Intragenic complementation at the Lotus japonicus CELLULOSE SYNTHASE-LIKE D1 locus rescues root hair defects.

Root hair cells form the primary interface of plants with the soil environment, playing key roles in nutrient uptake and plant defense. In legumes, they are typically the first cells to become infected by nitrogen-fixing soil bacteria during root nodule symbiosis. Here, we report a role for the CELLULOSE SYNTHASE-LIKE D1 (CSLD1) gene in root hair development in the legume species Lotus japonicus. CSLD1 belongs to the cellulose synthase protein family that includes cellulose synthases and cellulose synthase-like proteins, the latter thought to be involved in the biosynthesis of hemicellulose. We describe 11 Ljcsld1 mutant alleles that impose either short (Ljcsld1-1) or variable (Ljcsld1-2 to 11) root hair length phenotypes. Examination of Ljcsld1-1 and one variable-length root hair mutant, Ljcsld1-6, revealed increased root hair cell wall thickness, which in Ljcsld1-1 was significantly more pronounced and also associated with a strong defect in root nodule symbiosis. Lotus japonicus plants heterozygous for Ljcsld1-1 exhibited intermediate root hair lengths, suggesting incomplete dominance. Intragenic complementation was observed between alleles with mutations in different CSLD1 domains, suggesting CSLD1 function is modular and that the protein may operate as a homodimer or multimer during root hair development.

With an Ear Up against the Wall: An Update on Mechanoperception in Arabidopsis.

Cells interpret mechanical signals and adjust their physiology or development appropriately. In plants, the interface with the outside world is the cell wall, a structure that forms a continuum with the plasma membrane and the cytoskeleton. Mechanical stress from cell wall damage or deformation is interpreted to elicit compensatory responses, hormone signalling, or immune responses. Our understanding of how this is achieved is still evolving; however, we can refer to examples from animals and yeast where more of the details have been worked out. Here, we provide an update on this changing story with a focus on candidate mechanosensitive channels and plasma membrane-localized receptors.

Anno genominis XX: 20 years of Arabidopsis genomics.

Twenty years ago, the Arabidopsis thaliana genome sequence was published. This was an important moment as it was the first sequenced plant genome and explicitly brought plant science into the genomics era. At the time, this was not only an outstanding technological achievement, but it was characterized by a superb global collaboration. The Arabidopsis genome was the seed for plant genomic research. Here, we review the development of numerous resources based on the genome that have enabled discoveries across plant species, which has enhanced our understanding of how plants function and interact with their environments.

Missense mutations in a transmembrane domain of the Komagataeibacter xylinus BcsA lead to changes in cellulose synthesis.

BACKGROUND: Cellulose is synthesized by an array of bacterial species. Komagataeibacter xylinus is the best characterized as it produces copious amounts of the polymer extracellularly. Despite many advances in the past decade, the mechanisms underlying cellulose biosynthesis are not completely understood. Elucidation of these mechanisms is essential for efficient cellulose production in industrial applications. RESULTS: In an effort to gain a better understanding of cellulose biosynthesis and its regulation, cellulose crystallization was investigated in K. xylinus mutants resistant to an inhibitor of cellulose I formation, pellicin. Through the use of forward genetics and site-directed mutagenesis, A449T and A449V mutations in the K. xylinus BcsA protein were found to be important for conferring high levels of pellicin resistance. Phenotypic analysis of the bcsAA449T and bcsAA449V cultures revealed that the mutations affect cellulose synthesis rates and that cellulose crystallinity is affected in wet pellicles but not dry ones. CONCLUSIONS: A449 is located in a predicted transmembrane domain of the BcsA protein suggesting that the structure of the transmembrane domain influences cellulose crystallization either by affecting the translocation of the nascent glucan chain or by allosterically altering protein-protein interactions.

Effects of environmentally relevant metformin exposure on Japanese medaka (Oryzias latipes).

