Characterization of the role of glutamyl-tRNA synthetase in the protein subcellular localization

Nos organismos eucariotos, aproximadamente 50% das proteínas traduzidas no citoplasma são transportadas para as organelas, onde irão desempenhar suas funções. Com isso, surgiu um intricado sistema de transporte intracelular de proteínas. Nas plantas, a presença de uma segunda organela endossimbionte, o plastídio, tornou este sistema mais complexo e gerou demanda adicional por transporte. Ainda, grande maioria das proteínas mitocondriais e plastidiais são codificadas por genes nucleares e importadas do citosol. O dogma uma proteína-uma localização foi associado ao conceito de um gene-uma proteína na biologia celular. Entretanto, proteínas individuais podem ter mais de uma função, e mais recentemente, proteínas codificadas por um único gene foram identificadas em mais de um compartimento subcelular, o que deu origem ao conceito de duplo direcionamento (DD). Um exemplo bem estudado de DD vem das proteínas da família das aminoacil-tRNA sintetases (aaRS), que participam da síntese protéica ao acoplar o aminoácido ao seu tRNA cognato. Dentre as aaRSs, a glutamil-tRNA sintetase citosólica (GluRS), através de sua extensão N-terminal, parece estar envolvida com outras funções além da tradução. Em Arabidopsis thaliana, há dois genes nucleares que codificam a GluRS, um para uma proteína de duplo direcionamento (DD) e outro para uma proteína citosólica. Resultados recentes em nosso laboratório mostraram que a GluRS citosólica pode estar relacionada ao controle da localização subcelular de proteínas organelares em Arabidopsis. Para verificar um eventual papel desta proteína na localização subcelular de outras proteínas, foram realizados ensaios de duplo-híbrido em levedura, os quais mostraram interação entre a GluRS e a glutamina sintetase (GS) de Arabidopsis thaliana, proteína de DD para mitocôndrias e cloroplastos Esta interação foi confirmada in planta, sendo a sequência da GluRS responsável pela interação localizada na região N-terminal, do resíduo 207 ao 316. Análises filogenéticas apontam que esta região encontra-se ausente nas bactérias e que originou-se provavelmente em Archea, entre 2,6 e 1,8 bilhões de anos. Além disso, observa-se que esta sequência é conservada em fungos, musgos e plantas vaculares, tendo originado-se em Arabidopsis há cerca de 2 bilhões de anos.In eukaryotic organisms, about 50% of cytoplasmic translated proteins are transported to the organelles, where they can play their roles. Thus, a complex system for intracellular transport was established. In plants, the presence of a second endosymbiont organelle, the plastid, turned this system still more intricated and required an additional transport mechanism. Besides, most of organellar proteins are coded by nuclear genes and imported from the cytosol. The one protein-one localization was associated to the idea of one gene-one protein, which has long been established in molecular biology. However, individual proteins can show more than one function, and recently, proteins coded by one single gene were identified in more than one subcellular compartment, which has originated the concept of dual targeting. One of the most studied example of dual targeted proteins is the aminoacyl-tRNA synthetase (aaRS) family, which are related to protein synthesis by attaching the correct amino acid onto the cognate tRNA molecule. Among the aaRSs, cytosolic glutamyl-tRNA synthetase (GluRS), through its N-terminal extension, seems to be involved in other cellular role beyond translation. In Arabidopsis thaliana, there are two genes encoding GluRS, one for a dual-targeted protein and other for a cytosolic protein. Recent results in our laboratory showed that GluRS interacts with proteins destinated to other organelles, which suggest that this protein might have a role in interfering on protein localization in Arabidopsis. In order to gain some information on the role of this protein in subcellular localization, yeast two-hybrid assays were performed. These studies showed the interaction between GluRS and glutamine synthetase (GS), a mitochondrial and chloroplastic dual-targeted protein. This interaction was confirmed in planta. In addition, the GluRS sequence associated to protein interaction was localized at its N-terminal portion, between the residues 207 316. Phylogenetic analysis revealed that this region is absent in bacteria and it probably arose from Archea between 2.6 and 1.8 billion years ago. Also, this sequence is conserved in fungi, moss and all the green plants investigated. Finally, datation analysis showed that this sequence arose in Arabidopsis between 2 and 1.7 billion years ago

