Genetic Evidence of SpGH9A3 in Leaf Morphology Variation of Spathiphyllum ‘Mojo’

Abstract

Leaves play a crucial role as ornamental organs in Spathiphyllum, exhibiting distinct differences across various Spathiphyllum varieties. Leaf development is intricately linked to processes of cell proliferation and expansion, with cell morphology often regulated by plant cell walls, primarily composed of cellulose. Alterations in cellulose content can impact cell morphology, subsequently influencing the overall shape of plant organs. Although cellulases have been shown to affect cellulose levels in plant cells, genetic evidence linking them to the regulation of leaf shape remains limited. This study took the leaves of Spathiphyllum ‘Mojo’ and its somatic variants as the research objects. We screened four cellulase gene family members from the transcriptome and then measured the leaf cellulose content, cellulase activity, and expression levels of cellulase-related genes. Correlation analysis pinpointed the gene SpGH9A3 as closely associated with leaf shape variations in the mutant. Green fluorescent fusion protein assays revealed that the SpGH9A3 protein was localized to the cell membrane. Notably, the expression of the SpGH9A3 gene in mutant leaves peaked during the early spread stage, resulting in smaller overall leaf size and reduced cellulose content upon overexpression in Arabidopsis.

1. Introduction

The shape and structure of leaves play a crucial role in plant growth, directly impacting photosynthetic efficiency, gas exchange rate, and overall growth conditions. Plant leaf growth and development are intricate processes influenced by numerous genetic factors [1]. Understanding how cell proliferation and expansion contribute to the formation of complex organs, and how these processes are coordinated in time and space during pattern formation, are key questions in developmental biology [2]. Key structural characteristics of plant leaves include leaf base, edge, tip, and veins. Researchers have conducted comprehensive studies on various aspects such as appearance characteristics, internal organization, and cell structure [3]. Genetic research on model plants such as Arabidopsis thaliana has significantly contributed to our comprehension of the intricate process of leaf development. The utilization of bioinformatics, modern molecular biology, omics analysis, and other methodologies has enhanced the understanding of molecules involved in plant leaf development and morphogenesis mechanisms.

The morphogenesis of plant organs is closely related to processes such as growth and development, cell proliferation, and cell expansion, and these processes are regulated by the cell wall. Cellulose is the main component of plant cell walls. It is synthesized in the Golgi apparatus by the synthase complex and transported to the cell membrane under the action of related proteins to complete the basic synthesis of cellulose. The cellulose metabolic process is precisely regulated by a series of synthetases, hydrolases, and modification enzymes [4]. The glycoside hydrolase 9 gene family encodes cellulases that target β-1,4-glucan polymerization by breaking endo-β-1,4 glycosidic bonds between glucose groups within the polysaccharide chain (cellulose). Cellulase activity and expression levels may regulate cellulose biosynthesis by affecting cell expansion and division, plant mechanical strength, resistance to pathogens, and other aspects, thereby changing the composition and function of plant cells [5]. KORRIGAN (KOR), belonging to the GH9A subclass, is believed to impact cellulose biosynthesis and/or deposition in plant cell walls [6]. KOR1 plays a crucial role in primary and secondary cell wall formation in Arabidopsis; suppressing KOR gene expression results in Arabidopsis mutants displaying dwarf phenotypes and cell wall abnormalities [7]. Upregulation of the PttCel9A1 gene can enhance the amorphous cellulose content in transformed Arabidopsis plants and notably decrease cellulose crystallinity [8]. Cellulases also impact cellulose content in the cell walls of plants such as Arabidopsis, rice, poplar, and tomato [8,9,10,11,12]. Mutations in the Arabidopsis endoglucanase gene can result in significant leaf and organ shrinkage, abnormal cell wall structure, and alterations in overall plant morphology [13]. While these studies offer valuable insights into how cell wall-related functional genes influence phenotype by affecting cell number or size, there is still a scarcity of relevant research in ornamental plants.

