Patterned cell determination in a plant tissue: the secondary phloem of trees

The secondary vascular tissues (xylem and phloem) of woody plants originate from a vascular cambium and develop as radially oriented files of cells. The secondary phloem is composed of three or four cell types, which are organised into characteristic recurrent cellular sequences within the radial cell files of this tissue. There is a gradient of auxin (indole acetic acid) across both the cambium and the immediately postmitotic cells within the xylem and phloem domains, and it is believed that this morphogen, probably in concert with other morphogenic factors, is closely associated with the determination and differentiation of the different cells types in each tissue. A hypothesis is developed that, in conjunction with the positional values conferred by the graded radial distribution of morphogen, cell divisions at particular positions within the cambium are sufficient to determine not only each of the phloem cell types but also their recurrent pattern of differentiation within each radial cell file.     

Patterned cell development in the secondary phloem of dicotyledonous trees: a review and a hypothesis

The secondary phloem of dicotyledonous trees and shrubs is constructed of sieve tube cells (S) and their companion cells, as well as parenchyma (P) and fibre (F) cells. Different species have characteristic sequences of these S, P and F cells within the radial files of their phloem. The sequences are recurrent, and are evidence of rhythmic cell determination and differentiation. A model was devised to account for the sequences found in various dicot tree species. It is based on the pattern of radial displacement of cells through a gradient of morphogen which supports secondary phloem development. According to this model, each tree species shows a particular pattern of post-mitotic cellular displacement along each radial file as a result of a corresponding sequence of periclinal division in the cambial initial and its descendents. The divisions and displacements ensure that at each timestep (equivalent to an interdivisional interval) each cell resides in a specific location within the morphogenic gradient. Cells then emerge from the post-mitotic zone of cell determination, having acquired different final positional values. These values lie above a series of thresholds that permit the respective determination and subsequent differentiation of one or other of the three cell types S, P and F. The recurrent nature of the sequences of the three cell types within each radial cell file, as well as their tangential banding, are a consequence of a shared rhythmic spatio-temporal pattern of periclinal cambial divisions. With a single set of morphogen parameters required for cell determination, and using three positions for cambial cell divisions, all the cellular sequences of secondary phloem illustrated in the literature can be accounted for.This is an invited article.     

Repetitive cellular patterns in the secondary phloem of conifer and dicot trees,and a hypothesis for their development

Abstract

The radial fusiform cell files of the secondary phloem of conifers and dicots are composed of different cell types?–?fibres, parenchyma and sieve cells (in conifers), or sieve elements plus companion cells (in dicots). These cell types are arranged in characteristic, species-specific sequences along the radii of the files. The sequences are replicated in adjacent files and this leads to tangential bands of similar cell type. Moreover, the sequences are developed repetitively so that a sequence found in one year's growth increment of phloem is repeated in the next increment. In some species, many repetitions of the same sequence occur within one annual increment. A general hypothesis has been developed to account for the radial sequences of cell types. It is proposed that there is a gradient of a phloem-promoting morphogen, a series of morphogen thresholds for the determination of each phloem cell type, and a particular spatio-temporal pattern of periclinal cell division in the phloem domain of the vascular cambium that generates a corresponding pattern of cell displacement through the morphogen gradient in the immediately post-mitotic zone of cell determination. The feasibility of the hypothesis was supported by means of simulation which, using a constant set of initial conditions, could reproduce very nearly all the radial sequences of cell types found in the secondary phloem of a range of species of conifers and woody dicots. The tangential banding of the various cell types suggests that cell production and cell determination are events which occur synchronously across the radial files. The repeating blocks of cell types may constitute functional modules of phloem tissue, and the constituent cells probably have particular patterns of symplasmic connections and mechano-structural properties.     

Position dependent expression of GL2-type homeobox gene, Roc1: significance for protoderm differentiation and radial pattern formation in early rice embryogenesis

