different (P < 0.05) groups.
PCP expression requires genomic and intronic sequences
Since phylogenetic and structure-function analyses had not revealed any major differences in PCP and PCPL proteins, the question arises as to why the two mutants have such different phenotypes. Hypothesizing that the reason might reside in the transcriptional regulation of the genes, constructs expressing either the CDS (from start codon to stop codon), to which we refer as cPCP and cPCPL, respectively, or the genomic DNA (i.e., exons plus introns) of PCP (gPCP) and PCPL (gPCPL) under the regulation of the PCP promoter (pPCP) and terminator (tPCP) sequences were created. In addition, constructs overexpressing the CDS of either of these two genes under the control of the constitutive 35S promoter (S2 Table) were also generated.
These constructs were introduced into the pcp-1 mutant and primary root length and overall morphology were used as a measure of phenotypic rescue. Interestingly, at temperatures between 10°C to 23°C lines (#1-3) carrying the pPCP::cPCP::tPCP construct exhibited a pcp-1-like phenotype, which got more severe at lower temperatures (Fig 4A, S3 and S4 Figs). At the same time, p35S-mediated overexpression of the PCP CDS resulted in full phenotype restoration (Fig 4B, S3, and S4 Figs). Similarly, pPCP::gPCP::tPCP constructs (#7-9) fully rescued the pcp-1 phenotype (Fig 5, S3, and S4 Figs). Consistent with the phenotyping data, RT-qPCR showed that in the case of the gDNA and overexpression lines PCP expression was restored to approximately 70% of the WT levels and strongly overexpressed, respectively (Fig 6A). Importantly, the expression of pPCP::gPCP::tPCP also restored the temperature-dependent expression of PCP, which might indicate post-transcriptional regulation in addition to transcriptional regulation via cis-regulatory elements present in the genomic sequence of PCP. In contrast, no expression of PCP beyond the residual expression typical for the pcp-1 mutant could be detected in the lines expressing the PCP CDS under the control of pPCP and tPCP. Taken together, these findings support the idea that phenotype restoration correlates with the expression of PCP, and that the full gDNA sequence is essential for proper expression.
(A) Root phenotype of three independent lines carrying pPCP::cPCP::tPCP (#1-3) and pPCP::cPCPL::tPCP (#4-6) constructs in the pcp-1 background. Col-0, pcp-1, and pcpl-1 serve as controls. (B) Root phenotype of three independent lines carrying 35S::cPCPL::tRbcS (#14-16) constructs in the pcp-1 background. Col-0, pcp-1, pcpl-1, and previously established 35S::cPCP::tRbcS (#13) [32]. The length of the primary root of 10-day-old seedlings grown at 16°C (blue) and 23°C (light grey) is shown as violin plots (n = 20 to 25). Solid lines represent the median and dashed lines represent quartiles. One-way ANOVA test with Tukey correction was performed using GraphPad Prism. Letters represent significantly different (P < 0.05) groups.
Length of the primary root of three independent lines in the pcp-1 background transformed with pPCP::gPCP::tPCP (#7-9) and pPCP::gPCPL::tPCP (#10-12) constructs. Col-0, pcp-1, and pcpl-1 serve as controls. Root length was determined using 10-day-old seedlings grown at 16°C (blue) and 23°C (light grey). Root lengths are shown as violin plots (n = 20 to 25) where a solid line represents the median and dashed lines represent the quartiles. A one-way ANOVA test with Tukey correction was performed using GraphPad Prism. Letters represent significantly different (P < 0.05) groups.
(A) Expression of PCP and PCPL in Col-0, pcp-1, and lines carrying pPCP::cPCP::tPCP (#1), pPCP::gPCP::tPCP (#7), and 35S::cPCP::tRbcS (#13) [32] constructs in the pcp-1 background. (B) Expression of the PCP and PCPL in Col-0, pcp-1, and pcp-1 lines carrying pPCP::cPCPL::tPCP (#4), pPCP::gPCPL::tPCP (#11), and 35S::cPCPL::tRbcS (#14). Gene expression was determined in seedlings after 5 days of growth at 23°C followed by 24 h of growth at 10°C (light blue), 16°C (dark blue), 23°C (grey), and 27°C (orange). Bars show the mean expression calculated from three biological replicates with three technical replicates each. Error bars indicate the standard deviation (SD).
Transcription regulation differs between PCPL and PCP
Similarly, expression of the PCPL CDS under the control of the pPCP and tPCP (lines #4-6) was unable to rescue the pcp-1 phenotype at 16°C and 10°C (Fig 4A, S3, and S4 Figs). Except for line #4, which showed a moderate phenotype rescue, all lines were statistically indistinguishable from pcp-1 also at 23°C, while RT-qPCR analysis detected mild elevation in the PCPL expression at all temperature conditions (Fig 6B).
