Identification of XTH Family Genes and Expression Analysis of Endosperm Weakening in Lettuce (Lactuca sativa L.)

Abstract

Seed germination requires the relaxation of endosperm cap and radicle cell walls, with cell wall hydrolases playing a significant role in this process. Our study revealed that a type of cell wall hydrolase, xyloglucan endotransglucosylase, may significantly contribute to endosperm weakening during lettuce seed germination. Through bioinformatics analysis, the XTH gene family in lettuce was divided into five subfamilies localized on nine chromosomes. Notably, there were significant differences in gene structure among the members of the LsXTHs family containing 1–4 exons and 20 conserved motifs. Among these motifs, motif1, motif2, and motif3 encoded the XTH structural domain. The promoter regions of LsXTHs contained a large number of cis-acting elements responsive to various abiotic stresses, such as drought, anaerobiosis, low temperature, high temperature, and salt stress. Germination experiments showed that seeds imbibed in water and 5 μmol/L abscisic acid (ABA) were able to achieve typical germination with radicle protrusion from the endosperm cap, achieving germination of 100% and 36%, respectively. Conversely, in 0.3% sodium dichloroisocyanurate (SDIC), the swollen seeds were unable to germinate or complete atypical germination, resulting in a germination rate of 30%. Compared to the control, the mechanical strength of the endosperm cap of seeds imbibed in 0.3% SDIC for 8 h increased by 14%, indicating that SDIC may inhibit seed germination by enhancing the mechanical strength of the endosperm cap. Enzyme activity analysis revealed that during lettuce seed germination, XTH enzyme activity in the endosperm cap was significantly higher than in other tissues and increased gradually with imbibition. Transcriptome analysis of the endosperm cap detected the expression of 10 LsXTH genes. Among these, LsXTH43 exhibited the highest expression during germination and was significantly upregulated two-fold by high temperatures, suggesting a potential role in the high-temperature germination of lettuce seeds. Additionally, SDIC downregulated the expression of LsXTHs to varying degrees, with the expression of LsXTH15 reduced to only 6% of its original level. Low temperature, high temperature, drought, and salt stress all reduced the expression of most LsXTHs to different degrees; when seeds germinated under waterlogging and cadmium stress, LsXTH6, LsXTH7, LsXTH8, LsXTH32, and LsXTH33 were all upregulated to some extent.

1. Introduction

Lettuce (Lactuca sativa L.) is an annual or biennial herb of the Asteraceae family, which is widely planted and loved by people all over the world. Seeds are important reproductive organs in lettuce cultivation, and the quality of seed germination directly impacts seedling emergence and yield. However, lettuce seed germination is sensitive to high temperature and light [1,2]. Moreover, the seedlings exhibit weak resistance to various abiotic stresses such as waterlogging and salt stress during the emergence phase. Therefore, it is crucial to enhance seed germination and seedling emergence rates under various cultivation environments to ensure lettuce yield.

The weakening of the endosperm (coleorrhiza) and elongation of the radicle are essential events for seed germination, and the relaxation of the cell wall is a prerequisite for both processes [3]. Many enzymatic and non-enzymatic factors play an important role in cell wall relaxation during seed germination [4,5,6]. The plant cell wall primarily comprises polysaccharides such as cellulose, hemicellulose, and pectin. Cell wall relaxation is facilitated by the action of various hydrolytic enzymes such as cellulases, hemicellulases, xyloglucan endotransglycosylase/hydrolases (XTHs), and pectin methylesterases, which break the glycosidic bonds between polysaccharide molecules, converting polysaccharides into monosaccharides [7]. XTHs is a key enzyme involved in the degradation of hemicellulose in the cell wall. This process occurs by breaking the β-1,4-glycosidic bond in the main chain of the donor hemicellulose molecule, resulting in the generation of a reducing end and a non-reducing end [7]. Some members of the XTHs family possess the function of xyloglucan hydrolase, which integrates the newly generated reducing end onto water molecules [8]. Others exhibit the function of xyloglucan transglycosylase, which recombines the newly generated reducing end through the β-1,4-glycosidic bond onto the non-reducing end of another receptor xyloglucan molecule or subunit oligosaccharide molecule, thus completing the relaxation of the endosperm cell wall [9,10]. XTHs have been identified in various plants, and the number of family genes varies among different species. For example, there are 33 in Arabidopsis thaliana [11], 29 in rice [12], 56 in tobacco [13], 24 in barley [14], 25 in tomato [15], and 27 in peach [16]. In Brassica rapa L. and Brassica oleracea L., all XTHs can be classified into three groups: Group I/II, Group III, and the Early-Diverging Group. Gene structures and motif patterns have been found to be similar within each group. All XTHs in this study contained two characteristic conserved domains: Glyco_hydro and XET_C. These XTH proteins were mainly located in the cell wall, but some were also present in the cytoplasm [17].

Numerous studies have indicated that XTHs are closely associated with fruit ripening and softening [18], root growth [19], and rice jointing [20]. During seed germination, Chen et al. [21] cloned the LeXET4 gene from the endosperm at the bead pore end of tomato seeds and found that the expression of this gene gradually increased with seed imbibition and reached a peak just before germination. Silva et al. [22], while studying the molecular weight characteristics and mode of action of XTH in seeds of the Brazilian savanna plant, found that XTH degraded the storage hemicellulose in germinated seeds, relaxed the cell wall, and promoted the elongation growth of seedlings. During the germination of Podophyllum hexandrum seeds at high altitude, XTH facilitated germination by upregulating PhXET protein through GA-mediated endosperm weakening [23]. The transient expressions PavXTH14, PavXTH15, and PavPG38 in cherry fruits significantly reduced the fruit firmness. Additionally, the content of various cell wall components, including hemicellulose and pectin, was significantly altered in the transgenic fruit [24].

