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Xylogenesis: the birth of a corpse
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Xylogenesis: the birth of a corpse Keith Roberts* and Maureen C McCann† Xylogenesis is a complex developmental process culminating in programmed cell death as a truly terminal differentiation event. In Arabidopsis, the availability of vascular-patterning mutants, and the identification of genes and their products that are involved in cell specification, secondary-wall deposition and lignification, are providing clues to the functions of some of the sequences in the large expressed sequence tag databases derived from the xylem-rich tissues of trees. An in vitro system, the Zinnia mesophyll cell system, provides an alternative system for those cell-biological experiments that are difficult to tackle in intact plants. In particular, a combination of molecular-genetic and cellbiological approaches has made possible the elucidation of some of the features of plant programmed cell death. Addresses Department of Cell Biology, John Innes Centre, Norwich Research Park, Colney, Norwich, NR4 7UH, UK *e-mail: [email protected] † e-mail: [email protected] Current Opinion in Plant Biology 2000, 3:517–522 1369-5266/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations EST expressed sequence tag HR hypersensitive response ifl1 interfascicular fiber 1 nDNA nuclear DNA PCD programmed cell death scf scarface TE tracheary element
Introduction Plants, animals and fungi undergo processes of cell specialisation such that specific groups of cells are adapted to carry out particular functions. One of the more remarkable examples of cellular development in higher plants is the formation of long files of water-conducting cells that are capable of supporting a column of water that reaches from the roots to hundreds of feet in the air for some trees. The development of files of these cells is a critical feature of land plants that allows the delivery of water to every living cell. Each cell in a file must divide and elongate, before hoops of cell-wall material are deposited at right angles to the direction of cell elongation. This deposition reinforces the cells against the compressive forces of the surrounding tissues that are created by the suction forces of transpiration. The cell-wall material is stiffened and waterproofed by the deposition of phenolic compounds. Finally, the end walls of the cells are broken down and the cell contents are destroyed. The resulting hollow water-conducting tubes are called xylem vessels or tracheids, and the individual cells that form them are called vessel or tracheary elements (TEs) (Figure 1). The formation of TEs involves several processes
that are fundamental to plant development, including cell division, local cell signalling, cell elongation, cell specification, cell-wall synthesis and deposition, lignification and programmed cell death (PCD). Together, these processes involve many hundreds of genes [1]. Many of these genes have been identified in two large-scale screens involving cDNA sequencing of material derived from young xylem tissue from loblolly pine [2] and from poplar trees [3]. The large number of genes identified in this way is impressive, but it remains to be seen how many of them are really involved in xylogenesis itself, as these databases include cDNAs from a mixture of cell types at different developmental stages. Two alternative generic strategies are being used to try to identify genes involved in the various stages of xylem formation and to investigate their function: the use of Arabidopsis mutants and the use of a remarkable in vitro cell system, the Zinnia mesophyll cell system. The Zinnia system consists of cells isolated from the leaves of Zinnia elegans cv. Envy, an ornamental garden plant, that are put into liquid culture and supplied with two plant growth regulators, auxin and cytokinin. By 96 hours, 80% of the cells, which were already specialised as photosynthetic cells in the leaf, are induced to form TEs [4]. The Zinnia system is unique among plant systems for two reasons. First, the entry into a new developmental pathway is induced by adding plant growth regulators, which act like a molecular switch to turn on the process of trans-differentiation. Second, about 80% of the cells synchronously undergo trans-differentiation, making it possible to precisely stage the events involved in building a TE. Thus, one cell type can be reproducibly and synchronously switched by known external signals into a totally different cell type. The Zinnia system is simple and amenable to biochemical and molecular analyses, and offers a real hope of understanding the nature of the molecular machinery whereby plant cells become specialised to carry out particular functions.
The role of auxin in establishing patterns of vascular tissue in plant organs Polar auxin transport has long been known to be involved in the patterning of vascular strands within the plant, during both normal plant development and the creation of new strands in response to wounding [5]. In early embryonic development, this process is intimately tied up with establishing cell polarity and, subsequently, the apical–basal axis of the embryo. The polar movement of auxin depends on the progressive allocation and separation of the appropriate auxin influx and efflux carriers to the plasma membrane at opposite ends of the cell [6]. In the embryo, at least the efflux carriers become gradually restricted to the basal end of cells in the future vascular strands, a process that involves the activity of the MONOPTEROS and GNOM genes [7,8].
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Figure 1
Although polar auxin transport is now widely accepted as a mechanism by which vascular patterning is established, it has been difficult to identify cellular markers that reflect the acquisition of a fixed polarity by the future xylem element. Two recent examples help to redress this problem. In the first example, the phage display method is cleverly exploited to produce a recombinant antibody that crossreacts with a cell-wall polysaccharide, probably a hemicellulose. This polysaccharide is distributed asymmetrically with respect to the two ends of the element at an early stage, both in the plant and in isolated cells in the Zinnia system [13•]. The second example hints at a possible mechanism by which such localised secretion may come about. Three genes that encode different expansins were isolated from the Zinnia system. It was found that the mRNA corresponding to two of these three genes was localised to cytoplasm at the apical end of the differentiating cambial cell, whereas the third mRNA was localised to the basal end [14•]. It seems likely that local translation of the corresponding proteins is coupled in some way with their local secretion, a process that may underlie the polarised distribution of the auxin carriers.
Elucidating the signal transduction cascade that is involved in the specification of xylem cell fate
Scanning electron micrograph of xylem elements in a Zinnia stem. Courtesy of Kim Findlay.
The later elaboration of veins in patterns that are characteristic of the developing organs also appears to be under the control of polar auxin transport, at least in part. In two similar recent studies [9•,10•], the application of polar auxin transport inhibitors was found to disrupt the patterning and connectivity of secondary and tertiary veins. In addition, discontinuous groups of vessels were found near the leaf margin. The conclusion from both studies is that auxin flow, together with the early cellular organisation of the leaf, is important for the early patterning and connectivity of veins. In parallel with these studies, there have been a number of screens to look for mutants with altered or disrupted vein patterns in their cotyledons or leaves. Deyholos et al. reported a mutant called scarface (scf). Arabidopsis plants that are homozygous for a mutation in SCF have disrupted minor veins and an exaggerated response to auxin [11••]. A similar phenotype was found by Koizumi et al. [12••] who described seven non-allelic vascular network mutant loci (van1–7). These authors interpret their data as suggesting that the auxin canalisation model for vascular patterning may not explain adequately the placement of minor veins.
