Sticking to cellulose: exploiting Arabidopsis seed coat mucilage to understand cellulose biosynthesis and cell wall polysaccharide interactions.

The cell wall defines the shape of cells and ultimately plant architecture. It provides mechanical resistance to osmotic pressure while still being malleable and allowing cells to grow and divide. These properties are determined by the different components of the wall and the interactions between them. The major components of the cell wall are the polysaccharides cellulose, hemicellulose and pectin. Cellulose biosynthesis has been extensively studied in Arabidopsis hypocotyls, and more recently in the mucilage-producing epidermal cells of the seed coat. The latter has emerged as an excellent system to study cellulose biosynthesis and the interactions between cellulose and other cell wall polymers. Here we review some of the major advances in our understanding of cellulose biosynthesis in the seed coat, and how mucilage has aided our understanding of the interactions between cellulose and other cell wall components required for wall cohesion. Recently, 10 genes involved in cellulose or hemicellulose biosynthesis in mucilage have been identified. These discoveries have helped to demonstrate that xylan side-chains on rhamnogalacturonan I act to link this pectin directly to cellulose. We also examine other factors that, either directly or indirectly, influence cellulose organization or crystallization in mucilage.

Unidirectional movement of cellulose synthase complexes in Arabidopsis seed coat epidermal cells deposit cellulose involved in mucilage extrusion, adherence, and ray formation.

Cellulose synthase5 (CESA5) synthesizes cellulose necessary for seed mucilage adherence to seed coat epidermal cells of Arabidopsis (Arabidopsis thaliana). The involvement of additional CESA proteins in this process and details concerning the manner in which cellulose is deposited in the mucilage pocket are unknown. Here, we show that both CESA3 and CESA10 are highly expressed in this cell type at the time of mucilage synthesis and localize to the plasma membrane adjacent to the mucilage pocket. The isoxaben resistant1-1 and isoxaben resistant1-2 mutants affecting CESA3 show defects consistent with altered mucilage cellulose biosynthesis. CESA3 can interact with CESA5 in vitro, and green fluorescent protein-tagged CESA5, CESA3, and CESA10 proteins move in a linear, unidirectional fashion around the cytoplasmic column of the cell, parallel with the surface of the seed, in a pattern similar to that of cortical microtubules. Consistent with this movement, cytological evidence suggests that the mucilage is coiled around the columella and unwinds during mucilage extrusion to form a linear ray. Mutations in CESA5 and CESA3 affect the speed of mucilage extrusion and mucilage adherence. These findings imply that cellulose fibrils are synthesized in an ordered helical array around the columella, providing a distinct structure to the mucilage that is important for both mucilage extrusion and adherence.

Flying saucer1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed mucilage.

Pectins are complex polysaccharides that form the gel matrix of the primary cell wall and are abundant in the middle lamella that holds plant cells together. Their degree of methylesterification (DM) impacts wall strength and cell adhesion since unesterified pectin regions can cross-link via Ca(2+) ions to form stronger gels. Here, we characterize flying saucer1 (fly1), a novel Arabidopsis thaliana seed coat mutant, which displays primary wall detachment, reduced mucilage extrusion, and increased mucilage adherence. These defects appear to result from a lower DM in mucilage and are enhanced by the addition of Ca(2+) or completely rescued using alkaline Ca(2+) chelators. FLY1 encodes a transmembrane protein with a RING-H2 domain that has in vitro E3 ubiquitin ligase activity. FLY1 is orthologous to TRANSMEMBRANE UBIQUITIN LIGASE1, a Golgi-localized E3 ligase involved in the quality control of membrane proteins in yeast. However, FLY1-yellow fluorescent protein (YFP) fusions are localized in punctae that are predominantly distinct from the Golgi and the trans-Golgi network/early endosome in the seed coat epidermis. Wortmannin treatment, which induces the fusion of late endosomes in plants, resulted in enlarged FLY1-YFP bodies. We propose that FLY1 regulates the DM of pectin in mucilage, potentially by recycling pectin methylesterase enzymes in the endomembrane system of seed coat epidermal cells.

SALT-OVERLY SENSITIVE5 Mediates Arabidopsis Seed Coat Mucilage Adherence and Organization through Pectins.

