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One of the alternatives for reducing nitrogen fertilization is the use of plant growth-promoting bacteria (PGPB), such as Pseudomonas fluorescens and Azospirillum brasilense, which can provide a sustainable form of production, with increased profitability for rural producers, because bacteria are a low-cost technology compared to fertilizer. In view of the above, the objective of this study was to evaluate the effect of inoculation with plant growth-promoting bacteria (P. fluorescens and A. brasilense) combined with the supply or not of Si and doses of nitrogen (N) in zoysia grass sod production area. The experimental design was in randomized blocks with 12 treatments arranged in a 3 × 2 × 2 factorial scheme, with four replications, in plots of 9 m2. The treatments were: two inoculations with PGPB (A. brasilense and P. fluorescens) and non-inoculated, combined with two doses of N, with or without supply of Si through fertilization (through the commercial fertilizer known as Potasil). There was an increase in the shoot dry matter with the supply of Si, the standard dose of N and the inoculation with PGPB. The standard dose of N promoted an increase in the content of nitrogen (N), phosphorus (P) and potassium (K) in the shoot of Zoysia japonica. There was an effect of inoculation with plant growth-promoting bacteria combined with the supply of silicon and N rates in the zoysia grass production area, where the use of Pseudomonas combined with the application of Si with 75% of the N dose is recommended. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Zoysia japonica, a low growing grass, is tolerant to grazing and trampling. Kinkazan Island in northern Japan is inhabited by sika deer (Cervus nippon). The deer population increased in the 1970s at a shrine garden in the western part of the island, leading to expansion of Zoysia swards around the shrine garden and their colonization of remote open patches. The expansion around the garden is due to elongation of the rhizome, but expansion to remote places may be due to endozoochory by deer. This appears to be a good example of the “foliage is the fruit” hypothesis (the FF hypothesis) proposed by Janzen (1984; American Naturalist 123:338–353). To demonstrate this, we confirmed the expansion of the Zoysia swards and tested the traits of Zoysia by field surveys and indoor experiments. The Zoysia peduncles stood among the leaves, and sika deer fed on both the seeds and leaves. One deer fecal pellet contained about 20 seeds at its peak in June. In the feeding experiment, the survival rate through digestion was 38%. In the greenhouse experiment, the germination rate was 72%. An outdoor experiment showed that germination rates of the ingested seeds were 5% in a dark habitat and 58% in a bright habitat. Zoysia exhibited many of the traits presented by the FF hypothesis, and sika deer functioned as seed dispersal agents. Therefore, the Zoysia—sika deer relationship seems to support the FF hypothesis. [ABSTRACT FROM AUTHOR]

Salt stress severely inhibits plant growth. Understanding the mechanism of plant salt tolerance is highly important to improving plant salt tolerance. Previous studies have shown that nonselective cyclic nucleotide-gated ion channels (CNGCs) play an important role in plant salt tolerance. However, current research on CNGCs mainly focuses on CNGCs in glycophytic plants, and research on CNGCs in halophytes that exhibit special salt tolerance strategies is still scarce. This study used the halophilic plant Zoysia japonica, an excellent warm-season turfgrass, as the experimental material. Through bioinformatics analysis, 18 members of the CNGC family were identified in Zoysia japonica; they were designated ZjCNGC1 through ZjCNGC18 according to their scaffold-level chromosomal positions. ZjCNGCs are divided into four groups (I–IV), with the same groups having differentiated protein-conserved domains and gene structures. ZjCNGCs are unevenly distributed on 16 scaffold-level chromosomes. Compared with other species, the ZjCNGCs in Group III exhibit obvious gene expansion, mainly due to duplication of gene segments. The collinearity between ZjCNGCs, OsCNGCs, and SjCNGCs suggests that CNGCs are evolutionarily conserved among gramineous plants. However, the Group III ZjCNGCs are only partially collinear with OsCNGCs and SjCNGCs, implying that the expansion of Group III ZjCNGC genes may have been an independent event occurring in Zoysia japonica. Protein interaction prediction revealed that ZjCNGCs, calcium-dependent protein kinase, H+-ATPase, outwardly rectifying potassium channel protein, and polyubiquitin 3 interact with ZjCNGCs. Multiple stress response regulatory elements, including those involved in salt stress, are present on the ZjCNGC promoter. The qPCR results revealed differences in the expression patterns of ZjCNGCs in different parts of the plant. Under salt stress conditions, the expression of ZjCNGCs was significantly upregulated in roots and leaves, with ZjCNGC8 and ZjCNGC13 showing the greatest increase in expression in the roots. These results collectively suggest that ZjCNGCs play an important role in salt tolerance and that their expansion into Group III may be a special mechanism underlying the salt tolerance of Zoysia japonica. [ABSTRACT FROM AUTHOR]

It is naturally common that different proportions of ramets in a clone lose rooting conditions due to habitat stress or obstacles, which potentially affects the overall growth of the clonal plant to different extents. However, so far, little attention has been paid to such phenomena and much less to the underlying ecological mechanisms. Taking Zoysia japonica as material, through an experiment with two nutrition levels in the habitats and five rooting ramet proportions in the clones, the impacts of proportions of rooting ramets in the clone on the overall growth were tested and the ecological mechanisms were analyzed. The results showed that there was no significant difference in the total clonal biomasses among the clones with five rooting ramet proportions under high and low nutrition levels, except for that with 0% rooting ramet proportion. Under both high and low nutrition levels, the lower rooting ramet proportions (0% and 25%) in the clones significantly decreased the number of the so‐called A‐ and B‐ramets, root biomass, stolon length per unit biomass, and root–shoot ratio, but significantly increased the stolon biomass of the clones. Stolon elongation was promoted under high nutrient level, and biomass allocations to stolons and roots increased under low nutrition levels. A‐ramet biomasses accounted for about 50% and 30% of the total biomasses of the whole clone under high and low nutrition levels, respectively. These results might be reasonably explained in terms of clonal integration, compensatory growth, division of labor, and bet‐hedging strategy. [ABSTRACT FROM AUTHOR]

CRISPR/Cas9-based targeted gene editing has emerged as a versatile tool for deciphering gene function and improving traits in plants. However, this technique has not been applied to Zoysia japonica, a prominent warm-season turfgrass widely used for green spaces. Leaf senescence, a vital process affecting crop quality, occurs in Z. japonica during late growth, diminishing its aesthetic value and performance. In this study, we adeptly employed CRISPR/Cas9-mediated gene editing to create Z. japonica exhibiting delayed leaf senescence by targeting the ZjEIN2 gene, a crucial regulator of ethylene-mediated senescence. Precise gene editing, which generated knockout mutations in ZjEIN2, led to delayed leaf senescence in both dark and ethylene treatment conditions. This provided strong evidence for ZjEIN2's role in leaf senescence regulation. These findings highlight the potential of CRISPR/Cas9-mediated gene editing as a biotechnological strategy to enhance anti-senescence traits in Z. japonica and potentially other crops. This study carries significant implications for sustainable agriculture and turfgrass management, offering promising avenues for future applications and research. [ABSTRACT FROM AUTHOR]

