ABI4 activates DGAT1 expression in Arabidopsis seedlings during nitrogen deficiency

Abstract

Triacylglycerol (TAG) is the major seed storage lipid and is important for biofuel and other renewable chemical uses. Acyl-coenzyme A:diacylglycerol acyltransferase1 (DGAT1) is the rate-limiting enzyme in the TAG biosynthesis pathway, but the mechanism of its regulation is unknown. Here, we show that TAG accumulation in Arabidopsis (Arabidopsis thaliana) seedlings increased significantly during nitrogen deprivation (0.1 mm nitrogen) with concomitant induction of genes involved in TAG biosynthesis and accumulation, such as DGAT1 and OLEOSIN1. Nitrogen-deficient seedlings were used to determine the key factors contributing to ectopic TAG accumulation in vegetative tissues. Under low-nitrogen conditions, the phytohormone abscisic acid plays a crucial role in promoting TAG accumulation in Arabidopsis seedlings. Yeast one-hybrid and electrophoretic mobility shift assays demonstrated that ABSCISIC ACID INSENSITIVE4 (ABI4), an important transcriptional factor in the abscisic acid signaling pathway, bound directly to the CE1-like elements (CACCG) present in DGAT1 promoters. Genetic studies also revealed that TAG accumulation and DGAT1 expression were reduced in the abi4 mutant. Taken together, our results indicate that abscisic acid signaling is part of the regulatory machinery governing TAG ectopic accumulation and that ABI4 is essential for the activation of DGAT1 in Arabidopsis seedlings during nitrogen deficiency.

Figure 1.

Accumulation of storage oil in seedlings for different CN treatments. A, TAG content of 7-d-old Arabidopsis seedlings in Suc-free medium containing different N concentrations. Arabidopsis seedlings were grown in a growth chamber under a cycle of 16 h of light at 22°C and 8 h of dark at 20°C. Total lipids were extracted and separated by TLC. The arrow indicates the TAG bands. B, Phenotypes of 7-d-old Arabidopsis seedlings in different CN medium. Bars = 2 mm. C, Oil bodies in Arabidopsis cotyledons of seedlings grown in different CN medium. Bars = 20 μm. D, Total lipid was extracted from 7-d-old seedlings grown on different CN medium to determine the TAG content (arrow) by TLC.

Figure 2.

TAG biosynthesis is induced with 0.1 mm N. A, Total RNA was isolated from 7-d-old seedlings grown on medium with 0.1 and 60 mm N and different Suc concentrations. Relative mRNA levels of key genes in storage oil metabolism were determined by quantitative real-time RT-PCR using ACTIN1 as an internal control. Data represent three independent experiments, and the error bars represent sd. DGAT1, DGAT2, and PDAT1 are genes in the TAG biosynthesis pathway. OLEOSIN1 is essential for the production of seed-specific oil bodies. SDP1, ACX1, and ACX2 are genes involved in TAG degradation. B, Oil bodies in seedlings grown on 60 mm N for 7 d and then transferred to 0.1 mm N medium for another 7 d (c and d). Plants grown for 7 d on the 60–50 medium were used as controls (a and b). Seedlings were stained with Nile Red. Bars = 20 μm. [See online article for color version of this figure.]

Figure 3.

Influence of ABA on ectopic accumulation of storage oil during N deprivation. A, Phenotypes of 7-d-old seedlings on 60–50 medium without or with 10 μm final concentrations of different plant hormones. The 7-d-old seedling grown on 0.1–50 medium was chosen as a control. Bars = 2 mm. B, TAG content (arrow) of 7-d-old Arabidopsis seedlings grown on 60–50 (lanes 1 and 2) or 0.1–50 (lanes 3 and 4) medium in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of 10 μm final concentrations of 10 μm cytokinin (6-BA), GA₃, auxin (IAA), ethylene (ACC), or ABA. Total lipid was extracted and separated by TLC. C, Expression levels of key genes involved in ABA biosynthesis and signaling in 7-d-old seedlings grown on 60–50 or 0.1–50 medium. Relative mRNA levels were determined by quantitative real-time RT-PCR using ACTIN1 as an internal control. Data represent three independent experiments, and the error bars represent sd. D, Percentage of postgermination growth, defined as cotyledon expansion and greening, in 7-d-old seedlings grown on different CN medium. About 100 seeds were used in each experiment. Data represent three independent experiments, and average values are shown with sd. E, Germination rate of Arabidopsis seeds sown on MS medium with different CN concentrations. The percentage of germination (radicle emergence) was determined after 24 h of growth in light. About 100 seeds were used in each experiment. Data represent three independent experiments, and the error bars represent sd. [See online article for color version of this figure.]

