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.

Evaluation of strategies for improving the transgene expression in an oleaginous microalga Scenedesmus acutus.

BACKGROUND: Genetic transformation of microalgae has been hampered by inefficient transgene expression, limiting the progress of microalgal biotechnology. Many vector tools and strategies have been developed in recent years to improve transgene expression in the model microalga Chlamydomonas, but these were hardly applied to other microalgae. In this work, naturally-isolated oleaginous microalgae were accessed for genetic transformation, and various expression systems were evaluated in a selected microalga to circumvent inefficient transgene expression. RESULTS: Initially, a strain of Scenedesmus acutus was selected from the oleaginous microalgal collection based on its highest transformation rate and transgene stability. This strain, which had very low or no GFP reporter expression, was first tested to improve transgene expression by using intron-containing constructs and the transcript fusion using ble::E2A. The intron-containing constructs yielded 2.5-7.5% of transformants with 2-4-fold fluorescence signals, while the majority of the transformants of the transcript fusion had the fluorescence signals up to 10-fold. Subsequently, three UV-induced S. acutus mutants were isolated with moderate increases in the level and frequency of transgene expression (2-3-fold and 10-12%, respectively). Finally, a transcript fusion system was developed using psy white mutants with an expression vector containing PSY::E2A for complementation and light selection. Transformants with green colonies were selected under light exposure, and the transgene expression was detected at protein levels. Although the improvement using PSY::E2A was only minor (1-2-fold increase and ~ 7% of transformants), this system provides an alternative selectable marker that is compatible with large-scale culture. CONCLUSIONS: Here, the overall improvement of transgene expression using the Chlamydomonas tools was moderate. The most effective tool so far is the transcript fusion using ble::E2A system. This work demonstrates that, so far, genetic engineering of non-model microalgae is still a challenging task. Further development of tools and strategies for transgene expression in microalgae are critically needed.

Profiling of translatomes of in vivo-grown pollen tubes reveals genes with roles in micropylar guidance during pollination in Arabidopsis.

Transcriptome profiling has been used to identify genes expressed in pollen tubes elongating in vitro; however, little is known of the transcriptome of in vivo-grown pollen tubes due to the difficulty of collecting pollen that is elongating within the solid maternal gynoecium. Using a pollen-specific promoter (ProLAT52) to generate epitope-tagged polysomal-RNA complexes that could be affinity purified, we obtained mRNAs undergoing translation (the translatome) of in vivo-grown pollen tubes from self-pollinated gynoecia of Arabidopsis thaliana. Translatomes of pollen grains as well as in vivo- and in vitro-cultured pollen tubes were assayed by microarray analyses, revealing over 500 transcripts specifically enriched in in vivo-elongating pollen tubes. Functional analyses of several in vivo mutants (iv) of these pollination-enhanced transcripts revealed partial pollination/fertilization and seed formation defects in siliques (iv2, iv4, and iv6). Cytological observation confirmed the involvement of these genes in specialized processes including micropylar guidance (IV6 and IV4), pollen tube burst (IV2), and repulsion of multiple pollen tubes in embryo sac (IV2). In summary, the selective immunopurification of transcripts engaged with polysomes in pollen tubes within self-fertilized florets has identified a cohort of pollination-enriched transcripts that facilitated the identification of genes important in in vivo pollen tube biology.

Translational dynamics revealed by genome-wide profiling of ribosome footprints in Arabidopsis.

Translational regulation contributes to plasticity in metabolism and growth that enables plants to survive in a dynamic environment. Here, we used the precise mapping of ribosome footprints (RFs) on mRNAs to investigate translational regulation under control and sublethal hypoxia stress conditions in seedlings of Arabidopsis thaliana. Ribosomes were obtained by differential centrifugation or immunopurification and were digested with RNase I to generate footprint fragments that were deep-sequenced. Comparison of RF number and position on genic regions with fragmented total and polysomal mRNA illuminated numerous aspects of posttranscriptional and translational control under both growth conditions. When seedlings were oxygen-deprived, the frequency of ribosomes at the start codon was reduced, consistent with a global decline in initiation of translation. Hypoxia-up-regulated gene transcripts increased in polysome complexes during the stress, but the number of ribosomes per transcript relative to normoxic conditions was not enhanced. On the other hand, many mRNAs with limited change in steady-state abundance had significantly fewer ribosomes but with an overall similar distribution under hypoxia, consistent with restriction of initiation rather than elongation of translation. RF profiling also exposed the inhibitory effect of upstream ORFs on the translation of downstream protein-coding regions under normoxia, which was further modulated by hypoxia. The data document translation of alternatively spliced mRNAs and expose ribosome association with some noncoding RNAs. Altogether, we present an experimental approach that illuminates prevalent and nuanced regulation of protein synthesis under optimal and energy-limiting conditions.

