The rate of seeds germination and seedlings growth is extremely fast in pearl millet
In the studies of seed germination, we found that pearl millet seedlings grow very fast under suitable temperature and light conditions (Fig. 1, Figure S1 and Table S1). We speculated that a series of rapid physiological and biochemical reactions occurred during the very early stage of seedlings development due to which its growth increases. In order to study the reasons for the fast germination rate, we focused our attention on 5 time points in the early germination period: 1. the dry seed. 2. 2 hours after imbibition (still in seed form). 3–5. 24, 36 and 48 hours after imbibition (radicles and germs appeared). We measured the length of germs and radicles of seedlings at 24, 36 and 48 hours after imbibition to characterize the growth rate of seedlings.
The length of germ reaches one-half of the seed length and length of radicle reaches the seed length is regarded as germination. The results showed that the rate of germination in pearl millet seeds is very quick (Fig. 1, Table S2). At 24 h or even earlier than 24 h after seed imbibition, the length of germs and radicles have reached the germination standard, and growth rate of radicles was faster than that of germs, especially during the period from 24 h to 36 h (Fig. 1a). During the period of 24 h to 48 h, the seedlings experienced a very rapid growth process, and there were significant differences in the germ length and root length between among time points (Fig. 1b). These results suggested that the pearl millet seeds have gained some advantages after imbibition, which endows the seeds with ability of rapid germination and seedling growth. This indicates that we can study the transcriptome differences during this period in the next step, which will help to better understand the mechanism of pearl millet seed germination and seedling growth.
More DEGs in the radicles than in the germ
Through transcriptome sequencing, a total of 810,182,656 raw data were generated from 24 samples, ranging from 34402297 to 42543864. We uploaded the original data to NCBI database(PRJNA670183). After filtering the original data, we got 779,909,975 clean data, ranging from 33395607 to 41346841. The GC content of all samples ranges from 52.57–57.59%, Q20 ranges from 96.93–98.04%, and Q30 ranges from 92.01–94.66% (Table S3), indicating the high sequencing quality and the obtained data can be used for subsequent analysis.
We used the full-length transcriptome sequence of pearl millet as a reference sequence for alignment and gene expression calculation [6]. A great consistency between different biological replicates of each sample is a prerequisite to ensure the reliability of subsequent bioinformatics analysis results. Therefore, we calculated Pearson's correlation coefficient for different samples and found that the three biological replicates between each sample have good correlations, and the correlation coefficients are all greater than 0.9 (P value < 0.01) (Figure S2, Table S4). In order to understand the dynamics of gene expression changes in seed, germ and radicles at different time points, we divided the 16 comparison groups into four categories for differentially expressed genes analysis (Table 1). A total of 29,514 DEGs were identified in the germ, of which only 58 genes were shared among all time points, while a total of 30,263 DEGs were identified in the radicles, of which only 30 genes were shared among all time points (Fig. 2). It is worth noting that in both the radicles and the germ, in the 24HAI and 2HAI comparison groups (24HAIG:2HAIS and 24HAIR:2HAIS), there are more down-regulated genes than up-regulated genes, and the total number of DEGs were also the largest, while all other comparison groups have more up-regulated genes than down-regulated genes. This may indicate that the internal gene expression is very strong after the seed imbibition for 2 hours. We also found that, except for the 24HAI: 2HAI and the 48HAI: 36HAI comparison group after seed imbibition, the number of DEGs in the radicles was greater than that in the germ in other comparison groups. This indicated that the gene expression in the radicles was more active, which may explain why the radicles appear earlier than the germ, and the faster growth rate than germ. Since it was found in the morphological measurement that the elongation of the radicles was significantly greater than that of the germ at 36 h compared with 24 h, we checked the DEGs of the germ and the radicles in 36HAI:24HAI groups, and found that the number of DEGs in the radicles were 6644, which was higher than 3447 in the germ. Then we compared the germs and radicles at 3 time points and found that the number of DEGs between radicles and germ were 12697 in 36HAIG:36HAIR group, which was significantly higher than the 7239 and 8546 of 24HAI and 48HAI. This may explain why the elongation of the radicles is much greater than that of the germ at 36HAI.
