Cellulose Synthase Proteins Research Articles

Genome-wide analysis of cellulose synthase (CesA) and cellulose synthase-like (Csl) proteins in Cannabis sativa L

The cellulose and hemicellulose components of plant cell walls are synthesized by the cellulose synthase (CESA) and cellulose synthase-like (CSL) gene families and regulated in response to growth, development, and environmental stimuli. In this study, a total of 29 CESA/CSL family members were identified in Cannabis sativa and were grouped into seven subfamilies (CESA, CSLA, CSLB, CSLC, CSLD, CSLE and CSLG) according to phylogenetic relationships. The CESA/CESA proteins of C. sativa were closely related phylogenetically to the members of the subfamily of other species. The CESA/CSL subfamily members of C. sativa have unique gene structures. In addition, the expressions of four CESA and 10 CsCSL genes in flower, leaf, root, and stem organs of cannabis were detected using RT-qPCR. The results showed that CESA and CSL genes are expressed at varying levels in several organs. This detailed knowledge of the structural, evolutionary, and functional properties of cannabis CESA/CSL genes will provide a basis for designing advanced experiments for genetic manipulation of cell wall biogenesis to improve bast fibers and biofuel production.

WssI from the Gram-negative bacterial cellulose synthase is an O-acetyltransferase that acts on cello-oligomers with several acetyl donor substrates

In microbial biofilms, bacterial cells are encased in a self-produced matrix of polymers (e.g., exopolysaccharides) that enable surface adherence and protect against environmental stressors. For example, the wrinkly spreader phenotype of Pseudomonas fluorescens colonizes food/water sources and human tissue to form robust biofilms that can spread across surfaces. This biofilm largely consists of bacterial cellulose produced by the cellulose synthase proteins encoded by the wss (WS structural) operon, which also occurs in other species, including pathogenic Achromobacter species. Although phenotypic mutant analysis of the wssFGHI genes has previously shown that they are responsible for acetylation of bacterial cellulose, their specific roles remain unknown and distinct from the recently identified cellulose phosphoethanolamine modification found in other species. Here, we have purified the C-terminal soluble form of WssI from P.fluorescens and Achromobacter insuavis and demonstrated acetylesterase activity with chromogenic substrates. The kinetic parameters (kcat/KM values of 13 and 8.0 M-1 s-1, respectively) indicate that these enzymes are up to four times more catalytically efficient than the closest characterized homolog, AlgJ from the alginate synthase. Unlike AlgJ and its cognate alginate polymer, WssI also demonstrated acetyltransferase activity onto cellulose oligomers (e.g., cellotetraose to cellohexaose) with multiple acetyl donor substrates (p-nitrophenyl acetate, 4-methylumbelliferyl acetate, and acetyl-CoA). Finally, a high-throughput screen identified three low micromolar WssI inhibitors that may be useful for chemically interrogating cellulose acetylation and biofilm formation.

Identification, Classification and Expression Analysis of the CesA Gene Family from Pinus massoniana

Cellulose synthase (CESA) is a key enzyme in the synthesis of cellulose, which plays an important role in cell wall construction and plant growth and development. In this study, seven CesA genes of P. massoniana were identified by searching the transcriptome data. Bioinformatics analysis showed that the putative CESA proteins were composed of 984–1101 amino acids, each containing the typical motifs of CESA proteins. Phylogenetic analysis showed that Transcript4609, Tran-script2643 and Transcript1263 were clustered into three groups with proteins related to regulating secondary wall synthesis, while Transcript691, Transcript1283, Transcript418 and Transcript556 were categorized into three clades with those associated with the formation of the primary cell walls. RT-qPCR analysis showed that the CesA genes were differentially expressed in different tissues, and most of the genes were induced by different abiotic stress and hormones. Transcript4609, Tran-script2643 and Transcript1263 were mainly expressed in the xylem and could respond to drought and salt stress induced by ABA, MeJA, ETH and SA hormones, indicating that these three CesA genes may play an important role in the response to abiotic stress in P. massoniana. This study revealed the possible biochemical and physiological functions of the CesA gene in P. massoniana, which can provide a basis for further exploration of the function of the CesA gene in cell wall formation and the response to external stress.

