Intercellular communication in plants is carried out via plasmodesmata (PDs) to coordinate activities of cells and organs during development, to allow movement of nutrients required for metabolism, and to coordinate responses to changes in the environment.
PDs are microscopic channels that traverse the cell wall, which are bounded by a sleeve of plasma membrane, and have a tube of endoplasmic reticulum (ER) running through the middle.
Primary PDs are formed during cell division and act as sites for formation of secondary PDs during plasma membrane and cell wall expansion.
Essential structural components of PD include membranes, cytoskeleton (actin and myosin) and cell wall (callose) components.
PDs are highly dynamic and are tightly regulated by developmental and environmental signals.
Many plant development genes have been found to act non-cell autonomously. The developmental regulators (proteins and/or RNAs) encoded by these genes are in some cases transported between cells through PD.
While small molecules diffuse through PDs in a passive, nontargeted manner, larger molecules move by a selective pathway and may possess intrinsic trafficking signals.
Plant viruses hijack the PD pathway to spread their infection.
Forward genetic and proteomic screens have been effective methods to identify novel genes encoding PD proteins, while super-resolution electron and fluorescence microscopy are powerful tools for imaging PD structure and cell-to-cell trafficking.
KeywordsPlasmodesmata (PD) Function and regulation Cell-to-cell communication Plant development Non-cell autonomous
Intercellular communication in multicellular organisms is critical for proper development, metabolism, and environmental responses. This occurs in part via secreted signals and receptors in recipient cells, but both animals and plants also directly transfer molecules cell to cell, to coordinate cellular activity across diverse cell types. This kind of communication is especially important in plants, where development continues over the life span. Many plant developmental genes have been found to act non-cell autonomously. The developmental regulators (proteins and/or ribonucleic acids (RNAs)) encoded by a number of these genes are transported between cells through the microscopic channels called plasmodesmata (PDs, plural, singular = plasmodesma) (Xu and Jackson 2010; Burch-Smith and Zambryski 2012). PDs also facilitate systemic movement of protein and RNA molecules by providing access to the phloem vascular system. The PD pathway is hijacked by plant viruses to spread their own proteins and nucleic acids (Xu and Jackson 2010; Burch-Smith and Zambryski 2012; Pena et al. 2012).
PDs, however, are not just a hole through the cell wall but are complex structures and have a size exclusion limit (SEL). They allow free movement of molecules below this SEL, whereas larger molecules have to interact with the PD trafficking machinery, to open their channels and allow movement. The PD SEL is spatiotemporally controlled, to coordinate growth and development (Xu and Jackson 2010; Burch-Smith and Zambryski 2012). Although cell-to-cell transport is essential for plant development, many aspects of PD structure and trafficking mechanisms have remained a mystery to plant biologists. The recent publication of the PD proteome was a positive step toward understanding PD composition (Fernandez-Calvino et al. 2011). Other important discoveries provided hints on regulatory mechanisms of PD trafficking. For example, identification of a role of the chaperonin complex in KNOTTED1 (KN1) trafficking, and in stem cell maintenance, provides clues to how the KN1 protein successfully traffics cell to cell (Xu et al. 2011). Identification of GFP (green fluorescent protein) ARRESTED TRAFFICKING1 (GAT1) and INCREASED SIZE EXCLUSION LIMIT1/2 (ISE1/2) led to insights into the control of PD during plant development (Burch-Smith and Zambryski 2012). Here, information from these and other exciting discoveries that cover broad aspects of PD biology has been compiled.
Plasmodesmata Structure and Composition
These channels are formed during cell division, when strands of endoplasmic reticulum (ER) are trapped in the new plasma membrane and cell wall formed between daughter cells. Secondary PDs are formed after cell division, during cell wall expansion. The development of PDs has been studied using freeze-fracture EM and computational simulations. These data suggest that existing PDs serve as sites for the insertion of secondary PDs, resulting in twinning and branched PD channels (Xu and Jackson 2010; Burch-Smith and Zambryski 2012).
PD can be thought of as having two components: membranes and the spaces between them (Fig. 1a). Membranes form the boundaries of the channel. Spaces are the regions through which transport occurs. There are two membrane domains. The first is the plasma membrane between adjacent cells, which defines the outer boundary of the channel. The other membrane domain is a compressed piece of ER, called the desmotubule , which runs through the middle of the channel. The space between the plasma membrane and the desmotubule is called the cytoplasmic sleeve. This is the major path for cell-to-cell transport. However, there is also evidence of trafficking through the lumen of the desmotubule and along the lipid bilayers of the desmotubule (Burch-Smith and Zambryski 2012).
Recent development of the 3D-structured illumination light microscopy (3D-SIM) technique has provided super-resolution images of PDs. This technique allows resolution of different PD regions, such as the neck and central cavity. For example, in Fig. 1d, callose antibody staining marked the neck region, and the central cavity was visualized by a GFP-tagged viral movement protein (MP) (Fitzgibbon et al. 2010).
PDs are extremely dynamic, and their ability to change their aperture is important for their function. The fact that large molecules can pass selectively through PDs points to this dynamic behavior. General regulators of PD aperture include actin, myosin, and callose. Actin and myosin are thought to coat the desmotubule along the length of the PD and connect the desmotubule with the plasma membrane (Fig. 1a). Treatment with the actin-disrupting drug cytochalasin D results in increased cell-to-cell transport, while the actin-stabilizing reagent phalloidin reduces transport (Faulkner and Maule 2011; White and Barton 2011). Helical actin filaments are thought to connect the desmotubule to the plasma membrane lining the channel (Fig. 1a). Contraction and relaxation of these filaments could alter the aperture of the cytoplasmic sleeves, to increase or restrict transport. Factors that regulate actin/myosin assembly and disassembly, such as calreticulin and centrin, may also regulate PD size (White and Barton 2011).
PD aperture is also controlled by the amount of callose accumulated in the cell wall surrounding the PD. Increased levels of callose reduce the PD aperture and restrict movement through the PDs. In contrast, when callose levels are reduced, the PD loosens, resulting in increased cell-to-cell trafficking. Both the cytoskeleton components (actin/myosin) and cell wall components (callose) are modified by developmental or environmental signals, such as cellular patterning, seasonal changes, or viral infection, to fine-tune PD trafficking (Ueki and Citovsky 2011; Burch-Smith and Zambryski 2012). The role of callose in regulating PD trafficking is discussed in more detail in a later section.
Regulation of PD Trafficking
Our understanding of molecular transport through PDs is expanding, following discoveries of the mechanisms controlling PD dynamics. Critical aspects of PD regulation and macromolecular transport are discussed here. The SEL is an important parameter that controls the molecular flux through PD and is defined as the largest size of a molecule that can pass through PD pores without dilating them. The SEL is usually defined in terms of molecular mass, but it is important to note that physical size of a molecule (“stokes radius”), charge, and hydrophobicity are also critical for determining whether a particular molecule can pass through the PD.
Passive Versus Active Trafficking
Trafficking through PD is broadly classified either as a passive or an active process. Passive transport (also called nonregulated, or nontargeted, or nonselective) is the simple diffusion of molecules between cells, and probably occurs, without involving other cellular components. For example, the jellyfish GFP is able to move freely between cells, during embryogenesis, in meristems, or in the immature sink tissues of developing leaves (Pena et al. 2012). Passive diffusion of proteins is hindered when the molecular mass of the protein is increased or when it is targeted to intracellular compartments or structures, such as the nucleus, the ER, or the cytoskeleton. For example, in Arabidopsis embryos, three GFP molecules fused together (3×GFP, 81 kDa) are less free to move than 2×GFP (54 kDa), which is less mobile than a single GFP molecule (27 kDa). An endogenous protein that is known to move passively between cells is the TF LEAFY (LFY), which is required for floral meristem identity in Arabidopsis. LFY is thought to move between meristem layers in a nontargeted fashion, because its mobility is unaffected by deletion mutations in different parts of the protein (Ueki and Citovsky 2011; Pena et al. 2012).
