Transcriptome Analysis Identifies Novel Genes Associated with Low-Temperature Seed Germination in Sweet Corn

Typically, sweet corn, particularly sh2 sweet corn, has low seed vigor owing to its high sugar and low starch content, which is a major problem in sweet corn production, particularly at low temperatures. There is considerable variation in the germination rates among sweet corn varieties under low-temperature conditions, and the underlying mechanisms behind this phenomenon remain unclear. In this study, we screened two inbred sweet corn lines (tolerant line L282 and sensitive line L693) differing in their low-temperature germination rates; while no difference was observed in their germination rates at normal temperatures. To identify the specifically induced genes influencing the germination capacity of sweet corn at low temperatures, a transcriptome analysis of the two lines was conducted at both normal and low temperatures. Compared to the lines at a normal temperature, 3926 and 1404 differently expressed genes (DEGs) were identified from L282 and L693, respectively, under low-temperature conditions. Of them, 830 DEGs were common DEGs (cDEGs) that were identified from both L282 and L693, which were majorly enriched in terms of microtubule-based processes, histone H3-K9 modification, single-organism cellular processes, and carbohydrate metabolic processes. In addition, 3096 special DEGs (sDEGs), with 2199 upregulated and 897 downregulated, were detected in the tolerant line L282, but not in the sensitive line L693. These sDEGs were primarily related to plasma membranes and oxygen-containing compounds. Furthermore, electric conductivity measurements demonstrated that the membrane of L282 experienced less damage, which is consistent with its strong tolerance at low temperatures. These results expand our understanding of the complex mechanisms involved in the cold germination of sweet corn and provide a set of candidate genes for further genetic analysis.


Introduction
Sweet corn is one of the most important vegetables grown worldwide [1][2][3] and a good source of certain minerals, vitamins, phytonutrients, and dietary fiber, which makes sweet corn an ideal crop for maintaining good health [4]. Recently, it was reported that China produces approximately 530,000 ha of sweet corn, thereby surpassing the United States as the top sweet corn producer.
Sweet corn is derived from mutations in genes involved in the starch biosynthesis pathway. Several genes that modify the carbohydrate composition of its endosperm have been revealed to show increased sugar content when mutated [5]. The shrunken2 (sh2) gene encodes the large subunit of ADP-glucose pyrophosphorylase (AGPase), which is involved in the first rate-limiting step of the starch biosynthesis pathway [6]. The sh2 mutants exhibit remarkable reductions in their total number of carbohydrates, resulting in a large increase

Inbred Line L282 Showed Better Germination Resistance to Low Temperature Than L693
To test the low-temperature germination capacity of sh2 sweet corn, two inbred lines, L282 and L693, were evaluated in terms of their germination rate (GR) in paper rolls (Figure 1a). At a normal temperature, both lines had similar GRs of about 80% (Figure 1b). At low temperatures, L282 showed a GR similar to that at normal temperature ( Figure 1b); however, the GR of L693 reduced to 53% from a level of 80% for normal-temperature germination (Figure 1b). In addition, the root length (RL), shoot length (SL), and shoot weight (SW) of L282 were similar at normal and low temperatures (Figure 1c-e). Whereas L693 s RL, SW, and SL all reduced at low temperatures compared to normal temperature (Figure 1c-e). Root weight (RW) was reduced in both lines, but it decreased more significantly in L693 than in L282 (Figure 1f). These results indicate that L282 may have higher germination capacity at low temperature than L693.

Transcriptome Analysis of L282 and L693 Seeds Germinated at Normal and Low Temperatures
To investigate the differences in the genes involved in germination between the two sh2 sweet corn varieties L282 and L693, we performed a transcriptome analysis at normal and low temperatures. Eight RNA samples were prepared with two biological replicates from both lines that germinated under normal (L282 and L693) and cold (L282C and L693C) conditions. Our constructed principal component analysis (PCA) plots evidently separated (b-f) comparisons of germination rate (b), shoot length (c), root length (d), shoot weight (e), and root weight (f) of the two lines germinated at normal temperature (NT) and low temperature (LT). The asterisks indicate significant difference at p < 0.05 (n = 3). The "ns" indicates no significant difference.

