lAWA Journal, Vol. 17 (3),1996: 269-310

XYLOGENESIS, GENETIC AND ENVIRONMENTAL REGULATION - A REVIEW- by Rodney Arthur Savidge Faculty of Forestry & Environmental Management, University of New Brunswick, Fredericton, NB E3B 6C2, Canada

SUMMARY A critique is provided of the physical and chemical control of primary and secondary xylem development in terms of mechanics, genetics, phylogenetics, and the larger field of plant physiology. Strengths and weaknesses of the phytohormone theory of vascular development are analyzed. Homeobox genes, sub-cellular phytohormone localization, anatomical responses to varied phytohormone ratios and dosages, polar auxin transport, second messengers, radial fluxes in water potential, in• tercellular signalling, lignin biochemistry, and the phylogenetic position of bryophytes in relation to xylogenesis are identified as some areas for future research. Homeodomain proteins are addressed in terms of cambial initials and cell-fate determination, and other genetic and environmen• tal factors controlling differentiation of diverse cellular phenotypes are reviewed. As a 'continuum hypothesis', it is proposed that the extent of secondary wall sculpturing during tracheary element differentiation is a function of the duration of homeotic gene expression. Key words: Xylem, cambium, phytohormones, homeobox genes, second messenger, water potential, lignin, bryophytes, 'continuum hypothesis'.

INTRODUCTION

No plant meristem can be more abundantly obtained in pure form than the cambial zone from trees, and no developing tissue can be more readily had for molecular physi• ology studies than that comprising cambial derivatives on their way to becoming wood. Curiously, the opportunities for progress have not yet been fully appreciated in the field of plant biology and, consequently, much remains to be done before any solid conclusion can be drawn about how xylogenesis is regulated in any woody species. The objective here is not simply to provide a conceptual bridge from static anatomical observations of mature woody elements to the perceived molecular dynamics of xylo• genesis, but also and even more pointedly to provide students of wood anatomy with both an awareness of the limitations of current understanding and an appreciation of opportunities for future research. Wood is the most abundant form of biomass in the terrestrial biosphere; however, the vegetative sporophytic structures of higher plants comprise a variety of tissues in

*) Dedicated to the memory of Philip Frank Wareing (1914-1996), pioneer in cambial physology.

Downloaded from Brill.com10/09/2021 12:10:54AM via free access 270 IAWA Journal, Vol. 17 (3),1996 addition to xylem, including epidermis., endodermis, cortex, pith, phloem, photosyn• thetic tissues, rhytidome, root caps, and meristems, and the function of each is presum• ably just as essential as that of xylem for plant growth and survival. While it is clear that most plant tissues arise directly from meristems, knowledge about the factors initiating and controlling the formation of any kind of tissue development is still rudi• mentary. In most species, xylem comprises differentiated cells of more than one type; that is, it is a complex tissue. Explaining how adjoining cells, initially of similar ap• pearance and identical genetic constitution, come to appear fundamentally different presents one of the most fascinating challenges in biology. The rudiments of the theory that seasonal cambial growth and wood formation are under phytohormonal regulation arose at least a century ago (see Savidge & Wareing 198 I b for a historical review, Aloni 1995, and Little & Pharis 1995 for recent re• views). The theory can be stated to the effect that phytohormones (notably auxin, but also others) produced in and exported from non-vascular tissues, such as apical meristems and leaves, promote vascular development in those cells making up their transport corridors. Following more than a half century of research, there can be no doubt that the theory has substantial merit. On the other hand, it might be more correct to say that the phytohormone theory for vascular development has never had to be defended against serious alternatives, than to argue that it has solidly withstood re• peated challenges. Only by doing everything reasonable to disprove and displace a theory can it gain real credibility. Although opportunities for research with phytohormones are discussed below, my primary aim here is to re-examine some more fundamental considerations in order to develop a broader and deeper perspective about possible control mechanisms in xylem morphogenesis.

LOGIC UNDERLYING PHYSIOLOGICAL INVESTIGATIONS

Physiology is the study of the growth, development and functioning of a living organ• ism and how those processes are regulated by the organism itself as well as by its external environment. As indicated in Figure I, morphology, anatomy, cytology, ge• netics, biochemistry, and ultrastructural investigations all come under the umbrella of

1'llYSIOLOGy

MORPHOLOGY Anatomy Cytology Fine structure studies Genetics Molecular structure Biochemistry Thermodynamics Organic chemistry Mechanical theory Inorganic chemistry Electromagnetic theory Physical chemistry Nuclear physics

Quantum mechanical theory

Fig. 1. Physiology as a multi-disciplinary science concerned with explaining how morphogenesis occurs.

Downloaded from Brill.com10/09/2021 12:10:54AM via free access Savidge - Xylogenesis 271 physiology, each presenting a different physical or chemical 'window' on the control of morphogenesis. Plant physiology attempts to integrate this multi-disciplinary infor• mation into a larger, coordinated picture to explain how plants capture energy and assimilate carbon dioxide, water, inorganic ions and other substances into physically and chemically complex forms of biomass. The first principle of biology is that 'Laws of chemistry and physics govern living systems', and physiology ultimately reduces to considerations of the organism as all three states of matter (comprising thousands of different molecular species) and differ• ent kinds of energy interacting in space over time. Physical chemistry is concerned with very similar problems to those of nuclear physics, and phenomena addressed by both reduce to considerations that are explained in terms of quantum mechanical theory (Fig. 1). Thus, although a scientist may choose to see and analyze a phenomenon such as xylogenesis using a physical, chemical or genetical approach, biological phenom• ena per se must involve interactions of matter and energy that cannot occur in isola• tion. In this light, any singular physical, chemical or genetical explanation advanced for the regulation of xylogenesis (for example, control by pressure, auxin, or DNA binding proteins) can at best be regarded as merely a starting point in understanding; it should not discourage testing of new ideas and continuing integration of knowledge. Approaches toward explaining the control of physiological processes at tissue or finer levels can logically be separated into considerations of intrinsic and extrinsic factors (Fig. 2). Intrinsic factors are those generated internally by the plant itself, and extrinsic factors constitute everything else -living and non-living - affecting the plant. Not all extrinsic factors are exclusively external, nor do all intrinsic factors remain exclusively internal to the plant. For example, microorganisms as extrinsic factors occur commonly within plants (e.g., xylem-limited bacteria and endophytes) as well as externally, and parasites (e.g., mistletoe) or fungi (e.g., mycorrhizae) may simulta• neously reside in both locations (Ng et al. 1982; Fett et al. 1987). Intrinsically gener• ated factors such as ethylene may emanate from a plant and elicit a response as a component of the plant's extrinsic environment.

Factor (intrinsic or extrinsic)

role? I PROMOTER INHIBITOR I I nature? nature? I I I I PHYSICAL CHEMICAL PHYSICAL CHEMICAL I I I I identity? identity? identity? identity? I I I I mobility? mobility? mobility? mobility? Fig. 2. Flow diagram indicating questions that should be answered at the beginning of any physiological investigation.

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The investigative process to explain the control of morphogenesis traditionally has be• gun by determining whether intrinsic or extrinsic promoters or inhibitors exist (Fig. 2). To accomplish this, a bioassay simulating whatever aspect of morphogenesis happens to be under investigation must be devised. Five complimentary bioassay systems have been used to investigate the regulation of xylogenesis. I) Cell-free soluble, particulate and organellar systems have been investigated by biochemists and biophysicists to determine the enzymes, kinds of energy, and other factors affecting development at the sub-cellular level (Bevan & Northcote 1979; Demura & Fukuda 1993; Northcote 1993; Forster & Savidge 1995; Udagama-Randeniya & Savidge 1995). Enzymatic bioassays can, upon isolation of the enzyme, lead to characterization and manipulation of genes associated with cellular differentiation or morphogenesis (Demura & Fukuda 1994; Antosiewicz et al. 1995). Biophysical investigations can lead to a better understanding of the relationship between structure and function (Hasenstein & Rayle 1984; Roberts 1992; Wildon et al. 1992; Roberts & Haigler 1994). In addition, thermodynamic analyses can provide a rational basis for predicting the likelihood of a particular type of matter being produced during morphogenesis (Shigematsu et al. 1995). 2) The individual cell, as the fundamental unit of all living organisms, can be isolated from tissues and its capability for differentiation investigated in cell suspensions or as protoplasts lack• ing cell walls (Albinger & Beiderbeck 1983; Reynolds 1987; Church 1993; Leinhos & Savidge 1993). 3) Individual plant tissues can be explanted to defined environmental conditions in attempts to induce or inhibit differentiation (Minocha & Halperin 1976; Liskova 1985; Kutemozinskaet al. 1988, 1991; Wilson & Wilson 1991; Savidge 1983b, 1993b; Leitch & Savidge 1995).4) Plant organs, such as stem or root Guttings, have long served as convenient bioassays to investigate xylogenesis (Loomis & Torrey 1964; Torrey & Loomis 1967; Sheriff 1983; Zakrzewski 1983; Gersani 1987; Savidge 1994).5) Finally, development can be investigated in whole plants (Blum 1971; Sharma et al. 1979; Starbuck & Phelps 1986; McDaniel et al. 1990; Little & Sundberg 1991; Burrows et al. 1992; Ma & Steeves 1992; Wang et al. 1995). With the increasing tech• nological capability to transform plants genetically, the whole plant bioassay system is becoming an important one for determining the roles of particular genes in controlling morphogenesis (Li et al. 1992; Sitbon et al. 1992; Halpin et al. 1994; Klee & Romano 1994). Regardless of the bioassay system used, the 'control' for the investigation involves the same system except that the bioassay is not treated with the extrinsic or intrinsic factor which is under investigation. The bioassay response may be assessed either qualitatively or quantitatively, or both. When evidence for a promotor or inhibitor has been obtained, its identity can be pursued and, once known, that physical or chemical factor can be further characterized as being transmissible (i.e., cell-to-cell mobile, such as auxin or water), mobile between cells only when conduits such as plasmodesmata are present, or essentially non-mobile (e.g., chromosomal DNA). Systematic completion of the process indicated in Figure 2 sets the stage to perform ancillary investigations using the same system. For example, within the scope of the phytohormone theory, it might be asked whether a promoter or inhibitor is ever limit• ing by not being present in the cambium at some point during the annual cycle and, if

