BOTANY, VOLUME 2 - PLANT PHYSIOLOGY - 2007

7. DEVELOPMENTAL PHYSIOLOGY

7.4. Cell-to-Cell Communication during Development

Already in Introduction/4.html">Prokaryotic Cell communities based on a division of labor (e.g., heterocyst formation in nitrogen-fixing cyanobacteria) as well as in multicellular plants, cell determination and the resulting differentiation depend on The Cell's position within the cell community or Organism, respectively. For instance, conducting bundles in the SHOOT apex of monocots develop at a specific distance from the surface, whereas the epidermis typically originates directly at the surface. In the shoot axis and ROOT, tissue layers are invariably arranged in a characteristic sequence. These Examples confirm the presence of radial positional information in the axial Organs of higher plants (Fig. 7.26). In contrast, the differentiation of stomatal guard Cells and trichomes in the epidermis, or of root hairs in the rhizodermis, occurs at characteristic positions on the organism's surface. This can be viewed as an expression of tangential positional information operating in a plane, which differs—at least partially—from the radial positional signal. Thus, the rhytidiophyllum mutant of Epilobium hirsutum develops additional patches of epidermis in the inner tissue layers of the leaf, within which (albeit non-functional) stomatal guard cells are formed. Finally, recent successful micro-surgical experiments on roots of Arabidopsis thaliana also demonstrate the presence of longitudinal positional information established along the organ's axis, which dictates the cellular differentiation of daughter cells produced by meristematic initials (Fig. 7.26).

Class="center">Fig. 7.26. Cell determination in the root tip of Arabidopsis thaliana: A, B — Structure OF THE root tip (A) and root meristem (B) in radial longitudinal section; C — a series of laser ablation experiments (a–e) confirms that Cell Differentiation is determined by the cell's position within the tissue community. Dark gray: cell(s) destroyed (ablated) by UV-laser irradiation; other colors as in A and B, respectively. (a) Control (normal). Transverse division of the cortical initial cell yields a daughter cell, which subsequently divides periclinally and longitudinally to form a cortical parenchyma cell and an endodermal cell. (b) Destruction of the cortical initial cell. THE POSITION OF the destroyed cell is taken over by a pericycle initial cell. It alters its differentiation pathway and, following transverse and periclinal longitudinal divisions, produces a cortical parenchyma cell and an endodermal cell. This indicates that already-differentiated daughter cells exert an instructive influence on the differentiation of underlying, younger cells regardless of their origin—a Conclusion apparently contradicted by the result of the cortical daughter cell ablation experiment (c). However, experiment (d) proves that the hypothesis holds true. Evidently, positional information spreads not only along a cell file toward the directly adjacent initial cell beneath it, but also reaches it from both neighboring cells. That this is not an artifact caused by the destruction of three cells is demonstrated in turn by (e). If three cells in a radial file (epidermal, cortical, and pericycle initials) are destroyed via UV-laser irradiation, correct differentiation proceeds just as in (a) or (c), respectively.

In these experiments, individual cells were killed using point-focused, high-energy laser beams. Subsequently, the differentiation of cells growing into these regions was observed: pericycle cells migrating into the region of the killed cortical initials become cortical initials themselves and produce daughter cells, which give rise to endodermal and cortical parenchyma cells via periclinal divisions. However, if the daughter Cells of the cortical initials are also killed, the differentiation of subsequently formed daughter cells into endodermal and cortical parenchyma cells is suppressed. This demonstrates that a differentiating signal travels in the longitudinal direction from differentiated cells to the younger, underlying cells regardless of their origin, dictating the differentiation pathway of these cells (Fig. 7.26).

The Chemical Nature of these substances, the mechanisms by which plants generate positional information, and, ultimately, the precise impact of this information on differentiation remain unknown in detail.

Recently, particularly valuable insights have been gained from studies on developmental plant mutants, most of which were performed on Arabidopsis thaliana (see Box 7.1). Further descriptions of Embryogenesis (see 7.4.1), The formation of Meristems and organs in the shoot meristem (see 7.4.3), and tissue layer patterning (see 7.4.2) will focus primarily on this model organism. The MECHANISMS OF CELL communication involved in these processes are only partially understood.

Deviations from normal developmental pathways occurring under pathological conditions (e.g., gall formation, tumors) or during the establishment of symbioses (e.g., root nodules) should also be noted here. These are discussed in Chapter 9.

