Glycerol-3-phosphate acyltransferase 4 is essential for the normal development of reproductive organs and the embryo in Brassica napus

Abstract

The enzyme sn-glycerol-3-phosphate acyltransferase 4 (GPAT4) is involved in the biosynthesis of plant lipid poly-esters. The present study further characterizes the enzymatic activities of three endoplasmic reticulum-bound GPAT4 isoforms of Brassica napus and examines their roles in the development of reproductive organs and the embryo. All three BnGPAT4 isoforms exhibited sn-2 acyltransferase and phosphatase activities with dicarboxylic acid-CoA as acyl donor. When non-substituted acyl-CoA was used as acyl donor, the rate of acylation was considerably lower and phosphatase activity was not manifested. RNA interference (RNAi)-mediated down-regulation of all GPAT4 homologues in B. napus under the control of the napin promoter caused abnormal development of several reproductive organs and reduced seed set. Microscopic examination and reciprocal crosses revealed that both pollen grains and developing embryo sacs of the B. napus gpat4 lines were affected. The gpat4 mature embryos showed decreased cutin content and altered monomer composition. The defective embryo development further affected the oil body morphology, oil content, and fatty acid composition in gpat4 seeds. These results suggest that GPAT4 has a critical role in the development of reproductive organs and the seed of B. napus.

Keywords: Brassica napus; GPAT; cutin biosynthesis; embryo development; female fertility; reproductive organ..

Fig. 1.

Expression patterns of BnGPAT genes. The transcript abundance of individual BnGPAT genes was evaluated in different tissues and organs using qRT-PCR. DAF, days after flowering. Error bars denote the SE of three biological replicates.

Fig. 2.

Analysis of substrate specificity and product regiospecificity of BnGPAT4 enzyme assays. (A) Enzyme activities of three BnGPAT4 isoforms using 16:0-CoA as acyl donor. (B) Enzyme activities of three BnGPAT4 isoforms using 16:0 DCA-CoA as acyl donor. (C) Regiospecificities of the 16:0 DCA-MAGs produced in BnGPAT4 enzyme assays. The enzyme assays were performed using 20 μg of yeast microsomal protein incubated with 0.5mM [¹⁴C(U)]glycerol 3-phosphate (0.1 μCi) and 45 μM acyl-CoA in a buffer containing 37.5mM TRIS-HCl (pH 7.5), 2mM MgCl₂, 4mM NaF, 1mM DTT, and 0.1% BSA (w/v) at room temperature for 10min.

Fig. 3.

Transcript abundance of several GPAT family members in gpat4 RNAi lines and the wild type (WT) of B. napus. qRT-PCR was performed to compare the overall transcript abundance of BnGPAT4, 5, 6, and 9 homologues in gpat4 and WT lines. Compared with the WT line, only the transcription level of BnGPAT4 was significantly decreased (P<0.05). n=3. Error bars denote the SE.

Fig. 4.

Abnormal inflorescence development in gpat4 RNAi lines. (A) Comparison of inflorescence between a wild-type plant and a gpat4 RNAi line. (B) Close-up view of the gpat4 inflorescence. The development of the floral buds on the lower portion of the inflorescence was aborted in the gpat4 lines. (C) Close-up view of gpat4 axillary inflorescence primordia. (D) Close-up view of aborted flower buds in a later developmental stage of the axillary inflorescence. A similar phenotype was observed in all gpat4 RNAi lines. (E–G) Comparison of the pistils of the wild type (E and upper G) and gpat4 (F, lower G). Scale bar=1mm.

Fig. 5.

