Dynamic Light Regulation of Translation Status in Arabidopsis thaliana

Light, a dynamic environmental parameter, is an essential regulator of plant growth and development. Light-regulated transcriptional networks are well documented, whereas light-regulated post-transcriptional regulation has received limited attention. In this study, dynamics in translation of cytosolic mRNAs were evaluated at the genome-level in Arabidopsis thaliana seedlings grown under a typical light/dark diurnal regime, shifted to darkness at midday, and then re-illuminated. One-hour of unanticipated darkness reduced levels of polysomes by 17% in a manner consistent with inhibition of initiation of translation. This down-regulation of translation was reversed within 10 min of re-illumination. Quantitative comparison of the total cellular population of transcripts (the transcriptome) to those associated with one or more 80S ribosome (the translatome) identified over 1600 mRNAs that were differentially translated in response to light availability. Unanticipated darkness limited both transcription and translation of mRNAs encoding components of the photosynthetic machinery. Many mRNAs encoding proteins associated with the energy demanding process of protein synthesis were stable but sequestered in the dark, in a rapidly reversible manner. A meta-analysis determined these same transcripts were similarly and coordinately regulated in response to changes in oxygen availability. The dark and hypoxia translationally repressed mRNAs lack highly supported candidate RNA-regulatory elements but are characterized by G + C-rich 5′-untranslated regions. We propose that modulation of translation of a subset of cellular mRNAs functions as an energy conservation mechanism

Submergence and Waterlogging Stress in Plants: A Review Highlighting Research Opportunities and Understudied Aspects

Soil flooding creates composite and complex stress in plants known as either submergence or waterlogging stress depending on the depth of the water table. In nature, these stresses are important factors dictating the species composition of the ecosystem. On agricultural land, they cause economic damage associated with long-term social consequences. The understanding of the plant molecular responses to these two stresses has benefited from research studying individual components of the stress, in particular low-oxygen stress. To a lesser extent, other associated stresses and plant responses have been incorporated into the molecular framework, such as ion and ROS signaling, pathogen susceptibility, and organ-specific expression and development. In this review, we aim to highlight known or suspected components of submergence/waterlogging stress that have not yet been thoroughly studied at the molecular level in this context, such as miRNA and retrotransposon expression, the influence of light/dark cycles, protein isoforms, root architecture, sugar sensing and signaling, post-stress molecular events, heavy-metal and salinity stress, and mRNA dynamics (splicing, sequestering, and ribosome loading). Finally, we explore biotechnological strategies that have applied this molecular knowledge to develop cultivars resistant to flooding or to offer alternative uses of flooding-prone soils, like bioethanol and biomass production

Role of RNA Binding Proteins and Post-Transcriptional Regulation in Response to Environmental Changes in Arabidopsis thaliana

