CmNAC73 Mediates the Formation of Green Color in Chrysanthemum Flowers by Directly Activating the Expression of Chlorophyll Biosynthesis Genes HEMA1 and CRD1

Chrysanthemum is one of the most beautiful and popular flowers in the world, and the flower color is an important ornamental trait of chrysanthemum. Compared with other flower colors, green flowers are relatively rare. The formation of green flower color is attributed to the accumulation of chlorophyll; however, the regulatory mechanism of chlorophyll metabolism in chrysanthemum with green flowers remains largely unknown. In this study, we performed Illumina RNA sequencing on three chrysanthemum materials, Chrysanthemum vestitum and Chrysanthemum morifolium cultivars ‘Chunxiao’ and ‘Green anna’, which produce white, light green and dark green flowers, respectively. Based on the results of comparative transcriptome analysis, a gene encoding a novel NAC family transcription factor, CmNAC73, was found to be highly correlated to chlorophyll accumulation in the outer whorl of ray florets in chrysanthemum. The results of transient overexpression in chrysanthemum leaves showed that CmNAC73 acts as a positive regulator of chlorophyll biosynthesis. Furthermore, transactivation and yeast one-hybrid assays indicated that CmNAC73 directly binds to the promoters of chlorophyll synthesis-related genes HEMA1 and CRD1. Thus, this study uncovers the transcriptional regulation of chlorophyll synthesis-related genes HEMA1 and CRD1 by CmNAC73 and provides new insights into the development of green flower color in chrysanthemum and chlorophyll metabolism in plants.


Introduction
Color is an important ornamental trait in plants. Flower color plays an important role in the co-evolution of plants and insects, as flowers use their color to attract insects or birds for pollination [1]. In the tropics, plants often use bright colors to protect themselves from herbivores. In addition, at high altitudes, flowers usually accumulate high levels of anthocyanin, which protects the plants from damage by ultraviolet (UV) radiation [2]. In addition to anthocyanins, flower color is also influenced by carotenoids and chlorophyll. In nature, green flowers are relatively rare, which was disadvantageous for the recognition of insect pollinators. Since chlorophyll plays an important role in photosynthesis, a number of studies have focused on the mechanism of chlorophyll metabolism and regulation in leaves, which is of great importance in delaying leaf senescence and increasing the crop yield [3].
Chlorophyll biosynthesis and degradation are complex processes involving many enzymes. In the chlorophyll biosynthesis cycle, the conversion of glutamyl-tRNA (Glu-tRNA) to chlorophyll-a involves 14 steps, catalyzed by Glu-tRNA reductase (HEMA), glutamate 1-semialdehyde aminotransferase (GSA), porphobilinogen synthase (HEMB), hydroxymethyl-the synthesis of chlorophyll were be characterized, through transient overexpression of candidate genes in chrysanthemum leaves, transactivation of chlorophyll synthesis-related genes, and yeast one-hybrid assay. This work will help to uncover the transcriptional regulation of chlorophyll synthesis-related genes and provides a better understanding of the formation of green color in chrysanthemum flowers and the metabolism of chlorophyll in plants.

Plant Materials
Wild chrysanthemum (Chrysanthemum vestitum) and six cultivars of hybrid chrysanthemum (Chrysanthemum morifolium) with green flowers, 'Green peony', 'Green gemstone', 'Chunxiao', 'Greenlizard', 'Green anna' and 'Lv Dingdang', were grown in the greenhouse of Huazhong Agricultural University (Figure 1). The outer whorl of ray florets was sampled from the fully opened flowers of C. vestitum and C. morifolium cultivars 'Chunxiao' and 'Green anna' at the same time, with three biological replicates. The samples were quickly wrapped in foil, frozen in liquid nitrogen, transported to the laboratory and stored in a −80 • C freezer until needed for RNA extraction.

