Circadian clock factors regulate the first condensation reaction of fatty acid synthesis in Arabidopsis

Abstract

The circadian clock regulates temporal metabolic activities, but how it affects lipid metabolism is poorly understood. Here, we show that the central clock regulators LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) regulate the initial step of fatty acid (FA) biosynthesis in Arabidopsis. Triacylglycerol (TAG) accumulation in seeds was increased in LHY-overexpressing (LHY-OE) and decreased in lhycca1 plants. Metabolic tracking of lipids in developing seeds indicated that LHY enhanced FA synthesis. Transcript analysis revealed that the expression of genes involved in FA synthesis, including the one encoding β-ketoacyl-ACP synthase III (KASIII), was oppositely changed in developing seeds of LHY/CCA1-OEs and lhycca1. Chromatin immunoprecipitation, electrophoretic mobility shift, and transactivation assays indicated that LHY bound and activated the promoter of KASIII. Furthermore, phosphatidic acid, a metabolic precursor to TAG, inhibited LHY binding to KASIII promoter elements. Our data show a regulatory mechanism for plant lipid biosynthesis by the molecular clock.

Keywords: Arabidopsis; CP: Plants; Clock regulation of lipid metabolism; LHY/CCA1; Lipid-clock interconnection; b-ketoacyl-ACP synthase III (KASIII); circadian clock; fatty acid synthesis; lipid signaling; phosphatidic acid; seed oil; transcriptional regulation.

Figure 1.. Seed oil content in LHY/CCA1-altered plants

(A) Total TAG content in dry seeds. TAG from the indicated plant lines was measured by gas chromatography and expressed as % (w/w) of total seed weight. (B) Contents of individual FAs. The data in (A) are shown here as the amounts of individual species of FAs (number of carbons: number of double bonds). Values in both panels are mean ± SD with individual data points and p values less than 0.05 on the bar denoting statistical significance determined by Student’s t test (to WT; n = 5 groups of seeds). See also Figure S1F.

Figure 2.. TAG accumulation during seed development

(A) Thin-layer chromatography (TLC) separation of TAG. Total lipids were extracted from developing seeds of WT, lhycca1, and LHY-OE at the indicated DAF. The lipids were separated by TLC and visualized by iodine exposure. Arrowhead indicates the position of TAG. Std, authentic TAG standard. (B) Contents of total TAG. TAG spots in A were scraped out and quantified by gas chromatography for TAG contents. (C) Contents of individual FAs. The data in (B) are shown here as the amounts of individual species of FAs (number of carbons: number of double bonds). Values in (B) and ( are mean ± SD with asterisks (*) denoting statistical significance of the slope determined by Student’s t test (to WT; n = 3 groups of seeds; p < 0.05). See also Figure S2.

Figure 3.. Metabolic tracking of radiolabeled lipid precursors of TAG

(A) TAG biosynthetic pathways. Radioactive substrate used in this study is highlighted in red. Enzymes are in blue and the major lipid precursors (PC and DAG) and the final product (TAG) are in bold. CPT, choline phosphotransferase; DGAT, acyl-Co-A:DAG acyltransferase; G3P, glycerol-3-phosphate; GPAT, glycerol-3-phosphate acyltransferase; LPA, lysoPA; LPAT, LPA acyltransferase; LPC, lysoPC; LPCAT, LPC acyltransferase; PAH, PA phosphohydrolase; PDAT, PC:DAG acyltransferase; PDCT, PC:DAG cholinephosphotransferase. (B) Pulse labeling. The 10 DAF seeds of WT, lhycca1, and LHY-OE were incubated with [¹⁴C]-acetate for the indicated time. Total lipids were extracted and separated by thin-layer chromatography. Radioactivities of TAG, DAG, and PC were measured and are shown here as per microgram chlorophyll of the seeds. (C) Pulse-chase labeling. The 10 DAF seeds pre-incubated with [¹⁴C]-acetate for 3 h were washed to remove radioisotopes and further incubated for the indicated time. Lipids were processed and analyzed as in (B). Values in (B) and (C) are mean ± SD with asterisks (*) denoting statistical significance determined by Student’s t test (to WT; n = 5 groups of seeds; p < 0.05). See also Figures S3 and S4.

