RESUMO
Coffea canephora (2n = 2x = 22 chromosomes) is a species with extensive genetic diversity and desirable agronomic traits for coffee breeding programs. However, obtaining a new coffee cultivar through conventional breeding techniques may require more than 30 years of crossing cycles and selection, which hampers the effort of keeping up with market demands and rapidly proposing more resilient to climate change varieties. Although, the application of modern biotechnology tools such as precision genetic engineering technologies may enable a faster cultivar development process. Therefore, we aimed to validate the CRISPR/Cas9 system to generate mutations on a selected genotype of C. canephora, the clone 14. Embryogenic calli and a multiplex binary vector containing two sgRNAs targeting different exons of the CcPDS gene were used. The sgRNAs were under the C. canephora U6 promoter regulation. The target gene encodes phytoene desaturase, an enzyme essential for photosynthesis involved in ß-carotene biosynthesis. Somatic seedlings and embryos with albino, variegated and green phenotypes regenerated after Agrobacterium tumefaciens-mediated genetic transformation were analyzed by verifying the insertion of the Cas9 gene and later by sequencing the sgRNAs target regions in the genome of Robusta modified seedlings. Among them, 77% had the expected mutations, and of which, 50% of them had at least one target with a homozygous mutation. The genotype, temperature of co-cultivation with the bacteria, and light intensity used for subsequent embryo regeneration appeared to strongly influence the successful regeneration of plants with a mutated CcPDS gene in the Coffea genus.
Assuntos
Coffea , Sistemas CRISPR-Cas , Coffea/genética , Café , Edição de Genes , Oxirredutases , Melhoramento Vegetal , beta CarotenoRESUMO
BACKGROUND: Small auxin-up RNA (SAUR) genes form a wide family supposedly involved in different physiological and developmental processes in plants such as leaf senescence, auxin signaling and transport, hypocotyl development and tolerance to abiotic stresses. The transcription of SAUR genes is quickly induced by auxins, a group of phytohormones of major importance on embryo development. To better understand the distribution and expression profile of such still not explored family in Coffea sp., especially during the development of somatic embryogenesis (SE), SAUR members were characterized in silico using the available Coffea canephora genome data and analyzed for gene expression by RT-qPCR in C. arabica embryogenic samples. METHODS AND RESULTS: Over C. canephora genome 31 CcSAURs were distributed by 11 chromosomes. Out of these 31 gene members, 5 SAURs were selected for gene expression analysis in C. arabica embryogenic materials. CaSAUR12 and CaSAUR18 were the members highly expressed through almost all plant materials. The other genes had more expression in at least one of the developing embryo stages or plantlets. The CaSAUR12 was the only member to exhibit an increased expression in both non-embryogenic calli and the developing embryo stages. CONCLUSION: The identification of SAUR family on C. canephora genome followed by the analysis of gene expression profile across coffee somatic embryogenesis process on C. arabica represents a further additional step towards a better comprehension of molecular components acting on SE. Along with new research about this gene family such knowledge may support studies about clonal propagation methods via somatic embryogenesis to help the scientific community towards improvements into coffee crop.
Assuntos
Café , Ácidos Indolacéticos , Desenvolvimento Embrionário , Regulação da Expressão Gênica de Plantas/genética , Ácidos Indolacéticos/metabolismo , Técnicas de Embriogênese Somática de Plantas , RNA , TranscriptomaRESUMO
BACKGROUND: Coffee production relies on plantations with varieties from Coffea arabica and Coffea canephora species. The first, the most representative in terms of coffee consumption, is mostly propagated by seeds, which leads to management problems regarding the plantations maintenance, harvest and processing of grains. Therefore, an efficient clonal propagation process is required for this species cultivation, which is possible by reaching a scalable and cost-effective somatic embryogenesis protocol. A key process on somatic embryogenesis induction is the auxin homeostasis performed by Gretchen Hagen 3 (GH3) proteins through amino acid conjugation. In this study, the GH3 family members were identified on C. canephora genome, and by performing analysis related to gene and protein structure and transcriptomic profile on embryogenic tissues, we point a GH3 gene as a potential regulator of auxin homeostasis during early somatic embryogenesis in C. arabica plants. RESULTS: We have searched within the published C. canephora genome and found 17 GH3 family members. We checked the conserved domains for GH3 proteins and clustered the members in three main groups according to phylogenetic relationships. We identified amino acids sets in four GH3 proteins that are related to acidic amino acid conjugation to auxin, and using a transcription factor (TF) network approach followed by RT-qPCR we analyzed their possible transcriptional regulators and expression profiles in cells with contrasting embryogenic potential in C. arabica. The CaGH3.15 expression pattern is the most correlated with embryogenic potential and with CaBBM, a C. arabica ortholog of a major somatic embryogenesis regulator. CONCLUSION: Therefore, one out of the GH3 members may be influencing on coffee somatic embryogenesis by auxin conjugation with acidic amino acids, which leads to the phytohormone degradation. It is an indicative that this gene can serve as a molecular marker for coffee cells with embryogenic potential and needs to be further studied on how much determinant it is for this process. This work, together with future studies, can support the improvement of coffee clonal propagation through in vitro derived somatic embryos.
Assuntos
Coffea/genética , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Estudo de Associação Genômica Ampla , Proteínas de Plantas/genética , Sementes/crescimento & desenvolvimento , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Coffea/crescimento & desenvolvimento , Coffea/metabolismo , Homeostase , Ácidos Indolacéticos/metabolismo , Modelos Moleculares , Filogenia , Proteínas de Plantas/química , Conformação ProteicaRESUMO
The adaptation of crops to acid soils is needed for the maintenance of food security in a sustainable way, as decreasing fertilizers use and mechanical interventions in the soil would favor the reduction of agricultural practices' environmental impact. Phosphate deficiency and the presence of reactive aluminum affect vital processes to the plant in this soil, mostly water and nutrient absorption. From this, the understanding of the molecular response to these stresses can foster strategies for genetic improvement, so the aim was to broadly analyze the transcriptional variations in Poupulus spp. in response to these abiotic stresses, as a plant model for woody crops. A co-expression network was constructed among 3,180 genes differentially expressed in aluminum-stressed plants with 34,988 connections. Of this total, 344 genes presented two-fold transcriptional variation and the group of genes associated with those regulated after 246 hours of stress had higher number of connections per gene, with some already characterized genes related to this stress as main hubs. Another co-expression network was made up of 8,380 connections between 550 genes regulated by aluminum stress and phosphate deficiency, in which 380 genes had similar profile in both stresses and only eight with transcriptional variation higher than 20%. All the transcriptomic data are presented here with functional enrichment and homology comparisons with already characterized genes in another species that are related to the explored stresses, in order to provide a broad analysis of the co-opted responses for both the stresses as well as some specificity. This approach improves our understanding regarding the plants adaptation to acid soils and may contribute to strategies of crop genetic improvement for this condition that is widely present in regions of high agricultural activity.