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PubMed1997

3-Hydroxy-3-methylglutaryl-CoA reductase gene of Gibberella fujikuroi: isolation and characterization.

3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) is the first specific enzyme of the isoprenoid pathway, which leads to several classes of primary and secondary metabolites such as sterols, quinones, carotenoids and gibberellins. The structural gene of HMG-CoA reductase was isolated from the ascomycetous fungus Gibberella fujikuroi. Additionally, the most conserved region of this gene was also isolated from another plant pathogenic fungus, Sphaceloma manihoticola. Both ascomycetous fungi use the plant hormone gibberellin to induce an elongation of infected host plants, and in the case of S. manihoticola of plant tumors. Sequence analysis revealed a high degree of similarity between the deduced amino-acid sequences in the C-terminal catalytic domains of all known HMG-CoA reductases, but the highest degree was found between the sequences of both analysed ascomycetes. In contrast to Saccharomyces cerevisiae, Ustilago maydis and plants, G. fujikuroi and S. manihoticola possess only a single copy of this gene, although the product of HMGR (mevalonate) is the precursor for essential sterol and quinone biosynthesis and secondary metabolites such as gibberellins. RNA-blot and hybridization experiments showed that gene expression is not influenced by either glucose or ammonium excess.

Amino Acid Sequence↗

PubMed2005

Regulation of genes associated with auxin, ethylene and ABA pathways by 2,4-dichlorophenoxyacetic acid in Arabidopsis.

The chemical 2,4-dichlorophenoxyacetic acid (2,4-D) regulates plant growth and development and mimics auxins in exhibiting a biphasic mode of action. Although gene regulation in response to the natural auxin indole acetic acid (IAA) has been examined, the molecular mode of action of 2,4-D is poorly understood. Data from biochemical studies, (Grossmann (2000) Mode of action of auxin herbicides: a new ending to a long, drawn out story. Trends Plant Sci 5:506-508) proposed that at high concentrations, auxins and auxinic herbicides induced the plant hormones ethylene and abscisic acid (ABA), leading to inhibited plant growth and senescence. Further, in a recent gene expression study (Raghavan et al. (2005) Effect of herbicidal application of 2,4-dichlorophenoxyacetic acid in Arabidopsis. Funct Integr Genomics 5:4-17), we have confirmed that at high concentrations, 2,4-D induced the expression of the gene NCED1, which encodes 9-cis-epoxycarotenoid dioxygenase, a key regulatory enzyme of ABA biosynthesis. To understand the concentration-dependent mode of action of 2,4-D, we further examined the regulation of whole genome of Arabidopsis in response to a range of 2,4-D concentrations from 0.001 to 1.0 mM, using the ATH1-121501 Arabidopsis whole genome microarray developed by Affymetrix. Results of this study indicated that 2,4-D induced the expression of auxin-response genes (IAA1, IAA13, IAA19) at both auxinic and herbicidal levels of application, whereas the TIR1 and ASK1 genes, which are associated with ubiquitin-mediated auxin signalling, were down-regulated in response to low concentrations of 2,4-D application. It was also observed that in response to low concentrations of 2,4-D, ethylene biosynthesis was induced, as suggested by the up-regulation of genes encoding 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase. Although genes involved in ethylene biosynthesis were not regulated in response to 0.1 and 1.0 mM 2,4-D, ethylene signalling was induced as indicated by the down-regulation of CTR1 and ERS, both of which play a key role in the ethylene signalling pathway. In response to 1.0 mM 2,4-D, both ABA biosynthesis and signalling were induced, in contrast to the response to lower concentrations of 2,4-D where ABA biosynthesis was suppressed. We present a comprehensive model indicating a molecular mode of action for 2,4-D in Arabidopsis and the effects of this growth regulator on the auxin, ethylene and abscisic acid pathways.

2,4-Dichlorophenoxyacetic Acid↗

PubMed2002

Crystal structures of two homologous pathogenesis-related proteins from yellow lupine.

