Legume-rhizobia symbiosis: Translatome analysis.

Leguminous plants can establish endosymbiotic relationships with nitrogen-fixing soil rhizobacteria. Bacterial infection and nodule organogenesis are two independent but highly coordinated genetic programs that are active during this interaction. These genetic programs can be regulated along all the stages of gene expression. Most of the studies, for both eukaryotes and prokaryotes, focused on the transcriptional regulation level determining the abundance of mRNAs. However, it has been demonstrated that mRNA levels only sometimes correlate with the abundance or activity of the coded proteins. For this reason, in the past two decades, interest in the role of translational control of gene expression has increased, since the subset of mRNA being actively translated outperforms the information gained only by the transcriptome. In the case of legume-rhizobia interactions, the study of the translatome still needs to be explored further. Therefore, this review aims to discuss the methodologies for analyzing polysome-associated mRNAs at the genome-scale and their contribution to studying translational control to understand the complexity of this symbiotic interaction. Moreover, the Dual RNA-seq approach is discussed for its relevance in the context of a symbiotic nodule, where intricate multi-species gene expression networks occur.

Water deficit response in nodulated soybean roots: a comprehensive transcriptome and translatome network analysis.

BACKGROUND: Soybean establishes a mutualistic interaction with nitrogen-fixing rhizobacteria, acquiring most of its nitrogen requirements through symbiotic nitrogen fixation. This crop is susceptible to water deficit; evidence suggests that its nodulation status-whether it is nodulated or not-can influence how it responds to water deficit. The translational control step of gene expression has proven relevant in plants subjected to water deficit. RESULTS: Here, we analyzed soybean roots' differential responses to water deficit at transcriptional, translational, and mixed (transcriptional + translational) levels. Thus, the transcriptome and translatome of four combined-treated soybean roots were analyzed. We found hormone metabolism-related genes among the differentially expressed genes (DEGs) at the translatome level in nodulated and water-restricted plants. Also, weighted gene co-expression network analysis followed by differential expression analysis identified gene modules associated with nodulation and water deficit conditions. Protein-protein interaction network analysis was performed for subsets of mixed DEGs of the modules associated with the plant responses to nodulation, water deficit, or their combination. CONCLUSIONS: Our research reveals that the stand-out processes and pathways in the before-mentioned plant responses partially differ; terms related to glutathione metabolism and hormone signal transduction (2 C protein phosphatases) were associated with the response to water deficit, terms related to transmembrane transport, response to abscisic acid, pigment metabolic process were associated with the response to nodulation plus water deficit. Still, two processes were common: galactose metabolism and branched-chain amino acid catabolism. A comprehensive analysis of these processes could lead to identifying new sources of tolerance to drought in soybean.

Correction: Sainz et al. Analysis of Thioredoxins and Glutaredoxins in Soybean: Evidence of Translational Regulation under Water Restriction. Antioxidants 2022, 11, 1622.

In the original publication [...].

Analysis of Thioredoxins and Glutaredoxins in Soybean: Evidence of Translational Regulation under Water Restriction.

Soybean (Glycine max (L.) Merr.) establishes symbiosis with rhizobacteria, developing the symbiotic nodule, where the biological nitrogen fixation (BNF) occurs. The redox control is key for guaranteeing the establishment and correct function of the BNF process. Plants have many antioxidative systems involved in ROS homeostasis and signaling, among them a network of thio- and glutaredoxins. Our group is particularly interested in studying the differential response of nodulated soybean plants to water-deficit stress. To shed light on this phenomenon, we set up an RNA-seq experiment (for total and polysome-associated mRNAs) with soybean roots comprising combined treatments including the hydric and the nodulation condition. Moreover, we performed the initial identification and description of the complete repertoire of thioredoxins (Trx) and glutaredoxins (Grx) in soybean. We found that water deficit altered the expression of a greater number of differentially expressed genes (DEGs) than the condition of plant nodulation. Among them, we identified 12 thioredoxin (Trx) and 12 glutaredoxin (Grx) DEGs, which represented a significant fraction of the detected GmTrx and GmGrx in our RNA-seq data. Moreover, we identified an enriched network in which a GmTrx and a GmGrx interacted with each other and associated through several types of interactions with nitrogen metabolism enzymes.