Characterization of the circadian clock and its impact on sugarcane metabolism

O relógio biológico é um mecanismo molecular autossustentado gerador de ritmos. Ele integra vias de percepção das condições ambientais com um oscilador central para gerar respostas fisiológicas rítmicas em escalas diária e sazonal. Nas plantas, o relógio biológico está associado a vias metabólicas e fisiológicas importantes, como fotossíntese. Na cana-de-açúcar, uma gramínea de grande interesse econômico, estudos realizados em condições circadianas mostraram que o relógio biológico tem uma influência superior àquela vista em outras plantas. Assim, este trabalho visa a compreender os mecanismos de funcionamento do oscilador central do relógio biológico da cana-de-açúcar crescida em campo. Para tanto, foram investigados o transcriptoma de diferentes órgãos da cana-de-açúcar; a expressão de isoformas alternativas e de múltiplos alelos dos genes do relógio biológico da cana; e o efeito do sombreamento mútuo das plantas em campo sobre o funcionamento do relógio biológico. Os resultados obtidos sugerem que o relógio biológico é funcional e sincronizado entre os diferentes órgãos da cana-de-açúcar analisados. Os transcritos regulados sinergicamente pelo relógio biológico e pelo ambiente flutuante pertencem a vias metabólicas, fisiológicas e de regulação gênica e epigenéticas todas essenciais à produtividade da cana-de-açúcar. O sombreamento mútuo observado em campo parece alterar a fase de expressão de genes do relógio biológico da cana-de-açúcar. Além disso, eventos de splicing alternativo foram observados nos genes do relógio biológico em condições de baixa temperatura e múltiplos alelos dos genes do relógio biológico são expressos e a regulação de sua expressão parece ser sazonal.The circadian clock is a self-sustaining molecular mechanism that generates rhythms. It perceives the environmental conditions and connects this pathway with its central oscillator, generating daily and seasonal rhythms of physiological responses. In plants, the circadian clock is associated with major metabolic and physiological pathways. In sugarcane, an economically important grass, previous studies showed that the circadian clock has the largest influence on plants seen so far under circadian conditions. This work aims to understand how the central oscillator of the circadian clock works in field-grown sugarcane. Thus, the transcriptome from different sugarcane organs; the expression of alternative isoforms and multiple alleles of circadian clock genes; and the effect of mutual shading in the field on the circadian clock function were analyzed. The results suggest that there is a functional and synchronized circadian clock in the different sugarcane organs. The transcripts regulated synergistically by the circadian clock and the variable environment are related to metabolic, physiological, genetic or epigenetic pathways, all important to sugarcane productivity. Mutual shading observed in the field seems to change the phase of expression of the sugarcane circadian clock. Besides, alternative splicing events have been reported for circadian clock genes under low temperature conditions and multiple alleles of circadian clock genes are expressed and their expression is likely to be seasonally regulated

Circadian regulation of the transcriptome in a complex polyploid crop.

The circadian clock is a finely balanced timekeeping mechanism that coordinates programmes of gene expression. It is currently unknown how the clock regulates expression of homoeologous genes in polyploids. Here, we generate a high-resolution time-course dataset to investigate the circadian balance between sets of 3 homoeologous genes (triads) from hexaploid bread wheat. We find a large proportion of circadian triads exhibit imbalanced rhythmic expression patterns, with no specific subgenome favoured. In wheat, period lengths of rhythmic transcripts are found to be longer and have a higher level of variance than in other plant species. Expression of transcripts associated with circadian controlled biological processes is largely conserved between wheat and ; however, striking differences are seen in agriculturally critical processes such as starch metabolism. Together, this work highlights the ongoing selection for balance versus diversification in circadian homoeologs and identifies clock-controlled pathways that might provide important targets for future wheat breeding

