Functional Specialization of Cellulose Synthase Isoforms in a Moss Shows Parallels with Seed Plants.

The secondary cell walls of tracheary elements and fibers are rich in cellulose microfibrils that are helically oriented and laterally aggregated. Support cells within the leaf midribs of mosses deposit cellulose-rich secondary cell walls, but their biosynthesis and microfibril organization have not been examined. Although the Cellulose Synthase (CESA) gene families of mosses and seed plants diversified independently, CESA knockout analysis in the moss Physcomitrella patens revealed parallels with Arabidopsis (Arabidopsis thaliana) in CESA functional specialization, with roles for both subfunctionalization and neofunctionalization. The similarities include regulatory uncoupling of the CESAs that synthesize primary and secondary cell walls, a requirement for two or more functionally distinct CESA isoforms for secondary cell wall synthesis, interchangeability of some primary and secondary CESAs, and some CESA redundancy. The cellulose-deficient midribs of ppcesa3/8 knockouts provided negative controls for the structural characterization of stereid secondary cell walls in wild type P. patens Sum frequency generation spectra collected from midribs were consistent with cellulose microfibril aggregation, and polarization microscopy revealed helical microfibril orientation only in wild type leaves. Thus, stereid secondary walls are structurally distinct from primary cell walls, and they share structural characteristics with the secondary walls of tracheary elements and fibers. We propose a mechanism for the convergent evolution of secondary walls in which the deposition of aggregated and helically oriented microfibrils is coupled to rapid and highly localized cellulose synthesis enabled by regulatory uncoupling from primary wall synthesis.

Determining the subcellular location of synthesis and assembly of the cell wall polysaccharide (1,3; 1,4)-ß-D-glucan in grasses.

The current dogma for cell wall polysaccharide biosynthesis is that cellulose (and callose) is synthesized at the plasma membrane (PM), whereas matrix phase polysaccharides are assembled in the Golgi apparatus. We provide evidence that (1,3;1,4)-ß-D-glucan (mixed-linkage glucan [MLG]) does not conform to this paradigm. We show in various grass (Poaceae) species that MLG-specific antibody labeling is present in the wall but absent over Golgi, suggesting it is assembled at the PM. Antibodies to the MLG synthases, cellulose synthase-like F6 (CSLF6) and CSLH1, located CSLF6 to the endoplasmic reticulum, Golgi, secretory vesicles, and the PM and CSLH1 to the same locations apart from the PM. This pattern was recreated upon expression of VENUS-tagged barley (Hordeum vulgare) CSLF6 and CSLH1 in Nicotiana benthamiana leaves and, consistent with our biochemical analyses of native grass tissues, shown to be catalytically active with CSLF6 and CSLH1 in PM-enriched and PM-depleted membrane fractions, respectively. These data support a PM location for the synthesis of MLG by CSLF6, the predominant enzymatically active isoform. A model is proposed to guide future experimental approaches to dissect the molecular mechanism(s) of MLG assembly.

SnRK1 from Arabidopsis thaliana is an atypical AMPK.

SNF1-related protein kinase 1 (SnRK1) is the plant orthologue of the evolutionarily-conserved SNF1/AMPK/SnRK1 protein kinase family that contributes to cellular energy homeostasis. Functional as heterotrimers, family members comprise a catalytic α subunit and non-catalytic ß and γ subunits; multiple isoforms of each subunit type exist, giving rise to various isoenzymes. The Arabidopsis thaliana genome contains homologues of each subunit type, and, in addition, two atypical subunits, ß(3) and ßγ, with unique domain architecture, that are found only amongst plants, suggesting atypical heterotrimers. The AtSnRK1 subunit structure was determined using recombinant protein expression and endogenous co-immunoprecipitation, and six unique isoenzyme combinations were identified. Each heterotrimeric isoenzyme comprises a catalytic α subunit together with the unique ßγ subunit and one of three non-catalytic ß subunits: ß(1), ß(2) or the plant-specific ß(3) isoform. Thus, the AtSnRK1 heterotrimers contain the atypical ßγ subunit rather than a conventional γ subunit. Mammalian AMPK heterotrimers are phosphorylated on the T-loop (pThr175/176) within both catalytic a subunits. However, AtSnRK1 is insensitive to AMP and ADP, and is resistant to T-loop dephosphorylation by protein phosphatases, a process that inactivates other SNF1/AMPK family members. In addition, we show that SnRK1 is inhibited by a heat-labile, >30 kDa, soluble proteinaceous factor that is present in the lysate of young rosette leaves. Finally, none of the three SnRK1 carbohydrate-binding modules, located in the ß(1), ß(2) and ßγ subunits, associate with various carbohydrates, including starch, the plant analogue of glycogen to which AMPK binds in vitro. These data clearly demonstrate that AtSnRK1 is an atypical member of the SNF1/AMPK/SnRK1 family.