In contrast, active transport (also called targeted, or regulated, or selective transport) allows trafficking of molecules with a molecular mass higher than the SEL of PDs. Active transport involves the specific interaction of cargo with components of the transport machinery, which may help in docking cargo to PD, in dilating PD pores, or in delivering cargo into the neighboring cell. The first examples of active transport came from viral movement, using MP from Tobacco Mosaic Virus (TMV) (Pena et al. 2012). Subsequently, studies have shown that most plant viruses encode MPs that are able to alter the SEL of PD either directly or indirectly and help spread virus infection. Different experiments, including microinjections and tissue-specific expression of fusion proteins, have shown that many plant proteins, for example, KN1, SHORTROOT (SHR), and TRANSPARENT TESTA GLABRA1 (TTG1), are also able to dilate the PD and traffic between cells (Lee and Zhou 2012; Kragler 2013). Similar to nontargeted transport, targeted transport is also reduced when molecules are redirected to intracellular compartments or structures, such as the nucleus, ER, or cytoskeleton (Pena et al. 2012). However, KN1, SHR, and CAPRICE (CPC) mobility depends on their nuclear-cytoplasmic shuttling. These conflicting findings suggest that in some cases passage through the nucleus is important for targeted movement between cells. It is possible that by moving through the nucleus, the cargo is able to associate with factors that facilitate movement.
Regulation of PD Aperture by Callose Deposition
One of the best examples for reversible callose-mediated PD permeability comes from the birch tree. PD callose changes are associated with seasonal changes in growth and dormancy. As winter approaches, a short photoperiod induces accumulation of callose sphincters at the PD, leading to the closure of symplastic connections and isolation of the shoot apical meristem (SAM). As a result, morphogenesis stops, presumably due to restriction of nutrients, and the SAM enters dormancy. Following exposure to low winter temperatures, symplastic connectivity is reestablished in the spring, and dormancy is broken, which coincides with the altered levels and localization of BGs that can degrade callose (Zavaliev et al. 2011; Burch-Smith and Zambryski 2012).
Callose also controls the movement of viruses. For example, in order to dilate the PD pores, TMV MP interacts with an ankyrin repeat-containing cytoplasmic protein (ANK). The TMV MP-ANK complex localizes to PD, leading to reduced callose levels, increased SEL, and enhanced movement of TMV MP between cells (Ueki and Citovsky 2011; Pena et al. 2012).
A family of CalS/GSL genes has evolved in plants to fulfill the needs for callose synthesis in different developmental programs and in response to various physiological signals or biotic and abiotic stresses. Although there is no concrete evidence to show association of CalS/GSL enzymes with PD, some members of this gene family influence developmental processes by controlling callose-mediated PD regulation. For example, CalS10/GSL8, encoded by the CHORUS locus, is required to restrict the cell-to-cell trafficking of a bHLH TF SPEECHLESS (SPCH). SPCH is a positive regulator of the stomatal lineage and normally functions in a cell-autonomous manner during stomatal patterning. However, chorus (cals10/gsl8) mutants have less callose at PD, which results in abnormal SPCH transport into surrounding cells, leading to an increase in stomatal density in chorus mutants (Benitez-Alfonso et al. 2011; Ueki and Citovsky 2011; Zavaliev et al. 2011). Another example is provided by CalS7/GSL7, which is expressed in the phloem and is responsible for callose deposition at the sieve plates of developing phloem SEs. In cals7/gsl7 mutants, abnormal sieve plates are formed with fewer and narrower pores. Transport of photosynthates from source to sink, which normally takes place through sieve pores, is therefore less efficient in cals7 mutant plants (Xie and Hong 2011). A powerful application of the CALS system arose from the discovery that gain-of-function mutations in Cals3 affect the movement of factors controlling root patterning. These gain-of-function alleles have been made into an inducible system, which allows researchers to control PD aperture and test its effect on different signaling pathways (Carlsbecker et al. 2010).
Apart from CalS/GSL and BGs, various other proteins, including REVERSIBLY GLYCOSYLATED POLYPEPTIDE 2 (RGP2), PD-located proteins (PDLPs) and PD callose-binding proteins (PDCBs), ISEs, and GAT1, also regulate callose levels (Fig. 2). Plants with mutations in or overexpression of the genes encoding these proteins have altered trafficking, which is thought to be due to changes in PD callose levels (also see the section on discovery of PD-associated proteins).
ROS as a Regulator of PD Trafficking
Reactive oxygen species (ROS) are produced in plants in response to stress conditions and as by products of photosynthesis and photorespiration in chloroplast and mitochondria respectively. ROS impact several aspects of plant biology, including development and biotic and abiotic stress responses (Benitez-Alfonso et al. 2011; Burch-Smith and Zambryski 2012). Work in recent years has seen increasing evidence of ROS signaling in PD regulation. Interestingly, ROS appear to exert their effects on PD transport both positively and negatively, depending on the oxidative status of different cellular compartments, such as chloroplasts and mitochondria. This phenomenon was exemplified with the Arabidopsis mutants, gfp arrested trafficking1 (gat1) and increased size exclusion limit 1 and 2 (ise1, ise2). gat1 mutants carry lesions in a gene encoding thioredoxin-m3 (TRX-m3), and diffusion of GFP from phloem CCs was arrested in these mutants as a result of PD closure. The gat1 mutant plants have higher levels of ROS. Interestingly, these mutants accumulate higher levels of callose, which suggested a functional link between ROS, callose synthesis, and PD regulation (Benitez-Alfonso et al. 2011). TRX-m3 protein is found in plastids, therefore increased ROS resulted in oxidized plastids, and is believed to indirectly cause increased callose accumulation at PDs in gat1 mutants.
Changes in ROS levels in the ise1 mutant, however, have a different effect compared to gat1 mutants. ISE1 encodes a DEAD-box RNA helicase localized to mitochondria (Burch-Smith and Zambryski 2012; Zambryski et al. 2012). ise1 mutants, which show an increased size exclusion limit, overaccumulate ROS, leading to oxidized mitochondria and reduced chloroplasts. In contrast, ISE2 encodes a DEVH box RNA helicase localized to plastids. ise2 mutants, which also show an increased size exclusion limit, have a reduced chloroplast redox state but no effect on mitochondria. The observations in gat1 and ise mutants have shown that the redox state of the chloroplast or mitochondria modulates the PD permeability. Oxidized mitochondria or reduced chloroplasts lead to an increase in PD trafficking, while oxidized chloroplasts result in a decreased PD permeability. The outcomes of ROS signaling may also depend on ROS levels and on the particular ROS species that are generated (Burch-Smith and Zambryski 2012; Zambryski et al. 2012). Clearly the effects of ROS signaling on PD permeability are complex and warrant further investigation.
Discovery of PD-Associated Proteins
Despite more than 100 years since their discovery, one area of PD biology that has progressed slowly is an understanding of their protein composition. Although the list of non-cell-autonomous proteins and RNAs has been constantly increasing (Hyun et al. 2011; Lee and Zhou 2012; Ruiz-Medrano et al. 2012; Wu and Gallagher 2012), knowledge of their PD-associated interaction partners that presumably assist their trafficking is sparse. Efforts have been made to understand the structural and regulatory constituents of PDs, employing various approaches, some of which are described below (Faulkner and Maule 2011; Salmon and Bayer 2013). The basic structural constituents have been discussed in the PD structure and composition section of this article. Here the focus is on potential regulatory constituents of PDs.