Figure 4.. Expression of the genes required for FA synthesis

(A) FA biosynthetic pathway. Intermediate enzymes, cofactors, and byproducts are omitted for simplicity. The chain length (number of carbons) of FAs produced by each KAS is in parentheses. ACP, acyl carrier protein. (B) Subunits of heteromeric ACC. Note that relative size and position of the subunits are different from reality. See text for abbreviations. Numbers refer to isoforms in Arabidopsis. (C) Expression levels of plastidic ACC subunits. Total RNA was extracted from 10-DAF seeds of WT, lhycca1, and LHY/CCA1-OEs. Quantitative real-time PCR was performed with gene-specific primers. (D) Expression levels of cytosolic ACC and KAS. The experiments performed and data layout are as in (C). Values in (C) and (D) are mean ± SD with individual data points and p values < 0.05 on the bar denoting statistical significance determined by Student’s t test (to WT; n = 3 groups of seeds). (E) Time-course expression of KASIII. The data were generated from a public microarray database (Diurnal; diurnal.mocklerlab.org), where WT (Ler) and LHY-OE were grown under 8-h light/16-h dark cycles and are shown here as normalized to the level at 0 h. Pearson’s correlation coefficient: WT =0.872, LHY-OE = 0.979.

Figure 5.. LHY/CCA1 binding to KASIII promoter

(A) Upstream region of KASIII coding sequence. At 1,469 bps upstream from transcription start site (1,825 bps from start codon) omission of some intervening nucleotides is shown (••••). The 5′-untranslated region is underlined, and the start codon is in bold. EE and CBS are in red and blue, respectively. Arrows indicate primer binding sites for ChIP-PCR (red, P1; blue, P2; green, P3). (B) ChIP-PCR results. DNA immunoprecipitated with CCA1 was amplified by PCR using primers indicated on the right for the gene promoters on the left. TOC1 at the bottom is a positive control. C1 and C2, two independent CCA1-OEs; I, input DNA; C, ChIP DNA. (C) Purified LHY protein. LHY was expressed in and purified from E coli and subjected to SDS-PAGE followed by Coomassie blue staining. Size of marker proteins (M) is on the left. (D) EMSA. The 60-bp DNA probes containing WT or mutated (M) EE and CBS were incubated with the indicated amounts of the purified LHY and separated in an agarose gel. (E) PA effect on LHY binding to EE. EMSA was performed as in D with WT EEs, LHY (20 μg), and the indicated amounts of PA from egg yolk; 18:1 and 16:0 indicate the acyl chain at sn-1 position of PA species with 18:1 at sn-2 (10 μg each). See also Figure S5.

Figure 6.. LHY/CCA1 activation of KASIII promoter

(A) Schematic diagram of the reporter assay vectors. Effector (E) and reporter (R) genes and their promoters are shown. C, control vector; T, test vector. (B) LUC activity in the plants with different effector/reporter. Tobacco leaves were transformed with the indicated combinations of vectors. Gel images are PCR results from the transformed tobacco DNA with the primers indicated on the right. Total proteins extracted were measured for intensities of fluorescence (GFP) and luminescence (LUC). At the bottom is baseline (None)-subtracted values shown as LUC normalized to GFP. Values are mean ± SD with individual data points and p values less than 0.05 on the bar statistical significance determined by Student’s t test (to None for GFP and LUC; to test effector and test reporter (T_ET_R) for LUC/GFP; n = 5 infiltrated leaves). RFU, relative fluorescence unit; RLU, relative luminescence unit; None, non-transformant; N/A, not applicable. (C) Proposed model for LHY/CCA1-regulated seed oil accumulation. LHY and CCA1 enhance KASIII expression by binding to KASIII promoter through its EE and/or CBS. KASIII then catalyzes the synthesis of FAs that are incorporated into TAG during seed development.