Next Article in Journal
Characterization of Postharvest Changes in Fruit Quality Traits of Highbush Blueberry (Vaccinium corymbosum L.) Cultivars
Previous Article in Journal
Dynamics of Quality Traits During Cold Storage in ‘Annurca’ Apples: Impact of 1-MCP and the Traditional Melaio Reddening Process
Previous Article in Special Issue
Construction of SNP-PARMS Fingerprints and Analysis of Genetic Diversity in Taro (Colocasia esculenta)
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Horticulturae
Volume 11
Issue 10
10.3390/horticulturae11101248
horticulturae-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

RsNAC134 Regulates Taproot Skin Color via Positive Regulation of the Chlorophyll Degradation Pathway in Radish (Raphanus sativus)

by
Weifang Chen
1,2,
Chenghuan Yan
1,2,
Leifu Chen
1,2,
Lei Cui
1,2 and
Weiling Yuan
1,2,*
1
Hubei Key Laboratory of Vegetable Germplasm Innovation and Genetic Improvement, Institute of Economic Crops, Hubei Academy of Agricultural Sciences, Wuhan 430063, China
2
Key Laboratory of Vegetable Ecological Cultivation on Highland, Ministry of Agriculture and Rural Affairs, Institute of Economic Crops, Hubei Academy of Agricultural Sciences, Wuhan 430063, China
*
Author to whom correspondence should be addressed.
Horticulturae 2025, 11(10), 1248; https://doi.org/10.3390/horticulturae11101248
Submission received: 16 September 2025 / Revised: 11 October 2025 / Accepted: 14 October 2025 / Published: 16 October 2025
(This article belongs to the Special Issue Breeding by Design: Advances in Vegetables)
Browse Figures
Versions Notes

Abstract

The color of radish taproot skin is an important commercial quality trait that directly affects the visual judgment of consumers. The green/white coloration of radish taproots is caused by chlorophyll accumulation or fading; however, research on the mechanisms of color regulation in green/white variations remains limited. Therefore, we analyzed transcriptome data from the green radish ‘QZ-16’ and white radish ‘55’ and identified a key color-regulating gene, RsNAC134. The expression of RsNAC134 was significantly reduced in green radish ‘QZ-16’ but markedly increased in white radish ‘55’. Heterologous overexpression of RsNAC134 in transgenic tomatoes resulted in chlorotic phenotypes. Quantitative real-time polymerase chain reaction revealed significant upregulation of chlorophyll degradation pathway genes SlSGR and SlPAO in transgenic tomatoes. Similarly, in white radish, expression of the key chlorophyll degradation genes, RsSGR, RsPAO1, and RsPAO2, was notably increased. Yeast one-hybrid and luciferase assays demonstrated that RsNAC134 directly bound to the promoters of RsSGR, RsPAO1, and RsPAO2. These findings suggest that RsNAC134 regulates chlorophyll degradation by modulating RsSGR, RsPAO1, and RsPAO2 expression, ultimately influencing the radish color transition (loss of green pigmentation) or retention of green coloration. This work unravels novel regulatory factors of chlorophyll degradation and elucidates the molecular network governing chlorophyll degradation, providing crucial insights into the molecular basis of epidermal color variation in radish taproots.

1. Introduction

Chlorophyll is a crucial photosynthetic pigment that plays a vital role in plant growth and development, and involves three key processes: chlorophyll biosynthesis, chlorophyll cycle and chlorophyll degradation. The balance between chlorophyll synthesis and degradation is crucial, and chlorophyll degradation-related genes have been reported [1,2,3]. The chlorophyll degradation pathway involves seven steps from chlorophyll b to non-fluorescent chlorophyll catabolite, including chlorophyll b reductase [namely Non-Yellow Coloring 1 (NYC1) and NYC1-like (NOL)], 7-hydroxymethyl chlorophyll a reductase (HCAR), stay-green (SGR, also named NYE1), pheophytinase (PPH), pheophorbide a oxygenase (PAO), red chlorophyll catabolite reductase (RCCR) six catalytic enzymes, and one chemical decomposition process. SGR encodes a chloroplast protein that is essential for initiating chlorophyll breakdown in plastids. SGR was first proposed in the genetic mapping of Lolium temulentum [4], and was subsequently confirmed as the SGR gene in rice mutants [5]. In the same year, Mendel’s green cotyledon gene was found to be encoded by SGR [6]. Studies showed that SGR can accelerate chlorophyll degradation by recruiting chlorophyll metabolic enzymes [1,7,8], and has been identified as a magnesium chelatase [2,9]. The transient expression of SGR in Arabidopsis thaliana leads to chlorophyll degradation, resulting in leaf discoloration [2]. PAO is a monooxygenase that catalyzes the formation of red chlorophyll catabolites from pheophorbide A in the chlorophyll degradation pathway. It was first identified in maize and subsequently found in other higher plants, such as rice, wheat, tomato, soybean, and rapeseed [10,11,12,13,14,15]. PAO is a key enzyme in the chlorophyll degradation pathway and is involved in processes such as chlorophyll degradation and leaf senescence [16,17,18]. However, studies on the regulatory mechanisms of SGR and PAO are limited.
Transcription factors can regulate plant growth and development, as well as various stress responses, by modulating the expression of their target genes [19,20]. The plant-specific transcription factor NAC (no apical meristem [NAM], A. thaliana transcription activation factor 1/2 [ATAF1/2], and cup-shaped cotyledon 2 [CUC2]) is among the largest gene families in plants and can exert specific functions by binding to specific promoter cis-acting elements, such as regulating embryo and seed development, lateral root growth and development, secondary metabolites biosynthesis, and abiotic and biotic stress responses [21,22,23]. In trifoliate orange (Poncirus trifoliata), PtrNAC72 binds to the promoter of arginine decarboxylase (ADC) and inhibits its expression, thereby suppressing the biosynthesis of plant putrescine and negatively regulating drought resistance [24]. However, some studies have suggested that NAC transcription factors regulate the chlorophyll degradation pathway. ANAC046 can bind to the promoters of multiple genes in the chlorophyll degradation pathway (NYC1, SGR1, PAO, and SGR2), and NYC1, SGR1, PAO, and SGR2 expression levels are increased in plants overexpressing ANAC046, whereas the chlorophyll content is decreased and the phenotype shows premature aging [25]. ANAC032, as a transcriptional activator, can upregulate NYE1, SAG113, and SAUR36/SAG201 expression, positively modulating stress- and age-dependent senescence [22]. LcNAC002 directly binds to the LcSGR promoter and promotes its expression, thereby accelerating chlorophyll degradation [26].
Radish (Raphanus sativus L.) belongs to the Cruciferae family of vegetable crops and is cultivated globally. Radishes have a variety of uses; the enlarged taproot can be eaten raw or processed, the leaves can be grown as leafy greens, and the seeds are used medicinally for oil extraction. Radishes are also used as cover crops in the United States and Canada to improve soil quality and suppress weeds [27]. The skin color of radish taproots is an important aspect of their appearance, and directly affects consumer choice. Radishes possess a rich genetic germplasm resource with varying taproot colors, including white, green, red, and black. Red, green, and black colors are primarily regulated by chlorophyll and anthocyanins accumulation. Red-skinned radishes are primarily regulated by anthocyanins synthesis [28,29], black-skinned radishes by melanin accumulation [30], and green-skinned radishes by chlorophyll accumulation [31]. Chlorophyll accumulation in radish roots directly affects photosynthetic efficiency, providing energy and nutrients for plant growth, while also enhancing stress resistance. However, research on the regulatory mechanisms of radish skin color remains limited.
To explore the genes governing the green/white skin colors of radishes and analyze their skin coloration regulatory mechanisms. We analyzed the transcriptome data of the taproots of white radish ‘55’ and green radish ‘QZ-16’ at mature stage (84 DAG, day after germination) [32], and found that RsNAC134 was regularly expressed in the taproots and skins of white radish ‘55’ and green radish ‘QZ-16’; therefore, RsNAC134 is regarded as a candidate gene for regulating the white and green skin of radishes. RsNAC134 expression in white radish ‘55’ was significantly higher than that in green radish ‘QZ-16’ during the mature stage. Overexpression of RsNAC134 in transgenic tomato plants led to leaf chlorosis, with a significant increase in chlorophyll degradation-related gene expression. Yeast one-hybrid (Y1H) and luciferase (LUC) assays also demonstrated that RsNAC134 bound to the promoters of RsSGR and RsPAOs and acted as a positive regulator, thereby accelerating chlorophyll degradation.

