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Article

Transcriptional Regulation in Leaves of Cut Chrysanthemum (Chrysanthemum morifolium) ‘FenDante’ in Response to Post-Harvest Ethylene Treatment

by
Rui Liu
†,
Xuele Zuo
†,
Yu Chen
,
Ziyan Qian
,
Can Xu
,
Likai Wang
and
Sumei Chen
*
State Key Laboratory of Crop Genetics and Germplasm Enhancement, Key Laboratory of Flower Biology and Germplasm Innovation, Ministry of Agriculture and Rural Affairs, Key Laboratory of Biology of Ornamental Plants in East China, National Forestry and Grassland Administration, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China
*
Author to whom correspondence should be addressed.
†
These authors contribute equally to this work.
Horticulturae 2022, 8(7), 573; https://doi.org/10.3390/horticulturae8070573
Submission received: 13 April 2022 / Revised: 13 June 2022 / Accepted: 21 June 2022 / Published: 24 June 2022
(This article belongs to the Section Postharvest Biology, Quality, Safety, and Technology)
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Versions Notes

Abstract

:
The early wilting and yellowing of leaves in response to ethylene is the main limitation affecting the vase quality of cut chrysanthemums. Therefore, leaf senescence is the most difficult problem in the post-harvest and production of chrysanthemums. Nevertheless, the molecular mechanism of ethylene on the regulation of post-harvest senescence of cut chrysanthemum leaves is still unclear. In this study, we identified an ethylene-sensitive chrysanthemum ‘FenDante,’ which showed rapid chlorophyll content decrease under ethylene treatment, resulting in leaf yellowing and wilting before flower senescence. A new generation of Illumina sequencing platform was used to identify differentially expressed genes in the leaves in response to ethylene treatment in chrysanthemum. A total of 1.04 Gb of raw reads was obtained, including 753 and 2790 differentially expressed genes at 3 h and 24 h after ethylene treatment, respectively. KEGG analysis revealed that the differentially expressed genes are mainly involved in plant hormone synthesis and signal transduction, chlorophyll metabolism, aquaporins, and reactive oxygen species. The gene expression regulatory networks in the leaves of post-harvest cut chrysanthemums in response to ethylene treatment were studied, which lays the foundation for future research on the molecular mechanisms of ethylene-mediated leaf senescence in cut chrysanthemums.

1. Introduction

Ethylene is an important hormone that regulates plant growth and senescence, including seed germination, plant flowering, organ abscission and senescence, fruit ripening, and biotic and abiotic stresses [1]. Additionally, ethylene treatment accelerates leaf senescence and wilt, while inhibitors of ethylene biosynthesis or its receptor delay senescence [2]. In the early stage of aging, the genes encoding ethylene biosynthesis and other ethylene biosynthesis-related enzymes in aging tissues have been shown to be significantly induced [3]. In Arabidopsis and petunia, it has been found that an ethylene signaling pathway mutant etr1 delays leaf senescence [4,5], the ethylene signaling pathway genes EIN2 and EIN3 positively regulate leaf senescence, and EIN3 increases the expression of chlorophyll degradation genes (NYE1, NYC1, and PAO) by increasing their promoter activity [6]. In addition, EIN3 directly regulates ORE1, a positive regulator of leaf senescence in Arabidopsis. It has been reported that ORE1 elevates expression of the ethylene biosynthesis gene ACS2 during senescence, indicating a positive feedback effect of senescence on ethylene biosynthesis [6].
Senescence is the last stage of plant growth and development, which finally leads to the death of cells, tissues, organs, and the entire organism [7]. Senescence is not only a degradation process, but also a cyclic process, transporting nutrients from senescent cells to young leaves, developing seeds, or storage tissues [7]. At the same time, changes in chlorophyll content, photosynthetic capacities, protein content, malondialdehyde content, proline content, and other substances occur inside the leaves [8]. Lipid degradation is activated during leaf senescence, chloroplast lipids are depleted during senescence, and the degradation products are metabolized into sucrose for transport from senescent leaves to the phloem [9]. In the process of leaf senescence, degradation of existing proteins and new protein synthesis occur simultaneously, but the overall protein content is decreased. Protein degradation plays a key role in the nitrogen cycle during leaf senescence, including the involvement of serine, cysteine, aspartic acid, and threonine proteases in the degradation process [10,11]. A large amount of reactive oxygen species (ROS) are also produced during aging. These toxic ROS can oxidize proteins, membrane lipids, and nucleic acids, trigger membrane lipid peroxidation and ultimately lead to cell damage and death [12]. Moreover, when the antioxidant content in senescent leaves is reduced, the antioxidant enzyme activity and pyridine nucleotide activity also decrease [13], leading to the activation of proteolytic enzymes and protein degradation.
Leaf senescence is a process regulated by numerous genes. At the beginning of senescence, genes related to photosynthesis are downregulated, and the transcription level of senescence-associated genes (SAGs) is upregulated. Thousands of SAGs have been identified that regulate leaf senescence [8]. Transcription factors are a key component of the senescence signal transmission process. It is known that NAC, WRKY, AP2/EREBP, MYB, C2H2, bZIP, and GRAS family transcription factors are mostly upregulated during leaf senescence. The mechanism of ethylene signaling leading to leaf senescence has been extensively studied in Arabidopsis. AtNAP, an Arabidopsis NAC transcription factor, is upregulated during leaf senescence. AtNAP gene knockout significantly delays leaf senescence, while AtNAP-OX plants undergo early senescence [14,15]. As a downstream target gene of AtNAP, SAG113 regulates stomatal movement and water loss during abscisic acid (ABA)-induced leaf senescence [16]. Moreover, AtORE1/ANAC092 has been found to positively regulate leaf senescence [17]. Both ORE1 and AtNAP are downstream of the ethylene signaling proteins EIN2 and EIN3. EIN3 accelerates leaf senescence by promoting the expression of ORE1 and AtNAP but plays a different role in overlapping downstream signaling pathways [18]. WRKY family transcription factors also play an important role in regulating plant leaf senescence [19]. For example, WRKY53 positively regulates leaf senescence. Plants harboring the WRKY53 gene knockout exhibit significantly delayed senescence. WRKY53 transcription levels are upregulated in the early stage of leaf senescence and downregulated in the later stage [20]. Currently, 63 genes have been identified as targets of WRKY53, indicating that this transcription factor is an important upstream regulatory element in the signal transduction of leaf senescence [21]. However, the mechanism of ethylene-regulated leaf senescence in ornamental plants remains to be determined.
Chrysanthemums are globally the second most important ornamental plants in terms of socioeconomic importance [21]. Post-harvest life and quality are important factors for cut flowers. However, leaf yellowing and wilting often occur before the loss of ornamental value of the corolla in chrysanthemums, which greatly shortens vase life. A poor post-harvest environment may cause ethylene production in cut chrysanthemum leaves. Studies have shown that chrysanthemum sensitivity to ethylene varies among varieties [22,23]. The leaves of cut, ethylene-sensitive chrysanthemums are prone to yellowing before floret senescence [24]. Current research on ethylene’s effect on cut chrysanthemum leaf senescence is mainly focused on the physiological level. Nevertheless, the molecular mechanism of ethylene’s effects on the regulation of post-harvest senescence of cut chrysanthemum leaves is still unclear. To provide insight into the molecular mechanisms involved in ethylene-regulated leaf senescence, a new generation of Illumina sequencing platform was used to identify differentially expressed genes (DEGs) in the leaves in response to ethylene treatment. To provide insight into the molecular mechanisms involved in ethylene-regulated leaf senescence, a new generation of Illumina sequencing platform was used to identify differentially expressed genes in the leaves in response to ethylene treatment and the gene expression regulatory networks involved.

