Annals of Botany 81: 1–10, 1998

BOTANICAL BRIEFING

Plasmodesmata: Dynamics, Domains and Patterning

FRIEDRICH KRAGLER, WILLIAM J. LUCAS* and JAN MONZER Section of Plant Biology, DiŠision of Biological Sciences, UniŠersity of California, DaŠis, CA 95616, USA

Received: 30 June 1997 Returned for revision: 6 August 1997 Accepted: 8 September 1997

Although it has long been known that plasmodesmata establish cytoplasmic continuity between most cells within the body of the plant, it is only recently that these special structures have been viewed as dynamic intercellular organelles (Lucas and Wolf, Trends in Cell Biology 3: 308–315, 1993) involved in the transport of macromolecules. Ultrastructural studies have provided important information on the formation of plasmodesmata during cytokinesis (primary plasmodesmata) and as post-cytokinetic events, where the new cytoplasmic bridges are inserted within the existing wall (secondary plasmodesmata). Modifications to plasmodesmal frequency, and presumably composition probably reflect developmental and physiological requirements for symplasmic continuity\communication. Pioneering studies on viral movement proteins provided the first direct experimental evidence that proteins and protein-nucleic acid complexes could traffic cell to cell, via plasmodesmata. This knowledge paved the way for the identification and characterization of endogenous proteins that also possess a similar capacity for cell-to-cell transport. Such proteins range from plant transcription factors, involved in orchestration of plant development, to proteins present in the phloem sap of angiosperms which are probably involved in maintenance of the enucleate sieve tube system. Information from viral infection studies and plant development support the concept that plasmodesmata play an essential role in the formation of developmental and physiological domains in which regulated trafficking of macromolecules establishes a non-cell-autonomous control network. Finally, the role of plasmodesmal trafficking of informational molecules is discussed in terms of providing a novel means for controlling cell differentiation. This concept is illustrated using current knowledge on root hair and trichome pattern formation. # 1998 Annals of Botany Company

Key words: Plasmodesmata, macromolecular trafficking, developmental patterning, root hairs, trichomes.

INTRODUCTION PLASMODESMAL BIOGENESIS Plasmodesmata are unique intercellular transport complexes Keeping up with cell diŠision, expansion and differentiation that connect neighbouring cells (Robards and Lucas, 1990; Analyses of plasmodesmal distribution within such tissues Lucas, Ding and Van Der Schoot, 1993; Lucas and Wolf, as source and sink leaves provided important information 1993; Lucas, 1995). Extensive ultrastructural studies have on the symplasmic pathway available for the diffusion of provided important information on the basic structure of photosynthates (Van Bel and Oparka, 1995, Evert, Russin the plasmodesma (Robards and Lucas, 1990; Ding, Turgeon and Botha, 1996), but yielded little insight into the and Parthasarathy, 1992b; Botha, Hartley and Cross, 1993; underlying processes involved in the formation and regu- Overall and Blackman, 1996). It is now evident that, in lation of such patterns. Certainly it has long been known contrast to other multicellular organisms, the ability to that plasmodesmata are formed during the process of insert plasmodesmata across the cellulosic wall has allowed cytokinesis. The picture that has emerged is that this is a higher plants to develop an integrated cytoplasmic and highly orchestrated process in which strands of the endomembrane (endoplasmic reticulum-nuclear envelope) endoplasmic reticulum (ER) are first positioned across the continuum that extends throughout the body of the plant. plane of the advancing cell plate. Golgi vesicles that are Interestingly, exactly 20 years ago, Esau (1977) wrote, ‘The being delivered to this region fuse in the division plane to relation of the structural features to the function of provide both the initial matrix of the primary cell wall and plasmodesmata as connections between protoplasts is not establish the plasma membrane. Vesicle fusion in the vicinity yet fully understood’. As we will demonstrate in this review, of the ER provides the foundation for the establishment of plant biologists are currently providing the long-awaited primary plasmodesmata (Fig. 1). As the width of the new details concerning the functional roles played by plasmo- wall increases, individual plasmodesmata develop as simple, desmata. cylindrical-like structures: the central region is occupied by an appressed form of the ER (which is continuous with the fenestrated form of the ER within the cytoplasm of the daughter cells) and a cytoplasmic annulus forms a bridge * For correspondence. Fax 1 916 752 5410, e-mail wjlucas!ucdavis.edu between the daughter cells (Fig. 1; Hepler, 1982). 0305-7364\98\010001j10 $25.00 bo970522 # 1998 Annals of Botany Company 2 Kragler et al.—Plasmodesmata: Dynamics, Domains and Patterning

endoplasmic reticulum

cell plate assembly endoplasmic cell wall Primary cell wall reticulum formation vesicles

vesicles

vesicles Selective Macromolecule Transport

binding Secondary Modification formation plasma membrane

cell wall

endoplasmic release reticulum

Role in Development and Physiology

Site-specific modification: Closure of cell contacts: Sieve pores Occlusion and degradation sieve wall degraded pore apposition plasmodesmata

