Molecular insights into the mechanisms of a leaf color mutant in Anoectochilus roxburghii by gene mapping and transcriptome profiling based on PacBio Sequel II

Abstract

Plants with partial or complete loss of chlorophylls and other pigments are frequently occurring in nature but not commonly found. In the present study, we characterize a leaf color mutant 'arly01' with an albino stripe in the middle of the leaf, which is an uncommon ornamental trait in Anoectochilus roxburghii. The albino "mutant" middle portion and green "normal" leaf parts were observed by transmission electron microscopy (TEM), and their pigment contents were determined. The mutant portion exhibited underdevelopment of plastids and had reduced chlorophyll and other pigment (carotenoid, anthocyanin, and flavonoid) content compared to the normal portion. Meanwhile, comparative transcript analysis and metabolic pathways mapping showed that a total of 599 differentially expressed genes were mapped to 78 KEGG pathways, most of which were down-regulated in the mutant portion. The five most affected metabolic pathways were determined to be oxidative phosphorylation, photosynthesis system, carbon fixation & starch and sucrose metabolism, porphyrin and chlorophyll metabolism, and flavonoid biosynthesis. Our findings suggested that the mutant 'arly01' was a partial albinism of A. roxburghii, characterized by the underdevelopment of chloroplasts, low contents of photosynthetic and other color pigments, and a number of down-regulated genes and metabolites. With the emergence of ornamental A. roxburghii in southern China, 'arly01' could become a popular cultivar due to its unique aesthetics.

Figure 1

Phenotypes of the mutant ‘arly01’ (A) and wild type ‘Changtai’ (B) of A. roxburghii in the greenhouse. Overview of the chloroplast in the mutated portion (D) and normal portion (E) of the ‘arly01’ leaf (C) by transmission electron microscopic (TEM).

Figure 2

Chlorophyll (A) and other pigment contents (B) in the mutated portion (M) and the normal portion (N) of ‘arly01’ leaves (p-value < 0.01).

Figure 3

Volcano plots of gene expression profile between M portion versus N portion. Red points represent up-regulated DEGs. Blue points represent down-regulated DEGs. Gray points represent not changed genes. The identification thresholds were set as FDR < 0.01 and |log₂(fold change)|≥ 1. (A) 982 identified DEGs, including 366 up-regulated and 616 down-regulated genes (M portion vs. N portion); (B) 645 GO annotated DEGs, including 197 up-regulated and 448 down-regulated genes (M portion vs. N portion). The top three abundant GO terms were metabolic process (387 DEGs), cellular process (298 DEGs), and single-organism process (239 DEGs).

Figure 4

Comparison of the expression levels of six selected genes detected by transcriptome and RT-qPCR experiment. Results were presented as the mean of three repeated experiments.

Figure 5

GO enrichment analysis of the genes of mutation portion vs. normal portion from ‘arly01’ leaves. A total of 40,206 unigenes and 645 DEGs were divided into three categories: biological process (BP), cellular component (CC), and molecular function (MF). Red columns represent the number of unigenes involved in the GO terms, blue columns represent the number of DEGs.

Figure 6

Enriched and classified KEGG pathways of the DEGs between the M portion versus N portion of the ‘arly01’ leaves. Color and size of the dots indicated the p-value and the number of DEGs (numbers noted next to the dots) mapped to a certain pathway, respectively. The rich factor (x-axis) is the ratio of the DEG number to the total gene number in the same pathway, represented as dots position. The pathways were further classified into five major groups: metabolism, genetic information processing, environmental information processing, cellular processes, and organismal systems.

Figure 7

Overview of the differentially expressed genes (DEGs) that were involved in top-influenced metabolic pathways from the leaf color mutant ‘arly01’. Genes in red boxes and green boxes represented up-regulated genes and down-regulated genes, respectively. The metabolic processes include (A) photosynthesis system, (B) oxidative phosphorylation, (C) carbon fixation and starch and sucrose metabolism, (D) porphyrin and chlorophyll metabolism, and (E) flavonoid biosynthesis. Gene information is listed in Supplementary Table S3.