Microfibril orientation in wood cells: new angles on an old topic

trends in plant science Meeting Report Recent work1 could stimulate a reappraisal of the early work on elusive plant-derived mitogens. Perhaps neoplasm-inducing molecules more active than traumatin exist in pea or bean pods and, with modern methods, these substances might be more accessible. However, for the time being, bruchins might be among the first examples of potent lipid regulators of mitosis in plants and animals. This possibility should be borne in mind in future studies of these intriguing compounds. Edward E. Farmer Gene Expression Laboratory, Institute of Ecology, Biology, University of Lausanne, 1015 Lausanne, Switzerland (tel 141 21 692 4190; fax 141 21 692 4195; e-mail [email protected])

References 1 Doss, R.P. et al. (2000) Bruchins: insect-derived plant regulators that stimulate neoplasm formation. Proc. Natl. Acad. Sci. U. S. A. 97, 6128Ð6223 2 Shorthouse, J.D. and Rohrfritsch, O.E., eds (1992) Biology of Insect-Induced Galls, Oxford University Press 3 Doss, R.P. et al. (1995) Response of Np mutant of pea (Pisum sativum L) to pea weevil (Bruchus pisorum L) oviposition and extracts. J. Chem. Ecol. 21, 97Ð106 4 Kolattukudy, P.E. (1980) Cutin, suberin and waxes. In The Biochemistry of Plants (Vol. 4) (Stumpf, P.K., ed.), pp. 571Ð645, Academic Press 5 Bonner, J. and English, J. (1938) A chemical and physiological study of traumatin, a plant wound hormone. Plant Physiol. 13, 331Ð348

6 English, J. et al. (1939) The wound hormones of plants, II. The isolation of a crystalline active substance. Proc. Natl. Acad. Sci. U. S. A. 25, 323Ð329 7 English, J. et al. (1939) Structure and synthesis of a plant wound hormone. Science 96, 329 8 Wehnelt, B. (1927) Untersuchungen Ÿber das Wundhormon der Pflanzen. Jahrb. Wiss. Bot. 66, 773Ð813 9 Farmer, E.E. (1994) Fatty acid signalling in plants and their associated microorganisms. Plant Mol. Biol. 26, 1423Ð1437 10 Zimmerman, D.C. and Coudron, C.A. (1979) Identification of traumatin, a wound hormone, as 12-oxo-trans-10-dodecenoic acid. Plant Physiol. 63, 536Ð541

Microfibril orientation in wood cells: new angles on an old topic It is widely recognized that woods differ between trees; use of the terms ÔsoftwoodÕ and ÔhardwoodÕ is a crude acknowledgement of this phenomenon. It is also apparent that some woods are better for some purposes than others. This is because of both the heterogeneity of the cell types that make up the different woods and the structure of the individual cells. Chief among the structural features that determine the physical characteristics of the wood (and hence its ÔusefulnessÕ) are those concerned with cell walls. Wood cell walls are highly structured, with several layers within the secondary (S) cell wall, of which the S2 layer is the thickest and the major contributor to the overall properties of the wall. One of the most important aspects of the cell walls is the angle that the cellulose microfibrils make with the long axis of the cells; this Ôis now generally acknowledged as one of the main determinants of stiffness in woodÕ1. Aspects of the biology and importance of the microfibril angle (MFA) within wood fibres were considered recently at an international workshop*.

fact that the MFA might also have survival value for the tree. This was considered by Helga Lichtenegger (Austrian Academy of Sciences/University of Leoben, Austria), who used non-destructive small-angle X-ray scattering (SAXS) to measure the MFA, and also performed destructive tensile tests on the same blocks of wood. This work showed that, as the MFA increased (i.e. as the microfibrils diverged more from the vertical), both YoungÕs modulus (ÔstiffnessÕ) and tensile strength decreased, whereas cell wall extensibility increased. In the growing tree, the observed MFAs correlated well with the mechanical stresses most likely to be experienced at different stages of growth. For example, high MFAs (giving optimal cell wall extensibility) are a feature of young trees and branches (juvenile wood), which have to bend without breaking. By contrast, older trees (mature wood), which have to withstand compressive forces caused by the treeÕs own weight, have low MFAs, which tend to maximize stiffness.