Combined NanoSIMS and synchrotron X-ray fluorescence reveal distinct cellular and subcellular distribution patterns of trace elements in rice tissues.

The cellular and subcellular distributions of trace elements can provide important clues to understanding how the elements are transported and stored in plant cells, but mapping their distributions is a challenging task. The distributions of arsenic, iron, zinc, manganese and copper, as well as physiologically related macro-elements, were mapped in the node, internode and leaf sheath of rice (Oryza sativa) using synchrotron X-ray fluorescence (S-XRF) and high-resolution secondary ion mass spectrometry (NanoSIMS). Although copper and silicon generally showed cell wall localization, arsenic, iron and zinc were strongly localized in the vacuoles of specific cell types. Arsenic was highly localized in the companion cell vacuoles of the phloem in all vascular bundles, showing a strong co-localization with sulfur, consistent with As(III)-thiol complexation. Within the node, zinc was localized in the vacuoles of the parenchyma cell bridge bordering the enlarged and diffuse vascular bundles, whereas iron and manganese were localized in the fundamental parenchyma cells, with iron being strongly co-localized with phosphorus in the vacuoles. The highly heterogeneous and contrasting distribution patterns of these elements imply different transport activities and/or storage capacities among different cell types. Sequestration of arsenic in companion cell vacuoles may explain the limited phloem mobility of arsenite.

Gross morphological changes in thylakoid membrane structure are associated with photosystem I deletion in Synechocystis sp. PCC 6803.

Cells of Synechocystis sp. PCC 6803 lacking photosystem I (PSI-less) and containing only photosystem II (PSII) or lacking both photosystems I and II (PSI/PSII-less) were compared to wild type (WT) cells to investigate the role of the photosystems in the architecture, structure, and number of thylakoid membranes. All cells were grown at 0.5µmol photons m(-2)s(-1). The lumen of the thylakoid membranes of the WT cells grown at this low light intensity were inflated compared to cells grown at higher light intensity. Tubular as well as sheet-like thylakoid membranes were found in the PSI-less strain at all stages of development with organized regular arrays of phycobilisomes on the surface of the thylakoid membranes. Tubular structures were also found in the PSI/PSII-less strain, but these were smaller in diameter to those found in the PSI-less strain with what appeared to be a different internal structure and were less common. There were fewer and smaller thylakoid membrane sheets in the double mutant and the phycobilisomes were found on the surface in more disordered arrays. These differences in thylakoid membrane structure most likely reflect the altered composition of photosynthetic particles and distribution of other integral membrane proteins and their interaction with the lipid bilayer. These results suggest an important role for the presence of PSII in the formation of the highly ordered tubular structures.

Multi-scale assembly of hydrogels formed by highly branched arabinoxylans from Plantago ovata seed mucilage studied by USANS/SANS and rheology.

The structures of two hydrogels formed by purified brush-like polysaccharides from Plantago ovata seed mucilage have been characterised from the nanometre to micrometre scale by using a combination of SANS and USANS techniques. These two hydrogels have distinctly different melting and rheological properties, but the structure of their gel networks bears striking similarity as revealed by USANS/SANS experiments. Surprisingly, we find that the dramatic changes in the rheological properties induced by temperature or change in the solvent quality are accompanied by a small alteration of the network structure as inferred from scattering curves recorded above melting or in a chaotropic solvent (0.7 M KOD). These results suggest that, in contrast to most gel-forming polysaccharides for which gelation depends on a structural transition, the rheological properties of Plantago ovata mucilage gels are dependent on variations in intermolecular hydrogen bonding. By enzymatically cleaving off terminal arabinose residues from the side chains, we have demonstrated that composition of side-chains has a strong effect on intermolecular interactions, which, in turn, has a profound effect on rheological and structural properties of these unique polysaccharides.

