Dissertations / Theses on the topic 'Leaves – Morphology'

The epidermis from a range of species from the genus Allium was peeled from the base, middle, and tip of the adaxial and abaxial surfaces of leaf material. Epidermal peels were water mounted after being peeled using forceps, and photographed using Nomarski microscopy. The epidermis is composed of rows of cells which run parallel to one another, and to the long axis of the leaf. Guard cells are present on both surfaces of the leaf in most species, but some species lack guard cells on either the abaxial or adaxial surface. Guard cells are sunken to varying degrees in all species surveyed. Subsidiary cells are lacking in all species surveyed. End walls of nonstomatal cells are either even or oblique. Micropapilae, striations, or trichomes are present in some species, but most species lack epidermal structures. Epidermal cells range in size from two to three times greater in length than width to greater than fifteen times longer than wide. The majority of parallel walls are either straight or diamond-shaped while others are wrinkled, wavy, or broadened. Allium is separated into three groups according to the structure of the parallel walls. Group one includes those species with typically straight parallel walls; group two those species with diamond-shaped parallel walls; and group three is comprised of those species with wavy parallel walls.

Plant-based defence mutualisms involve aspects of plant morphology that influence the performance of plant parasites, their natural enemies and trophic interactions. Leaf domatia, small indentations on the underside of leaves, can be structurally complex, and are often inhabited by potentially beneficial mites and other arthropods. Plant morphological traits such as domatia that enhance mutualistic relationships may result in increased plant growth rates, and reproductive success. New Zealand supports ~60 plant species that have domatia, the most speciose genus being Coprosma. The aim of this thesis was to examine factors that affect the production of leaf domatia and their relationship with foliar mite assemblages. The three main objectives of this thesis are: First, to investigate the production of foliar domatia and their susceptibility to limited resources, particularly to carbon availability. Second, to test if domatia are inducible structures during leaf ontogeny in the presence of foliar mites and/or fungi. Finally, to explore the effect of domatia availability on foliar mite assemblages on leaves with and without resident mites. This thesis tested the stated objectives using C. lucida, C. ciliata, C. foetidissima and C. rotundifolia, with a combination of field investigations and controlled manipulative experiments. The cost of domatia production was investigated using two field surveys and two controlled experiments. Under natural conditions the relationship between leaf morphology and domatia were measured in situ and across an altitudinal gradient. The experimental manipulations used carbon and nutrient stress, induced by temperature, light and fertilizer application. The second objective was experimentally tested under field conditions by manipulating foliar mites and fungal densities on C. rotundifolia. The third objective was investigated by manipulating domatia availability on C. lucida shrubs across three different vegetation types. Under field conditions, the number of domatia per leaf was associated with leaf morphology in C. lucida and C. foetidissima, but not C. rotundifolia. Foliar carbon showed a positive, but weak association with domatia production in C. foetidissima and C. ciliata. Altitudinal induced-carbon stress on domatia production was ambiguous. Domatia production in C. foetidissima was positively associated to altitude in field survey (1), and negatively associated in the second survey, with no correlation found between carbon and altitude. Experimental C. rotundifolia shrubs held under elevated night-time temperatures showed a 2.5 fold increase in respiration, a 34% to 91% decrease in daily carbon gain, and 38% decrease in domatia per leaf mass. Domatia production showed no significant differences under nutrient stress. The results showed little evidence to support a role for induction of domatia. Domatia production in new leaves was similar across all experimental treatments. Diverse vegetation types supported 60% higher mite species. Leaves with domatia supported ~22 to 66% higher mite densities, greater colonisation success and more diverse mite assemblages, than those without domatia. In the pastoral vegetation, the absence of predatory mites on experimental shrubs resulted in no differences in fungivorous mite densities regardless of domatia availability. Plant investment in foliar domatia appears associated with the number of available sites on the leaf under field conditions. The role of carbon availability during leaf ontogeny suggests a complex and highly variable association with domatia production. Domatia are constitutive defence structures that influence mite assemblages, mediating both beneficial and antagonistic relationships. This thesis concludes that domatia are in part, carbon-based non-inducible structures that influence mite assemblages, plant-mite and mite-mite interactions, and increase the probability of successful colonisation.

