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The current study focuses on the vascular cambium and the reiterative formation of phloem fiber bundles in cotton stems. The role of the TDIF-PXY-WOX pathway was examined in regulating cambial activity and the differentiation of phloem fibers. A study was conducted to identify and characterize the cotton WOX family genes, focusing on WOX4 and WOX14, aiming to identify and analyze their phylogenetic relationships, tissue-specific expression profiles, functional roles, and metabolic consequences. Through a sequence analysis of the Gossypium hirsutum genome, 42 cotton loci were identified as WOX family members. GhWOX4 exhibited a close homology to 7 loci, while GhWOX14 displayed homology with 8 loci. Tissue-specific expression analysis revealed prominent expression patterns of GhWOX4 and GhWOX14 in cotton internodes and roots, suggesting their involvement in vascular tissue development. Functional studies utilizing VIGS (virus-induced gene silencing) demonstrated that the knockdown of GhWOX4 and GhWOX14 resulted in a significant reduction in stem diameter and bast fiber production. This result suggests that secondary phloem fiber development is regulated by GhWOX4 and GhWOX14 genes in cotton. Additionally, the metabolic profiling of VIGS plants revealed significant alterations in amino acids, organic acids, and sugars, with implications for primary metabolic pathways. These findings suggest that GhWOX4 and GhWOX14 play pivotal roles in cotton plant development, including vascular tissue growth and phloem fiber production, and metabolic regulation.

Lianas de Bignoniaceae são reconhecidas por apresentarem uma variação cambial em seus caules, que promove a formação de cunhas de floema que interrompem o xilema. Uma grande diversidade de formas anatômicas foram descritas para esta variação, assim como o floema resultante dela também foi descrito como sendo distinto do floema normal presente concomitantemente nestes caules. Entretanto, nada se sabe sobre a origem, evolução e diversificação das características anatômicas neste grupo. Por essa razão, o presente estudo teve como objetivo uma análise anatômica dos caules de Bignonieae num contexto filogenético, com o intuito de lançar hipóteses para a evolução da diversidade anatômica na tribo. Para tanto, foi realizada uma análise anatômica caulinar de 54 espécies de Bignonieae, representantes dos 21 gêneros atualmente reconhecidos. Nossos resultados apontam que, não obstante a grande diversidade anatômica presente nos caules de Bignonieae, todas compartilham estágios comuns de desenvolvimento, seguidos de adições terminais que promoveram o aumento da complexidade em seus caules. Além disso, vimos que as diferenças entre o floema normal e o variante tem aumentado ao longo da evolução e está presente em todos os tipos celulares do floema. Vimos ainda que o floema secundário em Bignonieae evolui em direções opostas em diferentes linhagens da tribo, evidenciando que a evolução do floema não segue uma única direção, mas várias. Por fim, este estudo demonstra que análises anatômicas dentro de um contexto filogenético são primordiais por permitirem um maior entendimento dos processos que promoveram a evolução e diversificação dos grupos. / Lianas in Bignoniaceae are well known for presenting a cambial variant in their stems, which develops into phloem wedges that deep furrows the xylem. An enormous diversity of anatomical forms were described as resulting from this cambial variant, as well as the phloem produced by the cambial variant was described as being distinct from the regular phloem concurrently present in these stems. However, nothing is known about the origin, evolution, and diversification of the anatomical traits in this group. Therefore, the present study aimed to provide an anatomical analysis of the stems of Bignonieae (Bignoniaceae) within a phylogenetic framework, in order to address questions on the evolution of anatomical diversity in this tribe. For that reason, here we analyzed the stems of 54 species of Bignonieae, representative of the 21 genera currently known for the tribe. Our results show that, despite the great anatomical diversity present in the stems of Bignonieae, all of them share common developmental stages, which are then followed by subsequent terminal additions that are though to have promoted an augment in the complexity of these stems. Furthermore, our results indicate that the differences found between regular and variant phloem is increasing along time and is present in all cell types of the phloem. Moreover, we found that the secondary phloem in Bignonieae is evolving in opposite directions in distinct lineages of the tribe, evidencing that the evolution of the phloem is not constraint to a single line of specialization. In conclusion, this study demonstrates the importance of anatomical analyses within a phylogenetic framework, allowing for the detection of the processes that have been involved in the evolution and diversification of plant groups.

