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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.


Related in: MedlinePlus

Specific enrichment of the epidermal, vascular and mesophyll tissues using the Meselect method. The different tissue types were separated using leaves of GAL4-GFP enhancer trap lines with GFP expression specifically in the epidermal (A,B), vascular (C,D) or mesophyll (E,F) tissue. The protein blots were probed with antibodies against GFP (lower panels in A,C,E) and HSP90 (upper panels in A,C,E) as loading control, and afterwards stained with Coomassie (B,D,F) to show protein loading. The black lines indicate where the membranes were cut. Lane 1, epidermal tissue proteins; 2, vascular tissue proteins; 3, mesophyll tissue proteins; and 4, total leaf proteins. At the left, the molecular weight is indicated.
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Figure 1: Specific enrichment of the epidermal, vascular and mesophyll tissues using the Meselect method. The different tissue types were separated using leaves of GAL4-GFP enhancer trap lines with GFP expression specifically in the epidermal (A,B), vascular (C,D) or mesophyll (E,F) tissue. The protein blots were probed with antibodies against GFP (lower panels in A,C,E) and HSP90 (upper panels in A,C,E) as loading control, and afterwards stained with Coomassie (B,D,F) to show protein loading. The black lines indicate where the membranes were cut. Lane 1, epidermal tissue proteins; 2, vascular tissue proteins; 3, mesophyll tissue proteins; and 4, total leaf proteins. At the left, the molecular weight is indicated.

Mentions: We developed Meselect (mechanical separation of leaf compound tissues) to specifically enrich the three main leaf tissue types. The method first utilizes the TAPE sandwich approach to separate the abaxial epidermis from the remainder of the leaf (Wu et al., 2009). Any attached mesophyll cells are released by rapid protoplasting, which leaves the epidermal cells intact. The tape is flash-frozen and the epidermal cells are collected from the tape with tweezers and scrapers. Mesophyll cells are released from the remainder of the leaf by rapid protoplasting. The vascular tissue embedded in the mesophyll tissue remains intact during protoplasting and is then isolated with a tweezer. To confirm the specificity of the enrichment we applied Meselect to leaves of Arabidopsis lines that express GFP specifically in epidermal, mesophyll and vascular tissues (Gardner et al., 2009) (Figure 1). In the tissue-type specific protein extracts of leaves from the KC464 line, which expresses GFP exclusively in the epidermis, the GFP protein was exclusively found in the epidermal protein extract but not in the protein extract of vasculature and mesophyll tissues (Figure 1A). Similarly, in the tissue type-specific protein extracts of leaves of the KC274 line, which expresses GFP only in the vasculature, GFP was specifically enriched in the vasculature protein extract (Figure 1C). We noticed small amount of GFP in the mesophyll but not the epidermal protein extract. In the JR11-2 line backcrossed with Col-0, GFP is expressed in the spongy mesophyll and GFP was detected only the mesophyll protein extract, confirming that both vasculature and epidermal protein extracts do not contain mesophyll proteins (Figure 1E). Together, the Meselect method effectively and efficiently separates the abaxial epidermal, mesophyll and vascular leaf tissues.


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

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

Specific enrichment of the epidermal, vascular and mesophyll tissues using the Meselect method. The different tissue types were separated using leaves of GAL4-GFP enhancer trap lines with GFP expression specifically in the epidermal (A,B), vascular (C,D) or mesophyll (E,F) tissue. The protein blots were probed with antibodies against GFP (lower panels in A,C,E) and HSP90 (upper panels in A,C,E) as loading control, and afterwards stained with Coomassie (B,D,F) to show protein loading. The black lines indicate where the membranes were cut. Lane 1, epidermal tissue proteins; 2, vascular tissue proteins; 3, mesophyll tissue proteins; and 4, total leaf proteins. At the left, the molecular weight is indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Specific enrichment of the epidermal, vascular and mesophyll tissues using the Meselect method. The different tissue types were separated using leaves of GAL4-GFP enhancer trap lines with GFP expression specifically in the epidermal (A,B), vascular (C,D) or mesophyll (E,F) tissue. The protein blots were probed with antibodies against GFP (lower panels in A,C,E) and HSP90 (upper panels in A,C,E) as loading control, and afterwards stained with Coomassie (B,D,F) to show protein loading. The black lines indicate where the membranes were cut. Lane 1, epidermal tissue proteins; 2, vascular tissue proteins; 3, mesophyll tissue proteins; and 4, total leaf proteins. At the left, the molecular weight is indicated.
Mentions: We developed Meselect (mechanical separation of leaf compound tissues) to specifically enrich the three main leaf tissue types. The method first utilizes the TAPE sandwich approach to separate the abaxial epidermis from the remainder of the leaf (Wu et al., 2009). Any attached mesophyll cells are released by rapid protoplasting, which leaves the epidermal cells intact. The tape is flash-frozen and the epidermal cells are collected from the tape with tweezers and scrapers. Mesophyll cells are released from the remainder of the leaf by rapid protoplasting. The vascular tissue embedded in the mesophyll tissue remains intact during protoplasting and is then isolated with a tweezer. To confirm the specificity of the enrichment we applied Meselect to leaves of Arabidopsis lines that express GFP specifically in epidermal, mesophyll and vascular tissues (Gardner et al., 2009) (Figure 1). In the tissue-type specific protein extracts of leaves from the KC464 line, which expresses GFP exclusively in the epidermis, the GFP protein was exclusively found in the epidermal protein extract but not in the protein extract of vasculature and mesophyll tissues (Figure 1A). Similarly, in the tissue type-specific protein extracts of leaves of the KC274 line, which expresses GFP only in the vasculature, GFP was specifically enriched in the vasculature protein extract (Figure 1C). We noticed small amount of GFP in the mesophyll but not the epidermal protein extract. In the JR11-2 line backcrossed with Col-0, GFP is expressed in the spongy mesophyll and GFP was detected only the mesophyll protein extract, confirming that both vasculature and epidermal protein extracts do not contain mesophyll proteins (Figure 1E). Together, the Meselect method effectively and efficiently separates the abaxial epidermal, mesophyll and vascular leaf tissues.

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.


Related in: MedlinePlus