Integrated physiological and transcriptomic analyses responses to altitude stress in oat (Avena sativa L.)

Abstract

Background The oat is an annual gramineous forage grass with the remarkable ability to survive under various stressful environments. However, understanding the effects of high altitude stresses on oats is poor. Therefore, the physiological and the transcriptomic changes were analyzed at two sites with different altitudes, low (ca. 2,080 m) or high (ca. 2,918 m) respectively.

Results It was indicated that oats at high altitude showed higher levels of antioxidant enzyme activities and significant decreases in the non-photochemical quenching coefficient,chlorophyll content and stomatal density. Furthermore, oat yields were severely suppressed at the high altitude. RNA-seq results showed that 11,639 differentially expressed genes were detected at both the low and the high altitudesin which 5,203 up-regulated and 6,436 down-regulated. GO and KEGG annotations suggested that many differentially expressed genes be involved in pathways of cellular processes, metabolic processes and plant-pathogen interactions. Furthermore, 125 differentially expressed genes encoding TFs were involved in signal transductions by regulating the resistance genes in oats at high altitude. Some differentially expressed genes related to pigment metabolisms and photosyntheses were differentially expressed suggesting that these genes probably play responsive and regulatory roles in oats under the high altitude stress. Meanwhile, 273 differentially expressed genes related to hormone signaling pathways were significantly enriched in gibberellin, auxin, jasmonic acid and abscisic acid pathways indicating that these genes participated in the processes of oats responding to high altitude stresses.

Conclusion In summary, our study generated genome-wide transcript profile and may be useful for understanding the molecular mechanisms of Avena sativa L. in response to high altitude stresses.These new findings contribute to our deeper relevant researches on altitude stresses and further exploring new candidategenes for adapting plateau environment oat molecular breeding.

Background

Extreme environments provide natural laboratories for studies on the processes of speciation and adaptive evolution of organisms [1]. The Qinghai-Tibetan Plateau, the highest plateau in the world, plays an important role in climate changes in Asia and even the world [2]. In particular, the Qinghai-Tibetan Plateau environment is known for its harsh conditions, characterized by severe coldness, intensive ultraviolet radiations, hypoxia, poor soils and low CO₂ pressure [3, 4]. Therefore, the survival in Qinghai-Tibetan Plateau is very challenging for most organisms. Nevertheless, many plant species can thrive in the cold and hypoxic conditions in high-alpine areas [5, 6]. Generally, in response to the bioclimatic conditions, plants can make corresponding molecular and physiological changes[7]. For example, the high-altitudinal gradient can restrict plant growth and reproduction via strong solar UV-B radiation, resulting in a reduction of photosynthetic rates by bleaching chlorophyll a (Chl a) and damaging the photosynthetic apparatus[8]. As such, some alpine plants are able to activate antioxidants such as APX, CAT, GR, proline and abscisic acid to confer plants with the tolerance to the alpine environments [9–11].

In recent years, transcriptome sequencing has been proven to be an effective and efficient method for determining adaptive evolutions and differential gene expressions in high-altitude plants, e.g. Kobresia pygmaea, Potentilla saundersiana, Lamiophlomis rotate and Lobelia. Some studies demonstrated that alpine plants had various morphological and physiological response strategies to adapt to high-elevation environments. For example, researchers had revealed that some genes were significantly involved in energy metabolism, hypoxia response under positive selection and rapid adaptation [12–14]. However, up to now, few transcriptome-based investigations have been devoted to the molecular mechanisms of high-altitude adaptation in oats.

Oats have not only high yield, rich nutrition and good palatability, but also the characteristics of cold tolerance, drought resistance and strong adaptability. Oats can germinate when the temperature is 3 ~ 4 °C and seedlings can resist the low temperature of -3ཞ-4 °C. Oats are the main forage species in the Qinghai-Tibet Plateau where they are high-quality forages for both winter and spring feeding and disaster-preserving livestock in local farming and pastoral areas. Planting oat grass is of great significance for solving short pasture supply in winter and spring grassland and developing herbivorous livestock and animal husbandry[15]. The oat has also been identified as an excellent species that can adapt to various environmental stresses including drought, salinity and pathogen attacks. Oats make morphological and physiological changes to adapt to environments under abiotic stress[16, 17]. However, the adaptation mechanisms of oats in high altitude environments have been rarely studied.

