Evolutionary Analysis of MYBs-bHLH-WD40 Protein Complexes Formation and Their Functional Relationship in Planta

It is well established that a network of three classes of proteins consisting of R2R3MYB, bHLH factors and WD40 repeat protein acted in concert as a ternary complex (i.g. MBW protein complex) to activate the flavonoid-based pigment biosynthetic pathway in most high plants. Several additional functions evolved in rosids: e.g. trichome patterning, root hair patterning and seed coat mucilage production in Arabidopsis (A. thaliana) or Arabis (A. alpina) and seed hair formation in cotton (G. hirsutum). New roles of MBW complexes controlling epidermal cell fate in rosids may have diverged since the evolutionary separation of rosid and asterid, although the details of this are still not clear. Previous studies in our lab revealed novel stereochemistry of MBW components, i.g. alternative dimers TTG1-GL3 and GL1-GL3, which revised the conventional TTG1-GL3-GL1 ternary model. However, it raises one major question: what are the evolutionary implications of such alternative dimers formation among MBW components in plants? In this study, we characterized the stereochemistry of MBW proteins in different plant species by triple LUMIER assay. Using the inter-relation of MBW components as the criterion, we achieved a highly accordant phylogenetic tree suggesting the evolutionary relevance of this novel stereochemistry of MBW components. Potential critical sites in bHLH proteins accounting for diversed MBW stereochemistry were predicted. In Arabidopsis, MBW genes which control trichome and root hair patterning traits are assumed to evolve from the duplication and diversification of flavonoid controlling genes, therefore trichome and root hair traits are considered as evolutionary current inventions. However, the exact evolving order of these traits still remains to be confirmed. To better define functional divergence of the MBW proteins in the five TTG1 related traits, we performed cross-species complementary assays with MBW homologs in Arabidopsis mutants. Among MBW protein complexes in Arabidopsis, AtTTG1-AtGL3-AtGL1 is considered to be the predominant regulatory complex in leaf trichome formation. This regulatory unit is not only represented by a single trimeric complex (synergetic inter-relation) but also by two alternative dimers (antagonistic inter-relation) that in turn regulate different downstream genes. Probably even more important is the finding that different promoters become activated depending on the relative concentration of these three proteins, as this should translate into different ratios of alternative dimers and trimers. In this study, we attempted to simulate regulatory models in the context of differential proportion of alternative dimers through quantitatively determining AtTTG1 and AtGL1 competing for the binding to AtGL3. These experimental studies were complemented by mathematical modeling by Anna Deneer, Waageningen

Local Grasses for the Control of the Invasive Vine <i>Mikania micrantha</i>

Abstract Aims Mikania micrantha (Asteraceae) is an invasive vine found in tropical and southern subtropical Asian and the Pacific Islands. The current methods used to control this vine are inadequate, which warrants the development of ecologically sustainable methods. Therefore, we investigated the ability of four grass species to prevent the invasion of M. micrantha, with an ultimate goal of developing ecologically sustainable control methods for widespread application. Methods The clumps of native grass species from China (Panicum incomtum, Pennisetum purpureum, Saccharum arundinaceum and Microstegium vagans) were established. We sowed M. micrantha seeds and transplanted the seedlings into the grass clumps to examine whether the clumps could eliminate the new M. micrantha plants. In addition, we transplanted M. micrantha into existing grass clumps to examine whether the grass clumps could prevent the re-invasion of M. micrantha. Furthermore, we grew M. micrantha with P. incomtum and P. purpureum in the field to examine whether the grasses could outcompete M. micrantha. Important Findings M. micrantha seeds had difficulty germinating in the grass clumps, and all seedlings died within 3 months. It was difficult for the vine to survive in the existing grass clumps. Our field experiment showed that the coverage of M. micrantha was significantly lower than that of the grass species in the first year, and the vine was outcompeted after 2 years. To our knowledge, this study is the first to reveal that tall grasses, particularly P. incomtum and P. purpureum, have potential to serve as bio-control agents for M. micrantha. </jats:sec

