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Article

Heterologous Grafting Improves Cold Tolerance of Eggplant

by
Duanhua Wang
1,
Shuanghua Wu
1,
Qian Li
1,
Xin Wang
1,
Xuefeng Li
1,
Feng Liu
2 and
Jianguo Yang
1,*
1
Hunan Vegetable Research Institute, Hunan Academy of Agricultural Science, Changsha 410125, China
2
College of Horticulture, Hunan Agricultural University, Changsha 410128, China
*
Author to whom correspondence should be addressed.
Sustainability 2022, 14(18), 11170; https://doi.org/10.3390/su141811170
Submission received: 22 July 2022 / Revised: 30 August 2022 / Accepted: 1 September 2022 / Published: 6 September 2022
(This article belongs to the Special Issue Advance in Plant Breeding and Plant Biotechnology for Sustainable Agriculture)
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Abstract

:
Grafting is commonly used to enhance the quality and confer biotic or abiotic stress tolerance to plants. There is, however, no clear understanding of how grafted eggplant responds to cold stress. Here, four grafting combinations of eggplant from cold-sensitive line J55 and cold-resistant line J65 were subjected to morpho-physiological experiments and transcriptome sequencing to compare their responses to cold stress. After being subjected to cold stress, a total of 5883,6608,6867 and 5815 differentially expressed genes (DEGs) were identified in J55-J55L0_vs_J55-J55L1 (C0), J55-J65_vs_J55-J65L1 (T2), J65-J55_vs_J65-J55L1 (T1), and J65-J65_vs_J65-J65L1 (C1), respectively. When comparing C0 and C1, there exist 4580 specifical DEGs which were differentially expressed either in C0 or C1 (C0_vs_C1), these DEGs are more likely to induce the difference of the two grafted combinations. There is a total of 5583 specifical DEGs in C0_vs_T1 and 5209 specifical DEGs in C0_vs_T2, respectively. GO functional analysis found specifical DEGs mainly enriched the cell and membrane, catalytic activity, metabolic process, and cellular process, which was the same in comparison to heterografted and self-grafted eggplant in C0_vs_C1, C0_vs_T1 and C0_vs_T2. KEGG analysis showed that the specifical DEGs were mainly enriched in plant hormone signal transduction in C0_vs_C1, C0_vs_T1, and C0_vs_T2. Therefore, we screened ten candidate genes associated with AUX/IAA, salicylic acid and other hormone regulations that were differentially expressed in C0_vs_C1 C0_vs_T1, and C0_vs_T2. We believe that plant hormones play a vital role in regulating the cold tolerance of grafted eggplant. We also found that 22 DEGs enriched in arginine and proline metabolism in comparison to self-and hetero-grafted eggplant C0 and T1, predicted that putrescine biosynthesis plays a certain role in improving the cold resistance of eggplant by heterologous grafting. Meanwhile, by the comparison of specifical DEGs on C0_vs_C1 and C0_vs_T2, the DEGs enriched in a similar KEGG pathway, it is considered that the better cold tolerance of J65 as a scion has a more important effect on the cold resistance of eggplant.

1. Introduction

Eggplant (Solanum melongena L.), as one of the world’s top ten vegetables, is rich in anthocyanins, vitamins, dietary fibers, phytonutrients, phenolic compounds, and flavonoids [1] that are highly beneficial for human health [2]. As an economically important vegetable crop, eggplant is globally cultivated, especially in Asia and Africa [3], but is sensitive to low temperatures [4]. After being exposed to cold, many cellular responses are activated; however, these responses adversely limit the plant’s growth and development, affect the yield and cause major losses to agricultural production [5]. Thus, enhancing the low-temperature tolerance of eggplant is imperative. Although breeding the low temperature resistance cultivar is the most effective approach to resistance to low temperatures, this approach is time- and labor-consuming.
Grafting is a powerful agronomical technique to increase tolerance to abiotic and biotic stress in agricultural production [6] and has long been performed in agriculture [7]. It can improve crop yield [8] and quality [9]. Cold tolerance can be promoted on grafted eggplant [10] and cucumber heterografts [11,12]. Vegetable grafting onto resistant rootstocks is widely adopted for large-scale applications of tomatoes, watermelons, melons, eggplants, cucumbers, and peppers [13]. A cold-tolerant rootstock can provide resistance to abiotic stresses and influence scion growth and performance [13]. The low temperature tolerance and the survival rate for overwintering of cotton could be evaluated by grafting [14]. It has been reported that under low temperature conditions, heterografting improves low temperature tolerance by altering photosynthesis, antioxidant enzyme activity and plant phytohormone. By using grafts on Capsicum annuum seedlings, lipid peroxidation could be effectively reduced, and low temperatures and weak light intensity could be alleviated [15]. Cold-tolerant rootstocks can maintain photosynthesis for strong metabolism and sharp signal transduction to stabilize the cell system in cucumbers [6]. Compared to self-grafting cucumber/cucumber (CS/CS) plants, heterografting CS/CF (Cucurbita ficifolia Bouche) plants exhibit higher antioxidant enzyme activity and less membrane peroxidation under chilling stress, the electron flux in photosystem II can be reduced to under the level of energy dissipation rate [16]. In the grafting of pumpkin (Cm) and cucumber (Cs), the low-temperature treatment of scion/rootstock (Cs/Cm) increases the SA content in leaves and the transport from roots. This phenomenon results in a higher salicylic acid (SA) content of Cs/Cm than that of Cs/Cs [12]. In spite of this, there is still some ambiguity about the role of grafting in the promotion of abiotic tolerance in plants.
The cold resistance of eggplant can effectively improve its yield and income, but in actual production, it is often faced with the problem of poor resistance of good-quality materials and poor quality of materials with good resistance. Grafting is an effective way to overcome the drawbacks of materials. In particular, it plays an important role in the stress resistance of plants. Although much has been known about the application and selection of rootstocks in a variety of plants, the molecular mechanisms of rootstock adaptation in different soils and the modification of rootstocks to scion phenotypes are still to be studied [17]. To analyze the reasons grafting improves cold tolerance, and also provide guidance for production, we selected two eggplant varieties showing differences in cold tolerance. In this study, we constructed four grafting combinations of eggplant and analyzed their cold tolerance and analyzed their cold tolerance after low-temperature treatment. On the one hand, the cold-tolerance materials were used as the rootstock to analyze the reasons for the cold tolerance of grafted eggplant, and on the other hand, the effect of the scion on the cold resistance was compared with the reverse grafting as the control, so as to provide guidance for cultivating cold-resistant seedlings.

2. Material and Methods

2.1. Plant Materials and Cultivation

Prior to this study, two eggplant inbred lines J65 and J55 were screened for cold tolerance from 10 inbred lines. In particular, J65 has significantly better cold tolerance than J55 (Supplementary Figure S1 and Supplementary Table S1). Thus, J65 and J55 were used as test materials for scion or rootstock. J65 and J55 seeds were soaked in 55 °C warm water for 40 min and then transferred to 28 °C for 3 days. Germinated seeds were placed in 50-cell plug trays containing peat:vermiculite:perlite (2:1:1) and grown in a normal growth temperature of eggplant (relative humidity 70%, 16 h/8 h light/dark, temperature 28 °C/25 °C day/night, and light intensity 200 µmol m−2s−1). The plants were grafted when three true leaves had expanded, and the grafting combinations (rootstock–scion) were designated as J55-J55, J55-J65, J65-J55, and J65-J65 (Figure 1). All of the grafted eggplants were grown in the dark in an artificial climate chamber at 85–90% relative humidity and 28°C for 7 days.

2.2. Chilling Treatment

At 14 days post-grafting, each of the four grafting combinations with three replicates (20 grafted eggplant every repeat) were transferred to a growth room with chilling conditions (relative humidity 70%, light, temperature 4 °C, and light intensity 200 µmol m−2 s−1). In addition, three biological replicates of each of the four grafting combinations remained at 28 °C. The first expanded leaf from thirty different plants with the same growth, divided into three different repeats each grafted eggplant after being treated for 12 h, collected for RNAseq and quantitative real-time PCR (qRT-PCR) analyses.

2.3. Chilling Injury Index Statistical

After 12 h of treatment at 4 °C, the degree of chilling damage of each variety was evaluated according to the following grades, referring to the method from Zhu [18]with a slight change: In grade 5, the plants had withered and could not recover under normal conditions. In grade 4, the edges of the heart leaves were dry. In grade 3, more than half of the leaves were affected by dry spots. In grade 2, half of the leaves were affected by dry spots, but not affected the heart leaves. In grade 1 it has mild dry spots, the area is less than half of the leaf area and in grade 0, the plant grows normally without any signs of disease. Chilling injury index = Σ (different grade × grade)/[total plants × 5]. Three biological replicates were performed.

2.4. Measurement of the Maximum Photochemical Efficiency of PSⅡ

PSII (Fv/Fm) in darkness at the first expanded leaf was determined using a handheld chlorophyll fluorometer Fluor Pen FP110 (FluorCam, Drásov, Czech Republic) following the manufacturer’s instructions. The seedlings were allowed to adapt in the dark for at least 20 min before the measurement.

2.5. RNA-Seq and Transcriptome Analysis

About 1g leaf sample was used to extract the total RNA following the manufacturer’s instructions of RNAprep Pure kit (Tiangen, Beijing, China). In brief, 1% agarose gel was used to monitor degradation and contamination and then check the purity of the extracted RNA with NanoPhotometer® spectrophotometer (IMPLEN, Westlake Village, CA, USA). Qubit® RNA Assay Kit in Qubit® 2.0 Flurometer (Life Technologies, Carlsbad, CA, USA) and RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara County, CA, USA) were applied to measure and evaluate RNA concentration and integrity, respectively [19]. Furthermore, the library quality generated from NEB Next® Ultra™ RNA Library Prep Kit for Illumina® (NEB, Ipswich, MA, USA) was evaluated with an Agilent Bioanalyzer 2100. The prepared library was sequenced on the Illumina Novaeq6000 and 150 bp paired-end reads were generated.
After the low-quality reads were filtered and removed, the clean reads were obtained, and the clean reads were aligned to the Solanum_melongena genome using HISAT2 [20]. The Fragments per kilobase of transcript per millions (FPKM) of each gene were then calculated based on the gene length and the counted reads mapped to this gene. Gene expression levels were all estimated by FPKM. Differential expression analysis was performed using the DESeq R package (1.10.1) with an adjusted p-value <0.05. The enrichment analysis of the differentially expressed genes (DEGs) was implemented using GOseq R packages based on Wallenius noncentral hypergeometric distribution [21].

