Open AccessArticle

Sucrose Promotes the Proliferation and Differentiation of Callus by Regulating ROS Intensity in Agapanthus praecox

Jianhua Yue

^1,*

Yan Dong

²,

Changmei Du

¹,

Chaoxin Li

^1,3,

Xinyi Wang

^1,4 and

Yan Zhang

^5,*

School of Horticulture, Xinyang Agriculture and Forestry University, Xinyang 464100, China

School of Forestry, Xinyang Agriculture and Forestry University, Xinyang 464100, China

College of Plant Science and Technology, Beijing University of Agriculture, Beijing 102206, China

⁴

School of Horticulture, Qingdao Agricultural University, Qingdao 266109, China

⁵

Department of Plant Science and Technology, Shanghai Vocational College of Agriculture and Forestry, Shanghai 201699, China

Authors to whom correspondence should be addressed.

Horticulturae 2024, 10(12), 1350; https://doi.org/10.3390/horticulturae10121350

Submission received: 6 November 2024 / Revised: 13 December 2024 / Accepted: 14 December 2024 / Published: 16 December 2024

(This article belongs to the Special Issue Plant Tissue and Organ Cultures for Crop Improvement in Omics Era)

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Abstract

The proliferation and differentiation of callus is the foundation for plant regeneration and propagation. The type of carbon sources in the medium significantly influences the efficacy of callus proliferation and differentiation in plants in vitro. Our study performed transcriptomic and physiological analyses utilizing sucrose, glucose, and maltose to understand the physiological and molecular characteristics of the proliferation and differentiation potential affected by carbon sources in Agapanthus praecox. Differentially expressed genes were notably associated with plant hormone signal transduction, glycolysis/gluconeogenesis, and MAPK signaling in the proliferation and differentiation of callus. The physiological indicators suggest glucose enhanced both callus and cell size by increasing endogenous indole-3-acetic acid (IAA), cytokinin, brassinosteroid, gibberellin (GAs), starch, and glucose levels, while concurrently reducing levels of reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂) and hydroxyl radical (·OH). Conversely, sucrose treatment promoted differentiation potential by elevating IAA oxidase activity alongside stress-related hormones such as abscisic acid and ethylene levels. Additionally, sucrose treatment led to increased accumulation of sucrose, fructose, H₂O₂, and ·OH within the callus tissue. Furthermore, sucrose influenced the regenerative capacity by modulating glycometabolism and osmoregulation. Our study posits that glucose facilitates callus proliferation via diminished ROS intensity while sucrose promotes callus differentiation by maintaining moderate ROS levels. Altogether, our results suggest carbon sources affected the regenerative capabilities of callus by regulating plant hormone signal and ROS intensity in A. praecox.

Keywords:

Agapanthus praecox; MAPK signaling; embryogenic callus; reactive oxygen species; differentiation; proliferation; somatic embryogenesis; organogenesis

1. Introduction

Whole plants can be regenerated from in vitro cultured plant cells either directly or indirectly through organogenesis or somatic embryogenesis (SE) [1], with indirect methods being more prevalent in plant propagation. In the indirect pathways, explants undergo the process of the callus stage [2]. Therefore, the induction, proliferation, and differentiation of callus laid the basis of plant regeneration and propagation in vitro in a majority of plants [3].

Explants can generate callus from various tissues and organs, including shoot and root tips, hypocotyls, young leaves, flower buds, and immature embryos [2,4,5,6], and callus is a transient tissue, but can be maintained for a long time under artificial conditions [1]. During the process of callus culture, the cell is capable of developing in three ways, such as elongation, division, or death [1], with some pluripotent or totipotent cells differentiating into organs and SE de novo. The proliferation and differentiation of callus is a complex developmental process. Its efficiency is affected by intrinsic factors such as genotype and culture age [7,8,9], as well as external conditions such as the basic culture medium [9], exogenous plant growth regulators (PGRs), and carbon sources [6,10].

Both SE and organogenesis occur in the process of callus differentiation, and this process relies on prior callus proliferation. SE involves cell differentiation characterized by the transformation of somatic cells into embryogenic callus (EC) [2]. It is a vital model for studying plant totipotency [11]. However, due to its inherent complexity, acquiring EC presents challenges that often result in randomness and inefficiency in many plants [2,6,9,12,13]. In contrast to SE, the acquisition of pluripotency via organogenesis occurs more frequently among various plants [5]. Within differentiating callus, only specific subsets of cells contribute to organ regeneration or embryogenesis, indicating a heterogeneous organization within the tissue [1]. In either case, the components of the regulators, including PGRs, along with carbon sources in the plant culture medium determine the regenerative potential of plant callus [2,10].

PGRs play critical roles in plant regeneration, and many reports indicate that plant hormones are crucial in callus proliferation and differentiation, such as indole-3-acetic acid (IAA) and cytokinins (CTKs) [2,14]. During these regeneration processes, appropriate hormonal gradients are established in the callus tissue, leading to stem cell niche formation and stem cell differentiation [15]. Auxins and CTKs are the most commonly used PGRs in plant in vitro culture due to their roles in cell division and differentiation [1]. The balance between these hormones is critical for the induction and maintenance of callus [3]. It was reported that auxin biosynthesis and signaling affect the proliferation and differentiation of callus [8], and CTKs have a critical role in triggering somatic cell dedifferentiation in Garcinia mangostana L. [16]. Moreover, it was reported that D-arginine could promote EC proliferation and increase the rate of somatic embryo induction of Litchi chinensis Sonn. by increasing indole-3-acetyl glycine (IAA-Gly) levels and reducing the production of hydrogen peroxide (H₂O₂), as well as promoting cell division and differentiation [17]. Furthermore, the balance between exogenous hormone application and endogenous hormonal levels is important for callus status [5,6]. For example, CTKs interacted with auxin, ethylene (ET), and brassinosteroid (BR) and participated in the process of callus differentiation, and the hormones above combined to regulate callus plant regeneration in Brassica juncea L. [5]. In Ginkgo biloba, the higher (IAA + ZR + iPA)/abscisic acid (ABA) and the lower IAA/(iPA + ZR) levels were critical in the SE process [14], and the content of IAA, ZR, and ABA were positively correlated with taro corm expansion in Colocasia esculenta L. [18].

Carbon sources are essential for synthesizing the carbon skeleton of cells and influencing the state of in vitro cultured cells by modulating energy metabolism, osmotic regulation, and sugar signaling [6]. An appropriate carbon source can facilitate the induction and proliferation of callus, serving as the foundation for an efficient regeneration system [6]. It was reported that sucrose metabolism is associated with callus regeneration in Sorghum bicolor L. [19]. The pretreatment of immature zygotic embryos with 0.5 M sucrose solution for 72 h efficiently induced somatic embryo initiation in Cinnamomum camphora L., suggesting that osmotic stress promotes SE, and sucrose-induced SE may share or partly share the mechanisms of SE induced by plant hormones [20]. Sugar signaling also plays a significant role in plant development, involving interactions with plant hormones, reactive oxygen species (ROS) bursts, and protein kinases [21,22].

Stresses have increasingly been recognized as playing a significant role during plant tissue culture, particularly at the callus stage. Plant hormones, including ABA, ET, and jasmonic acid (JA), are acknowledged as stress-related compounds within plant cells [6]. Additionally, it has been suggested that PGRs such as 2, 4-dichlorophenoxyacetic acid (2, 4-D) evoke stresses in plant regeneration in vitro. Stresses engage cascades of genetic triggers, turning on and off the expression of specific genes involved with the proliferation and differentiation ability [6]. Studies indicate that managing oxidative stress to prevent oxidative damage while maintaining cellular integrity is crucial for successful SE [6]. During the proliferation and differentiation process of callus, the antioxidant enzymatic system, including superoxide dismutase (SOD) and catalase (CAT) activities, exhibits significant changes [23,24].