Evolutionary Analysis of MBW Function by Phenotypic Rescue in Arabidopsis thaliana

The MBW complex consisting of the three proteins R2R3MYB, bHLH and WDR regulates five traits in Arabidopsis thaliana including trichome and root hair patterning, seed coat color, anthocyanidin production and seed coat mucilage release. The WDR gene TTG1 regulates each trait in specific combinations with different bHLH and R2R3MYB proteins. In this study we analyze to what extent the biochemical properties of the MBW proteins contribute to trait specificity by expressing them in appropriate A. thaliana mutants. We show that the rescue behavior of A. thaliana bHLH and R2R3MYB protein is sufficient to explain the function as derived previously from mutant analysis. When extending this rescue approach using MBW proteins from other species we find that proteins involved in anthocyanidin regulation typically show a rescue of the anthocyanidin phenotype but not of the other traits. Finally, we correlate the rescue abilities of MBW protein from different species with the A. thaliana proteins

TRANSPARENT TESTA GLABRA 1-Dependent Regulation of Flavonoid Biosynthesis

The flavonoid composition of various tissues throughout plant development is of biological relevance and particular interest for breeding. Arabidopsis thaliana TRANSPARENT TESTA GLABRA 1 (AtTTG1) is an essential regulator of late structural genes in flavonoid biosynthesis. Here, we provide a review of the regulation of the pathway’s core enzymes through AtTTG1-containing R2R3-MYELOBLASTOSIS-basic HELIX-LOOP-HELIX-WD40 repeat (MBW(AtTTG1)) complexes embedded in an evolutionary context. We present a comprehensive collection of A. thaliana ttg1 mutants and AtTTG1 orthologs. A plethora of MBW(AtTTG1) mechanisms in regulating the five major TTG1-dependent traits is highlighted

Quantitative analysis of MBW complex formation in the context of trichome patterning

Trichome patterning in Arabidopsis is regulated by R2R3MYB, bHLH and WDR (MBW) genes. These are considered to form a trimeric MBW protein complex that promotes trichome formation. The MBW proteins are engaged in a regulatory network to select trichome cells among epidermal cells through R3MYB proteins that can move between cells and repress the MBW complex by competitive binding with the R2R3MYB to the bHLHL protein. We use quantitative pull-down assays to determine the relative dissociation constants for the protein-protein interactions of the involved genes. We find similar binding strength between the trichome promoting genes and weaker binding of the R3MYB inhibitors. We used the dissociation constants to calculate the relative percentage of all possible complex combinations and found surprisingly low fractions of those complexes that are typically considered to be relevant for the regulation events. Finally, we predict an increased robustness in patterning as a consequence of higher ordered complexes mediated by GL3 dimerization

Evolutionary comparison of competitive protein-complex formation of MYB, bHLH, and WDR proteins in plants

A protein complex consisting of a MYB, basic Helix-Loop-Helix, and a WDR protein, the MBW complex, regulates five traits, namely the production of anthocyanidin, proanthocyanidin, and seed-coat mucilage, and the development of trichomes and root hairs. For complexes involved in trichome and root hair development it has been shown that the interaction of two MBW proteins can be counteracted by the respective third protein (called competitive complex formation). We examined competitive complex formation for selected MBW proteins from Arabidopsis thaliana, Arabis alpina, Gossypium hirsutum, Petunia hybrida, and Zea mays. Quantitative analyses of the competitive binding of MYBs and WDRs to bHLHs were done by pull-down assays using ProtA- and luciferase-tagged proteins expressed in human HEC cells. We found that some bHLHs show competitive complex formation whilst others do not. Competitive complex formation strongly correlated with a phylogenetic tree constructed with the bHLH proteins under investigation, suggesting a functional relevance. We demonstrate that this different behavior can be explained by changes in one amino acid and that this position is functionally relevant in trichome development but not in anthocyanidin regulation

The complete chloroplast genome sequence of Artabotrys pilosus (Annonaceae)

