Strain selection and initiation timing influence the cultivation period of <Emphasis Type="Italic">Saccharina japonica</Emphasis> and their impact on the abalone feed industry in Korea

Cultivations of the brown seaweed, Saccharina japonica, was developed, promoted, and industrialized in the 1970s and is highly valued in Korea both for human consumption and as a feed for the rapidly developing abalone farming industry. Production has continually increased, and approximately 442,637 tonnes wet weight was harvested in Korea in 2015. Abalone is a highly sought-after delicacy in Korea, and the abalone farming industry has been developed based on a stable production of seaweed. Korean abalone farmers prefer to feed their stock on locally cultured S. japonica; however, between August and November, the supply of farmed S. japonica declines because higher seawater temperatures reduce S. japonica productivity. In an attempt to overcome this temperature-induced period of low production, cultivation trials with a strain of S. japonica selected to withstand higher cultivation temperatures were undertaken. Strain selection involved using individual parent thalli that were found to remain productive under high seawater temperature (26 °C) at Wando. Male and female gametophytes were isolated through 3 cycles of free-living gametophyte culture to produce the F₃ strain used in the production trials. Production trials using the selected strain were initiated every month between December 2014 and March 2015. This delayed the initiation of culture beyond the latest initiation time currently used by farmers (December). Delaying initiation of cultivation resulted in delayed maximum growth compared to the control. Growth of the F₃ strain continued for up to 3 months longer than normally achieved on farms for non-selected thalli. The mean length, growth rate, and biomass were also greater than those achieved by the control strain. The use of the F₃ strain of S. japonica coupled with delayed initiation of culture can therefore be used to help to ensure a stable year round algal feed supply for abalone industry in Korea.     

Out-of-plane orientation of cellulose elementary fibrils on spruce tracheid wall based on imaging with high-resolution transmission electron microscopy

Main conclusion

A 3D model of the tracheid wall has been proposed based on high-resolution cryo-TEM where, in contrast to the current understanding, the cellulose elementary fibrils protrude from the cell wall plane. The ultrastructure of the tracheid walls of Picea abies was examined through imaging of ultrathin radial, tangential and transverse sections of wood by transmission electron microscopy and with digital image processing. It was found that the elementary fibrils (EFs) of cellulose were rarely deposited in the plane of the concentric cell wall layers, in contrast to the current understanding. In addition to the adopted concept of longitudinal fibril angle, EFs protruded from the cell wall plane in varying angles depending on the layer. Moreover, the out-of-plane fibril angle varied between radial and tangential walls. In the tangential S₂ layers, EFs were always out-of-plane whereas planar orientation was typical for the S₂ layer in radial walls. The pattern of protruding EFs was evident in almost all axial and transverse images of the S₁ layer. Similar out-of-plane orientation was found in the transverse sections of the S₃ layer. A new model of the tracheid wall with EF orientation is presented as a summary of this study. The outcome of this study will enhance our understanding of the elementary fibril orientation in the tracheid wall.     

Effects of Exogenous Phytohormones on Spore Germination and Morphogenesis of <Emphasis Type="Italic">Polystichum aculeatum</Emphasis> (L.) Roth Gametophyte in vitro Culture

The effects of exogenous phytohormones–gibberellic acid (GA3) and benzyl-aminopurine (BAP)–on the spore germination and morphogenesis of Polystichum aculeatum (L.) Roth gametophyte in vitro culture were studied. In the control, four stages of gametophyte morphogenesis were determined and their periods were established. Spore germination and protonema formation of P. aculeatum occurred according to the Vittaria-type and prothalium development according to the Aspidium-type. The spore germination percentage depended on the storage time duration. It was found that 80–95% of freshly collected spores germinated. Spore viability was within the range of 68–95% after 4–6-month storage under lab conditions and did not exceed 20% after 1.5 year of the storage period. High concentrations of exogenous GA3 (10^–5 and 10^–6 M) and BAP (10–5 M) significantly inhibited spore germination, whereas low concentrations (GA3 10^–7–10^–8 M) had an insignificant stimulating effect or did not affect germination at all (BAP 10^–6, 10^–7, 10^–8 M). All concentrations of exogenous BAP were demonstrated to inhibit the development of P. aculeatum gametophyte at the protonema stage, which might be due to the removal of apical dominance. The inhibiting effect directly depended on BAP concentrations. The formation of abnormal thalli of the P. aculeatum gametophyte in the response to exogenous GA3 treatments occurred as a result of impairment of cell growth by elongation. A direct interrelationship between GA3 concentrations and level of morphological abnormalities and grade of thalli underdevelopment was demonstrated.     