Lines #10-12, which expressed the gPCPL construct including all introns under the control of the PCP promoter and terminator (pPCP::gPCPL::tPCP) showed partial to full phenotypic rescue of the pcp-1 phenotype at 16°C and 23°C (Fig 5 and S3 Fig). However, irrespective of temperature (10°C to 23°C) these lines performed worse than their counterparts (lines #7-9) expressing gPCP under the control of the PCP promoter and terminator (Fig 5, S3, and S4 Figs). Expression of PCPL in line #11 was upregulated in comparison to Col-0 at all temperatures, especially at 10°C. PCPL expression did not differ significantly between #11 and pcp-1 except at 10°C (Fig 6B). Together, these findings suggest a discrepancy between the regulation of PCP and PCPL expression, likely due to different regulatory elements in the gDNA sequences of PCP and PCPL.
Overexpression of cPCPL (lines #14-16) partially rescued the pcp-1 phenotype, resulting in an overall root morphology that more closely resembled WT than pcp-1 (S4 Fig). However, the rescue of root length in these cPCPL overexpression lines was more variable than that observed in the WT plants and other rescue lines, often falling between WT and pcp-1 (Fig 4B). Possibly, this variability is a result of the very high overexpression of PCPL in these lines, which vastly exceeds the endogenous PCPL levels. Such misregulation of a central splicing factor can be expected to have unwanted effects on downstream genes, which could result in the observed high phenotypic variability. Interestingly, we also observed that the expression of PCP and PCPL in the overexpression lines was mildly temperature-dependent (Fig 6), confirming previous results that the 35S promoter is modulated by temperature [46].
PCP and PCPL proteins are functionally redundant
Having established a genomic PCP construct that not only rescued the phenotype of pcp-1 at low temperatures but also the temperature-responsive expression enabled us to address the role of the amino acid substitutions that discriminate PCP and PCPL proteins. To this end, the amino acid substitutions found in PCPL (N65K; T86A) were introduced into the gPCP genomic rescue construct (pPCP::gPCP_N65K_T86A::tPCP; lines #19-20) and introduced into pcp-1. We found that this construct fully restored the WT phenotype at 16°C (Fig 7) and 23°C, confirming our previous assumptions that the SmE1 and SmE2 proteins in A. thaliana were functionally redundant. Similar results were obtained using a construct in which the two amino acids that distinguish the two SmE proteins were changed to alanine and glycine (pPCP::gPCP_N65A_T86G::tPCP; lines #17-18).
Length of the primary root of plants carrying pPCP::gPCP_N65A_T86G::tPCP (#17-18) and pPCP::gPCP_N65K_T86A::tPCP (#19-20) rescue constructs in the pcp-1 background. Col-0 and pcp-1 served as controls. Root length was measured using 10-day-old seedlings grown at 16°C (blue) and 23°C (light grey). Root lengths are shown as violin plots (n = 20 to 25) where the solid line indicates the median and the dashed lines represent the quartiles. A two-way ANOVA test with Tukey correction was performed using GraphPad Prism. Letters represent significantly different (P < 0.05) groups.
Introns work together to regulate PCP expression
Since the full genomic sequence of PCP with 5’ promoter and 3’ UTRs was necessary for complete pcp-1 phenotype rescue, and expression of the PCP cDNA under the control of the PCP promoter and terminator did not result in expression of the gene, we assumed that PCP expression was linked to its introns. To test this hypothesis, several intron-deletion constructs were created in the context of gPCP (S2 and S3 Tables), introduced into pcp-1, and used to assess PCP expression and the extent of phenotypic rescue (Fig 8, S5, and S6 Figs).
(A) Schematic representation of the full genomic PCP construct used to perform the intron deletion studies. Teal boxes represent promoter and terminator regions, orange boxes represent exons, and solid lines represent non-coding sequences. i1 to i5 labels indicate introns 1 to 5. (B) Expression of PCP in the pcp-1 mutant transformed with various intron deletion constructs. Deletion lines and constructs are labeled as gPCP_ ∆ iX, where gPCP represents a full pPCP::gPCP::tPCP construct, ∆ stands for deletion, i stands for intron, and X indicates the number(s) of the deleted intron(s). Col-0, pcp-1, and a pcp-1 rescue line carrying the full-length pPCP::gPCP::tPCP construct served as controls. Gene expression was determined in seedlings after 5 days of growth at 23°C followed by 24 h of growth at 16°C (blue) or 23°C (grey). Bars show the mean expression calculated from one to three biological replicates with three technical replicates each. Error bars indicate the standard deviation (SD). A two-way ANOVA test with Tukey correction was performed using GraphPad Prism. Letters represent significantly different (P < 0.05) groups. (C) Average root-phenotype rescue relative to Col-0 (100%) and pcp-1 (0%) in the corresponding lines from panel (B).
