Molecular Genetic Insights into the Stress Responses and Cultivation Management of Zoysiagrass: Illuminating the Pathways for Turf Improvement

Abstract

Zoysiagrass (Zoysia spp.) and its hybrids are known for their low maintenance requirements and are widely utilized as warm-season turfgrass, which offers considerable ecological, environmental, and economic benefits in various environments. Molecular genetic approaches, including the identification and genetic engineering of valuable gene resources, present a promising opportunity to enhance the quality and performance of zoysiagrass. This review surveys the recent molecular genetic discoveries in zoysiagrass species, with a focus on elucidating plant responses to various abiotic and biotic stresses. Furthermore, this review explores the notable advancements in gene function exploration to reduce the maintenance demands of zoysiagrass cultivation. In addition, we discuss the achievements and potential of contemporary molecular and genetic tools, such as omics approaches and gene editing technologies, in developing zoysiagrass cultivars with desirable traits. Overall, this comprehensive review highlights future strategies that may leverage current molecular insights to accelerate zoysiagrass improvement and further promote sustainable turf management practices.

1. Introduction

Zoysiagrass (Zoysia spp.) is a warm-season perennial grass native to the temperate regions of Northeast Asia, including Korea and Japan, as well as tropical China and Southeast Asia. Zoysiagrass primarily propagates vegetatively through stolons and rhizomes and is well established in a wide range of environments. As one of the earliest grasses utilized for turf, zoysiagrass is valued for its sturdiness, aesthetic appeal, and low maintenance requirements, which makes it a popular choice for lawns, golf courses, and sports fields in diverse climates worldwide. Its dense growth habit, tolerance to foot traffic, and ability to sustain green coverage with minimal water and nutrient input make it an ideal candidate for sustainable landscaping. However, new challenges arising from increased urbanization and climate change, along with the demand for environmentally friendly landscape solutions, have prompted extensive research into the genetic and molecular basis of stress tolerance in turfgrasses, with a particular focus on zoysiagrass.

Zoysiagrass is renowned for its cost-effectiveness and superior stress tolerance when compared to other turfgrass species. This resilience is particularly important as the growth and development of zoysiagrass is influenced by various biotic and abiotic stresses, such as cold, salt, drought, wounding, diseases, pests, and senescence [1,2,3,4,5]. These stresses can adversely affect the health and appearance of the grass, thus highlighting the need to understand its stress response mechanisms to develop more robust cultivars.

Recent research has increasingly focused on determining the molecular genetic mechanisms that enable zoysiagrass to withstand these stresses. Advances in omics technologies, including genomics, transcriptomics, proteomics, metabolomics, and functional genomics, have expanded our understanding of the complex biological processes involved [6,7]. Substantial progress has been made in identifying the key regulatory genes, genetic pathways, and molecular networks that contribute to this stress resistance. Understanding the molecular and genetic mechanisms underlying the stress responses in zoysiagrass provides insights into how zoysiagrass can be genetically engineered or selectively bred to enhance this stress tolerance.

This review aims to summarize the latest findings in the molecular genetics of zoysiagrass and provide a comprehensive overview of the genetic determinants that enhance its resilience to cold, salinity, drought, wounding, pathogens, pests, and other environmental and developmental challenges. By compiling current research on gene identification, functional characterization, and the regulatory mechanisms of stress responses, we endeavor to improve our understanding of the stress resistance in zoysiagrass. This knowledge is essential to ensure its continued survival and ecological sustainability under varied and changing environmental conditions.

In addition, this review explores how recent advancements can facilitate zoysiagrass management by addressing areas such as herbicide selection, mowing-related properties, and propagation methods. Furthermore, it examines the potential of biotechnological interventions, such as transgenesis, targeted gene editing, and mutational breeding, in accelerating the development of superior zoysiagrass cultivars. The integration of these innovative techniques with traditional breeding approaches offers considerable potential to enhance the performance and sustainability of zoysiagrass, ultimately contributing to healthier and more resilient urban landscapes.

2. Molecular and Genetic Approaches in Zoysiagrass Investigation

Various molecular and genetic tools have revolutionized research on zoysiagrass, providing unprecedented insights into its genetic composition and offering new approaches for gene-based improvement [8,9]. The advent of next-generation sequencing (NGS) has facilitated rapid and cost-effective genome sequencing, which has enabled the identification of gene structures and sequences as well as the transcriptional regulation of genes associated with stress tolerance and growth characteristics in a genomic context [10,11,12,13,14,15,16].

2.1. Genomics Approaches

Genomics, encompassing the study of an organism’s entire genetic material, facilitates the identification and characterization of genes, regulatory elements, and their interactions, ultimately providing insights into the genetic basis of complex traits and enabling the development of molecular markers for breeding programs.

Pioneering studies for the NGS-based genome sequencing of zoysiagrass began with the analysis of three Zoysia species (Z. japonica variety (var.) “Nagirizaki”, Z. matrella var. “Wakaba”, and Z. pacifica var. “Zanpa”) using the HiSeq and MiSeq platforms [16]. The study reported draft genome sequences with 334 Gbp for Z. japonica, 563 Gbp for Z. matrella, and 397 Gbp for Z. pacifica, as well as 59,271 protein-coding sequences for Z. japonica. Further ortholog clustering analysis for these three Zoysia species revealed 28,469 core gene families that were shared among them and 13,720 orthogroups of species-specific gene families [1]. This comparative genome sequencing analysis revealed intriguing patterns of gene conservation and species-specific genetic adaptations within the Zoysia genus through evolutionary history analysis. Recent studies using PacBio long-read sequencing have provided more accurate and high-coverage genome information for Z. japonica var. “Yaji”, which features long contigs and the prediction of miRNA/target site pairs [17].

Reference-based whole genome NGS or cost-effective NGS techniques, such as whole exome sequencing or restriction site-associated DNA sequencing (RAD-Seq), have facilitated the discovery of single nucleotide polymorphisms (SNPs) and the construction of high-resolution genetic maps in sets of zoysiagrass accessions or crossed progeny lines [16,18]. RAD-seq analysis using an F1 mapping population of Z. japonica var. “Carrizo” and “El Toro” generated 2408 and 1230 RAD markers for the corresponding parental accessions [19]. Genome sequencing for three Z. japonica var. (“Nagirizaki”, “Kyoto”, and “Miyagi”) and two Z. matrella var. (“Wakaba” and “Chiba Fair Green”) identified more than 7.4 million SNPs and 0.85 million indels among the accessions [1]. A more recent RAD-seq analysis of a 95 F1 segregating population, generated from a cross between Z. matrella var. “Cavalier” and “Diamond”, resulted in the construction of high-resolution linkage maps of Z. matrella with 3563 and 2375 SNP markers for each accession, respectively, and identified six loci that were highly associated with fall armyworm (FAW) resistance [18]. The resulting high-resolution genetic maps can serve as a foundation for future gene mapping, quantitative trait locus (QTL) studies, and assigning genome sequences to chromosomes in Zoysia spp., as well as for comparative and evolutionary genome analysis within Panicoideae and Chloridoideae [15,18,20].

Collectively, these advances in genomics have significantly expanded the scope of molecular genetic studies in zoysiagrass biology, paving the way for the development of improved turfgrass varieties with desirable traits.

2.2. Transcriptomics Approaches

Transcriptomics, primarily through RNA sequencing (RNA-seq) technology, has become indispensable for deciphering the complex molecular mechanisms in plants [21]. By providing a comprehensive view of gene expression profiles, transcriptomics enables researchers to understand how plants respond to various environmental stimuli, developmental cues, and stress conditions. This understanding is crucial in identifying the molecular bases of traits that enhance plant resilience, productivity, and adaptability [22].

To date, more than 15 studies have been published on zoysiagrass biology and its responses to various stresses, including cold stress [3,23], salt stress [6,24], ethephon [25,26], senescence [27,28], and diseases [29,30]. The application of transcriptome analysis in zoysiagrass research is steadily increasing, reflecting its critical role in plant biology. These transcriptomic studies have revealed the intricate and dynamic gene expression patterns across different tissues, developmental stages, and under various environmental and stress conditions in Zoysiagrass. By elucidating the molecular regulation and pathways involved in these biological processes, transcriptomics provides vital insights that can inform breeding strategies and genetic improvements.

