Influência de temperatura, luz e giberelina na germinação de sementes de Thlaspi caerulescens J. Presl & C. Presl (Brassicaceae)

Thlaspi caerulescens é espécie hiperacumuladora de metais como Cd2+, Ni2+ e Zn2+, considerada como uma plantamodelo para estudar a acumulação e tolerância a metais pesados. No entanto, a baixa produção de sementes em nossas condições climáticas tornam necessária a determinação de condições que possam maximizar a germinação e o vigor de suas sementes. Para identificar as melhores condições para a germinação, sementes de T. caerulescens foram colocadas na presença (15 mmol.m-2.s-1, 8 h luz/16 h escuro) ou ausência de luz nas temperaturas de 10, 15, 20 e 25 ºC, em papel germitest previamente umedecido com solução de ácido giberélico (GA3) a 0,05% ou água destilada. Foram avaliados a porcentagem de germinação (%G) e o índice de velocidade de germinação (IVG). Maior porcentagem de germinação (66%) foi observada nos tratamentos com GA3 e temperaturas de 15 e 20 ºC, na presença de luz. Maiores valores do IVG foram obtidos com a utilização de GA3 nas temperaturas de 15 e 20 ºC, tanto na presença quanto na ausência de luz. Maiores germinação e IVG de T. caerulescens foram observados com uso de GA3 na presença de luz nas temperaturas de 15 e 20 ºC.

Cadmium hyperaccumulation and genetic differentiation of Thlaspi caerulescens populations

Although the knowledge on heavy metal hyperaccumulation mechanisms is increasing, the genetic basis of cadmium (Cd) hyperaccurnulation remains to be elucidated. Thlaspi caerulescens is an attractive model since Cd accumulation polymorphism observed in this species suggests genetic differences between populations with low versus high Cd hyperaccumulation capacities. In our study, a methodology is proposed to analyse at a regional scale the genetic differentiation of T. caerulescens natural populations in relation to Cd hyperaccumulation capacity while controlling for different environmental, soil, plant parameters and geographic origins of populations. Twenty-two populations were characterised with AFLP markers and cpDNA polymorphism. Over all loci, a partial Mantel test showed no significant genetic structure with regard to the Cd hyperaccumulation capacity. Nevertheless, when comparing the marker variation to a neutral model, seven AFLP fragments (9% of markers) were identified as presenting particularly high genetic differentiation between populations with low and high Cd hyperaccurnulation capacity. Using simulations, the number of outlier loci was showed to be significantly higher than expected at random. These loci presented a genetic structure linked to Cd hyperaccumulation capacity independently of the geography, environment, soil parameters and Zn, Pb, Fe and Cu concentrations in plants. Using a canonical correspondence analysis, we identified three of them as particularly related to the Cd hyperaccumutation capacity. This study demonstrates that populations with low and high hyperaccurnulation capacities can be significantly distinguished based on molecular data. Further investigations with candidate genes and mapped markers may allow identification and characterization of genomic regions linked to factors involved in Cd hyperaccumulation.

Genetics of cadmium hyperaccumulation variance in Thlaspi caerulescens natural populations from Switzerland