2. Materials and Methods

2.1. Plant Materials

The two radish varieties ‘QZ-16’ and ‘55’ in this study are representative Asian big radishes (Raphanus sativus var. hortensis), both of which were high-generation inbred lines. ‘QZ-16’ is a typical green taproot radish with green skin and flesh, and ‘55’ is a common white taproot radish. These two materials were planted in the greenhouse of the Yangjiayuan Experimental Base of the Agricultural Science Academy of Hubei Province (Wuhan, China). Harvesting and sampling were carried out during the maturity period of the two varieties (84 DAG, day after germination). The skin and flesh tissues of the taproots were collected separately and immediately placed in liquid nitrogen. All samples were stored at −80 °C for further use.

2.2. Gene Expression Analysis

Total RNA was extracted from radish and overexpressing tomato plants using the TRIzol reagent (Vazyme, Nanjing, China). The RNA was then reverse-transcribed into cDNA using a HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme). Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was performed using a CFX384 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) in conjunction with a ChamQ Universal SYBR® qPCR Master Mix (Vazyme). The radish samples were collected from the mature ‘55’ and ‘QZ16’ varieties, including two tissues of skin and flesh. Tomato samples were collected from the leaves of transgenic T0 seedlings. The relative expression levels of RsNAC134, genes related to chlorophyll degradation pathway (SGR, PAO, CHL, NYC and PPH) were measured. The primer sequences are listed in Supplementary Table S4. Three biological replicates were used for each experiment. The relative expression of specific genes was quantified using the 2−ΔΔCt method, and the radish PRⅡ (RNA polymerase-II transcription factor) gene was used as the internal control [33].

2.3. Vector Construction and Genetic Transformation

The full-length coding DNA sequence (CDS) of RsNAC134 was amplified via polymerase chain reaction (PCR) using Phanta polymerase (Vazyme, Nanjing, China) from radish cDNA using gene-specific primers (Supplementary Table S5). And the fragment was cloned into pHELLS GATE8 (Invitrogen) via recombination using Exnase II (Vazyme, Nanjing, China). The vector was transferred into Agrobacterium tumefaciens strain C58 for tomato transformation [34]. Subsequently, the construct was introduced by using A. tumefaciens to infect the cotyledons of Micro-Tom tomato, and transgenic plants were confirmed via PCR using genomic DNA from the leaves. The transgenic tomato materials were the positive material obtained by T0 generation transformation. The control plants were transgenic positive plants with extremely low expression of RsNAC134. The primers used are shown in Supplementary Table S5.

2.4. Subcellular Localization

The CDS of RsNAC134 without the stop codon was amplified and inserted into the p101-YFP vector [35] under the control of the CaMV 35S promoter. The recombinant plasmids CaMV35S::RsNAC134-YFP was transferred into A. tumefaciens strain GV3101 and infiltrated into the leaves of 4-week-old Nicotiana benthamiana plants together with the nuclear marker CaMV35S::H-mcherry (red fluorescent protein (RFP)) [36]. Two days later, the subcellular localization of the target proteins was visualized under a confocal laser scanning microscopy (Leica TCS SP8, Wetzlar, Germany). The primers used are shown in Supplementary Table S5.

2.5. Transactivation Assay

The full-length CDS of RsNAC134 or the truncated fragments were amplified and individually inserted into the pGBKT7 vector. These vectors were then transformed into the yeast strain AH109 and cultivated on synthetic dextrose (SD)/-Trp medium. Positive strains were diluted in sterilized distilled water (OD600 = 0.1), with a 2 μL suspension spotted onto SD/-Trp, SD/-Trp-His-Ade and SD/-Trp-His-Ade with X-α-gal (40 mg/L), and incubated at 30 °C for 3–5 days. The pGBKT7 vector was used as the negative control. All amplified primers are listed in Supplementary Table S5.

2.6. Dual-LUC Assay

The dual-LUC assay was conducted following Chen et al. (2020) [37]. The full-length CDS of RsNAC134 was cloned into the pGreen II 62-SK vector to obtain the effector construct. The promoter fragments of RsSGR, RsPAO1, and RsPAO2 were separately cloned into the pGreen II 0800-LUC vector as the reporter constructs. The reporter and effector constructs, together with the pSoup plasmid, were transformed into A. tumefaciens GV3101. The suspensions of cells containing either effector or reporter constructs were mixed and co-infiltrated into the leaves of 4-week-old N. benthamiana seedlings. The transient expression was evaluated by the activities of the LUC and REN ratio using a Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) and an Infinite M200 Pro instrument (Tecan, Männedorf, Switzerland). Six biological replicates were measured for each sample. All amplified primers are listed in Supplementary Table S5.

2.7. Y1H Assay

The Y1H assay was performed following the manufacturer’s protocol (Matchmaker One-Hybrid System; Clontech, CA, USA). The CDS of RsNAC134 was cloned into the pGADT7 vector containing a GAL4 transcriptional activation domain. The promoter fragments of RsSGR, RsPAO1, and RsPAO2 were separately cloned into the pAbAi vector. After confirming the integration of each bait vector into the yeast strain Y1H Gold, the prey vectors were added. The strains were diluted in sterilized distilled water (OD600 = 0.1), and 2 μL of suspension was spotted onto selective medium (SD/-Ura-Lue) containing different Aureobasidin A (AbA) concentrations. The medium was placed at 30 °C for 3–5 days to observe yeast plaque growth. All amplified primers are listed in Supplementary Table S5.

2.8. Determination of Chlorophyll

To assay chlorophyll levels, 1 mL of 80% (v/v) acetone was added to approximately 0.1 g of frozen powder (samples were collected and ground into fine powder in liquid nitrogen) in a 2 mL Eppendorf tube under low light intensity using the procedure described by Wellburn (1994) [38]. Finally, the absorbance at 646 nm and 663 nm were measured by an Infinite M200 Pro instrument (Tecan, Männedorf, Switzerland), chlorophyll content: mg/g (FW).

