Biology subjects

Anderson, C. T.

Publications and source records attributed to Anderson, C. T..

18 recordsLinked to original sources

bioRxiv2026

Mechanisms underlying the defective development of xyloglucan-deficient root hairs in Arabidopsis thaliana

Hemicellulosic xyloglucan is abundant in the primary cell walls of eudicots and non-gramineous monocots and contributes to their mechanical integrity. Its deficiency results in morphologically abnormal root hairs in Arabidopsis mutants lacking either two XYLOGLUCAN XYLOSYLTRANSFERASE (xxt1 xxt2) or five CELLULOSE SYNTHASE-LIKE C (cslc-{triangleup}5) genes, but the cellular and biomechanical mechanisms underlying these abnormalities have remained unexplored. We combined multiple imaging modalities and quantitative modeling to unmask these mechanisms, first observing that xyloglucan-deficient mutants produced fewer and shorter root hairs that grew slower than wild type. Xyloglucan-deficient trichoblasts showed root hair initiation defects, helping to explain the sparseness of root hairs in these mutants. The mutant root hairs also had lower turgor pressure, thicker walls, and wider cross-sections, that explain lack of anisotropic growth, but cellulose organization appeared normal in xyloglucan-deficient root hairs. Brillouin microscopy suggested that xyloglucan-deficient root hairs were softer than wild-type root hairs. Finite element modeling of mutant root hairs predicted lower surface stress and growth rates, which were directly proportional to turgor pressure and inversely proportional to wall thickness. Stress patterns on models hint at potential mechanisms of bulging in xyloglucan-deficient root hairs. We propose that though cellulose orientation specifies the axis of cell expansion, it cannot enforce it without xyloglucan that interlinks microfibrils into a load-bearing network, identifying matrix-cellulose interaction as a distinct and necessary determinant of anisotropic plant cell growth.

cell biology↗

bioRxiv2026

Natural isolates of Sinorhizobium meliloti with dry, as opposed to mucoid, colony morphologies have higher soil fitness, slightly lower host fitness, and reduced nitrogen fixation

Sinorhizobium meliloti is a nitrogen-fixing soil bacterium that forms a facultative mutualism with its host alfalfa. S. meliloti strains exhibit diverse colony morphologies on solid culture media that span a continuum from dry to mucoid. Studies on how this trait affects symbiosis rely on mutations in exopolysaccharide II production genes; however, whether natural variation in morphology confers a selective advantage on rhizobia in their natural habitats remains unknown. Here, we used 90 mucoid and 27 dry natural isolates to test whether colony morphology is associated with rhizobial fitness in the soil and in the host, or with the nitrogen-fixation benefits they provide to the plant. Colony morphologies were widely distributed across a phylogenetic tree, suggesting that morphological shifts occurred repeatedly and no single genetic variant was strongly associated with this trait. We inoculated soil mesocosms and plants with a mixture of dry and mucoid bacteria. We observed that dry rhizobia have higher fitness, particularly at 22{degrees}C and 32{degrees}C, but not under cold or drought conditions, independent of the initial rhizobial load. Conversely, mucoid bacteria have a modestly higher relative fitness in host nodules, and single inoculations showed that they tend to confer greater benefits to all three alfalfa varieties. Over time, we also observed rare, repeated conversion from mucoid to dry morphologies in soil and host plants. Taken together, our study reveals that morphological variation within a species in a single agricultural field is important for bacterial fitness in the soil and can have cascading effects on the rhizobia's plant benefits.

