Expression of a Chlorophyll b Reductase Gene from Zoysia japonica Causes Changes in Leaf Color and Chlorophyll Morphology in Agrostis stolonifera

The NYC-like (NOL) enzyme is considered as an essential enzyme for chlorophyll b degradation, which catalyzes the formation of 7-hydroxymethyl chlorophyll a from chlorophyll b. The ZjNOL gene was cloned from Zoysia japonica with a completed coding sequence of 981-bp in length, encoding 326 amino acids. ZjNOL was localized on the stroma side of the thylakoid membrane, and co-localized with ZjNYC in the chloroplasts. Multiple photoregulatory elements and hormone regulatory elements were identified in the promoter region of the ZjNOL gene, and the expression level of the ZjNOL gene was dramatically up-regulated in senescence leaves, which were regulated by a variety of plant hormones. ZjNOL’s ectopic expression in creeping bentgrass produced yellow leaves, thicker cortex, and smaller vascular column cells. Additionally, transgenic plants exhibited morphological alterations in their chloroplast structure, and the number of grana and thylakoids per grana stack reduced dramatically. Transgenic plants also had a lower photosynthetic rate and Fm/Fv than the control. The transgenic plants displayed a decreased chlorophyll content and a greater rate of ion leakage. The properties and activities of ZjNOL will serve as a foundation for future research into gene functions and regulatory processes.


Introduction
Leaf senescence is a dynamic process in which plants recover nutrients from senescent leaves. Leaf yellowing is a clear indicator of leaf senescence, due to chlorophyll breakdown and carotenoid color exposure. During senescence, cells must degrade free chlorophyll and its color-decomposed metabolites quickly in order to escape photooxidation toxicity [1]. In higher plants, chlorophyll is a component of the polysubunit thylakoid membrane protein complex, and it occurs in two forms, chlorophyll a (Chl a) and chlorophyll b (Chl b). Chl a is found in all chlorophyll protein complexes, but Chl b is found only in the lightharvesting complex I and II (LHCI and LHCII), which make up the photosystem I complex and photosystem II complex, respectively [2]. LHCII is mostly found in the grana in the thylakoid membrane's accumulation area, where it plays a critical role in the creation and maintenance of grana accumulation [3]. Chlorophyll degradation enzymes (CCEs) have been found to interact with photosynthetic organs. During leaf senescence, LHCII can interact with five chlorophyll-degrading enzymes (NYC, NOL, PPH, PAO, and RCCR) [4]. Chlorophyll b is converted to chlorophyll a via a two-step process involving chlorophyll b reductase and 7-hydroxymethyl chlorophyll a reductase (HCAR) (Figure 1) [5]. NYC (non-yellow coloring) and NOL (NYC1-like) are two distinct subtypes of chlorophyll  [5]. HMChl a, 7-hydroxymethyl chlorophyll a; HCAR, chlorophyll a reductase; NYE, non-yellowing 1; PPH, pheophytinase; PAO, pheophorbide a oxygenase; RCCR, red chlorophyll catabolite reductase; RCC, red chlorophyll catabolite; pFCC, primary blue-fluorescent chlorophyll catabolite; FCCs, fluorescent chlorophyll catabolites; NCCs, nonfluorescent chlorophyll catabolites; Pheide a, pheophorbide a; Phein a, Pheophytin a.
In Arabidopsis thaliana, overexpressed NOL plants had a considerably lower chlorophyll content than wild-type plants, and photosystem II had a reduced antenna size, indicating that NOL was engaged in photosystem II antenna size control [9]. A phylogenetic study of the rice (Oryza sativa) OsNOL gene revealed that the closest protein to OsNOL was OsNYC1. Rice nol mutants exhibited a phenotype similar to that of the nyc1 mutant, with significant suppression of chlorophyll b degradation. Additionally, LHCII was preserved selectively throughout senescence and thylakoid grana were retained even at a late stage of senescence [10]. The LpNOL RNA interference (NOLi) in perennial ryegrass (Lolium perenne) dramatically suppressed chlorophyll breakdown during leaf senescence and altered the leaf color of perennial ryegrass, implying that NOL functions as a chlorophyll b reductase and is also associated with the perennial herb's middle color phenotype [11].
Zoysia japonica is a typical warm-season turfgrass found around the world. Due to its drought and salt resistance, it is commonly utilized on golf courses, sports fields, and urban greening. Creeping bentgrass (Agrostis stolonifera) is a significant cool-season grass species that is extensively used on golf courses as turfgrass [12]. While the genes involved in chlorophyll breakdown have been identified, their regulatory roles in plant growth and development remain unknown. In this study, the ZjNOL gene from Zoysia japonica was isolated and identified, and its ectopic expression and functional studies in creeping bentgrass were carried out to gain a better understanding of the NOL gene's function and to lay the groundwork for future research on its role in chlorophyll degradation, growth, and development.