2.9. Tobacco Transient Expression

The CDS of RsSGR, RsPAO1 and RsPAO2 were amplified via polymerase chain reaction (PCR) using Phanta polymerase (Vazyme, Nanjing, China) from radish cDNA using gene-specific primers (Supplementary Table S5). And the fragments were cloned into pHELLS GATE8 (Invitrogen) via recombination using Exnase II (Vazyme, Nanjing, China). These vectors were transformed into A. tumefaciens GV3101. The suspensions of cells containing RsSGR-OE, RsPAO1-OE and RsPAO2-OE infiltrated into the leaves of 4-week-old N. benthamiana seedlings, respectively. Empty pHELLS GATE8 was used as the control. After 48 h, the phenotypic characteristics were observed and leaf samples were collected for subsequent experiments.

2.10. Phylogenetic Tree Analysis

The protein sequences from radish, Hirschfeldia incana, Brassica rapa, Brassica napus, Eutrema salsugineum, Capsella rubella, Brassica oleracea, tomato, A. thaliana, and Brassica oleracea were employed to construct a neighbor-joining tree. Using MEGA6 (v6.06) software, we performed multiple sequence alignments of these protein sequences. The resulting alignment was then utilized to build a phylogenetic tree based on the maximum parsimony method, with the bootstrap value set to 1000 to assess reliability.

2.11. Statistical Analysis

The International Business Machine-Statistical Package for Social Sciences (IBM-SPSS) and Microsoft excel software were used for statistical analyses. All dates were collected form 3 independent biological replicates and expressed as mean ± SD. And Student’s t-test were used to compare the means at p < 0.05 (*) or p < 0.01 (**).

3. Results

3.1. Differential Expression of RsNACs in ‘55’ and ‘QZ-16’

In previous studies, we analyzed the transcriptome data of white-skinned radish ‘55’ and green-skinned radish ‘QZ-16’ at mature stages, and identified NAC genes that may play an important role during the formation of green/white taproots in radishes [32]. However, the functions and regulatory mechanisms of RsNACs involved in regulating taproot color remain to be elucidated. In the present study, ‘55’ appeared as a white-skinned radish and ‘QZ-16’ was a green radish (Figure 1A). Through RT-qPCR technology, we found that RsNAC134 exhibited regular expression patterns in ‘55’ and ‘QZ-16’, with significantly higher expression in both the skin and flesh of ‘55’ compared to that in ‘QZ-16’ during the mature stage (Figure 1B). We speculate that the low expression of RsNAC134 may have maintained the green color in ‘QZ-16’, whereas high expression promoted chlorophyll degradation, resulting in a white appearance.
The sequence of RsNAC134 was analyzed using the National Center for Biotechnology Information database. It had a length of 966 nucleotides and encoded a polypeptide consisting of 321 amino acid residues, including a NAM-conserved domain (Figure 1C). The sequence exhibited high homology with those of other plant species (Figure 1C). We utilized the WoLF PSORT website to predict the subcellular localization of RsNAC134, and found a nuclear localization signal (NLS) sequence (PRDRKYP) at its N-terminus (Figure S1). A phylogenetic tree constructed using RsNAC134 and other NAC amino acid sequences from A. thaliana, potato, pepper, tobacco, and rice showed that RsNAC134 was closely related to other NAC proteins (Figure S1).

3.2. RsNAC134 Overexpression Caused Chlorosis in Tomatoes

To further analyze the functions of RsNAC134, we heterologously overexpressed it, and used Micro-Tom tomato as the background material for genetic transformation to obtain RsNAC134 overexpression (RsNAC134-OE) plants. Positive transgenic tomato plants were detected by PCR (Figure S2), and their phenotypes were observed. We found that overexpression of RsNAC134 caused leaf bleaching in tomato plants (Figure 2A). The RsNAC134 expression levels in these transgenic plants were analyzed by RT-qPCR, which showed that the expression levels of RsNAC134-OE-18, RsNAC134-OE-20, and RsNAC134-OE-22 lines were 16.5-, 15.3-, and 16.2-fold higher than that of the control (CK, which consisted of transgenic positive plants with low expression of RsNAC134) (Figure 2B). We observed that plants overexpressing RsNAC134 exhibited leaf chlorosis, which we speculated that leaf chlorosis might have been caused by chlorophyll degradation; the chlorophyll content detection results of the transgenic tomato plants confirmed this hypothesis. The results showed that compared to the control, the chlorophyll content of the heterologous overexpression tomato material of RsNAC134 was significantly lower (Figure 2C). This is consistent with the high RsNAC134 expression in white radish ‘55’, suggesting that RsNAC134 may positively regulate chlorophyll degradation in radish.

3.3. RsNAC134 Regulates the Expression of Chlorophyll Degradation Genes

We further analyzed the regulatory effect of RsNAC134 on genes related to the chlorophyll degradation pathway. RT-qPCR was used to detect the expression levels of SlSGR, SlPAO, SlNYC, SlCHL1, and SlPPH1 in CK and RsNAC134 transgenic tomato plants. The results showed that the expression levels of the chlorophyll degradation genes SlSGR and SlPAO were significantly increased in the RsNAC134 overexpression transgenic tomatoes, whereas the expression levels of SlNYC, SlCHL, and SlPPH were irregularly changed in the RsNAC134 overexpression transgenic tomatoes (Figure 3A–E). These results indicated that RsNAC134 promotes chlorophyll degradation in tomatoes by regulating the expression of SlSGR and SlPAO.
We also found that the chlorophyll content in the skin and flesh of ‘QZ-16’ was significantly higher than that of ‘55’ (Figure 3F). Furthermore, we analyzed the expression levels of genes related to the chlorophyll degradation pathway in the skin and flesh of white radish ‘55’ and green radish ‘QZ-16’. The identified genes were RsSGR, RsPAO1, RsPOA2, RsNYC, RsCHL1, and RsCHL2. We found that RsSGR, RsPAO1, and RsPOA2 showed higher expression in white radish ‘55’ than in green radish ‘QZ-16’. The expression levels of RsNYC, RsCHL1, and RsCHL2 were irregular in ‘55’ and ‘QZ-16’ (Figure 3G–L). Furthermore, we also found that the transient overexpression of RsSGR, RsPAO1, and RsPAO2 induced the chlorosis of tobacco leaves (Figure S3). These results suggested that the RsNAC134 expression influenced RsSGR, RsPAO1, and RsPAO2 expression, whereas the high expression of RsSGR, RsPAO1, and RsPAO2 promoted chlorophyll degradation.

3.4. RsNAC134 Is a Transcriptional Activator That Localized in the Nucleus

To verify whether RsNAC134 functions as a transcription factor, we performed a transcriptional activation assay using RsNAC134 in yeast cells. Based on its structural characteristics, the RsNAC134 amino acid sequence was divided into N- and C-terminal amino acids (Figure 4A). The full-length sequence of RsNAC134, as well as the sequences encoding amino acids 1-144 (N-terminal) and 19-321 (C-terminal), were introduced into the yeast strain AH109, and yeast cells growth was observed. The results showed that both the full-length and C-terminal amino acid sequence of RsNAC134 grew normally on SD/-Trp-His-Ade medium and activated α-galactopyranoside activity, resulting in a blue color. In contrast, the N-terminal and negative control did not grow (Figure 4B). Subsequently, we constructed an RsNAC134-YFP fusion protein to analyze the subcellular localization of RsNAC134. The RsNAC134-YFP fusion protein was co-transformed with the nuclear marker CaMV35S::H-mCherry in tobacco leaves, followed by transient expression and observation under confocal microscopy. The results showed that RsNAC134 exhibited fluorescence in the nucleus (Figure 4C), suggesting that RsNAC134 was localized in the nucleus and possessed transcriptional activation activity. These characteristics strongly suggested its functional roles in transcriptional regulation.