Cold shock protein 1 chaperones mRNAs during translation in Arabidopsis thaliana.

RNA binding proteins (RBPs) function post-transcriptionally to fine-tune gene regulation. Arabidopsis thaliana has four Gly-rich, zinc finger-containing RBPs called cold shock proteins 1-4 (CSP1-CSP4), that possess an evolutionary conserved cold shock domain. Here, we determined that CSP1 associates with polyribosomes (polysomes) via an RNA-mediated interaction. Both the abundance and polysomal co-fractionation of CSP1 was enhanced in the cold (4°C), but did not influence global levels of polysomes, which were minimally perturbed by above freezing cold temperatures. Using a polyclonal antiserum, CSP1 was co-immunopurified with several hundred transcripts from rosettes of plants cultivated at 23°C or transferred to 4°C for 12 h. CSP1-associated mRNAs were characterized by G+C-rich 5' untranslated regions and gene ontologies related to cellular respiration, mRNA binding and translation. The majority of the CSP1-associated mRNAs were constitutively expressed and stable in the cold. CSP1 abundance was correlated with improved translation of ribosomal protein mRNAs during cold stress and improved maintenance of homeostasis and translation of mRNAs under water-deficit stress. In summary, CSP1 selectively chaperones mRNAs, providing translational enhancement during stress.

Characteristics and significance of intergenic polyadenylated RNA transcription in Arabidopsis.

The Arabidopsis (Arabidopsis thaliana) genome is the most well-annotated plant genome. However, transcriptome sequencing in Arabidopsis continues to suggest the presence of polyadenylated (polyA) transcripts originating from presumed intergenic regions. It is not clear whether these transcripts represent novel noncoding or protein-coding genes. To understand the nature of intergenic polyA transcription, we first assessed its abundance using multiple messenger RNA sequencing data sets. We found 6,545 intergenic transcribed fragments (ITFs) occupying 3.6% of Arabidopsis intergenic space. In contrast to transcribed fragments that map to protein-coding and RNA genes, most ITFs are significantly shorter, are expressed at significantly lower levels, and tend to be more data set specific. A surprisingly large number of ITFs (32.1%) may be protein coding based on evidence of translation. However, our results indicate that these "translated" ITFs tend to be close to and are likely associated with known genes. To investigate if ITFs are under selection and are functional, we assessed ITF conservation through cross-species as well as within-species comparisons. Our analysis reveals that 237 ITFs, including 49 with translation evidence, are under strong selective constraint and relatively distant from annotated features. These ITFs are likely parts of novel genes. However, the selective pressure imposed on most ITFs is similar to that of randomly selected, untranscribed intergenic sequences. Our findings indicate that despite the prevalence of ITFs, apart from the possibility of genomic contamination, many may be background or noisy transcripts derived from "junk" DNA, whose production may be inherent to the process of transcription and which, on rare occasions, may act as catalysts for the creation of novel genes.

Posttranscriptional control of photosynthetic mRNA decay under stress conditions requires 3' and 5' untranslated regions and correlates with differential polysome association in rice.