The Isolation and Analysis of the Gene ZjNOL
The DNA fragment comprising the ZjNOL coding domain sequence (CDS) was cloned and ligated into cloning vector pMD19-T, according to the Zoysia Genome Database [13]. The ZjNOL gene has a 981-bp ORF that codes for a 326-amino-acid protein, which belongs to the SDR superfamily. The molecular weight of ZjNOL is 26.62 kD, and the theoretical isoelectric point is 10.76. ZjNOL had 12 exons (Supplementary Figure S1).
The upstream sequence was used to investigate the cis-regulatory elements of ZjNOL. Multiple photoresponsive elements, such as BOX4, G-box, Sp1, and GT1 motif, are found in the upstream region of ZjNOL ( Figure 2). Several hormone-related response elements (ABRE, AUXRR-core, P-box CGTCA-motif, TGACG-motif, TCA-element) are found, which are involved in the pathways of abscisic acid (ABA), 3-indoleacetic acid (IAA), gibberellin (GA), methyl jasmonate (MeJA), and salicylic acid (SA). In addition, various cis-acting elements have been found that are involved in growth and development as well as stress tolerance, such as ARE, MBS, and non-Box.

ZjNOL Expression Profiles and Expression Analysis of AsZDS, AsNCED, AsCLH1 and AsNYC Induced by Dark
Real-time PCR was used to detect ZjNOL expression patterns in various tissues and developmental phases. The results revealed that ZjNOL was expressed in root, stem, and leaf tissues, with leaf expression being substantially higher (Figure 3a). Among all the de-

ZjNOL Expression Profiles and Expression Analysis of AsZDS, AsNCED, AsCLH1 and AsNYC Induced by Dark
Real-time PCR was used to detect ZjNOL expression patterns in various tissues and developmental phases. The results revealed that ZjNOL was expressed in root, stem, and leaf tissues, with leaf expression being substantially higher (Figure 3a). Among all the detected tissues, ZjNOL was expressed highest in mature leaves, which was 9.67 and 12.61 times of those in young and fast-growing leaves, respectively (Figure 3b). ZjNOL expression was up-regulated by ABA or SA (Figure 3c-f). The level of ZjNOL expression was reduced after MeJA treatment (Figure 3c-f). The expression level of ZjNOL rose and then dropped after gibberellic acid (GA3) therapy. The expression of ZjNOL dropped initially and then rose in response to salt and drought treatments. ZjNOL may be involved in leaf growth, stress, and hormone response.  To understand the role of ZjNOL in plant growth and development, the expression profile of AsZDS (Z-carotene desaturase), AsNCED (9-cis-epoxyoid dioxygenase), As-CLH1 (chlorophyllase 1), and AsNYC (non-yellow coloring) after dark treatment was studied ( Figure 4). AsNYC and AsCLH1 were confirmed to play important roles in the degra- To understand the role of ZjNOL in plant growth and development, the expression profile of AsZDS (Z-carotene desaturase), AsNCED (9-cis-epoxyoid dioxygenase), AsCLH1 (chlorophyllase 1), and AsNYC (non-yellow coloring) after dark treatment was studied ( Figure 4). AsNYC and AsCLH1 were confirmed to play important roles in the degradation of chlorophyll [10,14]. AsZDS and AsNCED were reported to be involved in carotenoid metabolism [15,16]. After dark treatment, AsZDS did not change in the control plants, but in OE plants, the expression level of AsZDS increased from the fifth day, reaching three times of that in the first day. AsNCED in OE plants had a tendency of first reducing and then increasing, whereas it exhibited a trend of first increasing and then decreasing in the control plants. The expression pattern of AsCLH1 was similar in the control and OE plants. Under dark treatment, AsNYC expression in the control remained stable; however, AsNYC expression in OE had a declining trend.  The control (grey) and OE (white) columns were separately compa using one-way ANOVA with Duncan test. Significant differences (p < 0.05, n = 3) are shown different letters above the columns.

Analysis of the Protein ZjNOL
A modelled 3D tertiary structure analysis demonstrated that there was a nega potential area on the surface of the ZjNOL protein, primarily near the helix structure ( ure 5a,b). ZjNOL has a dinucleotide binding motif (TGXXXGXG) and SDR catalytic (YXXXK) at the position of amino acids 88 and 211 (Figure 5b). , AsCLH1 (c), AsNYC (d) in control and OE subjected to dark at 0-7 days. The control (grey) and OE (white) columns were separately compared using one-way ANOVA with Duncan test. Significant differences (p < 0.05, n = 3) are shown by different letters above the columns.