Pavlišta and Haber (1970) reported an atypical germination phenomenon in lettuce [25]. They found that the radicle of lettuce seeds treated with SDIC does not protrude from the tip of the micropylar endosperm (also called endosperm cap), but instead emerges from midway between the micropylar and cotyledonary ends. In some seeds, the embryos expanded without radicle protrusion, resulting in embryo buckling within the endosperm. Therefore, SDIC treatment provides an ideal model to investigate the mechanisms underlying the weakening of the endosperm cap and the elongation growth of the radicle, as SDIC can separate these two processes. Zhang et al. (2014) also confirmed the existence of endosperm weakening during seed germination with SDIC [4]. ABA is a well-known plant hormone that inhibits seed germination. Unlike SDIC, it not only inhibits radicle elongation, but also prevents endosperm weakening to some extent [5,26,27].

The weakening of the endosperm during seed germination is inseparable from the relaxation of the endosperm cell walls by hydrolytic enzymes. Currently, there is a lack of research on the identification of XTH family genes encoding cell wall hydrolytic enzymes in lettuce and their expression during seed endosperm weakening. This study aims to identify the members of the lettuce cell wall hydrolase XTH gene family and analyze their characteristics using bioinformatics methods. Additionally, it seeks to confirm the role of XTH in endosperm weakening through morphological, physiological, and other methods. Furthermore, the study intends to analyze the expression of XTHs in the seed endosperm cap during the germination process using transcriptome sequencing and qRT-PCR, thereby providing a theoretical basis for the investigation of the function of LsXTHs in lettuce seed germination.

2. Materials and Methods

2.1. Identification and Physicochemical Properties of LsXTHs

The genome sequences CDS and gff3, and the protein sequences of lettuce, Arabidopsis thaliana, rice, and Chinese cabbage were download from the Ensembl database (http://plants.ensembl.org/index.html accessed on 5 June 2023). From the Pfam database (http://pfam.xfam.org/ accessed on 5 June 2023), the conservative domain files PF00722 (Glyco_hydro_16) and PF06955 (XET_C) were obtained as templates. These sequences were then aligned using HMMER3.0 software with default parameters to identify potential members containing conservative domains. It is noteworthy that Glyco_hydro_16 exhibits hydrolase activity, while XET_C displays xylan endo-transglycosylase activity. To prevent missing target protein sequences, BlastP searches in the NCBI database (https://www.ncbi.nlm.nih.gov/ accessed on 5 June 2023) were used with the keyword “xyloglucan endotransglucosylase/hydrolase NOT putative AND plant” to retrieve XTH candidate members. Upon combining the results from both methods, duplicate sequences were eliminated and the conserved domains of the candidate protein sequences were validated using SMART (http://smart.embl.de/ accessed on 5 June 2023), NCBI CDD (https://www.ncbi.nlm.nih.gov/cdd/ accessed on 5 June 2023), and Pfam (https://www.ebi.ac.uk/interpro/ accessed on 5 June 2023). Finally, the candidate members lacking the XTH domain were excluded to obtain all members of the XTH gene family.

To predict the characteristics of lettuce XTH family proteins, we used ExPASy ProtParam (https://web.expasy.org/protparam/ accessed on 5 June 2023) to calculate the number of amino acids, relative molecular weight, theoretical isoelectric point, and total average hydrophobicity. Additionally, subcellular localization analysis was conducted with Cell-PLoc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/ accessed on 5 June 2023) under default parameters.

2.2. Phylogenetic Tree Construction, Genomic Structure, and Conserved Motif Analysis of LsXTHs

The XTH family protein sequences of lettuce, Arabidopsis, and rice were aligned to multiple sequence alignment using Clustal W. Subsequently, a phylogenetic tree was constructed using MEGA 7.0 software with the Neighbor-Joining method, selecting the poisson model, partial deletion, and 1000 repetitions. Finally, Evolview 2.0 (https://evolgenius.info//evolview-v2/ accessed on 5 June 2023) was utilized to beautify the visualization of the evolutionary tree.

The gene structure of lettuce was analyzed using the GSDS 2.0 database (http://gsds.gao-lab.org/ accessed on 5 June 2023). The distribution patterns of exons and introns within the XTH genes were depicted using TBtools software (TBtools (Toolbox for Biologists) v1.098765). To identify conserved motifs in the proteins encoded by the XTH gene, the online MEME tool v5.5.5. (http://meme-suite.org accessed on 5 June 2023) was employed, with a maximum of 20 motifs and default values for other parameters. Finally, the motif results were visualized using the TBtools software.

2.3. Chromosomal Localization and Collinearity Analysis of LsXTHs

The chromosomal location information of XTH was extracted from the gff3 file containing the lettuce gene information and visualized to delineate the chromosomal distribution details of LsXTHs using the MapChart mapping software (Mapchart 3.23). The MCScanX function of TBtools software was employed to align the lettuce whole-genome sequence and analyze the duplication events of LsXTH family members. The relationship between duplicate gene pairs was then visualized through the Circos function embedded within the TBtools software.

The Circos function of TBtools software was utilized to calculate and draw the tandem repeats of XTH on chromosomes, as well as the collinear genes between different chromosomes of lettuce. The MCScanX function in TBtools was employed to identify collinear genes between lettuce and the two comparative species, Arabidopsis and cabbage. The Multiple Synteny Plot function was then utilized to visualize the collinear relationship of XTH homologous genes.

Two homologous genes separated by five or fewer genes were identified as tandem duplicates, while two genes separated by more than five genes or located on different chromosomes were considered segmental duplicates. Further identification of homologous genes was conducted using a phylogenetic tree, with criteria for identification including a homologous sequence coverage > 75% and homology > 75% [28].

2.4. Protein Secondary and Tertiary Structure

The secondary structure of the lettuce XTH protein was predicted using the SOPMA program (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html accessed on 5 June 2023). Subsequently, to gain insights into its tertiary structure, the online tool Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index accessed on 5 June 2023) was employed. Finally, the PyMOL2.4 software was utilized for visual refinement of the predicted tertiary structure.

2.5. Cis-acting Element Analysis

The promoter sequences upstream of the ATG start codon in LsXTHs were extracted using TBtools software. The cis-acting elements were then analyzed using the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ accessed on 5 June 2023). Finally, the distribution of cis-acting elements in the promoter region of LsXTH members was visualized using TBtools software.

