The Role of Tissue Cell Polarity in Monocot Development Annis Richardson Thesis submitted for the Degree of Doctor of Philosophy University of East Anglia John Innes Centre September 2015 © This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that use of any information derived there from must be in accordance with current UK Copyright Law. In addition, any quotation or extract must include full attribution. Abstract Nature exhibits huge diversity in organ shape, and yet all organs start as small bud-like peripheral outgrowths. Combinations of different spatial and temporal developmental switches in shape determine final organ shape. In plants shape arises through growth, which is defined by axiality and growth rates. Here I tested three hypotheses for how developmental switches in shape could arise: (1) growth rates alone are altered, (2) axiality alone is altered (3) both growth rates and axiality are altered. Using a multidisciplinary approach I explored which of the hypotheses was true for developmental switches in shape during organ development in two monocot models: early grass leaf development and the Hooded barley mutant. Developmental switches in shape were first volumetrically described using 3D imaging. Using this framework, computational models were generated to formulate hypotheses which could account for the switches in shape. Model predictions were then tested using whole-mount immunolocalisation of SISTER OF PINFORMED 1 (SoPIN1), gene expression, and cell division and shape analyses. Synthetic biology was also used to generate a set of transgenic tools for future testing of the models. I found that a developmental switch in shape during early grass leaf development may arise through alterations in growth rates alone (hypothesis 1). In contrast, ectopic flower and wing formation in Hooded may arise through modulation of growth rates and axiality combined (hypothesis 2). In this case a single gene, BKn3, triggers the growth change, possibly through directly influencing tissue cell polarity (if axiality is defined by a polarity based axiality system), with differential effects on shape depending on where it is expressed. This suggests that novel developmental switches in shape could evolve due to single gene mutations, and that during evolution, modulation of growth may have been redeployed in different spatial and temporal patterns to trigger novel changes in shape, ultimately changing final form. 2 Contents Abstract ......................................................................................................................... 2 Contents ........................................................................................................................ 3 List of Tables .................................................................................................................. 8 List of Figures ................................................................................................................. 9 Acknowledgements ...................................................................................................... 13 Introduction ................................................................................................................. 15 1.1 Developmental switches ...................................................................... 15 1.2 Growth in plant tissues ........................................................................ 16 1.3 Growth and developmental switches in shape ...................................... 18 1.4 Axiality systems ................................................................................... 19 1.4.1 Polarity based axiality system ......................................................... 19 1.4.2 Stress based axiality system ............................................................ 23 1.4.3 Markers of axiality: PINs and hairs ................................................. 24 1.5 The contribution of different tissues within the organ ........................... 25 1.6 Computational models at different scales ............................................. 26 1.7 Exploring dicot and monocot development ........................................... 27 1.8 This work ............................................................................................. 31 2 Grass Leaf Development ................................................................................... 32 2.1 Leaf development in the grasses ........................................................... 32 2.2 Aim of this project ................................................................................ 37 2.3 Describing a developmental switch in shape during primordial grass leaf development ................................................................................................... 37 2.4 The formation of the hood from a ring primordium could be accounted for by enhanced anisotropic growth towards the midvein ................................. 50 2.5 Axial information in the early grass leaf primordium ............................. 55 2.6 A change in axial information and/ or growth rate pattern may be required for the next shape transition from a hood to a cone ............................ 60 2.7 Axial information is oriented towards the midvein tip after the hood stage consistent with both model predictions ................................................... 66 2.8 Exploring growth rate patterns across the grass leaf primordium .......... 69 2.9 Discussion ............................................................................................ 77 2.9.1 Characterising developmental switches in shape during primordial stages of grass leaf development ................................................................ 77 2.9.2 Modelling the primordial stages of grass leaf development .......... 77 3 2.9.3 Predicted changes in growth during grass leaf development ......... 78 2.9.4 The role of growth rate patterns in grass leaf development .......... 79 2.9.5 The role of axial information in grass leaf development ................ 80 2.9.6 Insights into the evolution of the grass leaf .................................... 81 2.9.7 Future work and concluding remarks .............................................. 82 3 How Can a Single Gene Induce a Developmental Switch in Shape? The Hooded Barley Mutant .............................................................................................................. 85 3.1 Barley floral development and the Hooded mutant ............................... 85 3.1.1 Morphology of wild-type barley ...................................................... 85 3.1.2 Morphology of the Hooded barley mutant ..................................... 87 3.1.3 Previous studies in the Hooded mutant. ......................................... 90 3.2 Aim of this project ............................................................................... 92 3.3 Characterising a developmental switch in shape in the barley flower: Staging ectopic flower development ................................................................. 92 3.4 Ectopic expression of BKn3 in the Hooded lemma precedes the formation of the ectopic meristem ................................................................................. 104 3.5 The ectopic expression of BKn3 in the Hooded lemma induces a reorientation in axial information at the cellular level ..................................... 110 3.6 The ectopic expression of BKn3 in the Hooded lemma induces changes in the expression pattern of candidate polarity organisers .................................. 123 3.7 Specific changes in growth in the lemma margin trigger the developmental switch in shape responsible for wing formation ...................... 136 3.7.1 Characterising a possible second developmental switch in shape in the Hooded mutant .................................................................................... 137 3.7.2 Modelling Hooded lemma wing development as a consequence of changes in growth ...................................................................................... 140 3.7.3 Axial information may specifically reorient at the margin of the Hooded lemma ........................................................................................... 144 3.7.4 BKn3 may act cell autonomously in the margins to form the wings in the Hooded mutant ................................................................................ 148 3.8 Discussion .......................................................................................... 151 3.8.1 Characterising the morphology of the wild-type and Hooded spike during development ................................................................................... 151 3.8.2 There may be two independent developmental switches in shape in the Hooded mutant .................................................................................... 152 3.8.3 Single genes are able to trigger developmental switches in shape through modulating growth rates and axiality .......................................... 153 4 3.8.4 BKn3 may influence axial information through modulating the expression of organiser components ......................................................... 154 3.8.5 Future work and concluding remarks ............................................ 156 4 Developing a Transgenic Toolkit in Barley ......................................................