The conserved KNOX domain mediates specificity of tobacco KNOTTED1-type homeodomain proteins.

Overproduction of the tobacco KNOTTED1-type homeodomain proteins NTH1, NTH15, and NTH23 in transgenic tobacco plants causes mild, severe, and no morphological alterations, respectively. The deduced amino acid sequences of the homeodomains and adjacent ELK domains are highly conserved, and the N-terminal KNOX domains also are moderately conserved. To investigate the contributions of both the conserved and divergent regions to the severity of morphological alterations, we generated chimeric proteins by exchanging different regions of NTH1, NTH15, and NTH23. The severity of the abnormal phenotype was dependent upon the synergistic action of both the N terminus, containing the KNOX domain, and the C terminus, containing the ELK homeodomain. Detailed analysis focusing on the C terminus revealed that the C-terminal half of the ELK domain is more effective in inducing the abnormal phenotypes than are the homeodomains. For the N terminus, severe morphological alterations were induced by exchanging a part of the KNOX domain of NTH1 with the corresponding region of NTH15. This limited region in the KNOX domain of all homeodomain proteins includes a predicted alpha-helical region, but only that in NTH15 is predicted to form a typical amphipathic structure. We discuss the possibility, based on these results, that the secondary structure of the KNOX domain is important for the induction of abnormal morphology in transgenic tobacco plants.     

GID1-mediated gibberellin signaling in plants

Gibberellin (GA) perception is mediated by GID1 (GA-INSENSITIVE DWARF1), a receptor that shows similarity to hormone-sensitive lipases. A key event in GA signaling is the degradation of DELLA proteins, which are negative regulators of GA response that interact with GID1 in a GA-dependent manner. This GID1-DELLA GA-perception system is conserved among vascular plants but is not found in the moss Physcomitrella patens. The identification of factors in GA signaling downstream of DELLA and the development of a new concept of DELLA function beyond its role as a repressor of GA signaling are important advances. DELLA proteins appear to have at least two other distinct roles: maintaining GA homeostasis and regulating cross-talk between GA and other plant hormones.     

Ectopic expression of KNOTTED1-like homeobox protein induces expression of cytokinin biosynthesis genes in rice

Some phytohormones such as gibberellins (GAs) and cytokinins (CKs) are potential targets of the KNOTTED1-like homeobox (KNOX) protein. To enhance our understanding of KNOX protein function in plant development, we identified rice (Oryza sativa) genes for adenosine phosphate isopentenyltransferase (IPT), which catalyzes the rate-limiting step of CK biosynthesis. Molecular and biochemical studies revealed that there are eight IPT genes, OsIPT1 to OsIPT8, in the rice genome, including a pseudogene, OsIPT6. Overexpression of OsIPTs in transgenic rice inhibited root development and promoted axillary bud growth, indicating that OsIPTs are functional in vivo. Phenotypes of OsIPT overexpressers resembled those of KNOX-overproducing transgenic rice, although OsIPT overexpressers did not form roots or ectopic meristems, both of which are observed in KNOX overproducers. Expression of two OsIPT genes, OsIPT2 and OsIPT3, was up-regulated in response to the induction of KNOX protein function with similar kinetics to those of down-regulation of GA 20-oxidase genes, target genes of KNOX proteins in dicots. However, expression of these two OsIPT genes was not regulated in a feedback manner. These results suggest that OsIPT2 and OsIPT3 have unique roles in the developmental process, which is controlled by KNOX proteins, rather than in the maintenance of bioactive CK levels in rice. On the basis of these findings, we concluded that KNOX protein simultaneously decreases GA biosynthesis and increases de novo CK biosynthesis through the induction of OsIPT2 and OsIPT3 expression, and the resulting high-CK and low-GA condition is required for formation and maintenance of the meristem.     