Immunological approaches have aided in identification of PD constituents. The association of cytoskeletal elements, such as actin and myosin, was discovered in this way. The association of pectin methylesterase (PME) , the calcium-sequestering protein calreticulin, and the calcium-binding contractile protein centrin with PDs are among other important findings. PME interaction with viral MPs has suggested a model where this enzyme may facilitate MP transport to PDs through the ER (Epel 2009). It is also suggested that PME is involved in altering the SEL, through changing the cell wall composition around PD. The role of centrin is thought to be in bridging the desmotubule and the PD plasma membrane. In many plant species, a high ATPase activity is also found at PD. For example, in barley roots, ATPase activity is found to localize at the PD neck region. Therefore, it is not surprising that PD SEL responds to cytoplasmic calcium and ATP levels (Pena et al. 2012).
Biochemical approaches, including advanced proteomics methods, have been successful in understanding additional components of PDs (Salmon and Bayer 2013). A major hurdle has been that the PDs are embedded in the tough cell wall, making it difficult to extract them without cell wall contamination. However, more careful fractionation successfully identified PD-associated proteins. In one example, a PD-associated protein kinase (PAPK) was identified from a plasmodesmal preparation using tobacco suspension culture cells (Faulkner and Maule 2011; Salmon and Bayer 2013). PAPK recognizes a subset of virus MPs, and a subset of endogenous mobile proteins, for example LFY, but not KN1. PAPK1, the Arabidopsis homolog of PAPK, was found to co-localize with TMV MP at the PD. Another example is a reversibly glycosylated polypeptide (RGP2), which was isolated from maize cell walls. Using fluorescent protein tagging, its Arabidopsis homologue, AtRGP2, was confirmed to be associated with the PD (Faulkner and Maule 2011; Salmon and Bayer 2013). Although the precise function of RGP2 is unknown, its expression has a negative effect on PD permeability, since RGP2 overexpression in tobacco resulted in increased callose deposition and reductions in photoassimilate flux and spread of TMV (Faulkner and Maule 2011). Later, fractionation approaches were improved using cell wall degrading enzymes to aid in separation of Arabidopsis PD from the cell wall. The proteins extracted from the relatively pure PDs included a glycophosphatidylinositol (GPI)-anchored protein, β-1, 3-glucanase (BG), which is a callose-degrading enzyme. This discovery was particularly significant, as it is well established that callose (β-1, 3-glucan) is deposited at the neck regions of PDs and callose dynamics regulates PD flux (discussed earlier). It is believed that BGs move to PD along the plasma membrane. However, their association with PD is not very stable, because they are not retained at the PD after plasmolysis (Levy et al. 2007). A more extensive study, using cell cultures, helped in defining an Arabidopsis “cell wall proteome” that consisted of about 500 proteins (Faulkner and Maule 2011; Salmon and Bayer 2013). Improvements in purification and proteomics later produced a “PD proteome” (Fernandez-Calvino et al. 2011). This proteome consists of approximately 1,300 proteins, of which about 30 % are predicted to be cytoplasmic contaminants, showing the difficulties that remain in purifying PD. The remainder includes soluble and membrane proteins, some of which were already known to be associated with PD. Many receptor-like proteins are among the predicted PD membrane proteins, suggesting a high level of receptor-mediated signaling functions in intercellular communication at PD (Maule et al. 2012). Candidates were screened for cellular localization, using GFP fusions. Two new classes of PD proteins, called PD-located proteins (PDLPs) and PD callose-binding proteins (PDCBs), were discovered using this approach (Burch-Smith and Zambryski 2012; Xu and Jackson 2010).
PDLPs make up a small family of eight members (PDLP1-8), with receptor-like properties. They have two “domains of unknown function” 26 (DUF26) in the extracellular space, a single transmembrane domain (TMD) and a short C-terminal tail in the cytoplasm. Strikingly, unlike previously described PD proteins, which also localize to other subcellular structures, PDLP1 only localizes to PDs when expressed under its own promoter, suggesting it functions specifically at PD. Due to this specific localization, the PDLPs have turned out to be useful markers to label PD (Lucas et al. 2009; Faulkner and Maule 2011). Interestingly, the predicted 21 amino acid TMD of PDLP1 is necessary and sufficient for targeting to PD, through the ER-Golgi-plasma membrane transport pathway. As they are predicted to act as receptors, the PDLPs are thought to selectively control trafficking through PD through a signal transduction mechanism. However, the short cytoplasmic tails of PDLPs are most likely insufficient for signaling. Therefore, they may render their function by interacting with membrane-localized kinases. In this regard, it is noteworthy that the Arabidopsis PD proteome contained more than 30 RLKs and several RLKs in rice localize to PDs (Burch-Smith and Zambryski 2012). Additional RLKs, such as maize CRINKLY4 (CR4)/Arabidopsis CR4 (ACR4) and CLAVATA1, which are critical for shoot and root meristem development, also localize to PD (Stahl and Simon 2013).
PDLP expression affects both active and passive movement through PDs. On the one hand, PDLPs promote spread of tubule-forming viruses, by helping their MPs to form tubules through the PD channels to spread their genetic material. On the other hand, PDLPs also affect GFP diffusion through PD. PDLP1 overexpression increases callose levels at the PD neck and reduces GFP movement. In contrast, combinations of pdlp knockout mutants show enhanced GFP movement (Amari et al. 2010; Pena et al. 2012). More details on PD structure and signaling are needed to understand how PDLPs modulate PD aperture.
PDCBs, the other class of proteins identified using the cell wall proteome, belong to the large family of X8 domain-containing proteins. The X8 domain allows these proteins to bind to callose, as well as to anchor the plasma membrane to the wall matrix. Overexpression of PDCB1 negatively regulates cell-to-cell GFP movement, by increasing callose levels at the PD neck region (Faulkner and Maule 2011; Xu and Jackson 2010). However, it is not clear whether this increase in callose is a result of increased callose synthesis or of reduced callose turnover.
More recently, members of the germin-like protein (GLP) family have also been found associated with PD (PDGLP) and are implicated in regulating protein trafficking (Ham et al. 2012; Salmon and Bayer 2013). In a Co-IP experiment using Cm-PP16 as bait, GLP1 was identified as an interaction partner. Arabidopsis has five putative orthologs of GLP1; however, only two of them, PDGLP1 and 2, co-localize with PD markers. It is interesting to note that the difference between PD-localized and non-PD-localized GLPs lies within the signal peptide that targets proteins to membranes. This novel signal peptide is both necessary and sufficient for PDGLP1/2 targeting to PD. Similar to PDLP1 and RGPs, PDGLP1 also exploits the ER-Golgi secretory pathway for its delivery to PD. However, PDGLP1 lacks any known membrane spanning or anchoring motifs. Therefore, retention of PDGLP1 at PD is thought to be through its N-terminal signal peptide. PDGLP1 also interacts with several proteins associated with PD trafficking, such as NON-CELL-AUTONOMOUS PATHWAY PROTEIN1 (NCAPP1), actin, and BGs (Pena et al. 2012). PDGLP1 and 2 are predominantly expressed in developing roots, but neither the single mutants nor the pdglp1/2 double mutants show any obvious phenotype. However, expression of C-terminally tagged versions of PDGLP1 and PDGLP2 causes a significant alteration in root architecture (Ham et al. 2012). In this dominant negative phenotype, the primary root length is reduced; however, this is compensated by an increase in lateral root length, keeping the overall root mass comparable to that of the wild type. The mutant plants also have a reduced root meristem size and altered phloem resource allocation. The root phenotypes might be due to defects in cell-to-cell trafficking of factors required for cell division or by changes in sink strength in lateral roots compared to primary roots (Ham et al. 2012).