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Extracts from the SurveyMonkey questionnaire SurveyMonkey.com (www.surveymonkey.com), LLC, Palo Alto, California, USA To conduct transient gene overexpression assay and ABA treatment, cut flowers of 'Lv Dingdang' were purchased from Nanhu flower market and transported to the laboratory within one hour. Flower stems were cut to a length of 40 cm and then placed in vases containing distilled water.

Determination of Chlorophyll, Carotenoid and Flavonoid Contents
The total chlorophyll content of ray florets was determined as described by Fu et al. [4]. Total carotenoid and total flavonoid contents were determined according to the methods of Potosí-Calvache et al. [20] and Cao et al. [21], respectively.

Quantitative Real-time PCR (qRT-PCR)
In total, 1 µg RNA of each sample was used to synthesize cDNA with the cDNA Synthesis SuperMix (AE311-03; TransGen Biotech, Beijing, China), according to the manufacturer's instructions. Subsequently, qRT-PCR was performed on LightCycler 96 (Roche, Basel, Switzerland) using the cDNA template and sequence-specific primers (Table S1). All reactions were performed with three biological replicates. The thermocycling program was as follows: 95°C for 5 min, followed by 40 cycles of three steps (95°C for 15 s, 60°C for 30 s, and 72°C for 30 s). In the end, there was a melting curve analysis: 95°C for 15 s, 60°C for 60 s, and 95°C for 15 s, which was used to ensure the specificity of the amplified product. The 2 −∆∆Ct method [22] was used to calculate relative gene expression levels. The chrysanthemum Ubiquitin (CmUBI) gene (accession no. EU862325) was used as the internal reference.

Gene Cloning and Vector Construction
The CmNAC73 gene sequence was obtained from the chrysanthemum genome [23], and the transcriptome data generated in this study and corrected with high-fidelity Phusion DNA Polymerase (Thermo Fisher Scientific, Waltham, MA, USA). The promoter sequences of chlorophyll synthesis-related genes were cloned according to the chrysanthemum genome, combined with the FPNI-PCR method, as described by Wang et al. [24].
To generate the CmNAC73 overexpression (OE) vector, the open reading frame (ORF) of CmNAC73, harboring the restriction sites Hind III and Kpn I, was amplified by PCR. The PCR product was digested and cloned into the pSuper1300 vector, which was derived from pCAMBIA1300 by the research group of Dr. Zhizhong Gong (China Agricultural University).
To analyze promoter activity, the promoters of candidate genes carrying the Hind III and BamH I restriction sites were amplified by PCR. The PCR products were digested and ligated, and the promoter with the appropriate restriction site was cloned into the PBI121 vector.

Transient Expression of CmNAC73 in Chrysanthemum Leaves
Agrobacterium tumefaciens GV3101 cells carrying the pSuper1300 empty vector or pSuper::CmNAC73 were harvested by centrifugation and resuspended in infiltration buffer (10 mM MgCl 2 , 150 µM acetosyringone [As], and 10 mM 2-morpholinoethanesulfonic acid [MES], pH 5.6), and the concentration of the cell suspension was adjusted to obtain an optical density (OD 600 ) of 0.8. Subsequently, the A. tumefaciens suspension was incubated in the dark, without shaking, for 2 h. Leaves of all three chrysanthemum accessions were agroinfiltrated under vacuum (0.5 atm). After agroinfiltration, the leaves were washed with deionized water and placed in a Petri dish lined with wet filter paper. The Petri dish was incubated in the dark at 8 • C for 3 days and then at 23 • C, 60% relative humidity and 5000 lux light intensity (time set to 0 days). The leaves were photographed every 2 days and used for RNA extraction after 4 days.

