NAC transcription factor JUNGBRUNNEN1 enhances drought tolerance in tomato

Abstract

Water deficit (drought stress) massively restricts plant growth and the yield of crops; reducing the deleterious effects of drought is therefore of high agricultural relevance. Drought triggers diverse cellular processes including the inhibition of photosynthesis, the accumulation of cell-damaging reactive oxygen species and gene expression reprogramming, besides others. Transcription factors (TF) are central regulators of transcriptional reprogramming and expression of many TF genes is affected by drought, including members of the NAC family. Here, we identify the NAC factor JUNGBRUNNEN1 (JUB1) as a regulator of drought tolerance in tomato (Solanum lycopersicum). Expression of tomato JUB1 (SlJUB1) is enhanced by various abiotic stresses, including drought. Inhibiting SlJUB1 by virus-induced gene silencing drastically lowers drought tolerance concomitant with an increase in ion leakage, an elevation of hydrogen peroxide (H₂ O₂ ) levels and a decrease in the expression of various drought-responsive genes. In contrast, overexpression of AtJUB1 from Arabidopsis thaliana increases drought tolerance in tomato, alongside with a higher relative leaf water content during drought and reduced H₂ O₂ levels. AtJUB1 was previously shown to stimulate expression of DREB2A, a TF involved in drought responses, and of the DELLA genes GAI and RGL1. We show here that SlJUB1 similarly controls the expression of the tomato orthologs SlDREB1, SlDREB2 and SlDELLA. Furthermore, AtJUB1 directly binds to the promoters of SlDREB1, SlDREB2 and SlDELLA in tomato. Our study highlights JUB1 as a transcriptional regulator of drought tolerance and suggests considerable conservation of the abiotic stress-related gene regulatory networks controlled by this NAC factor between Arabidopsis and tomato.

Keywords: Arabidopsis; DELLA; drought; reactive oxygen species; tomato; transcription factor.

Figure 1

SlJUB1 encodes a nuclear protein and is induced by various abiotic stresses. (a) Confocal microscope image showing nuclear localization of SlJUB1‐GFP fusion protein upon transient expression in N. benthamiana leaf cells. Scale bar, 5 μm. (b–d) SlJUB1 expression upon treatment with (b) H₂O₂ (5 mm), (c) NaCl (200 mm), (d) PEG 6000 (20% [w/v]). Three‐week‐old tomato seedlings were subjected to the stress treatments and harvested at the time points indicated at the x‐axes. (e) SlJUB1 expression upon dehydration treatment. Terminal leaflets (of leaf 2) were detached and subjected to 2 h and 3 h of desiccation, respectively. Transcript levels were measured using qRT‐PCR; numbers at the y‐axis indicate fold change (FCh; log2 basis) compared to controls (unstressed plants). Data represent means ± SD (two independent biological replications with three technical replications per assay).

Figure 2

Suppression of SlJUB1 leads to drought sensitivity in tomato. The role of SlJUB1 for drought sensitivity was assessed by VIGS. (a) Phenotypes of TRV2‐SlJUB1 and TRV2‐GUS (control) plants under control condition (well watered; left) and after drought stress (3 days: middle; 7 days: right). Note the more severe leaf wilting in the TRV2‐SlJUB1 plant. (b) Ion leakage of TRV2‐GUS and TRV2‐SlJUB1 leaves (leaf no. 2, terminal leaflet) 7 days after start of the drought treatment. Data represent means ± SD (n = 3). (c) Endpoint PCR analysis of SlJUB1 expression in TRV2‐GUS and TRV2‐SlJUB1 plants after 3 days of drought stress. (d) Phenotypes of detached terminal leaflets from leaf no. 2 (left) and DAB staining for visualization of ROS accumulation (right) of TRV2‐SlJUB1 (upper row) and pTRV2‐GUS plants (lower row) subjected to dehydration treatment for 3 h. (e) Ion leakage of TRV2‐GUS and TRV2‐SlJUB1 leaves after 10 h of dehydration treatment. (f) Water loss in detached leaves of TRV2‐GUS (grey columns) and TRV2‐SlJUB1 (black columns) plants. Data represent means ± SD (n = 3). Asterisk in panels (b), (e) and (f) represent statistically significant differences between TRV2‐SlJUB1 and TRV2‐GUS (Student's t‐test; P < 0.05). (g) Heatmap showing the fold change (log2 basis) difference in the expression of drought‐responsive genes and tomato orthologs of AtJUB1 direct target genes, compared between TRV2‐SlJUB1 and TRV2‐GUS plants after drought stress (2 h). Gene expression was determined by qRT‐PCR. Data represent the mean of two biological replications with three technical replications per assay.

