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Plant Physiology 148:1735-1736 (2008) © 2008 American Society of Plant Biologists On the InsideThe Proteome of Purified Peroxisomes Plant peroxisomes are dynamic organelles involved in a wide range of functions. In the seedlings of oil-rich seeds such as Arabidopsis (Arabidopsis thaliana), peroxisomes are mainly involved in the breakdown of fatty acids derived via β-oxidation during germination. In leaves, peroxisomes play another major role in photorespiration. Plant peroxisomes are also involved in the production of signaling compounds and hormones such as jasmonic acid and in the conversion of indole-3-butyric acid into indole-3-acetic acid. Due to the diversity of plant peroxisome functions, the proteome of the plant peroxisome may be quite different from that of its mammalian or fungal counterparts. The main obstacle to studying the proteomics of plant peroxisomes has been the ability to obtain purified organelles from model plants. The purification of peroxisomes from Arabidopsis has proven to be especially difficult due to the low yield of intact organelles and contamination with other cell organelles. To better elucidate the proteome of Arabidopsis peroxisomes, Eubel et al. (pp. 1809–1829) combined conventional centrifugation-based organelle isolation techniques with free-flow electrophoresis. They also used quantitative proteomics to identify proteins enriched in the peroxisome fractions relative to mitochondrial fractions. They provide evidence for the peroxisomal localization of 89 proteins, 36 of which have not previously been identified in other analyses of Arabidopsis peroxisomes. Chimeric GFP constructs of 35 proteins were used to confirm their localization in peroxisomes or to identify endoplasmic reticulum contaminants. A metabolic mapping approach was adopted to gauge the completeness of this peroxisomal proteome. By focusing on the metabolites representing the substrates, reactants, and products of the network, the authors were able to identify points of contact between the different functional categories of proteins, and also to predict the need for specific transporters in the peroxisomal membrane.
A common response of plants to drought stress is the production of osmolytes such as Suc, Pro, and betaine. A consequence of this accumulation is that the molecular density of the already crowded interior of the cell may double or triple. Under these cramped conditions, it is conceivable that proteins could undergo conformational changes. A possible mechanism for coping with these cramped conditions is the expression of stress proteins called dehydrins. The precise function of dehydrins is not established, but they do have a peculiar facet—they lack a fixed three-dimensional structure. It has been hypothesized that the functional structure of the dehydrins may be induced by changes in their hydration status. More specifically, it has been hypothesized that the disordered appearance of the dehydrins may be converted into active, three-dimensional structures during cellular dehydration. To test this hypothesis, Mouillon et al. (pp. 1925–1937) examined whether the addition of macromolecular crowding agents in vitro can force three types of dehydrins from Arabidopsis to fold. Cellular dehydration was mimicked by the application of polyethylene glycol, glycerol, or sugars (Suc and Glc) that plants naturally employ as compatible solutes. Macromolecular crowding was mimicked by the addition of the bulky polysaccharides ficoll and dextran. The results show that the dehydrins are remarkably stable in their disordered state and are only modestly affected by solvent alterations. These observations suggest that the dehydrin sequence is adapted to remain disordered under conditions of severe dehydration. In this respect, the dehydrins are different from other classes of disordered proteins that rely on folding to become functional. The authors propose that the function of the dehydrins likely lies in the interactions of the conserved segments with their specific biological targets.
In many plant species, including Arabidopsis, the triploid primary endosperm nucleus undergoes several rounds of free nuclear division, growing rapidly as a syncytium. In plants that have ephemeral endosperms, such as Arabidopsis, the embryo develops at the expense of the endosperm and absorbs these reserves, storing them in the cotyledons. Seeds that generate large endosperms during the early stages of development produce large embryos at maturity. The early proliferation of the endosperm is therefore associated with the growth of seed and final seed size. Indeed, the alteration of the rate and duration of cell division in the endosperm has been proposed as a strategy for altering seed size. Unfortunately, the small size of Arabidopsis seeds has made high-throughput molecular analysis of the early endosperm in this model species technically difficult. Day et al. (pp. 1964–1984) have bypassed this difficulty by using laser microdissection as a method for obtaining pure samples of syncytial Arabidopsis endosperm 4 d after pollination. Comparisons with publicly available microarray data revealed that 793 putative early seed-specific genes were preferentially expressed in the endosperm. Early seed expression was confirmed for 71 genes using quantitative reverse transcription-PCR with 27 transcription factors being confirmed as early seed specific. Gene ontology analyses revealed a developmental program dominated by the expression of genes associated with cell cycle, DNA processing, chromatin assembly, protein synthesis, cytoskeleton- and microtubule-related processes, and cell/organelle biogenesis and organization. The data generated provide novel insight into early seed development and identify new target genes for further characterization.
