Active Sites of Reduced Epidermal Fluorescence1 (REF1) Isoforms Contain Amino Acid Substitutions That Are Different between Monocots and Dicots

<div><p>Plant aldehyde dehydrogenases (ALDHs) play important roles in cell wall biosynthesis, growth, development, and tolerance to biotic and abiotic stresses. The Reduced Epidermal Fluorescence1 is encoded by the subfamily 2C of ALDHs and was shown to oxidise coniferaldehyde and sinapaldehyde to ferulic acid and sinapic acid in the phenylpropanoid pathway, respectively. This knowledge has been gained from works in the dicotyledon model species <i>Arabidopsis thaliana</i> then used to functionally annotate ALDH2C isoforms in other species, based on the orthology principle. However, the extent to which the ALDH isoforms differ between monocotyledons and dicotyledons has rarely been accessed side-by-side. In this study, we used a phylogenetic approach to address this question. We have analysed the <i>ALDH</i> genes in <i>Brachypodium distachyon</i>, alongside those of other sequenced monocotyledon and dicotyledon species to examine traits supporting either a convergent or divergent evolution of the ALDH2C/REF1-type proteins. We found that <i>B</i>. <i>distachyon</i>, like other grasses, contains more ALDH2C/REF1 isoforms than <i>A</i>. <i>thaliana</i> and other dicotyledon species. Some amino acid residues in ALDH2C/REF1 isoforms were found as being conserved in dicotyledons but substituted by non-equivalent residues in monocotyledons. One example of those substitutions concerns a conserved phenylalanine and a conserved tyrosine in monocotyledons and dicotyledons, respectively. Protein structure modelling suggests that the presence of tyrosine would widen the substrate-binding pocket in the dicotyledons, and thereby influence substrate specificity. We discussed the importance of these findings as new hints to investigate why ferulic acid contents and cell wall digestibility differ between the dicotyledon and monocotyledon species.</p></div

A slab view of the substrate channel of ZmALDH2C1 from the bottom (left panel).

<p>A similar view of the channel after the mutation F466Y to Y466 is shown in the middle panel. A superimposed picture of the two views is shown in the right panel. The crystal structure of ZmALDH2C1 (PDB file: 4PXL) was retrieved from the Protein Data Bank and used to analyse the effects of the mutation on the protein conformation. Residues F466 and Y466 are shown in cyan and magenta colors, respectively. Oxygen, nitrogen, and sulfur atoms are shown in red, blue, and yellow, respectively. Green discontinued lines represent H-bonds. The most plausible arrangement of the Y466 is shown (see text for details). Based on this arrangement, the mutation F466Y appears to widen the substrate channel and to create new H-bonds in its vicinity. Model manipulation and mutation analysis were performed in the Swiss-Pdb Viewer software V4.1.</p

Relative synonymous codon usage.

<p>Estimates were based on the protein coding sequences of 8 and 15 dicotyledonous and monocotyledonous ALDH2C/REF1 isoforms, respectively. Letters in brackets represent amino acids. I: isoleucine; N: asparagine; V: valine; A: alanine.</p

Frequencies of plant ALDH families within selected dicotyledonous and monocotyledonous species.

<p>Abbreviations and their corresponding species names: A.t: <i>Arabidopsis thaliana</i>; E.s: <i>Eutrema salsugineum</i>; G.m: <i>Glycine max</i>; V.v: <i>Vitis vinifera</i>; G.r: <i>Gossypium raimondii</i>; B.d: <i>Brachypodium distachyon</i>; O.s: <i>Oryza sativa</i>; S.b: <i>Sorghum bicolor</i>; S.i: <i>Setaria italica</i>; Z.m: <i>Zea mays</i>. Note that the ALDH families 5, 6, 12, 18, 22 were not found in <i>Glycine max</i> (G.m) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165867#pone.0165867.ref037" target="_blank">37</a>]. The bar representing <i>G</i>. <i>max</i> is therefore not displayed for those missing families in the plot.</p

Frequency of nucleotides in codons of ALDH2C proteins.

<p>Frequency of nucleotides in codons of ALDH2C proteins.</p

Phylogenetic analysis of the ALDH superfamily in selected monocotyledonous species.

<p>Sequences of the dicotyledon <i>A</i>. <i>thaliana</i> were used as outgroup. Sequences were aligned by using MUSCLE, and the unrooted phylogram was generated by using Maximum Likelihood statistical method (MEGA6 software). Bootstrap values from 1000 replicates are indicated at each branch. Prefixes and symbols were used to indicate the origin of the sequences: At and black square, <i>Arabidopsis thaliana</i>; Bd and blue circle, <i>Brachypodium distachyon</i>; Os and red circle, <i>Oryza sativa</i>; Sb and cyan upward triangle, <i>Sorghum bicolor</i>; Zm and magenta downward triangle, <i>Zea mays</i>. Asterisks (* and **) added to ZmALDH10A8 within the box of family 10 refer to the loci GRMZM2G146754 (Phytozome v10.2) and AC74867.1 (NCBI), respectively.</p

Polymorphic amino acid residues within and between dicotyledonous and monocotyledonous ALDH2C sequences.

<p>Polymorphic amino acid residues within and between dicotyledonous and monocotyledonous ALDH2C sequences.</p

Aldehyde dehydrogenase (ALDH) protein families in <i>Brachypodium distachyon</i>.

<p>Aldehyde dehydrogenase (ALDH) protein families in <i>Brachypodium distachyon</i>.</p

Comparison of lineage-specific amino acid polymorphisms within the ALDH2C/REF1 isoforms in three selected species.

<p>Comparison of lineage-specific amino acid polymorphisms within the ALDH2C/REF1 isoforms in three selected species.</p

Genome-Wide Identification and Functional Classification of Tomato (<i>Solanum lycopersicum</i>) Aldehyde Dehydrogenase (ALDH) Gene Superfamily

<div><p>Aldehyde dehydrogenases (ALDHs) is a protein superfamily that catalyzes the oxidation of aldehyde molecules into their corresponding non-toxic carboxylic acids, and responding to different environmental stresses, offering promising genetic approaches for improving plant adaptation. The aim of the current study is the functional analysis for systematic identification of <i>S</i>. <i>lycopersicum</i> ALDH gene superfamily. We performed genome-based ALDH genes identification and functional classification, phylogenetic relationship, structure and catalytic domains analysis, and microarray based gene expression. Twenty nine unique tomato ALDH sequences encoding 11 ALDH families were identified, including a unique member of the family 19 ALDH. Phylogenetic analysis revealed 13 groups, with a conserved relationship among ALDH families. Functional structure analysis of ALDH2 showed a catalytic mechanism involving <i>Cys-Glu</i> couple. However, the analysis of ALDH3 showed no functional gene duplication or potential neo-functionalities. Gene expression analysis reveals that particular ALDH genes might respond to wounding stress increasing the expression as ALDH2B7. Overall, this study reveals the complexity of <i>S</i>. <i>lycopersicum</i> ALDH gene superfamily and offers new insights into the structure-functional features and evolution of ALDH gene families in vascular plants. The functional characterization of ALDHs is valuable and promoting molecular breeding in tomato for the improvement of stress tolerance and signaling.</p></div