Transient Transactivation Assay
The pSuper::CmNAC73 effector plasmid, PBI121 reporter plasmid harboring the promoter of chlorophyll synthesis related genes, and corresponding empty vectors were separately transformed into A. tumefaciens strain GV3101. After overnight culture in Luria-Bertani (LB) broth, cells were collected by centrifugation at 5000× g for 8 min, and resuspended in infiltration solution (10 mM MES, 10 mM MgCl 2 , and 20 µM As [pH5.6]) (OD 600 = 1.6). Cells transformed with the effector and reporter plasmids were mixed at a 1:1 ratio, incubated at room temperature without shaking for 2 h, and then infiltrated into the abaxial surface of tobacco (Nicotiana benthamiana) leaves using a 1-mL needle-less syringe. The plants were incubated in the dark at 23 • C and 40-60% relative humidity for 3 days. Subsequently, the agroinfiltrated leaves were collected, immediately frozen in liquid nitrogen, and stored at −80 • C.

Determination of β-glucuronidase (GUS) Activity
The agroinfiltrated N. benthamiana leaves were ground in liquid nitrogen, and soluble proteins were extracted with the extraction buffer containing 0.05 M phosphate-buffered saline (PBS; pH 7.0), 0.1% sodium N-lauroylsarcosine, 10 mM EDTA (pH 8.0), 1/5 volume of methanol, 1/1000 volume of Triton X-100 and 1/1000 volume of β-mercaptoethanol. The protein extracts were centrifuged at 8000× g for 10 min at 4 • C. The protein concentration was determined with the Bradford method [25]. To measure GUS activity, 20 µL of the purified protein was mixed with 180 µL of 2 mM 4-methylumbelliferyl β-d-glucuronide hydrate (4-MUG; substrate) dissolved in extraction buffer. Then, half the volume of the mixture (100 µL) was combined with 900 µL of 0.2 M Na 2 CO 3 to immediately terminate the reaction, while the remaining 100 µL was incubated at 37 • C for 15 min before the reaction was terminated. The fluorescence of 4-methylumbelliferyl (4-MU; reaction product) was measured using a fluorescence spectrophotometer (F-4500; Hitachi, Tokyo, Japan) at 365-nm excitation and 455-nm emission. GUS activity was defined as the amount of 4-MU produced per mg protein per min (µM 4-MU mg −1 protein min −1 ).

Yeast One-Hybrid Assay
The yeast one-hybrid assay was performed as described previously [26]. Briefly, the CmNAC73 ORF was cloned into pGADT7, and the promoters of chlorophyll synthesisrelated genes were cloned into pHIS2.1. The recombinant pGADT7 and pHIS2.1 vectors were co-transfected into yeast (Saccharomyces cerevisiae) strain Y187. The transformed yeast cells were cultured on a synthetic-defined medium lacking leucine and tryptophan (SD/-Leu-Trp). Positive clones were verified by PCR and transferred to SD medium lacking Leu, Trp and histidine (SD/-Leu-Trp-His), supplemented with 0-80 mM 3-aminotriazole (3-AT).

Statistical Analysis
Statistical analysis of the data was carried out with SPSS 22.0 (IBM, Armonk, NY, USA). A two-tailed Student's t-test (* p < 0.05, ** p < 0.01) was used for the comparison of two groups, and one-way analysis of variance (ANOVA) with Duncan's multiple range tests (p < 0.05) was used for comparison of multiple groups.

Pigment Contents in Chrysanthemum Materials
To understand the basis of green flower color in chrysanthemum, we determined the contents of major pigments in C. vestitum and six C. morifolium cultivars. From C. vestitum to chrysanthemum 'Lv Dingdang', the chlorophyll and carotenoid contents of flowers increased gradually, consistent with the change in flower color, but the change in flavonoid contents was not obvious. Additionally, the colorimeter analysis showed that as the flower color deepened in the seven materials, the 'L' value decreased from 92.46 to 62.32, indicating that the flower color gradually became darker. Similarly, the 'a' value declined steadily from −1.66 to −12.48, indicating a gradual deepening of the green color, while the 'b' value (which represents the yellow color) gradually increased from C. vestitum to C. morifolium 'Greenlizard', and then declined in C. morifolium 'Green anna' and 'Lv Dingdang' (Figure 2). These results suggest that chlorophyll is the main color-forming pigment in chrysanthemums with green flowers, and both chlorophyll and carotenoid affect the flower color in these materials.
indicating that the flower color gradually became darker. Similarly, the 'a' value declined steadily from -1.66 to -12.48, indicating a gradual deepening of the green color, while the 'b' value (which represents the yellow color) gradually increased from C. vestitum to C. morifolium 'Greenlizard,' and then declined in C. morifolium 'Green anna' and 'Lv Dingdang' (Figure 2). These results suggest that chlorophyll is the main color-forming pigment in chrysanthemums with green flowers, and both chlorophyll and carotenoid affect the flower color in these materials.