High-mass-resolution MALDI mass spectrometry imaging reveals detailed spatial distribution of metabolites and lipids in roots of barley seedlings in response to salinity stress.

INTRODUCTION: Mass spectrometry imaging (MSI) is a technology that enables the visualization of the spatial distribution of hundreds to thousands of metabolites in the same tissue section simultaneously. Roots are below-ground plant organs that anchor plants to the soil, take up water and nutrients, and sense and respond to external stresses. Physiological responses to salinity are multifaceted and have predominantly been studied using whole plant tissues that cannot resolve plant salinity responses spatially. OBJECTIVES: This study aimed to use a comprehensive approach to study the spatial distribution and profiles of metabolites, and to quantify the changes in the elemental content in young developing barley seminal roots before and after salinity stress. METHODS: Here, we used a combination of liquid chromatography-mass spectrometry (LC-MS), inductively coupled plasma mass spectrometry (ICP-MS), and matrix-assisted laser desorption/ionization (MALDI-MSI) platforms to profile and analyze the spatial distribution of ions, metabolites and lipids across three anatomically different barley root zones before and after a short-term salinity stress (150 mM NaCl). RESULTS: We localized, visualized and discriminated compounds in fine detail along longitudinal root sections and compared ion, metabolite, and lipid composition before and after salt stress. Large changes in the phosphatidylcholine (PC) profiles were observed as a response to salt stress with PC 34:n showing an overall reduction in salt treated roots. ICP-MS analysis quantified changes in the elemental content of roots with increases of Na+ and decreases of K+ content. CONCLUSION: Our results established the suitability of combining three mass spectrometry platforms to analyze and map ionic and metabolic responses to salinity stress in plant roots and to elucidate tolerance mechanisms in response to abiotic stress, such as salinity stress.

Characterisation of the Fusarium graminearum-Wheat Floral Interaction.

Fusarium Ear Blight is a destructive fungal disease of cereals including wheat and can contaminate the crop with various trichothecene mycotoxins. This investigation has produced a new ß-glucuronidase (GUS) reporter strain that facilitates the quick and easy assessment of plant infection. The constitutively expressed gpdA:GUS strain of Fusarium graminearum was used to quantify the overall colonisation pattern. Histochemical and biochemical approaches confirmed, in susceptible wheat ear infections, the presence of a substantial phase of symptomless fungal growth. Separate analyses demonstrated that there was a reduction in the quantity of physiologically active hyphae as the wheat ear infection proceeded. A simplified linear system of rachis infection was then utilised to evaluate the expression of several TRI genes by RT-qPCR. Fungal gene expression at the advancing front of symptomless infection was compared with the origin of infection in the rachis. This revealed that TRI gene expression was maximal at the advancing front and supports the hypothesis that the mycotoxin deoxynivalenol plays a role in inhibiting plant defences in advance of the invading intercellular hyphae. This study has also demonstrated that there are transcriptional differences between the various phases of fungal infection and that these differences are maintained as the infection proceeds.

The infection biology of Fusarium graminearum: defining the pathways of spikelet to spikelet colonisation in wheat ears.

Fusarium graminearum is one of the main causal agents of Fusarium Ear Blight on wheat. How the pathogen colonises the entire ear is not known. There is controversy over whether this mycotoxin producing pathogenic fungus invades wheat floral tissue using a necrotrophic or another mode of nutrition. A detailed microscopic investigation has revealed how wild-type fungal hyphae, of the sequenced strain PH-1, colonised susceptible wheat ears and spread from spikelet to spikelet. At the advancing infection front, colonisation of the host cortex occurred ahead of any vascular colonisation and the hyphae adapted to the available intercellular space between host cells. Intercellular hyphae then became abundant and host cells lost their entire cellular contents just prior to intracellular colonisation. No host cells died ahead of the infection. However, while these deep cortex infections progressed, just below the surface the highly photosynthetic chlorenchyma cells were observed to have died prior to colonisation. Behind the infection front, hyphae were abundant in the vasculature and the cortex, often growing through the pit fields of thick walled cells. This high level of inter- and intracellular fungal colonisation resulted in the collapse of the non-lignified cell-types. In this middle zone of infection, hyphal diameters were considerably enlarged. Far behind the infection front inter- and intracellular hyphae were devoid of contents and had often collapsed. At later stages of infection, the pathogen switched from predominately vertical to lateral growth and accumulated below the surface of the rachis. Here the lignified host cell walls became heavily degraded and hyphae ruptured the epidermis and produced an aerial mycelium.

