Morphological identification and phenotype of Polygonatum cyrtonema
The plant morphology, flowers and tubers of Polygonatum cyrtonema Hua show in Fig. 1A, B, C. Figure 1B and Fig. 1C obtained from Plant Photo Bank of China, PPBC (http://ppbc.iplant.cn/). Polygonatum cyrtonema Hua is a perennial herb of the Liliaceae family, with thick rhizomes, alternate phyllotaxy and yellow-green perianth, flowering from May to June and fruiting from August to October. The medicinal part of Polygonatum is rhizome. In this study, the fruits and seeds (Fig. 1D, E) of Polygonatum cyrtonema Hua obtained from Chengdu Institute of Biology, Chinese Academy of Sciences. Germinated seeds (Fig. 1F) were collected for transcriptome sequencing and metabolome analysis after six months of planting.
RNA-seq de novo assembly and function annotation of Polygonatum cyrtonema
Six cDNA libraries, each for Huangjing seeds with germinated and control (G and CK) group, were characterized by Illumina HiSeq to detect the transcriptome level of gene expression information. A summary of RNA-Seq data is shown in Table 1, high-quality and clean reads were obtained by removing low-quality reads in per library. The Q30 of six cDNA libraries were more than 95% and GC was 52.33%. After de novo assembly, a total of 56,628 unigenes (UG) were detected. Among them, there were 29,688 (52.43%) annotated in NR, 27,901 (49.27%) were characterized from eggNOG database, 22,673 (40.04%) annotated in SwissProt, 11,611 (20.50%) annotated in KEGG, 20,543 (36.28%) annotated in GO and annotated in KOG were 17,965 (31.72%). A total of 33,705 unigenes > 500 bp and 15,223 unigenes > 1000 bp. The transcriptome assembly results revealed that the RNA-Seq datasets were reliable to further study.
Table 1
Statistics and functional annotations of unigenes in 6 RNA sequencing libraries
| Number of unigenes | Percentage (%) |
> 300 bp | 56628 | |
>=500 bp | 33705 | 59.5 |
>=1000 bp | 15223 | 26.9 |
N50 | 1127(bp) | - |
Max length | 11337 (bp) | - |
Min length | 301 (bp) | - |
Average length | 855 (bp) | - |
NR | 29688 | 52.4 |
EggNOG | 27901 | 49.3 |
SwissProt | 22673 | 40.1 |
KEGG | 11611 | 20.5 |
GO | 20543 | 36.3 |
KOG | 17965 | 31.7 |
Total | 56628 | |
Comparative Analysis And Qrt-pcr Verification
To elucidate the molecular basis for Huangjing seed germination, comparative transcriptome analysis was conducted. DEGs were analyzed using the RPKM method between the G and CK group. Compared with the CK, a total of 18,308 DEGs up-regulated and 9,709 DEGs down-regulated in the sprouted Huangjing seeds (Fig. 2A, Supplemental File 1–1). The results indicating that these 27,017 DEGs might be involved in Huangjing seed germination.
To evaluate the accuracy of transcriptome profiles from the RNA sequencing analysis, 18 genes related of seed germination were selected for qRT-PCR expression analysis. The correlation of RNA-Seq (FPKM) and qRT-PCR are shown in Fig. 2B (Supplemental File 1–2). The relative gene expression level of qRT-PCR were consistent with RNA-Seq profile, which indicated that the RNA-Seq data were reliable.
Kegg Analysis Of Differentially Expressed Unigenes (degs)
To identify the major functional terms and signal pathways between G and CK seeds, KEGG analysis was carried out of DEGs at a significance level of p < 0.05. The KEGG annotations indicated that plant hormone signal transduction (ko04075), phenylpropanoid biosynthesis (ko00940), starch and sucrose metabolism (ko00500),phenylalanine metabolism (ko00360), flavonoid biosynthesis (Ko00941) pathways were significantly enrichment in Huangjing seeds (Fig. 3, Supplemental File 2).
DEGs of phenylpropanoid and flavonoid biosynthesis related to seed germination
The DEGs involved in phenylpropanoid biosynthesis (ko00940) and flavonoid biosynthesis (Ko00941) were examined in this study, which involved in seed germination. A total of 114 DEGs (91 up-regulated, 23 down-regulated) were detect in phenylpropanoid biosynthesis, including PAL, CYP73A, CYP73A, 4CL and CCR, etc. Twenty-two DEGs were enriched in flavonoid biosynthesis and mostly up-regulated, such as CHS, FLS, F3Hand CHI et al. (Fig. 4A, Supplemental File 3). To validate the transcriptome expression data for genes involved in flavonoid biosynthesis, we randomly selected six genes for qPCR analysis. Including CHS (TRINITY_DN14701_c0_g2_i1_4, TRINITY_DN27324_c0_g1_i1_3), E5.5.1.6/Chalcone isomerase (TRINITY_DN39749_c0_g1_i5_3), F3H (TRINITY_DN16505_c0_g2_i1_4, TRINITY_DN16337_c0_g1_i1_4), and FLS (TRINITY_DN43663_c0_g1_i1_3). The fold changes of RNA_seq were all consistent with those from the qPCR analysis (Fig. 4B). Those results indicate that phenylpropanoid and flavonoid biosynthesis pathway are important for seed germination.