Understanding the dynamics of leaf litter inputs in streams involves assessment of retention, spatial distribution and processing of leaf litter inputs. However, leaf litter dynamics in streams has been mostly studied by determining the controlling factors of leaf litter processing, ignoring if leaf litter inputs are retained within the streams, and how these inputs are spatially distributed within the streambed. Furthermore, leaf litter processing in streams is not an isolated process because it can be linked with the dynamics of solutes of these streams. In fact, leachates from leaf litter provide to the streams with dissolved organic matter (DOM), as well as, dissolved nutrients (i.e., nitrogen [N] and phosphorous [P]). Also, the mineralization of leaf litter inputs implies the releasing of dissolved forms of C and nutrients to the stream water column. In addition, microbial communities inhabiting leaf litter uptake dissolved forms from water column during leaf litter decomposition. Despite the obvious linkage between leaf litter processing and in-stream solute dynamics, few studies provided information about the main drivers controlling this connection. In order to understand how leaf litter dynamics operates in stream ecosystems, the present thesis aims to examine (a) how the hydro-morphological characteristics of the recipient stream can influence the retention, spatial distribution, physical fragmentation and decomposition of leaf litter inputs. Moreover, we also examine (b) the interaction between leaf litter processing and in-stream dynamics of dissolved organic carbon and dissolved inorganic nitrogen and how it varies depending on the leaf litter species considered. To approach this aim, we specifically examine how the leaf litter quality of different riparian tree species can influence: (b1) the chemical-composition and bioavailability of leaf litter leachates to streams (b2) the uptake of DIN and DOC from water column by microbial decomposers during decomposition process and how it is related to the activity of microbial decomposers and, (b3) how microbially-driven leaf litter decomposition vary among streams which covered a wide gradient of inorganic nutrient concentrations. The present thesis reveals that heterogeneity of water velocity within a reach is a factor controlling different aspects of leaf litter dynamics in streams because not only influences the fraction of leaf litter retained but also how these inputs are spatially distributed and further processed within the streams. Therefore, we indicate that the relevance of leaf litter inputs as organic matter source to in-stream communities can be subjected to the hydro- morphological characteristics of the receiving stream reaches, since they determine the spatial heterogeneity of water velocity within the reach. In addition, we suggest that leaf litter quality influence on the chemical and structural properties of the leaf litter-leachates and also how these leaches are used by in-stream microbial assemblages. The quality of leaf litter can also dictate the microbial demands of dissolved C and N from the water column. Finally, we suggested that the influence of in-stream nutrient concentrations on leaf litter decomposition can be subjected to the leaf litter quality. Considering all our results together we suggest that riparian composition, which dictates the quality of leaf litter inputs, may control the linkage between leaf litter processing and in-stream C and N solute dynamics. However, the heterogeneity of water velocity may be the most relevant factor determining the importance of leaf litter to streams because it can control the probability that leaves contribute to the stream metabolism or to be exported downstream.
Para comprender cómo funciona la dinámica de la hojarasca en arroyos, la presente tesis pretende examinar (a) cómo las características hidro-morfológicas del lecho del arroyo pueden influir en la retención, distribución espacial, fragmentación física y descomposición de la hojarasca. Además, también examinamos (b) la interacción entre el procesamiento de hojarasca y la dinámica del carbono orgánico disuelto y nitrógeno inorgánico disuelto del arroyo y cómo varía dependiendo de las especies de hojarasca consideradas. La presente tesis revela que la heterogeneidad de la velocidad del agua dentro de un tramo es un factor que controla diferentes aspectos de la dinámica de hojarasca en arroyos, ya que no solo influye en la fracción de hojarasca retenida, sino también en cómo estas entradas se distribuyen espacialmente y se procesan en los arroyos. Por lo tanto, indicamos que la relevancia de las entradas de hojarasca como fuente de materia orgánica para las comunidades del arroyo puede estar sujeta a las características hidro-morfológicas de dicho ecosistema, ya que determinan la heterogeneidad espacial de la velocidad del agua dentro del mismo. Además, sugerimos que la calidad de la hojarasca influye en las propiedades químicas y estructurales de los lixiviados de hojarasca y también cómo estos lixiviados son utilizados por los microorganismos del arroyo. La calidad de la hojarasca también puede dictar las demandas microbianas de C y N disuelto de la columna de agua. Finalmente, sugerimos que la influencia de las concentraciones de nutrientes del arroyo en la descomposición de la hojarasca puede estar sujeta a la calidad de la hojarasca. Teniendo en cuenta todos nuestros resultados, sugerimos que la composición del bosque de ribera, que determina la calidad de las entradas de hojarasca, puede controlar el vínculo entre el procesamiento de la hojarasca y la dinámica de solutos C y N del arroyo. Sin embargo, la heterogeneidad de la velocidad del agua puede ser el factor más relevante para determinar la importancia de la hojarasca en las corrientes, ya que puede controlar la probabilidad de que las hojas queden retenidas en el arroyo o se exporten aguas abajo, perdiéndose como fuente de materia y energía.

INTRODUCTION The retina prefers to metabolize glucose through glycolysis rather than oxidative phosphorylation (OXPHOS) to meet their energy demand even when oxygen is abundant, known as the “Warburg effect”, despite having abundant functional mitochondria. We created transgenic mice with selective knockdowns of key enzymes in glycolysis and OXPHOS including hexokinase (HK) 2, lactate dehydrogenase (LDH) A and pyruvate dehydrogenase (PDH) E1α in rods to study the importance of glycolysis and OXPHOS in retinal metabolism and the role of metabolic derangement in the pathogenesis of retinal diseases. MATERIALS AND METHODS Immunohistochemistry and Western blots were performed to study the changes in protein expression and regulation. Scotopic electroretinography and optical coherence tomography were performed to study the retinal function and structure. Gas and liquid chromatography-mass spectrometry were employed to study the changes in13C-glucose-derived metabolites in the retina. RESULTS Knockdown of HK2 in rods led to photoreceptor degeneration, with reduction in the thickness of the retina and impaired retinal function. Knockdown of HK2 decreased pyruvate production but promoted the tricarboxylic acid (TCA) cycle in the retina. Knockdown of LDHA and PDHE1α in rods also led to retinal degeneration, with thinning of the retina and impaired retinal function. Deletion of LDHA and PDHE1α suppressed glycolysis and the TCA cycle in the retina. CONCLUSION We found that HK2/LDHA-mediated glycolysis and PDHE1α mediated-OXPHOS in rods are indispensable for the maintenance of photoreceptor structure and function. Disturbance in aerobic glycolysis or OXPHOS leads to metabolic remodelling in the retina and photoreceptor degeneration.

