Alternative oxidase (AOX) constitutes a small family of proteins in Citrus clementina and Citrus sinensis L. Osb

The alternative oxidase (AOX) protein is present in plants, fungi, protozoa and some invertebrates. It is involved in the mitochondrial respiratory chain, providing an alternative route for the transport of electrons, leading to the reduction of oxygen to form water. The present study aimed to characterize the family of AOX genes in mandarin (Citrus clementina) and sweet orange (Citrus sinensis) at nucleotide and protein levels, including promoter analysis, phylogenetic analysis and C. sinensis gene expression. This study also aimed to do the homology modeling of one AOX isoform (CcAOXd). Moreover, the molecular docking of the CcAOXd protein with the ubiquinone (UQ) was performed. Four AOX genes were identified in each citrus species. These genes have an open reading frame (ORF) ranging from 852 bp to 1150 bp and a number of exons ranging from 4 to 9. The 1500 bp-upstream region of each AOX gene contained regulatory cis-elements related to internal and external response factors. CsAOX genes showed a differential expression in citrus tissues. All AOX proteins were predicted to be located in mitochondria. They contained the conserved motifs LET, NERMHL, LEEEA and RADE-H as well as several putative post-translational modification sites. The CcAOXd protein was modeled by homology to the AOX of Trypanosona brucei (45% of identity). The 3-D structure of CcAOXd showed the presence of two hydrophobic helices that could be involved in the anchoring of the protein in the inner mitochondrial membrane. The active site of the protein is located in a hydrophobic environment deep inside the AOX structure and contains a diiron center. The molecular docking of CcAOXd with UQ showed that the binding site is a recessed pocket formed by the helices and submerged in the membrane. These data are important for future functional studies of citrus AOX genes and/or proteins, as well as for biotechnological approaches leading to AOX inhibition using UQ homologs. (Résumé d'auteur

HVA22 from citrus: A small gene family whose some members are involved in plant response to abiotic stress

International audienceThe HVA22 gene has been isolated for the first time from the aleurone layer of barley (Hordeum vulgare). Here, we characterized the HVA22 family from citrus (C. clementina and C. sinensis). Twelve genes, 6 in each species, were identified as well as duplication events for some of them. The ORF size ranged from 235 to 804 bp and the protein molecular weight from 94 to 267 kDa. All the citrus HVA22 protein presented transmembrane location and conserved TB2/DP1/HVA22 region. Phylogenetic and gene expression analyses suggested that some citrus HVA22 play a role in flower and fruit development, and that gene expression may be regulated by hormone or environmental conditions. Other regulation levels were also predicted, such as alternative splicing and post translational modifications. The overall data indicated that citrus HVA22 may be involved in vesicular traffic in stressed cells, and that CcHVA22d could be involved in dehydration tolerance

Expression of <i>CsAOX</i> genes in different <i>C</i>. <i>sinensis</i> tissues.

<p>Expression of <i>CsAOX</i> genes in different <i>C</i>. <i>sinensis</i> tissues.</p

<i>Cis</i>-elements present in the promoter region of citrus <i>AOX</i> genes.

<p>The <i>cis</i>-elements were analyzed in the upstream promoter region of the translation start site using the plantCARE database.</p

Post-translational modifications of citrus AOX proteins.

<p>* Protein resulting from the alternative transcript of the <i>CsAOXa</i> gene.</p

Characteristics of the AOX proteins present in the citrus genomes.

<p>GRAVY: grand average of hydropathicity; Mw: molecular weight; pI: isoeletric point; SP: signal peptide. *Protein resulting from the alternative transcript of the <i>CsAOXa</i> gene.</p

Phylogenetic tree obtained with the AOX proteins of <i>A</i>. <i>thaliana</i>, <i>C</i>. <i>sinensis</i> and <i>C</i>. <i>clementina</i>.

<p>Phylogenetic tree obtained with the AOX proteins of <i>A</i>. <i>thaliana</i>, <i>C</i>. <i>sinensis</i> and <i>C</i>. <i>clementina</i>.</p

Tridimensional structure of CcAOXd obtained by homology modeling with the <i>T</i>. <i>brucei</i> AOX (Pdb code 3VV9) as a template.

<p><b>A.</b> Alignment of TbAOX and CcAOXd proteins. Gaps introduced to get the best alignment are indicated by (-). Highly conserved domains related to protein structure and activity are indicated in grey. Identical amino acids are indicated by an asterisk (*), conservative substitutions by a colon (:) and semiconserved substitutions by a period (.). <b>B.</b> Representation of the 3-D structure of CcAOXd (in grey) containing 6 helices. Iron atoms are represented by orange spheres. <b>C.</b> Structural details of the CcAOXd catalytic center, showing the CcAOX diiron center. The diiron center contains 4 Glu and 2 His residues. Iron atoms are represented by orange spheres. <b>D.</b> Molecular surface position of the hydrophobic cavity during docking with UQ. UQ is shown in red. <b>E.</b> Structural details of UQ occupying the hydrophobic cavity. Iron atoms and UQ are represented by orange spheres and in red, respectively. Donors and acceptors H-bonds are indicated by a purple and green color gradient <b>F.</b> Predicted CcAOXd peptide sequence showing conserved domains and structurally important amino acids. The black triangle indicated the position of the redox-active Tyr (Y), and the 4 iron-binding sites are numbered from 1 to 4. The black arrows highlight the Glu (E) and His (H) residues, which are important for the coordination of the diiron center.</p

Characteristics of the <i>AOX</i> genes present in the <i>Citrus clementina</i> and <i>Citrus sinensis</i> genomes.

<p>ORF: open reading frame. (*) indicated the gene ID of the alternative transcript of the <i>CsAOXa</i> gene.</p