A Regulatory Mechanism on Pathways: Modulating Roles of MYC2 and BBX21 in the Flavonoid Network

doi:10.3390/plants13081156

. 2024 Apr 22;13(8):1156.

doi: 10.3390/plants13081156.

A Regulatory Mechanism on Pathways: Modulating Roles of MYC2 and BBX21 in the Flavonoid Network

Nan Li^{1

2}, Yunzhang Xu^{1

2

3}, Yingqing Lu^{1

2}

Affiliations

¹ State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China.
² University of Chinese Academy of Sciences, Beijing 100049, China.
³ State Key Laboratory of Plateau Ecology and Agriculture, Qinghai University, Xining 810016, China.

PMID: 38674565
PMCID: PMC11054080
DOI: 10.3390/plants13081156

A Regulatory Mechanism on Pathways: Modulating Roles of MYC2 and BBX21 in the Flavonoid Network

Nan Li et al. Plants (Basel). 2024.

. 2024 Apr 22;13(8):1156.

doi: 10.3390/plants13081156.

Authors

Nan Li^{1

2}, Yunzhang Xu^{1

2

3}, Yingqing Lu^{1

2}

Affiliations

¹ State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China.
² University of Chinese Academy of Sciences, Beijing 100049, China.
³ State Key Laboratory of Plateau Ecology and Agriculture, Qinghai University, Xining 810016, China.

PMID: 38674565
PMCID: PMC11054080
DOI: 10.3390/plants13081156

Abstract

Genes of metabolic pathways are individually or collectively regulated, often via unclear mechanisms. The anthocyanin pathway, well known for its regulation by the MYB/bHLH/WDR (MBW) complex but less well understood in its connections to MYC2, BBX21, SPL9, PIF3, and HY5, is investigated here for its direct links to the regulators. We show that MYC2 can activate the structural genes of the anthocyanin pathway but also suppress them (except F3'H) in both Arabidopsis and Oryza when a local MBW complex is present. BBX21 or SPL9 can activate all or part of the structural genes, respectively, but the effects can be largely overwritten by the local MBW complex. HY5 primarily influences expressions of the early genes (CHS, CHI, and F3H). TF-TF relationships can be complex here: PIF3, BBX21, or SPL9 can mildly activate MYC2; MYC2 physically interacts with the bHLH (GL3) of the MBW complex and/or competes with strong actions of BBX21 to lessen a stimulus to the anthocyanin pathway. The dual role of MYC2 in regulating the anthocyanin pathway and a similar role of BBX21 in regulating BAN reveal a network-level mechanism, in which pathways are modulated locally and competing interactions between modulators may tone down strong environmental signals before they reach the network.

Keywords: BBX21; F3′H activation; light signaling; molecular competition; pathway regulation.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