In this study, the first step was designed to characterize the impact of altitudes on oat physiology and agriculture. It was showed that high altitude influenced photosynthetic ability, antioxidant enzyme activity, stoma aperture and agricultural parameters of oats. The second step, RNA-seq was employed to study the changes at molecular levels between low and high altitudes. According to extensive data analyses, many DEGs and metabolic pathways were identified and characterized which were involved in adaption of oats to high altitude stresses.

Results

1. High altitude environments induced physiological changes in oats

MDA is an important marker for the lipid peroxidation due to overproduction of ROS in the cell. As shown in Fig. 1A, the MDA activities of the high altitude oats were higher than those of the low altitude. In order to further exploring the oxidative damage of oats, the activities of SOD were investigated. Under high altitude conditions, the SOD activities of the enzymes increased significantly compared to those at low altitude (Fig. 1B). In addition, the high altitude induced the accumulation of soluble sugars in oats (Fig. 1C). The results showed that oats could improve the ROS scavenging system, MDA homeostasis and osmotic regulation system which could decrease the jury caused by thehigh altitude environments.

Moreover, the chlorophyll content and the NPQ were determined to compare the photosynthesis changes of oats in two different altitudes. As shown in Fig. 1D, E, the chlorophyll content and NPQ at the high altitude decreased and were significantly lower than those at the low altitude. This result indicated that there was a strong photosynthetic apparatus damage in high altitude conditions. Stomata control the carbon dioxide absorption and play a crucial role in photosynthesis. The density of stomata was measured. This result suggested that the stoma density had significantly decreased at high altitude (Fig. 1F, G).

2. The agronomic traits of Oats were remarkably affected by altitudes

Oats displayed significant differences from two altitudes in terms of plant height, stem/leaf ratio, crude fat and total hay yield. Under high altitude conditions, the plant height of oats was 104.7 cm which was significantly shorter than 126.9 cm at the low altitude (Fig. 2A). The stem/leaf ratio and crude fat at the low altitude were 1.18 and 2.7%, respectively which were significantly different from 1.05 and 1.7% at the high altitude, (Fig. 2B, C). On the other hand, compared with the low altitude, the high altitude exhibited remarkable decreases in hay yield (Fig. 2D). It was clear that oats altered their agronomic traits in two different regions. All these differences between the two altitudes could be the results of distinct modifications under atmospheric pressure, UV radiation, day length, temperature or the combination among all or some of these factors. Therefore, further research may be needed to illuminate the specific functions of these climatic factors in oats.

3. Transcriptome Profiling

To identify the transcriptomes and gene expression profiles of oats at low and high altitudes, six cDNA samples from oat leaves were prepared and sequenced using Illumina HiSeq 2000 platform. A total of 47–50 million raw reads were harvested from each cDNA library. After the low quality reads were removed, 39.49 Gb clean reads were obtained with an average of 6.66 Gb reads for each sample. The percentage of bases higher than Q30 in each sample was not less than 90.91% (Table 1).

To predict and analyze the functions of the unigenes, we carried out functional annotations by using BLAST against multiple databases such as Nr, Nt, KEGG, Swissprot, KOG, Interpro, and GO. A total of 11,639 unigenes were successfully matched up in the databases mentioned above. Among them, 11,507 (12.12%), 38,504 (40.55%), 12,044 (12.68%), 11,801 (12.43%), 12,154 (12.80%), 15.199 (16.00%), 49,383 (52.00%) unigenes were found in Nr, Nt, KEGG, Swissprot, KOG, Interpro and GO databases, respectively (Additional file 1: Figue S1). Moreover, a species distribution map was drawn (Fig. 3A). The results indicated that oats showed the most similar to Aegilops tauschii subsp. tauschii, followed by Brachypodium distachyon and Hordeum vulgare subsp. vulgare.

4. The Analyses Of Differentially Expressed Genes (degs)

To obtain the differential expression genes response to high altitude, the Fragments Per Kilobase of transcript, FPKM method was used to analyze the expression abundance of unigenes. As a results, a total of 11,639 differentially expressed genes (log 2 Fold Change > 2) were differentially expressed between the low and the high altitudes (Additional file 2: Figue S2, Fig. 4A). Among them, 5,203 DEGs were found to be up-regulated and 6,436 DEGs were down-regulated (Fig. 4B). Interestingly, the number of down-regulation genes were more than the up-regulation ones. It indicated that more genes were down-regulated in response to high altitude stresses.