Thesis (MSc (Genetics. Plant Biotechnology)) --Stellenbosch University, 2008. / The main aim of the work presented in this thesis was to further our understanding of the role of Pyrrophosphate: fructose 6-phosphate 1-phosphotransferase (PFP) in sugarcane, by specifically investigating its potential contribution to phloem metabolism. PFP activity in sugarcane internodal tissue is inversely correlated to sucrose content across varieties that differ in their sucrose accumulation abilities. This apparent correlation is in contrast to previous studies that suggest PFP plays an insignificant role in metabolism. In the first part of this study an immunological characterisation of the two subunits of sugarcane PFP was conducted to establish whether it differ significantly from other plant species in terms of size and distribution. Both the alpha and beta subunit appears to be approximately sixty kilo Daltons in size and uniform in their relative distribution to each other in the various plant organs of sugarcane. Although the observed alpha subunit size is less than that predicted this could be explained at the hand of post translational modification, in essence the sugarcane PFP subunits appear similar than that described for other plants especially that of tobacco which was employed as a model system later on in this study. The only direct way to investigate PFP’s contribution to phloem metabolism is to alter its activity by recombinant DNA technologies. Therefore, in the second part of the study transformation systems were designed for both the constitutive and phloem specific downand up-regulation of PFP activity. For the down-regulation of activity a post transcriptional gene silencing system, i.e. a complementary strand intron hairpin RNA (ihpRNA) silencing system, was employed. A partial sequence of the PFP-beta subunit was isolated and used in vector construction. For the over-expression the Giardia lamblia PFP gene was used. The model plant tobacco was employed to investigate PFP’s effect on phloem metabolism and transport of assimilate. Transgene insertion was accomplished by means of Agobacterium mediated transformation and tissue specific manipulation of PFP activity was confirmed by in situ activity staining.

Lianas de Bignoniaceae são reconhecidas por apresentarem uma variação cambial em seus caules, que promove a formação de cunhas de floema que interrompem o xilema. Uma grande diversidade de formas anatômicas foram descritas para esta variação, assim como o floema resultante dela também foi descrito como sendo distinto do floema normal presente concomitantemente nestes caules. Entretanto, nada se sabe sobre a origem, evolução e diversificação das características anatômicas neste grupo. Por essa razão, o presente estudo teve como objetivo uma análise anatômica dos caules de Bignonieae num contexto filogenético, com o intuito de lançar hipóteses para a evolução da diversidade anatômica na tribo. Para tanto, foi realizada uma análise anatômica caulinar de 54 espécies de Bignonieae, representantes dos 21 gêneros atualmente reconhecidos. Nossos resultados apontam que, não obstante a grande diversidade anatômica presente nos caules de Bignonieae, todas compartilham estágios comuns de desenvolvimento, seguidos de adições terminais que promoveram o aumento da complexidade em seus caules. Além disso, vimos que as diferenças entre o floema normal e o variante tem aumentado ao longo da evolução e está presente em todos os tipos celulares do floema. Vimos ainda que o floema secundário em Bignonieae evolui em direções opostas em diferentes linhagens da tribo, evidenciando que a evolução do floema não segue uma única direção, mas várias. Por fim, este estudo demonstra que análises anatômicas dentro de um contexto filogenético são primordiais por permitirem um maior entendimento dos processos que promoveram a evolução e diversificação dos grupos. / Lianas in Bignoniaceae are well known for presenting a cambial variant in their stems, which develops into phloem wedges that deep furrows the xylem. An enormous diversity of anatomical forms were described as resulting from this cambial variant, as well as the phloem produced by the cambial variant was described as being distinct from the regular phloem concurrently present in these stems. However, nothing is known about the origin, evolution, and diversification of the anatomical traits in this group. Therefore, the present study aimed to provide an anatomical analysis of the stems of Bignonieae (Bignoniaceae) within a phylogenetic framework, in order to address questions on the evolution of anatomical diversity in this tribe. For that reason, here we analyzed the stems of 54 species of Bignonieae, representative of the 21 genera currently known for the tribe. Our results show that, despite the great anatomical diversity present in the stems of Bignonieae, all of them share common developmental stages, which are then followed by subsequent terminal additions that are though to have promoted an augment in the complexity of these stems. Furthermore, our results indicate that the differences found between regular and variant phloem is increasing along time and is present in all cell types of the phloem. Moreover, we found that the secondary phloem in Bignonieae is evolving in opposite directions in distinct lineages of the tribe, evidencing that the evolution of the phloem is not constraint to a single line of specialization. In conclusion, this study demonstrates the importance of anatomical analyses within a phylogenetic framework, allowing for the detection of the processes that have been involved in the evolution and diversification of plant groups.