**Figure 1**
Effects of MYC2 on the anthocyanin pathway of A. *thaliana*. (A) Phenotypes of mutant *myc2* and its complementary lines at day 3. The control line is Columbia (Col). The bar is 1 mm. (B) Accumulation of anthocyanins in seedlings. The lines in (A) were quantified for anthocyanin content during 48–72 h of growth. The unit is arbitrary and weighted by the fresh weight (FW) of seedlings. Each data point has two replicates, with each replicate containing 20–30 plants. 35S-1 is for 35S::MYC2-1, and 35S-2 for 35S::MYC2-2. The difference between 35S-1 and Col at 72 h is highly significant (t-test, one-tailed, p = 0.002), and that between 35S-2 and *myc2* is also significant (t-test, one-tailed, p = 0.03). (C) Real-time expressions of *MYC2* and *JAZ1* across the lines at day 3. Data are shown as mean ± standard error. Each mean is based on three biological replicates (n = 3) at the same sampling time, and each replicate was measured at least twice. The transcript levels of two complementary lines are significantly higher than that of *myc2* (one-tailed t-tests; *, p < 0.02; **, p < 0.01). (D) Activations of genes by MYC2 in dual LUC assays. Reporter is indicated by the x-axis, containing 5′ region of the target gene and designated by CHSpro for CHS_pro, etc., introduced along with pMYC2 (4 μg each). The promoter activity was measured by LUC/RUC. The background activity (blank) of each gene was shown by the treatment of reporters and empty effectors (4 μg each). The standard error bars include three to five biological replicates. Data were normalized. Compared to the reporter’s background level, the treatment of pMYC2 was significant for all reporters (one-tailed t-tests; *, p < 0.05; **, p < 0.01) except that of ANS (p = 0.40). (E) Effect of MYC2 on transcriptions of the MBW genes. Effectors and reporters (1:1) were mixed in 4 μg each for *PAP1*_pro and 5–10 μg each for *GL3*_pro or *TTG1*_pro. Each test had at least two biological replicates. Activation of *PAP1*_pro is highly significant per the t-test (one-tailed, **, p < 0.001, n = 28). (F) Transcript numbers of *PAP1* in the lines of (A) at day 3. Three biological replicates were taken. Format follows (C). (G) Combined effect of MYC2 and MBW on structural genes in dual LUC assays. Adding MYC2 caused significantly altered activity for all reporters (two-tailed t-tests; **, p < 0.002 in all cases). Data were normalized across tests (n ≥ 3). (H) Interactions of MYC2 with promoters of anthocyanin genes in Y1H. Each test had at least three biological replicates, with the interaction between P53 and the original pHIS2 as the positive control and the combination of empty pAD and a reporter vector as the negative one. (I) Interactions of MYC2 with probes based on 5′ regions of anthocyanin genes in EMSAs. Each of the probes with the sequences (5′ → 3′) listed by the numbers to the right was mixed with MYC2 (~10 μg) to test its binding capacity. The upper panel shows the DNA binding and the lower one the protein binding of the same gel (non-denaturing 8% polyacrylamide). The free probes are indicated by the black arrows. The expected *cis* elements are in bold and mutated ones in lower case.

**Figure 2**
Dual roles of *OsMYC2* in regulation of the anthocyanin pathway of *Oryza sativa*. (A) Activations of the local anthocyanin genes by OsMYC2 in living protoplasts of rice. In each treatment, a reporter (pOsC1pro, pOsCHSpro, pOsCHIpro, pOsF3Hpro, pOsF3′Hpro, pOsDFRpro, or pOsANSpro, 4 µg/each) was introduced along with 4 µg mock vectors (empty effectors) or pOsMYC2 to test background activation of *LUC* (corrected by RUC as inner reference) or the effect of OsMYC2, respectively. One standard error bar includes two to seven biological replicates. Data were normalized. The significant differences between the reporter background and the OsMYC2 treatment are shown (one-tailed t-tests; *, p < 0.05; **, p < 0.01). (B) Effects of OsMYC2 on the anthocyanin genes in the presence of OsC1/OsB2/OsTTG1. For each test, 2 µg reporter was introduced along with the OsMBW (2 µg of each component) in two biological replicates or further with 2 µg pOsMYC2 in three biological replicates. The format follows (A).

