[Show abstract][Hide abstract] ABSTRACT: [FeFe]-hydrogenase-like genes encode [Fe4 S4 ] containing proteins that are ubiquitous in eukaryotic cells. In human, IOP1 represses HIF1-α at normal atmospheric partial O2 pressure (normoxia, 21 kPa O2 ). In yeast, the nar1 mutant cannot grow at 21kPa O2, but can develop at lower O2 pressure (2kPa O2 ). We show here that plant [FeFe]-hydrogenase-like GOLLUM genes are essential for plant development and cell cycle progression. The mutant phenotypes of these plants are seen in normoxic condions, but not under conditions of mild-hypoxia (5kPa O2 ). Transcriptomic and metabolomic experiments showed that the mutation enhances the expression of some hypoxia-induced genes under normal atmospheric O2 conditions and changes the cellular content of metabolites related to energy metabolism. In conclusion, [FeFe]-hydrogenase-like proteins play a central role in eukaryotes including the adaptation of plants to the ambient O2 partial pressure.
Full-text · Article · May 2013 · Plant Cell and Environment
[Show abstract][Hide abstract] ABSTRACT: To extend our understanding of flowering time control in eudicots, we screened for mutants in the model legume Medicago truncatula (Medicago). We identified an early flowering mutant, spring1, in a T-DNA mutant screen, but spring1 was not tagged and was deemed a somaclonal mutant. We backcrossed the mutant to wild type R108. The F1 plants and the majority of F2 plants were early flowering like spring1, strongly indicating that spring1 conferred monogenic, dominant early flowering. We hypothesized that the spring1 phenotype resulted from over expression of an activator of flowering. Previously, a major QTL for flowering time in different Medicago accessions was located to an interval on chromosome 7 with six candidate flowering- time activators, including a CONSTANS gene, MtCO, and three FLOWERING LOCUS T (FT) genes. Hence we embarked upon linkage mapping using 29 markers from the MtCO/FT region on chromosome 7 on two populations developed by crossing spring1 with Jester. Spring1 mapped to an interval of ∼0.5 Mb on chromosome 7 that excluded MtCO, but contained 78 genes, including the three FT genes. Of these FT genes, only FTa1 was up-regulated in spring1 plants. We then investigated global gene expression in spring1 and R108 by microarray analysis. Overall, they had highly similar gene expression and apart from FTa1, no genes in the mapping interval were differentially expressed. Two MADS transcription factor genes, FRUITFULLb (FULb) and SUPPRESSOR OF OVER EXPRESSION OF CONSTANS1a (SOC1a), that were up-regulated in spring1, were also up-regulated in transgenic Medicago over-expressing FTa1. This suggested that their differential expression in spring1 resulted from the increased abundance of FTa1. A 6255 bp genomic FTa1 fragment, including the complete 5' region, was sequenced, but no changes were observed indicating that the spring1 mutation is not a DNA sequence difference in the FTa1 promoter or introns.
[Show abstract][Hide abstract] ABSTRACT: Medicago truncatula and Sinorhizobium meliloti form a symbiotic association resulting in the formation of nitrogen-fixing nodules. Nodule cells contain large numbers of bacteroids which are differentiated, nitrogen-fixing forms of the symbiotic bacteria. In the nodules, symbiotic plant cells home and maintain hundreds of viable bacteria. In order to better understand the molecular mechanism sustaining the phenomenon, we searched for new plant genes required for effective symbiosis. We used a combination of forward and reverse genetics approaches to identify a gene required for nitrogen fixation, and we used cell and molecular biology to characterize the mutant phenotype and to gain an insight into gene function. The symbiotic gene DNF2 encodes a putative phosphatidylinositol phospholipase C-like protein. Nodules formed by the mutant contain a zone of infected cells reduced to a few cell layers. In this zone, bacteria do not differentiate properly into bacteroids. Furthermore, mutant nodules senesce rapidly and exhibit defense-like reactions. This atypical phenotype amongst Fix(-) mutants unravels dnf2 as a new actor of bacteroid persistence inside symbiotic plant cells.