Transcriptome Analysis of C. vestitum and C. morifolium Cultivars
C. vestitum is a wild species of chrysanthemum with pure white flowers. C. morifolium cultivars 'Chunxiao' and 'Green anna' produce green flowers; however, the green color of 'Chunxiao' flowers gradually fades away with the flower opening, whereas that of 'Green anna' flowers is very stable. RNA-seq of the outer-whorl ray florets of these three accessions (three biological replicates per accession; nine samples total) generated 423,277,980 raw reads. After the removal of adaptor and low-quality sequences, a total of 403,791,250 clean reads were obtained (Q30 = 89.78%-91.48%) ( Table 1). After assembled with Trinity, a total of 262,601 unigenes were obtained (Table S2), with the minimum and maximum lengths of 201 and 14,698 bp, respectively, and the N50 value of 1,079 bp. Analysis of the DEGs showed that 19,764 unigenes were upregulated in 'Green anna' compared with 'Chunxiao,' while 33,057 and 24,118 unigenes were upregulated in group 'Green anna' vs. C. vestitum and 'Chunxiao' vs. C. vestitum, respectively. Additionally, 1,269 unigenes were upregulated in the above three groups ( Figure 3, Table S3). In addition, 14,910, 26,154, and 26,963 unigenes were downregulated in 'Green anna' vs. 'Chunxiao,' 'Green anna' vs. C. vestitum and 'Chunxiao' vs. C. vestitum comparisons, respectively, and 990 unigenes were significantly downregulated in all three groups ( Figure 3, Table S4).

Transcriptome Analysis of C. vestitum and C. morifolium Cultivars
C. vestitum is a wild species of chrysanthemum with pure white flowers. C. morifolium cultivars 'Chunxiao' and 'Green anna' produce green flowers; however, the green color of 'Chunxiao' flowers gradually fades away with the flower opening, whereas that of 'Green anna' flowers is very stable. RNA-seq of the outer-whorl ray florets of these three accessions (three biological replicates per accession; nine samples total) generated 423,277,980 raw reads. After the removal of adaptor and low-quality sequences, a total of 403,791,250 clean reads were obtained (Q30 = 89.78%-91.48%) ( Table 1). After assembled with Trinity, a total of 262,601 unigenes were obtained (Table S2), with the minimum and maximum lengths of 201 and 14,698 bp, respectively, and the N50 value of 1079 bp. Analysis of the DEGs showed that 19,764 unigenes were upregulated in 'Green anna' compared with 'Chunxiao', while 33,057 and 24,118 unigenes were upregulated in group 'Green anna' vs. C. vestitum and 'Chunxiao' vs. C. vestitum, respectively. Additionally, 1269 unigenes were upregulated in the above three groups (Figure 3, Table S3). In addition, 14,910, 26,154, and 26,963 unigenes were downregulated in 'Green anna' vs. 'Chunxiao', 'Green anna' vs. C. vestitum and 'Chunxiao' vs. C. vestitum comparisons, respectively, and 990 unigenes were significantly downregulated in all three groups ( Figure 3, Table S4).  Furthermore, KEGG pathway enrichment analysis showed that in the 'Green anna' vs C. vestitum comparison, a large number of upregulated genes were enriched in the photosynthesis, carotenoid biosynthesis, porphyrin and chlorophyll metabolism pathway; 94 unigenes were enriched in the photosynthesis pathway; 81 unigenes were enriched in the porphyrin and chlorophyll metabolism pathway ( Figure S1, Table S5).
Similarly, in the 'Chunxiao' vs C. vestitum comparison, a large number of upregulated genes were enriched in the ribosome, photosynthesis, chlorophyll and chlorophyll metabolism pathway, while 94 and 75 unigenes were enriched in the photosynthesis pathway and porphyrin and chlorophyll metabolism pathway, respectively ( Figure S2, Table S6). Photosynthesis and chlorophyll metabolism are closely related and may play important roles in the development of flower color in green chrysanthemum.