Transcriptome Analysis of L282 and L693 Seeds Germinated at Normal and Low Temperatures
To investigate the differences in the genes involved in germination between the two sh2 sweet corn varieties L282 and L693, we performed a transcriptome analysis at normal and low temperatures. Eight RNA samples were prepared with two biological replicates from both lines that germinated under normal (L282 and L693) and cold (L282C and L693C) conditions. Our constructed principal component analysis (PCA) plots evidently separated the replicates of different lines and treatments, suggesting the reliability of our RNA-seq data ( Figure 2a). Transcriptome sequencing revealed a total of 208,962,737 clean reads, with the reads for each sample ranging from 21,280,625 to 37,564,993 (Table 1). (b-f) comparisons of germination rate (b), shoot length (c), root length (d), shoot weight (e), and root weight (f) of the two lines germinated at normal temperature (NT) and low temperature (LT). The asterisks indicate significant difference at p < 0.05 (n = 3). The "ns" indicates no significant difference. After aligning the reads to the maize reference genome (Zm-B73-REFERENCE-G RAMENE-4.0), 88.19-90.11% (mean 89.11%) of the reads were mapped, with 73.93-80.55% (mean 78.19%) uniquely mapped ( Table 1). The genes with fragments per kilobase of exon  Hierarchical cluster analysis was performed to compare the gene expression patterns among the different samples, which showed great changes between the different lines and treatments (Figure 2c). Using the criteria of fold change ≥ 2 and false discovery rate (FDR) ≤ 0.05, the DEGs between L282C and L282 and those between L693C and L693 were defined. Compared to normal-temperature germination, low-temperature germination generated 3926 DEGs (2651 upregulated and 1275 downregulated) for L282 and 1404 DEGs (667 upregulated and 737 downregulated) for L693 ( Figure 3a). These DEGs were considered cold-responsive genes. Hierarchical cluster analysis was performed to compare the gene expression patte among the different samples, which showed great changes between the different lines treatments (Figure 2c). Using the criteria of fold change ≥ 2 and false discovery rate (F ≤ 0.05, the DEGs between L282C and L282 and those between L693C and L693 were fined. Compared to normal-temperature germination, low-temperature germination g erated 3926 DEGs (2651 upregulated and 1275 downregulated) for L282 and 1404 D (667 upregulated and 737 downregulated) for L693 (Figure 3a). These DEGs were con ered cold-responsive genes.

Identification of Common DEGs at Low Temperature
Of the identified cold germination-responsive DEGs, 830 (452 upregulated and downregulated) were shared by L282 and L693, which were regarded as common D (cDEGs) (Table S1) (Figure 3b,c). To further detect the functional pathways of the cDE gene ontology (GO) analysis was performed using the AgriGO-v2 software (Table S2 Regarding the upregulated cDEGs, we found that the enriched biological process categories were primarily associated with microtubule-based processes, histone H3 modification, single-organism cellular processes, and carbohydrate-metabolic proce ( Figure 4a, Table S2). The significant molecular function (MF) categories were relate microtubule motor activity, cytoskeletal protein binding, motor activity, and amygd beta-glucosidase activity (Figure 4a, Table S2). The enriched cellular component (CC)

Identification of Common DEGs at Low Temperature
Of the identified cold germination-responsive DEGs, 830 (452 upregulated and 378 downregulated) were shared by L282 and L693, which were regarded as common DEGs (cDEGs) (Table S1) (Figure 3b,c). To further detect the functional pathways of the cDEGs, gene ontology (GO) analysis was performed using the AgriGO-v2 software (Table S2).
Regarding the upregulated cDEGs, we found that the enriched biological process (BP) categories were primarily associated with microtubule-based processes, histone H3-K9 modification, single-organism cellular processes, and carbohydrate-metabolic processes ( Figure 4a, Table S2). The significant molecular function (MF) categories were related to microtubule motor activity, cytoskeletal protein binding, motor activity, and amygdalin beta-glucosidase activity ( Figure 4a, Table S2). The enriched cellular component (CC) categories were related to the cell periphery, the kinesin complex, polymeric cytoskeletal fiber, and microtubules ( Figure 4a, Table S2).

