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Basic helix–loop–helix (bHLH) proteins are one of the largest families of transcription factor (TF) in eukaryotes, and ~30% of all flowering plants’ bHLH TFs contain the aspartate kinase, chorismate mutase, and TyrA (ACT)-like domain at variable distances C-terminal from the bHLH. However, the evolutionary history and functional consequences of the bHLH/ACT-like domain association remain unknown. Here, we show that this domain association is unique to the plantae kingdom with green algae (chlorophytes) harboring a small number of bHLH genes with variable frequency of ACT-like domain’s presence. bHLH-associated ACT-like domains form a monophyletic group, indicating a common origin. Indeed, phylogenetic analysis results suggest that the association of ACT-like and bHLH domains occurred early in Plantae by recruitment of an ACT-like domain in a common ancestor with widely distributed ACT DOMAIN REPEAT ( ACR ) genes by an ancestral bHLH gene. We determined the functional significance of this association by showing that Chlamydomonas reinhardtii ACT-like domains mediate homodimer formation and negatively affect DNA binding of the associated bHLH domains. We show that, while ACT-like domains have experienced faster selection than the associated bHLH domain, their rates of evolution are strongly and positively correlated, suggesting that the evolution of the ACT-like domains was constrained by the bHLH domains. This study proposes an evolutionary trajectory for the association of ACT-like and bHLH domains with the experimental characterization of the functional consequence in the regulation of plant-specific processes, highlighting the impacts of functional domain coevolution.

The identification and characterization of cis-regulatory DNA sequences and how they function to coordinate responses to developmental and environmental cues is of paramount importance to plant biology. Key to these regulatory processes are cis-regulatory modules (CRMs), which include enhancers and silencers. Despite the extraordinary advances in high-quality sequence assemblies and genome annotations, the identification and understanding of CRMs, and how they regulate gene expression, lag significantly behind. This is especially true for their distinguishing characteristics and activity states. Here, we review the current knowledge on CRMs and breakthrough technologies enabling identification, characterization, and validation of CRMs; we compare the genomic distributions of CRMs with respect to their target genes between different plant species, and discuss the role of transposable elements harboring CRMs in the evolution of gene expression. This is an exciting time to study cis-regulomes in plants; however, significant existing challenges need to be overcome to fully understand and appreciate the role of CRMs in plant biology and in crop improvement.

Transcriptome profiling of maize inbred and hybrid seedlings subjected to heat or cold stress was used to identify key cis-regulatory elements and develop models to predict gene expression responses., Changes in gene expression are important for responses to abiotic stress. Transcriptome profiling of heat- or cold-stressed maize genotypes identifies many changes in transcript abundance. We used comparisons of expression responses in multiple genotypes to identify alleles with variable responses to heat or cold stress and to distinguish examples of cis- or trans-regulatory variation for stress-responsive expression changes. We used motifs enriched near the transcription start sites (TSSs) for thermal stress-responsive genes to develop predictive models of gene expression responses. Prediction accuracies can be improved by focusing only on motifs within unmethylated regions near the TSS and vary for genes with different dynamic responses to stress. Models trained on expression responses in a single genotype and promoter sequences provided lower performance when applied to other genotypes but this could be improved by using models trained on data from all three genotypes tested. The analysis of genes with cis-regulatory variation provides evidence for structural variants that result in presence/absence of transcription factor binding sites in creating variable responses. This study provides insights into cis-regulatory motifs for heat- and cold-responsive gene expression and defines a framework for developing models to predict expression responses across multiple genotypes.