2.6. Quantitative Real-Time PCR (qRT-PCR) Analysis

Six DEGs were selected at random for qRT-PCR analysis to validate the accuracy of the RNA-seq results, and SmEF-Iα was used as a reference gene for analysis. Gebious 2×SYBR Green Fast qPCR Mix (ABclonal) was employed for quantitative real-time qRT-PCR. Specific qRT-PCR primers were designed for specific unigenes and SmEF-Iα, which were show in Supplementary Table S2. Real-time PCR was conducted with a two-step qRT-PCR procedure (Applied BIOER FDQ-96A, China) with the following cycling parameters: 40 cycles of 95 °C for 5 min, 95 °C for 10 s, and 60 °C for 30 s; single-point signal detection at 72 °C; melting curve consisting of 95 °C for 15 s, 60 °C for 60 s, and 95 °C for 15 s.

3. Results

3.1. Morphological and Physiological Responses to Cold Stress

Four eggplant grafting combinations were prepared to investigate cold tolerance in eggplant. After 4 °C treatment for 12 h, we observed the phenotypes of the four grafting combinations, and the degree of leaf wilting of J55-J55 was more serious than that of the other three grafting combinations (Figure 2A). Meanwhile, we investigated the chilling injury index of four combinations, and there exists an obvious difference. More than 1/2 of the J55–J55 leaves wilted; by contrast, the wilted area in J55-J65, J65-J55, and J65-J65 leaves were less than 1/2 (Figure 2A,B). The first expanded leaf of these four grafted eggplants was measured by a portable chlorophyll fluorescence meter before and after the 4 °C treatment 12 h. The Fv/Fm significantly decreased after 4 °C treated, but in J55-J55 the Fv/Fm was the lowest among the four combinations (Figure 2C). These phenotypes showed that the use of cold-tolerant J65 as a rootstock or scion can notably increase the cold tolerance of eggplant.

3.2. Transcriptome Sequencing, Assembly, and Mapping

Twenty-four libraries were constructed, and samples were collected from four grafted eggplant J65-J65, J65-J55, J55-J65, and J55-J55 leaves at 28 °C and 4 °C for 12 h treatment and designed as J65-J65L0, J65-J55L0, J55-J65L0, J55-J55L0, J65-J65L1, J65-J55L1, J55-J65L1, and J55-J55L1. A total of 167.16 Gb data were obtained, the clean data produced from each sample was no more than 5.84 Gb, with Q30 > 92%, and GC content > 42% (Supplementary Table S2). Reference sequences mapped to the annotated genome of S. melongena L. from/ftp/genomes/Solanum melongena_V4.1/ (https://solgenomics.net/ftp/genomes/Solanum_melongena_V4.1/ (accessed on 2 January 2022)). The dimensionality reduction analysis using PCA found that there exists a large difference before and after low-temperature treatment. After low-temperature treatment, the difference between the J65-J65, J55-J65 and J65-J55 grafted combinations was not obvious, but it was significantly different from that of J55-J55, which was different from the no obvious difference between the materials before treatment (Figure 3A) and many different expression genes were obtained (Figure 3B).

3.3. Validation of Transcriptome Data

Six genes were selected from differentially expressed genes for qRT-PCR analysis to identify the accuracy of transcriptome results, including SMEL4.1_06g019660.1, SMEL4.1_01g038450.1, SMEL4.1_00g010170.1, QZ_newGene_1794, SMEL4.1_03g005310.1 and SMEL4.1_01g014640.1. The findings showed that SMEL4.1_03g005310.1, SMEL4.1_00g010170.1, QZ_newGene_1794, and SMEL4.1_06g019660.1 were highly expressed in J65–J55L1, J55–J65L1 and J65–J65L1 but lowly expressed in the four grafted eggplants under normal condition and J55–J55L1. SMEL4.1_01g038450.1, SMEL4.1_01g014640.1 and SMEL4.1_01g038450.1 was highly expressed only on the scion of J65 (Figure 4). The result showed by qRT-PCR was basically consistent with the expression pattern of the transcriptome, which confirmed the reliability of transcriptome results.

3.4. Identification of DEGs between the Grafting Combinations

The genes were tested for differential expression to explore how grafting improves the cold tolerance of eggplant under low-temperature treatment. In total, 11218 genes were induced from all four rootstock–scion combinations under 4 °C treatment. Compared with J55-J55L0, 5883 genes were differentially expressed in J55-J55L1, designed as control 0 (C0) for simple. There exist 6867 genes differentially expressed in J65-J55L0_vs_J65-J55L1, as test group 1 (T1), 6608 genes differentially expressed in J55-J65L0_vs_J55-J65L1, as test group 2 (T2), and 5815 genes differentially expressed in J65-J65L0_vs_J65-J65L1, as control 1 (C1). Then, we further analyzed these DEGs among different groups. Two comparisons were carried out: the first was to analyze the DEGs stimulated by cold stress in cold-tolerant (J65-J65) and cold-sensitive (J55-J55) self-grafted eggplant combinations, there exist 4580 genes specifical DEGs, which were differentially expressed either in C0 or C1 (C0_vs_C1), that we think these specifical DEGs are more likely to induce the difference of the two grafted combination. The second was to analyze the DEGs induced by heterografting including two modules: there were 5883 genes specifical DEGs in C0_vs_T1 with different rootstock and 5209 specifical DEGs in C0_vs_T2 with different scions (Figure 5).