Deletion of single introns 2, 3, and 5 (gPCP- ∆ i2, gPCP- ∆ i3, and gPCP- ∆ i5) resulted in a considerable but not complete loss of PCP expression (Fig 8B) and a corresponding reduction in the rescue of the primary root length phenotype (Fig 8C and S5 Fig). The combined deletion of introns 4 and 5 (gPCP-Δi4/5) largely resembled the deletion of intron 5, suggesting that intron 4 plays only a minor role in regulating PCP expression. Interestingly, PCP expression in these intron deletion lines still responded to temperature and was induced by 16°C, albeit at a reduced level, suggesting that these introns were not required for the cold-induced expression of PCP. For these intron deletion lines, PCP expression and phenotypic rescue correlated well and was consistent among independent rescue lines. In contrast, the deletion of intron 1 (gPCP- ∆ i1) gave more variable results (Fig 8 and S6 Fig). Of the two individual lines tested, one showed a low expression of PCP and phenotype rescue of the root phenotype (Fig 8). The second line showed an even weaker rescue and PCP expression was essentially undetectable (S6 Fig). However, when combined with the deletion of intron 3, the deletion of intron 1 (gPCP-Δi1/3) invariably leads to the complete loss of PCP expression, suggesting that intron 1 plays an important role in regulating PCP expression. On the other hand, the presence of intron 1 in the CDS alone (gPCP-Δi2/3/4/5) was insufficient to drive the expression and restore the pcp-1 phenotype (Fig 8 and S5B Fig). Finally, the deletion of more than two introns appears to have an additive effect, resulting in a complete loss of PCP expression and a pcp-1-like root phenotype at 16°C in all combinations tested (Fig 8 and S5 Fig). Taken together, our results show that introns are essential for the proper PCP expression, and they work in tandem. Whether introns 1 takes the role of the main expression switch or promotes PCP transcript accumulation by other means remains to be established.
Discussion
Throughout the course of evolution, plants underwent multiple genome duplications and preserved copies of paralogous genes. Through neo- and sub-functionalization these paralogues either acquired new functions or performed their original tasks under different conditions, thereby increasing the adaptability of plants to adverse environmental conditions [47].
Here we investigated SmE genes and proteins to shed light on the evolution of this important regulator of RNA splicing in the green lineage. Consistent with the work by Cao et al. 2011 [43] who had analyzed the relationship among Sm proteins in A. thaliana, we found that the duplication resulting in PCP/PCPL happened independently of SmE duplications in other plant clades during the most recent Brassicaceae-specific whole-genome duplication (WGD) event, namely At-α [48]. In general, reoccurring duplications of SmE could be beneficial for plant adaptation, as additional copies could ensure sufficient expression under specific conditions, which might explain a wide range of biotic and abiotic stresses that affect SmE expression across different species of the Viridiplantae. Considering the importance of SmE for plant survival, duplications also ensure that at least one functional copy of the gene is always active, while others are available for subfunctionalization [49].
Despite multiple independent duplication events in various clades of the Viridiplantae, the SmE CDS and the protein sequences exhibit a very high degree of sequence conservation. This is in line with a study by Chen and Cao 2014 [41], who reported a significant prevalence of synonymous over nonsynonymous mutations during SmE evolution. Interestingly, the same study also showed that the exon-intron structure of homologous Sm genes was conserved even between distantly related species. In agreement with this report, we observed the same 6 exon/ 5 intron structure found in AtPCP in euphyllophytes (ferns and seed plants). In contrast, the representatives of green algae, bryophytes and lycophytes were characterized by single-exon SmE coding sequences.
Cao et al. 2011 [43] also reported that duplicated Sm genes showed little similarity in their expression patterns despite the high conservation in their intron-exon structure. However, in the case of AtPCP and AtPCPL, analysis of publicly available expression data indicates their co-expression across a wide range of tissues and treatments. In line with this finding, we observed that the expression of PCP and PCPL in response to ambient temperatures ranging from 10°C to 27°C is quite similar. Despite these similarities in temperature-dependent gene regulation, we did not observe any temperature-sensitive phenotype in pcpl mutants (Fig 4, S3, and S4 Figs).
