CYP707A1 and CYP707A2, which encode abscisic acid 8'-hydroxylases, are indispensable for proper control of seed dormancy and germination in Arabidopsis

Abstract

Endogenous abscisic acid (ABA) levels are regulated by both biosynthesis and catabolism of the hormone. ABA 8'-hydroxylase is considered to be the key catabolic enzyme in many physiological processes. We have previously identified that four members of the Arabidopsis (Arabidopsis thaliana) CYP707A gene family (CYP707A1 to CYP707A4) encode ABA 8'-hydroxylases, and that the cyp707a2 mutants showed an increase in ABA levels in dry and imbibed seeds. In this study, we showed that the cyp707a1 mutant accumulated ABA to higher levels in dry seeds than the cyp707a2 mutant. Expression analysis showed that the CYP707A1 was expressed predominantly during mid-maturation and was down-regulated during late-maturation. Concomitantly, the CYP707A2 transcript levels increased from late-maturation to mature dry seed. Phenotypic analysis of single and double cyp707a mutants indicates that the CYP707A1 is important for reducing ABA levels during mid-maturation. On the other hand, CYP707A2 is responsible for the regulation of ABA levels from late-maturation to germination. Moreover, CYP707A1 and CYP707A3 were also shown to be involved in postgermination growth. Spatial expression analysis suggests that CYP707A1 was expressed predominantly in embryo during mid-maturation, whereas CYP707A2 expression was detected in both embryo and endosperm from late-maturation to germination. Our results demonstrate that each CYP707A gene plays a distinct role during seed development and postgermination growth.

Figure 1.

Phenotypic analysis of the cyp707a mutant seeds. A, Genomic structures of the cyp707a1-1 (SALK_069127) and cyp707a1-2 (WiscDsLox393-396J9) mutants. The cyp707a1-1 and cyp707a1-2 mutants harbor the T-DNA at the third and fifth exons, respectively. B, Dry seed ABA levels in the cyp707a mutants. Experiments were performed three times using independent seed batches and the averages are shown with ses. DW, Dry weight. C, Dormancy of the freshly harvested seeds in cyp707a mutants. Experiments were performed five times using independent seed batches and the averages are shown with ses.

Figure 2.

ABA biosynthesis and catabolism during seed development. A, Quantification of endogenous ABA, PA, and DPA levels in siliques during seed development. Experiments were performed three times using independent seed batches and the averages are shown with ses. FW, Fresh weight. B and C, Changes in the transcript levels of AtNCEDs and CYP707As in siliques determined by QRT-PCR. Experiments were performed four times and the averages are shown with ses. D and E, Quantification of endogenous ABA in siliques in cyp707a mutants 12 DAF (D) and 16 DAF (E). Experiments were performed three times and the averages are shown with ses.

Figure 3.

Localization of CYP707A transcripts during seed development. A, QRT-PCR quantification of mRNA levels of PDF1 and AtEPR1 in three silique fractions harvested at 10 DAF (left) and 15 DAF (right). QRT-PCR quantification of mRNA levels of CYP707A genes in three silique fractions harvested at 10 DAF (B) and 15 DAF (C). Results of QRT-PCR were confirmed in two independent assays. In situ hybridization of CYP707A1 mRNA in 10-DAF (D) and CYP707A2 mRNA in 15-DAF siliques (E). The arrows indicate site of strong signal in seeds (D and E).

Figure 4.

Phenotypic analysis of cyp707a double-mutant seeds. A, Dry seed ABA level in the cyp707a double mutants. Experiments were performed three times using independent seed batches and the averages are shown with ses. B, Dormancy of seeds after ripened for 1 week in cyp707a single and double mutants. Experiments were performed five times and the averages are shown with ses. C, Quantification of endogenous seed ABA levels in the cyp707a single and double mutants after seed imbibition. Two hundred milligrams of seeds were imbibed in 8.5-cm petri dishes containing two filter papers (approximately 7-cm diameter) and 4 mL of water. Experiments were performed twice using independent seed (after ripening for 1 week) batches.

Figure 5.

Localization of CYP707A mRNAs after seed imbibition. A, QRT-PCR quantification of mRNA levels of AtGA3ox1 and AtEPR1 (A) and CYP707As (B) in embryo or endosperm/testa of imbibed seeds at 6 h. Results of QRT-PCR were confirmed in two independent assays. C, In situ hybridization of CYP707A2 mRNA in imbibed seeds for 6 h. Longitudinal (left) and transverse (right) sections of imbibed seeds were hybridized with an antisense CYP707A2 probe.

Figure 6.

Germination and postgermination growth of the cyp707a single and double mutants in the presence of exogenous ABA. A, Germination was scored based on radicle emergence. B, Postgermination growth was scored based on greening of cotyledons. After-ripened seeds for 3 months were sown on agar plates containing several concentration of (+)-S-ABA and placed at 22°C under continuous light for 7 d after 4 d of stratification at 4°C. Experiments were performed four times and the averages are shown with ses.