Table 1
Summary of DEGs among 16 comparison groups
Sort
|
comparison groups
|
Number of up-regulated genes
|
Number of down-regulated genes
|
Total number of DEGs
|
Seeds
|
2HAIS:0
|
10595
|
5825
|
16420
|
Germs
|
24HAIG:0
|
8807
|
6468
|
15275
|
36HAIG:0
|
10026
|
7082
|
17108
|
48HAIG:0
|
11049
|
5879
|
16928
|
24HAIG:2HAIS
|
10043
|
11917
|
21960
|
36HAIG:24HAIG
|
2457
|
990
|
3447
|
48HAIG:36HAIG
|
805
|
513
|
1318
|
Radicles
|
24HAIR:0
|
9828
|
7324
|
17152
|
36HAIR:0
|
12646
|
6458
|
19104
|
48HAIR:0
|
12853
|
6806
|
19659
|
24HAIR:2HAIS
|
9703
|
11404
|
21107
|
36HAIR:24HAIR
|
4526
|
2118
|
6644
|
48HAIR:36HAIR
|
386
|
142
|
528
|
Germs:Radicles
|
24HAIG:24HAIR
|
3699
|
3540
|
7239
|
36HAIG:36HAIR
|
5538
|
7159
|
12697
|
48HAIG:48HAIR
|
4938
|
3608
|
8546
|
The regulation mechanism of seed germination is specific at different stages
In order to better identify the DEGs related to different time points and their expression trends in the radicles and germ, the standardized expression data of 24307 genes were analyzed by gene co-expression network from 24 samples (three biological replicates). A total of 19 modules were generated, and each color represents a module,which is a cluster of highly related genes (Fig. 3a and 3b). We found 4 modules with specific time or tissue expression, namely “midnightblue”, “cyan”, “turquoise” and “brown” (Fig. 3c). The brown module contains 1409 genes which are mainly expressed in dry seeds; the turquoise module contains 5577 genes which are mainly expressed at 2HAIS; the midnightblue module contains 902 genes which are mainly expressed in germ at 24, 36 and 48HAI; the cyan module contains 4498 genes which are mainly expressed in the radicles at 36 and 48HAI.
In order to further understand the biological functions of these 4 modules, we performed KEGG analysis on the genes in these 4 modules (Fig. 3d). The genes in the brown module are significantly enriched in alanine, aspartate and glutamate metabolism (ko00250), Pyrimidine metabolism (ko00240), Valine, leucine and isoleucine degradation (ko00280) and RNA polymerase (ko03020), etc. The genes in the turquois module are significantly enriched in 13 pathways including Plant hormone signal transduction (ko04075) and MAPK signaling pathway-plant (ko04016). The genes in the Midnightblue module are enriched in 11 pathways such as Photosynthesis-antenna proteins (ko00196), Porphyrin and chlorophyll metabolism (ko00860), Photosynthesis (ko00195), Carbon metabolism (ko01200) and Carbon fixation in photosynthetic organisms (ko00710). The genes in the Cyan module are significantly enriched in 17 pathways including Phenylpropanoid biosynthesis (ko00940), Flavonoid biosynthesis (ko00941) and Brassinosteroid biosynthesis (ko00905) (Fig. 3d, Table S4). It is worth noting that the pathways shared by genes in the four modules are very few (Figure S3, Table S5). The turquoise and cyan modules shared two pathways, namely taurine and hypotaurine metabolism (ko00430) and tryptophan metabolism (ko00380). Similarly, midnightblue and cyan modules also shared two pathways, namely biosynthesis of secondary metabolites (ko01110) and metabolic pathways (ko01100), while the brown module didn’t share pathways with any module. The above results indicated that the function of genes in the module has strong tissue and time specificity, and different genes are functioning at different stages. This indicated that exploring the regulation mechanism of each stage can help us better understand the reasons for rapid germination in pearl millet seeds.
Function analysis of candidate genes related to seeds germination and seedling growth in pearl millet
The signal transduction of gibberellin, auxin and cytokinin promotes the rapid germination of pearl millet seeds
Phytohormones are organic signaling molecules that are produced by plants through their own metabolism and induce obvious changes even at very low concentrations, and play a very important role in the regulation of plant growth and development. Plant hormones often activate the downstream gene expression through signal transduction pathways to regulate plant growth and development. Therefore, it is necessary to analyze the factors in these hormone signal transduction pathways.