Interestingly, rice SmE orthologs were shown to be induced in response to cold [41,50] reminiscent of PCP in A. thaliana. Similarly, it has been reported that SmE genes in tomato and Populus were also differentially expressed in response to cold [51–53]. In contrast, the SmE homolog in C. reinhardtii, which is encoded by a single-exon gene, was not induced in response to cold [54]. This finding could be interpreted as evidence that expression regulation by cold temperatures is a derived feature and might be conserved in higher land plants. However, considering that gene duplications provide a source for inter- and intraspecies expression and functional diversification [55,56] additional analyses, including the Brassicaceae SmE homologs, are required before any conclusions regarding the contribution of the SmE gene structure to expression in response to cold can be drawn.
Further investigation of the protein structure of the A. thaliana SmE proteins suggested that the two proteins did not functionally diversify. This notion is supported by our finding that substituting the two amino acids that discriminate PCP and PCPL in the context of the full genomic PCP rescue construct by either the amino acids found in PCPL or two other small amino acids resulted in the full rescue of the pcp-1 phenotype at 16 °C (Fig 7). This is in agreement with the observation that gene copies retained after the WGD show lower levels of structural and functional changes in the course of evolution than tandem duplications [57]. Taken together, these findings suggest that the main difference between PCP and PCPL resides in the differential regulation of the expression of these two genes. In A. thaliana, PCP might be the more important orthologue, especially at low temperatures, not only due to the differential expression in response to cold but also due to the downstream regulation of the transcripts (Fig 6). As mentioned above, shifts in expression patterns between duplicated genes contribute to the retention of the duplicates [49,58]. Furthermore, the fact that Brassica rapa and Brassica oleracea have lost their copy of PCPL and acquired an additional copy of PCP can be interpreted as indirect evidence for the functional redundancy of the two proteins, with PCP being the more important in A. thaliana. However, the observed seed abortion rate of 25% in the pcp-1 pcpl crossing progeny suggests that plants require at least one functional copy of SmE for survival (Fig 3). It has previously been demonstrated that PCP is important for proper splicing of pre-mRNA [33,34,59,60], but information regarding target preferences of PCPL is still lacking, and we cannot completely exclude the possibility that the two A. thaliana SmE proteins interact with different partners or are differentially regulated at some level.
In this context, it is important to note that our results indicate that the expression of PCP requires a full genomic sequence including promoter, 5’ UTR, gene region consisting of exons and introns, and downstream sequences. Only such a full genomic construct can rescue the pcp-1 phenotype at low ambient temperatures. At the same time, expression of the PCP cDNA under the control of the PCP promoter and terminator cannot (Figs 4A and 5). Expression of the PCPL gDNA under the control of the PCP promoter and terminator, despite being highly similar to PCP in exon-intron structure, was not sufficient to completely rescue the pcp-1 mutant (Fig 5). It should be noted that Wang et al. 2022 found that expression of a pPCP::gPCP-3xFLAG construct lacking the terminator was able to rescue the pcp-1 phenotype, which suggests that minimal sequence requirements for PCP expression do not include the 3’ region. Our intron deletion experiments clearly demonstrate that intronic sequences are required for PCP expression and function (Fig 8). Intron 1, in particular, appears to be important as its deletion can lead to complete loss of PCP expression. However, the 1st intron on its own (gPCP-Δi2/3/4/5) is not sufficient to drive the expression of PCP to rescue the pcp-1 mutant phenotype (Fig 8).
Introns show a very wide range of regulatory mechanisms that affect not only gene expression but also the processing of the transcript, its metabolism, and even translational efficiency, which complicates the study of intron-mediated expression regulation [61,62]. Introns can drive the expression of the gene, affect expression levels and organ-/tissue-specificity, activate transcription, contain cis-regulatory elements, provide miRNA-binding sites, and possibly contain elements that act as riboswitches [63–65]. First introns in particular have been shown to play important roles in these regulatory functions, in some cases turning them into the key regulators of gene expression [66–68]. In particular, the phenomenon of intron-mediated enhancement (IME) is strongly linked to the proximity of the given (first) intron to the 5’UTR [69,70].
Interestingly, lines expressing either the full genomic sequence of PCP or certain intron-deletion constructs with detectable PCP expression also exhibited cold-induced expression (Figs 6 and 8B). These findings suggest that temperature responsiveness is not mediated by these particular intronic sequences. The contribution of intron 1 to the temperature-regulated expression of PCP will need to be investigated in more detail as the deletion lines created in this study resulted in varying degrees of PCP expression and phenotypic rescue. However, our results nevertheless allow us to conclude that intronic sequences contribute to the cold-responsive expression of PCP in a complex and partially redundant manner.
