Expression of ZmLEC1 and ZmWRI1 increases seed oil production in maize

Abstract

Increasing seed oil production is a major goal for global agriculture to meet the strong demand for oil consumption by humans and for biodiesel production. Previous studies to increase oil synthesis in plants have focused mainly on manipulation of oil pathway genes. As an alternative to single-enzyme approaches, transcription factors provide an attractive solution for altering complex traits, with the caveat that transcription factors may face the challenge of undesirable pleiotropic effects. Here, we report that overexpression of maize (Zea mays) LEAFY COTYLEDON1 (ZmLEC1) increases seed oil by as much as 48% but reduces seed germination and leaf growth in maize. To uncouple oil increase from the undesirable agronomic traits, we identified a LEC1 downstream transcription factor, maize WRINKLED1 (ZmWRI1). Overexpression of ZmWRI1 results in an oil increase similar to overexpression of ZmLEC1 without affecting germination, seedling growth, or grain yield. These results emphasize the importance of field testing for developing a commercial high-oil product and highlight ZmWRI1 as a promising target for increasing oil production in crops.

Figure 1.

Transgenic overexpression of ZmLEC1 in maize. A, Effects of overexpression of ZmLEC1 under the control of the EAP1 promoter on embryo oil concentration and seed oil content. Each point represents an average of null seeds (white triangles) and transgenic seeds (black triangles) from each transgenic line. A total of 15 transgenic lines were analyzed. B, Seed oil content of T3 homozygous transgenic lines (black bars) and their null segregants (white bars). For each line, 10 seeds per ear, five homozygous transgenic or null ears, were analyzed. Data shown are means ± sd. All three transgenic lines showed a significant increase in seed oil content compared with their corresponding null segregants as determined by Student's t test (P < 0.01). C, Warm germination test of transgenic seeds. Transgenic and null seeds were placed between two sheets of filter paper and germinated at 25°C for 5 d. D, Transgenic and null plants at the three- to four-leaf stage. Seed was planted in soil mixture and grown in the greenhouse. The photograph shows a typical line 7 d after planting.

Figure 2.

ZmLEC1 activates the expression of GUS driven by a maize WRI1 promoter in maize BMS culture cells. ZmWRI1 promoter:GUS reporter or ZmWRI1 promoter:GUS reporter/Ubi promoter:ZmLEC1 was introduced into maize BMS culture cells via Agrobacterium transformation. Expression of GUS from the ZmWRI1 promoter was monitored 24, 48, 72, 96, and 120 h after transformation. The images show GUS staining of culture cells at 120 h post transformation.

Figure 3.

Transgenic overexpression of ZmWRI1 in maize. A, Effects of overexpression of ZmWRI1 under the control of the OLE promoter on embryo oil concentration and seed oil content. Each point represents an average of 10 null seeds (white triangles) and 10 transgenic seeds (black triangles) from each transgenic line. A total of 15 transgenic lines were analyzed. B, Seed oil content of T3 homozygous transgenic lines (black bars) and their corresponding null segregants (white bars). For each transgenic line, 10 seeds per ear, eight homozygous transgenic or null ears, were analyzed. Data shown are means ± sd. All five transgenic lines showed a significant increase in seed oil content compared with null segregants as determined by Student's t test (P < 0.01). C, Warm germination test of transgenic seeds. Transgenic and null seeds were placed between two sheets of filter paper and germinated at 25°C for 5 d. D, Transgenic and null plants at the three- to four-leaf stage. Seeds were planted in soil mixture and grown in the greenhouse. The photograph shows a typical line 7 d after planting.

Figure 4.

Protein and starch contents of ZmWRI1 transgenic embryos. The data represent means ± sd of three replicate samples. Each sample was run in triplicate in starch and protein assays. All three transgenic lines showed no significant difference in embryo protein content (P > 0.1 by Student's t test) but did show a significant reduction in embryo starch content as determined by Student's t test (P < 0.05).

Figure 5.

Early stand count and plant height of transgenic ZmWRI1 plants in the field. Six rows of transgenic and three rows of null were planted in the field for each transgenic line. Early stand count percentage was calculated by the number of plants divided by the total seeds planted at 3 weeks after planting. Plant height of 10 plants in the middle of each row was measured. Data shown are means ± sd. All five transgenic lines showed no significant difference from null as determined by Student's t test (P > 0.1).

Figure 6.

Yield test of transgenic ZmWRI1 hybrid. A total of five transgenic lines and their corresponding nulls were crossed to a male tester, PH1B5, to produce hybrid seeds. Yield was tested in eight locations in the United States with three repeats for each line at each location in 2007. Error bars represent 95% confidence intervals. All five transgenic lines showed no significant difference from nulls as determined by Student's t test (P > 0.10).