Arabidopsis LEAFY COTYLEDON1 Mediates Postembryonic Development via Interacting with PHYTOCHROME-INTERACTING FACTOR4

Abstract

Plants undergo postembryonic growth during the developmental transition from germinating seeds to seedlings. Recent studies suggest LEAFY COTYLEDON1 (LEC1), initially identified as a central regulator in embryogenesis and seed maturation in Arabidopsis thaliana, plays a distinct role in postembryonic development. However, the mechanism by which LEC1 regulates nonembryonic development still remains elusive. In this study, we observed etiolation-related phenotypes in early seedlings of lec1 mutants and inducible LEC1 overexpression transgenic lines. Consistent with this, LEC1 promotes the expression of hypocotyl elongation-related genes in a darkness-dependent manner in spite of the comparable LEC1 transcript levels in the light- and dark-grown seedlings. Furthermore, we show that LEC1 interacts with PHYTOCHROME-INTERACTING FACTOR4 (PIF4), a major transcription modulator in postgermination development, to interdependently regulate hypocotyl elongation-related genes via direct binding to G-box element in the dark. Moreover, loss of LEC1 function suppresses the elongated hypocotyl phenotype of PIF-overaccumulating plants; conversely, inducible overexpression of LEC1 does not rescue the short hypocotyl in pif4 mutants. Our findings reveal that LEC1 acts as a coactivator of PIFs in transcriptional regulation during postembryonic growth, providing a possible mechanism by which plants fine-tune morphological development for their survival during the transition from the embryonic phase to seedling establishment.

Figure 1.

Identification of a LEC1 Knockdown Allele in Arabidopsis. (A) Schematic diagram showing the T-DNA insertion site in lec1-4 (SALK_095699). Exons are represented by boxes and introns by lines between the boxes. Black boxes represent the LEC1 coding regions. (B) PCR of genomic DNA confirms that lec1-4 is a homozygous insertion mutant. The primers used are shown in (A). LB indicates the T-DNA left border primer. (C) LEC1 expression is low in lec1-4 in comparison to the wild type by RT-PCR using the primers F and R shown in (A). Total RNA was isolated from developing seeds (6 d after pollination) of lec1-4 and Col plants. (D) The lec1 mutants exhibit abnormal embryos in comparison to the wild type. Bar = 1 mm. (E) The lec1 mutants develop trichomes on their cotyledons or have shrunken cotyledons in comparison to the wild type. Five-day-old seedlings grown in the light were used for investigation. Bar = 1 mm. (F) Five-day-old seedlings of lec1-4 mutants grown in the light or dark show shorter hypocotyls than the wild type. Bar = 2 mm. (G) Hypocotyl length statistics of the wild type and lec1-4 seedlings shown in (F). Data represent mean ± sd of at least 30 seedlings. Asterisks indicate significant differences between Col and lec1-4 mutants (P < 0.05, by Student’s t test). (H) Germination investigation of the freshly harvested dry seeds from Col, lec1-4, Landsberg erecta (Ler), and lec1-3. Germination rate of seeds was measured after transfer to 22°C for 1 d from 4°C stratification. (I) Germination rate of the Col, lec1-4, Ler, and lec1-3 seeds with a 3-d, 1-month, or 3-month storage period after harvest.

Figure 2.

LEC1 Is Involved in Hypocotyl Elongation. (A) Quantitative RT-PCR analysis showing LEC1 expression in 5-d-old Col wild-type seedling grown in the dark (D) or in the light (L). Tissues from rosette leaves (R) and developing seeds (S) were used as negative and positive controls, respectively. The β-tubulin gene (TUB2) was amplified as an internal control. (B) Immunoblot analysis of LEC1-FLAG protein expressed in tissues indicated in (A). The lec1-4 pLEC1:LEC1-FLAG transgenic line was used to examine the LEC1 protein level. Coomassie blue staining (CBB) was used as a loading control. (C) GUS staining of pLEC1:GUS transgenic plants. Tissues detected are indicated in (A). (D) Quantitative RT-PCR analysis showing repression of hypocotyl elongation-related gene expression in lec1-4 in comparison to the wild type. Three-day-old lec1-4 and Col seedlings grown in the light (0 h) or then transferred to darkness for 6 h (6 h) were harvested. Relative gene expression was calculated by comparing the values to that of Col at 0 h. TUB2 was amplified as an internal control. Asterisks indicate significant differences between Col and lec1-4 mutant (P < 0.05, by Student’s t test). L to D, light to darkness. (E) Hypocotyl length of the pER10:LEC1-MYC transgenic line. Five-day-old Col and pER10:LEC1-MYC seedlings grown in the light with 10 μM estradiol (E) or mock treatment (M) were used for investigation. Bar = 2 mm. (F) Hypocotyl length statistics of the wild type and pER10:LEC1-MYC seedlings shown in (E). Data represent mean ± sd of at least 30 seedlings. Asterisk indicates significant difference between pER10:LEC1-MYC with estradiol and mock treatment (P < 0.05, by Student’s t test). (G) Estradiol-induced LEC1 overexpression promotes the expression of hypocotyl elongation-related genes. Three-day-old pER10:LEC1-MYC seedlings were treated with 10 μM estradiol or mock-treated and immediately transferred to darkness for the indicated time. Relative gene expression was calculated by comparing the values to that at 0 h. TUB2 was amplified as an internal control.

