Transcriptomic changes reveal hypoxic stress response in submerged seeds of maize ( Zea mays L.)

Maize is highly sensitive to waterlogging stress, and seeds fail to germinate under hypoxic conditions induced by sub-mergence, leading to severe yield losses. We conducted a comparative transcriptome analysis during the initial stages of seed germination, exploring aerobic and hypoxic conditions in two inbred lines, B73 and Okcheon Chal-1. Notably, significant differences emerged between aerobic and hypoxic conditions on the first day of germination, particularly in genes associated with fermentation and phytohormone regulation. However, consistent transcriptomic changes were observed in primary metabolic pathways such as glycolysis, the TCA cycle, and the pentose phosphate pathway. These differences strongly correlate with each other, illustrating the efficacy of the hypoxic response for survival in water. Furthermore, this suggests that germinating seeds serve as a promising model for studying plant hypoxia responses with controlled environmental conditions. Insights from this study contribute to understanding the fundamental mechanisms of hypoxia response and hold promise for developing strategies to cultivate waterlogging-tolerant maize cultivars.


Introduction
Maize (Zea mays L.) is sensitive to hypoxic stresses induced by waterlogging and flooding [1], resulting in significant yield losses during cultivation.Such hypersensitivity to hypoxia is specifically significant at early growing stages such as germination and seedling phases and severely limits the cultivation area of maize [2].
Most higher plants evolved as aerobic organisms with oxygen-dependent glycolysis pathways.Oxygen is essential for respiration and other metabolism, thus, oxygen deficiency leads to decreased ATP synthesis through the respiratory pathway and is related to the production of reactive oxygen species (ROS), which cause cell damages [3].Low oxygen levels inhibit plant growth and development across various stages of development and reduce mitochondrial respiration, and primary ATP synthesis becomes depending on fermentation [4].In hypoxia, most plants express adaptive mechanisms for low oxygen concentration including anoxic glycolysis, and fermentation.In this process, NADH is reoxidized to NAD + and produces lactate and ethanol.As a result, under the action of pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH), carbohydrates undergo fermentation, resulting in the conversion of CO 2 and ethanol [4].
Successful germination in hypoxia can be determined by whether it produces enzymes for starch degradation without oxygen or not [5].Unlike most cereals, including maize, wheat, barley, and oats, which require oxygen for germination, rice can germinate in anoxic conditions [6][7][8].During the last decade, many underlying molecular mechanisms enabling rice adaptation to anoxic conditions have been studied.An early response to hypoxic stress involves the differential regulation of transcription factors belonging to the ethylene response factor (ERF) gene family [9].Rice SNOKEL1 and SNOKEL2, belonging to the ERFs gene family, enhance tolerance to submergence conditions by inducing shoot elongation through the enhancement of GA levels and signaling under submerged conditions [10].EXPA7 and EXPA12 of the EXPA family, which are encoded by a large superfamily of Expansin, have been reported to elongate coleoptiles under hypoxia [11].Additionally, rice α-amylase (RAmy) gene is involved in submergence tolerance by inducing the accumulation of carbohydrate metabolism through encoding the α-amylase enzyme.
Similarly, diverse approaches have been applied to understanding hypoxia stress and response in maize and many related genes have been identified.Some of those genes, including ADH1 (alcohol dehydrogenase 1), PDC1 (pyruvate decarboxylase 1), and AOX1 (alternative oxidase 1), are related to cellular energy metabolism and have been revealed to play roles in hypoxia tolerance [1].In addition, stress-related transcription factors such as NAC, MYB, bZIP, and AP2/ERF are known to regulate physiological processes under hypoxic conditions, and their interactions contribute to stress tolerance.[12].In particular, NACs are key regulators in most abiotic stress responses, hypoxic tolerance is primarily induced through ERF and bHLH-related TF genes [13].
Understanding how maize seeds respond to hypoxia is crucial for identifying potential strategies to enhance their survival in such conditions.Waxy maize cultivars are widely cultivated especially in Asian countries and its cultivation area is expanding worldwide [14].While many studies focus on waterlogging stress, research using waxy maize seeds is limited, especially at the physiological and transcriptome levels.
In this study, we investigated the transcriptomic perspective of maize seed germination on the first day under hypoxic conditions and compared it to seeds germinating under normal conditions.By comparing the transcriptomic differences under hypoxic stress treatments in two maize inbreds, we aimed to gain insights into the genes and pathways involved in stress across comparative conditions, and to assess the expression of differentially expressed genes (DEGs) and metabolic changes in seeds under hypoxic stress.

