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Article

Overexpression of the SiLEA5 Gene in Saussurea involucrata Increases the Low-Temperature Tolerance of Transgenic Tomatoes

by
Xiaoyan Liu
1,†,
Wenwen Xia
1,2,3,†,
Xiaoli Zhang
1,
Aowei Li
1,
Jiawang Qin
1,
Huili Sun
1,
Jin Li
1,* and
Jianbo Zhu
1,*
1
Key Laboratory of Agricultural Biotechnology, College of Life Sciences, Shihezi University, Shihezi 832000, China
2
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China
3
Hainan Yazhou Bay Seed Laboratory, Sanya 572000, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Horticulturae 2022, 8(11), 1023; https://doi.org/10.3390/horticulturae8111023
Submission received: 11 September 2022 / Revised: 19 October 2022 / Accepted: 25 October 2022 / Published: 2 November 2022
(This article belongs to the Special Issue Abiotic Stress Responses of Vegetable Crops)
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Abstract

:
The late embryonic development abundant protein (LEA) is a family of proteins widely present in the body and related to osmoregulation. Saussurea involucrata is an extremely cold-tolerant plant. In our previous studies, we found that the LEAs gene in Saussurea involucrata has up-regulated expression under low temperature. To evaluate the biological function of SiLEA5 protein under low-temperature stress and its potential in agricultural breeding, we isolated the SiLEA5 gene from Saussurea involucrata, constructed a plant overexpression vector, and transformed tomato. We found that SiLEA5 protein significantly increased the yield of transgenic tomatoes by increasing their photosynthetic capacity, including net photosynthetic rate, stomatal conductance, and intercellular CO2 concentration. Under low-temperature stress, the SiLEA5 protein can regulate proline metabolism and oxidative stress, which confers transgenic tomatos with cold resistance. Thus, our work provided evidence for the role of SiLEA5 protein in low-temperature stress resistance in plants, as well as potential applications in crop breeding and cold stress resistance research.

1. Introduction

Climate change can have catastrophic impacts on ecosystems and socioeconomics, with lasting impacts on global agriculture [1], including droughts, storms, heat waves, and frost affecting agricultural pests and diseases, crop growth and development, yield, and quality [2,3]. Therefore, in addition to protecting the ecology of the environment, improving the resistance of crops to cope with stress is one of the main problems in the current agricultural production system [4]. Previous studies have shown that plant genetic engineering can be used to optimize crop improvement, especially in terms of yield and domestication [5,6,7].
In the long evolutionary process, plants have developed complex mechanisms to adapt to stress. One is stress avoidance mechanism and the other is stress tolerance mechanism [8]. In recent years, the study of the function of the LEA gene in plants has gradually attracted the interest of researchers. LEA proteins are highly hydrophilic glycine-rich proteins that accumulate mainly at late stages of seed maturation and gradually disappear after germination [9]. LEA proteins are a widespread class of proteins in organisms associated with osmoregulation [10]. Under environmental stresses, such as drought, low temperature, salt stress, ABA, UV radiation, and NaHCO3, the mRNA of LEA gene will accumulate in a large amount [11,12], which belongs to stress response proteins [13]. A total of 51 LEA proteins have been identified in the Arabidopsis genome. According to the characteristics of the conserved domain, it can be divided into eight subfamilies, which are LEA1, LEA2, LEA3, LEA4, LEA5, LEA6, Dehydrin, and SMP [14].
The LEA protein of Arabidopsis was found to be involved not only in plant drought stress, but also in response to chilling stress. Mowla S B et al. found that AtLEA5 enhances resistance by scavenging reactive oxygen species in yeast and Arabidopsis [15]. Expression of SiDHN gene could promote cold and drought tolerance in transgenic tomato plants by inhibiting cell membrane damage, protecting chloroplasts, and enhancing reactive oxygen species scavenging ability [16]. The cotton LEA2 gene enhances drought resistance in transgenic Arabidopsis by increasing root length and antioxidant enzyme activity [17]. Under cold stress, GeLEAs is expressed in E. coli, which plays a protective role in cells, exhibiting higher viability and tolerance to low temperature [18]. Although the research on LEA proteins is becoming more and more extensive, most of it has focused on model plants, and little research has been reported on the function and application of LEA proteins in Saussurea involucrata.
The Saussurea involucrata is a critically endangered Compositae species. It grows at an altitude of 4000 m in the Tianshan Mountains of Western Xinjiang. The Saussurea involucrata created and selected a physiological mechanism that could withstand the harsh environment [19]. It is also a well-known Chinese herbal medicine, which can maintain homeostasis and increase immunity [20]. Tomato (Solanum lycopersicum L.) is a typical temperature-loving plant and the most important vegetable crop in the world [21]. Improving the stress resistance of tomatoes is particularly crucial in extreme climate change.
In previous studies, our team completed the transcriptome and genome sequencing of Saussurea involucrata and found that SiLEA5 gene expression was significantly upregulated under low-temperature stress [22]. It laid a strong foundation for us to further explore the specific function of LEA protein. In this study, we cloned the SiLEA5 gene of Saussurea involucrata and obtained transgenic tomato lines by Agrobacterium-mediated method. Field experiments and low-temperature tolerance analysis were carried out. The results indicated that the SiLEA5 gene may have certain potential in the mining of tolerance genes and the breeding of new crop varieties.

2. Materials and Methods

2.1. Plant Growth Materials

The sterile seedlings of Saussurea involucrata are preserved by our laboratory and the seeds of the wild-type tomato variety “Yaxin 87-5” are provided by Xinjiang Huiyuan Seed Industry Co., Ltd. (Shihezi City, Xinjiang, China). After being sterilized with 10% NaClO for 10 min, tomato seeds were evenly seeded on 1/2MS solid medium. When the first true leaf grew, its hypocotils were cut into 1.0-cm-long stem segments, which were spread flat on MS solid medium and cultivated for 48 h under dark conditions for tomato transformation. After the transformation of tomato, the positive lines were screened on solid medium supplemented with 200 mg/L kanamycin (Kan). After transplanting, the transgenic positive lines were identified by phenotypic changes and PCR results. The growth conditions of tissue culture seedlings were as follows: day and night temperature was 23/25 °C, relative humidity was 70 ± 5%, photoperiod was 16/8 h, and light intensity was 5000 lx. The growth conditions of cultivated plants were as follows: temperature 28 ± 2 °C, relative humidity 70 ± 5%, photoperiod 16/8 h, and light intensity 8000 lx. MS medium was purchased from Shenggong Bioengineering Co., LTD. (Shanghai, China).

2.2. Bioinformatics Analysis of SiLEA5 Gene

The amino acid sequence of LEA protein was downloaded from the Arabidopsis database and BlastP in NCBI (https://www.ncbi.nlm.nih.gov/, accessed on 19 March 2022) was used to identify the homology of SiLEA5 gene. Sequence analysis was performed by DNAMAN software (https://www.lynnon.com/, accessed on 20 March 2022). Subsequently, multiple comparisons were performed with ClustalW. The phylogenetic tree was completed by MEGA 7.0 software (https://www.megasoftware.net/, accessed on 28 June 2022) and constructed using neighbor-joining (NJ) method with 1000 bootstrap replicates; sequences with bootstrap scores <50% were removed. Finally, the evolutionary tree was embellished using the iTOL online website (https://itol.embl.de/, accessed on 29 June 2022). Information such as molecular weight of SiLEA5 protein was predicted by the online website ExPASy (https://www.expasy.org/, accessed on 29 June 2022).

