Systematic Analysis and Expression Profiling of the Ginger FWL Gene Family Reveal Its Potential Functions in Rhizome Development and Response to Abiotic Stress

Abstract

Ginger (Zingiber officinale Roscoe) is a significant medicinal and culinary plant, with its growth influenced by various biotic and abiotic factors. The FWL gene, containing the PLAC8 motif, is prevalent in fungi, algae, higher plants, and animals. In plants, FWL primarily regulates fruit weight, cell division, and participates in heavy metal transport. However, the FWL family members in ginger have not been previously identified. This study identified 21 FWL members within the ginger genome, distributed across nine chromosomes. These 21 FWL genes were categorized into five subfamilies based on the phylogenetic analysis. Gene-structure and motif analyses revealed that ZoFWL has been conserved throughout evolution. Concurrently, the ZoFWL gene exhibits a homologous evolutionary relationship only with Musa acuminata. We identified three pairs of fragment-repeat events encompassing five genes, which likely represent the primary mechanism for amplification within the ZoFWL gene family. The promoter regions of the ZoFWL genes are enriched with numerous cis-acting elements implicated in plant growth, development, and responses to abiotic stress. These include elements responsive to low temperatures, anaerobic induction, MYB binding sites integral to defense and stress responses, and drought inducibility. Expression profiling revealed that the ZoFWL genes are responsive to a quartet of abiotic stressors, with ZoFWL18, in particular, demonstrating a pronounced response to osmotic, low-temperature, heat, and salinity stresses. This underscores the pivotal role of ZoFWLs in abiotic-stress responses. Our findings offer valuable insights into the potential of the ZoFWL gene family in modulating ginger rhizome development and the genes’ response to abiotic stressors, laying a foundational framework for future research into ginger’s resistance breeding.

1. Introduction

Studies have demonstrated that FW2.2 and its homologous gene FW2.2-like (FWL) are ubiquitous in plants, animals, and fungi, with a total of 136 FWL genes identified [1]. Among them, genes similar to tomato FW2.2 have been discovered in 14 plants, with 103 FWL genes identified [2]. FW2.2 and FWL proteins are cysteine-rich and possess a PLAC8 domain composed of N-terminal cysteine-rich motifs (CLXXXXCPC, CCXXXXCPC, CLXXXXFPC, or CCXXXXGPC) and a second conserved motif (QXXRELK) at the C-terminus [3,4]. The FW2.2 gene (fruit-weight-2.2) is a Quantitative Trait Locus (QTL) that significantly influences fruit weight. FW2.2, a gene governing tomato fruit size, was the first quantitative trait gene obtained through the map-based cloning method. Located on the tomato’s second chromosome, it is primarily responsible for the weight difference between domesticated tomatoes and their wild counterparts, accounting for 30% of the total fruit weight variation [3,5,6]. FW2.2 acts as a negative regulator of cell division, controlling fruit size by inhibiting carpel cell division. The expression level of FW2.2 is highly inversely correlated with fruit quality, indicating that a higher expression level corresponds to a lower cell division status [5,7]. There is evidence suggesting that FW2.2 also impacts the fruit number and photosynthate distribution. Furthermore, to better understand the mechanism of FW2.2-mediated cell division during fruit development, researchers performed yeast two-hybrid screening and discovered that the regulatory subunit of Casein Kinase II (CKII) interacts with FW2.2 [8]. The deletion of the CKII homologous protein results in the shortening of Arabidopsis siliques and affects the signal transduction of the plant cell cycle [9,10]. Therefore, FW2.2 may control the size and weight of tomato fruits by participating in the cell cycle control signal-transduction pathway through CKII.

In maize, 12 FW2.2 homologous genes were identified, all containing the PLAC8 domain and labeled Cell Number Regulators (CNRs). The natural expression and transgenic investigations of ZmCNR1 and ZmCNR2, which are closely related to FW2.2, were conducted. The overexpression of ZmCNR1 led to a significant reduction in maize plant height, as well as a decrease in the size of various organs, including tassels and leaves. Changes in the size of the whole maize plant and its organs were attributed to changes in cell number, not cell size. The expression of ZmCNR2 was negatively correlated with tissue growth activity and hybrid seedling activity [10]. In the soybean, GmFWL1 is specifically expressed in soybean root hairs and plays a crucial role in soybean nodule formation. Silencing GmFWL1 significantly reduces the number of nodules [11,12]. In rice, eight FWL genes were identified, and multiple OsFWL genes affected organ size by regulating cell division. OsFWL3 was negatively correlated with glume length and growth activity, and the grain length of OsFWL3 mutants was longer than that of the wild type. The expression pattern of OsFWL5 exhibits a negative correlation with the growth activity of rice leaves, suggesting that OsFWL5 acts as a negative regulator of rice plant height [13]. The orthologous gene PfCNR1 of FW2.2 in Physalis regulates the size of berries and seeds by affecting the cell division cycle, and its expression level is closely related to the interspecific variation in these organ sizes [14]. Researchers isolated a FW2.2 homologous gene PaFW2.2 with about a quarter of the coding region in avocado fruit. The transcription level of PaFW2.2 in small avocado fruit is significantly higher than that in normal avocado fruit, indicating that PaFW2.2 is a negative regulator of cell division in avocado fruit [15].