The Brassicaceae family comprises more than 3,700 species with a diversity of phenotypic characteristics, including seed oil content and composition. Recently, the global interest in Thlaspi arvense L. (pennycress) has grown as the seed oil composition makes it a suitable source for biodiesel and aviation fuel production. However, many wild traits of this species need to be domesticated to make pennycress ideal for cultivation. Molecular breeding and engineering efforts require the availability of an accurate genome sequence of the species. Here, we describe pennycress genome annotation improvements, using a combination of long- and short-read transcriptome data obtained from RNA derived from embryos of 22 accessions, in addition to public genome and gene expression information. Our analysis identified 27,213 protein-coding genes, as well as on average 6,188 biallelic SNPs. In addition, we used the identified SNPs to evaluate the population structure of our accessions. The data from this analysis support that the accession Ames 32872, originally from Armenia, is highly divergent from the other accessions, while the accessions originating from Canada and the United States cluster together. When we evaluated the likely signatures of natural selection from alternative SNPs, we found 7 candidate genes under likely recent positive selection. These genes are enriched with functions related to amino acid metabolism and lipid biosynthesis and highlight possible future targets for crop improvement efforts in pennycress.

Camelina (Camelina sativa) is an annual oilseed plant that is gaining momentum as a biofuel cover crop. Understanding gene regulatory networks is essential to deciphering plant metabolic pathways, including lipid metabolism. Here, we take advantage of a growing collection of gene expression datasets to predict transcription factors (TFs) associated with the control of Camelina lipid metabolism. We identified approximately 350 TFs highly co-expressed with lipid-related genes (LRGs). These TFs are highly represented in the MYB, AP2/ERF, bZIP, and bHLH families, including a significant number of homologs of well-known Arabidopsis lipid and seed developmental regulators. After prioritizing the top 22 TFs for further validation, we identified DNA-binding sites and predicted target genes for 16 out of the 22 TFs tested using DNA affinity purification followed by sequencing (DAP-seq). Enrichment analyses of targets supported the co-expression prediction for most TF candidates, and the comparison to Arabidopsis revealed some common themes, but also aspects unique to Camelina. Within the top potential lipid regulators, we identified CsaMYB1, CsaABI3AVP1-2, CsaHB1, CsaNAC2, CsaMYB3, and CsaNAC1 as likely involved in the control of seed fatty acid elongation and CsaABI3AVP1-2 and CsabZIP1 as potential regulators of the synthesis and degradation of triacylglycerols (TAGs), respectively. Altogether, the integration of co-expression data and DNA-binding assays permitted us to generate a high-confidence and short list of Camelina TFs involved in the control of lipid metabolism during seed development.

Improvement of agricultural species has exploited the genetic variation responsible for complex quantitative traits. Much of the functional variation is regulatory, in cis-regulatory elements and trans-acting factors that ultimately contribute to gene expression differences. However, the identification of gene regulatory network components that, when modulated, will increase plant productivity or resilience, is challenging, yet essential to provide increased predictive power for genome engineering approaches that are likely to benefit useful traits. Here, we discuss the opportunities and limitations of using data obtained from gene coexpression, transcription factor binding, and genome-wide association mapping analyses to predict regulatory interactions that impact crop improvement. It is apparent that a combination of information from these data types is necessary for the reliable identification and utilization of important regulatory interactions that underlie complex agronomic traits.

Plants respond to wounding stress by changing gene expression patterns and inducing the production of hormones including jasmonic acid. This wounding transcriptional response activates specialized metabolism pathways such as the glucosinolate pathways in Arabidopsis thaliana. While the regulatory factors and sequences controlling a subset of wound-response genes are known, it remains unclear how wound response is regulated globally. Here, we how these responses are regulated by incorporating putative cis-regulatory elements, known transcription factor binding sites, in vitro DNA affinity purification sequencing, and DNase I hypersensitive sites to predict genes with different wound-response patterns using machine learning. We observed that regulatory sites and regions of open chromatin differed between genes upregulated at early and late wounding time-points as well as between genes induced by jasmonic acid and those not induced. Expanding on what we currently know, we identified cis-elements that improved model predictions of expression clusters over known binding sites. Using a combination of genome editing, in vitro DNA-binding assays, and transient expression assays using native and mutated cis-regulatory elements, we experimentally validated four of the predicted elements, three of which were not previously known to function in wound-response regulation. Our study provides a global model predictive of wound response and identifies new regulatory sequences important for wounding without requiring prior knowledge of the transcriptional regulators.

