MsMYB62-like as a negative regulator of anthocyanin biosynthesis in Malus spectabilis

ABSTRACT

Crabapple is a valuable tree species in gardens due to its captivating array of flower and leaf colors, rendering it a favored choice in landscaping. The economic and ornamental values of Malus crabapple are closely associated with the biosynthesis of anthocyanin, a pigment responsible for its vibrant hues. The intricate regulation of anthocyanin biosynthesis involves the concerted activity of various genes. However, the specific mechanism governing this process in crabapple warrants in-depth exploration. In this study, we explored the inhibitory role of MsMYB62-like in anthocyanin biosynthesis. We identified MsDFR and MsANS as two downstream target genes of MsMYB62-like. These genes encode enzymes integral to the anthocyanin biosynthetic pathway. The findings demonstrate that MsMYB62-like directly binds to the promoters of MsDFR and MsANS, resulting in the downregulation of their expression levels. Additionally, our observations indicate that the plant hormone cytokinins exert a suppressive effect on the expression levels of MsMYB62-like, while concurrently upregulating MsDFR and MsANS. This study reveals that the MsMYB62-like-MsDFR/MsANS module plays an important role in governing anthocyanin levels in Malus crabapple. Notably, the regulatory interplay is modulated by the plant hormone cytokinins.

KEYWORDS:

• Malus spectabilis
• anthocyanin
• MsMYB62-like
• MsDFR and MsANS
• cytokinins

Introduction

Crabapple is an ornamental deciduous species known for its decorative value and aesthetic appeal in gardens and landscaping. The captivating colors of crabapple, including its fruits, flowers, and leaves, are attributed to the presence of anthocyanin. Anthocyanin, a type of water-soluble pigmented flavonoids, are widely distributed in higher plants.¹ Beyond enhancing the ornamental value of plants, anthocyanin plays a pivotal role in various physiological processes, safeguarding against damage from drought, pathogens, ultraviolet radiation, low temperatures, diseases, pests, and other adversities.^2–6 Furthermore, anthocyanins are the final products of flavonoid biosynthesis and play a crucial role in the purple or red pigmentation of plant leaves.⁷ These compounds have been suggested to offer protection against oxidative stress, coronary heart diseases, certain cancers, and other age-related diseases. However, the bioavailability of anthocyanins has been a subject of inquiry among some researchers.⁸

The biosynthesis of anthocyanin follows the conserved anthocyanin biosynthetic pathway, a branch of the phenylalanine pathway.^9,10 The original phenylpropanoid biosynthesis, under the regulation of a series of enzymes such as phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate:CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3’-hydroxylase (F3’H), Flavonoid 3‘,5’-hydroxylase (F3’5‘H), dihydroflavonol 4-reductase (DFR), anthocyanin synthase (ANS) and flavonoid 3-O-glu-cosyltransferase (UFGT), are responsible for anthocyanin biosynthesis. Simultaneously, enzymes such as β-glucosidase (BGLU), polyphenol oxidase (PPO), peroxidase (PER) and laccase (LAC) participate in anthocyanin degradation.^1,6,9 Moreover, these enzymes function sequentially to produce specific anthocyanin, and their activities are influenced by both internal and external factors, such as environmental signals and plant hormones.^1,11 Understanding these intricate processes is imperative for unraveling the vibrant realm of plant pigmentation.