Light and temperature are two of the most important factors that regulate plant growth and development. The adaptation to alterations in light and temperature involves programed changes in gene expression that are necessary for physiological and morphological adaptations. Transcript abundance is frequently used to monitor changes in gene expression in response to sub-optimal growth conditions. However, transcript accumulation may not accurately mirror gene expression due to extensive post-transcriptional and post-translational regulation. In this dissertation, the significance in post-transcriptional regulation in response to unanticipated alterations in light availability was evaluated in seedlings of Arabidopsis thaliana. Early darkness resulted in translational inhibition and sequestration of a subset of cellular mRNAs. The translationally regulated mRNAs were enriched in transcripts encoding chloroplastic and protein synthesis machinery. The reduced engagement of the majority of these transcripts with ribosomes was rapidly reversed upon re-illumination. These results suggest that regulation of the translational status of chloroplastic and protein synthesis mRNAs may aid in energy conservation during unanticipated darkness. Towards the elucidation of RNA binding proteins that control selective mRNA translation, the complexes of Arabidopsis Cold Shock Proteins (CSPs 1-4) were characterized. Transgenic lines over-expressing epitope-tagged CSPs were established and used for cellular fractionation and mass spectrometric protein identification of immunopurified CSP complexes. Fluorescently-tagged CSPs and associated proteins were localized in transiently transformed cells by confocal microscopy. Together, the results of this survey indicate Arabidopsis CSPs are involved in multiple processes of post-transcriptional regulation, including pre-mRNA/rRNA processing and mRNA translation. Of the four CSPs, CSP1 co-fractionated with ribosome. A mild RNase A treatment of ribosome complexes combined with sucrose gradient fractionation confirmed that CSP1 is a polysome-associated RNA binding protein. CSP1 accumulated under normal growth condition and was induced by low-temperature. A polyclonal antibody prepared to specifically recognize CSP1 protein was used to co-immunopurify native CSP1 complexes. DNA microarray hybridization was used to compare total (transcriptome), polysomal (translatome), and CSP1-associated mRNAs from plants grown under normal or low-temperature conditions. The results demonstrate that CSP1 preferentially associated with mRNAs involved in RNA processing and protein synthesis. Many of the CSP1-associated mRNA also have high 5'-UTR with a high G+C content. The transcriptome and translatome adjustments during low-temperature stress were highly correlated, in contrast to the findings with early darkness. This work provides new perspectives on post-transcriptional gene regulation in response to environmental cues inArabidopsis, as well as a foundation for future in-depth characterization of mRNA-RNP control networks in plants

Overexpression of Jatropha curcas ERFVII2 Transcription Factor Confers Low Oxygen Tolerance in Transgenic Arabidopsis by Modulating Expression of Metabolic Enzymes and Multiple Stress-Responsive Genes

Enhancing crop tolerance to waterlogging is critical for improving food and biofuel security. In waterlogged soils, roots are exposed to a low oxygen environment. The group VII ethylene response factors (ERFVIIs) were recently identified as key regulators of plant low oxygen response. Oxygen-dependent N-end rule pathways can regulate the stability of ERFVIIs. This study aims to characterize the function of the Jatropha curcas ERFVIIs and the impact of N-terminal modification that stabilized the protein toward low oxygen response. This study revealed that all three JcERFVII proteins are substrates of the N-end rule pathway. Overexpression of JcERFVII2 conferred tolerance to low oxygen stress in Arabidopsis. In contrast, the constitutive overexpression of stabilized JcERFVII2 reduced low oxygen tolerance. RNA-seq was performed to elucidate the functional roles of JcERFVII2 and the impact of its N-terminal modification. Overexpression of both wildtype and stabilized JcERFVII2 constitutively upregulated the plant core hypoxia-responsive genes. Besides, overexpression of the stabilized JcERFVII2 further upregulated various genes controlling fermentative metabolic processes, oxidative stress, and pathogen responses under aerobic conditions. In summary, JcERFVII2 is an N-end rule regulated waterlogging-responsive transcription factor that modulates the expression of multiple stress-responsive genes; therefore, it is a potential candidate for molecular breeding of multiple stress-tolerant crops

Towards sex identification of Asian Palmyra palm (Borassus flabellifer L.) by DNA fingerprinting, suppression subtractive hybridization and de novo transcriptome sequencing

Background Asian Palmyra palm, the source of palm-sugar, is dioecious with a long juvenile period requiring at least 12 years to reach its maturity. To date, there is no reliable molecular marker for identifying sexes before the first bloom, limiting crop designs and utilization. We aimed to identify sex-linked markers for this palm using PCR-based DNA fingerprinting, suppression subtractive hybridization (SSH) and transcriptome sequencing. Methods DNA fingerprints were generated between males and females based on RAPD, AFLP, SCoT, modified SCoT, ILP, and SSR techniques. Large-scale cloning and screening of SSH libraries and de novo transcriptome sequencing of male and female cDNA from inflorescences were performed to identify sex-specific genes for developing sex-linked markers. Results Through extensive screening and re-testing of the DNA fingerprints (up to 1,204 primer pairs) and transcripts from SSH (>10,000 clones) and transcriptome data, however, no sex-linked marker was identified. Although de novo transcriptome sequencing of male and female inflorescences provided ∼32 million reads and 187,083 assembled transcripts, PCR analysis of selected sex-highly represented transcripts did not yield any sex-linked marker. This result may suggest the complexity and small sex-determining region of the Asian Palmyra palm. To this end, we provide the first global transcripts of male and female inflorescences of Asian Palmyra palm. Interestingly, sequence annotation revealed a large proportion of transcripts related to sucrose metabolism, which corresponds to the sucrose-rich sap produced in the inflorescences, and these transcripts will be useful for further understanding of sucrose production in sugar crop plants. Provided lists of sex-specific and differential-expressed transcripts would be beneficial to the further study of sexual development and sex-linked markers in palms and related species

