Regulation of Arabidopsis embryo and endosperm development by the polypeptide signaling molecule CLE8

Abstract

The plant seed is a major nutritional source for humans as well as an essential embryo development and dispersal unit. To ensure proper seed formation, fine spatial and temporal coordination between the embryo, endosperm, and maternal seed components must be achieved. However, the intercellular signaling pathways that direct the synchronous development of these tissues are poorly understood. Here we show that the Arabidopsis thaliana peptide ligand CLAVATA3/embryo surrounding region-related8 (CLE8) is exclusively expressed in young embryos and endosperm, and that it acts cell and noncell autonomously to regulate basal embryo cell division patterns, endosperm proliferation, and the timing of endosperm differentiation. CLE8 positively regulates expression of the transcription factor gene Wuschel-like homeobox8 (WOX8), and together CLE8 and WOX8 form a signaling module that promotes seed growth and overall seed size. These results demonstrate that seed development is coordinated by a secreted peptide ligand that plays a key early role in orchestrating cell patterning and proliferation in the embryo and endosperm.

Figure 1.

CLE8 Expression Analysis in Developing Seed Tissues. (A) to (F) CLE8 mRNA expression pattern in a one-cell stage embryo (A), a four-cell stage embryo (B), a globular stage embryo (C), a triangular stage embryo (D), a heart stage embryo (E), and in the endosperm nuclei (F). (G) Control hybridization with a CLE8 sense probe. (H) to (P) CLE8 promoter-driven GUS activity in embryos ([H] to [M]) and endosperm ([N] to [P]). (I) shows a higher magnification image of the embryo boxed with the dotted lines in (H). White stars indicate background browning of the seed coat. Bars = 20 μm.

Figure 2.

cle8-1 and CLE8 amiRNA Seed Phenotypes. (A) Wild-type CLE8 CLE domain sequence (Top) and cle8-1 CLE domain sequence (Bottom). The mutated amino acid and its position in the protein sequence are indicated in red. (B) and (C) A wild-type (B) and a cle8-1 (C) open silique containing mature seeds. This cle8-1 silique contains more than the average number of abnormal seeds but illustrates the range of seed defects observed. (D) and (E) Length (D) and width (E) of wild-type Col and cle8-1 seeds (n = 100). (F) cle8-1 complementation experiment (n = 118 to 186). All transgenic lines are in the cle8-1 homozygous background. Graph values are means ± se, * indicates the difference is statistically significant (Z test, P < 0.05) when compared with the cle8-1 value but not when compared with the wild-type value. (G) Reciprocal crosses between wild-type Col and cle8-1 plants (n = 179 to 226). * indicates the values are statistically different from wild-type and cle8-1 values (Z test, P < 0.05). The difference in the percentage of defective seeds between the two reciprocal crosses is not significant. (H) RT-PCR analysis performed on RNA extracts from siliques of Col and three independent transgenic CLE8 amiRNA lines, using two technical replicates. (I) Quantification of defective seeds in Col, cle8-1, and the CLE8 amiRNA transgenic lines (n = 122 to 275). The difference between wild-type Col and the other genotypes is statistically significant (Z test, P < 0.01), whereas the CLE8 amiRNA transgenic line values are not significantly different from the cle8-1 values.

Figure 3.

cle8-1 and CLE8 amiRNA Embryo Phenotypes. (A) to (D) Wild-type embryos at the two-cell (A), 16-cell (B), globular (C), and triangular (D) stage. (E) to (H) cle8-1 aberrant embryos in the same developmental window. (I) Wild-type and cle8-1 suspensor length measured at the dermatogen embryo stage (n = 30). Graph values are means ± se, * indicates statistically significant values (Student’s t test, P < 0.05). (J) CLE8 amiRNA aberrant embryo. Red dashed lines outline the suspensors; white dashed lines outline the embryo proper; yellow arrowheads indicate unusual planes of cell division. Bars = 20 μm.

Figure 4.

cle8-1 and CLE8 amiRNA Endosperm Phenotypes. (A) to (C) Wild-type seed (A) and cle8-1 seeds ([B] and [C]). Free endosperm nuclei are highlighted in red, and the yellow arrow indicates a solitary endosperm nucleus near the suspensor. (D) and (E) CLE8 amiRNA seeds. (F) to (H) Wild-type (F) and (G) and cle8-1 (H) seeds expressing the ProMEA:GUS marker. (I) and (J) Wild-type (I) and cle8-1 (J) seeds expressing the ProFIS2:GUS marker. (F) to (J) Red dashed lines outline the embryos. Bars = 20 μm.

Figure 5.

Pro35S:CLE8 Seed Phenotypes. (A) CLE8 mRNA transcription levels in wild-type and three independent Pro35S:CLE8 transgenic lines determined by real-time quantitative RT-PCR. Graph values are means of three biological replicates ± se. All experimental values are statistically significant (Student’s t test, P < 0.05). (B) and (C) Mature wild-type (B) and Pro35S:CLE8 transgenic (C) seeds. (D) and (E) Length (D) and width (E) of wild-type and Pro35S:CLE8 seeds from three independent transgenic lines. Graph values are means ± se, * indicates statistically significant values (n = 100, Student’s t test, P < 0.01). Bars = 50 μm. [See online article for color version of this figure.]

Figure 6.

WOX8 Activity in cle8-1 and Pro35S:CLE8 Plants. (A) to (H) Merged Nomarski and fluorescence images of wild-type ([A] and [C] to [E]) and cle8-1 ([B] and [F] to [H]) seeds expressing WOX8gΔ:NLS-venusYFP₃. Insets show slightly older embryos displaying the same expression patterns. (I) WOX8 mRNA transcription levels in three independent Pro35S:CLE8 transgenic lines. Graph values are means of three biological replicates ± se, * indicates statistically significant values (Student’s t test, P < 0.05). (J) to (M) Wild-type (J), wox8-1 (K), Pro35S:CLE8 (L), and Pro35S:CLE8 wox8-1 (M) seeds. (N) and (O) Length (N) and width (O) of wild-type, wox8-1, Pro35S:CLE8, and Pro35S:CLE8 wox8-1 seeds from three independent transgenic lines. * indicates that the values of the Pro35S:CLE8 wox8-1 seeds are significantly different (n = 100, Student’s t test, P < 0.05) from those of the Pro35S:CLE8 seeds (gray bars) but not from those of the wox8-1 seeds (green bars). In this generation, Pro35S:CLE8 line 1 transgenic plants were silenced (n = 100). Bars in (A) to (H) = 20 μm; bars in (J) to (M) = 1 mm.