Sandra L. Stone

LEAFY COTYLEDON2 encodes a B3 domain transcription factor that induces embryo development.

The Arabidopsis LEAFY COTYLEDON2 (LEC2) gene is a central embryonic regulator that serves critical roles both early and late during embryo development. LEC2 is required for the maintenance of suspensor morphology, specification of cotyledon identity, progression through the maturation phase, and suppression of premature germination. We cloned the LEC2 gene on the basis of its chromosomal position and showed that the predicted polypeptide contains a B3 domain, a DNA-binding motif unique to plants that is characteristic of several transcription factors. We showed that LEC2 RNA accumulates primarily during seed development, consistent with our finding that LEC2 shares greatest similarity with the B3 domain transcription factors that act primarily in developing seeds, VIVIPAROUS1/ABA INSENSITIVE3 and FUSCA3. Ectopic, postembryonic expression of LEC2 in transgenic plants induces the formation of somatic embryos and other organ-like structures and often confers embryonic characteristics to seedlings. Together, these results suggest that LEC2 is a transcriptional regulator that establishes a cellular environment sufficient to initiate embryo development.

Arabidopsis LEAFY COTYLEDON2 induces maturation traits and auxin activity: Implications for somatic embryogenesis

LEAFY COTYLEDON2 (LEC2) is a central regulator of embryogenesis sufficient to induce somatic cells to form embryos when expressed ectopically. 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.

Comprehensive developmental profiles of gene activity in regions and subregions of the Arabidopsis seed

Significance Seeds are complex structures that are comprised of the embryo, endosperm, and seed coat. Despite their importance for food, fiber, and fuel, the cellular processes that characterize different regions of the seed are not known. We profiled gene activity genome-wide in every organ, tissue, and cell type of Arabidopsis seeds from fertilization through maturity. The resulting mRNA datasets provide unique insights into the cellular processes that occur in understudied seed regions, revealing unexpected overlaps in the functional identities of seed regions and enabling predictions of gene regulatory networks. This dataset is an essential resource for studies of seed biology. Seeds are complex structures that consist of the embryo, endosperm, and seed-coat regions that are of different ontogenetic origins, and each region can be further divided into morphologically distinct subregions. Despite the importance of seeds for food, fiber, and fuel globally, little is known of the cellular processes that characterize each subregion or how these processes are integrated to permit the coordinated development of the seed. We profiled gene activity genome-wide in every organ, tissue, and cell type of Arabidopsis seeds from fertilization through maturity. The resulting mRNA datasets offer the most comprehensive description of gene activity in seeds with high spatial and temporal resolution, providing unique insights into the function of understudied seed regions. Global comparisons of mRNA populations reveal unexpected overlaps in the functional identities of seed subregions. Analyses of coexpressed gene sets suggest that processes that regulate seed size and filling are coordinated across several subregions. Predictions of gene regulatory networks based on the association of transcription factors with enriched DNA sequence motifs upstream of coexpressed genes identify regulators of seed development. These studies emphasize the utility of these datasets as an essential resource for the study of seed biology.

Structural and Biochemical Changes in Loblolly Pine (Pinus taeda L.) Seeds during Germination and Early Seedling Growth. II. Storage Triacylglycerols and Carbohydrates

Triacylglycerols (TAGs) comprised 59% of the total storage reserve in mature loblolly pine (Pinus taeda L.) seeds; 80% of these TAGs were stored in the megagametophyte. The TAG breakdown in the seedling was initiated before radicle emergence (during germination), while in the megagametophyte breakdown occurred after radicle emergence (during early seedling growth). In both seed tissues, the majority of TAG breakdown took place during early seedling growth. Within the seedling, the most rapid rate of TAG breakdown occurred in the radicle and hypocotyl. Unlike TAGs, there was very little carbohydrate stored in loblolly pine seeds at maturity. Levels of 80% ethanol‐soluble carbohydrate in the megagametophyte and seedling decreased during germination and then increased during early seedling growth. This increase coincided with the period of rapid TAG depletion. Accompanying the increase in 80% ethanol‐soluble carbohydrate level was a corresponding increase in 80% ethanol‐insoluble carbohydrates, such as starch, in the seedling during early seedling growth. Accumulation of starch occurred in both the cotyledons and hypocotyl. Starch accumulation in the megagametophyte was more transient, occurring around germination. The megagametophyte was important for the growth and nutrition of the seedling. In the presence of the megagametophyte, the seedling accumulated sucrose during early seedling growth. However, in the absence of the megagametophyte, loblolly pine seedlings failed to accumulate carbohydrates to any great extent.