The U-box protein family of ubiquitin ligases is important in the biological processes of plant growth, development, and biotic and abiotic stress responses. Plants in the genus Zoysia are recognized as excellent warm-season turfgrass species with drought, wear and salt tolerance. In this study, we conducted the genome-wide identification of plant U-box (PUB) genes in Zoysia japonica based on U-box domain searching. In total, 71 ZjPUB genes were identified, and a protein tree was constructed of AtPUBs, OsPUBs, and ZjPUBs, clustered into five groups. The gene structures, characteristics, cis-elements and protein interaction prediction network were analyzed. There were mainly ABRE, ERE, MYB and MYC cis-elements distributed in the promoter regions of ZjPUBs. ZjPUBs were predicted to interact with PDR1 and EXO70B1, related to the abscisic acid signaling pathway. To better understand the roles of ZjPUBs under salt stress, the expression levels of 18 ZjPUBs under salt stress were detected using transcriptome data and qRT-PCR analysis, revealing that 16 ZjPUBs were upregulated in the roots under salt treatment. This indicates that ZjPUBs might participate in the Z. japonica salt stress response. This research provides insight into the Z. japonica PUB gene family and may support the genetic improvement in the molecular breeding of salt-tolerant zoysiagrass varieties. [ABSTRACT FROM AUTHOR]

Inadequate reference databases in RNA-seq analysis can hinder data utilization and interpretation. In this study, we have successfully constructed a high-quality reference transcript dataset, ZjRTD1.0, for Zoysia japonica, a widely-used turfgrass with exceptional tolerance to various abiotic stress, including low temperatures and salinity. This dataset comprises 113,089 transcripts from 57,143 genes. BUSCO analysis demonstrates exceptional completeness (92.4%) in ZjRTD1.0, with reduced proportions of fragmented (3.3%) and missing (4.3%) orthologs compared to prior datasets. ZjRTD1.0 enables more precise analyses, including transcript quantification and alternative splicing assessments using public datasets, which identified a substantial number of differentially expressed transcripts (DETs) and differential alternative splicing (DAS) events, leading to several novel findings on Z. japonica's responses to abiotic stresses. First, spliceosome gene expression influenced alternative splicing significantly under abiotic stress, with a greater impact observed during lowtemperature stress. Then, a significant positive correlation was found between the number of differentially expressed genes (DEGs) encoding protein kinases and the frequency of DAS events, suggesting the role of protein phosphorylation in regulating alternative splicing. Additionally, our results suggest possible involvement of serine/arginine-rich (SR) proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs) in generating inclusion/exclusion isoforms under low-temperature stress. Furthermore, our investigation revealed a significantly enhanced overlap between DEGs and differentially alternatively spliced genes (DASGs) in response to lowtemperature stress, suggesting a unique co-regulatory mechanism governing transcription and splicing in the context of low-temperature response. In conclusion, we have proven that ZjRTD1.0 will serve as a reliable and useful resource for future transcriptomic analyses in Z. japonica. [ABSTRACT FROM AUTHOR]

The spread of morning glory (Ipomoea spp.) in Japanese soybean (Glycine max (L.) Merr.) fields has severely decreased soybean yields. Field levees are believed to be a source of this invasion. The introduction of Zoysia japonica Steud. is expected to suppress weed growth and reduce the required labor for weed management on the levees. In this study, field experiments were conducted on the soybean levees of turf (Z. japonica), bare, and weed (mainly broad leaf weeds such as white clover) from 2017 to 2019 to compare its effects on the occurrence, growth, and seed production of I. hederacea (L.) Jacq. var. integriuscula A. Gray. The canopy height in the turf levee was the lowest, and the vegetation cover rate was the highest. At the flowering and fruiting stage of I. hederacea var. integriuscula (September), the largest number of I. hederacea var. integriuscula that survived were on the turf levee; however, the plant length, dry weight, and the number of fruits were the least. Additionally, the stems of the morning glory on the turf levee were slight and spindly, and their growth and seed productivity were suppressed. Consequently, vegetation conversion to Z. japonica on the soybean field levees can be an effective strategy to suppress the colonization and growth of morning glory, thereby, improving soybean yields. [ABSTRACT FROM AUTHOR]

Zoysia japonica (Zoysia japonica Steud.) is a kind of warm-season turfgrass with many excellent characteristics. However, the shorter green period and longer dormancy caused by cold stress in late autumn and winter are the most limiting factors affecting its application. A previous transcriptome analysis revealed that ethephon regulated genes in chlorophyll metabolism in Zoysia japonica under cold stress. Further experimental data are necessary to understand the effect and underlying mechanism of ethephon in regulating the cold tolerance of Zoysia japonica. The aim of this study was to evaluate the effects of ethephon by measuring the enzyme activity, intermediates content, and gene expression related to ethylene biosynthesis, signaling, and chlorophyll metabolism. In addition, the ethylene production rate, chlorophyll content, and chlorophyll a/b ratio were analyzed. The results showed that ethephon application in a proper concentration inhibited endogenous ethylene biosynthesis, but eventually promoted the ethylene production rate due to its ethylene-releasing nature. Ethephon could promote chlorophyll content and improve plant growth in Zoysia japonica under cold-stressed conditions. In conclusion, ethephon plays a positive role in releasing ethylene and maintaining the chlorophyll content in Zoysia japonica both under non-stressed and cold-stressed conditions. [ABSTRACT FROM AUTHOR]

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.

Cadmium (Cd) is a severe heavy metal pollutant globally. Zoysia japonica is an important perennial warm-season turf grass that potentially plays a role in phytoremediation in Cd-polluted soil areas; however, the molecular mechanisms underlying its Cd stress response are unknown. To further investigate the early gene response pattern in Z. japonica under Cd stress, plant leaves were harvested 0, 6, 12, and 24 h after Cd stress (400 μM CdCl2) treatment and used for a time-course RNA-sequencing analysis. Twelve cDNA libraries were constructed and sequenced, and high-quality data were obtained, whose mapped rates were all higher than 94%, and more than 601 million bp of sequence were generated. A total of 5321, 6526, and 4016 differentially expressed genes were identified 6, 12, and 24 h after Cd stress treatment, respectively. A total of 1660 genes were differentially expressed at the three time points, and their gene expression profiles over time were elucidated. Based on the analysis of these genes, the important mechanisms for the Cd stress response in Z. japonica were identified. Specific genes participating in glutathione metabolism, plant hormone signal and transduction, members of protein processing in the endoplasmic reticulum, transporter proteins, transcription factors, and carbohydrate metabolism pathways were further analyzed in detail. These genes may contribute to the improvement of Cd tolerance in Z. japonica. In addition, some candidate genes were highlighted for future studies on Cd stress resistance in Z. japonica and other plants. Our results illustrate the early gene expression response of Z. japonica leaves to Cd and provide some new understanding of the molecular mechanisms of Cd stress in Zosia and Gramineae species. [ABSTRACT FROM AUTHOR]

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Background: AUX/IAA is an essential signaling molecule and has great physiological importance in various plants, but its function in Zoysia japonica remains unknown. Methods and results: Genome-wide identification and analysis of AUX/IAA genes used bioinformatics methods to investigate the ZjIAA genes' expression of exogenous IAA hydroponics treatment for 2 h by qRT-PCR, control and exogenous IAA treated zoysia were subjected to transcriptome sequencing. ZjIAAs were distributed across the 13 subfamilies by phylogenetic analysis with Oryza sativa and Arabidopsis thaliana. Multiple sequence alignment revealed that the majority of genes were non-canonical ZjIAAs with incomplete domain. The optimal growth concentration of the IAA hormone was 0.05 mM, and the qRT-PCR analysis revealed that eight ZjIAAs were differentially expressed, with seven genes considerably upregulating and one gene significantly downregulating. The result of transcriptome sequencing revealed that 515 differentially expressed genes (DEGs) were identified, with 344 upregulated genes and 171 downregulated genes. A total of 18 genes were annotated as involved in the plant hormone signal transduction pathway. And 8 ZjIAAs exhibited distinct expressions, 7 upregulated, and only one downregulated, according to the qRT-PCR study. Conclusions: Genome-wide identification and analysis increased the understanding of the evolution and function of the IAA family in zoysia. DEGs of control and treatment with 0.05 mM exogenous IAA hormone were investigated by transcriptome sequencing. ZjIAAs had substantial variations in the expression of associated genes, with the majority of genes upregulated and 18 genes implicated in plant hormone signal transduction. [ABSTRACT FROM AUTHOR]