A MAPK pathway mediates ethylene signaling in plants

Ethylene signal transduction involves ETR1, a two-component histidine protein kinase receptor. ETR1 functions upstream of the negative regulator CTR1. The similarity of CTR1 to members of the Raf family of mitogen-activated protein kinase kinase kinases (MAPKKKs) suggested that ethylene signaling in plants involves a MAPK pathway, but no direct evidence for this has been provided. Here we show that distinct MAPKs are activated by the ethylene precursor aminocyclopropane-1-carboxylic acid (ACC) in Medicago and ARABIDOPSIS: In Medicago, the ACC-activated MAPKs were SIMK and MMK3, while in Arabidopsis MPK6 and another MAPK were identified. Medicago SIMKK specifically mediated ACC-induced activation of SIMK and MMK3. Transgenic Arabidopsis plants overexpressing SIMKK have constitutive MPK6 activation and ethylene-induced target gene expression. SIMKK overexpressor lines resemble ctr1 mutants in showing a triple response phenotype in the absence of ACC. Whereas MPK6 was not activated by ACC in etr1 mutants, ein2 and ein3 mutants showed normal activation profiles. In contrast, ctr1 mutants showed constitutive activation of MPK6. These data indicate that a MAPK cascade is part of the ethylene signal transduction pathway in plants.     

The transmembrane domain of acid trehalase mediates ubiquitin-independent multivesicular body pathway sorting

Trehalose serves as a storage source of carbon and plays important roles under various stress conditions. For example, in many organisms trehalose has a critical function in preserving membrane structure and fluidity during dehydration/rehydration. In the yeast Saccharomyces cerevisiae, trehalose accumulates in the cell when the nutrient supply is limited but is rapidly degraded when the supply of nutrients is renewed. Hydrolysis of trehalose in yeast depends on neutral trehalase and acid trehalase (Ath1). Ath1 resides and functions in the vacuole; however, it appears to catalyze the hydrolysis of extracellular trehalose. Little is known about the transport route of Ath1 to the vacuole or how it encounters its substrate. Here, through the use of various trafficking mutants we showed that this hydrolase reaches its final destination through the multivesicular body (MVB) pathway. In contrast to the vast majority of proteins sorted into this pathway, Ath1 does not require ubiquitination for proper localization. Mutagenesis analyses aimed at identifying the unknown targeting signal revealed that the transmembrane domain of Ath1 contains the information sufficient for its selective sequestration into MVB internal vesicles.     

Sexual dimorphism in snakes: different reproductive roles favour different body plans

Because snakes have a highly simplified morphology, and many species have a wide (and broadly overlapping) range of adult body sizes within each sex, they offer an excellent opportunity to compare body composition of males and females. Evolutionary theory predicts that particular body components should be differentially enlarged in the two sexes. For example, we might expect the reproductive success of females to be enhanced by enlargement of organ systems involved in the processing and storage of energy (e.g. alimentary tract, liver, fat stores) whereas males would benefit from the enlargement of systems important for mate-searching, male–male combat and sperm competition (e.g. larger mass of skeletal muscles, tail, and kidneys). Dissection of 243 specimens of three snake species (117 Vipera aspis, 43 Elaphe longissima, 83 Coluber viridflavus) broadly supported these predictions. Strong sex differences were apparent in relative sizes (masses) of all the non-gonadal body components that we weighed. For example, males consistently had more musculature (relative to body length) than did conspecific females. Dimorphism in relative muscle mass is likely to be one of the most fundamental and widespread morphological differences between males and females in the Animal Kingdom.     

Structural basis of Vta1 function in the multivesicular body sorting pathway

The MVB pathway plays essential roles in several eukaryotic cellular processes. Proper function of the MVB pathway requires reversible membrane association of the ESCRTs, a process catalyzed by Vps4 ATPase. Vta1 regulates the Vps4 activity, but its mechanism of action was poorly understood. We report the high-resolution crystal structures of the Did2- and Vps60-binding N-terminal domain and the Vps4-binding C-terminal domain of S. cerevisiae Vta1. The C-terminal domain also mediates Vta1 dimerization and both subunits are required for its function as a Vps4 regulator. Emerging from our analysis is a mechanism of regulation by Vta1 in which the C-terminal domain stabilizes the ATP-dependent double ring assembly of Vps4. In addition, the MIT motif-containing N-terminal domain, projected by a long disordered linker, allows contact between the Vps4 disassembly machinery and the accessory ESCRT-III proteins. This provides an additional level of regulation and coordination for ESCRT-III assembly and disassembly.     