In summary, recent advances in proteomics have provided important new discoveries of potential regulators of PD transport.
Intercellular Trafficking of Proteins
Many developmental regulatory proteins, including transcription factors (TFs), are found to act non-cell autonomously. Classic examples included the homeobox TF KN1, the GRAS TF SHR, the myeloblastosis (MYB) TF CPC, the WD40 protein TTG1, and the florigen protein FLOWERING LOCUS T (FT). While KN1 and SHR are required for proper development and morphogenesis in the shoot and root, respectively, TTG1 is important for trichome and root hair patterning, and FT regulates flowering induction. Intercellular trafficking of these proteins is required for their function (Wu and Gallagher 2012; Kragler 2013). Because movement of proteins through PD is essential, recent studies have focused on teasing out how this process is regulated. Some regulatory mechanisms are common for many proteins, while some are specific for certain proteins in certain biological contexts. PD trafficking of specific proteins is discussed below.
Although KN1 is a key regulator of meristem development, our understanding of KN1 trafficking came initially from studying its movement in the leaf, where dominant mutations leading to ectopic expression give rise to abnormal growth. KN1 moves from the inner cell layers into epidermal cells, but not in the opposite direction (Xu and Jackson 2012), suggesting that its cell-to-cell trafficking is a regulated process. KN1 mRNA can also traffic, though the biological significance of this is not well understood.
Studies of KN1 trafficking have provided significant insights into mechanism of targeted protein movement (Xu and Jackson 2012). KN1 contains an intrinsic PD-targeting sequence that was identified using a “trichome rescue system” (described later). The 60 amino acid homeodomain, including the nuclear localization sequence (NLS), was found to be necessary for KN1 trafficking in the leaf. This domain was also sufficient, since it could render mobility to a cell-autonomous protein. This means that the signal for PD targeting is “encoded” in the KN1 homeodomain, and this region may be a target for regulators of movement. In one such example of this, the homeodomain is bound by a plant-specific microtubule-associated factor, MOVEMENT PROTEIN BINDING PROTEIN 2C (MPB2C), which is a putative negative regulator of PD movement (Winter et al. 2007; Xu and Jackson 2010; Pena et al. 2012; Kragler 2013). Binding of MPB2C to the KN1 homeodomain could prevent other PD trafficking factors from interacting with it. Alternatively, MPB2C may sequester KN1 in a subcellular location away from PDs. Specific developmental signals might disrupt MPB2C-KN1 interactions, allowing KN1 to traffic. Strikingly, nuclear-cytoplasmic shuttling of KN1 appears to be required for its cell-to-cell movement (Kim et al. 2005); however, the reason for this is not clear. It is possible that KN1 needs to be modified by factors present only in the nucleus in order to move cell to cell.
After it is targeted to the PD trafficking pathway, the KN1 protein is transferred through the PD channel. It was hypothesized that proteins might be unfolded before trafficking, since an unfolded polypeptide could more easily fit through the narrow PD. Indeed, direct evidence for this model is the involvement of the chaperonin protein folding complex in KN1 trafficking. A chaperonin subunit, CHAPERONIN CONTAINING TAILLESS COMPLEX POLYPEPTIDE-1 8 (CCT8), is required for KN1 movement (Xu et al. 2011). CCT8 is a component of the chaperonin complex, which refolds proteins in an ATP-dependent manner (Clare et al. 2012; Nakagawa et al. 2014). In cct8 mutants, KN1 and STM proteins failed to move from the inner layers of the meristem to the outer layers (Fig. 3c, d). Trafficking was also impaired when the expression of other chaperonin subunits, such as CCT1 or CCT5, was reduced, suggesting that the complete CCT complex is required for PD movement (Xu et al. 2011). The chaperonin complex likely acts in the destination cell, to refold KN1 back to its functional structure (Fig. 3e). Interestingly, cct8 mutants also exhibit a leaf polarity phenotype, hinting that the CCT8/chaperonin complex may also be required for the correct functioning or trafficking of signals involved in leaf polarity (Xu et al. 2011). CCT8 is also required for the spread of viruses, further supporting the similarities between viral MP and KNOX protein trafficking (Kragler 2013). It is still unclear how the unfolded protein is transferred from the PD to the chaperonin to be refolded. Interestingly, CCT8 and other members of the CCT complex are present in a PD proteomic analysis (Fernandez-Calvino et al. 2011), hinting that the complex may be directly associated with the PD. This association would allow the chaperonin complex to grab the unfolded protein as it comes out of the PD channel.
The intrinsic PD-targeting sequences in SHR have also been explored (Gallagher and Benfey 2009). Single and combinations of different SHR domains were fused to GFP and expressed in Arabidopsis. Movement of the fusion proteins was monitored by confocal imaging of the GFP tag, and it was found that the GRAS domain was sufficient to promote movement. Similar to KN1 movement, SHR trafficking also requires its NLS, emphasizing the role of nuclear-cytoplasmic shuttling in PD trafficking of TFs (Gallagher and Benfey 2009). However, while trafficking of KN1 depends on a chaperonin, SHR trafficking does not, since it is not impaired in a chaperonin mutant (Xu et al. 2011). It remains to be determined if other protein folding machineries are required for movement of SHR.
Recent studies suggest a link between SHR movement and the endocytosis pathway (Koizumi et al. 2011; Wu and Gallagher 2013). A novel protein, SHORTROOT INTERACTING EMBRYONIC LETHAL (SIEL), was found to interact directly with SHR. siel mutant plants exhibited reduced SHR movement (Fig. 4b) and had severe root development defects. Thus, SIEL is important for SHR cell-to-cell movement, and because it localizes to both the nuclei and endosomes, it may serve as a shuttle (Wu and Gallagher 2012). By binding to SIEL, SHR could “hitch a ride” on endosomes to reach PDs. A similar mechanism has been proposed for viral protein movements (Lewis and Lazarowitz 2010). Interestingly, SIEL also interacts with other non-cell-autonomous proteins expressed in roots, including AGAMOUS-LIKE 21 (AGL21), TARGET OF MONOPTEROUS (TMO7), and CPC, but not with KN1 or STM (Wu and Gallagher 2012). This suggests that SIEL-dependent PD movement is a specific mechanism for a subset of non-cell-autonomous proteins.
Trichome and Root Hair Patterning Proteins
Trichomes are regularly spaced on the leaf, so that they are not adjacent to one another. This pattern is established by the expression of GL2 specifically in trichome cells, but not in the surrounding cells (Fig. 5a). GL2 expression in the leaf is moderated by TTG1 and TRY. TTG1 is expressed in all epidermal cells, but TTG1 protein accumulates in trichome cells and is depleted in the neighboring cells (Fig. 5b) (Bouyer et al. 2008; Balkunde et al. 2010; Kragler 2013). This suggests that TTG1 traffics from non-trichome cells into trichome cells, possibly via PDs. This idea was supported by microinjection experiments, where it was found that TTG1 could move out of the injected cell into the surrounding cells and also facilitated the mobility of a cell-autonomous fluorescent dye, suggesting TTG1 was able to dilate the PD. In trichome cells, TTG1 is sequestered in the nucleus by GL3. A protein complex containing TTG1 and GL3 promotes the expression of GL2 and TRY. TRY protein moves in the opposite direction, from trichome cells to the neighboring cells, where it suppresses GL2 expression and prevents trichome formation (Fig. 5b) (Ishida et al. 2008). As a result of this complex network, trichomes do not form next to each other.