Spathiphyllum, a perennial plant from the Araceae family, is native to Central and South America and Southeast Asia. It is recognized for its various leaf shapes, including lanceolate, oval, spoon-shaped, and egg-shaped, with prominent veins, long petioles, and glossy green to dark green leaves, making it highly ornamental. By the early 20th century, Spathiphyllum had become popular as a potted ornamental plant, playing a significant role in the global indoor foliage plant market. The growing demand for diverse leaf characteristics and new Spathiphyllum varieties has led to research efforts in traditional breeding methods such as screening for distinct traits, hybridization, and polyploid mutagenesis [14]. Some studies have also explored breeding through somatic cell hybridization [15,16,17]. While molecular biology and genomic technologies have revolutionized modern plant breeding, their application in ornamental plants such as Spathiphyllum is still limited. Recent research has demonstrated that the use of SSR and core primers can effectively differentiate Spathiphyllum germplasm resources and accurately identify hybrids, thereby expediting the breeding process and improving reliability [18]. The discovery of numerous SSR and SNP sites through the screening of Spathiphyllum leaf transcriptome data has provided valuable insights [19]. However, there is still a lack of understanding regarding the gene regulation mechanisms that govern leaf growth and development in Spathiphyllum.

As a foliage plant, the leaf traits of Spathiphyllum are essential for breeding and cultivation. Changes in Spathiphyllum leaves mainly involve size, length, and thickness. Despite qualitative descriptions and variety analysis, limited research has focused on the formation and growth of Spathiphyllum leaves. In a recent study by Li et al. [20], researchers explored post-budding leaf growth, developed a growth model using the Logistic growth curve equation, and identified key time nodes and growth characteristics. By segmenting growth time based on this model, the research team conducted transcriptome sequencing of Spathiphyllum leaves at different stages [19]. Transcriptome analysis revealed significant differences in cellulase expression during leaf growth in various Spathiphyllum varieties. This raises the following questions: do cellulase-related genes influence the leaf morphology of Spathiphyllum? Furthermore, how do these genes regulate the morphology of Spathiphyllum leaves? This study integrated the results of cellulose content and cellulase activity in Spathiphyllum leaves across three expansion stages, identifying SpGH9A3 closely associated with leaf morphological development. Cellulase likely plays a vital role in leaf expansion by regulating processes such as cell wall formation, impacting cell division, and overall volume growth. Investigating the functions of the candidate gene provides valuable insights into the role of cellulases and related genes in the development of morphological differences in Spathiphyllum leaves. Ultimately, this research contributes to understanding the regulatory mechanism of Spathiphyllum leaf growth.

2. Materials and Methods

2.1. Plant Materials

Spathiphyllum ‘Mojo’-Ssm-1 is a somatic mutant variety of Spathiphyllum ‘Mojo’, derived from bud mutation selection. We conducted tissue propagation of the bud mutants and subsequently transplanted some of the resulting seedlings to the greenhouse for cultivation. The Spathiphyllum ‘Mojo’(S) and its somatic variants (Spathiphyllum ‘Mojo’-Ssm-1)(T) were grown in a greenhouse under the following conditions: at a natural light intensity of 1500 μmol/m²s for 14 h/10 h (light/dark) photoperiod and temperature of 23 ± 2 °C. Spathiphyllum leaf buds are formed at the base of the leaves and are wrapped by membranous sheaths. As the buds grow, the leaves are exposed from the sheaths. Subsequently, the expansion process of the Spathiphyllum leaf was divided into three stages: (1) the curled leaf stage, (2) the early spread stage, and (3) the late spread stage according to the study by Li et al. [20] (

Table 1. The primary characteristics of Spathiphyllum leaf growth at three stages.

Stage	Number	Primary Growth and Morphological Characteristics
The curled leaf stage	W1/M1	Approximately ten days after the leaf buds transition to white, the tips of the leaves gradually change from white to yellow/white, while the base of the leaves remains white. During this period, curled leaves develop, with the leaves being enveloped in leaf sheaths.
The early spread stage	W2/M2	Approximately 10 to 13 days after the formation of curled leaves, the leaves emerge from the leaf sheath and begin to gradually unfold. During this process, the leaf color transitions from yellow/white to a delicate green.
The late spread stage	W3/M3	Approximately ten days after the onset of leaf color change, the leaves transition from light green to a deeper green. During this period, the leaves gradually expand, completing their growth. At this stage, the characteristics of the leaves stabilize and exhibit no further changes.

