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.

1. INTRODUCTION

The cell cycle consists of a series of molecular events that enable a cell to replicate its DNA, resulting in two daughter cells with identical genetic material. This process is highly regulated and energetically demanding (Inzé and De Veylder ²⁰⁰⁶; Rawat and Laxmi ²⁰²⁴).

Cyclins (Cyc) and cyclin‐dependent kinases (CDKs) are key protein families involved in regulating the cell cycle. Cyclins bind to CDKs to form active kinase complexes that modulate the activity of essential target proteins through phosphorylation. These target proteins are crucial for processes such as gene transcription, DNA synthesis, and sister chromatid separation (De Veylder et al., 2007, Xiao et al., 2021).

Three primary subfamilies of cyclins are directly involved in cell cycle regulation: CycDs, which act during the G1 phase to sense intrinsic and extrinsic conditions; CycAs, which regulate the S phase and the early G2 phase; and CycBs, which are involved in the transition from G2 to mitosis (Nieuwland et al., 2007). In maize, 17 genes encoding CycD have been identified (Buendía‐Monreal et al., 2011, Xiao et al., 2021).

CDKs are serine/threonine kinases whose activity is regulated by various factors. These include the type of Cyc to which they are associated, interactions with auxiliary proteins such as CDK subunits (Cks) (Faustova et al., 2021), or with inhibitors like CDK interacting protein/kinase inhibitory protein (CIP/KIP, known as Kip‐related proteins or KRPs in plants) (De Veylder et al., 2001); positive or negative phosphorylation events; and synthesis or degradation processes. In plants, two classes of CDKs involved in the cell cycle progression have been identified: CDKAs, which contain a canonical cyclin‐binding motif known as PSTAIRE, and CDKBs, which are plant‐specific and feature a divergent motif, either PPTALRE or PPTTLRE (Dudits et al., 2007, Banerjee et al., 2020). Other CDKs found in plants are CDKDs (also known as CAK) and CDKFs, involved in the phosphorylation of CDKA and the C‐terminal domain of RNA polymerase II, respectively (Umeda et al., 2000, Inzé and De Veylder, 2006).

In addition to their well‐known role in phosphorylating canonical targets related to cell cycle progression, recent research has revealed a connection between the Cyc/CDK heterodimer function and the modulation of central carbon metabolism (Harashima and Sugimoto, 2016; Siqueira et al., ²⁰¹⁸; Solaki and Ewald, ²⁰¹⁸). For instance, Harashima et al. (2016) identified various putative substrates of AtCDKA;1 involved in metabolic pathways such as starch digestion, glycolysis, the tricarboxylic acid (TCA) cycle, and amino acid metabolism. They demonstrated in vitro the phosphorylation of several enzymes, including pfkB‐like (phosphofructokinase), isopropylmalate dehydrogenase (IMD1), malate dehydrogenase (MDH), proline iminopeptidase (PIP), and aldehyde dehydrogenase (ALDH7B4), by CDKA;1/CycD2;1, though the effects on enzymatic activity were not assessed. In human cancer cells, CycD/CDK6 phosphorylates two key glycolytic enzymes, 6‐phosphofructokinase and pyruvate kinase, leading to a decrease in the activity of both enzymes. This reduction impairs the glycolytic flux and re‐directs intermediates into the pentose phosphate pathway and serine (Ser) biosynthesis (Wang et al., 2017).

Plant germination is a dynamic process that initiates with seed water uptake and concludes with embryo axes elongation (Bewley et al., 2013). According to Vázquez‐Ramos and Sánchez, germination begins with seed imbibition and concludes upon successful completion of the first cell cycle round (Vázquez‐Ramos and Sánchez, 2003).

Seed imbibition is categorized into three phases based on the water uptake rate. Phase I is characterized by a rapid water uptake, stabilizing in Phase II. During this phase, primary metabolism resumes, respiration activates, stored mRNAs from seed development are translated, and cell membranes and organelles undergo repair, among other crucial processes. Phase III marks another increase in the water uptake rate (Bewley et al., 2013). Events during this phase include radicle protrusion and mobilization of stored reserves, which are considered post‐germination processes (Carrera‐Castaño et al., 2020).

Baíza et al., analyzed the maize seed germination and its correlation with cell cycle timing. Using [³H]‐thymidine incorporation to track de novo DNA synthesis, they determined that DNA replication initiates approximately 14 to 16 h after imbibition (Baíza et al., 1989). Consequently, cells predominantly arrested at G1 before desiccation (Vázquez‐Ramos and Sánchez, 2003) remain in this cell cycle phase until the S phase onset, around 14 to 16 h. Labelled mitotic figures are observed around 28 h, indicating the occurrence of the G2 phase after the end of the S phase and prior to 28 h.

Adding sucrose or glucose to maize embryo axes positively affects germination (Lara‐Núñez et al., 2017). Providing glucose instead of sucrose in the imbibition media results in more cells at the root apical meristems committing to DNA duplication, suggesting a higher proliferation rate (Díaz‐Granados et al., 2020). In maize, three CDKA and two CDKB genes have been identified (Xiao et al., 2021). During germination, ZmCDKA1;1 and ZmCDKA2;1 gene expression is stimulated, and ZmCDKA protein abundance is stabilized by glucose in maize during early germination, with a peak at around 24 h of imbibition (Lara‐Núñez et al., 2017). The ZmCDKB protein was also identified during maize germination, and both ZmCDKA and ZmCDKB form active complexes with different D‐type cyclins (Godínez‐Palma et al., 2013, Garza‐Aguilar et al., 2017).

A metabolic map of mature kernels from maize has been constructed, correlating metabolomic, transcriptomic, and proteomic databases. This analysis highlights the presence of key central carbon metabolites, among them simple sugars, amino acids, organic acids, and intermediates of the TCA cycle (Rao et al., 2014). This suggests that mature kernels have the capability to reactivate their primary metabolic pathways upon imbibition.

