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. 2008 Mar;146(3):1368-85.
doi: 10.1104/pp.107.113738. Epub 2007 Dec 27.

High temperature-induced abscisic acid biosynthesis and its role in the inhibition of gibberellin action in Arabidopsis seeds

Shigeo Toh  1 , Akane Imamura, Asuka Watanabe, Kazumi Nakabayashi, Masanori Okamoto, Yusuke Jikumaru, Atsushi Hanada, Yukie Aso, Kanako Ishiyama, Noriko Tamura, Satoshi Iuchi, Masatomo Kobayashi, Shinjiro Yamaguchi, Yuji Kamiya, Eiji Nambara, Naoto Kawakami
Affiliations

High temperature-induced abscisic acid biosynthesis and its role in the inhibition of gibberellin action in Arabidopsis seeds

Shigeo Toh et al. Plant Physiol. 2008 Mar.

Abstract

Suppression of seed germination at supraoptimal high temperature (thermoinhibiton) during summer is crucial for Arabidopsis (Arabidopsis thaliana) to establish vegetative and reproductive growth in appropriate seasons. Abscisic acid (ABA) and gibberellins (GAs) are well known to be involved in germination control, but it remains unknown how these hormone actions (metabolism and responsiveness) are altered at high temperature. Here, we show that ABA levels in imbibed seeds are elevated at high temperature and that this increase is correlated with up-regulation of the zeaxanthin epoxidase gene ABA1/ZEP and three 9-cis-epoxycarotenoid dioxygenase genes, NCED2, NCED5, and NCED9. Reverse-genetic studies show that NCED9 plays a major and NCED5 and NCED2 play relatively minor roles in high temperature-induced ABA synthesis and germination inhibition. We also show that bioactive GAs stay at low levels at high temperature, presumably through suppression of GA 20-oxidase genes, GA20ox1, GA20ox2, and GA20ox3, and GA 3-oxidase genes, GA3ox1 and GA3ox2. Thermoinhibition-tolerant germination of loss-of-function mutants of GA negative regulators, SPINDLY (SPY) and RGL2, suggests that repression of GA signaling is required for thermoinibition. Interestingly, ABA-deficient aba2-2 mutant seeds show significant expression of GA synthesis genes and repression of SPY expression even at high temperature. In addition, the thermoinhibition-resistant germination phenotype of aba2-1 seeds is suppressed by a GA biosynthesis inhibitor, paclobutrazol. We conclude that high temperature stimulates ABA synthesis and represses GA synthesis and signaling through the action of ABA in Arabidopsis seeds.