Although GL3 is cell autonomous in the leaf, it is mobile in the root epidermis and plays a role there in regulating root hair patterning (Ishida et al. 2008). The root epidermis has alternating files of hair and non-hair cells. Hair cells overlie the junction between two cortical cell files, while non-hair cells overlie a single cortical cell file (Fig. 5c). In non-hair cells, GL2 is activated by a complex containing TTG1, GL3, and WEREWOLF (WER). This complex also promotes the expression of CPC in the non-hair cells. In the root hair cells, an unknown positional cue prevents WER from binding GL3. Although CPC transcript is found only in the non-hair cells, CPC protein moves into the root hair cells, where it competes with WER for binding to GL3. Next, the CPC-GL3-TTG1 complex suppresses GL2 expression and defines the non-hair cell fate. Moreover, GL3 traffics from the root hair cells to the non-hair cells, where it further activates GL2 expression, to reinforce the root hair fate (Ishida et al. 2008; Balkunde et al. 2010). Trichome and root hair patterning show the importance of reciprocal cell-to-cell movement of TFs in determining cell fate and spacing.
Analysis of trafficking domains in CPC has revealed that the N-terminus and MYB regions are required for its mobility. While CPC protein is mobile in the leaf and root epidermis, it is immobile when expressed in the stele, indicating that its mobility is tissue specific (Lee and Zhou 2012). CPC mobility is also influenced by its interaction partner, GL3. An elevated GL3 protein level reduces CPC mobility, likely by sequestering CPC in a protein complex (Wester et al. 2009).
FLOWERING LOCUS T (FT)
PDs not only mediate local intercellular communication but also facilitate systemic, long-distance movement by providing access to the phloem vascular system. An example of PD-dependent long-distance trafficking is the movement of the florigen (flowering inducer) protein, FLOWERING LOCUS T (FT). The transition from vegetative to reproductive development is a key process in the development of flowering plants and involves complex coordination between environmental and endogenous signals. FT is expressed specifically in the companion cells (CCs) of the leaf phloem vasculature, but the FT protein moves into sieve elements (SEs) and is delivered into the shoot apex from the phloem by a selective PD pathway (Wu and Gallagher 2012). After reaching the shoot apex, FT interacts with a transcription factor, FLOWERING LOCUS D (FD), to activate APETALA1 (AP1) expression (Abe et al. 2005; Wigge et al. 2005). AP1 triggers signaling pathways that convert the vegetative SAM into an inflorescence meristem, which makes flowers. Expression and long-distance trafficking of FT is regulated by various factors, including day length, temperature, plant age, and hormones, such as gibberellic acid. It was discovered recently that the FT INTERACTING PROTEIN 1 (FTIP1) is required for FT movement from phloem companion cells into sieve elements (Liu et al. 2012). FTIP1 is a putative membrane spanning protein that interacts directly with FT. Because FTIP1 is localized to the ER and PDs, it may associate with the desmotubule (Liu et al. 2012). It is proposed that FTIP1 may play a role in recruiting FT to PDs (Wu and Gallagher 2012; Stahl and Simon 2013). Indeed, loss of FTIP1 caused defects in FT movement and a late flowering phenotype (Liu et al. 2012; Wu and Gallagher 2012).
Roles of Chaperones in PD Trafficking
Chaperones are proteins that promote the proper 3D folding of other proteins and prevent newly synthesized proteins from aggregating. Chaperone machineries are also involved in transporting proteins into various cellular compartments, such as peroxisomes, chloroplasts, mitochondria, and ER (Eckardt 2010). As discussed earlier, the CCT chaperonin complex is important for KN1/STM and TTG1 cell-to-cell trafficking. Another example is HSP70 chaperones from the phloem, which have been shown to associate with PD and are implicated in regulating PD SEL. A C-terminal sequence specific to HSP70s from phloem, but absent from cytosolic HSP70s, makes them competent for cell-to-cell trafficking. Remarkably, an HSP70 homolog has also been found in closterovirus genomes and is essential for translocation of this virus through PD (Pena et al. 2012). HSP70 homologs in Arabidopsis are also found in the PD proteome (Fernandez-Calvino et al. 2011), suggesting that protein trafficking through PD may involve protein conformational changes in combination with increased PD SEL.
In summary, the list of non-cell-autonomous TFs and regulatory proteins is expanding, but the molecular mechanisms regulating protein trafficking through PDs are not fully understood. Based on recent studies, plant biologists have been able to propose and test different working models to gain insights into the mechanisms of this essential phenomenon.
Trafficking of RNAs
Plant viruses hijack the PD pathway to traffic and spread their genomes to promote infection. Recent work has also confirmed that some endogenous RNAs, including protein-coding mRNAs and regulatory sRNAs, function as non-cell-autonomous signaling molecules in plants (Lee and Cui 2009; Hyun et al. 2011; Gursanscky and Carroll 2012). Many recent studies have elegantly demonstrated the significant role of RNA trafficking in coordinating plant development, metabolism, and biotic and abiotic stress responses (Hyun et al. 2011; Gursanscky and Carroll 2012). Although evidence for cell-to-cell and long-distance signaling and regulation of gene expression by RNA molecules is expanding, the mechanisms underlying the trafficking of RNAs are poorly understood. Whether RNA trafficking is an active or a passive process and whether RNAs move alone or in a protein-bound form are active areas of research. Here, the current understanding of RNA trafficking in plants is summarized.
Trafficking of mRNAs
The vascular system in plants provides a pipeline to conduct water and nutrients throughout the plant body. Xylem and phloem are two major components of the vascular system. The xylem primarily transports water and minerals, whereas the phloem transports photosynthate and other organic and inorganic molecules. Asymmetric division of sieve mother cells results in the formation of two distinct cell types in the phloem, the enucleate sieve elements (SEs) and the companion cells (CCs). A special type of PD that is specific to the CC-SE junction connects these two cell types. It is generally believed that CCs supply macromolecules via the PDs that are required for the SEs’ function. The PDs connecting SEs to each other undergo modifications, resulting in an increase in the pore diameter, which helps in the mass flow of the phloem sap.
Support of the idea of mRNA trafficking from CCs to SEs came from the finding that mRNAs of SUCROSE TRANSPORTER 1 (SUT1) were observed in SEs of solanaceous plants, despite these cells lacking a nucleus (Lee and Cui 2009). This observation was further supported by the finding of SUT1 mRNAs in the phloem sap of other plant species. Subsequently, independent studies in Arabidopsis, castor bean, barley, and melon led to the identification of hundreds of mRNAs in the phloem sap (Lee and Cui 2009). Such mRNAs are predicted to encode proteins required for a wide range of functions, including metabolism, biotic and abiotic stress responses, signal transduction, and development. Several of these transcripts, when tested in grafting experiments, showed their capacity to cross the graft junction and even resulted in phenotypes in the specific target tissues and organs (Lee and Zhou 2012). For example, the regulation of leaf development by long-distance trafficking of Mouse ears (Me) mRNA has been demonstrated in grafted tomato plants (Lee and Zhou 2012; Ruiz-Medrano et al. 2012). In Me mutant tomato plants, the Me transcript arises from a spontaneous fusion of a PYROPHOSPHATE-DEPENDENT PHOSPHOFRUCTOKINASE (PFP) gene and LeT6, a KNOX gene. The strong activity of the native PFP promoter results in overexpression of the PFP-LeT6 fusion transcript. Indeed, the Me phenotype resembles the phenotype of transgenic tomato plants that overexpress LeT6. When wild-type shoots (scions) were grafted onto Me mutant rootstocks, the fusion transcript accumulated in the shoot apex and leaf primordia, and the scions displayed altered leaf morphology that was characteristic of the Me mutant (Lee and Zhou 2012; Ruiz-Medrano et al. 2012). The presence of Me mRNA in the phloem of the scion was also detected by in situ hybridization, suggesting that it was mobile.