Plants accumulate several thousand of phenolic compounds, including lignins and flavonoids, which are mainly synthesized through the phenylpropanoid pathway, and play important roles in plant growth and adaptation. A novel high-throughput ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) method was established to quantify the levels of 19 flavonoids and 15 other phenolic compounds, including acids, aldehydes, and alcohols. The chromatographic separation was performed in 10 min, allowing for the resolution of isomers such as 3-, 4-, and 5-chlorogenic acids, 4-hydroxybenzoic and salicylic acids, isoorientin and orientin, and luteolin and kaempferol. The linearity range for each compound was found to be in the low fmol to the high pmol. Furthermore, this UHPLC-MS/MS approach was shown to be very sensitive with limits of detection between 1.5 amol to 300 fmol, and limits of quantification between 5 amol to 1000 fmol. Extracts from maize seedlings were used to assess the robustness of the method in terms of recovery efficiency, matrix effect, and accuracy. The biological matrix did not suppress the signal for 32 out of the 34 metabolites under investigation. Additionally, the majority of the analytes were recovered from the biological samples with an efficiency above 75%. All flavonoids and other phenolic compounds had an intra- and inter-day accuracy within a ±20% range, except for coniferyl alcohol and vanillic acid. Finally, the quantification of flavonoids, free and cell wall-bound phenolics in seedlings from two maize lines with contrasting phenolic content was successfully achieved using this methodology.

Autofluorescence-based optical imaging methods have advanced cellular metabolism research as a technique to non-invasively measure metabolic shifts temporally and spatially. In this work, we present metabolic investigations using fluorescence anisotropy in two well-defined plant and animal models: 1) A set of flavonoid-deficient Arabidopsis thaliana mutants which shows unique fluorescence polarization properties with varying degrees of flavonoid accumulation. 2) The cancer metabolic deformities manifested in animal cells using real-time fluorescence anisotropy based metabolic readout (4fps). These studies demonstrated the potential of using fluorescence anisotropy in a systematic way to study metabolism and cellular heterogeneity.

Changes in gene expression are important for response to abiotic stress. Transcriptome profiling performed on maize inbred and hybrid genotypes subjected to heat or cold stress identifies many transcript abundance changes in response to these environmental conditions. Motifs that are enriched near differentially expressed genes were used to develop machine learning models to predict gene expression responses to heat or cold. The best performing models utilize the sequences both upstream and downstream of the transcription start site. Prediction accuracies could be improved using models developed for specific co-expression clusters compared to using all up- or down-regulated genes or by only using motifs within unmethylated regions. Comparisons of expression responses in multiple genotypes were used to identify genes with variable response and to identifycis-ortrans-regulatory variation. Models trained on B73 data have lower performance when applied to Mo17 or W22, this could be improved by using models trained on data from all genotypes. However, the models have low accuracy for correctly predicting genes with variable responses to abiotic stress. This study provides insights intocis-regulatory motifs for heat- and cold-responsive gene expression and provides a framework for developing models to predict expression response to abiotic stress across multiple genotypes.One sentence summaryTranscriptome profiling of maize inbred and hybrid seedlings subjected to heat or cold stress was used to identify key cis-regulatory elements and develop models to predict gene expression responses.

Camelina is an annual oilseed plant from the Brassicaceae family that is gaining momentum as a biofuel winter cover crop. However, a significant limitation in further enhancing its utility as a producer of oils that can be used as biofuels, jet fuels or bio-based products is the absence of a repository for all the gene expression and regulatory information that is being rapidly generated by the community. Here, we provide CamRegBase (https://camregbase.org/) as a one-stop resource to access Camelina information on gene expression and co-expression, transcription factors, lipid associated genes and genome-wide orthologs in the close-relative reference plant Arabidopsis. We envision this as a resource of curated information for users, as well as a repository of new gene regulation information.

