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Lateral organs
CUP-SHAPED COTYLEDONS (CUC 1 and 2)
genes
The CUC genes are NAC transcription factors. These genes are
expressed in the SAM at organ boundaries and may delimit
organs from each other and from the SAM. CUC 1 and 2 are
required for apical meristem formation in embryogenesis.
CUC expression pattern
CUC
STM
CUC
STM
CUC genes act upstream activating STM transcription, then CUC
genes are down-regulated in the SAM and specify organ
boundaries. CUC genes possibly act on cellular division
preventing growth at the boundaries
STM and CUC regulation
It was used an ethanol inducible version of STM or STM-VP16 or
STM-GR. After 1 week of treatment, lobuled leaves were
apparent. A microarrays analysis: induction of catabolic giberellin
enzymes and induction of IPT7, for synthesis of cytokinins.
Among genes induced more by STM-VP16 than by STM alone,
were CUC genes.
CUC1 is the direct target of STM
CUC1 is induced by DEX treatment in a STM-GR background even in
the presence of Cycloheximide, confirming that is a direct target. In
contrast, induction of CUC2 and CUC3 is prevented by Cyc treatment,
suggesting that are indirect targets. CUC1 promoter but not those of
CUC2 and CUC3 contains a STM binding site tested by gel shift and
one hybrid assays. In stm mutant, CUC1 is downregulated.
CUC genes are induced by STM
Axillary metistems
Axillary meristems (AM) are formed on the adaxial side of all lateral
organs. These are closely related to floral meristems (FM)
Different plant species contain a variable number of AMs and also differs in their
activity and dormant/activity cycle. LATERAL SUPRESSOR (LAS) is a GRAS
transcription factor involved in AM formation. las Arabidopsis mutants fail to
develop AM but not SAM. Closely homologues were found in tomato and rice. LAS
seems to active STM in leaf primordia.
Leaf development
Emergence of the leaf
primordia
Lateral organ formation is initiated when a small group of
cells in the PZ switch from an indeterminate to a
determinate fate. This commitment to organ fate is
associated with changes in gene expression and new
patterns of cell division and expansion. As organ founder
cells proliferate, new axes of growth are established –
away from the SAM and laterally – resulting in an
outgrowth (organ primordium) from the flanks of the
meristem. Model of inhibitory signalling.
The newly formed leaf becomes an auxin sink causing a local depletion. This
pattern of auxin depletion and accumulation largely account for the phyllotaxy
once it has been established. Phenotype of PIN1.
There are some evidences suggesting that auxin accumulation in organ
primordia activates organ gene markers like CUC or LFY
Organs in plants are patterned by two parallel
systems: one system defines the type of organ
that will develop, while the second system
defines the coordinates of the organ.
While the combination of factors that confer
organ identity tend to be unique for individual
organ types, the patterning system that
defines the coordinates of the organ tends to
be the same for all organs.
The only known marker for leaf initiation appears to be the
downregulation of Class I KNOX gene (KNAT1, and STM1)
expression in the P0 primordium in Arabidopsis
Phenotype of gain-of-function KNOX genes
Arabidopsis
Lettuce
Tomato
All compound leaf species show a reinitiation of transcription of KNOX 1 genes in
leaves creating a “meristem-leaf”. Class I KNOX genes have specific functions in
compound leaf development that are distinct from their ability to induce shoot
meristem formation.
Interactions between KNOX and BELL are essential for
proper SAM formation
KNOX proteins interact with another group of TALE proteins, the BEL1-like
homeodomain family, in a highly connected, complex network that determines not
only high-affinity KNOX target selection but also their subcellular localization. BEL
protein have been shown to drive nuclear localization of KNOX proteins (mainly
STM). This is similar to found in animals, through the MEINOX domain of HD
proteins.
PENNYWISE/BELLRINGER/REPLUMLESS. pny mutations enhance stm
phenotype and lost replum. PNY partially directs nuclear localzation of STM (by
GFP fusion proteins). A second component could be POUND-FOOLISH (PNF)
and or ATHOMEOBOX1 (ATH1). Triple mutants ath1 pny pnf phenocopy stm
mutant.
wt
ath1 pny pnf
Without SAM as stm
ASSYMETRIC LEAVES 1 (as1)
Wild type
as1 rosette
wt and as1 cauline leaves
as1 phenotype is similar to ectopic expression of STM and
KNAT1.
