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Erich Grotewold

Publications and source records attributed to Erich Grotewold.

At least 19 recordsLinked to original sources
PubMed2006

An ACT-like domain participates in the dimerization of several plant basic-helix-loop-helix transcription factors.

The maize basic-helix-loop-helix (bHLH) factor R belongs to a group of proteins with important functions in the regulation of metabolism and development through the cooperation with R2R3-MYB transcription factors. Here we show that in addition to the bHLH and the R2R3-MYB-interacting domains, R contains a dimerization region located C-terminal to the bHLH motif. This protein-protein interaction domain is important for the regulation of anthocyanin pigment biosynthesis by contributing to the recruitment of the C1 R2R3-MYB factor to the C1 binding sites present in the promoters of flavonoid biosynthetic genes. The R dimerization region bares structural similarity to the ACT domain present in several metabolic enzymes. Protein fold recognition analyses resulted in the identification of similar ACT-like domains in several other plant bHLH proteins. We show that at least one of these related motifs is capable of mediating homodimer formation. These findings underscore the function of R as a docking site for multiple protein-protein interactions and provide evidence for the presence of a novel dimerization domain in multiple plant bHLH proteins.

Amino Acid Motifs↗
PubMed2006

Apigenin-induced-apoptosis is mediated by the activation of PKCdelta and caspases in leukemia cells.

Apigenin, a flavone abundantly found in fruits and vegetables, exhibits antiproliferative, anti-inflammatory, and antimetastatic activities through poorly defined mechanisms. In the present study, the treatment of different cell lines with apigenin resulted in selective antiproliferative and apoptotic effect in monocytic and lymphocytic leukemias. Apigenin-induced-apoptosis was mediated by the activation of caspase-9 and caspase-3. Apigenin was found intracellularly and localized to the mitochondria. Treatment of monocytic cells with apigenin was accompanied by an increase in reactive oxygen species (ROS) and phosphorylation of the MAPKs, p38 and ERK. However, the inhibition of ROS, p38 or ERK failed to block apoptosis, suggesting that these cellular responses induced by apigenin are not essential for the induction of apoptosis. In addition, apigenin induced the activation of PKCdelta. Pharmacological inhibition of PKCdelta, the expression of dominant-negative PKCdelta and silencing of PKCdelta in leukemia cells showed that apigenin-induced-apoptosis requires PKCdelta activity. Together, these results indicate that this flavonoid provides selective activity to promote caspase-dependent-apoptosis of leukemia cells and uncover an essential role of PKCdelta during the induction of apoptosis by apigenin.

3T3 Cells↗
PubMed2006

AGRIS and AtRegNet. a platform to link cis-regulatory elements and transcription factors into regulatory networks.

Gene regulatory pathways converge at the level of transcription, where interactions among regulatory genes and between regulators and target genes result in the establishment of spatiotemporal patterns of gene expression. The growing identification of direct target genes for key transcription factors (TFs) through traditional and high-throughput experimental approaches has facilitated the elucidation of regulatory networks at the genome level. To integrate this information into a Web-based knowledgebase, we have developed the Arabidopsis Gene Regulatory Information Server (AGRIS). AGRIS, which contains all Arabidopsis (Arabidopsis thaliana) promoter sequences, TFs, and their target genes and functions, provides the scientific community with a platform to establish regulatory networks. AGRIS currently houses three linked databases: AtcisDB (Arabidopsis thaliana cis-regulatory database), AtTFDB (Arabidopsis thaliana transcription factor database), and AtRegNet (Arabidopsis thaliana regulatory network). AtTFDB contains 1,690 Arabidopsis TFs and their sequences (protein and DNA) grouped into 50 (October 2005) families with information on available mutants in the corresponding genes. AtcisDB consists of 25,806 (September 2005) promoter sequences of annotated Arabidopsis genes with a description of putative cis-regulatory elements. AtRegNet links, in direct interactions, several hundred genes with the TFs that control their expression. The current release of AtRegNet contains a total of 187 (September 2005) direct targets for 66 TFs. AGRIS can be accessed at http://Arabidopsis.med.ohio-state.edu.