Orientador: Michel Georges Albert Vincentz / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-09T01:42:08Z (GMT). No. of bitstreams: 1 Silveira_AmandaBortolini_M.pdf: 20873886 bytes, checksum: 0c34c8a141f7bb11f8f3176ba7a976e0 (MD5) Previous issue date: 2007 / Resumo: O crescimento e o desenvolvimento dos organismos são baseados na capacidade celular de expressão gênica diferencial que resulta, principalmente, do controle da taxa de iniciação da transcrição por fatores reguladores de transcrição (FTs). FTs do tipo Basic Leucine QQjJer(bZIP) fQram descritos em todos os eucariotos. Seu domínio conservado é constituído de uma região de ligação ao DNA rica em aminoácidos básicos, flanqueada a um zíper de leucinas responsável pela dimerização. Em angiospermas, os bZIPs são reguladores importantes de processos específicos como fotomorfogênese, desenvolvimento de órgãos, elongação celular, controle do balanço de carbono/nitrogênio, mecanismos de defesa, via de sinalização de hormônios e sacarose, controle osmótico e florescimento. Mostramos que os genomas de Arabidopsis thaliana e Orysa sativa codificam para um conjunto completo e não redundante de 76 e 113 fatores bZIP respectivamente, que foram organizados em 11 grupos de proteínas evolutivamente relacionadas e 33 Possíveis Grupos de Genes Ortólogos (PoGO) de mono e eudicotiledôneas, o que deve permitir racionalizar o processo de caracterização funcional destes fatores em angiospermas. O Grupo C, que inclui genes homólogos ao lócus de regulação Opaco-2 (02) de milho, está organizado em três PoGOS, que possivelmente desempenham três funções ancestrais de angiospermas. Em Arabidopsis estas três possíveis funções ancestrais estão representadas por quatro genes (bZIP' 02 !1omologous, Bzo2h), Bzo2h3/AtbZIP63 (PoGO C1), Bzo2h1/AtbZIP10 e Bzo2h4/ AtbZIP25 (PoGO C2) e Bzo2h2/AtbZIP9 (PoGO C3). Visando um melhor conhecimento sobre a evolução das funções dos fatores bZIP de angiospermas do Grupo C, iniciamos a caracterização funcional de~tes quatro reguladores, focando principalmente em AtbZIP9, um gene único representativo de uma função ancestral de angiospermas e cujo papel ainda é desconhecido. Notamos que a expressão de AtbZIP9 é restrita as células do floema e regulada por glicose, ácido abscísico e citocinina, sugerindo que este gene integra as vias de sinalização destes sinais metabólicos e hormonais no floema. Abordagens de genética reversa como RNAi, knockout e superexpressão não permitiram elucidar de maneira clara a atuação de AtbZIP9 no ciclo de vida de Arabidopsis, indicando que mecanismos de regulação pós-transcricional e/ou redundância genética atuam sobre este gene. Visando dar continuidade e ampliar o estudo funcional de AtbZIP9, foram obtidas linhagens transgênicas de Arabidopsis expressando versões modificadas deste gene que codificam para proteínas ativadoras constitutivas fortes da transcrição. Estas novas versões de AtbZIP9 são teoricamente capazes de ativar de maneira constitutiva a expressão dos genes alvos de AtbZIP9, contornando assim, as dificuldades decorrentes da análise de famílias gênicas que apresentam redundância funcional. Quando comparados a plantas selvagens, transformantes primários para ativadores constitutivos fortes apresentaram diversas alterações de morfologia foliar, além de mudanças metabólicas e fisiológicas como acúmulo de compostos fenólicos em folhas, sintomas de morte celular e senescência. A análise destes transformantes ainda sugere uma possível participação de AtbZIP9 no controle do desenvolvimento do sistema vascular de raízes e folhas. Suspeitamos que as alterações de morfologia foliar e fisiologia observadas possivelmente representem conseqüências de mudanças nas propriedades funcionais de transporte do floema, decorrentes de defeitos no processo de diferenciação e organização das células do cilindro vascular / Abstract: Transcriptional regulatory factors (TFs) play an important role in controlling growth and development of ali organisms. bZIPs TFs have been described in ali eukaryotes and are characterized by a basic aminoacid rich DNA binding domain and a leucine zipper, responsible for dimerization. bZIPs have been reported to act in several different plantspecific processes such as organ development, cell elongation, defense mechanism, hormones and sucrose signalization, light response, control of nitrogen/carbon balance, osmotic control and flowering. We showed that Arabidopsis thaliana and Orysa sativa genomes encode a complete and non-redundant set of 76 and 113 bZIP transcription factors, respectively, which were divided into 11 unique groups of homologous genes. More detailed phylogenetic analysis led to the identification of 33 Possible Groups of Monocot and Eudicot Orthologous Genes (PoGO), which allows rationalizing functional studies in angiosperms. Group C, which includes genes homologous to the maize Opaque-2 locus, is formed by three PoGOs, suggesting that this group represents three ancestral functions among angiosperms. In Arabidopsis these three possible ancestral functions may be represented by the bZIP Qpaque-2. homologous genes (Bzo2h), Bzo2h3/AtbZIP63 (PoGO C1), Bzo2h1/AtbZIP10 and Bzo2h4/AtbZIP25 (PoGO C2) and Bzo2h2/AtbZIP9 (PoGO C3). To get insight into the evolution pattern and function of Group C members, we have iniciated the functional characterization of the Bzo2h genes concentrating initially on AtbZIP9, a unique gene that represents an ancestral function and for which no functional informational is available. We showed that AtbZIP9 expression is restricted to phloem cells and regulated by glucose, abscisic acid and cytokinin, suggesting that this gene is an element of the signalization pathways of these metabolic and hormonal signals in the phloem. Reverse genetic approaches such as RNAi, knockout and superexpression failed to reveal the biological function of AtbZIP9 in Arabidopsis life cycle and suggested that post-transcriptional regulation and/or functional redundancy may act on AtbZIP9. In order to improve our Rnowledge on AtbZIP9 function, Arabidopsis transgenic lines expressing constitutive transcriptional activator versions of AtbZIP9 were obtained. Since such modified versions of AtbZIP9 are theoretically able to promote the superexpression of AtbZIP9 target genes, this strategy should be independent of functional redundancy. When compared to wild type plants, primary transformants for constitutive transcriptional activator versions of AtbZIP9 showed alterations of leaf morphology, as well as metabolic and physiologic modifications, such as phenolic compound accumulation in leaves, cell death and senescence symptoms. Analyses of this transformants also suggest that AtbZIP9 is possibly involved in the control of leaf and root vascular system development. We suspect that the alteration of leaf morphology and physiology observed in primary transformants possibly reflects consequences of changes in phloem transport functional properties, due to defects in vascular cylinder cell differentiation and organization / Mestrado / Genetica Vegetal e Melhoramento / Mestre em Genética e Biologia Molecular

Les phytovirus empruntent les vaisseaux du phloème pour envahir leur plante hôte de manière systémique. Ce mouvement étant très mal connu, l’objectif de cette étude était d’identifier par une approche transcriptomique, des gènes spécifiquement dérégulés dans les cellules compagnes (CC) suite à l’infection virale par un Polerovirus, le Turnip yellows virus (TuYV) ou par un Potyvirus, le Lettuce mosaic virus (LMV). Pour ce faire, des protoplastes de CC ont été préparés et triés par la technologie de FACS. Les ARN extraits ont ensuite été traités par RNAseq et hybridation sur puces CATMA. Malgré d’importantes variations entre les expériences, nous avons identifié des processus biologiques communs affectés par les infections virales : la voie d’assimilation du soufre et le mécanisme de résistance systémique acquise (SAR) pour le LMV, et la voie de biosynthèse des glucosinolates pour le TuYV. Pour compléter cette étude, une banque d’ADNc spécifique des CC a été construite et criblée en utilisant le domaine C-terminal de la protéine RT du TuYV. Une interaction avec la protéine CIPK7 a été détectée et le rôle potentiel de cette interaction dans le cycle viral a été étudié in planta. / Phytoviruses invade systemically their host plant through the phloem. As this viral step remains poorly understood, the aim of this work was to identify, using a