3.5. GO Enrichment Analysis of the Identified DEGs

In order to further analyze the function of these DEGs that may lead to differential changes in cold tolerance of grafted eggplant. GO functional annotation of DEGs was performed for the investigation of biological functions induced in the cold-sensitive and cold-tolerant grafting combinations under cold stress. All of these DEGs from C0, C1, T1 and T2 were categorized into 46 functional groups using GO classifications. The DEGs were mainly enriched in the cell, membrane, cell part, organelle, organelle part, and membrane part in the cellular component category. In molecular function, the DEGs were mainly enriched in catalytic activity and binding, and metabolic process, cellular process, response to stimulus, biological regulation in biological process (Supplementary Figure S2). These results indicate that cold treatment mainly affects cell and membrane parts, catalytic activity, metabolic process, and cellular process. We further analyzed the specifical DEGs in C0_vs_C1, C0_vs_T1, and C0_vs_T2. All the DEGs were also mainly enriched in cell, membrane, cell part, organelle, organelle part, membrane part in the cellular component category, catalytic activity and binding in molecular function, and metabolic process, cellular process, response to stimulus, biological regulation in biological process (Figure 6). These results showed that the DEGs between cold-sensitive and cold-tolerance involved in cold stress are also mainly enriched in cell and membrane part, catalytic activity, metabolic process, and cellular process, which were the same between heterografted eggplant and self-grafted eggplant.

3.6. KEGG Pathway Enrichment Analysis of Different Expression Genes

To further understand the biological function of DEGs, KEGG enrichment pathway analysis was conducted. The result showed that the DEGs in C0 (J55-J55L0_vs_J55-J55L1) were enriched in zeatin biosynthesis, photosynthesis, photosynthesis-antenna proteins, and MAPK signaling pathway. In C1 (J65-J65L0_vs_J65-J65L1), the DEGs were mainly enriched in photosynthesis, zeatin biosynthesis, plant hormone signal transduction, flavonoid biosynthesis, vitamin B6 metabolism, sesquiterpenoid and triterpenoid biosynthesis. In T1 (J65-J55L0_vs_J65-J55L1), the DEGs were mainly enriched in photosynthesis, plant hormone signal transduction, glyoxylate and dicarboxylate metabolism and so on. In T2 (J55-J65L0_vs_J55-J65L1), the DEGs were mainly enriched in photosynthesis, photosynthesis-antenna proteins, zeatin biosynthesis, vitamin B6 metabolism, plant hormone signal transduction (Supplementary Figure S3). We further analyzed the specifical DEGs of C0_vs_C1. The DEGs were mainly enriched in plant hormone signal transduction, MAPK signaling pathway, plant–pathogen interaction, and phenylpropanoid biosynthesis. We predicted that these pathways may play an important role in regulating cold tolerance (Figure 7A). When we compared C0 and T1, specifical DEGs were mainly enriched in photosynthesis, plant hormone signal transduction, plant-pathogen interaction, and oxidative phosphorylation (Figure 7B). Plant hormone signal transduction was a pathway existing in C0_vs_C1 and C0_vs_T1, which we cannot ignore in grafted eggplant to regulate cold tolerance. At the same time, 22 DEGs enriched in arginine and proline metabolism in comparison of self-and hetero-grated eggplant C0 and T1, which was different from C0 and C1 and associated with cold stress. The specifical DEGs of C0_vs_T2 were also analyzed, the DEGs were mainly enriched in plant hormone signal transduction, MAPK signaling pathway, plant–pathogen interaction, similar to C0_vs_C1 (Figure 7A,C).

3.7. Analysis of DEGs in Plant Hormone Signal Transduction, Arginine and Proline Metabolism

By analyzing the DEGs using the KEGG pathway, we predicted that the plant hormone signal transduction pathway is a vital method of regulating cold tolerance in eggplant. With cold tolerance material as rootstock, this pathway can be promoted to improve the cold tolerance of grafted eggplant. Meanwhile, arginine and proline metabolism can be activated to improve the cold tolerance in grafted eggplant. Therefore, these two pathways were chosen for further analysis.
In the plant hormone signal transduction pathway, ten significantly expressed candidate genes were screened and participated in the AUX/IAA, salicylic acid and other hormones regulation. SMEL4.1_06g008770.1 associated with abscisic acid were significantly downregulated in C0 but had no significant difference in C1 and T2, even upregulated in T1. Another gene associated with abscisic acid (SMEL4.1_09g020680.1) was upregulated by less than 1.0 times in C0, but more than 2.0 times in T1 and T2, and even nearly 4.0 times in C1. Auxin-responsive protein SAUR50 (SMEL4.1_04g022250.1 and SMEL4.1_01g004060.1), auxin-induced protein 15A (SMEL4.1_01g003840.1) and ethylene-responsive transcription factor (SMEL4.1_08g001940.1) expressed no significant difference in C0 but upregulated by more than 2.0 times in T1 and T2, even nearly seven times in C1. Probable xyloglucan endotransglucosylase/hydrolase protein 23 (SMEL4.1_05g025030.1) were downregulated less than by 1.0 times in C0, but 5–9 times in C1, T1 and T2. Two auxin-responsive proteins (SMEL4.1_01g004080.1 and SMEL4.1_03g020590.1) and transcription factor TGA9 (SMEL4.1_12g015670.1) were upregulated in C0 but downregulated by more than 2.0 times in C1, T1 and T2 (Figure 8A).
Analyzed the arginine and proline metabolism, arginine decarboxylase (SMEL4.1_01g004190.1), polyamine oxidase (SMEL4.1_02g020150.1) were upregulated in C0, C1, T1 and T2, but the times of upregulated in C1, T1 and T2 were much higher than in C0. Ornithine decarboxylase (SMEL4.1_04g023290.1 and SMEL4.1_00g007560.1) were downregulated in C0 but significantly upregulated in C1, T1 and T2. Meanwhile, amidase (SMEL4.1_03g008310.1) expressed no significant difference in C0, but significantly downregulated in C1, T1 and T2. All these genes regulated in C1, T1 and T2 were beneficial for putrescine biosynthesis (Figure 8B).