Identification of L282 Specifically Expressed Cold-Responsive DEGs
Compared to the sweet corn line L693, L282 showed strong tolerance to a low t perature ( Figure 1). Of the identified cold germination-responsive DEGs, 3096 (2199 regulated and 897 downregulated) were specifically identified from L282, but not f L693. These DEGs were considered L282 specifically expressed DEGs (sDEGs) (Fig  3b,c, Table S3). We compared the expression patterns of these sDEGs in L282 and L (Figure 5a,b) and found that they all showed dramatic changes in expression levels in L at low-temperature germination compared to normal-temperature germination, whil L693 they did not change in terms of expression patterns or change insufficiently (Fig  5a,b). To verify the reliability of gene expression characterized by transcriptome seque ing, the expression characteristics of seven sDEGs encoding heat shock protein, c chrome P450, and pectin methylesterase were quantified using qRT-PCR. All ge showed similar changes to the RNA-seq results in L282, and in L693, they did not cha significantly ( Figure 5c, Table S3). For the 378 downregulated cDEGs, the significant categories were primarily related to the response to high light intensity, the response to heat, and the response to osmotic stress ( Figure 4b, Table S2). These results showed that various processes, such as carbohydrate metabolism, the response to abiotic stimuli, histone H3-K9 modification, and single-organism cellular processes, can contribute to the low-temperature germination of both L282 and L693.

