Ontologies for increasing the FAIRness of plant research data.

The importance of improving the FAIRness (findability, accessibility, interoperability, reusability) of research data is undeniable, especially in the face of large, complex datasets currently being produced by omics technologies. Facilitating the integration of a dataset with other types of data increases the likelihood of reuse, and the potential of answering novel research questions. Ontologies are a useful tool for semantically tagging datasets as adding relevant metadata increases the understanding of how data was produced and increases its interoperability. Ontologies provide concepts for a particular domain as well as the relationships between concepts. By tagging data with ontology terms, data becomes both human- and machine- interpretable, allowing for increased reuse and interoperability. However, the task of identifying ontologies relevant to a particular research domain or technology is challenging, especially within the diverse realm of fundamental plant research. In this review, we outline the ontologies most relevant to the fundamental plant sciences and how they can be used to annotate data related to plant-specific experiments within metadata frameworks, such as Investigation-Study-Assay (ISA). We also outline repositories and platforms most useful for identifying applicable ontologies or finding ontology terms.

GXP: Analyze and Plot Plant Omics Data in Web Browsers.

Next-generation sequencing and metabolomics have become very cost and work efficient and are integrated into an ever-growing number of life science research projects. Typically, established software pipelines analyze raw data and produce quantitative data informing about gene expression or concentrations of metabolites. These results need to be visualized and further analyzed in order to support scientific hypothesis building and identification of underlying biological patterns. Some of these tools already exist, but require installation or manual programming. We developed "Gene Expression Plotter" (GXP), an RNAseq and Metabolomics data visualization and analysis tool entirely running in the user's web browser, thus not needing any custom installation, manual programming or uploading of confidential data to third party servers. Consequently, upon receiving the bioinformatic raw data analysis of RNAseq or other omics results, GXP immediately enables the user to interact with the data according to biological questions by performing knowledge-driven, in-depth data analyses and candidate identification via visualization and data exploration. Thereby, GXP can support and accelerate complex interdisciplinary omics projects and downstream analyses. GXP offers an easy way to publish data, plots, and analysis results either as a simple exported file or as a custom website. GXP is freely available on GitHub (see introduction).

Genetic and Molecular Analysis of Root Hair Development in Arabis alpina.

Root hair formation in Arabidopsis thaliana is a well-established model system for epidermal patterning and morphogenesis in plants. Over the last decades, many underlying regulatory genes and well-established networks have been identified by thorough genetic and molecular analysis. In this study, we used a forward genetic approach to identify genes involved in root hair development in Arabis alpina, a related crucifer species that diverged from A. thaliana approximately 26-40 million years ago. We found all root hair mutant classes known in A. thaliana and identified orthologous regulatory genes by whole-genome or candidate gene sequencing. Our findings indicate that the gene-phenotype relationships regulating root hair development are largely conserved between A. thaliana and A. alpina. Concordantly, a detailed analysis of one mutant with multiple hairs originating from one cell suggested that a mutation in the SUPERCENTIPEDE1 (SCN1) gene is causal for the phenotype and that AaSCN1 is fully functional in A. thaliana. Interestingly, we also found differences in the regulation of root hair differentiation and morphogenesis between the species, and a subset of root hair mutants could not be explained by mutations in orthologs of known genes from A. thaliana. This analysis provides insight into the conservation and divergence of root hair regulation in the Brassicaceae.

TRANSPARENT TESTA GLABRA 1 participates in flowering time regulation in Arabidopsis thaliana.

