The morphology of starch granules is a major determinant of the functional and nutritional properties of starch and is highly variable among cereal species. Much of this morphological variation stems from differences in the spatial and temporal patterns of starch granule initiation in amyloplasts during grain development. Simple granules are thought to arise from a single initiation per amyloplast (e.g. in Brachypodium distachyon), whereas compound granules develop from multiple initiations per amyloplast (e.g. in rice). We used live-cell imaging to visualise amyloplasts in the developing endosperm of Brachypodium, using transgenic lines expressing a fluorescent amyloplast reporter. We discovered that the simple-type starch granules in Brachypodium can arise from multiple initiations per amyloplast. The amyloplasts showed dynamic changes in their structure and formed two types of stromules: stable stromules that formed a stromal continuum between amyloplasts, and short-lived stromules that were more dynamic. We also observed actin-dependent movement of amyloplasts within endosperm cells, and movement of starch granules within the amyloplasts. Our results suggest complex and pleiomorphic amyloplast organisation and mobility that could influence granule formation. This goes beyond the existing 'one granule, one amyloplast' model for simple-type granules and advances our understanding of both amyloplast biogenesis and granule formation.

Molecular factors that contribute to the diverse spatial and temporal patterns of starch granule initiation between species and organs are poorly understood. Wheat (Triticum sp.) endosperm contains both large A-type granules initiated during early grain development and small B-type granules that initiate about 10 to 15 days later. Here, we identify that the MYOSIN-RESEMBLING CHLOROPLAST PROTEIN (MRC) is required for the correct timing of B-type granule initiation in wheat endosperm during grain development. MRC is expressed in the endosperm exclusively in early grain development, before B-type granule initiation. We isolated three independent TILLING mutants of tetraploid wheat (Triticum turgidum cv. 'Kronos') with premature stop or missense mutations in the A-genome homeolog, which we showed to be the only active homeolog in tetraploid wheat due to a disruption of the B-genome homeolog. The mrc mutants had significantly smaller A-type granules and a higher relative volume of B-type granules in the endosperm than the wild type. Whereas B-type granules initiated 15 to 20 days post-anthesis (dpa) in the wild type, they appeared as early as 10 dpa in the mrc-1 mutant. These results suggest a temporal role for MRC in repressing B-type granule initiation, providing insight into how the distinct biochemical mechanisms that control A- and B-type granule initiation are regulated. This role of MRC in the wheat endosperm is distinct from the previously described role of Arabidopsis (Arabidopsis thaliana) MRC in promoting granule initiation in leaves, providing an example of functional diversification among granule initiation proteins. A starch granule initiation protein in wheat expressed during early endosperm development represses B-type granule initiation.

Growth coordination between cell layers is essential for development of most multicellular organisms. Coordination may be mediated by molecular signaling and/or mechanical connectivity between cells, but how genes modify mechanical interactions between layers is unknown. Here we show that genes driving brassinosteroid synthesis promote growth of internal tissue, at least in part, by reducing mechanical epidermal constraint. We identified a brassinosteroid-deficient dwarf mutant in the aquatic plant Utricularia gibba with twisted internal tissue, likely caused by mechanical constraint from a slow-growing epidermis. We tested this hypothesis by showing that a brassinosteroid mutant in Arabidopsis enhances epidermal crack formation, indicative of increased tissue stress. We propose that by remodeling cell walls, brassinosteroids reduce epidermal constraint, showing how genes can control growth coordination between layers by means of mechanics.

Summary The determination of starch granule morphology in plants is poorly understood. The amyloplasts of wheat endosperm contain large discoid A‐type granules and small spherical B‐type granules. To study the influence of amyloplast structure on these distinct morphological types, we isolated a mutant in durum wheat ( Triticum turgidum ) defective in the plastid division protein PARC6, which had giant plastids in both leaves and endosperm. Endosperm amyloplasts of the mutant contained more A‐ and B‐type granules than those of the wild‐type. The mutant had increased A‐ and B‐type granule size in mature grains, and its A‐type granules had a highly aberrant, lobed surface. This morphological defect was already evident at early stages of grain development and occurred without alterations in polymer structure and composition. Plant growth and grain size, number and starch content were not affected in the mutants despite the large plastid size. Interestingly, mutation of the PARC6 paralog, ARC6, did not increase plastid or starch granule size. We suggest Tt PARC6 can complement disrupted Tt ARC6 function by interacting with PDV2, the outer plastid envelope protein that typically interacts with ARC6 to promote plastid division. We therefore reveal an important role of amyloplast structure in starch granule morphogenesis in wheat.

