Synchronous Arabidopsis suspension cultures for analysis of cell-cycle gene activity

Synchronized suspension cultures are powerful tools in plant cell-cycle studies. However, few Arabidopsis cell cultures are available, and synchrony extending over several sequential phases of the cell cycle has not been reported. Here we describe the first useful synchrony in Arabidopsis, achieved by selecting the rapidly dividing Arabidopsis cell suspensions MM1 and MM2d. Synchrony may be achieved either by removing and re-supplying sucrose to the growth media or by applying an aphidicolin block/release. Synchronization with aphidicolin produced up to 80% S-phase cells and up to 92% G2 cells, together with clear separation of different cell-cycle phases. These synchronization procedures can be used for analysis of gene expression and protein activity. We show that representatives of three CDK gene classes of Arabidopsis (CDKA, CDKB1 and CDKB2) show differential expression timing, and that three CDK inhibitor genes show strikingly different expression patterns during cell-cycle re-entry. We propose that ICK2 (KRP2) may have a specific role in this process.     

Cell cycle controls: genome-wide analysis in Arabidopsis

The first decade of molecular analysis of plant cell cycle control genes revealed how well the important regulators are conserved among eukaryotes. The recent completion of the Arabidopsis genome sequence, and the use of increasingly sophisticated biochemical assays and genetic approaches, heralds a period of more detailed functional analysis of cell cycle regulators aimed at resolving their role in plant growth and development.     

The effect of cadmium on cell cycle control in suspension culture cells of soybean

Heavy metals inhibit plant growth. This proces may be directly or indirectly connected with mechanisms regulating cell division. We analyzed the effect of Cd²⁺ on cell cycle progression in partially synchronized soybean (Glycine max) cell suspension culture and followed the expression of cell cycle genes (cyclin B1 and cyclin-dependent kinase A - CDK-A). We have checked the hypothesis that Cd²⁺-induced impairment of cell division is connected with DNA damage. The [³H]-thymidine incorporation in cell cultures synchronized either with hydroxyurea (HU) or phosphate starvation have shown, that Cd²⁺ strongly affects the S phase of soybean cell cycle, by causing the earlier entry of cells into S phase and by decreasing the rate of DNA synthesis. RT-PCR analysis indicated that Cd²⁺ decreases the level of cyclin B1 mRNA and has no effect on CDK-A mRNA. The result of comet assay indicated the damaging effect of Cd²⁺ on DNA of soybean cells. We suggest that Cd²⁺ affects plant cell cycle at two major checkpoints: the G1/S — by damaging of DNA, and G2/M - by decreasing the level of cyclin B1 mRNA     

Effects of brassinosteroid, auxin, and cytokinin on ethylene production in Arabidopsis thaliana plants

Inflorescence stalks produced the highest amount of ethylene in response to IAA as compared with other plant parts tested. Leaf age had an effect on IAA-induced ethylene with the youngest leaves showing the greatest stimulation. The highest amount of IAA-induced ethylene was produced in the root or inflorescence tip with regions below this producing less. Inflorescence stalks treated with IAA, 2,4-D, or NAA over a range of concentrations exhibited an increase in ethylene production starting at 1 microM with increasingly greater responses up to 100 microM, followed by a plateau at 500 microM and a significant decline at 1000 microM. Both 2,4-D and NAA elicited a greater response than IAA at all concentrations tested in inflorescence stalks. Inflorescence leaves treated with IAA, 2,4-D, or NAA exhibited the same trend as inflorescence stalks. However, they produced significantly less ethylene. Inflorescence stalks and leaves treated with 100 microM IAA exhibited a dramatic increase in ethylene production 2 h following treatment initiation. Inflorescence stalks showed a further increase 4 h following treatment initiation and no further increase at 6 h. However, there was a slight decline between 6 h and 24 h. Inflorescence leaves exhibited similar rates of IAA-induced ethylene between 2 h and 24 h. Light and high temperature caused a decrease in IAA-induced ethylene in both inflorescence stalks and leaves. Three auxin-insensitive mutants were evaluated for their inflorescence's responsiveness to IAA. aux2 did not produce ethylene in response to 100 microM IAA, while axr1-3 and axr1-12 showed reduced levels of IAA-induced ethylene as compared with Columbia wild type. Inflorescences treated with brassinolide alone had no effect on ethylene production. However, when brassinolide was used in combination with IAA there was a dramatic increase in ethylene production above the induction promoted by IAA alone.     