In addition to participating in the regulation of fruit size and cell number, proteins containing the PLAC8 domain also play a crucial role in the transport of heavy metals such as cadmium or zinc, and confer resistance to heavy metals such as cadmium [16,17]. OsFWL4 is involved in the transport of cadmium from root to stem, and cadmium stress induces significant expression of OsFWL4 in the rhizome tissues of rice seedlings [18]. OsFWL5/OsPCR1 and OsFWL2/OsPCR3 are closely related to cadmium accumulation. Overexpression of OsPCR1 and OsPCR3 increased the cadmium tolerance of plants and significantly reduced the accumulation of cadmium in different parts of rice plants [19]. Overexpression of OsFWL5/OsPCR1 increased Zn content and decreased Cd content in rice grains, maintaining metal ion homeostasis and rice grain weight [20]. Nonetheless, OsFWL5 not only exhibits a degree of resistance to heavy metals but also possesses the capacity to withstand biotic stressors. OsFWL5 plays a significant role in the defense against Xanthomonas oryzae pv. oryzae. Overexpression of OsFWL5 induces the production of reactive oxygen species and enhances resistance to Xanthomonas oryzae pv. oryzae. At the same time, the defense response of OsFWL5 in rice must be induced by cadmium [21]. AtPCR1 is a member of the PCR protein family in Arabidopsis thaliana. The Cd resistance of Arabidopsis thaliana plants overexpressing AtPCR1 is enhanced. At the same time, the homologous genes AtPCR2, AtPCR9, AtPCR10, and OsPCR11 were transformed into the yeast strain ycf1 and cultured on a plate containing cadmium. The strains grew better than the strains of the empty vector, and each gene could enhance the host’s tolerance to cadmium [16]. As a key protein for zinc transport, AtPCR2 plays an important role in maintaining the optimal zinc level in Arabidopsis plants [22]. AtMCA proteins containing the PLAC8 domain are identified as calcium permeability channels, and their abundance is negatively correlated with intracellular calcium accumulation [23]. AtMCA1 is a plasma membrane protein for Ca²⁺ influx and mechanical sensing in Arabidopsis thaliana. AtMCA2 is a duplicated sequence of AtMCA1. They both have high homology and share several identical structural features: the N-terminal half of the EF-hand region, the transmembrane TM fragment, the intermediate coil motif, and the C-terminal half of the PLAC8 motif [24,25]. Furthermore, the FWL gene family exhibits distinct responses to abiotic stresses in plants. In the context of tomato plants, an expression profile analysis of SlFWL genes revealed a universal response to abiotic stressors, with each gene subgroup demonstrating unique expression patterns [26].

Ginger (Zingiber officinale Roscoe), a plant with both medicinal and edible properties, is widely cultivated in China and holds high economic and nutritional value. Ginger exhibits antioxidant, anti-inflammatory, and antibacterial activities, often serving as an edible spice and medicinal resource [27,28]. However, environmental stress significantly impacts the growth and development of ginger. Ginger leaves are extremely susceptible to burns at high temperatures, and low temperatures can lead to a decline in ginger growth. The water shortage caused by drought first threatens the physiological processes and cell health of ginger, subsequently affecting its growth and yield [29]. Furthermore, ginger often encounters salt stress, which can notably diminish the dry and fresh weight of ginger [30], thereby affecting its quality. The underground rhizome of ginger, its principal edible part, is one of the significant export vegetables from China. The size of the ginger rhizome directly impacts the yield and is closely related to its appearance and commodity quality. Particularly, the high-end market places strict requirements on the size of the ginger rhizome [31].

Currently, our understanding of ginger FWL protein is limited. Given the importance of the FWL gene for plant fruit-cell division and weight regulation, along with its positive response to abiotic stress, studying the FWL gene family in ginger holds considerable significance. In this study, we identified 21 ginger FWL genes and comprehensively analyzed their protein physicochemical properties, subcellular localization prediction, phylogeny and homology analysis, gene duplication, and motif composition. We further analyzed their expression profiles in various growth stages and organs of ginger rhizomes and their expression under low-temperature, osmotic, heat, salt, and high-temperature and strong-light stress. This analysis aims to determine the different biological processes of ginger FWL gene-family members involved in ginger life activities, providing valuable clues for the functional verification of ginger FWL gene family members in ginger growth and development and the role of ginger plants in resisting abiotic stress.

2. Materials and Methods

2.1. Plant Material Growth and Treatment Conditions

The ginger cultivar employed in this study is a local variant known as bamboo root ginger, endemic to Chongqing. It is cultivated within the controlled environment of the greenhouse facilities at the Smart Agriculture College, Chongqing University of Arts and Sciences. Given Chongqing’s status as a predominant region for bamboo root-ginger cultivation, this variety is well-suited to the local growing conditions. A selection of ten bamboo root-ginger specimens was meticulously cultivated under specific conditions: a constant temperature of 25 °C, relative humidity maintained at 75%, a photosynthetic photon flux density of 1922.99 μmol m⁻² s⁻¹, and a photoperiod extending from 6:00 a.m. to 8:00 p.m., totaling 14 h of light exposure daily. Light intensity was carefully modulated using a combination of black shading nets and LED lighting systems. The cultivation was conducted over a period of 60 days. In order to best match the actual planting environment of ginger in Chongqing, we exposed two-month-old ginger seedlings to outdoor environments, with a maximum temperature of more than 40 °C and a light intensity of 1922.99 μmol m⁻² s⁻¹ (Supplementary Table S2). The complete functional leaves of ginger were collected at 8 a.m. and 3 p.m. on the first day; and at 3 p.m. on the second, third, and fourth days. The third-to-fifth expanded leaves from the top of the stem to the base of the stem. Each sample was subjected to three technical repetitions to ensure the accuracy of the results. The samples were then quickly frozen in liquid nitrogen and stored at −80 °C for a subsequent sequencing analysis.

2.2. Identification and Physicochemical Properties Analysis of Ginger FWL Gene-Family Members

Our group’s research project in 2021 obtained the genome data of ginger, including genome-annotation information and protein sequence. The hidden Markov model (PF04749) of the PLAC8 domain of FWL was downloaded from Pfam (http://pfam-legacy.xfam.org/, accessed on 2 June 2024) [32]. The hidden Markov model (HMM) was used to perform a hmmsearch search on the ginger genome protein sequence, and the E value of the PLAC8 family protein was predicted to be 1 × 10⁻⁵ [33]. In order to obtain a more accurate ZoFWL gene family, we downloaded the FWL protein sequence of tomato in NCBI. Through local Blast + alignment, the E value was set to 1 × 10⁻⁵ to remove duplicate and low-similarity members to obtain a comprehensive ZoFWL gene [34]. Then, the screened sequences were subjected to CDsearch (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 2 June 2024) to view the PLAC8 conserved domain [35], and 21 ZoFWL genes were finally identified. Using TBtools (v2.096) for chromosome localization [36], the physical and chemical properties (amino acid length, molecular weight, isoelectric point, etc.) of ZoFWL members were analyzed through the bioinformatics online website ExPASy (https://web.expasy.org/protparam/, accessed on 2 June 2024) [37]. Bioedit software (v7.0.9.1) [38] was used to compare and analyze the amino acid sequence of ZoFWL protein, and multiple alignments were performed on the conserved domains. Subcellular localization prediction of ZoFWL family members was performed using the Plant-mPLoc [39] online website (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/, accessed on 3 June 2024).