Plants respond to wounding stress by changing gene expression patterns and inducing jasmonic acid (JA), as well as other plant hormones. This includes activating some specialized metabolism pathways, including the glucosinolate pathways, in the case of Arabidopsis thaliana. We model how these responses are regulated by using machine learning to incorporate putative cis-regulatory elements (pCREs), known transcription factor binding sites from literature, in-vitro DNA affinity purification sequencing (DAP-seq) and DNase I hypersensitive sites to predict gene expression for genes clustered by their wound response using machine learning. We found temporal patterns where regulatory sites and regions of open chromatin differed between clusters of genes up-regulated at early and late wounding time points as well as clusters where JA response was induced relative to clusters where JA response was not induced. Overall, we identified pCREs that improved model predictions of expression clusters over known binding sites. We discovered 4,255 pCREs related to wound response at different time points and 2,569 pCREs related to differences between JA-induced and non-JA induced wound response. In addition, pCREs found to be important at different wounding time points were mapped to the promoters of genes in a glucosinolate biosynthesis pathway indicating regulation of this pathway under wounding stress. Finally, we experimentally validated a predicted cis-regulatory element, CCGCGT, showing that knock-out via CRISPR-Cas9 reduces gene expression in response to wounding.

In plants, the deoxy sugar l-rhamnose is widely present as rhamnose-containing polymers in cell walls and as part of the decoration of various specialized metabolites. Here, we review the current knowledge on the distribution of rhamnose, highlighting the differences between what is known in dicotyledoneuos compared to commelinid monocotyledoneous (grasses) plants. We discuss the biosynthesis and transport of UDP-rhamnose, as well as the transfer of rhamnose from UDP-rhamnose to various primary and specialized metabolites. This is carried out by rhamnosyltransferases, enzymes that can use a large variety of substrates. Some unique characteristics of rhamnose synthases, the multifunctional enzymes responsible for the conversion of UDP-glucose into UDP-rhamnose, are considered, particularly from the perspective of their ability to convert glucose present in flavonoids. Finally, we discuss how little is still known with regards to how plants rescue rhamnose from the many compounds to which it is linked, or how rhamnose is catabolized.

Gene co-expression analyses provide a powerful tool to determine gene associations. The interaction of transcription factors (TFs) with their target genes is an essential step in gene regulation, yet to what extent TFs-target gene associations are recovered in co-expression studies remains unclear. Using the wealth of data available for Arabidopsis, we show here that protein-DNA interactions are overall poor indicators of TF-target co-expression, yet the inclusion of TF-TF interaction information significantly enhance co-expression signals. These results highlight the impact of combinatorial gene control on such gene association networks. We integrated this information to predict higher-order regulatory complexes, which are difficult to identify experimentally. We demonstrate that genes strongly co-expressed with a TF are also enriched in indirect targets. Our results have significant implications on the empirical understanding of complex gene regulatory networks and transcription factor function, and the significance of co-expression from the perspective of protein-protein and protein-DNA interactions.

Anthocyanin pigments furnish a powerful visual output of the stress and metabolic status of Arabidopsis thaliana plants. Essential for pigment accumulation is TRANSPARENT TESTA19 (TT19), a glutathione S-transferase proposed to bind and stabilize anthocyanins, participating in their vacuolar sequestration, a function conserved across the flowering plants. Here, we report the identification of genetic suppressors that result in anthocyanin accumulation in the absence of TT19. We show that mutations in RDR6, SGS3, or DCL4 suppress the anthocyanin defect of tt19 by pushing carbon towards flavonoid biosynthesis. This effect is not unique to tt19 and extends to at least one other anthocyanin pathway gene mutant. This synergy between mutations in components of the RDR6-SGS3-DCL4 siRNA system and the flavonoid pathway reveals genetic/epigenetic mechanisms regulating metabolic fluxes., TRANSPARENT TESTA19 (TT19) encodes a glutathione S-transferase which functions in anthocyanin stabilization and vacuolar transport. Here, by tt19 suppressor screening, the authors show that RDR6/SGS3/DCL4 siRNA pathway constituents synergistically interact with components of the flavonoid pathway to control carbon metabolism.