2. Materials and Methods

2.1. Plant Materials and Ethylene Treatment

The chrysanthemum cultivars ‘FenDante,’ ‘Nannong Huanglongyu,’ and ‘Fen Luoli’ were obtained from the Chrysanthemum Germplasm Resource Preserving Center of Nanjing Agricultural University, China. Cut chrysanthemums were harvested when the diameter of the top-most bud was 3 cm. Disease-, pest-, and mechanical damage-free flowers and leaves were selected as the experimental materials.
The flower branches were placed in deionized water for rehydration treatment for 24 h before use. Then, the flowering branch was trimmed to 35–38 cm in length, and five leaves beneath the branch point of the flowering branches were kept. The environmental conditions for vase treatment were a temperature of 21 ± 1 °C, humidity of 70 ± 5%, and light intensity of 150 µmol·m−2 s−1.
The rehydrated flowering branches were placed in 350 mL deionized water (DW) + 200 mg·L−1 ethephon, 350 mL DW (control, CK), or 350 mL DW + 1 μL·L−1 1-MCP and sealed in a bag. One hundred milliliters of 1 mol·L−1 NaOH was also sealed in the bag to prevent CO2 accumulation [25]. After 24 h of pretreatment, the flower branches were placed in 350 mL of DW for vase treatment. There were three biological repeats per treatment. Each repeat included three stems, and the water was changed every 2 days during vase treatment.

2.2. Chlorophyll Content and Relative Water Content Measurements

For the determination of chlorophyll content in leaves, the leaves were ground in liquid nitrogen. The pulverized leaves (0.1× g) were transferred to a 10 mL centrifuge tube. A total of two milliliters of dimethyl sulfoxide (DMSO) was added to soak the plant tissue, which was extracted at 65 °C in the dark until the leaves became white or transparent. After cooling, 8 mL of 95% ethanol was added to dilute the DMSO, and the absorbance of the liquid was measured at 663.6 and 646.6 nm. The chlorophyll content (mg·g−1) was calculated as (8.04 × OD663 + 20.2 × OD645) × total volume of extract (L)/fresh weight of material (g).
For relative water content detection, the fresh flowering branches were first weighed, and then reweighed after oven drying. Specifically, the fresh weight measurement was recorded at 19:00 every day. After drying at 80 °C for more than 8 h, the dry weight was recorded. The relative water content was calculated as (fresh weight − dry weight)/fresh weight × 100%.

2.3. RNA Extraction, cDNA Library Construction, and Sequencing

Total RNA was isolated from each sample using RNAiso Plus reagent (TaKaRa Bio, Tokyo, Japan) following the manufacturer’s protocol. The fifth mature leaves treated with ethephon (ETP), 1-MCP, and DW for 0 h, 3 h, and 24 h were used for transcriptome sequencing. There were three biological repeats for each treatment. Each repeat included three leaves from three stems of flowers. In total, 21 samples were collected for sequencing.
For the construction of libraries, pooled RNA was collected by combining 1 µg from each biological replicate. Oligo (dT) magnetic beads were used to enrich the polyA+ mRNA, which was then randomly fragmented with divalent cations in NEB Fragmentation Buffer. Using the fragmented mRNA as a template and random oligonucleotides as primers, first strand cDNA was synthesized using the M-MuLV reverse transcriptase system. The RNA strands were then degraded with RNase H, and DNA polymerase I was used with dNTPs to synthesize the second strand of cDNA. The purified double-stranded cDNA was subjected to end-repair, A-tailing, and ligation of sequencing adapters. The 250–300 bp cDNA was screened with AMPure XP beads, PCR was performed, and the PCR products were purified again with AMPure XP beads to obtain the library. Finally, the cDNA library was sequenced using Illumina HiSeq™ 2500.

2.4. Sequencing Assembly and Annotation

We first removed the end-linker sequence and low-quality reads to obtain clean reads, and then employed the DESeq R package (1.10.1) to analyze the differential expression of the two groups. Fold change (multiple of difference) ≥ 1.5 and FDR < 0.05 were used as the differential gene screening criteria. The KEGG databases were subsequently used mainly for functional annotation and enrichment analyses of the differential genes, as well as prediction and functional annotation of new genes.

2.5. qRT-PCR Analysis

Twelve DEGs were selected from all of the DEGs to validate the reliability of the libraries. The primers that were used for these validations were designed using Primer 5.0 software, and qRT-PCR was performed, as previously described by Ren et al. [26]. A chrysanthemum gene (EF1α, GenBank accession number: KF305681) was selected as a reference gene for normalization. All gene-specific primers used in the qRT-PCR analysis are listed in Table S1. Three independent biological replicates were used for the qRT-PCR analysis.