Arabidopsis↗
PubMed2006

The genetics and biochemistry of floral pigments.

Three major groups of pigments, the betalains, the carotenoids, and the anthocyanins, are responsible for the attractive natural display of flower colors. Because of the broad distribution of anthocyanins (synthesized as part of the flavonoid pathway) among the flowering plants, their biosynthesis and regulation are best understood. However, over the past few years, significant progress has been made in understanding the synthesis and participation of carotenoids (derived from isoprenoids) and betalains (derived from tyrosine) in flower pigmentation. These three families of pigments play important ecological functions, for example in the attraction of pollinating animals. Anthocyanins in particular have also been the target of numerous biotechnological efforts with the objective of creating new, or altering the properties of existing, coloring compounds. The focus of this review is to examine the biosynthesis, regulation, and contribution to flower coloration of these three groups of pigments.

Flowers↗
PubMed2005

Covalent attachment of the plant natural product naringenin to small glass and ceramic beads.

BACKGROUND: Natural products have numerous medicinal applications and play important roles in the biology of the organisms that accumulate them. Few methods are currently available for identifying proteins that bind to small molecules, therefore the discovery of cellular targets for natural products with pharmacological activity continues to pose a significant challenge in drug validation. Similarly, the identification of enzymes that participate in the biosynthesis or modification of natural products remains a formidable bottleneck for metabolic engineering. Flavonoids are one large group of natural products with a diverse number of functions in plants and in human health. The coupling of flavonoids to small ceramic and glass beads provides a first step in the development of high-throughput, solid-support base approaches to screen complex libraries to identify proteins that bind natural products. RESULTS: The utilization of small glass and ceramic beads as solid supports for the coupling of small molecules was explored. Initial characterization of the beads indicated uniform and high capacity loading of amino groups. Once the beads were deemed adequate for the linking of small molecules by the coupling of NHS-fluorescein followed by microscopy, chemical hydrolysis and fluorometry, the flavonoid naringenin was modified with 1,4-dibromobutane, followed by the attachment of aminopropyltriethoxysilane. After NMR structural confirmation, the resulting 7-(4-(3-(triethoxysilyl)propylamino)butoxy) naringenin was attached to the ceramic beads. CONCLUSION: Our results demonstrate that ceramic and glass beads provide convenient solid supports for the efficient and facile coupling of small molecules. We succeeded in generating naringenin-coupled ceramic and glass beads. We also developed a convenient series of steps that can be applied for the solid-support coupling of other related flavonoids. The availability of solid-support coupled naringenin opens up new opportunities for the identification of flavonoid-binding proteins.

Journal Article↗
PubMed2005

Light-induced morphological alteration in anthocyanin-accumulating vacuoles of maize cells.

BACKGROUND: Plant pigmentation is affected by a variety of factors. Light, an important plant developmental signal, influences the accumulation of anthocyanins primarily through the activation of the transcription factors that regulate the flavonoid biosynthetic pathway. In this study, we utilized maize Black Mexican Sweet (BMS) cells expressing the R and C1 regulators of anthocyanin biosynthesis from a light-insensitive promoter as a means to investigate the existence of additional levels of control of pigmentation by light. RESULTS: BMS cells expressing the R and C1 regulators from the CaMV 35S constitutive promoter accumulate anthocyanins when grown in complete darkness, suggesting that the transcription factors R and C1 are sufficient for the transcription of the genes corresponding to the structural enzymes of the pathway, with no requirement for additional light-induced regulators. Interestingly, light induces a "darkening" in the color of the purple anthocyanin pigmentation of transgenic BMS cells expressing R and C1. This change in the pigment hue is not associated with a variation in the levels or types of anthocyanins present, or with an alteration of the transcript levels of several flavonoid biosynthetic genes. However, cytological observations show that light drives unexpected changes in the morphology and distribution of the anthocyanins-containing vacuolar compartments. CONCLUSION: By uncoupling the effect of light on anthocyanin accumulation, we have found light to induce the fusion of anthocyanin-containing vacuoles, the coalescence of anthocyanic vacuolar inclusion (AVI)-like structures contained, and the spread of anthocyanins from the inclusions into the vacuolar sap. Similar light-induced alterations in vacuolar morphology are also evident in the epidermal cells of maize floral whorls accumulating anthocyanins. Our findings suggest a novel mechanism for the action of light on the vacuolar storage of anthocyanin.

