Carotenoids are photosynthetic pigments and antioxidants that contribute to different plant colors. However, the involvement of TOPLESS (TPL/TPR)-mediated histone deacetylation in the modulation of carotenoid biosynthesis through ethylene-responsive element-binding factor-associated amphiphilic repression (EAR)-containing transcription factors (TFs) in apple (Malus domestica Borkh.) is poorly understood. MdMYB44 is a transcriptional repressor that contains an EAR repression motif. In the present study, we used functional analyses and molecular assays to elucidate the molecular mechanisms through which MdMYB44-MdTPR1-mediated histone deacetylation influences carotenoid biosynthesis in apples. We identified two carotenoid biosynthetic genes, MdCCD4 and MdCYP97A3, that were confirmed to be involved in MdMYB44-mediated carotenoid biosynthesis. MdMYB44 enhanced β-branch carotenoid biosynthesis by repressing MdCCD4 expression, whereas MdMYB44 suppressed lutein level by repressing MdCYP97A3 expression. Moreover, MdMYB44 partially influences carotenoid biosynthesis by interacting with the co-repressor TPR1 through the EAR motif to inhibit MdCCD4 and MdCYP97A3 expression via histone deacetylation. Our findings indicate that the MdTPR1-MdMYB44 repressive cascade regulates carotenoid biosynthesis, providing profound insights into the molecular basis of histone deacetylation-mediated carotenoid biosynthesis in plants. These results also provide evidence that the EAR-harboring TF/TPL repressive complex plays a universal role in histone deacetylation-mediated inhibition of gene expression in various plants.

Correct flower organ formation at the right timing is one of the most important strategies for plants to achieve reproductive success. Ectopic overexpression of LATE FLOWERING (LATE) is known to induce late flowering, partly through suppressing expression of the florigen-encoding gene FLOWERING LOCUS T (FT) in Arabidopsis. LATE is one of the C2H2 zinc finger transcription factors, and it has a canonical transcriptional repression domain called the ethylene-responsive element-binding factor-associated amphiphilic repression (EAR) motif at the end of its C terminus. Therefore, LATE is considered a transcriptional repressor, but its molecular function remains unclear. Our genome-edited late mutants exhibited no distinct phenotype, even in flowering, indicating the presence of redundancy from other factors. To reveal the molecular function of LATE and factors working with it, we investigated its transcriptional activity and interactions with other proteins. Transactivation activity assay showed that LATE possesses transcriptional repression ability, which appears to be attributable to both the EAR motif and other sequences. Yeast two-hybrid assay showed the EAR motif-mediated interaction of LATE with TOPLESS, a transcriptional corepressor. Moreover, LATE could also interact with CRABS CLAW (CRC), one of the most important regulators of floral meristem determinacy, through sequences in LATE other than the EAR motif. Our findings demonstrated the possibility that LATE can form a transcriptional repression complex with CRC for floral meristem determinacy.

Transcriptional corepressors form an ancient and essential layer of gene expression control in eukaryotes. TOPLESS and TOPLESS-RELATED (TPL/TPR) proteins constitute a conserved family of Groucho (Gro)/thymidine uptake 1 (Tup1)-type transcriptional corepressors and control diverse growth, developmental, and stress signaling responses in plants. Because of their central and versatile regulatory roles, they act as a signaling hub to integrate various input signaling pathways in the transcriptional responses. Recently, increasing pieces of evidence indicate the roles of TPL/TPR family proteins in the modulation of plant immunity. This is supported by studies on effectors of distantly related pathogens that target TPL/TPR proteins in planta. In this short review, we will summarize the latest findings concerning pathogens targeting plant TPL/TPR proteins to manipulate plant signaling responses for the successful invasion of their hosts. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The brown planthopper (BPH) is the most destructive pest of rice. The MYB transcription factors are vital for rice immunity, but most are activators. Although MYB22 positively regulates rice resistance to BPH and has an EAR motif associated with active repression, it remains unclear whether it is a transcriptional repressor affecting rice-BPH interaction. Genetic analyses revealed that MYB22 regulates rice resistance to BPH via its EAR motif. Several biochemical experiments (e.g. transient transcription assay, Y2H, LCA, and BiFC) indicated that MYB22 is a transcriptional repressor that interacts with the corepressor TOPLESS via its EAR motif and recruits HDAC1 to form a tripartite complex. Flavonoid-3\'-hydroxylase (F3\'H) is a flavonoid biosynthesis pathway-related gene that negatively regulates rice resistance to BPH. Based on a bioinformatics analysis and the results of EMSA and transient transcription assays, MYB22 can bind directly to the F3\'H promoter and repress gene expression along with TOPLESS and HDAC1. We revealed a transcriptional regulatory mechanism influencing the rice-BPH interaction that differs from previously reported mechanisms. Specifically, MYB22-TOPLESS-HDAC1 is a novel transcriptional repressor complex with components that synergistically and positively regulate rice resistance to BPH through the transcriptional repression of F3\'H.

The ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif, defined by the consensus sequence patterns LxLxL or DLNx(x)P, is found in a diverse range of plant species. It is the most predominant form of active transcriptional repression motif identified so far in plants. Despite its small size (5 to 6 amino acids), the EAR motif is primarily involved in the negative regulation of developmental, physiological and metabolic functions in response to abiotic and biotic stresses. Through an extensive literature review, we identified 119 genes belonging to 23 different plant species that contain an EAR motif and function as negative regulators of gene expression in various biological processes, including plant growth and morphology, metabolism and homeostasis, abiotic stress response, biotic stress response, hormonal pathways and signalling, fertility, and ripening. Positive gene regulation and transcriptional activation are studied extensively, but there remains much more to be discovered about negative gene regulation and the role it plays in plant development, health, and reproduction. This review aims to fill the knowledge gap and provide insights into the role that the EAR motif plays in negative gene regulation, and provoke further research on other protein motifs specific to repressors.

Red-skinned pears (Pyrus L.) are preferred to consumers for their attractive color and abundant anthocyanins. Pyrus ETHYLENE RESPONSE FACTOR 3 (PyERF3) positively regulates anthocyanin biosynthesis through interacting with Pyrus myeloblastosis family 114 (PyMYB114) and Pyrus basic helix-loop-helix 3 (PybHLH3) in red-skinned pears. However, the role of APETALA2/ethylene response factors (AP2/ERFs), which negatively regulate anthocyanin biosynthesis, remains unclear in red-skinned pears. Here, we validated that 2 AP2/ERFs, PyERF4.1 and PyERF4.2, screened from the transcriptome data of \'Starkrimson\' pear (Pyrus communis L.) and its green mutant, inhibit anthocyanin biosynthesis in transgenic pear calli, as well as in overexpression and gene-edited tomato (Solanum lycopersicum) fruits. Meanwhile, the co-transformation of PyERF4.1/PyERF4.2 with PyERF3-PyMYB114-PybHLH3 inhibited anthocyanin biosynthesis in pear fruits and strawberry (Fragaria vesca) receptacles. Further assays showed that PyMYB114 activated the transcription of PyERF4.1/PyERF4.2; PyERF4.1/PyERF4.2 then interacted with PyERF3 to affect the stability of the PyERF3-PyMYB114-PybHLH3 complex, thereby inhibiting the transcription of the anthocyanin biosynthesis gene Pyrus anthocyanidin synthase (PyANS). Furthermore, deletion of the ERF-associated-amphiphilic repression (EAR) motif eliminated the inhibitory effect of PyERF4.1/PyERF4.2 on anthocyanin biosynthesis, and a mutation of the PyERF4.2-EAR motif (LxLxM to LxLxL) strengthened the inhibitory effect, demonstrating that the EAR motif is indispensable for the inhibitory effect of PyERF4.1/PyERF4.2 on anthocyanin biosynthesis in pears. Our study has shed light on a feedback regulatory loop mechanism that balances the excessive accumulation of anthocyanins in red-skinned pears, providing insights into the regulatory mechanism of anthocyanin biosynthesis and the regulatory network of coloration in red-skinned pears.

APETALA2/ethylene responsive factors (AP2/ERF) are unique regulators in the plant kingdom and are involved in the whole life activity processes such as development, ripening, and biotic and abiotic stresses. In tomato (Solanum lycopersicum), there are 140 AP2/ERF genes; however, their functionality remains poorly understood. In this work, the 14th and 19th amino acid differences in the AP2 domain were used to distinguish DREB and ERF subfamily members. Even when the AP2 domain of 68 ERF proteins from 20 plant species and motifs in tomato DREB and ERF proteins were compared, the binding ability of DREB and ERF proteins with DRE/CRT and/or GCC boxes remained unknown. During fruit development and ripening, the expressions of 13 DREB and 19 ERF subfamily genes showed some regular changes, and the promoters of most genes had ARF, DRE/CRT, and/or GCC boxes. This suggests that these genes directly or indirectly respond to IAA and/or ethylene (ET) signals during fruit development and ripening. Moreover, some of these may feedback regulate IAA or ET biosynthesis. In addition, 16 EAR motif-containing ERF genes in tomato were expressed in many organs and their total transcripts per million (TPM) values exceeded those of other ERF genes in most organs. To determine whether the EAR motif in EAR motif-containing ERF proteins has repression function, their EAR motifs were retained or deleted in a yeast one-hybrid (YIH) assay. The results indicate that most of EAR motif-containing ERF proteins lost repression activity after deleting the EAR motif. Moreover, some of these were expressed during ripening. Thus, these EAR motif-containing ERF proteins play vital roles in balancing the regulatory functions of other ERF proteins by completing the DRE/CRT and/or GCC box sites of target genes to ensure normal growth and development in tomato.