Metformin is one of the most prevalent pharmaceuticals in both surface and waste waters, yet little is known about the bioavailability and/or effects of developmental exposure on early life stage (ELS) fish. Here, we demonstrate that embryo-larval stages of medaka are capable of taking up metformin from the aquatic environment, provided exposure occurs prior to chorion hardening (∼6-hpf). Once transferred to clean water, ELS medaka are able to completely depurate metformin in <24-hours. Furthermore, ELS medaka exposed to a range of relevant concentrations of waterborne metformin (from 6 hpf through 28-days post hatch) had significantly reduced growth metrics, altered metabolomes, and changes in the expression of genes associated with cell growth. The range of concentrations investigated were 1.0, 3.2, 10, 32, and 100 µg·L-1. To examine effects of chronic, low level metformin exposure across the full medaka life-cycle, we exposed newly fertilized embryos to 3.2 µg L-1 waterborne metformin for 165-days. The weight and length of adult fish were examined, as were effects on the production of some steroid hormones, specifically a significant increase (control females: 0.161 ± 0.023 pg/mg; metformin treated females: 3.42 ± 0.543) in the production of 11-ketotestosterone was observed in adult female medaka. Collectively, these results suggest that current environmental exposure scenarios may be sufficient to cause effects on developing fish.

Alleles Causing Resistance to Isoxaben and Flupoxam Highlight the Significance of Transmembrane Domains for CESA Protein Function.

The cellulose synthase (CESA) proteins in Arabidopsis play an essential role in the production of cellulose in the cell walls. Herbicides such as isoxaben and flupoxam specifically target this production process and are prominent cellulose biosynthesis inhibitors (CBIs). Forward genetic screens in Arabidopsis revealed that mutations that can result in varying degrees of resistance to either isoxaben or flupoxam CBI can be attributed to single amino acid substitutions in primary wall CESAs. Missense mutations were almost exclusively present in the predicted transmembrane regions of CESA1, CESA3, and CESA6. Resistance to isoxaben was also conferred by modification to the catalytic residues of CESA3. This resulted in cellulose deficient phenotypes characterized by reduced crystallinity and dwarfism. However, mapping of mutations to the transmembrane regions also lead to growth phenotypes and altered cellulose crystallinity phenotypes. These results provide further genetic evidence supporting the involvement of CESA transmembrane regions in cellulose biosynthesis.

Tertiary model of a plant cellulose synthase.

A 3D atomistic model of a plant cellulose synthase (CESA) has remained elusive despite over forty years of experimental effort. Here, we report a computationally predicted 3D structure of 506 amino acids of cotton CESA within the cytosolic region. Comparison of the predicted plant CESA structure with the solved structure of a bacterial cellulose-synthesizing protein validates the overall fold of the modeled glycosyltransferase (GT) domain. The coaligned plant and bacterial GT domains share a six-stranded ß-sheet, five α-helices, and conserved motifs similar to those required for catalysis in other GT-2 glycosyltransferases. Extending beyond the cross-kingdom similarities related to cellulose polymerization, the predicted structure of cotton CESA reveals that plant-specific modules (plant-conserved region and class-specific region) fold into distinct subdomains on the periphery of the catalytic region. Computational results support the importance of the plant-conserved region and/or class-specific region in CESA oligomerization to form the multimeric cellulose-synthesis complexes that are characteristic of plants. Relatively high sequence conservation between plant CESAs allowed mapping of known mutations and two previously undescribed mutations that perturb cellulose synthesis in Arabidopsis thaliana to their analogous positions in the modeled structure. Most of these mutation sites are near the predicted catalytic region, and the confluence of other mutation sites supports the existence of previously undefined functional nodes within the catalytic core of CESA. Overall, the predicted tertiary structure provides a platform for the biochemical engineering of plant CESAs.

Forward genetic screening for the improved production of fermentable sugars from plant biomass.

With their unique metabolism and the potential to produce large amounts of biomass, plants are an excellent bio-energy feedstock for a variety of industrial purposes. Here we developed a high-throughput strategy, using the model plant Arabidopsis thaliana, to identify mutants with improved sugar release from plant biomass. Molecular analysis indicates a variety of processes including starch degradation, cell wall composition and polar transport of the plant hormone auxin can contribute to this improved saccharification. To demonstrate translatability, polar auxin transport in maize was either genetically or chemical inhibited and this also resulted in increased sugar release from plant tissues. Our forward genetic approach using Arabidopsis not only uncovers new functions that contribute to cell wall integrity but also demonstrates that information gleaned from this genetic model can be directly translated to monocotyledonous crops such as maize to improve sugar extractability from biomass.

The FRIABLE1 gene product affects cell adhesion in Arabidopsis.