The metabolic events occurring during maize germination, as well as the impact of kinase activity from Cyc/CDK complexes on sugar metabolism in maize, are not well characterized. This study aims to provide comprehensive insights into the role of Cyc/CDK kinase activity in various pathways of central carbon metabolism during germination. The metabolomic profile of germinated maize embryo axes imbibed with glucose was compared to axes treated with RO‐3306, a competitive inhibitor of CDK kinase activity. RO‐3306 inhibited both ZmCDKA and ZmCDKB kinase activities in vitro when metabolic enzymes such as pyruvate kinase, phosphofructokinase (Lara‐Núñez et al., 2024), hexokinase 7 or glyceraldehyde‐3‐phosphate dehydrogenase (Vargas‐Cortez et al., 2023, Lara‐Núñez et al., 2024) were used as substrate. The important metabolite disparities among embryos with full or impaired CDK activity, as well as morphological alteration of the embryo axes with RO‐3306, provided evidence that CDK kinase activity played an important role in the coordination between cell cycle progress and central carbon metabolism.

Given the central role of CDKs in regulating both cell cycle progression and metabolic pathways, understanding how kinases modulate the central carbon metabolism during maize germination is crucial for elucidating the mechanisms that govern seedling establishment and early plant development.

2. MATERIALS AND METHODS

2.1. Materials

Protease inhibitor cocktail tablets (cOmplete) were from Roche. The Western chemiluminescent horseradish peroxidase (HRP) substrate and Immobilon polyvinylidene fluoride (PVDF) membranes were from Millipore. The anti‐rabbit IgG‐HRP conjugate was from Santa Cruz Biotechnology.

Seeds of Zea mays cv. Chalqueño (an open pollination genotype) were acquired at Chalco, Estado de México, Mexico, in 2019 and 2020.

2.2. Imbibition of maize embryo axes

Maize embryo axes were manually dissected from dry seeds and imbibed as previously reported (Lara‐Núñez et al., 2017). Embryo axes were incubated for 24, 72 h, or 7 days at 25°C in the dark and under sterile conditions with imbibition buffer (50 mM Tris–HCl pH 7.4, 50 mM KCl, 10 mM MgCl₂), plus either 120 mM glucose or without sugar, and with or without 50 μM RO‐3306 ((5Z)‐5‐quinolin‐6‐ylmethylene‐2‐[(thiophen‐2‐ylmethyl)‐amino]‐thiazol‐4‐one, Sigma‐Aldrich)). Four treatments were analyzed: (1) no sugar (NS); (2) no sugar plus RO‐3306 (NSR); (3) 120 mM glucose (G); and (4) 120 mM glucose plus RO‐3306 (GR). The optimal inhibitor concentration (50 μM) was determined from a dose–response curve (not shown): concentrations above 50 μM led to tissue oxidation, while lower concentrations did not impair growth.

RO‐3306 is expected to cross cell membranes and reach the cytoplasm of maize embryo axes cells due to its small size and hydrophobic nature. Nevertheless, vacuum was applied to ensure the entrance of the inhibitor into the tissue. This step consisted of a 5 min vacuum round followed by 30 s resting and was carried out twice. Procedures were performed under constant agitation. The embryo axes were transferred to Whatman filter paper wetted with imbibition media, including 50 μM RO‐3306, where applicable. The medium, including the inhibitor and glucose, as per the assigned treatment, was refreshed every 24 h.

2.3. Protein extraction

Protein extracts were obtained by grinding axes at 4°C with a Polytron homogenizer (Ultra‐Turrax, Janke & Kunkel,) on extraction buffer containing 25 mM Tris–HCl pH 7.5, 15 mM MgCl₂, 25 mM KCl, 250 mM NaCl, 5 mM EDTA, 1 mM DTT, 0.2% Triton X‐100, 30% (v v⁻¹) glycerol, 60 mM β‐glycerol phosphate, 50 mM NaF, 200 μM Na₃VO₄, 1 mM EGTA, and a mini tablet of protease inhibitor cocktail 15 mL⁻¹. Protein extracts were centrifuged for 1 h at 16 000 g and 4°C. Protein concentration was determined by the BCA method (Stoscheck 1990).

2.4. Electrochemical glucose determination

The glucose concentration in the media was quantified using electrochemical methodologies, in which glucose oxidase (GOX) was immobilized on glassy carbon. For this purpose, a catalytic ink was meticulously concocted. The ink included this enzyme, tetrabutylammonium bromide, Nafion, Vulcan carbon, and a pH 7.45 phosphate buffer solution. A minute volume of 2 μL from the catalytic ink was applied onto a glassy carbon electrode. The supporting electrolyte consisted of a buffer solution containing 1 mM ferrocene methanol; Ag/AgCl was the reference electrode. A Gamry Instruments Reference 3000 electrochemical workstation was utilized for these experiments. The oxidation–reduction kinetic of glucose was examined by cyclic voltammetry. Differential pulse voltammetry (DPV) was conducted with a scan speed of 0.025 V s⁻¹, a pulse width of 0.05 V, and a pulse duration of 0.05 s. This technique was crucial for constructing the calibration curve, employing different concentrations of glucose (0, 1, 2, 5, 10, 20, 50, 75, 100, and 150 mM), in a buffer solution with 1 mM ferrocene methanol. The glucose concentration in the medium was determined by averaging three measurements at each sampling evaluation time.

2.5. Metabolite extraction

Maize embryo axes (six per treatment) incubated for 24 h on imbibition media with or without glucose and with or without RO‐3306 were homogenized with 1 mL of ice‐cold methanol. The supernatant was recovered after 10 min of centrifugation at 7000 g.

2.6. Gas chromatography–mass spectrometry (GC–MS) analysis

The extract was thoroughly dried with a nitrogen flux, then derivatized with 80 μL of methoxyamine (Sigma) dissolved in pyridine (Sigma) (20 mg mL⁻¹) for 90 min at 37°C. After the incubation period, 80 μL of MBSTFA (N‐tert‐butyldimethylsilyl‐N‐methyltrifluoroacetamide, Sigma) with 1% trimethylchlorosilane (Sigma) were added to the mix and further incubated at 37°C for 30 min.

