Skip to main content
NCBI home page
Plant Communications logoLink to Plant Communications
. 2022 Aug 10;3(6):100420. doi: 10.1016/j.xplc.2022.100420

Convergent evolution of AP2/ERF III and IX subfamilies through recurrent polyploidization and tandem duplication during eudicot adaptation to paleoenvironmental changes

Liangyu Guo 1,3, Shuo Wang 1,3, Yuqi Nie 1,3, Yirong Shen 1, Xiaoxue Ye 2, Wenwu Wu 1,∗
PMCID: PMC9700204  PMID: 35949168

Abstract

Whole-genome duplication (WGD or polyploidization) has been suggested as a genetic contributor to angiosperm adaptation to environmental changes. However, many eudicot lineages did not undergo recent WGD (R-WGD) around and/or after the Cretaceous-Paleogene (K-Pg) boundary, times of severe environmental changes; how those plants survived has been largely ignored. Here, we collected 22 plants from major branches of the eudicot phylogeny and classified them into two groups according to the occurrence or absence of R-WGD: 12 R-WGD-containing plants (R-WGD-Y) and 10 R-WGD-lacking plants (R-WGD-N). Subsequently, we identified 496 gene-rich families in R-WGD-Y and revealed that members of the AP2/ERF transcription factor family were convergently over-retained after R-WGDs and showed exceptional cold stimulation. The evolutionary trajectories of the AP2/ERF family were then compared between R-WGD-Y and R-WGD-N to reveal convergent expansions of the AP2/ERF III and IX subfamilies through recurrent independent WGDs and tandem duplications (TDs) after the radiation of the plants. The expansions showed coincident enrichments in- times around and/or after the K-Pg boundary, when global cooling was a major environmental stressor. Consequently, convergent expansions and co-retentions of AP2/ERF III C-repeat binding factor (CBF) duplicates and their regulons in different eudicot lineages contributed to the rewiring of cold-specific regulatory networks. Moreover, promoter analysis of cold-responsive AP2/ERF genes revealed an underlying cis-regulatory code (G-box: CACGTG). We propose a seesaw model of WGDs and TDs in the convergent expansion of AP2/ERF III and IX genes that has contributed to eudicot adaptation during paleoenvironmental changes, and we suggest that TD may be a reciprocal/alternative mechanism for genetic innovation in plants that lack WGD.

Key words: polyploidization, tandem duplication, AP2/ERF family, adaptive evolution, global cooling, abiotic stress

This study shows convergent expansions of AP2/ERF III and IX subfamilies through recurrent independent duplications of WGDs in WGD-containing plants versus tandem duplications (TDs) in WGD-lacking plants during global cooling around and/or after the Cretaceous-Paleogene (K-Pg) boundary. It proposes a seesaw model of WGDs and TDs in the convergent expansion of AP2/ERF III and IX genes in eudicot adaptation to severe environmental changes.

Introduction

A century ago, the Chinese poet Mao Tse-Tung wrote: “Eagles cleave the air, fish glide in the limpid deep; Under freezing skies a million species contend in freedom.” Angiosperms (flowering plants) are one of the most diverse and abundant groups, with at least ∼350 000 known species, of which eudicots account for more than 75%. The wide distribution of angiosperms from tropical to polar terrestrial zones can be attributed to the evolution of advanced and sophisticated mechanisms of environmental adaptability. Among these mechanisms, whole-genome duplication (WGD, or polyploidization) is an extreme mechanism that replicates the entire genome and is likely to be associated with environmental stress (Panchy et al., 2016; Sessa, 2019).

The study of WGD spans more than 100 years (Soltis et al., 2014). Myriad studies have documented WGD as a critical evolutionary force for plant speciation, adaptation, and diversification (Van de Peer et al., 2009; Soltis and Soltis, 2016; Cai et al., 2019) and have suggested that WGD contributes to the wide distribution of angiosperms across the globe (Van de Peer et al., 2017; Rice et al., 2019). In nature, many extant plants are generally diploid, or have a polyploid ancestry with subsequent rediploidization; retention of WGD was thus suggested to be exceedingly rare, and polyploidy was proposed to be an evolutionary “blind alley” or “dead end” (Van de Peer et al., 2009, 2017; Arrigo and Barker, 2012; Mayrose et al., 2015). Recently, with more plant genomes being sequenced, a flock of concordant studies have shown that WGD events occurred independently in many different angiosperm lineages around the Cretaceous-Paleogene (K-Pg) boundary (∼66 Mya), suggesting that WGD may contribute to adaptive evolution at specific times, such as periods of severe environmental change (Vanneste et al., 2014; Novikova et al., 2018; Song et al., 2020; Wu et al., 2020; Van de Peer et al., 2021).

During the K-Pg boundary, global cooling was one of the major paleoenvironmental stressors (Schulte et al., 2010), and many angiosperm lineages underwent independent WGD events and convergently retained cold-related genes, which may have increased the robustness of plant cold tolerance (Song et al., 2020; Wu et al., 2020; Wang et al., 2022). Such retained cold-related genes include C-repeat binding factors (CBFs) and inducers of CBF expression (ICEs), which are the best-known key regulators of the major cold-signaling pathway for freezing tolerance (Barrero-Gil and Salinas, 2017; Shi et al., 2018), although Arabidopsis CBF1/2/3 were generated by tandem duplication (TD). Prior studies were generally performed on plants (e.g., Arabidopsis, Populus, Glycine, rice, and maize) that underwent WGD events around or after the K-Pg boundary; however, to our knowledge, many eudicot lineages (such as Rosa, Betula, and Vitis) did not undergo WGD events during a similar period. Not only did these lineages without WGD survive the severe paleoenvironmental changes, some of them also have a strong ability to adapt to severe cold stress. For example, Betula, primarily distributed in temperate and/or subarctic regions, can tolerate −40°C to −50°C (Lv et al., 2021). Thus, how these eudicot lineages survived the severe paleoenvironmental changes is an interesting story.

In addition to WGD, small-scale duplications (SSDs), such as tandem duplication, DNA- and RNA-based transposition, and dispersed duplication, also contribute to the expansion of gene families for genetic innovation (Panchy et al., 2016). Among the SSD mechanisms, TD (sometimes called local duplication) is the most striking mechanism, generating ∼16% of Arabidopsis genes and ∼14% of rice genes (Rizzon et al., 2006). In contrast to the preferential retention of transcription factors, protein kinases, and transporters after WGD (Freeling, 2009; Song et al., 2020; Wu et al., 2020), duplicates from TD are significantly enriched in membrane proteins or proteins that function in stress response and are depleted in transcription factors or other DNA- and RNA-binding proteins (Rizzon et al., 2006; Freeling, 2009). Some studies have shown that TD is an important mechanism that contributes to multigene family expansion in response to abiotic stresses (Hanada et al., 2008; Salojarvi et al., 2017). For instance, a study on Betula pendula, which did not undergo WGD during the K-Pg boundary, suggested that a suite of stress-responsive gene duplicates were not derived from polyploidy but instead from a TD process (Salojarvi et al., 2017). Accordingly, we asked whether TD served as an alternative and/or reciprocal mechanism to WGD in the replication and convergent retention of stress-related genes, making a genetic contribution to the adaptation of eudicots that lacked WGC during this period of severe stress.

To address this question, we collected 22 plants from major branches of the eudicot phylogeny (Supplemental Figure 1) and classified them into two groups according to the occurrence or absence of recent WGD (R-WGD) events around and/or after the K-Pg boundary: 12 R-WGD-containing plants (R-WGD-Y) and 10 R-WGD-lacking plants (R-WGD-N). In R-WGD-Y and R-WGD-N, we first identified 496 gene-rich families, analyzed their retention differences after R-WGD events in R-WGD-Y, revealed their cold responsiveness using RNA-seq datasets, and found that the over-retained AP2/ERF family were the most strikingly regulated transcription factors in response to cold stress. Next, we revealed that the most recent convergent expansions of AP2/ERF family genes occurred mainly through R-WGDs in R-WGD-Y but through TDs in R-WGD-N. We also investigated whether these expansions contributed to the rewiring of cold-regulatory networks in R-WGD-Y and R-WGD-N and studied the molecular basis of the cold response in AP2/ERF genes. Finally, we proposed a seesaw model of WGDs and TDs in the convergent expansion of AP2/ERF III and IX genes.