2.6. Data Analyses

Replicate data were used for all statistical analyses. Standard deviations, standard errors, and figures were calculated or constructed by Microsoft Excel 2013. Analysis of variance (ANOVA) was performed by SPSS statistical analysis software (IBM SPSS Statistics version 23, Chicago, IL, USA). Data are shown as means ± SD (standard deviation). Significant differences (p-value < 0.05) are denoted by different letters (a, b, c, d, and e) according to Duncan test.

3. Results

3.1. Phenotypic Characterization of Cut Chrysanthemum ‘FenDante’ with Ethylene and 1-MCP Treatments

We compared the morphological changes of cut chrysanthemum ‘FenDante’ treated with ethylene and 1-MCP. The leaves displayed a yellowing phenotype by day 7 and completely withered by the 17th day of ET treatment. The leaves of the control group (CK) yellowed by the 17th day, while no morphological changes were observed in the chrysanthemum leaves on the 17th day after 1-MCP treatment (Figure 1A).
The chlorophyll content of ‘FenDante’ with and without treatment of ethylene and 1-MCP was determined. In the vase, the chlorophyll content of ‘FenDante’ showed an overall downward trend; the chlorophyll content of the ET treatment group decreased rapidly and was only 50.9% of the CK group on day 7. The chlorophyll content of the CK group decreased to its lowest level after 17 days in the vase and was 52.5% of the 1-MCP-treated group value (Figure 1B).
During the entire vase senescence process, the relative water contents of cut chrysanthemum ‘FenDante’ after ET and 1-MCP treatments all showed a trend of rising and then falling. The relative water content in the ET treatment group had decreased by 3.28% on the 7th day, that of the CK group had decreased by 1.68%, and that of the 1-MCP treatment group had only decreased by 1.44%. By the 7th day, the relative water content was 85.10%, 86.59%, and 86.29% in the ET, CK and 1-MCP groups, respectively. The relative water content in the CK group had decreased by 4.17% after 17 days in the vase, and that in the 1-MCP treatment group had decreased by 2.65% (Figure 1C).

3.2. Transcriptome Sequencing and Data Analysis

Raw read data (1.04 billion) were generated by RNA-seq, and the Q20 and Q30 values of the original data reached 96.57% and 90.97%, respectively, indicating that the sequencing quality was reliable (Table S2). After filtering out low quality reads, a total of 1.00 billion clean reads were obtained, with an average of 47.68 million clean reads in each sequencing library.
Next, we assigned the genes with reads per kilobase per million mapped reads (RPKM) fold change ≥ 1.5 and false discovery rate (FDR) < 0.05 as DEGs between samples. After 3 h of treatment, 738 DEGs were detected in the ET vs. CK group, in which 498 genes were upregulated, and 240 genes were downregulated. In addition, 39 DEGs were detected in the ET vs. 1-MCP group, of which the expression of 19 genes was upregulated and that of 20 genes was downregulated. Only one gene was differentially expressed in the 1-MCP vs. CK group (Figure 2A). After 24 h of treatment, 196 DEGs were detected in the ET vs. CK group, of which 127 genes were upregulated and 69 genes were downregulated. In the ET vs. 1-MCP group, 2723 DEGs were detected, of which 992 genes were upregulated and 1731 genes were downregulated. In the 1-MCP vs. CK treatment group, 202 DEGs were detected, of which 111 genes were upregulated and 91 genes were downregulated (Figure 2B) (Table 1).