The specification of a xylem vessel or a TE is a long and complex process that requires a succession of signalling events and a progressive restriction of developmental fate to that of a TE. In the Zinnia cell system, the various signalling inputs that are known to influence different steps in the process include the initial wound signal, together with light, auxin, cytokinin, ethylene, brassinosteroids, and phytosulfokine (Figure 2). Even if the intervening signal-transduction steps remain mysterious, we can assume that these signalling inputs result in altered patterns of gene transcription, which in turn require the activity of specific transcription factors. Considerable progress has been made in understanding the roles of transcription factors in controlling TE specialisation. The first examples came from the MYB transcription factor family, whose members control, among other things, the biosynthetic pathways that lead to the production of the monolignols required for lignification. Many of these MYB proteins are produced specifically in xylem tissue [15]. Another class of transcription factors that has been centrally implicated in vascular development is the homeodomain-leucine zipper (HD-ZIP) family. Three members of this family, AtHB-8, AtHB-9 and AtHB-14, are closely related and expressed in vascular tissue, in particular in vascular cambium. AtHB-8 is expressed during the induction of xylem by wounding, and its expression is also induced by auxin [16]. More intriguing is the report that overexpression of the Arabidopsis AtHB-8 gene in tobacco plants stimulates the production of extra xylem cells [17]. Over- and underexpression of the gene in Arabidopsis plants did not, however, result in a similar response. There may be considerable redundancy in the action of these transcription factors.
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Figure 2 Schematic of the various signals known to regulate trans-differentiation in the Zinnia mesophyll cell system.
Maximum competence to respond to auxin and cytokinin Acquisition of competence 0h
Differentiation 48 h
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Brassinosteroids Current Opinion in Plant Biology
Similar transcriptional controls must operate on the differentiation pathways for the two kinds of lignified cells, xylem cells and fibre cells. The interfascicular fiber 1 (ifl1) mutant lacks the fiber bundles that are usually found between vascular bundles in the stem, and hence has a weak and floppy stem. The gene corresponding to this mutant has been isolated and found to encode an HD-ZIP protein, which is related to AtHB-8, that is expressed both in the expected interfascicular regions and in the vascular bundles [18•]. More recently, it has been found that ifl1 and a previously isolated mutant called revoluta have mutations in the same gene [19]. Their expression patterns suggest that the members of this family might have partially overlapping functions in vascular-tissue formation. Screens for stem-vascular mutants have also revealed an unusual semi-dominant phenotype shown by the amphivasal bundle 1 mutant. In this mutant, the usual collateral arrangement of xylem and phloem in a ring of connected vascular bundles in the stem is changed to an amphivasal arrangement. In this arrangement phloem tissue is surrounded sequentially by rings of cambium and xylem to form isolated vascular bundles [20]. The cloning of the cognate genes is awaited with interest. The Zinnia mesophyll cell culture must be at or above a minimum cell density for trans-differentiation to TEs to occur. At a late time-point, however, the culture can be diluted and differentiation is cell-autonomous, indicating the involvement of intercellular communication. It is likely that the synchrony of differentiation is underpinned by the secretion of signals into the culture medium, perhaps from a population of ‘nurse’ cells. A sulphated pentapeptide, called phytosulfokine-α, has been isolated from Zinnia culture medium and shown to promote TE differentiation in low-density cultures [21••]. This peptide was originally identified as a mitogen that promotes cell proliferation in
asparagus cell cultures [22]. Matsubayashi et al. [21••] demonstrate, however, that differentiation is enhanced independently of cell division. Auxin and cytokinin are both still required in the culture for differentiation to occur.
Genes involved in secondary-wall formation and lignification A range of xylem mutants in Arabidopsis has emerged from screens designed to look directly for vessel elements with an altered physical appearance [23]. The IRREGULAR XYLEM 3 locus encodes a cellulose synthase that is required to deposit the secondary-cell-wall cellulose, which thickens and reinforces the wall of the developing xylem-vessel elements [24•]. Other genes involved in the manufacture of the secondary wall, and its subsequent lignification, are likely to emerge as other mutants from this screen are analysed at the molecular level. A further screen has exploited the simple pattern of protoxylem elements in the seedling root of Arabidopsis to uncover a wide spectrum of mutant phenotypes, including changes in the timing of protoxylem differentiation and the number of protoxylem strands, and the formation of ectopic lignified cells. One of these mutants ectopic lignification 1 (eli1) shows disrupted protoxylem, and lignification in the stem-pith cells that appears to be related to the expansion of these cells. This is an intriguing connection, particularly as the authors have also found that some other cell-expansion mutants display ectopic lignification [25••]. Similar ectopic deposition of lignin, also in the pith of the stem of Arabidopsis, has been found in the elp1 (for ectopic deposition of lignin in pith 1) mutant [26•]. Although the tree expressed sequence tag (EST) databases [2,3] are rich sources of genes related to secondary-wall formation and lignification, establishing the xylem specificity
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of such genes is important as many come from large gene families that have roles in primary walls. A xylem-specific cellulose synthase from poplar has been shown to be upregulated in tension wood and during normal growth, but downregulated in compression wood, in which the relative proportion of crystalline cellulose to lignin is known to decrease [27•]. A technical problem that has hindered progress is the difficulty of doing reliable in situ localisations on tissue containing vascular elements. Cryofixation of the cambial meristem and its recent derivatives followed by embedding in a methacrylate resin has, however, resulted in more accurate in situ hybridisation analyses, diminishing non-specific binding of probes to secondary cell walls and improving mRNA retention in cells [28]. Such methodologies will allow cell-specific expression data to be gathered from the tree EST databases. Several laboratories, including our own, have characterised a handful of the genes involved in different stages of the developmental pathway leading to TE fate using the Zinnia system [1]. As molecular markers, these genes have proven extremely useful, and Igarashi et al. [29] were able to demonstrate that the promoter element from one such gene, TED3 (for TRACHEARY ELEMENT DIFFERENTIATION 3), was effective in promoting the xylem-specific expression of a β-glucuronidase (GUS) reporter gene in immature xylem cells of Arabidopsis. We have, however, recently applied a novel RNA-finger-printing technology to allow the detection of DNA fragments derived from RNA. This technology uses cDNA synthesis and subsequent PCR-amplified fragment length polymorphisms (cDNA-AFLP) to systematically characterise hundreds of the genes involved in xylogenesis. We have looked at the patterns of expression of about 25,000 genes, have selected over 600 genes whose transcription products show overt changes in abundance over time, and have obtained partial sequences of these selected genes. Comparison of these partial sequences with sequences in databases from the plant and animal genome sequencing projects has allowed us to assign an identity to about half of their predicted gene products. About 60 of the partial sequences have good sequence identities with a wide range of biosynthetic and hydrolytic cell-wall-related enzymes and structural proteins. It remains to be established, by in situ hybridisation, whether the 600 selected genes represent xylem-specific members of their respective gene families.
