Glycosyl transferase GT2 genes mediate the biosynthesis of an unusual (1,3;1,4)-ß-glucan exopolysaccharide in the bacterium Sarcina ventriculi.

Linear, unbranched (1,3;1,4)-ß-glucans (mixed-linkage glucans or MLGs) are commonly found in the cell walls of grasses, but have also been detected in basal land plants, algae, fungi and bacteria. Here we show that two family GT2 glycosyltransferases from the Gram-positive bacterium Sarcina ventriculi are capable of synthesizing MLGs. Immunotransmission electron microscopy demonstrates that MLG is secreted as an exopolysaccharide, where it may play a role in organizing individual cells into packets that are characteristic of Sarcina species. Heterologous expression of these two genes shows that they are capable of producing MLGs in planta, including an MLG that is chemically identical to the MLG secreted from S. ventriculi cells but which has regularly spaced (1,3)-ß-linkages in a structure not reported previously for MLGs. The tandemly arranged, paralogous pair of genes are designated SvBmlgs1 and SvBmlgs2. The data indicate that MLG synthases have evolved different enzymic mechanisms for the incorporation of (1,3)-ß- and (1,4)-ß-glucosyl residues into a single polysaccharide chain. Amino acid variants associated with the evolutionary switch from (1,4)-ß-glucan (cellulose) to MLG synthesis have been identified in the active site regions of the enzymes. The presence of MLG synthesis in bacteria could prove valuable for large-scale production of MLG for medical, food and beverage applications.

Phosphate starvation regulates cellulose synthesis to modify root growth.

In the model plant Arabidopsis (Arabidopsis thaliana), the absence of the essential macro-nutrient phosphate reduces primary root growth through decreased cell division and elongation, requiring alterations to the polysaccharide-rich cell wall surrounding the cells. Despite its importance, the regulation of cell wall synthesis in response to low phosphate levels is not well understood. In this study, we show that plants increase cellulose synthesis in roots under limiting phosphate conditions, which leads to changes in the thickness and structure of the cell wall. These changes contribute to the reduced growth of primary roots in low-phosphate conditions. Furthermore, we found that the cellulose synthase complex (CSC) activity at the plasma membrane increases during phosphate deficiency. Moreover, we show that this increase in the activity of the CSC is likely due to alterations in the phosphorylation status of cellulose synthases in low-phosphate conditions. Specifically, phosphorylation of CELLULOSE SYNTHASE 1 (CESA1) at the S688 site decreases in low-phosphate conditions. Phosphomimic versions of CESA1 with an S688E mutation showed significantly reduced cellulose induction and primary root length changes in low-phosphate conditions. Protein structure modeling suggests that the phosphorylation status of S688 in CESA1 could play a role in stabilizing and activating the CSC. This mechanistic understanding of root growth regulation under limiting phosphate conditions provides potential strategies for changing root responses to soil phosphate content.

Spatiotemporal deposition of cell wall polysaccharides in oat endosperm during grain development.

Oat (Avena sativa) is a cereal crop whose grains are rich in (1,3;1,4)-ß-D-glucan (mixed-linkage glucan or MLG), a soluble dietary fiber. In our study, we analyzed oat endosperm development in 2 Canadian varieties with differing MLG content and nutritional value. We confirmed that oat undergoes a nuclear type of endosperm development but with a shorter cellularization phase than barley (Hordeum vulgare). Callose and cellulose were the first polysaccharides to be detected in the early anticlinal cell walls at 11 days postemergence (DPE) of the panicle. Other polysaccharides such as heteromannan and homogalacturonan were deposited early in cellularization around 12 DPE after the first periclinal walls are laid down. In contrast to barley, heteroxylan deposition coincided with completion of cellularization and was detected from 14 DPE but was only detectable after demasking. Notably, MLG was the last polysaccharide to be laid down at 18 DPE within the differentiation phase, rather than during cellularization. In addition, differences in the spatiotemporal patterning of MLG were also observed between the 2 varieties. The lower MLG-containing cultivar AC Morgan (3.5% w/w groats) was marked by the presence of a discontinuous pattern of MLG labeling, while labeling in the same walls in CDC Morrison (5.6% w/w groats) was mostly even and continuous. RNA-sequencing analysis revealed higher transcript levels of multiple MLG biosynthetic cellulose synthase-like F (CSLF) and CSLH genes during grain development in CDC Morrison compared with AC Morgan that likely contributes to the increased abundance of MLG at maturity in CDC Morrison. CDC Morrison was also observed to have smaller endosperm cells with thicker walls than AC Morgan from cellularization onwards, suggesting the processes controlling cell size and shape are established early in development. This study has highlighted that the molecular processes influencing MLG content and deposition are more complex than previously imagined.

