1. Shoot Development A. Function and organization of the apical meristem
B. Origin of primary meristems
C. Gene regulation at the meristem
D. Leaf Initiation
E. Secondary Growth in stems?

2. Shoot organization - Phytomeres
modular unit
of the Shoot
Stem activity is an iterative process.
Modular units = phytomers

3. Shoot organization Apical Meristem Protoderm Primary Meristems
Procambium
Ground Meristem

4. Shoot apical meristem - Importance
Center of postembryonic growth & development
Source of all primary meristems
Protoderm, ground meristem & procambium
Source of
Leaves
Branches
Tendrils
Thorns
Self-renewing mass of cells  stem cells
Balance cell division and cell differentiation

5. Apical Meristem
Examples
Equisetum
Conifers
Angiosperms

6. Shoot apical meristem organization
Stem Cells
Central Zone
Organizing Center
L1 = tunica
L2 = tunica
Current understanding is that the central zone cells are a reservoir of meristematic cells that perpetuate themselves and contribute to formation of other SAM cells.
Peripheral zone: Rapidly dividing cells responsible for primary meristem and organ formation.
Each layer L1, L2 & L3 contains 1-3 stems cells (center of image) which contribute to formation of all cells in each layer.
L1  forms protoderm which forms epidermis
L2 & L3  forms procambium and ground meristem which form internal tissues  the ground and vascular tissues.
Peripheral Zone
L3 = corpus
Pith or Rib Meristem

7. See: http://www.public.iastate.edu/~bot.512/papers/Bowman.pdf

8. Chimeras & Organization of the Shoot Apical Meristem
Colchicine induced chimeras in Datura (Jimsonweed).

9. Shoot Development Organization of the Meristem
Periclinal chimeras at the SAM tell us that SAM layers have distinct lineages
Nuclear chimeras
Genetic markers
L1 marker: Arabidopsis thaliana meristem layer 1 (ATML1)
Mericlinal chimeras at the SAM tell us
Central zone stem cells are not permanent
A few cells (vs many) populate the central zone
Domains/tiers of cells may populate the SAM

10. Gene Expression in the Apical Embryo Domain
WUSCHEL (WUS), CLAVATA (CLV) AND SHOOT MERISTEMLESS (STM)
Figure 2. Development of the Apical Embryo Domain. The top row shows schemes of longitudinal median sections. The upper and lower thick lines represent clonal boundaries between the descendants of the apical and basal daughter cells of the zygote and between the apical and central embryo domains, respectively. The bottom row shows cross-sections of the same stages as indicated by the dashed line at left. CZ, central zone; PZ, peripheral zone; RZ, rib zone. The expression domains of early genes in the apical region are shown in color as indicated. See text for details.
Source:
Laux, T., et al. Plant Cell 2004;16:S190-S202
Copyright ©2004 American Society of Plant Biologists

11. Shoot Development II: Genetics WUSCHEL gene phenotype
Wild Type
Wild type SAM
Wuschel means “bushy looking” plant. Wuschel mutants have a bushy appearance due to a start and stop growth pattern of the shoot apical meristem. The apical meristem is initiated in shoot tips of wuschel mutants but it fails to develop into an organized SAM.
WUS gene mutations result in premature termination of vegetative meristems.
WUS expressing cells are required to identify overlying neighbors as stem cells
See:
See:
Older notes:
WUSCHEL gene is required to maintain the identity of shoot meristem cells.
Wus floral meristems terminate prematurely in a central stamen.
Wus stem cells appear to undergo differentiation.
WUS gene is expressed in inner portion of the central zone of the meristem.
Self perpetuation of the shoot meristem is essential for the repetitive initiation of shoot structures during plant development. In Arabidopsis shoot meristem maintenance is disrupted by recessive mutations in the WUSCHEL (WUS) gene. The defect is evident at all developmental stages and is restricted to shoot and floral meristems, whereas the root meristem is not affected. Results suggest that the WUS gene is specifically required for central meristem identity of shoot and floral meristems to maintain their structural and functional integrity. Source:
WUSCHEL is essential to properly organize a shoot meristem in the embryo. Postembryonically, wus mutants produce defective shoot meristems initiated repetitively, but terminate prematurely. Primordia initiate ectopically across the flat mutant apices and often new shoot meristems instead of organs are initiated.
Wus phenotype is plants that terminate growth after producing a few leaves. Flat thin meristems. Interpreted as a depletion of proliferative cells at the SAM.
Secondary meristems form resulting in hundreds of rosette leaves. STOP AND GO pattern of growth.
WUS normal gene restrains cell differentiation, and thus maintains pool of proliferative cells.
The wus mutant removes restraint of cell proliferation. This causes SAM meristem cells to be recruited before they can replenish themselves, leaving the SAM as a flat mass of cells. Wus mutant phenotype is a flat SAM lacking SAM organization… because the proliferative cells of L1, L2 and L3 have been used up for organ formation. In other words, the pace of organ formation is greater than the pace of SAM meristematic cell replacement in the L1, L2 and L3. WUSCHEL gene is required for meristem integrity in Arabidopsis. WUSCHEL gene (wild type or normal) restrains differentiation at the shoot apex. This allows SAM cell formation to keep pace with differentiation.
Wus mutant phenotype: Flat apical meristems, with periodic resumption of meristematic activity. Whole plant consists of bunches of leaves with floral stems with bunches of leaves with floral stems, etc. SAM integrity is lost with the wus mutant gene because L1, L2 and L3 cannot produce cells fast enough to keep up with the pace of organ formation.
wus SAM
wus mutant