Analysis of the Protein ZjNOL
A modelled 3D tertiary structure analysis demonstrated that there was a negative potential area on the surface of the ZjNOL protein, primarily near the helix structure (Figure 5a,b). ZjNOL has a dinucleotide binding motif (TGXXXGXG) and SDR catalytic site (YXXXK) at the position of amino acids 88 and 211 (Figure 5b).
NOL proteins were phylogenetically analyzed using the neighbor-joining method and protein sequences from various species obtained from the NCBI database (Supplementary Figure S2A,B). The phylogenetic tree reflects the valid taxonomy of the in-cluded species. ZjNOL has the most homology with NOL of Zea mays and all the other monocots above them. Although AtNOL does not have the highest resemblance with AtNOL from Arabidopsis thaliana, their motif architectures are similar (Supplementary Figure S2B). using one-way ANOVA with Duncan test. Significant differences (p < 0.05, n = 3) are shown by different letters above the columns.

Analysis of the Protein ZjNOL
A modelled 3D tertiary structure analysis demonstrated that there was a negative potential area on the surface of the ZjNOL protein, primarily near the helix structure (Figure 5a,b). ZjNOL has a dinucleotide binding motif (TGXXXGXG) and SDR catalytic site (YXXXK) at the position of amino acids 88 and 211 ( Figure 5b). NOL proteins were phylogenetically analyzed using the neighbor-joining method and protein sequences from various species obtained from the NCBI database (Supplementary Figure S2A,B). The phylogenetic tree reflects the valid taxonomy of the included species. ZjNOL has the most homology with NOL of Zea mays and all the other monocots above them. Although AtNOL does not have the highest resemblance with AtNOL from Arabidopsis thaliana, their motif architectures are similar (Supplementary Figure S2B).

Subcellular Localization of ZjNOL
After agrobacterium-mediated tobacco leaf infection, yellow fluorescent protein (YFP) fluorescence signal overlapped with chlorophyll self-fluorescence in the chloroplasts in the leaf tissue with transient expression of YFP and ZjNOL fusion protein, and the fluorescence signal was also found in the cytoplasm (Figure 6a-j). As the control, untargeted YFP was localized in the whole cell. According to subcellular localization assays, ZjNOL was mostly localized in the chloroplast and cytoplasm. ZjNOL was not equally distributed inside the chloroplast (Figure 6i,j).

Subcellular Localization of ZjNOL
After agrobacterium-mediated tobacco leaf infection, yellow fluorescent protein (YFP) fluorescence signal overlapped with chlorophyll self-fluorescence in the chloroplasts in the leaf tissue with transient expression of YFP and ZjNOL fusion protein, and the fluorescence signal was also found in the cytoplasm (Figure 6a-j). As the control, untargeted YFP was localized in the whole cell. According to subcellular localization assays, ZjNOL was mostly localized in the chloroplast and cytoplasm. ZjNOL was not equally distributed inside the chloroplast (Figure 6i,j).

ZjNOL Interacts with ZjNYC
For visualization, the conformation of ZjNOL and ZjNYC proteins with the highest negative energy was chosen. ZjNOL had a binding score of −310.52 kcal/mol to ZjNYC protein, and its binding sites comprised Arg-298, Tyr-306, Lys-304, Lys-223, Gln-230, Tyr-296, and other amino acid residues. Glu-359, Ser-355, Asp-289, Ser-344, Cys-347, Arg-308 and other amino acid residues are among the binding sites of ZjNYC protein. The contact residues of ZjNYC and ZjNOL proteins can create a number of interactions, including salt bridges, hydrogen bonds, hydrophobic interactions, and others ( Figure 7a). These interactions can help to increase the stability of the ZjNYC and ZjNOL protein complex. Furthermore, the binding surface diagrams of the two proteins revealed that the ZjNOL protein surface matched well with the ZjNYC protein surface, facilitating the creation of stable binding (Figure 7a).
The connection between ZjNOL and ZjNYC proteins was investigated using yeast two-hybrid and bimolecular fluorescence complementation (BiFC) methods. The results showed that the yeast cells pGADT7-ZjNYC and PGBKT7-ZjNOL developed normally and appeared blue on the QDO/X/A plate, indicating that they can interact in the yeast cell ( Figure 7b). ZjNOL and ZjNYC were fused to the C-terminal and N-terminal of YFP, respectively, and were expressed in tobacco leaf cells by co-transformation. After 3-day culture in the dark, a strong YFP signal was found in the chloroplasts and cytoplasm, showing how the two proteins physically interacted in living plant cells. These findings confirmed that ZjNOL interacts with the ZjNYC protein.

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R PEER REVIEW 8 of 20 The connection between ZjNOL and ZjNYC proteins was investigated using yeas two-hybrid and bimolecular fluorescence complementation (BiFC) methods. The results showed that the yeast cells pGADT7-ZjNYC and PGBKT7-ZjNOL developed normally and appeared blue on the QDO/X/A plate, indicating that they can interact in the yeas cell ( Figure 7b). ZjNOL and ZjNYC were fused to the C-terminal and N-terminal of YFP respectively, and were expressed in tobacco leaf cells by co-transformation. After 3-day culture in the dark, a strong YFP signal was found in the chloroplasts and cytoplasm showing how the two proteins physically interacted in living plant cells. These findings confirmed that ZjNOL interacts with the ZjNYC protein.

