RESULTS

Phenotypic analysis of galactosyltransferse (GALT) mutants for arabinogalactan-proteins in Arabidopsis thaliana

Shauni L. Bobbs, Debarati Basu, and Allan M. Showalter
Dept. of Environmental & Plant Biology, Molecular and Cellular Biology Program,
Ohio University Athens, OH 45701

Arabinogalactan proteins (AGPs) are plant specific glycoproteins that are found
in all plants as components of the cell wall, plasma membrane and cellular
secretions. AGPs function in a variety of cellular processes including cell
proliferation, cell expansion, somatic embryogenesis and cell death. They also
possess valuable adhesive and emulsification properties that are utilized for
commercial purposes. But little is known about the mechanisms and
galactosyltransferase enzymes (GALTs) responsible for glycosylation of AGPs.
Consequently, a bioinformatics approach was adopted to identify and
characterize putative GALTs responsible for synthesizing -(1, 3)-Gal linkages
and six putative GALT genes were selected bases on their galactosyltransferase
activity and Gal-binding lectin domain. These domain are known to be involved
in adding the first sugar in mammals. Here we have tried to characterize allelic
mutants for GALT6 and GALT3 as indicated in red in Table 1.
Gene Locus Mutant lines Location Full length
genomic
DNA (bp)
Coding
Region
(bp)
Amino
acids
GALT1 At1g26810 salk_006871 Promoter 3327

1932 643
CS 871760
Sail_170A08
Exon
GALT 2

At4g21060 salk_117233 Exon 4131 2053 741
salk_141126 Exon
GALT3 At3g06440 salk_085633
salk_005178
Promoter 3133 1860 619
GALT4 At1g27120 salk_136251 Exon 2839 2022 673
salk_131723 Exon
GALT5 At1g74800

At5g62620
salk_115741 Exon 3145 2019 672
Salk_080140 Exon
GALT6 CS 870637
(Sail_59 D08)
FLAG_107C5

Exon

RNAi line
3196 2046 681
Table 1. Six candidate AGP- GALTs with their mutant lines
Fig.1. Schematic representation of mutant characterization by PCR analysis. The
blue triangle represents T-DNA insertion. LP and RP are gene specific primers and
LBa1 and LB3 are T-DNA specific primers for Salk and Sail lines respectively. WT
denotes the wild type, HZ is the heterozygous and HM is the homozygous plants.
galt2/galt4 galt5/galt6
LP RP
LBa1/LB3
LP RP
LBa1/LB3
LP
LP RP
RP
LP RP
LP RP
LBa1/LB3
Wt
HZ
HM
INTRODUCTION
DISCUSSION
Six candidate GALTs have two allelic mutants per gene
(Table 1). Single mutants were generated using PCR analysis
and further confirmed by sequencing (Fig1 and 2A). galt6-2 is
a RNAi line and thus RT-PCR analysis was used to screen
knockout lines (Fig 2F).
Phenotypic characterization revealed no significant
differences between the wild type and the mutant plants when
grown under optimal growth condition in soil (Fig 2B, C, D
and E). But in response to mannitol (Fig 3A and 3B), and
ABA (Fig 5) the galt2 and galt 5 showed delayed germination
in contrast to the wild type. The germination frequency was
also significantly low in these mutants in varying mannitol
(Fig 4).
ACKNOWLEDGEMENTS
This work is funded by NSF (grant no. 0918661).
WT(Col 0) galt3-1 galt3-2
WT HM1 HZ HM2
WT
galt3-1
galt3-2
A
C
WT ( Ler) galt6-2
D
WT galt3-1 galt3-2
L3 WT L4 L5 L7 L6 L1
F
WT
galt5-1
galt5-2
galt2-1
galt2-2
WT
galt5-1
galt5-2
galt2-1
galt2-2
Fig.2. Screening of homozygous knockout mutants for GALT3 (salk_005178) and
GALT6 (FLAG_107C5) and phenotypic analysis of the mutants. A. PCR analysis of
galt3, B ,C and D. phenotypic analysis galt3, E. phenotypic analysis of galt6-2 and F.
RT-PCR analysis of galt6 , UBQ10 is the endogenous control and L1-L7 are individual
RNA i lines.
Time after stratification in days
0
10
20
30
40
50
60
70
80
90
100
WT galt2-1 galt2-2 galt5-1 galt5-2
0mM 100mM 300mM 500mM
F
r
e
q
u
e
n
c
y

o
f

g
e
r
m
i
n
a
t
i
o
n

(
%
)

B
UBQ 10
GALT6
0.5
1.5 3 3.5 2.5 1 2 4
0.5 1.5 3 3.5 2.5 1 2
Fig.3. Time course of germination as indicated by the emergence of radicle in MS
supplemented with A. 300mM and B. 500mM mannitol. In comparison to the wild type
seeds the mutants (galt2 and galt5) have delayed germination. Red arrow indicated the
radicle emergence. Scale bar 100m.
A
B
Fig.4. Rate of germination of wild type and mutant plants in
response to salt stress. A total of 25 seeds from both the wild type
and the mutant plants were grown in MS plates supplemented with
100mM,300mM and 500mM mannitol. Emergence of the radicle is
considered as the indication of germination. Each concentration set
is done in triplicates.
E
Fig.5. Time course of germination as indicated by the emergence of
radicle in MS supplemented with 5M ABA. In comparison to the
wild type seeds the mutants (galt2 and galt5) have delayed germination.
Red arrow indicated the radicle emergence. Scale bar 100m
WT
galt2-1
galt2-2
galt5-1
galt5-2
L2
Time after stratification in days
0.5 1.5 3 2.5 1 2