Transcriptional regulation of RAM1, a central regulator of arbuscule branching in arbuscular mycorrhiza symbiosis

Arbuscular mycorrhiza (AM) is an ancient symbiosis, established between 80% of land plants and obligate biotrophic fungi belonging to the class glomeromycotina. AM is an essential component in natural ecosystems, as it plays a major role in the global carbon cycle, enhances plant growth in nutrient deficient soil and is thus believed to sustain whole environ such as tropical rain forests. It also has a great fertilizing potential for sustainable practices in agriculture. Crucial for this symbiosis is the formation of highly branched tree-like structures called arbuscules by the fungus, inside root cortical cells of the host plant. These fungal structures deliver mineral nutrients after taking them up from the soil via extraradical hyphae, mainly phosphate and nitrogen, which are difficult to access for the plant. In turn, the fungus receives up to 20% of photosynthetically fixed carbon. Arbuscule formation is accompanied by massive transcriptional changes in the colonized cell. In addition, the cell undergoes subcellular rearrangements to accommodate the arbuscule. This is associated with the formation of a plant-derived membrane, called peri-arbuscular membrane, which surrounds the arbuscule and separates the fungal hyphae from the plant cytoplasm. The well-ordered and complex AM developmental steps, are regulated by the plant and depend on its nutritional status. Although arbuscule development is crucial for this symbiosis, the molecular basis of its development is poorly understood. A Lotus japonicus plant mutant reduced and degenerate arbuscules (red) found in a former study by forward genetics screen is perturbed in arbuscule development. To identify plant genes essential for arbuscule development, we investigated genes perturbed in red. Rough mapping indicated presence of two mutations in red, causative for the arbuscule phenotype. Complementation analysis confirmed causative mutations in a gene encoding a GRAS-type transcription factor named REDUCED ARBUSCULAR MYCORRHIZA 1 (RAM1) and in a gene encoding a lipid biosynthesis enzyme GLYCEROL 3-PHOSPHATE ACYL TRANSFERASE 6 (GPAT6/RAM2). In this doctoral thesis, I found that the AM symbiosis-specifically induced gene RAM1, is a principal regulator of arbuscule development. It is directly regulated by a complex of CYCLOPS and DELLA. CYCLOPS, is a DNA-binding transcription factor and a central regulator of symbiotic signaling and DELLA is a negative regulator of hormonal gibberellic acid (GA) signaling. The CYCLOPS-DELLA complex activates RAM1 expression via binding of CYCLOPS to a novel cis-element in the RAM1 promoter. Thus, we presented for the first time a target gene of CYCLOPS in AM symbiosis and a regulatory node integrating symbiosis (CYCLOPS) and hormonal GA signaling (DELLA). This direct connection may be important for the plant to connect symbiosis with its nutritional and therefore physiological status. Further, I revealed that RAM1 acts as a transcriptional activator of genes required for AM development, downstream of CCaMK and CYCLOPS. Ectopic expression of RAM1 induced AM-specific genes such as RAM2 in absence of AM-fungi. In frame of another thesis, they showed that RAM2 participate in an AM-specific lipid biosynthesis pathway and is essential for arbuscule development. RAM2 acts downstream of another lipid biosynthetic gene DIS (encoding ß-keto-acyl ACP synthase I), which is also indispensable for arbuscule development. RAM2 uses C16:0 fatty acids synthesized by DIS as substrates for synthesis of ß-monoacylglycerol. C16:0 is the predominant form of fatty acid found in AM fungi. Textbook knowledge exhibited carbohydrate as the only form of carbon supplied to the AM fungus, which is subsequently used to synthesis lipids. However, whole genome sequence analysis indicated that AM fungi lack genes encoding protein responsible for the de novo synthesis of C16:0 fatty acid. They further showed that the lipid containing C16:0 fatty acid synthesized by RAM2 is supplied to AM-fungi as a plant-derived carbon source. Arbuscule development can be conceptually divided into distinct steps by plant mutants, indicating that the respective gene product regulates the step-wise development of arbuscule. Accumulating evidences indicate transcriptional changes during arbuscule development occurs in successive but overlapping waves. For example, genes upregulated in the arbuscule containing cells might be also activated in neighboring cells preparing to accommodate arbuscule. These cells undergoing subcellular rearrangement forming a pre-penetration apparatus (PPA) do not have visible fungal structures. Transcriptomic analysis from cells containing only visible fungal structure, limit to relate the gene activation to individual stages of arbuscule development and PPA formation. To correlate the promoter activity of genes with the precise stages of arbuscule development, I designed a construct which allows visualization of the fungus in living roots due to accumulation of fluorescent protein mCherry in the apoplastic space surrounding the fungal hyphae. AM specific SbtM1 promoter used to drive mCherry is active across all stages of arbuscule development including cells undergoing rearrangement to form PPA. Using this construct, I showed that DIS and RAM2 promoters are activated during all the stages of arbuscule maturation, but become inactive during arbuscule degeneration