Comparative transcriptomics serves as a valuable technique for elucidating mechanisms underlying in vitro plant regeneration [25] and functional categorization of differentially expressed transcription factors (TFs) predominantly associated with plant hormone signal, starch and sucrose metabolism, and stress response in plant culture [1,2,9,18]. Numerous genes and TFs, including auxin response factor (ARF), SE receptor-like kinase (SERK), leafy cotyledon2 (LEC2), WUSCHEL (WUS), AGAMOUS-LIKE (AGL), CLAVATA (CLV) and PIN, have been identified as being involved in SE competence acquisition [5,13,14,16,26]. Meanwhile, some of the TFs, such as WUSCHEL-RELATED HOMEOBOX 11, establish the acquisition of pluripotency during callus formation and accomplish de novo shoot formation [27].

Agapanthus praecox is a monocotyledonous, herbaceous, and perennial plant, well known for its ornamental and medicinal qualities. Furthermore, A. praecox shows strong resistance to drought and high temperature in open-field cultivation. Due to its excellent characteristics and market demand, industrialized production of A. praecox becomes imperative at present. Both organogenesis and SE pathways were established in A. praecox; callus induction consistently occurred with picloram (PIC) as the auxin-like PGRs, along with sucrose serving as the carbon source. Subsequently, initial organogenesis-based callus (IOC) and initial embryogenic cell-originated callus (IEC) could be formed based on the process of callus proliferation [28]. Our previous studies indicated that PIC plays an indispensable role in both callus proliferation and differentiation, while varying carbon sources significantly influence these processes [10]. Conventionally, sucrose, glucose, maltose, and fructose are commonly used in research. Our prior investigations revealed that glucose, maltose, and sucrose can function as carbon sources for subculture, while the callus turned brown when fructose was used as the sole carbon source; conversely, callus incubated with maltose tended to yellow while efficiently inducing organogenesis (Supplementary Figure S1) [10]. In the research of A. praecox, it is of great significance to directionally control the proliferation and differentiation of callus. As an important component in the culture medium, carbon sources play a potential regulatory role in this process. However, despite numerous published studies addressing the effects of carbon sources on in vitro culture in various plants, limited information exists regarding the mechanisms underlying their specific efficacy, which is essential for callus regeneration.

The objective of this study is to elucidate the transcriptomic and physiological changes associated with callus proliferation and differentiation induced by different carbon sources in A. praecox. Specifically, we aim to distinguish the roles of these carbon sources in relation to plant hormone metabolism and signal regulation, glycolysis and gluconeogenesis, stress response, and osmoregulation. By gaining a comprehensive understanding of the transcriptional profiles and physiological characteristics linked to callus status, we hope to facilitate advancements in both callus proliferation and differentiation in A. praecox. Our findings will contribute to the establishment of more efficient and reliable callus-based propagation and differentiation protocols for A. praecox, facilitating its commercial cultivation and genetic improvement.

2. Materials and Methods

2.1. Plant Materials, Growth Conditions, and Treatments

Callus of A. praecox was induced using pedicel as explants with MS medium containing 2.0 mg·L⁻¹ PIC and 30 g·L⁻¹ sucrose [28]. Callus originating from a single plant was then chosen to establish a single callus cell line. The cell line possessing robust vitality and high proliferative ability was subsequently selected. Thereafter, the selected cell line was transferred monthly on MS medium containing 1.5 mg·L⁻¹ PIC and 30 g·L⁻¹ sucrose [28], and after six months, the callus was sub-cultured with sucrose (Suc), glucose (Glu), and maltose (Mal) separately. Next, callus treated with different carbon sources was cultured in a phytotron in the dark at 25 ± 1 °C and with relative humidity of 65%. In this experiment, environmental factors such as humidity and temperature were strictly controlled by the phytotron. The samples were collected after 30 days for morphological, physiological, and transcriptomic assay.

2.2. Morphological and Microscopic Observation

Morphological images of callus containing sucrose, glucose, and maltose were captured using Stereo Discovery V20 Macro Stereo (Carl Zeiss, Oberkochen, Germany).

For microscopic cell observation, fresh callus blocks (<5 mm³) of sucrose, glucose, and maltose were immediately transferred to a 1.5 mL Eppendorf tube, and the samples were suspended in 0.5 mL of 1% (w/v) acetocarmine for 30 min. Next, the callus suspensions were diluted with ddH₂O; the suspension, including callus cells (<0.5 mm³), was transferred to a slide; and a cover glass was slightly placed on the slide. Microscopic observations were performed using an Axio Scope A1 microscope (Carl Zeiss, Oberkochen, Germany), and three biological replicates were conducted.

2.3. Physiological Measurements

The physiological indices related to endogenous hormones, carbohydrate accumulation, ROS, and protective enzymes were detected by enzyme-linked immunosorbent assay (ELISA) [28,29], and the assay was conducted by Shanghai Enzyme-Linked Biotechnology Co., Ltd. (https://en.mlbio.cn/, accessed on 13 December 2024). Three biological replicates were performed with ELISA. A 0.5 g fresh specimen was rinsed in ice-cold phosphate-buffered saline (PBS) with a pH value of 7.4 and subsequently delicately positioned in a 5 mL homogenization tube. Thereafter, a volume of homogenization medium, quadruple the amount of the sample, was incorporated into the tube. The tissue was pulverized with a masher at a velocity of 12,000 rotations per minute. The sample was then centrifuged at 4000 rotations per minute for 10 min. Subsequently, the supernatant was extracted at 4 °C with the facilitation of a C-18 solid extraction column. Next, the samples were transferred to a 10 mL centrifuge tube and underwent freeze-drying treatment, trailed by dilution. The blank wells and sample wells were established in a 96-well ELISA plate. For the sake of standard curve measurement, the standard samples were diluted to formulate gradient concentrations. Fifty microliters of conjugate reagent were dispensed into each well, barring the blank wells. The plate was sealed with a closure plate membrane and then incubated at 37 °C for 30 min. Upon the completion of incubation, the liquid was removed. Each well was filled with the washing solution and incubated for 30 s, and then the washing solution was evacuated. This washing step was reiterated four times. Fifty microliters of chromogen solution A and chromogen solution B were added to the wells. The plate was gently stirred and then incubated at 37 °C for 10 min in the dark. Subsequently, 50 microliters of 2 moles per liter of sulfuric acid were introduced into each well to arrest the reaction. Finally, the optical density (OD) value at a specific wavelength of each well was gauged using a microtiter plate reader (Thermo Fisher Scientific, Waltham, MA, USA). The physiological indicators were reckoned in accordance with the standard curve.

2.4. Transcriptomics Sequence and Analysis

For transcriptome analysis, three biological replicates were utilized for each of the three samples (Suc, Glu, and Mal) and IEC. Total RNA was extracted using an RNAiso Plus kit (TaKaRa, Dalian, China) and the quality was verified using electrophoresis and spectrophotometry. The transcriptome analysis was conducted by Origingene Biotech Co., Ltd. (Shanghai, China, http://www.origin-gene.com/, accessed on 27 July 2022). Library construction adhered to Illumina’s standard instructions, and sequencing was performed on an Illumina HiSeq 2500 platform. The sequencing process was carried out simultaneously at both ends of the cDNA fragments, with each read length being approximately 150 base pairs. Since there is a lack of a reference genome, the transcriptome is a de novo transcriptome in this study. Raw reads were processed using Trimmomatic, and the reads containing ploy-N and lower-quality reads were removed to obtain clean reads. The clean reads were assembled into expressed sequence tag clusters and de novo assembled into a transcript by using Trinity (version: 2.4) with the paired-end method. The longest transcript was selected as a unigene based on the similarity and length of a sequence for subsequent analysis. The function of the unigenes was annotated by alignment of the unigenes with the NCBI database using Blastx with a threshold E-value of 10⁻⁵. Fragments per kilobase per million (FPKM) values were calculated to quantify the mapped reads. Genes were selected based on a false discovery rate (FDR) threshold of 0.05. Expression levels of the identified genes were then calculated using the FPKM method, and genes with a fold-change greater than 2 and an adjusted p-value of less than 0.05 were classified as differentially expressed genes (DEGs). Subsequently, the unigenes were mapped to the Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.kegg.jp/, accessed on 13 December 2024) database to annotate their potential metabolic pathways. Additionally, hierarchical clustering analyses were performed using Mev 4.9.0 (https://sourceforge.net/projects/mev-tm4/files/mev-tm4/, accessed on 13 December 2024).

