Os chamados ‘complexos de espécies’ são definidos como grupos de organismos que compartilham características morfológicas muito semelhantes. Os complexos de espécies representam um problema para os sistemas de classificação baseados apenas em caracteres morfológicos, uma vez que os critérios para delimitação de espécies são subjetivos e, por isso, variam de acordo com cada taxonomista. O complexo integrifolia, que reúne diversos taxa com características florais muito semelhantes à espécie Petunia integrifolia (Hook.) Schinz & Thell, é um exemplo dessa problemática taxonômica. A determinação de espécies dentro desse complexo, baseada apenas em caracteres morfométricos, é até hoje ainda muito controversa. Nesse trabalho, os espaçadores internos transcritos do DNA nuclear ribossomal (ITS1 e ITS2) e dois espaçadores intergênicos (trnS-trnG e psbA-trnH) do DNA plastidial (cpDNA) foram seqüenciados em 69 indivíduos pertencentes a cinco entidades taxonômicas do complexo integrifolia na tentativa de entender sua história evolutiva e melhor contribuir para a correta delimitação das espécies. Análises populacionais e filogeográficas dos três marcadores do cpDNA mostraram que apenas a entidade taxonômica descrita como Petunia interior pode ser considerada uma espécie distinta de Petunia integrifolia. As outras quatro entidades taxonômicas estão divididas em duas linhagens genéticas independentes e alopátricas, que surgiram mais ou menos na mesma época após um evento de diminuição populacional seguido de rápida expansão. Uma dessas linhagens está localizada na planície costeira do Rio Grande do Sul e Santa Catarina, enquanto a outra linhagem se distribui na porção continental do RS ao oeste da Lagoa dos Patos. Análises morfométricas mais detalhadas mostram que essas duas linhagens genéticas podem ser distinguidas taxonomicamente e, portanto, são definidas como duas subespécies de Petunia integrifolia. Há indícios de que um processo de especiação por adaptação a dois ambientes distintos (alta salinidade na planície costeira e baixa salinidade na porção continental) esteja envolvido na divergência dessas duas linhagens. No entanto, para confirmar essa hipótese, são necessários estudos adicionais. / “Species complex” are usually defined as group of species that are morphologically very similar and consequently are very difficult to distinguish. Species complexes, therefore, represent a serious problem to the classification systems based only in morphological characters, the criteria used to delimit species being subjective. The integrifolia complex, that congregates taxa with floral characteristics very similar to Petunia integrifolia (Hook.) Schinz & Thell, it is an example of this taxonomic challenge. The determination of the species inside of this complex, based only in morphometric characters, it is still very controversial. In this work, the internal transcribed spacers of the ribosomal nuclear DNA (ITS1 and ITS2) and two intergenic spacers (trnS-trnG and psbAtrnH) of the plastidial DNA (cpDNA) had been sequenced in 69 individuals pertaining to five taxonomic entities of the integrifolia complex, in the attempt to understand its evolutionary history and contribute to the better delimitation of the species. Populational and phylogeographic analyses of the three markers and of cpDNA had shown that only the taxonomic entity described as Petunia interior can be considered a distinct species of Petunia integrifolia. The four other taxonomic entities are divided in two independent and allopatric lineages, that diversified almost simultaneously after an event of population bottleneck followed by a fast expansion. One of these lineages is located in the coastal plain of the Rio Grande do Sul and Santa Catarina states, while to other lineage it’s distributed in the continental region of the Rio Grande do Sul to the west of the Lagoa dos Patos. Detailed morphometric analyses shown that these two lineages can be taxonomically distinguished and therefore, they may be considered as two subspecies of Petunia integrifolia. Some findings suggest that a process of adaptation to these two distinct environments (high salinity in the coastal plain and low salinity in the continental region) may be involved in the divergence of the two lineages. Additional studies are required to test this hypothesis.