Transcriptional regulation of RAM1, a central regulator of arbuscule branching in arbuscular mycorrhiza symbiosis

Arbuscular mycorrhiza (AM) is an ancient symbiosis, established between 80% of land plants and obligate biotrophic fungi belonging to the class glomeromycotina. AM is an essential component in natural ecosystems, as it plays a major role in the global carbon cycle, enhances plant growth in nutrient deficient soil and is thus believed to sustain whole environ such as tropical rain forests. It also has a great fertilizing potential for sustainable practices in agriculture. Crucial for this symbiosis is the formation of highly branched tree-like structures called arbuscules by the fungus, inside root cortical cells of the host plant. These fungal structures deliver mineral nutrients after taking them up from the soil via extraradical hyphae, mainly phosphate and nitrogen, which are difficult to access for the plant. In turn, the fungus receives up to 20% of photosynthetically fixed carbon. Arbuscule formation is accompanied by massive transcriptional changes in the colonized cell. In addition, the cell undergoes subcellular rearrangements to accommodate the arbuscule. This is associated with the formation of a plant-derived membrane, called peri-arbuscular membrane, which surrounds the arbuscule and separates the fungal hyphae from the plant cytoplasm. The well-ordered and complex AM developmental steps, are regulated by the plant and depend on its nutritional status. Although arbuscule development is crucial for this symbiosis, the molecular basis of its development is poorly understood. A Lotus japonicus plant mutant reduced and degenerate arbuscules (red) found in a former study by forward genetics screen is perturbed in arbuscule development. To identify plant genes essential for arbuscule development, we investigated genes perturbed in red. Rough mapping indicated presence of two mutations in red, causative for the arbuscule phenotype. Complementation analysis confirmed causative mutations in a gene encoding a GRAS-type transcription factor named REDUCED ARBUSCULAR MYCORRHIZA 1 (RAM1) and in a gene encoding a lipid biosynthesis enzyme GLYCEROL 3-PHOSPHATE ACYL TRANSFERASE 6 (GPAT6/RAM2). In this doctoral thesis, I found that the AM symbiosis-specifically induced gene RAM1, is a principal regulator of arbuscule development. It is directly regulated by a complex of CYCLOPS and DELLA. CYCLOPS, is a DNA-binding transcription factor and a central regulator of symbiotic signaling and DELLA is a negative regulator of hormonal gibberellic acid (GA) signaling. The CYCLOPS-DELLA complex activates RAM1 expression via binding of CYCLOPS to a novel cis-element in the RAM1 promoter. Thus, we presented for the first time a target gene of CYCLOPS in AM symbiosis and a regulatory node integrating symbiosis (CYCLOPS) and hormonal GA signaling (DELLA). This direct connection may be important for the plant to connect symbiosis with its nutritional and therefore physiological status. Further, I revealed that RAM1 acts as a transcriptional activator of genes required for AM development, downstream of CCaMK and CYCLOPS. Ectopic expression of RAM1 induced AM-specific genes such as RAM2 in absence of AM-fungi. In frame of another thesis, they showed that RAM2 participate in an AM-specific lipid biosynthesis pathway and is essential for arbuscule development. RAM2 acts downstream of another lipid biosynthetic gene DIS (encoding ß-keto-acyl ACP synthase I), which is also indispensable for arbuscule development. RAM2 uses C16:0 fatty acids synthesized by DIS as substrates for synthesis of ß-monoacylglycerol. C16:0 is the predominant form of fatty acid found in AM fungi. Textbook knowledge exhibited carbohydrate as the only form of carbon supplied to the AM fungus, which is subsequently used to synthesis lipids. However, whole genome sequence analysis indicated that AM fungi lack genes encoding protein responsible for the de novo synthesis of C16:0 fatty acid. They further showed that the lipid containing C16:0 fatty acid synthesized by RAM2 is supplied to AM-fungi as a plant-derived carbon source. Arbuscule development can be conceptually divided into distinct steps by plant mutants, indicating that the respective gene product regulates the step-wise development of arbuscule. Accumulating evidences indicate transcriptional changes during arbuscule development occurs in successive but overlapping waves. For example, genes upregulated in the arbuscule containing cells might be also activated in neighboring cells preparing to accommodate arbuscule. These cells undergoing subcellular rearrangement forming a pre-penetration apparatus (PPA) do not have visible fungal structures. Transcriptomic analysis from cells containing only visible fungal structure, limit to relate the gene activation to individual stages of arbuscule development and PPA formation. To correlate the promoter activity of genes with the precise stages of arbuscule development, I designed a construct which allows visualization of the fungus in living roots due to accumulation of fluorescent protein mCherry in the apoplastic space surrounding the fungal hyphae. AM specific SbtM1 promoter used to drive mCherry is active across all stages of arbuscule development including cells undergoing rearrangement to form PPA. Using this construct, I showed that DIS and RAM2 promoters are activated during all the stages of arbuscule maturation, but become inactive during arbuscule degeneration

