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John J Harada - One of the best experts on this subject based on the ideXlab platform.
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LEAFY COTYLEDON1, a key regulator of seed development, is expressed in vegetative and sexual propagules of Selaginella moellendorffii.
PLoS ONE, 2013Co-Authors: Ryan C. Kirkbride, Robert L. Fischer, John J HaradaAbstract:LEAFY COTYLEDON1 (LEC1) is a central regulator of seed development that plays a key role in controlling the Maturation Phase during which storage macromolecules accumulate and the embryo becomes tolerant of desiccation. We queried the genomes of seedless plants and identified a LEC1 homolog in the lycophyte, Selaginella moellendorffii, but not in the bryophyte, Physcomitrella patens. Genetic suppression experiments indicated that Selaginella LEC1 is the functional ortholog of Arabidopsis LEC1. Together, these results suggest that LEC1 originated at least 30 million years before the first seed plants appeared in the fossil record. The accumulation of Selaginella LEC1 RNA primarily in sexual and asexual reproductive structures suggests its involvement in cellular processes similar to those that occur during the Maturation Phase of seed development.
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lecs go crazy in embryo development
Trends in Plant Science, 2008Co-Authors: Siobhan A Braybrook, John J HaradaAbstract:Two fundamental aspects of plant development are the Maturation Phase of embryo development in seed plants and totipotency via somatic embryogenesis (SE). The LEAFY COTYLEDON (LEC) transcription factors (TFs) establish environments that promote cellular processes characteristic of the Maturation Phase and the initiation of somatic embryo formation. Based on recent studies, we and others propose that specific target genes activated by the LEC TFs underlie, in part, their roles in the Maturation Phase and SE. We also propose that the effect of LEC TFs on the balance of abscisic acid to gibberellic acid might link their roles in totipotency and the Maturation Phase.
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Arabidopsis LEAFY COTYLEDON2 induces Maturation traits and auxin activity: Implications for somatic embryogenesis
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Sandra L. Stone, Siobhan A Braybrook, Robert L. Fischer, Robert B. Goldberg, Stephanie L. Paula, Linda W. Kwong, Jonathan E. Meuser, Julie M. Pelletier, Tzung-fu Hsieh, John J HaradaAbstract: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.
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Genes directly regulated by LEAFY COTYLEDON2 provide insight into the control of embryo Maturation and somatic embryogenesis
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Siobhan A Braybrook, Robert L. Fischer, Sandra L. Stone, Soomin Park, Anhthu Q. Bui, Robert B. Goldberg, John J HaradaAbstract: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.
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role of arabidopsis leafy cotyledon genes in seed development
Journal of Plant Physiology, 2001Co-Authors: John J HaradaAbstract:Summary Embryogenesis is a critical period of the higher plant life cycle during which a single-celled zygote undergoes a series of differentiation events, leading to formation of a mature embryo that is generally desiccated and quiescent metabolically. Embryo development consists of two conceptually distinct Phases: the early morphogenesis Phase and the late Maturation Phase. Arabidopsis LEAFY COTYLEDON genes are central regulators of embryogenesis that play key roles in processes that occur during both the early and late Phases. Evidence is discussed which suggests that the LEAFY COTYLEDON genes coordinate the morphogenesis and Maturation Phases and confer embryogenic competence to cells.
Kazushige Yokota - One of the best experts on this subject based on the ideXlab platform.
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Comparison of pro-adipogenic effects between prostaglandin (PG) D2 and its stable, isosteric analogue, 11-deoxy-11-methylene-PGD2, during the Maturation Phase of cultured adipocytes.