In early plant embryogenesis, the determination of cell fate in the protodermal cell layer is considered to be the earliest event in radial pattern formation. To elucidate the mechanisms of epidermal cell fate determination and radial pattern formation in early rice embryogenesis, we have isolated a GL2-type homeobox gene Roc1 (Rice outermost cell-specific gene1), which is specifically expressed in the protoderm (epidermis). In early rice embryogenesis, cell division occurs randomly and the morphologically distinct layer structure of the protoderm cannot be observed until the embryo reaches more than 100 microm in length. Nonetheless, in situ hybridization analyses revealed that specific expression of Roc1 in the outermost cells is established shortly after fertilization, much earlier than protoderm differentiation. In the regeneration process from callus, the Roc1 gene is also expressed in the outermost cells of callus in advance of tissue and organ differentiation, and occurs independently of whether the cells will differentiate into epidermis in the future or not. Furthermore, this cell-specific Roc1 expression could be induced flexibly in the newly produced outermost cells when we cut the callus. These findings suggest that the expression of Roc1 in the outermost cells may be dependent on the positional information of cells in the embryo or callus prior to the cell fate determination of the protoderm (epidermis). Furthermore, the Roc1 expression is downregulated in the inner cells of ligule, which have previously been determined as protodermal cells, also suggesting that the Roc1 expression is position dependent and that this position dependent Roc1 expression is important also in post-embryonic protoderm (epidermis) differentiation.     

Cuttings of a tobacco mutant, rae, undergo cell divisions but do not initiate adventitious roots in response to exogenous auxin

Numerous studies have shown that auxin induces adventitious root initiation in stem explants from a variety of species, including tobacco. A dominant, monogenic mutation previously identified in tobacco ( Nicotiana tabacum cv. Xanthii), rac , confers tenfold auxin resistance to mesophyll-derived cell suspensions and an impaired primary root development phenotype to seedlings. Results presented here demonstrate that adventitious root formation does not occur when heterozygous and homozygous rac stem cuttings are treated in vitro with indole-3-butyric acid (IBA) concentrations ranging from 0.5 μ M to 500 μ M . Histological analysis showed that some phloem parenchyma or inner cortical parenchyma cells in wild-type stem cuttings undergo adventitious root morphogenesis when they are treated with 5 μ M IBA. The same cell types in heterozygous and homozygous rac stem cuttings undergo mitoses in response to auxin, but never form adventitious root meristems. The lack of adventitious root initiation in rac stem cuttings is phenotypically distinct from the aberrant primary root development in rac seedlings. The rac mutation appears to block an essential process for auxin induction of adventitious root initiation but not cell division in phloem parenchyma or inner cortical parenchyma cells. Comparisons of rac heterozygous and homozygous seedling primary root length and callus formation in response to auxin in stem cuttings indicate that rac copy number is correlated to the degree of expression of these two phenotypes.     

A novel Arabidopsis gene TONSOKU is required for proper cell arrangement in root and shoot apical meristems

Root apical meristem (RAM) and shoot apical meristem (SAM) are vital for the correct development of the plant. The direction, frequency, and timing of cell division must be tightly controlled in meristems. Here, we isolated new Arabidopsis mutants with shorter roots and fasciated stems. In the tonsoku (tsk) mutant, disorganized RAM and SAM formation resulted from the frequent loss of proper alignment of the cell division plane. Irregular cell division also occurred in the tsk embryo, and the size of cells in meristems and embryo in tsk mutant was larger than in the wild type. In the enlarged SAM of the tsk mutant, multiple centers of cells expressing WUSCHEL (WUS) were observed. In addition, expression of SCARECROW (SCR) in the quiescent center (QC) disappeared in the disorganized RAM of tsk mutant. These results suggest that disorganized cell arrangements in the tsk mutants result in disturbed positional information required for the determination of cell identity. The TSK gene was found to encode a protein with 1311 amino acids that possesses two types of protein-protein interaction motif, leucine-glycine-asparagine (LGN) repeats and leucine-rich repeats (LRRs). LGN repeats are present in animal proteins involved in asymmetric cell division, suggesting the possible involvement of TSK in cytokinesis. On the other hand, the localization of the TSK-GFP (green fluorescent protein) fusion protein in nuclei of tobacco BY-2 cells and phenotypic similarity of tsk mutants to other fasciated mutants suggest that the tsk mutation may cause disorganized cell arrangements through defects in genome maintenance.     

