Isolation of transcription factors binding auxin response elements using a yeast one-hybrid system

Plant hormones play an important role during higher plant embryogenesis. Auxin is central to the development of vascular tissues, formation of lateral and adventitious roots, control of apical dominance, and tropic responses. Auxin response element (AuxRE), present in the promoters of many auxin-induced genes, can confer auxin responsiveness. Using carrot somatic embryo under specific developmental phase, a cDNA expression library was constructed. Several plasmids were recombined containing the tetramer of AuxRE as a bait. After screening by a yeast one-hy-brid system, one positive clone was confirmed and characterized. Electrophoretic mobility shift assay showed that AxRF1 protein expressed in yeast cell could bind AuxRE in vitro. It suggests that AxRF1 participates in regulation of the expression of auxin responsive gene during carrot somatic embryogenesis.     

Auxin up-regulates MtSERK1 expression in both Medicago truncatula root-forming and embryogenic cultures

We have cloned a SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) gene from Medicago truncatula (MtSERK1) and examined its expression in culture using real time PCR. In the presence of the auxin 1-naphthaleneacetic acid (NAA) alone, root differentiation occurs from the proliferating calli in both the cultured highly embryogenic seed line (2HA) and a low to nonembryogenic seed line (M. truncatula cv Jemalong). Auxin stimulated MtSERK1 expression in both 2HA and M. truncatula cv Jemalong. Embryo induction in proliferating calli requires a cytokinin in M. truncatula and unlike root formation is substantively induced in 2HA, not M. truncatula cv Jemalong. On embryo induction medium containing NAA and the cytokinin 6-benzylaminopurine (BAP), expression of MtSERK1 is elevated within 2 d of initiation of culture in both M. truncatula cv Jemalong and 2HA. However, MtSERK1 expression is much higher when both NAA and BAP are in the medium. BAP potentiates the NAA induction because MtSERK1 expression is not up-regulated by BAP alone. The 2HA genotype is able to increase its embryo formation because of the way it responds to cytokinin, but not because of the cytokinin effect on MtSERK1. Although the studies with M. truncatula indicate that somatic embryogenesis is associated with high SERK expression, auxin alone does not induce somatic embryogenesis as in carrot (Daucus carota) and Arabidopsis. Auxin in M. truncatula induces roots, and there is a clear up-regulation of MtSERK1. Although our analyses suggest that MtSERK1 is orthologous to AtSERK1, which in Arabidopsis is involved in somatic embryogenesis, in legumes, MtSERK1 may have a broader role in morphogenesis in cultured tissue rather than being specific to somatic embryogenesis.     

Auxin-induced WUS expression is essential for embryonic stem cell renewal during somatic embryogenesis in Arabidopsis

Somatic embryogenesis requires auxin and establishment of the shoot apical meristem (SAM). WUSCHEL ( WUS ) is critical for stem cell fate determination in the SAM of higher plants. However, regulation of WUS expression by auxin during somatic embryogenesis is poorly understood. Here, we show that expression of several regulatory genes important in zygotic embryogenesis were up-regulated during somatic embryogenesis of Arabidopsis. Interestingly, WUS expression was induced within the embryonic callus at a time when somatic embryos could not be identified morphologically or molecularly. Correct WUS expression, regulated by a defined critical level of exogenous auxin, is essential for somatic embryo induction. Furthermore, it was found that auxin gradients were established in specific regions that could then give rise to somatic embryos. The establishment of auxin gradients was correlated with the induced WUS expression. Moreover, the auxin gradients appear to activate PIN1 polar localization within the embryonic callus. Polarized PIN1 is probably responsible for the observed polar auxin transport and auxin accumulation in the SAM and somatic embryo. Suppression of WUS and PIN1 indicated that both genes are necessary for embryo induction through their regulation of downstream gene expression. Our results reveal that establishment of auxin gradients and PIN1-mediated polar auxin transport are essential for WUS induction and somatic embryogenesis. This study sheds new light on how auxin regulates stem cell formation during somatic embryogenesis.     