2.3. SiLEA5 Gene Cloning and Plant Expression Vector Construction

Total RNA was isolated by RNAiso Plus kit (TaKaRa) and first-strand cDNA was synthesized on total RNA using a reverse transcription kit (TaKaRa) from low-temperature-treated Saussurea involucrata leaves. The specific primers of SiLEA5 gene were designed by Primer Premier 5.0 software (http://www.premierbiosoft.com/, accessed on 12 May 2020), and the SiLEA5 gene fragment was amplified from Saussurea involucrata cDNA by PCR. PCR products were identified by agarose gel electrophoresis, purified, and cloned into pMD19T vector (TaKaRa), then transformed into E. coli DH5-Alpha competent cells; positive clones were screened by PCR and verified by DNA sequencing. The correctly sequenced amplicons were inserted into the plant expression vector pCAMBIA2300 by double digestion (BamH Ι and Sal Ι). The plasmids were transformed into Agrobacterium tumefaciens GV3101 by electroshock. All data are provided in the supplementary material (Supplemental Figures S1–S3 and Table S1).

2.4. Transformation of Tomato and Identification of Positive Plants

To produce transgenic plants, we transformed wild-type plants with a plasmid containing SiLEA5. The specific method was as follows: hypocotyls of tomato sterile seedlings were cut into 1.0-cm-long stem segments and transferred to MS solid medium for dark culture for 48 h. Subsequently, the hypocotyls were placed in activated pCAMBIA2300-SiLEA5 bacterial solution, soaked for 10 min, then picked out, and the surface bacterial solution was blotted dry with sterile tissue paper. The stained explants were clamped onto MS medium supplemented with 2.0 mg/L 6-BA and 0.5 mg/L IAA (a sterile tissue paper was spread over the surface of the medium) and incubated in the dark for 2 days. After dark culture, explants were transferred to MS containing 2.0 mg/L 6-BA, 0.5 mg/L IAA, 200 mg/L Kan, and 200 mg/L Tim (Timentin). After 40 days of continuous culture, adventitious buds were differentiated at both ends of the stage callus (the medium was changed every 15 days). We cut adventitious buds with a surgical blade and transferred them onto 1/2 Ms rooting medium containing 0.3 mg/L IAA, 200 mg/L Kan, and 200 mg/L Tim. After 15 days, strong adventitious roots were induced. After 7 days of aeration, tomatoes were transplanted to mixed culture substrates (V (peat soil):V (vermiculite):V (perlite) = 3:1:2). They were allowed to grow under the conditions described in Section 2.1, and then normal cultivation and management proceeded.
According to the methods of plant genome extraction kit and RNAiso Plus kit (Takara), the genomic DNA and total RNA of tomato were extracted. Using DNA and cDNA as templates, the recombinant plasmid pCAMBIA2300-SiLEA5 was used as a positive control, and the non-transformed plants (WT) were negative. The transformed T0 tomato lines were validated by PCR and RT-PCR, and the transgenic lines from T1 generation were validated by qPCR. GAPDH was used as an internal reference, and the Roche LightCycler® 480 system (Salt Lake City, UT, USA) and SYBR Green Real-Time PCR Master Mix (KAPA Biosystems, Wilmington, DE, USA) were used for detection in 10 μL reactions, and the relative expression levels of each gene were detected by 2−ΔΔCt method calculation. Two independent T2 homologous lines were selected for subsequent experiments. All data are provided in the supplementary material (Supplemental Figures S4–S6).

2.5. Treatment of Low-Temperature Stress

To evaluate the effects of low-temperature stress on transgenic tomato plants, we selected wild-type and transgenic tomatoes with consistent growth at 4 weeks of age, which were treated with low temperature. In brief, wild-type and transgenic tomatoes grown at 25 °C were transferred to a 4 °C incubator and treated with cold for 8 h, then treated at 0 °C for 6 h. Freezing was carried out at −2 °C for 4 h. Tomato grown at 25 °C was used as a control. At the end of the cold and freezing treatments, the plants were moved to 25 °C for growth recovery. The growth state of tomato in each treatment stage was observed and recorded, and the camera was used to take photos. The above treatments were tested in three independent replicates.

2.6. Determination of Semilethal Temperature (LT50)

The relative conductivity of wild-type and transgenic tomatoes was evaluated at six temperature gradients (8, 6, 4, 2, and −2 °C) and at 25 °C as a control. During the treatment, the incubator was cooled at a rate of 2 °C/h and maintained for 2 h after reaching the set temperature. The low-temperature half-lethal temperature ( LT 50 = lnb k ) was used to calculate the inflection point temperature by fitting the observed relative conductivity using the Logstic equation model [23] y = a 1 + b e − k x . Three replications were performed for each treatment.

2.7. Physiological Determination of Stress Resistance

We tried to choose the same area of tomato leaves for the physiological and biochemical indication detection after each stress treatment. Relative water content (RWC) was determined by weighing method [24], and relative electrolyte leakage (REL) was determined using the EC215 conductivity meter (MarksonScienceInc., DelMar, CA, USA). Malondialdehyde (MDA) content was determined by the barbituric acid (TBA) method [25]. The soluble sugar, soluble protein, and proline content were determined by colorimetric method [26]. The superoxide dismutase activity (SOD) was determined by the nitrogen blue tetrazolium (NBT) photoreduction method. The peroxidase (POD) was determined by guaiacol method. The catalase (CAT) was determined by the ultraviolet absorption method [27]. In simple terms, 0.5 g fresh tissue leaves were taken, cleaned with tap water, put into a precooled mortar, 1.6 mL 50 mmol/L precooled phosphate buffer (pH7.8) was added and they were ground into homogenate (this step is performed on ice), transferred into a centrifuge tube and centrifuged at 12,000× g at 4 °C for 10 min, and the supernatant was the crude enzyme solution. For determination of SOD activity, in the experimental group, 50 μL enzyme solution and 3 mL NBT reaction solution were added into the test tube, and the test tube was placed under light for 20 min after being fully mixed. In the control tube, 50 µL PBS (without enzyme solution) and 3 mL NBT reaction solution were added, and the maximum photoreduction value was determined after mixing. The light absorption value at 560 nm was measured under the condition of dark-treated control tube after zero adjustment. For determination of POD enzyme activity, 1.7 mL PBS, 0.1 mL 1% guaiacol, 0.1 mL enzyme solution, and 0.1 mL 3% H2O2 were added into the test tube of the experimental group. In the control group, PBS was used instead of an enzyme solution. The absorbance at 470 nm was measured (1 min). For CAT enzyme activity assay, 3 mL of the reaction solution (200 mL 0.15 M, pH 7.0 PBS, and 0.3092 mL 30% H2O2) was added to 0.1 mL of the enzyme solution. The absorption value at 240 nm (1 min) was measured by zeroing PBS. Three biological replicates were used to calculate each index.

2.8. Analysis of Agronomic Characters and Yield

Two years of field trials were conducted on wild-type and transgenic tomatoes to examine the role and prospects of SiLEA5 protein in agricultural applications. The two-year trial planting area was 10.5 m2 (N44°20′, E85°30′, Shihezi, Xinjiang, China), and normal field management was followed during the period (according to the growth of crops having regular watering and appropriate fertilization, pests and diseases found timely treatment to avoid large-scale crop damage). At the start of flowering, the plant height and stem diameter were measured with a tape measure and vernier caliper. The ratio of fruit number, weight, transverse diameter, and longitudinal diameter of various tomato plants were measured during the harvest season, and the yield was calculated in accordance with planting density. For each indicator, at least 10 plants were measured (which the dots in the figure indicate) and the average value was calculated.
A portable photosynthesis system (GFS3000; Walz GmbH, Effeltrich, Germany) was used to quantify photosynthetic parameters, such as net photosynthetic rate, transpiration rate, intercellular CO2 concentration, and stomatal conductance. Water use efficiency (WUE) was calculated as the ratio of net photosynthetic rate to transpiration rate. Please refer to the operation manual of GSF-3000 for specific determination method. We determined 18 wild-type and 21 transgenic tomatoes (the dots in the figure indicate); the same location of the plant’s leaves was chosen for the analysis. All parameters were measured between 10:00 and 11:30.