Role of auxin and sucrose in the differentiation of sieve and tracheary elements in plant tissue cultures

The differentiation of sieve and tracheary elements was studied in callus culture of Daucus carota L., Syringa vulgaris L., Glycine max (L.) Merr., Helianthus annuus L., Hibiscus cannabinus L. and Pisum sativum L. By the lacmoid clearing technique it was found that development of the phloem commenced before that of the xylem. In not one of the calluses was differentiation of tracheary elements observed in the absence of sieve elements. The influence of indole-3-acetic acid (IAA) and sucrose was evaluated quantitatively in callus of Syringa, Daucus and Glycine. Low IAA levels resulted in the differentiation of sieve elements with no tracheary cells. High levels resulted in that of both phloem and xylem. IAA thus controlled the number of sieve and tracheary elements, increase in auxin concentration boosting the number of both cell types. Changes in sucrose concentration, while the IAA concentration was kept constant, did not have a specific effect on either sieve element differentiation, or on the ratio between phloem and xylem. Sucrose did, however, affect the quantity of callose deposited on the sieve plates, because increase in the sucrose concentration resulted in an increase in the amount of callose. It is proposed that phloem is formed in response to auxin, while xylem is formed in response to auxin together with some added factor which reaches it from the phloem.     

Expression of the phloem lectin is developmentally linked to vascular differentiation in cucurbits

The conducting elements of phloem in angiosperms are a complex of two cell types, sieve elements and companion cells, that form a single developmental and functional unit. During ontogeny of the sieve element/companion cell complex, specific proteins accumulate forming unique structures within sieve elements. Synthesis of these proteins coincides with vascular development and was studied in Cucurbita seedlings by following accumulation of the phloem lectin (PP2) and its mRNA by RNA blot analysis, enzyme-linked immunosorbent assay, immunocytochemistry and in␣situ hybridization. Genes encoding PP2 were developmentally regulated during vascular differentiation in hypocotyls of Cucurbita maxima Duch. Accumulation of PP2 mRNA and protein paralleled one another during hypocotyl elongation, after which mRNA levels decreased, while the protein appeared to be stable. Both PP2 and its mRNA were initially detected during metaphloem differentiation. However, PP2 mRNA was detected in companion cells of both bundle and extrafascicular phloem, but never in differentiating sieve elements. At later stages of development, PP2 mRNA was most often observed in extrafascicular phloem. In developing stems of Cucurbita moschata L., PP2 was immunolocalized in companion cells but not to filamentous phloem protein (P-protein) bodies that characterize immature sieve elements of bundle phloem. In contrast, PP2 was immunolocalized to persistent ␣ P-protein bodies in sieve elements of the extrafascicular phloem. Immunolocalization of PP2 in mature wound sieve elements was similar to that in bundle phloem. It appears that PP2 is synthesized in companion cells, then transported into differentiated sieve elements where it is a component of P-protein filaments in bundle phloem and persistent P-protein bodies in extrafascicular phloem. This differential accumulation in bundle and extrafascicular elements may result from different functional roles of the two types of phloem. Received: 31 July 1996 / Accepted: 27 August 1996     

Cells differentiated from mouse embryonic stem cells via embryoid bodies express renal marker molecules

Differentiation of mouse embryonic stem (ES) cells via embryoid bodies (EB) is established as a suitable model to study cellular processes of development in vitro. ES cells are known to be pluripotent because of their capability to differentiate into cell types of all three germ layers including germ cells. Here, we show that ES cells differentiate into renal cell types in vitro. We found that genes were expressed during EB cultivation, which have been previously described to be involved in renal development. Marker molecules characteristic for terminally differentiated renal cell types were found to be expressed predominantly during late stages of EB cultivation, while marker molecules involved in the initiation of nephrogenesis were already expressed during early steps of EB development. On the cellular level--using immunostaining--we detected cells expressing podocin, nephrin and wt-1, characteristic for differentiated podocytes and other cells, which expressed Tamm-Horsfall protein, a marker for distal tubule epithelial cells of kidney tissue. Furthermore, the proximal tubule marker molecules renal-specific oxido reductase, kidney androgen-related protein and 25-hydroxyvitamin D3alpha-hydroxylase were found to be expressed in EBs. In particular, we could demonstrate that cells expressing podocyte marker molecules assemble to distinct ring-like structures within the EBs. Because the differentiation efficiency into these cell types is still relatively low, application of fibroblast growth factor (FGF)-2 in combination with leukaemia inhibitory factor was tested for induction, but did not enhance ES cell-derived renal differentiation in vitro.     