7.4.1. Control of Embryogenesis

The Formation of the embryo following Fertilization exhibits many characteristic features (see 3.1, Fig. 3.1). The egg cell is already polarized—presumably under the Influence of the maternal plant—and the ovule is likewise polarized. The polarization of the zygote (Fig. 7.27, A) and the ESTABLISHMENT OF THE first, asymmetric division plane of the zygote resemble analogous processes in brown Algae. They also proceed via the Differentiation of the zygote wall, resulting in significant biochemical differences between the cell walls of the two daughter cells (Fig. 7.27, B). As in Fucus, these differences are required for the subsequent Determination of the daughter cells. The basal cell gives rise to the suspensor and hypophysis, whereas the apical cell produces the proembryo, with the exception of the Tissues derived from the hypophysis: the quiescent center and the central region of the root cap. Regions of the basal cell's Cell wall characterized by the presence of a specific glycoprotein (arabinogalactan) define the area from which subsequent divisions generate a 6- to 9-celled suspensor (Fig. 7.27, C, D), which ultimately undergoes programmed cell death at a late stage of embryogenesis (see 7.3.2), whereas the portion of the basal cell forming the hypophysis lacks this glycoprotein.

Fig. 7.27. Manifestation of polarity during the embryogenesis of Arabidopsis thaliana. The already polarized zygote (A) divides unequally (B). The vacuolated basal cell differentiates into the suspensor, whose uppermost cell—the hypophysis—subsequently becomes incorporated into the embryo proper, forming the quiescent center and the central portion of the root cap. The remaining suspensor disintegrates during embryo maturation via programmed cell death. These suspensor cells are characterized by the presence of arabinogalactan-associated Glycoproteins (gray) in their cell walls, which are already synthesized in the zygote. In the globular embryo (C — 16-celled, D — later stage), a new polarity axis (apical → basal) emerges as early as the 8-cell stage. The embryo is subdivided into three tiers (apical, central, basal) with distinct developmental fates. Subsequent differentiation (D → E → F) is largely directed by the polar distribution of auxin (gray arrows). Auxin flux depends on the redistribution of auxin transporters within the Plasma Membranes of embryonic cells. Evidence suggests that auxin transport operates similarly in the regions of both apical meristems during later post-germinative plant development (root tip; see Fig. 8.24). Further explanations are provided in the text.

The apical cell initially develops into a globular embryo, which undergoes a novel axial polarization at the 8-cell stage (octant stage). This polarization first becomes visible later in the roughly 100-celled globular embryo during the transition to The Heart stage, and becomes fixed with the formation of embryonic organs—cotyledons, hypocotyl, and embryonic root—as well as both apical meristems (see Fig. 3.1). Axial polarization allows the identification of three regions within the globular embryo (Fig. 7.27, C, D) that differentiate along distinct pathways: the apical layer forms the shoot meristem and cotyledons; the central layer forms the axial organs (hypocotyl and embryonic root) along with hypophysis-derived cells, namely the quiescent center and the central part of the root cap containing statenchyma¹ (Latin: columella) (Fig. 7.27, F; 7.26, A). The establishment of the polarity axis within the already multicellular globular embryo generates positional information within cellular boundaries.

¹ Here, statenchyma refers to a tissue rich in starch grains, which are presumed to function as statoliths. Removal of this root cap tissue leads to a disruption of root geotropism. The term "statenchyma" highlights the potential involvement of this tissue in gravity perception. — Ed. note.

It is highly probable that the phytohormone indole-3-acetic acid, belonging to the auxin group (see 7.6.1), plays a crucial role as this positional signal (Fig. 7.27, D–F). Experiments indicate a reorientation of transporter molecules that mediate the cellular efflux of the hormone (see 7.6.1.3), shifting from an initially uniform distribution in the Plasmalemma to a progressively targeted, directional distribution within the plasma membranes of the globular embryo.

In gnom mutants of Arabidopsis thaliana, the proper distribution of auxin transporters is disrupted, and longitudinal polarization along the embryonic axis fails to occur. The wild-type GNOM Gene (GN) encodes a protein involved in targeted vesicle transport. However, in gnom mutants, the first zygotic division is already defective because cell polarization is impaired; nevertheless, zygotic polarization itself is auxin-independent. Evidently, the GN protein is essential for mediating various directed transport processes at different phases of embryogenesis.