SUMMARY Heavy metal presence in the environment is a serious concern since some of them can be toxic to plants, animals and humans once accumulated along the food chain. Cadmium (Cd) is one of the most toxic heavy metal. It is naturally present in soils at various levels and its concentration can be increased by human activities. Several plants however have naturally developed strategies allowing them to grow on heavy metal enriched soils. One of them consists in the accumulation and sequestration of heavy metals in the above-ground biomass. Some plants present in addition an extreme strategy by which they accumulate a limited number of heavy metals in their shoots in amounts 100 times superior to those expected for a non-accumulating plant in the same conditions. Understanding the genetic basis of the hyperaccumulation trait - particularly for Cd - remains an important challenge which may lead to biotechnological applications in the soil phytoremediation. In this thesis, Thlaspi caerulescens J. & C. Presl (Brassicaceae) was used as a model plant to study the Cd hyperaccumulation trait, owing to its physiological and genetic characteristics. Twenty-four wild populations were sampled in different regions of Switzerland. They were characterized for environmental and soil parameters as well as intrinsic characteristics of plants (i.e. metal concentrations in shoots). They were as well genetically characterized by AFLPs, plastid DNA polymorphism and genes markers (CAPS and microsatellites) mainly developed in this thesis. Some of the investigated genes were putatively linked to the Cd hyperaccumulation trait. Since the study of the Cd hyperaccumulation in the field is important as it allows the identification of patterns of selection, the present work offered a methodology to define the Cd hyperaccumulation capacity of populations from different habitats permitting thus their comparison in the field. We showed that Cd, Zn, Fe and Cu accumulations were linked and that populations with higher Cd hyperaccumulation capacity had higher shoot and reproductive fitness. Using our genetic data, statistical methods (Beaumont & Nichols's procedure, partial Mantel tests) were applied to identify genomic signatures of natural selection related to the Cd hyperaccumulation capacity. A significant genetic difference between populations related to their Cd hyperaccumulation capacity was revealed based on somè specific markers (AFLP and candidate genes). Polymorphism at the gene encoding IRTl (Iron-transporter also participating to the transport of Zn) was suggested as explaining part of the variation in Cd hyperaccumulation capacity of populations supporting previous physiological investigations. RÉSUMÉ La présence de métaux lourds dans l'environnement est un phénomène préoccupant. En effet, certains métaux lourds - comme le cadmium (Cd) -sont toxiques pour les plantes, les animaux et enfin, accumulés le long de la chaîne alimentaire, pour les hommes. Le Cd est naturellement présent dans le sol et sa concentration peut être accrue par différentes activités humaines. Certaines plantes ont cependant développé des stratégies leur permettant de pousser sur des sols contaminés en métaux lourds. Parmi elles, certaines accumulent et séquestrent les métaux lourds dans leurs parties aériennes. D`autres présentent une stratégie encore plus extrême. Elles accumulent un nombre limité de métaux lourds en quantités 100 fois supérieures à celles attendues pour des espèces non-accumulatrices sous de mêmes conditions. La compréhension des bases génétiques de l'hyperaccumulation -particulièrement celle du Cd - représente un défi important avec des applications concrètes en biotechnologies, tout particulièrement dans le but appliqué de la phytoremediation des sols contaminés. Dans cette thèse, Thlaspi caerulescens J. & C. Presl (Brassicaceae) a été utilisé comme modèle pour l'étude de l'hyperaccumulation du Cd de par ses caractéristiques physiologiques et génétiques. Vingt-quatre populations naturelles ont été échantillonnées en Suisse et pour chacune d'elles les paramètres environnementaux, pédologique et les caractéristiques intrinsèques aux plantes (concentrations en métaux lourds) ont été déterminés. Les populations ont été caractérisées génétiquement par des AFLP, des marqueurs chloroplastiques et des marqueurs de gènes spécifiques, particulièrement ceux potentiellement liés à l'hyperaccumulation du Cd (CAPS et microsatellites). La plupart ont été développés au cours de cette thèse. L'étude de l'hyperaccumulation du Cd en conditions naturelles est importante car elle permet d'identifier la marque, éventuelle de sélection naturelle. Ce travail offre ainsi une méthodologie pour définir et comparer la capacité des populations à hyperaccumuler le Cd dans différents habitats. Nous avons montré que les accumulations du Cd, Zn, Fe et Cu sont liées et que les populations ayant une grande capacité d'hyperaccumuler le Cd ont également une meilleure fitness végétative et reproductive. Des méthodes statistiques (l'approche de Beaumont & Nichols, tests de Martel partiels) ont été utilisées sur les données génétiques pour identifier la signature génomique de la sélection naturelle liée à la capacité d'hyperaccumuler le Cd. Une différenciation génétique des populations liée à leur capacité d'hyperaccumuler le Cd a été mise en évidence sur certains marqueurs spécifiques. En accord avec les études physiologiques connues, le polymorphisme au gène codant IRT1 (un transporteur de Fe impliqué dans le transport du Zn) pourrait expliquer une partie de la variance de la capacité des populations à hyperaccumuler le Cd.