Sieve-element characters of Myristicaceae: Nuclear crystals, S-and P-type plastids, nacreous walls

The phloem of the Myristicaceae is composed of sieve elements, parenchymatous cells, and fibers. Within the metaphloem and secondary phloem parenchymatic layers including prominent secretory elements alternate with tangential bands of fibers and layers composed of sieve elements, companion cells and phloem-parenchyma cells. among the latter the sieve elements are most abundant and easily identified by the presence of thick (nacreous) walls. The most characteristic feature of the sieve elements of Myristicaceae (and found nowhere else among the Magnoliiflorae) are nuclear crystals, which are released into the lumen during nuclear degeneration and persist in the mature cell. P-and S-type sieve-element plastids were recorded for the 18 species investigated. Both types of the plastid are characterized by large diameters and many medium-sized starch grains. The sizes and contents (small protein crystals only) of the P-type plastids of the Myristicaceae do not conform to the tiny P-type plastids (with large protein crystals) of the Annonaceae, a family to which the Myristicaceae is traditionally allied.     

Primary Phloem Development in the Shoot Apex of Rhizophora mangle L. (Rhizophoraceae)

The primary phloem in the shoot apex of the mangrove Rhizophora mangle L. is largely confined to the comparatively condensed area between the first three leaf pairs. The main extension zone, surrounded by the stipular sheath of the third leaf pair, contains vascular bundles arranged in a procambial ring and characterized by a well-developed primary phloem and a less advanced xylem. The phloem consists of a great number of sieve elements, an equal number of associated companion cells, and a few phloem-parenchyma cells. The differentiation of the sieve-element protoplast (with e.g., chromatolytic nuclear degeneration, loss of the vacuole and most organelles) proceeds largely according to a well-known pattern. Their P-type plastids, however, form their protein crystals rather late and therefore cannot be used as an early cell marker. Lateral sieve-element walls are distinct from other wall parts and walls of other cells by their heavy nacreous thickenings, the formation of which is shown to be strictly correlated with the occurrence and orderly arrangement of cortical microtubules.     

Interphase chromosome arrangement in Arabidopsis thaliana is similar in differentiated and meristematic tissues and shows a transient mirror symmetry after nuclear division

Whole-mount fluorescence in situ hybridization (FISH) was applied to Arabidopsis thaliana seedlings to determine the three-dimensional (3D) interphase chromosome territory (CT) arrangement and heterochromatin location within the positional context of entire tissues or in particular cell types of morphologically well-preserved seedlings. The interphase chromosome arrangement was found to be similar between all inspected meristematic and differentiated root and shoot cells, indicating a lack of a gross reorganization during differentiation. The predominantly random CT arrangement (except for a more frequent association of the homologous chromosomes bearing a nucleolus organizer) and the peripheric location of centromeric heterochromatin were as previously observed for flow-sorted nuclei, but centromeres tend to fuse more often in nonendoreduplicating cells and NORs in differentiated cells. After mitosis, sister nuclei revealed a symmetric arrangement of homologous CTs waning with the progress of the cell cycle or in the course of differentiation. Thus, the interphase chromosome arrangement in A. thaliana nuclei seems to be constrained mainly by morphological features such as nuclear shape, presence or absence of a nucleolus organizer on chromosomes, nucleolar volume, and/or endopolyploidy level.     

An Auxin Gradient and Maximum in the Arabidopsis Root Apex Shown by High-Resolution Cell-Specific Analysis of IAA Distribution and Synthesis

Local concentration gradients of the plant growth regulator auxin (indole-3-acetic acid [IAA]) are thought to instruct the positioning of organ primordia and stem cell niches and to direct cell division, expansion, and differentiation. High-resolution measurements of endogenous IAA concentrations in support of the gradient hypothesis are required to substantiate this hypothesis. Here, we introduce fluorescence-activated cell sorting of green fluorescent protein–marked cell types combined with highly sensitive mass spectrometry methods as a novel means for analyses of IAA distribution and metabolism at cellular resolution. Our results reveal the presence of IAA concentration gradients within the Arabidopsis thaliana root tip with a distinct maximum in the organizing quiescent center of the root apex. We also demonstrate that the root apex provides an important source of IAA and that cells of all types display a high synthesis capacity, suggesting a substantial contribution of local biosynthesis to auxin homeostasis in the root tip. Our results indicate that local biosynthesis and polar transport combine to produce auxin gradients and maxima in the root tip.     

