Transcriptome analysis under different photoperiodic treatments
The results showed that in the 12 samples, more than 95.28% of the clean reads were successfully aligned to the reference genome (Shanghai Center for Plant Stress Biology and Center of Excellence in Molecular Plant Sciences, Chinese Academy of Sciences). The alignment efficiencies of these reads to the reference genome were approximately 87.86%. Furthermore, most of the mapped reads were concentrated in the exon region of the gene, indicating that both the sequencing data and the chosen reference genome are of high quality and suitable for analysis (Supplementary Table S2). Expression fold difference |log2FC| > 1 and P < 0.05 were used as criteria to screen DEGs for each comparison group. The results showed that a total of 1664 DEGs were identified in different Panicum miliaceum L. genotypes under the short-day treatment (SD), including 920 upregulated and 744 downregulated genes. Similarly, 2564 DEGs were identified under the long-day treatment (LD), including 1449 upregulated and 1115 downregulated genes. A total of 13017 DEGs were identified in the M51 Panicum miliaceum L. genotype under different photoperiod treatments, including 5915 up-regulated and 7102 down-regulated genes, and 15548 DEGs were identified in the D32 Panicum miliaceum L. genotype under different photoperiod treatments, consisting of 7116 upregulated and 8432 downregulated genes. The number of DEGs in the different Panicum miliaceum L. genotypes under the same light treatment was significantly lower than that in the same Panicum miliaceum L. genotype under different light treatments. Therefore, genetic mechanisms may be more responsive to changes in environmental factors (such as light) than to the materials themselves.
Analysis using Venn diagrams (Fig. 1) showed that there were 544 DEGs specific to LD-D vs. LD-M, 3822 DEGs specific to LD-D vs. SD-D, 1794 DEGs specific to LD-M vs. SD-M, 244 DEGs specific to SD-D vs. SD-M, and 112 common DEGs between these comparison treatment groups, indicating that these identified DEGs may be involved in the response to the photoperiod.
Functional enrichment analysis of the identified DEGs
Similarly, a 95% confidence interval (P < 0.05) was used as the standard to screen for significant GO terms and KEGG enrichment pathways. The identified DEGs of the LD-D vs. LD-M, SD-D vs. SD-M, LD-D vs. SD-D, and LD-M vs. SD-M groups were significantly enriched in 8, 21, 29, and 21 biological processes of GO terms, respectively. Furthermore, the DEGs between the M51 and D32 Panicum miliaceum L. genotypes involved in biological processes under the same light conditions were significantly different (Fig. 2). The DEGs between LD-D and LD-M were mainly enriched in cysteine-type peptidase activity, ubiquitin-like protein-specific protease activity, and UFM1 hydrolase activity under long sunlight, whereas the DEGs between SD-D and SD-M were primarily enriched in organonitrogen compound and protein metabolic processes and in an anchored component of the membrane. The enrichment pathways of the DEGs between the M51 and D32 Panicum miliaceum L. genotypes were similar to those under different light treatments (Table 1), and mainly included protein phosphorylation, phosphorus and phosphorus-containing compound metabolic processes, and phosphorylation. NEK (serine/threonine protein kinase) was involved in the phosphorylation of Panicum miliaceum L. protein, and the expression trends of NEK in photosensitive and photo-insensitive Panicum miliaceum L. were different, suggesting that the differential expression levels of NEK3 and NEK4 could distinguish the photoperiod sensitivity of the two types of Panicum miliaceum L..
In addition, the DEGs between the LD-D and LD-M, SD-D and SD-M, LD-D and SD-M, and LD-M and SD-M groups were significantly enriched in 17, 9, 35, and 34 KEGG pathways, respectively. The metabolic pathways of the DEGs between the M51 and D32 Panicum miliaceum L. genotypes were significantly different under the same light treatments (Fig. 3). Under the long-day treatment, the common pathway was mainly phenylalanine biosynthesis, while the main metabolic pathways were photosynthesis, carotenoid biosynthesis, and glycerophospholipid metabolism; psbM, psal, and psaJ were involved in photosystem I and II reactions. The photoperiod gene PRR95 was significantly expressed under the long but not under the short photoperiod. The main metabolic pathways under short-day conditions were the circadian rhythm plant, diterpenoid, and monobactam biosynthesis. The photoperiod genes HD3A, FT, and PRR37 were significantly enriched in the circadian-plant metabolic pathway under short-day conditions but not under long-day conditions. The metabolic pathways involved in the DEGs between M51 and D32 were similar under different light treatments, including photosynthesis, photosynthesis-antenna protein, and valine, leucine, and isoleucine degradation. The expression of LHCA6 (the harvesting complex gene 6) was downregulated, and the cytochrome psbE gene was upregulated in M51. The expression levels of the amino acid transferase At3g08860, acetaldehyde dehydrogenase ALDH2B4, psbA, psaI, and psaC were significantly upregulated on D32, while the expression levels of IBR3 and ALDH6B2 dehydrogenase were significantly downregulated.
