As one of the major environmental factors, low temperature can severely inhibit plant growth, development and productivity, and is also considered to be a principal determinant of biodiversity geographic distribution patterns (Humphreys and Linder, 2013). Low temperature can result in a variety of unfavorable changes in plant physiological processes by directly inhibiting metabolic reactions and indirectly causing osmotic and oxidative stresses (Diao et al., 2020). In response to this adverse environmental factor, plants successfully evolved a set of sophisticated mechanisms that allow them to withstand freezing (< 0°C) or chilling stress (0–15°C) (Ding et al., 2020). Therefore, unraveling the low temperature-adapted molecular mechanisms of plants may provide interesting targets for developing and selecting low temperature-tolerant genotypes using breeding or genomic approaches, which seems particularly important in high-latitude areas and high-altitude areas. Over the past two decades, much progress has been made in identifying the crucial components (e.g. messenger molecules, protein kinases, phosphatases and transcription factors) involved in low-temperature tolerance and dissecting their regulatory mechanisms (Ding et al., 2019). Accumulating evidence indicates that plants perceive cold signals at different sensory levels, including cell membrane fluidity hypothesis, calcium channels and phytochrome. After sensing the cold signals, the signals are transduced by second messengers (e.g. calcium, reactive oxygen species and nitric oxide). Following the transduction of the cold signals into the nucleus, the cold signaling pathways, including CBF-dependent and CBF-independent pathway, are activated and the expression levels of many genes are altered to mediate the low-temperature tolerance in plants (Ding et al., 2019; Zhang et al., 2020; Ding and Yang, 2022). The C-repeat/DREB binding factors (CBFs) have been identified as an important transcription factor that regulate the expression of low-temperature-responsive genes, and overexpressing CBF1 in Arabidopsis thaliana increased the expression of COR genes and enhanced freezing tolerance (Jaglo-Ottosen et al., 1998; Shi et al., 2018). Low-temperature stress responses are triggered to increase plant survival, but they generally sacrifice plant growth by repressing cell division and expansion (Zhang et al., 2020; Ding and Yang, 2022). Increasing evidence indicates that this low-temperature stress-specific sacrifice-for-survival mechanism is due to limit in energy/carbon supply which mainly results from the active suppression of growth by stress signaling pathways (Zhang et al., 2020). For example, jasmonate signaling, auxin signaling and other stress signaling pathways were identified in Antarctic moss Pohlia nutans and founded that these pathways might contribute to P. nutans acclimating to cold stress (Liu et al., 2022c).

Perennial ryegrass (Lolium perenne L.) is a wild perennial grass belonging to the family Pooideae, subfamily Pooideae, tribe Lolieae and is considered to be an important and widespread cool-season grass species (Förster et al., 2018). It is widely used as a forage species for ruminant animal production in temperate regions and as an alternative and renewable bioenergy source and is also widely cultivated for the establishment of lawns in urban areas (Dąbrowski et al., 2023). Pooideae, as one of the most species-rich grass subfamilies, occupy the coldest climate space, suggesting that they have successfully adapted to and diversified in cool climate ecosystems (Vigeland et al., 2013). Increasing evidence has inferred adaptation to cooler environments at the base of the Pooideae phylogeny (Edwards and Smith, 2010), and five gene families, including C-repeat-binding factors (CBF), dehydrins (DHN), chloroplast-targeted cold-regulated proteins (ctCOR), ice recrystallization inhibition proteins (IRIP) and fructosyl transferases (FST), may have important functions in response to cold stress and acclimation in core Pooideae (Schubert et al., 2019). As a member of the core Pooideae species, the understanding of its low-temperature tolerance of perennial ryegrass is still mainly focused on the physiological and molecular mechanisms, their underlying genetic basis of adaptation to low temperature at the whole genome level needs further exploration.

