EXO70A1-Mediated Vesicle Trafficking Is Critical for Tracheary Element Development in Arabidopsis

Exocysts are highly conserved octameric complexes that play an essential role in the tethering of Golgi-derived vesicles to target membranes in eukaryotic organisms. Genes encoding the EXO70 subunit are highly duplicated in plants. Based on expression analyses, we proposed previously that individual EXO70 members may provide the exocyst with functional specificity to regulate cell type– or cargo-specific exocytosis, although direct evidence is not available. Here, we show that, as a gene expressed primarily during tracheary element (TE) development, EXO70A1 regulates vesicle trafficking in TE differentiation in Arabidopsis thaliana. Mutations of EXO70A1 led to aberrant xylem development, producing dwarfed and nearly sterile plants with very low fertility, reduced cell expansion, and decreased water potential and hydraulic transport. Grafting of a mutant shoot onto wild-type rootstock rescued most of these aboveground phenotypes, while grafting of a wild-type shoot to the mutant rootstock did not rescue the short root hair phenotype, consistent with the role of TEs in hydraulic transport from roots to shoots. Histological analyses revealed an altered pattern of secondary cell wall thickening and accumulation of large membrane-bound compartments specifically in developing TEs of the mutant. We thus propose that EXO70A1 functions in vesicle trafficking in TEs to regulate patterned secondary cell wall thickening.     

Genome-Wide Characterization of Nonreference Transposons Reveals Evolutionary Propensities of Transposons in Soybean

Preferential accumulation of transposable elements (TEs), particularly long terminal repeat retrotransposons (LTR-RTs), in recombination-suppressed pericentromeric regions seems to be a general pattern of TE distribution in flowering plants. However, whether such a pattern was formed primarily by preferential TE insertions into pericentromeric regions or by selection against TE insertions into euchromatin remains obscure. We recently investigated TE insertions in 31 resequenced wild and cultivated soybean (Glycine max) genomes and detected 34,154 unique nonreference TE insertions mappable to the reference genome. Our data revealed consistent distribution patterns of the nonreference LTR-RT insertions and those present in the reference genome, whereas the distribution patterns of the nonreference DNA TE insertions and the accumulated ones were significantly different. The densities of the nonreference LTR-RT insertions were found to negatively correlate with the rates of local genetic recombination, but no significant correlation between the densities of nonreference DNA TE insertions and the rates of local genetic recombination was detected. These observations suggest that distinct insertional preferences were primary factors that resulted in different levels of effectiveness of purifying selection, perhaps as an effect of local genomic features, such as recombination rates and gene densities that reshaped the distribution patterns of LTR-RTs and DNA TEs in soybean.     

Patterning and Lifetime of Plasma Membrane-Localized Cellulose Synthase Is Dependent on Actin Organization in Arabidopsis Interphase Cells

The actin and microtubule cytoskeletons regulate cell shape across phyla, from bacteria to metazoans. In organisms with cell walls, the wall acts as a primary constraint of shape, and generation of specific cell shape depends on cytoskeletal organization for wall deposition and/or cell expansion. In higher plants, cortical microtubules help to organize cell wall construction by positioning the delivery of cellulose synthase (CesA) complexes and guiding their trajectories to orient newly synthesized cellulose microfibrils. The actin cytoskeleton is required for normal distribution of CesAs to the plasma membrane, but more specific roles for actin in cell wall assembly and organization remain largely elusive. We show that the actin cytoskeleton functions to regulate the CesA delivery rate to, and lifetime of CesAs at, the plasma membrane, which affects cellulose production. Furthermore, quantitative image analyses revealed that actin organization affects CesA tracking behavior at the plasma membrane and that small CesA compartments were associated with the actin cytoskeleton. By contrast, localized insertion of CesAs adjacent to cortical microtubules was not affected by the actin organization. Hence, both actin and microtubule cytoskeletons play important roles in regulating CesA trafficking, cellulose deposition, and organization of cell wall biogenesis.Plant cells are surrounded by a flexible yet durable extracellular matrix that makes up the cell wall. This structure offers mechanical strength that counters osmotically driven turgor pressure, is an important factor for water movement in plants, acts as a physical barrier against pathogens (Somerville et al., 2004), and is a determining factor for plant cell morphogenesis. Hence, the cell wall plays a central role in plant biology.Two main types of cell walls can typically be distinguished: the primary and the secondary cell wall. The major load-bearing component in both of these cell walls is the β-1,4-linked glucan polymer cellulose (Somerville et al., 2004). Cellulose polymers are synthesized by plasma membrane (PM)-localized cellulose synthase (CesA) complexes (Mueller and Brown, 1980), which contain several CesA subunits with similar amino acid sequences (Mutwil et al., 2008a). The primary wall CesA complexes are believed to be assembled in the Golgi and are subsequently delivered to the PM via vesicular trafficking (Gutierrez et al., 2009), sometimes associated with Golgi pausing (Crowell et al., 2009). Furthermore, the primary wall CesA complexes are preferentially inserted into the PM at sites that coincide with cortical microtubules (MTs), which subsequently guide cellulose microfibril deposition (Gutierrez et al., 2009). Hence, the cortical MT array is a determinant for multiple aspects of primary wall cellulose production.The actin cytoskeleton plays a crucial role in organized deposition of cell wall polymers in many cell types, including cellulose-related polymers and pectins in tip-growing cells, such as pollen tubes and root hairs (Hu et al., 2003; Chen et al., 2007). Thus, actin-depolymerizing drugs and genetic manipulation of ACTIN genes impair directed expansion of tip-growing cells and long-distance transport of Golgi bodies with vesicles to growing regions (Ketelaar et al., 2003; Szymanski, 2005). In diffusely growing cells in roots and hypocotyls, loss of anisotropic growth has also been observed in response to mutations to vegetative ACTIN genes and to actin-depolymerizing and -stabilizing drugs (Baluska et al., 2001; Kandasamy et al., 2009). While actin is clearly important for cell wall assembly, it is less clear what precise roles it plays.One well-known function of actin in higher plants is to support intracellular movement of cytoplasmic organelles via actomyosin-based motility (Geisler et al., 2008; Szymanski, 2009). During primary wall synthesis in interphase cells, treatment with the actin assembly inhibitor latrunculin B (LatB) led to inhibition of Golgi motility and pronounced inhomogenities in CesA density at the PM (Crowell et al., 2009; Gutierrez et al., 2009) that coincided with the density of underlying and immobile Golgi bodies (Gutierrez et al., 2009). These results suggested that Golgi motility is important for CesA distribution (Gutierrez et al., 2009). The actin cytoskeleton also appears to be important for secondary wall cellulose microfibril deposition. For example, longitudinal actin filaments (AFs) define the movement of secondary wall CesA-containing Golgi bodies in developing xylem vessels (Wightman and Turner, 2008). In addition, it has been proposed that the AFs also can regulate the delivery of the secondary wall CesA complex to the PM via pausing of the Golgi (Wightman and Turner, 2008). It is therefore clear that actin organization is important for CesA distribution and for the pattern of cellulose microfibril deposition.Despite the above findings, very few reports have undertaken detailed studies to elucidate the role of the actin cytoskeleton in the distribution and trafficking of specific proteins in plant cells. Here, we have investigated the intracellular trafficking of CesA-containing vesicles and delivery of CesAs to the PM, in the context of the actin cytoskeleton. We quantitatively demonstrate that the organization of the actin cytoskeleton regulates CesA-containing Golgi distribution and the exocytic and endocytic rate of the CesAs. However, actin organization has no effect on the localized insertion of CesAs at sites of MTs at the PM.     