Imaging Cell Wall Architecture in Single Zinnia elegans Tracheary Elements

The chemical and structural organization of the plant cell wall was examined in Zinnia elegans tracheary elements (TEs), which specialize by developing prominent secondary wall thickenings underlying the primary wall during xylogenesis in vitro. Three imaging platforms were used in conjunction with chemical extraction of wall components to investigate the composition and structure of single Zinnia TEs. Using fluorescence microscopy with a green fluorescent protein-tagged Clostridium thermocellum family 3 carbohydrate-binding module specific for crystalline cellulose, we found that cellulose accessibility and binding in TEs increased significantly following an acidified chlorite treatment. Examination of chemical composition by synchrotron radiation-based Fourier-transform infrared spectromicroscopy indicated a loss of lignin and a modest loss of other polysaccharides in treated TEs. Atomic force microscopy was used to extensively characterize the topography of cell wall surfaces in TEs, revealing an outer granular matrix covering the underlying meshwork of cellulose fibrils. The internal organization of TEs was determined using secondary wall fragments generated by sonication. Atomic force microscopy revealed that the resulting rings, spirals, and reticulate structures were composed of fibrils arranged in parallel. Based on these combined results, we generated an architectural model of Zinnia TEs composed of three layers: an outermost granular layer, a middle primary wall composed of a meshwork of cellulose fibrils, and inner secondary wall thickenings containing parallel cellulose fibrils. In addition to insights in plant biology, studies using Zinnia TEs could prove especially productive in assessing cell wall responses to enzymatic and microbial degradation, thus aiding current efforts in lignocellulosic biofuel production.The organization and molecular architecture of plant cell walls represent some of the most challenging problems in plant biology. Although much is known about general aspects of assembly and biosynthesis of the plant cell wall, the detailed three-dimensional molecular cell wall structure remains poorly understood. The highly complex and dynamic nature of the plant cell wall has perhaps limited the generation of such detailed structural models. This information is pivotal for the successful implementation of novel approaches for conversion of biomass to liquid biofuels, given that one of the critical processing steps in biomass conversion involves systematic deconstruction of cell walls. Therefore, a comprehensive understanding of the architecture and chemical composition of the plant cell wall will not only help develop molecular-scale models, but will also help improve the efficiency of biomass deconstruction.The composition and molecular organization of the cell wall is species and cell type dependent (Vorwerk et al., 2004). Thus, the development of a model plant system, which utilizes a single cell type, has enhanced our capacity to understand cell wall architecture. The ability to generate a population of single Zinnia elegans plant cells that were synchronized throughout cell wall deposition during xylogenesis was developed in the 1980s (Fukuda and Komamine, 1980). Mesophyll cells isolated from the leaves of Zinnia and cultured in the presence of phytohormones will transdifferentiate into tracheary elements (TEs), which are individual components of the xylem vascular tissue (Fukuda and Komamine, 1980). During this transdifferentiation process, TEs gradually develop patterned secondary wall thickenings, commonly achieving annular, spiral, reticulate, scalariform, and pitted patterns (Bierhorst, 1960; Falconer and Seagull, 1988; Roberts and Haigler, 1994). These secondary wall thickenings serve as structural reinforcements that add strength and rigidity to prevent the collapse of the xylem under the high pressure created by fluid transport. During the final stages of transdifferentiation, TEs accumulate lignin in their secondary walls and undergo programmed cell death, which results in the removal of all cell contents, leaving behind a “functional corpse” (Roberts and McCann, 2000; Fukuda, 2004).In broad terms, the primary cell wall of higher plants is mainly composed of three types of polysaccharides: cellulose, hemicelluloses, and pectins (Cosgrove, 2005). Cellulose is composed of unbranched β-1,4-Glc chains that are packed together into fibrils by intermolecular and intramolecular hydrogen bonding. Hemicelluloses and pectins are groups of complex polysaccharides that are primarily composed of xyloglucans/xylans and galacturonans, respectively. Hemicelluloses are involved in cross-linking and associating with cellulose microfibrils, while pectins control wall porosity and help bind neighboring cells together. The patterned deposits of secondary wall in Zinnia TEs primarily consist of cellulose microfibrils, along with hemicelluloses, and also lignin, a complex aromatic polymer that is characteristic of secondary walls and provides reinforcement (Turner et al., 2007). All the molecular components in the cell wall correspond to a multitude of different polysaccharides, phenolic compounds, and proteins that become arranged and modified in muro, yielding a structure of great strength and resistance to degradation.Currently, electron microscopy is the primary tool for structural studies of cell walls and has provided remarkable information regarding wall organization. Fast-freeze deep-etch electron microscopy in combination with chemical and enzymatic approaches have generated recent models of the architecture of the primary wall (McCann et al., 1990; Carpita and Gibeaut, 1993; Nakashima et al., 1997; Fujino et al., 2000; Somerville et al., 2004). Direct visualization of secondary wall organization has been focused toward the examination of multiple wall layers in wood cells (Fahlen and Salmen, 2005; Zimmermann et al., 2006). However, few studies have examined the secondary wall, so our knowledge regarding the higher order architecture of this type of wall is limited. Over the past few decades, atomic force microscopy (AFM) has provided new opportunities to probe biological systems with spatial resolution similar to electron microscopy techniques (Kuznetsov et al., 1997; Muller et al., 1999), with additional ease of sample preparation and the capability to probe living native structures. AFM has been successfully applied to studies of the high-resolution architecture, assembly, and structural dynamics of a wide range of biological systems (Hoh et al., 1991; Crawford et al., 2001; Malkin et al., 2003; Plomp et al., 2007), thus enabling the observation of the ultrastructure of the plant cell wall, which is of particular interest to us (Kirby et al., 1996; Morris et al., 1997; Davies and Harris, 2003; Yan et al., 2004; Ding and Himmel, 2006).To generate more detailed structural models, knowledge about the structural organization of the cell wall can be combined with spatial information about chemical composition. Instead of utilizing chromatography techniques to analyze cell wall composition by extracting material from bulk plant samples (Mellerowicz et al., 2001; Pauly and Keegstra, 2008), Fourier transform infrared (FTIR) spectromicroscopy can be used to directly probe for polysaccharide and aromatic molecules in native as well as treated plant material (Carpita et al., 2001; McCann et al., 2001). FTIR spectromicroscopy is not only able to identify chemical components in a specific system but also can determine their distribution and relative abundance. This technique also improves the sensitivity and spatial resolution of cellular components without the derivatization needed by chemical analysis using chromatography. Polysaccharide-specific probes, such as carbohydrate-binding modules (CBMs), can also be used to understand the chemical composition of the plant cell wall. CBMs are noncatalytic protein domains existing in many glycoside hydrolases. Based on their binding specificities, CBMs are generally categorized into three groups: surface-binding CBMs specific for insoluble cellulose surfaces, chain-binding CBMs specific for single chains of polysaccharides, and end-binding CBMs specific for the ends of polysaccharides or oligosaccharides. A surface-binding CBM with high affinity for the planar faces of crystalline cellulose (Tormo et al., 1996; Lehtio et al., 2003) has been fluorescently labeled and used to label crystals as well as plant tissue (Ding et al., 2006; Porter et al., 2007; Liu et al., 2009; Xu et al., 2009). The binding capacity of the CBM family has been further exploited for the detection of different polysaccharides, such as xylans and glucans, and can thus be used for the characterization of plant cell wall composition (McCartney et al., 2004, 2006).In this study, we used a combination of AFM, synchrotron radiation-based (SR)-FTIR spectromicroscopy, and fluorescence microscopy using a cellulose-specific CBM to probe the cell wall of Zinnia TEs. The Zinnia TE culture system proved ideal for observing the structure and chemical composition of the cell wall because it comprises a single homogeneous cell type, representing a simpler system compared with plant tissues, which may contain multiple cell types. Zinnia TEs were also advantageous because they were analyzed individually, and population statistics were generated based on specific conditions. Furthermore, cultured Zinnia TEs were used for the consistent production of cell wall fragments for analysis of the organization of internal secondary wall structures. In summary, we have physically and chemically dissected Zinnia TEs using a combination of imaging techniques that revealed primary and secondary wall structures and enabled the reconstruction of TE cell wall architecture.     