W—wild; M—mutant.

). In this experiment, the leaf of Spathiphyllum ‘Mojo’ was used as the control group (wild), and the leaf of Spathiphyllum ‘Mojo’-Ssm-1 was used as the treatment group (mutant). Leaf samples were used to measure leaf indicators, cellulose content, and cellulase activity, and perform transcriptome sequencing to screen for differential genes, as well as RNA extraction and cDNA reverse.

Arabidopsis was used for heterologous transgene verification, while Nicotiana benthamiana was used for subcellular localization of the target gene. Arabidopsis seeds were treated with 75% ethanol and 1% sodium hypochlorite for 30 s and 1 min, respectively, then washed with sterile water several times before sowing in 1/2 MS medium. The seeds were incubated in the culture chamber for 7 days and then transferred to nutrient soil and infected when the inflorescences emerged. Nicotiana seeds were sown in nutrient soil and injected with bacterial solution after they grew into young leaves. The infected Nicotiana leaves were placed into glass slides and observed under a confocal laser microscope for observation and imaging. The culture conditions of both plant materials were 25 ºC/20 ºC and 16 h/8 h (light/darkness).

The strains necessary for the construction of the expression vector include the DH5α Competent E. coli strain (C502; Vazyme) and GV3101 Agrobacterium tumefaciens strain (LM12-162; LMAI). DH5α is commonly used to transform plasmids into competent E. coli cells. The methodology is designed to maximize the efficiency of transformation, crucial for subsequent molecular biology experiments such as gene cloning and expression analysis [21]. A. tumefaciens strain GV3101 is widely utilized for plant transformation. The T-DNA binary vector system in A. tumefaciens is instrumental in facilitating this transformation process. T-DNA is engineered by removing oncogenes and crown gall synthase genes, which enhances the efficiency of gene transfer from A. tumefaciens to plants. This modification effectively disarms toxic strains, thereby preventing tumor induction [22]. The reagents and equipment used in this experiment are detailed in Supplementary Tables S2 and S3.

2.2. Morphological Traits of Leaves Measurement

Mature leaves from two varieties of Spathiphyllum were randomly selected for measurement. Five plants were sampled, and 3 to 5 leaves from each plant were measured. The leaf traits that were measured included leaf length, leaf width, petiole length, plant height, and crown width, all of which were measured using a ruler. Leaf area was calculated using Image-J (version 1.8.0) software.

2.3. Determination of Leaf Cellulose Content and Cellulase Activity

The determination of cellulose content and cellulase activity was completed using the cellulose (CLL) content detection kit (BC4280; Solarbio) and cellulase (CL) activity detection kit (BC2545; Solarbio) provided by Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China).

2.4. Analysis of Differential Gene and Quantitative Real-Time PCR Assays

Two plant leaf samples corresponding to the expansion period were collected and frozen in liquid nitrogen. Full-length transcriptome sequencing was completed by Beijing Biomarker Technologies Co., Ltd. (Beijing, China). The criteria for screening differentially expressed genes were a false discovery rate (FDR) of less than 0.01 and a fold change (FC) of 2 or greater. This was followed by GO function and KEGG enrichment analyses. The TBtools (version 2024.1.11) software was used to create a heatmap of the differentially expressed genes [23]. Candidate genes identified through screening were numbered in accordance with their gene ID order in the transcriptome.

Total RNA from leaves of Spathiphyllum was isolated using an RNA extraction kit (IVD3020-S-48; Guangzhou Magen Biotechnology Co., Ltd., Guangzhou, China). A commercially available kit (R312; Nanjing Vazyme Biotechnology Co., Ltd., Nanjing, China) was used for reverse transcription of Spathiphyllum RNA. Full-length SpGH9s cDNA sequence was isolated from Spathiphyllum using specific primers based on SpGH9s sequence in the Spathiphyllum transcriptome.