Polysome Purification from Soybean Symbiotic Nodules.

The aim of this protocol is to provide a strategy for studying the eukaryotic translatome of the soybean (Glycine max) symbiotic nodule. This paper describes methods optimized to isolate plant-derived polyribosomes and their associated mRNAs to be analyzed using RNA-sequencing. First, cytoplasmic lysates are obtained through homogenization in polysome- and RNA-preserving conditions from whole, frozen soybean nodules. Then, lysates are cleared by low-speed centrifugation, and 15% of the supernatant is used for total RNA (TOTAL) isolation. The remaining cleared lysate is used to isolate polysomes by ultracentrifugation through a two-layer sucrose cushion (12% and 33.5%). Polysome-associated mRNA (PAR) is purified from polysomal pellets after resuspension. Both TOTAL and PAR are evaluated by highly sensitive capillary electrophoresis to meet the quality standards of sequencing libraries for RNA-seq. As an example of a downstream application, after sequencing, standard pipelines for gene expression analysis can be used to obtain differentially expressed genes at the transcriptome and translatome levels. In summary, this method, in combination with RNA-seq, allows the study of the translational regulation of eukaryotic mRNAs in a complex tissue such as the symbiotic nodule.

Atomic Force Microscopy to Study the Physical Properties of Epidermal Cells of Live Arabidopsis Roots.

A method is described here to characterize the physical properties of the cell wall of epidermal cells of living Arabidopsis roots through nanoindentations with an atomic force microscope (AFM) coupled with an optical inverted fluorescence microscope. The method consists of applying controlled forces to the sample while measuring its deformation, allowing quantifying parameters such as the apparent Young's modulus of cell walls at subcellular resolutions. It requires a careful mechanical immobilization of the sample and correct selection of indenters and indentation depths. Although it can be used only in external tissues, this method allows characterizing mechanical changes in plant cell walls during development and enables the correlation of these microscopic changes with the growth of an entire organ.

The Arabidopsis TETRATRICOPEPTIDE THIOREDOXIN-LIKE 1 Gene Is Involved in Anisotropic Root Growth during Osmotic Stress Adaptation.

Mutations in the Arabidopsis TETRATRICOPEPTIDE THIOREDOXIN-LIKE 1 (TTL1) gene cause reduced tolerance to osmotic stress evidenced by an arrest in root growth and root swelling, which makes it an interesting model to explore how root growth is controlled under stress conditions. We found that osmotic stress reduced the growth rate of the primary root by inhibiting the cell elongation in the elongation zone followed by a reduction in the number of cortical cells in the proximal meristem. We then studied the stiffness of epidermal cell walls in the root elongation zone of ttl1 mutants under osmotic stress using atomic force microscopy. In plants grown in control conditions, the mean apparent elastic modulus was 448% higher for live Col-0 cell walls than for ttl1 (88.1 ± 2.8 vs. 16.08 ± 6.9 kPa). Seven days of osmotic stress caused an increase in the stiffness in the cell wall of the cells from the elongation zone of 87% and 84% for Col-0 and ttl1, respectively. These findings suggest that TTL1 may play a role controlling cell expansion orientation during root growth, necessary for osmotic stress adaptation.

Entering the Next Dimension: Plant Genomes in 3D.

After linear sequences of genomes and epigenomic landscape data, the 3D organization of chromatin in the nucleus is the next level to be explored. Different organisms present a general hierarchical organization, with chromosome territories at the top. Chromatin interaction maps, obtained by chromosome conformation capture (3C)-based methodologies, for eight plant species reveal commonalities, but also differences, among them and with animals. The smallest structures, found in high-resolution maps of the Arabidopsis genome, are single genes. Epigenetic marks (histone modification and DNA methylation), transcriptional activity, and chromatin interaction appear to be correlated, and whether structure is the cause or consequence of the function of interacting regions is being actively investigated.

Spectral phasor analysis reveals altered membrane order and function of root hair cells in Arabidopsis dry2/sqe1-5 drought hypersensitive mutant.