Pathogenesis-related class 10 (PR10) proteins are restricted to the plant kingdom where they are coded by multigene families and occur at high levels. In spite of their abundance, their physiological role is obscure although members of a distantly related subclass (cytokinin-specific binding proteins) are known to bind plant hormones. PR10 proteins are of special significance in legume plants where their expression patterns are related to infection by the symbiotic, nitrogen-fixing bacteria. Here we present the first crystal structures of classic PR10 proteins representing two homologues from one subclass in yellow lupine. The general fold is similar and, as in a birch pollen allergen, consists of a seven-stranded beta-sheet wrapped around a long C-terminal helix. The mouth of a large pocket formed between the beta-sheet and the helix seems a likely site for ligand binding. The shape of the pocket varies because, in variance with the rigid beta-sheet, the helix shows unusual conformational variability consisting in bending, disorder, and axial shifting. A surface loop, proximal to the entrance to the internal cavity, shows an unusual structural conservation and rigidity in contrast to the high glycine content in its sequence. The loop is different from the so-called glycine-rich P-loops that bind phosphate groups of nucleotides, but it is very likely that it does play a role in ligand binding in PR10 proteins.

Amino Acid Sequence↗

PubMed1997

Protein-protein interactions among the Aux/IAA proteins.

The plant hormone indoleacetic acid (IAA) transcriptionally activates early genes in plants. The Aux/IAA family of early genes encodes proteins that are short-lived and nuclear-localized. They also contain a putative prokaryotic betaalphaalpha DNA binding motif whose formation requires protein dimerization. Here, we show that the pea PS-IAA4 and Arabidopsis IAA1 and IAA2 proteins perform homo- and heterotypic interactions in yeast using the two-hybrid system. Gel-filtration chromatography and chemical cross-linking experiments demonstrate that the PS-IAA4 and IAA1 proteins interact to form homodimers in vitro. Deletion analysis of PS-IAA4 indicates that the betaalphaalpha containing acidic C terminus of the protein is necessary for homotypic interactions in the yeast two-hybrid system. Screening an Arabidopsis lambda-ACT cDNA library using IAA1 as a bait reveals heterotypic interactions of IAA1 with known and newly discovered members of the Arabidopsis Aux/IAA gene family. The new member IAA24 has similarity to ARF1, a transcription factor that binds to an auxin response element. Combinatorial interactions among the various members of the Aux/IAA gene family may regulate a variety of late genes as well as serve as autoregulators of early auxin-regulated gene expression. These interactions provide a molecular basis for the developmental and tissue-specific manner of auxin action.

Amino Acid Sequence↗

PubMed2004

LeMPK3 is a mitogen-activated protein kinase with dual specificity induced during tomato defense and wounding responses.

Mitogen-activated protein (MAP) kinase cascades are readily activated during the response of plants to avirulent pathogens or to pathogen-derived elicitors. Here we show that the tomato MAP kinase LeMPK3 is specifically induced at the mRNA level during elicitation of the hypersensitive response in resistant plants infected by avirulent strains of the phytopathogenic bacteria Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. tomato, as well as upon treatment with the fungal elicitor ethylene-inducing xylanase. LeMPK3 gene expression was also induced very rapidly by mechanical stress and wounding much earlier than upon pathogen infection, but not in response to the defense-related plant hormones ethylene and jasmonic acid. Moreover, in resistant tomato plants infected by X. campestris pv. vesicatoria, transcript accumulation was followed by an increase in LeMPK3 kinase activity. Biochemical characterization of a glutathione S-transferase-LeMPK3 fusion protein revealed that the LeMPK3 MAP kinase autophosphorylates in vitro mainly on tyrosine and less so on threonine and serine, whereas it phosphorylates myelin basic protein on serine and threonine. In vitro phosphorylation of a poly-(Glu-Tyr) copolymer by LeMPK3 demonstrated its capability to phosphorylate tyrosine residues on substrates as well. By mutagenesis and phosphoamino acid analysis, Tyr-201 in the kinase activation domain was identified as the main LeMPK3 autophosphorylation site and as critical for kinase activity. Finally, LeMPK3 autophosphorylation showed a preference for Mn(2+) cations and proceeded via an intramolecular mechanism with an estimated K(m) value for ATP of 9.5 microm. These results define LeMPK3 as a MAP kinase with dual specificity and strongly suggest that it represents a convergence point for different signaling pathways inducing the activation of defense responses in tomato.