Expression Pattern of Chlorophyll Synthesis and Degradation Related Genes
Based on the KEGG analysis, 35, 7 and 4 chlorophyll biosynthesis, cycling and 4 degradation-related unigenes, respectively, were differentially expressed in the 'Green anna' vs C. vestitum and 'Chunxiao' vs C. vestitum groups (Table S7).
Similarly, in the 'Chunxiao' vs C. vestitum comparison, a large number of upregulated genes were enriched in the ribosome, photosynthesis, chlorophyll and chlorophyll metabolism pathway, while 94 and 75 unigenes were enriched in the photosynthesis pathway and porphyrin and chlorophyll metabolism pathway, respectively ( Figure S2 and Table S6). Photosynthesis and chlorophyll metabolism are closely related and may play important roles in the development of flower color in green chrysanthemum.

Expression Pattern of Chlorophyll Synthesis and Degradation Related Genes
Based on the KEGG analysis, 35, 7 and 4 chlorophyll biosynthesis, cycling and 4 degradation-related unigenes, respectively, were differentially expressed in the 'Green anna' vs C. vestitum and 'Chunxiao' vs C. vestitum groups (Table S7).
In 'Chunxiao', the color of ray florets in the outer whorl gradually changes from green to white during flower opening. Our results showed that the chlorophyll content of the outer-whorl ray florets also decreased gradually during flower opening, and the expression of chlorophyll synthesis related unigenes HEMA1, CHLI1, CRD1 and PORA1 also showed a steady decline (Figure 4). sion levels of genes related to chlorophyll cycling and degradation showed no significant differences among the three genotypes ( Figure S4).
In 'Chunxiao,' the color of ray florets in the outer whorl gradually changes from green to white during flower opening. Our results showed that the chlorophyll content of the outer-whorl ray florets also decreased gradually during flower opening, and the expression of chlorophyll synthesis related unigenes HEMA1, CHLI1, CRD1 and PORA1 also showed a steady decline (Figure 4).  It has been shown that ABA promotes the degradation of chlorophyll and causes the yellowing of leaves [27]. Here, we treated cut 'Lv Dingdang' flowers with 20 mg/L ABA and examined the expression of chlorophyll synthesis-related genes. The results showed that the expression of HEMA1, CHLI1, CHLH1, CRD1 and PORA1 was significantly inhibited by ABA ( Figure S5).

Selection of Key Transcription Factors involved in Chlorophyll Synthesis
Analysis of DEGs showed that 45 and 58 transcription factor-encoding genes were significantly upregulated and downregulated in the three comparison groups, 'Green anna' vs. 'Chunxiao', 'Green anna' vs. C. vestitum, and 'Chunxiao' vs. C. vestitum (Tables S3 and S4).
The expression of a subset of the up-or down-regulated transcription factor-encoding genes was analyzed by qRT-PCR. The results showed that the expression of 10 genes increased gradually in C. vestitum, 'Chunxiao' and 'Green anna', while that of 4 genes, such as Cluster-35308.188936 (bHLH), decreased gradually in the three materials, consistent with the transcriptome data. Among these genes, Cluster-35308.177654, a NAC family gene, showed the most significant change in expression ( Figure 5). and examined the expression of chlorophyll synthesis-related genes. The results showed that the expression of HEMA1, CHLI1, CHLH1, CRD1 and PORA1 was significantly inhibited by ABA ( Figure S5).

