A Fraction of read count as read length B Fraction of unique reads as read length Length (nt) Read count fraction (%) Unique read fraction (%) Supplemental.

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A Fraction of read count as read length B Fraction of unique reads as read length Length (nt) Read count fraction (%) Unique read fraction (%) Supplemental Figure 1: Fraction of read count (A) and unique reads (B) as read length.

Read count fraction (%) Length (nt) sRNA origin: GP(w) Read count fraction (%) Length (nt) sRNA origin: GP(-w) Supplemental Figure 2: Length distribution of sRNAs derived from different origins.

Read Count Fraction (%) Read Length (nt) Supplemental Figure 3: Size fraction of miRNAs under water and drought conditions.

genes (HVGI) tRNAs barley chloroplast genes (NCBI) elements (RFAM) TEs sRNA origin: genes (HVGI) tRNAs barley chloroplast genes (NCBI) elements (RFAM) TEs sRNA origin: genes (HVGI) tRNAs barley chloroplast genes (NCBI) elements (RFAM) TEs sRNA origin: Supplemental Figure 4: Distribution of different classes of sRNAs. Top: Read count of different classes of sRNAs of up, down and non-regulation under drought stress. Middle: Read count fraction of different classes of sRNAs of up, down and non-regulation under drought stress. Bottom: Unique read fraction of different classes of sRNAs of up, down and non-regulation under drought stress.

tRNAs (barley chloroplast) elements (RFAM) TEs sRNA origin: tRNAs barley chloroplast (-tRNAs) barley miRNAs miRNA homologs Supplemental Figure 5: Fraction of non-consistently and consistently regulated sRNAs under two conditions (drought-treated and drought-tolenrant transgenic barley). tRNAs (barley chloroplast)-sRNAs were only derived from chloroplast tRNAs; Barley chloroplast (-tRNAs)-sRNAs were derived from chloroplast without those derived from tRNAs.

B Normalized RNA copies/µl (x10 6 ) A hvu-miR156 hvu-miR159b hvu-miR166a hvu-miR168-5p ath-miR169b ath-miR172a osa-miR393a hvu-miR444b hvu-miR5048 hvu-miR5049a hvu-miR5052 hvu-miRX33hvu-miRX34hvu-miRX35 Supplemental Figure 6: Quantitative real-time PCR of miRNAs (A) and siRNA (B) in leaf and root tissues of GP under water and drought conditions.

Normalized RNA copies/µl (x10 6 ) hvu-miR159b target: CV ath-miR169b target: TC248496osa-miR393a target: TC hvu-miR5048 target: TC hvu-miR5049a target: TC ath-miR169b target: TC254394osa-miR393a target: TC hvu-miR5048 target: TC Supplemental Figure 7: Quantitative real-time PCR of targets of drought-regulated miRNAs in leaf and root tissues of GP under water and drought conditions.