The indole-3-acetic acid gradient established within the embryo (Fig. 7.27, D–F) promotes differential gene activation depending on cellular concentration and sensitivity to auxin: the shoot meristem forms at the site of lowest auxin concentration; an elevated auxin concentration lateral to the shoot meristem is required for cotyledon primordia formation; and root tip tissues differentiate at the site of highest auxin concentration at the Base of the embryo. During subsequent plant development, auxin distribution in the meristematic region—likely mediated by polar auxin transport (see 7.6.1.3)—is vital for maintaining meristematic identity and organ differentiation: directly within the shoot meristem, auxin concentration is kept very low, and auxin is transported to regions just below the shoot meristem proper, which differentiate into leaf primordia. Conversely, through polar transport in the root tip region, auxin accumulates in the central cylinder toward the quiescent center and reaches its peak concentration in the cell layer directly beneath the quiescent center: the initial cells responsible for statenchyma formation (see Fig. 7.26, A; 7.27, D–F). Here, high auxin concentrations are presumably required to sustain meristematic function. However, auxin is evidently not the sole intercellular positional signal important for development; the conditions are likely more complex and also involve other phytohormones (e.g., Cytokinins, see 7.6.2).

Evidence suggests that during Cytology/cytology/16.html">Early stages of organ development, directed auxin transport is partially self-organizing. According to this concept, cells express more auxin-transporting Proteins the higher their internal auxin content is. Consequently, initially minor differences in auxin concentration and flux direction can be autocatalytically amplified and stabilized, ultimately establishing stable hormone gradients along the transport pathway. Such auxin "canalization processes" presumably occur, for instance, during Vascular System formation during leaf development (auxin-rich zones differentiate into conducting bundles), in the cambium (see Fig. 7.38), during embryogenesis (see above), in maintaining the differentiation of shoot and root apices, and during auxin-induced lateral root initiation.

7.4.2. Self-Organization Processes in Tissue Layers (Patterning)

Spatial patterning, which determines the positioning of differentiating cells, relies on self-organization processes, although the biochemical specifics of this patterning remain poorly understood.

A simple patterning model, as illustrated in Fig. 7.28, A, is based on a slowly diffusing or entirely immobile activator of the differentiation process, which is initially generated stochastically (randomly) in specific cells of a tissue layer. This activator autocatalytically enhances its own production while simultaneously inducing the synthesis of a rapidly diffusing inhibitor. Due to its greater range of action, the inhibitor suppresses activator formation in the surroundings of the "activated" cell.

Studies on Arabidopsis thaliana mutants with aberrant arrangements of differentiated cells have demonstrated that an activator-inhibitor system indeed underlies the patterning of leaf epidermal trichomes and rhizodermal root hairs (Fig. 7.28, B, C). However, because root hairs in Arabidopsis thaliana differentiate exclusively from rhizodermal cells positioned over more than one cortical parenchyma cell, a radial signal originating from the cortical parenchyma cells might also be involved here. In both cases (Fig. 7.28, B, C), the activators act strictly cell-autonomously, whereas the inhibitors also affect neighboring cells. Potential mechanisms for this type of Intercellular Communication are described in Section 7.4.4.

Fig. 7.28. Principles of tissue layer patterning: A — model of a self-organizing patterning process: a low-mobility activator that autocatalytically amplifies its own synthesis simultaneously induces The production of a faster-moving inhibitor; in the immediate vicinity of the activator-producing cell, the inhibitor suppresses further activator formation. Implementation of such systems during trichome development in the leaf epidermis (B) and root Hair formation in the rhizodermis (C) of Arabidopsis thaliana; B — activation of trichoblasts (gray: trichome precursor cells) occurs with the participation of the cell-autonomous METABOLISM/31.html">Transcription factor GL1 and its regulator TTG, both of which autocatalytically promote each other's synthesis while simultaneously inducing the production of the transcription factor TRY, which moves out of trichoblasts and inhibits GL1 formation in neighboring cells; C — a similar regulatory process underlies root hair differentiation. Here, the autocatalytic activation of the transcription factor WER and the aforementioned regulator TTG leads to the suppression of root hair differentiation and the Induction of the mobile inhibitor CPC (which is presumably also a transcription factor that inactivates WER synthesis). Cells that do not express WER differentiate into root Hair cells. Because these cells invariably overlie regions where two underlying cortical parenchyma cells contact one another, a positional influence of the cortical parenchyma on rhizodermal patterning (as yet uncharacterized) must be operational. The designations of the participating proteins derive from the mutant phenotypes that led to their discovery and are irrelevant here.