ecology↗

bioRxiv2026

Crop yields under simulated nuclear winter: a growth chamber experiment

A global nuclear war could inject soot into the stratosphere, blocking sunlight and causing rapid cooling. Assessments of the resulting agricultural collapse rely on crop models never validated under such conditions. We grew wheat, canola, and potato in growth chambers simulating the light and temperature of an extreme nuclear winter at tropical and temperate sites. In the tropical chamber (18-20 {degrees}C, 200 mol m-2 s-1 PAR), all three crops produced viable yields. Wheat yielded 2.1-2.3 t/ha (n=3 well-watered, n=3 water-stressed pots), 60% of the global average, and single-pot observations of canola and potato suggested biological yields comparable to global averages. In the temperate chamber simulating nuclear winter irradiance (60-360 mol m-2 s-1), wheat stems collapsed under their own weight. Although hand-harvesting recovered 0.6-2.8 t/ha of grain, mechanical field harvest of a flat canopy would recover substantially less. This failure mode was not observed in a higher-light control chamber and is not captured by existing crop models, which may therefore overestimate temperate cereal production under nuclear winter. Canola produced comparable yields under both temperate light regimes without lodging. Empirical screening of additional staples is needed to identify which remain viable under nuclear winter.

plant biology↗

bioRxiv2025

Mechanical constraint causes lower turgor, thicker walls, and faster growth in Arabidopsis root hairs

Root hairs absorb water and mineral nutrients while anchoring growing root tips. They must navigate through soils of varying mechanical properties. Mechanical changes in their microenvironment affect root hair shape and growth rate, but what underlies these responses has remained elusive. To uncover these mechanisms, we grew seedlings of Arabidopsis thaliana (Col-0) in media with increasing mechanical stiffness. Col-0 seedlings showed both shorter and fewer root hairs with increasing mechanical resistance. We used incipient plasmolysis to estimate turgor pressure in trichoblasts and found that it also decreased with increasing media stiffness. Because cellulose is the major load-bearing polymer in the cell wall and influences cell expansion, we quantified cellulose orientation in root hairs. We found that cellulose fibrils were oriented at steeper angles relative to the growth axis in root hairs grown in stiffer media, suggesting a response to mechanical stress. Cell wall thickness also increased with increasing media stiffness. Microtubule orientations followed patterns that were similar to those of cellulose fibrils, but at a smaller angle relative to the growth axis, whereas microtubule density decreased with increasing media stiffness. Unexpectedly, we observed that root hairs grew faster in stiffer media, implying that their growth is misregulated, potentially triggering wall integrity signaling that causes early growth arrest. Finite element modeling of root hairs predicted decreased surface stress, explaining these growth phenotypes. These findings help establish mechanistic links between mechanotransduction, cytoskeletal dynamics, and cell wall assembly during root hair growth.

cell biology↗

bioRxiv2025

Brassinosteroid treatment reveals the importance of xyloglucan transglucosylase/hydrolase (XTH) genes in growth habit determination of twining common bean vines

O_LIBrassinosteroids impact the development of G-fibers --specialized cells that generate tension in plants. To explore the functional and genetic relationships between G-fibers and twining stems of common bean, we applied an active brassinosteroid and a brassinosteroid inhibitor to perturb G-fiber development and probed these phenotypes through gene expression and anatomical analyses. C_LIO_LIBrassinosteroid treatment generated phenotypes that aKected the three key features of twining: elongation, circumnutation, and G-fiber development. We examined anatomical and biochemical changes in the G-fibers through cross-sections, macerations, and immunohistochemistry. RNA sequencing and differential gene expression analysis allowed us to identify unique gene expression patterns for each treatment. C_LIO_LIBrassinosteroid treatment led to significantly elongated internodes with disrupted circumnutation and long, thin-walled G-fibers. In contrast, inhibitor treatment produced short internodes with thick G-fibers. These phenotypes corresponded with significant differential expression of XTH genes, both at the onset of elongation and later, during G-layer deposition. Detection of xyloglucan epitopes in the G-layer, along with in situ hybridization, confirmed active xyloglucan remodeling after twining. C_LIO_LIOur results confirm the presence of xyloglucan in the G-layer of common bean, underscoring its importance in G-fiber function, and suggests a regulatory role for XTH genes in shaping the twining growth habit through modulation of cell wall properties. C_LI

plant biology↗

bioRxiv2025

The Carbohydrate Binding Module of TrCel7A Aids in Navigating the Complexity of Plant Cell Walls