Pleiotropic regulatory factors mediate concerted responses of the plant's trait network to endogenous and exogenous cues. TRANSPARENT TESTA GLABRA 1 (TTG1) is such a factor that has been predominantly described as a regulator of early developmental traits. Although its closest homologs LIGHT-REGULATED WD1 (LWD1) and LWD2 affect photoperiodic flowering, a role of TTG1 in flowering time regulation has not been reported. Here we reveal that TTG1 is a regulator of flowering time in Arabidopsis thaliana and changes transcript levels of different targets within the flowering time regulatory pathway. TTG1 mutants flower early and TTG1 overexpression lines flower late at long-day conditions. Consistently, TTG1 can suppress the transcript levels of the floral integrators FLOWERING LOCUS T and SUPPRESSOR OF OVEREXPRESSION OF CO1 and can act as an activator of circadian clock components. Moreover, TTG1 might form feedback loops at the protein level. The TTG1 protein interacts with PSEUDO RESPONSE REGULATOR (PRR)s and basic HELIX-LOOP-HELIX 92 (bHLH92) in yeast. In planta, the respective pairs exhibit interesting patterns of localization including a recruitment of TTG1 by PRR5 to subnuclear foci. This mechanism proposes additional layers of regulation by TTG1 and might aid to specify the function of bHLH92. Within another branch of the pathway, TTG1 can elevate FLOWERING LOCUS C (FLC) transcript levels. FLC mediates signals from the vernalization, ambient temperature and autonomous pathway and the circadian clock is pivotal for the plant to synchronize with diurnal cycles of environmental stimuli like light and temperature. Our results suggest an unexpected positioning of TTG1 upstream of FLC and upstream of the circadian clock. In this light, this points to an adaptive value of the role of TTG1 in respect to flowering time regulation.

Cryptochrome 2 competes with COP1 substrates to repress COP1 ubiquitin ligase activity during Arabidopsis photomorphogenesis.

In plants, the cryptochrome photoreceptors suppress the activity of the COP1/SPA ubiquitin ligase to initiate photomorphogenesis in blue light. Both CRY1 and CRY2 interact with the COP1/SPA complex in a blue light-dependent manner. The mechanisms underlying the inhibition of COP1 activity through direct interactions with photoactivated CRYs are not fully understood. Here we tested the hypothesis that CRY2 inhibits COP1 by displacing the degradation substrates from COP1. To this end, we analyzed the role of a conserved valine-proline (VP) motif in the C-terminal domain of CRY2 (CCT2), which resembles the core COP1-WD40-binding sequences present in the substrates of COP1. We show that the VP motif in CRY2 is essential for the interaction of CRY2 with COP1 in yeast two-hybrid assays and in planta. Mutations in the VP motif of CRY2 abolished the CRY2 activity in photomorphogenesis, indicating the importance of VP. The interaction between COP1 and its VP-containing substrate PAP2 was prevented in the presence of coexpressed CRY2, but not in the presence of CRY2 carrying a VP mutation. Thus, since both PAP2 and CRY2 engage VP motifs to bind to COP1, these results demonstrate that CRY2 outcompetes PAP2 for binding to COP1. We further found that the previously unknown interaction between SPA1-WD and CCT2 occurs via the VP motif in CRY2, suggesting structural similarities in the VP-binding pockets of COP1-WD40 and SPA1-WD40 domains. A VP motif present in CRY1 is also essential for binding to COP1. Thus, CRY1 and CRY2 might share this mechanism of COP1 inactivation.

Genetic and molecular analysis of trichome development in Arabis alpina.

The genetic and molecular analysis of trichome development in Arabidopsis thaliana has generated a detailed knowledge about the underlying regulatory genes and networks. However, how rapidly these mechanisms diverge during evolution is unknown. To address this problem, we used an unbiased forward genetic approach to identify most genes involved in trichome development in the related crucifer species Arabisalpina In general, we found most trichome mutant classes known in A. thaliana We identified orthologous genes of the relevant A. thaliana genes by sequence similarity and synteny and sequenced candidate genes in the A. alpina mutants. While in most cases we found a highly similar gene-phenotype relationship as known from Arabidopsis, there were also striking differences in the regulation of trichome patterning, differentiation, and morphogenesis. Our analysis of trichome patterning suggests that the formation of two classes of trichomes is regulated differentially by the homeodomain transcription factor AaGL2 Moreover, we show that overexpression of the GL3 basic helix-loop-helix transcription factor in A. alpina leads to the opposite phenotype as described in A. thaliana Mathematical modeling helps to explain how this nonintuitive behavior can be explained by different ratios of GL3 and GL1 in the two species.

