Arun Sampathkumar

Identification of a cellulose synthase-associated protein required for cellulose biosynthesis

Cellulose synthase-interactive protein 1 (CSI1) was identified in a two-hybrid screen for proteins that interact with cellulose synthase (CESA) isoforms involved in primary plant cell wall synthesis. CSI1 encodes a 2,150-amino acid protein that contains 10 predicted Armadillo repeats and a C2 domain. Mutations in CSI1 cause defective cell elongation in hypocotyls and roots and reduce cellulose content. CSI1 is associated with CESA complexes, and csi1 mutants affect the distribution and movement of CESA complexes in the plasma membrane.

POM-POM2/CELLULOSE SYNTHASE INTERACTING1 Is Essential for the Functional Association of Cellulose Synthase and Microtubules in Arabidopsis

Alignment of cellulose fibers during wall synthesis is crucial for anisotropic plant cell growth. Cortical microtubules guide and align cellulose synthases (CESAs) in the plasma membrane during cellulose synthesis. This study uses genetic and cell biological experiments to show that the CELLULOSE SYNTHASE INTERACTING1/POM2 protein is needed for the guidance of CESAs along cortical microtubules. In plants, regulation of cellulose synthesis is fundamental for morphogenesis and plant growth. Cellulose is synthesized at the plasma membrane, and the orientation of synthesis is guided by cortical microtubules; however, the guiding mechanism is currently unknown. We show that the conditional root elongation pom2 mutants are impaired in cell elongation, fertility, and microtubule-related functions. Map-based cloning of the POM-POM2 locus revealed that it is allelic to CELLULOSE SYNTHASE INTERACTING1 (CSI1). Fluorescently tagged POM2/CSI1s associated with both plasma membrane–located cellulose synthases (CESAs) and post-Golgi CESA-containing compartments. Interestingly, while CESA insertions coincided with cortical microtubules in the pom2/csi1 mutants, the microtubule-defined movement of the CESAs was significantly reduced in the mutant. We propose that POM2/CSI1 provides a scaffold between the CESAs and cortical microtubules that guide cellulose synthesis.

Subcellular and supracellular mechanical stress prescribes cytoskeleton behavior in Arabidopsis cotyledon pavement cells

Although it is a central question in biology, how cell shape controls intracellular dynamics largely remains an open question. Here, we show that the shape of Arabidopsis pavement cells creates a stress pattern that controls microtubule orientation, which then guides cell wall reinforcement. Live-imaging, combined with modeling of cell mechanics, shows that microtubules align along the maximal tensile stress direction within the cells, and atomic force microscopy demonstrates that this leads to reinforcement of the cell wall parallel to the microtubules. This feedback loop is regulated: cell-shape derived stresses could be overridden by imposed tissue level stresses, showing how competition between subcellular and supracellular cues control microtubule behavior. Furthermore, at the microtubule level, we identified an amplification mechanism in which mechanical stress promotes the microtubule response to stress by increasing severing activity. These multiscale feedbacks likely contribute to the robustness of microtubule behavior in plant epidermis. DOI: http://dx.doi.org/10.7554/eLife.01967.001

Live Cell Imaging Reveals Structural Associations between the Actin and Microtubule Cytoskeleton in Arabidopsis

This work investigates coordinated actin filament and microtubule activities. It shows that actin filaments and microtubules interact dynamically and that actin filaments depend on microtubules to recover following drug-induced depolymerization events. In eukaryotic cells, the actin and microtubule (MT) cytoskeletal networks are dynamic structures that organize intracellular processes and facilitate their rapid reorganization. In plant cells, actin filaments (AFs) and MTs are essential for cell growth and morphogenesis. However, dynamic interactions between these two essential components in live cells have not been explored. Here, we use spinning-disc confocal microscopy to dissect interaction and cooperation between cortical AFs and MTs in Arabidopsis thaliana, utilizing fluorescent reporter constructs for both components. Quantitative analyses revealed altered AF dynamics associated with the positions and orientations of cortical MTs. Reorganization and reassembly of the AF array was dependent on the MTs following drug-induced depolymerization, whereby short AFs initially appeared colocalized with MTs, and displayed motility along MTs. We also observed that light-induced reorganization of MTs occurred in concert with changes in AF behavior. Our results indicate dynamic interaction between the cortical actin and MT cytoskeletons in interphase plant cells.

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

The rate of insertion and lifetime of cellulose-synthesizing complexes at the plasma membrane is dependent on the organization of the actin cytoskeleton. 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.

