ANGUSTIFOLIA3 binds to SWI/SNF chromatin remodeling complexes to regulate transcription during Arabidopsis leaf development.

The transcriptional coactivator ANGUSTIFOLIA3 (AN3) stimulates cell proliferation during Arabidopsis thaliana leaf development, but the molecular mechanism is largely unknown. Here, we show that inducible nuclear localization of AN3 during initial leaf growth results in differential expression of important transcriptional regulators, including GROWTH REGULATING FACTORs (GRFs). Chromatin purification further revealed the presence of AN3 at the loci of GRF5, GRF6, CYTOKININ RESPONSE FACTOR2, CONSTANS-LIKE5 (COL5), HECATE1 (HEC1), and ARABIDOPSIS RESPONSE REGULATOR4 (ARR4). Tandem affinity purification of protein complexes using AN3 as bait identified plant SWITCH/SUCROSE NONFERMENTING (SWI/SNF) chromatin remodeling complexes formed around the ATPases BRAHMA (BRM) or SPLAYED. Moreover, SWI/SNF ASSOCIATED PROTEIN 73B (SWP73B) is recruited by AN3 to the promoters of GRF5, GRF3, COL5, and ARR4, and both SWP73B and BRM occupy the HEC1 promoter. Furthermore, we show that AN3 and BRM genetically interact. The data indicate that AN3 associates with chromatin remodelers to regulate transcription. In addition, modification of SWI3C expression levels increases leaf size, underlining the importance of chromatin dynamics for growth regulation. Our results place the SWI/SNF-AN3 module as a major player at the transition from cell proliferation to cell differentiation in a developing leaf.

The potential of text mining in data integration and network biology for plant research: a case study on Arabidopsis.

Despite the availability of various data repositories for plant research, a wealth of information currently remains hidden within the biomolecular literature. Text mining provides the necessary means to retrieve these data through automated processing of texts. However, only recently has advanced text mining methodology been implemented with sufficient computational power to process texts at a large scale. In this study, we assess the potential of large-scale text mining for plant biology research in general and for network biology in particular using a state-of-the-art text mining system applied to all PubMed abstracts and PubMed Central full texts. We present extensive evaluation of the textual data for Arabidopsis thaliana, assessing the overall accuracy of this new resource for usage in plant network analyses. Furthermore, we combine text mining information with both protein-protein and regulatory interactions from experimental databases. Clusters of tightly connected genes are delineated from the resulting network, illustrating how such an integrative approach is essential to grasp the current knowledge available for Arabidopsis and to uncover gene information through guilt by association. All large-scale data sets, as well as the manually curated textual data, are made publicly available, hereby stimulating the application of text mining data in future plant biology studies.

Type 1 diacylglycerol acyltransferases of Brassica napus preferentially incorporate oleic acid into triacylglycerol.

DGAT1 enzymes (acyl-CoA:diacylglycerol acyltransferase 1, EC 2.3.1.20) catalyse the formation of triacylglycerols (TAGs), the most abundant lipids in vegetable oils. Thorough understanding of the enzymology of oil accumulation is critical to the goal of modifying oilseeds for improved vegetable oil production. Four isoforms of BnDGAT1, the final and rate-limiting step in triacylglycerol synthesis, were characterized from Brassica napus, one of the world's most important oilseed crops. Transcriptional profiling of developing B. napus seeds indicated two genes, BnDGAT1-1 and BnDGAT1-2, with high expression and two, BnDGAT1-3 and BnDGAT1-4, with low expression. The activities of each BnDGAT1 isozyme were characterized following expression in a strain of yeast deficient in TAG synthesis. TAG from B. napus seeds contain only 10% palmitic acid (16:0) at the sn-3 position, so it was surprising that all four BnDGAT1 isozymes exhibited strong (4- to 7-fold) specificity for 16:0 over oleic acid (18:1) as the acyl-CoA substrate. However, the ratio of 18:1-CoA to 16:0-CoA in B. napus seeds during the peak period of TAG synthesis is 3:1. When substrate selectivity assays were conducted with 18:1-CoA and 16:0-CoA in a 3:1 ratio, the four isozymes incorporated 18:1 in amounts 2- to 5-fold higher than 16:0. This strong sensitivity of the BnDGAT1 isozymes to the relative concentrations of acyl-CoA substrates substantially explains the observed fatty acid composition of B. napus seed oil. Understanding these enzymes that are critical for triacylglycerol synthesis will facilitate genetic and biotechnological manipulations to improve this oilseed crop.

