Search

Duckweeds are amongst the fastest growing of higher plants, making them attractive high-biomass targets for biofuel feedstock production. Their fronds have high rates of fatty acid synthesis to meet the demand for new membranes, but triacylglycerols (TAG) only accumulate to very low levels. Here we report on the engineering of Lemna japonica for the synthesis and accumulation of TAG in its fronds. This was achieved by expression of an estradiol-inducible cyan fluorescent protein-Arabidopsis WRINKLED1 fusion protein (CFP-AtWRI1), strong constitutive expression of a mouse diacylglycerol:acyl-CoA acyltransferase2 (MmDGAT), and a sesame oleosin variant (SiOLE(*)). Individual expression of each gene increased TAG accumulation by 1- to 7-fold relative to controls, while expression of pairs of these genes increased TAG by 7- to 45-fold. In uninduced transgenics containing all three genes, TAG accumulation increased by 45-fold to 3.6% of dry weight (DW) without severely impacting growth, and by 108-fold to 8.7% of DW after incubation on medium containing 100 μm estradiol for 4 days. TAG accumulation was accompanied by an increase in total fatty acids of up to three-fold to approximately 15% of DW. Lipid droplets from fronds of all transgenic lines were visible by confocal microscopy of BODIPY-stained fronds. At a conservative 12 tonnes (dry matter) per acre and 10% (DW) TAG, duckweed could produce 350 gallons of oil/acre/year, approximately seven-fold the yield of soybean, and similar to that of oil palm. These findings provide the foundation for optimizing TAG accumulation in duckweed and present a new opportunity for producing biofuels and lipidic bioproducts.

The rates and patterns of somatic mutations in wild plants, as well as how they relate to longevity, are largely unknown1–3. Here, we examined the somatic mutation landscapes of slow- and fast-growing tropical species in central Borneo, Indonesia. Using newly-constructed genomes, we identified an average of 480 mutations in the slow-growing species (265-year-old, 44.1 m in height), which was five times greater than that observed in the fast-growing species (66-year-old, 43.9 m). The number of somatic mutations increased linearly with branch length. The somatic mutation rate per meter was higher in the slow-growing species, yet the rate per year remained constant across both species. The mutational spectra exhibited a dominance of spontaneous mutations, specifically cytosine-to-thymine substitutions at CpG sites. An analysis of nucleotide substitutions at both the intra- and inter-individual level revealed that somatic mutations are neutral within an individual, but those mutations transmitted to the next generation are subject to purifying selection. We developed a model to evaluate the relative contribution of cell division on mutational processes, and postulate that cell-division independent mutagenesis predominates. These findings deepen our understanding of mutational processes underlying the generation of genetic diversity in a tropical ecosystem.

Understanding how plants adapt to ambient temperatures has become a major challenge prompted by global climate change. This has led to the identification of several genes regulating the thermal plasticity of plant growth and flowering time. However, the mechanisms accounting for the natural variation and evolution of such developmental plasticity remain mostly unknown. In this study, we determined that natural variation at ICARUS2 (ICA2), which interacts genetically with its homolog ICA1, alters growth and flowering time plasticity in relation to temperature in Arabidopsis (Arabidopsis thaliana). Transgenic analyses demonstrated multiple functional effects for ICA2 and supported the notion that structural polymorphisms in ICA2 likely underlie its natural variation. Two major ICA2 haplogroups carrying distinct functionally active alleles showed high frequency, strong geographic structure, and significant associations with climatic variables related to annual and daily fluctuations in temperature. Genome analyses across the plant phylogeny indicated that the prevalent plant ICA genes encoding two tRNAHis guanylyl transferase 1 units evolved ∼120 million years ago during the early divergence of mono- and dicotyledonous clades. In addition, ICA1/ICA2 duplication occurred specifically in the Camelineae tribe (Brassicaceae). Thus, ICA2 appears to be ubiquitous across plant evolution and likely contributes to climate adaptation through modifications of thermal developmental plasticity in Arabidopsis., This work has been funded by the Agencia Estatal de Investigación of Spain and the Fondo Europeo de Desarrollo Regional (Unión Europea) (grant BIO2016-75754-P to C.A.-B.) and by the Australian Research Council (Discovery grant DP0983875 and ARC-Future Fellowship FT100100377 to S.B.).