3. Results

3.1. Bioinformatics Analysis of the SiLEA5 Gene

The full-length sequence (526bp) of SiLEA5 gene was cloned from Saussurea involucrata. The sequence contained a 402 bp open reading frame and encoded a protein with 133 amino acid residues. We used the Pfam database (http://Pfam-legacy.xfam.org/, accessed on 29 July 2022) to annotate the obtained protein sequences of the LEA family genes in Arabidopsis thaliana. Visual analysis was performed using the TBtools tool (https://github.com/CJ-Chen/TBtools/releases, accessed on 29 July 2022). The results showed that the SiLEA5 protein had the same conserved domain as the LEA5 family in Arabidopsis (Figure 1A). We constructed a phylogenetic tree (different colors represented different subfamilies) and showed that the SiLEA5 protein belongs to LEA5 subfamily (Figure 1B). Therefore, it was named SiLEA5. According to the prediction of ProtParam database (https://web.expasy.org/protparam/, accessed on 31 July 2022), the results showed that the protein has a molecular weight of 14.35 KD and an instability index of 49.34. It was an unstable protein.

3.2. Quantitative Real-Time PCR

QPCR results showed that the expression level of SiLEA5 gene in transgenic tomato was significantly higher than that in wild-type tomato, as shown in Supplementary material (Supplementary Table S1 and Figure S6). We selected OE-2 and OE-3 lines for subsequent experiments.

3.3. SiLEA5 Gene Increased the Low-Temperature Tolerance of Transgenic Tomato

To investigate whether the SiLEA5 gene plays a positive regulatory role in low temperature, we subjected transgenic tomatoes obtained to low-temperature stress. Transgenic tomato mostly maintained its pretreatment condition after an 8 h cold treatment at 4 °C, but wild-type tomato showed some wilting on the leaf edges that became worse as the treatment temperature was lowered. When the temperature decreased from 0 °C to −2 °C, the wild-type tomato almost lost its vitality, while the transgenic tomato leaves only slightly curled (Figure 2A–D). Wild-type tomato failed to recover after 24 h of room temperature recovery.
The ultimate degraded byproduct of membrane lipid peroxidation is malondialdehyde (MDA) and its concentration might indicate the severity of stress damage to plants. Malondialdehyde (MDA) and relative conductivity are crucial measures that assess the severity of bodily harm. Our findings demonstrated that malondialdehyde (MDA) and relative conductivity rose as treatment temperature decreased and that wild-type had a considerable advantage over transgenic tomato. The relative conductivity of the wild type reached 73.8% with a drop in temperature to 0 °C and 92.6% with a drop in temperature to −2 °C (Figure 2E,F). Water is the source of life; the relative water content of plant leaves represents the strength of metabolic activity and is one of the indicators to judge the strength of plant resistance. The relative water content (RWC) of tomato leaves decreased with the decrease in treatment temperature. However, the transgenic tomato was significantly higher than that of wild-type (Figure 2G).
Proline, soluble protein, and soluble sugar are commonly considered osmotic regulators, and their accumulation in plants is often used as one of the indicators for screening resistance. Our findings demonstrated that the soluble sugar, soluble protein, and proline content of wild-type and transgenic tomatoes gradually increased with the decrease in stress temperature, reaching the maximum at −2 °C. The accumulation of transgenic tomato was significantly higher than wild-type (Figure 3A–C). Plant antioxidant system is a major way to remove toxic substances of reactive oxygen species (ROS) in plants, which is significantly related to stress resistance. We found that the activities of catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) in wild-type and transgenic tomato increased with the decrease in treatment temperature. Moreover, the enzyme activity of transgenic tomato was significantly higher than that of wild-type. When the temperature decreased to −2 °C, the POD activity of wild-type and transgenic tomato increased by 2.815 and 7.185 folds, respectively, compared with that of room-temperature-growing tomato (Figure 3D–F).

3.4. Analysis of Semilethal Temperature (LT50)

A logistic equation was used to fit the electrolyte exudation rates under different low temperatures. The results showed that the LT50 of wild-type was 4.40 °C and that of transgenic tomato was −1.65 °C. These results indicated that transgenic tomato would suffer less damage under low-temperature treatment and showed strong low-temperature tolerance.

3.5. Transgenic Tomatoes with SiLEA5 Gene Overexpression Have Greater Photosynthetic Efficiency

Photosynthesis is one of the most important mechanisms in plant physiology and ecology. Our results showed that the transpiration rate of transgenic tomato was 9.95 mmol m−2 s−1, which was significantly higher than that of wild-type. Stomatal conductance was 1.65 times that of the wild type. The net photosynthetic rate was 18.60 μmol m−2 s−1, which was 1.27 times that of the wild type. The intercellular CO2 concentration was 1.07 times higher than that of the wild type. The water use efficiency (WUE) was 1.88 μmol mol−1, which was 1.16 times that of the wild type. There was no significant difference in saturated vapor pressure between wild-type and transgenic tomato (Figure 4A–F).

3.6. Analysis of Yields and Agronomic Characteristics

We monitored the field experiment for two years to acquire the yield data correctly. The findings demonstrated that both transgenic and wild-type tomato development states were consistently stable throughout the vegetative growth stage (Figure 5A,B). The plant height and stem diameter of different transgenic tomato lines were measured at flowering stage. In 2020 and 2021, the average plant height of transgenic tomato was 29.71 cm and 30.00 cm, respectively, which was significantly different from that of the wild type (Figure 5D). The average stem diameter of transgenic tomato was 33.72 mm and 35.41 mm, respectively, which was not significantly different from that of wild-type (Figure 5E). At the stage of fruit ripening, we measured the ratio of transverse diameter to longitudinal diameter of wild-type and transgenic tomato fruits. Our results showed that the transgenic tomatoes were significantly higher than the wild-type tomatoes (Figure 5C,F). Moreover, the number of fruits per plant and the average weight of fruit per plant of transgenic tomato were significantly higher than those of wild-type (Figure 5G,H). We evaluated the yield data for 2020 and 2021 and found that the yield of the transgenic tomato was 1.70 and 1.51 times higher than that of the wild type. Finally, we analyzed the data of agronomic characters and yield in these two years and found that there was no significant difference (p > 0.05) in plant height, stem diameter, and yield between the two years, and the whole tended to be stable.