3.8. Identification for Transcription Factors

To analyze the differential expression of transcription factors (TFs) induced by cold and grafting, the transcription factors different expressed among C0_vs_C1, C0_vs_T1 and C0_vs_T2 were screened. There were 355, 512 and 426 specifical differentially expressed transcription factors in C0_vs_C1, C0_vs_T1 and C0_vs_T2, respectively. Most transcription factors are concentrated in the MYB, NAC, bHLH and WRKY transcription factor family (Supplementary Date S1 and Supplementary Figure S4).
We further analyzed the common differentially expressed transcription factors in C0_vs_C1 and C0_vs_T1, there were129 transcription factors in C0_vs_C1 and C0_vs_T1, including MYB (17), C2H2 (10), WRKY (10), AP2/ERF (9), Bhlh (6), NAC (6) and others (Figure 9A). There exist 261 common differential transcription factors in C0_vs_C1 and C0_vs_T2, including MYB (32), bHLH (20), NAC (17), C2H2 (17), AP2/ERF (16), WRKY (14), and others (Figure 9B). These transcription factor families all play an important role in the regulation of cold tolerance. Six TFs were identified significantly differential expression in C0_vs_C1, C0_vs_T1 and C0_vs_T2, including AP2/ERF (SMEL4.1_00g004870.1, SMEL4.1_03g032200.1 and SMEL4.1_05g013060.1), MYB (SMEL4.1_11g009520.1), bHLH (SMEL4.1_01g012480.1) and WRKY (SMEL4.1_02g011200.1). SMEL4.1_00g004870.1 and SMEL4.1_05g013060.1 (AP2/ERF) significantly upregulated in C0, C1, T1 and T2, SMEL4.1_01g012480.1 (bHLH) and SMEL4.1_02g011200.1 (WRKY) were significantly downregulated in C0, C1, T1 and T2. However, the changes in C1, T1 and T2 are larger than in C0. SMEL4.1_11g009520.1 (MYB) upregulated by 12 times in C0 and downregulated in C1, T1 and T2 (Figure 9C). Indicating that these six transcriptional factor coding genes might be the main regulatory genes for improving the cold tolerance of grafted eggplant.

4. Discussion

4.1. Grafting Can Increase the Eggplant’s Cold Tolerance

Grafting is an important method to improve the cold resistance of plants. In this experiment, we found that when J65 was used as a rootstock material, the chilling injury index and the cold damage decreased significantly after being treated at 4 °C, indicating that the cold-resistant rootstock material improved the cold resistance of eggplant. On the other hand, when J65 was used as the scion and J55 was used as the rootstock, its cold resistance is also stronger than that of J55 self-grafting, indicating that the cold resistance of the scion itself has a greater impact on the cold resistance of the whole plant. High cold resistance material, the cold resistance of the scion cannot be ignored.

4.2. Plant Hormone Signal Transduction Is an Important Pathway Involved in Promoting the Eggplant Cold Tolerance

Many studies have reported that plant hormones play a vital role in regulating plant stress including AUX/IAA, salicylic acid, and ethylene [22]. Most early auxin response genes fall into three major families: AUX/IAA, GH3s and SAUR. AUX/IAA family members regulate genes in multiple ways by forming complexes with ARF or TIR and play different roles in plant growth and development [23]. In Arabidopsis, the Aux/IAAA 14 mutant exhibits cold sensitivity [24]. A large number of AUX/IAA genes were identified in chickpeas and soybeans and found to show significantly different expression patterns in desiccation, salinity and cold [24,25], showing crosstalk between auxin and abiotic stress signals [26].
In this study, we compared J55-J55 and J65-J65 and analyzed that the cold tolerance of J65 may be due to the fact that photosynthesis, photosynthesis-antenna proteins, plant hormone signal transduction and other pathways played an important role. After the comparative analysis of J55-J55 and J65-J55, it was found that a large number of differential genes were mainly concentrated in plant hormone signal transduction, indicating that cold-tolerant materials as rootstocks may change some of the hormone responses of grafted materials and improve the cold tolerance of plants. The AUX/IAA gene was identified in this study and showed significant up-regulation, indicating that it is a positive regulatory gene that affects the cold tolerance of eggplant grafting. In this study, a large number of SAUR-related genes were identified at the same time, showing different expression patterns, and it is predicted that there is also crosstalk between them and abiotic stress signals. Xyloglucan endotransglucosylase including xyloglucan endotransglucosylase (XET) and xyloglucan endohydrolase (XEH), is mainly involved in the catalytic hydrolysis of xyloglucan in the cell wall and plays a crucial role in relaxing or stiffening the cell wall [27], affecting cell wall rearrangement, altering cell wall homeostasis [28]. Many previous studies have shown that xyloglucan endotransglucosylase plays a positive regulatory role in plant cold resistance, drought tolerance and salt tolerance. An XTH19 was identified in Arabidopsis to improve freezing tolerance [29], A CaXTH3 was identified in pepper to positively regulate its tolerance to water deficit and high salinity [30]. However, negatively regulated genes XTH30 were also identified in Arabidopsis thaliana with increased salt tolerance after its loss of function [27]. In this study, the down-regulation of xyloglucan endotransglucosylase was significantly different in cold-tolerant grafted materials than in less cold-tolerant materials and showed that it may be a cold-tolerant-related negative regulatory gene.