CHITINASE-LIKE1/POM-POM1 and Its Homolog CTL2 Are Glucan-Interacting Proteins Important for Cellulose Biosynthesis in Arabidopsis

Cell wall and cellulose structure is imperative for proper cell elongation and, consequently, the architecture of plants, but components regulating cellulose structure are still elusive. This article shows that the secreted CTL1/POM1 and its close homolog CTL2 interact with glucan-based polymers and influence cellulose crystallinity and cell expansion. Plant cells are encased by a cellulose-containing wall that is essential for plant morphogenesis. Cellulose consists of β-1,4-linked glucan chains assembled into paracrystalline microfibrils that are synthesized by plasma membrane–located cellulose synthase (CESA) complexes. Associations with hemicelluloses are important for microfibril spacing and for maintaining cell wall tensile strength. Several components associated with cellulose synthesis have been identified; however, the biological functions for many of them remain elusive. We show that the chitinase-like (CTL) proteins, CTL1/POM1 and CTL2, are functionally equivalent, affect cellulose biosynthesis, and are likely to play a key role in establishing interactions between cellulose microfibrils and hemicelluloses. CTL1/POM1 coincided with CESAs in the endomembrane system and was secreted to the apoplast. The movement of CESAs was compromised in ctl1/pom1 mutant seedlings, and the cellulose content and xyloglucan structures were altered. X-ray analysis revealed reduced crystalline cellulose content in ctl1 ctl2 double mutants, suggesting that the CTLs cooperatively affect assembly of the glucan chains, which may affect interactions between hemicelluloses and cellulose. Consistent with this hypothesis, both CTLs bound glucan-based polymers in vitro. We propose that the apoplastic CTLs regulate cellulose assembly and interaction with hemicelluloses via binding to emerging cellulose microfibrils.

Physical forces regulate plant development and morphogenesis

Plant cells in tissues experience mechanical stress not only as a result of high turgor, but also through interaction with their neighbors. Cells can expand at different rates and in different directions from neighbors with which they share a cell wall. This in connection with specific tissue shapes and properties of the cell wall material can lead to intricate stress patterns throughout the tissue. Two cellular responses to mechanical stress are a microtubule cytoskeletal response that directs new wall synthesis so as to resist stress, and a hormone transporter response that regulates transport of the hormone auxin, a regulator of cell expansion. Shape changes in plant tissues affect the pattern of stresses in the tissues, and at the same time, via the cellular stress responses, the pattern of stresses controls cell growth, which in turn changes tissue shape, and stress pattern. This feedback loop controls plant morphogenesis, and explains several previously mysterious aspects of plant growth.

Laying down the bricks: logistic aspects of cell wall biosynthesis

Plant cell wall polysaccharides are synthesised at the plasma membrane and in the Golgi apparatus. Current research efforts mainly try to address how these molecules are synthesised or modified. However, it is clear that polysaccharide synthesis in the two compartments needs to be carried out in a coordinated fashion, and that carbohydrates and proteins that are delivered from the Golgi to the cell surface have to undergo a range of modifications. Consequently, there appears to be a need for a fine-tuned system that coalesces signals from the wall, synthesis of carbohydrate-based molecules and vesicle shuttling. Several recent papers have scratched the surface for an initial understanding of these linked processes. For example, the impairment of the proton pumping activity in the trans-Golgi network, which is part of the cells trafficking system, results in growth defects, changes in Golgi stack morphology and cellulose deficiency. An increased understanding of how cell wall synthesis is coordinated with the secretory machinery may facilitate avenues for modulating cell wall contents and therefore overall plant biomass.

Visualization of cellulose synthases in Arabidopsis secondary cell walls.

Secondary cell walls built with speed Plant cell walls provide the cellulose that is integral for wood, cotton fiber, and many biofuels. Cellulose is synthesized outside the cell membrane by cellulose synthase enzymes. Much of the secondary cell wall, responsible for the sturdiness of wood, is formed by xylem cells embedded in the core of the plant. Watanabe et al. leveraged ectopic expression to bring xylem-style cellulose synthase activity to the epidermal surface of the plant (see the Perspective by Schneider and Persson). Combining this improved accessibility with fluorescent tagging showed that secondary cell walls are built faster than primary cell walls, perhaps due to increased velocity and density of cellulose synthase complexes. Science, this issue p. 198, see also p. 156 Cellulose synthesis in secondary plant cell walls is focused in spiral wall thickenings during xylem cell development. [Also see Perspective by Schneider and Perssonn] Cellulose biosynthesis in plant secondary cell walls forms the basis of vascular development in land plants, with xylem tissues constituting the vast majority of terrestrial biomass. We used plant lines that contained an inducible master transcription factor controlling xylem cell fate to quantitatively image fluorescently tagged cellulose synthase enzymes during cellulose deposition in living protoxylem cells. The formation of secondary cell wall thickenings was associated with a redistribution and enrichment of CESA7-containing cellulose synthase complexes (CSCs) into narrow membrane domains. The velocities of secondary cell wall–specific CSCs were faster than those of primary cell wall CSCs during abundant cellulose production. Dynamic intracellular of endomembranes, in combination with increased velocity and high density of CSCs, enables cellulose to be synthesized rapidly in secondary cell walls.

An epidermis-driven mechanism positions and scales stem cell niches in plants

An epidermis control of plant shoot stem cells can explain the scaling and position of the niche expression domains. How molecular patterning scales to organ size is highly debated in developmental biology. We explore this question for the characteristic gene expression domains of the plant stem cell niche residing in the shoot apical meristem. We show that a combination of signals originating from the epidermal cell layer can correctly pattern the key gene expression domains and notably leads to adaptive scaling of these domains to the size of the tissue. Using live imaging, we experimentally confirm this prediction. The identified mechanism is also sufficient to explain de novo stem cell niches in emerging flowers. Our findings suggest that the deformation of the tissue transposes meristem geometry into an instructive scaling and positional input for the apical plant stem cell niche.