Lead tolerance and accumulation in initial sporophytes of Regnellidium diphyllum Lindm. (Marsileaceae).

Regnellidium diphyllum Lindm. is a heterosporous fern which grows in shallow waters and wetlands, and water pollution contributes to its vulnerability. Environmental lead contamination is mostly caused by industrial and agricultural residues as well as domestic sewage. Given its persistence in the environment, lead can cause important toxicity in living organisms. Megaspore germination and the initial growth of R. diphyllum sporophytes were assessed in Meyer's solution with lead nitrate (Pb(NO3)2) concentrations of 0 (control), 1, 5, 10 and 50 mg L(-1). The study was conducted in a growth chamber at 25 ± 1°C and a 12 hour photoperiod with a nominal irradiance of 100 µmol m(-2) s(-1), for 28 days. Lead concentration in sporophytes was assessed using atomic absorption spectrometry. In the absence of lead, 74% of spores germinated, while significantly lower germination percentages were observed in Pb(NO3)2 concentrations of 1, 10 and 50 mg L(-1). The presence of lead did not significantly influence root growth. At 28 days, primary leaf development was significantly lower in Pb(NO3)2 concentrations of 5 mg L(-1) and higher in relation to the control. The length of secondary leaves did not significantly differ between sporophytes exposed to different concentrations of lead and those of the control at 28 days. Sporophytes exposed to 10 and 50 mg L(-1) Pb(NO3)2 accumulated 1129 mg kg(-1) and 5145 mg kg(-1) of Pb, respectively. The presence of high levels of lead in R. diphyllum sporophytes did not prevent initial development. Future studies should investigate the ability of the species to accumulate and tolerate high levels of lead in advanced stages of its development and in environmental conditions.

Lead tolerance and accumulation in initial sporophytes of Regnellidium diphyllum Lindm. (Marsileaceae) / Tolerância e acumulação de chumbo em esporófitos jovens de Regnellidium diphyllum Lindm. (Marsileaceae)

Regnellidium diphyllum Lindm. is a heterosporous fern which grows in shallow waters and wetlands, and water pollution contributes to its vulnerability. Environmental lead contamination is mostly caused by industrial and agricultural residues as well as domestic sewage. Given its persistence in the environment, lead can cause important toxicity in living organisms. Megaspore germination and the initial growth of R. diphyllum sporophytes were assessed in Meyer's solution with lead nitrate (Pb(NO3)2) concentrations of 0 (control), 1, 5, 10 and 50 mg L–1. The study was conducted in a growth chamber at 25±1°C and a 12 hour photoperiod with a nominal irradiance of 100 μmol m–2 s–1, for 28 days. Lead concentration in sporophytes was assessed using atomic absorption spectrometry. In the absence of lead, 74% of spores germinated, while significantly lower germination percentages were observed in Pb(NO3)2 concentrations of 1, 10 and 50 mg L–1. The presence of lead did not significantly influence root growth. At 28 days, primary leaf development was significantly lower in Pb(NO3)2 concentrations of 5 mg L–1 and higher in relation to the control. The length of secondary leaves did not significantly differ between sporophytes exposed to different concentrations of lead and those of the control at 28 days. Sporophytes exposed to 10 and 50 mg L–1 Pb(NO3)2 accumulated 1129 mg kg–1 and 5145 mg kg–1 of Pb, respectively. The presence of high levels of lead in R. diphyllum sporophytes did not prevent initial development. Future studies should investigate the ability of the species to accumulate and tolerate high levels of lead in advanced stages of its development and in environmental conditions.