AUXIN RESPONSE FACTORS 6 and 17 control the flag leaf angle in rice by regulating secondary cell wall biosynthesis of lamina joints.

Flag leaf angle impacts the photosynthetic capacity of densely grown plants and is thus an important agronomic breeding trait for crop architecture and yield. The hormone auxin plays a key role in regulating this trait, yet the underlying molecular and cellular mechanisms remain unclear. Here, we report that two rice (Oryza sativa) auxin response factors (ARFs), OsARF6 and OsARF17, which are highly expressed in lamina joint tissues, control flag leaf angle in response to auxin. Loss-of-function double osarf6 osarf17 mutants displayed reduced secondary cell wall levels of lamina joint sclerenchymatous cells (Scs), resulting in an exaggerated flag leaf angle and decreased grain yield under dense planting conditions. Mechanical measurements indicated that the mutant lamina joint tissues were too weak to support the weight of the flag leaf blade, resembling the phenotype of the rice increased leaf angle1 (ila1) mutant. We demonstrate that OsARF6 and OsARF17 directly bind to the ILA1 promoter independently and synergistically to activate its expression. In addition, auxin-induced ILA1 expression was dependent on OsARF6 and OsARF17. Collectively, our study reveals a mechanism that integrates auxin signaling with the secondary cell wall composition to determine flag leaf angle, providing breeding targets in rice, and potentially other cereals, for this key trait.

Structure and formation of the perforated theca defining the Pelagophyceae (Heterokonta), and three new genera that substantiate the diverse nature of the class.

The pelagophytes, a morphologically diverse class of marine heterokont algae, have been historically united only by DNA sequences. Recently we described a novel perforated theca (PT) encasing cells from the Pelagophyceae and hypothesized it may be the first morphological feature to define the class. Here we consolidate that observation, describing a PT for the first time in an additional seven pelagophyte genera, including three genera new to science. We established clonal cultures of pelagophytes collected from intertidal pools located around Australia, and established phylogenetic trees based on nuclear 18S rDNA and plastid rbcL, psaA, psaB, psbA and psbC gene sequences that led to the discovery of three new species: Wyeophycus julieharrissiae and Chromopallida australis form a distinct lineage along with Ankylochrysis lutea within the Pelagomonadales, while Pituiglomerulus capricornicus is sister genus to Chrysocystis fragilis in the Chrysocystaceae (Sarcinochrysidales). Using fixation by high-pressure freezing for electron microscope observations, a distinctive PT was observed in the three new genera described in this paper, as well as four genera not previously investigated: Chrysoreinhardia, Sargassococcus, Sungminbooa and Andersenia. The mechanism of PT formation is novel, being fabricated from rafts in Golgi-derived vesicles before being inserted into an established PT. Extracellular wall and/or mucilage layers assemble exterior to the PT in most pelagophytes, the materials likewise secreted by Golgi-derived vesicles, though the mechanism of secretion is novel. Secretory vesicles never fuse with the plasma membrane as in classic secretion and deposition, but rather relocate extracellularly beneath the PT and disintegrate, the contents having to pass through the PT prior to wall and/or mucilage synthesis. This study substantiates the diverse nature of pelagophytes, and provides further evidence that the PT is a sound morphological feature to define the Pelagophyceae, with all 14 of the 20 known genera studied to date by TEM possessing a PT.

Arabidopsis DGD1 SUPPRESSOR1 Is a Subunit of the Mitochondrial Contact Site and Cristae Organizing System and Affects Mitochondrial Biogenesis.

Mitochondrial and plastid biogenesis requires the biosynthesis and assembly of proteins, nucleic acids, and lipids. In Arabidopsis (Arabidopsis thaliana), the mitochondrial outer membrane protein DGD1 SUPPRESSOR1 (DGS1) is part of a large multi-subunit protein complex that contains the mitochondrial contact site and cristae organizing system 60-kD subunit, the translocase of outer mitochondrial membrane 40-kD subunit (TOM40), the TOM20s, and the Rieske FeS protein. A point mutation in DGS1, dgs1-1, altered the stability and protease accessibility of this complex. This altered mitochondrial biogenesis, mitochondrial size, lipid content and composition, protein import, and respiratory capacity. Whole plant physiology was affected in the dgs1-1 mutant as evidenced by tolerance to imposed drought stress and altered transcriptional responses of markers of mitochondrial retrograde signaling. Putative orthologs of Arabidopsis DGS1 are conserved in eukaryotes, including the Nuclear Control of ATP Synthase2 (NCA2) protein in yeast (Saccharomyces cerevisiae), but lost in Metazoa. The genes encoding DGS1 and NCA2 are part of a similar coexpression network including genes encoding proteins involved in mitochondrial fission, morphology, and lipid homeostasis. Thus, DGS1 links mitochondrial protein and lipid import with cellular lipid homeostasis and whole plant stress responses.

