In Vitro Polyploidization of Brassolaeliocattleya Hybrid Orchid.

The Cattleya (Orchidaceae-Laeliinae subtribe) intergeneric hybrids, such as Brassolaeliocattleya (Blc.), have great ornamental value, due to their compact-size, with large and high color diversity of flowers. Artificial induction of polyploidy brings agronomic, ornamental and genetic benefits to plants. Polyploidization efficiency depends on factors, such as the type of antimitotic, polyploidization method, concentrations, exposure times and type of explant. This study aimed to develop a protocol to polyploidize Blc. orchids, by testing two types of explants (seeds and protocorms), concentrations and exposure times to colchicine. The effects of colchicine on the in vitro development of explants were also investigated. The responses of explants to colchicine depended on the concentrations, exposure time and the interaction of these factors. Flow cytometric analysis evidenced high endopolyploidy and allowed the separation of polyploidized (4C, 8C and 16C peaks) from non-polyploidized (only 2C and 4C peaks) plants. The highest percentage of polyploid plants was regenerated from protocorms (16.4%) treated with colchicine instead of seeds (3.2%). Protocorms treated with colchicine at 500-750 µM for 18 h resulted in the best percentage of polyploidization. Additionally, in vitro natural polyploidization using protocorms was reported (11.5%). Cytological analyses allowed an estimation of the number of chromosomes of the parents (≡70), polyploidized (≡140) and non-polyploidized progeny (≡70).

Stigma/Style Cell-Cycle Inhibitor 1, a Regulator of Cell Proliferation, Interacts With a Specific 14-3-3 Protein and Is Degraded During Cell Division.

The final shape and size of plant organs are determined by a network of genes that modulate cell proliferation and expansion. Among those, SCI1 (Stigma/style Cell-cycle Inhibitor 1) functions by inhibiting cell proliferation during pistil development. Alterations in SCI1 expression levels can lead to remarkable stigma/style size changes. Recently, we demonstrated that SCI1 starts to be expressed at the specification of the Nicotiana tabacum floral meristem and is expressed at all floral meristematic cells. To elucidate how SCI1 regulates cell proliferation, we screened a stigma/style cDNA library through the yeast two-hybrid (Y2H) system, using SCI1 as bait. Among the interaction partners, we identified the 14-3-3D protein of the Non-Epsilon group. The interaction between SCI1 and 14-3-3D was confirmed by pulldown and co-immunoprecipitation experiments. 14-3-3D forms homo- and heterodimers in the cytoplasm of plant cells and interacts with SCI1 in the nucleus, as demonstrated by Bimolecular Fluorescence Complementation (BiFC). Analyses of SCI1-GFP fluorescence through the cell-cycle progression revealed its presence in the nucleoli during interphase and prophase. At metaphase, SCI1-GFP fluorescence faded and was no longer detected at anaphase, reappearing at telophase. Upon treatment with the 26S proteasome inhibitor MG132, SCI1-GFP was stabilized during cell division. Site-directed mutagenesis of seven serines into alanines in the predicted 14-3-3 binding sites on the SCI1 sequence prevented its degradation during mitosis. Our results demonstrate that SCI1 degradation at the beginning of metaphase is dependent on the phosphorylation of serine residues and on the action of the 26S proteasome. We concluded that SCI1 stability/degradation is cell-cycle regulated, consistent with its role in fine-tuning cell proliferation.

Bacillus thuringiensis RZ2MS9, a tropical plant growth-promoting rhizobacterium, colonizes maize endophytically and alters the plant's production of volatile organic compounds during co-inoculation with Azospirillum brasilense Ab-V5.

