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.

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.

Effect of Photoperiod Duration on Flower Bud Differentiation and Related Gene Expression in Bougainvillea glabra 'Sao Paulo'.

BACKGROUND: Environmental conditions, such as photoperiod, affect the developmental response of plants; thus, plants have evolved molecular mechanisms to adapt to changes in photoperiod. In Bougainvillea spp., the mechanism of flower formation underlying flowering control techniques remains poorly understood, and the physiological changes that occur during flower bud formation and the expression of related genes are not yet fully understood. METHODS: In this study, we induced flowering of potted Bougainvillea glabra 'Sao Paulo' plants under light-control treatments and analyzed their effects on flowering time, number of flower buds, flowering quality, as well as quality of flower formation, which was analyzed using transcriptome sequencing. RESULTS: Light-control treatment effectively induced the rapid formation of flower buds and early flowering in B. glabra 'Sao Paulo', with the time of flower bud formation being 119 days earlier and the flowering period extended six days longer than those of the control plants. The light-control treatment caused the bracts to become smaller and lighter in color, while the number of flowers increased, and the neatness of flowering improved. Transcriptome sequencing of the apical buds identified 1235 differentially expressed genes (DEGs) related to the pathways of environmental adaptation, biosynthesis of other secondary metabolites, glycan biosynthesis and metabolism, and energy metabolism. DEGs related to gibberellin metabolism were analyzed, wherein five DEGs were identified between the control and treatment groups. Transcriptomic analysis revealed that the gibberellin regulatory pathway is linked to flowering. Specifically, GA and GID1 levels increased during this process, enhancing DELLA protein degradation. However, decreasing this protein's binding to CO did not halt FT upregulation, thereby advancing the flowering of B. glabra 'Sao Paulo'. CONCLUSIONS: The findings of our study have implications for future research on photoperiod and its role in controlling flowering timing of Bougainvillea spp.

Dissecting functions of KATANIN and WRINKLED1 in cotton fiber development by virus-induced gene silencing.

Most of the world's natural fiber comes from cotton (Gossypium spp.), which is an important crop worldwide. Characterizing genes that regulate cotton yield and fiber quality is expected to benefit the sustainable production of natural fiber. Although a huge number of expressed sequence tag sequences are now available in the public database, large-scale gene function analysis has been hampered by the low-efficiency process of generating transgenic cotton plants. Tobacco rattle virus (TRV) has recently been reported to trigger virus-induced gene silencing (VIGS) in cotton leaves. Here, we extended the utility of this method by showing that TRV-VIGS can operate in reproductive organs as well. We used this method to investigate the function of KATANIN and WRINKLED1 in cotton plant development. Cotton plants with suppressed KATANIN expression produced shorter fibers and elevated weight ratio of seed oil to endosperm. By contrast, silencing of WRINKLED1 expression resulted in increased fiber length but reduced oil seed content, suggesting the possibility to increase fiber length by repartitioning carbon flow. Our results provide evidence that the TRV-VIGS system can be used for rapid functional analysis of genes involved in cotton fiber development.

Integrative Transcriptome and Metabolome Analysis to Reveal Red Leaf Coloration in Shiya Tea (Adinandra nitida).

BACKGROUND: Adinandra nitida, commonly known as Shiya tea, is a healthcare drink enriched in several phenolic acids and flavonoids, with a purple-red leaf variety possessing a unique flavor and a higher economic value. However, the mechanisms underlying leaf coloration and senescence discoloration remain unknown. METHODS: Here, we compared both varieties of A. nitida (purple-red leaf, RL, and green leaf, GL) at two stages of development. To make sure the difference in leaf color in these four groups, several indexes, leaf colorimetric differences, H2O2 content in leaf cells, and antioxidant enzymes activities (superoxide dismutase (SOD), catalase (CAT)) were measured. With the integration of metabolome and transcriptome becoming a trend, metabolites in four groups were detected using an Ultra performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS) system, and the transcriptome was performed after the extraction of RNA in samples. Afterward, the activities of laccase (LAC) and peroxidase (POD) were measured for further analysis. RESULTS: The deeper or discoloration of leaf color was not caused by the reactive oxygen species (ROS) stress because the H2O2 content was similar for each group. And the SOD and CAT activities improved significantly in young leaves, especially RL_young. Metabolome data showed a large shift in four groups. By focusing on the variation of flavonoids and 1079 metabolites detected in both varieties, along with the accumulation of flavonoids and tannins, proanthocyanins (PAs) were mostly accumulated in young RL. Differential analysis of expressed genes (DEGs) revealed six genes associated with leaf discoloration as hub factors, of which ANRs (ANR1 and ANR2) were positively correlated with the accumulation of PA in RL. CONCLUSIONS: Using integrate analysis of metabolome and transcriptome, our results revealed that six structural genes found in proanthocyanin biosynthesis, two reductases (ANR), two oxidative polymerases (POD64, LAC17) and two TFs (bHLH3 and MYB4) related to biosynthesis and polymerization of proanthocyanins were associated with not only the difference of GL and RL but also the faded coloration in two RL groups (RL_young and RL_old), which provided a foundation for further research on an understanding of the regulatory genes and the enzymes specific for proanthocyanidin biosynthesis, facilitating the genetic engineering of crops for beneficial metabolite accumulation.