De nos jours, l’identification automatique des espèces de plantes par l’analyse d’images, devient incontournable pour faire perdurer, standardiser voire approfondir les connaissances relatives à la communauté végétale. Cette thèse aborde le problème d’identification automatique des espèces de plantes en utilisant les images de feuilles. Elle s’attaque à deux principaux challenges: Le premier challenge est le grand nombre et la large variabilité de la morphologie foliaire des espèces et le deuxième challenge est la variabilité intra-espèces qui se manifeste localement au niveau de régions particulières des feuilles. Pour pallier à ces deux problèmes, un retour à la botanique et notamment aux concepts botaniques foliaires a été établi pour définir une structuration automatique des feuilles à deux niveaux: Le premier niveau concerne un schéma de catégorisation selon les deux concepts botaniques “arrangement” et “lobation”. Le deuxième niveau consiste à définir les parties sémantiques qui composent la feuille. L’approche de la thèse s’articule autour de deux principaux volets: Dans le premier volet, nous nous intéressons à mettre en place cette structuration guidée par la sémantique botanique en définissant des propriétés géométriques simples corrélées avec les définitions et les observations botaniques. Dans le deuxième volet, nous étudions la faisabilité et la pertinence d’intégrer cette structuration dans la chaîne d’identification. Particulièrement, nous établissons des recherches ciblées dans les catégories et nous définissons des modèles de parties à significations botaniques. Nous établissons notre évaluation sur les deux bases d’images de Scans de feuilles ImageCLEF 2011 et ImageCLEF 2012. Nous comparons notre approche par rapport à un schéma d’identification de référence, appliqué sur la totalité de la base et en utilisant l’image entière, et par rapport à plusieurs méthodes référencées dans la littérature
Nowadays, automatic identification of plant species, by image analysis, has become crucial to maintain, standardize or deepen knowledge about the plant community. This thesis focus on the problem of automatic identification of plant species using leaf images. It addresses two main challenges: The first challenge is the large number and the high variability in foliar morphology across species. The second challenge is the intra-species variability which occurs locally at particular regions of leaves. To overcome these two problems, a return to botany and especially to leaf botanical concepts is established in order to define an automatic structuring of leaves at two levels: The first level concerns a categorisation scheme according to the botanical concepts “arrangement” and “lobation". The second level consists in decomposing leaves into semantic parts. The approach of the thesis is based on two key parts: In the first part, we focus on establishing this botanical-based structuring process by defining simple geometric properties correlated with botanical definitions and observations. In the second part, we investigate the feasibility and opportunities to integrate this structuring process in the identification scheme. Particularly, we make targeted researches in categories and we define specific part-based models.Experiments are conducted using the ImageCLEF 2011 and 2012 Scan images leaf databases. We compare our approach with respect to the reference identification scheme, applied on the whole databaseand using the entire images, and with respect to several methods referenced in the literature

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Bachelors
Burnett School of Biomedical Sciences
Molecular Biology & Microbiology

A synopsis of the southern African members of the genera Dicoma Cass. and Macledium Cass. (family Asteraceae, subfamily Cichorioidiae, tribe Mutiseae, subtribe Gochnatiinae) is presented. The most recent monograph of Dicoma was done by Wilson (1923). It included 34 species, of which 17 are southern African, The southern African species were last treated by Harvey (1865). Traditionally Dicoma included 50 to 65 species, of which 23 occur in southern Africa. Ortiz (2001) reinstated the genus Macledium Cass., which was first described by Cassini (1825), following a phylogenetic study of Dicoma and related genera (Ortiz, 2000). The species previously belonging to sections Macledium and Pterocoma of the genus Dicoma have been transferred to the genus Macledium by Ortiz (2001). In this study, characters relating to morphology of the involucral bracts, disc floret cuticle ornamentation, type of pappus and the type of achenes are used to distinguish the two genera. Differences were found between the grassland species and Cape species of Macledium. Type of florets in the capitulum and pappus morphology are used to distinguish sections within Dicoma sensu stricto. Characters such as habit, leaf morphology, shape and size of the capitulum, involucral bracts vestiture and details of the pappus could be reliably used to distinguish species. The genus Dicoma now consists of about 35 species, of which16 species and two subspecies occur in southern Africa. Four informal groups of D. anomala subsp. anomala, two of D. anomala subsp. gerrardii as well as two of D. montana are recognized to accommodate infraspecific variation. Macledium comprises about 20 species, of which seven species, three subspecies and three varieties occurr in this region. M. spinosum is divided into two informal forms and a new combination, Macledium zeyheri subsp. zeyheri var. thyrsiflorum, is made. In view of the above, a comprehensive taxonomic revision of all the southern African taxa was done. Morphological and anatomical data were used to investigate relationships among the species of Dicoma sensu stricto, using the genus Macledium as the sister group. A phylogenetic analysis of Macledium was also done to investigate relationships between the grassland species and the Cape species. The taxonomic treatment includes the nomenclature, synonymy, diagnostic characters, uses, common names (where possible) and geographical distribution for all southern African taxa belonging to the two genera. Keys to the genera, sections, species and subspecies are provided and the diagnostic characters of the two genera are illustrated.
Prof. B.E. van Wyk

