A multi-omics approach identifies bHLH71-like as a positive regulator of yellowing leaf pepper mutants exposed to high-intensity light

Abstract

Light quality and intensity can have a significant impact on plant health and crop productivity. Chlorophylls and carotenoids are classes of plant pigments that are responsible for harvesting light energy and protecting plants from the damaging effects of intense light. Our understanding of the role played by plant pigments in light sensitivity has been aided by light-sensitive mutants that change colors upon exposure to light of variable intensity. In this study, we conducted transcriptomic, metabolomic, and hormone analyses on a novel yellowing mutant of pepper (yl1) to shed light on the molecular mechanism that regulates the transition from green to yellow leaves in this mutant upon exposure to high-intensity light. Our results revealed greater accumulation of the carotenoid precursor phytoene and the carotenoids phytofluene, antheraxanthin, and zeaxanthin in yl1 compared with wild-type plants under high light intensity. A transcriptomic analysis confirmed that enzymes involved in zeaxanthin and antheraxanthin biosynthesis were upregulated in yl1 upon exposure to high-intensity light. We also identified a single basic helix-loop-helix (bHLH) transcription factor, bHLH71-like, that was differentially expressed and positively correlated with light intensity in yl1. Silencing of bHLH71-like in pepper plants suppressed the yellowing phenotype and led to reduced accumulation of zeaxanthin and antheraxanthin. We propose that the yellow phenotype of yl1 induced by high light intensity could be caused by an increase in yellow carotenoid pigments, concurrent with a decrease in chlorophyll accumulation. Our results also suggest that bHLH71-like functions as a positive regulator of carotenoid biosynthesis in pepper.

Figure 1

Phenotypes, agronomic traits, and hormone profiles of yl1 and 6421 plants exposed to different light intensities. (A) Images of yl1 and 6421 plants exposed to high (H), medium (M), or low (L) light conditions for 15 days. (B) Xanthophyll and chlorophyll contents in yl1 and 6421 plants exposed to L, M, and H light for 15 days. (C) Net photosynthetic rate (Pn), stomatal conductance (Cond), fruit set per plant, average fruit weight, and total fruit yield of yl1 and 6421 plants grown under field conditions with unfiltered light (HL) or 70% shade (LL). (D) Hormone concentrations of yl1 and 6421 plants exposed to L, M, and H light for 15 days. Bars with different letters are significantly different (Duncan’s test; P < .05).

Figure 2

. DEGs in yl1 and 6421 exposed to high, medium, or low light for 15 days. (A) Unsupervised fuzzy clustering of DEGs (numbers in parentheses are the total numbers of DEGs in each cluster). (B) Venn diagram of DEGs. (C) GO enrichment analysis of DEGs, showing biological process (BP), cellular component (CC), and molecular function (MF) terms. ROC, response to oxygen-containing compound; ACPM, anchored component of plasma membrane; HH, hydrolase activity, hydrolyzing O-glycosyl compounds; TS, transcription factor activity, sequence-specific DNA binding; CROC, cellular response to oxygen-containing compound.

Figure 3

. Comparative analysis of genes and metabolites related to carotenoid biosynthesis in yl1 and 6421 plants exposed to high (H), medium (M), or low (L) light intensity. CrtISO, carotene isomerase; CrtZ-2, β-carotene hydroxylase 2; GGDP, geranylgeranyl diphosphate; LCYB, lycopene β-cyclase; LCYE, lycopene ε-cyclase; LUT1, carotenoid epsilon hydroxylase; LUT5, β-ring hydroxylase; NXS, neoxanthin synthase; PDS, phytoene desaturase; PSY, phytoene synthase; VDE, violaxanthin de-epoxidase; ZDS, ζ-carotene desaturase; Z-ISO, ζ-carotene isomerase. Metabolites are in red text and were quantified using LC–MS/MS. Bar graphs show metabolite content (μg/g) and heat maps show gene expression data.

Figure 4

. Expression of bHLH transcription factor genes in yl1 and 6421 exposed to high (H), medium (M), or low (L) light intensity. (A) Heat map of bHLH transcription factor expression. (B) qRT–PCR validation of carotenoid-related and bHLH71-like transcription factor genes. (C) Expression of bHLH71-like genes in different pepper tissues measured by qRT–PCR.

Figure 5

. Subcellular localization of bHLH71-like and promoter binding analysis. (A) The nuclear marker HY5-mCherry was co-expressed with GFP-bHLH71-like in N. benthamiana leaf epidermal cells, and fluorescence microscopy was performed to determine whether the mCherry and GFP signals colocalized. GFP fluorescence, mCherry fluorescence, differential interference contrast (DIC), and merged images are shown. Three independent experiments were performed with similar results. Bar, 50 μm. (B) Yeast one-hybrid assay showing that bHLH71-like binds to the pCaVDE promoter. Serially diluted cell suspensions (OD₆₀₀ = 0.2, 0.02, 0.002, and 0.0002) were spotted onto selective (SD/−Leu/−Trp/−His) or non-selective (SD/−Leu/−Trp) medium. AD-REC2-P53 and p53HIS2 were used as a positive control and AD+pHIS2.1-pCaVDE as a negative control. (C) Reporter and effector vectors used in the dual-luciferase assay. (D) LUC images of tobacco leaves after transient infiltration. (E) Ratio of LUC to REN activity. Data are means ± standard deviation from at least three biological replicates. The asterisk indicates a significant difference relative to the vector control (Duncan’s test, ^*P < .05).

Figure 6

. Carotenoid-related gene expression and metabolite concentrations in yl1 plants with reduced expression of CaVDE and bHLH71-like. (A) Phenotypes of yl1 plants inoculated with an empty vector (EV) VIGS construct and constructs targeting PDS, CaVDE, and bHLH71-like. (B) Relative expression levels of carotenoid-related genes in CaVDE-silenced plants. (C) Relative expression levels of carotenoid-related genes in bHLH71-like-silenced plants. (D) Concentrations of carotenoid metabolites. Error bars represent standard deviations. Asterisks indicate significant differences (Duncan’s test, ^*P < .05). ns, no significant difference.

Figure 7

Schematic diagram of the regulatory network that controls the yellowing phenotype in yl1 plants under high-intensity light. Arrows beside gene and metabolite names indicate increases (upward) or decreases (downward) in gene expression and metabolite content. The red dotted arrows represent light conditions. HL, high light. ML, medium light. LL, low light.