Point mutations in the catalytic domain disrupt cellulose synthase (CESA6) vesicle trafficking and protein dynamics.

Cellulose, the main component of the plant cell wall, is synthesized by the multimeric cellulose synthase (CESA) complex (CSC). In plant cells, CSCs are assembled in the endoplasmic reticulum or Golgi and transported through the endomembrane system to the plasma membrane (PM). However, how CESA catalytic activity or conserved motifs around the catalytic core influence vesicle trafficking or protein dynamics is not well understood. Here, we used yellow fluorescent protein (YFP)-tagged AtCESA6 and created 18 mutants in key motifs of the catalytic domain to analyze how they affected seedling growth, cellulose biosynthesis, complex formation, and CSC dynamics and trafficking in Arabidopsis thaliana. Seedling growth and cellulose content were reduced by nearly all mutations. Moreover, mutations in most conserved motifs slowed CSC movement in the PM as well as delivery of CSCs to the PM. Interestingly, mutations in the DDG and QXXRW motifs affected YFP-CESA6 abundance in the Golgi. These mutations also perturbed post-Golgi trafficking of CSCs. The 18 mutations were divided into two groups based on their phenotypes; we propose that Group I mutations cause CSC trafficking defects whereas Group II mutations, especially in the QXXRW motif, affect protein folding and/or CSC rosette formation. Collectively, our results demonstrate that the CESA6 catalytic domain is essential for cellulose biosynthesis as well as CSC formation, protein folding and dynamics, and vesicle trafficking.

CAGEs are Golgi-localized GT31 enzymes involved in cellulose biosynthesis in Arabidopsis.

SUMMARYCellulose is the main structural component in the plant cell walls. We show that two glycosyltransferase family 31 (GT31) enzymes of Arabidopsis thaliana, here named cellulose synthesis associated glycosyltransferases 1 and 2 (CAGE1 and 2), influence both primary and secondary cell wall cellulose biosynthesis. cage1cage2 mutants show primary cell wall defects manifesting as impaired growth and cell expansion in seedlings and etiolated hypocotyls, along with secondary cell wall defects, apparent as collapsed xylem vessels and reduced xylem wall thickness in the inflorescence stem. Single and double cage mutants also show increased sensitivity to the cellulose biosynthesis inhibitor isoxaben. The cage1cage2 phenotypes were associated with an approximately 30% reduction in cellulose content, an approximately 50% reduction in secondary cell wall CELLULOSE SYNTHASE (CESA) protein levels in stems and reduced cellulose biosynthesis rate in seedlings. CESA transcript levels were not significantly altered in cage1cage2 mutants, suggesting that the reduction in CESA levels was caused by a post‐transcriptional mechanism. Both CAGE1 and 2 localize to the Golgi apparatus and are predicted to synthesize β‐1,3‐galactans on arabinogalactan proteins. In line with this, the cage1cage2 mutants exhibit reduced levels of β‐Yariv binding to arabinogalactan protein linked β‐1,3‐galactan. This leads us to hypothesize that defects in arabinogalactan biosynthesis underlie the cellulose deficiency of the mutants.

Cellulose-synthesizing machinery in bacteria

Cellulose is produced by all plants and a number of other organisms, including bacteria. The most representative cellulose-producing bacterial species is Gluconacetobacter xylinus, an acetic acid bacterium. Cellulose produced by G. xylinus, commonly referred to as bacterial cellulose (BC), has exceptional physicochemical properties resulting in its use in a variety of applications. All cellulose-producing organisms that synthesize cellulose microfibrils have membrane-localized protein complexes (also called terminal complexes or TCs) that contain the enzyme cellulose synthase and other proteins. The bacterium G. xylinus is a prolific cellulose producer and a model organism for studies on cellulose biosynthesis. The widths of cellulose fibers produced by Gluconacetobacter are 50–100 nm, suggesting that cellulose-synthesizing complexes are nanomachines spinning a nanofiber. At least four different proteins (BcsA, BcsB, BcsC, and BcsD) are included in TC from Gluconacetobacter, and the proposed function of each is as follows: BcsA, synthesis of a glucan chain through glycosyl transfer from UDP-glucose; BcsB, complexes with BcsA for cellulose synthase activity; BcsC, formation of a pore in the outer membrane through which a glucan chain is extruded; BcsD, regulates aggregation of glucan chains through four tunnel-like structures. In this review, we discuss structures and functions of these four and a few other proteins that have a role in cellulose biosynthesis in bacteria.

