**1. Introduction**

In eukaryotic, nucleosome is largely comprised of 146–147 base pairs of DNA and a histone octamer, including four types of histones (namely H2A, H2B, H3 and H4) [1]. The N-terminal tails of these histones can be subjected to post-translational and covalent modifications, including methylation, acetylation, phosphorylation, glycosylation, ADPribosylation, sumoylation and ubiquitination, designated as histone codes that regulate gene expression epigenetically through various mechanisms [2]. These histone codes have been demonstrated to not only directly affect and change the structure of chromatin but also extensively involve the regulation of gene expression [3]. It has been revealed that

**Citation:** Ling, Q.; Liao, J.; Liu, X.; Zhou, Y.; Qian, Y. Genome-Wide Identification of Maize Protein Arginine Methyltransferase Genes and Functional Analysis of *ZmPRMT1* Reveal Essential Roles in *Arabidopsis* Flowering Regulation and Abiotic Stress Tolerance. *Int. J. Mol. Sci.* **2022**, *23*, 12793. https://doi.org/ 10.3390/ijms232112793

Academic Editors: Zhiyong Li and Jian Zhang

Received: 27 September 2022 Accepted: 20 October 2022 Published: 24 October 2022

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the levels of histone methylation can be altered in abiotic and biotic stress responses of plants, and the methylation of some specific residues at the N-terminal tails of histone is closely related to the upregulated or downregulated expression of stress response genes [4]. Therefore, histone methylation has become one of the hot topics in epigenetic regulation research in recent years.

Various arginine (R) and lysine (K) residues at the N-terminal tails of histone can be methylated via protein arginine and lysine methyltransferases, respectively. Protein arginine methyltransferases (PRMTs) can methylate histone H3 at R2 (H3R2), R8 (H3R8), R17 (H3R17), R26 (H3R26) and H4 at R3 (H4R3) through transferring the methyl group from S-adenosylmeth ionine (Ado-Met) to the nitrogen atom of arginine side chain [5]. Generally, there are three main forms of methylated arginine: monomethylarginines (MMA), asymmetric dimethylarginines (ADMA) and symmetric dimethylarginines (SDMA). Based on various methylated arginine forms, PRMTs can be categorized into four major types, namely types I, II, III or IV enzymes [6,7]. The type I and II PRMTs can regulate gene expression through the methylation of histone tails. Among them, the type I PRMTs include PRMT1, PRMT2, PRMT3, PRMT4, PRMT6 and PRMT10 and usually methylate H3R2 and H4R3 residues to generate ADMA, leading to transcriptional activation and ribosomal biosynthesis [8]. In contrast, the type II PRMTs, including PRMT5 and PRMT9, are required for the formation of SDMA at H3R8 and H4R3 residues, resulting in transcriptional repression [9,10]. However, the type III PRMTs (mainly PRMT7) only catalyze the generation of MMA [11]. In addition, the type IV PRMTs can methylate secondary amine on arginine residues, which has only been revealed in yeast [11–13]. Since PRMTs are involved in regulating diverse biological processes in animals and yeast, their significance of PRMTs in the model plant *Arabidopsis* has been paid great attention to in recent years [14].

In plants, genes encoding PRMTs have been identified and analyzed in several species, including *Arabidopsis thaliana* [15], *Oryza sativa* [14], *Eucalyptus grandis* [16] and *Glycine max* [17]. Studies have revealed that PRMTs share rather conserved features in eukaryotic cells and play crucial roles in chromatin structure, RNA processing, altered gene transcription, transport and translation, DNA repair, cellular differentiation and signal transduction [18,19]. Previous studies have demonstrated that the absence of AtPRMT3 can lead to multiple developmental defects in *Arabidopsis*, including unbalanced polyribosome spectra and abnormal rRNA preprocessing, where rRNA precursor processing is required for ribosomal biogenesis [20]. In *Arabidopsis*, AtPRMT4a and AtPRMT4b, two orthologs of human PRMT4/CARM1 protein, can methylate histone H3R2, H3R17 and H3R26 in vitro and are required for the methylation at H3R17 in vivo. The double mutant of *AtPRMT4a* and *AtPRMT4b* genes exhibited an FLC-dependent late flowering phenotype [21]. In addition, AtPRMT5/Skb1 (Shk1binding protein 1) belongs to a type II PRMT and can result in the generation of SDMA at H4R3 residue in vitro, and the *atprmt5* mutant exhibited pleiotropic phenotypes, such as growth retardation, curly and dark green leaves and late flowering in FLC-dependent manner in *Arabidopsis* [22,23]. Increasing evidence has suggested that the PRMT5-mediated arginine methylation plays a crucial role in alterative splicing of normal pre-mRNA in plants and animals [24,25] and that the late-flowering phenotype shown in *atprmt5-1* and *atprmt5-2* mutants may be resulted from alterative splicing of flowering time regulatory genes associated with RNA processing [24]. Previous studies have demonstrated that PRMT5 acts as a key determinant of circadian period in *Arabidopsis* and Drosophila, which may link circadian cycle with alternative splicing [25]. It has also been revealed that the expression level of *FLOWERING LOCUS C (FLC)* gene is upregulated, which might be resulted from alternative splicing of *FLK (Flowering Locus C)* in *atprmt5* mutant [26]. In addition, AtPRMT5/Skb1 has been revealed to regulate gene transcription and involve alterative splicing of pre-mRNA through symmetrically dimethylating H4R3 residues of histone and small nuclear ribonucleoprotein LSM4 and thereby confer a high salt stress tolerance in *Arabidopsis* [27]. Furthermore, AtPRMT10, a plant-specific type I PRMT, has been revealed to play divergent roles in flowering time control [15]. A mutation in the *AtPRMT10* gene resulted in late flowering by upregulating the transcript level of