2.3. Phylogenetic Analysis of ZoFWL Gene Family

The identified ZoFWL protein sequence was aligned by ClustalW, and then the aligned sequence was used to construct a phylogenetic tree using IQ-TREE [40], using the maximum likelihood method (ML), setting the bootstrap value to 1000, the best model LG + G. In addition, we also constructed a multi-species phylogenetic tree and obtained the FWL protein sequences of Arabidopsis thaliana, tomato, maize, and rice from the NCBI (https://www.ncbi.nlm.nih.gov/, accessed on 3 June 2024) database. The phylogenetic tree was constructed using the same method. The maximum likelihood method (ML) was used to set the bootstrap value to 1000 and the best model JTT + R4. The multi-species phylogenetic tree was optimized using iTOL (https://itol.embl.de/, accessed on 4 June 2024) [41].

2.4. Analysis of Factorial Structure, Conserved Motifs, and Cis-Acting Elements

TBtools software (v2.096) was used to analyze and visualize the ZoFWL gene structure (exon-intron) in the ginger genome-annotation file (GFF) [36]. Then, the conserved motifs of ZoFWL members were predicted by the MEME (https://meme-suite.org, accessed on 5 June 2024) online website [42] and set to retrieve 10 motifs. The first 2000 bp in the ginger FWL gene file was extracted as the promoter region sequence, and these sequences were submitted to PlantCARE [43] (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 7 June 2024) for analysis. Then, the results were visualized using TBtools (v2.096) [36].

2.5. Gene Replication, Collinearity Analysis, and Protein Interaction Network Prediction

The gene replication events of ginger FWL members were analyzed using the multi-collinear scanning toolkit (MCScanX), and the One Step MCScanX-Superfast of TBtools software (v2.096) [36] was used to reveal the homologous relationship between ginger FWL gene and small-fruited wild banana, tomato, and maize. The genome data were obtained from the Ensembl Plants (https://plants.ensembl.org/index.html, accessed on 7 June 2024) database. The online website STRING (https://cn.string-db.org/, accessed on 13 June 2024) was used to predict the protein interaction network of ginger FWL gene family members. Arabidopsis thaliana was used as the reference plant, and the credibility was set to 0.600. Then Cytoscape was used to visualize the protein interaction network, with Degree value as the standard [44].

2.6. Expression Pattern and qRT-PCR Analysis of ZoFWL

Our research group conducted transcriptome sequencing of various organs of ginger, including ginger subjected to abiotic stresses such as low temperature, heat, salt, and osmosis. In this study, we aimed to investigate the expression pattern of the ZoFWL gene family in ginger rhizomes. To achieve this, we selected transcriptome data from ginger roots, stems, and five different bud stages. Furthermore, we subjected two-month-old ginger seedlings to four types of abiotic-stress treatments, with each treatment group consisting of ten ginger plants. The seedlings were exposed to low-temperature and heat stress treatments at 4 °C and 40 °C, respectively, in a temperature-controlled incubator. For osmotic stress treatment, the ginger roots were immersed in a 15% PEG6000 culture medium. We also treated the ginger plants under salt stress using a 200 mM NaCl solution, ensuring that the roots were fully exposed to the NaCl solution. Leaf samples treated with osmosis, salt, and low-temperature stress were harvested at 0, 1, 3, 6, 12, 24, and 48 h after treatment, and leaves were harvested at 0, 1, 3, 6, 12, and 24 h after treatment under heat stress. To ensure biological accuracy, each reaction was repeated three times. The collected leaf drugs were immediately frozen in liquid nitrogen and stored at −80 °C. The transcriptome data obtained by sequencing were visualized using HeatMap of TBtools [36].

Total RNA was extracted from samples under different treatments using the TRIzol kit (Invitrogen, Waltham, CA, USA), and cDNA was synthesized using the MonScriptTM RTIII ALL-in-mixed dsDNase kit (Monad, Suzhou, China) according to manufacturer’s instructions. The primers of ZoFWL genes were designed by Primer 5.0 software (Supplementary Table S1), and the response of ZoFWL to high temperature and strong-light stress was analyzed by qRT-PCR. The ZoTUB2 gene was used as the internal reference control, and the relative gene expression level was evaluated by 2^−∆∆Ct method. The qRT-PCR-specific experimental protocol involved an initial denaturation step of 30 s at 95 °C, followed by a series of 40 cycles. Each cycle comprised denaturation for 10 s at 95 °C and annealing for 30 s at 60 °C. All qRT-PCR assays were performed using three biological and technical replicates [45].

2.7. Statistical Analyses

We used Microsoft Excel 2016 to organize data, SPSS (IBM SPSS Statistics 27.0.1) software to perform data analysis under Duncan’s test (p < 0.05), and GraphPad (v10.1.2) for plotting.

3. Results

3.1. Identification and Sequence Analysis of the Ginger FWL Gene Family

The conserved domain PLAC8 (PF04749) of the FWL gene family was downloaded from the Pfam database. We used hmmsearch to search the bamboo ginger genome protein database, followed by sequence alignment using Blastp. The obtained sequences were then subjected to CD-Search, and we ultimately identified 21 ZoFWL genes with PLAC8 conserved domains in bamboo ginger. These genes were named ZoFWL1-ZoFWL21 based on their location on the chromosome. We analyzed the basic characteristics of these genes and their corresponding proteins (

Table 1. Information on the FWL family genes in Zingiber officinale.