Cytokinin transport is not simple. The translocation of cytokinins from roots to shoots via the xylem and their reflux occurring in the phloem support the hypothesis that they serve as long-distance signaling molecules. On the other hand, the distinct sites of expression of genes for cytokinin biosynthesis, degradation, and signaling have led to the hypothesis that cytokinins function locally in a paracrine-like fashion. It is known that cell cultures rapidly take up and inactivate free cytokinin bases by glycosylation and store them in the vacuole, indicating the presence of cellular uptake systems. Recently, low-affinity transporters for cytokinin nucleobases belonging to the PUP (purine permease) family in Arabidopsis have been identified. PUP1 mediates high-affinity transport of adenine and other nucleobases. Cedzich et al. (pp. 1857–1867) have used radiolabeled trans-zeatin to reveal that the uptake of trans-zeatin into Arabidopsis cells in culture cells is multiphasic, indicating the presence of both low- and high-affinity transport systems. A protonophore inhibited cytokinin uptake, consistent with H+-mediated uptake. Other cytokinins such as isopentenyladenine and benzylaminopurine were effective competitors of trans-zeatin uptake. Adenine also competes for zeatin uptake, indicating that the degradation product of cytokinin oxidases is transported by the same systems. These research findings are consistent with the idea that members of the plant-specific PUP family play a role in adenine transport for scavenging extracellular adenine, and may in addition be involved in low-affinity cytokinin uptake.
Two articles in this issue focus respectively on the role of genome duplication (polyploidy) and retrotransposon insertion on the evolution of legumes, particularly soybean (Glycine max). The impact of polyploidy events is poorly understood, as is the fate of most duplicated genes. Recent data indicate, however, that large-scale genetic and epigenetic alterations occur following polyploidy. Some of these genome-altering processes affect duplicated gene copies and may be associated with the acquisition of adaptive traits. The soybean genome has undergone at least two rounds of whole-genome duplication, one estimated to have occurred 10 to 14 million years ago and a second, more ancient event estimated to have occurred 50 to 60 million years ago. Important questions in genome evolution, particularly about the evolution of gene families and genome structure, can be addressed most effectively by the analysis of large contiguous blocks of DNA sequence from multiple species. With the goal of assessing the impact of polyploidy on the evolution of disease resistance genes, which are among the most rapidly evolving and polymorphic genes known in plants, Innes et al. (pp. 1740–1759) compared a 1-Mb region centered on the Rpg1-b disease resistance gene of soybean to homologous regions in three other legume species. These comparisons enabled them to determine how each of the duplicated regions (homoeologues) in soybean has changed following polyploidy. The biggest change was in retroelement content. Despite this accumulation of retroelements, more than 77% of the duplicated low-copy genes have been retained in the same order and appear to be functional—considerably higher than in maize (Zea mays). In contrast to low-copy genes, the disease resistance gene clusters have undergone dramatic duplications and losses. Wawrzynski et al. (pp. 1760–1771) present further analyses of the effects of retrotransposons in altering the soybean genome. Transposable elements are abundant components of plant genomes and have had major impacts on genome structure, not only promoting mutations of genes and affecting gene regulatory sequences but also playing a substantial role in the creation of new genes by "exon-shuffling" and retrotransposition. Recent studies, however, have indicated that the expansion of genomes by retrotransposon activity is counteracted by spontaneous deletions resulting from unequal homologous recombination and illegitimate recombination events. As a result of their analysis of 3.7 Mb of genomic sequence, the authors have uncovered 45 intact long terminal repeat retrotransposons in soybean. Both autonomous and nonautonomous retrotransposons appear to be active and abundant in Glycine and Phaseolus vulgaris. The general conclusion reached by the authors is that the impact of nonautonomous retrotransposon replication on genome size appears to be much greater than previously appreciated. Moreover, the removal of retrotransposons by homologous recombination between long terminal repeats is occurring more slowly in soybean than in previously characterized plant species.
Division of Health Professions and Natural Sciences FOOTNOTES www.plantphysiol.org/cgi/doi/10.1104/pp.104.900278 Related articles in Plant Physiol.:
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