2.1 Global analysis of dynamic changes during quinoa seed germination by transcriptomic and metabolic profiling.
For plant, seed germination is a critical event in life cycle which is comprised of multiple steps. To better understand physiological changes and establish the platform for further omics studies, we performed the seed germination assay in quinoa dry seeds, and the dry seed was designed as the first stage in our experiment. Seed germination begins with a rapid water uptake indicating by increase of fresh weight at first 12 hours after seed imbibition. Subsequently, the seed size along with the fresh weight continuously increased, promoting radical emergence in the third stage of seed germination (Fig. 1A and Fig S1A). Following the emergence of quinoa seed radical, hypocotyl was elongated to next stage. In the final stage, cotyledons gradually unfolded to facilitate the establishment and growth of green seedling (Fig. 1A). Based on the physiological and morphological divergence during seed germination, we collected quinoa seed germinating sample at distinct five stages including dry seed (stage Ⅰ), imbibed seed (stage Ⅱ), radical emergence (stage Ⅲ), hypocotyl elongation (stage Ⅳ) and cotyledon expansion (stage Ⅴ), and performed omic studies to study molecular dynamics behind (Fig. 1A).
To identify key genes involved in regulating quinoa seed germination, we firstly carried out RNA-seq analysis in these five stages samples. After filtering out low-quality reads, we obtain a transcriptomic data with high quality with average 20.65 million paired-end clean reads, among ~ 90% of which successfully mapped to reference genome. The GC contents (43.63%-47.49%) and high Q30 value (92.02%-94.93%) enabled the next differential expression analysis (Table S1). Firstly, we used principal component analysis (PCA) to verify the relationship of the distinct five stages during quinoa seed germination. The PCA result showed a high similarity between three biological replicates, and presented significant large variations among different stages, expect for a partial overlap between stage Ⅱ and Ⅲ (Fig. 1B). Secondly, by setting a P value (< 0.05) and log2 of fold change (> 1 or < -1), we identified thousands of differentially expressed genes (DEGs) involved in quinoa seed germination. A Venn diagram showed the identified DEGs between any two neighboring stages, indicating the stage specificity of quinoa seed germination (Fig S1B). Finally, we counted the numbers of up- or down-DEGs by comparation between any two neighboring stages. Thousands of up- or down-regulated genes are identified between different stages of seed germination indicates specificity of each stage, while only a small number of DEGs identified in stage Ⅱ versus stage Ⅲ suggesting the similarity of the two stages (Fig. 1C).
In order to better explore the dynamics during quinoa seed germination from metabolic level, we next perform a metabolomic profiling analysis by Gas Chromatography-Mass Spectrometry (GC-MS) in the five distinct stages samples. PCA was conducted and revealed a significant separation among the different stages, indicating the metabolic changes occurred in transition of seed germination (Fig. 1D). The number of detected metabolite peaks in each stage and differentially metabolites in any two stages were counted after processing of the raw GC-MS data (Fig. 1E). A Venn diagram showed the differentially metabolites between any two neighboring stages indicated the stage specificity of quinoa germination (Fig S2A). The significantly differentially metabolites were imported into KEGG enriched analysis to generate the metabolome view (Fig S2B). KEGG analysis of differentially metabolites between neighboring stages exhibited largely difference, provides important information with transcriptome data in further exploration of dynamics of quinoa seed germination.