Prediction of auxin response elements based on data fusion in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The plant hormone “auxin” is a key regulator of plant development and environmental responses. Many genes in Arabidopsis thaliana are known to be up-regulated in response to auxin. Auxin response factors activate or repress the expression of genes by binding at their promoter regions within auxin response elements (AuxRE) that are key regulatory cis-acting motives. Therefore, the identification of auxin-response elements is among the most important issues to understand the auxin regulation mechanisms. Thus, searching the TGTCTC motif is an unreliable method to identify AuxRE because many AuxRE variants may also be functional. In the present study, we perform an In-silico prediction of AuxREs in promoters of primary auxin responsive genes. We exploit microarray data of auxin response in Arabidopsis thaliana seedlings, in order to provide biological annotation to AuxRE. We apply a data fusion method based on the combined use of evidence theory and fuzzy sets to scan upstream sequences of response genes.     

Isolation of putative glycoprotein gene from early somatic embryo of carrot and its possible involvement in somatic embryo development

Somatic embryogenesis is a unique process in plant cells. For example, embryogenic cells (EC) of carrot (Daucus carota) maintained in a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) regenerate whole plants via somatic embryogenesis after the depletion of 2,4-D. Although some genes such as C-ABI3 and C-LEC1 have been found to be involved in somatic embryogenesis, the critical molecular and cellular mechanisms for somatic embryogenesis are unknown. To characterize the early mechanism in the induction of somatic embryogenesis, we isolated genes expressed during the early stage of somatic embryogenesis after 2,4-D depletion. Subtractive hybridization screening and subsequent RNA gel blot analysis suggested a candidate gene, Carrot Early Somatic Embryogenesis 1 (C-ESE1). C-ESE1 encodes a protein that has agglutinin and S-locus-glycoprotein domains and its expression is highly specific to primordial cells of somatic embryo. Transgenic carrot cells with reduced expression of C-ESE1 had wide intercellular space and decreased polysaccharides on the cell surface and showed delayed development in somatic embryogenesis. The importance of cell-to-cell attachment in somatic embryogenesis is discussed.     

Formation of embryogenic cell clumps from carrot epidermal cells is suppressed by 5-azacytidine, a DNA methylation inhibitor

Using a direct somatic embryogenesis system in carrot, we examined the role of DNA methylation in the change of cellular differentiation state, from somatic to embryogenic. 5-Azacytidine (aza-C), an inhibitor of DNA methylation suppressed the formation of embryogenic cell clumps from epidermal carrot cells. Aza-C also downregulated the expression of DcLEC1c, a LEC1-like embryonic gene in carrot, during morphogenesis of embryos. A carrot DNA methyltransferase gene, Met1-5 was expressed transiently after the induction of somatic embryogenesis by 2,4-dichlorophenoxyacetic acid (2,4-D), before the formation of embryogenic cell clumps. These findings suggested the significance of DNA methylation in acquiring the embryogenic competence in somatic cells in carrot.     

Arginine and ornithine decarboxylases in embryogenic and non-embryogenic carrot cell suspensions

The in vivo activities of arginine and ornithine decarboxylases, key enzymes in the biosynthesis of putrescine and thus polyamines, were measured in three different cell lines of carrot (Daucus carota) during growth and somatic embryogenesis. The activities of these two enzymes differed in the different cell lines in the presence of various levels of auxin (2,4 dichlorophenoxy acetic acid), but was highest during periods of active cell division. During somatic embryo development, the activities of both enzymes were highest during globular stage formation. Thus, both enzymes were found to be active during growth and somatic embryogenesis and could contribute to polyamine biosynthesis.     

2004 SIVB Congress Symposium Proceeding: Mechanisms of somatic embryogenesis in carrot suspension cultures—Morphology,physiology, biochemistry,and molecular biology

Summary Various aspects of somatic embryogenesis in carrot suspension cultures were reviewed on the basis of results obtained in our laboratory. We have established high-frequency and synchronous somatic embryogenesis systems needed for biochemical and molecular analysis. Using these systems, four phases of somatic embryogenesis were identified. The importance of expression of polarities in these phases, particularly from single cells to embryogenic cell clusters, in determining somatic embryogenesis, is emphasized. At the molecular level, genes expressed during somatic embryogenesis were described, and they were classified into three categories: (1) genes involved in cell division, (2) genes involved in organ formation and (3) genes specific for the process of somatic embryogenesis. From the results obtained, it is concluded that discrete developmental phases in carrot somatic embryogenesis are characterized by distinct biochemical and molecular events, but much remains to be understood.     

