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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.
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