The U-box protein family of ubiquitin ligases is important in the biological processes of plant growth, development, and biotic and abiotic stress responses. Plants in the genus Zoysia are recognized as excellent warm-season turfgrass species with drought, wear and salt tolerance. In this study, we conducted the genome-wide identification of plant U-box (PUB) genes in Zoysia japonica based on U-box domain searching. In total, 71 ZjPUB genes were identified, and a protein tree was constructed of AtPUBs, OsPUBs, and ZjPUBs, clustered into five groups. The gene structures, characteristics, cis-elements and protein interaction prediction network were analyzed. There were mainly ABRE, ERE, MYB and MYC cis-elements distributed in the promoter regions of ZjPUBs. ZjPUBs were predicted to interact with PDR1 and EXO70B1, related to the abscisic acid signaling pathway. To better understand the roles of ZjPUBs under salt stress, the expression levels of 18 ZjPUBs under salt stress were detected using transcriptome data and qRT-PCR analysis, revealing that 16 ZjPUBs were upregulated in the roots under salt treatment. This indicates that ZjPUBs might participate in the Z. japonica salt stress response. This research provides insight into the Z. japonica PUB gene family and may support the genetic improvement in the molecular breeding of salt-tolerant zoysiagrass varieties.

The Zoysia japonica plant is a warm-season turfgrass and a horticultural crop used in various places and an important genetic resource because it is more resilient to stress from high temperature, drought, coastal salt, disease and pests, and trampling. Genome research on Z. japonica is necessary to find molecular markers related to useful traits. This study analyzed the genome of the Z. japonica 'Yaji' cultivar plant by PacBio-based long-read sequencing and assembly and annotation. Before performing the PacBio-based long-read NGS analysis, the 'Yaji' cultivar was analyzed by the external morphological classification method and the molecular identification method, and it was confirmed that the 'Yaji' cultivar was identified as a Z. japonica plant. Construction of genomic DNA libraries and sequencing with the PacBio RSII platform were performed. As a result of the DNA sequencing, 40 SMRT cells were sequenced, and about 38.6 Gb of reads were produced. De novo assembly was done by the Canu assembler. The resulting assembly consisted of 1,350 contigs. The BUSCO analysis results showed that 1390 (96.5%) of 1440 conserved orthologous are present as complete genes in the Z. japonica 'Yaji' cultivar genome. The PacBio-based assembly found that the predicted putative gene sequences and the number of the genes showed high identity to the previously reported individual genes. Gene predictions and annotation of Z. japonica 'Yaji' cultivar were done with the Augustus gene prediction software and NCBI-nr database. As a result, 50,140 gene sets of the Z. japonica 'Yaji' cultivar were obtained. Target prediction of the 'Yaji' cultivar was performed for conserved miRNAs using psRNATarget. As a result, 8207 miRNA/target site pairs of the 'Yaji' cultivar were predicted. The transposon element of the 'Yaji' cultivar genome was predicted with the REPET package. As a result, 2656 transposon elements of LARD, TRIM of class I (RXX), and TIR of class II (DXX) were predicted. Genomic data of Korean Z. japonica plants were obtained by PacBio long-read sequencing. These genome data of Z. japonica plant will provide valuable basic data for developing molecular markers and molecular identification of Zoysia turfgrass research. [ABSTRACT FROM AUTHOR]

Zoysia japonica is an important warm-season turfgrass used worldwide. The decreased aesthetic quality and functionality during leaf senescence hamper its further utilization. However, information about the transcriptional mechanism and genes involved in leaf senescence in Z. japonica needs to be more extensive. Therefore, to better understand leaf senescence in Z. japonica, we investigated the integrated analysis of chlorophyll fluorescence test (JIP-test) and RNA sequencing (RNA-seq) of mature and senescent leaves. First, we identified 22,049 genes, of which 4038 were differentially expressed genes (DEGs). The results for gene expression profiles were evaluated using quantitative real-time PCR. A total of 2515 genes have homologous genes in other plants. The matched known-function SAGs are mainly involved in chlorophyll degradation and plant hormone response. A total of 539 differentially expressed transcription factor genes, including AP2/ERF-ERF, NAC, WRKY, bHLH, and MYB, were identified to be associated with leaf senescence. Next, senescence represses chlorophyll biosynthesis while upregulating chlorophyll degradation. Senescence harms the integrity and functionality of PSII, PSI, and the intersystem electron transport chain. In addition, IAA biosynthesis was inhibited, whereas ABA and ET biosynthesis were activated in leaf senescence, and senescence activates signal transduction of IAA, ABA, and ET. These findings add to our understanding of the regulatory mechanism of leaf senescence. The senescence-associated genes are candidate targets for providing new insight into leaf senescence modeling in Z. japonica. They provided a theoretical foundation to reveal the functions of senescence-associated genes and chlorophyll catabolic genes involved in leaf senescence. [ABSTRACT FROM AUTHOR]

Abstract Background Zoysia japonica is an important warm-season turfgrass used worldwide. Although the draft genome sequence and a vast amount of next-generation sequencing data have been published, the current genome annotation and complete mRNA structure remain incomplete. Therefore, to analyze the full-length transcriptome of Z. japonica, we used the PacBio single-molecule long-read sequencing method in this study. Results First, we generated 37,056 high-confidence non-redundant transcripts from 16,005 gene loci. Next, 32,948 novel transcripts, 913 novel gene loci, 8035 transcription factors, 89 long non-coding RNAs, and 254 fusion transcripts were identified. Furthermore, 15,675 alternative splicing events and 5325 alternative polyadenylation sites were detected. In addition, using bioinformatics analysis, the underlying transcriptional mechanism of senescence was explored based on the revised reference transcriptome. Conclusion This study provides a full-length reference transcriptome of Z. japonica using PacBio single-molecule long-read sequencing for the first time. These results contribute to our knowledge of the transcriptome and improve the knowledge of the reference genome of Z. japonica. This will also facilitate genetic engineering projects using Z. japonica.

Cadmium (Cd) is a severe heavy metal pollutant globally. Zoysia japonica is an important perennial warm-season turf grass that potentially plays a role in phytoremediation in Cd-polluted soil areas; however, the molecular mechanisms underlying its Cd stress response are unknown. To further investigate the early gene response pattern in Z. japonica under Cd stress, plant leaves were harvested 0, 6, 12, and 24 h after Cd stress (400 μM CdCl2) treatment and used for a time-course RNA-sequencing analysis. Twelve cDNA libraries were constructed and sequenced, and high-quality data were obtained, whose mapped rates were all higher than 94%, and more than 601 million bp of sequence were generated. A total of 5321, 6526, and 4016 differentially expressed genes were identified 6, 12, and 24 h after Cd stress treatment, respectively. A total of 1660 genes were differentially expressed at the three time points, and their gene expression profiles over time were elucidated. Based on the analysis of these genes, the important mechanisms for the Cd stress response in Z. japonica were identified. Specific genes participating in glutathione metabolism, plant hormone signal and transduction, members of protein processing in the endoplasmic reticulum, transporter proteins, transcription factors, and carbohydrate metabolism pathways were further analyzed in detail. These genes may contribute to the improvement of Cd tolerance in Z. japonica. In addition, some candidate genes were highlighted for future studies on Cd stress resistance in Z. japonica and other plants. Our results illustrate the early gene expression response of Z. japonica leaves to Cd and provide some new understanding of the molecular mechanisms of Cd stress in Zosia and Gramineae species.

