Anatomical and Transcriptomic Analyses Revealed the Key Genes Associated with Tuber Expansion in Cyperus esculentus L

TL;DR

This study identifies key genes involved in tuber expansion in Cyperus esculentus, a tuber oil crop, using transcriptomic and anatomical analyses.

Contribution

The study identifies 822 differentially expressed genes and 57 transcription factors linked to tuber expansion in Cyperus esculentus.

Findings

Tuber expansion in Cyperus esculentus is primarily driven by cell expansion rather than cell number.

Five genes encoding expansin proteins are significantly associated with cell wall loosening during tuber expansion.

822 differentially expressed genes and 57 transcription factors were identified as key regulators of tuber expansion.

Abstract

Background: Cyperus esculentus L. is a unique tuber oil crop, in which tuber size directly determines both yield and oil storage capacity. It is crucial to clarify the tuber expansion pattern and explore the key genes associated with tuber expansion in Cyperus esculentus for crop improvement. Methods: This study conducted comprehensive morphological and cytological observations as well as transcriptomic analysis of tubers at multiple developmental stages. Tubers at 1, 5, 10, and 15 d were collected for transcriptome sequencing to identify differentially expressed genes (DEGs) and differentially expressed transcription factors. Gene ontology (GO) enrichment analysis was used to determine key functional categories. RT-qPCR was employed to verify the expression patterns of key genes. Results: Cyperus esculentus tubers expanded rapidly from 1 d to 15 d after initial tuber formation, and the expansion rate exhibited a trend of increasing first (1~5 d) and then decreasing (5~15 d). Cell expansion, rather than number, mainly contributed to tuber expansion. By combining the analysis of differential expression and the variation pattern of tuber expansion rate, 822 DEGs were identified to be associated with tuber expansion. GO enrichment analysis revealed that 20 genes were significantly enriched in GO:0043231 (cell wall), especially five remarkable genes encoding expansin, which exercise the function of cell wall loosening and have been proven to be associated with cell expansion. In addition, 57 differentially expressed TFs were further identified to be associated with tuber expansion. Conclusions: This study revealed the tuber expansion pattern of Cyperus esculentus and identified several key genes and TFs, which will facilitate the construction of the regulatory network and the analysis of the mechanism of tuber expansion in Cyperus esculentus.

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1. Introduction

Cyperus esculentus is a C4 plant belonging to the Cyperaceae family. It is widely distributed in tropical, temperate, and colder zones due to its drought resistance, salt tolerance, and barren tolerance [1,2]. As a new characteristic oil crop, Cyperus esculentus is propagated through underground tubers, with high yield and rich nutritional value. The oil content is as high as 20~30%. It also contains 4.2% protein, 32.7% starch, 6.9% sugar, and 8.26–15.47% dietary fiber [3,4]. As the vegetative reproductive organ and main economic organ of Cyperus esculentus, the tuber develops from an underground stem leaf bud to a stolon, and then the top of the stolon expands and develops. Compared with potato, sweet potato, and other tuber crops, Cyperus esculentus tuber is unique for its small size and short expansion cycle. Tuber size directly determines its yield and oil storage capacity as an oil crop. Therefore, it is of direct and important breeding application value to analyze the expansion mechanism of the Cyperus esculentus tuber.

