Luiz Felipe Valter de Oliveira

New Insights into Aluminum Tolerance in Rice: The ASR5 Protein Binds the STAR1 Promoter and Other Aluminum-Responsive Genes

Aluminum (Al) toxicity in plants is one of the primary constraints in crop production. Al³⁺, the most toxic form of Al, is released into soil under acidic conditions and causes extensive damage to plants, especially in the roots. In rice, Al tolerance requires the ASR5 gene, but the molecular function of ASR5 has remained unknown. Here, we perform genome-wide analyses to identify ASR5-dependent Al-responsive genes in rice. Based on ASR5_RNAi silencing in plants, a global transcriptome analysis identified a total of 961 genes that were responsive to Al treatment in wild-type rice roots. Of these genes, 909 did not respond to Al in the ASR5_RNAi plants, indicating a central role for ASR5 in Al-responsive gene expression. Under normal conditions, without Al treatment, the ASR5_RNAi plants expressed 1.756 genes differentially compared to the wild-type plants, and 446 of these genes responded to Al treatment in the wild-type plants. Chromatin immunoprecipitation followed by deep sequencing identified 104 putative target genes that were directly regulated by ASR5 binding to their promoters, including the STAR1 gene, which encodes an ABC transporter required for Al tolerance. Motif analysis of the binding peak sequences revealed the binding motif for ASR5, which was confirmed via in vitro DNA-binding assays using the STAR1 promoter. These results demonstrate that ASR5 acts as a key transcription factor that is essential for Al-responsive gene expression and Al tolerance in rice.

Identifying conserved and novel microRNAs in developing seeds of Brassica napus using deep sequencing.

MicroRNAs (miRNAs) are important post-transcriptional regulators of plant development and seed formation. In Brassica napus, an important edible oil crop, valuable lipids are synthesized and stored in specific seed tissues during embryogenesis. The miRNA transcriptome of B. napus is currently poorly characterized, especially at different seed developmental stages. This work aims to describe the miRNAome of developing seeds of B. napus by identifying plant-conserved and novel miRNAs and comparing miRNA abundance in mature versus developing seeds. Members of 59 miRNA families were detected through a computational analysis of a large number of reads obtained from deep sequencing two small RNA and two RNA-seq libraries of (i) pooled immature developing stages and (ii) mature B. napus seeds. Among these miRNA families, 17 families are currently known to exist in B. napus; additionally 29 families not reported in B. napus but conserved in other plant species were identified by alignment with known plant mature miRNAs. Assembled mRNA-seq contigs allowed for a search of putative new precursors and led to the identification of 13 novel miRNA families. Analysis of miRNA population between libraries reveals that several miRNAs and isomiRNAs have different abundance in developing stages compared to mature seeds. The predicted miRNA target genes encode a broad range of proteins related to seed development and energy storage. This work presents a comparative study of the miRNA transcriptome of mature and developing B. napus seeds and provides a basis for future research on individual miRNAs and their functions in embryogenesis, seed maturation and lipid accumulation in B. napus.

Heavy metal‐associated isoprenylated plant protein (HIPP): characterization of a family of proteins exclusive to plants

Metallochaperones are key proteins for the safe transport of metallic ions inside the cell. HIPPs (heavy metal‐associated isoprenylated plant proteins) are metallochaperones that contain a metal binding domain (HMA) and a C–terminal isoprenylation motif. In this study, we provide evidence that proteins of this family are found only in vascular plants and may be separated into five distinct clusters. HIPPs may be involved in (a) heavy metal homeostasis and detoxification mechanisms, especially those involved in cadmium tolerance, (b) transcriptional responses to cold and drought, and (c) plant–pathogen interactions. In particular, our results show that the rice (Oryza sativa) HIPP OsHIPP41 gene is highly expressed in response to cold and drought stresses, and its product is localized in the cytosol and the nucleus. The results suggest that HIPPs play an important role in the development of vascular plants and in plant responses to environmental changes.