Os chamados ‘complexos de espécies’ são definidos como grupos de organismos que compartilham características morfológicas muito semelhantes. Os complexos de espécies representam um problema para os sistemas de classificação baseados apenas em caracteres morfológicos, uma vez que os critérios para delimitação de espécies são subjetivos e, por isso, variam de acordo com cada taxonomista. O complexo integrifolia, que reúne diversos taxa com características florais muito semelhantes à espécie Petunia integrifolia (Hook.) Schinz & Thell, é um exemplo dessa problemática taxonômica. A determinação de espécies dentro desse complexo, baseada apenas em caracteres morfométricos, é até hoje ainda muito controversa. Nesse trabalho, os espaçadores internos transcritos do DNA nuclear ribossomal (ITS1 e ITS2) e dois espaçadores intergênicos (trnS-trnG e psbA-trnH) do DNA plastidial (cpDNA) foram seqüenciados em 69 indivíduos pertencentes a cinco entidades taxonômicas do complexo integrifolia na tentativa de entender sua história evolutiva e melhor contribuir para a correta delimitação das espécies. Análises populacionais e filogeográficas dos três marcadores do cpDNA mostraram que apenas a entidade taxonômica descrita como Petunia interior pode ser considerada uma espécie distinta de Petunia integrifolia. As outras quatro entidades taxonômicas estão divididas em duas linhagens genéticas independentes e alopátricas, que surgiram mais ou menos na mesma época após um evento de diminuição populacional seguido de rápida expansão. Uma dessas linhagens está localizada na planície costeira do Rio Grande do Sul e Santa Catarina, enquanto a outra linhagem se distribui na porção continental do RS ao oeste da Lagoa dos Patos. Análises morfométricas mais detalhadas mostram que essas duas linhagens genéticas podem ser distinguidas taxonomicamente e, portanto, são definidas como duas subespécies de Petunia integrifolia. Há indícios de que um processo de especiação por adaptação a dois ambientes distintos (alta salinidade na planície costeira e baixa salinidade na porção continental) esteja envolvido na divergência dessas duas linhagens. No entanto, para confirmar essa hipótese, são necessários estudos adicionais. / “Species complex” are usually defined as group of species that are morphologically very similar and consequently are very difficult to distinguish. Species complexes, therefore, represent a serious problem to the classification systems based only in morphological characters, the criteria used to delimit species being subjective. The integrifolia complex, that congregates taxa with floral characteristics very similar to Petunia integrifolia (Hook.) Schinz & Thell, it is an example of this taxonomic challenge. The determination of the species inside of this complex, based only in morphometric characters, it is still very controversial. In this work, the internal transcribed spacers of the ribosomal nuclear DNA (ITS1 and ITS2) and two intergenic spacers (trnS-trnG and psbAtrnH) of the plastidial DNA (cpDNA) had been sequenced in 69 individuals pertaining to five taxonomic entities of the integrifolia complex, in the attempt to understand its evolutionary history and contribute to the better delimitation of the species. Populational and phylogeographic analyses of the three markers and of cpDNA had shown that only the taxonomic entity described as Petunia interior can be considered a distinct species of Petunia integrifolia. The four other taxonomic entities are divided in two independent and allopatric lineages, that diversified almost simultaneously after an event of population bottleneck followed by a fast expansion. One of these lineages is located in the coastal plain of the Rio Grande do Sul and Santa Catarina states, while to other lineage it’s distributed in the continental region of the Rio Grande do Sul to the west of the Lagoa dos Patos. Detailed morphometric analyses shown that these two lineages can be taxonomically distinguished and therefore, they may be considered as two subspecies of Petunia integrifolia. Some findings suggest that a process of adaptation to these two distinct environments (high salinity in the coastal plain and low salinity in the continental region) may be involved in the divergence of the two lineages. Additional studies are required to test this hypothesis.