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of requirements for the degree of Master of Science. August 2015.
iii ABSTRACT Global changes are likely to have negative impacts on many ecosystems including savannas. Semi-arid environments are notable for the wide range of seasonal patterns of leaf display in the tree communities. The environmental cues of leaf out and leaf drop are not consistent across species, and are not always directly linked to water availability, indicating that some species might be particularly sensitive to changes in climate. Strategies employed by trees which leaf early or drop their leaves late are likely to impact other aspects of their life-history and functioning so I expect particular plant functional types to be associated with particular vegetation functional traits. I assessed how variable savanna leafing strategies are among 28 species at a semi-arid savanna site at Nylsvley, and used this information to group species into plant functional types (PFTs). These PFTs were then assessed in terms of key vegetative traits to explore the life history consequences of different leafing strategies. Leaf phenology was monitored throughout one growing season and quantified using 8 key phenological metrics. The timing of leaf display tracks the timing of seasonal rainfall but with wide variation, with some species retaining their leaves throughout dry season. Other species loss some leaves throughout the growing season, some species only flushed their leaves after the first rains, and other flush before the first rains. I identified 4 clear PFTs using the MClust clustering integrated with subjective procedure. Four vegetative traits were measured: specific leaf area, leaf nitrogen, maximum stomatal conductance and wood density. I identified some clear trade-offs between vegetative traits and phenological strategies. There was also a positive relationship between degree of rain stimulated flushing metric and wood density. Using objective clustering methods to determine plant functional types has some clear advantages over more subjective methods but depends on good input data. Identifying plant functional types at Nylsvley has led to some insights into functioning of these savannas, but as there appear to be strong links between plant traits and particular leafing strategies it might be more appropriate to explore syndromes of vegetation functional traits when modelling responses to global change.

A thesis submitted to the Faculty of Science, in partial fulfilment of the requirements for the degree of Master of Science, 2018
Since its discovery in India many centuries ago, Moringa oleifera has been used largely in the tropics and subtropics where it is native for nutritional, medicinal and industrial benefits. These benefits are attributed to the antioxidant, antifungal and anti-inflammatory properties in the phytochemical compounds of M. oleifera. Agriculture of M. oleifera in South Africa is currently minimal despite its multiple benefits. Naturalisation of M. oleifera in the country is important in the aid to eliminate the shortage of nutritional food reserves, and utilise the plant as a source for realising and developing new medicinal compounds. Global warming and consequent climate change have caused extreme high and low temperature seasons in the past in South Africa. Studying the effects of extreme high and low temperatures on the growth, performance and phytochemistry of M. oleifera at controlled conditions is vital in understanding its responses and adaptability. M. oleifera grown for three months in a greenhouse of the University of the Witwatersrand was subjected to two extreme temperature conditions of 15/10°C and 47/29°C for seven days (144 hours). The control was kept at ambient temperature (25/22°C). The morphology, histology, total phenolic contents, total flavonoid contents, quercetin and kaempferol concentrations were assessed using dissecting microscope, light electron microscope, UV-Vis spectroscopy and HPLC-UV respectively. The effect of high temperatures showed increased senescence. However, plants that were treated with low temperature appeared to be more tolerant by indicating a delay in leaf senescence. Yellowing, browning, curling and drying of the leaves worsened after 144 hours at high temperature treatment. The histological examination at high temperatures showed intact cellular structures such as upper epidermis, lower epidermis, spongy mesophyll, palisade mesophyll and intercellular spaces in green leaves after 24 hours. However, leaf size decreased over the seven days treatment. The cellular structures in leaves that became yellow had collapsed and showed evidence of necrosis. In the low temperature treatment, 1% of leaves showed signs of leaf curling after VII 48 hours while others remained green and intact. After 144 hours, 5% of leaves showed signs of senescence while others turned dark green and remained intact. Leaves cells such as palisade mesophyll had elongated and thus increased the thickness of the leaves. This was evidence of acclimation to cold temperatures. The total phenolic content increased by 10.1% after 48 hours under high temperatures. However, a decrease of 3.5% and 3.1% was observed after 96 and 144 hours respectively. Quercetin increased by 8.5% after 48 hours then after 96 and 144 hours a decrease of 41.7% and 61.6% was observed respectively. A significant increase in kaempferol concentration of 203.5% after 48 hours was noted. This was followed by a decline after 96 and 144 hours of 43.0% and 24.8% respectively. Concentration of total flavonoid content increased by 11.6% and 33.8% after 48 and 96 hours, and after 144 hours, a decline of 0.4% was observed. The combined evidence from the present study showed that the three months old M. oleifera plants are sensitive and cannot withstand temperatures as high as 47/29°C. However, the species has showed to be positively more tolerant to cold temperatures (15/10°C). This evidence may have meaningful contribution towards the industry, Moringa oleifera farming practices as well as advance the understanding of the effects of extreme temperatures on the species.
XL2019

