Function and Expression Analysis on StFLA4 in Response to Drought Stress and Tuber Germination in Potato

Abstract

Drought stress is one of the main factors limiting the high yield and quality of potatoes. Arabinogalactan proteins (AGPs) are an important class of glycoproteins widely present in the cell walls, plasma membranes, and extracellular matrices of higher plants. Among them, fasciclin-like arabinogalactan proteins (FLAs) are involved in plant development, stress responses, and hormone signal regulation. However, little is known about the FLAs gene in potatoes. Based on transcriptome sequencing data, this study screened a drought stress-related candidate FLA gene (StFLA4) through bioinformatics and expression analysis in potatoes. qRT-PCR analysis showed that StFLA4 was induced by drought stress, and its expression decreased with the extension of stress time. Moreover, the relative expression level of StFLA4 in the drought-resistant variety “Kexin 1” was lower than in the drought-sensitive variety “Atlantic”. The StFLA4 protein was located in the cell membrane and interacted with nineteen proteins, mainly related to response to environmental stimulus, cellular response to abiotic stimulus, and cell maturation. After heterologous overexpression of StFLA4 in tobacco, the transgenic plants showed more withered leaves than the wild-type tobacco under drought stress. During the drought stress period, the expression level of StFLA4 in the transgenic plants significantly decreased, and the activity of SOD and POD was significantly lower than that of WT. However, the MDA content was higher than that of WT. These results indicated that StFLA4 negatively regulates the response to drought stress. In addition, in the germination test of potato “Variety V7” tubers, it was found that the variation tendency of StFLA4 expression was along with the concentration of arabinogalactan proteins, and it may participate in the regulation of potato tuber germination. This study lays the foundation for elucidating the function and expression pattern of StFLA4 response to drought stress and tuber germination in potatoes.

1. Introduction

Potato (Solanum tuberosum L.) has become the fourth largest food crop in the world due to its short growth cycle, wide adaptability, rich nutritional content, and high practical value [1]. It is also an important industrial raw material recognized by the Food and Agriculture Organization of the United Nations (FAOSTAT) as a food security crop [2]. In addition to providing starch, potato tubers are rich in vitamin C, potassium, and seven other essential nutrients for the human body [3]. Currently, European and American countries have completed the staple foodization of potatoes, and the consumption of potatoes in Asian countries is also increasing daily. Arabinogalactan-proteins (AGPs) are a class of structurally complex glycoproteins. They are rich in hydroxyproline or proline and widely distributed on cell walls, protoplasts, or plasma membranes. AGPs are involved in the construction and development of plant root morphology and control of cell proliferation, differentiation, cell–cell signalling, and cell–matrix interactions during plant tissue development and embryogenesis [4,5]. FLAs (fasciclin-like arabinogalactan proteins) are a type of arabinogalactan protein that contain AGP-like glycosylation regions and fasciclin (FAS) domains [6].

FLAs play an important role in cell support and cell-to-cell communication, and they respond to abiotic stress, including salt stress, low-temperature stress, and reactive oxygen species stress [7]. After treating hemp with 200 mM NaCl and detecting gene expression patterns by real-time quantitative PCR (qRT-PCR), the results showed that genes involved in secondary cell wall biosynthesis (CesA4, FLA10, FLA8) were all significantly upregulated [8]. At the same time, based on isotope labeling relative and absolute quantification (iTRAQ) proteomics technology, proteins in the early stage of cotton subjected to salt stress were identified. The results showed that under NaCl stress, the expression abundance of FLA proteins was significantly induced [9]. In Arabidopsis, AtFLA4 (SOS5) was involved in ABA-mediated salt stress response; it was essential for normal cell expansion, and the cell wall was thinner in its mutants [10,11]. Under salt stress, AtFLA4 is necessary to maintain the normal growth of Arabidopsis roots. Spraying abscisic acid (ABA) inhibits the response of AtFLA4 to salt stress. At the same time, AtFLA4 positively regulates the response of roots to low concentrations of ABA and is necessary for the normal expression of ABA-related stress-induced genes. In addition, AtFLA4 positively regulates cell wall synthesis and root growth through the ABA pathway. Further research has shown that AtFLA4 is essential for ABA-mediated stress responses [12]. Other studies have shown that AtFLA4 can mediate root growth and seed adhesion through cell wall receptor-like kinases (FEI1/2) and may interact with the ETH/ABA pathway to regulate cell wall integrity [13].