Selection of Key Transcription Factors involved in Chlorophyll Synthesis
Analysis of DEGs showed that 45 and 58 transcription factor-encoding genes were significantly upregulated and downregulated in the three comparison groups, 'Green anna' vs. 'Chunxiao,' 'Green anna' vs. C. vestitum, and 'Chunxiao' vs. C. vestitum (Table S3,  Table S4).
The expression of a subset of the up-or down-regulated transcription factor-encoding genes was analyzed by qRT-PCR. The results showed that the expression of 10 genes increased gradually in C. vestitum, 'Chunxiao' and 'Green anna,' while that of 4 genes, such as Cluster-35308.188936 (bHLH), decreased gradually in the three materials, consistent with the transcriptome data. Among these genes, Cluster-35308.177654, a NAC family gene, showed the most significant change in expression ( Figure 5). The RNA-seq data of Cluster-35308.177654 (NAC) and chlorophyll synthesis-related genes were used to analyze their Pearson correlation coefficients with SPSS 22.0 (IBM, Armonk, NY, USA). The results showed that the expression of HEMA1, CRD1, PORA1, CHLI1, CHLM1 were significantly correlated with Cluster-35308.177654 (P < 0.05), for HEMA1, CRD1and PORA1, the correlation was highly significant (P < 0.01) ( Table S8).
The expression of Cluster-35308.177654 (NAC) was further examined in the flowers of C. morifolium cultivars 'Chunxiao' (at different opening stages) and 'Lv Dingdang' The RNA-seq data of Cluster-35308.177654 (NAC) and chlorophyll synthesis-related genes were used to analyze their Pearson correlation coefficients with SPSS 22.0 (IBM, Armonk, NY, USA). The results showed that the expression of HEMA1, CRD1, PORA1, CHLI1, CHLM1 were significantly correlated with Cluster-35308.177654 (p < 0.05), for HEMA1, CRD1 and PORA1, the correlation was highly significant (p < 0.01) ( Table S8).
The expression of Cluster-35308.177654 (NAC) was further examined in the flowers of C. morifolium cultivars 'Chunxiao' (at different opening stages) and 'Lv Dingdang' (treated with or without ABA). In 'Chunxiao', the expression of Cluster-35308.177654 (NAC) increased significantly from stage 1 to stage 2 and then declined gradually from stage 2 to stage 4. In 'Lv Dingdang', ABA treatment significantly decreased the expression of Cluster-35308.177654 ( Figure 6).
(treated with or without ABA). In 'Chunxiao,' the expression of Cluster-35308.177654 (NAC) increased significantly from stage 1 to stage 2 and then declined gradually from stage 2 to stage 4. In 'Lv Dingdang,' ABA treatment significantly decreased the expression of Cluster-35308.177654 ( Figure 6).

Characterization of CmNAC73
Our results showed that the expression of Cluster-35308.177654 (NAC) was highly correlated with chlorophyll synthesis (Figure 2,4,5,6). To investigate the role of Cluster-35308.177654 in chlorophyll synthesis, we first cloned this gene from C. morifolium cultivar 'Chunxiao.' The full-length CDS of Cluster-35308.177654 (NAC) is 876 bp in length and is predicted to encode a protein of 291 amino acids. Phylogenetic analysis revealed that the protein encoded by Cluster-35308.177654 (NAC) showed high homology to NAC73 genes in many other species. Therefore, we named the Cluster-35308.177654 (NAC) gene as CmNAC73 (Genbank accession: MW916171) ( Figure S6).