Death, a necessary end… In the past few years, the surge of interest in apoptotic death in animal cells has prompted consideration of the mechanism of cell death involved in xylogenesis, senescence and the hypersensitive response (HR) in plant cells. PCD is distinguished from necrotic death by involving cell-autonomous, active and ordered suicide in which specific proteases are recruited to destroy a limited number of key cellular proteins [30]. The detection of fragmented nuclear DNA (nDNA) in the vessel elements of pea indicated that TE formation might share features of apoptotic
cell death, in which nDNA is cleaved into fragments by specific nucleases rather than being degraded randomly [31]. Animal PCD is mediated by the family of cysteinyl aspartate-specific proteinases, known as caspases, which require an aspartic-acid residue to the immediate left and at least four amino acids on the amino-terminal side of the scissile bond. Specific consensus sequences within substrate proteins are required for cleavage by caspases, limiting the number of cellular targets. Both cysteine and serine proteases have been implicated in the HR, in which plant cells die surrounding sites of pathogen ingress [32], and also in TE formation in the Zinnia system [33–35]. Two cDNAs, p48h-17 [33] and ZCP4 [34], encoding cysteine proteases have been isolated from transdifferentiating Zinnia cells in culture and are associated with differentiating xylem in stems. Although no obvious orthologs of mammalian caspases have been found in plants, it seems likely that other families of cysteine and possibly even serine and other proteinases form a comparable battery of cell-death effectors [36]. The expression of components of the death machinery is constitutive in all animal cells, and death is held in check by negative regulators. Procaspases are activated by cleavage at sites that resemble their own substrate sites. On receipt of either intracellular (e.g. cytochrome c release from damaged mitochondria) or extracellular (e.g. Fas ligands, tumour necrosis factors and a serine protease called granzyme B) signals, the cascade is initiated by adaptor proteins that bring about the aggregation of procaspases into a complex in which residual proteolytic activity induces autoor trans-activation. Once begun, the cascade is self-amplifying and irreversible. In plants, there are mutants that exhibit constitutive initiation of HR-like cell death in the absence of pathogens [37–39] and, thus, are candidates for direct negative control of cell death. Ectopic expression of cystatin, a cysteine-protease-inhibitor gene, blocks PCD triggered either by pathogen or by oxidative stress [40••]. In the solanaceous plant brinjal, a transcript encoding a cysteine proteinase has been shown to be upregulated during xylogenesis, anther senescence and ovule development, thus showing that members of the death machinery can be recruited into several, rather than specific, events in developmentally regulated PCD [41]. The transient expression of cysteine-protease-encoding genes in Zinnia, and their specificity for xylem cells, may, however, indicate that they have a specific role in trans-differentiation. TE formation may represent a specialised form of PCD in which death is an integral part of differentiation in a pre-programmed developmental pathway. The most appropriate parallel with animals may be in the differentiation of keratinocytes in which the terminal stage is to die and form a layer of corpses (squames) on the surface of the skin [42]. Caspases are activated during normal human keratinocyte differentiation [43], but the outcome is different from that during classical apoptosis, as only the organelles are degraded.
Xylogenesis: the birth of a corpse Roberts and McCann
In animals, diverse signals can be transduced into a common mechanism of death. Similarly, different hormones have been implicated in the induction of different examples of plant PCD: ethylene in triggering the formation of aerenchyma, GA3 in aleurone cells, and auxin in all examples of TE formation. Uniconazole, an inhibitor of brassinosteroid biosynthesis, blocks a late stage of TE formation, and prevents the expression of genes involved in secondary-wall thickening and also in cell death, in particular, the ZCP4 cysteine protease gene [44]. In tissues, survival factors secreted by neighbouring cells can suppress apoptosis by binding to cell-surface receptors that maintain the activities of the inhibitors of apoptosis (IAP) family. Such factors are necessary to keep cells alive, but have also been implicated as mitogens or differentiation signals. The phytosulfokine-α would appear to be such a factor [21••]. Calcium has been implicated in the regulation both of secondary-wall formation and of the final commitment to cell death. Groover and Jones have shown that the collapse of the vacuole is preceded by a large influx of Ca2+ into the cell [45•]. The cysteine proteinases are optimally active at vacuolar pH [34] and autolysis occurs rapidly after vacuolar disruption, when other hydrolytic enzymes are also released to mix with the cytoplasm [46]. About 6 hours after the appearance of visible cell-wall thickenings, there is a rapid disruption of the tonoplast (occurring within 3 minutes). This is succeeded by the swelling and disruption of, first, the single-membraned organelles (i.e. the endoplasmic reticulum and Golgi) and then the doublemembraned organelles (i.e. chloroplasts, mitochondria and the nucleus) within hours of vacuole collapse [46]. Nuclear DNA is degraded and can be assayed in individual cells using cytochemistry [45•,46]. Reducing calcium influx protects against nDNA fragmentation. Addition of trypsin to the cell cultures initiates cell death, suggesting that specific proteolysis of the extracellular matrix triggers Ca2+ influx, vacuole collapse, cell death and chromatin degradation [45•]. A 40-kiloDalton serine protease is secreted during secondary-cell-wall synthesis [45•], and soybean trypsin inhibitor inhibits both PCD in the Zinnia system and the activity of this serine protease. Interestingly, granzyme B is a serine protease, delivered by cytotoxic T lymphocytes, that mediates cell suicide by activating endogenous caspases within the target cell. The mechanism outlined above is consistent with the dependence of TE differentiation on cell concentration in the Zinnia system until a late stage.
Conclusions The use of mutants and transgenic plants, coupled with large EST and genome-sequencing projects, has begun to make a significant impact on xylem biology. This impact has been felt most in two intimately connected areas: the mechanisms that orchestrate the ordered patterning of vascular tissues and the signalling pathways that specify xylem cell fate. The Zinnia mesophyll system is proving to be both an engine for gene discovery for understanding
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xylogenesis in planta and a useful cell-culture system for dissecting the roles of individual signalling molecules. Candidate cysteine and serine proteases have been implicated in the PCD that terminates xylem formation.
Acknowledgements Maureen McCann thanks the Royal Society of London for a University Research Fellowship, and Keith Roberts’ work is funded by the Biotechnology and Biological Sciences Research Council (BBSRC).
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9. Sieburth LE: Auxin is required for leaf vein pattern in Arabidopsis. • Plant Physiol 1999, 121:1179-1190. This paper, together with [10•], provides physiological evidence for the role of polar auxin transport in early vascular patterning events in the leaf, and for the establishment of connectivity between the cellular elements within the vascular tissue. The authors of both papers test the effects of a range of polar auxin transport inhibitors on vascular patterning events in Arabidopsis cotyledons and leaves, and find disrupted leaf patterning with coalesced mid-veins and discontinuous groups of vascular elements at the leaf margins. 10. Mattsson J, Sung ZR, Berleth T: Responses of plant vascular • systems to auxin transport inhibition. Development 1999, 126:2979-2991. See annotation [9•]. 11. Deyholos MK, Cordner G, Beebe D, Sieburth LE: The SCARFACE •• gene is required for cotyledon and leaf vein patterning. Development 2000, 127:3205-3213. Arabidopsis plants that are homozygous for a mutation in the SCARFACE gene on chromosome 5 show early defects in vein patterning in aerial organs. The secondary and tertiary veins of these mutants become discontinuous and the mutants show an exaggerated response to auxin. SCF also appears to have overlapping functions with MONOPTEROS (see also [12••]). 12. Koizumi K, Sugiyama M, Fukuda H: A series of novel mutants of •• Arabidopsis thaliana that are defective in the formation of continuous vascular network: calling the auxin signal flow canalization hypothesis into question. Development 2000, 127:3197-3204. A screen in Arabidopsis revealed recessive mutations in seven genes (VAN1–VAN7) that result in the fragmentation of the secondary and tertiary veins in cotyledons and young leaves. The phenotype leads the authors to support a diffusion-reaction model for minor vein patterning in preference to the auxin canalisation model (see also [11••]).