F. 1. Biogenesis and function of plasmodesmata. The biogenesis and function of plasmodesmata are closely linked to plant development and differentiation. Their capability for selective macromolecule transport (central box) provides a highly specialized pathway for information exchange that establishes non-cell-autonomous control over plant development. Macromolecules able to move cell-to-cell may act as transmitters of information themselves, or traffic other information molecules such as mRNA. Primary plasmodesmata are established during cytokinesis (top). Cytoplasmic bridges containing strands of endoplasmic reticulum are retained during cell plate assembly, leaving cytoplasmic connections between the daughter cells (top left). Plasmodesmata can be modified during differentiation (left). Branching increases the total cross sectional area available for macromolecular transport and diffusion of metabolites. Sieve pores (lower left) are established as a site-specific modification to primary plasmodesmata. Secondary plasmodesmata can arise from arrays of endoplasmic reticulum and vesicles at the surface of neighbouring cells (top right) that fuse across thinned wall areas to establish usually branched connections (right). Considerable numbers can be formed during elongation growth in primary differentiation. Secondary plasmodesmata are thought to provide specific pathways for macromolecule transport and for non-cell-autonomous regulation. In terminal differentiation, plasmodesmal communication can be down regulated (lower right). For example, plasmodesmata in the differentiating xylem are sealed by apposition of wall material before maturation.