Cell adhesion in plants is mediated predominantly by pectins, a group of complex cell wall associated polysaccharides. An Arabidopsis mutant, friable1 (frb1), was identified through a screen of T-DNA insertion lines that exhibited defective cell adhesion. Interestingly, the frb1 plants displayed both cell and organ dissociations and also ectopic defects in organ separation. The FRB1 gene encodes a Golgi-localized, plant specific protein with only weak sequence similarities to known proteins (DUF246). Unlike other cell adhesion deficient mutants, frb1 mutants do not have reduced levels of adhesion related cell wall polymers, such as pectins. Instead, FRB1 affects the abundance of galactose- and arabinose-containing oligosaccharides in the Golgi. Furthermore, frb1 mutants displayed alteration in pectin methylesterification, cell wall associated extensins and xyloglucan microstructure. We propose that abnormal FRB1 action has pleiotropic consequences on wall architecture, affecting both the extensin and pectin matrices, with consequent changes to the biomechanical properties of the wall and middle lamella, thereby influencing cell-cell adhesion.

Negative regulation of CCaMK is essential for symbiotic infection.

One of the earliest responses of legumes to symbiotic signalling is oscillation of the calcium concentration in the nucleoplasm of root epidermal cells. Integration and decoding of the calcium-spiking signal involve a calcium- and calmodulin-dependent protein kinase (CCaMK) and its phosphorylation substrates, such as CYCLOPS. Here we describe the Lotus japonicus ccamk-14 mutant that originated from a har1-1 suppressor screen. The ccamk-14 mutation causes a serine to asparagine substitution at position 337 located within the calmodulin binding site, which we determined to be an in vitro phosphorylation site in CCaMK. We show that ccamk-14 exerts cell-specific effects on symbiosis. The mutant is characterized by an increased frequency of epidermal infections and significantly compromised cortical infections by Mesorhizobium loti and also the arbuscular mycorrhiza fungus Rhizophagus irregularis. The S337 residue is conserved across angiosperm CCaMKs, and testing discrete substitutions at this site showed that it participates in a negative regulation of CCaMK activity, which is required for the cell-type-specific integration of symbiotic signalling.

Accelerating forward genetics for cell wall deconstruction.

The elucidation of the genes involved in cell wall synthesis and assembly remains one of the biggest challenges of cell wall biology. Although traditional genetic approaches, using simple yet elegant screens, have identified components of the cell wall, many unknowns remain. Exhausting the genetic toolbox by performing sensitized screens, adopting chemical genetics or combining these with improved cell wall imaging, hold the promise of new gene discovery and function. With the recent introduction of next-generation sequencing technologies, it is now possible to quickly and efficiently map and clone genes of interest in record time. The combination of a classical genetics approach and cutting edge technology will propel cell wall biology in plants forward into the future.

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 CESA1(A903V) and CESA3(T942I) in Arabidopsis thaliana. Using (13)C 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 CESA1(A903V) and CESA3(T942I) 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 CESA1(A903V) and CESA3(T942I) have modified microfibril structure in terms of crystallinity and suggest that in plants, as in bacteria, crystallization biophysically limits polymerization.

Characterization of pellicle inhibition in Gluconacetobacter xylinus 53582 by a small molecule, pellicin, identified by a chemical genetics screen.

Pellicin ([2E]-3-phenyl-1-[2,3,4,5-tetrahydro-1,6-benzodioxocin-8-yl]prop-2-en-1-one) was identified in a chemical genetics screen of 10,000 small molecules for its ability to completely abolish pellicle production in Gluconacetobacter xylinus. Cells grown in the presence of pellicin grew 1.5 times faster than untreated cells. Interestingly, growth in pellicin also caused G. xylinus cells to elongate. Measurement of cellulose synthesis in vitro showed that cellulose synthase activity was not directly inhibited by pellicin. Rather, when cellulose synthase activity was measured in cells that were pre-treated with the compound, the rate of cellulose synthesis increased eight-fold over that observed for untreated cells. This phenomenon was also apparent in the rapid production of cellulose when cells grown in the presence of pellicin were washed and transferred to media lacking the inhibitor. The rate at which cellulose was produced could not be accounted for by growth of the organism. Pellicin was not detected when intracellular contents were analyzed. Furthermore, it was found that pellicin exerts its effect extracellularly by interfering with the crystallization of pre-cellulosic tactoidal aggregates. This interference of the crystallization process resulted in enhanced production of cellulose II as evidenced by the ratio of acid insoluble to acid soluble product in in vitro assays and confirmed in vivo by scanning electron microscopy and powder X-ray diffraction. The relative crystallinity index, RCI, of pellicle produced by untreated G. xylinus cultures was 70% while pellicin-grown cultures had RCI of 38%. Mercerized pellicle of untreated cells had RCI of 42%, which further confirms the mechanism of action of pellicin as an inhibitor of the cellulose I crystallization process. Pellicin is a useful tool for the study of cellulose biosynthesis in G. xylinus.