Changes in the pattern of phospholipid synthesis during the induction by cytokinin of cell division in soybean suspension cultures

A method is described for preparing fully viable, cytokinin-starved soybean (Glycine max (L.) Merr. cv. Acme) cells from a suspension-culture of callus tissue. The cells respond to kinetin treatment by re-initiating cell division. We present evidence, from the pattern of incorporation of ³²P-labelled inorganic phosphate into individual phospholipids during the first hour of this response, that the synthesis of phosphatidylinositol (PI) and of phosphatidic-acid head-groups is affected within 15 min. The polyphosphoinositide phosphatidylinositol 4-phosphate, but not phosphatidylinositol 4,5-bisphosphate, was detected in the tissue. The characteristics of cytokinin-induced PI synthesis in cytokinin-starved soybean cells appear to resemble the PI response of animal cells.Abbreviations DPG diphosphatidylglycerol - PA phosphatidic acid - PC phosphatidylcholine - PE phosphatidylethanolamine - PG phosphatidylglycerol - PI phosphatidylinositol - PIP phosphatidylinositol 4-phosphate - PIP₂ phosphatidylinositol 4,5-bisphosphate - PS phosphatidylserine - Pi inorganic phosphate - TLC thin-layer chromatography     

Transient expression in Arabidopsis thaliana protoplasts derived from rapidly established cell suspension cultures

Arabidopsis thaliana has emerged as a model species for the analysis of genes controlling plant development. However, its small size has impaired biochemical analyses, and the absence of a transient expression system has hampered promoter analysis. Here, we report a method for rapidly establishing A. thaliana suspension cultures that yield protoplasts that can be readily transfected. We have optimized transient expression conditions using a modified polyethylene glycol / calcium nitrate transformation protocol and a Cauliflower Mosaic Virus 35S promoter-luciferase reporter gene construct. Our methods permit isolation of large quantities of rapidly growing cells and analysis of Arabidopsis promoters in vivo in a homologous system.Abbreviations CaMV Cauliflower Mosaic Virus - 2,4D 2,4-dichlorophenoxyacetic acid - MES 2-(N-morpholino)ethanesulfonic acid - PEG polyethylene glycol     

Zunahme von cytokinin und auxin in verwundetem speichergewebe von Solanum tuberosum

“Butanol-soluble” cytokinins increase in grated and in lighted cut potato tissue. In slices, an increase of “water soluble” cytokinins (darkness and of tRNA-cytokinins can be found. Together with auxin, the cytokinin factor stimulates cell division in wound tissue.     

Roles of GIG1 and UVI4 in genome duplication in Arabidopsis thaliana

Endomitosis and endoreplication are atypical modes of cell cycle that results in genome duplication in single nucleus. Because the cell size of given cell type is generally proportional to the nuclear DNA content, endoreplication and endomitosis are effective strategy of cell growth, which are widespread in multicellular organisms, especially those in plant kingdom. We found that these processes might be differently regulated by GIGAS CELL1 (GIG1) and its paralog UV-INSENSITIVE4 (UVI4) in Arabidopsis thaliana. GIG1 and UVI4 may negatively regulate activities of anaphase-promoting complex or cyclosome (APC/C) ubiquitin ligase that acts as an important mitotic regulator. The gig1 mutation induced ectopic occurrence of endomitosis during somatic cell division, while it has been reported that uvi4 mutation resulted in premature occurrence of endoreplication during organ development. Overexpression of GIG1 and UVI4 dramatically increased the amount of mitotic cyclin, CYCB1;1, a well-known substrate of APC/C. Ectopic endomitosis in gig1 was enhanced by mutation in CYCB2;2 and suppressed by downregulation of APC10 encoding a core subunit of APC/C. Overexpression of CDC20.1, an activator protein of APC/C, further promoted the ectopic endomitosis in gig1. These findings suggest that endomitosis and endoreplication are regulated by similar molecular mechanisms, in which two related proteins, GIG1 and UVI4, may inhibit APC/C in different ways.     

