Regulation of Cell Division in Arabidopsis

The study of cell cycle control in plants is expected to contribute to the understanding of plants' unique developmental features. The principal regulators of the eukaryotic cell cycle, namely, cyclin-dependent kinases (CDKs) and cyclins, are also conserved in plants. This review is concerned with our present knowledge on cell cycle regulation in Arabidopsis thaliana, which is widely accepted as a model plant for the study of a broad range of biological questions. Up to the present, 2 CDKs and 11 cyclins have been identified in Arabidopsis. While the expression of one of these CDKs has been found to be positively correlated with the competence of cells to divide, cyc1A1 expression of the cyclin has been almost exclusively confined to dividing cells. Although much remains to be studied concerning upstream regulators of these genes, the successful introduction of mutant CDKs into plants demonstrates the potential of using such an approach to intentionally modulate the plant cell cycle and development.     

Identification of proteins interacting with the Arabidopsis Cdc2aAt protein

Cyclin-dependent kinases (CDK5) control the progression throughthe cell cycle. Using a two-hybrid approach, two clones encodingproteins interacting with the Arabidopsis thailana CDK Cdc2aAtwere identified. One clone encoded a novel putative substrateof Cdc2aAt, whereas the second clone was identified as a D-typecyclin (cycDl;1). Key words: Arabidopsis thaliana, cell cycle, cyclin, cyclindependent kinases, yeast two-hybrid screening     

The Interaction of Ultraviolet-B Radiation and Water Deficit in Two Arabidopsis thaliana Genotypes

It has been demonstrated, in both herbaceous and woody species,that tissue hydration resulting from exposure to drought isless pronounced if plants are concurrently exposed to ultraviolet-Bradiation (UV-B). An explanation for the mechanisms underlyingthis phenomenon has been elusive. Arabidopsis thaliana(L.) Heynh.genotypes, defective in specific defences against UV-B exposure,may permit more insightful study of drought-UV-B interactionsthan is possible with genetically uniform plants. Arabidopsishas a rosette stature and has predominantly abaxial stomata.Thus, it is difficult to investigate its stomatal behaviourand gas exchange using conventional techniques and instrumentation.In this study, the relative abundance of¹³C and¹²C in leaf tissue(¹³C) was used as a means of determining water use efficiency(WUE) and the relative balance, at the site of carbon fixation,between CO₂supply and demand. UV-B insensitive (L er) and sensitive(fah1)Arabidopsis genotypes were raised in a growth chamberand exposed to 6 kJ m^-2 d^-1UV-B irradiation and subjected todrought. In both genotypes, leaf desiccation was less pronouncedthan that of control plants that were subjected to drought butnot exposed to UV-B. The relatively low (more negative) leaf¹³C values (indicating low WUE), but high dry matter productionof the UV-B exposed plants suggest that their higher leaf watercontent was not primarily due to stomatal closure. We proposethat the mechanisms underlying the maintenance of higher leafwater content involved UV-B and water stress induced biosynthesisof stress proteins and compatible osmolytes. Copyright 2000Annals of Botany Company Arabidopsis thaliana, ultraviolet-B, water deficit, stable carbon isotopes, ¹³C, stomatal opening, tissue dehydration, dehydrin     

Analysis of the genes encoding the largest subunit of RNA polymerase II in Arabidopsis and soybean

We have cloned and sequenced the gene encoding the largest subunit of RNA polymerase II (RPB1) from Arabidopsis thaliana and partially sequenced genes from soybean (Glycine max). We have also determined the nucleotide sequence for a number of cDNA clones which encode the carboxyl terminal domains (CTDs) of RNA polymerase II from both soybean and Arabidopsis. The Arabidopsis RPB1 gene encodes a polypeptide of approximately 205 kDa, consists of 12 exons, and encompasses more than 8 kb. Predicted amino acid sequence shows eight regions of similarity with the largest subunit of other prokaryotic and eukaryotic RNA polymerases, as well as a highly conserved CTD unique to RNA polymerase II.The CTDs in plants, like those in most other eukaryotes, consist of tandem heptapeptide repeats with the consensus amino acid sequence PTSPSYS. The portion of RPB1 which encodes the CTD in plants differs from that of RPB1 of animals and lower eukaryotes. All the plant genes examined contain 2–3 introns within the CTD encoding regions, and at least two plant genes contain an alternatively spliced intron in the 3 untranslated region. Several clustered amino acid substitutions in the CTD are conserved in the two plant species examined, but are not found in other eukaryotes. RPB1 is encoded by a multigene family in soybean, but a single gene encodes this subunit in Arabidopsis and most other eukaryotes.     

