Background

Seed germination is a crucial stage for plant development and agricultural production, which is a complex process involving various physical and biochemical cues, determined by exogenous (e.g. temperature, water, oxygen, light) and endogenous (phytohormones) factors [1]. The physical, physiological and biochemical processes of seed germination have been described in detail for several species [2]. In the process of seed germination, water is absorbed by the embryo, resulting in the rehydration and expansion of the cells. In general, the imbibition of water by seeds before germination can be divided into three phases: phase I, which is denoted by a rapid initial water uptake (imbibition), where a rapid activation of respiration and protein synthesis (utilizing stored mRNAs already present in the seed) is observed; phase II, which is marked by a plateau in water uptake, where massive degradation of food reserves (storage proteins, fats and carbohydrates) occurs; and phase III, which comprises a further increase in water uptake at the end of germination, as the embryonic axis elongates [2, 3].

It is widely accepted that hormones are involved in the regulation of seed dormancy and germination [4, 5]. Abscisic acid (ABA) is an essential repressor of seed germination, while gibberellins (GAs) are a promoter of seed germination, and it is apparent that ABA and GAs antagonistically regulate seed germination [6, 7]. During seed germination, GA increases the growth potential of the embryo and is necessary to overcome the mechanical restraint conferred by the seed-covering layers by weakening the tissues surrounding the radicle [7]. Conversely, ABA can prevent the weakening of the cell wall induced by GA [4]. It has been demonstrated that ABA-insensitive factors, including ABI3 and ABI5, are involved in the promotion of seed dormancy and inhibition of seed germination [8, 9]. Moreover, recent studies have pointed out that DELAY OF GERMINATION-1 (DOG1), which is a master regulator of primary dormancy (PD), acts in concert with ABA to delay germination [10, 28]. Late embryogenesis abundant (LEA) proteins are defined by the pronounced increase in their expression in embryos during late embryogenesis and their disappearance during subsequent germination stages [29]. Interestingly, it is worth noting that the expression of SSP- and LEA-related genes was significantly down-regulated by cyanide pretreatment (Table 1). The gene expression of SSPs in cyanide-pretreated seeds, including 2S, 11S, and 12S proteins, was 3- to 8-fold lower than that of the control (KCN12 vs. CK12) (Table 1). The differences were more pronounced in KCN24 vs. CK24 (Table 1), indicating that cyanide pretreatment helps mobilize the utilization of stored protein. In addition, the results showed that cyanide pretreatment also significantly down-regulated the gene expression of LEAs (Table 1). Compared with the control seeds, the expression of most LEA genes in cyanide-pretreated seeds was decreased by 4 to 16 times (Table 1), indicating that LEA proteins were involved in cyanide-mediated tomato seed germination.

Table 1 DEGs related to storage protein and late embryogenesis abundant protein
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Cyanide pretreatment accelerates glucose metabolism and energy conversion

Since GO and KEGG analyses showed that energy metabolism pathways were regulated by cyanide, the DEGs were further analyzed in KCN12 vs. CK12 and KCN24 vs. CK24. As shown in Fig. 5, the DEGs assigned to glycolysis and TCA were significantly up-regulated by cyanide pretreatment, and more pronounced in KCN24 vs. CK24 (Fig. 5A, B). In particular, 96 DEGs assigned to the oxidative phosphorylation pathway were enriched in both KCN12 vs. CK12 and KCN24 vs. CK24, and most of them showed up-regulated gene expression after cyanide pretreatment (Fig. 5C). However, it should be noted that the cyanide-resistant pathway, i.e., AOX pathway related gene expression was down-regulated after cyanide pretreatment (Fig. 5D). These findings indicate that pretreatment with a lower concentration of cyanide (10 μM KCN) did not damage cellular respiration but helped to accelerate glucose metabolism and energy conversion.

Fig. 5
figure 5

Expression profile of glucose metabolism and cell respiration related genes in response to cyanide pretreatment. Heatmap shows the expression of genes related to glycolysis (A), TCA (B), oxidative phosphorylation (C) and AOX (D) pathways. FC, fold change. DEGs, differentially expressed genes. TCA, tricarboxylic acid cycle. AOX, alternative oxidase

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Cyanide pretreatment inhibits ABA but promotes GA biosynthesis and perception