Many bermudagrass (Cynodon sp.) and zoysiagrass (Zoysia sp.) cultivars are not available as seed and are commonly planted vegetatively using sprigs, especially for sod production or in sand-based systems. Sprig planting is typically done in late spring or early summer, but this can result in an extended grow-in period and delay the use of the turf in the first growing season. The objective of this study was to determine if sprigs of bermudagrass and zoysiagrass could be planted earlier in the year, during the dormancy phase, to hasten establishment. A field study was carried out in Fayetteville, AR, in 2014 and 2016 using ‘Tifway’ hybrid bermudagrass (Cynodon dactylon × Cynodon transvaalensis) and ‘Meyer’ zoysiagrass (Zoysia japonica), and in Guangzhou, China, in 2015, using ‘Tifway’ hybrid bermudagrass and ‘Lanyin III’ zoysiagrass (Z. japonica). Sprigs were planted in March (dormant), May (spring) and July (summer) in Fayetteville, and in January (dormant), March (spring) and May (summer) in Guangzhou. Sprigging rates of 30, 60, and 90 m3·ha−1 were tested at both locations and across all planting dates. Bermudagrass was less affected by planting date, with dormant, spring or summer plantings effectively establishing full cover in the first growing season. Zoysiagrass that was sprigged in the dormant season was successfully established by the end of the first growing season while a full zoysiagrass cover was not achieved with either spring or summer plantings in Arkansas. Dormant sprigging reached full coverage as fast or faster than traditional spring or summer planting dates at both locations, indicating that bermudagrass and zoysiagrass establishment can be achieved earlier in the growing season using dormant sprigging methods.

The ZjHSP18.9 gene was isolated from Zoysiagrass by homologous cloning based on publicly available transcriptome database. Sequence alignment analysis revealed that ZjHSP18.9 protein contains a classic Hsp23/ACD domain. Phylogenetic analysis and subcellular localization prediction suggested that ZjHSP18.9 belongs to cytosolic class VI subfamily. The expression profiles of ZjHSP18.9 in leaf and root after abiotic stress or Abscisic Acid (ABA) treatment was investigated using quantitative real‐time polymerase chain reaction. The results showed that the expression level of ZjHSP18.9 in leaf was higher than that in root. The expression of ZjHSP18.9 was strongly induced by high temperature in leaf; the ZjHSP18.9 expression level was up‐regulated in both leaf and root by the treatment of low temperature, salt and heavy metal, while down‐regulated under the treatment of drought and ABA. The gene expression profiles indicated that ZjHSP18.9 may play an important role in Zoysiagrass response to environmental stress including extreme temperature, salinity, water deficiency and heavy metal through ABA dependent and/or independent pathway. [ABSTRACT FROM AUTHOR]

Background: Zoysia japonica is an important warm-season turfgrass used worldwide. Although the draft genome sequence and a vast amount of next-generation sequencing data have been published, the current genome annotation and complete mRNA structure remain incomplete. Therefore, to analyze the full-length transcriptome of Z. japonica, we used the PacBio single-molecule long-read sequencing method in this study. Results: First, we generated 37,056 high-confidence non-redundant transcripts from 16,005 gene loci. Next, 32,948 novel transcripts, 913 novel gene loci, 8035 transcription factors, 89 long non-coding RNAs, and 254 fusion transcripts were identified. Furthermore, 15,675 alternative splicing events and 5325 alternative polyadenylation sites were detected. In addition, using bioinformatics analysis, the underlying transcriptional mechanism of senescence was explored based on the revised reference transcriptome. Conclusion: This study provides a full-length reference transcriptome of Z. japonica using PacBio single-molecule long-read sequencing for the first time. These results contribute to our knowledge of the transcriptome and improve the knowledge of the reference genome of Z. japonica. This will also facilitate genetic engineering projects using Z. japonica. [ABSTRACT FROM AUTHOR]

The degradation of chlorophyll is of great significance to plant growth. The chlorophyll b reductase NOL (NYC1-like) is in charge of catalyzing the degradation of chlorophyll b and maintaining the stability of the photosystem. However, the molecular mechanisms of NOL-mediated chlorophyll degradation, senescence, and photosynthesis and its functions in other metabolic pathways remain unclear, especially in warm-season turfgrass. In this study, ZjNOL was cloned from Zoysia japonica. It is highly expressed in senescent leaves. Subcellular localization investigation showed ZjNOL is localized in the chloroplast and the bimolecular fluorescence complementation (BiFC) results proved ZjNOL interacts with ZjNYC1 in vivo. ZjNOL promoted the accumulation of abscisic acid (ABA) and carbohydrates, and the increase of SAG14 at the transcriptional level. ZjNOL simultaneously led to the excessive accumulation of reactive oxygen species (ROS), the activation of antioxidant enzymes, and the generation of oxidative stress, which in turn accelerated senescence. Chlorophyll fluorescence assay (JIP-test) analysis showed that ZjNOL inhibited photosynthetic efficiency mainly through damage to the oxygen-evolving complex. In total, these results suggest that ZjNOL promotes chlorophyll degradation and senescence and negatively affects the integrity and functionality of the photosystem. It could be a valuable candidate gene for genome editing to cultivate Z. japonica germplasm with prolonged green period and improved photosynthesis efficiency. [ABSTRACT FROM AUTHOR]

The degradation of chlorophyll is of great significance to plant growth. The chlorophyll b reductase NOL (NYC1-like) is in charge of catalyzing the degradation of chlorophyll b and maintaining the stability of the photosystem. However, the molecular mechanisms of NOL-mediated chlorophyll degradation, senescence, and photosynthesis and its functions in other metabolic pathways remain unclear, especially in warm-season turfgrass. In this study, ZjNOL was cloned from Zoysia japonica. It is highly expressed in senescent leaves. Subcellular localization investigation showed ZjNOL is localized in the chloroplast and the bimolecular fluorescence complementation (BiFC) results proved ZjNOL interacts with ZjNYC1 in vivo. ZjNOL promoted the accumulation of abscisic acid (ABA) and carbohydrates, and the increase of SAG14 at the transcriptional level. ZjNOL simultaneously led to the excessive accumulation of reactive oxygen species (ROS), the activation of antioxidant enzymes, and the generation of oxidative stress, which in turn accelerated senescence. Chlorophyll fluorescence assay (JIP-test) analysis showed that ZjNOL inhibited photosynthetic efficiency mainly through damage to the oxygen-evolving complex. In total, these results suggest that ZjNOL promotes chlorophyll degradation and senescence and negatively affects the integrity and functionality of the photosystem. It could be a valuable candidate gene for genome editing to cultivate Z. japonica germplasm with prolonged green period and improved photosynthesis efficiency.