Then, 1 μL was injected (splitless mode) into a GC–MS system (Agilent 5977A/7890B) with a HP‐5MS column (30 m × 250 μm × 0.25 μm – Agilent). The mobile phase was high‐purity helium (99.9995%) at a 1 mL min⁻¹ flow rate. The oven temperature was set to 60°C for 1 min with increments of 10°C min⁻¹ until reaching 325°C. Other parameters were as follows: 200°C inlet temperature, 200°C source temperature, and 250°C interface temperature. All the samples were injected randomly, and a quality control was injected into every four samples to check for differences in retention time and signal intensities across all the injections.

The raw data was converted to.mzdata using the Chemstation software (Agilent) to then perform the spectral deconvolution and alignment with Mzmine2 (Pluskal et al., 2010). Peak heights were employed in all the data transformations, and the rule of 80 was applied to all the variables. The National Institute of Standards and Technology (NIST) 2.0 library was used to identify the peak‐associated spectra (probability >70%, match and reverse match >700). Metabolites without a match were marked as unknown. Only those variables with RSD (relative standard deviation) values lower than 30% in all the pooled samples were included. Finally, Metaboanalyst 6.0 was used to perform the sum normalization, log transformation, and autoscaling prior to multivariate partial least‐squares discriminant analysis (PLS‐DA). Sum normalization was implemented for principal component analysis (PCA) and univariate analysis.

2.7. Western Blotting

Protein samples (25 μg) were fractionated by SDS‐PAGE (12%). Gels were blotted onto PVDF membranes, blocked with 5% dry milk, and then incubated with the anti‐mitotic protein antibody (MPM‐2, ab14581, Abcam, 1:1000 dilution) or anti‐P‐Thr‐Pro‐101 (9391S, Cell Signaling Technology, antibody, 1:5000). Briefly, transferred membranes were incubated overnight at 4°C with the antibody, washed once with PBS, then a second wash with PBS plus 0.5 M NaCl, 1% Triton X‐100, and once again with PBS (15 min each wash). Membranes were then incubated for 1 h with peroxidase‐conjugated anti‐rabbit antibody at a 1:20 000 dilution (Santa Cruz). Subsequently, membranes were washed three times with PBS for 15 min each. The peroxidase reaction was detected by enhanced chemiluminescence (ECL, Pierce kit, Thermo Fisher Scientific). The signal was detected in a ChemiDoc system (Bio‐Rad), and the densitometric analysis was performed by using the Image Lab software (Bio‐Rad). Transferred membranes were stained with Red Congo solution. The phosphorylation signal for each time measurement and treatment was normalized to its corresponding band in the dry seed (0 h), which was used as the reference unit and further adjusted against the corresponding band on the Red Congo‐stained membrane, serving as the loading control.

Alternatively, the SDS‐PAGE gel was rinsed with deionized water and incubated with Phospho‐Tag™ gel staining solution and imaged in ChemiDoc after the staining and washing steps, according to the provider instructions (ABP Biosciences).

2.8. Determination of Enzyme Activity

To measure PFK activity (EC 2.7.1.11), protein extracts were added to an assay mixture containing 50 mM HEPES buffer (pH 7.0), 5 mM MgCl₂, 1 mM EDTA, 1.5 mM ATP, 0.2 mM NADH, 1 U mL⁻¹ aldolase, 10 U mL⁻¹ triose phosphate isomerase, and 1 U mL⁻¹ α‐glycerophosphate dehydrogenase. The reaction was initiated by adding fructose‐6‐phosphate to a final concentration of 6 mM. The assay volume was 240 μL, and the decrease in absorbance was monitored at 340 nm using a microplate reader (Epoch, BioTek).

For the glyceraldehyde‐3‐phosphate dehydrogenase (G3PDH, EC 1.1.5.3) activity (measurement, protein extracts were added to an assay mixture containing 50 mM TEA‐HCl (pH 8.8), 5 mM fructose‐16 bisphosphate and, 0.5 U mL⁻¹ aldolase. The reaction was started by adding NADP⁺ to a final concentration of 0.7 mM. The assay volume was 240 μL, and the increase in absorbance was tracked at 340 nm using a microplate reader (Epoch, BioTek).

Pyruvate kinase (PK, EC 2.7.1.40) activity was assessed using a mixture containing protein extracts, 80 mM TEA buffer pH 7.5, 8 mM MgCl₂, 5 mM ADP, 0.2 mM NADH, and 2 U mL⁻¹ lactate dehydrogenase (LDH). The reaction was initiated by adding PEP to a final concentration of 1 mM. The assay volume was 240 μL, and the decrease in absorbance was measured at 340 nm using a microplate reader (Epoch, BioTek®). In this assay, the disappearance of PEP is coupled with lactate production via LDH.

To measure citrate synthase (CS, EC 2.3.3.1) activity, protein extracts were added to an assay mixture containing 100 mM Tris–HCl (pH 8.0), 1 mM DTNB (5,5′‐dithio‐bis‐[1‐nitrobenzoic acid]), and 0.5 mM acetyl‐CoA. The reaction was initiated by adding oxaloacetate to a final concentration of 0.1 mM. The assay volume was 240 μL, and the increase in absorbance was monitored at 412 nm using a microplate reader (Epoch, BioTek).

Malate dehydrogenase (MDH, EC 1.1.1.37) activity was determined in a mixture containing 250 mM HEPES‐KOH pH 8.0, 2 mM MgCl₂, and 0.25 mM NAD⁺. The reaction was started by adding malate to a final concentration of 2.5 mM. The assay volume was 240 μL, and the increase in absorbance was monitored at 340 nm with a microplate reader (Epoch, BioTek).

