Natalia Balbinott

Rice (Oryza sativa) is a major staple crop that feeds over half of the world’s population. However, its cultivation depends heavily on nitrogen fertilizers, which increase both environmental impacts and production costs. Enhancing the sustainable use of nitrogen is therefore essential for maintaining global food security. Previously, we characterized an aquaporin (VgPIP1;2) from the bromeliad Vriesea gigantea that transports ammonium and has great biotechnological potential. Here, we investigated the effect of VgPIP1;2 heterologous expression on rice, particularly in root development and nitrogen metabolism. Transgenic plants cultivated in hydroponics exhibited a larger root network area compared to wild type plants. Biochemical and metabolomic analyses revealed that the roots of VgPIP1;2 overexpressing plants have higher contents of nitrogen, free amino acids and sugars. In line with these results, the transcriptional profile showed that genes involved with nitrogen uptake and assimilation, amino acid biosynthesis and sugar metabolism are upregulated in transgenic plants. These findings indicate that VgPIP1;2 overexpression positively modulates nitrogen and carbon metabolism, altering root development in rice. Thus, the expression of VgPIP1;2 would represent a potential strategy to develop new rice cultivars with improved root architecture suited to enhance nitrogen absorption and assimilation.

Phytocystatins are proteinaceous inhibitors found in plants that competitively target various classes of cysteine proteinases, including papain-like enzymes, cathepsins, and legumains. Based on structural characteristics and gene organization, phytocystatins can be classified into four subtypes: intronless (I1 and I2), intron-containing (IwI), and multidomain cystatins containing more than one inhibitory region (II). This work presents PhyCysID, a dedicated web server designed for the rapid classification of phytocystatin subtypes. PhyCysID uses a set of 21 features derived from amino acid composition, in combination with 15 distinct machine learning algorithms, to classify phytocystatin sequences into one of the four subtypes. Initially, the input sequence is analyzed to verify if it comprises a true phytocystatin sequence. If so, the input sequence is further analyzed using a specialized classification pipeline called PhyCysID 12M, which integrates 12 machine learning models to assign it to one of the four defined phytocystatin classes. As a case study, a curated dataset of phytocystatin sequences from the UniProt database was used to evaluate the algorithm's performance. The PhyCysID web server enables rapid classification of both individual and batch-submitted sequences in less than 15 s, providing high-throughput analysis for an accurate identification of phytocystatin class and function. PhyCysID is freely available at https://www.ufrgs.br/labec/phycysid.

Understanding the evolution and the effect of plasticity in plant responses to environmental changes is crucial to combat global climate change. It is particularly interesting in species that survive in distinct environments, such as Eugenia uniflora, which thrives in contrasting ecosystems within the Atlantic Forest (AF). In this study, we combined transcriptome analyses of plants growing in nature (Restinga and Riparian Forest) with greenhouse experiments to unveil the DEGs within and among adaptively divergent populations of E. uniflora. We compared global gene expression among plants from two distinct ecological niches. We found many differentially expressed genes between the two populations in natural and greenhouse-cultivated environments. The changes in how genes are expressed may be related to the species’ ability to adapt to specific environmental conditions. The main difference in gene expression was observed when plants from Restinga were compared with their offspring cultivated in greenhouses, suggesting that there are distinct selection pressures underlying the local environmental and ecological factors of each Restinga and Riparian Forest ecosystem. Many of these genes engage in the stress response, such as water and nutrient transport, temperature, light intensity, and gene regulation. The stress-responsive genes we found are potential genes for selection in these populations. These findings revealed the adaptive potential of E. uniflora and contributed to our understanding of the role of gene expression reprogramming in plant evolution and niche adaptation.