Gene duplication is an important evolutionary mechanism and is considered a major driving force for expanding the functionality of a multigene family and providing new genes for evolutionary novelty and ecological adaptation (Zhou et al., 2019). Whole-genome duplication (WGD), tandem duplication, duplication mediated by transposable elements, segmental duplication and retroduplication are proposed as the main mechanisms for gene duplication (Panchy et al., 2016). Among them, tandem duplication resulting from unequal crossing over is a prevalent phenomenon that occurs multiple times in all angiosperms and plays significant roles in conferring plant adaptation to changing environments (Yu et al., 2015; Das Laha et al., 2020). For example, tandem duplication events contributed to eudicot adaptation during paleoenvironmental changes (Guo et al., 2022), were involved in tolerance to salt stress in poplar (Ma et al., 2013), and 27% of tandem element-mediated duplicates were responsive to abiotic stress in Arabidopsis (Wu et al., 2012). Research about the influence of tandem duplication on duplicate retention indicated that those genes involved in stress responses generally have an elevated probability of retention following tandem duplication, and new tandem gene paralogs are continuously generated with the occurrence of duplication events, likely providing a pool of high dynamic targets for adaptive evolution to rapidly changing environments (Hanada et al., 2008; Zhong et al., 2018). For example, gene collinearity and phylogeny analyses uncovered that the C-repeat binding factors/dehydration-responsive element binding protein 1 (CBF/DREB1) is an innovation resulted from tandem duplication-derived DREB III gene, and subsequent ϵ-whole genome duplication led to Clades I and II of CBF/DREB1 in ancient angiosperms. Among them, Clades I and their parent DREB III genes showed cold-insensitivity, while Clade II genes evolved into cold-sensitive response and underwent independent expansions by convergent evolution (conserved in cold induction) in eudicots and monocots, suggesting that the duplicated CBF/DREB1 genes mediated the rewiring of CBFs/DREB1s-regulatory network for cold tolerance (Nie et al., 2022). Similarly, a tandem array of CBF/DREB1 genes located in a major freezing tolerance QTL region were identified on Medicago truncatula chromosome 6 (Tayeh et al., 2013), and the expanded gene families (e.g. CBF and LEA) might drive Pooideae grasses from tropical to temperate regions (Zhong et al., 2018). Moreover, expansion of the early light-induced proteins (ELIPs) was previously reported in some plant species, including Boea hydrometrica (Xiao et al., 2015), Selaginella lepidophylla (VanBuren et al., 2018b), Lindernia subracemosa (VanBuren et al., 2018a) and biocrust moss Syntrichia caninervis (Silva et al., 2021). As the subfamily of the light-harvesting chlorophyll a/b-binding protein (Lhc) superfamily, ELIPs encode proteins act as photoprotectants by binding to chlorphylls and carotenoids to protect them against photooxidative damage involved in high light stress (Silva et al., 2021), as well as participate in response to desiccation, cold and drought stresses (Adamska and Kloppstech, 1994; VanBuren et al., 2019). For example, overexpression of a M. truncatula ELIP in Nicotiana benthamiana increased resistance to freezing and chilling, and overexpression of a Craterostigma plantagineum ELIP in M. truncatula increased drought tolerance (Araújo et al., 2013). Recently, comparative genomic and transcriptomic analyses uncovered that ELIPs expanded in resurrection plants through tandem gene duplication and the increased abundance of ELIPs help facilitated the rapid recovery for most resurrection plants under desiccation and rehydration conditions (VanBuren et al., 2019). In addition, tandem duplication events also play important roles in plant growth, development and metabolic processes (Hofberger et al., 2013; Xu et al., 2020b). Unfortunately, tandem duplication genes (TDGs) and their possible contributions to the genetic basis of low-temperature adaptation in L. perenne are still ambiguous.