Os chamados ‘complexos de espécies’ são definidos como grupos de organismos que compartilham características morfológicas muito semelhantes. Os complexos de espécies representam um problema para os sistemas de classificação baseados apenas em caracteres morfológicos, uma vez que os critérios para delimitação de espécies são subjetivos e, por isso, variam de acordo com cada taxonomista. O complexo integrifolia, que reúne diversos taxa com características florais muito semelhantes à espécie Petunia integrifolia (Hook.) Schinz & Thell, é um exemplo dessa problemática taxonômica. A determinação de espécies dentro desse complexo, baseada apenas em caracteres morfométricos, é até hoje ainda muito controversa. Nesse trabalho, os espaçadores internos transcritos do DNA nuclear ribossomal (ITS1 e ITS2) e dois espaçadores intergênicos (trnS-trnG e psbA-trnH) do DNA plastidial (cpDNA) foram seqüenciados em 69 indivíduos pertencentes a cinco entidades taxonômicas do complexo integrifolia na tentativa de entender sua história evolutiva e melhor contribuir para a correta delimitação das espécies. Análises populacionais e filogeográficas dos três marcadores do cpDNA mostraram que apenas a entidade taxonômica descrita como Petunia interior pode ser considerada uma espécie distinta de Petunia integrifolia. As outras quatro entidades taxonômicas estão divididas em duas linhagens genéticas independentes e alopátricas, que surgiram mais ou menos na mesma época após um evento de diminuição populacional seguido de rápida expansão. Uma dessas linhagens está localizada na planície costeira do Rio Grande do Sul e Santa Catarina, enquanto a outra linhagem se distribui na porção continental do RS ao oeste da Lagoa dos Patos. Análises morfométricas mais detalhadas mostram que essas duas linhagens genéticas podem ser distinguidas taxonomicamente e, portanto, são definidas como duas subespécies de Petunia integrifolia. Há indícios de que um processo de especiação por adaptação a dois ambientes distintos (alta salinidade na planície costeira e baixa salinidade na porção continental) esteja envolvido na divergência dessas duas linhagens. No entanto, para confirmar essa hipótese, são necessários estudos adicionais. / “Species complex” are usually defined as group of species that are morphologically very similar and consequently are very difficult to distinguish. Species complexes, therefore, represent a serious problem to the classification systems based only in morphological characters, the criteria used to delimit species being subjective. The integrifolia complex, that congregates taxa with floral characteristics very similar to Petunia integrifolia (Hook.) Schinz & Thell, it is an example of this taxonomic challenge. The determination of the species inside of this complex, based only in morphometric characters, it is still very controversial. In this work, the internal transcribed spacers of the ribosomal nuclear DNA (ITS1 and ITS2) and two intergenic spacers (trnS-trnG and psbAtrnH) of the plastidial DNA (cpDNA) had been sequenced in 69 individuals pertaining to five taxonomic entities of the integrifolia complex, in the attempt to understand its evolutionary history and contribute to the better delimitation of the species. Populational and phylogeographic analyses of the three markers and of cpDNA had shown that only the taxonomic entity described as Petunia interior can be considered a distinct species of Petunia integrifolia. The four other taxonomic entities are divided in two independent and allopatric lineages, that diversified almost simultaneously after an event of population bottleneck followed by a fast expansion. One of these lineages is located in the coastal plain of the Rio Grande do Sul and Santa Catarina states, while to other lineage it’s distributed in the continental region of the Rio Grande do Sul to the west of the Lagoa dos Patos. Detailed morphometric analyses shown that these two lineages can be taxonomically distinguished and therefore, they may be considered as two subspecies of Petunia integrifolia. Some findings suggest that a process of adaptation to these two distinct environments (high salinity in the coastal plain and low salinity in the continental region) may be involved in the divergence of the two lineages. Additional studies are required to test this hypothesis.

Citrus spp. are known for the accumulation of flavanone glycosides (e.g., naringin comprises up to 70% of the dry weight of very young grapefruit). In contrast, petunia utilizes relatively more naringenin for production of flavonol glycosides and anthocyanins. This investigation addressed whether or not petunia is capable of glucosylation of naringenin and if so, what are the characteristics of this flavanone glucosylating enzyme. Petunia leaf tissue contains some flavanone-7-O-glucosyltransferase (E.C. 2.4.1.185) activity, although at 90-fold lower levels than grapefruit leaves. This activity was partially purified 89-fold via ammonium sulfate fractionation followed by FPLC on Superose 12 and Mono Q yielding three chromatographically separate peaks of activity. The enzymes in the peak fractions glucosylated flavanone, flavonol, and flavone substrates. Enzymes in Mono Q peaks I and II were relatively more specific toward flavanone substrates and peak I was significantly more active. Enzyme activity was not effected by Ca2+, Mg2+, AMP, ADP, or ATP. The petunia enzyme was over 10,000 times more sensitive to UDP inhibition (Ki 0.89 μM) than the flavanone-specific 7GT in grapefruit. These and other results suggest that different flavonoid accumulation patterns in these two plants may be partially due to the different relative levels and biochemical properties of their flavanone glucosylating (7GT) enzymes.

Petunia hybrida and Citrus paradisi have significantly different flavonoid accumulation patterns. Petunia sp. tend to accumulate flavonol glycosides and anthocyanins while Citrus paradisi is known for its accumulation of flavanone diglycosides. One possible point of regulation of flavanone metabolism is flavanone 3-hydroxylase (F3H) expression. To test whether this is a key factor in the different flavanone usage by Petunia hybrida and Citrus paradisi, F3H mRNA expression in seedlings of different developmental stages was measured using semi-quantitative RT-PCR. Primers were designed to conserved regions of F3H and used to amplify an approximately 350 bp segment for quantitation by PhosphorImaging. Primary leaves of 32 day old grapefruit seedlings and a grapefruit flower bud had the highest levels of F3H mRNA expression. Petunia seedlings had much lower levels of F3H mRNA expression relative to grapefruit. The highest expression in petunia was in primary leaves and roots of 65 day old seedlings. These results indicate that preferential use of naringenin for production of high levels of flavanone glycosides in young grapefruit leaves cannot be attributed to decreased F3H mRNA expression.

