The last stage of xylem development is programmed death of the cells, which is followed by complete autolysis of the cell contents. Two cell types predominate in the xylem, the vessel elements and the fibres, and my earlier research in Populus trees has revealed that both of these cell types display programmed cell death (PCD), but in a very different manner. I am interested in why the xylem fibres have to die and the underlying molecular mechanism. From the biotechnological point of view, the fibres should stay alive as long as possible, assince extending the lifetime of the fibres is expected to result in thicker cell walls and therefore higher biomass yields of wood.

Touminen Hannele 1150 2Identification of key regulators of xylem cell death should enable modifications in the cell death process using transgenic approaches, in order to test how they affect wood properties. This is especially tractable in xylem fibres, which constitute the main bulk of biomass.
We have taken three different approaches to identify key genes in the regulation of xylem cell death: one is based on knowledge from other PCD processes, mainly in Arabidopsis thaliana; one on sequencing of ESTs from Populus stem tissues undergoing fibre cell death, and the third isone based on microarray analyses of transcripts from various vascular tissues of Populus stems and comparative genom- ics approaches.

tuominen_1 tuominen_2
A longitudinal section of Populus wood is shown here after staining with DAPI, which stains nuclear DNA. The contours of the longitudinal xylem fibers and the radially oriented xylem rays are revealed by unspecific staining of the cell walls. The nuclei of the xylem fibers are appressed against the cell wall due to the high pressure from the vacuole in the living cells. When the fibers die, the vacuole bursts and the remaining cellular contents are degraded by hydrolytic enzymes released from the vacuole. We have studied xylem maturation in a cell culture system where xylem vessel like structures, called tracheary elements (TEs), differentiate in a semi-synchronous manner after hormonal stimulus. This cell culture system has allowed us to define a critical role for ethylene in xylem maturation by using various pharmacological agents. The figure shows three mature TEs with spiral-type secondary cell wall thickenings.

tuominen_3We have identified a metacaspase gene that is specifically expressed in the xylem elements of both /Populus /and Arabidopsis. The figure shows a confocal microscope image of green fluorescent protein (GFP) expression that is driven by the Arabidopsis /metacaspase 9/ promoter in the Arabidopsis root. The GFP signal is located in the nuclei of the vessel elements that can be recognised by the spiral cell wall thickenings.These analyses have revealed novel putative regulators of xylem PCD, as well as several homologues of known PCD regulators, such as vacuolar processing enzymes, autophagy-related genes, Bcl-2-associated athanogene (BAG) genes and a metacaspase gene. Three candidate genes were selected from these genes for detailed functional and molecular characterisation, encoding: a metacaspase, a bifunctional nuclease, and a polyamine biosynthetic ACL5. The functions of the metacaspase and bifunctional nuclease genes are currently being studied with reverse genetics approaches in both Arabidopsis plants and Populus trees.
The polyamine biosynthetic ACL5 gene has been found to be specifically expressed in the early developing vessels, and a mutation in ACL5 resulted in altered xylem development. Results of xylem morphology analysis and experiments with the xylogenic Zinnia elegans cell culture (see below) lead us to conclude that ACL5 prevents premature death of the developing xylem vessels to allow complete differentiation. This model is supported by the finding that transgenic Arabidopsis plants expressing the DT-A toxin gene under the control of the ACL5 promoter display similar alterations in xylem development to the acl5 mutant.
We have also studied the role of the plant hormone ethylene in xylem differentiation. In an in vitro tracheary element (TE) differentiation system of Zinnia elegans, we showed that ethylene has an important role in the control of lignification and cell death of TEs, since application of ethylene signalling inhibitors blocked both of these processes. We also created suppressive subtractive hybridisation (SSH) libraries in Zinnia elegans, in order to identify genes that were
1) active during the cell death phase of vessel differentiation and 2) that were dependent on the cell-death stimulatory effect of ethylene. One gene fulfilling these criteria was a PIRIN gene that also showed activity during the xylem cell death stage in Populus. There are four PIRIN genes in Arabidopsis, and we have started functional characterisation of this whole gene family.

