Table_4_Multi-omics analysis reveals the molecular changes accompanying heavy-grazing-induced dwarfing of Stipa grandis.xlsx

Heavy grazing significantly reduces Stipa grandis growth. To enhance our understanding of plant responses to heavy grazing, we conducted transcriptomic, proteomic, and metabolic analyses of the leaves of non-grazed plants (NG) and heavy-grazing-induced dwarf plants (HG) of S. grandis. A total of 101 metabolites, 167 proteins, and 1,268 genes differed in abundance between the HG and NG groups. Analysis of Kyoto Encyclopedia of Genes and Genomes pathways among differentially accumulated metabolites (DAMs) revealed that the most enriched pathways were flavone and flavonol biosynthesis, tryptophan metabolism, and phenylpropanoid biosynthesis. An integrative analysis of differentially expressed genes (DEGs) and proteins, and DAMs in these three pathways was performed. Heavy-grazing-induced dwarfism decreased the accumulation of DAMs enriched in phenylpropanoid biosynthesis, among which four DAMs were associated with lignin biosynthesis. In contrast, all DAMs enriched in flavone and flavonol biosynthesis and tryptophan metabolism showed increased accumulation in HG compared with NG plants. Among the DAMs enriched in tryptophan metabolism, three were involved in tryptophan-dependent IAA biosynthesis. Some of the DEGs and proteins enriched in these pathways showed different expression trends. The results indicated that these pathways play important roles in the regulation of growth and grazing-associated stress adaptions of S. grandis. This study enriches the knowledge of the mechanism of heavy-grazing-induced growth inhibition of S. grandis and provides valuable information for restoration of the productivity in degraded grassland.</p

Data_Sheet_1_Multi-omics analysis reveals the molecular changes accompanying heavy-grazing-induced dwarfing of Stipa grandis.zip

Heavy grazing significantly reduces Stipa grandis growth. To enhance our understanding of plant responses to heavy grazing, we conducted transcriptomic, proteomic, and metabolic analyses of the leaves of non-grazed plants (NG) and heavy-grazing-induced dwarf plants (HG) of S. grandis. A total of 101 metabolites, 167 proteins, and 1,268 genes differed in abundance between the HG and NG groups. Analysis of Kyoto Encyclopedia of Genes and Genomes pathways among differentially accumulated metabolites (DAMs) revealed that the most enriched pathways were flavone and flavonol biosynthesis, tryptophan metabolism, and phenylpropanoid biosynthesis. An integrative analysis of differentially expressed genes (DEGs) and proteins, and DAMs in these three pathways was performed. Heavy-grazing-induced dwarfism decreased the accumulation of DAMs enriched in phenylpropanoid biosynthesis, among which four DAMs were associated with lignin biosynthesis. In contrast, all DAMs enriched in flavone and flavonol biosynthesis and tryptophan metabolism showed increased accumulation in HG compared with NG plants. Among the DAMs enriched in tryptophan metabolism, three were involved in tryptophan-dependent IAA biosynthesis. Some of the DEGs and proteins enriched in these pathways showed different expression trends. The results indicated that these pathways play important roles in the regulation of growth and grazing-associated stress adaptions of S. grandis. This study enriches the knowledge of the mechanism of heavy-grazing-induced growth inhibition of S. grandis and provides valuable information for restoration of the productivity in degraded grassland.</p

Table_3_Multi-omics analysis reveals the molecular changes accompanying heavy-grazing-induced dwarfing of Stipa grandis.xlsx

Heavy grazing significantly reduces Stipa grandis growth. To enhance our understanding of plant responses to heavy grazing, we conducted transcriptomic, proteomic, and metabolic analyses of the leaves of non-grazed plants (NG) and heavy-grazing-induced dwarf plants (HG) of S. grandis. A total of 101 metabolites, 167 proteins, and 1,268 genes differed in abundance between the HG and NG groups. Analysis of Kyoto Encyclopedia of Genes and Genomes pathways among differentially accumulated metabolites (DAMs) revealed that the most enriched pathways were flavone and flavonol biosynthesis, tryptophan metabolism, and phenylpropanoid biosynthesis. An integrative analysis of differentially expressed genes (DEGs) and proteins, and DAMs in these three pathways was performed. Heavy-grazing-induced dwarfism decreased the accumulation of DAMs enriched in phenylpropanoid biosynthesis, among which four DAMs were associated with lignin biosynthesis. In contrast, all DAMs enriched in flavone and flavonol biosynthesis and tryptophan metabolism showed increased accumulation in HG compared with NG plants. Among the DAMs enriched in tryptophan metabolism, three were involved in tryptophan-dependent IAA biosynthesis. Some of the DEGs and proteins enriched in these pathways showed different expression trends. The results indicated that these pathways play important roles in the regulation of growth and grazing-associated stress adaptions of S. grandis. This study enriches the knowledge of the mechanism of heavy-grazing-induced growth inhibition of S. grandis and provides valuable information for restoration of the productivity in degraded grassland.</p

