AOX1-Subfamily Gene Members in Olea europaea cv. "Galega Vulgar"-Gene Characterization and Expression of Transcripts during IBA-Induced in Vitro Adventitious Rooting

Abstract

Propagation of some Olea europaea L. cultivars is strongly limited due to recalcitrant behavior in adventitious root formation by semi-hardwood cuttings. One example is the cultivar "Galega vulgar". The formation of adventitious roots is considered a morphological response to stress. Alternative oxidase (AOX) is the terminal oxidase of the alternative pathway of the plant mitochondrial electron transport chain. This enzyme is well known to be induced in response to several biotic and abiotic stress situations. This work aimed to characterize the alternative oxidase 1 (AOX1)-subfamily in olive and to analyze the expression of transcripts during the indole-3-butyric acid (IBA)-induced in vitro adventitious rooting (AR) process. OeAOX1a (acc. no. MF410318) and OeAOX1d (acc. no. MF410319) were identified, as well as different transcript variants for both genes which resulted from alternative polyadenylation events. A correlation between transcript accumulation of both OeAOX1a and OeAOX1d transcripts and the three distinct phases (induction, initiation, and expression) of the AR process in olive was observed. Olive AOX1 genes seem to be associated with the induction and development of adventitious roots in IBA-treated explants. A better understanding of the molecular mechanisms underlying the stimulus needed for the induction of adventitious roots may help to develop more targeted and effective rooting induction protocols in order to improve the rooting ability of difficult-to-root cultivars.

Keywords: IBA; adventitious rooting; alternative oxidase; alternative polyadenylation; auxins; gene expression; olive; plant mitochondria; transposable elements; vegetative propagation.

Figure 1

Neighbor-Joining (NJ) tree showing the relationships among deduced AOX sequences from 56 plant species, including monocot and eudicot plant species. Putative peptide sequences corresponding to the isolated AOX1-subfamily members of Olea europaea L. were included (shown in red). 206 AOX sequences from higher plants were included (correspondence of accession numbers and the plant species is included in supplementary Tables S3 and S4). The NJ tree was obtained using the complete peptide sequences. The alignments were bootstrapped with 1000 replicates by the NJ method using the MEGA 7 software. AOX sequence from Neurospora crassa and two sequences of Chlamydomonas reinhardtii were used as outgroups. The scale bar indicates the relative amount of change along branches. In green: the branch corresponding to the AOX2-subfamily members. AOX1d members are in red and AOX1 members from monocot plant species are in the branch colored in light blue. Clusters grouping olive AOX members are in yellow and accessions corresponding to AOX from cv. ”Galega vulgar” are in red (OeAOX1a, acc. no. MF410314; OeAOX1d, acc. no. MF410315 and JX912721; OeAOX2, acc. no. JX912722).

Figure 2

Alignment of the six different isolated sequences corresponding to the 3′-UTR of OeAOX1a gene. The sequences are presented starting at the stop codon TGA shown in red. The reverse primers used in RT-qPCR analysis for each transcript variant are shown in different colors (green: variant X1, acc. no. MF410314; grey: variant X2, acc. no. MG208095) (for primers sequence see Table S2).

Figure 3

Multiple alignment of putative amino acid translated sequences of previously reported AOX proteins from A. thaliana (AtAOX1a_AT3G22370, AtAOX1b_AT3G22360, AtAOX1c_AT3G27620, AtAOX1d_AT1G32350) and AOX from O. europaea L. cv. ”Galega vulgar” (OeAOX1a_transcript variant X1_MF410314 and OeAOX1a_transcript variant X2_MG208095, OeAOX1d_transcript variant X1_ MF410315 and OeAOX1d_ transcript variant X2_JX91272). The alignment was performed using CLC Main Workbench 6.7.1 software. The data were retrieved from public web-based database Plaza v2.5, freely available at http://bioinformatics.psb.ugent.be/plaza/versions/plaza_v2_5/. Amino acid residues differing are shown in red, deletions are shown by minus signs. The putative mitochondrial transit peptides (mTP) are shown in blue boxes. The sites of two conserved cysteins (CysI and CysII) involved in dimerization of the AOX protein by S–S bond formation [37] are indicated in dark grey boxes. Helices α1 and α4, which form the hydrophobic region on the AOX molecular surface and are involved in membrane binding, are shown in red; helices α2, α3, α5, and α6, which form the four-helix bundle accommodating the diiron center, are shown in green [38]. Amino acids residues that coordinate the diiron center (E, glutamate and H, histidine) and those that interact with the inhibitor are in yellow and light pink boxes, respectively.

Figure 4

Structural mapping of the sequence diversity of OeAOX1d. The homology-based model of (a) OeAOX1d_transcript variant X2 and the structure of (b) AOX from T. brucei are displayed using a cartoon representation, with the helices shown as cylinders. The two identical functional subunits are colored in yellow (subunit A) and grey (subunit B), and the iron atoms that form the diiron center are represented by orange spheres, with the coordinating residues displayed using sticks. The region that corresponds to the sequence change in OeAOX1d_transcript variant X2 is highlighted in pink.

Figure 5

Relative mRNA expression of (a) OeAOX1a and (b) OeAOX1d in stem basal segments of O. europaea L. microcuttings during IBA-induced adventitious rooting. OeACT and OeEF1a were used as reference genes in data normalization. The relative expression values are depicted as the mean ± standard deviation of four biological replicates for each time point. The bars represent the fold-change related to the time point 0 hours after microcuttings treatment and inoculation, which was set to 1. Statistical significances (* p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001) between the two means were determined by the t-test using IBM^® SPSS^® Statistics version 22.0 (SPSS Inc., Armonk, NY, USA), h: hours, d: days.

Figure 6

Relative mRNA expression of (a) OeAOX1a_transcript variant X1, (b) OeAOX1a_transcript variant X2, (c) OeAOX1d_transcript variant X1, and (d) OeAOX1d_transcript variant X2 (D) in stem basal segments of O. europaea L. microcuttings during IBA-induced adventitious rooting. OeACT and OeEF1a were used as reference genes in data normalization. The relative expression values are depicted as the mean ± standard deviation of four biological replicates for each time point. The bars represent the fold-change related to the time point 0 hours after stem microcuttings treatment and inoculation, which was set to 1. Statistical significances (* p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001) between the two means were determined by the t-test using IBM^® SPSS^® Statistics version 22.0 (SPSS Inc., Armonk, NY, USA), h: hours, d: days, tv: transcript variant.

Figure 6

Relative mRNA expression of (a) OeAOX1a_transcript variant X1, (b) OeAOX1a_transcript variant X2, (c) OeAOX1d_transcript variant X1, and (d) OeAOX1d_transcript variant X2 (D) in stem basal segments of O. europaea L. microcuttings during IBA-induced adventitious rooting. OeACT and OeEF1a were used as reference genes in data normalization. The relative expression values are depicted as the mean ± standard deviation of four biological replicates for each time point. The bars represent the fold-change related to the time point 0 hours after stem microcuttings treatment and inoculation, which was set to 1. Statistical significances (* p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001) between the two means were determined by the t-test using IBM^® SPSS^® Statistics version 22.0 (SPSS Inc., Armonk, NY, USA), h: hours, d: days, tv: transcript variant.