A Spatiotemporal DNA Endoploidy Map of the Arabidopsis Root Reveals Roles for the Endocycle in Root Development and Stress Adaptation.

Somatic polyploidy caused by endoreplication is observed in arthropods, molluscs, and vertebrates but is especially prominent in higher plants, where it has been postulated to be essential for cell growth and fate maintenance. However, a comprehensive understanding of the physiological significance of plant endopolyploidy has remained elusive. Here, we modeled and experimentally verified a high-resolution DNA endoploidy map of the developing Arabidopsis thaliana root, revealing a remarkable spatiotemporal control of DNA endoploidy levels across tissues. Fitting of a simplified model to publicly available data sets profiling root gene expression under various environmental stress conditions suggested that this root endoploidy patterning may be stress-responsive. Furthermore, cellular and transcriptomic analyses revealed that inhibition of endoreplication onset alters the nuclear-to-cellular volume ratio and the expression of cell wall-modifying genes, in correlation with the appearance of cell structural changes. Our data indicate that endopolyploidy might serve to coordinate cell expansion with structural stability and that spatiotemporal endoreplication pattern changes may buffer for stress conditions, which may explain the widespread occurrence of the endocycle in plant species growing in extreme or variable environments.

Recombinant IgA production for mucosal passive immunization, advancing beyond the hurdles.

Vaccination is a successful strategy to proactively develop immunity to a certain pathogen, but most vaccines fail to trigger a specific immune response at the mucosal surfaces, which are the first port of entry for infectious agents. At the mucosal surfaces, the predominant immunoglobulin is secretory IgA (SIgA) that specifically neutralizes viruses and prevents bacterial colonization. Mucosal passive immunization, i.e. the application of pathogen-specific SIgAs at the mucosae, can be an effective alternative to achieve mucosal protection. However, this approach is not straightforward, mainly because SIgAs are difficult to obtain from convalescent sources, while recombinant SIgA production is challenging due to its complex structure. This review provides an overview of manufacturing difficulties presented by the unique structural diversity of SIgAs, and the innovative solutions being explored for SIgA production in mammalian and plant expression systems.

Arabidopsis ULTRAVIOLET-B-INSENSITIVE4 maintains cell division activity by temporal inhibition of the anaphase-promoting complex/cyclosome.

The anaphase-promoting complex/cyclosome (APC/C) is a multisubunit ubiquitin ligase that regulates progression through the cell cycle by marking key cell division proteins for destruction. To ensure correct cell cycle progression, accurate timing of APC/C activity is important, which is obtained through its association with both activating and inhibitory subunits. However, although the APC/C is highly conserved among eukaryotes, no APC/C inhibitors are known in plants. Recently, we have identified ULTRAVIOLET-B-INSENSITIVE4 (UVI4) as a plant-specific component of the APC/C. Here, we demonstrate that UVI4 uses conserved APC/C interaction motifs to counteract the activity of the CELL CYCLE SWITCH52 A1 (CCS52A1) activator subunit, inhibiting the turnover of the A-type cyclin CYCA2;3. UVI4 is expressed in an S phase-dependent fashion, likely through the action of E2F transcription factors. Correspondingly, uvi4 mutant plants failed to accumulate CYCA2;3 during the S phase and prematurely exited the cell cycle, triggering the onset of the endocycle. We conclude that UVI4 regulates the temporal inactivation of APC/C during DNA replication, allowing CYCA2;3 to accumulate above the level required for entering mitosis, and thereby regulates the meristem size and plant growth rate.

Phosphorylation of a mitotic kinesin-like protein and a MAPKKK by cyclin-dependent kinases (CDKs) is involved in the transition to cytokinesis in plants.

Cytokinesis in eukaryotes involves specific arrays of microtubules (MTs), which are known as the "central spindle" in animals, the "anaphase spindle" in yeasts, and the "phragmoplast" in plants. Control of these arrays, which are composed mainly of bundled nonkinetochore MTs, is critically important during cytokinesis. In plants, an MAPK cascade stimulates the turnover of phragmoplast MTs, and a crucial aspect of the activation of this cascade is the interaction between the MAPKKK, nucleus- and phragmoplast-localized protein kinase 1 (NPK1) and the NPK1-activating kinesin-like protein 1 (NACK1), a key regulator of plant cytokinesis. However, little is known about the control of this interaction at the molecular level during progression through the M phase. We demonstrated that cyclin-dependent kinases (CDKs) phosphorylate both NPK1 and NACK1 before metaphase in tobacco cells, thereby inhibiting the interaction between these proteins, suggesting that such phosphorylation prevents the transition to cytokinesis. Failure to inactivate CDKs after metaphase prevents dephosphorylation of these two proteins, causing incomplete mitosis. Experiments with Arabidopsis NACK1 (AtNACK1/HINKEL) revealed that phosphorylated NACK1 fails to mediate cytokinesis. Thus, timely and coordinated phosphorylation by CDKs and dephosphorylation of cytokinetic regulators from prophase to anaphase appear to be critical for the appropriate onset and/or progression of cytokinesis.