4.3. Putrescine Biosynthesis Improve the Cold Tolerance of Grafted Eggplants

The polyamine putrescine is a ubiquitous polycationic aliphatic compound formed through the decarboxylation of arginine by arginine decarboxylase or under the direct catalysis of ornithine decarboxylase and then converted to generate spermidine and spermine [31,32]. Involved in several physiological processes, such as response to chilling, salt, and other abiotic stresses [31,32]. Putrescine can regulate the biosynthesis of ABA and act as a signal triggering the antioxidant system to increase the cold tolerance of melon [32] or improve the freezing tolerance and cold acclimation of Arabidopsis [32,33]. It is reported that putrescine can be initiated through increasing CBF genes expression level to improve the cold acclimation freezing tolerance of potato plants [33] and inhibit the superoxide anion production rate and increase the activities of superoxide dismutase (SOD) and catalase [34]. Grafting improves plants’ cold tolerance mainly by increasing their antioxidant metabolism. In common clementines (scion), tetraploid Carrizo citrange (rootstock) affects the antioxidant system which improves chilling tolerance [35]. A study on the changes in antioxidative enzyme activities on own-rooted and grafted Capsicum annuum subjected to low temperature for 1–7 days found that during the early stress days, the activities of SOD and peroxidase (POD) increased in the scion and roots of own-rooted and grafted seedlings [16]. however, the activities of SOD, POD and root were higher during chilling compared with the own-rooted seedlings. CS/CF plants showed higher antioxidant enzyme activity than CS/CS plants after chilling [17]. Grafting with pumpkin rootstock increases the regulation of antioxidant metabolism to improve chilling tolerance in watermelon [36].
In the present work, we found that when J65 as rootstock grafted with J55, the genes on its arginine and proline metabolism had undergone great changes, and the up-regulation of these genes was beneficial to the synthesis of putrescine. As an important antioxidant, putrescine may play a role in improving the cold resistance of eggplant by grafting.

4.4. MYB, AP2/ERF Transcription Factor Family Involved in the Cold Tolerance Improving of Grafted Eggplant Combination

As important regulatory genes, transcription factors are particularly important for plants. They participate in the transcription of a range of cold-responsive genes to regulate the cold tolerance of crops. A large number of reports have shown that MYB, AP2/ERF, WRKY, bHLH, bZIP, NAC, and C2H2 family all are involved in the regulation of cold stress tolerance [37,38,39,40,41]. We analyzed the transcription factors expression level between cold sensitive and cold tolerant grafted eggplant combination after cold stress induced by material, and the different transcription factors mainly focused on MYB, AP2/ERF, bHLH and WRKY family. Predicted that the cold-tolerant eggplant can increase the expression of transcription factor families including MYB, AP2/ERF, bHLH and WRKY to be included in the cold tolerance of grafted eggplant combination.

5. Conclusions

Heterografting can increase the cold tolerance of eggplant, but the cold resistance is also related to the material (scion or rootstock). A cold-resistant material is applied to improve the resistance of the grafted material by changing the plant hormone level. On the other hand, heterologous grafting can also improve putrescine biosynthesis and improve the antioxidant capacity of eggplant, thereby enhancing the cold tolerance of eggplant. At the same time, through the analysis of hetero-grafting, it was found that the cold resistance of the scion itself has a great influence on the cold resistance of eggplant. Further functional analyses could be conducted on the DEGs identified in this study and further studies of the molecular mechanisms underlying grafted eggplant’s enhanced cold tolerance may be conducted using these findings.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/su141811170/s1, Table S1, Evaluation by membership function; Table S2, The primer of genes; Table S3, The alignment of transcript date; Figure S1, Plant morphology of 10 eggplant varieties grown in normal temperature for 7 days after treated in 5 °C~0 °C 3 days. Two rows for each varieties; Figure S2, GO functional analysis of the DEGs from four comparison including J55-J55_vs_ J55-J55L1 (A), J65-J65L0_vs_J65-J65L1 (B), J65-J55L0_vs_J65-J55L1 (C), and J65-J65L0_vs_J65-J65L1(D); Figure S3, The KEGG enrichment analysis of DEGs. A, the DEGs enriched in KEGG in J55-J55L0_vs_J55-J55L1 (A) ,J65-J65L0_vs_J65-J65L1 (B), J65-J55L0_vs_J65-J55L1 (C) and J55-J65L0_vs_J55-J65L1 (D). The size of the circle indicates the quantity of genes, and the different colors mean the difference in q value. The redder the circle, the smaller the q value. The Y-axis represents the terms of KEGG pathway cluster, and the X-axis represents the enrichment factor. Figure S4, The analyze of specifical different expressed transcription factors. (A) the specifical differentially ex pressed transcription factors in C0_vs_C1; (B) the specifical differentially expressed transcription factors in C0_vs_T1; (C) the specifical differentially expressed transcription factors in C0_vs_T2. Date S1, The differentical TFs in C0_vs_T1, The differentical TFs in C0_vs_C1, The differentical TFs in C0_vs_T1.

