An 82 bp tandem repeat family typical of 3' non-coding end of Gypsy/TAT LTR retrotransposons is conserved in Coffea spp. pericentromeres.

Coffea spp. chromosomes are very small and accumulate a variety of repetitive DNA families around the centromeres. However, the proximal regions of Coffea chromosomes remain poorly understood, especially regarding the nature and organisation of the sequences. Taking advantage of the genome sequences of C. arabica (2n = 44), C. canephora, and C. eugenioides (C. arabica progenitors with 2n = 22) and good coverage genome sequencing of dozens of other wild Coffea spp., repetitive DNA sequences were identified, and the genomes were compared to decipher particularities of pericentromeric structures. The searches revealed a short tandem repeat (82 bp length) typical of Gypsy/TAT LTR retrotransposons, named Coffea_sat11. This repeat organises clusters with fragments of other transposable elements, comprising regions of non-coding RNA production. Cytogenomic analyses showed that Coffea_sat11 extends from the pericentromeres towards the middle of the chromosomal arms. This arrangement was observed in the allotetraploid C. arabica chromosomes, as well as in its progenitors. This study improves our understanding of the role of the Gypsy/TAT LTR retrotransposon lineage in the organisation of Coffea pericentromeres, as well as the conservation of Coffea_sat11 within the genus. The relationships between fragments of other transposable elements and the functional aspects of these sequences on the pericentromere chromatin were also evaluated. Highlights: A scattered short tandem repeat, typical of Gypsy/TAT LTR retrotransposons, associated with several fragments of other transposable elements, accumulates in the pericentromeres of Coffea chromosomes. This arrangement is preserved in all clades of the genus and appears to have a strong regulatory role in the organisation of chromatin around centromeres.

IGS sequences in Cestrum present AT- and GC-rich conserved domains, with strong regulatory potential for 5S rDNA.

The 35S and 5S ribosomal DNA (rDNA) organized in thousands of copies in genomes, have been widely used in numerous comparative cytogenetic studies. Nevertheless, several questions related to the diversity and organization of regulatory motifs in 5S rDNA remain to be addressed. The 5S rDNA unit is composed of a conserved 120 bp length coding region and an intergenic spacer (IGS) containing potential regulatory motifs (Poly-T, AT-rich and GC-rich) differing in number, redundancy and position along the IGS. The Cestrum species (Solanaceae) have large genomes (about 10 pg/1C) and conserved 2n = 16 karyotypes. Strikingly, these genomes show high diversity of heterochromatin distribution, variability in 35S rDNA loci and the occurrence of B chromosomes. However, the 5S rDNA loci are highly conserved in the proximal region of chromosome 8. Comparison of seventy-one IGS sequences in plants revealed several conserved motifs with potential regulatory function. The AT- and GC-rich domains appeared highly conserved in Cestrum chromosomes. The 5S genic and the GC-rich IGS probe produced FISH signals in both A (pair 8) and B chromosomes. The GC-rich domain presented a strong potential for regulation because it may be associated with CpG islands organization, as well as to hairpin and loop organization. Another interesting aspect was the ability of AT- and GC-rich motifs to produce non-heterochromatic CMA/DAPI signals. While the length of the 5S rDNA IGS region varied in size between the Cestrum species, the individual sequence motifs seem to be conserved suggesting their regulatory function. The most striking feature was the conserved GC-rich domain in Cestrum, which is recognized as a signature trait of the proximal region of chromosome pair 8.

Comparative adventitious root development in pre-etiolated and flooded Arabidopsis hypocotyls exposed to different auxins.

Adventitious roots (ARs) emerge from stems, leaves or hypocotyls, being strategic for clonal propagation. ARs may develop spontaneously, upon environmental stress or hormonal treatment. Auxins strongly influence AR development (ARD), depending on concentration and kind. However, the role of different types of auxin is rarely compared at the molecular level. Rooting triggered by light exposure and flooding was examined in intact etiolated Arabidopsis thaliana hypocotyls treated with distinct auxin types. Morphological aspects, rooting-related gene expression profiles, and IAA immunolocalization were recorded. NAA and 2,4-D effects were highly dose-dependent; at higher concentrations NAA inhibited root growth and 2,4-D promoted callus formation. NAA yielded the highest number of roots, but inhibited elongation. IAA increased the number of roots with less interference in elongation, yielding the best overall rooting response. IAA was localized close to the tissues of root origin. Auxin stimulated ARD was marked by increased expression of PIN1 and GH3.3. NAA treatment induced expression of CYCB1, GH3.6 and ARF8. These NAA-specific responses may be associated with the development of numerous shorter roots. In contrast, expression of the auxin action inhibitor IAA28 was induced by IAA. Increased PIN1 expression indicated the relevance of auxin efflux transport for focusing in target cells, whereas GH3.3 suggested tight control of auxin homeostasis. IAA28 increased expression during IAA-induced ARD differs from what was previously reported for lateral root development, pointing to yet another possible difference in the molecular programs of these two developmental processes.

Comparative transcriptional analysis provides new insights into the molecular basis of adventitious rooting recalcitrance in Eucalyptus.

