Genome-wide analysis of R2R3-MYB transcription factors in poplar and functional validation of PagMYB147 in defense against Melampsora magnusiana.

MAIN CONCLUSION: Transcription of PagMYB147 was induced in poplar infected by Melampsora magnusiana, and a decline in its expression levels increases the host's susceptibility, whereas its overexpression promotes resistance to rust disease. Poplars are valuable tree species with diverse industrial and silvicultural applications. The R2R3-MYB subfamily of transcription factors plays a crucial role in response to biotic stresses. However, the functional studies on poplar R2R3-MYB genes in resistance to leaf rust disease are still insufficient. We identified 191 putative R2R3-MYB genes in the Populus trichocarpa genome. A phylogenetic analysis grouped poplar R2R3-MYBs and Arabidopsis R2R3-MYBs into 33 subgroups. We detected 12 tandem duplication events and 148 segmental duplication events, with the latter likely being the main contributor to the expansion of poplar R2R3-MYB genes. The promoter regions of these genes contained numerous cis-acting regulatory elements associated with response to stress and phytohormones. Analyses of RNA-Seq data identified a multiple R2R3-MYB genes response to Melampsora magnusiana (Mmag). Among them, PagMYB147 was significantly up-regulated under Mmag inoculation, salicylic acid (SA) and methyl jasmonate (MeJA) treatment, and its encoded product was primarily localized to the cell nucleus. Silencing of PagMYB147 exacerbated the severity of Mmag infection, likely because of decreased reactive oxygen species (ROS) production and phenylalanine ammonia-lyase (PAL) enzyme activity, and up-regulation of genes related to ROS scavenging and down-regulation of genes related to PAL, SA and JA signaling pathway. In contrast, plants overexpressing PagMYB147 showed the opposite ROS accumulation, PAL enzyme activity, SA and JA-related gene expressions, and improved Mmag resistance. Our findings suggest that PagMYB147 acts as a positive regulatory factor, affecting resistance in poplar to Mmag by its involvement in the regulation of ROS homeostasis, SA and JA signaling pathway.

Identification of R2R3-MYB family in blueberry and its potential involvement of anthocyanin biosynthesis in fruits.

BACKGROUND: Blueberries (Vaccinium corymbosum) are regarded as "superfoods" attributed to large amounts of anthocyanins, a group of flavonoid metabolites, which provide pigmentation in plant and beneficial effects for human health. MYB transcription factor is one of vital components in the regulation of plant secondary metabolism, which occupies a dominant position in the regulatory network of anthocyanin biosynthesis. However, the role of MYB family in blueberry responding to anthocyanin biosynthesis remains elusive. RESULTS: In this study, we conducted a comprehensive analysis of VcMYBs in blueberry based on the genome data, including phylogenetic relationship, conserved motifs, identification of differentially expressed MYB genes during fruit development and their expression profiling, etc. A total of 437 unique MYB sequences with two SANT domains were identified in blueberry, which were divided into 3 phylogenetic trees. Noticeably, there are many trigenic and tetragenic VcMYBs pairs with more than 95% identity to each other. Meanwhile, the transcript accumulations of VcMYBs were surveyed underlying blueberry fruit development, and they showed diverse expression patterns, suggesting various functional roles in fruit ripening. More importantly, distinct transcript profiles between skin and pulp of ripe fruit were observed for several VcMYBs, such as VcMYB437, implying the potential roles in anthocyanin biosynthesis. CONCLUSIONS: Totally, 437 VcMYBs were identified and characterized. Subsequently, their transcriptional patterns were explored during fruit development and fruit tissues (skin and pulp) closely related to anthocyanin biosynthesis. These genome-wide data and findings will contribute to demonstrating the functional roles of VcMYBs and their regulatory mechanisms for anthocyanins production and accumulation in blueberry in the future study.

Analysis of flavonol regulator evolution in the Brassicaceae reveals MYB12, MYB111 and MYB21 duplications and MYB11 and MYB24 gene loss.