transcriptomic approach, genes specifically deregulated in companion cell (CC) during infection with the Polerovirus Turnip yellows virus (TuYV) and the Potyvirus Lettuce mosaic virus (LMV). CC protoplasts were prepared and sorted by FACS technology. Extracted RNA were further analyzed by RNAseq andCATMA microarrays. Although considerable variations between the experiments were observed,we were able to identify common biological processes affected by viral infections: sulfate assimilation and systemic acquired resistance (SAR) mechanism for LMV and glucosinolate biosynthesis for TuYV. To complete this study on systemic viral movement, a CC-specific cDNA library was constructed and screened using the TuYV RT C-terminal domain as a bait. An interaction with the AtCIPK7 protein was retrieved, a protein kinase interacting with calcineurin Blike proteins. The potential role of this interaction in the viral cycle in planta was further investigated in planta.

Potato is one of the most important agricultural crops and there is an attempt to increase and improve yields of tubers, among other things, by elucidation of the mechanisms that regulate the process of tuber induction. Potato tuberization is a morphogenetic process in which the tubers are formed from the underground parts of the stem - stolons. The correct timing of this process is controlled by a complex regulatory network and influenced by many internal and external factors. Under favourable conditions, an inductive signal is generated in the leaves and it is transported to the stolon by a "phloem information superhighway" driven by carbohydrates flow. The signal triggers cell division, expansion, and changes in the cell growth orientation in the stolon. The development of tubers is influenced by number of biochemical and morphological processes driven by a regulatory network of genes that are expressed in different parts of plants. This work was focused on Solanum tuberosum, Lada cultivar and its derived D69 mutant line with lacking isoform of manganese-stabilizing protein (MSP), which is so far the only dissimilarity identified under all tested conditions. I aimed to map the processes related to the production of carbohydrates in leaves (photosynthetic characteristics - rate of photosynthesis...

El virus de la tristeza de los cítricos (Citrus tristeza virus; CTV) es el agente causal de unas de las enfermedades virales de los árboles cítricos más devastadoras en el mundo. CTV está restringido al floema en su huésped cítrico natural, y ha desarrollado tres proteínas supresoras de silenciamiento que actúan a nivel intra-(p23 y p20) e intercelular (p20 y p25) para superar la fuerte defensa antiviral del huésped. La interferencia de RNA, una aproximación basada en el uso de dsRNA para desencadenar el silenciamiento de RNA, ha sido utilizada ampliamente para generar plantas transgénicas resistentes a virus. Considerando el importante papel de p23, p20 y p25 en la patogénesis de CTV, hemos transformado plantas de lima Mexicana con un vector intrón-horquilla que porta la secuencia completa en versión no traducible de los genes p25, p20, p23 y el extremo 3¿-UTR de la cepa T36 de CTV, para intentar silenciar su expresión en células infectadas. Se ha observado resistencia completa a la infección viral en tres líneas transgénicas, manteniéndose todas sus propagaciones asintomáticas y libres de virus tras ser inoculadas mediante injerto con CTV-T36, tanto en el portainjertos no transgénico como directamente sobre la variedad transgénica. La acumulación de siRNA derivados del transgén fue necesaria pero no suficiente para lograr resistencia frente a CTV en las plantas. Al inocular propagaciones de las líneas transgénicas inmunes con una cepa de CTV divergente, la resistencia fue parcialmente superada, destacando la importancia de la identidad de secuencia en el mecanismo subyacente a la interferencia de RNA. Este trabajo es el primero en que se consigue resistencia completa a CTV en un huésped cítrico muy sensible, actuando simultáneamente sobre los tres supresores virales de silenciamiento mediante interferencia de RNA. La proteína p23 codificada por el virus es además un importante factor de patogenicidad. La expresión ectópica de p23 en plantas