CCS52 and DEL1 genes are key components of the endocycle in nematode-induced feeding sites.

The establishment of galls and syncytia as feeding sites induced by root-knot and cyst nematodes, respectively, involves a progressive increase in nuclear and cellular size. Here we describe the functional characterization of endocycle activators CCS52A, CCS52B and a repressor of the endocycle, DEL1, during two types of nematode feeding site development in Arabidopsis thaliana. In situ hybridization analysis showed that expression of CCS52A1 and CCS52B was strongly induced in galls and syncytia and DEL1 was stably but weakly expressed throughout feeding site development. Down-regulation and over-expression of CCS52 and DEL1 in Arabidopsis drastically affected giant cell and syncytium growth, resulting in restrained nematode development, illustrating the need for mitotic activity and endo-reduplication for feeding site maturation. Exploiting the mechanism of endo-reduplication may be envisaged as a strategy to control plant-parasitic nematodes.

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.

The DNA replication checkpoint aids survival of plants deficient in the novel replisome factor ETG1.

Complete and accurate chromosomal DNA replication is essential for the maintenance of the genetic integrity of all organisms. Errors in replication are buffered by the activation of DNA stress checkpoints; however, in plants, the relative importance of a coordinated induction of DNA repair and cell cycle-arresting genes in the survival of replication mutants is unknown. In a systematic screen for Arabidopsis thaliana E2F target genes, the E2F TARGET GENE 1 (ETG1) was identified as a novel evolutionarily conserved replisome factor. ETG1 was associated with the minichromosome maintenance complex and was crucial for efficient DNA replication. Plants lacking the ETG1 gene had serrated leaves due to cell cycle inhibition triggered by the DNA replication checkpoints, as shown by the transcriptional induction of DNA stress checkpoint genes. The importance of checkpoint activation was highlighted by double mutant analysis: whereas etg1 mutant plants developed relatively normally, a synthetically lethal interaction was observed between etg1 and the checkpoint mutants wee1 and atr, demonstrating that activation of a G2 cell cycle checkpoint accounts for survival of ETG1-deficient plants.

Model-based analysis of Arabidopsis leaf epidermal cells reveals distinct division and expansion patterns for pavement and guard cells.

To efficiently capture sunlight for photosynthesis, leaves typically develop into a flat and thin structure. This development is driven by cell division and expansion, but the individual contribution of these processes is currently unknown, mainly because of the experimental difficulties to disentangle them in a developing organ, due to their tight interconnection. To circumvent this problem, we built a mathematic model that describes the possible division patterns and expansion rates for individual epidermal cells. This model was used to fit experimental data on cell numbers and sizes obtained over time intervals of 1 d throughout the development of the first leaf pair of Arabidopsis (Arabidopsis thaliana). The parameters were obtained by a derivative-free optimization method that minimizes the differences between the predicted and experimentally observed cell size distributions. The model allowed us to calculate probabilities for a cell to divide into guard or pavement cells, the maximum size at which it can divide, and its average cell division and expansion rates at each point during the leaf developmental process. Surprisingly, average cell cycle duration remained constant throughout leaf development, whereas no evidence for a maximum cell size threshold for cell division of pavement cells was found. Furthermore, the model predicted that neighboring cells of different sizes within the epidermis expand at distinctly different relative rates, which could be verified by direct observations. We conclude that cell division seems to occur independently from the status of cell expansion, whereas the cell cycle might act as a timer rather than as a size-regulated machinery.

APC/C-CCS52A complexes control meristem maintenance in the Arabidopsis root.

Plant organs originate from meristems where stem cells are maintained to produce continuously daughter cells that are the source of different cell types. The cell cycle switch gene CCS52A, a substrate specific activator of the anaphase promoting complex/cyclosome (APC/C), controls the mitotic arrest and the transition of mitotic cycles to endoreduplication (ER) cycles as part of cell differentiation. Arabidopsis, unlike other organisms, contains 2 CCS52A isoforms. Here, we show that both of them are active and regulate meristem maintenance in the root tip, although through different mechanisms. The CCS52A1 activity in the elongation zone of the root stimulates ER and mitotic exit, and contributes to the border delineation between dividing and expanding cells. In contrast, CCS52A2 acts directly in the distal region of the root meristem to control identity of the quiescent center (QC) cells and stem cell maintenance. Cell proliferation assays in roots suggest that this control involves CCS52A2 mediated repression of mitotic activity in the QC cells. The data indicate that the CCS52A genes favor a low mitotic state in different cell types of the root tip that is required for meristem maintenance, and reveal a previously undescribed mechanism for APC/C mediated control in plant development.