Gene Name	Gene ID	Chromosome	Localization (bp)	Animo Acid Number	Molecular Weight (kDa)	PI	Subcellular Localization
ZoFWL1	Maker00025051	Chr06	70661613–70663046(+)	477	53.43	8.46	Cell membrane Nucleus
ZoFWL2	Maker00026518	Chr06	107790889–107801973(+)	239	26.76	5.03	Cell membrane Chloroplast
ZoFWL3	Maker00016179	Chr08	118773164–118778419(−)	551	62.51	8.02	Cell membrane
ZoFWL4	Maker00009473	Chr10	15788317–15794441(+)	239	26.58	4.88	Cell membrane
ZoFWL5	Maker00032137	Chr10	133341584–133342428(−)	123	13.24	4.46	Cell membrane
ZoFWL6	Maker00032080	Chr10	133355752–133356594(+)	203	21.90	6.34	Cell membrane
ZoFWL7	Maker00032144	Chr10	133496230–133497188(+)	204	22.30	5.69	Cell membrane
ZoFWL8	Maker00030347	Chr10	139765351–139766271(−)	204	22.36	5.87	Cell membrane
ZoFWL9	Maker00030271	Chr10	139875528–139876205(−)	167	18.08	6.37	Cell membrane
ZoFWL10	Maker00020626	Chr12	3849932–3854545(−)	357	40.03	6.92	Cell membrane Nucleus
ZoFWL11	Maker00042686	Chr12	14606275–14607401(+)	217	23.71	4.87	Cell membrane
ZoFWL12	Maker00035127	Chr14	122859462–122864489(−)	273	30.00	4.93	Cell membrane
ZoFWL13	Maker00014483	Chr14	135425628–135429693(−)	277	30.37	5.43	Cell membrane
ZoFWL14	Maker00004172	Chr16	2000297–2001809(+)	369	40.34	4.92	Cell membrane Nucleus
ZoFWL15	Maker00009037	Chr16	113827519–113842946(−)	737	83.68	6.11	Cell membrane Nucleus
ZoFWL16	Maker00012794	Chr18	40089638–40092331(+)	244	26.93	5.36	Cell membrane
ZoFWL17	Maker00028616	Chr18	82462280–82465007(+)	228	25.25	4.52	Cell membrane
ZoFWL18	Maker00022350	Chr20	4671208–4672823(−)	182	19.83	6.83	Cell membrane
ZoFWL19	Maker00022475	Chr20	4717902–4718711(−)	170	18.76	6.75	Cell membrane
ZoFWL20	Maker00026904	Chr20	24062166–24063058(+)	176	19.06	7.47	Cell membrane
ZoFWL21	Maker00001158	Chr22	173270323–173283255(+)	222	24.11	4.97	Cell membrane

The term “(−)” refers to the antisense strand of the chromosome; The term “(+)” refers to the sense strand of the chromosome.

).

The length of the protein encoded by the FWL gene family members ranges from 123 to 737 aa, with most falling within the 100–400 aa range. The isoelectric point range was 4.46–8.46, and the molecular weight range was 13.24–83.68 kDa. Among the ZoFWLs’ proteins, 18 are acidic proteins and 3 are alkaline proteins. The protein subcellular localization prediction results showed that most of the ZoFWLs family members are located on the cell membrane, with ZoFWL1, ZoFWL10, ZoFWL14, and ZoFWL15 located in both the cell membrane and nucleus, and ZoFWL2 located in the cell membrane and chloroplast. The subcellular localization results suggest that ZoFWL protein may perform various functions on the cell membrane.

To study the domain of ginger FWL protein, we compared the protein sequence of ZoFWLs, AtPCR1, OsFWL1, OsFWL4, SlFWL2.2, and ZmCNR1 (Figure 1). The results showed that, within the PLAC8 domain, all FWL domains were relatively conserved and consisted of two common protein motifs: CXXXXXCPC and QXXRELK. However, the CXXXXXCPC motif is incomplete in ZoFWL1, 3, 7, 8, 11, 12, 13, and 14. This may be due to the natural variation of different species in evolution, adapting to different natural environments.

3.2. Chromosome Localization Analysis of ZoFWL Genes

Utilizing the genome information of ginger, we mapped the chromosome localization (Figure 2). The chromosome localization analysis of the members of the ZoFWL gene family demonstrated that the 21 FWL genes of ginger are evenly distributed across nine chromosomes. There are six genes on chromosome Chr10; only two FWL genes on chromosomes Chr06, Chr12, Chr14, Chr16, and Chr18; one FWL gene on chromosomes Chr08 and Chr22; and three FWL genes on chromosome Chr20. ZoFWL5, ZoFWL6, ZoFWL7, ZoFWL8, and ZoFWL9 on chromosome Chr10, and ZoFWL18 and ZoFWL19 on chromosome Chr20 are positioned very closely to each other.

3.3. Phylogenetic Analysis of the ZoFWL Gene Family

In order to more clearly reveal the homology and evolutionary relationship of FWL proteins among different species, a total of 75 protein sequences were used to construct a phylogenetic tree using the maximum likelihood method, using 21 members of the FWL gene family, 12 members of the Arabidopsis PLAC8 family, 21 members of the tomato FWL family, 11 members of the rice FWL family, and 12 members of the maize CNR family (Figure 3). According to the classification results of the phylogenetic tree, FWL proteins can be divided into five subfamilies, which are divided into group A–group E. The FWL family members from the same species were clustered in different subfamilies, and each subfamily contained both monocots and dicots, indicating that they expanded after the common ancestor differentiation, and the emergence of these five plants was later than the FWL gene family. The subfamily D contains multiple genes that control cell division and affect the size of plant organs such as fruits, including typical SIFWL2.2, ZmCNR1, OsFWL1, and OsFWL3.

Simultaneously, to more distinctly elucidate the homology and evolutionary relationships among FWL proteins across various species, we employed the maximum likelihood method to construct a phylogenetic tree. This construction involved a total of 75 protein sequences. These sequences comprised 21 members from the FWL gene family, 12 from the Arabidopsis PLAC8 family, 21 from the tomato FWL family, 11 from the rice FWL family, and 12 from the maize CNR family (Figure 3). According to the classification results of the phylogenetic tree, FWL proteins can be divided into five subfamilies, labeled group A to group E. The FWL family members from the same species were clustered into different subfamilies, and each subfamily contained both monocots and dicots. This indicates that they expanded after the differentiation of the common ancestor, and the emergence of these five plants was later than that of the FWL gene family.

Subfamily D contains multiple genes that control cell division and affect the size of plant organs such as fruits, including typical SIFWL2.2, ZmCNR1, OsFWL1, and OsFWL3. This subfamily also contains seven ginger FWL proteins, namely ZoFWL6, ZoFWL7, ZoFWL8, ZoFWL9, ZoFWL18, ZoFWL19, and ZoFWL20. In subfamily B and subfamily E, there is only one ginger FWL member each, ZoFWL15 and ZoFWL5, respectively. In subfamily A, there are 10 ginger FWL family members, which are closely related to tomato FWL family members. Subfamily C contains two members, ZoFWL11 and ZoFWL14, which are similar to the homologous sequences of tomato.