Identification of L282 Specifically Expressed Cold-Responsive DEGs
Compared to the sweet corn line L693, L282 showed strong tolerance to a low temperature ( Figure 1). Of the identified cold germination-responsive DEGs, 3096 (2199 upregulated and 897 downregulated) were specifically identified from L282, but not from L693. These DEGs were considered L282 specifically expressed DEGs (sDEGs) (Figure 3b,c, Table S3). We compared the expression patterns of these sDEGs in L282 and L693 (Figure 5a,b) and found that they all showed dramatic changes in expression levels in L282 at low-temperature germination compared to normal-temperature germination, while in L693 they did not change in terms of expression patterns or change insufficiently (Figure 5a,b). To verify the reliability of gene expression characterized by transcriptome sequencing, the expression characteristics of seven sDEGs encoding heat shock protein, cytochrome P450, and pectin methylesterase were quantified using qRT-PCR. All genes showed similar changes to the RNA-seq results in L282, and in L693, they did not change significantly (Figure 5c, Table S3). As the main characteristic of sweet corn is a change in carbohydrate composition, we focused on the sDEGs relating to carbohydrate metabolism and transport ( Table 2). Zm00001d025943 is a gene encoding fructofuranosidase, while Zm00001d014866 encodes glycosyl transferase (Table 2). Zm00001d031303 and Zm00001d037480 are two genes encoding raffinose synthases, and Zm00001d017502 and Zm00001d029371 encode trehalose 6-phosphate phosphatase ( Table 2). All six of these genes were significantly upregulated in the L282 seeds in response to low temperature, whereas their expression levels were not detected in the L693 seeds at both normal and low-temperature conditions (Table 2). Moreover, the expressions of eight sugar transporters-ZmSWEET1a, ZmSWEET4a, ZmSWEET4b, ZmSWEET4c, ZmSWEET13a, ZmSWEET13b, ZmSWEET14a, ZmSWEET14b, and ZmSWEET17a-were also significantly upregulated in L282C but were not detected in L693C ( Table 2). The expression levels of six ZmSWEET genes were also confirmed by qRT-PCR analysis ( Figure 6). As the main characteristic of sweet corn is a change in carbohydrate composition, we focused on the sDEGs relating to carbohydrate metabolism and transport ( Table 2). Zm00001d025943 is a gene encoding fructofuranosidase, while Zm00001d014866 encodes glycosyl transferase (Table 2). Zm00001d031303 and Zm00001d037480 are two genes encoding raffinose synthases, and Zm00001d017502 and Zm00001d029371 encode trehalose 6-phosphate phosphatase ( Table 2). All six of these genes were significantly upregulated in the L282 seeds in response to low temperature, whereas their expression levels were not detected in the L693 seeds at both normal and low-temperature conditions (Table 2). Moreover, the expressions of eight sugar transporters-ZmSWEET1a, ZmSWEET4a, Zm-SWEET4b, ZmSWEET4c, ZmSWEET13a, ZmSWEET13b, ZmSWEET14a, ZmSWEET14b, and ZmSWEET17a-were also significantly upregulated in L282C but were not detected in L693C ( Table 2). The expression levels of six ZmSWEET genes were also confirmed by qRT-PCR analysis ( Figure 6).   A GO analysis was performed to detect the functional pathways of the sDEGs (T S4). The enriched categories of upregulated sDEGs were primarily associated with sin organism metabolic processes, the response to stimulus, and membrane and catalyti tivity (Figure 7). Similarly, the downregulated sDEGs were also assigned to single-or ism metabolic processes and cellular processes (Figure 7). This indicated that these gories may contribute to the low-temperature tolerance concerning seed germinatio the inbred line L282. A GO analysis was performed to detect the functional pathways of the sDEGs (Table S4). The enriched categories of upregulated sDEGs were primarily associated with singleorganism metabolic processes, the response to stimulus, and membrane and catalytic activity (Figure 7). Similarly, the downregulated sDEGs were also assigned to singleorganism metabolic processes and cellular processes (Figure 7). This indicated that these categories may contribute to the low-temperature tolerance concerning seed germination of the inbred line L282.
At low temperature, the plasma membrane system plays a crucial role in the response to cold stress [23]. In our study, the membrane category was enriched according to the GO analysis and 586 genes were specially upregulated in L282 at a low temperature (Table S4). Three genes, which are related to lipid metabolism membranes (Zm00001d023768, Zm00001d023774 and Zm00001d045660), were significantly upregulated in L282 at a low temperature, which was verified by qRT-PCR analysis (Figure 8a). Electric conductivity is an important index used to measure membrane permeability [24]. The electric conductivity and relative electric conductivity were both significantly lower in L282 than that in L693 at a low temperature (Figure 8b,c), indicating that the membrane of L282 experienced less damage, which is consistent with its strong tolerance. Taken together, this indicated that membrane genes may contribute to the low-temperature tolerance in the inbred line L282.  ity and relative electric conductivity were both significantly lower in L282 than tha L693 at a low temperature (Figure 8b,c), indicating that the membrane of L282 experien less damage, which is consistent with its strong tolerance. Taken together, this indica that membrane genes may contribute to the low-temperature tolerance in the inbred l L282.

Discussion
The absence of starch accumulation has resulted in lower seed vigor, which is a ma problem with respect to the seed production of sweet corn, particularly in super-sw corn (sh2) [1]. Although it is caused by the sh2 mutation, the seed vigor properties of ferent sh2 sweet corn varieties show considerable differences [12]; however, the unde ing mechanism behind this is unclear.