12. 1. early termination of SAM  wus defective in maintaining SAM
Shoot Development II: Genetics
WUSCHEL (WUS) gene Maintains Stem Cell Population
wus mutants result in
1. early termination of SAM
 wus defective in maintaining SAM
2. aberrant SAM organization
 wus defective in maintaining SAM integrity
Localization of WUS gene product in organizing center (OC) of shoot L1 L2 L3
Fig 6
WUS function:
1. WUS Protein product is a homeodomain transcription factor
2. Gene regulation
3. Positional influence of once cell type by another
WUS gene mutations result in premature termination of vegetative meristems.
WUS expressing cells are required to identify overlying neighbors as stem cells (arrow) in the Figure 6 above
These observations are consistent with a gene responsible for maintaining the SAM as well as maintaining the organization/integrity of the SAM.
See:
See:
See:

13. Shoot Development II: Genetics CLAVATA gene phenotype
Normal SAM
clavata SAM
CLV localization in Central Zone of SAM
1. CLV localization in Central Zone of SAM
2. Mutant phenotype: Huge apical meristems
 CLV wild type restricts stem cell accumulation
CLAVATA genes promote progression of meristem cells toward organ initiation.
CLAVATA genes play a role in limiting size of the SAM.
Overexpression of CLAVATA-3 causes depletion of the vegetative and floral shoot meristems.
NOTE: Ectopic expression of Clavata causes proliferation of meristematic cells in the apex.
Mutant clavata genes result in massive meristems with undifferentiated cells.
Source for a great paper online:
Mutation in clavata leads to delayed organ initiation, leading to an increase in meristem cells and to an increase in the size of the shoot apical meristem (SAM).
See:

14. Shoot Development II: Genetics CLAVATA gene mechanism
Protein-binding motif
Signal transduction pathway
CLAVATA GENE Characteristics
CLV1 – Extracellular polypeptide: 96 amino acids
Restricted to L1, L2 of SAM Central Zone
CLV2 – Membrane-bound protein receptor with a protein-binding motif
CLV3 – Membrane-bound protein receptor with a protein-binding motif and
Kinase activity… signaling… Kinase cascade
Inhibitory to WUS expression
See:
See:

15. Gene Interaction: WUSCHEL and CLAVATA Initiation of an Organizing Center in the shoot apical meristem
CLV3 expression
WUS expression
1. OC precursor lineage established in 4 subepidermal cells of 16 cell proembryo as indicated by expression of WUS (red)
2. Stem cells of Central Zone induced by heart stage as indicated by expression of CLV3 gene (blue)
See:

