Effects of sand storage on the seed germinatio n rate of P. cyrtonema Hua
The seeds of P. cyrtonema Hua treated with sand storage at 4 °C in winter for 30 d. (Fig.1). The rate of seeds germination was significantly higher (3.69 times) after the treatment with sand storage as compared to non-sand storage (control). . These results showed that treatment of sand storage plays an important role in the germination of P. cyrtonema Hua seeds.
Metabolic profiling of the seed coat and peeled seeds of P. cyrtonema Hua at different storage time
The metabolic profiles of the seed coat and peeled seeds during four sand storage stages were investigated using GC-MS. Metabolome-wide expression profiling showed highest metabolite expression during the treatment of sand storage. Through comparative analysis, 85 and 74 metabolites were identified in seed coat and peeled seeds, respectively (Fig.3). In the metabolite profiles were significant differences existed between the seed coat and peeled seeds. There are 65 metabolites detected in both the seed coat and peeled seeds, but 20 metabolites were only detected in the seed coat and 9 metabolites were only detected in peeled seeds (Fig.3B). The specific metabolites of seed coat included four carbohydrates, six alcohols, four organic acids, four nitrogenous compounds, and two other compounds, as well as, the nine metabolites specific to peeled seeds included two carbohydrates, five organic acids, and two other metabolites were presented (Fig.3A).
Changes of metabolites in seed coat and peeled seeds of P. cyrtonema Hua in metabolic pathways during different sand storage time
Our study sought to obtain a more detailed overview of the abundance of the identified substances and, similarities and differences in each storage time of both organs (Fig.4). In starch and sucrose metabolism, trehalose showed a similar trend in seed coat and peeled seeds during sand storage. Trehalose increased at 30 d of sand storage, decreased significantly at 60 d, and slightly increased at 90 d. The sucrose, fructose, and maltose in peeled seeds were significantly higher than those in other periods when stored in the sand for 30 d. The sucrose and maltose of seed coat decreased significantly after 60 d of sand storage. In the TCA cycle, seed coat and peeled seeds had the same metabolites, but the changing trend of metabolites was different in seed coat and peeled seeds. During sand storage, the content of pyruvate, succinate, and malate in peeled seeds showed similar trends, and the metabolites of sand storage at 60 d were significantly higher than those in the other three periods, indicating that the TCA cycle was relatively active at this time. Succinate of seed coat showed a significant downward trend in the sand storage process and increased slightly at 90 d. Pyruvate of seed coat was significantly higher than that in the other three periods during 30 d of sand storage. Malate of seed coat and peeled seeds changed in opposite directions.
These results indicating that, most of the sugar and glycosides content in the seed coat increased at 30 d, indicating that the macromolecular nutrients in the seed coat began to decompose into soluble sugar, and the other sand storage time changed differently. Most of the sugars and glycosides in peeled seeds increased at 30 d of sand storage and decreased at 60 d of sand storage, which indicated that the macromolecular nutrients in seeds began to decompose into soluble sugars in preparation for seeds to break dormancy. After 60 d of sand storage, sugar and glycosides began to degrade, indicating that the seeds have been in a relatively active state, began to consume sugar and glycosides to prepare for seed germination. The changing trend of amino acids in seed coat and peeled seeds were also different. The content of most amino acids in seed coat decreased with the extension of sand storage time, and most of the amino acids in peeled seeds were the highest at 60 d of sand storage. Proline was an important product of dormancy release . The content of proline in the seed coat decreased after sand storage, while the content of proline in peeled seeds increased after 60 d of sand storage. Benzoic acid is only detected in the seed coat, and benzoic acid is considered to be an endogenous inhibitor .
Morphological changes of P. cyrtonema Hua seeds at different germination stages
Through the dynamic observation of the germination process (Fig.5), it was found that the seeds of P. cyrtonema Hua started to germinate on 14 d, and the seeds began to show small buds, and the corms started outgrowth after the germination of 21 d. Then the corm began to germinate and grow to form radicle and hypocotyl.
Analysis of metabolites during seed germination of P. cyrtonema Hua based on GC-MS
GC-MS was used to analyze the metabolites during the germination process of P. cyrtonema Hua. A total of 96 metabolites were isolated and identified, including 22 amino acids, 20 sugars and glycosides, 22 organic acids, 13 alcohols and esters, 8 nitrogen compounds, 4 fatty acids, and 7 other compounds.
Firstly, the samples were analyzed by using PCA to detect the distribution of metabolites at different germination stages of P. cyrtonema Hua seeds (Fig.6). The first principal component covered 22.3%, and the second principal component covered 30.9%. From the dispersion trend of the score map samples, it can be seen that the seed germination 0 d can be obviously distinguished from other germination periods, 21 d, 28 d, 35 d cannot be distinguished obviously.
The OPLS-DA, a supervised pattern recognition method, was further employed to identify the metabolites during the germination of P. cyrtonema Hua seeds (Fig.7, VIP>1.0, p<0.05). There are 17, 27, 9, 7, 1 metabolites that showed significantly changed and greatly contributed to typing from 0 d vs. 7 d, 7 d vs. 14 d, 14 d vs. 21 d, 21 d vs. 28 d, 28 d vs.35 d, respectively(Additional file1: Table S1).