Regnellidium diphyllum Lindm. é uma samambaia heterosporada que se desenvolve em águas rasas ou em solos úmidos, sendo que a poluição da água contribui para sua vulnerabilidade. A contaminação ambiental por chumbo ocorre principalmente por resíduos industriais e agrícolas, bem como por efluentes domésticos. Devido à sua persistência no ambiente, esse metal pode apresentar importante toxicidade aos organismos vivos. A germinação de megásporos e o desenvolvimento inicial de esporófitos de R. diphyllum foram avaliados em solução de Meyer com concentrações de 0 (controle), 1, 5, 10 e 50 mg L–1 de nitrato de chumbo (Pb(NO3)2). O estudo foi conduzido em câmara de germinação a 25±1 °C e fotoperíodo de 12 horas sob irradiância nominal de 100 μmol m–2 s–1, por 28 dias. A concentração de chumbo em esporófitos foi analisada por espectrometria de absorção atômica. Na ausência de chumbo, 74% dos esporos germinaram, enquanto que porcentagens de germinação significantemente menores foram observadas nas concentrações de 1, 10 e 50 mg L–1 de Pb(NO3)2. A presença de chumbo não influenciou significativamente o crescimento das raízes. O desenvolvimento das folhas primárias foi significativamente menor em relação ao controle a partir de 5 mg L–1 de Pb(NO3)2 aos 28 dias. O comprimento das folhas secundárias não diferiu significativamente entre esporófitos expostos às diferentes concentrações de chumbo e aqueles do controle, aos 28 dias. Esporófitos expostos a 10 e 50 mg L–1 de Pb(NO3)2 acumularam 1129 mg kg–1 e 5145 mg kg–1 de Pb, respectivamente. A presença de altas concentrações de chumbo nos esporófitos de R. diphyllum não impediu seu desenvolvimento inicial. Estudos futuros deverão investigar a capacidade de a espécie acumular e tolerar altas concentrações de chumbo em estádios avançados de desenvolvimento e também em condições ambientais.

Effects of nickel on the fern Regnellidium diphyllum Lindm. (Marsileaceae) / Efeitos do níquel sobre a filicínea Regnellidium diphyllum Lindm. (Marsileaceae)

The heterosporous fern Regnellidium diphyllum occurs in southern Brazil and some adjoining localities in Uruguay and Argentina. Currently it is on the list of threatened species in the state of Rio Grande do Sul. Anthropic alterations such as the conversion of wetlands into agricultural areas or water and soil contamination by pollutants may compromise the establishment and survival of this species. Nickel (Ni) is an essential nutrient for plants but increasing levels of this metal due to pollution can cause deleterious effects especially in aquatic macrophytes. Megaspore germination tests were performed using Meyer's solution, at concentrations of 0 (control), 0.05, 0.5, 1, 5, 10, 20, 30, 50 and 100 mg L-1 of Ni. The initial development of apomictic sporophytes was studied using solutions containing 0 (control) to 4.8 mg L-1 of Ni. A significant negative relation was observed between the different Ni concentrations and the megaspore germination/sporophyte formation rates. Primary roots, primary leaves and secondary leaves were significantly shorter at 3.2 and 4.8 mg L-1 of Ni, when compared with the treatment without this metal. At 4.8 mg L-1, leaves also presented chlorosis and necrosis. The introduction of pollutants with Ni in the natural habitat of Regnellidium diphyllum may inhibit the establishment of plants in the initial stage of development, a problem to be considered in relation to the conservation of this species.

A samambaia heterosporada Regnellidium diphyllum Lindm. ocorre no Sul do Brasil e em algumas localidades vizinhas no Uruguai e na Argentina. Atualmente, consta da lista de espécies ameaçadas do Estado do Rio Grande do Sul. Alterações antrópicas - tais como a conversão de áreas úmidas em áreas de agricultura ou a contaminação de água e solo por poluentes - podem comprometer o estabelecimento e a sobrevivência da espécie. Níquel (Ni) é um nutriente essencial às plantas, mas níveis aumentados desse metal, em razão da poluição, podem causar efeitos deletérios, especialmente em macrófitas aquáticas. Testes de germinação de megásporos foram realizados usando-se solução de Meyer, em concentrações de 0 (controle); 0,05; 0,5; 1; 5; 10; 20; 30; 50; 100 mg L-1 de Ni. O desenvolvimento inicial de esporófitos apomíticos foi estudado usando-se soluções contendo 0 (controle) a 4,8 mg L-1 de Ni. Relação negativa significante foi observada entre diferentes concentrações de Ni e as taxas de germinação de megásporos/formação de esporófitos. Raízes primárias e folhas primárias e secundárias foram significantemente mais curtas a 3,2 e 4,8 mg L-1 de Ni, quando comparadas ao tratamento sem esse metal. Em 4,8 mg L-1, folhas também apresentaram clorose e necrose. A introdução de poluentes com Ni no habitat natural de Regnellidium diphyllum pode inibir o estabelecimento de plantas em estádio inicial de desenvolvimento, um problemaa a ser considerado em relação à conservação dessa espécie.

