Cytoplasmic Linker Protein-Associating Protein at the Nexus of Hormone Signaling, Microtubule Organization, and the Transition From Division to Differentiation in Primary Roots.

The transition from cell division to differentiation in primary roots is dependent on precise gradients of phytohormones, including auxin, cytokinins and brassinosteroids. The reorganization of microtubules also plays a key role in determining whether a cell will enter another round of mitosis or begin to rapidly elongate as the first step in terminal differentiation. In the last few years, progress has been made to establish connections between signaling pathways at distinct locations within the root. This review focuses on the different factors that influence whether a root cell remains in the division zone or transitions to elongation and differentiation using Arabidopsis thaliana as a model system. We highlight the role of the microtubule-associated protein CLASP as an intermediary between sustaining hormone signaling and controlling microtubule organization. We discuss new, innovative tools and methods, such as hormone sensors and computer modeling, that are allowing researchers to more accurately visualize the belowground growth dynamics of plants.

20 years of Developmental Cell: Looking forward.

In our 20th anniversary year, we reflect on how fields have changed since our first issue and here look to the future. In this collection of Voices, our writers speculate on the future: in terms of philosophy, cell states, cell processes, and then how to model cell systems.

The Microtubule-Associated Protein CLASP Is Translationally Regulated in Light-Dependent Root Apical Meristem Growth.

The ability for plant growth to be optimized, either in the light or dark, depends on the intricate balance between cell division and differentiation in specialized regions called meristems. When Arabidopsis (Arabidopsis thaliana) seedlings are grown in the dark, hypocotyl elongation is promoted, whereas root growth is greatly reduced as a result of changes in hormone transport and a reduction in meristematic cell proliferation. Previous work showed that the microtubule-associated protein CLASP sustains root apical meristem size by influencing microtubule organization and by modulating the brassinosteroid signaling pathway. Here, we investigated whether CLASP is involved in light-dependent root growth promotion, since dark-grown seedlings have reduced root apical meristem activity, as observed in the clasp-1 null mutant. We showed that CLASP protein levels were greatly reduced in the root tips of dark-grown seedlings, which could be reversed by exposing plants to light. We confirmed that removing seedlings from the light led to a discernible shift in microtubule organization from bundled arrays, which are prominent in dividing cells, to transverse orientations typically observed in cells that have exited the meristem. Brassinosteroid receptors and auxin transporters, both of which are sustained by CLASP, were largely degraded in the dark. Interestingly, we found that despite the lack of protein, CLASP transcript levels were higher in dark-grown root tips. Together, these findings uncover a mechanism that sustains meristem homeostasis through CLASP, and they advance our understanding of how roots modulate their growth according to the amount of light and nutrients perceived by the plant.

Plant Cell Biology: Shifting CORDs to Fine-Tune Phragmoplast Microtubule Turnover.

A new study provides insight into microtubule turnover during plant cell division. Using clever molecular-genetic and imaging strategies, the authors demonstrate that the recently discovered CORD4 and 5 proteins associate with phragmoplast microtubules and control recruitment and activity of the microtubule-severing protein katanin.

Cellulose-rich secondary walls in wave-swept red macroalgae fortify flexible tissues.