Prostaglandins & other lipid mediators, 2018Co-Authors: Mohammad Shahidur Rahman, Kohji Nishimura, Mitsuo Jisaka, Pinky Karim Syeda, Michael Nii N. Nartey, Fumiaki Shono, Mazharul Islam Chowdhury, Hidehisa Shimizu, Kazushige YokotaAbstract:Abstract Prostaglandin (PG) D2 is relatively unstable and dehydrated non-enzymatically into PGJ2 derivatives, which are known to serve as pro-adipogenic factors by activating peroxisome proliferator-activated receptor (PPAR) γ, a master regulator of adipogenesis. 11-Deoxy-11-methylene-PGD2 (11d-11m-PGD2) is a novel, chemically stable, isosteric analogue of PGD2 in which the 11-keto group is replaced by an exocyclic methylene. Here we attempted to investigate pro-adipogenic effects of PGD2 and 11d-11m-PGD2 and to compare the difference in their ways during the Maturation Phase of cultured adipocytes. The dose-dependent study showed that 11d-11m-PGD2 was significantly more potent than natural PGD2 to stimulate the storage of fats suppressed in the presence of indomethacin, a cyclooxygenase inhibitor. These pro-adipogenic effects were caused by the up-regulation of adipogenesis as evident with higher gene expression levels of adipogenesis markers. Analysis of transcript levels revealed the enhanced gene expression of two subtypes of cell-surface membrane receptors for PGD2, namely the prostanoid DP1 and DP2 (chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2)) receptors together with lipocalin-type PGD synthase during the Maturation Phase. Specific agonists for DP1, CRTH2, and PPARγ were appreciably effective to rescue adipogenesis attenuated by indomethacin. The action of PGD2 was attenuated by specific antagonists for DP1 and PPARγ. By contrast, the effect of 11d-11m-PGD2 was more potently interfered by a selective antagonist for CRTH2 than that for DP1 while PPARγ antagonist GW9662 had almost no inhibitory effects. These results suggest that PGD2 exerts its pro-adipogenic effect principally through the mediation of DP1 and PPARγ, whereas the stimulatory effect of 11d-11m-PGD2 on adipogenesis occurs preferentially by the interaction with CRTH2.
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Stimulation of fat storage by prostacyclin and selective agonists of prostanoid IP receptor during the Maturation Phase of cultured adipocytes.
Cytotechnology, 2016Co-Authors: Ferdous Khan, Kohji Nishimura, Mitsuo Jisaka, Pinky Karim Syeda, Michael Nii N. Nartey, Mohammad Shahidur Rahman, Mohammad Safiqul Islam, Fumiaki Shono, Kazushige YokotaAbstract:We have previously shown that cultured adipocytes have the ability to biosynthesize prostaglandin (PG) I2 called alternatively as prostacyclin during the Maturation Phase by the positive regulation of gene expression of PGI synthase and the prostanoid IP receptor. To clarify how prostacyclin regulates adipogenesis, we investigated the effects of prostacyclin and the specific agonists or antagonists for the IP receptor on the storage of fats during the Maturation Phase of cultured adipocytes. Exogenous PGI2 and the related selective agonists for the IP receptor including MRE-269 and treprostinil rescued the storage of fats attenuated by aspirin, a cyclooxygenase inhibitor. On the other hand, selective antagonists for IP such as CAY10441 and CAY10449 were effective to suppress the accumulation of fats as GW9662, a specific antagonist for peroxisome proliferator-activated receptor (PPAR)γ. Thus, pro-adipogenic action of prostacyclin can be explained by the action mediated through the IP receptor expressed at the Maturation stage of adipocytes. Cultured adipocytes incubated with each of PGI2 and MRE-269 together with troglitazone, an activator for PPARγ, exhibited additively higher stimulation of fats storage than with either compound alone. The combined effect of MRE-269 and troglitazone was almost abolished by co-incubation with GW9662, but not with CAY10441. Increasing concentrations of troglitazone were found to reverse the inhibitory effect of CAY10441 in a dose-dependent manner while those of MRE-269 failed to rescue adipogenesis suppressed by GW9662, indicating the critical role of the PPARγ activation as a downstream factor for the stimulated adipogenesis through the IP receptor. Treatment of cultured adipocytes with cell permeable stable cAMP analogues or forskolin as a cAMP elevating agent partly restored the inhibitory effect of aspirin. However, excess levels of cAMP stimulated by forskolin attenuated adipogenesis. Supplementation with H-89, a cell permeable inhibitor for protein kinase A (PKA), had no effect on the promoting action of PGI2 or MRE-269 along with aspirin on the storage of fats, suggesting that the promotion of adipogenesis mediated by the IP receptor does not require the PKA activity.