It is also important to note that induction of PCP has been reported to be attenuated in a cbf123 triple mutant background in response to prolonged cold treatment [34], indicating that these key regulators of plant temperature responses contribute to PCP regulation. Notably, this regulation appears to be direct as EMSA and ChIP experiments indicated interaction between the promoter region of PCP and CBFs at a CRT/DRE cis‐regulatory element located approx. 1 kb upstream of the PCP start codon [34]. Importantly, the genomic rescue construct used in the current study employed a shorter promoter fragment and lacked the CRT/DRE cis-regulatory element. Furthermore, no additional CRT/DRE consensus sequences could be identified in the gPCP rescue construct, suggesting that cold responsiveness, in this case, is not mediated by CBFs but rather by an unknown mechanism. It is also worthwhile noting that the CRT/DRE cis-regulatory element in the PCP upstream region is contained within a recently discovered lncRNA locus, FLAIL (At2g18735) [71]. Whether the CRT/DRE element regulates the expression of FLAIL, PCP, or both remains to be resolved.
In summary, our work provides evidence that SmE is a protein essential for plant growth and development and that the two copies of the SmE protein present in A. thaliana perform the same function. However, in contrast to PCP, the deletion of PCPL does not affect plant growth and development under any of the conditions tested. We attribute these differences to divergence in gene expression as PCP is, in general, the more highly expressed of the two genes. Interestingly, our results indicate that intronic sequences are central to PCP expression. The molecular mechanism underlying intron-dependent regulation of PCP is, however, currently unclear and will need to be investigated further in the future.
Materials and methods
Phylogenetic analysis
At2g18740 (AtPCP/AtSmE1) was used to query the Phytozome v13 [72] database to identify potential homologues among the available genomes of Viridiplantae. Species were chosen to represent the major groups of the green plants as best as possible (S1 Table). Genomic DNA, coding DNA, and protein sequences of selected SmE orthologs were downloaded from the NCBI GenBank database (www.ncbi.nlm.nih.gov/genbank/).
The retrieved SmE amino acid sequences of the selected species were aligned using MAFFT v7 (L-INS-i algorithm) [73] and manually checked for mistakes in the alignment using Geneious Prime 2023.0.4 software (https://www.geneious.com/). The corrected amino acid alignment was reverse translated into a corresponding cDNA alignment using Pal2Nal v14 [74]. The output cDNA alignment was used to build a phylogenetic tree using IQ-TREE v2.2.0 [75,76]. The tree was rooted on the green alga Chlamydomonas reinhardtii. Command lines used to perform the tree calculation are provided in Supplementary Method S1 Method. Trees were visualized using the iTOL online tool v6 [77].
Protein structure prediction
Protein domain search and secondary structure prediction were performed on the consensus sequence reconstructed after protein alignment using NCBI Conserved Domains Search [78–80] and Jpred 4 tool [81], respectively.
3D protein structures of PCP (SmE1) and PCPL (SmE2) based on AlphaFold2 predictions [82] were retrieved from the BAR ePlant (https://bar.utoronto.ca/eplant/) [83]. Protein structures were visualized using PyMOL, version 3.0.2 (Schrödinger, LLC).
Plant material and growth conditions
All work was done using Arabidopsis thaliana plants of Col-0 accession. The pcp-1 T-DNA insertion line (SALK_089521) has previously been described [32]. The pcpl-1 and pcpl-2 deletion mutants were generated as part of this study. Briefly, A. thaliana Col-0 plants were transformed by floral dipping with constructs for CRISPR/Cas9-mediated mutagenesis (described below) [84]. Primary transformants (T1) carrying deletions in PCPL were identified, and homozygous transgene-free pcpl-1 mutants were isolated in subsequent T2/T3 generations.
Seeds were stratified at 4°C in the dark for 48 h before sowing on soil or plates. Seeds grown in sterile conditions were sown on ½ MS media [85] plates with 1.6% agar. Plates for the selection of transformants were additionally supplemented with 10 ug/mL phosphinotricin (Duchefa Biochemie) (BASTA). Seeds sown on plates were surface sterilized beforehand with 0.015% Triton X-100 solution in 70% EtOH for 10 minutes, washed once with 96% EtOH and three times with MQ water.
Plants were grown in long day (LD) conditions (16 h light/8h dark) in Percival chambers equipped with full range LED illumination with the following conditions: light intensity of 120 μmol * m-2s-1, constant humidity (RH 65%) at either 10°C, 16°C, 23°C, or 27°C.