Annotation of metabolites
A total of 822 metabolites were identified under different treatments (Fig. 4), including 538 and 284 metabolites in the positive and negative ion modes, respectively. Among these, 29.6% were lipids and lipid-like molecules, 16.8% were undefined, 8.9% were organic acids and derivatives, 8% were organoheterocyclic compounds, 7.3% were benzenoids, and 3.2% were nucleosides, nucleotides, and analogs.
Statistical analysis of differential metabolites
Differential metabolites in each comparison group were screened using the criteria VIP > 1 and P < 0.05. It was found between the LD-D and LD-M groups, there were 8 differential metabolites in the negative ion mode (such as L-malic and fumaric acids and protodioscin, Supplementary Table S3), which were produced by synthesis and decomposition of organic acids and lipids; and 23 differential metabolites (such as isoreserpin, olivil 4′-O-glucoside, and ellipticine) were screened in the positive ion mode (Supplementary Table S3), generated by the synthesis and decomposition of ketones, alkaloids, and lipids compounds. Between the SD-M and SD-D groups, in the negative ion mode(Supplementary Table S3), 10 differential metabolites (such as protodioscin, L-malic acid, and flavone base + 4O, C-Hex-dHex) were found, which were involved in the synthesis and decomposition of lipids, organic acids, and flavonoids, while in the positive ion mode (Supplementary Table S3), 20 differential metabolites (such as cephaeline, cholestenone, and betaine) were involved in the biosynthesis and decomposition of alkaloids and lipids. Between the SD-D and LD-D groups, 21 differential metabolites (such as L-malic, fumaric, and glyceric acids) were detected in the negative ion mode (Supplementary Table S3), which were generated by the synthesis and decomposition of organic acids, lipids, and oxides, and 28 differential metabolites (such as euphol, loperamide, and brucine) were detected in the positive ion mode (Supplementary Table S3), which were mainly produced through the synthesis and decomposition of lipids, benzene ring compounds, and alkaloids. Furthermore, between the SD-M and LD-M groups, there were 14 differential metabolites (including L-malic and fumaric acids and D-glucose) in the negative ion mode (Supplementary Table S3), which were generated through the synthesis and decomposition of organic acids and oxides, and 15 different metabolites (including loperamide, violanthin, and euphol) in the positive ion mode (Supplementary Table S3), which were produced by the synthesis and decomposition of benzenoid compounds, ketones, and lipids. Taken together, the differential metabolites identified in negative mode (L-malic acid and fumaric acid) were mainly produced by the synthesis and decomposition of organic acids, whereas those identified in positive mode (brucine and loperamide) were primarily generated through the synthesis and decomposition of lipids.
As shown in the Venn diagram (Fig. 5), there were 15, 6, 15, and 1 unique differential metabolites between the LD-M and LD-D groups, between the SD-M and SD-D groups, between the SD-D and LD-D groups, and between the SD-M and LD-M in the positive ion mode, respectively. In negative ion mode, there were three unique differential metabolites between the LD-D and LD-M groups, three between the SD-M and LD-M groups, 12 between the SD-D and LD-D groups, four between the SD-M and LD-M groups, and one common differential metabolite between each comparison group, L-malic acid.
KEGG analysis of the identified differential metabolites
Differential metabolites identified between the different groups were subjected to KEGG enrichment analysis (Fig. 6). The main metabolic pathways of the differential metabolites in the LD-M and LD-D groups were associated with the citrate cycle (TCA cycle), pyruvate metabolism, and carbon fixation in prokaryotes. Differential metabolites between the SD-D and LD-D groups were primarily enriched in the TCA cycle, pyruvate metabolism, and carbon fixation in prokaryotes. Additionally, the differential metabolites in the SD-M and LD-M groups were closely related to carbon metabolism, TCA cycle, and pyruvate metabolism. Moreover, carbon metabolism, TCA cycle, and carbon fixation in photosynthetic organisms were observed in the differential metabolites between SD-M and SD-D. Interestingly, there was a common metabolic pathway among the different groups: the TCA cycle.