Author Contributions

Conceptualization, D.W. and J.Y.; methodology, S.W.; software, S.W.; validation, D.W., S.W., and Q.L.; formal analysis, X.L.; investigation, X.W.; resources, D.W. and J.Y.; data curation, S.W.; writing—original draft preparation, S.W.; writing—review and editing, D.W. and S.W.; visualization, J.Y.; supervision, F.L.; project administration, F.L.; funding acquisition, X.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by [The National Key Research and Development Program of China] grant number [Grant No. 2019YFD1000300].

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. The experimental design of graft eggplant combination, four grafted combinations were designed, including rootstock (J55)-scion (J55), rootstock (J55)-scion (J65), rootstock (J65)-scion (J55) and rootstock (J65)-scion (J65).
Figure 1. The experimental design of graft eggplant combination, four grafted combinations were designed, including rootstock (J55)-scion (J55), rootstock (J55)-scion (J65), rootstock (J65)-scion (J55) and rootstock (J65)-scion (J65).
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Figure 2. Phenotype of four combinations of grafted eggplant after cold stress: (A) phenotype of plant after 4 °C treatment 12 h, the wilting degree from J55-J55 is more seriously; (B) the chilling injury index of four grafted combinations; (C) the value of Fv/Fm from the four grafted combinations under 28 °C and 4 °C, the white bars represent the value of Fv/Fm under 28 °C and the black bars represent the value of Fv/Fm under 4 °C, a and b represent the significance.
Figure 2. Phenotype of four combinations of grafted eggplant after cold stress: (A) phenotype of plant after 4 °C treatment 12 h, the wilting degree from J55-J55 is more seriously; (B) the chilling injury index of four grafted combinations; (C) the value of Fv/Fm from the four grafted combinations under 28 °C and 4 °C, the white bars represent the value of Fv/Fm under 28 °C and the black bars represent the value of Fv/Fm under 4 °C, a and b represent the significance.
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Figure 3. Transcriptome differential analysis of grafted combinations. (A) The PCA analysis of transcriptome data, groups can be divided into three categories: J55-J55L0, J55-J65L0, J65-J55L0 and J65-J65L0 groups before cold stress get together, J55-J55L1 together alone; and J55-J65L1, J65-J55L1, J65-J65L1 groups get together. (B) the heatmap of differentially expressed genes in four grafted eggplants before and after cold stress.
Figure 3. Transcriptome differential analysis of grafted combinations. (A) The PCA analysis of transcriptome data, groups can be divided into three categories: J55-J55L0, J55-J65L0, J65-J55L0 and J65-J65L0 groups before cold stress get together, J55-J55L1 together alone; and J55-J65L1, J65-J55L1, J65-J65L1 groups get together. (B) the heatmap of differentially expressed genes in four grafted eggplants before and after cold stress.
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Figure 4. Validation of transcriptome data by qRTPCR analysis. A total of six genes were validated including SMEL4.1_06g019660.1 (A); SMEL4.1_01g038450.1 (B); SMEL4.1_00g010170.1 (C); QZ_newGene_1794 (D); SMEL4.1_03g005310.1 (E); and SMEL4.1_01g014640.1 (F). The black bars represent the relative expression of genes in four grafted eggplant leaves before and after cold stress by qRT-PCR analysis, and the red line represents the value of FPKM from RNA−Seq.
Figure 4. Validation of transcriptome data by qRTPCR analysis. A total of six genes were validated including SMEL4.1_06g019660.1 (A); SMEL4.1_01g038450.1 (B); SMEL4.1_00g010170.1 (C); QZ_newGene_1794 (D); SMEL4.1_03g005310.1 (E); and SMEL4.1_01g014640.1 (F). The black bars represent the relative expression of genes in four grafted eggplant leaves before and after cold stress by qRT-PCR analysis, and the red line represents the value of FPKM from RNA−Seq.
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Figure 5. The number of the differentially expressed genes. (A) Venn diagram of the differentially expressed genes (logfold change > 1) on four grafted eggplant combinations under cold stress; (B) Number of the differentially expressed genes in each comparison group, J55-J55L0_vs_ J55-J55L1 as C0, J65-J55L0_vs_J65-J55L1 as T1, J65-J65L0_vs_J65-J65L1 as C1, and J55-J65L0_vs_J55-J65L1 as T2, C0_vs_C1-S represent the special DEGs in C0_vs_C1, C0_vs_T1-S represent the special DEGs in C0_vs_T1, C0_vs_T2-S represent the special DEGs in C0_vs_T2.
Figure 5. The number of the differentially expressed genes. (A) Venn diagram of the differentially expressed genes (logfold change > 1) on four grafted eggplant combinations under cold stress; (B) Number of the differentially expressed genes in each comparison group, J55-J55L0_vs_ J55-J55L1 as C0, J65-J55L0_vs_J65-J55L1 as T1, J65-J65L0_vs_J65-J65L1 as C1, and J55-J65L0_vs_J55-J65L1 as T2, C0_vs_C1-S represent the special DEGs in C0_vs_C1, C0_vs_T1-S represent the special DEGs in C0_vs_T1, C0_vs_T2-S represent the special DEGs in C0_vs_T2.