The mechanism of plant organ size control is a long-standing biological problem. At the cellular level, the two main potential factors that determine plant organ size are cell number and cell size, and changing either of them may affect plant organ size [5]. Cell number is determined by the rate and duration of cell division, regulated by a series of enzymes involved in DNA replication and mitosis in the nucleus, while cell size is determined by cell wall loosening and de novo synthesis of cell wall components [6]. At present, the research on plant seed size is the most extensive and in-depth, and the signal pathways known to control seed size are mainly the IKU pathway, DA1 ubiquitin–proteasome pathway, G protein signaling pathway, and mitogen-activated protein kinase (MAPK) signaling pathway, plant hormones, and transcriptional regulators. Arabidopsis thaliana, iku1, iku2, mini3, and shb1 mutants all exhibit reduced seed size phenotypes; MINI3 controls seed size by recruiting SHB1 to form a complex, and the MINI3-SHB1 complex binds to IKU2 and MINI3 promoter regions and induces their transcription, thereby promoting endosperm cell development [7,8,9]. The ubiquitin–proteasome pathway is composed of key enzymatic components, including the ubiquitin receptor protein DA1, E3 ubiquitin ligases DA2, and EOD1/BB (enhancer of DA1/BIG BROTHER), as well as deubiquitinating enzymes UBP15 (ubiquitin-specific protease 15) and SOD2 (SUPPRESSOR2 of DA1), and E3 ubiquitin ligases DA2 and EOD1/BB interact with DA1 to further ubiquitinate DA1 and activate the protease activity of DA1, thereby cleaving downstream substrates, such as UBP15, and controlling seed size [10,11]. G protein signal pathway plays a molecular switch role in signal transduction. Heterotrimeric G protein complex consists of Gα, Gβ, and Gγ subunits [12]. Overexpression of Arabidopsis Gγ protein (AGG3) can promote seed growth by promoting cell proliferation, while the AGG3 function loss mutation leads to seed size reduction [13]. In addition, hormones have been found to play a crucial role in the growth and expansion of tubers and roots [14]. For example, GA at low concentrations promotes tuber formation in potatoes, while GA at high concentrations inhibits tuber development [15]. External application of cytokinins (CK) promotes tuber expansion by promoting cell division and inhibiting cell elongation [16]. Overexpression of potato StJAZ1-like inhibits tuber formation in potatoes [17]. However, there are few reports on the regulatory network and related genes of tuber expansion in tubers.

In fact, the expansion of plant organs (such as seeds, fruits, and tubers) fundamentally results from coordinated cellular proliferation and expansion, which phenotypically manifests as progressive increases in length and width. Therefore, the relative expression level of genes regulating organ size is closely related to the growth rate of organ length and width. In view of this, this study comprehensively analyzed the tuber expansion pattern by combining phenotypic and anatomical observations, and then explored the key genes associated with tuber expansion by comparative transcriptomic analysis of tubers at multiple stages. Our findings would provide basic data for analyzing the molecular mechanism of tuber expansion and a theoretical basis for further cultivation of high-yielding Cyperus esculentus.

2. Materials and Methods

2.1. Plant Materials

The experimental material used in this study was Cyperus esculentus variety “Yuyousha 2”, which was purified and identified by Dr. Li Chunxin (Industrial Crops Research Institute of the Henan Academy of Agricultural Sciences) from the mixed local landraces collected from Minquan County, Shangqiu City, Henan Province (Collection ID: H0002). The “Yuyousha 2” variety was stored in the Henan Provincial Center for Crop Germplasm Resources Protection and Utilization (Pingyuan New District, Xinxiang City, Henan Province; Storage Number: IIA0300002). Access to experimental materials is permissible under the relevant institutional agreements.

2.2. Dynamic Observation on the Growth and Development of Cyperus esculentus Tubers

Uniform tubers of Cyperus esculentus were selected and sterilized with 10% sodium hypochlorite for 5 min, and then germinated in 72-cell trays. After about 10 days of germination, the seedlings with consistent growth were selected and transplanted into a double-layer sand culture device (inner-layer was transparent) for pot cultivation. By observing the transparent inner-layer, the initially formed tubers (about 3 mm) were marked as developing 1 d tubers. With the growth and development, tubers at different developmental stages (including 1d, 5 d, 10 d, 15 d, 20 d, 30 d, 40 d, and 50 d) were monitored. Tuber dimensions were measured as length (parallel to stolon axis) and width (perpendicular to stolon). Considering the small size of tubers, 10 tubers were selected for measurement at each stage to reduce the measurement error and ensure the accuracy of phenotypic values. The phenotypic values of each index were the average values of 10 tubers. All plants were grown in controlled chambers at Henan Academy of Agricultural Sciences under 14 h light (30 °C)/10 h dark (30 °C) cycles.

2.3. Paraffin Section Observation

The tubers in the period of rapid expansion (1 d, 5 d, 10 d, and 15 d) were selected for cytological observation, with an individual tuber per period. The entire central part of the tuber was cut into small pieces (2 mm thick) horizontally. The small piece was fixed in formaldehyde–acetic acid–ethanol (FAA) solution (50% ethanol: formaldehyde: acetic acid, 9: 1: 1), and then prepared into a paraffin section with Safranin O and Fast Green staining, which could show the cell wall contour and clearly observe the cell size and number. Through a slice panoramic scanner, the entire central transverse section of the tubers was photographed. The cell morphology was observed by using the CaseViewer 2.4 software. The total area of the transverse section was measured by the “Annotations” option of the CaseViewer 2.4 software. Moreover, three local areas of the same size were marked by a circle in the transverse section, and their cell numbers were counted. Avoid the central area (small cells), and select the upper, middle, and lower three local areas as a representative. Based on this, the total cell numbers and the average areas of a single cell were estimated for the transverse section.