To better illustrate the molecular evolutionary mechanisms of TDGs underlying low-temperature stress in L. perenne, we performed a comprehensive study to identify the TDG signatures in the L. perenne genome and analyze their evolutionary contributions. Subsequently, GO and KEGG enrichment analyses were performed to investigate the functions of the TDGs specific for L. perenne and Pooideae lineage species. Moreover, the expression patterns of the TDGs in different tissues and their response to low-temperature stress were analyzed. Finally, the potential and important gene family involved in adaptation to low-temperature stress in L. perenne was also investigated. The information generated in this study facilitates the understanding of low-temperature adaptation and provides valuable genetic resources for further studies on low-temperature-related traits in L. perenne.

Numerous studies have confirmed that Pooideae have successfully adapted to and diversified in cool climate ecosystems (Edwards and Smith, 2010; Vigeland et al., 2013). However, as a member of the core Pooideae species, the potential genetic mechanism underlying low-temperature adaptation in L. perenne is not well understood, and there is a lack of evidence at the genomic level. In this study, comprehensive comparative genomic and transcriptomic analyses were performed to illustrate the genomic basis of low-temperature adaptation in L. perenne. A total of 3,770 TDGs were identified in the L. perenne genome (Table S1), and chromosomes 4 and 5 contained the largest (728 TDGs, 19.31% of total TDGs) and lowest (396 TDGs, 10.50% of total TDGs) numbers of TDGs, respectively (Figure 1A; Table S2). Large-scale TDG cluster (more than five genes in the same cluster) analysis showed that 19 large-scale clusters (containing 99 TDGs) were identified, and 9 large-scale clusters were found to be associated with abiotic stress (Table S4). Among them, three clusters contain F-box genes, which encode proteins that play crucial roles in regulating various biological processes and abiotic stress responses by integrating almost all phytohormone signaling pathways (Yan et al., 2011; Jain et al., 2023). One cluster contains the AP2/ERF transcription factor, which has largely been implicated in abiotic stress responses by activating the expression of abiotic stress-responsive genes (Mizoi et al., 2012). In our study, 16 F-box and 5 AP2/ERF genes were identified resulting from tandem duplication events, suggesting that large-scale tandem clusters might participate in abiotic stress responses in L. perenne (Table S4). In addition, although the large-scale TDG clusters included uncharacterized proteins, some genes, including V3.Lp_chr3_0G5746.1, V3.Lp_chr3_0G5750.1 and V3.Lp_chr3_0G5754.1, were induced by low temperature (Table S10), suggesting that these genes also contribute to low temperature resistance in L. perenne and could serve as potential novel genes related to low temperature resistance.

GO and KEGG enrichment analyses may provide valuable information for understanding the high-level functions and utilities of biological processes (Kanehisa et al., 2017). GO enrichment analysis showed that 3,770 L. perenne TDGs were enriched in 148 species-specific GO terms compared with H. vulgare, B. distachyon, A. splendens and O. sativa (Table S6). These unique GO terms included plant-type cell wall organization or biogenesis, inorganic anion transport and organic cation transport (Figure 2C; Table S6), suggesting that these GO terms participate in the modification of cell wall composition and promote intracellular ion homeostasis to avoid osmotic stress caused by abiotic stress (Ambroise et al., 2020; Xu et al., 2020a). In comparison with rice, 26 Pooideae-specific GO terms, including photoprotection and cellular response to light intensity, were identified (Figure 2D; Table S7), implying that Pooideae plants (such as L. perenne, H. vulgare, B. distachyon and A. splendens) can activate a number of highly dynamic photoprotective strategies depending on the light intensity under low temperature stress (Velitchkova et al., 2020). Photosynthesis-antenna proteins that play indispensable roles in the capture of solar energy as well as photoprotection under various stress conditions (Zhao et al., 2020), such as avoiding photooxidative damage in overwintering plants caused by low temperature (Oquist and Huner, 2003). Under chilling stress, 7.38% photosynthesis–antenna proteins were significantly induced in rice leaves (Li et al., 2022), and overexpression tomato LHC antenna protein gene (LeLhcb2) enhanced transgenic tobacco tolerance to chilling stress by alleviating photo-oxidation of PSII (Deng et al., 2014). Similarly, overexpression of Rhododendron ELIP in Arabidopsis conferred plant tolerance to freezing stress through rescuing photosystem (Liu et al., 2022a). The cytochrome P450 proteins (CYPs) participate in various metabolic pathways and play crucial roles in multiple processes, particularly roles in stress responses (Zeng et al., 2019). In osmanthus fragrans, 67 tandem duplicated CYPs were identified, and some of them were significantly induced by cold stress (Liu et al., 2022b). Integrating genomic and transcriptomic analyses revealed expanded cytochrome P450 contribute to stress adaptation for Pistachio (Zeng et al., 2019). In addition, tandem duplicated auxin response factor genes (ARFs) have been reported in Arabidopsis (Okushima et al., 2005) and peach (Shen et al., 2015), and the duplicated Aux/IAA14 regulates microRNA-mediated cold stress response in Arabidopsis (Okushima et al., 2005; Aslam et al., 2020). In our study, photosynthesis-antenna proteins pathway was identified to be a unique KEGG pathway for L. perenne, and TDGs were also enriched in drug metabolism-cytochrome P450 and plant hormone signal transduction pathway (Figure 3C; Table S8), suggesting that TDGs might play important roles in the response of L. perenne to environmental stimuli, particularly roles in low-temperature responses.