Quantitative real-time PCR (qPCR) assays were performed according to Liu et al. [24]. Analyses were conducted following the minimum information for publication of qPCR experiments guidelines. Spathiphyllum TUB genes were used as internal controls for qPCR analysis. The data reported in the results represent the relative expression values calculated by the 2^−ΔΔCt approximation method. All experiments were performed with three biological replicates and three technical replicates.

2.5. Correlation Analysis

The cellulase gene expression results of differential genes obtained from transcriptome screening were correlated with the cellulose content and cellulase activity measurement results (p < 0.05). The gene most closely associated with morphological differences in the mutant leaf samples was selected as the candidate gene for subsequent bioinformatics analysis and functional validation.

2.6. Sequence Analysis

Alignments were performed, and a phylogenetic tree was generated using the DNAMAN (version 7.0) and MEGA (version 8.0) software. The physicochemical properties of the target protein were analyzed using ProtParam. Signal peptide analysis of the gene was conducted using the online software SignalP (version 5.0). The secondary and tertiary structures of the protein were predicted using Sopm and Swiss-Model, respectively.

2.7. Subcellular Localization

Using specific primers, a SpGH9A3 (GenBank: WYV98059.1) full-length cDNA sequence was inserted into the GFP N-terminus of the pC18 vector. The construct was sequenced to ensure that coding sequences were fused in the frame and no mutations occurred. The construct was transformed into A. Tumefaciens (strain GV3101), which was then infiltrated into Nicotiana leaf epidermal cells. After 48 h of incubation under a 16 h light/8 h dark cycle at 25 °C, the GFP fluorescence signal was observed under a confocal microscope.

2.8. Phenotypic Analysis of Transgenic Plants

35S::CAMBIA1300-SpGH9A3 was constructed by amplifying the full length of SpGH9A3 using specific primers and cloning it into the pCAMBIA1300 vector. This construct was introduced into A. tumefaciens (strain GV3101) and transformed into Arabidopsis (Col-0) using the floral dip method. For gene overexpression experiments, 10 Arabidopsis plants were inoculated with the vector. The inoculated plants were grown under greenhouse conditions as aforementioned.

Leaf samples from both the wild-type and transgenic plants were collected for quantitative real-time PCR analysis. The phenotypic differences of the natural extension state of T2 generation transgenic Arabidopsis in terms of leaf length, leaf width, leaf area, petiole length, pod length, and plant height were photographed and compared with the wild-type.

2.9. Statistical Analysis

The presented data were statistically analyzed using GraphPad Prism (version 9.0.0) software. The significance of the data was investigated through a t-test or analysis of variance (ANOVA) (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

3. Results

3.1. Comparison of the Leaf Morphologies of Spathiphyllum ‘Mojo’ (Wild) and ‘Mojo’-Ssm-1 (Mutant)

Spathiphyllum ‘Mojo’ produces light green, lanceolate-shaped leaves. The leaf has a neat margin, an elongated tip, and a relatively narrow leaf base. ‘Mojo’-Ssm-1, on the other hand, has ovate, dark green leaves. The leaf has a tail-like tip and frequently has a curved surface (Figure 1). In terms of petiole length, plant height, leaf area, length-width ratio, and crown width, Spathiphyllum ‘Mojo’ considerably outperforms the mutant plants; however, its leaf width is noticeably smaller (Supplementary Table S1). The findings of the analysis show that the morphology of the leaves of Spathiphyllum ‘Mojo’ and ‘Mojo’-Ssm-1 differs significantly (Figure S1).

3.2. Comparison of Cellulose Content and Cellulase Activity

During the growth and development of leaves, the cellulose content in wild leaves continued to increase and was significantly higher than in mutants. The cellulose content in mutants reached its lowest value in the early spread stage, showing a trend of initially decreasing and then increasing. The cellulase activity in wild leaves gradually decreased, whereas the cellulase activity in mutants first increased and then decreased, peaking in the early spread stage. Cellulase plays a crucial role in the synthesis of cellulose essential for the expansion of different types of Spathiphyllum leaves, underscoring its importance in leaf growth and morphological development (Figure 2).

