Tackling the plant proteome: practical approaches, hurdles and experimental tools

Rose, Jocelyn K. C.; Bashir, Sajid; Giovannoni, James J.; Jahn, Molly M.; Saravanan, Ramu S.

doi:10.1111/j.1365-313x.2004.02182.x

Cited by 291 publications

(193 citation statements)

References 139 publications

(143 reference statements)

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“…High-resolution 2-DE has recently been shown to be a powerful tool for studying complex patterns of protein expression in various tissues of higher plants (Roberts, 2002;Rose et al, 2004). Thus, to identify possible target proteins of CaPUB1, 2-DE analysis was carried out to compare the protein profiles of the 35STCaPUB1 transgenic and wild-type Arabidopsis plants.…”

Section: Discussionmentioning

confidence: 99%

Heterologous Expression and Molecular and Cellular Characterization of CaPUB1 Encoding a Hot Pepper U-Box E3 Ubiquitin Ligase Homolog

et al. 2006

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The U-box motif is a conserved domain found in the diverse isoforms of E3 ubiquitin ligase in eukaryotes. From water-stressed hot pepper (Capsicum annuum L. cv Pukang) plants, we isolated C. annuum putative U-box protein 1 (CaPUB1), which encodes a protein containing a single U-box motif in its N-terminal region. In vitro ubiquitination and site-directed mutagenesis assays revealed that CaPUB1 possessed E3 ubiquitin ligase activity and that the U-box motif was indeed essential for its enzyme activity. RNA gel-blot analysis showed that CaPUB1 mRNA was induced rapidly by a broad spectrum of abiotic stresses, including drought, high salinity, cold temperature, and mechanical wounding, but not in response to ethylene, abscisic acid, or a bacterial pathogen, suggesting its role in the early events in the abiotic-related defense response. Because transgenic work was extremely difficult in hot pepper, in this study we overexpressed CaPUB1 in Arabidopsis (Arabidopsis thaliana) to provide cellular information on the function of this gene in the development and plant responses to abiotic stresses. Transgenic Arabidopsis plants that constitutively expressed the CaPUB1 gene under the control of the cauliflower mosaic virus 35S promoter had markedly longer hypocotyls and roots and grew more rapidly than the wild type, leading to an early bolting phenotype. Microscopic analysis showed that 35STCaPUB1 roots had increased numbers of small-sized cells, resulting in disordered, highly populated cell layers in the cortex, endodermis, and stele. In addition, CaPUB1-overexpressing plants displayed increased sensitivity to water stress and mild salinity. These results indicate that CaPUB1 is functional in Arabidopsis cells, thereby effectively altering cell and tissue growth and also the response to abiotic stresses. Comparative proteomic analysis showed that the level of RPN6 protein, a non-ATPase subunit of the 26S proteasome complex, was significantly reduced in 35STCaPUB1 seedlings as compared to the wild type. Pull-down and ubiquitination assays demonstrated that RPN6 interacted physically with CaPUB1 and was ubiquitinated in a CaPUB1-dependent manner in vitro. Although the physiological function of CaPUB1 is not yet clear, there are several possibilities for its involvement in a subset of physiological responses to counteract dehydration and high-salinity stresses in transgenic Arabidopsis seedlings.

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Section: Discussionmentioning

confidence: 99%

Heterologous Expression and Molecular and Cellular Characterization of CaPUB1 Encoding a Hot Pepper U-Box E3 Ubiquitin Ligase Homolog

et al. 2006

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“…Plant proteins are notoriously difficult to extract (Rose et al, 2004). This is mainly due to the low protein content of plant tissue and the low solubility of plant proteins.…”