Interactions between cell wall polymers are critical for establishing cell wall integrity and cell-cell adhesion. Here, we exploit the Arabidopsis (Arabidopsis thaliana) seed coat mucilage system to examine cell wall polymer interactions. On hydration, seeds release an adherent mucilage layer strongly attached to the seed in addition to a nonadherent layer that can be removed by gentle agitation. Rhamnogalacturonan I (RG I) is the primary component of adherent mucilage, with homogalacturonan, cellulose, and xyloglucan constituting minor components. Adherent mucilage contains rays composed of cellulose and pectin that extend above the center of each epidermal cell. CELLULOSE SYNTHASE5 (CESA5) and the arabinogalactan protein SALT-OVERLY SENSITIVE5 (SOS5) are required for mucilage adherence through unknown mechanisms. SOS5 has been suggested to mediate adherence by influencing cellulose biosynthesis. We, therefore, investigated the relationship between SOS5 and CESA5. cesa5-1 seeds show reduced cellulose, RG I, and ray size in adherent mucilage. In contrast, sos5-2 seeds have wild-type levels of cellulose but completely lack adherent RG I and rays. Thus, relative to each other, cesa5-1 has a greater effect on cellulose, whereas sos5-2 mainly affects pectin. The double mutant cesa5-1 sos5-2 has a much more severe loss of mucilage adherence, suggesting that SOS5 and CESA5 function independently. Double-mutant analyses with mutations in MUCILAGE MODIFIED2 and FLYING SAUCER1 that reduce mucilage release through pectin modification suggest that only SOS5 influences pectin-mediated adherence. Together, these findings suggest that SOS5 mediates adherence through pectins and does so independently of but in concert with cellulose synthesized by CESA5.

Plant virus diversity in bee and pollen samples from apple (Malus domestica) and sweet cherry (Prunus avium) agroecosystems.

Introduction: Honey bee (Apis mellifera) pollination is widely used in tree fruit production systems to improve fruit set and yield. Many plant viruses can be associated with pollen or transmitted through pollination, and can be detected through bee pollination activities. Honey bees visit multiple plants and flowers in one foraging trip, essentially sampling small amounts of pollen from a wide area. Here we report metagenomics-based area-wide monitoring of plant viruses in cherry (Prunus avium) and apple (Malus domestica) orchards in Creston Valley, British Columbia, Canada, through bee-mediated pollen sampling. Methods: Plant viruses were identified in total RNA extracted from bee and pollen samples, and compared with profiles from double stranded RNA extracted from leaf and flower tissues. CVA, PDV, PNRSV, and PVF coat protein nucleotide sequences were aligned and compared for phylogenetic analysis. Results: A wide array of plant viruses were identified in both systems, with cherry virus A (CVA), prune dwarf virus (PDV), prunus necrotic ringspot virus (PNRSV), and prunus virus F (PVF) most commonly detected. Citrus concave gum associated virus and apple stem grooving virus were only identified in samples collected during apple bloom, demonstrating changing viral profiles from the same site over time. Different profiles of viruses were identified in bee and pollen samples compared to leaf and flower samples reflective of pollen transmission affinity of individual viruses. Phylogenetic and pairwise analysis of the coat protein regions of the four most commonly detected viruses showed unique patterns of nucleotide sequence diversity, which could have implications in their evolution and management approaches. Coat protein sequences of CVA and PVF were broadly diverse with multiple distinct phylogroups identified, while PNRSV and PDV were more conserved. Conclusion: The pollen virome in fruit production systems is incredibly diverse, with CVA, PDV, PNRSV, and PVF widely prevalent in this region. Bee-mediated monitoring in agricultural systems is a powerful approach to study viral diversity and can be used to guide more targeted management approaches.

Identification and analysis of an outer-seed-coat-specific promoter from Arabidopsis thaliana.

Differentiation of the Arabidopsis thaliana (Arabidopsis) seed coat epidermal cells involves pronounced changes highlighted by the synthesis and secretion of copious amounts of dispensable, pectinaceous mucilage followed by a thick cellulosic secondary cell wall. This cell type, therefore, represents an excellent molecular-genetic model to study the biosynthesis and modification of cell wall components, particularly pectin. To support such research, we sought to identify a promoter that drives expression specifically in the Arabidopsis seed coat epidermis. Arabidopsis seed coat microarray data was analysed for genes expressed in the wild type seed coat but not the seed coat of the apetala2 mutant where the epidermal cells fail to differentiate. Of 14 candidate genes, 9 showed a seed-specific expression pattern by reverse transcriptase-PCR. Transcriptional regulatory region-ß-glucuronidase (GUS) reporter gene fusions introduced into Arabidopsis identified one promoter, that of the DIRIGENT PROTEIN1 (DP1) gene, as seed coat specific. The specificity of the expression was confirmed using a second reporter gene, Citrine YFP. Expression of both reporter genes was limited to the epidermal and palisade cell layers of the seed coat. Quantitative PCR data using wild type seed coat RNA suggested that the promoter is particularly active at 7 days post anthesis. The DP1 promoter was able to direct transcription of GUS in a similar pattern in the Brassica napus seed coat. Thus, in addition to its application in studying the plant cell wall, this promoter will provide an experimental tool for expressing high-valued recombinant proteins as well as modifying seed coat traits in economically important crops.