Efficient enzymatic deconstruction of plant cell walls is critical for utilization of lignocellulose biomass. Key enzymes in this process are cellobiohydrolases, a class of cellulases that processively degrade crystalline cellulose. Many cellobiohydrolases possess a carbohydrate-binding module (CBM), yet the importance of CBMs in substrate interaction remains unclear. Here, we use single-molecule fluorescence microscopy to investigate how CBM1 of Trichoderma reesei Cel7A influences enzyme binding and motility on cellulose substrates of varying complexity. We compare wild-type Cel7A with a truncated variant lacking CBM1 (Cel7A{Delta}CBM) on bacterial cellulose (BC), phosphoric acid swollen cellulose (PASC), delignified milkweed cellulose (MWC), and holocellulose nanofibrils (hCNF). While both variants showed similar steady-state binding densities on BC and PASC, Cel7A{Delta}CBM exhibited reduced binding on MWC and hCNF, with the greatest reduction on the hemicellulose-rich hCNF. Alkali removal of hemicellulose partially restored Cel7A{Delta}CBM binding, suggesting a role for CBM1 in substrate navigation and productive binding sites recognition. Kinetic analyses revealed that CBM1 enables a rapid binding mode absent in the truncated variant. Comparisons with isolated CBM3 further showed that CBMs are capable of fast substrate association. These findings demonstrate that CBMs enhance cellulase-substrate interactions by accelerating binding, enabling navigation of the complex environment of plant cell walls. Our results emphasize the importance of CBMs in natural cellobiohydrolase function and highlight their value in the design of improved cellulases for industrial biomass conversion.

biochemistry↗

bioRxiv2025

Design principles of the common Gly-X6-Gly membrane protein building block

Protein behavior in lipid is poorly understood and inadequately represented in current computational models. Design and prediction abilities for bilayer-embedded molecular structures may be improved by characterizing membrane proteins most frequent, favored structural features to glean both context-specific and general principles. We used protein design to proactively interrogate the sequence-structure relationship and stabilizing atomic details of two highly prevalent antiparallel transmembrane (TM) motifs with Small-X6-Small consensus sequences. A fragment-based data-mining and sequence statistical inference method including cross-evolutionary structure-aligned covariance enabled engineering of de novo multi-span TM protein assemblies by successfully encoding Gly-X6-Gly and Ala-X6-Ala building blocks. A highly stable glycine-based designs X-ray structure hosts C-H{middle dot}{middle dot}{middle dot}O=C H-bonding alongside extensive backbone-directed van der Waals packing, idealizing features of this motif in Nature. Data-driven design navigates sequence space to directly inquiry upon how to encode and stabilize vital membrane protein structural elements, facilitating efficacious construction of lipid-embedded architectures of increasing complexity. SignificanceMembrane proteins comprised of -helices pack together within lipid bilayers, establishing stabilities and architectures that brace function and guide evolution. De novo design was used to clarify the consensus sequences and molecular features encoding of one exceedingly common TM helix packing architecture ([~]10% of those in membrane folds). Small-X6-Small residue patterns were proven to reliably drive minimal proteins into these antiparallel TM geometries, revealing glycine mainchain hydrogen bonding or via fully apolar interfaces can encode the motif. Optimizing steric packing was the most decisive feature amongst the synthetic proteins. New membrane-specific design methods and model molecules are validated in route to outlining this important sequence-structure relationship broadly impacting the membrane proteome and revealing generalized structure-energetic imperatives governing interactions in lipid.

biophysics↗

bioRxiv2025

Impacts of sea level rise on an endemic butterfly and its freshwater wetland habitat in the Florida Keys