The regulation of gene expression is central to many biological processes. Gene regulatory networks (GRNs) link transcription factors (TFs) to their target genes and represent maps of potential transcriptional regulation. Here, we analyzed a large number of publically available maize (Zea mays) transcriptome data sets including >6000 RNA sequencing samples to generate 45 coexpression-based GRNs that represent potential regulatory relationships between TFs and other genes in different populations of samples (cross-tissue, cross-genotype, and tissue-and-genotype samples). While these networks are all enriched for biologically relevant interactions, different networks capture distinct TF-target associations and biological processes. By examining the power of our coexpression-based GRNs to accurately predict covarying TF-target relationships in natural variation data sets, we found that presence/absence changes rather than quantitative changes in TF gene expression are more likely associated with changes in target gene expression. Integrating information from our TF-target predictions and previous expression quantitative trait loci (eQTL) mapping results provided support for 68 TFs underlying 74 previously identified trans-eQTL hotspots spanning a variety of metabolic pathways. This study highlights the utility of developing multiple GRNs within a species to detect putative regulators of important plant pathways and provides potential targets for breeding or biotechnological applications.

Flavones, one of the largest groups of flavonoids, have beneficial effects on human health and are considered of high nutritional value. Previously, we demonstrated that maize type I flavone synthase (ZmFNSI) is one of the enzymes responsible for the synthesis of O-glycosyl flavones in floral tissues. However, in related species such as rice and sorghum, type II FNS enzymes also contribute to flavone biosynthesis. In this work, we provide evidence that maize has both one FNSI and one FNSII flavone synthases. Arabidopsis transgenic plants expressing each FNS enzyme were generated to validate the role of flavones in protecting plants against UV-B radiation. Here, we demostrate that ZmCYP93G7 (FNSII) has flavone synthase activity and is able to complement the Arabidopsis dmr6 mutant, restoring the susceptibility to Pseudomonas syringae. ZmFNSII expression is controlled by the C1/PL1 + R/B anthocyanin transcriptional complexes, and both ZmFNSI and ZmFNSII are regulated by UV-B. Arabidopsis transgenic plants expressing ZmFNSI or ZmFNSII that accumulate apigenin exhibit less UV-B-induced damage than wild-type plants. Together, we show that maize has two FNS-type enzymes that participate in the synthesis of apigenin, conferring protection against UV-B radiation.

Arabidopsis thaliana, a model system for plant research, serves as the ideal organism for teaching a variety of basic genetic concepts including inheritance, genetic variation, segregation, and dominant and recessive traits. Rapid advances in the field of genetics make understanding foundational concepts, such as Mendel's laws, ever more important to today's biology student. Coupling these concepts with hands-on learning experiences better engages students and deepens their understanding of the topic. In our article, we present a teaching module from the Arabidopsis Biological Resource Center as a tool to engage students in lab inquiry exploring Mendelian genetics. This includes a series of protocols and assignments that guide students through growing two generations of Arabidopsis, making detailed observations of mutant phenotypes, and determining the inheritance of specific traits, thus providing a hands-on component to help teach genetics at the middle and high school level.

Coffee organoleptic properties are based in part on the quality and chemical composition of coffee beans. The presence of defective beans during processing and roasting contribute to off flavors and reduce overall cup quality. A multipronged approach was undertaken to identify specific biochemical markers for defective beans. To this end, beans were split into defective and non-defective fractions and biochemically profiled in both green and roasted states. A set of 17 compounds in green beans, including organic acids, amino acids and reducing sugars; and 35 compounds in roasted beans, dominated by volatile compounds, organic acids, sugars and sugar alcohols, were sufficient to separate the defective and non-defective fractions. Unsorted coffee was examined for the presence of the biochemical markers to test their utility in detecting defective beans. Although the green coffee marker compounds were found in all fractions, three of the roasted coffee marker compounds (1-methylpyrrole, 5-methyl- 2-furfurylfuran, and 2-methylfuran) were uniquely present in defective fractions.

