Genes directly regulated by LEAFY COTYLEDON2 provide insight into the control of embryo maturation and somatic embryogenesis

Abstract

The B3 domain protein LEAFY COTYLEDON2 (LEC2) is required for several aspects of embryogenesis, including the maturation phase, and is sufficient to induce somatic embryo development in vegetative cells. Here, we demonstrate that LEC2 directly controls a transcriptional program involved in the maturation phase of seed development. Induction of LEC2 activity in seedlings causes rapid accumulation of RNAs normally present primarily during the maturation phase. Several RNAs encode proteins with known roles in maturation processes, including seed-storage and lipid-body proteins. Clustering analyses identified other LEC2-induced RNAs not previously shown to be involved in the maturation phase. We show further that genes encoding these maturation RNAs all possess in their 5' flanking regions RY motifs, DNA elements bound by other closely related B3 domain transcription factors. Our finding that recombinant LEC2 specifically binds RY motifs from the 5' flanking regions of LEC2-induced genes provides strong evidence that these genes represent transcriptional targets of LEC2. Although these LEC2-induced RNAs accumulate primarily during the maturation phase, we show that a subset, including AGL15 and IAA30, accumulate in seeds containing zygotes. We discuss how identification of LEC2 target genes provides a potential link between the roles of LEC2 in the maturation phase and in the induction of somatic embryogenesis.

Fig. 1.

Profiles of RNAs induced by LEC2 that are prevalent during the maturation phase. Representation of mean normalized expression data for 14 LEC2-induced RNAs in LEC2 induction experiments (Left) and during seed development (Right). Gene names and AGI loci are listed. Color scale shows relative RNA levels, with gray representing RNAs not present. L2GR, seedlings treated with Dex for the indicated period; NT, nontransgenic seedlings; Zyg, seeds containing zygotes; Cot, embryos at the cotyledon stage; MatGr, mature green stage; Post MatGr, postmature green stage; Str, steroleosin; CysProt, cysteine proteinase; Expr Pr, expressed protein.

Fig. 2.

qPCR data validate DNA microarray results and reveal an early role for several LEC2-induced RNAs. Normalized RNA levels from DNA microarray experiments (bars) and relative RNA levels from qPCR experiments (lines) in LEC2 induction experiments (A and B) and during seed development (C and D). (A and C) AGL15, IAA30, LOB40, and EEL. (B and D) AT2S2, AT2S3, AT2SL, and CRA1.

Fig. 3.

LEC2 target genes contain upstream RY motifs. Representation of the location of RY motifs (CATGCA) present within 1 kb of the transcription start site of 14 LEC2 target genes. White boxes, RY motifs on sense strand; gray boxes, RY motifs on antisense strand with respect to the gene.

Fig. 4.

LEC2 binds specifically to RY motifs upstream of LEC2 target genes. (A) Binding of the 2SRY probe DNA oligonucleotides with recombinant LEC2 in the presence and absence of the indicated unlabeled competitor DNA oligonucleotides. LegRY contains a RY motif, whereas the RY motif in mLegRY is mutated. UAS2 does not contain a RY motif. Free, no added LEC2 protein; 0x, no added competitor. (B) DNA oligonucleotides containing RY motifs upstream of LEC2 target genes compete for binding of the 2SRY probe with LEC2. Competitors: EEL; Str, steroleosin; AGL, AGL15; CP, cysteine proteinase. Competition experiments were performed with 500- and 2,000-fold molar excesses of competitor, except for the 2SRY self-competition experiments in B in which 100-, 500-, and 2,000-fold molar excesses were used. Competition experiments with oligonucleotides lacking a RY motif were done with a 2,000-fold molar excess.