Abiotic stress, including drought, salinity, and temperature extremes, regulates gene expression at the transcriptional and posttranscriptional levels. Expression profiling of total messenger RNAs (mRNAs) from rice (Oryza sativa) leaves grown under stress conditions revealed that the transcript levels of photosynthetic genes are reduced more rapidly than others, a phenomenon referred to as stress-induced mRNA decay (SMD). By comparing RNA polymerase II engagement with the steady-state mRNA level, we show here that SMD is a posttranscriptional event. The SMD of photosynthetic genes was further verified by measuring the half-lives of the small subunit of Rubisco (RbcS1) and Chlorophyll a/b-Binding Protein1 (Cab1) mRNAs during stress conditions in the presence of the transcription inhibitor cordycepin. To discern any correlation between SMD and the process of translation, changes in total and polysome-associated mRNA levels after stress were measured. Total and polysome-associated mRNA levels of two photosynthetic (RbcS1 and Cab1) and two stress-inducible (Dehydration Stress-Inducible Protein1 and Salt-Induced Protein) genes were found to be markedly similar. This demonstrated the importance of polysome association for transcript stability under stress conditions. Microarray experiments performed on total and polysomal mRNAs indicate that approximately half of all mRNAs that undergo SMD remain polysome associated during stress treatments. To delineate the functional determinant(s) of mRNAs responsible for SMD, the RbcS1 and Cab1 transcripts were dissected into several components. The expressions of different combinations of the mRNA components were analyzed under stress conditions, revealing that both 3' and 5' untranslated regions are necessary for SMD. Our results, therefore, suggest that the posttranscriptional control of photosynthetic mRNA decay under stress conditions requires both 3' and 5' untranslated regions and correlates with differential polysome association.

Correlation Between Relative Gene Expression Patterns of Two Flowering locus T (MeFT1 and MeFT2) in Cassava Leaf and Flowering Traits Under Different Flowering Induction Conditions.

<b>Background and Objective:</b> <i>Flowering locus T</i> (<i>FT</i>) genes are involved in the flower induction mechanism in plants as florigen signals. The objective of this study was to study the relationship between the expression of <i>Flowering locus T</i> genes (<i>MeFTs</i>) in cassava and flowering traits under the different flowering induction conditions. <b>Materials and Methods:</b> The experimental design for flowering induction was RCBD for 4 replications. There were 5 treatment factors which were control, red light set from 5 pm to 7 am, 0.5 mM 6-benzyladenine (BA) with 2 mM silver thio-sulfate (STS), paclobutrazol for 6 g/plant and potassium chlorate (KCIO₃) for 250 g/plant. The number of plants with flower bunches and the average number of bunches per plant in two cassava varieties were collected each month from 5-9 months after planting (MAP). The leaf samples were collected from HB80 and R9 varieties at 5-7 MAP for RNA extraction to study <i>MeFT1</i> and <i>MeFT2</i> expression. <b>Results:</b> The results show that <i>MeFT1</i> expression level positively correlated with flowering traits in the same month, while <i>MeFT2</i> expression level positively correlated with flowering traits in the following months. <b>Conclusion:</b> Therefore, expression of <i>MeFT2</i> can be used for the prediction of cassava flowering in the following month which will assist the breeder for the crossing management.

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.

Genome-Wide Association Study of Local Thai Indica Rice Seedlings Exposed to Excessive Iron.

Excess soluble iron in acidic soil is an unfavorable environment that can reduce rice production. To better understand the tolerance mechanism and identify genetic loci associated with iron toxicity (FT) tolerance in a highly diverse indica Thai rice population, a genome-wide association study (GWAS) was performed using genotyping by sequencing and six phenotypic data (leaf bronzing score (LBS), chlorophyll content, shoot height, root length, shoot biomass, and root dry weight) under both normal and FT conditions. LBS showed a high negative correlation with the ratio of chlorophyll content and shoot biomass, indicating the FT-tolerant accessions can regulate cellular homeostasis when encountering stress. Sixteen significant single nucleotide polymorphisms (SNPs) were identified by association mapping. Validation of candidate SNP using other FT-tolerant accessions revealed that SNP:2_21262165 might be associated with tolerance to FT; therefore, it could be used for SNP marker development. Among the candidate genes controlling FT tolerance, RAR1 encodes an innate immune responsive protein that links to cellular redox homeostasis via interacting with abiotic stress-responsive Hsp90. Future research may apply the knowledge obtained from this study in the molecular breeding program to develop FT-tolerant rice varieties.

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.

Comparative Transcriptome Analysis of Waterlogging-Sensitive and Tolerant Zombi Pea (Vigna Vexillata) Reveals Energy Conservation and Root Plasticity Controlling Waterlogging Tolerance.