Updating Insights into the Catalytic Domain Properties of Plant Cellulose synthase (CesA) and Cellulose synthase-like (Csl) Proteins.

The wall is the last frontier of a plant cell involved in modulating growth, development and defense against biotic stresses. Cellulose and additional polysaccharides of plant cell walls are the most abundant biopolymers on earth, having increased in economic value and thereby attracted significant interest in biotechnology. Cellulose biosynthesis constitutes a highly complicated process relying on the formation of cellulose synthase complexes. Cellulose synthase (CesA) and Cellulose synthase-like (Csl) genes encode enzymes that synthesize cellulose and most hemicellulosic polysaccharides. Arabidopsis and rice are invaluable genetic models and reliable representatives of land plants to comprehend cell wall synthesis. During the past two decades, enormous research progress has been made to understand the mechanisms of cellulose synthesis and construction of the plant cell wall. A plethora of cesa and csl mutants have been characterized, providing functional insights into individual protein isoforms. Recent structural studies have uncovered the mode of CesA assembly and the dynamics of cellulose production. Genetics and structural biology have generated new knowledge and have accelerated the pace of discovery in this field, ultimately opening perspectives towards cellulose synthesis manipulation. This review provides an overview of the major breakthroughs gathering previous and recent genetic and structural advancements, focusing on the function of CesA and Csl catalytic domain in plants.

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.

The role of ion-transporting proteins in the evolution of salt tolerance in charophyte algae.

Species within the genus Chara have variable salinity tolerance. Their close evolutionary relationship with embryophytes makes their study crucial to understanding the evolution of salt tolerance and key evolutionary processes shared among the phyla. We examined salt-tolerant Chara longifolia and salt-sensitive Chara australis for mechanisms of salt tolerance and their potential role in adaptation to salt. We hypothesize that there are shared mechanisms similar to those in embryophytes, which assist in conferring salt tolerance in Chara, including a cation transporter (HKT), a Na+ /H+ antiport (NHX), a H+ -ATPase (AHA), and a Na+ -ATPase (ENA). Illumina transcriptomes were created using cultures grown in freshwater and exposed to salt stress. The presence of these candidate genes, identified by comparing with genes known from embryophytes, has been confirmed in both species of Chara, with the exception of ENA, present only in salt-tolerant C.longifolia. These transcriptomes provide evidence for the contribution of these mechanisms to differences in salt tolerance in the two species and for the independent evolution of the Na+ -ATPase. We also examined genes that may have played a role in important evolutionary processes, suggested by previous work on the Chara braunii genome. Among the genes examined, cellulose synthase protein (GT43) and response regulator (RRB) were confirmed in both species. Genes absent from all three Chara species were members of the GRAS family, microtubule-binding protein (TANGLED1), and auxin synthesizers (YUCCA, TAA). Results from this study shed light on the evolutionary relationship between Chara and embryophytes through confirmation of shared salt tolerance mechanisms, as well as unique mechanisms that do not occur in angiosperms.

