Expression of miR159 Is Altered in Tomato Plants Undergoing Drought Stress

Abstract

In a scenario of global climate change, water scarcity is a major threat for agriculture, severely limiting crop yields. Therefore, alternatives are urgently needed for improving plant adaptation to drought stress. Among them, gene expression reprogramming by microRNAs (miRNAs) might offer a biotechnologically sound strategy. Drought-responsive miRNAs have been reported in many plant species, and some of them are known to participate in complex regulatory networks via their regulation of transcription factors involved in water stress signaling. We explored the role of miR159 in the response of Solanum lycopersicum Mill. plants to drought stress by analyzing the expression of sly-miR159 and its target SlMYB transcription factor genes in tomato plants of cv. Ailsa Craig grown in deprived water conditions or in response to mechanical damage caused by the Colorado potato beetle, a devastating insect pest of Solanaceae plants. Results showed that sly-miR159 regulatory function in the tomato plants response to distinct stresses might be mediated by differential stress-specific MYB transcription factor targeting. sly-miR159 targeting of SlMYB33 transcription factor transcript correlated with accumulation of the osmoprotective compounds proline and putrescine, which promote drought tolerance. This highlights the potential role of sly-miR159 in tomato plants' adaptation to water deficit conditions.

Keywords: Colorado potato beetle; MYB transcription factors; P5CS; Solanum lycopersicum; drought; miR159; proline; putrescine.

Figure 1

RT-qPCR analysis of sly-miR159 expression and its MYB predicted targets in tomato plants undergoing drought stress. (A) RT-qPCR analysis of sly-miR159 expression in control tomato plants and tomato plants following 7-day water deprivation. (B) Nucleotide sequence of sly-miR159-binding sites in tomato GAMYB-like transcripts. Nucleotides in the cleavage site are underlined, lower-case red letters indicate mismatches to sly-miR159, and G:U pairing is shown in uppercase green letters. (C) RT-qPCR analysis of SlMYB33, SlMYB65, SlMYB104, SlMYB97, and SlMYB120 genes expression in control tomato plants and tomato plants following 7-day water deprivation. In panels (A) and (C), data shown are the mean of three independent experiments ± standard error (SE). Asterisk indicates that differences between means of control and undergoing drought stress tomato plants were statistically significant (Student’s t-test, p < 0.05).

Figure 2

RT-PCR amplification of SlMYB33 mRNA fragments. (A) Nucleotide sequence of sly-miR159-binding sites in SlMYB33 transcripts. Bar in red depicts the putative cleavage site and arrows indicate the annealing positions of primer pair OFw and ORv, outside the sly-miR159-binding region, and primer pair FFw and FRv, flanking the putative cleavage site in sly-miR159-binding region. (B) RT-PCR analysis of SlMYB33 small RNA fragments in control tomato plants and tomato plants following 7-day water deprivation using primers OFw and ORv, or FFw and FRv. RPS18 gene expression was used as normalization control. For each sample, three biological replicates were pooled and analyzed.

Figure 3

Analysis of SlP5CS gene expression, and amino acid and polyamines in tomato plants undergoing drought stress. (A) RT-qPCR analysis of SlP5CS expression in control tomato plants and tomato plants following 7-day water deprivation. (B) Amino acids levels upon drought treatment. Amino acids levels are expressed in µg/g DW. (C) Polyamines levels upon drought treatment. Polyamines levels are expressed in µg/g DW. Put (putrescine), Spd (Spermidine), Spn (Spermine). Tomato leaves were collected from plants that were properly irrigated (Control) or deprived of water 1 week (Drought). Data shown are the mean of three independent experiments ± standard error (SE). Asterisk indicates that differences between means of control and undergoing drought stress tomato plants were statistically significant (Student’s t-test, p < 0.05).

Figure 4

Analysis of SlP5CS gene expression, and amino acid and polyamines in tomato plants infested by Colorado potato beetle (CPB) larvae. (A) RT-qPCR analysis of SlP5CS expression in control tomato plants and tomato plants infested by CPB larvae. (B) Amino acids levels upon CPB infestation. Tomato leaves were collected from non-infested plants (Control) or plants infested by CPB. Amino acids levels are expressed in µg/g DW. (C) Polyamines levels upon CPB larvae infestation. Tomato leaves were collected from non-infested plants (Control) or plants infested by CPB. Polyamines levels are expressed in µg/g DW. Put (putrescine), Spd (Spermidine), Spn (Spermine). Data shown are the mean of three independent experiments ± standard error (SE). Asterisk indicates that differences between means of control and undergoing drought stress tomato plants were statistically significant (Student´s t-test, p < 0.05).

Figure 5

RT-qPCR analysis of sly-miR159 expression and its MYB predicted targets in tomato plants infested by CPB larvae. (A) RT-qPCR analysis of sly-miR159 expression in control tomato plants and tomato plants infested by CPB larvae. (B) RT-qPCR analysis of SlMYB33, SlMYB65, SlMYB104, SlMYB97, and SlMYB120 genes expression in control tomato plants and tomato plants upon CPB larvae infestation. In panels (A) and (B), data shown are the mean of three independent experiments ± standard error (SE). Asterisk indicates that differences between means of control tomato plants and tomato plants infested by CPB larvae were statistically significant (Student´s t-test, p < 0.05).