Regulation of cyclin-dependent kinases in Arabidopsis thaliana

In plants, different families of cyclin-dependent kinases (CDKs) and cyclins have been identified, indicating that also in plants the progression through the cell cycle is regulated by CDKs. In all eukaryotes, CDKs exert their activity through well-controlled phosphorylations of specific substrates on serine/threonine residues. Such post-translational modifications are universal mechanisms in signal transduction pathways. They allow the organism to differentiate, regulate growth and/or adapt to environmental changes, the latter being crucial for plants because of their sedentary life-style. This adaptation might explain the occurrence of a special CDK type with plant-specific features. This review focuses on the involvement of plant CDKs in different phases of the cell cycle in Arabidopsis thaliana and outlines their regulation by binding to other proteins, and by phosphorylation and dephosphorylation.     

Identification of clones carrying minisatellite-like loci in an Arabidopsis thaliana YAC library

Minisatellite-like DNA elements occur in the Arabidopsis thalianagenome in low copy and are weakly polymorphic between ecotypes.YAC clones from the EG-Arabidopsis library were identified withhomology to minisatellite 33.15 and bacteriophage M13 repeatelements. Other highly repeated A. thaliana DNA elements tendnot to be found in YAC clones carrying the minisatellite elementssuggesting that the elements are dispersed in the Arabidopsisgenome in regions of low complexity. The minisatellite elementsare represented at low copy in the EG-YAC library reflectingtheir frequency in the Arabidopsis genome. Key words: Minisatellite elements, Arabidopsis thaliana, YAC library screening     

Farnesylation is involved in meristem organization in Arabidopsis

 Although studies in plant and animal cell culture systems indicate farnesylation is required for normal cell cycle progression, how this lipid modification of select proteins translates into whole-organism developmental decisions involving cell proliferation or differentiation is largely unknown. The era1 mutant of the higher plant Arabidopsis thaliana (L.) Heynh. offers a unique opportunity to understand the role farnesylation may play in regulating various processes during the development of a multicellular organism. Loss of farnesylation affects many aspects of Arabidopsis growth and development. In particular, apical and axillary meristem development is altered and these phenotypes are contingent on the growth conditions. Received: 25 October 1999 / Accepted: 22 December 1999     

Glutathione reductase gene expression depends on chloroplast signals in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Glutathione reductase (EC 1.6.4.2) is one of the main antioxidant enzymes of the plant cell. In Arabidopsis thaliana, glutathione reductase is encoded by two genes: the gr1 gene encodes the cytosolic-peroxisomal form, and the gr2 gene encodes the chloroplast-mitochondrial form. Little is known about the regulation of expression of plant glutathione reductase genes. In the present work, we have demonstrated that gr2 (but not gr1) gene expression in Arabidopsis leaves changes depending on changes in redox state of the photosynthetic electron transport chain. Expression of both the gr1 and gr2 genes was induced by reactive oxygen species. In heterotrophic suspension cell culture of Arabidopsis, expression of both studied genes did not depend on H₂O₂ level or on changes in the redox state of the mitochondrial electron transport chain. Our data indicate that chloroplasts are involved in the regulation of the glutathione reductase gene expression in Arabidopsis.     

An expression atlas of miRNAs in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

MicroRNAs (miRNAs) are small non-coding RNAs that regulate a variety of biological processes. MiRNA expression often exhibits spatial and temporal specificity. However, genome-wide miRNA expression patterns in different organs during development of Arabidopsis thaliana have not yet been systemically investigated. In this study, we sequenced small RNA libraries generated from 27 different organ/tissue types, which cover the entire life cycle of Arabidopsis. Analysis of the sequencing data revealed that most miRNAs are ubiquitously expressed, whereas a small set of miRNAs display highly specific expression patterns. In addition, different miRNA members within the same family have distinct spatial and temporal expression patterns. Moreover, we found that some miRNAs are produced from different arms of their hairpin precursors at different developmental stages. This work provides new insights into the regulation of miRNA biogenesis and a rich resource for future investigation of miRNA functions in Arabidopsis.     