Relevance of microfibril angle

One of the great dogmas of 20th century biology was DNA ® RNA ® protein. Another is that cytoplasmic microtubules direct the orientation of cellulose microfibrils within the growing cell wall. This view has been widely promoted since the suggestion, almost 40 years ago, that Ôit may be of some significance that the tubules just beneath the surface

How is microfibril angle created?

With the emphasis on the end-user requirements for wood, it is easy to overlook the *The European Community COST Action E20 (Wood Fibre Cell Wall Structure) Workshop on Fibre Wall and Microfibril Angle, Athens, Greece, 11Ð13 May 2000. 360

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of the protoplast mirror the orientation of the cellulose microfibrils of the adjacent cell wallsÕ2. However, although there are many instances of co-orientation of these two structural features within plant cells, there is a body of evidence that challenges the universal validity of this dogma. In view of the importance of the MFA to the overall mechanical properties of wood cells, deciding whether this ÔmicrotubuleÐmicrofibril paradigmÕ is correct or not is of great importance, and is highly relevant to any attempts to modify the MFA by influencing microtubule angles. Theory

The case against microtubule involvement in orientation of microfibrils during S-wall formation was elegantly made in the talks by Anne Mie Emons (Wageningen University, The Netherlands) and Bela Mulder (FOM Institute for Atomic and Molecular Physics, Amsterdam, The Netherlands). Their relatively simple mathematical model relies on the geometry of the cell, rather than biological influence from cytoplasmic structures, to produce the observed arrangement of microfibrils3. Although the model was developed from the special case of root hairs of Equisetum hyemale, it is considered to be applicable to S-wall formation in all cells. However, it has yet to be fully tested, particularly in the wood-forming system of trees.

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trends in plant science Meeting Report Practice choice for most participants at Nigel Chaffey (IACR Long Ashton this workshop. X-ray analysis is Research Station, Long Ashton, UK) extremely useful from a materials presented the latest information on the science point of view, but it only role of the microtubular and microfilgives historical information. If we ament cytoskeletons during developwant to know the orientation of the ment of normal wood, gelatinous microfibrils that are currently being and phloem fibres in hardwood trees. deposited and also want to know the Microfilaments remain largely axial orientation of the microtubules throughout the development of then we need techniques that can normal wood and gelatinous fibres, distinguish these nascent microwhereas microtubules adopt a wide fibrils from older ones. Immuvariety of angles in normal wood nolocalization is an obvious candifibres (Fig. 1a) but were axially oridate but, although such procedures ented in gelatinous fibres (Fig. 1b). are well advanced for microtubules, Phloem and normal wood fibres have a similar breakthrough is not yet similar arrangements of microtubules available for cellulose. and microfilaments. ThereÕs more to wood than Notwithstanding the demonstrated cellulose co-orientation of microtubules and Notwithstanding the focus on microfibril-like features in the fibres, microfibrils at the workshop, celluit is necessary to show that such wall lose is only one of a wide variety of features are in fact cellulose microfibwood cell wall components. Katia rils before conclusions can be drawn Ruel (CERMAV, Grenoble, France) about the validity of the micropresented recent work on the tubuleÐmicrofibril dogma in such immunolocalization of lignins and cells. However, it suggests that microdemonstrated the complicated topotubule angles can vary markedly from chemistry of the distribution of varicell to cell along a radial file (Fig. 1), ous lignins within and between the which might correspond to the walls of different wood cells. Lignins changes in microfibril orientation are of particular interest in wood with successive lamellae of the S-wall pulping, because they must be relayers that have been reported in the moved from the fibres to produce past. Furthermore, the axial arrangehigh-quality paper, and in the contriment of microtubules in gelatinous bution they make in planta to the fibres corresponds to the near-axial overall physical strength of the wood. orientation of microfibrils recorded However, there is also a link here for this cell type in the literature. with the MFA, because the orienSerendipitously, experimental eviFig. 1. Confocal laser-scanning micrographs of fluorestation of the microfibrils might well dence supporting the view that cently immunolocalized microtubules (green) within dictate the deposition patterns of this microtubules are required to change radial longitudinal sections of wood fibres in the stem of complex polymer. cellulose microfibril angle in the hybrid aspen (Populus tremula 3 Populus tremuloides). (a) S-wall of tracheids of Pinus radiata Normal wood fibres. (b) Gelatinous fibres from tension Benefits of age was presented in a poster by Anna wood. Notice the variety of microtubule angles (arrows) As demand for wood continues Wilkes (University of Canterbury, in normal wood fibres compared with the near-axial orientation within gelatinous fibres. Scale bar 5 25 mm. to increase5 and as new trees are New Zealand). Furthermore, recent developed that grow faster than their work on fixed material from gympredecessors6, the proportion of juvenosperms has also identified parallels between the orientation of cortical micro- and in determining the physical or morpho- nile wood (wood produced during the first tubules and of cellulose microfibrils during logical characteristics of fibres. The new age 5Ð25 years of a treeÕs life) in the harvested of wood microscopy was brought into focus timber increases. One of the characteristics of the differentiation of tracheids4. with presentations on acoustic microscopy by juvenile wood (compared with mature wood) Historical perspective HŒkan Lindstršm (University of Canterbury, is a relatively high MFA, which generally In a timely summary of the historical per- Christchurch, New Zealand) and atomic-force leads to such wood being considered ÔinferiorÕ spective of studies of the wood cell wall microscopy (also known as scanning-probe to mature wood. This view was underlined (which is at least 150 years old), Uwe Schmitt microscopy) by Jouko Peltonen (•bo by Marc Herman (Catholic University of (Institute for Wood Biology and Wood Akademi University, Turku, Finland) and Louvain, Belgium), who examined the differProtection, Hamburg, Germany) reminded us Monika …sterberg (Helsinki University of ences between fast- and slow-grown Norway of the great debt we owe to those early pio- Technology, Finland). spruce (Picea abies). Although growth rate neers, whose seminal discoveries were made Over the years, many methods have been did not affect the morphological characteriswith relatively unsophisticated microscopes. proposed for measuring the MFA (e.g. orien- tics of the tracheids, those from fast-grown It is gratifying to see that microscopy still has tation of pits, iodine staining, polarized light). trees had a significantly higher MFA and as important role to play in modern attempts All methods have their drawbacks but X-ray so were predicted to have lower tensile and to understand wood cell structure and biology, analysis was evidently the current method of tear strength. September 2000, Vol. 5, No. 9

