A phosphorylation-deficient ribosomal protein eS6 is largely functional in Arabidopsis thaliana, rescuing mutant defects from global translation and gene expression to photosynthesis and growth.

The eukaryote-specific ribosomal protein of the small subunit eS6 is phosphorylated through the target of rapamycin (TOR) kinase pathway. Although this phosphorylation event responds dynamically to environmental conditions and has been studied for over 50 years, its biochemical and physiological significance remains controversial and poorly understood. Here, we report data from Arabidopsis thaliana, which indicate that plants expressing only a phospho-deficient isoform of eS6 grow essentially normally under laboratory conditions. The eS6z (RPS6A) paralog of eS6 functionally rescued a double mutant in both rps6a and rps6b genes when expressed at approximately twice the wild-type dosage. A mutant isoform of eS6z lacking the major six phosphorylatable serine and threonine residues in its carboxyl-terminal tail also rescued the lethality, rosette growth, and polyribosome loading of the double mutant. This isoform also complemented many mutant phenotypes of rps6 that were newly characterized here, including photosynthetic efficiency, and most of the gene expression defects that were measured by transcriptomics and proteomics. However, compared with plants rescued with a phospho-enabled version of eS6z, the phospho-deficient seedlings retained a mild pointed-leaf phenotype, root growth was reduced, and certain cell cycle-related mRNAs and ribosome biogenesis proteins were misexpressed. The residual defects of the phospho-deficient seedlings could be understood as an incomplete rescue of the rps6 mutant defects. There was little or no evidence for gain-of-function defects. As previously published, the phospho-deficient eS6z also rescued the rps6a and rps6b single mutants; however, phosphorylation of the eS6y (RPS6B) paralog remained lower than predicted, further underscoring that plants can tolerate phospho-deficiency of eS6 well. Our data also yield new insights into how plants cope with mutations in essential, duplicated ribosomal protein isoforms.

Prioritization of the concepts and skills in quantitative education for graduate students in biomedical science.

Substantial guidance is available on undergraduate quantitative training for biologists, including reports focused on biomedical science. Far less attention has been paid to the graduate curriculum and the particular challenges of the diversity of specialization within the life sciences. We propose an innovative approach to quantitative education that goes beyond recommendations of a course or set of courses or activities, derived from analysis of the expectations for students in particular programs. Due to the plethora of quantitative methods, it is infeasible to expect that biomedical PhD students can be exposed to more than a minority of the quantitative concepts and techniques employed in modern biology. We collected key recent papers suggested by the faculty in biomedical science programs, chosen to include important scientific contributions that the faculty consider appropriate for all students in the program to be able to read with confidence. The quantitative concepts and methods inherent in these papers were then analyzed and categorized to provide a rational basis for prioritization of those concepts to be emphasized in the education program. This novel approach to prioritization of quantitative skills and concepts provides an effective method to drive curricular focus based upon program-specific faculty input for science programs of all types. The results of our particular application to biomedical science training highlight the disconnect between typical undergraduate quantitative education for life science students, focused on continuous mathematics, and the concepts and skills in graphics, statistics, and discrete mathematics that arise from priorities established by biomedical science faculty. There was little reference in the key recent papers chosen by faculty to classic mathematical areas such as calculus which make up a large component of the formal undergraduate mathematics training of graduate students in biomedical areas.

A Miniature Sucrose Gradient for Polysome Profiling.

Polysome profiling by sucrose density gradient centrifugation is commonly used to study the overall degree of translation (messenger RNA to protein synthesis). Traditionally, the method begins with synthesis of a 5-10 mL sucrose gradient onto which 0.5-1 mL of cell extract is layered and centrifuged at high speed for 3-4 h in a floor-model ultracentrifuge. After centrifugation, the gradient solution is passed through an absorbance recorder to generate a polysome profile. Ten to twelve fractions (0.8-1 mL each) are collected for isolating different RNA and protein populations. The overall method is tedious and lengthy (6-9 h), requires access to a suitable ultracentrifuge rotor and centrifuge, and requires a substantial amount of tissue material, which can be a limiting factor. Moreover, there is often a dilemma over the quality of RNA and protein populations in the individual fractions due to the extended experiment times. To overcome these challenges, here we describe a miniature sucrose gradient for polysome profiling using Arabidopsis thaliana seedlings that takes ~1 h centrifugation time in a tabletop ultracentrifuge, reduced gradient synthesis time, and also less tissue material. The protocol described here can be easily adapted to a wide variety of organisms and polysome profiling of organelles, such as chloroplasts and mitochondria. Key Features • Mini sucrose gradient for polysome profiling that requires less than half the processing time vs. traditional methods. • Reduced starting tissue material and sample volume for sucrose gradients. • Feasibility of RNA and protein isolation from polysome fractions. • Protocol can be easily modified to a wide variety of organisms (and even polysome profiling of organelles, such as chloroplast and mitochondria). Graphical Overview.