3.3. Screening of Differential Genes for Leaf Morphology

In transcriptome, 20,499 DEGs in all had KEGG annotation. Sixty-six DEGs associated with pathways such as photosynthetic, carbon metabolism, peroxidase, plant hormone signal transduction, and starch and sucrose metabolism were enriched during the curled leaf stage. Two hundred DEGs associated with pathways such as starch and sucrose metabolism, plant-pathogen interaction, endoplasmic reticulum protein processing, and spliceosome were found during the early spread stage. One hundred and sixteen DEGs that are involved in pathways such as the metabolism of starch and sucrose, the interaction between plants and pathogens, the processing of proteins in the endoplasmic reticulum, spliceosomes, and plant hormone signal transduction were found to be enriched during the late spread stage. The starch and sucrose metabolism pathway’s genes were notably enriched at all three stages, with the early spread stage showing significant enrichment, and the late spread stage showing the highest level of enrichment, far exceeding other pathways (Figure S2).

Four cellulase genes were analyzed in starch and sucrose metabolic pathways, revealing distinct expression patterns in different leaf types. F01_transcript_17438, F01_transcript_22687, and F01_transcript_36346 (GenBank: WYV98059.1) exhibited a gradual increase in expression levels in wild-type leaves, while in mutants, their expression initially rose and then declined, peaking during early spread stage. Conversely, F01_transcript_38749 showed significantly higher expression in mutant leaves during the curled leaf stage compared to wild-type leaves, with expression levels gradually decreasing (Figure 3).

Fluorescence quantitative PCR not only validated the reliability of the transcriptome data but also highlighted the expression variances of these genes in the two leaves at different expansion stages. During the early spread stage, the expression levels of F01_transcript_17438, F01_transcript_22687, and F01_transcript_36346 were significantly higher in the mutant compared to the wild-type. In the wild-type, the expression levels of F01_transcript_17438 and F01_transcript_36346 exhibited a gradual increase, while the expression level of F01_transcript_22687 remained relatively stable. On the other hand, F01_transcript_38749 showed a peak expression at the curled leaf stage, followed by a gradual decrease as the leaves expanded. These findings indicate notable differences in gene expression between the wild-type and mutant at various expansion stages, with the most pronounced disparities observed during the early spread stage. Such differences in gene expression could potentially play a crucial role in regulating leaf morphology in Spathiphyllum (Figure 4).

These four genes are annotated as cellulases in the transcriptome and are classified under the GH9 gene family. To compare, we retrieved the GH9 family amino acid sequences of Arabidopsis and Oryza sativa L. from NCBI (National Center for Biotechnology Information) and compared them with the amino acid sequences of the Spathiphyllum GH9 family. Then, a phylogenetic tree was constructed using the NJ (Neighbor-Joining) method in MEGA 7 (version 7.0) software with 1000 bootstrap replicates. GH9A, GH9B, and GH9C are the three subfamilies into which the GH9 family genes can be broadly subdivided. The GH9A subfamily in Spathiphyllum is comprised of the genes F01_transcript_17438, F01_transcript_22687, and F01_transcript_36346, whereas the GH9B subfamily is made up of F01_transcript_38749. F01_transcript_17438 was named SpGH9A1, F01_transcript_22687 was named SpGH9A2, F01_transcript_36346 was named SpGH9A3, and F01_transcript_38749 was named SpGH9B1, according to the gene ID order in the transcriptome of Spathiphyllum.