Distribution of Transglutaminase in Pear Pollen Tubes in Relation to Cytoskeleton and Membrane Dynamics

Transglutaminases (TGases) are ubiquitous enzymes that take part in a variety of cellular functions. In the pollen tube, cytoplasmic TGases are likely to be involved in the incorporation of primary amines at selected peptide-bound glutamine residues of cytosolic proteins (including actin and tubulin), while cell wall-associated TGases are believed to regulate pollen tube growth. Using immunological probes, we identified TGases associated with different subcellular compartments (cytosol, membranes, and cell walls). Binding of cytosolic TGase to actin filaments was shown to be Ca²⁺ dependent. The membrane TGase is likely associated with both Golgi-derived structures and the plasma membrane, suggesting a Golgi-based exocytotic delivery of TGase. Association of TGase with the plasma membrane was also confirmed by immunogold transmission electron microscopy. Immunolocalization of TGase indicated that the enzyme was present in the growing region of pollen tubes and that the enzyme colocalizes with cell wall markers. Bidimensional electrophoresis indicated that different TGase isoforms were present in distinct subcellular compartments, suggesting either different roles or different regulatory mechanisms of enzyme activity. The application of specific inhibitors showed that the distribution of TGase in different subcellular compartments was regulated by both membrane dynamics and cytoskeleton integrity, suggesting that delivery of TGase to the cell wall requires the transport of membranes along cytoskeleton filaments. Taken together, these data indicate that a cytoplasmic TGase interacts with the cytoskeleton, while a different TGase isoform, probably delivered via a membrane/cytoskeleton-based transport system, is secreted in the cell wall of pear (Pyrus communis) pollen tubes, where it might play a role in the regulation of apical growth.Transglutaminases (TGases [EC 2.3.2.13]; protein-Gln γ-glutamyltransferase) are a family of ubiquitous Ca²⁺-activated enzymes that are involved in animal cell morphogenesis and differentiation, apoptosis, cell death, inflammation, cell migration, and wound healing (Griffin et al., 2002; Mehta et al., 2006; Beninati et al., 2009). TGases are associated with different subcellular compartments, such as cytosol, plasma membrane, nucleus, mitochondria, and extracellular matrix. The specific localization of TGases is likely to determine both the biochemical activity and the type of proteins and/or substrates with which TGases react (Park et al., 2010). The distribution profile of TGase is affected by Ca²⁺, since the enzyme is preferentially associated with the lysosome compartment of liver cells in the absence of Ca²⁺ (Juprelle-Soret et al., 1984).TGase was initially detected in association with the cytosol, with the particulate (probably the microsomal) fraction (Birckbichler et al., 1976), and with the nucleus of animal cells (Remington and Russell, 1982). The association of TGase with the plasma membrane was related to its activity in promoting cell adhesion and to the interaction of cells with the extracellular matrix, while the presence of TGase in the nucleus is likely related to cell apoptosis (Griffin et al., 2002). How TGase is delivered to its final destination in animal cells remains to be clarified. Since the cytoskeleton is essential for the correct positioning of proteins in the cells, this interplay has often been studied in terms of potential substrates of TGase activity (Griffin et al., 2002). For example, the TGase-mediated incorporation of polyamines (PAs) stimulates actin polymerization (Takashi, 1988; Griffin et al., 2002). TGase was also found to associate with myosin in stress fibers of vascular smooth cells (Chowdhury et al., 1997). The association between TGase and microtubules (MTs) was initially studied in view of the importance of MTs in Alzheimer’s disease (Griffin et al., 2002), whereas the dynamics of MTs is also likely to be controlled by TGase (Al-Jallad et al., 2011). Interestingly, MTs are also a substrate of TGase activity in cells committed to apoptosis (Piredda et al., 1999). TGase was also shown to posttranslationally modify MT-associated proteins such as tau (Griffin et al., 2002).Information about the localization and function of TGases in plant cells is limited. Following the early evidence of an enzyme-based incorporation of PAs in plants (Serafini-Fracassini et al., 1988), a number of reports described the presence and role of TGase in nonphotosynthetic/photosynthetic tissues and in isolated chloroplasts (Serafini-Fracassini and Del Duca, 2008, and refs. therein). Attempts have also been made to examine the differences and similarities between plant and animal TGases. For example, a tobacco (Nicotiana tabacum) TGase was proposed to be involved in the programmed cell death (PCD) of the flower corolla (Della Mea et al., 2007); in such a case, TGase is likely to be released into the cell wall by a Golgi vesicle-based transport. Plant TGases might also be involved in protection against viruses (Del Duca et al., 2007) and in the self-incompatibility (SI) response involving pollen and stigma during sexual reproduction (Del Duca et al., 2010). Recently, different TGase isoforms were detected in meristematic apices of Jerusalem artichoke (Helianthus tuberosus) tuber sprouts (Beninati et al., 2013).The pollen tube is a widely investigated tip-growing plant cell (Lee and Yang, 2008). Studies are generally aimed at clarifying the many aspects related either to its growth or to rejection by the stigma/style. Early evidence for a role of PAs during pollen tube emergence (Bagni et al., 1981) was confirmed through the detection of PA binding via a Ca²⁺-activated TGase activity (Del Duca et al., 1997) and later by the identification of actin and tubulin as substrates of purified pollen TGase (Del Duca et al., 2009). In pollen, the enzyme affected the polymerization state and activity of actin filaments (AFs) and MTs (Del Duca et al., 2009) and existed as both soluble and cell wall associated (Di Sandro et al., 2010). Visualization of fluorescently labeled TGase products indicated that the cross-linking activity of TGase occurred at the apex of pollen tubes, in a basal region close to the pollen grain and within the pollen grain itself (Iorio et al., 2008). The enzyme was found as a soluble cytoplasmic form likely involved in the regulation of unspecified physiological processes (possibly associated with the cytoskeleton; Del Duca et al., 2009).Although the association of pollen TGases with organelles/vesicles has not been reported, an extracellular form of a Ca²⁺-dependent TGase was shown to be involved in pollen tube growth (likely as a modulator of cell wall building and strengthening). Moreover, pollen TGase was secreted in the incubation medium during germination, where it might catalyze the cross linking of PAs with secreted proteins (Di Sandro et al., 2010). This suggests that pollen TGase may be secreted through a vesicle-based mechanism. Finally, a TGase activity was also observed in planta, consistent with a possible role of TGase during tube migration through the style (Di Sandro et al., 2010) or in the SI response of pollen tubes (Del Duca et al., 2010).The pollen tube is an excellent model to study how a given plant protein is either secreted or delivered to its final destination. Although we know that actin and tubulin are substrates of TGase activity, and that the active enzyme is located in the cell wall and released outside, how TGase is distributed in the cells and how this process is dependent on cytoskeleton and membrane dynamics remain unknown. Here, we wanted to study in detail the localization and distribution of TGase in growing pollen tubes of pear (Pyrus communis) in relation to both cytoskeleton and membrane dynamics. The aim was to shed light on the mechanism by which TGase is transported and secreted, a process that is still not well understood even in animal cells. Specific antibodies that cross react with the TGase of pollen tubes were used to localize the enzyme in different membrane compartments and in the cell wall. The use of specific inhibitors indicated that the delivery of extracellular TGase is dependent on both AFs and membrane dynamics. Analysis by bidimensional electrophoresis (2-DE) showed that distinct TGase isoforms are associated with different cell compartments, suggesting that TGase might be differently regulated according to its position in the cell. Together, these data may contribute to our understanding of the mechanisms underlying pollen tube growth, an essential aspect of fertilization processes.     

Glycosylphosphatidylinositol (GPI) Modification Serves as a Primary Plasmodesmal Sorting Signal

