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Plant roots integrate environmental signals with development using exquisite spatiotemporal control. This is apparent in the deposition of suberin, an apoplastic diffusion barrier, which regulates flow of water, solutes and gases, and is environmentally plastic. Suberin is considered a hallmark of endodermal differentiation but is absent in the tomato endodermis. Instead, suberin is present in the exodermis, a cell type that is absent in the model organism Arabidopsis thaliana. Here we demonstrate that the suberin regulatory network has the same parts driving suberin production in the tomato exodermis and the Arabidopsis endodermis. Despite this co-option of network components, the network has undergone rewiring to drive distinct spatial expression and with distinct contributions of specific genes. Functional genetic analyses of the tomato MYB92 transcription factor and ASFT enzyme demonstrate the importance of exodermal suberin for a plant water-deficit response and that the exodermal barrier serves an equivalent function to that of the endodermis and can act in its place., (© 2024. The Author(s).)

Light regulates chlorophyll homeostasis and photosynthesis via various molecular mechanisms in plants. The light regulation of transcription and protein stability of nuclear-encoded chloroplast proteins have been extensively studied, but how light regulation of mRNA metabolism affects abundance of nuclear-encoded chloroplast proteins and chlorophyll homeostasis remains poorly understood. Here we show that the blue light receptor cryptochrome 2 (CRY2) and the METTL16-type m 6 A writer FIONA1 (FIO1) regulate chlorophyll homeostasis in response to blue light. In contrast to the CRY2-mediated photo-condensation of the mRNA adenosine methylase (MTA), photoexcited CRY2 co-condenses FIO1 only in the presence of the CRY2-signalling protein SUPPRESSOR of PHYTOCHROME A (SPA1). CRY2 and SPA1 synergistically or additively activate the RNA methyltransferase activity of FIO1 in vitro, whereas CRY2 and FIO1, but not MTA, are required for the light-induced methylation and translation of the mRNAs encoding multiple chlorophyll homeostasis regulators in vivo. Our study demonstrates that the light-induced liquid-liquid phase separation of the photoreceptor/writer complexes is commonly involved in the regulation of photoresponsive changes of mRNA methylation, whereas the different photo-condensation mechanisms of the CRY/FIO1 and CRY/MTA complexes explain, at least partially, the writer-specific functions in plant photomorphogenesis., (© 2023. The Author(s).)

Cell surface pattern recognition receptors sense invading pathogens by binding microbial or endogenous elicitors to activate plant immunity. These responses are under tight control to avoid excessive or untimely activation of cellular responses, which may otherwise be detrimental to host cells. How this fine-tuning is accomplished is an area of active study. We previously described a suppressor screen that identified Arabidopsis thaliana mutants with regained immune signaling in the immunodeficient genetic background bak1-5, which we named modifier of bak1-5 (mob) mutants. Here, we report that bak1-5 mob7 mutant restores elicitor-induced signaling. Using a combination of map-based cloning and whole-genome resequencing, we identified MOB7 as conserved binding of eIF4E1 (CBE1), a plant-specific protein that interacts with the highly conserved eukaryotic translation initiation factor eIF4E1. Our data demonstrate that CBE1 regulates the accumulation of respiratory burst oxidase homolog D, the NADPH oxidase responsible for elicitor-induced apoplastic reactive oxygen species production. Furthermore, several mRNA decapping and translation initiation factors colocalize with CBE1 and similarly regulate immune signaling. This study thus identifies a novel regulator of immune signaling and provides new insights into reactive oxygen species regulation, potentially through translational control, during plant stress responses., Competing Interests: Conflict of interest The authors declare no conflict of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Flooded plants experience impaired gas diffusion underwater, leading to oxygen deprivation (hypoxia). The volatile plant hormone ethylene is rapidly trapped in submerged plant cells and is instrumental for enhanced hypoxia acclimation. However, the precise mechanisms underpinning ethylene-enhanced hypoxia survival remain unclear. We studied the effect of ethylene pretreatment on hypoxia survival of Arabidopsis (Arabidopsis thaliana) primary root tips. Both hypoxia itself and re-oxygenation following hypoxia are highly damaging to root tip cells, and ethylene pretreatments reduced this damage. Ethylene pretreatment alone altered the abundance of transcripts and proteins involved in hypoxia responses, root growth, translation, and reactive oxygen species (ROS) homeostasis. Through imaging and manipulating ROS abundance in planta, we demonstrated that ethylene limited excessive ROS formation during hypoxia and subsequent re-oxygenation and improved oxidative stress survival in a PHYTOGLOBIN1-dependent manner. In addition, we showed that root growth cessation via ethylene and auxin occurred rapidly and that this quiescence behavior contributed to enhanced hypoxia tolerance. Collectively, our results show that the early flooding signal ethylene modulates a variety of processes that all contribute to hypoxia survival., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Plant species have evolved myriads of solutions, including complex cell type development and regulation, to adapt to dynamic environments. To understand this cellular diversity, we profiled tomato root cell type translatomes. Using xylem differentiation in tomato, examples of functional innovation, repurposing, and conservation of transcription factors are described, relative to the model plant Arabidopsis. Repurposing and innovation of genes are further observed within an exodermis regulatory network and illustrate its function. Comparative translatome analyses of rice, tomato, and Arabidopsis cell populations suggest increased expression conservation of root meristems compared with other homologous populations. In addition, the functions of constitutively expressed genes are more conserved than those of cell type/tissue-enriched genes. These observations suggest that higher order properties of cell type and pan-cell type regulation are evolutionarily conserved between plants and animals., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Gene transcription is counterbalanced by messenger RNA decay processes that regulate transcript quality and quantity. We show here that the evolutionarily conserved DHH1/DDX6-like RNA hellicases of Arabidopsis thaliana control the ephemerality of a subset of cellular mRNAs. These RNA helicases co-localize with key markers of processing bodies and stress granules and contribute to their subcellular dynamics. They function to limit the precocious accumulation and ribosome association of stress-responsive mRNAs involved in auto-immunity and growth inhibition under non-stress conditions. Given the conservation of this RNA helicase subfamily, they may control basal levels of conditionally regulated mRNAs in diverse eukaryotes, accelerating responses without penalty.