sweden_greySvensk sammanfattning

Publication list

  1. Extracellular peptide Kratos restricts cell death during vascular development and stress in Arabidopsis
    J Exp Bot. 2019 Feb 7 [Epub ahead of print]
  2. Ethylene-Related Gene Expression Networks in Wood Formation
  3. Transcriptional roadmap to seasonal variation in wood formation of Norway spruce
    Plant Physiol. 2018, 176(4):2851-2870
  4. A multi-omics approach reveals function of Secretory Carrier-Associated Membrane Proteins in wood formation of​ ​​Populus​​ ​trees
    BMC Genomics. 2018, 19(1)
  5. The function of two type II metacaspases in woody tissues of Populus trees
    New Phytol. 217 (4):1551-1565
  6. A collection of genetically engineered Populus trees reveals wood biomass traits that predict glucose yield from enzymatic hydrolysis
    Sci Rep 2017, 7(1):15798
  7. Metacaspases versus caspases in development and cell fate regulation
    Cell Death Differ. 2017, 24(8):1314-1325
  8. AspWood: High-spatial-resolution transcriptome profiles reveal uncharacterized modularity of wood formation in Populus tremula
    Plant Cell. 2017, 29 (7):1585-1604
  9. NorWood: a gene expression resource for evo-devo studies of conifer wood development
    New Phytol. 2017, 216(2):482-494
  10. Quick Histochemical Staining Methods to Detect Cell Death in Xylem Elements of Plant Tissues
    Methods Mol Biol. 2017;1544:27-36
  11. Contribution of cellular autolysis to tissular functions during plant development
    Curr Opin Plant Biol. 2017, 35:124-130
  12. METACASPASE9 modulates autophagy to confine cell death to the target cells during Arabidopsis vascular xylem differentiation
    Biol Open. 2016, 5(2):122-129
  13. A bHLH-Based Feedback Loop Restricts Vascular Cell Proliferation in Plants
    Dev Cell. 2015, 35(4):432-443
  14. Cooperative Lignification of Xylem Tracheary Elements
    Plant Signal Behav. 2015;10(4):e1003753
  15. GRIM REAPER peptide binds to receptor kinase PRK5 to trigger cell death in Arabidopsis
    EMBO J. 2015, 31(1):55-66
  16. PIRIN2 stabilizes cysteine protease XCP2 and increases susceptibility to the vascular pathogen Ralstonia solanacearum in Arabidopsis
    Plant J. 2014, 79(6):1009-1019
  17. Programmes of cell death and autolysis in tracheary elements: when a suicidal cell arranges its own corpse removal
    J Exp Bot. 2014; 65(5):1313-1321
  18. Thermospermine levels are controlled by an auxin-dependent feedback loop mechanism in Populus xylem
    The Plant Journal 2013, 75:685–698
  19. Post mortem function of AtMC9 in xylem vessel elements
    New Phytol. 2013, 200(2):498-510