Plasmodesmata (Pd) are membranous channels that serve as a major conduit for cell-to-cell communication in plants. The Pd-associated β-1,3-glucanase (BG_pap) and CALLOSE BINDING PROTEIN1 (PDCB1) were identified as key regulators of Pd conductivity. Both are predicted glycosylphosphatidylinositol-anchored proteins (GPI-APs) carrying a conserved GPI modification signal. However, the subcellular targeting mechanism of these proteins is unknown, particularly in the context of other GPI-APs not associated with Pd. Here, we conducted a comparative analysis of the subcellular targeting of the two Pd-resident and two unrelated non-Pd GPI-APs in Arabidopsis (Arabidopsis thaliana). We show that GPI modification is necessary and sufficient for delivering both BG_pap and PDCB1 to Pd. Moreover, the GPI modification signal from both Pd- and non-Pd GPI-APs is able to target a reporter protein to Pd, likely to plasma membrane microdomains enriched at Pd. As such, the GPI modification serves as a primary Pd sorting signal in plant cells. Interestingly, the ectodomain, a region that carries the functional domain in GPI-APs, in Pd-resident proteins further enhances Pd accumulation. However, in non-Pd GPI-APs, the ectodomain overrides the Pd targeting function of the GPI signal and determines a specific GPI-dependent non-Pd localization of these proteins at the plasma membrane and cell wall. Domain-swap analysis showed that the non-Pd localization is also dominant over the Pd-enhancing function mediated by a Pd ectodomain. In conclusion, our results indicate that segregation between Pd- and non-Pd GPI-APs occurs prior to Pd targeting, providing, to our knowledge, the first evidence of the mechanism of GPI-AP sorting in plants.Plant cells are interconnected with cross-wall membranous channels called plasmodesmata (Pd). Recent studies have shown that the region of the plasma membrane (PM) lining the Pd channel is a specialized membrane microdomain whose lipid and protein composition differs from the rest of the PM (Tilsner et al., 2011, 2016; Bayer et al., 2014; González-Solís et al., 2014; Grison et al., 2015). In a similar manner, the cell wall domain surrounding the Pd channel is specialized and, unlike the rest of the cell wall, is devoid of cellulose, rich in pectin, and contains callose (an insoluble β-1,3-glucan; Zavaliev et al., 2011; Knox and Benitez-Alfonso, 2014). In response to physiological signals, callose can be transiently deposited and degraded at Pd, which provides a mechanism for controlling the Pd aperture in diverse developmental and stress-related processes (Zavaliev et al., 2011). Control of Pd functioning is mediated by proteins that are specifically targeted to Pd. Plasmodesmal proteins localized to the PM domain of Pd can be integral transmembrane proteins, such as Pd-localized proteins (Thomas et al., 2008), the receptor kinase ARABIDOPSIS CRINKLY4 (Stahl et al., 2013), and callose synthases (Vatén et al., 2011). Alternatively, Pd proteins can associate with the membrane through a lipid modification like myristoylation (e.g. remorins; Raffaele et al., 2009) or be attached by a glycosylphosphatidylinositol (GPI) anchor (e.g. Pd-associated β-1,3-glucanases [BG_pap]; Levy et al., 2007; Rinne et al., 2011; Benitez-Alfonso et al., 2013), Pd-associated callose-binding proteins (PDCBs; Simpson et al., 2009), and LYSIN MOTIF DOMAIN-CONTAINING PROTEIN2 (LYM2; Faulkner et al., 2013).Among the known Pd proteins involved in Pd-specific callose degradation is BG_pap, a cell wall enzyme carrying a glycosyl hydrolase family 17 (GH17) module as its functional domain (Levy et al., 2007). Another group of proteins controlling callose dynamics at Pd are PDCBs that harbor a callose-binding domain termed carbohydrate-binding module 43 (CBM43), implicated in stabilizing callose at Pd (Simpson et al., 2009). Some β-1,3-glucanases may combine the two callose-modifying activities by harboring both GH17 and CBM43 functional domains, and several such proteins were shown to localize to Pd (Rinne et al., 2011; Benitez-Alfonso et al., 2013; Gaudioso-Pedraza and Benitez-Alfonso, 2014). A distinct feature of BG_pap and PDCBs is that both are predicted glycosylphosphatidylinositol-anchored proteins (GPI-APs). The GPI anchor is a form of posttranslational modification common to many cell surface proteins in all eukaryotes. GPI-APs are covalently attached to the outer leaflet of the PM through the GPI anchor. The basic structure of the anchor consists of ethanolamine phosphate, followed by a glycan chain of three Man residues and glucosamine, followed by phosphatidylinositol lipid moiety (EtNP-6Manα1-2Manα1-6Manα1-4GlcNα1-6myoinositol-1-P-lipid; Ferguson et al., 2009). All predicted GPI-APs carry an N-terminal secretion signal peptide (SP) similar to other secreted proteins. Distinctly, GPI-APs also carry a structurally conserved 25- to 30-residue C-terminal GPI attachment signal, which typically begins with a small amino acid (e.g. Ala, Asn, Asp, Cys, Gly, or Ser) termed omega, followed by a spacer region of five to 10 polar residues, and ending with a transmembrane segment of 15 to 20 hydrophobic residues (Ferguson et al., 2009). The entire region between the N-terminal and the C-terminal signals of a GPI-AP is termed the ectodomain and carries the protein’s functional domain(s). The GPI modification process takes place in the lumenal face of the endoplasmic reticulum (ER) in a cotranslational manner. Upon translocation into the ER, a GPI-AP is stabilized in the ER membrane by its C-terminal signal, which is concurrently cleaved after the omega amino acid, and a preassembled GPI anchor is covalently attached to the C terminus of the omega amino acid. After attachment to a protein, the GPI anchor undergoes a series of modifications (remodeling), both at the glucan chain and at the lipid moiety. Such remodeling is crucial for the sorting of GPI-APs in the secretory pathway and the subsequent lateral heterogeneity at the PM (Kinoshita, 2015). In particular, the addition of saturated fatty acid chains to the lipid moiety of the anchor leads to the enriched accumulation of GPI-APs in the PM microdomains, also termed lipid rafts (Muñiz and Zurzolo, 2014). In Arabidopsis (Arabidopsis thaliana), GPI modification has been predicted for 210 proteins of diverse functions at the PM or the cell wall or both (Borner et al., 2002). Despite extensive research on the GPI modification pathway and the function of GPI-APs in mammalian and yeast cells, such knowledge in plant systems is scarce. In particular, despite an emerging role of GPI-APs in the regulation of the cell wall domain of Pd, their subcellular targeting and compartmentalization mechanism have not been studied. In addition, it is not known how the targeting mechanism of Pd-resident GPI-APs is different from that of other classes of GPI-APs, which are not localized to Pd.In this study, we investigated the subcellular targeting mechanism of Pd-associated callose-modifying GPI-APs, BG_pap and PDCB1, and compared it with that of two unrelated non-Pd GPI-APs, ARABINOGALACTAN PROTEIN4 (AGP4) and LIPID TRANSFER PROTEIN1 (LTPG1). Using sequential fluorescent labeling of protein domains, we found that the C-terminal GPI modification signal present in both Pd- and non-Pd GPI-APs can function as a primary signal in targeting proteins to the Pd-enriched PM domain. Moreover, we show that while the GPI signal is sufficient for Pd targeting, the ectodomains in BG_pap and PDCB1 further enhance their accumulation at Pd. In contrast, the ectodomains in non-Pd GPI-APs mediate exclusion of the proteins from the Pd-enriched targeting pathway. The Pd exclusion effect was found to be dominant over the Pd-targeting function of the GPI signal and the Pd-enhancing function of the Pd ectodomain, and it possibly occurs prior to PM localization. Our findings thus uncover a novel Pd-targeting signal and provide, to our knowledge, the first evidence of the cellular mechanism that regulates the sorting of GPI-APs in plants.     