4. Discussion and Conclusions

One of the key elements limiting agricultural growth is abiotic stress [28]. Stress response in plants is a very intricate life process. With the development of molecular biology and plant genetic engineering, various new technologies have been gradually applied to the study of plant stress resistance [29,30,31]. We cloned SiLEA5 protein from Saussurea involucrata and constructed a plant overexpression vector. To study the protective effect of SiLEA5 protein on tomato under stress.
Our results revealed that transgenic tomato showed greater tolerance under low-temperature stress. Previous studies have found that the NtLEA gene in tobacco has the potential function of enhancing the resistance of tobacco to abiotic stresses [32]. BPC2 negatively regulates LEA4-5 expression to participate in osmotic stress in Arabidopsis [33]. These results agree with our findings. Under adversity stress, it is simple to harm plants, which may both stunt their natural physiological development and lead them to perish. Because of adversity stress, the plant cell’s steady-state balance is disrupted, resulting in the cell’s disordered state [34]. The introduction of some stress-resistant genes may essentially resolve this issue and enhance the plant’s capacity to withstand stress, such as the SiLEA5 gene.
Initially, it was discovered that cotton seed late embryos accumulated LEA protein [35]. LEA protein plays an essential role in abiotic stress of plants, which can stabilize the membrane system and enhance the resistance of antioxidant enzyme activity to various stresses [36,37]. In general, plants produce large amounts of ROS in response to stress. Lower levels of ROS can transmit cellular signals as a signaling molecule [38]. However, excessive ROS may adversely harm the antioxidant system and cell membrane structure [39].
In this study, MDA accumulation was found to be significantly lower in transgenic tomato than in wild-type under increased low-temperature stress. This finding suggested that the expression of SiLEA5 reduced the destruction and oxidative damage of cell membrane structure and relatively maintained the stability of cell membrane. Osmotic regulation is a key mechanism by which plants overcome adversity. In a stress environment, cells regulate the osmotic potential by accumulating various organic and inorganic substances to increase the concentration of cell fluid to maintain the normal metabolic process [40]. In this investigation, we discovered a strong and favorable correlation between the amount of osmotic regulator accumulation and the stress level. The accumulation of proline was approximately 2.51 times higher in transgenic tomato. These osmoregulatory substances typically co-ordinate plant osmotic equilibrium and cellular water content, which supports cells’ regular metabolic processes [41]. Proline serves as a protective component for cell membranes and enzymes, as well as a free radical scavenger and excellent osmotic regulator [42]. As a result, proline protects plant development under osmotic stress. Previous studies have found that controlling the interaction between the antioxidant system and proline metabolism in cucumber leaves allowed the plant to respond to salt stress [43]. Additionally, in response to abiotic stress, plants regulate gas signaling molecules, including NO, CO, and HS, to participate in plant proline metabolism [44]. The GmDREB6 gene is overexpressed in soybean, increasing the proline concentration and giving transgenic soybean salt tolerance [45]. When LbDREB is overexpressed, the proline and soluble proteins are present at higher concentrations. LbDREB may also increase plants’ resistance to copper by activating several stress-related genes, including LEA, Cu/ZN-SOD, and PODs, thus mediating physiological processes related to plant stress tolerance [46]. The study by Weidong Wang et al. elucidated the potential regulatory network of Camellia sinensis CsLEA genes in resistance to stress response to adversity and found that the expression of CsLEA genes is directly regulated by transcription factors, such as DREB/CBF and bZIP [47,48]. Our study also revealed that, when transgenic tomatoes were exposed to low-temperature stress, antioxidant enzymes, such as CAT, POD, and SOD, were significantly increased compared to wild-type tomatoes. This finding suggests that overexpressing the SiLEA5 gene enhances the capacity to scavenge ROS as a means of regulating oxidative stress capacity and maintaining normal cellular metabolism [49]. Additionally, previous research has shown that LEA proteins act as molecular chaperones to scavenge excessive ROS accumulation [50,51,52]. This suggests that transgenic tomatoes produce and scavenge reactive oxygen species in a dynamic equilibrium under low-temperature stress, minimizing the damage caused by abiotic pressure and preserving the organism’s regular metabolic processes. According to Binghao Du et al. [53], overexpression of MSGSTU8 increased free radical scavenging ability and decreased the severity of cell damage in tobacco. This agrees with our results. Therefore, we speculate that SiLEA5 protein may be involved in ABA signaling [54,55,56] through the expression regulation of the CBF/DREB1 transcription factor family [47,57,58,59] and further involved in the response to stress signals [60,61,62].
At present, the light energy utilization efficiency of field crops is still very low, far from reaching the theoretical value [63]. To tap the potential of light energy transfer and transformation at present, it depends on in-depth research on the molecular mechanism of photosynthesis and its mechanism breakthrough [64]. Water transport efficiency of plants decreases under stress, resulting in insufficient water supply, one of the raw materials of photosynthesis, which leads to inhibition of photosynthesis [65]. In this study, we discovered that transgenic tomato had significantly higher net photosynthetic rate, transpiration rate, stomatal conductance, and intercellular CO2 concentration than wild-type. These results indicated that SiLEA5 could improve photosynthesis rate and water use efficiency by regulating intercellular CO2 concentration and stomatal conductance. This allows the plant to gain more water-absorbing power through transpiration, lower the leaf surface temperature, and prevent high-temperature burns. The body also began to collect the byproducts of cellular absorption at the same time, which further explained why the output of transgenic tomatoes increased. Plant aquaporins have been shown to have considerable promise for enhancing crop development, boosting agricultural production, and adversity protection, according to recent research [66]. Additionally, Barbara karpinska et al. also verified that AtLEA5 expression increased barley seed output by boosting photosynthesis in response to stress [67]. Consequently, new genes and metabolic routes may enhance photosynthesis and biomass output [68]. We hypothesized that the enhanced photosynthetic capacity of transgenic plants may be due to SiLEA5 protein, as a signaling molecule, regulating chlorophyll content and ensuring the level of photosynthesis [69]. As a result, crop yields are raised.
Due to adverse climate change and an increasing global population, more productive and tolerant crops are urgently needed [70]. Traditional methods of crop improvement may have reached their limits; therefore, the use of genetic engineering promises to further improve crop productivity [68]. Tomato is one of the most influential edible vegetable crops in the world, with high nutritional value and popularity among consumers. In addition, it also has important economic significance [71]. Our research shows that the SiLEA5 protein can increase the yield of transgenic tomato. The possible reason is that the introduction of SiLEA5 gene may promote cell division and growth, leading to the increase in fruit size and, ultimately, increased yield. Fruit quality is an essential link in crop breeding. However, it has not been clarified for fruit quality and its molecular mechanism, and it deserves our further investigation. Therefore, our research indicates that SiLEA5 can improve crop yield while improving crop stress resistance. It may be an excellent biological resource and has great application potential in crop breeding.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/horticulturae8111023/s1, Figure S1: Cloning the LEA5 gene from Saussurea involucrate; Figure S2: Identification of recombinant plasmids by enzyme digestion; Figure S3: Identification of Agrobacterium tumefaciens by PCR; Figure S4: Characterization of transgenic tomato through DNA PCR; Figure S5: Characterization of transgenic tomato expressing SiLEA5 through RT-PCR; Figure S6: Real-time PCR was used to detect wild type and transgenic tomatoes; Table S1: List of primers used in this study.

Author Contributions

X.L. and W.X. were co-first authors of the present paper. J.L. and J.Z. conceived and designed the experiments. X.L. wrote the paper. W.X. completed the data acquisition of the paper. X.Z., A.L., J.Q. and H.S. completed the preliminary preparation and data analysis of the experiment. All authors have read and agreed to the published version of the manuscript.

Funding

Natural Science Foundation of China (32160061) (Entry name: Study on improving water use efficiency of Saussurea involucrata (SIPIP2;7) through chloride ion signal pathway).

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary material; further inquiries can be directed to the corresponding authors [email protected].

