Lectures in Plant Developmental Physiology, 3 cr.

1. Lectures in Plant Developmental Physiology, 3 cr. Kurt Fagerstedt Department of Biological and Environmental Sciences Plant Biology Viikki Biocenter Spring 2006

2. Cell intrinsic information:The cell and the cell cycleLecture 2

3. Contents

4. References / Further Reading • Dewitte, W. & Murray, J.A.H. 2003. The Plant Cell Cycle. Annu. Rev. Plant Biol. 54: 235-264. • Buchanan, Gruissem & Jones (eds.) 2000. Biochemistry & Molecular Biology of Plants. Chapter 11. Cell Division Regulation.

5. ’Omnis cellula e cellula’ • similar fundamental mechanisms are operational at the core of the cell-division cycle of all eukaryotes. • the mechanisms of pattern formation have evolved independently in plants and animals.

6. How crucial is the plant cell cycle as a point of control in plant development? • Cell proliferation – meristems in some trees. How do they avoid the cell cycle running out of control? • Cell cycle genes –the complement of cell cycle genes in plants is more complex than in animals • plants’ sessile lifestyle i.e. well adapted to adjust their development in response to changing environmental conditions? Developmental fine tuning? • Cell cycle and development • cellular theory, organismal theory > new and more complex concepts will emerge • The cell wall

7. Cell Lineage and Cell Position • Since plant cells do not migrate within the tissues, their position is of vital importance in the development of the tissues. However, lineage does not restrict development to a specific fate. • Examples can be found in mutation studies in developing leaves or in ablation experiments in the root tip.

8. Examples: tangled1 mutant of maize and fass mutant of Arabidopsis

9. Mitotic cell cycle • Within the shoot apices of intact plants cell division is essentially asynchronous with little or no coordination of division timing between cells. • Most suitable for detailed analysis are in vitro suspension cultures of plant cells i.e. cell division is removed from any developmental context. • cultures capable for various degrees of synchronization have been developed for Acer, Catharanthus, alfalfa, Nicotiana. • the best levels of synchronization – tobacco Bright Yellow-2 (BY-2) cell line.

10. Mitotic cell cycle Control point • The gap phases allow the operation of controls that ensure that the previous phase has been accurately and fully completed. The major regulatory points are G1/S, G2/M & metaphase/anaphase boundaries. Control point

11. Control points of the Cell Cycle

12. DNA replication is strictly controlled during the cell cycle • Initiation of DNA synthesis is inhibited in wild-type cells during G2, M and G1 • DNA synthesis in S-phase is initiated at discrete origins of replication distributed at regular intervals throughout the genome. They occur on average every 36 kb in yeast, 66 kb in dicots and 47 kb in monocots.

13. DNA replication is strictly controlled during the cell cycle A large number of proteins interact directly and indirectly with the origins to control progression through the chromosomal cycle. ORC = origin recognition complex

14. DNA replication is strictly controlled during the cell cycle • During late M & G1, the assembly of the protein complexes that mediate initiation of DNA synthesis is promoted = the cells become competent to initiate DNA synthesis.

15. DNA replication is strictly controlled during the cell cycle • To prevent premature DNA replication, the replication proteins that associate with ORC are assembled in steps. • Cdc6 protein binds first • then MCM and Cdc45 • prereplication complex is activated by protein phosphorylation at the restriction point (START of S phase) • phosphorylation is achieved by Cdc7/Dbf4p protein kinase complex > MCM complex releases from ORC & facilitates access of DNA polymerases to the template. • if mitotic kinase activity is suppressed in S or G2 phase, rereplication will occur without intervening mitosis

17. Mitosis • Mitosis is suppressed during G1, S and G2 • Onset of M-phase / initiation of chromosome condensation and the disassembly of the nuclear matrix from the cytoplasm • the cells are not yet competent for chromosome segregation

