4Shoot apical meristem - Importance Center of postembryonic growth & developmentSource of all primary meristemsProtoderm, ground meristem & procambiumSource ofLeavesBranchesTendrilsThornsSelf-renewing mass of cells stem cellsBalance cell division and cell differentiation
6Shoot apical meristem organization Stem CellsCentral ZoneOrganizing CenterL1 = tunicaL2 = tunicaCurrent 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 epidermisL2 & L3 forms procambium and ground meristem which form internal tissues the ground and vascular tissues.Peripheral ZoneL3 = corpusPith or Rib Meristem
8Chimeras & Organization of the Shoot Apical Meristem Colchicine induced chimeras in Datura (Jimsonweed).
9Shoot Development Organization of the Meristem Periclinal chimeras at the SAM tell us that SAM layers have distinct lineagesNuclear chimerasGenetic markersL1 marker: Arabidopsis thaliana meristem layer 1 (ATML1)Mericlinal chimeras at the SAM tell usCentral zone stem cells are not permanentA few cells (vs many) populate the central zoneDomains/tiers of cells may populate the SAM
11Shoot Development II: Genetics WUSCHEL gene phenotype Wild TypeWild type SAMWuschel 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 cellsSee: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 SAMwus mutant
121. early termination of SAM wus defective in maintaining SAM Shoot Development II: GeneticsWUSCHEL (WUS) gene Maintains Stem Cell Populationwus mutants result in1. early termination of SAM wus defective in maintaining SAM2. aberrant SAM organization wus defective in maintaining SAM integrityLocalization of WUS gene product in organizing center (OC) of shootL1L2L3Fig 6WUS function:1. WUS Protein product is a homeodomain transcription factor2. Gene regulation3. Positional influence of once cell type by anotherWUS 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 aboveThese observations are consistent with a gene responsible for maintaining the SAM as well as maintaining the organization/integrity of the SAM.See:See:See:
13Shoot Development II: Genetics CLAVATA gene phenotype Normal SAMclavata SAMCLV localization in Central Zone of SAM1. CLV localization in Central Zone of SAM2. Mutant phenotype: Huge apical meristems CLV wild type restricts stem cell accumulationCLAVATA 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:
14Shoot Development II: Genetics CLAVATA gene mechanism Protein-binding motifSignal transduction pathwayCLAVATA GENE CharacteristicsCLV1 – Extracellular polypeptide: 96 amino acidsRestricted to L1, L2 of SAM Central ZoneCLV2 – Membrane-bound protein receptor with a protein-binding motifCLV3 – Membrane-bound protein receptor with a protein-binding motif andKinase activity… signaling… Kinase cascadeInhibitory to WUS expressionSee:See:
15Gene Interaction: WUSCHEL and CLAVATA Initiation of an Organizing Center in the shoot apical meristemCLV3 expressionWUS expression1. 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:
16mRNA Expression Domains and gene interaction for CLV1 CLV3 and WUSwus mutantWild typeOverexpressedWUS mutantclv mutantWild-type mRNA expression domains illustrate location of gene expression.1. WUS – under stem cells of Central Zone2. 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:
17Gene interaction CLAVATA and WUSCHEL in the shoot apical meristem WUS geneWhere? Organizing Center of Central Zone (just a few cells)Function? Molecular: Encodes homeodomain proteinFunction? Molecular Genetic: Induces Expression of CLV3Function? 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 CenterFunction? Molecular: Encodes peptide secreted in extracellular spaceFunction? 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:
18Shoot Meristemless (stm) phenotype Gene Regulation: stm mutation STM wild-type prevents cell differentiation in Peripheral ZoneWild type shoot apexWild type SAMstm mutantShoot Meristemless (stm) phenotype1. SAM terminates prematurely2. rapid depletion of stem cells, faster than they are replenishedArabidopsis shoot apex. Shoot meristemless (stm) mutantSam 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 onlineSee:A “knot” caused by the Knotted gene mutant is an ectopic center of meristematic activity.STM geneWhere? Stem cells of Central Zone and peripheral Zone.Function? Molecular: Molecular:Encodes homeodomain protein – KNOTTED ClassFunction? Molecular Genetic: Transcription factorFunction? Developmental: Prevents premature differentiation of cells from Peripheral Zone.stm mutant SAM
19WUS, CLV and STM expression in the shoot apex WUS geneWhere? Organizing Center of Central Cells (just a few cells)Function? Molecular: Encodes homeodomain proteinFunction? Molecular Genetic: Induces Expression of CLV3Function? Developmental: WUS specifies stem cells of the SAM,i.e. maintains stem cells and maintains their identity.CLV3 geneWhere? Stem cells of Central ZoneFunction? Molecular: Encodes peptide secreted in extracellular spaceFunction? Molecular Genetic: Inhibits WUS expression.Function? Developmental: CLV3 restricts size of Central Cells, i.e. CLV3 restricts size of the stem cell population.STM geneWhere? Through SAM apical “dome” of cells: central zone and peripheral zone.Function? Molecular:Encodes homeodomain proteinFunction? 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 p364Downregulation 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.
20Summary: Shoot Apical Meristem Genetic Influences on Development 1. Organizing Center (OC): Maintains Stem Cellsa. cells expressing WUS gene confers stem cell fate to overlying stem cellsb. cells above OC target stem cells by preferred connections through plasmodesmata2. Stem Cells control boundaries of stem cellsa. Surgical expts. Demonstrate “release” from inhibition of differentiating “daughter” cellsb. CLV3 gene inhibits WUS gene expression3. Daughter cells of stem kept in undifferentiated statea. STM gene keeps daughter cells in an undifferentiated state.b. Daughter cells increase to sufficient numbers before organ formationSurgical destruction of stem cells in SAM results in other cells taking on activity of stem cells.See: