Fig 1.
Physiological and biochemical characterization of in vitro sugarcane plantlets under osmotic stress induced by PEG 8000.
Four physiological parameters and one biochemical parameter were measured in sugarcane plantlets to characterize the effect of the osmotic stress imposition. A) Plantlets exposed to osmotic stress by PEG 8000 (40%). The time of stress exposure (from top to bottom) is indicated by an arrow showing the severity of the stress treatment increased according to time (0, 24, 48, and 72 hours respectively). Physiological measurement of B) Relative water content, C) Osmotic potential, D) Assimilation rate, E) Transpiration rate, and F) Biochemical measurement of proline content in roots and leaves of sugarcane.
Table 1.
Summary of transcriptome data for sugarcane var. Mex 69–290.
Fig 2.
Summary of the annotated unigenes of S. officinarum var. Mex 69–290 transcriptome.
A) Gene Ontology (GO) classification of the unigenes annotated. B) Distribution lengths of the assembled unigenes. C) Distribution of BLASTX-hits for annotated unigenes among the 10 principal plant species represented in the PlantRef protein database. D) Distribution of the closest homology matches (Top BLASTX-hits) for mapped unigenes in the ten principal species represented in the PlantRef protein database. E) Venn diagram is showing BLAST results of unique and shared unigenes among CDD, GO, Pfam, Sprot, TrEMBL, and PlantRef databases. F) Venn diagram showing shared unigenes among GO, Pfam, Sprot, and PlantRef databases. G) Venn diagram showing the BLAST results for the three principal protein databases, Pfam, Sprot, and CDD. Plant species names were coded for C) and D) as follows: Bd = Brachypodium distachyon, Eg = Elaeis guineensis, Ma = Musa acuminata subsp. Malaccensis, Ob = Oryza brachyantha, Os = Oryza sativa Japonica, Pd = Phoenix dactylifera, Sb = Sorghum bicolor, Si = Setaria italica, and Zm = Zea mays.
Fig 3.
Unigenes involved in the starch and sucrose metabolism pathway according to KEGG database.
Predicted coded enzymes for starch and sucrose metabolism are marked as colored boxes (S2 File). Colors are used simply to differentiate each enzyme (one color for each EC). Reprinted from Kanehisa Laboratories under a CC BY license, with permission from Miwako Matsumoto, original copyright 5/9/16 (S3 File).
Fig 4.
Correlation of sample replicates and hierarchical clustering analysis of gene expression levels in each sample (0, 24, 48, and 72 h) under osmotic stress treatment.
A) Sample relatedness is plotted using the first two principal component (PCs) showing the variability between replicates and different treatments. Non-stressed leaf and root samples are in blue shade background. In dark-green and in red background colors are the samples corresponding to stressed leaves and roots (24, 48, 72 h ASI), respectively. C = control, D = Drought. B) Correlation dendrogram of gene expression in all samples. Correlation r values are coded into key colors where green indicates lower r values; reds indicates higher r above the mean. Blue squares in the nodes represent non-stressed samples, green stars represent leaf stressed samples, and the red stars represent root stressed samples. L = leaf, R = root, L0 and R0 = non-stress treatment, L1 and R1 = 24 h treatment ASI, L2 and R2 = 48 h treatment ASI, L3 and R3 = 72 h treatment ASI. * = biological replicate.
Fig 5.
Correlation and clustering analysis of the sugarcane DEGs subjected to osmotic stress treatments.
A) Tissue-specific heatmap clustering of 12,662 significant DEGs (FDR ≤ 0.001 and FC ≥2) for the 0, 24, 48, and 72 h treatments ASI. B-G) Osmotic stress DEG profiles for the organ specific behavior clusters. Clusters were built according to their expression patterns during the stress treatments. Gray lines indicate the behavior of each gene into the treatment and the mean expression profile for each cluster is plotted as blue lines. Bottom number indicates the number of time series genes. L = leaf, R = root. L0 and R0 = non-stress treatment, L1 and R1 = 24 h treatment ASI, L2 and R2 = 48 h treatment ASI, L3 and R3 = 72 h treatment ASI. * = biological replicate.
Fig 6.
Differentially expressed gene analysis along the osmotic stress treatments in sugarcane.
A) Volcano plots show DEGs in each of the stress treatments for leaf and root tissues. The x-axis represents the log2FoldChange of genes, and the y-axis represents the statistical significance degree by–log10 (PValue). DEGs are shown in red dots and were detected using their statistical significance differences (FDR<0.001 and a log2FoldChange>2). B) Comparison of the up- and down-regulated DEGs for leaf and root tissues in all three stress treatments and comparison of shared genes among all the treatments (CORE gene sets).
Fig 7.
Comparative analysis of the unique and shared DEGs among the osmotic stress treatments for sugarcane var. Mex 69–290.
The Venn diagrams show the overlap of the DEGs for leaves and roots submitted to a 3 time-series of osmotic stress treatments. A and C) Comparison among the genes differentially up-regulated for the stress treatments for leaf and root tissues respectively. D and F) Comparison among the genes differentially down-regulated for the stress treatments for leaf and root tissues respectively. B and E) Comparison among the core set of up- and down-regulated genes respectively for both tissues. CULG = CORE of Up-regulated Leaf Genes, CURG = CORE of Up-regulated Root genes, COSUG = CORE of Osmotic Stress Up-regulated Genes, CDLG = CORE of Down-regulated Leaf Genes, CDRG = CORE of Down-regulated Root Genes, and COSDG = CORE of Osmotic Stress Down-regulated Genes. The Venn diagrams were done by using Vennerable R package.
Table 2.
Stress-related DEGs detected in the CORE sets (CULG and CDLG) of regulated genes in leaves during the osmotic stress treatments.
Table 3.
Stress-related DEGs detected in the CORE sets (CURG and CDRG) of regulated genes in roots during the osmotic stress treatments.
Fig 8.
Functional enrichment analysis for the CORE set of DEGs regulated in the osmotic stress treatments for sugarcane var. Mex 69–290.
Significant groups (FDR<0.05) for COSUG and COSDG groups are plotted according to their enriched adjusted p-value (x axis). The size is proportional to the number of enriched genes among the total number of their GO term associated unigenes. COSUG = CORE of Osmotic Stress Up-regulated Genes for both leaf and root tissues, COSDG = CORE of Osmotic Stress Down-regulated Genes for both leaf and root tissues.
Fig 9.
Principal findings of the analysis of sugarcane transcriptome under osmotic stress.
A) Genes regulated under osmotic stress treatments. Inside blue square are the DEGs regulated in the three treatments. The green square represents the DEG regulated only in the T1 treatment, within the yellow square are the DEGs expressed only in T2 treatment, and within the red square are the DEGs regulated only in T3 treatment. Inside gray circles are DEGs shared among treatments. Letters in black color represent the up-regulated DEGs and letters in orange color represent the down-regulated DEGs per treatment. B) Schematic depiction of the physiological results obtained in sugarcane var. Mex 69–290, imposed to an osmotic stress treatment by PEG 8000 for 24, 48, and 72 h. The green arrow under the depiction indicates the directions of the time-stress treatments. RCW = relative content water, OsPot = osmotic potential, AR = assimilation rate. Complete name of the genes in A) are listed in the S4 File.