Regulation of Auxin Homeostasis and Gradients in Arabidopsis Roots through the Formation of the Indole-3-Acetic Acid Catabolite 2-Oxindole-3-Acetic Acid

The native auxin, indole-3-acetic acid (IAA), is a major regulator of plant growth and development. Its nonuniform distribution between cells and tissues underlies the spatiotemporal coordination of many developmental events and responses to environmental stimuli. The regulation of auxin gradients and the formation of auxin maxima/minima most likely involve the regulation of both metabolic and transport processes. In this article, we have demonstrated that 2-oxindole-3-acetic acid (oxIAA) is a major primary IAA catabolite formed in Arabidopsis thaliana root tissues. OxIAA had little biological activity and was formed rapidly and irreversibly in response to increases in auxin levels. We further showed that there is cell type–specific regulation of oxIAA levels in the Arabidopsis root apex. We propose that oxIAA is an important element in the regulation of output from auxin gradients and, therefore, in the regulation of auxin homeostasis and response mechanisms.     

Control of Cell Proliferation,Organ Growth,and DNA Damage Response Operate Independently of Dephosphorylation of the Arabidopsis Cdk1 Homolog CDKA;1

Entry into mitosis is universally controlled by cyclin-dependent kinases (CDKs). A key regulatory event in metazoans and fission yeast is CDK activation by the removal of inhibitory phosphate groups in the ATP binding pocket catalyzed by Cdc25 phosphatases. In contrast with other multicellular organisms, we show here that in the flowering plant Arabidopsis thaliana, cell cycle control does not depend on sudden changes in the phosphorylation pattern of the PSTAIRE-containing Cdk1 homolog CDKA;1. Consistently, we found that neither mutants in a previously identified CDC25 candidate gene nor plants in which it is overexpressed display cell cycle defects. Inhibitory phosphorylation of CDKs is also the key event in metazoans to arrest cell cycle progression upon DNA damage. However, we show here that the DNA damage checkpoint in Arabidopsis can also operate independently of the phosphorylation of CDKA;1. These observations reveal a surprising degree of divergence in the circuitry of highly conserved core cell cycle regulators in multicellular organisms. Based on biomathematical simulations, we propose a plant-specific model of how progression through the cell cycle could be wired in Arabidopsis.     

Coordinated Activation of the Origin Licensing Factor CDC6 and CDK2 in Resting Human Fibroblasts Expressing SV40 Small T Antigen and Cyclin E