Plant materials
Two inbred lines, B73 and Okcheon Chal-1 (OkC1), and a commercial cultivar, 'Sinhwangok' were used for this study.B73 is a typical dent type maize used for the first maize reference genome sequencing [15] and OkC1, a waxy maize, is a landrace in Korea [16].'Sinhwangok' is a flint type F1 hybrid cultivar [17].Freshly harvested and dried seeds were used for the all experiment.Seeds were obtained from self-pollination of plants.
Seeds were sterilized with 0.5% sodium hypochlorite for a minute at room temperature and washed with autoclaved distilled water five times.Seeds were imbibed at 20 °C for 24 h and moved to 25 °C on petri-dish with wet filter paper or in 50 ml conical tube with half of water for a day which represent air or hypoxic condition, respectively.For imbibition and hypoxia treatment, 25 ml TDW was applied for 10 seeds in 50 ml conical tube.Seeds were sampled at right after finishing imbibition (0D) and 1 days after moving to 25 °C petri-dish (A1D) or in water (W1D).Each sampling was triplicated for pooling of five seeds.

Seed germination and viability test
Seed vigor was tested according to protocol of International Seed Testing Association (ISTA) [18].Seed were put on double layers of wet filter paper at 25 °C for 7 days, and counted daily for germination.
The viability of embryos after hypoxic conditions was tested using 2,3,5-triphenyl tetrazolium chloride solution, following the protocol of ISTA [18].We observed sets of 5 OkC1 seeds and 5 Sinhwangok seeds for each condition, the seeds were initially soaked in distilled water for 24 (0D), 48 (W1D), and 72 (W2D) hours, respectively.After that, three-fourths of the embryo was longitudinally incised, they were placed in a conical tube filled with 0.1% 2, 3, 5-triphenyl tetrazolium chloride.The conical tubes were wrapped with aluminum foil to prevent light penetration and then incubated at 30 °C for 2 h.The surface of the seed section was pictured using a camera (Progress Gryphax, Jenopitk, Germany) and a Leica M80 Stereo Microscope (Leica, Germany).The average color of the embryo area of the pictures was obtained with the Adobe Photoshop PS software (Adobe, USA), and the RGB values of each embryo region were extracted using the color picker tool.The redness of the embryo region was calculated using as follows: Red ratio = R / (R + G + B) which is between 0 and 1, where values closer to 1 indicate that the color closes to red [19].

RNA extraction
The 18 samples (2 genotypes × 3 treatments × 3 biological replicates) were collected and immediately frozen in liquid nitrogen.Total RNA was extracted from the excised embryo of the seeds with RNeasy Plant mini kit (QIAGEN, Germany) with on-column DNase treatment according to the manufacturer's protocol.The quality and quantity of the RNA was measured using Nano Drop 2000 (ThermoFisher, USA) and Bioanalyzer 2100 (Agilent, USA).

Transcriptome analysis
RNA sequencing was performed with commercial service by Macrogen (South Korea).Sequencing libraries were generated using the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, USA), and the resulting fragments were amplified and sequenced using a NovaSeq 6000 platform (Illumina, USA) to generate 101 bp paired-end read data for each sample.The raw reads were filtered by Trimmomatic software (Ver.0.36) [20] with default parameters (ILLUMINACLIP:adapter:2:30:10 LEAD-ING:3 TRAILING:3 SLIDINGWINDOW:4:15 MIN-LEN:36).The clean reads were subsequently mapped on the reference genome version 4 (GCF_000005005.2_B73_RefGen_v4) with HISAT2 software v2.2.1 [21].The resulting sam files were converted into bam files and sorted using SAMtools software v1.17 [22].The transcripts were assembled by StringTie software v2.2.1 [23], and prepDE.pypython script was used for generating the count matrix for all identified transcript assemblies by samples.The read count for each sample was obtained from read mapping results and normalized to transcripts per million (TPM).The annotated genes and expression profiles from the transcriptome analysis are available as the supplementary data.