Adenosine Triphosphate↗

PubMed1996

The excessive production of indole-3-acetic acid and its significance in studies of the biosynthesis of this regulator of plant growth and development.

Because of the importance of indole-3-acetic acid (IAA) in the growth and development of plants, extensive studies of the biosynthesis of IAA have been performed during the four decades since the discovery of IAA as a plant hormone. The pathway for the biosynthesis of IAA in plants remains, however, to be unelucidated, even though studies within the past decade have revealed unexpected aspects of such biosynthesis. By contrast, two pathways to IAA have been characterized in bacteria at the molecular level: the indole-3-acetamide (IAM) pathway (L-tryptophan-->IAM-->IAA); the indole-3-pyruvic acid pathway (L-tryptophan-->indole-3-pyruvic acid-->indole-3-acetaldehyde-->IAA) (Fig. 1). In both pathways, the details of the biosynthesis of IAA were clarified using IAA-overproducing bacteria. After a description of recent advances of the studies of the biosynthesis of IAA in plants, this review focuses on the excessive production of IAA in several organisms and its significance in the studies of the biosynthesis of IAA.

Forecasting↗

PubMed2005

Salicylic acid accumulation under O3 exposure is regulated by ethylene in tobacco plants.

Ozone (O3), a major photochemical oxidant, induces leaf injury concomitant with salicylic acid (SA) synthesis. In pathogen-infected leaves, SA is synthesized via two pathways, involving phenylalanine or isochorismate. SA biosynthesis under O3 fumigation is not well understood. When we applied 14C-labeled benzoic acid (a precursor of SA in the pathway via phenylalanine) to O3-exposed tobacco leaves, it was effectively metabolized to SA. However, the activity and mRNA level of isochorismate synthase (ICS) were not increased. In contrast, ICS activity was increased in O3-exposed Arabidopsis thaliana L. These results suggest that SA is synthesized via benzoic acid from phenylalanine in O3-exposed tobacco leaves but via isochorismate in Arabidopsis. Ethylene is a plant hormone that promotes leaf damage in O3-exposed plants. During O3 exposure, transgenic plants with a phenotype of reduced O3-induced ethylene production accumulated less SA than did wild-type plants. O3 increased the activity of phenylalanine ammonia-lyase (PAL) and the transcript levels of the chorismate mutase (CM) and PAL genes in wild-type tobacco, but their induction was suppressed in the transgenic plants. These results indicate that ethylene promotes SA accumulation by regulating the expression of the CM and PAL genes in O3-exposed tobacco.

Amino Acid Sequence↗

PubMed2005

ABR1, an APETALA2-domain transcription factor that functions as a repressor of ABA response in Arabidopsis.

The plant hormone abscisic acid (ABA) plays an important role in plant development and stress responses. An important step of ABA action is activation or inactivation of gene expression. Although several transcription factors are identified to function as positive regulators of ABA-induced gene expression, little is known about the negative regulators of ABA-regulated gene expression. Here, we have identified an APETALA2 (AP2) domain transcription factor that serves as a repressor of ABA response during seed germination and ABA- and stress-induced gene expression in Arabidopsis (Arabidopsis thaliana). The expression of the AP2-like ABA repressor 1 (ABR1) gene itself was responsive to ABA and stress conditions including cold, high salt, and drought. Disruption of ABR1 led to hypersensitive response to ABA in seed germination and root growth assays. The mutant plants were also hypersensitive to osmotic stress conditions, such as high salt and high concentrations of mannitol. Further analyses indicated that increased stress sensitivity may result from hypersensitivity to ABA as ABA biosynthesis inhibitor rescued the stress hypersensitivity phenotype. The abr1 mutant plants accumulated significantly higher levels of ABA- and stress-inducible gene transcripts as compared to the wild-type plants, supporting the hypothesis that this AP2 domain protein serves as a repressor of ABA-regulated gene expression.