3.3. DEGs Related to Plant Hormone Biosynthesis and Signaling

To gain insight into the types of transcripts regulated temporally and with different treatments, we performed Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Plant hormones play an important role in senescence, and different hormones affect and regulate plant senescence comprehensively. In order to clarify the preliminary changes in the expression patterns of hormone-related genes during the senescence of cut chrysanthemum ‘FenDante’ leaves treated with ethylene, all DEGs were compared to the plant hormone database, and a total of 75 hormone-related genes were identified. DEGs of the ET biosynthesis pathway include the ACC oxidase synthesis gene ACO (evm.TU.scaffold_1307.49, evm.TU.scaffold_1005.154, evm.TU.scaffold_693.12), ethylene precursor methionine metabolism pathway gene CGS1 (evm.TU.scaffold_397.4), MTK (evm.TU.scaffold_558.34), and HMT2 (evm.TU.scaffold_906.121). The abovementioned six genes were all significantly upregulated after 3 h of ET treatment, and most of them were downregulated after 3 h of 1-MCP treatment. All six DEGs were upregulated after 24 h of ET treatment, while their expressions were downregulated after 24 h of 1-MCP treatment (Figure 3A).
There were six DEGs involved in the biosynthetic pathway of indole-3-acetic acid (IAA), namely YUC (evm.TU.scaffold_1473.122, evm.TU.scaffold_858.112) and the cytochrome CYP83B (evm.TU.scaffold_6797.99, evm.TU.scaffold_10615.103, evm.TU.scaffold_1759.395, evm.TU.scaffold_1759.359). All these DEGs were upregulated after 3 h of ET treatment, and three of them were still upregulated after 24 h of ET treatment; however, the six DEGs were all downregulated by 24 h of 1-MCP treatment (Figure 3B).
A total of three genes involved in the jasmonate (JA) biosynthesis pathway, namely AOS (evm.TU.scaffold_598.578), ACX (evm.TU.scaffold_1573.604), OPCL1 (evm.TU.scaffold_11614.18), were found to be differentially expressed. They were all upregulated after 3 h and 24 h of ET treatment, while AOS and OPCL1 were downregulated and ACX was upregulated after 24 h of 1-MCP treatment (Figure 3C).
Twenty-three DEGs were involved in the ET signal transduction pathway, including seven ETR (ethylene receptor), ten EBF1/2, one EIN2, two ERF1 (ethylene response factor), and three CTR1 genes. The EBF homologous genes and ERF1 were all significantly upregulated after 3 h of ET treatment, EIN2 was downregulated after 3 h of ET treatment. Two EBF homologous genes (evm.TU.scaffold_1260.358, evm.TU.scaffold_821.114) and EIN2 were downregulated and the remaining 20 DEGs were all significantly upregulated after 24 h of ET treatment. The expression pattern at 24 h of 1-MCP treatment was exactly the opposite. Only two EBF homologous genes (evm.TU.scaffold_1260.358, evm.TU.scaffold_821.114) and EIN2 were upregulated, while ETR, CTR1, eight EBF homologous genes, and ERF1 were all downregulated (Figure 3D).
The DEGs of the IAA signal transduction pathway include the auxin input vector AUX1 (evm.TU.scaffold_3576.25), the auxin/indole acetate protein synthesis gene AUX/IAA (evm.TU.scaffold_11460.11, evm.TU.scaffold_1546.90, evm.TU.scaffold_1590.15, evm.TU.scaffold_1537.72, evm.TU.scaffold_1047.68), GH3 (evm.TU.scaffold_409.13, evm.TU.scaffold_10628.77), and the auxin response gene SAUR (evm.TU.scaffold_8912.124, evm.TU.scaffold_591.88, evm.TU.scaffold_1461.20, evm.TU.scaffold_878.26, evm.TU.scaffold_2951.63, evm.TU.scaffold_9187.12). AUX1, two AUX/IAA homologous genes (evm.TU.scaffold_1537.72 and evm.TU.scaffold_1047.68), one GH3 homologous gene (evm.TU.scaffold_409.13), and three SAUR homologous genes (evm.TU.scaffold_8912.124, evm.TU.scaffold_591.88, evm.TU.scaffold_1461.20) were significantly upregulated after 3 h of ET treatment. One GH3 homologous gene and three SAUR homologous genes were downregulated after 3 h of ET treatment. Five AUX/IAA homologous genes and six SAUR homologous genes were upregulated and one GH3 was downregulated after 3 h of 1-MCP treatment. After 24 h of ET treatment, AUX1 and two AUX/IAA homologous genes (evm.TU.scaffold_11460.11 and evm.TU.scaffold_1546.90), one GH3 homologous gene (evm.TU.scaffold_10628.77), and two SAUR homologous genes (evm.TU.scaffold_591.88 and evm.TU.scaffold_1461.20) were downregulated. Three AUX/IAA genes (evm.TU.scaffold_1590.15, evm.TU.scaffold_1537.72, evm.TU.scaffold_1047.68), one GH3 homologous gene (evm.TU.scaffold_409.13), and two SAUR homologs (evm.TU.scaffold_878.26 and evm.TU.scaffold_2951.63) were upregulated after 24 h of ET treatment. The gene expression patterns under 1-MCP treatment were exactly the opposite of those under ET treatment (Figure 3E).
The DEGs of the JA signal transduction pathway include JAR1 (evm.TU.scaffold_225.470, evm.TU.scaffold_3417.27, evm.TU.scaffold_362.152, evm.TU.scaffold_24.21, evm.TU.scaffold_841.30), JAZ (evm.TU.scaffold_10085.36, evm.TU.scaffold_11623.7, evm.TU.scaffold_790.182, evm.TU.scaffold_10243.115, evm.TU.scaffold_436.182, evm.TU.scaffold_911.171), and MYC2 (evm.TU.scaffold_782.119). JAR1 and two JAZ homologous genes (evm.TU.scaffold_10243.115 and evm.TU.scaffold_911.171) were significantly upregulated after 3 h of ET treatment, while the other four JAZ homologous genes were downregulated. Three JAR1 were upregulated after 3 h of 1-MCP treatment. After 24 h of ET treatment, JAR1 and two JAZ homologous genes (evm.TU.scaffold_10243.115 and evm.TU.scaffold_911.171) were upregulated, while the other four JAZ homologous genes and MYC2 were downregulated. The gene expression patterns under 1-MCP treatment were exactly the opposite of those under ET treatment (Figure 3F).

3.4. DEGs Related to Chlorophyll Synthesis and Degradation and the ROS Signaling Pathway

Leaf senescence is accompanied by chlorophyll degradation, manifested as leaf yellowing. Based on the literature, the DEGs related to chlorophyll synthesis and degradation were analyzed. These genes are the iron chelator HEmH (evm.TU.scaffold_8644.141), magnesium chelator subunit CHLI (evm.TU.scaffold_1489.582, evm.TU.scaffold_1266.202), the magnesium-protoporphyrin IX monomethyl ester oxidase CRD1 (evm.TU.scaffold_831.87, evm.TU.scaffold_297.143), REM41 (evm.TU.scaffold_1626.2), the glutamyl-tRNA reductase HEM11 (evm.TU.scaffold_12108.48), the chlorophyll oxygenase CAO (evm.TU.scaffold_234.430, evm.TU.scaffold_749.326), and the chlorophyll reductase POR (evm.TU.scaffold_1265.237). HEM11 and POR were significantly downregulated after 3 h of ET treatment, but there were no significant changes after 3 h of 1-MCP treatment. The DEGs of the porphyrin and chlorophyll synthesis pathway were all downregulated after 24 h of ET treatment, and all were upregulated after 24 h of 1-MCP treatment (Figure 4A).
The DEGs in the chlorophyll degradation pathway include SGR (evm.TU.scaffold_5200.41, evm.TU.scaffold_2875.14, evm.TU.scaffold_4190.92), the chlorophyll b reductase NYC1/NOL (evm.TU.scaffold_11816.158), the pheophytinase PPH (evm.TU.scaffold_234.138, evm.TU.scaffold_2712.47), and the pheophytinase PAO (evm.TU.scaffold_94.182, evm.TU.scaffold_1785.133). These DEGs showed no changes in their expression levels after 3 h of ET treatment, and PAO was downregulated after 3 h of 1-MCP treatment. After 24 h of ET treatment, two SGRs homologous genes, NYC1/NOL, and PPH were all significantly downregulated and PAO was upregulated. Two SGRs homologous genes, NYC1/NOL, and PPH were upregulated after 24 h of 1-MCP treatment, while one SGR homologous gene and PAO were downregulated (Figure 4A).
ROS are by-products of aerobic metabolism in plants. The DEGs of the ROS metabolic pathway include peroxidase POD genes (evm.TU.scaffold_991.119, evm.TU.scaffold_1298.159, evm.TU.scaffold_6083.15, evm.TU.scaffold_1678.120, evm.TU.scaffold_9340.552, evm.TU.scaffold_1598.26, evm.TU.scaffold_2548.395, evm.TU.scaffold_2548.395, evm.TU.scaffold_320.66, evm.TU.scaffold_1146.444, evm.TU.scaffold_322.246, evm.TU.scaffold_1730.51, evm.TU.scaffold_10665.38), the calcium-dependent protein kinase gene CDPK (evm.TU.scaffold_6371.10, evm.TU.scaffold_1817.3), and the polyphenol oxidase gene PPO (evm.TU.scaffold_448.480). Seven POD homologous genes, the CDPKs, and PPO were upregulated and three POD homologous genes were downregulated after 3 h of ET treatment. Seven POD homologous genes, the CDPKs, and PPO were upregulated after 24 h of ET treatment. The gene expression patterns under 1-MCP treatment were exactly the opposite of those under ET treatment (Figure 4B).