The cystatin superfamily includes proteins crucial for inhibiting cysteine proteases, enzymes involved in many biological processes. In plants, cystatins regulate seed germination, development, and pathogen defense. In humans, inhibiting legumain-type cysteine proteases offers a promising cancer treatment strategy, as this enzyme’s expression often rises during tumor progression. We evaluated a novel rice-derived chimeric legumain inhibitor using in silico and in vitro methods. Computational simulations confirmed the inhibitor’s stability and nanomolar affinity for legumain’s active site. Post-expression and purification assays determined its kinetics and demonstrated its efficacy in reducing HT29 tumor cell migration and viability. Our findings suggest the chimeric Oryzacystatin I mutant with SNSL motifs is a promising candidate for cancer drug development.

Chloroplast ascorbate peroxidases exert an important role in the maintenance of hydrogen peroxide levels in chloroplasts by using ascorbate as the specific electron donor. In this work, we performed a functional study of the stromal APX in rice (OsAPX7) and demonstrated that silencing of OsAPX7 did not impact plant growth, redox state, or photosynthesis parameters. Nevertheless, when subjected to drought stress, silenced plants (APX7i) show a higher capacity to maintain stomata aperture and photosynthesis performance, resulting in a higher tolerance when compared to non-transformed plants. RNA-seq analyses indicate that the silencing of OsAPX7 did not lead to changes in the global expression of genes related to reactive oxygen species metabolism. In addition, the drought-mediated induction of several genes related to the proteasome pathway and the down-regulation of genes related to nitrogen and carotenoid metabolism was impaired in APX7i plants. During drought stress, APX7i showed an up-regulation of genes encoding flavonoid and tyrosine metabolism enzymes and a down-regulation of genes related to phytohormones signal transduction and nicotinate and nicotinamide metabolism. Our results demonstrate that OsAPX7 might be involved in signaling transduction pathways related to drought stress response, contributing to the understanding of the physiological role of chloroplast APX isoforms in rice.

Abiotic stresses such as nutritional imbalance, salt, light intensity, and high and low temperatures negatively affect plant growth and development. Through the course of evolution, plants developed multiple mechanisms to cope with environmental variations, such as physiological, morphological, and molecular adaptations. Epigenetic regulation, transcription factor activity, and post-transcriptional regulation operated by RNA molecules are mechanisms associated with gene expression regulation under stress. Epigenetic regulation, including histone and DNA covalent modifications, triggers chromatin remodeling and changes the accessibility of transcription machinery leading to alterations in gene activity and plant homeostasis responses. Soybean is a legume widely produced and whose productivity is deeply affected by abiotic stresses. Many studies explored how soybean faces stress to identify key elements and improve productivity through breeding and genetic engineering. This review summarizes recent progress in soybean gene expression regulation through epigenetic modifications and circRNAs pathways, and points out the knowledge gaps that are important to study by the scientific community. It focuses on epigenetic factors participating in soybean abiotic stress responses, and chromatin modifications in response to stressful environments and draws attention to the regulatory potential of circular RNA in post-transcriptional processing.

Myrtaceae is a large and species-rich family of woody eudicots, with prevalent distribution in the Southern Hemisphere. Classification and taxonomy of species belonging to this family is quite challenging, sometimes with difficulty in species identification and producing phylogenies with low support for species relationships. Most of the current knowledge comes from few molecular markers, such as plastid genes and intergenic regions, which can be difficult to handle and produce conflicting results. Based on plastid protein-coding sequences and nuclear markers, we present a topology for the phylogenetic relationships among Myrtaceae tribes. Our phylogenetic estimate offers a contrasting topology over previous analysis with fewer markers. Plastome phylogeny groups the tribes Syzygieae and Eucalypteae and individual chloroplast genes produce divergent topologies, especially among species within Myrteae tribe, but also in regard to the grouping of Syzygieae and Eucalypteae. Results are consistent and reproducible with both nuclear and organellar datasets. It confronts previous data about the deep nodes of Myrtaceae phylogeny.

