ABA sensitivity in seedlings of two novel mutants with reduced dormancy of a common wheat cultivar ‘Norin 61’

Fuminori Kobayashi¹, Kazuhide Rikiishi², Chiharu Nakamura¹ and Shigeo Takumi¹

¹: Laboratory of Plant Genetics, Department of Biological and Environmental Science, Faculty of Agriculture, and Graduate School of Science and Technology, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan

²: Research Institute of Bioresources, Okayama University, Kurashiki, Okayama 710-0046, Japan

corresponding author: Shigeo Takumi

Laboratory of Plant Genetics, Department of Biological and Environmental Science, Faculty of Agriculture, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan

E-mail: takumi@kobe-u.ac.jp

Abstract

Abscisic acid (ABA) plays important roles in mediating stress responses and in acquiring dormancy of seeds.  To study ABA sensitivity in two non-dormant mutants of common wheat, seedling growth in ABA-containing water, ABA-responsibility of Cor (cold-responsive)/Lea (late-embryogenesis-abundant) gene expression and freezing tolerance after cold acclimation were analyzed in the mutant lines and their parental cultivar ‘Norin 61’ (N61).  In spite of their non-dormant phenotype, no significant difference was observed in post-germination growth of the parental and the mutant lines.  Our results indicated that the two mutations of ABA sensitivity mainly affect developing seeds.

Abscisic acid (ABA) regulates important aspects of plant growth and development including environmental stress tolerance and seed maturation and dormancy (Leung and Giraudat 1998; Finkelstein et al. 2002).  Regulatory mechanisms of ABA-dependent gene expression have been studied using a vp1 (viviparous1) mutant of maize and abi (ABA-insensitive) mutants of Arabidopsis thaliana, both of which showed reduced levels of seed dormancy and sensitivity to exogenous ABA for inhibition of germination (Leung and Giraudat 1998).  ABA biosynthesis is required for seed maturation and dormancy during seed development, while in vegetative tissues ABA is synthesized de novo mainly in response to drought and high salinity stresses (Xiong and Zhu 2003; Shinozaki et al. 2003).  Many genes as components of stress signaling pathways are induced by exogenous ABA in Arabidopsis and rice (Xiong et al. 2002; Rabbani et al. 2003).  LT and ABA regulatory pathways are not completely independent.  Several Cor/Lea genes are in fact responsive to exogenous ABA, and their promoter sequences commonly contain ABRE (ABA responsive element) (Lång and Palva 1992; Shinozaki and Yamaguchi-Shinozaki 2000). 

However, information on roles of ABA in the regulation of ABRE-containing genes under LT conditions is still limited in wheat and its related species.  An ABA-insensitive, non-dormant line of common wheat, EH47-1, was derived from an ABA sensitive and dormant line, ‘Kitakei-1354’ (Kitakei), a single dominant mutant by EMS (ethylmethan sulfonate) mutagenesis (Kawakami et al. 1997).  Embryos of the mutant line loose sensitivity to ABA during the later half process of seed maturation, while embryos of the parental line maintain the sensitivity even after maturity.  Comparative studies of freezing tolerance after cold acclimation and Cor/Lea gene expression between Kitakei and EH47-1 suggested that ABA sensitivity contributes to determine the basal level of freezing tolerance in wheat (Kobayashi et al. unpublished data).  To obtain more information on the relationship between ABA and cold/freezing tolerance, two novel wheat mutant lines of reduced seed dormancy were identified and analyzed in this study.

Two mutant lines (RSD16-1 and RSD32) of common wheat (Triticum aestivum L.) were selected through NaN3-induced mutagenesis of a strong dormant cultivar ‘Norin 61’ (N61) based on the increased germination rate at DAP40 (40 days-after-pollination) (Fig. 1A).   N61 seeds at DAP40 showed a very low germination rate thus strong dormancy, whereas both mutant lines showed dramatically reduced dormancy at this stage.  Half seeds with embryos germinated normally without exogenous supply of ABA and thus completely lost dormancy in both N61 and the mutants (Fig. 1B), indicating that ABA was supplied from the endosperm.  Exogenous ABA (100µM) reduced the germination rate of the half seeds of N61.  Both mutant lines showed lower levels of sensitivity to the inhibitory effect of exogenous ABA than N61. 

In bioassay for ABA sensitivity based on post-germination growth, 10 seeds from each line were placed in plastic petri dish with distilled water or 20 µM ABA solution and incubated at 20˚C in the darkness.  On the sixth day, lengths of shoots and primary roots were recorded.  In our previous study, this bioassay could efficiently monitor differences in ABA sensitivity among wheat accessions based on seedling growth (Kobayashi et al. unpublished result; Fig. 2A).  According to this parameter, exogenous ABA greatly reduced shoot and root lengths in both N61 and the mutants, but no significant differences were observed between them (Fig. 2B, C).  This result indicated that ABA sensitivity estimated based on the germination rate of developing seeds was not necessarily corresponding to that based on the post-germination growth. 