碩士
國立嘉義大學
園藝學系研究所
99
Leaf morphology and anatomy, pollen grains morphology and pollen viability as well as inter simple sequence repeat (ISSR) molecular markers were carried out on the identification , classification and genetic diversity of 21 coleus cultivars. In the study of leaf morphology, 5 leaf shapes with ovate, lance-ovate, widely ovate, very widely ovate and cordate etc. were classified. Four leaf apexes were with acuminate, acute, obtuse and rounded. Six leaf bases were with narrowly cuneate, cuneate, obtuse, rounded, truncate and cordate etc. Five margin type of leaves in 21 coleus cultivars were with crenate, crenulate, pinnately cleft, pinnately parted and pinnately divided etc . The anatomical transverse sectional structure of leaf in 21 coleus cultivars were also studied. Upper epidermis was a neat single layer of epidermal cell. The lower epidermis wasn’t as neat as upper epidermis, it appeared curved. The palisade tissue consisted of one or two arrangement of neat layers. The spongy cells were irregular round shapes mingled with intercellular spaces. There were some significant difference also exited in the transverse section structure of leaf and could be used to separate in the 21 coleus cultivars .The pollen grain morphology of 16 coleus cultivars was investigated by LM and SEM. The major aperture shape of coleus pollen grains were 6-colpate, with 7-colpate and 8-colpate occasionally. The aperture situated at the equator, the colpus with granules. The sculpture was reticulate. the external aperture of pollen in the 16 coleus cultivars were similar , but percentage of pollen aperture with 6-colpate, 7-colpate and 8-colpate in the 16 coleus cultivars were different . The pollen viability of six coleus cultivars (CH007, CH010, CH011, CH017, CH021 and CH022) were tested by Alexander solution. The pollen sampled at 1-7 days after anthesis in summer and winter. In summer, the pollen viability of CH021 and CH011 were highest with 23.9% and 20.0%; that of CH010 was lowest (1.6%). In winter, the pollen ability of CH007 was highest (74.7%); that of CH010 was lowest (4.9%).The pollen ability in winter was higher than those of summer. According to ISSR analysis, six ISSR primers with polymorphic pattern were UBC830, UBC836, UBC841, UBC861, UBC885 and UBC900 and used for the trial. In 21 coleus cultivars, 82 bands were amplified and they were all polymorphism, the percentage of polymorphism were 100%. The similarity coefficient were between 0 and 0.6885. Cluster analysis by using UPGMA method showed that 21 coleus cultivars could be divided to nine groups based on similarity coefficient with 0.43 as the critical point. The group 1 consisted of CH001, CH017, CH020, CH018, CH028, CH012, CH016, CH023, CH009 and CH024; the group 2 consisted of CH004 and CH006; the group 3 was CH025; the group 4 consisted of CH007 and CH011; the group 5 was CH021; the group 6 was CH002; the group 7 consisted of CH022 and CH027; the group 8 was CH010; the group 9 was CH003.

