Genome-wide analysis, transcription factor network approach and gene expression profile of GH3 genes over early somatic embryogenesis in Coffea spp

Abstract

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.

Keywords: Auxin homeostasis; Baby Boom; Coffee clonal propagation; Gretchen Hagen 3; Phylogenetics.

Fig. 1

Phylogenetic relationship and genomic structure of C. canephora putative GH3 genes. Exons are represented by yellow ellipses, introns by black lines and upstream/downstream untranslated regions by blue rectangles

Fig. 2

Phylogenetic tree with GH3 proteins from A. thaliana, Z. mays, O. sativa and C. canephora. The branches in red, green and blue colors represent the groups I, II and III, respectively

Fig. 3

Alignment view of the CcGH3 proteins sections containing the conserved amino acid residues involved in auxin conjugation by acidic amino acids. a Alignment of five amino acid residue sets (numbered from 1 to 5) related to acyl acid-binding specificity. The yellow shade represents key residue positions for specificity and residues written in red match with patterns for auxin binding; b Set of two amino acid sequences related to amino acid-binding specificity in which red shade represents the pattern for acidic amino acid binding and blue shade represents nonpolar amino acid binding

Fig. 4

Tridimensional structure of CcGH3.15. a overview of the protein structure; b close-up to the ligands adenosine monophosphate (AMP, green arrow) and 1H-indol-3-Yacetic acid (IAC, red arrow)

Fig. 5

Motif-binding network for selected CcGH3s genes (red ellipses) and their related transcription factors (rectangles). The color scale from white to black refers to the number of CcGH3 genes (one to four) in which a specific transcription factor can bind. Arrow width refers to the number of binding sites for one transcription factor at the promoter region of some GH3 gene (Additional file 6: Table S1)

Fig. 6

Visual and histological aspects of NEC, EC and ECS. a clusters of non-embryogenic cells; b clusters of embryogenic cells (the yellow cells are those with embryogenic potential); c bottom view of an erlenmeyer containing ECS; Histology of d NEC; e EC; and f ECS, stained with toluidine blue

Fig. 7

Gene expression patterns of a CaGH3.9, b CaGH3.13, c CaGH3.15 and d CaGH3.16 measured by RT-qPCR in NEC, EC and ECS. Data represent mean ± SD. Different letters above columns represent statistical differences among treatments by Tukey test at 5% of significance (P < 0.05)

Fig. 8

Expression profile of CaBBM and its regulatory relationships with CaGH3.9, CaGH3.13, CaGH3.15 and CaGH3.16. a Expression pattern of CaBBM in non-embryogenic cells (NEC), embryogenic cells (EC) and embryogenic cell suspension (ECS) measured by RT-qPCR; b Co-expression analysis of CaBBM gene with CaGH3.9, CaGH3.13, CaGH3.15 and CaGH3.16, inferred by Spearman’s method; dark blue means significant positive Spearman’s correlation, light blue non-significant and light red non-significant negative Spearman’s correlation