Transcriptional coordination between leaf cell differentiation and chloroplast development established by TCP20 and the subgroup Ib bHLH transcription factors.

The establishment of the photosynthetic apparatus during chloroplast development creates a high demand for iron as a redox metal. However, iron in too high quantities becomes toxic to the plant, thus plants have evolved a complex network of iron uptake and regulation mechanisms. Here, we examined whether four of the subgroup Ib basic helix-loop-helix transcription factors (bHLH38, bHLH39, bHLH100, bHLH101), previously implicated in iron homeostasis in roots, also play a role in regulating iron metabolism in developing leaves. These transcription factor genes were strongly up-regulated during the transition from cell proliferation to expansion, and thus sink-source transition, in young developing leaves of Arabidopsis thaliana. The four subgroup Ib bHLH genes also showed reduced expression levels in developing leaves of plants treated with norflurazon, indicating their expression was tightly linked to the onset of photosynthetic activity in young leaves. In addition, we provide evidence for a mechanism whereby the transcriptional regulators SAC51 and TCP20 antagonistically regulate the expression of these four subgroup Ib bHLH genes. A loss-of-function mutant analysis also revealed that single mutants of bHLH38, bHLH39, bHLH100, and bHLH101 developed smaller rosettes than wild-type plants in soil. When grown in agar plates with reduced iron concentration, triple bhlh39 bhlh100 bhlh101 mutant plants were smaller than wild-type plants. However, measurements of the iron content in single and multiple subgroup Ib bHLH genes, as well as transcript profiling of iron response genes during early leaf development, do not support a role for bHLH38, bHLH39, bHLH100, and bHLH101 in iron homeostasis during early leaf development.

Ethylene Response Factor6 acts as a central regulator of leaf growth under water-limiting conditions in Arabidopsis.

Leaf growth is a complex developmental process that is continuously fine-tuned by the environment. Various abiotic stresses, including mild drought stress, have been shown to inhibit leaf growth in Arabidopsis (Arabidopsis thaliana), but the underlying mechanisms remain largely unknown. Here, we identify the redundant Arabidopsis transcription factors ETHYLENE RESPONSE FACTOR5 (ERF5) and ERF6 as master regulators that adapt leaf growth to environmental changes. ERF5 and ERF6 gene expression is induced very rapidly and specifically in actively growing leaves after sudden exposure to osmotic stress that mimics mild drought. Subsequently, enhanced ERF6 expression inhibits cell proliferation and leaf growth by a process involving gibberellin and DELLA signaling. Using an ERF6-inducible overexpression line, we demonstrate that the gibberellin-degrading enzyme GIBBERELLIN 2-OXIDASE6 is transcriptionally induced by ERF6 and that, consequently, DELLA proteins are stabilized. As a result, ERF6 gain-of-function lines are dwarfed and hypersensitive to osmotic stress, while the growth of erf5erf6 loss-of-function mutants is less affected by stress. Besides its role in plant growth under stress, ERF6 also activates the expression of a plethora of osmotic stress-responsive genes, including the well-known stress tolerance genes STZ, MYB51, and WRKY33. Interestingly, activation of the stress tolerance genes by ERF6 occurs independently from the ERF6-mediated growth inhibition. Together, these data fit into a leaf growth regulatory model in which ERF5 and ERF6 form a missing link between the previously observed stress-induced 1-aminocyclopropane-1-carboxylic acid accumulation and DELLA-mediated cell cycle exit and execute a dual role by regulating both stress tolerance and growth inhibition.

Pause-and-stop: the effects of osmotic stress on cell proliferation during early leaf development in Arabidopsis and a role for ethylene signaling in cell cycle arrest.