Characterization of CmNAC73
Our results showed that the expression of Cluster-35308.177654 (NAC) was highly correlated with chlorophyll synthesis (Figures 2 and 4-6). To investigate the role of Cluster-35308.177654 in chlorophyll synthesis, we first cloned this gene from C. morifolium cultivar 'Chunxiao'. The full-length CDS of Cluster-35308.177654 (NAC) is 876 bp in length and is predicted to encode a protein of 291 amino acids. Phylogenetic analysis revealed that the protein encoded by Cluster-35308.177654 (NAC) showed high homology to NAC73 genes in many other species. Therefore, we named the Cluster-35308.177654 (NAC) gene as CmNAC73 (Genbank accession: MW916171) ( Figure S6).

Regulation of Chlorophyll Synthesis-Related Genes by CmNAC73
Based on the published reference genome of chrysanthemum, the promoters of HEMA1, CHLI1, CRD1 and PORA1 (1648, 1245, 1580, and 1192 bp, respectively) were cloned. Transient transactivation assays in N. benthamiana leaves showed that CmNAC73 significantly induced the promoters of HEMA1, CHLI1 and CRD1 but did not have a significant effect on the promoter activity of PORA1 (Figure 8a).

Regulation of Chlorophyll Synthesis-related Genes by CmNAC73
Based on the published reference genome of chrysanthemum, the promoters of HEMA1, CHLI1, CRD1 and PORA1 (1,648, 1,245, 1,580, and 1,192 bp, respectively) were cloned. Transient transactivation assays in N. benthamiana leaves showed that CmNAC73 significantly induced the promoters of HEMA1, CHLI1 and CRD1 but did not have a significant effect on the promoter activity of PORA1 (Figure 8a).
Previous studies have shown that NAC transcription factors bind to the core sequence CACG or CGTG [28,29]. In this study, 13, 5, 6 and 1 putative NAC-binding sites were found in the promoters of HEMA1, CHLI1, CRD1 and PORA1, respectively ( Figure  8b). To test whether CmNAC73 binds to the promoters of HEMA1, CHLI1, CRD1 and PORA1, we performed a yeast one-hybrid assay. When pHEMA1-His and pGAD-CmNAC73 were co-transferred into yeast, the positive clone of yeast cells could grow normally on SD/-Leu-Trp-His, supplied with 4 mM 3-AT, while the yeast carrying pGAD and pHEMA1-His could not grow, which suggested CmNAC73 have a weak binding to the HEMA1 promoter. Additionally, CmNAC73 showed strong binding to the promoter of CRD1, and 70 mM 3-AT can completely inhibit the expression of HIS gene in the combination of GAD + pCRD1, whereas CmNAC73 + pCRD1 could grow normally (Figure 8c and d).  Previous studies have shown that NAC transcription factors bind to the core sequence CACG or CGTG [28,29]. In this study, 13, 5, 6 and 1 putative NAC-binding sites were found in the promoters of HEMA1, CHLI1, CRD1 and PORA1, respectively (Figure 8b). To test whether CmNAC73 binds to the promoters of HEMA1, CHLI1, CRD1 and PORA1, we performed a yeast one-hybrid assay. When pHEMA1-His and pGAD-CmNAC73 were co-transferred into yeast, the positive clone of yeast cells could grow normally on SD/-Leu-Trp-His, supplied with 4 mM 3-AT, while the yeast carrying pGAD and pHEMA1-His could not grow, which suggested CmNAC73 have a weak binding to the HEMA1 promoter. Additionally, CmNAC73 showed strong binding to the promoter of CRD1, and 70 mM 3-AT can completely inhibit the expression of HIS gene in the combination of GAD + pCRD1, whereas CmNAC73 + pCRD1 could grow normally (Figure 8c,d).