Early pioneer studies using transcriptome profiling focused on cold and salt stress responses in Z. japonica using de novo assembly of NGS reads [13,31]. Cold stress-related transcriptomic analyses performed at short (2 h) and prolonged (72 h) periods of cold treatment in leaves identified 756 and 5327 differentially expressed genes (DEGs), respectively, with 15 DEGs confirmed as cold-response markers [31]. The molecular responses to cold stress indicated that cold exposure induces desiccation and oxidative stress, inhibits photosynthesis and substance transport, and affects gibberellin (GA) metabolism, abscisic acid (ABA), and jasmonic acid (JA) stimulus responses. Furthermore, this study reported approximately 46,000 genes as unigenes in Z. japonica.

Another study focused on the salt-mediated root transcriptome and identified candidate unigenes related to early responses when exposed to salt stress [13]. They identified 32,849 unigenes, a relatively reduced gene number from leaf tissue, and 1455 DEGs, which were enriched in responses to chemical or abiotic stimulus and stress-related hormones including ABA, JA, and salicylic acid (SA). Overall, these studies provided the first substantial dataset of sequence information and gene expression profiles when zoysiagrass was exposed to cold and salt stress, enhancing the understanding of zoysiagrass responses to these stresses.

Recently, a high-quality ZjRTD1.0 reference transcript dataset for zoysiagrass has been established, which incorporates data from 10 transcriptome resources [32]. This dataset includes 113,089 transcripts from 57,143 genes, which provides a more precise analysis of the zoysiagrass transcriptome. Further research beyond these studies is detailed in Section 3 and Section 4 of this review. These transcriptional analyses have elucidated the molecular responses of zoysiagrass, revealing the crucial regulatory pathways and key genes involved in stress responses and developments. Moreover, RNA-seq data was particularly advanced through long-read RNA-seq and complemented the genomic sequencing by elucidating the gene structures, identifying alternative splicing events, and refining the gene annotations in zoysiagrass [14,27].

Given their powerful and comprehensive capabilities, transcriptomic approaches will undoubtedly play an increasingly vital role in revealing the pathways for zoysiagrass improvement, ultimately contributing to the development of superior turf varieties with enhanced stress tolerance and quality traits.

2.3. Proteomics Approaches

Proteomics is a robust method for exploring molecular mechanisms at the protein level, although its development and application tend to lag behind NGS-based genomics and transcriptomics [33]. As proteins are the primary effectors of biological functions, understanding their expression, modifications, and interactions is crucial in deciphering the physiological responses of plants to various environmental stresses [34]. By examining the proteome, researchers can gain insights into the dynamic regulatory modules that govern plant growth, development, and stress adaptation.

Proteomic approaches have effectively been applied to the biology of zoysiagrass (Zoysia spp.) [7,35,36]. One notable study analyzed the differential proteomic responses in the stolons of Z. japonica var. Meyer (cold-tolerant) and Z. matrella var. Diamond (cold-sensitive) using classic 2D gel electrophoresis (2-DE) followed by mass spectrometry (MS) [35]. This analysis identified 70 differential protein spots and cold stress-responsive protein networks related to the processes of reactive oxygen species (ROS) balancing, protein homeostasis, and energy supply. These activities were more pronounced in the cold-tolerant Z. japonica.

Another significant study investigated the proteomic responses involved in cold acclimation-dependent freezing tolerance in the meristematic tissue of Z. japonica var. Meyer (freeze-tolerant) and var. Victoria (freeze-susceptible) using the same 2-DE and MS approach [36]. This research identified 62 protein spots with differential accumulation during cold acclimation, with 9 and 22 differentially expressed proteins in each cultivar, respectively. These proteins were associated with protein storage and energy production, which highlights the protein responses that underlie the differences in freezing tolerance among zoysiagrass germplasms.

The most recent study employed data-independent acquisition proteomics with nano-HPLC–MS/MS analysis to determine the molecular mechanisms behind enhanced salt tolerance in transgenic Z. matrella overexpressing gene-encoding protein disulfide isomerase (ZmPDI-OX) [7]. This advanced proteomic approach identified 550 differentially expressed proteins with high sensitivity in the wild-type (WT) and ZmPDI-OX in response to salt stress for 24 h, with gene ontology (GO) processes related to the microtubule cytoskeleton and phagosomes being affected in ZmPDI-OX.

These proteomic approaches have substantially contributed to our understanding of zoysiagrass biology at the protein level, particularly in the context of stress responses and cultivar differences. Although there are limited reports on proteomic approaches in zoysiagrass, their extensive and detailed capabilities make them capable of elucidating pathways for zoysiagrass improvement.

2.4. Functional Validation Approaches

The introduction of advanced high-throughput analysis techniques, including transcriptomics and proteomics, in addition to the completion of genome drafts, has identified numerous candidate regulatory genes for trait improvement in zoysiagrass. However, the functional validation of these genes remains a considerable bottleneck in molecular improvement efforts. Establishing the precise roles of these candidate genes is essential in translating genomic data into practical applications, enabling targeted breeding and biotechnological approaches for enhancing zoysiagrass varieties.

Transient expression techniques in protoplasts have emerged as a rapid method for the functional characterization of target genes, offering versatile molecular assays in model and crop plants [37,38]. Initially, transient expression methods were developed using zoysiagrass protoplasts derived from calli, facilitating planta transformation through regeneration processes [39,40]. Recent studies have established efficient and convenient protocols for the use of protoplasts derived from the leaf tissue of zoysiagrass [14,41]. These have been instrumental in the functional analysis of Zoysia genes such as Zj_B TYPE RESPONSE REGULATOR (ZjRR_B), Zj_NON-YELLOW COLORING 1 (ZjNYC1) and Zj_NYC1-like (ZjNOL), utilizing molecular techniques including subcellular localization, immunoblot-based protein detection, or bimolecular fluorescence complementation. This protoplast system provides a convenient and effective platform to study gene function and regulatory mechanisms in zoysiagrass research, thereby accelerating the functional validation process and facilitating the molecular improvement of this valuable turfgrass.

Developments in transformation techniques have provided new possibilities for genetic improvement and functional genomics studies in zoysiagrass [42]. The generation of valuable zoysiagrass plants using gene resources requires transgenesis for ectopic expression or targeted loss of genes of interest. Several independent trials have created transgenic Z. japonica plants using polyethylene glycol (PEG)-mediated transformation in protoplasts [39] and Agrobacterium-mediated transformation in calli or stolons, followed by regeneration into plants [43,44,45]. In addition, Agrobacterium-mediated transformation using calli has become a popular method for use with Z. japonica and has been applied to other Zoysia spp., such as Z. sinica [46], Z. tenuifolia [47], and Z. matrella [48]. Recently, clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9)-based gene editing has successfully been established to generate loss-of-function mutants, which were used for the functional validation of Zj_ETHYLENE INSENSITIVE 2 (ZjEIN2) in Z. japonica [49] and ZjNYC1 in Z. matrella [50]. These genetic modification technologies, which enable the precise control of gene function in either direction, could offer more targeted trait development for the generation of valuable zoysiagrass varieties.

Overall, the integration of these molecular and genetic tools has significantly advanced our understanding and ability to manipulate zoysiagrass genes. By leveraging these technologies, researchers aim to develop resilient cultivars that can thrive under diverse environmental conditions, thereby ensuring the sustainability and broader application of zoysiagrass for landscaping and turf management.

3. Current State of Research on Abiotic and Biotic Stress in Zoysiagrass

Zoysiagrass is valued for its aesthetic and functional qualities in turf management but faces a range of abiotic and biotic stresses that can significantly impact its health and performance. Understanding and mitigating these stresses are essential for improving the resilience and longevity of zoysiagrass in diverse environments. This section explores the current state of research on the various stresses that affect zoysiagrass, including cold, salt, and drought stresses, as well as its response to wounding, pathogens, pests, and senescence (Figure 1;

Table 1. Valuable genes regulating stress resilence and cultivation management in zoysiagrass.