Modulation of GCN2 activity under excess light stress by osmoprotectants and amino acids.

The protein kinase GCN2 (General Control Nonderepressible2) and its phosphorylation target, the eukaryotic translation initiation factor (eIF)2α represent the core module of the plant's integrated stress response, a signaling pathway widely conserved in eukaryotes that can rapidly regulate translation in response to stressful conditions. Recent findings indicate that the Arabidopsis thaliana GCN2 protein operates under the command of reactive oxygen species (ROS) emanating from the chloroplast under a variety of abiotic stresses such as excess light. To get deeper insights into the mechanism of GCN2 activation under excess light, we assessed the role of amino acids in view of the classic function of GCN2 as a sensor of amino acid status. Additionally, given that osmoprotectants can counteract ROS-related stresses, we tested their ability to mitigate GCN2 activity. Our results demonstrate that certain amino acids and osmoprotectants attenuate eIF2α-phosphorylation under excess light stress to some degree. Future investigations into the biochemical mechanisms of these natural compounds on GCN2 signaling activity will provide better insights into the GCN2-eIF2α regulation.

Review: Emerging roles of the signaling network of the protein kinase GCN2 in the plant stress response.

The pan-eukaryotic protein kinase GCN2 (General Control Nonderepressible2) regulates the translation of mRNAs in response to external and metabolic conditions. Although GCN2 and its substrate, translation initiation factor 2 (eIF2) α, and several partner proteins are substantially conserved in plants, this kinase has assumed novel functions in plants, including in innate immunity and retrograde signaling between the chloroplast and cytosol. How exactly some of the biochemical paradigms of the GCN2 system have diverged in the green plant lineage is only partially resolved. Specifically, conflicting data underscore and cast doubt on whether GCN2 regulates amino acid biosynthesis; also whether phosphorylation of eIF2α can in fact repress global translation or activate mRNA specific translation via upstream open reading frames; and whether GCN2 is controlled in vivo by the level of uncharged tRNA. This review examines the status of research on the eIF2α kinase, GCN2, its function in the response to xenobiotics, pathogens, and abiotic stress conditions, and its rather tenuous role in the translational control of mRNAs.

Early Detection of Daylengths with a Feedforward Circuit Coregulated by Circadian and Diurnal Cycles.

Light-entrained circadian clocks confer rhythmic dynamics of cellular and molecular activities to animals and plants. These intrinsic clocks allow stable anticipations to light-dark (diel) cycles. Many genes in the model plant Arabidopsis thaliana are regulated by diel cycles via pathways independent of the clock, suggesting that the integration of circadian and light signals is important for the fitness of plants. Previous studies of light-clock signal integrations have focused on moderate phase adjustment of the two signals. However, dynamical features of integrations across a broad range of phases remain elusive. Phosphorylation of ribosomal protein of the small subunit 6 (eS6), a ubiquitous post-translational modification across kingdoms, is influenced by the circadian clock and the light-dark (diel) cycle in an opposite manner. To understand this striking phenomenon and its underlying information processing capabilities, we built a mathematical model for the eS6 phosphorylation (eS6-P) control circuit. We found that the dynamics of eS6-P can be explained by a feedforward circuit with inputs from both circadian and diel cycles. Furthermore, the early day response of this circuit with dual rhythmic inputs is sensitive to the changes in daylength, including both transient and gradual changes observed in realistic light intervals across a year, because of weather and seasons. By analyzing published gene expression data, we found that the dynamics produced by the eS6-P control circuit can be observed in the expression profiles of a large number of genes. Our work provides mechanistic insights into the complex dynamics of a ribosomal protein, and it proposes a previously underappreciated function of the circadian clock, which not only prepares organisms for normal diel cycles but also helps to detect both transient and seasonal changes with a predictive power.

Light-Dependent Activation of the GCN2 Kinase Under Cold and Salt Stress Is Mediated by the Photosynthetic Status of the Chloroplast.