Plasmodesmata can also be established, de noŠo, across to be formed by a process in which the matrix of the wall is existing cell walls. As such plasmodesmata are post- modified to allow a thinning to occur (Fig. 1). In concert cytokinetic in origin, they are normally referred to as with this thinning, ER cisternae begin to aggregate in this secondary plasmodesmata (Ding and Lucas, 1996). The area and, simultaneously, Golgi-derived vesicles begin to underlying mechanism(s) responsible for the establishment fuse around the ER to deliver new plasma membrane and of these additional cytoplasmic bridges appears to depend deposit the supporting wall material. A loosening of the upon cell position and\or developmental programme. In original wall matrix then allows the formation of continuous young, developing tissues, secondary plasmodesmata appear cytoplasmic connections (Monzer, 1991). Secondary plas- Kragler et al.—Plasmodesmata: Dynamics, Domains and Patterning 3 modesmata that form by this mechanism are often irregular ER and membranes is precisely organized and always leads and complex in morphology (Fig. 1). to a cytoplasmic annulus of the same dimension; (e) Formation of secondary plasmodesmata across the plasmodesmata appear to undergo rapid assembly into a periclinal walls located between the outer layers of the plant supramolecular structure, as intermediate stages in their meristem(s) plays a vital role in plant development. These formation have rarely been observed. Unfortunately, little special intercellular transport complexes provide an essential information is currently available on the cellular mechan- pathway for the exchange of information molecules involved isms involved in vesicle targeting, membrane fusion and in non-cell-autonomous orchestration of developmental protein integration\assembly to form the plasmodesmal cascades (Lucas, 1995; Lucas et al., 1995). In addition, as a supramolecular complex. This area clearly warrants im- consequence of elongation growth, site-specific insertion of mediate attention. secondary plasmodesmata also appears necessary to main- Any analysis of plasmodesmal dynamics during plant tain an adequate level of symplasmic continuity within differentiation would be incomplete without a discussion of tissues as they advance towards physiological competence the processes involved in the sealing or removal\degradation (Robards and Lucas, 1990). An excellent example of such a of these cytoplasmic bridges. Analysis of plasmodesmal role for secondary plasmodesmal formation was recently frequencies, within specific cells, indicates that plant cells provided by Volk, Turgeon and Beebe (1996). Here the have the capacity to regulate the degree to which individual formation of secondary plasmodesmata was correlated with cells are symplasmically connected to neighbouring cells. the sink-to-source transition in the minor veins of squash Thus, the plant must have the ability to remove, or seal off leaves. Furthermore, this form of plasmodesmata generally (temporarily or permanently), existing plasmodesmata. represents a significant proportion of the total number of Insight into this process was recently provided by studies plasmodesmata that interconnect most of the cells within performed on secondary plasmodesmal formation in regen- the body of the plant (Ding et al., 1992a; Ding and Lucas, erating protoplasts. Immunocytochemical studies revealed 1996). Thus, the role of secondary plasmodesmata in the presence of high levels of ubiquitin in aborted developmental and physiological processes cannot be plasmodesma that failed to establish secondary contacts overstated. between neighbouring cells (Ehlers, Schulz and Kollmann, 1996). Ubiquitination is well known as a signal for protein degradation and, thus, its accumulation within these half- plasmodesma may well reflect a central role for this enzyme A complex symplasmic continuum: primary, modified in the targeted removal and subsequent elimination of primary and secondary plasmodesmata specific cytoplasmic bridges between cells (Fig. 1). It is interesting that the formation of primary and A further important example of site-specific control over secondary plasmodesmata had often been considered to plasmodesmal structure\function can be found in differen- represent fundamentally different processes. The concept tiating xylem tissues. Immature xylem vessels and tracheids that plasmodesmata can undergo quite extensive structural are connected to adjacent parenchyma cells by pits that modifications during the process of tissue differentiation is contain a high density of plasmodesmata. As these water- fully supported by ultrastructural studies (Ding et al., conducting cells enter the final stage of programmed cell 1992a, 1993; Lucas et al., 1993). However, until recently, death, plasmodesmata within the pits become sealed these changes were thought primarily to reflect modifications (Lachaud and Maurousset, 1996). This process is carefully to secondary plasmodesmata (Ding and Lucas, 1996). An orchestrated, and is accomplished by the apposition of new elegant study by Ehlers and Kollmann (1996) has now wall material across the orifice at both ends of the demonstrated that this viewpoint is in need of revision. plasmodesmata (Fig. 1). At present nothing is known about Their studies on primary plasmodesmal formation in cells of the exchange of information between these special cells Solanum nigrum revealed a dynamic aspect associated with (Savidge, 1996), nor of the underlying molecular events that these structures. Primary plasmodesmata were shown to orchestrate this truncation of cytoplasmic continuity. undergo branching and this process was initiated from a Genetic and molecular information on this and related region within the appressed ER which subsequently became processes may be forthcoming from studies on the sucrose bifurcated. Aggregation of vesicles at the plasmodesmal export deficient 1 (sxd1) mutant of maize (Russin et al., orifice, with the subsequent separation of plasmodesmal 1996). In this mutant, plasmodesmata located within the branches, gave rise to branched primary plasmodesmata bundle sheath-vascular parenchyma cell wall become (Fig. 1). blocked by wall apposition, thereby inhibiting sucrose Collectively, the ultrastructural studies performed on movement into the loading region within these minor veins. primary and secondary plasmodesmata suggest that a Cloning of sxd1 should allow a dissection of the molecular common mechanism may well be involved in the formation events underlying the development of this intriguing mutant of these cytoplasmic bridges. This mechanism probably phenotype. As a similar process appears to be involved in involves the following steps: (a) strands of ER co-assemble the sealing of plasmodesmata between guard cells and with Golgi-derived vesicles; (b) the membrane from these epidermal cells it may well be that both processes share vesicles forms the outer plasma membrane lining of the common elements. Obviously, identification of the genes plasmodesmal channels; (c) wall material, delivered from involved in the truncation of plasmodesmata would poten- within the lumen of these vesicles, reinforces the newly tiate a wide range of physiological and developmental established plasmodesmal strands; (d) the arrangement of studies. 