2.5. Confirmatory Experiment

For the validation experiment, callus samples were cultured on MS medium containing 1.5 mg·L⁻¹ PIC and 30 g·L⁻¹ carbon source as the elementary culture medium. Then, the additive was added to the medium for each treatment. If it is not specified, the medium contained 1.5 mg·L⁻¹ PIC and 30 g·L⁻¹ sucrose as the basic components of the medium. Approximately one gram of callus was accurately weighed on a clean bench, after which the callus was equally divided into nine smaller clusters. Five Petri dishes of samples were used in one replicate, and three biological replicates were conducted. The data regarding the size of callus were examined after 60 days.

Morphological images of the callus were captured by LX100M2 (Panasonic, Toyko, Japan). For microscopic cell observation, images of callus cells were created according to Section 2.2.

2.6. Detection of the Osmotic Regulatory Characteristics

The relative water content of callus treated by different combinations of carbon source and polyethylene glycol (PEG) 6000 was determined by the drying method [30]. The water content (fresh weight − dry weight) of the callus tissues was expressed as a percentage of fresh to dry weight at 60 °C in a dry oven for 24 h. The water potential of the callus and medium was examined by the ψs = −CiRT method [31]. Three replications were performed for each experiment, and callus in one Petri dish was tested for each replication.

2.7. Determination of ROS Intensity and Antioxidant Enzymes in Osmotic Regulation by PEG 6000

Fresh tissues (0.5 g) were ground in a 5 mL precooled centrifuge tube. Then, the supernatant was collected for ROS intensity and antioxidant enzyme activity detection. The contents of malondialdehyde (MDA, D799761-0050), H₂O₂ (D799773-0050), and ascorbic acid (AsA, D799271-0050), and the activities of ascorbate peroxidase (APX, D799461-0050), CAT (D799597-0050), and SOD (D799593-0050), were determined using reagent kits (Sangon Biotech Co., Ltd., Shanghai, China, https://store.sangon.com/, accessed on 13 December 2024). The reaction solution was examined by ultraviolet spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Each test was performed in triplicate.

2.8. Statistical Analysis

SPSS software (v26.0, SPSS Inc., Chicago, IL, USA) was used for the statistical analysis of the data. The data were statistically tested using a one-way analysis of variance and are shown as mean ± SE. The statistical significance among means was calculated by post-hoc Duncan’s multiple range test at p < 0.05.

3. Results

3.1. Overview of Morphological and Transcriptomic Differences

An overview of samples related to callus proliferation and differentiation affected by different carbon sources is demonstrated in Figure 1. Callus is usually semitransparent, slightly yellowish, and watery. The morphological characteristics showed that glucose increased the size of the callus compared to sucrose, while maltose promoted organogenesis evidently (Figure 1A). Cell microscopy showed that the callus cultured with sucrose exhibited a pellet shape and thick cytoplast, while the callus cultured with glucose showed an elliptic shape, thin cytoplast, and bigger size. Meanwhile, the callus cultured with maltose showed a pellet shape and thin cytoplast (Figure 1B). The information on the clean reads is provided in Supplementary Table S1. Correlation analysis among samples showed that the callus cultured with glucose had a closer relationship with the callus cultured with maltose (Figure 1C). The Venn analysis revealed 11,588, 9622 and 6257 DEGs between different comparison groups (Suc/Glu, Suc/Mal, and Glu/Mal), and 4875 (1929 up/2946 down), 3575 (2181 up/1394 down), and 1131 (568 up/563 down) DEGs were specifically expressed among Suc/Glu, Suc/Mal, and Glu/Mal separately (Figure 1D).

The KEGG analysis revealed that different pathways were enriched among three comparisons (Suc/Glu, Suc/Mal, and Glu/Mal), and the DEGs were mainly involved in pathways (level 2) including plant hormone signal transduction, glycolysis/gluconeogenesis, starch and sucrose metabolism, flavonoid biosynthesis, cell cycle, MAPK signaling pathway-plant, phenylpropanoid biosynthesis, fructose and mannose metabolism, longevity regulating pathway, glutathione metabolism, nitrogen metabolism, and signaling pathways regulating the pluripotency of stem cells. The top terms between the compared pairs of Suc/Glu were plant hormone signal transduction (p-value 5.78 × 10⁻⁶), glycolysis/gluconeogenesis (p-value 1.44 × 10⁻³), flavonoid biosynthesis (p-value 6.91 × 10⁻³), starch and sucrose metabolism (p-value 1.11 × 10⁻²), cell cycle (p-value 1.96 × 10⁻²), and MAPK signaling pathway-plant (p-value 5.38 × 10⁻²) (Figure 1E). Meanwhile, pathways including glycolysis/gluconeogenesis (p-value 8.23 × 10⁻⁴), plant hormone signal transduction (p-value 9.47 × 10⁻⁴), flavonoid biosynthesis (p-value 7.83 × 10⁻³), starch and sucrose metabolism (p-value 1.98 × 10⁻²), phenylpropanoid biosynthesis (p-value 3.89 × 10⁻²), and fructose and mannose metabolism (p-value 2.88 × 10⁻²) were quite different between sucrose and maltose treatment (Figure 1F). Also, starch and sucrose metabolism (p-value 1.19 × 10⁻³), glycolysis/gluconeogenesis (p-value 2.87 × 10⁻³), plant hormone signal transduction (p-value 5.37 × 10⁻³), phenylpropanoid biosynthesis (p-value 1.30 × 10⁻²), fructose and mannose metabolism (p-value 6.21 × 10⁻²), and MAPK signaling pathway-plant (p-value 6.94 × 10⁻²) were enriched in the comparison of Glu/Mal (Figure 1G).

3.2. Transcriptomic Analyses of Genes Concerning Callus Proliferation and Differentiation

Higher-expression DEGs (FPKM > 100) were analyzed by hierarchical clustering, and four clusters were separated by gene expression patterns (Figure 2A). Among them, 298 DEGs, including TCH4, heat shock protein 90 (HSP 90), showed a similar expression pattern in the callus samples (subcluster1, SC1) (Figure 2B); six DEGs, such as histone H1/5, fructose-bisphosphate aldolase [EC:4.1.2.13], were downregulated in sucrose-treated callus (SC2) (Figure 2C); and 10 DEGs, including DNA-directed RNA polymerase II subunit RPB1 [EC:2.7.7.6], basic endochitinase B [EC:3.2.1.14], and two pore calcium channel protein (TPC1, CCH1) were highly expressed in sucrose treatment (SC3) (Figure 2D), while four DEGs, such as large subunit ribosomal protein L18e, MPBQ/MSBQ methyltransferase [EC:2.1.1.295], were highly expressed in IEC and Suc, which indicates that the differentiation stage was accompanied by methylated modifications (SC4) (Figure 2E, Supplementary Table S2).

