La caracterització funcional de dos gens en la peridermis, la ω hidroxilasa d'àcids grassos CYP86A33 -candidata per la funcionalització del carboni ω-terminal dels monòmers alifàtics de la suberina- i la ketoacyl-CoA sintasa StKCS6 -candidata per elongar àcids grassos o derivats llargs de suberina i ceres- es realitza per silenciament per RNA d'interferència en patata. La deficiència de CYP86A33 comporta una gran reducció dels monòmers principals de la suberina, l'àcid gras ω-hidroxilat i l'α,ω-diàcid C18:1, juntament amb una reducció total de la quantitat de suberina del 60%. Aquesta deficiència altera l'estructura lamel·lar típica de la suberina, així com també la funció barrera de la peridermis. La deficiència en StKCS6 comporta que els monòmers de la suberina de 28 carbonis o més llargs es redueixin i que els de 26 carbonis o més curts s'incrementin. Aquesta deficiència suggereix que la llargada dels compostos alifàtics pot contribuir a les propietats impermeabilitzants de la peridermis. / The functional characterization of two genes in the periderm, the ω-hydroxylase CYP86A33 -candidate for the functionalization of the ω-terminal carbon of suberin aliphatic compounds- and the putative ketoacyl-CoA synthase StKCS6 -candidate for the elongation of VLCFA and derivatives of suberin and waxes of periderm- is performed by RNA interference-mediated silencing in potato The CYP86A33 deficiency leads to a great reduction of the main suberin monomers, the C18:1 ω-hydroxyacid and α,ω-diacid, together with an overall decrease of the suberin total amount by 60%. The deficiency in these ω-oxidized fatty acids alters the typical suberin lamellar structure as well as the periderm water barrier function.StKCS6 deficiency leads to a decrease of suberin and wax compounds with chain-length C28 and higher and an increase of those with chain-length C26 and lower. This deficiency suggests that the aliphatics chain-length can contribute to the sealing properties of periderm.

Activation-tagging is a functional genomics technique where strong enhancers are inserted randomly into target genomes to over-activate endogenous genes. Phenotypes of interest can be selected for investigation of genetic factors contributing to the mutant phenotype. From initial screens of a population of activation-tagged potato, a mutant with chocolate-coloured tuber skin has been identified. In this thesis, a novel sequence capture method for identifying T-DNA loci in activation tagged potato was used to characterize chocolate’s single T-DNA insertion locus. Transcriptome analysis of tuber periderm tissue was used to identify major processes occurring in the chocolate mutant. Our data suggest activation of a chitin-binding receptor-like kinase located 65 kb from T-DNA insert may cause activation of immune signaling pathways in chocolate. The present work explores a putative model of transcriptional and cellular responses involved in gain-of-function immune receptor activation. Selectively, these findings illustrate the periderm tissue as an important area of defense charged against biotic and abiotic stresses. Periderm development and anatomy are highly important for tuber storage. Further characterization of potato tuber periderm may contribute knowledge to model periderm systems and have implications for molecular breeding strategies to improve tuber storage quality. / Thesis (Master, Biology) -- Queen's University, 2012-09-27 11:45:16.478