Anthocyanins↗
PubMed2005

Genome wide analysis of Arabidopsis core promoters.

BACKGROUND: Core promoters are the gene regulatory regions most proximal to the transcription start site (TSS), central to the formation of pre-initiation complexes and for combinatorial gene regulation. The DNA elements required for core promoter function in plants are poorly understood. To establish the sequence motifs that characterize plant core promoters and to compare them to the corresponding sequences in animals, we took advantage of available full-length cDNAs (FL-cDNAs) and predicted upstream regulatory sequences to carry out the analysis of 12,749 Arabidopsis core promoters. RESULTS: Using a combination of expectation maximization and Gibbs sampling methods, we identified several motifs overrepresented in Arabidopsis core promoters. One of them corresponded to the TATA element, for which an in-depth analysis resulted in the generation of robust TATA Nucleotide Frequency Matrices (NFMs) capable of predicting Arabidopsis TATA elements with a high degree of confidence. We established that approximately 29% of all Arabidopsis promoters contain TATA motifs, clustered around position -32 with respect to the TSS. The presence of TATA elements was associated with genes represented more frequently in EST collections and with shorter 5' UTRs. No cis-elements were found over-represented in TATA-less, compared to TATA-containing promoters. CONCLUSION: Our studies provide a first genome-wide illustration of the composition and structure of core Arabidopsis promoters. The percentage of TATA-containing promoters is much lower than commonly recognized, yet comparable to the number of Drosophila promoters containing a TATA element. Although several other DNA elements were identified as over-represented in Arabidopsis promoters, they are present in only a small fraction of the genes and they represent elements not previously described in animals, suggesting a distinct architecture of the core promoters of plant and animal genes.

5' Untranslated Regions↗
PubMed2005

Flavonoids as developmental regulators.

Flavonoids, usually regarded as dispensable phytochemicals derived from plant secondary metabolism, play important roles in the biology of plants by affecting several developmental processes. Bioactive flavonoids also signal to microbes, serve as allelochemicals and are important nutraceuticals in the animal diet. Despite the significant progress made in identifying flavonoid pathway genes and regulators, little is currently known about the protein targets of flavonoids in plant or animal cells. Recently, there have been advances in our understanding of the roles that flavonoids play in developmental processes of plants. The multiple cellular roles of flavonoids can reflect their chemical diversity, or might suggest the existence of cellular targets shared between many of these seemingly disparate processes.

Antineoplastic Agents, Phytogenic↗
PubMed2005

Plant metabolic diversity: a regulatory perspective.

Plants accumulate an amazing diversity of phytochemicals that play important roles in the interaction of plants with the environment. Mechanisms have been proposed to describe the evolution of phytochemicals from the perspective of the biosynthetic enzymes. However, it is not known how the transcription factors that regulate these pathways have evolved to ensure the coordinate expression of all the genes in a pathway. A model is provided here to explain how duplication and divergence of regulatory genes result in the control of new pathways. In this model, the purported ability of recently duplicated regulatory genes to activate new metabolic pathways is a consequence of mutations that partially impair function, resulting in the loss of activation of one or several steps in a metabolic pathway. Consequently, pathway intermediates accumulate and are then converted into new compounds by broad-specificity enzymes. In contrast to the resilience of developmental regulatory circuits, this model provides an explanation for the rapid evolution of new metabolic pathways from existing ones.

Enzymes↗
PubMed2005

Cloning and characterization of a flavanone 3-hydroxylase gene from Saussurea medusa.