Understanding how plants adapt to ambient temperatures has become a major challenge prompted by global climate change. This has led to the identification of several genes regulating the thermal plasticity of plant growth and flowering time. However, the mechanisms accounting for the natural variation and evolution of such developmental plasticity remain mostly unknown. In this study, we determined that natural variation at

Lack of genetic tools in the Lemnaceae (duckweed) has impeded full implementation of this organism as model for biological research, despite its rapid doubling time, simple architecture and unusual metabolic characteristics. Here we present technologies to facilitate high-throughput genetic studies in duckweed. We developed a fast and efficient method for producing Lemna minor stable transgenic fronds via agrobacterium-mediated transformation and regeneration from tissue culture. Additionally, we engineered an artificial microRNA (amiRNA) gene silencing system. We identified a Lemna gibba endogenous miR166 precursor and used it as a backbone to produce amiRNAs. As a proof of concept we induced the silencing of CH42, a Magnesium Chelatase subunit, using our amiRNA platform. Expression of CH42 in transgenic Lemna minor fronds was significantly reduced, which resulted in reduction of chlorophyll pigmentation. The techniques presented here will enable tackling future challenges in the biology and biotechnology of Lemnaceae.

Summary In Arabidopsis thaliana, mutations in genes encoding ribosomal proteins (r-proteins) perturb various developmental processes. Whether these perturbations are caused by overall ribosome insufficiency or partial dysfunction of the ribosome caused by deficiency of a particular ribosomal protein is not known. To distinguish these possibilities, a comparative study using several r-protein mutants was required. Here, we identified mutations in 11 r-protein genes from previously isolated denticulata and pointed-leaves mutants. Most of these mutations were associated with pointed leaves, with reduced growth due to a decrease in the number or size of palisade mesophyll and pavement cells. In addition, leaf abaxialization was usually observed when these r-protein mutations were combined with asymmetric leaves1 (as1) and as2 mutations. These results suggest that the establishment of leaf polarity is highly sensitive to ribosome functionality in general. However, several r-protein mutants showed a preference towards a specific developmental defect. For example, rpl4d mutations did not affect cell proliferation but caused strong abaxialization of leaves in the as1 and as2 backgrounds. On the other hand, rps28b enhanced leaf abaxialization of as2 to a weaker extent than expected on the basis of its negative effect on cell proliferation. In addition, hypomorphic rps6a alleles had the strongest effects on most of the phenotypes examined. These findings suggest that deficiencies in these three r-protein genes lead to production of dysfunctional ribosomes. Depending on their structural abnormalities, dysfunctional ribosomes may affect translation of specific transcripts involved in the regulation of some leaf developmental processes.

*† ‡ § SUMMARY Arabidopsis thaliana reticulate mutants exhibit differential pigmentation of the veinal and interveinal leaf regions, a visible phenotype that often indicates impaired mesophyll development. We performed a metabolomic analysis of one ven6 (venosa6) and three ven3 reticulate mutants that revealed altered levels of arginine precursors, namely increased ornithine and reduced citrulline levels. In addition, the mutants were more sensitive than the wild-type to exogenous ornithine, and leaf reticulation and mesophyll defects of these mutants were completely rescued by exogenous citrulline. Taken together, these results indicate that ven3 and ven6 mutants experience a blockage of the conversion of ornithine into citrulline in the arginine pathway. Consistent with the participation of VEN3 and VEN6 in the same pathway, the morphological phenotype of ven3 ven6 double mutants was synergistic. Map-based cloning showed that the VEN3 and VEN6 genes encode subunits of Arabidopsis carbamoyl phosphate synthetase (CPS), which is assumed to be required for the conversion of ornithine into citrulline in arginine biosynthesis. Heterologous expression of the Arabidopsis VEN3 and VEN6 genes in a CPS-deficient Escherichia coli strain fully restored bacterial growth in minimal medium, demonstrating the enzymatic activity of the VEN3 and VEN6 proteins, and indicating a conserved role for CPS in these distinct and distant species. Detailed study of the reticulate leaf phenotype in the ven3 and ven6 mutants revealed that mesophyll development is highly sensitive to impaired arginine biosynthesis.