Plant transformation and selection of transformants
Electro-competent Agrobacterium tumefaciens GV3101 cells containing the pSOUP plasmid required for the replication of pGreen-IIS-based vectors [86,87] were transformed with the plasmids described below and grown on selective media using the appropriate antibiotics. A. thaliana pcp-1 mutants were grown at 23°C till flowering stage before siliques started to develop. Plants were transformed by floral dipping using Agrobacterium tumefaciens-mediated gene transfer [84]. T1 seeds were selected by growing on ½ MS media plates with 1.6% agar supplemented with 10 ug/mL BASTA. Seedlings exhibiting BASTA-resistant phenotype were transferred to soil and the presence of insertion or deletion was confirmed by PCR (S4 Table). Alternatively, the selection of transformants expressing the mCherry or GFP seed marker was performed by picking T1 fluorescent-positive seeds using an epifluorescent stereomicroscope.
To remove the CRISPR/Cas9 cassette, plants were backcrossed to Col-0 and transgene-free plants with deletions in PCPL were established (S1 Fig).
Plant phenotyping
Phenotypes are reported for homozygous mutants and transgenic lines for which segregation analysis indicated the presence of a single T-DNA insertion unless otherwise stated. Phenotypic analyses were performed using plants grown on ½ MS plates at 10°C, 16°C, 23°C, and 27°C for 25, 10, 10, and 7 days, respectively. Rescue assays of pcp-1 plants transformed with PCP intron deletions were performed in seedlings grown on ½ MS plates at 16°C for 10 days. Plates for root growth assays were scanned using an EPSON Expression 12000XL scanner equipped with SilverFast® 8 software at 1800 dpi resolution or photographed. In addition, pictures were taken using a digital camera to document rosette phenotypes.
Embryo lethality in self-pollinated pcp-1/pcpl-1 (het/het), pcp-1/pcpl-2 (het/het) lines was calculated as the proportion of aborted embryos relative to the total amount of healthy seeds and aborted embryos on one side of the silique.
Cloning of DNA fragments and assembly of plasmids
Genomic PCP and PCPL fragments were amplified by PCR using primers listed in S4 Table from DNA extracted from Col-0 using the protocol described by [88] with the following modifications: the volume of the extraction buffer added to the sample before the grinding step was reduced to 500 μl and the incubation time at 95°C was reduced to 1 minute. Intron deletion constructs in PCP were created by overlap extension PCR [89] using the primers listed in S4 Table. PCP and PCPL open reading frames (ORFs) were amplified from cDNA. For this, total RNA was isolated from Col-0 seedlings using the RNeasy® Plant Mini Kit (Qiagen) following the manufacturer’s protocol. Purified RNA was treated with DNase I (Thermo Scientific) followed by first-strand cDNA synthesis using the RevertAid RT kit (Thermo Scientific) with oligo(dT)18 primer. The primers used for amplification of PCP and PCPL Coding Sequences (CDSs) by PCR are listed in S4 Table.
PCP and PCPL genomic sequences and ORFs were cloned into GreenGate C-module entry plasmids as previously described [87]. PCP promoter (232 bp upstream of the start codon, covering the 5’-UTR and intergenic region) and PCP terminator (423 bp downstream the stop codon, including the 3’-UTR and intergenic region) sequences were amplified from Col-0 DNA using primers listed in S4 Table and cloned into GreenGate entry vectors A and E, respectively. For the generation of PCP and PCPL overexpression lines, existing A- and E-modules carrying 35S promoter and pea RbcS terminator sequences, respectively, were used [87].
The plant binary plasmids were assembled in destination vector pGGZ003 using GreenGate cloning as previously described [87]. Empty B- and D-modules were used in the assembly reaction. BASTA resistance was used as a selection marker in module F in all constructs except PCP intron deletion lines, in which case a seed-specific mCherry selection marker was used [90–92].
Point mutations to exchange amino acids in positions 65 and 86 in PCP and PCPL were generated by PCR using assembled plasmids with gPCP (pVD_007) and gPCPL (pVD_008) insertions as a template (S5 Table) and primers listed in S4 Table.
The final plasmids assembled by GreenGate cloning are listed in S2 Table. The remaining plasmids were generated by amplifying the inserts using overlap fusion PCR to simultaneously introduce the desired mutations/deletions and A- and G-compatible GreenGate sites, followed by GreenGate cloning into the pGGZ003 destination (S3 Table).
