Transcript WUS

Homeodomain Proteins
Plant Homeodomain containing proteins
The first gene known encoded homeodomain related sequences in plants
was KNOTTED1 from Z. mays. (1991), identified by transposon tagging.
Plant homeodomain protein families
Plant Homeodomain genes encode modular DNA-binding proteins
similar to animal homeodomain proteins
HD-Zip
N
HD Z
C
GLABRA
N
HD Z
Z
PHD finger
N
BEL
N
ZF-HD
N
WUSCHEL
N
HD
KNOX class I
N
KNOX
C
PHD
BEL
HD
C
C
HD
C
HD
C
HD
C
TALE class
Plant homeodomain protein families
HD-Zip
Class I. Response to light, hormones and environmental factors.
HAT or ATHB proteins
Class II.
Class III. Leaf and vascular development. Bilateral symmetry.
Polarity. REVOLUTA, PHABULOSA, PHAVOLUTA, CORONA
GLABRA
Required for epidermal cell fate. Trichomes.
GLABRA 1 and 2 proteins
PHD-finger
Required for PR regulation. HOX1 and 2 proteins
BEL
Ovule development. Tuber formation in
potato.BELL1 protein. RPL: important for replum
KNOX
Class I. Required for SAM identity. STM, KNAT 1(BP), 2.
Class II. Expressed in several tissues. Unknown function.
WUSCHEL
Required for shoot apical meristem (SAM) identity.
Regulates downstream homeogenes. WUSCHEL
itself and WUSCHEL related protein (WOX genes).
ZF-HD
Required for some photosynthetic enzyme regulation.
Involved in developed tissues and important for abiotic
stress tolerance
Shoot Apical Meristem
(SAM) Development
Shoot Apical Meristem
SAM is the source of all above-ground post-embryonic organs in higher
plants. Light dot: stem cells. Dark dot: next organ primordia
Just as generally you can distinguish between two different types
of tissue, there generally also exists two types of dividing tissue.
The first type of tissue is that from which the entire organ is
originally formed, and which will also remain active later,
sometimes for the entire life; I propose to name this ‘‘Meristem’’.
The other type of tissue has long been known as cambium.
Recently all developing tissue has been referred to as cambium by
Schleiden, Schacht etc. However, there is no reason for this
generalization and the use of the same word for all tissues, as this
would simply result in the need to distinguish later between
different types of cambium and describe these as a special type of
tissue which was simply called cambium in the past. Nägeli
‘‘Beiträge zur Wissenschaftlichen Botanik” (1858)
CZ: central zone, contains a core of stem cells
PZ: peripheral zone, site of production of lateral organ primordia
RZ: rib zone, give rise to differentiated cells of the growing stem
SAM mutants
Wild type
stm
wus
SHOOT MERISTEMLESS and WUSCHEL encode for homeodomain
proteins. wus mutants contains partially differentiated cells that are
incapable of sustained organogenesis producing short bushy plants.
WUSCHEL is expressed at the earliest stages of meristem initiation.
The pattern of WUS expression
suggests that stem cells in the shoot
meristem are specified by an underlying cell group which is established
very early during Arabidopsis embryogenesis - in the 16-cell embryo and
becomes progressively localized to an inner portion of the central zone of
the meristem.
STM expression pattern
STM expression commences slightly later than WUS, in the
globular stage embryo and accumulates in both, the central
and peripheral zones.
stm phenotype
The phenotype of stm mutants depends on the ecotype and severity of the
allele. Plants carrying the stm-1 allele in a Landsberg erecta background
produce cotyledons but no further components of the shoot system. The
cotyledons are also fused at their base. This phenotype suggests that STM
is required to maintain and/or initiate the shoot apical meristem. It also
suggests a requirement for defining the boundaries between organs.
WUSCHEL and SHOOT MERISTEMLESS play complementary
roles in maintaining the shoot meristem.
(i) The STM gene is thought to be necessary for stem cells to
proliferate and to prevent premature recruitment of cells into
differentiation pathways.
(ii) The WUS gene is required for initial specification of stem cell
identity and to maintain this pool of stem cells in the central zone of
the meristem.
(iii) Combined expression of both WUS and STM can trigger the
initiation of ectopic meristems and organogenesis even in
differentiated tissues.