[Show abstract][Hide abstract] ABSTRACT: Epithelial tubes perform functions that are essential for the survival of multicellular organisms. Understanding how their polarised features are maintained is therefore crucial. By analysing the function of the clathrin adaptor AP-1 in the C. elegans intestine, we found that AP-1 is required for epithelial polarity maintenance. Depletion of AP-1 subunits does not affect epithelial polarity establishment or the formation of the intestinal lumen. However, the loss of AP-1 affects the polarised distribution of both apical and basolateral transmembrane proteins. Moreover, it triggers de novo formation of ectopic apical lumens between intestinal cells along the lateral membranes later during embryogenesis. We also found that AP-1 is specifically required for the apical localisation of the small GTPase CDC-42 and the polarity determinant PAR-6. Our results demonstrate that AP-1 controls an apical trafficking pathway required for the maintenance of epithelial polarity in vivo in a tubular epithelium.
[Show abstract][Hide abstract] ABSTRACT: The root system architecture is crucial to adapt plant growth to changing soil environmental conditions and consequently to maintain crop yield. In addition to root branching through lateral roots, legumes can develop another organ, the nitrogen-fixing nodule, upon a symbiotic bacterial interaction. A mutant, cra1, showing compact root architecture was identified in the model legume Medicago truncatula. cra1 roots were short and thick due to defects in cell elongation, whereas densities of lateral roots and symbiotic nodules were similar to the wild type. Grafting experiments showed that a lengthened life cycle in cra1 was due to the smaller root system and not to the pleiotropic shoot phenotypes observed in the mutant. Analysis of the cra1 transcriptome at a similar early developmental stage revealed few significant changes, mainly related to cell wall metabolism. The most down-regulated gene in the cra1 mutant encodes a Caffeic Acid O-Methyl Transferase, an enzyme involved in lignin biosynthesis; accordingly, whole lignin content was decreased in cra1 roots. This correlated with differential accumulation of specific flavonoids and decreased polar auxin transport in cra1 mutants. Exogenous application of the isoflavone formononetin to wild-type plants mimicked the cra1 root phenotype, whereas decreasing flavonoid content through silencing chalcone synthases restored the polar auxin transport capacity of the cra1 mutant. The CRA1 gene, therefore, may control legume root growth through the regulation of lignin and flavonoid profiles, leading to changes in polar auxin transport.
[Show abstract][Hide abstract] ABSTRACT: We have identified an active Medicago truncatula copia-like retroelement called Medicago RetroElement1-1 (MERE1-1) as an insertion in the symbiotic NSP2 gene. MERE1-1 belongs to a low-copy-number family in the sequenced Medicago genome. These copies are highly related, but only three of them have a complete coding region and polymorphism exists between the long terminal repeats of these different copies. This retroelement family is present in all M. truncatula ecotypes tested but also in other legume species like Lotus japonicus. It is active only during tissue culture in both R108 and Jemalong Medicago accessions and inserts preferentially in genes.
[Show abstract][Hide abstract] ABSTRACT: The symbiotic association between legumes and nitrogen-fixing bacteria collectively known as rhizobia results in the formation of a unique plant root organ called the nodule. This process is initiated following the perception of rhizobial nodulation factors by the host plant. Nod factor (NF)-stimulated plant responses, including nodulation-specific gene expression, is mediated by the NF signaling pathway. Plant mutants in this pathway are unable to nodulate. We describe here the cloning and characterization of two mutant alleles of the Medicago truncatula ortholog of the Lotus japonicus and pea (Pisum sativum) NIN gene. The Mtnin mutants undergo excessive root hair curling but are impaired in infection and fail to form nodules following inoculation with Sinorhizobium meliloti. Our investigation of early NF-induced gene expression using the reporter fusion ENOD11::GUS in the Mtnin-1 mutant demonstrates that MtNIN is not essential for early NF signaling but may negatively regulate the spatial pattern of ENOD11 expression. It was recently shown that an autoactive form of a nodulation-specific calcium/calmodulin-dependent protein kinase is sufficient to induce nodule organogenesis in the absence of rhizobia. We show here that MtNIN is essential for autoactive calcium/calmodulin-dependent protein kinase-induced nodule organogenesis. The non-nodulating hcl mutant has a similar phenotype to Mtnin, but we demonstrate that HCL is not required in this process. Based on our data, we suggest that MtNIN functions downstream of the early NF signaling pathway to coordinate and regulate the correct temporal and spatial formation of root nodules.