Another example of mRNA trafficking through phloem is seen for the GRAS family transcription factor GIBBERELLIC ACID-INSENSITIVE (GAI) (Lee and Cui 2009; Lee and Zhou 2012). GAI protein plays an important role as a negative regulator in the gibberellic acid (GA) response. A semidominant gain-of-function phenotype is observed when a mutant version of GAI, lacking the DELLA domain and designated here as gai, is overexpressed (Lee and Zhou 2012; Ruiz-Medrano et al. 2012). The DELLA domain is essential for GA-dependent proteosomal degradation of GAI proteins (Daviere and Achard 2013). Grafting experiments involving tomato and Arabidopsis rootstocks overexpressing gai revealed that gai transcripts could move into wild-type scions. Furthermore, this mobility led to mutant leaf phenotypes in the scion, suggesting gai mRNA exited from the phloem and was translated (Lee and Cui 2009; Lee and Zhou 2012; Ruiz-Medrano et al. 2012). The 3′-UTR and part of the coding region of GAI mRNA are required for GAI mRNA mobility. This region forms a stem-loop structure, suggesting that secondary structure within the RNA facilitates its mobility (Ruiz-Medrano et al. 2012; Hannapel 2013).
In another example, in potato, the BEL1-like TF StBEL5 and its KNOX partner, Potato Homeobox1 (POTH1), regulate hormone levels and tuber formation. StBEL5 and POTH1 bind to the GA20 OXIDASE1 (GA20ox1) promoter and repress its expression (Ruiz-Medrano et al. 2012). StBEL5 overexpression results in increased cytokinin levels and increased tuber yields. Interestingly, StBEL5 mRNA was detected in the phloem, suggesting a function as a long-distance signaling molecule. Grafting experiments confirmed that, indeed, StBEL5 mRNA regulates tuberization by moving through the phloem from leaves to the root. StBEL5 mRNA expression in leaves is induced by light, and its mobility is induced by a short-day photoperiod, suggesting a mechanism by which tuber formation can be controlled by environmental cues (Lee and Cui 2009; Hyun et al. 2011; Hannapel 2013).
Small RNA Biogenesis and Intercellular Movement
Recently, extensive research has been conducted on RNA silencing guided by small regulatory RNAs (sRNAs) . RNA silencing plays essential roles in genome integrity, silencing of transposable elements (TEs), regulation of endogenous gene expression, and defense against viruses (Ruiz-Ferrer and Voinnet 2009; Calarco and Martienssen 2011). A range of experimental approaches, including grafting, virus-induced gene silencing, microinjection, and particle bombardment, have demonstrated that RNA silencing can function in a non-cell-autonomous manner, due to intercellular movement of sRNAs (Voinnet and Baulcombe 1997; Gursanscky and Carroll 2012; Skopelitis et al. 2012). Movement of sRNAs in plants can be either cell to cell (short range) or systemic (long range). sRNA biogenesis and action in plants are mediated by the combined action of DNA-dependent RNA polymerases (RNA Pol II, IV and V), RNA-dependent RNA polymerases (RDRs), dicer-like (DCL) proteins, and argonaute (AGO) proteins (Chen 2009; Molnar et al. 2011; Gursanscky and Carroll 2012; Axtell 2013). All four classes of proteins are represented by multigene families in plants, and the biogenesis of different types of sRNA depends on which specific members from these protein families are involved. MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are the two major classes of sRNAs in plants, and these are classified based on their modes of biogenesis and functions (Chen 2009; Hyun et al. 2011; Molnar et al. 2011).
MicroRNAs are produced from MIR genes, and they function in the regulation of protein-coding genes (Lee and Cui 2009; Hyun et al. 2011; Gursanscky and Carroll 2012). Like protein-coding genes, MIR genes have their own promoters and 3′ terminator regions and are transcribed by Pol II. However, MIR transcripts fold back on themselves to produce an imperfect double-stranded RNA (dsRNA) hairpin structure that is processed by DCL1 into 21–22 nt miRNA duplexes. These duplexes are further processed into mature, single-stranded miRNAs that usually associate with AGO1 to guide silencing of target mRNAs.
In contrast, siRNAs are derived from a longer dsRNA produced by RDRs or by Pol II transcription of inverted repeats (Lee and Cui 2009; Hyun et al. 2011; Gursanscky and Carroll 2012). The primary role of siRNAs is to protect the genome from viruses and transposons. RDRs use single-stranded viral and transposon RNA to synthesize perfectly complementary dsRNAs that are processed into siRNAs by DCL2, DCL3, and DCL4 to produce 22, 24, and 21 nucleotide (nt) siRNAs, respectively. DCL4 and DCL2 produce siRNAs associated with posttranscriptional gene silencing (PTGS), whereas the siRNAs produced by DCL3 are responsible for transcriptional gene silencing (TGS), involving DNA methylation and chromatin modifications. siRNA duplexes associate with one of the ten members of the AGO protein family, where they are converted to mature, single-stranded siRNAs, to guide gene silencing. Silencing of gene expression at the transcriptional level by 24 nt siRNAs usually involves AGO4, whereas PTGS is usually guided by siRNAs in association with AGO1, and involves cleavage of complementary mRNA or blocking of translation (Chen 2009; Lee and Cui 2009; Hyun et al. 2011; Gursanscky and Carroll 2012; Axtell 2013; Dunoyer et al. 2013).
Transacting siRNAs (ta-siRNAs) are a plant-specific class of siRNAs that are produced by components of both the miRNA and siRNA pathways (Yoshikawa 2013). Like miRNAs, ta-siRNAs are involved in the developmental regulation of endogenous gene expression. The source of ta-siRNAs is non-coding TAS transcripts that have been cleaved by a miRNA. The cleaved TAS transcripts are then converted into dsRNA by RDR6, followed by processing into 21 nt ta-siRNAs by DCL4 (Gursanscky and Carroll 2012; Skopelitis et al. 2012). In Arabidopsis, there are eight TAS loci, which are grouped into four families: TAS1–4. While ta-siRNAs derived from TAS1 and TAS2 target several different mRNAs, the ta-siRNAs derived from TAS3 and TAS4 target AUXIN RESPONSE FACTOR (ARF) and MYB TF mRNAs, respectively (Gursanscky and Carroll 2012; Axtell 2013). ta-siRNA production from the TAS3 locus requires the specific action of a miR390/AGO7 complex to cleave the TAS3 transcript, making it a template for dsRNA synthesis by RDR6. This TAS3 dsRNA is then processed by DCL4 to generate tasiR-ARFs, which are 21 nt ta-siRNAs that target ARF2, ARF3, and ARF4 mRNAs (Gursanscky and Carroll 2012; Skopelitis et al. 2012).
The analysis of phloem sap from pumpkin and other species revealed that along with protein-coding mRNAs, small RNAs including miRNAs and siRNAs were also abundant (Lee and Cui 2009; Kehr 2012). This finding suggested a possible role of sRNAs as non-cell-autonomous signaling molecules. Several studies have shown that sRNAs are indeed capable of moving between cells and through the vasculature (Gursanscky and Carroll 2012; Kehr 2012). A reporter system was developed to follow movement of silencing signals out of the phloem companion cells (CCs). As mentioned above, production of 21 nt siRNAs in Arabidopsis requires DCL4. Expression of DCL4 specifically in CCs in a dcl4 mutant background was sufficient to maintain spread of the silencing of target gene expression into mesophyll cells surrounding the vasculature. However, when the viral silencing suppressor protein p19 was expressed in CCs, the intercellular spread of silencing was blocked. p19 is cell autonomous and specifically sequesters 21 nt siRNAs, preventing their movement. These experiments showed that 21 nt siRNAs generated in the CCs can move out of their expression domain, into the surrounding mesophyll cells to silence their mRNA targets (Skopelitis et al. 2012). To ask which form of sRNA was mobile, single- or double-stranded sRNAs were bombarded into leaves, and their ability to induce silencing was assayed. Double- but not single-stranded sRNAs were effective in this assay, suggesting they form the mobile silencing signal (Lee and Cui 2009; Hyun et al. 2011; Gursanscky and Carroll 2012; Axtell 2013; Dunoyer et al. 2013). However, there is also evidence from grafting experiments that single-stranded RNAs could be mobile and their mobility was suggested to transfer epigenetic information through the plant (Hyun et al. 2011; Molnar et al. 2010).
