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Leaf has 3 axes:1) proximodistal, 2) centrolateral, 3) ab-adaxial Primordium starts out as a peglike outgrowth that is radially symmetrical Figure 5-1 Figure 1-7 1) Proximodistal axis: •cell divisions end first at the tip, then stop proximally • cell differentiation is complete first at the tip •petiole is distinct from the blade 2) Centrolateral •Midrib is a thickened region surrounding the midvein •Leaf margin is at the leaf edge 1 3) ab-adaxial •Growth rate is different - cells on adaxial side divide more, resulting in the leaf flattening •Epidermal cells are different on ab and adaxial sides (trichomes, stomata, cell size and shape) •Internal cell arrangement is polarized (palisade mesophyll on adaxial, spongy on abaxial, xylem adaxial, phloem abaxial)_ •Incision is made isolating I1 from SAM •Radially symmetric leaf, with abaxialized epidermis, uniform parencyhyma, core of vascular tissue Signal from SAM is necessary to adaxialize tissue Figure 5-2 Bowman et al., 2002 phantastica •Most extreme phenotype is needle-like, radially symmetrical leaves •Adaxial cells are replaced by abaxial Phantastica •PHAN is a MYB-transcription factor •Expressed uniformly throughout the leaf primordia •May respond to putative adaxializing signal from the SAM Bowman et al., 2002 2 •ago and phn (ago1/ago1 pnh/+) mutants unable to maintain a meristem •Mutants in AGO show a reversal of leaf polarity Figure 5-4 Kidner and Martienssen, 2004 •DICER cuts primiRNA and premiRNA in plants to generate a duplex DNA •Duplex DNA is unwound and loaded onto RISC, including AGO •RISC is guided to mRNA •mRNA cleaved Kidner and Martinssen, 2005 •AGO - PAZ and PIWI domains - common in eukaryotes •Required for RNA interference (part of RNA interference complex) that targets mRNA for degradation •PNH (also PAZ and PIWI domains - overlaps AGO function) expressed in leaves - first throughout, then in adaxial •Coordination between regions of cell division and cell differentiation Wild type + phb phb phb McConnell and Barton, 1998 phabulosa (dominant) plants are severely adaxialized 3 Ectopic buds form on abaxial side of phb leaves Wild type Ad ab phb phb adaxialized phb phb adaxialized phb + Seedling Wild type McConnell and Barton, 1998 phb + Radially symmetrical McConnell and Barton, 1998 Adaxial cell fate promotes axillary bud development Kidner and Martienssen, 2004 •PHB encodes homeodomain, leucine zipper (HD-ZIPIII) containing protein •Loss of function mutations have no phenotype (redundant with PHAVOLUTA (PHV) and REVOLUTA (REV) - triple mutants are abaxialized) •Also has a sterol/lipid-binding domain •PHB is expressed throughout leaf •May be activated by sterol/lipid ligand only in adaxial region (ligand identity unknown) - could be a ligand secreted by SAM •Dominant mutations result in constitutive activation •In addition, PHB undergoes miRNA induced degradation on abaxial side, dominant mutant alleles inhibit miRNA induced degradation •miRNA specific to PHB, PHV and REV is localized initially in meristem, then on leaf abaxial side - signal from meristem? •PHB mutations disrupt the miRNA binding site - no degradation on abaxial side •Alleles of AGO that affect the PIWI domain look like PHB mutants •In ago mutants, PHB is ectopically expressed •ago mutants enhance the phb phenotype •In ago mutants, miRNA is ectopically expressed, possibly because it never gets into degradation pathway 4 KANADI loss-of-function morphological phenotypes KANADI genes (family of 3 transcription factors) are redundant •Double and triple mutants have only adaxial tissue trichomes, ectopic axillary meristems •Genes are normally expressed on abaxial side of organs (e.g. cotyledons) Figure 5-4 •KANADI genes necessary for YABBY expression(6 gene