Discussion
Chrysanthemum is an important flower, owing to its diverse flower shapes, bright colors and a long ornamental period. Chrysanthemum has more than 30,000 cultivars [30]. Depending on the flower color, 811 varieties of chrysanthemum were divided into nine groups, including brown, orange, pink, purple, red, white, yellow, yellow-green and dark red; green varieties are very rare and are therefore classified into the yellow-green group [31]. It has been speculated that the ancestry of modern chrysanthemum includes C. vestitum (2n = 54), C. indicum (2n = 18, 36), C. lavandulifolium (2n = 18), C. nankingense (2n = 18) and C. zawadskii (2n = 54) [32]. Interestingly, none of these ancestral species produce green flowers. Moreover, the evolution of green chrysanthemums remains unclear.
The flower is an important organ of angiosperms derived from leaves [33]. One of the important functions of leaves is photosynthesis, which takes place in chloroplasts. These photosynthetic organelles have also been observed in some green flowers by transmission electron microscopy [4,34]. In transgenic Arabidopsis and Nicotiana tabacum lines overexpressing SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) or SOC1-like, heat-stress induced the biogenesis of chloroplasts and the formation of green petals [35]. In many plants, such as lily and chrysanthemum, flowers are green at the early stages of blooming, and their chlorophyll content decreases during the opening process [4,34,36]. This phenomenon was also observed in C. morifolium cultivar 'Chunxiao' (Figure 4). A previous study reported that white flowers are formed partly as a result of chlorophyll degradation [37]. It has been speculated that the maintenance of green color in green chrysanthemum may be related to the continuous synthesis and reduced degradation of chlorophyll. The results of the current study also showed that the content of chlorophyll and expression of chlorophyll synthesis-related genes increased steadily in C. vestitum and C. morifolium cultivars 'Chunxiao' and 'Green anna', which was closely related to the change in their flower color (Figure 2, Figure S3).
During ABA-induced leaf senescence, several NAC genes are induced, which inhibits the accumulation of chlorophyll, partly by promoting the expression of genes related to chlorophyll degradation [15,16]. Conversely, in rice (Oryza sativa), OsNAC2 accelerated leaf senescence by promoting ABA biosynthesis [51]. A few NAC genes have been shown to negatively regulate leaf senescence. Overexpression of a grape (Vitis vinifera L.) NAC gene, DRL1, in N. benthamiana inhibited ABA biosynthesis and significantly inhibited leaf senescence [18,52]. However, whether the genes regulate chlorophyll metabolism remains unclear. During the ripening process of citrus (Citrus reticulata Blanco) fruits, chlorophyll degradation was accompanied by fruit color changes, and two ethylene-responsive genes, CitERF6 and CitERF13, promoted chlorophyll degradation, leading to fruit degreening [53]. Some studies have also reported the regulation of chlorophyll metabolism in green flowers. In chrysanthemum, CmCOL16, CmERF and CmbHLH transcription factors were found to be closely related to chlorophyll synthesis [4]. In petunia, overexpression of PhCOL16a, a homolog of the CmCOL16 gene, significantly promoted the synthesis of chlorophyll [19]. In this study, we found a novel chrysanthemum NAC gene, CmNAC73, whose expression was repressed by ABA, and its transient overexpression in chrysanthemum leaves delayed leaf senescence by activating several chlorophyll synthesis-related genes, including HEMA1, CHLI1, CHLM1, CRD1 and PORA1 (Figures 6 and 7). Furthermore, transient transactivation and yeast one-hybrid assays showed that CmNAC73 binds to the promoters of HEMA1 and CRD1, thus activating their expression (Figure 8).

Conclusions
In short, we carried out transcriptome analysis of three chrysanthemum accessions, including C. vestitum and C. morifolium cultivars 'Chunxiao' and 'Green anna', with white, light-green and dark-green colored flowers, respectively, and identified a novel NAC gene, CmNAC73, which acts as a positive regulator of chlorophyll biosynthesis, at least in part, through the direct activation of chlorophyll synthesis-related genes HEMA1 and CRD1. These results provide a better understanding of the formation of a green color in chrysanthemum flowers and the metabolism of chlorophyll in plants and guide the breeding of more green-flower chrysanthemum cultivars.

Conflicts of Interest:
The authors declare no conflict of interest.