In addition to salt stress, FLA has been proven to be involved in the response to low temperature and drought stress, but the specific mechanisms still need further research [14]. In rice, the expression pattern of OsFLA1 and OsFLA4 was downregulated under cold stress, and OsFLA5, OsFLA18, OsFLA19, and OsFLA27 were downregulated under drought and salt stress [15]. In Arabidopsis, AtFLA4 was related to drought stress-related signals [16], and the expression level of AthFLA9 was reduced in drought stress [17]. In wheat, the expression abundance of TaFLA9/12/14 was increased when the roots were subjected to cold or dehydration stress, while the expression of TaFLA3/4/9 was decreased after heat, dehydration, or NaCl treatment [18]. In addition, analysis of the promoter elements of the FLAs gene in potatoes, the results showed that there were many hormone response elements in the promoter of the StFLAs gene family, including salicylic acid response element (TCA-element), abscisic acid response element (ABRE), gibberellin response element (TATC-box), it may play a crucial role in regulating stress resistance and tuber formation [7].

The planting area of potatoes in our country has been stable at around 70 million mu, and the output is nearly 90 million tons, accounting for about a quarter of the world’s total, with the output ranking first in the world for many years. However, more than 60% of the potato growing areas in China are located in arid or semi-arid areas, which are vulnerable to high temperatures and drought stress, thus adversely affecting potato growth [19]. Drought has a greater impact on plant growth than any other environmental stress; it severely affects physiological and biochemical processes such as photosynthesis, secondary metabolism, and osmotic regulation in plants [20]. It has been reported that the area of global drought accounts for about 41% of the total land area of the world, and in the total area of global arable land, arid and semi-arid areas account for 43% [3]. Drought stress inhibits the growth and development of potatoes and can even lead to plant death in severe cases. Ensuring the yield and quality of potatoes and improving their ability to cope with drought stress is crucial. Therefore, studying the drought resistance mechanisms and drought-resistant traits of plants is of great significance for potato genetic improvement [21]. However, there were only a few reports on the FLAs gene family in potatoes, and their involvement mechanism in the drought stress response was unclear.

In this study, a StFLA4 gene involved in the response to drought stress and participated in the regulation of tuber germination in potatoes was studied. Our results found that the StFLA4 was induced by drought stress and overexpressed StFLA4 in tobacco. The transgenic tobacco showed more withered leaves than the wild-type tobacco under drought stress. Further analysis indicated that StFLA4 and its interaction proteins negatively regulated the response to drought stress. Furthermore, our studies also proved that StFLA4 participates in the regulation of tuber germination in potatoes. This research has laid a theoretical foundation for elucidating the mechanism of the StFLA4 in potato drought stress resistance and tuber germination.

2. Materials and Methods

2.1. Verification of StFLA4 Expression Pattern in Potatoes After Drought Stress

In this study, based on potato tuber RNA-Seq data (NCBI ID: PRJNA742170) (Unpublished), the StFLA4 gene (Gene ID: Soltu.DM.06G031480) involved in the response to drought stress in potatoes was screened out using bioinformatics methods [7]. “Atlantic” is a drought-sensitive variety, and “Kexin 1” is a drought-resistant variety; hence, we used these two varieties for studying the drought resistance of StFLA4. “Atlantic” and “Kexin 1” were grown in pots at 25–30 °C during the day and 20–25 °C at night (16/8-h light/dark cycle) in a greenhouse and treated with 500 mL 25% PEG-6000 (w/v) liquid to simulate drought stress when the “Atlantic” and “Kexin 1” were with eight leaves. All the above materials were treated with three replicates, and total RNA was extracted from potato leaves (mixed triad) at different time gradients after treatment for 1 h, 3 h, 6 h, 12 h, and 24 h to detect the expression patterns of the StFLA4 under simulated drought stress. Quantitative real-time PCR (qRT-PCR) was used to detect the expression pattern of StFLA4 during different treatments, and qRT-PCR was performed using MonAmpTM SYBR^® Green qPCR Mix reagent (Monad Biotech Co, Ltd., Suzhou, China) according to the manufacturer’s instructions with four replicates for each period. The constitutively expressed gene U6 was used to normalize gene expression [22]. The relative expression levels of the genes were quantified using the 2^−∆∆CT method [23].