**Figure 3**
Interaction and competition between MYC2 and GL3. (A) Protein interaction between MYC2 and GL3 in co-IP. The upper panel shows expressed proteins (HA-labeled) detected by mouse antibodies (anti-HA) in input solution (Input) or from HA-agarose beads (IP). The lower panel shows the detections of MYC-label proteins in the same input (Input) and IP solutions (CoIP) by mouse anti-MYC. Only interacting proteins are present in CoIP. The known interaction (underlined) is shown as positive control and the targeted interaction in bold. The results had at least two biological duplicates (n ≥ 2). (B) Interaction of MYC2 and GL3 in BiFC. The nucleus indicated by the arrow emits yellow fluorescence as a result of the physical interaction of NE-labeled MYC2 and CE-labeled GL3. Pictures were taken of the epidermis of N. *benthamiana* under visible or fluorescent lights. No signal was detected in co-transformations of pMYC2-NE and pUC-SPYCE (in place of pGL3-CE). (C) Responses of MYC2 and GL3 to mutated G-boxes in dual LUC assays. Four reporters hosting the promoter of *DFR* (*DFR*_pro) and its mutated versions (–m1, –m2, and –m1&2), as shown in partial sequences here (5′ → 3′), were driven by effectors pGL3 or pMYC2. Each treatment had three biological replicates, shown in the standard error bar. Significant changes in promoter activity are shown (two-tailed t-tests; **, p < 0.01). (D) Binding preferences between MYC2 and GL3 in EMSAs. Probes were labeled by numbers, with sequences shown (5′ → 3′). The known *cis* element is in red, and mutated sites are underlined. A non-denaturing polyacrylamide gel (10%) was used. The upper gel shows results of DNA-binding, while the lower one shows protein-binding. (E) Competition of MYC2 with GL3 in DNA binding in EMSAs. The probe is *DFR*-based. Different quantities of MYC2, shown by the lane numbers (1–3), were mixed with the same quantity (0.2 μg) of GL3 (bHLH domain) and exposed to the same quantity of probe. Controls are in lanes 5–8. A non-denaturing polyacrylamide (8%) gel was used. The black arrow indicates free probes. The white arrows indicate the strengths of binding under different quantities of MYC2. The binding tests were duplicated and results were the same.

**Figure 4**
Features of regulations of *MYB*s and *HY5* by MYC2, BBX21, and SPL9. (A) Responses of the reporter (pPAP1_pro) to TF effectors in dual LUC assays. The Y-axis shows the background activity (PAP1_pro) of the reporter with empty effectors (4 μg, as mock) co-transformed (1:1) and activities of the same reporter co-transformed with effectors (4 μg/each) as indicated. The standard error bars are based on biological replicates varying from 2 (PIF3 or HY5) to 27 (BBX21 + MYC2). Data were normalized. All effectors are significant (t-tests; **, p < 0.005), except HY5. (B) Activation of *MYBL2* by MYC2, BBX21, and SPL9. The experimental conditions and data format followed (A) and the standard error bars represent at least three biological replicates per treatment. (C) Activation of *TT2* by MYC2, SPL9, and BBX21. The format follows (B) and the standard error bars contain at least two biological replicates per trial. (D) Activation of *HY5* by MYC2, SPL9, and BBX21. As in (B), the standard error bar contains at least three biological replicates (one-tailed t-tests; *, p < 0.05; **, p < 0.01). (E) Quantifications of *HY5* transcripts. Transcript copy number estimated for *myc2* is significantly smaller than that of Col for day-3 seedlings, with three biological replicates (one-tailed t-tests; **, p = 0.004). (F) Co-IP tests on possible protein interaction between SPL9 and MYC2. As in Figure 3A, confirmed protein functions are underlined and the interaction at focus is in bold. (G) Co-IP tests on possible interaction between BBX21 and MYC2. Proteins are labeled as in (F). (H) Co-IP tests on possible interaction between BBX21 and SPL9. Presentation follows (F).

**Figure 5**
Regulations of BBX21 and SPL9 on structural genes of the flavonoid network with or without an MBW complex in A. *thaliana*. (A) Single effects of BBX21 in dual LUC assays. Each test (4 µg/each vector) was done with at least three biological replicates, shown by the standard error. Data were normalized. Significant activations (relative to empty effectors) are shown (one-tailed t-tests; *, p < 0.05; **, p < 0.01). (B) Combined regulation of BBX21 with the MBW complex. All vectors were introduced in 2 µg, with at least three biological replicates performed. Only reporter of *F3′H* shows a significantly lower activity for combined regulation than for BBX21 only (one-tailed t-test, *, p = 0.007). (C) Single effects of SPL9 in dual LUC assays. The format follows those in (A). (D) Regulation of SPL9 with the MBW complex. The tests follow those in (B), with *F3H* showing a higher activation by combined regulation than by SPL9 alone (one-tailed t-test, **, p = 0.004). (E) Phenotype of *spl9* at day 3. Pigmentation is indicated by the arrow. The control line is Columbia (Col) shown in Figure 1A. (F) Anthocyanin content of seedlings from day 2 to day 3. The protocol follows Figure 1B. (G) Effects of BBX21, SPL9, and MYC2 on promoter of *BAN*. The activation by BBX21 is significant relative to the background (empty effectors) by one-tailed t-test (**, p = 0.006). The activation of TT2/TT8/TTG1 complex is significantly lower when BBX21 is present (one-tailed t-test, *, p = 0.049), following settings of (A,B) here. Sample sizes are at least two biological replicates per treatment.