For CRISPR/Cas9-mutagenesis of PCPL, pairs of gene-specific sgRNAs were designed using CRISPR-P 2.0 (http://crispr.hzau.edu.cn/CRISPR2/) to simultaneously target two positions in the gene (S1 Fig). Selected pairs of sgRNAs were introduced into forward and reverse primers (S1 Fig and S4 Table), which were used to amplify a dual-guideRNA cassette via the pCBC-DT1T2 vector [93] thereby introducing a sgRNA scaffold to the first guide RNA sequence, a U6-29 Promoter to the second guide RNA sequence and flanking BsaI restriction sites, making it compatible with GreenGate-based CRISPR vectors. The PCR amplicons were purified using the E.Z.N.A.® Gel Extraction Kit (Omega Bio-tek) and cloned into BsaI-compatible sites of the vector CF588 via the GreenGate reaction.
All constructs were transformed and propagated in DH5α Escherichia coli cells. Single colonies were selected on solid LB media supplemented with the appropriate antibiotic, propagated in liquid LB media, and plasmids were purified using the E.Z.N.A.® Plasmid DNA Mini Kit I (Omega Bio-tek). All plasmids generated in this study were verified by Sanger sequencing using insert-specific primers (S4 Table) and are listed in S2 and S3 Tables.
All primers used in this study were synthesized by Eurofins Genomics.
RNA extraction and RT-qPCR analysis
For RT-qPCR plants were grown on ½ MS media plates with 1.6% agar at 23°C for 5 days and then transferred to the temperature of interest (10°C, 16°C, 23°C and 27°C) for one more day of growth. Approximately 10 seedlings per biological replicate were collected in Eppendorf tubes®, immediately flash-frozen in liquid nitrogen, and stored at -80°C. The frozen plant material was ground to fine powder using a Mixer Mill MM 400 (Retsch) and stainless-steel beads. All steps were performed with cooled-down equipment to prevent premature thawing of the samples. Extraction of RNA was performed using the RNeasy® Plant Mini Kit (Qiagen) following manufacturer’s protocol. RNA concentration and quality were measured using a NanoDrop™ 2000 Spectrophotometer (Thermo Scientific) device and RNA was stored at -80°C till further use.
Reverse transcription was carried out from 1 μg of purified RNA using the RevertAid RT kit (Thermo Scientific) with oligo(dT)18 primer. Single-strand cDNA was stored at -20°C. RT-qPCR reactions were prepared using the LightCycler® 480 SYBR Green I Master mix. All RT-qPCR assays were performed on CFX™ Real-Time PCR Detection System (Bio-Rad, Inc., Hercules, CA, USA) instruments equipped with either 96- or 384-well blocks using the following program: 5 min at 95°C, followed by 40 cycles of 15 sec at 95°C (denaturation), 30 sec at 55°C (annealing), 20 sec at 72°C (elongation), followed by a melting curve analysis from 55°C to 95°C using 1°C increments. Relative gene expression was calculated using the 2–△△Ct method. with TUB2 (AT5G62690) as a reference gene. All RT-qPCR assays were performed using 3 biological replicates with 3 technical repetitions each except lines expressing pNR_060_5-2, pNR_121_4-1-6, pNR_157_1-2 constructs that lacked material for 3 biological replicates. All primers used for RT-qPCR analyses can be found in the S6 Table.
Image processing
Figures were processed and prepared for publication using Affinity Designer v1.10.6 (Serif Ltd, https://affinity.serif.com/en-us/).
Statistical analysis
Root length was calculated using Fiji 2.14.0 (ImageJ) [94]. Calculation of the average root length, one-way ANOVA with Tukey’s test to estimate the difference between genotypes and data plotting was performed with the GraphPad Prism version 10.2.2 for Windows, GraphPad Software, Boston, Massachusetts USA, www.graphpad.com. Similarly, calculation of the average, Tukey test to eliminate the outliers and data plotting was performed for abortion rates in the mutants.
Supporting information
S1 Method. Command line instructions used for phylogenetic analysis.
https://doi.org/10.1371/journal.pone.0318163.s001
(DOCX)
S1 Fig. Arabidopsis thaliana SmE protein structure.
Overlay of PCP (SmE1) (teal) and PCPL (SmE2) (orange) illustrates the high similarity of the predicted structures. N- and C-terminals of the proteins and amino acids in positions 65 and 86 are marked on the protein structures with the corresponding colors.
https://doi.org/10.1371/journal.pone.0318163.s002
(TIF)
S2 Fig. CRISPR/Cas9-generated PCPL mutant alleles.
