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Article

Functional Validation and Promoter DNA Methylation Analysis of the OfPAO Gene of Osmanthus fragrans ‘Yinbi Shuanghui’

by
Rui Wang
1,2,
Yixiao Zhou
1,2,
Xuan Chen
3,
Hao Wei
1,2,
Dong Zheng
1,2,
Wuwei Zhu
1,2,
Lianggui Wang
1,2,* and
Xiulian Yang
1,2,*
1
Key Laboratory of Landscape Architecture, College of Landscape Architecture, Nanjing Forestry University, No. 159 Longpan Road, Nanjing 210037, China
2
Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China
3
College of Fine Arts, Nanjing Normal University of Special Education, No. 1 Shennong Road, Nanjing 210038, China
*
Authors to whom correspondence should be addressed.
Forests 2024, 15(1), 11; https://doi.org/10.3390/f15010011
Submission received: 14 November 2023 / Revised: 9 December 2023 / Accepted: 11 December 2023 / Published: 20 December 2023
(This article belongs to the Section Genetics and Molecular Biology)
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Abstract

:
Osmanthus fragrans ‘Yinbi Shuanghui’ is a colored leaf variety of O. fragrans. To study the mechanism of leaf color formation in O. fragrans ‘Yinbi Shuanghui’, we selected green and colored leaves with two different parts, namely yellow and green, as our research materials. We analyzed the expression changes related to leaf color in genes by performing qRT-PCR in the different leaf parts, finding that OfPAO was significantly up-regulated in the yellow part of colored leaves, and we initially determined that OfPAO was the key gene involved in the formation of colored leaves. Then, we constructed an OfPAO overexpression vector, before transforming it into tobacco through an Agrobacterium-mediated transformation to obtain transgenic plants. We found that the transgenic tobacco leaf color of OfPAO was lighter than that of the null carrier, the chlorophyll content in leaves decreased, and the expression of genes involved in the chlorophyll degradation pathway in OfPAO transgenic tobacco was up-regulated, suggesting that OfPAO regulates chlorophyll degradation, leading to changes in leaf color. According to the results of transcriptome sequencing and the genome data of O. fragrans ‘Rixianggui’, we cloned CDS and the promoter sequence of OfPAO, and the promoter regions 901-1307 of the OfPAO were sequenced through bisulfite genomic sequencing PCR (BSP), finding that the methylation level of CHH in the yellow part of colored leaves was lowest in colored and green leaves at 145 bp. The methylation of CHH in the promoter of OfPAO in O. fragrans ‘Yinbi Shuanghui’ was negatively correlated with the gene expression level, suggesting that the methylation of the promoter of OfPAO may regulate the expression of OfPAO, affecting chlorophyll degradation in the leaves.

1. Introduction

Osmanthus fragrans ‘Yinbi Shuanghui’ is a leaf color mutant of the O. fragrans ‘Sijigui’ plant, known for its yellow and green variegated leaves. Leaf color mutants are widely found in higher plants, which are ideal materials for studying physiological and metabolic processes, such as plant photosynthesis, photochemical functions, plant hormones, and disease-resistance mechanisms [1,2,3], and they also provide a new direction for future garden plant breeding. In the preliminary research carried out by our group, transcriptome sequencing and physiological indexes were performed on the green and colored leaves of this plant. The two related data items were analyzed via a WGCNA (weighted gene co-expression network); the differentially expressed genes of chlorophyll metabolism, carotenoid metabolism, secondary metabolism, and chloroplast development (related to leaf color) were screened; and the correlations between physiological indexes and the differentially expressed genes were analyzed [4]. We found significant differences in the chlorophyll contents of leaves found in different leaf color parts, and, at the same time, we found that the OfPAO expression was significantly different in leaf parts with different leaf colors when using qRT-PCR.
Chlorophyll enables plants to perform absorption from light energy during photosynthesis and a key substance involved in determining the plant leaf color phenotype, and the synthesis and degradation of chlorophyll is considered to be a key pathway for the study of the formation of leaf color [5,6], while the chlorophyll degradation pathway has been less thoroughly studied. Pheophorbide a oxidase (PAO) is a key enzyme involved in in chlorophyll degradation, as it can catalyze the degradation of demagnesium chlorophyll a into chlorophyll catabolites, the reduction in green color, and the formation of colorless material [7,8]. PAO encodes a demagnesium chlorophyll a mono-oxygenase localized in the cyst-like membranes of the chloroplasts, and it is organized to contain a Rieske-type structural domain, an N-terminal chloroplast transit peptide sequence and a mononuclear iron-binding structural domain, the structure of which is highly conserved in plants [8]. In a past study, leaves of the eas1 mutant in rice showed a yellowish or brownish color, while Chl levels were greatly reduced in eas1 leaves compared to wild-type leaves, and it was found that the eas1 mutant phenotype was created via a mutation in Os03g0146400, which encodes the PAO [9]. From this result, we concluded that PAO plays an important role in the formation of leaf color in plants. DNA methylation is an important form of an epigenetic modification widely found in plant genomes, and it regulates the expression of genes in different growth periods and tissues of plants [10,11,12,13]. The dynamic regulation of DNA methylation and DNA demethylation in plants is controlled by DNA methyltransferases and demethylases, with high levels of methylation inhibiting gene transcription and low methylation levels promoting gene expression [14,15,16,17]. Currently, studies of the relationship between the expression of PAO, a key gene for chlorophyll degradation, and DNA methylation are not reported in the literature.
In this study, we cloned and analyzed the sequence bioinformatics of OfPAO, constructed an overexpression vector, and clarified the role played by this gene in the formation of leaf color in O. fragrans ‘Yinbi Shuanghui’ through the genetic transformation of tobacco. In addition, the 901-1307 bp region of the PAO promoter was selected to investigate the DNA methylation level and gene expression of OfPAO in different parts of the green and colored leaves. This study validates the function of PAO in O. fragrans and outlines the relationship between the expression and DNA methylation of the mechanisms involved in leaf color formation.

2. Materials and Methods

2.1. Plant Material

All the leaves used in this study were collected from an O. fragrans ‘Yinbi Shuanghui’ tree grown in a field at Liyang, China (31°43′ N, 119°48′ E). Considering leaves from plants with both green leaves (GL) and colored leaves (CL), we collected green leaves and different parts, namely green (CG) and yellow (CY) parts, of the colored leaves for used in this study, and all leaves were sampled in their upper and middle parts using a perforator with a diameter of 8 mm, as shown in Figure 1A. Three biological replicates were used for each sample.

2.2. DNA and RNA Extraction, cDNA Preparation, and qRT-PCR Analysis

Firstly, we weighed 100 mg from leaves of different parts of O. fragrans ‘Yinbi Shuanghui’ at maturity, ground them with liquid nitrogen, and extracted the DNA by referring to the operating instructions included with the DNA extraction kit (Tiangen Biotech Co., Beijing, China). We also extracted the total RNA by referring to the instructions of the EASYspin Plus Plant RNA Rapid Extraction Kit (Aidlab Biotechnologies Co., Ltd., Beijing, China). cDNA was obtained from total RNA using the one-step de-genomization instructions included with the RNA Reverse Transcription Kit FastKing (Tiangen Biotech Co., Beijing, China). The integrity of the RNA was verified with RNase-free agarose gel electrophoresis and the concentration was measured using a Nano Drop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). The cDNA first-strand synthesis premix reagent (Tiangen Biotech Co., Beijing, China) was used to reverse-transcribe the extracted total RNA to obtain cDNA. Primers were designed using Premier 5.0 software, and the primer sequences obtained are shown in Table S1. Combined with the results of the previous study, 25 genes related to leaf color were screened via qRT-PCR, as performed using ABI StepOnePlus Systems (Applied Biosystems, Foster City, CA, USA). The qRT PCR reaction kit used was TB Green ™ Premium Ex Taq (Takara, Kusatsu, Japan), reaction system was TB Green Premix Ex Taq II 5 μL, forward primer 0.4 μL, reverse primer 0.4 μL, ROX reference dye 0.2 μL, cDNA 1 μL, and ddH2O 3 μL. The reaction procedure was as follows: pre denaturation at 95 °C for 3 min, denaturation at 95 °C for 45 s, annealing at 60 °C for 30 s, and extension at 95 °C for 15 s; then, 40 cycles later, annealing at 60 °C for 1 min, extension at 95 °C for 15 s, and insulation at 16 °C. The PCR instrument used was ABI StepOnePlus Systems (Applied Biosystems, Foster City, CA, USA).

2.3. Transient Overexpression of OfPAO in Tobacco

To explore the role played by OfPAO in the mechanism of leaf color formation in Osmanthus fragrans ‘Yinbi Shuanghui’, the overexpression vector of OfPAO was used for the transient transformation of tobacco via a published method, albeit with some modifications [18,19,20,21]. Specific primers were designed for OfPAO, and these primer sequences are shown in Table S2. The cloned region was inserted into the pSuper1300-GFP vector, and the vector was then transformed into Agrobacterium tumefaciens strain GV3101.
After the overnight incubation of Agrobacterium, the culture products were poured into tubes and centrifuged at 2716× g for 10 min at 4 °C. Agrobacterium cultures were resuspended using a solution of 10 mM MgCl2, 10 mM MES, and 150 mM acetosyringone until an OD600 of 0.6–0.8 was reached. After leaving it at room temperature for 1 h, the Agrobacterium rhizogenes resuspension solution was inoculated on the surfaces of the tobacco leaves using a syringe.