Recent work has identified several proteins involved in starch granule initiation, the first step of starch synthesis. However, the degree of conservation in the granule initiation process remains poorly understood, especially among grass species differing in patterns of carbohydrate turnover in leaves, and granule morphology in the endosperm. We therefore compared mutant phenotypes of Hordeum vulgare (barley), Triticum turgidum (durum wheat), and Brachypodium distachyon defective in PROTEIN TARGETING TO STARCH 2 (PTST2), a key granule initiation protein. We report striking differences across species and organs. Loss of PTST2 from leaves resulted in fewer, larger starch granules per chloroplast and normal starch content in wheat, fewer granules per chloroplast and lower starch content in barley, and almost complete loss of starch in Brachypodium. The loss of starch in Brachypodium leaves was accompanied by high levels of ADP-glucose and detrimental effects on growth and physiology. Additionally, we found that loss of PTST2 increased granule initiation in Brachypodium amyloplasts, resulting in abnormal compound granule formation throughout the seed. These findings suggest that the importance of PTST2 varies greatly with the genetic and developmental background and inform the extent to which the gene can be targeted to improve starch in crops.

The spatial and temporal patterns by which starch granules initiate vary greatly between species and organs, but molecular factors that contribute to these diverse patterns are poorly understood. We reveal distinct organ-specific roles of the MYOSIN-RESEMBLING CHLOROPLAST PROTEIN (MRC) in regulating granule initiation in the endosperm and leaves of wheat. We isolated three independent TILLING mutants of tetraploid wheat ( Triticum turgidum cv. Kronos) with premature stop or missense mutations in the A-genome homeolog, which we showed to be the only active homeolog in tetraploid wheat due to a disruption of the B-genome homeolog. Wheat endosperm contains both large A-type granules initiated during early grain development, and small B-type granules that initiate about 10 – 15 days later. The mrc mutants had significantly smaller A-type granules and a higher relative volume of B-type granules in the endosperm than the wild type. Whereas B-type granules initiated 15 - 20 days post anthesis (dpa) in the wild-type, they appeared as early as 10 dpa in the mrc-1 mutant, suggesting a role for MRC in suppressing B-type granule initiation during early grain development. By contrast, MRC promotes granule initiation in leaves: mutants carrying premature stop mutations in MRC had fewer granules per chloroplast than the wild type. These contrasting roles of MRC among wheat organs provide new insight into functional diversification of granule initiation proteins, and suggest that they may facilitate the diverse patterns of granule initiation observed across species and organs.

Ammonia-oxidising archaea (AOA) are environmentally important microorganisms involved in the biogeochemical cycling of nitrogen. Routine cultivation of AOA is exclusively performed in liquid cultures and reports on their growth on solid medium are scarce. The ability to grow AOA on solid medium would be beneficial for not only the purification of enrichment cultures but also for developing genetic tools. The aim of this study was to develop a reliable method for growing individual colonies from AOA cultures on solid medium. Three phylogenetically distinct AOA strains were tested: 'Candidatus Nitrosocosmicus franklandus C13', Nitrososphaera viennensis EN76 and 'Candidatus Nitrosotalea sinensis Nd2'. Of the gelling agents tested, agar and Bacto-agar severely inhibited growth of all three strains. In contrast, both 'Ca. N. franklandus C13' and N. viennensis EN76 tolerated Phytagel™ while the acidophilic 'Ca. N. sinensis Nd2' was completely inhibited. Based on these observations, we developed a Liquid-Solid (LS) method that involves immobilising cells in Phytagel™ and overlaying with liquid medium. This approach resulted in the development of visible distinct colonies from 'Ca. N. franklandus C13' and N. viennensis EN76 cultures and lays the groundwork for the genetic manipulation of this group of microorganisms.

The control of starch granule number and morphology in plastids is poorly understood. Here, we demonstrate that At FZL, a protein involved in thylakoid membrane organisation, is required for correct starch granule morphology in Arabidopsis. Leaves of mutants lacking At FZL had the same starch content as wild-type leaves, but their starch granules were smaller and had a distinct, uneven surface morphology. Most chloroplasts in the mutant were larger than those of the wild type. However, the difference in chloroplast size could not explain the difference in granule size and shape in the Atfzl mutants, since other mutants with larger chloroplasts than the wild type ( arc mutants) had granules that were similar in size and shape to wild-type granules. As reported previously, the Atfzl mutant had aberrant thylakoid organisation. We found that this phenomenon was particularly pronounced in regions surrounding starch granules. The location of the thylakoid-bound granule initiation protein MFP1 was unaffected in the Atfzl mutant. We propose that At FZL affects starch granule size and shape by influencing thylakoid organisation at the periphery of starch granules. Our results are consistent with an important role for thylakoid architecture in determining granule morphology.