5. Go And Kegg Analyses Of Degs

To investigate the functions of these DEGs under high altitude stresses, we performed GO annotation enrichment analysis using the software package top GO (Additional file 3: Figue S3, Fig. 5A). The results showed that 2,879 transcripts were characterized and annotated with the top GO terms for classified genes being “cellular process” (1,956 transcripts) for biological processes. Other categories with high numbers of genes included “metabolic process” (1,819 transcripts) and “biological regulation” (887 transcripts). These showed that substance synthesis and catabolism, cellular processes and physiological regulation responses to external stresses were involved in responses of oats to high altitude environmental stresses. In addition, 3,308 transcripts were mapped to interior cell components in which “cell” (2,348 transcripts) and “cell part” (2,314 transcripts) were the most represented categories. The mechanism by which oats responding to high altitude was closely related to the structural composition of their cells, cell membranes and organelles. As for molecular functions, 4,011 transcripts were significantly annotated with the “binding” (2,533 transcripts) and “catalytic activity” (2,478 transcripts) pathways having the most DEGs, respectively. These indicated that the non-covalent bonding of molecules and the catalytic activity of enzymes in biochemical reactions played important roles in the physiological adaptability of oats to high altitude stresses.

At the same time, the DEGs and the biological processes involved in altitude stresses were illuminated and all of the DEGs were queried in KEGG databases. In this study 4,831 transcripts were allocated to 30 pathways in KEGG databases (Additional file 4: Figue S4, Fig. 5B) while “Biosynthesis of secondary metabolites” (1,060 transcripts), “plant-pathogen interaction” (541 transcripts) and “RNA transport” (504 transcripts) pathways were mostly enriched followed by “phenylpropanoid biosynthesis” (390 transcripts), “plant hormone signal transduction” (259 transcripts), MAPK sigaling pathway” (253 transcripts) and “starch and sucrose metabolism” (215 transcripts). Interestingly most of the genes mapped in the first seven significantly enriched pathways had the trend in down-regulation. These results above indicated that various pathways were involved in responses of oats to different altitudes.

6. Analyses Of Transcription Factors (tf) In The Degs

TFs control the expression of numerous genes and play crucial functions in stress-induced signal transduction pathways. In this study, 125 TF genes were differentially expressed between two different altitudes (Additional file 5: Figue S5). Among them, 53 bHLH transcripts, 36 AP2-EREBP transcripts, 11 C₂C₂-CO-like transcripts were the top three most TF families. The rest of the transcription factors were ABI3VP1, C₂C₂-Dof, C₂C₂-GATA, bZIP, C₂H₂, ARF, ARR-B, BSD and C₂C₂-YABBY with the numbers of transcripts were 6, 6, 3, 3, 3, 1, 1, 1 and 1, respectively.These results indicated that the TFs above played critical roles in oats response to high altitude stresses.

8. Relative Degs Measured By Qrt-pcr

To further verify the accuracy of RNA-seq data and confirm the level of differential expression genes, we performed randomly qRT-PCR from ten genes induced by the high altitude and concluded two chlorophyll metabolism pathways and eight hormone-related DEGs as was shown in Table 2 and Additional file 10. The results revealed that the ten DEGs were up-regulated in accordance with the results of the RNA-seq analysis Fig. 6. The results suggested that RNA-Seq data be reliable and the above genes were involved in high altitude stresses.

Discussion

The Qinghai-Tibet Plateau itself has unique climate and geographical features which pose huge challenges to the survival of plants. Although plants cannot escape from unfavorable environments, they can change their morphological structures, physiological and molecular processes in which changes happen in endogenous secondary metabolite levels, ion homeostasis, gene expressions and protein activity regulations to adapt to different environmental conditions. Oats are an important source of food and feed in the Qinghai-Tibet Plateau and are beneficial to human health because they are rich in protein and minerals[18]. Now the data available on the molecular basis of oats response to Qinghai-Tibet Plateau conditions stress is not limited any longer with the development of high-throughput sequencing.