3.3. Transcriptomic and Physiological Analyses of Plant Hormone Signal Transduction

Physiological characteristics indicated that the content of free IAA and conjugated IAA was higher in glucose treatment than in sucrose treatment; meanwhile, the activity of IAA oxidase with sucrose treatment was the highest among the three samples (Figure 3A). Consistently, the contents of GA₃, GA₄, BR, and CTK showed the highest levels with glucose treatment (Figure 3A). On the contrary, the concentrations of ABA, ET, and JA showed the lowest levels with glucose treatment (Figure 3A). In the plant hormone signal transduction pathway, auxin signal transduction-related DEGs such as auxin/indoleacetic acids (AUX/IAAs), small auxin-upregulated RNA (SAUR), transport inhibitor response1 (TIR1), GH3, and ARF, and transcripts of CTK signal transduction-related genes (ARR-A, ARR-B, AHK2_3_4), were changed considerably (Figure 3B,C). GA signal transduction-related gene gibberellin-insensitive dwarf1 (GID1) was downregulated in the comparison of Suc/Glu, while the expression level of DELLA was upregulated in the comparison of Suc/Glu and Suc/Mal (Figure 3D). Transcripts, including brassinosteroid insensitive 2 (BIN2), BR-signaling kinase [EC:2.7.11.1] (BSK), brassinosteroid insensitive 1 (BRI1), brassinosteroid resistant 1/2 (BZR1_2), and cyclin D3 (CYCD3), were changed significantly with carbon source treatment (Figure 3D). Correspondingly, ABA signal transduction-related genes (e.g., PP2C, and SNRK2), ET signal transduction-related genes (e.g., EIN), and JA signal transduction receptor JAZ were changed significantly (Figure 3B–D). Combined morphological, transcriptomic, and physiological analyses revealed that plant hormones control cell proliferation of callus by regulating the expression of plant signal transduction-related genes and plant hormone metabolism. Hormones such as IAA, GA, CTK, and BR, which are beneficial to the cell division cycle and cell enlargement, were increased in callus samples, while ABA, ET, and JA, which are harmful to the process of cell proliferation, were decreased in callus. To validate the effect of plant hormones, PGRs in gradient, including PIC, GA₄, HBL, and ABA, were added to the culture medium. As expected, PIC and GA₄ promoted the size of the callus (Figure 3E,F). Interestingly, HBL showed an inhibitory effect on callus proliferation (Figure 3G). The addition of ABA decreased the size of the callus significantly (Figure 3H).

3.4. Transcriptomic and Physiological Analyses of Starch and Sucrose Metabolism

The result of the ELISA show that glucose treatment increased the contents of starch and glucose, sucrose treatment enhanced the levels of fructose, and maltose treatment enhanced the concentration of maltose in callus significantly (Figure 4A). The physiological analyses suggested that the exogenous carbon source affected saccharides metabolism in the in vitro culture directly. In the starch and sucrose metabolism pathway, the expression levels of DEGs, including alpha-glucosidase (malZ) [EC:3.2.1.20], beta-glucosidase [EC:3.2.1.21], trehalose 6-phosphate synthase/phosphatase (TPS) [EC:2.4.1.15/3.1.3.12], and sucrose synthase (SUS) [EC:2.4.1.13], are quite different among the compared pairs of Suc/Glu, Suc/Mal, and Glu/Mal (Figure 4B–D). Notably, the expression level of beta-fructofuranosidase (INV) [EC:3.2.1.26] increased in sucrose compared with glucose (Log₂ FC 5.28) (Figure 4B), while the expression of TPS and malZ decreased sharply in the comparison of Suc/Glu (Figure 4C), and the transcriptional level of SUS decreased significantly in sucrose- compared with glucose-treated samples (Log₂ FC −8.20) (Figure 4D). The expression level of sucrose-6-phosphatase (SPP) [EC:3.1.3.24] and 4-alpha-glucanotransferase (malQ) decreased in Suc/Glu and increased in Glu/Mal, while the expression level of fructokinase (FRK) [EC:2.7.1.4] increased in Suc/Glu and decreased in Glu/Mal (Figure 4E). The expression levels of malQ, TPS, and SUS decreased in Suc/Glu, Suc/Mal and increased in Glu/Mal (Figure 4F). The validated results show that 30 g·L⁻¹ sucrose was the suitable dosage in callus culture (Figure 4G). The addition of glucose increased the size of callus significantly (Figure 4H), while a higher concentration of fructose was harmful to the proliferation of callus (Figure 4I). Maltose showed a positive effect on callus proliferation, and the addition of 20 g·L⁻¹ maltose increased the size of callus remarkably compared with the control (Figure 4J).

3.5. Transcriptomic and Physiological Analyses of MAPK Signaling Pathway

Sucrose treatment led to a higher level of ROS, including H₂O₂ and ·OH, in the callus. Lower activity of peroxidase (POD), SOD, and CAT was observed in sucrose treatment compared with glucose treatment, while ROS and protectant enzyme activity showed a moderate level with maltose treatment (Figure 5A). In total, 82 DEGs were enriched in the MAPK signaling pathway. The DEGs were related to the key members in the MAPK signaling pathway, including MAP kinase kinases (MKKs) and MAPKs; respiratory burst oxidase homologues (RBOHs); TFs such as WRKYs; plant hormone signal transduction-related genes PP2C, EIN, SNRK2, and peroxisomal membrane protein 2 (PXMP2); and the protective enzyme CAT. Transcripts including mitogen-activated protein kinase 7 (MAPK7), RBOH, and WRKY33 were upregulated in sucrose compared with glucose and maltose, and the transcriptional level of RBOH increased significantly in sucrose compared with glucose (Log₂ FC 3.88) and maltose (Log₂ FC 3.07) (Figure 5B). Meanwhile, the expression level of PXMP2 showed significant differences among samples (Figure 5C,D). On the contrary, CAT was downregulated in sucrose compared to glucose and maltose, and the transcriptional level of CAT decreased significantly in sucrose compared with glucose (Log₂ FC −10.26) (Figure 5E). The results above suggest that the carbon source changed the MAPK signaling pathway, and MAPK signaling evoked the ROS metabolism and response of stress-related plant hormones (ABA, ET), which affected the proliferation and differentiation of callus in A. praecox. The addition of H₂O₂ inhibited the proliferation of callus, and the size of the callus decreased in relation with the dosage of H₂O₂ (Figure 5F). When 2, 4-D was the auxin-like PGRs, the callus size decreased sharply, suggesting that PIC was indispensable in the callus culture of A. praecox (Figure 5G). Meanwhile, the osmotic stress from 50 g·L⁻¹ PEG 6000 decreased the callus size (Figure 5H). Furthermore, caspase 3 inhibitor CHO significantly promoted callus proliferation, while programmed cell death (PCD) promoter CCCP decreased callus size in A. praecox (Figure 5I).

3.6. The Effects of Carbon Source Combination on the Proliferation and Differentiation of Callus

The results show that the hydrolysate of sucrose (glucose and fructose) reduced the size of callus slightly compared with sucrose; however, there were no significant differences among the size of the callus (p > 0.05), and the size of the callus was close to the level of sucrose treatment with increasing dosage of glucose (Figure 6A,B). The addition of 20 g·L⁻¹ sucrose in the medium led to a smaller callus size, and the addition of glucose increased the size of callus when the dosage of the carbon source reached 30 g·L⁻¹ (Figure 6C,D). Unlike glucose, the addition of fructose decreased the proliferation of callus when 20 g·L⁻¹ sucrose was selected as the basic carbon source (Figure 6E,F).

Compared with sucrose, the hydrolysate of sucrose showed quite different efficacy in callus proliferation. The combination of sucrose (glucose and fructose) and glucose showed a similar trend with sucrose (Figure 6D,F) in that the size of the callus decreased when the dosage of the carbon source was 40 g·L⁻¹ compared with 30 g·L⁻¹; however, the 60 g·L⁻¹ carbon source elevated the callus size dominantly (Figure 6G,H). In contrast to sucrose, the addition of fructose increased the callus size when the hydrolysate of sucrose was employed as the basic carbon source (Figure 6I,J), while the addition of maltose showed a similar pattern to that of glucose in that the hydrolysate of sucrose was the basic carbon source (Figure 6K,L).

The results above suggest that the type and dosage of carbon sources affected the callus proliferation and differentiation. In general, sucrose and its hydrolysate showed a quite different effect on callus proliferation; a higher concentration of carbon source was harmful to callus proliferation. Glucose promoted the size of the callus when sucrose was the basic carbon source, and fructose promoted the size of the callus when the hydrolysate of sucrose was the basic carbon source. Our investigation also showed that a certain dosage and a lower ratio of glucose and fructose promoted the generation of EC (Figure 6A, Supplementary Figure S2).