A comparison of the Thlaspi caerulescens and Thlaspi arvense shoot transcriptomes

Whole-genome transcriptome profiling is revealing how biological systems are regulated at the transcriptional level. This study reports the development of a robust method to profile and compare the transcriptomes of two nonmodel plant species, Thlaspi caerulescens, a zinc (Zn) hyperaccumulator, and Thlaspi arvense, a nonhyperaccumulator, using Affymetrix Arabidopsis thaliana ATH1-121501 GeneChip (R) arrays (Affymetrix, Santa Clara, CA, USA). Transcript abundance was quantified in the shoots of agar- and compost-grown plants of both species. Analyses were optimized using a genomic DNA (gDNA)-based probe-selection strategy based on the hybridization efficiency of Thlaspi gDNA with corresponding A. thaliana probes. In silico alignments of GeneChip (R) probes with Thlaspi gene sequences, and quantitative real-time PCR, confirmed the validity of this approach. Approximately 5000 genes were differentially expressed in the shoots of T. caerulescens compared with T. arvense, including genes involved in Zn transport and compartmentalization. Future functional analyses of genes identified as differentially expressed in the shoots of these closely related species will improve our understanding of the molecular mechanisms of Zn hyperaccumulation.

Altered Zn Compartmentation in the Root Symplasm and Stimulated Zn Absorption into the Leaf as Mechanisms Involved in Zn Hyperaccumulation in Thlaspi caerulescens

We investigated Zn compartmentation in the root, Zn transport into the xylem, and Zn absorption into leaf cells in Thlaspi caerulescens, a Zn-hyperaccumulator species, and compared them with those of a related nonaccumulator species, Thlaspi arvense. 65Zn-compartmental analysis conducted with roots of the two species indicated that a significant fraction of symplasmic Zn was stored in the root vacuole of T. arvense, and presumably became unavailable for loading into the xylem and subsequent translocation to the shoot. In T. caerulescens, however, a smaller fraction of the absorbed Zn was stored in the root vacuole and was readily transported back into the cytoplasm. We conclude that in T. caerulescens, Zn absorbed by roots is readily available for loading into the xylem. This is supported by analysis of xylem exudate collected from detopped Thlaspi species seedlings. When seedlings of the two species were grown on either low (1 μm) or high (50 μm) Zn, xylem sap of T. caerulescens contained approximately 5-fold more Zn than that of T. arvense. This increase was not correlated with a stimulated production of any particular organic or amino acid. The capacity of Thlaspi species cells to absorb 65Zn was studied in leaf sections and leaf protoplasts. At low external Zn levels (10 and 100 μm), there was no difference in leaf Zn uptake between the two Thlaspi species. However, at 1 mm Zn2+, 2.2-fold more Zn accumulated in leaf sections of T. caerulescens. These findings indicate that altered tonoplast Zn transport in root cells and stimulated Zn uptake in leaf cells play a role in the dramatic Zn hyperaccumulation expressed in T. caerulescens.