Dissertação de Mestrado em Biodiversidade e Biotecnologia Vegetal apresentada à Faculdade de Ciências e Tecnologia
A familía Ericaceae é constítuida por várias espécies economicamente importantes, tais como as pertencentes ao género Rhododendron and Vaccinium. Entre elas, o arbusto mediterrânico Arbutus unedo L. destaca-se do ponto de vista pomológico e ornamental. Para aprofundar o conhecimento sobre esta espécie, analisaram-se as alterações morfológicas e composicionais que ocorrem na ontogenia dos frutos, com o objetivo de identificar fatores e eventos relacionados com a sua maturação. Deste modo, os resultados da execução de FTIR revelaram alterações ao nível dos polissacarídeos estruturais da parede celular, entre frutos imaturos e maduros. Investigações espetroscópicas adicionais com biomassa intacta dos frutos, revelaram que existem variações composicionais, principalmente decorrentes de polissacarídeos da matriz, tais como as pectinas e hemicelulloses, e também celulose. No decorrer da maturação verificou-se também um aumento no teor de sólidos solúveis totais (˚Brix), e um decréscimo na acidez dos frutos. Por outro lado, a degradação de açúcares estruturais que advém da hidrólise e/ou do processo oxidativo, resulta num decréscimo acentuado da firmeza dos frutos, que leva ao seu amolecimento, e também a variações do seu diâmetro polar e equatorial. Além disso, traçou-se um perfil fenólico preliminar dos frutos recorrendo a RP-HPLC-PDA, concluindo-se que ocorrem poucas variações desses compostos durante a maturação. Por sua vez, no que diz respeito à vitrificação de tecidos, decorrente da sua cultura em meio líquido, investigou-se a parede celular de folhas e caules vitrificados, comparando depois com tecidos não vitrificados. As diferenças nos teores de celulose, polissacarídeos da matriz e lenhina, entre material in vitro não vitrificado e vitrificado, foram reveladas através de técnicas como FTIR., lenhina solúvel em brometo de acetil e quantificação de carbohidratos totais. Contrariamente ao que poderia ser expectável e ao que é normalmente indicado na literatura, os resultados obtidos mostraram, de forma consistente, um teor de lenhina mais elevado nas folhas do que nos caules. Por outro lado, através de microscopia ótica de campo claro, fluorescência e varrimento, revelaram-se informações meticulosas sobre a anatomia de A. unedo, através de comparações entre folhas e caules de um arbusto e de material in vitro, vitrificado e não vitrificado. Em comparação com folhas normais, as vitrificadas evidenciaram uma epiderme desorganizada e fina, com imensos complexos estomáticos anormais presentes na página inferior da folha. O mesófilo apresentava grandes espaços lacunares e não existia um parênquima em paliçada bem definido. No que diz respeito aos caules, as principais diferenças estão relacionadas com as células corticais muito espaçadas devido à presença de pronunciados espaços intercelulares. A presente dissertação contribui para um melhor conhecimento desta espécie economicamente importante, mas pouco conhecida, não só pelo melhor conhecimento do processo de maturação dos frutos, mas também por ajudar a perceber as alterações morfológicas e químicas num fenótipo vitrificado.
The Ericaceae family comprises a wide number of economic relevant species, such as the plants belonging to the Rhododendron and Vaccinium genera. Among them, the Mediterranean evergreen shrub Arbutus unedo L. outstands from the pomological and ornamental point of view. For an in-depth knowledge of this plant, the ripening-derived composition and morphology was monitored, aiming at identifying factors and events related with fruit maturation. Accordingly, to explore the ripening-derived compositional differences, FTIR spectroscopy was performed, founding chemical variations related to the structural polysaccharides of the cell wall, between immature and fully ripen fruits. Further spectroscopic investigations using the fruits intact biomass, revealed more variations, mainly due to matrix polysaccharides like pectins and hemicelluloses, and cellulose. The process of maturation was also found to be related to an increase of total soluble solids content (˚Brix), and a decrease in fruits acidity. Notwithstanding, the hydrolysis and/or oxidative derived-degradation of structural sugars, results in an extreme decline of fruit firmness, leading to fruit softening, and variations in the polar and equatorial diameters. Furthermore, a preliminary phenolic profile of the fruits was also investigated by RP-HPLC-PDA, and only slight variations of the phenolic compounds were observed during fruit maturation. On the other hand, concerning the vitrification (hyperhydricity) phenomenon that outcomes through the culture of axillary shoots in liquid medium, the cell wall of non-vitrified and vitrified in vitro shoots of A. unedo was evaluated, comparing leaves and stems. Regarding on the differences in the amounts of cellulose, matrix polysaccharides and lignin, compositional differences were uncovered in leaves and stems from in vitro shoots, using FTIR spectroscopy, acetyl-bromide soluble lignin method and total carbohydrates estimation. Unexpectedly, our results showed higher amounts of lignin in leaves, comparing to stems. Additionally, meticulous information about the A unedo anatomy was assessed by light, fluorescence and scanning electron microscopy, making comparisons between leaves and stems of non-vitrified and vitrified in vitro shoots, and tissues from a strawberry tree. Comparing with normal leaves, the vitrified ones showed to have a delicate and disorganized epidermis, with many abnormal stomata present in the abaxial surface. The mesophyll has wide lacunar spaces, and lack of palisade parenchyma. Concerning vitrified stems, the major differences relied on the the broad-spaced cortical cells. In the end, the present dissertation contributed to better characterization of this economic relevant species but largely unknown, not only for a better understanding on the fruits ripening events, but also by helping to unveil the chemical and anatomy-related factors of the vitrified phenotype.
Outro - This master thesis was supported by the Project “RENATURE – Valorization of the Natural Endogenous Resources of the Centro Region” (CENTRO-01-0145-FEDER-000007), funded by the Comissão de Coordenação da Região Centro (CCDR-C) and subsidized by the European Regional Developmental Fund (FEDER)