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trends in plant science Comment Designer wood?

Testing times

Another common feature of fast-growing trees, such as poplars, is the occurrence of gelatinous fibres (Ôtension woodÕ)7, which compromise the commercial value of such timber8. However, gelatinous fibres are characterized by a particularly low MFA, especially in their largely cellulosic innermost gelatinous (G) wall layer. This, together with the wide variety of MFAs that naturally exists within any tree, indicates that the wood-forming system has the capacity to generate almost any MFA desired by the end-user. A low MFA is generally considered to be more ÔusefulÕ and, because it can be achieved even in juvenile wood (albeit in specific cell types), the opportunity for developing Ôdesigner woodÕ9 with particularly useful qualities might yet become a reality. Of course, such applications are a long way off, but their possibility must act as a further incentive to study the cell biology of wood formation. However, the MFA is but one aspect of the biology of wood and, although physical characteristics of wood cells are intensively studied, Pekka SaranpŠŠ (Finnish Forest Research Institute, Vantaa, Finland) reminded us of the dearth of information on the biological aspects of wood formation. Accordingly, he presented work dealing with the effect of growth rate on fibre properties in Norway spruce. Among the findings was a reduction in wood density at higher growth rates, which was correlated with a decrease in cell-wall proportion.

Although it is essential to obtain experimental evidence about any role for microtubules in microfibril orientation, the difficulty of accessing the developing wood cells in trees has to date required the use of excised, fixed material. A system in which cell-biological events can be followed, and experimentally manipulated, in vivo is clearly desirable and will permit a much better understanding of all aspects of wood-cell differentiation. Arabidopsis, which also undergoes substantial wood-formation10, might have a role to play here, in tandem with poplar, the model hardwood tree, of course! Nigel Chaffey Dept of Plant Sciences, IACR-Long Ashton Research Station, University of Bristol, Long Ashton, Bristol, UK BS41 9AF (tel 144 1275 392181; fax 144 1275 394281; e-mail [email protected]) References 1 Butterfield, B.G. (1998) Preface. In Microfibril Angle in Wood (Butterfield, B.G., ed.), pp. 11Ð12, IAWA/IUFRO, Christchurch, New Zealand 2 Ledbetter, M.C. and Porter, K.R. (1963) A ÔÔmicrotubuleÕÕ in plant cell fine structure. J. Cell Biol. 19, 239Ð250 3 Emons, A.M.C. and Mulder, B.M. (2000) How the deposition of cellulose microfibrils builds cell wall architecture. Trends Plant Sci. 5, 35Ð40