2.6. Typical and Atypical Seed Germination

In this study, the experimental material was “Gua Si Hong”, and the research was carried out at South China Agricultural University and Guangdong Academy of Agricultural Sciences. Lettuce seeds were placed in a 10 cm diameter culture dish lined with filter paper, and 10 mL of double-distilled water, 5 μmol/L ABA, or 0.3% SDIC solution was added. The seeds were incubated in a constant-temperature incubator at 22 °C (Shanghai, China), with a light intensity of 10,000 lux and equal light and dark periods of 12 h each. The rupture of endosperm and seed germination was observed using a stereomicroscope (Zeiss, Oberkochen, Germany). Seeds with radicle emergence from the endosperm cap were considered typical germination, while those with emergence from other locations were considered atypical germination.

2.7. XTH Enzyme Activity Determination

To collect samples of theendosperm cap, cotyledon tip endosperm, cotyledon, and radicle, lettuce seeds were soaked in water for 6, 12, 18, and 24 h. Lettuce tissues were then added to a pre-cooled 40 mmol/L sodium acetate extraction buffer at a ratio of 0.5:1 (w/v), ground into a homogenate on ice, transferred to a 2 mL centrifuge tube, and were extracted through ice bath shaking for 18 h. After centrifugation at 4 °C, 12,000 rpm, for 15 min, the supernatant was carefully aspirated to obtain the crude extract enzyme. The protein content of the crude extract enzyme solution was determined using the Coomassie Brilliant Blue method. XTH activity was measured using a modified version of the Miedes and Lorences methods [29]. First, the substrate solution was preheated to 37 °C in a constant-temperature incubator. Then, 200 μL of the crude extract enzyme solution was mixed with the substrate solution, and 0.2 mL of this mixture was aspirated with a pipette tip to record the initial time T_o for the outflow of 100 μL. The mixture was incubated at 37 °C for 24 h to ensure a sufficient reaction. The time T required for 100 μL to flow out was calculated again using the same method as before. The relative XTH enzyme activity was represented by [(1 − T/T_o) × 100% ÷ protein content].

2.8. Viability and Mechanical Strength Determination of the Endosperm Cap

After being soaked in water and 0.3% SDIC solution for 12 h, the endosperm cap of lettuce seeds was separated. The endosperm cap was then immersed in a 0.5% TTC solution at a temperature of 35 °C for 1 h to stain it. The viability of the endosperm cap was evaluated based on the intensity of staining.

The mechanical strength of the endosperm cap was determined following the methods described by Steinbrecher and Leubner-Metzger [30] and Zhang et al. [4]. Seeds swollen in water, 5 μmol/L ABA, and 0.3% SDIC for 6, 8, 10, 12, 14, and 18 h were used to isolate the endosperm cap. Subsequently, a fruit and vegetable hardness tester (Instron, Norwood, MD, USA) was used to measure the mechanical force required to penetrate the endosperm cap with a steel needle. This measurement provided a quantitative assessment of the mechanical resilience of the endosperm cap.

2.9. Transcriptome Analysis and Gene Expansion during Lettuce Seed Germination

In 2022, transcriptome sequencing was performed on the endosperm caps of lettuce seeds that had been swollen in water for 6, 12, and 18 h at Wuhan (Majorbio Bio-pharm Technology Co., Ltd., Wuhan, China). The transcript abundance of XTH genes was represented as FPKM (fragments per kilobase of exon model per million mapped fragments) values. The data were normalized, and heat maps were generated using the Omic Studio tool (https://www.omicstudio.cn accessed on 12 May 2022).

Lettuce seeds were germinated under different conditions: water, 0.3% SDIC, 5 μmol/L ABA, low temperature (4 °C), high temperature (28 °C), waterlogging (100 mL ddH₂O), drought (10% PEG 6000), cadmium stress (500 mg/L CdCl₂), and salt stress (100 mmol/L NaCl). After swelling for 6, 12, and 18 h, respectively, the endosperm caps were isolated from the seeds. The total RNA of lettuce seed embryos was extracted using a plant total RNA rapid extraction kit (Bioteke Gorporation, Beijing, China), and the concentration and quality of the RNA were evaluated using a NanoDrop spectrophotometer (Therm, Waltham, MA, USA). First-strand cDNA was synthesized using ReverTra Ace Qpcr RT MasterMix with a gDNA Remover kit (Toyobo, Shanghai, China). qRT-PCR was employed to detect the relative expression levels of lettuce seeds under different germination conditions. The reaction system (20.0 μL) included 10.0 μL 2 × RealStar Green Fast Mixture (GenStar, Beijing, China) kits, 0.8 μL each of 10 μmol/L forward and reverse primers, 2.0 μL of 5 ng/μL cDNA template, and ddH₂O up to 20.0 μL. The amplification program included 40 cycles of 95 °C for 2 min, 95 °C for 15 s, 58 °C for 30 s, and 72 °C for 30 s. Each experiment was repeated three times, and the relative quantification was calculated using the 2^−ΔΔCt method. The primer sequences are shown in

Table 1. Primer sequence of qRT-PCR.

Gene	Forward Primer	Reverse Primer
LsXTH2	5′-AGCTTTGGTTTGACCCGACT-3′	5′-TCTCGTAGTCGGCGTTCTTG-3′
LsXTH6	5′-CCGATGATTGGGCGACAAGA-3′	5′-GGGACAGAACAGCCCTCAAT-3′
LsXTH7	5′-CCTCCCAAAACTCGGAGCAT-3′	5′-TCCCGCCGGTGAAAACATTA-3′
LsXTH8	5′-GACCAAGCGATGGGTGTGTA-3′	5′-GGTAGGATGCGACAAACGGA-3′
LsXTH14	5′-AGCGATGAAATGGGAAGCGA-3′	5′-CCACCATTGGTAGCCCAAGT-3′
LsXTH15	5′-TACACAAGGGGGTCGAGTCA-3′	5′-CTAGAGCACCGCCTCATGTT-3′
LsXTH22	5′-TCTTGTGGAGCCCAAACGAT-3′	5′-TAGCCCATGAAGAAGCGTCC-3′
LsXTH32	5′-ACTCAAGCTCCCTTTACGGC-3′	5′-CCTGATTGCCACCGGAAGAT-3′
LsXTH33	5′-TCTGGCTCCGGCTTTGAATC-3′	5′-ATCCCAGTTTGACCCCTTCG-3′
LsXTH43	5′-TCATGCCGATAAAGGGGTGG-3′	5′-TTACCTCCGTTTGTTGCCCA-3′
Ls18S	5′-CCTGCGGCTTAATTTGACTC-3′	5′-AACTAAGAACGGCCATGCAC-3′

.

