Cristiane Paula Gomes Calixto.

Bryophytes sensu lato were the first embryophytes that faced the typical adversities of the terrestrial environment. The mechanisms of tolerance to desiccation and UV radiation ensured the success in this new environment. Many of these plants, including species of Polytrichaceae, are characterized as " "resurrection species", which means they can remain viable for long periods of anabiosis and return their vital processes in a few seconds. Resurrection species has already been reported in four genera of Polythricaceae, one of them Polytrichum. In this context, the main goals of this project are (i) to verify the presence of the resurrection phenomenon in native species of Polytrichaceae that have not yet been investigated, (ii) to evaluate whether this characteristic can influence the repair mechanism to the increase in UV-B radiation, using a already known resurrection species (Polytrichum juniperinum Hedw.) and a non-resurrection species (SP1 - to be defined). To determine the resurrectiion phenomenon, gametophytes of Polytrichaceae species will be collected in the state of São Paulo and will be evaluate through physiological and anatomical parameters in anabiosis and hydrated conditions. The physiological parameters will be: (a) determination of relative water content (CRA), (b) fluorescence measurements (effective quantum yield (PSII) and maximum quantum yield (Fv/Fm) and apparent electron transport rate (ETR), (c) pigment content (chlorophyll a, b and carotenoids), (d) lipid peroxidation level (MDA), (e) activity of enzymatic (CAT, APX, GR and SOD) and non-enzymatic (ascorbic acid and glutathione) antioxidants and, (f) DPPH radical scavenging capacity. Anatomical investigation will be conducted through optical microscopy. To investigate the influency of resurrection capacity in the tolerance to another stress (eg increased UV-B radiation) gametophytes of P. juniperinum and SP1 will be transferred to pots and kept in a growth chamber under controlled conditions for two weeks. Then, the pots will be arranged into 4 groups: (1) control; (2) water restriction for six days followed by rehydration and sample collection at T=0, T=15 min and T=30 min; (3) increased UV- B for 3 hours/day and sample collection after 5 and 10 days; and (4) increased radiation UV-B for 3 hours/day for 5 and 10 days after a water restriction cycle. Before samples collections, fluorescence measurements will be carried out and then the samples will be collected and stored properly until the analyzes described above are carried out. In addition, samples will be collected for analysis of differentially expressed transcripts using the RNA-seq technique. Data from each experiment will be statistically treated and compared with control. In this way, the analysis of the parameters proposed in this project can help in the understanding, from a broad and robust view, of several events involved in the responses of these plants to environmental factors, besides opening a range of new research possibilities due to molecular global analysis.

To identify mechanistic solutions to improve rice yield under rising temperatures, molecular responses of heat stress tolerance must be understood. Transcriptional and post-transcriptional controls are involved in a wide range of plant environmental responses. Alternative splicing (AS), in particular, is a widespread mechanism impacting the stress defence in plants but it has been, to our knowledge, completely overlooked in rice genome-wide heat-stress studies. Our project aims at understanding the complexity of dynamic regulation of gene expression in response to heat stress by integrating AS responses to identify key genes regulating heat tolerance. For this, we will perform a high-resolution time-series transcriptome analysis of rice plants exposed to high temperatures. We will also investigate the sensitivity and speed of expression/AS behaviour of key heat-sensitive genes in different heat stress conditions, as well as its regulation in rice cultivars with contrasting heat tolerance/sensitivity. Lastly, we will investigate putative heat-affected regulators of AS by generating over-expression and loss-of-function (non-transgenic CRISPR-Cas9) lines in candidate genes, which will be monitored for heat stress phenotypes. The resulting knowledge will provide insight and strategies to rapidly advance rice breeding in response to changing environments.

Global rising temperatures negatively impact crop development and yield. To identify mechanistic solutions that improve plant heat adaptation, we need to understand plant molecular responses to heat stress (HS). The mechanisms underlying the formation, extent, and resetting of thermotolerance and HS memories in plants is largely unknown. Both post-transcriptional and epigenetic regulations are involved in a wide range of plant environmental responses and recent evidence suggest they may play an important role in temperature memory. To investigate this hypothesis, our project aims to explore alternative RNA processing events, epigenetic modifications, and the crosstalk between the two mechanisms in rice plants for generating a molecular response to temperature. Firstly, we will analyse the transcriptome and epigenome of rice cultivars contrasting for thermotolerance to investigate both basal and acquired thermotolerance responses, which will be examined with state-of-the-art analysis methods established by our team1,2. Secondly, we will perform robust integrative data analysis of the transcriptome and epigenome data for the understanding of gene regulatory networks at the transcriptional, post-transcriptional and epigenetic levels. Lastly, we will carry out functional analysis of candidate genes, selected by our preliminary analysis3, to identify putative epigenetic regulators of heat-induced alternative RNA processing events. Our project fosters interdisciplinary research at an international level to provide insights into plant heat adaptation and mitigation strategies for climate-resilient agriculture.