The Apoplastic Copper AMINE OXIDASE1 Mediates Jasmonic Acid-Induced Protoxylem Differentiation in Arabidopsis Roots

Polyamines are involved in key developmental processes and stress responses. Copper amine oxidases oxidize the polyamine putrescine (Put), producing an aldehyde, ammonia, and hydrogen peroxide (H₂O₂). The Arabidopsis (Arabidopsis thaliana) amine oxidase gene At4g14940 (AtAO1) encodes an apoplastic copper amine oxidase expressed at the early stages of vascular tissue differentiation in roots. Here, its role in root development and xylem differentiation was explored by pharmacological and forward/reverse genetic approaches. Analysis of the AtAO1 expression pattern in roots by a promoter::green fluorescent protein-β-glucuronidase fusion revealed strong gene expression in the protoxylem at the transition, elongation, and maturation zones. Methyl jasmonate (MeJA) induced AtAO1 gene expression in vascular tissues, especially at the transition and elongation zones. Early protoxylem differentiation was observed upon MeJA treatment along with Put level decrease and H₂O₂ accumulation in wild-type roots, whereas Atao1 loss-of-function mutants were unresponsive to the hormone. The H₂O₂ scavenger N,N¹-dimethylthiourea reversed the MeJA-induced early protoxylem differentiation in wild-type seedlings. Likewise, Put, which had no effect on Atao1 mutants, induced early protoxylem differentiation in the wild type, this event being counteracted by N,N¹-dimethylthiourea treatment. Consistently, AtAO1-overexpressing plants showed lower Put levels and early protoxylem differentiation concurrent with H₂O₂ accumulation in the root zone where the first protoxylem cells with fully developed secondary wall thickenings are found. These results show that the H₂O₂ produced via AtAO1-driven Put oxidation plays a role in MeJA signaling leading to early protoxylem differentiation in root.Root development is affected by several environmental stresses that may result in the inhibition of root growth and/or the modulation of differentiation pattern. It is not surprising, then, that a complex network of hormonal signals control root architecture under either physiological or stress growth conditions, since in changing environments plants take advantage of root developmental plasticity. Thus, it is reasonable that root growth and vascular development can be either conveniently coordinated or selectively modulated in growing roots depending on specific plant needs, in order to ensure the appropriate water absorption and nutrient uptake in heterogenous soils with varying resource availability.During root vascular development, pericycle/vascular meristematic stem cells differentiate into procambial cell lineages, including protoxylem and metaxylem, intervening procambium, phloem, and pericycle (Mähönen et al., 2006; Petricka et al., 2012). Coordinated events of secondary cell wall deposition and programmed cell death (PCD) characterize the last stage of both protoxylem and metaxylem vessel maturation (Ohashi-Ito and Fukuda, 2010). It is well known that, under physiological conditions, an array of auxin, cytokinin, and brassinosteroid signaling pathways participate in root tissue differentiation (Petricka et al., 2012; Mähönen et al., 2014). Specifically, it has been proposed that vascular patterning is finely regulated by a feedback loop between auxin and cytokinin signaling pathways occurring through mutual inhibition (Bishopp et al., 2011; Perilli et al., 2012; Petricka et al., 2012). Brassinosteroids have also been shown to induce root growth and promote xylem differentiation by driving the entry of xylem precursors into the final stage of tracheary element differentiation (Yamamoto et al., 1997). Recently, reactive oxygen species (ROS) have been described to play a key role in the transition from cell proliferation to tissue differentiation in the root (Tsukagoshi et al., 2010), independently from the auxin/cytokinin feedback loop mentioned above. Indeed, while superoxide anion is required to maintain cell proliferation in the meristem, hydrogen peroxide (H₂O₂) is required for tissue differentiation in the elongation/differentiation zone.However, less attention has been devoted to xylem differentiation under stress growth conditions, when resource availability and/or water supply may be restrictive, creating the need for a rearrangement of root architecture and vascular differentiation. In this regard, an alteration of the temporal pattern of xylem differentiation was observed in roots of soybean (Glycine max) plants upon saline stress, with a delay in primary xylem differentiation and a precocious formation of secondary xylem (Hilal et al., 1998). Moreover, significant anatomical changes were observed to occur in roots of Agave salmiana under water stress, among them a reduction of vessel number and an increase of xylem diameter and wall thickness (Peña-Valdivia and Sánchez-Urdaneta, 2009). The rearrangement of root vascular tissues has also been reported to occur as a defense reaction against pathogen invasion, such as the regeneration of xylem vessels observed in a Fusarium spp. wilt-resistant carnation (Dianthus caryophyllus ‘Novada’) upon fungal infection to compensate for local vascular dysfunction (Baayen, 1986) as well as the vascular tissue redifferentiation revealed in Arabidopsis (Arabidopsis thaliana) plants following nematode invasion in order to counteract mechanical pressure (Møller et al., 1998). Moreover, xylem regeneration around a wound has been described in maize (Zea mays) seedling stems (Aloni and Plotkin, 1985).Of note, previous studies reported that the stress signaling hormone jasmonic acid (JA), while inducing root growth inhibition (Ren et al., 2009), behaves as a promoter of early vascular tissue differentiation (Cenzano et al., 2003) and xylogenesis (Fattorini et al., 2009). The role of JA in vascular tissue differentiation was first revealed in stolons of potato (Solanum tuberosum) during the tuberization process. In particular, exogenous JA accelerated potato tuber formation via the induction of both cell expansion and early differentiation of protoxylem vessels with ring-shaped secondary wall thickenings, leading to increased movement of nutrients toward the stolon tip (Cenzano et al., 2003). Moreover, exogenous methyl jasmonate (MeJA) was reported to enhance the formation of adventitious roots and the development of xylogenic nodules in tobacco (Nicotiana tabacum) thin layers under root-inductive hormonal conditions (Fattorini et al., 2009).The polyamines (PAs) putrescine (Put), spermidine (Spd), and spermine (Spm) are small aliphatic polycations ubiquitous in living organisms and essential for cell growth, proliferation, and differentiation (Tavladoraki et al., 2012). In plants, PAs have been involved in a multiplicity of developmental processes as well as stress responses and tolerance strategies, their intracellular and extracellular levels varying in response to different physiological and pathological conditions (Mattoo et al., 2010). A fine regulation of their metabolism and/or transport ensures the occurrence of the appropriate PA levels depending on the specific cell needs (Tavladoraki et al., 2012). Oxidative deamination of PAs is catalyzed by amine oxidases (AOs) in a multistep mechanism, with the release of the removed amine moiety and amino aldehydes in the oxidative phase and the production of H₂O₂ in the reoxidation step of the reduced enzyme (Tavladoraki et al., 2012). Although AOs are a heterogenous class of enzymes varying in subcellular localization, tissue expression pattern, substrate specificity, and mode of catalysis, they share roles in both the homeostasis of PAs and the production of H₂O₂, the latter representing a common product in the AO-driven oxidative catabolism of PAs (Cona et al., 2006; Tavladoraki et al., 2012). On the basis of the cofactor involved, AOs can be classified into two subclasses: the copper amine oxidases (CuAOs), showing high affinity for Put, and the FAD-dependent polyamine oxidases (PAOs), whose preferred substrates are Spd, Spm, and/or their acetyl derivatives (Cona et al., 2006; Tavladoraki et al., 2012). In Arabidopsis, five PAO genes (AtPAOs) and 10 CuAO genes (AtCuAOs) were identified by database search and in some cases characterized at the protein level (Fincato et al., 2011; Planas-Portell et al., 2013; Ahou et al., 2014; Kim et al., 2014). Among CuAO genes, At4g14940 (The Arabidopsis Information Resource [TAIR] accession no. 2129519), here designed as AtAO1 (formerly ATAO1; Møller and McPherson, 1998), encodes an extracellular protein found in apoplastic fluids of Arabidopsis rosettes, as demonstrated by mass spectrometry analysis (Boudart et al., 2005).H₂O₂ derived from the extracellular catabolism of PAs by cell wall-localized AOs has been shown to be involved in both developmental processes, such as the light-induced inhibition of mesocotyl growth (Cona et al., 2003) and the PCD occurring in differentiating tracheary elements (Tisi et al., 2011b), as well as defense responses during wound healing (Angelini et al., 2008), salt stress (Moschou et al., 2008), and pathogen attack (Moschou et al., 2009). In this regard, AOs have also been suggested to act as stress-responsive genes whose expression strongly increases in response to both pathogen infection and abiotic stresses (Moschou et al., 2008; Tavladoraki et al., 2012). During the plant response to stresses, a faster apoplastic oxidation of PAs has been supposed to occur, allowed by the concurrent increase of PA secretion and catabolism in the cell wall, and the PA-derived H₂O₂ has been demonstrated to trigger signal transduction pathways leading to the induction of defense gene expression, stress tolerance, or PCD (Moschou et al., 2008; Tisi et al., 2011a). Recently, the dual role of PAs as either signaling compounds or the source of the second messenger H₂O₂ has been highlighted, and it has been hypothesized that AOs may have a role in PA/H₂O₂ balance (Moschou et al., 2008; Tisi et al., 2011a, 2011b). In fact, the coordinated modulation of PA metabolism and secretion in the cell wall may represent a crucial mechanism in the control of the PA-H₂O₂ ratio, which has been suggested to be a significant player in fixing cell fate and behavior under stress conditions (Moschou et al., 2008; Tisi et al., 2011a).It is worth noting that the H₂O₂ derived from the apoplastic PA catabolism has been shown to be involved in JA-dependent wound signaling pathways, behaving as a mediator of cell wall-stiffening events during wound healing (Cona et al., 2006; Angelini et al., 2008). Moreover, it has been reported recently that PA-derived H₂O₂ inhibits root growth and promotes xylem differentiation, inducing both cell wall-stiffening events and developmental PCD (Tisi et al., 2011a, 2011b). Indeed, Spd treatment in maize or overexpression of maize PAO (ZmPAO) in the cell wall of tobacco plants induced early differentiation and precocious cell death of xylem precursors along with enhanced in vivo H₂O₂ production in xylem tissues of maize and tobacco root apex, respectively (Tisi et al., 2011a, 2011b). Owing to the high rate of apoplastic Spd catabolism supposed to occur upon Spd supply or PAO overexpression, it has been suggested that, in such unphysiological status, plants may experience stress-like conditions under which the AO-driven H₂O₂ production may have a role in promoting xylem differentiation (Tisi et al., 2011a).Taking into account that AtAO1 is expressed at the early stages of vascular tissue development in Arabidopsis roots (Møller et al., 1998; Møller and McPherson, 1998), we explored the possibility that the cell wall-localized AtAO1 could be involved in JA signaling, leading to the induction of root xylem differentiation by means of both pharmacological and forward/reverse genetic approaches. Our results show that Atao1 loss-of-function mutants (TAIR accession nos. 1005841762 and 4284859) are unresponsive to MeJA signaling leading to root protoxylem differentiation. Conversely, AtAO1 overexpression leads to early protoxylem differentiation along with enhanced H₂O₂ production in the root zone where the first protoxylem cells with fully developed secondary wall thickenings can be observed. Overall, our data show that H₂O₂ produced via AtAO1-driven Put oxidation behaves as a mediator in JA-induced root xylem differentiation.Moreover, the data presented here suggest that Put-derived H₂O₂ may play a role in xylem differentiation under stress growth conditions such as those signaled by MeJA or simulated by either Put treatment or AtAO1 overexpression.     