*FLC* gene. Moreover, the correlation between the *FLC* expression and flowering time and vernalization has been determined, which indicates that the *FLC* gene acts as an important determinant in natural variation of flowering time [28,29]. Further, the *Arabidopsis* FLC protein has been demonstrated to function as a flowering repressor, which may be involved in regulating related genes in autonomous or vernalization pathways. Previous studies have demonstrated that the *FLC* gene can function in repressing expression of the flowering regulatory genes *SOC1 (Super of overexpression COI)* and *FT (Flowering Locus T)* through genetic and transgenic methods [30].

Furthermore, recent studies have revealed that many epigenetic factors are involved in various abiotic stress responses, and distinct chromatin modifications can be altered when plants are exposed to adverse environmental conditions, resulting in a dynamic chromatin environment to regulate gene expression [31]. At present, the cross-talk between diverse abiotic stress response pathways and epigenetic regulatory pathways has been thoroughly studied in plants [31]. However, the underlying mechanisms of epigenetic regulation of plant responses to heat stress remain to be elucidated, especially in regulating the dynamic histone arginine methylation patterns of stress-responsive genes, which partly depends on the catalytic function of PRMTs in plants. Moreover, proline is an essential multifunctional amino acid, which plays an important role in abiotic stress tolerance of plants. However, little is known about the biological functions of proline metabolism in plant responses to heat stress. Previous studies have revealed that short-term heat shock at 42 ◦C can result in proline accumulation in plant seedlings, and the external application of proline can induce the level of endogenous free proline and activities of antioxidant enzymes, followed by enhancing heat tolerance of plant seedlings [32]. Moreover, proline accumulation is mainly caused by the following three aspects: increasing the synthesis of proline, reducing the oxidation and degradation of proline and reducing the utilization of protein synthesis [33].

So far, much is well-known regarding proline metabolism in plants. The metabolic pathway of proline includes synthetic pathway and catabolic pathway. The synthetic pathway involves two enzymes, P5CS (pyrroline-5-carboxylate synthetase) and P5CR (P5C reductase). The degradation pathway involves other two enzymes, ProDH (proline dehydrogenase) and P5CDH (pyrroline-5-carboxylate dehydrogenase). [34]. Proline accumulation has been demonstrated to play adaptive roles in abiotic stress tolerance of plants [35]. Accumulated free proline could be implicated in adjusting cytosolic osmotic potential in order to save water [36], protect the membrane structure, sustain the structures of soluble proteins and activities of enzymes [37], act as reactive oxygen species (ROS) scavenger [38] and retain storage of carbon and nitrogen [39]. For example, overexpression of bean *P5CS1* gene in tobacco can slow down the decline of osmotic potential of transgenic plants under water stress [36].

Maize (*Zea mays* L.), as one of the most important crop species in the world and is vulnerable to environmental factors with climate change. Thus, how to use the methods of molecular biology to improve stress resistance and yield of maize is one of the hot topics of current biological research. Moreover, it has been demonstrated that plant *PRMT* genes are extensively involved in the regulation of growth and development and responses to various abiotic stresses. However, little is known regarding identification and function analysis of *PRMT* gene family in maize. In the present study, we carried out a comprehensive identification and functional analysis of *PRMT* genes in maize, including their phylogenetic relationships, gene and protein structures, conserved domain and motif architecture, chromosome location, gene duplication events and diverse expression profiles, which will facilitate further studies to unravel the exact biological roles of *ZmPRMT* genes in maize. Furthermore, the roles of *ZmPRMT1* in regulating flowering time and conferring heat stress tolerance were further clarified in transgenic *Arabidopsis*.

In this study, we selected transgenic *Arabidopsis* lines that overexpress maize *Zm-PRMT1* gene to further reveal its functional roles in plants based on the conserved evolutionary relationship among ZmPRMT1 protein and some orthologous PRMT proteins in *Arabidopsis,* rice and sorghum. Although the direct use of *Arabidopsis* as a model plant for

maize still has limitations, the current performance of *Arabidopsis* in plants is better than all other models. For example, when the purpose of the research is to gain a fundamental understanding of the specific growth process of plants, such as the regulation of flowering time, the *Arabidopsis* model system is still of reference significance [40]. Of course, it is also necessary to further verify the function of genes in maize through the overexpression and latest CRISPR/Cas9 gene editing techniques. In the case of flowering time regulation, the factors that advance the flowering time of *Arabidopsis* may inhibit the flowering time of rice (*Oryza sativa*) [41], and the network variation that affects the flowering time is significant even in gramineous plants [42,43]. Taken together, this study may contribute to an in-depth comprehension of the evolution of *ZmPRMT* genes and their crucial roles in maize growth, development and responses to abiotic stresses.