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Pretreatment of cultured preadipocytes with arachidonic acid during the differentiation Phase without a cAMP-elevating agent enhances fat storage after the Maturation Phase.
Prostaglandins & other lipid mediators, 2016Co-Authors: Ferdous Khan, Kohji Nishimura, Mitsuo Jisaka, Pinky Karim Syeda, Michael Nii N. Nartey, Mohammad Shahidur Rahman, Mohammad Safiqul Islam, Fumiaki Shono, Kazushige YokotaAbstract:Arachidonic acid (AA) and the related prostanoids exert complex effects on the adipocyte differentiation depending on the culture conditions and life stages. Here, we investigated the effect of the pretreatment of cultured 3T3-L1 preadipocytes with exogenous AA during the differentiation Phase without 3-isobutyl-1-methylxanthine (IBMX), a cAMP-elevating agent, on the storage of fats after the Maturation Phase. This pretreatment with AA stimulated appreciably adipogenesis after the Maturation Phase as evident with the up-regulated gene expression of adipogenic markers. The stimulatory effect of the pretreatment with AA was attenuated by the co-incubation with each of cyclooxygenase (COX) inhibitors. Among exogenous prostanoids and related compounds, the pretreatment with MRE-269, a selective agonist of the IP receptor for prostaglandin (PG) I2, strikingly stimulated the storage of fats in adipocytes. The gene expression analysis of arachidonate COX pathway revealed that the transcript levels of inducible COX-2, membrane-bound PGE synthase-1, and PGF synthase declined more greatly in cultured preadipocytes treated with AA. By contrast, the expression levels of COX-1, cytosolic PGE synthase, and PGI synthase remained constitutive. The treatment of cultured preadipocytes with AA resulted in the decreased synthesis of PGE2 and PGF2α serving as anti-adipogenic PGs although the biosynthesis of pro-adipogenic PGI2 was up-regulated during the differentiation Phase. Moreover, the gene expression levels of EP4 and FP, the respective prostanoid receptors for PGE2 and PGF2α, were gradually suppressed by the supplementation with AA, whereas that of IP for PGI2 remained relatively constant. Collectively, these results suggest the predominant role of endogenous PGI2 in the stimulatory effect of the pretreatment of cultured preadipoccytes with AA during the differentiation Phase without IBMX on adipogenesis after the Maturation Phase.
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Endogenous synthesis of prostacyclin was positively regulated during the Maturation Phase of cultured adipocytes
Cytotechnology, 2013Co-Authors: Mohammad S. Rahman, Kohji Nishimura, Mitsuo Jisaka, Tsutomu Nagaya, Ferdous Khan, Pinky Karim Syeda, Fumiaki Shono, Kazushige YokotaAbstract:Prostacyclin alternatively called prostaglandin (PG) I2 is an unstable metabolite synthesized by the arachidonate cyclooxygenase pathway. Earlier studies have suggested that prostacyclin analogues can act as a potent effector of adipose differentiation. However, biosynthesis of PGI2 has not been determined comprehensively at different life stages of adipocytes. PGI2 is rapidly hydrolyzed to the stable product, 6-keto-PGF1α, in biological fluids. Therefore, the generation of PGI2 can be quantified as the amount of 6-keto-PGF1α. In this study, we attempted to develop a solid-Phase enzyme-linked immunosorbent assay (ELISA) using a mouse antiserum specific for 6-keto-PGF1α. According to the typical calibration curve of our ELISA, 6-keto-PGF1α can be quantified from 0.8 pg to 7.7 ng in an assay. The evaluation of our ELISA revealed the higher specificity of our antiserum without the cross-reaction with other related prostanoids while it exhibited only the cross-reaction of 1.5 % with PGF2α. The resulting ELISA was applied to the quantification of 6-keto-PGF1α generated endogenously by cultured 3T3-L1 cells at different stages. The cultured cells showed the highest capability to generate 6-keto-PGF1α during the Maturation Phase of 4–6 days, which was consistent with the coordinated changes in the gene expression of PGI synthase and the IP receptor for PGI2. Following these events, the accumulation of fats was continuously promoted up to 14 days. Thus, our immunological assay specific for 6-keto-PGF1α is useful for monitoring the endogenous levels of the unstable parent PGI2 at different life stages of adipogenesis and for further studies on the potential association with the up-regulation of adipogenesis in cultured adipocytes.