The main objective of the research was to study the effect of the Stenotaphrum secundatum and Zoysia japonica grasses on the higher and lower environment temperature and lower relative humidity; the secondary objective was to compare whether the Stenotaphrum secundatum grass has a greater impact on the environment parameters of comfort than the Zoysia japonica species. Six materials were used for the extensive green roof, each one forming a layer of the system, which were placed on the concrete slab and in an upward direction, including: PVC geomembrane, Polyester asphalt carpet, Pumice stone, Planar geodren, Prepared soil with guano, compost, muss, and Substrate. In order to make measurements of the higher and lower ambient temperature, a digital thermometer and lower relative humidity meter was used. Stenotaphrum secundatum and Zoysia japonica were used as grass species, as they were the most representative of the grasses used in extensive green roofs. The experimentation was carried out for 2 months from September to October of 2021, having built 3 modules of 1000x600mm roofs, including 1module of the concrete roof with ceramic covering and 2 modules of extensive green roof with two types of grass: Stenotaphrum secundatum and Zoysia japonica. The readings of the environment temperature and relative humidity of the higher and lower part were taken in six points of each module to have a greater number of representative measurements. The watering of the 2 green roof modules with grass was carried out twice a week, applying 5L of water per module. The results indicate that the Zoysia japonica grass is the one that presents a better behavior before the higher ambient temperature and that the Stenotaphrum secundatum grass behaves better before the lower ambient temperature and lower relative humidity. The conclusions indicate that the Stenotaphrum secundatum grass behaves better temperature and relative humidity; the two grass types exhibit different behavior. [ABSTRACT FROM AUTHOR]

Physiological integration may be an important ecological strategy for clonal plants living in heterogeneous environments. The physiological integration between Zoysia japonica parent and offspring ramets under different experimental water conditions was examined. We applied four treatments: (1) both the parents and offspring were well watered, (2) both experienced drought, (3) the parents were well watered and the offspring experienced drought, and (4) the parents experienced drought and the offspring were well watered. The growth, chlorophyll content, photosynthesis, chlorophyll fluorescence, and antioxidant enzymes of Zoysia japonica were measured. The chlorophyll contents, chlorophyll a/b ratio, and photosynthetic parameters (Pn, Gs) of offspring ramets were not reduced when parents under well-watered conditions and offspring ramets under drought conditions suggested that the physiological support provided by parents enabled their offspring to maintain photosynthetic efficiencies. The support provided to the offspring by their parents was also obvious in the antioxidant systems and proline accumulation of Zoysia japonica in heterogeneous water environments. Physiological integration was not detected when the parents experienced drought and the offspring were well watered. The parents provided physiological support to their offspring and did not expense significant benefits through physiological integration when parents were well watered and offspring experienced drought. When the opposite conditions were true, physiological integration did not exist. [ABSTRACT FROM AUTHOR]

Transient expression of genes in protoplasts is a versatile technique for rapid functional characterization of genes by assessing protein localization or effector–reporter responses. In addition, protoplasts have been widely used for generating transgenic or gene-edited plants in model or crop plants. Zoysiagrass (Zoysia japonica Steud.) is one of the most economically important turf plants used in many living places or natural fields, but its management is labor-intensive. Therefore, genetic manipulation using valuable genes is highly demanded in zoysiagrass. Although transient expression systems in zoysiagrass facilitates the identification of valuable zoysiagrass genes, transient expression use in the zoysiagrass protoplasts is yet to be reported. Here we describe the methodology and feasibility for transient expression of genes in the zoysiagrass protoplasts isolated from green leaves. We obtained more than 70% of transfection efficiency in the zoysiagrass protoplasts using polyethylene glycol-mediated transfection. Additionally, we showed the feasibility of cellular, biochemical, and molecular approaches using the zoysiagrass protoplasts transiently expressing genes of interest through fluorescent microscopic observation, immunoblot analysis, and luciferase-based promoter activity assay. Along with the genome draft of the Zoysia family, transient expression will be a valuable tool to rapidly investigate gene functions. An initial assessment of gene function using protoplasts facilitate the identification of valuable genes that can improve the economic value of the Z. japonica or its closely related species by gene manipulation. [ABSTRACT FROM AUTHOR]

Zoysia japonica is an important warm-season turfgrass used worldwide. The decreased aesthetic quality and functionality during leaf senescence hamper its further utilization. However, information about the transcriptional mechanism and genes involved in leaf senescence in Z. japonica needs to be more extensive. Therefore, to better understand leaf senescence in Z. japonica, we investigated the integrated analysis of chlorophyll fluorescence test (JIP-test) and RNA sequencing (RNA-seq) of mature and senescent leaves. First, we identified 22,049 genes, of which 4038 were differentially expressed genes (DEGs). The results for gene expression profiles were evaluated using quantitative real-time PCR. A total of 2515 genes have homologous genes in other plants. The matched known-function SAGs are mainly involved in chlorophyll degradation and plant hormone response. A total of 539 differentially expressed transcription factor genes, including AP2/ERF-ERF, NAC, WRKY, bHLH, and MYB, were identified to be associated with leaf senescence. Next, senescence represses chlorophyll biosynthesis while upregulating chlorophyll degradation. Senescence harms the integrity and functionality of PSII, PSI, and the intersystem electron transport chain. In addition, IAA biosynthesis was inhibited, whereas ABA and ET biosynthesis were activated in leaf senescence, and senescence activates signal transduction of IAA, ABA, and ET. These findings add to our understanding of the regulatory mechanism of leaf senescence. The senescence-associated genes are candidate targets for providing new insight into leaf senescence modeling in Z. japonica. They provided a theoretical foundation to reveal the functions of senescence-associated genes and chlorophyll catabolic genes involved in leaf senescence.

How vegetation change caused by ungulates affects other ungulate species has been well studied, yet how grazing the vegetation of the habitat affects a species own food habits is not known. Long‐term studies on the effects of sika deer (Cervus nippon) on vegetation have been performed, but not in the context of the species own food habits. I monitored grassland vegetation and deer food habits on an island in northern Japan for 25 years from 1975 to 2000. During the first decade, deer grazing caused the replacement of Miscanthus sinensis tussocks by a Zoysia japonica sward. Deer diets in 1976 were composed of equal proportions of M. sinensis, Pleioblastus chino, and Z. japonica, but after the 1980s, Z. japonica became their major food source. This is because of the high productivity of Z. japonica afforded by its morphology and physiology, as well as by the warm and humid summer climate of the Japanese archipelago. Although the vegetational changes were gradual, changes in the deer food habits were abrupt and the compositions were predominated by Z. japonica after the first decade. This study showed that an ungulate changed its own diet because of its effect on the vegetation by grazing, and that the changes in vegetation and deer diet were not synchronized over a 25‐year monitoring period. [ABSTRACT FROM AUTHOR]

Pheophytinase (PPH), the phytol hydrolase, plays important roles in chlorophyll degradation. Nevertheless, little attention has been paid to the PPHs in warm-season grass species; neither its detailed function in photosynthesis has been systematically explored to date. In this study, we isolated ZjPPH from Zoysia japonica , an excellent warm-season turfgrass species. Quantitative real-time PCR analysis and promoter activity characterization revealed that the expression of ZjPPH could be induced by senescence, ABA, and dark induction. Subcellular localization observation proved that ZjPPH was localized in the chloroplasts. Overexpression of ZjPPH accelerated the chlorophyll degradation and rescued the stay-green phenotype of the Arabidopsis pph mutant. Moreover, ZjPPH promoted senescence with the accumulation of ABA and soluble sugar contents, as well as the increased transcriptional level of SAG12 and SAG14. Transmission electron microscopy investigation revealed that ZjPPH caused the decomposition of chloroplasts ultrastructure in stable transformed Arabidopsis. Furthermore, chlorophyll a fluorescence transient measurement analysis suggested that ZjPPH suppressed photosynthesis efficiency by mainly suppressing both photosystem II (PSII) and photosystem I (PSI). In conclusion, ZjPPH plays an important role in chlorophyll degradation and senescence. It could be a valuable target for genetic editing to cultivate new germplasms with stay-green performance and improved photosynthetic efficiency. [ABSTRACT FROM AUTHOR]