To investigate the phylogenetic relationships of the SpGH9s gene family members in Spathiphyllum, we selected the GH9 gene families of Arabidopsis and O. sativa as references and jointly constructed a phylogenetic tree. The findings demonstrated the considerable diversity of Arabidopsis, O. sativa, and Spathiphyllum genes by demonstrating their intermixture and lack of independent clustering branches. Three groups comprised the four SpGH9s proteins. Among them, SpGH9A1 and SpGH9A2 belonged to group IV, SpGH9B1 to group I, and SpGH9A3 to group V. The support for the phylogenetic relationship between the homologous genes of Arabidopsis and O. sativa and members of the SpGH9s gene family in Spathiphyllum was relatively low, indicating that these GH9s members may have developed new functions during the course of evolution (Figure 5).

This study investigated the relationship between cellulose content, cellulase activity, and the expression level of GH9s genes, which encode cellulose endoglucanase, in the leaves of wild and mutant plants. The results revealed significant or extremely significant correlations between these parameters in both types of leaves. The correlation coefficients were 0.9690** for SpGH9A1, 0.8075** for SpGH9A3, and 0.9422** for SpGH9B1 in wild plants, indicating a highly significant correlation with cellulose content. On the other hand, SpGH9A2 showed a significant correlation with a coefficient of 0.4424*. Interestingly, cellulase activity and GH9 gene expression did not show a significant correlation.

The study examined the correlation between GH9 gene expression levels in the mutant and cellulose content. Results showed that SpGH9A3 had a highly significant negative correlation (correlation coefficient of −0.8603**) with cellulose content, while SpGH9A2 and SpGH9B1 had significant negative correlations (correlation coefficients of −0.6086* and −0.4964*, respectively). However, the correlation of SpGH9A1 (correlation coefficient of −0.1837) with cellulose content was not statistically significant. Additionally, there were varying degrees of correlation between cellulase activity and GH9 gene expression levels. SpGH9A1, SpGH9A2, and SpGH9A3 showed significant correlations with cellulase activity, with correlation coefficients of 0.4002*, 0.5393*, and −0.5761*, respectively. On the other hand, the correlation coefficient between SpGH9B1 and cellulase activity was −0.01463, indicating no significant relationship. Further analysis revealed significant correlations between the cellulose content of both leaf types and the expression levels of these genes. This suggests that increased cellulase activity may lead to reduced cellulose content in the mutant, potentially impacting leaf growth and morphogenesis. Notably, all three genes from the SpGH9As subfamily showed significant correlations with cellulase activity in the mutant, indicating higher cellulase activity compared to the wild-type. Specifically, SpGH9A3 exhibited stronger correlations with cellulose content and cellulase activity in the mutant leaves when compared to other genes in the family. Based on the data analysis above, it was found that SpGH9A3 showed stronger correlation coefficients with cellulose content and cellulase activity in mutant leaves compared to other genes in the same family when wild was used as a control. As a result, further investigation including sequence analysis, gene cloning, and functional verification is planned for SpGH9A3, which has been provisionally identified as a potential candidate gene involved in the morphogenesis of mutants (Figure 6).

3.4. Bioinformatics Study of SpGH9A3 in Spathiphyllum

In the phylogenetic tree, the amino acid sequences of AtKOR1 (Arabidopsis) (AAC83240.1), AtGH9A1 (AED95850.1), AtGH9A2 (NP_176738.1), and AtGH9A4 (AEE77836.1) were found to be taxonomically congruent with SpGH9A3 of Spathiphyllum. A multi-sequence alignment using DNAMAN 8 (version 7.0) software revealed that SpGH9A3 shares a conserved GH9 (glycoside hydrolase family 9) domain with other proteins (Figure S3).