Abscisic Acid↗

PubMed2000

Trivalent ions activate abscisic acid-inducible promoters through an ABI1-dependent pathway in rice protoplasts.

The plant hormone abscisic acid (ABA) mediates many vital processes in plant growth and development, including seed dormancy, cell division, water use efficiency, and adaptation to drought, salinity, chilling, pathogen attack, and UV light. Our understanding of ABA signal transduction is fragmentary and would benefit from specific and facile probes of the process. Protoplasts from rice (Oryza sativa L. cv IR54) embryonic suspension cultures cotransformed with effector plasmids encoding the maize (Zea mays) VIVIPAROUS1 cDNA and/or the Arabidopsis dominant negative mutant (abi1-1) ABA-insensitive cDNA demonstrated genetic interactions of VIVIPAROUS1 and abi1-1 in transactivation of the ABA-inducible HVA1 promoter from barley (Hordeum vulgare), suggesting the mechanisms of these effectors are conserved among monocots and dicots. Trivalent ions have been shown to act as an effector of gene expression in plants and animals, although the mechanism of action is unknown. We show in two complementary transient ABA-inducible gene expression assays (beta-glucuronidase and luciferase enzymatic activities and quantitative flow cytometry of green fluorescent protein) that trivalent ions specifically interact with an ABI1-dependent ABA-signaling pathway leading to gene expression. Trivalent ions mimic ABA effects on gene expression and may be a useful tool to study ABA signaling.

Abscisic Acid↗

PubMed2005

[Iron from soil to plant products].

As an essential mineral, iron plays an important role in fundamental biological processes such as photosynthesis, respiration, nitrogen fixation and assimilation, and DNA synthesis. Iron is also a co-factor of many enzymes involved in the synthesis of plant hormones. The latter are involved in many pathways controling plant development or adaptative responses to environmental conditions. Iron reactivity with oxygen leads to its insolubility (responsible for deficiency) and potential toxicity, and complicates iron use by aerobic organisms. If plants lacked an active root system with which to acquire iron from the soil, most would experience iron deficiency and show physiological changes. In contrast, an excess of soluble iron, which can occur in flooded acidic soils, can lead to ferrous iron toxicity due to iron reactivity with reduced forms of oxygen and subsequent free radical production. An optimal iron concentration is thus required for a plant to grow and develop normally. This concentration depends on multiple regulatory mechanisms controlling iron uptake from soil by the roots, as well as iron transport and distribution to the various plant organs. Optimized seed iron content is a major biotechnological challenge identified by the World Health Organization, and it is therefore crucial to understand the underlying mechanisms. Iron delivery to seeds is tightly controlled, and depends on the nature of iron speciation in specific chelates, and their transport.

Iron↗

PubMed2000

The Rhodococcus fascians-plant interaction: morphological traits and biotechnological applications.

Rhodococcus fascians is a Gram-positive bacterium that infects dicotyledonous and monocotyledonous plants, leading to an alteration in the normal growth process of the host. The disease results from the modulation of the plant hormone balances, and cytokinins are thought to play an important role in the induction of symptoms. Generally, on the aerial parts of the plants, existing meristems were found to be most sensitive to the action of R. fascians, but, depending on the infection procedure, differentiated tissues as well gave rise to shoots. Similarly, in roots not only actively dividing cells, but also cells with a high competence to divide were strongly affected by R. fascians. The observed symptoms, together with the determined hormone levels in infected plant tissue, suggest that auxins and molecules of bacterial origin are also involved in leafy gall formation. The complexity of symptom development is furthermore illustrated by the necessary and continuous presence of the bacteria for symptom persistence. Indeed, elimination of the bacteria from a leafy gall results in the further development of the multiple embryonic buds of which it consists. This interesting characteristic offers novel biotechnological applications: a leafy gall can be used for germplasm storage and for plant propagation. The presented procedure proves to be routinely applicable to a very wide range of plants, encompassing several recalcitrant species.