2.2 Phytohormones signals play important roles in early quinoa seed germination.
To understand the global expression patterns during quinoa seed germination, we tried to map the processes by functional annotating the DEGs against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. In this study, the KEGG analysis of DEGs between neighboring stages exhibited largely difference, suggesting multiple stage specific processes arose during the rapid transition of quinoa seed germination (Fig S1C). Within the KEGG analysis between Stage Ⅰ versus Ⅱ, the first period that dry seed began to germinate, “plant hormone signal transduction” was significantly enriched, following by other processes such as “starch and sucrose metabolism” (Fig. 2A). It has been well demonstrated that abscisic acid (ABA) plays a central role in regulating seed germination in plant , therefore we firstly analyzed the content of ABA in germinating quinoa seeds by enzyme linked immunosorbent assay (ELISA), and the result showed that dry seed (stage Ⅰ) obtained a highest level of ABA, and significantly decreased in the stages afterward during germination (Fig. 2B), suggesting that ABA may play a conserved role in inhibiting seed germination as in other plant species. According to the genetic and functional studies in Arabidopsis, the important components in ABA biosynthetic pathway contain ZEP/ABA1 encoding zeaxanthin epoxidase which catalyzes zeaxanthin to all-trans‐violaxanthin, NCEDs encoding 9‐cis‐epoxycarotenoid dioxygenase that oxidase both 9′‐cis‐neoxanthin and 9′‐cis‐violaxanthin, and cytosolic ABA2 and AAO3 encoding short‐chain alcohol dehydrogenase and abscisic aldehyde oxidase respectively to convert xanthoxin into ABA [18, 19]. Catabolism of ABA mediated by CYP707As encoding cytochrome P450 monooxygenase also determines the ABA level . We used a heatmap analysis to see the transcript pattern of DEGs involved in ABA metabolism, several genes annotated as CqABA1, CqNCED5 and CqNCED6 display a higher expression level in dry seed comparing to germinating seed, though an increase of transcript level of CqABA1 and CqNCED5 were observed in final stage during seed germination (Fig. 2C, Table S3). Besides, CqCYP707A1 exhibited a lower expression level in dry seed but increased in the after stages, which may contribute to the decrease of ABA content during seed germination (Fig. 2C, Table S3). Other components like CqABA2, CqCYP707A2 possibly have distinct functions in regulating ABA metabolism based on their different expression patterns (Fig. 2C, Table S3). Among these genes, the transcript level of two important ones in this process, CqABA1 (AUR62001926) and CqCYP707A1 (AUR62030408) were further confirmed by qRT-PCR analysis (Fig. 2D). Finally, we identified the DEGs encoding ABA signaling core components, CqPYLs encoding ABA receptors, CqHAIs / CqAHG1 encoding PP2C protein phosphatases, and CqSnRK2s encoding SnRK2 protein kinases . The relative low transcript levels of CqPYLs and high level of CqPP2C suggesting that the ABA signaling pathway tends to be silenced in dry seed although a high content of ABA in seeds. Moreover, the differential of expression level or patterns of CqPYLs suggesting the diversity of PYLs, which was consistent with cases in other plant species (Fig. 2E, Table S3) [22, 23].
Besides ABA, we also analyzed DEGs associated with other plant hormones, including gibberellin (GA) (Fig S3A), auxin (Fig S3B) and cytokinin (Fig S3C). Gibberellin is another central hormone that has been widely recognized in promoting seed germination . The transcript levels of genes participating in gibberellin biosynthetic pathway like CqGA20OX and CqGA3OX and gibberellin receptor CqGID1 were significantly up-regulated in the stage of radicle emergence, indicated an important role of GA in promoting seed germination (Fig S3A). A cluster of genes involved in other hormones like auxin and cytokinin were also enriched, such as auxin related IAA and GH3 family genes, cytokinin synthase and ARR regulators, suggesting these hormones possibly play functions in regulating quinoa seed germination though it was not thought as important as ABA or GA in this process (Fig S3). These results indicate that ABA and GA signaling and their potential integration with other hormonal signaling are required and essential during seed germination in quinoa.