Improving the Germination Capacity of Sweet Corn Seeds at Low Temperature Is Crucial Sweet Corn Production
Seed quality is the main factor affecting the emergence of seedlings in crop prod tion, and this parameter is mostly reflected by seed vigor [25]. Seed vigor is a compreh sive concept which refers to a seed's emergence capacity in a wide range of environme [26]. At present, field corn with high vigor can be sown with single-seed precision, wh has greatly improved production efficiency and saved labor in recent years [27]. In c trast, the seed vigor of sweet corn is typically low due to its low starch content [1]. obtain the optimal population density, sweet corn is usually planted with 4-5 seeds hole, followed by hand thinning at the 3-leaves-stage, which is very time-consuming a laborious [1,2,11]. Therefore, the improvement of seed vigor is crucial for sweet corn p duction.
Low-temperature stress is one of the main environmental factors limiting crop p duction [16]. Maize, whose origins lie in tropical and subtropical regions, is extrem sensitive to cold stress during the seed germination stage, which consistently causes layed germination and reduces the seedling emergence rate [28]. Compared with fi corn, the germination capacity of sweet corn is usually lower at low temperatures Adding salicylic acid or fungi to sweet corn seeds as a coating material can improv certain degree of their germination capacity at low temperatures [29,30]. Meanwhile, g mination capacity is significantly different among different sweet corn lines, implying t

Discussion
The absence of starch accumulation has resulted in lower seed vigor, which is a major problem with respect to the seed production of sweet corn, particularly in super-sweet corn (sh2) [1]. Although it is caused by the sh2 mutation, the seed vigor properties of different sh2 sweet corn varieties show considerable differences [12]; however, the underlying mechanism behind this is unclear.

Improving the Germination Capacity of Sweet Corn Seeds at Low Temperature Is Crucial for Sweet Corn Production
Seed quality is the main factor affecting the emergence of seedlings in crop production, and this parameter is mostly reflected by seed vigor [25]. Seed vigor is a comprehensive concept which refers to a seed's emergence capacity in a wide range of environments [26]. At present, field corn with high vigor can be sown with single-seed precision, which has greatly improved production efficiency and saved labor in recent years [27]. In contrast, the seed vigor of sweet corn is typically low due to its low starch content [1]. To obtain the optimal population density, sweet corn is usually planted with 4-5 seeds per hole, followed by hand thinning at the 3-leaves-stage, which is very time-consuming and laborious [1,2,11]. Therefore, the improvement of seed vigor is crucial for sweet corn production.
Low-temperature stress is one of the main environmental factors limiting crop production [16]. Maize, whose origins lie in tropical and subtropical regions, is extremely sensitive to cold stress during the seed germination stage, which consistently causes delayed germination and reduces the seedling emergence rate [28]. Compared with field corn, the germination capacity of sweet corn is usually lower at low temperatures [1]. Adding salicylic acid or fungi to sweet corn seeds as a coating material can improve a certain degree of their germination capacity at low temperatures [29,30]. Meanwhile, germination capacity is significantly different among different sweet corn lines, implying that there are genetic factors controlling germination capacity [1]. Hence, the screening of cold-tolerance lines and the cloning of low-temperature-tolerant genes constitute an important technique for improving seeds' germination capacity [1]. In this study, we screened two inbred sweet corn lines, which had the same germination capacity at a normal temperature but showed significantly different germination capacities at low temperatures ( Figure 1). Owing to these characteristics, they were excellent materials with which to analyze the differences in the molecular mechanisms of sweet corn at low temperatures.