A Developmental Framework for Complex Plasmodesmata Formation Revealed by Large-Scale Imaging of the Arabidopsis Leaf Epidermis

Plasmodesmata (PD) form tubular connections that function as intercellular communication channels. They are essential for transporting nutrients and for coordinating development. During cytokinesis, simple PDs are inserted into the developing cell plate, while during wall extension, more complex (branched) forms of PD are laid down. We show that complex PDs are derived from existing simple PDs in a pattern that is accelerated when leaves undergo the sink–source transition. Complex PDs are inserted initially at the three-way junctions between epidermal cells but develop most rapidly in the anisocytic complexes around stomata. For a quantitative analysis of complex PD formation, we established a high-throughput imaging platform and constructed PDQUANT, a custom algorithm that detected cell boundaries and PD numbers in different wall faces. For anticlinal walls, the number of complex PDs increased with increasing cell size, while for periclinal walls, the number of PDs decreased. Complex PD insertion was accelerated by up to threefold in response to salicylic acid treatment and challenges with mannitol. In a single 30-min run, we could derive data for up to 11k PDs from 3k epidermal cells. This facile approach opens the door to a large-scale analysis of the endogenous and exogenous factors that influence PD formation.     

Cytoplasmic Nucleation and Atypical Branching Nucleation Generate Endoplasmic Microtubules in Physcomitrella patens

The mechanism underlying microtubule (MT) generation in plants has been primarily studied using the cortical MT array, in which fixed-angled branching nucleation and katanin-dependent MT severing predominate. However, little is known about MT generation in the endoplasm. Here, we explored the mechanism of endoplasmic MT generation in protonemal cells of Physcomitrella patens. We developed an assay that utilizes flow cell and oblique illumination fluorescence microscopy, which allowed visualization and quantification of individual MT dynamics. MT severing was infrequently observed, and disruption of katanin did not severely affect MT generation. Branching nucleation was observed, but it showed markedly variable branch angles and was occasionally accompanied by the transport of nucleated MTs. Cytoplasmic nucleation at seemingly random locations was most frequently observed and predominated when depolymerized MTs were regrown. The MT nucleator γ-tubulin was detected at the majority of the nucleation sites, at which a single MT was generated in random directions. When γ-tubulin was knocked down, MT generation was significantly delayed in the regrowth assay. However, nucleation occurred at a normal frequency in steady state, suggesting the presence of a γ-tubulin-independent backup mechanism. Thus, endoplasmic MTs in this cell type are generated in a less ordered manner, showing a broader spectrum of nucleation mechanisms in plants.     

Dynamics and Organization of Cortical Microtubules as Revealed by Superresolution Structured Illumination Microscopy