Overexpression of <Emphasis Type="Italic">PSP1</Emphasis> enhances growth of transgenic Arabidopsis plants under ambient air conditions

Cellulose biosynthesis is mediated by cellulose synthases (CesAs), which constitute into rosette-like cellulose synthase complexe (CSC) on the plasma membrane. Two types of CSCs in Arabidopsis are believed to be involved in cellulose synthesis in the primary cell wall and secondary cell walls, respectively. In this work, we found that the two type CSCs participated cellulose biosynthesis in differentiating xylem cells undergoing secondary cell wall thickening in Populus. During the cell wall thickening process, expression of one type CSC genes increased while expression of the other type CSC genes decreased. Suppression of different type CSC genes both affected the wall-thickening and disrupted the multilaminar structure of the secondary cell walls. When CesA7A was suppressed, crystalline cellulose content was reduced, which, however, showed an increase when CesA3D was suppressed. The CesA suppression also affected cellulose digestibility of the wood cell walls. The results suggest that two type CSCs are involved in coordinating the cellulose biosynthesis in formation of the multilaminar structure in Populus wood secondary cell walls.     

Plastid Genome of <Emphasis Type="Italic">Dictyopteris divaricata</Emphasis> (Dictyotales,Phaeophyceae): Understanding the Evolution of Plastid Genomes in Brown Algae

Dictyotophycidae is a subclass of brown algae containing 395 species that are distributed worldwide. A complete plastid (chloroplast) genome (ptDNA or cpDNA) had not previously been sequenced from this group. In this study, the complete plastid genome of Dictyopteris divaricata (Okamura) Okamura (Dictyotales, Phaeophyceae) was characterized and compared to other brown algal ptDNAs. This plastid genome was 126,099 bp in size with two inverted repeats (IRs) of 6026 bp. The D. divaricata IRs contained rpl21, making its IRs larger than representatives from the orders Fucales and Laminariales, but was smaller than that from Ectocarpales. The G + C content of D. divaricata (31.19%) was the highest of the known ptDNAs of brown algae (28.94–31.05%). Two protein-coding genes, rbcR and rpl32, were present in ptDNAs of Laminariales, Ectocarpales (Ectocarpus siliculosus), and Fucales (LEF) but were absent in D. divaricata. Reduced intergenic space (13.11%) and eight pairs of overlapping genes in D. divaricata ptDNA made it the most compact plastid genome in brown algae so far. The architecture of D. divaricata ptDNA showed higher similarity to that of Laminariales compared with Fucales and Ectocarpales. The difference in general features, gene content, and architecture among the ptDNAs of D. divaricata and LEF clade revealed the diversity and evolutionary trends of plastid genomes in brown algae.     

