Castor bean (Ricinus communis L.) is an oilseed species globally recognized for an
uncounted number of industrial applications and high market value of the oil extracted
from its seeds. Besides, it has significant socioeconomic importance concerning its
production being traditional and mostly carried out in Brazil by family farmers in the
region of the Brazilian Northeastern semiarid region, where rudimentary crops
predominate under adverse conditions typical of the region. Therefore, the present study
aimed the characterization and overexpression of castor bean genes associated with
better tolerance to abiotic stresses during germination and initial seedling development.
Three families of genes related to superoxide dismutase (SOD), small heat shock
proteins (sHSP), and the nuclear transcription factor Y subunit B (NF-YB) were
analyzed, according to the importance of these genes in the literature, and their
selection by complementary analysis of within a castor bean microarray for genes
expressed under heat stress. The identification and characterization of these families in
castor bean was compared with angiosperm genomes, obtaining the profile of gene
expression under conditions of abiotic stresses during imbibition, radicle protrusion
(germination per se) and post-germination (young seedlings), and functional
characterization of the target genes for tolerance to abiotic stresses through
overexpression in Arabidopsis thaliana. In CHAPTER 1 (published article), we identified
the SOD gene family in castor bean (RcSOD), and the orthologous genes in
angiosperms, showing the expression profile of RcSOD genes in embryos during
imbibition and germination under different osmotic potentials (water restriction). The
RcCuZnSOD1 and RcFeSOD8 genes were induced during imbibition by osmotic stress
that induces ‘priming’ in Castor bean seeds as being associated with beneficial
responses to seeds and seedlings vigor in different Castor bean genotypes. In
CHAPTER 2 (article to be submitted), we identified possible regulatory elements of
RcSOD genes under abiotic stresses and regulation by ABA, and the mechanism of
regulation by the microRNA 398 for the RcCCuSOD4 and RcCuZnSOD3 genes. We
identified the RcCuZnSOD1 and RcFeSOD8 genes as responsive to heat stress (35 °C)
during imbibition and germination. Besides, we identified that other RcSOD genes
(RcCCuSOD4, RcFeSOD7, RcCuZnSOD3) were induced in seeds under heat stress
during radicle protrusion and in seedlings with 2 cm roots. It was possible to observe the
subcellular localization of the RcSOD genes in Nicotiana benthamiana leaves, in which
RcSOD genes (RcMnSOD5 and RcFeSOD8) may have differences compared to the
subcellular location in A. thaliana. In CHAPTER 3 (published article), we initially
identified 41 genes of the heat shock protein family predicted in Castor bean (RcsHSP),
showing that tandem duplication seems to be one of the possible causes for the largest
number of castor genes compared to A. thaliana. Besides, we showed the pattern of
XI
gene expression of 10 RcsHSP genes induced during radicle protrusion and early
seedling stage. However, demonstrating the specificity of expression in roots,
cotyledons, and leaves at different stages of seedling development under heat stress.
Finally, we performed the functional characterization through the overexpression of two
RcsHSP genes (RcsHSP12 and RcsHSP19) in A. thaliana, resulting in (a) higher
percentage of seed germination under heat, osmotic and saline stresses; (b) greater
enzymatic antioxidant potential of SOD; (c) and higher concentration of protective
carbohydrates (sucrose and raffinose). In CHAPTER 4 (submitted article), we identified
the family of genes of the nuclear transcription factor subunit B (NF-YB) in Castor bean
and made the functional characterization of the RcNF-YB8 gene regarding the induction
of early flowering in A. thaliana. The phylogenetic comparison allowed us to identify
orthologous genes in angiosperms and motif patterns that may be associated with
differences between the RcNF-YB subfamilies. The induction of RcNF-YB genes was
observed to be greater during imbibition and germination compared to the postgerminative phase, also showing different profiles of induction and suppression by heat
stress. The RcNF-YB8 gene demonstrated suppression by heat stress and a pattern of
greater expression in leaves, while the overexpression in A. thaliana also demonstrated
to induce early flowering, therefore, impacting the size of the plant and fruits, and
consequent productivity. The results involve a broad characterization of genes from
three important families in the response of seeds and seedlings to abiotic stresses and
subsequent plant development, in which the characterization of the genes showed
significant relevance indicating target genes potentially useful in breeding programs
towards varieties of Castor bean with superior vigor aimed at better stand stablishment
and crop development, and consequently better productivity of Castor bean around the
world and by the family farmers in the Brazilian Northeast semiarid.
