Over-expression of SINAL7 increases biomass and drought tolerance, and also delays senescence in Arabidopsis.

The seven in absentia like 7 gene (At5g37890, SINAL7) from Arabidopsis thaliana encodes a RING finger protein belonging to the SINA superfamily that possesses E3 ubiquitin-ligase activity. SINAL7 has the ability to self-ubiquitinate and to mono-ubiquitinate glyceraldehyde-3-P dehydrogenase 1 (GAPC1), suggesting a role for both proteins in a hypothetical signaling pathway in Arabidopsis. In this study, the in vivo effects of SINAL7 on plant physiology were examined by over-expressing SINAL7 in transgenic Arabidopsis plants. Phenotypic and gene expression analyses suggest the involvement of SINAL7 in the regulation of several vegetative parameters, essentially those that affect the aerial parts of the plants. Over-expression of SINAL7 resulted in an increase in the concentrations of hexoses and sucrose, with a concommitant increase in plant biomass, particularly in the number of rosette leaves and stem thickness. Interestingly, using the CAB1 (chlorophyll ab binding protein 1) gene as a marker revealed a delay in the onset of senescence. Transgenic plants also displayed a remarkable level of drought resistance, indicating the complexity of the response to SINAL7 over-expression.

Applications of Bioinformatics to Plant Biotechnology.

Bioinformatics encompasses many tools and techniques that today are essential for all areas of research in the biological sciences. New databases with a wealth of information about genomes, proteins, metabolites, and metabolic pathways appear almost daily. Particularly, for scientists who carry out research in plant biology, the amount of information has multiplied exponentially due to the large number of databases available for many individual plant species. In this sense, bioinformatics together with next generation sequencing and 'omics' approaches, can provide tools for plant breeding and the genetic engineering of plants. In addition, these technologies enable a better understanding of the processes and mechanisms that can lead to plants with increased tolerance to different abiotic stress conditions and resistance to pathogen attack, as well as the development of crop varieties with improved nutritional quality of seeds and fruits.

The targeting of starch binding domains from starch synthase III to the cell wall alters cell wall composition and properties.

KEY MESSAGE: Starch binding domains of starch synthase III from Arabidopsis thaliana (SBD123) binds preferentially to cell wall polysaccharides rather than to starch in vitro. Transgenic plants overexpressing SBD123 in the cell wall are larger than wild type. Cell wall components are altered in transgenic plants. Transgenic plants are more susceptible to digestion than wild type and present higher released glucose content. Our results suggest that the transgenic plants have an advantage for the production of bioethanol in terms of saccharification of essential substrates. The plant cell wall, which represents a major source of biomass for biofuel production, is composed of cellulose, hemicelluloses, pectins and lignin. A potential biotechnological target for improving the production of biofuels is the modification of plant cell walls. This modification is achieved via several strategies, including, among others, altering biosynthetic pathways and modifying the associations and structures of various cell wall components. In this study, we modified the cell wall of A. thaliana by targeting the starch-binding domains of A. thaliana starch synthase III to this structure. The resulting transgenic plants (E8-SDB123) showed an increased biomass, higher levels of both fermentable sugars and hydrolyzed cellulose and altered cell wall properties such as higher laxity and degradability, which are valuable characteristics for the second-generation biofuels industry. The increased biomass and degradability phenotype of E8-SBD123 plants could be explained by the putative cell-wall loosening effect of the in tandem starch binding domains. Based on these results, our approach represents a promising biotechnological tool for reducing of biomass recalcitrance and therefore, the need for pretreatments.

The E3 ubiquitin-ligase SEVEN IN ABSENTIA like 7 mono-ubiquitinates glyceraldehyde-3-phosphate dehydrogenase 1 isoform in vitro and is required for its nuclear localization in Arabidopsis thaliana.

