Genome-Wide Analyses of Aspartic Proteases on Potato Genome (Solanum tuberosum): Generating New Tools to Improve the Resistance of Plants to Abiotic Stress

Abstract

Aspartic proteases are proteolytic enzymes widely distributed in living organisms and viruses. Although they have been extensively studied in many plant species, they are poorly described in potatoes. The present study aimed to identify and characterize S. tuberosum aspartic proteases. Gene structure, chromosome and protein domain organization, phylogeny, and subcellular predicted localization were analyzed and integrated with RNAseq data from different tissues, organs, and conditions focused on abiotic stress. Sixty-two aspartic protease genes were retrieved from the potato genome, distributed in 12 chromosomes. A high number of intronless genes and segmental and tandem duplications were detected. Phylogenetic analysis revealed eight StAP groups, named from StAPI to StAPVIII, that were differentiated into typical (StAPI), nucellin-like (StAPIIIa), and atypical aspartic proteases (StAPII, StAPIIIb to StAPVIII). RNAseq data analyses showed that gene expression was consistent with the presence of cis-acting regulatory elements on StAP promoter regions related to water deficit. The study presents the first identification and characterization of 62 aspartic protease genes and proteins on the potato genome and provides the baseline material for functional gene determinations and potato breeding programs, including gene editing mediated by CRISPR.

Keywords: CIS-elements; MEME; abiotic stress; aspartic proteases; intronless; phylogeny.

Figure 1

Phylogenetic tree of representative aspartic proteases. The phylogenetic tree was built with a total of 62 and 51 AP aminoacid sequences from S. tuberosum and A. thaliana, respectively, and 6 reference sequences from O. sativa L. (OsAP2), S. tuberosum L. (StAP3), A. thaliana L., C. cardunculos L. (Cardosin A), N. gracilis (Nepenthesin I), and H. vulgare L. signed in bold. Cluster names include typical (a), atypical (b, c, e, f, g, and h), and nucellin-like (d) aspartic proteases.

Figure 2

Phylogenetic tree of 62 representative aspartic proteases from S. tuberosum. The tree includes 8 reference aspartic proteases from O. sativa L. (OsAP2), S. tuberosum L. (StAP3), A. thaliana L. (NP_565219.1 and NP_563851.1), C. cardunculos. (Procardosin A), N. gracilis (NepenthesinI), and H. vulgare (Phytepsin) indicated in red. Group names and branches are indicated with different colors. (A) Pfam Domains; (B) MEME motifs (specified in Table S6).

Figure 3

Chromosome distribution of 62 StAP genes. Potato physical map (in bp) built by Agrobiotech tools based on PGSC DM3.4 sequence [50]. Blue lines connect genes in segmental duplications blocks previously reported [80], and green fine lines represent possible cases of segmental duplication detected.

Figure 4

Aspartic proteases exonic and intronic structure and gene expression heatmap. The phylogenetic tree was built with 62 StAP amino acid sequences. (a) Exon and intron structures were built based on the predicted primary longest transcript structure, and (b) RNA-Seq data from different libraries from double monoploid S. tuberosum group Phureja DM1-3 516 R44 clone (DM) were indicated as “number of fragments per kilobase of exon per million fragments mapped” (FPKM) by color scale [50]. The phylogram was built with the iTol program [81,82]. Names of genes are colored based on the group membership in Figure 1.

Figure 5

In silico identification of cis-acting elements in promoters of StAP. Colored boxes represent each element position in the coding strand.