Figure 3

SlJUB1 binds to the promoters of SlDREB2, SlDREB1 and SlDELLA . (a) Schematic representation of the position of the AtJUB1 binding sites in the promoters of SlDREB2, SlDREB1 and SlDELLA (relative to the translation start codon; numbers indicate the start position of the binding sites). Binding sites are located on the forward strand in the case of SlDREB2 and SlDELLA , and on the reverse strand in the case of SlDREB1. (b) EMSA showing binding of SlJUB1 to SlDREB2, SlDREB1 and SlDELLA promoter regions harbouring the JUB1 binding site; 1, labelled probe (5′‐DY682‐labelled double‐stranded oligonucleotide) only; 2, labelled probe plus SlJUB1‐GST protein; 3, labelled probe, SlJUB1‐GST protein and 100× competitor DNA (unlabelled oligonucleotide containing SlJUB1 binding site). (c) Transcript levels of SlJUB1, SlDREB1, SlDELLA and SlDREB2 in TRV2‐SlJUB1 plants 3 days after water withholding compared with TRV2‐GUS . Expression was analysed by qRT‐PCR. Data represent the means of three independent experiments.

Figure 4

Ectopically expressed AtJUB1‐GFP in tomato confers tolerance to water deprivation. (a) Phenotype of AtJUB1‐expressing ( OX1) and wild‐type tomato cv. Moneymaker (MM) plants under well‐watered control (left) and water‐deficit conditions (right): 42‐day‐old plants were subjected to drought for 7, 14 and 21 days. Note the more severe wilting in MM plants. (b) Relative water content of terminal leaflets (leaf no. 2) of MM and OX1 plants measured during drought treatment. Data represent the means ± SD (n = 4 independent experiments). (c) Shoot fresh weight of MM and OX1 plants after 21 days of drought. (d) Malondialdeyhde (MDA) content of MM and OX1 plants during water deprivation. (e) Wilting phenotype and DAB staining for ROS accumulation in detached leaves of MM and OX1 plants, 10 h after start of the dehydration treatment. (f) Percent water loss in detached leaves of MM and OX1 plants. Data in (c), (d) and (f) represent the means ± SD (n = 3). Asterisks (*) indicate statistically significant differences between MM and OX1 according to Student's t‐test (P < 0.05).

Figure 5

AtJUB1 directly regulates SlDREB2, SlDREB1 and SlDELLA . (a) Expression of SlDREB2, SlDREB1 and SlDELLA in MM and AtJUB1‐GFP ( OX1) plants upon 7 days of withholding water. Expression was analysed by qRT‐PCR. Values were normalized to those determined in the well‐watered controls. Data represent the means ± SD (n = 3). Asterisks represent statistically significant differences between MM and OX1 plants according to Student's t‐test (P < 0.05). (b) EMSA showing binding of AtJUB1 to SlDREB2, SlDREB1 and SlDELLA promoter regions harbouring the AtJUB1 binding site; 1, labelled probe (5′‐DY682‐labelled double‐stranded oligonucleotides) only; 2, labelled probe plus AtJUB1‐GST protein; 3, labelled probe, AtJUB1‐GST protein and 100× competitor (unlabelled oligonucleotide containing SlJUB1 binding site); 4, labelled probe plus GST protein. (c) ChIP‐qPCR shows enrichment of SlDREB2, SlDREB1 and SlDELLA promoter regions containing the AtJUB1 binding site. For ChIP experiments, terminal leaflets (from leaf no. 2) of AtJUB1‐GFP tomato plants were harvested after drought treatment (7 days). Values were normalized to the values for Solyc01G090460 (promoter lacking an AtJUB1 binding site). qPCR was used to quantify the enrichment of SlDREB2, SlDREB1 and SlDELLA promoter regions. Data represent means ± SD (n = 3). FC, fold change.

Figure 6

Model for the action of JUNGBRUNNEN1 (JUB1) in conferring tolerance to drought in tomato. Water deprivation triggers elevated expression of SlJUB1, which leads to activation of DELLA and the stress‐related genes DREB2 and DREB1. This, together with reduced ROS levels, increases drought tolerance.