DEG identification and analysis
The DEG identification and analysis were conducted using the edgeR R package v4.0.16 [23].The read counts in the matrix were normalized by library size, and MDS plot were generated by plotMDS function.The genes with |log 2 fold change|≥ 1 and FDR ≤ 0.05 were extracted as DEGs for each comparison.To conduct DEG analysis with Entrez IDs, we loaded maize profile database from the AnnotationHub R package v3.10.0 [24].The two functions embedded in the clusterProfiler R package v4.10.0 [25] such as enrichGO and enrichKEGG were used for comparing enriched gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) between B73 and OkC1, respectively.The log 2 fold change values of enriched Entrez IDs were mapped into corresponding Z. mays KEGG pathway entries by pathview R package v1.42.0 [26].

qRT-PCR
Gene expressions of transcriptome data were validated using qRT-PCR.About 500 ng of RNA from each sample was used for reverse transcription with Superscript IV Reverse Transcriptase (Invitrogen, USA).The qRT-PCR was conducted on the Rotor-Gene Q (Qiagen, Germany) with the SYBR Green RT-PCR Kit (Qiagen, Germany) in 10 μl reaction volumes.The reaction conditions were as follows: initial pre-incubation at 95 ℃ for 3 min, followed by 45 cycles of amplification consisting of denaturation at 95 ℃ for 15 s, annealing at 60 ℃ for 15 s, and extension at 72 ℃ for 30 s; the melting curve was generated with a cooling step at 45 ℃ for 30 s and following heating up to 99 ℃.The gene expression level was calculated using the relative 2 − CT method.
The tested genes were randomly selected from DEG lists that were commonly expressed in the 'W1D vs A1D' of both inbred lines, and primers were designed using Primer3 (https:// prime r3.ut.ee/).GRMZM2G102471 (ubiquitin-conjugating enzyme) was used as a control for housekeeping gene [1].Primers used for qRT-PCR analysis are listed in Supplementary Table S1.

Maize seeds maintain germination ability in water for two days
We first tested the germination rates of the two inbred lines and the F1 cultivar.The soaked seeds in aerobic condition were started to germinate at 2 days and fully germinated at three or four days for both inbred lines and the F1 cultivar.However, in submerged condition (hypoxia), seeds were completely failed to germinate for a week and started to be decay thereafter.Maize seeds germinated in aerobic but not in hypoxic condition.
There were slight differences for germination time within individual seeds of each inbreds.To synchronize the seed condition, we tried to decide the duration of imbibition by checking the weight gaining during water absorption of the seeds.By measuring seeds weight gaining during absorption time it reached 80% of the maximum at 24 h (Fig. 1A; Supplementary Fig S1A).The weight gaining ratio was high until 24 h and the rate was slowed down.The seed weight reached at maximum after 72 h.Interestingly weight gaining was clearly different depending on maize lines.The flint F1 hybrid seeds absorbed water 43% of their dry weight and the two inbreds gained 58% and 69% of their dry weight.Since water content in dry seeds were quite similar the gaining weight differs largely depending on kernel texture.The waxy OkC1 absorbed more water than the dent B73 and the flint Sinhwangok.According to the water absorbing time 24 h imbibition was regarded as 0D control.Seeds were imbibed for 24 h to synchronize their further response.
Next, we tested how long the seeds can survive in water.For that we submerged seeds in different days and move the seeds to aerobic condition to check whether the seeds able to germinate or not.When the seeds in water for a day and were moved to aerobic condition, most of them were germinated in two days showing the seeds in hypoxic condition were still able to germinate at least a day (Fig. 1B; Supplementary Fig S1B).Germination ability was dramatically decreased after three days in water showing the seeds are severely damaged in three days of hypoxic condition (Fig. 1B).
When embryos were stained with TZ solution, the colors, indicating viability, were significantly decreased (Fig. 1C, D; Supplementary Fig S1C, D).The TZ test showed that even in W1D and W2D seeds are still able to germinate if they come back to aerobic condition but the viabilities of the embryo are started to be affected.Thus we decided to pick W1D sample for further analysis, in which dramatic physiological changes for surviving could be observed by comparing A1D seeds samples.

