A Pair of Arabidopsis Diacylglycerol Kinases Essential for Gametogenesis and Endoplasmic Reticulum Phospholipid Metabolism in Leaves and Flowers

Abstract

Phosphatidic acid (PA) is a key phospholipid in glycerolipid metabolism and signaling. Diacylglycerol kinase (DGK) produces PA by phosphorylating diacylglycerol, a crucial step in PA metabolism. Although DGK activity is known to be involved in plant development and stress response, how specific DGK isoforms function in development and phospholipid metabolism remains elusive. Here, we showed that Arabidopsis (Arabidopsis thaliana) DGK2 and DGK4 are crucial for gametogenesis and biosynthesis of phosphatidylglycerol and phosphatidylinositol in the endoplasmic reticulum (ER). With comprehensive transcriptomic data of seven DGKs and genetic crossing, we found that dgk2-1/- dgk4-1/- plants were gametophyte lethal, although parental single homozygous plants were viable. The dgk2-1/+ dgk4-1/+ double heterozygote showed defective pollen tube growth and seed development because of nonviable mutant gametes. DGK2 and DGK4 were localized to the ER and were involved in PA production for pollen tube growth. Transgenic knockdown lines of DGK2 and DGK4 confirmed the gametophyte defect and also revealed defective leaf and root growth. Glycerolipid analysis in the knockdown lines showed that phosphatidylglycerol and phosphatidylinositol metabolism was affected differently in floral buds and leaves. These results suggest that DGK2 and DGK4 are essential during gametogenesis and are required for ER-localized phospholipid metabolism in vegetative and reproductive growth.

Figure 1.

Characterization of Arabidopsis DGK2, DGK4, and DGK6. NPC, nonspecific phospholipase C; PC, nonspecific phospholipase; PIP₂, phosphatidylinositol 4,5-bisphosphate; PIPLC, phosphoinositide-specific phospholipase C. (A) Schematic representation of DGK function in plant PA signaling. (B) Heatmap of tissue-specific expression pattern of DGK genes. Data were analyzed with GENEVESTIGATOR. (C) Positions of T-DNA and primers used for PCR-based genotyping. Black bars, exons; gray bars, introns; white bars, untranslated regions. (D) RT-PCR analysis to detect transcripts of DGK2, DGK4, and DGK6 in homozygous dgk2-1, dgk4-1, and dgk6-1 mutants. WT, wild type.

Figure 2.

Defective Gametogenesis in the Double Heterozygous Mutant of dgk2-1 and dgk4-1 (dgk2-1/+ dgk4-1/+). (A) and (B) Pollen viability in 5-week-old plants by Alexander staining (A) and morphology of pollen grains by scanning electron microscopy imaging (B). Bars in (A) = 200 μm and bars in (B) = 20 μm. White asterisks indicate aborted pollen. The first flower was avoided for observation. WT, wild type. (C) Developing seeds in a mature silique. Shrunken seeds were marked with a black asterisk. Bars = 500 μm. (D) Percentage of viable (black bars) or nonviable (grey bars) pollen observed in (A). More than 800 pollen grains were counted for each line. (E) Percentage of normal (black bars) or shrunken (grey bars) seeds (left) and silique length (right). More than 500 seeds were counted, and at least eight siliques were measured for each line. Statistical significance was analyzed by Student’s t test: ***, P < 0.001.

Figure 3.

Tissue-Specific Localization of DGK2 and DGK4. DGK2_pro:DGK2-GUS line #23 (see [A] to [G] and [O] to [Q]) and DGK4_pro:DGK4-GUS line #12 (see [H] to [N] and [R] to [T]) were subjected to histochemical GUS staining. (A) to (E) and (H) to (L) Time-course GUS staining profile of germinating seedlings. Seeds were stratified in sterile water for 1 d and placed on MS agar plates. Seedlings on day 1 (see [A] and [H]), day 2 (see [B] and [I]), day 3 (see [C] and [J]), day 7 (see [D] and [K]), and day 14 (see [E] and [L]) after plating. (F) and (M) Cauline leaves. (G) and (N) Rosette leaves. (O) and (R) Inflorescence stems. (P) and (S) Flowers at different developmental stages. (Q) and (T) Developing siliques. Bars in (A) to (C), (H), (I), and (J) = 500 μm; bars in (P), (Q), (S), and (T) = 1 mm; and bars in (D) to (G), (K) to (O), and (R) = 2 mm.

Figure 4.

ER Localization of DGK2-Ven and DGK4-Ven in N. benthamiana Leaf Epidermal Cells. (A) and (B) N. benthamiana leaves were bombarded with particles carrying the constructs DGK2_pro:DGK2-Ven (A) or DGK4_pro:DGK4-Ven (B). Fluorescence signals for Ven (green) and ER marker (ER-rk, magenta) are merged. Bars = 20 μm.

Figure 5.