18. Regulation of mitosis • condensins and cohesins > assemble long chromatin fibres into chromosomes > replicated DNA is able to be segregated without damage • cohesion proteins Scc1p & Smc1p are synthesized during S phase (sister chromatids need to be joined in S phase) • Inhibitory proteins such as Pds1p accumulate and bind and antagonize to Cut1p protein • Cut1p breaks the linkage between sister chromatids • anaphase-promoting complex activated by protein phosphorylation tags the mitosis inhibitor Pds1p for proteolysis > destruction of cohesion proteins

19. Chromosome condensation and kinetochore complex

20. Chromosome segregation • segregation in suppressed during metaphase by Pds1p (inhibitor of Cut1p) • metaphase –anaphase transition is activated by phosphorylation of APC which is catalyzed by CDKs & CDC5 • Activation of APC results in ubiquination of Pds1p, which is recognized by the 26S proteasome and will be degraded ………

21. Cell cycle is controlled in multiple points by CDKs • cyclin dependent kinases • yeast have a single CDK which has PSTAIRE sequence within its cyclin-binding domain • In higher eukaryotes there are multiple additional CDKs that have roles at different points in the cell cycle • In plants CDKA to CDKE

22. Cyclins • diverse group of proteins with low homology that share a large, poorly conserved region (cyclin core), which is responsible for their interaction with CDKs. • In plants there are cyclins A, B, C, D & H.

23. Cyclins • A-type cyclins (also known as S cyclins) appear at the beginning of S-phase and will be destroyed at G2/M transition. Cyclic expression / abundance. • B-type cyclins (also known as M cyclins, mitotic cyclins) appear at G2, control G2/M and are destroyed at anaphase. Cyclic expression / abundance. • D-type cyclins control progression through G1 and S-phase. Presence depends on extracellular signals that stimulate or maintain division.

24. CDK activity is regulated at multiple levels Activity depends on several factors: • levels of cyclins and CDK (transcription, translation, protein turnover, sequestration, intracellular localization) • CAK activates phosphorylation • inhibitory WEE1-mediated phosphorylation

26. CDK inhibitors have a key role in controlling cell cycle progress • ICK = CKI =inhibitors of CDK or KRP (Kip-related proteins) which bind both CDK and cyclin subunits. • ICKs are used by the cell to control CDK-cyclin complex activity before undergoing cell cycle transitions and to temporarily arrest the cell cycle in response to DNA damage or to other signaling pathways. • In Arabidopsis ICK1 gene is induced by treatment with ABA and probably mediates the cell cycle arrest.

27. Cell Cycle Inhibitors and Root Tip Development

28. CDK activity is regulated at multiple levels CDK subunit proteins (CKS) scaffold interactions with target substrates by WEE1 kinase CAK = CDK activating kinase by binding with inhibitory proteins e.g. KRP

29. Cell cycle control • domino model -”clock” model > combination model • In living systems, biochemical reactions do not always proceed to completion and a cell undergoing division can experience adverse conditions that could damage DNA or spindle apparatus > checkpoints at which cell can monitor completion of specific reactions are important.

30. The Cell Cycle

31. Once cell division is completed, how does the cell regulate its entry into a new cycle? • In somatic cells G1 cyclins (D-type) play an important role and their synthesis is coupled with growth. • cyclin D dependent CDK activity increases.

32. E2Ftranscription factor family & Rb = retinoblastoma gene family are accessory proteins required to enforce CDK control of cell cycle progression • E2F essential during DNA replication, critical effectors of the decision to pass the G1-to-S and allow the cell to procede into S phase. • autocatalytic transcription loop creates a need for an inhibitor =Rb to inactivate E2F.

33. E2F transcription factor activation

34. E2F transcription factor activation

35. Intercellular communication controls the cell cycle during growth and development • Nutrition limitation is the most important factor restricting cell growth in single-cell organisms and the cell size is the major cue for division. • In plants only a few stem cells organized into meristems divide to produce the plant body. • In most multicellular organisms, social control prevails over nutritional control of the cell cycle.

36. Intercellular communication controls the cell cycle during growth and development • Cell cycle control is integrated with the regulation of cell expansion, differentiation and cell death. • Cells in multicellular organisms are normally quiescent and they do not proliferate without stimulation. • Stimuli provide the cell with information about the status of the whole organism (rather than just its individual cells). • If only a few cells permanently retain the capacity to divide, the majority of cells that have lost this capacity must be instructed how to do so.