Area Wide Monitoring of Plant and Honey Bee (Apis mellifera) Viruses in Blueberry (Vaccinium corymbosum) Agroecosystems Facilitated by Honey Bee Pollination.

Healthy agroecosystems are dependent on a complex web of factors and inter-species interactions. Flowers are hubs for pathogen transmission, including the horizontal or vertical transmission of plant-viruses and the horizontal transmission of bee-viruses. Pollination by the European honey bee (Apis mellifera) is critical for industrial fruit production, but bees can also vector viruses and other pathogens between individuals. Here, we utilized commercial honey bee pollination services in blueberry (Vaccinium corymbosum) farms for a metagenomics-based bee and plant virus monitoring system. Following RNA sequencing, viruses were identified by mapping reads to a reference sequence database through the bioinformatics portal Virtool. In total, 29 unique plant viral species were found at two blueberry farms in British Columbia (BC). Nine viruses were identified at one site in Ontario (ON), five of which were not identified in BC. Ilarviruses blueberry shock virus (BlShV) and prune dwarf virus (PDV) were the most frequently detected viruses in BC but absent in ON, while nepoviruses tomato ringspot virus and tobacco ringspot virus were common in ON but absent in BC. BlShV coat protein (CP) nucleotide sequences were nearly identical in all samples, while PDV CP sequences were more diverse, suggesting multiple strains of PDV circulating at this site. Ten bee-infecting viruses were identified, with black queen cell virus frequently detected in ON and BC. Area-wide bee-mediated pathogen monitoring can provide new insights into the diversity of viruses present in, and the health of, bee-pollination ecosystems. This approach can be limited by a short sampling season, biased towards pollen-transmitted viruses, and the plant material collected by bees can be very diverse. This can obscure the origin of some viruses, but bee-mediated virus monitoring can be an effective preliminary monitoring approach.

Subfunctionalization of cellulose synthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment.

Arabidopsis (Arabidopsis thaliana) epidermal seed coat cells follow a complex developmental program where, following fertilization, cells of the ovule outer integument differentiate into a unique cell type. Two hallmarks of these cells are the production of a doughnut-shaped apoplastic pocket filled with pectinaceous mucilage and the columella, a thick secondary cell wall. Cellulose is thought to be a key component of both these secondary cell wall processes. Here, we investigated the role of cellulose synthase (CESA) subunits CESA2, CESA5, and CESA9 in the seed coat epidermis. We characterized the roles of these CESA proteins in the seed coat by analyzing cell wall composition and morphology in cesa mutant lines. Mutations in any one of these three genes resulted in lower cellulose content, a loss of cell shape uniformity, and reduced radial wall integrity. In addition, we found that attachment of the mucilage halo to the parent seed following extrusion is maintained by cellulose-based connections requiring CESA5. Hence, we show that cellulose fulfills an adhesion role between the extracellular mucilage matrix and the parent cell in seed coat epidermal cells. We propose that mucilage remains attached to the seed coat through interactions between components in the seed mucilage and cellulose. Our data suggest that CESA2 and CESA9 serve in radial wall reinforcement, as does CESA5, but CESA5 also functions in mucilage biosynthesis. These data suggest unique roles for different CESA subunits in one cell type and illustrate a complex role for cellulose biosynthesis in plant developmental biology.

Tomato brown rugose fruit virus: An emerging and rapidly spreading plant RNA virus that threatens tomato production worldwide.

