The Cyclin-Dependent Kinase activity modulates the central carbon metabolism in maize during germination

Abstract

The cell cycle is predominantly controlled by Cyclins/Cyclin-Dependent Kinases (Cyc/CDK) complexes, which phosphorylate targets involved in cellular proliferation. Evidence suggests that Cyc/CDK targets extend beyond traditional proteins and include enzymes that regulate the central carbon metabolism. Maize embryo axes rapidly internalize and metabolize glucose. After 24 h of imbibition in glucose-rich media, axes exhibited increased length and weight, with more pronounced effects at 72 h. This morphology enhancement was impaired when RO-3306, a specific CDK inhibitor, was added. The protein profile of maize embryo extracts at 18 and 24 h indicated altered phosphorylation patterns following CDK activity inhibition. Metabolomic analysis at 24 h of imbibition revealed that maize embryos without sugar in the media, with or without RO-3306, had a decreased sugar and amino acid content. Conversely, axes exposed to glucose demonstrated increased conversion into various mono and di-saccharides such as fructose, mannitol, galactose, and maltose but not sucrose. This pattern was reversed upon the addition of RO-3306. Glucose promoted the accumulation of amino acids such as cysteine, valine, leucine, and intermediates of the tricarboxylic acid (TCA) cycle, such as malate and citrate. The CDK inhibitor redirected the glucose metabolism toward increased serine levels, followed by other amino acids like phenylalanine, valine, and leucine. Additionally, TCA cycle intermediates and sterols significantly decreased. Overall, these results contribute to understanding the role of CDK in maize morphogenesis during germination and underscore its impact on modulating various central carbon pathways, including glycolysis, amino acid catabolism/anabolism, TCA cycle, and sterols biosynthesis.

FIGURE 1

Early glucose uptake during maize germination. (A) Reaction scheme representing the electron transfer process involved in glucose detection. Ferrocene methanol re‐oxidizes glucose oxidase (GOX), facilitating the conversion of β‐D‐glucose to D‐gluconolactone. This mediated electron transfer simplifies detection. (B) Glucose detection via cyclic voltammetry using the GOX‐modified electrode. (C) Differential pulse voltammetry at various glucose concentrations. (D) The calibration curve for the determination of glucose concentration ranged from 0 to 150 mM (R² = 0.99149 with a detection limit of 0.073 mM L⁻¹). (E) Glucose uptake during the initial imbibition period of maize embryo axes (0 to 6 h). Glucose levels were assessed in the residual media of ten embryo axes imbibed in 2 mL, measuring at various time points. The glucose concentration at the start of imbibition was 120 mM. Each determination represents the average of three independent measurements. Bars represent the error deviation for each data point.

FIGURE 2

Morphology of maize embryo axes imbibed in the presence of glucose and RO‐3306. (A) Axes imbibed without sugar (NS); without sugar and with 50 μM RO‐3306 (NSR), with 120 mM glucose (G), and with 120 mM glucose plus 50 μM RO‐3306 (GR) for 24 and 72 h, or 7 days. White bar = 25 mm. Images of embryo axes at 7 days of imbibition without sugar (NS) are presented in Figure S2. (B) Weight (mg) and (C) length (cm) of embryo axes. Measurements were made for 20 embryo axes per treatment. A, b and c in bars indicate significant statistical differences (p < 0.01).

FIGURE 3

Phosphorylated protein profile of maize embryo axes. Protein extracts (25 μg) obtained from maize embryo axes imbibed with glucose (G) or glucose plus RO‐3306 (GR) at 18 and 24 h. Proteins were resolved by SDS‐PAGE (12%). (A) Phosphorylated protein bands were detected using the Phospho‐Tag™‐Phosphoprotein Gel Stain (ABP Biosciences). (B) Proteins transferred to a PVDF membrane were stained with Congo Red (CRS) as a loading control. Densitometry analysis in panel A was performed on seven selected bands, normalized against the corresponding band from dry seed (0 h) and then against the same band in CRS. The blue and green charts represent the G/GR ratio associated with each band.

FIGURE 4

Metabolomic analysis in maize embryo axes at 24 h of imbibition. The treatments were no sugar (NS, red), no sugar plus RO‐3306 (NSR, blue), 120 mM glucose (G, green), and 120 mM glucose plus 50 μM RO‐3306 (GR, cyan). (A) Clustering analysis of metabolomic data. The heatmap displays the intensities of differential metabolites, with each row representing the abundance and each column showing the metabolic patterns for different treatments. (B) Partial least square discriminant Analysis (PLS‐DA) plot of maize embryo axes metabolite content. (C) Variable importance in projections (VIP) score for identified metabolites, with the Metabolic map and relative metabolite composition. Error bars represent the standard error from four independent experiments, with different symbols above error bars indicating statistical differences among treatments (p < 0.05). Grey arrows represent biochemical steps in central carbon metabolism. Abbreviations: GAL, galactose; SUC, sucrose; FRU, fructose; N‐Ac Glu: N‐acetyl glucosamine; G6P, glucose‐6‐phosphate; F6P, fructose‐6‐phosphate; SHI, shikimate; PYR, pyruvate; AcCoA, acetylCoA; CIT, citrate; isoCIT, isocitrate; AKG, a‐cetoglutarate; SUCC, succinate; FUM, fumarate; MAL, malate; OAA, oxaloacetate; and 3PGA, 3‐phosphoglycerate.

FIGURE 5

Glycolysis and TCA cycle enzymatic activity rates during germination. Activities were assessed in dry seeds (0 h) and maize embryo axes imbibed for 6, 15, 18, and 24 h under four different conditions: no sugar (NS), no sugar plus RO‐3306 (NSR), 120 mM glucose (G), and 120 mM glucose plus 50 μM RO‐3306 (GR). The enzymatic activities included: (A) phosphofructokinase (PFK), (B) glyceraldehyde‐3‐phosphate dehydrogenase (G3PDH), (C) pyruvate kinase (PK), (D) citrate synthase (CS) and (E) malate dehydrogenase (MDH). Data represents the average of three biologically independent samples, with each measurement performed in duplicate. Asterisks denote statistical differences among time points with p < 0.01. Error bars indicate the standard error from four independent experiments. F) Correlation analysis of enzymatic activities for each treatment at 24 h was assessed using the SRplot platform (Tang et al., 2023). The white dots inside the circles highlight statistical differences with p < 0.05 (Pearson correlation).

FIGURE 6

Metabolic model illustrating the effects of CDK activity on various cellular and metabolic processes in maize during germination. “P” in orange circles denotes phosphorylation. Green indicates processes enhanced by glucose and full CDK activity, red corresponds to processes affected by CDK activity inhibition by RO‐3306. The figure was created using Biorender.com.