TOOTH/MIR160A regulates leaf margin outgrowth in Arabidopsis thaliana Unlike animals, a striking aspect of the plant development is that they have evolved a flexible pattern of post embryonic development. This exposes them to the challenges of many biotic and abiotic signals throughout their life. So, plants have to evolve/regulate various mechanisms to modulate their growth and development for accomplishing a successful life cycle in the prevailing environmental conditions. Auxin is involved in the initiation of lateral organs at the meristem and serration development along the leaf margin (Bilsborough et al., 2011, Hay et al., 2006). These two developmental mechanisms share common molecular players. For example, CUC2 is required for the boundary formation at the SAM and also is shown to be essential for serration formation at the leaf margin. Similarly, tth shows increased leaf serration phenotype as well as defects in the positioning of flowers at the meristem. This demonstrates the functional significance of TTH-regulated ARFs in controlling auxin mediated developmental pathways. Leaves originate as small lumps of undifferentiated cells at the flanks of the shoot apical meristem which undergo several rounds division and expansion to generate the mature leaf with characteristic size, shape and leaf margin. Both, endogenous as well as environmental factors modulate the growth and development of a leaf. This is evident from the plasticity in leaf form, observed during the life time of a single plant, as well as from the diversity among closely related species living in different habitats. It is well known that pathways controlling leaf form are subjected to the effects of selection and adaptation. Leaf margin is a key feature of the final leaf shape and it contributes to the abundant diversity in leaf form. Leaf margin architecture varies quite significantly from smooth or entire margin to margins with large outgrowths (lobed margins). The evolution and ecological advantages of this diversity is a subject of intense investigation. It also provides a wonderful system to study the mechanistic details of iterative generation of repeated units, which is a common feature in producing many biological shapes. Recent advances in molecular technologies and the availability of genomic resources ushered the identification of new factors involved in leaf margin development. Our current knowledge of this developmental programme is that CUC2 establishes auxin maxima at the leaf margin by reorienting an auxin efflux carrier PIN1 which ultimately results in serration outgrowth (Bilsborough et al., 2011, Hay et al., 2006). A few missing links in this pathway are the mechanistic details of CUC2 function in reorienting PIN1 and the molecular details of auxin mediated serration outgrowth. Forward genetic screens have been valuable in characterizing a genetic pathway even in the post genomic era. An EMS mutagenesis screen was performed in this context to identify novel factors that can improve our understanding of this intricate mechanism. tooth was identified in the M2 population based on its increased leaf serration phenotype. Genetic analysis showed that tth phenotype is due to a monogenic recessive mutation. Along with increased leaf serration, tth also shows various developmental defects such as aberrant phyllotaxy, narrower cotyledons and narrower leaves. Positional cloning and sequencing analysis showed a G to A transition at the AT2G39175 locus which codes for MIR160A. The mutation is at the 7th base position of the mature miRNA sequence. Functional characterization of miRNAs by isolating mutations is hampered by their small genomic sizes. Till now, only a few miRNAs have been characterized by mutational analysis in plants (Allen et al., 2007, Baker et al., 2005, Cartolano et al., 2007, Chuck et al., 2007, Knauer et al., 2013, Nag et al., 2009, Nikovics et al., 2006). miR160-ARF10 regulatory module is shown to be required for leaf blade out growth and serration, but not leaf complexity in tomato (Hendelman et al., 2012). miR160 is coded by 3 loci in Arabidopsis, MIR160A, B and C. All three loci encode identical mature miRNA that targets 3 Auxin response factors, ARF10, 16 and 17. ARFs are the effector molecules of auxin mediated developmental programmes. Genetic analysis showed that enhanced serration outgrowth in tth is due to the up-regulation of its target genes. Here, we have identified a miRNA that negatively regulates serration outgrowth by repressing ARF10, 16 and 17 whose functional significance in regulating leaf margin development was not known previously. Extensive genetic interaction studies have shown that TTH acts in parallel to SAW-BP and MIR164-CUC pathways in regulating leaf margin development. We have also shown that CUC2 and PIN1 are absolutely essential for serration development in tth. CUC2 establishes a pattern required for the expression of ARF10 at the leaf margin. In the absence of CUC2, downstream effector molecules such as ARFs can not perform their function. arf10-2 arf16-2 could reduce, but not suppress serration outgrowth in various mutants suggesting their functional redundancy with other ARF family members. CUC2 establishes auxin maxima at the leaf margin that triggers the degradation of AUX/IAA repressors thereby relieving ARF proteins which mediate serration outgrowth. Whereas, TTH acts at the post transcriptional level for maintaining normal ARF transcript levels Role of SPYINDLY in Arabidopsis leaf margin development SPYNDLY encodes an O-linked N-acetyl glucosamine transferase that acts as a negative regulator of GA response. Consistent with its role in GA response, spy mutants show several GA dependent phenotypes such as early flowering and hyper branched trichomes. spy mutants also show several GA independent phenotypes such as aberrant phyllotaxy and smooth leaf margin. We have studied its role in regulating Arabidopsis leaf serration development. Reporter analysis of ARF10::GUS and CUC2::GUS in spy-3 revealed that SPY is not involved in establishing serration pattern. The spy-3 leaves did not show any defects during the early stages of serration development, but the mature leaves display smooth leaf margin indicating that SPY function is required for serration outgrowth. As shown in the present study, TTH regulated ARFs are also involved in serration outgrowth. Analysis of leaf margin phenotype in tth spy-3 showed that SPY activity is not required for ARF mediated serration outgrowth. Similar genetic interaction studies with SAW-BP pathway mutants showed that leaf margin out growth mediated by meristematic genes is not dependent on SPY function. Genetic interaction studies with MIR164-CUC pathway genes showed that SPY is required for serration outgrowth in these mutants. Interestingly, the cuc2-3 mutant is defective at both patterning and outgrowth of serration. The spy-3 could suppress serration out growth in cuc2-D suggesting that CUC2 mediated serration out growth is dependent on SPY activity. Protein-protein interaction studies between SPY and CUC2 are in progress to demonstrate whether SPY directly interacts with CUC2 or CUC2 derived signal to regulate serration out outgrowth. It is interesting to examine how mutations at SPY locus can abolish serration out growth mediated by CUC2, but does not affect the serration pattern, even though CUC2 is reported to be essential for both the patterning and outgrowth of serration.