2.4. Transformation of OfPAO in Tobacco

The stable transformation of O. fragrans was performed using a previously published method, albeit with some modifications [22,23,24]. The young leaves of tobacco cv. K326 plants were first washed with pure water and then sterilized on an ultra-clean workbench. The OfPAO coding region was amplified using specific primers (Table S3) and inserted into the PBI121-GUS vector, and the vector was then transformed into the Agrobacterium tumefaciens strain GV3101. The Agrobacterium cultures used were identical to the Agrobacterium medium used for a transient transformation, as described above. When the medium reached an OD600 of 0.4, it was used for the transformation. The exosomes were first soaked in 75% ethanol for 30 s and immediately washed three times with sterile purified water. After washing, the exosomes were placed into 5% NaClO for 6 min and rinsed five to six times with sterile pure water. The leaf margins and veins of the explants were excised using a sterile scalpel, and the remaining leaves were cut into 1 cm diameter disks and infiltrated with Agrobacterium for 10 min.
After 72 h of dark co-cultivation using the symbiotic medium, the leaf disks were transferred to the selection medium containing 2.0 mg/L of 6-benzylaminopurine, 0.1 mg/L of 1-naphthaleneacetic acid, 100 mg/L of kanamycin, and 400 mg/L of cefotaxime. The medium was maintained at 25 °C in 16 h of light per day and changed every 15 days to ensure that there was a nutrient supply. When bud regeneration occurred and mature leaves appeared, independent kanamycin-resistant tobacco plants were selected and transferred to the rooting medium. When the fibrous roots of the plants developed, the plants were removed from the medium, planted in pots, and grown in a greenhouse at the Nanjing Forestry University. Control plants were transformed using an empty vector and regenerated under the same conditions.

2.5. qRT-PCR Analysis of Transgenic Tobacco

The above-mentioned transgenic tobacco leaves were taken, and RNA was extracted after grinding with liquid nitrogen. The integrity of the RNA was verified by RNase-free agarose gel electrophoresis and the concentration was measured using a Nano Drop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). cDNA, from reverse transcription, was diluted 10-fold as a template; NbL25 was used as an internal reference gene for the transiently overexpressed tobacco, while NtEFα-1 was used as an internal reference gene for the transformed tobacco, and the relative expression of the genes was calculated using −2△△CT. Three technical replicates were set up for each reaction. The qRT PCR reaction kit used was TB Green ™ Premium Ex Taq (Takara), reaction system was TB Green Premix Ex Taq II 5 μL, forward primer 0.4 μL, reverse primer 0.4 μL, ROX reference dye 0.2 μL, cDNA 1 μL, and ddH2O 3 μL. The reaction procedure was as follows: pre denaturation at 95 °C for 3 min, denaturation at 95 °C for 45 s, annealing at 60 °C for 30 s, and extension at 95 °C for 15 s; then, 40 cycles later, annealing at 60 °C for 1 min, extension at 95 °C for 15 s, and insulation at 16 °C. The PCR instrument used was ABI StepOnePlus Systems (Applied Biosystems, Foster city, CA, USA).

2.6. Promoter Cloning Ligation Transformation Sequencing

The PCR amplification reaction was performed using the colored leaf laurel DNA as a template, and the primers were designed using Primer Premier, the primer sequences of which are shown in Table S4. The PCR products were purified and recovered. The qualified PCR products were ligated using a pEASY-Blunt vector and sent to Nanjing Jie Li Sequencing Company for sequencing after bacterial inspection. The PCR reaction procedure was as follows: 1 μL cDNA as a template, pre denaturation at 94 °C for 4 min, denaturation at 98 °C for 10 s, annealing at 59 °C for 25 s, extension at 72 °C for 90 s, and holding at 16 °C after 35 cycles [25].

2.7. Promoter Bisulfite Sequencing

Following the instructions of the Quick Bisulfite Transformation Kit (QIAGEN, Hilden, Germany), 1 μg of genomic DNA was taken for sulfite treatment, and primers were designed using Methyl Primer Express v1.0 software. The primer sequences are shown in Table S5. The PCR reaction was then performed, and PCR products were purified and recovered. The qualified PCR product was ligated using pTG19-T (Generay) as a vector and transformed into competence (Generay), and the positive colonies were selected for sequencing (Generay Biotechnology Co., Shanghai, China).

2.8. Bioinformatic Analysis of Gene Sequences

The cloned sequences were translated and compared to the genomic sequences using DNAMAN software (DNAMAN 6.0 software), and molecular weight and isoelectric point analyses were conducted using an online tool (http://web.expasy.org/protparam/, accessed on 12 September 2020). An amino acid sequence homology comparison was performed using Blastp in NCBI, while multiple sequence comparison was performed using MEGA5.2 software. A phylogenetic tree was constructed using the neighbor-joining (NJ) method with a bootstrap value of 1000. The phylogenetic tree was predicted and analyzed using the online tool Plant CARE (http//bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 13 September 2020), providing a predictive analysis of promoter sequence cis-acting elements, and the prediction of the methylated regions of the gene promoter sequences was performed using MethPrimer 2.0 software.

3. Results

3.1. Expression Patterns of Leaf Color-Related Genes

To further screen genes associated with the formation of colored leaves in the O. fragrans ‘Yinbi Shuanghui’ plant, thus combining transcriptome data and WGCNA analysis, 25 genes associated with leaf color were selected for use in this study [4], and the expression of these genes in different parts of the leaf was analyzed via qRT-PCR. Our results suggested that (Figure 1B) the OfCRD1 gene encoding Mg-protoporphyrin IX methylester cyclase (CRD1) related to chlorophyll synthesis significantly differed between the green and yellow parts of colored leaves, while the expression recorded in the green leaves was not significantly different from that recorded in the green parts of colored leaves. We also found that OfCRD1 was down-regulated in the yellow part of the colored leaves, likely due to a decrease in chlorophyll synthesis in these leaves. The OfPAO gene associated with chlorophyll degradation was significantly up-regulated in colored leaves, in contrast to its expression level in green leaves. The expression of OfGLK, related to chloroplast development, in the yellow parts of colored leaves was significantly lower than those in the green parts and green leaves.