The beneficial features of Bacillus thuringiensis (Bt) are not limited to its role as an insecticide; it is also able to promote plant growth interacting with plants and other plant growth-promoting rhizobacterium (PGPR). The PGPR Bt strain RZ2MS9 is a multi-trait maize growth promoter. We obtained a stable mutant of RZ2MS9 labelled with green fluorescent protein (RZ2MS9-GFP). We demonstrated that the Bt RZ2MS9-GFP successfully colonizes maize's roots and leaves endophytically. We evaluated whether RZ2MS9 has an additive effect on plant growth promotion when co-inoculated with Azospirillum brasilense Ab-V5. The two strains combined enhanced maize's roots and shoots dry weight around 50% and 80%, respectively, when compared to the non-inoculated control. However, non-differences were observed comparing RZ2MS9 alone and when co-inoculated with Ab-V5, In addition, we used co-inoculation experiments in glass chambers to analyse the plant's volatile organic compounds (VOCs) production during the maize-RZ2MS9 and maize-RZ2MS9-Ab-V5 interaction. We found that the single and co-inoculation altered maize's VOCs emission profile, with an increase in the production of indoles in the co-inoculation. Collectively, these results increase our knowledge about the interaction between the Bt and maize, and provide a new possibility of combined application with the commercial inoculant A. brasilense Ab-V5.

A Heterochromatic Knob Reducing the Flowering Time in Maize.

Maize flowering time is an important agronomic trait, which has been associated with variations in the genome size and heterochromatic knobs content. We integrated three steps to show this association. Firstly, we selected inbred lines varying for heterochromatic knob composition at specific sites in the homozygous state. Then, we produced homozygous and heterozygous hybrids for knobs. Second, we measured the genome size and flowering time for all materials. Knob composition did not affect the genome size and flowering time. Finally, we developed an association study and identified a knob marker on chromosome 9 showing the strongest association with flowering time. Indeed, modelling allele substitution and dominance effects could offer only one heterochromatic knob locus that could affect flowering time, making it earlier rather than the knob composition.

Karyotype structure and NOR activity in Brazilian Smilax Linnaeus, 1753 species (Smilacaceae).

The genus Smilax Linnaeus, 1753 (Smilacaceae) is a large genus of dioecious plants distributed in tropical, subtropical and temperate regions. Some Smilax species have medicinal importance and their identification is important for the control of raw material used in the manufacture of phytotherapeutical products. The karyotypes of seven Brazilian Smilax species were investigated. Mitotic metaphases of roots from young plants were analysed in Feulgen-stained preparations. The karyotypes were asymmetric and modal with 2n = 2x = 32 chromosomes gradually decreasing in size. In S. goyazana A De Candolle & C De Candolle, 1878, a polyploid species, 2n = 4x = 64. In all the species, the large and medium-sized chromosomes were subtelocentric and submetacentric and the small chromosomes were submetacentric or metacentric. Their karyotypes were quite similar, with differences in the arm ratio of some chromosomes. S. fluminensis Steudel, 1841 differed from the other species by having a large metacentric chromosome 1. These findings suggest that evolution occurred without drastic changes in the chromosomal structure in the species analyzed. Terminal secondary constrictions were visualized on the short arm of some chromosomes, but they were detected only in one homologue of each pair. Due to the terminal location and the degree of chromosome condensation, secondary constrictions were not visualized in some species. The nucleolus organizer regions (NORs) were mapped by silver-staining and fluorescent in situ hybridization (FISH) in S. rufescens Grisebach, 1842 and S. fluminensis. Silver-staining and FISH signals were colocalized on the short arms of six chromosomes in S. rufescens and four chromosomes in S. fluminensis. In FISH preparations, one of the largest chromosomes had the secondary constrictions highly decondensed in some cells. This finding and the heteromorphism observed in Feulgen-stained chromosomes suggest that differential rRNA gene expression between homologous rDNA loci can occur in some cells, resulting in different degrees of ribosomal chromatin decondensation. The presence of a heteromorphic chromosome pair in S. rufescens, S. polyantha Grisebach, 1842 and S. goyazana suggests a chromosomal sex determination in these dioecious species.

Cadmium toxicity and its relationship with disturbances in the cytoskeleton, cell cycle and chromosome stability.

