Distinct roles of LAFL network genes in promoting the embryonic seedling fate in the absence of VAL repression

Abstract

The transition between seed and seedling phases of development is coordinated by an interaction between the closely related ABSCISIC ACID-INSENSITIVE3 (ABI3), FUSCA3 (FUS3), and LEAFY COTYLEDON2 (LEC2; AFL) and VIVIPAROUS1/ABI3-LIKE (VAL) clades of the B3 transcription factor family that respectively activate and repress the seed maturation program. In the val1 val2 double mutant, derepression of the LEC1, LEC1-LIKE (L1L), and AFL (LAFL) network is associated with misexpression of embryonic characteristics resulting in arrested seedling development. We show that while the frequency of the embryonic fate in val1 val2 seedlings depends on the developmental timing of seed rescue, VAL proteins repress LAFL genes during germination, but not during seed development. Quantitative analysis of LAFL mutants that suppress the val1 val2 seedling phenotype revealed distinct roles of LAFL genes in promoting activation of the LAFL network. LEC2 and FUS3 are both essential for coordinate activation of the network, whereas effects of LEC1, L1L, and ABI3 are additive. Suppression of the val1 val2 seedling phenotype by the B3 domain-deficient abi3-12 mutation indicates that ABI3 activation of the LAFL network requires the B3 DNA-binding domain. In the VAL-deficient background, coordinate regulation of the LAFL network is observed over a wide range of genetic and developmental conditions. Our findings highlight distinct functional roles and interactions of LAFL network genes that are uncovered in the absence of VAL repressors.

Figure 1.

The embryonic seedling phenotype of the val1 val2 double mutant. Col-0, val1, val2, and val1 val2 seeds were rescued at 1-d increments between 7 and 15 DAF. Sterilized seeds were sown on Murashige and Skoog phytagel media and stratified by incubation at 4°C in the dark for 3 d. After treatment, they were grown for 12 d at 23°C to 25°C under continuous light, and the seedling phenotypes were recorded. A, Based on the observed variation, the phenotypes of val1 val2 mutant seedling are classified into three types: embryonic, partial, and normal. Bar = 1 cm. B, Representative phenotypes of Col-0, val1, val2, and val1 val2 seedlings rescued at different stages of seed development (7–15 DAF). Bar = 1 cm. C, Histogram of the three phenotypic classes of val1 val2 seedlings grown from seed rescued at different seed developmental stages (7–15 DAF). Values are means ± se of the mean (n = 6–10).

Figure 2.

Expression of LAFL network genes in wild-type and rescued val mutant seedlings (A) and developing seeds (B). Quantitative real-time reverse transcription PCR (Q-PCR) analysis was performed to quantify the transcript level of LAFL network genes (LEC1, L1, LEC2, FUS3, and ABI3) and downstream targets (At2S1 and CRC). Bars show copies of mRNA per nanogram total RNA. Values are means ± se of the mean (n = 3). Asterisks indicate significant differences (Student’s t test; P < 0.05) in the transcript levels of the wild type, val1, and val2 compared with the corresponding val1 val2 double mutant mean. A, Marker gene expression in 6-d-old wild-type and val (val1, val2, and val1 val2) mutant seedlings grown from seeds rescued at different developmental stages (9, 11, 13, and 15 DAF). B, Marker gene expression in wild-type and val (val1, val2, and val1 val2) mutant developing seeds (9 and 11 DAF). n.d., Not detected.

Figure 3.

Seedling phenotypes of LAFL-VAL triple mutants. Triple mutant seeds were rescued between 7 and 15 DAF. The culture conditions are described in Figure 1. A, Representative phenotypes of triple mutant (val1 val2 l1l, val1 val2 lec1, val1 val2 abi3-6, val1 val2 abi3-12, val1 val2 fus3-3, and val1 val2 lec2) seedlings from seeds rescued at 7 to 15 DAF. Bar = 1 cm. B, Histogram of the three phenotypic classes of triple mutant seedlings grown from seeds rescued at different developmental stages (7–15 DAF). The phenotypic classes are described in Figure 1. Values are means ± se of the mean (n = 6–10).

Figure 4.