Identifying MicroRNAs and Transcript Targets in Jatropha Seeds

MicroRNAs, or miRNAs, are endogenously encoded small RNAs that play a key role in diverse plant biological processes. Jatropha curcas L. has received significant attention as a potential oilseed crop for the production of renewable oil. Here, a sRNA library of mature seeds and three mRNA libraries from three different seed development stages were generated by deep sequencing to identify and characterize the miRNAs and pre-miRNAs of J. curcas. Computational analysis was used for the identification of 180 conserved miRNAs and 41 precursors (pre-miRNAs) as well as 16 novel pre-miRNAs. The predicted miRNA target genes are involved in a broad range of physiological functions, including cellular structure, nuclear function, translation, transport, hormone synthesis, defense, and lipid metabolism. Some pre-miRNA and miRNA targets vary in abundance between the three stages of seed development. A search for sequences that produce siRNA was performed, and the results indicated that J. curcas siRNAs play a role in nuclear functions, transport, catalytic processes and disease resistance. This study presents the first large scale identification of J. curcas miRNAs and their targets in mature seeds based on deep sequencing, and it contributes to a functional understanding of these miRNAs.

Metatranscriptomic analysis of small RNAs present in soybean deep sequencing libraries

A large number of small RNAs unrelated to the soybean genome were identified after deep sequencing of soybean small RNA libraries. A metatranscriptomic analysis was carried out to identify the origin of these sequences. Comparative analyses of small interference RNAs (siRNAs) present in samples collected in open areas corresponding to soybean field plantations and samples from soybean cultivated in greenhouses under a controlled environment were made. Different pathogenic, symbiotic and free-living organisms were identified from samples of both growth systems. They included viruses, bacteria and different groups of fungi. This approach can be useful not only to identify potentially unknown pathogens and pests, but also to understand the relations that soybean plants establish with microorganisms that may affect, directly or indirectly, plant health and crop production.

The Wall-associated Kinase gene family in rice genomes

The environment is a dynamic system in which life forms adapt. Wall-Associated Kinases (WAK) are a subfamily of receptor-like kinases associated with the cell wall. These genes have been suggested as sensors of the extracellular environment and triggers of intracellular signals. They belong to the ePK superfamily with or without a conserved arginine before the catalytic subdomain VIB, which characterizes RD and non-RD WAKs. WAK is a large subfamily in rice. We performed an extensive comparison of WAK genes from A. thaliana (AtWAK), O. sativa japonica and indica subspecies (OsWAK). Phylogenetic studies and WAK domain characterization allowed for the identification of two distinct groups of WAK genes in Arabidopsis and rice. One group corresponds to a cluster containing only OsWAKs that most likely expanded after the monocot-dicot separation, which evolved into a non-RD kinase class. The other group comprises classical RD-kinases with both AtWAK and OsWAK representatives. Clusterization analysis using extracellular and kinase domains demonstrated putative functional redundancy for some genes, but also highlighted genes that could recognize similar extracellular stimuli and activate different cascades. The gene expression pattern of WAKs in response to cold suggests differences in the regulation of the OsWAK genes in the indica and japonica subspecies. Our results also confirm the hypothesis of functional diversification between A. thaliana and O. sativa WAK genes. Furthermore, we propose that plant WAKs constitute two evolutionarily related but independent subfamilies: WAK-RD and WAK-nonRD. Recognition of this structural division will further provide insights to understanding WAK functions and regulations.

Cold tolerance in rice germinating seeds revealed by deep RNAseq analysis of contrasting indica genotypes

Rice productivity is largely affected by low temperature, which can be harmful throughout plant development, from germination to grain filling. Germination of indica rice cultivars under cold is slow and not uniform, resulting in irregular emergence and small plant population. To identify and characterize novel genes involved in cold tolerance during the germination stage, two indica rice genotypes (sister lines previously identified as cold-tolerant and cold-sensitive) were used in parallel transcriptomic analysis (RNAseq) under cold treatment (seeds germinating at 13 °C for 7 days). We detected 1,361 differentially expressed transcripts. Differences in gene expression found by RNAseq were confirmed for 11 selected genes using RT-qPCR. Biological processes enhanced in the cold-tolerant seedlings include: cell division and expansion (confirmed by anatomical sections of germinating seeds), cell wall integrity and extensibility, water uptake and membrane transport capacity, sucrose synthesis, generation of simple sugars, unsaturation of membrane fatty acids, wax biosynthesis, antioxidant capacity (confirmed by histochemical staining of H2O2), and hormone and Ca(2+)-signaling. The cold-sensitive seedlings respond to low temperature stress increasing synthesis of HSPs and dehydrins, along with enhanced ubiquitin/proteasome protein degradation pathway and polyamine biosynthesis. Our findings can be useful in future biotechnological approaches aiming to cold tolerance in indica rice.