Programmed cell death plays a crucial role in plant development and disease defense. Here, we report that the expression of StERF3, a potato EAR motif-containing transcription factor, promotes Phytophthora infestans colonization in Nicotiana benthamiana. Transient overexpression of StERF3 induces cell death in N. benthamiana leaves. The substitution of two key amino acids (14th and 19th) in its ERF domain (the DNA binding domain) dramatically altered its cell death-inducing ability. In addition, StERF3△EAR EAR motif-deletion or StERF3AAA mutation abolished the cell death-inducing ability. StERF3 interacted with the co-repressors Topless-related protein 1 (StTPL1) and Topless-related protein 3 (StTPL3) via the EAR motif. Moreover, cell death induced by StERF3 was facilitated by co-expression with StTPL1 or StTPL3. Virus-induced gene silencing (VIGS) of NbTPL1 and NbTPL3 in N. benthamiana compromised the cell death-inducing ability of StERF3. Furthermore, StERF3-induced cell death accompanied with ROS bursts and the upregulation of the respiratory burst oxidase homolog (Rboh) genes NbRbohA and NbRbohC. In addition, several cell death regulator genes, including NbCRTD, NbNCBP, and NbBCPL, and a hypersensitive cell death marker gene Hin1 were upregulated. StERF3 may positively regulate cell death through its EAR motif-mediated transcriptional repressor activity by inhibiting the expression of genes potentially coding the repressor of cell death (CD).

Purple-fleshed sweetpotato (Ipomoea batatas（L.）Lam.) is rich in anthocyanins. R2R3-type MYB transcription factors（TFs）with EAR motifs inhibiting anthocyanin biosynthesis have been reported, and there is still a lack of information on how mutations in the EAR motifs of MYBs affect anthocyanin accumulation. In this study, we obtained three IbMYB44 TFs by bioinformatics. Among these TFs, IbMYB44.1, IbMYB44.3 with a complete EAR motif and IbMYB44.2 with a single amino acid mutant in the EAR motif caused an amino acid substitution from leucine to valine. RT-qPCR analysis showed that IbMYB44s was expressed at lower levels in the purple-fleshed sweetpotato than in nonpurple-fleshed sweetpotato (P < 0.01). Transient expression assays showed that the inhibitory effect of IbMYB44.1/3 was stronger than IbMYB44.2 in tobacco leaves and red-skinned pears. RT-qPCR analysis further proved that IbMYB44.1/3 significantly inhibited the expression of anthocyanin biosynthesis-related genes compared with IbMYB44.2 in tobacco leaves and red-skinned pears. A dual luciferase reporter assay showed that IbMYB44s cannot directly activate the IbANS promoter, and the result was also verified by yeast one-hybrid (Y1H) experiments. Moreover, we identified the interaction of IbMYB340 with IbMYB44.1, IbMYB44.2 and IbMYB44.3 via yeast two-hybrid (Y2H) assays. Thus, IbMYB44.1/3 could interact with IbMYB340 to negatively regulate anthocyanin biosynthesis. This study enriched the regulatory network of anthocyanins and also provided a theoretical basis for a single amino acid mutant from leucine to valine in the EAR motif of IbMYB44.2 affecting anthocyanin biosynthesis in the purple-fleshed sweetpotato.

Both anthocyanins and lignins are essential secondary metabolites in plant growth and development. Their biosynthesis is metabolically interconnected and diverges in the central metabolite 4-coumaroyl CoA of the phenylpropanoid pathway. Considerable progress has been made in understanding transcriptional regulation of genes involved in lignin and anthocyanin synthesis pathways, but the concerted regulation of these pathways is not yet fully understood. Here, we functionally characterized PtrMYB120, a R2R3-MYB transcription factor from Populus trichocarpa. Overexpression of PtrMYB120 in a hybrid poplar (i.e., 35S::PtrMYB120) was associated with increased anthocyanin (i.e., cyanidin 3-O-glucoside) accumulation and upregulation of anthocyanin biosynthetic genes. However, transgenic poplars with dominant suppression of PtrMYB120 function achieved by fusing the ERF-associated amphiphilic repression motif to PtrMYB120 (i.e., 35S::PtrMYB120-SRDX) had a dramatic decrease in not only anthocyanin but also Klason lignin content with downregulation of both anthocyanin and lignin biosynthetic genes. Indeed, 35S::PtrMYB120-SRDX poplars had irregularly shaped xylem vessels with reduced S-lignin content in stems, which was proportionally related to the level of the introduced PtrMYB120-SRDX gene. Furthermore, protoplast-based transcriptional activation assay using the PtrMYB120-GR system suggested that PtrMYB120 directly regulates genes involved in both anthocyanin and lignin biosynthesis, including chalcone synthase and ferulate-5 hydroxylase. Interestingly, the saccharification efficiency of line #6 of 35S::PtrMYB120-SRDX poplars, which had slightly reduced lignin content with a normal growth phenotype, was dramatically enhanced (>45%) by NaOH treatment. Taken together, our results suggest that PtrMYB120 functions as a positive regulator of both anthocyanin and lignin biosynthetic pathways and can be targeted to enhance saccharification efficiency in woody perennials.