During the evolution of the eukaryotic cell, plastids, and mitochondria arose from an endosymbiotic process, which determined the presence of three genetic compartments into the incipient plant cell. After that, these three genetic materials from host and symbiont suffered several rearrangements, bringing on a complex interaction between nuclear and organellar gene products. Nowadays, plastids harbor a small genome with ∼130 genes in a 100–220 kb sequence in higher plants. Plastid genes are mostly highly conserved between plant species, being useful for phylogenetic analysis in higher taxa. However, intergenic spacers have a relatively higher mutation rate and are important markers to phylogeographical and plant population genetics analyses. The predominant uniparental inheritance of plastids is like a highly desirable feature for phylogeny studies. Moreover, the gene content and genome rearrangements are efficient tools to capture and understand evolutionary events between different plant species. Currently, genetic engineering of the plastid genome (plastome) offers a number of attractive advantages as high-level of foreign protein expression, marker gene excision, gene expression in operon and transgene containment because of maternal inheritance of plastid genome in most crops. Therefore, plastid genome can be used for adding new characteristics related to synthesis of metabolic compounds, biopharmaceutical, and tolerance to biotic and abiotic stresses. Here, we describe the importance and applications of plastid genome as tools for genetic and evolutionary studies, and plastid transformation focusing on increasing the performance of horticultural species in the field.

Background Podocarpus lambertii (Podocarpaceae) is a native conifer from the Brazilian Atlantic Forest Biome, which is considered one of the 25 biodiversity hotspots in the world. The advancement of next-generation sequencing technologies has enabled the rapid acquisition of whole chloroplast (cp) genome sequences at low cost. Several studies have proven the potential of cp genomes as tools to understand enigmatic and basal phylogenetic relationships at different taxonomic levels, as well as further probe the structural and functional evolution of plants. In this work, we present the complete cp genome sequence of P. lambertii.Methodology/Principal FindingsThe P. lambertii cp genome is 133,734 bp in length, and similar to other sequenced cupressophytes, it lacks one of the large inverted repeat regions (IR). It contains 118 unique genes and one duplicated tRNA (trnN-GUU), which occurs as an inverted repeat sequence. The rps16 gene was not found, which was previously reported for the plastid genome of another Podocarpaceae (Nageia nagi) and Araucariaceae (Agathis dammara). Structurally, P. lambertii shows 4 inversions of a large DNA fragment ∼20,000 bp compared to the Podocarpus totara cp genome. These unexpected characteristics may be attributed to geographical distance and different adaptive needs. The P. lambertii cp genome presents a total of 28 tandem repeats and 156 SSRs, with homo- and dipolymers being the most common and tri-, tetra-, penta-, and hexapolymers occurring with less frequency.ConclusionThe complete cp genome sequence of P. lambertii revealed significant structural changes, even in species from the same genus. These results reinforce the apparently loss of rps16 gene in Podocarpaceae cp genome. In addition, several SSRs in the P. lambertii cp genome are likely intraspecific polymorphism sites, which may allow highly sensitive phylogeographic and population structure studies, as well as phylogenetic studies of species of this genus.

BackgroundPerforming chloroplast DNA (cpDNA) isolation is considered a major challenge among different plant groups, especially conifers. Isolating chloroplasts in conifers by such conventional methods as sucrose gradient and high salt has not been successful. So far, plastid genome sequencing protocols for conifer species have been based mainly on long-range PCR, which is known to be time-consuming and difficult to implement.Methodology/Principal FindingsWe developed a protocol for cpDNA isolation using three different conifer families: Araucaria angustifolia and Araucaria bidwilli (Araucariaceae), Podocarpus lambertii (Podocarpaceae) and Pinus patula (Pinaceae). The present protocol is based on high salt isolation buffer followed by saline Percoll gradient. Combining these two strategies allowed enhanced chloroplast isolation, along with decreased contamination caused by polysaccharides, polyphenols, proteins, and nuclear DNA in cpDNA. Microscopy images confirmed the presence of intact chloroplasts in high abundance. This method was applied to cpDNA isolation and subsequent sequencing by Illumina MiSeq (2×250 bp), using only 50 ng of cpDNA. Reference-guided chloroplast genome mapping showed that high average coverage was achieved for all evaluated species: 24.63 for A. angustifolia, 135.97 for A. bidwilli, 1196.10 for P. lambertii, and 64.68 for P. patula.ConclusionResults show that this improved protocol is suitable for enhanced quality and yield of chloroplasts and cpDNA isolation from conifers, providing a useful tool for studies that require isolated chloroplasts and/or whole cpDNA sequences.

5-Azacytidine combined with 2,4-D increases the number of Acca sellowiana somatic embryos. Global DNA methylation is directly affected by these compounds.

Climate change will inevitably lead to environmental variations, thus plant drought tolerance will be a determinant factor in the success of plantations and natural forestry recovery. Some metabolites, such as soluble carbohydrates and amino acids, have been described as being the key to both embryogenesis efficiency and abiotic stress response, contributing to phenotypic plasticity and the adaptive capacity of plants. For this reason, our main objectives were to evaluate if the temperature during embryonal mass initiation in radiata pine was critical to the success of somatic embryogenesis, to alter the morphological and ultrastructural organization of embryonal masses at cellular level and to modify the carbohydrate, protein, or amino acid contents. The first SE initiation experiments were carried out at moderate and high temperatures for periods of different durations prior to transfer to the control temperature of 23°C. Cultures initiated at moderate temperatures (30°C, 4 weeks and 40°C, 4 days) showed significantly lower initiation and proliferation rates than those at the control temperature or pulse treatment at high temperatures (50°C, 5 min). No significant differences were observed either for the percentage of embryogenic cell lines that produced somatic embryos, or for the number of somatic embryos per gram of embryonal mass. Based on the results from the first experiments, initiation was carried out at 40°C 4 h; 50°C, 30 min; and a pulse treatment of 60°C, 5 min. No significant differences were found for the initiation or number of established lines or for the maturation of somatic embryos. However, large morphological differences were observed in the mature somatic embryos. At the same time, changes observed at cellular level suggested that strong heat shock treatments may trigger the programmed cell death of embryogenic cells, leading to an early loss of embryogenic potential, and the formation of supernumerary suspensor cells. Finally, among all the differences observed in the metabolic profile, it is worth highlighting the accumulation of tyrosine and isoleucine, both amino acids involved in the synthesis of abiotic stress response-related secondary metabolites.

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