2.4. RNA Extraction and Sequencing

Fresh tubers of Cyperus esculentus at different developmental stages (1 d, 5 d, 10 d, and 15 d) were collected, wrapped in aluminum foil, and immediately frozen in liquid nitrogen, with three replicates per treatment. Total RNA was extracted by using the RNAprep Pure Plant Kit (TIANGEN, Beijing, China), following the manufacturer’s protocol. RNA quality and concentration were assessed using NanoDrop 2000, while integrity was verified by agarose gel electrophoresis. Qualified RNA samples were submitted to Biomarker Technologies (Beijing, China) for transcriptome sequencing. Eukaryotic mRNA was enriched by using Oligo(dT) magnetic beads, then fragmented into approximately 300 bp segments via ion disruption. The fragmented mRNA was reverse transcribed into cDNA, and sequencing libraries were prepared through adapter ligation and PCR amplification. Finally, paired-end sequencing (150 bp) was performed on the Illumina NovaSeq 6000 platform (San Diego, CA, USA), generating approximately 6 Gb of raw data per sample.

2.5. Transcriptome Gene Identification and Functional Annotation

Raw reads were processed using Trimmomatic 0.36 [18] to remove low-quality sequences and adapter-contaminated reads at the 3′ end, yielding high-quality clean reads. The Q20 and Q30 values of clean reads were calculated to evaluate sequencing quality. Clean reads from each sample were then aligned to the Cyperus esculentus reference genome (https://db.cngb.org/ (accessed on 22 December 2021), CNP0003839) [19] using Hisat2 2.0.1 [20] software to obtain mapped reads, with the mapped ratio being subsequently calculated. Subsequently, Stringtie v3.0.0 [21] software was employed for genome-wide gene identification and quantification analysis of the mapped reads, where gene expression levels were represented by FPKM values (fragments per kilobase of exon model per million mapped fragments). Based on the gene expression matrix of all samples, correlation analysis and principal component analysis (PCA) were performed to assess the suitability of the sequencing data. Functional annotation of the identified genes was conducted through the Biomarker Cloud Platform (https://international.biocloud.net/ (accessed on 16 March 2025)), with GO (gene ontology), NR (non-redundant protein sequence), and Swiss-Prot databases.

2.6. Differential Expression Analysis

Differential expression analysis was performed by the edgeR R package 2.11 between two treatments [22]. The screening criteria for significantly differentially expressed genes (DEGs) were established as follows: genes with fold changes (FPKM) ≥ 2 and p-value < 0.05. Combined with the trend of tuber expansion, the DEGs associated with tuber expansion were further screened out.

2.7. GO Enrichment Analysis

The GO enrichment analysis of tuber expansion-associated DEGs was performed based on cellular component (CC) through the STRING online platform (https://string-db.org/ (accessed on 25 June 2025)), with significantly enriched GO terms (Fisher’s exact test, p-value < 0.05) being identified. The results were subsequently visualized through the Bioinformatics Cloud Platform (https://www.bioinformatics.com.cn/ (accessed on 28 June 2025)). Then, the DEGs involved in these significantly enriched GO terms were picked out for further analysis.

2.8. Predictive Analysis of TFs

Through the PlantTFDB website (http://planttfdb.gao-lab.org/prediction.php (accessed on 8 July 2025)), TFs were predicted from the Cyperus esculentus protein sequences based on the family assignment rules and thresholds determined by established methods (see details in the website). Furthermore, the interested TFs would be extracted from the tuber expansion-associated DEGs.

2.9. RT-qPCR Analysis

Gene-specific primers for RT-qPCR (Table S1) were designed using Primer 5.0 software. Reactions were performed in a 20 μL system containing SYBR Green PCR Master Mix (Vazyme, Nanjing, China) on a Roche LightCycler 480 real-time PCR system (Basel, Switzerland) using cDNA templates prepared from tubers at four developmental stages (1 d, 5 d, 10 d, and 15 d). The CeUCE2 (CESC_21309) served as the internal control for Cyperus esculentus [23]. Three biological replicates and three technical replicates were included for each analysis. Relative expression levels of these genes were calculated using the 2^−ΔΔCt^ method. Student’s test was used to assess the significance of differences in gene expression levels.

