René Schneider

Cellulose and callose synthesis and organization in focus, what's new?

Plant growth and development are supported by plastic but strong cell walls. These walls consist largely of polysaccharides that vary in content and structure. Most of the polysaccharides are produced in the Golgi apparatus and are then secreted to the apoplast and built into the growing walls. However, the two glucan polymers cellulose and callose are synthesized at the plasma membrane by cellulose or callose synthase complexes, respectively. Cellulose is the most common cell wall polymer in land plants and provides strength to the walls to support directed cell expansion. In contrast, callose is integral to specialized cell walls, such as the cell plate that separates dividing cells and growing pollen tube walls, and maintains important functions during abiotic and biotic stress responses. The last years have seen a dramatic increase in our understanding of how cellulose and callose are manufactured, and new factors that regulate the synthases have been identified. Much of this knowledge has been amassed via various microscopy-based techniques, including various confocal techniques and super-resolution imaging. Here, we summarize and synthesize recent findings in the fields of cellulose and callose synthesis in plant biology.

Golgi-localized STELLO proteins regulate the assembly and trafficking of cellulose synthase complexes in Arabidopsis

As the most abundant biopolymer on Earth, cellulose is a key structural component of the plant cell wall. Cellulose is produced at the plasma membrane by cellulose synthase (CesA) complexes (CSCs), which are assembled in the endomembrane system and trafficked to the plasma membrane. While several proteins that affect CesA activity have been identified, components that regulate CSC assembly and trafficking remain unknown. Here we show that STELLO1 and 2 are Golgi-localized proteins that can interact with CesAs and control cellulose quantity. In the absence of STELLO function, the spatial distribution within the Golgi, secretion and activity of the CSCs are impaired indicating a central role of the STELLO proteins in CSC assembly. Point mutations in the predicted catalytic domains of the STELLO proteins indicate that they are glycosyltransferases facing the Golgi lumen. Hence, we have uncovered proteins that regulate CSC assembly in the plant Golgi apparatus.

BRASSINOSTEROID INSENSITIVE2 negatively regulates cellulose synthesis in Arabidopsis by phosphorylating cellulose synthase 1

Significance Cellulose is the most abundant biopolymer on Earth and is a critical component for plants to grow and develop. Cellulose is synthesized by large cellulose synthase complexes containing multiple cellulose synthase A (CESA) subunits; however, how cellulose synthesis is regulated remains unclear. In this study, we identify BRASSINOSTEROID INSENSITIVE2 (BIN2) as a protein kinase that directly phosphorylates Arabidopsis CESA1 and further demonstrate that this phosphorylation event negatively regulates CESA activity, and thus cellulose biosynthesis, in Arabidopsis. Therefore, this study provides a clear link between cell wall biosynthesis and hormonal signal transduction pathways that regulate plant growth and development. The deposition of cellulose is a defining aspect of plant growth and development, but regulation of this process is poorly understood. Here, we demonstrate that the protein kinase BRASSINOSTEROID INSENSITIVE2 (BIN2), a key negative regulator of brassinosteroid (BR) signaling, can phosphorylate Arabidopsis cellulose synthase A1 (CESA1), a subunit of the primary cell wall cellulose synthase complex, and thereby negatively regulate cellulose biosynthesis. Accordingly, point mutations of the BIN2-mediated CESA1 phosphorylation site abolished BIN2-dependent regulation of cellulose synthase activity. Hence, we have uncovered a mechanism for how BR signaling can modulate cellulose synthesis in plants.

Connecting two arrays: the emerging role of actin-microtubule cross-linking motor proteins

The cytoskeleton of plant cells, consisting of actin filaments (AFs) and microtubules (MTs), is a central structure for various intracellular processes, such as cell division, isotropic and polar growth, vesicle transport, cell shape, and morphogenesis. Pharmaceutical and genetic studies have provided indications for interdependent cross-talk between the cytoskeletal components. Recent live-cell imaging studies have cemented this notion, in particular when the cytoskeleton rearranges. However, the proteins that directly mediate this cross-talk have remained largely elusive. Recent data indicate that certain proteins can interact with both cytoskeletal arrays at the same time, and hence connecting them. In this review, we summarize the recent literature of the AF- and MT-interactors, mainly focusing on a plant-specific mediator of cytoskeletal cross-talk: the calponin homology (CH) domain-containing kinesin-14 motor proteins (KCHs).

