Report on the Office of Treasurer of State for the year ended June 30, 2016.

Report on the Office of Treasurer of State for the year ended June 30, 2016

Additional file 4: Figure S3. of Eobowenia gen. nov. from the Early Cretaceous of Patagonia: indication for an early divergence of Bowenia?

Isolated cuticles of Ceratozamia mexicana (A, B), and Stangeria eriopus (C, D), Microcycas calocoma (E, F) and Zamia portoricensis (G, H) stained with Auramine O. Scale bar: 100 μm. (PNG 5239 kb

Additional file 2: Figure S1. of Eobowenia gen. nov. from the Early Cretaceous of Patagonia: indication for an early divergence of Bowenia?

Maximum parsimony reconstruction of character evolution on the combined molecular-morphological Bayesian tree for character 50 (A), 53 (B) and 89 (C). (PDF 97 kb

Supplementary_material

Supplementary_materia

PROTEIN TARGETING TO STARCH Is Required for Localising GRANULE-BOUND STARCH SYNTHASE to Starch Granules and for Normal Amylose Synthesis in Arabidopsis

<div><p>The domestication of starch crops underpinned the development of human civilisation, yet we still do not fully understand how plants make starch. Starch is composed of glucose polymers that are branched (amylopectin) or linear (amylose). The amount of amylose strongly influences the physico-chemical behaviour of starchy foods during cooking and of starch mixtures in non-food manufacturing processes. The GRANULE-BOUND STARCH SYNTHASE (GBSS) is the glucosyltransferase specifically responsible for elongating amylose polymers and was the only protein known to be required for its biosynthesis. Here, we demonstrate that PROTEIN TARGETING TO STARCH (PTST) is also specifically required for amylose synthesis in Arabidopsis. PTST is a plastidial protein possessing an N-terminal coiled coil domain and a C-terminal carbohydrate binding module (CBM). We discovered that Arabidopsis <i>ptst</i> mutants synthesise amylose-free starch and are phenotypically similar to mutants lacking GBSS. Analysis of granule-bound proteins showed a dramatic reduction of GBSS protein in <i>ptst</i> mutant starch granules. Pull-down assays with recombinant proteins <i>in vitro</i>, as well as immunoprecipitation assays <i>in planta</i>, revealed that GBSS physically interacts with PTST via a coiled coil. Furthermore, we show that the CBM domain of PTST, which mediates its interaction with starch granules, is also required for correct GBSS localisation. Fluorescently tagged Arabidopsis GBSS, expressed either in tobacco or Arabidopsis leaves, required the presence of Arabidopsis PTST to localise to starch granules. Mutation of the CBM of PTST caused GBSS to remain in the plastid stroma. PTST fulfils a previously unknown function in targeting GBSS to starch. This sheds new light on the importance of targeting biosynthetic enzymes to sub-cellular sites where their action is required. Importantly, PTST represents a promising new gene target for the biotechnological modification of starch composition, as it is exclusively involved in amylose synthesis.</p></div

Localisation of fluorescently tagged PTST and GBSS in tobacco leaves.

<p>(A) PTST-YFP and GBSS-CFP were individually or co-expressed in tobacco epidermal cells and imaged using confocal microscopy. Note that the localisation of GBSS-CFP on starch granules depends on PTST-YFP co-expression. Bar = 10 μm. (B) Same as (A), but additionally using the W217A/W255A non-starch-binding variant of PTST. Bar = 4 μm.</p

Maximum likelihood phylogenetic tree of PTST proteins from the MAFFT alignment.

<p>Angiosperm sequences are shown in red, mosses in blue, while chlorophyte sequences are shown in green. Bootstrap values that are >50 are shown over the branches. The alignment used to generate this tree is available as <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002080#pbio.1002080.s001" target="_blank">S1 Data</a>.</p

The CBM48 domain is required for glucan-binding in PTST.

<p>Binding of GST-PTST recombinant protein to intact wild-type (WT) and waxy (<i>wx</i>) maize starch granules was assessed <i>in vitro</i>. Unbound proteins are in the soluble fraction (S), while bound proteins are in the pellet (P). No protein was detected in the final wash (W_f). Mutating both Trp217 and Trp255 in the CBM48 domain abolished the interaction with starch.</p

The abundance of granule-bound GBSS protein is greatly reduced in <i>ptst</i>.

<p>(A) Silver-stained SDS-PAGE gels of granule-bound proteins extracted from purified starch granules. Lanes were loaded according to equivalent mass of starch (1.3 mg starch). The band corresponding to GBSS is indicated. (B) Immunoblot detection of GBSS in granule-bound protein extracts. Loading was according to equivalent mass of starch (0.7 mg). (C) Same as (B), but more extract was loaded (equivalent to1.3 mg starch) and exposure time was greatly extended. (D) Immunoblot detection of PTST in granule-bound protein extracts. (E) Soluble (S) and insoluble (I) protein fractions of leaves were subject to immunoblot analysis with GBSS and PTST antibodies. Starch-bound proteins are contained in the insoluble fraction.</p

<i>ptst</i> knockout mutants produce amylose-free starch.

<p>(A) Schematic illustration of the exon-intron structure of the <i>PTST</i> gene. Exons are represented by blue boxes. Pale blue boxes represent the 5′ and 3′ UTRs. Translation start (ATG) and stop (TAG) codons are indicated with arrows. Red arrows indicate T-DNA insertion sites. (B) Immunoblot detection of PTST in soluble protein extracts from leaves. The corresponding wild types to <i>ptst-1</i> and <i>ptst-2</i> are Columbia (Col) and Wassilewskija (Ws), respectively. (C) Representative images of 4-week-old rosettes. Plants harvested at the end of the photoperiod were cleared of chlorophyll and iodine-stained to visualise starch. Amylose-free mutants (<i>gbss</i>, <i>ptst-1</i>, and <i>ptst-2</i>) produce a brown staining that is distinct from the wild types.</p