3. Results

3.1. Analysis of Tuber Expansion Pattern in Cyperus esculentus

From the initial expansion of tubers (marked as 1 d), the morphology of Cyperus esculentus tubers at different developmental stages was observed continuously. The results showed that the expansion rate of tubers was very fast, but the duration was short (Figure 1). From 1 d to 15 d, the length and width of tubers increased rapidly, and the color of the epidermis gradually deepened (Figure 1A). After 15 d, the tuber size (length and width) tended to be stable, and the morphology was almost indistinguishable (Figure 1A,B). In addition, during the tuber development from 1 d to 15 d, the growth rate of tuber length and width showed a trend of increasing first (15 d) and then decreasing (515 d) (Figure 1C,D). That is, since initial tuber formation, Cyperus esculentus tubers expanded rapidly in a short period of time, and then the expansion rate gradually decreased and the size gradually stabilized after 15 d.

3.2. Cytological Observation of Tubers at Different Developmental Stages in Cyperus esculentus

The size of plant organs is determined by the number and size of cells. To explore the changes in cell number and size in the process of tuber expansion, the cell morphology of tubers from 1 d to 15 d was observed by paraffin section. Through a slice panoramic scanner, the entire central transverse section of the tubers from 1 d to 15 d was observed and presented, and the total area of the transverse section was calculated (Figure 2A). In the fixed local area (304,557.6 μm^2^), the average cell numbers of the tubers from 1 d to 15 d were 465.33, 118.67, 84.33, and 72.67, respectively (Figure 2A,B; Table S2). Correspondingly, the total cell numbers of the entire central transverse section of the tubers from 1 d to 15 d were 14,515.04, 14,884.10, 14,869.76, and 15,007.78, respectively (Figure 2C; Table S2); meanwhile, the average areas of single cells were 654.66, 2569.16, 3612.14, and 4193.43 μm^2^, respectively (Figure 2D; Table S2). The total cell numbers of tubers from 1 d to 15 d appeared to be similar, but the cell sizes showed dramatic changes. Furthermore, the growth rate of the single cell area showed a trend of increasing first and then decreasing (Figure 2E), which is consistent with the trend of tuber expansion rate. These data suggested that cell expansion may be the main driver of tuber expansion from 1 d to 15 d.

3.3. Transcriptome Sequencing of Tubers at Different Developmental Stages in Cyperus esculentus

Transcriptome sequencing was performed on tubers of 1 d, 5 d, 10 d, and 15 d in Cyperus esculentus. The clean reads and clean bases of all samples met the requirements, and the base quality Q20 and Q30 values were higher than 93% (Table S3). Based on the Cyperus esculentus genome, the mapped ratio of the samples is very high, ranging from 89.42% to 91.02% (Table S3). Furthermore, the correlation analysis showed that the correlation coefficient of gene expression levels among biological replicates is extremely high (Figure 3A; Table S4). The PCA showed that the expression levels among biological replicates at the same developmental stage are grouped together, while the expression levels among different developmental stages are dispersed (Figure 3B). Overall, the sequencing data are highly reproducible and realistic, and can be used for further in-depth analysis.

3.4. Screening of DEGs Associated with Tuber Expansion

Based on the above tuber expansion pattern, the differential expression analysis was carried out in three groups (5 d vs. 1 d, 5 d vs. 10 d, and 5 d vs. 15 d) with the 5 d tuber (the highest expansion rate) as the hub. A total of 4408, 4858, and 5624 DEGs were screened in the three groups, respectively (Figure 4; Table S5). By Venn analysis, 1672 overlapped DEGs were identified among the three groups (Figure 4A). Considering the trend of tuber expansion rate, 822 DEGs were screened out for their appropriate expression trends, which showed a positive pattern of increasing first and then decreasing, or a negative pattern of decreasing first and then increasing (Figure 4B). These DEGs should probably be associated with tuber expansion.