Ectopic Expression of ZjNOL Resulted in Leaf Yellowing and Sluggish Development
Transgenic creeping bentgrass with ZjNOL was obtained by the Agrobacterium tumefaciens mediated method. The control group was named control, while the ZjNOL ectopic overexpression creeping bentgrass was named OE. The transgenic plants had yellow leaves, indicating that ectopic expression of ZjNOL may result in a reduction in chlorophyll concentration. Additionally, the root length of OE was less than that of the control. Those findings imply that ZjNOL has negative effect on the development of cholophyll biosynthesis and leaf color (Figure 8).
Transgenic creeping bentgrass with ZjNOL was obtained by the Agrobacterium tumefaciens mediated method. The control group was named control, while the ZjNOL ectopic overexpression creeping bentgrass was named OE. The transgenic plants had yellow leaves, indicating that ectopic expression of ZjNOL may result in a reduction in chlorophyll concentration. Additionally, the root length of OE was less than that of the control. Those findings imply that ZjNOL has negative effect on the development of cholophyll biosynthesis and leaf color ( Figure 8).

Morphological Changes of Transgenic Plants
Ferro green was used to stain the leaves of the control and transgenic plants, while ferro red was used to stain the cell walls, which were rich in cellulose. In the leaves of the transgenic plants, cortex tissue thickness and cell length were increased. The cortex of transgenic plants was thicker and the vascular column cells were smaller than those in the

Morphological Changes of Transgenic Plants
Ferro green was used to stain the leaves of the control and transgenic plants, while ferro red was used to stain the cell walls, which were rich in cellulose. In the leaves of the transgenic plants, cortex tissue thickness and cell length were increased. The cortex of transgenic plants was thicker and the vascular column cells were smaller than those in the control (Figure 9). In the cross sections of the control and transgenic stems, the average length of vascular column cells was 54.52 µm and 31.71 µm, respectively. Additionally, the average pith cell length of the control and transgenic plants was 27.14 µm and 17.34 µm, respectively (Supplementary Figure S3). The leaves and stems of the transgenic plants were more likely to be colored green by ferro solid green staining in the cross sections, indicating that they had developed more cellulose in their cell walls.  Figure S3). The leaves and stems of the transgenic plants were more likely to be colored green by ferro solid green staining in the cross sections, indicating that they had developed more cellulose in their cell walls.

Expression of ZjNOL Gene Results in Chloroplast Morphological Changes
The chloroplasts of OE shifted from a typical spindle shape to an ellipsoidal shape. In comparison to the chloroplasts of the control, the length and width of chloroplasts in OE grew shorter and wider. Furthermore, the average number of grana stacks (GS) in OE cells was significantly lower than those in the control cells ( Figure 10). The grana in the chloroplasts of the control leaf cells were well developed and highly stacked, with an average of 13.85 thylakoids per grana layer (Figure 10c). In the chloroplasts of OE, the

Expression of ZjNOL Gene Results in Chloroplast Morphological Changes
The chloroplasts of OE shifted from a typical spindle shape to an ellipsoidal shape. In comparison to the chloroplasts of the control, the length and width of chloroplasts in OE grew shorter and wider. Furthermore, the average number of grana stacks (GS) in OE cells was significantly lower than those in the control cells ( Figure 10). The grana in the chloroplasts of the control leaf cells were well developed and highly stacked, with an average of 13.85 thylakoids per grana layer (Figure 10c). In the chloroplasts of OE, the thylakoid network was plainly incomplete. The number of GS in chloroplasts was reduced to 4-14, and each GS layer contained an average of 6.45 thylakoids (Figure 10f). Some thylakoids developed into long bundles of pseudograna, resulting in essentially the unstacking of the grana and an incompact thylakoid structure that was loose in the chloroplast of OE leaves.

Net Photosynthetic Rate, Fv/Fm, Chlorophyll Content, Membrane Ion Leakage of Control and OE
The leaves of OE plants had lower net photosynthetic efficiency and Fv/Fm values than the control plants. The net photosynthetic rate of the control and OE plants declined by 75.91% and 86.83%, respectively, after 9 days of dark treatment ( Figure 11). The Fv/Fm values of the control and OE plant reduced by 10.86% and 18.72%, respectively. After 8 days of dark treatment, the chlorophyll content of the OE reduced by 75.90%, whereas the control only decreased by 62.13%. Furthermore, OE plants demonstrated a higher rate of ion leakage. This meant that following shading, the photosynthetic level of OE plants reduced faster.

Hormone Content
There were no significant variations in the hormone content of ABA, IPA, and GA7 between the control and OE plants ( Figure 12). The IAA and SA concentrations in OE were 5.89 and 4.60 times more than in the control, respectively. Simultaneously, the MeJA contents decreased drastically in the transgenic plants, only 4% of the control.

