Cytokinins (CK) are plant growth regulators involved in multiple physiological processes in plants. One less studied aspect is CK homeostasis (HM). The primary genes related to HM are involved in biosynthesis (IPT), degradation (CKX), and signaling (ARR). This paper demonstrates the effect of auxin (Aux) and CK and their cross talk in a Coffea canephora embryogenic system. The transcriptome and RT-qPCR suggest that Aux in pre-treatment represses biosynthesis, degradation, and signal CK genes. However, in the induction, there is an increase of genes implicated in the CK perception/signal, indicating perhaps, as in other species, Aux is repressing CK, and CK are inducing per se genes involved in its HM. This is reflected in the endogenous concentration of CK; pharmacology experiments helped study the effect of each plant growth regulator in our SE system. We conclude that the Aux–CK balance is crucial to directing somatic embryogenesis in C. canephora.
Auxin plays a central role in growth and plant development. To maintain auxin homeostasis, biological processes such as biosynthesis, transport, degradation, and reversible conjugation are essential. The Gretchen Hagen 3 (GH3) family genes codify for the enzymes that esterify indole-3-acetic acid (IAA) to various amino acids, which is a key process in the induction of somatic embryogenesis (SE). The GH3 family is one of the principal families of early response to auxin genes, exhibiting IAA-amido synthetase activity to maintain optimal levels of free auxin in the cell. In this study, we carried out a systematic identification of the GH3 gene family in the genome of Coffea canephora, determining a total of 18 CcGH3 genes. Analysis of the genetic structures and phylogenetic relationships of CcGH3 genes with GH3 genes from other plant species revealed that they could be clustered in two major categories with groups 1 and 2 of the GH3 family of Arabidopsis. We analyzed the transcriptome expression profiles of the 18 CcGH3 genes using RNA-Seq analysis-based data and qRT-PCR during the different points of somatic embryogenesis induction. Furthermore, the endogenous quantification of free and conjugated indole-3-acetic acid (IAA) suggests that the various members of the CcGH3 genes play a crucial role during the embryogenic process of C. canephora. Three-dimensional modeling of the selected CcGH3 proteins showed that they consist of two domains: an extensive N-terminal domain and a smaller C-terminal domain. All proteins analyzed in the present study shared a unique conserved structural topology. Additionally, we identified conserved regions that could function to bind nucleotides and specific amino acids for the conjugation of IAA during SE in C. canephora. These results provide a better understanding of the C. canephora GH3 gene family for further exploration and possible genetic manipulation.
Auxin is involved in almost every aspect of plant growth and development, from embryogenesis to senescence. Indole-3-acetic acid (IAA) is the main known natural auxin that is synthesized by enzymes tryptophan aminotransferase of arabidopsis (TAA) and YUCCA (YUC) of the flavin-containing monooxygenases family (FMO) from one of the tryptophan-dependent pathways. Genome-wide identification and comprehensive analysis of the YUC-protein family have been conducted in Coffea canephora in the present study. A total of 10 members CcYUC gene family were identified in C. canephora. Phylogenetic analysis revealed that the CcYUC protein family is evolutionarily conserved, and they consist of four groups. In contrast, bioinformatic analysis predicted a hydrophobic transmembrane helix (TMH) for one CcYUC (YUC10) member only. Isoelectric point (pI), molecular mass (Ms), signal peptide, subcellular localization, and phosphorylation sites were predicted for CcYUC proteins.YUC enzymes require the prosthetic group flavin adenine dinucleotide (FAD) and the cofactor nicotinamide adenine dinucleotide phosphate (NADPH) for their enzymatic activity. Therefore, we include the molecular docking for CcYUC2-FAD-NADPH-IPyA and yucasin, which is a specific inhibitor for YUC activity. The docking results showed FAD and NADPH binding at the big and small domain sites, respectively, in CcYUC2. IPyA binds very close to FAD along the big domain, and yucasin competes for the same site as IPA, blocking IAA production. Furthermore, in silico point mutations affect the stability of the CcYUC2-4 proteins.
Fruits of species of the genus Bromelia contain compounds with health benefits and potential biotechnological applications. For example, Bromelia karatas fruits contain antioxidants and proteins with bactericidal activity, but studies regarding the activity of these metabolites and potential benefits are required. We evaluated the bactericidal activity of the methanolic extract (treated and not treated with activated charcoal) and its fractions (hexane, ethyl acetate, and methanol) from ripe B. karatas fruit (8 °Brix) against Escherichia coli, Enterococcus faecalis, Salmonella enteritidis, and Shigella flexneri. The methanolic extract (ME) minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined at eight concentrations. The methanolic extract MIC was 5 mg/mL for E. faecalis and 10 mg/mL for the other bacteria; the MBC was 20 mg/mL for E. coli and E. faecalis, and 40 mg/mL for S. enteritidis and S. flexneri. Through gas chromatography–mass spectrometry, 131 compounds were identified, some of which had previously been reported to have biological activities, such as bactericidal, fungicide, anticancer, anti-inflammatory, enzyme inhibiting, and anti-allergic properties. The most abundant compounds found in the ME of B. karatas fruits were maleic anhydride, 5-hydroxymethylfurfural, and itaconic anhydride. This study shows that B. karatas fruits contain metabolites that are potentially beneficial for health.
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