3.5. RsNAC134 Regulated RsSGR and RsPAO Expression by Directly Binding to Their Promoters

Based on the transcriptional activity of RsNAC134 and its regulatory role in SGR and PAOs expression, we hypothesized that it regulates chlorophyll degradation through SGR and PAOs. To verify this hypothesis, we analyzed the cis-elements in the 3 kb promoter sequences of RsSGR and RsPAOs using the PLACE and PlantCARE databases, respectively (Tables S1–S3). The results showed that there were NAC-binding element (TTTATCCAA) at 238 bp upstream of the RsSGR promoter (Table S1), a NAC-binding element (TTGGATTGA) at 587 bp upstream of the RsPAO1 promoter (Table S2), and a NAC-binding element (TTGATCCAA) at 216 bp upstream of the RsPAO2 promoter (Table S3).
We performed dual-luciferase assays to verify that RsNAC134 recognized and bound to the cis-elements in the promoters of RsSGR, RsPAO1, and RsPAO2. RsNAC134 was mixed with the promoters of RsSGR, RsPAO1, and RsPAO2, and was transiently expressed in tobacco leaves. The empty vector pGreen II-62SK was used as the negative control. When the combinations co-infiltrated with the RsNAC134 and RsSGR promoter, the RsNAC134 and RsPAO1 promoter, the RsNAC134 and RsPAO2 promoter showed significantly higher LUC activity than that of the control (Figure 5).
The Y1H assay was performed to demonstrated that RsNAC134 bound to the promoters of RsSGR, RsPAO1, and RsPAO2. The results showed that yeast cells containing the RsSGR promoter and RsNAC134, the RsPAO1 promoter and RsNAC134, and the RsPAO2 promoter and RsNAC134 grew normally on SD/-Ura-Leu with AbA medium, whereas the corresponding negative controls did not grow normally (Figure 6). These results indicated that RsNAC134 regulated the expression of these genes by binding to the promoters of RsSGR, RsPAO1, and RsPAO2.

4. Discussion

The color of the radish taproot determines its commercial value and nutritional quality. Taproots of different colors contain different secondary metabolites; red and purple radishes are rich in anthocyanins, black radishes are rich in melanin, and green radishes accumulate more chlorophyll. Numerous studies have shown that MYB transcription factors play an important role in regulating radish color. For example, in a study of 56 natural populations and RsMYB haplotypes, three homologous RsMYBs were found to undergo functional differentiation during radish domestication, with RsMYB1 regulating the red skin of ‘new Limei’ radishes and RsMYB2 and RsMYB3 regulating the red skin of the East Asian large long radish (R. sativus var. hortensis), and European small radish (R. sativus var. sativus). It was further discovered that RsMYB1-H1, RsMYB2-H10, and RsMYB3-H6 are the main haplotypes that regulate anthocyanin synthesis [39]. In black radish, RsMYB48/RsMYB97 acts as a transcriptional activator that promotes melanin accumulation [30]. The regulation of radish color by other transcription factors requires further exploration, and the regulatory mechanisms underlying green/white radish formation remain unclear. We analyzed the transcriptome data of white radish ‘55’ and green radish ‘QZ-16’ and found that RsNAC134 showed regular expression (Figure 1). Furthermore, RsNAC134 heterologous overexpression in transgenic tomato plants resulted in chlorosis (Figure 2), consistent with the higher RsNAC134 expression in white radishes.
Chlorophyll degradation occurs during leaf senescence and fruit ripening, and genes related to this pathway have been identified [40]. SGR encodes Mg-dechelatase, which is the first step in the catalytic degradation of chlorophyll a [2,9]. PAO is a key gene in the chlorophyll degradation pathway and can catalyze the conversion of pheophorbide A into red chlorophyll catabolite. Owing to its importance, the chlorophyll degradation pathway is also known as the PAO pathway [41,42]. Overexpression of SGR in A. thaliana leads to a significant reduction in the chlorophyll content, resulting in a slow growth phenotype [2]. Furthermore, transient expression of SGR can induce jasmonic acid synthesis and chlorophyll degradation [7]. Heterologous expression of poplar SGR genes effectively triggers rapid chlorophyll catabolism and stimulates ethylene biosynthesis [42]. Moreover, in pepper, virus-induced gene silencing targeting CaPAO significantly delays chlorophyll degradation [43]. Leaves of N. benthamiana transiently overexpressing RsSGR, RsPAO1, and RsPAO2 showed bleaching compared to the control, and the bleaching effect was stronger in leaves overexpressing RsPAOs than in those overexpressing RsSGR (Figure S3). In tomato plants overexpressing RsNAC134, expression levels of both SlSGR and SlPAO were significantly increased, accompanied by striking leaf whitening. Similarly, the white radish ‘55’ demonstrated markedly elevated RsSGR and RsPAOs expression levels compared to those of the green radish ‘QZ-16’ (Figure 3). The precise regulation of SlSGR and SlPAO, but not other genes like SlNYC and SlPPH, by RsNAC134 suggests a high degree of target specificity. This specificity could be attributed to the unique presence of high-affinity NAC binding elements in the promoters of SlSGR and SlPAO.
NAC transcription factors play an important role in leaf senescence and chlorophyll degradation; however, research on the regulatory mechanisms of taproot coloration is limited. In A. thaliana, NAC019, NAC055, and NAC072 positively regulate leaf senescence by activating AtSGR expression [44,45]. Overexpression of OsNAC2 promotes OsSGR expression [46], whereas overexpression of MpSNAC67 leads to increased expression of PAO-like, HCAR-like and NYC/NOL-like genes [47]. SlNAP2 positively regulates the expression of SlSGR1 and SlPAO expression to promote senescence in tomato leaves [48]. In lychee, LcNAC002 regulates LcSGR expression by directly binding to its promoter, thereby promoting chlorophyll degradation [26]. RsNAC134 is a member of the NAC transcription factor family that contains a conserved NAM domain and exhibits nuclear transcriptional characteristics as a transcription factor (Figure 4). In the present study, we found that RsNAC134 significantly enhanced the expression of RsSGR, RsPAO1, and RsPAO2 by binding to their promoters (Figure 5 and Figure 6), subsequently modulating the chlorophyll degradation pathway, and ultimately leading to a color difference between green and white radishes.