GA is a type of diterpene compound, which plays an important role in the growth and development of plants, and has been proven by a large number of studies to regulate the process of seed germination. When GA works through the signal transduction pathway, the receptor GID1 first senses gibberellin, and then combines with DELLA protein to form a GID1/GA/DELLA complex, which relieves the inhibition of DELLA on key downstream regulatory factors such as PIF3 and PIF4, and then regulates various biological processes [31, 32]. Our results found that the expression of the two GID1 genes (i1_LQ_LWC_c23529/f1p0/1967 and i2_LQ_LWC_c18562/f1p4/2746) reached the highest at 2 h after seeds imbibition, and the same expression trend was also observed in the two PIF3 genes (i2_LQ_LWC_c21636/f1p4 /2470 and i2_LQ_LWC_c36807/f1p4/2205) and 3 PIF4 genes (i0_LQ_LWC_c2048/f1p68/923, i1_LQ_LWC_c35538/f1p6/1792 and i2_LQ_LWC_c83983/f1p0/2153) (Fig. 4b). Considering GA as the main hormone for seed germination, and the signal transduction pathway of this hormone is so highly active, we determined the GA content in the early stage after seed imbibition. We found that the content of GA in dry seeds was the lowest, began to rise after imbibition, and arrived the highest at 4HAIS. The difference between every two time points was extremely significant (P < 0.01) (Fig. 5, Table S6). This result showed that a large amount of GA was synthesized immediately after seed imbibition, and the downstream signal transduction pathway was also stimulated to promote seed germination rapidly.
Furthermore, auxin is the earliest discovered member of the plant hormones family. It is a general term for a class of compounds that include indole acetic acid (IAA) and have similar physiological effects to indole acetic acid. It is involved in many biological and physiological processes including growth and development, such as root and leaf [15, 17, 33, 34]. When auxin works through the signal transduction pathway, auxin receptor TIR1 first senses auxin, then mediates the degradation of Aux/IAA protein, and then mediates the expression of downstream genes by regulating ARFs transcription factors to promote cell growth and regulate plant growth and development. In the current study, we found that the TIR1 gene (i5_LQ_LWC_c8187/f1p3/5450) and two ARF genes (i2_LQ_LWC_c82062/f1p4/3002 and i2_LQ_LWC_c126242/f1p4/2422) were highly expressed at 2HAIS. In addition, some studies have shown that the downstream gene GH3 of this pathway was up-regulated by the action of auxin, but it involved the degradation of auxin, so it showed a negative feedback effect on auxin. It is generally believed that GH3 gene plays an important role in auxin homeostasis [35]. The SAUR gene is thought to be involved in auxin-regulated cell expansion, and it has also been found to be highly expressed in the elongated hypocotyl [36, 37]. In our results, we also found that two GH3 genes (i2_HQ_LWC_c98146/f9p2/2256 and i2_LQ_LWC_c131239/f1p1/2110) and two SAUR genes (i1_LQ_LWC_c13114/f1p10/1086 and i1_LQ_LWC_c13186/f1p28/1222) have similar expression patterns (Fig. 4a).
CTK has been involved in the regulation of plant cell division, growth and development of tissues, organs and individuals. The signal transduction pathway of cytokinin is firstly sensed by the histidine receptor kinase CRE1 (AHK2_3_4) to autophosphorylate histidine, and then the phosphate group is transferred to the aspartic acid residue in the self-receptive region. Then the phosphate group on the aspartic acid residue of the receptor is transferred to the histidine residue of the cytoplasmic histidine phosphorylation transfer protein (AHP). Finally, the phosphorylated histidine transfer protein enters the nucleus and transfers phosphate groups to A or B response factors (ARRs), among which B response factors (ARR-B) have transcription factor activity and can initiate downstream gene expression after phosphorylation [38–40]. Our research found that the expression levels of three CRE1 genes (i4_HQ_LWC_c31467/f3p1/4211, i4_LQ_LWC_c5323/f1p3/4657 and i4_LQ_LWC_c16458/f1p0/4224) and three ARR-B genes (i2_HQ_LWC_c64881/f4p3/2784, i2_HQ_LWC_c72282/f2p2/2653 and i3_HQ_LWC_c29456/f2p0/3081) reached their peak at 2HAIS (Fig. 4c).
In summary, we found that in the signal transduction pathways of gibberellin, auxin, and cytokinin, the expression level of genes that play a positive role reached the highest level at 2HAIS in the pearl millet. Therefore, we speculated that after the seeds absorb water for a short time, the hormone signal transduction in the seeds became active, which promoted the germination of pearl millet seeds.
