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The role of proteomics in progressing insights into plant secondary metabolism.

Martínez-Esteso MJ, Martínez-Márquez A, Sellés-Marchart S, Morante-Carriel JA, Bru-Martínez R - Front Plant Sci (2015)

Bottom Line: Here we focus on the particular contribution that proteomic technologies have made in progressing knowledge and characterising plant secondary metabolism (SM) pathways since early expectations were created 15 years ago.We analyzed how three major issues in the proteomic analysis of plant SM have been implemented in various research studies.We also examine to what extent the most-advanced technologies have been incorporated into proteomic research in plant SM and highlight some cutting edge techniques that would strongly benefit the progress made in this field.

View Article: PubMed Central - PubMed

Affiliation: Plant Proteomics and Functional Genomics Group, Department of Agrochemistry and Biochemistry, Multidisciplinary Institute for Environmental Studies "Ramon Margalef", University of Alicante , Alicante, Spain.

ABSTRACT
The development of omics has enabled the genome-wide exploration of all kinds of biological processes at the molecular level. Almost every field of plant biology has been analyzed at the genomic, transcriptomic and proteomic level. Here we focus on the particular contribution that proteomic technologies have made in progressing knowledge and characterising plant secondary metabolism (SM) pathways since early expectations were created 15 years ago. We analyzed how three major issues in the proteomic analysis of plant SM have been implemented in various research studies. These issues are: (i) the selection of a suitable plant material rich in secondary metabolites of interest, such as specialized tissues and organs, and in vitro cell cultures; (ii) the proteomic strategy to access target proteins, either a comprehensive or a differential analysis; (iii) the proteomic approach, represented by the hypothesis-free discovery proteomics and the hypothesis-driven targeted proteomics. We also examine to what extent the most-advanced technologies have been incorporated into proteomic research in plant SM and highlight some cutting edge techniques that would strongly benefit the progress made in this field.

No MeSH data available.


Related in: MedlinePlus

Major issues in the proteomic analysis of plant secondary metabolism. Three major issues have been considered in the proteomic analysis of plant secondary metabolism. Two are common to any type of proteomic analysis, i.e., the strategy to find proteins of interest, and the technical approach to achieve it. In the scheme above, we have included the ways in which such issues have been resolved to date and the corresponding number of representative studies (figure in brackets). So the first and specific issue for accessing the plant secondary metabolism proteome is the selection of suitable plant material, which is rich in secondary metabolites of interest. If using whole plants as a source, collection of specialised tissues-roots, fruit exocarp and mesocarp-, organs –trichomes-, fluids –milky sap- or preparation of organelles –chromoplasts- before starting protein extraction has been a successful strategy to access the target proteome. Alternatively, in vitro cell culture has been a smart option to easily generate an abundant population of homogeneous cells that produce SM whenever they were stimulated through different treatments, such as elicitation, precursor feeding or physical stress. A second issue is the proteomic strategy to find target proteins; i.e., enzymes and transporters specifically involved in the metabolic pathway of interest. One is a comprehensive analysis in which the identification of the largest possible number of proteins is intended. The other typical strategy is differential proteomics. In this case, the proteome complements obtained from two experimental groups or more, which differ in secondary metabolite content, are compared. Proteins with differential abundance are selected. In both cases, a bioinformatics-based analysis of the protein lists follows to classify proteins according to their molecular and (potential) biological function, and to select the candidate proteins involved in SM for further functional characterization using biochemical and genetic tools. Eventually, a third major issue is the proteomic approach. As the initial goal is to find the new enzymes and transporters involved in secondary metabolite synthesis and biology, a hypothesis-free type discovery proteomics approach, either top-down or bottom-up, is usually undertaken. A number of applications of classical and advanced gel-based and gel-free proteomic techniques to investigate plant SM pathways have been reported. Having identified the proteins of interest, a hypothesis-driven targeted proteomics approach is the next step to profoundly characterize the pathway under different experimental conditions. For this purpose, proteomic workflows have utilized MRM. Indeed a number of technological developments of immediate applicability that are currently used in proteomics from which SM proteomic research would very much benefit are suggested. These may introduce advantages in handling plant material to obtain cleaner and higher yield protein or peptide samples, and to provide improvements in analytical times, protein identification rates, and quantification of protein changes at either the whole or targeted proteome level.
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Figure 1: Major issues in the proteomic analysis of plant secondary metabolism. Three major issues have been considered in the proteomic analysis of plant secondary metabolism. Two are common to any type of proteomic analysis, i.e., the strategy to find proteins of interest, and the technical approach to achieve it. In the scheme above, we have included the ways in which such issues have been resolved to date and the corresponding number of representative studies (figure in brackets). So the first and specific issue for accessing the plant secondary metabolism proteome is the selection of suitable plant material, which is rich in secondary metabolites of interest. If using whole plants as a source, collection of specialised tissues-roots, fruit exocarp and mesocarp-, organs –trichomes-, fluids –milky sap- or preparation of organelles –chromoplasts- before starting protein extraction has been a successful strategy to access the target proteome. Alternatively, in vitro cell culture has been a smart option to easily generate an abundant population of homogeneous cells that produce SM whenever they were stimulated through different treatments, such as elicitation, precursor feeding or physical stress. A second issue is the proteomic strategy to find target proteins; i.e., enzymes and transporters specifically involved in the metabolic pathway of interest. One is a comprehensive analysis in which the identification of the largest possible number of proteins is intended. The other typical strategy is differential proteomics. In this case, the proteome complements obtained from two experimental groups or more, which differ in secondary metabolite content, are compared. Proteins with differential abundance are selected. In both cases, a bioinformatics-based analysis of the protein lists follows to classify proteins according to their molecular and (potential) biological function, and to select the candidate proteins involved in SM for further functional characterization using biochemical and genetic tools. Eventually, a third major issue is the proteomic approach. As the initial goal is to find the new enzymes and transporters involved in secondary metabolite synthesis and biology, a hypothesis-free type discovery proteomics approach, either top-down or bottom-up, is usually undertaken. A number of applications of classical and advanced gel-based and gel-free proteomic techniques to investigate plant SM pathways have been reported. Having identified the proteins of interest, a hypothesis-driven targeted proteomics approach is the next step to profoundly characterize the pathway under different experimental conditions. For this purpose, proteomic workflows have utilized MRM. Indeed a number of technological developments of immediate applicability that are currently used in proteomics from which SM proteomic research would very much benefit are suggested. These may introduce advantages in handling plant material to obtain cleaner and higher yield protein or peptide samples, and to provide improvements in analytical times, protein identification rates, and quantification of protein changes at either the whole or targeted proteome level.