5. Conclusions

In this study, transcriptome data analysis of the green radish ‘QZ-16’ and white radish ‘55’ identified the key regulatory gene RsNAC134 controlling radish green/white coloration. RsNAC134 bound to the promoters of key genes in the chlorophyll degradation pathway, including RsSGR, RsPAO1, and RsPAO2, thereby regulating chlorophyll degradation. Compared with the green radish ‘QZ-16’, the expression levels of RsNAC134 and chlorophyll degradation pathway key genes were significantly elevated in the white radish ‘55’. This discovery expands our understanding of the function of NAC transcription factor, reveals a new regulatory node of radish color formation, lays a foundation for further study of the molecular mechanism of chloroplast development and pigment synthesis, and provides a theoretical basis for improving radish quality.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae11101248/s1, Figure S1. Amino acid sequence and phylogenetic analysis of RsNAC134. (A) Amino acid sequence analysis of RsNAC134. The red underline indicates the NAM conserved domain, and the blue font represents the nuclear localization signal (NLS). (B) Phylogenetic analysis of RsNAC134. The protein sequences are from radish (R120332790), Hirschfeldia incana (KAJ0242295, KAJ0244216), Brassica rapa (XP_009147651, XP_009122872), Brassica napus (XP_048637727, NP_001302941), Eutrema salsugineum (XP_006392856), Capsella rubella (XP_006305156), Brassica oleracea (VDD60482), tomato (Solyc10g006880, Solyc04g005610), Arabidopsis (AT3G15510, AT1G52880), Brassica oleracea (XP_013591746). Figure S2. The positive detection of RsNAC134-OE transgenic material was amplified by PCR. gDNA was extracted from young leaves and used as amplification template. Vector forward primers (35S) and gene direction primers were used for amplification. Marker strips from top to bottom are: 2000, 1500, 750, 500, 200 and 100 bp, respectively. Figure S3. RsSGR, RsPAO1, and RsPAO2 were transiently overexpressed in tobacco leaves. (A) The phenotypes of RsSGR, RsPAO1, and RsPAO2 overexpression in tobacco leaves. Relative expression levels of RsSGR (B), RsPAO1 (C), and RsPAO2 (D) in tobacco leaves. Table S1. The number and functions of cis-elements in radish RsSGR promoter. Table S2. The number and functions of cis-elements in radish RsPAO1 promoter. Table S3. The number and functions of cis-elements in radish RsPAO2 promoter. Table S4. The RT-qPCR primer sequences were used in the study. Table S5. The primer sequences for vector construction in this study.

Author Contributions

Conceptualization, W.C. and W.Y.; methodology, W.C. and W.Y.; validation, W.C., L.C. (Leifu Chen) and C.Y.; formal analysis, W.C. and C.Y.; investigation, W.C. and L.C. (Lei Cui); data curation, W.C. and L.C. (Leifu Chen); writing—original draft preparation, W.C.; writing—review and editing, W.C. and W.Y.; supervision, W.Y.; funding acquisition, W.C. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by grants from the National Natural Science Foundation of China (32202469); International Science and Technology Cooperation Project of Hubei Province (2025EHA010); Hubei Provincial Agricultural Science and Technology Innovation Center Project (2025-620-000-001-007); Hubei Provincial Central Government Program for Guiding Local Science and Technology Development (2024EIA016).