1. The adaptation of the oat physiological characteristics to high altitude environments

The increased levels of protective antioxidant enzymes could maintain the homeostasis of ROS. In this study, oats could regulate many physiological processes to adapt to high altitude stresses, e.g. increasing the activities of SOD (Fig. 1B) which suggested that oats can improve their ability to resist damages by establishing a better antioxidant enzyme system under high altitude stresses. The membrane lipid peroxidation product MDA was also one of the important indicators of cell damages which could bind to proteins and cause membrane protein denaturation [19]. Our result showed that oats suffered more severe damages to the cell membrane and antioxidant system in high altitude environments than those at low altitude (Fig. 1A). In an alpine environment, low temperatures could affect the osmotic pressure of plant cells. As shown in Fig. 1C, high altitude stresses induced the accumulation of soluble sugars in oats. Therefore, soluble sugars might play important roles in oat growth and development and responses to abiotic stresses, and eventually affect yield and quality. In addition, sugars acted as not only an essential source of carbon but also signaling molecules in response to the integration of information from environments as well as developmental and metabolic cues in plants [20–22]. Therefore, it could further improve the resistance of oats under low temperatures at high altitude by increasing the content of soluble sugars to avoid protein coagulation. Meanwhile, oat plants showed sharp decreases in chlorophyll content and NPQ at high altitude (Fig. 1D, E). The decreases of chlorophyll content might be because the enhancement of radiation could exacerbate the photooxidation process of photosynthetic pigments or restrain some synthetic processes to decrease the light energy absorbed by leaves at high altitude to avoid or reduce the radiation damages on plants [23]. The decrease in NPQ indicated that the plant did not dissipate excess excitation energy in the form of heat, thereby increased the damage on PSII by the accumulation of the excitation energy under high altitude stresses [24]. Moreover, the chlorophyll content and NPQ as photosynthesis markers were closely linked to yield which was probably ascribed to the reductions of both plant height and the ratio of stem/leaf, subsequently led to marked decreases in hay yield index (Fig. 2). In addition, as was shown on Fig. 2 high altitude conditions could regulate the development of oat plants, e.g. decreased the stomatal density to reduce water loss to adapt to high altitude stress [25]. This mightbe due to the water shortage caused by low temperatures which was also the result of adaptation to low temperature and strong radiation in high mountains.

When subjected to abiotic stresses, TFs could play an important role by binding to cis-regulated DNA elements in gene promoters and activating or inhibiting their expressions[26]. bHLH had been reported to directly bond the phytochrome action factor in the photoreceptor signaling network which specifically binded to phytochrome responsive gene promoter G-box regulatory elements in vitro[27]. In addition, overexpression of Arabidopsis bHLH TFs enhanced chilling or osmotic tolerance by reducing malondialdehyde content and accumulating ROS[28, 29]. AP2-EREBP and ABI3 TF could participate in the process of plant growth, development and respond to stresses by regulating the expression of downstream target genes[30, 31]. In our study, bHLH, AP2-EREBP and ABI3VP1 were the three most abundant transcription factors identified as DEGs in response to high altitude stresses. Moreover, many transcription factors containing C₂C₂ zinc finger domain were found including C₂C₂-CO-like, C₂C₂-Dof, C₂C₂-GATA and C₂C₂-YABBY. Researches had shown that they played vital biological functions in vegetative tissue development and multiple stress responses [32]. It could be inferred that bHLH, AP2-EREBP, ABI3VP1 and C₂C₂ TFs might play important roles in response to the high altitude stress. In addition, many other TF families were also identified to be differentially expressed such as bZIP, C₂H₂, ARF, ARR and BSD. This also explained that numerous TFs participated actively in the regulationof oats in response to high altitude conditions through different pathways.