Improvement of cell wall digestibility in tall fescue by <Emphasis Type="Italic">Oryza sativa</Emphasis> SECONDARY WALL NAC DOMAIN PROTEIN2 chimeric repressor

Forage digestibility is one of the most important factors in livestock performance. As grasses grow and mature, dry matter increases but they become fibrous with secondary cell wall deposition and lignification of sclerenchyma cells, and forage quality drops. In rice (Oryza sativa), the SECONDARY WALL NAC DOMAIN PROTEIN2 fused with the modified EAR-like motif repression domain (OsSWN2-SRDX) reduces secondary cell wall thickening in sclerenchyma cells. We introduced OsSWN2-SRDX under the control of the OsSWN1 promoter into tall fescue (Festuca arundinacea Schreb.) to increase cell wall digestibility. Of 23 transgenic plants expressing OsSWN2-SRDX, nine had brittle internodes that were easily broken by bending. Their secondary cell walls were significantly thinner than those of the wild type in interfascicular fibers of internodes and in cortical fiber cells between leaf epidermal cells and vascular bundles. The dry matter digestibility increased by 11.8% in stems and by 6.8% in leaves compared with the wild type, and therefore forage quality was improved. In stem interfascicular fibers, acid detergent fiber and acid insoluble lignin were greatly reduced. Thus, the reduction of indigestible fiber composed of cellulose and lignin increased the degradability of sclerenchyma cell walls. OsSWN2-SRDX plants offer great potential in the genetic improvement of forage digestibility.     

Characterization of 25 full-length <Emphasis Type="Italic">S-RNase</Emphasis> alleles,including flanking regions,from a pool of resequenced apple cultivars

Key message

Our work focuses on understanding the lifetime and thus stability of the three main cellulose synthase (CESA) proteins involved in primary cell wall synthesis of Arabidopsis. It had long been thought that a major means of CESA regulation was via their rapid degradation. However, our studies here have uncovered that AtCESA proteins are not rapidly degraded. Rather, they persist for an extended time in the plant cell.

Abstract

Plant cellulose is synthesized by membrane-embedded cellulose synthase complexes (CSCs). The CSC is composed of cellulose synthases (CESAs), of which three distinct isozymes form the primary cell wall CSC and another set of three isozymes form the secondary cell wall CSC. We determined the stability over time of primary cell wall (PCW) CESAs in Arabidopsis thaliana seedlings, using immunoblotting after inhibiting protein synthesis with cycloheximide treatment. Our work reveals very slow turnover for the Arabidopsis PCW CESAs in vivo. Additionally, we show that the stability of all three CESAs within the PCW CSC is altered by mutations in individual CESAs, elevated temperature, and light conditions. Together, these results suggest that CESA proteins are very stable in vivo, but that their lifetimes can be modulated by intrinsic and environmental cues.

     

Wall texture in the spore of a vesicular-arbuscular mycorrhizal fungus

Summary InGlomus epigaeum Daniels and Trappe, a vesicular-arbuscular mycorrhizal fungus, the mature spore has a complex multi-layered wall containing a regular pattern of wall subunits.The outer wall (2–4 m thick) consists of a simple layer of parallel microfibrils. The inner wall (5–6 m thick) is built from two layers possessing different organization. The innermost layer, near the plasmalemma has a texture of apparently dispersed fibrils, whereas the second layer is regularly organized with an arced texture. Ten to twelve bundles of fibrils connected by apparently bow-shaped fibrils are consistently observed. The appearance of this arced organization depends on the section plane and on the angle of observation in the electron microscope as confirmed by tilting experiments. Wall subunits are evident as straight electron transparent fibrils; particularly well-defined in negatively stained frozen sections: their diameter is about 3.5nm.The regular pattern of wall subunits in this fungal cell wall is compared with the textures shown by cellulose fibrils in algae or higher plants and by chitin fibrils in arthropod cuticle.Research work supported by CNR, Italy. Special grant I.P.R.A.—Sub-project 1. Paper No. 55.     

Chemical and enzymatic fractionation of cell walls from Fucales: insights into the structure of the extracellular matrix of brown algae

Background and Aims

Brown algae are photosynthetic multicellular marine organisms evolutionarily distant from land plants, with a distinctive cell wall. They feature carbohydrates shared with plants (cellulose), animals (fucose-containing sulfated polysaccharides, FCSPs) or bacteria (alginates). How these components are organized into a three-dimensional extracellular matrix (ECM) still remains unclear. Recent molecular analysis of the corresponding biosynthetic routes points toward a complex evolutionary history that shaped the ECM structure in brown algae.

Methods

Exhaustive sequential extractions and composition analyses of cell wall material from various brown algae of the order Fucales were performed. Dedicated enzymatic degradations were used to release and identify cell wall partners. This approach was complemented by systematic chromatographic analysis to study polymer interlinks further. An additional structural assessment of the sulfated fucan extracted from Himanthalia elongata was made.

Key Results

The data indicate that FCSPs are tightly associated with proteins and cellulose within the walls. Alginates are associated with most phenolic compounds. The sulfated fucans from H. elongata were shown to have a regular α-(1→3) backbone structure, while an alternating α-(1→3), (1→4) structure has been described in some brown algae from the order Fucales.

Conclusions

The data provide a global snapshot of the cell wall architecture in brown algae, and contribute to the understanding of the structure–function relationships of the main cell wall components. Enzymatic cross-linking of alginates by phenols may regulate the strengthening of the wall, and sulfated polysaccharides may play a key role in the adaptation to osmotic stress. The emergence and evolution of ECM components is further discussed in relation to the evolution of multicellularity in brown algae.     