The role of gibberellin in regulation of dwarf plants development

The effect of exogenously applied gibberellic (GA₃) acid on developmental processes in dwarf pea and dwarf maize seedlings was studied. Plants responded to the phytohormone by accelerated longitudinal growth rate and apparent shortening of developmental phases. Poly(A)-mRNA population isolated from gibberellin-treated pea or maize seedlings exhibited much higher translational activity per mRNA unit in the cell-free wheat germ system when compared with control, untreated plants. Analysis of in vitro translation products made by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS—PAGE) followed by autoradiography and densitometry revealed markedly increased overall intensity of the labelled polypeptide bands in addition to the new protein bands which started to appear in gibberellin-treated pea and maize seedlings while were still not detectable in the control plants of the same age. The banding pattern of translation products programmed by poly(A)-mRNA extracted from 2 days older untreated pea plants resembled that of the gibberellin-treated 2 days younger seedlings. It is concluded that gibberellic acid applied to dwarfs accelerates not exclusively the longitudinal growth of plants but also promotes their transition to the next developmental phases.     

Gibberellin homeostasis in tobacco is regulated by gibberellin metabolism genes with different gibberellin sensitivity

Gibberellins are phytohormones that regulate growth and development of plants. Gibberellin homeostasis is maintained by feedback regulation of gibberellin metabolism genes. To understand this regulation, we manipulated the gibberellin pathway in tobacco and studied its effects on the morphological phenotype, gibberellin levels and the expression of endogenous gibberellin metabolism genes. The overexpression of a gibberellin 3-oxidase (biosynthesis gene) in tobacco (3ox-OE) induced slight variations in phenotype and active GA(1) levels, but we also found an increase in GA(8) levels (GA(1) inactivation product) and a conspicuous induction of gibberellin 2-oxidases (catabolism genes; NtGA2ox3 and -5), suggesting an important role for these particular genes in the control of gibberellin homeostasis. The effect of simultaneous overexpression of two biosynthesis genes, a gibberellin 3-oxidase and a gibberellin 20-oxidase (20ox/3ox-OE), on phenotype and gibberellin content suggests that gibberellin 3-oxidases are non-limiting enzymes in tobacco, even in a 20ox-OE background. Moreover, the expression analysis of gibberellin metabolism genes in transgenic plants (3ox-OE, 20ox-OE and hybrid 3ox/20ox-OE), and in response to application of different GA(1) concentrations, showed genes with different gibberellin sensitivity. Gibberellin biosynthesis genes (NtGA20ox1 and NtGA3ox1) are negatively feedback regulated mainly by high gibberellin levels. In contrast, gibberellin catabolism genes which are subject to positive feedback regulation are sensitive to high (NtGA2ox1) or to low (NtGA2ox3 and -5) gibberellin concentrations. These two last GA2ox genes seem to play a predominant role in gibberellin homeostasis under mild gibberellin variations, but not under large gibberellin changes, where the biosynthesis genes GA20ox and GA3ox may be more important.     

The interaction between L1-type proteins and ankyrins - a master switch for L1-type CAM function

L1-type cell adhesion molecules (CAMs) are important mediators of neural differentiation, including axonal outgrowth and pathfinding and also of synapse formation and maintenance. In addition, their interactions with cytoskeletal components are highly conserved and regulated. How these different aspects of CAM functionality relate to each other is not well understood. Based on results from our and other laboratories we propose that ankyrin-binding to L1-type CAMs provides a master switch. The interaction with ankyrins directs L1-type adhesive proteins into different functional contexts, either ankyrin-independent functions, such as neurite outgrowth and axonal pathfinding or into ankyrin-dependent functions, such as L1’s role at axon initial segments (AIS), paranodal regions, synapses and in dendrites. The content of this Mini review was first presented in a shortened form at the 12th Mejbaum-Katzenellenbogen Seminar “Membrane Skeleton. Recent Advances and Future Research Directions”, June 15–18, 2008, Zakopane, Poland     

The isoprostanoid pathway in plants

Higher plants are generally unable to synthesize arachidonic acid, and thus, do neither form prostaglandins nor C20-isoprostanes. Instead, plants utilize linolenic acid for the synthesis of prostaglandin-like compounds of the jasmonate type via the lipoxygenase/allene oxide synthase pathway and C18-isoprostanoids, termed phytoprostanes, via a nonenzymatic, free radical catalyzed pathway analogous to the isoprostane pathway in animals. Both pathways are constitutively present in many if not all plants. Formation of jasmonates can be triggered by specific stimuli interacting with membrane receptors while phytoprostane synthesis can be induced by ROS and heavy metals. Jasmonates are established plant signal compounds that induce defense responses including accumulation of antimicrobial secondary metabolites (phytoalexins). Preliminary data indicates that phytoprostanes also induce phytoalexins in a variety of plant species suggesting a possible function of phytoprostanes as mediators of defense reactions in response to oxidative stress in plants.     