Section: Application Of Cpll To Arabidopsis Leaf Extracts Following Tmentioning

confidence: 99%

Looking Deep Inside: Detection of Low-Abundance Proteins in Leaf Extracts of Arabidopsis and Phloem Exudates of Pumpkin

et al. 2012

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The field of proteomics suffers from the immense complexity of even small proteomes and the enormous dynamic range of protein concentrations within a given sample. Most protein samples contain a few major proteins, which hamper in-depth proteomic analysis. In the human field, combinatorial hexapeptide ligand libraries (CPLL; such as ProteoMiner) have been used for reduction of the dynamic range of protein concentrations; however, this technique is not established in plant research. In this work, we present the application of CPLL to Arabidopsis (Arabidopsis thaliana) leaf proteins. One-and two-dimensional gel electrophoresis showed a decrease in high-abundance proteins and an enrichment of less abundant proteins in CPLL-treated samples. After optimization of the CPLL protocol, mass spectrometric analyses of leaf extracts led to the identification of 1,192 proteins in control samples and an additional 512 proteins after the application of CPLL. Upon leaf infection with virulent Pseudomonas syringae DC3000, CPLL beads were also used for investigating the bacterial infectome. In total, 312 bacterial proteins could be identified in infected Arabidopsis leaves. Furthermore, phloem exudates of pumpkin (Cucurbita maxima) were analyzed. CPLL prefractionation caused depletion of the major phloem proteins 1 and 2 and improved phloem proteomics, because 67 of 320 identified proteins were detectable only after CPLL treatment. In sum, our results demonstrate that CPLL beads are a time-and cost-effective tool for reducing major proteins, which often interfere with downstream analyses. The concomitant enrichment of less abundant proteins may facilitate a deeper insight into the plant proteome.

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“…This is at least partly for technical reasons. While custom-made or commercial arrays are available for transcript profiling and widely used techniques like gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry are available for metabolite profiling, it is still a technical challenge to obtain quantitative information about large numbers of proteins (Rose et al, 2004;Baerenfaller et al, 2008) or enzyme activities (Mitchell-Olds and Pedersen, 1998;Prioul et al, 1999;Thevenot et al, 2005;Cross et al, 2006). While there have been many studies of the developmental changes of small sets of enzymes in tomato fruits Yelle et al, 1988), medium-size surveys are limited to a study of 13 enzymes during fruit development in S. lycopersicum 'MicroTom' (Obiadalla-Ali et al, 2004).…”

mentioning

confidence: 99%

Enzyme Activity Profiles during Fruit Development in Tomato Cultivars andSolanum pennellii

Steinhauser

Koehl

et al. 2010

Plant Physiology

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Enzymes interact to generate metabolic networks. The activities of more than 22 enzymes from central metabolism were profiled during the development of fruit of the modern tomato cultivar Solanum lycopersicum 'M82' and its wild relative Solanum pennellii (LA0716). In S. pennellii, the mature fruit remains green and contains lower sugar and higher organic acid levels. These genotypes are the parents of a widely used near introgression line population. Enzymes were also profiled in a second cultivar, S. lycopersicum 'Moneymaker', for which data sets for the developmental changes of metabolites and transcripts are available. Whereas most enzyme activities declined during fruit development in the modern S. lycopersicum cultivars, they remained high or even increased in S. pennellii, especially enzymes required for organic acid synthesis. The enzyme profiles were sufficiently characteristic to allow stages of development and cultivars and the wild species to be distinguished by principal component analysis and clustering. Many enzymes showed coordinated changes during fruit development of a given genotype. Comparison of the correlation matrices revealed a large overlap between the two modern cultivars and considerable overlap with S. pennellii, indicating that despite the very different development responses, some basic modules are retained. Comparison of enzyme activity, metabolite profiles, and transcript profiles in S. lycopersicum 'Moneymaker' revealed remarkably little connectivity between the developmental changes of transcripts and enzymes and even less between enzymes and metabolites. We discuss the concept that the metabolite profile is an emergent property that is generated by complex network interactions.

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Tackling the plant proteome: practical approaches, hurdles and experimental tools

Cited by 291 publications

References 139 publications

Heterologous Expression and Molecular and Cellular Characterization of CaPUB1 Encoding a Hot Pepper U-Box E3 Ubiquitin Ligase Homolog

Heterologous Expression and Molecular and Cellular Characterization of CaPUB1 Encoding a Hot Pepper U-Box E3 Ubiquitin Ligase Homolog

Looking Deep Inside: Detection of Low-Abundance Proteins in Leaf Extracts of Arabidopsis and Phloem Exudates of Pumpkin

Enzyme Activity Profiles during Fruit Development in Tomato Cultivars andSolanum pennellii

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