Vigna vexillata (zombi pea) is an underutilized legume crop considered to be a potential gene source in breeding for abiotic stress tolerance. This study focuses on the molecular characterization of mechanisms controlling waterlogging tolerance using two zombi pea varieties with contrasting waterlogging tolerance. Morphological examination revealed that in contrast to the sensitive variety, the tolerant variety was able to grow, maintain chlorophyll, form lateral roots, and develop aerenchyma in hypocotyl and taproots under waterlogging. To find the mechanism controlling waterlogging tolerance in zombi pea, comparative transcriptome analysis was performed using roots subjected to short-term waterlogging. Functional analysis indicated that glycolysis and fermentative genes were strongly upregulated in the sensitive variety, but not in the tolerant one. In contrast, the genes involved in auxin-regulated lateral root initiation and formation were expressed only in the tolerant variety. In addition, cell wall modification, aquaporin, and peroxidase genes were highly induced in the tolerant variety under waterlogging. Our findings suggest that energy management and root plasticity play important roles in mitigating the impact of waterlogging in zombi pea. The basic knowledge obtained from this study can be used in the molecular breeding of waterlogging-tolerant legume crops in the future.

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.

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.

De novo transcriptome analysis and gene expression profiling of an oleaginous microalga Scenedesmus acutus TISTR8540 during nitrogen deprivation-induced lipid accumulation.

Nitrogen deprivation (-N) has been used as a technique to promote lipid accumulation in various microalgae. Scenedesmus acutus is a promising oleaginous green microalga that can be cultivated in organic wastewater for biodiesel production. Nevertheless, the molecular mechanisms controlling S. acutus lipid accumulation in response to -N remain unidentified. Physiological study determined that -N reduced cell growth and photosynthetic pigments. On the other hand, it promoted carbohydrate and neutral lipid accumulation. To find the mechanisms underlying lipid accumulation, we performed de novo transcriptome profiling of the non-model S. acutus in response to -N. The transcriptome analysis revealed that glycolysis and starch degradation were up-regulated; on the contrary, gluconeogenesis, photosynthesis, triacylglycerol (TAG) degradation and starch synthesis were down-regulated by -N. Under -N, the carbon flux was shifted toward fatty acid and TAG synthesis, and the down regulation of TAG lipase genes may contribute to TAG accumulation. A comparative analysis of the -N transcriptomes of oleaginous microalgae identified that the down-regulation of multiple lipase genes was a specific mechanism found only in the -N transcriptome of S. acutus. Our study unraveled the mechanisms controlling -N-induced lipid accumulation in S. acutus, and provided new perspectives for the genetic manipulation of biodiesel-producing microalgae.

Increasing the Triacylglycerol Content in Dunaliella tertiolecta through Isolation of Starch-Deficient Mutants.

The production cost of biodiesel from microalgae is still not competitive, compared with that of petroleum fuels. The genetic improvement of microalgal strains to increase triacylglycerol (TAG) accumulation is one way to reduce production costs. One of the most promising approaches is the isolation of starch-deficient mutants, which have been reported to successfully increase TAG yields. To date, such a stable mutant is not available in an oleaginous marine microalga, despite several advantages of using marine species for biodiesel production. Algae in the genus Dunaliella are known to tolerate high salt concentration and other environmental stresses. In addition, the cultivation processes for large-scale outdoor commercialization have been well established for this genus. In this study, Dunaliella tertiolecta was used to screen for starch-deficient mutants, using an iodine vapor-staining method. Four out of 20,016 UV-mutagenized strains showed a substantial reduction of starch content. A significantly higher TAG content, up to 3-fold of the wild-type level, was observed in three of the mutants upon induction by nitrogen depletion. The carotenoid production and growth characteristics of these mutants, under both normal and oxidative stress conditions, were not compromised, suggesting that these processes are not necessarily affected by starch deficiency. The results from this work open up new possibilities for exploring Dunaliella for biodiesel production.

Transcriptomic analysis of submergence-tolerant and sensitive Brachypodium distachyon ecotypes reveals oxidative stress as a major tolerance factor.