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20. Zhong R, Taylor JJ, Ye Z-H: Transformation of the collateral vascular bundles into amphivasal vascular bundles in an Arabidopsis mutant. Plant Physiol 1999, 120:53-64. 21. Matsubayashi Y, Takagi L, Omura N, Morita A, Sakagami Y: The •• endogenous sulfated pentapeptide phytosulfokine-alpha stimulates tracheary element differentiation of isolated mesophyll cells of Zinnia. Plant Physiol 1999, 120:1043-1048. If Zinnia cells are cultured at too low a cell density, then TE formation is suppressed even in the presence of auxin and cytokinin. However, addition of phytosulfokine promotes TE formation in low-density cultures in a concentration-dependent fashion. 22. Matsubayashi Y, Morita A, Matsunaga E, Furuya A, Hanai N, Sakagami Y: Physiological relationships between auxin, cytokinin, α, in stimulation of and a peptide growth factor, phytosulfokine-α asparagus cell proliferation. Planta 1999, 207:559-565. 23. Turner SR, Somerville CR: Collapsed xylem phenotype of Arabidopsis identifies mutants deficient in cellulose deposition in the secondary cell wall. Plant Cell 1997, 9:689-701.
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Dietrich RA, Richberg MH, Schmidt R, Dean C, Dangl JL: A novel zinc finger protein is encoded by the Arabidopsis LSD1 gene and functions as a negative regulator of plant cell death. Cell 1997, 88:685-694.
38. Tanaka Y, Makishima T, Sasabe M, Ichinose Y, Shiraishi T, Nishimoto T, Yamada T: dad-1, a putative programmed cell death suppressor gene in rice. Plant Cell Physiol 1997, 38:379-383. 39. Gray J, Close PS, Briggs SP, Johal GS: A novel suppressor of cell death in plants encoded by the Lls1 gene of maize. Cell 1997, 89:25-31. 40. Solomon M, Belenghi B, Delledonne M, Menachem E, Levine A: The •• involvement of cysteine proteases and protease inhibitor genes in the regulation of programmed cell death in plants. Plant Cell 1999, 11:431-443. PCD-activating oxidative stress induces a set of cysteine proteases in soybean cells. Ectopic expression of cystatin, an endogenous cysteine-proteaseinhibitor gene, inhibited the activity of cysteine proteases and PCD.
24. Taylor NG, Scheible WR, Cutler S, Somerville CR, Turner SR: The • irregular xylem3 locus of Arabidopsis encodes a cellulose synthase required for secondary cell wall synthesis. Plant Cell 1999, 11:769-779. Stems of irx3 plants have less than 20% of the cellulose content of wild-type stems. A cellulose synthase gene was identified at the irx3 map location, and this wild-type gene complemented the mutation by restoring cellulose deposition in the secondary walls of xylem vessels. Other irregular xylem (irx) genes are currently being cloned.
41. Xu F-X, Chye M-L: Expression of cysteine proteases during developmental events associated with programmed cell death in brinjal. Plant J 1999, 17:321-327.
25. Cano-Delgado A, Metzlaff K, Bevan MW: The eli1 mutation reveals a •• link between cell expansion and secondary cell wall formation in Arabidopsis thaliana. Development 2000, 127:3395-3405. Another screen for vascular mutants, but this time taking advantage of the simplicity of the seedling root xylem, has identified many mutants, of which one displaying ectopic lignification (eli1) is described in detail. The mutant shows disorganised xylem with discontinuous elements and cells, possibly connected with altered expansion growth, that develop inappropriately secondary walls that become lignified. (See [26•].)
44. Yamamoto R, Demura T, Fukuda H: Brassinosteroids induce entry into the final stage of tracheary element differentiation in cultured Zinnia cells. Plant Cell Physiol 1997, 38:980-983.
26. Zhong R, Ripperger A, Ye Z-H: Ectopic deposition of lignin in the • pith of stems of two Arabidopsis mutants. Plant Physiol 2000, 123:59-69. Another screen for altered lignification patterns has identified two mutant alleles of the ELP1 gene on chromosome 1. The wild-type function of this gene may be to repress lignin deposition in pith tissues.
42. Jacobson MD, Weil M, Raff MC: Programmed cell death in animal development. Cell 1997, 88:347-354. 43. Weil M, Raff MC, Braga VMM: Caspase activation in the terminal differentiation of human epidermal keratinocytes. Curr Biol 1999, 9:361-364.
45. Groover A, Jones AM: Tracheary element differentiation uses a • novel mechanism coordinating programmed cell death and secondary cell wall synthesis. Plant Physiol 1999, 119:375-384. The authors present evidence that a serine protease is secreted into the culture medium during TE differentiation. This protease may act upon a substrate produced in the cell wall during secondary-wall formation, producing a signalling molecule that triggers programmed cell death and thus co-ordinating the cessation of wall deposition with PCD. 46. Groover A, DeWitt NG, Heidel A, Jones AM: Programmed cell death of plant tracheary elements differentiating in vitro. Protoplasma 1997, 196:197-211.