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Individual cells

Fig. 3. Possible corroborative linkages between the five types of bioassay systems. so, what the cambial response is to that limitation. In addition to undertaking ancillary investigations, attempts can be made to corroborate the research findings using other bioassay systems, as indicated in Figure 3. Corroborative research using a particular species should ideally proceed reduc• tionistically from whole plants to cell-free systems (counterclockwise in Fig. 3). Sys• tematically reductive investigation into the control of xylem morphogenesis has not been usual, however. Evidently, the bioassay system and choice of species have rarely if ever been based on a perceived need to corroborate reported findings generated from another bioassay system. The findings based on a bioassay system are, more often than not, projected directly to the whole plant and, perhaps too often, to other species. In early stages of physiological investigation near the end of the 19th and beginning of the 20th centuries, singUlar endogenous regulators were sought by extracting sub• stances from plant tissues using solvents of different polarities (such as methanol, ether and chloroform) followed by bioassaying, fractionating, re-bioassaying, purifying, and chemically characterizing the active component(s). Through these highly focussed ef• forts, a number of regulatory molecules (phytohormones and biochemical regulators) were identified in plants. Many can now be chemically synthesized and are readily available for further investigation. These achievements set the stage for immense pro• gress in understanding how endogenous regulators control morphogenesis and cellular differentiation in plants. It cannot be stated too strongly, however, that the discovered regulatory factors have, for the most part, been found by macroscopic analysis and only subsequently tested for anatomical effects (Savidge 1983a). Although anatomi• cally based bioassays are tedious and slow compared to the macroscopic approach, it may be that some regulators of plant cellular differentiation - such as those affecting bordered pit, perforation plate, or trabecula development - will await discovery until the focus becomes microscopic (Savidge 1985, 1994). Endeavours to discover such regulators will require cross disciplinary research, combining at a minimum the talents of plant anatomists, physiologists, and chemists.

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1 Increasing bioassay response No Promotion Inhibition effect I 0

----Increasing amount of factor under investigation ... Fig. 4. Theoretical dose response curve generated in a bioassay by investigating different molarities of a 'promotory' factor.

Only a small fraction ofbioactive compounds already isolated have yet been inves• tigated for effects on xylogenesis, and considering the immense range of biodiversity existing among vascular plants, they have been tested on a very inadequate number of species. Dicots, followed by conifers, have received the most attention (Savidge & Wareing 1981 b; Roberts et a1. 1988). Almost nothing has been done with horsetails, lycopods, and ferns, and very little with monocots. Moreover, dose response curves are virtually non-existent for xylogenesis. As Figure 4 indicates, testing a putative regula• tor at only one concentration in a bioassay and finding no evidence for a response does not permit the conclusion that the factor is biologically inactive. The absence of activ• ity could be due to either insufficient or excessive amounts having been tested. This possibility can be investigated by systematically varying the concentration, as implied in Figure 4. Note that it is possible for a promoter to also inhibit if present in excess (Sheriff 1983; Savidge 1993b). The converse, however, evidently does not apply. What Figure 4 does not indicate is the possibility for related but nevertheless distinct re• sponses occurring within a single tissue over the active range of the tested factor (Savidge 1983b; Leitch & Savidge 1995). Different species may respond in fundamentally distinct ways to the identical treat• ment. For example, exogenous gibberellic acid applied to stem cuttings promotes cam• bial activity in hardwoods and not in most conifers (Little & Savidge 1987), and auxin applied to a fern stimulates parenchymatous leaf gaps rather than vascular tissue to develop (Ma & Steeves 1992). With current knowledge, each tissue of each plant spe• cies merits its own dose response curve. Once lists of promoters and inhibitors are available and the dose response curves for those factors are known, complex multiple-factor experiments become possible. The combined effect of two promoters might be investigated over a range of molarity ratios in order to discover possible interactions. If the quantitative response to two or more promoters (or inhibitors) is greater than the sum of the responses to the indi• vidual factors, the factors are said to be synergistic in relation to the type of morphogenesis under investigation. Antagonistic effects between promoters and in• hibitors can also be investigated. For example, one might want to look at the response

Downloaded from Brill.com10/09/2021 12:10:54AM via free access Savidge - Xylogenesis 275 of the active cambium to a combination of short days (an established inhibitor) and exogenous auxin (a known promoter). Only a very limited amount of multiple factor research has yet been undertaken into the regulation of xylogenesis. Over the last two decades there has been a major shift in research emphasis within plant physiology, from discovering effects of externally provided factors to observing development in transgenic plants. By far, the predominant effort currently is directed at determining, localizing and manipulating DNA sequences in Zea mays (corn), Ni• cotiana tabacum (tobacco) and Arabidopsis thaliana (a small weed of the mustard family). Although tobacco and Arabidopsis exhibit only limited secondary growth (Lev• Yadun 1996), and corn none, the information coming from these angiosperms will nevertheless provide a starting point for addressing the problems of cambial develop• ment and wood formation in trees.

MECHANISTIC PERSPECTIVES

The ordered inner structure of plants was emphasized by Antoni van Leeuwenhoek, Nehemiah Grew and Marcello Malpighi in the 1600's (Baas 1982). These early inves• tigators described the structure of annual rings in trees and recorded the difference between early- and latewood for the first time. Malpighi evidently was the first to call xylem elements 'tracheae' because of similarities he noted between tracheary elements and the respiratory tubes of insects. Grew used the term 'parenchyma' to refer to the rela• tively thin-walled more or less isodiametric cells in plants, and he also decided that the whole body of a tree is "truly continuous by means of the parenchyma" (Grew 1682). Grew adopted the term 'cambium' from the same Latin word, meaning a place of commercial exchange, because he perceived the cambium to be some kind of sap, rather than living cells, having the ability to secrete materials both inward and out• ward, to form wood and bark, respectively. Plausible as such a mechanism for produc• tion of woody material may be, no evidence has yet been provided that wood (or inner bark) originates in any species other than through highly ordered 'secretion' of radial files of living cells, some of which die after producing woody walls, and others survive for many years despite having been incorporated into otherwise dead xylem. In general, the mechanistic processes underlying any type of morphogenesis com• prise phenomena of cellular differentiation. 'Cellular differentiation' sometimes con• veys preconceived anatomical considerations of undifferentiated meristematic cells reaching their ultimate destiny; however, in the final analysis there is no such thing as an 'undifferentiated' cell with which to compare differentiated cell types. Thus, cellu• lar differentiation can be more fundamentally defined simply as a cell becoming struc• turally and/or biochemically different from what it was. Cellular differentiation phenomena can be designated as primary, secondary, and terminal (Savidge 1983a, 1985). These terms applied at the cellular level should not be confused with primary (extension) and secondary (diameter) growth as traditional• ly used when referring to whole organs. Primary cellular differentiation phenomena include cell division, cell expansion or elongation, and making or breaking of intercel• lular bonds, all processes occurring within or on the margins of the cambium. Cell

Downloaded from Brill.com10/09/2021 12:10:54AM via free access 276 IAWA Journal, Vol. 17 (3),1996 division serves to multiply cell number and reduce cell size, and cell expansion in• creases cell size. The extent of intercellular bonding either prevents or facilitates cell positional changes. Although they sometimes exhibit temporal overlap within the same cell, each of these three processes is, from a biochemical perspective, a distinct phe• nomenon involving specific enzymes and correspondingly specific gene expression. Each process is, therefore, conceivably subject to unique regulatory control. As ex• plained in more detail below, increasing evidence indicates that meristems per se are regulated by homeodomain proteins (McHale 1993; Hake et al. 1995; Klinge & Werr 1995; Matsuoka et al. 1995); however, it remains unclear whether those proteins func• tion in determining the formation of meristematic tissues or, alternatively, in control• ling the primary cellular differentiation activities occurring within meristems. Existing models of higher plant morphogenesis propose that cortical microtubules playa key regulatory role. The preprophase band is considered to be the factor deter• mining the plane of cell division, and parallel-ordered cortical arrays of microtubules are thought to control whether a cell expands or elongates by determining the orienta• tion of cellulose microfibril deposition. Recent findings with mutant plants have indi• cated, however, that organogenesis and cellular differentiation patterns can be estab• lished in the absence of specific cytoskeletal arrangements (Traas et al. 1995). Numerous studies have shown that cortical microtubules are arranged in parallel association with developing secondary-wall microfibrils during xylogenesis (Inomata et al. 1992; Prodhan et al. 1995; Uhnak & Roberts 1995); however, there is also contradictory evi• dence concerning this (critique: Savidge & Barnett 1993). Precisely what the role of cortical microtubules may be during xylem-cell differentiation remains uncertain. Plants can produce virtually any form, or multi-cellular structure, using mechanisms of primary cellular differentiation, provided that the cells remain turgid (Kutschera 1989). However, secondary and terminal cellular differentiation events must occur if the fashioned, or marginally fashioned, forms are to be maintained against variable forces of nature, as when water becomes limiting within the plant. Secondary cellular differentiation phenomena follow upon or overlap with other meristem-associated processes and result in cells becoming specialized as still-living and distinguishable, but not necessarily stable, cell types. A cell in a state of secondary cellular differentia• tion usually is distinct from those in states of primary differentiation by being spatially removed from meristems (but, compare Savidge 1994), and the secondary state can be distinguished from a terminally differentiated cell in that the cell retains its nucleus and the capacity to resume cell division, cell expansion or intercellular bonding activ• ity under favourable circumstances (e.g., when an axial parenchyma cell contributes to tylosis development). When a cell has lost the capacity for either de-differentiation or further differentiation (e.g., sieve or vessel elements), the cell can be considered to be terminally differentiated (Savidge 1983a). Secondary and terminal cellular differentiation phenomena are normally but not in• variably suppressed in meristems. The explanation for this suppression remains uncer• tain. In the case of the conifer cambium, a continuing basipetal flux of auxin evidently is needed to prevent the fusiform cambial cells from differentiating directly into axial parenchyma (Savidge & Wareing 1981b; Savidge & Barnett 1993) or into tracheids