Mentions: One first issue is to select suitable plant material that is rich in secondary metabolites of interest (Figure 1). In a literature survey about proteomics-based research into plant SM, the first impression is that major families, i.e., phenolics, alkaloids, and terpenes, have been investigated to some extent. However, the high diversity of the metabolic pathways within them is quite under-represented, thus it is necessary to broaden the proteomic research scope in this field. The success of a proteomic approach in finding and discovering new potential pathway enzymes very much relies on the choice of a suitable plant part where the target pathway is over-represented. In grape berry skin, a flesh proteomic analysis has provided extensive coverage of not only shikimate, phenylpropanoid and flavonoid pathway enzymes, but also of their relative abundance profiles during berry development (Martínez-Esteso et al., 2011a,b, 2013). Isolation and comprehensive analyses of trichomes in tobacco (Van Cutsem et al., 2011) and tomato (Schilmiller et al., 2010) have led to the identification of the enzymes involved in the methylerythritol phosphate (MEP) synthesis pathway of terpenoid precursors, terpenoid synthesis and modification, and also potential transporters. The study of tomato, which was combined with a transcriptomic analysis, has also revealed pathways of flavonoid and volatile aldehydes synthesis which occur in trichomes; most interestingly, morphologically identical trichomes from different plant parts appeared to be specialized in the terpenoid metabolite type produced by a sesquiterpene synthase, only found at the protein level in leaf, but not stem, trichomes (Schilmiller et al., 2010). Parts of complex biosynthetic pathways, such as that for alkaloids, may occur in conducting fluids (e.g., phloem and latex). An analysis of latex has revealed the presence of enzymes of SM. Yet a major factor for their successful identification is the availability of the extensive structural annotation of sequences in the databases of the target species. For Euphorbia kansui (Zhao et al., 2014), only four of the 19 identified proteins had a functional description, while several hundreds of proteins, including various morphine synthesis steps, have been identified and described for opium poppy (Onoyovwe et al., 2013). The preparation of subcellular fractions specialized in secondary metabolite synthesis, such as chromoplasts from orange fruit pulp (Zeng et al., 2011), has allowed the identification of most of the enzymes of the MEP pathway and lycopene synthesis, and also one enzyme involved in vitamin E. However, it has been noted that it was not possible to identify the enzymes that catalyze the regulated steps in each pathway.