Factors (*)	Genes	Gene Description	Gene Source	Reference
		Phenotype/Regulation	Approaches	Species Analyzed
Abiotic and Biotic Stress
Cold	ZjICE1	MYC-type bHLH transcription factor	Z. japonica	[51]
		Increases cold tolerance	Overexpression	Arabidopsis
	ZjICE2	MYC-type bHLH transcription factor	Z. japonica	[52]
		Improves cold resistance and ROS scavenging ability	Overexpression	Arabidopsis
	ZjCIGR1	GRAS family gene	Z. japonica	[53]
		Increases cold stress resistance and expression of COR genes	Overexpression	Z. japonica
Salt	ZmVP1	Vacuolar H+-pyrophosphatase (VP) family gene	Z. matrella	[54]
		Improves salt tolerance	Overexpression	Arabidopsis
	ZmPDI	Protein disulfide isomerase gene	Z. matrella	[7]
		Improves salt tolerance	Overexpression	Z. matrella
	ZjGRP	Glycine-rich RNA-binding protein gene	Z. japonica	[55]
		Reduces salt tolerance	Overexpression	Arabidopsis
	ZjZFN1	C2H2-type zinc finger gene	Z. japonica	[56]
		Increases salt tolerance	Overexpression	Arabidopsis
	ZjABR1	Ethylene-responsive factor	Z. japonica	[57]
		Increases salt resistance, lower seed setting rate and dwarfism	Overexpression	Oryza sativa
Drought	ZjICE2	MYC-type bHLH transcription factor	Z. japonica	[52]
		Increases cold, drought, and salt tolerance	Overexpression	Arabidopsis
	ZjZFN1	C2H2-type zinc finger gene	Z. japonica	[58]
		Reduces plant adaptability to drought stress	Overexpression	Arabidopsis
Pathogens	Zjchi2	Class II chitinase gene	Z. japonica	[59]
		Enhances antifungal resistance	Overexpression	Z. japonica
Sene- scence	ZjSGR	Magnesium-dechelatase	Z. japonica	[60]
		Accelerates chlorophyll degradation and reduces photosynthesis	Overexpression	Arabidopsis
	ZjPPH	Pheophytinase gene	Z. japonica	[61]
		Accelerates chlorophyll degradation	Overexpression	Arabidopsis
	ZjNYC1	Short-chain dehydrogenase/reductase	Z. japonica	[62]
		Promotes chlorophyll degradation and senescence	Overexpression	Arabidopsis
	ZmNYC1	Short-chain dehydrogenase/reductase	Z. matrella	[50]
		Retains an extended greening phenotype	Knock-out	Z. matrella
	ZjEIN2	Integral membrane protein	Z. japonica	[49]
		Delays leaf senescence	Knock-out	Z. japonica
Cultivation Management
Growth Control	phyA (S599A)	Phytochrome A (Serine 599 to Alanine)	Z. japonica	[63]
		Suppresses shade avoidance response and induces dwarfism	Overexpression	Avena sativa
	ZjHCT4	Hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase 4 gene	Z. japonica	[64]
		Increases stem elongation	Overexpression	Agrostis stolonifera
	ZjPSY	Phytoene synthase gene	Z. japonica	[65]
		Affects plant height and carotenoid synthesis	Overexpression	Arabidopsis
Propagations	ZjWRKY10	WRKY transcription factor 10	Z. japonica	[66]
		Triggers early flowering	Overexpression	Arabidopsis
	ZjLsL	GRAS TF family gene	Z. japonica	[67]
		Induces axillary meristem initiation and tiller formation	Overexpression	Arabidopsis, A. stolonifera

(*) For wound/traffic stress, insect pests, and herbicide selection, no functional candidate genes were identified in zoysiagrass.

).

3.1. Cold Stress

Cold stress poses a substantial threat to plants, adversely affecting their growth, development, and overall survival [3]. At the cellular level, low temperatures can cause membrane rigidification, which can result in cellular leakage and disruption of metabolic processes [68,69]. On a broader scale, prolonged exposure to cold impairs nutrient uptake, diminishes photosynthetic efficiency, and disrupts hormone balances, thus leading to stunted growth, leaf senescence, and reduced crop yields in agricultural settings. To mitigate the impact of cold stress, plants have evolved sophisticated regulatory mechanisms, including cold acclimation, which increases cold resistance through exposure to sub-lethal, low temperatures [36,70,71,72,73]. During cold acclimation, plants accumulate carbohydrates and antioxidant substances and upregulate specific regulatory genes, including the C-repeat binding factor (CBF), which promotes the synthesis of protective proteins and enhances cold resistance [74].

Zoysiagrass originates from subtropical regions and exhibits limited freezing tolerance [75]. This poor cold tolerance can lead to significant damage or mortality during winter frosts, thus substantially diminishing its aesthetic and functional value [3]. Consequently, sensitivity to low temperatures is a key limiting factor for the wide distribution and use of zoysiagrass. Improving the cold resistance of zoysiagrass is essential for sustaining its usage in existing regions and enabling its expansion into areas with varying seasonal climates.

The application of advanced RNA-seq technology has substantially enhanced our understanding of cold stress responses in zoysiagrass. In a transcriptome analysis of various transgenic and WT zoysiagrass strains exposed to cold stress, researchers identified numerous DEGs associated with cold resistance [3,23,31,35,76]. These DEGs are involved in various critical biological processes and signaling pathways. The key pathways include those regulated by proline synthesis, antioxidant enzymes such as superoxide dismutase (SOD) and peroxidase (POD), and stress-related metabolites, including malondialdehyde (MDA) [3,76]. Notably, SOD and POD, as key ROS scavengers, also play crucial roles in other stress-response mechanisms, such as salt [24] and drought [58], further emphasizing that zoysiagrass can improve tolerance to a wide range of abiotic stresses through enhanced ROS scavenging. In addition, significant expression has been observed in pathways related to chlorophyll metabolism and hormone regulation, specifically ABA and JA [31]. The critical role of specific time points and tissues, such as leaves and stolons, in cold stress responses has also been highlighted [31,35], with potential cold tolerance genes associated with photosynthesis, protein biosynthesis, and ROS scavenging pathways being further identified [23,31,35]. These findings provide valuable insights into the molecular mechanisms underlying cold resistance in zoysiagrass.

Fundamental research on cold stress in zoysiagrass employed association analysis to evaluate cold resistance in Z. japonica and Z. matrella varieties [77,78]. Significant variation in cold resistance was observed among the different zoysiagrass varieties, with Z. japonica generally exhibiting greater cold tolerance than Z. matrella. The improved cold tolerance across the populations was positively correlated with the level of dehydrin proteins, which suggests the role of these proteins in enhancing cold tolerance [77]. Further investigations explored the physiological and metabolic responses to low temperatures in zoysiagrass varieties that were native to high and low latitudes [78]. The enhanced cold resistance in the high-latitude varieties may be attributed to their higher carbohydrate content, which serves both as an energy reserve and a protective agent against adverse conditions. In addition, the increased levels of stress hormones, such as ABA, SA, and JA, as well as the reduced levels of growth hormones, such as auxin, GA, and cytokinin, were observed in the high-latitude lines. These stress hormones induced the upregulation of cold-responsive genes, including ZjCBF, Zj_DEHYDRATION-RESPONSIVE ELEMENT BINDING 1 (ZjDREB1), and Zj_LATE EMBRYOGENESIS ABUNDANT (ZjLEA), thereby enhancing the cold tolerance of zoysiagrass.

The advent of molecular marker technology and the construction of molecular linkage maps have enabled QTL mapping research in zoysiagrass [79]. Various methods and populations have been employed to identify the QTLs related to cold resistance in zoysiagrass. One study used an F1 population of 86 individuals, derived from two Z. japonica var., “Muroran” and “Tawarayama Kita”, to analyze the semi-lethal temperatures (LT50), soluble protein and sugar contents, and SOD activity [80]. This study revealed a negative correlation between LT50 and the levels of protein, sugar contents, and SOD activity, and further identified single QTLs related to each biochemical parameter. These findings suggest a genetic basis for cold stress-related traits. Another investigation employed association mapping with a total of 83 simple sequence repeats and sequence-related amplified polymorphism markers in 96 zoysiagrass accessions or varieties. This approach revealed four molecular markers that were significantly associated with cold tolerance, with effect values ranging from 37% to 58% [81]. A more comprehensive study identified 56 QTLs related to winter damage, establishment, and turf quality across six environments, using a pseudo-F2 hybrid population from the freeze-tolerant Z. japonica var. “Meyer” and the freeze-susceptible Z. japonica var. “Victoria” [15]. Further research on the Meyer × Victoria population focused on additional cold stress recovery traits, including the post-freezing surviving green tissue and regrowth traits [82], which revealed 31 and 30 QTLs, which accounted for 6.4–12.2% of the phenotype variations, respectively. Sequencing for QTL regions suggested several abiotic response candidate genes, including transcription factors (TFs) and genes related to cell wall modification or defense signal transduction. These findings have provided valuable genetic and molecular resources for further identification of cold regulatory genes and molecular breeding to enhance cold stress responses in zoysiagrass.