Branch regeneration induced by sever damage in the brown alga <Emphasis Type="Italic">Dictyota dichotoma</Emphasis> (dictyotales,phaeophyceae)

Tissue wounds are mainly caused by herbivory, which is a serious threat for macro-algae, and brown algae are known to regenerate branches or buds in response to wounding. In the present paper, we describe a branch regeneration system, induced by sever damage, in the brown alga Dictyota dichotoma. Segmentations of juvenile thalli induced branch regenerations unless explants possessed apical cells. Apical excisions in distinct positions elucidated that disruption of an apical cell or disconnection of tissue with an apical cell triggered the branch regeneration. Furthermore, spatial positions of regenerated branches seemed to be regulated by the apical region, which was assumed to generate inhibitory effects for lateral branch regeneration. Mechanical incision, which disrupted tissue continuity with the apical region, induced branch regeneration preferentially below the incision. Although we were unable to identify the candidate inhibitory substance, our results suggested that the apical region may have an inhibitory effect on lateral branch regeneration. Additionally, observations of branch regeneration showed that all epidermal cells in D. dichotoma possess the ability to differentiate into apical cells, directly. This may be the first report of algal transdifferentiation during the wound-stress response.     

Seasonal variations of the photosynthetic activity and pigment concentrations in different reproductive phases of <Emphasis Type="Italic">Gigartina skottsbergii</Emphasis> (Rhodophyta,Gigartinales) in the Magellan region,sub-Antarctic Chile

Seasonal environmental changes may significantly influence macroalgal diversity and biomass. Cryptogam species richness increases towards the poles, especially in sub-Antarctic environments. Yet, subpolar seaweed biodiversity and ecophysiology remain understudied even though it is essential for the management and sustainability of endemic species of significant economic interest (e.g., Gigartina skottsbergii). We evaluate the seasonality and ecophysiology of the different life phases of the rhodophyte G. skottsbergii by analyzing variation in fluorescence yield and photosynthetic pigment composition. There were significant seasonal differences in maximum relative electron transport rate (rETR_max) between gametophyte and tetrasporophyte phase, and between reproductive and vegetative specimens. Photosynthetic efficiency (α) was not significantly different between reproductive states of G. skottsbergii. We found significant differences in mean concentrations of allophycocyanin (APC), phycocyanin (PC), and chlorophyll a (Chl a) between gametophyte and tetrasporophyte phases. Results obtained provide new insight into seasonal acclimation patterns of an ecologically important species, which can be used for the design of appropriate management and cultivation strategies of G. skottsbergii towards the restoration of natural populations in fragile, subpolar regions where some of the last, relatively undisturbed communities of G. skottsbergii still remain.     

Real-Time Imaging of Cellulose Reorientation during Cell Wall Expansion in Arabidopsis Roots

