SeeDev-binary@ldeleger:SeeDev-binary-11573014-4 JSONTXT

Role of LEC2 in Seed Development. Two consequences of expressing the LEC2 gene postembryonically provide insight into its role in embryo development and strongly support the conclusion that LEC2 is a key regulator of embryogenesis. First, although seedlings expressing the 35S∷LEC2 gene exhibited a range of morphologies (Fig. 5), approximately one-third possessed embryonic rather than postgerminative characteristics. Cotyledons of these seedlings remained fleshy and did not expand, and their roots and hypocotyls did not extend, suggesting that ectopic LEC2 expression can extend embryonic development. 35S∷LEC2 seedlings at the other end of the phenotypic spectrum initially resembled wild type. The reason for this range of phenotypes is not clear, but one possibility is that it relates to differences in transgene expression levels. Second, regardless of their initial morphology, 35S∷LEC2 seedlings gave rise to somatic embryos as indicated by their morphological similarities with zygotic embryos, their expression of embryo-specific genes, and their establishment of functional apical meristems (Figs. 5 and 6). Together, these results suggest that ectopic LEC2 expression is sufficient to establish an embryonic environment that promotes somatic embryo formation. In this regard, it is possible that LEC2 RNA accumulation is regulated posttranscriptionally, thereby accounting for the ability of somatic embryos to “germinate” and, in some cases, give rise to masses of vegetatively growing plantlets. Transgenic seedlings ectopically expressing a different LEC gene, LEC1, have embryonic characteristics that are substantially similar to those of 35S∷LEC2 embryo-like seedlings (12). Moreover, postembryonic expression of LEC1 is sufficient to induce somatic embryogenesis in transgenic plants as occurs with 35S∷LEC2 seedlings, although somatic embryo formation is more robust with LEC2. Recessive mutations in the Arabidopsis PICKLE (PKL) gene also induce somatic embryogenesis in postgerminative roots cultured on hormone-free medium (40). However, LEC1 is expressed in the roots of pkl mutant but not wild-type seedlings (41). This result suggests that the chromodomain protein PKL acts normally to repress LEC1 in postgerminative roots, and therefore that LEC1, at least in part, mediates somatic embryo formation in pkl mutants. Given our results, we hypothesize that LEC2 may also be derepressed in pkl mutants. The ability of LEC2 and LEC1 to induce somatic embryogenesis suggests a role for the genes in the establishment of embryogenic competence. Somatic plant cells generally must be induced to become competent for somatic embryogenesis (42–44). Embryogenic competence is often induced by culturing cells with the hormone auxin and, sometimes cytokinin, although completion of somatic embryogenesis usually requires removal of the hormone(s). LEC2 and LEC1 obviate the need for hormone treatments in the acquisition of embryogenic competence, suggesting the two LEC transcription factors activate genes that play roles in the initiation of somatic embryogenesis. Our finding that both LEC2 and LEC1 RNAs are detected at the earliest embryonic stages tested (Fig. 4; ref. 12) opens the possibility that both genes are involved in establishing embryogenic competence during zygotic embryogenesis. Thus, the role of LEC2 and LEC1 in somatic embryogenesis may reflect their function in zygotic embryogenesis. Similarities in the expression patterns and overexpression phenotypes of LEC2 and LEC1 suggest that they may have partially overlapping roles early in zygotic embryogenesis to induce embryo formation. This interpretation is consistent with analyses showing that lec1 lec2 double mutants arrest at an earlier embryonic stage than either single mutant, which indicates partial genetic redundancy (12, 15). Although LEC1 and LEC2 are each sufficient to induce embryogenic competence in somatic cells, they have similar but not identical functions. Mutations in each gene result in distinct phenotypes, and the double mutant displays a synergistic phenotype (12, 15, 16). Furthermore, the vast majority of 35S∷LEC1 seedlings arrest as embryo-like seedlings and fail to develop further, although cotyledon-like organs sometimes form in place of the first true leaves (12). By contrast, 35S∷LEC2 embryo-like seedlings continued to proliferate, producing callus, cotyledon-like and leaf-like organs in addition to somatic embryos. Thus, LEC1 and LEC2 may have complementary but partially redundant functions in embryo formation. The precise roles of LEC1 and LEC2 in embryo development remain to be determined.