Evolution of Hominin Detoxification: Neanderthal and Modern Human Ah Receptor Respond Similarly to TCDD.

In studies of hominin adaptations to fire use, the role of the aryl hydrocarbon receptor (AHR) in the evolution of detoxification has been highlighted, including statements that the modern human AHR confers a significantly better capacity to deal with toxic smoke components than the Neanderthal AHR. To evaluate this, we compared the AHR-controlled induction of cytochrome P4501A1 (CYP1A1) mRNA in HeLa human cervix epithelial adenocarcinoma cells transfected with an Altai-Neanderthal or a modern human reference AHR expression construct, and exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). We compared the complete AHR mRNA sequences including the untranslated regions (UTRs), maintaining the original codon usage. We observe no significant difference in CYP1A1 induction by TCDD between Neanderthal and modern human AHR, whereas a 150-1,000 times difference was previously reported in a study of the AHR coding region optimized for mammalian codon usage and expressed in rat cells. Our study exemplifies that expression in a homologous cellular background is of major importance to determine (ancient) protein activity. The Neanderthal and modern human dose-response curves almost coincide, except for a slightly higher extrapolated maximum for the Neanderthal AHR, possibly caused by a 5'-UTR G-variant known from modern humans (rs7796976). Our results are strongly at odds with a major role of the modern human AHR in the evolution of hominin detoxification of smoke components and consistent with our previous study based on 18 relevant genes in addition to AHR, which concluded that efficient detoxification alleles are more dominant in ancient hominins, chimpanzees, and gorillas than in modern humans.

The Medicago truncatula nodule identity gene MtNOOT1 is required for coordinated apical-basal development of the root.

BACKGROUND: Legumes can utilize atmospheric nitrogen by hosting nitrogen-fixing bacteria in special lateral root organs, called nodules. Legume nodules have a unique ontology, despite similarities in the gene networks controlling nodule and lateral root development. It has been shown that Medicago truncatula NODULE ROOT1 (MtNOOT1) is required for the maintenance of nodule identity, preventing the conversion to lateral root development. MtNOOT1 and its orthologs in other plant species -collectively called the NOOT-BOP-COCH-LIKE (NBCL) family- specify boundary formation in various aerial organs. However, MtNOOT1 is not only expressed in nodules and aerial organs, but also in developing roots, where its function remains elusive. RESULTS: We show that Mtnoot1 mutant seedlings display accelerated root elongation due to an enlarged root apical meristem. Also, Mtnoot1 mutant roots are thinner than wild-type and are delayed in xylem cell differentiation. We provide molecular evidence that the affected spatial development of Mtnoot1 mutant roots correlates with delayed induction of genes involved in xylem cell differentiation. This coincides with a basipetal shift of the root zone that is susceptible to rhizobium-secreted symbiotic signal molecules. CONCLUSIONS: Our data show that MtNOOT1 regulates the size of the root apical meristem and vascular differentiation. Our data demonstrate that MtNOOT1 not only functions as a homeotic gene in nodule development but also coordinates the spatial development of the root.

Cis-regulatory PLETHORA promoter elements directing root and nodule expression are conserved between Arabidopsis thaliana and Medicago truncatula.

Nodules are unique organs formed on roots of legumes by soil-borne bacteria, collectively known as rhizobium. Recently, we have shown that orthologs of the AINTEGUMENTA-like (AIL) AP2 transcription factors PLETHORA (PLT) 1 to 4, that redundantly regulate Arabidopsis thaliana root development are involved in root and nodule growth in Medicago truncatula. Hence, it is conceivable that rhizobium has co-opted these genes for nodule development. Whether this co-option requires the presence of specific cis-elements in the promoters and/or specialization of PLT protein function is not clear. Here, we analyzed the qualitative expression patterns of the Arabidopsis PLT1 to 4 promoters in Medicago roots and nodules and compared these with the described expression patterns of the Medicago PLT genes. Our studies reveal that the expression patterns of the investigated promoters and their Medicago orthologs are very similar, indicating that at least all cis-elements regulating spatial PLT expression are conserved among the Arabidopsis and Medicago PLT1 to 4 promoters.

Root developmental programs shape the Medicago truncatula nodule meristem.

Nodules on the roots of legume plants host nitrogen-fixing Rhizobium bacteria. Several lines of evidence indicate that nodules are evolutionarily related to roots. We determined whether developmental control of the Medicago truncatula nodule meristem bears resemblance to that in root meristems through analyses of root meristem-expressed PLETHORA genes. In nodules, MtPLETHORA 1 and 2 are preferentially expressed in cells positioned at the periphery of the meristem abutting nodule vascular bundles. Their expression overlaps with an auxin response maximum and MtWOX5, which is a marker for the root quiescent center. Strikingly, the cells in the central part of the nodule meristem have a high level of cytokinin and display MtPLETHORA 3 and 4 gene expression. Nodule-specific knockdown of MtPLETHORA genes results in a reduced number of nodules and/or in nodules in which meristem activity has ceased. Our nodule gene expression map indicates that the nodule meristem is composed of two distinct domains in which different MtPLETHORA gene subsets are expressed. Our mutant studies show that MtPLETHORA genes function redundantly in nodule meristem maintenance. This indicates that Rhizobium has recruited root developmental programs for nodule formation.