In plants, MYB transcription factors emerge as pivotal players in the regulation of anthocyanin production. These factors exert influence by activating or inhibiting the expression of anthocyanin structural genes, directly or indirectly binding to their promoters.^9,12,13 Apple MYB transcription factors, such as MdMYB1, MdMYB10, MdMYB9, MdMYB11, MdMYB110a, MdMYB308 and MdMYBPA1, promote anthocyanin accumulation by activating the expression of anthocyanin structural genes.¹⁴ Conversely, MYB transcription factors, including MdMYB16, MdMYB17, MdMYB111, MdMYB308 and MdMYB306-like, suppress anthocyanin production by downregulating the expression of anthocyanin structural genes.¹⁰ Additionaly, PbMYB120 in pears inhibits UFGT promoter activity, thereby suppressing anthocyanin biosynthesis.¹⁵ In blueberries, VcMYB1 positively regulates the transcription of VcDFR, contributing to increased anthocyanin levels.¹⁶ Meanwhile, PpMYB39 in peaches activates PpDFR, thereby promoting anthocyanin biosynthesis.¹⁷ In bananas, MaMYB4 represses the expression of MaCHS, MaANS, and MaDFR, effectively hindering anthocyanin accumulation.¹⁸ The recognized influence of MYB transcription factors in anthocyanin biosynthesis has prompted a surge in reported mechanisms governing their regulatory role.¹ Nevertheless, the intricacies of MYB transcription factor-mediated regulation of anthocyanin biosynthesis pose a complex biological puzzle, underscoring the ongoing importance of exploring downstream genes subject to their regulation.

In our previous work, we demonstrated the role of Malus spectabilis MYB62-like in regulating anthocyanin biosynthesis under low-nitrogen conditions.¹⁹ However, the functions and mechanisms of MsMYB62-like under normal environmental conditions remain largely unknown. In the present study, we found that overexpression of MsMYB62-like led to a reduction in anthocyanin levels under normal growth conditions. Further investigation revealed that MsMYB62-like could directly bind to the promoters of the anthocyanin structural genes MsDFR and MsANS, resulting in a decrease in their expression levels. Intriguingly, cytokinins, known enhancers of anthocyanin levels,^20,21 exhibited a dual effect by enhancing the expression of MsDFR and MsANS, while simultaneously downregulating the expression of MsMYB62-like. These findings propose a potential mechanism wherein cytokinins suppress the expression of MsMYB62-like, disrupting the MsMYB62-like-MsDFR/MsANS module and ultimately leading to an increase in anthocyanin biosynthesis.

Plant material and methods

Plant material

Malus spectabilis shoots were cultivated in vitro, and the sterile leaves from four-week-old shoots were collected as plant materials. The culture conditions for M. spectabilis growth were performed as previously described.²²

Measurement of total anthocyanin

The fresh leaves were weight and ground in a pre-cooled mortar with ‘Anthocyanin Aaaay Buffer’ (Saint-Bio, China). Mixtures were incubated at 4°C for 20 min, and samples were inverted gently for 2–3 times during the incubation. After that, mixtures were centrifuged at 8000 r/min for 3 min, and supernatants were collected. Absorbance was measured at 530 nm. Relative anthocyanin content was determined according to the instruction manual.

Agroinfiltration of M. spectabilis leaves

The MsMYB62-like overexpression plasmid was constructed using the pCHF70 vector.²³ Agroinfiltration in M. spectabilis leaves of MsMYB62-like overexpression plasmid and empty vector were performed according to procedures described previously with some modifications.²⁴ Sterile leaves were placed in the bacterial suspension solution, and infiltration was performed by vacuum at −0.08 MPa for 15 s. Leaves were then washed in deionized water for two times and placed in the selection plates. Afterwards, the plates were kept in the dark for 24 h and then under a 16/8 h (light/dark) photoperiod at 25°C for 3 days.

Quantitative real-time PCR analysis

Total RNA was extracted with ‘RNeasy PowerPlant Kit’ (QIAGEN, Germany) by following the manufacturer’s instruction. Approximately 1 μg of total RNA was used for reverse transcription to synthesize cDNA according to the protocol of the supplier with the ‘Evo M-MLV RT Premix for qPCR’ (Accurate Biology, China). The resulting cDNA was used for RT-qPCR analysis with ‘SYBR Green Premix Pro Taq HS qPCR Kit’ (Accurate Biology, China) on the CFX96TM (BIO-RAD, USA). 18S rRNA was used as a reference gene. The primer sequences used are shown in the Supplementary Table. All experiments were performed with three technical replicates and three independent biological replicates.