3.5. GO Enrichment Analysis of DEGs Associated with Tuber Expansion

To clarify the crucial GO terms related to tuber expansion, these DEGs associated with tuber expansion were selected to perform the GO enrichment analysis. The result revealed that 14 GO terms were significantly enriched (Table S6; Figure 5A), including GO:0110165 (cellular anatomical entity), GO:0005622 (intracellular anatomical structure), GO:0043229 (intracellular organelle), GO:0043231 (intracellular membrane-bounded organelle), GO:0071944 (cell periphery), GO:0005618 (cell wall), and so on. As is well known, plant cells are surrounded by cell walls, so cell expansion is closely related to cell wall loosening. From this perspective, GO:0005618 (cell wall) was the key GO term related to tuber expansion. In this GO term, a total of 20 genes were included (Table S6; Figure 5B). Among them, there were five remarkable genes (CESC_04660, CESC_21910, CESC_13871, CESC_06110, and CESC_23082), which encode expansin (EXP), exercising the function of cell wall loosening. In addition, several glycosylhydrolase genes involved in the modification of cell wall polysaccharides (CESC_07699, CESC_09851, and CESC_18627) were identified, and these enzymes may affect cell wall extensibility by regulating hemicellulose metabolism [24]. And pectin acetylesterase (CESC_14765), which is involved in the synthesis and modification of pectin components of a cell wall, affects cell wall extensibility by affecting the remodeling and physicochemical form of cell wall polysaccharides [25]. Moreover, their expression patterns were positively correlated with the trend of tuber expansion rate, suggesting that they should play a role in cell wall loosening and then regulate tuber expansion of Cyperus esculentus.

3.6. Identification of Transcription Factors Associated with Tuber Expansion

Transcription factors (TFs) play crucial roles in plant growth and development by directly regulating the expression of some key genes. Through transcription factor prediction analysis, a total of 683 TFs belonging to multiple families were identified (Table S7), such as MYB, bHLH, and NAC families. Among them, 57 differentially expressed TFs were identified, which exhibited special expression patterns suitable for the change in tuber expansion rate (Figure 6; Table S7). The MYB TF family was the most abundant, with seven genes identified, followed by five genes each from the bHLH, NAC, and ERF families. The other genes were identified from the MADS, LBD, ARF, TALE, AP2, and other families. It is widely known that MYB, NAC, MADS, and other transcription factors play an important role in xylem and phloem cell differentiation [26], and specifically, members of the NAC family and the MYB family have been identified as the primary and secondary master switches of lignin biosynthesis [27]. Therefore, these screened TFs might be important regulatory factors involved in the tuber expansion of Cyperus esculentus.

3.7. RT-qPCR Validation of Key Genes Involved in Tuber Expansion

In order to verify the accuracy of RNA-seq data, a total of eight key genes associated with tuber expansion, including five EXPs (CESC_04660, CESC_21910, CESC_13871, CESC_06110, and CESC_23082) and three TFs (CESC_18928, CESC_13541, and CESC_20022), were selected for examining their expression patterns by RT-qPCR. The CeUCE2 gene was used as an internal reference gene for RT-qPCR analysis. The result showed that the expression levels of the eight genes increase first and then decrease (Figure 7), which is highly consistent with RNA-seq data and the trend of tuber expansion rate, confirming the reproducibility of the RNA-seq data.

4. Discussion

Cyperus esculentus is a unique tuber crop. The formation of tubers originates from the expansion of the top of the stolon, similar to that of a potato [28]. However, the tuber size of Cyperus esculentus (length: about 1 cm; width: about 1 cm) is much smaller than that of the potato (length: about 10 cm; width: about 310 cm). To find the growth pattern of Cyperus esculentus tubers is the key to analyzing their tuber expansion mechanism. This study compared tuber sizes of Cyperus esculentus from 1 d to 50 d after tuber initiation and found that its tubers expand rapidly from 1 d to 15 d, with a relatively short duration. Moreover, the growth rate of tuber size reaches the peak at about 5 d. In contrast, the expansion of potato tubers lasts for a longer period [29,30], which may lead to the formation of large tubers. In addition, cytological observation showed that the increase in cell size is the main driving force for the expansion of Cyperus esculentus tubers from 1 d to 15 d. Notably, the average diameter of a single cell in Cyperus esculentus tubers (15 d) is about 75 μm (area: 4193.43 μm^2^), which is much smaller than that of a potato (usually in the range of 100300 μm). Therefore, the expansion of Cyperus esculentus tubers is more driven by the expansion of cells and has great potential for genetic improvement.