Copyright of Revista Brasileira de Engenharia Agricola e Ambiental - Agriambi is the property of Revista Brasileira de Engenharia Agricola e Ambiental and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Pheophytinase (PPH), the phytol hydrolase, plays important roles in chlorophyll degradation. Nevertheless, little attention has been paid to the PPHs in warm-season grass species; neither its detailed function in photosynthesis has been systematically explored to date. In this study, we isolated ZjPPH from Zoysia japonica, an excellent warm-season turfgrass species. Quantitative real-time PCR analysis and promoter activity characterization revealed that the expression of ZjPPH could be induced by senescence, ABA, and dark induction. Subcellular localization observation proved that ZjPPH was localized in the chloroplasts. Overexpression of ZjPPH accelerated the chlorophyll degradation and rescued the stay-green phenotype of the Arabidopsis pph mutant. Moreover, ZjPPH promoted senescence with the accumulation of ABA and soluble sugar contents, as well as the increased transcriptional level of SAG12 and SAG14. Transmission electron microscopy investigation revealed that ZjPPH caused the decomposition of chloroplasts ultrastructure in stable transformed Arabidopsis. Furthermore, chlorophyll a fluorescence transient measurement analysis suggested that ZjPPH suppressed photosynthesis efficiency by mainly suppressing both photosystem II (PSII) and photosystem I (PSI). In conclusion, ZjPPH plays an important role in chlorophyll degradation and senescence. It could be a valuable target for genetic editing to cultivate new germplasms with stay-green performance and improved photosynthetic efficiency.

Abstract Zoysiagrass is an important ornamental and turfgrass cultivated in different countries. Recently, damage to this plant, characterized by a type of witch’s broom and stunting were observed in southern Brazil, suggesting possible attack by mites. A detailed examination of these plants showed the presence of a new mite species, Abacarus zoysiae Flechtmann & Tassi sp. nov., as responsible for the damage. The objective of this publication is to describe that new species, based on adult females and males.

Various cultivars of Zoysia japonica Steud. have been developed as genetically modified organisms for commercial purposes. The chloroplast genome was completed to investigate genetic diversity in Z. japonica isolated in Korea. The chloroplast genome of Z. japonica is 135,884 bp long (GC ratio is 38.4%) and has four subregions: 81,378 bp of large single copy (36.3%) and 12,582 bp of small single copy (32.7%) regions are separated by 20,962 bp of inverted repeat (44.1%) regions including 130 genes (83 protein-coding genes (PCGs), eight rRNAs, 38 tRNAs, and one pseudogene). 68 SNPs and 24 INDEL regions were identified from the two Z. japonica chloroplast genomes, similar to those of Z. matrella. 12 SNPs located in PCGs can be good candidates for developing molecular markers of Z. japonica. Based on nucleotide diversity analysis, atpB/rbcL, rpl16/rps3, and rpl32/trnL regions exhibited the high nucleotide diversity, which can be used for developing species-specific marker sequences to distinguish their species. Phylogenetic analysis presented that two Z. japonica and Z. sinica were clustered together, indicating that they have been used for developing cultivars of Z. japonica. [ABSTRACT FROM AUTHOR]

Zoysia japonica is a commonly used turfgrass species around the world. Seed germination is a crucial stage in the plant life cycle and is particularly important for turf establishment and management. Experiments have confirmed that melatonin can be a potential regulator signal in seeds. To determine the effect of exogenous melatonin administration and explore the its potential in regulating seed growth, we studied the concentrations of several hormones and performed a transcriptome analysis of zoysia seeds after the application of melatonin. The total antioxidant capacity determination results showed that melatonin treatment could significantly improve the antioxidant capacity of zoysia seeds. The transcriptome analysis indicated that several of the regulatory pathways were involved in antioxidant activity and hormone activity. The hormones concentrations determination results showed that melatonin treatment contributed to decreased levels of cytokinin, abscisic acid and gibberellin in seeds, but had no significant effect on the secretion of auxin in early stages. Melatonin is able to affect the expression of IAA (indoleacetic acid) response genes. In addition, melatonin influences the other hormones by its synergy with other hormones. Transcriptome research in zoysia is helpful for understanding the regulation of melatonin and mechanisms underlying melatonin-mediated developmental processes in zoysia seeds. [ABSTRACT FROM AUTHOR]

Genetically modified (GM) plants have been mainly advanced for the mass production of agricultural plants. However, there are concerns about GM crops which cause side effects on ecosystems and human beings. Zebrafish is a better in vivo model and possess the advantages; small size, large clutches, transparency, low cost, and physiological similarity to mammals. The objective of this study was to assess the safety of non-GM and GM grasses (Zoysia japonica) including JG21, JG21-MS1, JG21-MS2 in zebrafish embryos by examining the survival rates, heart-beating rate, yolk sac edema size, cell death, and morphology. The survival rates, heart-beating rates, and yolk sac edema sizes in non-GM and GM grass-treated zebrafish were similar change with the control group. And, all the groups did not show a significant difference within less than 1.0-fold in cell death by fluorescence levels. These results indicate that the zebrafish model is a useful and simple tool and can be used for risk assessment of GM plants. [ABSTRACT FROM AUTHOR]

Weed control can be most effectively achieved through the use of herbicide-resistance transgene. A preliminary study bar-transgenic Zoysia japonica posed no serious risk on the unintended escape of the transgene from its cultivation site. The present follow-up investigation of the dispersion of pollen and its short-distance escape outside of the bar-transgenic Zoysia japonica habitats were ascertained in terms of environmental factors affecting anthesis and pollen viability. In a 24-h day cycle, zoysiagrass pollen was released predominantly between 08:00 and 10:00, and the pollen was most viable during the same time interval. Optimal temperature and humidity for pollen viability was 15–20 °C and 80–90%, respectively. The pollen germinated in 120 min after anthesis, but under cloudy conditions germination time doubled. No differences in pollen viability/longevity between the transgenic and non-transgenic plants were observed. The pollen-mediated gene flow of transgenic Zoysia japonica to wild-type non-transgenic zoysiagrass species was monitored by measuring the cross-over rate of the bar gene in the context of three different models. At distances within 5 m, the rate of gene flow ranged from 3 to 5.7% according to the models used. The greater the distance from the transgenic plant site, the lower the gene flow rate. The furthest transgene detected was 38 m away and exhibited a 0.25% gene flow rate. The radial model yielded a 3.7% escape rate within a 3 m radius and was wind direction dependent. The distance- and direction-dependent gene flow events were influenced by wind direction and velocity during flowering season. [ABSTRACT FROM AUTHOR]

With the increasing popularization of football (soccer), new technologies have arrived in Brazil that assist in the maintenance of sports fields, making it possible to increase the quality of turfgrass in both professional and amateur fields. Thus, the revitalization process has become fundamental in sports fields installed with emerald grass to improve game performance and turfgrass development. The objective of this study was to revitalize an amateur sports field in the interior of São Paulo state, Brazil, planted with emerald grass. The conditions of the field were initially assessed and a set of revitalization procedures were carried out from November 2019 to March 2020 to improve turfgrass quality, including: chemical analysis of the soil and liming; mowing of the grass and disposal of clippings; soil decompaction through aeration with solid tines; application of sand (top dressing); pest and weed control; planting of new sod; fertilization and maintenance. Technical training with employees was also conducted for subsequent field maintenance. All tested operations were essential and necessary to improve the aesthetics and quality of the amateur sports field, so that the turfgrass could be revitalized and ready for use in soccer matches.

