title: LEAFPROCESSOR: a new leaf phenotyping tool using contour bending energy and shape cluster analysis.
abstract: *Significant progress has been made in the identification of the genetic factors controlling leaf shape. However, no integrated solution for the quantification and categorization of leaf form has been developed. In particular, the analysis of local changes in margin growth, which define many of the differences in shape, remains problematical. *Here, we report on a software package (LEAFPROCESSOR) which provides a semi-automatic and landmark-free method for the analysis of a range of leaf-shape parameters, combining both single metrics and principal component analysis. In particular, we explore the use of bending energy as a tool for the analysis of global and local leaf perimeter deformation. *As a test case for the implementation of the LEAFPROCESSOR program, we show that this integrated analysis leads to deeper insights into the morphogenic changes underpinning a series of previously identified Arabidopsis leaf-shape mutants. Our analysis reveals that many of these mutants which, at first sight, show similar leaf morphology, can be distinguished via our shape analysis. *The LEAFPROCESSOR program provides a novel integrated tool for the analysis of leaf shape.
authors.affiliation: Department of Animal and Plant Sciences, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
authors.forename: Andreas
authors.initials: A
authors.lastname: Backhaus
authors.affiliation: Department of Animal and Plant Sciences, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
authors.forename: Asuka
authors.initials: A
authors.lastname: Kuwabara
authors.affiliation: Department of Animal and Plant Sciences, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
authors.forename: Marion
authors.initials: M
authors.lastname: Bauch
authors.affiliation: School of Mathematical Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK.
authors.forename: Nick
authors.initials: N
authors.lastname: Monk
authors.affiliation: Department of Computer Science, University of Sheffield, Regent Court, 211 Portobello, Sheffield S1 4DP, UK.|Department of Chemical and Process Engineering, University of Sheffield, Mappin Street, Sheffield S1 3JD, UK.
authors.forename: Guido
authors.initials: G
authors.lastname: Sanguinetti
authors.affiliation: Department of Animal and Plant Sciences, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
authors.forename: Andrew
authors.initials: A
authors.lastname: Fleming
mesh_terms: D017360:Arabidopsis / Q000033:anatomy & histology* / Q000502:physiology*; D016000:Cluster Analysis; D016002:Discriminant Analysis; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D009154:Mutation / Q000235:genetics; D009929:Organ Size; D010641:Phenotype; D018515:Plant Leaves / Q000033:anatomy & histology* / Q000502:physiology*; D025341:Principal Component Analysis; D012984:Software; D013816:Thermodynamics
references.citation: Anastasiou E, Kenz S, Gerstung M, MacLean D, Timmer J, Fleck C, Lenhard M. 2007. Control of plant organ size by KLUH/CYP78A5-dependent intercellular signaling. Developmental Cell 13: 843-856.
references.citation: Bartlett M. 2001. Face image analysis by unsupervised learning. Boston, MA, USA: Kluwer Academic Publishers.
references.citation: Bensmihen S, Hanna AI, Langlade NB, Micol JL, Bangham A, Coen ES. 2008. Mutational spaces for leaf shape and size. HFSP Journal 2: 110-120.
references.citation: Blake A, Isard M. 1998. Active contours. London, UK: Springer-Verlag.
references.citation: Blein T, Pulido A, Vialette-Guiraud A, Nikovics K, Morin H, Hay A, Johansen IE, Tsiantis M, Laufs P. 2008. A conserved molecular framework for compound leaf development. Science 322: 1835-1839.
references.citation: Bowie JE, Young IT. 1977. An analysis technique for biological shape-II. Acta Cytologica 21: 455-464.
references.citation: Bylesjö M, Segura V, Soolanayakanahally RY, Rae AM, Trygg J, Gustafsson P. 2008. Lamina: a tool for rapid quantification of leaf size and shape parameters. BMC Plant Biology 8: 82.
references.citation: Canny J. 1986. A computational approach to edge detection. IEEE Transactions on Pattern Analysis and Machine Intelligence 8: 679-714.
references.citation: Castleman K. 1996. Digital image processing. Englewood Cliffs, NJ, USA: Prentice-Hall, Inc.
references.citation: Cesar RM, Fontoura Costa LDA. 1997. Application and assessment of multiscale bending energy for morphometric characterization of neural cells. Review of Scientific Instruments 68: 2177-2186.
references.citation: Davies DL, Bouldin DW. 1979. A cluster separation measure. IEEE Transactions on Pattern Analysis and Machine Intelligence 1: 224-227.
references.citation: Hay A, Tsiantis M. 2006. The genetic basis for differences in leaf form between Arabidopsis thaliana and its wild relative Cardamine hirsuta. Nature Genetics 38: 942-947.
references.citation: Huff PM, Wilf P, Azumar EJ. 2003. Digital future for paleoclimate estimation from fossil leaves? Preliminary results Palaios 18: 266-274.
references.citation: Jähne B. 2002. Digital image processing. Berlin, Germany: Springer-Verlag.
references.citation: Kass M, Witkin A, Terzopoulos D. 1987. Snakes - active contour models. International Journal of Computer Vision 1: 321-331.