MAIN CONCLUSION: Cellulosic secondary walls evolved convergently in coralline red macroalgae, reinforcing tissues against wave-induced breakage, despite differences in cellulose abundance, microfibril orientation, and wall structure. Cellulose-enriched secondary cell walls are the hallmark of woody vascular plants, which develop thickened walls to support upright growth and resist toppling in terrestrial environments. Here we investigate the striking presence and convergent evolution of cellulosic secondary walls in coralline red algae, which reinforce thalli against forces applied by crashing waves. Despite ostensible similarities to secondary wall synthesis in land plants, we note several structural and mechanical differences. In coralline red algae, secondary walls contain three-times more cellulose (~ 22% w/w) than primary walls (~ 8% w/w), and their presence nearly doubles the total thickness of cell walls (~ 1.2 µm thick). Field emission scanning electron microscopy revealed that cellulose bundles are cylindrical and lack any predominant orientation in both primary and secondary walls. His-tagged recombinant carbohydrate-binding module differentiated crystalline and amorphous cellulose in planta, noting elevated levels of crystalline cellulose in secondary walls. With the addition of secondary cell walls, Calliarthron genicular tissues become significantly stronger and tougher, yet remain remarkably extensible, more than doubling in length before breaking under tension. Thus, the development of secondary walls contributes to the strong-yet-flexible genicular tissues that enable coralline red algae to survive along wave-battered coastlines throughout the NE Pacific. This study provides an important evolutionary perspective on the development and biomechanical significance of secondary cell walls in a non-model, non-vascular plant.

Primary wall cellulose synthase regulates shoot apical meristem mechanics and growth.

How organisms attain their specific shapes and modify their growth patterns in response to environmental and chemical signals has been the subject of many investigations. Plant cells are at high turgor pressure and are surrounded by a rigid yet flexible cell wall, which is the primary determinant of plant growth and morphogenesis. Cellulose microfibrils, synthesized by plasma membrane-localized cellulose synthase complexes, are major tension-bearing components of the cell wall that mediate directional growth. Despite advances in understanding the genetic and biophysical regulation of morphogenesis, direct studies of cellulose biosynthesis and its impact on morphogenesis of different cell and tissue types are largely lacking. In this study, we took advantage of mutants of three primary cellulose synthase (CESA) genes that are involved in primary wall cellulose synthesis. Using field emission scanning electron microscopy, live cell imaging and biophysical measurements, we aimed to understand how the primary wall CESA complex acts during shoot apical meristem development. Our results indicate that cellulose biosynthesis impacts the mechanics and growth of the shoot apical meristem.

Differential regulation of TNL-mediated immune signaling by redundant helper CNLs.

Higher plants utilize nucleotide-binding leucine-rich repeat domain proteins (NLRs) as intracellular immune receptors to recognize pathogen-derived effectors and trigger a robust defense. The Activated Disease Resistance 1 (ADR1) family of coiled-coil NLRs (CNLs) have evolved as helper NLRs that function downstream of many TIR-type sensor NLRs (TNLs). Close homologs of ADR1s form the N REQUIREMENT GENE 1 (NRG1) family in Arabidopsis, the function of which is unclear. Through CRISPR/Cas9 gene editing methods, we discovered that the tandemly repeated NRG1A and NRG1B are functionally redundant and operate downstream of TNLs with differential strengths. Interestingly, ADR1s and NRG1s function in two distinct parallel pathways contributing to TNL-specific immunity. Synergistic effects on basal and TNL-mediated defense were detected among ADR1s and NRG1s. An intact P-loop of NRG1s is not required for mediating signals from sensor TNLs, whereas auto-active NRG1A exhibits autoimmunity. Importantly, NRG1s localize to the cytosol and endomembrane network regardless of the presence of effectors, suggesting a cytosolic activation mechanism. Taken together, different sensor TNLs differentially use two groups of helper NLRs, ADR1s and NRG1s, to transduce downstream defense signals.

The Microtubule-Associated Protein CLASP Sustains Cell Proliferation through a Brassinosteroid Signaling Negative Feedback Loop.

The capacity for sustained cell division within the plant meristem is a critical determinant of organ structure and performance. This capacity is diminished in mutants lacking the microtubule-associated protein CLASP and when brassinosteroid signaling is increased. Here, we discovered that CLASP is both targeted by and promotes activity of the brassinosteroid pathway in Arabidopsis root apical meristems. We show that enhanced brassinosteroid signaling reduces CLASP transcript and protein levels, dramatically shifts microtubule organization, and reduces the number of cells in the meristem. In turn, CLASP, which tethers sorting nexin 1 vesicles to microtubules, sustains brassinosteroid signaling by fostering retrieval of endocytosed BRI1 receptors to the plasma membrane. clasp-1 null mutants have dampened brassinosteroid (BR)-mediated transcriptional activity and responses. Global transcript profiling confirmed the collapse of cell-cycle activity in clasp-1 and identified CLASP-mediated hormone crosstalk. Together, these findings reveal an unprecedented form of negative feedback supporting meristem homeostasis.