3.7. The Effects of the Osmotic Regulatory Substance on the Proliferation and Differentiation of Callus

The dosage of the carbon source showed important effects on the proliferation and differentiation of callus in A. praecox, suggesting osmotic regulation performed critical roles in this process. Compared to sucrose, mannitol, and ABA, PEG was found to be more effective in the proliferation of callus in A. praecox (Supplementary Figure S3). Subsequently, the effects of the combinations of carbon sources and PEG on callus proliferation were investigated. When 30 g·L⁻¹ sucrose was used as the basic carbon source, the callus size decreased slightly with the increase in the levels of PEG (Figure 7A,B). The size of callus reached the biggest value with the addition of 40 g·L⁻¹ PEG (without significant difference), and the cells had small diameters and dense cytoplasm (Figure 7A,B). Interestingly, the treatment with 40 g·L⁻¹ PEG significantly reduced the size of single cells (p < 0.05). Physiological indexes showed that PEG 6000 influenced the oxidative balance of callus significantly (Supplementary Figure S4). The content of MDA and H₂O₂ and the activity of CAT were the lowest with 40 g·L⁻¹ PEG treatment, and the content of AsA and the activity of SOD and APX increased significantly with the 40 g·L⁻¹ PEG treatment compared with the control (Supplementary Figure S4). However, the callus size decreased significantly with the 40 g·L⁻¹ PEG treatment, with 30 g·L⁻¹ glucose as the basic carbon source (Figure 7C,D). Notably, the callus size increased slightly with the addition of PEG when fructose was the carbon source, and the callus was brown, with large cell diameter and thin cytoplasm (Figure 7E,F). The size of the callus decreased continuously with the increment in PEG, with 15 g·L⁻¹ glucose and 15 g·L⁻¹ fructose as the basic carbon source (Figure 7G,H). The addition of 10 g·L⁻¹ PEG increased the callus size, with 15 g·L⁻¹ sucrose and 15 g·L⁻¹ glucose as the carbon source (Figure 7I,J), and the addition of 40 g·L⁻¹ PEG increased the callus size compared with 30 g·L⁻¹ PEG with 15 g·L⁻¹ sucrose and 15 g·L⁻¹ fructose as the basic carbon source (Figure 7K,L). The results indicate that the osmotic regulatory substance enhances callus proliferation when carbon sources are present at a specific concentration. Furthermore, PEG plays a positive role in promoting callus proliferation in A. praecox. Intriguingly, the type of carbon source has a minor impact on the relative water content of the callus, but it significantly influences the water potential of both the medium and the callus (Supplementary Figure S5).

4. Discussion

The proliferation and differentiation of callus constitute the foundation for plant regeneration and propagation. In the present study, we compared the effects of sucrose, glucose, and maltose on the proliferation and differentiation of callus in A. praecox. Herein, the transcriptomic and physiological analyses revealed DEGs and physiological indicators involved in the proliferation and differentiation of callus. The results imply potential regulatory roles of plant hormone metabolisms and signals, glycometabolism, MAPK signaling, oxidative stress, and osmotic stress in these processes. Additionally, our findings indicate optimal carbon sources in the proliferation and differentiation of callus in A. praecox (Figure 8A).

4.1. Plant Hormone Metabolisms and Signals Are Critical in Callus Proliferation and Differentiation

Callus can proliferate and differentiate in vitro by exposure to plant growth regulators (PGRs), indicating that plant hormone metabolisms and signals have crucial roles in plant tissue culture. In the present study, the plant hormone metabolisms as well as transcriptional profiles were affected by carbon sources, and the underlying regulatory mechanisms governing these processes were elucidated in A. praecox. Our study demonstrated that high levels of endogenous indole-3-acetic acid (IAA), cytokinins (CTKs), brassinosteroid (BR), and gibberellins (GAs) were accompanied by a larger size of callus, while endogenous abscisic acid (ABA), ethylene (ET), and jasmonic acid (JA) showed an opposite proliferation efficacy (Figure 3). The interplay between IAA and CTK highlights the intricate hormonal crosstalk that governs callus proliferation and differentiation in many plants [32,33]. For instance, 1-naphthaleneacetic acid (NAA) combined with 6-benzylaminopurine (BAP) was proven to be essential for the in vitro callus induction and proliferation in Artemisia annua [33], while NAA, BAP, and 2, 4-dichlorophenoxyacetic acid (2, 4-D) promoted the increase in callus biomass with suspension culture in Siraitia grosvenorii [23]. 2, 4-D and picloram (PIC) regulate the endogenous IAA biosynthesis and auxin signaling, emphasizing the pivotal role in the expression of key factors involved in callus differentiation and epigenetic regulatory networks [32], and CTK is known to induce IAA biosynthesis [34]. In Passiflora edulis Sims, the alternative induction of de novo shoot organogenesis or SE is modulated by the ratio between auxin and CTK in the medium [35]. In A. praecox, the combination of PIC and BAP promoted organogenesis and EC proliferation effectively [28,36].

Transcriptomic analysis has highlighted the effects of plant hormones on in vitro culture in many plants, such as Cucumis sativus L. [2], Ginkgo biloba [14], and Passiflora edulis Sims [35]. An intricate balance exists between the intracellular hormone and auxin-related PGR-induced stress signals [37]. Auxin-related PGRs such as 2, 4-D trigger the burst of ROS and alter the plant hormone signals in plants [25,38]. The differentiation of callus is a prerequisite for auxin-related PGR-induced chromatin accessibility and transcriptome alteration [37]. The balance in auxin and CTK levels is critical for cell differentiation in different plants due to the regulation of plant hormone signaling-related genes and methylation levels [39]. In this study, auxin- and CTK-signal transduction-related transcripts such as auxin/indoleacetic acids (AUX/IAAs), small auxin-upregulated RNA (SAUR), transport inhibitor response1 (TIR1), Gretchen Hagen 3 (GH3), auxin response factor (ARF), type-A authentic response regulator (ARR-A), ARR-B, and Arabidopsis histidine kinase 2/3/4 (AHK2_3_4), and methylation-related transcripts such as MPBQ/MSBQ methyltransferase, significantly changed their expression levels, and the accumulation of endogenous IAA and CTK considerably increased, suggesting the important roles of auxin and CTK-signal transduction in callus proliferation and differentiation. Furthermore, it seemed that GAs promoted in vitro culture in A. praecox, and the expression levels of gibberellin-insensitive dwarf1 (GID1) and DELLA were altered considerably with the different treatment of carbon sources. Furthermore, our previous study suggested that GAs promoted SE in A. praecox [40].

4.2. Glycometabolism Plays Fundamental and Crucial Roles in Callus Proliferation and Differentiation

Previous studies have reported crosstalk between plant hormone signals and sugar metabolism in Cinnamomum camphora L., with DEGs indicating that sucrose-induced cell proliferation may fully or partly share the mechanisms of cell proliferation induced by plant hormones [20]. Sugar signaling interacts with plant hormones to regulate plant development. In our previous study, BAP promoted EC proliferation through the regulation of both plant hormones and sugar metabolism [36]. This study showed that the regulation of carbon sources within the culture medium significantly affected cell proliferation and differentiation, suggesting a synergistic regulatory role for plant hormone signals and sugar metabolism.

Sugars play essential roles in energy supply and osmotic regulation in in vitro culture. Integrated transcriptomic and physiological analyses suggest that sugar and energy metabolism have essential roles by supporting cell proliferation and differentiation [12]. Our physiological assessments revealed that carbon sources within the medium directly impacted intracellular sugar metabolism in callus (Figure 4A and Figure 8B), suggesting that sucrose, glucose, and maltose in the medium directly influenced cell proliferation and differentiation by modulating glycometabolism [41].

The effect of carbon sources on plant cell culture is attributed to variations in hydrolysis rates, component composition, and utilization efficiency. Fructose has been identified as an unsuitable carbon source for promoting callus proliferation in A. praecox as well as differentiation to SE in Larix kaempferi × Larix olgensis [6], while a lower ratio of fructose and glucose promoted the formation of EC in A. praecox. In the present study, glucose was proven to promote the proliferation of callus effectively. However, glucose failed to induce SE and organogenesis in A. praecox. Maltose hydrolyzes into glucose at a slower rate than sucrose, resulting in a lower efficiency of energy supply. Conversely, a combination of sucrose and maltose was found to simultaneously maintain robust callus growth and cellular activity. Sucrose rapidly hydrolyzes into glucose and fructose, and sucrose is the most commonly used carbon source in in vitro culture. In A. praecox, sucrose showed positive efficacy in callus differentiation (Figure 8A) [28]. Starch typically serves as a form of storage of carbohydrates [42]. The correlation between the size of the callus and the sugar content suggests that starch content reflects the state of cellular sugar metabolism and utilization.