NO way back: nitric oxide and programmed cell death in Arabidopsis thaliana suspension cultures

Recent research has implicated nitric oxide (NO) in the induction of the hypersensitive response (HR) during plant-pathogen interactions. Here we demonstrate that Arabidopsis suspension cultures generate elevated levels of NO in response to challenge by avirulent bacteria, and, using NO donors, show that these elevated levels of NO are sufficient to induce cell death in Arabidopsis cells independently of reactive oxygen species (ROS). We also provide evidence that NO-induced cell death is a form of programmed cell death (PCD), requiring gene expression, and has a number of characteristics of PCD of mammalian cells: NO induced chromatin condensation and caspase-like activity in Arabidopsis cells, while the caspase-1 inhibitor, Ac-YVAD-CMK, blocked NO-induced cell death. A well-established second messenger mediating NO responses in mammalian cells is cGMP, produced by the enzyme guanylate cyclase. A specific inhibitor of guanylate cyclase blocked NO-induced cell death in Arabidopsis cells, and this inhibition was reversed by the cell-permeable cGMP analogue, 8Br-cGMP, although 8Br-cGMP alone did not induce cell death or potentiate NO-induced cell death. This suggests that cGMP synthesis is required but not sufficient for NO-induced cell death in Arabidopsis. In-gel protein kinase assays showed that NO activates a potential mitogen-activated protein kinase (MAPK), although a specific inhibitor of mammalian MAPK activation, PD98059, which blocked H2O2-induced cell death, did not inhibit the effects of NO.     

周期蛋白和周期蛋白依赖性激酶及相关激酶抑制剂在细胞周期进程中的调控机制研究进展

在细胞发育过程中,细胞周期起着至关重要的作用。细胞周期进程主要受细胞周期蛋白依赖性激酶(cyclin dependent kinase, CDK)、周期蛋白和内源性CDK抑制剂(cyclin-dependent kinase inhibitors,CKI)调控。其中,CDK是主要的细胞周期调节因子,可与周期蛋白结合形成周期蛋白-CDK复合物,从而使数百种底物磷酸化,调控分裂间期和有丝分裂进程。各类细胞周期蛋白的活性异常,可引起不受控制的癌细胞增殖,导致癌症的发生与发展。因此,了解CDK的活性变化情况、周期蛋白-CDK的组装以及CKI的作用,将有助于了解细胞周期进程中潜在的调控过程,为癌症与疾病的治疗和CKI治疗药物的研发提供基础。本文关注了CDK激活和灭活的关键事件,并总结了周期蛋白-CDK在特定时期及位置的调控过程,以及相关CKI治疗药物在癌症及疾病中的研究进展,最后简单阐述了细胞周期进程研究面临的问题和存在的挑战,以期为后续细胞周期进程的深入研究提供参考和思路。     

Control of cell cycle gene expression in bone development and during c-Fos-induced osteosarcoma formation

We have used c-Fos transgenic mice which develop osteosarcomas to determine the expression patterns of cyclins, cyclin-dependent kinases (CDKs), and cyclin-dependent kinase inhibitors (CKIs) in different bone cell populations in order to define the potential mechanisms of c-Fos transformation. Immunohistochemical analysis in embryonic and early postnatal bone demonstrated that cyclin E and its kinase partner CDK2 were expressed specifically in bone-forming osteoblasts. Cyclin D1 expression was absent despite high levels of CDK4 and CDK6, and the CKI p27 was expressed in chondrocytes, osteoclasts, and at lower levels in osteoblasts. Following activation of the c-fos transgene in vivo and before overt tumor formation, cyclin D1 expression increased dramatically and was colocalized with exogenous c-Fos protein specifically in osteoblasts and chondrocytes, but not in osteoclasts. Prolonged activation of c-Fos resulted in osteosarcoma formation wherein the levels of cyclin D1, cyclin E, and CDKs 2, 4, and 6 were high in a wide spectrum of malignant cell types, especially in transformed osteoblasts. The CKI p27 was expressed at very high levels in bone-resorbing osteoclasts, and to a lesser extent in chondrocytes and osteoblasts. These in vivo observations suggest that cyclin D1 may be a target for c-Fos action and that elevation of cyclin D1 in osteoblasts which already express cyclin E/CDK2 and the cyclin D1 partners CDKs 4 and 6, may predispose cells to uncontrolled cell growth leading to osteosarcoma development. This study implicates altered cell cycle control as a potential mechanism through which c-Fos causes osteoblast transformation and bone tumor formation. Dev. Genet. 22:386–397, 1998. © 1998 Wiley-Liss, Inc.     

