SI legends
Figure S1. Libraries reproducibility in well-watered (WW) and drought-stressed (DS) seedlings.
(a) Relative water content (RWC) of WW and DS seedlings, respectively. Error bars represent
standard deviation of three biological replicates. Three independent experiments were conducted.
Student’s t-test was used for hypothesis testing. Triple asterisks indicate P-value < 0.0001. (b) The
reproducibility of RNA-seq libraries (black spots) in WW (right) and DS (left) seedlings. (c) The
reproducibility of ribosome profiling libraries (red spots) from WW (right) and DS (left)
seedlings.
Figure S2. The characteristics of ribosome profiling data in WW seedling. (a) The length
distribution of ribosome protected fragments (RPF) in WW seedlings. The grey and white bars
refer to two biological replicates. (b) 3-nt periodicity of ribosome protected reads at the first 50 nt
and the last 40 nt of CDS in WW seedlings.
Figure S3. A diagram of A site calibration based on ribosome protected fragment (RPF) length. (a)
Schematic for relevant position of start (left) and stop codons (right) on mRNA to A/P sites of
ribosomes. “A” indicated aminoacyl-tRNA binding site and “P” suggested peptidyl-tRNA binding
site. (b) Read density distribution of different length of RPFs at 5′ end (left) and 3′ end (right) of
CDSs. X axis indicates the relevant position of mRNA based on figure S3a while Y axis represents
the number of RPF. (c) The exhibition of relative codon usage at first 150 codons of CDS.
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Figure S4. MapMan functional categories of drought responsive genes enriched at transcriptional
and translational levels. Solid triangles in the left indicate enriched metabolic pathways and other
process. The four columns parallel display pathways being transcriptionally up-regulated,
transcriptionally down-regulated, translationally up-regulated and translationally down-regulated
and are indicated by the upper numbers. The blue color represents underrepresented, and the red
color represents overrepresented. Only significant categories (FDR < 0.05) were displayed.
Figure S5. Gene expression patterns at transcriptional and translational levels. (a) The expression
patterns of GRMZM2G155216 in WW (red) and DS (blue) seedlings with two replicates. (b) The
expression patterns of AC190609.3_FG002. The first track indicates gene’s location on
corresponding chromosome. Eight tracks in the middle display read density of RNA-seq and
ribosome profiling in WW (red) and DS (blue) seedlings with two biological replicates,
respectively. Gene model was supplied at bottom tracks and transcription direction is indicated by
arrows. For mRNA structure, the thin lines represent introns and blue boxes refer to exons.
Figure S6. GO annotation of discordantly regulated genes under drought condition. (a) GO
annotation of genes down-regulated at transcriptional level and unchanged at translational level
(Class D in figure 4). (b) GO annotation of genes up-regulated at transcriptional level and
unchanged at translational level (Class F in figure 4). (c) GO annotation of genes down-regulated
at translational level and unchanged at transcriptional level (Class H in figure 4). (d) GO
annotation of genes up-regulated at translational level and unchanged at transcriptional level
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(Class B in figure 4). Blue, green and orange bars represent biological process, molecular function
and cellular component. P-value of GO terms was under the control of FDR < 0.05.
Figure S7. The comparison of simulated 30 bp single end (SE) and 100 bp pair-end RNA-seq data
(PE). (a) Pairwise pearson correlation of transcriptional abundance (FPKM) between simulated 30
bp SE and 100 bp PE RNA-seq data. (b) The distribution of transcriptional abundance (FPKM)
between 30 bp SE (blue curve) and 100 bp PE RNA-seq data (red curve). (c) Pairwise pearson
correlation of translational efficiency (TE) between 100 bp PE (x axis) and simulated 30 bp SE
RNA-seq data (y axis).
Figure S8. Global distribution of translational efficiency (TE) in WW and DS seedlings. (a) An
overview of the distribution of log2-transformed TE and corresponding mRNA abundance. Red
and blue spots indicate genes in WW and DS seedlings respectively. The density of TE and mRNA
abundance is shown along the axes. (b) The distribution of gene with decreased TE in 9 responsive
patterns. (c) The distribution of genes with increased TE in 9 responsive patterns. The numbers of
gene with increased and decreased TE were derived from figure 5b. Nine responsive groups (from
group A to group I) were derived from figure 4.
Figure S9. The TE of genes was affected by sequence features. NMFE (a), sequence length (b)
and GC content (c) of 3′ UTR in four TE groups based on log2(TE) value (< -1, -1 to 1, 0 to 1, > 1)
in WW seedlings. Figure d, e and f displayed NMFE, sequence length and GC content features of
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5′ UTR in different TE groups in WW seedlings, respectively. The exhibition of NMFE, sequence
length and GC content features of the CDS (g), 3′ UTR (h) and 5′ UTR (i) in four TE groups in DS
seedlings, respectively. The letters a, b and c indicate significant differences according to Student’s
t-test (P-value < 0.05). (g) The difference of the GC content of 5′ UTR between accordantly (red)
and discordantly (blue) regulated genes. The statistical difference was determined by the
Kolmogorov-Smirnov test.
Figure S10. Genome-wide prediction of upstream open reading frames (uORFs). (a) The length
distribution of all predicted uORFs. (b) Statistic for the number of predicted uORFs per gene. (c)
The length distribution of translated uORFs in WW seedlings. Figure (d) shows Lc expression
patterns. The first track indicates gene’s location on corresponding chromosome. Eight tracks in
the middle display read density of RNA-seq and ribosome profiling in WW (red) and DS (blue)
seedlings with two biological replicates, respectively. Gene model and predicted uORFs are
supplied at bottom tracks. Transcription direction is indicated by arrows. For mRNA structure, the
thin lines represent introns and blue boxes refer to exons.
Figure S11. The sequence features of uORFs. The difference between translated and untranslated
uORFs in uORF length (a), 5′ UTR length (b), uORF NMFE (c), normalized distance from uORFs
to CDS ATG start codon (d) and normalized distance from uORFs to transcription start site (TSS)
in DS seedlings (e). White and grey boxes indicate translated and untranslated uORFs respectively.
P-values were determined by Student’s t-test. (f) Sequence logo indicates the flanking sequence
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around ATG start codon of all maize genes. (g) GC content of flanking sequence of translated (red
line) and untranslated (blue line) uORFs’ ATG start codons, respectively.
Table S1. Alignment statistics for Ribosome profiling reads.
Table S2. Alignment statistics for RNA-seq reads.
Table S3. List of genes in four main discordantly responsive groups.
Table S4. Numbers of gene analyzed in 4 different TE (translational efficiency)
groups.
Table S5. List of genes with translated uORFs in well-watered seedlings.
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