4 Kragler et al.—Plasmodesmata: Dynamics, Domains and Patterning increase in the size exclusion limit (SEL) of the plasmodesma. Identification of plasmodesmal constituents Normally, plasmodesmata permit the free diffusion of small The isolation and biochemical characterization of the molecules in the range of 850 Da, but this value increases to molecular constituents of plasmodesmata remains a major more than 10 kDa during the trafficking of macromolecules. challenge. A number of workers have attempted to purify Here it should be noted that in microinjection studies plasmodesmal components from cell wall preparations performed on trichomes, the TMV MP was reported to (Monzer and Kloth, 1991; Yahalom et al., 1991; Kotlizky move along the length of the trichome without inducing a et al., 1992; Turner, Wells and Roberts, 1994; Epel et al., detectable increase in SEL (Waigmann and Zambryski, 1996b). Unfortunately, to our knowledge no published 1995). This finding is consistent with the notion that specific report has provided unequivocal evidence for the local- plasmodesmata can mediate in tissue-specific processes. ization of a cloned plasmodesmal protein within the Although much has been learned in terms of the way in cytoplasmic boundaries of the plasmodesma. The appli- which MPs interact with plasmodesmata, there is still a cation of novel preparative techniques may yield the long- great deal yet to be elucidated, especially with respect to awaited breakthrough in this area. For example, as chimeric both the molecular constituents of the plasmodesmata viral movement protein:GFP constructs have been shown involved in this transport as well as the nature of the cellular to accumulate in mesophyll plasmodesmata during viral factors that restrict viral movement to particular domains infection (Heinlein et al., 1995; Epel et al., 1996a), these within the plant (Wang, Gilbertson and Lucas, 1996). fluorescently-tagged proteins may be of use in future The finding that viral MPs could move through plasmo- attempts to isolate plasmodesmal components that interact desmata raised the important question as to whether with viral movement proteins (MPs). Our laboratory is also endogenous proteins were also able to move between cells working on the development of an in Šitro assay that will via plasmodesmata. That this was indeed the case was utilize the binding of endogenous transcription factors established by experiments performed with the maize and\or viral MPs to plasmodesmal components in an KNOTTED1 (KN1) homeobox protein. Expression of KN1 attempt to identify binding motifs. Finally, the inhibitory within the maize meristem leads to the presence of KN1 effects of the herbicide, 2,6-dichlorobenzonitrile, on cellulose mRNA within L2 cells, whereas KN1 is detected in almost biosynthesis may also prove useful. Under the influence of all cells including the L1 layer (Sinha, Williams and Hake, this herbicide, newly formed division walls exist within cells 1993; Jackson, Veit and Hake, 1994; Lucas et al., 1995), as incomplete sheets that contain disoriented plasmo- suggesting that KN1 can move through the secondary desmata (Vaughn et al., 1996). Thus, cell fractionation plasmodesmata that interconnect the L1 and L2 layers of procedures could well be developed that would allow the the meristem. Direct experimental proof that KN1 has the isolation of these sheets of wall material which could then capacity to interact with and traffic through plasmodesmata serve as a source for highly purified plasmodesmal proteins. was gained through microinjection studies. Lucas et al. (1995) showed that KN1 behaves in a manner almost identical to viral MPs, in that it increased plasmodesmal PLASMODESMATA TRAFFIC SEL (to approx. 40 kDa), mediated its own cell-to-cell MACROMOLECULES movement and potentiated the trafficking of its own mRNA but not viral RNA; i.e. with KN1, mRNA trafficking was Viral MPs and plant proteins traffic through found to be sequence specific. plasmodesmata Additional evidence that endogenous proteins are trans- It has long been known that plant viruses spread throughout ported cell-to-cell, via plasmodesmata, has been gained the plant by passing through plasmodesmata (Robards and through studies on the phloem. In angiosperms, the Lucas, 1990). It is now clear that viruses encode proteins enucleate sieve tube members are thought to be maintained that mediate in the cell-to-cell trafficking of the infectious by proteins synthesized in neighbouring companion cells form of the virus. Based on microinjection studies, it has and then transported through plasmodesmata into the now been established that certain plant viruses, such as the functional sieve tube system (Lucas and Wolf, 1993; Lucas, tobacco mosaic virus (TMV), red clover necrotic mosaic 1995). Direct evidence has now been obtained that proteins virus and cucumber mosaic virus use a dedicated protein, within the phloem sap also have the capacity to increase termed the movement protein (MP) to traffic their infectious plasmodesmal SEL and mediate their own cell-to-cell viral RNA through mesophyll plasmodesmata (Fujiwara et transport (Ishiwatari et al., 1996). al., 1993; Waigmann et al., 1994; Ding et al., 1995). An Recent studies performed using tobacco, potato and extensive analysis of this viral MP literature can be found in tomato demonstrated that the mRNA for the phloem recent reviews by Lucas and Gilbertson (1994), Gilbertson specific sucrose transporter1 (SUT1) could be detected and Lucas (1996) and Ghoshroy et al. (1997). The salient within the plasmodesmata connecting the companion cells features from this work are that viral MPs can interact with to the functional sieve tube system (Ku$ hn et al., 1997). This certain plasmodesmata, located in various plant tissues, to critical finding suggests that proteins within the companion mediate their own cell-to-cell transport. In addition, these cell can interact with SUT1 mRNA to allow it to gain entry MPs have the ability to bind, in a non sequence-specific into the sieve tube member where this integral membrane manner, to vRNA\DNA and presumably it is this MP- protein may be synthesized on residual ribosomes attached vRNA complex that moves through receptive plasmodes- to the SER. In any event, these studies on phloem proteins mata. During this trafficking process, MPs induce an and KN1 have provided insight into the extent to which the Kragler et al.