Non-syndromic orofacial clefting (nsOFC) is among the most common congenital birth defects occurring up to 1 in 800 live births, with genetic and environmental causes. Genome wide association studies (GWAS) have identified several genetic loci that confer risk for nsOFC. However, more than half the heritable risk for nsOFC remains unknown and is considered ‘missing’. Moreover, continued sequencing of nsOFC patient DNA by whole exome sequencing and whole exome sequencing identify hundreds of single nucleotide polymorphism (SNPs). The identification of causal SNPs, however, continues to be a challenge in the OFC community. This is fueled partly by a lack of understanding of: (i) molecular mechanism and, (i) the gene regulatory network (GRN) governing differentiation of the relevant tissue, the embryonic superficial epithelia, also known as the periderm. Research has demonstrated that aberrant differentiation of the periderm, particularly the oral periderm results in pathological adhesions of surfaces within the developing oral cavity resulting in OFC. Further these adhesions can extend to the limbs which is a hallmarks feature in some forms of syndromic OFC (sOFC). In zebrafish, our model system of choice, knock-out of interferon regulatory factor 6 (irf6) ablated periderm marker expression and subsequently induces early embryonic lethality. The ortholog of IRF6 is a major genetic locus of Van der Woude syndrome (VWS) the most common form of sOFC and variants of IRF6 elevate risk for nsOFC. Therefore, we hypothesize that GRN of zebrafish periderm differentiation under the control of irf6 is a tool that can be used to identify novel OFC loci. Supporting this view, we have recently demonstrated that knock-down of an irf6 dependent gene encoding transcription factor Grainy-head like 3 (Grhl3) results in aberrant zebrafish periderm differentiation and GRHL3 was recently discovered as a novel VWS genetic locus. Hence it is likely that orthologs of genes encoding additional members of the periderm GRN harbor mutations in OFC patients. To identify cis–regulatory and transcriptional components in the periderm GRN, we performed: (i) a screen for periderm enhancers through in vivo green fluorescent protein (GFP) reporter assays, and, (ii) irf6 RNA-seq, followed by irf6 ChIP-seq to identify direct targets. From our screen for cis-regulatory elements we have identified a candidate human ZNF750 enhancer that directs GFP reporter expression in the zebrafish periderm. From our screen for irf6 direct targets we have identified several transcription factors including klf17, tfap2a and grhl3, all of which have variants in the human orthologs found in OFC patients. We further resolve the structure of the periderm differentiation GRN in zebrafish by assessing loss of function profiles for klf17, tfap2a and grhl3. Additionally, among the irf6 direct targets is a gene encoding another transcription factor, Zinc finger protein 750 (Znf750). We provide evidence to show that znf750 is expressed weakly in the zebrafish periderm. Further, we sequenced DNA in 500 nsOFC patient samples and identify a novel missense Ser160Pro ZNF750 variant which phenocopies the early embryonic lethality observed in irf6 mutants. Therefore, investigation of the zebrafish periderm GRN structure has facilitated the identification of OFC-associated risk loci.

Orientador: Carmen Regina Marcati / Resumo: Avaliar a estrutura anatômica de plantas que crescem em diferentes ambientes é uma maneira de compreender como as plantas se adaptam às variações destes ambientes. Algumas destas adaptações influenciam no transporte de água e de fotoassimilados, na proteção dos tecidos internos, na força mecânica e na capacidade de armazenamento dos tecidos, que são funções associadas ao caule das plantas. Assim, neste trabalho, avaliamos a estrutura caulinar de duas espécies, Moquiniastrum polymorphum e Zanthoxylum rhoifolium que ocorrem simultaneamente em diferentes tipos vegetacionais: o cerrado sensu stricto, o cerradão, a floresta estacional semidecídua e a floresta ombrófila densa. Os três primeiros tipos vegetacionais têm um período de seca durante o ano, enquanto que na floresta ombrófila densa o regime pluviométrico é relativamente constante ao longo do ano. Os solos de cada local apresentam diferentes propriedades físicas e químicas e no cerrado sensu stricto o fogo é um fator ambiental que pode ocorrer naturalmente. Estes fatores podem influenciar a estrutura anatômica dos tecidos vegetais. Para a descrição anatômica coletamos amostras do caule (a 1,30 m do solo) contendo xilema secundário e casca, pelo método não destrutivo, de cinco indivíduos de cada tipo vegetacional, que foram processadas conforme técnicas usuais em anatomia da madeira. Para verificar as diferenças entre os tipos vegetacionais, nós comparamos as características anatômicas por meio de uma análise de variância. ... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