Generation of nonvernal-obligate, faster-cycling Noccaea caerulescens lines through fast neutron mutagenesis

Noccaea caerulescens (formerly Thlaspi caerulescens) is a widely studied metal hyperaccumulator. However, molecular genetic studies are challenging in this species because of its vernal-obligate biennial life cycle of 7-9 months. Here, we describe the development of genetically stable, faster cycling lines of N. caerulescens which are nonvernal-obligate. A total of 5500 M(0) seeds from Saint Laurent Le Minier (France) were subjected to fast neutron mutagenesis. Following vernalization of young plants, 79 of plants survived to maturity. In all, 80 000 M(2) lines were screened for flowering in the absence of vernalization. Floral initials were observed in 35 lines, with nine flowering in < 12 wk. Two lines (A2 and A7) were selfed to the M(4) generation. Floral initials were observed 66 and 87 d after sowing (DAS) in A2 and A7, respectively. Silicle development occurred for all A2 and for most A7 at 92 and 123 DAS, respectively. Floral or silicle development was not observed in wild-type (WT) plants. Leaf zinc (Zn) concentration was similar in WT, A2 and A7 lines. These lines should facilitate future genetic studies of this remarkable species. Seed is publicly available through the European Arabidopsis Stock Centre (NASC).

Cellular Compartmentation of Zinc in Leaves of the Hyperaccumulator Thlaspi caerulescens1

Cellular compartmentation of Zn in the leaves of the hyperaccumulator Thlaspi caerulescens was investigated using energy-dispersive x-ray microanalysis and single-cell sap extraction. Energy-dispersive x-ray microanalysis of frozen, hydrated leaf tissues showed greatly enhanced Zn accumulation in the epidermis compared with the mesophyll cells. The relative Zn concentration in the epidermal cells correlated linearly with cell length in both young and mature leaves, suggesting that vacuolation of epidermal cells may promote the preferential Zn accumulation. The results from single-cell sap sampling showed that the Zn concentrations in the epidermal vacuolar sap were 5 to 6.5 times higher than those in the mesophyll sap and reached an average of 385 mm in plants with 20,000 μg Zn g−1 dry weight of shoots. Even when the growth medium contained no elevated Zn, preferential Zn accumulation in the epidermal vacuoles was still evident. The concentrations of K, Cl, P, and Ca in the epidermal sap generally decreased with increasing Zn. There was no evidence of association of Zn with either P or S. The present study demonstrates that Zn is sequestered in a soluble form predominantly in the epidermal vacuoles in T. caerulescens leaves and that mesophyll cells are able to tolerate up to at least 60 mm Zn in their sap.

Metallomics and chemical speciation: towards a better understanding of metal-induced stress in plants

Most metal ions are toxic to plants, even at low concentrations, despite the fact that some are essential for growth and play key roles in metabolism. The majority of metals induce the formation of reactive oxygen species, which require the synthesis of additional antoxidant compounds and enzymes for their removal. New techniques that have greatly improved the identification, localisation and quantification of metals within plant tissues have led to the science of metallomics. This advancement in knowledge should eventually allow the characterisation of plants used in the process of phytoremediation of soils contaminated with toxic metals.

Zinc in plants

Zinc (Zn) is an essential component of thousands of proteins in plants, although it is toxic in excess. In this review, the dominant fluxes of Zn in the soil-root-shoot continuum are described, including Zn inputs to soils, the plant availability of soluble Zn(2+) at the root surface, and plant uptake and accumulation of Zn. Knowledge of these fluxes can inform agronomic and genetic strategies to address the widespread problem of Zn-limited crop growth. Substantial within-species genetic variation in Zn composition is being used to alleviate human dietary Zn deficiencies through biofortification. Intriguingly, a meta-analysis of data from an extensive literature survey indicates that a small proportion of the genetic variation in shoot Zn concentration can be attributed to evolutionary processes whose effects manifest above the family level. Remarkable insights into the evolutionary potential of plants to respond to elevated soil Zn have recently been made through detailed anatomical, physiological, chemical, genetic and molecular characterizations of the brassicaceous Zn hyperaccumulators Thlaspi caerulescens and Arabidopsis halleri.

