Arabidopsis LEAFY COTYLEDON2 induces maturation traits and auxin activity: Implications for somatic embryogenesis

Abstract

LEAFY COTYLEDON2 (LEC2) is a central regulator of embryogenesis sufficient to induce somatic cells to form embryos when expressed ectopically. Here, we analyze the cellular processes induced by LEC2, a B3 domain transcription factor, that may underlie its ability to promote somatic embryogenesis. We show auxin-responsive genes are induced after LEC2 activation in seedlings. Genes encoding enzymes involved in auxin biosynthesis, YUC2 and YUC4, are activated within 1 h after induction of LEC2 activity, and YUC4 appears to be a direct transcriptional target of LEC2. We also show ectopic LEC2 expression induces accumulation of seed storage protein and oil bodies in vegetative and reproductive organs, events that normally occur during the maturation phase of embryogenesis. Furthermore, LEC2 activates seed protein genes before an increase in RNAs encoding LEC1 or FUS3 is observed. Thus, LEC2 causes rapid changes in auxin responses and induces cellular differentiation characteristic of the maturation phase. The relevance of these changes to the ability of LEC2 to promote somatic embryogenesis is discussed.

Fig. 1.

Auxin responses are enhanced by ectopic LEC2 activity. (A–J) DR5:GUS plants were stained for GUS activity. Seedling without (A) and with (B) 35S:LEC2 grown in the light on hormone-free media. Seedlings lacking 35S:LEC2 were grown in the dark to reduce endogenous auxin levels in the presence (C) or absence (D) of 5 μM 2,4-D. Temporal changes in LEC2-induced auxin responses are shown. (E–J) Eight-day 35S:LEC2-GR seedlings were grown in the presence (H–J) or absence (E–G) of Dex for 7 h (E and H), 3 days (F and I), and 7 days (G and J). Arrows indicate young vegetative leaves stained differently for GUS activity. (K) DR5:GUS activities in 8-day 35S:LEC2-GR seedlings treated with Dex for 3 or 7 days. Control seedlings contained the DR5:GUS but not 35S:LEC2-GR. (L) Relative levels of three auxin-responsive RNAs in 8-day 35S:LEC2-GR seedlings untreated or treated with Dex for 1 and 4 h as determined by qRT-PCR. IAA1 and ACS4 RNA levels differed in Dex-treated and untreated samples at the 0.05 significance level. (M) Relative RNA levels in shoots of nontransgenic and 35S:LEC2-GR seedlings either mock or Dex-treated for 7 h. RNAs from Dex-treated and mock-treated samples differed at the 0.05 significance level. (Scale bars: B and C, 0.5 mm; A and D–J, 1 mm.)

Fig. 2.

LEC2 activates YUC2 and YUC4 genes. (A) Relative levels of YUC2 and YUC4 RNA in 35S:LEC2-GR seedlings either not treated with Dex or Dex-treated for 1 or 4 h and in nontransgenic seedlings Dex-treated for 4 h. Bars show relative RNA levels determined by qRT-PCR, and lines show normalized RNA levels determined previously (9). (B) ChIP of 35S:FLAG-LEC2-GR seedlings treated with Dex for 8.5 h. Fold DNA enrichment represents the ratios of DNA amplicons for the indicated genes in samples immunoprecipitated with anti-FLAG and anti-GST (control) antibodies. Oleosin and 2S3 storage protein genes are known LEC2 targets, and actin and Ta3 are negative controls not induced by LEC2.

Fig. 3.

Macromolecular reserves characteristic of seeds accumulate in unpollinated, enlarged 35S:LEC2 ovules. (A) Maturing seeds in the upper third of a nontransgenic (NT) silique and enlarged 35S:LEC2 ovules from an unpollinated pistil. (B) 35S:LEC2 ovules 20 days after flower emasculation (DAE). Embryo sac contents (*) have degenerated and integument cells have divided and enlarged. (C–E) Lipid staining (black) of an enlarged, unfertilized 35S:LEC2 ovule (C) with oil reserves, a nontransgenic ovule at 1 DAE (D) lacking oil reserves, and a nontransgenic mature green-stage seed (E) with oil reserves in embryonic cotyledons (c), axis (a) and endosperm (en) but not seed coat (sc). (F–H) Protein bodies (blue) and insoluble carbohydrate-stained (pink) tissues. (F) Protein bodies in nontransgenic seeds are detected only in the endosperm (en) and embryo (c), whereas starch grains (pink) are visible in the seed coat (sc). (G) Enlarged 35S:LEC2 ovule. (H) Higher-magnification view of region from G showing the presence of protein bodies and starch grains in ovule integument cells. (I) Fatty acid methyl ester analysis showing profiles from enlarged 35S:LEC2 ovules, nontransgenic rosette leaves, and seeds. For clarity, only fatty acids that constitute >0.1% of the total are shown. Minor components are either not fatty acids or fatty acids of unknown identity. (Scale bars: A, 1 mm; B–H, 50 μm.)

Fig. 4.

Seed RNAs accumulate in postembryonic organs of 35S:LEC2 plants. RT-PCR detection of RNAs for genes expressed predominantly during embryogenesis and a control RNA encoding a ribosomal protein (TIN) in organs from two independent 35S:LEC2 transformants and nontransgenic (NT) tissues. Forty PCR cycles were used to detect LEC2, LEC1, and FUS3 RNAs, 35 cycles for CRA1 12S storage protein and TIN RNA, and 30 cycles for oleosin (OLE) RNA. DAE, day after emasculation; DAP, day after pollination; SE, somatic embryos.