Molecular Basis Underlying Leaf Variegation of a Moth Orchid Mutant (Phalaenopsis aphrodite subsp. formosana)

Abstract

Leaf variegation is often the focus of plant breeding. Here, we studied a variegated mutant of Phalaenopsis aphrodite subsp. formosana, which is usually used as a parent of horticultural breeding, to understand its anatomic and genetic regulatory mechanisms in variegation. Chloroplasts with well-organized thylakoids and starch grains were found only in the mesophyll cells of green sectors but not of yellow sectors, confirming that the variegation belongs to the chlorophyll type. The two-dimensional electrophoresis and LC/MS/MS also reveal differential expressions of PsbP and PsbO between the green and yellow leaf sectors. Full-length cDNA sequencing revealed that mutant transcripts were caused by intron retention. When conditioning on the total RNA expression, we found that the functional transcript of PsbO and mutant transcript of PsbP are higher expressed in the yellow sector than in the green sector, suggesting that the post-transcriptional regulation of PsbO and PsbP differentiates the performance between green and yellow sectors. Because PsbP plays an important role in the stability of thylakoid folding, we suggest that the negative regulation of PsbP may inhibit thylakoid development in the yellow sectors. This causes chlorophyll deficiency in the yellow sectors and results in leaf variegation. We also provide evidence of the link of virus CymMV and the formation of variegation according to the differential expression of CymMV between green and yellow sectors.

Keywords: alternative polyadenylation; alternative splicing; chloroplast structure; differential expression protein; proteome analysis; variegation mutant.

FIGURE 1

Plant phenotype of Phalaenopsis aphrodite subsp. formosana variegated line. (A) Plant, (B) stalk, (C) flower bud, (D) pedicle, and (E) young leaf. All scale bars: 1 cm.

FIGURE 2

Comparison of leaf structure between the yellow (Y) and green sectors (G) of a variegated leaf of P. aphrodite subsp. formosana. Inspection of cross sections of yellow sectors using (A) light microscopy and (B) confocal microscopy does not reveal chloroplasts filled with chlorophyll. Scale bars: 100 μm.

FIGURE 3

The comparative ultrastructure of plastids in a variegated leaf. (A) The green leaf area with typical chloroplasts, including well-organized thylakoids, grana, and starch grains. (B) The yellow leaf area with poor developed plastids, with few unstacked and loose thylakoids (open arrows), rich of plastoglobuli (arrows), but no starch grains. M: mitochondrion, G: granum, S: starch grain, Pg: plastoglobulus.

FIGURE 4

2-DE gel image of acidic range (pH 4–7) of total protein fraction from green sectors and yellow sectors. Two-dimensional electrophoresis analysis of green and yellow P. aphrodite proteins. Proteins were separated by gel electrophoresis followed by Coomassie blue staining. The pH and molecular weight standards are labeled. PsbO and PsbP spots are delineated by boxes. All experiments were repeated three times. pI, isoelectric points.

FIGURE 5

Reverse transcription PCR (RT-PCR) analyses of PsbP and PsbO polymorphic and 18S rRNA gene products of total RNA from green and yellow sectors of P. aphrodite subsp. formosana. Green: green sector; yellow: yellow sector; PA: all type; PB: PsbP wild-type; PC: PsbP mutant type; OA: wild-type; OB: all type.

FIGURE 6

Reverse transcription-PCR (A) and Real-time PCR (B) analysis of gene expression in PsbP, PsbO and 18S rRNA gene products of total RNA from green and yellow sector of variegated leaf from P. aphrodite subsp. formosana. PA: all type; OB: all type. ^∗p < 0.05 compare between green and yellow sectors.

FIGURE 7

Real-time PCR analyses of PsbP and PsbO polymorphic and 18S rRNA gene products of total RNA from green and yellow sectors of P. aphrodite subsp. formosana. Green: green sector; yellow: yellow sector; PB: PsbP wild-type; PC: PsbP mutant type; OA: PsbO wild-type; OC: PsbO mutant type; CymMV: Cymbidium mosaic virus. ^∗p < 0.05 compare between green and yellow sectors.