de cítricos induce aberraciones fenológicas semejantes a síntomas de CTV. Para estudiar en más detalle el papel de p23 en la patogénesis de CTV, se ha sobre-expresado en lima Mexicana el gen p23 de CTV T36 y tres versiones truncadas del mismo bajo el control del promotor 35S del virus del mosaico de la coliflor (Cauliflower mosaic virus). Solo la versión truncada, que expresa los aminoácidos del 1 al 157 (p23-¿157) indujo síntomas similares a los producidos por CTV, aunque más suaves que los inducidos por la expresión de la proteína p23 entera (209 aminoácidos), permitiendo delimitar la región responsable de la patogénesis de p23 en cítricos a un fragmento de 157 aminoácidos que incluye el dedo de zinc y los motivos básicos flanqueantes de la proteína. La actividad de p23 como supresor de silenciamiento de RNA en N. benthamiana se perdía en todos los mutantes de p23 probados, lo cual indica que la supresión de silenciamiento implica a la mayoría de las regiones de la proteína. Para profundizar más en el papel de p23 en la patogénesis, en un siguiente paso hemos restringido la expresión de transgenes derivados de p23 a células asociadas al floema de lima Mexicana mediante el uso del promotor especifico de floema del virus del moteado amarillo de la comelina (Commelina yellow mottle virus, CoYMV). Se transformó lima Mexicana con construcciones que portaban el gen p23 completo, ya sea de la cepa agresiva de CTV T36 o de la suave T317, o con un fragmento que comprende el dedo de zinc y los motivos básicos flanqueantes de la primera, todas ellas bajo el control bien del promotor de CoYMV o bien del promotor constitutivo 35S. La expresión de estas construcciones en el floema dio lugar a aberraciones semejantes a los síntomas específicos de CTV, pero no a los síntomas inespecíficos observados cuando se expresaba p23 de forma constitutiva. Por otra parte, la apariencia e intensidad de las aberraciones fenotípicas más notorias similares a síntomas inducidos por CTV generadas por la expresión específica en floema del gen p23 se relacionó positivamente con la agresividad de la cepa origen utilizada. Además, la expresión en tejidos floemáticos del fragmento de p23 que comprende el dominio de dedo de zinc y los motivos básicos flanqueantes fue suficiente para inducir síntomas semejantes a los producidos por la infección con CTV, confirmando así que la región N-terminal delimitada por los aminoácidos 1 y 157 podría determinar, al menos en parte, la patogénesis de CTV en lima Mexicana. / Citrus tristeza virus (CTV) is the causal agent of one of the most devastating viral diseases of citrus trees in the world. CTV is phloem-restricted in natural citrus hosts, and has evolved three silencing suppressor proteins acting at intra- (p23 and p20) and inter-cellular level (p20 and p25) to overcome strong host antiviral defense in citrus. RNA interference (RNAi), an approach based on using dsRNA to trigger RNA silencing, has been widely used for generating transgenic plants resistant against viruses. Considering the important role of p23, p20 and p25 in CTV pathogenesis, we have transformed Mexican lime plants with an intron-hairpin vector carrying full untranslatable versions of genes p25, p20, p23 and the 3¿-UTR from the CTV strain T36, to attempt silencing their expression in CTV-infected cells. Complete resistance to viral infection was observed in three transgenic lines, with all their propagations remaining symptomless and virus-free after graft-inoculation with CTV-T36, either in the non-transgenic rootstock or directly in the transgenic scion. Accumulation of transgene-derived siRNAs was necessary but not sufficient for CTV resistance. Challenging immune transformants with a divergent CTV strain resulted in partial breakage of the resistance, stressing the importance of sequence identity in the underlying RNAi mechanism. This is the first evidence that it is possible to achieve full resistance to CTV in a highly sensitive citrus host by targeting simultaneously its three viral silencing suppressors through RNAi. The p23 protein encoded by the virus is additionally an important pathogenicity factor. Ectopic expression of p23 in transgenic citrus plants induces developmental aberrations resembling CTV