Rhizobium Lipo-chitooligosaccharide Signaling Triggers Accumulation of Cytokinins in Medicago truncatula Roots.

Legume rhizobium symbiosis is initiated upon perception of bacterial secreted lipo-chitooligosaccharides (LCOs). Perception of these signals by the plant initiates a signaling cascade that leads to nodule formation. Several studies have implicated a function for cytokinin in this process. However, whether cytokinin accumulation and subsequent signaling are an integral part of rhizobium LCO signaling remains elusive. Here, we show that cytokinin signaling is required for the majority of transcriptional changes induced by rhizobium LCOs. In addition, we demonstrate that several cytokinins accumulate in the root susceptible zone 3 h after rhizobium LCO application, including the biologically most active cytokinins, trans-zeatin and isopentenyl adenine. These responses are dependent on calcium- and calmodulin-dependent protein kinase (CCaMK), a key protein in rhizobial LCO-induced signaling. Analysis of the ethylene-insensitive Mtein2/Mtsickle mutant showed that LCO-induced cytokinin accumulation is negatively regulated by ethylene. Together with transcriptional induction of ethylene biosynthesis genes, it suggests a feedback loop negatively regulating LCO signaling and subsequent cytokinin accumulation. We argue that cytokinin accumulation is a key step in the pathway leading to nodule organogenesis and that this is tightly controlled by feedback loops.

Fate map of Medicago truncatula root nodules.

Legume root nodules are induced by N-fixing rhizobium bacteria that are hosted in an intracellular manner. These nodules are formed by reprogramming differentiated root cells. The model legume Medicago truncatula forms indeterminate nodules with a meristem at their apex. This organ grows by the activity of the meristem that adds cells to the different nodule tissues. In Medicago sativa it has been shown that the nodule meristem is derived from the root middle cortex. During nodule initiation, inner cortical cells and pericycle cells are also mitotically activated. However, whether and how these cells contribute to the mature nodule has not been studied. Here, we produce a nodule fate map that precisely describes the origin of the different nodule tissues based on sequential longitudinal sections and on the use of marker genes that allow the distinction of cells originating from different root tissues. We show that nodule meristem originates from the third cortical layer, while several cell layers of the base of the nodule are directly formed from cells of the inner cortical layers, root endodermis and pericycle. The latter two differentiate into the uninfected tissues that are located at the base of the mature nodule, whereas the cells derived from the inner cortical cell layers form about eight cell layers of infected cells. This nodule fate map has then been used to re-analyse several mutant nodule phenotypes. This showed, among other things, that intracellular release of rhizobia in primordium cells and meristem daughter cells are regulated in a different manner.

Assessment of technological options and economical feasibility for cyanophycin biopolymer and high-value amino acid production.

Major transitions can be expected within the next few decades aiming at the reduction of pollution and global warming and at energy saving measures. For these purposes, new sustainable biorefinery concepts will be needed that will replace the traditional mineral oil-based synthesis of specialty and bulk chemicals. An important group of these chemicals are those that comprise N-functionalities. Many plant components contained in biomass rest or waste stream fractions contain these N-functionalities in proteins and free amino acids that can be used as starting materials for the synthesis of biopolymers and chemicals. This paper describes the economic and technological feasibility for cyanophycin production by fermentation of the potato waste stream Protamylassetrade mark or directly in plants and its subsequent conversion to a number of N-containing bulk chemicals.

Medicago truncatula ENOD40-1 and ENOD40-2 are both involved in nodule initiation and bacteroid development.

The establishment of a nitrogen-fixing root nodule on legumes requires the induction of mitotic activity of cortical cells leading to the formation of the nodule primordium and the infection process by which the bacteria enter this primordium. Several genes are up-regulated during these processes, among them ENOD40. Here it is shown, by using gene-specific knock-down of the two Medicago truncatula ENOD40 genes, that both genes are involved in nodule initiation. Further, during nodule development, both genes are essential for bacteroid development.

ENOD40 affects elongation growth in tobacco Bright Yellow-2 cells by alteration of ethylene biosynthesis kinetics.

Plant developmental processes are controlled by co-ordinated action of phytohormones and plant genes encoding components of developmental response pathways. ENOD40 was identified as a candidate for such a plant factor with a regulatory role during nodulation. Although its mode of action is poorly understood, several lines of evidence suggest interaction with phytohormone response pathways. This hypothesis was investigated by analysing cytokinin-, auxin-, and ethylene-induced responses on cell growth and cell division in transgenic 35S:NtENOD40 Bright Yellow-2 (BY-2) tobacco cell suspensions. It was found that cell division frequency is controlled by the balance between cytokinin and auxin in wild-type cells and that this regulation is not affected in 35S:NtENOD40 lines. Elongation growth, on the other hand, is reduced upon overexpression of NtENOD40. Analysis of ethylene homeostasis shows that ethylene accumulation is accelerated in 35S:NtENOD40 lines. ENOD40 action can be counteracted by an ethylene perception blocker, indicating that ethylene is a negative regulator of elongation growth in 35S:NtENOD40 cells, and that the NtENOD40-induced response is mediated by alteration of ethylene biosynthesis kinetics.