The E3 ubiquitin-protein ligases are associated to various processes such as cell cycle control and diverse developmental pathways. Arabidopsis thaliana SEVEN IN ABSENTIA like 7, which has ubiquitin ligase activity, is located in the nucleus and cytosol and is expressed at several stages in almost all plant tissues suggesting an important role in plant functions. However, the mechanism underlying the regulation of this protein is unknown. Since we found that the SEVEN IN ABSENTIA like 7 gene expression is altered in plants with impaired mitochondria, and in plants deficient in the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase 1, we decided to study the possible interactions between both proteins as potential partners in plant signaling functions. We found that SEVEN IN ABSENTIA like 7 is able to interact in vitro with glyceraldehyde-3-phosphate dehydrogenase and that the Lys231 residue of the last is essential for this function. Following the interaction, a concomitant increase in the glyceraldehyde-3-phosphate dehydrogenase catalytic activity was observed. However, when SEVEN IN ABSENTIA like 7 was supplemented with E1 and E2 proteins to form a complete E1-E2-E3 modifier complex, we observed the mono-ubiquitination of glyceraldehyde-3-phosphate dehydrogenase 1 at the Lys76 residue and a dramatic decrease of its catalytic activity. Moreover, we found that localization of glyceraldehyde-3-phosphate dehydrogenase 1 in the nucleus is dependent on the expression SEVEN IN ABSENTIA like 7. These observations suggest that the association of both proteins might result in different biological consequences in plants either through affecting the glycolytic flux or via cytoplasm-nucleus relocation.

Frataxin Is Localized to Both the Chloroplast and Mitochondrion and Is Involved in Chloroplast Fe-S Protein Function in Arabidopsis.

Frataxin plays a key role in eukaryotic cellular iron metabolism, particularly in mitochondrial heme and iron-sulfur (Fe-S) cluster biosynthesis. However, its precise role has yet to be elucidated. In this work, we studied the subcellular localization of Arabidopsis frataxin, AtFH, using confocal microscopy, and found a novel dual localization for this protein. We demonstrate that plant frataxin is targeted to both the mitochondria and the chloroplast, where it may play a role in Fe-S cluster metabolism as suggested by functional studies on nitrite reductase (NIR) and ferredoxin (Fd), two Fe-S containing chloroplast proteins, in AtFH deficient plants. Our results indicate that frataxin deficiency alters the normal functioning of chloroplasts by affecting the levels of Fe, chlorophyll, and the photosynthetic electron transport chain in this organelle.

An optimized InCell Western screening technique identifies hexachlorophene as a novel potent TDP43 targeting drug.

TAR DNA binding protein (TDP43) is a DNA- and RNA-binding protein that is implicated in several neurodegenerative disorders termed as "TDP43 proteinopathies" including Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS) and fronto-temporal lobe dementia (FTLD). We have developed an InCell Western (ICW) technique for screening TDP targeting drugs in 96 well plates. We tested 281 compounds and identified a novel compound hexachlorophene (referred to as B10) that showed potent reduction in TDP43 levels. The effect of B10 on TDP protein level was validated in two different cellular models: endogenous TDP43 expressing N9 microglial cells and TDP43-over-expressing HEK293 and HeLa cells. We also analyzed effect of B10 on various pathological forms of TDP such as the C25 cleaved fragment that localizes to the cytosol, insoluble high molecular weight species, and ALS-linked mutants. Our data suggest that B10 effectively reduces all forms of TDP. Overall, our data suggest that B10 could serve as a potential drug molecule for the treatment of AD, ALS and other TDP43 proteinopathies.

Characterization of the Arabidopsis thaliana E3 ubiquitin-ligase AtSINAL7 and identification of the ubiquitination sites.

Protein ubiquitination leading to degradation by the proteasome is an important mechanism in regulating key cellular functions. Protein ubiquitination is carried out by a three step process involving ubiquitin (Ub) activation by a E1 enzyme, the transfer of Ub to a protein E2, finally an ubiquitin ligase E3 catalyzes the transfer of the Ub peptide to an acceptor protein. The E3 component is responsible for the specific recognition of the target, making the unveiling of E3 components essential to understand the mechanisms regulating fundamental cell processes through the protein degradation pathways. The Arabidopsis thaliana seven in absentia-like 7 (AtSINAL7) gene encodes for a protein with characteristics from a C3HC4-type E3 ubiquitin ligase. We demonstrate here that AtSINAL7 protein is indeed an E3 protein ligase based on the self-ubiquitination in vitro assay. This activity is dependent of the presence of a Lys residue in position 124. We also found that higher AtSINAL7 transcript levels are present in tissues undergoing active cell division during floral development. An interesting observation is the circadian expression pattern of AtSINAL7 mRNA in floral buds. Furthermore, UV-B irradiation induces the expression of this transcript indicating that AtSINAL7 may be involved in a wide range of different cell processes.