Cell dynamics and gene expression control in tissue homeostasis and development

During tissue and organ development and maintenance, the dynamic regulation of cellular proliferation and differentiation allows cells to build highly elaborate structures. The development of the vertebrate retina or the maintenance of adult intestinal crypts, for instance, involves the arrangement of newly created cells with different phenotypes, the proportions of which need to be tightly controlled. While some of the basic principles underlying these processes developing and maintaining these organs are known, much remains to be learnt from how cells encode the necessary information and use it to attain those complex but reproducible arrangements. Here, we review the current knowledge on the principles underlying cell population dynamics during tissue development and homeostasis. In particular, we discuss how stochastic fate assignment, cell division, feedback control and cellular transition states interact during organ and tissue development and maintenance in multicellular organisms. We propose a framework, involving the existence of a transition state in which cells are more susceptible to signals that can affect their gene expression state and influence their cell fate decisions. This framework, which also applies to systems much more amenable to quantitative analysis like differentiating embryonic stem cells, links gene expression programmes with cell population dynamics.     

The dynamic plant stem cell niches

Stem cells exist in specific locations called niches, where extracellular signals maintain stem cell division and prevent differentiation. In plants, the best characterised niches are within the shoot and root meristems. Networks of regulatory genes and intercellular signals maintain meristem structure in spite of constant cell displacement by division. Recent works have improved our understanding of how these networks function at the cellular and molecular levels, particularly in the control of the stem cell population in the shoot meristem. The meristem regulatory genes have been found to function partly through localised control of widely used signals such as cytokinin and auxin. The retinoblastoma protein has also emerged as a key regulator of cell differentiation in the meristems.     

Technical advance: near-infrared femtosecond laser pulses as a novel non-invasive means for dye-permeation and 3D imaging of localised dye-coupling in the Arabidopsis root meristem

We have used near-infrared femtosecond Titanium: Sapphire laser pulses as novel non-invasive means for dye loading into various cell types of the Arabidopsis root meristem, and by 3D imaging have assessed the extent of dye coupling between the meristematic cells. The post-embryonic primary root of Arabidopsis thaliana has an invariant ontogeny and fixed cellular organisation which makes it an attractive model system to study developmental events involving cell fate determination, cellular differentiation and pattern formation. Local intercellular communication and local transmission of positional signals are likely to play a pivotal role in cell proliferation and regulation of differentiation. We have therefore examined the extent to which the constituent cells in the root meristem are symplastically coupled. Following laser-assisted loading of membrane impermeate fluorescent dye propidium iodide (PI) in single cells, we show by time-lapse and 3D imaging that in the root tip all undifferentiated cells are dye-coupled. When PI is permeated into the central cells, it rapidly moved into the adjacent initials of the columella, cortex, pericycle and stele. Interestingly, when only either of the initials were loaded with the dye, it never moved into any of the central cells. Amongst the epidermal cells, the differentiated hair cells are symplastically isolated. Our data provide evidence (1) for differential dye-coupling behaviour between quiescent centre cells and the neighbouring initials; (2) that cells in the root are coupled during stages at which the cell-lineage pattern is formed and that it becomes progressively secluded as they differentiate and the pattern is fixed. Taken together, our NIR-laser mediated approach is highly efficient and has numerous potential applications for non-invasive permeation of dyes in different cell types.     

Generation of asymmetry during development. Segregation of type-specific proteins in Caulobacter.

An essential event in developmental processes is the introduction of asymmetry into an otherwise undifferentiated cell population. Cell division in Caulobacter is asymmetric; the progeny cells are structurally different and follow different sequences of development, thus providing a useful model system for the study of differentiation. Because the progeny cells are different from one another, there must be a segregation of morphogenetic and informational components at some time in the cell cycle. We have examined the pattern of specific protein segregation between Caulobacter stalked and swarmer daughter cells, with the rationale that such a progeny analysis would identify both structurally and developmentally important proteins. To complement the study, we have also examined the pattern of protein synthesis during synchronous growth and in various cellular fractions. We show here, for the first time, that the association of proteins with a specific cell type may result not only from their periodicity of synthesis, but also from their pattern of distribution at the time of cell division. Several membrane-associated and soluble proteins are segregated asymmetrically between progeny stalked and swarmer cells. The data further show that a subclass of soluble proteins becomes associated with the membrane of the progeny stalked cells. Therefore, although the principal differentiated cell types possess different synthetic capabilities and characteristic proteins, the asymmetry between progeny stalked and swarmer cells is generated primarily by the preferential association of specific soluble proteins with the membrane of only one daughter cell. The majority of the proteins which exhibit this segregation behavior are synthesized during the entire cell cycle and exhibit relatively long, functional messenger RNA half-lives.