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Fig. 5. Direct differentiation of cambial fusiform cells of Pinus resinosa into tracheary elements at the junction of a short-shoot trace with the cambium. - A: Cross section showing a radial file fully differentiated into tracheary elements. Note that some fusiform cells (arrows) did not undergo radial expansion before differentiation whereas others enlarged somewhat. - B: Radial section immediately basal to the junction showing the primary-xylem-like tracheary elements that differentiated in the cambium and axial parenchyma (a) that formed alongside the new tracheids. - p = phloem; x = latewood. Unstained hand sections, Nomarski birefringence. For further details, see Savidge (1994).

(Savidge & Wareing 1981a; Savidge 1994) (Fig. 5). Complete maturation of the cambium in angiosperms is also common, particularly in flowering stems (Larson 1994). In considering cell fate, no clear temporal correlation exists between when a cell is produced and undergoes primary-wall enlargement and when the onset of xylem-cell differentiation begins. For example, during primary-xylem development in roots of seed plants, the process of terminal differentiation proceeds inwardly, or centripetally. However, the first cells to enlarge and become vacuolated are in the central region of the elongating root. Those enlarged vacuolated cells ultimately become metaxylem tracheary elements, but before doing so they exist for a prolonged spell as primary• walled cells in states of secondary cellular differentiation. After the first phloem ele-

Downloaded from Brill.com10/09/2021 12:10:54AM via free access 278 IAWA Journal, Vol. 17 (3),1996 ments have matured, xylogenic secondary-wall deposition commences first in small• diameter protoxylem elements next to the pericycle. Then, the enlarged centrally lo• cated cells which were produced before the predecessors of the protoxylem begin to deposit secondary walls and, ultimately, mature as metaxylem elements (Esau 1960). From such anatomical observations, it is evident that terminal differentiation of meta• xylem elements during root development must be controlled either through active in• hibition or because some factor essential for the differentiation process is limiting. Primary-xylem development in stems usually proceeds centrifugally, beginning on the margins of the pith; however, centripetal progression has also been noted in stems of lycopods and Psi/otum (Fig. 6). During secondary-xylem development within temperate-zone conifers, cell produc• tion and enlargement of cambial derivatives may occur concomitantly with or much earlier than the commencement of terminal differentiation, depending on the position in the stem in relation to the tree's live crown. The cambium in stem regions well below the foliage typically produces a large number of primary-walled radially ex• panded cells before any initiates bordered pit development or S 1 deposition, whereas little or no delay in the commencement of xylogenesis accompanies cambial reactiva• tion in foliated regions (Savidge & Wareing 1981b, 1984; Savidge 1990; Savidge & Udagama-Randeniya 1992). As in roots of angiosperms, there is no clear temporal correlation between either production of a cambial derivative or its subsequent pri• mary-wall enlargement and the onset of terminal differentiation. Nevertheless, when secondary-wall development does begin in the basal regions of conifer stems, the en• larged cells adjoining the mature latewood are the first to commence, and terminal differentiation then progresses centrifugally in a sequential manner. The rate at which the woody, or prosenchyma, component of developing xylem proceeds from primary through secondary to terminal cellular differentiation varies from weeks to months (Nix & Villiers 1985). In contrast, the living parenchyma cells immediately adjoining, or at least in close proximity to, the prosenchyma remain in a state of secondary cellular differentiation for one to many years. Terminal differentia• tion of prosenchyma occurs usually through protoplasmic autolysis; however, autolysis does not necessarily follow directly upon the completion of lignification and may be delayed for months or even years (Dumbroff & Elmore 1977; Nix & Villiers 1985). Current understanding of how protoplasmic autolysis may be controlled has been re• viewed elsewhere (Savidge, in press). Death of xylem parenchyma is frequently asso• ciated with heartwood formation and evidently does not involve protoplasmic autolysis (Hillis 1985; Yang et al. 1994). A reductionistic analysis ultimately has individual cells in multi-cellular organisms functioning as discrete entities, or closed systems, where change occurs independently of other cells. Rapid growth is probably always accompanied by tissue stresses, how• ever (Kutschera 1989). The linking of vessel members into longitudinal vessels, of axial parenchyma and epithelial cells into resin canals, of ray cells into rays, and of pits in walls of adjoining cells into perfectly symmetrical pit pairs are all manifesta• tions of intercellular communication. Under unusual circumstances the ray system of the cambium can function independently of the fusiform cells (Lev-Yadun 1994), an

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Fig. 6. Aerial stem of Psilotum, an extant genus frequently described as being similar to some of the most primiti ve fossilized vascular plants in that it lacks both roots and vascularized leaves, but probably more closely related to ferns (see Kolukisaoglu et aJ. 1995). - A: Young stem region showing early stage of development of the protostele; x 1000. - B: Older stem location showing unorganized multiplication of cells, development of an actinostele, and evidence for additional polyphenolic material in the secondary walls of some of the first produced tracheary elements; x 1333. - Brightfield, 15 J.llTI cross sections stained with safranin, fast green.

Downloaded from Brill.com10/09/2021 12:10:54AM via free access 280 IAWA Journal, Vol. 17 (3), 1996 indication that ray- and fusiform-cell derivatives probably communicate during nor• mal development as they become part of the xylem or phloem. These and additional phenomena of wood formation indicate that, within the whole plant, the cambium and its derivative cells function not independently but in concert to give rise to the hetero• geneous yet highly ordered tissue we know as wood. It can be deduced, therefore, that cell-to-cell physical and/or chemical messages, some of which are transmissible over long distances and others of which perhaps are transmitted only between two adjoin• ing cells, have roles in controlling wood morphogenesis. The concept that chemical messengers influence cell fate and, ultimately, morphogenesis finds support in the ob• servations that laser ablation of specific cells in root tips changes the fate of adjacent cells (Van den Berg et al. 1995) and that proteins regulating transcription can move from cell to cell through plasmodesmata and can also mediate intercellular transfer of mRNA through those same structures (Lucas et al. 1995). Plasmodesmata are abun• dantly present in angiosperms (Sauter & Kloth 1986); however, it remains unclear whether plasmodesmata are generally present in the fusiform cells of gymnosperm cambia (Savidge & Barnett 1993; Dute 1994). A tree comprises an autotrophic crown with heterotrophic stem, branches and roots through which transport must occur if the crown is to function in carbon assimilation and export and the root to provide the crown with water, inorganic nutrients, amino acids, and other substances (Fig. 7). Hence, transport through the xylem, the phloem, and the rays are forms of long-distance communication, and the possible roles of these transport corridors in conveying messengers regulating wood formation have been discussed (Savidge 1989, 1993a, 1994). The cambium and its derivative cells act as a fourth axial transport corridor, at least for the basipetal transport of auxin (Sabnis et al. 1969; Lachaud & Bonnemain 1984; Zaj'!czkowski et al. 1984; Savidge 1988; Kurczyn• ska & Micha1czuk 1989; Lim & Tamas 1989; Morris & Johnson 1990; Botla et al. 1992). In general, bioassay systems such as stem cuttings or tissue cultures which have been provided with water, inorganic nutrients, amino acids and dissolved sugars - the main substances known to be coursing through xylem and phloem - have not yielded tracheary elements unless the provided nutriment has been supplemented with auxin. These observations frequently are interpreted as evidence that nutritional factors have little or no regulatory role in xylogenesis. It should be considered, however, that bioassay systems based on isolated organs, tissues or cells do not perfectly simulate the whole plant, not only because of wound effects (Wildon et al. 1992), but also because trans• port of nutriment through the stem, rather than the mere availability of it, may affect xylogenesis. For example, there is evidence indicating that sucrose concentration, which is known to vary seasonally, can influence the nature of xylem-cell differentiation (Savidge & Wareing 1981 b; Zakrzewski 1983; Roberts et al. 1988; Savidge 1991; Wil• son et al. 1994b). Radial transport from xylem to phloem and vice versa undoubtedly serves as a mecha• nism for accumulating starch and other reserves in ray cells (Sauter & Kloth 1986). However, with current knowledge it remains uncertain how important symplastic trans• port of solutes through the rays - either centrifugally or centripetally - is for occur• rence of cambial growth and/ or wood formation. Conceivably, the process is non-