—Plasmodesmata: Dynamics, Domains and Patterning 5 plant is capable of utilizing plasmodesmata to traffic genetic and microinjection studies conducted on the MADS- macromolecules between cells to regulate developmental box carrying, homeotic proteins DEFICIENS (DEF) and and physiological processes. GLOBOSA (GLO) of Antirrhinum majus. DEF and GLO act as heterodimers and control their own transcription as well as transcription of other genes. Perbal et al. (1996) Plasmodesmal trafficking and the plant cytoskeleton employed periclinal chimeras expressing DEF and GLO in different cell layers to recover wild-type flower development. Experiments with a TMV MP::green fluorescent protein Here it is important to note that establishment of DEF and (GFP) construct provided interesting results consistent with GLO expression in the L2 and L3 layers resulted in recovery the hypothesis that this MP interacts with either micro- of petaloid identity of the epidermal (L1) layer (derived filaments (actin) or microtubules during the process of viral from a def and glo mutant plant line). Immunocytochemical infection. Plants or protoplasts inoculated with infectious studies confirmed that both DEF and GLO were present in TMV MP::GFP clones were found to accumulate L1, whereas in situ analyses failed to detect the presence of MP::GFP within regions of the cytoplasm where tubulin DEF and GLO mRNA (Perbal et al., 1996). Finally, and actin were also co-localized. These findings suggested microinjection experiments performed on tobacco meso- that the delivery of proteins and protein-RNA complexes to phyll cells demonstrated that both DEF and GLO have the plasmodesmata may utilize the plant cytoskeleton (Heinlein capacity to increase SEL and traffic through plasmodesmata et al., 1995; McLean, Zupan and Zambryski, 1995). (Mezitt and Lucas, 1996). Thus, with the basic concept of Additional evidence that the cytoskeleton may be involved SCPs in place, the challenge ahead will be the establishment in plasmodesmal function has been provided by micro- of appropriate screens that will permit the identification of injection studies using cytochalasin D and profilin. Co- these SCP cofactors. injection of either cytochalasin D or profilin with fluor- escently labelled 20 kDa dextran allowed cell-to-cell move- ment of the dextran thus indicating that actin microfilaments ROLE OF PLASMODESMATA IN may be involved in maintaining the structural integrity of DIFFERENTIATION plasmodesmata (White et al., 1994; Ding, Kwon and Warnberg, 1996). The challenge remains to identify the Root deŠelopment: A model system for plasmodesmal molecular components that function in linking the cyto- studies skeleton to this intercellular macromolecular trafficking The primary root of Arabidopsis thaliana has a simple pathway. organization consisting of single-cell layers of the epidermis, cortex, endodermis and pericycle, which surround the central vascular cylinder (Fig. 2). The cortical and en- DeŠelopmental domains established by protein trafficking dodermal layers of the primary root consist of eight cells. through plasmodesmata This simple cellular architecture of the primary root is The concept of non-cell-autonomous differentiation, established in the embryo. Here, the first asymmetric cell during organogenesis, seems to hold for all multicellular division produces a smaller apical and a larger basal cell. organisms. At the molecular level, the intra- and in- The basal cell forms the hypophysis, which is located at the tercellular mechanisms involved in controlling differenti- top of the suspensor. Later in embryogenesis the hypophysis ation share striking homologies between animals and plants. divides to form a lens-shaped cell, the progenitor to four Support for this viewpoint comes from the recent finding central cells (quiescent centre) and the initial cells directly that, in maize, CRINKLY4, a homologue of the membrane- adjacent to the meristem region (Fig. 2). The rest of the bound tumor necrosis factor receptor, appears to be involved embryonic root derives from the apical cell. These two cell in epidermal differentiation (Becraft, Stinard and McCarty, populations, having different clonal origins, act in concert 1996). However, plants are clearly different from animals in to form the embryonic root, thus suggesting that cell-to-cell one important aspect: they have cell walls and plasmo- interaction is occurring to orchestrate the development of desmata through which regulators of differentiation can be the root. exchanged. Obviously, this unique plant pathway for At first sight, the cellular architecture of the Arabidopsis information exchange must have evolved highly selective primary root appears consistent with the hypothesis that mechanisms to regulate the trafficking of such proteins, cell fate is lineage dependent i.e. the cells of the epidermis otherwise the formation of developmentally different do- and lateral root cap would appear to descend from a mains could not be maintained. A model for such regulation common set of initial cells, and the underlying cortical and was recently proposed by Mezitt and Lucas (1996). Proteins, endodermal cells appear to be derived from discrete initials such as KN1, that can traffic through plasmodesmata and (Dolan and Roberts, 1995). However, insightful laser act as bona fide regulators of cellular development\ ablation experiments performed on this root system pro- differentiation were termed supracellular control proteins vided unambiguous evidence that positional dependent (SCPs). Cellular elements that act to regulate the delivery, information plays a critical role in the establishment binding, or plasmodesmal transport of such SCPs were (maintenance) of cell fate. Following ablation of an called SCP cofactors. epidermal initial, a neighbouring cortical cell alters it fate Support for this SCP concept was provided by studies on and becomes a functional epidermal initial. Similarly, KN1, the homeobox protein of maize, and more recently by ablation of a cortical cell initial leads to the reversion of a 6 Kragler et al.—Plasmodesmata: Dynamics, Domains and Patterning significantly altered by killing adjacent daughter cells. Thus, it would appear that these ablation studies provide support for the involvement of signalling via the plasmodesmal pathway. This conclusion gains further support from an