New pelagophytes show a novel mode of algal colony development and reveal a perforated theca that may define the class.

Pelagophytes (Heterokonta) are a morphologically diverse class of marine algae historically united only by DNA sequences. We established clonal cultures of sand-dwelling pelagophytes collected from intertidal pools around Australia. Phylogenetic trees based on nuclear 18S rDNA and plastid rbcL, psaA, psaB, psbA, and psbC sequences revealed two new genera, Gazia and Glomerochrysis, related to Aureoumbra in a distinct lineage within the Sarcinochrysidaceae (Pelagophyceae). The three new species (Gazia saundersii, Gazia australica, and Glomerochrysis psammophila), along with an Australian strain of Aureoumbra geitleri, are characterized by dominant benthic stages that differ significantly from one another, while occasionally producing classic heterokont zoospores. The benthic stage of Ga. saundersii has a novel development not observed in any other colonial alga, consisting of large, spherical colonies (up to 140 µm in diameter) containing c. 2,500 cells that eventually differentiate and segregate into a large number of daughter colonies that are subsequently liberated. Alternatively, colonies may differentiate into a mass of zoospores that escape and settle to develop into new colonies. In Gl. psammophila, cubic packets of cells form large sticky clusters that bind sand together, while Ga. australica and A. geitleri are unicellular species. Using fixation by high-pressure freezing, a distinctive perforated theca was observed by TEM in all genera of this lineage, and we hypothesize this unique covering may be the first morphological feature to characterize most, if not all, pelagophytes. This study substantiates the diverse nature of sand-dwelling pelagophytes as well as their mechanisms for thriving in a dynamic habitat.

The golden paradox - a new heterokont lineage with chloroplasts surrounded by two membranes.

A marine, sand-dwelling, golden-brown alga is described from clonal cultures established from a high intertidal pool in southeastern Australia. This tiny, unicellular species, which we call the "golden paradox" (Chrysoparadoxa australica gen. et sp. nov.), is benthic, surrounded by a multilayered cell wall and attached to the substratum by a complex adhesive plug. Each vegetative cell gives rise to a single, naked zoospore with heterokont flagella that settles and may become briefly amoeboid prior to dividing. Daughter cells are initially amoeboid, then either permanently attach and return to the benthic stage or become motile again prior to final settlement. Two deeply lobed chloroplasts occupy opposite ends of the cell and are surrounded by only two membranes. The outer chloroplast membrane is continuous between the two chloroplasts via the outer membrane of the nuclear envelope. Only two membranes occupy the chloroplast-nucleus interface, the inner membrane of the nuclear envelope and the inner chloroplast membrane. A small pyrenoid is found in each chloroplast and closely abuts the nucleus or protrudes into it. It contains an unusual, membrane-bound inclusion that stains with SYBR green but is unlikely to be a nucleomorph. Phylogenies inferred from a 10-gene concatenated alignment show an early-branching position within the PX clade. The unusual morphological features and phylogenetic position indicate C. australica should be classified as a new class, Chrysoparadoxophyceae. Despite an atypical plastid, exploration of the C. australica transcriptome revealed typical heterokont protein targeting to the plastid.

KNS4/UPEX1: A Type II Arabinogalactan ß-(1,3)-Galactosyltransferase Required for Pollen Exine Development.