Transcriptome Profiling with Respect to Sweet Corn Kernels' Response to Low Temperature
Low temperatures typically cause damage to the membrane system, cell dehydration, ROS accumulation, and protein denaturation [16]. At the molecular level, dynamic changes in transcription levels contribute to the adaptability of plants to environmental changes during seed germination [31]. In addition, genes involved in protein degradation, lipid-related activities, transport, redox balance, and hormone response can also improve cold tolerance in field corn [32,33]. RNA-seq technology has recently been used to study global changes at the transcript level in field corn. Li et al. (2021) used two freeze-tolerant and freeze-sensitive lines each to perform RNA-seq analysis at the seedling stage and found 948 cold-stress-contributable DEGs that were associated with binding functions, protein kinase activity, and peptidase activity [34]. Li et al. (2022) used a low-temperaturetolerant line, L220, and its introgressed lines to reveal the cell division process and plasma membrane-related categories associated with low-temperature germination [21].
Compared with field corn, there are few studies on the low-temperature tolerance of sweet corn using transcriptome analysis. In this study, we conducted a transcriptome analysis of seed embryos at the germination stage under normal-and low-temperature conditions to identify the DEGs involved in the response to low temperatures. Of the identified cold-germination-responsive DEGs, 830 (452 upregulated and 378 downregulated) common DEGs (cDEGs) were shared by L282 and L693, which were associated with microtubule-based processes, histone H3-K9 modification, single-organism cellular processes, carbohydrate-metabolic processes, protein binding, and so on (Figures 3 and 4). In addition, we also identified 3096 (2199 upregulated and 897 downregulated) coldgermination-responsive sDEGs from the cold-tolerance line L282 ( Figure 5). These sDEGs were primarily associated with single-organism metabolic processes, the response to stimulus, and membrane and catalytic activity ( Figure 6). These biological processes of cDEGs and sDEGs were partially consistent with the results of similar studies conducted on field corn, indicating that there is similarity between field corn and sweet corn with respect to their responses to low temperature.

The Genes Related to Plasma Membrane Contribute to Low-Temperature Tolerance of sh2 Sweet Corn
The plasma membrane is an essential component in cells; it can separate internal and external spaces and plays an important role in the exchange of metabolites and signal transduction [35][36][37]. Low temperatures can inactivate the functions of the plasma membrane, including destroying its lipid bilayer structure and negating its transport activities as well as basic metabolism [38][39][40]. To cope with low-temperature stress, plant membranes have evolved a variety of adaptive mechanisms, including changing their lipid composition and increasing sugar and soluble protein content [41][42][43]. In this process, plasma membrane proteins and lipids have been well described [41][42][43].
The SWEET families are primary carbohydrate transporters in the plasma membrane that play roles in phloem loading and organogenesis [44,45]. In recent years, the SWEET family has also been reported to have a potential capacity for stress tolerance through controlling sugar concentrations [46][47][48]. In Arabidopsis, the overexpression of AtSWEET16 not only facilitated cold adaption but also promoted seed germination [49]. The heterologous expression of the HfSWEET17 gene led to higher cold tolerance than that of wild-type tobacco [50]. In this study, eight SWEET genes were found to be significantly upregulated in cold-tolerance line L282, which may represent potential candidate genes that contribute to the low-temperature tolerance of sh2 sweet corn.

Plant Materials and Trait Evaluation
The sh2 sweet corn inbred lines L282 and L693 were bred in our lab. Their seeds were harvested and stored in a controlled storage facility at 4 • C.
For the standard germination test, 50 seeds were surface sterilized for 5 min in 0.1% sodium hypochlorite, rinsed three times with distilled water, and then sown in germination paper (Anchor Ltd., St. Paul, MN, USA). The paper was vertically rolled in a sealed plastic bag, and the paper rolls were cultured in a 25 • C chamber under a 16 h/8 h light/dark photoperiod.
For the cold test, 50 seeds were surface-sterilized for 5 min in 0.1% sodium hypochlorite and rinsed three times with distilled water. The seeds were soaked in water at 4 • C for 3 days and then dried again to the normal moisture content. Finally, the seeds were subjected to standard germination test mentioned above.
The SL and RL were measured using a ruler at 7 days after sowing. The SW and RW were measured at the same time, and the seedlings were dried at 85 • C to a constant weight. For each trait's evaluation, an average of 10 seedlings served as the trait value for each line. All germination tests were repeated three to four times.