Genetic aspects of potato resistance to phytophthorosis

Оомицет Рhytophthora infestans Mont. de Bary – основной патоген сельскохозяйственных культур семейства Пасленовые, особенно картофеля (Solanum tuberosum). С учетом того, что картофель – четвертая культура в мире по масштабам выращивания, ежегодные потери от фитофтороза огромны. Исследования базовых механизмов взаимодействия между картофелем и возбудителем фитофтороза не только расширяют фундаментальные знания в этой области, но и открывают новые возможности для влияния на эти взаимодействия с целью повышения резистентности к патогену. Взаимодействие картофеля и возбудителя фитофтороза можно рассматривать с генетической точки зрения, причем интересны как ответ картофеля на процесс колонизации со стороны P. infestans, так и изменение активности генов у фитофторы при заражении растения. Можно исследовать этот процесс через изменение профиля вторичных метаболитов хозяина и патогена. Помимо фундаментальных исследований в этой области, не меньшее значение имеют и прикладные работы в виде создания новых препаратов для защиты картофеля. Представленный обзор кратко описывает основные этапы исследований устойчивости картофеля к фитофторозу, начиная с самых первых работ. Большое внимание уделяется ключевым моментам по изменению профиля вторичных метаболитов (фитоалексинов). Отдельный раздел посвящен описанию как качественных, так количественных признаков устойчивости картофеля к возбудителю фитофтороза: их вкладу в общую резистентность, картированию и возможности регуляции. Оба вида признаков важны для селекции картофеля: качественная устойчивость за счет R-генов быстро преодолевается патогеном, в то время как пирамидирование локусов количественных признаков способствует созданию высокоустойчивых сортов. Новейшие подходы молекулярной биологии дают возможность изучать и транслятомные профили, что позволяет посмотреть на взаимодействие картофеля и возбудителя фитофтороза. Показано, что процесс колонизации картофеля отражается не только на активности различных генов и профиле вторичных метаболитов, выявлены также белки-маркеры ответа на заражение со стороны картофеля – это патоген-зависимые белки и пластидная углекислая ангидраза. Маркерами заражения от P. infestans были белки грибной целлюлозо-синтазы и гаусторий-специфический мембранный белок. В данном обзоре приведена информация по наиболее актуальным комплексным исследованиям генетических механизмов устойчивости картофеля к фитофторозу.

The molecular basis of plant cellulose synthase complex organisation and assembly.

The material properties of cellulose are heavily influenced by the organisation of β-1,4-glucan chains into a microfibril. It is likely that the structure of this microfibril is determined by the spatial arrangement of catalytic cellulose synthase (CESA) proteins within the cellulose synthase complex (CSC). In land plants, CESA proteins form a large complex composed of a hexamer of trimeric lobes termed the rosette. Each rosette synthesises a single microfibril likely composed of 18 glucan chains. In this review, the biochemical events leading to plant CESA protein assembly into the rosette are explored. The protein interfaces responsible for CESA trimerization are formed by regions that define rosette-forming CESA proteins. As a consequence, these regions are absent from the ancestral bacterial cellulose synthases (BcsAs) that do not form rosettes. CSC assembly occurs within the context of the endomembrane system, however the site of CESA assembly into trimers and rosettes is not determined. Both the N-Terminal Domain and Class Specific Region of CESA proteins are intrinsically disordered and contain all of the identified phosphorylation sites, making both regions candidates as sites for protein-protein interactions and inter-lobe interface formation. We propose a sequential assembly model, whereby CESA proteins form stable trimers shortly after native folding, followed by sequential recruitment of lobes into a rosette, possibly assisted by Golgi-localised STELLO proteins. A comprehensive understanding of CESA assembly into the CSC will enable directed engineering of CESA protein spatial arrangements, allowing changes in cellulose crystal packing that alter its material properties.

Analysis of a cellulose synthase catalytic subunit from the oomycete pathogen of crops Phytophthora capsici

Phytophthora capsici Leonian is an important oomycete pathogen of crop vegetables, causing significant economic losses each year. Its cell wall, rich in cellulose, is vital for cellular integrity and for interactions with the host organisms. Predicted cellulose synthase (CesA) proteins are expected to catalyze the polymerization of cellulose, but this has not been biochemically demonstrated in an oomycete. Here, we present the properties of the four newly identified CesA proteins from P. capsici and compare their domain organization with that of CesAs from other lineages. Using a newly constructed glucosyltransferase-deficient variant of Saccharomyces cerevisiae with low residual background activity, we have achieved successful heterologous expression and biochemical characterization of a CesA protein from P. capsici (PcCesA1). Our results demonstrate that the individual PcCesA1 enzyme produces cellobiose as the major reaction product. Co-immunoprecipitation studies and activity assays revealed that several PcCesA proteins interact together to form a complex whose multiproteic nature is most likely required for cellulose microfibril formation. In addition to providing important insights into cellulose synthesis in the oomycetes, our data may assist the longer term identification of cell wall biosynthesis inhibitors to control infection by pathogenic oomycetes.