The motif analysis of five plant FWL proteins showed that almost every member contains motif1, motif3, motif4, and motif5, indicating that these motifs were highly conserved. The main motif composition of the members in the same subfamily is similar, and there is a highly conserved motif structure. These results indicate that FWL proteins are widely present and diverse in plants, and FWL proteins maintain relatively high conservation during plant evolution. This suggests that FWL proteins may play similar biological functions in different plants.

3.4. Phylogenetic Evolution, Gene Structure, and Conserved Motif Analysis of ZoFWL Gene

To analyze the conserved domains of ZoFWL family protein members, we used the MEME online tool to search for the conserved domains of ZoFWL members and identified 10 conserved motifs (Figure 4B). Among the 21 members of the ZoFWL family, each member possesses motif1 and 2. Interestingly, the distribution of 1 and motif2 on each protein is similar. Motif1 is located at the N-terminus, motif2 is located at the C-terminus, and both motif1 and motif2 are identified as the characteristic domains of the PLAC8 protein. Most ZoFWL proteins have motif1, 2, 3, and 4. These results suggest that the PLAC8 domain is highly conserved in the evolution of ZoFWL proteins.

According to the phylogenetic tree analysis in Figure 3 and Figure 4A, each subfamily member has similar conserved motifs and distribution positions, and there are significant differences between different subfamilies. This phenomenon suggests that the ZoFWL protein may have differentiated during evolution and assumed different functions.

To study the structural composition of the ZoFWL gene, we performed a visual analysis of its gene structure (Figure 4C). The exon and intron structures of the ZoFWL gene indicate that most members of the ZoFWL gene family have 3–12 exons, but their exon lengths are relatively short. ZoFWL15 has 12 exons and 11 introns, the highest number. ZoFWL1 and ZoFWL3 have only one exon, and the number of exons of other members is evenly distributed at three to five. Although the number of conserved motifs, the length, and number of exons and introns of ZoFWL family members are different, the conserved motifs and gene structures of members in the same subfamily are highly conserved. This suggests that the biological functions of ZoFWL proteins have potential similarities.

3.5. Analysis of Cis-Acting Elements of the ZoFWL Gene Family

The cis-acting elements of gene family promoters play a crucial role in regulating gene expression. To investigate the potential role of the ZoFWL gene family in the growth and development of ginger, we analyzed the promoter region of the ginger FWL gene family and identified 19 intriguing cis-acting elements (Figure 5). These primarily include environmental-response elements and hormone-response elements, mainly studied for their regulatory mechanism in the growth and development of ginger and abiotic-stress response.

The results showed that most promoters of the ZoFWL gene contained hormone-response elements such as the abscisic acid-response element, gibberellin-response element, auxin-response element, jasmonic acid-response element, and salicylic acid-response element. Additionally, there are light-response elements and MYB binding sites involved in light response. Simultaneously, there are cis-acting elements responsive to meristematic activity and endosperm development. The genes ZoFWL1, 7, 8, 16, and 21 contain cis-acting elements responsive to endosperm development, while ZoFWL5, 7, 9, 11, 12, 15, and -21 contain cis-acting elements responsive to meristematic activity. Furthermore, it also contains low-temperature-response elements, anaerobic-inducible elements, MYB binding sites involved in defense from stress, and osmotic induction. These cis-acting elements of ZoFWL suggest that members of the ZoFWL gene family may play a significant role in regulating ginger growth and development, metabolic activities, and response to abiotic stresses.

3.6. Genome-Wide Duplication Events and Collinearity Analysis of ZoFWLs

In an effort to further explore the evolutionary process of the ginger FWL family, we analyzed the genome-wide duplication events of the ZoFWL gene family, with the results presented as follows (Figure 6A). The analysis revealed that no tandem duplication event was detected, but a total of three pairs of ZoFWL genes were found to have segmental duplication activities, namely ZoFWL17 and ZoFWL10, ZoFWL18 and ZoFWL5, and ZoFWL8. This suggests that these genes may have originated from segmental duplication events, underlining the significant role of these events in the differentiation and amplification of the ZoFWL gene family.

To study the phylogenetic mechanism of the ginger FWL gene family, we constructed the gene collinearity relationships between ginger and model plants such as tomato, small-fruited wild banana, and maize. The results are as follows (Figure 6B). The gray line signifies all genes with collinear relationships between the two plants, and the red line represents the FWL genes. We analyzed the FWL gene of ginger with most plants in the Ensembl Plants database. Ginger showed homology only with 18 ZoFWL genes in Musa microcarpa and had no collinearity with model plants such as Arabidopsis thaliana, tomato, rice, and maize.

3.7. Expression Patterns of ZoFWL Genes in Different Ginger Organs

We examined the expression patterns of ZoFWL genes across various developmental stages of ginger rhizomes. Utilizing RNA-seq data, we analyzed the expression profile of ZoFWL (Figure 7). ZoFWL13 was highly expressed in ginger roots and stems, primary rhizome buds, secondary rhizome buds, tertiary rhizome buds, fourth rhizome buds, and fifth rhizome buds. ZoFWL3, ZoFWL6, ZoFWL9, ZoFWL16, and ZoFWL18 exhibited higher expression levels in stems, while ZoFWL2, ZoFWL4, and ZoFWL21. ZoFWL2, ZoFWL3, ZoFWL6, ZoFWL9, ZoFWL15, and ZoFWL16 demonstrated varying expression levels across the five ginger rhizome buds. Genes that were not expressed in organs may be pseudogenes or may not be expressed in the underground rhizomes of ginger.

Interestingly, the genes expressed across the five rhizome bud stages, from the first to the fifth rhizome bud, exhibited a gradual increase in expression level, with the expression level in the fifth rhizome bud being significantly higher than that in the first rhizome bud. The expression of ZoFWL genes in rhizome buds suggests that they may play a role in the growth and development of ginger tubers and regulate rhizome bud growth. The expression patterns of ZoFWL genes in different ginger rhizome indicated their potential functions in plant growth and development, laying the groundwork for further investigation into the functions of ZoFWL genes and FWL genes in other species.