4 Funada, R. et al. (2000) The role of cytoskeleton in secondary xylem differentiation in conifers. In Cell and Molecular Biology of Wood Formation (Savidge, R.A. et al., eds), pp. 255Ð264, Bios Scientific Publishers, Oxford, UK 5 Haygreen, J.G. and Bowyer, J.K. (1996) Forest Products and Wood Science (3rd edn), Iowa State University Press, Ames, IA, USA 6 Bues, C.T. (1990) Wood quality of fast and normal growing trees. In Fast Growing and Nitrogen Fixing Trees (Werner, D. and MŸller, P., eds), pp. 340Ð353, Gustav Fischer Verlag, Stuttgart, NY, USA 7 Isebrands, J.G. and Bensend, D.W. (1972) Incidence and structure of gelatinous fibers within rapid-growing eastern cottonwood. Wood Fiber 4, 61Ð71 8 Peszlen, I. (1996) Gelatinous fibers in Populus 3 euramericana clones. In Recent Advances in Wood Anatomy (Donaldson, L.A. et al., eds), pp. 327Ð333, New Zealand Forest Research Institute, Rotorua, New Zealand 9 Chaffey, N.J. (2000) Cytoskeleton, cell walls, and cambium: new insights into secondary xylem differentiation. In Cell and Molecular Biology of Wood Formation (Savidge, R.A. et al., eds), pp. 31Ð42, Bios Scientific Publishers, Oxford, UK 10 Chaffey, N.J. (1999) Wood formation in forest trees: from Arabidopsis to Zinnia. Trends Plant Sci. 4, 203Ð204

Viral sequences integrated into plant genomes There are two groups of DNA viruses that infect plants. The single-stranded DNA (ssDNA) Geminiviridae replicate via a rolling circle mechanism. The double-stranded DNA (dsDNA) Caulimoviridae, comprising the caulimoviruses and the badnaviruses, replicate by reverse transcription. Unlike viruses of vertebrates and bacteria, the infection cycle of plant viruses is not known to involve integration; because the Caulimoviridae lack features associated with the retrovirus integration phase, they are called pararetroviruses. However, virus sequences are found integrated into plant genomes and, in some cases, have recently been implicated in causing episomal viral infection. The first reports of integration were of multiple direct repeats of partial geminivirus sequence, found in Nicotiana tabacum1. These sequences included only the origin of replication and the adjacent viral replication protein, transcription was not detectable and there was no associated virus infection. 362

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The caulimovirus petunia vein-clearing virus (PVCV) is vertically transmitted and hybridizes to the petunia genome2. Integrated sequences and virus infection have been linked2, although there is no evidence that the entire viral genome integrates, and there are no details of the relationship between integrated and episomal virus sequences. Similarly, caulimovirus-related sequences (tobacco-pararetrovirus-like, TPVL) have been found in the Nicotiana tabacum genome but no related episomal virus has been detected3. However, there are two cases in which the integration of virus sequences is strongly correlated with virus infection: banana streak virus (BSV) and tobacco vein-clearing virus (TVCV). Banana streak virus

BSV is a typical badnavirus, with a circular dsDNA genome of ~7.4 kb (Ref. 4). There have been serious outbreaks of BSV infection in a significant proportion of progeny from

different breeding and tissue culture programmes to improve Musa (banana and plantain). Modern Musa contains various ploidies and combinations of two original genomes, Musa accuminata (A genome) and Musa balbisiana (B genome), many of the widely used varieties being sterile triploids. The infected progeny of these programmes were derived from parents or mother plants showing no sign of infection. The polymerase chain reaction, using specific primers, amplifies BSV sequences from a wide range of Musa progeny, including some from asymptomatic plants. By contrast, detection methods based on viral symptoms and serology indicate a much lower incidence of the virus5. One explanation for these observations is the integration of BSV sequences into the host genome, which has recently been confirmed by two different approaches6,7. A genomic DNA library of plantain Obino lÕEwai (genome AAB), the maternal parent used in breeding programmes, was screened

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