Results

Intragenomic analysis shows two groups of eudicots either with or without R-WGD events around and/or after the K-Pg boundary

WGD events generate a rich group of similar synonymous distances between gene duplicates, which is often reflected in a Gaussian distribution of synonymous substitution (Ks) values (Blanc and Wolfe, 2004; Kagale et al., 2014). In each of 22 eudicots, we detected intragenomic collinear blocks and calculated Ks values for collinear paralogous pairs to create a Ks distribution (see section “methods”). As expected, except for Nelumbo nucifera, the plants in both R-WGD-Y and R-WGD-N groups have a common peak around Ks = ∼1.5, with a range from 1.2 to 2.2 (Figure 1), which corresponds to the γ-whole-genome triplication (WGT; ∼120 Mya) event in the common ancestor of core eudicots after the divergence of Proteales (e.g., Nelumbo) (Jiao et al., 2012; Vekemans et al., 2012). Despite one or two more ancient WGD events (ζ- and/or ε-WGD) before the radiation of angiosperms (Jiao et al., 2011; Ruprecht et al., 2017), no observed peaks were detected with Ks > 2.2 in R-WGD-Y and R-WGD-N (Figure 1). This is probably because of synonymous substitution saturation and low gene retention rates for ancient WGD-derived duplicates.

Figure 1.

Figure 1

Open in a new tab

Two groups of eudicots classified on the basis of R-WGD events around and after the K-Pg boundary.

(A and B) Distributions of synonymous substitution values (Ks) for intragenomic collinear gene pairs reveal the two groups R-WGD-Y and R-WGD-N, with (A) or without (B) R-WGD events around and after the K-Pg boundary. The evolutionary relationships among the plants are based on the accepted topology from the Angiosperm Phylogeny Website, and their divergence times were obtained from TimeTree (Kumar et al., 2017). Well-acknowledged WGD/T events (Supplemental Table 1) are shown on the branches of the phylogeny, and peaks of Ks values were recorded corresponding to these events. A phylogenetic tree of all the studied plants is shown in Supplemental Figure 1.

Notably, after the radiation of core eudicots, the plants in R-WGD-Y have lineage- or species-specific peaks at Ks < 1.2 (Figure 1A) that agree with well-acknowledged WGD events that occurred independently around and/or after the K-Pg boundary (Vanneste et al., 2014; Wu et al., 2020) (Supplemental Table 1). The observation of different Ks peak values of β-WGDs among R-WGD-Y plants from similar times near the K-Pg boundary can be explained by different synonymous substitution rates among different plant lineages (Tuskan et al., 2006; Fawcett et al., 2009). By contrast, the plants in R-WGD-N have an even distribution of Ks values with Ks < 1.2, implying that no R-WGD events occurred during this time (Figure 1B). These findings demonstrate that after the radiation of core eudicots, plants underwent divergent evolutionary paths either with or without R-WGD events mainly around and/or after the K-Pg boundary.

Over-retained gene families after R-WGD events are significantly associated with cold stimulation

The predominant fate of most duplicates after WGD is loss, but gene families that contribute to plant fitness are likely to be retained (Panchy et al., 2016; Wu et al., 2020). To explore the retained families, we first identified a total of 496 gene-rich families, each with 10 or more members in at least half of the 22 eudicots (Supplemental Table 2; see section “methods”). Subsequently, we investigated retention differences in the 496 families after independent R-WGD events in R-WGD-Y and identified the top 20 families with the significantly highest retention (Figure 2A; Supplemental Table 3). Consistent with previous studies (Maere et al., 2005; Freeling, 2009; Wu et al., 2020), the over-retained families included transcription factor (TF) genes such as MYB, AP2/ERF, Homeobox, bHLH, WRKY, bZIP, AUX_IAA, and NAC family genes, which are mainly involved in diverse developmental processes and response to environmental stresses. We also found some other over-retained families, including Ras, RRM, PP2C, IQ, and TPT genes (Figure 2A).

Figure 2.

Figure 2

Open in a new tab

Retention patterns of 496 gene-rich families after R-WGD events in R-WGD-Y plants and their expression patterns in response to cold stress.

(A) Comparative genomes show retention patterns of 496 gene-rich families following independent R-WGD events in R-WGD-Y plants. A total of 496 gene-rich families were investigated, and the top 20 over-retained families (sorted by average retentions in R-WGD-Y) after R-WGD events were enlarged. The abundance of retained genes after R-WGD was compared between each of the 496 families and other genomic genes via Fisher’s exact test, and their significance levels (−log10(P value)) were scaled as retention scores in the column direction (Supplemental Table 3).

(B) Comparative transcriptomes show expression patterns of 496 gene-rich families in response to cold stress in R-WGD-Y plants. Four different plant lineages (A. thaliana, C. illinoinensis, G. max, and P. trichocarpa) were selected for study of the cold-responsive transcriptome. The top 20 significantly cold-responsive families were enlarged. The eight families marked with purple stars overlapped with the top 20 over-retained families. The abundance of cold-induced genes was compared between each of the 496 families and other genomic genes via Fisher's exact test, and their significance levels (−log10(P value)) represent the cold responsiveness of the families (Supplemental Table 4).

(C) The statistically significant overlap between over-retained families and cold-responsive families. Statistical significance was assessed by Fisher’s exact test.

The over-retained TF genes in angiosperms after WGD events have been suggested to be critical genetic contributors to survival during environmental changes around the K-Pg boundary, when global cooling was a major environmental stressor (Schulte et al., 2010; Wu et al., 2020). Accordingly, we assumed that TF families or genes that have higher cold responsiveness and regulate cold-resistance genes would make a greater contribution to plant survival during global cooling. To identify such TF families or genes, we selected four different lineages (Glycine max, Populus trichocarpa, Carya illinoinensis, and Arabidopsis thaliana) from R-WGD-Y for investigating differences in cold responsiveness of the 496 families (Figure 2B and Supplemental Table 4). Notably, among the top 20 families with the highest cold responsiveness, eight were also among the aforementioned top 20 families with the significantly highest retention, showing a statistically significant association (P = 1.4e−7) between gene over-retention and cold responsiveness (Figure 2C). These findings suggest that cold-stimulated genes were preferentially retained following R-WGD events.

Among the eight overlapping families, the AP2/ERF family was the most strikingly responsive TF family to cold stimulation, followed by the WRKY, EF-hand_5, and Helicase_C families (Figure 2B). Considering the widely recognized roles of AP2/ERF genes in abiotic stresses, especially the key role of AP2/ERF members such as CBF genes in cold acclimation and freezing tolerance (Barrero-Gil and Salinas, 2017; Shi et al., 2018), we were struck by the convergent over-retention and exceptional cold stimulation of AP2/ERF genes in different eudicot lineages of R-WGD-Y plants. This finding suggests that such genes may have made a substantial genetic contribution to the survival of R-WGD-Y plants during global cooling.

AP2/ERF family genes were convergently expanded by WGD in R-WGD-Y and by TD in R-WGD-N after the radiation of core eudicots

In this section, the expansion history of AP2/ERF family genes was studied and compared between R-WGD-Y and R-WGD-N. To this end, we classified two periods of AP2/ERF gene expansion, before and after the radiation of the 22 plants (Supplemental Table 5; see section “methods”). Before radiation, ancestral WGDs (A-WGDs), including ζ-/ε- and γ-WGD/T events, occurred and were thus shared by the studied core eudicots. After radiation, the plants underwent divergent evolution, either with recent WGDs (R-WGDs) that occurred independently in R-WGD-Y or without R-WGDs in R-WGD-N. As expected, AP2/ERF genes descended from A-WGD events showed roughly similar retention patterns in R-WGD-Y and R-WGD-N (Figure 3A). More AP2/ERF duplicates from A-WGDs in R-WGD-Y than R-WGD-N may have resulted from R-WGDs, which led to more complicated collinearities and overestimation of A-WGD-derived duplicates in R-WGD-Y. After radiation of the core eudicot plants, AP2/ERF genes were further expanded after R-WGDs in R-WGD-Y, whereas their counterparts in R-WGD-N plants did not undergo WGD-mediated expansion (Figure 3A). Interestingly, R-WGD-Y and R-WGD-N contain comparable numbers of AP2/ERF family genes, except for 200 more AP2/ERF genes in G. max and Brassica oleracea due to at least two R-WGDs in these plants (Figure 3B). This finding implies that an alternative duplication mechanism may have expanded AP2/ERF genes in R-WGD-N plants.

Figure 3.

Figure 3

Open in a new tab

WGD and TD exhibit convergent roles in expansion of the AP2/ERF III and IX subfamilies in R-WGD-Y and R-WGD-N.

(A) Retentions of AP2/ERF duplicates following WGD events in R-WGD-Y and R-WGD-N. The size of the circle represents the number of AP2/ERF duplicates. AP2/ERF duplicates retained from WGD events in extant plants were classified into two periods: before (i.e., A-WGD) and after (i.e., R-WGD) the radiation of the plants (see section “methods”).

(B) Both R-WGD-Y and R-WGD-N groups have a large family of AP2/ERF genes with more than 100 members in each plant.

(C) The proportions of AP2/ERF genes generated by different duplication modes. TSP indicates DNA- or RNA-mediated transposed duplication. The statistical difference of duplication modes in producing AP2/ERF genes between R-WGD-Y and R-WGD-N was assessed by the nonparametric Mann–Whitney test.