2.2. Subcellular Localization

To verify the mode of action of the StFLA4, an expression vector driven by the 35S promoter and fused with a green fluorescent protein (EGFP) was constructed (pCAMBIA1302-StFLA4-EGFP). The plasmid of empty vector pCAMBIA1302-EGFP and recombinant vector pCAMBIA1302-StFLA4-EGFP were transformed into Agrobacterium Tumefaciens GV3101 strain (Weidi, AC1001, Shanghai, China). Then, the A. tumefaciens were cultured until reaching an OD600 value of 0.6 and suspended in bacterial liquid (10 mmol/L MgCl₂, 10 mmol/L MES, and 200 μmol/L acetosyringone) with an OD600 adjusted to around 0.6 [24]. After being left standing at room temperature for about 2 h, it is injected into tobacco leaves. In order to open the stomata of the tobacco leaves to facilitate the injection of the infection liquid, the well-grown tetraploid tobacco was exposed to incandescent light for about 1 h. The third leaf from the bottom was chosen as the infection leaf, and the lower epidermis of the tobacco was “minimally invasively” injected with a medical sterile syringe (avoiding the veins). The area of infection on the leaves was marked with a marker pen. The leaves were moistened and cultured under 14 h of light/10 h of darkness for 2 days. The specific localization of the pCAMBIA1302-StFLA4-EGFP fusion protein was observed using a laser confocal microscope (FV10-ASW; Olympus, Tokyo, Japan).

2.3. Overexpressed StFLA4 Vector Construction and Tobacco Transformation

Total RNA from potato leaves was extracted using TRNzol Universal Reagent (Tiangen Biotech, DP424, Beijing, China). Subsequently, the concentration and purity of the RNA were detected using a NanoPhotometer spectrophotometer (IMPLEN, Westlake Village, CA, USA), and the RNA was reverse transcribed into cDNA using the FastQuant RT Kit (Tiangen biotech, KT106, Beijing, China), serving as a cloning template. The cloning process utilized primers StFLA4-F/R (Table S1), and the amplification system and procedure followed the steps outlined in Table S2. The purified StFLA4 target fragment was recovered using the TIANgel Midi Purification Kit (Tiangen Biotech, DP209, Beijing, China). Subsequent ligation of the StFLA4 target fragment and the cloning vector was carried out following the guidelines of the pEASY-Blunt Simple Cloning Kit (Transgen, CB111, Beijing, China). Positive clones were subjected to verification using specific primers (StFLA4-F/R). The amplification band of the target sequence was 1275 bp. The pCAMBIA1302 vector plasmid (Miaoling biology, Wuhan, China) and StFLA4 target fragment were ligated using the double enzymes digestion method (Nco I/Spe I). The overexpression recombinant plasmid of pCAMBIA1302-StFLA4 (Figure S1) was transformed into tetraploid tobacco SR1 (Nicotiana tabacum cv. Petit Havana, 2n = 4X = 48) via the leaf discs method [25]. Then, the seedlings with kanamycin resistance (5 mg/L) were transferred into the soil under suitable conditions. Reverse transcription PCR (RT-PCR) and quantitative real-time PCR (qRT-PCR) detection were used to further test and screen the transgenic tobacco lines.

2.4. Drought Stress Treatment and the Expression Patterns of Target Gene Detection

Well-grown wild-type (WT) and transgenic tobacco seedlings with uniform plant height and canopy were placed in an artificial climate chamber and subjected to drought stress treatment (7 days without watering). Total RNA was isolated from the young leaves of WT and transgenic tobacco (at 0 days and 7 days without watering) and reverse-transcribed into cDNA. The qRT-PCR technique was used to detect the expression level of the StFLA4 in transgenic tobacco under drought stress for 7 days, with L25 as the internal reference gene for tobacco. The reaction system and program were used as described by Nie et al. [26]. All the primers for qRT-PCR detection are listed in Table S3.