ABA sensitivity in wheat seedlings was next studied based on the level of ABA-induced gene expression.  For this, ABA treatment was performed by spraying 7-day-old seedlings of N61 and the mutants grown under standard conditions (Ohno et al. 2001) with a solution of 20 µM ABA containing 0.1% (w/v) Tween 20.  Total RNA was extracted 2 hour after the ABA treatment.  Steady state levels of transcripts of two wheat Cor/Lea genes, Wdhn13 and Wrab17, were studied by northern blot analysis using the corresponding cDNA clones as ³²P-labelled probes.  These cDNA clones were previously characterized (Tsuda et al. 2000; Ohno et al. 2003; Kobayashi et al. 2004).  The northern blots showed that Wdhn13 and Wrab17 were clearly induced by exogenous ABA in the mutant lines as well as in Norin 61 (Fig. 3).  Bases on the amount of transcripts, Wdhn13 was more sensitive to ABA than Wrab17, but there were no significant differences between N61 and the mutant lines.

These results indicate that the two mutations of ABA sensitivity mainly affect developing seeds similar to Arabidopsis abi3 mutation (Finkelstein et al. 2002).  Arabidopsis ABI3, which is an ortholog of a maize VP1, has pleiotropic effects on seed maturation, regulation of sensitivity to ABA inhibition of germination, expression of some seed-specific genes, acquisition of desiccation tolerance, and dormancy (Giraudat et al. 1992; Parcy et al. 1994).  Transcripts of wheat Vp-1 genes are alternatively spliced (Mckibbin et al. 2002).  Further expression study of the wheat Vp-1 genes should be required to clarify the relationship between the three mutations and the Vp-1 loci.

For evaluation of freezing tolerance, 7-day-old seedlings of N61 and the mutants were subjected to bioassay according to Kobayashi et al. (2004).  No significant difference however was found in cold acclimated seedlings at 4˚C for 21 days between N61 and the mutants.  Association of ABA sensitivity with cold/freezing tolerance therefore remained unclear.  Isolation of additional mutations affecting ABA sensitivity should be needed to study roles of ABA in abiotic stress responses in wheat.

References

Finkelstein RR, Gampala SS, Rock CD (2002) Abscisic acid signaling in seeds and seedlings. Plant Cell 14 (Suppl): S15-S45.

Giraudat J, Hauge B, Valon C, Smalle J, Parcy F, Goodman H (1992) Isolation of the Arabidopsis ABI3 gene by positional cloning. Plant Cell 4: 1251-1261.

Kawakami N, Miyake Y, Noda K (1997) ABA insensitivity and low ABA levels during seed development of non-dormant wheat mutant. J Exp Bot 48: 1415-1421.

Kobayashi F, Takumi S, Nakata M, Ohno R, Nakamura T, Nakamura C (2004) Comparative study of the expression profiles of the Cor/Lea gene family in two wheat cultivars with contrasting levels of freezing tolerance. Physiol Plant 120: 585-594.

Leung J, Giraudat J (1998) Abscisic acid signal transduction. Annu Rev Plant Physiol Plant Mol Biol 49: 199-222.

Lång V, Palva ET (1992) The expression of a rab-related gene, rab18, is induced by abscisic acid during the cold acclimation process of Arabidopsis thaliana (L.) Heynh. Plant Mol Biol 20: 951-962.

Mckibbin RS, Wilkinson MD, Bailey PC, Flintham JE, Andrew LM, Lazzeri PA, Gale MD, Lenton JR, Holdsworth MJ (2002) Transcripts of Vp-1 homoeologues are miss spliced in modern wheat and ancestral species. Proc Natl Acad Sci USA 99: 10203-10208.

Ohno R, Takumi S, Nakamura C (2001) Expression of a cold-responsive Lt-Cor gene and development of freezing tolerance during cold acclimation in wheat (Triticum aestivum L.). J Exp Bot 52: 2367-2374.

Ohno R, Takumi S, Nakamura C (2003) Kinetics of transcript and protein accumulation of a low-molecular-weight wheat LEA D-11 dehydrin in response to low temperature. J Plant Physiol 160: 193-200.

Parcy F, Valon C, Raynal M, Gaubier-Comella P, Deleny M, Giraudat J (1994) Regulation of gene expression programs during Arabidopsis seed development: Roles of the ABI3 locus and of endogenous abscisic acid. Plant Cell 6: 1567-1582.

Rabbani MA, Maruyama K, Abe H, Khan MA, Katsura K, Ito Y, Yoshiwara K, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2003) Monitoring expression profiles of rice genes under cold, drought, and high-salinity stresses and abscisic acid application using cDNA microarray and RNA gel-blot analyses. Plant Physiol 133: 1755-1767.

Shinozaki K, Yamaguchi-Shinozaki K (2000) Molecular responses to dehydration and low temperature: differences and cross-talk between two stress signaling pathways. Curr Opin Plant Biol 3: 217-223.

Shinozaki K, Yamaguchi-Shinozaki K, Seki M (2003) Regulatory network of gene expression in the drought and cold responses. Curr Opin Plant Biol 6: 410-417.

Tsuda K, Tsvetanov S, Takumi S, Mori, N, Atanassov, A, Nakamura C (2000) New members of a cold-responsive group-3 Lea/Rab-related Cor gene family from common wheat (Triticum aestivum L.). Genes Genet Syst 75: 179-188.

Xiong L, Schumaker KS, Zhu JK (2002) Cell signaling during cold, drought, and salt stress. Plant Cell 14 (Suppl): S165-S183.

Xiong L, Zhu JK (2003) Regulation of abscisic acid biosynthesis. Plant Physiol 133: 29-36.