Identification and Evolution of Functional Alleles of the Previously Described Pollen Specific Myrosinase Pseudogene AtTGG6 in Arabidopsis thaliana

Abstract

Myrosinases are β-thioglucoside glucohydrolases and serve as defense mechanisms against insect pests and pathogens by producing toxic compounds. AtTGG6 in Arabidopsis thaliana was previously reported to be a myrosinase pseudogene but specifically expressed in pollen. However, we found that AlTGG6, an ortholog to AtTGG6 in A. lyrata (an outcrossing relative of A. thaliana) was functional, suggesting that functional AtTGG6 alleles may still exist in A. thaliana. AtTGG6 alleles in 29 A. thaliana ecotypes were cloned and sequenced. Results indicate that ten alleles were functional and encoded Myr II type myrosinase of 512 amino acids, and myrosinase activity was confirmed by overexpressing AtTGG6 in Pichia pastoris. However, the 19 other ecotypes had disabled alleles with highly polymorphic frame-shift mutations and diversified sequences. Thirteen frame-shift mutation types were identified, which occurred independently many times in the evolutionary history within a few thousand years. The functional allele was expressed specifically in pollen similar to the disabled alleles but at a higher expression level, suggesting its role in defense of pollen against insect pests such as pollen beetles. However, the defense function may have become less critical after A. thaliana evolved to self-fertilization, and thus resulted in loss of function in most ecotypes.

Keywords: AlTGG6; AtTGG6; evolution; frame-shift mutation; myrosinase; pollen specific; β-thioglucosidase.

Figure 1

Chromosomal locations and phylogenetic analysis of Myr II members in Arabidopsis lyrata and A. thaliana. (A) Chromosomal locations of Myr II genes in A. thaliana; (B) Chromosomal locations of Myr II genes in A. lyrata; (C) Phylogeny inferred from cDNAs of Myr II members in A. thaliana and A. lyrata, using Myr I myrosinases AtTGG1 (At5g26000), AtTGG2 (At5g25980), and AtTGG3 (At5g48375) as outgroups. Note: AtTGG4 (At1g47600) and AtTGG5 (At1g51470) cDNA sequences were from ecotype Col-0, AtTGG6 (At1g51490) sequence was from ecotype Tsu-1 (GenBank accession number KU301834). AlTGG cDNA sequences were obtained by annotating a genomic scaffold (NW_003302555) in A. lyrata, and partially confirmed by sequencing the PCR-amplified cDNAs. The sequences were deposited in the GenBank database under accession numbers KU301856, KU301857, KU301858, and KU301859 for AlTGG4, AlTGG5, AlTGG6, and AlTGG45, respectively.

Figure 2

Myrosinase activity of recombinant AtTGG6 from Arabidopsis ecotype Tsu-1. (A) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of recombinant protein overexpressed in Pichia pastoris GS115; Lane 1, molecular weight marker, the mass of each band in kilodalton (kD) is shown to the left; Lane 2, purified recombinant AtTGG6; (B) Myrosinase activity of purified AtTGG6 as visualized using a glucose test reagent; AtTGG6 + Vc, AtTGG6 (50 ng) plus 0.8 mM ascorbic acid (Vc); AtTGG6 − Vc, AtTGG6 (50 ng) only, Vc not added; CK, AtTGG6 (50 ng) disabled by heating at 95 °C for 5 min before use.

Figure 3

Phylogenetic analysis of AtTGG6 genomic (A) and cDNA (B) sequences by Maximum Likelihood method. The evolutionary history was inferred based on the Tamura–Nei model [23]. The bootstrap values over 50% are shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically. The tree is drawn to scale, with branch lengths measured in million years (MYA) and substitutions per site (SPS). Evolutionary analyses were conducted in MEGA6 [24]. Functional alleles are highlighted in green or blue. The two functional alleles in Tsu-1 and Mr-0 which are clustered different in genomic and cDNA phylogenies are highlighted in blue.

Figure 4

Expression pattern of functional (A–H) and disabled (I–L) alleles of AtTGG6 in Arabidopsis thaliana. Functional Prom::GUS and disabled Prom::GUS were transformed into Col-0, and stained with X-gluxuronide solution. (A) Rosette; (B) stem; (C) leaves; and (D) root of transgenic plant with functional Prom::GUS; (E) a flower bud opened with a needle showing no GUS staining; (F) a flower one day before flowering opened with a needle showing the early expression; (G) a fully opened flower showing the high expression level; (H) an anther showing predominant expression in pollen; (I) a flower bud of disabled Prom::GUS opened with a needle showing no GUS staining; (J) a flower of disabled Prom::GUS one day before flowering showing weak expression; (K) a fully opened flower of disabled Prom::GUS showing expression at moderate level; (L) an anther showing disabled AtTGG6 predominantly transcribed in pollen.