The source of the river as a nursery for microbial diversity.

Bacteria are highly diverse and ubiquitous organisms that play a key role as drivers for ecosystem processes. The application of NGS (next-generation sequencing technologies) for 16S analysis has been broadly used for understanding bacterioplankton composition and structure. Most of studies conducted on aquatic ecosystems with 16S NGS have been in seawater and lakes. A few studies using NGS have been conducted in river environments and have suggested the presence of a bacterial seed-bank. We performed 16S highly variable V4 region high-throughput analysis in the Sinos River, which is located in one of most important Brazilian industrial centers. This region has several contrasts in its environmental characteristics, presenting a longitudinal gradient of eutrophication and making it a remarkable study site for observing the dynamics of bacterioplankton. We demonstrated consistent evidence for the existence of a bacterial seed-bank and its longitudinal persistence. Seasonal shifts reinforce the importance of the source of the river in maintaining the bacterial seed-bank that spreads throughout the river. Therefore, the preservation of the source of the river is important not only for hydrologic reasons but also to maintain the microbial composition and the ecological integrity of the river.

Unusual RNA plant virus integration in the soybean genome leads to the production of small RNAs.

Horizontal gene transfer (HGT) is known to be a major force in genome evolution. The acquisition of genes from viruses by eukaryotic genomes is a well-studied example of HGT, including rare cases of non-retroviral RNA virus integration. The present study describes the integration of cucumber mosaic virus RNA-1 into soybean genome. After an initial metatranscriptomic analysis of small RNAs derived from soybean, the de novo assembly resulted a 3029-nt contig homologous to RNA-1. The integration of this sequence in the soybean genome was confirmed by DNA deep sequencing. The locus where the integration occurred harbors the full RNA-1 sequence followed by the partial sequence of an endogenous mRNA and another sequence of RNA-1 as an inverted repeat and allowing the formation of a hairpin structure. This region recombined into a retrotransposon located inside an exon of a soybean gene. The nucleotide similarity of the integrated sequence compared to other Cucumber mosaic virus sequences indicates that the integration event occurred recently. We described a rare event of non-retroviral RNA virus integration in soybean that leads to the production of a double-stranded RNA in a similar fashion to virus resistance RNAi plants.

ASR5 is involved in the regulation of miRNA expression in rice

Key messageThe work describes an ASR knockdown transcriptomic analysis by deep sequencing of rice root seedlings and the transactivation of ASR cis-acting elements in the upstream region of a MIR gene.AbstractMicroRNAs are key regulators of gene expression that guide post-transcriptional control of plant development and responses to environmental stresses. ASR (ABA, Stress and Ripening) proteins are plant-specific transcription factors with key roles in different biological processes. In rice, ASR proteins have been suggested to participate in the regulation of stress response genes. This work describes the transcriptomic analysis by deep sequencing two libraries, comparing miRNA abundance from the roots of transgenic ASR5 knockdown rice seedlings with that of the roots of wild-type non-transformed rice seedlings. Members of 59 miRNA families were detected, and 276 mature miRNAs were identified. Our analysis detected 112 miRNAs that were differentially expressed between the two libraries. A predicted inverse correlation between miR167abc and its target gene (LOC_Os07g29820) was confirmed using RT-qPCR. Protoplast transactivation assays showed that ASR5 is able to recognize binding sites upstream of the MIR167a gene and drive its expression in vivo. Together, our data establish a comparative study of miRNAome profiles and is the first study to suggest the involvement of ASR proteins in miRNA gene regulation.