3.5. DEGs Related to Aquaporins

Aquaporins (AQPs) are a family of water-transporting proteins expressed in the cell membranes of many prokaryotic/eukaryotic organisms and tissues [27]. In response to ethylene treatment, three types of AQPs, namely the tonoplast intrinsic protein gene TIP (evm.TU.scaffold_4532.3, evm.TU.scaffold_1537.62, evm.TU.scaffold_1172.150), the plasma membrane intrinsic protein PIP gene (evm.TU.scaffold_4503.19, evm.TU.scaffold_9053.40, evm.TU.scaffold_9088.143, evm.TU.scaffold_1056.136, evm.TU.scaffold_1012.92), and the small molecule basic membrane intrinsic protein SIP (evm.TU.scaffold_977.233) were differentially expressed. TIP was downregulated and SIP was upregulated after 3 h and 24 h of ET treatment; in contrast, TIP was significantly upregulated after 24 h of 1-MCP treatment, and SIP was downregulated after 3 h and 24 h of 1-MCP treatment (Figure 4C).

3.6. Identification of Differentially Expressed Transcription Factors (TFs) with Ethylene and 1-MCP Treatment

Leaf senescence is a complex gene regulation process, and transcription factors play an important role in this regulatory network. To further analyze the gene regulatory network of chrysanthemum leaves in response to ethylene signals, the TFs among the DEGs were examined, and 476 differentially expressed TFs were identified. Among the differentially expressed TF family, AP2/ERF had the most members, followed by bHLH, orphans, and MYB (Figure 5A).
Heat map analysis of the transcription factors identified AP2/ERF, NAC, WRKY, MYB, AUX/IAA, and bHLH as differentially expressed in response to ethylene. Nineteen NAC genes were upregulated and the other three NAC members were downregulated by ethylene (Figure 5B). Four WRKY genes (evm.TU.scaffold_4861.88, evm.TU.scaffold_9028.9, evm.TU.scaffold_1403.116, evm.TU.scaffold_1298.48) were upregulated after ET treatment. Four additional WRKY genes (evm.TU.scaffold_611.105, evm.TU.scaffold_4488.44, evm.TU.scaffold_1089.137, evm.TU.scaffold_1064.274) were upregulated only after 24 h of ET treatment, and nine WRKY genes (evm.TU.scaffold_138.51, evm.TU.scaffold_3800.95, evm.TU.scaffold_1749.144, evm.TU.scaffold_566.439, evm.TU.scaffold_811.89, evm.TU.scaffold_11341.15, evm.TU.scaffold_1198.298, evm.TU.scaffold_1425.29, evm.TU.scaffold_1097.232) were upregulated only after 3 h of ET treatment. In addition, there were three WRKY genes (evm.TU.scaffold_566.407, evm.TU.scaffold_956.8, evm.TU.scaffold_858.536) that were decreased after 3 h of ET treatment (Figure 5B). Of the 32 MYB transcription factors, 11 were significantly upregulated after 24 h of ET treatment, nine were significantly upregulated after 24 h of 1-MCP treatment, and four were significantly upregulated after 3 h of ET treatment (Figure 5B). Among the 55 AP2/ERF genes with differential expression, 41 were upregulated and 14 were downregulated after 3 h of ET treatment (Figure 5B). Among the 16 AUX/IAA transcription factors, 12 were upregulated by ET treatment (Figure 5B). Among the 43 bHLH transcription factors, 26 were upregulated and 17 were downregulated after 24 h of 1-MCP treatment (Figure 5B).

3.7. Validation of the DEGs by Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR)

To validate expression of the DEGs, 12 genes were randomly selected and quantified by using qRT-PCR. The results showed that these genes demonstrated expression profiles highly consistent with the expression levels generated from the RNA-seq analysis, suggesting that the RNA-seq data are reliable (Figure 6).