Associations between phytohormones and cellulose biosynthesis in land plants.

Phytohormones are small molecules that regulate virtually every aspect of plant growth and development, from basic cellular processes, such as cell expansion and division, to whole plant environmental responses. While the phytohormone levels and distribution thus tell the plant how to adjust itself, the corresponding growth alterations are actuated by cell wall modification/synthesis and internal turgor. Plant cell walls are complex polysaccharide-rich extracellular matrixes that surround all plant cells. Among the cell wall components, cellulose is typically the major polysaccharide, and is the load-bearing structure of the walls. Hence, the cell wall distribution of cellulose, which is synthesized by large Cellulose Synthase protein complexes at the cell surface, directs plant growth. Here, we review the relationships between key phytohormone classes and cellulose deposition in plant systems. We present the core signalling pathways associated with each phytohormone and discuss the current understanding of how these signalling pathways impact cellulose biosynthesis with a particular focus on transcriptional and post-translational regulation. Because cortical microtubules underlying the plasma membrane significantly impact the trajectories of Cellulose Synthase Complexes, we also discuss the current understanding of how phytohormone signalling impacts the cortical microtubule array. Given the importance of cellulose deposition and phytohormone signalling in plant growth and development, one would expect that there is substantial cross-talk between these processes; however, mechanisms for many of these relationships remain unclear and should be considered as the target of future studies.

Cellulose biosynthesis in plants - the concerted action of CESA and non-CESA proteins

Cellulose is the most abundant polysaccharide produced by plants. In the form of rigid microfibrils surrounding the cells, cellulose constitutes the load-bearing cell wall element that controls cell growth and shape. Cellulose microfibrils are laid down outside the cell by the multimeric plasma membrane-inserted cellulose synthase complexes (CSCs), which move along underlying cortical microtubules (CMTs). In plants, CSCs are shaped as rosettes with six lobes symmetrically arranged in a hexagonal structure. In Arabidopsis, the CSC is composed of at least three functionally non-redundant cellulose synthase (CESA) glycosyltransferases in both primary and secondary cell walls. The number, organization, and interactions of CESA proteins within the CSC have been debated for many years on the basis of numerous lines of evidence provided by electron microscopy, biochemical and genetic approaches, spectroscopic techniques, as well as computational modeling. The Arabidopsis thaliana model was extremely useful in elucidating the molecular composition of CSC and enabled to elucidate the specialized functions of distinct AtCESA isoforms. Several additional, non-CESA proteins involved in cellulose synthesis and its regulation were also identified in Arabidopsis. This review outlines the latest findings on CSC organization, trafficking, and plant-specific proteins directly associated with the complex and interconnecting CESAs with CMTs.

In silico structure prediction of full-length cotton cellulose synthase protein (GhCESA1) and its hierarchical complexes