Despite its relevance for agricultural production, environmental stress-induced growth inhibition, which is responsible for significant yield reductions, is only poorly understood. Here, we investigated the molecular mechanisms underlying cell cycle inhibition in young proliferating leaves of the model plant Arabidopsis thaliana when subjected to mild osmotic stress. A detailed cellular analysis demonstrated that as soon as osmotic stress is sensed, cell cycle progression rapidly arrests, but cells are kept in a latent ambivalent state allowing a quick recovery (pause). Remarkably, cell cycle arrest coincides with an increase in 1-aminocyclopropane-1-carboxylate levels and the activation of ethylene signaling. Our work showed that ethylene acts on cell cycle progression via inhibition of cyclin-dependent kinase A activity independently of EIN3 transcriptional control. When the stress persists, cells exit the mitotic cell cycle and initiate the differentiation process (stop). This stop is reflected by early endoreduplication onset, in a process independent of ethylene. Nonetheless, the potential to partially recover the decreased cell numbers remains due to the activity of meristemoids. Together, these data present a conceptual framework to understand how environmental stress reduces plant growth.

CORNET 2.0: integrating plant coexpression, protein-protein interactions, regulatory interactions, gene associations and functional annotations.

To enable easy access and interpretation of heterogeneous and scattered data, we have developed a user-friendly tool for data mining and integration in Arabidopsis, named CORNET. This tool allows the browsing of microarray data, the construction of coexpression and protein-protein interaction (PPI) networks and the exploration of diverse functional annotations. Here, we present the new functionalities of CORNET 2.0 for data integration in plants. First of all, CORNET allows the integration of regulatory interaction datasets accessible through the new transcription factor (TF) tool that can be used in combination with the coexpression tool or the PPI tool. In addition, we have extended the PPI tool to enable the analysis of gene-gene associations from AraNet as well as newly identified PPIs. Different search options are implemented to enable the construction of networks centered around multiple input genes or proteins. New functional annotation resources are included to retrieve relevant literature, phenotypes, plant ontology and biological pathways. We have also extended CORNET to attain the construction of coexpression and PPI networks in the crop species maize. Networks and associated evidence of the majority of currently available data types are visualized in Cytoscape. CORNET is available at https://bioinformatics.psb.ugent.be/cornet.

A reciprocal 15N-labeling proteomic analysis of expanding Arabidopsis leaves subjected to osmotic stress indicates importance of mitochondria in preserving plastid functions.

Plants respond to environmental stress by dynamically reprogramming their growth. Whereas stress onset is accompanied by rapid growth inhibition leading to smaller organs, growth will recover and adapt once the stress conditions become stable and do no threaten plant survival. Here, adaptation of growing Arabidopsis thaliana leaves to mild and prolonged osmotic stress was investigated by means of a complete metabolic labeling strategy with the (15)N-stable isotope as a complement to a previously published transcript and metabolite profiling. Global analysis of protein changes revealed that plastidial ATPase, Calvin cycle, and photorespiration were down-regulated, but mitochondrial ATP synthesis was up-regulated, indicating the importance of mitochondria in preserving plastid functions during water stress. Although transcript and protein data correlated well with the stable and prolonged character of the applied stress, numerous proteins were clearly regulated at the post-transcriptional level that could, at least partly, be related to changes in protein synthesis and degradation. In conclusion, proteomics using the (15)N labeling helped understand the mechanisms underlying growth adaptation to osmotic stress and allowed the identification of candidate genes to improve plant growth under limited water.

Targeted interactomics reveals a complex core cell cycle machinery in Arabidopsis thaliana.

Cell proliferation is the main driving force for plant growth. Although genome sequence analysis revealed a high number of cell cycle genes in plants, little is known about the molecular complexes steering cell division. In a targeted proteomics approach, we mapped the core complex machinery at the heart of the Arabidopsis thaliana cell cycle control. Besides a central regulatory network of core complexes, we distinguished a peripheral network that links the core machinery to up- and downstream pathways. Over 100 new candidate cell cycle proteins were predicted and an in-depth biological interpretation demonstrated the hypothesis-generating power of the interaction data. The data set provided a comprehensive view on heterodimeric cyclin-dependent kinase (CDK)-cyclin complexes in plants. For the first time, inhibitory proteins of plant-specific B-type CDKs were discovered and the anaphase-promoting complex was characterized and extended. Important conclusions were that mitotic A- and B-type cyclins form complexes with the plant-specific B-type CDKs and not with CDKA;1, and that D-type cyclins and S-phase-specific A-type cyclins seem to be associated exclusively with CDKA;1. Furthermore, we could show that plants have evolved a combinatorial toolkit consisting of at least 92 different CDK-cyclin complex variants, which strongly underscores the functional diversification among the large family of cyclins and reflects the pivotal role of cell cycle regulation in the developmental plasticity of plants.