Effect of DGK2 and DGK4 on Pollen Tube Growth. (A) and (B) Percentage of germinated pollen (A) and length of pollen tube (B) of the wild type (WT; Col-0), dgk2-1/+ dgk4-1/+, and WT treated with n-butanol (n-BuOH) or tert-butanol (t-BuOH). (C) Percentage of germinated pollen in the wild type (WT), dgk2-1/–, dgk4-1/–, dgk2-1/+ dgk4-1/+, and DGK2_pro:DGK2 dgk2-1 dgk4-1. (D) Chemical complementation of defective pollen germination in dgk2-1/+ dgk4-1/+ by dioleoyl PA (di18:1-PA) and dilinoleoyl PA (di18:2-PA) in vitro. More than 50 pollen grains were counted in each assay. Statistical significance was analyzed by Student’s t test: *, P < 0.05; ****, P < 0.0001.

Figure 6.

Construction and Screening of the 35S_pro:amiDGK2 dgk4-1 Plants. (A) Schematic representation of the 35S_pro:amiDGK2-I and 35S_pro:amiDGK2-II constructions. Two target sites of amiRNAs designed are indicated. LB, left border; RB, right border; ter, transcriptional terminator. (B) Relative transcript levels of DGK2 in 7-d-old seedlings of the wild type (WT) and four independent transgenic lines: 35S_pro:amiDGK2-I dgk4-1 (lines #1 and #7) and 35S_pro:amiDGK2-II dgk4-1 (lines #2 and #4). Values are mean ± sd of three biological replicates and with three technical replicates. (C) Percentage of viable (black bars) or nonviable (grey bars) pollen by Alexander staining. More than 800 pollen grains were counted for each line. (D) Percentage of normal (black bars) or aborted (grey bars) seeds (left) and length of silique (right). More than 500 seeds were counted, and at least eight siliques were measured for each line. Statistical significance was analyzed by Student’s t test: **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure 7.

Vegetative Growth of the Wild Type, 35S_pro:amiDGK2-I dgk4-1, and 35S_pro:amiDGK2-II dgk4-1. (A) Detached rosette leaves of 24-d-old plants. Bars = 2 cm. WT, wild type. (B) and (C) Seedling size (B) and fresh weight (FW) of the shoot part (C) of 24-d-old, soil-grown wild-type (WT) and 35S_pro:amiDGK2 plants. Values are mean ± sd of 14 plants for each. (D) Primary root length of 14-d-old wild-type (WT) and 35S_pro:amiDGK2 seedlings grown on MS medium. Values in (A) to (D) are mean ± sd of eight plants. (E) Flowering time under long-day (16-h-light/8-h-dark cycle) conditions. The number of rosette leaves was counted when plants were bolting (n = 14). WT, wild type. Statistical significance was analyzed by Student’s t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure 8.

Glycerolipid Contents in Flower Buds of the Wild Type and 35S_pro:amiDGK2-II dgk4-1. (A) Amount of major polar glycerolipid classes normalized by tissue dry weight (DW). WT, wild type. (B) Contents of major polar glycerolipid classes shown in mol % fractions. WT, wild type. (C) Fatty acid composition (mol%) of the polar glycerolipid classes. WT, wild type. Data are mean ± sd from at least three biological replicates. The asterisks indicate significance by Student’s t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 9.

Glycerolipid Contents in Rosette Leaves of the 24-d-Old Wild Type and 35S_pro:amiDGK2-II dgk4-1. (A) Amount of major polar glycerolipid classes normalized by tissue dry weight (DW). WT, wild type. (B) Contents of major polar glycerolipid classes shown in mol % fractions. WT, wild type. (C) Fatty acid composition (mol %) of the polar glycerolipid classes. WT, wild type. Data are mean ± sd from at least three biological replicates. The asterisks indicate significance by Student’s t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 10.

Pulse-Chase Radiolabeling Experiments in Rosette Leaves of the 24-d-Old Wild Type and 35S_pro:amiDGK2-II dgk4-1. (A) Schematic illustration of two distinct radiolabeling methods used in this experiment. [¹⁴C]Acetate is incorporated into the fatty acid synthesis pathway (F.A.S), which produces ¹⁴C-labeled 18:1-CoA to be incorporated into glycerol backbone and thus radiolabel lipid metabolites indicated in the territory of the green dashed square. [γ-³²P]ATP is a substrate of DGK; hence, immediately radiolabeled compound is a product PA. Since PG and PI are derived from PA in ER-localized phospholipid metabolism, metabolites within the territory of the red dashed square will be radiolabeled. G3P, glycerol 3-phosphate; LPA, lysophosphatidic acid. (B) and (C) Pulse-chase radiolabeling analysis of polar glycerolipid metabolism using [¹⁴C]acetate (B) or [γ-³²P]ATP (C). Rosette leaves of the wild type (khaki) and lines 35S_pro:amiDGK2-II dgk4-1 #2 (blue) and #4 (dark brown) were labeled for 1 h with [¹⁴C]acetate or 30 min with [γ-³²P]ATP, and radiolabeled polar glycerolipids were analyzed at different times after chasing. Data are mean ± sd from three biological replicates. The asterisks indicate significance by Student’s t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001.