Transcriptome Analysis on Key Metabolic Pathways in Rhodotorula mucilaginosa Under Pb(II) Stress

Abstract

Rhodotorula mucilaginosa shows adaption to a broad range of Pb²⁺ stress. In this study, three key pathways, i.e., glycolysis (EMP), the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation (OXPHOS), were investigated under 0-2,500 mg · L^-1 Pb stress, primarily based on biochemical analysis and RNA sequencing. R. mucilaginosa cells showed similar metabolic response to low/medium (500/1000 mg · L^-1) Pb²⁺ stress. High (2,500 mg · L^-1) Pb²⁺ stress exerted severe cytotoxicity to R. mucilaginosa. The downregulation of HK under low-medium Pb²⁺ suggested a correlation with the low hexokinase enzymatic activity in vivo. However, IDH3, regulating a key step of circulation in TCA, was upregulated to promote ATP feedstock for downstream OXPHOS. Then, through activation of complex I & IV in the electron transport chain (ETC) and ATP synthase, ATP production was finally enhanced. This mechanism enabled fungal cells to compensate for ATP consumption under low-medium Pb²⁺ toxicity. Hence, R. mucilaginosa tolerance to such a broad range of Pb²⁺ concentrations can be attributed to energy adaption. In contrast, high Pb²⁺ stress caused ATP deficiency. Then, the subsequent degradation of intracellular defense systems further intensified Pb toxicity. This study correlated responses of EMP, TCA, and OXPHOS pathways in R. mucilaginosa under Pb stress, hence providing new insights into the fungal resistance to heavy metal stress. IMPORTANCE Glycolysis (EMP), the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation (OXPHOS) are critical metabolism pathways for microorganisms to obtain energy during the resistance to heavy metal (HM) stress. However, these pathways at the genetic level have not been elucidated to evaluate their cytoprotective functions for Rhodotorula mucilaginosa under Pb stress. In this study, we investigated these three pathways based on biochemical analysis and RNA sequencing. Under low-medium (500-1,000 mg · L^-1) Pb²⁺ stress, ATP production was stimulated mainly due to the upregulation of genes associated with the TCA cycle and the electron transport chain (ETC). Such an energy compensatory mechanism could allow R. mucilaginosa acclimation to a broad range of Pb²⁺ concentrations (up to 1000 mg · L^-1). In contrast, high (2500 mg · L^-1) Pb²⁺ stress exerted its excessive toxicity by provoking ATP deficiency and damage to intracellular resistance systems. This study provided new insights into R. mucilaginosa resistance to HM stress from the perspective of metabolism.

Keywords: Rhodotorula mucilaginosa; TCA; lead; metabolism; transcriptome.

FIG 1

Pb sorption and ATP production after incubation. (A) The sorption concentrations (mg · L⁻¹) and rate (%) of Pb²⁺ after 3 days of incubation are marked as gray and red, respectively. The initial Pb²⁺ concentrations varied from 500–2,500 mg · L⁻¹. (B) The ATP production of yeast cells at 0–2.500 mg · L⁻¹ Pb²⁺ (mmol·L⁻¹).

FIG 2

The enzyme activities of hexokinase, pyruvate kinase, and phosphofructokinase in Rhodotorula mucilaginosa cells at Pb²⁺ levels of 0, 500, 1,000, 1,500, 2,000, and 2,500 mg · L⁻¹ (U·mL⁻¹). The significance was compared within each enzyme group, denoted by lowercase letters.

FIG 3

Principal component analysis (PCA) based on 41,422 differentially expressed genes. The batch-effect bias was removed from the input data. ck, PbH, PbM, and PbL represented 0, 500, 1,000, and 2,500 mg · L⁻¹ Pb²⁺, respectively. Three independent biological replicates with the same treatments are designated with identical colors. PC1 and PC2 provided 96% variation interpretation of all sample data.

FIG 4

Characterization of the transcriptome in the six comparison groups (ck-PbH, ck-PbM, ck-PbL, PbH-PbM, PbH-PbL, PbM-PbL). (A) Numbers of differentially expressed genes among the six treatment comparisons (|log₂ (fold change)| > 1 and adjusted P value < 0.01). (B) Upset plot of common DEGs among the different comparison sets. The left bars represent the total number of DEGs in each set. Black dots with interconnecting vertical lines represent the intersections between selected sets. Other unfilled light gray dots represent the sets that are not part of the intersection. The top bars in image B display the number of DEGs within the intersection. The intersection of ck-PbH, PbH-PbM, and PbH-PbL is highlighted in orange.

FIG 5

GO and KEGG enrichment analysis. The sizes of markers represent the percentage of genes in each term to the overall genes (>0.2). The colors of markers represent the significance of enrichment (–log₂) in each term through the hypergeometric test. (A) GO enrichment analysis of DEGs in molecular function, biological process, and cellular component. The shapes of markers represent different GO ontologies. (B) KEGG enrichment analysis of DEGs in metabolism.

FIG 6

Differentially expressed genes involved in key carbohydrate and bioenergy metabolism pathways of glycolysis (EMP), the TCA cycle, and oxidative phosphorylation (OXPHOS). Control and different Pb treatments are labeled at the bottom of each column. Independent biological replicates with the same treatments are grouped with identical colors. Gene names are labeled on the right side, with their corresponding pathway terms marked by different colors, respectively. Euclidean distance and Pearson clustering method are applied to group the treatments. Filled shades indicate gene expression in the form of FPKM standardized by row.

FIG 7

Carbohydrate and bioenergy metabolism network (EMP: glycolysis; TCA cycle; OXPHOS: oxidative phosphorylation) in Rhodotorula mucilaginosa. Relevant DEGs were mapped in the above metabolic pathways. Treatments are labeled as ck, PbL, PbM, and PbH from left to right. The filled square denotes average FPKM normalized by row. Glucose was gradually oxidized to pyruvate and converted to acetyl-CoA before entering the TCA cycle. Through multiple stages of enzymatic reactions, carbohydrate metabolites were completely oxidized, and then produced declined equivalents NADH and FADH₂ for OXPHOS on the inner mitochondrial membrane. NAD⁺ and FAD were replenished by complex I and II in the electron transfer chain (ETC), driving the operation of the TCA cycle. Electrons were transferred though enzyme complexes, coupling with ATP synthase to generate ATP for cell physiological activities.