3. Results

3.1. Identification and Characteristics of XTHs

Through whole-genome alignment analysis, a total of 43 lettuce XTH genes were identified. Based on their positions on the chromosome, these genes were designed LsXTH1 through LsXTH43 (

Table 2. Protein member information of XET in lettuce.

Gene Name	Gene ID *	Protein ID	Length of Amino Acids (aa)	Molecular Weight (Da)	PI	Instability Index	Aliphatic Index	Grand Average of Hydropathicity	Alpha Helix (%)	Extended Strand (%)	Beta Turn (%)	Random Coil (%)	Subcell Location
LsXTH1	gene-LSAT_1X64420	cds-PLY70468.1	290	33,109.20	9.03	53.07	63.55	−0.517	11.72	28.97	6.55	52.76	Cell wall
LsXTH2	gene-LSAT_1X127501	cds-PLY66370.1	302	35,264.33	5.03	26.97	54.24	−0.590	13.58	29.14	5.63	51.66	Cell wall
LsXTH3	gene-LSAT_2X41960	cds-PLY75087.1	186	21,921.66	9.06	39.09	57.58	−0.799	9.14	31.72	7.53	51.61	Cell wall
LsXTH4	gene-LSAT_2X40980	cds-PLY75084.1	189	22,350.60	9.81	45.01	62.33	−0.781	7.94	31.75	6.35	53.97	Cell wall
LsXTH5	gene-LSAT_2X45920	cds-PLY90799.1	280	32,134.30	9.18	26.63	67.21	−0.417	17.50	30.36	5.71	46.43	Cell wall
LsXTH6	gene-LSAT_2X80380	cds-PLY94495.1	293	33,776.20	5.90	32.59	72.53	−0.365	14.68	30.36	5.80	49.15	Cell wall
LsXTH7	gene-LSAT_3X33121	cds-PLY85478.1	292	33,954.51	8.76	44.23	64.11	−0.455	13.36	31.16	4.79	50.68	Cell wall Cytoplasm
LsXTH8	gene-LSAT_3X43161	cds-PLY67568.1	292	32,873.60	5.55	35.44	68.08	−0.336	13.7	29.79	5.82	50.68	Cell wall
LsXTH9	gene-LSAT_3X47880	cds-PLY68852.1	314	35,339.39	8.81	49.76	73.25	−0.208	14.65	28.66	6.05	50.64	Cell wall
LsXTH10	gene-LSAT_3X84840	cds-PLY84990.1	294	33,717.06	6.24	41.68	70.95	−0.366	13.27	32.31	5.78	48.64	Cell wall
LsXTH11	gene-LSAT_4X6921	cds-PLY72098.1	318	36,136.19	6.56	44.42	79.94	−0.181	15.41	29.25	5.66	49.69	Cell wall
LsXTH12	gene-LSAT_4X22721	cds-PLY81384.1	349	39,896.97	7.65	39.84	71.23	−0.444	11.46	28.94	7.16	52.44	Cell wall
LsXTH13	gene-LSAT_4X32600	cds-PLY99886.1	311	35,700.90	4.65	45.57	68.01	−0.348	13.18	28.30	8.36	50.16	Cell wall
LsXTH14	gene-LSAT_4X42380	cds-PLY72301.1	347	40,252.60	8.41	36.23	73.05	−0.525	19.60	26.80	8.36	45.24	Cell wall
LsXTH15	gene-LSAT_4X69800	cds-PLY93082.1	290	33,327.67	6.29	27.35	69.17	−0.333	15.86	30.69	6.90	46.55	Cell wall
LsXTH16	gene-LSAT_4X159341	cds-PLY91589.1	293	33,464.79	6.70	34.81	67.51	−0.388	16.04	30.03	5.12	48.81	Cell wall
LsXTH17	gene-LSAT_5X144400	cds-PLY68715.1	322	36,620.56	8.88	37.75	72.70	−0.323	17.39	28.88	6.21	47.52	Cell wall
LsXTH18	gene-LSAT_5X144420	cds-PLY68721.1	322	36,616.56	9.00	36.69	72.39	−0.343	18.01	29.50	6.21	46.27	Cell wall
LsXTH19	gene-LSAT_5X144460	cds-PLY68709.1	322	36,629.55	8.88	37.06	73.91	−0.324	17.39	30.12	5.9	46.58	Cell wall
LsXTH20	gene-LSAT_5X147421	cds-PLY68720.1	190	21,576.27	10.16	42.61	49.21	−0.914	15.79	21.05	8.95	54.21	Cell wall
LsXTH21	gene-LSAT_6X3400	cds-PLY69844.1	311	36,614.11	8.86	51.82	61.80	−0.694	13.18	28.62	8.68	49.52	Cell wall
LsXTH22	gene-LSAT_6X24740	cds-PLY64636.1	259	29,875.69	9.42	44.86	57.26	−0.549	7.72	31.27	8.11	52.90	Cell wall
LsXTH23	gene-LSAT_6X44180	cds-PLY61830.1	274	31,173.46	6.12	41.80	55.15	−0.754	7.3	32.12	8.39	52.19	Cell wall
LsXTH24	gene-LSAT_7X54320	cds-PLY75482.1	206	23,466.41	6.75	38.17	71.99	−0.457	11.17	29.13	11.65	48.06	Cell wall Cytoplasm
LsXTH25	gene-LSAT_7X54340	cds-PLY75422.1	276	31,374.56	8.26	20.82	71.01	−0.323	9.78	36.96	7.97	45.29	Cell wall Cytoplasm
LsXTH26	gene-LSAT_7X56941	cds-PLY69337.1	281	31,985.02	5.40	38.34	71.10	−0.311	13.88	31.32	6.05	48.75	Cell wall Cytoplasm
LsXTH27	gene-LSAT_7X83821	cds-PLY64715.1	257	28,923.24	9.15	29.95	64.16	−0.535	9.73	34.24	6.61	49.42	Cell wall Cytoplasm
LsXTH28	gene-LSAT_7X83841	cds-PLY64682.1	287	31,965.73	8.95	29.96	71.74	−0.370	15.33	31.01	6.97	46.69	Cell wall Cytoplasm
LsXTH29	gene-LSAT_8X67401	cds-PLY87542.1	288	32,352.09	5.32	34.97	67.74	−0.392	7.99	35.76	5.21	51.04	Cell wall Cytoplasm
LsXTH30	gene-LSAT_8X67381	cds-PLY87535.1	288	32,413.16	5.31	28.70	68.40	−0.405	16.67	31.94	5.21	46.16	Cell wall Cytoplasm
LsXTH31	gene-LSAT_8X67360	cds-PLY87501.1	288	32,234.95	5.32	39.28	67.40	−0.402	10.42	35.07	4.51	50.00	Cell wall Cytoplasm
LsXTH32	gene-LSAT_8X67301	cds-PLY87522.1	288	32,285.09	5.69	34.86	68.44	−0.384	15.97	29.51	5.9	48.61	Cell wall Cytoplasm
LsXTH33	gene-LSAT_8X67581	cds-PLY87527.1	287	32,043.72	5.30	40.61	65.92	−0.387	17.07	33.10	5.23	44.60	Cell wall Cytoplasm
LsXTH34	gene-LSAT_8X67261	cds-PLY87515.1	289	33,847.57	8.47	34.11	76.26	−0.402	12.11	33.91	5.88	48.10	Cell wall
LsXTH35	gene-LSAT_8X96260	cds-PLY90696.1	291	33,447.96	8.87	33.88	67.01	−0.301	15.12	31.27	5.5	48.11	Cell wall
LsXTH36	gene-LSAT_8X109641	cds-PLY70758.1	163	18,573.94	9.22	45.11	60.37	−0.801	30.06	14.72	6.13	49.08	Cell wall Cytoplasm
LsXTH37	gene-LSAT_8X148840	cds-PLY93123.1	286	33,327.74	9.10	37.59	64.72	−0.513	11.89	32.87	6.29	48.95	Cell wall Cytoplasm
LsXTH38	gene-LSAT_9X12461	cds-PLY70929.1	193	22,713.32	6.58	33.19	53.06	−0.877	10.36	30.57	6.22	52.85	Cell wall
LsXTH39	gene-LSAT_9X13241	cds-PLY70871.1	186	21,825.54	8.93	44.34	54.46	−0.792	10.22	30.65	7.53	51.61	Cell wall
LsXTH40	gene-LSAT_9X13221	cds-PLY70888.1	186	21,815.50	8.76	44.47	54.46	−0.763	10.22	31.72	7.53	50.54	Cell wall
LsXTH41	gene-LSAT_9X13201	cds-PLY70906.1	184	21,514.10	8.27	35.45	57.66	−0.748	9.78	33.15	6.52	50.54	Cell wall
LsXTH42	gene-LSAT_9X13181	cds-PLY70878.1	115	13,519.14	9.16	56.01	41.57	−0.911	23.48	20.00	6.09	50.43	Cell wall
LsXTH43	gene-LSAT_9X112641	cds-PLY65450.1	294	34,640.12	6.45	38.21	70.00	−0.411	15.65	29.25	5.10	50.00	Cell wall