Building on the genetic insights from QTL analysis, recent studies have revealed specific genetic regulators that are involved in the cold responses of zoysiagrass. In the regulation of cold responses in plants, the CBF/DREB family represents a low-temperature signaling pathway. This pathway regulates the expression of approximately 12% of cold-responsive genes in Arabidopsis that function in cold acclimation and freezing tolerance in plants [83,84,85]. The expression of CBF is regulated by the CBF inducer of expression (ICE1) protein [86]. Studies in Z. japonica have identified and characterized stress-responsive ICE homologs, specifically ZjICE1 and ZjICE2 [51,52,87]. Both ZjICE1 and ZjICE2 bind to MYC cis-acting elements in the promoters of DREB1 genes, which suggests a comparable regulatory mechanism. Transgenic Z. japonica plants that overexpressed ZjICE1 and ZjICE2 exhibited enhanced cold tolerance, with increased levels of SOD, POD, and free proline, reduced MDA content, and elevated expression of cold-responsive genes, such as ZjCBF and ZjDREB1 [51,52]. Another gene studied in zoysiagrass is the chitin-inducible gibberellins-responsive1 (ZjCIGR1), a member of the plant-specific GRAS TF protein family from zoysiagrass [53]. Transgenic zoysiagrass overexpressing ZjCIGR1 displayed enhanced cold stress tolerance, exhibiting morphological phenotypes characteristic of stress resistance and increased expression of cold-regulated genes under cold stress conditions. These findings suggest that ICE genes (ZjICE1 and ZjICE2) and ZjCIGR1 are promising candidates for molecular breeding programs to develop cold-tolerant zoysiagrass lines.

Warm-season turfgrass species are particularly sensitive to cold stress, which is the primary environmental limitation that they encounter. Exposure to low temperatures can result in significant turf damage and widespread lawn die-off. Therefore, it is essential to systematically investigate the molecular and physiological response mechanisms of zoysiagrass to cold stress. Such research aims to identify key genes and metabolic pathways related to cold resistance, which would provide a foundation for developing new strains with enhanced cold resistance.

3.2. Salt Stress

Salt stress is becoming a serious problem in turfgrass management across various regions, such as in coastal regions, urban landscapes, sports fields, and parks, because of rising ambient temperatures and water resource limitations [88]. This stress affects seed germination, growth and development, nutrient accumulation, and metabolism in turfgrass [89,90,91]. To maintain ideal turf quality in areas with increasing levels of salinity, it is essential to understand the physiological responses, as well as the molecular and genetic mechanisms of salt tolerance in turfgrasses, including zoysiagrass.

The detrimental effects of salt stress are primarily related to cellular water loss caused by high osmotic pressure and ion toxicity that result from K⁺/Na⁺ homeostasis disruption [92]. Sodium ion toxicity is a significant issue caused by salt stress, particularly with exposure to NaCl [93]. A crucial regulatory mechanism in mitigating these effects is the salt overly sensitive (SOS) signal pathway, which comprises three key genes: SOS1, SOS2, and SOS3. Under salt stress, SOS3 senses increased intracellular calcium levels, which subsequently activates SOS2. Activated SOS2 kinase phosphorylates and activates SOS1, a plasma membrane Na⁺/H⁺ antiporter that transports Na⁺ out of the plant cells, thereby maintaining K⁺/Na⁺ homeostasis. In addition, SOS2 activates multiple subunits of vacuolar H⁺-ATPase pumps (VPs) through direct interaction. These pumps, which are activated under high salt conditions, generate a proton gradient that drives the Na⁺/H⁺ antiport activity, thereby facilitating the sequestration of sodium ions in the vacuoles [94,95]. In addition to the SOS pathway, which is essential for sodium ion elimination and maintaining ion balance, zoysiagrass activates various ROS scavenging mechanisms to reduce oxidative stress induced by salt exposure [24]. Effective ROS management is crucial not only for resilience to salt stress but also for enhancing tolerance to other abiotic stresses, including drought and cold. This overlap illustrates a shared defense strategy among plants in response to salt, cold, and drought stress, underscoring the comprehensive nature of their responses to diverse environmental challenges.

The application of RNA-seq technology, including the pioneering study mentioned above [13], significantly advanced our understanding of the molecular mechanism in zoysiagrass. Through the comparative analysis of time-series transcriptomes of various Zoysia species and varieties exposed to salt stress, researchers have identified numerous DEGs implicated in salt tolerance [6,7,13,24,27,96,97]. These DEGs are involved in a wide array of critical biological processes and signaling pathways [13]. These key pathways include those regulated by ABA, JA, SA, and calcium ions. In addition, pathways related to ROS scavenging, ion regulation, osmotic regulation, and protein folding were also significantly represented. The study highlighted the importance of the 24 h post-treatment period and root tissue in the salt stress response [6]. Further investigation revealed potential salt tolerance genes associated with the mitochondrial membrane translocase pathway and RNA metabolism [97]. A study comparing the transcriptomes between salt-sensitive and salt-tolerant varieties identified DEGs that are predominantly involved in auxin and ABA signaling pathways as crucial for salt tolerance [24]. These findings enhance our understanding of molecular salt stress responses and have revealed both unique and common elements of this response across different plant species.

In zoysiagrass, several strategies have been employed to identify potential candidate genes that confer salt tolerance, such as functional studies of genes that are homologous to salt stress regulators, screening for genes that rescue salt-sensitive yeasts, and RNA-seq analysis under salt conditions or in salt-tolerant varieties or species [6,24,56,97,98]. From literature-based approaches, ZmVP1 was selected as one of these candidates. The overexpression of Arabidopsis VP1 has been shown to enhance salt tolerance in barley and cotton as well as in Arabidopsis [99,100]. The ectopic expression of ZmVPI1, isolated from halophytic turfgrass Z. matrella, in Arabidopsis promotes Na⁺/K⁺ assimilation and vacuole H⁺ transport enzyme activities [54]. Furthermore, transgenic lines overexpressing ZmVP1 exhibited more vigorous growth under salt stress, which suggests that ZmVP1 could be a valuable gene resource for improving salt tolerance in zoysiagrass.

In addition, several salt tolerance genes were identified by screening a cDNA library from the halophyte Z. matrella for genes that could rescue salt-sensitive yeast under high salt conditions, including ZmPDI (protein disulfide isomerase) and ZjGRP (glycine-rich RNA-binding proteins) [97]. Transcriptome and proteome analysis in transgenic Z. matrella plants overexpressing ZmPDI revealed that ZmPDI can enhance the salt tolerance of Z. matrella, potentially by regulating the expression of TUBB2, PXG4, PLD α2, PFK4, and 4CL1 [7]. Another significant gene, ZjGRP, plays a crucial role in post-transcriptional regulation by affecting the plant’s response to environmental stress signals [55]. ZjGRP has been identified as a salt-inducing gene, and its overexpression in Arabidopsis has been found to reduce salt tolerance. Transgenic Arabidopsis overexpressing ZjGRP exhibited reduced salt tolerance, possibly due to effects on ion transport, osmosis, and antioxidant properties. This suggests that ZjGRP functions as a negative regulator of salt tolerance, which indicates that ZjGRP loss-of-function mutations may enhance salt tolerance in zoysiagrass.