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I III Auxin Sucrose from from leaves leaves 'Po « 0 and CPo ~ 0 'Pp '" 0 shoots 'Pp '" 0 III L == 1,1 == " © @ == 0 @ == i'" ~ 1,0 == © == 0 © 00 ::::::;== © 0 ;::::=: © .~ ~ .!.r_p~ !..n..!!'!! __ _ ------== .. III III .~ ~ O© @ @ III III ~ ~\ © III III ~ i\p © 0 © $ ~ ~ O© © I:: '-p-hl~oe-m-JI'7"'';:,\----'111'-\;.A-!""7''--ln-iti-at-ioJnO~f--=~J-G-en-e-ra-I s-ecA..o-nd-a-ry---'-..A.J..-·'+'{rItT-J Enlarging Cambial tracheid wall formation and cambial zone differentiation lignification Xylem derivatives Enlarging (bordered-pit sap cambial development) derivatives Developing earlywood Fig. 7. Diagrammatic representation of developing xylem in a conifer as seen in radial section, to show movement of xylem sap (water), phloem sap (sucrose from leaves), auxin, and the radial water potential flux lines. -

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During the growing season, the foliage of transpiring trees exerts a strong pull on xylem water; consequently, vascular plant act to mobilize water in the radial as well as the axial direction. There are few experimental data yet available (Wignall & Browning 1988; Van Bel 1990), but it can be imagined that a perpetual tug-of-war for available water occurs through the rays, between the phloem and xylem (Fig. 7). When

DNA, PROTEINS, AND THE REGULATION OF GENE EXPRESSION

The phenotype is the outward appearance of an organism, or of an individual cell within a multi-cellular organism. The phenotype can be viewed as the final outcome of a number of interactions between the genotype and its environment. When members of a clone (i.e., perfectly homogeneous genotypes) are grown in a singular environ• ment, the plants (or cells) develop similar phenotypes. However, when grown in var• ied environments, clonal individuals appear differently, indicating environmental con• trol of phenotypic expression.

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Proteins, in the form of enzymes, are the catalysts of life processes determining the phenotype. Thus, understanding the regulation of enzyme activity is central to under• standing how varied xylem phenotypes arise. There are many different kinds of pro• teins, and the amino-acid sequence of each is encoded by a corresponding nucleotide sequence in its DNA. Each higher plant contains thousands of genes within its genome, but they are not all expressed (i.e., transcribed into RNA, edited into messenger RNA [mRNA] and translated into protein) simultaneously. Selective gene expression is be• lieved to be a major part of the explanation for particular biochemical reactions being catalyzed, hence different sub-cellular products being produced, at discrete locations and at different points in time in support of cellular differentiation and organogenesis. Upstream from a gene sequence are regions on the chromosome that regulate when and where transcription of that gene occurs. These upstream sequences are known as 'promoters'; they should not, however, be confused with intercellularly transmissible promoters, such as phytohormones. The upstream 'promoter' region of a DNA strand activates gene expression and mayor may not be inducible (Meshi & Iwabuchi 1995; Mett et al. 1993). When it is inducible, the promoter serves as a mechanism for con• trolling when and where gene expression occurs in the plant in relation to morphogenesis (Yang & Russel 1990). In addition to there being structural genes encoding both enzymes and storage pro• teins, plant chromosomes also contain 'homeobox' genes encoding homeodomain pro• teins. Homeobox genes, like homeotic genes in animals, are considered to act as 'mas• ter' regulatory genes orchestrating the coordinated activity of numbers of structural genes which, in turn, are essential for development. A homeodomain protein is one capable of binding to the promoter region upstream of a structural gene; the 'homeo• domain' is that part ofthe protein enabling it to bind to that DNA. Increasing evidence indicates that binding of homeodomain proteins to promoter regions is the explanation for selective activation or repression of structural gene expression during morphogenesis. It follows that the regulation of homeobox-gene transcription/translation and conse• quent production of homeodomain proteins is one mechanism for morphogenetic con• trol. Another possible mechanism by which gene expression may be regulated during plant development is through DNA methylation. For example, the cytosines in plant DNA are extensively methylated, and there is evidence for an inverse relationship existing between DNA methylation and gene expression (Ngernprasirtsiri & Akazawa 1990). The genotype varies between species and can also be different within a species from one organism to the next. Chromosomes are replicated with perfect fidelity every time a cell undergoes mitosis; hence, following fertilization of the haploid ovule, the genetic makeup of the single-celled diploid zygote is maintained constant throughout vegetative development. In theory, epigenetic changes such as inactivation of gene expression through methylation of portions of DNA can nevertheless occur (Matzke & Matzke 1990; Renkawitz 1990; Ngernprasirtsiri & Akazawa 1990). Somatic mutation also may occur at any point during vegetative development; however, the frequency is difficult to estimate because eukaryotic organisms, in general, possess a number of mechanisms for rapidly correcting mutations. The phenomenon of totipotency (i.e.,

Downloaded from Brill.com10/09/2021 12:10:54AM via free access 284 IAWA Journal, Vol. 17 (3), 1996 the ability of every living cell, whether present in leaf, stem, root, or other vegetative organ, to regenerate a whole plant of identical genotype) has given strong credence to the concept of genotypic constancy. Totipotent expression by cells from the cambial region has been achieved (Kumar et al. 1991). Throughout the woody plant, the vegetative structure comprises a clone of potentially totipotent cells, all having identical genotype. In vitro, totipotent expres• sion requires hormonal activation, and it is well established that phytohormones di• rectly affect gene expression (McClure et al. 1989; Kikuchi et al. 1989). On the other hand, there can be no doubt that the cambial region is enriched by the same phytohormones required for totipotent expression (see below). How is it, then, that cambial cells within stems or roots do not normally express their totipotency, rather remain part of the cambium and produce only new cambial cells, or derivatives that differentiate into elements of xylem and phloem? The biological basis for there being constraints on totipotent expression within intact organs is unknown, but it presum• ably involves controlled expression of only particular genes. The existence of such constraints is fundamental to any consideration of how plant morphogenesis, includ• ing wood formation, is regulated. As noted above, the cambium comprises a clone of cells, but in a tree the deriva• tives nevertheless become a number of distinct xylem cell types - phenotypes at the cellular level - at any point in root, stem, or branch during wood formation. As shown in Figure 8, additional, often abnormal, phenotypes are evoked in response to severe stresses or wounding (Rademacher et al. 1984; White & Lovell 1984; Kuroda 1986; Lowerts et al. 1986). Experimentally, the ratio of auxin to gibberellic acid also clearly controls the nature of the wood produced (Digby & Wareing 1966; Zhong & Savidge 1995a). The fact that different cell types arise side by side within radial files points to the internal environment, in the vicinity of and within individual cells, having a major role in determining what kind of woody, or non-woody, element a cambial derivative becomes (Savidge 1983a, 1985, 1994). This internal environment evidently comprises absolute amounts and ratios of phytohormones, other intrinsic factors such as those generated by wounding and stress, and a host of extrinsic factors. In view of the cam• bium's competence to generate variable and unexpected cell types (Fig. 8), it seems highly improbable that the fates of cambial derivatives are genetically predetermined when those derivatives first originate. Although the cell fate of a cambial derivative evidently is predetermined by little more than the overall form (e.g., elongated fusiform vs. isodiametric ray cell) of the cambial mother cell, biochemical evidence has been obtained indicating that the cambium itself, at least that in conifers, possesses an element of commitment to lignification, hence prosenchyma production (Savidge 1989, 1991, 1994; Forster & Savidge 1995). Coniferin, the glucoside of coniferyl alcohol and a molecule well es• tablished to be utilized in support of lignification, is not present in the cambium except during the period of cambial growth, nor is coniferin present in other tissues. In spring• time, coniferin begins accumulating in the cambium soon after fusiform-cell vacuolation and well before the first cambial derivatives have begun primary-wall radial expansion (Savidge 1989, 1991). Coniferin remains present throughout the duration of seasonal

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Fig. 8. Cross section of Pinus strobus stem showing aberrant cell types induced to differentiate in the earlywood in response to nearby wounding of the cambium. Tyloses can also be seen in the l&tewood (arrow). Brightfield, 20 Illll cross section stained with safranin, fast green. - Fig. 9. Cross section showing a radial file of the cambial zone of Pinus contorta. Two distinct locations can be seen where fusiform cells initiated new radial files through anticlinal divisions (arrows). The xylem was at the bottom of the photo. Brightfield, 20 Illll cross section stained with safranin, fast green; x 2000.

cambial growth and disappears with the onset of cambial dormancy (Savidge 1989, 1991). Biochemical investigations revealed that the activity of the enzyme catalyzing coniferin biosynthesis was detectable only when coniferin was present in the cambium (Forster & Savidge 1995). The fact that coniferin is produced only during the growing season points to some mechanism for seasonal regulation of gene expression; hence, further investigations into this enzyme and its gene(s) may provide insight into the control of cambial dormancy. That coniferin biosynthesis occurs only in the cambium indicates that the cambium is epigenetically committed to producing monolignols, but not necessarily to production of particular cell types.