Medicinal plants are an excellent source of structurally diverse, bio-active compounds with potential in the fight against cancer. One of the most promising is Scutellaria barbata, prescribed traditionally for the treatment of cancers. Scutebarbatine A is the major diterpenoid, produced in specialized large, peltate trichomes on leaves of S. barbata. It induces dose-dependent apoptosis, specifically in cancer cells. The major class of proteins down-regulated are pro-survival proteins, the Inhibitors of Apoptosis (IAPs), and IAP regulating proteins. We propose that scutebarbatine A works by releasing the molecular brakes (the IAPs) on apoptosis in cell death-evading cancer cells. Comparison between the cytotoxicity of methanolic extracts of S. barbata leaves and decoctions (Ban Zhi Lian) prepared traditionally, showed substantially different chemical compositions and differential induction of apoptosis. Analyses suggest polyvalency between the constituents in both extracts, and ways to produce enhanced chemopreventive preparations for the treatment of cancer.

Fungi have evolved an array of spore discharge and dispersal processes. Here, we developed a theoretical model that explains the ejection mechanics of aeciospore liberation in the stem rust pathogen Puccinia graminis . Aeciospores are released from cluster cups formed on its Berberis host, spreading early-season inoculum into neighboring small-grain crops. Our model illustrates that during dew or rainfall, changes in aeciospore turgidity exerts substantial force on neighboring aeciospores in cluster cups whilst gaps between spores become perfused with water. This perfusion coats aeciospores with a lubrication film that facilitates expulsion, with single aeciospores reaching speeds of 0.053 to 0.754 m · s −1 . We also used aeciospore source strength estimates to simulate the aeciospore dispersal gradient and incorporated this into a publicly available web interface. This aids farmers and legislators to assess current local risk of dispersal and facilitates development of sophisticated epidemiological models to potentially curtail stem rust epidemics originating on Berberis .

Starch granule initiation is poorly understood at the molecular level. The glucosyltransferase, STARCH SYNTHASE 4 (SS4), plays a central role in granule initiation in Arabidopsis leaves, but its function in cereal endosperms is unknown. We investigated the role of SS4 in wheat, which has a distinct spatiotemporal pattern of granule initiation during grain development. We generated TILLING mutants in tetraploid wheat (Triticum turgidum) that are defective in both SS4 homoeologs. The morphology of endosperm starch was examined in developing and mature grains. SS4 deficiency led to severe alterations in endosperm starch granule morphology. During early grain development, while the wild-type initiated single 'A-type' granules per amyloplast, most amyloplasts in the mutant formed compound granules due to multiple initiations. This phenotype was similar to mutants deficient in B-GRANULE CONTENT 1 (BGC1). SS4 deficiency also reduced starch content in leaves and pollen grains. We propose that SS4 and BGC1 are required for the proper control of granule initiation during early grain development that leads to a single A-type granule per amyloplast. The absence of either protein results in a variable number of initiations per amyloplast and compound granule formation.

Flower development is an important determinant of grain yield in crops. In wheat (Triticum spp.), natural variation for the size of spikelet and floral organs is particularly evident in Triticum turgidum ssp. polonicum (also termed Triticum polonicum), a tetraploid subspecies of wheat with long glumes, lemmas, and grains. Using map-based cloning, we identified VEGETATIVE TO REPRODUCTIVE TRANSITION 2 (VRT2), which encodes a MADS-box transcription factor belonging to the SHORT VEGETATIVE PHASE family, as the gene underlying the T. polonicum long-glume (P1) locus. The causal P1 mutation is a sequence rearrangement in intron-1 that results in ectopic expression of the T. polonicum VRT-A2 allele. Based on allelic variation studies, we propose that the intron-1 mutation in VRT-A2 is the unique T. polonicum subspecies-defining polymorphism, which was later introduced into hexaploid wheat via natural hybridizations. Near-isogenic lines differing for the P1 locus revealed a gradient effect of P1 across spikelets and within florets. Transgenic lines of hexaploid wheat carrying the T. polonicum VRT-A2 allele show that expression levels of VRT-A2 are highly correlated with spike, glume, grain, and floral organ length. These results highlight how changes in expression profiles, through variation in cis-regulation, can affect agronomic traits in a dosage-dependent manner in polyploid crops.