(D) A significant proportion of the AP2/ERF III and IX subfamilies were convergently expanded by WGD in R-WGD-Y (left) and by TD in R-WGD-N (right). The green and red dots indicate AP2/ERF genes generated by WGD and by TD, respectively. The circular trees were constructed using IQ-TREE (Nguyen et al., 2015) for maximum likelihood (ML) analysis.

Like WGD, TD and transposed duplication (TSP) can contribute to gene family expansion (Panchy et al., 2016). To explore the mechanisms underlying the expansion of AP2/ERF genes in R-WGD-N plants, we analyzed the proportions of AP2/ERF genes generated by these duplication modes in R-WGD-N versus R-WGD-Y (Figure 3C; Supplemental Table 6). As shown in Figure 3A, the proportion of WGD-derived AP2/ERF duplicates was significantly lower in R-WGD-N than in R-WGD-Y (Figure 3C). However, TD showed the opposite trend, producing a significantly higher proportion of AP2/ERF duplicates in R-WGD-N than in R-WGD-Y. For the TSP mode, there was no significant difference between the two groups (Figure 3C). Together, these findings demonstrate that before the radiation of these plants, AP2/ERF genes had been expanded by at least two waves of A-WGDs (ζ/ε and γ events), and after their radiation, AP2/ERF genes in R-WGD-Y plants were further expanded after independent R-WGD events, whereas their counterparts in R-WGD-N plants were alternatively expanded by independent TD events.

AP2/ERF III and IX subfamilies were especially expanded by WGD in R-WGD-Y and by TD in R-WGD-N

AP2/ERF genes have an ancient evolutionary diversity with more than 15 subfamilies (Nakano et al., 2006; Mizoi et al., 2012). Convergent expansion of specific subfamilies probably implies functional roles in adaptation to the same or similar environments. We explored whether any subfamilies of AP2/ERF genes were especially expanded by WGD or TD in R-WGD-Y and R-WGD-N. As expected, the AP2/ERF genes in R-WGD-Y were mainly derived from WGD events, and interestingly, they were mainly clustered in subfamilies III and IX in addition to the AP2 subfamily (Figure 3D and Supplemental Figure 2). Despite the small number of TD-derived AP2/ERF genes in R-WGD-Y, we still observed that they were clustered mainly in the III and IX subfamilies. Similarly, in the R-WGD-N group, the III and IX subfamilies were also expanded but in a different way; they were expanded mainly by TD events and secondarily by A-WGDs (Figure 3D and Supplemental Figure 2). These findings show that the III and IX subfamilies of AP2/ERF genes were especially expanded by WGD in R-WGD-Y but by TD in R-WGD-N.

Convergent expansions of AP2/ERF III and IX subfamilies were enriched around severe paleoenvironmental changes

Consistent with prior studies (Vanneste et al., 2014; Song et al., 2020; Wu et al., 2020), independent WGD events were observed around the K-Pg boundary (∼66 Mya) and/or during the Late Cenozoic Icehouse (<25 Mya). Following these WGD events, III and IX subfamily duplicates were significantly retained in R-WGD-Y, despite a relatively small number of TD-derived duplicates (Figure 4A). On the other hand, in R-WGD-N, the inferred times of recent TD (R-TD) events in the expanding III and/or IX subfamilies were also enriched around and/or after the K-Pg boundary (Figure 4A; Supplemental Table 7; see section “methods”), when the paleoclimate underwent continuous global cooling (∼17°C) (McElwain and Punyasena, 2007; Schulte et al., 2010; Scotese et al., 2021).

Figure 4.

Figure 4

Open in a new tab

Convergent expansions of AP2/ERF III and IX subfamilies were enriched around periods of global cooling.

(A) Enrichment of WGD and TD events that expanded AP2/ERF III and IX subfamilies during paleotemperature changes. (Upper) The blue curve indicates global average temperature (GAT) fluctuations over the past 150 Mya. When the GAT is below 18°C (red dashed line), large polar icecaps can form (Scotese et al., 2021). (Middle) Expansion times of AP2/ERF III and IX subfamilies from R-WGDs and R-TDs in R-WGD-Y and R-WGD-N are shown along the timeline. (Lower) Geological times are shown.

(B) GO enrichment analysis of R-WGD- and R-TD-derived III and IX genes in R-WGD-Y and R-WGD-N. The significant enrichment value is shown as the −log10-transformed false discovery rate (FDR).

(C) A significantly higher proportion of R-WGD- and R-TD-derived III and IX genes are cold induced compared with other genomic genes. The difference was assessed by performing Fisher’s exact test on the number of R-WGD- and R-TD-derived III and IX genes affected by cold stress compared with other genomic genes. Cold transcriptome data from four (G. max, P. trichocarpa, C. illinoinensis, and A. thaliana) R-WGD-Y species and one (B. pendula) R-WGD-N species were used to extract the genes and perform the analysis.

(D and E) An example of the alternative and/or reciprocal relationship of WGD and TD events in the convergent expansion of CBFs in R-WGD-Y (D) and R-WGD-N (E) around and after the K-Pg boundary. The number on the branches indicates the bootstrap value (%). The three letters in brackets after each gene accession indicate the abbreviation of the species name.

Gene Ontology (GO) term enrichment analysis suggested that both R-WGD- and R-TD-derived III and IX genes were significantly enriched in cold acclimation, response to temperature stimulus, and ethylene-activated signaling pathway (Figure 4B). Cold-transcriptome analysis confirmed a significantly higher proportion of cold-induced genes in the III and IX subfamilies than in other genomic genes (Figure 4C). It is thus reasonable to infer that the expansion of III and IX genes in these plants might be associated with global cooling. For instance, CBFs, key regulators of cold acclimation for freezing tolerance (Barrero-Gil and Salinas, 2017; Shi et al., 2018), were convergently expanded by either independent R-WGD events in R-WGD-Y or independent R-TD events in R-WGD-N (Figures 4D and 4E). We speculate that the convergent expansion of CBFs may have contributed to the rewiring of cold-specific regulatory networks for plant adaptation during paleotemperature cooling.

Convergent expansion and co-retention of CBF duplicates and CBF regulons contributed to the rewiring of cold-specific regulatory networks

To confirm the potential rewiring of CBF-dependent cold-specific regulatory networks, we selected five plants from different eudicot lineages (G. max, P. trichocarpa, B. pendula, C. illinoinensis, and A. thaliana) and generated cold-transcriptome experimental datasets (see section “methods”). In the phylogeny of the five plants, independent WGD and/or TD events occurred, producing CBF inparalogs (Figure 5A). Compared with other plants, a WGD event followed by two successive TD events in A. thaliana led to the extant CBF1/2/3/4 genes. Notably, the expression of these CBF duplicates was significantly induced in response to cold stress and showed a highly conserved cold-induced pattern in eudicots (Figure 5A).

Figure 5.

Figure 5

Open in a new tab

Convergent expansions and co-retentions of CBF genes and their targets have rewired the cold-specific regulatory network.

(A) Independent gains of CBF duplicates from R-WGD and/or R-TD events in five different lineages and their conserved cold-induced expression. The number on the branches indicates the bootstrap value (%). (Middle) Genomic collinearity analysis supports R-WGD- and R-TD-derived CBF duplicates and their convergent retention. (Right) Expression values of the CBF duplicates were extracted from the cold-responsive transcriptomes of five species at different time points (0 h, 2 h, and 24 h) of cold stress (4°C).

(B) The rewiring of cold-specific CBF-dependent regulatory networks after WGD and/or TD events. For space limitation, the CBF-dependent regulatory networks of A. thaliana (left), B. pendula (top right), and C. illinoinensis (bottom right) are shown, and those of the other two species are shown in Supplemental Figure 4. Some well-known targets of CBF1/2/3 that were identified as having been generated by WGD and/or TD events around and after the K-Pg boundary are highlighted with gene names and different colors.

We constructed CBF-dependent cold-specific regulatory networks in the five plants (Figure 5B, Supplemental Figure 3; Supplemental Tables 8 and 9). In the A. thaliana network, many genes were directly regulated by CBF1/2/3, and parts were specifically regulated by a subset of CBFs, indicating neofunctionalization or subfunctionalization of CBF duplicates after duplication events. Importantly, many well-acknowledged cold-regulated targets of A. thaliana CBFs (also named CBF regulons) were duplicated through R-TD (e.g., LTI29/COR47, COR15A/COR15B, and RLP22/RLP33) or through R-WGD events (e.g., ZAT6/ZAT10 and GOLS2/GOLS3) during similar periods of global cooling (Supplemental Table 10). Likewise, in the other four plants, we also observed convergent expansion of CBFs and their significantly co-expressed genes through WGD and/or TD events during this time (Figure 5B, Supplemental Figure 3; and Supplemental Table 10). These findings strongly suggest that convergent expansion and co-retention of key components of the CBF-dependent pathway through independent WGD and/or TD events may have together rewired stress-related regulatory networks during adaptation to global cooling.