3.8. Expression Patterns of Transcription Factor DEGs in Leaves of Different Ethylene-Sensitive Cultivars

In order to determine if the expression patterns of the DEGs are conserved across different cultivars, the expression patterns of representative DGEs were detected in the ethylene-sensitive cut chrysanthemum ‘Nannong Huanglongyu’ and ethylene-insensitive ‘Fen Luoli’ subjected to ET or 1-MCP treatment (Figure 7 and Figure S1). In general, the DEGs can be divided into three categories: upregulated at the early (3 h) stage after ethylene treatment, upregulated at both early and late stages showing a “double peak”, and downregulated after ethylene treatment.
After ET treatment, the ethylene signaling pathway-related genes EBF2, CTR1, and EIN2 and transcription factor NAC100 were significantly upregulated at the early stage (3 h after treatment). In ethylene-sensitive ‘Nannong Huanglongyu,’ the EBF2 level was 14.8 times, the CTR1 level was 9.8 times, the EIN2 level was 10.7 times, and the NAC100 level was 5.7 times that in the CK group after 3 h of ET treatment. However, the expression levels of these four genes in ethylene-insensitive ‘Fen Luoli’ were slightly elevated and were 4.9 times, 3.9 times, 1.2 times, and 4 times that in the CK group after 3 h of ET treatment, respectively. After 18 h of treatment, the expression levels of EBF2, CTR1, EIN2, and NAC100 tended to stabilize. In summary, the four genes EBF2, CTR1, EIN2, and NAC100 could be induced in both ethylene-sensitive and -insensitive cut chrysanthemum varieties at the early stage (3 h), and their expression levels tended to plateau at the later stage.
The expression patterns of the transcription factors ERF92, RAP2-2, and NAC72 in response to ethylene treatment showed a “double peak”. In the ethylene-sensitive chrysanthemum ‘Nannong Huanglongyu,’ the expression levels of ERF92, RAP2-2, and NAC72 reached peaks after 3 h of ET treatment, which were 9.8 times, 4.4 times, and 3.8 times the levels in the CK group, respectively. After 12 h of ET treatment, RAP2-2 and NAC72 reached their second peaks, which were 3.7 times and 3.6 times the levels in the CK group, respectively. ERF92 expression reached its second peak after 18 h of ET treatment, which was 10 times the level in the CK group. In the ethylene-insensitive ‘Fen Luoli,’ the expression levels of ERF92, RAP2-2, and NAC72 reached their peaks at 6 h of ET treatment, which were 7.4 times, 2.2 times, and 2.1 times the levels in the CK group respectively. After ET treatment for 18 h, ERF92, RAP2-2, and NAC72 reached their second peak, which was 5.5 times, 1.6 times, and 1.6 times the level in the CK group, respectively. The above results indicate that the transcription factors ERF92, RAP2-2, and NAC72 in the ethylene-sensitive variety ‘Nannong Huanglongyu’ have a rapid response at the early stage (3 h) of ET treatment, and then, under the continuous effect of ethylene, the expression level increased again at the later stage. However, in the ethylene-insensitive cultivar ‘Fen Luoli,’ the ethylene response was delayed, and only a slight increase in the expression was observed.
The expression of bHLH63 and PIP1 in ‘Nannong Huanglongyu’ increased significantly after 1-MCP treatment. bHLH63 reached its highest peak at 12 h of 1-MCP treatment, which was 12.4 times that of the CK group; it reached its lowest peak after 12 h of ET treatment, which was 0.4 times that of the CK group. PIP1 in ‘Nannong Huanglongyu’ reached its highest peak at 6 h of 1-MCP treatment, which was 2.6 times that of the CK group; after 3 h of ET treatment, it reached its lowest peak, which was 0.2 times that of the CK group. In ‘Fen Luoli,’ bHLH63 reached its highest level at 12 h of 1-MCP treatment, which was 1.2 times that of the CK group, while PIP1 reached its highest peak at 18 h of 1-MCP treatment, which was 1.9 times that of the CK group. In summary, bHLH63 and PIP1 might be negatively responsive to ethylene. ET treatment inhibits the transcriptional activity of bHLH63 and PIP1 in both ethylene-sensitive and -insensitive cut chrysanthemum varieties, while 1-MCP induces their expression.

4. Discussion

The preservation of cut flowers after harvesting has always been an urgent problem for ornamental horticulture. Separating the flowers from the plant and poor environmental conditions during post-harvest storage and transportation may also cause the production of ethylene in the cut flowers, which affects their vase quality. Studies have shown that different varieties of chrysanthemums are differentially sensitive to ethylene [22,23]. The leaves of ethylene-sensitive cut chrysanthemums are prone to turn yellow before the florets senescence [24]. Our previous research found that the chrysanthemum ‘FenDante’ is sensitive to ethylene, leaf yellowing and wilting is induced by ET, and the ethylene inhibitor 1-MCP could effectively delay leaf senescence. Therefore, we used RNA-seq technology to reveal the gene expression changes in the ethylene-sensitive chrysanthemum ‘FenDante’ in response to ET and 1-MCP treatment after cutting.

4.1. DEGs Involved in Chlorophyll Synthesis and Degradation

The senescence process of cut flowers after harvest is manifested as leaf chlorophyll degradation, yellowing, and withering. Leaf chlorophyll degradation is an important physiological indicator of cut chrysanthemum senescence. There is a significant negative correlation between chlorophyll content and senescence [28]. In this study, the chlorophyll content in the cut chrysanthemum ‘FenDante’ leaves was significantly reduced in the vase after ethylene treatment. The expression of most chlorophyll synthesis-related genes, such as the glutamyl-tRNA reductase HEM11 and chlorophyll reductase POR, was also significantly reduced, and the expression of some chlorophyll degradation-related genes, such as SGRs and the chlorophyll b reductase NYC1/NOL, increased. Arabidopsis SGR1 and SGRL overexpression induces an early leaf-yellowing phenotype, while sgr1/nye1-1 and sgrl mutants display a stay-green phenotype under abiotic stress conditions [29,30]. Ethylene may accelerate chlorophyll degradation and inhibit chlorophyll synthesis by regulating chlorophyll synthesis and degradation-related genes to accelerate the post-harvest senescence process of cut chrysanthemum ‘FenDante.’

4.2. DEGs Related to Aquaporins

Aquaporins are considered to play an important role in the regulation of plant water transport, increasing the water permeability of cell membranes and promoting water transport [31,32]. Studies have indicated that ethylene promotes water transport in poplar roots under hypoxic stress, and it has been speculated that ethylene enhances root aquaporin activity [33]. In cut roses, ethylene was found to inhibit petal aquaporin activity at the transcriptional level, thereby significantly inhibiting petal cell elongation, reducing petal water content, and promoting flower opening [34,35]. In Arabidopsis, it was found that ethylene inhibits aquaporin activity at the transcriptional level [36], but at the post-translational level, it enhances aquaporin phosphorylation to improve aquaporin activity, leading to accelerated expansion/contraction of protoplasts in response to water loss [37,38]. The above studies show that the response of aquaporins to ethylene differs in different species and organs. In this study, the water content of the flower branches of cut chrysanthemum ‘FenDante’ increased under different treatments during the early vase stage, indicating that water transport in the flower branches was smooth at this time and the water absorption rate was greater than the leaf transpiration loss. Subsequently, the water content of the flowering branches in the ethylene treatment group decreased significantly, and the three types of AQPs found in this study and two types of TIP and PIP were downregulated. It is speculated that ethylene inhibits the transcription levels of TIP and PIP. Therefore, the permeability of the cell membrane prevents water transport and accelerates water loss in leaves of the cut chrysanthemum ‘FenDante.’