AbstractFlower development is a major determinant of yield in crops. In wheat, natural variation for the size of spikelet and floral organs is particularly evident inTriticum polonicum, a tetraploid subspecies of wheat with long glumes, lemmas, and grains. Using map-based cloning, we identifiedVRT2, a MADS-box transcription factor belonging to theSVPfamily, as the gene underlying theT. polonicumlong-glume (P1)locus. The causalP1mutation is a sequence re-arrangement in intron-1 that results in both increased and ectopic expression of theT. polonicum VRT-A2allele. Based on allelic variation studies, we propose that the intron-1 mutation inVRT-A2is the uniqueT. polonicumspecies defining polymorphism, which was later introduced into hexaploid wheat via natural hybridizations. Near-isogenic lines differing for theP1locus revealed a gradient effect ofP1across florets. Transgenic lines of hexaploid wheat carrying theT. polonicum VRT-A2allele show that expression levels ofVRT-A2are highly correlated with spike, glume, grain, and floral organ length. These results highlight how changes in expression profiles, through variation incis-regulation, can impact on agronomic traits in a dosage-dependent manner in polyploid crops.One-sentence summaryAn intron-1 rearrangement in the MADS-box transcription factorVRT-A2leads to its misexpression and defines the long-glume phenotype of Polish wheat (T. polonicum).

Inulin nanoparticles (INNPs) are a biocompatible material which has a potential application for enhancing solubility and preventing degradation of compounds. In this work, we demonstrated that INNPs could be synthesized from sucrose using inulosucrase from Lactobacillus reuteri 121. Noticeably, dynamic light scattering (DLS) analysis showed that the derived INNPs exhibited uniformity in size, which was easily controlled by the reaction temperature. The effect of enzyme and sucrose concentration, as well as reaction time, was explored. Moreover, the solubility of INNPs in various organic solvents was also investigated, and we found that the INNPs were freely regenerated in water even though they had precipitated by organic solvents. Essentially, we demonstrated that the derived INNPs could be applied for flavonoid encapsulation. The solubility and stability of quercetin and fisetin in the INNPs complexes was higher than those of free compounds. These results make the INNPs very promising for many applications.

Prymnesium parvum is a toxin-producing haptophyte that causes harmful algal blooms globally, leading to large-scale fish kills that have severe ecological and economic implications. For the model haptophyte, Emiliania huxleyi, it has been shown that large dsDNA viruses play an important role in regulating blooms and therefore biogeochemical cycling, but much less work has been done looking at viruses that infect P. parvum, or the role that these viruses may play in regulating harmful algal blooms. In this study, we report the isolation and characterization of a lytic nucleo-cytoplasmic large DNA virus (NCLDV) collected from the site of a harmful P. parvum bloom. In subsequent experiments, this virus was shown to infect cultures of Prymnesium sp. and showed phylogenetic similarity to the extended Megaviridae family of algal viruses.

Prymnesium parvum is a toxin-producing haptophyte that causes harmful algal blooms globally, leading to large-scale fish kills that have severe ecological and economic implications. For the model haptophyte, Emiliania huxleyi, it has been shown that large dsDNA viruses play an important role in regulating blooms and therefore biogeochemical cycling, but much less work has been done looking at viruses that infect P. parvum, or the role that these viruses may play in regulating harmful algal blooms. In this study, we report the isolation and characterization of a lytic nucleo-cytoplasmic large DNA virus (NCLDV) collected from the site of a harmful P. parvum bloom. In subsequent experiments, this virus was shown to infect cultures of Prymnesium sp. and showed phylogenetic similarity to the extended Megaviridae family of algal viruses.

GlgE is a maltosyltransferase involved in α-glucan biosynthesis in bacteria that has been genetically validated as a target for tuberculosis therapies. Crystals of the Mycobacterium tuberculosis enzyme diffract at low resolution so most structural studies have been with the very similar Streptomyces coelicolor GlgE isoform 1. Although the donor binding site for α-maltose 1-phosphate had been previously structurally defined, the acceptor site had not. Using mutagenesis, kinetics, and protein crystallography of the S. coelicolor enzyme, we have now identified the +1 to +6 subsites of the acceptor/product, which overlap with the known cyclodextrin binding site. The sugar residues in the acceptor subsites +1 to +5 are oriented such that they disfavor the binding of malto-oligosaccharides that bear branches at their 6-positions, consistent with the known acceptor chain specificity of GlgE. A secondary binding site remote from the catalytic center was identified that is distinct from one reported for the M. tuberculosis enzyme. This new site is capable of binding a branched α-glucan and is most likely involved in guiding acceptors toward the donor site because its disruption kinetically compromises the ability of GlgE to extend polymeric substrates. However, disruption of this site, which is conserved in the Streptomyces venezuelae GlgE enzyme, did not affect the growth of S. venezuelae or the structure of the polymeric product. The acceptor subsites +1 to +4 in the S. coelicolor enzyme are well conserved in the M. tuberculosis enzyme so their identification could help inform the design of inhibitors with therapeutic potential.