Regulation of cytosolic mRNA translation is a key node for rapid adaptation to environmental stress conditions. In yeast and animals, phosphorylation of the α-subunit of eukaryotic translation initiation factor eIF2 is the most thoroughly characterized event for regulating global translation under stress. In plants, the GCN2 kinase (General Control Nonderepressible-2) is the only known kinase for eIF2α. GCN2 is activated under a variety of stresses including reactive oxygen species (ROS). Here, we provide new evidence that the GCN2 kinase in Arabidopsis is also activated rapidly and in a light-dependent manner by cold and salt treatments. These treatments alone did not repress global mRNA ribosome loading in a major way. The activation of GCN2 was accompanied by a more oxidative environment and was attenuated by inhibitors of photosynthetic electron transport, suggesting that it is gated by the redox poise or the reactive oxygen status of the chloroplast. In keeping with these results, gcn2 mutant seedlings were more sensitive than wild type to both cold and salt in a root elongation assay. These data suggest that cold and salt stress may both affect the status of the cytosolic translation apparatus via the conserved GCN2-eIF2α module. The potential role of the GCN2 kinase pathway in the global repression of translation under abiotic stress is discussed.

Translational gene regulation in plants: A green new deal.

The molecular machinery for protein synthesis is profoundly similar between plants and other eukaryotes. Mechanisms of translational gene regulation are embedded into the broader network of RNA-level processes including RNA quality control and RNA turnover. However, over eons of their separate history, plants acquired new components, dropped others, and generally evolved an alternate way of making the parts list of protein synthesis work. Research over the past 5 years has unveiled how plants utilize translational control to defend themselves against viruses, regulate translation in response to metabolites, and reversibly adjust translation to a wide variety of environmental parameters. Moreover, during seed and pollen development plants make use of RNA granules and other translational controls to underpin developmental transitions between quiescent and metabolically active stages. The economics of resource allocation over the daily light-dark cycle also include controls over cellular protein synthesis. Important new insights into translational control on cytosolic ribosomes continue to emerge from studies of translational control mechanisms in viruses. Finally, sketches of coherent signaling pathways that connect external stimuli with a translational response are emerging, anchored in part around TOR and GCN2 kinase signaling networks. These again reveal some mechanisms that are familiar and others that are different from other eukaryotes, motivating deeper studies on translational control in plants. This article is categorized under: Translation > Translation Regulation RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.

Light Activates the Translational Regulatory Kinase GCN2 via Reactive Oxygen Species Emanating from the Chloroplast.

Cytosolic mRNA translation is subject to global and mRNA-specific controls. Phosphorylation of the translation initiation factor eIF2α anchors a reversible regulatory switch that represses cytosolic translation globally. The stress-responsive GCN2 kinase is the only known kinase for eIF2α serine 56 in Arabidopsis (Arabidopsis thaliana). Here, we show that conditions that generate reactive oxygen species (ROS) in the chloroplast, including dark-light transitions, high light, and the herbicide methyl viologen, rapidly activated GCN2 kinase, whereas mitochondrial and endoplasmic reticulum stress did not. GCN2 activation was light dependent and mitigated by photosynthesis inhibitors and ROS quenchers. Accordingly, the seedling growth of multiple Arabidopsis gcn2 mutants was retarded under excess light conditions, implicating the GCN2-eIF2α pathway in responses to light and associated ROS. Once activated, GCN2 kinase preferentially suppressed the ribosome loading of mRNAs for functions such as mitochondrial ATP synthesis, the chloroplast thylakoids, vesicle trafficking, and translation. The gcn2 mutant overaccumulated transcripts functionally related to abiotic stress, including oxidative stress, as well as innate immune responses. Accordingly, gcn2 displayed defects in immune priming by the fungal elicitor, chitin. Therefore, we provide evidence that reactive oxygen species produced by the photosynthetic apparatus help activate the highly conserved GCN2 kinase, leading to eIF2α phosphorylation and thus affecting the status of the cytosolic protein synthesis apparatus.

ErbB-3 BINDING PROTEIN 1 Regulates Translation and Counteracts RETINOBLASTOMA RELATED to Maintain the Root Meristem.