3.2. OfPAO Cloning and Sequence Analysis

The CDS of OfPAO was 1632 bp in length, encoding 543 amino acids, with a theoretical molecular weight of 61.31 kD and a theoretical isoelectric point of 6.71. The cloned OfPAO sequence was translated into an amino acid sequence via DNAMAN and compared to the sequence in the genome of O. fragrans ‘Rixianggui’, and we found that this sequence was not consistent with that recorded in the genome of O. fragrans ‘Rixianggui’ (Figure 2B), while the Osmanthus fragrans ‘Rixianggui’ genome sequence was 1668 bp in length, encoding a total of 555 amino acids, with a CDS sequence similarity of 80.33% between each acid. The deletion and the increase in sequence may be related to the formation of the leaf color.
We compared the homology between the amino acid sequence encoded by OfPAO and the amino acid sequence encoded by PAO in other plants (Figure 3). The MEGA software was used to compare OfPAO to the sequences of O. europaea, CAA3018646.1, Phtheirospermum japonicum, GFP93526.1, C.sinensis, KAF5931044.1, C.sinensis, XP_028064144.1, Erythranthe guttata, XP_012838005.1, Ipomoea triloba, XP_031123497.1, Quercus lobata, XP_030958501.1, Actinidia chinensis, PSR89917.1, Actinidia rufa, GFZ20391.1, A. chinensis, PSR86546.1, Trema orientale, PON37730.1, Nicotiana attenuata, XP_019258776.1, Sesamum indicum, XP_011088801.1, Juglans regia, XP_018833132.2, C. arabica, XP_027084853.1, C.eugenioides, and XP_027157621.1. These comparisons revealed that the amino acids encoded by the OfPAO gene were 94.84% similar to O. europaea, and we found that they had more than 80% similarity to C. sinensis, Actinidia chinensis, and Trema orientale, indicating that all have similar PAO functions.
These sequences were used to construct a phylogenetic tree (Figure 4), and the amino acid sequences encoded by the OfPAO gene were clustered within the same branch as O. europaea and CAA3018646.1, which had the closest relatives. This result was consistent with the results obtained for the O. fragrans ‘Rixianggui’ genome.

3.3. Potential Regulatory Roles of OfPAO in Tobacco with Transient Expression

Using a pre-established plant genetic transformation system, the Agrobacterium tumefaciens strain GV3101 containing 35S OfPAO recombinant plasmids was injected into tobacco leaves, and multiple transient expression (TE) leaves were obtained. Moreover, the expression of the target genes was identified via PCR using 35S promoters and target gene-specific primers (Figure 5C). Our phenotypic observation showed that the leaf color of TE was lighter than that of the wild type (WT) (Figure 5B). The expression level of transiently expressed tobacco was detected (Figure 5C), and our results showed that the expression level of TE was significantly higher than that of WT. At the same time, the expression level of OfPAO was significantly different in each TE, with higher expression levels of P1, P3, and P5, of which P3 had the highest expression level of OfPAO.
To study the regulation of the relative expression of chlorophyll pathway genes, different transiently expressed tobacco types were used as templates and analyzed via qRT-PCR (Figure 5D). The results showed that the relative expression levels of the genes regulating the chlorophyll pathway significantly differed between different TEs. In transiently expressed tobacco, the expressions of the NbPPH and NbSGR genes involved in the chlorophyll degradation pathway were up-regulated compared to that of WT. In P3, which had the highest level of OfPAO expression, the NbCAO, NbPOR1, and NbCHLH genes involved in the chlorophyll synthesis pathway had the lowest expression levels.

3.4. The Overexpression of OfPAO Promotes the Degradation of Chlorophyll in Tobacco

To further confirm the function of OfPAO, we ectopically expressed OfPAO in multiple transgenic lines of tobacco. Positive transgenic lines were screened via RT-PCR, indicating that OfPAO was integrated into the tobacco genome and successfully expressed (Figure 6A). P3, P4, and P6 were selected for the following analysis because of their consistent growth statuses. Compared to the CK, there were significant differences between the relative expression of OfPAO (Figure 6C), lighter leaf color in the positive plants, no significant differences in leaf shape, and significantly lower chlorophyll and carotenoid contents in leaves (Figure 6D,E).