This study aimed to investigate the mode of action of cadmium (Cd) toxicity at cell level, especially at early stages of plant exposure. Tomato seedlings were cultivated in growth media containing from 0.1 to 70 µM CdCl2 for 24 h. Mitotic index, chromosome abnormality, DNA integrity and organization of tubulin-based structures were assessed in root cells. As higher the Cd concentration in the growth media, higher was the DNA damage intensity and the occurrence of chromosomal abnormalities that included chromosome lost, bridges, stickiness, C-metaphase and polyploidy. The profile of chromosomal aberrations also varied with elevated Cd concentration, being observed increases in the frequency of chromosome stickiness. The mitotic index was reduced at the lowest Cd concentration, but such reduction was statistically similar to that detected at the highest concentration, suggesting that mitotic depression is a rapid outcome and, at same time, a Cd-induced effect that is limited at the first 24 h of direct root exposure to this metal. Under exposure to 20 µM CdCl2, heterogenous distribution of the spindle fibers, formation of two spindle complexes in both of the cell poles, absence of centrosome center, polarization of the spindle fibers during cell division, and non-uniform tubulin deposition in microtubule and phragmoplast were noticed. The results indicate that the tubulin-dependent components of cytoskeleton are Cd targets, and the sensitivity of tubulin-based structures to Cd exposure depends on cell cycle phase. Moreover, DNA damage intensity and chromosomal abnormality profile can be employed as markers of Cd toxicity level.

Histone acetylation and methylation marks in chromatin of Panstrongylus megistus (Hemiptera, Reduviidae).

Panstrongylus megistus, a potential vector of Chagas disease, currently occupies a wider geographic distribution in Brazil than Triatoma infestans, another member of the hemipteran Reduviidae family and a vector of the same disease. A small heterochromatic body (chromocenter) formed by the Y chromosome is evident in the somatic cells of P. megistus, differing in size and chromosome type contribution from the well-studied chromocenters present in T. infestans. While the overall distribution of histone epigenetic marks differ when comparing the heterochromatin and euchromatin territories in T. infestans, no similar data have been established for other hemipteran reduviids, including P. megistus. In the present work, histone acetylation and methylation marks were investigated in cells of Malpighian tubules of P. megistus 5th instar nymphs using immunocytochemical assays and compared to previously published data for T. infestans. Although similarities between these species were found regarding absence of acetylated H3K9, H4K8 and H4K16, and H3K9me and H3K9me2 in the chromocenter, presence of these marks in euchromatin, and presence of H3K9me3 in the chromocenter, no intimate association of acetylated H4K8 and 18S rDNA was revealed in the chromocenter of P. megistus. The elevated abundance of H3K9me2 marks at the nuclear periphery in P. megistus cells, differing from data for T. infestans, is suggested to reflect differences in the interaction of lamina-associated chromatin domains with the nuclear lamina, methyl-transferase modulation and/or association with the last DNA endoreplication step in 5th instar nymphs, which is a matter for further investigation.

Alterations in Heterochromatic Knobs in Maize Callus Culture by Breakage-Fusion-Bridge Cycle and Unequal Crossing Over.

The meiotic and mitotic behavior of regenerated plants derived from a long-term callus culture, designated 12-F, was analyzed. This culture was heterozygous for an amplification of the heterochromatic knob on the long arm of chromosome 7 (K7L). We aimed to investigate if the amplification resulted from a breakage-fusion-bridge (BFB) cycle or from unequal sister chromatid recombination. Therefore, C-banded mitotic metaphases and pachytene, diakinesis, and anaphase I of regenerated plants were analyzed. Additionally, the occurrence of alterations in K7L was investigated in C-banded metaphases from short-term callus cultures derived from lines related to the donor genotype of the 12-F culture. As a result, plants homozygous and heterozygous for the amplification were detected. Meiosis was normal with few abnormalities, such as a low frequency of univalents at diakinesis. In the callus cultures a chromosome 7 with knobs of different sizes in the sister chromatids was detected and interpreted as a result of unequal crossing over. Other chromosomal alterations were consistent with the occurrence of BFB cycles. The finding of unequal crossing over in the cultures supports the conclusion that the amplification in the culture 12-F would be derived from this mechanism. If the amplification was derived from a BFB cycle, the terminal euchromatic segment between knob and the telomere would be deleted, and possibly, homozygous plants would not be viable.

Screening of tropically derived, multi-trait plant growth- promoting rhizobacteria and evaluation of corn and soybean colonization ability.

