1. Role of Arabidopsis ABF genes during det1 germination
in salt and osmotic stress conditions
V. C. Dilukshi Fernando, Mark F. Belmonte and Dana F. Schroeder
Dept. of Biological Sciences, University of Manitoba, Winnipeg, MB, Canada R3T 2N2
Abstract
While DE-ETIOLATED 1 (DET1) is well known as a negative regulator of light development, det1 mutants also exhibit altered responses to salt and osmotic stress, such as salt and mannitol resistant germination. LONG HYPOCOTYL 5 (HY5) positively regulates both light and ABA signalling. hy5 suppressed the det1 salt and mannitol resistant germination phenotype, thus, det1 stress resistant germination requires HY5. Publically available microarray data were analyzed to identify genes downstream of HY5 and that are differentially expressed in det1 mutants. This analysis revealed that ABA regulated genes, including ABA RESPONSIVE ELEMENT BINDING FACTOR 3 (ABF3), are downregulated in det1. The role of ABF3 and homologues ABF4 and ABF1 in det1 stress phenotypes was investigated. Molecular analysis revealed that while ABF3 is induced by salt in wildtype seeds, ABF4 and ABF1 are repressed, and that all three of these genes are underexpressed in det1 seeds. Double mutant analysis indicated that abf3, abf4, and abf1 suppress the det1 salt/osmotic stress resistant germination phenotype. In det1 abf1 and det1 abf4 double mutant seeds ABF3 levels are elevated, consistent with the reduced germination observed in these lines. Thus regulation of the ABF genes appears to contribute to det1 salt/osmotic stress response phenotypes. b a c
Fig. 6. ABF4 is required for det1 salt/osmotic stress resistant and ABA sensitive germination
Germination (%) on (a) control (b) 100 mM NaCl (c) 200 mM Mannitol and (d) 2.5 μM ABA. Values are means ± SE of 2 replicates of seeds.
Fig. 3. Identification of candidate genes contributing to det1 germination phenotypes
(a) Summary of double mutant analysis of det1 germination phenotypes. (b) Heat map of enriched GO terms in det1 dark grown seedlings in publicly available microarray data. Downregulated GO terms and upregulated GO terms are represented in left and right columns respectively. GO terms statistically enriched at 10-3 (P<0.001) are blue in colour. (c) Analysis of promoter elements and transcription factors bound to these elements in “response to ABA stimulus” GO term revealed an ABF3 transcriptional module.
Fig. 1. det1 ABA sensitive germination requires HY5 and ABI5
det1 mutants exhibit ABA sensitive germination relative to wild type Col-0. Germination of (a) hy5 det1 and (b) abi5 det1 after 2 days. hy5 and abi5 rescue det1 ABA sensitive germination. Values are means ± SE of 2 replicates of seeds, *=P< 0.05 of single mutants vs wildtype, doubles vs det1. a b
Fig. 7. ABF1 is required for det1 salt/osmotic stress resistant and ABA sensitive germination
Germination (%) on (a) control (b) 100mM NaCl (c) 200mM Mannitol and (d) 2.5 μM ABA. Values are means ± SE of 2 replicates of seeds. a b c
Fig. 2. det1 salt/osmotic stress resistant germination requires HY5 but not ABI5
det1 mutants exhibit salt and osmotic stress resistant germination after 3 days. Germination of (b) hy5 det1 and (c) abi5 det1 on 100 mM NaCl and 200 mM Mannitol after 2 days. hy5 suppresses det1 salt/mannitol resistant germination. However, abi5 det1 and det1 in the Ws-2 background are not significantly different from each other indicating that ABI5 is not required for det1 salt/mannitol resistant germination. Values are means ± SE of 2 replicates of seeds, *=P< 0.05 of single mutants vs wildtype, doubles vs det1. c a b
Fig. 4. In seeds ABF3 is induced by salt while ABF4 and ABF1 are repressed, and all three genes are underexpressed in det1
Real-time PCR analysis of (a) ABF3, (b) ABF4, and (c) ABF1 mRNA levels in Col-0 and det1 seeds imbibed in liquid media in the presence or absence of 150 mM NaCl for 48 h during cold stratification at 4oC. Values are normalized relative to the reference gene EF1α. Error bars indicate SE of 6 technical replicates. *=P≤ 0.05 of det1 vs Col-0 and + =P≤ 0.05 of + NaCl vs – NaCl. a b
Fig. 5. ABF3 is required for det1 salt/osmotic stress resistant and ABA sensitive germination
Germination (%) on (a) control (b) 100mM NaCl (c) 200mM Mannitol and (d) 2.5 μM ABA. Values are means ± SE of 2 replicates of seeds.
Fig. 8. ABF3 transcript levels are upregulated in salt treated det1 abf1 and det1 abf4 seeds
Real-time PCR analysis of ABF3 mRNA levels in (a) det1 abf1 and (2) det1 abf4 seeds imbibed in liquid media in the presence or absence of 150 mM NaCl for 48 h during cold stratification at 4oC. Values are normalized relative to the reference gene EF1α. Error bars indicate SE of 3 technical replicates. *=P≤ 0.05 of single mutants vs Col-0 and double mutants vs det1.
Conclusions
det1 ABA sensitive germination requires both HY5 and ABI5 while det1 salt and osmotic stress resistant germination requires HY5 but not ABI5. ABF3, ABF4, and ABF1 are downregulated in det1 seeds and required for both det1 salt and osmotic stress resistant and ABA sensitive germination. ABF3 is upregulated in det1 abf4 and det1 abf1 double mutants, suggesting regulation of the ABF genes contributes to det1 stress phenotypes.