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.

Sub-epidermal Expression of ENHANCER OF TRIPTYCHON AND CAPRICE1 and Its Role in Root Hair Formation Upon Pi Starvation.

Root hair patterning is best studied in Arabidopsis thaliana. A pattern of root hair and non-root hair files is governed by a gene-regulatory network of activators and inhibitors. Under phosphate starvation conditions, extra root hairs are formed in non-root hair positions. This raises the question, whether and how this environmental stimulus is mediated by the known root hair gene network. In this study, we provide genetic and molecular data on the role of ETC1 in the phosphate starvation induced ectopic root hair formation. We show that the expression in the epidermis is irregular and reduced and that a new expression domain is induced in the sub-epidermis. By expressing ETC1 in the sub-epidermis, we show that this is sufficient to induce extra root hair formation in N-files. This suggests that the phosphate induced expressional switch from epidermal to epidermal plus sub-epidermal expression of ETC1 is one environmental input to the underlying patterning network.

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.

A fast and simple LC-MS-based characterization of the flavonoid biosynthesis pathway for few seed(ling)s.

BACKGROUND: (Pro)anthocyanidins are synthesized by the flavonoid biosynthesis pathway with multi-layered regulatory control. Methods for the analysis of the flavonoid composition in plants are well established for different purposes. However, they typically compromise either on speed or on depth of analysis. RESULTS: In this work we combined and optimized different protocols to enable the analysis of the flavonoid biosynthesis pathway with as little as possible biological material. We chose core substances of this metabolic pathway that serve as a fingerprint to recognize alterations in the main branches of the pathway. We used a simplified sample preparation, two deuterated internal standards, a short and efficient LC separation, highly sensitive detection with tandem MS in multiple reaction monitoring (MRM) mode and hydrolytic release of the core substances to reduce complexity. The method was optimized for Arabidopsis thaliana seeds and seedlings. We demonstrate that one Col-0 seed/seedling is sufficient to obtain a fingerprint of the core substances of the flavonoid biosynthesis pathway. For comparative analysis of different genotypes, we suggest the use of 10 seed(lings). The analysis of Arabidopsis thaliana mutants affecting steps in the pathway revealed foreseen and unexpected alterations of the pathway. For example, HY5 was found to differentially regulate kaempferol in seeds vs. seedlings. Furthermore, our results suggest that COP1 is a master regulator of flavonoid biosynthesis in seedlings but not of flavonoid deposition in seeds. CONCLUSIONS: When sample numbers are high and the plant material is limited, this method effectively facilitates metabolic fingerprinting with one seed(ling), revealing shifts and differences in the pathway. Moreover the combination of extracted non-hydrolysed, extracted hydrolysed and non-extracted hydrolysed samples proved useful to deduce the class of derivative from which the individual flavonoids have been released.

Rapid identification of a natural knockout allele of ARMADILLO REPEAT-CONTAINING KINESIN1 that causes root hair branching by mapping-by-sequencing.

In Arabidopsis (Arabidopsis thaliana), branched root hairs are an indicator of defects in root hair tip growth. Among 62 accessions, one accession (Heiligkreuztal2 [HKT2.4]) displayed branched root hairs, suggesting that this accession carries a mutation in a gene of importance for tip growth. We determined 200- to 300-kb mapping intervals using a mapping-by-sequencing approach of F2 pools from crossings of HKT2.4 with three different accessions. The intersection of these mapping intervals was 80 kb in size featuring not more than 36 HKT2.4-specific single nucleotide polymorphisms, only two of which changed the coding potential of genes. Among them, we identified the causative single nucleotide polymorphism changing a splicing site in ARMADILLO REPEAT-CONTAINING KINESIN1. The applied strategies have the potential to complement statistical methods in high-throughput phenotyping studies using different natural accessions to identify causative genes for distinct phenotypes represented by only one or a few accessions.