Transcriptional and post-transcriptional regulation of a NAC1 transcription factor in Medicago truncatula roots.

• Legume roots develop two types of lateral organs, lateral roots and nodules. Nodules develop as a result of a symbiotic interaction with rhizobia and provide a niche for the bacteria to fix atmospheric nitrogen for the plant. • The Arabidopsis NAC1 transcription factor is involved in lateral root formation, and is regulated post-transcriptionally by miRNA164 and by SINAT5-dependent ubiquitination. We analyzed in Medicago truncatula the role of the closest NAC1 homolog in lateral root formation and in nodulation. • MtNAC1 shows a different expression pattern in response to auxin than its Arabidopsis homolog and no changes in lateral root number or nodulation were observed in plants affected in MtNAC1 expression. In addition, no interaction was found with SINA E3 ligases, suggesting that post-translational regulation of MtNAC1 does not occur in M. truncatula. Similar to what was found in Arabidopsis, a conserved miR164 target site was retrieved in MtNAC1, which reduced protein accumulation of a GFP-miR164 sensor. Furthermore, miR164 and MtNAC1 show an overlapping expression pattern in symbiotic nodules, and overexpression of this miRNA led to a reduction in nodule number. • This work suggests that regulatory pathways controlling a conserved transcription factor are complex and divergent between M. truncatula and Arabidopsis.

CORNET: a user-friendly tool for data mining and integration.

As an overwhelming amount of functional genomics data have been generated, the retrieval, integration, and interpretation of these data need to be facilitated to enable the advance of (systems) biological research. For example, gathering and processing microarray data that are related to a particular biological process is not straightforward, nor is the compilation of protein-protein interactions from numerous partially overlapping databases identified through diverse approaches. However, these tasks are inevitable to address the following questions. Does a group of differentially expressed genes show similar expression in diverse microarray experiments? Was an identified protein-protein interaction previously detected by other approaches? Are the interacting proteins encoded by genes with similar expression profiles and localization? We developed CORNET (for CORrelation NETworks) as an access point to transcriptome, protein interactome, and localization data and functional information on Arabidopsis (Arabidopsis thaliana). It consists of two flexible and versatile tools, namely the coexpression tool and the protein-protein interaction tool. The ability to browse and search microarray experiments using ontology terms and the incorporation of personal microarray data are distinctive features of the microarray repository. The coexpression tool enables either the alternate or simultaneous use of diverse expression compendia, whereas the protein-protein interaction tool searches experimentally and computationally identified protein-protein interactions. Different search options are implemented to enable the construction of coexpression and/or protein-protein interaction networks centered around multiple input genes or proteins. Moreover, networks and associated evidence are visualized in Cytoscape. Localization is visualized in pie charts, thereby allowing multiple localizations per protein. CORNET is available at http://bioinformatics.psb.ugent.be/cornet.

Developmental stage specificity and the role of mitochondrial metabolism in the response of Arabidopsis leaves to prolonged mild osmotic stress.

When subjected to stress, plants reprogram their growth by largely unknown mechanisms. To provide insights into this process, the growth of Arabidopsis (Arabidopsis thaliana) leaves that develop under mild osmotic stress was studied. Early during leaf development, cell number and size were reduced by stress, but growth was remarkably adaptable, as division and expansion rates were identical to controls within a few days of leaf initiation. To investigate the molecular basis of the observed adaptability, leaves with only proliferating, exclusively expanding, and mature cells were analyzed by transcriptomics and targeted metabolomics. The stress response measured in growing and mature leaves was largely distinct; several hundred transcripts and multiple metabolites responded exclusively in the proliferating and/or expanding leaves. Only a few genes were differentially expressed across the three stages. Data analysis showed that proliferation and expansion were regulated by common regulatory circuits, involving ethylene and gibberellins but not abscisic acid. The role of ethylene was supported by the analysis of ethylene-insensitive mutants. Exclusively in proliferating cells, stress induced genes of the so-called "mitochondrial dysfunction regulon," comprising alternative oxidase. Up-regulation for eight of these genes was confirmed with promoter:beta-glucuronidase reporter lines. Furthermore, mitochondria of stress-treated dividing cells were morphologically distinct from control ones, and growth of plants overexpressing the alternative oxidase gene was more tolerant to osmotic and drought stresses. Taken together, our data underline the value of analyzing stress responses in development and demonstrate the importance of mitochondrial respiration for sustaining cell proliferation under osmotic stress conditions.