Zoysia japonica grass has commercial importance to Brazil due to its use in golf courses and football stadiums. The main concern about its commercial cultivation is the unevenness on seed germination as well as the ideal seed harvesting time. Therefore, the objective was to measure the effect of temperature and potassium nitrate on dormancy overcoming, and of substrate and harvesting time on germination of zoysia grass seeds (Zoysia japonica Steud). There were three experiments: 1) temperature and potassium nitrate treatments, with six temperature conditions (constants at 20 ˚C, 25 ˚C, 30 ˚C, and 35 ˚C, and alternating at 20-30 ˚C and 20- 35 ˚C), under absence or presence of potassium nitrate; 2) temperature and substrate treatments, with two temperature conditions (alternating at 20-30 ˚C and 20-35 ˚C) and three types of substrates (on paper, between paper, and on sand); and 3) harvesting time treatments, which seeds were obtained by manual harvesting comprising 11 harvesting periods. Seeds germinated more rapidly on the sand and paper, at the alternating temperature of 20-35 °C. The best period for seed harvesting was from 18 to 19 days after ear emergence in the production field.

Zoysia japonica is a warm-season turfgrass with a good tolerance and minimal maintenance requirements. However, its use in Northern China is limited due to massive chlorophyll loss in early fall, which is the main factor affecting its distribution and utilization. Although ethephon treatment at specific concentrations has reportedly improved stress tolerance and extended the green period in turfgrass, the potential mechanisms underlying this effect are not clear. In this study, we evaluated and analyzed chlorophyll changes in the physiology and transcriptome of Z. japonica plants in response to cold stress (4 °C) with and without ethephon pretreatment. Based on the transcriptome and chlorophyll content analysis, ethephon pretreatment increased the leaf chlorophyll content under cold stress by affecting two processes: the stimulation of chlorophyll synthesis by upregulating ZjMgCH2 and ZjMgCH3 expression; and the suppression of chlorophyll degradation by downregulating ZjPAO, ZjRCCR, and ZjSGR expression. Furthermore, ethephon pretreatment increased the ratio of chlorophyll a to chlorophyll b in the leaves under cold stress, most likely by suppressing the conversion of chlorophyll a to chlorophyll b due to decreased chlorophyll b synthesis via downregulation of ZjCAO. Additionally, the inhibition of chlorophyll b synthesis may result in energy redistribution between photosystem II and photosystem I.

The AP2/ERF (AP2, APETALA2; ERF, ethylene responsive factor) transcription factors regulate various important processes in plants, including growth, development, metabolism and stress responses. In the zoysiagrass genome (Zoysia japonica), 147 AP2/ERF genes were identified, which were classified into three families based on phylogenetic analysis. By analysis of RNA-sequencing data, the expression profiles of 46 members were assessed, and their regulation patterns in response to cold stress were investigated. Meanwhile, a zoysiagrass genetic regulation network (GRN) was reconstructed based on rice interaction data using the homology search method. Two GRN modules containing AP2/ERF transcription factors were characterized as having important functions in exocytosis and sucrose metabolism processes, which suggested that AP2/ERF transcription factors play critical roles in these biological processes, exerting control over plant growth and stress responses. These results provide useful information for future functional analyses of the AP2/ERF family in zoysiagrass.

Zoysia japonica Steud. is a native turfgrass in the Korean peninsula and is used worldwide. Abnormal conditions of soil salinity inhibit plant growth and adversely affect the quality of the turfgrass. AT-hook motif nuclear-localized (AHL) family proteins known to co-regulate the transcription of genes as a chromatin remodeling factor play a role in plant developmental processes and stress responses. In this study, AT-Hook Protein of Genomine 1 (ATHG1)/AT-Hook motif nuclear-Localized protein 23 (AHL23) and AT-hook Protein of Genomine 3 (ATPG3)/AT-Hook motif nuclear-Localized protein 20 (AHL20), two AHL genes from A. thaliana, were transformed into Z. japonica under the control of a constitutive ubiquitin promoter. Southern blot analysis proved that ATHG1/AHL23 and ATPG3/AHL20 were introduced into the ATHG1/AHL23-transgenic plants and ATPG3/AHL20-transgenic plants, respectively. Overexpression of each ATHG1/AHL23 and ATPG3/AHL20 in all of the transgenic plants was confirmed by quantitative real-time PCR (qRT-PCR). To evaluate a tolerant response to salt stress of the transgenic plants, 4 transgenic plants including ATHG1-overexpressing line 1 (ATHG1-OE1) and ATHG1-OE2, and ATPG3-overexpressing line 1 (ATPG3-OE1) and ATPG3-OE2 were selected, respectively. All the transgenic plants showed higher salt-tolerant phenotype with higher chlorophyll and lower malondialdehyde (MDA) contents under salt treatment, compared to the wild types. Also, under salt treatment, the transgenic plants revealed higher activities of catalase (CAT) and peroxidase (POD), reactive oxygen species (ROS)-scavenging enzymes, than those of the wild-type plant. These results suggest that overexpression of ATHG1/AHL23 or ATPG3/AHL20 belonging to the AHL gene family confers salt tolerance to the transgenic zoysiagrass plants. [ABSTRACT FROM AUTHOR]

Key message: ZjICE2 works as a positive regulator in abiotic stress responses and ZjICE2 is a valuable genetic resource to improve abiotic stress tolerance in the molecular breeding program of Zoysia japonica. The basic helix-loop-helix (bHLH) family transcription factors (TFs) play an important role in response to biotic or abiotic stresses in plants. However, the functions of bHLH TFs in Zoysia japonica, one of the warm-season turfgrasses, remain poorly understood. Here, we identified ZjICE2 from Z. japonica, a novel MYC-type bHLH transcription factor that was closely related to ICE homologs in the phylogenetic tree, and its expression was regulated by various abiotic stresses. Transient expression of ZjICE2-GFP in onion epidermal cells revealed that ZjICE2 was a nuclear-localized protein. Also, ZjICE2 bound the MYC cis-element in the promoter of dehydration responsive element binding 1 of Z. japonica (ZjDREB1) using yeast one-hybrid assay. A phenotypic analysis showed that overexpression of the ZjICE2 in Arabidopsis enhanced tolerance to cold, drought, and salt stresses. The transgenic Arabidopsis and Z. japonica accumulated more transcripts of cold-responsive DREB/CBFs and their downstream genes than the wild type (WT) after cold treatment. Furthermore, the transgenic plants exhibited an enhanced Reactive oxygen species (ROS) scavenging ability, which resulted in an efficient maintenance of oxidant–antioxidant homeostasis. In addition, overexpression of the ZjICE2 in Z. japonica displayed intensive cold tolerance with increases in chlorophyll contents and photosynthetic efficiency. Our study suggests that ZjICE2 works as a positive regulator in abiotic stress responses and the ICE-DREB/CBFs response pathway involved in cold stress tolerance is also conserved in Z. japonica. These results provide a valuable genetic resource for the molecular breeding program especially for warm-season grasses as well as other leaf crop plants. [ABSTRACT FROM AUTHOR]