3.5. OfPAO Promoter Sequence Cis-Acting Element Prediction

Using Osmanthus fragrans ‘Yinbi Shuanghui’ DNA as a template, primers were designed according to the 1500 bp sequence upstream of the OfPAO of the O. fragrans ‘Rixianggui’ genome, and the length of the amplified product was the same as that of the target fragment product (Figure 7A). The amplified fragments were connected to the T carrier, the positive clones were screened and sequenced, and the promoter sequences of the OfPAO of O. fragrans ‘Yinbi Shuanghui’ and the O. fragrans ‘Rixianggui’ genome were compared using DNAMAN software. Our results showed that the homology between the promoters of the two genes was 92.43% (Figure 7B).
The function and number of cis-acting elements were predicted using the online software Plant CARE using the promoter sequence of the 1532 bp of OfPAO (Table 1). Three cis-acting elements were involved in the photo response, three cis-acting regulatory elements were involved in the reactivity of methyl jasmonate, and one cis-acting element was involved in the low-temperature response. Moreover, one cis-regulatory element was related to meristem expression and one cis-acting regulatory element was essential for anaerobic induction, with transcription factor binding sites such as MYB, ARE, and WRE being recorded. We inferred that OfPAO gene expression was regulated by light, a low temperature, and plant hormones.

3.6. DNA Methylation CpG Island Prediction of the OfPAO Promoter

The methylated region of the OfPAO promoter was predicted to be located at 901–1307 bp using MethPrimer 2.0 software (red boxed region in Figure 8A). The primers were designed using Methyl Primer Express v1.0 software (Figure 8B), and the amplification length was 407 bp, with a total of nine CpG sites present within this region. WRE3, TCT-motif, I-box, CAAT-box, TATA-box, and OSE2 elements were predicted to be present in the promoter region of the OfPAO gene using the online software Plant CARE, with TCT-motif and I-box being associated with a response to light. The amplification of genomic DNA from different parts of Osmanthus fragrans ‘Yinbi Shuanghui’ via PCR after the bisulfite transformation showed a band at 363 bp (Figure 8C), and the results indicated that the sequence length of the promoter CpG region of OfPAO was correct.

3.7. Relationship between the Methylation Degree and the Gene Expression Level of the OfPAO Promoter

We recorded three types of methylation in the promoter region of the OfPAO gene in the leaves of ‘Yinbi Shuanghui’ (Figure 9A). The methylation levels of CG and CHG in the promoter of the OfPAO gene were not significantly different in three parts of the leaves, the methylation sites of the CHH type were significantly higher than those of CG and CHG, and the most methylation sites of the CHH type were present in the yellow part of the colored leaves. The above results suggest that the difference in the CHH methylation level may be the main reason for the difference in the leaf color.
In total, 38 CHH type methylation sites were present in the promoter region of the OfPAO, and a further study of the CHH methylation levels revealed that there were 33 sites with non-significant differences in 3 types of leaves, with 5 CHH methylation sites differing significantly (Figure 9B). Through analysis, we found that the CHH methylation levels at 145 bp in the yellow parts of the colored leaves were lower than in the green parts of colored leaves; although the difference was not significant, it was significantly different and significantly lower in green leaves. Through qRT-PCR, we found that OfPAO had the highest expression level in the yellow parts of colored leaves. By combining the CHH methylation level and OfPAO expression in different parts of the leaves, by using Pearson’s correlation analysis by taking the logarithm of these two sets of values, we found that the promoter of OfPAO in Osmanthus fragrans ‘Yinbi Shuanghui’ was significantly lower than that in the green leaves. CHH methylation was negatively correlated with the gene expression level (Figure 9C,D).

4. Discussion

4.1. OfPAO Is a Key Gene for Leaf Color Formation

Chlorophyll is a key component involved in photosynthesis, as it absorbs light energy and determines the color of plant leaves; at the same time, it is precisely because of its absorbance properties that it has potential intracellular phototoxicity. When plants are stimulated via external conditions, they can directly or indirectly affect the metabolic pathway of chlorophyll, changing the chlorophyll content present in the plant body, ultimately leading to leaf color mutations [26,27,28,29]. Thus, the synthesis and degradation of chlorophyll are tightly regulated during plant development [30,31]. Compared to the regulation of chlorophyll synthesis, there are a few reports on the regulation of chlorophyll degradation. Changes in any of the genes during chlorophyll synthesis and degradation can potentially affect chlorophyll’s biosynthesis and degradation efficiency [32,33,34,35]. The chlorosis of leaves occurs due to the oxidation and opening of the porphyrin ring of demagnesified chlorophyllin, which is then reduced to produce fluorescent chlorophyll decomposition metabolites. This step represents an interaction of two enzymes, namely PAO and red chlorophyll catabolite metabolite reductase [8,36,37,38]. PAO is a key gene involved in the regulation of chlorophyll degradation, which catalyzes demagnesyl chlorophyll a for the production of chlorophyll catabolites, the reduction in the green color, and the formation of colorless material [3,8,39]. A previous study reported a significant negative correlation between PAO expression and chlorophyll levels in leaves during fruit ripening in pollinated figs, along with a high expression level in yellow leaves, highlighting the key role played by PAO in the degradation of chlorophyll in fig leaves [40,41]. The lls in maize can encode PAO, and the maize lls1 spotted leaf mutant, a mutant defective in PAO, exhibited a phenotype that allowed it to remain green in the dark and accumulate Pheide a, leading to light-dependent premature cell death and leaf spotting [8]. The old leaves of the eas1 mutant in rice were light yellow or brownish yellow in appearance. Moreover, compared to the wild-type leaves, the Chl level of eas1 leaves was significantly reduced, showing that the eas1 mutant phenotype is produced by a mutation in the Os03g0146400 gene, thus encoding the PAO [9]. By observing the phenotype of O. fragrans ‘Yinbi Shuanghui’ (with leaves that are green in the middle and have yellow margins) and by analyzing the chlorophyll contents of colored and green leaves, we found that chlorophyll levels in colored leaves were significantly lower than those in green leaves. Furthermore, through the analysis of the transcriptome data and WGCNA, we screened for the presence of PAO, a chlorophyll degradation gene related to the metabolism of chlorophyll, and the expression of the Unigene0029892-encoding PAO in the colored leaves was significantly higher than that in the green leaves [4]. The analysis of gene expression in different parts of the colored leaves and green leaves revealed that the OfPAO genes were also different in the yellow and green parts of the colored leaves.

