The olive DGAT2 gene is developmentally regulated and shares overlapping but distinct expression patterns with DGAT1

Abstract

Diacylglycerol acyltransferases (DGATs) catalyse the final step of the triacylglycerol (TAG) biosynthesis of the Kennedy pathway. Two major gene families have been shown to encode DGATs, DGAT1 (type-1) and DGAT2 (type-2). Both genes encode membrane-bound proteins, with no sequence homology to each other. In this study, the molecular cloning and characterization of a type-2 DGAT cDNA from olive is presented. Southern blot analysis showed that OeDGAT2 is represented by a single copy in the olive genome. Comparative transcriptional analysis revealed that DGAT1 and DGAT2 are developmentally regulated and share an overall overlapping but distinct transcription pattern in various tissues during vegetative growth. DGAT2 is highly expressed in mature or senescing olive tissues. In flowers, the expression of DGAT1 was almost undetectable, while DGAT2 transcripts accumulated at the later stages of both anther and ovary development. Differential gene regulation was also detected in the seed and mesocarp, two drupe compartments that largely differ in their functional roles and mode of lipid accumulation. DGAT1 appears to contribute for most of the TAG deposition in seeds, whereas, in the mesocarp, both DGAT1 and DGAT2 share an overlapping expression pattern. During the last stages of mesocarp growth, when TAGs are still accumulating, strong up-regulation of DGAT2 but a marked decline of DGAT1 transcript levels were detected. The present results show overlapping gene expression for olive DGATs during mesocarp growth, with a more prominent implication of DGAT2 in floral bud development and fruit ripening.

Fig. 1.

Nucleotide sequence and deduced amino acid sequence, in single-letter code, of the olive type-2 diacylglycerol acyltransferase cDNA (OeDGAT2). The stop codon is denoted by an asterisk. Numbers represent the position of nucleotides and amino acids. Arrows indicate the position of primers used. A predicted ER-retrieval motif is denoted with a discontinuous lined box at the protein C-terminus. The amino acid sequences corresponding to the predicted transmenbrane domains are underlined.

Fig. 2.

Phylogenetic relationships among deduced amino acid sequences of OeDGAT1, OeDGAT2, and other plant diacylglycerol acyltransferases (DGATs). The tree was constructed according to the Neighbor–Joining algorithm. GenBank accession numbers are given in parentheses. Numbers at branch points are bootstrap percentages derived from 1000 replicates. Only values ≥50% are presented. Numbers within brackets correspond to % amino acids sequence homologies between olive and other species within each group.

Fig. 3.

Southern analysis of olive genomic DNA digested with EcoRI (E), EcoRI/HindIII (E/H), or HindIII (H) and probed with a cDNA fragment of OeDGAT2. Hybridization was performed at 65 °C (0.745 M Na⁺). Blot was subjected to high stringency wash conditions (3× in 0.21 M Na⁺ and 3× in 0.075 M Na⁺ at 65 °C). Numbers indicate molecular size markers in kb.

Fig. 4.

Northern blot analysis of DGAT1 and DGAT2 expressions during olive fruit development. Total RNA was extracted from drupes, embryos, endosperms, and mesocarps at different developmental stages. Drupes lanes: 5 (1–2 mm in diameter drupes), 7 (3–4 mm), 9 (5–8 mm), and 11 (9–11 mm) weeks after flowering (WAF). Embryo lanes: 13 (early torpedo stage), 16 (early-mid torpedo), 19 (mid-late torpedo), and 22 (late torpedo) WAF. Endosperm lanes: 13, 16, 19, and 22 WAF. Mesocarp lanes: 13, 16, 19, 22, 25, and 28 WAF. Equivalent amounts of rRNA were loaded onto a gel and stained with ethidium bromide to evaluate equal loading in each lane (lower panel).

Fig. 5.

Localization of OeDGAT1 and OeDGAT2 transcripts in young drupes at 7 weeks after flowering (WAF). Longitudinal sections were processed for in situ hybridization with DIG-labelled antisense RNA probes of OeDGAT1 (C, D) and OeDGAT2 (E, F). Negative controls were included by using two respective DIG-labelled riboprobes sense probes. Only representatives using OeDGAT1 sense-probe are presented here (A, B). DGAT1 expression was detected in mesocarp (me), the developing seed coat tissues (sc), the perisperm (pe), and the globular embryo (em), while the expression was relatively lower in endosperm (en) and the integuments (in). Sites of positive hybridization signals are shown as blue/violet regions. Scale bar represents 300 μm.

Fig. 6.

Semi-quantitative RT-PCR expression analysis of olive DGAT1 and OeDGAT2 genes. First-strand cDNAs were synthesized from total RNA extracted from roots (R), hypocotyls (H), cotyledons (C), and shoot tips (ST) of olive seedlings; expanding (EL), young (YL), and mature (ML) leaves; embryos (E) at 13 weeks after flowering (WAF) and mesocarps (M) at 22 WAF; flower buds 1.0 mm (B1) and 2.0 mm (B2) in length; anthers (A) and ovaries (O) of flowers 2.5–3.0 mm in length. To ensure equal amounts of template, olive β-tubulin was used as a reference gene.

Fig. 7.

In situ localization of DGAT1 and DGAT2 transcript accumulation in transverse sections of expanding olive leaves. A representative negative control using the OeDGAT1 sense-probe is presented here (A). Signals of DGAT1 expression are prominent in almost every cell type including palisade parenchyma (pp), spongy parenchyma (sp), and phloem (ph) of the central vein, while DGAT2 mRNA is mostly localized to the phloem tissue of the vascular bundle. Sites of positive hybridization signals are shown as blue/violet regions. Scale bar represents 50 μm.

Fig. 8.

In situ localization of OeDGAT1 and OeDGAT2 transcript accumulation in longitudinal sections of anthers (A–C) and pistils (D–F) from buds of about 2.5 mm in length. A representative negative control using the OeDGAT2 sense-probe is presented (A, D). DGAT1 exhibited no labelling or negligible background reaction in pollen grains (p) (B) or in ovaries (E). DGAT2 transcripts are largely localized in the tapetum cells (t) of anthers (C) and in the stigma (s) and the vascular bundles (vb) of ovaries (F). Sites of positive hybridization signals are shown as blue/violet regions. Scale bar represents 500 μm.