The physicochemical characteristics of SpGH9A3 were analyzed using ProtParam software (https://web.expasy.org/protparam/). The full-length cDNA of the SpGH9A3 gene spans 858 bp and encodes 285 amino acids. The amino acid composition was predominantly alanine (211, 24.6%), glycine (230, 26.8%), and threonine (172, 20.0%). The theoretical isoelectric point (pI) of the protein was determined to be 8.79 with an approximate molecular weight of 31.7 KDa. A signal peptide sequence consisting of 29 amino acids and a cleavage site at VTT-EI was identified at the N-terminal of SpGH9A3. The secondary structure prediction of SpGH9A3 protein revealed the presence of random coils (132, 46.32%), extended strands (58, 20.35%), α-helices (86, 30.18%), and β turns (9, 3.16%). The three-dimensional structure of SpGH9A3 was analyzed using SWISS-MODEL, with a Global Model Quality Estimation (GMQE) score of 0.72, indicating a relatively stable protein structure. The predicted model showed a 77.15% similarity rate with the template, suggesting a reliable prediction. The gene structure of SpGH9A3 mainly consists of α-helices and random coils, as supported by both secondary and tertiary structure predictions. These findings provide valuable insights into the function of SpGH9A3, paving the way for further research (Figure S4).

3.5. SpGH9A3 Protein Is Localized to the Cell Membrane

The full-length SpGH9A3 protein was fused to GFP at the C-terminus and transiently expressed in N. benthamiana leaves under the control of the 35S promoter (35S::SpGH9A3-GFP) to investigate the localization of SpGH9A3 in plant cells. The SpGH9A3-GFP fusion proteins were observed in the cell membrane of N. benthamiana leaves, indicating that SpGH9A3 is localized to the cell membrane and suggesting that cellulose hydrolysis occurred in this site (Figure 7).

3.6. Overexpressing SpGH9A3 Changed the Leaf Morphology and Decreased Cellulose Content

An overexpression system utilizing the pCAMBIA1300 vector was employed to enhance the expression of SpGH9A3 in Arabidopsis. The complete gene sequence of SpGH9A3 was inserted into pCAMBIA1300 to generate the pCAMBIA1300-SpGH9A3 vector, with wild-type Arabidopsis serving as the control group. Ten to fifteen Arabidopsis plants were inoculated with the vector.

After one month post-infection, transgenic plants displayed a notable increase in SpGH9A3 expression, with leaves showing an 8-fold rise compared to the control group. Further examination of cellulose content in the leaves of transgenic SpGH9A3 and wild-type plants revealed a significant decrease in cellulose content in the transgenic plants, accounting for only 49.00% of the control group (Figure 8).

The statistical analysis revealed that overexpressing SpGH9A3 plants exhibited significantly shorter leaf length, smaller leaf area, shorter pods, and shorter plants, amounting to 75%, 78%, 68%, and 69% of the control group, respectively. In contrast, leaf width remained unchanged at 98% of the control group with no observable differences. Additionally, there was a notable change in leaf morphology, with petiole length and leaf length–width ratio measuring 121% and 77% of the control group, respectively. These results suggest that the plant’s morphological characteristics, including leaves, pods, and overall plant structure, are greatly influenced by the overexpression of SpGH9A3 (Figure 9 and Figure 10 and

Table 2. Growth variation in leaves with overexpression of the SpGH9A3 gene.

	WT	OE of SpGH9A3	SpGH9A3/WT (%)
Leaf length (cm)	1.36 ± 0.05	1.02 ± 0.10 *	75.00%
Leaf width (cm)	0.49 ± 0.03	0.48 ± 0.05	97.96%
Length–width ratio	2.78 ± 0.19	2.14 ± 0.28 *	76.98%
Petiole length (cm)	0.57 ± 0.03	0.69 ± 0.04 *	121.05%
Leaf area (cm2)	0.42 ± 0.03	0.33 ± 0.05 *	78.57%
Silique length (cm)	1.25 ± 0.06	0.86 ± 0.06 *	68.80%
Plant height (cm)	25.49 ± 1.42	17.63 ± 1.00 *	69.16%

The asterisks indicate different levels of significant differences between clusters (ns, not statically significant; *, p < 0.05).

).

4. Discussion

Leaf morphology plays a crucial role in determining the aesthetic aspect of plants, with cell proliferation and cell expansion being key biological processes that influence it [25]. The growth and development of plants are closely tied to cellulose, a β-1,4-linked glucan that is a vital component of plant cell walls. Cellulose biosynthesis impacts cell expansion, division, mechanical strength, pathogen resistance, and various other aspects of plant growth [26]. The GH9 family is essential in plant cellulose synthesis and hydrolysis [7]. The overexpression of SpGH9A3 resulted in a decrease in leaf cellulose content, leading to a smaller leaf phenotype.