3. High altitude affected the expression of genes related to pigment metabolism and photosynthesis

Plant pigments determine color variations of leaves which are complex biological processes. Among them, chlorophyll and carotenoid are two important antenna pigments in the photosynthetic system. Chlorophyll plays a central role in the process of absorbing and transmitting energy[33]. Carotenoids can protect chlorophyll from degradation as a major component of the photosynthetic apparatus and photoprotection system^[38]. Plateau plants can adapt to UV-B radiation by changing the content of photosynthetic pigments in leaves such as reducing the chlorophyll and carotenoid content[34]. Chlorophyll and carotenoid metabolisms are complex processes involving multiple enzymes. Comparative transcriptome profiling data for the low altitude and the high altitude showed that the expression of numbers of DEGs were significantly regulated including uroporphyrinogen decarboxylase, copropor phyrinogen III oxidase, protochlorophyllide reductase, chlorophyll(ide) b reductase and cytochrome c oxidase assembly protein subunit 15, which were key enzymes associated with the process of chlorophyll synthesis[35]. Meanwhile, our comparison of the DEGs involved in carotenoid biosynthesis showed that 32 genes were significantly down-regulated in high altitude (Additional file 3: Figue S3). It might imply that oats can adapt to UV-B radiation stress by changing the content of chlorophyll and carotenoids in leaves.

Chloroplasts are the main sites for photosynthesis which are comprised of membranes, thylakoid system, and stroma with functional units in it. In higher plants, the multi-subunit pigment–protein complexes (i.e., photosystem (PS) I, PSII, light harvesting complexes, cytochrome b6/f, and ATP synthase) are embedded in the highly folded thylakoid membrane where they are responsible for light absorption and energy transfer [36]. Strong radiation in high-altitude environments might have been related to thylakoid development and photosynthesis at the two different altitudes. Based on transcriptome data it was found that genes encoding photosystem II CP47 chlorophyll apoprotein, photosystem II oxygen-evolving enhancer protein, photosystem I P700 chlorophyll a apoprotein and photosystem I subunit PsaO were significantly regulated at high altitude. In higher plants, light-harvesting complex II chlorophyll a/b binding proteins that accumulate in the thylakoid membranes, where their proposed function is in photoprotection [37–39]. The chlorophyll a/b-binding proteins are correlated with photodamage in the PSII reaction centers [40]. We found that the proteins were significantly enriched suggesting that strong photosynthetic apparatus damage occur in high altitude (Table 3). These results were consistent with our previous photosynthetic physiological index analysis (Fig. 2B) which further demonstrated that the genes related to pigment metabolism and photosynthesis in oats could respond to high altitude stresses.

4. Identification of signal transduction genes responsible for high altitude stress

The different expression of genes involved in the signal cascade mechanism can affect the expression of genes participating in the formation of plant hormones such as auxin, ABA, ET, SA and JA. Theses hormones may amplify the cascades or initiate some new signaling pathways[41, 42]. It is now known that these phytohormones extensively regulate all aspects of plant stress responses ranging from signal cascade transduction to modifications in plant developmental processes[43]. In this experiment, GA, IAA and JA were the top three responsive hormone signal transduction pathways in oats and there were a number of DEGs involved in them. Studies had shown that the gibberellin receptor sensing the GA signal could activate the signaling pathway, thereby regulating the expression of downstream genes to affect plant growth and morphogenesis[44]. Totally 60 DEGs involved in the response to GA signal transduction pathway were identified as being differentially expressed, which encoded gibberellin receptor GID and DELLA proteins. This indicated that high altitude-induced expression of oats appeared to be GA dependent.

Auxin is a plant hormone extensively involved in regulating cell growth, cell division and cell-specific differentiations to participate in a series of growth and development processes [45]. ARF and SAUR family protein could bind to the cis-acting element of auxin-inducible gene and regulate its transcriptional expression[46, 47]. Forty-eight DEGs encoding three key proteins (transport inhibitor response, auxin-responsive and SAUR family protein) of IAA signaling pathway were identified in our study. It could infer that both ABF and SAUR can activate the down-stream gene expressions to respond tohigh altitude stresses.

JAs are a group of important hormones that play pivotal roles in a variety of plant growth, developmental and stress response processes[48]. In this study, 41 DEGs encoding jasmonate ZIM domain-containing protein and transcription factor MYC2 were enriched in the JA signaling pathway. It had been shown that JAZ gene and MYC2 were the major regulators of jasmonic acid signaling pathway in which JAZ protein degradation could release MYC2 transcription factor and activate the expression of downstream genes of jasmonic acid signaling pathway [49].