3.8. Expression Patterns of ZoFWL Genes under Abiotic Stress

In order to investigate the potential function of the ZoFWL gene family under abiotic stress, we subjected ginger to four types of abiotic stresses. We analyzed the RNA-seq data from ginger at 0, 1, 3, 6, 12, 24, and 48 h after cold, osmotic, and salt stress, and at 0, 1, 3, 6, 12, and 24 h after heat treatment (Figure 8). The ZoFWL genes exhibited diverse expression patterns following the different stress treatments.

Under heat-stress treatment (Figure 8A), ZoFWL2, 4, 6, 9, 16, 18, and 21 shared a similar expression trend, showing an overall upward trajectory. They peaked at 6 h, decreased at 12 h, and reached a maximum peak at 24 h. Notably, the expression levels of ZoFWL6, 9, and 18 under heat stress were significantly higher than those of other members, while ZoFWL5, 7, 8, and 11 were almost not expressed at each time point.

Under cold stress (Figure 8C), ZoFWL genes exhibited a downward trend. Compared to the control group at 0 h, ZoFWL2, 3, 4, 6, 9, 15, 16, 19, and 21 at 48 h were significantly lower than at 0 h. ZoFWL2, 6, 9, 15, and 19 expression increased significantly after 1 h of cold treatment; almost all increased by 2–3 times, reaching the maximum peak of the whole process. ZoFWL12, 13, 14, and 18 showed an upward trend under cold stress, peaking at 48 h.

The ZoFWL genes showed a less pronounced response to osmotic stress (Figure 8B). ZoFWL2, 3, 4, 12, 15, 16, and 21 displayed a downward trend, reaching their lowest at 48 h. ZoFWL6, 9, 3, 18, and 19 showed an upward trend, with ZoFWL6, 9, 13, and 19 reaching their maximum peak at 24 h, almost 2–3 times higher than at 0 h, and then decreased.

Under salt-stress treatment (Figure 8D), most ZoFWL genes showed an upward trend, with ZoFWL1, 2, 3, 4, 6, 9, 13, 14, 16, 18, 19, and 21 showing varying degrees of expression increase. ZoFWL2, 6, 9, 13, and 16 peaked at 12 h, increasing by 2–3 times. ZoFWL10, 12, 15, and 17 showed a downward trend. Interestingly, ZoFWL18 showed an upward trend under all four stresses, increasing by 13 times at 24 h compared to 0 h under heat stress. We speculate that ZoFWL18 plays a positive regulatory role in ginger’s response to abiotic stress, aiding ginger in resisting such stresses.

We also examined the expression patterns of ginger under natural high-temperature and intense-light stress by qRT-PCR (Figure 9). Most ZoFWL genes, such as ZoFWL3, 4, 6, 9, 14, and 18, showed a downward trend under high-temperature and intense-light stress. They all followed the same expression trend, showing a decrease initially, then an increase, reaching the lowest value on the second day of stress treatment, and gradually increasing on the third and fourth days. ZoFWL2, 13, 16, and 21 were higher than the control group on the fourth day after treatment.

These findings suggest that, in the early stages of stress treatment, gene expression is inhibited by stress. Over time, plants initiate their own protective mechanisms to adapt to and even resist environmental stress. This research may help develop strategies to improve ginger’s tolerance to abiotic stress and provide a foundation for subsequent molecular breeding of ginger.

3.9. Prediction of ZoFWL Protein Interaction Network

In order to further investigate the function of ZoFWL genes, we utilized String to predict proteins interacting with ZoFWLs (the species model was Arabidopsis thaliana), and the entire ginger genome was used to construct a differentially expressed protein interaction network (Figure 10). The results showed that a total of eight members from the ZoFWL gene family were detected to have protein interactions. ZoFWL15 had strong interactions with 38 proteins; ZoFWL5 interacted with 34 proteins; ZoFWL19 and ZoFWL21 interacted with 22 and 20 proteins, respectively; ZoFWL14 and ZoFWL17 interacted with 18 proteins; and ZoFWL4 and ZoFWL20 interacted with 14 proteins. The prediction of interaction networks is of great significance for studying gene functions. It can be speculated that members of the ZoFWL gene family may interact with these proteins in regulating certain physiological functions, providing a reference for subsequent research on ZoFWL proteins.

4. Discussion

Currently, substantial progress has been made by researchers in understanding the genetic regulation of the fruit-weight-2.2-like gene on fruit size in model plants such as tomato [26], Arabidopsis [22], rice [13], and maize [1]. The plant FWL gene family comprises fruit-weight-2.2-like genes and a protein family with a PLAC8 domain, which is a crucial component controlling cell division and organ growth [2]. A genome-wide analysis of the FWL gene family has been broadly conducted in many genome-sequenced species [46], but the number of FWL gene family members varies among different species [47].

In the present study, we identified 21 ZoFWL members in the ginger genome, which are rich in Cys and contain a PLAC8 structure. These are distributed across nine chromosomes of ginger and are named ZoFWL1 to ZoFWL21 according to their location on the chromosome (Figure 2). The shortest and longest ZoFWL proteins are 123aa and 737aa, respectively. Subcellular localization prediction indicated that ZoFWLs are mainly localized on the cell membrane, which aligns with the results from tomato and rice studies [13,26]. However, in this study, ZoFWLs are not entirely located on the cell membrane (Table 1). ZoFWL1, 10, 14, and 15 are predicted to be both membrane proteins and nucleoproteins, and ZoFWL2 is predicted to be both a membrane protein and a chloroplast protein. It is more common to find proteins with dual localizations [48]. Due to the limited accuracy of prediction software, the exact location of ZoFWL members needs to be determined experimentally. In tomato, FW2.2 is identified as a membrane protein [8], which can interact with the regulatory subunit of CKII near the plasma membrane, thereby affecting the transduction of plant cell-cycle signals [10,49]. Furthermore, GmFWL1 is located in the membrane microstructure domain [11], which has biological functions such as intercellular communication and signal transduction [50]. The growth and development of ginger’s underground rhizomes is a complex biological process. The development of the root system is closely related to signal transduction on the cell membrane, particularly the regulatory mechanism of plant hormone-signaling pathways and environmental factor responses [51]. Therefore, it is speculated that members of ZoFWLs may be involved in mediating intercellular signaling activities, and the specific regulatory mechanism requires further experimental analysis.