The release transcriptional profiles provide a great opportunity to understand the expression patterns in different tissues and in response to stress responses (Farrell et al., 2014; Abeynayake et al., 2015). Duplication events, which occur frequently in most plants, contribute to species diversification and functional innovation and play crucial roles in plant adaptation to stressful habitats (Murat et al., 2010; Wang et al., 2021). In our study, the differential expression analysis revealed that 9.91% (6,582 out of 66,405) of genes were identified as DEGs in L. perenne, while a higher proportion (17.48%, 659 out of 3,770) of TDGs were significantly affected by low temperature (Figure 5C), suggesting that TDGs might play more important roles in contributing to low temperature tolerance for L. perenne. Among these 659 differentially expressed TDGs, 39.8% TDGs (262 out of 659) were shared at all time points under low-temperature stress (Figure 5B), implying that a considerable proportion of TDGs display conservative functions and retain stress responsiveness. A total of 12.4% (82 out 659), 11.4% (75 out of 659) and 8.5% (56 out of 659) differentially expressed TDGs were specifically expressed in 9, 13 and 17 d low temperature-stressed leaves, respectively (Figure 5B), suggesting that these TDGs in L. perenne underwent functional divergence in the process of evolution. Determining the number of synonymous substitutions per synonymous site (Ks) between paralogs allows us to trace the history of duplication events and detect the main duplication events that occurred in plants (Vanneste et al., 2015; Ren et al., 2018). Genomic synteny analysis for 2,042 gene pairs (3,770 TDGs) indicated that L. perenne might has undergone a duplication event approximately 7.69 Mya (Ks = 0.10) (Figure 1B), while the distribution of Ks for 262 shared differentially expressed TDGs at all time points under low-temperature stress revealed that these stress-responsive TDGs might result from duplication events that occurred approximately 20-31 Mya (Ks = 0.36) (Figure 5D). These results suggested that recent duplication events led to the expansion of TDGs, and stress-responsive TDGs mainly originated from earlier duplication events and played a more important role in facilitating the adaptation of L. perenne to low temperature.