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Cultured Preadipocytes Undergoing Stable Transfection with Cyclooxygenase-1 in the Antisense Direction Accelerate Adipogenesis During the Maturation Phase of Adipocytes
Applied biochemistry and biotechnology, 2013Co-Authors: Mohammad S. Rahman, Kohji Nishimura, Mitsuo Jisaka, Tsutomu Nagaya, Ferdous Khan, Pinky Karim Syeda, Fumiaki Shono, Kazushige YokotaAbstract:The arachidonate cyclooxygenase (COX) pathway is involved in the generation of several types of endogenous prostaglandins (PGs) with opposite effects on adipogenesis at different life stages of adipocytes. However, the specific role of COX isoforms, the rate-limiting enzymes for the pathway, remains elusive in the regulation of the endogenous synthesis of PGs. This study was aimed at the selective suppression of the constitutive COX-1 in cultured preadipocytes by the isolation of cloned preadipocytes transfected stably with a mammalian expression vector harboring cDNA encoding mouse COX-1 in the antisense direction. The gene expression analysis revealed that the transcript and protein levels of the constitutive COX-1 were substantially suppressed in the isolated cloned transfectants with antisense COX-1. By contrast, the expression of the inducible COX-2 was not affected in the stable transfectants with antisense COX-1. All of the cloned stable transfectants with antisense COX-1 exhibited a significant reduction in the immediate synthesis of PGE2 serving as an anti-adipogenic factor. The sustained expression of COX-1 in the antisense direction induced the appreciable stimulation of fat storage in adipocytes during the Maturation Phase, which was associated with the higher expression levels of adipocyte-specific genes, indicating the positive regulation of adipogenesis program. Moreover, the up-regulation of adipogenesis is accompanied by a higher production of J2 series PGs including 15-deoxy-Δ12,14-PGJ2 and Δ12-PGJ2, known as pro-adipogenic factors by the transfectants with antisense COX-1. The results suggest that the inducible COX-2 can contribute to the endogenous synthesis of PGJ2 derivatives acting as autocrine mediators to simulate adipogenesis during the Maturation Phase by way of compensation for the suppressed expression of the constitutive COX-1.
Mariko Sugiyama - One of the best experts on this subject based on the ideXlab platform.