Exploring Microtubule-Dependent Cellulose-Synthase-Complex Movement with High Precision Particle Tracking.

Cellulose synthesis at the plasma membrane is a critical process in plant growth and development. The displacement of cellulose synthase complexes (CSCs) by the rigid cellulose polymers they produce is a measure of enzyme activity. Connections between cortical microtubules and CSCs have been identified but it remains unclear how these affect CSC displacement speed. In this study, we applied a high throughput automated particle tracking method using near-total internal reflection fluorescence microscopy to measure the speed of CSCs. We found CSC speeds did not vary according to their proximity to microtubules, and that inhibiting microtubule polymerization could have opposite effects on CSC speed, depending on the nature of inhibition. While CSC speed increased in the temperature-sensitive mor1-1 mutant, it decreased after treatment with the drug oryzalin. Moreover, introducing the mor1-1 mutation into the CesA1 mutant any1 increased CSC speed, suggesting that microtubule dynamics affect CSC speed by a mechanism other than Cellulose Synthase A (CesA) catalytic activity. CSC speed varied widely in a range of mutants with reduced growth anisotropy, indicating that the relationship between CSC speed and anisotropy is complex. We conclude that microtubules affect CSC speed by finely tuned mechanisms that are independent of their physical association with CSCs.

Plant Cytokinesis: Terminology for Structures and Processes.

Plant cytokinesis is orchestrated by a specialized structure, the phragmoplast. The phragmoplast first occurred in representatives of Charophyte algae and then became the main division apparatus in land plants. Major cellular activities, including cytoskeletal dynamics, vesicle trafficking, membrane assembly, and cell wall biosynthesis, cooperate in the phragmoplast under the guidance of a complex signaling network. Furthermore, the phragmoplast combines plant-specific features with the conserved cytokinetic processes of animals, fungi, and protists. As such, the phragmoplast represents a useful system for understanding both plant cell dynamics and the evolution of cytokinesis. We recognize that future research and knowledge transfer into other fields would benefit from standardized terminology. Here, we propose such a lexicon of terminology for specific structures and processes associated with plant cytokinesis.

The ARM Domain of ARMADILLO-REPEAT KINESIN 1 is Not Required for Microtubule Catastrophe But Can Negatively Regulate NIMA-RELATED KINASE 6 in Arabidopsis thaliana.

Microtubules are dynamic filaments, the assembly and disassembly of which are under precise control of various associated proteins, including motor proteins and regulatory enzymes. In Arabidopsis thaliana, two such proteins are the ARMADILLO-REPEAT KINESIN 1 (ARK1), which promotes microtubule disassembly, and the NIMA-RELATED KINASE 6 (NEK6), which has a role in organizing microtubule arrays. Previous yeast two-hybrid and in vitro pull-down assays determined that NEK6 can interact with ARK1 through the latter protein's Armadillo-repeat (ARM) cargo domain. To explore the function of the ARM domain, we generated fluorescent reporter fusion proteins to ARK1 lacking the ARM domain (ARK1ΔARM-GFP) and to the ARM domain alone (ARM-GFP). Both of these constructs strongly associated with the growing plus ends of microtubules, but only ARK1ΔARM-GFP was capable of inducing microtubule catastrophe and rescuing the ark1-1 root hair phenotype. These results indicate that neither the ARM domain nor NEK6's putative interaction with it is required for ARK1 to induce microtubule catastrophe. In further exploration of the ARK1-NEK6 relationship, we demonstrated that, despite evidence that NEK6 can phosphorylate ARK1 in vitro, the in vivo distribution and function of ARK1 were not affected by the loss of NEK6, and vice versa. Moreover, NEK6 and ARK1 were found to have overlapping but non-identical distribution on microtubules, and hormone treatments known to affect NEK6 activity did not stimulate interaction. These findings suggest that ARK1 and NEK6 function independently in microtubule dynamics and cell morphogenesis. Despite the results of this functional analysis, we found that overexpression of the ARM domain led to complete loss of NEK6 transcription, suggesting that the ARM domain might have a regulatory role in NEK6 expression.

