**2.3 The endocytic pathway**

*Electrodialysis*

identified thus far.

*2.2.1 RAB GTPases*

The diverse functions of plant vacuoles are fulfilled through tight regulation of trafficking to and from the vacuoles, which involves evolutionarily conserved machinery components including Rab GTPases [79]. However, the basic framework of the Rab GTPase action is well conserved in eukaryotic cells [80]. Recent comparative genomic studies suggest that each eukaryotic lineage has acquired a unique repertoire of Rab GTPases during evolution [81] Autophagy-related and Golgi-independent transport from the ER to the vacuole is another example of such trafficking pathway [82]. This pathway also involves an exocyst subcomplex, although Rab and SNARE molecules associated with this pathway have not been

RAB GTPases constitute the largest family of small GTPases; 57 members are encoded in the *Arabidopsis* genome [83]. Based on their similarity to animal RAB GTPases, RAB GTPases are grouped into eight clades, i.e., RAB1/RABD, RAB2/ RABB, RAB5/RABF, RAB6/RABH, RAB7/ RABG, RAB8/RABE, RAB11/RABA, and RAB18/RABC in angiosperms [84]. Plants also harbor a unique set of Rab GTPases partly characterized by diversification of the RAB5 group acting in endosomal/ vacuolar trafficking pathways [85]. RAB7 is also proposed to regulate vacuolar traffic in plants [86]. In animal cells, a sequential action of RAB5 and RAB7 mediated by the effector complex HOPS [18] and a guanine nucleotide exchange factor for RAB7 consisting of SAND1/Mon1 and CCZ1 is responsible for the maturation from

Future studies on the molecular mechanisms of these plant-specific vacuolar trafficking pathways will reveal how plants have used unique vacuolar trafficking routes and how plants have developed their unique vacuolar trafficking pathways during evolution. The tethering of transport vesicles to the target membranes is mediated by the interaction between RAB GTPases and specific sets of tethering factors, many of which have been shown to be RAB effectors, which bind to specific RABs at the GTP-bound active state in yeast and animal systems [80]. After the tethering of transport vesicles by the tethering factors, soluble N-ethylmaleimidesensitive-factor attachment protein receptors lead to the membrane fusion [80]. Tethering factors comprise long coiled-coil proteins and protein complexes called

*RAB and the other proteins in intracellular trafficking (modified from Malaria Parasite Metabolic* 

early to late endosomal compartments (**Figure 4**) [86].

**30**

**Figure 4.**

*Pathways [47]).*

Endocytosis in plant cells has an essential role for basic cellular functions and communication with the environment. Through the formation of closed membrane vesicles (60–120 nm), the uptake of extracellular molecules or the internalization of plasma membrane lipids and proteins is achieved [37]. During the life cycle of the plant, endosomes have vital importance for different processes including lateral organ differentiation, hormone signal transduction, root hair formation, embryo patterning, and plant immunity [36, 87–92]. Transportation of various cargo molecules involved in a broad range of physiological processes from the plasma membrane into the cytoplasm is achieved by this pathway. **Figure 5** shows the general organization of the endosomal trafficking system in plants [36].

As in animal cells, endocytosis in plant cells is mediated by (i) *clathrin-mediated* (CME) and (ii) *clathrin-independent pathways* (CIE) [93].

## *2.3.1 Clathrin-mediated pathway*

In plants, the major endocytic mechanism depends on the coat protein clathrin. This pathway starts at the plasma membrane by clathrin-coated vesicle formation. CME is important for different physiological processes involving cell signaling, cell adhesion, nutrient uptake, developmental regulation, etc. The pathway starts by clathrin-coated vesicle formation at the plasma membrane; in the cytosolic parts of different transmembrane cargo molecules, the clathrin coat binds to specific binding sites. The recruitment of the pioneer proteins to the plasma membrane is ensured by the cargo molecules and enhanced by the initiation of an endocytic pathway. Clathrin involved in a variety of other processes such as the salt stress response, the defense response, cryptogein-induced signaling, and cytokinesis [94–96].