Plants employ acentrosomal mechanisms to organize cortical microtubule arrays essential for cell growth and differentiation. Using structured illumination microscopy (SIM) adopted for the optimal documentation of Arabidopsis (Arabidopsis thaliana) hypocotyl epidermal cells, dynamic cortical microtubules labeled with green fluorescent protein fused to the microtubule-binding domain of the mammalian microtubule-associated protein MAP4 and with green fluorescent protein-fused to the alpha tubulin6 were comparatively recorded in wild-type Arabidopsis plants and in the mitogen-activated protein kinase mutant mpk4 possessing the former microtubule marker. The mpk4 mutant exhibits extensive microtubule bundling, due to increased abundance and reduced phosphorylation of the microtubule-associated protein MAP65-1, thus providing a very useful genetic tool to record intrabundle microtubule dynamics at the subdiffraction level. SIM imaging revealed nano-sized defects in microtubule bundling, spatially resolved microtubule branching and release, and finally allowed the quantification of individual microtubules within cortical bundles. Time-lapse SIM imaging allowed the visualization of subdiffraction, short-lived excursions of the microtubule plus end, and dynamic instability behavior of both ends during free, intrabundle, or microtubule-templated microtubule growth and shrinkage. Finally, short, rigid, and nondynamic microtubule bundles in the mpk4 mutant were observed to glide along the parent microtubule in a tip-wise manner. In conclusion, this study demonstrates the potential of SIM for superresolution time-lapse imaging of plant cells, showing unprecedented details accompanying microtubule dynamic organization.Plant cell growth and differentiation depend on dynamic cortical microtubule organization mechanisms (Ehrhardt, 2008). Such mechanisms include branched microtubule formation and release (Murata et al., 2005; Nakamura et al., 2010; Fishel and Dixit, 2013), microtubule-templated microtubule growth (Chan et al., 2009), angle-of-contact microtubule bundling or catastrophe induction (Dixit and Cyr, 2004; Tulin et al., 2012), severing at microtubule crossovers (Wightman and Turner, 2007), and unique dynamic behavior between steady-state treadmilling and dynamic instability (Shaw et al., 2003).Cortical microtubule dynamics have been studied in vivo and in vitro with total internal reflection microscopy (TIRFM; Vizcay-Barrena et al., 2011), variable-angle emission microscopy (VAEM; Wan et al., 2011), spinning-disc microscopy (SD; Shaw and Lucas, 2011), and confocal laser scanning microscopy (CLSM; Shaw et al., 2003). TIRFM and VAEM provide sufficient resolution and speed but at limited depth of imaging (approximately 200 nm; Martin-Fernandez et al., 2013) and inevitably a very narrow field of view when used for in vivo studies (Mattheyses et al., 2010). Dynamic CLSM imaging suffers from field-of-view limitations while also introducing phototoxicity to the imaged sample. Furthermore, CLSM is based on a speed-to-resolution tradeoff that will necessitate computational extrapolation to bring resolution to affordable levels (Rosero et al., 2014). Finally, SD can provide sufficient depth and speed but otherwise poor resolution, owing to aberrations arising from the sample and the properties of the optics commonly used (Shaw and Ehrhardt, 2013).Microtubule research evolved concomitant with optical microscopy and the development of fluorescent proteins markers, allowing the resolution of microtubule dynamics and organization at video rates (Marc et al., 1998; Shaw and Ehrhardt, 2013). However, the bulk of plant cells organized in tissues and the optical properties of cell walls hamper microscopic observations, so that the delineation of fine details of microtubule organization still relies on laborious transmission electron microscopy (Kang, 2010).Alternatively, in vitro assays using total internal reflection (TIRFM) or Allen’s video-enhanced contrast-differential interference contrast microscopy (Allen et al., 1981) with purified components have advanced the understanding of microtubule-microtubule-associated protein (MAP) interactions while providing mechanistic insight on the function of MAP65 proteins (Tulin et al., 2012; Portran et al., 2013; Stoppin-Mellet et al., 2013), kinesin motors (Song et al., 1997), katanin-mediated microtubule severing (Stoppin-Mellet