Transcriptome-Wide Gene Expression Plasticity in Stipa grandis in Response to Grazing Intensity Differences

Abstract

Organisms have evolved effective and distinct adaptive strategies to survive. Stipa grandis is a representative species for studying the grazing effect on typical steppe plants in the Inner Mongolia Plateau. Although phenotypic (morphological and physiological) variations in S. grandis in response to long-term grazing have been identified, the molecular mechanisms underlying adaptations and plastic responses remain largely unknown. Here, we performed a transcriptomic analysis to investigate changes in gene expression of S. grandis under four different grazing intensities. As a result, a total of 2357 differentially expressed genes (DEGs) were identified among the tested grazing intensities, suggesting long-term grazing resulted in gene expression plasticity that affected diverse biological processes and metabolic pathways in S. grandis. DEGs were identified in RNA-Seq and qRT-PCR analyses that indicated the modulation of the Calvin-Benson cycle and photorespiration metabolic pathways. The key gene expression profiles encoding various proteins (e.g., ribulose-1,5-bisphosphate carboxylase/oxygenase, fructose-1,6-bisphosphate aldolase, glycolate oxidase, etc.) involved in these pathways suggest that they may synergistically respond to grazing to increase the resilience and stress tolerance of S. grandis. Our findings provide scientific clues for improving grassland use and protection and identifying important questions to address in future transcriptome studies.

Keywords: Calvin–Benson cycle; Stipa grandis; comparative transcriptomic analysis; differentially expressed gene; gene expression plasticity; grazing adaptation; photorespiration.

Figure 1

Expression patterns and functional annotations of the differentially expressed genes (DEGs). (A) K-means clustering of DEGs. The red-to-blue gradient indicates high-to-low expression levels. Prior to clustering, the expression level for each transcript over the four grazing treatments was standardized using z-scores. (B) KEGG pathway enrichment analysis. (C) GO enrichment analysis. In panels B and C, only the top five enriched GO terms and KEGG pathways are shown, respectively; the x-axis indicates the number of the unigenes, and clusters are separated by left-extended short black bars.

Figure 2

Expression patterns of differentially expressed genes (DEGs) in the Calvin–Benson cycle (CBC). The line and symbol chart next to each enzyme represents the expression profiles of DEGs shown in Table S6. The lines with different colors represent the assembled transcripts for an enzyme in the CBC. According to the gene expression levels under CK conditions, these transcripts were listed next to each chart in descending order. The grazing gradient is shown on the x-axis, and the gene expression level (the mean FPKM value of three biological replicates) is shown on the y-axis. The carboxylation reaction catalyzed by Rubisco fixes CO₂ into the acceptor molecule RuBP, forming 3-PGA. The reductive phase of the cycle follows with two reactions catalyzed by PGKase and GAPDHase, producing GAP. The GAP enters the regenerative phase catalyzed by ALDase and either FBPase or SBPase, producing F6P (fructose-6-phosphate) and S7P (sedoheptulose-7-phosphate). The F6P and S7P are then used in reactions catalyzed by TKase, RPIase, and RPEase, producing Ru5P (ribulose 5-phosphate). The final step, which is catalyzed by PRKase, converts Ru5P to RuBP. Rubisco is the initiating enzyme for the Calvin–Benson cycle and the photorespiratory cycle, fixing O₂ into the acceptor molecule RuBP to form 2-PG, which is then metabolized via the photorespiratory pathway.

Figure 3

Expression patterns of differentially expressed genes (DEGs) in the photorespiratory pathway. The line and symbol chart next to each enzyme represents the expression profiles of DEGs shown in Table S7. The lines with different colors represent the assembled transcripts for an enzyme in the photorespiratory pathway. According to the gene expression levels under CK conditions, these transcripts were listed next to each chart in descending order. The grazing gradient is shown on the x-axis and the gene expression level (the mean FPKM value of three biological replicates) is shown on the y-axis. The photorespiratory cycle is a process in photosynthetic cells involving the chloroplasts, peroxisomes, mitochondria, and the cytosol. In chloroplasts, Rubisco catalyzes the oxygenation of RuBP, which generates one molecule of 3-PGA and one molecule of 2-PG. The 2-PG is first dephosphorylated to glycolate by PGLPase, after which it diffuses into the peroxisome. In the peroxisome, the O₂-dependent glycolate is oxidized to glyoxylate by GOXase to produce H₂O₂, which is quickly detoxified by CATase. Glyoxylate is transaminated to glycine by the parallel action of GGTase or SGTase. Glycine then moves into the mitochondrion, wherein the GDCase multienzyme system and SHMTase convert two molecules of glycine to one molecule of serine, NH₃, and CO₂. After being transported from the mitochondrion to the peroxisome, serine is converted by SGTase to hydroxypyruvate, which is reduced to glycerate by HPRase. The glycerate returns to the chloroplast to be phosphorylated by GLYKase (glycerate 3-kinase), and the resulting 3-PGA is converted to RuBP in the Calvin–Benson cycle. * represents transcripts that were not DEGs among the four grazing treatments.

Figure 4

Expression patterns of selected genes measured using qRT-PCR. Nine genes were selected for validating observations of gene expression plasticity in various grazing intensities in two years of S. grandis samples. The x-axis represents four different grazing intensities, and the y-axis indicates fold change of genes’ relative expression levels. The color curves represent the gene expression patterns of the selected genes in 2018 and 2019, respectively. The error bars represent mean standard deviations (± SD) of three biological replicates.

Figure 5

Vegetation status of plots of the four grazing intensity treatments (Photographed 28 July 2018).