Plant cell expansion is a very complex biological event, which is precisely regulated by a series of genes [31,32]. In this study, during the rapid expansion period of tubers (from 1 d to 15 d), transcriptome sequencing was used to identify the key genes associated with tuber expansion. Based on the analysis of differential expression and the variation pattern of tuber expansion rate, 822 DEGs were considered to be related to tuber expansion. Furthermore, GO enrichment analysis revealed that these DEGs were significantly enriched in GO:0005618 (cell wall), which might be closely related to cell expansion. For plant cells with a cell wall, cell wall loosening can allow cell expansion [33,34]. In this GO term (cell wall), 20 genes were involved, particularly including five EXPs (CESC_04660, CESC_21910, CESC_13871, CESC_06110, and CESC_23082), which have been proven to be associated with cell expansion. EXP facilitates cell wall loosening by breaking hydrogen bonds between cell wall polysaccharides and promotes cell expansion [35]. In addition, some other genes (CESC_07699, CESC_09851, and CESC_18627) encode a class of glycosyl hydrolases involved in cell wall modification, and their expression levels may be related to hemicellulose metabolism, potentially influencing cell wall extensibility and thereby regulating cell size [24]. Therefore, these identified cell wall-related genes may play a significant role in tuber expansion in Cyperus esculentus. Although some of these genes have been shown to be involved in cell expansion or cell wall regulation in other species, their specific functions in Cyperus esculentus still need to be verified by subsequent experiments.

Gene expression is often regulated by TFs, and they work together to play a role in certain biological processes. In this study, 57 differentially expressed TFs showed a trend consistent with the tuber expansion rate, among which MYB, bHLH, and NAC family members were the most abundant. MYB and NAC TFs are widely known as key regulators of secondary cell wall (SCW) biosynthesis in plants [27]. In potato, silencing StMYB168 and StWRKY20 suppressed the expression of StPAL5 and StCAD14 and significantly reduced lignin accumulation in damaged potato tubers [36]. Additionally, MYB TFs can interact with other TFs to negatively regulate SCW biosynthesis. For example, in Arabidopsis, AtMYB6 physically interacts with the KNOX TF member KANT7, forming a complex that suppresses SCW development [37]. Beyond their role as master switches for SCW synthesis [38], NAC TFs are also involved in cell expansion. Silencing rose RhNAC100 significantly increases petal size and promotes the expansion of petal abaxial hypodermis cells, and thus promotes petal expansion [39]. In potato, StbHLH93 binds to the TIC56 promoter to regulate stolon swelling and tuber initiation during proplastid differentiation [40]. These findings suggested that these TFs may perform similar functions in Cyperus esculentus by regulating secondary cell wall biosynthesis, cell wall loosening, and cell expansion to modulate tuber expansion. Furthermore, the identification of hormone-responsive TFs, such as ARF and ERF, links tuber expansion to plant hormone signaling. For instance, an identified ARF homolog (CESC_20022) is a core component of the auxin signaling pathway. In tomato, SlARF9 regulates cell division/expansion during early fruit development via auxin signaling [41]. Arabidopsis AtERF19 activates MYB21/24 through auxin signaling to promote cell division/expansion of flower organs to increase flower organ size [42]. The above data indicated that the tuber expansion of Cyperus esculentus is regulated by multiple functional genes and TFs, and the results of this study can provide an important reference for the construction of the regulatory network of genes related to tuber expansion in Cyperus esculentus.

It should be noted that our conclusion regarding the predominance of cell expansion over cell proliferation is based on observations starting from 1 d after tuber initiation. The possibility that early cell division (before 1 d) contributes to tuber establishment cannot be ruled out. The deficiency of this study is that the genes that regulate the cell number are not identified, which is also an important factor affecting tuber expansion. This study found that the cell numbers in Cyperus esculentus tubers did not change significantly after 1 d, suggesting that the significant change in cell numbers in tubers may occur significantly before 1 d. Actually, tubers from initial formation to 1 d (marked as such) cannot be accurately obtained under sand culture conditions. In the future, it may be possible to explore the use of hydroponic methods to determine the process of initial tuber formation from stolons in Cyperus esculentus. During the initial period of tuber formation, transcriptomics analysis will reveal the genes that regulate the cell number and tuber expansion, which further contribute to the molecular mechanism analysis and genetic improvement of tuber expansion in Cyperus esculentus.

5. Conclusions

This study reveals that tuber expansion from 1 d to 15 d in Cyperus esculentus is primarily driven by cell expansion. Integrated morphological and transcriptomic analyses identified 822 DEGs associated with this process, including five expansin genes likely facilitating cell wall loosening, and 57 transcription factors (such as MYB, NAC, and bHLH) as potential regulators. These findings provide the cytological and molecular framework for understanding tuber growth in this species. While this work clarifies the post-initiation growth phase (after 1d), earlier regulatory events remain to be explored. Functional validation of candidate genes and network construction will be essential for future molecular breeding of high-yield varieties.