Using the Person-Based Approach to Develop a Digital Intervention Targeting Diet and Physical Activity in Pregnancy: Development Study

Background: In pregnancy, eating well, keeping active, and avoiding excessive weight gain are associated with better maternal and fetal health outcomes. Dietary and physical activity (PA) interventions can be effective in changing behaviors and managing weight gain. The comparatively lower cost and greater accessibility of digital interventions make them an attractive alternative to in-person interventions. Baby Buddy is a free pregnancy and parenting app from the charity Best Beginnings. Designed to support parents, improve health outcomes, and reduce inequalities, the app is actively used within the UK National Health Service. It offers an ideal platform for delivering and evaluating a new prenatal dietary and PA intervention./ Objective: The aim of this study was to create a theory-based intervention within Baby Buddy to empower, encourage, and support expectant parents to develop healthier dietary and PA habits for pregnancy and parenthood. Methods: The intervention’s development process was guided by the Behavior Change Wheel, with the person-based approach used to create and test its design. Three stages of qualitative research with pregnant and recently pregnant parents guided the intervention design. Study 1 (n=30), comprising 4 web-based focus groups and 12 telephone interviews, gauged response to the rudimentary concept and generated ideas for its development. Results were analyzed thematically. At this stage, the guiding principles for the intervention development were established, and regular team meetings ensured that the intervention design remained aligned with Best Beginnings’ objectives, evidence-based approach, and feasibility criteria. Study 2 (n=29), comprising web-based individual and couple interviews, explored design ideas using wireframes and scripts and generated iterative feedback on the intervention content, branding, and tone. A table of changes analysis tracked design amendments. Study 3 (n=19) tested an app prototype using think-aloud interviews with current Baby Buddy users. A patient and public involvement and engagement activity (n=18) and other expert contributors (n=14) provided ad hoc input into the research process and design development./ Results: Study 1 confirmed the appeal and relevance of the intervention concept and its novel approach of including partners. The identified themes underpinned the development of the intervention design. Iterative feedback from study 2, in conjunction with patient and public involvement and engagement and expert contributor input, helped refine the intervention design and ensure its relevance and appeal to a diverse target user group. Study 3 highlighted functionality, content, and design issues with the app prototype and identified ways of improving the user experience./ Conclusions: This study illustrates the value of combining a theoretical method for intervention development with the person-based approach to create a theory-based intervention that is also user-friendly, appealing, and engaging for its target audience. Further research is needed to evaluate the effectiveness of the intervention in improving diet, PA, and weight management in pregnancy

Independent signalling cues underpin arbuscular mycorrhizal symbiosis and large lateral root induction in rice.