Vascular cylinderEndodermisCortex Epidermis analysis of plasmodesmal distribution in the root meristem of Arabidopsis. As illustrated schematically in Fig. 2, cells within the ablation zone are highly interconnected by plasmodesmata. In addition, the transverse walls for cell files of common origin contain high numbers of plasmo- desmata, presumably to ensure symplasmic continuity and the capacity to traffic information molecules within such cell layers. Furthermore, the density of plasmodesmata between epidermal, cortical, and the endodermal cell layers was found to be low, which would restrict symplasmic exchange but still permit cell-to-cell communication (Zhu, Lucas and Rost, unpubl. res.). These interpretations of symplasmic connectivity are supported by dye-loading experiments in which it was Meristematic zone shown that longitudinal movement of carboxyfluorescein, across transverse walls, was preferred over radial movement between different cell layers (Duckett et al., 1994; Zhu, Stelar initials Lucas and Rost, unpubl. res.). It is interesting to note that Cortical/endodermal the observed slow radial movement of carboxyfluorescein initial was correlated with the density of secondary plasmodesmata within the longitudinal walls. Lateral root cap cell Quiescent centre

Epidermal/lateral Columella root root initials Root hair cell patterning: Role for plasmodesmal cap cell trafficking In the Brassicaceae, root hair cells are arranged in files Columella root cap exclusively located over anticlinal walls of the underlying initial cortical cells (Fig. 3A). Root hair differentiation can be followed from a very early stage, as the progenitor cells are F. 2. Cellular arrangement and degree of symplasmic continuity within the meristematic root of Arabidopsis thaliana. Model developed shorter and exhibit delayed vacuolation compared to their from plasmodesmal frequency and dye loading experiments (Zhu, neighbouring non-hair epidermal cells (Benfey and Schiefel- Lucas and Rost, unpubl. res.). *, High number of plasmodesmata\ bein, 1994). As discussed above, a reduction in symplasmic symplasmic connections; , low number of plasmodesmata\ continuity occurs later in development. Microinjection symplasmic connections. experiments performed on mid-to-late differentiating epi- dermal cells provided strong support for this change in symplasmic state, as carboxyfluorescein and Lucifer yellow pericyle cell which then replaces the dead cell, leading to the failed to move into adjacent cells (Duckett et al., 1994). progression of normal layered root tissue development. Hence, pattern formation occurs early while the epidermal Additional evidence that continuous cell-to-cell communi- cells are still symplasmically connected by plasmodesmata. cation is essential for cellular\tissue\organ development The critical unresolved question is how does the root was provided by isolating a cortical initial cell from its epidermis establish the observed regular pattern of root hair cortical daughter cells. This isolated cell was shown to be differentiation? Clearly, at least two signals must be competent to undergo cell division, but was now unable to involved, one coming from the underlying cortical layer, divide asymmetrically to generate cortical and endodermal which determines the exact positioning over the anticlinal cell files. Thus, daughter cells within a cell layer appear to be cell walls, and a second, involved in suppression of the involved in sending positional signals back to their initial neighbouring non-hair cells. At a very early stage in cells to control cell fate and, hence, tissue differentiation epidermal cell differentiation, cells are considered to exist in (Van Den Berg et al., 1995). a default state for root hair identity. An important gene Two pathways are available for the transmission of these involved in controlling (suppressing) this default state is the putative regulatory signals: across the apoplasmic space of homeobox protein GLABRA2 (GL2) of Arabidopsis thali- the adjacent cell walls, or cell-to-cell through plasmodes- ana; this gene is expressed in non-root hair epidermal cells mata. Ablation experiments should cause little perturbation (Masucci et al., 1996). Another allele affecting the de- to the transmission of an apoplasmic signal, as the distance velopment of root hairs is the putative transcription factor, over which diffusion of the signal would occur is very short. TRANSPARENT TESTA GLABRA (TTG). Mutants in In contrast, direct cell-to-cell communication would be either allele (gl2 or ttg) develop hair cells irrespective of their Kragler et al.—Plasmodesmata: Dynamics, Domains and Patterning 7