La peridermis és una estructura complexa que protegeix els òrgans vegetals madurs (secundaris) i les zones que han sofert ferides de la pèrdua d'aigua i dels patògens. Aquesta funció barrera és deguda al fel·lema o súber, un teixit format per cèl·lules suberificades. Tant el fel·lema com la suberina són crucials per la vida de les plantes terrestres, però pràcticament no es coneix res dels processos moleculars que regulen la seva formació, probablement degut a la manca de models adequats. En aquesta tesi s'han identificat i caracteritzat gens induïts al fel·lema mitjançant la combinació de dues plantes models. L'escorça d'alzina surera (Quercus suber) s'ha utilitzat per aïllar gens candidats de la formació del fel·lema i per investigar el comportament d'alguns d'aquests gens durant l'estació de creixement, mentre que la pela de la patata (Solanum tuberosum) s'ha utilitzat en estudis de genètica reversa per demostrar la funció d'alguns gens reguladors al fel·lema. / The periderm is a complex structure that protects plant mature (secondary) organs and wounded tissues from water loss, injuries and pathogens. This barrier capacity is accomplished by the cork layer of the periderm, a tissue made of dead cells with suberin deposited into cell walls. Although cork and suberin are critical for the survival of land plants, very few is known about the molecular processes involved in their biosynthesis and differentiation, probably due to the lack of appropriate plant models. Here we developed a strategy to identify and characterize cork candidate genes using a combination of two model plants for periderm studies. The bark of cork oak (Quercus suber) was used to identify candidate genes and to analyze the seasonal behaviour of some of these genes. The potato (Solanum tuberosum) tuber was used to demonstrate the role of some selected candidates in the regulation of cork by reverse genetic analyses.

Orientador: Carmen Regina Marcati / Resumo: A casca é um sistema biológico complexo que desempenha diversas funções na planta, incluindo condução de fotoassimilados, suporte mecânico, armazenamento de substâncias e proteção contra herbívoros, patógenos e intempéries como o fogo. A casca é composta, principalmente, pelo floema secundário e pela periderme, e reveste tanto caules quanto raízes. O caule está exposto à atmosfera e tem funções de elevação e suporte da planta, enquanto a raiz está exposta ao solo e tem funções de fixar a planta ao solo, armazenar substâncias e absorver e conduzir água e nutrientes. Contudo, pouco se sabe se as diferentes funções de caules e raízes indicam diferentes funções na casca de cada órgão. Neste trabalho, comparamos a casca de caules e raízes de 15 espécies representativas do cerrado paulista e testamos se a casca do caule apresentaria funções de suporte e proteção, enquanto a casca da raiz apresentaria função de armazenamento de substâncias. Também testamos se encontraríamos maior eficiência na condução de fotoassimilados na casca da raiz. Para tanto, selecionamos 15 espécies de árvores e arbustos do cerrado sensu stricto e amostramos a casca do caule e da raiz. Analisamos a estrutura (espessura e anatomia), a densidade e a química (água, açúcares solúveis, amido, nitrogênio, fósforo e carbono) e relacionamos com as funções da casca em cada órgão. Na casca do caule, encontramos maior espessura da periderme, devido ao felema mais largo com células maiores e mais espessas, e menor dens... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Bark is a complex biological system that performs multiple functions in plant, including photoassimilates conduction, mechanical support, storage and protection against herbivores, pathogens and fire. Bark comprises mainly the secondary phloem and the periderm, and covers both trunks and roots. The trunk is exposed to the atmosphere and has the main functions of plant elevation and plant support, whereas the root is exposed to the ground and has the main functions of fix the plant to the ground, store substances, provide water and nutrients to the plant. However, remain unclear whether the different functions of trunk s and roots indicate different functions in the bark of each organ. In this work, we compared the bark of trunk and roots of species in the cerrado of São Paulo, tested whether the trunk bark presents both support and protection functions, whereas the root bark presents storage function. We also tested whether we would find higher efficiency of photoassimilates conduction in the root bark. For this purpose, we selected 15 representative species of trees and shrubs in the cerrado sensu stricto and sample both trunk and root barks. We analyzed the structure (thickness and anatomy), density and chemistry (water, soluble sugars, starch, nitrogen, phosphorus and carbon) and associated to the bark functions in each organ. In the trunk bark, we found thicker periderm, due to thicker phellem with larger cell and thicker wall cells, and lower periderm density, suggesting... (Complete abstract click electronic access below) / Doutor