Expression of LAFL network genes in LAFL-VAL triple mutant seedlings. Q-PCR analysis was performed to quantify transcript levels of LAFL network genes (LEC1, L1L, LEC2, FUS3, and ABI3) and downstream targets (At2S1 and CRC) in 6-d-old triple mutant (val1 val2 l1l, val1 val2 lec1, val1 val2 abi3-6, val1 val2 abi3-12, val1 val2 fus3-3, and val1 val2 lec2) seedlings grown from seeds rescued at 9, 11, 13, and 15 DAF. Bars show copies of mRNA per nanogram total RNA. Values are means ± se of the mean (n = 3). n.d., Not detected.

Figure 5.

Seedling phenotypes and LAFL network expression in val1 val2 val3 triple and val1 val2 val3 abi3-6 quadruple mutants. The val1 val2 val3 triple mutants were confirmed by genotyping seedlings grown from val1 val2 val3/+ triple mutant plants harvested at maturity. val1 val2 val3 abi3-6 quadruple mutant seeds were rescued at intervals between 7 and 15 DAF. The culture conditions are as described in Figure 1. A, Representative phenotypes of val1 val2 val3 triple mutant seedlings grown from mature seeds. Bar = 1 cm. B, Representative phenotypes of quadruple mutant val1 val2 val3 abi3-6 seedlings grown from seeds rescued at 7 to 15 DAF. Bar = 1 cm. C, Histogram of the three phenotypic classes of val1 val2 val3 abi3-6 mutant seedlings grown from seeds rescued at 7 to 15 DAF. The phenotypic classes are as described in Figure 1. Values are means ± se of the mean (n = 6–10). D, Expression of seed-specific marker genes in val1 val2 abi3-6 triple and val1 val2 val3 abi3-6 quadruple mutant seedlings. Q-PCR analysis was performed to quantify the transcript levels of LAFL network genes (LEC1, L1L, LEC2, FUS3, and ABI3) and downstream targets (At2S1 and CRC) in 6-d-old mutant (val1 val2 abi3-6 and val1 val2 val3 abi3-6) seedlings grown from seeds rescued at 9, 11, 13, and 15 DAF. Bars show copies of mRNA per nanogram total RNA. Values are means ± se of the mean (n = 3).

Figure 6.

Seedling phenotypes of LAFL-VAL quadruple mutants. Quadruple mutant seeds were rescued from 7 to 14 DAF using culture conditions described in Figure 1. All quadruple mutants except val1 val2 abi3-6 l1l that were rescued after 14 DAF failed to germinate. A, Representative phenotypes of quadruple mutant (val1 val2 abi3-6 l1l, val1 val2 abi3-6 lec1, val1 val2 abi3-6 fus3-3, and val1 val2 abi3-6 lec2) seedlings from seeds rescued at 7 to 14 DAF. Bar = 1 cm. B, Histogram of the three phenotypic classes of val1 val2 abi3-6 l1l and val1 val2 abi3-6 lec1 quadruple mutant seedlings grown from seeds rescued at different developmental stages (7–15 DAF) using phenotypic classes described in Figure 1. Values are means ± se of the mean (n = 6–10).

Figure 7.

Expression of LAFL network genes in LAFL-VAL quadruple mutant seedlings. Q-PCR analysis was performed to quantify the transcript levels of LAFL network genes (LEC1, L1L, LEC2, FUS3, and ABI3) and downstream targets (At2S1 and CRC) in 6-d-old quadruple mutant (val1 val2 abi3-6 l1l, val1 val2 abi3-6 lec1, val1 val2 abi3-6 fus3-3, and val1 val2 abi3-6 lec2) seedlings grown from seeds rescued at 9, 11, 13, and 15 DAF. Note that with the exception of abi3-6 l1l, all quadruple mutants failed to germinate after 14 DAF. Bars show copies of mRNA per nanogram total RNA. Values are means ± se of the mean (n = 3). n.d., Not detected.

Figure 8.

Correlation of LAFL gene expression under various genetic and developmental treatments. A, Matrix of pairwise correlations (R²) of LAFL gene expression data across all genotypes and treatments, excluding genotypes where one of the two genes is mutant. Red, R² ≥ 0.90; yellow, R² < 0.90. B, Scatterplot of LEC2 expression versus each of the other LAFL genes. Regression equations and R² are indicated for data from each comparison.