Conclusion

In order to understand the mechanisms of oats adapting to high altitudes, we accomplished physiological and agronomical characterizations and transcriptome sequence analysis. The physiological and agronomical results showed that oats displayed significant increases in MDA, SOD and soluble sugar contents, accompanied by decreases in chlorophyll, NPQ, stomatal density and hay yield. The transcriptomic profiles showed apparent differences between low and high altitudes where many pathways associated with high altitude stresses were identified including photosynthetic pigment synthesis, photosynthesis, and plant hormone signaling transduction pathways. Moreover, large amount of differentially expressed TFs between low and high altitudes was identified to participate in the response to altitude stress of oats, which can serve as candidate genes for altitude stresses molecular breeding project of oat. In general, this results provided the basis for understanding better the biological regulation on oats under two scenarios of altitudes and thus contributed to breeding efforts aimed at increasing yield under such situations.

Methods

1. Plant Materials And Growth Conditions

Seeds of Avena sativa L. Qingyin No.1 were collected from the Qinghai with the permission of the Qinghai Academy of Animal Science and Veterinary Medicine in the Qinghai Province of China. Animal Science and Veterinary Medicine in the Qinghai Province of China undertook the formal identification of the samples and provided detailsof specimen deposited. In order to investigate the effects of altitudes on the physiology and the transcriptions, oat leaf samples of the cultivar (cv.) “Qingyin No.1” were collected at heating stage in April, 2015 in two regions with significant differences in altitude in Qinghai Province, China, County of Minhe, (36°14′, 102°41′) at the altitude ca. 2080 m (defined as the low altitude) and County of Huangzhong (36°21′,101°44′) at ca. 2918 m (defined as the high altitude). During the heating period, the climate data in the two altitude regions were recorded (Additional file 10: Figue S10). The field plots were arranged as a completely randomized block design and adopted line sowing method. The distance of row spacing and seeding depth of were 20 cm and 5 cm, respectively. The experiment was carried out without fertilization and irrigation and weeding manually. The whole flag leaf was collected randomly and immediately frozen in liquid nitrogen. Samples were kept at − 80℃for further analyses.

2. The Measurements Of Agronomic Traits

Plant heights were measured at heading stage of oats. Each of the 5 oat plants in each plot was selected randomly to measure its height to calculate their mean. At the same stage, the ratio of stem to leaf was measured. About 0.5 kg of oat samples were weighed in each plot, and stems, leaves and inflorescences were separated into two parts. After air dried, samples were weighed and the ratio of stem to leaf was calculated (repeated with above). The leaf sheath of the oat grasses was collected into stems and the ears into leaves. The fresh grass samples were naturally air-dried for the dry weights which were converted into hay yield per hectare. After weighed, the hay samples were crushed and sieved with 40-mesh then the crude fat (EE) was determined by the soxhlet extraction.

3. Measurements Of The Stomatal Density And Physiological Analyses

The stomatal density of oats grown at the two altitudes were observed by SEM.

SOD activity was detected by recording the decrease in optical density of NBT dye by the enzyme. MAD content was measured using a modified TBA method. The two methods above were described by Tang et al.

Approximate 0.1 g of each leaf sample was incubated on ice for 30 min and immersed in 10 ml of 95% ethanol in 25 ml brown flask at room temperature. Until the green color disappeared, the solution was set to volume, shaken and stored in the dark. A series of 100 µl aliquots were taken every 10 min after initial immersion, and subjected to spectrophotometry (absorption measured at 647 nm and 664 nm) to quantify the amount of chlorophyll leached.

Before Fv/Fm measurement, whole oat plants were dark-adapted for 20 min, and Fv/Fm was measured using a steady-state gas-exchange system with an integrated fluorescence chamber head (Heinz Walz GmbH,Effeltrich༌Germany). Fv/Fm values were calculated as Fv/Fm = (Fm – Fo)/Fm, NPQ = F m /F m ’⁻¹.