We have previously shown that SV40 small t antigen (st) cooperates with deregulated cyclin E to activate CDK2 and bypass quiescence in normal human fibroblasts (NHF). Here we show that st expression in serum-starved and density-arrested NHF specifically induces up-regulation and loading of CDC6 onto chromatin. Coexpression of cyclin E results in further accumulation of CDC6 onto chromatin concomitantly with phosphorylation of CDK2 on Thr-160 and CDC6 on Ser-54. Investigation of the mechanism leading to CDC6 accumulation and chromatin loading indicates that st is a potent inducer of cdc6 mRNA expression and increases CDC6 protein stability. We also show that CDC6 expression in quiescent NHF efficiently promotes cyclin E loading onto chromatin, but it is not sufficient to activate CDK2. Moreover, we show that CDC6 expression is linked to phosphorylation of the activating T loop of CDK2 in serum-starved NHF stimulated with mitogens or ectopically expressing cyclin E and st. Our data suggest a model where the combination of st and deregulated cyclin E result in cooperative and coordinated activation of both an essential origin licensing factor, CDC6, and an activity required for origin firing, CDK2, resulting in progression from quiescence to S phase.Upon mitogenic stimulation mammalian G1 CDKs⁴ trigger passage through the restriction point and the transition into DNA replication. In particular, cyclin E/CDK2 is activated in mid to late G1 and phosphorylates a variety of substrates that play critical roles in these processes. CDK2 cooperates with D-type cyclin/CDKs to inactivate E2F/pocket protein repressor complexes inducing the expression of DNA synthesis factors and other cell cycle regulators (reviewed in Refs. 1 and 2). CDK2 also phosphorylates DNA replication factors facilitating prereplication complex assembly and origin firing and plays additional roles in centrosome duplication and histone synthesis (reviewed in Ref. 1). In particular, it has been proposed that CDK2 phosphorylates the essential origin licensing factor CDC6 promoting its stabilization prior to inactivation of the APC^Cdh1 ubiquitin ligase (3). This is thought to ensure that CDC6 accumulation precedes accumulation of other APC substrates that inhibit origin licensing. Moreover, CDK2-independent cyclin E functions have also been reported to be important for prereplication complex assembly in cells in transit from G0 into G1 (4, 5). In keeping with its role as positive regulator of major G1 transitions, deregulation of the cyclin E via gene amplification or defective protein turnover is commonly seen in primary tumors and is associated with poor prognosis (6–8). In normal fibroblasts, ectopic expression of cyclin E has been associated with shortening of the G1 phase of the cell cycle (9, 10), and with induction of DNA damage (reviewed in Ref. 8). Cyclin E deregulation in certain human tumor cell lines and immortalized rat fibroblasts is associated with mitogen-independent cell cycle entry and progression through the cell cycle (11). However, when cyclin E is ectopically expressed in quiescent normal human fibroblasts (NHF), cells remain in G0 (12).We have recently reported that coexpression of SV40 small t antigen (st) in quiescent NHF with deregulated cyclin E expression is sufficient to trigger mitogen-independent cell cycle progression, proliferation beyond cell confluence, and foci formation. The bypass of quiescence induced by the expression of st and cyclin E is dependent on CDK2 activation (12). Thus, contrary to what is seen in normal murine cells (13), CDK2 activity appears essential for cell cycle progression when it is oncogenically driven by cyclin E and st expression (12). Because st is known to target pathways uniquely required for the transformation of human cells (14, 15), tumor cells with altered pathways that mimic st/cyclin E expression could predictably be sensitive to selective inhibition of CDK2 activity.Given the critical role of CDK2 activity in cyclin E and st cooperation in inducing cell proliferation and transformation of NHF, we sought to determine the factors and mechanisms by which st modulates CDK2 activation. In this report we have identified the CDC6 replication licensing factor as a cellular target of st. We also uncover CDC6 as a participant in the events leading to chromatin association of cyclin E and CDK2 and in phosphorylation of CDK2 on its activating T loop both in response to mitogenic stimulation, as well as expression of cyclin E and st in NHF.     

Telomere elongation upon transfer to callus culture reflects the reprogramming of telomere stability control in Arabidopsis

Key message

Standard pathways involved in the regulation of telomere stability do not contribute to gradual telomere elongation observed in the course of A. thaliana calli propagation.

Abstract

Genetic and epigenetic changes accompanying the culturing of plant cells have frequently been reported. Here we aimed to characterize the telomere homeostasis during long term callus propagation. While in Arabidopsis thaliana calli gradual telomere elongation was observed, telomeres were stable in Nicotiana tabacum and N. sylvestris cultures. Telomere elongation during callus propagation is thus not a general feature of plant cells. The long telomere phenotype in Arabidopsis calli was correlated neither with changes in telomerase activity nor with activation of alternative mechanisms of telomere elongation. The dynamics of telomere length changes was maintained in mutant calli with loss of function of important epigenetic modifiers but compromised in the presence of epigenetically active drug zebularine. To examine whether the cell culture-induced disruption of telomere homeostasis is associated with the modulated structure of chromosome ends, epigenetic properties of telomere chromatin were analysed. Albeit distinct changes in epigenetic modifications of telomere histones were observed, these were broadly stochastic. Our results show that contrary to animal cells, the structure and function of plant telomeres is not determined significantly by the epigenetic character of telomere chromatin. Set of differentially transcribed genes was identified in calli, but considering the known telomere- or telomerase-related functions of respective proteins, none of these changes per se was apparently related to the elongated telomere phenotype. Based on our data, we propose that the disruption in telomere homeostasis in Arabidopsis calli arises from the interplay of multiple factors, as a part of reprogramming of plant cells to long-term culture conditions.