Coastal and island ecosystems are disproportionately vulnerable to sea level rise and other impacts of anthropogenic climate change. Using monitoring data collected between 2013 and 2024, we explore habitat shifts, population dynamics, and phenology of an endemic butterfly in the Florida Keys, Klots sawgrass skipper (Euphyes pilatka klotsi). Range-wide surveys of the butterfly and its associated habitat demonstrated that the butterflys sole host plant is decreasing in abundance at lower elevations, likely due to increased salinization. We also find declines in butterfly population size over an eleven-year period using distance sampling. Despite these declines, we find that Klots sawgrass skippers range has not contracted significantly over the study period. This study demonstrates that monitoring a single taxon and its host is useful for understanding how sea level rise impacts a keystone plant species in freshwater wetlands which are vital for the continued survival of a suite of rare and endemic species found in the Florida Keys. Projections of near-future sea level rise indicate that most or all of this habitat will be lost within several decades; the continued study of low-lying islands is critical to gain insight into the global phenomenon of sea level rise.

ecology↗

bioRxiv2024

Gelatinous fibers develop asymmetrically for posture support of bends and coils in common bean vine

Gelatinous (G)-fibers are common in the stems of twining vines (twiners), but their role remain unclear given the lack of developmental insights. Here, we characterize the developmental anatomy of G-fiber formation in common bean stems (Phaseolus vulgaris L., Fabaceae). G-fibers in common bean exhibit cell wall organization comparable to other species, consisting of cellulose and Rhamnogalacturonan-I pectins, with possible traces of lignin. We show that G-fibers are absent in the actively circumnutating stems, thus these tensile fibers are not associated with the dynamic searching movements characteristics of twiners. Instead, we found that after a subtle bend or dramatic coil is formed, G-fibers form asymmetrically on the concave side of the stem for posture maintenance. Therefore, G-fibers do not drive movement, but provide support for existing bends, thus stabilizing the helical conformation of twiners around its host to avoid slippage. Finally, we present common bean as an emergent system to study twiners and growth form diversity given its easy cultivation, self-pollination, fast growth, and habit diversity arising from plant breeding, and the ability to induce habit shifts through simple modifications to light conditions.

plant biology↗

bioRxiv2024

Young guard cells function dynamically despite low mechanical anisotropy but gain efficiency during stomatal maturation in Arabidopsis thaliana

Stomata are pores at the leaf surface that enable gas exchange and transpiration. The signaling pathways that regulate the differentiation of stomatal guard cells and the mechanisms of stomatal pore formation have been characterized in Arabidopsis thaliana. However, the process by which stomatal complexes develop after pore formation into fully mature complexes is poorly understood. We tracked the morphogenesis of young stomatal complexes over time to establish characteristic geometric milestones along the path of stomatal maturation. Using 3D-nanoindentation coupled with finite element modeling of young and mature stomata, we found that despite having thicker cell walls than young guard cells, mature guard cells are more energy efficient with respect to stomatal opening, potentially attributable to the increased mechanical anisotropy of their cell walls and smaller changes in turgor pressure between the closed and open states. Comparing geometric changes in young and mature guard cells of wild-type and cellulose-deficient plants revealed that although cellulose is required for normal stomatal maturation, mechanical anisotropy appears to be achieved by the collective influence of cellulose and additional wall components. Together, these data elucidate the dynamic geometric and biomechanical mechanisms underlying the development process of stomatal maturation. SignificanceWe examined how young stomata develop into mature complexes and the functional consequences of stomatal maturation, establishing geometric milestones and detecting mechanical changes in wall anisotropy and turgor pressure during stomatal maturation. We show that mature guard cells exert less energy and require a lower increase in turgor pressure to open the pore, likely due to higher wall anisotropy that results from the patterned deposition and/or remodeling of cellulose and other wall components.

plant biology↗

bioRxiv2024

Xylan inhibition of cellulase binding and processivity observed at single-molecule resolution

