Figure 1.
Water deficit assay of sugarcane cv. SP70-1143 colonized with the beneficial endophytic diazotrophic bacteria G. diazotrophicus strain PAL5.
(a) Simplified pipeline for water deficit assay. (b) left panel shows qRT-PCR quantification of sugarcane colonization by G.diazotrophicus 7 days after inoculation in hydroponic solution. Bacterial 23S rRNA levels are presented relative to rice 28 S rRNA levels. Right panel shows root and shoot fresh weight measurements 14 days after inoculation and 1 day before treatments. (c) Phenotype of sugarcane cv. SP70-1143 inoculated or not with G. diazotropicus, 3 days after withholding water. Senescent edges are indicated by arrows and are shown in the amplified image from a WD+GD plant. (d) qRT-PCR quantification of sugarcane colonization by G.diazotrophicus after 3 days under water deficit assay. Bacterial 23S rRNA levels are presented relative to rice 28 S rRNA levels. (e) Phenotype of sugarcane cv. SP70-1143 inoculated or not with G. diazotropicus, after 40 days withholding water. Bar = 5 cm. Error bars indicate standard error of the mean. Asterisk mark statistical significance between GD-R vs CT-R (*p<0.05) and WD+GD-R vs GD-R (**p<0.01), performed by statistical t-test (unpaired).
Table 1.
Abbreviations used for 8 sample types and dataset comparisons.
Figure 2.
Simplified pipeline of the construction and analyses of the sugarcane reference RT2.
(a) Construction of the sugarcane reference transcriptome RT2. The total numbers of transcripts or loci/gene quantified in each step is shown. (b) Generation of differentially expressed transcriptomes. Numbers represent a total of up and downregulated DEG in each dataset, details are presented in Table 4. (c) and (d) represent TRAPID-based analysis of (c) sequence length meta annotation and (d) number of Gene families, Protein domains and GO terms found in RT2, and the percentage of genes that were annotated in at least one of the tree functional categories.
Table 2.
Summary of Illumina transcriptome sequence data and quality control.
Table 3.
Summary of reads and genes (loci) mapped in each generated library.*
Table 4.
Overall summary of the differentially regulated genes in Sugarcane SP70-1143 in the tree datasets comparisons.
Figure 3.
MapMan overview of differentially expressed genes in sugarcane SP70-1143 colonized or not with G. diazotrophicus and subjected to water deficit.
MapMan ‘regulation overview’ of differentially expressed genes (DEG) from roots and shoots in two dataset comparisons: Bacteria&Drought and Drought. Blue stands for upregulated and red stands for downregulated loci. Only differentially expressed genes are shown, with p<0.05 and at least 2-fold difference from control.
Figure 4.
Validation of differentially expressed genes by qRT-PCR analysis compared to RNA-seq analysis.
Pattern of expression of stress-responsive genes in (a) shoots and (b) roots of Drought and Bacteria&Drought datasets. qRT-PCR results are presented as the ratio of expression of each gene (relative to 28S rRNA) in the treatments WD or WD+GD, compared to controls CT or WD respectively, and transformed in log2. Each biological replicate represents a pool of tree plants. Error bars indicate the standard error of the mean (n = 2).
Table 5.
Genes known to be upregulated in response to water deprivation and their expression in Drought dataset comparison for root and shoot tissues.
Figure 5.
Gene set enrichment analysis of differentially expressed genes in sugarcane SP70-1143 colonized or not with G. diazotrophicus and subjected to water deficit.
PageMan analysis of Drought and Bacteria&Drought dataset comparisons, for root and shoot tissues, using Bin-wise Wilcoxon test with Benjamini-Hochberg FDR multiple testing correction (ORA cutoff = 1.0). Blue stands for upregulated/increased classes; Red stands for downregulated/decreased classes. For a complete overview, see Table S1.
Figure 6.
GO enrichment analysis of non-inoculated SP70-1143 plants under water deficit.
TRAPID system calculated GO enrichment based on the Drought dataset compared to a background (p-value<0.05). (a) Number of GOs highly represented as up or downregulated, in shoots and roots. Graphs in (b) and (c) show enrichment level and p-value of selected GOs indicating drought pattern in shoots of Drought dataset, corroborating PageMan analysis (Table S1). GO enriched terms are shown as (b) up and (c) downregulated in shoots of SP70-1143. For a complete overview, see Table S2.
Figure 7.
Hormonal responses to bacteria colonization and drought stress in sugarcane SP70-1143.
Only differentially expressed genes (DEG) related to ABA, ET and auxin biosynthesis, signaling and response, present in Drought and Bacteria&Drought datasets, are indicated in the schemes of each hormone pathway. Expresssion levels (log2 fold change) are represented as colors and symbols. Red stands for downregulated and blue stands for upregulated genes. For each hormone pathway, the DEG are identified with numbers. For more detailed function of each regulated gene, see Table S3.
Figure 8.
Hierarchical clustering of drought responsive transcription factors in cv. SP70-1143.
Expression pattern of (a) MYB, (b) WRKY, (c) ERF/AP2 and (d) bZIP gene families, in both root and shoot of sugarcane Drought and Bacteria&Drought datasets. Both genes and datasets were clustered in Genesis software, using Pearson correlation and Average linkage. For specific analysis of each gene after hierarchical clustering, see Table S4.