symptoms. To explore in more detail the role of p23 in CTV pathogenesis, the p23 gene from CTV T36 and three truncated versions thereof under the control of the Cauliflower mosaic virus 35S promoter were used to transform Mexican lime. Only the truncated version expressing amino acids 1 to 157 (p23¿158-209) elicited CTV-like symptoms, similar to, albeit milder than, those incited by expressing the whole p23 protein (209 amino acids), thus delimiting the region responsible for p23 pathogenesis in citrus to a 157 amino acid fragment including the Zn finger and flanking basic motifs of the protein. RNA silencing suppressor activity of p23 in N. benthamiana was abolished by all mutants tested, indicating that silencing suppression involves most p23 regions. To better define the role of p23 in CTV pathogenesis, we next restricted the expression of p23-derived transgenes to phloem-associated cells in Mexican lime plants by means of using the phloem-specific promoter from Commelina yellow mottle virus (CoYMV). Constructions carrying the complete gene p23 from either the severe T36 or the mild T317 CTV strains, or a fragment comprising the zinc-finger and flanking basic motifs from the former, either under the control of the CoYMV promoter or the constitutive 35S promoter were used for genetic transformation of Mexican lime. Expression of these constructs in the phloem incited aberrations resembling CTV-specific symptoms, but not the unspecific symptoms observed when p23 was constitutively expressed. Moreover, appearance and intensity of the most notorious CTV-like phenotypic aberrations induced by the phloem-specific expression of the p23 gene were positively related with the aggressiveness of the source CTV strain used. Additionally, expression in phloem-tissues of the p23 fragment comprising the zinc-finger domain and flanking basic motifs was sufficient to induce CTV-like symptoms, corroborating that the N-terminal region (delimited by amino acids 1 and 157) determines, at least in part, CTV pathogenesis in Mexican lime. / Soler Calvo, N. (2013). Transgenic resistance against Citrus tristeza virus (CTV) and analysis of the viral p23 protein as pathogenicity determinant in citrus [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/31631

Phloem loading is the process by which sugars are loaded into the phloem of source leaves and then subsequently transported to sink organs via bulk flow driven by hydrostatic pressure. Three loading mechanisms are described: passive, polymer trap, and apoplastic loading. In passive loading, sucrose diffuses from mesophyll through plasmodesmata into the phloem. The two energized loading mechanisms are the polymer trap and apoplastic loading. In the polymer trap, sucrose moves into intermediary cells and is synthesized into oligosaccharides that become "trapped." In apoplastic loading, sucrose is transported into the apoplast by SWEETs, and subsequently taken up by SUTs in a proton-sucrose symport mechanism, concentrating sucrose in companion cells. Herbaceous species tend to use active loading, while woody species tend to use passive loading. Confirming either passive or energized loading is not without ambiguity. Cotton was investigated as a model because its phloem loading mechanism is ambiguous. Cotton was expected to use passive loading. However, experiments showed that active sucrose accumulation occurs in leaves through GhSUT1-L2, suggesting plasmodesmata are not always a reliable indicator of passive loading and passive loading should not be assumed for woody plants. Genetic manipulation of carbohydrate transport could prove helpful for improving productivity and challenging the passive loading hypothesis. To test this, constitutive and phloem-specific AtSUC2 expression in poplar was used to (1) test the conservation of AtSUC2 expression and (2) test for apoplastic phloem loading. Poplar expressing AtSUC2 were expected to show conserved expression and apoplastic loading. Poplar expressing AtSUC2 shared a conserved vascular-specific pattern with Arabidopsis but did not load from the apoplast. These results suggest that there is conservation of companion cell identity between poplar and Arabidopsis, passive loading is the loading mechanism in poplar.