RNA Extraction and Sequencing
Twenty-four hours after seeds were subjected to standard germination and cold germination tests, germinated embryos were collected for RNA sequencing, with two replicates for each inbred line. Total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). The RNA concentration and quality were checked using the NanoDrop ND-2000 (Thermo Scientific, New York, USA). mRNAs were isolated using oligo (dT) magnetic beads (Illumina, San Diego, CA, USA). RNA fragmentation and PCR amplification were performed according to the RNA-seq protocol [51]. cDNA libraries were sequenced with a read length of 150 bp (paired-end) using DNBSEQ-T7 (Table S5) (Annoroad Gene Technology, Beijing, China).

Sequence Data Analysis
All sequenced reads from each sample were aligned to the maize B73 reference genome (AGPv4) using HISAT2 [52]. The number of mapped reads for each gene was counted from the unique mapping reads using HTSeq-count version 1.92.2 [53]. The number of raw reads was used to calculate the FPKM values using the following formula: FPKM = read counts / (mapped reads (Millions) × exon length (KB)). A gene was considered an expressed gene if the FPKM was equal to or more than 1 in at least one sample. DEGs were identified using the DESeq2 R package with |log2fold change| ≥1 and FDRadjusted p-value < 0.05 [54]. All expressed genes from different samples were submitted for PCA calculation in the R software by using the prcomp function with default settings [55].

Cluster Analysis and Functional Annotation Enrichment Analysis
Hierarchical clustering was performed using the hclust function in R with settings corresponding to the ward.D method. For functional annotation enrichment analysis, the agriGO online website (http://systemsbiology.cau.edu.cn/agriGOv2/, accessed on 3 July 2017) was used to perform gene ontology analysis under threshold of FDR < 0.05.

Quantitative RT-PCR Analysis
First-strand cDNA was synthesized using the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China) with total RNA as template. Quantitative RT-PCR was performed using Taq Pro Universal SYBR qPCR Master Mix (Vazyme) in triplicate on the Q6 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Relative quantification was performed using the 2 −∆ Ct method, and the ACTIN gene (Zm00001d010159) was used as control. The primers used in qRT-PCR analysis are listed in Table S6.

Electric Conductivity Measurement
Electrical conductivity (EC) was measured using a DDSJ-306A conductivity meter (Shanghai Precision & Scientific instrument Co., Ltd., Shanghai, China). For each line, 60 seeds were randomly selected and divided into 3 replicates. Each replicate of 20 seeds was weighed and then washed with deionized water thrice. The seeds were dried with filter paper and placed in 250 mL of deionized water. Then, EC was measured and recorded as initial EC d1. The seeds were soaked in water at 4 • C for 3 days; then, the EC was measured and recorded as d2. The final EC (µS cm -1 /g -1 ) = (d2 − d1) /seed weight. Then, the solution (containing the seeds) was boiled for 20 min and cooled to room temperature.

Conclusions
We screened two inbred lines of sweet corn differing in terms of low-temperature germination capacity and conducted a transcriptome analysis to identify the genes involved in their seed germination capacity in response to low temperatures. We found that the specially expressed genes in the cold germination-tolerant line L282 were primarily related to the plasma membrane and oxygen-containing compounds, constituting a result that was partially consistent with the results of similar studies conducted on field corn. The results indicate that improving the universal low-temperature tolerance genes in field corn, such as plasma membrane-encoding genes, could effectively compensate for the low tolerance caused by starch reduction in the breeding of sweet corn. This study lays a foundation for determining and cloning key genes affecting the germination of sweet corn seeds under low-temperature conditions. Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/plants12010159/s1, Table S1: common DEGs in L282 and L693 at low temperature; Table S2: GO analysis of common DEGs; Table S3: specially expressed DEGs in L282 at low temperature; Table S4: GO analysis of specially expressed DEGs in L282 at low temperature; Table S5: Summary of RNA-Seq; Table S6: primer for qRT-PCR.