Tomato brown rugose fruit virus (ToBRFV) is an emerging and rapidly spreading RNA virus that infects tomato and pepper, with tomato as the primary host. The virus causes severe crop losses and threatens tomato production worldwide. ToBRFV was discovered in greenhouse tomato plants grown in Jordan in spring 2015 and its first outbreak was traced back to 2014 in Israel. To date, the virus has been reported in at least 35 countries across four continents in the world. ToBRFV is transmitted mainly via contaminated seeds and mechanical contact (such as through standard horticultural practices). Given the global nature of the seed production and distribution chain, and ToBRFV's seed transmissibility, the extent of its spread is probably more severe than has been disclosed. ToBRFV can break down genetic resistance to tobamoviruses conferred by R genes Tm-1, Tm-2, and Tm-22 in tomato and L1 and L2 alleles in pepper. Currently, no commercial ToBRFV-resistant tomato cultivars are available. Integrated pest management-based measures such as rotation, eradication of infected plants, disinfection of seeds, and chemical treatment of contaminated greenhouses have achieved very limited success. The generation and application of attenuated variants may be a fast and effective approach to protect greenhouse tomato against ToBRFV. Long-term sustainable control will rely on the development of novel genetic resistance and resistant cultivars, which represents the most effective and environment-friendly strategy for pathogen control. TAXONOMY: Tomato brown rugose fruit virus belongs to the genus Tobamovirus, in the family Virgaviridae. The genus also includes several economically important viruses such as Tobacco mosaic virus and Tomato mosaic virus. GENOME AND VIRION: The ToBRFV genome is a single-stranded, positive-sense RNA of approximately 6.4 kb, encoding four open reading frames. The viral genomic RNA is encapsidated into virions that are rod-shaped and about 300 nm long and 18 nm in diameter. Tobamovirus virions are considered extremely stable and can survive in plant debris or on seed surfaces for long periods of time. DISEASE SYMPTOMS: Leaves, particularly young leaves, of tomato plants infected by ToBRFV exhibit mild to severe mosaic symptoms with dark green bulges, narrowness, and deformation. The peduncles and calyces often become necrotic and fail to produce fruit. Yellow blotches, brown or black spots, and rugose wrinkles appear on tomato fruits. In pepper plants, ToBRFV infection results in puckering and yellow mottling on leaves with stunted growth of young seedlings and small yellow to brown rugose dots and necrotic blotches on fruits.

Molecular Identification of Prune Dwarf Virus (PDV) Infecting Sweet Cherry in Canada and Development of a PDV Full-Length Infectious cDNA Clone.

Prune dwarf virus (PDV) is a member of ilarviruses that infects stone fruit species such as cherry, plum and peach, and ornamentally grown trees worldwide. The virus lacks an RNA silencing suppressor. Infection by PDV either alone, or its mixed infection with other viruses causes deteriorated fruit marketability and reduced fruit yields. Here, we report the molecular identification of PDV from sweet cherry in the prominent fruit growing region of Ontario, Canada known as the Niagara fruit belt using next generation sequencing of small interfering RNAs (siRNAs). We assessed its incidence in an experimental farm and determined the full genome sequence of this PDV isolate. We further constructed an infectious cDNA clone. Inoculation of the natural host cherry with this clone induced a dwarfing phenotype. We also examined its infectivity on several common experimental hosts. We found that it was infectious on cucurbits (cucumber and squash) with clear symptoms and Nicotiana benthamiana without causing noticeable symptoms, and it was unable to infect Arabidopsis thaliana. As generating infectious clones for woody plants is very challenging with limited success, the PDV infectious clone developed from this study will be a useful tool to facilitate molecular studies on PDV and related Prunus-infecting viruses.

Altered gene expression changes in Arabidopsis leaf tissues and protoplasts in response to Plum pox virus infection.