Light-harvesting chlorophyll a/b-binding (LHC) proteins play indispensable roles in capturing solar energy during photosynthesis, photoprotection of photosystem II (PSII) and alleviation of oxidative stress caused by stress conditions (de Bianchi et al., 2008; Luo et al., 2022). To study their contributions to low temperature stress, we identified a total of 45 LpLhc superfamily proteins in the L. perenne genome (Table S12). Comparative analysis with other species found that the ELIP subfamily from L. perenne was significantly expanded (Figure 6A; Table S12). Increasing evidence has confirmed that ELIPs accumulate in photosynthetic tissue under various abiotic stresses, including cold, drought and heat (Adamska and Kloppstech, 1994; VanBuren et al., 2019), and play an important role in protecting against photooxidative damage by chlorophyll binding and stabilization of the photosynthetic complex (Hutin et al., 2003). For example, ELIP3 showed significant accumulation in Chlamydomonas reinhardtii under cold stress and helped survival of the cell under photooxidative stress, and the phenotype results of mutant and overexpression plants revealed that ELIP3 plays an important role in protecting the photosystem under photooxidative stress at low temperatures by regulating the redox state of the cell (Lee et al., 2020). Overexpression of a M. truncatula ELIP in N. benthamiana enhanced the resistance to freezing, chilling and osmotic stress by protecting the chloroplast against photooxidative damage (Araújo et al., 2013). In the present study, 13 out of all 14 LpELIPs were significantly induced in leaves by low temperature (Figures 6B, C), suggesting that LpELIPs actively responded to low temperature. Expansion of ELIPs was previously reported in B. hydrometrica (Xiao et al., 2015), S. lepidophylla (VanBuren et al., 2018b) and L. subracemosa (VanBuren et al., 2018a). A comparative genomics analysis of 75 sequenced land plants showed that massive tandem proliferation of ELIPs supports convergent evolution of desiccation tolerance across land plants, and expression analysis revealed that ELIPs had low or undetectable expression under well-watered conditions but exhibited higher expression levels under dehydration stress and that expression increased throughout the progression of dehydration stress (VanBuren et al., 2019). In our study, the expression of all TDGs was significantly lower than that of all genes, and 14 LpELIPs had low or undetectable expression in all six tissues under normal conditions (Figure 4B; Table S15), while the expression level of LpELIPs rapidly increased and continuously maintained higher expression during all of the sampled stress time points (Figure 6B), implying that ELIPs might be involved in various stress responses and play an important role in regulating the low temperature tolerance of L. perenne. In addition, comparative genomics analysis revealed that most ELIPs (76.6%, 49 out of 64 ELIPs) resulted from tandem duplication events, and Pooideae has more ELIPs than Panicoideae and Oryzoideae, particularly in core-Pooideae species (Figure 6D; Table S14), suggesting that tandem duplication might increase the absolute transcript abundance of ELIPs, improve photoprotective capacity, and contribute to low temperature adaptation in L. perenne. Our results provide insights into the roles of tandem duplication in the evolution and low-temperature adaptation of L. perenne and provide candidate gene resources for molecular breeding in L. perenne, although the specific functionality of ELIPs needs further verification.

In this study, we examined the TDG signatures and analyzed their contributions to adaptive evolution in L. perenne, including L. perenne, which might has undergone a duplication event approximately 7.69 Mya, and TDGs might contribute to the environmental adaptability of L. perenne. By transcriptomic analysis, we also found that these TDGs had lower expression than all genes in all six different tissues, while a higher proportion of TDGs were significantly affected by low-temperature stress, and those stress-responsive TDGs mainly resulted from the duplication event that occurred approximately 20-31 Mya. In addition, ELIPs could rapidly respond and continuously maintain higher expression levels during all of the sampled stress time points, suggesting that the expanded ELIPs, which were mainly caused by tandem duplication events, participate in low temperature responses and help facilitate the adaptation to low temperature in L. perenne. Our results provide an important and valuable basis for understanding L. perenne adaptation to low-temperature stress and facilitate the genetic improvement of molecular breeding in L. perenne.

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

WW and YL conceived the experiments and wrote the manuscript, WW, YL, YH and MW performed the experiments and analyzed the data, XL and JW assisted in data analysis, SF and YY revised the manuscript. All authors contributed to the article and approved the submitted version.