[Show abstract][Hide abstract] ABSTRACT: Molecular genetic studies in model plants have been facilitated by the use of inser-tion mutagenesis with transposable elements
or T-DNA. We have initiated an inser-tion mutagenesis program in M. truncatula using the tobacco retrotransposon
Tnt1, an autonomous 5.3 kb long copia-like LTR element (Grandbastien et al., Nature, 337, 376-80, 1989) as mutagen. We
demonstrated (d’Erfurth et al. Plant J., 34, 95-106, 2003) that Tnt1 transposes actively only during in vitro culture, generating
from 4 to 40 insertions per plant. Sequencing of a large number of Tnt1 insertion sites indi-cated the lack of insertion
site specificity for this element in Medicago. Tnt1 transposed preferentially into genes and its multiplication by transposition
can be re-induced in tissue culture making this element an attractive tool for large scale insertion mutagenesis in M. truncatula.
In the frame of the European Grain Legume Integrated Project (www.eugrainlegumes.org), ten laboratories are joining
efforts to prepare a large M. truncatula Jemalong-Tnt1 tagged population. We recently isolated an endogenous copia type
mobile retrotransposon also activated by the tissue culture process in Medicago. The discovery of this element add value to
the collection already produced.
We will describe the work done and the objectives achieved in the GLIP mutagenesis project.
[Show abstract][Hide abstract] ABSTRACT: Tag1 is an element from the hAT (hobo, Ac, Tam) family of DNA transposable elements present in Arabidopsis thaliana ecotype Landsberg. We have compared its behavior in the A. thaliana ecotype Columbia and in the legume model plant Medicago truncatula. As previously described Tag1 transposed efficiently in A. thaliana ecotype Columbia. In contrast, only somatic excision, but no germinal excision or transposition events could be detected in M. truncatula, indicating that Tag1 is poorly active in this plant. These differences in activity correlated with the absence of two minor Tag1 transcripts in M. truncatula transgenic lines but not with the structure of the T-DNA loci or the methylation of the element. These results indicate that Tag1 is not suitable for insertion mutagenesis in M. truncatula.
[Show abstract][Hide abstract] ABSTRACT: The development of efficient transformation protocols in several legume species has opened the possibility to use T-DNA insertional mutagenesis to investigate legume-specific processes such as symbiosis. A T-DNA mutagenesis approach was initiated in the model legume Medicago truncatula by constructing a small collection of 1000 M. truncatula T-DNA lines, using in vitro-mediated transformation-regeneration protocols. In this T-DNA collection, mutant phenotypes were observed but generally did not result from the disruption of genes by the T-DNA but most probably from somaclonal mutations induced by the in vitro regeneration process or by unsuccess-ful T-DNA integration events resulting in small (undetectable) insertions or deletions in the genome of the regenerated plants. In addition, a promoter trap approach was also not very successful for gene discovery because aberrant gene fusions were observed in this collection. Therefore, the T-DNA mutagenesis approach in M. truncatula seems to be limited by the lack of an efficient in planta transformation protocol that would allow the generation of a large mutant collection suitable for reverse genetic studies. Thus, there is a need for the development of alternative tools for efficient gene disruption in legumes.
No preview · Article · Jun 2006 · CAB Reviews Perspectives in Agriculture Veterinary Science Nutrition and Natural Resources
[Show abstract][Hide abstract] ABSTRACT: The organogenesis of nitrogen-fixing nodules in legume plants is initiated in specific root cortical cells and regulated by long-distance signaling and carbon allocation. Here, we explore cell-to-cell communication processes that occur during nodule initiation in Medicago species and their functional relevance using a combination of fluorescent tracers, electron microscopy, and transgenic plants. Nodule initiation induced symplasmic continuity between the phloem and nodule initials. Macromolecules such as green fluorescent protein could traffic across short or long distances from the phloem into these primordial cells. The created symplasmic field was regulated throughout nodule development. Furthermore, Medicago truncatula transgenic plants expressing a viral movement protein showed increased nodulation. Hence, the establishment of this symplasmic field may be a critical element for the control of nodule organogenesis.