Arabidopsis↗

PubMed1969

A protein intermediary in the interaction of a hormone with the genome.

The increased rate of RNA synthesis by target cells caused by the plant hormone auxin has been studied as an example of hormonal regulation of transcription. The hormone does not interact directly with chromatin but requires a protein mediator. In the presence of this mediator, auxin increases the rate of RNA synthesis both by isolated plant nuclei and by isolated chromatin. This increased rate of RNA synthesis occurs even in the presence of saturating amounts of RNA polymerase. The hormone and protein do not affect the rate of RNA synthesis if pure DNA is used as the template. The results suggest that auxin plus the protein increase the rate of RNA synthesis by making an increased portion of the genome available for transcription.

Culture Techniques↗

PubMed2002

ABA-based chemical signalling: the co-ordination of responses to stress in plants.

There is now strong evidence that the plant hormone abscisic acid (ABA) plays an important role in the regulation of stomatal behaviour and gas exchange of droughted plants. This regulation involves both long-distance transport and modulation of ABA concentration at the guard cells, as well as differential responses of the guard cells to a given dose of the hormone. We will describe how a plant can use the ABA signalling mechanism and other chemical signals to adjust the amount of water that it loses through its stomata in response to changes in both the rhizospheric and the aerial environment. The following components of the signalling process can play an important part in regulation: (a) ABA sequestration in the root; (b) ABA synthesis versus catabolism in the root; (c) the efficiency of ABA transfer across the root and into the xylem; (d) the exchange of ABA between the xylem lumen and the xylem parenchyma in the shoot; (e) the amount of ABA in the leaf symplastic reservoir and the efficiency of ABA sequestration and release from this compartment as regulated by factors such as root and leaf-sourced changes in pH; (f) cleavage of ABA from ABA conjugates in the leaf apoplast; (g) transfer of ABA from the leaf into the phloem; (h) the sensitivity of the guard cells to the [ABA] that finally reaches them; and lastly (i) the possible interaction between nitrate stress and the ABA signal.

Journal Article↗

PubMed2006

A novel cell division factor from tobacco 2B-13 cells that induced cell division in auxin-starved tobacco BY-2 cells.

Effects of auxin as plant hormones are widespread; in fact in almost all aspects of plant growth and development auxin plays a pivotal role. Although auxin is required for propagating cell division in plant cells, its effect upon cell division is least understood. If auxin is depleted from the culture medium, cultured cells cease to divide. It has been demonstrated in this context that the addition of auxin to auxin-starved nondividing tobacco BY-2 cells induced semisynchronous cell division. On the other hand, there are some cell lines, named habituated cells, that can grow without auxin. The cause and reason for the habituated cells have not been clarified. A habituated cell line named 2B-13 is derived from the tobacco BY-2 cell line, which has been most intensively studied among plant cell lines. When we tried to find the difference between two cell lines of BY-2 and 2B-13 cells, we found that the addition of culture filtrated from the auxin-habituated 2B-13 cells induced semisynchronous cell division in auxin-starved BY-2 cells. The cell division factor (CDF) that is responsible for inducing cell division in auxin-starved BY-2 cells was purified to near-homogeneity by sequential passage through a hydroxyapatite column, a ConA Sepharose column and a Sephadex gel filtration column. The resulting purified fraction appeared as a single band of high molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels by silver staining and was able to induce cell division in auxin-starved BY-2 cells. Identification of the protein by MALD-TOF-MS/MS revealed that it is structurally related to P-glycoprotein from Gossypioides kirkii, which belongs to ATP-binding cassette (ABC)-transporters. The significance of CDF as a possible ABC-transporter is discussed in relationship to auxin-autotrophic growth and auxin-signaling pathway.