Establishment and characterisation of a rapidly dividing diploid cell suspension culture of Arabidopsis thaliana suitable for cell cycle synchronisation

Polyploid plants often have altered gene expression, biochemistry, and metabolism compared to their diploid predecessors. Therefore cultured diploid cells have distinct benefits over cultured polyploid cells for the study of gene regulation and metabolism of the parent plant. Here we report methods for establishing and maintaining a rapidly dividing diploid Arabidopsis thaliana cell suspension culture, and subsequent cell cycle synchronisation. Rapid growth of homogeneous cell populations was achieved after 3 months of initiation of cultures from leaf calluses. The cells were grown in the dark on an orbital shaker (110 rpm, 50 mm orbit) at 24 °C. Continued maintenance of the culture required the use of late-exponential stage cells for subculture at weekly intervals using careful subculturing techniques to achieve accurate biomass transfer. Cell cycle synchronisation was achieved following sucrose starvation, phosphate starvation, hydroxyurea treatment, aphidicolin treatment, and a combination of phosphate starvation and aphidicolin treatment. Inhibition of the cell cycle and accumulation of cells in specific phases was monitored by microscopy to determine the metaphase/anaphase index, and by flow cytometry. The cell cycle was partially and reversibly blocked by sucrose or phosphate starvation and by hydroxyurea (2.5 mM) treatment. A complete block at G1/S interphase was achieved after aphidicolin treatment or phosphate starvation combined with aphidicolin treatment. Release from the aphidicolin block achieved ca. 78% cell cycle synchronisation in the cell population. Endoreduplication was evident after release from the block in all treatments but after one cycle (24 h) the cells returned to the diploid state. This diploid culture is currently being used in our laboratory for the genetic analysis of cell death.     

Cryopreservation of transformed and wild-type Arabidopsis and tobacco cell suspension cultures

We have recently described Arabidopsis cell suspension cultures that can be effectively synchronised. Here, we describe procedures that allow clonal-transformed cell suspension lines to be produced using Agrobacterium-mediated transformation, and an optimised and straightforward procedure for the cryopreservation and recovery of both parental and transformed lines. Frozen cultures show 90% viability and rapid re-growth after recovery. We show that the cryopreservation procedure is equally applicable to the frequently used tobacco bright yellow (BY)2 cell suspension culture, and that cell cycle synchronisation capacity of parental lines is maintained after both transformation and recovery from cryopreservation. The techniques require no specialised equipment, and are suitable for routine laboratory use, greatly facilitating the handling and maintenance of cell cultures and providing security against both contamination and cumulative somaclonal variation. Finally, the ability to store easily large numbers of transformed lines opens the possibility of using Arabidopsis cell suspension cultures for high-throughput analysis.     

蛋白激酶C与细胞周期

近年的研究表明,PKC涉及到细胞的周期调节。在酵母细胞和哺乳动物细胞均发现PKC参与细胞周期调控,从而提示PKC可能在进化上是一种保守的细胞周期调节子。一般认为PKC在两个点上对细胞周期起作用,即G1期和G2期到M期的过渡期（G2／M）。在G1期,PKC分别在早G1期和晚G1期作用有所不同,主要作用表现在使细胞停留在G1期的中末阶段,这一过程,主要涉及到抑制肿瘤抑制因子－成视网膜细胞瘤（Rb)蛋白的磷酸化。PKC的主要作用是降低周期素依赖激酶CDK2的活性、降低周期素E和A的表达和增加周期素依赖的周期抑制蛋白p21^WAF1和p27^KIP1的表达;在G2／M期,PKC对细胞周期的调节主要与Cdc2(CDK1)的活性抑制有关。