The dynamic changes of metabolites during the seed germination stages were analyzed by HCA. As shown in Fig.8, the metabolites during seed germination can be divided into three clusters. Cluster I mainly consists of two organic acids (succinic acid and 2, 4, 6-trihydroxybenzoic acid), three amino acids (glycine, 4-aminobutyric acid, β-alanine), and a glycoside compound. The contents of these metabolites decreased gradually during the seed germination process. Cluster II mainly includes 9 amino acids (proline, alanine, glutamic acid, etc.), 10 organic acids (citric acid, mandelic acid, glyceric acid, etc.), 7 sugars and glycosides (maltose, trehalose, moschose, etc.), 4 alcohols (inositol, glycerol, squalinositol, etc.), one fatty acid, four nitrogen-containing compounds, and four other metabolites after seed germination for 7 d, these metabolites contents firstly increase, and most of them decreased after 14 d of germination. Cluster III mainly consists of 12 sugars and glycosides (sucrose, cellobiose, fructose, etc.), 10 amino acids (tryptophan, L-lysine, tyrosine, etc.), 11 organic acids (malic acid, lactic acid, niacin, etc.), 9 alcohols and esters, 5 nitrogen compounds, 3 fatty acids (linoleic acid, stearic acid, palmitic acid) and 4 other compounds. The changing trend of cluster III was partly similar to that of cluster II. These metabolite contents gradually increased at 14 d of seed germination, and decreased at 21 d. However, compared with cluster I, the change was not significant. In summary, from the results of thermographic analysis of metabolites, it was found that the content of most compounds began to increase at 7 d of germination, and to decrease at 14 d.
Changes of metabolites during seeds germinationof P. cyrtonema Hua
Through the analysis of KEGG pathway of differential metabolites, the differential metabolites were enriched to 32, 32, 4, and 9 pathways from 0 d vs. 7 d, 7 d vs. 14 d, 14 d vs. 21 d, 21 d vs. 28 d, respectively. There was only one differential metabolite in 28 d vs. 35 d, and on the other side, there was no differential metabolic pathway (Additional file2: Table S2). The significantly enriched metabolic pathway was mainly related to amino acid metabolism, sugar metabolism, inositol phosphate metabolism and citric acid cycle. The metabolic pathways with significant enrichment were citric acid cycle, arginine and proline metabolism, starch and sucrose metabolism, etc. (Additional file2: Table S2).
A pair-wise comparison between all the stages was performed. We observed significant metabolite changes (p<0.05) between 0 d and 7 d as depicted on the metabolic map (Fig.9A). In 7 d，we observed significant increase in sucrose (2.02), sorbitol (1.75), glycine (1.28), serine (1.85), phenylalanine (1.97), carbamate (2.10), citric acid (1.70), malic acid (1.57), glutamic acid (6.27), proline (1.38), aspartic acid (2.05), asparagine (2.66), palmitic acid (1.89) and stearic acid (2.63) , accompanied by a significant decrease in inositol (0.14), tryptophan (0.59) and succinic acid (0.46)
We also observed significant metabolite changes (p<0.05) between stages 7 d and 14 d (Fig.9B). In 14 d, our study showed significant increase in sucrose (1.27), glucose (1.90), carbamate (1.14), valine (1.33), isoleucine (1.36), proline (1.38), glycine (1.28), serine (1.27), malic acid (1.30), L-hydroxyproline (1.34), phenylalanine (1.97), palmitic acid (1.30) and ethanolamine (1.24) accompanied by significant decrease in inositol (0.90) ,tryptophan (0.9) ,Glycolic acid (0.76).
When the seeds germinated at 21 d, the differential metabolities contents (mainly were amino acids and inositol) were decreased. On the 28 d of germination, the content of most of the differential metabolism decreased significantly, except for isoleucine and 4-hydroxypyridine, the content of other differential metabolites decreased significantly, including sugar, niacin and sorbitol. In summary, it can be concluded that the seed germination of P. cyrtonema Hua may be affected by a variety of metabolic pathways.
Correlation analysis of metabolites during seed germination of P. cyrtonema Hua
In this study, the changes of metabolic network among metabolites (r ≥ 0.8, p < 0.001) during the germination of P. cyrtonema Hua were analyzed. As shown in Fig.10, a total of 250 pairs of metabolites were significantly correlated, and all of them were positively correlated. It mainly existed between amino acids, organic acids, sugars, glycosides and alcohols, accounting for 48% of the total correlation, such as proline and aspartic acid (r = 0.87, p = 5.41E-12), maltose (r = 0.963, p = 5.45E-21), inositol (r = 0.843, p = 1.08E-10), phosphate (r = 0.914, p = 6.89E-15). Glutamine and lysine (r = 0.836, p = 2.24E-10). In addition, malic acid and serine (r = 0.82, p = 9.79E-10), sucrose (r = 0.819, p = 9.91E-10). Succinic acid and glycine (r = 0.928, p = 4.05E-16), γ-aminobutyric acid (r = 0.875, p = 3.12E-12), β-alanine (r = 0.91, p = 1.52E-14), 2 O-glycerol-α-D-galactose (r = 0.894, p = 2.08E-13). It shows that the metabolic pathways of amino acids, sugars and organic acids act together during seed germination（Fig.10）.