Three GH9A subfamily members, SpGH9A1, SpGH9A2, and SpGH9A3 (GenBank: WYV98059.1), have been identified in Spathiphyllum. These genes display similar expression patterns during leaf growth and development, showing a high degree of sequence similarity and containing typical GH9-conserved domains. Previous studies have shown that cellulose is synthesized by the cellulose synthase complex, which is formed in the Golgi apparatus and then transported to the plasma membrane to build the cell wall structure [27,28]. The current study observed the presence of the SpGH9A3 protein on the cell wall, aligning with prior findings and suggesting that cellulose hydrolysis in Spathiphyllum leaves takes place within the cell wall.

The GH9 family is a critical component of the hydrolase family involved in both cellulose synthesis and hydrolysis [29]. Mutations in the KOR gene have been observed to induce dwarfing in Arabidopsis plants and a significant reduction in cellulose content. Inhibition of KOR gene expression in Arabidopsis mutant plants also results in dwarf phenotypes and cell wall wrinkles, indicating the gene’s involvement in cellulose synthesis and modification [30]. Moreover, mutation of the KOR gene, which is homologous to Cel3 in Arabidopsis kor-1, leads to organ shrinkage [31]. The overexpression of cottonwood PdeKOR in Eucalyptus tenuifolia promotes significant growth in stem segments [32]. Similarly, the expression of poplar endoglucanase in transgenic Arabidopsis plants accelerates growth and increases cellulose content [3]. Overexpressing the Arabidopsis cel1 gene in poplar plants also enhances growth and cellulose content [33]. Mutations in GH9A1 in Arabidopsis result in reduced cellulose content and play a crucial role in cellulose synthesis in the primary cell wall [13]. This study investigated the leaf phenotype following the overexpression and transformation of the SpGH9A3 gene in Arabidopsis plants. The leaves of the overexpressed plants displayed a small-leaf phenotype, along with reduced growth and leaf cellulose content compared to the control. This experimental outcome slightly deviates from previous research, possibly due to the stringent regulation of cellulase activity across different species and stages of transcription, translation, and post-translation [34].

Morphological differences were observed in the leaves of wild and mutant plants, with varying cellulose content and cellulase activity trends. Transcriptome analysis revealed significant differences in the expression levels of cellulase SpGH9s. While the cellulase activity of mutant leaves was similar to wild leaves during certain stages, it notably increased in the early spread stage.

Further verification of the potential functions of SpGH9s in Spathiphyllum using additional molecular biology techniques and tools is essential. These findings lay the groundwork for further exploration of SpGH9s, providing fresh insights into its biological functions. The intricate chemical basis and regulatory mechanisms of cell wall polysaccharide biosynthesis remain complex and warrant further comprehensive investigation. To enhance our comprehension, a combination of experimental and computational approaches should be employed to isolate target proteins, develop enzyme analysis methods, and construct multi-omics regulatory networks to establish detailed molecular models.

5. Conclusions

This study focused on Spathiphyllum ‘Mojo’ and its mutant leaves as research materials, with morphological index measurements revealing significant differences between the two types of leaves. Four GH9 family genes were identified from the transcriptome data of Spathiphyllum. By analyzing the correlation coefficient between gene expression, leaf cellulose content, and cellulase activity, the study preliminarily identified SpGH9A3 as the most relevant gene for the expasion of mutant leaves. Overexpression of the SpGH9A3 gene in transformed Arabidopsis plants resulted in smaller leaves and reduced leaf cellulose content compared to the control group. These findings suggest that differential expression of the SpGH9A3 gene in the leaves of different Spathiphyllum varieties may impact cellulase activity, subsequently influencing leaf cellulose content at various stages of expansion and ultimately leading to differences in leaf morphology. This research enhances our comprehension of the roles of glycoside hydrolase family genes and lays a foundation for future investigations.