Transcriptome profiles reflect stress conditions and genotypes
We obtained 58.6 million reads on average ranging from 44.5 to 69.9 million reads of raw data for the 18 samples.The portion of Q20 reads were 98.54% on average with a minimum of 97.94% which was good enough for the further analysis (Supplementary Table S2).The average mapping rate was 94.14% in which B73 samples (96.87%) have significantly (p < 0.01) higher mapping rate than OkC1 (91.40%).This result is caused by genetic difference of OkC1 compared to B73, however the mapping rate is still high enough for the following transcriptome analysis (Supplementary Table S3) [16].
When the 18 samples were plotted with PCA for their transcriptome profile, the three biological replicates tend to be located closely each other.B73 samples were located lower part of the plot whereas the OkC1 located upper part of the plot showing genetic distinction of the two inbred lines.In addition, A1D and W1D treated samples were located to the left compared to the 0D samples on the right suggesting different treatment caused similar transcriptome changes for both inbreds (Fig. 2A).Treatment causes more distinction among samples rather than the genetic distance of the samples since the same treatment samples cluster closer rather than the same inbreds.Number of DEGs were commonly higher in ' A1D vs 0D' compared to 'W1D vs 0D' for both inbreds (Fig. 2B).DEGs between two inbred lines having same treatment showed relatively small number of DEGs showing different treatment have more effect on transcriptome changes rather than genetic difference.There were higher number of DEGs for ' A1D vs 0D' compared to 'W1D vs 0D' showing likely reflecting more active physiological changes in aerobic than hypoxic condition.The commonly detected DEGs in both inbreds for ' A1D vs 0D' and 'W1D vs 0D' which is 3,597 and 1,609, respectively, were focused on further analysis (Fig. 2C, D; Supplementary Fig S2 ; Supplementary Data).In addition, 276 DEGs from the comparison between W1D and A1D were also investigated (Fig. 2E; Supplementary Table S4).

Hypoxia response initiated during imbibition of maize seeds and enhanced in water
GO enrichment test showed significantly enriched GO terms both for A1D and W1D and additional GO terms enriched for both of the two inbred (Fig. 3).The presence of 'aerobic respiration' and 'response to reactive oxygen species' indicate that the imbibed seeds have already responded to hypoxic stress and after moved to aerobic condition, A1D samples reflect normoxia response (Fig. 3A; Supplementary Fig S3A ).Lipid reserve mobilization is known to be an important process for seed germination [27], in this study we could detect many genes have been changed during a day of germination even though the actual germination were shown at the next day suggesting lipid mobilization is one the early change during germination.The other significant change is an amino acid metabolism.Amino acid is important not only for building blocks for newly differentiated tissue during germination but also precursors for the phytohormones such as GA and ABA.In W1D samples, prolonged exposure to hypoxia triggers enhanced fermentation, as well as the conversion of polysaccharide and lipid metabolism (Fig. 3B; Supplementary Fig S3B).Notably, lipid metabolism emerges as a significantly affected pathway under hypoxic conditions.Additionally, glossy13 and callose synthase were identified as differentially expressed genes (DEGs) in both inbreds.DEGs associated with DNA damage recovery were also identified.Further KEGG enrichment analysis confirms that the activated pathways primarily involve key metabolic processes, including glycolysis, amino acid biosynthesis, and others (see Supplementary Fig S4).
Transcriptome profiles of the A1D suggest that even in air condition there were some amount of anaerobic respiration is underway for germination.It suggests that oxygen supply is not enough to produce energy for germination.It is also possible that partial distribution of aerobic and anaerobic respiration is required for large amount of energy production during seed germination.In W1D sample, anaerobic metabolism seems to be more prevalent.Three fermentation related genes pyruvate decarboxylase, alcohol dehydrogenase, and lactate dehydrogenase are detected as DEGs (Fig. 4A).
Ethylene production was already started during imbibition period before treating plants to W1D and A1D.Under A1D, the expression levels of ETR (ethylene receptor) show similar levels of up/down-regulations, suggesting a moderation of ethylene production (Fig. 4B).Under W1D, a substantial increase in ethylene production could Fig. 4 Heat map illustrating changes of DEGs involved in hypoxia-related pathways in each condition and inbred lines.Expression levels of genes involved in the (A) fermentation pathway and (B) ethylene-related pathways in maize seeds.The heat map color gradient from white to red indicates expression levels ranging from minimum to maximum of the indicated gene be expected by the activation of the ethylene-responsive pathway.These alterations are supported by the up-regulation of ERF (Ethylene Response Factors), ETR, and related transcription factors like ABI4, RAP2 (Fig. 4B).
Exposure of plants to various stresses, such as waterlogging leads to the generation of reactive oxygen species (ROS), which in turn causes various imbalances affecting growth, cell cycle, and other cellular processes.DEGs were enriched in 'response to reactive oxygen species' and 'response to hydrogen peroxide' under A1D both B73 and OkC1, indicating an increase in ROS production after 24 h of imbibition.However, these GO terms were not enriched under W1D, suggesting that there were no significant changes in ROS production under continuous hypoxic condition.Under A1D, numerous peroxidase genes were significantly up-regulated, indicating their role in decomposing substrates using H 2 O 2 as a hydrogen acceptor.In contrast, the expression levels of peroxidase genes were lower under W1D, with OkC1 showing more downregulation than B73.Under W1D, SOD (superoxide dismutase) was down-regulated in B73, whereas no significant regulation was observed in OkC1.
The common DEGs in two inbreds were further investigated.Among 276 DEGs, 210 genes were down-regulated and remaining genes were up-regulated (Fig. 2C; Supplementary Table S4).In there, ABA, GA, and ethylene responsive genes are included.GA and ABA are related with germination and ethylene is hypoxia responsive plant hormone (Fig. 4B; Supplementary Fig S5).Three expansin-related genes and LNG2 [28] are all downregulated showing quantitative growth is started in A1D whereas W1D is not.
Collectively, the seeds in A1D normally shift to germination process even though it affected anaerobic conditions in imbibition stage.However, the seeds in W1D is continuously affected by hypoxia condition showed retarded growth and stress responses.