Non-cell-autonomous regulation of root hair patterning genes by WRKY75 in Arabidopsis.

In Arabidopsis (Arabidopsis thaliana), root hairs are formed in cell files over the cleft of underlying cortex cells. This pattern is established by a well-known gene regulatory network of transcription factors. In this study, we show that WRKY75 suppresses root hair development in nonroot hair files and that it represses the expression of TRIPTYCHON and CAPRICE. The WRKY75 protein binds to the CAPRICE promoter in a yeast one-hybrid assay. Binding to the promoter fragment requires an intact WRKY protein-binding motif, the W box. A comparison of the spatial expression of WRKY75 and the localization of the WRKY75 protein revealed that WRKY75 is expressed in the pericycle and vascular tissue and that the WRKY75 RNA or protein moves into the epidermis.

Analysis of TTG1 function in Arabis alpina.

BACKGROUND: In Arabidopsis thaliana (A. thaliana) the WD40 protein TRANSPARENT TESTA GLABRA1 (TTG1) controls five traits relevant for the adaptation of plants to environmental changes including the production of proanthocyanidin, anthocyanidin, seed coat mucilage, trichomes and root hairs. The analysis of different Brassicaceae species suggests that the function of TTG1 is conserved within the family. RESULTS: In this work, we studied the function of TTG1 in Arabis alpina (A. alpina). A comparison of wild type and two Aattg1 alleles revealed that AaTTG1 is involved in the regulation of all five traits. A detailed analysis of the five traits showed striking phenotypic differences between A. alpina and A. thaliana such that trichome formation occurs also at later stages of leaf development and that root hairs form at non-root hair positions. CONCLUSIONS: The evolutionary conservation of the regulation of the five traits by TTG1 on the one hand and the striking phenotypic differences make A. alpina a very interesting genetic model system to study the evolution of TTG1-dependent gene regulatory networks at a functional level.

MIDGET cooperates with COP1 and SPA1 to repress flowering in Arabidopsis thaliana.

The life cycle of plants is strictly regulated by light, which directly influences the initiation of developmental programs such as photomorphogenesis of seedlings and induction of flowering. When environmental conditions are unsuitable, both processes are actively repressed by the action of COP1/SPA protein complexes which participate in ubiquitylation and subsequent degradation of transcription factors. We have shown recently that MIDGET (MID), a regulator of the TOPOISOMERASE VI complex, physically interacts with COP1 and is required for its function as suppressor of photomorphogenesis. Here we show that in Arabidopsis thaliana, the MID protein similarly plays a role in COP1/SPA1-controlled repression of flowering under short-day conditions.

Semi-automated 3D leaf reconstruction and analysis of trichome patterning from light microscopic images.

Trichomes are leaf hairs that are formed by single cells on the leaf surface. They are known to be involved in pathogen resistance. Their patterning is considered to emerge from a field of initially equivalent cells through the action of a gene regulatory network involving trichome fate promoting and inhibiting factors. For a quantitative analysis of single and double mutants or the phenotypic variation of patterns in different ecotypes, it is imperative to statistically evaluate the pattern reliably on a large number of leaves. Here we present a method that enables the analysis of trichome patterns at early developmental leaf stages and the automatic analysis of various spatial parameters. We focus on the most challenging young leaf stages that require the analysis in three dimensions, as the leaves are typically not flat. Our software TrichEratops reconstructs 3D surface models from 2D stacks of conventional light-microscope pictures. It allows the GUI-based annotation of different stages of trichome development, which can be analyzed with respect to their spatial distribution to capture trichome patterning events. We show that 3D modeling removes biases of simpler 2D models and that novel trichome patterning features increase the sensitivity for inter-accession comparisons.