family = transcription factors, includes FILAMENTOUS FLOWER (FIL)) •triple KAN mutants do not express YAB •YABBY gene expression is on abaxial side Eshed, Y. et al. Development 2004;131:2997-3006 •If express KAN throughout leaf via AS1 promoter, leaves are abaxialized •Expression of YAB3 throughout leaf also results in abaxialization, suggesting ecotpic KAN phentoype is the result of ectopic YAB •Expression of YAB and KAN downregulate PHB and PHV •Activated PHB downregulates YAB and KAN +miRNA Bowman et al., 2002 Bowman et al., 2002 5 Figure 5.5 •lam1 tobacco mutants are normal until P2 •In lamina, adaxial cell types are replaced by abaxial •Blade does not grow •In lam1 L1 chimera, adaxial epidermis develops normally, suggesting LAM1 L2 signals to mutant L1, telling it to be adaxial •In lam1 L1/L2 chimera, palisade develops normally, but epidermis does not normal L3 enough to inform interior L2, but not L1, subepidermal L2 Laminar outgrowth requires juxtaposition of ab- and adaxial cell types •Leaves that are either adaxialized or abaxialized are needle-like (no blade outgrowth Figure 5-6 Phan mutants are temperature sensitive: •High temp - radially symmetrical leaves •Low temp - normal leaves •Intermediate leaves, but adaxial surface has patches of abaxial cells within it •Where patch of abaxial meets adaxial, laminar outgrowth occurs •Similar phenomenon occurs in leafbladeless mutant of maize D/V compartment specification in wing imaginal disk Adaxial cell fate ad/ab compartment specification in leaf primordium Margin cells form between leaf and wing dorsal and ventral compartments 6 Sterol? miRNA? PHB KAN, YAB •in phantastica mutants, ectopic abaxial cells in adaxial compartment causes ectopic margin formation and laminar outgrowth •Signal from SAM to leaf •Sterol ligand to activate PHB, PHV and REV in adaxial domain? •miRNA may move from SAM to abaxial domain, where it degrades PHB, PHV and REV mRNA •At 15C, phan mutant SAM arrests •Ectopic expression of YAB or FIL can arrest SAM •Suggest that adaxial cell fate is required to maintain meristem Proximal/distal axis - the role of KNOTTED Figure 5-8 •In maize leaf, sheaf is proximal, blade is distal •Ligule and auricles mark boundary •Dominant kn1 mutations cause mesophyll cells along the lateral veins to continue division resulting in knots •Cells between and over knots develop into sheath •KN1 - homeodomain protein •mRNA can go through plasmodesmata •Wild type expressed in SAM •Mutant ectopically expressed in lateral veins Figure 5-9 Models for leaf P/D patterning: a) Morphogen gradient, produced from SAM is high proximally and low distally; KN1 is required for morphogen activity, ectopic KN1 causes ectopic morphogen and proximal fate b) distal cells are recruited first into the leaf primordium, proximal cells last; cells can measure time in leaf - older cells = distal, younger = proximal; KN1 (normally in SAM) resets timing 7 Determinate vs. indeterminate development Figure 5-10 •Lack of KN1 results in no SAM •Ectopic KN1 in leaves results in meristem activity in leaves (knots in maize, shoots in tobacco) •PHAN and its homologues RS2 (maize) and AS1 (Arabidopsis) act to repress KN1 in the leaf (determinate) Figure 5-10 Compound leaves: •In tomato, KN1 is expressed not only in SAM, but also in developing leaves •Constitutive expression of KN1 results in super-compound leaves •Compound leaves in pea develop acropetally - requires the pea homologue of LEAFY, UNIFOLIATA (UNI) Figure 5-10 Figure 5-11 •Chimeras with albino L2 sectors •In b), albino sector is also slowly dividing •In this case, the L2 contribution to the edge is much less •there must be competition between cells •Leaf achieves normal size •If cell size is altered (e.g. ABP1), cell division alters to compensate and vice versa 8