SUMMARY
Drought stress induces anthocyanin biosynthesis in many plant species, but the underlying molecular mechanism remains unclear. Ethylene response factors (ERFs) play key roles in plant growth ...and various stress responses, including affecting anthocyanin biosynthesis. Here, we characterized an ERF protein, MdERF38, which is involved in drought stress‐induced anthocyanin biosynthesis. Biochemical and molecular analyses showed that MdERF38 interacted with MdMYB1, a positive modulator of anthocyanin biosynthesis, and facilitated the binding of MdMYB1 to its target genes. Therefore, MdERF38 promoted anthocyanin biosynthesis in response to drought stress. Furthermore, we found that MdBT2, a negative modulator of anthocyanin biosynthesis, decreased MdERF38‐promoted anthocyanin biosynthesis by accelerating the degradation of the MdERF38 protein. In summary, our data provide a mechanism for drought stress‐induced anthocyanin biosynthesis that involves dynamic modulation of MdERF38 at both transcriptional and post‐translational levels.
Significance Statement
MdERF38 promotes anthocyanin biosynthesis by interacting with MdMYB1 and enhancing the binding of MdMYB1 to its target genes in response to drought stress. MdBT2 decreases drought‐induced anthocyanin accumulation by accelerating the degradation of MdERF38.
Summary
Cold stress severely affects plant growth and yield. C‐repeat binding factors (CBFs) play important roles in the response to cold stress. In the present study, we identified an R2R3‐MYB ...transcription factor (TF) MdMYB23 from apple (Malus × domestic) using transcriptome analyses, which was notably induced in response to cold stress. Transgenic apple calli and Arabidopsis with overexpression of MdMYB23 exhibited increased cold tolerance. Electrophoretic mobility shift assay (EMSA) and transient expression assays indicated that MdMYB23 directly bound to the promoters of MdCBF1 and MdCBF2 and activated their expression. MdMYB23 interacted with the promoter of MdANR, a key modulator of proanthocyanidin biosynthesis, and activated its expression to promote proanthocyanidin accumulation and reactive oxygen species (ROS) scavenging. MdBT2 was identified as an MdMYB23‐interacting protein using yeast two‐hybrid (Y2H), pull‐down, and bimolecular fluorescence complementation (BiFC) assays. MdBT2 repressed cold tolerance and proanthocyanidin accumulation by promoting the degradation of MdMYB23 protein. Our findings shed light on the functions of MYB TFs and underlying mechanism in the modulation of plant cold tolerance.
Significance Statement
An apple R2R3‐MYB TF MdMYB23 increases cold tolerance and proanthocyanidin accumulation by directly activating the expression of MdCBF1/2 and MdANR. MdBT2 represses cold tolerance and proanthocyanidin accumulation by promoting the degradation of MdMYB23 protein.
Summary
The plant hormone jasmonic acid (JA) is involved in the cold stress response, and the inducer of CBF expression 1 (ICE1)‐ C‐repeat binding factor (CBF) regulatory cascade plays a key role in ...the regulation of cold stress tolerance. In this study, we showed that a novel B‐box (BBX) protein MdBBX37 positively regulates JA‐mediated cold‐stress resistance in apple.
We found that MdBBX37 bound to the MdCBF1 and MdCBF4 promoters to activate their transcription, and also interacted with MdICE1 to enhance the transcriptional activity of MdICE1 on MdCBF1, thus promoting its cold tolerance.
Two JA signaling repressors, MdJAZ1 and MdJAZ2 (JAZ, JAZMONATE ZIM‐DOMAIN), interacted with MdBBX37 to repress the transcriptional activity of MdBBX37 on MdCBF1 and MdCBF4, and also interfered with the interaction between MdBBX37 and MdICE1, thus negatively regulating JA‐mediated cold tolerance. E3 ligase MdMIEL1 (MIEL1, MYB30‐Interacting E3 Ligase1) reduced MdBBX37‐improved cold resistance by mediating ubiquitination and degradation of the MdBBX37 protein.
The data reveal that MIEL1 and JAZ proteins co‐regulate JA‐mediated cold stress tolerance through the BBX37‐ICE1‐CBF module in apple. These results will aid further examination of the post‐translational modification of BBX proteins and the regulatory mechanism of JA‐mediated cold stress tolerance.
The molecular mechanism of ABA‐promoted anthocyanin accumulation and fruit coloration is less known. Here, an apple bZIP transcription factor MdbZIP44 was identified to be a positive regulator in ...ABA‐promoted anthocyanin accumulation by interacting with MdMYB1 and enhancing its binding capacity to the promoters of downstream target genes. MdBT2 decreased ABA‐promoted anthocyanin accumulation by degrading MdbZIP44 protein.
Phytohormone abscisic acid (ABA) induces anthocyanin biosynthesis; however, the underlying molecular mechanism is less known. In this study, we found that the apple MYB transcription factor MdMYB1 activated anthocyanin biosynthesis in response to ABA. Using a yeast screening technique, we isolated MdbZIP44, an ABA‐induced bZIP transcription factor in apple, as a co‐partner with MdMYB1. MdbZIP44 promoted anthocyanin accumulation in response to ABA by enhancing the binding of MdMYB1 to the promoters of downstream target genes. Furthermore, we identified MdBT2, a BTB protein, as an MdbZIP44‐interacting protein. A series of molecular, biochemical, and genetic analysis suggested that MdBT2 degraded MdbZIP44 protein through the Ubiquitin‐26S proteasome system, thus inhibiting MdbZIP44‐modulated anthocyanin biosynthesis. Taken together, we reveal a novel working mechanism of MdbZIP44‐mediated anthocyanin biosynthesis in response to ABA.