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.

Transcriptome and metabolome analyses provide insights into the relevance of pericarp thickness variations in Camellia drupifera and Camellia oleifera.

Camellia fruit is a woody edible oil source with a recalcitrant pericarp, which increases processing costs. However, the relevance of pericarp thickness variations in Camellia species remains unclear. Therefore, this study aimed to identify pericarp differences at the metabolic and transcription levels between thick-pericarp Camellia drupifera BG and thin-pericarp Camellia oleifera SG. Forty differentially accumulated metabolites were screened through non-targeted UHPLC-Q-TOF MS-based metabolite profiling. S-lignin was prominently upregulated in BG compared with SG, contributing to the thick pericarp of BG. KEGG enrichment and coexpression network analysis showed 29 differentially expressed genes associated with the lignin biosynthetic pathway, including 21 genes encoding catalysts and 8 encoding transcription factors. Nine upregulated genes encoding catalysts potentially led to S-lignin accumulation in BG pericarp, and transcription factors NAC and MYB were possibly involved in major transcriptional regulatory mechanisms. Conventional growth-related factors WRKYs and AP2/ERFs were positively associated while pathogenesis-related proteins MLP328 and NCS2 were negatively associated with S-lignin content. Thus, Camellia balances growth and defense possibly by altering lignin biosynthesis. The results of this study may guide the genetic modifications of C. drupifera to optimize its growth-defense balance and improve seed accessibility.

Integrative Analysis of the Metabolome and Transcriptome of a Cultivated Pepper and Its Wild Progenitor Chiltepin (Capsicum annuum L. var. glabriusculum) Revealed the Loss of Pungency During Capsicum Domestication.

Pungency is a unique characteristic of chili peppers (Capsicum spp.) caused by capsaicinoids. The evolutionary emergence of pungency is thought to be a derived trait within the genus Capsicum. However, it is not well-known how pungency has varied during Capsicum domestication and specialization. In this study, we applied a comparative metabolomics along with transcriptomics analysis to assess various changes between two peppers (a mildly pungent cultivated pepper BB3 and its hot progenitor chiltepin) at four stages of fruit development, focusing on pungency variation. A total of 558 metabolites were detected in two peppers. In comparison with chiltepin, capsaicinoid accumulation in BB3 was almost negligible at the early stage. Next, 412 DEGs associated with the capsaicinoid accumulation pathway were identified through coexpression analysis, of which 18 genes (14 TFs, 3 CBGs, and 1 UGT) were deemed key regulators due to their high coefficients. Based on these data, we speculated that downregulation of these hub genes during the early fruit developmental stage leads to a loss in pungency during Capsicum domestication (from chiltepin to BB3). Of note, a putative UDP-glycosyltransferase, GT86A1, is thought to affect the stabilization of capsaicinoids. Our results lay the foundation for further research on the genetic diversity of pungency traits during Capsicum domestication and specialization.

Identification of the Trichome Patterning Core Network Using Data from Weak ttg1 Alleles to Constrain the Model Space.

The regular distribution of trichomes on leaves in Arabidopsis is a well-understood model system for two-dimensional pattern formation. It involves more than 10 genes and is governed by two patterning principles, the activator-inhibitor (AI) and the activator-depletion (AD) mechanisms, though their relative contributions are unknown. The complexity of gene interactions, protein interactions, and intra- and intercellular mobility of proteins makes it very challenging to understand which aspects are relevant for pattern formation. In this study, we use global mathematical methods combined with a constraining of data to identify the structure of the underlying network. To constrain the model, we perform a genetic, cell biological, and biochemical study of weak ttg1 alleles. We find that the core of trichome patterning is a combination of AI and AD mechanisms differentiating between two pathways activating the long-range inhibitor CPC and the short-range inhibitor TRY.

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. thalianattg1 mutants and AtTTG1 orthologs. A plethora of MBW(AtTTG1) mechanisms in regulating the five major TTG1-dependent traits is highlighted.

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.

Musa paradisica RCI complements AtRCI and confers Na+ tolerance and K+ sensitivity in Arabidopsis.

The mechanisms involved in Na⁺/K⁺ uptake and extrusion are important in plant salt tolerance. In this study, we investigated the physiological role of a plasma membrane (PM)-localized protein, MpRCI, from plantain in transgenic Arabidopsis under NaCl and KCl stress and determined its effect on PM fluidity and H⁺-ATPase activity. The MpRCI gene exhibited high homology to the AtRCI2 gene family in Arabidopsis and was therefore able to complement for loss of the yeast AtRCI2-related PMP3 gene. Results of phenotypic espial and atomic emission spectrophotometer (AES) assays indicated that MpRCI overexpression in the AtRCI2A knockout mutant with reduced shoot Na⁺ and increased K⁺ exhibited increased Na⁺-tolerance and K⁺-sensitivity under NaCl or KCl treatments, respectively. Furthermore, comparisons of PM fluidity and H⁺-ATPase activity in shoots, with expression or absence of MpRCI/AtRCI2A expression under NaCl or KCl stress, showed MpRCI maintained PM fluidity and H⁺-ATPase activity under stress conditions. Results suggest that MpRCI plays an essential role in Na⁺/K⁺ flux in plant cells.