Download Barley `orange lemma` is a mutant in the CAD gene

Barley ‘orange lemma’ is a mutant in the CAD gene
Jennifer Stephens and Claire Halpin
Division of Environmental & Applied Biology,
University of Dundee at SCRI, Invergowrie DD2 5DA
Email: [email protected]
Introduction
Orange lemma phenotype
Lignin analysis
Lignin is an important component of the plant cell
wall. It provides support and structure as well as
acting as a defence barrier against external
stresses.
A number of orange lemma mutants have been
identified following screening of the EMS-mutant
TILLING population at SCRI.
Chemical analysis of lignin, which has been
extracted from cell walls by thioacidolysis shows
that the orange lemma mutants have reduced lignin
content (10 to 15% less) when compared to wild
type barley.
Figure 3a) Flower heads
from mutant (left) and
wild type (right) barley.
Most of the genes in the lignin biosynthesis pathway
have been characterized in many species including
Arabidopsis, tobacco and maize.
% klason lignin in extract-free samples of barley wild type and
'orange lemma' mutants
Genetic manipulation of lignin genes can lead to
structural and compositional changes in lignin.
Some of these changes affect the extractability or
digestibility of the lignin. Commercially, this is
beneficial to the paper and pulp industries and also
the animal feed industry.
105
% klason lignin
100
95
90
85
80
75
WT Bowman
Emir 894A
WT Optic
Optic mutant
Lignin is mainly composed of two types - syringyl (S)
and guaiacyl (G) lignin. A reduction in the S/G ratio
was found in the orange lemma mutants when
compared to the wild type. It has been reported that
a decrease in the S/G ratio can result in lignin with
greater digestibility.
Figure 3b) Stems from
mutant (left) and wild
type (right) barley.
b
Characterization of orange lemma
mutants
1.4
Biochemical analysis of barley stem tissue from
natural mutants (Bowman and Emir) and EMS
mutants (Optic) show reduced activity of CAD when
compared to their wild type counterparts.
S/G ratio of lignin in barley wild type and 'orange lemma'
mutants
1.2
1
S:G
Expression profile of CAD in barley
WT Emir
Figure 6. Amount of klason lignin in extract-free samples
from wild type and mutant stems shown as a percentage
of wild type lignin.
a
The barley orange lemma (rob1) natural mutant was
first described over 80 years ago and resembles the
brown midrib (bmr) mutants in maize.
We have characterized a number of natural and
ethyl methane sulfonate (EMS) orange lemma
mutants and have now identified them as mutations
in the cinnamyl alcohol dehydrogenase (CAD) gene.
This enzyme catalyzes the final step in the lignin
biosynthesis pathway.
Rob1
plant line
Barley (Hordeum vulgaris) is the second most
important arable crop in the UK with an annual
production of approximately 7 million tonnes.
Currently this is utilized mainly by the brewing and
distilling industries and as animal feed. Barley also
has the potential to be an important source for
biofuels.
0.8
0.6
0.4
0.2
0
WT Bowman
Rob1
WT Emir
Emir 894A
WT Optic
Optic mutant
plant line
Expression profile of HvCAD2 in different tissue types
CAD activity in different orange lem m a m utants of barley and their
corresponding wild type
6000
Figure 7. Ratio of S/G lignin in extract-free samples from
wild type and mutant stems.
5000
4000
100
3000
80
%wt activity
RAW value
120
2000
1000
60
40
20
0
pistel, bef ore caryopsis, caryopsis, caryopsis, endosperm, embryo, germinating germinating germinatingseedling, leaf seedling, seedling, root immature
anthers, f loral bracts,
anthesis
5dap
10dap
16dap
22dap
22dap seed, radicle
seed, seed, radicle
crow n
inf lorescence bef ore
bef ore
coleoptyle
anthesis
anthesis
tissue type
Figure 1. Expression profile of Barley CAD (HvCAD2) from
microarray analysis showing CAD is highly expressed in seedling
roots and floral bracts.
0
WT Bowman
Rob1 862
Rob1 869
WtEmir
Emir894A
WtOptic
Optic89
Optic180
plant line
Figure 4. CAD enzyme activity in wild type (shaded) and orange
lemma mutants showing reduced activity in the mutants.
Western analysis shows reduced levels of CAD in
orange lemma mutants when probed with antiserum
raised against the tobacco CAD protein.
Conclusion
We have identified the first lignin mutant in
barley. The detected mutations altered the
sequence of the gene encoding cinnamyl alcohol
dehydrogenase (CAD). This is the same gene
that was mutated in one of the brown midrib
maize mutants.
The barley orange lemma mutants have a
reduction in lignin content and a reduction in the
ratio of S/G lignin.
These changes in lignin may aid digestibility of
the plant fibres and help promote its usage as a
biofuel source.
WTBowman
Figure 2. Phylogenetic tree of all known CADs in Arabidopsis, Rice and
Barley highlighting the CAD genes involved in lignin biosynthesis (blue)
which all cluster together.
Rob1-862
Rob1-869
WTEmir
Emir894A
WTOptic
Optic89
Optic96
Optic180
Figure 5. Western blot of proteins from wild type and orange lemma
stems probed with tobacco CAD antiserum showing CAD is less
abundant in the mutants.
Acknowledgements
Arnis Druka, Brian Forster, David Leader, David
Marshall, Robbie Waugh (SCRI)
Catherine Lapierre (INRA, Grignon, France)