**Figure 6**
Interactive relationships of MYC2 with SPL9 and BBX21 in dual LUC assays in A. *thaliana* leaf cells. (A) Additive relationship between SPL9 and MYC2 in activation of *PAP1*. Results are shown as mean ± standard error, based on three biological replicates (n = 3). (B) Competition test between BBX21 and MYC2 at *PAP1*_pro. The effectors are indicated by the x-axis, with pBBX21 (4 µg) in every trial and varied amounts of pMYC2 shown after + sign across trials. The same reporter (pPAP1_pro) in 4 μg is provided across tests. The standard error bar includes at least three biological replicates. The comparison between treatments (a and b) is significant (one-tailed t-test, p = 0.016, n = 12). (C) Tests of effects of *cis* elements of *PAP1*_pro on regulations of BBX21 and MYC2. Four reporters are shown in the upper panel with mutations (M1 & M2) indicated in the partial sequences. Their activations were examined under effector pMYC2 or pBBX21 (4 µg/each), with standard error bars shown (n = 3). Significant reductions in promoter activity are shown (one-tailed t-tests; *, p < 0.05; **, p < 0.01). (D) Competition between BBX21 and MYC2 at *F3′H*_pro. Effectors and reporters (4 µg each) were provided as 1:1 for each test (n = 3). Significant activations (relative to empty effectors) are shown (one-tailed t-tests; **, p < 0.01). (E) Competition between BBX21 and MYC2 at *CHI*_pro. Significant activations (relative to empty effectors) are shown (one-tailed t-tests; *, p < 0.05; **, p < 0.01). Data were normalized.

**Figure 7**
Effects of HY5 and PIF3 on the anthocyanin pathway and related genes. (A) Impact of HY5 on the anthocyanin genes with MBW. Dual LUC assays show the activations of the promoter regions when exposed to two sets of effectors in at least two biological replicates. Effector and reporter were provided in the same ratio (2 μg/each). Data were normalized. Significantly reduced activity was shown for *CHS*_pro, *CHI*_pro, and *F3H*_pro (one-tailed t-tests; ***, p < 0.0001 in all cases). (B) Phenotype of *hy5* at day 3 in water. The bar is for 0.5 mm. The control line is Columbia (Col) shown in Figure 1A. The anthocyanin content was undetectable using the same protocol as in Figure 1B. (C) Quantifications of transcript copy numbers of *MYBL2* between lines of day-3 seedlings. The difference is not significant between the wild type (Col) and *hy5* (two-tailed t-test, p = 0.56). (D) Transcript levels of structural genes between Col and *hy5*. Significant differences between lines are based on one-tailed t-tests after Bonferroni’s correction for multiple comparisons (experimental error rate α = 0.05, * p < 0.05). (E) Transcript levels of regulators between Col and *hy5*. Standard errors are based on three biological replicates (n = 3). Significant differences (*) are based on one-tailed t-tests (p = 0.04 for *BBX21* and p = 0.03 for *MYC2*). (F) Responses of pSPL9_pro to pHY5 or pPIF3 in dual LUC assays. Results show activities of the reporter pSPL9_pro with 4 μg empty TF-vector (SPL9pro, n = 4), 4 μg pPIF3 (+PIF3pro, n = 6), or 4 μg pHY5 (+HY5pro, n = 6). Significantly reduced responses (*) are shown (one-tailed t-tests; p < 0.04 for both TF effectors). (G) Response of pBBX21_pro to pHY5 or pPIF3 in dual LUC assays. As in (F), results show the activity of reporter with empty effectors (n = 3), PIF3 (+PIF, n = 6), or HY5 (+HY5, n = 6). One-tailed t-test is significant for HY5 only (p = 0.04). (H) Phenotype of *pif3* at day 3. The control line is Columbia (Col) shown in Figure 1A. (I) Accumulation of anthocyanins in *pif3* seedlings over 24-h period from day 2 to day 3. Format follows that of Figure 1B. (J) Transcript levels of TF genes between line Col and line *pif3*. TF transcripts increased significantly in *pif3* (one-tailed t-tests; *, p < 0.05; **, p < 0.05). (K) Transcript levels of structural genes between Col and *pif3*. Significantly increased transcripts are shown (one-tailed t-tests; all ** p < 0.002; after Bonferroni’s correction, ** p < 0.05).