Auxin synthesized by the YUCCA flavin monooxygenases is essential for embryogenesis and leaf formation in Arabidopsis

Auxin plays a key role in embryogenesis and seedling development, but the auxin sources for the two processes are not defined. Here, we demonstrate that auxin synthesized by the YUCCA (YUC) flavin monooxygenases is essential for the establishment of the basal body region during embryogenesis and the formation of embryonic and postembryonic organs. Both YUC1 and YUC4 are expressed in discrete groups of cells throughout embryogenesis, and their expression patterns overlap with those of YUC10 and YUC11 during embryogenesis. The quadruple mutants of yuc1 yuc4 yuc10 yuc11 fail to develop a hypocotyl and a root meristem, a phenotype similar to those of mp and tir1 afb1 afb2 afb3 auxin signaling mutants. We further show that YUC genes play an essential role in the formation of rosette leaves by analyzing combinations of yuc mutants and the polar auxin transport mutants pin1 and aux1. Disruption of YUC1, YUC4, or PIN1 alone does not abolish leaf formation, but the triple mutant yuc1 yuc4 pin1 fails to form leaves and flowers. Furthermore, disruption of auxin influx carrier AUX1 in the quadruple mutant yuc1 yuc2 yuc4 yuc6, but not in wild-type background, phenocopies yuc1 yuc4 pin1, demonstrating that auxin influx is required for plant leaf and flower development. Our data demonstrate that auxin synthesized by the YUC flavin monooxygenases is an essential auxin source for Arabidopsis thaliana embryogenesis and postembryonic organ formation.     

Mechanisms of somatic embryogenesis in cell cultures: Physiology,biochemistry, and molecular biology

Summary One of the most characteristic cell functions in plants is totipotency. Somatic embryogenesis can be regarded as a model system for the investigation of mechanisms of totipotency, because a high frequency and synchronous embryogenic system from single somatic cells has been established in carrot suspension cultures. Four phases are recognized in this process, and several molecular markers, viz. polypeptides, mRNAs, antigens against monoclonal antibodies, can be detected during the expression of totipotency, but they disappear during its loss. Four organ-specific genes have been isolated from hypocotyls and roots by differential screening. They were expressed preferentially after the globular-heart stages of embryogenesis, and were strongly suppressed by auxin. A CEM 1 gene was isolated by differential screening of embryogenic cell clusters. This gene was expressed strongly and transiently during the proglobular and globular stages. The sequence of CEM 1 was found to encode a polypeptide showing high homology to the elongation factor isolated from eucaryotic cells. Thus good progress is being made in understanding the basic mechanisms of somatic embryogenesis. Presented in the Session-in-Depth Developmental Biology of Embryogenesis at the 1991 World Congress on Cell and Tissue Culture, Anaheim, California, June 16–20, 1991.     

The role of auxin in hairy root induction

Summary We have investigated the relative role of auxin and of Agrobacterium rhizogenes T-DNA in the induction of hairy roots. By infecting carrot discs with suitably constructed bacterial strains containing different T-DNA complements, we have shown that both auxin and the presence of T-DNA in the carrot cells are required for root growth on the discs. Auxin added alone or in combination with cytokinin is not sufficient to induce rooting on uninfected discs. Also cells transformed by T-DNA containing only auxin synthetic genes very rarely differentiate into roots. On the other hand auxin is necessary for hairy root induction since A. rhizogenes devoid of T-DNA-borne auxin genes is not capable of eliciting symptoms in the absence of hormone. Auxin is not required for either T-DNA transfer or T-DNA expression in the transformed host. Cells infected in the absence of auxin, which do not respond by rooting, do contain T-DNA whose expression is shown by the synthesis of hairy root opines; subsequent addition of auxin to these quiescent transformed cells results in root development. A model for hairy root induction where the action of T-DNA is envisaged as conferring auxin responsiveness to the transformed cells is discussed.     

The retromer protein VPS29 links cell polarity and organ initiation in plants

A key feature of plants (as opposed to animals) is their ability to establish new organs not only during embryogenesis, but also throughout their development. A master regulator of organ initiation in plants is the phytohormone auxin. Auxin acts locally as a morphogen and is directionally transported from cell to cell by polarized auxin efflux carriers, termed PIN-FORMED (PIN) proteins. Here we report that the Arabidopsis ortholog of the yeast and mammalian vacuolar protein sorting 29 (VPS29), a member of the retromer complex, mediates the formation of new axes of development. Furthermore, we show that VPS29 is required for endosome homeostasis, PIN protein cycling, and dynamic PIN1 repolarization during development. We propose a model that links VPS29 function, PIN1 polarity, and organ initiation in plants.