#### **Figure 5.**

*The general organization of the endosomal trafficking system in plants. Current models of retromer localization and function place the retromer recycling complex in the TGN, in the MVB, or both [36].*

Many viruses act as endocytic cargoes for the entry into the cell [97]. A large number of "coat-associated clathrin adaptor proteins" and "scaffold proteins" serve as cargo adaptors by interaction with specific cargoes. Short linear sequence motifs or covalent modifications such as phosphorylation or ubiquitylation in cargo proteins are important for cargo adaptor interactions [98]. Most cargo adaptors also interact directly with lipids and with other coat proteins. These complex interactions cause the initiation of clathrin coat assembly and its further expansion [99]. Binding of the clathrin coat proteins to the cytosolic sites of different transmembrane cargo molecules is essential for the cargo recruitment to the region of the plasma membrane that will form the vesicle.

Because of the difficulty of visualizing and manipulating, studies on cargo and lipids are very difficult [100]. The most widely studied one is the maintenance of polar localization of auxin transporters, namely, the PIN proteins. Polar localization of PIN proteins involves three steps: (i) nonpolar secretion, (ii) clathrin-dependent endocytosis, and (iii) polar recycling [91]. Dynamic regulation of PIN polarity provided with this mechanism is important in response to environmental and developmental stimuli [101, 102]. Mutations in Rab5 or clathrin cause endocytosis disruption and auxin-related developmental defects [91]. Endocytosis works as a negative feedback of the signaling process. After internalization, flagellin-sensitive 2 (FLS2) is targeted for degradation by ubiquitination which then terminates the signal transduction process [103, 104]. The polarly localized *Arabidopsis* boron transporter 1(BOR1), the tomato ethylene-inducing xylanse receptor (LeEIX2), and the iron transporter 1 (IRT1) are the other plasma membrane cargoes in plant cells [92, 105]. For accurate development and growth regulation, the equilibrium of cargo localization in the endomembrane system and the dynamic trafficking machinery is essential during the plant life cycle.

#### *2.3.2 Clathrin-independent pathway*

Several endocytic pathways that do not use clathrin-coated vesicles are involved in a CI pathway that was mediated by caveolae. Some of these pathways are constitutive, whereas others are activated by specific signals or by pathogens [106]. Furthermore, their mechanisms and kinetics of endocytic vesicle formation, associated molecular machinery, and cargo direction are different. Some members of the ARF and Rho subfamilies of small GTPases have been suggested to have key roles in regulating different pathways of CI endocytosis [107]. CI pathways are grouped in terms of those that use a "dynamin-mediated scission mechanism" (dynamindependent) and those that require other processes (dynamin-independent). A second characteristic is a contribution of small GTPases in several CI pathways [108].

#### **2.4 The endomembrane system in plant development and plant defense**

The dysfunction of the endomembrane system can affect plant development and signal transduction [109, 110]. The interaction between the actin cytoskeleton and the endomembrane system involves various aspects of plant cell function and development [111–113].

The actin cytoskeleton is involved for the dynamic feature of the ER [114]. Microtubules have been reported to also influence the mobility of the ER, but to a lesser degree or at a much slower rate [115]. ARF1 plays an essential role in normal cell growth, plant development, and cell polarity and is ubiquitously expressed in all organs of *Arabidopsis* [116, 117]. In de-etiolated pea shoots, ARF1 was concentrated mainly in the crude Golgi fractions [38]. Antisense RNA studies show that ARF also affects cell expansion and cell size in *Arabidopsis* [118]. BFA-visualized

**33**

**3. Conclusions**

*Endomembrane Trafficking in Plants*

*DOI: http://dx.doi.org/10.5772/intechopen.91642*

ing the activity of the ARF GTPase [122].

exocytic trafficking defective1 (BEX1) has been reported to require for recycling of PIN transporters and auxin-mediated development in *Arabidopsis* [52]. BEX1 encodes ARF1A1C which localizes to the TGN/EE and Golgi apparatus. For normal venation patterning, polar auxin transport by PIN1 is required [118, 119]. Vascular network defective 4 (VAN4) is required for cellular growth and venation development [120]. VAN4 encodes a putative TRS120 subunit of the TRAPPII complex protein that functions as a Rab-GEF and/or tethering factor [121]. VAN4 is involved in polar localization and the recycling of PIN proteins. VAN3/SFC, ARF-GAP, and VAN7/GNOM ARF-GEF have been reported to regulate venation pattern by regulat-

Another relation with endomembrane trafficking and plant development was revealed by the continuous vascular ring mutants (COV1). Parker et al. have reported that the COV1 mutant is involved in ectopic differentiation of vascular *Arabidopsis* cells [123]. Afterward, COV1 has been reported as a TGN-localized membrane protein that is required for Golgi morphology and vacuolar protein traf-