Effects of nickel on the fern Regnellidium diphyllum Lindm. (Marsileaceae).

The heterosporous fern Regnellidium diphyllum occurs in southern Brazil and some adjoining localities in Uruguay and Argentina. Currently it is on the list of threatened species in the state of Rio Grande do Sul. Anthropic alterations such as the conversion of wetlands into agricultural areas or water and soil contamination by pollutants may compromise the establishment and survival of this species. Nickel (Ni) is an essential nutrient for plants but increasing levels of this metal due to pollution can cause deleterious effects especially in aquatic macrophytes. Megaspore germination tests were performed using Meyer's solution, at concentrations of 0 (control), 0.05, 0.5, 1, 5, 10, 20, 30, 50 and 100 mg L(-1) of Ni. The initial development of apomictic sporophytes was studied using solutions containing 0 (control) to 4.8 mg L(-1) of Ni. A significant negative relation was observed between the different Ni concentrations and the megaspore germination/sporophyte formation rates. Primary roots, primary leaves and secondary leaves were significantly shorter at 3.2 and 4.8 mg L(-1) of Ni, when compared with the treatment without this metal. At 4.8 mg L(-1), leaves also presented chlorosis and necrosis. The introduction of pollutants with Ni in the natural habitat of Regnellidium diphyllum may inhibit the establishment of plants in the initial stage of development, a problem to be considered in relation to the conservation of this species.

Effects of 2,4-D on the germination of megaspores and initial development of Regnellidium diphyllum Lindman (Monilophyta, Marsileaceae) / Efeitos do 2,4-D sobre a germinação de megásporos e o desenvolvimento inicial de Regnellidium diphyllum Lindman (Monilophyta, Marsileaceae)

Regnellidium diphyllum is considered as endangered, occurring in the State of Rio Grande do Sul, Brazil, and a few adjoining localities in Uruguay, Argentina and the State of Santa Catarina. It grows in wetlands frequently altered for agricultural activities. Herbicides based on 2,4-dichlorophenoxyacetic acid (2,4-D) are widely used in these fields. The effects of 2,4-D on the germination of megaspores and initial sporophytic development of R. diphyllum were investigated. Six concentrations of 2,4-D (0.32; 0.64; 1.92; 4.80; 9.60 and 19.20 mg.L-1), and the control (0.00 mg.L-1), were tested in vitro, using Meyer's medium. Cultures were maintained in a growth chamber at 24 ± 1 °C, under artificial light with nominal irradiance of 110 µmol.m-2/s and 16 hours photoperiod. Megaspore germination was lower at 9.60 and 19.20 mg.L-1 of 2,4-D (56 and 48 percent, respectively), compared with the control (68 percent). Herbicide concentrations of up to 1.92 mg.L-1 did not significantly decrease the number of sporophytes formed. At 19.20 mg.L-1, no sporophytes were formed. The lengths of the primary root, primary and secondary leaves were greater at concentrations of 0.32 and 0.64 mg.L-1 of 2,4-D. Low concentrations of 2,4-D do not affect germination rates and initial development of R. diphyllum in a significant way. However, higher concentrations (9.60 and 19.20 mg.L-1) affect substantially the germination of the megaspores and interfere with the establishment of the species.

Regnellidium diphyllum é considerada ameaçada, ocorrendo no Estado do Rio Grande do Sul, Brasil, e em algumas localidades vizinhas no Uruguai, na Argentina e no Estado de Santa Catarina. Cresce em áreas alagáveis, frequentemente alteradas para atividades agrícolas. Herbicidas baseados em ácido 2,4-diclorofenoxiacético (2,4-D) são largamente utilizados nestas plantações. Os efeitos do 2,4-D sobre a germinação de megásporos e o desenvolvimento esporofítico inicial de R. diphyllum foram investigados. Seis concentrações de 2,4-D (0,32; 0,64; 1,92; 4,80; 9,60 e 19,20 mg.L-1), além do controle (0,00 mg.L-1) foram testadas in vitro, utilizando meio de Meyer. As culturas foram mantidas em câmara de germinação a 24 ± 1 °C, sob luz artificial, com irradiância nominal de 110 mmol.m-2/s e fotoperíodo de 16 horas. A germinação de megásporos foi menor em 9,60 e 19,20 mg.L-1 de 2,4-D (56 e 48 por cento, respectivamente), comparada ao controle (68 por cento). Concentrações até 1,92 mg.L-1 não diminuíram significativamente o número de esporófitos formados. Em 19,20 mg.L-1, não houve formação de esporófitos. Os comprimentos da raiz primária e das folhas primária e secundária foram maiores em concentrações de 0,32 e 0,64 mg.L-1 de 2,4-D. Baixas concentrações de 2,4-D não afetam significantemente as taxas de germinação e o desenvolvimento inicial de R. diphyllum. Entretanto, maiores concentrações (9,60 e 19,20 mg.L-1) afetam substancialmente a germinação de megásporos e interferem no estabelecimento da espécie.

