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Growth polarity in leaves – a final discussion Insights into the growth processes of leaf lamina have come from studies on several species including Arabidopsis, Antirrhinum, tobacco and maize. A feature common to the growth of leaf in these distantly related species is the existence of a pronounced growth gradient in the proximo-distal axis -growth at the tip (distal part) is arrested at an early stage while the basal region (proximal part) continues to grow for the longest duration. This is because the cell division is arrested first at the tip at an early stage of development and the arrest progressively spreads towards the base. Along with the strong proximo-distal growth gradient, a milder growth gradient also exists in the medio-lateral axis, such that the cell division arrest travels slightly faster on the leaf margins imparting an overall convex shape to the arrest front. The temporal and spatial progression of the arrest front has not only been implicated in shaping up of a leaf but is also of paramount importance in the maintenance of a flat surface during leaf growth. Although the patterning mechanisms described above seem to operate during leaf growth in many6 species, the molecular mechanisms governing these processes is still in its infancy. Moreover, patterning of leaf growth has been studied only in a handful of model species and, therefore, the information from the vast body of natural variation remains neglected. Proximo-distal growth patterning by CINCINNATA Mutant leaves with altered rates or shapes of the arrest front progression deviate significantly from the normal shape and overall flat structure. Mutation in the CIN gene in Antirrhinum and its orthologues in Arabidopsis cause buckling of the leaf due to excess cell proliferation, which in turn is caused by a delayed progression of the arrest front. CIN-like genes code for TCP transcription factors and are expressed in a broad zone of a growing leaf somewhat distal to the proliferation zone. Even though several direct and indirect targets of CIN-like genes have been identified in various plant species, their role in regulating leaf maturity and surface curvature has remained unclear. The comparison of global transcription profile of wild type and cincinnata mutant of Antirrhinum showed that the expression of genes involved in either signaling or biosynthesis of the major growth hormones were altered in the mutant. By combining DNA-protein interaction, expression analysis, chromatin immuno-precipitation and RNA in situ hybridization, we show that CIN maintains surface flatness by regulating the signaling or level of major plant hormones in developing leaves. CIN promotes cytokinin signaling by directly binding to and thereby promoting the expression of a cytokinin receptor, AmHK4, in a spatio¬temporal manner. Furthermore, it also seems to affect GA level indirectly in young leaves by regulating the spatio-temporal as well as levels of GA-biosynthetic and GA-degrading enzymes. Thus, CIN seems to accelerate maturity in leaf cells along the tip-to-base direction through its effect on the cytokinin and GA signaling pathways. In addition, CIN suppresses auxin signaling more at the margin than in the centre by establishing a margin-to-medial expression gradient of a homologue of the auxin suppressor IAA3, thereby suppressing excess cell proliferation on the margin. Our results uncover an underlying mechanism in a developing leaf that controls curvature of the leaf surface by promotion of timely exit from cell proliferation in the proximo-distal as well as the medio-lateral axes via multiple hormone pathways. Divergent growth polarities in the proximo-distal axis of leaves The morphogenetic gradient in the proximo-distal axis of a leaf is brought about by the dynamic expression of several heterochronic regulators which can include TCP and GRF classes of transcription factors. Interestingly, many of these transcription factors are also regulated post-transcriptionally by micro RNAs. In case of the studied model species, these factors seem to be associated with basipetal growth. The early arrest in cell proliferation at the tip and continued cell division at the base has served as a paradigm in studying leaf growth and has been used to conceptualize the growth of leaves with different shapes. However, the possibility of the existence of different patterning mechanisms during leaf growth in the highly diverse plant kingdom remains unexplored. Our survey of leaf growth patterns in 75 dicot species reveals the existence of four distinct proximo-distal polarities in growth patterns. Using the law of simple allometry, we also show that the differentially growing regions of leaves bear a constant relationship between them during growth. A combination of cell-size studies, histochemical staining and expression analysis reveals a strong correlation among growth pattern, cell size and the cell proliferation status. The cell size studies also indicate that there is a wide variation in the final cell sizes of leaves and the relative contribution of cell division and cell expansion to the final leaf size can be highly variable. Furthermore, we find that the varying growth patterns are linked to changes in the expression pattern of miR396, which controls the expression pattern of cell division regulatory transcription factors, the GRFs. Mis-expressing miR396 at the base of the young Arabidopsis leaf caused an early exit from cell division while reducing the expression of the miR396 at the tip allowed cell division to continue for a longer duration near the tip. Our results demonstrate that leaves with similar shapes can be differently patterned and that this divergent patterning is linked to the expression differences in the regulatory micro RNA, miR396 In conclusion, this study shows that regulators like CIN maintain surface flatness of the Antirrhinum leaf during growth by promoting timely exit from cell division along the proximo-distal and the medio-lateral axes; and it does so by regulating multiple hormone pathways. Although the basic mechanism of patterned cell division and differentiation seems to be conserved among species, the polarities of growth can vary. The variability in the growth polarities could be brought about by changes in the trans-regulation or cis-regulatory changes in the patterning genes.