Heterologous expression and protein purification

The MsMYB62-like gene was amplified from cDNA extracted from M. spectabilis leaves and subsequently cloned into the pGEX-6P-1 vector.²⁵ The recombinant pGEX-6P-1-MsMYB62-like plasmid was transformed into E. coli BL21(DE3) cells. LB medium supplemented with 100 µg/ml ampicillin was inoculated with the transformed strain at 37°C overnight while shaking at 180 rpm. Then, 1 L of TB medium supplemented with 100 µg/ml ampicillin was inoculated with 10 ml of the pre-culture and grown to an OD₆₀₀ of 0.5–0.6. The culture was induced by adding 0.3 mM IPTG and incubated overnight at 18°C under shaking at 150 rpm. After that, cells were harvested by centrifugation at 4000 g for 15 min. For protein purification, cells were resuspended in binding buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO_4, pH 7.4) containing 0.1 mg/ml DNase and lysed by ultrasonication (1 s pulse, 2 s pause; 4 min pulse time; 2 times). After that, the cell lysate was centrifuged at maximum speed for 30 min. The cleared lysate was loaded onto a 1 ml Glutathione Beads 4FF column (Smart-Lifesciences, China) pre-equilibrated with binding buffer. Unspecifically bound proteins were removed from the column by washing with 15 column volumes of binding buffer. Then, GST-tagged protein was eluted using the elution buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO_4, 10 mM reduced glutathione, pH 7.4). The most concentrated protein fraction was flash-frozen in liquid nitrogen and stored at −80°C.

Electrophoretic mobility shift assay

Electrophoretic mobility shift assays (EMSAs) were done as described in Li et al.²⁵ with some modification. Biotin-labeled probes were generated by annealing complementary oligonucleotides using primers listed in the Supplementary Table. The annealing process involved heating the mixture to 95°C for 5 min, followed by a 5 min incubation on ice. To begin the assay, unlabeled DNA (cold competitor) at a final concentration of 5 μM and 3 μg of GST-MsMYB62 protein were pre-incubated in 20 μl of 1× binding buffer (10 mM Tris-HCl pH 8.0, 150 mM KCl, 0.5 mM EDTA pH 8.0, 0.1% Triton-X 100, 12.5% glycerol, 0.2 mM DTT, freshly added before use) at 24°C for 20 min. Subsequently, biotin-labeled probes at a final concentration of 50 nM were added to the respective binding reactions, followed by another 20 min incubation at 24°C. The samples were then analyzed on 6% non-denaturing polyacrylamide gels, which were run in 0.5× Tris-Borate-EDTA buffer at 120 V. After electrophoresis, the gels were transferred onto a nylon membrane at 380 mA (~100 V) for 30 min, and the DNA was immediately crosslinked to the membrane at 120 mJ cm⁻² using a UV crosslinker. Next, the membrane was blocked for 30 min at room temperature in 30 ml of 0.5% BSA buffer. It was then incubated under gentle shaking for an additional 30 min at room temperature in 0.5% BSA buffer containing Stabilized Streptavidin-Horseradish Peroxidase Conjugate. Following two washes with PBST and one wash with PBS, ECL detection reagent was applied to the membrane, and luminescence signals were detected using a CCD camera.

The dual luciferase reporter assay

Agrobacterium tumefaciens (GV3101) carrying the respective plasmids were co-infiltrated into leaves of 3- to 5-week-old Nicotiana benthamiana plants, following the protocol described by Grefen et al..²⁶ The effectors used were p35S: EYFP-MsMYB62 and p35S: EYFP (empty vector, negative control).²⁵ The promoter regions of the target genes were fused with the firefly luciferase (LUC) gene in the pGreenII0800-LUC vector, generating reporter plasmids; these reporter plasmids also contained the Renilla luciferase (REN) gene driven by the 35S promoter, which served as an internal control.²⁷ After co-expression for 2 and 3 days, leaf discs were harvested, and the LUC and REN activities were determined using a dual-LUC reporter assay kit on a GloMax 20/20 luminometer (Promega).²⁷

Results and discussion

Overexpression of MsMYB62-like inhibits anthocyanin accumulation in Malus spectabilis leaves

In our previous work, we found that MsMYB62-like from Malus spectabilis plays a role in suppressing anthocyanin production under low nitrogen conditions.¹⁹ In this study, we employed Malus spectabilis leaves and introduced transient genetic modifications by overexpressing MsMYB62-like using the cauliflower mosaic virus (CaMV) 35S promoter (35S:EYFP-MsMYB62-like). After placing these modified leaves in a culture box, we measured anthocyanin levels in the leaves overexpressing MsMYB62-like and compared them with control leaves carrying an empty vector, three days later. Interestingly, we observed a lower anthocyanin content in the leaves overexpressing MsMYB62-like compared to the control (Figure 1a, b), suggesting that MsMYB62-like could reduce anthocyanin levels.