A partial loss-of-function mutation in an Arabidopsis RNA polymerase III subunit leads to pleiotropic defects.

Plants employ five DNA-dependent RNA polymerases (Pols) in transcription. One of these polymerases, Pol III, has previously been reported to transcribe 5S rRNA, tRNAs, and a number of small RNAs. However, in-depth functional analysis is complicated by the fact that knockout mutations in Pol subunits are typically lethal. Here, we report the characterization of the first known viable Pol III subunit mutant,nrpc7-1 This mutant was originally isolated from a forward genetic screen designed to identify enhancers of the autoimmune mutantsnc1, which contains a gain-of-function mutation in a nucleotide-binding leucine-rich repeat (NLR) immune receptor-encoding gene. Thenrpc7-1mutation occurs in an intron-exon splice site and results in intron retention in someNRPC7transcripts. There is a global disruption in RNA equilibrium innrpc7-1, exemplified by the altered expression of a number of RNA molecules, some of which are not reported to be transcribed by Pol III. There are developmental defects associated with the mutation, as homozygous mutant plants are dwarf, have stunted roots and siliques, and possess serrated leaves. These defects are possibly due to altered small RNA stability or activity. Additionally, thenrpc7-1mutation confers anNLR-specific alternative splicing defect that correlates with enhanced disease resistance, highlighting the importance of alternative splicing in regulating NLR activity. Altogether, these results reveal novel roles for Pol III in maintaining RNA homeostasis, adjusting the expression of a diverse suite of genes, and indirectly modulating gene splicing. Future analyses using thenrpc7-1mutant will be instrumental in examining other unknown Pol III functions.

High Light Intensity Leads to Increased Peroxule-Mitochondria Interactions in Plants.

Peroxules are thin protrusions from spherical peroxisomes produced under low levels of reactive oxygen species (ROS) stress. Whereas, stress mitigation favors peroxule retraction, prolongation of the ROS stress leads to the elongation of the peroxisome into a tubular form. Subsequently, the elongated form becomes constricted through the binding of proteins such as dynamin related proteins 3A and 3B and eventually undergoes fission to increase the peroxisomal population within a cell. The events that occur in the short time window between peroxule initiation and the tubulation of the entire peroxisome have not been observed in living plant cells. Here, using fluorescent protein aided live-imaging, we show that peroxules are formed after only 4 min of high light (HL) irradiation during which there is a perceptible increase in the cytosolic levels of hydrogen peroxide. Using a stable, double transgenic line of Arabidopsis thaliana expressing a peroxisome targeted YFP and a mitochondrial targeted GFP probe, we observed sustained interactions between peroxules and small, spherical mitochondria. Further, it was observed that the frequency of HL-induced interactions between peroxules and mitochondria increased in the Arabidopsis anisotropy1 mutant that has reduced cell wall crystallinity and where we show accumulation of higher H2O2 levels than wild type plants. Our observations suggest a testable model whereby peroxules act as interaction platforms for ROS-distressed mitochondria that may release membrane proteins and fission factors. These proteins might thus become easily available to peroxisomes and facilitate their proliferation for enhancing the ROS-combating capability of a plant cell.

Microtubules in Plant Cells: Strategies and Methods for Immunofluorescence, Transmission Electron Microscopy, and Live Cell Imaging.

