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Proteasome targeting of proteins in Arabidopsis leaf mesophyll, epidermal and vascular tissues.

Svozil J, Gruissem W, Baerenfaller K - Front Plant Sci (2015)

Bottom Line: SylA treatment of leaves resulted in the accumulation of 225 proteins and identification of 519 ubiquitylated proteins.Epidermis enzymes of the TCA cycle and cell wall biosynthesis specifically accumulated after proteasome inhibition, and in the vascular tissue several enzymes involved in glucosinolate biosynthesis were found to be ubiquitylated.Our results demonstrate that protein level changes and UPS protein targets are characteristic of the individual leaf tissues and that the proteasome is relevant for tissue-specific functions.

View Article: PubMed Central - PubMed

Affiliation: Plant Biotechnology, Department of Biology, Swiss Federal Institute of Technology Zurich Zurich, Switzerland.

ABSTRACT
Protein and transcript levels are partly decoupled as a function of translation efficiency and protein degradation. Selective protein degradation via the Ubiquitin-26S proteasome system (UPS) ensures protein homeostasis and facilitates adjustment of protein abundance during changing environmental conditions. Since individual leaf tissues have specialized functions, their protein composition is different and hence also protein level regulation is expected to differ. To understand UPS function in a tissue-specific context we developed a method termed Meselect to effectively and rapidly separate Arabidopsis thaliana leaf epidermal, vascular and mesophyll tissues. Epidermal and vascular tissue cells are separated mechanically, while mesophyll cells are obtained after rapid protoplasting. The high yield of proteins was sufficient for tissue-specific proteome analyses after inhibition of the proteasome with the specific inhibitor Syringolin A (SylA) and affinity enrichment of ubiquitylated proteins. SylA treatment of leaves resulted in the accumulation of 225 proteins and identification of 519 ubiquitylated proteins. Proteins that were exclusively identified in the three different tissue types are consistent with specific cellular functions. Mesophyll cell proteins were enriched for plastid membrane translocation complexes as targets of the UPS. Epidermis enzymes of the TCA cycle and cell wall biosynthesis specifically accumulated after proteasome inhibition, and in the vascular tissue several enzymes involved in glucosinolate biosynthesis were found to be ubiquitylated. Our results demonstrate that protein level changes and UPS protein targets are characteristic of the individual leaf tissues and that the proteasome is relevant for tissue-specific functions.

No MeSH data available.


Schematic representation of the experimental workflow.
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Figure 2: Schematic representation of the experimental workflow.

Mentions: We adopted the workflow in Figure 2 to investigate tissue-specific differences in UPS targeting. For this we extracted total proteins from the three tissues of mock-treated leaves or leaves that were treated with Syringolin A (SylA), which specifically and effectively inhibits the UPS (Groll et al., 2008; Svozil et al., 2014). Together, we identified a total of 1799 distinct proteins and 1114 proteins that were identified with at least 5 spectra in at least one tissue (Supplemental Table 1). Of these proteins only those were considered to change in abundance if one of the following criteria was met: (i) the fold-change between SylA and mock-treated samples was >1.5 with a p < 0.05, (ii) the fold-change was large enough to be considered an outlier according to boxplot statistics, or (iii) the protein was identified in at least two of three biological replicates in one condition but not another condition (Table 1, Supplemental Table 2). Additionally, for tissue specificity we required that the protein was not identified in the other tissues (Table 1, Supplemental Table 3). In total, 225 distinct proteins were found that had increased and 30 that had decreased in the different leaf tissues after inhibition of the proteasome. As discussed previously, reduced protein levels could result from reduced transcription, translation or stabilization of the proteins after SylA treatment, or from proteasome-independent protein degradation (Svozil et al., 2014). In contrast, the proteins that increased after inhibition of the proteasome are likely targets of the UPS or participate in pathways that respond to SylA treatment.


Proteasome targeting of proteins in Arabidopsis leaf mesophyll, epidermal and vascular tissues.

Svozil J, Gruissem W, Baerenfaller K - Front Plant Sci (2015)

Schematic representation of the experimental workflow.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4446536&req=5

Figure 2: Schematic representation of the experimental workflow.
Mentions: We adopted the workflow in Figure 2 to investigate tissue-specific differences in UPS targeting. For this we extracted total proteins from the three tissues of mock-treated leaves or leaves that were treated with Syringolin A (SylA), which specifically and effectively inhibits the UPS (Groll et al., 2008; Svozil et al., 2014). Together, we identified a total of 1799 distinct proteins and 1114 proteins that were identified with at least 5 spectra in at least one tissue (Supplemental Table 1). Of these proteins only those were considered to change in abundance if one of the following criteria was met: (i) the fold-change between SylA and mock-treated samples was >1.5 with a p < 0.05, (ii) the fold-change was large enough to be considered an outlier according to boxplot statistics, or (iii) the protein was identified in at least two of three biological replicates in one condition but not another condition (Table 1, Supplemental Table 2). Additionally, for tissue specificity we required that the protein was not identified in the other tissues (Table 1, Supplemental Table 3). In total, 225 distinct proteins were found that had increased and 30 that had decreased in the different leaf tissues after inhibition of the proteasome. As discussed previously, reduced protein levels could result from reduced transcription, translation or stabilization of the proteins after SylA treatment, or from proteasome-independent protein degradation (Svozil et al., 2014). In contrast, the proteins that increased after inhibition of the proteasome are likely targets of the UPS or participate in pathways that respond to SylA treatment.

Bottom Line: SylA treatment of leaves resulted in the accumulation of 225 proteins and identification of 519 ubiquitylated proteins.Epidermis enzymes of the TCA cycle and cell wall biosynthesis specifically accumulated after proteasome inhibition, and in the vascular tissue several enzymes involved in glucosinolate biosynthesis were found to be ubiquitylated.Our results demonstrate that protein level changes and UPS protein targets are characteristic of the individual leaf tissues and that the proteasome is relevant for tissue-specific functions.

View Article: PubMed Central - PubMed

Affiliation: Plant Biotechnology, Department of Biology, Swiss Federal Institute of Technology Zurich Zurich, Switzerland.

ABSTRACT
Protein and transcript levels are partly decoupled as a function of translation efficiency and protein degradation. Selective protein degradation via the Ubiquitin-26S proteasome system (UPS) ensures protein homeostasis and facilitates adjustment of protein abundance during changing environmental conditions. Since individual leaf tissues have specialized functions, their protein composition is different and hence also protein level regulation is expected to differ. To understand UPS function in a tissue-specific context we developed a method termed Meselect to effectively and rapidly separate Arabidopsis thaliana leaf epidermal, vascular and mesophyll tissues. Epidermal and vascular tissue cells are separated mechanically, while mesophyll cells are obtained after rapid protoplasting. The high yield of proteins was sufficient for tissue-specific proteome analyses after inhibition of the proteasome with the specific inhibitor Syringolin A (SylA) and affinity enrichment of ubiquitylated proteins. SylA treatment of leaves resulted in the accumulation of 225 proteins and identification of 519 ubiquitylated proteins. Proteins that were exclusively identified in the three different tissue types are consistent with specific cellular functions. Mesophyll cell proteins were enriched for plastid membrane translocation complexes as targets of the UPS. Epidermis enzymes of the TCA cycle and cell wall biosynthesis specifically accumulated after proteasome inhibition, and in the vascular tissue several enzymes involved in glucosinolate biosynthesis were found to be ubiquitylated. Our results demonstrate that protein level changes and UPS protein targets are characteristic of the individual leaf tissues and that the proteasome is relevant for tissue-specific functions.

No MeSH data available.