Xylogenesis: the birth of a corpse Keith Roberts* and Maureen C McCann† Xylogenesis is a complex developmental process culminating in programmed cell death as a truly terminal differentiation event. In Arabidopsis, the availability of vascular-patterning mutants, and the identification of genes and their products that are involved in cell specification, secondary-wall deposition and lignification, are providing clues to the functions of some of the sequences in the large expressed sequence tag databases derived from the xylem-rich tissues of trees. An in vitro system, the Zinnia mesophyll cell system, provides an alternative system for those cell-biological experiments that are difficult to tackle in intact plants. In particular, a combination of molecular-genetic and cellbiological approaches has made possible the elucidation of some of the features of plant programmed cell death. Addresses Department of Cell Biology, John Innes Centre, Norwich Research Park, Colney, Norwich, NR4 7UH, UK *e-mail: [email protected] † e-mail: [email protected] Current Opinion in Plant Biology 2000, 3:517–522 1369-5266/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations EST expressed sequence tag HR hypersensitive response ifl1 interfascicular fiber 1 nDNA nuclear DNA PCD programmed cell death scf scarface TE tracheary element
Introduction Plants, animals and fungi undergo processes of cell specialisation such that specific groups of cells are adapted to carry out particular functions. One of the more remarkable examples of cellular development in higher plants is the formation of long files of water-conducting cells that are capable of supporting a column of water that reaches from the roots to hundreds of feet in the air for some trees. The development of files of these cells is a critical feature of land plants that allows the delivery of water to every living cell. Each cell in a file must divide and elongate, before hoops of cell-wall material are deposited at right angles to the direction of cell elongation. This deposition reinforces the cells against the compressive forces of the surrounding tissues that are created by the suction forces of transpiration. The cell-wall material is stiffened and waterproofed by the deposition of phenolic compounds. Finally, the end walls of the cells are broken down and the cell contents are destroyed. The resulting hollow water-conducting tubes are called xylem vessels or tracheids, and the individual cells that form them are called vessel or tracheary elements (TEs) (Figure 1). The formation of TEs involves several processes
that are fundamental to plant development, including cell division, local cell signalling, cell elongation, cell specification, cell-wall synthesis and deposition, lignification and programmed cell death (PCD). Together, these processes involve many hundreds of genes [1]. Many of these genes have been identified in two large-scale screens involving cDNA sequencing of material derived from young xylem tissue from loblolly pine [2] and from poplar trees [3]. The large number of genes identified in this way is impressive, but it remains to be seen how many of them are really involved in xylogenesis itself, as these databases include cDNAs from a mixture of cell types at different developmental stages. Two alternative generic strategies are being used to try to identify genes involved in the various stages of xylem formation and to investigate their function: the use of Arabidopsis mutants and the use of a remarkable in vitro cell system, the Zinnia mesophyll cell system. The Zinnia system consists of cells isolated from the leaves of Zinnia elegans cv. Envy, an ornamental garden plant, that are put into liquid culture and supplied with two plant growth regulators, auxin and cytokinin. By 96 hours, 80% of the cells, which were already specialised as photosynthetic cells in the leaf, are induced to form TEs [4]. The Zinnia system is unique among plant systems for two reasons. First, the entry into a new developmental pathway is induced by adding plant growth regulators, which act like a molecular switch to turn on the process of trans-differentiation. Second, about 80% of the cells synchronously undergo trans-differentiation, making it possible to precisely stage the events involved in building a TE. Thus, one cell type can be reproducibly and synchronously switched by known external signals into a totally different cell type. The Zinnia system is simple and amenable to biochemical and molecular analyses, and offers a real hope of understanding the nature of the molecular machinery whereby plant cells become specialised to carry out particular functions.
The role of auxin in establishing patterns of vascular tissue in plant organs Polar auxin transport has long been known to be involved in the patterning of vascular strands within the plant, during both normal plant development and the creation of new strands in response to wounding [5]. In early embryonic development, this process is intimately tied up with establishing cell polarity and, subsequently, the apical–basal axis of the embryo. The polar movement of auxin depends on the progressive allocation and separation of the appropriate auxin influx and efflux carriers to the plasma membrane at opposite ends of the cell [6]. In the embryo, at least the efflux carriers become gradually restricted to the basal end of cells in the future vascular strands, a process that involves the activity of the MONOPTEROS and GNOM genes [7,8].
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Figure 1
Although polar auxin transport is now widely accepted as a mechanism by which vascular patterning is established, it has been difficult to identify cellular markers that reflect the acquisition of a fixed polarity by the future xylem element. Two recent examples help to redress this problem. In the first example, the phage display method is cleverly exploited to produce a recombinant antibody that crossreacts with a cell-wall polysaccharide, probably a hemicellulose. This polysaccharide is distributed asymmetrically with respect to the two ends of the element at an early stage, both in the plant and in isolated cells in the Zinnia system [13•]. The second example hints at a possible mechanism by which such localised secretion may come about. Three genes that encode different expansins were isolated from the Zinnia system. It was found that the mRNA corresponding to two of these three genes was localised to cytoplasm at the apical end of the differentiating cambial cell, whereas the third mRNA was localised to the basal end [14•]. It seems likely that local translation of the corresponding proteins is coupled in some way with their local secretion, a process that may underlie the polarised distribution of the auxin carriers.
Elucidating the signal transduction cascade that is involved in the specification of xylem cell fate
Scanning electron micrograph of xylem elements in a Zinnia stem. Courtesy of Kim Findlay.
The later elaboration of veins in patterns that are characteristic of the developing organs also appears to be under the control of polar auxin transport, at least in part. In two similar recent studies [9•,10•], the application of polar auxin transport inhibitors was found to disrupt the patterning and connectivity of secondary and tertiary veins. In addition, discontinuous groups of vessels were found near the leaf margin. The conclusion from both studies is that auxin flow, together with the early cellular organisation of the leaf, is important for the early patterning and connectivity of veins. In parallel with these studies, there have been a number of screens to look for mutants with altered or disrupted vein patterns in their cotyledons or leaves. Deyholos et al. reported a mutant called scarface (scf). Arabidopsis plants that are homozygous for a mutation in SCF have disrupted minor veins and an exaggerated response to auxin [11••]. A similar phenotype was found by Koizumi et al. [12••] who described seven non-allelic vascular network mutant loci (van1–7). These authors interpret their data as suggesting that the auxin canalisation model for vascular patterning may not explain adequately the placement of minor veins.
The specification of a xylem vessel or a TE is a long and complex process that requires a succession of signalling events and a progressive restriction of developmental fate to that of a TE. In the Zinnia cell system, the various signalling inputs that are known to influence different steps in the process include the initial wound signal, together with light, auxin, cytokinin, ethylene, brassinosteroids, and phytosulfokine (Figure 2). Even if the intervening signal-transduction steps remain mysterious, we can assume that these signalling inputs result in altered patterns of gene transcription, which in turn require the activity of specific transcription factors. Considerable progress has been made in understanding the roles of transcription factors in controlling TE specialisation. The first examples came from the MYB transcription factor family, whose members control, among other things, the biosynthetic pathways that lead to the production of the monolignols required for lignification. Many of these MYB proteins are produced specifically in xylem tissue [15]. Another class of transcription factors that has been centrally implicated in vascular development is the homeodomain-leucine zipper (HD-ZIP) family. Three members of this family, AtHB-8, AtHB-9 and AtHB-14, are closely related and expressed in vascular tissue, in particular in vascular cambium. AtHB-8 is expressed during the induction of xylem by wounding, and its expression is also induced by auxin [16]. More intriguing is the report that overexpression of the Arabidopsis AtHB-8 gene in tobacco plants stimulates the production of extra xylem cells [17]. Over- and underexpression of the gene in Arabidopsis plants did not, however, result in a similar response. There may be considerable redundancy in the action of these transcription factors.
Xylogenesis: the birth of a corpse Roberts and McCann
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Figure 2 Schematic of the various signals known to regulate trans-differentiation in the Zinnia mesophyll cell system.