Microtubules (MTs) are required throughout plant development for a wide variety of processes, and different strategies have evolved to visualize and analyze them. This chapter provides specific methods that can be used to analyze microtubule organization and dynamic properties in plant systems and summarizes the advantages and limitations for each technique. We outline basic methods for preparing samples for immunofluorescence labeling, including an enzyme-based permeabilization method, and a freeze-shattering method, which generates microfractures in the cell wall to provide antibodies access to cells in cuticle-laden aerial organs such as leaves. We discuss current options for live cell imaging of MTs with fluorescently tagged proteins (FPs), and provide chemical fixation, high-pressure freezing/freeze substitution, and post-fixation staining protocols for preserving MTs for transmission electron microscopy and tomography.

Analysis of Microtubule-Associated-Proteins during IBA-Mediated Adventitious Root Induction Reveals KATANIN Dependent and Independent Alterations of Expression Patterns.

Adventitious roots (AR) are post embryonic lateral organs that differentiate from non-root tissues. The understanding of the molecular mechanism which underlies their differentiation is important because of their central role in vegetative plant propagation. Here it was studied how the expression of different microtubule (MT)-associated proteins (MAPs) is affected during AR induction, and whether expression differences are dependent on MT organization itself. To examine AR formation when MTs are disturbed we used two mutants in the MT severing protein KATANIN. It was found that rate and number of AR primordium formed following IBA induction for three days was reduced in bot1-1 and bot1-7 plants. The reduced capacity to form ARs in bot1-1 was associated with altered expression of MAP-encoding genes along AR induction. While the expression of MAP65-4, MAP65-3, AURORA1, AURORA2 and TANGLED, increased in wild-type but not in bot1-1 plants, the expression of MAP65-8 and MDP25 decreased in wild type plants but not in the bot1-1 plant after two days of IBA-treatment. The expression of MOR1 was increased two days after AR induction in wild type and bot1-1 plants. To examine its expression specifically in AR primordium, MOR1 upstream regulatory sequence was isolated and cloned to regulate GFP. Expression of GFP was induced in the primary root tips and lateral roots, in the pericycle of the hypocotyls and in all stages of AR primordium formation. It is concluded that the expression of MAPs is regulated along AR induction and that reduction in KATANIN expression inhibits AR formation and indirectly influences the specific expression of some MAPs.

Dissecting the contribution of microtubule behaviour in adventitious root induction.

Induction of adventitious roots (ARs) in recalcitrant plants often culminates in cell division and callus formation rather than root differentiation. Evidence is provided here to suggest that microtubules (MTs) play a role in the shift from cell division to cell differentiation during AR induction. First, it was found that fewer ARs form in the temperature-sensitive mutant mor1-1, in which the MT-associated protein MOR1 is mutated, and in bot1-1, in which the MT-severing protein katanin is mutated. In the two latter mutants, MT dynamics and form are perturbed. By contrast, the number of ARs increased in RIC1-OX3 plants, in which MT bundling is enhanced and katanin is activated. In addition, any1 plants in which cell walls are perturbed made more ARs than wild-type plants. MT perturbations during AR induction in mor1-1 or in wild-type hypocotyls treated with oryzalin led to the formation of amorphous clusters of cells reminiscent of callus. In these cells a specific pattern of polarized light retardation by the cell walls was lost. PIN1 polarization and auxin maxima were hampered and differentiation of the epidermis was inhibited. It is concluded that a fine-tuned crosstalk between MTs, cell walls, and auxin transport is required for proper AR induction.

CLASP: a microtubule-based integrator of the hormone-mediated transitions from cell division to elongation.