Effects of 2,4-D on the germination of megaspores and initial development of Regnellidium diphyllum Lindman (Monilophyta, Marsileaceae).

Regnellidium diphyllum is considered as endangered, occurring in the State of Rio Grande do Sul, Brazil, and a few adjoining localities in Uruguay, Argentina and the State of Santa Catarina. It grows in wetlands frequently altered for agricultural activities. Herbicides based on 2,4-dichlorophenoxyacetic acid (2,4-D) are widely used in these fields. The effects of 2,4-D on the germination of megaspores and initial sporophytic development of R. diphyllum were investigated. Six concentrations of 2,4-D (0.32; 0.64; 1.92; 4.80; 9.60 and 19.20 mg.L-1), and the control (0.00 mg.L-1), were tested in vitro, using Meyer's medium. Cultures were maintained in a growth chamber at 24 +/- 1 degrees C, under artificial light with nominal irradiance of 110 micromol.m-2/s and 16 hours photoperiod. Megaspore germination was lower at 9.60 and 19.20 mg.L-1 of 2,4-D (56 and 48%, respectively), compared with the control (68%). Herbicide concentrations of up to 1.92 mg.L-1 did not significantly decrease the number of sporophytes formed. At 19.20 mg.L-1, no sporophytes were formed. The lengths of the primary root, primary and secondary leaves were greater at concentrations of 0.32 and 0.64 mg.L-1 of 2,4-D. Low concentrations of 2,4-D do not affect germination rates and initial development of R. diphyllum in a significant way. However, higher concentrations (9.60 and 19.20 mg.L-1) affect substantially the germination of the megaspores and interfere with the establishment of the species.

Germination and sporophytic development of Regnellidium diphyllum Lindman (Marsileaceae) in the presence of hexavalent chromium.

Regnellidium diphyllum Lindman is a heterosporous fern, growing in aquatic environments and surrounding wetlands, which is assumed to be threatened by increasing water pollution and disappearance of its natural habitats. Among contaminants, hexavalent chromium - Cr(VI) - is known to be present in effluents from some leather tanning factories. Megaspore germination tests were performed using Meyer's solution, at concentrations 0 (control), 0.1, 0.5, 1, 5, 10, 15, 20, 30, 50, and 80 mg.L⁻¹, from a standard solution of Titrisol® 1000 mg.L⁻¹. The primary development of apomictic sporophytes was studied using solutions containing 0.025 to 4.8 mg.L⁻¹ of Cr(VI). The experiments were conducted in a growth chamber at 24 ± 1 ºC and for a 12-hour photoperiod under fluorescent lights, providing a nominal irradiance of 77 µmol.m⁻²/s. Significant differences in megaspore germination, with subsequent sporophytic development, were verified from 0.5 mg.L⁻¹ Cr(VI) concentration onwards. Growth of primary root and primary and secondary leaves was significantly reduced at 3.2 mg.L⁻¹ Cr(VI) concentration or higher. Considering the pollution from Cr(VI) in some areas of R. diphyllum natural occurrence, these data indicate that low reproductive rates and disappearance of populations are likely to occur in these situations.

Mago nashi is essential for spermatogenesis in Marsilea.