Hormone Content
There were no significant variations in the hormone content of ABA, IPA, and GA7 between the control and OE plants ( Figure 12). The IAA and SA concentrations in OE were 5.89 and 4.60 times more than in the control, respectively. Simultaneously, the MeJA contents decreased drastically in the transgenic plants, only 4% of the control.

The Transcriptome Analysis
Transcriptome sequencing was applied and a total of 296 differentially expressed genes (DEGs) was identified between the transgenic and control samples, of which 133 were up-regulated and 163 were down-regulated. For example, senily-associated protein gene DIN1 (C153676.graph_c0) and leucine tRNA ligase gene (C163435. Graph_c1) were significantly up-regulated. NADH-ubiquinone oxidoreductase chain 4 gene (c152156.graph_c1), pheophytinase gene (c159122.graph_c0) in the chloroplast part (GO:0044434), chloroplast stroma (GO:0009570), chloroplast thylakoid membrane (GO:0009535) and chloroplast thylakoid (GO:0009534) were significantly down-regulated. The up-regulated DEGs were assigned into 28 pathways and the most enriched pathways were "starch and sucrose metabolism" and "plant hormone signal transduction". Eight pathways were enriched for down-regulated DEGs, with "MAPK signaling pathway" being the most enriched ( Figure 13). A total of ten genes were selected for quantitative RT-PCR assays. The expression patterns of the genes acquired from the RNA-seq data were compared to those obtained from qRT-PCR, and the findings show that the expression trends of these 10 genes in RT-qPCR were compatible with those found by RNA-Seq analysis (Supplementary Table S1).

The Transcriptome Analysis
Transcriptome sequencing was applied and a total of 296 differentially express genes (DEGs) was identified between the transgenic and control samples, of which 1 were up-regulated and 163 were down-regulated. For example, senily-associated prote gene DIN1 (C153676.graph_c0) and leucine tRNA ligase gene (C163435. Graph_c1) we significantly up-regulated. NADH-ubiquinone oxidoreductase chain 4 ge (c152156.graph_c1), pheophytinase gene (c159122.graph_c0) in the chloroplast p (GO:0044434), chloroplast stroma (GO:0009570), chloroplast thylakoid membra (GO:0009535) and chloroplast thylakoid (GO:0009534) were significantly down-regulate The up-regulated DEGs were assigned into 28 pathways and the most enriched pathwa were "starch and sucrose metabolism" and "plant hormone signal transduction". Eig pathways were enriched for down-regulated DEGs, with "MAPK signaling pathway" b ing the most enriched ( Figure 13). A total of ten genes were selected for quantitative R PCR assays. The expression patterns of the genes acquired from the RNA-seq data we compared to those obtained from qRT-PCR, and the findings show that the expressi trends of these 10 genes in RT-qPCR were compatible with those found by RNA-Seq an ysis (Supplementary Table S1).

Discussion
The ZjNOL gene encodes a SDR superfamily protein. The SDR family contains a significant number of proteins that are split into 464 groups [17]. SDR proteins have a wide range of functions and low consistency, typically between 20% and 30% [18]. The SDR family of proteins are all based on the Rossmann-fold structure, with an N-terminal dinucleotide cofactor binding motif and an active site with a catalytical residue motif [19]. The three-dimensional structure prediction revealed that ZjNOL protein contains Rossmannfold, a structure that the central β folded and surrounded by three α helixes. At amino acid 80, ZjNOL has the dinucleotide binding motif (TGXXXGXG) and SDR catalytic site (YXXXK) at amino acids 88 and 211. Dinucleotide binding motifs can act as a coenzyme binding region's binding motif [20]. It is less likely that the ZjNOL protein's N-terminal region contains a transmembrane domain (Supplementary Figure S4). To determine ZjNOL's intracellular localization, we fused YFP to ZjNOL and performed subcellular localization of tobacco. The results indicated that ZjNOL was found in the stromal side of the thylakoid membrane. In addition, the heterologous expression of NOL may impact plant growth and development, which may be a result of the lower photosynthetic efficiency resulting from chlorophyll degradation, which requires further investigation [9,11].
Proteins in the SDR family commonly form dimers or tetramers [19]. Studies in rice confirmed that NYC1 and NOL may form a complex and function as chlorophyll b reductase ( Figure 14) [10]. Only until Chl b is converted to HMChl a by the NYC-NOL complex can LHCII be degraded by protease, and then chlorophyllase can hydrolyze chlorophyll

