Table 1.
ARF gene family in Eucalyptus.
Figure 1.
Gene structure of the EgrARF family.
The information on exon–intron structure was extracted from the Phytozome database and visualized by using the FancyGene software (http://bio.ieo.eu/fancygene/). The sizes of exons and introns are indicated by the scale at the top. The domains of EgrARF gene were predicted by Pfam (http://pfam.xfam.org/) and are indicated by different colours. The B3 together with ARF subdomains constitute the DNA binding domain (DBD). The CTD contains two sub-domains III and IV. The TAS3 and microRNA target sites are marked on the corresponding target genes. The triangles underline the insertion sites of additional short amino-acids segments between the B3 and ARF subdomains.
Table 2.
Summary of ARF gene content in angiosperm species.
Figure 2.
Phylogenetic relationships of ARF proteins between Eucalyptus and other species.
(A) Phylogenetic relationships between ARF proteins from Arabidopsis, Populus and Eucalyptus. Full-length protein sequences were aligned by using the Clustal_X program. The phylogenetic tree was constructed by using the MEGA5 program and the neighbor-joining method with predicted ARF proteins. Bootstrap support is indicated at each node. The blue shade highlights the activators, and the green shade indicates the distinct likely woody preferential clade containing EgrARF24. (B) Phylogenetic relationships between the orthologs of EgrARF24 in other species. EgrARF24 proteins were used to blast 33 species genomes in Phytozome. An E-value of 1.0E-50 as used as a cut off to select the ARF potential orthologs from each species. A phylogenetic tree was constructed used the procedure as in (A) and using AtARF2 was used as an outgroup. The species containing putative orthogs of EgrARF24 were the followings: 1 Aquilegia coerulea, 2 Glycine max, 1 Phaseolus vulgaris, 1 Carica papaya, 2 Malus domestica, 1 Prunus persica, 1 Fragaria vesca, 1 Vitis vinifera, 2 Populus trichocarpa, 1 Citrus sinensis, 1 Citrus clementine, 2 Gossypium raimondii, 1 Theobroma cacao.
Figure 3.
Expression profiles of 16 EgrARF genes in various organs and tissues.
The heat map was constructed by using the relative expression values determined by qRT-PCR of 16 EgrARF genes (indicated on the right) in 13 tissues and organs (indicated at the top) normalized with a control sample (in vitro plantlets). In the heat map, red and green indicate relatively high and lower expression (log2ratios) than in the control, respectively. Each measurement is the mean of three independent samples. The heat map and the hierarchical clustering were generated by MultiExperiment Viewer (MEV).
Figure 4.
Effect of environmental cues and developmental stages on EgrARF expression.
The heat map was constructed by using the relative expression values determined by qRT-PCR of EgrARF genes (indicated on the right) in various tissues and conditions (indicated at the top) normalized with a control sample (in vitro plantlets). In the heat map, red and green indicates relatively higher expression and lower expression (log2ratios) than in the control, respectively. The heat map and the hierarchical clustering were generated by MultiExperiment Viewer (MEV).
Figure 5.
EgrARF genes expression levels in young tree stems after long term hormones treatments.
Hormone treatments are detailed in the Methods section. NAA, 1-naphthaleneacetic acid, a synthetic auxin usually used in in vitro culture. ACC, a precursor of ethylene biosynthesis. GA, gibberellic acid. Relative mRNA abundance was compared to expression in mock-treated young tree stems. Error bars indicated the SE of mean expression values from three independent experiments.
Figure 6.
EgrARF transcriptional activities in tobacco protoplasts.
(A) Schemes of the effector and reporter constructs used to analyse the function of EgrARFs in auxin-responsive gene expression. The effector constructs express the EgrARF of interest driven by the 35S promoter. The reporter construct consists of a reporter gene expressing GFP driven by the auxin-responsive promoter DR5 (DR5::GFP). (B) Effector and reporter constructs were co-expressed in tobacco protoplasts in the presence or absence of a synthetic auxin (50 µM 2, 4-D). GFP fluorescence was quantified 16 h after transfection by flow cytometry. A mock effector construct (empty vector) was used as a control. In each experiment, protoplast transformations were performed in independent biological triplicates. Two independent experiments were performed and similar results were obtained; the figure indicates the data from one experiment. Error bars represent SE of mean fluorescence. Significant statistical differences (student T test, P<0.001) to control are marked with **.