Increased leaf size: different means to an end.

The final size of plant organs, such as leaves, is tightly controlled by environmental and genetic factors that must spatially and temporally coordinate cell expansion and cell cycle activity. However, this regulation of organ growth is still poorly understood. The aim of this study is to gain more insight into the genetic control of leaf size in Arabidopsis (Arabidopsis thaliana) by performing a comparative analysis of transgenic lines that produce enlarged leaves under standardized environmental conditions. To this end, we selected five genes belonging to different functional classes that all positively affect leaf size when overexpressed: AVP1, GRF5, JAW, BRI1, and GA20OX1. We show that the increase in leaf area in these lines depended on leaf position and growth conditions and that all five lines affected leaf size differently; however, in all cases, an increase in cell number was, entirely or predominantly, responsible for the leaf size enlargement. By analyzing hormone levels, transcriptome, and metabolome, we provide deeper insight into the molecular basis of the growth phenotype for the individual lines. A comparative analysis between these data sets indicates that enhanced organ growth is governed by different, seemingly independent pathways. The analysis of transgenic lines simultaneously overexpressing two growth-enhancing genes further supports the concept that multiple pathways independently converge on organ size control in Arabidopsis.

Comparison of developmental and stress-induced nodule senescence in Medicago truncatula.

Mature indeterminate Medicago truncatula nodules are zonated with an apical meristem, an infection zone, a fixation zone with nitrogen-fixing bacteroids, and a "developmental" senescence zone that follows nodule growth with a conical front originating in the center of the fixation zone. In nitrogen-fixing cells, senescence is initiated coincidently with the expression of a family of conserved cysteine proteases that might be involved in the degradation of symbiotic structures. Environmental stress, such as prolonged dark treatment, interferes with nodule functioning and triggers a fast and global nodule senescence. Developmental and dark stress-induced senescence have several different structural and expression features, suggesting at least partly divergent underlying molecular mechanisms.

The da1 mutation in wheat increases grain size under ambient and elevated CO2 but not grain yield due to trade-off between grain size and grain number.

Grain size is potentially yield determining in wheat, controlled by the ubiquitin pathway and negatively regulated by ubiquitin receptor DA1. We analyzed whether increased thousand grain weight in wheat da1 mutant is translated into higher grain yield and whether additional carbon provided by elevated (e)CO2 can be better used by the da1, displaying higher grain sink strength and size. Yield-related, biomass, grain quality traits, and grain dimensions were analyzed by two-factorial mixed-model analysis, regarding genotype and eCO2. da1 increased grain size but reduced spikes and grains per plant, grains per spike, and spikelets per spike, independent of eCO2 treatment, leaving total grain yield unchanged. eCO2 increased yield and grain number additively and independently of da1 but did not overcome the trade-off between grain size and number observed for da1. eCO2 but not da1 impaired grain quality, strongly decreasing concentrations of several macroelement and microelement. In conclusion, intrinsic stimulation of grain sink strength and grain size, achieved by da1, is not benefitting total yield unless trade-offs between grain size and numbers can be overcome. The results reveal interactions of yield components in da1-wheat under ambient and eCO2, thereby uncovering limitations enhancing wheat yield potential.

Predicting protein-protein interactions in Arabidopsis thaliana through integration of orthology, gene ontology and co-expression.