Spermatogenesis in Marsilea vestita is a rapid process that is activated by placing dry microspores into water. Nine division cycles produce seven somatic cells and 32 spermatids, where size and position define identity. Spermatids undergo de novo formation of basal bodies in a particle known as a blepharoplast. We are interested in mechanisms responsible for spermatogenous initial formation. Mago nashi (Mv-mago) is a highly conserved gene present as stored mRNA and stored protein in the microspore. Mv-mago protein increases in abundance during development and it localizes at discrete cytoplasmic foci (Mago-dots). RNA interference experiments show that new Mv-mago protein is required for development. With Mv-mago silenced, asymmetric divisions become symmetric, cell fate is disrupted, and development stops. The alpha-tubulin protein distribution, centrin translation, and Mv-PRP19 mRNA distribution are no longer restricted to the spermatogenous cells. Centrin aggregations, resembling blepharoplasts, occur in jacket cells. Mago-dots are undetectable after the silencing of Mv-mago, Mv-Y14, or Mv-eIF4AIII, three core components of the exon junction complex (EJC), suggesting that Mago-dots are either EJCs in the cytoplasm, or Mv-mago protein aggregations dependent on EJCs. Mv-mago protein and other EJC components apparently function in cell fate determination in developing male gametophytes of M. vestita.

Abscisic acid regulation of heterophylly in Marsilea quadrifolia L.: effects of R-(-) and S-(+) isomers.

The plant hormone abscisic acid (ABA) induces a developmental switch in the aquatic fern Marsilea quadrifolia, causing the formation of aerial type characteristics, including the elongation of petioles and roots, a change in leaf morphology, the expansion of leaf surface area, and the shortening of the internodes. A number of ABA-responsive heterophylly (ABRH) genes are induced early during the transition. Using optically pure isomers of ABA, it was found that both the natural S-(+)-ABA and the unnatural R-(-)-ABA are capable of inducing a heterophyllous switch and regulating ABRH gene expression. When dose responses are compared, the unnatural ABA gives stronger morphogenic effects than the natural ABA at the same concentration, it is effective at lower concentrations, and its optimal concentration is also lower compared with the natural ABA. Deuterium-labelled ABA enantiomers were used to trace the fate of applied ABA and to distinguish the applied compound and its metabolites from the endogenous counterparts. In tissues, the supplied (+)-ABA was metabolized principally to dihydrophaseic acid, while the supplied (-)-ABA was converted at a slower rate to 7'-hydroxy abscisic acid. Treatment with either enantiomer resulted in increased biosynthesis of ABA, as reflected in the accumulation of endogenous dihydrophaseic acid. Taken together, these results suggest two distinct mechanisms of action for (-)-ABA: either (-)-ABA is intrinsically active, or its activity is due to the stimulation of ABA biosynthesis.

Changes in the abundance and distribution of conserved centrosomal, cytoskeletal and ciliary proteins during spermiogenesis in Marsilea vestita.

Spermiogenesis in the male gametophytes of the water fern Marsilea vestita is a precise and rapid process resulting in the production of ciliated gametes. Development begins from a single cell within the microspore wall that undergoes nine rapid cell division cycles in distinct planes to produce 32 spermatids that are surrounded by 7 sterile cells. Thereafter, the de novo formation of basal bodies occurs in a discrete cytoplasmic particle known as a blepharoplast, with the subsequent formation of a complex ciliary apparatus in elongating spermatids. The rate and extent of development appear to be controlled at a post-transcriptional level, where the sudden translation of specific stored mRNAs (e.g., centrin) results in the formation of particular structures in the cells (e.g., blepharoplasts). We show here that additional centrosomal and cytoskeletal antigens known as SF assemblin, p95 kDa protein, delta tubulin, gamma tubulin, Xgrip109, Aik, CTR453, RanBPM, BX63, RSP6, and alpha tubulin each exhibit specific localization patterns both on immunoblots of gametophyte protein isolates and in fixed cells. BAp90, PP4, and RLC exhibit specific localization patterns in fixed cells. We show that the antigens exhibit complex patterns of abundance during spermiogenesis. In an attempt to identify regulatory agents involved in spermiogenesis, we employed a RNAi-based screen of 41 randomly selected gametophyte cDNAs on developing populations of synchronously growing gametophytes. The gametophytes treated with each of the RNAi probes exhibited arrest at a specific stage of development. A consequence of anomalous development was the block to assembly of the ciliary apparatus, an effect highlighted by altered staining with anti-centrin, anti-beta-tubulin, and anti-RSP6 antibodies. Our results show that complex, integrated processes of translation and protein partitioning apparently underlie the assembly of the ciliary apparatus during spermiogenesis in male gametophytes of M. vestita.