Amino Acid Sequence↗

PubMed2005

Ectopic expression of EFFECTOR OF TRANSCRIPTION perturbs gibberellin-mediated plant developmental processes.

The plant hormone gibberellin (GA) is known to modulate various aspects of plant cell differentiation and development. The current model of GA-mediated regulation is based on a de-repressible system and includes specific protein modification and degradation. HRT, a zinc finger protein from barley has been shown to have GA-dependent transcriptional repressing activity on the seed-specific alpha-amylase promoter [Raventos, D., Skriver, K., Schlein, M., Karnahl, K., Rogers, S.W., Rogers, J.C. and Mundy, J. 1998. J. Biol. Chem. 273: 23313-23320]. Here we report the characterization of a dicot homologue from Brassica napus (BnET) and provide evidence for its role in GA response modulation suggesting that this could be a conserved feature of this gene family. When BnET is ectopically expressed in either Arabidopsis or tobacco the phenotypes include dwarfism due to shorter internodes and late flowering, reduced germination rate, increased anthocyanin content and reduced xylem lignification as a marker for terminal cell differentiation. Transient expression in protoplasts supports the notion that this most likely is due to a transcriptional repression of GA controlled genes. Finally, histological analysis showed that in contrast to other GA deficient mutants the shorter internodes were due to fewer but not smaller cells, suggesting a function of BnET in GA-mediated cell division control.

Amino Acid Sequence↗

PubMed2005

Plant development is regulated by a family of auxin receptor F box proteins.

The plant hormone auxin has been implicated in virtually every aspect of plant growth and development. Auxin acts by promoting the degradation of transcriptional regulators called Aux/IAA proteins. Aux/IAA degradation requires TIR1, an F box protein that has been shown to function as an auxin receptor. However, loss of TIR1 has a modest effect on auxin response and plant development. Here we show that three additional F box proteins, called AFB1, 2, and 3, also regulate auxin response. Like TIR1, these proteins interact with the Aux/IAA proteins in an auxin-dependent manner. Plants that are deficient in all four proteins are auxin insensitive and exhibit a severe embryonic phenotype similar to the mp/arf5 and bdl/iaa12 mutants. Correspondingly, all TIR1/AFB proteins interact with BDL, and BDL is stabilized in triple mutant plants. Our results indicate that TIR1 and the AFB proteins collectively mediate auxin responses throughout plant development.

Arabidopsis↗

PubMed2001

Mitogen-activated protein [MAP] kinase pathways in plants: versatile signaling tools.

Mitogen-activated protein kinases (MAPKs) are important signaling tools in all eukaryotes, and function in mediating an enormous variety of external signals to appropriate cellular responses. MAPK pathways have been studied extensively in yeast and mammalian cells, and a large body of knowledge on their functioning has accumulated, which is summarized briefly. Plant MAPK pathways have attracted increasing interest, resulting in the isolation of a large number of different components of MAPK cascades. Studies on the functions of these components have revealed that MAPKs play important roles in the response to a broad variety of stresses, as well as in the signaling of most plant hormones and in developmental processes. Finally, the involvement of various plant phosphatases in the inactivation of MAPKs is discussed.

Cell Cycle↗

PubMed2003

Transgenic plants with altered ethylene biosynthesis or perception.

The plant hormone ethylene is an essential signaling molecule involved in many plant processes including: germination, flower development, fruit ripening and responses to many environmental stimuli. Moreover, large increases in ethylene levels occur during plant stress responses, fruit ripening and flower wilting. Manipulation of ethylene biosynthesis or perception allows us to modulate these processes and thereby create plants with more robust and/or desirable traits, giving us a glimpse into the role of ethylene in the plant. Here, recent and landmark advances in genetic alteration of members of the ethylene pathway in plants and the physiological consequences of these alterations are examined.

Arabidopsis↗