NOTE: * Database: EnsembIPlant, Lsat_Salinas_v7, 12, 2021.

). The amino acid lengths of LsXTHs ranged from 115 to 349 aa, with molecular weights between 13.519 and 40.252 KDa. The isoelectric points of these proteins ranged from 5.03 to 10.16. Of these, 18 proteins had an isoelectric point below 7, indicating acidic proteins, while the remaining proteins had an isoelectric point above 7, indicating basic proteins. The instability index of LsXTH proteins ranged from 20.82 to 56.01, while the aliphatic indices ranged from 41.57 to 79.94, indicating significant differences in thermal stability. The amino acid hydrophilicity values ranged from −0.208 to −0.914, all of which were less than 0, indicating that the majority of the proteins were hydrophilic. Secondary structure prediction analysis revealed that the XTH family was dominated by random coils, with a minimum proportion of 44.6%. In XTH22, XTH23, and XTH24, the proportion of α-helices was the smallest, while in all other proteins, the proportion of β-turns was the smallest. Protein subcellular localization prediction analysis indicated that LsXTH7, LsXTH24, LsXTH25, LsXTH26, LsXTH27, LsXTH28, LsXTH29, LsXTH30, LsXTH31, LsXTH32, LsXTH33, LsXTH36, and LsXTH37 were located in both the cell wall and cytoplasm, while the remaining proteins were all located in the cell wall.

3.2. Phylogenetic Analysis of LsXTH Gene Family

To understand the phylogenetic relationships of the LsXTH gene family, a phylogenetic tree was constructed using 43 lettuce XTH members, 33 Arabidopsis XTH members, and 29 rice XTH members. Employing the gene grouping methodology employed for the XTH family in Arabidopsis [9] and grape [31], the LsXTH gene family members were divided into five subfamilies: Group I, Group II, Group IIIA, Group IIIB, and Ancestral Group (Figure 1). Among these, Group II was the largest subfamily, with 18 LsXTH members, followed by Group I, while Group IIIA had the fewest members. It is worth mentioning than the Ancestral subfamily exclusively encompassed genes from Arabidopsis and lettuce, with no inclusion of rice XTH genes, indicating the conservation of XTHs during the evolution of monocotyledons and dicotyledons. Additionally, a significant degree of similarity can be observed among certain family members, and some XTH genes had formed relative sister pairs, such as AtXTH8 and LsXTH2, AtXTH10 and LsXTH43, AtXTH32 and LsXTH22, LsXTH9 and LsXTH1, LsXTH23 and LsXTH1, LsXTH15 and LsXTH8, and LsXTH27 and LsXTH28. These findings suggest a closer evolutionary relationship between lettuce and Arabidopsis XTH family members.

