Plant cytokinesis and the construction of new cell wall.

Cytokinesis in plants is fundamentally different from that in animals and fungi. In plant cells, a cell plate forms through the fusion of cytokinetic vesicles and then develops into the new cell wall, partitioning the cytoplasm of the dividing cell. The formation of the cell plate entails multiple stages that involve highly orchestrated vesicle accumulation, fusion and membrane maturation, which occur concurrently with the timely deposition of polysaccharides such as callose, cellulose and cross-linking glycans. This review summarizes the major stages in cytokinesis, endomembrane components involved in cell plate assembly and its transition to a new cell wall. An animation that can be widely used for educational purposes further summarizes the process.

Isolation and Glycomic Analysis of Trans-Golgi Network Vesicles in Plants.

The dynamic endomembrane system facilitates sorting and transport of diverse cargo. Therefore, it is crucial for plant growth and development. Vesicle proteomic studies have made substantial progress in recent years. In contrast, much less is known about the identity of vesicle compartments that mediate the transport of polysaccharides to and from the plasma membrane and the types of sugars they selectively transport. In this chapter, we provide a detailed description of the protocol used for the elucidation of the SYP61 vesicle population glycome. Our methodology can be easily adapted to perform glycomic studies of a broad variety of plant cell vesicle populations defined via subcellular markers or different treatments.

AtTRAPPC11 is involved in TRAPPIII mediated control of post-Golgi protein trafficking.

The plant trans-Golgi Network/Early Endosome (TGN/EE), as an organizer of vesicle trafficking, fulfills a crucial role for plant development and adaptation. Because it coordinates the transport of cell material along different routes, it is expected that a number of TGN/EE associated factors function in the rapid organization of post-Golgi trafficking to ensure that proteins reach their destination. The roles of Transport Protein Particle (TRAPP) complexes in the regulation of plant post-Golgi trafficking start to emerge. We previously demonstrated that the plant TRAPPIII complex is involved in maintenance of TGN organization and function and has a role in endocytic trafficking mediated by the SYP61 TGN/EE compartment. Here we show that attrappc11 mutants display accumulation of the plasma membrane resident proteins CESA6, BRI1 and PIP1;4 in aberrant intracellular compartments. This adds further insights into the functions of TRAPPIII as a regulators of post-Golgi/endosomal traffic.

Cytokinin functions as an asymmetric and anti-gravitropic signal in lateral roots.

Directional organ growth allows the plant root system to strategically cover its surroundings. Intercellular auxin transport is aligned with the gravity vector in the primary root tips, facilitating downward organ bending at the lower root flank. Here we show that cytokinin signaling functions as a lateral root specific anti-gravitropic component, promoting the radial distribution of the root system. We performed a genome-wide association study and reveal that signal peptide processing of Cytokinin Oxidase 2 (CKX2) affects its enzymatic activity and, thereby, determines the degradation of cytokinins in natural Arabidopsis thaliana accessions. Cytokinin signaling interferes with growth at the upper lateral root flank and thereby prevents downward bending. Our interdisciplinary approach proposes that two phytohormonal cues at opposite organ flanks counterbalance each other's negative impact on growth, suppressing organ growth towards gravity and allow for radial expansion of the root system.

AtTRAPPC11/ROG2: A Role for TRAPPs in Maintenance of the Plant Trans-Golgi Network/Early Endosome Organization and Function.