The present study assessed the plant growth-promoting (PGP) traits and diversity of culturable rhizobacteria associated with guarana (Paullinia cupana), a typical tropical plant. Ninety-six bacteria were isolated, subjected to biochemical tests, and identified by partial or total 16S rDNA sequencing. Proteobacteria and Firmicutes were the dominant rhizospheric phyla found, and Burkholderia and Bacillus were the most abundant genera. Thirteen strains exhibited the four PGP traits evaluated, and most of them belonged to the genus Burkholderia. Two multi-trait PGP strains, RZ2MS9 (Bacillus sp.) and RZ2MS16 (Burkholderia ambifaria), expressively promoted corn and soybean growth under greenhouse conditions. Compared to the non-inoculated control, increases in corn root dry weight of 247.8 and 136.9% were obtained with RZ2MS9 and RZ2MS16 inoculation, respectively, at 60days after seeding. The dry weights of corn and soybean shoots were significantly higher than those of non-inoculated plants, showing increases of more than 47% for both strains and crops. However, soybean root dry weight did not increased after bacterial inoculation with either strain. The colonization behavior of RZ2MS16 was assessed using GFP-labeling combined with fluorescence microscopy and a cultivation-based approach for quantification. RZ2MS16:gfp was able to colonize the roots and shoots of corn and soybean, revealing an endophytic behavior.

Histone epigenetic marks in heterochromatin and euchromatin of the Chagas' disease vector, Triatoma infestans.

Triatoma infestans, a vector of Chagas' disease, shows several particular cell biology characteristics, including the presence of conspicuous heterochromatic bodies (chromocenters) where DNA methylation has not been previously detected. Whether histone modifications contribute to the condensed state of these bodies has not yet been studied. Here, we investigated epigenetic modifications of histones H3 and H4 and presence of the non-histone heterochromatin protein (HP1-α) in the chromocenters and euchromatin of T. infestans cell nuclei, using immunocytochemistry. The effect of different concentrations of the histone deacetylase inhibitors valproic acid (VPA) and sodium butyrate (NaBt) on chromocenter condensation was visually examined; in VPA-treated specimens, this effect was also analyzed by image analysis. Trimethylated H3K9 signals, which were revealed in chromocenter and non-chromocenter areas, were strongest in chromocenters, whereas selected acetylated histone marks and mono- and dimethylated H3K9 and H4K20 signals were detected only in euchromatin. Weak trimethylated H4K20 signals and variable distribution of HP1-α were detected in chromocenters of part of the cellular population analyzed. Although specific VPA and NaBt treatment conditions affected the heterochromatin condensation pattern, they did not induce a decrease in survival and molting rates of the T. infestans nymphs. The VPA-induced chromatin remodeling was not accompanied by induction of H3K9 acetylation in chromocenters. Present findings regarding histone modifications and effects following VPA or NaBt treatments did not yet solve the question of which factors are responsible for maintenance of the condensed state of chromocenters in T. infestans. A possibility requiring further investigation remains on histone methylation marks and/or non-histone proteins.

Karyotype variability in tropical maize sister inbred lines and hybrids compared with KYS standard line.

Maize karyotype variability has been extensively investigated. The identification of maize somatic and pachytene chromosomes has improved with the development of fluorescence in situ hybridization (FISH) using tandemly repeated DNA sequences as probes. We identified the somatic chromosomes of sister inbred lines that were derived from a tropical flint maize population (Jac Duro [JD]), and hybrids between them, using FISH probes for the 180-bp knob repeat, centromeric satellite (CentC), centromeric satellite 4 (Cent4), subtelomeric clone 4-12-1, 5S ribosomal DNA and nucleolus organizing region DNA sequences. The observations were integrated with data based on C-banded mitotic metaphases and conventional analysis of pachytene chromosomes. Heterochromatic knobs visible at pachynema were coincident with C-bands and 180-bp FISH signals on somatic chromosomes, and most of them were large. Variation in the presence of some knobs was observed among lines. Small 180-bp knob signals were invariant on the short arms of chromosomes 1, 6, and 9. The subtelomeric 4-12-1 signal was also invariant and useful for identifying some chromosomes. The centromere location of chromosomes 2 and 4 differed from previous reports on standard maize lines. Somatic chromosomes of a JD line and the commonly used KYS line were compared by FISH in a hybrid of these lines. The pairing behavior of chromosomes 2 and 4 at pachytene stage in this hybrid was investigated using FISH with chromosome-specific probes. The homologues were fully synapsed, including the 5S rDNA and CentC sites on chromosome 2, and Cent4 and subtelomeric 4-12-1 sites on chromosome 4. This suggests that homologous chromosomes could pair through differential degrees of chromatin packaging in homologous arms differing in size. The results contribute to current knowledge of maize global diversity and also raise questions concerning the meiotic pairing of homologous chromosomes possibly differing in their amounts of repetitive DNA.