4.2. The Methylation of the OfPAO Promoter Is Involved in Regulating Its Expression

DNA methylation is an important form of an epigenetic modification widely found in plant genomes, and it regulates the expression of genes in various developmental periods, as well as in different plant tissues [42,43]. There are three main forms of sequence methylation in plant genomic DNA, CG, CHG, and CHH (H denotes non-G bases) [14,44]. Typically, the dynamic regulation of DNA methylation and DNA demethylation in plants is controlled by both DNA methyltransferases and demethylases, with high methylation levels repressing gene transcription and low methylation levels promoting gene expression [15,45]. The differential distribution of pigments on the surfaces of plant organs is the main reason for the striped phenotype found in plants, and the formation of this phenomenon has previously been studied at an epigenetic level. Through the study of the mechanism behind the formation of stripes on the pericarp of the ‘Honeycrisp’ apple, we found that the transcription level of the MdMYB10 transcription factor, which regulates the synthesis of apple anthocyanins, was negatively correlated with the methylation level of its promoter in the red and green pericarp stripes, and we believe that the difference in the methylation of the MdMYB10 promoter altered the transcript level of MdMYB10. This, in turn, caused changes in the expression of genes related to anthocyanin synthesis, ultimately leading to the differential accumulation of anthocyanin and the production of red and yellow stripes on apple pericarps [46]. Similarly, previous reports found that differences in the methylation of the PcMYB10 promoter were a key reason for the formation of stripes and blotches on pear pericarps [47]. In tomato, differences in the methylation level of the MADS-box family member SlTAGL1 affects the formation of pericarp stripes by regulating the development of chloroplasts and the accumulation of carotenoids in fruits [48]. Meanwhile, the relationship between DNA methylation and leaf stripe formation has also been explored in crops such as maize, in which the expression of Zmpl-bh (purple plant 1), a member of the MYB-like transcription factor group that regulates anthocyanin synthesis, was negatively correlated with its DNA methylation level between maize stripe and normal tissues, and the authors suggested that the hypermethylation of the Zmpl-bh promoter in leaf stripe tissues was the key reason for this correlation [49]. In ornamental plants, Clivia miniata var. variegata (Cmvv) is a variegated leaf mutant of Clivia miniata, and the formation of a white–green variegated leaf was associated with DNA methylation modification through methylation-specific probe amplification (MSPA) [50]. In this study, the OfPAO promoter was amplified via BSP treatment to have a 407 bp sequence, in which the CG methylation in the yellow part of the colored leaves was low, the differences in the methylation levels between the three parts of the colored leaves were not significant, and the methylation levels of CHH in the yellow parts of the colored leaves were not significantly different from those in the green parts of the colored leaves at 145 bp but were significantly different from those in the green leaves. We also performed a correlation analysis of the methylation levels of different parts of the leaves, and the expression of OfPAO showed that the promoter methylation of OfPAO in colored leaves was negatively correlated with its expression level, which is consistent with the previous study; therefore, the promoter methylation of OfPAO might regulate its expression.

5. Conclusions

In summary, we analyzed the expression levels of genes in several metabolic pathways related to leaf color and combined them with the results of previous studies, before screening out the key gene OfPAO related to chlorophyll degradation. The OfPAO overexpression vector was constructed and transferred into transgenic tobacco plants. The role played by OfPAO in chlorophyll degradation in transgenic plants was verified, and we speculated that OfPAO influenced the mechanism regulating leaf color. Subsequently, we combined the expression level of BSP and OfPAO to conduct a correlation analysis, and our results showed that the CHH methylation level in different parts of the laurel was negatively correlated with the expression level of OfPAO. These results suggest that the methylation of the OfPAO promoter may affect the regulation of leaf color formation via chlorophyll degradation in Osmanthus fragrans ‘Yinbi Shuanghui’ leaves by regulating OfPAO expression.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/f15010011/s1, Table S1. Sequences for primers used in qRT-PCR to analyse chlorophyll metabolism-related genes. Table S2. Sequences for primers used to study target gene subcellular localization. Table S3. Sequences for primers used to amplify the PAO gene promoter. Table S4. Sequences for primers used to analyse the BSP methylation. Table S5. Expression pattern of leaf color related genes. Table S6. Regulate the relative expression of chlorophyll pathway genes in transgenic tobacco and the relative expression of OfPAO. Table S7. Relative expression of OfPAO in transgenic tobacco transformed with the empty vector and overexpressing the OfPAO. Table S8. Chlorophyll and carotenoid content in transgenic tobacco leaves overexpressing the OfPAO. Text S1. Sequence.