Cellulose synthase (CESA) polymerizes glucose into β-1,4-glucan chains that assemble to form cellulose microfibrils. Cellulose is the most abundant natural polymer in the world and a major structural component of the plant cell wall. An understanding of cellulose synthesis in plants is crucial for advancement in biofuels research and requires high-resolution 3D CESA structures. However, the determination of 3D structures of plant CESAs and their specific hierarchical arrangement into cellulose synthesis complexes (CSCs) has been a challenge. The prediction of CESA structures using computational methods presents a challenge due to poor sequence homology with resolved structures, long sequence, and structural complexity due to a mixture of globular, transmembrane, and intrinsically disordered regions. Herein, we present a 3D atomic-resolution model of a full-length (974-aa) cotton CESA (GhCESA1) structure using a variety of computational techniques with a reasonable ProSA-web z-score of − 8.32 (PDB available in SI). The overall fold of the CESA model indicates that there are similarities to BcsA bacterial cellulose synthase, such as the transmembrane topology and the internalization of conserved catalytic motifs. The plant-specific regions (CSR, P-CR, and N-term) fold into distinct subdomains, indicating the importance of these regions in CESA assembly into plant CSCs. We further examined possible assemblies of CESA monomers forming trimers and 18-mer CSCs, and we compared our results to those obtained by freeze fracture transmission electron microscopy. We observed that there are numerous competing ways in which CESAs may be arranged into homotrimers and CSCs. Our predicted structure can be used to probe CESA structure–activity relationships, select and subsequently test possible mutants, and investigate CESA aggregation into CSCs and microfibril formation to optimize biomass properties.

Endosidin20 Targets the Cellulose Synthase Catalytic Domain to Inhibit Cellulose Biosynthesis

Plant cellulose is synthesized by rosette-structured cellulose synthase (CESA) complexes (CSCs). Each CSC is composed of multiple subunits of CESAs representing three different isoforms. Individual CESA proteins contain conserved catalytic domains for catalyzing cellulose synthesis, other domains such as plant-conserved sequences, and class-specific regions that are thought to facilitate complex assembly and CSC trafficking. Because of the current lack of atomic-resolution structures for plant CSCs or CESAs, the molecular mechanism through which CESA catalyzes cellulose synthesis and whether its catalytic activity influences efficient CSC transport at the subcellular level remain unknown. Here, by performing chemical genetic analyses, biochemical assays, structural modeling, and molecular docking, we demonstrate that Endosidin20 (ES20) targets the catalytic site of CESA6 in Arabidopsis (Arabidopsis thaliana). Chemical genetic analysis revealed important amino acids that potentially participate in the catalytic activity of plant CESA6, in addition to previously identified conserved motifs across kingdoms. Using high spatiotemporal resolution live cell imaging, we found that inhibiting the catalytic activity of CESA6 by ES20 treatment reduced the efficiency of CSC transport to the plasma membrane. Our results demonstrate that ES20 is a chemical inhibitor of CESA activity and trafficking that represents a powerful tool for studying cellulose synthesis in plants.

Biochemical and Genetic Analysis Identify CSLD3 as a beta-1,4-Glucan Synthase That Functions during Plant Cell Wall Synthesis.

In plants, changes in cell size and shape during development fundamentally depend on the ability to synthesize and modify cell wall polysaccharides. The main classes of cell wall polysaccharides produced by terrestrial plants are cellulose, hemicelluloses, and pectins. Members of the cellulose synthase (CESA) and cellulose synthase-like (CSL) families encode glycosyltransferases that synthesize the β-1,4-linked glycan backbones of cellulose and most hemicellulosic polysaccharides that comprise plant cell walls. Cellulose microfibrils are the major load-bearing component in plant cell walls and are assembled from individual β-1,4-glucan polymers synthesized by CESA proteins that are organized into multimeric complexes called CESA complexes, in the plant plasma membrane. During distinct modes of polarized cell wall deposition, such as in the tip growth that occurs during the formation of root hairs and pollen tubes or de novo formation of cell plates during plant cytokinesis, newly synthesized cell wall polysaccharides are deposited in a restricted region of the cell. These processes require the activity of members of the CESA-like D subfamily. However, while these CSLD polysaccharide synthases are essential, the nature of the polysaccharides they synthesize has remained elusive. Here, we use a combination of genetic rescue experiments with CSLD-CESA chimeric proteins, in vitro biochemical reconstitution, and supporting computational modeling and simulation, to demonstrate that Arabidopsis (Arabidopsis thaliana) CSLD3 is a UDP-glucose-dependent β-1,4-glucan synthase that forms protein complexes displaying similar ultrastructural features to those formed by CESA6.

Structural Conservation and Diversity of PilZ-Related Domains.