Data Availability Statement

The data used and presented in this paper are available upon request from the corresponding authors.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Sakuraba, Y.; Schelbert, S.; Park, S.Y.; Han, S.H.; Lee, B.D.; Andrès, C.B.; Kessler, F.; Hörtensteiner, S.; Paek, N.C. STAY-GREEN and chlorophyll catabolic enzymes interact at light-harvesting complex Ⅱ for chlorophyll detoxification during leaf senescence in Arabidopsis. Plant Cell 2012, 24, 507–518. [Google Scholar] [CrossRef]
  2. Shimoda, Y.; Ito, H.; Tanaka, A. Arabidopsis STAY-GREEN, Mendel’s green cotyledon gene, encodes magnesium-dechelatase. Plant Cell 2016, 28, 2147–2160. [Google Scholar] [CrossRef]
  3. Hörtensteiner, S. Update on the biochemistry of chlorophyll breakdown. Plant Mol. Biol. 2013, 82, 505–517. [Google Scholar] [CrossRef] [PubMed]
  4. Armstead, I.; Donnison, I.; Aubry, S.; Harper, J.; Hörtensteiner, S.; James, C.; Mani, J.; Moffet, M.; Ougham, H.; Roberts, L.; et al. From crop to model to crop: Identifying the genetic basis of the staygreen mutation in the Lolium/Festuca forage and amenity grasses. New Phytol. 2006, 172, 592–597. [Google Scholar] [CrossRef] [PubMed]
  5. Park, S.Y.; Yu, J.W.; Park, J.S.; Li, J.; Yoo, S.C.; Lee, N.Y.; Lee, S.K.; Jeong, S.W.; Seo, H.S.; Koh, H.J.; et al. The senescence-induced staygreen protein regulates chlorophyll degradation. Plant Cell 2007, 19, 1649–1664. [Google Scholar] [CrossRef] [PubMed]
  6. Armstead, I.; Donnison, I.; Aubry, S.; Harper, J.; Hörtensteiner, S.; James, C.; Mani, J.; Moffet, M.; Ougham, H.; Roberts, L.; et al. Cross-species identification of Mendel’s I locus. Science 2007, 315, 73. [Google Scholar] [CrossRef]
  7. Ono, K.; Kimura, M.; Matsuura, H.; Tanaka, A.; Ito, H. Jasmonate production through chlorophyll a degradation by Stay-Green in Arabidopsis thaliana. J. Plant Physiol. 2019, 238, 53–62. [Google Scholar] [CrossRef]
  8. Sato, T.; Shimoda, Y.; Matsuda, K.; Tanaka, A.; Ito, H. Mg-dechelation of chlorophyll a by Stay-Green activates chlorophyll b degradation through expressing Non-Yellow Coloring 1 in Arabidopsis thaliana. J. Plant Physiol. 2018, 222, 94–102. [Google Scholar] [CrossRef]
  9. Matsuda, K.; Shimoda, Y.; Tanaka, A.; Ito, H. Chlorophyll a is a favorable substrate for chlamydomonas Mg-dechelatase encoded by STAY-GREEN. Plant Physiol. Biochem. 2016, 109, 365–373. [Google Scholar] [CrossRef]
  10. Chung, D.W.; Pruzinska, A.; Hörtensteiner, S.; Ort, D.R. The role of pheophorbide a oxygenase expression and activity in the canola green seed problem. Plant Physiol. 2006, 142, 88–97. [Google Scholar] [CrossRef]
  11. Gray, J.; Close, P.S.; Briggs, S.P.; Johal, G.S. A novel suppressor of cell death in plants encoded by the Lls1 gene of maize. Cell 1997, 89, 25–31. [Google Scholar] [CrossRef]
  12. Li, P.; Ma, Y.; Li, X.; Zhang, L.; Wang, Y.; Wang, N. Cloning and expressional characterization of soybean GmLls1 gene. Chin. Sci. Bull. 2006, 51, 1210–1218. [Google Scholar] [CrossRef]
  13. Ma, N.; Ma, X.; Li, A.; Cao, X.; Kong, L. Cloning and expression analysis of wheat pheophorbide a oxygenase gene TaPaO. Plant Mol. Biol. Rep. 2012, 30, 1237–1245. [Google Scholar] [CrossRef]
  14. Spassieva, S.; Hille, J. A lesion mimic phenotype in tomato obtained by isolating and silencing an Lls1 homologue. Plant Sci. 2002, 162, 543–549. [Google Scholar] [CrossRef]
  15. Tang, Y.; Li, M.; Chen, Y.; Wu, P.; Wu, G.; Jiang, H. Knockdown of OsPAO and OsRCCR1 cause different plant death phenotypes in rice. J. Plant Physiol. 2011, 168, 1952–1959. [Google Scholar] [CrossRef] [PubMed]
  16. Hörtensteiner, S. Chlorophyll degradation during senescence. Annu. Rev. Plant Biol. 2006, 57, 55–77. [Google Scholar] [CrossRef]
  17. Hörtensteiner, S.; Krautler, B. Chlorophyll breakdown in higher plants. Biochim. Biophys. Acta. 2011, 1807, 977–988. [Google Scholar] [CrossRef]
  18. Xie, Q.; Liang, Y.; Zhang, J.; Zheng, H.; Dong, G.; Qian, Q.; Zuo, J. Involvement of a putative bipartite transit peptide in targeting rice pheophorbide a oxygenase into chloroplasts for chlorophyll degradation during leaf senescence. J. Genet. Genom. 2016, 43, 145–154. [Google Scholar] [CrossRef]
  19. Chen, W.; Provart, N.J.; Glazebrook, J.; Katagiri, F.; Chang, H.S.; Eulgem, T.; Mauch, F.; Luan, S.; Zou, G.; Whitham, S.A.; et al. Expression profile matrix of Arabidopsis transcription factor genes suggests their putative functions in response to environmental stresses. Plant Cell 2002, 14, 559–574. [Google Scholar] [CrossRef]
  20. Zafar, Z.; Fatima, S.; Bhatti, M.F. Comprehensive analyses of NAC transcription factor family in almond (Prunus dulcis) and their differential gene expression during fruit development. Plants 2021, 10, 2200. [Google Scholar] [CrossRef]
  21. He, X.J.; Mu, R.L.; Cao, W.H.; Zhang, Z.G.; Zhang, J.S.; Chen, S.Y. AtNAC2, a transcription factor downstream of ethylene and auxin signaling pathways, is involved in salt stress response and lateral root development. Plant J. 2005, 44, 903–916. [Google Scholar] [CrossRef] [PubMed]
  22. Mahmood, K.; Xu, Z.; El-Kereamy, A.; Casaretto, J.A.; Rothstein, S.J. The Arabidopsis transcription factor ANAC032 represses anthocyanin biosynthesis in response to high sucrose and oxidative and abiotic stresses. Front. Plant Sci. 2016, 7, 1548. [Google Scholar] [CrossRef]
  23. Zhong, R.; Demura, T.; Ye, Z.H. SND1, a NAC domain transcription factor, is a key regulator of secondary wall synthesis in fibers of Arabidopsis. Plant Cell 2006, 18, 3158–3170. [Google Scholar] [CrossRef]
  24. Wu, H.; Fu, B.; Sun, P.; Xiao, C.; Liu, J.H. A NAC transcription factor represses putrescine biosynthesis and affects drought tolerance. Plant Physiol. 2016, 172, 1532–1547. [Google Scholar] [CrossRef]
  25. Oda-Yamamizo, C.; Mitsuda, N.; Sakamoto, S.; Ogawa, D.; Ohme-Takagi, M.; Ohmiya, A. The NAC transcription factor ANAC046 is a positive regulator of chlorophyll degradation and senescence in Arabidopsis leaves. Sci. Rep. 2016, 6, 23609. [Google Scholar] [PubMed]
  26. Zou, S.C.; Zhuo, M.G.; Abbas, F.; Hu, G.B.; Wang, H.C.; Huang, X.M. Transcription factor LcNAC002 coregulates chlorophyll degradation and anthocyanin biosynthesis in litchi. Plant Physiol. 2023, 192, 1913–1927. [Google Scholar] [CrossRef]
  27. Weil, R.; Kremen, A. Thinking across and beyond disciplines to make cover crops pay. J. Sci. Food Agric. 2006, 87, 551–557. [Google Scholar] [CrossRef]
  28. Fan, L.; Wang, Y.; Xu, L.; Tang, M.; Zhang, X.; Ying, J.; Li, C.; Dong, J.; Liu, L. A genome-wide association study uncovers a critical role of the RsPAP2 gene in red-skinned Raphanus sativus L. Hortic. Res. 2020, 7, 164. [Google Scholar] [CrossRef]
  29. Luo, X.; Xu, L.; Wang, Y.; Dong, J.; Chen, Y.; Tang, M.; Fan, L.; Zhu, Y.; Liu, L. An ultra-high-density genetic map provides insights into genome synteny, recombination landscape and taproot skin colour in radish (Raphanus sativus L.). Plant Biotechnol. J. 2020, 18, 274–286. [Google Scholar] [CrossRef]
  30. Zhang, S.; Yuan, G.; Peng, Z.; Li, X.; Huang, Y.; Yin, C.; Cui, L.; Xiao, G.; Jiao, Z.; Wang, L.; et al. Chemical composition analysis and transcriptomics reveal the R2R3-MYB genes and phenol oxidases regulating the melanin formation in black radish. Int. J. Biol. Macromol. 2024, 271 Pt 1, 132627. [Google Scholar] [CrossRef] [PubMed]
