Tissue mechanics have been shown to play a key role in the regulation of morphogenesis in animals [1-4] and may have an equally important role in plants [5-9]. The aerial organs of plants are formed at the shoot apical meristem following a specific phyllotactic pattern [10]. The initiation of an organ from the meristem requires a highly localized irreversible surface deformation, which depends on the demethylesterification of cell wall pectins [11]. Here, we used atomic force microscopy (AFM) to investigate whether these chemical changes lead to changes in tissue mechanics. By mapping the viscoelasticity and elasticity in living meristems, we observed increases in tissue elasticity, correlated with pectin demethylesterification, in primordia and at the site of incipient organs. Measurements of tissue elasticity at various depths showed that, at the site of incipient primordia, the first increases occurred in subepidermal tissues. The results support the following causal sequence of events: (1) demethylesterification of pectin is triggered in subepidermal tissue layers, (2) this contributes to an increase in elasticity of these layers-the first observable mechanical event in organ initiation, and (3) the process propagates to the epidermis during the outgrowth of the organ.

The B3 domain protein LEAFY COTYLEDON2 (LEC2) is required for several aspects of embryogenesis, including the maturation phase, and is sufficient to induce somatic embryo development in vegetative cells. Here, we demonstrate that LEC2 directly controls a transcriptional program involved in the maturation phase of seed development. Induction of LEC2 activity in seedlings causes rapid accumulation of RNAs normally present primarily during the maturation phase. Several RNAs encode proteins with known roles in maturation processes, including seed-storage and lipid-body proteins. Clustering analyses identified other LEC2-induced RNAs not previously shown to be involved in the maturation phase. We show further that genes encoding these maturation RNAs all possess in their 5 flanking regions RY motifs, DNA elements bound by other closely related B3 domain transcription factors. Our finding that recombinant LEC2 specifically binds RY motifs from the 5 flanking regions of LEC2-induced genes provides strong evidence that these genes represent transcriptional targets of LEC2. Although these LEC2-induced RNAs accumulate primarily during the maturation phase, we show that a subset, including AGL15 and IAA30, accumulate in seeds containing zygotes. We discuss how identification of LEC2 target genes provides a potential link between the roles of LEC2 in the maturation phase and in the induction of somatic embryogenesis.Arabidopsis ͉ B3 domain E mbryogenesis in higher plants can be divided conceptually into two distinct phases. Early in embryogenesis, during the morphogenesis phase, the basic body plan of the plant is established with regional specification of apical-basal and radial domains from which morphological structures derive, fixation of polarity from specification of the shoot-root axis, and formation of embryonic tissue and organ systems (1-3). The morphogenesis phase is followed temporally by the maturation phase, although the two phases can overlap (4, 5). During the maturation phase, embryo cell-division rates decline markedly, embryo cells acquire the ability to withstand desiccation, and embryo cell growth occurs, with the accumulation of storage reserves that comprise lipids and proteins in Arabidopsis (6, 7). At the end of the maturation phase, the embryo becomes quiescent metabolically as the seed desiccates.The maturation phase can be viewed as an interruption of an ancestral life cycle, as occurs in lower plants, in which there are no periods of maturation or dormancy separating the end of embryogenesis and the beginning of postembryonic development (4). Evolution of this unique mode of embryogenesis has enabled higher plants to make seeds. The ability to make seeds has provided tremendous selective advantages that, in part, account for the success of the angiosperms (8, 9). Little is known at a mechanistic level about the processes by which the maturation phase has been integrated into the higher plant life cycle. LEAFY COTYLEDON2 (LEC2), along with ABA INSEN-SITIVE3 (ABI3), and FUSCA3 (FUS3), have been im...

LEAFY COTYLEDON2 (LEC2) is a central regulator of embryogenesis sufficient to induce somatic cells to form embryos when expressedectopically. 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.seed development ͉ totipotency A n outstanding characteristic of plants is their totipotency. That is, a variety of cells can be induced to regenerate the adult organism, and several cell types do so by undergoing embryogenesis. For example, the fertilized egg cell undergoes zygotic embryogenesis, a number of differentiated cells of the sporophyte can be induced to undergo somatic embryogenesis, microspores can be diverted from their development into pollen grains to enter microspore embryogenesis, and a number of ovule cell types undergo asexual embryo development in a suite of processes known collectively as apomixis (1-4). Evidence suggests the morphological pathways of development used by these different types of embryos are similar to those of zygotic embryos (5, 6). However, the processes that induce a cell to change its fate and enter an embryonic program of development are not known, nor is it known whether a common pathway is used to initiate embryonic development in all of these diverse cell types.To gain insight into these questions, we focused on Arabidopsis LEAFY COTYLEDON2 (LEC2). LEC2 regulates many distinct aspects of embryogenesis (7,8). For example, during the early morphogenesis phase of embryogenesis in which the basic body plan of the embryo is established, loss-of-function mutations in LEC2 affect the maintenance of embryonic cell fate and specification of cotyledon identity. Later in embryogenesis, lec2 mutants have cotyledon tips that do not accumulate storage reserves nor acquire desiccation tolerance, indicating defects in the initiation and/or maintenance of the maturation phase. Consistent with the pleiotropic effects of the lec2 mutation, LEC2 encodes a transcription factor with a B3 domain, a DNA binding region found thus far only in plant proteins (8-10). Two transcription factors most closely related to LEC2, ABA INSENSITIVE3 (ABI3) and another LEC protein, FUSCA3 (FUS3), also play criti...

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