Leaf photosynthetic measurements using a portable 14Co2 field system were carried out and correlative leaf relative growth rates, RGR, were determined at different leaf positions of three alpine plant species throughout the growing season. Initially there was a period of high leaf RGR associated with a period of increasing photosynthetic activity. Following this stage was a long period of no net change in length of the living leaf. During this period, photosynthetic activity generally increased to a maximum level and then decreased steadily. The final ontogenetic stage was a period of negative leaf RGR denoting leaf senescence which was associated with a marked decline in leaf Co2 uptake. Ontogenetic timing of these alpine species is geared with the surge and decline of individual leaf photosynthetic activity so that one to several leaves operating at near maximal photosynthetic capacity are always maintained during the growing season for each plant. These findings are discussed in relation to their adaptive significance for these species.

The maize leaf has three main axes of growth, with an asymmetric distribution of tissue types along each axis. This study focuses on three mutants, Wavy auricle in blade1-R (Wab 1-R), liguleless1-R (lg1-R) and milkweed pod1-R (mwp1-R) that disrupt axial patterning of maize leaves. Dominant Wab1 mutations disrupt both medial-lateral and proximal-distal patterning. Wab1 leaf blades are narrow and ectopic auricle and sheath-like tissues extend into the leaf blade. Previous analyses have shown that Lg1 acts cell-autonomously to specify ligule and auricle tissues. The current study reveals additional roles in defining leaf shape. The recessive lg1-R mutation exacerbates the Wab1-R phenotype; in the double mutants, most of the proximal blade is deleted and sheath tissue extends along the residual blade. A mosaic analysis of Wab1-R was conducted in Lg1 and lg1-R backgrounds to determine if Wab1-R affects leaf development in a cell-autonomous manner. Normal tissue identity was restored in all wab1/- sectors in a lg1-R mutant background, and in three quarters of sectors in a Lg1 background. These results suggest that Lg1 can influence the autonomy of Wab1-R. In both genotypes, leaf-halves with wab1/- sectors were significantly wider than non-sectored leaf-halves, suggesting that Wab1-R acts cell-autonomously to affect lateral growth. mwp1-R is a recessive mutation that specifically affects patterning of sheath tissue. Characterisation of the mwp1-R phenotype revealed that mwp1-R husk leaves and the sheaths of vegetative leaves develop pairs of outgrowths on the abaxial surface associated with regions of adaxialised tissue. In situ hybridisation confirmed that disruptions to adaxial-abaxial patterning are correlated with misexpression of leaf polarity genes. Leaf margins and fused organs such as the prophyll are most severely affected by mwp1-R. The first two husk leaves normally fuse along adjacent margins to form the bi-keeled prophyll. In the most severe cases the mwp1-R prophyll is reduced to an unfused, two-pronged structure and keel outgrowth is significantly reduced. We speculate that the adaxial-abaxial patterning system has been co-opted during evolution to promote outgrowth of the keels in normal prophyll development. The results of this study place Mwp1, wab1 and Lg1 in a network of genes that regulate leaf polarity and axial patterning.