Biological membranes allow the regulation of numerous cellular processes, which are affected when unfavorable environmental factors are perceived. Lipids and proteins are the principal components of biological membranes. Each lipid has unique biophysical properties, and, therefore the lipid composition of the membrane is critical to maintaining the bilayer structure and functionality. Membrane composition and integrity are becoming the focus of studies aiming to understand how plants adapt to its environment. In this study, using a combination of di-4-ANEPPDHQ fluorescence and spectral phasor analysis, we report that the drought hypersensitive/squalene epoxidase (dry2/sqe1-5) mutant with reduced major sterols such as sitosterol and stigmasterol in roots presented higher membrane fluidity than the wild type. Moreover, analysis of endomembrane dynamics showed that vesicle formation was affected in dry2/sqe1-5. Further analysis of proteins associated with sterol rich micro domains showed that dry2/sqe1-5 presented micro domains function altered.

Metabolic fingerprinting of Arabidopsis thaliana accessions.

In the post-genomic era much effort has been put on the discovery of gene function using functional genomics. Despite the advances achieved by these technologies in the understanding of gene function at the genomic and proteomic level, there is still a big genotype-phenotype gap. Metabolic profiling has been used to analyze organisms that have already been characterized genetically. However, there is a small number of studies comparing the metabolic profile of different tissues of distinct accessions. Here, we report the detection of over 14,000 and 17,000 features in inflorescences and leaves, respectively, in two widely used Arabidopsis thaliana accessions. A predictive Random Forest Model was developed, which was able to reliably classify tissue type and accession of samples based on LC-MS profile. Thereby we demonstrate that the morphological differences among A. thaliana accessions are reflected also as distinct metabolic phenotypes within leaves and inflorescences.

Unraveling the signal scenario of fruit set.

Long-term goals to impact or modify fruit quality and yield have been the target of researchers for many years. Different approaches such as traditional breeding,mutation breeding, and transgenic approaches have revealed a regulatory network where several hormones concur in a complex way to regulate fruit set and development,and these networks are shared in some way among species with different kinds of fruits. Understanding the molecular and biochemical networks of fruit set and development could be very useful for breeders to meet the current and future challenges of agricultural problems.

Toward understanding the role of CYP78A9 during Arabidopsis reproduction.

After fertilization in Arabidopsis, auxin response in ovules triggers fruit development through the stimulation of gibberellin metabolism. In a recent work, we showed that this model could not explain why CYP78A9 overexpression can uncouple these processes. The specific expression pattern of CYP78A9 suggests its involvement during reproductive development. Moreover, controlled pollination showed that CYP78A9 responds to fertilization. The genetic evidence supports the idea that CYP78A9 and its closest paralogs participate in a pathway that control floral organ size and ovule integuments development as denoted by the phenotypes of es1-D overexpression and cyp78a8 cyp78a9 double mutants. Furthermore, according to previous predictions, perturbations in the flavonol biosynthesis pathway were detected in cyp78a9, cyp78a8 cyp78a9 and es1-D mutants. However, they do not cause the observed phenotypes. Our results add new insights into the role of CYP78A9 in plant reproduction and present the first characterization of metabolite differences between mutants in this gene family.

Cytochrome P450 CYP78A9 is involved in Arabidopsis reproductive development.

Synchronized communication between gametophytic and sporophytic tissue is crucial for successful reproduction, and hormones seem to have a prominent role in it. Here, we studied the role of the Arabidopsis (Arabidopsis thaliana) cytochrome P450 CYP78A9 enzyme during reproductive development. First, controlled pollination experiments indicate that CYP78A9 responds to fertilization. Second, while CYP78A9 overexpression can uncouple fruit development from fertilization, the cyp78a8 cyp78a9 loss-of-function mutant has reduced seed set due to outer ovule integument development arrest, leading to female sterility. Moreover, CYP78A9 has a specific expression pattern in inner integuments in early steps of ovule development as well as in the funiculus, embryo, and integuments of developing seeds. CYP78A9 overexpression did not change the response to the known hormones involved in flower development and fruit set, and it did not seem to have much effect on the major known hormonal pathways. Furthermore, according to previous predictions, perturbations in the flavonol biosynthesis pathway were detected in cyp78a9, cyp78a8 cyp78a9, and empty siliques (es1-D) mutants. However, it appeared that they do not cause the observed phenotypes. In summary, these results add new insights into the role of CYP78A9 in plant reproduction and present, to our knowledge, the first characterization of metabolite differences between mutants in this gene family.