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A long-standing and controversial concept in the field of vascular development is that each radial file within the cambial zone contains a cambial 'initial' which is epigenetically different from other cambial cells, which is indefinitely self-perpetuat• ing, which can divide anticlinally to produce new radial files within the cambial zone, and which somehow determines the ultimate destiny of daughter cells at least in terms of whether they become part of xylem or phloem. The controversy has centered on whether each radial file contains one or two initials (Larson 1994). A third possibility is that initials do not really exist at all (Savidge 1985) and that the destiny of cambial derivatives therefore cannot be predetermined (Savidge 1990). As reviewed above, cambial cells may mature so that the cambium disappears; thus, it is clear that cambial cells are not indefinitely self-perpetuating. A key defining fea• ture of putative cambial initials is their supposedly unique ability to initiate new radial files within the cambial zone. Observations such as that shown in Figure 9, where fusi• form cells distantly separated from one another more or less concomitantly undergo anticlinal division to initiate new radial files, demonstrate clearly that more than one cell in each radial file has the capability to divide anticlinally. Elsewhere, it has been considered that every cell within the cambial zone may be equally competent, and that their differential behaviour could be explained by the microenvironment experienced by each cell (Savidge 1985, 1989, 1990). Investigations with putative homeodomain proteins and home otic genes hold forth hope for resolving the issue of cambial initials. Already in one study with Arabidopsis thaliana, it has been reported that expression of a particular homeotic gene is restricted to procambial cells of embryo and developing organs and that expression of this gene is modulated by auxin (Baima et al. 1995). In a study with rice, the expression of a homeotic gene was localized to vascular strands of the stem (Matsuoka et al. 1995), and putative regulatory genes associated with vascular cells have also been reported in other species (Wissenbach et al. 1993; Demura & Fukuda 1994). Apparently distinct homeotic genes have been found to be expressed specifically in association with other meristems during vegetative development of plants (McHale 1993; Jackson et al. 1994; Hake et al. 1995; Klinge & Werr 1995; Meissner & Theres 1995; Smith et al. 1995). It is still very early, but there is as yet no report of the expression of a homeotic gene being restricted to only a single cell of the cambium, as might be expected if cambial initials are real. A great many woody perennial plants can be accurately identified on the basis of wood anatomical traits alone. The existence of intraspecific homogeneity in wood anatomy manifests the underlying constancy, or relatedness, of the species' genome and its environment at the cellular level. Nevertheless, at the tissue level substantial variation exists between individuals of the same species, in terms of overall diameter growth rate, earlywood-Iatewood proportions, dimensions attained by individual xylem elements, etc. This variability evidently derives more from primary than from secondary and terminal cellular differentiation activities, although there is evidence that terminal differentiation can also be affected (Van Buijtenen 1958; Bissing 1982; Antonova & Stasova 1993). Correlative and factor-withholding experimental studies have repeatedly shown that the seasonal availability of extrinsic factors such as sun-

Downloaded from Brill.com10/09/2021 12:10:54AM via free access Savidge - Xylogenesis 287 light, water, nutrients and warmth can greatly modify the xylem phenotype at the tis• sue level (Davis 1949; Denne & Dodd 1981; Bissing 1982; Carlquist 1988; Downes & Turvey 1990; Dtinisch & Bauch 1994; Lev-Yadun & Aloni 1995). Clearly, the cambial genome is plastic in terms of primary cellular differentiation activity yet less flexible in those ways that permit species to be keyed on the basis of their differing wood anatomies. If the assumption of only limited flexibility of the cambial genome at the secondary and terminal cellular differentiation stages is correct, it points to the possibility that whole families of genes are not only co-expressed but also orchestrated during dif• ferentiation. For example, as shown in Figure 7, the production of a normal tracheid in a pine tree involves a series of sequential activities: primary-wall radial expansion and extension growth with associated making and breaking of intercellular bonds, ini• tiation of pit development through modifications of the primary wall, torus develop• ment, secondary-wall polysaccharide deposition around the pit circumference, initia• tion oflignification at cell corners and in the compound middle lamella region, Sl poly• saccharide deposition closely followed by lignification of the Sl layer, S2, S3 pro• duction, and protoplasmic autolysis. These structural considerations do not take into account the possibilities of homeotic control of gene expression, mRNA editing, the need for post-translational modification of proteins (Yan et al. 1989), or the trafficking through endoplasmic reticula, dictyosomes and membranes of materials destined for exocytosis (Banfield et al. 1995). Nevertheless, a simplified biochemical analysis yields the requirement for a number of gene families to account for the variety of enzymatic• ally catalyzed reactions and products needed for this type of cellular differentia• tion: several are necessary for the primary cellular differentiation component, at least one for bordered-pit development, a large family for polysaccharide deposition, a smaller family for lignification, and an immense family of hydro lases for protoplasmic autolysis. During differentiation of cells into woody types, the cumulative expressions of these gene families are separated in space and sequentially expressed over time. How fine spatio-temporal control can be exerted remains to be elucidated. Plausibly, additio• nal families of regulatory genes coordinate the structural gene families. Thus, when a particular type of xylem-cell differentiation is initiated, it could be that activation of a regulatory gene determines the final outcome of the cellular differentiation process. Based on anatomical considerations alone, it was predicted that the gene families underlying polysaccharide and lignin deposition were loosely coupled and, therefore, that they could be manipulated independently of one another (Savidge 1985). This possibility has now been clearly substantiated in various ways (Smart & Amrhein 1985; Ingold et al. 1990; Pissarra et al. 1990; Taylor et al. 1992; Halpin et al. 1994; Shedletzky et al. 1994). The question thus arises whether every cell, during its differ• entiation, is constructed by distinct sub-processes, such as cellulose and lignin produc• tion, occurring more or less concomitantly but with each being independently regu• lated. Alternatively, the overall process of cellular differentiation may be regulated, but in a non-rigid manner such that sub-processes can be removed without otherwise affecting the outcome.

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The absence of variation in wood anatomical traits, such as the nature of cross-field pitting, between individuals of the same species provides a valuable clue for under• standing the regulation of wood morphogenesis. On the other hand, the concept of the wood anatomical phenotype being diagnostically constant among individuals within a species has practical value only when like is compared with like. Branch wood and stem wood of the same tree tend to be unlike, and both are different from root wood despite the cambium being a singular genotype (Lebedenko 1962; Patel 1965). The xylem in the juvenile core frequently is anatomically and chemically different from the later-produced mature wood of the stem, and so forth (Paul 1963). Cambial posi• tion and / or age clearly are associated with altered phenotypic expression during wood morphogenesis, again pointing to either intrinsic or extrinsic environmental factors, or both, as controlling factors.

PHYLOGENETIC CONSIDERATIONS

Excepting algae, mosses, liverworts and hornworts, all extant land plants are regarded as vascular plants. For a plant (or fossilized specimen) to be designated as a 'vascular' plant, or member of the Tracheophyta, requires that it exhibit discernible tracheary ele• ments. The oldest recorded vascular plant in the fossil record is Cooksonia (Fig. 10), a plant evidently more like a moss than most vascular plants as we know them today (Gensel & Andrews 1987). If a plant is classified as vascular, it is customary to rank it phylogenetically based upon the type of stele present. The stelar concept originated with Van.Tieghem and Douliot (1886) and is based upon the assumption that the primary structures of stem and root are fundamentally similar in that each consists of a central column, or stele, of vascular tissues, enveloped by the cortex. Most traditional botanists evidently agreed

BRYOPHYTES GREEN LYCOPODS SEED FERNS HORSETAILS ALGAE PLANTS

Cooksonia

Fig. 10. Currently perceived lines of Primitive green evolution of vascular and non-vascu• algae lar terrestrial plants from green algae.

Downloaded from Brill.com10/09/2021 12:10:54AM via free access Savidge - Xylogenesis 289 that characteristics of the stele, as viewed in cross section or three dimensions, pro• vided a useful approach for distinguishing primitive from more advanced vascular plants, thus for ranking plants phylogenetic ally. The proto stele, characterized by the absence of a central column of pith, is consid• ered to be the most primitive type, and it is what is seen in the stems of the earliest fossilized vascular plants as well as in some extant plants (Fig. 6). However, protosteles also occur in the roots of all extant seed plants, and it seems implicit within this logic, therefore, that roots have evolved less than shoots, despite xylem development in roots being highly complex as described above. The root system being more primitive than the shoot system is plausible, but it would seem improbable because both root and shoot systems originate from a single cell and are of identical genotype. Cambial ini• tiation and vascular development in detached roots can be induced by phytohormone application (Loomis & Torrey 1964; Torrey & Loomis 1967); hence, a phytohormone explanation for the varying nature of stelar morphogenesis is conceivable. Wardlaw (1949) found that the type of stele developing could be manipulated to be more or less 'primitive' by making incisions in the region of the shoot apex, or merely by forcing an upright branch to grow horizontally. Ma and Steeves (1992) have made related observations. Referring to the atactosteles in monocots, Jeffrey (1917) noted that the number of vascular bundles in the stem is directly correlated with the number of traces in the leaves. Esau (1960) was also of the opinion that "the vascular system of the stem, to be properly understood, must be studied with reference to its connection with the vascular system of the leaves," and recent observations by Ma and Steeves (1995) support this view. The nature of primary-xylem morphogenesis, whether in root or shoot, can be viewed in terms of genetically predetermined structures or, alternatively, as non-determined developmental responses to transmissible regulatory factors. Based on successful ex• perimental manipulation, stelar morphology itself appears not to be predetermined genetically. Its predictable character within the whole plant may nevertheless be a function of genetic factors in other organs, such as those controlling development of leaves. The fossil record indicates that early vascular plants were devoid of cambium, indi• cating that differentiation into tracheary elements preceded cambial development (Barghoorn 1964). The origin of competence for this type of terminal differentiation probably cannot be explained in terms of a singular mutation event occurring in a primitive non-vascular terrestrial plant because, as already noted, the biochemical proces• ses underlying differentiation into a tracheary element are multi-faceted and, undoubt• edly, require coordinated expression of a large contingent of genes. If antecedent cell division and cell expansion/ elongation and ensuing protoplasmic autolysis are removed from consideration, production of the first hypothetical simple tracheary element, one having annular ribs consisting only of cellulose and a single kind of lignin, would nevertheless have required that the genome had become competent to 1) localize where in the cortical protoplasm cellulose was produced, such that annulets of secondary• wall material were deposited, 2) produce lignin, 3) localize the deposition of the lignin to the cellulosic ribs, and 4) control the timing and sequential order of cellulose and