**Figure 8**
*MYC2* as a modulator for the anthocyanin pathway. (A) Transcript levels of *MYC2* between lines. The estimated numbers of transcripts are from day-3 seedlings and the standard errors from three biological replicates. The mutant lines contain fewer transcript copies than Col (one-tailed t-test, *, p < 0.03). (B) Activation of *MYC2*_pro by different regulators in dual LUC assays. Results are based on four to eight biological replicates for each treatment. The activation effect of SPL, PIF3, or BBX21 is significant (one-tailed t-test, n = 5–8; *, p < 0.03, **, p < 0.001). The difference between a (with empty effectors added) and b is also significant (one-tailed t-test, p = 0.005, n = 12). Data were normalized. (C) Transcript levels of *MYBL2* across lines of day-3 seedlings. Details of the lines follow Figure 1C. The lines of 35S-MYC2 and *myc2* have significantly fewer copies of *MYBL2* transcripts than Col (one-tailed t-tests; **, p < 0.01). (D) A summary of major relationships among the regulators and the anthocyanin pathway. The upper plate is for environmental signals, which regulate PIF3 and *HY5* (black lines show inferences from literature), which in turn regulate *SPL9*, *MYC2*, and *BBx21* (colored lines having evidence from this study). Actions of SPL9, MYC2, and BBx21 on *MYB* genes (*PAP1* and *MYBL2*) are indicated in the middle plate and their regulations of the structural genes in the bottom plate. A single activation (without MBW) is presented by an arrow in the color of the regulator in the middle plate, thickness of which roughly indicates activation strength. Double arrows indicate protein-protein interactions. White bars in the bottom plate designate the scope of genes under the regulation of the PAP1/GL3/TTG1 complex, which is also suppressed by MYC2 when coupling with the complex.

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References

1. Shi M.-Z., Xie D.-Y. Biosynthesis and metabolic engineering of anthocyanins in Arabidopsis thaliana. Recent Pat. Biotechnol. 2014;8:47–60. doi: 10.2174/1872208307666131218123538. - DOI - PMC - PubMed
1. Kitashova A., Adler S.O., Richter A.S., Eberlein S., Dziubek D., Klipp E., Nagele T. Limitation of sucrose biosynthesis shapes carbon partitioning during plant cold acclimation. Plant Cell Environ. 2023;46:464–478. doi: 10.1111/pce.14483. - DOI - PubMed
1. Motten A.F., Stone J.L. Heritability of stigma position and the effect of stigma-anther separation on outcrossing in a predominantly self-fertilizing weed, Datura stramonium (Solanaceae) Am. J. Bot. 2000;87:339–347. doi: 10.2307/2656629. - DOI - PubMed
1. Lan J.X., Li A.L., Chen C.X. Effect of transient accumulation of anthocyanin on leaf development and photoprotection of Fagopyrum dibotrys mutant. Biol. Plant. 2011;55:766–770. doi: 10.1007/s10535-011-0184-6. - DOI
1. Luo H.H., Li W.J., Zhang X., Deng S.F., Xu Q.C., Hou T., Pang X.Q., Zhang Z.Q., Zhang X.L. In planta high levels of hydrolysable tannins inhibit peroxidase mediated anthocyanin degradation and maintain abaxially red leaves of Excoecaria cochinchinensis. BMC Plant Biol. 2019;19:20. doi: 10.1186/s12870-019-1903-y. - DOI - PMC - PubMed