4. Library Preparation For Transcriptome Sequencing

RNA extraction was performed according to the manufacturer’s protocol of the RNA prep Pure Plant kit (KangWei Biotech Co. Ltd., Beijing, China). RNA purity and integrality were checked before the cDNA library construction. The mRNA was enriched from the total RNA through the magnetic beads with oligo (dT). Afterward, mRNA was randomly broken into fragments. Catalyzed by reverse transcriptase, double-stranded cDNA was synthesized through mRNA by combining with random primers. After ds-cDNA purified, the adapters and poly (A) tails were ligated at both ends of ds-cDNA. With the adapter sequences, ds-cDNA fragments were amplified through PCR. After ds-cDNA quantification, the cDNA libraries were ready for sequencing. using the Illumina HiSeq Xten (single strands for sequencing).

5. Transcriptome Assembly And Gene Function Annotations

The clean data were The clean data were obtained by removing reads containing adapters and low quality ones from raw data. Transcriptome assemblies were performed using Trinity[50] based on clean data. The sequences of unigenes were compared with Nr (NCBI non-redundant), Nt (Nucleotide Sequence Database), SwissProt (a manually annotated and reviewed protein sequence database) and GO (Gene Ontology), KOG (euKaryotic Orthologous Groups) databases by BLAST and the KEGG (Kyoto Encyclopedia of Genes and Genomes). Orthology results were obtained by comparing with KEGG using KOBAS2.0[51].

6. Differential Expression Analyses

The EdgeR program package was used to adjust read counts of each sequenced library and the RSEM package for differential expression level analyses of two samples. The p value was adjusted using the q value. In this study, q value < 0.01 and |log2 (fold change)| >2 were set as the thresholds for significantly differential expressions.

Abbreviations

NPQ:non-photochemical quenching coefficient; DEGs:differentially expressed genes; GO:Gene ontology; KEGG:Kyoto Encyclopedia of Genes and Genomes; Nr:NCBI non-redundant; Nt:Nucleotide Sequence Database; SwissProt:a manually annotated and reviewed protein sequence database; KOG:euKaryotic Orthologous Groups; TFs:transcription factors; Chl a:chlorophyll a; APX: ascorbate oxidase; CAT: catalase; GR: glutathione reductase; MDA: Malondialdehyde; SOD:superoxide dismutase; ROS: Reactive oxygen species; qRT-PCR:Quantificational real-time polymerase chain reaction; RNA-seq:RNA sequencing; SEM:scanning electron microscopy; IAA:Auxin; ABA:Abscisic acid; ET:Ethylene; SA:salicylicacid; JA:jasmonic acid; ARF:Auxin response factor; NBT:nitro-blue tetrazolium; TBA:thiobarbituric acid; FPKM:per Million mapped reads;RSEM: RNA sequencing by expectation maximization

Declarations

Ethics approval and consent to participate

Not applicable. 

Consent for publication

Not applicable. 

Availability of data and materials

The raw sequencing data have been submitted to the NCBI Sequence Read Archive database with accession number PRJNA639320. 

Competing interests

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 

Funding

The RNAseq analysis was supported by National Natural Science Foundation of China (31872998). The funding body had no influence on the experimental design, data analysis and interpretation, or writing of the manuscript. 

Authors’ contributions

JQY ,GWC and YJC conceived and designed the experiments. JQY, BL, WHH and LL performed the experiments. JQY,BL,TTS,JLH and ZLKL analyzed and interpreted the sequence data. JQY wrote the manuscript. All authors readand approved the final manuscript. 

Acknowledgments

We thank the Biomarker Technologies (Beijing, China) for the RNA-seq and for the raw data analysis.

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Additional Files

Additional file 1:Figue S1. Function annotation results of oat.

Additional file 2:Figue S2. Annotation of 11,639 significantly differentially-expressed genes transcripts.

Additional file 3:Figue S3. Gene Ontology (GO) functional annotation of transcripts.

Additional file 4:Figue S4. KEGG pathway mapping

Additional file 5:Figue S5. Genotypic differences in expressions of DEGs associated with TFs between low and high altitude.

Additional file 6:Figue S6. Differently-expressed genes related to porphyrin and chlorophyll metabolism.

Additional file 7:Figue S7. Differently-expressed genes related to carotenoid biosynthesis.

Additional file 8:Figue S8. Differently-expressed genes related to photosynthesis.

Additional file 9:Figue S9. Differently-expressed genes related to hormone signal transduction pathway.

Additional file 10:Figue S10. Meteorological data of the growing season of Minhe and Huangzhong County (April-October 2015).