Flavanone 3-hydroxylase (F3H) is a key enzyme in the flavonoid biosynthetic pathway, providing a branching point for the biosynthesis of different flavonoids, including the formation of 3-deoxy and 3-hydroxy flavonoids found in the silks of maize. Here, we report the cloning and characterization of a F3H gene (Smf3h) from a cDNA library derived from a red line callus of Saussurea medusa, a traditional Chinese medicinal plant. The cDNA contains a 1032 bp open reading frame (ORF) encoding a protein of 343 amino acid residues, a 149 bp long 5'untranslated regions (UTR) and a 163 bp long 3'UTR containing three putative polyadenylation signals (AATAAA) and an ATTTA element. The secondary structure of the mRNA predicted by MFOLD is very complex, suggesting a role in a post-transcriptional mechanism of regulation of Smf3h. The genomic structure of Smf3h includes four exons and three introns within the coding region, with all the splice donor/acceptor site sequences in accordance with the "GU-AG" consensus rule. The deduced SmF3H protein is 343 amino acid residues in length and has 40% and 39% identity and 60% and 58% similarity to the F3H of Arabidopsis and rice, respectively. Strikingly, the identity of SmF3H is higher to the H6H (hyoscyamine 3beta-hydroxylase, 45%) from Atropa belladonna. However, the analysis of the active center and the predicted protein secondary structure are more related to F3H than H6H. Together, our studies provide the first identification of a S. medusa flavonoid gene and its similarities to metabolic enzymes from other plants.

3' Untranslated Regions↗
PubMed2004

The tomato early fruit specific gene Lefsm1 defines a novel class of plant-specific SANT/MYB domain proteins.

We describe here a novel plant-specific gene, Lefsm1 (fruit SANT/MYB-like 1) harboring a single SANT/MYB domain. The expression of Lefsm1 is specific to the very early stages of tomato (Lycopersicon esculentum) fruit development. Ectopic expression of Lefsm1 results in severe developmental alterations manifested in retarded growth, and reduced apical dominance during tomato and Arabidopsis seedling development. A promoter sequence residing 1.0 kb upstream to the translation initiation codon confers the organ-specific expression of the gene. Lefsm1 belongs to a novel small gene family consisting of five to six members in tomato, Arabidopsis and rice. The SANT/MYB domain of LeFSM1 and its orthologs in Arabidopsis and rice differs from that of all other plant or animal MYB proteins and from the SANT domains found in part of the chromatin remodeling proteins. Together, our results indicate that Lefsm1 is a founding member of a small family of proteins containing a novel MYB/SANT domain which is likely to participate in the regulation of a plant-specific developmental program.

Amino Acid Sequence↗
PubMed2004

Different mechanisms participate in the R-dependent activity of the R2R3 MYB transcription factor C1.

The R2R3 MYB transcription factor C1 requires the basic helix-loop-helix factor R as an essential co-activator for the transcription of maize anthocyanin genes. In contrast, the R2R3 MYB protein P1 activates a subset of the C1-regulated genes independently of R. Substitution of six amino acids in P1 with the C1 amino acids results in P1(*), whose activity on C1-regulated and P1-regulated genes is R-dependent or R-enhanced, respectively. We have used P1(*) in combination with various promoters to uncover two mechanisms for R function. On synthetic promoters that contain only C1/P1 binding sites, R is an essential co-activator of C1. This function of R is unlikely to simply be the result of an increase in the C1 DNA-binding affinity, since transcriptional activity of a C1 mutant that binds DNA at a higher affinity, comparable with P1, remains R-dependent. The differential transcriptional activity of C1 fusions with the yeast Gal4 DNA-binding domain in yeast and maize cells suggests that part of the function of R is to relieve C1 from a plant-specific inhibitor. A second function of R requires cis-regulatory elements in addition to the C1/P1 DNA-binding sites for R-enhanced transcription of a1. We hypothesize that R functions in this mode by binding or recruiting additional factors to the anthocyanin regulatory element conserved in the promoters of several anthocyanin genes. Together, these findings suggest a model in which combinatorial interactions with co-activators enable R2R3 MYB factors with very similar DNA binding preferences to discriminate between target genes in vivo.