Cellulose forms the major load-bearing network of the plant cell wall, which simultaneously protects the cell and directs its growth. Although the process of cellulose synthesis has been observed, little is known about the behavior of cellulose in the wall after synthesis. Using Pontamine Fast Scarlet 4B, a dye that fluoresces preferentially in the presence of cellulose and has excitation and emission wavelengths suitable for confocal microscopy, we imaged the architecture and dynamics of cellulose in the cell walls of expanding root cells. We found that cellulose exists in Arabidopsis (Arabidopsis thaliana) cell walls in large fibrillar bundles that vary in orientation. During anisotropic wall expansion in wild-type plants, we observed that these cellulose bundles rotate in a transverse to longitudinal direction. We also found that cellulose organization is significantly altered in mutants lacking either a cellulose synthase subunit or two xyloglucan xylosyltransferase isoforms. Our results support a model in which cellulose is deposited transversely to accommodate longitudinal cell expansion and reoriented during expansion to generate a cell wall that is fortified against strain from any direction.The walls of growing plant cells must fulfill two simultaneous and seemingly contradictory requirements. First, they must expand to accommodate cell growth, which is anisotropic in many tissues and determines organ morphology. Second, they must maintain their structural integrity, both to constrain the turgor pressure that drives cell growth and to provide structural rigidity to the plant. These requirements are met by constructing primary cell walls that can expand along with growing cells, whereas secondary cell walls are deposited after cell growth has ceased and serve the latter function.One of the major constituents of both types of cell walls is cellulose, which exists as microfibrils composed of parallel β-1,4-linked glucan chains that are held together laterally by hydrogen bonds (Somerville, 2006). Microfibrils are 2 to 5 nm in diameter, can extend to several micrometers in length, and exhibit high tensile strength that allows cell walls to withstand turgor pressures of up to 1 MPa (Franks, 2003). In vascular plants, cellulose is synthesized by a multimeric cellulose synthase (CESA) complex composed of at least three types of glycosyl transferases arranged into a hexameric rosette (Somerville, 2006). After delivery to the plasma membrane, CESA initially moves in alignment with cortical microtubules (Paredez et al., 2006), but its trajectory can be maintained independently of microtubule orientation. For example, in older epidermal cells of the root elongation zone in Arabidopsis (Arabidopsis thaliana), cellulose microfibrils at the inner wall face are oriented transversely despite the fact that microtubules reorient from transverse to longitudinal along the elongation zone (Sugimoto et al., 2000), suggesting that microtubule orientation and cellulose deposition are independent in at least some cases.Depending on species, cell type, and developmental stage, cellulose microfibrils may be surrounded by additional networks of polymers, including hemicelluloses, pectins, lignin, and arabinogalactan proteins (Somerville et al., 2004). Hemicelluloses are composed of β-1,4-linked carbohydrate backbones with side branches and include xyloglucans, mannans, and arabinoxylans. Xyloglucan is thought to interact with the surface of cellulose and form cross-links between adjacent microfibrils (Vissenberg et al., 2005). In some cell types, pectin or lignin may also participate in cross-linking or entrapment of other cell wall polymers. It is unclear how the associations between networks of different cell wall components are relaxed to allow for cell wall expansion during growth.Several models have been proposed for the behavior of cell wall components during wall expansion. The passive reorientation hypothesis (also called the multinet growth hypothesis; Preston, 1982) postulates that in longitudinally expanding cells, cellulose microfibrils are synthesized in a transverse pattern and are then reoriented toward the longitudinal axis due to the strain generated by turgor pressure (Green, 1960). This phenomenon has been observed in the multicellular alga Nitella (Taiz, 1984). In higher plants, there is less direct evidence for passive reorientation, and another hypothesis holds that wall expansion involves active, local, and controlled remodeling of cellulose microfibrils along a diversity of orientations (Baskin, 2005). Such remodeling could be achieved by proteins such as xyloglucan endotransglycosylases (XETs), which break and rejoin xyloglucan chains, and expansins, which loosen cell walls in vitro in a pH-dependent manner (Cosgrove, 2005). Marga et al. measured cellulose microfibril orientation at the innermost layer of the cell wall before and after in vitro extension and did not observe reorientation (Marga et al., 2005). This suggests that processes other than microfibril reorientation might be involved in wall expansion, at least under certain circumstances or in some wall layers. Thus, the degree to which cellulose microfibrils are reoriented after their synthesis during wall expansion has remained unclear.One difficulty in resolving this problem has been the inability to directly image cellulose microfibrils in the growing cell wall. Existing methods to assess cellulose structure and orientation in plant cell walls are limited by the low contrast of cellulose in transmission electron microscopy, the ability to image only the surface of the wall using field emission scanning electron microscopy, and the use of polarized light microscopy in combination with dyes such as Congo red to measure only the bulk orientation of cellulose microfibrils (Baskin et al., 1999; Sugimoto et al., 2000; Verbelen and Kerstens, 2000; MacKinnon et al., 2006). In addition, the sample manipulation required for the former two methods has the potential to introduce artifacts (Marga et al., 2005). Although cellulose microfibril orientation differs at the inner and outer surfaces of the cell wall (Sugimoto et al., 2000) and presumably changes over time, the dynamics of cellulose reorientation during cell wall expansion have not been observed to date.In this study, we tested fluorescent dyes for their potential to allow imaging of cellulose distribution in the walls of Arabidopsis seedlings by confocal microscopy. We used one of these dyes to characterize the distribution of cellulose in wild-type root cells and in mutants with reduced cellulose or xyloglucan. By directly observing the fine structure of cellulose over time in growing wild-type root cells, we concluded that cellulose microfibrils in these cells reorient in a transverse to longitudinal direction as predicted by the passive reorientation hypothesis.     

New rhythmic changes in mitosis and growth in low differentiated green and red marine macroalgae

The cell division and vegetative growth of the thalli of simply differentiated macroalgae with a diffuse growth type—Ulva pseudocurvata (Chlorophyta) and Porphyra umbilicalis (Rhodophyta)-have been studied under natural and laboratory conditions. For this purpose the mitotic index and growth rate of algae were measured over 18 days. A diurnal rhythm of the mitotic index was revealed: the minimal mitotic index was registered in morning and daylight hours (for U. pseudocurvata 1–4%, for P. umbilicalis 0.5–2%), in the afternoon the index grew and reached its maximum 1 hour before dark (for U. pseudocurvata 12%, for P. umbilicalis 7%), then it slowly decreased during the night. In the studied algal species 2–3-and 6-day rhythms of mitotic index and growth rate were found for the first time both under natural and laboratory conditions. With constant white light these rhythms persisted for 9 days, this confirms the endogenous regulation of these rhythmic variations.     

Promotion of Efficient Saccharification of Crystalline Cellulose by Aspergillus fumigatus Swo1