Plant cells can be adjusted in feedback according to the demand of sugar metabolism. Sugar composition and starch in plants undergo interconversions under the action of enzymes such as sucrose synthase (SUS) and amylase. SUS is widely considered a key enzyme participating in sucrose metabolism in higher plants [43]. Notably, the expression level of neutral invertase (IN) increased, and trehalose 6-phosphate synthase/phosphatase (TPS), alpha-glucosidase (malZ), SUS, sucrose-6-phosphatase (SPP), and 4-alpha-glucanotransferase (malQ) decreased in the comparative group of Suc/Glu, suggesting a feedback regulation model for sucrose and starch metabolism and an increase in reducing sugars such as glucose during callus proliferation and differentiation to support the high frequencies of cell divisions [41]. Subsequently, TPS, which has pivotal roles in sensing sucrose status, regulating plant growth, and mediating sugar signaling and homeostasis in a plant cell [44], was specifically accumulated in the comparison of Glu/Mal and Suc/Mal. Altogether, carbon sources affected plant cell proliferation and differentiation potentially by regulating basal glycometabolism [12].

4.3. Oxidative Stress and Osmotic Stress Play Critical Roles in Callus Proliferation and Differentiation

Transcriptomic analysis highlights the effects of MAPK signaling on in vitro culture in A. praecox. The MAPK signaling cascade plays a pivotal role in modulating stress-resistance genes and hormones, thereby initiating cellular stress resistance. However, it can also lead to the burst of ROS and alter the oxidation-reduction state of the cell.

The intricate balance between ROS production and scavenging within cells acts as a fundamental regulatory mechanism influencing cell proliferation, differentiation, and survival [6]. Studies have demonstrated that carefully managing H₂O₂ content in hybrid larch can improve the efficiency of SE [6]. However, excessive accumulation of ROS such as H₂O₂ can become detrimental, resulting in membrane lipid peroxidation, electrolyte imbalance, and alterations in glycoprotein cross-linking, ultimately compromising cell membrane integrity and impeding cell expansion and division [6]. Moderate levels of ROS, including H₂O₂, typically facilitate in vitro culture and redifferentiation of plant cells [45]. In A. praecox, ROS exhibit a dual nature; moderate levels of ROS promoted callus proliferation and differentiation [28]. As signaling molecules that influence cell proliferation and differentiation, antioxidant enzymes and metabolic enzymes, such as POD, SOD, CAT, and APX, are employed for intracellular ROS clearance [41,45]. The addition of 0.5 g·L⁻¹ ascorbic acid (AsA) and 1.0 g·L⁻¹ choline has been shown to elevate the induction rate from 0.75% to 68% and enhance the antioxidant enzyme activities of loose EC, and the antioxidant enzymes showed significantly increasing activities in EC compared to those in non-EC of Siraitia grosvenorii [23].

Our results demonstrate a correlation between ROS intensity, antioxidant enzyme activity, and callus status under different carbon source treatments (Figure 5). Previous studies have shown that glucose represses stress-related transcription factor genes such as dehydration-responsive element-binding (DREB), C-repeat binding factor (CBF), NAC, WRKY, and bHLH in cut flowers of Paeonia suffruticosa Andrews [46]. In this study, glucose treatment resulted in lower levels of ·OH and H₂O₂, coupled with higher antioxidant enzyme activities, larger callus size, and increased cell size. When cultured with maltose, the intensity of ROS, activities of antioxidant enzymes, and cell size exhibited moderate levels among the three treatments. Respiratory burst oxidase homologue (RBOH) plays a pivotal role in ROS production and is involved in plant growth, development, and stress adaptation [47]. Notably, the expression level of RBOH was significantly upregulated in sucrose compared to glucose and maltose, accompanied by a higher level of H₂O₂ and smaller cell size. Plant RBOHs, also known as NADPH oxidases, are well characterized ROS-producing systems. RBOHs are integral plasma membrane proteins, catalyzing formation of the superoxide anion (O₂⁻) by transferring an electron from cytosolic NADPH to an apoplastic O₂, followed by dismutation of O₂⁻ to H₂O₂ [48]. The RBOH-dependent H₂O₂ burst, functioning as a pivotal signaling cascade, triggers the activation of MAPK signaling pathways and elicits the expression of TFs, including MYB and WRKY. Concurrently, it expedites the transcription and translation processes of antioxidant enzyme genes such as SOD, CAT, and POD [49]. This orchestrated activation mechanism forms a comprehensive defense network within the plant cell, enabling it to counteract various environmental stresses effectively and maintain cellular homeostasis [48,49]. In L. chinensis Sonn., polyamine and its metabolite H₂O₂ play a key role in the formation of EC, which reduces the activity of diamine oxidase (DAO) and polyamine oxidase (PAO) by the exogenous addition of D-Arg [17]. Abiotic stresses trigger sugar signaling by inducing ROS burst, and abiotic stresses target sugar signaling to regulate PCD in plants. Sugar deprivation disrupts the homeostasis of ROS and induces oxidative damage through the stimulation of ROS production. This phenomenon is closely intertwined with the diminished expressions of sucrose transporters (SUTs) and hexokinases (HXKs) within the sugar signaling pathway [50]. Intriguingly, sucrose is the optimal carbon source for callus differentiation via SE and organogenesis pathways in A. praecox, compared to glucose and maltose (Figure 8A). This is partly attributed to the metabolism of ROS (particularly H₂O₂) in cells, which is crucial for cell reprogramming. In conclusion, our findings indicate a balance between cell proliferation and differentiation, regulated by ROS metabolism in in vitro cells (Figure 8A,B).

Furthermore, osmotic stress induced by sucrose and PEG may play a significant role in cell proliferation and differentiation in callus. H₂O₂ and MDA accumulation was significantly decreased in the 40 g·L⁻¹ PEG treatment compared to the control, CAT activity showed a lower level, and SOD, AsA, and APX maintained moderate levels, reflecting the important role of oxidative stress in callus proliferation (Supplementary Figure S4). Furthermore, sucrose supplementation leads to the enhancement of AsA content. Sucrose acts as precursors for AsA biosynthesis through the L-galactose pathway. This increase in AsA content due to sucrose addition is especially significant in callus differentiation. In L. kaempferi × L. olgensis, ABA and PEG promote somatic embryo formation in unique and complementary ways [6], suggesting a crosstalk between oxidative stress and osmotic stress. In the present study, the surface of the callus became watery with the increase in the dosage of PEG, and the water potential of the callus and medium was decreased significantly with 40 g·L⁻¹ PEG treatment (Supplementary Figure S5), suggesting a powerful uptake potential with higher osmotic stress. Because glucose can provide more powerful osmotic pressure than sucrose at the same concentration, the results can explain why glucose treatment increased cell size and callus size while the cytoplasm was thinner (Figure 1A). Furthermore, cyclin D3 (CYCD3) was upregulated in Glu/Mal and downregulated in Suc/Glu, suggesting the cell population may contribute to the biomass of callus by glucose treatment. As for sucrose, lower osmotic pressure induced a smaller cell size and a thick cytoplast (Figure 1A). No matter which carbon source was used in the medium, a higher PEG concentration induced lower water potential in the callus (Supplementary Figure S5), which suggests that the cell of the callus maintain proliferation by adjusting osmotic pressure and metabolism. In A. praecox, the enhancement of osmotic stress by PEG 6000 elevated the callus size and decreased the cell size of the callus by triggering the variation in stress responses. This suggests that a higher PEG dosage stimulates cell division capacity during callus proliferation.

Carbon sources and their combinations exert a substantial influence on the in vitro cultivation of A. praecox. Glucose proves advantageous for callus proliferation, whereas maltose facilitates long-term subculturing and organogenesis. Sucrose holds a greater propensity for promoting callus differentiation. When it comes to the precise modulation of callus growth, the combination of sucrose and glucose confers enhanced benefits for callus proliferation and differentiation (SE), while the blend of sucrose and maltose is beneficial for the subculture maintenance of callus.