The translation of the genotype into phenotype, represented for example by the expression of genes encoding enzymes required for the biosynthesis of phytochemicals that are important for interaction of plants with the environment, is largely carried out by transcription factors (TFs) that recognize specific cis-regulatory elements in the genes that they control. TFs and their target genes are organized in gene regulatory networks (GRNs), and thus uncovering GRN architecture presents an important biological challenge necessary to explain gene regulation. Linking TFs to the genes they control, central to understanding GRNs, can be carried out using gene- or TF-centered approaches. In this study, we employed a gene-centered approach utilizing the yeast one-hybrid assay to generate a network of protein-DNA interactions that participate in the transcriptional control of genes involved in the biosynthesis of maize phenolic compounds including general phenylpropanoids, lignins, and flavonoids. We identified 1100 protein-DNA interactions involving 54 phenolic gene promoters and 568 TFs. A set of 11 TFs recognized 10 or more promoters, suggesting a role in coordinating pathway gene expression. The integration of the gene-centered network with information derived from TF-centered approaches provides a foundation for a phenolics GRN characterized by interlaced feed-forward loops that link developmental regulators with biosynthetic genes.

Combinatorial gene regulation provides a mechanism by which relatively small numbers of transcription factors can control the expression of a much larger number of genes with finely tuned temporal and spatial patterns. This is achieved by transcription factors assembling into complexes in a combinatorial fashion, exponentially increasing the number of genes that they can target. Such an arrangement also increases the specificity and affinity for the cis-regulatory sequences required for accurate target gene expression. Superimposed on this transcription factor combinatorial arrangement is the increasing realization that histone modification marks expand the regulatory information, which is interpreted by histone readers and writers that are part of the regulatory apparatus. Here, we review the progress in these areas from the perspective of plant combinatorial gene regulation, providing examples of different regulatory solutions and comparing them to other metazoans. This article is part of a Special Issue entitled: Plant Gene Regulatory Mechanisms and Networks, edited by Dr. Erich Grotewold and Dr. Nathan Springer.

Many plant species display remarkable developmental plasticity and regenerate new organs after injury. Local signals produced by wounding are thought to trigger organ regeneration but molecular mechanisms underlying this control remain largely unknown. We previously identified an AP2/ERF transcription factor WOUND INDUCED DEDIFFERENTIATION1 (WIND1) as a central regulator of wound-induced cellular reprogramming in plants. In this study, we demonstrate that WIND1 promotes callus formation and shoot regeneration by upregulating the expression of the ENHANCER OF SHOOT REGENERATION1 (ESR1) gene, which encodes another AP2/ERF transcription factor in Arabidopsis thaliana The esr1 mutants are defective in callus formation and shoot regeneration; conversely, its overexpression promotes both of these processes, indicating that ESR1 functions as a critical driver of cellular reprogramming. Our data show that WIND1 directly binds the vascular system-specific and wound-responsive cis-element-like motifs within the ESR1 promoter and activates its expression. The expression of ESR1 is strongly reduced in WIND1-SRDX dominant repressors, and ectopic overexpression of ESR1 bypasses defects in callus formation and shoot regeneration in WIND1-SRDX plants, supporting the notion that ESR1 acts downstream of WIND1. Together, our findings uncover a key molecular pathway that links wound signaling to shoot regeneration in plants.