Colonization of Madagascar periwinkle (Catharanthus roseus), by endophytes encoding gfp marker.

This study reports the introduction of gfp marker in two endophytic bacterial strains (Pantoea agglomerans C33.1, isolated from cocoa, and Enterobacter cloacae PR2/7, isolated from citrus) to monitor the colonization in Madagascar perinwinkle (Catharanthus roseus). Stability of the plasmid encoding gfp was confirmed in vitro for at least 72 h of bacterial growth and after the colonization of tissues, under non-selective conditions. The colonization was observed using fluorescence microscopy and enumeration of culturable endophytes in inoculated perinwinkle plants that grew for 10 and 20 days. Gfp-expressing strains were re-isolated from the inner tissues of surface-sterilized roots and stems of inoculated plants, and the survival of the P. agglomerans C33:1gfp in plants 20 days after inoculation, even in the absence of selective pressure, suggests that is good colonizer. These results indicated that both gfp-tagged strains, especially P. agglomerans C33.1, may be useful tools to deliver enzymes or other proteins in plant.

Contribution of AT-, GC-, and methylated cytidine-rich DNA to chromatin composition in Malpighian tubule cell nuclei of Panstrongylus megistus (Hemiptera, Reduviidae).

The Malpighian tubule cell nuclei of male Panstrongylus megistus, a vector of Chagas disease, contain one chromocenter, which is composed solely of the Y chromosome. Considering that different chromosomes contribute to the composition of chromocenters in different triatomini species, the aim of this study was to determine the contribution of AT-, GC-, and methylated cytidine-rich DNA in the chromocenter as well as in euchromatin of Malpighian tubule cell nuclei of P. megistus in comparison with published data for Triatoma infestans. Staining with 4',6-diamidino-2-phenylindole/actinomycin D and chromomycin A(3)/distamycin, immunodetection of 5-methylcytidine and AgNOR test were used. The results revealed AT-rich/GC-poor DNA in the male chromocenter, but equally distributed AT and GC DNA sequences in male and female euchromatin, like in T. infestans. Accumulation of argyrophilic proteins encircling the chromocenter did not always correlate with that of GC-rich DNA. Methylated DNA identified by immunodetection was found sparsely distributed in the euchromatin of both sexes and at some points around the chromocenter edge, but it could not be considered responsible for chromatin condensation in the chromocenter, like in T. infestans. However, unlike in T. infestans, no correlation between the chromocenter AT-rich DNA and nucleolus organizing region (NOR) DNA was found in P. megistus.

Heterochromatin patterns and ribosomal DNA loci distribution in diploid and polyploid Crotalaria species (Leguminosae, Papilionoideae), and inferences on karyotype evolution.

Most Crotalaria species display a symmetric karyotype with 2n = 16, but 2n = 14 is found in Chrysocalycinae subsection Incanae and 2n = 32 in American species of the section Calycinae. Seven species of the sections Chrysocalycinae, Calycinae, and Crotalaria were analyzed for the identification of heterochromatin types with GC- and AT-specific fluorochromes and chromosomal location of ribosomal DNA loci using fluorescent in situ hybridization (FISH). A major 45S rDNA locus was observed on chromosome 1 in all the species, and a variable number of minor ones were revealed. Only one 5S rDNA locus was observed in the species investigated. Chromomycin A(3) (CMA) revealed CMA(+) bands colocalized with most rDNA loci, small bands unrelated to ribosomal DNA on two chromosome pairs in Crotalaria incana, and CMA(+) centromeric bands that were quenched by distamycin A were detected in species of Calycinae and Crotalaria sections. DAPI(+) bands were detected in C. incana. The results support the species relationships based on flower specialization and were useful for providing insight into mechanisms of karyotype evolution. The heterochromatin types revealed by fluorochromes suggest the occurrence of rearrangements in repetitive DNA families in these heterochromatic blocks during species diversification. This DNA sequence turnover and the variability in number/position of rDNA sites could be interpreted as resulting from unequal crossing over and (or) transposition events. The occurrence of only one 5S rDNA locus and the smaller chromosome size in the polyploids suggest that DNA sequence losses took place following polyploidization events.