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tanycyte like cells derived from mouse embryonic stem culture show hypothalamic neural stem progenitor cell functions
Endocrinology, 2019Co-Authors: Mayuko Kano, Hidetaka Suga, Takeshi Ishihara, Mayu Sakakibara, Mika Soen, Tomiko Yamada, Hajime Ozaki, Kazuki Mitsumoto, Takatoshi Kasai, Mariko SugiyamaAbstract:Tanycytes have recently been accepted as neural stem/progenitor cells in the postnatal hypothalamus. Persistent retina and anterior neural fold homeobox (Rax) expression is characteristic of tanycytes in contrast to its transient expression of whole hypothalamic precursors. In this study, we found that Rax+ residual cells in the Maturation Phase of hypothalamic differentiation in mouse embryonic stem cell (mESC) cultures had similar characteristics to ventral tanycytes. They expressed typical neural stem/progenitor cell markers, including Sox2, vimentin, and nestin, and differentiated into mature neurons and glial cells. Quantitative RT-PCR analysis showed that Rax+ residual cells expressed Fgf-10, Fgf-18, and Lhx2, which are expressed by ventral tanycytes. They highly expressed tanycyte-specific genes Dio2 and Gpr50 compared with Rax+ early hypothalamic progenitor cells. Therefore, Rax+ residual cells in the Maturation Phase of hypothalamic differentiation were considered to be more differentiated and similar to late progenitor cells and tanycytes. They self-renewed and formed neurospheres when cultured with exogenous FGF-2. Additionally, these Rax+ neurospheres differentiated into three neuronal lineages (neurons, astrocytes, and oligodendrocytes), including neuropeptide Y+ neuron, that are reported to be differentiated from ventral tanycytes toward the arcuate nuclei. Thus, Rax+ residual cells were multipotent neural stem/progenitor cells. Rax+ neurospheres were stably passaged and retained high Sox2 expression even after multiple passages. These results suggest the successful induction of Rax+ tanycyte-like cells from mESCs [induced tanycyte-like (iTan) cells]. These hypothalamic neural stem/progenitor cells may have potential in regenerative medicine and as a research tool.
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Tanycyte-Like Cells Derived From Mouse Embryonic Stem Culture Show Hypothalamic Neural Stem/Progenitor Cell Functions.
Endocrinology, 2019Co-Authors: Mayuko Kano, Hidetaka Suga, Takeshi Ishihara, Mayu Sakakibara, Mika Soen, Tomiko Yamada, Hajime Ozaki, Kazuki Mitsumoto, Takatoshi Kasai, Mariko SugiyamaAbstract:Tanycytes have recently been accepted as neural stem/progenitor cells in the postnatal hypothalamus. Persistent retina and anterior neural fold homeobox (Rax) expression is characteristic of tanycytes in contrast to its transient expression of whole hypothalamic precursors. In this study, we found that Rax+ residual cells in the Maturation Phase of hypothalamic differentiation in mouse embryonic stem cell (mESC) cultures had similar characteristics to ventral tanycytes. They expressed typical neural stem/progenitor cell markers, including Sox2, vimentin, and nestin, and differentiated into mature neurons and glial cells. Quantitative RT-PCR analysis showed that Rax+ residual cells expressed Fgf-10, Fgf-18, and Lhx2, which are expressed by ventral tanycytes. They highly expressed tanycyte-specific genes Dio2 and Gpr50 compared with Rax+ early hypothalamic progenitor cells. Therefore, Rax+ residual cells in the Maturation Phase of hypothalamic differentiation were considered to be more differentiated and similar to late progenitor cells and tanycytes. They self-renewed and formed neurospheres when cultured with exogenous FGF-2. Additionally, these Rax+ neurospheres differentiated into three neuronal lineages (neurons, astrocytes, and oligodendrocytes), including neuropeptide Y+ neuron, that are reported to be differentiated from ventral tanycytes toward the arcuate nuclei. Thus, Rax+ residual cells were multipotent neural stem/progenitor cells. Rax+ neurospheres were stably passaged and retained high Sox2 expression even after multiple passages. These results suggest the successful induction of Rax+ tanycyte-like cells from mESCs [induced tanycyte-like (iTan) cells]. These hypothalamic neural stem/progenitor cells may have potential in regenerative medicine and as a research tool.
Geoffrey F. Gibbons - One of the best experts on this subject based on the ideXlab platform.