Atypical E2F activity restrains APC/CCCS52A2 function obligatory for endocycle onset.

The endocycle represents an alternative cell cycle that is activated in various developmental processes, including placental formation, Drosophila oogenesis, and leaf development. In endocycling cells, mitotic cell cycle exit is followed by successive doublings of the DNA content, resulting in polyploidy. The timing of endocycle onset is crucial for correct development, because polyploidization is linked with cessation of cell division and initiation of terminal differentiation. The anaphase-promoting complex/cyclosome (APC/C) activator genes CDH1, FZR, and CCS52 are known to promote endocycle onset in human, Drosophila, and Medicago species cells, respectively; however, the genetic pathways governing development-dependent APC/C(CDH1/FZR/CCS52) activity remain unknown. We report that the atypical E2F transcription factor E2Fe/DEL1 controls the expression of the CDH1/FZR orthologous CCS52A2 gene from Arabidopsis thaliana. E2Fe/DEL1 misregulation resulted in untimely CCS52A2 transcription, affecting the timing of endocycle onset. Correspondingly, ectopic CCS52A2 expression drove cells into the endocycle prematurely. Dynamic simulation illustrated that E2Fe/DEL1 accounted for the onset of the endocycle by regulating the temporal expression of CCS52A2 during the cell cycle in a development-dependent manner. Analogously, the atypical mammalian E2F7 protein was associated with the promoter of the APC/C-activating CDH1 gene, indicating that the transcriptional control of APC/C activator genes by atypical E2Fs might be evolutionarily conserved.

Systematic analysis of cell-cycle gene expression during Arabidopsis development.

The steady-state distribution of cell-cycle transcripts in Arabidopsis thaliana seedlings was studied in a broad in situ survey to provide a better understanding of the expression of cell-cycle genes during plant development. The 61 core cell-cycle genes analyzed were expressed at variable levels throughout the different plant tissues: 23 genes generally in dividing and young differentiating tissues, 34 genes mostly in both dividing and differentiated tissues and four gene transcripts primarily in differentiated tissues. Only 21 genes had a typical patchy expression pattern, indicating tight cell-cycle regulation. The increased expression of 27 cell-cycle genes in the root elongation zone hinted at their involvement in the switch from cell division to differentiation. The induction of 20 cell-cycle genes in differentiated cortical cells of etiolated hypocotyls pointed to their possible role in the process of endoreduplication. Of seven cyclin-dependent kinase inhibitor genes, five were upregulated in etiolated hypocotyls, suggesting a role in cell-cycle arrest. Nineteen genes were preferentially expressed in pericycle cells activated by auxin that give rise to lateral root primordia. Approximately 1800 images have been collected and can be queried via an online database. Our in situ analysis revealed that 70% of the cell-cycle genes, although expressed at different levels, show a large overlap in their localization. The lack of regulatory motifs in the upstream regions of the analyzed genes suggests the absence of a universal transcriptional control mechanism for all cell-cycle genes.

The DP-E2F-like gene DEL1 controls the endocycle in Arabidopsis thaliana.

Endoreduplication or DNA replication without mitosis is widespread in nature. Well-known examples are fruit fly polytene chromosomes and cereal endosperm. Although endocycles are thought to be driven by the same regulators as those that control the G1-S transition of the mitotic cell cycle, the molecular mechanisms that differentiate mitotically dividing cells from endoreduplicating ones are largely unknown. A novel class of atypical E2F-like proteins has recently been identified and is designated E2F7 in mammals and DP-E2F-like (DEL) in Arabidopsis thaliana . We demonstrate that loss of DEL1 function resulted in increased ploidy levels, whereas ectopic expression of DEL1 reduced endoreduplication. Ploidy changes were correlated with altered expression of a subset of E2F target genes encoding proteins necessary for DNA replication. Because DEL1 proteins were postulated to antagonize the E2F pathway, we generated DEL1-E2Fa-DPa triple transgenics. DEL1 inhibited the endoreduplication phenotype, but not the ectopic cell divisions that resulted from the overexpression of both E2Fa and DPa, illustrating that DEL1 specifically represses the endocycle. Because DEL1 transcripts were detected exclusively in mitotically dividing cells, we conclude that DEL1 is an important novel inhibitor of the endocycle and preserves the mitotic state of proliferating cells by suppressing transcription of genes that are required for cells to enter the DNA endoreduplication cycle.

What if higher plants lack a CDC25 phosphatase?