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Fig. II. Coleochaete, a sessile green alga that begins as single cells or cell aggregates and forms a parenchymatous disk of tissue. Frequently considered as a possible forbearer of terrestrial plants. Note the thickened secondary walls (arrow). Brightfield, 12 !ll11 section stained with safranin, fast green; x 400. - Fig. 12. Base of the antheridial sac of a Marchantia (liverwort) sporophyte, to show the secondary-wall thickenings of the elaters (arrow). Nomarski, 12 !ll11 section stained with safranin, fast green; x 1200. lignin production and deposition in relation to concomitant cellular activities. On this basis, as discussed above, it is difficult to imagine how fewer than four (and probably many more) mutation events can account for the genetic competence needed for this type of cellular differentiation to arise. The Chlorophyta (green algae) from which land plants are believed to have evolved were present in the seas at least a billion years before the origin of land plants (Fig. 10). Many extant green algae closely resemble ancient fossilized specimens, and compe• tence for cellulose production is widespread among the living species. Green algae such as Coleochaete, a presumed predecessor of land plants (Delwiche et al. 1989), produce secondary-like walls (Fig. 11). Therefore, the genetic information for produc• ing massive secondary walls of cellulose may have arisen long before terrestrial plant

Downloaded from Brill.com10/09/2021 12:10:54AM via free access Savidge - Xylogenesis 291 ecosystems began to develop. There are spiralled cellulosic structures in the elaters of liverworts, such as Marchantia (Fig. 12), and annulets in the pseudoelaters of hom worts, such as Anthoceros. These structures are not identical to those found in primary-xylem elements (Taylor et a1. 1974); however, they are examples of how non-vascular land plants utilize genes, perhaps those inherited from algae, to control cortical positioning of secondary-wall-like structures. Thus, it may be unnecessary to invoke the occur• rence of a mutation in a land plant in order to explain acquisition of capability for localized deposition of secondary wall cellulosic ribs during vascular development. Evidence by Taylor et a1. (1992) indicates that the localization of lignin within the wall of Zinnia elegans tracheary elements is somehow dependent on the localization of cellulose. If this angiosperm evidence applies to primitive vascular plants, then the requirement for yet another mutation is eliminated and the focus can be on the capabil• ity for lignification alone. Barghoom (1964) considered that "the mutation providing the chemical pathways for lignin formation arose only once in the chemical evolution of plants and triggered the mechanism for evolution and selection of entirely new aspects of morphological expression and specialization." Lignin is what makes polysacchardic cell walls woody, strong and hydrophobic; thus, the formation of lignin is considered to have been the crucial achievement in the adaption of plants to terrestrial environments, the assump• tion being that only with lignin did it become possible to produce rigid cell walls capable of conducting water under tension over long distances. The phylogenetic ori• gin of lignin, however, is uncertain. As indicated in Figure 13, biochemical investi• gations with extant vascular plants indicate that lignin arises from diversion of glucose

Erythrose 4-phosphate =-----I~~Phenylalanine PAL Pentose Phos!.ates ;~;~::; / andNADPH PROTEINS oxidative pentose phosphate pathway LIGNIN glycolysis SUCROSE • Glucose 6-phosphate, phosphoenolpyruvate CELLULOSE phospho- \ I glucomutase

Glucose I-phosphate • Uridine 1,5-diphosphoglucose / Uridine triphosphate

Fig. 13. Overview of current biochemical knowledge about how photosynthate (sucrose) is diverted to lignin and cellulose.

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p-Hydroxyphenyl Guaiacyl Syringyl lignin lignin lignin

CH 0H E4t Ert E4t 2 CH20H CH 20H

p_coumaryl alcohol coniferyi alcohol sinapyl ~ alcohol ~I ~I ~ ~ 0. 1 6 0. OCH) CH 30 OCH) OH OH ~OH

EJ CHO E3t CHO E 3 "t CHO

p_coumaryl aldehyde coniferYI aldehyde SinapaldehYde ~I ~I ~~I 0. S:--- OCH3 CH 0 :--- ' OCH3 6 3 OH OH OH

EJ CO-SCoA E2.t CO-SCoA E 2"t CO-SCoA

p_coumarOYI CoA ferulOYI CoA SinapOYI CoA ~I ~~I ~I ~ 6 :--- OCH3 CH30 40. OCH3 OH OH OH

L-tyrosine p-coumaric acid caffeic ferulic acid acid COOH Est COOH Enzymes of lignification: PAL = phenylalanine ammonia lyase II;: ! TAL = tyrosine ammonia lyase O~O Ef, E}', Ej" = hydroxycinnamate .- CoA ligases L-phenylalanine trans-cinnamic E 2 , E2', E2" = hydroxycinnamoyl-CoA reductases acid E 3 , E 3 " E 3 " = cinnamyl alcohol dehydrogenases E 4 , E 4 " E 4 " = oxidative enzymes Es = cinnamate-4-hydroxylase E6 = p-coumarate-3-hydroxylase E7 & E9 = hydroxycinnamate O-methyltransferases E8 = ferulate-5-hydroxylase

Fig, 14, The three biochemical pathways currently recognized as sources of lignin precursors ('monolignols' = p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol). The enzymes catalyzing the molecular transformations are indicated.

6-phosphate into the oxidative pentose phosphate pathway, followed by successive chemical transformations catalyzed by enzymes within the shikimate and phenylpro• panoid pathways. The oxidative pentose phosphate and shikimate pathways underlie production of phenylalanine, tyrosine and tryptophan, aromatic amino acids essential

Downloaded from Brill.com10/09/2021 12:10:54AM via free access Savidge - Xylogenesis 293 for protein biosynthesis in bacteria and other primitive as well as all higher lifeforms. It can be assumed, therefore, that these pathways were present long before the origin of vascular plants. On the other hand, the enzyme phenylalanine ammonia lyase (PAL) evidently emerged much later, as most algal species so far investigated have yielded no PAL activity. Loffelhardt et al. (1973) did report evidence for PAL in the green alga Dunaliella, indicating the need for more investigations into algae. PAL catalyzes the deamination of phenylalanine to trans-cinnamic acid as the starting point in the biosynthesis of lignin and a vast array of additional phenolic substances. Figure 14 indicates the three biochemical pathways currently regarded as the imme• diate predecessors of lignin in vascular plants (Lewis & Yamamoto 1990). Three types of lignin are recognized, p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S), de• pending on whether the lignin polymer is generated from p-coumaryl, coniferyl, or sinapyl alcohols, respectively. Monocots have a mixture of all three types of lignin whereas hardwoods have essentially no H and conifers essentially no S lignin. Lycopods are similar to conifers in lignin composition, whereas ferns contain predominantly G and a small amount of S lignin. In hardwoods, G lignin tends to predominate in vessel elements whereas S lignin is more abundant in fibres. Experimentally, these cell types and lignin distributions can be manipulated by using different ratios of auxin to gibberellic acid (Aloni et al. 1990; Zhong & Savidge 1995b). In conifers, H lignin is primarily associated with compressionwood formation and G lignin with normal wood. Mosses are not included in the Tracheophyta because they, in the words of many botanists, "contain neither tracheids nor true lignin." Such an interpretation implies that the tracheid, or tracheary element, is a chemically and structurally well defined readily diagnosed singular entity, which in fact is not the case. As Sphagnum leaves mature, the larger cells develop annulets or spiralled secondary thickenings, and pit• like openings also appear in the primary walls. Although an argument could be made that such cells are just as much tracheary elements as those described in Cooksonia (Edwards et al. 1992) or other primitive vascular plants, the fact is that they are not recognized as tracheary elements because they occur in a moss species and not in the location (stem axis of the gametophyte) where expected. The central region of the gametophytic axis of a moss contains elongated water-conducting hydroids; however, annular and spiral ribbed elements are not conspicuous (Fig. 15). Hebant (1975) de• scribed moss hydroids in both sporophytes and gametophytes as being devoid of pro• toplasm and having partially hydroyzed end walls. Karl Freudenberg, a pioneer in lignin biochemistry, evidently was of the opinion that lignin occurs in mosses: "It is very difficult to estimate and to characterize the lignin of lycopodia and of mosses because they contain hardly any methoxyl groups" (Zimmermann 1964: 13). Some evidence indicates that moss hydroids contain H lignin or a closely related polymeric material (Farmer & Morrison 1964; Bland et al. 1968; Miksche & Yasuda 1978), and there is biochemical evidence for pheny lpropanoid metabolism in mosses (Rasmussen et al. 1995). Using definitive analytical technology, Wilson et al. (1989) concluded that mosses actually do not contain significant amounts of H lignin, rather a polymer de• rived from hydroxybenzene. Be this as it may, the results from all of these investiga• tors have implicated PAL or TAL activity (Fig. 14), subsequent phenolic metabolism,