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Shahzad, A.; Ullah, S.; Dar, A.A.; Sardar, M.F.; Mehmood, T.; Tufail, M.A.; Shakoor, A.; Haris, M. Nexus on climate change: Agriculture and possible solution to cope future climate change stresses. Environ. Sci. Pollut. Res. Int. 2021, 28, 14211–14232. [Google Scholar] [CrossRef] [PubMed]
  2. Shao, J.; Li, G.; Li, Y.; Zhou, X. Intraspecific responses of plant productivity and crop yield to experimental warming: A global synthesis. Sci. Total Environ. 2022, 840, 156685. [Google Scholar] [CrossRef] [PubMed]
  3. Filho, W.L.; Setti, A.F.F.; Azeiteiro, U.M.; Lokupitiya, E.; Donkor, F.K.; Etim, N.N.; Matandirotya, N.; Olooto, F.M.; Sharifi, A.; Nagy, G.J.; et al. An overview of the interactions between food production and climate change. Sci. Total Environ. 2022, 838, 156438. [Google Scholar] [CrossRef] [PubMed]
  4. Garcia-Ruiz, H.; Szurek, B.; Van den Ackerveken, G. Stop helping pathogens: Engineering plant susceptibility genes for durable resistance. Curr. Opin. Biotechnol. 2021, 70, 187–195. [Google Scholar] [CrossRef]
  5. Sami, A.; Xue, Z.; Tazein, S.; Arshad, A.; He Zhu, Z.; Ping Chen, Y.; Hong, Y.; Tian Zhu, X.; Jin Zhou, K. CRISPR-Cas9-based genetic engineering for crop improvement under drought stress. Bioengineered 2021, 12, 5814–5829. [Google Scholar] [CrossRef]
  6. Nowicka, B.; Kruk, J. Genetic engineering as a method for the improvement of photosynthesis. Postepy. Biochem. 2018, 64, 13–20. [Google Scholar] [CrossRef]
  7. Xia, W.; Liu, X.; Xin, H.; Wu, X.; Liu, R.; Li, J.; Zhu, J. Saussurea involucrata PIP2;7 improves photosynthesis and drought resistance by decreasing the stomatal density and increasing intracellular osmotic pressure. Environ. Exp. Bot. 2021, 191, 10406. [Google Scholar] [CrossRef]
  8. Zandalinas, S.I.; Fichman, Y.; Devireddy, A.R.; Sengupta, S.; Azad, R.K.; Mittler, R. Systemic signaling during abiotic stress combination in plants. Proc. Natl. Acad. Sci. USA 2020, 117, 13810–13820. [Google Scholar] [CrossRef]
  9. Battaglia, M.; Covarrubias, A.A. Late Embryogenesis Abundant (LEA) proteins in legumes. Front. Plant Sci. 2013, 4, 190. [Google Scholar] [CrossRef] [Green Version]
  10. Xu, M.; Tong, Q.; Wang, Y.; Wang, Z.; Xu, G.; Elias, G.K.; Li, S.; Liang, Z. Transcriptomic Analysis of the Grapevine LEA Gene Family in Response to Osmotic and Cold Stress Reveals a Key Role for VamDHN3. Plant Cell Physiol. 2020, 61, 775–786. [Google Scholar] [CrossRef]
  11. Wu, X.; Gong, F.; Yang, L.; Hu, X.; Tai, F.; Wang, W. Proteomic analysis reveals differential accumulation of small heat shock proteins and late embryogenesis abundant proteins between ABA-deficient mutant vp5 seeds and wild-type Vp5 seeds in maize. Front. Plant Sci. 2014, 5, 801. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  12. Zamora-Briseño, J.A.; de Jiménez, E.S. A LEA 4 protein up-regulated by ABA is involved in drought response in maize roots. Mol. Biol. Rep. 2016, 43, 221–228. [Google Scholar] [CrossRef] [PubMed]
  13. Abdul Aziz, M.; Sabeem, M.; Mullath, S.K.; Brini, F.; Masmoudi, K. Plant Group II LEA Proteins: Intrinsically Disordered Structure for Multiple Functions in Response to Environmental Stresses. Biomolecules 2021, 11, 1662. [Google Scholar] [CrossRef] [PubMed]
  14. Hong-Bo, S.; Zong-Suo, L.; Ming-An, S. LEA proteins in higher plants: Structure, function, gene expression and regulation. Colloids Surf. B Biointerfaces 2005, 45, 131–135. [Google Scholar] [CrossRef]
  15. Mowla, S.B.; Cuypers, A.; Driscoll, S.P.; Kiddle, G.; Thomson, J.; Foyer, C.H.; Theodoulou, F.L. Yeast complementation reveals a role for an Arabidopsis thaliana late embryogenesis abundant (LEA)-like protein in oxidative stress tolerance. Plant J. 2006, 48, 743–756. [Google Scholar] [CrossRef]
  16. Guo, X.; Zhang, L.; Wang, X.; Zhang, M.; Xi, Y.; Wang, A.; Zhu, J. Overexpression of Saussurea involucrata dehydrin gene SiDHN promotes cold and drought tolerance in transgenic tomato plants. PLoS ONE 2019, 14, e0225090. [Google Scholar] [CrossRef]
  17. Magwanga, R.O.; Lu, P.; Kirungu, J.N.; Dong, Q.; Hu, Y.; Zhou, Z.; Cai, X.; Wang, X.; Hou, Y.; Wang, K.; et al. Cotton Late Embryogenesis Abundant (LEA2) Genes Promote Root Growth and Confer Drought Stress Tolerance in Transgenic Arabidopsis thaliana. G3 (Bethesda) 2018, 8, 2781–2803. [Google Scholar] [CrossRef] [Green Version]
  18. Zeng, X.; Ling, H.; Yang, J.; Li, Y.; Guo, S. LEA proteins from Gastrodia elata enhance tolerance to low temperature stress in Escherichia coli. Gene 2018, 646, 136–142. [Google Scholar] [CrossRef]
  19. Gong, G.; Huang, J.; Yang, Y.; Qi, B.; Han, G.; Zheng, Y.; He, H.; Chan, K.; Tsim, K.W.; Dong, T.T. Saussureae Involucratae Herba (Snow Lotus): Review of Chemical Compositions and Pharmacological Properties. Front. Pharmacol. 2019, 10, 1549. [Google Scholar] [CrossRef]
  20. Kuo, C.L.; Agrawal, D.C.; Chang, H.C.; Chiu, Y.T.; Huang, C.P.; Chen, Y.L.; Huang, S.H.; Tsay, H.S. In vitro culture and production of syringin and rutin in Saussurea involucrata (Kar. et Kir.)—An endangered medicinal plant. Bot. Stud. 2015, 56, 12. [Google Scholar] [CrossRef]
  21. Wang, J.; Hu, H.; Wang, W.; Wei, Q.; Hu, T.; Bao, C. Genome-Wide Identification and Functional Characterization of the Heat Shock Factor Family in Eggplant (Solanum melongena L.) under Abiotic Stress Conditions. Plants 2020, 9, 915. [Google Scholar] [CrossRef] [PubMed]
  22. Li, J.; Liu, H.; Xia, W.; Mu, J.; Feng, Y.; Liu, R.; Yan, P.; Wang, A.; Lin, Z.; Guo, Y.; et al. De Novo Transcriptome Sequencing and the Hypothetical Cold Response Mode of Saussurea involucrata in Extreme Cold Environments. Int. J. Mol. Sci. 2017, 18, 1155. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  23. Chung, S.W.; Rho, H.; Lim, C.K.; Jeon, M.K.; Kim, S.; Jang, Y.J.; An, H.J. Photosynthetic response and antioxidative activity of ’Hass’ avocado cultivar treated with short-term low temperature. Sci. Rep. 2022, 12, 11593. [Google Scholar] [CrossRef] [PubMed]
  24. Dong, S.; Jiang, Y.; Dong, Y.; Wang, L.; Wang, W.; Ma, Z.; Yan, C.; Ma, C.; Liu, L. A study on soybean responses to drought stress and rehydration. Saudi J. Biol. Sci. 2019, 26, 2006–2017. [Google Scholar] [CrossRef]
  25. Dai, L.; Feng, Z.; Pan, X.; Xu, Y.; Li, P.; Lefohn, A.S.; Harmens, H.; Kobayashi, K. Increase of apoplastic ascorbate induced by ozone is insufficient to remove the negative effects in tobacco, soybean and poplar. Environ. Pollut. 2019, 245, 380–388. [Google Scholar] [CrossRef]
