Rampant Nuclear Transfer and Substitutions of Plastid Genes in Passiflora

Abstract

Gene losses in plastid genomes (plastomes) are often accompanied by functional transfer to the nucleus or substitution of an alternative nuclear-encoded gene. Despite the highly conserved gene content in plastomes of photosynthetic land plants, recent gene loss events have been documented in several disparate angiosperm clades. Among these lineages, Passiflora lacks several essential ribosomal genes, rps7, rps16, rpl20, rpl22, and rpl32, the two largest plastid genes, ycf1 and ycf2, and has a highly divergent rpoA. Comparative transcriptome analyses were performed to determine the fate of the missing genes in Passiflora. Putative functional transfers of rps7, rpl22, and rpl32 to nucleus were detected, with the nuclear transfer of rps7, representing a novel event in angiosperms. Plastid-encoded rps7 was transferred into the intron of a nuclear-encoded plastid-targeted thioredoxin m-type gene, acquiring its plastid transit peptide (TP). Plastid rpl20 likely experienced a novel substitution by a duplicated, nuclear-encoded mitochondrial-targeted rpl20 that has a similar gene structure. Additionally, among rosids, evidence for a third independent transfer of rpl22 in Passiflora was detected that gained a TP from a nuclear gene containing an organelle RNA recognition motif. Nuclear transcripts representing rpoA, ycf1, and ycf2 were not detected. Further analyses suggest that the divergent rpoA remains functional and that the gene is under positive or purifying selection in different clades. Comparative analyses indicate that alternative translocon and motor protein complexes may have substituted for the loss of ycf1 and ycf2 in Passiflora.

Keywords: ycf1/ycf2; gene loss; plastid-encoded RNA polymerase); plastid-encoded ribosomal genes; transit peptide.

Fig. 1.

aa alignments of Passiflora RPS7 and TRX-m3. (A) Arabidopsis thaliana (A.thal) plastid RPS7 aa alignment with nuclear RPS7 in six species of Passiflora (P.). (B) Comparison of TRX-m3 in Populus trichocarpa (Po_tri) and Populus alba (Po_alb) with the consensus TRX-m3 sequence for six Passiflora species. (C) Alignment of nuclear RPS7 against TRX-m3 among six Passiflora species with only the first 150 aa of sequence alignment shown. The aa identity for the TP between RPS7 and TRX-m3 for each species is 100%. Black triangles denote TP cleavage site predicted by TargetP. Abbreviations: P. pit, P. pittieri; P. con, P. contracta; P. oer, P. oerstedii; P. ten, P. tenuiloba; P. aur, P. auriculata; P. bif, P. biflora.

Fig. 2.

Integration of plastid rps7 into the intron of nuclear-encoded thioredoxin gene in Passiflora. (A) Schematic diagram (not to scale) depicts the insertion of plastid rps7 into the intron of thioredoxin (trx-m3) that contains TP known for plastid localization. Gray boxes indicate the exons of the trx-m3 gene and the black line in between indicates the intron. The first exon of trx-m3 gene contains TP. White box represents the plastid rps7. Alternative splicing is shown in dotted arrows. Blue and red arrows represent the gene product of alternative splicing and localization of the product to the plastid, respectively. Arrows (a, b, and c) below the chimeric rps7-trx-m3 indicate the location annealing sites of primers designed to amplify the gene product. The figure is not drawn to scale. (B) PCR amplifications of the chimeric rps7-trx-m3 in Passiflora pittieri with the primers designed in figure (A). Lane 1, 1 kb DNA ladder (N3232L New England Biolabs, Inc); Lane 2, PCR product with primer set a and b; and Lane 3, PCR product with primer set a and c as indicated in (A). (C) Passiflora pittieri chimeric rps7-trx-m3 as a representation for all other Passiflora species. The three exons of the gene are annotated in yellow. Intron 5′ and 3′ splice sites are boxed in gray. Abbreviations, Nu, nucleus; Pt, plastid, Mt, mitochondrion.

Fig. 3.

aa alignments of Passiflora nuclear RPL22. (A) aa comparison of Passiflora nuclear RPL22 with plastid RPL22 in Arabidopsis thaliana. (B) Comparison of the organelle RNA recognition motif (ORRM) protein sequence among Populus trichocarpa (Po_triORRM), P. contracta (P.conORRM), and P. contracta RPL22 (P.conRPL22). Passiflora contracta RPL22 is used to represent RPL22 identified in all Passiflora species. The predicted TP of the Populus ORMM along with the ORRM and RPL22 sequences are labeled. Abbreviations: A. thal, Arabidopsis thaliana; P. pit, Passiflora pittieri; P. con, P. contracta; P. oer, P. oerstedii; P. ten, P. tenuiloba; P. aur, P. auriculata; P. bif, P. biflora.

Fig. 4.

Nuclear-encoded RPL20 isoforms in Passiflora. The NCBI CD database was used for CD prediction. (A) Putative mitochondrial RPL20 (RPL20-1) in Passiflora containing RNA-binding site as well as binding sites for other ribosomal subunits. (B) Putative plastid RPL20 (RPL20-2) in Passiflora with predicted binding sites for ribosomal subunits. (C) Mapping of P. oerstedii rpl20-2 transcript against the P. edulis BAC clone Pe84M23 indicates the presence of an intron. (D and E) PCR amplifications to verify intron presence in rpl20-1 and rpl20-2 genes. Lane 1, 1 kb DNA ladder (N3232L New England Biolabs, Inc); Lane 2, Passiflora pittieri; Lane 3, P. contracta; Lane 4, P. oerstedii; Lane 5, P. tenuiloba; Lane 6, P. auriculata; and Lane 7, P. biflora.

Fig. 5.

Phylogenetic distribution of nuclear transfer or substitution of plastid genes in Passiflora. The cladogram depicts the subgeneric relationships within Passiflora based on Shrestha et al. (2019) with Salicaceae as a outgroup. Distribution of plastid gene transfers to the nucleus (solid bar) and substitutions by nuclear genes (open bar) are plotted on the tree.

Fig. 6.

Schematic representation of two alternative scenarios for the origin of the nuclear-encoded plastid-targeted rpl20 gene in Passiflora. (A) Duplication of nuclear-encoded mitochondrial rpl20 followed by gain of a plastid-targeted TP by the duplicated copy, followed by the loss of rpl20 from the plastome. (B) Transfer of plastid rpl20 to nucleus that includes acquisition of a TP and an intron. A possible scenario for intron gain could be intron transfer from nuclear-encoded mitochondrial rpl20 due to sequence homology with nuclear-transferred rpl20, which is shown with dotted lines. Gain of a TP by nuclear-transferred plastid rpl20 facilitates plastid localization of its product. Gray and white boxes represent exons for the nuclear and plastid genes, respectively. Black lines between the exons represent introns. Dotted lines with arrowheads indicate proteins that are targeted either to mitochondria or plastids. Major evolutionary events are shown in thick black arrows and descriptions are provided. The figure is not drawn to scale. Abbreviations, Nu, nucleus; Mt, mitochondrion; Pt, Plastid.