A A

Epidermis +–– – – +

TCIF Hair cells TRIC Cortex TCSF TCSF TCSF TCSF TCSF ? GL1 TCIF TCIF TCIF TCIF TCIF

Anticlinal cell walls B

Signal produced by differentiating Periclinal trichome cell wall + Suppression of – trichome differentiation B – Induction of trichome – differentiation HAIR HAIR HCSF HCSF HCSF HCSF – +

HCSF HCSF Concentration of regulators HCIF GL2 GL2 GL2 HCIF GL2

Threshold concentration TCSF TCIF for trafficking

? ? F. 3. Root hair differentiation in the Arabidopsis root. A, Cross section through the zone of root hair differentiation illustrating that C root hair cells develop (grey shading) adjacent to the anticlinal walls of the inner cortical cells. B, Model for non-cell-autonomous, positional dependent control over root hair patterning. An unidentified signal (?) First derived from the inner root tissue activates expression of a putative hair primary trichome TRIC cell inducing factor (HCIF) which activates both root hair differen- tiation and expression of a hair cell suppressor factor (HCSF). The HCSF has the capacity to traffic through plasmodesmata that interconnect epidermal cells (double-headed arrows indicate direction of movement), to active GLABRA2 (GL2; single-headed arrows indicate activation of GL2) thereby giving rise to the suppressed state. position relative to the underlying cortical cells (Masucci TRIC and Schiefelbein, 1996). These results are consistent with the hypothesis that regulatory information must be exchanged between neigh- bouring cells, within or between these two layers. A model illustrating a likely scenario is presented in Fig. 3B. In this model, an as yet unidentified signal, generated from the F. 4. Role for plasmodesmal trafficking in trichome patterning in the cortex, activates the expression of a hair cell inducing factor epidermis of Arabidopsis leaf primordia. A trichome inducing factor (HCIF) which potentiates root hair differentiation. In (TCIF) activates the differentiation of the first priming trichome (A). addition, this HCIF controls the production of a hair cell The TCIF also activates expression of a trichome suppressing factor (TCSF) which can move cell-to-cell through epidermal plasmodesmata suppressor factor (HCSF); note that HCSF would not have (A and B). In addition, this TCSF can mediate the plasmodesmal suppressor function in cells in which the HCIF programme transport of TCIF mRNA (bold arrows indicate movement into has been activated. Cell fate of neighbouring cells is then neighbouring cells). Note that when the TCSF and TCIF are present in controlled by HCSF, which in this model would have the the same cell, TCIF function is suppressed (indicated by U). As TCSF moves out from the first priming trichome, its concentration is lowered capacity to mediate its own cell-to-cell transport through due to binding to suppressor elements (B). Eventually the level of free the plasmodesmata located within the radial, anticlinal TCSF falls below the threshold for plasmodesmal trafficking and only walls of the epidermis. In the presence of TTG, this TCSF-mRNA (TCIF) moves into the next cell (A and B). Dissociation trafficking event would allow HCSF to induce GL2 of the TCSF-mRNA (TCIF) complex allows translation of the TCIF expression, thereby leading to the suppressed state. Once mRNA which leads to induction of the next trichome (TRIC) and the activation of a second cycle (C). (Shading indicates the relative change this putative HCSF has been identified and cloned this in concentrations of TCSF and TCIF.) hypothesis can be tested by in situ (mRNA), immuno- localization (protein) and microinjection (cell-to-cell move- ment) studies. 8 Kragler et al.—Plasmodesmata: Dynamics, Domains and Patterning