Combined analysis of transcriptomic and metabolomic data
A combined analysis was conducted based on differential metabolites and DEGs. The differential metabolites of the different photosensitive genotypes (M51 and D32) of Panicum miliaceum L. were significantly different in the negative mode (Fig. 7). The differential metabolites of the M51 Panicum miliaceum L. genotype were mainly d-glucose and those of the D32 Panicum miliaceum L. genotype were primarily L-malic and fumaric acids, which could assist in identifying the photoperiod sensitivity of Panicum miliaceum L..
The number of common KEGG pathways for both differential metabolites and DEGs in the comparison groups of LD-D vs SD-D and LD-M vs SD-M were 16 (Table 2), with two common metabolic pathways in the LD-D vs. SD-D comparison group, which included carbon fixation in photosynthetic organisms as well as alanine, aspartate, and glutamate metabolism. There were five common metabolic pathways between the LD-M and SD-M groups: alanine, aspartate, and glutamate metabolism; pyruvate metabolism; glycolysis/gluconeogenesis; the pentose phosphate pathway; and butanoate metabolism. In summary, the DEGs and differential metabolites of the photosensitive M51 Panicum miliaceum L. and photoinsensitive D32 Panicum miliaceum L. genotypes were both significantly enriched in alanine, aspartate, and glutamate metabolism; and the DEGs and differential metabolites in the photosensitive (M51) Panicum miliaceum L. were enriched in four unique pathways, containing pyruvate metabolism, glycolysis/gluconeogenesis, pentose phosphate pathway, and butanoate metabolism; as well as the DEGs and differential metabolites in the photoinsensitive (D32) Panicum miliaceum L. were associated with the unique pathway of carbon fixation in photosynthetic organisms.
Correlation analysis of the DEGs and differential metabolites in the pathway enriched by photosensitive M51 and photoinsensitive D32 showed that these metabolites were involved in the synthesis and decomposition of organic acids in the negative ion mode, and the main metabolites were L-malic and fumaric acids. In the positive mode, the main metabolites were brucine and loperamide, which are involved in lipid synthesis and decomposition. Additionally, LHCA6 (light-harvesting complex I chlorophyll a/b binding protein) was down-regulated in the M51 Panicum miliaceum L. genotype, while the cytochrome psbE gene was up-regulated; as well as in the D32 Panicum miliaceum L. genotype, the expression levels of amino acid transferase At3g08860, acetaldehyde dehydrogenase ALDH2B4, psbA, psaI, and psaC genes were significantly up-regulated, whereas the expression levels of IBR3 and ALDH6B2 dehydrogenase were significantly down-regulated.
To screen the differentially upregulated and downregulated genes in the LD-D vs. SD-D and LD-M vs. SD-M comparison groups, the DEGs and differential metabolites that could accurately identify different photoperiod-sensitive Panicum miliaceum L. were mined and combined with the metabolomic data. A total of 18 genes were found to be significantly involved in the differential expression between the photoinsensitive D32 and photosensitive M51 Panicum miliaceum L. genotypes (Fig. 8), and candidate gene36259 (name: C2845_PM11G01290), which was annotated as V-type H+-transporting ATPase subunit B (ATP6B), significantly increased the levels of L-malic and fumaric acids.
qRT-PCR validation
To verify the reliability of the transcriptomic data, 10 genes involved in photosynthesis, carbon fixation in photosynthetic organisms, and glycerophospholipid, pyruvate, alanine, aspartate, and glutamate metabolism were randomly selected for qRT-PCR analysis. The expression trends of the 10 genes in the different combinations were consistent with those of the transcriptomic data (Fig. 9A), with a consistency rate of 100%, indicating the reliability of the transcriptome sequencing results.
In addition, the expression of the candidate gene (C2845_PM11G01290) over a 24 h time-period was determined in two types of Panicum miliaceum L.. It was found that with an increase in light time, the expression of C2845_PM11G01290 first increased, decreased, and then increased again, and the degree of photosensitivity in Panicum miliaceum L. (M51) was more significant than that of photo-insensitive Panicum miliaceum L. (D32) (P < 0.05, Fig. 9B). In the 24 h time-period expression analysis, the peak expression levels primarily occur between 18 and 21 h. Furthermore, the gene expression level of D32 is significantly lower than that of M51, suggesting that the impact on plants is mainly concentrated within the 18 h-21 h time frame. Although the ATP6B gene expression level of D32 (light-insensitive) is higher than that of M51(light-sensitive) at 0 h, 3 h, and 12 h, its influence on the growth and development of different materials is relatively minimal. Throughout the entire 24 h candidate gene expression trend, the magnitude of change in D32 is significantly lower than that of M51, which is consistent with its phenotypic changes (Table 3).