Download Heartwood extractives – from phenotype to candidate genes

Heartwood extractives – from phenotype to candidate genes
Jukka-Pekka Vertaa, Teemu Teeria, Katri Kärkkäinenb, Anni M. Harjuc, Martti Venäläinenc
a
c
Dept. Applied Biology, University of Helsinki, b Finnish Forest Research Institute, Muhos Research Unit
Finnish Forest Research Institute, Punkaharju Research Unit
Variation in heartwood decay resistance: possibilities for breeding of wood quality
Pinosylvin is fluorescent in UV-light, making
its visualisation straightforward (picture to the
left, Eija Matikainen).
The heartwood timber of Scots pine (Pinus sylvestris L.)
is moderately resistant against biological degradation,
which provides an opportunity to use it in harsh service
conditions. Ample phenotypic and genetic (heritability
0.4) variation in the decay resistance against brown rot
fungus Coniophora puteana has been found (Harju &
Venäläinen 2002).
Mass loss due to cellar fungus, %
At phenotypic level, the variation is to a large degree explained by concentration of total phenolics (correlation –
0.82), mainly stilbenes (Harju & Venäläinen 2006). The
concentration of heartwood extractives, like resistance itself, is a characteristic of mature trees.
Heartwood decay resistance is a heritable
trait. However, the heartwood of Scots pine
doesn´t form untill the trees mature (at 30-50
years). Because resistance to decay cannot
be determined before, conventional breeding
for heartwood decay resistance becomes
unrealistic.
Mass loss of heartwood samples correlates
with stilbene concentration (figure to the
left).
60
50
40
To determine the molecular basis of
variation
in
heartwood
pinosylvin
concentration, we sampled trees from the
two extremes of the distribution (sampled
trees shown with yellow and red triangles).
R2 = 0,85
30
20
10
0
0
Scots pine stilbenes are pinosylvin (PS) and
its monomethyl- and dimethylethers (PSM,
PSD, figure above, from Celimene et al.
1999).
2
4
6
8
10
Sum of stilbenes (PS + PSM), mg/g
12
From these trees we studied the genetic
variation of the gene PST-1. Our goal was
to determine whether polymorfic loci in the
gene correlate with heartwood stilbene
concentration.
The search for candidate genes: Scots pine pinosylvin synthase gene PST-1
shikimate pathway
PAL is the branchpoint
between primary metabolism
and phenylpropanoid
biosynthesis.
According to earlier studies, the pinosylvin synthase gene is present in five copies in the Scots pine
genome (PST-1 through PST-5; Preisig-Müller et al. 1999). All gene family members have two exons
and a single intron in a conserved site. PST-1 was identified as the most active gene, which
according to Preisig-Müller et al. (1999) is responcible for pinosylvin production both in the heartwood
and in stress-induced responces.
phenylalanine
PAL
C4H
lignins
cinnamic acid
Using PCR-cloning, we sequenced the PST-1 gene from nine individuals with low, or high heartwood
pinosylvin concentration. The coding region of one PST-1 allele contains a 10 bp deletion, causing a
frameshift and an early stop codon. This deletion resembles a deletion observed in P. densiflora
PDSTS3 gene, coding for a homologous stilbene synthase (Kodan et al. 2002).
4CL
anthocyanins,
flavonoids, etc.
CHS
cinnamoyl-CoA
The transcript of PDSTS3 is fully functional, but lacks a C-terminal extension (shown in ClustalW
alignment below). This causes the emancipation of the enzyme from wild-type end product-inhibition
and subsequent high activity (Kodan et al. 2002). Whether the observed PST-1 allele transcript
behaves similarly remains to be determined. If so, this finding may have applications for molecular
breeding.
The pine stilbene, pinosylvin, is the
end product of pinosylvin synthase.
PST
pinosylvin
Pinosylvin is modified to its
monomethylether, and
possibly also to its
dimethylether, by pinosylvin
monomethyltransferase.
PMT
wt15 (Lv83)
w
w
t8
wt2
wt5 (Lv251
)
D3 (L
v251
D2
)
(Lv
D6 555)
(Lv
55
5)
w
D2
t1
-1
3
(L
(L
v5
v5
68
68
)
)
D15 (Lv83)
v83)
D14 (L
)
)
51
v2
(L
D9 55)
v5
(L
-7
68
5
Lv
D
D
16
(L
v5
wt
55
15
(L )
D1
v5
-4
(Lv 55)
55
5)
D1-5
(Lv555 D1
)
D1-3 (Lv424)
4(
wt
1
)
68
v5
(L
4
t1
w
)
28
)
v6
628
(L
(Lv
-8
D1 wt8-1
2
(Lv6
wt1
8)
D1-9
)
71
v1
(L
1)
1
t1 Lv25
(
7
1)
D
v25
(L
51)
D8
(Lv2
wt12
628)
wt10 (Lv
According to promoter sequences from
seven individuals, the PST-1 gene is
present in at least six copies in the Scots
pine genome. The roughly 120 sequences
analysed group into 50 haplotypes, each
individual
posessing
6-12
different
haplotypes of the PST-1 promoter.
Fylogenetic analysis of the sequences
indicate that the newly found PST-1 gene
family is comparatively young.
Figure on the left illustrates a fylogenetic
tree of 47 different promoter sequences (ME
-bootstrap consensus tree according to
Tajima-Nei distances).
The promoter area (-1000 bp) of PST-1 is
highly polymorfic. The area contains at least
four SNP´s and an abundant number of
indel mutations.
25,
0
Heartwood stilbene concen tration, mg/g
-1
PST 8)
62 )
(Lv 24 )
1-8 Lv4 68
wt
(
2 Lv5
1(
wt
-7
t1
w
0.005
D1
-1
(L
v4
-4
(Lv 24)
D1-2
4
(Lv 24)
424
)
wt1-3
(Lv424
)
wt1
wt3-1 (Lv171)
wt3 (Lv17
1)
wt9
(Lv1
71)
wt6
(Lv
D4
568
(Lv
)
25
w
t7
1)
D
(L
5
v1
(L
71
v2
)
51
)
pinosylvin monomethylether
Lv568
20,
0
For example a complete GCC-box, located
-534 bp from the transcription strart,
contains three indel alleles. Polymorphism
in this locus correlates with heartwood
pinosylvin concentration (p=0,02). This
indicates, that the box maybe functionally
important and that mutations in it may affect
the transcriptional regulation of the gene.
15,
0
10,
0
5,
0
0,
0
0
v4
D12 (L
1
Locus -534 genotype
4
Figure on the left shows how heartwood
stilbene concentration is dependent on a
polymorfic loci in the PST-1 promoter. 0:wt,
1:insertion, 4:deletion, (N=54, additive
nature of haplotypes assumed)
24)
D 11
28)
(Lv6
Correspondence: [email protected]
Celimene et. al 1999, Holtzforchung, 53(5):491
Harju & Venäläinen 2002, Scand. J. For. Res. 17:199
Harju & Venäläinen 2006, Can. J. For. Res. 36:1797
Kodan et. al 2002, PNAS, 99(5):3335
Preisig-Müller et. al 1999, Plant Mol Biol, 39(2):221