RESUMO
KEY MESSAGE: Global DNA methylation changes caused by in vitro conditions are associated with the subculturing and phenotypic variation in Agave angustifolia Haw. While the relationship between the development of albinism and in vitro culture is well documented, the role of epigenetic processes in this development leaves some important questions unanswered. During the micropropagation of Agave angustifolia Haw., we found three different phenotypes, green (G), variegated (V) and albino (A). To understand the physiological and epigenetic differences among the somaclones, we analyzed several morphophysiological parameters and changes in the DNA methylation patterns in the three phenotypes during their in vitro development. We found that under in vitro conditions, the V plantlets maintained their CAM photosynthetic capacity, while the A variant showed no pigments and lost its CAM photosynthetic ability. Epigenetic analysis revealed that global DNA methylation increased in the G phenotype during the first two subcultures. However, after that time, DNA methylation levels declined. This hypomethylation correlated with the appearance of V shoots in the G plantlets. A similar correlation occurred in the V phenotype, where an increase of 2 % in the global DNA methylation levels was correlated with the generation of A shoots in the V plantlets. This suggests that an "epigenetic stress memory" during in vitro conditions causes a chromatin shift that favors the generation of variegated and albino shoots.
Assuntos
Agave/genética , Agave/fisiologia , Metilação de DNA/genética , Técnicas de Cultura de Tecidos/métodos , Agave/anatomia & histologia , Agave/ultraestrutura , Carotenoides/metabolismo , Clorofila/metabolismo , Segregação de Cromossomos , Células Clonais , Malatos/metabolismo , Fenótipo , Fotoperíodo , Estômatos de Plantas/anatomia & histologia , Estômatos de Plantas/metabolismo , Estômatos de Plantas/ultraestruturaRESUMO
Polyploidy has been widely described in many Agave L. species, but its influence on environmental response to stress is still unknown. With the objective of knowing the morphological adaptations and regulation responses of genes related to biotic (LEA) and abiotic (NBS-LRR) stress in species of Agave with different levels of ploidy, and how these factors contribute to major response of Agave against environmental stresses, we analyzed 16 morphological trials on five accessions of three species (Agave tequilana Weber, Agave angustifolia Haw. and Agave fourcroydes Lem.) with different ploidy levels (2n=2x=60 2n=3x=90, 2n=5x=150, 2n=6x=180) and evaluated the expression of NBS-LRR and LEA genes regulated by biotic and abiotic stress. It was possible to associate some morphological traits (spines, nuclei, and stomata) to ploidy level. The genetic characterization of stress-related genes NBS-LRR induced by pathogenic infection and LEA by heat or saline stresses indicated that amino acid sequence analysis in these genes showed more substitutions in higher ploidy level accessions of A. fourcroydes Lem. 'Sac Ki' (2n=5x=150) and A. angustifolia Haw. 'Chelem Ki' (2n=6x=180), and a higher LEA and NBS-LRR representativeness when compared to their diploid and triploid counterparts. In all studied Agave accessions expression of LEA and NBS-LRR genes was induced by saline or heat stresses or by infection with Erwinia carotovora, respectively. The transcriptional activation was also higher in A. angustifolia Haw. 'Chelem Ki' (2n=6x=180) and A. fourcroydes 'Sac Ki' (2n=5x=150) than in their diploid and triploid counterparts, which suggests higher adaptation to stress. Finally, the diploid accession A. tequilana Weber 'Azul' showed a differentiated genetic profile relative to other Agave accessions. The differences include similar or higher genetic representativeness and transcript accumulation of LEA and NBS-LRR genes than in polyploid (2n=5x=150 and 2n=6x=180) Agave accessions, thus suggesting a differentiated selection pressure for overcoming the lower ploidy level of the diploid A. tequilana Weber 'Azul'.