BACKGROUND: Flavonols are the largest subgroup of flavonoids, possessing multiple functions in plants including protection against ultraviolet radiation, antimicrobial activities, and flower pigmentation together with anthocyanins. They are of agronomical and economical importance because the major off-taste component in rapeseed protein isolates is a flavonol derivative, which limits rapeseed protein use for human consumption. Flavonol production in Arabidopsis thaliana is mainly regulated by the subgroup 7 (SG7) R2R3-MYB transcription factors MYB11, MYB12, and MYB111. Recently, the SG19 MYBs MYB21, MYB24, and MYB57 were shown to regulate flavonol accumulation in pollen and stamens. The members of each subgroup are closely related, showing gene redundancy and tissue-specific expression in A. thaliana. However, the evolution of these flavonol regulators inside the Brassicaceae, especially inside the Brassiceae, which include the rapeseed crop species, is not fully understood. RESULTS: We studied the SG7 and SG19 MYBs in 44 species, including 31 species of the Brassicaceae, by phylogenetic analyses followed by synteny and gene expression analyses. Thereby we identified a deep MYB12 and MYB111 duplication inside the Brassicaceae, which likely occurred before the divergence of Brassiceae and Thelypodieae. These duplications of SG7 members were followed by the loss of MYB11 after the divergence of Eruca vesicaria from the remaining Brassiceae species. Similarly, MYB21 experienced duplication before the emergence of the Brassiceae tribe, where the gene loss of MYB24 is also proposed to have happened. The members of each subgroup revealed frequent overlapping spatio-temporal expression patterns in the Brassiceae member B. napus, which are assumed to compensate for the loss of MYB11 and MYB24 in the analysed tissues. CONCLUSIONS: We identified a duplication of MYB12, MYB111, and MYB21 inside the Brassicaceae and MYB11 and MYB24 gene loss inside the tribe Brassiceae. We propose that polyploidization events have shaped the evolution of the flavonol regulators in the Brassicaceae, especially in the Brassiceae.

Automatic identification and annotation of MYB gene family members in plants.

BACKGROUND: MYBs are among the largest transcription factor families in plants. Consequently, members of this family are involved in a plethora of processes including development and specialized metabolism. The MYB families of many plant species were investigated in the last two decades since the first investigation looked at Arabidopsis thaliana. This body of knowledge and characterized sequences provide the basis for the identification, classification, and functional annotation of candidate sequences in new genome and transcriptome assemblies. RESULTS: A pipeline for the automatic identification and functional annotation of MYBs in a given sequence data set was implemented in Python. MYB candidates are identified, screened for the presence of a MYB domain and other motifs, and finally placed in a phylogenetic context with well characterized sequences. In addition to technical benchmarking based on existing annotation, the transcriptome assembly of Croton tiglium and the annotated genome sequence of Castanea crenata were screened for MYBs. Results of both analyses are presented in this study to illustrate the potential of this application. The analysis of one species takes only a few minutes depending on the number of predicted sequences and the size of the MYB gene family. This pipeline, the required bait sequences, and reference sequences for a classification are freely available on github: https://github.com/bpucker/MYB_annotator . CONCLUSIONS: This automatic annotation of the MYB gene family in novel assemblies makes genome-wide investigations consistent and paves the way for comparative studies in the future. Candidate genes for in-depth analyses are presented based on their orthology to previously characterized sequences which allows the functional annotation of the newly identified MYBs with high confidence. The identification of orthologs can also be harnessed to detect duplication and deletion events.

TCP3 interacts with R2R3-MYB proteins, promotes flavonoid biosynthesis and negatively regulates the auxin response in Arabidopsis thaliana.