Next-generation mapping of Arabidopsis genes.

Next-generation genomic sequencing technologies have made it possible to directly map mutations responsible for phenotypes of interest via direct sequencing. However, most mapping strategies proposed to date require some prior genetic analysis, which can be very time-consuming even in genetically tractable organisms. Here we present a de novo method for rapidly and robustly mapping the physical location of EMS mutations by sequencing a small pooled F2 population. This method, called Next Generation Mapping (NGM), uses a chastity statistic to quantify the relative contribution of the parental mutant and mapping lines to each SNP in the pooled F2 population. It then uses this information to objectively localize the candidate mutation based on its exclusive segregation with the mutant parental line. A user-friendly, web-based tool for performing NGM analysis is available at http://bar.utoronto.ca/NGM. We used NGM to identify three genes involved in cell-wall biology in Arabidopsis thaliana, and, in a power analysis, demonstrate success in test mappings using as few as ten F2 lines and a single channel of Illumina Genome Analyzer data. This strategy can easily be applied to other model organisms, and we expect that it will also have utility in crops and any other eukaryote with a completed genome sequence.

Abscisic acid inhibits type 2C protein phosphatases via the PYR/PYL family of START proteins.

Type 2C protein phosphatases (PP2Cs) are vitally involved in abscisic acid (ABA) signaling. Here, we show that a synthetic growth inhibitor called pyrabactin functions as a selective ABA agonist. Pyrabactin acts through PYRABACTIN RESISTANCE 1 (PYR1), the founding member of a family of START proteins called PYR/PYLs, which are necessary for both pyrabactin and ABA signaling in vivo. We show that ABA binds to PYR1, which in turn binds to and inhibits PP2Cs. We conclude that PYR/PYLs are ABA receptors functioning at the apex of a negative regulatory pathway that controls ABA signaling by inhibiting PP2Cs. Our results illustrate the power of the chemical genetic approach for sidestepping genetic redundancy.

Sentinels at the wall: cell wall receptors and sensors.

The emerging view of the plant cell wall is of a dynamic and responsive structure that exists as part of a continuum with the plasma membrane and cytoskeleton. This continuum must be responsive and adaptable to normal processes of growth as well as to stresses such as wounding, attack from pathogens and mechanical stimuli. Cell expansion involving wall loosening, deposition of new materials, and subsequent rigidification must be tightly regulated to allow the maintenance of cell wall integrity and co-ordination of development. Similarly, sensing and feedback are necessary for the plant to respond to mechanical stress or pathogen attack. Currently, understanding of the sensing and feedback mechanisms utilized by plants to regulate these processes is limited, although we can learn from yeast, where the signalling pathways have been more clearly defined. Plant cell walls possess a unique and complicated structure, but it is the protein components of the wall that are likely to play a crucial role at the forefront of perception, and these are likely to include a variety of sensor and receptor systems. Recent plant research has yielded a number of interesting candidates for cell wall sensors and receptors, and we are beginning to understand the role that they may play in this crucial aspect of plant biology.

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.

The ABSCISIC ACID INSENSITIVE 3 (ABI3) gene is modulated by farnesylation and is involved in auxin signaling and lateral root development in Arabidopsis.

Genetic screens have identified a number of genes that regulate abscisic acid (ABA) responsiveness in Arabidopsis. Using a combination of suppressor screens and double mutant analysis, we have determined a genetic relationship for a number of these ABA response loci. Based on germination in the presence of exogenous ABA, the ABI1 and ABI2 phosphatases act at or upstream of the ERA1 farnesyl transferase and the ABI3 and ABI5 transcription factors act at or downstream of ERA1. In contrast with ABI3 and ABI5, the ABI4 transcription factor appears to act at or upstream of ERA1. Based on reporter gene constructs, the upstream regulation of ABI3 by ERA1 occurs at least partially at the level of transcription, suggesting that this lipid modification is required to attenuate ABI3 expression. Similar experiments also indicate that ABI3 is auxin inducible in lateral root primordia. Related to this, loss-of-function abi3 alleles show reduced lateral root responsiveness in the presence of auxin and an auxin transport inhibitor, and era1 mutants have increased numbers of lateral roots. These results suggest the possibility that genes identified through ABA responsive germination screens such as ERA1 and ABI3 have functions in auxin action in Arabidopsis.