1. Homeodomain Proteins

2. The first gene known encoded homeodomain related sequences in plants was KNOTTED1 from Z. mays. (1991), identified by transposon tagging. Plant Homeodomain containing proteins

3. Plant Homeodomain genes encode modular DNA-binding proteins similar to animal homeodomain proteins HD Z Z Z PHD BEL KNOX HD-Zip GLABRA PHD finger BEL ZF-HD WUSCHEL KNOX class I N N N N N N N C C C C C C C Plant homeodomain protein families

4. 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 TALE class

5. Shoot Apical Meristem (SAM) Development

6. 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

7. 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)

8. 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

9. 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.

10. 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.

11. STM expression commences slightly later than WUS, in the globular stage embryo and accumulates in both, the central and peripheral zones. STM expression pattern

12. 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.

13. 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?

14. 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).

15. Mutations in clavata affect floral meristem

16. Mutation of any of the CLV genes results in the accumulation of undifferentiated cells and fails to promote differentiation on the flanks.

17. 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.

18. 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. WT35S::KNAT1

19. 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-REPLUMLESS- RPL), POUND-FOOLISH (PNF) and ARABIDOPSIS THALIANA HOMEOBOX 1 (ATH1), enhance the stm phenotype STM-GFP STM-GFP + PNY-cmyc

20. First Break

21. CLAVATA1 encodes a receptor kinase protein expressed in the shoot meristem. What kind of proteins encode CLAVATA genes? CLAVATA2 encodes a receptor-like protein partner for CLAVATA1. CLAVATA3 encodes a secreted peptide ligand for the CLV1/CLV2 receptor complex.

22. WUS and CLV1,2, 3 expression pattern CLV1

23. Overexpression of CLV3 shows a wus phenotype Expression pattern in SAM mutants

24. POLTERGEIST (POL) pol suppresses clv phenotype and interacts with wus.

25. 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.

26. 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

27. 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. stm ult stm

28. 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

29. 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

30. WUS transcription persists in ult mutants longer than in wild type This implies that ULT negatively regulates WUS to establish floral determinacy wt ult wt ult

31. 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.

32. homeodomain WUSCHEL structure Acidic domain WUS box EAR like box Gene repression Transcriptional activator DNA binding domain C-terminal domain

33. wus-1 ·35S::WUS  -GR A C-terminal truncated version of WUSCHEL is a dominant negative allele

34. C-terminal WUSCHEL interacts with two co-repressors WUS WSIP1 WSIP1 and 2 contain LisH and WD domains common to repressors, The complex then could repress genes avoiding differentiation and thus maintaining meristem characteristics.

35. 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 Secretory signal SSCLE motif

36. clv314aa CLV3p The CLE motif and the SS are sufficient to complement clv-3 phenotype

37. CLV3 Peptide Directly Binds CLV1 Ectodomain CLV1 ectodomain was expressed in tobacco BY- 2 cells and treated with 3 H CLV3 peptide competed with unlabelled CLV3. Other Cle proteins could also compete this binding

38. 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

39. 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.

40. 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.

41. 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.

42. 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 co- localization 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.

43. LFY + WUS AG WUS Stem cells are inhibited because WUS is inhibited by AG and the meristem is determinate The number of stem cells is maintained and the meristem is indeterminate 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.

44. LFY::WUS flowers, 4 whorls of carpels and/or staminoids carpels