Trichome patterning: Role for plasmodesmal trafficking A Additional insight into the possible role of plasmodesmal trafficking of information molecules in cell patterning can + +/– – – – +/– be gained by examining data on trichome patterning in tcsf tcsf TCIF TRIC TCIF tcsf tcsf tcsf leaves. The positional distribution of trichomes on Arabi- TRIC dopsis leaves is not random, since newly initiated trichomes ? GL1 TRIC TCIF TCIF TCIF TCIF are normally separated by three to five cells. Furthermore, clusters of trichomes have been found to be extremely rare. B In addition, in leaves, an ordered distribution of trichomes, derived from cell lineages, can be excluded based on Increased signal produced transposon-induced GUS expression within cells from a by differentiating common origin (Larkin et al., 1996; Marks, 1997). trichome A model for non-random trichome patterning, based on the hypothesis that cells immediately bordering trichomes Suppression of receive a non-translocatable suppressor signal, was proposed + +/– trichome by Korn (1994). Recent advances in our understanding of differentiation the role played by plasmodesmata in the trafficking of plant – Induction transcription factors (Lucas et al., 1995) now provide a of trichome – foundation for the development of a novel model to account differentiation – for trichome patterning. This situation is highly analogous +/– Concentration of regulators to root hair patterning, in that at least two antagonistic signals must be involved (Fig. 4A). One signal must induce trichome development (trichome inducing factor, TCIF), Threshold concentration tcsf TCIF whereas the second signal suppresses trichome development for trafficking (trichome suppressing factor, TCSF). These players act on F. 5. Model for the formation of trichome clusters in Arabidopsis the stage of the developing leaf primordia. The nature of the leaves. Details as in Fig. 4, except that plants express a tcsf allele in signal molecule responsible for initiating the first priming which tcsf exhibits decreased TCIF mRNA binding activity. See text trichome remains to be identified. Thus, for the present for a full description. discussion, we will make the assumption that the actual location of the first priming trichome within the epidermis is therefore acts to propagate the next developmental field. As random. illustrated in Fig. 4C, the progression of this wave would Within the first priming trichome, TCIF is produced and then build an ordered pattern of trichomes across the initiates trichome differentiation. During this initiation surface of the leaf consistent with the non-random dis- process, the TCIF acts in concert with GLABRA1 (GL1, tribution of trichomes reported by Larkin et al. (1996). see below), GL2 and a number of other, as yet unidentified, The model presented in Fig. 4 can be tested against recent regulators. Based on our studies with KN1, we propose that findings with mutant plants that displayed clustering of the putative TCSF binds and mediates the cell-to-cell trichomes. Here it is important to note that TTG and GL1, transport of mRNA encoding TCIF. The stable form of this which is a myc-like transcription factor expressed only in TCSF-mRNA (TCIF) complex is unavailable for trans- trichomes (Larkin et al., 1994), function as major elements lation. Both the TCSF-mRNA (TCIF) and free TCSF can in the regulation of trichome formation. Mutations in either interact with plasmodesmata to traffic cell-to-cell. As TTG or GL1 significantly alter the number and distribution illustrated in Fig. 4A and B, the TCSF suppresses trichome of trichomes. Clustering of trichomes was observed in development within a field of cells. The number of cells heterozygous plants expressing ttg1\TTG and over-ex- within a given field is determined by the threshold level of pressing GL1 (as a 35S::GL1 construct). Similar phenotypes TCSF (free and complexed with TCIF mRNA) required for were observed in homozygous plants expressing either the plasmodesmal transport (see, for example, Balachandran et weak allele of ttg10 (relatively high numbers of trichomes) al., 1997). or the strong allele of ttg1 (low number of trichomes) During the course of this movement, the concentration of (Larkin et al., 1994; Marks, 1997). TCSF declines as a result of its binding to either a nuclear The formation of trichome clusters in plants carrying the or a cytoplasmic receptor involved in suppressor function. ttg allele can be explained on the basis of an imbalance in At the outer margin of this field of epidermal cells, a the activities of TCSF and TCIF (Fig. 5). One scenario situation develops where free TCSF falls below the threshold might be that ttg plants produce an altered form of TCSF for plasmodesmal transport; however, the stability (and (tcsf) (note that TTG may be the TCSF) having decreased level) of the TCSF-mRNA (TCIF) complex permits its entry TCIF mRNA binding activity; however, tcsf would still into the next cell (Fig. 4B). Within this cell, the absence of retain both suppressor and plasmodesmal transport func- free TCSF allows the dissociation of TCSF from the tions. In the rare event that tcsf mediates trafficking of TCIF complex, thereby releasing the TCIF mRNA, which is mRNA to the next cell, its reduced mRNA binding capacity immediately followed by translation to produce TCIF. would potentiate the dissociation of the tcsf-mRNA (TCIF) Consequently, this cell is induced to form a trichome and complex, thereby resulting in translation of TCIF. This Kragler et al.—Plasmodesmata: Dynamics, Domains and Patterning 9 interpretation is consistent with the observation that these and function contributed greatly to studies on plasmodesmal ttg mutants exhibit a decreased number of trichomes and biology. the formation of clusters. Research on plasmodesmata in our laboratory was The influence of a weak allele, such as ttg10, on trichome supported by grants from the National Science Foundation patterning can be explained on the basis of this plasmo- (IBN-94-06974), the Department of Energy Biosciences desmal macromolecular trafficking model. In this mutant, (DE-FG03-94ER20134) and the Human Frontier Science clusters of trichomes are found superimposed on a relatively Program. FK was supported by an Erwin Schro$ dinger normal background of trichome distribution. Relative to Fellowship from the Austrian FWF (J1249). JM was the situation with the ttg1 allele, binding of tcsf to TCIF supported by a research fellowship from the German DFG mRNA would be less impaired. As a consequence, the (Mo 639\4-1). clustering phenotype, as seen with the ttg1 allele, would still develop for all the same reasons advanced above. However, in the ttg10 mutants, a cluster of trichomes might cause a LITERATURE CITED localized increase in both tcsf and the tcsf-mRNA (TCIF) Balachandran S, Xiang Y, Schobert C, Thompson G, Lucas WJ. 1997. complex. This would create a unique situation in which the Phloem sap proteins from Cucurbita maxima and Ricinus communis high level of tcsf would stabilize the tcsf-mRNA (TCIF) have the capacity to traffic cell to cell through plasmodesmata. complex, thereby reestablishing a near wild-type condition, Proceedings of the National Academy of Sciences USA (in press). but with clusters (Fig. 5). Becraft PW, Stinard PS, McCarty DR. 1996. CRINKLY4: A TNFR- Our explanation of the ttg10 phenotype can also account like receptor kinase involved in maize epidermal differentiation. Science 273: 1406–1409. for the clustering that develops in plants constitutively Benfey PN, Schiefelbein JW. 1994. Getting to the root of plant expressing GL1, in a background of lower levels of development: the genetics of Arabidopsis root development. Trends functional TTG (i.e. heterozygous 35S::GL1\wt GL1, wt in Genetics 10: 84–88. TTG\ttg1). Consistent with the arguments developed by Botha CEJ, Hartley BJ, Cross RHM. 1993. The ultrastructure and Larkin et al. (1994), GL1 in these heterozygous plants could computer-enhanced digital image analysis of plasmodesmata at sensitize epidermal cells for trichome development and, in the Kranz mesophyll-bundle sheath interface of Themeda triandra var imberbis Retz A. Camus in conventionally-fixed leaf blades. conjunction with perturbed TTG levels, establishes a Annals of Botany 72: 255–261. concentration-dependent positional signalling system that Ding B, Haudenshield JS, Hull RJ, Wolf S, Beachy RN, Lucas WJ. would lead to clustering of trichomes. 1992a. Secondary plasmodesmata are specific sites of localization of the tobacco mosaic virus movement protein in transgenic CONCLUSIONS tobacco plants. Plant Cell 4: 915–928. Ding B, Haudenshield JS, Willmitzer L, Lucas WJ. 1993. 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