O objetivo deste projeto é organizar eventos bienais para despertar nas pessoas o fascínio pelas plantas e lembrá-los da importância da investigação científica em plantas para a paisagem social e ambiental, agora e no futuro. plantday18may.org/

We have recently demonstrated that the SCI1 gene is expressed in floral meristem cells since its specification and mainly in proliferative cells. These results were obtained by in situ hybridization and by analysis of SCI1prom::SCI1-GFP transgenic plants. Thus, this gene was renamed fSCI1 (floral Stem Cell-cycle Inhibitor 1). We also showed that the AGAMOUS transcription factor binds to the SCI1 gene promoter in Y1H experiments, while bioinformatics analyzes additionally suggest the binding of WUSCHEL and AINTEGUMENTA-Like 6 (AIL6) in the SCI1 genomic sequence. These results led us to ask about the importance of the SCI1 gene in the floral meristem and its transcriptional regulation. Moreover, we need to deepen our study of how SCI1 acts at the molecular level to regulate cell proliferation, and try to understand the origin and evolution of this protein. Therefore, the objectives of this project are: 1) To verify if the SCI1 gene is target of the transcription factors AG, WUS and AIL6; 2) Identify transcription factors that bind to the genomic sequence of SCI1; 3) Test the hypothesis that SCI1 and/or its interaction partner, NtDDX35, a DEAD-box RNA helicase, directly bind to RNA; 4) Identify NtDDX35 interaction partner proteins; 5) Produce and analyze transgenic NtDDX35 overexpression and silencing plants, including knock-out plants by CRISPR; 6) To produce tomato plants with the SCI1 gene silenced by RNAi and knocked out by CRISPR, and to analyze the floral meristem development in these plants; 7) Study the evolution of the SCI1 gene; and 8) Analyze whether the putative SCI1 homolog in Aspergillus nidulans affects cell proliferation in this fungus.

A chefia do DB e as representantes do DB na CCEx-IB estão realizando um projeto para preservar o nosso legado e aprender mais sobre nossos funcionários. Veja aqui (https://www.ib.usp.br/botanica/cultura-e-extensao/projeto-memoria.html) as entrevistas com cada um dos nossos colegas, onde eles(as) nos contam sobre sua trajetória profissional.

The circadian clock provides a mechanism through which plants can anticipate the predictable cycle of light and dark imposed by life on a rotating earth and adjust many aspects of their physiology. There is strong evidence that possession of a circadian clock provides a darwinian fitness advantage. Understanding the operation and control of the clock is therefore a crucial objective of modern plant biology. The clock involves a set of interlocked gene expression feedback loops controlled by several different mechanisms operating at multiple levels, including protein phosphorylation, protein turnover, gene expression and chromatin remodelling. Recent work has added a new dimension by showing that alternative splicing (AS) plays a significant role in determining the level of functional transcripts of key clock genes, and thence the levels of the clock proteins, and mediates responses of the clock to changes in temperature. Our objectives are to illuminate the function of AS of clock genes by defining the scope and extent of the AS events, the conditions that give rise to AS and the underlying mechanisms and factors that control temperature-sensitive AS.

O processamento alternativo regula extensivamente os genes do relógio em Arabidopsis. O objetivo deste projeto era determinar se os genes do relógio e seus modos de regulação são conservados em outras plantas superiores, como a cevada. Através do uso de uma análise in silico robusta e seqüência nucleotídica de 27 genes do relógio de Arabidopsis, foi identificado 21 genes de cevada, 60% dos quais são ortólogos de Arabidopsis. A maioria dos genes do relógio da cevada tem um ritmo diário definido que é mantido em condições de luz constantes. Mutações dos genes HvPPDH1 (ortólogo de AtPRR7) e HvELF3 do relógio de cevada afetam fortemente o tempo de florescimento e aumentaram a distribuição geográfica onde a cevada é cultivada. Nós mostramos que ambas as mutações afetam a expressão de genes do relógio: a mutação Hvppdh1 afeta moderadamente os níveis de expressão e fase, enquanto a mutação Hvelf3 causa arritmicidade da maioria desses genes, o que ajuda a explicar o fenótipo de florescimento precoce. Alguns dos ortólogos do relógio da cevada possuem processamento alternativo dependente da temperatura. O foco desta parte da análise foi com os genes HvLHY e HvPPDH1. Apesar de eventos específicos de processamento alternativo serem mal conservados, observou-se um comportamento semelhante em termos de diminuição de mRNA funcional. Acreditamos que esta nova camada de controle do relógio, observado em duas espécies diferentes, uma planta modelo e uma espécie de cultura, pode ajudar a nossa compreensão da adaptação da planta em diferentes ambientes e, finalmente, pode oferecer uma nova gama de alvos para o melhoramento da planta.

SnoRNAs órfãos, i.e. sem complementaridade a nenhum pre-rRNA, foram identificados em Arabidopsis. Análises de bioinformática com estes snoRNAs identificaram complementariedades em diversos mRNAs, o qual poderia afetar o processamento ou atuar como miRNAs. Além disso, dois mutantes em um destes snoRNAs haviam sido obtidos e a mutação confirmada. Estes mutantes mostraram diferenças fenotípicas quando comparados com as plantas selvagens e análises de expressões iniciais sugeriram que a mutação no snoRNA afeta o nível de vários transcritos, incluindo os alvos apontados pelas análises de bioinformatica. Os objetivos do projeto eram identificar e caracterizar mRNAs alvos dos snoRNAs órfãos em Arabidopsis e investigar o mecanismo pela qual tais snoRNAs poderiam regular a expressão gênica de seus alvos. Análises realizadas com os supostos mutantes snor146 e plantas selvagens possibilitou a identificação de mudança nos níveis de transcrição de 2/3 dos genes alvos apontados pelas análises de bioinformática. Quatro destes alvos tinham seus padrões de processamento alterados. Estes foram então sequenciados para identificar a mudança no processamento. Infelizmente, as sequências resultantes não mostraram diferenças no processamento, mas sim polimorfismos a nível de DNA, sugerindo que o ?background? genético dos mutantes não correspondia ao mesmo ?background? da planta selvagem. O cenário ficou ainda mais complicado quando descobrimos que as linhagens mutantes eram inesperadamente descendentes F2 ou F3 do cruzamento entre dois cultivares diferentes e estavam segregando vários polimorfismos. Assim, este projeto teve que ser descontinuado.