SummaryGlucan, water dikinase (GWD) is a key enzyme of starch metabolism but the physico-chemical properties of starches isolated from GWD-deficient plants and their implications for starch metabolism have so far not been described.Transgenic Arabidopsis thaliana plants with reduced or no GWD activity were used to investigate the properties of starch granules. In addition, using various in vitro assays, the action of recombinant GWD, b-amylase, isoamylase and starch synthase 1 on the surface of native starch granules was analysed.The internal structure of granules isolated from GWD mutant plants is unaffected, as thermal stability, allomorph, chain length distribution and density of starch granules were similar to wild-type. However, short glucan chain residues located at the granule surface dominate in starches of transgenic plants and impede GWD activity. A similarly reduced rate of phosphorylation by GWD was also observed in potato tuber starch fractions that differ in the proportion of accessible glucan chain residues at the granule surface.A model is proposed to explain the characteristic morphology of starch granules observed in GWD transgenic plants. The model postulates that the occupancy rate of single glucan chains at the granule surface limits accessibility to starch-related enzymes.

) is a positive regulator of light-dependent chlorophyll biosynthesis. GUN4 activates Mg chelatase (MgCh) that catalyzes the insertion of an Mg 2+ ion into protoporphyrin IX. We show that Arabidopsis (Arabidopsis thaliana) GUN4 is phosphorylated at Ser 264 (S264), the penultimate amino acid residue at the C terminus. While GUN4 is preferentially phosphorylated in darkness, phosphorylation is reduced upon accumulation of Mg porphyrins. Expression of a phosphomimicking GUN4(S264D) results in an incomplete complementation of the white gun4-2 null mutant and a chlorotic phenotype comparable to gun4 knockdown mutants. Phosphorylated GUN4 has a reduced stimulatory effect on MgCh in vitro and in vivo but retains its protein stability and tetrapyrrole binding capacity. Analysis of GUN4 found in oxygenic photosynthetic organisms reveals the evolution of a C-terminal extension, which harbors the phosphorylation site of GUN4 expressed in angiosperms. Homologs of GUN4 from Synechocystis and Chlamydomonas lack the conserved phosphorylation site found in a C-terminal extension of angiosperm GUN4. Biochemical studies proved the importance of the C-terminal extension for MgCh stimulation and inactivation of GUN4 by phosphorylation in angiosperms. An additional mechanism regulating MgCh activity is proposed. In conjunction with the dark repression of 5-aminolevulinic acid synthesis, GUN4 phosphorylation minimizes the flow of intermediates into the Mg branch of the tetrapyrrole metabolic pathway for chlorophyll biosynthesis.

Photosynthesis in plant cells would not be possible without the supportive role of mitochondria. However, isolating mitochondria from plant cells for physiological and biochemical analyses is a lengthy and tedious process. Established isolation protocols require multiple centrifugation steps and substantial amounts of starting material. To overcome these limitations, we tagged mitochondria in Arabidopsis (Arabidopsis thaliana) with a triple hemagglutinin tag for rapid purification via a single affinity-purification step. This protocol yields a substantial quantity of highly pure mitochondria from 1 g of Arabidopsis seedlings. The purified mitochondria were suitable for enzyme activity analyses and yielded sufficient amounts of proteins for deep proteomic profiling. We applied this method for the proteomic analysis of the Arabidopsis bou-2 mutant deficient in the mitochondrial Glu transporter À BOUT DE SOUFFLE (BOU) and identified 27 differentially expressed mitochondrial proteins compared with tagged Col-0 controls. Our work sets the stage for the development of advanced mitochondria isolation protocols for distinct cell types.