TCP proteins belong to the plant-specific bHLH transcription factor family, and function as key regulators of diverse developmental processes. Functional redundancy amongst family members and post-transcriptional down-regulation by miRJAW of several TCP genes complicate their functional characterization. Here, we explore the role of TCP3 by analyzing transgenic plants expressing miRJAW-resistant mTCP3 and dominant-negative TCP3SRDX. Seedlings and seeds of mTCP3 plants were found to hyper-accumulate flavonols, anthocyanins and proanthocyanidins, whereas levels of proanthocyanidins were slightly reduced in TCP3SRDX plants. R2R3-MYB proteins control not only early flavonoid biosynthetic steps but also activate late flavonoid biosynthetic genes by forming ternary R2R3-MYB/bHLH/WD40 (MBW) complexes. TCP3 interacted in yeast with R2R3-MYB proteins, which was further confirmed in planta using BiFC experiments. Yeast three-hybrid assays revealed that TCP3 significantly strengthened the transcriptional activation capacity of R2R3-MYBs bound by the bHLH protein TT8. Transcriptome analysis of mTCP3 and TCP3SRDX plants supported a role for TCP3 in enhancing flavonoid biosynthesis. Moreover, several auxin-related developmental abnormalities were observed in mTCP3 plants. Transcriptome data coupled with studies of an auxin response reporter and auxin efflux carriers showed that TCP3 negatively modulates the auxin response, probably by compromising auxin transport capacity. Genetic experiments revealed that the chalcone synthase mutant tt4-11 lacking flavonoid biosynthesis abrogated the auxin-related defects caused by mTCP3. Together, these data suggest that TCP3 interactions with R2R3-MYBs lead to enhanced flavonoid production, which further negatively modulates the auxin response.

Identification and functional analysis of three new anthocyanin R2R3-MYB genes in Petunia.

We identified three novel members of the R2R3-MYB clade of anthocyanin regulators in the genome of the purple flowering Petunia inflata S6 wild accession, and we called them ANTHOCYANIN SYNTHESIS REGULATOR (ASR). Two of these genes, ASR1 and ASR2, are inactivated by two different single base mutations in their coding sequence. All three of these genes are absent in the white flowering species P. axillaris N and P. parodii, in the red flowering P. exserta, and in several Petunia hybrida lines, including R27 and W115. P. violacea and other P. hybrida lines (M1, V30, and W59) instead harbor functional copies of the ASR genes. Comparative, functional and phylogenic analysis of anthocyanin R2R3-MYB genes strongly suggest that the ASR genes cluster is a duplication of the genomic fragment containing the other three R2R3-MYB genes with roles in pigmentation that were previously defined, the ANTHOCYANIN4-DEEP PURPLE-PURPLE HAZE (AN4-DPL-PHZ) cluster. An investigation of the genomic fragments containing anthocyanin MYBs in different Petunia accessions reveals that massive rearrangements have taken place, resulting in large differences in the regions surrounding these genes, even in closely related species. Yeast two-hybrid assays showed that the ASR proteins can participate in the WMBW (WRKY, MYB, B-HLH, and WDR) anthocyanin regulatory complex by interacting with the transcription factors AN1 and AN11. All three ASRs can induce anthocyanin synthesis when ectopically expressed in P. hybrida lines, but ASR1 appeared to be the most effective. The expression patterns of ASR1 and ASR2 cover several different petunia tissues with higher expression at early stages of bud development. In contrast, ASR3 is only weakly expressed in the stigma, ovary, and anther filaments. The characterization of these novel ASR MYB genes completes the picture of the MYB members of the petunia anthocyanin regulatory MBW complex and suggests possible mechanisms of the diversification of pigmentation patterns during plant evolution.

Review: ABA, flavonols, and the evolvability of land plants.

There is evidence that the ABA signaling pathway has greatly contributed to increase the complexity of land plants, thereby sustaining their ability to adapt in an ever-changing environment. The regulatory functions of the ABA signaling pathway go well beyond the movements of stomata and the dormancy of seeds. For instance, the ABA signaling regulates the flavonoid biosynthesis, consistent with the high integration of ABA and light signaling pathways, which occurs at the level of key signaling components, such as the bZIP transcription factors HY5 and ABI5. Here we focus on the regulation of 'colorless' (UV-absorbing) flavonol biosynthesis by the ABA signaling and, about how flavonols may regulate, in turn, the ABA signaling network. We discuss very recent findings that quercetin regulates the ABA signaling pathway, and hypothesize this might occur at the level of second messenger and perhaps of primary signaling components as well. We critically review old and recent suggestions of the primary roles played by flavonols, the ancient class of flavonoids already present in bryophytes, in the evolution of terrestrial plants. Our reasoning strongly supports the view that the ABA-flavonol relationship may represent a robust trait of land plants, and might have contributed to their adaptation on land.