Actinomycetes, such as mycobacteria and streptomycetes, synthesize α-glucan with α-1,4 linkages and α-1,6 branching to help evade immune responses and to store carbon. α-Glucan is thought to resemble glycogen except for having shorter constituent linear chains. However, the fine structure of α-glucan and how it can be defined by the maltosyl transferase GlgE and branching enzyme GlgB were not known. Using a combination of enzymolysis and mass spectrometry, we compared the properties of α-glucan isolated from actinomycetes with polymer synthesized in vitro by GlgE and GlgB. We now propose the following assembly mechanism. Polymer synthesis starts with GlgE and its donor substrate, α-maltose 1-phosphate, yielding a linear oligomer with a degree of polymerization (∼16) sufficient for GlgB to introduce a branch. Branching involves strictly intrachain transfer to generate a C chain (the only constituent chain to retain its reducing end), which now bears an A chain (a nonreducing end terminal branch that does not itself bear a branch). GlgE preferentially extends A chains allowing GlgB to act iteratively to generate new A chains emanating from B chains (nonterminal branches that themselves bear a branch). Although extension and branching occur primarily with A chains, the other chain types are sometimes extended and branched such that some B chains (and possibly C chains) bear more than one branch. This occurs less frequently in α-glucans than in classical glycogens. The very similar properties of cytosolic and capsular α-glucans from Mycobacterium tuberculosis imply GlgE and GlgB are sufficient to synthesize them both.

The GlgE pathway is thought to be responsible for the conversion of trehalose into a glycogen-like α -glucan polymer in bacteria. Trehalose is first converted to maltose, which is phosphorylated by maltose kinase Pep2 to give α -maltose 1-phosphate. This is the donor substrate of the maltosyl transferase GlgE that is known to extend α -1,4-linked maltooligosaccharides, which are thought to be branched with α -1,6 linkages. The genome of Streptomyces venezuelae contains all the genes coding for the GlgE pathway enzymes but none of those of related pathways, including glgC and glgA of the glycogen pathway. This provides an opportunity to study the GlgE pathway in isolation. The genes of the GlgE pathway were upregulated at the onset of sporulation, consistent with the known timing of α -glucan deposition. A constructed Δ glgE null mutant strain was viable but showed a delayed developmental phenotype when grown on maltose, giving less cell mass and delayed sporulation. Pre-spore cells and spores of the mutant were frequently double the length of those of the wild-type, implying impaired cross-wall formation, and spores showed reduced tolerance to stress. The mutant accumulated α -maltose 1-phosphate and maltose but no α -glucan. Therefore, the GlgE pathway is necessary and sufficient for polymer biosynthesis. Growth of the Δ glgE mutant on galactose and that of a Δ pep2 mutant on maltose were analysed. In both cases, neither accumulation of α -maltose 1-phosphate/ α -glucan nor a developmental delay was observed. Thus, high levels of α -maltose 1-phosphate are responsible for the developmental phenotype of the Δ glgE mutant, rather than the lack of α -glucan.

Herbivore selection of plant hosts and plant responses to insect colonization have been subjects of intense investigations. A growing body of evidence suggests that, for successful colonization to occur, (effector/virulence) proteins in insect saliva must modulate plant defense responses to the benefit of the insect. A range of insect saliva proteins that modulate plant defense responses have been identified, but there is no direct evidence that these proteins are delivered into specific plant tissues and enter plant cells. Aphids and other sap-sucking insects of the order Hemiptera use their specialized mouth parts (stylets) to probe plant mesophyll cells until they reach the phloem cells for long-term feeding. Here, we show, by immunogold-labeling of ultrathin sections of aphid feeding sites, that an immuno-suppressive aphid effector localizes in the cytoplasm of mesophyll cells near aphid stylets but not in cells further away from aphid feeding sites. In contrast, another aphid effector protein localizes in the sheaths composed of gelling saliva that surround the aphid stylets. Thus, insects deliver effectors directly into plant tissue. Moreover, different aphid effectors locate extracellularly in the sheath saliva or are introduced into the cytoplasm of plant cells.