Shown are the region of the genomic PCPL sequence (PCPL gDNA) into which a mutation (deletion) was introduced. Boxes mark exon sequence and dashes indicate the deleted nucleotides. Lines above the gDNA indicate the position of sgRNAs. Underlined letters mark the in-frame stop codons generated in the truncated PCPL coding sequence (CDS). The predicted protein sequence is shown at the bottom with bold letters indicating the first altered amino acid.
https://doi.org/10.1371/journal.pone.0318163.s003
(TIF)
S3 Fig. Plant phenotypes at 27°C and 10°C.
Representative images of the 7-day-old seedlings transformed with various rescue constructs grown at 27°C (A) and 25-day-old seedlings grown at 10°C (B). Three independent lines are shown for each rescue construct generated in this study: #1-3: pPCP::cPCP::tPCP; #4-6: pPCP::cPCPL::tPCP; #7-9: pPCP::gPCP::tPCP; #10-12: pPCP::gPCPL::tPCP; #13: 35S::cPCP::tRbcS [32]; #14-16: 35S::cPCPL::tRbcS. All constructs were expressed in the pcp-1 background.
https://doi.org/10.1371/journal.pone.0318163.s004
(TIF)
S4 Fig. Plant phenotypes at 23°C and 16°C.
Representative images of the 10-day-old seedlings transformed with various rescue constructs grown at 23°C (A) and 16°C (B). Three independent lines are shown for each rescue construct generated in this study: #1-3: pPCP::cPCP::tPCP; #4-6: pPCP::cPCPL::tPCP; #7-9: pPCP::gPCP::tPCP; #10-12: pPCP::gPCPL::tPCP; #13: 35S::cPCP::tRbcS [32]; #14-16: 35S::cPCPL::tRbcS. All constructs were expressed in the pcp-1 background.
https://doi.org/10.1371/journal.pone.0318163.s005
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S5 Fig. Root length of PCP intron-deletion lines.
Length of the primary root of three independent lines in the pcp-1 background transformed with various intron deletion constructs. Deletion lines and constructs are labeled as gPCP_ ∆ iX, where gPCP represents a full pPCP::gPCP::tPCP construct, ∆ stands for deletion, i stands for intron, and X indicates the number(s) of the deleted intron(s). Col-0, pcp-1, and a pcp-1 rescue line carrying the full-length pPCP::gPCP::tPCP construct serve as controls. Root length was determined using 10-day-old seedlings grown at 16°C (blue) and 23°C (light grey) for three independent transgenic lines per intron-deletion construct. Bars show the mean of the root length measurement (n = 10 to 37), and error bars indicate the standard deviation (SD). A and B show independent measurements. A two-way ANOVA test with Tukey correction was performed using GraphPad Prism. Letters represent significantly different (P < 0.05) groups.
https://doi.org/10.1371/journal.pone.0318163.s006
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S6 Fig. PCP expression and root phenotype rescue in independent gPCP_ ∆ i1 lines.
A) Difference in the length of the primary root of independent gPCP_ ∆ i1 lines in the pcp-1 background. Col-0 and pcp-1 serve as controls. Root length was determined using 10-day-old seedlings grown at 16°C (blue) and 23°C (light grey). Bars show the mean of the root length measurement (n = 10 to 28), and error bars indicate the standard deviation (SD). B) Corresponding expression of PCP. Gene expression was determined in seedlings after 5 days of growth at 23°C followed by 24 h of growth at 16°C (blue) or 23°C (grey). Bars show the mean expression calculated from three biological replicates with three technical replicates each. Error bars indicate the standard deviation (SD). A two-way ANOVA test with Tukey correction was performed using GraphPad Prism. Letters represent significantly different (P < 0.05) groups.
https://doi.org/10.1371/journal.pone.0318163.s007
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S1 Table. SmE genes used in phylogenetic analysis.
https://doi.org/10.1371/journal.pone.0318163.s008
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S2 Table. GreenGate plant binary plasmids used in this study.
https://doi.org/10.1371/journal.pone.0318163.s009
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S3 Table. Summary of plasmids cloned by overlap PCR used in this study.
https://doi.org/10.1371/journal.pone.0318163.s010
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S4 Table. Oligonucleotides used in this study.
https://doi.org/10.1371/journal.pone.0318163.s011
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S5 Table. Summary of GreenGate entry modules generated in this study, including information on PCR primers used for amplification.
https://doi.org/10.1371/journal.pone.0318163.s012
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Acknowledgments
We would like to thank the group of Prof. Wolf L. Eiserhardt from Aarhus University for support in phylogenetic analysis and GenomeDK cluster for providing computational resources to perform necessary calculations and support that contributed to these research results. Prof. Christian Fankhauser, Lausanne University, Lausanne, Switzerland, for providing the CF588 vector. Dmitry Malyshev, PhD, for help with language editing.
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