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Figures

Figure 1.
Figure 1.
ABA biosynthesis in imbibed seeds is required for thermoinhibition. After-ripened Col seeds were imbibed at 22°C (white circles), 34°C (black squares), and at 34°C in the presence of 10 μm fluridone (black triangles) under continuous illumination. A, Effect of ABA biosynthesis inhibitor fluridone on thermoinhibition. Error bars show sd (n = 3). B, Effects of high temperature and ABA biosynthesis inhibitor fluridone on endogenous ABA levels in imbibed seeds. ABA levels were quantified by GC-MS using two independent seed batches and similar results were obtained.
Figure 2.
Figure 2.
Effect of high temperature on expression of ABA biosynthesis and catabolism genes in imbibed seeds. Total RNA was prepared from after-ripened Col seeds imbibed at 22°C (white diamonds) or at 34°C (black squares) under continuous illumination. Transcript levels were quantified by quantitative RT-PCR as described in “Materials and Methods.” Experiments were repeated twice with different seed batches and similar results were obtained. Data from one of the replicates are shown. Values are means with sds from three measurements.
Figure 3.
Figure 3.
NCED2, NCED5, and NCED9 are unequally redundant on thermoinhibition and ABA synthesis in the seeds. A, Thermoinhibition resistance of single and multiple mutants of nced2, nced5, and nced9. Seeds were imbibed at 22°C (white bars), 33°C (gray bars), and 34°C (black bars) under continuous illumination for 5 d. Error bars show sd (n = 3). Germination assays were confirmed in three independent batches. B, ABA content in seeds of nced single and multiple mutants. ABA was extracted from dry seeds (white bars) and the seeds imbibed at 34°C for 24 h (gray bars) and 36 h (black bars) under continuous illumination. ABA was quantified by liquid chromatography-tandem mass spectrometry. Dotted line indicates ABA content in wild-type seeds imbibed at 22°C for 24 h. Error bars show sd (n = 3).
Figure 4.
Figure 4.
Expression of NCED2, NCED5, and NCED9 in embryo and endosperm of thermoinhibited seeds. Total RNA was prepared from embryo and endosperm/testa separated from Col seeds imbibed at 22°C or at 34°C under continuous illumination for 24 h. Transcript levels of NCED2 (white bars), NCED5 (gray bars), and NCED9 (black bars) were quantified by quantitative RT-PCR with a set of primers and a Taq-Man probe specific to each gene. To compare transcript levels across different NCED genes, standard curves were generated using a series of known concentration of target sequences (genomic DNA). Results for each tissue were normalized to the amplification of the 18S rRNA control and the amount of total RNA extracted from each tissue of one seed. Experiments were repeated three times with different seed batches and similar results were obtained. Data from one of the replicates are shown. Values are means with sds from three measurements.
Figure 5.
Figure 5.
Alleviation of thermoinhibition by exogenous GA and suppression of bioactive GA levels by high temperature. A, Effect of exogenous GA on thermoinhibition. After-ripened Col seeds were imbibed at 22°C (white circles), 34°C (black squares), and 34°C with 50 μm GA3 (black triangles) under continuous illumination. Error bars show sd (n = 3). B, Effect of temperature on bioactive GA levels in seeds. GA levels were quantified by GC-MS using two independent seed batches and similar results were obtained.
Figure 6.
Figure 6.
Expression of GA biosynthesis and deactivation genes was suppressed at high temperature. Total RNA was prepared from after-ripened Col seeds imbibed at 22°C (white diamonds) or at 34°C (black squares) under continuous illumination. Transcript levels were quantified by quantitative RT-PCR as described in “Materials and Methods.” Experiments were repeated twice with similar results. Data from one of the replicates are shown. Values are means with sds from three measurements.
Figure 7.
Figure 7.
Thermoinhibition-resistant germination of spy-4 and rgl2 seeds and expression of the GA negative regulator genes at high temperature. A, T-DNA insertion sites in the RGL2 gene. Arrow indicates 5′ to 3′ direction of the gene. B, Effect of imbibition temperature on germination of spy and rgl2 mutant seeds. Seeds were imbibed at 22°C (white bars), 32°C (light gray bars), 33°C (dark gray bars), and 34°C (black bars) under continuous illumination for 5 d. Error bars show sd (n = 3). C, Effect of temperature on expression of SPY and RGL2 genes. Total RNA was prepared from after-ripened Col seeds imbibed at 22°C (white diamonds) or at 34°C (black squares) under continuous illumination. Transcript levels were quantified by quantitative RT-PCR as described in “Materials and Methods.” Experiments were repeated twice with similar results. Data from one of the replicates are shown. Values are means with sds from three measurements.
Figure 8.
Figure 8.
Modulation of GA synthesis and signaling by ABA in thermoinhibited seeds. A, Effect of GA biosynthesis inhibitor PAC on thermoinhibition-tolerant germination of ABA-deficient seeds. The seeds of aba2-1 were imbibed at 22°C and at 33°C under continuous illumination for 6 d in the presence or absence of PAC and GA3. All the imbibition solutions contained 10 μm fluridone to ensure ABA deficiency. Asterisk denotes that the germination test at 22°C in the presence of both PAC and GA3 was not done. Error bars show sd (n = 3). B, Expression of GA synthesis and signaling genes in ABA-deficient mutant seeds. Seeds of wild type (Col) and aba2-2 were imbibed at 22°C (white bars) and 33°C (black bars) under continuous illumination. Transcript levels were quantified by quantitative RT-PCR at 12 h (GA20ox) or at 24 h (GA3ox and SPY) after the start of imbibition. Experiments were repeated three times with different seed batches and similar results were obtained. Data from one of the replicates are shown. Values are means with sds from three measurements.
Figure 9.
Figure 9.
Regulation of ABA synthesis, GA synthesis, and GA signaling by high temperature in Arabidopsis seeds. High temperature enhances expression of specific ABA biosynthesis genes (ABA1/ZEP, NCED2, NCED5, and NCED9) under continuous illumination. High temperature also enhances expression of a GA negative regulator gene (SPY) and suppresses expression of GA biosynthesis genes (GA20ox1, GA20ox2, GA20ox3, GA3ox1, and GA3ox2) not directly, but through the action of ABA. Expression of GA20ox2 and GA20ox3 may be regulated by both ABA and high temperature. Transcripts of DELLA protein genes are not increased by high temperature, but their protein activity may be enhanced by SPY. A different set of NCED genes (NCED6 and NCED9) works during seed development and contributes to dormancy development. Expression of NCED6 is induced by far-red (FR) light, and those of GA3ox1 and GA3ox2 are induced by red (R) light. Expression of a set of GA biosynthesis genes (GA20ox1, GA20ox2, and GA3ox1) is induced by low temperature and suppressed by high temperature and may contribute to germination control in a wide range of temperatures. [See online article for color version of this figure.]
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