Remaining question: what factors determine how meristem arise in
specific locations? How are adaptative responses to external cues
mediated?
Mutations in clavata affect SAM development
WT
clv1-1
clv3-2
Both, the central stem cell zone of the Shoot Apical and Flower
meristems are maintained by the same genes, WUSCHEL (WUS)
SHOOT MERISTEMLESS (STM) and CLAVATA (CLV).
Mutations in clavata affect floral meristem
Mutation of any of the CLV genes results in the accumulation of
undifferentiated cells and fails to promote differentiation on the
flanks.
KNAT gene expression pattern
KNAT 1 (BP) and 2 expression, two other homeodomain
encoding genes, is restricted to the peripheral zone and to the
boundaries of organ primordia.
WT
35S::KNAT1
Arabidopsis, KNOX (STM, KNAT 1 and 2) genes are expressed in shoot
meristems but not in leaf founder cells. Recessive, loss-of-function
mutations in these genes impair the generation of organs from the shoot
apical meristem (SAM), whereas their over-expression leads to the
production of meristems on leaves. In dissected or compound leaves,
KNOX genes are also expressed in leaf primordia, suggesting that they
may play a role in the diversity of leaf form.
Members of the KNOX family interact with other
homeodomain proteins called BELL..There are evidences
that BELL proteins causes KNOX to localize in the nucleus.
This translocation/retention in the nucleus is fundamental to
maintain the SAM.
BELL class proteins PENNYWISE (PNY-REPLUMLESSRPL), POUND-FOOLISH (PNF) and ARABIDOPSIS
THALIANA HOMEOBOX 1 (ATH1), enhance the stm
phenotype
STM-GFP
STM-GFP +
PNY-cmyc
First Break
What kind of proteins encode CLAVATA genes?
CLAVATA1 encodes a receptor kinase protein expressed in the shoot
meristem.
CLAVATA2 encodes a receptor-like protein partner for CLAVATA1.
CLAVATA3 encodes a secreted peptide ligand for the CLV1/CLV2
receptor complex.
WUS and CLV1 ,2, 3 expression pattern
CLV1
Expression pattern in SAM mutants
Overexpression of CLV3 shows a wus phenotype
POLTERGEIST (POL)
pol suppresses clv phenotype and interacts with wus.
Other genes that code for proteins interacting with WUS are
ULTRAPETALA 1 and 2 (ULT 1 and 2) that seems to regulate
negatively WUS
HANABATARANAU (HAN), a GATA transcription factor,
which is expressed on the boundary between CZ and PZ,
appears to maintain a focused pattern of WUS expression. han
clv double mutants show an enlarged meristem bigger in
comparison to either single mutant class. This synergistic
interaction suggests that HAN and CLV influence WUS
expression by distinct pathways.
DORNRÖSCHEN (DRN), encodes an AP2 transcription factor
that is normally expreesed at the tip of SAM. Overexpression
of DRN leads to a shift of WUS and STM expression to deeper
regions and suppress lateral organ development.
ULTRAPETALA 1 and 2 are a negative regulator of stem cell
accumulation
ULT 1 and 2 encode Cys rich proteins expressed at shoot and floral meristems
ult 1 and ult 2 cause the inflorescence meristem to produce more floral meristems
and extra organs form each floral meristem. Cause also a partial loss of floral
meristem determinacy, supernumerary whorls are observed in the center of ult 1
gynoecia.
ult1 clv3 double mutants show bigger meristems than single mutants indicating
that act in separate genetic pathways. These double mutants often develop new
meristem in the center of the flower suggesting that ULT 1 and CLV are involved
in floral meristem determinacy.
wt
ult 1
Gene candidates for action of both CLV and ULT that could size
change and floral determincy are WUS and STM
Double mutants ult stm, are partiallly restored with leaves and
abnormal flowers although the typical spiral phyllotaxy is lost.
ult stm
stm
ult stm
ult wus double mutants are similar to wus single mutants,
indicating that ult mutation does not restore wus vegetative
phenotype but is able to restore some functions to wus
inflorescence meristem. Neither wus single mutants nor ult wus
double mutants are able to form carpels
wus flower
ult wus flower
WUS and ULT play antagonistic roles concerning floral
meristem determinacy, wus mutants lack carpels and terminates
development while ult mutants contain extra whorls with extra
carpels
wt silique
ult silique
Extra carpels
WUS transcription persists in ult mutants longer than in wild
type
wt
ult
wt
ult
This implies that ULT negatively regulates WUS to establish
floral determinacy
WUS and CLV3 expression throughout
embryogenesis
(1) WUS expression is sufficient to induce meristem cell identity and
the expression of the stem cell marker CLV3.