Discussion
The ZjNOL gene encodes a SDR superfamily protein. The SDR family contains a significant number of proteins that are split into 464 groups [17]. SDR proteins have a wide range of functions and low consistency, typically between 20% and 30% [18]. The SDR family of proteins are all based on the Rossmann-fold structure, with an Nterminal dinucleotide cofactor binding motif and an active site with a catalytical residue motif [19]. The three-dimensional structure prediction revealed that ZjNOL protein contains Rossmann-fold, a structure that the central β folded and surrounded by three α helixes. At amino acid 80, ZjNOL has the dinucleotide binding motif (TGXXXGXG) and SDR catalytic site (YXXXK) at amino acids 88 and 211. Dinucleotide binding motifs can act as a coenzyme binding region's binding motif [20]. It is less likely that the ZjNOL protein's N-terminal region contains a transmembrane domain (Supplementary Figure S4). To determine ZjNOL's intracellular localization, we fused YFP to ZjNOL and performed subcellular localization of tobacco. The results indicated that ZjNOL was found in the stromal side of the thylakoid membrane. In addition, the heterologous expression of NOL may impact plant growth and development, which may be a result of the lower photosynthetic efficiency resulting from chlorophyll degradation, which requires further investigation [9,11].
Proteins in the SDR family commonly form dimers or tetramers [19]. Studies in rice confirmed that NYC1 and NOL may form a complex and function as chlorophyll b reductase ( Figure 14) [10]. Only until Chl b is converted to HMChl a by the NYC-NOL complex can LHCII be degraded by protease, and then chlorophyllase can hydrolyze chlorophyll produced from LHCII. In addition, the degradation of LHCII promoted the degradation of the grana structure and thylakoid (Figures 10 and 14). The findings of the ZjNOL and ZjNYC interaction research revealed that the two proteins were able to develop stable binding and maintain good matching. In this study, the BiFC and transmission electron microscopy results revealed that ZjNYC and ZjNOL co-locate and the ectopic expression of ZjNOL may also induce the deterioration of the grana structure and thylakoid. In transgenic plants, the vascular bundle cells shrank considerably, which may be connected to morphological changes caused by chlorophyll degradation. produced from LHCII. In addition, the degradation of LHCII promoted the degradation of the grana structure and thylakoid (Figures 10 and 14). The findings of the ZjNOL and ZjNYC interaction research revealed that the two proteins were able to develop stable binding and maintain good matching. In this study, the BiFC and transmission electron microscopy results revealed that ZjNYC and ZjNOL co-locate and the ectopic expression of ZjNOL may also induce the deterioration of the grana structure and thylakoid. In transgenic plants, the vascular bundle cells shrank considerably, which may be connected to morphological changes caused by chlorophyll degradation. Figure 14. NOL regulates the degradation of chlorophyll and LHCII during leaf senescence [10].
Although the promoter element analysis revealed that the ZjNOL promoter contains cis-elements linked with ABA, IAA, GA, MeJA, and SA, the hormone content analysis revealed no significant variation in ABA and GA7 content between the transgenic and control plants. The IAA and SA concentrations were substantially greater in the transgenic plants than in the control, while MeJA concentrations were significantly lower in transgenic plants. To investigate the effect of ZjNOL ectopic expression on leaf color, we examined the effect of darkness treatment on the expression of the AsZDS, AsNCED, AsCLH, and AsNYC genes. AtZDS regulates the catalytic impact of Z-carotene on tetracis-lycopene and is also a key gene for carotenoids synthesis [15]. AsNCED, a carotene lyase, is a critical enzyme for stress-induced ABA biosynthesis [16]. AsCLH catalyzes the ester bond hydrolysis of chlorophyll and chlorophyl, the initial step in the breakdown of chlorophyll [14]. AsNYC participates in the breakdown of the LHCII [6]. The relative expression of AsCLH1 between the control and OE plants have similar trends on different days. Conversely, the trends of overexpression of AsZDS, AsNCED, and AsNYC are predominantly distinct with the control plants. The impact of ectopic expression of ZjNOL on plant leaf color may be due to a variety of biological processes, not just chlorophyll. Fewer differentially expressed genes were filtered out in the transcriptome, which may be attributable to the fact that the plant samples for transcriptome analysis had not yet passed the senescence stage. This shows that NOL performs a major regulatory function exclusively in mature leaves, which is congruent with the findings of expression pattern analysis. Even yet, numerous genes associated with chlorophyll degradation were screened by transcriptome sequencing, and DIN1 and LARS (leucine tRNA ligase) were considerably up-regulated in transgenic plants. Mt-nd4 (NADH-ubiquinone oxidoreductase chain 4) and PPH (pheophytinase) Figure 14. NOL regulates the degradation of chlorophyll and LHCII during leaf senescence [10].
Although the promoter element analysis revealed that the ZjNOL promoter contains cis-elements linked with ABA, IAA, GA, MeJA, and SA, the hormone content analysis revealed no significant variation in ABA and GA 7 content between the transgenic and control plants. The IAA and SA concentrations were substantially greater in the transgenic plants than in the control, while MeJA concentrations were significantly lower in transgenic plants. To investigate the effect of ZjNOL ectopic expression on leaf color, we examined the effect of darkness treatment on the expression of the AsZDS, AsNCED, AsCLH, and AsNYC genes. AtZDS regulates the catalytic impact of Z-carotene on tetracis-lycopene and is also a key gene for carotenoids synthesis [15]. AsNCED, a carotene lyase, is a critical enzyme for stress-induced ABA biosynthesis [16]. AsCLH catalyzes the ester bond hydrolysis of chlorophyll and chlorophyl, the initial step in the breakdown of chlorophyll [14]. AsNYC participates in the breakdown of the LHCII [6]. The relative expression of AsCLH1 between the control and OE plants have similar trends on different days. Conversely, the trends of overexpression of AsZDS, AsNCED, and AsNYC are predominantly distinct with the control plants. The impact of ectopic expression of ZjNOL on plant leaf color may be due to a variety of biological processes, not just chlorophyll. Fewer differentially expressed genes were filtered out in the transcriptome, which may be attributable to the fact that the plant samples for transcriptome analysis had not yet passed the senescence stage. This shows that NOL performs a major regulatory function exclusively in mature leaves, which is congruent with the findings of expression pattern analysis. Even yet, numerous genes associated with chlorophyll degradation were screened by transcriptome sequencing, and DIN1 and LARS (leucine tRNA ligase) were considerably up-regulated in transgenic plants.
Mt-nd4 (NADH-ubiquinone oxidoreductase chain 4) and PPH (pheophytinase) were dramatically down-regulated. DIN1 is a dark-induced senescence-related gene encoding the synthesis of senily-associated protein DIN1. DIN1 encoded peptides may be imported by isolated chloroplasts and subsequently localized to the stromal fraction in Raphanus sativus, and it was thought to be the first gene producing a chloroplast protein that is negatively regulated by light [21]. LARS is targeted to chloroplasts. Chloroplast protein hydrolysis increased the amounts of amino acids during the dark treatment in Camellia sinensis [22]. The research suggested that aminoacyl tRNA synthetases of Oryza sativa may govern amino acid metabolism and the homeostasis of chloroplast thylakoid growth via regulating the protein synthesis process [23]. The NADH-ubiquinone oxidoreductase complex is the principal entry point of electrons into the respiratory chain and it is regarded to be directly associated with sustaining effective flow underambient (photorespiratory) circumstances in Arabidopsis thaliana [24]. PPH of Solanum lycopersicum in particular dephytylates pheophytin, leading to the creation of pheophorbide a, a crucial step in the chlorophyll breakdown process [25]. PPH catalyzes the hydrolysis of chlorophyll to chlorophyllide and phytol in Arabidopsis thaliana, which was considered to be the rate-limiting step of the pathway [26]. Heterologously expressed PPH dephytylates phein to pheide but does not accept chlorophyll as a substrate. PPH can only dehydrogenate phenol without taking chlorophyll as a substrate and has no activity with chlorophyll [26]. This indicates that PPH must conduct its chlorophyll degrading function after NOL has fulfilled its role. However, the underlying chemical mechanism driving the high substrate selectivity of PPH towards pheophytin remains unexplained in Arabidopsis thaliana [27].