More recently, intercellular movement of sRNAs has been shown to be critical for many functions in higher plants. For example, sRNA mobility plays a role in radial patterning of cell types in roots, in lateral root formation, in leaf polarity, in germline genome integrity, and in gametophyte development, as discussed below (Chen 2009; Xu and Jackson 2010; Calarco and Martienssen 2011; Gursanscky and Carroll 2012; Skopelitis et al. 2012).
Roles of sRNA Mobility in Root and Leaf Development
The class III homeodomain-leucine zipper (HD-ZIP III) TFs are regulators of vascular patterning and development of roots, stems, leaves, and the SAM. In Arabidopsis, this class consists of five members, PHABULOSA (PHB), PHAVOLUTA (PHV), REVOLUTA (REV), CORONA/ATHB15 (CNA), and ATHB8, which have both distinct and overlapping roles in tissue specification. HD-ZIP III TFs are made up of conserved domains, including a DNA-binding homeodomain and a leucine zipper domain involved in dimerization. Crucially, all five HD-ZIP III genes are regulated posttranscriptionally by microRNAs165/166 (miR165/166) (Yamaguchi et al. 2012; Bishopp et al. 2013).
Lateral root formation is also influenced by mobile sRNAs (Gursanscky and Carroll 2012), involving the coordinated function of miR390 and tasiR-ARFs working in concert with auxin signals. miR390 and tasiR-ARFs are produced in the central vascular cylinder of the root, where their expression domains overlap. They then move outwards, in order to repress ARF2, ARF3, and ARF4, clearly showing their non-cell-autonomous function during lateral root development (Gursanscky and Carroll 2012).
While it is clear that tasiR-ARFs move cell to cell to establish leaf polarity, it is not clear whether miR165/166 are also non-cell autonomous. However, considering that miR165/166 are mobile in roots, it is likely that cell-to-cell movement is a necessary component of their function during leaf development.
Further evidence for the role of mobile ta-siRNAs in leaf development came from experiments involving ectopic expression of DCL4, a key component of the ta-siRNA pathway. Mutations in genes required for ta-siRNA production, e.g., in RDR6 or DCL4, result in an epinastic (downward bending) leaf phenotype (Gursanscky and Carroll 2012). The importance of mobility of ta-siRNAs in leaf development was confirmed by an experiment where DCL4 was expressed specifically in the phloem of a dcl4 mutant using the companion cell (CC)-specific SUC2 promoter. This expression was sufficient to rescue the epinastic leaf phenotype, suggesting that ta-siRNAs moved out of the phloem into the leaf primordia to direct leaf development (Gursanscky and Carroll 2012).
Role of sRNA Mobility in Nutrient Signaling
Long-distance signaling by sRNAs is one way in which plants sense the deficiency of certain nutrients. Under sulfate, copper, or phosphate deprivation, there is an elevated level of miR395, miR398, or miR399, respectively, in the phloem (Chen 2009; Hyun et al. 2011; Kehr 2012; Skopelitis et al. 2012), suggesting an involvement of these miRNAs in long-range signaling. Mineral uptake from the soil is achieved by roots, followed by transport through the vascular system to the shoot. However, roots need to respond to deficiencies of these minerals in the shoot and adjust their physiology and development accordingly. For example, there is growing evidence that miR399 functions as a long-distance signal for conveying a phosphate deficiency message from the shoot to the root (Kehr 2012). In Arabidopsis, the PHOSPHATE2 (PHO2) gene is a negative regulator of inorganic phosphate (Pi) uptake; pho2 mutants have an increased uptake and translocation of Pi from roots to the shoot. PHO2 mRNA is a target of miR399, and when Pi starvation occurs in the shoot, miR399 expression is induced, and miR399 moves from the shoot to the root. There, it degrades PHO2 transcripts, thereby increasing Pi uptake and translocation (Hyun et al. 2011; Kehr 2012; Skopelitis et al. 2012). This conclusion is supported by the observation that in response to Pi deficiency, miR399 accumulates in the phloem sap at a much higher level than found in leaves, stems, or roots. Furthermore, miR399 accumulated in rootstocks that were grafted onto shoots overexpressing miR399, suggesting that miR399 moves from shoot to root in this context (Lee and Cui 2009; Skopelitis et al. 2012). Movement of miRNAs from the shoot to target genes regulating uptake of minerals in the roots is an ingenious way in which plants coordinate their systemic mineral needs.
Role of sRNA Signaling During Gamete Formation
Cell-to-cell movement of siRNA has also been shown to play an important role in maintenance of genome integrity in the male germline of Arabidopsis. The male microgametophytes (pollen) contain two sperm cells within a larger vegetative cell. During the process of double fertilization, one of the sperm cells fertilizes the egg cell to produce the embryo, and the second sperm cell fertilizes the central cell in the female gametophyte, to give rise to the endosperm of the seed. Therefore, the vegetative nucleus does not contribute any DNA to the plant body in next generation; nevertheless, it does play a vital role in silencing transposable element (TEs) and maintaining genome integrity in the sperm cells.
DECREASED DNA METHYLATION 1 (DDM1) is a gene required for DNA methylation and maintenance of epigenetic silencing of TEs and heterochromatin in plants (Calarco and Martienssen 2011; Gursanscky and Carroll 2012). During pollen development, DDM1 expression is downregulated specifically in the vegetative nucleus, resulting in the activation of transcription of TEs (Calarco and Martienssen 2011; Gursanscky and Carroll 2012). The abundant transposon-derived transcripts become substrates for PTGS and the generation of 21 nt siRNAs. Remarkably, these siRNAs move from the vegetative cell to sperm cells, to reinforce epigenetic silencing of transposons in these cells, which make up the male germline (Fig. 7b). How the TE siRNAs move from the vegetative cell into the sperm cells is not known. Mobile siRNAs produced in the endosperm during seed development may also contribute to maintenance of genome integrity and TE silencing in the developing embryo (Calarco and Martienssen 2011; Gursanscky and Carroll 2012; Skopelitis et al. 2012).
There is also evidence of cell-to-cell movement of sRNAs contributing to female reproductive development. Female gametophytes (megagametophytes) are formed in a two-step process. First, a single subepidermal cell at the distal tip of the developing ovule differentiates into a megaspore mother cell (MMC). Next, the MMC undergoes meiosis to produce four megaspores, but only one of these survives to produce the functional megaspore, which undergoes three rounds of mitosis to produce the mature female gametophyte (Calarco and Martienssen 2011). Restricting the number of MMCs to one per ovule appears to require cell-to-cell mobility of 24 nt siRNAs associated with ARGONAUTE 9 (AGO9). This role of AGO9 was revealed from the finding that ago9 mutant ovules produce multiple functional megaspores (Chevalier et al. 2011; Gutierrez-Marcos and Dickinson 2012). AGO9 preferentially binds to 24 nt siRNAs produced from TEs. These siRNAs are thought to then move into the subepidermal cells of the ovule with the help of AGO9. It is unclear how this siRNA signaling pathway operates, but it may involve silencing components of the genome and/or heterochromatin reprogramming (Fig. 7c) (Calarco and Martienssen 2011; Gursanscky and Carroll 2012; Skopelitis et al. 2012). These findings have potential applications in the development of apomixis, or clonal reproduction through seeds, which could have remarkable impacts on agriculture.