Cold-responsive AP2/ERF genes reveal a conserved cis-regulatory code

As described above, the AP2/ERF family shows exceptional cold stimulation. To reveal the regulatory code of cold-responsive AP2/ERF genes, we classified AP2/ERF genes into 15 subfamilies based on a prior study (Nakano et al., 2006) and examined their expression patterns under cold stress (Figure 6A; Supplemental Table 11). As expected, the AP2/ERF III subfamily, including A. thaliana CBF1/2/3 genes, showed the most striking cold induction in the plants. Some other subfamilies that have close evolutionary relationships with subfamily III, such as II, IV, VIII, and IX, also showed considerable cold-responsive expression patterns (Figure 6A).

Figure 6.

Figure 6

Open in a new tab

Cold-responsive AP2/ERF genes reveal a conserved cis-regulatory code.

(A) Conservation and diversity of cold-induced activities of the 15 AP2/ERF subfamilies in eudicots. The size of the circle indicates the number of corresponding subfamily genes, and its color (from white to purple) indicates the maximum cold responsiveness (2 h or 24 h) relative to normal conditions (0 h). The relative expression was obtained by normalization of the average trimmed mean of the M value (aTMM) for each subfamily divided by the maximum aTMM.

(B) A significant cis-regulatory motif in the promoters of cold-responsive AP2/ERF genes. (Upper) A significant motif peak (CACGTG) across the promoters (1000 bp upstream of the annotated translation initiation site [TIS]) of cold-responsive AP2/ERF genes. (Lower) Motif loci (blue thick lines) around the peak in each cold-responsive AP2/ERF gene; the number in the dark green circle on the right indicates the occurrences of the elements in the region.

(C) Three examples of Arabidopsis CBF1/2/3 promoters with multiple CACGTG elements experimentally confirmed to be recognized by several cold-relevant transcription factors. The motif in blue indicates that the motif-containing region is recognized and bound by the corresponding transcription factors, which were obtained from previous chromatin immunoprecipitation (ChIP)-PCR, EMSA, and/or ChIP-seq experiments (Kim et al., 2015b; Eremina et al., 2016; Li et al., 2017; Song et al., 2021).

(D) A higher motif number is correlated with a higher expression value. The boxplot shows more CACGTG motifs (left) and higher expression (right) in the DREB than the ERF group. The higher expression value of the 2 h and 24 h cold-treatment points for each cold-responsive AP2/ERF gene was used to calculate the fold change (FC) relative to normal conditions (0 h). Statistical differences in motif numbers and expression between the DREB and ERF groups were calculated by the nonparametric Mann–Whitney test.

Next, we performed motif-enrichment analysis and identified specific cis-regulatory elements (CACGTG) enriched ∼280 bp upstream of the translation initiation site (TIS) of the cold-responsive AP2/ERF genes (Figure 6B). The palindromic CACGTG element, also known as the G-box, has been widely demonstrated to be an essential element of many stimulus-responsive promoters that are bound by transcription factors from the basic helix-loop-helix (bHLH) and basic leucine zipper (bZIP) families (Menkens et al., 1995; Ezer et al., 2017). Experimentally, inducer of CBF expression 1 (ICE1) encodes an MYC-like bHLH transcriptional activator that binds to a degenerate sequence (CACATG) of the G-box in the CBF3 promoter, significantly inducing CBF3 expression (Chinnusamy et al., 2003). Moreover, chromatin immunoprecipitation sequencing (ChIP-seq) analysis further revealed ICE1 binding peaks that shared the G-box or its degenerate sequences in all the three CBF1/2/3 promoters (Tang et al., 2020). Interestingly, by literature data mining, we found the that same cis-regulatory G-box loci in CBF1/2 promoters can also be bound by other HLH transcription factors such as Brassinazole resistant 1 (BZR1) (Li et al., 2017) and CESTA (CES) (Eremina et al., 2016). These experimental datasets together suggest that G-box loci are hot spots bound cooperatively or competitively by such transcription factors to regulate CBF expression and freezing tolerance (Figure 6C). Moreover, there seem to be more G-box elements across the promoters in the DREB group than in the ERF group (Figure 6B), suggesting that there are potentially more G-boxes and greater expression. In support of this notion, statistical analysis showed that the DREB group had a greater number of G-boxes in their promoters and significantly higher expression induced by cold stress than the ERF group (Figure 6D). Together, these results suggest that G-box elements serve as a conserved cis-regulatory code underlying the response of AP2/ERF family genes to cold stress in eudicots.

Discussion

A handful of WGD events have generally been detected in angiosperm evolution over the past 100 million years (Van de Peer et al., 2009, 2017). Recently, several studies have documented that waves of WGD events were associated with severe paleoenvironmental changes in many different angiosperms and made a genetic contribution to plant adaptive evolution at specific times (Vanneste et al., 2014; Song et al., 2020; Wu et al., 2020). However, many eudicot plants had no R-WGD events around or after the K-Pg boundary, and it is thus fascinating to investigate how such plants survived those specific times. In this study, we compared 10 plants without R-WGD events (R-WGD-N) with 12 plants with independent R-WGD events (R-WGD-Y) in major eudicot plant lineages. First, we detected common over-retained gene families in R-WGD-Y plants after independent R-WGD events and found that many were associated with cold stimulation. Among these families, we noted that AP2/ERF genes were the most strikingly induced TF factors in response to cold stimulation. We then traced and compared the evolutionary trajectories of AP2/ERF genes in R-WGD-Y plants and R-WGD-N plants, revealing waves of convergent expansion in the AP2/ERF III and IX subfamilies through recurrent and independent WGDs and/or TDs during evolution. Interestingly, the expansions of these subfamilies were enriched around severe paleoenvironmental changes. Around and after the K-Pg boundary, global cooling was one of the major paleoenvironmental stressors (Schulte et al., 2010), and convergent expansions and co-retentions of CBF duplicates and their regulons through independent WGD and/or TD events in different plants rewired and upgraded cold-specific regulatory networks, probably for plant adaptation to global cooling. Moreover, we found that many AP2/ERF genes showed conserved responses to cold stress, and we revealed a cis-regulatory code underlying their cold responsiveness. In light of these findings, we propose a seesaw model in which convergent expansion of AP2/ERF III and IX subfamilies is widespread and robust, recurring independently through WGD and/or TD events in different eudicot lineages during adaptation to severe paleoenvironmental changes. In this model, TD seems to serve as an alternative and/or reciprocal mechanism to WGD, especially in replicating stress-related AP2/ERF genes for genetic contribution to plants that lack WGD events (Figure 7). Our research illuminates the importance of convergent expansions of AP2/ERF III and IX genes and provides new insights into the survival of plants that lack R-WGD during global cooling around and/or after the K-Pg boundary.

Figure 7.

Figure 7

Open in a new tab

The seesaw model of WGD and TD in convergent expansion of AP2/ERF III and IX genes during adaptation of eudicots to severe environmental changes.

The relative locus of each point indicates the proportions contributed by TD and WGD events to expanding subfamilies III and IX in each species.

Interestingly, many other gene families (e.g., WRKY, EF-hand_5, and NAC) showed high cold responsiveness, not far from that of AP2/ERF genes. We questioned whether they were also convergently expanded by WGD and TD events in R-WGD-Y and R-WGD-N plants. We traced the evolution of the other 19 top cold-responsive gene families and identified 16 families that were over-retained after R-WGD events in R-WGD-Y plants (Supplemental Figure 4), again supporting the biased retention of cold-relevant gene families after R-WGD events. Of the 16 cold-responsive and over-retained families in R-WGD-Y plants, including the aforementioned WRKY, EF-hand_5, and WD40 families, three families (AAA, NAC, and LEA_2) were significantly compensated by TD events in R-WGD-N plants (Supplemental Figure 4). This result suggests that TD may effectively contribute to the expansion of specific gene families. On the other hand, some other families over-retained after R-WGD events, including the MYB, Homeobox, and bHLH families, showed no significant cold responsiveness (Figure 2). It may be that global darkness (or low light), another major environmental stressor during the K-Pg boundary (Schulte et al., 2010), contributed to over-retention of such TF genes, including phytochrome interacting factors of the bHLH family and homeodomain-leucine zipper genes of the Homeobox family involved in the shade-avoidance pathway (Wu et al., 2020). Thus, the evolution of gene families involved in shade avoidance also needs to be studied in plants that lack R-WGD in the near future.

Methods

Identification of 496 gene-rich families in eudicot genomes

We downloaded proteome sequences and gene annotations for 21 core eudicot plants and one basal eudicot, N. nucifera, from the Ensembl Plants (Aken et al., 2016), Phytozome (Goodstein et al., 2012), CoGe, and NCBI genome (Kitts et al., 2016) public databases. To identify gene families in eudicots, we downloaded all the Hidden Markov Model (HMM) profiles (>19 000) of protein domains from the Pfam database (Finn et al., 2016) and searched them against the plant proteome sequences using HMMER 3.1b2 with an E value <1e−5 (Eddy, 1998). In this study, given that our focus was on the expanded gene families retained after WGD events, we retained those gene-rich families that had more than 10 members in at least half of the 22 species. To reduce redundancy, if two families shared >60% overlapped genes, only the larger family was retained. We thus obtained a total of 496 gene-rich families in the eudicots (Supplemental Table 2).