Plant Materials and Growth Conditions
The plants of Zoysia japonica (cv. Compadre), Agrostis stolonifera (cv. Penn A-4), Arabidopsis thaliana and Nicotiana benthamiana were cultivated in pots, which were incubated in a plant growth chamber (26/20 • C, 50% humidity, 16 h/8 h photoperiod). All the cultivated species are from and kept in a laboratory. Transgenic plants of 4-weeks-old were transferred to dark environments for 8 d consecutively to perform dark treatments [28].

Gene Cloning and Vector Construction
Healthy Zoysia japonica was selected as the experimental material. Total RNA was isolated from 3-month-old leaves according to the manufacturer's recommendations (Promega, Madison, WI, USA, plant RNA isolation kit). The full-length cDNAs of ZjNOL were amplified according to the Zoysia genome database [13], and then ligated into the vectors pDM19-T, 3302Y, 3302YUBI, pGBKT7 and YCE4 with the primers listed in Supplemental  Table S2. The plasmid 3302Y-ZjNOL was subsequently transformed into the Agrobacterium tumefaciens strain 'GV3101'. The coding sequences of ZjNYC were cloned into pGADT7 and YNE vectors according to the Zoysia genome database.

Generation of Transgenic Plants
The plant expression vector 3302YUBI-ZjNOL was transformed into Agrobacterium EHA105 [29]. The Agrobacterium-mediated method was conducted following the protocol described by Luo et al. [30]. The transgenic seedlings of Agrostis stolonifera were identified on selective agar plates and transferred to the controlled growth chamber conditions and subsequent experiments were conducted. Transgenic plants carrying 3302YUBI were used as a negative control.