The ErbB-3 BINDING PROTEIN 1 (EBP1) drives growth, but the mechanism of how it acts in plants is little understood. Here, we show that EBP1 expression and protein abundance in Arabidopsis (Arabidopsis thaliana) are predominantly confined to meristematic cells and are induced by sucrose and partially dependent on TARGET OF RAPAMYCIN (TOR) kinase activity. Consistent with being downstream of TOR, silencing of EBP1 restrains, while overexpression promotes, root growth, mostly under sucrose-limiting conditions. Inducible overexpression of RETINOBLASTOMA RELATED (RBR), a sugar-dependent transcriptional repressor of cell proliferation, depletes meristematic activity and causes precocious differentiation, which is attenuated by EBP1. To understand the molecular mechanism, we searched for EBP1- and RBR-interacting proteins by affinity purification and mass spectrometry. In line with the double-stranded RNA-binding activity of EBP1 in human (Homo sapiens) cells, the overwhelming majority of EBP1 interactors are part of ribonucleoprotein complexes regulating many aspects of protein synthesis, including ribosome biogenesis and mRNA translation. We confirmed that EBP1 associates with ribosomes and that EBP1 silencing hinders ribosomal RNA processing. We revealed that RBR also interacts with a set of EBP1-associated nucleolar proteins as well as factors that function in protein translation. This suggests EBP1 and RBR act antagonistically on common processes that determine the capacity for translation to tune meristematic activity in relation to available resources.

What makes ribosomes tick?

In most organisms, gene expression over the course of the day is under the control of the circadian clock. The canonical clock operates as a gene expression circuit that is controlled at the level of transcription, and transcriptional control is also a major clock output. However, rhythmic transcription cannot explain all the observed rhythms in protein accumulation. Although it is clear that rhythmic gene expression also involves RNA processing and protein turnover, until two years ago little was known in any eukaryote about diel dynamics of mRNA translation into protein. A recent series of studies in animals and plants demonstrated that diel cycles of translation efficiency are widespread across the tree of life and its transcriptomes. There are surprising parallels between the patterns of diel translation in mammals and plants. For example, ribosomal proteins and mitochondrial proteins are under translational control in mouse liver, human tissue culture, and Arabidopsis seedlings. In contrast, the way in which the circadian clock, light-dark changes, and other environmental factors such as nutritional signals interact to drive the cycles of translation may differ between organisms. Further investigation is needed to identify the signaling pathways, biochemical mechanisms, RNA sequence features, and the physiological implications of diel translation.

Graduate Training at the Interface of Computational and Experimental Biology: An Outcome Report from a Partnership of Volunteers between a University and a National Laboratory.

Leading voices in the biological sciences have called for a transformation in graduate education leading to the PhD degree. One area commonly singled out for growth and innovation is cross-training in computational science. In 1998, the University of Tennessee (UT) founded an intercollegiate graduate program called the UT-ORNL Graduate School of Genome Science and Technology in partnership with the nearby Oak Ridge National Laboratory. Here, we report outcome data that attest to the program's effectiveness in graduating computationally enabled biologists for diverse careers. Among 77 PhD graduates since 2003, the majority came with traditional degrees in the biological sciences, yet two-thirds moved into computational or hybrid (computational-experimental) positions. We describe the curriculum of the program and how it has changed. We also summarize how the program seeks to establish cohesion between computational and experimental biologists. This type of program can respond flexibly and dynamically to unmet training needs. In conclusion, this study from a flagship, state-supported university may serve as a reference point for creating a stable, degree-granting, interdepartmental graduate program in computational biology and allied areas.

Phosphorylation of Ribosomal Protein RPS6 Integrates Light Signals and Circadian Clock Signals.

The translation of mRNA into protein is tightly regulated by the light environment as well as by the circadian clock. Although changes in translational efficiency have been well documented at the level of mRNA-ribosome loading, the underlying mechanisms are unclear. The reversible phosphorylation of RIBOSOMAL PROTEIN OF THE SMALL SUBUNIT 6 (RPS6) has been known for 40 years, but the biochemical significance of this event remains unclear to this day. Here, we confirm using a clock-deficient strain of Arabidopsis thaliana that RPS6 phosphorylation (RPS6-P) is controlled by the diel light-dark cycle with a peak during the day. Strikingly, when wild-type, clock-enabled, seedlings that have been entrained to a light-dark cycle are placed under free-running conditions, the circadian clock drives a cycle of RPS6-P with an opposite phase, peaking during the subjective night. We show that in wild-type seedlings under a light-dark cycle, the incoherent light and clock signals are integrated by the plant to cause an oscillation in RPS6-P with a reduced amplitude with a peak during the day. Sucrose can stimulate RPS6-P, as seen when sucrose in the medium masks the light response of etiolated seedlings. However, the diel cycles of RPS6-P are observed in the presence of 1% sucrose and in its absence. Sucrose at a high concentration of 3% appears to interfere with the robust integration of light and clock signals at the level of RPS6-P. Finally, we addressed whether RPS6-P occurs uniformly in polysomes, non-polysomal ribosomes and their subunits, and non-ribosomal protein. It is the polysomal RPS6 whose phosphorylation is most highly stimulated by light and repressed by darkness. These data exemplify a striking case of contrasting biochemical regulation between clock signals and light signals. Although the physiological significance of RPS6-P remains unknown, our data provide a mechanistic basis for the future understanding of this enigmatic event.