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[1] Shi M.-Z., Xie D.-Y. Biosynthesis and metabolic engineering of anthocyanins in Arabidopsis thaliana. Recent Pat. Biotechnol. 2014;8:47–60. doi: 10.2174/1872208307666131218123538. - DOI - PMC - PubMed

[2] Shi M.-Z., Xie D.-Y. Biosynthesis and metabolic engineering of anthocyanins in Arabidopsis thaliana. Recent Pat. Biotechnol. 2014;8:47–60. doi: 10.2174/1872208307666131218123538. - DOI - PMC - PubMed

[3] Kitashova A., Adler S.O., Richter A.S., Eberlein S., Dziubek D., Klipp E., Nagele T. Limitation of sucrose biosynthesis shapes carbon partitioning during plant cold acclimation. Plant Cell Environ. 2023;46:464–478. doi: 10.1111/pce.14483. - DOI - PubMed

[4] Kitashova A., Adler S.O., Richter A.S., Eberlein S., Dziubek D., Klipp E., Nagele T. Limitation of sucrose biosynthesis shapes carbon partitioning during plant cold acclimation. Plant Cell Environ. 2023;46:464–478. doi: 10.1111/pce.14483. - DOI - PubMed

[5] Motten A.F., Stone J.L. Heritability of stigma position and the effect of stigma-anther separation on outcrossing in a predominantly self-fertilizing weed, Datura stramonium (Solanaceae) Am. J. Bot. 2000;87:339–347. doi: 10.2307/2656629. - DOI - PubMed

[6] Motten A.F., Stone J.L. Heritability of stigma position and the effect of stigma-anther separation on outcrossing in a predominantly self-fertilizing weed, Datura stramonium (Solanaceae) Am. J. Bot. 2000;87:339–347. doi: 10.2307/2656629. - DOI - PubMed

[7] Lan J.X., Li A.L., Chen C.X. Effect of transient accumulation of anthocyanin on leaf development and photoprotection of Fagopyrum dibotrys mutant. Biol. Plant. 2011;55:766–770. doi: 10.1007/s10535-011-0184-6. - DOI

[8] Lan J.X., Li A.L., Chen C.X. Effect of transient accumulation of anthocyanin on leaf development and photoprotection of Fagopyrum dibotrys mutant. Biol. Plant. 2011;55:766–770. doi: 10.1007/s10535-011-0184-6. - DOI

[9] Luo H.H., Li W.J., Zhang X., Deng S.F., Xu Q.C., Hou T., Pang X.Q., Zhang Z.Q., Zhang X.L. In planta high levels of hydrolysable tannins inhibit peroxidase mediated anthocyanin degradation and maintain abaxially red leaves of Excoecaria cochinchinensis. BMC Plant Biol. 2019;19:20. doi: 10.1186/s12870-019-1903-y. - DOI - PMC - PubMed

[10] Luo H.H., Li W.J., Zhang X., Deng S.F., Xu Q.C., Hou T., Pang X.Q., Zhang Z.Q., Zhang X.L. In planta high levels of hydrolysable tannins inhibit peroxidase mediated anthocyanin degradation and maintain abaxially red leaves of Excoecaria cochinchinensis. BMC Plant Biol. 2019;19:20. doi: 10.1186/s12870-019-1903-y. - DOI - PMC - PubMed

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A Regulatory Mechanism on Pathways: Modulating Roles of MYC2 and BBX21 in the Flavonoid Network

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A Regulatory Mechanism on Pathways: Modulating Roles of MYC2 and BBX21 in the Flavonoid Network

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Abstract

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