Maximum competence to respond to auxin and cytokinin Acquisition of competence 0h
Differentiation 48 h
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Brassinosteroids Current Opinion in Plant Biology
Similar transcriptional controls must operate on the differentiation pathways for the two kinds of lignified cells, xylem cells and fibre cells. The interfascicular fiber 1 (ifl1) mutant lacks the fiber bundles that are usually found between vascular bundles in the stem, and hence has a weak and floppy stem. The gene corresponding to this mutant has been isolated and found to encode an HD-ZIP protein, which is related to AtHB-8, that is expressed both in the expected interfascicular regions and in the vascular bundles [18•]. More recently, it has been found that ifl1 and a previously isolated mutant called revoluta have mutations in the same gene [19]. Their expression patterns suggest that the members of this family might have partially overlapping functions in vascular-tissue formation. Screens for stem-vascular mutants have also revealed an unusual semi-dominant phenotype shown by the amphivasal bundle 1 mutant. In this mutant, the usual collateral arrangement of xylem and phloem in a ring of connected vascular bundles in the stem is changed to an amphivasal arrangement. In this arrangement phloem tissue is surrounded sequentially by rings of cambium and xylem to form isolated vascular bundles [20]. The cloning of the cognate genes is awaited with interest. The Zinnia mesophyll cell culture must be at or above a minimum cell density for trans-differentiation to TEs to occur. At a late time-point, however, the culture can be diluted and differentiation is cell-autonomous, indicating the involvement of intercellular communication. It is likely that the synchrony of differentiation is underpinned by the secretion of signals into the culture medium, perhaps from a population of ‘nurse’ cells. A sulphated pentapeptide, called phytosulfokine-α, has been isolated from Zinnia culture medium and shown to promote TE differentiation in low-density cultures [21••]. This peptide was originally identified as a mitogen that promotes cell proliferation in
asparagus cell cultures [22]. Matsubayashi et al. [21••] demonstrate, however, that differentiation is enhanced independently of cell division. Auxin and cytokinin are both still required in the culture for differentiation to occur.
Genes involved in secondary-wall formation and lignification A range of xylem mutants in Arabidopsis has emerged from screens designed to look directly for vessel elements with an altered physical appearance [23]. The IRREGULAR XYLEM 3 locus encodes a cellulose synthase that is required to deposit the secondary-cell-wall cellulose, which thickens and reinforces the wall of the developing xylem-vessel elements [24•]. Other genes involved in the manufacture of the secondary wall, and its subsequent lignification, are likely to emerge as other mutants from this screen are analysed at the molecular level. A further screen has exploited the simple pattern of protoxylem elements in the seedling root of Arabidopsis to uncover a wide spectrum of mutant phenotypes, including changes in the timing of protoxylem differentiation and the number of protoxylem strands, and the formation of ectopic lignified cells. One of these mutants ectopic lignification 1 (eli1) shows disrupted protoxylem, and lignification in the stem-pith cells that appears to be related to the expansion of these cells. This is an intriguing connection, particularly as the authors have also found that some other cell-expansion mutants display ectopic lignification [25••]. Similar ectopic deposition of lignin, also in the pith of the stem of Arabidopsis, has been found in the elp1 (for ectopic deposition of lignin in pith 1) mutant [26•]. Although the tree expressed sequence tag (EST) databases [2,3] are rich sources of genes related to secondary-wall formation and lignification, establishing the xylem specificity
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of such genes is important as many come from large gene families that have roles in primary walls. A xylem-specific cellulose synthase from poplar has been shown to be upregulated in tension wood and during normal growth, but downregulated in compression wood, in which the relative proportion of crystalline cellulose to lignin is known to decrease [27•]. A technical problem that has hindered progress is the difficulty of doing reliable in situ localisations on tissue containing vascular elements. Cryofixation of the cambial meristem and its recent derivatives followed by embedding in a methacrylate resin has, however, resulted in more accurate in situ hybridisation analyses, diminishing non-specific binding of probes to secondary cell walls and improving mRNA retention in cells [28]. Such methodologies will allow cell-specific expression data to be gathered from the tree EST databases. Several laboratories, including our own, have characterised a handful of the genes involved in different stages of the developmental pathway leading to TE fate using the Zinnia system [1]. As molecular markers, these genes have proven extremely useful, and Igarashi et al. [29] were able to demonstrate that the promoter element from one such gene, TED3 (for TRACHEARY ELEMENT DIFFERENTIATION 3), was effective in promoting the xylem-specific expression of a β-glucuronidase (GUS) reporter gene in immature xylem cells of Arabidopsis. We have, however, recently applied a novel RNA-finger-printing technology to allow the detection of DNA fragments derived from RNA. This technology uses cDNA synthesis and subsequent PCR-amplified fragment length polymorphisms (cDNA-AFLP) to systematically characterise hundreds of the genes involved in xylogenesis. We have looked at the patterns of expression of about 25,000 genes, have selected over 600 genes whose transcription products show overt changes in abundance over time, and have obtained partial sequences of these selected genes. Comparison of these partial sequences with sequences in databases from the plant and animal genome sequencing projects has allowed us to assign an identity to about half of their predicted gene products. About 60 of the partial sequences have good sequence identities with a wide range of biosynthetic and hydrolytic cell-wall-related enzymes and structural proteins. It remains to be established, by in situ hybridisation, whether the 600 selected genes represent xylem-specific members of their respective gene families.
Death, a necessary end… In the past few years, the surge of interest in apoptotic death in animal cells has prompted consideration of the mechanism of cell death involved in xylogenesis, senescence and the hypersensitive response (HR) in plant cells. PCD is distinguished from necrotic death by involving cell-autonomous, active and ordered suicide in which specific proteases are recruited to destroy a limited number of key cellular proteins [30]. The detection of fragmented nuclear DNA (nDNA) in the vessel elements of pea indicated that TE formation might share features of apoptotic
cell death, in which nDNA is cleaved into fragments by specific nucleases rather than being degraded randomly [31]. Animal PCD is mediated by the family of cysteinyl aspartate-specific proteinases, known as caspases, which require an aspartic-acid residue to the immediate left and at least four amino acids on the amino-terminal side of the scissile bond. Specific consensus sequences within substrate proteins are required for cleavage by caspases, limiting the number of cellular targets. Both cysteine and serine proteases have been implicated in the HR, in which plant cells die surrounding sites of pathogen ingress [32], and also in TE formation in the Zinnia system [33–35]. Two cDNAs, p48h-17 [33] and ZCP4 [34], encoding cysteine proteases have been isolated from transdifferentiating Zinnia cells in culture and are associated with differentiating xylem in stems. Although no obvious orthologs of mammalian caspases have been found in plants, it seems likely that other families of cysteine and possibly even serine and other proteinases form a comparable battery of cell-death effectors [36]. The expression of components of the death machinery is constitutive in all animal cells, and death is held in check by negative regulators. Procaspases are activated by cleavage at sites that resemble their own substrate sites. On receipt of either intracellular (e.g. cytochrome c release from damaged mitochondria) or extracellular (e.g. Fas ligands, tumour necrosis factors and a serine protease called granzyme B) signals, the cascade is initiated by adaptor proteins that bring about the aggregation of procaspases into a complex in which residual proteolytic activity induces autoor trans-activation. Once begun, the cascade is self-amplifying and irreversible. In plants, there are mutants that exhibit constitutive initiation of HR-like cell death in the absence of pathogens [37–39] and, thus, are candidates for direct negative control of cell death. Ectopic expression of cystatin, a cysteine-protease-inhibitor gene, blocks PCD triggered either by pathogen or by oxidative stress [40••]. In the solanaceous plant brinjal, a transcript encoding a cysteine proteinase has been shown to be upregulated during xylogenesis, anther senescence and ovule development, thus showing that members of the death machinery can be recruited into several, rather than specific, events in developmentally regulated PCD [41]. The transient expression of cysteine-protease-encoding genes in Zinnia, and their specificity for xylem cells, may, however, indicate that they have a specific role in trans-differentiation. TE formation may represent a specialised form of PCD in which death is an integral part of differentiation in a pre-programmed developmental pathway. The most appropriate parallel with animals may be in the differentiation of keratinocytes in which the terminal stage is to die and form a layer of corpses (squames) on the surface of the skin [42]. Caspases are activated during normal human keratinocyte differentiation [43], but the outcome is different from that during classical apoptosis, as only the organelles are degraded.