Genetic variation influences the responses of maize seeds to hypoxia
Similar to the common DEG numbers, or even higher numbers of inbred-specific DEGs were also detected (Fig. 2C-E).While enriched GO terms were quite similar to each other, some inbred-specific GO terms were also detected.Notably, we found that several fatty acid-associated genes, including GRMZM2G099696 (fatty acid biosynthesis1), GRMZM2G152105 (putative 3-oxoacylsynthase), GRMZM2G007757 (putative β-ketoacyl synthase), were particularly responsive to hypoxic treatment, showing higher expression in OkC1 compared to B73.These differences are predicted to be due to variations in kernel composition between the two lines.Unlike other varieties, waxy maize contains minimal amylose content, which could alter sugar levels and impact energy metabolism differently.
Previous studies have demonstrated the involvement of peroxisomes in energy generation through fatty acid metabolism, with increased β-oxidation activity and activation of peroxisome-related enzymes observed during glucose starvation [29,30].Thus, we further explored other DEGs in fatty acid-related pathways in our transcriptome data.Within the peroxisome pathway, DEGs such as GRMZM5G864319 and GRMZM2G052389, which function as peroxisomal acyl-coenzyme A oxidases, were relatively highly expressed in OkC1.DEGs like GRMZM2G071288, associated with the peroxisomal membrane family protein, were also identified.These findings suggest that in waxy maize, characterized by a deficiency in amylose compared to B73, lipids and proteins may act as essential processes.In addition, autophagy-related genes and GO terms were detected only in OkC1.It shows a significant impact of hypoxia on OkC1 compared to B73.

Confirmation of transcriptome data with qRT-PCR
Six genes were selected from the DEGs (W1D vs A1D), and their expressions were profiled with quantitative RT-PCR for both B73 and OkC1 samples.The selected genes included GRMZM2G133885, GRMZM2G075775, GRMZM2G168898, GRMZM2G359070, GRMZM2G306345, and GRMZM2G125775 (Supplementary Table S1).When the qRT-PCR expression profiles were compared with the RNA-seq data, the patterns were quite similar to each other, confirming the that RNA-seq data is reproducible with qRT-PCR (Fig. 5).