The ubiquitin-proteasome system (UPS) is important for the cytosolic and nuclear protein degradation, whereas certain proteins are degraded by autophagic degradation. De-ubiquitylating enzymes (DUBs) are essential for endosomal trafficking by affecting the fate of endocytosed cargo [125]. Endosomal sorting complexes required for transport (ESCRT) components are crucial for plant growth and development. Mutations of ESCRT or ESCRT-associated proteins in plants lead to ubiquitin accumulation, embryonic and seedling lethality, and misregulation of different signaling pathways, which can be associated with endosomal sorting defects in *Arabidopsis* [10, 126]. ESCRT mutations in *Arabidopsis* cause it to die at different developmental stages [127]. Under optimal growth conditions, autophagy seems to be unessential for plant life cycle. But a lack of autophagy can be the reason of the carcinogenesis and neurodegenerative diseases in the mammalian system [128]. Plants protect themselves with the help of small RNA-dependent immune system in response to biotic stress [129]. sRNAs are short regulatory RNAs (20–30 nucleotides) that silence genes with complementary sequences [130]. Against pathogens, several groups of plant sRNAs have important roles in plant defense. Plants send sRNAs in extracellular vesicles (exosomes) to the pathogen to silence virulence genes [130–132]. Host *Arabidopsis* cells have been shown to secrete exosome-like extracellular vesicles to deliver sRNAs into fungal pathogen *Botrytis cinerea*. These sRNA-containing vesicles accumulate at the infection sites of plant and are occupied up by the fungal cells. Transferred host sRNAs cause silencing of virulence-related genes critical for pathogenicity. Plant extracellular vesicles, mainly exosomes, have been reported to play a crucial role in cross-kingdom sRNA

trafficking between *Arabidopsis* and the fungal pathogen *B. cinerea* [129].

The functional organization of eukaryotic cells requires the exchange of proteins, lipids, and polysaccharides between membrane compartments through transport intermediates. Transport from one compartment of this pathway to another is mediated by vesicular carriers, which are formed by the controlled assembly of coat protein complexes (COPs) on donor organelles. The plant endomembrane system is mostly conserved among eukaryotes but shows complex features. The structural organization of the endomembrane system is important for correct membrane trafficking and plant physiology. The trans-Golgi network (TGN) is a unique subcellular structure, which is a sorting center that integrates upstream cargoes

ficking and for the development of myrosin cells in leaves [124].

#### *Endomembrane Trafficking in Plants DOI: http://dx.doi.org/10.5772/intechopen.91642*

*Electrodialysis*

brane that will form the vesicle.

machinery is essential during the plant life cycle.

*2.3.2 Clathrin-independent pathway*

development [111–113].

Many viruses act as endocytic cargoes for the entry into the cell [97]. A large number of "coat-associated clathrin adaptor proteins" and "scaffold proteins" serve as cargo adaptors by interaction with specific cargoes. Short linear sequence motifs or covalent modifications such as phosphorylation or ubiquitylation in cargo proteins are important for cargo adaptor interactions [98]. Most cargo adaptors also interact directly with lipids and with other coat proteins. These complex interactions cause the initiation of clathrin coat assembly and its further expansion [99]. Binding of the clathrin coat proteins to the cytosolic sites of different transmembrane cargo molecules is essential for the cargo recruitment to the region of the plasma mem-

Because of the difficulty of visualizing and manipulating, studies on cargo and lipids are very difficult [100]. The most widely studied one is the maintenance of polar localization of auxin transporters, namely, the PIN proteins. Polar localization of PIN proteins involves three steps: (i) nonpolar secretion, (ii) clathrin-dependent endocytosis, and (iii) polar recycling [91]. Dynamic regulation of PIN polarity provided with this mechanism is important in response to environmental and developmental stimuli [101, 102]. Mutations in Rab5 or clathrin cause endocytosis disruption and auxin-related developmental defects [91]. Endocytosis works as a negative feedback of the signaling process. After internalization, flagellin-sensitive 2 (FLS2) is targeted for degradation by ubiquitination which then terminates the signal transduction process [103, 104]. The polarly localized *Arabidopsis* boron transporter 1(BOR1), the tomato ethylene-inducing xylanse receptor (LeEIX2), and the iron transporter 1 (IRT1) are the other plasma membrane cargoes in plant cells [92, 105]. For accurate development and growth regulation, the equilibrium of cargo localization in the endomembrane system and the dynamic trafficking

