Frederik Coppens

Gibberellin Signaling Controls Cell Proliferation Rate in Arabidopsis

Plant growth involves the integration of many environmental and endogenous signals that together with the intrinsic genetic program determine plant size. At the cellular level, growth rate is regulated by the combined activity of two processes: cell proliferation and expansion. Gibberellins (GA) are plant-specific hormones that play a central role in the regulation of growth and development with respect to environmental variability. It is well established that GA promote growth through cell expansion by stimulating the destruction of growth-repressing DELLA proteins (DELLAs); however, their effects on cell proliferation remain unknown. Kinematic analysis of leaf and root meristem growth revealed a novel function of DELLAs in restraining cell production. Moreover, by visualizing the cell cycle marker cyclinB1::beta-glucuronidase in GA-signaling mutants, we show that GA modulate cell cycle activity in the root meristem via a DELLA-dependent mechanism. Accordingly, expressing gai (a nondegradable DELLA protein) solely in root meristem reduced substantially the number of dividing cells. We also show that DELLAs restrain cell production by enhancing the levels of the cell cycle inhibitors Kip-related protein 2 (KRP2) and SIAMESE (SIM). Therefore, DELLAs exert a general plant growth inhibitory activity by reducing both cell proliferation and expansion rates, enabling phenotypic plasticity.

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.

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.

ANGUSTIFOLIA3 Binds to SWI/SNF Chromatin Remodeling Complexes to Regulate Transcription during Arabidopsis Leaf Development

The transcriptional coactivator ANGUSTIFOLIA3 (AN3) stimulates cell division during Arabidopsis leaf development. It is shown that AN3 associates with SWI/SNF chromatin remodeling complexes to regulate the expression of important downstream transcription factors and that the module SWI/SNF-AN3 is a major player in the transition from cell division to cell expansion in developing leaves. 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.

Systems analysis of the responses to long-term magnesium deficiency and restoration in Arabidopsis thaliana

*Unravelling mechanisms that control plant growth as a function of nutrient availability presents a major challenge in plant biology. This study reports the first transcriptome response to long-term (1 wk) magnesium (Mg) depletion and restoration in Arabidopsis thaliana. *Before the outbreak of visual symptoms, genes responding to Mg starvation and restoration were monitored in the roots and young mature leaves and compared with the Mg fully supplied as control. *After 1 wk Mg starvation in roots and leaves, 114 and 2991 genes were identified to be differentially regulated, respectively, which confirmed the later observation that the shoot development was more affected than the root in Arabidopsis. After 24 h of Mg resupply, restoration was effective for the expression of half of the genes altered. We emphasized differences in the expression amplitude of genes associated with the circadian clock predominantly in leaves, a higher expression of genes in the ethylene biosynthetic pathway, in the reactive oxygen species detoxification and in the photoprotection of the photosynthetic apparatus. Some of these observations at the molecular level were verified by metabolite analysis. *The results obtained here will help us to better understand how changes in Mg availability are translated into adaptive responses in the plant.

Early transcriptomic changes induced by magnesium deficiency in Arabidopsis thaliana reveal the alteration of circadian clock gene expression in roots and the triggering of abscisic acid‐responsive genes

*Plant growth and development ultimately depend on environmental variables such as the availability of essential minerals. Unravelling how nutrients affect gene expression will help to understand how they regulate plant growth. *This study reports the early transcriptomic response to magnesium (Mg) deprivation in Arabidopsis. Whole-genome transcriptome was studied in the roots and young mature leaves 4, 8 and 28 h after the removal of Mg from the nutrient solution. *The highest number of regulated genes was first observed in the roots. Contrary to other mineral deficiencies, Mg depletion did not induce a higher expression of annotated genes in Mg uptake. Remarkable responses include the perturbation of the central oscillator of the circadian clock in roots and the triggering of abscisic acid (ABA) signalling, with half of the up-regulated Mg genes in leaves being ABA-responsive. However, no change in ABA content was observed. *The specificity of the response of some Mg-regulated genes was challenged by studying their expression after other mineral deficiencies and environmental stresses. The possibility to develop markers for Mg incipient deficiency is discussed here.