Pollen exine is essential for protection from the environment of the male gametes of seed-producing plants, but its assembly and composition remain poorly understood. We previously characterized Arabidopsis (Arabidopsis thaliana) mutants with abnormal pollen exine structure and morphology that we named kaonashi (kns). Here we describe the identification of the causal gene of kns4 that was found to be a member of the CAZy glycosyltransferase 31 gene family, identical to UNEVEN PATTERN OF EXINE1, and the biochemical characterization of the encoded protein. The characteristic exine phenotype in the kns4 mutant is related to an abnormality of the primexine matrix laid on the surface of developing microspores. Using light microscopy with a combination of type II arabinogalactan (AG) antibodies and staining with the arabinogalactan-protein (AGP)-specific ß-Glc Yariv reagent, we show that the levels of AGPs in the kns4 microspore primexine are considerably diminished, and their location differs from that of wild type, as does the distribution of pectin labeling. Furthermore, kns4 mutants exhibit reduced fertility as indicated by shorter fruit lengths and lower seed set compared to the wild type, confirming that KNS4 is critical for pollen viability and development. KNS4 was heterologously expressed in Nicotiana benthamiana, and was shown to possess ß-(1,3)-galactosyltransferase activity responsible for the synthesis of AG glycans that are present on both AGPs and/or the pectic polysaccharide rhamnogalacturonan I. These data demonstrate that defects in AGP/pectic glycans, caused by disruption of KNS4 function, impact pollen development and viability in Arabidopsis.

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.

Reduction in circulating bile acid and restricted diffusion across the intestinal epithelium are associated with a decrease in blood cholesterol in the presence of oat ß-glucan.

Although oat (1,3:1,4)-ß-glucan (BG) has been shown to decrease blood cholesterol in intervention trials, the detailed mechanism is not yet defined, but restricted reabsorption of bile acids (BAs) has been hypothesized. Using pigs as a model for humans we demonstrated that, compared to the control, BG added to the diet for 26 d caused decreases of 24% in blood total BAs (TBAs), 34% in total cholesterol (TC), and 57% in LDL cholesterol (LDL-C) (P < 0.01); decreases of 20% TBA in the midjejunum and terminal ileum (P < 0.01); increases of 80% in cecal total neutral sterols (TNSs) including cholesterol (P < 0.01); a 50% reduction in BA active transport across ex vivo ileum after 40 min (P < 0.001); and 32% decrease in jejunal microvillus heights with apparent increased goblet cell activity. The results suggest that BG not only physically hinders the active reabsorption of BAs and uptake of cholesterol, but also changes the BAs profile with lower circulating levels without excess excretion in the feces, thus resulting in reduced blood TC and LDL-C. Fermentation of sterols reaching the colon enhanced production of therapeutic ursodeoxycholic acid, suppressed toxic lithocholic acid, and decreased the possibility of cholesterol absorption by transforming the latter into coprostanol, a nonabsorbable NS.-Gunness, P., Michiels, J., Vanhaecke, L., De Smet, S., Kravchuk, O., Van de Meene, A., Gidley, M. J. Reduction in circulating bile acid and restricted diffusion across the intestinal epithelium are associated with a decrease in blood cholesterol in the presence of oat ß-glucan.

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.

Plant perception of ß-aminobutyric acid is mediated by an aspartyl-tRNA synthetase.

Specific chemicals can prime the plant immune system for augmented defense. ß-aminobutyric acid (BABA) is a priming agent that provides broad-spectrum disease protection. However, BABA also suppresses plant growth when applied in high doses, which has hampered its application as a crop defense activator. Here we describe a mutant of Arabidopsis thaliana that is impaired in BABA-induced disease immunity (ibi1) but is hypersensitive to BABA-induced growth repression. IBI1 encodes an aspartyl-tRNA synthetase. Enantiomer-specific binding of the R enantiomer of BABA to IBI1 primed the protein for noncanonical defense signaling in the cytoplasm after pathogen attack. This priming was associated with aspartic acid accumulation and tRNA-induced phosphorylation of translation initiation factor eIF2α. However, mutation of eIF2α-phosphorylating GCN2 kinase did not affect BABA-induced immunity but relieved BABA-induced growth repression. Hence, BABA-activated IBI1 controls plant immunity and growth via separate pathways. Our results open new opportunities to separate broad-spectrum disease resistance from the associated costs on plant growth.

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.

Immunolocalization of hordein synthesis and transport in developing barley endosperm.

The spatial accumulation of hordeins in the developing endosperm of barley grains was examined by immunofluorescence microscopy (immunolight microscopy [iLM]) and immunoelectron microscopy (iEM) to establish the timing and subcellular pattern of hordein synthesis and deposition. The pattern seen for hordeins was compared to other abundant grain proteins, such as serpin Z4 and lipid transfer protein 1 (LTP1). Hordein accumulates throughout grain development, from 6 to 37 days post-anthesis (DPA). In contrast, serpin Z4 was present at 6 DPA, but the greatest synthesis and accumulation occurred during the middle of seed development, from 15 to 30 DPA. LTP1 accumulated later in seed development, from 15 to 30 DPA. Hordeins accumulated within the lumen of the endoplasmic reticulum (ER), were exocytosed from the ER membrane, and accumulated in protein bodies, which then fused either with the protein storage vacuoles or with other protein bodies, which also later fused with the protein storage vacuoles. iEM showed hordein, and LTP1 appeared not to traverse the Golgi apparatus (GA). Hordein, LTP1, and serpin Z4 colocalized to the same protein bodies and were co-transported to the protein storage vacuole in the same protein bodies. It is likely that this represents a general transport mechanism common to storage proteins in developing grains.