For the CDK (2.7.11.22) activity measurement and inhibition assessment using RO‐3306, CDKB immunocomplexes were isolated from 1 mg of protein extract derived from maize embryo imbibed for 12 and 24 h. Immunoprecipitation was performed using α‐ZmCDKB specific antibodies, and histone H1 (HH1) served as the target for CDKB phosphorylation. Increasing concentrations of RO‐3306 (0–100 or 200 μM) were added 30 mins prior to the enzyme assay. [γ‐³²P]‐ATP was used as the gamma‐phosphate donor.

In vitro kinase activity assays of the ZmCDKA;1 or ZmCDKB1;1/ZmCycD2;2 complexes were performed as follows. CycD2;2 and CDKA;1 or CDKB1;1 recombinant proteins (250 ng of each) were mixed to form the complex in kinase buffer (Lara‐Núñez et al., 2024) and 500 ng of recombinant ZmHXK7 (hexokinase 7) served as phosphorylation target (Vargas‐Cortez et al., 2023). Thio‐ATP was used as a gamma‐P donor and alkylated by p‐nitrobenzylmesylate (PNBM) to create a chemical signature recognized by an anti‐thiophosphate ester antibody (RabMab, ab92570).

2.9. Statistical analysis

Data from embryo axes growth and length were analyzed using one‐way ANOVA and then pairwise compared by Tukey's test to assess significant differences at p < 0.05 or less. Measurements were made from 20 embryo axes per treatment. Data for glucose in the imbibition medium represents the average of three independent measurements. The metabolite quantification was performed across four independent experiments, analyzed using one‐way ANOVA, and subsequently compared pairwise with Tukey's test.

3. RESULTS

3.1. Glucose uptake by maize embryo axes during germination

Simple sugars within the embryo axes are gradually metabolized once the seed is imbibed. During imbibition, maize embryo axes exhibit an increasing glucose uptake rate from 0 to 6 h (Figure 1), followed by a consistent glucose uptake rate from 12 to 18 h (Sánchez‐Linares et al., 2012). The glucose uptake by maize embryo axes was evaluated over time using the electrochemical determination of glucose in the media. This is an indirect measurement of glucose internalization that relies on its oxidation via glucose oxidase, which is bound to the working electrode. The electrochemical method developed for glucose detection proved effective, reliable, and sensitive (Figure 1).

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.

Glucose depletion from the media occurred after 6 h of imbibition (Figure 1E). To evaluate whether the glucose uptake rate remained high after the initial imbibition period, the medium was refreshed with 120 mM glucose at 12 h. The rate of glucose depletion from the refreshed media was slower compared to the initial uptake from 0 to 6 h. Nevertheless, the embryo axes continued to consume glucose (Figure S1). Supplying this simple sugar was sufficient for the maize embryo axes to increase in size over the analysis period (from 0 h to 7 days, with 120 mM glucose refreshed every 24 h; Figure 2). Without glucose, the maize embryo axes were unable to gain weight or increase in size (Figures 2 and 3 and Figure S2).

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.

3.2. CDK kinase activity inhibition affects the growth of the maize embryo axes

RO‐3306, a selective ATP‐competitive inhibitor of CDK, inhibits human CDK activity with inhibitory constant (Ki) values ranging from 0.020 to 2 μM (Vassilev et al., 2006). However, there is no information regarding the sensitivity of CDKB, a CDK found only in plants and algae (Inzé and De Veylder, 2006), toward RO‐3306. To address this gap, we assessed the effectiveness of RO‐3306 in inhibiting the kinase activity of CDKB complexes isolated from maize embryo axes imbibed for 12 and 24 h by immunoprecipitation (IP), using Histone H1 (HH1) as the substrate for phosphorylation. Figure S3A shows that RO‐3306 inhibited the kinase activity of CDKB complexes isolated from maize extracts, but a high concentration of 100 μM was required to achieve approximately 50% inhibition of HH1 phosphorylation. In vitro kinase activity of maize CDKA;1/CycD2;2 or CDKB1;1/CycD2;2 complexes were also inhibited by RO‐3306 when maize hexokinase 7 was used as substrate (Figure S3B). This indicates that RO‐3306 is less effective against maize CDKs compared to mammalian CDKs.

When 120 mM glucose was included in the imbibition media, embryo axes tended to twist, increase in length, and gain weight (Lara‐Núñez et al., 2017). However, the addition of 50 μM RO‐3306 to the glucose media resulted in fewer twisted axes (Figure 2A), reduced elongation (Figure 2C), and a lower weight gain compared to embryos in glucose media (Figure 2B). The differences in length and weight between glucose (G) and glucose plus RO‐3306 (GR) treatments were statistically significant at 24 and 48 h (Figure 2B and C). From 72 h and up to 7 days, the differences were no longer significant, although fewer twists were observed at 72 h (Figure 2A) in GR treatment compared to G. After 7 days, the negative effect of RO‐3306 on primary and seminal root growth was noticeable, and the hypocotyl was thinner and less developed compared to axes with no RO‐3306 (Figure 2).

These results suggest that maize embryo axes partially overcome the effect of CDK inhibition on the morphogenetic program through a compensatory mechanism. Our findings confirm that, as previously demonstrated, embryo axes cannot grow without the supply of a simple sugar (Figure 2). Therefore, the presence of the CDK inhibitor had a negligible impact after germination (72 h and 7 days, Figure 2).

3.3. Phosphorylation profile of CDK targets affected by RO‐3306

Differences in the morphology of embryo axes, imbibed with G and GR treatments, indicate changes in the Cyc/CDK total kinase activity and, consequently, in the phosphorylation patterns of CDKs targets. To assess the effect of RO‐3306 on CDK activity at the molecular level during germination, the total protein phosphorylation profile was analyzed. Protein extracts from maize embryo axes imbibed with G or GR for 18 or 24 h were analyzed by SDS‐PAGE; the signals of no specific phosphorylated proteins were identified using the Phospho‐Tag™ kit. Well‐defined bands were identified (Figure 3). Seven bands were selected for densitometric analysis to estimate differences between the G and GR treatments. Each band was first normalized against its corresponding counterpart in dry seed (0 h) and then against the protein band in the Congo Red stained membrane (CRS, loading control). The G/GR ratio was determined for each band (bar charts in Figure 3). The inhibitor negatively affected the phosphorylation profile of bands 1 and 4 at both 18 and 24 h of imbibition. Bands 2 and 3 exhibited a slight decrease at 18 h and a marked reduction at 24 h. Band 6 decreased only at 18 h, while bands 5 and 7 remained unaffected by the inhibitor.

