High-throughput discovery of plastid genes causing albino phenotypes in ornamental chimeric plants

Abstract

Chimeric plants composed of green and albino tissues have great ornamental value. To unveil the functional genes responsible for albino phenotypes in chimeric plants, we inspected the complete plastid genomes (plastomes) in green and albino leaf tissues from 23 ornamental chimeric plants belonging to 20 species, including monocots, dicots, and gymnosperms. In nine chimeric plants, plastomes were identical between green and albino tissues. Meanwhile, another 14 chimeric plants were heteroplasmic, showing a mutation between green and albino tissues. We identified 14 different point mutations in eight functional plastid genes related to plastid-encoded RNA polymerase (rpo) or photosystems which caused albinism in the chimeric plants. Among them, 12 were deleterious mutations in the target genes, in which early termination appeared due to small deletion-mediated frameshift or single nucleotide substitution. Another was single nucleotide substitution in an intron of the ycf3 and the other was a missense mutation in coding region of the rpoC2 gene. We inspected chlorophyll structure, protein functional model of the rpoC2, and expression levels of the related genes in green and albino tissues of Reynoutria japonica. A single amino acid change, histidine-to-proline substitution, in the rpoC2 protein may destabilize the peripheral helix of plastid-encoded RNA polymerase, impairing the biosynthesis of the photosynthesis system in the albino tissue of R. japonica chimera plant.

Figure 1

Chimeric plant materials and variation in their plastomes between green and albino sectors. a, Representative chimeric leaves of the 14 plants with plastome polymorphisms identified in this study. 1, Reynoutria japonica; 2, Ficus benjamina; 3, Ficus natalensis; 4, Spiraea japonica; 5, Hibiscus syriacus; 6, Hedera helix; 7, Syngonium podophyllum; 8, Cymbidium hybrid; 9, Cymbidium sinense; 10, Hosta longipes; 11, Chlorophytum comosum; 12, Dracaena sanderiana; 13, Juniperus chinensis; 14, Chamaecyparis pisifera. b, Local DNA sequence alignment showing the polymorphism and associated amino acid change between green and albino tissues collected from chimeric leaves. PEP-related genes are shown in gray, ATP synthase-related genes in yellow, and photosystem-related genes in green. c, Representative images of the eight chimeric plant species with identical plastid or intronic variation between green and albino leaf tissues. From the left: 15, Euonymus japonicus; 16, Euonymus hamiltonianus; 17, E. hamiltonianus “Snow”; 18, H. helix with big leaves; 19, H. helix with small leaves; 20, Hoya carnosa; 21, Polygonatum odoratum; 22, Aglaonema costatum; 23, Epipremnum aureum.

Figure 2

Consensus plastome maps and albino genes in 14 chimeric plants. Each arrow indicates the location of a polymorphism between green and albino plastomes; the numbers refer to the species, as in in Fig. 1a. Red, SNP; blue, InDel. a, Complete representative plastome map for the 21 angiosperm species. b, Complete representative plastome map of the two gymnosperm species Chamaecyparis pisifera and Juniperus chinensis.

Figure 3

Morphology, chlorophyll contents, and cell structures of the green and albino leaf sectors of Reynoutria japonica. a, Representative R. japonica leaf showing three types of chimeric tissue: Normal green, pale green, and albino were denoted as GLT/GLT, GLT/ALT, and ALT/ALT for adaxial and abaxial sides, respectively. b, Chlorophyll contents in green and albino leaf sectors. Data are shown as means ± standard deviation from three biological samples. c, Gel electrophoresis of an allele-specific marker to validate the SNP detected in R. japonica. GLT, green sector only; ALT, albino sector only; Mix, DNA from mixed leaf tissues containing both green and albino sections. d, Transmission electron microscopy analysis of the plastids in green (GLT) and albino (ALT) leaf sectors. C, chloroplasts in GLT. P, protoplasts in ALT. Albino tissues had abnormal cell structures with a large vacuole (V; right). Scale bars in each picture represent 0.5 μm for GLT, 0.2 μm for ALT, and 2 μm for ALT cells.

Figure 4

Structural prediction model of the plastid-encoded RNA polymerase of Reynoutria japonica. a, Structure of Thermus thermophilus RNA polymerase (PDB code: 1IW7), shown as a ribbon diagram. The individual subunits are colored as follows: αI subunit (green), αII subunit (blue), β subunit (magenta), β’ subunit (yellow), ω subunit (salmon), and σ70 subunit (gray). The β’ subunit used for modeling the R. japonica RNA polymerase is highlighted by the dashed box. b, Structural model of the β’ subunit of R. japonica RNA polymerase, shown as a ribbon diagram. The α-helix harboring the His-to-Pro substitution is shown in a dashed box, while the proline residue is shown as a space-filling model. c, Magnification of the α-helix containing the polymorphism between the green and albino leaf sectors. His114 in green tissues and Pro114 in albino tissues are shown using the space-filling model, and the steric clash between Pro114 and Ala100 in the α-helix is highlighted by a yellow dashed oval. d, Amino acid frequencies of plant RpoC2s near the R. japonica His114 residue. The WebLogo was generated using 159 plant RpoC2 proteins retrieved from GenBank. The yellow-shaded region is the His114 residue.

Figure 5

Transcript levels of 14 chloroplast genes in the green and albino leaf tissues in a chimeric Reynoutria japonica plant. All RT-qPCR experiments were repeated three times with biological replicates. Data are shown as means ± standard deviation. Asterisks highlight significant differences between the two genotypes, determined by an unpaired Student’s t-test (^**p < 0.01, ^*p < 0.05).