(2) Expression of CLV genes represses meristem maintenance and
WUS activity.
WUSCHEL structure
C-terminal domain
homeodomain
DNA binding domain
EAR like box
Acidic domain
WUS box
Transcriptional
activator
Gene repression
A C-terminal truncated version of WUSCHEL is a
dominant negative allele
·35S::WUS-GR
wus-1
C-terminal WUSCHEL interacts with two
co-repressors
WSIP1
WUS
WSIP1 and 2 contain LisH and WD domains common to repressors,
The complex then could repress genes avoiding differentiation and
thus maintaining meristem characteristics.
CLAVATA-3 is a member of CLE proteins
CLE proteins are small proteins acting in a non-cell
autonomous fashion
Present in plants and Nematodes
SS
Secretory signal
CLE motif
The CLE motif and the SS are sufficient to
complement clv-3 phenotype
clv3
14aa CLV3p
CLV3 Peptide Directly Binds CLV1 Ectodomain
CLV1 ectodomain was
expressed in tobacco BY2 cells and treated with
3H CLV3 peptide
competed with unlabelled
CLV3.
Other Cle proteins could
also compete this binding
FANTASTIC FOUR (FAF) genes are involved in meristem size
Plant specific genes, four in Arabidopsis and a
putative fifth member with homology with genes
present in monocotyledoneous plants.
FAF2 and FAF4 are expressed in the centre of
the meristem
35S::FAF2 plants show
arrest in development,
show a strong repression
of WUS without
alteration of CLV3
expression. However,
FAF2 and 4 expression is
enhanced in clv3 mutants
Dynamic studies using live imaging
35S::WUS-GR x CLV3-GFP ER plants upon
dexamethasone induction together with an
yellow marker of cell division.
CLV3 expression is increasing after DEX
treatment radially through the PZ, however,
even WUS is expressed ubiquitously, CLV3 is
not, increasing with the time radially
converting pluripotent cells into stem cells.
CLV3::LhG4 x 6xOp::WUS-GR in a
background CLV3-GFP-ER and yellow
marker of cell division.
In the progeny, a dramatic increase of
SAM was observed with the lack of
differentiating new organs.
Inducible activation of WUS within the
CZ results in transformation of outer
peripheral zone cells (OPZ) into stem
cells, which induces CLV3, and in this
case induces WUS-GR, which maintains
stem cell fate.
CLV1-CLV3 interactions
A CLV1::CLV1-GFP
accumulates in the plasma
membrane (PM) in the clv1clv3
mutant background but it is
internalized in clv1CLV3
background. These data
indicate that in the absence of
CLV3, CLV1 accumulates at
the PM, whereas it is found in
the vacuole in the presence of
CLV3 protein. This suggests
that CLV3 drives CLV1
endocytosis from the PM
followed by subsequent
targeting to the vacuole.
Internalized CLV1 co-localizes with lytic vacuole markers
pCLV1::CLV1-GFP into the
background clv1CLV3 was stained
with two known markers o f
vacuoles. Merge indicate colocalization of CLV1 in lytic
vacuoles
In the backgroud clv1clv3
background, CLV1-GFP co-localized
with PM markers
CLV1 trafficking mediated by CLV3 is observed only in SAM but not in other tissues
suggesting SAM co-factor should be needed. It is proposed that CLV1 is degraded in these
lytic vacuoles.
Maintenance of the shoot meristem depends on the coordination of
two antagonistic processes, organ initiation and self-renewal of the
stem cell population. WUS gene is required for stem cell identity,
whereas the CLV1, 2, and 3 genes promote organ initiation.
AG
LFY +
WUS
The number of stem cells is
maintained and the meristem is
indeterminate
WUS
Stem cells are inhibited because
WUS is inhibited by AG and the
meristem is determinate
LFY::WUS flowers, 4 whorls of carpels and/or staminoids carpels