Abstract
Aim of this work was a comprehensive analysis of the cell wall proteome of Medicago truncatula suspension cell cultures. Three different tasks included the establishment of an optimised protocol for the extraction of the cell wall proteins (CWPs), development of an optimised method to establish the cell wall proteome map of M. truncatula and comparative proteome profiling of CWPs during elicitation with yeast invertase and suppression with Sinorhizobium meliloti lipopolysaccharides (LPS), representing the pathogenic and symbiotic interactions respectively. The CWPs extracted using living cells and/or cell wall fragments were analysed using different techniques. Using a combination of either one-dimensional, preparative isoelectric focussing, two-dimensional gel electrophoresis (2-DE) or LC-MS/MS and MALDI-TOF-MS analysis, established the proteome reference map of M. truncatula CWPs. In addition to establishing methods for proteome analysis of CWPs of the model plant M. truncatula, the presented work highlights the different composition of several protein classes in cell culture. 2D-PAGE analysis for the CWPs extracted from living cells enabled to characterise approximately 48 CWPs. An alternative approach using a preparative free-flow electrophoresis technique for first dimension separation to obtain 20 fractions with proteins differing in their isoelectric points which were subsequently separated by SDS-PAGE was established. This resulted in identification of 59 out of 100 additional proteins, resulting in 107 identified proteins in total. The proteins analyzed were related to defence, oxidative stress, cell wall modification and signal mediation. In order to overcome the limitations caused because of the nature of the CWPs in proteome analysis, a new extraction protocol (using cell wall fragments) and alternate method (LC-MS/MS) to identify more proteins was established. Furthermore CWPs extracted from purified cell wall fragments and using 2D-PAGE or LC-MS/MS approach, 46 and 65 proteins were identified respectively with a total of 111 proteins. Among the identified were the proteins involved in various processes like cell wall modifications, signaling, defence mechanism, membrane transport, protein synthesis & processing, as well as wall construction processes. Also the comparative proteome analysis using yeast elicitor (Invertase) in response to pathogen defence and suppressor using S. meliloti were also established. 2D-PAGE analysis for the CWP samples extracted with elicitor and LPS treated cells resembled the same as the proteome map of the elicitation with invertase alone, with the few upregulated proteins involved in defence and from LPS treated cell wall proteome map there was no significant difference. Using this approach, proteins involved in defence like L-ascorbate peroxidase, specifically targeted proteins to the cell wall during defence response which include glyceraldehydes-3-phosphate dehydrogenase and proteins that play an important role during growth and development were identified. Also some of this defence related proteins are absent in the same gel after elicitation conferring that oxidant protection is regulated by these proteins. For most CWPs the biological functions are yet to be identified. Bioinformatic analysis helps to have a clue to design an experiment, to understand the biochemical and biological functions of these proteins.
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Claims 15
Claim · model-written summary
The study established a reference map of Medicago truncatula cell-wall proteins.
- Taxon
- Medicago truncatula
Medicago truncatulat0 - Outcome
- proteome reference map of M. truncatula CWPse0
Not reportedComparatorValue
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“established the proteome reference map of M. truncatula CWPs”
t0“Medicago truncatula”
t1“Medicago truncatula”
Claim · model-written summary
Analysis of proteins from living cells characterized approximately 48 cell-wall proteins.
- Taxon
- Medicago truncatulaMedicago truncatulat0
- Outcome
- CWPs extracted from living cellse0
- Value
- approximately 48 CWPse0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“2D-PAGE analysis for the CWPs extracted from living cells enabled to characterise approximately 48 CWPs”
t0“Medicago truncatula”
t1“Medicago truncatula”
Claim · model-written summary
A preparative free-flow electrophoresis approach yielded 20 fractions.
- Taxon
- Medicago truncatulaMedicago truncatulat0
- Outcome
- fractionse0
- Value
- 20 fractionse0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“a preparative free-flow electrophoresis technique for first dimension separation to obtain 20 fractions”
t0“Medicago truncatula”
t1“Medicago truncatula”
Claim · model-written summary
The alternative approach identified 59 additional proteins among 100.
- Taxon
- Medicago truncatulaMedicago truncatulat0
- Outcome
- additional proteinse0
- Value
- 59 out of 100 additional proteinse0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“This resulted in identification of 59 out of 100 additional proteins”
t0“Medicago truncatula”
t1“Medicago truncatula”
Claim · model-written summary
The total number of identified proteins was 107.
- Taxon
- Medicago truncatulaMedicago truncatulat0
- Outcome
- identified proteins in totale0
- Value
- 107 identified proteinse0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“resulting in 107 identified proteins in total”
t0“Medicago truncatula”
t1“Medicago truncatula”
Claim · model-written summary
The proteins analyzed were associated with defence, oxidative stress, cell-wall modification and signaling.