Phylogenetic analysis of the AP2/ERF gene family

From the 496 gene-rich families above, we extracted the AP2/ERF family and examined the presence and architecture of the AP2/ERF domain in the retrieved proteins using SMART (Letunic et al., 2021). Proteins with at least one AP2 domain were considered to be members of the AP2/ERF family (Supplemental Table 12). To evaluate the accuracy of AP2/ERF gene identification, we compared the retrieved A. thaliana AP2/ERF genes with those reported previously (Nakano et al., 2006), and the results were fully consistent.

Using AP2/ERF proteins from R-WGD-Y and R-WGD-N, we performed multiple alignments using ClustalW 2.1 with default parameters (Larkin et al., 2007). Phylogenetic trees of the AP2/ERF family were then constructed using IQ-TREE v1.6.12 for maximum likelihood (ML) analysis with 1000 bootstrap replicates and the best model, JTT + F + R10, selected from 545 candidates according to the Bayesian information criterion score (Nguyen et al., 2015).

Genome collinearity analysis and inferred duplication modes of eudicot genes

In each eudicot, we used BLASTP (E value <1e-10, -max_target_seqs 5) (Altschul et al., 1990) and MCScanX (-k 50, -g -1, -e 1e-5, -s 5) (Wang et al., 2012) to detect intra-genome synteny and collinear blocks. At least five pairs of homologous genes were required to define a collinear block. Subsequently, we ran the script duplicate_gene_classifier (Wang et al., 2012) to classify the generation modes of the duplicate genes into whole genome/segmental (WGD), tandem (TD), and others (including proximal and dispersed duplications). To further explain the others, we identified DNA- or RNA-based transposition events (TSP) using DupGen_finder (Qiao et al., 2019) with Nelumbo nucifera as the outgroup for core eudicot plants and Nymphaea colorata as the outgroup for N. nucifera. The transposed duplicate pairs were identified on the basis of the following criterion: one copy was at its ancestral locus, and the other was at a non-ancestral locus. Intraspecific colinear genes and interspecific colinear genes can be regarded as ancestral loci. Accordingly, we extracted AP2/ERF genes with their duplication modes from eudicot plants.

Timing of AP2/ERF gene duplications and the history of paleotemperature changes

We used ParaAT 2.0 (Zhang et al., 2012) and KaKs_Calculator 2.0 (Wang et al., 2010) to calculate Ks values for each pair of collinear paralogous genes obtained from the above collinear blocks in each plant; Ks values were estimated with Yang-Nielsen (YN) method. We designated ancestral ζ-/ε- and γ-WGD/T events before the radiation of core eudicots (Jiao et al., 2011, 2012) simply as ancestral WGD events (A-WGDs) and thus considered collinear paralogous genes with Ks > 1.2 to be derived from A-WGDs. After the radiation of core eudicots, the plants had either no or recent WGD events (R-WGDs) that occurred independently in different eudicot lineages, mainly around the K-Pg boundary (∼66 Mya) and/or during the Late Cenozoic Icehouse (<25 Mya) (Vanneste et al., 2014; Wu et al., 2020). Given the different synonymous substitution rates (r) among different plant lineages (Tuskan et al., 2006; Fawcett et al., 2009), the Ks ranges from K-Pg boundary R-WGDs varied among different plant lineages. Accordingly, we carefully assigned collinear gene duplicates with different Ks ranges to R-WGD events in different plant lineages (Supplemental Table 1).

For timing of TD-derived AP2/ERF duplicates after the radiation of core eudicots, tandem AP2/ERF duplicates with Ks < 1 were considered to be recent TD (R-TD)-derived duplicates. Next, we inferred the detailed times of these R-TDs in different plant lineages. Given the variation in r values, which is related to generation time, between different plant lineages after the radiation of core eudicots, we computed the r value for each R-WGD-Y plant species using the formula r = Ks/2T (Thomson et al., 2000), where Ks indicates the peak value (with 95% confidence interval), and T indicates the time of the R-WGD event from previous well-acknowledged studies (Supplemental Figure 5; Supplemental Table 1). The computed r values were highly consistent with previous reports. For instance, in A. thaliana, we obtained r = ∼6.7–8.27 × 10−9, which is very close to the 7 × 10−9 that was calculated using the number of spontaneous mutations that accumulated over 30 generations in an Arabidopsis population descended from a single seed (Ossowski et al., 2010). On the other hand, for R-WGD-N plants without R-WGD events, we directly adopted corresponding r values from their closely related plants (Supplemental Table 13). Using the corresponding r value for each plant species, we used the formula T = Ks/2r to calculate the times of R-TD-derived AP2/ERF duplicates.

To explore the potential relationship between AP2/ERF duplicates in the plants and paleoenvironmental changes, we collected data on global average temperature (GAT) fluctuations over the past 150 million years (Scotese et al., 2021).

GO annotations and enrichment analysis

GO-term annotations for A. thaliana were downloaded directly from The Arabidopsis Information Resource (TAIR) (Rhee et al., 2003). The proteome sequences of other studied species were searched against the A. thaliana proteins (-evalue 1e-10 -num_alignments 3) with BLASTP (Altschul et al., 1990). Subsequently, we assigned the GO terms from the most similar A. thaliana gene hit to the other species’ genes. GO enrichment analysis of the interesting AP2/ERF duplicates was performed using OmicShare tools (https://www.omicshare.com/tools).

RNA-seq and expression analysis of five selected plants under cold stress

We performed RNA-seq experiments to examine gene expression in leaf tissues of G. max, P. trichocarpa, B. pendula, C. illinoinensis, and A. thaliana under cold stress (4°C) at 0 h, 2 h, 24 h, and 168 h. Before cold-stress treatment, all plant seedlings were cultured in an artificial climate chamber at 25°C with a 16-h/8-h light/dark cycle. For A. thaliana, 3-week old seedlings were used for cold-stress treatments. For other species, young seedlings up to 30 cm in height were used, including 1-month-old G. max, 2-month-old P. trichocarpa and B. pendula, and 6-month-old C. illinoinensis. Total RNA was isolated and purified from leaves of each species and assessed with a NanoDrop ND-1000 spectrophotometer and an Agilent 2100 Bioanalyzer. Poly(A)-RNA was enriched for cDNA library construction and sequencing on the Illumina NovaSeq 6000 platform (2 × 150-bp paired-end reads).

RNA-seq reads from cold-treated samples were aligned to the corresponding genomes and reference genes using HISAT2 2.1.0 (Kim et al., 2015a) with the parameters (--min-intronlen 20 --max-intronlen 5000 --rna-strandness RF). We then used StringTie v2.0.3 (Pertea et al., 2015) with default parameters to calculate gene expression and obtain expression values (trimmed mean of M value [TMM]) of all genes in the five cold-treated species at four time points (0, 2, 24, and 168 h). Genes upregulated more than two fold between the cold treatment and the normal conditions were identified as cold-induced genes. To obtain more reliable cold-induced genes, those genes whose highest expression over the four time points was less than 5 were filtered out.

DAP-seq and ChIP-seq analysis of CBF target genes

To determine the binding peaks of CBFs across the genome, we downloaded DNA affinity purification sequencing (DAP-seq) datasets of A. thaliana CBF1, CBF2, and CBF3 proteins (CBFs) from the NCBI BioProject database (accession: PRJNA257556) (O'Malley et al., 2016). Recently, Song et al. published ChIP-seq datasets of CBFs (Song et al., 2021), and we also downloaded their data (accession: PRJNA732005). Subsequently, we aligned the DAP-seq and ChIP-seq reads to the A. thaliana genome (TAIR10) using Bowtie 2 (v2.3.5) with default parameters (Langmead and Salzberg, 2012), performed peak calling using MACS2 (v2.7.1) with custom parameters (--keep-dup 1 -g 119481543 -p 0.05) for the Arabidopsis genome, and finally annotated the peaks with the nearest genes using ChIPseeker (v1.20) (Yu et al., 2015). The genes with at least one binding peak in the 1 kb upstream of the TIS from the DAP-seq and/or ChIP-seq analysis were considered to be CBF-binding genes.