Amino Acid Sequence↗
PubMed2004

Two cysteines in plant R2R3 MYB domains participate in REDOX-dependent DNA binding.

Plant R2R3 MYB domain proteins comprise one of the largest known families of transcription factors. Discrete evolutionary steps have shaped the plant-specific R2R3 MYB family from the broadly distributed R1R2R3 MYB proteins. R1R2R3 MYB domains have a single Cys residue (Cys-130) that needs to be reduced for DNA binding and transcriptional activity. In contrast, most R2R3 MYB domains contain two cysteines, Cys-49 and Cys-53, with Cys-53 at the equivalent position as Cys-130 in R1R2R3 MYB. Using the maize P1 regulator of flavonoid biosynthesis as a typical R2R3 MYB-domain protein, we investigated here the in vitro REDOX requirement for DNA binding by P1. We show that the C53S mutation requires reducing conditions for DNA-binding, whereas C53A binds DNA under oxidizing and reducing conditions. Neither mutation impairs the in vivo regulatory activity of P1. The C49S and C49A mutants bind DNA in vitro irrespective of the REDOX conditions. A C49I mutant, which simulates the MYB domain of c-MYB, binds DNA only under reducing conditions, and its binding is significantly affected by the C53S replacement. It is interesting that under non-reducing conditions, Cys-49 and Cys-53 form a disulfide bond that prevents the R2R3 MYB domain from binding DNA. Together, our results suggest that the evolutionary origin of Cys-49 within the plants has provided R2R3 MYB domains with a regulatory feature not present in animal MYB domains, highlighting fundamental structural and functional differences between similar DNA-binding domains from plants and animals.

Amino Acid Sequence↗
PubMed2004

RNase P as a tool for disruption of gene expression in maize cells.

RNase P, a ribonucleoprotein responsible for the 5' maturation of precursor tRNAs (ptRNAs) in all organisms, can be enticed to cleave any target mRNA that forms a ptRNA-like structure and sequence-specific complex when bound to an RNA, termed the EGS (external guide sequence). In the present study, F3H (flavanone 3-hydroxylase), a key enzyme in the flavonoid biosynthetic pathway that participates in the formation of red-coloured anthocyanins, was used as a target for RNase P-mediated gene disruption in maize cells. Transient expression of an EGS complementary to the F3H mRNA resulted in suppression of F3H to 29% of the control, as indicated by a reduced number of anthocyanin-accumulating cells. This decrease was not observed in experiments where a disabled mutant EGS was expressed. Our results demonstrate the potential of employing plant RNase P, in the presence of an appropriate gene-specific EGS, as a tool for targeted degradation of mRNAs.

Gene Expression Regulation, Plant↗
PubMed2004

Comparison of ESTs from juvenile and adult phases of the giant unicellular green alga Acetabularia acetabulum.

BACKGROUND: Acetabularia acetabulum is a giant unicellular green alga whose size and complex life cycle make it an attractive model for understanding morphogenesis and subcellular compartmentalization. The life cycle of this marine unicell is composed of several developmental phases. Juvenile and adult phases are temporally sequential but physiologically and morphologically distinct. To identify genes specific to juvenile and adult phases, we created two subtracted cDNA libraries, one adult-specific and one juvenile-specific, and analyzed 941 randomly chosen ESTs from them. RESULTS: Clustering analysis suggests virtually no overlap between the two libraries. Preliminary expression data also suggests that we were successful at isolating transcripts differentially expressed between the two developmental phases and that many transcripts are specific to one phase or the other. Comparison of our EST sequences against publicly available sequence databases indicates that ESTs from the adult and the juvenile libraries partition into different functional classes. Three conserved sequence elements were common to several of the ESTs and were also found within the genomic sequence of the carbonic anhydrase1 gene from A. acetabulum. To date, these conserved elements are specific to A. acetabulum. CONCLUSIONS: Our data provide strong evidence that adult and juvenile phases in A. acetabulum vary significantly in gene expression. We discuss their possible roles in cell growth and morphogenesis as well as in phase change. We also discuss the potential role of the conserved elements found within the EST sequences in post-transcriptional regulation, particularly mRNA localization and/or stability.