Phenolic compounds are among the most diverse and widespread of specialized plant compounds and underly many important agronomic traits. Our comprehensive analysis of the maize genome unraveled new aspects of the genes involved in phenylpropanoid, monolignol, and flavonoid production in this important crop. Remarkably, just 19 genes accounted for 70 % of the overall mRNA accumulation of these genes across 95 tissues, indicating that these are the main contributors to the flux of phenolic metabolites. Eighty genes with intermediate to low expression play minor and more specialized roles. Remaining genes are likely undergoing loss of function or are expressed in limited cell types. Phylogenetic and expression analyses revealed which members of gene families governing metabolic entry and branch points exhibit duplication, subfunctionalization, or loss of function. Co-expression analysis applied to genes in sequential biosynthetic steps revealed that certain isoforms are highly co-expressed and are candidates for metabolic complexes that ensure metabolite delivery to correct cellular compartments. Co-expression of biosynthesis genes with transcription factors discovered connections that provided candidate components for regulatory modules governing this pathway. Our study provides a comprehensive analysis of maize phenylpropanoid related genes, identifies major pathway contributors, and novel candidate enzymatic and regulatory modules of the metabolic network.

International audience; Seed development is a complex regulatory process that includes a transition from gametophytic to sporophytic program. The synchronized development of different seed compartments (seed coat, endosperm and embryo) depends on a balance in parental genome contributions. In the most severe cases, the disruption of the balance leads to seed abortion. This represents one of the main obstacles for the engineering of asexual reproduction through seeds (apomixis), and for generating new interspecies hybrids. The repression of auxin synthesis by the Polycomb Repressive Complex 2 (PRC2) is a major mechanism contributing to sensing genome balance. However, current efforts focusing on decreasing PRC2 or elevating auxin levels have not yet resulted in the production of apomictic seed. Here, we show that EMSY-Like Tudor/Agenet H3K36me3 histone readers EML1 and EML3 are necessary for early stages of seed development to proceed at a normal rate in Arabidopsis. We further demonstrate that both EML1 and EML3 are required to prevent the initiation of seed development in the absence of fertilization. Based on the whole genome expression analysis, we identify auxin transport and signaling genes as the most enriched downstream targets of EML1 and EML3. We hypothesize that EML1 and EML3 function to repress and soften paternal gene expression by fine-tuning auxin responses. Discovery of this pathway may contribute to the engineering of apomixis and interspecies hybrids.

The visual phenotypes afforded by flavonoid pigments have provided invaluable tools for modern genetics. Many Arabidopsis transparent testa (tt) mutants lacking the characteristic proanthocyanidin (PA) seed coat pigmentation and often failing to accumulate anthocyanins in vegetative tissues have been characterized. These mutants have significantly contributed to our understanding of flavonoid biosynthesis, regulation, and transport. A comprehensive screening for tt mutants in available large T-DNA collection lines resulted in the identification of 16 independent lines lacking PAs and anthocyanins, or with seed coat pigmentation clearly distinct from wild type. Segregation analyses and the characterization of second alleles in the genes disrupted by the indexed T-DNA insertions demonstrated that all the lines contained at least one additional mutation responsible for the tt phenotypes. Using a combination of RNA-Seq and whole genome re-sequencing and confirmed through complementation, we show here that these mutations correspond to novel alleles of ttg1 (two alleles), tt3 (two alleles), tt5 (two alleles), ban (two alleles), tt1 (two alleles), and tt8 (six alleles), which harbored additional T-DNA insertions, indels, missense mutations, and large genomic deletion. Several of the identified alleles offer interesting perspectives on flavonoid biosynthesis and regulation.