3.3. Chromosomal Distribution of LsXTH Genes

Based on the annotation information from the whole lettuce genome, a chromosome mapping of lettuce XTH genes was created using TBtools software. The 43 lettuce XTH genes were positioned on nine chromosomes (Figure 2), with the most distribution found on chromosome 8, containing nine members, accounting for 20.93% of the total genes. In contrast, chromosome 1 had the fewest members, with only two genes (LsXTH1, LsXTH2), accounting for 4.65%. Additionally, the genes in this family were clustered on Chr5, Chr7, Chr8, and Chr9, which may be related to the expansion and functional differentiation of genes in this subfamily.

3.4. Gene Structure and Motif Analysis in LsXTH

Using the online software GSGD2.0 and MEME, gene structure and protein-conserved motif analyses were conducted on the lettuce LsXTH gene family (Figure 3). Notably, the LsXTH35 gene was the longest within the family, spanning 38.7 kb, while the LsXTH4 gene was 6694 bp in length. Both genes had a very high proportion of introns, with the remaining genes had lengths less than 3000 bp. All LsXTH genes contained 1–4 exons, with 2 genes containing 1 exon, 9 genes containing 2 exons, 14 genes containing 3 exons, and 18 genes containing 4 exons. This showed that there were significant differences in gene structure among members of the LsXTHs family in lettuce, suggesting diverse and differentiated functions of the LsXTH genes. The lettuce XTH family proteins were found to contain a total of 20 conserved motifs. The majority of the genes (83.7%) in the 43 LsXTHs contained between 6 and 10 motifs, while only 16.3% of the genes contained 1–5 motifs. Notably, all genes except LsXTH24 contained motif2; all genes except LsXTH20 and LsXTH24 contained motif 4. Additionally, 90.7% of the genes contained motif5, and 79.1% of the genes contained motif3. In contrast, motifs 11, 12, 16, and 19 were present in only a few genes (2–3); for instance, motif 19 was present exclusively in LsXTH9 and LsXTH11. An annotation analysis of the conserved motifs in the Interpro database revealed that motif1, motif2, and motif3 encoded XTH domains, indicating that these three motifs were the most conserved sequences in the lettuce XTH family proteins.

3.5. Collinearity Analysis of XTH Genes

To further understand the evolutionary mechanism of the lettuce XTHs family, a collinearity relationship map of the XTHs family in lettuce, Arabidopsis, and cabbage was constructed (Figure 4). The results of the collinearity analysis revealed 24 pairs of collinear genes between lettuce and Arabidopsis, and 18 pairs of collinear genes between lettuce and cabbage. Notably, 10 genes showed collinearity with both Arabidopsis and cabbage, indicating their conserved roles in the evolution of the XTH gene family. Additionally, gene LsXTH29 displayed collinearity relationships with three genes in Arabidopsis and cabbage, indicating its potential significant role in the evolution of the XTH gene family. Moreover, genes LsXTH6, LsXTH10, LsXTH16, LsXTH23, LsXTH25, LsXTH26, and LsXTH38 each had two collinear genes in Arabidopsis or cabbage, indicating that these genes had undergone expansion.

Further investigation of the collinearity among XTH genes, as shown in Figure 5, revealed significant collinear relationships between LsXTH1, LsXTH6, LsXTH10, LsXTH16, LsXTH17, LsXTH23, LsXTH24, LsXTH29, and LsXTH35. A total of six pairs of homologous relationships were identified among these genes, whereas no collinearity was observed between other members. This indicated gene duplication within the LsXTH gene family. It was speculated that, during evolution, XTH genes may have undergone an expansion of their family members through duplication events.

3.6. Protein Structure Analysis of LsXTHs

Using homology-based modeling methods, the three-dimensional structures of LsXTH family proteins were further predicted. The majority of the modeled structures for LsXTH proteins exhibited a root mean square deviation (RMSD) of less than 1Å when aligned with their respective homologous templates, indicating reliable predictions. The results showed that LsXTH36 and LsXTH42 comprised α-helix and random coils, whereas LsXTH24 was composed of β-sheets alongside random coils, and LsXTH20 was primarily made up of α-helices. The remaining 39 LsXTH proteins had similar three-dimensional structures, all composed of α-helices, β-sheets, and random coils (Figure 6). The upper part of the structure was composed of one α-helix and several β-sheets and random coils, while the lower part mainly consisted of random coils, creating a hydrophobic cavity within the protein.

3.7. Cis-Acting Elements Analysis in LsXTHs

To gain insight into the regulatory functions of the lettuce XTH gene family members, the promoter sequences located 2000 bp upstream of the coding sequences (CDS) were analyzed for all members. In addition to the common cis-regulatory elements (TATA box and CAAT box) found in eukaryotes, a large number of non-biological stress response elements were also identified (Figure 7). The most abundant element was the drought response element, accounting for 45.4%; this was followed by the anaerobic induction response element, accounting for 18%; the high-temperature stress and flooding stress response elements, accounting for 12.6% each; the low-temperature stress response element accounting for 6.5%; the defense stress response element accounting for 3.4%; and the remaining seed-specific regulatory elements, salt stress elements, and cadmium stress elements, all accounting for less than 1%.

Through an in-depth analysis of the cis-regulatory elements in the promoters of each gene, it was discovered that the LsXTH36 gene promoter contained the largest number of cis-regulatory elements, totaling 26, while the LsXTH40 promoter contained the smallest number of cis-regulatory elements, with only 3 cis-regulatory elements. The promoters of 28 genes had between 10 and 20 cis-acting elements, accounting for 65.1% of the entire gene family. Notably, LsXTH1, LsXTH9, and LsXTH10 were the only genes containing cadmium stress response elements; LsXTH9 and LsXTH38 were the only genes containing salt stress response elements; and LsXTH9, LsXTH35, LsXTH21, and LsXTH23 were the only genes with seed-specific response elements. The distribution of these response elements across different genes suggested their involvement in different functions under non-biological stresses.