et al., 2007), and microtubule dynamics (Moore et al., 1997). However, it is explicitly acknowledged that such in vitro assays should be addressed in biologically coherent systems with physiological relevance to microtubule dynamics (Gardner et al., 2013; Zanic et al., 2013). Thus, an ideal approach would be to address microtubule dynamics in the complex cellular environment at spatiotemporal resolutions achieved by in vitro assays.Subdiffraction optical microscopy techniques allow subcellular observations below Abbe’s resolution threshold (Verdaasdonk et al., 2014), complementing the use of transmission electron microscopy. Such approaches permit dynamic subcellular tracking of appropriately tagged structures within living cells (Tiwari and Nagai, 2013). Practically, two superresolution strategies exist. The first involves patterned light illumination, allowing superresolution acquisitions by two fundamentally different methods, stimulated emission depletion (STED; Hell, 2007) and structured illumination microscopy (SIM; Gustafsson, 2000). The second interrogates the precision of fluorophore localization and includes stochastic optical reconstruction microscopy (STORM; Kamiyama and Huang, 2012) and photoactivation localization microscopy (PALM; Sengupta et al., 2012). The above regimes differ in translational and axial resolution, and their temporal efficiency depends on the size of the imaged area. SIM is probably the best compromise for superresolution live imaging, as it offers reasonable lateral resolution (approximately 100 nm; Gustafsson, 2000), which may be reduced to 50 nm (Rego et al., 2012), and sufficient depth of imaging combined with a reasonable axial resolution (approximately 200 nm). SIM allows dynamic imaging in a broader field of view than STED, at biologically meaningful rates compared with PALM and STORM (Kner et al., 2009), and with deeper imaging capacity compared with other superresolution regimes and with TIRFM/VAEM (Leung and Chou, 2011). Superresolution approaches have received limited attention in the plant cell biology field (Fitzgibbon et al., 2010; Kleine-Vehn et al., 2011), and their resolution potential during live imaging was not quantified previously.Here, high-numerical aperture (NA) objectives were combined with SIM for the acquisition and systematic quantification of subdiffraction details of cortical microtubules labeled either with GFP fused to the microtubule-binding domain of mammalian MAP4 (GFP-MBD; Marc et al., 1998) or with GFP fused to alpha tubulin6 (GFP-TUA6; Shaw et al., 2003). For such studies, wild-type plants and a mitogen-activated protein kinase4 (mpk4) mutant, exhibiting extensive microtubule bundling due to the overexpression and underphosphorylation of MAP65-1 (Beck et al., 2010), were used.     

The Phragmoplast-Orienting Kinesin-12 Class Proteins Translate the Positional Information of the Preprophase Band to Establish the Cortical Division Zone in Arabidopsis thaliana

The preprophase band (PPB) is a faithful but transient predictor of the division plane in somatic cell divisions. Throughout mitosis the PPBs positional information is preserved by factors that continuously mark the division plane at the cell cortex, the cortical division zone, by their distinct spatio-temporal localization patterns. However, the mechanism maintaining these identity factors at the plasma membrane after PPB disassembly remains obscure. The pair of kinesin-12 class proteins PHRAGMOPLAST ORIENTING KINESIN1 (POK1) and POK2 are key players in division plane maintenance. Here, we show that POK1 is continuously present at the cell cortex, providing a spatial reference for the site formerly occupied by the PPB. Fluorescence recovery after photobleaching analysis combined with microtubule destabilization revealed dynamic microtubule-dependent recruitment of POK1 to the PPB during prophase, while POK1 retention at the cortical division zone in the absence of cortical microtubules appeared static. POK function is strictly required to maintain the division plane identity factor TANGLED (TAN) after PPB disassembly, although POK1 and TAN recruitment to the PPB occur independently during prophase. Together, our data suggest that POKs represent fundamental early anchoring components of the cortical division zone, translating and preserving the positional information of the PPB by maintaining downstream identity markers.