  26. Cheng, L.; Li, X.; Huang, X.; Ma, T.; Liang, Y.; Ma, X.; Peng, X.; Jia, J.; Chen, S.; Chen, Y.; et al. Overexpression of sheepgrass R1-MYB transcription factor LcMYB1 confers salt tolerance in transgenic Arabidopsis. Plant Physiol. Biochem. 2013, 70, 252–260. [Google Scholar] [CrossRef]
  27. Batool, T.; Ali, S.; Seleiman, M.F.; Naveed, N.H.; Ali, A.; Ahmed, K.; Abid, M.; Rizwan, M.; Shahid, M.R.; Alotaibi, M.; et al. Plant growth promoting rhizobacteria alleviates drought stress in potato in response to suppressive oxidative stress and antioxidant enzymes activities. Sci. Rep. 2020, 10, 16975. [Google Scholar] [CrossRef]
  28. Yoshida, T.; Yamaguchi-Shinozaki, K. Metabolic engineering: Towards water deficiency adapted crop plants. J. Plant Physiol. 2021, 258–259, 153375. [Google Scholar] [CrossRef]
  29. Xia, X.; Cheng, X.; Li, R.; Yao, J.; Li, Z.; Cheng, Y. Advances in application of genome editing in tomato and recent development of genome editing technology. Theor. Appl. Genet. 2021, 134, 2727–2747. [Google Scholar] [CrossRef]
  30. Hefferon, K. Plant biotechnology patents: Applications in agriculture and medicine. Recent. Pat. Biotechnol. 2010, 4, 136–152. [Google Scholar] [CrossRef]
  31. Kumar, V.; Khare, T.; Sharma, M.; Wani, S.H. Engineering Crops for the Future: A Phosphoproteomics Approach. Curr. Protein. Pept. Sci. 2018, 19, 413–426. [Google Scholar] [CrossRef] [PubMed]
  32. Geng, W.; Wang, Y.; Zhang, J.; Liu, Z.; Chen, X.; Qin, L.; Yang, L.; Tang, H. Genome-wide identification and expression analyses of late embryogenesis abundant (LEA) gene family in tobacco (Nicotiana tabacum L.) reveal their function in abiotic stress responses. Gene 2022, 836, 146665. [Google Scholar] [CrossRef]
  33. Li, Q.; Wang, M.; Fang, L. BASIC PENTACYSTEINE2 negatively regulates osmotic stress tolerance by modulating LEA4-5 expression in Arabidopsis thaliana. Plant Physiol. Biochem. 2021, 168, 373–380. [Google Scholar] [CrossRef]
  34. Mareri, L.; Parrotta, L.; Cai, G. Environmental Stress and Plants. Int. J. Mol. Sci. 2022, 23, 5416. [Google Scholar] [CrossRef] [PubMed]
  35. Dure, L., 3rd; Greenway, S.C.; Galau, G.A. Developmental biochemistry of cottonseed embryogenesis and germination: Changing messenger ribonucleic acid populations as shown by in vitro and in vivo protein synthesis. Biochemistry 1981, 20, 4162–4168. [Google Scholar] [CrossRef] [PubMed]
  36. Popova, A.V.; Rausch, S.; Hundertmark, M.; Gibon, Y.; Hincha, D.K. The intrinsically disordered protein LEA7 from Arabidopsis thaliana protects the isolated enzyme lactate dehydrogenase and enzymes in a soluble leaf proteome during freezing and drying. Biochim. Biophys. Acta. 2015, 1854, 1517–1525. [Google Scholar] [CrossRef] [PubMed]
  37. He, C.; Liu, X.; Teixeira da Silva, J.A.; Wang, H.; Peng, T.; Zhang, M.; Si, C.; Yu, Z.; Tan, J.; Zhang, J.; et al. Characterization of LEA genes in Dendrobium officinale and one Gene in induction of callus. J. Plant. Physiol. 2021, 258–259, 153356. [Google Scholar] [CrossRef]
  38. Shi, K.; Gao, Z.; Shi, T.Q.; Song, P.; Ren, L.J.; Huang, H.; Ji, X.J. Reactive Oxygen Species-Mediated Cellular Stress Response and Lipid Accumulation in Oleaginous Microorganisms: The State of the Art and Future Perspectives. Front. Microbiol. 2017, 8, 793. [Google Scholar] [CrossRef]
  39. Bobrovskikh, A.; Zubairova, U.; Kolodkin, A.; Doroshkov, A. Subcellular compartmentalization of the plant antioxidant system: An integrated overview. Peer J. 2020, 8, e9451. [Google Scholar] [CrossRef]
  40. Bouzroud, S.; Gasparini, K.; Hu, G.; Barbosa, M.A.M.; Rosa, B.L.; Fahr, M.; Bendaou, N.; Bouzayen, M.; Zsögön, A.; Smouni, A.; et al. Down Regulation and Loss of Auxin Response Factor 4 Function Using CRISPR/Cas9 Alters Plant Growth, Stomatal Function and Improves Tomato Tolerance to Salinity and Osmotic Stress. Genes 2020, 11, 272. [Google Scholar] [CrossRef]
  41. Furlan, A.L.; Bianucci, E.; Giordano, W.; Castro, S.; Becker, D.F. Proline metabolic dynamics and implications in drought tolerance of peanut plants. Plant Physiol. Biochem. 2020, 151, 566–578. [Google Scholar] [CrossRef] [PubMed]
  42. Lin, Z.; Wang, Y.L.; Cheng, L.S.; Zhou, L.L.; Xu, Q.T.; Liu, D.C.; Deng, X.Y.; Mei, F.Z.; Zhou, Z.Q. Mutual regulation of ROS accumulation and cell autophagy in wheat roots under hypoxia stress. Plant Physiol. Biochem. 2021, 158, 91–102. [Google Scholar] [CrossRef]
  43. Naliwajski, M.; Skłodowska, M. The Relationship between the Antioxidant System and Proline Metabolism in the Leaves of Cucumber Plants Acclimated to Salt Stress. Cells 2021, 10, 609. [Google Scholar] [CrossRef] [PubMed]
  44. He, H.; He, L.F. Regulation of gaseous signaling molecules on proline metabolism in plants. Plant Cell Rep. 2018, 37, 387–392. [Google Scholar] [CrossRef]
  45. Nguyen, Q.H.; Vu, L.T.K.; Nguyen, L.T.N.; Pham, N.T.T.; Nguyen, Y.T.H.; Le, S.V.; Chu, M.H. Overexpression of the GmDREB6 gene enhances proline accumulation and salt tolerance in genetically modified soybean plants. Sci. Rep. 2019, 9, 19663. [Google Scholar] [CrossRef] [Green Version]
  46. Ban, Q.; Liu, G.; Wang, Y. A DREB gene from Limonium bicolor mediates molecular and physiological responses to copper stress in transgenic tobacco. J. Plant Physiol. 2011, 168, 449–458. [Google Scholar] [CrossRef]
  47. Wang, W.; Gao, T.; Chen, J.; Yang, J.; Huang, H.; Yu, Y. The late embryogenesis abundant gene family in tea plant (Camellia sinensis): Genome-wide characterization and expression analysis in response to cold and dehydration stress. Plant Physiol. Biochem. 2019, 135, 277–286. [Google Scholar] [CrossRef]
  48. Shibuya, T.; Itai, R.; Maeda, M.; Kitashiba, H.; Isuzugawa, K.; Kato, K.; Kanayama, Y. Characterization of PcLEA14, a Group 5 Late Embryogenesis Abundant Protein Gene from Pear (Pyrus communis). Plants 2020, 9, 1138. [Google Scholar] [CrossRef]
  49. Mittler, R.; Vanderauwera, S.; Gollery, M.; Van Breusegem, F. Reactive oxygen gene network of plants. Trends Plant Sci. 2004, 9, 490–498. [Google Scholar] [CrossRef]
  50. Vessal, S.; Siddique, K.H.; Atkins, C.A. Comparative proteomic analysis of genotypic variation in germination and early seedling growth of chickpea under suboptimal soil-water conditions. J. Proteome Res. 2012, 11, 4289–4307. [Google Scholar] [CrossRef]
  51. Khurana, P.; Vishnudasan, D.; Chhibbar, A.K. Genetic approaches towards overcoming water deficit in plants-special emphasis on LEAs. Physiol. Mol. Biol. Plants 2008, 14, 277–298. [Google Scholar] [CrossRef] [Green Version]