Xylogenesis: the birth of a corpse Roberts and McCann
In animals, diverse signals can be transduced into a common mechanism of death. Similarly, different hormones have been implicated in the induction of different examples of plant PCD: ethylene in triggering the formation of aerenchyma, GA3 in aleurone cells, and auxin in all examples of TE formation. Uniconazole, an inhibitor of brassinosteroid biosynthesis, blocks a late stage of TE formation, and prevents the expression of genes involved in secondary-wall thickening and also in cell death, in particular, the ZCP4 cysteine protease gene [44]. In tissues, survival factors secreted by neighbouring cells can suppress apoptosis by binding to cell-surface receptors that maintain the activities of the inhibitors of apoptosis (IAP) family. Such factors are necessary to keep cells alive, but have also been implicated as mitogens or differentiation signals. The phytosulfokine-α would appear to be such a factor [21••]. Calcium has been implicated in the regulation both of secondary-wall formation and of the final commitment to cell death. Groover and Jones have shown that the collapse of the vacuole is preceded by a large influx of Ca2+ into the cell [45•]. The cysteine proteinases are optimally active at vacuolar pH [34] and autolysis occurs rapidly after vacuolar disruption, when other hydrolytic enzymes are also released to mix with the cytoplasm [46]. About 6 hours after the appearance of visible cell-wall thickenings, there is a rapid disruption of the tonoplast (occurring within 3 minutes). This is succeeded by the swelling and disruption of, first, the single-membraned organelles (i.e. the endoplasmic reticulum and Golgi) and then the doublemembraned organelles (i.e. chloroplasts, mitochondria and the nucleus) within hours of vacuole collapse [46]. Nuclear DNA is degraded and can be assayed in individual cells using cytochemistry [45•,46]. Reducing calcium influx protects against nDNA fragmentation. Addition of trypsin to the cell cultures initiates cell death, suggesting that specific proteolysis of the extracellular matrix triggers Ca2+ influx, vacuole collapse, cell death and chromatin degradation [45•]. A 40-kiloDalton serine protease is secreted during secondary-cell-wall synthesis [45•], and soybean trypsin inhibitor inhibits both PCD in the Zinnia system and the activity of this serine protease. Interestingly, granzyme B is a serine protease, delivered by cytotoxic T lymphocytes, that mediates cell suicide by activating endogenous caspases within the target cell. The mechanism outlined above is consistent with the dependence of TE differentiation on cell concentration in the Zinnia system until a late stage.
Conclusions The use of mutants and transgenic plants, coupled with large EST and genome-sequencing projects, has begun to make a significant impact on xylem biology. This impact has been felt most in two intimately connected areas: the mechanisms that orchestrate the ordered patterning of vascular tissues and the signalling pathways that specify xylem cell fate. The Zinnia mesophyll system is proving to be both an engine for gene discovery for understanding
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xylogenesis in planta and a useful cell-culture system for dissecting the roles of individual signalling molecules. Candidate cysteine and serine proteases have been implicated in the PCD that terminates xylem formation.
Acknowledgements Maureen McCann thanks the Royal Society of London for a University Research Fellowship, and Keith Roberts’ work is funded by the Biotechnology and Biological Sciences Research Council (BBSRC).
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
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Allona I, Quinn M, Shoop E, Swope K, Cyr SS, Carlis J, Riedl J, Retzel E, Campbell MM, Sederoff R, Whetten RW: Analysis of xylem formation in pine by cDNA sequencing. Proc Natl Acad Sci USA 1998, 95:9693-9698.
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McCann MC, Domingo C, Stacey NJ, Milioni D, Roberts K: Tracheary element formation in an in vitro system. In Cambium: the Biology of Wood Formation. Edited by Savidge R, Barnett J, Napier R. Oxford: BIOS Scientific Publishers Ltd; 2000:457-470.
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9. Sieburth LE: Auxin is required for leaf vein pattern in Arabidopsis. • Plant Physiol 1999, 121:1179-1190. This paper, together with [10•], provides physiological evidence for the role of polar auxin transport in early vascular patterning events in the leaf, and for the establishment of connectivity between the cellular elements within the vascular tissue. The authors of both papers test the effects of a range of polar auxin transport inhibitors on vascular patterning events in Arabidopsis cotyledons and leaves, and find disrupted leaf patterning with coalesced mid-veins and discontinuous groups of vascular elements at the leaf margins. 10. Mattsson J, Sung ZR, Berleth T: Responses of plant vascular • systems to auxin transport inhibition. Development 1999, 126:2979-2991. See annotation [9•]. 11. Deyholos MK, Cordner G, Beebe D, Sieburth LE: The SCARFACE •• gene is required for cotyledon and leaf vein patterning. Development 2000, 127:3205-3213. Arabidopsis plants that are homozygous for a mutation in the SCARFACE gene on chromosome 5 show early defects in vein patterning in aerial organs. The secondary and tertiary veins of these mutants become discontinuous and the mutants show an exaggerated response to auxin. SCF also appears to have overlapping functions with MONOPTEROS (see also [12••]). 12. Koizumi K, Sugiyama M, Fukuda H: A series of novel mutants of •• Arabidopsis thaliana that are defective in the formation of continuous vascular network: calling the auxin signal flow canalization hypothesis into question. Development 2000, 127:3197-3204. A screen in Arabidopsis revealed recessive mutations in seven genes (VAN1–VAN7) that result in the fragmentation of the secondary and tertiary veins in cotyledons and young leaves. The phenotype leads the authors to support a diffusion-reaction model for minor vein patterning in preference to the auxin canalisation model (see also [11••]).
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13. Shinohara N, Demura T, Fukuda H: Isolation of a vascular cell wall• specific monoclonal antibody recognizing a cell polarity by using a phage display subtraction method. Proc Natl Acad Sci USA 2000, 97:2585-2590. Exploiting the antibody phage display method, the authors identify an epitope in the cell-wall hemicellulose fraction that is distributed in a polarised way in immature tracheids both in planta and in the Zinnia cell system.