3.8. Structure of Lettuce Seed and XTH Enzyme Activity during Lettuce Germination

Lactuca sativa seeds are typical dicotyledonous seeds with endosperm. As shown in Figure 8A, during seed germination, the radicle first broke through the endosperm cap and eventually broke through the seed coat to complete germination. Since the seed coat is dead tissue, it will physically expand as imbibition proceeds. Therefore, lettuce seeds require the dual forces of weakening the endosperm at the micropylar end and elongation of the radicle to initiate germination [32,33]. XTH was one of the key hydrolytic enzymes that relaxed the endosperm cell wall to promote endosperm weakening [26,27]. As shown in Figure 8B, during seed germination, the activity of XTH gradually increased in the endosperm cap and radicle, peaking at 24 h after imbibition. Notably, the activity of XTH in the endosperm cap was significantly higher than its activity in the radicle. In contrast, there was a slow upward trend in the activity of XTH in the chalazal endosperm, while the activity of this enzyme in the radicle remained relatively unchanged.

3.9. Typical and Atypical Germination of Lettuce Seeds

Upon imbibition in water, lettuce seeds initiated germination at 12 h and the majority of seeds completed germination within a brief period, with germination rates approaching nearly 100% by 26 h. In contrast, treatment with 5 μmol/L ABA and 0.3% SDIC significantly inhibited seed germination rates, with germination rates of 36% and 30%, respectively (Figure 9A). Seeds imbibed in water and ABA exhibited typical germination patterns, with the radicles breaking through the endosperm cap, similar to dicotyledonous plants like tomato and chili peppers (Figure 9B). However, for seeds imbibed in SDIC solution, the endosperm cap failed to rupture, and as the embryonic root elongated, the entire embryo exhibited a distorted shape within the endosperm, preventing germination. In some seeds, the embryonic root ruptured at other locations within the endosperm until seedlings emerged (Figure 9C). The mechanism by which SDIC causes atypical germination in lettuce seeds needs further exploration.

3.10. Endosperm Cap Vitality and Endosperm-Weakening Degree of Lettuce Seeds

To investigate the effect of SDIC on the viability of lettuce seed endosperm caps, endosperm caps from seeds imbibed in water and SDIC solution for 12 h were separated and stained with TTC to observe changes in their viability. As shown in Figure 10, the entire endosperm cap of seeds imbibed in water could be stained red, indicating high viability; however, endosperm caps from seeds imbibed in 0.3% SDIC could not be stained or only partially stained a light red color, indicating a significant reduction in viability.

Further, the piercing force of the probe on the endosperm reflects the mechanical strength and weakening degree of the endosperm. As shown in Figure 11A, during the measurement process, before the endosperm was broken by the steel needle, the compressive load increased slowly, reached a peak, and then decreased sharply, indicating that the endosperm was already punctured at this time. With the progress of germination, the mechanical strength of the endosperm cap gradually decreased. The mechanical strength of the basal endosperm was higher than that of the endosperm cap, and its puncture force also decreased slowly as germination progressed (Figure 11B). In ABA-soaked seeds, the puncture force of the endosperm cap gradually decreased, similar to that in water; for SDIC-soaked seeds, the mechanical strength of the endosperm cap was 41% higher than that when soaked in water and ABA. Before soaking for 10 h, the puncture force of the endosperm cap showed an increasing trend, followed by a slow decreasing trend (Figure 11C).

3.11. Expression Analysis of LsXTH Family Genes in Lettuce Seeds under Different Germination Conditions

To explore the function of XTH genes in the softening of lettuce seed endosperm, transcriptome sequencing was performed on endosperm caps of seeds at 6 h, 12 h, and 18 h after imbibition. The data analysis revealed that only LsXTH2, LsXTH6, LsXTH7, LsXTH8, LsXTH14, LsXTH15, LsXTH22, LsXTH32, LsXTH33, and LsXTH43 were transcriptionally expressed during lettuce seed germination. Based on the gene expression trend, the above genes were clustered. Among them, LsXTH6, LsXTH32, LsXTH22, and LsXTH43 exhibited relatively low and consistent expression levels, and were therefore categorized into a single group. The remaining genes displayed relatively high expression levels and were grouped together. Furthermore, distinct subgroups were identified within each major group based on variations in gene expression (Figure 12). The gene expression heatmap showed that the transcriptional expression levels of these genes, except for LsXTH5, gradually increased as seed germination progressed (Figure 12).

To further investigate the expression of XTH family genes in the weakening process of lettuce seed endosperm, the endosperm caps of seeds imbibed for 12 h were collected and subjected to various stress conditions. qRT-PCR was used to analyze the expression levels of the 10 LsXTH genes identified in the transcriptome sequencing. As shown in Figure 13, during lettuce seed germination in water, the transcription expression level of LsXTH43 was the highest, followed by LsXTH7, while LsXTH6 and LsXTH14 had the lowest expression levels. Each gene exhibited various expression patterns under different stress conditions. Compared with the control, both SDIC and ABA treatments decreased the transcription expression of LsXTHs to varying degrees. Among them, SDIC reduced the expression levels of LsXTH15, LsXTH33, and LsXTH43 to 6%, 11.4%, and 13.8% of the control group, respectively. The most significantly downregulated gene under ABA treatment was LsXTH32, with an expression level of 28% of the control group. Waterlogging stress significantly upregulated the expression levels of LsXTH2 and LsXTH15, while other stress conditions inhibited the expression of the two genes to varying degrees. Under waterlogging and cadmium stresses, the expression levels of LsXTH6, LsXTH7, LsXTH8, LsXTH32, and LsXTH33 were significantly upregulated, while other adversities inhibited their expression to some extent. Notably, LsXTH8 expression under low temperature was only 3.3% of the control group. The expression of LsXTH14 was significantly downregulated under low temperature, high temperature, drought, and salt stresses. The LsXTH22 gene was upregulated only under cadmium stress, while other adverse conditions significantly inhibited its expression level. The expression of LsXTH43 was twice as high as the control under high-temperature treatment, and cadmium stress slightly increased its expression. Under other stress conditions, the expression of this gene remained almost unchanged or slightly decreased.