Phytoene synthase (PSY) is a key limiting enzyme in the carotenoid biosynthesis pathway for regulating phytoene synthesis. In this study, ZjPSY was isolated and identified from Zoysia japonica, an important lawn grass species. ZjPSY cDNA was 1230 bp in length, corresponding to 409 amino acids. ZjPSY showed higher expression in young leaves and was downregulated after GA3, ABA, SA, and MeJA treatments, exhibiting a sensitivity to plant hormones. Regulatory elements of light and plant hormone were found in the upstream of ZjPSY CDS. Expression of ZjPSY in Arabidopsis thaliana protein led to carotenoid accumulation and altered expression of genes involved in the carotenoid pathway. Under no-treatment condition, salt treatment, and drought treatment, transgenic plants exhibited yellowing, dwarfing phenotypes. The carotenoid content of transgenic plants was significantly higher than that of wild-type under salt stress and no-treatment condition. Yeast two-hybrid screening identified a novel interacting partner ZjJ2 (DNAJ homologue 2), which encodes heat-shock protein 40 (HSP40). Taken together, this study suggested that ZjPSY may affect plant height and play an important role in carotenoid synthesis. These results broadened the understanding of carotenoid synthesis pathways and laid a foundation for the exploration and utilization of the PSY gene.

Inadequate reference databases in RNA-seq analysis can hinder data utilization and interpretation. In this study, we have successfully constructed a high-quality reference transcript dataset, ZjRTD1.0, for Zoysia japonica, a widely-used turfgrass with exceptional tolerance to various abiotic stress, including low temperatures and salinity. This dataset comprises 113,089 transcripts from 57,143 genes. BUSCO analysis demonstrates exceptional completeness (92.4%) in ZjRTD1.0, with reduced proportions of fragmented (3.3%) and missing (4.3%) orthologs compared to prior datasets. ZjRTD1.0 enables more precise analyses, including transcript quantification and alternative splicing assessments using public datasets, which identified a substantial number of differentially expressed transcripts (DETs) and differential alternative splicing (DAS) events, leading to several novel findings on Z. japonica's responses to abiotic stresses. First, spliceosome gene expression influenced alternative splicing significantly under abiotic stress, with a greater impact observed during low-temperature stress. Then, a significant positive correlation was found between the number of differentially expressed genes (DEGs) encoding protein kinases and the frequency of DAS events, suggesting the role of protein phosphorylation in regulating alternative splicing. Additionally, our results suggest possible involvement of serine/arginine-rich (SR) proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs) in generating inclusion/exclusion isoforms under low-temperature stress. Furthermore, our investigation revealed a significantly enhanced overlap between DEGs and differentially alternatively spliced genes (DASGs) in response to low-temperature stress, suggesting a unique co-regulatory mechanism governing transcription and splicing in the context of low-temperature response. In conclusion, we have proven that ZjRTD1.0 will serve as a reliable and useful resource for future transcriptomic analyses in Z. japonica., (© 2024 Scandinavian Plant Physiology Society.)

Copyright of Acta Prataculturae Sinica is the property of Acta Prataculturae Sinica Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Numerous genes are known to regulate the cold response in plants. The ICE1 gene is a regulator of coldinduced transcriptome playing an important role in cold tolerance in Arabidopsis thaliana. Here, the Arabidopsis ICE1 gene was cloned and transformed into A. thaliana and Korean lawn grass (Zoysia japonica) respectively, under the control of a maize ubiquitin promoter. The transgenic Arabidopsis plants showed higher survival rate, less MDA content, and higher levels of proline, SOD, and POD after freezing treatment. In the case of Z. japonica, 43 transgenic plants were produced by Agrobacterium-mediated transformation. Southern blot analysis showed that 9 transgenic lines selected by phosphinothricin (PPT) were transformed with ICE1 independently. Cold treatment significantly induced the transcripts abundant in the up-regulated ZjCBF and ZjDREB1 in transgenic lines OE-18 and OE-19. Meanwhile, transgenic plants showed increased proline level but decreased MDA content and with higher activities of SOD and POD compared with wild type under cold stress. These results suggest that overexpression of the ICE1 gene could improve the antioxidative capacity in conferring cold tolerance to the transgenic zoysiagrass plants. [ABSTRACT FROM AUTHOR]

Growing evidence indicates that some grass species are more tolerant to various abiotic and biotic stresses than many crops. Zinc finger proteins play important roles in plant abiotic and biotic stresses. Although genes coding for these proteins have been cloned and identified in various plants, their function and underlying transcriptional mechanisms in the halophyte Zoysia japonica are barely known. In the present study, ZjZFN1 was isolated from Z. japonica using RACE method. Quantitative real time PCR results revealed that the expression of ZjZFN1 was much higher in leaf than in root and stem tissues, and induced by salt, cold or ABA treatment. The subcellular localization assay demonstrated that ZjZFN1 was localized to the nucleus. Expression of the ZjZFN1 in Arabidopsis thaliana improved seed germination and enhanced plant adaption to salinity stress with improved percentage of green cotyledons and growth status under salinity stress. Physiological and transcriptional analyses suggested that ZjZFN1 might, at least in part, influence reactive oxygen species accumulation and regulate the transcription of salinity responsive genes. Furthermore, RNA-sequencing analysis of ZjZFN1-overexpressing plants revealed that ZjZFN1 may serve as a transcriptional activator in the regulation of stress responsive pathways, including phenylalanine metabolism, α-linolenic acid metabolism and phenylpropanoid biosynthesis pathways. Taken together, these results provide evidence that ZjZFN1 is a potential key player in plants’ tolerance to salt stress, and it could be a valuable gene in Z. japonica breeding projects.

Graphical abstract Highlights • ZjDREB1.4 is belongs to a DREB1 branch in the grass family that is more highly represented in warm-season species. • The over-expression of ZjDREB1.4 in Arabidopsis induced the expression of multiple genes including a subset of the CBF regulon. • Plants over-expressing ZjDREB1.4 showed enhanced tolerance to high and freezing temperature stresses with only a few days delay in bolting. Abstract The DREB (dehydration-responsive element binding) protein family comprises transcription factors that can increase the survivability of a plant under abiotic stresses by regulating expression of multiple genes and altering downstream metabolism at the cost of growth retardation and developmental delay. In this study, a gene for the DREB1-type transcription factor, designated ZjDREB1.4 , was isolated from zoysiagrass (Zoysia japonica Steud.), a popular warm-season turfgrass. This gene contains a conserved AP2/ERF DNA-binding domain flanking the signature sequence of DREB1 and belongs to a DREB1 branch in the grass family that expands in the warm-season species. The expression of ZjDREB1.4 was significantly induced by chilling stress (4–15 °C), moderately induced by salt stress, and only slightly induced by drought stress. The product of ZjDREB1.4 was targeted to the nucleus and showed strong transactivation activity but weak binding to the DRE with ACCGAC as the core sequence. The ZjDREB1.4 protein bound to GCCGAC more preferentially than to ACCGAC. Overexpression of ZjDREB1.4 in Arabidopsis induced the expression of multiple genes including a part of the CBF-regulon, and moderately increased the levels of proline and soluble sugars under normal growth conditions. The transgenic Arabidopsis plants showed an increase in tolerance to high and freezing temperature stresses without obvious growth inhibition and with only a few days delay in bolting. ZjDREB1.4 is potentially useful for producing transgenic plants that are tolerant to high temperature and/or cold stresses with few negative effects. [ABSTRACT FROM AUTHOR]