Progression through the cell cycle is regulated by cyclin-dependent kinases (CDKs). Plants possess a unique class of CDKs, designated B-type CDKs, but seem to lack a functional CDC25 phosphatase, which is a crucial activator of the onset of mitosis in non-plant species. Based on a striking number of functional parallels between the Arabidopsis thaliana CDKB1;1 and the Drosophila melanogaster CDC25 (string), we hypothesize that the acquisition of B-type CDKs and the disappearance of CDC25 in plants might have been associated; in these coupled events, the CDC25-controlled onset of mitosis might have been evolutionarily replaced by a B-type CDK-dominated pathway, eventually resulting in the loss of the CDC25 gene.

Root hydrotropism is controlled via a cortex-specific growth mechanism.

Plants can acclimate by using tropisms to link the direction of growth to environmental conditions. Hydrotropism allows roots to forage for water, a process known to depend on abscisic acid (ABA) but whose molecular and cellular basis remains unclear. Here we show that hydrotropism still occurs in roots after laser ablation removed the meristem and root cap. Additionally, targeted expression studies reveal that hydrotropism depends on the ABA signalling kinase SnRK2.2 and the hydrotropism-specific MIZ1, both acting specifically in elongation zone cortical cells. Conversely, hydrotropism, but not gravitropism, is inhibited by preventing differential cell-length increases in the cortex, but not in other cell types. We conclude that root tropic responses to gravity and water are driven by distinct tissue-based mechanisms. In addition, unlike its role in root gravitropism, the elongation zone performs a dual function during a hydrotropic response, both sensing a water potential gradient and subsequently undergoing differential growth.

CDKB1;1 forms a functional complex with CYCA2;3 to suppress endocycle onset.

The mitosis-to-endocycle transition requires the controlled inactivation of M phase-associated cyclin-dependent kinase (CDK) activity. Previously, the B-type CDKB1;1 was identified as an important negative regulator of endocycle onset. Here, we demonstrate that CDKB1;1 copurifies and associates with the A2-type cyclin CYCA2;3. Coexpression of CYCA2;3 with CDKB1;1 triggered ectopic cell divisions and inhibited endoreduplication. Moreover, the enhanced endoreduplication phenotype observed after overexpression of a dominant-negative allele of CDKB1;1 could be partially complemented by CYCA2;3 co-overexpression, illustrating that both subunits unite in vivo to form a functional complex. CYCA2;3 protein stability was found to be controlled by CCS52A1, an activator of the anaphase-promoting complex. We conclude that CCS52A1 participates in endocycle onset by down-regulating CDKB1;1 activity through the destruction of CYCA2;3.

The plant-specific cyclin-dependent kinase CDKB1;1 and transcription factor E2Fa-DPa control the balance of mitotically dividing and endoreduplicating cells in Arabidopsis.

Transgenic Arabidopsis thaliana plants overproducing the E2Fa-DPa transcription factor have two distinct cell-specific phenotypes: some cells divide ectopically and others are stimulated to endocycle. The decision of cells to undergo extra mitotic divisions has been postulated to depend on the presence of a mitosis-inducing factor (MIF). Plants possess a unique class of cyclin-dependent kinases (CDKs; B-type) for which no ortholog is found in other kingdoms. The peak of CDKB1;1 activity around the G2-M boundary suggested that it might be part of the MIF. Plants that overexpressed a dominant negative allele of CDKB1;1 underwent enhanced endoreduplication, demonstrating that CDKB1;1 activity was required to inhibit the endocycle. Moreover, when the mutant CDKB1;1 allele was overexpressed in an E2Fa-DPa-overproducing background, it enhanced the endoreduplication phenotype, whereas the extra mitotic cell divisions normally induced by E2Fa-DPa were repressed. Surprisingly, CDKB1;1 transcription was controlled by the E2F pathway, as shown by its upregulation in E2Fa-DPa-overproducing plants and mutational analysis of the E2F binding site in the CDKB1;1 promoter. These findings illustrate a cross talking mechanism between the G1-S and G2-M transition points.

B1-type cyclin-dependent kinases are essential for the formation of stomatal complexes in Arabidopsis thaliana.

Cyclin-dependent kinases (CDKs) are key regulators of the cell cycle. In yeasts, only one CDK is sufficient to drive cells through the cell cycle, whereas higher eukaryotes developed a family of related CDKs. Curiously, plants contain a unique class of CDKs (B-type CDKs), whose function is still unclear. We show that the CDKB1;1 gene of Arabidopsis (Arabidopsis thaliana) is highly expressed in guard cells and stomatal precursor cells of cotyledons, suggesting a prominent role for B-type CDKs in stomatal development. In accordance, transgenic Arabidopsis plants with reduced B-type CDK activity had a decreased stomatal index because of an early block of meristemoid division and inhibition of satellite meristemoid formation. Many aberrant stomatal cells were observed, all of them blocked in the G2 phase of the cell cycle. Although division of stomatal precursors was inhibited, cells still acquired stomatal identity, illustrating that stomatal cell differentiation is independent of cellular and nuclear division.