RNA-seq analysis has revealed several candidate genes involved in the regulation of salt responses in zoysiagrass, including ZjZFN1 and Zj_AP2-LIKE ABA REPRESSOR 1 (ZjABR1) [6,13,27]. ZjZFN1, a C2H2-type zinc finger gene, is induced by salt, cold, and ABA exposure [56]. The overexpression of ZjZFN1 in Arabidopsis enhanced the plant’s adaptation to salt stress, which resulted in healthier growth by influencing ROS accumulation and inducing several salt tolerance-related genes under salt stress [56]. The ethylene-responsive factor (ERF) TFs largely participate in stress responses by regulating various stress-related genes [101]. ZjABR1/ERF10, one of the ERF TF family members in Z. japonica, is induced by salt stress as well as stress hormones such as ABA and ethylene [57]. The overexpression of ZjABR1 in rice demonstrated increased salt resistance but exhibited additional phenotypes, such as lower seed setting rate and dwarfism, potentially by mediating the signal transduction of GA-ABA crosstalk through its interaction with ZjCIGR1. Overall, these insights emphasize the complexity of genetic responses to environmental stresses and underscore the potential of genetic engineering in developing stress-resilient zoysiagrass varieties.

Salt stress is a significant environmental factor that affects the growth and development of zoysiagrass. Studies on the response of zoysiagrass to salt stress have laid a solid foundation for future genetic improvement and biotechnology applications by identifying key genes, including TFs, and elucidating molecular mechanisms. These findings enhance our understanding of plant stress physiology and provide potential targets for the development of salt-tolerant crop varieties.

3.3. Drought Stress

Drought stress, caused by a water deficit, is a major environmental stressor that limits the growth, development, and establishment of turfgrasses. The efficient water management of Z. japonica is an important issue in urban landscape design and sports field management [102]. Given the increasing demand for sustainable and cost-effective turfgrass management practices, understanding the adaptive mechanisms of these grasses under drought stress conditions is essential.

Drought resistance in plants is governed by both complex molecular mechanisms and morphological traits. On the molecular level, the synthesis of osmolytes, such as proline, glycine betaine, mannitol, and sorbitol, plays a crucial role in osmotic adjustment, allowing cells to retain water and maintain function under drought stress. Additionally, the expression of LEA proteins and heat shock proteins (HSPs) helps stabilize cellular structures and proteins during stress [103,104]. Detoxifying enzymes, such as superoxide dismutase, catalase, and glutathione, are essential in reducing oxidative damage caused by ROS, which accumulate under drought conditions. Another key mechanism is the activation of the ABA signaling pathway, which regulates various stress-responsive genes and enzymes, leading to stomatal closure and improved water use efficiency.

For the physiological adaptations, drought resistance in plants operates through two major mechanisms: drought avoidance and physiological adaptations [105,106]. Drought avoidance involves physical traits that help maintain tissue hydration and turgidity during water-limited conditions, such as deeper and more extensive rooting systems and stomatal closure for less transpiration [107]. A study on 27 cultivars of Z. japonica, Z. matrella, and their interspecific hybrids conducted an association analysis among the turf quality, leaf water content, and drought avoidance traits, including stomatal conductance and root mass/length [108]. The findings revealed that turf quality under drought conditions was strongly associated with leaf water content, stomatal conductance, and root biomass. This indicates that both underground rooting traits and aboveground factors that affect transpiration play crucial roles in the drought resistance of zoysiagrass.

In addition, the physiological responses to drought stress in zoysiagrass have been investigated [108,109]. The drought-resistant line Z. tenuifolia exhibits distinct physiological responses under drought conditions compared to drought-sensitive Z. japonica. These responses include a prolonged peak duration of ascorbic acid content, higher POD activity and MDA content, and higher levels of rubisco and chlorophyll [109]. These results suggest that zoysiagrass reduces drought-induced oxidative stress by increasing and sustaining ascorbic acid content and POD activity in leaves, thereby protecting rubisco and chlorophyll from degradation and enhancing drought resistance.

Cell signaling and growth hormones also play a critical role in reducing drought stress in zoysiagrass. In particular, calcium signaling plays a crucial role in drought resistance [110,111]. Moderate concentrations of calcium chloride have been shown to improve various physiological parameters in Z. japonica under water-deficit stress, including increased biomass, chlorophyll content, photosynthetic rate, proline content, and antioxidant enzyme activities. This result also further emphasizes the important role of proline as an osmotic regulator to maintain cell filling under water-deficient conditions [112]. In addition, plant growth regulators have demonstrated positive effects on drought resistance [113]. Pretreatment with SA enhances the activities of antioxidant enzymes, such as SOD, POD, and catalase (CAT), and increases the biomass and chlorophyll content [114]. Meanwhile, paclobutrazol and uniconazole, GA biosynthesis inhibitors, reduce evapotranspiration rates and vertical growth [114,115]. These cellular regulators maintain turf quality under water-limited conditions.

Recent research has identified candidate genes that are related to the regulation of drought resistance, although when compared with other plants, the molecular mechanism of drought resistance in zoysiagrass remains less understood. The key mechanisms of drought stress responses involve TF-mediated regulation of downstream effectors, including LEA and DREB1 genes, as well as ABA-responsive genes and antioxidant genes such as POD and SOD. These genes, also involved in responses to other stresses, such as cold and salt stress, contribute to cellular protection and stress regulation under cold, salt, and drought conditions, highlighting the interconnected nature of plant stress responses [116,117]. Genetic regulators, including TFs, of resilience to other environmental stresses are also implicated in drought tolerance, potentially through common stress regulatory pathways. For example, ZjICE2, a cold stress regulator with an MYC-like bHLH domain, is also regulated by dehydration [52]. The overexpression of ZjICE2 in Arabidopsis enhances drought tolerance through stomatal closure, in addition to increasing resistance to cold and salt stress. In addition, the promoter activity of ZjZFN1, a salt stress regulator with a C₂H₂ zinc finger domain, is upregulated by mannitol treatment, which mimics drought conditions [58]. Interestingly, the overexpression of ZjZFN1 in Arabidopsis reduces plant adaptability to drought stress and impairs plant growth under drought stress because of the reduced expression of POX, SOD, and pyrroline-5-carboxylate synthase (P5CS) genes. These TFs likely regulate numerous common stress response genes, although their specific targets and regulatory molecular mechanisms in zoysiagrass require further investigation.

Drought stress is an important environmental challenge for turfgrass, yet the molecular mechanisms underlying its response to drought conditions remain poorly understood. Although several drought-related genes have been identified, their specific functions and regulatory pathways are not fully elucidated, which highlights the need for further investigation.

3.4. Wound Stress

Turfgrass is widely used in parks, sports fields, golf courses, and courtyards and is often exposed to traffic stress, which includes wounding caused by direct pressure, tearing, and scuffing of the leaves, stems, and crowns [118,119]. Understanding the impact of traffic stress on zoysiagrass is critical for its maintenance and the selection of varieties with strong resistance to such stress. Light traffic stress can promote tillering and slow growth in zoysiagrass, thus enhancing the lawn quality and utility [120]. However, excessive traffic stress crushes the blades, stems, and crowns, which leads to discoloration, bare patches, and an increased susceptibility to pathogens and insects [121,122]. Traffic stress decreases chlorophyll content and photosynthetic capacity and causes soil compaction, which hinders root vitality and the capacity for nutrient absorption, thereby ultimately inhibiting the overall growth of zoysiagrass [123,124].

The TF network in plant wound signaling involves the coordinated activation of defense and repair mechanisms. The key TF families in wound signaling include APETALA2/ethylene-responsive element binding protein (AP2/EREBP), NAM/ATAF/CUC (NAC), and WRKY [125,126,127]. These TFs regulate the expression of wound-response genes by binding to specific promoter regions, thereby coordinating local and systemic defense responses through interactions with signaling molecules like JA-responsive genes, calcium ions, and ROS [72,125,128,129]. A time-series transcriptome analysis of wounded Z. japonica leaves revealed differential expression of genes within the TFs of AP2/EREBP, NAC, and WRKY and identified 13 AP/EREBP family genes, 8 NAC family genes, and 11 WRKY-family genes [130]. Although further functional validation is required for these genes as wound-regulatory TFs, comparing these findings with transcriptome analyses under other stresses will allow them to be classified as either unique wounding stress regulators or common stress regulators.