STM and KNAT1 expression in as1 mutants
Wild type
as1
KNAT1 expression
AS1 expression pattern
AS1 encodes for a MYB transcription factor expressed in
cotyledons and in leaf founder cells and young leaves.
stm mutant
AS1 is ectopically expressed in stm mutant (1) but STM is not
misexpressed in as1 mutant. KNAT 1 is ectopically expressed
in as1 mutant (2), as1stm double mutants are able to form stem
cells presumably by the action of KNAT 1 (3) suggesting that:
1) STM negatively regulates AS1, thus preventing differentiation
2) AS1 negatively regulates KNAT1
3) KNAT 1 is able to replace STM in the absence of AS1.
Model?
Expression patterns
CLAVATA
WUSCHEL
STM/KNAT1
AS1, LEAFY
AS1
TERMINAL EAR
KNOX3
CUC 1 and 2
Regulation of KNOX genes
Upstream regulators control the expression of Knotted1-like homeobox (KNOX) genes. KNOX proteins function
as heterodimers with BELL protein co-factors to activate or repress target genes, thus producing a cellular
read-out. The mechanistic basis for KNOX gene regulation is either direct, mediated through chromatin
modifications, or is unknown (figure key). KNOX proteins directly bind to promoters of the biosynthetic gene GA
20-oxidase1 (GA20ox1) and the catabolic gene GA 2-oxidase1 (GA2ox1) to reduce gibberellin (GA) levels, and
the lignin biosynthetic genes caffeic acid-O-methyltransferase1 (COMT1), caffeoyl-CoA O-methyltransferase
(CCoAOMT) and Arabidopsis thaliana peroxidase12a (AtP12a) to reduce lignin levels. KNOX proteins activate
the biosynthetic gene ISOPENTENYL TRANSFEREASE7 (IPT7) to increase cytokinin (CK) levels. KNOX
proteins probably regulate these target genes as KNOX-BELL heterodimers, and this activity is antagonized by
KNOX repression
To determine if AS1 acts as a repressor or an activator, chimaeric constructs
consisting in LFY DNA binding domain and no-MYB motifs of AS1 were introduced
in wild type plants. lfy phenocopies indicate that AS1 acts as a repressor. By ChIP
on a AS1::AS1-HA background, two cis-acting elements on KNAT1 promoter were
identified.
KNOX repression
The two cis-regularory
elements where AS1-AS2
binds were confirmed in a
background KNAT1::GUS
with different promoter
truncations and by gel shift.
KNAT1
In addition KNOX genes are repressed by auxin, consistent with that
an auxin maxima triggers new organ formation
Controversial
Not all the phenotypic features shown by as1as2 mutants are due to ectopic
expression of KNAT 1, 2 and 6, Notably the leaflet and asymmetric leaf lobing
formation should be the effect of as yet unidentified AS1-AS2 regulated gene.
as1-as2-knat1-knat2-knat6
quintuple mutants still
show leaf lobes and leaflet
like structures as well as
adaxialization of leaves.
First break
Adaxial-abaxial axis
(dorsoventral axis)
•adaxial-abaxial asymmetry.
•Dicot leaf primordium is initiated as a
radially symmetric outgrowth that rapidly
acquires adaxial-abaxial asymmetry:
– In tobacco P1 (the youngest visible leaf
primordium) is cylindrical whereas P2 has a
flattened adaxial surface
•adaxial-abaxial polarity in the leaf depends
on the radial axis of the shoot apical
meristem.
Adaxial-abaxial axis
(dorsoventral axis)
Symmetry development in the
leaf
Adaxial-abaxial polarity
•adaxial-abaxial polarity in the leaf depends on the
radial axis of the shoot apical meristem.
•PHANTASTICA (Antirrhinum)
•PINHEAD
•PHABULOSA
•YABBY
PHANTASTICA encodes a MYB-type
transcription factor
•loss-of-function phan mutants in Antirrhinum develop
leaves with variable loss of adaxial-abaxial asymmetry.
The extreme is completely abaxialized round leaves.
•it is expressed in apical meristems at the future sites of
leaf initiation and in leaf primordia up until the P3 stage.
•However, PHAN expression is uniform along the adaxialabaxial axis.
•the Arabidopsis homologous as1 mutants, the leaves
develop with essentially normal polarity.
Wild type
Radially symmetric phan leaf
In phan mutants, the leaf has a rod-like shape with abaxial
characters. Thus, adaxial characters are lost and replaced by
abaxial markers.