When excessive amounts of water accumulate around roots and aerial parts of plants, submergence stress occurs. To find the integrated mechanisms of tolerance, we used ecotypes of the monocot model plant Brachypodium distachyon to screen for genetic material with contrasting submergence tolerance. For this purpose, we used a set of previously studied drought sensitive/tolerant ecotypes and the knowledge that drought tolerance is positively associated with submergence stress. We decided to contrast aerial tissue transcriptomes of the ecotype Bd21 14-day-old plants as sensitive and ecotype Bd2-3 as tolerant after 2 days of stress under a long-day photoperiod. Gene ontology and the grouping of transcripts indicated that tolerant Bd2-3 differentially down-regulated NITRATE REDUCTASE and ALTERNATIVE OXIDASE under stress and constitutively up-regulated HAEMOGLOBIN, when compared with the sensitive ecotype, Bd21. These results suggested the removal of nitric oxide, a gaseous phytohormone and concomitant reactive oxygen species as a relevant tolerance determinant. Other mechanisms more active in tolerant Bd2-3 were the pathogen response, glyoxylate and tricarboxylic acid cycle integration, and acetate metabolism. This data set could be employed to design further studies on the basic science of plant tolerance to submergence stress and its biotechnological application in the development of submergence-tolerant crops.

Ribosome profiling: a tool for quantitative evaluation of dynamics in mRNA translation.

Translational regulation is important for plant growth, metabolism, and acclimation to environmental challenges. Ribosome profiling involves the nuclease digestion of mRNAs associated with ribosomes and mapping of the generated ribosome-protected footprints to transcripts. This is useful for investigation of translational regulation. Here we present a detailed method to generate, purify, and high-throughput-sequence ribosome footprints from Arabidopsis thaliana using two different isolation methods, namely, conventional differential centrifugation and the translating ribosome affinity purification (TRAP) technology. These methodologies provide researchers with an opportunity to quantitatively assess with high-resolution the translational activity of individual mRNAs by determination of the position and number of ribosomes in the corresponding mRNA. The results can provide insights into the translation of upstream open reading frames, alternatively spliced transcripts, short open reading frames, and other aspects of translation.

Translating Ribosome Affinity Purification (TRAP) followed by RNA sequencing technology (TRAP-SEQ) for quantitative assessment of plant translatomes.

Translating Ribosome Affinity Purification (TRAP) is a technology to isolate the population of mRNAs associated with at least one 80S ribosome, referred as the translatome. TRAP is based on the expression of an epitope-tagged version of a ribosomal protein and the affinity purification of ribosomes and associated mRNAs using antibodies conjugated to agarose beads. Quantitative assessment of the translatome is achieved by direct RNA sequencing (RNA-SEQ), which provides accurate quantitation of ribosome-associated mRNAs and reveals alternatively spliced isoforms. Here we present a detailed procedure for TRAP, as well as a guide for preparation of RNA-SEQ libraries (TRAP-SEQ) and a primary data analysis. This methodology enables the study of translational dynamic by assessing rapid changes in translatomes, at organ or cell-type level, during development or in response to endogenous or exogenous stimuli.

Elucidation of the molecular responses to waterlogging in Jatropha roots by transcriptome profiling.

Jatropha (Jatropha curcas) is a promising oil-seed crop for biodiesel production. However, the species is highly sensitive to waterlogging, which can result in stunted growth and yield loss. To date, the molecular mechanisms underlying the responses to waterlogging in Jatropha remain elusive. Here, the transcriptome adjustment of Jatropha roots to waterlogging was examined by high-throughput RNA-sequencing (RNA-seq). The results indicated that 24 h of waterlogging caused significant changes in mRNA abundance of 1968 genes. Comprehensive gene ontology and functional enrichment analysis of root transcriptome revealed that waterlogging promoted responses to hypoxia and anaerobic respiration. On the other hand, the stress inhibited carbohydrate synthesis, cell wall biogenesis, and growth. The results also highlighted the roles of ethylene, nitrate, and nitric oxide in waterlogging acclimation. In addition, transcriptome profiling identified 85 waterlogging-induced transcription factors including members of AP2/ERF, MYB, and WRKY families implying that reprogramming of gene expression is a vital mechanism for waterlogging acclimation. Comparative analysis of differentially regulated transcripts in response to waterlogging among Arabidopsis, gray poplar, Jatropha, and rice further revealed not only conserved but species-specific regulation. Our findings unraveled the molecular responses to waterlogging in Jatropha and provided new perspectives for developing a waterlogging tolerant cultivar in the future.