BACKGROUND: Large-scale identification of the interrelationships between different components of the cell, such as the interactions between proteins, has recently gained great interest. However, unraveling large-scale protein-protein interaction maps is laborious and expensive. Moreover, assessing the reliability of the interactions can be cumbersome. RESULTS: In this study, we have developed a computational method that exploits the existing knowledge on protein-protein interactions in diverse species through orthologous relations on the one hand, and functional association data on the other hand to predict and filter protein-protein interactions in Arabidopsis thaliana. A highly reliable set of protein-protein interactions is predicted through this integrative approach making use of existing protein-protein interaction data from yeast, human, C. elegans and D. melanogaster. Localization, biological process, and co-expression data are used as powerful indicators for protein-protein interactions. The functional repertoire of the identified interactome reveals interactions between proteins functioning in well-conserved as well as plant-specific biological processes. We observe that although common mechanisms (e.g. actin polymerization) and components (e.g. ARPs, actin-related proteins) exist between different lineages, they are active in specific processes such as growth, cancer metastasis and trichome development in yeast, human and Arabidopsis, respectively. CONCLUSION: We conclude that the integration of orthology with functional association data is adequate to predict protein-protein interactions. Through this approach, a high number of novel protein-protein interactions with diverse biological roles is discovered. Overall, we have predicted a reliable set of protein-protein interactions suitable for further computational as well as experimental analyses.

And then there were many: MADS goes genomic.

During the past decade, MADS-box genes have become known as key regulators in both reproductive and vegetative plant development. Traditional genetics and functional genomics tools are now available to elucidate the expression and function of this complex gene family on a much larger scale. Moreover, comparative analysis of the MADS-box genes in diverse flowering and non-flowering plants, boosted by bioinformatics, contributes to our understanding of how this important gene family has expanded during the evolution of land plants. Therefore, the recent advances in comparative and functional genomics should enable researchers to identify the full range of MADS-box gene functions, which should help us significantly in developing a better understanding of plant development and evolution.

Gibberellins and DELLAs: central nodes in growth regulatory networks.

Gibberellins (GAs) are growth-promoting phytohormones that were crucial in breeding improved semi-dwarf varieties during the green revolution. However, the molecular basis for GA-induced growth stimulation is poorly understood. In this review, we use light-regulated hypocotyl elongation as a case study, combined with a meta-analysis of available transcriptome data, to discuss the role of GAs as central nodes in networks connecting environmental inputs to growth. These networks are highly tissue-specific, with dynamic and rapid regulation that mostly occurs at the protein level, directly affecting the activity and transcription of effectors. New systems biology approaches addressing the role of GAs in growth should take these properties into account, combining tissue-specific interactomics, transcriptomics and modeling, to provide essential knowledge to fuel a second green revolution.

Transcript abundance on its own cannot be used to infer fluxes in central metabolism.

An attempt has been made to define the extent to which metabolic flux in central plant metabolism is reflected by changes in the transcriptome and metabolome, based on an analysis of in vitro cultured immature embryos of two oilseed rape (Brassica napus) accessions which contrast for seed lipid accumulation. Metabolic flux analysis (MFA) was used to constrain a flux balance metabolic model which included 671 biochemical and transport reactions within the central metabolism. This highly confident flux information was eventually used for comparative analysis of flux vs. transcript (metabolite). Metabolite profiling succeeded in identifying 79 intermediates within the central metabolism, some of which differed quantitatively between the two accessions and displayed a significant shift corresponding to flux. An RNA-Seq based transcriptome analysis revealed a large number of genes which were differentially transcribed in the two accessions, including some enzymes/proteins active in major metabolic pathways. With a few exceptions, differential activity in the major pathways (glycolysis, TCA cycle, amino acid, and fatty acid synthesis) was not reflected in contrasting abundances of the relevant transcripts. The conclusion was that transcript abundance on its own cannot be used to infer metabolic activity/fluxes in central plant metabolism. This limitation needs to be borne in mind in evaluating transcriptome data and designing metabolic engineering experiments.

A guide to CORNET for the construction of coexpression and protein-protein interaction networks.

To enable easy access and interpretation of heterogenous and scattered data, we have developed a user-friendly tool for data mining and integration in Arabidopsis thaliana, designated CORrelation NETworks (acronym CORNET), allowing browsing of microarray data, construction of coexpression and protein-protein interactions (PPIs), analysis of gene association and transcription factor (TF) regulatory networks, and exploration of diverse functional annotations. CORNET consists of three tools that can be used individually or in combination, namely, the coexpression tool, the PPI tool, and the TF tool. Different search options are implemented to enable the creation of networks centered around multiple input genes or proteins. Functional annotation resources are included to retrieve relevant literature, phenotypes, localization, gene ontology, plant ontology, and biological pathways. Networks and associated evidence of the majority of the currently available data types are visualized in Cytoscape. CORNET is available at https://bioinformatics.psb.ugent.be/cornet.