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Fig, 15, Gametophytic axis ofa true moss (Mnium spp,), Note that although there is no obvious stele, cells both internally and near the margins exhibit secondary thickening and polyphenolic deposition in the walls, Brightfield, 12 Iilll cross section stained with safranin, fast green; x 530, and deposition of polyphenolics into cell walls during development of moss stems, The current thinking, where three pathways of phenolic metabolism leading to three kinds of chemically distinct polyphenolic materials are classified as lignin whereas a fourth is not, appears incongruous and difficult to justify. The reasoning that mosses should not be considered primitive kinds of vascular plants appears to be based more on imposed classification and other assumptions than on thorough investigation and clear definition. All classification systems are, of course, subjective; however, the tradi• tional phylogenetic treatment of mosses may be diverting researchers from addressing key questions in vascular plant development, viz. how both lignin and the cambium originated. Moreover, knowing that at least some moss species have the genetic com• petence to produce tracheid-like cells, it might be revealing to investigate the control of this type of cellular differentiation further. Methods for axenic culturing ofbryophytes have been developed (Sargent 1988). Current fossil evidence places mosses no older than 350 x 106 years whereas Cooksonia and related primitive vascular plants were present on Earth more than 400 x 10 6 years ago (Edwards et al. 1992). This temporal gap has been interpreted as evi• dence that bryophytes represent merely a specialized evolutionary offshoot from green algae, away from the main stream which led to vascular plants (Fig. 10). However, recent comparative investigations of molecular phylogeny based upon the amino acid sequence of phytochrome (a light-sensitive protein involved in photomorphogenetic

Downloaded from Brill.com10/09/2021 12:10:54AM via free access Savidge - Xylogenesis 295 responses) indicated that mosses are closely related to lower vascular plants (Ko• lukisaoglu et al. 1995). Moreover, bryophyte-like spores pre-dating Cooksonia have been described (Taylor 1995). In addition, the evidence for lignin in fossil specimens of the most primitive 'vascular' plants cannot be described as solid; conceivably, the secondary wall ribs found in those fossils were devoid of lignin. It remains possible that bryophytes were the ancestors of vascular plants.

PHYTOHORMONE REGULATION

Knowledge about the roles of phytohormones in xylogenesis is extensive but can be summarized to the effect that auxin and cytokinin in gymnosperms, and auxin, cytokinin and gibberellins in angiosperms appear to be the endogenously generated long-distance transmissible factors promoting, and possibly also inhibiting, xylogenesis (Aloni 1993, 1995; Little & Pharis 1995). Very little is known about seedless vascular plants. In the case of ferns, Ma and Steeves (1992) have questioned the concept that auxin directly promotes xylogenesis. In hardwoods, auxin and gibberellin appear to synergistically promote cambial growth; auxin and cytokinin are necessary for PAL induction; and the auxin-gibberellin ratio not only synergistically promotes xylem production but also determines vessel and fibre frequencies, depending on the ratio (see below). Some data indicate that abscisic acid may be inhibitory to tracheid differentiation (Minocha 1984; Pissarra et al. 1986). Accumulating evidence indicates that ethylene may have a controlling role in several aspects of wood formation (Nelson & Hillis 1978; Roberts et al. 1988; Koritsas 1988; Savidge 1988; Menon & Babu 1989). It remains uncertain whether cambium-associated ethylene (or one of its biosynthetic precursors) is trans• mitted from cell to cell, as is probable with auxins, cytokinins, and gibberellins, or if ethylene arises intracellularly in response to intercellularly mobile factors, such as auxin, that promote biosynthesis of ethylene. In addition to the above classes of phytohormones, there is increasing evidence for calcium ion (Ca++) and calcium-binding proteins having key regulatory roles in the control of tracheary element differentiation (Kobayashi & Fukuda 1994; Antosiewicz et al. 1995), possibly mediated through a second messenger such as inositol 1,4,5- trisphosphate (Savidge 1994). Addressing vascular differentiation in transgenic angiosperms, Klee and Romano (1994) stated that "Auxin-overproducing plants contain more xylem elements than control plants. The cells are, however, smaller and more lignified. Conversely, plants with lowered IAA levels contain fewer and larger xylem elements that are less lignified?' These observations are at least in partial agreement with the hypothesis of Aloni and Zimmermann (1983) that vessel diameter is inversely proportional to auxin concentra• tion; however, detailed dose response curves would clarify this supposition. Genetic transformations leading to elevations in endogenous cytokinin contents in leaves and stems of angiosperms were apparently without effect on vascular development (Medford et al. 1989; Smigocki 1991). Photomicrographs published by Medford et al. (1989) suggest, however, that xylem produced in cytokinin-overproducing Arabidopsis plants is the more porous, evidently due to increased vessel production or enlargement. In

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Fig. 16. Stem of Sedum spp. showing hypertrophic response caused by Agrobacterium tume• facien.~, a bacterium which donates a plasmid (single-stranded loop of DNA) encoding auxin and cytokinin biosynthesis to the plant genome. - A: Cross section showing how stem cells proliferated and produced a zone of sclerenchyma (arrow) at the site of wounding inoculation. Pre-existing xylem can also be seen on the left, top and bottom. Brightfield, x 200. - B: Nomarski photograph on the edge of the sclerenchymatous zone (arrowed in A) to show the tracheary elements induced to differentiate; x 1300; l21illl section stained with safranin, fast green. transgenic tobacco engineered to overproduce cytokinins in the stem, the number of cortical and pith cells were reduced and the dimensions of cortical cells were increased substantially (Li et al. 1992). Consequently, the xylem of transgenic tobacco stems was more centralized than that of wild type, but the prosenchyma volume of the stem and the nature of the stele were evidently unaffected by elevated cytokinin con• tent. The observations to date on xylem development in transgenic whole plants which overproduce auxin or cytokinin appear to be at variance with a substantial body of physiological research findings, including the observation (Fig. 16) that increased auxin and cytokinin production following Agrobacterium tumefaciens inoculation of stems

Downloaded from Brill.com10/09/2021 12:10:54AM via free access Savidge - Xylogenesis 297 accelerates localized cambial growth and induces woody tumours (Aloni et al. 1995). More detailed anatomical analyses of development in hormone-transgenic plants are needed. Most knowledge about phytohormone regulation of wood formation derives from applications of phytohormones, their synthetic analogues, and inhibitors of phytohor• mone biosynthesis and/ or transport to cut surfaces of stems or cells in culture, follow• ed by microscopy to determine the bioassay response days to weeks later. In compar• ing treatment and control responses, the intrinsic regulatory roles of phytohormones in xylogenesis are deduced upon the assumption that these imposed extrinsic agents elicit responses by the same mechanisms used by endogenous phytohormones within intact plants. This reasoning remains suspect, however, because exogenous phytohormones frequently are applied as spot applications to cuttings or whole plants in near milli• gram quantities that may well evoke inhibitory or otherwise abnormal, hyperphysio• logical responses. Endogenous phytohormones commonly occur in nanogram (10- 9 g) or lower amounts in a gram of fresh plant tissue. Higher contents have been reported in developing em• bryos and other seed parts, tissues where xylogenesis rarely occurs. In developing xylem of tree stems, auxin is present at nanogram to microgram (10- 6 g) levels per gram of tissue (Savidge et al. 1982; Wignall & Browning 1988); gibberellins are at nanogram or lower levels (Ridoutt et al. 1995); abscisic acid occurs at sub-microgram levels (Savidge & Wareing 1984; Wignall & Browning 1988). Ethylene levels in the cambial region are higher than those reported for most other plant tissues (Savidge 1988). Cytokinins in plant tissues occur at nanogram or lower levels (Taylor et al. 1990). Cytokinins are known to occur in the cambium (Funada et al. 1992); however, accurate estimates of their content remain to be made. Published estimates of endogenous phytohormone content have been based on diverse quantitative or semi-quantitative methods of analysis. In addition, a variety of solvents has been employed to extract phytohormones from plant tissues, and the pro• portion of phytohormone extracted into the solvent undoubtedly varies depending on the nature of the tissue, how it is prepared, what volume and temperature of solvent is used, the solubility of the phytohormone in the solvent, etc. The plant physiology community recognizes that the accuracy of the estimate depends very much on the methodology and, regardless, all estimates tend to be of uncertain precision in relation to how much physiologically active phytohormone actually exists in differentiating cells. On the assumption that all of the endogenous phytohormone that can be extracted from a tissue is physiologically active, even accurate physico-chemical quantitation of endogenous phytohormones can be difficult to interpret if insufficient attention is given to the composition of the tissue being analyzed. For example, if auxin in the cambium is the focus of investigation, but the auxin also present in phloem or developing xylem is extracted together with that from the cambium, it is difficult to decide the role of auxin in the cambium. Even when singular tissues are carefully separated and analyzed, attempts to correlate endogenous phytohormones directly with a particular type of cellular differentiation tend to be crude because of the complex nature of the tissues.