Initial characterization of the photosynthetic apparatus of "Candidatus Chlorothrix halophila," a filamentous, anoxygenic photoautotroph.

"Candidatus Chlorothrix halophila" is a recently described halophilic, filamentous, anoxygenic photoautotroph (J. A. Klappenbach and B. K. Pierson, Arch. Microbiol. 181:17-25, 2004) that was enriched from the hypersaline microbial mats at Guerrero Negro, Mexico. Analysis of the photosynthetic apparatus by negative staining, spectroscopy, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the photosynthetic apparatus in this organism has similarities to the photosynthetic apparatus in both the Chloroflexi and Chlorobi phyla of green photosynthetic bacteria. The chlorosomes were found to be ellipsoidal and of various sizes, characteristics that are comparable to characteristics of chlorosomes in other species of green photosynthetic bacteria. The absorption spectrum of whole cells was dominated by the chlorosome bacteriochlorophyll c (BChl c) peak at 759 nm, with fluorescence emission at 760 nm. A second fluorescence emission band was observed at 870 nm and was tentatively attributed to a membrane-bound antenna complex. Fluorescence emission spectra obtained at 77 K revealed another complex that fluoresced at 820 nm, which probably resulted from the chlorosome baseplate complex. All of these results suggest that BChl c is present in the chlorosomes of "Ca. Chlorothrix halophila," that BChl a is present in the baseplate, and that there is a membrane-bound antenna complex. Analysis of the proteins in the chlorosomes revealed an approximately 6-kDa band, which was found to be related to the BChl c binding protein CsmA found in other green bacteria. Overall, the absorbance and fluorescence spectra of "Ca. Chlorothrix halophila" revealed an interesting mixture of photosynthetic characteristics that seemed to have properties similar to properties of both phyla of green bacteria when they were compared to the photosynthetic characteristics of Chlorobium tepidum and Chloroflexus aurantiacus.

Pigment analysis of "Candidatus Chlorothrix halophila," a green filamentous anoxygenic phototrophic bacterium.

The pigment composition of "Candidatus Chlorothrix halophila," a filamentous anoxygenic phototrophic bacterium found in Baja California Sur, Mexico, was determined. Previous work showed that bacteriochlorophyll c (BChl c) was the major pigment in "Ca. Chlorothrix halophila," but it was not clear if this bacterium also contains BChl a (J. A. Klappenbach and B. K. Pierson, Arch. Microbiol. 181:17-25, 2004). Here we show that in addition to BChl c, a small amount of a pigment that is spectrally indistinguishable from BChl a is present in cell extracts of "Ca. Chlorothrix halophila." Nevertheless, the BChl a-like pigment from "Ca. Chlorothrix halophila" has a different molecular weight and a different high-performance liquid chromatography elution time than BChl a from other photosynthetic bacteria. Based on mass spectrometry and other spectroscopic analysis, we determined that the BChl a-like pigment in "Ca. Chlorothrix halophila" contains a tetrahydrogeranylgeraniol tail rather than the phytol tail that is present in BChl a. The carotenoids and major BChl c homologs in "Ca. Chlorothrix halophila" were also identified. BChls c were found to be farnesol esterified and geranylgeraniol esterified.

The three-dimensional structure of the cyanobacterium Synechocystis sp. PCC 6803.

