Ectopic expression of WRINKLED1 in rice improves lipid biosynthesis but retards plant growth and development

Abstract

WRINKLED1 (WRI1) is a transcription factor which is key to the regulation of seed oil biosynthesis in Arabidopsis. In the study, we identified two WRI1 genes in rice, named OsWRI1a and OsWRI1b, which share over 98% nucleotide similarity and are expressed only at very low levels in leaves and endosperms. The subcellular localization of Arabidopsis protoplasts showed that OsWRI1a encoded a nuclear localized protein. Overexpression of OsWRI1a under the control of the CaMV 35S promoter severely retarded plant growth and development in rice. Expressing the OsWRI1a gene under the control of the P1 promoter of Brittle2 (highly expressed in endosperm but low in leaves and roots) increased the oil content of both leaves and endosperms and upregulated the expression of several genes related to late glycolysis and fatty acid biosynthesis. However, the growth and development of the transgenic plants were also affected, with phenotypes including smaller plant size, later heading time, and fewer and lighter grains. The laminae (especially those of flag leaves) did not turn green and could not unroll normally. Thus, ectopic expression of OsWRI1a in rice enhances oil biosynthesis, but also leads to abnormal plant growth and development.

Fig 1. Neighbor-joining unrooted tree.

Bootstrap values were calculated for 100 replicates, and values are indicated at the corresponding nodes. The database accession numbers of sequences are indicated in brackets.

Fig 2. Expression analysis of the OsWRI1a/OsWRI1b genes.

qRT-PCR applied to total RNA samples was used to measure OsWRI1a/OsWRI1b gene expression. Relative expression was normalized to that of the reference gene ubiquitin (internal control). The 10 d represent endosperms and embryos were sampled from seeds at 10 days after flowering. Bars show means ± SD of three biological replicates.

Fig 3. Subcellular localization of OsWRI1a.

Transiently expressed the GFP (upper) and OsWRI1a-GFP fusion protein (lower) in Arabidopsis protoplasts which observed under a laser scanning confocal microscope. Bars = 10 μm.

Fig 4. The phenotypes of P_Bt2P1::OsWRI1a plants.

(A) Plants at the jointing-booting stage. (B) Plants at heading stage. (C) Plants at the mature stage. (D) Expression analysis of the OsWRI1a gene in leaves of plants at heading stage. The experiment included three biological replicates, each with two technical replicates. Values represent means of n = 6 ± SD (Duncan test: **, P < 0.01). (E) and (F) Lengths of internodes and panicles of the main stems. Values in (F) represent means of n = 30 ± SD. P1-6, -7, -8, and -10, P_Bt2P1::OsWRI1a transgenic lines.

Fig 5. The abnormal phenotypes of leaves in P_Bt2P1:: OsWRI1a plants.

(A) to (D) The developing course of abnormal lamina of flag leaves in P_Bt2P1:: OsWRI1a plants. P1-6, -7, -8, and -10, P_Sh2P1:: OsWRI1a transgenic lines. (E) The lamina of the second leaf of heading stage plants. Bar = 10 cm. (F) The chlorophyll content in the second leaves of heading stage plants. Values represent means of n = 6 ± SD (Duncan test: **, P < 0.01). (G) The FA and TAG content in leaves of heading stage plants. Values represent means of n = 3 ± SD (Duncan test: **, P < 0.01). DW = dry weight. (H) and (I) FA composition in free FA (H) and TAG (I) in leaf blades of heading stage plants. Values represent means of n = 3 ± SD (Duncan test: **, P < 0.01). C16:0, palmitic acid; C16:1 palmitoleic acid; C18:0, stearic acid; C18:1, oleic acid; C18:2, linoleic acid; C18:3, linolenic acid. DW = dry weight. P1-6 and -7, P_Sh2P1::OsWRI1a transgenic lines.

Fig 6. Changes in TAG accumulation in endosperm and embryo.

(A) The TAG content in endosperm of mature seeds. (B) FA composition in TAG of endosperm. Values represent means of n = 3 ± SD (Duncan test: **, P < 0.01; *, P < 0.05). DW = dry weight. P1-6, and -7, P_Sh2P1::OsWRI1a transgenic lines. C16:0, palmitic acid; C18:0, stearic acid; C18:1, oleic acid; C18:2, linoleic acid; C18:3 arachidic acid.

Fig 7. Expression of genes predicted to encode proteins involved in the later stages of glycolysis and in fatty acid biosynthetic pathways in developing endosperms.

Total RNA was isolated from endosperms of the seeds of plants 5 and 10 days after flowering (5 d, 10 d). The experiment included three biological replicates, each with two technical replicates. Values represent means of n = 6 ± SD (Duncan test: **, P < 0.01; *, P < 0.05). P1-6, -7, -8, and -10, P_Bt2P1::OsWRI1a transgenic lines. The gene accession numbers are: OsACP, LOC_Os08g43580; OsENR1, LOC_Os08g23810; OsFatA, LOC_Os09g32760; OsKASIII, LOC_Os04g55060; OsPDH-E1α, LOC_Os04g02900; OsPDH-E1β, LOC_Os12g42230; OsPDH-E2, LOC_Os12g08170; OsPK, LOC_Os10g42100; OsRUB1, LOC_Os06g46770.