37. Cells need stimulation to proceed with proliferation

38. Plant growth regulators and cell cycle • Plant hormones affect cell proliferation. • Because most hormones also provoke morphogenetic effects, the cell-cycle consequences may be direct or part of the morphogenetic response.

39. Plant growth regulators and cell cycle • Auxins and cytokinins have direct roles in cell proliferation: • withdrawal of auxin from tobacco cell cultures arrests cell cycle in G1. • withdrawal of cytokinin from tobacco cell cultures arrests cell cycle in G2. • In Arabidopsis cytokinin is required to stimulate CYCD. Auxins and cytokinins have direct roles in cell proliferation. • Cytokinins have effects on G1/S and G2/M as well as progression through S phase. • ABA – expression of the CDK-cyclin complex inhibitor (ICK1) is induced in the root during water stress and probably mediates the cell cycle arrest in the apical meristem.

40. Plant hormones and cell cycle control Sucrose, auxin and cytokinin promote accumulation of CYCD, and therefore support entry into new cell cycle. Cytokinin promotes entry into M-phase. ICK = CKI = inhibitors of KRP (Kip- related proteins) which bind both CDK and cyclin subunits.

41. Auxin and cell cycle control

42. Endoreduplication • Dewitte, W. & Murray, J.A.H. 2003. The Plant Cell Cycle. Annu. Rev. Plant Biol. 54: 235-264. • Sugimoto-Shirasu, K. & Roberts, K. 2003. Current Opinion in Plant Biology 6: 544-553.

43. An alternative cycleendoreduplication • in differentiating plant cells. • characterized by an increase in the nuclear ploidy level that results from repeated S phases with no intervening mitosis. • This occurs only after cells have ceased normal mitotic cycles. • e.g. in Arabidopsis this produces ploidy levels up to 32 C in the final stages of leaf development. • takes place also in trichomes and in leguminous nodules.

44. An alternative cycleendoreduplication • The entire complement of chromosomes is usually re-replicated. • chromosomes go through condensation and decondensation stages after replication and sister chromatids separate > polyploidy • chromosomes replicate without undergoing such condensation stages and sister chromatids remain closely associated > polyteny • The term polyploidy is often used more generally to describe all types of endoreduplicated chromosomes.

45. Endoreduplication in • Arabidopsis (from Sugimoto-Shirasu, K. & Roberts, K. 2003.) • a pair of stomatal • quard cells 2C • b. a small epidermal cell • 4C • c. a large epidermal • cell 8C • d. trichome 32C • heterochromatic • centromeric areas • are few in number > • polytene chromosomes

46. Ploidy and cell size Datura stramonium Different cell layers have different ploidies green nuclei 8C yellow nuclei 2C

47. Switching from cell cycle to endocycle • During the normal mitotic cell cycle, cells have a mechanism that licenses chromosomes to replicate only once each cycle, after an intervening mitosis. • The key step in the switch to endoreduplication is to allow cells to start another round of DNA replication while at the same time inhibiting mitosis. • G1-S transition; endocycle appears to use much of the same machinery to re-enter S-phase.

48. ORC = origin of recognition complex Cdc6 = cell division cycle 6 protein MCM = minichromosome maintenance complex when replication starts all proteins dissociate except ORC

49. In yeast, mitotic cyclin B (CycB/Cdc2) then binds to replication origins via ORCs during G2 and early mitosis thus preventing rereplication. In mitotic cell cycle CycB is degradaded in late metaphase > reassembly of prereplication complex & licensing of next round of DNA replication.

50. An alternative cycleendoreduplication • a general view is the downregulation of CYCA1, CYCA2,CYCBs and CDKB in endoreduplicating cells. • CYCB1 overexpression induced mitosis i.e. this may be a limiting factor for entry into mitosis. • The loss of mitotic cyclins is involved in the switch from mitotic cycles to endocycles but the upstream mechanisms are still unknown.