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When supposedly physiological amounts of native phytohormones are applied, xylogenic responses are rarely seen in stem cuttings (Savidge 1994). However, in vitro cultures do produce tracheary elements in response to microgram amounts of phytohormone in the growth medium, possibly an indication that massive doses must be. provided to organ-based in vivo bioassay systems if sufficient phytohormone is to gain entry into the system. In the case of auxin, agar has been found to be 100 times more effective than lanolin in inducing a xylogenic response (Gersani 1987). Gaining an appreciation of the internal process within whole plants is complicat• ed by the fact that phytohormones tend to be both unstable and readily metabolized molecules. Rarely is a commercially supplied phytohormone even 95% pure, yet pre• liminary investigations have seldom been undertaken to characterize (i. e., confirm the identity of) either the composition of the chemical substance( s} applied or the molecu• lar changes which may occur on the surface of or within the plant or in the culture medium. Investigations to determine how much exogenous phytohormone actually gains entry into a cell or tissue in relation to differentiation or development, and how the internal amount changes with time or correlates with development, are needed. Inves.tigations of endogenous phytohormone content in non-wounded and non-treated plants, for-purposes of corroborating data generated by applying exogenous compounds, would provide additional understanding. Many investigators have presented evidence that it is the active polar transport of auxin through a tissue, and not merely the presence of it within cells, that induces tracheary elements to differentiate (Sachs 1981, 1993; Zajltczkowski et a1. 1984; Gersani 1987; Kurczyriska & Michalczuk 1989; Kuternozinskaet al. 1991; Wilson et a1. 1994a; Moni 1995). This agrees with the fact that basipetal auxin transport occurs preferen• tially in the cambium and its derivatives (Sabnis et al. 1969; Savidge et al. 1982; Savidge & Wareing 1984; Lachaud & Bonnemain 1984; Little & Savidge 1987). The ability of cells. to transport auxin basipetaUy is thought to be a function of specific but distinct uptake and efflux carriers localized to the plasma membrane, and the process evi• dently is regulated by auxin itself (Morris & Johnson 1990; Wodzicki 1993; Wilson & Wilson 1993) as wen as by other phytohonnones and second messengers such as Ca++ (Savidge 1988, 1994). Significant progress has been made toward understanding phytohormonal regula• tion of differentiation using the Zinnia-mesophyll suspension culture system (Fukuda 1992; Church 1993). In that system, up to 65% of isolated mesophyll cells from young leaves have been reported to differentiate into 'tracheary elements' more or less syn• chronously and directly, without preceding cell division, in response to exogenous auxin and cytokinin. In the sapwood of most woody species, parenchyma cells occupy a relatively small volume of the tissue, but they nevertheless outnumber secondary• waned and lignified prosenchyma elements considerably. Hence, differentiation of 65% of isolated mesophyll cells into prosenchyma in response to auxin and cytokinin ap• pears to be an indisputable indication that those phytohormones control production of woody cells. On the other hand, an equimolar (0.5-1.5 JIDl) auxin to cytokinin ratio is needed to induce differentiation in the Zinnia system, and it is doubtful that either the

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absolute amounts (0.5 J.lIIl I-naphthaleneacetic acid is equivalent to 93 j.lg per litre of culture solution, a high dosage) or the ratio approximates the endogenous environ• ment. The naturally occurring auxin indol-3-ylacetic acid (IAA) is less effective in the Zinnia system than synthetic NAA, and the cytokinins N6-benzyladenine (BA, syn• thetic) and 2-isopentenyladenine (natural) are more potent than zeatin (natural) or syn• thetic kinetin (Church & Galston 1988). Treatment of cotyledons of Solanum aviculare with an auxin--<:ytokinin mixture has also resulted in mesophyll cells differentiating into tracheary elements, sclereids and vessel elements (Gahan et al. 1994). It remains unclear whether the initiating event for induction of differentiation in the Zinnia system is phytohormones, wounding, or some other factor such as DNA repair occurring in the wounded mesophyll (Fukuda 1992; Sugiyama et al. 1995). An addi• tional problem with the Zinnia system is that the mesophyll cells have abundant chloroplasts. Vascular development in the whole plant is linked to the presence of leaves (Esau 1943; Savidge & Wareing 1981b; Sachs 1993; Savidge 1994), and it is conceivable that the Zinnia bioassay responds only indirectly to auxin and cytokinin through the mesophyll cells producing a leaf factor which initiates differentiation of prosenchyma (Savidge 1994). It would be useful to know if mesophyll cells from etiolated leaves respond similarly to green mesophyll when treated with auxin and cytokinin. A number of studies have indicated that it is the ratio of one class of phytohormone to another, as well as the absolute amount of PGR provided, that determines the nature of cellular differentiation. Although an auxin--<:ytokinin combination promotes differ• entiation of cells into tracheary elements, Zinnia mesophyll cells cultured first in a medium lacking either the auxin or the cytokinin component, and then transferred to an auxin--<:ytokinin inductive medium, generally exhibit severely reduced competence for tracheary element differentiation (Sugiyama & Komarnine 1990). Reynolds (1987) found that a 20: 1 ratio of auxin to cytokinin was most effective in inducing tracheary element differentiation in suspension cultures of Solanum carolinense. An auxin-cyto• kinin combination also appears to be essential for induction of phenylalanine ammo• nia lyase (PAL) activity in support of lignification (Bevan & Northcote 1979; Northcote 1993). The auxin-gibberellin ratio controls whether vessel elements or fibres are pro• duced (Digby & Wareing 1966; Zhong & Savidge 1995a). True xylogenesis in the whole plant almost always involves co-differentiation of parenchyma and prosenchyma, sometimes with other cell types. If auxin and cytokinin regulate xylogenesis in vivo as well as in vitro, then it could be that there are one or more additional, still unknown, regulators that counteract the auxin--<:ytokinin in• ductive effect to yield the non-prosenchymatic component of the tissue. Plausibly, an inhibitory factor exists. Alternatively, it may be neither inhibitory nor promotory, rather a factor that somehow adjusts the auxin-cytokinin ratio within individual cells. Albinger and Beiderbeck (1983) working with suspension cultures observed that high cyto• kinin concentrations with low phosphate concentrations were conducive to tracheary elements differentiating, whereas lowered cytokinin concentrations yielded paren• chyma.

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AN ATTEMPT TO INTEGRATE CURRENT KNOWLEDGE - THE CONTINUUM HYPOTHESIS -

Annular, spiralled, reticulated, and scalariform primary xylem elements, bordered-pit• ted secondary xylem elements, and vessel elements are all physically different and, at first glance for the novice, they are not obviously related cell types. It nevertheless is common to refer collectively to these woody, or prosenchymatous, types as 'tracheary elements' (TEs) because they are all perceived to function in water conduction. Rarely do any two TEs of even the same sub-type appear perfectly identical, although there are common structural themes associated with particular sub-types. Experimentally, a continuum of decreasingly less complex TE sub-types was in• duced to differentiate concomitantly in the cambial zone of conifers with increasing distance of the cambial cells from the junction of the short -shoot trace with the cambium (Savidge & Wareing 198Ia). This was interpreted as evidence that a singular needle• produced tracheid-differentiation factor controlled the extent of differentiation by its abundance (Savidge & Wareing 1981a, 1981b; Savidge 1994). The possibility that a differentiation continuum exists from annular-ribbed TEs to bordered-pitted second• ary xylem TEs is supported by additional observations. In culture, individual 'Zinnia'

Fig. 17. Radial section near the pith of a stem of Pinus contorta showing how overarching pit borders (ar• row) sometimes develop in direct association with an• nular or spiralled ribs of secondary wall in tracheary elements of the primary xylem. SEM photomicro• graph, x 1500.

Downloaded from Brill.com10/09/2021 12:10:54AM via free access Savidge - Xylogenesis 301 cells were observed to elaborate increasingly more complex secondary walls from relatively simple rib structures (Falconer & Seagull 1988). In nature, bordered pits frequently are found in association with primary-xylem TEs otherwise containing only annular or spiralled ribs of secondary-wall material (Fig. 17). Moreover, during differ• entiation of secondary-xylem TEs, isolated ribs of secondary-wall materials are the last to be deposited in many woody species. At the level of gene expression, the existence of a continuum in TE differentiation has relevance to the number of distinct families of genes which must be up- or down• regulated to account for the presence of each sub-type and where and when that sub• type differentiates. As indicated in Figure 18, a differentiation continuum can be per• ceived as the requisite families of structural genes being repeatedly co-expressed dur• ing cellular differentiation to generate increasingly more complex sub-types. Conceiv• ably, this co-expression and its spatio-temporal coordination could be achieved by each gene, or family of structural genes, being independently controlled by a specific regulator, as previously proposed (Savidge 1985); however, this requires not only that there be a multitude of regulatory factors but also that all of those hypothetical regula• tors somehow be orchestrated in space and time during differentiation. A more readily envisaged alternative is that the activation, duration and synchronization of structural gene expression is regulated by one or only a few regulatory factors - possibly pro• teins produced from homeotic genes - which activate families of structural genes in general. This raises the question of how regulatory genes are themselves regulated. Very little is yet known, but evidence has been presented that auxin has a role (Baima et al. 1995).

Bordered• pitted

Scalariform

Reticulated

Spiralled

Annular

------Duration of gene expression

Fig. 18. Continuum hypothesis to explain how different TEs arise as a function of duration of expression of a singular regulatory gene controlling expression of families of structural genes.

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If the continuum hypothesis is correct, it follows that apparently distinct sub-cellu• lar anatomical phenomena, such as bordered-pit versus simple-pit development and various types of ribbed secondary walls versus S 1> S 2 and S 3 massive secondary-wall deposition, are all under a common form of regulation. The concept of a cellular differ• entiation continuum based on the duration of expression of regulatory genes, hence structural genes, is also compatible with there being additional factors which, upon entering the cell, influence cell fate. For example, which monolignol pathway contrib• utes to lignification may be determined by a mobile intrinsic factor influencing gene expression, in terms of transcription, translation, post-translational processing or enzymatic activity, of one of the members of a structural gene family. The continuum hypothesis can be projected beyond tracheary elements. The 'fibre tracheid' conceivably bridges the gulf between tracheary elements and sclerenchyma because a factor (such as gibberellic acid) directly influences how one or more fami• lies of structural genes is expressed, without modifying the expression of the regula• tory gene(s). Similarly, secondary-walled and lignified 'parenchyma' (otherwise seen as living sclerenchyma, or lignified collenchyma) could manifest a point on the con• tinuum between thin-walled ray parenchyma cells and secondary-walled and lignified ray tracheids or sclereids.

ACKNOWLEDGEMENTS

My thanks to P. Baas (editor) and the reviewers for pointing out a number of deficiencies in the first submission.

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