Castor bean (Ricinus communis L.) is an oilseed species globally recognized for an
uncounted number of industrial applications and high market value of the oil extracted
from its seeds. Besides, it has significant socioeconomic importance concerning its
production being traditional and mostly carried out in Brazil by family farmers in the
region of the Brazilian Northeastern semiarid region, where rudimentary crops
predominate under adverse conditions typical of the region. Therefore, the present study
aimed the characterization and overexpression of castor bean genes associated with
better tolerance to abiotic stresses during germination and initial seedling development.
Three families of genes related to superoxide dismutase (SOD), small heat shock
proteins (sHSP), and the nuclear transcription factor Y subunit B (NF-YB) were
analyzed, according to the importance of these genes in the literature, and their
selection by complementary analysis of within a castor bean microarray for genes
expressed under heat stress. The identification and characterization of these families in
castor bean was compared with angiosperm genomes, obtaining the profile of gene
expression under conditions of abiotic stresses during imbibition, radicle protrusion
(germination per se) and post-germination (young seedlings), and functional
characterization of the target genes for tolerance to abiotic stresses through
overexpression in Arabidopsis thaliana. In CHAPTER 1 (published article), we identified
the SOD gene family in castor bean (RcSOD), and the orthologous genes in
angiosperms, showing the expression profile of RcSOD genes in embryos during
imbibition and germination under different osmotic potentials (water restriction). The
RcCuZnSOD1 and RcFeSOD8 genes were induced during imbibition by osmotic stress
that induces ‘priming’ in Castor bean seeds as being associated with beneficial
responses to seeds and seedlings vigor in different Castor bean genotypes. In
CHAPTER 2 (article to be submitted), we identified possible regulatory elements of
RcSOD genes under abiotic stresses and regulation by ABA, and the mechanism of
regulation by the microRNA 398 for the RcCCuSOD4 and RcCuZnSOD3 genes. We
identified the RcCuZnSOD1 and RcFeSOD8 genes as responsive to heat stress (35 °C)
during imbibition and germination. Besides, we identified that other RcSOD genes
(RcCCuSOD4, RcFeSOD7, RcCuZnSOD3) were induced in seeds under heat stress
during radicle protrusion and in seedlings with 2 cm roots. It was possible to observe the
subcellular localization of the RcSOD genes in Nicotiana benthamiana leaves, in which
RcSOD genes (RcMnSOD5 and RcFeSOD8) may have differences compared to the
subcellular location in A. thaliana. In CHAPTER 3 (published article), we initially
identified 41 genes of the heat shock protein family predicted in Castor bean (RcsHSP),
showing that tandem duplication seems to be one of the possible causes for the largest
number of castor genes compared to A. thaliana. Besides, we showed the pattern of
XI
gene expression of 10 RcsHSP genes induced during radicle protrusion and early
seedling stage. However, demonstrating the specificity of expression in roots,
cotyledons, and leaves at different stages of seedling development under heat stress.
Finally, we performed the functional characterization through the overexpression of two
RcsHSP genes (RcsHSP12 and RcsHSP19) in A. thaliana, resulting in (a) higher
percentage of seed germination under heat, osmotic and saline stresses; (b) greater
enzymatic antioxidant potential of SOD; (c) and higher concentration of protective
carbohydrates (sucrose and raffinose). In CHAPTER 4 (submitted article), we identified
the family of genes of the nuclear transcription factor subunit B (NF-YB) in Castor bean
and made the functional characterization of the RcNF-YB8 gene regarding the induction
of early flowering in A. thaliana. The phylogenetic comparison allowed us to identify
orthologous genes in angiosperms and motif patterns that may be associated with
differences between the RcNF-YB subfamilies. The induction of RcNF-YB genes was
observed to be greater during imbibition and germination compared to the postgerminative phase, also showing different profiles of induction and suppression by heat
stress. The RcNF-YB8 gene demonstrated suppression by heat stress and a pattern of
greater expression in leaves, while the overexpression in A. thaliana also demonstrated
to induce early flowering, therefore, impacting the size of the plant and fruits, and
consequent productivity. The results involve a broad characterization of genes from
three important families in the response of seeds and seedlings to abiotic stresses and
subsequent plant development, in which the characterization of the genes showed
significant relevance indicating target genes potentially useful in breeding programs
towards varieties of Castor bean with superior vigor aimed at better stand stablishment
and crop development, and consequently better productivity of Castor bean around the
world and by the family farmers in the Brazilian Northeast semiarid.