Construction of CBF-dependent regulatory network in multiple plants

To determine the CBF regulon of cold induction in A. thaliana, we downloaded two RNA-seq datasets of wild-type and mutant cbfs from the NCBI BioProject database (PRJNA316458 and PRJNA541229): the CRISPR/Cas9-generated cbfs mutant with a deletion of ∼7000 bp covering CBF1, CBF3, and part of CBF2 (Zhao et al., 2016), and another cbfs mutant with CRISPR/Cas9-mediated insertion of a nucleotide (thymine) in both the CBF1 and CBF2 coding regions in the cbf3 T-DNA insertion mutant (Jia et al., 2016). For RNA-seq analysis, we used the same methods described above to map and calculate gene expression. Subsequently, we used DESeq2 (Love et al., 2014) to identify differentially expressed genes (DEGs) between the cbfs mutants and the wild type under cold stress, and from the DEGs, we extracted the CBF regulon of cold induction from at least one of the two RNA-seq datasets.

In addition, we used our RNA-seq data from cold-treated A. thaliana samples with four time points and three replications (a total of 12 samples) to calculate Pearson's correlation coefficients (PCCs) and P values of CBFs with other expressed genes using the psych package in R (Revelle, 2021). We integrated the direct CBF target genes from DAP-seq and/or ChIP-seq analysis, the CBF regulon of cold induction from RNA-seq analysis of cbfs mutants, and the CBF co-expressed genes (PCC > 0.8 and P < 3e−4) from RNA-seq analysis of multiple cold-treatment time points to construct a comprehensive, cold-specific CBF-dependent regulatory network for A. thaliana. For G. max, P. trichocarpa, B. pendula, and C. illinoinensis, we directly used the top 300 CBF co-expressed genes from the RNA-seq analysis of multiple cold-treatment time points to construct cold-specific, CBF-related regulatory networks. The networks were plotted with Cytoscape 3.8.2 (Shannon et al., 2003).

cis-regulatory code analysis of cold-responsive AP2/ERF genes

To reveal the underlying cis-regulatory code of AP2/ERF genes in response to cold stress, we developed the following procedure. First, we collected those AP2/ERF genes whose expression was upregulated at least four fold at either one of the early cold-treatment time points (2 h and 24 h) in the RNA-seq analysis of five cold-treated plant species. Second, we extracted the 1-kb promoter sequences of these cold-responsive AP2/ERF genes from their corresponding genomes. We also collected 985 published A. thaliana cis-regulatory motifs of 587 transcription factors (Franco-Zorrilla et al., 2014; O'Malley et al., 2016). Using the motifs as the query, we searched the promoters with MEME-ChIP v5.3.3 (Bailey et al., 2015) and obtained a statistically significant motif (CACGTG, E value < 0.01). Subsequently, we scanned for motif degenerates in the promoters using FIMO (Grant et al., 2011) to refine the cis-regulatory motif. Finally, we identified a significant preference in the motif locations around ∼200–300 bp upstream in the promoters using CentriMo (Bailey et al., 2015).

Statistical analysis

Differences between two groups were assessed by the nonparametric Mann–Whitney test, and the significance of enrichment was calculated with Fisher’s exact test. All statistical tests were performed in R.

Funding

We thank the members of W.W.’s laboratory at Zhejiang A&F University for suggestions to improve the manuscript and Shuke Chen for her inspiration of Mao Tse-Tung’s poems. This work was supported by the National Natural Science Foundation of China (grant number 31871233) and the National Key R&D Program of China (2018YFD1000604).

Author contributions

W.W. designed the research. L.G. and Y.N. performed the study. S.W. and S.Y. designed the RNA-seq experiments, and Y.X. performed the analysis. W.W. wrote the manuscript.

Acknowledgments

No conflict of interest is declared.

Published: August 10, 2022

Footnotes

Published by the Plant Communications Shanghai Editorial Office in association with Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and CEMPS, CAS.

Supplemental information is available at Plant Communications Online.

Accession numbers

The RNA-seq datasets of the five eudicot plants, A. thaliana, G. max, P. trichocarpa, B. pendula, and C. illinoinensis, under cold stress have been deposited to NCBI BioProject (accession: PRJNA767196) and CNGBdb (accession: CNP0002243).

Supplemental information

Document S1. Supplemental Figures 1–5
mmc1.pdf (971.4KB, pdf)
Supplemental Tables 1–7
mmc2.xlsx (2.2MB, xlsx)
Supplemental Tables 8 and 9
mmc3.xlsx (126.2KB, xlsx)
Supplemental Table 10. Duplicates in the cold-specific CBF-dependent regulatory network that arose through TD and/or WGD events during a similar period of global cooling
mmc4.xlsx (12.5KB, xlsx)
Supplemental Table 11. RNA-seq expression values of AP2/ERF genes in the five cold-treated species at four time points (0, 2, 24, and 168 h) of cold stress
mmc5.xlsx (156.3KB, xlsx)
Supplemental Tables S12 and S13
mmc6.xlsx (61.3KB, xlsx)
Document S2. Article plus supplemental information
mmc7.pdf (16.8MB, pdf)