Arabidopsis thaliana reticulate mutants exhibit differential pigmentation of the veinal and interveinal leaf regions, a visible phenotype that often indicates impaired mesophyll development. We performed a metabolomic analysis of one ven6 (venosa6) and three ven3 reticulate mutants that revealed altered levels of arginine precursors, namely increased ornithine and reduced citrulline levels. In addition, the mutants were more sensitive than the wild-type to exogenous ornithine, and leaf reticulation and mesophyll defects of these mutants were completely rescued by exogenous citrulline. Taken together, these results indicate that ven3 and ven6 mutants experience a blockage of the conversion of ornithine into citrulline in the arginine pathway. Consistent with the participation of VEN3 and VEN6 in the same pathway, the morphological phenotype of ven3 ven6 double mutants was synergistic. Map-based cloning showed that the VEN3 and VEN6 genes encode subunits of Arabidopsis carbamoyl phosphate synthetase (CPS), which is assumed to be required for the conversion of ornithine into citrulline in arginine biosynthesis. Heterologous expression of the Arabidopsis VEN3 and VEN6 genes in a CPS-deficient Escherichia coli strain fully restored bacterial growth in minimal medium, demonstrating the enzymatic activity of the VEN3 and VEN6 proteins, and indicating a conserved role for CPS in these distinct and distant species. Detailed study of the reticulate leaf phenotype in the ven3 and ven6 mutants revealed that mesophyll development is highly sensitive to impaired arginine biosynthesis.

In Arabidopsis thaliana, mutations in genes encoding ribosomal proteins (r-proteins) perturb various developmental processes. Whether these perturbations are caused by overall ribosome insufficiency or partial dysfunction of the ribosome caused by deficiency of a particular ribosomal protein is not known. To distinguish these possibilities, a comparative study using several r-protein mutants was required. Here, we identified mutations in 11 r-protein genes from previously isolated denticulata and pointed-leaves mutants. Most of these mutations were associated with pointed leaves, with reduced growth due to a decrease in the number or size of palisade mesophyll and pavement cells. In addition, leaf abaxialization was usually observed when these r-protein mutations were combined with asymmetric leaves1 (as1) and as2 mutations. These results suggest that the establishment of leaf polarity is highly sensitive to ribosome functionality in general. However, several r-protein mutants showed a preference towards a specific developmental defect. For example, rpl4d mutations did not affect cell proliferation but caused strong abaxialization of leaves in the as1 and as2 backgrounds. On the other hand, rps28b enhanced leaf abaxialization of as2 to a weaker extent than expected on the basis of its negative effect on cell proliferation. In addition, hypomorphic rps6a alleles had the strongest effects on most of the phenotypes examined. These findings suggest that deficiencies in these three r-protein genes lead to production of dysfunctional ribosomes. Depending on their structural abnormalities, dysfunctional ribosomes may affect translation of specific transcripts involved in the regulation of some leaf developmental processes.

Este trabajo se ha realizado básicamente para cumplir dos objetivos principalmente: A) La realización del Proyecto Final de Carrera que permitirá la obtención del título de Licenciado en Ciencias Ambientales, y al mismo tiempo proporcionará una cierta familiarización con el mundo de los Sistemas de gestión medioambientales. B) La Implantación de un SGMA, basándose en la norma UNE-EN-ISO- 14001, y cuyos objetivos más concretos son: 1. Revisión del estado actual de la empresa escogida, atendiendo a la legislación que aplican. 2. Actuaciones necesarias para la correcta implantación del sistema según la norma. 3. Obtención de un modelo de comportamiento de los trabajadores que facilite una buena gestión ambiental. 4. Identificación del proceso necesario para obtener la certificación de SGMA.

The C-LytA protein constitutes the choline-binding module of the LytA amidase from Streptococcus pneumoniae. Owing to its affinity for choline and analogs, it is regularly used as an affinity tag for the purification of proteins in a single chromatographic step. In an attempt to build a robust variant against thermal denaturation, we have engineered several salt bridges on the protein surface. All the stabilizing mutations were pooled in a single variant, C-LytAm7, which contained seven changes: Y25K, F27K, M33E, N51K, S52K, T85K and T108K. The mutant displays a 7 degrees C thermal stabilization compared with the wild-type form, together with a complete reversibility upon heating and a higher kinetic stability. Moreover, the accumulation of intermediates in the unfolding of C-LytA is virtually abolished for C-LytAm7. The differences in stability become more evident when the proteins are bound to a DEAE-cellulose affinity column, as most of wild-type C-LytA is denatured at approximately 65 degrees C, whereas C-LytAm7 may stand temperatures up to 90 degrees C. Finally, the change in the isoelectric point of C-LytAm7 enhances its solubility at acidic pHs. Therefore, C-LytAm7 behaves as an improved affinity tag and supports the engineering of surface salt bridges as an effective approach for protein stabilization.