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).
Table 1
Overview of the RNA-sequencing reads generated from each sample.
Sample | Total Raw Reads (Mb) | Total Clean Reads (Mb) | Total Clean Bases (Gb) | Clean Reads Q20 (%) | Clean Reads Q30 (%) | Clean Reads Ratio (%) |
Low1 | 50.62 | 44.36 | 6.65 | 96.63 | 91.03 | 87.64 |
Low 2 | 50.62 | 44.63 | 6.69 | 96.59 | 90.91 | 88.16 |
Low 3 | 50.61 | 44.48 | 6.67 | 96.41 | 90.99 | 87.88 |
High1 | 50.62 | 45.01 | 6.75 | 96.77 | 91.30 | 88.92 |
High2 | 47.92 | 42.68 | 6.40 | 96.86 | 91.51 | 89.06 |
High3 | 50.62 | 45.19 | 6.78 | 96.78 | 91.34 | 89.27 |
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.
7. Analyses of DEGs related to altitudinal responses
To further identify the key genes related to responses at different altitudes, the DEGs in two pathways for the chlorophyll metabolism and the carotenoid biosynthesis were compared in detail between the low and the high altitudes. The results demonstrated that 23 genes in the porphyrin and the chlorophyll metabolism pathways were detected. Among them, 14 genes had been up-regulated and only 9 genes were down-regulated at high altitude. The DEGs played important roles during the chlorophyll biosynthesis and encoding chlorophyllase(Additional file 6: Figue S6, Table 2). Moreover, the comparison of the DEGs involved in the carotenoid biosynthesis showed that 32 genes were significantly down-regulated at high altitude (Additional file 7: Figue S7). The results indicated that the photosynthetic pigment biosynthesis was an important process for oats under the high altitude stress.
Table 2
Genotypic differences in expressions of DEGs associated with pigment metabolism.
Group | Gene ID | Regulation | Gene description |
| CL3626.Contig4_All | up | uroporphyrinogen decarboxylase |
| Unigene13229_All | up | coproporphyrinogen III oxidase |
| CL8939.Contig6_All | up | glutamyl-tRNA reductase |
| CL9433.Contig7_All | up | glutamyl-tRNA synthetase |
Chlorophyll and prophyrin metabolism | CL900.Contig8_All CL25634.Contig3_All CL8418.Contig1_All CL22519.Contig2_All CL22519.Contig1_All CL5496.Contig2_All CL10250.Contig3_All CL10437.Contig1_All CL10437.Contig2_All CL23991.Contig6_All CL5813.Contig5_All CL8939.Contig2_All CL9433.Contig14_All Unigene4721_All Unigene7840_All Unigene16352_All Unigene41659_All Unigene36472_All CL20435.Contig2_All | up up up up up up up up up up down down down down down down down down down | Glucuronosyltransferase Glucuronosyltransferase protochlorophyllide reductase chlorophyllase chlorophyllase chlorophyll(ide) b reductase geranylgeranyl diphosphate geranylgeranyl diphosphate geranylgeranyl diphosphate cytochrome c oxidase assembly protein subunit 15 porphobilinogen synthase glutamyl-tRNA reductase glutamyl-tRNA synthetase glucuronosyltransferase glucuronosyltransferase glucuronosyltransferase geranylgeranyl-bacteriochlorophyllide a reductase geranylgeranyl-bacteriochlorophyllide a reductase geranylgeranyl-bacteriochlorophyllide a reductase |
High altitude stresses might have an impact on plant photosynthesis. In this study, we identified 15 DEGs were significantly regulated in photosynthesis which played a part in photosystem I and II. Meanwhile, 9 genes that mapped to the photosynthesis-antenna protein pathways exhibited differential expressions at high altitude, where the genes encoded light-harvesting complex II chlorophyll a/b binding protein (Additional file 8: Figue S8, Table 3). These results indicated that changes at the expression levels of the genes above might have blocked photosynthesis, thereby influencing the chlorophyll biosynthesis of oats at high altitude.
Table 3
Genotypic differences in expressions of DEGs associated with photosynthesis.
Group | | Gene ID | Regulation | Gene description |
Photosynthesis | | Unigene34665_All Unigene16087_All CL7675.Contig8_All CL10733.Contig3_All CL11120.Contig6_All CL10733.Contig2_All CL9061.Contig5_All Unigene24290_All CL25957.Contig11_All Unigene24271_All CL399.Contig3_All Unigene56697_All Unigene13484_All Unigene10050_All CL10048.Contig11_All | up up up up up up up up up up up up down down down | photosystem II CP47 chlorophyll apoprotein photosystem II CP47 chlorophyll apoprotein photosystem II oxygen-evolving enhancer protein 2 photosystem II oxygen-evolving enhancer protein 3 photosystem II oxygen-evolving enhancer protein 3 photosystem II oxygen-evolving enhancer protein 3 photosystem II oxygen-evolving enhancer protein 3 photosystem I P700 chlorophyll a apoprotein A1 photosystem I P700 chlorophyll a apoprotein A1 photosystem I P700 chlorophyll a apoproteinA1/ A2 photosystem I subunit PsaO ferredoxin photosystem II CP47 chlorophyll apoprotein photosystem II CP47 chlorophyll apoprotein ferredoxin–NADP + reductase |
Photosynthesis-antenna protein | | Unigene13588_All Unigene16609_All Unigene23029_All Unigene15222_All CL25362.Contig4_All Unigene14215_All Unigene21557_All Unigene75195_All CL23904.Contig9_All | up up up up up up down down down | light-harvesting complex II chlorophyll a/b binding protein 1 light-harvesting complex II chlorophyll a/b binding protein 1 light-harvesting complex II chlorophyll a/b binding protein 1 light-harvesting complex II chlorophyll a/b binding protein 1 light-harvesting complex II chlorophyll a/b binding protein 1 light-harvesting complex II chlorophyll a/b binding protein 1 light-harvesting complex II chlorophyll a/b binding protein 1 light-harvesting complex II chlorophyll a/b binding protein 5 light-harvesting complex II chlorophyll a/b binding protein 7 |
Hormones are pivotal to plants in stress adaptive signaling cascades and act as central integrators to connect and reprogram different responses. In this experiment, we identified 273 hormone-related DEGs between the low and the high altitude conditions. Among them, 60 genes were identified to encode DELLA protein and acted as a part of gibberellin receptor and phytochrome-interacting factor. Forty-eight genes were identified in the auxin signal transduction pathway in which some encoded auxin-responsive protein IAA and others participated in the process of auxin synthesis and metabolism. In jasmonic acid signal transduction pathway, 41 genes encoded jasmonate ZIM domain-containing protein and transcription factor MYC2. The three pathways above were the most abundant phytohormone signal transduction pathways. In addition, 33 genes in cytokinin pathway, 31 genes in abscisic acid signal pathway, 24 genes in brassinosteroid pathway, 22 genes in ethylene pathway and 14 genes in salicylic acid pathway were differentially expressed (Additional file 9: Figue S9). The results above suggested that these hormone-related genes can be involved in cell division, stem elongation, stomata aperture, fruit maturation and disease prevention processes in response to high altitude conditions.
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.