No description available.

No description available.

No description available.

Using gentian and petunia as the experimental systems, potential alternative post-harvest treatments for cut flowers were explored in this project. Pulsing with GA₃ (1 to 100 µM) or sucrose (3%, w/v) solutions delayed the rate of senescence of flowers on cut gentian stems. The retardation of flower senescence by GA₃ in both single flower and half petal systems was accompanied by a delay in petal discoloration. The delay in ion leakage increase or fresh weight loss was observed following treatment with 5 or 10 µM GA₃ of the flowers at the unopen bud stage. Ultrastructural analysis showed that in the cells of the lower part of a petal around the vein region, appearance of senescence-associated features such as degradation of cell membranes, cytoplasm and organelles was faster in water control than in GA₃ treatment. In particular, degeneration of chloroplasts including thylakoids and chloroplast envelope was retarded in response to GA₃ treatment. In the cells of the top part of a petal, more carotenoids-containing chromoplasts were found after GA₃ application than in water control. In petunia, treatment with 6% of ethanol or 0.3 mM of STS during the flower opening stage was effective to delay senescence of detached flowers. The longevity of isolated petunia petals treated with 6% ethanol was nearly twice as long as when they were held in water. Senescence-associated petal membrane damage, weight decline, ovary growth and decrease in protein and total RNA levels were counteracted in ethanol-treated petals. The accumulation of ROS, particularly superoxide and hydrogen peroxide, was also inhibited or delayed by ethanol application. Anti-senescence mechanisms, particularly the changes of oxidative / antioxidant metabolism involved in petal senescence, were investigated. In gentian, activities of AP and SOD but not POD in the GA₃-treated petals were significantly higher than those of the control. In isolated petunia petals, the decreased trends of antioxidative SOD and AP activities during senescence were apparently prevented in response to ethanol treatment although the levels of ascorbate and photo-protective carotenoids were not affected. Furthermore, by optimizing a range of critical PCR parameters such as primer combinations, cDNA concentrations and annealing temperatures, a reliable protocol has been established for quantifying the expression level of Cu-Zn SOD gene in petunia petals using SYBR Green I based real-time RT-PCR. A 228 bp gene fragment of Cu-Zn SOD was isolated from petunia (var. 'hurrah') using RT-PCR. It was found that the mRNA level (relative to 18S rRNA level) of Cu-Zn SOD decreased significantly after 6 days in water. However, there was about a 55-fold increase in Cu-Zn mRNA level after 6 days of ethanol treatment when compared to water-treated petals. Similarly, down-regulation of the mRNA level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also observed during senescence of petunia petals. Increased vase life of petunia petals by ethanol treatment was correlated with promotion of GAPDH expression by a factor of about 16 on day 6. Taking together, the anti-senescence effects of GA₃ and ethanol are at least partially associated with an increased efficiency of petal system utilizing ROS since the selected antioxidants were significantly maintained when compared to the corresponding values for the control.