2.5. Physiological Index Measurement of Transgenic Tobacco Under Drought Stress

Wild-type and transgenic tobacco seedlings (OE-2, OE-7) treated with drought stress for 0 days and 7 days were used. At 9:00 AM, tobacco leaves from the same position were picked, placed in cryogenic tubes, quickly frozen in liquid nitrogen, and then stored in a −80 °C refrigerator. Tobacco seedlings not subjected to drought stress treatment were used as a control. The SOD reagent kit (A001-3), POD reagent kit (A084-3), and MDA reagent kit (A003-1) from Nanjing Jiancheng Bioengineering Research Institute (Nanjing, China) were used to measure the content of superoxide dismutase (SOD), peroxidase (POD), and malondialdehyde (MDA) in WT and transgenic tobacco under drought stress for 7 days. The test procedures strictly adhered to the instructions provided with each kit.

2.6. Determination of Arabinogalactan Protein Content and Analysis of StFLA4 Expression During Potato Tuber Germination

The mid-maturing potato “Variety V7” has a high good quality yield. The bud eyes are rare and extremely shallow; hence, they are storable. In order to study the relationship between StFLA4 expression and tuber germination, the “Variety V7” was planted in the experimental field of the Inner Mongolia Academy of Agricultural and Animal Husbandry Sciences (111°40′33″ E, 40°46′45″ N). Forty-five uniform, undamaged, and pest-free “Variety V7” tubers were selected and divided into five groups, with nine potatoes per group. After marking the skin of the tubers (1~45 numbers), they were placed in a 37 °C dark constant temperature incubator. The experimental period was 28 days, and the weight changes and germination conditions were recorded on days 0, 7th, 14th, 21st, and 28th. According to the group number, five tubers were randomly sampled from each group each time, and three buds were randomly selected from each tuber for sampling. On days 0, 7th, 14th, 21st, and 28th of storage, 2 g of tuber flesh was taken from three random bud eyes using a sterilized stainless steel knife centered on the bud eye to determine the content of arabinogalactan proteins [27].

1 g of fresh-weight potato tuber was homogenized in ice-cold PBS buffer (pH = 7.2–7.4, 0.01 mol/L), and the homogenate ratio was selected to be 10%, with 9 mL of PBS buffer added to 1 g of tissue for homogenization. After centrifugation (speed of 2000–3000 rpm for 20 min) to obtain the supernatant, the AGPs enzyme-linked immunoassay kit (produced by Jiangsu Jingmei Biotechnology Co., Ltd., Jiangsu, China) was used to measure the concentration of arabinogalactan proteins in the supernatant, with an enzyme marker wavelength of 600.

On days 0, 7th, 14th, 21st, and 28th of storage, total RNA was extracted from three bud eyes and reverse transcribed into cDNA. The qRT-PCR technique was used to detect the expression level of the StFLA4 gene during potato tuber germination, with four replicates for each period. The reaction system and program were used as described above.

2.7. Bioinformatic Analysis

In order to predict the function, StFLA4 was screened against the Uniprot database (http://www.uniprot.org, URL: accessed on 14 October 2024) by inputting its gene index accompanied by Uniprot accessions. Known and predicted physical and functional protein–protein interactions were obtained from the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database (http://string-db.org/, URL: accessed on 14 October 2024) by “Single/Multiple Proteins by Sequence”. The StFLA4 and its interaction proteins were functionally categorized by the Gene Ontology (GO) (http://www.geneontology.org/, URL: accessed on 14 October 2024) database using the default parameters, and they were classified into different categories according to the predicted biological functions using the WEGO 2.0 website (https://wego.genomics.cn/, URL: accessed on 14 October 2024). Parts of the transcriptome data used in this study were obtained by our laboratory, and these data have been uploaded to NCBI (accession number: PRJNA742170 and PRJNA1136141), but the article has not yet been published. In addition, we also used the website of Spud DB Potato Genomics Resource (https://spuddb.uga.edu/index.shtml, URL: accessed on 7 November 2024) to obtain public expression profile data, and the gene expression values (TPM) for different potato RNA-seq libraries from the SRA were generated from DM_1-3_516_R44_potato.v6.1.TPM_gene_expression_matrix.xlsx (https://spuddb.uga.edu/dm_v6_1_download.shtml, URL: accessed on 7 November 2024), the specific references are Sprenger et al., 2016. In this part, the variety “RH89-039-16” is the material for transcriptome sequencing of roots, stems, vitro plant, petiole, young tuber, and mature tube [28]. In addition, we analyzed the expression pattern of StFLA4 and its interaction proteins between the two drought-sensitive cultivars, Milva and Alegria, and two more tolerant European potato reference cultivars, Desiree and Saturna, which also from the public transcriptome results [28]. Promoter element analysis was performed using the Plantcare website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, URL: accessed on 8 November 2024).