Efficient cellulose degradation by cellulase enzymes is crucial for using lignocellulosic biomass in bioenergy production. In the cell wall of plants, cellulose is bound by lignin and hemicellulose, which are key factors contributing to the recalcitrance of plant biomass. These non-cellulosic cell wall components are known to interfere with the function of cellulolytic enzymes. While the effects of lignin have been studied extensively, the contribution of xylan, the major hemicellulose in the secondary cell walls of plants, is often overlooked. To study those effects, we generated model cell wall composites by growing bacterial cellulose supplemented with varying concentrations of purified xylan. We used single-molecule microscopy to image and track fluorescently labeled TrCel7A, a commonly used model cellulase, as it binds and hydrolyses cellulose in these synthetic composites. We found that minute amounts of xylan are sufficient to significantly inhibit the binding of Cel7A to cellulose. The inclusion of xylan also reduced considerably the proportion of moving enzyme molecules, without affecting their velocity and run length. We suggest that, when available at low concentrations, xylan thinly coats cellulose fibrils, and incorporates as continuous patches when available at higher concentrations. Non-productive binding of Cel7A to xylan was not found to be a major inhibition mechanism. Our results highlight the importance of targeting xylan removal during biomass processing and demonstrate the potential of using single-molecule imagining to study the activity and limitations of cellulolytic enzymes. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=165 SRC="FIGDIR/small/577775v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1c7afbeorg.highwire.dtl.DTLVardef@e30ce4org.highwire.dtl.DTLVardef@635358org.highwire.dtl.DTLVardef@f4bdc9_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Lignin impairs Cel7A degradation of cellulose by impeding enzyme movement and not by acting as a sink

BackgroundCellulose degradation by cellulases has been studied for decades due to the potential of using lignocellulosic biomass as a sustainable source of bioethanol. In plant cell walls, cellulose is bonded together and strengthened by the polyphenolic polymer, lignin. Because lignin is tightly linked to cellulose and is not digestible by cellulases, is thought to play a dominant role in limiting the efficient enzymatic degradation of plant biomass. Removal of lignin via pretreatments currently limits the cost-efficient production of ethanol from cellulose, motivating the need for a better understanding of how lignin inhibits cellulase-catalyzed degradation of lignocellulose. Work to date using bulk assays has suggested three possible inhibition mechanisms: lignin blocks access of the enzyme to cellulose, lignin impedes progress of the enzyme along cellulose, or lignin binds cellulases directly and acts as a sink. ResultsWe used single-molecule fluorescence microscopy to investigate the nanoscale dynamics of Cel7A from Trichoderma reesei, as it binds to and moves along purified bacterial cellulose in vitro. Lignified cellulose was generated by polymerizing coniferyl alcohol onto purified bacterial cellulose, and the degree of lignin incorporation into the cellulose meshwork was analyzed by optical and electron microscopy. We found that Cel7A preferentially bound to regions of cellulose where lignin was absent, and that in regions of high lignin density, Cel7A binding was inhibited. With increasing degrees of lignification, there was a decrease in the fraction of Cel7A that moved along cellulose rather than statically binding. Furthermore, with increasing lignification, the velocity of processive Cel7A movement decreased, as did the distance that individual Cel7A molecules moved during processive runs. ConclusionsIn an in vitro system that mimics lignified cellulose in plant cell walls, lignin did not act as a sink to sequester Cel7A and prevent it from interacting with cellulose. Instead, lignin both blocked access of Cel7A to cellulose and impeded the processive movement of Cel7A along cellulose. This work implies that strategies for improving biofuel production efficiency should target weakening interactions between lignin and cellulose surface, and further suggest that nonspecific adsorption of Cel7A to lignin is likely not a dominant mechanism of inhibition.

plant biology↗

bioRxiv2023

Altering the substitution and crosslinking of glucuronoarabinoxylans affects cell wall porosity and assembly in Brachypodium distachyon.

