Molecular Hydrogen Improves Blueberry Main Fruit Traits via Metabolic Reprogramming

Abstract

Fruit yield and quality improvement are challenges for researchers and farmers. This study reveals that the main fruit traits of blueberry (Vaccinium ashei 'Bluegem') were significantly improved after hydrogen (H₂)-based irrigation, assessed by the increased single fruit weight (14.59 ± 6.66%) and fruit equatorial diameter (4.19 ± 2.39%), decreased titratable acidity, increased solid-acid and sugar-acid ratios. The enhancement of fruit quality was confirmed by the increased total volatiles, vitamin C contents, and antioxidant capacity. Using weighted protein co-expression network analysis (WPCNA), proteomic interrogation revealed that serine carboxypeptidase-like proteins I/II (SCPLI/II), ADP ribosylation factor 1/2 (ARF1/2), and UDP-glucosyltransferase 85A (UGT85A) might be functionally associated with the increased fruit weight and size driven by H₂. Reduced organic acid accumulation was caused by the regulation of the specific enzymes involved in sucrose metabolism (e.g., α-amylase, endoglucanase, β-glucosidase, etc.). H₂ regulation of fatty acid degradation (e.g., acyl CoA oxidase 1 (ACX1), acetyl CoA acyltransferase 1 (ACAA1), etc.) and phenylpropanoid metabolism were used to explain the improved fruit aroma and anthocyanin accumulation. Meanwhile, the upregulated heat shock protein 20/70 matched with the enhanced antioxidant activity. Together, this study provides a novel approach for yield and quality improvement in horticultural crops.

Keywords: blueberry; fruit size; hydrogen nanobubble water; metabolism; proteome; quality.

Figure 1

Growth and fruit of blueberry after hydrogen nanobubble water (HNW) irrigation. Photos of blueberry plants, flowers, and fruits were taken on 7 May 2022 and 15 June 2022, respectively (A). Changes in fruit equatorial diameter (B), polar diameter (C), fruit shape index (D), single-fruit weight (E), firmness (F), and fruit water content (G) in response to HNW. The number of samples in (B–E), (F), and (G) is 198 fruits (66 × 3), 30 fruits (10 × 3), and 15 fruits (5 × 3), respectively. * and *** indicate p < 0.05 and 0.001, respectively (t-test).

Figure 2

Effects of hydrogen nanobubble water (HNW) on contents of total volatiles (A), representative volatiles (B), total phenols (C), vitamin C (D), total anthocyanins (E), and anthocyanin biosynthesis-related genes expression profiles (F) in blueberry fruits. Volatile concentration is expressed as μg g⁻¹ of fresh weight, equivalent to ethyl decanoate. Values are expressed as mean ± SD (three replicates and ten fruits per each). * p < 0.05, ** p < 0.01, and *** p < 0.001 (t-test). 4CL: 4-coumarate:CoA ligase; ANS: anthocyanidin synthase; C4H: cinnamic acid-4-hydroxylase; CHI: chalcone isomerase; CHS: chalcone synthase; DFR: dihydroflavonol 4-reductase; F3H: flavanone-3-hydroxylase; F3′H: flavonoid 3′-hydroxylase; F3′5′H: flavonoid 3′,5′-hydroxylase; GSTU: tau class glutathione S-transferase; PAL: phenylalanine ammonia lyase; UFGT: UDP-glycose flavonoid glycosyltransferase.

Figure 3

Effects of hydrogen nanobubble water (HNW) on antioxidant capacity and multivariate analysis of quality-related characters in blueberry fruits. The activities of superoxide dismutase (SOD; (A)), peroxidase (POD; (B)), catalase (CAT; (C)), ascorbate peroxidase (APX; (D)), and glutathione reductase (GR; (E)). The scavenging abilities of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical (ABTS·⁺; (F)) and 1,1-diphenyl-2-picryl-hydrazyl radical (DPPH·; (G)). Values are expressed as mean ± SD (three replicates and ten fruits per each). * p < 0.05 and ** p < 0.01 (t-test). Score plot of partial least squares-discriminant analysis (PLS-DA; (H)). Variable importance in projection (VIP; (I)) scores listed importance of quality-related characters. The colored boxes on the right indicate the relative abundances of the corresponding characters.

Figure 4

The proteomic response of blueberry fruits after hydrogen nanobubble water (HNW) irrigation. Correlation between modules and blueberry yield and quality traits (A). Each row of the left heatmap represents a module, and each column represents a character of blueberry yield and quality. The value in each cell represents the Pearson correlation coefficient between the module and the trait, and the value in parentheses in each cell represents the p-value. Red color represents the positive correlation between module and trait, and blue color represents the negative correlation. ABTS: ABTS·⁺ scavenging ability; DPPH: DPPH· scavenging ability; SFW: single-fruit weight; ED: equatorial diameter; TA: titratable acidity; TV: total volatile content. KEGG enrichment analysis of proteins in the corresponding modules that were highly positively correlated with traits (B); p-value ranks of the 20 top.

Figure 5

Co-expression networks of proteins in MEblue and MEturquoise modules (A) and the expression levels of genes coding differentially expressed proteins (B). Nodes represent the top 20 proteins for connectivity (K value) in each module, labeled by NCBI accession number, and lines represent the linear relationship. ACAA1: acetyl-CoA acyltransferase 1; ACX1: acyl-CoA oxidase; ADH: alcohol dehydrogenase; ALDH: alcohol dehydrogenase; AMY: α-amylase; ARF1: ADP-ribosylation factor 1; BGL: β-glucosidase; CHIB: endochitinase B; EG: endoglucanase; ExgA: glucan 1,3-β-glucosidase; HSP20/70: heat shock 20/70kDa protein; PGM: phosphoglucomutase; SCPLI/II: serine carboxypeptidase-like protein I/II; UGT85A/85K: UDP-glucosyltransferase 85A/85K; VAMP72: vesicle-associated membrane protein 72. * p < 0.05, ** p < 0.01 and *** p < 0.001 (t-test; n = 3).

Figure 6

Schematic model describing hydrogen nanobubble water (HNW)-improved yield and quality of blueberries. 4CL: 4 coumarate CoA ligase; ACAA1: acetyl-CoA acyltransferase 1; ACX1: acyl-CoA oxidase; ADH: alcohol dehydrogenase; ALDH: alcohol dehydrogenase; AMY: α-amylase; ANS: anthocyanidin synthase; ARF1: ADP-ribosylation factor 1; BGL: β-glucosidase; CAD: cinnamyl alcohol dehydrogenase; CHI: chalcone isomerase; CHIB: endochitinase B; CHS: chalcone synthase; C12RT1: flavanone 7-O-glucoside 2′-O-β-L-rhamnosyltransferase; EG: endoglucanase; ExgA: glucan 1,3-β-glucosidase; F3H: flavanone 3-hydroxylase; GST: glutathione S-transferase; GSTU: tau class glutathione S-transferase; HSP20/70: heat shock 20/70kDa protein; PGM: phosphoglucomutase; PGT1: phlorizin synthase; POD: peroxidase; SCPLI/II: serine carboxypeptidase-like protein I/II; UGT85A/85K: UDP-glucosyltransferase 85A/85K; VAMP72: vesicle-associated membrane protein 72. The red and blue arrows indicate the upregulation and downregulation of gene exression, respectvely.