The OXI1 kinase pathway mediates Piriformospora indica-induced growth promotion in Arabidopsis

Piriformospora indica is an endophytic fungus that colonizes roots of many plant species and promotes growth and resistance to certain plant pathogens. Despite its potential use in agriculture, little is known on the molecular basis of this beneficial plant-fungal interaction. In a genetic screen for plants, which do not show a P. indica- induced growth response, we isolated an Arabidopsis mutant in the OXI1 (Oxidative Signal Inducible1) gene. OXI1 has been characterized as a protein kinase which plays a role in pathogen response and is regulated by H₂O₂ and PDK1 (3-PHOSPHOINOSITIDE-DEPENDENT PROTEIN KINASE1). A genetic analysis showed that double mutants of the two closely related PDK1.1 and PDK1.2 genes are defective in the growth response to P. indica. While OXI1 and PDK1 gene expression is upregulated in P. indica-colonized roots, defense genes are downregulated, indicating that the fungus suppresses plant defense reactions. PDK1 is activated by phosphatidic acid (PA) and P. indica triggers PA synthesis in Arabidopsis plants. Under beneficial co-cultivation conditions, H₂O₂ formation is even reduced by the fungus. Importantly, phospholipase D (PLD)α1 or PLDδ mutants, which are impaired in PA synthesis do not show growth promotion in response to fungal infection. These data establish that the P. indica-stimulated growth response is mediated by a pathway consisting of the PLD-PDK1-OXI1 cascade.     

The DELLA domain of GA INSENSITIVE mediates the interaction with the GA INSENSITIVE DWARF1A gibberellin receptor of Arabidopsis

Gibberellic acid (GA) promotes seed germination, elongation growth, and flowering time in plants. GA responses are repressed by DELLA proteins, which contain an N-terminal DELLA domain essential for GA-dependent proteasomal degradation of DELLA repressors. Mutations of or within the DELLA domain of DELLA repressors have been described for species including Arabidopsis thaliana, wheat (Triticum aestivum), maize (Zea mays), and barley (Hordeum vulgare), and we show that these mutations confer GA insensitivity when introduced into the Arabidopsis GA INSENSITIVE (GAI) DELLA repressor. We also demonstrate that Arabidopsis mutants lacking the three GA INSENSITIVE DWARF1 (GID1) GA receptor genes are GA insensitive with respect to GA-promoted growth responses, GA-promoted DELLA repressor degradation, and GA-regulated gene expression. Our genetic interaction studies indicate that GAI and its close homolog REPRESSOR OF ga1-3 are the major growth repressors in a GA receptor mutant background. We further demonstrate that the GA insensitivity of the GAI DELLA domain mutants is explained in all cases by the inability of the mutant proteins to interact with the GID1A GA receptor. Since we found that the GAI DELLA domain alone can mediate GA-dependent GID1A interactions, we propose that the DELLA domain functions as a receiver domain for activated GA receptors.     

The expression of tobacco knotted1-type class 1 homeobox genes correspond to regions predicted by the cytohistological zonation model

We have isolated and characterized four tobacco homeobox genes, NTH1, NTH9, NTH20, NTH22 (Nicotiana tabacum homeobox) which belong to the class 1 knotted1-type family of homeobox genes. Comparison of the inferred amino acid sequences of the ELK homeodomains of these genes and previously reported kn1-type class 1 proteins has revealed that the four new tobacco genes belong to distinct subclasses, suggesting that each NTH gene may have distinct functions. Using in situ hybridization and by analysing the distribution of GUS activity in tobacco plants transformed with NTH promoter::GUS constructs, localized expression of the three NTH genes was observed in the shoot apical meristem (SAM). In the vegetative SAM, NTH1 and NTH15 showed overlapping expression in the corpus, NTH20 was expressed in the peripheral zone, and NTH9 was predominantly expressed in the rib zone. The expression patterns of the different NTH genes correspond to regions predicted by the cytohistological zonation model, suggesting that each NTH gene specifies the function of the SAM zone with which it is associated.     