The role of proteomics in progressing insights into plant secondary metabolism.

Martínez-Esteso MJ, Martínez-Márquez A, Sellés-Marchart S, Morante-Carriel JA, Bru-Martínez R - Front Plant Sci (2015)

Major issues in the proteomic analysis of plant secondary metabolism. Three major issues have been considered in the proteomic analysis of plant secondary metabolism. Two are common to any type of proteomic analysis, i.e., the strategy to find proteins of interest, and the technical approach to achieve it. In the scheme above, we have included the ways in which such issues have been resolved to date and the corresponding number of representative studies (figure in brackets). So the first and specific issue for accessing the plant secondary metabolism proteome is the selection of suitable plant material, which is rich in secondary metabolites of interest. If using whole plants as a source, collection of specialised tissues-roots, fruit exocarp and mesocarp-, organs –trichomes-, fluids –milky sap- or preparation of organelles –chromoplasts- before starting protein extraction has been a successful strategy to access the target proteome. Alternatively, in vitro cell culture has been a smart option to easily generate an abundant population of homogeneous cells that produce SM whenever they were stimulated through different treatments, such as elicitation, precursor feeding or physical stress. A second issue is the proteomic strategy to find target proteins; i.e., enzymes and transporters specifically involved in the metabolic pathway of interest. One is a comprehensive analysis in which the identification of the largest possible number of proteins is intended. The other typical strategy is differential proteomics. In this case, the proteome complements obtained from two experimental groups or more, which differ in secondary metabolite content, are compared. Proteins with differential abundance are selected. In both cases, a bioinformatics-based analysis of the protein lists follows to classify proteins according to their molecular and (potential) biological function, and to select the candidate proteins involved in SM for further functional characterization using biochemical and genetic tools. Eventually, a third major issue is the proteomic approach. As the initial goal is to find the new enzymes and transporters involved in secondary metabolite synthesis and biology, a hypothesis-free type discovery proteomics approach, either top-down or bottom-up, is usually undertaken. A number of applications of classical and advanced gel-based and gel-free proteomic techniques to investigate plant SM pathways have been reported. Having identified the proteins of interest, a hypothesis-driven targeted proteomics approach is the next step to profoundly characterize the pathway under different experimental conditions. For this purpose, proteomic workflows have utilized MRM. Indeed a number of technological developments of immediate applicability that are currently used in proteomics from which SM proteomic research would very much benefit are suggested. These may introduce advantages in handling plant material to obtain cleaner and higher yield protein or peptide samples, and to provide improvements in analytical times, protein identification rates, and quantification of protein changes at either the whole or targeted proteome level.
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Related In: Results  -  Collection

