JUNGBRUNNEN1 Confers Drought Tolerance Downstream of the HD-Zip I Transcription Factor AtHB13

Abstract

Low water availability is the major environmental factor limiting growth and productivity of plants and crops and is therefore considered of high importance for agriculture affected by climate change. Identifying regulatory components controlling the response and tolerance to drought stress is thus of major importance. The NAC transcription factor (TF) JUNGBRUNNEN1 (JUB1) from Arabidopsis thaliana extends leaf longevity under non-stress growth conditions, lowers cellular hydrogen peroxide (H₂O₂) level, and enhances tolerance against heat stress and salinity. Here, we additionally find that JUB1 strongly increases tolerance to drought stress in Arabidopsis when expressed from both, a constitutive (CaMV 35S) and an abiotic stress-induced (RD29A) promoter. Employing a yeast one-hybrid screen we identified HD-Zip class I TF AtHB13 as an upstream regulator of JUB1. AtHB13 has previously been reported to act as a positive regulator of drought tolerance. AtHB13 and JUB1 thereby establish a joint drought stress control module.

Keywords: Arabidopsis; HB13; JUB1; drought; transcription factor.

FIGURE 1

JUB1 confers tolerance to drought. (A) Four-week-old jub1-1, WT, JUB1Ox, and RD29A:JUB1 plants were grown in soil and subjected to drought stress by withholding water for 18 days. Photographs were taken 8 days after start of the drought stress experiment (8DD), 16DD, 18DD, and 18DD + 6 days of re-watering (18DD+6DRW). The experiment was repeated more than three times, and a representative result is shown here. (B) Ion leakage of the first six leaves of WT and transgenic lines after 8, 12, and 16 days of drought stress. (C) Relative water content (RWC) of leaves (%). Means ± SD are shown (n = 3). Asterisks represent statistically significant difference from WT; Student’s t-test (^∗p < 0.05).

FIGURE 2

Identification of an upstream regulator of JUB1. (A) A series of 5′ deletions of the JUB1 promoter (including the 5′- untranslated region up to the ATG start codon) were transcriptionally fused to the β-GLUCURONIDASE (GUS) reporter gene and the constructs were transformed into Arabidopsis. (B) Two-week-old seedlings of different Pro_JUB1:GUS deletion lines (1 kb as well as 0.73, 0.68, 0.31, and 0.21 kb) were subjected to H₂O₂ (10 mM) treatment overnight and then incubated at 37°C in GUS buffer. Arrows indicate induced GUS staining. Note the lack of induction of GUS activity in the –0.31-kb and –0.21-kb deletion lines. (C) Four-week-old Pro_JUB1:GUS deletion lines were subjected to drought for 6 days (6DD). Following GUS staining, the lines expressing GUS from the 1, 0.73, and 0.68 kb JUB1 promoter fragments showed higher GUS activity than the corresponding well-watered (control) plants, while no GUS staining was visible in the –0.31-kb and –0.21-kb deletion lines. (D) Yeast-one-hybrid (Y1H) assay demonstrates interaction between the functional 373-bp JUB1 promoter fragment and transcription factor (TF) AtHB13. The JUB1 promoter fragment contains the common binding site of HD-Zip I TFs at positions –618 to –610 bp upstream of the translational start site (ATG). Upon interaction of AtHB13-GAL4AD fusion protein with the binding site, transcription of the yeast HIS3 reporter gene is activated and diploid yeast cells grow on SD medium lacking the three essential amino acids Trp, Leu, and His. The yeast one-hybrid assay was performed three times giving the same result. NC, negative control containing the pTUY1H-JUB1-373 plasmid but no TF as a test for autoactivation. (E) Schematic representation of the HD-Zip I binding site (BS) within the JUB1 promoter. The sequence of the BS as well as the surrounding nucleotides are indicated. (F) EMSA showing binding of purified AtHB13-GST protein to the JUB1 promoter region harboring the HD-Zip I BS. DNA binding reactions were performed with a 40-bp long wild-type fragment derived from the JUB1 promoter containing the HD-Zip I BS. 1, 5′-DY682-labeled, double-stranded oligonucleotide; 2, labeled probe plus AtHB13-GST protein; 3, labeled probe plus AtHB13-GST and 200× competitor (unlabeled oligonucleotide).