Figure 3.

LEC1 Physically Interacts with PIF4 in Vitro and in Vivo. (A) Sketches showing the domain structures of LEC1 and PIF4 and various deletions. (B) Yeast two-hybrid assays showing the interactions between LEC1, PIF4, and their derivatives. Transformed yeast cells were grown on SD/-Trp/-Leu/-His/-Ade and SD/-Trp/-Leu medium. (C) Pull-down assay showing direct interaction between His-LEC1 and GST-PIF4 fusion proteins in vitro. His-LEC1 protein was incubated with immobilized GST or GST-PIF4 proteins, and immunoprecipitated fractions were detected by anti-His antibody. (D) BiFC assay showing LEC1-EYFP^C and PIF4-EYFP^N interact to form a functional EYFP in the nucleus. DAPI, fluorescence of 4′,6-diamidino-2-phenylindole; Merge, merge of EYFP and DAPI. (E) In vivo interaction of PIF4 and LEC1 in Arabidopsis. Plant nuclear extracts from 5-d-old pLEC1:LEC1-FLAG 35S:PIF4-HA seedling grown in the dark were immunoprecipitated by either anti-HA antibody or preimmune serum (IgG). The coimmunoprecipitated proteins were detected by anti-FLAG antibody.

Figure 4.

LEC1 and PIF4 Coregulate the Hypocotyl Elongation-Related Genes via Directly Binding to the G-Box. (A) Schematic diagram of the IAA19 and YUC8 genomic regions. P1 to P3 indicate fragments for ChIP-qPCR amplification. Numbers indicate the positions of G-box elements in IAA19 and YUC8 promoters relative to ATG. (B) ChIP analysis of PIF4-HA binding to G-box containing region in IAA19 and YUC8 genes upon precipitation with anti-HA antibody. Three-day-old of 35S:PIF4-HA and Col seedlings were transferred to darkness for an additional 2 d and harvested for ChIP assay. Data represent mean ± sd of triplicates. Asterisks indicate significant changes in ChIP enrichment in 35S:PIF4-HA compared with the Col sample (P < 0.05, by Student’s t test). (C) ChIP analysis of LEC1-MYC binding to the G-box containing region in IAA19 and YUC8 genes upon precipitation with anti-MYC antibody. Three-day-old of pER10:LEC1-MYC seedlings were treated with 10 μM estradiol or mock and immediately transferred to darkness for an additional two days and harvested for ChIP assay. Data represent mean ± sd of triplicates. Asterisks indicate significant changes in ChIP enrichment in estradiol-treated sample compared with the mock-treated sample (P < 0.05, by Student’s t test). (D) EMSA assay of the PIF4-LEC1 complex binding to the G-box in IAA19 promoter (P3 region). The biotinylated probe containing the G-box element was incubated with GST-PIF4 (Lane 10), His-LEC1 (Lane 9), or their mixture (lanes 3 to 8), while the probe incubated with no protein (lane 1) or GST protein (lane 2) was used as negative control. Nonlabeled probes in 5- and 50-fold molar excess relative to the biotinylated probe containing G-box (G-wt, CACGTG) or mutated G-box (G-mut, CACGGG), respectively, were used as cold competitors. White and black arrowheads indicate the specific bindings of PIF4 protein and PIF4-LEC1 complex to the biotinylated probe, respectively, while arrow indicates nonspecific bands. (E) Transient expression assays of IAA19 transcriptional activity modulated by LEC1 and PIF4 in Arabidopsis mesophyll protoplasts. Various constructs used in transient expression assays are shown in the upper panel. Either pIAA19:GUS or mpIAA19:GUS was cotransformed with effectors or empty vector (Control) into Col mesophyll protoplasts. Relative GUS activity (GUS/Luciferase) that indicates the level of IAA19 expression activated by various effectors is shown in the lower panel. Values are mean ± sd of five biological replicates.