Gene regulation is a dynamic process involving changes ranging from the remodeling of chromatin to preferential translation. To understand integrated nuclear and cytoplasmic gene regulatory dynamics, we performed a survey spanning the epigenome to translatome of Arabidopsis ( Arabidopsis thaliana ) seedlings in response to hypoxia and reoxygenation. This included chromatin assays (examining histones, accessibility, RNA polymerase II [RNAPII], and transcription factor binding) and three RNA assays (nuclear, polyadenylated, and ribosome-associated). Dynamic patterns of nuclear regulation distinguished stress-induced and growth-associated mRNAs. The rapid upregulation of hypoxia-responsive gene transcripts and their preferential translation were generally accompanied by increased chromatin accessibility, RNAPII engagement, and reduced Histone 2A.Z association. Hypoxia promoted a progressive upregulation of heat stress transcripts, as evidenced by RNAPII binding and increased nuclear RNA, with polyadenylated RNA levels only elevated after prolonged stress or reoxygenation. Promoters of rapidly versus progressively upregulated genes were enriched for cis -elements of ethylene-responsive and heat shock factor transcription factors, respectively. Genes associated with growth, including many encoding cytosolic ribosomal proteins, underwent distinct histone modifications, yet retained RNAPII engagement and accumulated nuclear transcripts during the stress. Upon reaeration, progressively upregulated and growth-associated gene transcripts were rapidly mobilized to ribosomes. Thus, multilevel nuclear regulation of nucleosomes, transcript synthesis, accumulation, and translation tailor transient stress responses.plantcell;31/11/2573/FX1F1fx1., (© 2019 American Society of Plant Biologists. All rights reserved.)

Timely perception of adverse environmental changes is critical for survival. Dynamic changes in gases are important cues for plants to sense environmental perturbations, such as submergence. In Arabidopsis thaliana, changes in oxygen and nitric oxide (NO) control the stability of ERFVII transcription factors. ERFVII proteolysis is regulated by the N-degron pathway and mediates adaptation to flooding-induced hypoxia. However, how plants detect and transduce early submergence signals remains elusive. Here we show that plants can rapidly detect submergence through passive ethylene entrapment and use this signal to pre-adapt to impending hypoxia. Ethylene can enhance ERFVII stability prior to hypoxia by increasing the NO-scavenger PHYTOGLOBIN1. This ethylene-mediated NO depletion and consequent ERFVII accumulation pre-adapts plants to survive subsequent hypoxia. Our results reveal the biological link between three gaseous signals for the regulation of flooding survival and identifies key regulatory targets for early stress perception that could be pivotal for developing flood-tolerant crops.

