Transcriptome analysis of microRNA156 overexpression alfalfa roots under drought stress

Abstract

Drought is one of the major abiotic stresses that negatively impact alfalfa growth and productivity. The role of microRNA156 (miR156) in drought has been demonstrated in plants. To date, there are no published studies investigating the role of miR156 in regulating global gene expression in alfalfa under drought. In our study, alfalfa genotypes overexpressing miR156 (miR156OE) exhibited reduced water loss, and enhanced root growth under drought. Our RNA-seq data showed that in response to drought, a total of 415 genes were upregulated and 169 genes were downregulated specifically in miR156OE genotypes. Genotypic comparison revealed that 285 genes were upregulated and 253 genes were downregulated in miR156OE genotypes relative to corresponding WT under drought. Gene Ontology enrichment analysis revealed that the number of differentially expressed genes belonging to biological process, molecular function and cell component functional groups was decreased in miR156OE genotypes under drought. Furthermore, RNA-Seq data showed downregulation of a gene encoding WD40 repeat in a miR156-specific manner. 5' RACE experiments verified cleavage of WD40-2 transcript under drought. Moreover, alfalfa plants overexpressing WD40-2 showed drought sensitive, whereas those with silenced WD40-2 exhibited drought tolerant phenotypes. These findings suggest that miR156 improves drought tolerance in alfalfa by targeting WD40-2.

Figure 1

Physiological response of WT and miR156OE genotypes to drought. (a) Root length, (b) water loss, measurements in drought stressed and well-watered control WT and miR156OE plants at day 13. An asterisk (*) shows statistical significance at p < 0.05 while double asterisk (**) indicates significance at p < 0.01 where n = 3–8 (ANOVA).

Figure 2

Summarized statistics of de novo assembly of Medicago sativa transcriptome using the Trinity program. (a) Summary of de novo assembly of M. sativa root transcriptome, (b) assembled transcript length distribution.

Figure 3

Comparison of significant DEGs found in WT and two miR156OE genotypes in response to drought stress. Venn diagram shows statistically significant DEGs in (a) total, (b–d) upregulated, and (e–g) downregulated, in WT, A8 and A16b under drought stress conditions relative to corresponding well-watered control plants.

Figure 4

Comparison of significant DEGs found in two miR156OE genotypes relative to corresponding WT. Venn diagram shows statistically significant DEGs (a) total, (b) upregulated and (c) downregulated in A8 and A16b under well-watered control (A8-C, A16b-C) or drought stress (A8-D, A16b-D) conditions relative to corresponding WT.

Figure 5

Distribution of gene ontology (GO) categories involved in drought stress responses among three GO domains. Left panel (a,c,e) indicates number of downregulated whereas right panel (b,d,f) shows upregulated DEGs in WT and miR156OE genotypes in response to drought stress.

Figure 6

WD40-2 cleavage in root of miR156OE genotypes. (a) Validation of WD40-2 cleavages by 5′ RACE in transgenic alfalfa overexpressing miR156. The predicted miR156 target sequences are highlighted in yellow. The WD40-2 sequences are shown and cleavage sites are highlighted in green. The denominator refers to the number of clones sequenced whereas the numerator represents the number of clones cleaved. (b) qRT-PCR analysis of WD40-2 expression in roots of WT and miR156OE genotypes under well-watered control and drought stress conditions.

Figure 7

Effect of drought stress on water conservation and shoot biomass of alfalfa. WT and WD40-2 overexpression, and RNAi genotypes (a) well-watered control conditions, (b) under drought stress, (c) leaf water potential, (d) water loss, and (e) shoot biomass. Data are average of 3-5 biological replications. Single asterisk (*) shows significance of WD40-2 RNAi and overexpression plants with WT at P < 0.05 and double asterisk (**) indicates P < 0.01 (ANOVA).

Figure 8

Effect of drought stress on root growth and root biomass of alfalfa. WT and WD40-2 overexpression, and RNAi genotypes (a) well-watered control, (b) drought stress, (c) root length and (d) root biomass. Data are average of 3-5 biological replications. Single asterisk (*) shows significance of WD40-2 RNAi and overexpression plants with WT at P < 0.05 and double asterisk (**) indicates P < 0.01 (ANOVA).