Kinesin-1 Motors Can Circumvent Permanent Roadblocks by Side-Shifting to Neighboring Protofilaments

Obstacles on the surface of microtubules can lead to defective cargo transport, proposed to play a role in neurological diseases such as Alzheimers. However, little is known about how motor proteins, which follow individual microtubule protofilaments (such as kinesin-1), deal with obstacles on the molecular level. Here, we used rigor-binding mutants of kinesin-1 as roadblocks to permanently obstruct individual microtubule binding sites and studied the movement of individual kinesin-1 motors by single-molecule fluorescence and dark-field scattering microscopy in vitro. In the presence of roadblocks, kinesin-1 often stopped for ∼ 0.4 s before either detaching or continuing to move, whereby the latter circumvention events occurred in >30% after a stopping event. Consequently, and in agreement with numerical simulations, the mean velocity, mean run length, and mean dwell time of the kinesin-1 motors decreased upon increasing the roadblock density. Tracking individual kinesin-1 motors labeled by 40 nm gold particles with 6 nm spatial and 1 ms temporal precision revealed that ∼ 70% of the circumvention events were associated with significant transverse shifts perpendicular to the axis of the microtubule. These side-shifts, which occurred with equal likelihood to the left and right, were accompanied by a range of longitudinal shifts suggesting that roadblock circumvention involves the unbinding and rebinding of the motors. Thus, processive motors, which commonly follow individual protofilaments in the absence of obstacles, appear to possess intrinsic circumvention mechanisms. These mechanisms were potentially optimized by evolution for the motors specific intracellular tasks and environments.

Using a quartz paraboloid for versatile wide-field TIR microscopy with sub-nanometer localization accuracy

Illumination based on objective-type total internal reflection (TIR) is nowadays widely used in high-performance fluorescence microscopy. However, the desirable application of such setups for dark-field imaging of scattering entities is cumbersome due to the spatial overlap of illumination and detection light, which cannot be separated spectrally. Here, we report a novel TIR approach based on a parabolically shaped quartz prism that allows for the detection of single-molecule fluorescence as well as single-particle scattering with high signal-to-noise ratios. We demonstrate homogeneous and spatially invariant illumination profiles in combination with a convenient control over a wide range of illumination angles. Moreover, we quantitatively compare the fluorescence performance of our setup to objective-type TIR and demonstrate sub-nanometer localization accuracies for the scattering of 40 nm gold nanoparticles (AuNPs). When bound to individual kinesin-1 motors, the AuNPs reliably report on the characteristic 8 nm stepping along microtubules.

Two Complementary Mechanisms Underpin Cell Wall Patterning during Xylem Vessel Development

The CELLULOSE SYNTHASE INTERACTING1 protein directs secondary wall patterning during the early phases of xylem vessel development. The evolution of the plant vasculature was essential for the emergence of terrestrial life. Xylem vessels are solute-transporting elements in the vasculature that possess secondary wall thickenings deposited in intricate patterns. Evenly dispersed microtubule (MT) bands support the formation of these wall thickenings, but how the MTs direct cell wall synthesis during this process remains largely unknown. Cellulose is the major secondary wall constituent and is synthesized by plasma membrane-localized cellulose synthases (CesAs) whose catalytic activity propels them through the membrane. We show that the protein CELLULOSE SYNTHASE INTERACTING1 (CSI1)/POM2 is necessary to align the secondary wall CesAs and MTs during the initial phase of xylem vessel development in Arabidopsis thaliana and rice (Oryza sativa). Surprisingly, these MT-driven patterns successively become imprinted and sufficient to sustain the continued progression of wall thickening in the absence of MTs and CSI1/POM2 function. Hence, two complementary principles underpin wall patterning during xylem vessel development.