The formation of photosynthetic system promotes the rapid growth of pearl millet seedlings
Photosynthesis plays a very important role in the growth and development of plants. It converts solar energy into chemical energy and inorganic matter into organic matter and provides energy to plants needed for growth. It includes two light system. Photosystem I mostly produces the negative oxidation-reduction reaction in nature, and to a large extent determines the amount of global enthalpy in the living system. Photosystem II produces an oxidant whose redox potential is sufficient to oxidize H2O, which is a very abundant substrate that can ensure an almost unlimited source of electrons for life on earth [41]. Both systems are multi-subunit supramolecular complexes, including a core complex and a peripheral antenna system [41, 42]. In plants, the peripheral antennas are all made up of the light-harvesting complex (LHC). LHCIs (Lhca) and PSI core form a PSI-LHCI complex, and LHCIIs (Lhcb) and PSII core form a PSII-LHCII complex. The antenna system has different pigment composition, so it has different light absorption characteristics [25]. The light-harvesting complex (LHCII) in PSII is the most abundant membranous protein on earth. It participates in the first step of photosynthesis, absorbs and transmits solar energy for photosynthesis on the chloroplast membrane, and plays a role in regulating photosynthesis and photoprotection [43, 44]. Our results found that in the photosynthesis pathway, a total of 23 genes were identified to exist in different photosystems, and their expression levels reached the highest at 36HAIG (Fig. 6a). In the photosynthesis-antenna protein pathway, in addition to LHCA5, the other four light-harvesting complexes in LHCIs (LHCA1,i0_LQ_LWC_c2012/f1p117/375; LHCA2, i1_LQ_LWC_c27204/f1p0/1130; LHCA3, i1_HQ_LWC_c39810/f24p5/1138; LHCA4, i1_LQ_LWC_c34601/f1p0/1459)were significantly enriched and expressed the highest at 36 h after seeds imbibition in the germ. LHCB1 (i1_LQ_LWC_c19196/f1p0/1725, i1_LQ_LWC_c40686/f1p0/1092 and i1_LQ_LWC_c42565/f1p0/1078), LHCB4 (i1_HQ_LWC_c36891/f14p0/1217) and LHCB5 (i1_LQ_LWC_c26257/f1p0/1255) in LHCⅡs also showed the same expression pattern (Fig. 6b). These results showed that the genes involved in photosynthesis and antenna protein genes in the germs have been highly expressed at 36 hours after the seeds imbibition. They generated a lot of energy for plant growth and utilization, and the antenna protein system promoted the transmission of solar energy and improved photosynthesis effectiveness of action. Therefore, we speculated that the rapid formation of the photosynthesis system helped the pearl millet to more effectively produce energy for growth and development, which is an important reason for the rapid growth of the seedlings in pearl millet.
Brassinosteroids promote radicles elongation
Brassinosteroid is a kind of widely distributed plant hormone, which plays an important role in almost all the growth process of plants, regulating the elongation and division of cells [45]. When rice lacks BR, its growth and development was affected, showing the stunted growth of plant [46]. The BR-insensitive mutants of Arabidopsis thaliana showed many defects during growth and development, including short plants and reduced apical dominance [45]. It was also found in Arabidopsis roots that the interaction of BR and auxin mediated by BRX (BREVIS RADIX), necessary for optimal root growth. The phenotype of the brx mutant is caused by the lack of root-specific BR. This defect affects about 15% of the root gene expression levels of all Arabidopsis genes, but the expression levels of these genes can be restored by BR treatment [47]. This suggested that the normal level of BR is essential for the development of plants, especially roots. In our research, we found that the key enzymes for biosynthesis with BR include CPD (i2_HQ_LWC_c41220/f2p0/2056 and i2_LQ_LWC_c11071/f1p0/2085), DET (i3_LQ_LWC_c34585/f1p1/4026) and CYP92A6 (i1_LQ_LWC_c8195/f1p3/1783 and i1_LQ_LWC_c36100/f1p0/1831) are all enriched and expressed in the radicles (Figure S4). In addition, D2 (i1_LQ_LWC_c2765/f2p1/2032) and CYP734A1 (i2_LQ_LWC_c108886/f1p0/2200), which inactivate BR through hydroxylation to maintain the steady state of BR, also had higher expression levels in radicles (Figure S4). In summary, the genes that synthesize BR and maintain BR homeostasis in the radicles were very active, which ensured the normal level of BR. We speculated that this may explain why the elongation of radicles was significantly higher than that of the germs.