Artabotrys pilosus (Annonaceae) is endemic to China, this plant has high medicinal value and broad application prospect. In this study, we assembled and systematically analyzed the chloroplast genome of A. pilosus on the basis of DNA sequencing using high-throughput techniques. The chloroplast sequence of A. pilosus was 178,195 bp in length, including two inverted repeat regions of 42,150 bp, a large single-copy region of 90,797 bp and a small single-copy region of 3098 bp. It was predicted to contain 142 genes, of which 96 are coding, 38 are tRNA genes, and eight are rRNA genes. The overall GC content was 38.8%; this was higher in the IRs (40.4%) when compared to the LSC (37.6%) and the SSC (32%) regions. Phylogenetic analysis showed that A. pilosus is in subfamily Annonoideae

The Second Intron Is Essential for the Transcriptional Control of the Arabidopsis thaliana GLABRA3 Gene in Leaves

The GLABRA3 gene is a major regulator of trichome patterning in Arabidopsis thaliana. The regulatory regions important for the trichome-specific expression of GL3 have not been characterized yet. In this study, we used a combination of marker and rescue constructs to determine the relevant promoter regions. We demonstrate that a 1 kb 5' region combined with the second intron is sufficient to rescue the trichome mutant phenotype of gl3 egl3 mutants. Swap experiments of the second intron suggest that it is not sufficient to generally enhance the expression level of GL3. This implies that the second intron contains regulatory regions for the temporal and spatial regulation of GL3. The corresponding GUS-marker constructs revealed trichome-specific expression in young trichomes

DataSheet_1_Quantitative analysis of MBW complex formation in the context of trichome patterning.pdf

Trichome patterning in Arabidopsis is regulated by R2R3MYB, bHLH and WDR (MBW) genes. These are considered to form a trimeric MBW protein complex that promotes trichome formation. The MBW proteins are engaged in a regulatory network to select trichome cells among epidermal cells through R3MYB proteins that can move between cells and repress the MBW complex by competitive binding with the R2R3MYB to the bHLHL protein. We use quantitative pull-down assays to determine the relative dissociation constants for the protein-protein interactions of the involved genes. We find similar binding strength between the trichome promoting genes and weaker binding of the R3MYB inhibitors. We used the dissociation constants to calculate the relative percentage of all possible complex combinations and found surprisingly low fractions of those complexes that are typically considered to be relevant for the regulation events. Finally, we predict an increased robustness in patterning as a consequence of higher ordered complexes mediated by GL3 dimerization.</p

Metabolomic Response to Drought Stress in <i>Belosynapsis ciliata</i> (Blume) ‘Qiuhong’

The drought stress responses of plants are complex regulatory mechanisms that include various physiological responses reflected by the global metabolic status. Metabolomics is an effective, analytical, and instrumental technique for informatics/statistics for the acquisition of comprehensive information on metabolites. We investigate the effect of drought stress on a Belosynapsis ciliata cultivar, ‘Qiuhong’ (a drought-tolerant cultivar), using liquid chromatography-mass spectrometry based on a widely targeted metabolomic approach. ‘Qiuhong’ leaves are subjected to 15- and 30-day drought treatments and are then compared to a control group without drought stress and a rehydration group. In total, 290 differentially accumulated metabolites were detected between drought and normal conditions through multivariate statistical analyses, of which 65 metabolites (36 upregulated and 29 downregulated) were highlighted for their significant contribution to drought tolerance, including an anthocyanin (peonidin 3-O-galactoside) that caused the purple-red hue in leaves under drought stress. In addition, we found that two significantly altered pathways (citrate cycle and purine metabolism) were related to enhanced drought tolerance in plants. Notably, the synthesis of three compounds (p-coumaroyl putrescine, apigenin 6-C-glucoside, and β-nicotinamide mononucleotide) was specifically induced in the drought-treated ‘Qiuhong’, indicating their critical roles in drought resistance. Our results provide a foundation for further research on drought-resistant mechanisms in B. ciliata