Myrteae species from Brazil (^{Salvador et al., 2011}; ^{Souza Moreira et al., 2019}). Thus, due to its plethora of roles, a growing interest has been addressed to this group as a model for evolutionary, ecological and applied studies. For this study, leaves from Eugenia brasiliensis, Eugenia pyriformis, Eugenia nitida, Myrcianthes pungens, Plinia edulis and Psidium cattleianum trees were from a private in vivo collection in Gravataí, RS, Brazil (latitude (S): 29°51’52"; longitude (W): 50°53’53") and used to isolate chloroplasts by the modified high salt method, followed by cpDNA extraction with the CTAB method (^{Vieira et al., 2014}). DNA quality was evaluated by electrophoresis in a 1% agarose gel, and DNA quantity was determined using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). For each species, one genomic paired-end library of 150 nt length was generated using an Illumina HiSeq2000 platform (Macrogen). The bases with quality below Q30 and adapter contamination were trimmed using Trim Galore!0.4.2, with a 50 bases minimum allowed. Paired-end sequence reads were filtered against 28 Myrtaceae plastomes (Table S1) using BWA software (^{Li and Durbin, 2009}) with two mismatches allowed. Mapped reads were used for the de novo assembly with ABySS software (^{Jackman et al., 2017}). The plastome scaffolds were orientated by MUMmer (^{Delcher et al., 2003}) using Eugenia uniflora (NC_026450.1), Plinia trunciflora (NC_034801.1) or Psidium guajava (NC_033355.1) as reference genomes for species of the same or closer genus. Genes were annotated using GeSeq (^{Tillich et al., 2017}) by BLAST searches with 80% similarity. Circular plastome maps were drawn using the OGDRAW web toolkit (^{Lohse et al., 2013}). IRa and IRb boundaries were analyzed using IRScope (^{Amiryousefi et al., 2018}). For each species, local mVISTA (^{Frazer et al., 2004}) was used to pairwise align plastomes with their respective reference. An overall genome comparison was performed with BLAST Ring Image Generator (BRIG) (^{Alikhan et al., 2011}). Krait v0.11.4 (^{Du et al., 2018}) was used to search and annotate perfect SSRs using the genomes and their annotated GFF3 file. The parameters for minimum repeat numbers were 8, 4, 3, 3, 3, 3 for mono-, di-, tri-, tetra-, penta- and hexanucleotide SSRs, respectively. DNA sequencing libraries were produced for each species, and these comprised 34.1-48.7 M raw Illumina paired-end reads (summarized in Table S2). The percentage of removed reads due to trimming ranged from 1.17-1.45%. The number of filtered reads was 1.1-2.2 M reads. The obtained reads were de novo assembled into scaffolds that completely covered each plastome, without any gaps. The number of assembled scaffolds obtained ranged from four to eight. The minimum coverage ranged from 13 to 46 reads and the maximum coverage, 1,508-3,620 reads. The complete sequences were submitted to GenBank at accession numbers MN095407 to MN095411 and MN095413 (Table 1). The complete plastomes of six Myrteae have a narrow size range, from 157,683 bp in E. nitida to 159,631 bp in P. edulis, similar to the size of Myrtaceae species plastomes (^{Eguiluz et al., 2017 a},^b; ^{Machado et al., 2017}). Figures S1-S6 present the genome maps for each species. Four well-defined regions are present in all newly assembled genomes. Inverted regions (IR) ranged from ~26.3 to 26.4 kbp and had the smallest size variation, up to 78 bp. Short single copy (SSC) sections have ~18.2-18.5 kbp, while long single copy (LSC) sections have ~86.4-88.2 kbp (Table 1). Protein coding sequences comprise ~50% of the genome, rRNAs and tRNAs comprise ~7%, and non-coding regions, such as introns, pseudogenes and intergenic spacers correspond to ~43% (Table 1). Genome structure analysis showed a high degree of synteny among evaluated species (Figure 1). Genomes contained 129 genes in total, corresponding to 78 single-copy protein-coding genes, 30 transfer RNA (tRNA) genes, four ribosomal genes (rRNA) and one pseudogene (ycf1) (Figure 1, Table 1). In general, genomic features, such as size, structure, and gene abundance are similar to previously