Ryan Gutierrez

Arabidopsis cortical microtubules position cellulose synthase delivery to the plasma membrane and interact with cellulose synthase trafficking compartments.

Plant cell morphogenesis relies on the organization and function of two polymer arrays separated by the plasma membrane: the cortical microtubule cytoskeleton and cellulose microfibrils in the cell wall. Studies using in vivo markers confirmed that one function of the cortical microtubule array is to drive organization of cellulose microfibrils by guiding the trajectories of active cellulose synthase (CESA) complexes in the plasma membrane, thus orienting nascent microfibrils. Here we provide evidence that cortical microtubules also position the delivery of CESA complexes to the plasma membrane and interact with small CESA-containing compartments by a mechanism that permits motility driven by microtubule depolymerization. The association of CESA compartments with cortical microtubules was greatly enhanced during osmotic stress and other treatments that limit cellulose synthesis. On recovery from osmotic stress, delivery of CESA complexes to the plasma membrane was observed in association with microtubule-tethered compartments. These results reveal multiple functions for the microtubule cortical array in organizing CESA in the cell cortex.

Morlin, an inhibitor of cortical microtubule dynamics and cellulose synthase movement

Morlin (7-ethoxy-4-methyl chromen-2-one) was discovered in a screen of 20,000 compounds for small molecules that cause altered cell morphology resulting in swollen root phenotype in Arabidopsis. Live-cell imaging of fluorescently labeled cellulose synthase (CESA) and microtubules showed that morlin acts on the cortical microtubules and alters the movement of CESA. Morlin caused a novel syndrome of cytoskeletal defects, characterized by cortical array reorientation and compromised rates of both microtubule elongation and shrinking. Formation of shorter and more bundled microtubules and detachment from the cell membrane resulted when GFP::MAP4-MBP was used to visualize microtubules during morlin treatment. Cytoskeletal effects were accompanied by a reduction in the velocity and redistribution of CESA complexes labeled with YFP::CESA6 at the cell cortex. Morlin caused no inhibition of mouse myoblast, bacterial or fungal cell proliferation at concentrations that inhibit plant cell growth. By contrast, morlin stimulated microtubule disassembly in cultured hippocampal neurons but had no significant effect on cell viability. Thus, morlin appears to be a useful new probe of the mechanisms that regulate microtubule cortical array organization and its functional interaction with CESA.

A Mechanism for Reorientation of Cortical Microtubule Arrays Driven by Microtubule Severing

Introduction Organization of the cortical cytoskeleton guides the growth and morphogenesis of organisms, from bacteria to higher plants, that depend on cell walls. By positioning wall-building enzymes, the cytoskeleton acts as an interior scaffold to direct construction of the cell’s exterior. In plants, environmental and hormonal signals that modulate cell growth cause reorganization of cortical microtubule arrays. These arrays do not appear to be remodeled by moving individual microtubules, but rather by rules that govern how microtubules are assembled or disassembled. In this Research Article, we investigate the mechanism by which blue light, an important signal from the environment, causes a rapid 90° reorientation of cortical arrays in growing cells of the plant axis. Blue light perception stimulates generation of a cascade of newly oriented microtubules by katanin severing. A confocal microscopy time series of the cortical microtubule array (white, preexisting; blue, newly assembled) in an Arabidopsis epidermal cell is shown. Perception of blue light by phototropin receptors has stimulated severing at microtubule intersections. Growth of the new ends creates new and co-oriented microtubules. Together, the organization of the preexisting array and the statistical behavior of severing favor the growth of longitudinal microtubules, driving array reorientation. Methods We used spinning-disk confocal microscopy to image the reorganization of cortical microtubule arrays in real time and visualize functional proteins tagged with fluorescent proteins. We developed image-analysis methods to measure changes in array organization and behaviors of individual microtubules during array reorientation. To test hypotheses about signaling and reorganizational mechanisms, we analyzed mutants in light-perception pathways and in activity of the microtubule-severing protein katanin. Finally, we conducted photomorphogenesis assays in plant seedlings to place our findings in a physiological context. Results We discovered a mechanism, based on microtubule severing by the protein katanin, that reorients cortical microtubule arrays in response to perception of blue light. Specifically, we observed that katanin localized to microtubule crossovers, where it was required to preferentially catalyze the severing of the newer microtubule, an activity that was stimulated by the function of phototropin blue light receptors. New plus ends created by severing were stabilized and immediately grew at a high frequency, resulting in the effective creation of new microtubules. Most microtubules generated during reorientation were created by this mechanism, producing ~83% of new longitudinal microtubules. Cortical arrays failed to reorient in a mutant lacking the katanin protein. Microtubules produced by severing at crossovers can make new crossovers and, thus, opportunities for further rounds of severing and initiation, constituting a molecular amplifier that rapidly builds a new population of microtubules orthogonal to the initial array. Further experiments put this mechanism in a physiological context by revealing that katanin function is required for directional blue light to stimulate bending of the plant axis toward the light source. Discussion Cortical microtubule arrays in higher plants are being recognized as systems with self-organizing properties arising from rules governing the outcomes of microtubule interactions. In this Research Article, we present evidence that one outcome of microtubule interaction, katanin-mediated severing at crossover sites, is regulated by light perception and acts to reorient the array. Severing is thought to help build microtubule arrays in neurons and meiocytes, but it has been difficult to test this idea directly because of imaging limitations. With live imaging of plant cell cortical arrays, we have been able to investigate the cellular function of severing at the level of individual molecular events, revealing how generation of microtubules by severing is used to construct a new array. Light Turns the Array The organization of cortical microtubule arrays in higher plant cells is essential for organizing cell and tissue morphogenesis, but it is not clear how specific architectures are acquired and reconfigured in response to environmental cues. Lindeboom et al. (10.1126/science.1245533, published online 7 November; see the Perspective by Roll-Mecak) used live-cell imaging and genetic studies to show that the microtubule-severing protein, katanin, plays a crucial role in reorienting cortical arrays from transverse to longitudinal in Arabidopsis seedlings in response to blue light perception. Katanin localized to microtubule intersections where, stimulated by blue light receptors, it preferentially catalyzed the severing of the newer microtubule. The microtubule “plus” end created by severing were observed to grow preferentially, effectively building a new population of microtubules orthogonal to the initial array. The net effect of this process steers the growing seedling toward light. A self-organizing system makes the microtubule array in plants rearrange in order for the shoot to turn toward blue light. Environmental and hormonal signals cause reorganization of microtubule arrays in higher plants, but the mechanisms driving these transitions have remained elusive. The organization of these arrays is required to direct morphogenesis. We discovered that microtubule severing by the protein katanin plays a crucial and unexpected role in the reorientation of cortical arrays, as triggered by blue light. Imaging and genetic experiments revealed that phototropin photoreceptors stimulate katanin-mediated severing specifically at microtubule intersections, leading to the generation of new microtubules at these locations. We show how this activity serves as the basis for a mechanism that amplifies microtubules orthogonal to the initial array, thereby driving array reorientation. Our observations show how severing is used constructively to build a new microtubule array.