2.8. Statistical Analysis

Data were collated, and pie and bar charts were drawn using Excel 2010 (Microsoft, Los Angeles, CA, USA). Data were statistically analyzed by one-way analysis of variance (ANOVA) by Duncan’s multiple range tests in SPSS 19.0 (IBM Inc., NYSE, Armonk, NY, USA) software, and differences were regarded as significant at p < 0.05.

3. Results

3.1. StFLA4 Expression Abundance Is Downregulated Under Drought Stress in Potatoes

According to the results of RNA-Seq related to tuber development (PRJNA742170) (Unpublished), we found that with the extension of the potato growth period, the expression abundance of StFLA4 genes was significantly downregulated (Figure S2A). Through analysis of the expression pattern of StFLA4 under different materials and treatment conditions, the results showed a significant difference between the control group (CK) and different drought treatment times. The expression levels of the StFLA4 were lower than those in the CK at all drought treatment times, and the expression level dropped to the lowest level at 24 h of drought stress treatment. In the drought-sensitive variety “Atlantic”, the expression level of StFLA4 was about 13.7 times higher under normal cultivation conditions than after drought stress treatment (Figure 1), and in the drought-resistant variety “Kexin 1”, the difference in expression levels was 50-fold (Figure 1). These results indicate that while treating potatoes with 25% PEG-6000 (w/v) simulated drought, the StFLA4 gene negatively regulates the response to drought stress by downregulating its expression.

After 1 h of 25% PEG-6000 (w/v) simulated drought stress treatment, the expression level of the StFLA4 in the drought-resistant variety “Kexin 1” significantly decreased. However, the expression patterns in the drought-sensitive variety “Atlantic” have hardly changed (Figure 1). The result showed that the gene response to drought stress was faster in the “Kexin 1” than in the “Atlantic”. At all drought treatment times, the expression level of the StFLA4 in the drought-resistant variety “Kexin 1” was significantly lower than that in the drought-sensitive variety “Atlantic”. In summary, before drought treatment, there was no significant difference in gene StFLA4 expression between the two materials. In contrast, the expression difference was significant under drought treatment, indicating that the StFLA4 responds to drought stress and is more pronounced in drought-resistant varieties. Analysis of the promoter elements of the StFLA4 gene in potatoes showed that there were many stress response elements, such as drought-induced MYB binding site elements (MBS), anaerobic stress response elements (ARE), defense and stress response elements (TC-rich repeats), and cis-acting elements involved in the abscisic acid responsiveness (ABRE) (Figure S2B). These results further indicate that StFLA4 plays important roles in the stress response defense.

3.2. StFLA4 Located on Cell Membrane and Nucleus for Function

The subcellular localization experiment indicated that the StFLA4 protein was mainly localized in the cell membrane of the tobacco lower epidermal cells (Figure 2). The results are consistent with those predicted by bioinformatics (Figure S3; PlantmPLoc.cgi; https://www.uniprot.org/uniprot/, URL: accessed on 14 October, 2024), indicating that StFLA4 functions in the cell membrane to exert regulatory effects.

3.3. Overexpressing StFLA4 in Tobacco Reduces the Drought Resistance of the Plants

To verify the molecular function of StFLA4, the overexpression vector (pCAMBIA1302-StFLA4) was constructed, and the transgenic tobacco was obtained via the leaf disc method (Figure S4). Through PCR and qRT-PCR identification, seven transgenic plants were obtained (Figure 3A and Figure S5). Among them, there were two transgenic tobacco lines, OE2 and OE7, with the highest expression levels of the target gene, and these two lines were selected as the research materials for subsequent experiments. Drought stress treatment was performed on the negative control materials (CK) and the transgenic lines. Results showed that the CK developed well compared with the transgenic lines after fifteen days of drought stress (Figure 3B,C). Overexpressed StFLA4 in tobacco inhibited plant growth under drought stress, and the transgenic lines exhibited more wilted leaves than those of CK (Figure 3C). The expression pattern of StFLA4 in the overexpressing tobacco OE2 and OE7 under 7 days of drought stress showed a downward trend (Figure 4A), which is consistent with the expression results of StFLA4 in different time gradients of potato tubers under 25% PEG-6000 (w/v) simulated drought stress.