4.3. DEGs Involved in Plant Hormones ET, JA, and IAA Biosynthesis and Signaling

After ET treatment, the expression of ACO related to ethylene biosynthesis and the ethylene precursor methionine metabolism pathway genes CGS1, MTK, and HMT2 increased, which is inferred to be related to the significant increase in ethylene production in leaves with ethylene treatment. Studies have reported that ORE1, a direct target of the ethylene signaling pathway gene EIN3 in Arabidopsis thaliana, can induce the expression of the ethylene biosynthesis gene ACS2 during leaf senescence, indicating that aging also has a positive feedback on ethylene biosynthesis. Similarly, the expression of ethylene signaling pathway genes promotes ethylene biosynthesis [6]. JA is also a typical plant hormone that promotes senescence. The JA biosynthesis genes AOS, ACX, and OPCL1 are significantly upregulated after ethylene treatment, indicating that ET induces JA synthesis and accelerates the senescence of cut chrysanthemum leaves. It has been reported that in the absence of JA, JAZ protein binds to the MYC2 transcription factor to form a complex through the repressor protein TPL to inhibit the transcription of early JA response genes. In the presence of JA, JAZ protein is subsequently degraded when it binds to COI1, and the NINJA-TPL complex separates from the MYC2 transcription factor to initiate the transcription of JA response genes [39,40]. Therefore, in the JA signaling pathway, JAR1 plays a positive regulatory role in the leaf senescence process in response to ET, while downregulated JAZ and MYC2 play a negative regulatory role. The JA pathway may play a key role in the leaf senescence process in response to ET. A previous study found that, compared with wild-type Arabidopsis, transgenic Arabidopsis overexpressing YUCCA6 showed a phenotype of increased auxin levels and delayed leaf senescence [41]. Consistent with these reports, our results showed that the expression of the YUC (evm.TU.scaffold_1473.122) gene was downregulated under 24 h of ET treatment (Figure 8A).

4.4. DEGs of Transcription Factors

In addition, the differentially expressed transcription factors identified in response to ethylene treatment included NAC72, NAC100, ERF92, RAP2-2, bHLH63, and bHLH130. Grapevine VvNAC72 negatively modulates detoxification of methylglyoxal through repression of VvGLYI-4, thus enhancing resistance to downy mildew [42]; ghr-miR164 can also directly cleave GhNAC100 mRNA in the post-transcriptional process to positively regulate Verticillium dahliae resistance [43]. ERF92 is selective for temperature stress tolerance in peas (Pisum sativum L.) [44]. In Arabidopsis, resistance to submergence-induced hypoxia involves upregulation of RAP2.2 through interaction of WRKY33 and WRKY12 [45,46]. However, the functions of these six differentially expressed transcription factors in the leaf senescence of ornamental plants remain unclear. In the future, analyses of these candidate genes are required to reveal the ET-mediated molecular mechanisms underlying the leaf senescence process.

5. Conclusions

Using cut chrysanthemum ‘FenDante’ as the material, through RNA-Seq analysis, we discovered that ethylene affects the expression of pathway-related genes such as plant hormone biosynthesis and signal transduction, chlorophyll synthesis and degradation, aquaporin and reactive oxygen species, such as plant hormone pathway genes (e.g., ACO, CGS1, and ERF1), chlorophyll synthesis and degradation (e.g., HEMM11, POP, and PAO), aquaporin (e.g., PIP1), ROS (e.g., PER51). Especially, senescence-related transcription factors (e.g., NAC100, ERF92, RAP2-2, bHLH63 and NAC72) are conserved in different varieties in response to ET treatment (Figure 8B). The gene expression regulatory networks in the leaves of post-harvest cut chrysanthemums in response to ethylene treatment were studied, which provides valuable information about research on the molecular mechanisms of ethylene-mediated leaf senescence in cut chrysanthemums.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae8070573/s1, Figure S1: Vase quality of ethylene-sensitive cut chrysanthemum ‘Nannong Huanglongyu’ and ethylene-insensitive ‘Fen Luoli’; Table S1: Primer sequence for qRT-PCR; Table S2: RNA sequencing data output; Table S3: The aligning result of sequencing data output.

Author Contributions

Conceptualization, S.C.; Investigation, X.Z., C.X., R.L., Y.C. and Z.Q.; Supervision, S.C.; Writing–original draft, R.L. and X.Z.; Writing–review and editing, S.C. and L.W. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Key Research and Development Program of China, 2018YFD1000400; the National Natural Science Foundation of China, 32030098.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The datasets generated during the current study were submitted to the NCBI repository, bioproject PRJNA809838. NCBI. https://dataview.ncbi.nlm.nih.gov/object/PRJNA809838 (accessed on 2 April 2022).

