1. The Basis of ABA phenotypes in Arabidopsis det1 mutants
V.C. Dilukshi Fernando and Dana F. Schroeder
Dept. of Biological Sciences, University of Manitoba, Winnipeg, MB, Canada A B
Figure 3. Effect of drought stress on Germination and relative primary root growth of the wild type, det1 and 35S:DET1 mutants.
Germination assay showing germination rates under 400 mM Sorbitol & Mannitol 5 days after stratification. det1 mutants are resistant to drought stress in terms of seed germination.
Comparative analysis of root lengths of wild type and mutants at 5 days, after being transferred to plates with or without Sorbitol or Mannitol (200mM and 400mM). det1 seedlings are resistant to both Sorbitol and Mannitol.
Water loss from detached leaves of the wild type, det1 and 35S:DET1 mutants.. Results are from 2 replicates and values are means ± SD.
Statistically significant differences are indicated by asterisks.
Figure 1. Effect of ABA on Germination and relative primary root growth of the wild type, det1 and 35S:DET1 mutants.
Germination assay showing germination rates under 3 different concentrations of ABA 3 days after stratification. det1 mutants are sensitive to ABA inhibition of seed germination.
Comparative analysis of root lengths of wild type and mutants after ABA treatment (0.5, 2.5, 5 M) in the dark for 5 days.
Statistically significant differences are indicated by asterisks. A B
Abstract
Seed germination and seedling development in plants are regulated by both light and phytohormones. Recent studies indicate light and hormone signalling pathways are connected with each other at the molecular level by means of signalling integrators such as LONG HYPOCOTYL 5 (HY5) in Arabidopsis thaliana. The phytohormone abscisic acid (ABA), plays an essential role in plant growth, development and as an endogenous messenger in stress signal transduction pathways. Thus, understanding ABA signalling is essential in improving plant performance. Genetic studies of ABA regulation of gene expression and seed germination have identified a number of Arabidopsis mutants with altered ABA sensitivities. Preliminary studies reveal that the light signalling mutant de-etiolated 1 (det1) shows altered sensitivity to ABA. Several genes including HY5, ABI5, ABI3, DWA1 and DWA2 potentially interact with both DET1 and ABA and we are currently investigating which of these intermediates are essential for the det1ABA response. The findings of this research will allow us to investigate a novel phenotype involved in ABA signalling in Arabidopsis which will ultimately pave the way to improving abiotic stress performance of crop plants.
Introduction
Due to recent evidence of interactions between light signaling and hormone response, we are interested in the role of DET1, a negative regulator of light signaling; abiotic stress tolerance, particularly those mediated by ABA signaling.
To asses the abiotic responses in det1 mutants to ABA, salt and osmotic stresses we used partial loss of function mutant det1-1 as well as gain of function 35S:Myc-DET1 line, an overexpression line of DET1 in Col-0; in the preliminary experiments.
Figure 2. Effect of salt stress on Germination, relative primary root growth and survival of the wild type, det1 and 35S:DET1 mutants.
Germination assay showing germination rates under 2 different concentrations of NaCl 3 days after stratification. det1 mutants are resistant to salt stress in terms of seed germination.
Comparative analysis of root lengths of wild type and mutants at 5 days after being transferred to plates with or without NaCl (100mM and 200mM).
Statistically significant differences are indicated by asterisks.
(C) Survival assay showing the percentage of surviving individuals 7 days after seedlings were transferred to plates with indicated concentrations of NaCl. det1 seedlings are hypersensitive to salt and do not survive in NaCl media. B A C
Discussion
Based on the altered ABA sensitivity in the det1 mutants, we propose that det1 mutants by pass the effect of further downstream effects like NaCl and osmotic stress due to its precautious germination strategy. Therefore, det1 is not actually salt or drought tolerant but it has a germination phenotype.
The overall results show (Table 01) that early germination in det1 is not inhibited by exogenous NaCl or osmotic stress. Early germination in det1 is also light-independent (Chory et al., 1989). However, early germination in det1 is still regulated by ABA and also Gibberellic acid because det1 mutants require Gibberellic acid synthesis for germination (Nambara et al., 1991).
The root growth of det1 is resistant to inhibition of ABA, Mannitol and Sorbitol. Genetic studies indicate that promotion of primary root growth by drought stress and ABA are adaptive responses of roots to drought stress as well as drought tolerance of the whole plant (Xiong, et a., 2006). Interestingly, det1 is not significantly affected by NaCl in the root assays and also det1 is highly sensitive to higher concentrations of salt stress and do not survive after being transferred to 200 mM NaCl media (Fig. 2C). In the overexpression line of DET1 there is no consistent significant effect of ABA salt or osmotic stress on its germination In contrast, it shows some significant sensitivity to Sorbitol and Mannitol and resistance to ABA in the root assays.
There are several intermediate genes in the det1 ABA response that interact either genetically or biochemically. In future studies we will be investigating the genetic interactions of these intermediates by assessing the double mutants in the det1 background.
Materials and methods
Seed germination percentage was determined for an average of 70 seeds from two independent experiments. Seeds were plated on 1 LS media with 0.6% sucrose, 8.6% Phytoagar enriched with different concentrations of ABA, NaCl, Sorbitol and Mannitol. The plates were stratified in 40C for 2 days and then transferred to 200C and long day conditions.
For Root Growth experiments seedlings were initially grown on 1 LS media and then transferred to different concentrations of indicated stress media after 1 week. Root Lengths were measured 5 days after transfer.
Percentage survival was determined by the number of individuals with green cotyledons 1 week after transfer.
Rosette leaves were detached from 5 weeks old plants and their fresh weights were measured at indicated times to determine water loss. Water loss shows the percentage of weight loss versus initial fresh weight.