Swollenin is a protein from Trichoderma reesei that has a unique activity for disrupting cellulosic materials, and it has sequence similarity to expansins, plant cell wall proteins that have a loosening effect that leads to cell wall enlargement. In this study we cloned a gene encoding a swollenin-like protein, Swo1, from the filamentous fungus Aspergillus fumigatus, and designated the gene Afswo1. AfSwo1 has a bimodular structure composed of a carbohydrate-binding module family 1 (CBM1) domain and a plant expansin-like domain. AfSwo1 was produced using Aspergillus oryzae for heterologous expression and was easily isolated by cellulose-affinity chromatography. AfSwo1 exhibited weak endoglucanase activity toward carboxymethyl cellulose (CMC) and bound not only to crystalline cellulose Avicel but also to chitin, while showing no detectable affinity to xylan. Treatment by AfSwo1 caused disruption of Avicel into smaller particles without any detectable reducing sugar. Furthermore, simultaneous incubation of AfSwo1 with a cellulase mixture facilitated saccharification of Avicel. Our results provide a novel approach for efficient bioconversion of crystalline cellulose into glucose by use of the cellulose-disrupting protein AfSwo1.Cellulose is the primary polysaccharide of plant cell wall and the most abundant renewable biomass resource. Biological degradation of cellulose to soluble sugars has long been considered an alternative to the use of starch feedstocks for bioethanol production. Natural cellulose is an ordered, linear polymer of thousands of d-glucose residues linked by β-1,4-glucosidic bonds. Spontaneous crystallization of cellulose molecules due to chemical uniformity of glucose units and the high degree of hydrogen bonding in cellulose can often result in the formation of tightly packed microfibrils (8), which remain inaccessible to cellulolytic enzymes. No single enzyme is able to hydrolyze crystalline cellulose microfibrils completely. Synergistic effects of cellulase mixtures on crystalline cellulose degradation are well known (1, 7, 21). Nevertheless, cost-competitive technology for overcoming the recalcitrance of cellulosic biomass to enhance enzymatic saccharification is still a major impediment to the utilization of cellulosic materials in bioenergy generation.Expansins are plant cell wall proteins that cause cell wall enlargement by a unique loosening effect in an acid-induced manner (15, 20). They are also involved in many physiological processes where cell wall extension occurs, such as pollination, fruit ripening, organ abscission, and seed germination (13, 14). It has been proposed that plant expansins disrupt hydrogen bonding between cellulose microfibrils and other cell wall polysaccharides without hydrolytic activity, causing sliding of cellulose fibers or expansion of the cell wall (18, 19, 27). Swollenin, an expansin-like protein, was isolated and characterized from the cellulolytic filamentous fungus Trichoderma reesei. It has a bimodular structure consisting of a carbohydrate-binding module family 1 (CBM1) domain and an expansin-like domain connected by a linker region rich in serine and threonine. Swollenin exhibits disruption activity on cellulosic materials such as cotton and algal cell walls without releasing any detectable reducing sugars (23). However, effects of cellulose disruption activity on degradation/saccharification of crystalline cellulose have not yet been reported.Here, we report cloning a swollenin-like gene (designated Afswo1) from the filamentous fungus Aspergillus fumigatus. We also report its production by Aspergillus oryzae and characterization of the purified AfSwo1.     

SIDECAR POLLEN suggests a plant-specific regulatory network underlying asymmetric microspore division in Arabidopsis

Asymmetric cell division is a universal strategy to generate diverse cell types necessary for patterning and proliferation of all eukaryotes. The development of haploid male gametophytes (pollen grains) in flowering plants is a remarkable example in which division asymmetry governs the functional specialization and germline differentiation essential for double fertilization. The male gametophyte is patterned via two mitotic divisions resulting in three highly differentiated daughter cells at maturity, a vegetative cell and two sperm cells. The first asymmetric division segregates a unique male germ cell from an undetermined haploid microspore and is executed in an elaborate sequence of cellular events. However the molecular mechanisms governing the division asymmetry in microspores are poorly understood. Recently we studied the phenotype of sidecar pollen (scp) mutants in detail, and demonstrated a requirement of SCP for both the correct timing and orientation of microspore division. SCP is a microspore-specific member of the LOB/AS2 domain family (LBD27/ASL29) showing that a plant-specific regulator plays a key role in oriented division of polarized microspores. Identification of SCP will serve as a new platform to further explore the largely unknown molecular networks regulating division asymmetry in microspores that establishes the male germline in flowering plants.Key words: sidecar pollen, microspore division, division asymmetry, male gametophyte development, male germline, LBD/ASL family proteinUnlike animals, flowering plants do not set aside a distinct germline from an early stage of the life cycle. Instead the angiosperm germline or germ cells are only segregated in the male and female gametophytes by a limited number of post-meiotic mitoses.¹ However, in common with their metazoan cousins, angiosperms utilize division asymmetry for cellular patterning and differentiation of their germlines. Through the unique patterning of a ‘cell-within-a-cell’ structure with three highly differentiated cells, the male gametophyte (pollen grains) serves its biological role to deliver two sessile male gametes to the female gametophyte. Two sequential but different modes of mitotic divisions pattern the male gametophyte (Fig. 1).² The first division (of the microspore) is asymmetric giving rise to two completely different daughter cells, a larger vegetative cell that will form the pollen tube and a smaller germ cell that is engulfed within the vegetative cell cytoplasm. The second division (of the germ cell) usually appears symmetric^† and produces a pair of linked sperm cells. Microspores artificially induced to undergo symmetric division using microtubule inhibitors lack the germ cell and fail to form the typical three-celled structure showing that asymmetry in microspore division is critical for patterning of the male gametophyte.⁴Open in a separate windowFigure 1Male gametophyte development in Arabidopsis (upper part) and mutations that block germ cell formation (lower part). (Upper part) Male gametophyte development involves two rounds of mitotic division. Prior to the first division the centrally positioned microspore nucleus migrates towards the radial wall (the future germ cell pole marked with an asterisk). At this eccentric site the polarized microspores undergo oriented mitosis and cytokinesis giving rise to highly unequal daughter cells, a vegetative cell and a germ cell of which the later produces a pair of sperm cells by symmetric division. (Lower part) Mutants that fail to establish a distinct germ cell arising from specific defects are illustrated. Arrows in red indicate the developmental origin of the phenotypic defects in mutants. Note that two daughter nuclei in the mutants are in grey to show that their cell fates have not yet been thoroughly investigated. n, nucleus; Vn, vegetative nucleus; Gn, generative nucleus; Gc, generative (or germ) cell; Sc, sperm cell; WT, wild type; gem1, gemini pollen1; scp, sidecar pollen; tio, two-in-one; hik/tes, hinkel/tetraspore 12a/12b, kinesin-12a/kinesin-12b.     