7.4.3. Control of Meristem and Organ Formation in the Shoot Meristem

The processes discussed here, much like many events in developmental biology, have been investigated in exceptional detail using numerous developmental mutants of Arabidopsis thaliana. A model organism from another family—snapdragon (Antirrhinum majus, Scrophulariaceae)—exhibits developmental patterns identical to those of Arabidopsis, matching even in fine detail. It is therefore widely assumed that data obtained from Arabidopsis thaliana can be generalized to all angiosperms.

In its vegetative state, the shoot meristem gives rise to the shoot axis and leaves. Often influenced by environmental factors such as day length, it can transition into a floral meristem to form the floral axis and organs, a process during which the meristem is consumed (thereby shifting from an indeterminate to a determinate state). The developmental program of floral organs within the floral meristem is repressed (its execution is blocked) by a complex of genes until an inductive signal intervenes. This is evidenced by loss-of-function Mutations in the repressor gene complex, which trigger Flower Formation even in the absence of inductive environmental factors. The embryonic flower (emf) mutant of Arabidopsis thaliana forms a solitary flower immediately upon germination, bearing only two cotyledons and no leaves.¹ Environmental factors that induce flowering act by suppressing EMF activity. When the concentration of the (as yet uncharacterized) EMF gene product in the shoot meristem drops below a critical threshold, the meristematic switch is flipped, and the floral development program is activated. This partially hypothetical model explains why floral induction in Arabidopsis thaliana occurs gradually: the organism is a quantitative long-day plant (Table 7.6), meaning it transitions to flowering significantly faster under long days than under short days.

¹ The Description of the emf mutant flower provided here is somewhat schematic. For instance, it lacks petals, and structures resembling sepals undergo epidermal differentiation identical to that of vegetative leaves. Atypical carpels are present, the number of stamens is reduced, and the "flower" itself develops in a terminal position (whereas a normal flower is lateral). It remains unclear whether this structure constitutes a true flower. — Transl./Ed. note

The differentiation of floral organ primordia—that is, Organogenesis—is governed by a group of genes that can be divided into four classes: A, B, C, and D. These are classified as homeotic genes because their loss of function or misexpression in inappropriate locations (ectopic expression) leads to alterations in normal organ development (homeosis). In developmental genetics, homeotic mutants are defined as those that bear an alternative organ in place of the one normally formed (e.g., developing a carpel instead of a sepal).¹

¹ In mutants, homeotic replacement of one organ by another is often accompanied by broader alterations in overall floral architecture (such as organ number and arrangement). This indicates that many genes also play a role in patterning the floral meristem and participate in self-organization processes (see Section 7.4.2). — Ed. note

Arabidopsis thaliana possesses 7 genes responsible for floral organ formation:

✵ class A gene APETALA1 (AP1);

✵ two class B genes, APETALA3 (AP3) and PISTILLATA (PI);

✵ class C gene AGAMOUS (AG);

✵ 3 class D genes, SEPALLATA1, 2, and 3 (SEP1, SEP2, SEP3).²

² In reality, the number of genes determining floral organ identity is greater. For instance, the text omits APETALA2, which belongs to class A. Class D is still a matter of debate; petunia genes FBP1 and FBP11 are assigned to it, while the SEPALLATA genes are now classified under class E. — Ed. note

All these genes encode transcription factors. During the Regulation of Gene activity, many of them interact directly with one another and bind in various combinations to the promoters of target genes. Depending on the specific gene combination, cells differentiate into sepals, petals, stamens, or carpels (Fig. 7.29). The precise spatial and temporal expression of floral organ identity genes within the meristem is ensured, on the one hand, by mutual Cross-Regulation (e.g., A-activity represses C-class Gene Expression, and C-activity represses A-class gene expression) and, on the other hand, by additional genes that help regulate the expression of classes A, B, and C within the floral meristem (for instance, B-activity in the first and fourth whorls is suppressed by the products of three other genes that do not possess organ-forming Functions themselves).

