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?

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

Shoot organization Apical Meristem Protoderm Primary Meristems Procambium Ground Meristem

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

Apical Meristem Examples Equisetum Conifers Angiosperms

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. http://www.plantsci.cam.ac.uk/Haseloff/teaching/PDFlists/to1998_PDFs/Mayer98.pdf http://www.plantcell.org/cgi/reprint/16/suppl_1/S190 http://www.biologie.uni-freiburg.de/data/bio3/laux/publications-Dateien/Gross-Hardt.pdf Peripheral Zone L3 = corpus Pith or Rib Meristem

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

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

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

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: http://www.plantcell.org/cgi/content/full/16/suppl_1/S190 Laux, T., et al. Plant Cell 2004;16:S190-S202 Copyright ©2004 American Society of Plant Biologists

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: http://www.plantsci.cam.ac.uk/Haseloff/teaching/PDFlists/to1998_PDFs/Mayer98.pdf See: http://dev.biologists.org/cgi/reprint/122/1/87 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: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=8565856&dopt=Abstract 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

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: http://www.plantsci.cam.ac.uk/Haseloff/teaching/PDFlists/to1998_PDFs/Mayer98.pdf See: http://dev.biologists.org/cgi/reprint/122/1/87 See: http://www.biologie.uni-freiburg.de/data/bio3/laux/publications-Dateien/Gross-Hardt.pdf

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: http://www.biologie.uni-freiburg.de/data/bio3/laux/publications-Dateien/Schoof2000.pdf 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: http://www.nap.edu/openbook/0309084466/html/8.html

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: http://www.nap.edu/openbook/0309084466/html/8.html See: http://www.nap.edu/openbook/0309084466/html/9.html

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: http://www.biologie.uni-freiburg.de/data/bio3/laux/publications-Dateien/Gross-Hardt.pdf

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: http://images.google.com/imgres?imgurl=http://www.plantsci.cam.ac.uk/Haseloff/teaching/PlantSciPart2/2004Part2WebNotes_4_files/image039.gif&imgrefurl=http://www.plantsci.cam.ac.uk/Haseloff/teaching/PlantSciPart2/2004Part2WebNotes_4.htm&h=167&w=223&sz=5&tbnid=zX1e4y6ZMCkJ:&tbnh=76&tbnw=102&hl=en&start=1&prev=/images%3Fq%3Dclavata%2Bgene%26svnum%3D10%26hl%3Den%26lr%3D%26sa%3DG

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: http://www.biologie.uni-freiburg.de/data/bio3/laux/publications-Dateien/Gross-Hardt.pdf See also: http://www.public.iastate.edu/~zool.533/lectures/SamReview.pdf

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: http://biology.kenyon.edu/courses/biol114/Chap12/Chapter_12A.html Source for image of localized STM transcription product: http://dev.biologists.org/cgi/reprint/125/16/3027.pdf full article free online See: http://dev.biologists.org/cgi/reprint/129/13/3207 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

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 16.28. 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.

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: http://www.biologie.uni-freiburg.de/data/bio3/laux/publications-Dateien/Gross-Hardt.pdf

End Shoot Development