Structural and Biochemical Changes in Loblolly Pine (Pinus taeda L.) Seeds During Germination and Early-Seedling Growth. I. Storage Protein Reserves

Quantitative and qualitative changes in the storage proteins of loblolly pine (Pinus taeda L.) seeds were followed during germination and early-seedling growth and were correlated with light-microscopic observations. In both the megagametophyte and the embryo, the cells of tissues from fully stratified seeds appeared very similar to the cells of tissues from mature desiccated seeds. A change in the appearance of protein vacuoles, resulting from the hydrolysis of storage proteins, occurred in the seedling prior to the completion of germination (denoted by radicle emergence from the seed coat) and continued during early-seedling growth. Within the seedling, storage proteins were mobilized more rapidly in the root pole, including the hypocotyl and radicle, than in the shoot pole, including the cotyledonary whorl, and shoot apex or epicotyl. In both parts of the seedling, protein hydrolysis was first observed in the procambial and epidermal tissue. In contrast to the seedling, changes in the appearance of protein vacuoles were not evident in the megagametophyte until after germination was completed. Changes in protein vacuoles in the megagametophyte occurred in two directional waves, relative to corrosion cavity proximity.

Patterns of storage protein and triacylglycerol accumulation during loblolly pine somatic embryo maturation

Conifer somatic embryo germination and early seedling growth are fundamentally different than in their zygotic counterparts in that the living maternal megagametophyte tissue surrounding the embryo is absent. The megagametophyte contains the majority of the seed storage reserves in loblolly pine and the lack of the megagametophyte tissue poses a significant challenge to somatic embryo germination and growth. We investigated the differences in seed storage reserves between loblolly pine mature zygotic embryos and somatic embryos that were capable of germination and early seedling growth. Somatic embryos utilized in this study contained significantly lower levels of triacylglycerol and higher levels of storage proteins relative to zygotic embryos. A shift in the ratio of soluble to insoluble protein present was also observed. Mature zygotic embryos had roughly a 3:2 ratio of soluble to insoluble protein whereas the somatic embryos contained over 5-fold more soluble protein compared to insoluble protein. This indicates that the somatic embryos are not only producing more protein overall, but that this protein is biased more heavily towards soluble protein, indicating possible differences in metabolic activity at the time of desiccation.

Leafy Cotyledon Genes and the Control of Embryo Development

Zygotic embryogenesis begins with the double fertilization event in which the egg cell of the female gametophyte fuses with one sperm nucleus to form the zygote, and the central cell fuses with another sperm nucleus to form the endosperm mother cell (Russell, 1993). The single-celled zygote then undergoes a series of differentiation events, resulting in the formation of a mature embryo. The basic body plan of the plant is established during the early morphogenesis phase of embryogenesis. During this period, regional specification events establish morphological domains within the developing embryo, the polarity of the embryo is expressed as a shoot-root axis, the embryonic tissue and organ systems are formed, and the rudimentary shoot and root apices develop (Goldberg et al., 1994; Jurgens, 2001; West and Harada, 1993). In seed plants, this early embryonic period is followed by the maturation phase in which the embryo acquires the ability to withstand desiccation, storage reserves in the form of proteins, lipids, and starch accumulate in the embryo and/or endosperm, and the embryo becomes metabolically quiescent as a result of desiccation (Bewley, 1997; Harada, 1997; Koornneef and Karssen, 1994). The seed generally remains in a quiescent state until environmental conditions signal the embryo to germinate.