Perception of arbuscular mycorrhizal fungi (AMF) triggers distinct plant signalling responses for parallel establishment of symbiosis and induction of lateral root formation. Rice receptor kinase CHITIN ELICITOR RECEPTOR KINASE 1 (CERK1) and α/β-fold hydrolase DWARF14-LIKE (D14L) are involved in pre-symbiotic fungal perception. After 6 wk post-inoculation with Rhizophagus irregularis, root developmental responses, fungal colonization and transcriptional responses were monitored in two independent cerk1 null mutants; a deletion mutant lacking D14L, and with D14L complemented as well as their respective wild-type cultivars (cv Nipponbare and Nihonmasari). Here we show that although essential for symbiosis, D14L is dispensable for AMF-induced root architectural modulation, which conversely relies on CERK1. Our results demonstrate uncoupling of symbiosis and the symbiotic root developmental signalling during pre-symbiosis with CERK1 required for AMF-induced root architectural changes.Leverhulme Early Career Fellowship. Grant Number: ECF‐2016‐39

Bio-nanotechnology application in wastewater treatment

The nanoparticles have received high interest in the field of medicine and water purification, however, the nanomaterials produced by chemical and physical methods are considered hazardous, expensive, and leave behind harmful substances to the environment. This chapter aimed to focus on green-synthesized nanoparticles and their medical applications. Moreover, the chapter highlighted the applicability of the metallic nanoparticles (MNPs) in the inactivation of microbial cells due to their high surface and small particle size. Modifying nanomaterials produced by green-methods is safe, inexpensive, and easy. Therefore, the control and modification of nanoparticles and their properties were also discussed

Lipid transfer from plants to arbuscular mycorrhiza fungi

Arbuscular mycorrhiza (AM) symbioses contribute to global carbon cycles as plant hosts divert up to 20% of photosynthate to the obligate biotrophic fungi. Previous studies suggested carbohydrates as the only form of carbon transferred to the fungi. However, de novo fatty acid (FA) synthesis has not been observed in AM fungi in absence of the plant. In a forward genetic approach, we identified two Lotus japonicus mutants defective in AM-specific paralogs of lipid biosynthesis genes (KASI and GPAT6). These mutants perturb fungal development and accumulation of emblematic fungal 16:1 omega 5 FAs. Using isotopolog profiling we demonstrate that C-13 patterns of fungal FAs recapitulate those of wild-type hosts, indicating cross-kingdom lipid transfer from plants to fungi. This transfer of labelled FAs was not observed for the AM-specific lipid biosynthesis mutants. Thus, growth and development of beneficial AM fungi is not only fueled by sugars but depends on lipid transfer from plant hosts

Evolutionary Analysis of DELLA-Associated Transcriptional Networks

[EN] DELLA proteins are transcriptional regulators present in all land plants which have been shown to modulate the activity of over 100 transcription factors in Arabidopsis, involved in multiple physiological and developmental processes. It has been proposed that DELLAs transduce environmental information to pre-wired transcriptional circuits because their stability is regulated by gibberellins (GAs), whose homeostasis largely depends on environmental signals. The ability of GAs to promote DELLA degradation coincides with the origin of vascular plants, but the presence of DELLAs in other land plants poses at least two questions: what regulatory properties have DELLAs provided to the behavior of transcriptional networks in land plants, and how has the recruitment of DELLAs by GA signaling affected this regulation. To address these issues, we have constructed gene co-expression networks of four different organisms within the green lineage with different properties regarding DELLAs: Arabidopsis thaliana and Solanum lycopersicum (both with GA- regulated DELLA proteins), Physcomitrella patens (with GA- independent DELLA proteins) and Chlamydomonas reinhardtii (a green alga without DELLA), and we have examined the relative evolution of the subnetworks containing the potential DELLA-dependent transcriptomes. Network analysis indicates a relative increase in parameters associated with the degree of interconnectivity in the DELLA-associated subnetworks of land plants, with a stronger effect in species with GA- regulated DELLA proteins. These results suggest that DELLAs may have played a role in the coordination of multiple transcriptional programs along evolution, and the function of DELLAs as regulatory 'hubs' became further consolidated after their recruitment by GA signaling in higher plants.Work in the laboratories was funded by grants BFU2016-80621-P and BIO2014-52425-P of the Spanish Ministry of Economy, Industry and Competitiveness, and H2020-MSCA-RISE-2014-644435 of the European Union. AB-M and JH-G hold Fellowships of the Spanish Ministry of Education, Culture and Sport FPU14/01941 and FPU15/01756, respectively.Briones-Moreno, A.; Hernández-García, J.; Vargas-Chávez, C.; Romero-Campero FJ; Romero, J.; Valverde, F.; Blazquez Rodriguez, MA. (2017). Evolutionary Analysis of DELLA-Associated Transcriptional Networks. Frontiers in Plant Science. 8(626):1-11. https://doi.org/10.3389/fpls.2017.00626S111862