5. Conclusions

In summary, our comprehensive analysis of transcriptomic and physiological data has illuminated the intricate interplay between plant hormone metabolisms and signals, sugar metabolism, and stress responses in the proliferation and differentiation of callus. These findings underscore the significance of selecting an optimal carbon source for tissue culture to enhance the growth and development of plant cells. Furthermore, our results provide a valuable foundation for a deeper understanding of the mechanisms underlying SE and organogenesis in A. praecox. Notably, our study indicates that sucrose promotes the proliferation and differentiation of callus in A. praecox by maintaining an appropriate level of ROS intensity (Figure 8A). The results also provide a theoretical basis for plant tissue culture in other ornamental and crop plants. The regulatory mechanisms related to the modulation of plant hormone and ROS metabolism can optimize the in vitro culture conditions of other plants.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae10121350/s1, Figure S1: Effects of exogenous carbon sources on callus differentiation direction; Figure S2: Effects of exogenous carbon sources on callus proliferation and differentiation; Figure S3: Effects of different osmotic regulatory substance on the proliferation of callus; Figure S4: Effect of PEG 6000 on physiological indexes related to oxidative stress; Figure S5: The osmotic regulatory characteristics of carbon sources and PEG; Table S1: Basic information for transcriptome sequencing; Table S2: Genes and expression levels in hierarchical clustering analyses.

Author Contributions

Conceptualization, J.Y. and Y.Z.; methodology, Y.D.; software, C.D.; validation, J.Y., Y.D. and Y.Z.; investigation, X.W.; resources, J.Y. and Y.Z.; data curation, C.L.; writing—original draft preparation, J.Y.; writing—review and editing, J.Y.; visualization, J.Y.; supervision, Y.Z.; project administration, J.Y.; funding acquisition, J.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Project of Science and Technology in Henan Province (232102110184), the Key Research Project of Higher Education Institutions in Henan Province (23B180005), the Academic Core Teachers Program in Xinyang Agriculture and Forestry University (2022), the Young Key Teachers Training Program in Xinyang Agriculture and Forestry University (2021), the Program for Innovative Research Team of Horticultural Plant Resources and Utilization in Xinyang Agriculture and Forestry University (XNKJTD-012), and the Program of Research Fund for Young Teachers in Xinyang Agriculture and Forestry University (QN2022016).

Data Availability Statement

The data that support the findings of this study are openly available at https://www.ncbi.nlm.nih.gov/ (accessed on 13 December 2024) with accession number PRJNA800607.

Acknowledgments

We are grateful to Mengfan Liu and Man Zuo for the experiments of osmotic regulatory characteristics.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Morphological and transcriptomic differences of callus cultured by sucrose, glucose, and maltose. (A) Morphology of samples including callus cultured by sucrose, glucose, and maltose; the bar represents 1.0 cm. The white arrow indicates an adventitious bud and a hairy root. (B) Cell micromorphology of callus, bar = 100 μm. (C) Correlation analysis among samples; the horizontal axis represents the sample clusters, and colors from blue to red indicate the correlation index from low to high. (D) Venn diagram of DEGs among three compared pairs, including Suc/Mal, Suc/Glu, and Glu/Mal. The red arrows indicate upregulation, and the green arrows indicate downregulation. (E) KEGG pathway enrichment of the comparison of Suc/Glu. The bubble size represents the number of members detected in the KEGG pathway, and the color of the bubble represents the p-value, the same as below. (F) KEGG pathway enrichment of the comparison of Suc/Mal. (G) KEGG pathway enrichment of the comparison of Glu/Mal.

Figure 2. Hierarchical clustering analyses of DEGs among samples of sucrose, glucose, maltose, and IEC. (A) Hierarchical clustering analyses of DEGs between samples including Suc, Glu, Mal, and IEC. (B) Hierarchical clustering analyses of DEGs in subcluster 1. (C) Hierarchical clustering analyses of DEGs in subcluster 2. (D) Hierarchical clustering analyses of DEGs in subcluster 3. (E) Hierarchical clustering analyses of DEGs in subcluster 4.

Figure 3. Differential analyses of plant hormone signal transduction and metabolism. (A) Analyses of the contents and enzymatic activity of plant hormones. The data are means, n = 3. Means marked by the same letter in the column are not significantly different according to Duncan’s multiple range test at p < 0.05. Table marked in red, yellow, and green indicating high, middle, and low values with different carbon source treatments. (B) DEGs with higher expression levels with sucrose. (C) DEGs with lower expression levels with sucrose. (D) Hierarchical clustering analyses of DEGs. (E) Size of callus treated by PIC, n = 3. Means marked by the same letter on the bar are not significantly different according to Duncan’s multiple range test at p < 0.05, and the same hereinafter. (F) Size of callus treated by GA₄. (G) Size of callus treated by homobrassinolide (HBL). (H) Size of callus treated by ABA. Abbreviations: BIN2: brassinosteroid insensitive2; TGA: TGACG binding TFs; IAA: auxin/indole-3-acetic acid; PP2C: type 2C protein phosphatases; JAZ: jasmonate ZIM domain protein; SAUR: small auxin-up RNA; ARR-A: type-A authentic response regulator; ARR-B: type-B authentic response regulator; NPR1: nonexpressor of pathogenesis-related genes 1; BZR1_2: brassinosteroid-resistant 1/2; AHP: histidine-containing phosphotransfer protein; PIF3: phytochrome-interacting factor 3; SNRK2: sucrose nonfermenting 1-related protein kinase 2; EIN3: ethylene-insensitive protein 3; TIR1: transport inhibitor response 1; TCH4: xyloglucan: xyloglucosyl transferase TOUCH4; BSK: BR-signaling kinase; PIF4: phytochrome-interacting factor 4; GH3: Gretchen Hagen 3; AHK2_3_4: Arabidopsis histidine kinase 2/3/4 (cytokinin receptor); PR1: pathogenesis-related protein 1; DELLA: DELLA transcriptional regulatory proteins; ABF: ABA responsive element binding factor; CTR1: constitutive triple response1; AUX1, LAX: auxin influx carrier (AUX1/LAX family); ARF: auxin response factor; CYCD3: cyclin D3; PYL: pyrabactin resistance 1-like protein; GID1: gibberellin insensitive dwarf1; MPK6: mitogen-activated protein kinase 6; BRI1: brassinosteroid insensitive 1.

Figure 4. Differential analyses of starch and sucrose metabolism. (A) Analyses of the contents of starch and soluble sugars. The data are means, n = 3. Means marked by the same letter in the column are not significantly different according to Duncan’s multiple range test at p < 0.05. Table marked in red, yellow, and green indicates high, middle, and low values with different carbon source treatments. (B) DEGs with higher expression levels in Suc/Glu and Suc/Mal. (C) DEGs with lower expression levels in Suc/Glu and higher expression levels in Glu/Mal. (D) DEGs with lower expression levels in Suc/Glu and higher expression levels in Glu/Mal. (E) Hierarchical clustering analyses of DEGs involved in starch and sucrose metabolism. (F) DEGs with lower expression levels in Suc/Glu and Suc/Mal. (G) Size of callus treated by different concentrations of sucrose, n = 3. Means marked by the same letter on the bar are not significantly different according to Duncan’s multiple range test at p < 0.05, and the same hereinafter. (H) Size of callus treated by the combination of sucrose and glucose. (I) Size of callus treated by the combination of sucrose and fructose. (J) Size of callus treated by the combination of sucrose and maltose. Abbreviations: GBE1, glgB: 1,4-alpha-glucan branching enzyme; SUS: sucrose synthase; glgC: glucose-1-phosphate adenylyltransferase; INV, sacA: beta-fructofuranosidase; PYG, glgP: glycogen phosphorylase; scrK: fructokinase; malZ: alpha-glucosidase; TREH, treA, treF: alpha-trehalase; TPS: trehalose 6-phosphate synthase/phosphatase; ISA, treX: isoamylase; otsB: trehalose 6-phosphate phosphatase; GPl, pgi: glucose-6-phosphate isomerase; ENPP1_3, CD203: ectonucleotide pyrophosphatase/phosphodiesterase family member 1/3; malQ: 4-alpha-glucanotransferase; UGP2, galU, galF: UTP--glucose-1-phosphate uridylyltransferase; SPP: sucrose-6-phosphatase; HK: hexokinase; NV, sacA: beta-fructofuranosidase; TREH, treA, treF: alpha,alpha-trehalase.