PLAC8 is a conserved domain of the FWL gene family, and its function is highly related to PLAC8. It consists of CXXXXXCPC and QXXRELK, but alterations in certain amino acids in these two motifs might lead to changes in protein function [52]. Currently, researchers mainly analyze the function of CXXXXXCPC, and there are few studies on the function of QXXRELK. AtPCR1, located on the plasma membrane, has a CXXXXCPC that confers AtPCR1 resistance to Cd, while AtPCR8, which lacks CXXXXCPC, shows no Cd resistance [16]. OsFWL4, which also possesses this motif, is involved in the regulation of rice-plant tillering and yield, and also participates in the transport of the heavy metal cadmium [18,53]. OsFWL1 can regulate the length of rice grains [53], and ZmCNR1 can regulate the number of cells to affect the size of maize organs [54]. They also contain a CXXXXXCPC structure. The functions of the genes in the branch of the multi-species phylogenetic tree are similar [55]. In the multi-species phylogenetic tree that we constructed, ZoFWL6, 9, 18, 19 show close homology to OsFWL1, while ZoFWL20 is closely related to ZmCNR1. ZoFWL5, AtPCR1, and OsFWL4 are in the same subfamily, and they all possess a CXXXXXXCPC motif (Figure 1). We speculate that they potentially function in regulating ginger cell division and heavy metal resistance. The specific regulatory functions need further experiments, such as yeast hybridization experiments. In the genus Physalis, PfCNR1 has the CWXXXXCPC motif, which controls the size of berries and seeds by regulating the cell cycle [56]. In ginger, ZoFWL2, 4, 10, and 17 have this motif, which can be used to explore their effects on the ginger cell cycle in future studies.

To study the evolutionary relationship between the ginger FWL gene and other plant FWLs, we constructed an interspecific phylogenetic tree of ginger FWL with two dicotyledonous plants (Arabidopsis thaliana and tomato) and two monocotyledonous plants (maize and rice) and analyzed their conserved motifs (Figure 3). Each branch contains both monocotyledonous and dicotyledonous plants, indicating that the FWL gene existed in these two types of plants before differentiation and shares a common ancestor [57]. A motif analysis of five plant FWL proteins showed that almost every member contained motif1, motif3, motif4, and motif5, suggesting that FWL is conserved in both monocots and dicots. However, our collinearity analysis (Figure 6B) showed that the ZoFWL gene was most closely related to the genome of Musa acuminata, resulting in 18 pairs of collinearity genes and 0 pairs of collinearity genes with other plants in the Ensembl Plants database. This phenomenon is quite common. Due to species morphology and the need to adapt to the growth environment, homologous genes have mutated during evolution. Homologous genome regions may lose gene collinearity due to sequence changes and the insertion of different transposable elements. In addition, gene replication events can generate new gene copies, which are mutated or functionally divergent due to evolutionary pressures, disrupting their collinearity on chromosomes [58,59]. In summary, the ZoFWL gene may have undergone family expansion or gene rearrangement events during long-term evolution, resulting in ZoFWL having no collinearity with most plants.

Genome duplication, tandem duplication, and segmental duplication are considered to be the main driving forces for the expansion of gene families, and they are the primary factors that increase the number and functional complexity of gene family members [60,61]. In this study, we identified three pairs of duplicate genes: ZoFWL17 and ZoFWL10, ZoFWL18 and ZoFWL5, and ZoFWL8 (Figure 6A). The expansion of the ZoFWL gene family is mainly driven by segmental duplication, with no events of tandem duplication found. These genes are located in the same subfamily in the phylogenetic tree, but their motif compositions differ. ZoFWL5 lacks motif2, 7, and 9, and ZoFWL17 and ZoFWL10 differ in regard to motif3 (Figure 4B). Combined with an expression-pattern analysis, the expression patterns of these duplicated ZoFWLs in ginger organs were found to be distinct. ZoFWL18 is highly expressed in ginger stems, but ZoFWL5 is expressed at a very low level, or not at all, in stems (Figure 7). The expression level of ZoFWL17 in various organs of ginger rhizome is higher than that of ZoFWL10, indicating the redundant cellular functions of these duplicate genes during ginger development [62]. The motif composition of ZoFWL18 and ZoFWL8 is the same, but their expression patterns in ginger differ. This may be due to subtle gene mutations during gene amplification, and changes in motif composition may also lead to functional differences [63].

There is a close relationship between gene function and gene structure [64]. Analyses of gene structure and conserved motifs are important for understanding gene function and classification. The structural distribution of ZoFWLs in the same subfamily is very similar. Most members have from 3 to 12 exons, which are relatively evenly distributed (Figure 4C). ZoFWL15 is the longest gene and has the most introns and exons, suggesting that ZoFWL15 may be an early member of ZoFWLs. Through processes such as alternative splicing, functional diversification and complexity are achieved, increasing the regulatory flexibility and diversity of ZoFWL15 expression [65]. Conserved motif analysis showed that almost every member had motif1 and 2, and genes with the same motif arrangement were clustered in the same subgroup (Figure 4B), which was consistent with the evolutionary classification. In conclusion, these unique gene structures have a significant influence on protein function.

Cis-acting elements are critical factors in plant growth, development [66], and environmental adaptation, significantly influencing the expression of downstream genes. We analyzed the cis-acting elements of the 2000 bp region upstream of the coding region of ZoFWL gene family members, identifying a variety of cis-acting elements (Figure 5). These include low-temperature-response elements, anaerobic-induction elements, MYB binding sites involved in defense and stress responses, and drought-induction elements. Additionally, we found light-response elements and various hormone-response elements, such as abscisic acid-response elements, gibberellin-response elements, auxin-response elements, jasmonic acid-response elements, and salicylic acid-response elements. The role of abscisic acid in responding to various abiotic stresses like drought has been well-documented [67]. Concurrently, jasmonic acid is a crucial signaling molecule that regulates plant resistance to biotic stress [68]. The ZoFWL gene family also possesses meristem regulatory elements and endosperm-expression regulatory elements, with the promoter carrying endosperm-expression regulatory elements involved in regulating the specific expression of genes related to plant germination [69]. Thus, we speculate that ZoFWLs not only participate in plant growth and development but also actively contribute to ginger’s resistance to abiotic and biotic stresses.