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Figure 1: Major issues in the proteomic analysis of plant secondary metabolism. Three major issues have been considered in the proteomic analysis of plant secondary metabolism. Two are common to any type of proteomic analysis, i.e., the strategy to find proteins of interest, and the technical approach to achieve it. In the scheme above, we have included the ways in which such issues have been resolved to date and the corresponding number of representative studies (figure in brackets). So the first and specific issue for accessing the plant secondary metabolism proteome is the selection of suitable plant material, which is rich in secondary metabolites of interest. If using whole plants as a source, collection of specialised tissues-roots, fruit exocarp and mesocarp-, organs –trichomes-, fluids –milky sap- or preparation of organelles –chromoplasts- before starting protein extraction has been a successful strategy to access the target proteome. Alternatively, in vitro cell culture has been a smart option to easily generate an abundant population of homogeneous cells that produce SM whenever they were stimulated through different treatments, such as elicitation, precursor feeding or physical stress. A second issue is the proteomic strategy to find target proteins; i.e., enzymes and transporters specifically involved in the metabolic pathway of interest. One is a comprehensive analysis in which the identification of the largest possible number of proteins is intended. The other typical strategy is differential proteomics. In this case, the proteome complements obtained from two experimental groups or more, which differ in secondary metabolite content, are compared. Proteins with differential abundance are selected. In both cases, a bioinformatics-based analysis of the protein lists follows to classify proteins according to their molecular and (potential) biological function, and to select the candidate proteins involved in SM for further functional characterization using biochemical and genetic tools. Eventually, a third major issue is the proteomic approach. As the initial goal is to find the new enzymes and transporters involved in secondary metabolite synthesis and biology, a hypothesis-free type discovery proteomics approach, either top-down or bottom-up, is usually undertaken. A number of applications of classical and advanced gel-based and gel-free proteomic techniques to investigate plant SM pathways have been reported. Having identified the proteins of interest, a hypothesis-driven targeted proteomics approach is the next step to profoundly characterize the pathway under different experimental conditions. For this purpose, proteomic workflows have utilized MRM. Indeed a number of technological developments of immediate applicability that are currently used in proteomics from which SM proteomic research would very much benefit are suggested. These may introduce advantages in handling plant material to obtain cleaner and higher yield protein or peptide samples, and to provide improvements in analytical times, protein identification rates, and quantification of protein changes at either the whole or targeted proteome level.
Mentions: One first issue is to select suitable plant material that is rich in secondary metabolites of interest (Figure 1). In a literature survey about proteomics-based research into plant SM, the first impression is that major families, i.e., phenolics, alkaloids, and terpenes, have been investigated to some extent. However, the high diversity of the metabolic pathways within them is quite under-represented, thus it is necessary to broaden the proteomic research scope in this field. The success of a proteomic approach in finding and discovering new potential pathway enzymes very much relies on the choice of a suitable plant part where the target pathway is over-represented. In grape berry skin, a flesh proteomic analysis has provided extensive coverage of not only shikimate, phenylpropanoid and flavonoid pathway enzymes, but also of their relative abundance profiles during berry development (Martínez-Esteso et al., 2011a,b, 2013). Isolation and comprehensive analyses of trichomes in tobacco (Van Cutsem et al., 2011) and tomato (Schilmiller et al., 2010) have led to the identification of the enzymes involved in the methylerythritol phosphate (MEP) synthesis pathway of terpenoid precursors, terpenoid synthesis and modification, and also potential transporters. The study of tomato, which was combined with a transcriptomic analysis, has also revealed pathways of flavonoid and volatile aldehydes synthesis which occur in trichomes; most interestingly, morphologically identical trichomes from different plant parts appeared to be specialized in the terpenoid metabolite type produced by a sesquiterpene synthase, only found at the protein level in leaf, but not stem, trichomes (Schilmiller et al., 2010). Parts of complex biosynthetic pathways, such as that for alkaloids, may occur in conducting fluids (e.g., phloem and latex). An analysis of latex has revealed the presence of enzymes of SM. Yet a major factor for their successful identification is the availability of the extensive structural annotation of sequences in the databases of the target species. For Euphorbia kansui (Zhao et al., 2014), only four of the 19 identified proteins had a functional description, while several hundreds of proteins, including various morphine synthesis steps, have been identified and described for opium poppy (Onoyovwe et al., 2013). The preparation of subcellular fractions specialized in secondary metabolite synthesis, such as chromoplasts from orange fruit pulp (Zeng et al., 2011), has allowed the identification of most of the enzymes of the MEP pathway and lycopene synthesis, and also one enzyme involved in vitamin E. However, it has been noted that it was not possible to identify the enzymes that catalyze the regulated steps in each pathway.

Bottom Line: Here we focus on the particular contribution that proteomic technologies have made in progressing knowledge and characterising plant secondary metabolism (SM) pathways since early expectations were created 15 years ago.We analyzed how three major issues in the proteomic analysis of plant SM have been implemented in various research studies.We also examine to what extent the most-advanced technologies have been incorporated into proteomic research in plant SM and highlight some cutting edge techniques that would strongly benefit the progress made in this field.

View Article: PubMed Central - PubMed

Affiliation: Plant Proteomics and Functional Genomics Group, Department of Agrochemistry and Biochemistry, Multidisciplinary Institute for Environmental Studies "Ramon Margalef", University of Alicante , Alicante, Spain.

ABSTRACT
The development of omics has enabled the genome-wide exploration of all kinds of biological processes at the molecular level. Almost every field of plant biology has been analyzed at the genomic, transcriptomic and proteomic level. Here we focus on the particular contribution that proteomic technologies have made in progressing knowledge and characterising plant secondary metabolism (SM) pathways since early expectations were created 15 years ago. We analyzed how three major issues in the proteomic analysis of plant SM have been implemented in various research studies. These issues are: (i) the selection of a suitable plant material rich in secondary metabolites of interest, such as specialized tissues and organs, and in vitro cell cultures; (ii) the proteomic strategy to access target proteins, either a comprehensive or a differential analysis; (iii) the proteomic approach, represented by the hypothesis-free discovery proteomics and the hypothesis-driven targeted proteomics. We also examine to what extent the most-advanced technologies have been incorporated into proteomic research in plant SM and highlight some cutting edge techniques that would strongly benefit the progress made in this field.

No MeSH data available.


Related in: MedlinePlus