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Profiling and localization of phenolics in Arabidopsis lignin mutants
Oana Dima, Kris Morreel, Ruben Vanholme, Bartel Vanholme and Wout Boerjan
VIB Department of Plant Systems Biology; UGent Department of Plant biotechnology
and Genetics, Technologiepaerk 927 - 9052 Gent, Belgium
Lignin, an aromatic polymer in the cell wall, allows plants to grow upward and to transport water and solutes through the vascular system.
However, lignin obstructs agro-industrial processes such as the production of pulp and paper or biofuels from wood. A more easily
degradable lignin structure, e.g. by introducing easily cleavable units, would alleviate these problems. This could be done by rerouting
metabolites that can potentially co-polymerize into lignin, to the cell wall. We profiled the phenolics in a set of Arabidopsis lignin mutants.
Additionally, because most phenolics are transported to the apoplast or the vacuole, we are in the process of profiling the protoplasts and
the vacuoles isolated from these mutants to reveal the subcellular site of accumulation of these phenolics.
CCR phenolic profiling
Comparative leaf profiling
Arabidopsis WT and 7 lignin mutants
Phenolic profiling
FT-ICR-MS based identification
Subcellular localization of interesting
compounds
Vacuole isolation
vacuole buffer
4% ficoll
interphase
with purified
vacuoles
protoplasts in lysis
buffer with 10% ficoll
35 days old
Arabidopsis
rosette leaves
(2 grams)
4h with cellulase
and macerozyme
Washing cell walls and
tissue away from
protoplasts
Lysis of protoplasts
and ficoll gradient
GFP
Col-0/FM4-64
GFP/FM4-64
Phenolic extraction
SPE steps
Phenolic profiling
LLE vs SPE
Glucosinolates
12.3
2.9
12.4
Load
3.0
4.0
4.3
LLE
2.8
9.7 11.0
13.9
16.3
3.2
Wash
4.3
7.1
6.5
5.9
13.2
7.3
9.8 11.5
14.1
15.5
Protoplasts
26.3
19.4
20.0
22.4
10.5
2.6
30.4
4.1
4.4
5.1
2.0
SPE
11.8
9.2 9.9
5.2
24.2
15.4
28.2
32.4
26.9
30.4
13.7
9.9
3.1
2.5
Leaves
19.8
3.1
2.9
3.3
Sinapoyl malates
Kaempherol glycosides
13.1
5.9 6.5
7.1
2.2
Ultracentrifugation
4.0
8.5
8.1
Elution
13.3
14.5
19.6 19.7
19.0
16.6 17.4
26.5
22.1
24.4
29.6
29.3
30.8
31.4 32.8
8.5 9.9
3.1
4.0
19.7 20.5
11.4
19.6
12.9
Vacuoles
29.6
13.7 14.5
22.8
11.4
26.9
22.8
16.6
21.1
26.5
24.4
29.2
28.5
30.8
31.4 32.8
Conclusion
Perturbation of the lignin biosynthesis pathway in Arabidopsis
affects flavonoid and glucosinolate metabolism. Furthermore,
several of the accumulating
aromatic compounds are
candidate lignin monomers. To trace their localization, the
phenolic profiling of vacuoles was optimized. Currently, this
data is being explored.
Profiling of CCR vacuoles