O_LIThe Poaceae family of plants provides cereal crops that are critical for human and animal nutrition and also they are an important source of biomass. Interacting plant cell wall components give rise to recalcitrance to digestion, thus understanding the wall molecular architecture is important to improve biomass properties. Xylan is the main hemicellulose in grass cell walls. Recently, we reported structural variation in grass xylans, suggesting functional specialisation and distinct interactions with cellulose and lignin. Here, we investigated the functions of these xylans by perturbing the biosynthesis of specific xylan types. C_LIO_LIWe generated CRISPR/Cas9 knockout mutants in Brachypodium distachyon XAX1 and GUX2 genes involved in xylan biosynthesis. Using carbohydrate gel electrophoresis we identified biochemical changes in different xylan types. Saccharification, cryo-SEM, subcritical water extraction and ssNMR were used to study wall architecture. C_LIO_LIBdXAX1A and BdGUX2 enzymes modify different types of grass xylan. Brachypodium mutant walls are more porous, suggesting the xylan substitutions directed by both BdXAX1A andGUX2 enzymes influence xylan-xylan and/or xylan-lignin interactions. C_LIO_LISince xylan substitutions influence wall architecture and digestibility, our findings open new avenues to improve cereals for food and to use grass biomass for feed and the production of bioenergy and biomaterials. C_LI

biochemistry↗

bioRxiv2023

Single-molecule tracking reveals dual front door/back door inhibition of Cel7A cellulase by its product cellobiose

Degrading cellulose is a key step in the processing of lignocellulosic biomass into bioethanol. Cellobiose, the disaccharide product of cellulose degradation, has been shown to inhibit cellulase activity, but the mechanisms underlying product inhibition are not clear. We combined single-molecule imaging and biochemical investigations with the goal of revealing the mechanism by which cellobiose inhibits the activity of Trichoderma reesei Cel7A, a well-characterized exo-cellulase. We find that cellobiose slows the processive velocity of Cel7A and shortens the distance moved per encounter; effects that can be explained by cellobiose binding to the product release site of the enzyme. Cellobiose also decreases the binding rate of Cel7A to immobilized cellulose but does not slow the binding rate of an isolated carbohydrate-binding module, suggesting that cellobiose inhibits binding of the catalytic domain of Cel7A to cellulose. In support of this, cellopentaose, which is considerably larger than cellobiose, also slows the binding rate of Cel7A to cellulose without affecting the velocity and run length. Together, these results suggest that cellobiose inhibits Cel7A activity both by binding to the back door product release site to slow activity and to the front door substrate binding tunnel to inhibit interaction with cellulose. These findings point to new strategies for engineering cellulases to reduce product inhibition and enhance cellulose degradation, supporting the growth of a sustainable bioeconomy. SignificanceCellulose, a polymer of repeating glucose subunits, is the primary component of plant cell walls. A promising route to reducing petrochemical use is digesting plant biomass to glucose and fermenting glucose to bioethanol. Cel7A is a model cellulase enzyme that degrades cellulose from one end to generate the disaccharide product, cellobiose. Because industrial-scale bioethanol generation generates high concentrations of cellobiose, product inhibition is a significant concern. We investigated product inhibition of Cel7A by cellobiose at the single-molecule level and found that cellobiose both slows the movement of Cel7 along cellulose and inhibits the initial binding of Cel7 to cellulose. These results suggest that cellobiose binds to the enzyme at more than one site and achieves its inhibition by multiple mechanisms.

plant biology↗

bioRxiv2021

An integrated multi-wavelength SCATTIRSTORM microscope combining TIRFM and IRM modalities for imaging cellulases and other processive enzymes

We describe a multimodal SCATTIRSTORM microscope for visualizing processive enzymes moving on immobilized substrates. The instrument combines Interference Reflection Microscopy (IRM) with multi-wavelength Total Internal Reflectance Fluorescence Microscopy (TIRFM). The microscope can localize quantum dots with a precision of 2.8 nm at 100 frames/s, and was used to image the dynamics of the cellulase, Cel7a interacting surface-immobilized cellulose. The instrument, which was built with off-the-shelf components and controlled by custom software, is suitable for tracking other degradative enzymes such as collagenases, as well as motor proteins moving along immobilized tracks.