4. Discussion

XTH is a key enzyme that degrades xyloglucan in plants and plays an important role in the relaxation and degradation of cell walls, widely participating in various biological processes such as fruit ripening and softening [34], internode elongation, root hair development, organ abscission [35], seed germination, and response to abiotic stress [36]. The XTH family consists of multiple genes, and the number of genes varies across different species. The gene structures and system evolution characteristics of XTHs have been reported in various species, including Arabidopsis thaliana, rice, tobacco, barley, tomato, apple, persimmon, kiwi fruit, pear, strawberry, banana, peach, and black goji berry [11,12,13,14,15,16,36,37,38,39,40,41,42]. It has been reported that there are 33 XTH genes in Arabidopsis thaliana, which can be divided into three subfamilies based on their phylogenetic branches and topological structures: XTH subfamily I, XTH subfamily II, and XTH subfamily III [9]. In the present study, we identified a total of 43 XTH members in lettuce. The difference in the number of homologous XTH genes compared to other species may be attributed to variations in the occurrence of gene duplication events in lettuce. When conducting evolutionary analysis on XTH family genes in lettuce, Arabidopsis, and rice together, we found that LsXTHs were divided into five subfamilies. Group II contained the largest number of LsXTH family genes, while other branches had fewer. In the Ancestral subfamily, only some genes from the two dicotyledonous plants, Arabidopsis and lettuce, were present, but no rice XTH genes were found. This suggests that these genes are more conserved during evolution. Promoter analysis indicated that the promoters of LsXTHs contained a large number of regulatory elements responsive to drought, high temperature, anaerobic conditions, waterlogging, low temperature, high temperature, cadmium stress, salt stress, and seed-specific regulation. This suggests that this gene family is widely involved in the non-biological stress response in lettuce. Of interest, seed-specific response elements were found in LsXTH9, LsXTH35, LsXTH21, and LsXTH23; however, transcriptome sequencing and qRT-PCR did not detect the expression of several genes in the micropylar endosperm of germinating seeds. This may be due to the tissue-specific nature of the genes, suggesting that they may function in the embryo during germination or during seed development [43].

The weakening of the endosperm and the elongation of the radicle are prerequisites in the completion of lettuce seed germination. These two biological processes occur simultaneously and require the involvement of hydrolases, reactive oxygen species, and expansion proteins to loosen the endosperm and promote radicle elongation [4,5,44]. In our research, we found a gradual increase in XTH enzyme activity in the endosperm cap during lettuce seed germination, suggesting that this enzyme may play a role in promoting germination by facilitating the weakening of the endosperm. However, the XTH activity in the endosperm cap was significantly higher than that in the radicle, a result similar to that of Zhong et al. (2010) [45]. Using SDIC solution, we further confirmed that even if the radicle can elongate without endosperm cap weakening, the seed will exhibit abnormal germination (atypical germination) or an inability to germinate. SDIC reduced the degree of endosperm weakening by reducing the vitality of the endosperm cap; however, additional physiological and molecular mechanisms still require further investigation. Previous studies have used penetration force tests to confirm endosperm weakening during germination in tomato [46], melon [47], coffee [48] and Ludwigia grandiflora [49] seeds. We employed this method to measure the degree of endosperm weakening in lettuce seeds, consistent with previous research results. Although ABA significantly inhibits lettuce seed germination, it does not have a noticeable effect on endosperm cap weakening, possibly due to the fact that ABA inhibits embryo growth [50,51]. Puncture force testing can also be applied to study the weakening degree of coleoptile in monocotyledon plants such as corn and wheat.

During lettuce seed germination, transcriptome sequencing could only detect the expression of 10 LsXTH genes in the endosperm caps, which may be determined by spatial, temporal, and tissue-specific gene expression [52]. As imbibition progresses, the expression levels of these genes gradually increase, suggesting their potential involvement in regulating endosperm relaxation and seed germination. Similar findings have been observed in transcriptome data from the tomato seed germination process, where genes encoding hydrolytic enzymes such as XTH16, MAN2, and 1,4-GLU showed a gradual increase in expression during imbibition, suggesting that they play key roles in controlling endosperm relaxation and seed germination [37]. As previously mentioned, SDIC diminished the degree of endosperm cap weakening, and at the gene expression level, it also downregulates the expression of LsXTHs to varying extents, particularly affecting the expression of LsXTH15. This gene may serve as a candidate gene for studying endosperm weakening in lettuce. The analysis of the promoter region suggested that the LsXTH gene family may play an important role in responding to abiotic stress. The gene expression results indicated that low temperature, high temperature, drought, and salt stress all reduced the expression of most LsXTHs to varying degrees during the germination process. It is worth noting that among the 10 candidate XTH genes, LsXTH43 had the highest expression level and was significantly upregulated by high temperature, suggesting that this gene may play a role in lettuce germination under high-temperature conditions. During germination under waterlogging and cadmium stress, LsXTH6, LsXTH7, LsXTH8, LsXTH32, and LsXTH33 were all upregulated to some extent. The question of whether these two non-biological stresses share similar regulatory mechanisms in these genes warrants further investigation. Uncovering these mechanisms could provide valuable insights into the adaptive responses of lettuce seeds to diverse environmental challenges during germination.

5. Conclusions

In summary, the lettuce xyloglucan endo transglucosylase/hydrolase (XTH) gene family was divided into 5 subfamilies, comprising a total of 43 homologous genes located on 9 chromosomes. Members of the LsXTHs family exhibited significant differences in gene structure, and their promoters contained numerous regulatory elements responsive to various abiotic stresses, indicating the widespread involvement of XTH family genes in multiple abiotic stress responses in lettuce. Endosperm weakening is a prerequisite for lettuce seed germination, and enhanced XTH enzyme activity in the endosperm cap may play a significant role in loosening the endosperm cap cell wall and promoting seed germination. LsXTH genes display diverse expression patterns under adverse conditions during lettuce seed germination. LsXTH15 may be associated with endosperm weakening, while LsXTH43 may potentially be involved in seed germination under high temperature.