3.4. StFLA4 Inhibits ROS Scavenging and Protection Under Drought Stress

High levels of ROS (oxidative stress) are deleterious, and the major scavenging mechanisms include superoxide dismutase (SOD) and peroxidase (POD). In our study, there was not much difference in the SOD activity between the transgenic plants and the WT plants under normal watering conditions. However, after 7 days of drought stress, the difference between the two materials reached a highly significant level. The SOD activity of the WT plants was 1.28 and 1.22 times higher than that of the overexpressing transgenic tobacco OE2 and OE7, respectively (Figure 4B). Similarly, the POD activity of the WT plants was 1.37 and 1.18 times higher than that of the OE2 and OE7 after 7 days of drought stress (Figure 4C). Under normal watering conditions, the content of MDA in the transgenic plants was not much different from that in the WT plants. After 7 days of drought stress, the content of MDA in the transgenic tobacco was 65% and 68% higher than that in the WT tobacco, respectively (Figure 4D), indicating that the membrane system of the transgenic plants was damaged more severely under drought stress conditions.

3.5. StFLA4 Is Largely Implicated in Critical Proteins Related to Drought Stress

Based on the functional analysis from screening against the Uniprot database, we found that StFLA4 mainly participated in the abscisic acid-activated signaling pathway. This pathway comprises cellular response to stimulus, oxygen-containing compound, response to the hormone, and response to abscisic acid. The above-mentioned biological processes play key roles in drought stress (Figure 5A). In order to further reveal the function, we constructed a protein–protein interaction network of StFLA4 using the STRING database. In the network, most of the protein species were FAS1 domain-containing proteins. Others include DIOX_N domain-containing protein, galectin domain-containing protein, Ser/Thr protein kinase, and hexosyltransferase (Figure 5B). Gene Ontology (GO) analysis showed that these proteins mainly participated in response to osmotic stress, response to water deprivation, oxidoreductase activity, and response to stimulus (Figure 5C). In order to elucidate the mechanism of these proteins in response to drought stress, we analyzed the gene expression patterns in different stages or conditions using transcriptome data already available in our lab. Results showed that with the development of potato tubers, eleven genes presented the same decreasing expression pattern as StFLA4 (Figure 5D and Figure S2 NCBI ID of transcriptome data: PRJNA742170). Furthermore, after 6 h of drought stress treatment with 25% PEG-6000 (w/v) of potato, most of these genes were downregulated (Figure 5E, NCBI ID of transcriptome data: PRJNA1136141). Using public transcriptome data [28], we analyzed the expression pattern of StFLA4 and its interaction proteins between the two drought-sensitive cultivars, Milva and Alegria, and two more tolerant European potato reference cultivars, Desiree and Saturna. From the results in Figure 5F and Figure S7, StFLA4 and its interaction proteins present a higher expression abundance in the drought-sensitive cultivars (Milva/Alegria) than in the drought-tolerant cultivars (Desiree/Saturna) under drought and control conditions in the greenhouse (G3/G2/G1) and field (F4/F3/F1). This phenomenon is more obvious in the case of drought stress. In addition, most of these proteins were downregulated after experiencing drought stress in the greenhouse and the field, especially in the drought-tolerant cultivars (Desiree/Saturna) (Figure 5F). The above results further proved that these genes negatively regulated drought resistance in potatoes, along with StFLA4.