<i>De novo</i> Assembly and Transcriptomic Profiling of the Grazing Response in <i>Stipa grandis</i>

<div><p>Background</p><p><i>Stipa grandis</i> (Poaceae) is one of the dominant species in a typical steppe of the Inner Mongolian Plateau. However, primarily due to heavy grazing, the grasslands have become seriously degraded, and <i>S</i>. <i>grandis</i> has developed a special growth-inhibition phenotype against the stressful habitat. Because of the lack of transcriptomic and genomic information, the understanding of the molecular mechanisms underlying the grazing response of <i>S</i>. <i>grandis</i> has been prohibited.</p><p>Results</p><p>Using the Illumina HiSeq 2000 platform, two libraries prepared from non-grazing (FS) and overgrazing samples (OS) were sequenced. <i>De novo</i> assembly produced 94,674 unigenes, of which 65,047 unigenes had BLAST hits in the National Center for Biotechnology Information (NCBI) non-redundant (nr) database (E-value < 10^-5). In total, 47,747, 26,156 and 40,842 unigenes were assigned to the Gene Ontology (GO), Clusters of Orthologous Group (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, respectively. A total of 13,221 unigenes showed significant differences in expression under the overgrazing condition, with a threshold false discovery rate ≤ 0.001 and an absolute value of log₂Ratio ≥ 1. These differentially expressed genes (DEGs) were assigned to 43,257 GO terms and were significantly enriched in 32 KEGG pathways (q-value ≤ 0.05). The alterations in the wound-, drought- and defense-related genes indicate that stressors have an additive effect on the growth inhibition of this species.</p><p>Conclusions</p><p>This first large-scale transcriptome study will provide important information for further gene expression and functional genomics studies, and it facilitated our investigation of the molecular mechanisms of the <i>S</i>. <i>grandis</i> grazing response and the associated morphological and physiological characteristics.</p></div

Identification of the DEGs (differentially expressed genes) between FS and OS.

<p>The DEGs were determined using a threshold of FDR ≤ 0.001 and an absolute value of log₂Ratio ≥ 1. The red, green and blue spots represent the upregulated, the downregulated DEGs and the genes without obvious changes in response to grazing, respectively.</p

The DEGs related to abiotic stress.

<p>The prefix “CL” represents clusters, and “unigene” represents singletons. FPKM indicates the FPKM values of the unigenes in FS or OS. “Fold change” is equal to log₂ (OS-FPKM / FS-FPKM). “+” indicates upregulated transcription, and “-” represents downregulated transcription.</p><p>The DEGs related to abiotic stress.</p

Histogram of the GO (gene ontology) classification of the <i>S</i>. <i>grandis</i> unigenes.

<p>All 47,747 unigenes were grouped into three ontologies: molecular function, cellular component and biological process. The y-axis indicates the percentage (left) and number (right) of genes in each term.</p

The GO term assignments for the DEGs related to the stress response under the overgrazing and non-grazing conditions.

<p>The red bars represent the upregulated DEGs, the black bars represent the downregulated DEGs.</p

Unigene homology searches against the nr database.

<p>(A) The proportional frequency of the E-value distribution. (B) The proportional frequency of the sequence similarity distribution. (C) The proportional species distribution of <i>S</i>. <i>grandis</i> among other plant species.</p

qPCR validation of the gene expression patterns.

<p>A total of 30 genes, including (A) 10 downregulated, (B) 10 upregulated, and (C) 10 unigenes with no difference in expression and with an absolute value of log₂Ratio < 1, were selected, and the expression levels were confirmed using qPCR. The gray bars represent the changes in transcript abundance determined by the FPKM method. The black bars represent the relative expression levels estimated using qPCR and the 2^-ΔΔCT method. The error bars indicate the standard deviation (n = 3).</p