  20. The Norway spruce genome sequence and conifer genome evolution
    Nature 2013; 497(7451):579-584
  21. Pesquet E, Zhang B, Gorzsás A, Puhakainen T, Serk H, Escamez S, Barbier O, Gerber L, Courtois-Moreau C, Alatalo E, Paulin L, Kangasjärvi J, Sundberg B, Goffner D, Tuominen H
    Non-Cell-Autonomous Postmortem Lignification of Tracheary Elements in Zinnia elegans
    Plant Cell. 2013; 25(4):1314-1328
  22. Bollhöner B, Prestele J, Tuominen H
    Xylem cell death: emerging understanding of regulation and function
    Journal of Experimental Botany 2012 63(3):1081-94
  23. Pesquet E, Tuominen H
    Ethylene stimulates tracheary element differentiation in Zinnia elegans cell cultures
    New Phytologist: 2011 190:138-149
  24. Vera-Sirera F, Minguet EG, Singh SK, Ljung K, Tuominen H, Blázquez MA, Carbonell J
    Role of polyamines in plant vascular development
    Plant Physiology and Biochemistry: 2010 48:534-539
  25. Fracheboud Y, Luquez V, Björkén L, Sjödin A, Tuominen H, Jansson S
    The control of autumn senescence in European aspen
    Plant Physiology: 2009 149:1982-1991
  26. Courtois-Moreau CL, Pesquet E, Sjödin A, Muniz L, Bollhöner B, Kaneda M, Samuels L, Jansson S, Tuominen H
    A unique program for cell death in xylem fibers of Populus stem
    The Plant Journal: 2009 58(2):260-274
  27. Overmeyer K, Kollist H, Tuominen H, Betz, C, Langebartels C, Wingsle G, Kangasjärvi S, Brader G, Mullineaux P, Kangasjärvi J
    Complex phenotypic profiles leading to ozone sensitivity in Arabidopsis thaliana mutants
    Plant, Cell and Environment: 2008 31: 1237-1249
  28. Muñiz L, Minguet EG, Singh SK, Pesquet E, Vera-Sirera F, Moreau-Courtois CL, Carbonell J, Blázquez MA, Tuominen H
    ACAULIS5 controls Arabidopsis xylem specification through the prevention of premature cell death
    Development: 2008 135:2573-2582
  29. Keech O, Pesquet E, Ahad A, Askne A, Nordvall D, Vodnala SM, Tuominen H, Hurry V, Dizengremel P, Gardeström P
    The different fates of mitochondria and chloroplasts during dark-induced senescence in Arabidopsis leaves
    Plant, Cell and Environment: 2007 30:1523-1534
  30. Ruonala R, Rinne PLH, Baghour M, Moritz T, Tuominen H, Kangasjarvi J
    Transitions in the functioning of the shoot apical meristem in birch (Betula pendula) involve ethylene
    Plant Journal: 2006 46:628-640
  31. Moreau C, Aksenov N, Lorenzo MG, Segerman B, Funk C, Nilsson P, Jansson S, Tuominen H
    A genomic approach to investigate developmental cell death in woody tissues of Populus trees
    Genome Biology: 2005 6:R34
  32. Overmyer K, BroschE M, Pellinen R, Kuittinen T, Tuominen H, Ahlfors R, Keinanen M, Saarma M, Scheel D, Kangasjarvi J
    Ozone-induced programmed cell death in the Arabidopsis radical-induced cell death1 mutant
    Plant Physiol: 2005 137:1092-1104
  33. Ahlfors R, Lång S, Overmyer K, Jaspers P, BroschE M, Tauriainen A, Kollist H, Tuominen H, Boix EB, Piippo M, InzE D, Palva ET, Kangasjarvi J
    Arabidopsis RADICAL-INDUCED CELL DEATH1 belongs to the WWE protein-protein interaction domain protein family and modulates abscisic acid, ethylene, and methyl jasmonate responses
    Plant Cell: 2004 16:1925-1937
  34. Tuominen H, Overmyer K, Keinanen M, Kollist H, Kangasjarvi J
    Mutual antagonism of ethylene and jasmonic acid regulates ozone-induced spreading cell death in Arabidopsis
    Plant J: 2004 39:59-69
  35. Vahala J, Ruonala R, Keinanen M, Tuominen H, Kangasjarvi J
    Ethylene insensitivity modulates ozone-induced cell death in birch
    Plant Physiol: 2003 132:185-195
  36. Ahlfors R, Keinanen M, Kollist H, Kuusela T, Lang S, Overmyer K, Pulkkinen P, Tuominen H, Kangasjarvi J
    Hormonal interactions and ROS-dependent cell death
    Free Radical Research: 2003 37:6-7
  37. Tuominen H, Puech L, Regan S, Fink S, Olsson O, Sundberg B
    Cambial-region-specific expression of the Agrobacterium iaa genes in transgenic aspen visualized by a linked uidA reporter gene
    Plant Physiology: 2000 123:531-541
  38. Overmyer K, Tuominen H, Kettunen R, Betz C, Langebartels C, Sandermann H, Kangasjarvi J
    Ozone-sensitive Arabidopsis rcd1 mutant reveals opposite roles for ethylene and jasmonate signaling pathways in regulating superoxide-dependent cell death
    Plant Cell: 2000 12:1849-1862
  39. Regan S, Bourquin V, Tuominen H, Sundberg B
    Accurate and high resolution in situ hybridization analysis of gene expression in secondary stem tissues
    Plant J: 1999 19:363-369
  40. Tuominen H, Puech L, Fink S, Sundberg B
    A Radial Concentration Gradient of Indole- 3- Acetic Acid Is Related to Secondary Xylem Development in Hybrid Aspen
    Plant Physiol: 1997 115:577-585
  41. Nilsson O, Tuominen H, Sundberg B, Olsson O
    The Agrobacterium rhizogenes rolB and rolC promoters are expressed in pericycle cells competent to serve as root initials in transgenic hybrid aspen
    Physiologia Plantarum: 1997 100:456-462
  42. Tuominen H, Sitbon F, Jacobsson C, Sandberg G, Olsson O, Sundberg B
    Altered Growth and Wood Characteristics in Transgenic Hybrid Aspen Expressing Agrobacterium -Tumefaciens T -DNA Indoleacetic -Acid Biosynthetic Genes
    Plant Physiology: 1995 109:1179-1189
  43. Tuominen H, Ostin A, Sandberg G, Sundberg B
    A Novel Metabolic Pathway for Indole- 3- Acetic- Acid in Apical Shoots of Populus- Tremula (L) X Populus-Tremuloides (Michx)
    Plant Physiology: 1994 106:1511-1520
  44. Sundberg B, Tuominen H, Little CHA
    Effects of the Indole- 3- Acetic- Acid (Iaa) Transport Inhibitors N- 1- Naphthylphthalamic Acid and Morphactin on Endogenous Iaa Dynamics in Relation to Compression Wood Formation in 1- Year- Old Pinus- Sylvestris (L) Shoots
    Plant Physiology: 1994 106:469-476
  45. Rinne P, Tuominen H, Sundberg B
    Growth-Patterns and Endogenous Indole- 3- Acetic- Acid Concentrations in Current- Year Coppice Shoots and Seedlings of 2 Betula Species
    Physiologia Plantarum: 1993 88:403-412
  46. Rinne P, Tuominen H, Junttila O
    Arrested Leaf Abscission in the Nonabscising Variety of Pubescent Birch - Developmental, Morphological and Hormonal Aspects
    Journal of Experimental Botany: 1992 43:975-982