MIDGET connects COP1-dependent development with endoreduplication in Arabidopsis thaliana.

In Arabidopsis thaliana, loss of CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) function leads to constitutive photomorphogenesis in the dark associated with inhibition of endoreduplication in the hypocotyl, and a post-germination growth arrest. MIDGET (MID), a component of the TOPOISOMERASE VI (TOPOVI) complex, is essential for endoreduplication and genome integrity in A. thaliana. Here we show that MID and COP1 interact in vitro and in vivo through the amino terminus of COP1. We further demonstrate that MID supports sub-nuclear accumulation of COP1. The MID protein is not degraded in a COP1-dependent fashion in darkness, and the phenotypes of single and double mutants prove that MID is not a target of COP1 but rather a necessary factor for proper COP1 activity with respect to both, control of COP1-dependent morphogenesis and regulation of endoreduplication. Our data provide evidence for a functional connection between COP1 and the TOPOVI in plants linking COP1-dependent development with the regulation of endoreduplication.

Light and the E3 ubiquitin ligase COP1/SPA control the protein stability of the MYB transcription factors PAP1 and PAP2 involved in anthocyanin accumulation in Arabidopsis.

Anthocyanins are natural pigments that accumulate only in light-grown and not in dark-grown Arabidopsis plants. Repression of anthocyanin accumulation in darkness requires the CONSTITUTIVELY PHOTOMORPHOGENIC1/SUPPRESSOR OF PHYA-105 (COP1/SPA) ubiquitin ligase, as cop1 and spa mutants produce anthocyanins also in the dark. Here, we show that COP1 and SPA proteins interact with the myeloblastosis (MYB) transcription factors PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP)1 and PAP2, two members of a small protein family that is required for anthocyanin accumulation and for the expression of structural genes in the anthocyanin biosynthesis pathway. The increased anthocyanin levels in cop1 mutants requires the PAP1 gene family, indicating that COP1 functions upstream of the PAP1 gene family. PAP1 and PAP2 proteins are degraded in the dark and this degradation is dependent on the proteasome and on COP1. Hence, the light requirement for anthocyanin biosynthesis results, at least in part, from the light-mediated stabilization of PAP1 and PAP2. Consistent with this conclusion, moderate overexpression of PAP1 leads to an increase in anthocyanin levels only in the light and not in darkness. Here we show that SPA genes are also required for reducing PAP1 and PAP2 transcript levels in dark-grown seedlings. Taken together, these results indicate that the COP1/SPA complex affects PAP1 and PAP2 both transcriptionally and post-translationally. Thus, our findings have identified mechanisms via which the COP1/SPA complex controls anthocyanin levels in Arabidopsis that may be useful for applications in biotechnology directed towards increasing anthocyanin content in plants.

MIDGET unravels functions of the Arabidopsis topoisomerase VI complex in DNA endoreduplication, chromatin condensation, and transcriptional silencing.

The plant homologs of the archaeal DNA topoisomerase VI complex are required for the progression of endoreduplication cycles. Here, we describe the identification of MIDGET (MID) as a novel component of topoisomerase VI. We show that mid mutants show the same phenotype as rhl1, rhl2, and top6B mutants and that MID protein physically interacts with RHL1. The phenotypic analysis revealed new phenotypes, indicating that topoisomerase VI is involved in chromatin organization and transcriptional silencing. In addition, genetic evidence is provided suggesting that the ATR-dependent DNA damage repair checkpoint is activated in mid mutants, and CYCB1;1 is ectopically activated. Finally, we demonstrate that overexpression of CYCB1;2 can rescue the endoreduplication defects in mid mutants, suggesting that in mid mutants, a specific checkpoint is activated preventing further progression of endoreduplication cycles.