Fig. 7.29. Genetic control of organ formation during Flower Development in Arabidopsis thaliana. Four gene classes (A, B, C, and D) govern organogenesis within the floral meristem. The products of genes controlling floral organ differentiation occupy distinct domains within the meristem; the expression domains of classes A and B, as well as classes B and C, partially overlap, whereas class D is expressed In the second, third, and fourth whorls. Class A is represented by APETALA1 (AP1). Cells in which only class A is active differentiate into sepals. When both class A and class B activities are present, petals develop. Class B is represented by APETALAS [sic: APETALA3] (AP3) and PISTILLATA (PI); class C is represented by AGAMOUS (AG). The co-activation of class B and C genes results in the differentiation of stamens, while the exclusive activity of class C genes leads to the formation of carpels. However, the second, third, and fourth whorls additionally require class D activity, which is encoded by SEPALLATA1, 2, and 3 (SEP1, SEP2, SEP3) (In recent literature, these genes are assigned to class E. — Ed. note). Mutations in genes controlling floral organ formation result in characteristic homeotic transformations. If class A activity is lost, class C activity expands into all four whorls (since class A genes normally repress class C expression); consequently, carpels form in the first and fourth whorls, and stamens in the second and third. Because these mutants lack petals, they were named apetala. If class C activity is lost, class class A activity [sic] expands into all whorls (since class C genes normally repress class A expression). These mutants develop sepals in the first and fourth whorls and petals in the second and third, resulting in sterile flowers; hence they were named agamous. The loss of class B activity does not affect classes A and C; sepals form in the First and Second whorls, and carpels in the third and fourth. Because these mutants lack petals or develop additional pistils in place of stamens, they were named apetala or pistillata [sic], respectively. When mutations affect class D, sepals form in all four whorls; thus, these mutants are called sepallata. Class D gene activity is therefore essential for executing the functions of classes A, B, and C in the floral meristem during the formation of organs in the second, third, and fourth whorls.

To the best of our knowledge, corresponding homologous genes (genes sharing a common evolutionary origin and typically conserved functions)¹ have been discovered in other angiosperms. This suggests that floral organogenesis follows universal principles across angiosperms, which are also largely evolutionarily conserved. This model holds true, for example, for the zygomorphic flower of snapdragon. However, dorsoventral Asymmetry is conferred by an additional gene, CYCLOIDEA. When this gene's function is disrupted (in cycloidea mutants), snapdragon produces radially symmetrical flowers.

¹ These are referred to as orthologs (see Section 7.2.2.3 above). — Ed. note

7.4.4. Mechanisms of Cell Communication

Positional control over developmental processes—which, as described previously (see 7.3.3; 7.4.1–7.4.3), is of paramount importance in plants—requires The ability to directionally transport developmental regulatory molecules beyond the confines of the cell. Based on current knowledge, this can occur via several pathways:

✵ targeted secretion of regulatory macromolecules into The cell wall matrix, such as during zygote polarization; the COMPOSITION OF THE cell walls determines the determination of daughter cells via contact with the protoplast (e.g., thallus and rhizoid cells in Fucus, see 7.3.3);

✵ polar transport of low-molecular-weight regulators; this is the mechanism by which Auxins are transported (see 7.6.1), for instance, during embryogenesis in Arabidopsis thaliana (see 7.4.1);

✵ local synthesis followed by diffusion (through the symplast and/or apoplast) from the site of synthesis into surrounding tissues; this is how Gibberellins travel via the apoplast from the embryo to the aleurone layer in cereal grains (see 7.6.3.3);

✵ transport through The vascular system: a process crucial for systemic developmental control (correlations) (see 7.5);

✵ cell-to-cell transport of regulatory macromolecules via plasmodesmata (see 7.4.4.1).

7.4.4.1. Macromolecular Exchange Between Cells

The plant cell wall is permeable to ions, small Water-soluble molecules, and small proteins with a Molecular Weight of up to approximately 5 kDa, but it prevents the free diffusion of larger macromolecules. Plasmodesmata (for structure, see 2.2.7.3, Fig. 2.70), which interconnect cells integrated into the symplastic system, were also long considered to be merely pores for low-molecular-weight metabolites with a size exclusion limit of up to 1 kDa. Therefore, it came as a surprise when a recent discovery revealed that plasmodesmata are likewise involved in intercellular macromolecular exchange, functioning as regulated pores through which specific macromolecules, proteins, or even ribonucleoprotein complexes are transported from Cell to Cell.