The widespread bacterial second messenger cyclic diguanylate (c-di-GMP) regulates a variety of processes, including protein secretion, motility, cell development, and biofilm formation. c-di-GMP-dependent responses are often mediated by its binding to the cytoplasmic receptors that contain the PilZ domain. Here, we present comparative structural and sequence analysis of various PilZ-related domains and describe three principal types of them: (i) the canonical PilZ domain, whose structure includes a six-stranded beta-barrel and a C-terminal alpha helix, (ii) an atypical PilZ domain that contains two extra alpha helices and forms stable tetramers, and (iii) divergent PilZ-related domains, which include the eponymous PilZ protein and PilZN (YcgR_N) and PilZNR (YcgR_2) domains. We refine the second c-di-GMP binding motif of PilZ as [D/N]hSXXG and show that the hydrophobic residue h of this motif interacts with a cluster of conserved hydrophobic residues, helping maintain the PilZ domain fold. We describe several novel PilZN-type domains that are fused to the canonical PilZ domains in specific taxa, such as spirochetes, actinobacteria, aquificae, cellulose-degrading clostridia, and deltaproteobacteria. We propose that the evolution of the three major groups of PilZ domains included (i) fusion of pilZ with other genes, which produced Alg44, cellulose synthase, and other multidomain proteins; (ii) insertion of an ∼200-bp fragment, which resulted in the formation of tetramer-forming PilZ proteins; and (iii) tandem duplication of pilZ genes, which led to the formation of PilZ dimers and YcgR-like proteins.IMPORTANCE c-di-GMP is a ubiquitous bacterial second messenger that regulates motility, biofilm formation, and virulence of many bacterial pathogens. The PilZ domain is a widespread c-di-GMP receptor that binds c-di-GMP through its RXXXR and [D/N]hSXXG motifs; some PilZ domains lack these motifs and are unable to bind c-di-GMP. We used structural and sequence analysis to assess the diversity of PilZ-related domains and define their common features. We show that the hydrophobic residue h in the second position of the second motif is highly conserved; it may serve as a readout for c-di-GMP binding. We describe three principal classes of PilZ-related domains, canonical, tetramer-forming, and divergent PilZ domains, and propose the evolutionary pathways that led to the emergence of these PilZ types.

Imaging the delivery and behavior of cellulose synthases in Arabidopsis thaliana using confocal microscopy.

Confocal microscopy has been a key tool for characterizing the behavior of cellulose synthase (CESA) proteins as they extrude cellulose into the apoplast to help construct plant cell walls. While other microscopy techniques like electron microscopy can achieve higher resolution images of CESAs, confocal microscopy is still the most accessible way to image these proteins in living plants as they are trafficked to and from the cell surface and move through the plasma membrane. Here, we describe a method for imaging fluorescently tagged CESA proteins in seedlings of Arabidopsis thaliana using spinning disk confocal microscopy, with a focus on quantifying the speed, density, and delivery rate of CESA particles. Many of these techniques can be adapted and applied to imaging other membrane-localized proteins and other plant species. In addition to imaging techniques, we describe several options for image analysis that can be optimized for different datasets.

Bioinformatics Analysis of Plant Cell Wall Evolution.

In the past hundreds of millions of years, from green algae to land plants, cell walls have developed into a highly complex structure that is essential for plant growth and survival. Plant cell wall diversity and evolution can be directly investigated by chemically profiling polysaccharides and lignins in the cell walls of diverse plants and algae. With the increasingly low cost and high throughput of DNA sequencing technologies, cell wall evolution can also be studied by bioinformatics analysis of the occurrence of cell wall synthesis-related enzymes in the genomes and transcriptomes of different species. This chapter presents a bioinformatics workflow running on a Linux platform to process genomic data for such gene occurrence analysis. As a case study, cellulose synthase (CesA) and CesA-like (Csl) protein families are mined for in two newly sequenced organisms: the charophyte green alga Klebsormidium flaccidum (renamed as Klebsormidium nitens) and the fern Lygodium japonicum.