Discussion
Hypoxic stress can have detrimental effects on plant growth, development, and physiological processes [31].Modification of gene expression in plants under stress condition often confers resistance or adaptation mechanisms.Hypoxic condition significantly impacts most plant developmental stages and shifts the energy-related metabolisms from aerobic respiration to anaerobic fermentation [32].Previous studies have analyzed gene expression and hypoxic stress resistant loci in maize under abiotic stress using various methods [33,34].Experiments using seedlings have shown that, generally, genes associated with energy metabolism, including sucrose metabolism (sucrose synthase 4 and phosphoglucomutase), glycolysis (phosphofructokinase and triosephosphate isomerase), lactate fermentation (lactate dehydrogenase), and ethanol fermentation (pyruvate decarboxylase and ADH1), are strongly expressed under waterlogging condition [35].Furthermore, Feng et al. [33] demonstrated that genes such as PDC, ADH, and ALDH maintain high expression levels during alcohol fermentation in sweetcorn seedlings under waterlogging stress, thereby proving their role in regulating carbohydrate levels within the plant to enhance resilience under stressful conditions.In response to hypoxic stress in maize, there is an increase in ethylene production within the plant, and many ethylene-related genes and transcription factors have been shown to function as transcriptional activators to upregulate ADH and PDC [36].Unlike most studies that utilize maize seedlings for experimentation, this study employed seeds.By obtaining similar results to hypoxic stress-related research conducted with seedlings, it enabled a more efficient experimental process in terms of time, labor, and cost savings.Most importantly, hypoxic stress can be easily applied with minimizing unintended conditional variation during treatment.
The fermentation reaction is likely activated even under aerobic conditions during imbibition.We determined the imbibition time for 24 h based on the water absorption ratio.Generally, maize cultivation does not require imbibition or pretreatment as a priming process before sowing the seed on the field.However, for experimental purposes, seeds need to be stimulated to synchronize their response to environmental changes.Previous studies have imbibed maize seeds for as short a duration as possible, but there was no consensus for the duration of imbibition time [37,38].In this study, we estimate the duration by observing water absorption during imbibition.However, transcriptome analysis showed that 24 h slightly impacts their hypoxic stress response, indicating that imbibition should be shorter than 24 h.Further analysis is required for determine proper timing of imbibition.
Many genes involved in hormone response are detected in DEGs suggesting hormonal interaction plays a major role in hypoxia during germination.In addition, several transcription factors (TFs) related with hormone signaling pathways that induce hypoxic stress tolerance were detected in this study.TFs serve as key regulators of most abiotic stresses, including hypoxia.The majority of TFs belong to the NAC, MYB, bZIP, WRKY, and AP2/ERF families.It was observed that many ERF-related genes were up-regulated in W1D compared to A1D, while bHLH-related genes were down-regulated.
In this study, hypoxic conditions were induced by simply submerging seeds.By comparing these conditions with aerobic conditions, we could detect hypoxic response of the seeds' embryos.In the A1D sample, transcriptomic changes during germination were clearly observed, including alterations in cell proliferation, hormonal responses, glycolysis, among other factors.In contrast, seeds under hypoxic conditions exhibited distinct transcriptomic changes, primarily characterized by an enhanced fermentation metabolism.These Fig. 5 The qRT-PCR validation of the RNA-seq data.Expression of six randomly selected genes from the DEGs (W1D vs A1D).A Gene expression levels in the RNA-seq data.The y-axis represents the expression level (TPM) in two inbred lines under each condition, respectively.B Gene expression levels in the real-time PCR analysis changes were supported by the enriched GO terms (Fig. 3).In addition, hypoxia-related GO terms such as GO:0036293 (response to decreased oxygen levels) and GO:0070482 (response to oxygen levels) were also detected as the enriched GO terms.Overall transcriptomic changes in seeds show very similar to seedling or plants in hypoxia suggesting observing hypoxia response in germination stage could represent that of mature plant stage.Using seeds for studying hypoxia response would be efficient to make consistent environment repeatedly.From this study, we assume that the sensitivity to hypoxia in maize seeds is responsible for hormonal changes and fermentation metabolism.The effort to convert these metabolisms would be beneficial to improve resistance to submerging stress of maize.

Fig. 1
Fig. 1 Maize seeds were damaged after 1 day in water.A Weight gaining by water absorption rates of two maize inbreds for 5 days.B Germination rates were decreased as duration of hypoxic treatments were increased.C Visualization of viability in control and hypoxia-treated seeds.As the duration of the waterlogging treatment increased, the viability of the embryos decreased.Bar = 1 cm.D Quantification of seed viability.Red ratio = R / (R + G + B), the closer the value is to 1, the redder.Data are shown as mean ± SE and letters above bar indicate significant difference using one-way ANOVA and Tukey's HSD (p < 0.05)

Fig. 2
Fig. 2 Principal component analysis (PCA) and statistical analysis of differentially expressed genes (DEGs) (A) PCA of 18 RNA-seq samples based on their expression profiles.B The number of up-and down regulated genes identified in DEGs under each condition.Venn diagram (C, D) for aerobic condition and hypoxic condition specific DEGs and (E) for hypoxic condition specific DEGs compared to aerobic condition

Fig. 3
Fig. 3 Gene Ontology analysis of hypoxia-related DEGs in each condition.The enriched GO terms in Biological Process with the |log 2 fold change|≥ 1 and FDR ≤ 0.05.The number of genes and p-values for the enrichment test were indicated as colors.A A1D vs 0D, B W1D vs 0D