Meeting report: processing, translation, decay - three ways to keep RNA sizzling.

This meeting report highlights key trends that emerged from a conference entitled Post-Transcriptional Gene Regulation in Plants, which was held 14-15 July 2016, as a satellite meeting of the annual meeting of the American Society of Plant Biologists in Austin, Texas. The molecular biology of RNA is emerging as an integral part of the framework for plants' responses to environmental challenges such as drought and heat, hypoxia, nutrient deprivation, light and pathogens. Moreover, the conference illustrated how a multitude of customized and pioneering omics-related technologies are being applied, more and more often in combination, to describe and dissect the complexities of gene expression at the post-transcriptional level.

The Circadian Clock Modulates Global Daily Cycles of mRNA Ribosome Loading.

Circadian control of gene expression is well characterized at the transcriptional level, but little is known about diel or circadian control of translation. Genome-wide translation state profiling of mRNAs in Arabidopsis thaliana seedlings grown in long day was performed to estimate ribosome loading per mRNA. The experiments revealed extensive translational regulation of key biological processes. Notably, translation of mRNAs for ribosomal proteins and mitochondrial respiration peaked at night. Central clock mRNAs are among those subject to fluctuations in ribosome loading. There was no consistent phase relationship between peak translation states and peak transcript levels. The overlay of distinct transcriptional and translational cycles can be expected to alter the waveform of the protein synthesis rate. Plants that constitutively overexpress the clock gene CCA1 showed phase shifts in peak translation, with a 6-h delay from midnight to dawn or from noon to evening being particularly common. Moreover, cycles of ribosome loading that were detected under continuous light in the wild type collapsed in the CCA1 overexpressor. Finally, at the transcript level, the CCA1-ox strain adopted a global pattern of transcript abundance that was broadly correlated with the light-dark environment. Altogether, these data demonstrate that gene-specific diel cycles of ribosome loading are controlled in part by the circadian clock.

Analysis of mRNA translation states in Arabidopsis over the diurnal cycle by polysome microarray.

Gene regulation at the level of translation occurs in response to environmental perturbation and is increasingly recognized as a factor affecting plant development. Despite extensive knowledge of transcriptional control, very little is known about translational regulation of genes in response to the daily light/dark cycles. Here we describe the experimental layout designed to address how the translation states of genes change at various times during a diurnal cycle in Arabidopsis thaliana seedlings. We have adopted a strategy combining sucrose-gradient profiling of ribosomes and high-throughput microarray analysis of the ribosome-associated mRNA to investigate the translational landscape of the Arabidopsis genome. This is a powerful technique that can be easily extended to study translation regulation in different genetic backgrounds and under various environmental conditions.

Translational control of Arabidopsis meristem stability and organogenesis by the eukaryotic translation factor eIF3h.

Essentially all aboveground plant tissues develop from the stem cells in the primary shoot apical meristem. Proliferation of the stem cell population in the Arabidopsis shoot apical meristem is tightly controlled by a feedback loop formed primarily by the homeodomain transcription factor WUSCHEL (WUS) and the CLAVATA ligand-receptor system. In this study, it is shown that mutation of a translation initiation factor, eIF3h, causes a tendency to develop a strikingly enlarged shoot apical meristem with elevated and ectopic expression of WUS and CLAVATA3 (CLV3). Many of the mRNAs that function in apical meristem maintenance possess upstream open reading frames (uORFs), translational attenuators that render translation partially dependent on eIF3h. Specifically, the mRNA for the receptor kinase, CLV1, is undertranslated in the eif3h mutant as shown by transient and transgenic expression assays. Concordant phenotypic observations include defects in organ polarity and in translation of another uORF-containing mRNA, ASYMMETRIC LEAVES 1 (AS1), in eif3h. In summary, the expression of developmental regulatory mRNAs is attenuated by uORFs, and this attenuation is balanced in part by the translation initiation factor, eIF3h. Thus, translational control plays a key role in Arabidopsis stem cell regulation and organogenesis.