About a third of the plant basic helix–loop–helix (bHLH) transcription factors harbor a C-terminal aspartate kinase, chorismate mutase, and TyrA (ACT)-like domain, which was originally identified in the maize R regulator of anthocyanin biosynthesis, where it modulates the ability of the bHLH to dimerize and bind DNA. Characterization of other bHLH ACT-like domains, such as the one in the Arabidopsis R ortholog, GL3, has not definitively confirmed dimerization, raising the question of the overall role of this potential regulatory domain. To learn more, we compared the dimerization of the ACT-like domains of R (RACT) and GL3 (GL3ACT). We show that RACT dimerizes with a dissociation constant around 100 nM, over an order of magnitude stronger than GL3ACT. Structural predictions combined with mutational analyses demonstrated that V568, located in a hydrophobic pocket in RACT, is important: when mutated to the Ser residue present in GL3ACT, dimerization affinity dropped by almost an order of magnitude. The converse S595V mutation in GL3ACT significantly increased the dimerization strength. We cloned and assayed dimerization for all identified maize ACT-like domains and determined that 12 of 42 formed heterodimers in yeast two-hybrid assays, irrespective of whether they harbored V568, which was often replaced by other aliphatic amino acids. Moreover, we determined that the presence of polar residues at that position occurs only in a small subset of anthocyanin regulators. The combined results provide new insights into possibly regulatory mechanisms and suggest that many of the other plant ACT-like domains associate to modulate fundamental cellular processes.

Summary Anthocyanins are flavonoid pigments that accumulate in most seed plants. They are synthesized in the cytoplasm but accumulate inside the vacuoles. Anthocyanins are pigmented at the lower vacuolar pH, but in the cytoplasm they can be visualized based on their fluorescence properties. Thus, anthocyanins provide an ideal system for the development of new methods to investigate cytoplasmic pools and association with other molecular components. We have analyzed the fluorescence decay of anthocyanins by fluorescence lifetime imaging microscopy (FLIM), in both in vitro and in vivo conditions, using wild type and mutant Arabidopsis thaliana seedlings. Within plant cells, the amplitude-weighted mean fluorescence lifetime (τm) correlated with distinct subcellular localizations of anthocyanins. The vacuolar pool of anthocyanins exhibited shorter τm than the cytoplasmic pool. Consistently, lowering the pH of anthocyanins in solution shortened their fluorescence decay. We propose that FLIM is a useful tool for understanding the trafficking of anthocyanins and potentially, for estimating vacuolar pH inside intact plant cells. This article is protected by copyright. All rights reserved.

Anthocyanins provide ideal visual markers for the identification of mutations that disrupt molecular responses to abiotic stress. We screened Arabidopsis mutants of ABC (ATP‐Binding Cassette) and MATE (Multidrug And Toxic compound Extrusion) transporter genes under nutritional stress and identified four genes (ABCG25,ABCG9,ABCG5, and MATE45) required for normal anthocyanin pigmentation. ABCG25 was previously demonstrated to encode a vascular‐localized cellular exporter of abscisic acid (ABA). Our results show that MATE45 encodes an aerial meristem‐ and a vascular‐localized transporter associated with the trans‐Golgi, and that it plays an important role in controlling the levels and distribution of ABA in growing aerial meristems and non‐meristematic tissues. MATE45 promoter‐GUS reporter fusions revealed the activity localized to the leaf and influorescence meristems and the vasculature. Loss‐of‐function mate45 mutants exhibited accelerated rates of aerial organ initiation suggesting at least partial functional conservation with the maize ortholog bige1. The aba2‐1 mutant, which is deficient in ABA biosynthesis, exhibited a number of phenotypes that were rescued in the mate45‐1 aba2‐1 double mutant. mate45 exhibited enhanced the seed dormancy, and germination was hypersensitive to ABA. Enhanced frequency of leaf primordia growth in mate45 seedlings grown in nutrient imbalance stress was ABA‐dependent. The ABA signaling reporter construct pRD29B::GUS revealed elevated levels of ABA signaling in the true leaf primordia of mate45 seedlings grown under nutritional stress, and gradually reduced signaling in surrounding cotyledon and hypocotyl tissues concomitant with reduced expressions of ABCG25. Our results suggest a role of MATE45 in reducing meristematic ABA and in maintaining ABA distribution in adjacent non‐meristematic tissues.