Plants use robust mechanisms to optimize organ size to prevailing conditions. Modulating the transition from cell division to elongation dramatically affects morphology and size. Although it is well established that auxin, cytokinin and brassinosteroid mediate these transitions, recent works show that the cytoskeleton, which is normally thought to act downstream of these hormones, plays a key role in this regulatory process. In particular, the microtubule-associated protein CLASP has a dual role in meristem maintenance. CLASP modulates levels of the auxin efflux carrier PIN2 by tethering SNX1 endosomes to cortical microtubules, which in turn fine tunes auxin maxima in the root apical meristem. CLASP is also required for transfacial microtubule bundle formation at the sharp cell edges, a feature strongly associated with maintaining the capacity for further cell division.

The microtubule plus-end tracking protein ARMADILLO-REPEAT KINESIN1 promotes microtubule catastrophe in Arabidopsis.

Microtubule dynamics are critically important for plant cell development. Here, we show that Arabidopsis thaliana ARMADILLO-REPEAT KINESIN1 (ARK1) plays a key role in root hair tip growth by promoting microtubule catastrophe events. This destabilizing activity appears to maintain adequate free tubulin concentrations in order to permit rapid microtubule growth, which in turn is correlated with uniform tip growth. Microtubules in ark1-1 root hairs exhibited reduced catastrophe frequency and slower growth velocities, both of which were restored by low concentrations of the microtubule-destabilizing drug oryzalin. An ARK1-GFP (green fluorescent protein) fusion protein expressed under its endogenous promoter localized to growing microtubule plus ends and rescued the ark1-1 root hair phenotype. Transient overexpression of ARK1-RFP (red fluorescent protein) increased microtubule catastrophe frequency. ARK1-fusion protein constructs lacking the N-terminal motor domain still labeled microtubules, suggesting the existence of a second microtubule binding domain at the C terminus of ARK1. ARK1-GFP was broadly expressed in seedlings, but mutant phenotypes were restricted to root hairs, indicating that ARK1's function is redundant in cells other than those forming root hairs.

Microtubule initiation from the nuclear surface controls cortical microtubule growth polarity and orientation in Arabidopsis thaliana.

The nuclear envelope in plant cells has long been known to be a microtubule organizing center (MTOC), but its influence on microtubule organization in the cell cortex has been unclear. Here we show that nuclear MTOC activity favors the formation of longitudinal cortical microtubule (CMT) arrays. We used green fluorescent protein (GFP)-tagged gamma tubulin-complex protein 2 (GCP2) to identify nuclear MTOC activity and GFP-tagged End-Binding Protein 1b (EB1b) to track microtubule growth directions. We found that microtubules initiate from nuclei and enter the cortex in two directions along the long axis of the cell, creating bipolar longitudinal CMT arrays. Such arrays were observed in all cell types showing nuclear MTOC activity, including root hairs, recently divided cells in root tips, and the leaf epidermis. In order to confirm the causal nature of nuclei in bipolar array formation, we displaced nuclei by centrifugation, which generated a corresponding shift in the bipolarity split point. We also found that bipolar CMT arrays were associated with bidirectional trafficking of vesicular components to cell ends. Together, these findings reveal a conserved function of plant nuclear MTOCs and centrosomes/spindle pole bodies in animals and fungi, wherein all structures serve to establish polarities in microtubule growth.

Characean internodal cells as a model system for the study of cell organization.

Giant internodal cells of characean green algae have been widely used for studying cellular physiology. This review emphasizes their significance for understanding cytoarchitecture and cytoplasmic reorganization. The cytoarchitecture of internodal cells undergoes pronounced, cytoskeleton-dependent changes during development and in response to environmental cues. Under bright light, internodes develop alternating bands of acid and alkaline pH at their surface that correlate with the differential size and abundance of cortical organelles and, in the genus Chara, with the size and distribution of convoluted plasma membrane domains known as charasomes. Wounding induces responses ranging from chloroplast detachment to deposition of wound walls. These properties and the possibility for mechanical manipulation make the internodal cell ideal for exploring plasma membrane domains, organelle interactions, vesicle trafficking, and local cell wall deposition. The significance of this model system will further increase with the application of molecular biological methods in combination with metabolomics and proteomics.