Acetabularia↗
PubMed2003

Sub-cellular trafficking of phytochemicals explored using auto-fluorescent compounds in maize cells.

BACKGROUND: Little is known regarding the trafficking mechanisms of small molecules within plant cells. It remains to be established whether phytochemicals are transported by pathways similar to those used by proteins, or whether the expansion of metabolic pathways in plants was associated with the evolution of novel trafficking pathways. In this paper, we exploited the induction of green and yellow auto-fluorescent compounds in maize cultured cells by the P1 transcription factor to investigate their targeting to the cell wall and vacuole, respectively. RESULTS: We investigated the accumulation and sub-cellular localization of the green and yellow auto-fluorescent compounds in maize BMS cells expressing the P1 transcription factor from an estradiol inducible promoter. We established that the yellow fluorescent compounds accumulate inside the vacuole in YFBs that resemble AVIs. The green fluorescent compounds accumulate initially in the cytoplasm in large spherical GFBs. Cells accumulating GFBs also contain electron-dense structures that accumulate initially in the ER and which later appear to fuse with the plasma membrane. Structures resembling the GFBs were also observed in the periplasmic space of plasmolized cells. Ultimately, the green fluorescence accumulates in the cell wall, in a process that is insensitive to the Golgi-disturbing agents BFA and monensin. CONCLUSIONS: Our results suggest the presence of at least two distinct trafficking pathways, one to the cell wall and the other to the vacuole, for different auto-fluorescent compounds induced by the same transcription factor in maize BMS cells. These compartments represent two of the major sites of accumulation of phenolic compounds characteristic of maize cells. The secretion of the green auto-fluorescent compounds occurs by a pathway that does not involve the TGN, suggesting that it is different from the secretion of most proteins, polysaccharides or epicuticular waxes. The yellow auto-fluorescent compounds accumulate in a vacuolar compartment, in structures that resemble the AVIs present in many cells accumulating anthocyanins. Together, our studies suggest that the accumulation of auto-fluorescent compounds can provide a powerful tool to dissect the trafficking of phytochemicals, knowledge necessary for the efficient engineering of plant metabolism.

Biological Transport↗
PubMed2003

AGRIS: Arabidopsis gene regulatory information server, an information resource of Arabidopsis cis-regulatory elements and transcription factors.

BACKGROUND: The gene regulatory information is hardwired in the promoter regions formed by cis-regulatory elements that bind specific transcription factors (TFs). Hence, establishing the architecture of plant promoters is fundamental to understanding gene expression. The determination of the regulatory circuits controlled by each TF and the identification of the cis-regulatory sequences for all genes have been identified as two of the goals of the Multinational Coordinated Arabidopsis thaliana Functional Genomics Project by the Multinational Arabidopsis Steering Committee (June 2002). RESULTS: AGRIS is an information resource of Arabidopsis promoter sequences, transcription factors and their target genes. AGRIS currently contains two databases, AtTFDB (Arabidopsis thaliana transcription factor database) and AtcisDB (Arabidopsis thaliana cis-regulatory database). AtTFDB contains information on approximately 1,400 transcription factors identified through motif searches and grouped into 34 families. AtTFDB links the sequence of the transcription factors with available mutants and, when known, with the possible genes they may regulate. AtcisDB consists of the 5' regulatory sequences of all 29,388 annotated genes with a description of the corresponding cis-regulatory elements. Users can search the databases for (i) promoter sequences, (ii) a transcription factor, (iii) a direct target genes for a specific transcription factor, or (vi) a regulatory network that consists of transcription factors and their target genes. CONCLUSION: AGRIS provides the necessary software tools on Arabidopsis transcription factors and their putative binding sites on all genes to initiate the identification of transcriptional regulatory networks in the model dicotyledoneous plant Arabidopsis thaliana. AGRIS can be accessed from http://arabidopsis.med.ohio-state.edu.

3' Untranslated Regions↗