Figure 5.

LEC1 Is Required for PIF4-Mediated Hypocotyl Elongation. (A) Hypocotyl lengths of the wild-type, 35S:PIF4, and lec1-4 35S:PIF4 plants. Five-day-old seedlings grown in the light were used for investigation. Bar = 2 mm. (B) Hypocotyl length statistics of the wild-type, 35S:PIF4, and lec1-4 35S:PIF4 seedlings shown in (A). Data represent mean ± sd of at least 30 seedlings. Asterisks indicate significant difference between 35S:PIF4 and lec1-4 35S:PIF4 (P < 0.05, by Student’s t test). (C) Quantitative RT-PCR analysis showing hypocotyl elongation-related genes expression in 35S:PIF4 and lec1-4 35S:PIF4. Three-day-old seedlings grown in the light were transferred to darkness for 6 h and then harvested for RNA extraction and further analysis. Relative gene expression levels were normalized against the expression of TUB2 and those in 35S:PIF4 seedlings were designated as 100%. (D) ChIP analysis of PIF4-HA binding to G-box containing region in IAA19 and YUC8 genes upon precipitation with anti-HA antibody. Three-day-old 35S:PIF4-HA and lec1-4 35S:PIF4-HA seedlings were transferred to darkness for an additional 2 d and harvested for ChIP assay. Data represent mean ± sd of triplicates. Asterisks indicate significant changes in ChIP enrichment in lec1-4 35S:PIF4-HA compared with 35S:PIF4-HA sample (P < 0.05, by Student’s t test). (E) Hypocotyl lengths of pER10:LEC1-MYC and pif4 pER10:LEC1-MYC transgenic line. Five-day-old seedlings grown in the light with 10 μM estradiol or mock treatment were used for investigation. Bar = 2 mm. (F) Hypocotyl length statistics of pER10:LEC1-MYC and pif4 pER10:LEC1-MYC shown in (E). The percentage indicates the relative hypocotyl length with estradiol treatment against that with mock treatment (designated as 100%). Data represent mean ± sd of at least 30 seedlings. Asterisks indicate significant difference of relative hypocotyl length in pif4 pER10:LEC1-MYC compared with pER10:LEC1-MYC seedlings (P < 0.05, by Student’s t test). (G) Quantitative RT-PCR analysis showing expression of hypocotyl elongation-related genes in pER10:LEC1-MYC and pif4 pER10:LEC1-MYC. Three-day-old seedlings were treated with 10 μM estradiol and immediately transferred to darkness for 3 h and then harvested for RNA extraction and further analysis. Relative gene expression levels were normalized against the expression of TUB2 and those in pER10:LEC1-MYC seedlings were designated as 100%. (H) ChIP analysis of LEC1-MYC binding to G-box-containing region in IAA19 and YUC8 genes upon precipitation with anti-HA antibody. Three-day-old pER10:LEC1-MYC and pif4 pER10:LEC1-MYC seedlings were treated with 10 μM estradiol and immediately transferred to darkness for additional 2 d and harvested for ChIP experiment. Data represent mean ± sd of triplicates. Asterisks indicate significant changes in ChIP enrichment in pif4 pER10:LEC1-MYC compared with pER10:LEC1-MYC sample (P < 0.05, by Student’s t test).

Figure 6.

A Model Illustrating the Proposed Role of LEC1 and PIF4 in the Transcriptional Regulation during Postembryonic Growth. In the light, photoactivated phytochromes (Phy) interact with PIF4 and trigger their codegradation by the 26S proteasome, thus abolishing the expression of the etiolation-related genes and promoting photomorphogenesis. In the dark, LEC1 interacts with stabilized PIF4 to coregulate the etiolation-related genes via direct binding to the G-box, thus promoting skotomorphogenic growth.