Several endocytic pathways that do not use clathrin-coated vesicles are involved in a CI pathway that was mediated by caveolae. Some of these pathways are constitutive, whereas others are activated by specific signals or by pathogens [106]. Furthermore, their mechanisms and kinetics of endocytic vesicle formation, associated molecular machinery, and cargo direction are different. Some members of the ARF and Rho subfamilies of small GTPases have been suggested to have key roles in regulating different pathways of CI endocytosis [107]. CI pathways are grouped in terms of those that use a "dynamin-mediated scission mechanism" (dynamindependent) and those that require other processes (dynamin-independent). A second characteristic is a contribution of small GTPases in several CI pathways [108].

**2.4 The endomembrane system in plant development and plant defense**

The dysfunction of the endomembrane system can affect plant development and signal transduction [109, 110]. The interaction between the actin cytoskeleton and the endomembrane system involves various aspects of plant cell function and

The actin cytoskeleton is involved for the dynamic feature of the ER [114]. Microtubules have been reported to also influence the mobility of the ER, but to a lesser degree or at a much slower rate [115]. ARF1 plays an essential role in normal cell growth, plant development, and cell polarity and is ubiquitously expressed in all organs of *Arabidopsis* [116, 117]. In de-etiolated pea shoots, ARF1 was concentrated mainly in the crude Golgi fractions [38]. Antisense RNA studies show that ARF also affects cell expansion and cell size in *Arabidopsis* [118]. BFA-visualized

**32**

exocytic trafficking defective1 (BEX1) has been reported to require for recycling of PIN transporters and auxin-mediated development in *Arabidopsis* [52]. BEX1 encodes ARF1A1C which localizes to the TGN/EE and Golgi apparatus. For normal venation patterning, polar auxin transport by PIN1 is required [118, 119]. Vascular network defective 4 (VAN4) is required for cellular growth and venation development [120]. VAN4 encodes a putative TRS120 subunit of the TRAPPII complex protein that functions as a Rab-GEF and/or tethering factor [121]. VAN4 is involved in polar localization and the recycling of PIN proteins. VAN3/SFC, ARF-GAP, and VAN7/GNOM ARF-GEF have been reported to regulate venation pattern by regulating the activity of the ARF GTPase [122].

Another relation with endomembrane trafficking and plant development was revealed by the continuous vascular ring mutants (COV1). Parker et al. have reported that the COV1 mutant is involved in ectopic differentiation of vascular *Arabidopsis* cells [123]. Afterward, COV1 has been reported as a TGN-localized membrane protein that is required for Golgi morphology and vacuolar protein trafficking and for the development of myrosin cells in leaves [124].

The ubiquitin-proteasome system (UPS) is important for the cytosolic and nuclear protein degradation, whereas certain proteins are degraded by autophagic degradation. De-ubiquitylating enzymes (DUBs) are essential for endosomal trafficking by affecting the fate of endocytosed cargo [125]. Endosomal sorting complexes required for transport (ESCRT) components are crucial for plant growth and development. Mutations of ESCRT or ESCRT-associated proteins in plants lead to ubiquitin accumulation, embryonic and seedling lethality, and misregulation of different signaling pathways, which can be associated with endosomal sorting defects in *Arabidopsis* [10, 126]. ESCRT mutations in *Arabidopsis* cause it to die at different developmental stages [127]. Under optimal growth conditions, autophagy seems to be unessential for plant life cycle. But a lack of autophagy can be the reason of the carcinogenesis and neurodegenerative diseases in the mammalian system [128].

Plants protect themselves with the help of small RNA-dependent immune system in response to biotic stress [129]. sRNAs are short regulatory RNAs (20–30 nucleotides) that silence genes with complementary sequences [130]. Against pathogens, several groups of plant sRNAs have important roles in plant defense. Plants send sRNAs in extracellular vesicles (exosomes) to the pathogen to silence virulence genes [130–132]. Host *Arabidopsis* cells have been shown to secrete exosome-like extracellular vesicles to deliver sRNAs into fungal pathogen *Botrytis cinerea*. These sRNA-containing vesicles accumulate at the infection sites of plant and are occupied up by the fungal cells. Transferred host sRNAs cause silencing of virulence-related genes critical for pathogenicity. Plant extracellular vesicles, mainly exosomes, have been reported to play a crucial role in cross-kingdom sRNA trafficking between *Arabidopsis* and the fungal pathogen *B. cinerea* [129].