- Taxon
- Medicago truncatulaMedicago truncatulat0
- Outcome
- related to defence, oxidative stress, cell wall modification and signal mediatione0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“The proteins analyzed were related to defence, oxidative stress, cell wall modification and signal mediation”
t0“Medicago truncatula”
t1“Medicago truncatula”
Claim · model-written summary
A new extraction protocol using cell-wall fragments and LC-MS/MS was established to identify more proteins.
- Taxon
- Medicago truncatulaMedicago truncatulat0
- Outcome
- identify more proteinse0
Not reportedComparatorValue
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“a new extraction protocol (using cell wall fragments) and alternate method (LC-MS/MS) to identify more proteins was established”
t0“Medicago truncatula”
t1“Medicago truncatula”
Claim · model-written summary
2D-PAGE of purified cell-wall fragments identified 46 proteins.
- Taxon
- Medicago truncatulaMedicago truncatulat0
- Outcome
- proteins were identifiede0
- Value
- 46e0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“CWPs extracted from purified cell wall fragments and using 2D-PAGE or LC-MS/MS approach, 46 and 65 proteins were identified respectively with a total of 111 proteins”
t0“Medicago truncatula”
t1“Medicago truncatula”
Claim · model-written summary
LC-MS/MS of purified cell-wall fragments identified 65 proteins.
- Taxon
- Medicago truncatulaMedicago truncatulat0
- Outcome
- proteins were identifiede0
- Value
- 65 proteinse0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“CWPs extracted from purified cell wall fragments and using 2D-PAGE or LC-MS/MS approach, 46 and 65 proteins were identified respectively with a total of 111 proteins”
t0“Medicago truncatula”
t1“Medicago truncatula”
Claim · model-written summary
The purified-fragment approaches identified 111 proteins in total.
- Taxon
- Medicago truncatulaMedicago truncatulat0
- Outcome
- proteinse0
- Value
- 111 proteinse0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“with a total of 111 proteins”
t0“Medicago truncatula”
t1“Medicago truncatula”
Claim · model-written summary
The identified proteins included classes involved in cell-wall modification, signaling and defence.
- Taxon
- Medicago truncatulaMedicago truncatulat0
- Outcome
- proteins involved in various processes like cell wall modifications, signaling, defence mechanism, membrane transport, protein synthesis & processing, as well as wall construction processese0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Among the identified were the proteins involved in various processes like cell wall modifications, signaling, defence mechanism, membrane transport, protein synthesis & processing, as well as wall construction processes”
t0“Medicago truncatula”
t1“Medicago truncatula”
Claim · model-written summary
The combined elicitor and LPS protein map resembled the invertase elicitation map, with a few defence proteins upregulated.
- Taxon
- Medicago truncatulaMedicago truncatulat0
- Treatment
- elicitor and LPS treated cellse0
- Outcome
- few upregulated proteins involved in defencee0
- Direction
- upregulatede0
- Comparator
- elicitation with invertase alonee0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“CWP samples extracted with elicitor and LPS treated cells resembled the same as the proteome map of the elicitation with invertase alone, with the few upregulated proteins involved in defence”
t0“Medicago truncatula”
t1“Medicago truncatula”
Claim · model-written summary
The LPS treated cell-wall proteome map showed no significant difference.
- Taxon
- Medicago truncatulaMedicago truncatulat0
- Treatment
- LPS treatede0
- Outcome
- cell wall proteome mape0
- Direction
- no significant differencee0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“from LPS treated cell wall proteome map there was no significant difference”
t0“Medicago truncatula”
t1“Medicago truncatula”
Claim · model-written summary
Defence-related cell-wall proteins identified included L-ascorbate peroxidase and glyceraldehydes-3-phosphate dehydrogenase.
- Taxon
- Medicago truncatulaMedicago truncatulat0
- Gene
- L-ascorbate peroxidasee0
- Outcome
- proteins involved in defencee0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“proteins involved in defence like L-ascorbate peroxidase, specifically targeted proteins to the cell wall during defence response which include glyceraldehydes-3-phosphate dehydrogenase”
t0“Medicago truncatula”
t1“Medicago truncatula”
Claim · model-written summary
Some defence-related proteins were absent after elicitation.
- Taxon
- Medicago truncatulaMedicago truncatulat0
- Treatment
- elicitatione0
- Outcome
- defence related proteins are absente0
Not reportedDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“some of this defence related proteins are absent in the same gel after elicitation”
t0“Medicago truncatula”
t1“Medicago truncatula”
The same questions in other papers
lipopolysaccharide · molecular: expression · Medicago truncatula (barrel medic)Positive and negative results
1 in the opposite direction
- positive
Claims are grouped by organism, treatment and what was measured or targeted. A paper counts as positive when any of its claims in the group is positive.