  52. Lv, A.; Wen, W.; Fan, N.; Su, L.; Zhou, P.; An, Y. Dehydrin MsDHN1 improves aluminum tolerance of alfalfa (Medicago sativa L.) by affecting oxalate exudation from root tips. Plant J. 2021, 108, 441–458. [Google Scholar] [CrossRef]
  53. Du, B.; Zhao, W.; An, Y.; Li, Y.; Zhang, X.; Song, L.; Guo, C. Overexpression of an alfalfa glutathione S-transferase gene improved the saline-alkali tolerance of transgenic tobacco. Biol. Open 2019, 8, bio043505. [Google Scholar] [CrossRef] [Green Version]
  54. Rubio, S.; Noriega, X.; Pérez, F.J. Abscisic acid (ABA) and low temperatures synergistically increase the expression of CBF/DREB1 transcription factors and cold-hardiness in grapevine dormant buds. Ann. Bot. 2019, 123, 681–689. [Google Scholar] [CrossRef]
  55. Li, M.; Duan, X.; Gao, G.; Liu, T.; Qi, H. Running title: ABA pathway meets CBF pathway at CmADC. Hortic. Res. 2022, 9, uhac002. [Google Scholar] [CrossRef]
  56. Xie, Z.; Lin, W.; Yu, G.; Cheng, Q.; Xu, B.; Huang, B. Improved cold tolerance in switchgrass by a novel CCCH-type zinc finger transcription factor gene, PvC3H72, associated with ICE1-CBF-COR regulon and ABA-responsive genes. Biotechnol. Biofuels 2019, 12, 224. [Google Scholar] [CrossRef]
  57. Vyse, K.; Schaarschmidt, S.; Erban, A.; Kopka, J.; Zuther, E. Specific CBF transcription factors and cold-responsive genes fine-tune the early triggering response after acquisition of cold priming and memory. Physiol. Plant 2022, 174, e13740. [Google Scholar] [CrossRef]
  58. Morran, S.; Eini, O.; Pyvovarenko, T.; Parent, B.; Singh, R.; Ismagul, A.; Eliby, S.; Shirley, N.; Langridge, P.; Lopato, S. Improvement of stress tolerance of wheat and barley by modulation of expression of DREB/CBF factors. Plant Biotechnol. J. 2011, 9, 230–249. [Google Scholar] [CrossRef]
  59. Feng, H.L.; Ma, N.N.; Meng, X.; Zhang, S.; Wang, J.R.; Chai, S.; Meng, Q.W. A novel tomato MYC-type ICE1-like transcription factor, SlICE1a, confers cold, osmotic and salt tolerance in transgenic tobacco. Plant Physiol. Biochem. 2013, 73, 309–320. [Google Scholar] [CrossRef]
  60. Ali, N.; Hadi, F. CBF/DREB transcription factor genes play role in cadmium tolerance and phytoaccumulation in Ricinus communis under molybdenum treatments. Chemosphere 2018, 208, 425–432. [Google Scholar] [CrossRef]
  61. Cao, S.; Wang, Y.; Li, X.; Gao, F.; Feng, J.; Zhou, Y. Characterization of the AP2/ERF Transcription Factor Family and Expression Profiling of DREB Subfamily under Cold and Osmotic Stresses in Ammopiptanthus nanus. Plants 2020, 9, 455. [Google Scholar] [CrossRef] [Green Version]
  62. Hassan, S.; Berk, K.; Aronsson, H. Evolution and identification of DREB transcription factors in the wheat genome: Modeling, docking and simulation of DREB proteins associated with salt stress. J. Biomol. Struct. Dyn. 2021, 40, 7191–7204. [Google Scholar] [CrossRef]
  63. Wang, R.; Yin, J.; Li, S.; Zhang, W.; Zhang, J.; Dong, Z.; Li, X. Crop Cultivation Science, 2nd ed.; Higher Education Press: Beijing, China, 2015; p. 15. ISBN 978-7-04-041881-1. [Google Scholar]
  64. Nuccio, M.L.; Potter, L.; Stiegelmeyer, S.M.; Curley, J.; Cohn, J.; Wittich, P.E.; Tan, X.; Davis, J.; Ni, J.; Trullinger, J.; et al. Strategies and tools to improve crop productivity by targeting photosynthesis. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2017, 372, 1730. [Google Scholar] [CrossRef]
  65. Zhang, Y.; Kaiser, E.; Marcelis, L.F.M.; Yang, Q.; Li, T. Salt stress and fluctuating light have separate effects on photosynthetic acclimation, but interactively affect biomass. Plant Cell Environ. 2020, 43, 2192–2206. [Google Scholar] [CrossRef]
  66. Chen, X.; Ma, J.; Wang, X.; Lu, K.; Liu, Y.; Zhang, L.; Peng, J.; Chen, L.; Yang, M.; Li, Y.; et al. Functional modulation of an aquaporin to intensify photosynthesis and abrogate bacterial virulence in rice. Plant J. 2021, 108, 330–346. [Google Scholar] [CrossRef]
  67. Karpinska, B.; Razak, N.; Shaw, D.S.; Plumb, W.; Van De Slijke, E.; Stephens, J.; De Jaeger, G.; Murcha, M.W.; Foyer, C.H. Late Embryogenesis Abundant (LEA)5 Regulates Translation in Mitochondria and Chloroplasts to Enhance Growth and Stress Tolerance. Front. Plant Sci. 2022, 13, 875799. [Google Scholar] [CrossRef]
  68. Nowicka, B.; Ciura, J.; Szymańska, R.; Kruk, J. Improving photosynthesis, plant productivity and abiotic stress tolerance-current trends and future perspectives. J. Plant Physiol. 2018, 231, 415–433. [Google Scholar] [CrossRef]
  69. Juszczak, I.; Bartels, D. LEA gene expression, RNA stability and pigment accumulation in three closely related Linderniaceae species differing in desiccation tolerance. Plant Sci. 2017, 255, 59–71. [Google Scholar] [CrossRef]
  70. Snowdon, R.J.; Wittkop, B.; Chen, T.W.; Stahl, A. Crop adaptation to climate change as a consequence of long-term breeding. Theor. Appl. Genet. 2021, 134, 1613–1623. [Google Scholar] [CrossRef]
  71. García-Estrada, R.S.; Diaz-Lara, A.; Aguilar-Molina, V.H.; Tovar-Pedraza, J.M. Viruses of Economic Impact on Tomato Crops in Mexico: From Diagnosis to Management-A Review. Viruses 2022, 14, 1251. [Google Scholar] [CrossRef]
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Figure 1. Conserved domain and phylogenetic analysis of SiLEA5 protein. (A) Conserved domain analysis of SiLEA5 protein. The Arabidopsis LEA protein and SiLEA5 protein were annotated using the Pfam database about conserved structural domains. The visual structure map of conserved structural domains was constructed using TBtools. The different colored squares represent the conserved domains of the LEA protein. (B) SiLEA5 and Arabidopsis thaliana LEA protein rootless phylogenetic tree. The different colored areas represent the different subfamilies of LEA. (The phylogenetic tree was built using MEGA 7.0 by the neighbor-joining method with 1000 bootstrap replicates).
Figure 1. Conserved domain and phylogenetic analysis of SiLEA5 protein. (A) Conserved domain analysis of SiLEA5 protein. The Arabidopsis LEA protein and SiLEA5 protein were annotated using the Pfam database about conserved structural domains. The visual structure map of conserved structural domains was constructed using TBtools. The different colored squares represent the conserved domains of the LEA protein. (B) SiLEA5 and Arabidopsis thaliana LEA protein rootless phylogenetic tree. The different colored areas represent the different subfamilies of LEA. (The phylogenetic tree was built using MEGA 7.0 by the neighbor-joining method with 1000 bootstrap replicates).