References

  1. Aken B.L., Ayling S., Barrell D., Clarke L., Curwen V., Fairley S., Fernandez B.J., Billis K., Garcia G.C., Hourlier T., et al. The Ensembl Gene Annotation System. Database. 2016;2016:w93. doi: 10.1093/database/baw093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Altschul S.F., Gish W., Miller W., Myers E.W., Lipman D.J. Basic local alignment search tool. J. Mol. Biol. 1990;215:403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  3. Arrigo N., Barker M.S. Rarely successful polyploids and their legacy in plant genomes. Curr. Opin. Plant Biol. 2012;15:140–146. doi: 10.1016/j.pbi.2012.03.010. [DOI] [PubMed] [Google Scholar]
  4. Bailey T.L., Johnson J., Grant C.E., Noble W.S. The MEME suite. Nucleic Acids Res. 2015;43:W39–W49. doi: 10.1093/nar/gkv416. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Barrero-Gil J., Salinas J. CBFs at the crossroads of plant hormone signaling in cold stress response. Mol. Plant. 2017;10:542–544. doi: 10.1016/j.molp.2017.03.004. [DOI] [PubMed] [Google Scholar]
  6. Blanc G., Wolfe K.H. Widespread paleopolyploidy in model plant species inferred from age distributions of duplicate genes. Plant Cell. 2004;16:1667–1678. doi: 10.1105/tpc.021345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cai L., Xi Z., Amorim A.M., Sugumaran M., Rest J.S., Liu L., Davis C.C. Widespread ancient whole-genome duplications in Malpighiales coincide with Eocene global climatic upheaval. New Phytol. 2019;221:565–576. doi: 10.1111/nph.15357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chinnusamy V., Ohta M., Kanrar S., Lee B.H., Hong X., Agarwal M., Zhu J.K. ICE1: a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis. Genes Dev. 2003;17:1043–1054. doi: 10.1101/gad.1077503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Eddy S.R. Profile hidden Markov models. Bioinformatics. 1998;14:755–763. doi: 10.1093/bioinformatics/14.9.755. [DOI] [PubMed] [Google Scholar]
  10. Eremina M., Unterholzner S.J., Rathnayake A.I., Castellanos M., Khan M., Kugler K.G., May S.T., Mayer K.F.X., Rozhon W., Poppenberger B. Brassinosteroids participate in the control of basal and acquired freezing tolerance of plants. Proc. Natl. Acad. Sci. USA. 2016;113:E5982–E5991. doi: 10.1073/pnas.1611477113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ezer D., Shepherd S.J.K., Brestovitsky A., Dickinson P., Cortijo S., Charoensawan V., Box M.S., Biswas S., Jaeger K.E., Wigge P.A. The G-Box transcriptional regulatory code in Arabidopsis. Plant Physiol. 2017;175:628–640. doi: 10.1104/pp.17.01086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fawcett J.A., Maere S., Van de Peer Y. Plants with double genomes might have had a better chance to survive the Cretaceous-Tertiary extinction event. Proc. Natl. Acad. Sci. USA. 2009;106:5737–5742. doi: 10.1073/pnas.0900906106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Finn R.D., Coggill P., Eberhardt R.Y., Eddy S.R., Mistry J., Mitchell A.L., Potter S.C., Punta M., Qureshi M., Sangrador-Vegas A., et al. The Pfam protein families database: towards a more sustainable future. Nucleic Acids Res. 2016;44:D279–D285. doi: 10.1093/nar/gkv1344. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Franco-Zorrilla J.M., López-Vidriero I., Carrasco J.L., Godoy M., Vera P., Solano R. DNA-binding specificities of plant transcription factors and their potential to define target genes. Proc. Natl. Acad. Sci. USA. 2014;111:2367–2372. doi: 10.1073/pnas.1316278111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Freeling M. Bias in plant gene content following different sorts of duplication: tandem, whole-genome, segmental, or by transposition. Annu. Rev. Plant Biol. 2009;60:433–453. doi: 10.1146/annurev.arplant.043008.092122. [DOI] [PubMed] [Google Scholar]
  16. Goodstein D.M., Shu S., Howson R., Neupane R., Hayes R.D., Fazo J., Mitros T., Dirks W., Hellsten U., Putnam N., et al. Phytozome: a comparative platform for green plant genomics. Nucleic Acids Res. 2012;40:D1178–D1186. doi: 10.1093/nar/gkr944. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Grant C.E., Bailey T.L., Noble W.S. FIMO: scanning for occurrences of a given motif. Bioinformatics. 2011;27:1017–1018. doi: 10.1093/bioinformatics/btr064. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hanada K., Zou C., Lehti-Shiu M.D., Shinozaki K., Shiu S.H. Importance of lineage-specific expansion of plant tandem duplicates in the adaptive response to environmental stimuli. Plant Physiol. 2008;148:993–1003. doi: 10.1104/pp.108.122457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jia Y., Ding Y., Shi Y., Zhang X., Gong Z., Yang S. The cbfs triple mutants reveal the essential functions of CBFs in cold acclimation and allow the definition of CBF regulons in Arabidopsis. New Phytol. 2016;212:345–353. doi: 10.1111/nph.14088. [DOI] [PubMed] [Google Scholar]
  20. Jiao Y., Leebens-Mack J., Ayyampalayam S., Bowers J.E., McKain M.R., McNeal J., Rolf M., Ruzicka D.R., Wafula E., Wickett N.J., et al. A genome triplication associated with early diversification of the core eudicots. Genome Biol. 2012;13:R3. doi: 10.1186/gb-2012-13-1-r3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jiao Y., Wickett N.J., Ayyampalayam S., Chanderbali A.S., Landherr L., Ralph P.E., Tomsho L.P., Hu Y., Liang H., Soltis P.S., et al. Ancestral polyploidy in seed plants and angiosperms. Nature. 2011;473:97–100. doi: 10.1038/nature09916. [DOI] [PubMed] [Google Scholar]
  22. Kagale S., Robinson S.J., Nixon J., Xiao R., Huebert T., Condie J., Kessler D., Clarke W.E., Edger P.P., Links M.G., et al. Polyploid evolution of the brassicaceae during the cenozoic era. Plant Cell. 2014;26:2777–2791. doi: 10.1105/tpc.114.126391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lv K., Wu W., Wei H., Liu G. A systems biology approach identifies a regulator, BplERF1, of cold tolerance in Betula platyphylla. Forestry Research. 2021;1:1–10. [Google Scholar]
  24. Kim D., Langmead B., Salzberg S.L. HISAT: a fast spliced aligner with low memory requirements. Nat. Methods. 2015;12:357–360. doi: 10.1038/nmeth.3317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kim Y.S., Lee M., Lee J.H., Lee H.J., Park C.M. The unified ICE-CBF pathway provides a transcriptional feedback control of freezing tolerance during cold acclimation in Arabidopsis. Plant Mol. Biol. 2015;89:187–201. doi: 10.1007/s11103-015-0365-3. [DOI] [PubMed] [Google Scholar]
  26. Kitts P.A., Church D.M., Thibaud-Nissen F., Choi J., Hem V., Sapojnikov V., Smith R.G., Tatusova T., Xiang C., Zherikov A., et al. Assembly: a resource for assembled genomes at NCBI. Nucleic Acids Res. 2016;44:D73–D80. doi: 10.1093/nar/gkv1226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kumar S., Stecher G., Suleski M., Hedges S.B. TimeTree: a resource for timelines, timetrees, and divergence times. Mol. Biol. Evol. 2017;34:1812–1819. doi: 10.1093/molbev/msx116. [DOI] [PubMed] [Google Scholar]
  28. Langmead B., Salzberg S.L. Fast gapped-read alignment with Bowtie 2. Nat. Methods. 2012;9:357–359. doi: 10.1038/nmeth.1923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Larkin M.A., Blackshields G., Brown N.P., Chenna R., McGettigan P.A., McWilliam H., Valentin F., Wallace I.M., Wilm A., Lopez R., et al. Clustal W and clustal X version 2.0. Bioinformatics. 2007;23:2947–2948. doi: 10.1093/bioinformatics/btm404. [DOI] [PubMed] [Google Scholar]
  30. Letunic I., Khedkar S., Bork P. SMART: recent updates, new developments and status in 2020. Nucleic Acids Res. 2021;49:D458–D460. doi: 10.1093/nar/gkaa937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Li H., Ye K., Shi Y., Cheng J., Zhang X., Yang S. BZR1 positively regulates freezing tolerance via CBF-dependent and CBF-independent pathways in Arabidopsis. Mol. Plant. 2017;10:545–559. doi: 10.1016/j.molp.2017.01.004. [DOI] [PubMed] [Google Scholar]
  32. Love M.I., Huber W., Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15:550. doi: 10.1186/s13059-014-0550-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Maere S., De Bodt S., Raes J., Casneuf T., Van Montagu M., Kuiper M., Van de Peer Y. Modeling gene and genome duplications in eukaryotes. Proc. Natl. Acad. Sci. USA. 2005;102:5454–5459. doi: 10.1073/pnas.0501102102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Mayrose I., Zhan S.H., Rothfels C.J., Arrigo N., Barker M.S., Rieseberg L.H., Otto S.P. Methods for studying polyploid diversification and the dead end hypothesis: a reply to Soltis et al. (2014) New Phytol. 2015;206:27–35. doi: 10.1111/nph.13192. [DOI] [PubMed] [Google Scholar]
  35. McElwain J.C., Punyasena S.W. Mass extinction events and the plant fossil record. Trends Ecol. Evol. 2007;22:548–557. doi: 10.1016/j.tree.2007.09.003. [DOI] [PubMed] [Google Scholar]
  36. Menkens A.E., Schindler U., Cashmore A.R. The G-box: a ubiquitous regulatory DNA element in plants bound by the GBF family of bZIP proteins. Trends Biochem. Sci. 1995;20:506–510. doi: 10.1016/s0968-0004(00)89118-5. [DOI] [PubMed] [Google Scholar]
  37. Mizoi J., Shinozaki K., Yamaguchi-Shinozaki K. AP2/ERF family transcription factors in plant abiotic stress responses. Biochim. Biophys. Acta. 2012;1819:86–96. doi: 10.1016/j.bbagrm.2011.08.004. [DOI] [PubMed] [Google Scholar]
  38. Nakano T., Suzuki K., Fujimura T., Shinshi H. Genome-wide analysis of the ERF gene family in Arabidopsis and rice. Plant Physiol. 2006;140:411–432. doi: 10.1104/pp.105.073783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Nguyen L.T., Schmidt H.A., von Haeseler A., Minh B.Q. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 2015;32:268–274. doi: 10.1093/molbev/msu300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Novikova P.Y., Hohmann N., Van de Peer Y. Polyploid Arabidopsis species originated around recent glaciation maxima. Curr. Opin. Plant Biol. 2018;42:8–15. doi: 10.1016/j.pbi.2018.01.005. [DOI] [PubMed] [Google Scholar]