L'endosymbiose racinaire entre les plantes et les champignons mycorhiziens à arbuscules (CMA) permet aux plantes d'avoir un meilleur accès aux nutriments du sol. Pour cette raison, cette endosymbiose joue un rôle majeur dans les écosystèmes et pour l'agriculture durable. Les étapes clés de la colonisation des racines par les CMA sont: 1) la pénétration des CMA dans le système racinaire à travers les cellules de l'épiderme et du cortex externe, et 2) la formation dans les cellules du cortex interne d'une structure ramifiée appelée arbuscule, qui permet des échanges entre les cellules végétales et les hyphes fongiques. L'établissement de cette symbiose implique une communication entre les deux partenaires. Les plantes produisent des hormones, les strigolactones qui induisent chez les CMA la production de signaux symbiotiques : des lipo-chitooligosaccharides (Myc-LCO) et des chitooligosaccharides courts (Myc-COs). Les Myc-LCO et les Myc-CO induisent des réponses moléculaires et physiologiques chez les plantes qui sont capables de former des mycorhizes à arbuscules. Cependant, leur rôle exact dans l'établissement des mycorhizes à arbuscules n'est pas connu. La difficulté à cultiver et transformer le partenaire fongique de cette symbiose rend la recherche compliquée du côté fongique. Du côté des plantes, on sait que des membres de la famille des récepteurs kinases à domaines lysin (LysM-RLK) perçoivent des LCO et des CO produits par divers microorganismes et sont donc de bons candidats pour percevoir des Myc-LCO et des Myc-CO. La plupart des recherches sur les mycorhizes à arbuscules sont réalisées chez des légumineuses, espèces chez lesquelles plusieurs duplications de gènes codant les LysM-RLK ont eu lieu. J'ai donc utilisé lors de mon doctorat des Solanaceae (Solanum lycopersicum, Petunia hybrida et Nicotiana benthamiana) pour étudier le rôle de deux récepteurs putatifs de Myc- LCO, codés par les gènes LYK10 et LYK4. Ces deux gènes, physiquement proches l'un de l'autre dans les génomes de la plupart des dicotylédones, proviennent probablement d'une ancienne duplication en tandem. En utilisant une approche biochimique, nous avons montré que SlLYK10 de S. lycopersicum est capable de lier des LCO avec une haute affinité. De plus, j'ai montré que le promoteur de SlLYK10 est exprimé dans l'épiderme et le cortex externe avant la colonisation par les CMA, puis dans des cellules contenant des arbuscules au cours de la colonisation par les CMA. Enfin, des approches de génétique inverse chez la tomate et le pétunia ont permis de démontrer que LYK10 contrôle la pénétration des CMA dans les racines et la formation des arbuscules. Ces résultats suggèrent que LYK10 perçoit les LCO et active chez les plantes la machinerie nécessaire à la pénétration de CMA dans les cellules végétales et à la formation des arbuscules. / The root endosymbiosis between plants and arbuscular mycorrhizal fungi (AMF) allows the plants to have a better access to soil nutrients. For this reason this endosymbiosis plays a major role in ecosystems and in sustainable agriculture. The key steps for AMF colonization are: 1) the AMF penetration in the root system through crossing epidermal/outer cortical cells, and 2) the formation of a branched inner cortex structure called arbuscules that permits exchanges between plant cells and fungal hyphae. The establishment of this symbiosis involves communication between the two partners of the symbiosis. Plants produce strigolactones, hormones that induce in AMF the production of symbiotic signals : lipo-chitooligosaccharides (Myc-LCOs) and short chitooligosaccharides (Myc-COs). Both Myc-LCOs and Myc-COs induce plant molecular and physiological responses known to be associated with the formation of arbuscular mycorrhiza (AM). However, theit exact role in AM establishment is unknown. The difficulty to grow and transform the fungal partner of this symbiosis makes the research complicated on the fungal side. On the plant side, members of the lysin motif receptor-like kinase (LysM-RLK) family are known to perceive LCOs and COs produced by various microorganisms and are thus good candidates to perceive Myc-LCOs and Myc-COs. Most of the laboratory researches on AM conducted worldwide are performed on legumes where the LysM-RLK family has encountered several gene duplications. During my PhD I used Solanaceae species (Solanum lycopersicum, Petunia hybrida and Nicotiana benthamiana) to study the role of two candidate Myc-LCO receptors encoded by the genes LYK10 and LYK4. These two genes are physically close to each other in genomes of most of the dicotyledons and likely originate from of an ancient tandem duplication. By using a biochemical approach, we showed that S. lycopersicum SlLYK10 is able to bind LCOs with high affinity. Moreover, I showed that SlLYK10 promoter is expressed in epidermis/outer cortex before AMF colonization and also in arbuscule-containing cells during colonization. Finally, reverse genetic approaches in tomato and petunia allowed demonstrating that LYK10 controls AMF penetration into the roots and arbuscule formation. Taken together, these results suggest that LYK10 perceive LCOs and induce/activate the plant machinery required for AMF penetration into plant cells. Altogether this strongly suggests that LCOs play a role in AMF perception by plant during AM establishment. By using the same approaches, we found that N. benthamiana NbLYK4, as its orthologs in legumes and other dicotyledons, also binds LCOs with high affinity and is involved in AM establishment and plant defence. NbLYK4-silenced plants showed reduced responses to defence elicitors and increased colonization by pathogens and AMF. This led to the hypothesis that LYK4 perceives LCOs and locally inhibits plant defence during AMF colonization. This strongly suggests that Myc-LCOs are able to regulate plant defence. In conclusion, at least two proteins are involved non-redundantly in LCO perception in Solanaceae, LYK10 and LYK4 and regulates complementary plant machineries required for AMF colonization.