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insulin inhibits the Maturation Phase of vldl assembly via a phosphoinositide 3 kinase mediated event
Arteriosclerosis Thrombosis and Vascular Biology, 2001Co-Authors: Anna-marie Brown, Geoffrey F. GibbonsAbstract:LY 294002 (80 μmol/L), an inhibitor of phosphoinositide 3-kinase, was used to investigate the involvement of this enzyme in the insulin-mediated regulation of very low density lipoprotein (VLDL) apolipoprotein B (apoB) output from cultured rat hepatocytes. Newly synthesized apoB was pulse-labeled with [35S]methionine and was then allowed to assemble, via an intermediate precursor stage, into mature VLDL during subsequent chase periods. Brefeldin A (BFA, 0.2 μg/mL) was used to discriminate between the role of insulin in the regulation of the early, compared with the later, events of VLDL assembly, including apoB degradation. Insulin (78 nmol/L), when present during the pulse-labeling and subsequent chase periods, inhibited the secretion of apoB-100 and apoB-48 as VLDL by 53% and 56%, respectively. Degradation of both was concomitantly increased. Secretion of high density lipoprotein apoB, derived from VLDL precursors, was relatively unaffected under these conditions, as was the net synthesis of apoB-100 an...
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Insulin Inhibits the Maturation Phase of VLDL Assembly via a Phosphoinositide 3-Kinase—Mediated Event
Arteriosclerosis thrombosis and vascular biology, 2001Co-Authors: Anna-marie Brown, Geoffrey F. GibbonsAbstract:LY 294002 (80 μmol/L), an inhibitor of phosphoinositide 3-kinase, was used to investigate the involvement of this enzyme in the insulin-mediated regulation of very low density lipoprotein (VLDL) apolipoprotein B (apoB) output from cultured rat hepatocytes. Newly synthesized apoB was pulse-labeled with [35S]methionine and was then allowed to assemble, via an intermediate precursor stage, into mature VLDL during subsequent chase periods. Brefeldin A (BFA, 0.2 μg/mL) was used to discriminate between the role of insulin in the regulation of the early, compared with the later, events of VLDL assembly, including apoB degradation. Insulin (78 nmol/L), when present during the pulse-labeling and subsequent chase periods, inhibited the secretion of apoB-100 and apoB-48 as VLDL by 53% and 56%, respectively. Degradation of both was concomitantly increased. Secretion of high density lipoprotein apoB, derived from VLDL precursors, was relatively unaffected under these conditions, as was the net synthesis of apoB-100 an...
Siobhan A Braybrook - One of the best experts on this subject based on the ideXlab platform.
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lecs go crazy in embryo development
Trends in Plant Science, 2008Co-Authors: Siobhan A Braybrook, John J HaradaAbstract:Two fundamental aspects of plant development are the Maturation Phase of embryo development in seed plants and totipotency via somatic embryogenesis (SE). The LEAFY COTYLEDON (LEC) transcription factors (TFs) establish environments that promote cellular processes characteristic of the Maturation Phase and the initiation of somatic embryo formation. Based on recent studies, we and others propose that specific target genes activated by the LEC TFs underlie, in part, their roles in the Maturation Phase and SE. We also propose that the effect of LEC TFs on the balance of abscisic acid to gibberellic acid might link their roles in totipotency and the Maturation Phase.
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Arabidopsis LEAFY COTYLEDON2 induces Maturation traits and auxin activity: Implications for somatic embryogenesis
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Sandra L. Stone, Siobhan A Braybrook, Robert L. Fischer, Robert B. Goldberg, Stephanie L. Paula, Linda W. Kwong, Jonathan E. Meuser, Julie M. Pelletier, Tzung-fu Hsieh, John J HaradaAbstract: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.
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Genes directly regulated by LEAFY COTYLEDON2 provide insight into the control of embryo Maturation and somatic embryogenesis
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Siobhan A Braybrook, Robert L. Fischer, Sandra L. Stone, Soomin Park, Anhthu Q. Bui, Robert B. Goldberg, John J HaradaAbstract: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.