Wu LG, Joshi SP, Chiang VL: A xylem-specific cellulose synthase gene from aspen (Populus tremuloides) is responsive to mechanical stress. Plant J 2000, 22:495-502. The ratio of cellulose to lignin differs between tension and compression woods. The observation that a particular cellulose-synthase-encoding gene is turned off in compression wood tissues provides tentative evidence for a mechanism whereby wall composition can be altered in response to biomechanical signals.
14. Im KH, Cosgrove DT, Jones AM: Subcellular localization of • expansin mRNA in xylem cells. Plant Physiol 2000, 123:463-470. Three Zinnia expansin genes are described. The mRNA corresponding to each gene is localised in stem-tissue cells, two being localised to the apical end and one to the basal end of putative cambial cells.
28. Regan S, Bourquin V, Tuominen H, Sundberg B: Accurate and high resolution in situ hybridization analysis of gene expression in secondary stem tissues. Plant J 2000, 19:363-369.
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29. Igarashi M, Demura T, Fukuda H: Expression of the Zinnia TED3 promoter in developing tracheary elements of transgenic Arabidopsis. Plant Mol Biol 1998, 36:917-927.
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16. Sessa G, Steindler C, Morelli G, Ruberti I: The Arabidopsis ATHB-8, 9 and 14 genes are members of a small gene family coding highly related HD-Zip proteins. Plant Mol Biol 1998, 38:609-622.
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33. Ye Z-H, Varner JE: Induction of cysteine and serine proteases during xylogenesis in Zinnia elegans. Plant Mol Biol 1996, 30:1233-1246.
18. Zhong R, Ye Z-H: IFL1, a gene regulating interfascicular fiber • differentiation in Arabidopsis encodes a homeodomain-leucine zipper protein. Plant Cell 1999, 11:2139-2152. Mutations in the IFL1 gene abolish the formation of the fibre bundles normally located between vascular bundles in the Arabidopsis stem, and in addition affect normal xylem development. IFL1 encodes a member of the HD-ZIP class of transcription factors and is expressed in vascular tissue. (The ifl1 mutant is allelic to the revoluta mutation, see below.)
34. Minami A, Fukuda H: Transient and specific expression of a cysteine endopeptidase during differentiation of Zinnia mesophyll cells into tracheary elements. Plant Cell Physiol 1995, 36:1599-1606.
19. Ratcliffe OJ, Riechmann JL, Zhang JZ: INTERFASCICULAR FIBERLESS 1 is the same gene as REVOLUTA. Plant Cell 2000, 12:315-317.
36. Iliev I, Savidge R: Proteolytic activity in relation to seasonal cambial growth and xylogenesis in Pinus banksiana. Phytochemistry 1999, 50:953-960.
20. Zhong R, Taylor JJ, Ye Z-H: Transformation of the collateral vascular bundles into amphivasal vascular bundles in an Arabidopsis mutant. Plant Physiol 1999, 120:53-64. 21. Matsubayashi Y, Takagi L, Omura N, Morita A, Sakagami Y: The •• endogenous sulfated pentapeptide phytosulfokine-alpha stimulates tracheary element differentiation of isolated mesophyll cells of Zinnia. Plant Physiol 1999, 120:1043-1048. If Zinnia cells are cultured at too low a cell density, then TE formation is suppressed even in the presence of auxin and cytokinin. However, addition of phytosulfokine promotes TE formation in low-density cultures in a concentration-dependent fashion. 22. Matsubayashi Y, Morita A, Matsunaga E, Furuya A, Hanai N, Sakagami Y: Physiological relationships between auxin, cytokinin, α, in stimulation of and a peptide growth factor, phytosulfokine-α asparagus cell proliferation. Planta 1999, 207:559-565. 23. Turner SR, Somerville CR: Collapsed xylem phenotype of Arabidopsis identifies mutants deficient in cellulose deposition in the secondary cell wall. Plant Cell 1997, 9:689-701.
35. Beers EP, Freeman TB: Proteinase activity during tracheary element differentiation in Zinnia mesophyll cultures. Plant Physiol 1997, 113:873-880.
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Dietrich RA, Richberg MH, Schmidt R, Dean C, Dangl JL: A novel zinc finger protein is encoded by the Arabidopsis LSD1 gene and functions as a negative regulator of plant cell death. Cell 1997, 88:685-694.
38. Tanaka Y, Makishima T, Sasabe M, Ichinose Y, Shiraishi T, Nishimoto T, Yamada T: dad-1, a putative programmed cell death suppressor gene in rice. Plant Cell Physiol 1997, 38:379-383. 39. Gray J, Close PS, Briggs SP, Johal GS: A novel suppressor of cell death in plants encoded by the Lls1 gene of maize. Cell 1997, 89:25-31. 40. Solomon M, Belenghi B, Delledonne M, Menachem E, Levine A: The •• involvement of cysteine proteases and protease inhibitor genes in the regulation of programmed cell death in plants. Plant Cell 1999, 11:431-443. PCD-activating oxidative stress induces a set of cysteine proteases in soybean cells. Ectopic expression of cystatin, an endogenous cysteine-proteaseinhibitor gene, inhibited the activity of cysteine proteases and PCD.
24. Taylor NG, Scheible WR, Cutler S, Somerville CR, Turner SR: The • irregular xylem3 locus of Arabidopsis encodes a cellulose synthase required for secondary cell wall synthesis. Plant Cell 1999, 11:769-779. Stems of irx3 plants have less than 20% of the cellulose content of wild-type stems. A cellulose synthase gene was identified at the irx3 map location, and this wild-type gene complemented the mutation by restoring cellulose deposition in the secondary walls of xylem vessels. Other irregular xylem (irx) genes are currently being cloned.
41. Xu F-X, Chye M-L: Expression of cysteine proteases during developmental events associated with programmed cell death in brinjal. Plant J 1999, 17:321-327.
25. Cano-Delgado A, Metzlaff K, Bevan MW: The eli1 mutation reveals a •• link between cell expansion and secondary cell wall formation in Arabidopsis thaliana. Development 2000, 127:3395-3405. Another screen for vascular mutants, but this time taking advantage of the simplicity of the seedling root xylem, has identified many mutants, of which one displaying ectopic lignification (eli1) is described in detail. The mutant shows disorganised xylem with discontinuous elements and cells, possibly connected with altered expansion growth, that develop inappropriately secondary walls that become lignified. (See [26•].)
44. Yamamoto R, Demura T, Fukuda H: Brassinosteroids induce entry into the final stage of tracheary element differentiation in cultured Zinnia cells. Plant Cell Physiol 1997, 38:980-983.
26. Zhong R, Ripperger A, Ye Z-H: Ectopic deposition of lignin in the • pith of stems of two Arabidopsis mutants. Plant Physiol 2000, 123:59-69. Another screen for altered lignification patterns has identified two mutant alleles of the ELP1 gene on chromosome 1. The wild-type function of this gene may be to repress lignin deposition in pith tissues.
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