DEGs related to starch and sucrose metabolism in seed germination
The significant enriched pathway was found in G vs CK seeds, a total of 59 DEGs were detect in starch and sucrose metabolism (ko00500), 41 up- and 18 down-regulated (Supplemental File 4) in Huangjing. We found that α-amylase synthesize gene and MGAM enzyme genes significantly up-regulated during seed germination (Fig. 5A). Therefore, we determined the α-amylase activity and related gene expression in this pathway. The results show that the highest α-amylase activity was found in germination seeds, followed by the CK seeds and radicle (Fig. 5B). The enzyme genes of α-amylase, E2.4.1.15, E3.2.1.20, E3.2.1.39 and E3.2.1.21 in starch and sucrose metabolism were determined by qRT-PCR (Fig. 5C) and all DEGs were significantly up-regulated in germination seeds. The expression levels of unigenes were consistent with the trends of α-amylase activity, indicating that the activity of α-amylase and sucrose metabolism might play an important roles in promoting seed germination.
Plant Hormone Related Degs In Seed Germination
Plant hormone affect the germination of plant seeds, thus we found that plant hormone signal transduction (ko04075) were significantly enriched in this study. DEGs involved in the auxin synthesis regulation pathway, such as AUX1 and TIR1 were significantly up-regulated in germinated seeds (Fig. 6A, Supplemental File 5). The GID2 and TF in GA related pathway were also up-regulated. However, compare with CK, the DEGs were almost down-regulated in ABA signal pathway, including PP2C, SnRK2 and ABF et al. In addition, we found that the DEGs were significantly changed in the upstream of salicylic acid synthesis (ko00360, ko00362), while some genes in the downstream of salicylic acid pathway were also significantly different, such as NPR1, PR1. To validate the transcriptome expression data, three genes of plant hormone signal transduction, AUX1 (TRINITY_DN27428_c0_g1_i1_3), AUX/IAA (TRINITY_DN32072_c0_g1_i1_2) and A-ARR (TRINITY_DN21518_c0_g1_i1_2) were randomly selected for qPCR analysis (Fig. 6B). The fold changes for the qPCR analysis indicated the relative expression of the germinated and the control seeds, and the fold changes of RNA_seq were consist with the qPCR analysis. Therefore, we suggest that auxin, GA and ABA were involved in seed germination.
Metabolic Analysis And Association Of Transcriptomic Analysis
To verify the accuracy of transcriptome sequencing results, we conducted metabolic analysis. In this study, a total of 637 metabolites were detected in Huangjing seeds by LC-MS, including certain primary and secondary metabolites, such as organic acids, amino acids, flavonoids, lipids etc. Compared with CK, 230 measured different metabolites (96 up-regulated and 134 down-regulated) were significantly to be identified in germination seeds (Fig. 7, Supplemental File 6). To study the association between transcriptomic and metabolic in seeds, we analyzed the connection of DEGs expression and metabolites. Certain key enzyme encoding DEGs were related to produce metabolites, this result reveal that these enzyme encoding DEGs might be involved in seeds germination.
Flavonoids And Hydroxycinnamyl Related To Seed Germination
We conducted a detailed analysis of the different metabolites in G vs CK seeds. The result shown that 42 flavonoids significantly accumulated during the germination of seeds (Fig. 8A, Supplemental File 7 − 1). Compare with CK, All 42 flavonoids were significantly increased in G group, including flavanone, flavonol and anthocyanins, etc. The result is consistent with the RNA_seq results. Therefore, we suggesting that the synthesis of flavonoids may play an important roles to the germination of Huangjing seeds.
In addition, we found that hydroxycinnamyl showed a significant down-regulated trend (Fig. 8B, Supplemental File 7 − 2). Hydroxycinnamyl are precursors in the lignin synthesis, and the metabolites in this pathway may negatively regulate the germination of seeds. Therefore, we further analyzed the flavonoid synthesis and the lignin synthesis pathway, and found that both pathways are derived from the phenylalanine metabolism pathway. Based on the KEGG pathway, we mapped metabolic pathways related to germination of Huangjing. The metabolites of flavonoid synthesis pathway naringenin chlcone, naringenin, aromadedrin, taxifolin and cyaniding were significantly increased in germinated seeds. However, compared with CK, the metabolites cinnamic acid, caffeic acid, caffeic aldehyde, coniferyl aldehyde, coniferin and sinapic acid were significantly decreased in phenylpropanoid pathway (Fig. 8C, Supplemental File 6). The results showed that the metabolites flow of phenylalanine pathway was mainly to promote flavonoids synthesis and to inhibit the lignin synthesis during the seeds germination, which was beneficial to the germination of Polygonatum cyrtonema seeds.
Organic acids related to germination of Polygonatum cyrtonema
In present study, we found that organic acids significant down-regulate with the germination of Polygonatum cyrtonema seeds (Fig. 9, Supplemental File7-3). The results showed that organic acids could significantly inhibit seed germination. We further analyzed and found that benzoic acid, ferulic acid, and coumaric acid were the most significant organic acid metabolites during germination. Therefore, we speculated that these metabolites have a major inhibitory effect on the germination of Polygonatum cyrtonema seeds.