Protein–protein interaction networks can often predict unknown functional proteins, aiding in the understanding of biological activities and dynamic interaction networks between biomolecules [70]. In this study, we constructed a predictive ZoFWL protein interaction network (Figure 10). The results indicate that, besides ZoFWL itself, TPR10 and MSL2 are the most central nodes. TPR proteins mediate protein–protein interactions in a variety of biological systems, including cell-cycle control, transcription, protein transport, and protein folding [71]. In Arabidopsis, AtTPR10 is induced by heat in roots and stems [72]. Concurrently, studies have shown that AtTPR10 is highly expressed under heat shock, acting as a molecular chaperone to protect intracellular proteins against heat stress [73]. MSL2, located on the plastid membrane, maintains plastid osmotic homeostasis and plays a role in communication between plastids and mitochondria, thus influencing plant seedling development [74]. Moreover, MSL2 can control the size and shape of the plastid by altering ion flux [75]. In summary, ZoFWL genes may have the potential function of resisting heat stress and regulating plant growth and development, consistent with the function of FW2.2. This also suggests that ZoFWL may regulate the growth of ginger. However, the specific regulatory mechanisms need to be confirmed through experimental verification.

The FWL gene family and the protein family containing the PLAC8 structure are pivotal in regulating plant cell division and organ growth [1]. AtMCA2 is expressed in the primary-root vascular tissue and shoot apical meristem [76]. ZmCNR1 governs maize plant height and panicle and leaf size [8]. The rice FWL gene family controls rice plant height and grain length [13]. The underground rhizome of ginger, its main harvest and edible part, has its degree of rhizome expansion as an important index to evaluate its economic value. Therefore, we analyzed the expression patterns of ZoFWL in different developmental stages and organs of ginger rhizomes (Figure 7). We observed that ZoFWL13 was significantly expressed in ginger roots and fifth-order rhizome buds, with a low expression level in stems. Its FPKM was significantly higher than other ZoFWL members, reaching up to 120,000. The expression level of ZoFWL13 in roots and rhizome buds increased gradually with the growth order. The expression level in rhizome buds was higher than in roots, and it increased progressively in first-order rhizome buds, second-order rhizome buds, and third-order rhizome buds; then, it gradually decreased in fourth-order and fifth-order rhizome buds. The expression level in stems was much lower than in roots and rhizome buds. The expression levels of ZoFWL2, 3, 6, 9, 15, and 16 in buds were higher than those of other members. Overall, the expression levels in ginger rhizome buds showed an upward trend. Additionally, we also found endosperm-expression regulatory elements in the promoter region of ZoFWL16, and ZoFWL9 and 15 had meristem regulatory elements. This finding suggests that ZoFWL2, 3, 6, 9, 15, 13, and 16 may be involved in regulating the development of ginger rhizome buds, especially ZoFWL13, a highly expressed gene in ginger rhizome buds. The specific regulatory mechanism of this gene requires further experimental research.

Plants are highly susceptible to abiotic stresses, such as osmotic, salinity, extreme-temperature, and heavy-metal stress. These stressors greatly limit the growth and development of plants and reduce crop productivity. Abiotic stress severely threatens agricultural production and leads to extreme deterioration of the ecological environment. Currently, the research on the abiotic stress of the FWL gene family mainly focuses on resistance to heavy metals [77]; there are precious few reports on extreme gas, osmotic, and salt stress. In this study, we performed low-temperature-, heat-, osmotic-, and salt-stress treatments on ginger and obtained their transcriptome data. Under heat stress, ZoFWL2, 4, 6, 9, 16, 18, and 21 were significantly upregulated. Under low-temperature stress, ZoFWL2, 3, 4, 6, 9, 15, 16, 19, and 21 were significantly downregulated. The expression trends of ZoFWL2, 4, 6, 9, 16, and 21 under low-temperature and heat stress were opposite. Under osmotic stress, ZoFWL2, 3, 4, 12, 15, 16, and 21 showed a downward trend and reached the lowest at 48 h. ZoFWL6, 9, 13, 18, and -19 showed an upward trend. In salt stress, the vast majority of members, including ZoFWL1, 2, 3, 4, 6, 9, 13, 14, 16, 18, 19, and 21, showed an upward trend. Among them, we found that the expression of ZoFWL18 was much higher than that of other genes in four stresses, and the expression showed an upward trend in all four treatments, especially in heat stress, where the expression level increased by 13 times at 24 h. We speculate that ZoFWL18 is a key regulatory factor in the response of ginger to abiotic stress. It is the result of ginger’s selection of various abiotic stresses in the long-term evolutionary process, which enhances the survival and reproduction of ginger. Additionally, high-temperature and strong-light qRT-PCR analyses showed that the expression of the ZoFWL13 gene was significantly upregulated under high-temperature and strong-light stress. At the same time, the expression of ZoFWL13 in ginger buds was much higher than that of other genes. The ZoFWL13 gene may have important regulatory functions and protective effects in the adaptability and development of ginger.

5. Conclusions

In conclusion, our study successfully mapped 21 FWL genes within the ginger genome that are spread across nine distinct chromosomes. The phylogenetic analysis categorized ZoFWLs into five subfamilies, with a conserved gene structure observed within each subfamily. Through a comprehensive examination of cis-regulatory elements, organ-specific expression patterns, and responses to abiotic stress, we observed a progressive increase in the expression of most ZoFWL genes in ginger rhizome buds, with ZoFWL13 exhibiting a notably higher expression compared to its counterparts. The upsurge in the expression of ZoFWL13 under conditions of high temperature and intense light suggests its potential role in the development of ginger rhizome buds and in the plant’s adaptive response to these stressors. Moreover, our findings indicate that ZoFWL genes are broadly responsive to abiotic stresses, particularly ZoFWL18, which demonstrated a significant and consistent upregulation across various stress conditions, hinting at its central role in abiotic-stress resistance.

Our research contributes to a deeper understanding of the molecular functions of ZoFWL genes in ginger’s growth, development, and stress-response mechanisms. It lays the groundwork for future work on gene cloning and functional studies to further elucidate the role of ZoFWL genes in ginger rhizome development and stress resistance. This knowledge provides a valuable foundation for the breeding of ginger varieties with enhanced resistance to environmental stresses, with potential applications in agricultural biotechnology and crop improvement strategies.