To advance our knowledge of the model cyanobacterium Synechocystis sp. PCC 6803 we investigated the three-dimensional organization of the cytoplasm using standard transmission electron microscopy and electron tomography. Electron tomography allows a resolution of ~5 nm in all three dimensions, superior to the resolution of most traditional electron microscopy, which is often limited in part by the thickness of the section (70 nm). The thylakoid membrane pairs formed layered sheets that followed the periphery of the cell and converged at various sites near the cytoplasmic membrane. At some of these sites, the margins of thylakoid membranes associated closely along the external surface of rod-like structures termed thylakoid centers, which sometimes traversed nearly the entire periphery of the cell. The thylakoid membranes surrounded the central cytoplasm that contained inclusions such as ribosomes and carboxysomes. Lipid bodies were dispersed throughout the peripheral cytoplasm and often juxtaposed with cytoplasmic and thylakoid membranes suggesting involvement in thylakoid maintenance or biogenesis. Ribosomes were numerous and mainly located throughout the central cytoplasm with some associated with thylakoid and cytoplasmic membranes. Some ribosomes were attached along internal unit-membrane-like sheets located in the central cytoplasm and appeared to be continuous with existing thylakoid membranes. These results present a detailed analysis of the structure of Synechocystis sp. PCC 6803 using high-resolution bioimaging techniques and will allow future evaluation and comparison with gene-deletion mutants.

Application of electron tomography to fungal ultrastructure studies.

Access to structural information at the nanoscale enables fundamental insights into many complex biological systems. The development of the transmission electron microscope (TEM) has vastly increased our understanding of multiple biological systems. However, when attempting to visualize and understand the organizational and functional complexities that are typical of cells and tissues, the standard 2-D analyses that TEM affords often fall short. In recent years, high-resolution electron tomography methods, coupled with advances in specimen preparation and instrumentation and computational speed, have resulted in a revolution in the biological sciences. Electron tomography is analogous to medical computerized axial tomography (CAT-scan imaging) except at a far finer scale. It utilizes the TEM to assemble multiple projections of an object which are then combined for 3-D analyses. For biological specimens, tomography enables the highest 3-D resolution (5 nm spatial resolution) of internal structures in relatively thick slices of material (0.2-0.4 microm) without requiring the collection and alignment of large numbers of thin serial sections. Thus accurate and revealing 3-D reconstructions of complex cytoplasmic entities and architecture can be obtained. Electron tomography is now being applied to a variety of biological questions with great success. This review gives a brief introduction into cryopreservation and electron tomography relative to aspects of cytoplasmic organization in the hyphal tip of Aspergillus nidulans.

Myxoxanthophyll is required for normal cell wall structure and thylakoid organization in the cyanobacterium Synechocystis sp. strain PCC 6803.

Myxoxanthophyll is a carotenoid glycoside in cyanobacteria that is of unknown biological significance. The sugar moiety of myxoxanthophyll in Synechocystis sp. strain PCC 6803 was identified as dimethyl fucose. The open reading frame sll1213 encoding a fucose synthetase orthologue was deleted to probe the role of fucose and to determine the biological significance of myxoxanthophyll in Synechocystis sp. strain PCC 6803. Upon deletion of sll1213, a pleiotropic phenotype was obtained: when propagated at 0.5 micromol photons m(-2) s(-1), photomixotrophic growth of cells lacking sll1213 was poor. When grown at 40 micromol photons m(-2) s(-1), growth was comparable to that of the wild type, but cells showed a severe reduction in or loss of the glycocalyx (S-layer). As a consequence, cells aggregated in liquid as well as on plates. At both light intensities, new carotenoid glycosides accumulated, but myxoxanthophyll was absent. New carotenoid glycosides may be a consequence of less-specific glycosylation reactions that gained prominence upon the disappearance of the native sugar moiety (fucose) of myxoxanthophyll. In the mutant, the N-storage compound cyanophycin accumulated, and the organization of thylakoid membranes was altered. Altered cell wall structure and thylakoid membrane organization and increased cyanophycin accumulation were also observed for deltaslr0940K, a strain lacking zeta-carotene desaturase and thereby all carotenoids but retaining fucose. Therefore, lack of myxoxanthophyll and not simply of fucose results in most of the phenotypic effects described here. It is concluded that myxoxanthophyll contributes significantly to the vigor of cyanobacteria, as it stabilizes thylakoid membranes and is critical for S-layer formation.