Physiochemical changes, metabolite discrepancies of brown seaweed-derived sulphated polysaccharides in the upper gastrointestinal tract and their effects on bioactive expression.

Brown seaweed-derived polysaccharides, notably fucoidan and laminarin, are known for their extensive array of bioactivities and physicochemical properties. However, the effects of upper digestive tract modification on the bioactive performance of fucoidan and laminarin fractions (FLFs) sourced from Australian native species are largely unknown. Here, the digestibility and bioaccessibility of FLFs were evaluated by tracking the dynamic changes in reducing sugar content (CR), profiling the free monosaccharide composition using LC-MS, and comparing high-performance gel permeation chromatography profile variation via LC-SEC-RI. The effects of digestive progression on bioactive performance were assessed by comparing the antioxidant and antidiabetic potential of FLFs and FLF digesta. We observed that molecular weight (Mw) decreased during gastric digestion indicating that FLF aggregates were disrupted in the stomach. During intestinal digestion, Mw gradually decreased and CR increased indicating cleavage of glycosidic bonds releasing free sugars. Although the antioxidant and antidiabetic capacities were not eliminated by the digestion progression, the bioactive performance of FLFs under a digestive environment was reduced contrasting with the same concentration level of the undigested FLFs. These data provide comprehensive information on the digestibility and bioaccessibility of FLFs, and shed light on the effects of digestive progression on bioactive expression.

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.

A candidate transporter allowing symbiotic dinoflagellates to feed their coral hosts.

The symbiotic partnership between corals and dinoflagellate algae is crucial to coral reefs. Corals provide their algal symbionts with shelter, carbon dioxide and nitrogen. In exchange, the symbiotic algae supply their animal hosts with fixed carbon in the form of glucose. But how glucose is transferred from the algal symbiont to the animal host is unknown. We reasoned that a transporter resident in the dinoflagellate cell membrane would facilitate outward transfer of glucose to the surrounding host animal tissue. We identified a candidate transporter in the cnidarian symbiont dinoflagellate Breviolum minutum that belongs to the ubiquitous family of facilitative sugar uniporters known as SWEETs (sugars will eventually be exported transporters). Previous gene expression analyses had shown that BmSWEET1 is upregulated when the algae are living symbiotically in a cnidarian host by comparison to the free-living state [1, 2]. We used immunofluorescence microscopy to localise BmSWEET1 in the dinoflagellate cell membrane. Substrate preference assays in a yeast surrogate transport system showed that BmSWEET1 transports glucose. Quantitative microscopy showed that symbiotic B. minutum cells have significantly more BmSWEET1 protein than free-living cells of the same strain, consistent with export during symbiosis but not during the free-living, planktonic phase. Thus, BmSWEET1 is in the right place, at the right time, and has the right substrate to be the transporter with which symbiotic dinoflagellate algae feed their animal hosts to power coral reefs.

Colocalization and potential interactions of Endozoicomonas and chlamydiae in microbial aggregates of the coral Pocillopora acuta.

Corals are associated with a variety of bacteria, which occur in the surface mucus layer, gastrovascular cavity, skeleton, and tissues. Some tissue-associated bacteria form clusters, termed cell-associated microbial aggregates (CAMAs), which are poorly studied. Here, we provide a comprehensive characterization of CAMAs in the coral Pocillopora acuta. Combining imaging techniques, laser capture microdissection, and amplicon and metagenome sequencing, we show that (i) CAMAs are located in the tentacle tips and may be intracellular; (ii) CAMAs contain Endozoicomonas (Gammaproteobacteria) and Simkania (Chlamydiota) bacteria; (iii) Endozoicomonas may provide vitamins to its host and use secretion systems and/or pili for colonization and aggregation; (iv) Endozoicomonas and Simkania occur in distinct, but adjacent, CAMAs; and (v) Simkania may receive acetate and heme from neighboring Endozoicomonas. Our study provides detailed insight into coral endosymbionts, thereby improving our understanding of coral physiology and health and providing important knowledge for coral reef conservation in the climate change era.