Acknowledgments

This work was supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions, PAPD.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Phenotypic characterization of cut chrysanthemum ‘FenDante’ with ethylene and 1-MCP treatment. (A) Vase quality of cut chrysanthemum ‘FenDante’ with ethylene and 1-MCP treatment. (a) Vase quality after 7 days of vase treatment; (b) Vase quality after 17 days of vase treatment. Black squares indicate the corresponding magnified images. Chlorophyll content (B) and relative water content (C) of cut chrysanthemum ‘FenDante’ with ethylene or 1-MCP treatment. 1-MCP, 1-methylcyclopropene.
Figure 1. Phenotypic characterization of cut chrysanthemum ‘FenDante’ with ethylene and 1-MCP treatment. (A) Vase quality of cut chrysanthemum ‘FenDante’ with ethylene and 1-MCP treatment. (a) Vase quality after 7 days of vase treatment; (b) Vase quality after 17 days of vase treatment. Black squares indicate the corresponding magnified images. Chlorophyll content (B) and relative water content (C) of cut chrysanthemum ‘FenDante’ with ethylene or 1-MCP treatment. 1-MCP, 1-methylcyclopropene.
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Figure 2. Venn diagrams of differentially expressed genes. (A) Numbers of differentially expressed genes after 3 h of ethylene or 1-MCP treatment. (B) Numbers of differentially expressed genes after 24 h of ethylene or 1-MCP treatment. 1-MCP, 1-methylcyclopropene.
Figure 2. Venn diagrams of differentially expressed genes. (A) Numbers of differentially expressed genes after 3 h of ethylene or 1-MCP treatment. (B) Numbers of differentially expressed genes after 24 h of ethylene or 1-MCP treatment. 1-MCP, 1-methylcyclopropene.
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Figure 3. DEGs of plant hormone biosynthesis and signaling. (A) Ethylene biosynthesis-related genes. (B) IAA biosynthesis-related genes. (C) JA biosynthesis-related genes. (D) Ethylene signaling-related genes. (E) IAA signaling-related genes. (F) JA signaling-related genes. Different color bands represent the different expression levels of the individual samples. The red bands represent high expression levels and the blue bands represent low expression levels. DEGs, differentially expressed genes; IAA, indole-3-acetic acid; JA, jasmonate.
Figure 3. DEGs of plant hormone biosynthesis and signaling. (A) Ethylene biosynthesis-related genes. (B) IAA biosynthesis-related genes. (C) JA biosynthesis-related genes. (D) Ethylene signaling-related genes. (E) IAA signaling-related genes. (F) JA signaling-related genes. Different color bands represent the different expression levels of the individual samples. The red bands represent high expression levels and the blue bands represent low expression levels. DEGs, differentially expressed genes; IAA, indole-3-acetic acid; JA, jasmonate.
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Figure 4. Heatmap analysis of DEGs involved in various processes. Chlorophyll synthesis and degradation (A), ROS (reactive oxygen species) (B), and aquaporin signaling pathway (C) are shown. DEGs, differentially expressed genes.
Figure 4. Heatmap analysis of DEGs involved in various processes. Chlorophyll synthesis and degradation (A), ROS (reactive oxygen species) (B), and aquaporin signaling pathway (C) are shown. DEGs, differentially expressed genes.
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Figure 5. Identification of transcription factors (TFs) differentially expressed with ethylene and 1-MCP treatment. (A) Distribution of transcription factor family members in differentially expressed genes. (B) Heatmap analysis of transcription factor family genes in post-harvest cut chrysanthemum in response to ethylene treatment. (a) NAC; (b) WRKY; (c) AP2/ERF; (d) MYB; (e) AUX/IAA; and (f) bHLH. Red represents high expression and green represents low expression.
Figure 5. Identification of transcription factors (TFs) differentially expressed with ethylene and 1-MCP treatment. (A) Distribution of transcription factor family members in differentially expressed genes. (B) Heatmap analysis of transcription factor family genes in post-harvest cut chrysanthemum in response to ethylene treatment. (a) NAC; (b) WRKY; (c) AP2/ERF; (d) MYB; (e) AUX/IAA; and (f) bHLH. Red represents high expression and green represents low expression.
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Figure 6. Quantitative reverse transcription PCR validation and RNA-seq data of selected DEGs in cut chrysanthemum ‘FenDante’. DEGs, differentially expressed genes.
Figure 6. Quantitative reverse transcription PCR validation and RNA-seq data of selected DEGs in cut chrysanthemum ‘FenDante’. DEGs, differentially expressed genes.
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Figure 7. Analysis of DEG expression patterns between ethylene-sensitive and-insensitive varieties. DEG, differentially expressed gene.
Figure 7. Analysis of DEG expression patterns between ethylene-sensitive and-insensitive varieties. DEG, differentially expressed gene.
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Figure 8. Working model of ethylene (ET)-mediated chrysanthemum leaf senescence. (A) Ethylene-induced leaf senescence module by regulating other plant hormones. (B) ET may promote senescence of chrysanthemum leaves by regulating genes that are mainly related to plant hormone biosynthesis and signaling (e.g., ACO, CGS1, and ERF1), chlorophyll synthesis and degradation (e.g., HEMM11, POP, and PAO), aquaporin (e.g., PIP1), ROS (e.g., PER51), and senescence-associated transcription factors (TFs) (e.g., NAC72, NAC100, ERF92, RAP2-2, bHLH63, and bHLH130). Red arrows represent upregulation, and green arrows represent downregulation. ROS, reactive oxygen species.
Figure 8. Working model of ethylene (ET)-mediated chrysanthemum leaf senescence. (A) Ethylene-induced leaf senescence module by regulating other plant hormones. (B) ET may promote senescence of chrysanthemum leaves by regulating genes that are mainly related to plant hormone biosynthesis and signaling (e.g., ACO, CGS1, and ERF1), chlorophyll synthesis and degradation (e.g., HEMM11, POP, and PAO), aquaporin (e.g., PIP1), ROS (e.g., PER51), and senescence-associated transcription factors (TFs) (e.g., NAC72, NAC100, ERF92, RAP2-2, bHLH63, and bHLH130). Red arrows represent upregulation, and green arrows represent downregulation. ROS, reactive oxygen species.
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Table
Table 1. Gene number statistics of different expression gene.
Table 1. Gene number statistics of different expression gene.
DEG SetAll DEGUp-RegulatedDown-Regulated
3 h ET vs. 3 h CK738498240
3 h 1-MCP vs. 3 h CK110
3 h ET vs. 3 h 1-MCP391920
24 h ET vs. 24 h CK19612769
24 h 1-MCP vs. 24 h CK20211191
24 h ET vs. 24 h 1-MCP27239921731
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Liu, R.; Zuo, X.; Chen, Y.; Qian, Z.; Xu, C.; Wang, L.; Chen, S. Transcriptional Regulation in Leaves of Cut Chrysanthemum (Chrysanthemum morifolium) ‘FenDante’ in Response to Post-Harvest Ethylene Treatment. Horticulturae 2022, 8, 573. https://doi.org/10.3390/horticulturae8070573

AMA Style

Liu R, Zuo X, Chen Y, Qian Z, Xu C, Wang L, Chen S. Transcriptional Regulation in Leaves of Cut Chrysanthemum (Chrysanthemum morifolium) ‘FenDante’ in Response to Post-Harvest Ethylene Treatment. Horticulturae. 2022; 8(7):573. https://doi.org/10.3390/horticulturae8070573

Chicago/Turabian Style

Liu, Rui, Xuele Zuo, Yu Chen, Ziyan Qian, Can Xu, Likai Wang, and Sumei Chen. 2022. "Transcriptional Regulation in Leaves of Cut Chrysanthemum (Chrysanthemum morifolium) ‘FenDante’ in Response to Post-Harvest Ethylene Treatment" Horticulturae 8, no. 7: 573. https://doi.org/10.3390/horticulturae8070573

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