In turfgrass management, mowing is a straightforward and valuable practice for maintaining aesthetic appeal, but it also causes the most common wounding stress. The ability of organs to regenerate after cutting is a critical aspect of resistance to wounding. The organ regeneration of turf grass is associated with the activity of shoot apical meristem (SAM) [131]. In Arabidopsis, PIN-FORMED 1 (PIN1) and WUSCHEL (WUS) are key regulators in the maintenance and proliferation of SAM by regulating auxin and cytokinin [132,133,134,135]. A time-series transcriptome analysis of Z. japonica stems containing SAM was performed at 2, 6, and 24 h after cutting and examined the global expression patterns of genes related to the PIN1 and WUS networks [136]. Gene ontology analysis of the DEGs revealed significant enrichment in cell proliferation, auxin- and cytokinin-related processes, and phytohormone signal transduction. Within the PIN1 and WUS networks, 87.5% (21 out of 24 genes) exhibited dynamic changes. Specifically, ZjPIN1 was highly expressed shortly after wounding, while auxin levels were reduced, and ZjWUS was significantly upregulated at 2 and 24 h post-wounding. These findings suggest that ZjPIN1 and ZjWUS play crucial roles in organ regeneration in zoysiagrass after wounding, although their functional roles need further investigation. They also highlight the dynamic changes of the auxin and cytokinin signaling networks in zoysiagrass in response to wounding, which provides valuable insights for further studies on the post-wounding organ regeneration mechanism in zoysiagrass.

Zoysiagrass exhibits a greater sensitivity to wounding stress than other species [136]. This turfgrass is susceptible to damage from high foot traffic and mechanical stress, which causes its growth and regeneration abilities to be considerably affected. Despite the important role of zoysiagrass in turfgrass management, there remains a relative lack of research on its molecular mechanisms under traffic damage stress. To better utilize the excellent characteristics of zoysiagrass, it is particularly important to further study its response mechanism to wounding stress. By revealing the physiological and molecular changes in zoysiagrass under wounding conditions, we can provide a scientific basis for its management and improve the tolerance and recovery ability of zoysiagrass.

3.5. Pathogen Disease

Plant diseases, including those affecting turfgrass, are predominantly caused by pathogens such as bacteria, fungi, nematodes, and viruses. Among these, fungi are the primary culprits that are responsible for significant declines in turfgrass health, causing diseases such as rust, large patch, brown patch, dollar spot, and Pythium blight. Recent molecular and genetic analyses have focused on the two prevalent fungal diseases in zoysiagrass: large patch disease caused by Rhizoctonia solani AG2-2 and rust disease caused by Puccinia zoysiae Diet.

Large patch disease is caused by R. solani AG2-2 and is the most devastating disease in zoysiagrass [137,138]. This disease primarily manifests in late spring, progressing more rapidly and extensively than other fungal diseases, thereby complicating control efforts [139]. The development of disease-resistant zoysiagrass through genetic engineering is in high demand. Chitinases, which are well-known defense-related proteins that hydrolyze chitin—a major component of fungal cell walls—play a critical role in plant defense against fungal diseases. In Z. japonica, the genes Zjchi1 and Zjchi2 encode chitinases with broad-spectrum antifungal activity, including activity against R. solani AG2-2 [140]. Upon infection by R. solani AG2-2, the expression of these genes increases significantly, with Zjchi2 showing a 20-fold increase in inducible levels compared to Zjchi1, which indicates that Zjchi2 plays a more significant role during R. solani AG2-2 infection [139]. Transgenic Z. japonica plants overexpressing Zjchi2 exhibit resistance to the R. solani AG2-2 pathogen, with no morphological changes and generally enhanced antifungal activity [59]. These findings confirmed that Zjchi2 is a valuable gene for enhancing antifungal resistance and suggest that its overexpression could significantly enhance the plant’s defense against a broad range of fungal infections.

In addition, Streptomyces spp. has demonstrated significant potential in biological control because of its ability to produce a variety of secondary metabolites that have antimicrobial activity, including raw materials for anticancer drugs, antiviral drugs, disinfectants, herbicides, and insecticides [141,142]. Streptomyces sp. S8 has been shown to inhibit the density of pathogenic fungi and has excellent potential as a biological control agent for large-area patches of turfgrass [143]. Genome sequencing analysis of Streptomyces sp. S8 identified a valinomycin biosynthetic gene cluster, in which vlm1 and vlm2 produce an antifungal compound [144]. The deletion of vlm1 and vlm2 from Streptomyces sp. S8 resulted in the loss of antifungal activity against pathogens. This result suggests that the biological control of large patch disease may be possible by generating turfgrass resistant to the disease through the introduction of the valinomycin biosynthesis gene cluster into zoysiagrass via metabolic engineering. This approach could reduce the use of chemical pesticides in turf management.

Rust is also prevalent as well as the longest-enduring disease in zoysiagrass, which seriously affects the turf quality. Rust pathogens, such as Puccinia zoysiae, absorb nutrients from living plant cells, destroy chlorophyll, and reduce photosynthesis and respiration rates [145], thereby adversely affecting plant growth, physiology, and overall health. Severe rust infections can cause the death of entire turf areas and delay regrowth [146], thereby reducing turf value and causing substantial economic losses [147,148].

Comparative analyses of rust-resistant and rust-susceptible Z. japonica varieties, derived from natural population screening [148,149], have provided insights into the molecular basis of rust resistance. Rust-resistant varieties exhibit a higher wax content in the leaf epidermis, a thicker spongy parenchyma and palisade tissue, and a greater number of stomata [145]. In addition, these resistant varieties demonstrated a quicker post-inoculation recovery of antioxidant enzyme activities, such as SOD, CAT, and ascorbate peroxidase (APX), which indicates a positive correlation between rust resistance and antioxidant enzyme activity [145]. Further transcriptome analysis has revealed that the plant–pathogen interaction and galactose metabolism pathways are upregulated, but the biosynthetic pathways of zeatin and unsaturated fatty acids are downregulated post-infection in rust-resistant lines [30]. Potential candidate genes implicated in fungal infection protection include the bZIP TF, cysteine-rich RLK 25 (CRK25), and glutathione synthetase 2 (GSH2). Further research on these genes is required to elucidate their specific regulatory mechanisms and biological functions to provide a theoretical basis for the breeding of disease-resistant plants or developing new control strategies.

3.6. Insect Pests

Zoysiagrass, like most warm-season grasses, is susceptible to various insect and mite pests [150]. The FAW, Spodoptera frugiperda, is a particularly destructive pest that affects more than 60 plant species, including both major cool- and warm-season turfgrasses [151]. While insecticides remain the primary method for controlling FAW, their efficacy is increasingly compromised by narrow profit margins, decreased pesticide availability, environmental concerns, and rising insect resistance [152,153,154]. Consequently, there is a growing need for alternative strategies to manage FAW in zoysiagrass.

Molecular tools offer significant potential to enhance the breeding efforts for FAW resistance in turfgrasses, particularly in zoysiagrass. An initial QTL analysis of the F1 Z. matrella hybrid, derived from a cross between the FAW-susceptible cv. “Diamond” and the FAW-resistant cv. “Cavalier”, identified the major control regions for FAW resistance. This analysis revealed a coarse linkage with a 6–9 cm distance to the resistance locus Zfawr1, which is a homologous gene locus that has been previously reported in maize [155]. Furthermore, a high-resolution genetic map of Z. matrella using RAD-seq revealed six loci that were significantly associated with FAW resistance, thus providing primary targets for cloning FAW resistance genes in zoysiagrass [18].

The protein toxins produced by Bacillus thuringiensis (Bt) are the most widely used natural insecticides and are lethal to certain herbivorous pests, including FAW [156]. Bt toxins are formed by aggregated protein crystals produced by the Cry and Cyt genes during sporulation, which cause toxicity in caterpillars by creating pores in the membranes of cells that line their gut, thereby disrupting the ion balance [157]. The cryIA(b) gene has been successfully introduced into various crop species, including rice, maize, cotton, and tobacco, which has resulted in transgenic lines that are highly toxic to target insects while having little to no direct effect on non-target species [158]. In Z. japonica, a synthetic Bt cryIA(b) gene under the ubiquitin promoter was introduced, and the expression of the CryIA protein was detected in several transgenic lines [43]. Although the functional activity of these insecticidal proteins and the resistance of Bt-expressing lines against insect pests remain unanswered, this approach will establish an effective insect management strategy for Zoysia species.