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Figure 2. SiLEA5 gene endows transgenic tomato with low-temperature tolerance. (A) Wild-type and transgenic tomatoes were grown in a greenhouse at 25 °C for 4 weeks of age. (B) Growth state of tomato plants treated with 4 °C for 8 h, (C) 0 °C for 6 h, (D) −2 °C for 4 h. (E) MDA content. (F) Relative conductivity content. (G) Relative water content. Data are means ± SD of three replicates. Ns indicate no significant difference between the wild-type and transgenic lines. Asterisk(s) indicate significant difference between the wild-type and transgenic plants by Student’s t-tests: ** p < 0.01, **** p < 0.0001. Bar = 4 cm.
Figure 2. SiLEA5 gene endows transgenic tomato with low-temperature tolerance. (A) Wild-type and transgenic tomatoes were grown in a greenhouse at 25 °C for 4 weeks of age. (B) Growth state of tomato plants treated with 4 °C for 8 h, (C) 0 °C for 6 h, (D) −2 °C for 4 h. (E) MDA content. (F) Relative conductivity content. (G) Relative water content. Data are means ± SD of three replicates. Ns indicate no significant difference between the wild-type and transgenic lines. Asterisk(s) indicate significant difference between the wild-type and transgenic plants by Student’s t-tests: ** p < 0.01, **** p < 0.0001. Bar = 4 cm.
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Figure 3. Accumulation of osmotic regulators and change in antioxidant enzyme activity in transgenic tomato. (A) Soluble sugar content. (B) Soluble protein content. (C) Proline content. (D) CAT activity. (E) SOD activity. (F) POD activity. Data are means ± SD of three replicates. Ns indicate no significant difference between the wild-type and transgenic lines. Asterisk(s) indicate significant difference between the wild-type and transgenic plants by Student’s t-tests: *** p < 0.001, **** p < 0.0001.
Figure 3. Accumulation of osmotic regulators and change in antioxidant enzyme activity in transgenic tomato. (A) Soluble sugar content. (B) Soluble protein content. (C) Proline content. (D) CAT activity. (E) SOD activity. (F) POD activity. Data are means ± SD of three replicates. Ns indicate no significant difference between the wild-type and transgenic lines. Asterisk(s) indicate significant difference between the wild-type and transgenic plants by Student’s t-tests: *** p < 0.001, **** p < 0.0001.
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Figure 4. Photosynthetic parameters of wild-type and transgenic tomato leaves in the field. (A) Transpiration rate. (B) Stomatal conductance. (C) Net photosynthetic rate. (D) Intercellular CO2 concentration. (E) Water use efficiency. (F) Saturated vapor pressure. Data are means ± SD of three replicates. Each red dot represents a sample. Ns indicate no significant difference between the wild-type and transgenic lines. Asterisk(s) indicate significant difference between the wild-type and transgenic plants by Student’s t-tests: ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 4. Photosynthetic parameters of wild-type and transgenic tomato leaves in the field. (A) Transpiration rate. (B) Stomatal conductance. (C) Net photosynthetic rate. (D) Intercellular CO2 concentration. (E) Water use efficiency. (F) Saturated vapor pressure. Data are means ± SD of three replicates. Each red dot represents a sample. Ns indicate no significant difference between the wild-type and transgenic lines. Asterisk(s) indicate significant difference between the wild-type and transgenic plants by Student’s t-tests: ** p < 0.01, *** p < 0.001, **** p < 0.0001.
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Figure 5. Agronomic characters analysis of wild-type and transgenic tomato. (A) Overall growth status of tomato plants during vegetative growth period. Bar = 25 cm. (B) Overall growth status of tomato plants during fruit ripening. Bar = 25 cm. (C) Yield comparison between wild-type and transgenic tomatoes in 2020 and 2021. (D) Plant height. (E) Stem diameter. (F) The ratio of transverse diameter to the longitudinal diameter of fruits. (G) Number of fruits per plant. (H) Weight per fruit. Data are means ± SD of three replicates. Each red dot represents a sample. Ns indicate no significant difference between the wild-type and transgenic lines. Asterisk(s) indicate significant difference between the wild-type and transgenic plants by Student’s T-tests: ** p < 0.01 and **** p < 0.0001.
Figure 5. Agronomic characters analysis of wild-type and transgenic tomato. (A) Overall growth status of tomato plants during vegetative growth period. Bar = 25 cm. (B) Overall growth status of tomato plants during fruit ripening. Bar = 25 cm. (C) Yield comparison between wild-type and transgenic tomatoes in 2020 and 2021. (D) Plant height. (E) Stem diameter. (F) The ratio of transverse diameter to the longitudinal diameter of fruits. (G) Number of fruits per plant. (H) Weight per fruit. Data are means ± SD of three replicates. Each red dot represents a sample. Ns indicate no significant difference between the wild-type and transgenic lines. Asterisk(s) indicate significant difference between the wild-type and transgenic plants by Student’s T-tests: ** p < 0.01 and **** p < 0.0001.
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Liu, X.; Xia, W.; Zhang, X.; Li, A.; Qin, J.; Sun, H.; Li, J.; Zhu, J. Overexpression of the SiLEA5 Gene in Saussurea involucrata Increases the Low-Temperature Tolerance of Transgenic Tomatoes. Horticulturae 2022, 8, 1023. https://doi.org/10.3390/horticulturae8111023

AMA Style

Liu X, Xia W, Zhang X, Li A, Qin J, Sun H, Li J, Zhu J. Overexpression of the SiLEA5 Gene in Saussurea involucrata Increases the Low-Temperature Tolerance of Transgenic Tomatoes. Horticulturae. 2022; 8(11):1023. https://doi.org/10.3390/horticulturae8111023

Chicago/Turabian Style

Liu, Xiaoyan, Wenwen Xia, Xiaoli Zhang, Aowei Li, Jiawang Qin, Huili Sun, Jin Li, and Jianbo Zhu. 2022. "Overexpression of the SiLEA5 Gene in Saussurea involucrata Increases the Low-Temperature Tolerance of Transgenic Tomatoes" Horticulturae 8, no. 11: 1023. https://doi.org/10.3390/horticulturae8111023

APA Style

Liu, X., Xia, W., Zhang, X., Li, A., Qin, J., Sun, H., Li, J., & Zhu, J. (2022). Overexpression of the SiLEA5 Gene in Saussurea involucrata Increases the Low-Temperature Tolerance of Transgenic Tomatoes. Horticulturae, 8(11), 1023. https://doi.org/10.3390/horticulturae8111023

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