Les feuilles des plantes sont des organes importants pour la production de biomasse dans la nature car elles sont le siège principal de la photosynthèse, qui permet de transformer la matière minérale en matière organique. Identifier les mécanismes responsables de la morphogenèse, i.e. la genèse de la forme pendant le développement, est donc une question d'intérêt. Pour être analysée, la morphogenèse doit être appréhendée tout au long de la croissance car la forme finale d'une feuille est le résultat de mécanismes coordonnés dans l'espace et le temps. Pour comprendre ce type de processus complexes, la modélisation est une approche de choix. L'objectif de cette thèse était donc de développer des stratégies de quantification et de modélisation de la morphogenèse pour mieux comprendre le développement des feuilles. Pour quantifier la morphogenèse, ma première contribution a été de développer des méthodes pour dater précisément l'apparition des feuilles sur la plante et celle des dentelures sur la marge foliaire, ce qui permet de recaler dans le temps et comparer différentes feuilles en croissance. En calculant les trajectoires de croissance de feuilles moyennes, il est alors possible de préciser où et quand le développement de feuilles peuvent différer, au niveau global ou des dentelures, pendant la croissance. En analysant des feuilles de formes différentes de la plante modèle Arabidopsis thaliana, j'ai ainsi pu montrer que malgré des différences importantes en taille et forme globale, il y a une similarité dans le développement des dentelures. Ces résultats suggèrent qu'il existe des processus identiques qui gouvernent l'apparition et la croissance des dentelures. J'ai ensuite proposé un modèle de développement des feuilles, à partir duquel il est possible de simuler la croissance d'une feuille. Il est basé sur des mécanismes biologiques qui on été identifiés comme étant importants dans la mise en place de la forme. Pour paramétrer le modèle, une approche d'optimisation a été mise au point pour déterminer les paramètres optimaux du modèle. Les résultats obtenus montrent que l'apparition séquentielle des dents ainsi que certains paramètres morphologiques peuvent être bien reproduits par le modèle. / Plant leaves are important for the production of biomass in nature, because they are the main site of photosynthesis, They have various shapes and it has been shown that their morphology influences photosynthesis efficiency. Identifying the mechanisms responsible for morphogenesis, i.e. the genesis of the shape during development, is therefore a matter of interest. To be analyzed, morphogenesis must be apprehended throughout the whole growth because the leaf final form is the result of coordinated mechanisms in space and time. To understand this type of complex processes, modeling is an approach of choice. Consequently, the objective of this thesis was to develop strategies for the quantification and modeling of morphogenesis to better understand leaf development. To quantify morphogenesis, my first contribution was to develop methods to precisely date the appearance of the leaves on the plant, and of the serrations at the leaf margin, allowing to register in time and to compare different growing leaves. Besides, based on mean growth trajectories, it is possible to specify where and when the developments of different leaves differ, at global and serration scales, during growth.By analyzing the development of leaves of the plant model Arabidopsis thaliana that have different shapes, in wild type or in mutants, it has been shown that, despite significant differences in leaf size and shape, there is a similarity in the development of all serrations. These results suggest that there are identical processes that control the appearance and growth of serrations. I proposed two leaf development models, based on biological mechanisms that have been identified, in the literature, as important for the leaf shaping, and also on the quantification of leaf morphogenesis performed in this work. The simulation module, that generates growth trajectories from the model, makes it possible to compare simulated and real developments. To parameterize the model, an optimization approach has been proposed to determine optimal parameters, which minimizes the differences between simulation and real growths. The results showed that the sequential appearance of the teeth as well as important morphological characteristics can be well reproduced by the models.

Leaves of Rumex obtusifolius L. and R. patientia L.were exposed to combinations of mild water stress and enhanced ultraviolet-B irradiation during their ontogeny. Two UV-B treatments (enhanced UV-B and control) and three water stress treatments (-0.0 MPa, -0.2 MPa and -0.4 MPa rooting medium matric potentials) were employed. The impact of the stress interaction was assessed on the basis of changes in leaf area, average adaxial epidermal cell size, and total number of adaxial epidermal cells per leaf. Although the level of UV-B irradiation applied was insufficient to significantly alter leaf growth at any given water stress, UV-B did interact with water stress to alter the pattern o= plant response to water stress. The interaction was only apparent when the water stress was greater than -0.2 MPa root matric potential. For both species UV-B irradiation exacerbated the depression of leaf growth due to -0.4 MPa water stress. For R. obtusifolius the basis of the reduction in leaf growth was likely a reduction in the rate of cell division during the early phase of leaf growth. For R. patientia the effect of the interaction on cell division was less clear. Cell expansion was not directly affected by UV-B irradiation in either species, although the reduction in cell size with increasing water stress was apparent. In terrestrial ecosystems, mild water stress is a common occurrence and with predicted anthropogenic modifications of the atmospheric ozone layer, UV-B radiation reaching the earth's surface can be expected to increase. The effect or. higher plants of the stress interaction may thus be of considerable significance under natural conditions.