Abiotic stresses in plants are often transient, and the recovery phase following stress removal is critical. Flooding, a major abiotic stress that negatively impacts plant biodiversity and agriculture, is a sequential stress where tolerance is strongly dependent on viability underwater and during the postflooding period. Here we show that in Arabidopsis thaliana accessions (Bay-0 and Lp2-6), different rates of submergence recovery correlate with submergence tolerance and fecundity. A genome-wide assessment of ribosome-associated transcripts in Bay-0 and Lp2-6 revealed a signaling network regulating recovery processes. Differential recovery between the accessions was related to the activity of three genes: RESPIRATORY BURST OXIDASE HOMOLOG D , SENESCENCE-ASSOCIATED GENE113 , and ORESARA1 , which function in a regulatory network involving a reactive oxygen species (ROS) burst upon desubmergence and the hormones abscisic acid and ethylene. This regulatory module controls ROS homeostasis, stomatal aperture, and chlorophyll degradation during submergence recovery. This work uncovers a signaling network that regulates recovery processes following flooding to hasten the return to prestress homeostasis., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Eukaryotic transcriptomes contain a major non-protein-coding component that includes precursors of small RNAs as well as long noncoding RNA (lncRNAs). Here, we utilized the mapping of ribosome footprints on RNAs to explore translational regulation of coding and noncoding RNAs in roots of Arabidopsis thaliana shifted from replete to deficient phosphorous (Pi) nutrition. Homodirectional changes in steady-state mRNA abundance and translation were observed for all but 265 annotated protein-coding genes. Of the translationally regulated mRNAs, 30% had one or more upstream ORF (uORF) that influenced the number of ribosomes on the principal protein-coding region. Nearly one-half of the 2,382 lncRNAs detected had ribosome footprints, including 56 with significantly altered translation under Pi-limited nutrition. The prediction of translated small ORFs (sORFs) by quantitation of translation termination and peptidic analysis identified lncRNAs that produce peptides, including several deeply evolutionarily conserved and significantly Pi-regulated lncRNAs. Furthermore, we discovered that natural antisense transcripts (NATs) frequently have actively translated sORFs, including five with low-Pi up-regulation that correlated with enhanced translation of the sense protein-coding mRNA. The data also confirmed translation of miRNA target mimics and lncRNAs that produce trans -acting or phased small-interfering RNA ( tasi RNA/phasiRNAs). Mutational analyses of the positionally conserved sORF of TAS3a linked its translation with tasi RNA biogenesis. Altogether, this systematic analysis of ribosome-associated mRNAs and lncRNAs demonstrates that nutrient availability and translational regulation controls protein and small peptide-encoding mRNAs as well as a diverse cadre of regulatory RNAs., Competing Interests: The authors declare no conflict of interest., (Copyright © 2017 the Author(s). Published by PNAS.)

Proteomic plasticity undergirds stress responses in plants, and understanding such responses requires accurate measurement of the extent to which proteins levels are adjusted to counter external stimuli. Here, we adapt bioorthogonal noncanonical amino acid tagging (BONCAT) to interrogate protein synthesis in vegetative Arabidopsis ( Arabidopsis thaliana ) seedlings. BONCAT relies on the translational incorporation of a noncanonical amino acid probe into cellular proteins. In this study, the probe is the Met surrogate azidohomoalanine (Aha), which carries a reactive azide moiety in its amino acid side chain. The azide handle in Aha can be selectively conjugated to dyes and functionalized beads to enable visualization and enrichment of newly synthesized proteins. We show that BONCAT is sensitive enough to detect Arabidopsis proteins synthesized within a 30-min interval defined by an Aha pulse and that the method can be used to detect proteins made under conditions of light stress, osmotic shock, salt stress, heat stress, and recovery from heat stress. We further establish that BONCAT can be coupled to tandem liquid chromatography-mass spectrometry to identify and quantify proteins synthesized during heat stress and recovery from heat stress. Our results are consistent with a model in which, upon the onset of heat stress, translation is rapidly reprogrammed to enhance the synthesis of stress mitigators and is again altered during recovery. All experiments were carried out with commercially available reagents, highlighting the accessibility of the BONCAT method to researchers interested in stress responses as well as translational and posttranslational regulation in plants., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