Considering that plant hormones are involved in the regulation of seed germination, the DEGs in KCN12 vs. CK12 and KCN24 vs. CK24 were further analyzed to reveal the effects of cyanide pretreatment on hormone biosynthesis and perception. The results showed that cyanide pretreatment down-regulated the gene expression of ABA biosynthesis and signal transduction (Fig. 6), but up-regulated the gene expression of GA biosynthesis and signal transduction (Fig. 7). As shown in Fig. 6, the gene expression of key enzymes of ABA biosynthesis such as 9-cis-epoxycarotenoid dioxygenase (NCED) and abscisic-aldehyde oxidase (AO) was down-regulated after cyanide pretreatment, especially in KCN24 vs. CK24. In addition, the gene expression of protein kinases such as SNF1-related protein kinase (SnRK) and ABA responsive factors including ABA-insensitive 3 (ABI3) and ABA-insensitive 5 (ABI5) was also significantly down-regulated by cyanide pretreatment in both KCN12 vs. CK12 and KCN24 vs. CK24 (Fig. 6). Moreover, it is worth noting that cyanide pretreatment down-regulated the gene expression of DELAY OF GERMINATION1 (DOG1) (Table 2), which is the main regulator of dormancy and was recently identified as a unique ABA signal component in seeds [10, 39, 40]. Likewise, LEA proteins are enriched in the embryo during late embryonic development and disappear in the subsequent germination stage [29]. Previous studies have shown that SSPs and LEA proteins respond to ABA signaling during seed development. For example, the ABI3 and B3 factors FUS3 (ABI3 homolog-like protein) have been demonstrated to directly activate SSPs gene expression in Arabidopsis thaliana [41]. In addition, it has been observed that the gene expression of LEA proteins is regulated by ABI3, which is suppressed in abi3 mutants [29]. In contrast, GA has been proposed to stimulate the mobilization of SSPs and LEA proteins by suppressing the expression of these genes while promoting the transcription of hydrolysis-related genes [42]. Consequently, these findings led to the hypothesis that cyanide promoted tomato seed germination by accelerating the mobilization of storage reserves, thus promoting the supply of energy and nutrients to help seed germination. However, whether the effect of cyanide on the storage substance is direct (i.e., as a signal molecule) or indirect (i.e., by regulating ABA and GA signals) remains to be confirmed by further studies in the future.

Conclusions

In summary, our study suggests that cyanide-stimulated tomato seed germination occurs in a dose- and time- dependent manner. It is worth noting that the appropriate concentration of cyanide pretreatment contributes to respiration metabolism and the degradation of storage materials required for seed germination. In addition, cyanide may act as a signaling molecule to control the balance of ABA and GA by regulating their biosynthesis and signal perception. Combined with previous studies, we believe that the effective concentration and duration of cyanide differ in controlling the germination of different plant seeds. In any case, our research provides useful resources for further understanding and elucidating the molecular mechanism of cyanide-mediated seed germination.

Methods

Plant materials and chemical treatment

In this study, the seeds of tomato (Solanum lycopersicum cv. Alisa Craig) were used, which originally obtained from Thompson and Morgan (https://www.thompson-morgan.com/).

For germination, seeds were surface-sterilized with 0.1% mercury bichloride for 10 min and then were washed with ddH2O for at least three times. After that, some seeds were pretreated with different concentrations of potassium cyanide (5, 10, 50, 100 μM) for 6 h, 12 h, and 24 h at 25 °C. The other seeds were pretreated with ddH2O (defined as controls) under the same conditions. In each pretreatment experiment, approximately 100 sterilized seeds were used. All pretreatments had three biological replicates in each experiment, and three or more independent experiments were carried out for the whole study.

After pretreatment, cyanide was removed and the seeds were placed on sterile filter paper moistened with deionized water for germination and growth under the conditions of 16 h of light (approx. 150 μmol m− 2 s− 1) and 8 h of dark, at 25 °C, 70% relative humidity.

Germination test

Germination was scored over time, and the initiation of the germination process was calculated from the moment that the seeds were treated with cyanide or water; a seed was considered germinated when the radicle protruded from the teguments by 1 ~ 2 mm. The germination percentage (%) and germination speed (T50) were computed as described previously [43]. T50, time to obtain 50% of germinated seeds.

Respiration measurement

Respiration of seeds was performed as described previously [44] with some modification. In this experiment, ten seeds were collected and transferred into air-tight cuvettes containing 2 ml of phosphate buffered saline (pH 7.5), and oxygen uptake was measured as a decrease of oxygen concentration in the dark using a Clark-type electrode (Chorolab-2; Hansatech, King’s Lynn, UK). Total respiration rate (Vt) was achieved when the measurement without any inhibitors. Then, 2 mM salicylhydroxamic acid (SHAM, an AOX inhibitor) [45, 46], was added to inhibit cyanide-resistant respiration rate (Valt), and the values of cytochrome pathway respiration (Vcyt) and residual respiration rate (Vres) were obtained. Valt = Vt-Vcty-Vres.