Arbovírus Ilheus em aves silvestres (Sporophila caerulescens e Molothrus bonariensis)

OBJETIVO: Relatar o isolamento do vírus Ilheus no Estado de São Paulo e avaliar o seu impacto para a saúde pública. MÉTODOS: O isolamento de vírus foi realizado em camundongos albinos Swiss, a partir de sangue de aves silvestres, capturadas com redes de espera tipo mist net, armadas no nível do solo, no Parque Ecológico do Tietê, São Paulo. A identificação das cepas isoladas foi feita pelos testes de inibição da hemaglutinação, fixação de complemento e neutralização em camundongos. Amostras de plasma de aves e de mamíferos silvestres foram submetidas à pesquisa sorológica para detecção de anticorpos inibidores de hemaglutinação. RESULTADOS: Foram isoladas duas cepas do vírus Ilheus em sangue de aves das espécies Sporophila caerulescens e Molothrus bonariensis e detectados anticorpos em aves das espécies Columbina talpacoti, Geopelia cuneata, Molothrus bonariensis e Sicalis flaveola, em sagüis das espécies Callithrix jacchus e Callithrix penicillata e no quati Nasua nasua. CONCLUSÕES: O isolamento do vírus Ilheus e a detecção de anticorpos específicos em aves residentes, migratórias e de cativeiro, em sagüis e quatis, comprovam a presença desse agente no Parque Ecológico do Tietê. O comportamento migratório de aves silvestres pode determinar a introdução do vírus em outras regiões. Considerando-se a patogenicidade para o homem e a confirmação da circulação desse agente viral em área urbana, freqüentada para atividade de lazer e de educação, o risco de ocorrência de infecção na população humana não pode ser descartado.

Three new species of Isospora Schneider, 1881 (Apicomplexa: Eimeriidae) from the double-collared seed eater, Sporophila caerulescens (Passeriformes: Emberizidae), from Eastern Brazil

Three isosporan species are described from the double-collared seedeater, Sporophila caerulescens from Eastern Brazil. Isospora sporophilae n. sp. oocysts spherical to subspherical; oocyst wall bi-layered, smooth, inner layer colorless to pale yellowish, 21.6 × 20.9 (19.20-23.20 × 18.40-22.60) µm, shape-index 1.03 ± 0.02 (1-1.10), with no micropyle or oocyst residuum. Polar bodies splinter-like or comma-like. Sporocysts ovoidal, 15.2 × 10.6 (17.40-12.80 × 12.60-8.40) µm, shape-index 1.43 ± 0.14 (1.17-1.81), with knob-like Stieda body and residuum. Large crystalloid body in the center of the sporocyst. Isospora flausinoi n. sp. oocysts spherical to subspherical, oocyst wall bi-layered, smooth, colorless, 17.30 x 16.53 (14-20 × 13.60-20) µm, shape-index 1.05 ± 0.04 (1-1.21). Micropyle and oocyst residuum absent; presence of a large polar body. Sporocyst piriform, 14.88 x 10.70 (11.80-18 × 8-12.40) µm, shape-index 1.40 ± 0.18 (1.07-1.77), with smooth, thin, single-layered wall. Sporocyst with rounded Stieda body with no substieda body, and residuum composed of granular material. Isospora teixeirafilhoi n. sp. oocysts spherical to subspherical, oocyst wall bi-layered, smooth, colorless, 17.41 x 16.81 (15.60 19.40 × 14.20-18.80) µm. Shape-index 1.04 ± 0.08 (1-1.12). Micropyle and oocyst residuum absent; presence of a small double-lobuled polar body. Sporocyst ovoid, 11.74 × 8.12 (9-14.20 × 6.20-9.40) µm. Shape-index 1.46 ± 0.23 (1.06-1.88). Sporocyst with knob-like Stieda body, no sub-Stieda body and residuum composed of granular material.