The evolutionarily conserved arginyltransferase 1 mediates a pVHL-independent oxygen-sensing pathway in mammalian cells

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The N-acylethanolamine-mediated regulatory pathway in plants

While cannabinoids are secondary metabolites synthesized by just a few plant species, N-acylethanolamines (NAEs) are distributed widely in the plant kingdom, and are recovered in measurable, bioactive quantities in many plant-derived products. NAEs in higher plants are ethanolamides of fatty acids with acyl-chain lenghts of C12-C(18) and zero to three C=C bonds. Generally, the most-abundant NAEs found in plants and vertebrates are similar, including NAE 16 : 0, 18 : 1, 18 : 2, and 18 : 3. Like in animal systems, NAEs are formed in plants from N-acylphosphatidylethanolamines (NAPEs), and they are hydrolyzed by an amidase to yield ethanolamine and free fatty acids (FFA). Recently, a homologue of the mammalian fatty acid amide hydrolase (FAAH-1) was identified in Arabidopsis thaliana and several other plant species. Overexpression of Arabidopsis FAAH (AtFAAH) resulted in plants that grew faster, but were more sensitive to biotic and abiotic insults, suggesting that the metabolism of NAEs in plants resides at the balance between growth and responses to environmental stresses. Similar to animal systems, exogenously applied NAEs have potent and varied effects on plant cells. Recent pharmacological approaches combined with molecular-genetic experiments revealed that NAEs may act in certain plant tissues via specific membrane-associated proteins or by interacting with phospholipase D-alpha, although other, direct targets for NAE action in plants are likely to be discovered. Polyunsaturated NAEs can be oxidized via the lipoxygenase pathway in plants, producing an array of oxylipin products that have received little attention so far. Overall, the conservation of NAE occurrence and metabolic machinery in plants, coupled with the profound physiological effects of elevating NAE content or perturbing endogenous NAE metabolism, suggest that an NAE-mediated regulatory pathway, sharing similarities with the mammalian endocannabinoid pathway, indeed exists.     

The mRNA export pathway in plants

A double lipid bilayer separating the nucleus from the cytoplasm, termed the nuclear envelope, is a defining feature of eukaryotes. Nucleocytoplasmic transport of macromolecules through the nuclear pores enables fine-tuned regulation of biologic processes. All mature mRNAs are delivered to the cytoplasm from the nucleus via an mRNA export pathway. Much work has been done in yeast and animals to study the machinery of mRNA export. However, until recently, research on plant mRNA export has been quite limited. Genetic, bioinformatic, and biochemical investigations have expanded our understanding of the mRNA export process in plants. Here, we review recent progress that has been made elucidating the components of the mRNA export pathway in plants. MOS3 (MODIFIER OF SNC1, 3) /AtNup96 and AtNup160 are both components of the highly conserved Nup107–160 nucleoporin complex and were shown to play key roles in mRNA export. MOS11 (MODIFIER OF SNC1, 11), which is homologous to the RNA helicase enhancer CIP29 in human, was recently found to be involved in the same pathway as MOS3. A DEAD Box RNA helicase, LOS4 (low expression of osmotically responsive genes 4) was also found to play a role in the mRNA export process, putatively by carrying mRNA molecules through the nuclear envelope. Recently, a protein complex homologous to the yeast TREX-2 complex was also found to play important roles in mRNA export in plants. It appears that most players in the mRNA export pathway are highly conserved among plants, yeast and animals.     

The gibberellin signaling pathway is regulated by the appearance and disappearance of SLENDER RICE1 in nuclei

The slender rice1 mutant (slr1) shows a constitutive gibberellin (GA) response phenotype. To investigate the mode of action of SLR1, we generated transgenic rice expressing a fusion protein consisting of SLR1 and green fluorescent protein (SLR1-GFP) and analyzed the phenotype of the transformants and the subcellular localization of GFP in vivo. SLR1-GFP worked in nuclei to repress the GA signaling pathway; its overproduction caused a dwarf phenotype. Application of GA(3) to SLR1-GFP overproducers induced GA actions such as shoot elongation, downregulation of GA 20-oxidase expression, and upregulation of SLR1 expression linked with the disappearance of the nuclear SLR1-GFP protein. We also performed domain analyses of SLR1 using transgenic plants overproducing different kinds of truncated SLR1 proteins. The analyses revealed that the SLR1 protein can be divided into four parts: a GA signal perception domain located at the N terminus, a regulatory domain for its repression activity, a dimer formation domain essential for signal perception and repression activity, and a repression domain at the C terminus. We conclude that GA signal transduction is regulated by the appearance or disappearance of the nuclear SLR1 protein, which is controlled by the upstream GA signal.