Despite the progress in developing zoysiagrass germplasm with enhanced insect resistance, numerous challenges remain. The response mechanisms of zoysiagrass to specific insect pests are not well understood. Future research should focus on elucidating these response mechanisms, which will be essential in breeding more resilient zoysiagrass varieties and improving the overall pest management strategies in zoysiagrass.

3.7. Senescence

Plant senescence is the final stage of plant development, which is marked by a color change from green to yellow because of the gradual degradation of chlorophyll and the decline of cellular, tissue, and organ functions [159]. In addition to aging, various environmental factors, including biotic and abiotic stresses, along with endogenous factors such as growth status, carbohydrate levels, and hormones, influence senescence onset and progression [160,161]. The aesthetic appeal of zoysiagrass is significantly affected by senescence, making it essential to understand the senescence mechanisms in zoysiagrass to prevent premature senescence [27].

RNA-seq analysis during senescence revealed crucial insights into the transcriptional regulation of senescence [14,27,28]. Comparative transcriptomic analysis was performed to investigate early senescence responses triggered by age, darkness, and salt. The study identified the up- and downregulated senescence markers for senescence and putative regulators that triggered the common senescence pathways. Furthermore, protoplast-based functional analysis revealed that ZjNAP, ZjWRKY75, ZjARF2, ZjNAC1, ZjNAC083, ZjARF1, and ZjPIL5 affected the promoter activity of ZjPCAP and Zj_STAY GREEN (ZjSGR). Another transcriptome study on age-induced leaf senescence revealed that indole-3-acetic acid biosynthesis was inhibited, while ethylene and ABA biosynthesis were induced, and the signaling pathways of auxin, ethylene, and ABA were also activated during leaf senescence [28]. These findings provide a molecular understanding of the biological process during senescence and identify regulatory candidate genes for senescence regulation.

The fundamental and distinct feature of leaf senescence is the degradation of chlorophyll, which is a process that is executed by a series of chlorophyll catabolic enzymes [162]. The genes encoding chlorophyll catabolism enzymes discovered in higher plants include SGR, pheophytinase (PPH), NYC1, NOL, and red chlorophyll catabolite reductase (RCCR) [163]. Among these, the SGR gene, known as Mendel’s I locus for the green trait, encodes magnesium-dechelatase, a key regulatory gene for chlorophyll degradation [164], and has been extensively studied for its role in the chlorophyll degradation process during senescence in various plants [165,166]. In Z. japonica, the SGR gene is upregulated in natural senescence and artificially induced senescence, including conditions such as darkness and treatment with ABA and methyl JA [60]. The overexpression of ZjSGR in Arabidopsis accelerates chlorophyll degradation and reduces photosynthesis, which results in a rapidly yellowing phenotype. In addition, ZjSGR can rescue the stay green phenotype of nye1-1 mutants, which suggests that ZjSGR is a functional homolog of AtNYE1 from Arabidopsis. RNA-seq analysis in Arabidopsis lines that overexpress ZjSGR revealed that ZjSGR may play multiple roles in senescence and chlorophyll degradation by regulating hormone signal transduction and the expression of numerous senescence and environmental stress-related genes. Moreover, the PPH gene, encoding a phytol hydrolase, is induced during senescence and is involved in the chlorophyll decomposition mechanism in Arabidopsis [167] and rice [168]. The overexpression of ZjPPH in Arabidopsis accelerates chlorophyll degradation and rescues the green phenotype in the Arabidopsis pph mutant [61]. ZjPPH also promotes senescence, along with the induction of ABA and soluble sugar contents as well as the transcript levels of SENESCENCE-ASSOCIATED GENE 12 (SAG12) and SAG14. Another significant gene for the inhibition of yellowing during senescence is ZjNYC1, which encodes short-chain dehydrogenase/reductase, an enzyme that is involved in chlorophyll catabolism [62]. The overexpression of ZjNYC1 in Arabidopsis accelerated chlorophyll degradation, promoted senescence, and was accompanied by the accumulation of ABA and ROS [169]. The expression of ZjNYC1 rescued the stay green phenotype in the Arabidopsis nyc1 mutant. Combined analysis of the JIP-test and RNA-seq indicated that ZjNYC1 negatively affected photosystem (PS) II, PS I, and the efficiency of the electron transport chain. Functional analysis studies of zoysiagrass genes involved in chlorophyll degradation elucidated their role in chlorophyll degradation and senescence and further maintained aesthetic appeal by blocking leaf yellowing during senescence as well as under multiple stress conditions.

Authentic function needs to be addressed in zoysiagrass using a loss-of-functional analysis of these genes. In addition, developing zoysiagrass varieties through gene editing of positive senescence regulators can help extend the green period of zoysiagrass by inhibiting senescence. The CRISPR/Cas9 system-based gene editing of zoysiagrass in senescence research has recently been applied [49,50]. Loss-of-function mutants of the ZmNYC1 gene were obtained by targeting the Z. matrella “Wakaba” NYC1 locus using the Agrobacterium-mediated CRISPR/Cas9 system [50]. Under dark conditions and winter simulation treatments, these mutants retained an extended green phenotype. The ZjEIN2 gene, a crucial regulator of ethylene-mediated senescence through the trifurcate feed-forward senescence regulation involving EIN2-miRNA164-ORESARA1, was also targeted [170]. By successfully applying CRISPR/Cas9-based gene editing technology to knock out the ZjEIN2 gene, researchers generated a Z. japonica mutant that exhibited delayed senescence under dark and ethylene treatment conditions [49]. This provides strong evidence for the role of ZjEIN2 in regulating functional leaf senescence beyond just the color change.

Understanding the molecular mechanisms of senescence in zoysiagrass is essential in improving its resilience and extending its utility. Research has identified key genes and regulatory pathways involved in chlorophyll degradation and senescence processes, which has provided valuable insights for genetic improvement. Future studies should focus on integrating these molecular insights with practical breeding strategies to cultivate superior zoysiagrass lines with extended greening periods.

4. Research on Cultivation Management of Zoysiagrass

Zoysiagrass is a popular choice for commercial and residential landscapes because of its aesthetic appeal and functional benefits. Effective management of zoysiagrass is vital in optimizing its performance and longevity. This section provides an overview of the key management practices, including herbicide selection, mowing-related properties, and propagation strategies.

4.1. Herbicide Selection

Zoysiagrass is widely used in various settings, from commercial landscapes to personal lawns, which makes its management a crucial aspect of turf improvement. In natural environments, lawn grasses often compete with weeds, whose proliferation can significantly depreciate the aesthetic and functional value of the turf. Therefore, effective weed control has become an important aspect of turf management.

Herbicides play a critical role in preserving the visual appeal of zoysiagrass by eliminating unwanted weeds. Genes that confer herbicide resistance, such as the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene for glyphosate resistance (commonly known by the trade name Roundup) and the bialaphos resistance (BAR) or phosphinothricin acetyltransferase (PAT) gene for glufosinate resistance (commercially known as Basta), are extensively utilized to select genetically modified plants. These genes are also applied to commercial crops, which facilitates the removal of unwanted vegetation. In zoysiagrass, the BAR gene was first introduced into Z. japonica via Agrobacterium-mediated transformation. The resulting transgenic plants were successfully selected by applying 2 g/L bialaphos for a week, which effectively eliminated untransformed zoysiagrass and invading weeds [44]. Bialaphos and its derivative, phosphinothricin, are non-selective and environmentally safe herbicides that are quickly absorbed by the soil, making them highly effective for weed control [171,172]. This can reduce serious water and soil pollution that is caused by residual herbicides on lawns, thereby protecting the environment [173].