  41. O'Malley R.C., Huang S.S.C., Song L., Lewsey M.G., Bartlett A., Nery J.R., Galli M., Gallavotti A., Ecker J.R. Cistrome and epicistrome features shape the regulatory DNA landscape. Cell. 2016;165:1280–1292. doi: 10.1016/j.cell.2016.04.038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Ossowski S., Schneeberger K., Lucas-Lledó J.I., Warthmann N., Clark R.M., Shaw R.G., Weigel D., Lynch M. The rate and molecular spectrum of spontaneous mutations in Arabidopsis thaliana. Science. 2010;327:92–94. doi: 10.1126/science.1180677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Panchy N., Lehti-Shiu M., Shiu S.H. Evolution of gene duplication in plants. Plant Physiol. 2016;171:2294–2316. doi: 10.1104/pp.16.00523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Pertea M., Pertea G.M., Antonescu C.M., Chang T.C., Mendell J.T., Salzberg S.L. StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat. Biotechnol. 2015;33:290–295. doi: 10.1038/nbt.3122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Qiao X., Li Q., Yin H., Qi K., Li L., Wang R., Zhang S., Paterson A.H. Gene duplication and evolution in recurring polyploidization-diploidization cycles in plants. Genome Biol. 2019;20:38. doi: 10.1186/s13059-019-1650-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Revelle W. 2021. Psych: Procedures for Psychological, Psychometric, and Personality Research. [Google Scholar]
  47. Rhee S.Y., Beavis W., Berardini T.Z., Chen G., Dixon D., Doyle A., Garcia-Hernandez M., Huala E., Lander G., Montoya M., et al. The Arabidopsis Information Resource (TAIR): a model organism database providing a centralized, curated gateway to Arabidopsis biology, research materials and community. Nucleic Acids Res. 2003;31:224–228. doi: 10.1093/nar/gkg076. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Rice A., Šmarda P., Novosolov M., Drori M., Glick L., Sabath N., Meiri S., Belmaker J., Mayrose I. The global biogeography of polyploid plants. Nat. Ecol. Evol. 2019;3:265–273. doi: 10.1038/s41559-018-0787-9. [DOI] [PubMed] [Google Scholar]
  49. Rizzon C., Ponger L., Gaut B.S. Striking similarities in the genomic distribution of tandemly arrayed genes in Arabidopsis and rice. PLoS Comput. Biol. 2006;2:e115. doi: 10.1371/journal.pcbi.0020115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Ruprecht C., Lohaus R., Vanneste K., Mutwil M., Nikoloski Z., Van de Peer Y., Persson S. Revisiting ancestral polyploidy in plants. Sci. Adv. 2017;3:e1603195. doi: 10.1126/sciadv.1603195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Salojärvi J., Smolander O.P., Nieminen K., Rajaraman S., Safronov O., Safdari P., Lamminmäki A., Immanen J., Lan T., Tanskanen J., et al. Genome sequencing and population genomic analyses provide insights into the adaptive landscape of silver birch. Nat. Genet. 2017;49:904–912. doi: 10.1038/ng.3862. [DOI] [PubMed] [Google Scholar]
  52. Schulte P., Alegret L., Arenillas I., Arz J.A., Barton P.J., Bown P.R., Bralower T.J., Christeson G.L., Claeys P., Cockell C.S., et al. The Chicxulub asteroid impact and mass extinction at the Cretaceous-Paleogene boundary. Science. 2010;327:1214–1218. doi: 10.1126/science.1177265. [DOI] [PubMed] [Google Scholar]
  53. Scotese C.R., Song H., Mills B.J., van der Meer D.G. Phanerozoic paleotemperatures: the earth's changing climate during the last 540 million years. Earth Sci. Rev. 2021;215:103503. [Google Scholar]
  54. Sessa E.B. Polyploidy as a mechanism for surviving global change. New Phytol. 2019;221:5–6. doi: 10.1111/nph.15513. [DOI] [PubMed] [Google Scholar]
  55. Shannon P., Markiel A., Ozier O., Baliga N.S., Wang J.T., Ramage D., Amin N., Schwikowski B., Ideker T. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003;13:2498–2504. doi: 10.1101/gr.1239303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Shi Y., Ding Y., Yang S. Molecular regulation of CBF signaling in cold acclimation. Trends Plant Sci. 2018;23:623–637. doi: 10.1016/j.tplants.2018.04.002. [DOI] [PubMed] [Google Scholar]
  57. Soltis D.E., Visger C.J., Soltis P.S. The polyploidy revolution then.And now: stebbins revisited. Am. J. Bot. 2014;101:1057–1078. doi: 10.3732/ajb.1400178. [DOI] [PubMed] [Google Scholar]
  58. Soltis P.S., Soltis D.E. Ancient WGD events as drivers of key innovations in angiosperms. Curr. Opin. Plant Biol. 2016;30:159–165. doi: 10.1016/j.pbi.2016.03.015. [DOI] [PubMed] [Google Scholar]
  59. Song X.M., Wang J.P., Sun P.C., Ma X., Yang Q.H., Hu J.J., Sun S.R., Li Y.X., Yu J.G., Feng S.Y., et al. Preferential gene retention increases the robustness of cold regulation in Brassicaceae and other plants after polyploidization. Hortic. Res. 2020;7:20. doi: 10.1038/s41438-020-0253-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Song Y., Zhang X., Li M., Yang H., Fu D., Lv J., Ding Y., Gong Z., Shi Y., Yang S. The direct targets of CBFs: in cold stress response and beyond. J. Integr. Plant Biol. 2021;63:1874–1887. doi: 10.1111/jipb.13161. [DOI] [PubMed] [Google Scholar]
  61. Tang K., Zhao L., Ren Y., Yang S., Zhu J.K., Zhao C. The transcription factor ICE1 functions in cold stress response by binding to the promoters of CBF and COR genes. J. Integr. Plant Biol. 2020;62:258–263. doi: 10.1111/jipb.12918. [DOI] [PubMed] [Google Scholar]
  62. Thomson R., Pritchard J.K., Shen P., Oefner P.J., Feldman M.W. Recent common ancestry of human Y chromosomes: evidence from DNA sequence data. Proc. Natl. Acad. Sci. USA. 2000;97:7360–7365. doi: 10.1073/pnas.97.13.7360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Tuskan G.A., Difazio S., Jansson S., Bohlmann J., Grigoriev I., Hellsten U., Putnam N., Ralph S., Rombauts S., Salamov A., et al. The genome of black cottonwood, Populus trichocarpa (Torr. & Gray) Science. 2006;313:1596–1604. doi: 10.1126/science.1128691. [DOI] [PubMed] [Google Scholar]
  64. Van de Peer Y., Ashman T.L., Soltis P.S., Soltis D.E. Polyploidy: an evolutionary and ecological force in stressful times. Plant Cell. 2021;33:11–26. doi: 10.1093/plcell/koaa015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Van de Peer Y., Maere S., Meyer A. The evolutionary significance of ancient genome duplications. Nat. Rev. Genet. 2009;10:725–732. doi: 10.1038/nrg2600. [DOI] [PubMed] [Google Scholar]
  66. Van de Peer Y., Mizrachi E., Marchal K. The evolutionary significance of polyploidy. Nat. Rev. Genet. 2017;18:411–424. doi: 10.1038/nrg.2017.26. [DOI] [PubMed] [Google Scholar]
  67. Vanneste K., Baele G., Maere S., Van de Peer Y. Analysis of 41 plant genomes supports a wave of successful genome duplications in association with the Cretaceous-Paleogene boundary. Genome Res. 2014;24:1334–1347. doi: 10.1101/gr.168997.113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Vekemans D., Proost S., Vanneste K., Coenen H., Viaene T., Ruelens P., Maere S., Van de Peer Y., Geuten K. Gamma paleohexaploidy in the stem lineage of core eudicots: significance for MADS-box gene and species diversification. Mol. Biol. Evol. 2012;29:3793–3806. doi: 10.1093/molbev/mss183. [DOI] [PubMed] [Google Scholar]
  69. Wang D., Zhang Y., Zhang Z., Zhu J., Yu J. KaKs_Calculator 2.0: a toolkit incorporating gamma-series methods and sliding window strategies. Dev. Reprod. Biol. 2010;8:77–80. doi: 10.1016/S1672-0229(10)60008-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Wang S., Shen Y., Guo L., Tan L., Ye X., Yang Y., Zhao X., Nie Y., Deng D., Liu S., et al. Innovation and emerging roles of populus trichocarpa TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR transcription factors in abiotic stresses by Whole-Genome duplication. Front. Plant Sci. 2022;13:850064. doi: 10.3389/fpls.2022.850064. [DOI] [PMC free article] [PubMed] [Google Scholar]
  71. Wang Y., Tang H., Debarry J.D., Tan X., Li J., Wang X., Lee T.H., Jin H., Marler B., Guo H., et al. MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res. 2012;40:e49. doi: 10.1093/nar/gkr1293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  72. Wu S., Han B., Jiao Y. Genetic contribution of paleopolyploidy to adaptive evolution in angiosperms. Mol. Plant. 2020;13:59–71. doi: 10.1016/j.molp.2019.10.012. [DOI] [PubMed] [Google Scholar]
  73. Yu G., Wang L.G., He Q.Y. ChIPseeker: an R/Bioconductor package for ChIP peak annotation, comparison and visualization. Bioinformatics. 2015;31:2382–2383. doi: 10.1093/bioinformatics/btv145. [DOI] [PubMed] [Google Scholar]
  74. Zhang Z., Xiao J., Wu J., Zhang H., Liu G., Wang X., Dai L. ParaAT: a parallel tool for constructing multiple protein-coding DNA alignments. Biochem. Biophys. Res. Commun. 2012;419:779–781. doi: 10.1016/j.bbrc.2012.02.101. [DOI] [PubMed] [Google Scholar]
  75. Zhao C., Zhang Z., Xie S., Si T., Li Y., Zhu J.K. Mutational evidence for the critical role of CBF transcription factors in cold acclimation in Arabidopsis. Plant Physiol. 2016;171:2744–2759. doi: 10.1104/pp.16.00533. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Supplemental Figures 1–5
mmc1.pdf (971.4KB, pdf)
Supplemental Tables 1–7
mmc2.xlsx (2.2MB, xlsx)
Supplemental Tables 8 and 9
mmc3.xlsx (126.2KB, xlsx)
Supplemental Table 10. Duplicates in the cold-specific CBF-dependent regulatory network that arose through TD and/or WGD events during a similar period of global cooling
mmc4.xlsx (12.5KB, xlsx)
Supplemental Table 11. RNA-seq expression values of AP2/ERF genes in the five cold-treated species at four time points (0, 2, 24, and 168 h) of cold stress
mmc5.xlsx (156.3KB, xlsx)
Supplemental Tables S12 and S13
mmc6.xlsx (61.3KB, xlsx)
Document S2. Article plus supplemental information
mmc7.pdf (16.8MB, pdf)

Articles from Plant Communications are provided here courtesy of Elsevier

RESOURCES