FIGURE 3

AtHB13 directly regulates JUB1. (A) Expression of JUB1 in 10-, 20-, and 50-day-old WT, AtHB13Ox, and athb13-1 and athb13-2 plants in well-watered condition. Transcript levels were determined by qRT-PCR; values are expressed as the difference between an arbitrary value of 40 and dCt, so that high 40-dCt value indicates high gene expression level. Means ± SD calculated from three independent biological experiments (each with nine leaves pooled from three plants). Expression levels were normalized against the expression level of ACTIN2. DAS, days after sowing. Asterisk indicates statistically significant difference (Student’s t-test (^∗p < 0.05) from WT. (B) Expression of JUB1 in 35S:AtHB13 (AtHB13Ox) and athb13-1 plants compared to WT upon drought treatment. For drought treatment, 4-week-old plants were subjected to water withholding for 6 days. Whole rosettes of drought-treated and well-watered (control) plants were harvested for gene expression analysis by qRT-PCR. Data represent the means of three biological repetitions ± SD. FCh, fold change. (C) Confocal microscope image showing nuclear localization of the AtHB13-GFP fusion protein in transgenic 35S:AtHB13-GFP Arabidopsis plants. Left, GFP signal; middle, chlorophyll autofluorescence merged with GFP fluorescence; right, chlorophyll autofluorescence. (D) Expression of JUB1 in 35S:AtHB13-GFP plants compared to WT upon drought stress and at a later stage of development (50-day-old plants). For drought treatment, 4-week-old plants were subjected to drought by withholding water for 6 days. Whole rosettes of drought-treated and well-watered (control) plants were harvested for gene expression analysis by qRT-PCR. Data represent the means of three biological repetitions ± SD. FCh, fold change. Asterisks indicate statistically significant difference (p < 0.01; Student’s t-test) from the non-stress control at 34 DAS. (E) ChIP-qPCR showing enrichment of the JUB1 promoter region containing the HD-Zip I binding site, quantified by qPCR. For the ChIP experiment rosettes of 35S:AtHB13-GFP and WT plants were harvested as follows: from 4-week-old control plants (well watered; ‘control’); from plants grown for 4 weeks in well-watered condition and then subjected for 6 d to drought stress by withholding water (‘drought’); and from 50-day-old plants grown under well-watered condition (‘50 DAS’). As negative controls, primers annealing to promoter regions of two Arabidopsis genes lacking an HD-Zip I binding site, i.e., AT3G18040 (Neg. 1) and AT2G22180 (Neg. 2), were used. Data represent the means of three biological repetitions ± SD.

FIGURE 4

AtHB13Ox/jub1-1 plants behave similar to jub1-1 plants during drought stress. (A) Kinetics of relative water loss of leaves form WT, AtHB13Ox, jub1-1, and AtHB13Ox/jub1-1 plants. Plants were grown under well-watered condition for 20 days; thereafter, irrigation was stopped, gradually leading to severe drought stress. (B) Relative water loss in leaves during 9 days of the treatment. For each genotype, leaves from five plants (one leaf per plant) were analyzed. Bars represent SD. The asterisk indicates significant difference from AtHB13Ox/jub1-1 plants (Student’s t-test, p < 0.05).

FIGURE 5

Model for AtHB13-JUB1 regulation of drought stress tolerance. Drought stress induces the expression of both, AtHB13 and JUB1. Increased levels of JUB1 confer enhanced tolerance to drought, in part by lowering cellular reactive oxygen species (hydrogen peroxide) level and by restricting growth via the GA/BR/DELLA pathway. AtHB13 also indirectly induces the expression of glucanase (PR2 and GLU) and chitinase (PR4) genes, each of which enhances drought tolerance when overexpressed. However, control of the PR and GLU genes appears to be independent of JUB1.