Brassinosteroid production and signaling differentially control cell division and expansion in the leaf

Brassinosteroid (BR) hormones control plant growth through acting on both cell expansion and division. Here, we examined the role of BRs in leaf growth using the Arabidopsis BR-deficient mutant constitutive photomorphogenesis and dwarfism (cpd). We show that the reduced size of cpd leaf blades is a result of a decrease in cell size and number, as well as in venation length and complexity. Kinematic growth analysis and tissue-specific marker gene expression revealed that the leaf phenotype of cpd is associated with a prolonged cell division phase and delayed differentiation. cpd-leaf-rescue experiments and leaf growth analysis of BR biosynthesis and signaling gain-of-function mutants showed that BR production and BR receptor-dependent signaling differentially control the balance between cell division and expansion in the leaf. Investigation of cell cycle markers in leaves of cpd revealed the accumulation of mitotic proteins independent of transcription. This correlated with an increase in cyclin-dependent kinase activity, suggesting a role for BRs in control of mitosis.

Developmental regulation of CYCA2s contributes to tissue‐specific proliferation in Arabidopsis

In multicellular organisms, morphogenesis relies on a strict coordination in time and space of cell proliferation and differentiation. In contrast to animals, plant development displays continuous organ formation and adaptive growth responses during their lifespan relying on a tight coordination of cell proliferation. How developmental signals interact with the plant cell‐cycle machinery is largely unknown. Here, we characterize plant A2‐type cyclins, a small gene family of mitotic cyclins, and show how they contribute to the fine‐tuning of local proliferation during plant development. Moreover, the timely repression of CYCA2;3 expression in newly formed guard cells is shown to require the stomatal transcription factors FOUR LIPS/MYB124 and MYB88, providing a direct link between developmental programming and cell‐cycle exit in plants. Thus, transcriptional downregulation of CYCA2s represents a critical mechanism to coordinate proliferation during plant development.

Leaf Responses to Mild Drought Stress in Natural Variants of Arabidopsis

Arabidopsis accessions show different phenotypes in response to mild drought, yet a robust transcriptome response is conserved between the accessions. Although the response of plants exposed to severe drought stress has been studied extensively, little is known about how plants adapt their growth under mild drought stress conditions. Here, we analyzed the leaf and rosette growth response of six Arabidopsis (Arabidopsis thaliana) accessions originating from different geographic regions when exposed to mild drought stress. The automated phenotyping platform WIWAM was used to impose stress early during leaf development, when the third leaf emerges from the shoot apical meristem. Analysis of growth-related phenotypes showed differences in leaf development between the accessions. In all six accessions, mild drought stress reduced both leaf pavement cell area and number without affecting the stomatal index. Genome-wide transcriptome analysis (using RNA sequencing) of early developing leaf tissue identified 354 genes differentially expressed under mild drought stress in the six accessions. Our results indicate the existence of a robust response over different genetic backgrounds to mild drought stress in developing leaves. The processes involved in the overall mild drought stress response comprised abscisic acid signaling, proline metabolism, and cell wall adjustments. In addition to these known severe drought-related responses, 87 genes were found to be specific for the response of young developing leaves to mild drought stress.

SHORT-ROOT and SCARECROW regulate leaf growth in Arabidopsis by stimulating S-phase progression of the cell cycle

SHORT-ROOT (SHR) and SCARECROW (SCR) are required for stem cell maintenance in the Arabidopsis (Arabidopsis thaliana) root meristem, ensuring its indeterminate growth. Mutation of SHR and SCR genes results in disorganization of the quiescent center and loss of stem cell activity, resulting in the cessation of root growth. This paper reports on the role of SHR and SCR in the development of leaves, which, in contrast to the root, have a determinate growth pattern and lack a persistent stem cell niche. Our results demonstrate that inhibition of leaf growth in shr and scr mutants is not a secondary effect of the compromised root development but is caused by an effect on cell division in the leaves: a reduced cell division rate and early exit of the proliferation phase. Consistent with the observed cell division phenotype, the expression of SHR and SCR genes in leaves is closely associated with cell division activity in most cell types. The increased cell cycle duration is due to a prolonged S-phase duration, which is mediated by up-regulation of cell cycle inhibitors known to restrain the activity of the transcription factor, E2Fa. Therefore, we conclude that, in contrast to their specific roles in cortex/endodermis differentiation and stem cell maintenance in the root, SHR and SCR primarily function as general regulators of cell proliferation in leaves.