  31. Liu, T.; Zhang, Y.; Zhang, X.; Sun, Y.; Wang, H.; Song, J.; Li, X. Transcriptome analyses reveal key genes involved in skin color changes of ‘Xinlimei’ radish taproot. Plant Physiol. Biochem. 2019, 139, 528–539. [Google Scholar] [CrossRef] [PubMed]
  32. Huang, Y.; Cui, L.; Chen, W.; Liu, Z.; Yuan, W.; Zhu, F.; Jiao, Z.; Zhang, Z.; Deng, X.; Wang, L.; et al. Comprehensive analysis of NAC transcription factors and their expressions during taproot coloration in radish (Raphanus sativus L.). Sci. Hortic. 2022, 299, 111047. [Google Scholar] [CrossRef]
  33. Xu, Y.; Zhu, X.; Gong, Y.; Xu, L.; Wang, Y.; Liu, L. Evaluation of reference genes for gene expression studies in radish (Raphanus sativus L.) using quantitative real-time PCR. Biochem. Biophys. Res. Commun. 2012, 424, 398–403. [Google Scholar] [CrossRef]
  34. Jones, B.; Frasse, P.; Olmos, E.; Zegzouti, H.; Li, Z.G.; Latché, A.; Pech, J.C.; Bouzayen, M. Down-regulation of DR12, an auxin-response-factor homolog, in the tomato results in a pleiotropic phenotype including dark green and blotchy ripening fruit. Plant J. 2002, 32, 603–613. [Google Scholar] [CrossRef] [PubMed]
  35. Tzfira, T.; Tian, G.W.; Lacroix, B.t.; Vyas, S.; Li, J.; Leitner-Dagan, Y.; Krichevsky, A.; Taylor, T.; Vainstein, A.; Citovsky, V. pSAT vectors: A modular series of plasmids for autofluorescent protein tagging and expression of multiple genes in plants. Plant Mol. Biol. 2005, 57, 503–516. [Google Scholar] [CrossRef]
  36. Zheng, F.; Cui, L.; Li, C.; Xie, Q.; Ai, G.; Wang, J.; Yu, H.; Wang, T.; Zhang, J.; Ye, Z.; et al. Hair interacts with SlZFP8-like to regulate the initiation and elongation of trichomes by modulating SlZFP6 expression in tomato. J. Exp. Bot. 2022, 73, 228–244. [Google Scholar] [CrossRef]
  37. Chen, W.; Hu, T.; Ye, J.; Wang, B.; Liu, G.; Wang, Y.; Yuan, L.; Li, J.; Li, F.; Ye, Z.; et al. A CCAAT-binding factor, SlNFYA10, negatively regulates ascorbate accumulation by modulating the D-mannose/L-galactose pathway in tomato. Hortic. Res. 2020, 7, 200. [Google Scholar] [CrossRef]
  38. Wellburn, A.R. The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. J. Plant Physiol. 1994, 144, 307–313. [Google Scholar] [CrossRef]
  39. Wang, Q.; Wang, Y.; Wu, X.; Shi, W.; Chen, N.; Pang, Y.; Zhang, L. Sequence and epigenetic variations of R2R3-MYB transcription factors determine the diversity of taproot skin and flesh colors in different cultivated types of radish (Raphanus sativus L.). Theor. Appl. Genet. 2024, 137, 133. [Google Scholar] [CrossRef]
  40. Kuai, B.; Chen, J.; Hörtensteiner, S. The biochemistry and molecular biology of chlorophyll breakdown. J. Exp. Bot. 2018, 69, 751–767. [Google Scholar] [CrossRef]
  41. Christ, B.; Egert, A.; Sussenbacher, I.; Krautler, B.; Bartels, D.; Peters, S.; Hörtensteiner, S. Water deficit induces chlorophyll degradation via the ‘PAO/phyllobilin’ pathway in leaves of homoio- (Craterostigma pumilum) and poikilochlorophyllous (Xerophyta viscosa) resurrection plants. Plant Cell Environ. 2014, 37, 2521–2531. [Google Scholar] [CrossRef] [PubMed]
  42. Ito, H.; Saito, H.; Fukui, M.; Tanaka, A.; Arakawa, K. Poplar leaf abscission through induced chlorophyll breakdown by Mg-dechelatase. Plant Sci. 2022, 324, 111444. [Google Scholar] [CrossRef] [PubMed]
  43. Xiao, H.J.; Liu, K.K.; Li, D.W.; Arisha, M.H.; Chai, W.G.; Gong, Z.H. Cloning and characterization of the pepper CaPAO gene for defense responses to salt-induced leaf senescence. BMC Biotechnol. 2015, 15, 100. [Google Scholar] [CrossRef] [PubMed]
  44. Broda, M.; Khan, K.; O’Leary, B.; Pružinská, A.; Lee, C.P.; Millar, A.H.; Van Aken, O. Increased expression of ANAC017 primes for accelerated senescence. Plant Physiol. 2021, 186, 2205–2221. [Google Scholar] [CrossRef]
  45. Zhu, X.; Chen, J.; Xie, Z.; Gao, J.; Ren, G.; Gao, S.; Zhou, X.; Kuai, B. Jasmonic acid promotes degreening via MYC2/3/4- and ANAC019/055/072-mediated regulation of major chlorophyll catabolic genes. Plant J. 2015, 84, 597–610. [Google Scholar] [CrossRef]
  46. Singh, S.; Koyama, H.; Bhati, K.K.; Alok, A. The biotechnological importance of the plant-specific NAC transcription factor family in crop improvement. J. Plant Res. 2021, 134, 475–495. [Google Scholar] [CrossRef]
  47. Tak, H.; Negi, S.; Gupta, A.; Ganapathi, T.R. A stress associated NAC transcription factor MpSNAC67 from banana (Musa x paradisiaca) is involved in regulation of chlorophyll catabolic pathway. Plant Physiol. Biochem. 2018, 132, 61–71. [Google Scholar] [CrossRef]
  48. Ma, X.; Zhang, Y.; Turečková, V.; Xue, G.P.; Fernie, A.R.; Mueller-Roeber, B.; Balazadeh, S. The NAC transcription factor SlNAP2 regulates leaf senescence and fruit yield in tomato. Plant Physiol. 2018, 177, 1286–1302. [Google Scholar] [CrossRef]
Horticulturae 11 01248 g001
Figure 1. Expression levels of RsNAC134 in green/white radish and its sequence alignment with corresponding sequences in plants. (A) Phenotypes of white radish ‘55’ and green radish ‘QZ-16’. (B) Relative expression levels of RsNAC134 in the skin and flesh of ‘55’ and ‘QZ-16’. (C) Multiple sequence alignment of RsNAC134 and its counterparts from different species. The protein sequences are from radish (R120332790), Hirschfeldia incana (KAJ0242295), Brassica rapa (XP_009147651), Brassica napus (XP_048637727), Eutrema salsugineum (XP_006392856), Capsella rubella (XP_006305156). The red line refers to the NAM conservative domain. Asterisks indicate significant differences (** p < 0.01).
Figure 1. Expression levels of RsNAC134 in green/white radish and its sequence alignment with corresponding sequences in plants. (A) Phenotypes of white radish ‘55’ and green radish ‘QZ-16’. (B) Relative expression levels of RsNAC134 in the skin and flesh of ‘55’ and ‘QZ-16’. (C) Multiple sequence alignment of RsNAC134 and its counterparts from different species. The protein sequences are from radish (R120332790), Hirschfeldia incana (KAJ0242295), Brassica rapa (XP_009147651), Brassica napus (XP_048637727), Eutrema salsugineum (XP_006392856), Capsella rubella (XP_006305156). The red line refers to the NAM conservative domain. Asterisks indicate significant differences (** p < 0.01).
Horticulturae 11 01248 g001
Horticulturae 11 01248 g002
Figure 2. Overexpression of RsNAC134 causes color changes in tomato plants. (A) Phenotype of RsNAC134-overexpression lines and control (CK) in tomato. (B) Relative expression level of RsNAC134 in tomato leaves of overexpression lines and CK. (C) Chlorophyll content of RsNAC134-overexpression lines and CK. Asterisks indicate significant differences (** p < 0.01).
Figure 2. Overexpression of RsNAC134 causes color changes in tomato plants. (A) Phenotype of RsNAC134-overexpression lines and control (CK) in tomato. (B) Relative expression level of RsNAC134 in tomato leaves of overexpression lines and CK. (C) Chlorophyll content of RsNAC134-overexpression lines and CK. Asterisks indicate significant differences (** p < 0.01).
Horticulturae 11 01248 g002
Horticulturae 11 01248 g003
