Unraveling the Molecular Mechanisms of Blueberry Root Drought Tolerance Through Yeast Functional Screening and Metabolomic Profiling

Abstract

Blueberry plants are among the most important fruit-bearing shrubs, but they have shallow, hairless roots that are not conducive to water and nutrient uptake, especially under drought conditions. Therefore, the mechanism underlying blueberry root drought tolerance should be clarified. Hence, we established a yeast expression library comprising blueberry genes associated with root responses to drought stress. High-throughput sequencing technology enabled the identification of 1475 genes potentially related to drought tolerance. A subsequent KEGG enrichment analysis revealed 77 key genes associated with six pathways: carbon and energy metabolism, biosynthesis of secondary metabolites, nucleotide and amino acid metabolism, genetic information processing, signal transduction, and material transport and catabolism. Metabolomic profiling of drought-tolerant yeast strains under drought conditions detected 1749 differentially abundant metabolites (DAMs), including several up-regulated metabolites (organic acids, amino acids and derivatives, alkaloids, and phenylpropanoids). An integrative analysis indicated that genes encoding several enzymes, including GALM, PK, PGLS, and PIP5K, modulate key carbon metabolism-related metabolites, including D-glucose 6-phosphate and β-D-fructose 6-phosphate. Additionally, genes encoding FDPS and CCR were implicated in terpenoid and phenylalanine biosynthesis, which affected metabolite contents (e.g., farnesylcysteine and tyrosine). Furthermore, genes for GST and GLT1, along with eight DAMs, including L-γ-glutamylcysteine and L-ornithine, contributed to amino acid metabolism, while genes encoding NDPK and APRT were linked to purine metabolism, thereby affecting certain metabolites (e.g., inosine and 3',5'-cyclic GMP). Overall, the yeast functional screening system used in this study effectively identified genes and metabolites influencing blueberry root drought tolerance, offering new insights into the associated molecular mechanisms.

Keywords: blueberry root; drought tolerance; genes; metabolomic profiling; molecular mechanisms; yeast functional screening.

Figure 1

Blueberry plant growth changes following treatments with different PEG-6000 concentrations. (A) Control; (B) 5% PEG-6000 treatment for 48 h; (C) 10% PEG-6000 treatment for 48 h; (D) 15% PEG-6000 treatment for 48 h; (E) 20% PEG-6000 treatment for 48 h. Bar: 2 cm.

Figure 2

Trends in the changes in the relative water content of ‘Emerald’ blueberry leaves after treatments with different PEG-6000 concentrations. * indicates a significant difference (p < 0.05) relative to the control group.

Figure 3

Yeast library was treated with different PEG-4000 concentrations (0, 50, 80, 100, and 120 mM) to select the appropriate simulated drought concentration.

Figure 4

Top 10 GO terms assigned to drought tolerance-related genes.

Figure 5

Enriched KEGG pathways among drought tolerance-related genes. The dot area represents the relative number of isolated genes in the pathway, whereas the dot color represents the Q value.

Figure 6

Verification of drought-tolerant yeast clones. The number above is the number of positive yeast clones.

Figure 7

Metabolite classifications and proportions.

Figure 8

Overview of the identified DAMs. (A) Venn diagram of the results of the comparisons of three groups (i.e., A, B, and C); (B–D) Heat maps of DAMs between different groups.

Figure 9

Association analysis of drought tolerance-related genes and DAMs in carbohydrate metabolism pathways. (A) Inositol phosphate metabolism, glycolysis/gluconeogenesis, and pentose phosphate pathway; (B) qRT-PCR results for four drought tolerance-related genes involved in carbohydrate metabolism; (C) Heat map of DAMs involved in carbohydrate metabolism. Colors reflect the regulation of metabolites under drought conditions (indicated in the scale bar). * and ** represented significant difference under p < 0.05 and p < 0.01, respectively.

Figure 10

Association analysis of drought tolerance-related genes and DAMs in secondary metabolite biosynthesis pathways. (A) Terpenoid backbone biosynthesis pathway and phenylpropanoid biosynthesis pathway; (B) qRT-PCR results for two drought tolerance-related genes involved in secondary metabolite biosynthesis; (C) Heat map of DAMs involved in secondary metabolite biosynthesis. Colors reflect the regulation of metabolites under drought conditions (indicated in the scale bar). * and ** represented significant difference under p < 0.05 and p < 0.01, respectively.

Figure 11

Association analysis of drought tolerance-related genes and DAMs in amino acid metabolism pathways. (A) Alanine, aspartate, and glutamate metabolism and glutathione metabolism; (B) qRT-PCR results for six drought tolerance-related genes involved in amino acid metabolism; (C) Heat map of DAMs involved in amino acid metabolism. Colors reflect the regulation of metabolites under drought conditions (indicated in the scale bar). * and ** represented significant difference under p < 0.05 and p < 0.01, respectively.

Figure 12

Association analysis of drought tolerance-related genes and DAMs in a nucleotide metabolism pathway. (A) Purine metabolism; (B) qRT-PCR results for six drought tolerance-related genes involved in nucleotide metabolism; (C) Heat map of DAMs involved in nucleotide metabolism. Colors reflect the regulation of metabolites under drought conditions (indicated in the scale bar). * and ** represented significant difference under p < 0.05 and p < 0.01, respectively.

Figure 13

A conceptual model of key genes and metabolites affecting blueberry root drought resistance. This model identifies key genes and metabolites involved in carbon metabolism, secondary metabolite biosynthesis, and amino acid and nucleotide metabolism. The squares represent the genes, while the ellipses represent the metabolites. Solid arrows indicate the direct regulation of metabolites by genes, whereas dotted arrows suggest metabolites that are presumed to ultimately have functional roles.