Author Contributions

L.W., X.Y. and R.W. designed and funded the experiment; R.W. analyzed the data and drafted the manuscript; Y.Z. and X.C. prepared the materials and performed the bioinformatics analysis; R.W. and D.Z. conducted the qRT-PCR analyses; and H.W. and W.Z. revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (grant numbers 32071828 and 31870695) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. The expression pattern of leaf color-related genes. (A) CG: green parts of colored leaves; CY: yellow parts of colored leaves; G: green leaves. (B) The presence of different lowercase letters above the relative expressions of genes in different parts of the leaves indicates that the difference was significant when the significance level was 0.05. The expressions of the genes in different parts of the leaves were not significant. The presence of different lowercase letters above the relative expressions of genes in different parts of the leaves indicate that the difference was significant when the significance level was 0.05. Red square is stand for the gene we selected.
Figure 1. The expression pattern of leaf color-related genes. (A) CG: green parts of colored leaves; CY: yellow parts of colored leaves; G: green leaves. (B) The presence of different lowercase letters above the relative expressions of genes in different parts of the leaves indicates that the difference was significant when the significance level was 0.05. The expressions of the genes in different parts of the leaves were not significant. The presence of different lowercase letters above the relative expressions of genes in different parts of the leaves indicate that the difference was significant when the significance level was 0.05. Red square is stand for the gene we selected.
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Figure 2. The cloning of the OfPAO gene: (A) the electropherogram of the PCR product of OfPAO and (B) the comparison of the cloned OfPAO sequence to the ‘Rixianggui’ genome.
Figure 2. The cloning of the OfPAO gene: (A) the electropherogram of the PCR product of OfPAO and (B) the comparison of the cloned OfPAO sequence to the ‘Rixianggui’ genome.
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Figure 3. Multiple sequence alignment of OfPAO.
Figure 3. Multiple sequence alignment of OfPAO.
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Figure 4. Phylogenetic tree of PAO.
Figure 4. Phylogenetic tree of PAO.
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Figure 5. The identification of the transgenic phenotype and the expression of related genes in transgenic tobacco with OfPAO. (A) The identification of transgenic tobacco with OfPAO; (B) the phenotype of the transgenic tobacco with OfPAO; (C) the analysis of OfPAO gene expression in transgenic tobacco; and (D) the regulation of the relative expression of chlorophyll pathway genes in transgenic tobacco and the expression of chlorophyll pathway genes in transgenic tobacco with OfPAO. Nb is for tobacco. Lowercase letters represent significant differences, and the same letter means the difference is not significant, and different letters mean the opposite.
Figure 5. The identification of the transgenic phenotype and the expression of related genes in transgenic tobacco with OfPAO. (A) The identification of transgenic tobacco with OfPAO; (B) the phenotype of the transgenic tobacco with OfPAO; (C) the analysis of OfPAO gene expression in transgenic tobacco; and (D) the regulation of the relative expression of chlorophyll pathway genes in transgenic tobacco and the expression of chlorophyll pathway genes in transgenic tobacco with OfPAO. Nb is for tobacco. Lowercase letters represent significant differences, and the same letter means the difference is not significant, and different letters mean the opposite.
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Figure 6. The phenotype, positive identification, and relative expression analysis of transgenic tobacco with OfPAO. (A) The phenotype of the empty vector and transgenic tobacco; (B) semi-quantitative electropherogram of the OfPAO gene in an empty vector and transgenic tobacco; (C) relative expression level analysis of the OfPAO gene in an empty vector and transgenic tobacco. The presence of different lowercase letters on different data indicates that different transgenic tobacco leaves have different expression levels and that different transgenic tobacco leaves have differences at a significance level of 0.05. (D) Chlorophyll contents in transgenic tobacco leaves with OfPAO; (E) carotenoid contents in transgenic tobacco leaves with OfPAO. The presence of different lowercase letters indicates differences at a significance level of 0.05.
Figure 6. The phenotype, positive identification, and relative expression analysis of transgenic tobacco with OfPAO. (A) The phenotype of the empty vector and transgenic tobacco; (B) semi-quantitative electropherogram of the OfPAO gene in an empty vector and transgenic tobacco; (C) relative expression level analysis of the OfPAO gene in an empty vector and transgenic tobacco. The presence of different lowercase letters on different data indicates that different transgenic tobacco leaves have different expression levels and that different transgenic tobacco leaves have differences at a significance level of 0.05. (D) Chlorophyll contents in transgenic tobacco leaves with OfPAO; (E) carotenoid contents in transgenic tobacco leaves with OfPAO. The presence of different lowercase letters indicates differences at a significance level of 0.05.