16. mRNA Expression Domains and gene interaction
for CLV1 CLV3 and WUS
wus mutant
Wild type
Overexpressed
WUS mutant
clv mutant
Wild-type mRNA expression domains illustrate location of gene expression.
1. WUS – under stem cells of Central Zone
2. CLV3 – stem cells of Central Zone above OC (produces extracellular protein)
3. CLV1 – Organizing Center (OC) & vicinity (produces membrane-bound protein)
A feedback look between WUS and CLV exists because:
1. Expansion of the WUS expression domain in a clv mutant suggests a feedback loop between the two genes.
2. wus mutants have downregulated (smaller) CLV3 expression domain.
3. WUS overexpressed mutants have a broader CLV3 expression domain.
See:

17. Gene interaction CLAVATA and WUSCHEL in the shoot apical meristem
WUS gene
Where? Organizing Center of Central Zone (just a few cells)
Function? Molecular: Encodes homeodomain protein
Function? Molecular Genetic: Induces Expression of CLV3
Function? Developmental: WUS specifies stem cells of the SAM,
i.e. maintains stem cells and maintains stem cell identity.
CLV3 gene:
Where? Stem cells above Organizing Center
Function? Molecular: Encodes peptide secreted in extracellular space
Function? Molecular Genetic: Inhibits WUS expression.
Function? Developmental: CLV3 restricts size of Central Zone, i.e. CLV3 restricts size of the stem cell population.
See:
See also:

18. Shoot Meristemless (stm) phenotype
Gene Regulation: stm mutation STM wild-type prevents cell differentiation in Peripheral Zone
Wild type shoot apex
Wild type SAM
stm mutant
Shoot Meristemless (stm) phenotype
1. SAM terminates prematurely
2. rapid depletion of stem cells, faster than they are replenished
Arabidopsis shoot apex. Shoot meristemless (stm) mutant
Sam mutant lacks a shoot apical meristem because proliferative cells fail to proliferate. With a finite population of cells, the SAM fails to produce leaves and the shoot in general.
Seedlings normal and wus mutants:source:
Source for image of localized STM transcription product: full article free online
See:
A “knot” caused by the Knotted gene mutant is an ectopic center of meristematic activity.
STM gene
Where? Stem cells of Central Zone and peripheral Zone.
Function? Molecular: Molecular:Encodes homeodomain protein – KNOTTED Class
Function? Molecular Genetic: Transcription factor
Function? Developmental: Prevents premature differentiation of cells from Peripheral Zone.
stm mutant SAM

19. WUS, CLV and STM expression in the shoot apex
WUS gene
Where? Organizing Center of Central Cells (just a few cells)
Function? Molecular: Encodes homeodomain protein
Function? Molecular Genetic: Induces Expression of CLV3
Function? Developmental: WUS specifies stem cells of the SAM,
i.e. maintains stem cells and maintains their identity.
CLV3 gene
Where? Stem cells of Central Zone
Function? Molecular: Encodes peptide secreted in extracellular space
Function? Molecular Genetic: Inhibits WUS expression.
Function? Developmental: CLV3 restricts size of Central Cells, i.e. CLV3 restricts size of the stem cell population.
STM gene
Where? Through SAM apical “dome” of cells: central zone and peripheral zone.
Function? Molecular:Encodes homeodomain protein
Function? Molecular Genetic: Blocks organ formation genes (AS1, AS2)
Function? Developmental: Prevents premature differentiation of cells from Peripheral Zone… thus prevents premature organ initiation.
Source: Taiz & Zeiger 3rd Fig p364
Downregulation of CLV3 expression is an early indicator of cell differentiation.
Downregulation of STM expression in cells already determined (i.e. “anlagen”)is followed by organ development.
Of German origin the term anlagen refers to any group of relatively undifferentiated cells that is destined to become a particular group of differentiated cells at a later time in development.

20. Summary: Shoot Apical Meristem Genetic Influences on Development
1. Organizing Center (OC): Maintains Stem Cells
a. cells expressing WUS gene confers stem cell fate to overlying stem cells
b. cells above OC target stem cells by preferred connections through plasmodesmata
2. Stem Cells control boundaries of stem cells
a. Surgical expts. Demonstrate “release” from inhibition of differentiating “daughter” cells
b. CLV3 gene inhibits WUS gene expression
3. Daughter cells of stem kept in undifferentiated state
a. STM gene keeps daughter cells in an undifferentiated state.
b. Daughter cells increase to sufficient numbers before organ formation
Surgical destruction of stem cells in SAM results in other cells taking on activity of stem cells.
See:

21. End Shoot Development