Figure 1. Functional characterization of MsMYB62-like in Malus spectabilis leaves.

(a) Leaf phenotype in transiently transgenic Malus spectabilis leaves overexpressing the MsMYB62-like and empty vector. (b) Total anthocyanin content in transiently transgenic Malus spectabilis leaves overexpressing the MsMYB62-like and empty vector. Error bars represent standard deviation (SD) of three biological replicates. Different lowercase letters indicate significant differences (p < 0.05, Student’s t-test). (c) RT-qPCR analysis of MsCHS, MsDFR, MsANS, and MsUFGT expression in transiently transgenic Malus spectabilis leaves overexpressing the MsMYB62-like relative to overexpressing the empty vector. 18S rRNA was used as an internal control. Error bars represent standard deviation (SD) of three biological replicates. Different lowercase letters indicate significant differences (p < 0.05, Student’s t-test).

To understand how MsMYB62-like inhibits anthocyanin accumulation, we investigated its impact on several anthocyanin biosynthesis genes. We extracted RNA from the modified leaves, performed reverse transcription, and conducted quantitative real-time polymerase chain reaction (qPCR) experiments. The RT-qPCR results showed a significant decrease in the expression levels of four anthocyanin structural genes, namely, MsCHS, MsDFR, MsANS, and MsUFGT, in the transiently transformed Malus spectabilis leaves (Figure 1c). These findings suggest that MsMYB62-like may inhibit anthocyanin biosynthesis by downregulating the expression of genes encoding anthocyanin biosynthetic enzymes.

MsMYB62-like directly binds to the promoters of MsDFR and MsANS

MsMYB62-like was found to suppress the expression of MsF3’H, leading to the negative regulation of anthocyanin biosynthesis.¹⁹ As mentioned earlier, MsMYB62-like suppresses the expression levels of MsCHS, MsDFR, MsANS, and MsUFGT. To investigate whether MsMYB62-like inhibits anthocyanin biosynthesis by directly suppressing the expression of these genes, we focused on two genes, MsDFR and MsANS, for further investigation. To determine whether the promoters of MsDFR and MsANS are directly targeted by MsMYB62-like, we conducted a search for putative binding sites of MsMYB62-like in the promoters of MsDFR and MsANS. In the MsDFR promoter, we identified a TGGTTAG motif at positions −710 to −704, previously recognized as the motif bound by AtMYB2 in Arabidopsis.²⁸ Additionally, we found four potential binding sites (TGGTTT) in the MsANS promoter, which are known as MYB binding sites.²⁹

To evaluate the binding interaction between MsMYB62-like and the promoters of MsDFR and MsANS, we constructed biotin-labeled probes containing the TGGTTAG motif in the MsDFR promoter (−718…-694) and the TGGTTT motif in the MsANS promoter (−474…-416) (Figure 2a, b). An unlabeled probe was used as a competitor. We expressed MsMYB62-like as GST fusion proteins in E. coli, purified it, and conducted electrophoretic mobility shift assay (EMSA) experiments. The results of the EMSA experiments demonstrated that MsMYB62-like caused an upward shift in the promoter fragments of both MsDFR and MsANS (Figure 2c, d), indicating that MsMYB62-like binds to them in vitro. To further support the role of MsMYB62-like in suppressing the expression of MsDFR and MsANS, we conducted a transactivation assay in infiltrated tobacco leaves. 35S:EYFP-MsMYB62-like or 35S:EYFP alone were co-expressed with MsDFR and MsANS promoter: luciferase reporter constructs. As a control, we also used the MsF3‘H promoter: luciferase construct, which has previously been shown to be inhibited by MsMYB62-like.¹⁹ The expression of MsMYB62-like led to a reduction in the activation of the MsDFR and MsANS promoters compared to the control, which was the empty 35S:EYFP vector (Supplementary Figure). These findings suggest that MsMYB62-like forms a regulatory module with MsDFR/MsANS. Through this MsMYB62-like-MsDFR/MsANS module, MsMYB62-like directly downregulates the expression levels of MsDFR and MsANS, thereby inhibiting the accumulation of anthocyanin.