H2O2 content estimation

The H2O2 content was measured as described previously [47]. In this experiment, ten seeds were homogenized with liquid nitrogen and then mixed with 5 mL 0.1% (w/v) trichloroacetic acid (TCA). The homogenate was centrifuged at 12000 g for 20 min at 4 °C. In addition, 0.5 mL of the supernatant was added to 0.5 mL 10 mM potassium phosphate buffer (pH 7.0) and 1 mL 1 M KI. The absorbance of the supernatant was read at 390 nm.

HPLC for plant hormone determination

The plant hormones include GA, ABA, and IAA were measured by HPLC. For GA extraction and measurement, samples were ground carefully and 0.2 g of the powdered sample was extracted overnight at 4 °C with 1.5 mL 70% (V/V) acetonitrile. After vortex for 30 s and centrifugation at 14,000 rpm for 10 min, the supernatants (1.0 mL) were collected and then evaporated to dryness under nitrogen gas stream at room temperature, constituted in 100 μL 80% (V/V) methanol, diluted to 800 μl with water. The extracts were passed through the SPE cartridge (CNWBOND Carbon-GCB SPE Cartridge, 200 mg, 3 mL; Anpel, Shanghai, China) and evaporated to dryness under nitrogen gas stream at room temperature. Following the sample were reconstituted in 200 μl 80% (V/V) methanol and filtrated (PTFE, 0.22 μm; Anpel, Shanghai, China) before LC–MS/MS analysis [48].

For ABA and IAA extraction and measurement, 50 mg samples were ground with liquid nitrogen and extracted with 0.5 mL methanol/water/formic acid (15:4:4, V/V/V) at 4 °C. The extract was vortexed for 10 min and centrifuged at 14,000 rpm for 5 min at 4 °C. The supernatants were collected and repeated the steps above. The combined extracts were evaporated to dryness under nitrogen gas stream, reconstituted in 80% methanol (V/V), ultraphoniced (1 min) and filtrated (PTFE, 0.22 μm; Anpel, Shanghai, China) before LC-MS/MS analysis.

All of the standards were purchased from Olchemim Ltd. (Olomouc, Czech Republic) and Sigma (St. Louis, MO, USA). More details about the LC-MS/MS analysis can be found in Supplementary Methods.

RNA extraction and transcriptome sequencing

For RNA-SEQ analysis, tomato seeds pretreated with 10 μM KCN for 12 h (labeled KCN-12) were used in this study. In addition, after cyanide pretreatment, the seeds were placed under normal conditions for another 12 h (labeled KCN-24) were also used for analysis. The seeds without cyanide pretreatment for 12 h (CK-12) and 24 h (CK-24) were used as the control. Subsequent handling of tomato samples includes extraction, purification, analysis and sequencing of total RNA performed by Novogene Bioinformatics Technology Co. Ltd. (Bei**g, China). Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) according the manufacturer’s instructions, and index codes were added to attribute sequences to each sample.

Quantification of gene expression levels

HISAT2 was used to count the number of reads mapped to each gene. In addition, the FPKM (fragments per kilobase of exon model per million mapped reads) of each gene was calculated based on the length of the gene and the number of reads mapped to the gene.

Differential expression analysis

Differential expression analysis was performed using the DESeq2 R package (1.16.1). DESeq2 provides statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P-values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate. Genes with an adjusted P < 0.05 found by DESeq2 were assigned as differentially expressed. In addition, the gene IDs are shown in the table and figures, and the gene name is the gene IDs that contain the prefix “LOC”.

GO and KEGG enrichment analysis

Gene Ontology (GO) enrichment analysis of differentially expressed genes was implemented by the cluster Profiler R package, in which gene length bias was corrected. GO terms with corrected P-value less than 0.05 were considered significantly enriched by differential expressed genes.

Kyoto Encyclopedia of Genes and Genomes (KEGG) is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-through put experimental technologies (http://www.genome.jp/kegg/). We used cluster Profiler R package to test the statistical enrichment of differential expression genes in KEGG pathways.

Real-time quantitative PCR analysis

In order to validate the results from transcriptome sequencing analysis, part of genes was confirmed by quantitative real-time PCR (qRT-PCR) and Actin (Accession number: AB158612) gene was used as internal control. All the Primers are listed in Supplementary Material: Table S1. qRT-PCR reactions were prepared with the SYBR Green Master Mix Reagent (Applied Biosystems, MA, USA), following the manufacturer’s instruction. Reactions were carried out in Applied Real-Time System (ABI7500). All samples were performed in triplicate and relative expression levels were calculated using the delta-delta Ct method of the system.

Statistical analysis

Statistical analysis of the results from three independent experiments. The averages and standard deviations (SD) of all results were calculated, and one-way analysis of variance (ANOVA) were performed to generate P values. The difference was considered to be statistically significant when *P < 0.05, ** P < 0.01, and *** P < 0.001.