2.3 Nutrient and energy metabolism was activated during quinoa germination.
Metabolic activities in dormant seeds are weak or silenced until seed imbibition occurs to broke dormancy and began seed germination . Once the germination was activated, seed stored reserves including starch, sucrose, proteins, and lipids are mobilized during the transition to enable seedling establishment, and starch and sucrose metabolism likely happened in the early stages during quinoa seed germination to provide nutrient and energy (Fig. 2A). In order to identify important genes functioning in promoting seed germinating, we searched the major components in starch and sucrose metabolism pathway (Fig. 3A). Genes annotated as CqAMY1 and CqBAM3 encoding alpha- and beta-amylase respectively were identified and showed significantly increasing after seed germinating, indicating their roles in leading starch converse to glucose and maltose respectively (Fig. 3B, Table S3). Three invertase candidates that degraded sucrose to glucose and fructose displayed similar pattern, and the up-regulated transcript level of CqSUTs encoding sucrose-proton symporter suggesting that exportation of sucrose is also activated in the beginning of seed germination, since sucrose is participating in other process and function in the normal energy provision (Fig. 3B, Table S3). The increase of transcript levels of CqBAM3 (AUR62007199), CqINVB (AUR62041914), CqFRK4 (AUR62023862), and CqSUT1 (AUR62004667) were further verified by qRT-PCR assay (Fig. 3C). Consistent with expression data, starch content decreased gradually during germination, and sucrose content changed rapidly, used for energy provision in time (Fig. 3D). Glucose and fructose content were significantly enhanced at the imbibed seeds, glucose-6-phosphate and fructose-6-phosphate content were continues increased during quinoa germination, used for glycolysis and tricarboxylic acid (TCA) cycle metabolism (Fig. 3D). The result demonstrated that starch and sucrose metabolism provided the main energy at the very early stages during quinoa seed germination.
2.4 Remodeling and modification of cell walls are important during seed germination
To further study the molecular dynamics occurring during quinoa seed germination, Gene ontology (GO) analysis was conducted, and cell-wall remodeling and modification associating processes were enriched in the intervals of dry seeds versus seeds imbibition (Fig S4). Within this GO analysis, cell wall remodeling associated processes including “glucan metabolic process” and “cellular glucan metabolic process” in biological process group, together with “xyloglucan-xyloglucosyl transferase activity” and “transferase activity” in molecular function group, as well as “cell wall” in cellular components group were significantly enriched (Fig S4). Plant cell walls are composed of complex matrices including cellulose, glucans, hemicelluloses and pectins, which provide the mechanical properties of cell and tissue . Besides, the primary walls controls cell growth and contributes to the energy metabolism for its high carbon fixation, so the cell-wall recycling by degradation, reorganization, and modification is important during fast growth phase such as seed germination .
In order to better verify the molecular regulation of cell-wall remodeling during quinoa seed germination, we examined the DEGs involved in the multiple processes including genes functioning in degradation of primary cell wall. DEGs annotated as CqFUC95A encoding α-fucosidase and CqBGAL encoding β-galactosidase are significantly up-regulated right after seed imbibition, which has been characterized as xyloglucans degradation associating enzymes in arabidopsis (Fig. 4A, Table S3). Another cluster of DEGs related to pectin degradation shows similar pattern during this transition, including genes encoding pectin methylesterase (PME), pectin methylesterase inhibiter (PMEI) and pectin lyase (PEL) (Fig. 4A, Table S3). Besides, genes associated with cellular xyloglucan metabolic such as xyloglucan hydrolase, xyloglucan endotransglycosylase, and xyloglucan:xyloglucosyl transferase were highly expressed within the intervals of radicle emergence, which catalyze the remodeling and mobilization of xyloglucan chains in hemicellulose, weaken cellulose microfibrils, promote radical protrusion (Fig. 4A, Table S3) . Genes encoding UDP-glycosyltransferases (CqUGTs), catalyzed the formation of glycosidic bonds by using nucleotide sugars as monosaccharide donor, promoting the synthesis of cell wall polysaccharides, was also significantly enriched within the intervals of radicle emergence (Fig. 4A, Table S3) . These result from global transcriptomic aspect demonstrated cell-wall remodeling processes are significantly active, contributing to the rapid transition of early seed germination.
According to further confirm to transcriptomic analysis associating with cell-wall remodeling, four related genes including CqPMEI (AUR62041471), CqUGT85A2 (AUR62027785), CqXTH8 (AUR62012652) and CqXTH23 (AUR62018298) were chosen for qRT-PCR verification, which showed significantly up-regulated at the imbibed seeds (Fig. 4B). Consistent with expression data, our metabolic profiling analysis showed cell wall sugars are increased during seed germination, such as fucose, galactose and xylose (Fig. 4C). The dynamic change of these metabolites suggested that activation of cell-wall remodeling pathway not only speeds the construction of cell wall but also participates in metabolic recycling associating with energy metabolism and multiple primary and secondary metabolism (Fig S1B).