RNA-Seq Reveals Waterlogging-Triggered Root Plasticity in Mungbean Associated with Ethylene and Jasmonic Acid Signal Integrators for Root Regeneration

Global climate changes increase the frequency and intensity of heavy precipitation events, which result in flooding or soil waterlogging. One way to overcome these low-oxygen stresses is via modifying the plant root system to improve internal aeration. Here, we used a comparative RNA-seq based transcriptomic approach to elucidate the molecular mechanisms of waterlogging-triggered root plasticity in mungbean (Vigna radiata), a major grain legume cultivated in Asia. Two mungbean varieties with contrasting waterlogging tolerance due to the plasticity of the root system architecture were subjected to short-term and long-term waterlogging. Then, RNA-seq was performed. Genes highly expressed in both genotypes under short-term waterlogging are related to glycolysis and fermentation. Under long-term waterlogging, the expression of these genes was less induced in the tolerant variety, suggesting it had effectively adapted to waterlogging via enhancing root plasticity. Remarkably, under short-term waterlogging, the expression of several transcription factors that serve as integrators for ethylene and jasmonic acid signals controlling root stem cell development was highly upregulated only in the tolerant variety. Sequentially, root development-related genes were more expressed in the tolerant variety under long-term waterlogging. Our findings suggest that ethylene and jasmonic acids may contribute to waterlogging-triggered root plasticity by relaying environmental signals to reprogram root regeneration. This research provides the basis for the breeding and genetic engineering of waterlogging-tolerant crops in the future

Submergence Stress Alters the Expression of Clock Genes and Configures New Zeniths and Expression of Outputs in Brachypodium distachyon

Plant networks of oscillating genes coordinate internal processes with external cues, contributing to increased fitness. We hypothesized that the response to submergence stress may dynamically change during different times of the day. In this work, we determined the transcriptome (RNA sequencing) of the model monocotyledonous plant, Brachypodium distachyon, during a day of submergence stress, low light, and normal growth. Two ecotypes of differential tolerance, Bd21 (sensitive) and Bd21-3 (tolerant), were included. We submerged 15-day-old plants under a long-day diurnal cycle (16 h light/8 h dark) and collected samples after 8 h of submergence at ZT0 (dawn), ZT8 (midday), ZT16 (dusk), ZT20 (midnight), and ZT24 (dawn). Rhythmic processes were enriched both with up- and down-regulated genes, and clustering highlighted that the morning and daytime oscillator components (PRRs) show peak expression in the night, and a decrease in the amplitude of the clock genes (GI, LHY, RVE) was observed. Outputs included photosynthesis-related genes losing their known rhythmic expression. Up-regulated genes included oscillating suppressors of growth, hormone-related genes with new late zeniths (e.g., JAZ1, ZEP), and mitochondrial and carbohydrate signaling genes with shifted zeniths. The results highlighted genes up-regulated in the tolerant ecotype such as METALLOTHIONEIN3 and ATPase INHIBITOR FACTOR. Finally, we show by luciferase assays that Arabidopsis thaliana clock genes are also altered by submergence changing their amplitude and phase. This study can guide the research of chronocultural strategies and diurnal-associated tolerance mechanisms