3.4. CDKs inhibition alters the metabolic profile of maize embryo axes at 24 h

An untargeted metabolic approach was used to determine whether CDK kinase activity affects glucose utilization or mobilization of stored materials and to assess its overall impact on germination metabolism. Maize embryo axes were imbibed in the four treatments (NS, NSR, G, and GR) for 24 h and their metabolomic profiles were analyzed. Intracellular methanolic extracts were analyzed by gas‐chromatography tandem mass spectrometry (GC–MS). The main profile included 39 different metabolites that encompass amino acids, sterols, TCA cycle intermediates, sugars, and lipids. Our analytical method seems robust, as noted by the compact clustering of the pooled samples used as quality control (which covered all the injected samples, Figure S4).

To visualize the main metabolic changes resulting from the treatments, a heat map diagram with hierarchical clustering analysis was used (Figure 4A). NS and NSR treatments did not show distinct separation patterns, revealing similar profiles for sugars, amino acids, sterols, and TCA cycle intermediates. Conversely, the G and GR treatments were separately clustered.

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.

For further differentiation of the groups based on their metabolic profiles, partial least squares discrimination analysis (PLS‐DA) was conducted (Figure 4B). This supervised method effectively segregated all groups (R² = 0.90. Q² = 0.739, Accuracy = 0.75). The variable importance in projections (VIP) analysis (Figure 4C) highlighted several key metabolites relevant for group categorization, including amino acids, sterols, TCA cycle intermediates, and sugars.

Overall, embryo axes imbibed with glucose exhibited high levels of various sugars, cysteine, threonine, and TCA cycle intermediates. In contrast, the addition of RO‐3306 led to a significant increase in aliphatic amino acids and Ser, while sugars decreased notably. The most pronounced differences in metabolite concentrations between the G and GR treatments (Figure 4C) suggest that CDK activity may play a critical role in regulating central carbon metabolism, including sugars, glycolysis, and TCA cycle intermediates, among others. Particular attention should be given to Ser and ribitol (a ribulose derivative), which had the most divergent concentrations when comparing the G and GR treatments.

A detailed analysis of the relative abundance profile across the four treatments revealed significant changes in sugar levels. In the absence of sugar in the imbibition media, only low levels of galactose, fructose, and allose were detected (Figure 4D). These sugars increased with glucose treatment but were found at lower levels in the presence of the CDK inhibitor (GR treatment). A similar pattern was observed for N‐acetyl glucosamine (a glucose derivative) and glycerol. In contrast, the levels of sucrose, maltose, and ribitol were similar in embryo axes imbibed in NS, NSR and G media but decreased significantly in the GR treatment.

Glycolysis intermediates may serve as amino acid precursors. For example, the abundance of Ser, a derivative of 3‐phosphoglycerate, was lower in the G treatment compared to NS but accumulated in the GR treatment where CDK activity was inhibited (Figure 4D). Phenylalanine (Phe), valine (Val), and leucine (Leu), hydrophobic amino acids, were present at low levels in both NS and NSR treatments, increased in G, and reached their highest levels in GR after CDK activity inhibition. 3‐Hydroxybutanoic acid, an intermediate of lipid metabolism and a derivate of succinate, exhibited similar behaviour. TCA cycle intermediaries such as citrate and succinate, along with their derivatives (glutamate and α‐aminoadipate, a lysin precursor), also showed the opposite trend: elevated levels in both no‐sugar treatments, a decrease in G and even lower levels in GR. Malate displayed a slightly different pattern, with higher levels in G but a notable decrease in GR. Finally, three plant‐abundant sterols were detected at a high concentration on embryo axes without sugar. This was reduced in NSR and G at similar proportions, but the lowest level was found in the GR treatment (Figure 4D).

3.5. Interrelationship of CDK activity with glycolysis and TCA cycle enzyme activities in maize germination

To assess whether the enzyme activities for glycolysis and TCA cycle depend on the cell and are influenced by CDK kinase activity, the activity of several enzymes was evaluated. Figure 5A shows that phosphofructokinase (PFK) activity remained low and constant from dry seed up to 15 h in all treatments. However, in the presence of glucose, PFK activity significantly increased to approximately twice that observed for dry seeds at 18 and 24 h of imbibition. Glyceraldehyde‐3‐phosphate dehydrogenase (G3PDH), although not a metabolic regulator, plays a crucial role in glycolysis and the pentose phosphate pathway in non‐photosynthetic tissues. G3PDH activity showed a contrasting behaviour: at 6 h of imbibition, the activity in the G treatment was nearly twice that of dry seeds. For NS‐treated seeds, the activity was 1.2 times larger. Notably, the presence of the inhibitor eliminated the effect of glucose at 6 h. Thus, the enzyme activity decreased in all treatments except GR, which showed a significant increase at 24 h (Figure 5B). Pyruvate kinase (PK), which regulates the final reaction of glycolysis to produce pyruvate, maintained a stable activity from dry seed up to 24 h in all treatments except NSR, in which the activity significantly increased at 24 h (Figure 5C).

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).