Spatial distribution of AT- and GC-rich DNA within interphase cell nuclei of Triatoma infestans Klug.

Heterochromatin bodies in single- and multichromocentered interphase cell nuclei of Triatoma infestans, a vector of Chagas disease, have been suggested to contain AT-rich DNA, based on their positive response to Q-banding and Hoechst 33248 treatment. No information exists on whether GC-rich DNA is also present in these nuclei and whether it plays a role on chromatin condensation. Considering that methodologies more precise than those previously used to determine DNA base composition in situ are currently available, and that the spatial distribution of chromatin areas differing in composition in interphase cell nuclei of different species is a matter of interest, the localization of AT- and GC-rich DNA in T. infestans nuclei is revisited here. The methodologies used included DAPI/AMD and CMA(3)/Distamycin differential staining, Feulgen-DNA image analysis following Msp I and Hpa II enzymatic digestion, 5-methylcytidine immunodetection, AgNOR response, confocal microscopy, and the 5-aza-2'-deoxycytidine (5-AZA) demethylation assay. The results identified the presence of AT-rich/GC-poor DNA in chromocenters and evenly distributed AT and GC sequences in euchromatin. A GC-rich DNA zone encircling the chromocenters was also found but it could not be associated with NOR regions. To corroborate the DNA AT-richness in T. infestans nuclei, bioinformatic analyses were also performed. Methylated cytosine was evident at some points of the chromocenters' edge in single- and multichromocentered nuclei and at the euchromatin of multichromocentered nuclei and could be transiently affected by the 5-AZA treatment. The present results suggest that in the particular case of chromocenters of the hemipteran T. infestans, cytosine methylation is not a relevant factor involved in chromatin condensation.

Age-related association of rDNA and telomeres with the nuclear matrix in mouse hepatocytes.

Transcribed sequences have been suggested to be associated with the nuclear matrix, differing from non-transcribing sequences, which have been reported to be contained in DNA loops. However, although a dozen of genes have their expression level affected by aging, data on chromatin-nuclear matrix interactions under this physiological condition are still scarce. In the present study, liver imprints from young, adult and old mice were subjected to FISH (fluorescence in situ hybridization) for 45S rDNA and telomeric sequences, with or without a lysis treatment to produce extended chromatin fibres. There was an increased amount of 45S rDNA sequences located in DNA loops as the animals grow older, while telomeric sequences were always observed in DNA loops irrespective of the animal age. We assume that active rRNA genes associate with the nuclear matrix, while DNA loops contain silent sequences. Transcription of each 45S rDNA repeat unit is suggested to be dependent on its interaction with the nuclear matrix.

Karyotype characterization of Crotalaria juncea (L) by chromosome banding and physical mapping of 18S-5.8S-26S and 5S rRNA gene sites

The chromosomes of Crotalaria juncea, a legume of agronomic interest with a 2n = 16 karyotype composed of metacentric chromosomes, were analyzed using several cytogenetic techniques. C-banding revealed heterochromatic regions around the centromeres in all chromosomes and adjacent to the secondary constriction on the chromosome 1 short arm. Fluorescent staining with the GC-specific chromomycin A3 (CMA) highlighted these heterochromatic regions and a tiny site on the chromosome 1 long arm while the AT-specific stain 4'-6-diamidino-2-phenylindole (DAPI) induced a reversed pattern. Staining with CMA combined with AT-specific distamycin A (DA) counterstaining quenched the pericentromeric regions of all chromosomes, but enhanced fluorescence was observed at the heterochromatic regions around the secondary constriction and on the long arms of chromosomes 1 and 4. Fluorescence in situ hybridization (FISH) revealed 18S-5.8S-26S rRNA gene sites (45S rDNA) on chromosomes 1 and 4, and one 5S rDNA locus on chromosome 1. All the rDNA sites were co-located with the positive-CMA/DA bands, suggesting they were very rich in GC. Silver staining revealed signals at the main 45S rDNA locus on chromosome 1 and, in some cells, chromosome 4 was labeled. Two small nucleoli were detected in a few interphase cells, suggesting that the minor site on chromosome 4 could be active at some stages of the cell cycle.