Aborted development of seeds in gpat4 RNAi lines. (A) Comparison between a wild-type plant and a gpat4 line during silique development. A reduced number of siliques was observed in the gpat4 lines. (B) Close-up view of the developing siliques of a gpat4 line. (C, D) Comparison of the developing siliques from Arabidopsis wild-type and gpat4 gpat8 lines. Black arrows in (C) indicate the aborted siliques. Scale bar in (D), 1mm. (E) Seeds inside a developing gpat4 silique. Scale bar=1mm. (F–I) Before and after opening of the developing siliques of the wild type (F, G) and gpat4 lines (H, I). Scale bars in (F, G), 1cm. Scale bars in (H, I), 0.5cm. (J, K) Comparison of the 10 days after flowering (DAF; J) and 25 DAF (K) developing seeds of the wild type (left) and gpat4 lines (right). Scale bar in (J), 1mm. Scale bar in (K), 1cm.

Fig. 6.

Abnormal pollen grains of the gpat4 RNAi lines. (A, B) Pollen grains of the wild type (A) and gpat4 (B) lines under light microscopy. White arrows indicate the deformed pollen grains. (C) Transmission electron microscopy (TEM) image of the wild-type pollen grains. Black arrows indicate the exine deposited on the pollen coat. (D–F) TEM images of the gpat4 pollen grains. Reduced deposition of exine was observed in some of the gpat4 pollen grains. (G, H) Aniline blue staining showing the in vivo pollen tube growth (as indicated by black arrows) inside the pistils of the self-pollinated wild type (G) and gpat4 (H) lines. Scale bars in (G, H), 0.1mm.

Fig. 7.

Reciprocal cross result and examination of the embryo sacs of gpat4. (A, B) Siliques and seeds from reciprocal crosses between wild-type and gpat4 plants. Siliques and seeds from wild-type plants that were pollinated with gpat4 pollen grains were as normal as those from the self-pollinated wild-type plants. Pollination of gpat4 plants with wild-type pollen grains resulted in fewer seeds per silique, similar to the self-pollinated gpat4 plants. (C, D) Unfertilized wild-type (C) and gpat4 (D) ovules collected on the first day of stamen adhesion. The embryo sacs of the wild type and gpat4 exhibited no morphological difference at this stage. Scale bars=0.1mm. (E) A wild-type ovule at 4 d post-pollination. Scale bar=0.1mm. (F–H) Ovules of gpat4 at 4 d post-pollination. The embryo sac inside the wild-type ovule was different from that of gpat4. Scale bars=0.1mm.

Fig. 8.

Down-regulation of GPAT4 homologues resulted in decreased cutin monomer load. (A) The gpat4 lines had decreased cutin content by >30% (P<0.05) in the mature embryos compared with the wild-type (WT) lines; n=3. (B) The cutin monomer profile of the gpat4 and WT lines; n=3. The error bar denotes the SD. (This figure is available in colour at JXB online.)

Fig. 9.

Analysis of the seed oil fatty acid profile of wild-type and T₂ gpat4 lines. (A) Relative mole fraction (mol%) of fatty acids in the total seed oil. (b) A detailed chart of the mole fractions of oleic (18:1), linoleic (18:2), and α-linolenic (18:3) acids of wild-type and gpat4 lines. Asterisks (*) indicate the significant differences in 18:1 contents between the wild type and the gpat4 lines as determined by t-test. Black circles indicate significant differences in the total contents of 18:2 and α-18:3 between the wild type and gpat4 lines as determined by t-test. One asterisk or black circle indicates P<0.05; double asterisks or black circles indicate P<0.01; triple asterisks or black circles indicate P<0.001. n=3 or 4. Error bars denote the SD.

Fig. 10.

Mature embryo sections of the wild type and gpat4. (A, B) Sections of wild-type embryo cotyledons. (C. D) Sections of gpat4 embryo cotyledons. More protein bodies were present in the gpat4 cotyledon cells than in the wild-type cotyledons. (E–G) Transmission electron microscopy (TEM) images of the wild-type cotyledons. The T₂ wild-type segregant line exhibited the same cellular morphology. (H–J) TEM images of the gpat4 cotyledons. PB, protein body. OB, oil body. The oil bodies in wild-type cotyledon cells were compacted and connected to each other; in contrast, the oil bodies in gpat4 cotyledon cells were disconnected from each other and appeared to be smaller and rounder than those observed in the wild type.