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.

Cellulose microfibril crystallinity is reduced by mutating C-terminal transmembrane region residues CESA1A903V and CESA3T942I of cellulose synthase

The mechanisms underlying the biosynthesis of cellulose in plants are complex and still poorly understood. A central question concerns the mechanism of microfibril structure and how this is linked to the catalytic polymerization action of cellulose synthase (CESA). Furthermore, it remains unclear whether modification of cellulose microfibril structure can be achieved genetically, which could be transformative in a bio-based economy. To explore these processes in planta, we developed a chemical genetic toolbox of pharmacological inhibitors and corresponding resistance-conferring point mutations in the C-terminal transmembrane domain region of CESA1A903V and CESA3T942I in Arabidopsis thaliana. Using 13C solid-state nuclear magnetic resonance spectroscopy and X-ray diffraction, we show that the cellulose microfibrils displayed reduced width and an additional cellulose C4 peak indicative of a degree of crystallinity that is intermediate between the surface and interior glucans of wild type, suggesting a difference in glucan chain association during microfibril formation. Consistent with measurements of lower microfibril crystallinity, cellulose extracts from mutated CESA1A903V and CESA3T942I displayed greater saccharification efficiency than wild type. Using live-cell imaging to track fluorescently labeled CESA, we found that these mutants show increased CESA velocities in the plasma membrane, an indication of increased polymerization rate. Collectively, these data suggest that CESA1A903V and CESA3T942I have modified microfibril structure in terms of crystallinity and suggest that in plants, as in bacteria, crystallization biophysically limits polymerization.

Nonmotile Cellulose Synthase Subunits Repeatedly Accumulate within Localized Regions at the Plasma Membrane in Arabidopsis Hypocotyl Cells following 2,6-Dichlorobenzonitrile Treatment

2,6-Dichlorobenzonitrile (DCB; [Fig. 1A][1] ) was reported to inhibit cellulose synthesis more than 30 years ago ([Hogetsu et al., 1974][2]) and has subsequently been used in numerous studies (e.g. [Montezinos and Delmer, 1980][3]; [Edelmann and Fry, 1992][4]; [Shedletzky et al., 1992][5]; [Suzuki

RNR1, a 3′–5′ exoribonuclease belonging to the RNR superfamily, catalyzes 3′ maturation of chloroplast ribosomal RNAs in Arabidopsis thaliana

Arabidopsis thaliana chloroplasts contain at least two 3′ to 5′ exoribonucleases, polynucleotide phosphorylase (PNPase) and an RNase R homolog (RNR1). PNPase has been implicated in both mRNA and 23S rRNA 3′ processing. However, the observed maturation defects do not affect chloroplast translation, suggesting that the overall role of PNPase in maturation of chloroplast rRNA is not essential. Here, we show that this role can be largely ascribed to RNR1, for which homozygous mutants germinate only on sucrose-containing media, and have white cotyledons and pale green rosette leaves. Accumulation of chloroplast-encoded mRNAs and tRNAs is unaffected in such mutants, suggesting that RNR1 activity is either unnecessary or redundant for their processing and turnover. However, accumulation of several chloroplast rRNA species is severely affected. High-resolution RNA gel blot analysis, and mapping of 5′ and 3′ ends, revealed that RNR1 is involved in the maturation of 23S, 16S and 5S rRNAs. The 3′ extensions of the accumulating 5S rRNA precursors can be efficiently removed in vitro by purified RNR1, consistent with this view. Our data suggest that decreased accumulation of mature chloroplast ribosomal RNAs leads to a reduction in the number of translating ribosomes, ultimately compromising chloroplast protein abundance and thus plant growth and development.

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.

Opportunities to explore plant membrane organization with super-resolution microscopy.

Electron microscopy and light microscopy both have been essential tools for investigating molecular distribution and cell structure. While electron microscopy is capable of much higher resolution compared to light microscopy, it is prone to artifacts introduced by sample preparation and it produces