Low-temperature and circadian signals are integrated by the sigma factor SIG5

Chloroplasts are a common feature of plant cells and aspects of their metabolism, including photosynthesis, are influenced by low-temperature conditions. Chloroplasts contain a small circular genome that encodes essential components of the photosynthetic apparatus and chloroplast transcription/translation machinery. Here, we show that in Arabidopsis, a nuclear-encoded sigma factor that controls chloroplast transcription (SIGMA FACTOR5) contributes to adaptation to low-temperature conditions. This process involves the regulation of SIGMA FACTOR5 expression in response to cold by the bZIP transcription factors ELONGATED HYPOCOTYL5 and ELONGATED HYPOCOTYL5 HOMOLOG. The response of this pathway to cold is gated by the circadian clock, and it enhances photosynthetic efficiency during long-term cold and freezing exposure. We identify a process that integrates low-temperature and circadian signals, and modulates the response of chloroplasts to low-temperature conditions

Low-temperature and circadian signals are integrated by the sigma factor SIG5.

Acknowledgements: This research was funded by Biotechnology & Biological Sciences Research Council (UK) (BB/I005811/2, BB/J014400/1, BB/T003030/1, Institute Strategic Programme GEN BB/P013511/1 to A.N.D.; studentship 1518540 awarded to P.E.P.), Norwich Research Park Doctoral Training Partnership (BB/T008717/1, to R.D. and A.B.), The Leverhulme Trust (RPG-2018-216, to A.N.D.), the Bristol Centre for Agricultural Innovation (to A.N.D.), the Wolfson Foundation (to A.N.D.), NAGASE Science Technology Foundation (Japan), the Ministry of Education, Culture, Sports, Science and Technology (Japan) (Grants-in-Aid 17K07438, to K.T. and S.I.) and Tokyo Institute of Technology World Research Hub Initiative Program of Institute of Innovative Research (to K.T.). D.L.C.-R. is grateful to the Consejo Nacional de Ciencia y Tecnología (Mexico) for granting a PhD scholarship. We thank the University of Bristol Genomics Facility and Z. Song for experimental support; G. Jenkins for seed donation; S. Samwald, E. Tee, J. Sallmen and N. Holmes for help with protein analysis; C. Faulkner, T. Oyama and T. Muranaka for advice about transient expression; and M. Knight, Y. Yoshitake and M. Shimojima for technical advice. Figure 5 created with BioRender.com.Funder: Bristol Centre for Agricultural Innovation Consejo Nacional de Ciencia y Tecnología (Mexico)Funder: BBSRC (Durham) Studentship 1518540 The Leverhulme Trust RPG-2018-216Funder: NAGASE Science Technology Foundation Tokyo Institute of Technology World Research Hub InitiativeFunder: The Leverhulme Trust RPG-2018-216 Bristol Centre for Agricultural InnovationChloroplasts are a common feature of plant cells and aspects of their metabolism, including photosynthesis, are influenced by low-temperature conditions. Chloroplasts contain a small circular genome that encodes essential components of the photosynthetic apparatus and chloroplast transcription/translation machinery. Here, we show that in Arabidopsis, a nuclear-encoded sigma factor that controls chloroplast transcription (SIGMA FACTOR5) contributes to adaptation to low-temperature conditions. This process involves the regulation of SIGMA FACTOR5 expression in response to cold by the bZIP transcription factors ELONGATED HYPOCOTYL5 and ELONGATED HYPOCOTYL5 HOMOLOG. The response of this pathway to cold is gated by the circadian clock, and it enhances photosynthetic efficiency during long-term cold and freezing exposure. We identify a process that integrates low-temperature and circadian signals, and modulates the response of chloroplasts to low-temperature conditions