For the TCA cycle, two key enzymes were evaluated: citrate synthase (CS), involved in the initial carbon reaction, and malate dehydrogenase (MDH), which catalyzes the final reaction and regeneration. For the G treatment, CS activity increased at 6 h, remained steady at 15 h, and then steadily decreased until 24 h (Figure 5D). In contrast, for the GR treatment, the CS activity remained constant at the dry seed level, up until 6 h, then dropped significantly thereafter. An interesting pattern was observed for the CS activity in the NS treatment; it decreased at 6 h, then increased until reaching dry‐seed levels at 15 h, then decreased again at 18 h, and returned to its initial levels at 24 h. The inhibitor eliminated the increasing trend; the activity decreased from 0 to 6 h and remained low from 6 to 24 h. For both the G and GR treatments, the MDH activity (Figure 5E) showed a more than 2‐fold increase at 6 h, similar to the CS behaviour. In NSR, the activity increased 1.5 times, and it remained at the dry seed levels in the NS treatment. At 15 h, the activity showed similar levels for G, GR, and NSR treatments, but the NS treatment dropped below dry seed levels. At 18 and 24 h, the MDH activity in the G and GR treatments was constant, whereas in the NS medium, this activity decreased to half at 18 h and then significantly increased (fourfold) at 24 h. In the NSR medium, a low but consistent increase in activity was observed from 18 to 24 h.

Figure 5F presents a correlation analysis of enzymatic activities at 24 h for the four media. In the NS treatment, only a positive correlation between PFK and MDH was observed. The inclusion of RO‐3306 (NSR) increases the frequency of positive associations, particularly between CS and all enzyme activities except G3PDH. G3PDH showed an anti‐correlation with the rest of the enzymes in the NSR treatment. The presence of glucose further enhanced positive correlations, suggesting a coordinated metabolic regulation. The addition of the inhibitor extended the positive correlations to all enzymes but introduced a negative correlation between CS and the other enzymes, indicating the relevance of the role of CDK in regulating CS activity.

4. DISCUSSION

4.1. Glucose and the effect of CDK inhibition in germinating maize embryos

Glucose is rapidly internalized and metabolized once the maize embryo axes are in contact with the imbibition media (Figure 1E and Figure S1). After 6 h of imbibition, glucose levels were depleted. At this stage, proliferating cells are likely in the G1 phase of the cell cycle phase, progressing into the S phase by 24 h (Baíza et al., 1989, Lara‐Núñez et al., 2017). Consequently, morphological differences between treatments are feasible at this point. Size and weight parameters showed notable differences, particularly between G and GR treatments (Figure 2), suggesting that glucose, along with CDK activity, plays a crucial role in the morphogenetic program of maize embryo axes during germination.

By 48 and 72 h of imbibition (post‐germination), morphological changes become more pronounced. Embryo axes deprived of glucose (NS and NSR treatments) exhibited a minor weight gain and enlargement, failing to grow significantly by 7 days. In contrast, embryo axes exposed to glucose were longer, heavier, and more coiled (Figure 2), which is consistent with previous reports (Lara‐Núñez et al., 2017, Díaz‐Granados et al., 2020). These morphological features were affected by the presence of RO‐3306, i.e. by the inhibition of CDK; in GR, they were less coiled and showed reduced weight gain and enlargement. RO‐3306 partially inhibited CDKB and CDKA activity (Figure S3). This partial CDK inhibition was sufficient to influence growth parameters, particularly after 24 and 48 h of imbibition (Figure 2). By 7 days (post‐germination), the embryo axes showed reduced responsiveness to RO‐3306, likely due to metabolic changes, a compensatory mechanism countering the decreased CDK activity, or a diminished role of CDKs. Additionally, it is plausible that the inhibitor concentration decreased due to cell degradation or that the effective cell concentration was lower compared to shorter imbibition times. This could be due to the formation of new tissues, such as lateral roots, which may have different inhibitor permeability, or due to the dilution of the inhibitor in a larger seedling. Given that the most significant differences in length and weight between G and GR treatments were observed at 24 h, this imbibition time was emphasized in subsequent analyses. Overall, the presence of RO‐3306 impaired the growth rate of maize embryo axes imbibed with glucose, indicating an impact on the morphological program due to reduced CDK kinase activity.

4.2. CDK inhibitor RO‐3306 altered the protein phosphorylation profile in maize embryo axes

Various approaches have been employed to identify CDK phosphorylation targets. The in vitro phosphorylation of five proteins by the CDKA;1‐CycD2 heterodimer was demonstrated using a chemical genetic approach in Arabidopsis. These proteins are involved in diverse cellular and molecular mechanisms and vary widely in size and isoelectric points (Harashima et al., 2016). Similarly, a study on CDK1‐phosphorylation targets in budding yeast identified approximately 200 candidate proteins. Some of those were less abundant, while others were well‐represented, and their phosphorylation was readily detectable (Ubersax et al., 2003).

In the present study, we examined the phosphorylation profile of proteins from maize embryo axes extracts imbibed for 18 and 24 h, with either glucose or glucose plus the inhibitor RO‐3306. With this approach, we aimed to determine if RO‐3306 was internalized by the embryos and if its inhibitory effect on CDK was reflected at the molecular level. Seven phosphorylated proteins, identified using Phospho‐Tag™ technology, were analyzed. The presence of RO‐3306 in the imbibition media caused alterations in the phosphorylation profiles of five not‐yet identified proteins. It is possible that the less phosphorylated proteins in the GR treatment were direct CDK targets, but other kinases might also be affected directly or indirectly by RO‐3306 or through upstream effects on CDKS.

Eight human kinases were analyzed by Vassilev and collaborators to assess the specificity of RO‐3306 inhibition. They found all of these kinases were less sensitive to RO‐3306 compared to CDK1 sensitivity, which was 15 times more responsive to this inhibitor (Vassilev et al., 2006). While this broad approach did not allow the identification of specific phosphorylated proteins, it indicated that RO‐3306 partially inhibits CDK kinase activity (Figure S3) as previously demonstrated (Vassilev et al., 2006, Vargas‐Cortez et al., 2023). This inhibitor has implications at the molecular, morphological, and likely physiological levels.

Active heterodimeric complexes of CDKA/B and CycD/B have been identified during maize germination (Garza‐Aguilar et al., 2017, Lara‐Núñez et al., 2021) and endosperm development (Dante et al., 2014), but their specific targets are not well understood. This analysis suggests several potential substrates for these complexes, which warrant further detailed investigation. Ongoing research aims to elucidate these targets.

