Inactivation of the phloem-specific Dof zinc finger gene DAG1 affects response to light and integrity of the testa of Arabidopsis seeds

Abstract

We show here that seeds from the knockout mutant of the Arabidopsis DAG1 gene encoding a Dof zinc finger transcription factor have an altered response to red and far-red light. Mutant dag1 seeds are induced to germinate by much lower red light fluence rates, and germination reaches more quickly a point where it is independent of phytochrome signaling. Moreover, although microscopic analysis reveals no obvious structural alterations in the seed coat (testa) of dag1 seeds, staining assays with different dyes point to an abnormal fragility of the testa. By extensive in situ mRNA hybridization analysis we show here that the gene, which is not expressed in the embryo, is specifically expressed in the phloem of all organs of the mother plant.

Figure 1

Germination of dag1 mutant seeds is more sensitive to red light. Ws (white squares) and dag1 (black circles) seeds were irradiated with far red light for 30 min and germination in the dark was scored after a pulse (5 min) of red light (660 nm) of different fluence rates (A); a pulse (5 min) of monochromatic light of different wavelengths (4 μmol m⁻² s⁻¹; B); a pulse (5 min) of red light (660 nm, 20 μmol m⁻² s⁻¹) and a subsequent pulse of saturating long-wavelength far-red light after different intervals of time (the dotted and dashed line indicate the germination of, respectively, Ws and dag1 seeds in the absence of the long-wavelength far-red pulse; C). Means of at least 50 seeds sown in triplicate are shown. Error bars represent ses.

Figure 2

The seed coat of dag1 seeds shows histochemical alterations. A and B, Toluidine blue-stained sections of a Ws and, respectively, dag1 seed coat (ml, mucilage layer; c, columella; ep, epidermis; p, palisade layer; cp, crushed parenchymatic layers; e, endothelium layer; a, aleurone layer; bar = 40 μm); C and D, epidermis of Ws and, respectively, dag1 seeds as viewed with Nomarski differential interference optics (c, columella; bar = 40 μm); E and F, Ws and, respectively, dag1 seed stained for 15 min with ruthenium red after a 3-min imbibition (bars = 200 μm); G and H, Ws and, respectively, dag1 seeds stained with ruthenium red for 15 s without prior imbibition (bar = 1,000 μm); I and J, Ws and, respectively, dag1 seeds stained with tetrazolium salts (bar = 1000 μm); K and L, immature Ws and, respectively, dag1 seeds stained with vanillin (bar = 500 μm).

Figure 3

DAG1 is expressed in the phloem of all organs of Arabidopsis plants. Dark-field images of in situ mRNA hybridizations with tritium-labeled antisense DAG1 riboprobe on sections of: mature seed (A; bar = 100 μm); primary root including the apex (B; longitudinal section, bar = 100 μm), arrow points to where differentiation of the protophloem begins and the autoradiographic signal becomes visible; primary root (C; transverse section, bar = 50 μm), arrow points to the phloem region, where the autoradiographic signal is localized; primary root at the site where a secondary root is formed (D; transverse section, bar = 100 μm); upper part of the floral stem including the main apex (E; longitudinal section, bar = 200 μm); floral stem (F; longitudinal section, bar = 100 μm); floral stem (G; transverse section, bar = 200 μm); floral stem and attached leaf (H; transverse section, bar = 200 μm), arrows point to leaf midrib bundle and to secondary veins; floral bud (I; longitudinal section, bar = 200 μm), arrow points to the autoradiographic signal in the immature carpel.