Molecular analysis of transcription factors involved in Medicago truncatula nodulation

Thanks to the symbiotic interaction with rhizobia many legumes are important natural fertilizers and pioneer plants on eroded soils. This plant-bacteria interaction leads to formation of root nodules in which differentiated rhizobia, alias bacteroids, fix nitrogen in return for C- and energy sources. Medicago truncatula and Lotus japonicus are considered as model legumes for the research on indeterminate en determinate nodule formation and development respectively. In chapter 1, an overview is given of the current knowledge on the nodulation process in model legumes, covering molecular players in nodule initiation and nitrogen fixation, description of the different stages of nodulation, involvement of hormones, autoregulation mechanisms and tools to study legumes. Based on the numerous transcription factors present in plant genomes, transcriptional gene regulation might constitute an important element to control plant processes including nodulation and identifying their role and target genes will help to elucidate complex gene networks. Within the nodulation process, several TFs were identified which regulate pathways leading to nodule formation and nodule functioning. In this study, the role of three transcription factors in nodulation and other plant developmental pathways was investigated in Medicago truncatula. In chapter 2 we focus on an AP2 domain containing transcription factor, MtRAP2.4, which was upregulated during nodule senescence and is a member of the DREB (dehydratation responsive elelement binding) subgroup of AP2 transcription factors. We performed an expression analysis, functional analysis and a micro-array analysis in order to define its role in the (later stages of the) nodulation process. Moreover, its role during nodulation in relation to stress is discussed. In chapter 3, the analysis of a basic leucine zipper (bZIP) transcription factor, MtATB2, is presented and discussed. Because this gene has a close homologue in A. thaliana from which the expression is strongly regulated by sucrose, we analyzed its expression not only during nodule senescence but also upon sucrose application. Moreover, the function of the gene is analyzed by modulating its expression level by ectopic expression and RNAi analysis. Whereas previous transcription factors were selected based on their upregulation in aging nodules, a third transcription factor, MtNAC1 was investigated based on its homology with AtNAC1 and the similarities between lateral root formation - in which AtNAC1 was involved - and nodule formation. The role of MtNAC1 in both processes is analyzed and discussed in chapter 4. Throughout this study, we frequently used the A. rhizogenes transformation method in order to obtain transgenic roots. Based on our experiences with this technique, we summarized tips and tricks in chapter 5 which might increase the efficiency of this transformation method

Transcription factor MtATB2: about nodulation, sucrose and senescence

The symbiotic interaction between legumes and rhizobia results in root nodules with nitrogen-fixing bacteroids. Throughout the lifespan of the nodules, the exchange of C sources and N compounds between the host plant and the bacteria is tightly balanced. Sucrose plays a major role in the provision of C skeletons and energy to the bacteroids. Transcription of MtATB2, encoding a bZIP transcription factor, is shown to be regulated by sucrose and is enhanced during nodule senescence. Transcripts occur in the nodule apex and in the vascular tissue of nodules and roots. Ectopic expression of the gene diminished nodule formation and affected root growth. Presumably, MtATB2 controls processes that are under sucrose homeostasis and are important for nodule and root growth

CLE Peptides Control Medicago truncatula Nodulation Locally and Systemically1[C][W][OA]

The CLAVATA3/embryo-surrounding region (CLE) peptides control the fine balance between proliferation and differentiation in plant development. We studied the role of CLE peptides during indeterminate nodule development and identified 25 MtCLE peptide genes in the Medicago truncatula genome, of which two genes, MtCLE12 and MtCLE13, had nodulation-related expression patterns that were linked to proliferation and differentiation. MtCLE13 expression was up-regulated early in nodule development. A high-to-low expression gradient radiated from the inner toward the outer cortical cell layers in a region defining the incipient nodule. At later stages, MtCLE12 and MtCLE13 were expressed in differentiating nodules and in the apical part of mature, elongated nodules. Functional analysis revealed a putative role for MtCLE12 and MtCLE13 in autoregulation of nodulation, a mechanism that controls the number of nodules and involves systemic signals mediated by a leucine-rich repeat receptor-like kinase, SUNN, which is active in the shoot. When MtCLE12 and MtCLE13 were ectopically expressed in transgenic roots, nodulation was abolished at the level of the nodulation factor signal transduction, and this inhibition involved long-distance signaling. In addition, composite plants with roots ectopically expressing MtCLE12 or MtCLE13 had elongated petioles. This systemic effect was not observed in transgenic roots ectopically expressing MtCLE12 and MtCLE13 in a sunn-1 mutant background, although nodulation was still strongly reduced. These results suggest multiple roles for CLE signaling in nodulation

Occurence of speech-language disorders in the acute phase following pediatric acquired brain injury : results from the Ghent University Hospital

Aims: This study investigated the occurrence of speech-language disorders during the acute phase of recovery in children with acquired brain injury (ABI) with an age between 0 and 16 years. Methods: A retrospective chart analysis was performed including 228 children (n=118 boys, n=110 girls) who consecutively presented with ABI over a 10-year period (2006-2016) at the children’s rehabilitation center at Ghent University Hospital. Descriptive statistical analyses were applied. Results: 71.1% (162/228) of the children who were admitted to the rehabilitation center presented with a speech-language disorder. Within this sample (n=162), results demonstrated the occurrence of acquired disorders in language (48.9%), speech (35.1%), learning (33.3%), swallowing (21.5%), and early communicative functions (17.4%). The proportion of children presenting with disturbances in early communicative functions differed by ABI cause. More than half (10/18, 58.8%) of the children who presented with ABI following inflammatory processes demonstrated disorders in early communicative functions. Conclusions: Especially in young children who present with inflammatory processes as the ABI cause, speech-language pathologists (SLPs) must be aware of disorders in early speech-language development. The present findings allow the SLP to appropriately plan research, education, and clinical management

The impact of palatal repair before and after 6 months of age on speech characteristics

Introduction: Optimal timing of palatal repair is still subject of discussion. The purpose of this study was to describe and compare the articulation and resonance characteristics after early (≤6m) and later (>6m) palatal repair with the Sommerlad technique. Methods: Articulation analyses and perceptual and objective evaluations of resonance were carried out in 12 Ugandan children with early repaired cleft lip and palate (mean age 3m) and 12 Belgian patients (matched for cleft type, age and gender) with later palatal closure (mean age 11m). A Ugandan and Belgian age- and gender-matched non-cleft control group was included to control for language, culture and other environmental factors. Results: Comparison of the experimental groups revealed no clinically relevant significant group differences for consonant inventory or phonological processes. Phonetic analysis showed significantly more (inter)dental productions of alveolar consonants in Belgian patients. Perceptual consensus ratings of resonance and nasal emission/turbulence and objective mean nasalance values for oral speech samples revealed no significant group differences (p>0.05). Conclusion: Articulation and resonance characteristics of young children following palatal repair before and after 6 months of age seem to be at least comparable. Considering additionally the financial, cultural and social aspects of clefts in resource-poor countries, performing early (≤6m) synchronous lip and palatal closure is justified