Wounding stress leads to anthocyanin accumulation. However, the underlying molecular mechanism remains elusive. In this study, MdWRKY40 was found to promote wounding-induced anthocyanin biosynthesis ...in association with MdMYB1 and undergo MdBT2-mediated degradation in apple.
We found that MdMYB1, a positive regulator of anthocyanin biosynthesis, was essential for the wounding-induced anthocyanin biosynthesis in apple. MdWRKY40 was identified as an MdMYB1-interacting protein, and enhanced the binding of MdMYB1 to its target genes in response to wounding.
We found that MdBT2 interacted physically with MdWRKY40 and was involved in its degradation through the 26S proteasome pathway.
Our results demonstrate that MdWRKY40 is a key modulator in the wounding-induced anthocyanin biosynthesis, which provides new insights into the regulation of wounding-induced anthocyanin biosynthesis at both the transcriptional and post-translational levels in apple.
Summary
MYB transcription factors (TFs) have been demonstrated to play diverse roles in plant growth and development through interaction with basic helix‐loop‐helix (bHLH) TFs. MdbHLH33, an apple ...bHLH TF, has been identified as a positive regulator in cold tolerance and anthocyanin accumulation by activating the expressions of MdCBF2 and MdDFR. In the present study, a MYB TF MdMYB308L was found to also positively regulate cold tolerance and anthocyanin accumulation in apple. We found that MdMYB308L interacted with MdbHLH33 and enhanced its binding to the promoters of MdCBF2 and MdDFR. In addition, an apple RING E3 ubiquitin ligase MYB30‐INTERACTING E3 LIGASE 1 (MdMIEL1) was identified to be an MdMYB308L‐interacting protein and promoted the ubiquitination degradation of MdMYB308L, thus negatively regulated cold tolerance and anthocyanin accumulation in apple. These results suggest that MdMYB308L acts as a positive regulator in cold tolerance and anthocyanin accumulation in apple by interacting with MdbHLH33 and undergoes MdMIEL1‐mediated protein degradation. The dynamic change in MYB‐bHLH protein complex seems to play a key role in the regulation of plant growth and development.
SUMMARY
Jasmonate (JA) induces the biosynthesis of anthocyanin and proanthocyanidin. MdMYB9 is essential for modulating the accumulation of both anthocyanin and proanthocyanidin in apple, but the ...molecular mechanism for induction of anthocyanin and proanthocyanidin biosynthesis by JA is unclear. In this study, we discovered an apple telomere‐binding protein (MdTRB1) to be the interacting protein of MdMYB9. A series of biological assays showed that MdTRB1 acted as a positive modulator of anthocyanin and proanthocyanidin accumulation, and is dependent on MdMYB9. MdTRB1 interacted with MdMYB9 and enhanced the activation activity of MdMYB9 to its downstream genes. In addition, we found that the JA signaling repressor MdJAZ1 interacted with MdTRB1 and interfered with the interaction between MdTRB1 and MdMYB9, therefore negatively modulating MdTRB1‐promoted biosynthesis of anthocyanin and proanthocyanidin. These results show that the JAZ1–TRB1–MYB9 module dynamically modulates JA‐mediated accumulation of anthocyanin and proanthocyanidin. Taken together, our data further expand the functional study of TRB1 and provide insights for further studies of the modulation of anthocyanin and proanthocyanidin biosynthesis by JA.
Significance Statement
The apple telomere‐binding protein MdTRB1 positively modulates JA‐mediated accumulation of anthocyanin and proanthocyanidin by interacting with MdMYB9 and enhancing the activation activity of MdMYB9 to MdANS and MdANR. The JA signaling repressor MdJAZ1 interacted with MdTRB1 and interfered with the interaction between MdTRB1 and MdMYB9, therefore negatively modulating MdTRB1‐promoted biosynthesis of anthocyanin and proanthocyanidin.
The MYB proteins comprise one of the largest families of transcription factors (TFs) in plants. Although several MYB genes have been characterized to play roles in secondary metabolism, the MYB ...family has not yet been identified in apple. In this study, 229 apple MYB genes were identified through a genome-wide analysis and divided into 45 subgroups. A computational analysis was conducted using the apple genomic database to yield a complete overview of the MYB family, including the intron-exon organizations, the sequence features of the MYB DNA-binding domains, the carboxy-terminal motifs, and the chromosomal locations. Subsequently, the expression of 18 MYB genes, including 12 were chosen from stress-related subgroups, while another 6 ones from other subgroups, in response to various abiotic stresses was examined. It was found that several of these MYB genes, particularly MdoMYB121, were induced by multiple stresses. The MdoMYB121 was then further functionally characterized. Its predicted protein was found to be localized in the nucleus. A transgenic analysis indicated that the overexpression of the MdoMYB121 gene remarkably enhanced the tolerance to high salinity, drought, and cold stresses in transgenic tomato and apple plants. Our results indicate that the MYB genes are highly conserved in plant species and that MdoMYB121 can be used as a target gene in genetic engineering approaches to improve the tolerance of plants to multiple abiotic stresses.
The molecular mechanism of leaf senescence in apple (Malus domestica) is still not fully understood. We used gene expression analysis and protein–protein interactions to decipher the relationships of ...abscisic acid (ABA) and two proteins, MdbHLH93 and MdBT2, in the senescence process.
We found that MdbHLH93 promoted leaf senescence and the expression of senescence-related genes, which exhibited similar effects to ABA on leaf senescence. MdbHLH93 activated directly the transcription of MdSAG18.
We also found that an ABA-responsive protein, MdBT2, interacted directly with MdbHLH93, and induced the ubiquitination and degradation of the MdbHLH93 protein, and thus delayed leaf senescence.
Our findings provide new insights into the regulatory network of leaf senescence through the functional interactions among ABA, MdbHLH93 and MdBT2.
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