biophysics↗

bioRxiv2021

Nanoscale dynamics of cellulase TrCel7A digesting cellulose

Understanding how cellulases catalyze the digestion of lignocellulose is a major goal of bioenergy research. Cel7A from Trichoderma reesei is a model exoglucanase that degrades cellulose strands from their reducing ends by processively cleaving individual cellobiose units. Despite being one of the most studied cellulases, the binding and hydrolysis mechanisms of Cel7A are still debated. We used single-molecule tracking to analyze the dynamics of 11,116 quantum dot-labeled TrCel7A binding to and moving processively along immobilized Gluconoacetobacter cellulose. Enzyme molecules were localized with a spatial precision of a few nanometers and followed for hundreds of seconds. Most enzymes bound into a static state and dissociated without detectable movement. Processive enzymes moved an average distance of 39 nm at an average speed of 3.2 nm/s. Static binding episodes preceding and following processive runs were of similar duration to static binding events that lacked any processive movement. Transient jumps of >20 nm were observed, but no diffusive behavior indicative of a diffusive search of the enzyme for a free cellulose strand end was observed. These data were integrated into a three-state model in which TrCel7A molecules can bind from solution into either a static or a processive state, and can reversibly switch between static and processive states before dissociating. From these results, we conclude that the rate-limiting step for cellulose degradation by Cel7A is the transition out of the static state either by dissociation from the cellulose surface or initiation of a processive run.

plant biology↗

bioRxiv2020

Cell wall remodeling and vesicle trafficking mediate the root clock in Arabidopsis

In Arabidopsis, lateral roots initiate along the primary root in a process preceded by periodic gene expression, a phenomenon known as the root clock. Many genes involved in lateral root initiation have been identified. However, very little is known about the structural changes underlying the initiation process nor about how root clock function is regulated. In genetic screens, we identified the vesicle trafficking regulators, GNOM and its suppressor, AGD3, as critical to root clock function. We show that GNOM is required for the proper distribution of pectin, a mediator of intercellular adhesion, and that pectin esterification state is essential for a functional root clock. We found that in sites of lateral root primordia emergence, both esterified and de-esterified pectin are differentially distributed. Using a reverse genetic approach, we identified significant enrichment of GO terms associated with pectin modifying enzymes in the oscillation zone were the root clock is established. In agreement with a recent study on the function of pectin in pavement cell morphogenesis, our results indicate that the balance between esterified and de-esterified pectin is essential for proper root clock function and the subsequent initiation of lateral root primordia.

plant biology↗

bioRxiv2019

The role of post-Golgi transport pathways and sorting motifs in the plasmodesmal targeting of the movement protein (MP) of Ourmia melon virus (OuMV)

Plants have a highly sophisticated endomembrane system targeted by plant viruses for cell-to-cell movement. The movement protein (MP) of ourmia melon virus (OuMV) is delivered to plasmodesmata (PD) and forms tubules to facilitate cell-to-cell movement. Although several functionally important regions for correct subcellular localization of OuMV MP have been identified, little is known about the pathways OuMV MP hijacks to reach the PD. Here, we demonstrate that OuMV MP localizes to the trans-Golgi network (TGN), but not to the multivesicular body/prevacuolar compartment or Golgi, and carries two putative sorting motifs, a tyrosine (Y) and a dileucine (LL) motif, near its N-terminus. Glycine substitutions in these motifs result in loss of OuMV infectivity in Nicotiana benthamiana and Arabidopsis. Live cell imaging of GFP-labeled sorting motif mutants shows that Y motif mutants fail to localize to the TGN, plasma membrane, and PD. Mutations in the LL motif do not impair plasma membrane targeting of MP but affect its ability to associate with callose deposits at the PD. Taken together, these results suggest that both motifs are indispensable for targeting OuMV MP to PD and for efficient systemic infection but show differences in functionality. Co-immunoprecipitation assays coupled with mass spectrometry identified a series of host factors that could interact with the OuMV MP and link the MP with various pathways, in particular vesicle trafficking and membrane lipids. This study provides new insights into the intracellular targeting of MPs and pathways that plant viruses hijack for cell-to-cell movement.

plant biology↗