To produce plant material for this thesis, Trifolium repens (white clover) (genotype 10F) and Trifolium occidentale (genotype 18Z) were propagated to produce individual stolons trained over a plastic matrix to inhibit nodal root formation. These stolons comprised leaf tissue representative of all developmental stages, from leaf initiation, maturation through to senescence. The developmental pattern for both species in terms of leaf ontogeny was generally reproducible between vegetatively propagated clones. Three distinct 1-aminocyclopropane-1-carboxylatc (ACC) oxidase genes expressed during leaf ontogeny in white clover (Trifolium repens L.) have been identified (Hunter et al., 1999). Of the three ACC oxidase genes identified, one designated TR-ACO2 is expressed in newly initiated and mature green leaves while TR-ACO3 is expressed predominantly in the senescent leaf tissue. In order to further characterize the protein products of these genes, a series of FPLC columns was used to partially purify isoforms of ACC oxidase from leaf tissue of white clover at different developmental stages, followed by 2D gel electrophoresis to obtain further purification. Two distinct isoforms of ACC oxidase were identified and partially purified from newly initiated green (designated the NIGI isoform) and senescent (designated the SEI isoform) leaf tissue. Both purified NIGI and SEI proteins were recognized by western analysis using an anti-(Trifolium repens) TR-ACO2 antibody after SDS-PAGE or 2D gel electrophoresis. To determine whether NIGI is coded for by TR-ACO2 and SEI is coded for by gene TR-ACO3, protein spots (after 2D gel electrophoresis) were digested with trypsin and the masses of the peptide determined by matrix-assisted laser desorption ionization-timc of flight (MALDI-TOF) mass spectrometric analysis. For NIGI, the coverage of the putative protein sequence (TR-ACO2) by tryptic digestion ranged from 24.5% to 37.6%, while the observed pI (5.1) and molecular mass (37 kDa) were close to the theoretical pI (5.3) and computed mass (35.7 kDa). For SEI, the percentage coverage of the putative protein sequence (TR-ACO3) from the peptides identified ranged from 13.4% to 18.0%, while the observed pI (5.2) and molecular masses (35.0-35.5 kDa) were also close to the theoretical pI (5.5) and computed mass (35.2 kDa). These data suggest that the NIGI isoform is encoded by TR-ACO2, while the SEI isoform is encoded by TR-ACO3. ACC oxidase activity in vitro and ACC oxidase protein accumulation over 24 h in mature green leaf tissue extracts during both short and long days has been shown to be under circadian control. There are two ACC oxidase activity peaks observed, in which the pattern of fluctuation in ACC oxidase activity resulted in a high level of enzyme activity at 12:00 am (0.18-0.27 nmol ethylene/h/mg), and maximum activity at 12:00 pm (0.24-031 nmol ethylene/h/mg). Lowest activity was observed in both long and short days at 9:00 pm (0.09-0.10 nmol ethylene/h/mg). In addition, northern analysis indicated that the TR-ACO2 mRNA level also displayed a circadian pattern of expression. Investigation of the effect of protein phosphorylation and dephosphorylation on ACC oxidase activity indicated that ACC oxidase activity in vitro during the periods of maximum activity increased 36% (at 12:00 am) and 56% (at 12:00 pm) after dephosphorylation, respectively. However, there was only 21% increase in enzyme activity at the time point with lowest activity (9:00 pm) in the dephosphorylated extracts. SDS-PAGE using a mini-protein gel system or a gradient gel system showed that the molecular mass of ACC oxidase decreased after dephosphorylation when compared with phosphorylation of the enzyme. These results suggest that the phosphorylation and dephosphorylation of the ACC oxidase proteins occurs in vitro and the state does affect enzyme activity. In the second part of this thesis, the coding regions of putative ACC oxidase gene transcripts were generated from leaf tissue of genotype 18Z of T. occidentale using RT-PCR. Sequence alignments indicated that the sequences could be grouped into two distinct classes, and these coding regions were designated TO-ACO2 (Trifolium occidentale ACC oxidase 2) and TO-ACO3 (Trifolium occidentale ACC oxidase 3). TO-ACO2 and TO-ACO3 share 82% similarity in nucleotide sequence and 84% similarity in amino acid sequence. The TO-ACO2 and TO-ACO3 sequences were validated as encoding ACC oxidases by comparison with other ACC oxidases in the GenBank database and both TO-ACO2 and TO-ACO3 deduced amino acid sequences contain all the residues hitherto shown to be important for maximal activity of the enzyme. Further, TO-ACO2 had 97% identity with TR-ACO2 at the nucleotide level, and 98% identity at the amino acid level. TO-ACO3 had 97% identity with TR-ACO3 at the nucleotide level, and 96% identity at the amino acid level. Genomic Southern analysis, using 3'-UTRs of TR-ACO2 and TR-ACO3 as probes, could not confirm that TO-ACO2 and TO-ACO3 arc encoded for by distinct genes. Expression studies of TO-ACO2 and TO-ACO3 genes during leaf maturation and senescence of T. occidentale were examined using northern analysis. TO-ACO2 is expressed predominantly in newly initiated and at the onset of the mature-green leaf stage, while TO-ACO3 shows maximal expression in senescent leaf tissue. The changes of ACC oxidase activity during leaf ontogeny of T. occidentale coincided with the pattern observed for ACC oxidase protein accumulation using western analysis and image analysis.