4.3. Impairment of CDK kinase activity altered the central carbon metabolism

The metabolomic analysis provides insight into the global metabolic events occurring in a specific tissue at a given time. In this study, the metabolomic profile of maize embryo axes was assessed at 24 h. This time was selected based on the weight and length differences observed between G and GR treatments and because the first round of cell cycle at meristem cells has not yet been completed at this point (Baíza et al., 1989).

Methanol‐soluble metabolites were extracted from maize embryo axes and analyzed by GC–MS. The clear separation between treatment groups in PLS‐DA may be the result of disruptions in basal metabolism and sugar signalling (Figure 4B). However, the presence of the CDK kinase inhibitor led to a different metabolite profile compared to the no‐sugar treatment (Figures 4B and C). An overlapping zone between NS and NSR may indicate minor metabolic changes due to a lack of sugar signalling. A more pronounced separation was observed when comparing the PLS‐DA plots of the G and GR treatments (Figure 4B). This suggests that glucose at the onset of imbibition was sufficient to activate various metabolic processes in the embryo axes, leading to metabolite changes parallel to the initial cell cycle phases in root meristems (Baíza et al., 1989, Lara‐Núñez et al., 2017, 2021) and probably other proliferating tissues. RO‐3306 significantly altered the metabolic profile. The inhibition of CDK activity resulted in the accumulation of certain metabolites and the decrease of others. Figure 4D summarizes the metabolites with statistically significant changes between the G and GR treatments, organized by metabolite type.

4.3.1. Simple sugars

The results indicate that glucose consumed by the embryo axes underwent epimerization, isomerization, and dimerization but not sucrose. This conclusion is supported by the fact that maltose, galactose (a glucose epimer), fructose, allose (another glucose epimer), and ribitol (a ribose derivative or precursor) accumulated only in axes imbibed in the G medium. Conversely, RO‐3306 disrupted this trend, reducing the concentration level of these sugars. Notably, sucrose levels are similar in the NS, NSR, and G treatments but were significantly reduced in the GR treatment. CDK kinase activity facilitated the conversion of glucose and helped maintain a steady sucrose level. Reduced CDK kinase activity led to an imbalance in glucose conversion and simple sugar accumulation, potentially affecting signalling pathways related to cell proliferation and embryo growth (Lara‐Núñez et al., 2017), resulting in smaller embryo axes (Figures 2 and 6).

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.

As previously mentioned, glucose triggers the morphogenetic program in embryo axe cells and central metabolism, as evidenced by sugar interconversion (excluding sucrose). After 24 h of imbibition, glucose‐derived carbon backbones were directed towards the biosynthesis of different metabolites, such as free amino acids, while cells were transitioning from the S phase to mitosis.

4.3.2. Amino acids derived from glycolysis intermediates

Ser exhibited the highest fold‐change across treatments, followed by ribitol and two sterols. Compared to imbibition in NS and NSR media, the presence of glucose (G treatment) led to a reduction in Ser (approximately 30%). However, CDK activity inhibition (GR treatment) resulted in a significant accumulation of Ser, about 2.5 times higher than what was found in the G treatment. This suggests that CDKs play a critical role in regulating Ser levels, potentially by either constraining the Se synthesis pathways or stimulating its conversion into one‐carbon metabolism (as a folates precursor essential for purine biosynthesis) (Smith and Atkins, 2002), glycine, phospholipids, or its incorporation into proteins (Ros et al., 2014). Additionally, Ser can accumulate in response to stress in plants (Igamberdiev and Kleczkowski, 2018), which could be induced by RO‐3306. This stress might also occur in the NSR treatment. The results showed a mild increase in Ser in NSR that did not statistically differ from the NS treatment Figure 4D), suggesting that Ser accumulation likely reflects the reduced CDK kinase activity and lower phosphorylation of key Ser regulators, potentially affecting cell proliferation during germination. Further research is needed to assess this.

Our results contrast with those reported by Wang et al., in cancer cells, where the CycD3/CDK complex phosphorylates rate‐limiting glycolytic enzymes, thus diverting glucose‐derived metabolites into the pentose phosphate pathway or the Ser pathway. The inhibition of kinase activity led to Ser depletion (Wang et al., 2017). Recent findings identified hexokinase 7 and G3PDH from maize as targets of CycD2/CDKA or B. Phosphorylation by, or interaction with, CDKB suppressed the catalytic activity of these glycolytic enzymes (Vargas‐Cortez et al., 2023).

Conversely, Phe, Val, and Leu accumulated in glucose‐treated axes after 24 h of imbibition. CDK inhibition led to a significant increase in the levels of these amino acids, indicating possible phospho‐regulation of one or more enzymes involved in their de novo synthesis or removal. Increased Phe, Val, and Leu could affect physiological processes, as elevated Val and Leu reduce Arabidopsis germination rates (Gipson et al., 2017), while high Phe positively impacts proliferation (Perkowski and Warpeha, 2019). The imbalance in amino acid homeostasis due to low CDK kinase activity warrants further investigation.

4.3.3. Sterols

Changes in sterol content can impact seed germination, as reduced sterol levels delay this process (Du et al., 2022). Three key plant sterols were detected, stigmasterol, β‐sitosterol and campesterol, showing significant reductions in embryo axes with decreased CDK activity, both in control (no sugar) and glucose‐treated axes. Campesterol and sitosterol, the major sterols found in most higher plants, are precursors of brassinosteroids, phytoregulators synthesized in the primary root of maize (Kim et al., 2005). These compounds are known to induce antioxidant defence and regulate root growth and development in maize (Yan et al., 2015). The absence or improper metabolism of sugar might induce oxidative stress during imbibition, leading to sterol accumulation.

The drastic reduction in sterol levels may account for the impaired growth in embryo axes, particularly in the GR treatment, suggesting a direct or indirect modulation of sterol metabolism by CDK. To the best of our knowledge, this is the first report proposing CDK involvement in sterol metabolism.