Three AtCesA6-like members enhance biomass production by distinctively promoting cell growth in Arabidopsis

Summary Cellulose is an abundant biopolymer and a prominent constituent of plant cell walls. Cellulose is also a central component to plant morphogenesis and contributes the bulk of a plants biomass. While cellulose synthase (CesA) genes were identified over two decades ago, genetic manipulation of this family to enhance cellulose production has remained difficult. In this study, we show that increasing the expression levels of the three primary cell wall AtCesA6‐like genes (AtCesA2, AtCesA5, AtCesA6), but not AtCesA3, AtCesA9 or secondary cell wall AtCesA7, can promote the expression of major primary wall CesA genes to accelerate primary wall CesA complex (cellulose synthase complexes, CSCs) particle movement for acquiring long microfibrils and consequently increasing cellulose production in Arabidopsis transgenic lines, as compared with wild‐type. The overexpression transgenic lines displayed changes in expression of genes related to cell growth and proliferation, perhaps explaining the enhanced growth of the transgenic seedlings. Notably, overexpression of the three AtCesA6‐like genes also enhanced secondary cell wall deposition that led to improved mechanical strength and higher biomass production in transgenic mature plants. Hence, we propose that overexpression of certain AtCesA genes can provide a biotechnological approach to increase cellulose synthesis and biomass accumulation in transgenic plants.

The cellulose synthase companion proteins act non-redundantly with CELLULOSE SYNTHASE INTERACTING1/POM2 and CELLULOSE SYNTHASE 6.

ABSTRACT Cellulose is a cell wall constituent that is essential for plant growth and development, and an important raw material for a range of industrial applications. Cellulose is synthesized at the plasma membrane by massive cellulose synthase (CesA) complexes that track along cortical microtubules in elongating cells of Arabidopsis through the activity of the protein CELLULOSE SYNTHASE INTERACTING1 (CSI1). In a recent study we identified another family of proteins that also are associated with the CesA complex and microtubules, and that we named COMPANIONS OF CELLULOSE SYNTHASE (CC). The CC proteins protect the cellulose synthesising capacity of Arabidopsis seedlings during exposure to adverse environmental conditions by enhancing microtubule dynamics. In this paper we provide cell biology and genetic evidence that the CSI1 and the CC proteins fulfil distinct functions during cellulose synthesis. We also show that the CC proteins are necessary to aid cellulose synthesis when components of the CesA complex are impaired. These data indicate that the CC proteins have a broad role in aiding cellulose synthesis during environmental changes and when core complex components are non-functional.

Cellulose synthase complexes display distinct dynamic behaviors during xylem transdifferentiation

Significance Cellulose, the most abundant biopolymer on earth, is the major constituent of plant cell walls and is ubiquitously used by industry. This biopolymer is made by plasma membrane-localized CELLULOSE SYNTHASE (CESA) enzymes. To transit from deposition of a growing primary cell wall to a strong secondary cell wall, xylem cells must remodel the CESA machinery to express a new set of CESA isoforms specific to secondary cell wall synthesis. We outline a detailed framework for how this change in cellulose synthesis occurs. Our work provides the principles for how plants change their capacity to produce cellulose and therefore plant biomass. In plants, plasma membrane-embedded CELLULOSE SYNTHASE (CESA) enzyme complexes deposit cellulose polymers into the developing cell wall. Cellulose synthesis requires two different sets of CESA complexes that are active during cell expansion and secondary cell wall thickening, respectively. Hence, developing xylem cells, which first undergo cell expansion and subsequently deposit thick secondary walls, need to completely reorganize their CESA complexes from primary wall- to secondary wall-specific CESAs. Using live-cell imaging, we analyzed the principles underlying this remodeling. At the onset of secondary wall synthesis, the primary wall CESAs ceased to be delivered to the plasma membrane and were gradually removed from both the plasma membrane and the Golgi. For a brief transition period, both primary wall- and secondary wall-specific CESAs coexisted in banded domains of the plasma membrane where secondary wall synthesis is concentrated. During this transition, primary and secondary wall CESAs displayed discrete dynamic behaviors and sensitivities to the inhibitor isoxaben. As secondary wall-specific CESAs were delivered and inserted into the plasma membrane, the primary wall CESAs became concentrated in prevacuolar compartments and lytic vacuoles. This adjustment in localization between the two CESAs was accompanied by concurrent decreased primary wall CESA and increased secondary wall CESA protein abundance. Our data reveal distinct and dynamic subcellular trafficking patterns that underpin the remodeling of the cellulose biosynthetic machinery, resulting in the removal and degradation of the primary wall CESA complex with concurrent production and recycling of the secondary wall CESAs.