2.5 Photosynthesis process was highly activated after the stage of hypocotyl elongation.
Quinoa cotyledons gradually unfolded after the hypocotyl elongation. Cluster and KEGG enrichment analysis showed that “Photosynthesis proteins”, “Photosynthesis - antenna proteins”, “photosynthesis”, and “Carbon fixation in photosynthetic organisms” were significantly enriched not only in transition of radicle emergence (stage Ⅲ) to hypocotyl elongation (stage Ⅳ) but also in the last cotyledon expensed stage (Fig. 5A). Most genes involved in photosynthesis including light reaction and calvin cycle were induced at the stage of cotyledon expansion (Fig. 5B, Table S3). In the post germination stages, the up-regulated DEGs associating light reaction includes genes encoding the subunit and light harvest of photosystem Ⅰ and photosystem Ⅱ respectively, and also several genes are annotated linking to ATP synthase complex and electron carrier or transfer (Fig. 5B, Table S3). Similarly, DEGs annotated as functional components in calvin cycle reaction showed highly expressed at the last stage (Fig. 5B, Table S3). These indicated that subsequent quinoa growth depends not only on the mobilization of storage reserves, but also on photosynthesis. However, the transcript levels of most genes involved in this process exhibited a significant difference between stage Ⅲ and Ⅳ, suggesting the activation of photosynthesis is likely taken place in the stage closely followed radicle emergence. We selected six genes involved in photosynthesis process to do the qRT-PCR analysis, and the substantial increase of these gene in seedling stage comparing stage before (Fig S5), indicating that photosynthesis play a critical role in quinoa post germination and the following green seedling establishment.
2.6 Dynamic metabolism of amino acids happened along with quinoa seed germination.
Amino acids, as one of main metabolic products, serve as important nutritional resources during quinoa germination, provide sufficient raw material for protein synthesis . Quinoa has been recognized as an excellent source of dietary proteins because of the high level of protein and well-balanced amino acids composition, it exhibited relative higher lysine concentration than other cereals [31, 32]. Besides, quinoa is rich in histidine, which is an essentially amino acid for infant . Here, lysine and histidine were significantly accumulated at the stage of hypocotyl elongation. Moreover, most other amino acids content including glycine, alanine, asparagine, glutamine, serine, threonine, cysteine, methionine, isoleucine, leucine, valine, phenylalanine, and tyrosine was significantly increased at the stage of cotyledon expansion, which was necessary for protein biosynthesis during the rapid seedling development (Fig. 6A).
As a non-protein amino acid, γ-aminobutyrate (GABA) was widely involved in signal transduction or regulatory pathways, taking multiple functions under non-stressed and stressed conditions in plants [34, 35]. GABA metabolism through the GABA shunt provides a source for carbon skeletons and energy for down-stream biosynthetic pathways, its accumulation is associated with the activity of glutamate decarboxylase (GAD), GABA transaminases (GABA-T), glutamate dehydrogenase (GLDH), and polyamine oxidase (PAO) [36, 37]. In this study, we first analyzed the content of GABA and found it was significantly accumulated in the late stages during seed germination, while it showed not different in the early stages (Fig. 6B). Then we analyzed the DEGs associating with GABA metabolism and found 10 genes including CqGAD, CqGABA-T, and CqPAO, which were significantly up-regulated at the stage of hypocotyl elongation (Fig. 6C, Table S3). Subsequently, two genes annotated as CqGAD1.1 (AUR62013119) and CqGAD1.2 (AUR62010123) were chosen for expression verification, which were highly expressed at the stages of hypocotyl elongation and cotyledon expansion (Fig. 6D). The result suggested that GABA may play an important role in the regulation of quinoa germination and the transition to seedling establishment.