Figure 5. Differential analyses of MAPK signaling pathway. (A) Analyses of the contents and enzymatic activity involved in ROS metabolism. The data are means, n = 3. Means marked by the same letter in the column are not significantly different according to Duncan’s multiple range test at p < 0.05. Table marked in red, yellow, and green indicates high, middle, and low values with different carbon source treatments. (B) DEGs with higher expression levels in Suc/Glu and Suc/Mal. (C) DEGs with higher expression levels in Suc/Glu and lower expression levels in Glu/Mal. (D) DEGs with lower expression levels in Suc/Glu and higher expression levels in Glu/Mal. (E) DEGs with lower expression levels in Suc/Glu and Suc/Mal. (F) Size of callus treated by H₂O₂, n = 3. Means marked by the same letter on the bar are not significantly different according to Duncan’s multiple range test at p < 0.05, and the same hereinafter. (G) Size of callus treated by 2, 4-D. (H) Size of callus treated by PEG 6000. (I) Size of callus treated by Ac-DEVD-CHO (CHO) and carbonyl cyanide m-chlorophenylhydrazone (CCCP). Abbreviations: MAPK7: mitogen-activated protein kinase 7; RBOH: respiratory burst oxidase; IRAK1: interleukin-1 receptor-associated kinase 1; CALM: calmodulin; WRKY33: WRKY DNA-binding protein 33; CTSL: cathepsin L; IDH1, IDH2, icd: isocitrate dehydrogenase; CYC: cytochrome c; PR1: pathogenesis-related protein 1; ACsL, fadD: long-chain acyl-CoA synthetase; CTSH: cathepsin H; PEX12, PAF3: peroxin-12; HAO: (S)-2-hydroxy-acid oxidase; VIP1: transcription factor VIP1; ACAA1: acetyl-CoA acyltransferase 1; PXMP2, PMP22: peroxisomal membrane protein 2; PEX10: peroxin-10; FLS2: LRR receptor-like serine/threonine-protein kinase FLS2; EIN3: ethylene-insensitive protein 3; FBXL2_20: F-box and leucine-rich repeat protein 2/20; PP2C: type 2C protein phosphatases; PYL: abscisic acid receptor PYR/PYL family; NRK2: sucrose nonfermenting 1-related protein kinase 2; ANP1: mannan polymerase II complex ANP1 subunit; copA, ATP7: P-type Cu+ transporter; katE, CAT, catB, srpA: catalase; MKK9: mitogen-activated protein kinase kinase 9; PARP: poly (ADP-ribose) polymerases; ATF4, CREB2: cyclic AMP-dependent transcription factor ATF-4; EIF2S1: translation initiation factor 2 subunit 1.

Figure 6. Analyses of the effects of carbon source combination on the proliferation and differentiation of callus. (A) Morphological differences of callus treated by the combination of sucrose and the hydrolysate of sucrose (glucose and fructose). The bars in the morphology and micromorphology represent 1.0 cm and 100 μm, respectively, and the same hereinafter. (B) Size of callus (left Y-axis) and single cell (right Y-axis) treated by the combination of sucrose and hydrolysate of sucrose. Data on the bars marked without the same lowercase letter indicate significant differences at p < 0.05, n = 3, and the same hereinafter. (C) Morphological differences of callus treated by the combination of sucrose and glucose. (D) Size of callus (left Y-axis) and single cell (right Y-axis) treated by the combination of sucrose and glucose. (E) Morphological differences of callus treated by the combination of sucrose and fructose. (F) Size of callus (left Y-axis) and single cell (right Y-axis) treated by the combination of sucrose and fructose. (G) Morphological differences of callus treated by the combination of hydrolysate of sucrose and glucose. (H) Size of callus (left Y-axis) and single cell (right Y-axis) treated by the combination of hydrolysate of sucrose and glucose. (I) Morphological differences of callus treated by the combination of hydrolysate of sucrose and fructose. (J) Size of callus (left Y-axis) and single cell (right Y-axis) treated by the combination of hydrolysate of sucrose and fructose. (K) Morphological differences of callus treated by the combination of hydrolysate of sucrose and maltose. (L) Size of callus (left Y-axis) and single cell (right Y-axis) treated by the combination of hydrolysate of sucrose and maltose.

Figure 7. Analyses of the effects of osmotic regulatory substance (ORS) on the proliferation and differentiation of callus. (A) Morphological differences of callus treated by the combination of sucrose and PEG. The bars in the morphology and micromorphology represent 1.0 cm and 100 μm, respectively, and the same hereinafter. (B) Size of callus (left Y-axis) and single cell (right Y-axis) treated by the combination of sucrose and PEG. Data on the bars marked without the same lowercase letter indicate significant differences at p < 0.05, n = 3, and the same hereinafter. (C) Morphological differences of callus treated by the combination of glucose and PEG. (D) Size of callus (left Y-axis) and single cell (right Y-axis) treated by the combination of glucose and PEG. (E) Morphological differences of callus treated by the combination of fructose and PEG. (F) Size of callus (left Y-axis) and single cell (right Y-axis) treated by the combination of fructose and PEG. (G) Morphological differences of callus treated by the combination of glucose, fructose, and PEG. (H) Size of callus (left Y-axis) and single cell (right Y-axis) treated by the combination of glucose, fructose, and PEG. (I) Morphological differences of callus treated by the combination of sucrose, glucose, and PEG. (J) Size of callus (left Y-axis) and single cell (right Y-axis) treated by the combination of sucrose, glucose, and PEG. (K) Morphological differences of callus treated by the combination of sucrose, fructose, and PEG. (L) Size of callus (left Y-axis) and single cell (right Y-axis) treated by the combination of sucrose, fructose, and PEG.

Figure 8. Hypothesized model diagram of the acquisition of regenerative potential induced by carbon sources in A. praecox. (A) Carbon sources affected the proliferation and differentiation of callus, and the intensity of ROS determined the cell fate of callus. (B) Schematic diagram about the influence of carbon sources on hormone metabolism, sugar content, ROS, and protective enzymes. Since maltose treatment usually results in moderate levels of physiological indicators, maltose is used as a control. The short horizontal line indicate control, the red arrows indicate a significant increase, and the green arrows a indicate significant decrease.

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MDPI and ACS Style

Yue, J.; Dong, Y.; Du, C.; Li, C.; Wang, X.; Zhang, Y. Sucrose Promotes the Proliferation and Differentiation of Callus by Regulating ROS Intensity in Agapanthus praecox. Horticulturae 2024, 10, 1350. https://doi.org/10.3390/horticulturae10121350

AMA Style

Yue J, Dong Y, Du C, Li C, Wang X, Zhang Y. Sucrose Promotes the Proliferation and Differentiation of Callus by Regulating ROS Intensity in Agapanthus praecox. Horticulturae. 2024; 10(12):1350. https://doi.org/10.3390/horticulturae10121350

Chicago/Turabian Style

Yue, Jianhua, Yan Dong, Changmei Du, Chaoxin Li, Xinyi Wang, and Yan Zhang. 2024. "Sucrose Promotes the Proliferation and Differentiation of Callus by Regulating ROS Intensity in Agapanthus praecox" Horticulturae 10, no. 12: 1350. https://doi.org/10.3390/horticulturae10121350

APA Style

Yue, J., Dong, Y., Du, C., Li, C., Wang, X., & Zhang, Y. (2024). Sucrose Promotes the Proliferation and Differentiation of Callus by Regulating ROS Intensity in Agapanthus praecox. Horticulturae, 10(12), 1350. https://doi.org/10.3390/horticulturae10121350

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