4.3.4. TCA cycle intermediaries and derivatives

In maize dry seeds, mitochondria are non‐functional due to poorly developed internal membranes. By 24 h of imbibition, mitochondria develop normal membranous structures and become fully functional (Logan et al., 2001). Thus, TCA cycle enzymes such as citrate synthase, succinate dehydrogenase, and malate dehydrogenase become active and help maintain the metabolic homeostasis necessary for cell growth and proliferation.

The TCA cycle intermediates detected in this study ‐citrate, succinate, and malate‐ were significantly reduced in the GR treatment compared to the G treatment, indicating a role of CDKs in maintaining the TCA cycle balance. Reduced CDK activity led to TCA cycle depletion, which could impact the production of metabolites derived from these intermediates, such as glutamic acid (Glu) and α‐aminoadipate (a lysine precursor), suggesting a systemic nitrogen and carbon imbalance. In contrast, 3‐hydroxybutyric acid, a derivative of succinic acid, showed significant accumulation in the GR treatment. Overproduction of this metabolite, and potentially other undetected metabolites, could sequester carbon backbones from the TCA cycle following CDKs inhibition, revealing an imbalance controlled by CDK kinase activity in glucose‐treated maize embryo axes.

Finally, our activity assays and correlation analysis provide insight into how glucose enhances central metabolism and how CDK activity modulates key enzymes in glycolysis and the TCA cycle, which is consistent with the observed metabolic changes. Interestingly, this modulation appears to be time‐specific during germination, as indicated by the down‐regulation of the G3PDH activity at 6 h and its up‐regulation at 24 h. Conversely, CDK activity seems to positively regulate the citrate synthase activity.

Overall, our results suggest that Cyc/CDKs complexes were active in the cytosol during maize germination and may present kinase activity against key regulatory enzymes of different pathways, directly or indirectly, creating metabolic fluxes, favouring the accumulation of simple sugars, attenuating the glycolysis intermediates and derivatives accumulation, and activating the TCA cycle to probably provide NADH to energize the mitochondria or to generate other metabolites and intermediaries. This Warburg‐like behaviour of maize embryo axes may create favourable conditions to enhance DNA synthesis in cell proliferation during germination since the meristems in maize embryo axes at 24 h are mainly in the S‐phase. To our knowledge, this is the first report that shows an association between CDK's kinase activity and the modulation of many metabolic pathways in germination.

5. CONCLUSION

The main contribution of this paper is demonstrating that CDK (cyclin‐dependent kinase) activity, traditionally known for its role in cell cycle regulation, also plays a crucial role in modulating central carbon metabolism during maize germination. The study highlights how partial inhibition of CDK activity, using the inhibitor RO‐3306, disrupts various metabolic pathways, including sugar, amino acid, and sterol biosynthesis and degradation. These metabolic changes are linked to impaired cell proliferation and morphogenetic processes in maize embryos. The findings suggest that CDK/Cyc complexes are active not only in the nucleus but also in the cytosol, where they influence metabolic enzymes and create a link between energy production and cellular growth. This research deepens the understanding of CDKs role beyond cell cycle control, suggesting a broader involvement in metabolism regulation, particularly during the early stages of germination.

Previously, Harashima and colleagues identified a mitochondrial malate dehydrogenase (MDH) in Arabidopsis as a direct target of AtCDKA;1/CycD2;1, detecting phosphorylated sites by mass spectrometry (Harashima et al., 2016). This finding, along with the identification of other mitochondrial and cytosol enzymes, highlights the significance of cell cycle regulation in energetic metabolism. Moreover, it emphasizes the role of Cyc/CDKs in channelling carbon through various pathways to support cellular growth and proliferation. For instance, the CycB1/CDK1 heterodimer phosphorylates five Complex I subunits in the respiratory chain, enhancing ATP production in mammals (Wang et al., 2014).

In yeast, trehalase, which regulates the conversion of trehalose to glucose, is controlled by CDK (CDC28) phosphorylation (Zhao et al., 2016, Ewald et al., 2016). These findings illustrate the broad range of potential phosphorylation targets for Cyc/CDKs, which may contribute to the dramatic metabolic changes observed in this study. Further research is needed to elucidate how the cell cycle machinery finely tunes both central and secondary carbon metabolism, providing a deeper understanding of the interplay between the cell cycle and central carbon metabolism regulation in plants.

Additional regulatory features and processes governed by Cyc/CDKs complexes, such as transcription, mTOR, and SnRK1, have been documented elsewhere and extend beyond the scope of this paper (Siqueira et al., 2018). Notably, the role of CDK/Cyc complexes in modulating processes outside the cell cycle appears to be conserved across eukaryotes.

AUTHOR CONTRIBUTIONS

A. Lara‐Núñez: Conceptualization, Data curation, Funding acquisition, Writing‐ Reviewing and Editing, Visualization, Investigation. S.M. Garza‐Aguilar: Writing‐ Reviewing and Editing, Visualization. J.C. Páez‐Franco: Methodology, Validation, Visualization, Investigation. J.D Galindo‐de‐la‐Rosa: Methodology, Visualization, Investigation. V. Vallejo‐Becerra: Validation, Supervision.

FUNDING INFORMATION

This work was supported by Consejo Nacional de Ciencia y Tecnología (CONACyT, A1‐S‐9076); Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica, UNAM (PAPIIT IN211423); Programa de Apoyo a la Investigación y al Posgrado, Facultad de Química, UNAM (PAIP 5000–9130).

DECLARATION OF INTERESTS

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Supporting information

Data S1

Lara‐Núñez, A. , Garza‐Aguilar, S.M. , Páez‐Franco, J.C. , de Dios Galindo‐de‐la‐Rosa, J. & Vallejo‐Becerra, V. (2025) The Cyclin‐Dependent Kinase activity modulates the central carbon metabolism in maize during germination. Physiologia Plantarum, 177(1), e70119. Available from: 10.1111/ppl.70119

DATA AVAILABILITY STATEMENT

The data that supports the findings of this study are available from the corresponding author upon reasonable request.