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Figure 7. Promoter amplification and sequence alignment of OfPAO: (A) the electropherogram of the PCR product of the promoter of OfPAO and (B) the promoter sequence alignment of OfPAO.
Figure 7. Promoter amplification and sequence alignment of OfPAO: (A) the electropherogram of the PCR product of the promoter of OfPAO and (B) the promoter sequence alignment of OfPAO.
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Figure 8. The methylation analysis of the OfPAO promoter. (A) The CpG islands in the promoter of OfPAO. (B) The BSP amplification sequence of the OfPAO promoter. The yellow sequence in the figure indicates the sequence of the upstream and downstream primers, ::: indicates the CG methylation site, * indicates non-CpG, and C is converted to T. (C) The BSP amplification product of the OfPAO promoter. CG: the green parts of colored leaves; CY: the yellow parts of colored leaves; G: green leaves.
Figure 8. The methylation analysis of the OfPAO promoter. (A) The CpG islands in the promoter of OfPAO. (B) The BSP amplification sequence of the OfPAO promoter. The yellow sequence in the figure indicates the sequence of the upstream and downstream primers, ::: indicates the CG methylation site, * indicates non-CpG, and C is converted to T. (C) The BSP amplification product of the OfPAO promoter. CG: the green parts of colored leaves; CY: the yellow parts of colored leaves; G: green leaves.
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Figure 9. Analysis of the promoter region methylation degree of OfPAO: (A) the methylation type of the OfPAO promoter. CG: the green parts of colored leaves; CY: the yellow parts of colored leaves; G: green leaves. Different letters present in the same part denote significant differences, according to Duncan’s test, at the 0.05 level. (B) The degree of CHH methylation in the OfPAO promoter. CG: the green parts of colored leaves; CY: the yellow parts of colored leaves; G: green leaves. The presence of different letters in the same site denote significant differences, according to Duncan’s test, at the 0.05 level. (C) A correlation analysis between the methylation level of the OfPAO promoter and the relative expression of OfPAO. (D) A correlation analysis of the relative expression of OfPAO.
Figure 9. Analysis of the promoter region methylation degree of OfPAO: (A) the methylation type of the OfPAO promoter. CG: the green parts of colored leaves; CY: the yellow parts of colored leaves; G: green leaves. Different letters present in the same part denote significant differences, according to Duncan’s test, at the 0.05 level. (B) The degree of CHH methylation in the OfPAO promoter. CG: the green parts of colored leaves; CY: the yellow parts of colored leaves; G: green leaves. The presence of different letters in the same site denote significant differences, according to Duncan’s test, at the 0.05 level. (C) A correlation analysis between the methylation level of the OfPAO promoter and the relative expression of OfPAO. (D) A correlation analysis of the relative expression of OfPAO.
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Table 1. The number of cis-acting elements in the OfPAO promoter.
Table 1. The number of cis-acting elements in the OfPAO promoter.
FunctionNumberElement NameSequence
Cis-acting elements involved in the cryogenic response1LTRCCGAAA
Cis-acting regulatory elements essential for anaerobic induction1AREAAACCA
Cis-acting regulatory elements involved in MeJA reactivity3CGTCA-motifCGTCA
TGACG-motifTGACG
Cis-regulatory elements associated with meristematic tissue expression1CAT-boxGCCACT
Common cis-acting elements in promoter and enhancer regions16CAAT-boxCAAAT
CAAT-boxTGCCAAC
Core promoter elements around the transcription promoter (i.e., about −30)14TATA-boxTATA
TATA-boxATATAT
TATA-boxATATAA
TATA-boxATTATA
Part of a conserved DNA module involved in light responsiveness1Box 4ATTAAT
Part of a light-responsive element2I-boxAGATAAGG
TCT-motifTCTTAC
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Wang, R.; Zhou, Y.; Chen, X.; Wei, H.; Zheng, D.; Zhu, W.; Wang, L.; Yang, X. Functional Validation and Promoter DNA Methylation Analysis of the OfPAO Gene of Osmanthus fragrans ‘Yinbi Shuanghui’. Forests 2024, 15, 11. https://doi.org/10.3390/f15010011

AMA Style

Wang R, Zhou Y, Chen X, Wei H, Zheng D, Zhu W, Wang L, Yang X. Functional Validation and Promoter DNA Methylation Analysis of the OfPAO Gene of Osmanthus fragrans ‘Yinbi Shuanghui’. Forests. 2024; 15(1):11. https://doi.org/10.3390/f15010011

Chicago/Turabian Style

Wang, Rui, Yixiao Zhou, Xuan Chen, Hao Wei, Dong Zheng, Wuwei Zhu, Lianggui Wang, and Xiulian Yang. 2024. "Functional Validation and Promoter DNA Methylation Analysis of the OfPAO Gene of Osmanthus fragrans ‘Yinbi Shuanghui’" Forests 15, no. 1: 11. https://doi.org/10.3390/f15010011

APA Style

Wang, R., Zhou, Y., Chen, X., Wei, H., Zheng, D., Zhu, W., Wang, L., & Yang, X. (2024). Functional Validation and Promoter DNA Methylation Analysis of the OfPAO Gene of Osmanthus fragrans ‘Yinbi Shuanghui’. Forests, 15(1), 11. https://doi.org/10.3390/f15010011

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