Morfoanatomia e ontogênese do pericarpo de Manihot caerulescens Pohl e M. tripartita Müll. Arg. (Euphorbiaceae)

Manihot caerulescens e M. tripartita são espécies arbustivas de cerrado, conhecidas como mandioca-brava. Não são encontrados muitos trabalhos sobre estrutura do pericarpo de Euphorbiaceae e muitas das descrições morfológicas existentes apresentam interpretações dúbias, especialmente sobre a deiscência. O presente trabalho objetivou descrever a morfologia, anatomia e ontogênese do pericarpo das duas espécies de Manihot, comparando-as entre si e relacionando aspectos estruturais do pericarpo à deiscência. Verificou-se que o desenvolvimento do pericarpo enquadra-se em quatro estágios, típicos de frutos secos. Dois meristemas são formados: um subadaxial, que produz o mesocarpo interno; outro adaxial, que forma o endocarpo, homogêneo e colenquimatoso em M. caerulescens, e heterogêneo em M. tripartita, com fibroesclereídes gelatinosas externas e uma porção colenquimatosa interna. Observou-se a formação de tecido de separação, composto por parênquima laxo, em direção aos feixes dorsais dos três carpelos e aos septos, o qual é mais evidente em M. tripartita. Por fim, verificou-se variação na composição do estrato esclerenquimático: em M. caerulescens, há uma faixa de fibroesclereídes e uma de braquiesclereídes, ambas do mesocarpo; em M. tripartita, ocorrem essas duas faixas mesocárpicas, com maior espessura, acrescidas do endocarpo externo, também composto por fibroesclereídes. Os frutos de ambas as espécies são cápsulas, cujo tipo varia em função do desenvolvimento do tecido de separação e da amplitude do estrato esclerenquimático. Assim, M. caerulescens apresenta cápsulas indeiscentes, enquanto M. tripartita forma cápsulas septi-loculicidas, uma vez que a deiscência, embora passiva, ocorre nas regiões dorsais e em direção aos septos.

Tandem quadruplication of HMA4 in the zinc (Zn) and cadmium (Cd) hyperaccumulator Noccaea caerulescens

Zinc (Zn) and cadmium (Cd) hyperaccumulation may have evolved twice in the Brassicaceae, in Arabidopsis halleri and in the Noccaea genus. Tandem gene duplication and deregulated expression of the Zn transporter, HMA4, has previously been linked to Zn/Cd hyperaccumulation in A. halleri. Here, we tested the hypothesis that tandem duplication and deregulation of HMA4 expression also occurs in Noccaea. A Noccaea caerulescens genomic library was generated, containing 36,864 fosmid pCC1FOS (TM) clones with insert sizes similar to 20-40 kbp, and screened with a PCR-generated HMA4 genomic probe. Gene copy number within the genome was estimated through DNA fingerprinting and pooled fosmid pyrosequencing. Gene copy numbers within individual clones was determined by PCR analyses with novel locus specific primers. Entire fosmids were then sequenced individually and reads equivalent to 20-fold coverage were assembled to generate complete whole contigs. Four tandem HMA4 repeats were identified in a contiguous sequence of 101,480 bp based on sequence overlap identities. These were flanked by regions syntenous with up and downstream regions of AtHMA4 in Arabidopsis thaliana. Promoter-reporter beta-glucuronidase (GUS) fusion analysis of a NcHMA4 in A. thaliana revealed deregulated expression in roots and shoots, analogous to AhHMA4 promoters, but distinct from AtHMA4 expression which localised to the root vascular tissue. This remarkable consistency in tandem duplication and deregulated expression of metal transport genes between N. caerulescens and A. halleri, which last shared a common ancestor > 40 mya, provides intriguing evidence that parallel evolutionary pathways may underlie Zn/Cd hyperaccumulation in Brassicaceae.

Morfoanatomia e ontogênese do pericarpo de Manihot caerulescens Pohl e M. tripartita Müll. Arg. (Euphorbiaceae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Morfologia, anatomia e desenvolvimento do fruto e semente de Manihot caerulescens Pohl. e M. tripartita Mull. Arg. (Euphorbiaceae)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)