HD-ZIP III genes are adaxial genes
The HD-ZIPIII transcription factor family also plays a major
role in polarity. The HD-ZIPIII proteins PHAVOLUTA (PHV),
PHABULOSA (PHB), and REVOLUTA (REV) specify adaxial
cell fate.
Wild type HD-ZIPIII genes are expressed on the adaxial side
All three HD-ZIPIII
genes and two other
homologues share the
adaxial expression
pattern from
emrbyonic cotyledons
and in each lateral
organ
YABBY
genes
•YABBY gene family is required for the development of
abaxial leaf tissue in Arabidopsis:
–
–
–
–
FILAMENTOUS FLOWER (FIL)
JAGGED
YABBY2 (YAB2)
YABBY3 (YAB3)
Enhancer trap
YAB3::GUS showing
abaxial expression
YABBY genes
The YABBY family of abaxially expressed genes, which encode
presumptive transcription factors with high-mobility group and
zinc-finger domains, promote abaxial cell fates in the lateral
organs of Arabidopsis. YABBY genes act, directly or indirectly, to
downregulate meristematic genes during lateral organ
development in wild-type plants in addition to promoting abaxial
cell fate in the lateral organs.
KANADI genes
Four GARP TFs, named the KANADI genes
(KAN1-4), are present in the Arabidopsis genome,
all of which are capable of inducing abaxial cell fate
upon uniform expression KAN1 is expressed in the
abaxial regions of all lateral organs At the heart
stage of embryogenesis, KAN1, KAN2 and KAN3
displayed a similar expression pattern in the abaxial
basal portion of emerging cotyledon primordia
KANADI genes
Only multiple mutants exhibit narrow and partial abaxialized
leaves, however, trichomes are still adaxial, and also outgrowth,
ectopic meristem at the abaxial side
KANADI genes
Triple mutants kan123 supress
expression of YABBY genes
Trple mutants kan123
alter HD ZIP III adaxial
expression, however
there is still a gradient.
That means that KAN genes induce
YABBY genes and repress HD ZIP
genes abaxially most likely indirectly.
KANADI genes
Leaves become
completely radialized
and abaxialized when
KAN genes are
uniformely express.
KANADI and YABBY genes
Compromising most of the activity of
both KANADI and YABBY genes, as in
the kan1-2 kan2-1 fil-5 yab3-1 quadruple
mutants, further reduces the polar nature
of lateral organs, as evidenced by
formation of trichomes on both sides of
the first leaves. Partial adaxialization
How could be the ad/ab pattern first
established?
Further roles of YABBY genes
The fruit-leaf connection
Leaves
AS1 AS2
fruit
class I STM
KNOX KNAT1
genes
JAG
FIL
YAB3
PNY/RPL
CUC1
CUC2
CUC3
Replum and valves appear to partially mimic the
antagonistic relationships between meristem and
leaves.
French flag model.
STM
KNAT1
PNY/RPL
Organ size
ant
wt
Although plant growth is influenced greatly by external
environmental factors, it appears that the intrinsic size of plant
organs is determined by internal developmental factors.
AINTEGUMENTA (ANT) regulates growth and cell number
during organogenesis. ant mutant shows reduced size of lateral
organs throughout the shoot by decreasing cell number. ANT
encodes a transcription factor of the AP2 family
ant
wild type
The systemic reduction in size of ant-1 organs is associated
with a decrease in cell number but not a decrease in cell size.
35S::ANT phenotype
Ectopic ANT expression is sufficient to increase organ size and
mass by enhancing organ growth that is usually coordinated with
organ morphogenesis in Arabidopsis and tobacco plants.
How does ANT control cell number during
organogenesis?
Petal cells
Ant-1
wild type
35S::ANT
In ant mutant, cells are competent to differentiate early than
wild type. In 35S::ANT, there are more division prior to
differentiation (consistent with prolonged Cyclin expression in
mature organs and thus the overall organ size is enlarged.
Leaf curvature
CINCINNATA (CIN) is an Antirrhinum TCP transcription
factor required for making a leaf flat (zero Gaussian
curvature).
cin phenotype
Cell division arrest zones
H4 expression,
a marker of
dividing cells
CIN seems to act differentially regulating the cell cycle or
making cells competent to the arrest of cell cycle. Thus, cin
mutant cells do not arrest properly and overgrow, changing
the leaf shape and curvature.