Ultrastructural changes in the epidermis of petals of the sweet orange infected by <Emphasis Type="Italic">Colletotrichum acutatum</Emphasis>

Postbloom fruit drop (PFD) is an important disease caused by the fungus Colletotrichum acutatum. PFD is characterised by the formation of necrotic lesions on the petals and stigmas of flowers as well as premature abscission of the fruit in Citrus spp. We compare the ultrastructure of the epidermis of uninoculated Citrus sinensis petals with that of petals inoculated with the fungus to understand the changes that occur upon C. acutatum infection. Healthy petals have a cuticle with parallel striations covering the uniseriate epidermis. This pattern consists of vacuolated parietal cells whose cytoplasm contains mitochondria, plastids with an undeveloped endomembrane system and a slightly dense stroma, a poorly developed rough endoplasmic reticulum, polysomes, few lipid droplets, and a nucleus positioned near the inner periclinal wall. In damaged regions, the cytoplasm of some cells is densely packed with well-developed endoplasmic reticulum, a large number of hyperactive dictyosomes, numerous mitochondria, and many lipid droplets. The plastids have an electron-dense stroma, starch grains, and a large amount of electron-dense lipid droplets, which can be released into vacuoles or the endoplasmic reticulum. Multivesicular bodies and myelin bodies are frequently observed in the vacuole, cytoplasm, and periplasmic space. Vesicles migrate through the cell wall and are involved in the deposition of cuticular material. In the later stages of infection, there is deposition of new cuticle layers in plaques. The outer periclinal walls can be thick. These observations indicate that epidermal cells respond to the pathogen, resulting in cuticular and parietal changes, which may limit further infection.     

An Arabidopsis Cell Wall Proteoglycan Consists of Pectin and Arabinoxylan Covalently Linked to an Arabinogalactan Protein

Plant cell walls are comprised largely of the polysaccharides cellulose, hemicellulose, and pectin, along with ∼10% protein and up to 40% lignin. These wall polymers interact covalently and noncovalently to form the functional cell wall. Characterized cross-links in the wall include covalent linkages between wall glycoprotein extensins between rhamnogalacturonan II monomer domains and between polysaccharides and lignin phenolic residues. Here, we show that two isoforms of a purified Arabidopsis thaliana arabinogalactan protein (AGP) encoded by hydroxyproline-rich glycoprotein family protein gene At3g45230 are covalently attached to wall matrix hemicellulosic and pectic polysaccharides, with rhamnogalacturonan I (RG I)/homogalacturonan linked to the rhamnosyl residue in the arabinogalactan (AG) of the AGP and with arabinoxylan attached to either a rhamnosyl residue in the RG I domain or directly to an arabinosyl residue in the AG glycan domain. The existence of this wall structure, named ARABINOXYLAN PECTIN ARABINOGALACTAN PROTEIN1 (APAP1), is contrary to prevailing cell wall models that depict separate protein, pectin, and hemicellulose polysaccharide networks. The modified sugar composition and increased extractability of pectin and xylan immunoreactive epitopes in apap1 mutant aerial biomass support a role for the APAP1 proteoglycan in plant wall architecture and function.     

Anther ontogeny in <Emphasis Type="Italic">Brachypodium distachyon</Emphasis>

Brachypodium distachyon has emerged as a model plant for the improvement of grain crops such as wheat, barley and oats and for understanding basic biological processes to facilitate the development of grasses as superior energy crops. Brachypodium is also the first species of the grass subfamily Pooideae with a sequenced genome. For obtaining a better understanding of the mechanisms controlling male gametophyte development in B. distachyon, here we report the cellular changes during the stages of anther development, with special reference to the development of the anther wall. Brachypodium anthers are tetrasporangiate and follow the typical monocotyledonous-type anther wall formation pattern. Anther differentiation starts with the appearance of archesporial cells, which divide to generate primary parietal and primary sporogenous cells. The primary parietal cells form two secondary parietal layers. Later, the outer secondary parietal layer directly develops into the endothecium and the inner secondary parietal layer forms an outer middle layer and inner tapetum by periclinal division. The anther wall comprises an epidermis, endothecium, middle layer and the secretory-type tapetum. Major documented events of anther development include the degradation of a secretory-type tapetum and middle layer during the course of development and the rapid formation of U-shaped endothecial thickenings in the mature pollen grain stage. The tapetum undergoes degeneration at the tetrad stage and disintegrates completely at the bicellular stage of pollen development. The distribution of insoluble polysaccharides in the anther layers and connective tissue through progressive developmental stages suggests their role in the development of male gametophytes. Until sporogenous cell stage, the amount of insoluble polysaccharides in the anther wall was negligible. However, abundant levels of insoluble polysaccharides were observed during microspore mother cell and tetrad stages and gradually declined during the free microspore and vacuolated microspore stages to undetectable level at the mature stage. Thus, the cellular features in the development of anthers in B. distachyon share similarities with anther and pollen development of other members of Poaceae.