Formation of organelle-like N2-fixing symbiosomes in legume root nodules is controlled by DMI2.

In most legume nodules, the N2-fixing rhizobia are present as organelle-like structures inside their host cells. These structures, named symbiosomes, contain one or a few rhizobia surrounded by a plant membrane. Symbiosome formation requires the release of bacteria from cell-wall-bound infection threads. In primitive legumes, rhizobia are hosted in intracellular infection threads that, in contrast to symbiosomes, are bound by a cell wall. The formation of symbiosomes is presumed to represent a major step in the evolution of legume-nodule symbiosis, because symbiosomes facilitate the exchange of metabolites between the two symbionts. Here, we show that the genes, which are essential for initiating nodule formation, are also actively transcribed in mature Medicago truncatula nodules in the region where symbiosome formation occurs. At least one of these genes, encoding the receptor kinase DOES NOT MAKE INFECTIONS 2 (DMI2) is essential for symbiosome formation. The protein locates to the host cell plasma membrane and to the membrane surrounding the infection threads. A partial reduction of DMI2 expression causes a phenotype that resembles the infection structures found in primitive legume nodules, because infected cells are occupied by large intracellular infection threads instead of by organelle-like symbiosomes.

Reduction of cell size induced by enod40 in Arabidopsis thaliana.

An extensive analysis of organ and cell size was performed in three different Arabidopsis lines transformed with the early nodulin gene enod40 under control of the CaMV35S promoter. All three transgenic lines presented a significant decrease in the mean size of both epidermal internode and leaf mesophyll cells. Flow cytometric and image analysis of enod40-transfected protoplasts prepared from wild-type Arabidopsis cell suspensions showed that transient expression of the gene resulted in reduced forward light scattering (a factor correlated with particle size) and cell size. The direct administration of ENOD40 peptide to fresh protoplasts also resulted in reduced forward scattering with respect to the control and to the administration of unrelated peptides. As far as is known this is the first report documenting a biological effect of enod40 at the cellular level in non-legume plants.

RNA interference in Agrobacterium rhizogenes-transformed roots of Arabidopsis and Medicago truncatula.

RNA interference (RNAi) is a powerful reverse genetic tool to study gene function. The data presented here show that Agrobacterium rhizogenes-mediated RNAi is a fast and effective tool to study genes involved in root biology. The Arabidopsis gene KOJAK, involved in root hair development, was efficiently knocked down. A. rhizogenes-mediated root transformation is a fast method to generate adventitious, genetically transformed roots. In order to select for co-transformed roots a binary vector was developed that enables selection based on DsRED1 expression, with the additional benefit that chimaeric roots can be discriminated. The identification of chimaeric roots provided the opportunity to examine the extent of systemic spread of the silencing signal in the composite plants of both Arabidopsis and Medicago truncatula. It is shown that RNA silencing does not spread systemically to non-co-transformed (lateral) roots and only inefficiently to the non-transgenic shoot. Furthermore, evidence is presented which shows that RNAi is cell autonomous in the root epidermis.

Identification and characterization of a Zea mays line carrying a transposon-tagged ENOD40.

In Zea mays, two ENOD40 homologous were identified that show only 30% of sequence homology to each other. We identified line e40-mum1 carrying a Mu transposon inserted in ZmENOD40-1, the maize gene that has the highest homology to leguminous ENOD40. The insertion causes a dramatic reduction of the ZmENOD40-1 transcript level. Irrespective of this, homozygous e40-mum1 plants are still able to interact with mycorrhizal fungi. Furthermore, no phenotypic aberrations correlated to the presence of e40-mum1 have been identified and therefore it is suggested that Z. mays ENOD40 genes are functionally redundant despite their strikingly low homology.

Expression of ENOD40 during tomato plant development.

In legumes, ENOD40 expression is increased upon interaction of plants with rhizobia. Little is known of the expression pattern of ENOD40 during other stages of the plant life cycle. Studies of ENOD40 expression in non-legume development may give an indication of the function of the gene. To investigate the ENOD40 expression pattern during plant development, a fusion between the beta-glucuronidase (GUS) reporter gene and 150 bp of the 5' untranslated region plus 3,000 bp of 5' untranscribed tomato ENOD40 sequence was constructed and introduced into Lycopersicon esculentum Miller. Based on the observed GUS expression patterns in transgenic tomato we speculate that ENOD40 in tomato has a role in counteracting ethylene-provoked responses.