Proteome of amyloplasts isolated from developing wheat endosperm presents evidence of broad metabolic capability*

Abstract

By contrast to chloroplasts, our knowledge of amyloplasts--organelles that synthesize and store starch in heterotrophic plant tissues--is in a formative stage. While our understanding of what is considered their primary function, i.e. the biosynthesis and degradation of starch, has increased dramatically in recent years, relatively little is known about other biochemical processes taking place in these organelles. To help fill this gap, a proteomic analysis of amyloplasts isolated from the starchy endosperm of wheat seeds (10 d post-anthesis) has been conducted. The study has led to the identification of 289 proteins that function in a range of processes, including carbohydrate metabolism, cytoskeleton/plastid division, energetics, nitrogen and sulphur metabolism, nucleic acid-related reactions, synthesis of various building blocks, protein-related reactions, transport, signalling, stress, and a variety of other activities grouped under 'miscellaneous'. The function of 12% of the proteins was unknown. The results highlight the role of the amyloplast as a starch-storing organelle that fulfills a spectrum of biosynthetic needs of the parent tissue. When compared with a recent proteomic analysis of whole endosperm, the current study demonstrates the advantage of using isolated organelles in proteomic studies.

A complete ferredoxin/thioredoxin system regulates fundamental processes in amyloplasts

“…By applying 2D gel electrophoresis and MS analysis, we obtained proteomic evidence for the presence of FNR, ferredoxin, and FTR among the 284 proteins identified in amyloplasts isolated from wheat endosperm (68). As summarized in Table 1, we also confirmed the presence of the two above-mentioned enzymes of the oxidative pentose phosphate pathway that generate the NADPH needed to reduce ferredoxin: glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase (13).…”

“…However, despite these indications, evidence for the presence of a complete Trx system in amyloplasts is lacking. This problem has been addressed in a recent proteomic analysis (68) and in the experiments below.…”

“…Spots were excised by using an automated spot picker (Investigator, Genomics Solution), destained, reduced, alkylated with iodoacetamide, and digested with trypsin by using a DigestPro gel-spot-processing robot (Intavis, Langenfeld, Germany). LC͞tandem MS of tryptic peptides of proteins was carried out by using a QSTAR Pulsar i quadrupole TOF mass spectrometer (Applied Biosystems͞MDS Sciex, Toronto) equipped with a Proxeon Biosystems (Odense, Denmark) nanoelectrospray source to perform ESI-MS of the tryptic peptides as described (68). Matching of tandem MS spectra to known protein or nucleotide sequences was carried out by using a locally installed copy of the spectrum modeler X!TANDEM, an open source program that is a part of the Global Proteome Machine (64,65).…”

“…Version 2004.09.01.2 of X!TANDEM was used, with one missed tryptic cleavage and unanticipated cleavages allowed. Each search was against a f lat file containing amino acid sequences of all plant proteins in HARVEST:WHEAT 1.04 (http:͞͞harvest.ucr.edu), the National Center for Biotechnology Information nonredundant green plant database, National Center for Biotechnology Information T. aestivum UniGene Build 37, and the wEST Database (http:͞͞wheat.pw.usda.gov͞wEST) (68). Further analysis and examination of the data were accomplished by using a locally installed copy of the Global Proteome Machine (www.thegpm.org).…”

Proteomic Analysis of Chloroplast-to-Chromoplast Transition in Tomato Reveals Metabolic Shifts Coupled with Disrupted Thylakoid Biogenesis Machinery and Elevated Energy-Production Components

“…Most of the data available were related to chloroplasts (Armbruster et al, 2011;van Wijk and Baginsky, 2011), although some information was also available for nonphotosynthetic plastid structures such as amyloplasts (Andon et al, 2002;Balmer et al, 2006), etioplasts (von Zychlinski et al, 2005, proplastids (Baginsky et al, 2004), and embryoplasts (Demartini et al, 2011). The present inventory also provides information that complements previous articles published on the chromoplast proteome (bell pepper: Siddique et al, 2006;tomato: Barsan et al, 2010;and sweet orange: Zeng et al, 2011).…”