Figure 2. MsMYB62-like directly binds to the promoter of MsDFR and MsANS.

(a)-(b) Diagram depicting the MsDFR and MsANS promoters, and promoter fragments used in the EMSAs. (c)-(d) EMSAs. Biotin-labeled MsDFR and MsANS promoter fragments (pMsDFR −718 … −694 and pMsANS −474 … −416) were incubated with GST-MsMYB62-like, or GST alone (negative control). FP, free probe; Cold, excess of unlabeled probes. Experiments were repeated at least three times with similar results.

Cytokinins inhibit the expression of MsMYB62-like

Cytokinins have been reported to enhance the biosynthesis of anthocyanin.^20,21 In our study, we exposed Malus spectabilis leaves to a 20 μM concentration of the cytokinins analog 6-benzylaminopurine (6-BA) for a period of 3 days. Subsequently, we measured the content of anthocyanin in the leaves, and extracted total RNA from the leaves to perform RT-qPCR experiments. After treatment with 6-BA, we observed a higher content of anthocyanin compared to the control group (Figure 3a), indicating that cytokinins can enhance the anthocyanin content in Malus spectabilis leaves.

Figure 3. Cytokinins regulate MsMYB62-like and anthocyanin structural genes expression.

(a) Total anthocyanin content in Malus spectabilis leaves treatment with (CTK) or without (CK) 20 μM cytokinins analog 6-benzylaminopurine (6-BA). Error bars represent standard deviation (SD) of three biological replicates. Different lowercase letters indicate significant differences (p < 0.05, Student’s t-test). (b) RT-qPCR analysis of MsMYB62-like, MsCHS, MsDFR and MsANS expression in Malus spectabilis leaves treatment with (CTK) or without (CK) 20 μM cytokinins analog 6-benzylaminopurine (6-BA). 18S rRNA was used as an internal control. Error bars represent standard deviation (SD) of three biological replicates. Different lowercase letters indicate significant differences (p < 0.05, Student’s t-test).

The RT-qPCR experiments revealed a significant decrease in the expression level of MsMYB62-like in the presence of 6-BA, while the expression levels of MsCHS, MsDFR, and MsANS were increased (Figure 3b). This implies that cytokinins can elevate anthocyanin levels by upregulating the expression of MsCHS, MsDFR, and MsANS. The downregulated expression of MsMYB62-like by cytokinins further suggests that cytokinins can also increase anthocyanin content by suppressing the expression of MsMYB62-like. Based on these observations, we can infer that cytokinins may regulate, or at least partially regulate, anthocyanin by disrupting the MsMYB62-like-MsDFR/MsANS module.

Overall, we have uncovered that MsMYB62-like can directly bind to the promoters of the anthocyanin structural genes MsDFR and MsANS, thereby reducing their expression levels (Figure 4). Furthermore, our study has demonstrated that cytokinins have the ability to enhance the expression of MsDFR and MsANS while concurrently reducing the expression of MsMYB62-like (Figure 4). These findings suggest a potential mechanism by which cytokinins disrupt the MsMYB62-like-MsDFR/MsANS module, ultimately resulting in an increase in anthocyanin biosynthesis.

Figure 4. A proposed working model for how MsMYB62-like inhibits anthocyanin biosynthesis in Malus spectabilis.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15592324.2024.2318509

Additional information

Funding

This work was supported by the [Doctoral Startup Fund of Northwest A&F University] under Grant [number 2452023033]; [Chinese Universities Scientific Fund] under Grant [number 2452023085].