Comparative phylogenomic and structural analysis of canonical secretory PLA2 and novel PLA2-like family in plants

Abstract

Plant secretory phospholipase A₂ (sPLA₂) is a family of lipolytic enzymes involved in the sn-2 hydrolysis of phospholipid carboxyester bonds, characterized by the presence of a conserved PA2c domain. PLA₂ produces free fatty acids and lysophospholipids, which regulate several physiological functions, including lipid metabolism, plant growth and development, signal transduction, and response to various environmental stresses. In the present work, we have performed a comparative analysis of PA2c domain-containing genes across plants, focusing on gene distribution, phylogenetic analysis, tissue-specific expression, and homology modeling. Our data revealed the widespread occurrence of multiple sPLA₂ in most land plants and documented single sPLA₂ in multiple algal groups, indicating an ancestral origin of sPLA₂. We described a novel PA2c-containing gene family present in all plant lineages and lacking secretory peptide, which we termed PLA₂-like. Phylogenetic analysis revealed two independent clades in canonical sPLA₂ genes referred to as α and β clades, whereas PLA₂-like genes clustered independently as a third clade. Further, we have explored clade-specific gene expressions showing that while all three clades were expressed in vegetative and reproductive tissues, only sPLA₂-β and PLA₂-like members were expressed in the pollen and pollen tube. To get insight into the conservation of the gene regulatory network of sPLA₂ and PLA₂-like genes, we have analyzed the occurrence of various cis-acting promoter elements across the plant kingdom. The comparative 3D structure analysis revealed conserved and unique features within the PA2c domain for the three clades. Overall, this study will help to understand the evolutionary significance of the PA2c family and lay the foundation for future sPLA₂ and PLA₂-like characterization in plants.

Keywords: PLA2-like; modelling; phylogeny; plant; pollen; sPLA2.

Figure 1

(A) Distribution of secretory PLA₂ and PLA₂-like genes in the different plant taxonomic groups, including Algae, Bryophytes, Pteridophytes, Gymnosperms, and Angiosperms. The species tree was elucidated using the NCBI taxonomy database and edited in the Interactive Tree of Life (iTOL) web tool. Each histogram corresponds to the number of sPLA₂ and PLA₂-like in each species. Black, histogram-sPLA₂; Gray, histogram-PLA2-like genes. (B) Schematic representation of PLA₂ protein with N and C terminal, conserved PA2c domain, Ca²⁺ binding motif, and HD catalytic dyad.

Figure 2

Phylogenetic relationships between sPLA₂ and PLA₂ -like members were constructed using the Maximum likelihood (ML) with the ultrafast bootstrap method. The evolution Whelan and Goldman model with Invariable and gamma (WAG+I+G4) was selected based on the Bayesian information criterion (BIC) score. Color nodes correspond to the bootstrap support. The scale bar indicates the rates of substitutions/site. Atha, Arabidopsis thaliana; Afil, Azolla filiculoides; Aang, Anthocerus angustus; Atri, Amborella trichopoda; Brap, Brassica rapa; Bdic, Brachypodium distachyon; Cann, Capsicum annuum; Crei, Chlamydomonas reinhardtii; Csor, Chlorella sorokiniana; Cric, Ceratopteris_richardii; Dcar, Daucus carota; Egra, Eucalyptus grandis; Gbil, Ginkgo biloba; Gmax, Glycine max; Gkin, Gonatozygon_kinahanii; Knit, Klebsormidium nitens; Mneg, Monoraphidium neglectum; Mpol, Marchantia polymorpha; Mtru, Medicago truncatula; Mend, Mesotaenium endlicherianum; Ntab, Nicotiana tabacum; Ncol, Nymphaea colorata; Osat, Oryza sativa; Pdru, Phylloglossum drummondii; Ptri, Populus trichocarpa; Pmar, Penium margaritaceum; Ppat, Physcomitrium patens; Ppro; Pycnococcus provasolii; Slyc, Solanum lycopersicum; Stub, Solanum tuberosum; Sbic, Sorghum bicolor; Tpli, Thuja plicata; Rsub, Raphidocelis subcapitata; Scon, Staurodesmus_convergens; Swal, Selaginella_wallacei; Sfal, Sphagnumfallax; Scus, Salvinia cucullata; Tsoc, Tetrabaena socialis; Tpra, Trifolium pratense; Vvin, Vitis vinifera; Vcar, Volvox carteri; Zmay, Zea mays.

Figure 3

(A) Vegetative and reproductive tissue-specific gene expression data of sPLA₂ and PLA₂-like members were collected for the selected plant species and arranged on a phylogenetic tree. All collected data were exploited to generate the heatmap using the CIMminer web tool. The expression data was represented as a transcript per million (TPM). The phylogenetic relationships between sPLA2- α, β, and PLA₂-like members were determined using an ML tree with 100 bootstrap replications in the MEGA X software with the default model. (B) Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showing the expression of tobacco sPLA2 and PLA2-like members in different tissues and during pollen germination (Pollen30) and pollen tube elongation (Pollen90).

Figure 4

In-silico prediction of cis-acting elements in the promoter region (5’ upstream region about 2kbp) using PlantCARE database. The number of elements in the promoter regions was depicted by a heatmap using the CIMminer web tool. The color code represents the number of cis-elements in the promoter regions; Cyan- 0 (absent), Red- 5 copies of the element.

Figure 5

A comparative analysis of predicted Arabidopsis PLA₂ structural models. (A) Cartoon representation of sPLA₂-α, PLA₂-β, and PLA₂-like protein models. (B) A comparative analysis of the Coulombic electrostatic potential distribution among PLA₂ members, calculated with the ChimeraX software. (C) Analysis of conserved residues on the sPLA₂ and PLA₂ surfaces, calculated using the Consurf server.