Another transgenic Z. japonica line, Jeju GREEN 21, developed with the BAR gene, underwent rigorous regulatory approval processes. These included testing for selection efficiency, environmental introgression, and human health and insect safety, all of which passed [63,65]. Another herbicide resistance gene, EPSPS, has been proposed for dual selection or for potentially reducing the combined use of two herbicides in turfgrass management. While EPSPS has been successfully implemented in creeping bentgrass (Agrostis stolonifera), its integration into Zoysia species remains under development. Furthermore, efforts are underway to create transgenic turfgrass plants that express the dicamba monooxygenase gene (DMO) in various laboratories [174]. Dicamba (3,6-dichloro-2-methoxybenzoic acid) is an active auxin analog, but its overdose leads to cell and plant death. Other potential target genes for herbicide resistance include the bromoxynil nitrilase gene (BXN) for bromoxynil, the dihydropteroate synthase gene (DHPS) for asulam, and acetolactate synthase gene (ALS) for sulfonylureas, which could be employed for multiple selection processes [63].

4.2. Growth Control

Mowing is a widely used method for managing lawns, aimed at enhancing aesthetics by controlling overgrowth and removing weeds. Despite its popularity, mowing requires frequent manual effort and causes wound stress in the grass. This stress can lead to decreased photosynthetic efficiency, inhibited root development, and reduced carbohydrate reserves that are essential for regrowth.

To reduce the frequency of mowing, certain traits in zoysiagrass are desirable, including growth retardation, roughness, and wounding tolerance. Among these, dwarfism or growth retardation is particularly beneficial, as it requires less frequent mowing and reduced fertilization. Various strategies have been explored to develop growth retardation traits in zoysiagrass, such as knowledge-based studies and mutational breeding [169,170,174,175,176]. For instance, the overexpression of phyA(S599A) in Z. japonica suppresses shade avoidance responses and induces dwarfism [63]. In addition, hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT), an essential enzyme for regulating the biosynthesis and composition of lignin [174], has been studied for its role in growth regulation. The overexpression of ZjHCT4 increased stem elongation with lignin composition changes in creeping bentgrass, which suggests that the editing of this gene might cause stunted and slower growth in zoysiagrass [64]. PSY, which encodes phytoene synthase, a key limiting enzyme in the carotenoid biosynthesis pathway for regulating phytoene synthesis, has also been a focus of research [175]. The overexpression of ZjPSY in Arabidopsis exhibited dwarfing phenotypes but also resulted in yellowing, which potentially limits its use for growth retardation [65].

Moreover, mutation breeding has yielded valuable germplasm with dwarfism in Z. japonica, such as “Halla Green 1” and “Halla Green 2”. These lines, generated from a gamma ray-induced mutation pool, exhibited significantly reduced height, shorter leaves and internodes, and a lack of trichomes at the abaxial leaf surface, which enhanced its commercial viability [176,177]. Furthermore, another study isolated a dwarf mutant in the Z. matrella genetic background by screening the dwarf line from regenerated plants from γ-irradiated calli. This mutant exhibited a short height with shorter stems, wider leaves, a lower canopy height, and a darker green color, likely due to reduced auxin contents and signaling [178]. These findings indicate that a combination of genetic manipulation and mutation breeding can effectively produce dwarf zoysiagrass varieties, which are desirable for their reduced maintenance requirements and enhanced aesthetic appeal.

4.3. Propagation

For the initial establishment of grass plugs or recovery from stress-induced damage in lawns, zoysiagrass must rapidly propagate to expand its coverage through clonal reproduction via runner formation and sexual reproduction via seed formation for long-term sustainability. Consequently, traits such as the growth and development of stolons or rhizomes for vegetative spreading, flowering initiation, tiller formation, and branching for seed production are highly desirable for zoysiagrass propagation. Zoysiagrass primarily expands its coverage through lateral spreading with runner formation rather than seed production. Stolons and rhizomes, two organ types of runners, are modified stems for vegetative reproduction. While stolons grow above the ground, rhizomes grow beneath the ground [179].

In rhizomatous wild rice, the BLADE-ON-PETIOLE (BOP) gene promotes a sheath-to-blade ratio that confers rhizome tip stiffness and supports underground growth. In Z. matrella, the ZmaBOP gene is highly expressed at the apex of rhizomes or stolons with a pattern that is similar to that of BOP in wild rice, which indicates that the BOP-mediated repression of leaf blade growth may be a common characteristic in both rhizomes and stolons. Therefore, the overexpression of this gene likely promotes proper rhizome development in zoysiagrass [180] and mediates the rapid spatial propagation of zoysiagrass. Further studies regarding lateral spreading will be conducted for the rapid propagation of zoysiagrass.

The flowering of zoysiagrass is mainly influenced by day length, temperature, and nutrient conditions [181]. Flowering in Z. japonica, Z. matrella, and Z. pacifica is induced by short days (8–10 h) and warm temperatures (27–30 °C). In addition, a combination of low light intensity and low nitrogen levels can trigger flowering. Despite the limited research on the molecular mechanisms of flowering initiation in zoysiagrass, some research suggests the involvement of specific genes. For example, the overexpression of the Z. japonica ZjWRKY10 gene in Arabidopsis triggers early flowering through activation of FLOWERING LOCUS T [66]. In contrast, inhibition of flowering, such as the generation of never-flowering lines, can be used to control unwanted propagation or gene flow through pollination with the same or closely related species. The generation of unbolting lines through gamma ray irradiation mutagenesis has been applied to commercially valuable herbicide-tolerant genetically modified (GM) zoysiagrass to prevent transgene escape in GM plants [182]. As flowering is an important trait for seed propagation and controlling gene flow in zoysiagrass, it is essential to explore the in-depth genetic and molecular mechanisms of flowering initiation in this genus.

The LATERAL SUPPRESSOR (LS) gene, a member of the TF GRAS family, is a key regulator in the formation of axillary meristems and shoot branching and is highly conserved among plant kingdoms [183]. The Z. japonica LS-like (ZjLsL) gene is highly expressed in culm and nodes, where axillary meristems are initiated [67]. The overexpression of ZjLsL in Arabidopsis increases axillary shoot formation, and its overexpression in creeping bentgrass promotes tiller formation, reduced height, and wider leaf width, although the impact on seed formation remains to be investigated. These traits are valuable for turf management, which suggests that ZjLsL could be a promising gene resource for turf improvement. In addition, the NYC gene is necessary for chlorophyll degradation during leaf senescence. A recent study has indicated that ZmNYC1 gene knockout enhances the retention of chlorophyll content and also reduces tiller production in Z. matrella [50]. This enhances our understanding of the zoysiagrass propagation molecular mechanisms.

Molecular understanding of the physiological processes related to zoysiagrass propagation currently lags behind that of other physiological traits. However, these are valuable traits for turf management, and advancing our understanding and application of these genetic insights is necessary for the development of improved zoysiagrass varieties.

5. Conclusions and Future Perspectives

This review highlights the significant advancements in the molecular genetics of zoysiagrass, emphasizing the critical genetic findings that have implications for improving zoysiagrass as a turfgrass. Comprehensive genomic analyses, including NGS and single-molecule long-read sequencing, have provided valuable insights into the genetic makeup of zoysiagrass. The application of RNA-seq technology has advanced our understanding of gene expression patterns under various stress conditions. In addition, genetic transformation systems, such as Agrobacterium-mediated methods and CRISPR/Cas9 editing, have facilitated targeted genetic modifications to enhance stress tolerance and other desirable traits.

Despite these advancements, there are limitations to the current molecular genetic methods in zoysiagrass research. The complexity of the zoysiagrass genome and the challenges associated with its transformation efficiency and regeneration rates pose significant difficulties. In addition, the translation of laboratory findings to field applications remains a critical challenge, which necessitates robust validation and testing under diverse environmental conditions.

Looking toward future applications, the prospects for advancing turfgrass genetics are promising. Continued improvements in sequencing technologies and genetic engineering tools will enable more precise and efficient modifications. The integration of multi-omics approaches, such as genomics, transcriptomics, and proteomics, provides a holistic understanding of the underlying genetic and molecular mechanisms of zoysiagrass traits.

In conclusion, while significant progress has been made in the molecular genetics of zoysiagrass, addressing the current limitations and exploring new research avenues is essential to realizing the full potential of zoysiagrass improvement. By adopting a strategic and collaborative approach, future research can contribute to the sustainable management and enhancement of zoysiagrass, which ultimately benefits both the ecological and economic aspects of turfgrass cultivation.