Due to two different mutations in the gene that encodes the a1A subunit of voltage-activated CaV 2.1 calcium ion channels, the compound heterozygous tottering/leaner (tg/tgla) mouse exhibits numerous neurological deficits. Human disorders that arise from mutations in this voltage dependent calcium channel are familial hemiplegic migraine, episodic ataxia-2, and spinocerebellar ataxia 6. The tg/tgla mouse exhibits ataxia, movement disorders and memory impairment, suggesting that both the cerebellum and hippocampus are affected. To gain greater understanding of the many neurological abnormalities that are exhibited by the 90-120 day old tg/tgla mouse the following aspects were investigated: 1) the morphology of the cerebellum and hippocampus, 2) proliferation and death in cells of the hippocampal dentate gyrus and 3) changes in basic biochemical parameters in granule cells of the cerebellum and hippocampus. This study revealed no volume abnormalities within the hippocampus of the mutant mice, but a decrease in cell density with the pyramidal layer of CA3 and the hilus of the dentate gyrus. Cell size in the CA3 region was unaffected, but cell size in the hilus of the dentate gyrus did not exhibit the gender difference seen in the wild type mouse. The cerebellum showed a decrease in volume without any decrease in cerebellar cellular density. Cell proliferation and differentiation in the subgranular zone of the hippocampal dentate gyrus remained normal. This region also revealed a decrease in cell death in the tg/tgla mice. Basal intracellular calcium levels in granule cells show no difference within the hippocampus, but an increase in the tg/tgla male cerebellum compared to the wild type male cerebellum. There was no significant difference in granule cell mitochondrial membrane potential within the wild type and mutant animals in either the hippocampus or cerebellum. The rate of reactive oxygen species (ROS) production in granule cells revealed no variation within the hippocampus or cerebellum. The amount of ROS was decreased in cerebellar granule cells, but not granule cells of the hippocampus. Inducing ROS showed no alteration in production or amount of ROS produced in the hippocampus, but did show a ceiling in the amount of ROS produced, but not rate of production, in the cerebellum.

Tottering and leaner mutant mice carry mutations in the pore-forming subunit (1A) of P/Q-type (CaV 2.1) voltage-gated calcium ion (Ca2+) channels that result in reduced Ca2+ current density. Since Ca2+ influx via voltage-dependent Ca2+ channels regulates important Ca2+-dependent neuronal processes including neurotransmitter release and synaptogenesis, we assessed effects of these mutations on hippocampus volume, neuronal density, neuronal morphology of hippocampal pyramidal cells in adult (six-month-old) mice, and adult neurogenesis in three-week-old and six-month-old mice. Hippocampal volume and neuronal density were assessed using hematoxylin and eosin stained serial sections. Neuronal morphology was assessed using Golgi-Cox staining as well as ultrastructural assessment using transmission electron microscopy. Adult hippocampal neurogenesis was assessed using standard 5-bromo-2’-deoxyuridine (BrdU) labeling with fluorescent immunohistochemistry (IHC) and proliferating cell nuclear antigen (PCNA) with diaminobenzidine IHC. To determine neuron and astrocyte survival, we used fluorescent double labeling for neurons with BrdU-neuronal nuclei IHC or astrocytes using BrdU-glial fibrillary acidic protein, respectively. Fluoro-Jade histochemistry was used to assess numbers of degenerating cells in the dentate gyrus subgranular zone. Decreased hippocampus volume was observed in tottering female mice and increased dentate hilar and CA1 cell density in mutant mice compared to wild type mice. Cell proliferation was increased in the hilus and combined CA3, CA2 and CA1 regions of mutant mice compared to wild type mice. Decreased total dendritic length and decreased number of dendritic intersections was observed in tottering mice compared to wild type mice. The decrease in dendritic arborization of tottering mice occurred at the concentric circles close to the neuronal cell body indicating that basal dendrites of CA1 pyramidal neurons are reduced. Taken together, P/Q-type voltage gated calcium channel mutation has age variable influence on adult hippocampal cell proliferation, and it altered neuronal morphology in terms of dendritic complexity in tottering mice, while the leaner mutation reduced mitochondrial density.

碩士
國立臺灣大學
森林學研究所
91
The objectives of this study were to investigate the changes of seedling growth and leaf morphology in different simulated light environments on 3 kinds of native broad-leaved species , namely, Ring-couple oak（Cyclobalanopsis glauca）,Camphor tree （Cinnamomum camphora）and Formosan ash（Fraxinus griffithii）.The simulated relative light intensities were 15%, 30%, 60% and 100%, respectively. The R/FR ratios were 0.6 and 1.3. Results showed that growth of these 3 species were affected by both light intensities and R/FR ratio. And the seedling growth were affected significantly by R/FR ratio in low light intensity. The height, ground diameter, dry weight accumulation, Dickson seedling quality, stomatal density and leaf thickness of these 3 species were increased with light intensity. However, the height of Camphor tree was decreased with light intensity. The T/R ratio, slender index and specific stem length were higher in lower light intensity than those in higher light. The ground diameter and stem weight of these 3 species were decreased in low R/FR ratio. The height growth of Ring-couple oak and Formosan ash were lower in lower R/FR ratio than those in higher ratio. The stem of Camphor tree grew suddenly and the specific stem length was higher in lower R/FR ratio than in higher ratio. All of these 3 species was showed slender growth pattern in lower R/FR ratio. The mean leaf area and specific leaf area were higher in lower light intensity and lower R/FR ratio than those in higher light intensity and higher R/FR ratio the leaf thickness and stomatal density were in the contrast. The ratio of chloroplast, starch grain and oil body area in palisade cell was increased with light intensity. But the amounts of chloroplast, starch grain and oil body in Formosan ash were decreased in higher light intensity. As light intensity being increased, Ring-couple oak and Formosan ash grew badly than those in lower light intensity and R/FR ratio but Camphor tree didn’t.