Stefanie De Bodt

The gain and loss of genes during 600 million years of vertebrate evolution

BackgroundGene duplication is assumed to have played a crucial role in the evolution of vertebrate organisms. Apart from a continuous mode of duplication, two or three whole genome duplication events have been proposed during the evolution of vertebrates, one or two at the dawn of vertebrate evolution, and an additional one in the fish lineage, not shared with land vertebrates. Here, we have studied gene gain and loss in seven different vertebrate genomes, spanning an evolutionary period of about 600 million years.ResultsWe show that: first, the majority of duplicated genes in extant vertebrate genomes are ancient and were created at times that coincide with proposed whole genome duplication events; second, there exist significant differences in gene retention for different functional categories of genes between fishes and land vertebrates; third, there seems to be a considerable bias in gene retention of regulatory genes towards the mode of gene duplication (whole genome duplication events compared to smaller-scale events), which is in accordance with the so-called gene balance hypothesis; and fourth, that ancient duplicates that have survived for many hundreds of millions of years can still be lost.ConclusionBased on phylogenetic analyses, we show that both the mode of duplication and the functional class the duplicated genes belong to have been of major importance for the evolution of the vertebrates. In particular, we provide evidence that massive gene duplication (probably as a consequence of entire genome duplications) at the dawn of vertebrate evolution might have been particularly important for the evolution of complex vertebrates.

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.

Exit from proliferation during leaf development in **Arabidopsis thaliana** : a not-so-gradual process

Early leaf growth is sustained by cell proliferation and subsequent cell expansion that initiates at the leaf tip and proceeds in a basipetal direction. Using detailed kinematic and gene expression studies to map these stages during early development of the third leaf of Arabidopsis thaliana, we showed that the cell-cycle arrest front did not progress gradually down the leaf, but rather was established and abolished abruptly. Interestingly, leaf greening and stomatal patterning followed a similar basipetal pattern, but proliferative pavement cell and formative meristemoid divisions were uncoordinated in respect to onset and persistence. Genes differentially expressed during the transition from cell proliferation to expansion were enriched in genes involved in cell cycle, photosynthesis, and chloroplast retrograde signaling. Proliferating primordia treated with norflurazon, a chemical inhibitor of retrograde signaling, showed inhibited onset of cell expansion. Hence, differentiation of the photosynthetic machinery is important for regulating the exit from proliferation.

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:β-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.

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

This research assesses how plant leaf growth is regulated under water-limiting conditions at the cellular and molecular level. It demonstrates that growth and, more specifically, cell division responds to stress in a highly dynamic manner. Growth inhibition is mediated by ethylene signaling followed by adaptation and recovery. 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.

Nonrandom divergence of gene expression following gene and genome duplications in the flowering plant Arabidopsis thaliana

BackgroundGenome analyses have revealed that gene duplication in plants is rampant. Furthermore, many of the duplicated genes seem to have been created through ancient genome-wide duplication events. Recently, we have shown that gene loss is strikingly different for large- and small-scale duplication events and highly biased towards the functional class to which a gene belongs. Here, we study the expression divergence of genes that were created during large- and small-scale gene duplication events by means of microarray data and investigate both the influence of the origin (mode of duplication) and the function of the duplicated genes on expression divergence.ResultsDuplicates that have been created by large-scale duplication events and that can still be found in duplicated segments have expression patterns that are more correlated than those that were created by small-scale duplications or those that no longer lie in duplicated segments. Moreover, the former tend to have highly redundant or overlapping expression patterns and are mostly expressed in the same tissues, while the latter show asymmetric divergence. In addition, a strong bias in divergence of gene expression was observed towards gene function and the biological process genes are involved in.ConclusionBy using microarray expression data for Arabidopsis thaliana, we show that the mode of duplication, the function of the genes involved, and the time since duplication play important roles in the divergence of gene expression and, therefore, in the functional divergence of genes after duplication.

ETHYLENE RESPONSE FACTOR6 acts as a central regulator of leaf growth under water-limiting conditions in Arabidopsis

ETHYLENE RESPONSE FACTOR6 is a central regulator of both leaf growth inhibition and stress tolerance under osmotic stress conditions. 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.

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.

Genomewide Structural Annotation and Evolutionary Analysis of the Type I MADS-Box Genes in Plants

The type I MADS-box genes constitute a largely unexplored subfamily of the extensively studied MADS-box gene family, well known for its role in flower development. Genes of the type I MADS-box subfamily possess the characteristic MADS box but are distinguished from type II MADS-box genes by the absence of the keratin-like box. In this in silico study, we have structurally annotated all 47 members of the type I MADS-box gene family in Arabidopsis thaliana and exerted a thorough analysis of the C-terminal regions of the translated proteins. On the basis of conserved motifs in the C-terminal region, we could classify the gene family into three main groups, two of which could be further subdivided. Phylogenetic trees were inferred to study the evolutionary relationships within this large MADS-box gene subfamily. These suggest for plant type I genes a dynamic of evolution that is significantly different from the mode of both animal type I (SRF) and plant type II (MIKC-type) gene phylogeny. The presence of conserved motifs in the majority of these genes, the identification of Oryza sativa MADS-box type I homologues, and the detection of expressed sequence tags for Arabidopsis thaliana and other plant type I genes suggest that these genes are indeed of functional importance to plants. It is therefore even more intriguing that, from an experimental point of view, almost nothing is known about the function of these MADS-box type I genes.