The dynamic trans-Golgi network/early endosome (TGN/EE) facilitates cargo sorting and trafficking and plays a vital role in plant development and environmental response. Transport protein particles (TRAPPs) are multi-protein complexes acting as guanine nucleotide exchange factors and possibly as tethers, regulating intracellular trafficking. TRAPPs are essential in all eukaryotic cells and are implicated in a number of human diseases. It has been proposed that they also play crucial roles in plants; however, our current knowledge about the structure and function of plant TRAPPs is very limited. Here, we identified and characterized AtTRAPPC11/RESPONSE TO OLIGOGALACTURONIDE2 (AtTRAPPC11/ROG2), a TGN/EE-associated, evolutionarily conserved TRAPP protein in Arabidopsis (Arabidopsis thaliana). AtTRAPPC11/ROG2 regulates TGN integrity, as evidenced by altered TGN/EE association of several residents, including SYNTAXIN OF PLANTS61, and altered vesicle morphology in attrappc11/rog2 mutants. Furthermore, endocytic traffic and brefeldin A body formation are perturbed in attrappc11/rog2, suggesting a role for AtTRAPPC11/ROG2 in regulation of endosomal function. Proteomic analysis showed that AtTRAPPC11/ROG2 defines a hitherto uncharacterized TRAPPIII complex in plants. In addition, attrappc11/rog2 mutants are hypersensitive to salinity, indicating an undescribed role of TRAPPs in stress responses. Overall, our study illustrates the plasticity of the endomembrane system through TRAPP protein functions and opens new avenues to explore this dynamic network.

Post-Golgi Trafficking and Transport of Cell Wall Components.

The cell wall, a complex macromolecular composite structure surrounding and protecting plant cells, is essential for development, signal transduction, and disease resistance. This structure is also integral to cell expansion, as its tensile resistance is the primary balancing mechanism against internal turgor pressure. Throughout these processes, the biosynthesis, transport, deposition, and assembly of cell wall polymers are tightly regulated. The plant endomembrane system facilitates transport of polysaccharides, polysaccharide biosynthetic and modifying enzymes and glycoproteins through vesicle trafficking pathways. Although a number of enzymes involved in cell wall biosynthesis have been identified, comparatively little is known about the transport of cell wall polysaccharides and glycoproteins by the endomembrane system. This review summarizes our current understanding of trafficking of cell wall components during cell growth and cell division. Emerging technologies, such as vesicle glycomics, are also discussed as promising avenues to gain insights into the trafficking of structural polysaccharides to the apoplast.

Plant TGN in the stress response: a compartmentalized overview.

The cellular responses to abiotic and biotic stress rely on the regulation of vesicle trafficking to ensure the correct localization of proteins specialized in sensing stress stimuli and effecting the response. Several studies have implicated the plant trans-Golgi network (TGN)-mediated trafficking in different types of biotic and abiotic stress responses; however, the underlying molecular mechanisms are poorly understood. Further, the identity, specialization and stress-relevant cargo transported by the TGN subcompartments involved in stress responses await more in depth characterization. This review presents TGN trafficking players implicated in stress and discusses potential avenues to understand the role of this dynamic network under such extreme circumstances.

PIN7 Auxin Carrier Has a Preferential Role in Terminating Radial Root Expansion in Arabidopsis thaliana.

Directional growth of lateral roots is critical for radial expansion and soil coverage. Despite its importance, almost nothing is known about its molecular determinants. Initially, young lateral roots (LRs) grow away from the parental root, maintaining the angle acquired shortly after emergence. A second downwards bending response to gravity terminates the so-called plateau phase and thereby limits radial root expansion. Here, we show that the exit from the plateau phase correlates with an increase in auxin signalling at the tip of the LRs. Moreover, the increase in auxin levels induces the termination of the plateau phase, which requires PIN-FORMED (PIN) auxin efflux carriers. Our data suggests that the developmental increase in auxin triggers the preferential derepression of PIN7 in gravity-sensing columella cells. The subsequent polarization of PIN7 heralds the bending towards gravity and, hence, the exit from the plateau phase. This developmental framework reveals the distinct roles of PIN auxin efflux carriers in controlling the radial growth of root systems.

Halotropism: turning down the salty date.

Plants have a deep-rooted trust in gravity, but it is not unconditional. A new study shows that, if plant roots sense high doses of salt coming up from below, they dump gravity responses and grow away from the salt contamination.

An auxin transport mechanism restricts positive orthogravitropism in lateral roots.