How Do RNA Molecules Move?
Although it is generally understood that mobile RNAs move cell to cell via PDs, knowledge of the underlying mechanisms is limited. Initial information on viral RNA mobility came from pioneering studies on TMV (Pena et al. 2012). It is well known that the MP of TMV is targeted to PD and is required for the virus to spread from cell to cell. Furthermore, it was demonstrated that TMV MP increases the PD SEL, thereby facilitating the spread of the viral genome as a viral ribonucleoprotein (vRNP) complex (Pena et al. 2012). Subsequent work has shown that viral MPs and/or coat proteins mediate spread of most viruses in plants. While MPs from many virus groups facilitate viral RNA movement through PD, they share little if any sequence similarity. MP-mediated viral genome trafficking can take place by forming tubules through the PD channels, possibly replacing the PD desmotubule, as seen with grapevine fanleaf virus (GFLV) and cauliflower mosaic virus (CaMV) (Schoelz et al. 2011; Burch-Smith and Zambryski 2012; Pena et al. 2012). Alternatively, MPs can facilitate movement via an interaction with ER and severing the actin filaments attached to PD, as has been observed with TMV (Schoelz et al. 2011; Pena et al. 2012).
More recently, it has been found that some viral RNAs, such as those from brome mosaic virus (BMV), can spread throughout plants without the assistance of a coat protein or MP (Ruiz-Medrano et al. 2012). This finding hinted that some viruses might spread solely by exploiting the host trafficking machinery, perhaps by binding to endogenous mobile RNA-binding proteins (RBPs). It is known that structural features in the viral RNA can facilitate intercellular mobility. For example, bamboo mosaic potexvirus (BMP) RNA has a knot-like structure in the 3′ untranslated region (UTR) that resembles tRNAs. This structure might serve as a binding motif for endogenous proteins that facilitate cell-to-cell movement. Indeed, mutations in a gene encoding a chloroplast enzyme, phosphoglycerate kinase, which binds to the 3′ UTR of BMP RNA, resulted in a drastic reduction in viral spread (Ruiz-Medrano et al. 2012).
There is additional evidence that motifs within mRNAs can facilitate their movement through phloem. For example, photoperiod-induced movement of StBEL5 mRNA from leaves to root in potato is regulated by a motif in its 3′ UTR, which is most likely bound by an RNA-binding protein. This motif can promote the mobility of a less mobile homolog of StBEL5, indicating that it is sufficient to promote phloem RNA movement (Takeda and Ding 2009; Hyun et al. 2011).
The picture is not as clear for sRNA, but some studies have indicated that their mobility can be facilitated by specific proteins (Fig. 8). For example, identification of PHLOEM SMALL RNA BINDING PROTEIN 1 (CmPSRP1) in pumpkin phloem provided the first hint of how sRNA molecules are transported into and through the phloem (Gursanscky and Carroll 2012). Microinjection experiments showed that PSRP1 facilitates the intercellular mobility of single-stranded siRNAs. This finding, together with the finding that sRNAs are present in pumpkin phloem, suggests that PSRP1 promotes active transport between CCs and SEs (Gursanscky and Carroll 2012; Ruiz-Medrano et al. 2012). However, PSRP1 homologs have not been found in other plant species, suggesting that additional factors remain to be discovered. The molecular weight of single-stranded and double-stranded their is more than 7 kDa and 14 kDa, respectively. This suggests that an active process is involved in sRNA trafficking, as the SEL of PD is generally thought to be less than this range (Gursanscky and Carroll 2012). However, it is important to note that GFP, with a molecular weight of 27 kDa, is able to move passively between some types of cells, suggesting that there is a possibility of sRNAs diffusing through PD channels (Gursanscky and Carroll 2012).
In summary, while it is known that RNA mobility is required for the physiology and development of plants, research to understand the underlying mechanism is ongoing.
Plasmodesmata in Defense Responses
As discussed earlier, plant viruses exploit the host PD system to facilitate their intercellular spread. The host plant, on the other hand, launches defense responses to viral infection by blocking PD trafficking. Callose deposition at the PD neck is a nonspecific defense mechanism in response to stress. Upon infection, the defense-related hormone salicylic acid (SA) induces deposition of callose in cell walls around PDs, thereby reducing the channel and blocking the spread of viruses (Burch-Smith and Zambryski 2012).
In a recent study, the Arabidopsis protein PDLP5 was shown to mediate cross talk between PD regulation and SA-dependent defense responses. PDLP5 is found in the central region of PDs, where it acts as an inhibitor of PD trafficking, potentially by modulating callose deposition. This ability of PDLP5 to regulate PD permeability in SA-dependent manner correlates with enhanced innate immunity against bacterial pathogens (Lee et al. 2011).
Gene-silencing pathways can also provide a defense mechanism against viruses (Gursanscky and Carroll 2012). It has long been known that plants respond to local virus infection by developing resistance in distant, systemic tissues. Once silencing against a virus is induced, the associated siRNAs can act as mobile silencing signals and confer resistance against the virus throughout the whole plant (Gursanscky and Carroll 2012).
Assays to Analyze PD Trafficking
PD permeability can be assessed by a dye-loading assay using dyes such as carboxy-fluorescein diacetate (CFDA). CFDA is a membrane-permeable and nonfluorescent molecule that easily penetrates the cell membrane. Once inside the cell, CFDA is modified by esterase enzymes, to form carboxy-fluorescein (CF), which is membrane impermeable and fluorescent. Because it is membrane impermeable, any intercellular spread of CF can only be through the symplastic connections provided by PDs. In the Drop-ANd-See (DANS) dye-loading assay, CFDA is loaded onto the upper epidermal surface of an intact leaf. The diffusion of CF is then observed in the lower epidermis, and the diameter of the CF fluorescent area in the lower epidermis can be used as a readout for PD permeability (Lee et al. 2011).
Alternatively, membrane-impermeable fluorescent dyes, such as Lucifer yellow CH (LYCH), can be microinjected into a particular cell (Rinne and van der Schoot 1998). The movement of the dye reveals both the rate and direction of PD transport. This method was used to define the symplasmic fields in the epidermal layer of the birch seedling meristem (Rinne and van der Schoot 1998).
Bombardment of GFP-Fusion Expression Constructs
During viral infection, viral MPs interact with and traffic through PDs. Recombinant GFP-tagged viral MP (GFP-MP) serves as a tool to study PD transport capacity. The DNA encoding GFP-MP is bombarded into a single cell, where the fusion protein is expressed. If PD transport occurs, GFP-MP will spread to neighboring cells. If PD trafficking is blocked, GFP-MP remains in the original cell (Oparka and Boevink 2007). Similar methods have been used to assay movement of endogenous plant proteins, such as KN1, or of GFP, which can move passively through PDs in some tissues.
Trichome Rescue System
Intercellular communication via PD trafficking is essential for plant signaling and development. Studies in recent years have started to reveal important PD structural and regulatory components. Examples discussed in this review demonstrate a great diversity in PD forms and functions in different tissue types and stages of development. Understanding how such a high complexity is achieved will keep researchers busy for many years. Forward genetic and proteomic screens will continue to be effective methods to identify novel PD genes, while advances in super-resolution electron and fluorescence microscopy techniques will provide powerful tools for imaging PD structure and cell-to-cell trafficking.
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