Regulation of plant translation by upstream open reading frames.

We review the evidence that upstream open reading frames (uORFs) function as RNA sequence elements for post-transcriptional control of gene expression, specifically translation. uORFs are highly abundant in the genomes of angiosperms. Their negative effect on translation is often attenuated by ribosomal translation reinitiation, a process whose molecular biochemistry is still being investigated. Certain uORFs render translation responsive to small molecules, thus offering a path for metabolic control of gene expression in evolution and synthetic biology. In some cases, uORFs form modular logic gates in signal transduction. uORFs thus provide eukaryotes with a functionality analogous to, or comparable to, riboswitches and attenuators in prokaryotes. uORFs exist in many genes regulating development and point toward translational control of development. While many uORFs appear to be poorly conserved, and the number of genes with conserved-peptide uORFs is modest, many mRNAs have a conserved pattern of uORFs. Evolutionarily, the gain and loss of uORFs may be a widespread mechanism that diversifies gene expression patterns. Last but not least, this review includes a dedicated uORF database for Arabidopsis.

Translational Regulation of Cytoplasmic mRNAs.

Translation of the coding potential of a messenger RNA into a protein molecule is a fundamental process in all living cells and consumes a large fraction of metabolites and energy resources in growing cells. Moreover, translation has emerged as an important control point in the regulation of gene expression. At the level of gene regulation, translational control is utilized to support the specific life histories of plants, in particular their responses to the abiotic environment and to metabolites. This review summarizes the diversity of translational control mechanisms in the plant cytoplasm, focusing on specific cases where mechanisms of translational control have evolved to complement or eclipse other levels of gene regulation. We begin by introducing essential features of the translation apparatus. We summarize early evidence for translational control from the pre-Arabidopsis era. Next, we review evidence for translation control in response to stress, to metabolites, and in development. The following section emphasizes RNA sequence elements and biochemical processes that regulate translation. We close with a chapter on the role of signaling pathways that impinge on translation.

The global translation profile in a ribosomal protein mutant resembles that of an eIF3 mutant.

BACKGROUND: Genome-wide assays performed in Arabidopsis and other organisms have revealed that the translation status of mRNAs responds dramatically to different environmental stresses and genetic lesions in the translation apparatus. To identify additional features of the global landscape of translational control, we used microarray analysis of polysomal as well as non-polysomal mRNAs to examine the defects in translation in a poly(A) binding protein mutant, pab2 pab8, as well as in a mutant of a large ribosomal subunit protein, rpl24b/shortvalve1. RESULTS: The mutation of RPL24B stimulated the ribosome occupancy of mRNAs for nuclear encoded ribosomal proteins. Detailed analysis yielded new insights into the translational regulon containing the ribosomal protein mRNAs. First, the ribosome occupancy defects in the rpl24b mutant partially overlapped with those in a previously analyzed initiation factor mutant, eif3h. Second, a group of mRNAs with incomplete coding sequences appeared to be uncoupled from the regulon, since their dependence on RPL24B differed from regular mRNAs. Third, different sister paralogs of the ribosomal proteins differed in their translation state in the wild-type. Some sister paralogs also differed in their response to the rpl24b mutation. In contrast to rpl24b, the pab2 pab8 mutant revealed few gene specific translational defects, but a group of seed storage protein mRNAs were stimulated in their ribosome occupancy. In the course of this work, while optimizing the statistical analysis of ribosome occupancy data, we collected 12 biological replicates of translation states from wild-type seedlings. We defined 20% of mRNAs as having a high variance in their translation state. Many of these mRNAs were functionally associated with responses to the environment, suggesting that subtle variation in the environmental conditions is sensed by plants and transduced to affect the translational efficiency of hundreds of mRNAs. CONCLUSIONS: These data represent the first genome-wide analysis of translation in a eukaryote defective in the large ribosomal subunit. RPL24 and eIF3h play similar but non-identical roles in eukaryotic translation. The data also shed light on the fine structure of the regulon of ribosomal protein mRNAs.