described Myrtaceae species (^{Eguiluz et al., 2017a},b). Despite the similarity in genomic features, the mVISTA comparison against each respective reference genome showed that some regions display lower similarity (Figure S7 – S9). Non-coding regions, particularly the intergenic, had lower conservation and, therefore, more variation, such as psbI-trnS, trnT-psbD, trnS-psbZ-trnG, accD-psaI, and ndhF-rp132 in Eugenia and Myrcianthes; trnS-trnR, atpF-atpH, trnT-trnL, and rpl32-trnL in Plinia; and most intergenic regions of LSC in Psidium. Regarding protein-coding genes, we observed a conservation decrease in accD and ccsA in Eugenia species and M. pungens; and rpoC2 in Plinia and Psidium. Protein-coding genes matK, ndhF and ycf1, showed more nucleotide diversity (4.6 to 6.1%) in all analyzed species (Figure S7 – S10). This diversity corroborates previous studies based on plastidial genes and non-coding regions with contrasting substitution rates (^{Thornhill et al., 2015}; ^{Machado et al., 2017}). Some structural changes in the IRa and IRb boundaries were found for the evaluated species (Figure 2). Within the IRb-LSC boundaries, the boundaries of the rps19 gene were located on the left side. In the IRb region, except for M. pungens, the IRb-LSC boundary was embedded in rps19 and had a length of three bp in Eugenia (Figure 2A), ~30 bp in Plinia (Figure 2B), and 31 bp in Psidium, contained in the IRb (Figure 2C). The IRb-SSC boundaries were embedded in the ycf1 pseudogene, ranging from one to eight bp in Eugenia/Myrcianthes, one and two bp in Plinia, and only one bp in Psidium species. The ndhF gene was located on the right side of the IRb-SSC at a distance from the boundary of 10 bp in E. uniflora, 36 to 121 bp in other Eugenia, 72 bp in M. pungens, 109 bp and 120 bp in Plinia, and 111 bp and 124 bp in Psidium. The SSC-IRa boundary was embedded in ycf1, with a length of 1047 to 1080 bp in Eugenia/Myrcianthes, 1080 and 1011 bp in Plinia, and 1071 and 1079 bp in Psidium in the IRa region. The trnH-GUG gene was located on the right side of the IRa-LSC boundary ranging from 11 to 52 bp in Eugenia/Myrcianthes, from 3 to 10 bp in Plinia and 10 to 14 bp in Psidium. The contraction and expansion of IR regions are measurable events of plastome evolution. These results demonstrate a genus-specific IR conservation, which can be considered one of the reasons for genome size variation among species. In this work, we present, for the first time, a characterization of IR boundaries from species of the same genus in Myrteae because they compared different genera, previous studies could not report a significant variation in IRb-SSC border within Myrteae species (^{Eguiluz et al., 2017a},b; ^{Machado et al., 2017}). All plastomes presented a similar number of SSRs. In total, over 315 SSRs were identified for each species (Table 2). The mononucleotide SSRs of A/T were the most frequent, varying in number from 85/98 in E. brasiliensis/E. pyriformis to 93/103 in P. edulis/P. cattleianum (Table S3). This AT richness was already demonstrated in previous studies and reflects the lower GC content in these plastid genomes (^{Eguiluz et al., 2017a},b). The secondmost common were the trinucleotide SSRs, ranging in number from 61 in E. nitida to 71 in P. edulis. In addition, the number of SSRs located in different regions were similar: in intergenic regions ranging from 171 in E. nitida to 185 in P. edulis; in genes, ranging from 96 in E. brasiliensis, E. pyriformis, M. pungens, and P. cattleianum, and 98 in E. nitida and P. edulis; and in introns, ranging from 43 in E. brasiliensis to 47 in E. nitida, P. edulis, and P. cattleianum (Table 2). Hexanucleotide SSRs could not be found in the genomes. All found SRRs are listed in Table S4. These SSR results provide more information on molecular markers that could be used to evaluate intra- and interspecific diversity. This work provides reference genomes for six Neotropical Myrtaceae species, increasing the genetic information available for the Myrteae tribe, and allowing the improvement of taxa sampling in further investigations into Myrtaceae evolution.