As soon as a seed germinates, plant growth relates to gravity to ensure that the root penetrates the soil and the shoot expands aerially. Whereas mechanisms of positive and negative orthogravitropism of primary roots and shoots are relatively well understood, lateral organs often show more complex growth behavior. Lateral roots (LRs) seemingly suppress positive gravitropic growth and show a defined gravitropic set-point angle (GSA) that allows radial expansion of the root system (plagiotropism). Despite its eminent importance for root architecture, it so far remains completely unknown how lateral organs partially suppress positive orthogravitropism. Here we show that the phytohormone auxin steers GSA formation and limits positive orthogravitropism in LR. Low and high auxin levels/signaling lead to radial or axial root systems, respectively. At a cellular level, it is the auxin transport-dependent regulation of asymmetric growth in the elongation zone that determines GSA. Our data suggest that strong repression of PIN4/PIN7 and transient PIN3 expression limit auxin redistribution in young LR columella cells. We conclude that PIN activity, by temporally limiting the asymmetric auxin fluxes in the tip of LRs, induces transient, differential growth responses in the elongation zone and, consequently, controls root architecture.

A novel putative auxin carrier family regulates intracellular auxin homeostasis in plants.

The phytohormone auxin acts as a prominent signal, providing, by its local accumulation or depletion in selected cells, a spatial and temporal reference for changes in the developmental program. The distribution of auxin depends on both auxin metabolism (biosynthesis, conjugation and degradation) and cellular auxin transport. We identified in silico a novel putative auxin transport facilitator family, called PIN-LIKES (PILS). Here we illustrate that PILS proteins are required for auxin-dependent regulation of plant growth by determining the cellular sensitivity to auxin. PILS proteins regulate intracellular auxin accumulation at the endoplasmic reticulum and thus auxin availability for nuclear auxin signalling. PILS activity affects the level of endogenous auxin indole-3-acetic acid (IAA), presumably via intracellular accumulation and metabolism. Our findings reveal that the transport machinery to compartmentalize auxin within the cell is of an unexpected molecular complexity and demonstrate this compartmentalization to be functionally important for a number of developmental processes.

Cellular auxin homeostasis: gatekeeping is housekeeping.

The phytohormone auxin is essential for plant development and contributes to nearly every aspect of the plant life cycle. The spatio-temporal distribution of auxin depends on a complex interplay between auxin metabolism and cell-to-cell auxin transport. Auxin metabolism and transport are both crucial for plant development; however, it largely remains to be seen how these processes are integrated to ensure defined cellular auxin levels or even gradients within tissues or organs. In this review, we provide a glance at very diverse topics of auxin biology, such as biosynthesis, conjugation, oxidation, and transport of auxin. This broad, but certainly superficial, overview highlights the mutual importance of auxin metabolism and transport. Moreover, it allows pinpointing how auxin metabolism and transport get integrated to jointly regulate cellular auxin homeostasis. Even though these processes have been so far only separately studied, we assume that the phytohormonal crosstalk integrates and coordinates auxin metabolism and transport. Besides the integrative power of the global hormone signaling, we additionally introduce the hypothetical concept considering auxin transport components as gatekeepers for auxin responses.

PCR-mediated whole genome amplification of phytoplasmas.

A method was developed for genome analysis of phytoplasmas, bacterial plant pathogens that cannot be cultivated in vitro in cell-free media. The procedure includes a CsCl-bisbenzimide gradient buoyant centrifugation followed by polymerase chain reaction (PCR)-mediated whole genome amplification. The latter step involves digestion of the DNA by a restriction enzyme with an A/T-rich recognition sequence. Due to the different A/T content in the DNA of the pathogen and its plant host, the fragments originating from phytoplasma are shorter and are preferentially amplified in the PCR reaction. Products obtained were cloned and screened by dot-blot hybridization. Results showed that about 90% of recombinant clones appeared to harbor phytoplasma specific DNA inserts. Sequencing of randomly selected clones was carried out and comparison with the NCBI database confirmed the bacterial origin for the sequences, which have been assigned a putative function. The origin of the recombinant clones was further confirmed by the generation of specific amplicons from the phytoplasma-infected plant and not from the healthy control, using PCR primers devised from the sequences of the recombinant clones. This method could be used for genome-wide comparisons between phytoplasmas.