Subcellular Localization and BiFC Analysis
The YFP plasmid, ZjNOL and YFP recombinant plasmid 3302Y-ZjNOL were respectively introduced into agrobacterium strain EHA105, then infiltrated young leaves of tobacco as previously described [31]. The interaction between ZjNOL and ZjNYC was verified using the BiFC approach [32]. The plasmids YCE4-ZjNOL and NE6-ZjNYC, which contain THE C-terminal and N-terminal of YFP protein, were transformed with Agrobacterium and co-infected tobacco. A Leica TCS SP 8 confocal microscope was used to capture the YFP fluorescence signal.

Yeast Two-Hybrid Assay
The PGBKT7-ZjNOL and PGADT7-ZjNYC recombinant plasmids were transformed into yeast strains Y2H and Y187, respectively, using the Yeastmaker™ Yeast Transformation System (Clontech Laboratories, Mountain View, CA, USA). The MATCHMAKER GAL4 yeast two-hybrid system was set up according to the manufacturer's instructions for the Matchmaker TM Gold Yeast Two-Hybrid System (Clontech Laboratories).

Measurement of Net Photosynthesis of Leaves
The net photosynthetic rate was measured using the Li-6400 portable photosynthetic system (Li-Cor Biosciences, Lincoln, NE, USA). The artificial light utilized for the measurement was 1200 µmol m −2 s −1 , and three repeats were carried out to verify the uniformity of the test data. All measurements were taken between 10 and 12 a.m.

Photosynthetic Pigment Analysis and Electrolyte Leakage (EL) Measurement
Pigment and ion permeability were measured in the leaves treated with darkness for 0-7 days. Total chlorophyll was extracted from the 2nd leaves of the control and transgenic plants, quantified spectrophotometrically as described previously [33]. Leaf membrane stability was assessed by measuring EL following the protocol described earlier [34].

Transmission Electron Microscopy
Transmission electron microscopy experiments were performed on the leaves of the control and transgenic plants. The second leaf of the transgenic and wild-type plants was chosen and immersed in a fixed buffer solution for 24 h. The specimens were subsequently stained and observed as described previously [35].

Morphological Observation
The second leaf and stem of the transgenic plants of 30-days-old were selected for ferro red solid-green staining. The walls of lignified cells were stained red, while the walls of fibrotic cells were stained green. Under a light microscope, the samples were observed and the images were captured [36].

Expression Levels of ZjNOL Gene and Pigment-Related Genes
RT-PCR (real-time quantitative PCR) was used to evaluate the expression pattern of the ZjNOL. Different tissues (roots, stems, and leaves) of 2-month-old Zoysia japonica, as well as leaves from Zoysia japonica at various growth stages (young, fast growing, mature), were collected. A total of 10 µmol/L ABA, 10 µmol/L MeJA, 200 µmol/L ETH, 300 mmol/L NaCl, and 20% PEG4000 were used to treat Zoysia japonica. RNA was extracted and cDNA was produced from the leaves at 0 h, 1 h, 3 h, 6 h, 12 h, and 24 h after treatment. The Z. japonica beta-actin was used as an internal reference gene (GenBank accession number: GU290546) [43]. RNA was isolated from OE and control plants that had been exposed to darkness for 0-7 days. To evaluate the relative expression levels of AsZDS, AsNCED, AsCLH1, and AsNYC, the actin gene (GenBank accession number: DY543529) was utilized as an internal control [44]. Three biological replicates were set for each treatment. The sequences of related primers used above are shown in Supplementary Table S2. The relative expression levels were calculated using the 2 −∆∆Ct method [45].

Hormone Contents Assay
One-month-old leaves of the control and OE plants were collected and ground after quick-freezing with liquid nitrogen. The metabolites were extracted and concentrated and redissolved with 80% methanol/aqueous solution for LC-MS/MS analysis. The experiment was carried out using an AB SCIEX 6500+ Qtrap mass spectrometer [46,47].

Experimental Validation of DEGs by RT-PCR
The transcriptome analysis-derived DEGs were validated using RT-PCR. Ten genes (AsFEH, AsTPS, AsCK1, AsYLS3, AsJRL9, AsWRKY23, AsLOX, AsERF9, AsFAR1, AsERF4) were chosen at random for RT-PCR, and the experimental procedure was as described before. The results of the gene expression levels were then analyzed by the corresponding standard deviations repeated by the three techniques.

Conclusions
The NYC-like enzyme (NOL) is a key enzyme in the degradation of chlorophyll b. The NOL gene of Zoysia japonica codes a protein with 326 amino acids and has a molecular mass of 26.62 kD. ZjNOL is found on the stromal side of the thylakoid membrane, and both ZjNOL and ZjNYC proteins are capable of forming stable structures and interacting among each other. Ectopically expressed ZjNOL resulted in yellow leaves, a larger cortex, and smaller vascular column cells in creeping bentgrass. Additionally, the transgenic plants revealed morphological changes in their chloroplast structure, with a significant reduction in the number of grana and thylakoids per grana stack. The features and activities of ZjNOL will serve as a starting point for further study into the gene's functions and regulatory mechanisms.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.