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Figure 6. Gene ontology (GO) statistics and enrichment analysis of identified specifical differentially expressed genes (DEGs) of self-grafted cold sensitive and cold tolerance eggplant induced by cold stress, C0_vs_C1 (A); self-grafted and heterografted eggplant induced by cold stress with different rootstock, C0_vs_T1 (B); and with different scions, C0_vs_T2 (C). Different colors represent different GO terms. The numbers on the bar graph indicate the number of differential genes.
Figure 6. Gene ontology (GO) statistics and enrichment analysis of identified specifical differentially expressed genes (DEGs) of self-grafted cold sensitive and cold tolerance eggplant induced by cold stress, C0_vs_C1 (A); self-grafted and heterografted eggplant induced by cold stress with different rootstock, C0_vs_T1 (B); and with different scions, C0_vs_T2 (C). Different colors represent different GO terms. The numbers on the bar graph indicate the number of differential genes.
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Figure 7. The KEGG enrichment analysis of DEGs. Top 20 enriched KEGG pathways of identified specifical DEGs of C0 _vs_C1 (A), C0_vs_T1 (B), and C0_vs_T2 (C). The size of the circle indicates the quantity of genes, and the different colors mean the difference in q value. The redder the circle, the smaller the q value. The Y-axis represents the terms of KEGG pathway cluster, and the X-axis represents the enrichment factor.
Figure 7. The KEGG enrichment analysis of DEGs. Top 20 enriched KEGG pathways of identified specifical DEGs of C0 _vs_C1 (A), C0_vs_T1 (B), and C0_vs_T2 (C). The size of the circle indicates the quantity of genes, and the different colors mean the difference in q value. The redder the circle, the smaller the q value. The Y-axis represents the terms of KEGG pathway cluster, and the X-axis represents the enrichment factor.
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Figure 8. DEGs changes in the expression of plant hormone transduction (A) and arginine and proline metabolism (B). From left to right represent J55-J55L0, J55-J55L1, J55-J65L0, J55-J65L1, J65-J55L0, J65-J55L1, J65-J65L0 and J65-J65L1. From blue to red represent the value of the relative expression is higher.
Figure 8. DEGs changes in the expression of plant hormone transduction (A) and arginine and proline metabolism (B). From left to right represent J55-J55L0, J55-J55L1, J55-J65L0, J55-J65L1, J65-J55L0, J65-J55L1, J65-J65L0 and J65-J65L1. From blue to red represent the value of the relative expression is higher.
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Figure 9. The analyze of common different expressed transcription factors: (A) the common differential expression transcription factors in C0_vs_C1 and C0_vs_T1; (B) the common differential expression transcription factors in C0_vs_C1 and C0_vs_T2, the numbers mean the numbers of TF genes; (C) the expression of six significantly differentially expressed transcription factors in J55-J55L0, J55-J55L1. J55-J65L0, J55-J65L1, J65-J55L0, J65-J55L1, J65-J65L0 and J65-J65L1, different colors mean different relative expressions, from blue to red represent the value of relative expression from low to high.
Figure 9. The analyze of common different expressed transcription factors: (A) the common differential expression transcription factors in C0_vs_C1 and C0_vs_T1; (B) the common differential expression transcription factors in C0_vs_C1 and C0_vs_T2, the numbers mean the numbers of TF genes; (C) the expression of six significantly differentially expressed transcription factors in J55-J55L0, J55-J55L1. J55-J65L0, J55-J65L1, J65-J55L0, J65-J55L1, J65-J65L0 and J65-J65L1, different colors mean different relative expressions, from blue to red represent the value of relative expression from low to high.
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Wang, D.; Wu, S.; Li, Q.; Wang, X.; Li, X.; Liu, F.; Yang, J. Heterologous Grafting Improves Cold Tolerance of Eggplant. Sustainability 2022, 14, 11170. https://doi.org/10.3390/su141811170

AMA Style

Wang D, Wu S, Li Q, Wang X, Li X, Liu F, Yang J. Heterologous Grafting Improves Cold Tolerance of Eggplant. Sustainability. 2022; 14(18):11170. https://doi.org/10.3390/su141811170

Chicago/Turabian Style

Wang, Duanhua, Shuanghua Wu, Qian Li, Xin Wang, Xuefeng Li, Feng Liu, and Jianguo Yang. 2022. "Heterologous Grafting Improves Cold Tolerance of Eggplant" Sustainability 14, no. 18: 11170. https://doi.org/10.3390/su141811170

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