Adventitious rooting (AR) is essential in clonal propagation. Eucalyptus globulus is relevant for the cellulose industry due to its low lignin content. However, several useful clones are recalcitrant to AR, often requiring exogenous auxin, adding cost to clonal garden operations. In contrast, E. grandis is an easy-to-root species widely used in clonal forestry. Aiming at contributing to the elucidation of recalcitrance causes in E. globulus, we conducted a comparative analysis with these two species differing in rooting competence, combining gene expression and anatomical techniques. Recalcitrance in E. globulus is reversed by exposure to exogenous indole-3-acetic acid (IAA), which promotes important gene expression modifications in both species. The endogenous content of IAA was significantly higher in E. grandis than in E. globulus. The cambium zone was identified as an active area during AR, concentrating the first cell divisions. Immunolocalization assay showed auxin accumulation in cambium cells, further indicating the importance of this region for rooting. We then performed a cambium zone-specific gene expression analysis during AR using laser microdissection. The results indicated that the auxin-related genes TOPLESS and IAA12/BODENLOS and the cytokinin-related gene ARR1may act as negative regulators of AR, possibly contributing to the hard-to-root phenotype of E. globulus.

The mitochondrial glutathione peroxidase GPX3 is essential for H2O2 homeostasis and root and shoot development in rice.

Glutathione (GSH) peroxidases (GPXs: EC 1.11.1.9 and EC1.11.1.12) are non-heme thiol peroxidases that catalyze the reduction of H2O2 or organic hydroperoxides to water, and they have been identified in almost all kingdoms of life. The rice glutathione peroxidase (OsGPX) gene family is comprised of 5 members spread throughout a range of sub cellular compartments. The OsGPX gene family is induced in response to exogenous H2O2 and cold stress. In contrast, they are down regulated in response to drought and UV-B light treatments. Transgenic rice plants have been generated that lack mitochondrial OsGPX3. These GPX3s plants showed shorter roots and shoots compared to non-transformed (NT) plants, and higher amounts of H2O2 mitochondrial release were observed in the roots of these plants cultivated under normal conditions. This accumulation of H2O2 is positively associated with shorter root length in GPX3s plants compared to NT ones. Moreover, GPX3 promoter analysis indicated that it is mainly expressed in root tissue. These results suggest that silencing the mitochondrial OsGPX3 gene impairs normal plant development and leads to a stress-induced morphogenic response via H2O2 accumulation.

Occurrence and chromosome distribution of retroelements and NUPT sequences in Copaifera langsdorffii Desf. (Caesalpinioideae).

Copaifera langsdorffii possesses 2n = 24 large meta- and submetacentric chromosomes (5.97-2.60 microm) in comparison with other Caesalpinioideae trees. Chromosome banding revealed an abundance of GC-rich blocks with a few differences in the size and location of bands between different populations. Polymerase chain reaction and digestion with restriction enzyme RsaI were carried out in order to isolate repetitive DNAs, yielding three fragments of different size: (1) cp-rDNA-like, 109 bp (pCl03 clone); (2) Ty1-copia-like retroelement, 185 bp (pCl23 clone); and (3) Ty3-gypsy-like retroelement, 269 bp (pCl08 clone). The first clone produced unmistakable hybridization signals at subterminal and intercalary positions, coinciding with or adjacent to most of the GC-rich bands. The second clone (pCl23 clone) showed dispersed signals distributed throughout several chromosomes, while the pCl08 clone exhibited hybridization signals scattered and organized in blocks in all chromosomes of the complement. Fluorescent in situ hybridization and chromosome banding results allowed the detection of translocation events and unequal crossing-over involving 45S rDNA regions (pairs 7, 8, and 11). However, the most intriguing result was the insertion and subsequent overamplification of a cp-rDNA-like fragment and its distribution over the chromosomes of C. langsdorffii. Additionally, the results suggest that this segment behaves like satellite DNA. These different chromosome markers produced by cytomolecular techniques show that samples from different locations, although isolated, retain more karyotypic similarities than differences.

Distribution of a Ty3/gypsy-like retroelement on the A and B-chromosomes of Cestrum strigilatum Ruiz & Pav. and Cestrum intermedium Sendtn. (Solanaceae)

Retroelements are a diversified fraction of eukaryotic genomes, with the Ty1/copia and Ty3/gypsy groups being very common in a large number of plant genomes. We isolated an internal segment of the Ty3/gypsy retroelement of Cestrum strigilatum (Solanaceae) using PCR amplification with degenerate primers for a conserved region of reverse transcriptase. The isolated segment (pCs12) was sequenced and showed similarity with Ty3/gypsy retroelements of monocotyledons and dicotyledons. This segment was used as probe in chromosomes of C. strigilatum and Cestrum intermedium. Diffuse hybridization signals were observed along the chromosomes and more accentuated terminal signals in some chromosome pairs, always associated with nucleolus organizer regions (NORs). The physical relationship between the hybridization sites of pCs12 and pTa71 ribosomal probes was assessed after sequential fluorescence in situ hybridization (FISH). Hybridization signals were also detected in the B chromosomes of these species, indicating an entail among the chromosomes of A complement and B-chromosomes.