This was first demonstrated using The transport of phytopathogenic Viruses (such as tobacco mosaic virus) as an example. In virus-infected plants, the size exclusion limit for substances passing through plasmodesmata is greater than 10 kDa, whereas in uninfected plants it is less than 1 kDa. This process is mediated by virus-encoded movement proteins with a molecular weight of ~30 kDa. These proteins form a ribonucleoprotein complex with the viral nucleic acid (single-stranded RNA in tobacco mosaic virus), which moves through plasmodesmata from cell to cell, thereby enabling the virus to spread throughout the plant and causing disease symptoms—viral mosaic (affected interveinal areas). It was only much later discovered that viruses merely hijack a transport mechanism that plays a vital role in healthy plants as well, serving for the transport of proteins (Fig. 7.16) and ribonucleoprotein complexes (see below). For instance, in angiosperms, enucleate and ribosome-free sieve elements import proteins through plasmodesmata from companion cells, where these proteins are synthesized. Various models have been proposed to explain the Mechanisms of Protein transport through plasmodesmata (Fig. 7.30). Since only specific proteins are transported, they must bear (details remain unknown) topogenic structural elements¹ that interact with specific receptors (export receptors on the exporting cell side and import receptors on the importing cell side), which either direct the protein into the transport pathway or remove it accordingly. Evidence suggests that small proteins are transported through plasmodesmata in a folded state (Model I), while larger proteins are partially or completely unfolded (Model II). However, many details remain hypothetical.

¹ By analogy with Intracellular Transport (see 7.3.1). — Ed. note.

Fig. 7.30. Model of protein translocation through plasmodesmata. According to this concept, the protein to be transported (TP), either in a folded (A, Model I) or unfolded state (B, Model II), binds to export receptors (R_e) of the exporting cell and is released into its Cytoplasm via import receptors (R₁) of the importing cell. Chaperones are likely involved in the unfolding and refolding of proteins in Model II.

Among the proteins found to undergo intercellular transport are several development-regulating transcription factors that move from the deeper layers of the shoot meristem, where they are synthesized, into the outer layers (see 3.1.1.1, Fig. 3.5). One such transcription factor is the maize KN1 protein. KN1 is the product of the KNOTTED gene, which maintains the meristematic state of cells and is not expressed in non-meristematic cells. However, in maize knotted mutants, the transcription factor is expressed outside its normal domain: in the growth zone of the leaf blade. This results in the formation of abnormal, nodular structures on the leaf surface, caused by excessive cell proliferation, from which these mutants derived their name (English *knotted*). It is hypothesized that the KN1 protein may move through plasmodesmata as a complex with its own mRNA. Furthermore, evidence indicates that mRNA molecules move from companion cells into sieve elements and thus could potentially carry long-distance information via the phloem. Such intercellular movements of macromolecules likely play a crucial role in implementing positional information (intercellular communication) during cell differentiation and patterning, and could potentially participate in the systemic correlation of developmental processes (see 7.5).

It has recently become clear that the permeability of plasmodesmata depends on developmental changes. Only the complex, branched plasmodesmata of differentiated tissues (primarily source tissues¹, see 6.8.3) presumably function as regulated pores through which macromolecules pass only when they can activate the transport machinery. In contrast, simple, unbranched plasmodesmata of growing sink tissues allow macromolecules with a molecular weight of up to 50–70 kDa to pass unimpeded. The complex plasmodesmata between companion cells and sieve elements also appear to be permanently permeable to macromolecules of at least 25–30 kDa. Consequently, upon entering the sieve elements, proteins can undergo long-distance transport and be distributed via the symplast into sink tissues. It remains unclear how the selectivity of protein transport through these plasmodesmata is ensured. For instance, thioredoxin is efficiently translocated from companion cells into sieve elements, whereas ubiquitin is not transported at all, even though the molecular weights of both proteins are well below the size exclusion limit.

¹ The terminology used here is adapted from English-language literature. Tissues that act as Donors of photoassimilates are termed sources, and acceptor tissues are termed sinks. — Ed. note.