3.6. The Expression Pattern of StFLA4 Changed with the Tuber Germination in Potato

As shown in Figure S7, the expression level (TPM: Transcripts Per Million) of the StFLA4 gene in roots, stems, vitro plant, petiole, young tuber, and mature tuber in the cultivar “RH89-039-16” were 13.2686, 42.463, 34.88, 32.2597, 42.7939 and 6.73584 respectively [28]. This result is consistent with our result, in which the StFLA4 was downregulated as the tuber developed (Figure S2). Although the TPM value of StFLA4 in mature tuber is only 6.73584, the value in tuber sprout was raised to 15.418 (Figure S8) [28], which indicated that StFLA4 might respond to tuber germination. As the storage time extended, the germination rate of potatoes showed an increasing trend. At 7 days of storage, the buds of potato tubers were about 1–2 mm long, and at 28 days of storage, the buds of potato tubers were about 1–2 cm long (Figure 6A). After measuring the concentration of arabinogalactan proteins, it was found that the concentration of arabinogalactan proteins remained at high levels before germination for 14 days and reached a peak, which was 1.91 times the initial value. Then, the concentration began to decline, and by the 28th day, it had decreased by 1.44 times compared to the initial value of the start of storage (Figure 6B). Throughout the germination experiment, the concentration of arabinogalactan proteins in potato flesh increased and then decreased, indicating that the arabinogalactan proteins positively correlated with the germination vigor in potato buds. It is worth noting that the expression level of StFLA4 gradually increased before reaching a peak at 14 days of germination, which was 1.6 times compared to the initial value. Then the expression level began to decline (Figure 6C). Interestingly, in addition to responding to drought stress, StFLA4 and its interaction proteins also participated in seed coat development, cell differentiation, cell maturation, and cell growth (Figure 5C and Figure S5). This result indicated that the StFLA4 expression was consistent with AGP content during potato tuber germination and that StFLA4 was involved in the regulation of tuber germination.

4. Discussion

4.1. Differential Expression Pattern of StFLA4 Was Related to Drought Stress

As a class of glycoproteins related to cell wall structure, FLA proteins are a subfamily of arabinogalactans (AGPs) that play an important role in growth and development, stress response, secondary metabolism, and hormone signal regulation. Many FLA genes exist in plants, such as Arabidopsis, cotton, soybean, and pepper, but little is known about the function of FLA in potatoes. Analysis of the promoter elements of the FLA genes in potatoes showed that the StFLA gene family may be involved in the growth and development, stress response, hormone regulation, and photoperiod regulation of potato tuber [7]. This study screened the FLA genes based on transcriptome sequencing data and identified 11 members of the potato FLA gene family [7]. The StFLA4, which responds to drought stress, was selected as the target gene for verification. StFLA4 mainly participated in the mucilage biosynthetic process, abscisic acid-activated signaling pathway, seed coat development, and response to abiotic stress (Figure 5 and Figure S6). Studies have shown that genes induced by specific abiotic stress were likely to play an important role in regulating this stress tolerance [29]. To verify whether the StFLA4 was induced by drought stress, the drought-sensitive variety “Atlantic” and the drought-resistant variety “Kexin 1” were treated with 25% PEG-6000 (w/v) simulated drought stress, and the expression of the StFLA4 was analyzed at different times after drought stress using the qRT-PCR method. The results showed that the expression levels of the two potato varieties were lower than that of the CK at all time gradients, showing an overall downward trend. Moreover, the expression level of the StFLA4 in the drought-resistant variety “Kexin 1” decreased more seriously than that in the drought-sensitive variety “Atlantic”. Therefore, the StFLA4 has a negative regulatory function in drought resistance and enhances drought tolerance by reducing its expression level in potatoes.

4.2. Potato StFLA4 Negative Response to Drought Stress by Inhibiting ROS Scavenging

Due to their immovable characteristics, plants must face various harsh environments during their growth cycle. With the continuous evolution of plants, various stress-responsive genes have been produced to adapt to harsh living environments [30,31]. The research on FLA genes in the stress resistance of plants is extensive. In rice, the expression of OsFLA1 and OsFLA4 was downregulated under cold stress, and that of OsFLA5, OsFLA18, OsFLA19, and OsFLA27 was downregulated under drought and salt stress [32]. In wheat, the expression abundance of TaFLA9/12/14 was increased when the roots were subjected to cold or dehydration stress. In contrast, the expression of TaFLA3/4/9 was decreased after heat, dehydration, or NaCl treatment [12], and the expression of FLA in tobacco was downregulated after salt stress [11]. In this study, we found that in normal plants, the expression level of StFLA4 and its interaction proteins was decreased with the extension of drought stress time. In transgenic tobacco plants that overexpressed StFLA4, the expression pattern of StFLA4 was also downregulated under the induction of drought stress, indicating that StFLA4 negatively regulates drought stress in potatoes.