Figure 3. Relative expression levels of genes related to the chlorophyll degradation pathway in RsNAC134-overexpression tomatoes and radishes. Relative expression levels of SlSGR (A), SlPAO (B), SlNYC (C), SlCHL1 (D), and SlPPH1 (E) in tomato leaves of RsNAC134-overexpression lines and CK. (F) The chlorophyll content of the skin and flesh of ‘55’ and ‘QZ-16’ radishes. Relative expression levels of RsSGR (G), RsPAO1 (H), RsPAO2 (I), RsNYC (J), RsCHL1 (K), and RsCHL2 (L) in the skin and flesh of radish varieties ‘55’ and ‘QZ-16’. Asterisks indicate significant differences (* p < 0.05, ** p < 0.01).
Figure 3. Relative expression levels of genes related to the chlorophyll degradation pathway in RsNAC134-overexpression tomatoes and radishes. Relative expression levels of SlSGR (A), SlPAO (B), SlNYC (C), SlCHL1 (D), and SlPPH1 (E) in tomato leaves of RsNAC134-overexpression lines and CK. (F) The chlorophyll content of the skin and flesh of ‘55’ and ‘QZ-16’ radishes. Relative expression levels of RsSGR (G), RsPAO1 (H), RsPAO2 (I), RsNYC (J), RsCHL1 (K), and RsCHL2 (L) in the skin and flesh of radish varieties ‘55’ and ‘QZ-16’. Asterisks indicate significant differences (* p < 0.05, ** p < 0.01).
Horticulturae 11 01248 g003
Horticulturae 11 01248 g004
Figure 4. RsNAC134 is a transcriptional activator. (A) Schematic representation of the construct vectors used for transcriptional activity assays. Full-length and truncated fragments of RsNAC134 were introduced downstream of the galactose-specific transcription enhancing factor binding domain (GAL4BD) in the pGBKT7 vector. RsNAC134 N- and C- represent the deletion of the C- and N-termini of RsNAC134 proteins, respectively. Numbers above the shaded boxes indicate the nucleotide positions. (B) Growth of the yeast strain AH109 transformed with the vectors, along with the negative control (pGBKT7), on SD/-Trp, SD/-Trp-His-Ade, and SD/-Trp-His-Ade supplemented with X-α-gal. (C) Subcellular localization of RsNAC134 based on the visualization of the YFP signal. The fusion construct RsNAC134-YFP was co-transformed with the nuclear marker CaMV35S::H-mcherry into Nicotiana benthamiana leaves. 35S::YFP was co-transformed with the nuclear marker CaMV35S::H-mcherry as positive control. YFP: Yellow fluorescence, RPF: Red fluorescence. Confocal microscopic images were obtained 48 h after Agrobacterium injection. Scale bars = 50 µm.
Figure 4. RsNAC134 is a transcriptional activator. (A) Schematic representation of the construct vectors used for transcriptional activity assays. Full-length and truncated fragments of RsNAC134 were introduced downstream of the galactose-specific transcription enhancing factor binding domain (GAL4BD) in the pGBKT7 vector. RsNAC134 N- and C- represent the deletion of the C- and N-termini of RsNAC134 proteins, respectively. Numbers above the shaded boxes indicate the nucleotide positions. (B) Growth of the yeast strain AH109 transformed with the vectors, along with the negative control (pGBKT7), on SD/-Trp, SD/-Trp-His-Ade, and SD/-Trp-His-Ade supplemented with X-α-gal. (C) Subcellular localization of RsNAC134 based on the visualization of the YFP signal. The fusion construct RsNAC134-YFP was co-transformed with the nuclear marker CaMV35S::H-mcherry into Nicotiana benthamiana leaves. 35S::YFP was co-transformed with the nuclear marker CaMV35S::H-mcherry as positive control. YFP: Yellow fluorescence, RPF: Red fluorescence. Confocal microscopic images were obtained 48 h after Agrobacterium injection. Scale bars = 50 µm.
Horticulturae 11 01248 g004
Horticulturae 11 01248 g005
Figure 5. RsNAC134 activated RsSGR, RsPAO1, and RsPAO2 promoters in a dual-luciferase assay. (A) Schematic representation of the dual-luciferase reporter assay constructs used to assess the transactivation of the RsSGR, RsPAO1, and RsPAO2 promoters by RsNAC134. Relative luciferase (LUC/REN) activity of RsSGR pro-LUC co-transformed with RsNAC134 (B), RsPAO1 pro-LUC co-transformed with RsNAC134 (C), and RsPAO2 pro-LUC co-transformed with RsNAC134 (D) compared to with those of empty vector in tobacco leaves. pGreen II-62SK empty vector as Empty, with six biological replicates per combination. Asterisks indicate significant differences (** p < 0.01, * p < 0.05).
Figure 5. RsNAC134 activated RsSGR, RsPAO1, and RsPAO2 promoters in a dual-luciferase assay. (A) Schematic representation of the dual-luciferase reporter assay constructs used to assess the transactivation of the RsSGR, RsPAO1, and RsPAO2 promoters by RsNAC134. Relative luciferase (LUC/REN) activity of RsSGR pro-LUC co-transformed with RsNAC134 (B), RsPAO1 pro-LUC co-transformed with RsNAC134 (C), and RsPAO2 pro-LUC co-transformed with RsNAC134 (D) compared to with those of empty vector in tobacco leaves. pGreen II-62SK empty vector as Empty, with six biological replicates per combination. Asterisks indicate significant differences (** p < 0.01, * p < 0.05).
Horticulturae 11 01248 g005
Horticulturae 11 01248 g006
Figure 6. Interaction of RsNAC134 with the RsSGR, RsPAO1, and RsPAO2 promoters in Y1H assays. Schematic representation of NAC-binding sites on the promoters of RsSGR (A), RsPAO1 (C), and RsPAO2 (E). Yeast one-hybrid assay results demonstrated the interaction of RsNAC134 with the RsSGR (B), RsPAO1 (D), and RsPAO2 (F) promoters. Prey and bait vectors were co-transformed into Y1H Gold strain cells and cultured on SD/-Ura-Leu medium containing different AbA concentrations. p53-AbAi/pGAD-p53 and bait-pGADT7 were used as positive (‘+’) and negative controls (‘−’), respectively.
Figure 6. Interaction of RsNAC134 with the RsSGR, RsPAO1, and RsPAO2 promoters in Y1H assays. Schematic representation of NAC-binding sites on the promoters of RsSGR (A), RsPAO1 (C), and RsPAO2 (E). Yeast one-hybrid assay results demonstrated the interaction of RsNAC134 with the RsSGR (B), RsPAO1 (D), and RsPAO2 (F) promoters. Prey and bait vectors were co-transformed into Y1H Gold strain cells and cultured on SD/-Ura-Leu medium containing different AbA concentrations. p53-AbAi/pGAD-p53 and bait-pGADT7 were used as positive (‘+’) and negative controls (‘−’), respectively.
Horticulturae 11 01248 g006
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Chen, W.; Yan, C.; Chen, L.; Cui, L.; Yuan, W. RsNAC134 Regulates Taproot Skin Color via Positive Regulation of the Chlorophyll Degradation Pathway in Radish (Raphanus sativus). Horticulturae 2025, 11, 1248. https://doi.org/10.3390/horticulturae11101248

AMA Style

Chen W, Yan C, Chen L, Cui L, Yuan W. RsNAC134 Regulates Taproot Skin Color via Positive Regulation of the Chlorophyll Degradation Pathway in Radish (Raphanus sativus). Horticulturae. 2025; 11(10):1248. https://doi.org/10.3390/horticulturae11101248

Chicago/Turabian Style

Chen, Weifang, Chenghuan Yan, Leifu Chen, Lei Cui, and Weiling Yuan. 2025. "RsNAC134 Regulates Taproot Skin Color via Positive Regulation of the Chlorophyll Degradation Pathway in Radish (Raphanus sativus)" Horticulturae 11, no. 10: 1248. https://doi.org/10.3390/horticulturae11101248

APA Style

Chen, W., Yan, C., Chen, L., Cui, L., & Yuan, W. (2025). RsNAC134 Regulates Taproot Skin Color via Positive Regulation of the Chlorophyll Degradation Pathway in Radish (Raphanus sativus). Horticulturae, 11(10), 1248. https://doi.org/10.3390/horticulturae11101248

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop