Download Control of GL2 expression in Arabidopsis leaves and trichomes

Control of GL2 expression in Arabidopsis leaves
and trichomes
Daniel B. Szymanski, Ross A. Jilk, Susan M. Pollock and M.
David Marks
Bjarni Már Óskarsson,
Magnús Eysteinsson
Sævar Ingþórsson
Arabidopsis thaliana
• A small flowering plant
– A part of the mustard family along with
species such as cabbage and radish
• Discovered by Johannes Thal, in the
Harz mountains in the sixteenth
century
• Has many advantages for being a
model organism in the study of plant
biology/genetics
Arabidopsis as a model organism
• Many Benefits
–
–
–
–
–
Small genome (125mb)
Rapid life cycle (6 weeks)
Easy cultivation
Prolific seed production
Efficient transformation
• Using Agrobacterium tumefaciens
– Large numer of mutant lines
– A very large research community
Trichomes
•
•
Definition: A hairlike or
bristlelike outgrowth, as from
the epidermis of a plant
Many shapes and sizes that
depend on the plant species
– Unicellular trichomes
– Multicellular trichomes
– Secretory trichomes
•
Root hairs are specialized
trichomes found on roots
The development of Arabidopsis
unicellular trichomes
1.
2.
3.
4.
5.
6.
radial expansion of the trichome
precursor in the plane of the leaf
stalk emergence and expansion
formation of branch structures
expansion of the stalk and
branches
continued expansion of the stalk
and branches, which develop
pointed tips
mature trichome with papillate
surface
What Genes control the
development of trichomes?
• More than 20 genes are required
– The initial selection (stage 1-2) of trichomal precursors
depends on 2 genes.
• GLABROUS1 (GL1) a myb-class transcription factor
• TRANSPARENT TESTA GLABROUS (TTG)
WT
gl1
ttg
What Genes control the
development of trichomes?
• GLABROUS2
(GL2) is required
for subsequent
development of
trichomes
WT later stages
WT, early stages
gl2
Later
stages
The expression pattern of the
GL2 gene
• In order to look at where the GL2 gene was being translated,
the researchers made a special T-DNA construct
• It was composed of a 5’UTR 2,1kb fragment of the GL2 gene
and the b-glucuronidase gene (GUS)
– GL2::GUS, (essentially the GUS gene under the control of the GL2
promoter region)
•
This construct was then used to transform Wild type
Arabidopsis plants
– The plants were then treated for GUS staining at different
developmental stages and GUS activity was analyzed
A.
Developing Leaves
B.
Transverse Section through a
developing leaf primorida
C.
Apaxial surface of a
developing leaf
D.
Cross section through the
apical third of a developing
leaf
E.
Emerging trichomes at the
bse of a mature leaf
F.
Transverse section through
leaf base with developing
trichomes
G.
Developing leaf with
trichomes at several
developmental stages
H.
Mature leaf
Immunolocalization of the GL2
protein
• To confirm the expression pattern of GL2, antiGL2 antibodies were used to localize the GL2
protein
– WT and gl2-2 plants were used and prepared for
immunocytochemical staining using a polyclonal
antibody against an epitope in the C terminus of GL2.
• gl2-2 was used as a negative control as it contains a T-DNA
unit insertion consisting of randomly linked T-DNA units in the
coding sequence upstream from the epitope.
A.
TS through gl2-2
developing leaf
B.
TS through WT
developing leaf
C.
TS through a mature
WT leaf
Dependence of GL2::GUS
activity on genes that affect
trichome development
• Other researchers have shown that GL2
dependant on the presence of GL1 and TTG
– Analyzed by looking at GL2::GUS activity in
different mutation backgrounds
• gl1, ttg, and gl1 ttg plants
Dependence of GL2::GUS
activity on genes that affect
trichome development
• In order to quantify the relative levels of
GL2 transcription, the total GUS activity of
leaves and cotyledons at the four leaf stage
was measured in vitro
Deletion analysis of the GL2
promoter
• To look at the promoter regions of the GL2
gene several deletion constructs using
restriction enzymes were made, and fused to
GUS and used to transform plants
Relationship between GL1 and
GL2
• Both the GL1 and TTG loci are required for
trichome initiation, while GL2 is required for the
earliest morphogenetic events of trichome growth
– The Promoter region of GL2 contains a myb-class
binding site
• Remember that GL1 is a myb-class transcription factor...
• A comparison between GL1::GUS and GL2::GUS
plants was made
A. GL1::GUS
B. GL2::GUS
C. TS of apical section of a GL1::GUS plant leaf
D. TS of apical section of a GL2::GUS plant leaf
Ectopic expression analysis
• The Cauliflower mosaic virus promoter 35S can be used to
induce ectopic expression of genes
– Plants that ectopically express GL1 and the Maize R Gene were
analyzed for ectopic GL2 expression
• The R gene can compensate for the lack of TTG (ttg plants)
– However, the combination of 35S::GL1 35S::R is lethal, unless the
R gene contains an N-terminal fusion of the vertebrate
glucocorticoid receptor (35S::R-GR)
• The plants must also have the genotype ttg/ttg in order to survive
– Gene expression can then be controlled by adding the
Glucocorticoid homolog Dexamethasone to the growth medium
Ectopic expression analysis
•
To rule out the possibility that the ectopic
activation of the GL2 promoter in the 35S::GL1
and 35S::R-GR background represents a
nonspecific transcriptional activation, two
additional GL2 reporter constructs were
analyzed in that genetic background
A.
B.
The DXHp construct (3), which lacks the GL2
promoter regions required for detectable trichome
expression
The DRI construct(5), which contains both
putative shoot transcription domains
A Complex Question...
• Because both GL1 and R are required to ectopically activate GL2, it is
possible that GL1 and R function as a complex
• A construct containing the GL1 gene with a myc epitope as an Nterminal fusion was generated
– This same epitope fusion was made to a truncated version of the GL1
protein that expressed only the N-terminal 122 amino acids, which
contains the entire myb homologous domain
• These two protein fusions, as well as the full length untagged R
protein, were cloned into expression vectors for the in vitro production
of mRNA and subsequent reticulocyte translations in the presence of
[35S]methionine.
A.
B.
C.
SDS Page gel containing the
results of in vitro translations
1.
GL1 myc full length
protein
2.
N terminal GL1 myc
protein
3.
mock translation
4.
Full length R
Anti myc immunoprecipitations
1.
tagged GL1
2.
Tagged N terminal GL1
3.
R
Co-immunoprecipitations
1.
Full length tagged GL1
and R
2.
N terminal tagged GL1
and R
Conclusions
•
In Arabidopsis the TTG and GL1 genes are required for trichome initiation.
– GL2 function does not appear to be required for initiation since gl2 leaves have
approximately normal numbers of trichomes.
•
•
However gl2 trichome morphology is variable, and shows defects from stage 2
to stage 6
In leaf primordia, the GL2 gene is expressed in all cell layers.
– Because the number of cell layers and leaf morphology of developing gl2 leaves
are not obviously different from wild-type, the nontrichome function of GL2 in
developing leaves is unknown.
•
•
GL2 protein appears to show celltype specific subcellular localization in
developing leaves, and is localized in the nucleus in developing trichomes
Both GL1 and TTG may directly bind to the GL2 promoter, either alone or as
part of a larger complex, but the interaction between GL1 and R somehow
eliminates the requirement for TTG
– Further studies [Since 1998] of the mechanisms of GL2 regulation will aid in the
understanding of how complex and integrated gene networks control plant
development.