Biology Reference
In-Depth Information
TABLE III
Effect of Mutations in Stem-Specific Laccases on the Lignification and Saccharification
of Mature Stems as Compared to the Wild Type (Col0)
Lignin content relative to
the wild-type level
(number of cultures)
Saccharification
(nmol sugars/
mg h)
Thioacidolysis S/
G ratio
Lines
WT
100
0.40
0.00
63.2
4.6
lac2
105.0
5.7 (2)
0.40
0.02
63.2
3.5
3.5 (8)
a
3.8
a
lac4-1
87.9
0.44
0.00
76.7
3.4 (6)
a
0.3
a
lac4-2
90.5
0.43
0.02
82.3
lac10
96.3
5.5 (2)
0.40
0.01
61.8
3.6
lac11
104.0
10.3 (4)
-
-
1.8 (5)
a
lac12
96.5
0.40
0.02
64.8
3.1
4.8 (8)
a
0.01
a
0.3
a
lac17
89.7
0.48
72.3
5.3 (6)
a
0.02
a
4
a
lac4-1 lac17
83.0
0.54
79.6
2.2 (5)
a
0.03
a
3.7
a
lac4-2 lac17
63.0
0.67
94.1
84.1
1.7 (2)
a
0.55
0.02
a
74.6
2.9
a
lac12 lac17
The data represent mean values (
standard deviations) between replicates. The relative lignin
content of the mutant lines is measured by the Klason protocol (
Dence, 1992
) and is expressed
relative to the corresponding control arbitrarily set at 100% and grown together with the mutant
(the number of different cultures is given between brackets; each Klason determination is run on
extract-free samples and in duplicates). The thioacidolysis S/G ratio is measured from one
culture of all the mutants grown together and with analytical duplicates. Saccharification (run
in triplicates) corresponds to the amount of sugars released after enzymatic treatment of
pretreated stem segments (alkaline pretreatment NaOH 0.5 mol L
1
,90
8
C, 30 min) and is
expressed in nanomoles of sugars released per milligram of dry material and per hour.
a
Significantly different from the corresponding control when p
0.05.
temporal correlation of the enzyme with active lignification, (2) altered lignin
content and/or structure in plants with modified levels of the enzyme, and (3) a
substrate specificity of the enzyme for monolignols. Up to now, the laccases
encoded by AtLAC4 and AtLac17 have fulfilled the first two criteria. However,
the last criteria still remains to be verified and will require purification and
enzymatic characterization of the AtLAC4 and AtLAC17 proteins. Neverthe-
less, perhaps the most conclusive demonstration that AtLAC17 participates to
the formation of G units in supportive tissues (fibres) was provided by comple-
mentation experiments with the lac17 mutant. When the full LAC17 construct
was reintroduced in the lac17 line under the control of its endogenous promot-
er, both the S/G ratio and the Klason lignin content of the complemented line
were restored to WT levels (
Berthet et al.,2011
).
Deciphering the role of other stem-specific laccases needs further investiga-
tions. AtLAC11 belongs to the same functional cluster as AtLAC4 and
AtLAC17 (
Fig. 1
A). In addition, the expression study of promoter-uidA gene
(beta-glucuronidase, GUS) fusion constructs revealed that AtLAC11 was