references.citation: Kim GT, Shoda K, Tsuge T, Cho KH, Uchimiya H, Yokoyama R, Nishitani K, Tsukaya H. 2002. The ANGUSTIFOLIA gene of Arabidopsis, a plant CtBP gene, regulates leaf-cell expansion, the arrangement of cortical microtubules in leaf cells and expression of a gene involved in cell-wall formation. EMBO Journal 21: 1267-1279.
references.citation: Kim GT, Tsukaya H, Uchimiya H. 1998. The ROTUNDIFOLIA3 gene of Arabidopsis thaliana encodes a new member of the cytochrome P-450 family that is required for the regulated polar elongation of leaf cells. Genes and Development 12: 2381-2391.
references.citation: Langlade N, Feng X, Dransfield T, Copsey L, Hanna A, Thebaud C, Bangham A, Hudson A, Coen E. 2005. Evolution through genetically controlled allometry space. Proceedings of the National Academy of Sciences, USA, 102: 10221-10226.
references.citation: MacQueen JB. 1967. Some methods for classification and analysis of multivariate observations. In: LML, Neyman, J, eds. Proceedings of the fifth Berkeley Symposium on Mathematical Statistics and Probability. Berkeley, USA: University of California Press, 1: 281-297.
references.citation: McLellan T, Endler JA. 1998. The relative success of some methods for measuring and describing the shape of complex objects. Systematic Biology 47: 264-281.
references.citation: Nelissen H, Fleury D, Bruno L, Robles P, Veylder LD, Traas J, Micol JL, Van Montagu M, Inze D, Van Lijsebettens M. 2005. The elongata mutants identify a functional elongator complex in plants with a role in cell proliferation during organ growth. Proceedings of the National Academy of Sciences, USA 102: 7754-7759.
references.citation: Ohno CK, Reddy GV, Heisler MGB, Meyerowitz EM. 2004. The Arabidopsis jagged gene encodes a zinc finger protein that promotes leaf tissue development. Development 131: 1111-1122.
references.citation: Osborne CP, Beerling DJ, Lomax BH, Chaloner WG. 2004. Biophysical constraints on the origin of leaves inferred from the fossil record. Proceedings of the National Academy of Sciences, USA 101: 10360-10362.
references.citation: Prigge MJ, Wagner DR. 2001. The Arabidopsis serrate gene encodes a zinc-finger protein required for normal shoot development. Plant Cell 13: 1263-1279.
references.citation: Reinhardt B, Hanggi E, Muller S, Bauch M, Wyrzykowska J, Kerstetter R, Poethig S, Fleming AJ. 2007. Restoration of DWF4 expression to the leaf margin of a dwf4 mutant is sufficient to restore leaf shape but not size: the role of the margin in leaf development. Plant Journal 52: 1094-1104.
references.citation: Royer DL, McElwain JC, Adams JM, Wilf P. 2008. Sensitivity of leaf size and shape to climate within Acer rubrum and Quercus kelloggii. New Phytologist 179: 808-817.
references.citation: Sanguinetti G. 2008. Dimensionality reduction of clustered data set. IEEE Transactions on Pattern Analysis and Machine Intelligence 30: 535-540.
references.citation: Sato M, Tsutsumi M, Ohtsubo A, Nishii K, Kuwabara A, Nagata T. 2008. Temperature-dependent changes of cell shape during heterophyllous leaf formation in Ludwigia arcuata (Onagraceae). Planta 228: 27-36.
references.citation: Savaldi-Goldstein S, Peto C, Chory J. 2007. The epidermis both drives and restricts plant shoot growth. Nature 446: 199-202.
references.citation: Scarpella E, Marcos D, Friml J, Berleth T. 2006. Control of leaf vascular patterning by polar auxin transport. Genes and Development 20: 1015-1027.
references.citation: Slice DE. 2007. Geometric morphometrics. Annual Review of Anthropology 36: 261-281.
references.citation: Tsuge T, Tsukaya H, Uchimiya H. 1996. Two independent and polarized processes of cell elongation regulate leaf blade expansion in Arabidopsis thaliana (L.) Heynh. Development 122: 1589-1600.
references.citation: Tsukaya H. 2006. Mechanism of leaf-shape determination. Annual Review of Plant Biology 57: 477-496.
references.citation: Ullah H, Chen JG, Young JC, Im KH, Sussman MR, Jones AM. 2001. Modulation of cell proliferation by heterotrimeric G protein in Arabidopsis. Science 292: 2066-2069.
references.citation: van Vliet L, Verbeek P. 1993. Curvature and bending energy in digitized 2D and 3D images. In: Hogda KA, Braathen B, Heia K, eds. Sciaâ93, Proceedings of the 8th Scandinavian conference on image analysis. Tromso, Norway: 1403-1410.
references.citation: Weight C, Parnham D, Waites R. 2008. LeafAnalyser: a computational method for rapid and large-scale analyses of leaf shape variation. Plant Journal 53: 578-586.
references.citation: Young JP, Dickinson TA, Dengler NG. 1995. A morphometric analysis of heterophyllous leaf development in Ranunculus flabellaris. International Journal of Plant Sciences 156: 590-602.
references.citation: Young IT, Walker JE, Bowie JE. 1974. An analysis technique for biological shape. Information and Control 25: 357-370.