BACKGROUND: Virus infection induces the activation and suppression of global gene expression in the host. Profiling gene expression changes in the host may provide insights into the molecular mechanisms that underlie host physiological and phenotypic responses to virus infection. In this study, the Arabidopsis Affymetrix ATH1 array was used to assess global gene expression changes in Arabidopsis thaliana plants infected with Plum pox virus (PPV). To identify early genes in response to PPV infection, an Arabidopsis synchronized single-cell transformation system was developed. Arabidopsis protoplasts were transfected with a PPV infectious clone and global gene expression changes in the transfected protoplasts were profiled. RESULTS: Microarray analysis of PPV-infected Arabidopsis leaf tissues identified 2013 and 1457 genes that were significantly (Q < or = 0.05) up- (> or = 2.5 fold) and downregulated (< or = -2.5 fold), respectively. Genes associated with soluble sugar, starch and amino acid, intracellular membrane/membrane-bound organelles, chloroplast, and protein fate were upregulated, while genes related to development/storage proteins, protein synthesis and translation, and cell wall-associated components were downregulated. These gene expression changes were associated with PPV infection and symptom development. Further transcriptional profiling of protoplasts transfected with a PPV infectious clone revealed the upregulation of defence and cellular signalling genes as early as 6 hours post transfection. A cross sequence comparison analysis of genes differentially regulated by PPV-infected Arabidopsis leaves against uniEST sequences derived from PPV-infected leaves of Prunus persica, a natural host of PPV, identified orthologs related to defence, metabolism and protein synthesis. The cross comparison of genes differentially regulated by PPV infection and by the infections of other positive sense RNA viruses revealed a common set of 416 genes. These identified genes, particularly the early responsive genes, may be critical in virus infection. CONCLUSION: Gene expression changes in PPV-infected Arabidopsis are the molecular basis of stress and defence-like responses, PPV pathogenesis and symptom development. The differentially regulated genes, particularly the early responsive genes, and a common set of genes regulated by infections of PPV and other positive sense RNA viruses identified in this study are candidates suitable for further functional characterization to shed lights on molecular virus-host interactions.

Transfection of Arabidopsis protoplasts with a Plum pox virus (PPV) infectious clone for studying early molecular events associated with PPV infection.

The development of novel strategies against plant viral diseases relies on a better understanding of molecular virus-host interactions. Here, we report an easy, efficient and reproducible protocol for Arabidopsis protoplast isolation and transfection to study the infection and replication of a potyvirus, Plum pox virus (PPV). Macerozyme and cellulose were used to release protoplasts from Arabidopsis leaf tissues, and polyethylene glycol-mediated DNA uptake was employed for transfection of a PPV infectious clone. Protoplast viability was monitored by fluorescein diacetate staining, and transfection efficiency was estimated by transient expression of the green fluorescent protein. The protocol allowed production of 95% viable mesophyll protoplasts and a successful transfection rate of 35%. The system was used further in a time-course experiment to investigate PPV viral RNA accumulation. It was found that 3 h post-transfection (hpt) in the transfected protoplasts viral RNA increased by about 150-fold and progressively accumulated to reach the maximum at 12 hpt. Viral RNA then decreased dramatically at 24 hpt reaching 40% of its peak level. Considering the availability of the whole genome microarrays, and other genomic resources of Arabidopsis, the synchronized single-cell (protoplast) infection system will be useful for elucidating early molecular events associated with PPV infection.

Dissecting Seed Mucilage Adherence Mediated by FEI2 and SOS5.

The plant cell wall is held together by the interactions between four major components: cellulose, pectin, hemicellulose, and proteins. Mucilage is a powerful model system to study the interactions between these components as it is formed of polysaccharides that are deposited in the apoplast of seed coat epidermal cells during seed development. When seeds are hydrated, these polysaccharides expand rapidly out of the apoplastic pocket, and form an adherent halo of mucilage around the seed. In Arabidopsis, mutations in multiple genes have similar loss of mucilage adherence phenotypes including CELLULOSE SYNTHASE 5 (CESA5)/MUCILAGE-MODIFIED 3 (MUM3), MUM5/MUCI21, SALT-OVERLY SENSITIVE 5 (SOS5), and FEI2. Here, we examine the interactions between these factors to better understand how they participate to control mucilage adherence. Double mutant phenotypes indicated that MUM5 and CESA5 function in a common mechanism that adheres pectin to the seed through the biosynthesis of cellulose and xylan, whereas SOS5 and FEI2, encoding a fasciclin-like arabinogalactan protein or a receptor-like kinase, respectively, function through an independent pathway. Cytological analyses of mucilage indicates that heteromannans are associated with cellulose, and not in the pathway involving SOS5 or FEI2. A SOS5 fluorescent protein fusion (SOS5-mCITRINE) was localized throughout the mucilage pocket, consistent with a structural role in pectin adhesion. The relationship between SOS5 and FEI2 mediated mucilage adherence was examined in more detail and while sos5 and fei2 mutants show similar phenotypes, key differences in the macromolecular characteristics and amounts of mucilage polymers were observed. FEI2 thus appears to have additional, as well as overlapping functions, with SOS5. Given that FEI2 is required for SOS5 function, we propose that FEI2 serves to localize SOS5 at the plasma membrane where it establishes interactions with mucilage polysaccharides, notably pectins, required for mucilage adherence prior to SOS5 being released into the apoplast.