Reactive oxygen species (ROS) are the result of the partial reduction in atmospheric O₂ [33,34]. Both high levels of ROS (oxidative stress) and excessively low levels of ROS (reductive stress) are deleterious [35]. In order to cope with continuous ROS production, plants have a battery of enzymatic and non-enzymatic antioxidants, which function as an extremely efficient cooperative system [33]. Superoxide dismutase (SOD) and peroxidase (POD) can eliminate superoxide anion free radicals to protect cells from damage [36], and they play a crucial role in the oxidation and antioxidant balance of plants. When plants are subjected to drought stress, the balance of reactive oxygen species (ROS) in the plant body is disrupted, leading to a large accumulation of ROS, which are strong oxidizing compounds that can damage the plasma membrane and intracellular membrane systems [37,38,39]. In general, drought stress induces the overproduction of ROS, leading to oxidative stress that can damage the fatty acids in the cell membrane and produce small hydrocarbons such as malondialdehyde (MDA). MDA is a product of lipid peroxidation and represents the degree of cellular membrane damage [40]. The measurement of MDA is often used in conjunction with the measurement of SOD, where the high or low activity of SOD indirectly reflects the body’s ability to eliminate oxygen free radicals, and the high or low level of MDA indirectly reflects the severity of cell damage by free radicals [41]. When plants are in a state of drought, the plant membrane system is damaged, and after lipid peroxidation of the membrane, the final product is malondialdehyde. Therefore, MDA has become an indicator for evaluating drought severity [42]. In this experiment, through the analysis of drought resistance in transgenic tobacco and wild-type tobacco, it was found that under drought stress treatment, the wild-type tobacco with fewer withered leaves but overexpressed StFLA4 transgenic tobacco showed more withered leaves and looked even more wilted. After drought stress treatment, the SOD activity and POD activity in the overexpressing transgenic plants were significantly lower than those in the WT, and the MDA content was higher than that in the WT. These results collectively indicated that the drought resistance of the overexpressing StFLA4 transgenic tobacco was significantly reduced compared to the wild-type tobacco after the induction of drought stress. This confirms that the StFLA4 gene has a negative regulatory function in potato drought resistance.

4.3. StFLA4 Regulation of Potato Tuber Germination

After harvesting, as the storage time of potatoes increases, the tubers complete the ripening process, and the germination rate of potatoes shows an increasing trend under suitable conditions. In this study, we found that the concentration of arabinogalactan proteins in potato tubers remained at a high level before germination for 14 days and reached a peak. Then, the concentration of arabinogalactan proteins began to decline, and by the 28th day, it had decreased by 1.44 times compared to the initial value of the start of storage. Throughout the entire germination process of potatoes, the concentration of arabinogalactan proteins first increased and then decreased, indicating that the utilization degree of arabinogalactan proteins in potato flesh was positively correlated with the germination of buds, and arabinogalactan proteins affected the germination of potato tubers. The higher the germination index of potato tubers, the longer and larger buds grow, and the faster the concentration of arabinogalactan proteins in potato flesh decreases.

Analysis of the expression pattern of the StFLA4 gene during the germination process revealed that not only did the concentration of arabinogalactan proteins affect the germination of potato tubers, but the expression level of the StFLA4 gene also changed with the variation in germination time. When the concentration of arabinogalactan proteins was high, the expression level of the StFLA4 gene also increased accordingly. It is inferred that arabinogalactan proteins are closely related to the germination of potato tubers, and the StFLA4 gene also plays a critical role in the germination process of potato tubers. Throughout the germination experiment, the expression level of the StFLA4 increased firstly and then decreased, consistent with the change in the concentration of arabinogalactan proteins. This indicates that the StFLA4 is involved in the regulation of arabinogalactan protein synthesis in potatoes, promoting bud germination.

5. Conclusions

Fasciclin-like arabinogalactan proteins (FLAs) are involved in plant’s abiotic stress responses and cell proliferation, but there is little report about the FLAs gene in potatoes. This study found that the StFLA4 was induced by drought stress, and its expression decreased with the extension of stress time. Overexpressed StFLA4 in tobacco, the transgenic tobacco showed more withered leaves than wild-type tobacco under drought stress. Further analysis indicated several enzymes related to the removal of oxidative stress were decreased in transgenic tobacco. The above solid experimental results have validated that the StFLA4 negatively responded to drought stress by increasing oxidative stress. Knock-out StFLA4 in drought-sensitive varieties may be beneficial in developing drought-resistant potatoes. Furthermore, our results also proved that StFLA4 participates in the regulation of tuber germination in potatoes. These findings lay the foundation for elucidating the function and expression pattern of StFLA4 response to drought stress and tuber germination in potatoes.