Figure 4.

Tobacco transient assay for the interaction between ABI4 and the DGAT1 promoter. A, Analysis of the Arabidopsis DGAT1 promoter sequence, showing the core sequence of the CE1-like element. B, Schematic of the ABI4 plant expression constructs and the 5′ deletion of the DGAT1 promoter used in the tobacco transient assay. The effector plasmid contained the cauliflower mosaic virus 35S promoter fused to ABI4 cDNA. The reporter plasmid contained the DGAT1 −1,000 to +226 or −117 to +226 promoter region fused to the GUS gene. C, Histochemical GUS assays of Agrobacterium infiltrated with different constructs. Bars = 5 mm. D, Relative GUS activity directed by pD1000 and pD117 alone or together with 35S:ABI4. Data represent three independent experiments, and the error bars represent sd. [See online article for color version of this figure.]

Figure 5.

Interaction between DGAT1 CE1-like elements and ABI4. A, Interaction of ABI4 and the DGAT1 promoter in yeast cells. The β-galactosidase activity indicates the LacZ expression level. Data represent three independent experiments, and the error bars represent sd. B, Growth of yeast cells on 45 mm 3-aminotriazole (3-AT) His^– SD medium. Cells were grown in liquid medium to an optical density at 600 nm of 1.0. The numbers at the top indicate the dilutions. C, Electrophoretic mobility shift analysis of interactions between ABI4 and DGAT1 CE1-like elements. The sequence of the 165-bp DGAT1 fragment is shown at the top. Labeled CE1-CE1 DGAT1 promoter sequence was incubated with 20 ng (lane 2) or 100 ng (lane 3) of purified ABI4 protein, and the DNA probe incubated with GST served as the negative control (lane 1). Nonlabeled DGAT1 promoter sequences CE1-CE1 (lane 4), mCE1-CE1 (lane 5), and CE1-mCE1 (lane 6) were used at a 50-fold molar excess as competitors. The numerals 1 and 2 indicate nonmutated sequences, whereas m1 and m2 indicate mutated sequences. [See online article for color version of this figure.]

Figure 6.

Analysis of the TAG content and the DGAT1 expression levels in abi mutants. A to C, The 7-d-old Col-0 (a−d) and abi4 (e−h) seedlings grown on 60–50 (a, b, e, and f) or 0.1–50 (c, d, g, and h) medium in the absence (a, c, e, and g) or presence (b, d, f, and h) of 10 μm ABA. Bars = 1 mm. A, The phenotypes of the seedlings. B, Total lipid was isolated from seedlings and analyzed by TLC to show TAG content (arrow). C, GUS expression of the DGAT1 promoter in 7-d-old seedlings. D, The transcription level of DGAT1 in 7-d-old Col-0 and abi4 seedlings grown on 60–50 or 0.1–50 medium. E, Levels of DGAT1 mRNA in 7-d-old abi3, abi5, and their wild-type (Landsberg erecta [Ler] and Ws) seedlings grown on 0.1–50 or 60–50 medium with or without 10 μm ABA. F, Levels of OLEOSIN1 mRNA in 7-d-old abi3, abi4, abi5, and wild-type (Landsberg erecta, Col-0, and Ws) seedlings grown on 60–50 or 0.1–50 medium. Relative mRNA levels were determined by quantitative real-time RT-PCR using ACTIN1 as an internal control. Data represent three independent experiments, and the error bars represent sd.