1. Lateral organs

2. 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.

3. CUC expression pattern
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

4. 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.

5. 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

6. Axillary metistems
Axillary meristems (AM) are formed on the adaxial side of all lateral organs. These are closely related to floral meristems (FM)

7. 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.

8. Leaf development

9. Emergence of the leaf primordia

10. 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.

11. The newly formed leaf becomes an auxin sink causing a local depletion
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

13. 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.

14. 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

15. 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.

16. 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.
Without SAM as stm wt
ath1 pny pnf

17. ASSYMETRIC LEAVES 1 (as1)
Wild type as1 rosette wt and as1 cauline leaves
as1 phenotype is similar to ectopic expression of STM and KNAT1.

18. STM and KNAT1 expression in as1 mutants
Wild type as1
KNAT1 expression

19. AS1 expression pattern
AS1 encodes for a MYB transcription factor expressed in cotyledons and in leaf founder cells and young leaves.

20. STM negatively regulates AS1, thus preventing differentiation
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:
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?

22. Expression patterns CLAVATA WUSCHEL STM/KNAT1 AS1, LEAFY AS1
TERMINAL EAR
KNOX3
CUC 1 and 2

23. 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 the interaction of mini-KNOX proteins with BELL partners (see key).

24. 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.

25. 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

26. 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.

27. First break

28. 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.

29. Adaxial-abaxial axis (dorsoventral axis)

30. Symmetry development in the leaf

31. Adaxial-abaxial polarity
adaxial-abaxial polarity in the leaf depends on the radial axis of the shoot apical meristem.
PHANTASTICA (Antirrhinum)
PINHEAD
PHABULOSA
YABBY

32. 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 adaxial- abaxial axis.
the Arabidopsis homologous as1 mutants, the leaves develop with essentially normal polarity.

33. 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.

35. 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

36. 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

37. 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.

39. 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

40. 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

41. 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.

42. KANADI genes
Leaves become completely radialized and abaxialized when KAN genes are uniformely express.

43. 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

44. How could be the ad/ab pattern first established?

45. Further roles of YABBY genes

46. The fruit-leaf connection
Leaves
fruit
AS1 AS2
class I KNOX genes
STM KNAT1
STM KNAT1
JAG
FIL
YAB3
PNY/RPL
PNY/RPL
CUC1
CUC2
CUC3
Replum and valves appear to partially mimic the antagonistic relationships between meristem and leaves.
French flag model.

47. 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

48. 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.

49. 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.

50. How does ANT control cell number during organogenesis?
Petal cells
Ant wild type S::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.

51. Leaf curvature
CINCINNATA (CIN) is an Antirrhinum TCP transcription factor required for making a leaf flat (zero Gaussian curvature).

52. cin phenotype

53. 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.