Climate change has increased the frequency and severity of flooding events, with significant negative impact on agricultural productivity. These events often submerge plant aerial organs and roots, limiting growth and survival due to a severe reduction in light reactions and gas exchange necessary for photosynthesis and respiration, respectively. To distinguish molecular responses to the compound stress imposed by submergence, we investigated transcriptomic adjustments to darkness in air and under submerged conditions using eight Arabidopsis (Arabidopsis thaliana) accessions differing significantly in sensitivity to submergence. Evaluation of root and rosette transcriptomes revealed an early transcriptional and posttranscriptional response signature that was conserved primarily across genotypes, although flooding susceptibility-associated and genotype-specific responses also were uncovered. Posttranscriptional regulation encompassed darkness- and submergence-induced alternative splicing of transcripts from pathways involved in the alternative mobilization of energy reserves. The organ-specific transcriptome adjustments reflected the distinct physiological status of roots and shoots. Root-specific transcriptome changes included marked up-regulation of chloroplast-encoded photosynthesis and redox-related genes, whereas those of the rosette were related to the regulation of development and growth processes. We identified a novel set of tolerance genes, recognized mainly by quantitative differences. These included a transcriptome signature of more pronounced gluconeogenesis in tolerant accessions, a response that included stress-induced alternative splicing. This study provides organ-specific molecular resolution of genetic variation in submergence responses involving interactions between darkness and low-oxygen constraints of flooding stress and demonstrates that early transcriptome plasticity, including alternative splicing, is associated with the ability to cope with a compound environmental stress., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

The central vasculature of plant roots is protected by a hydrophobic ring of endodermal cells that are enclosed by lamellae of suberin. Barberon et al. demonstrate that endodermal suberization plasticity facilitates ion homeostasis, with antithetical regulation of suberin deposition and degradation by the phytohormones abscisic acid and ethylene., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The response of Arabidopsis thaliana to low-oxygen stress (hypoxia), such as during shoot submergence or root waterlogging, includes increasing the levels of ∼50 hypoxia-responsive gene transcripts, many of which encode enzymes associated with anaerobic metabolism. Upregulation of over half of these mRNAs involves stabilization of five group VII ethylene response factor (ERF-VII) transcription factors, which are routinely degraded via the N-end rule pathway of proteolysis in an oxygen- and nitric oxide-dependent manner. Despite their importance, neither the quantitative contribution of individual ERF-VIIs nor the cis-regulatory elements they govern are well understood. Here, using single- and double-null mutants, the constitutively synthesized ERF-VIIs RELATED TO APETALA2.2 (RAP2.2) and RAP2.12 are shown to act redundantly as principle activators of hypoxia-responsive genes; constitutively expressed RAP2.3 contributes to this redundancy, whereas the hypoxia-induced HYPOXIA RESPONSIVE ERF1 (HRE1) and HRE2 play minor roles. An evolutionarily conserved 12-bp cis-regulatory motif that binds to and is sufficient for activation by RAP2.2 and RAP2.12 is identified through a comparative phylogenetic motif search, promoter dissection, yeast one-hybrid assays, and chromatin immunopurification. This motif, designated the hypoxia-responsive promoter element, is enriched in promoters of hypoxia-responsive genes in multiple species., (© 2016 American Society of Plant Biologists. All rights reserved.)

Arabidopsis thaliana cytosolic ribosomes are large complexes containing eighty-one distinct ribosomal proteins (r-proteins), four ribosomal RNAs (rRNA) and a plethora of associated (non-ribosomal) proteins. In plants, r-proteins of cytosolic ribosomes are each encoded by two to seven different expressed and similar genes, forming an r-protein family. Distinctions in the r-protein coding sequences of gene family members are a source of variation between ribosomes. We performed proteomic investigation of actively translating cytosolic ribosomes purified using both immunopurification and a classic sucrose cushion centrifugation-based protocol from plants of different developmental stages. Both 1D and 2D LC-MS(E) with data-independent acquisition as well as conventional data-dependent MS/MS procedures were applied. This approach provided detailed identification of 165 r-protein paralogs with high coverage based on proteotypic peptides. The detected r-proteins were the products of the majority (68%) of the 242 cytosolic r-protein genes encoded by the genome. A total of 70 distinct r-proteins were identified. Based on these results and information from DNA microarray and ribosome footprint profiling studies a re-annotation of Arabidopsis r-proteins and genes is proposed. This compendium of the cytosolic r-protein proteome will serve as a template for future investigations on the dynamic structure and function of plant ribosomes., Biological Significance: Translation is one of the most energy demanding processes in a living cell and is therefore carefully regulated. Translational activity is tightly linked to growth control and growth regulating mechanism. Recently established translational profiling technologies, including the profiling of mRNAs associated with polysomes and the mapping of ribosome footprints on mRNAs, have revealed that the expression of gene expression is often fine-tuned by differential translation of gene transcripts. The eukaryotic ribosome, the hub of these important processes, consists of close to eighty different proteins (depending on species) and four large RNAs assembled into two highly conserved subunits. In plants and to lesser extent in yeast, the r-proteins are encoded by more than one actively transcribed gene. As r-protein gene paralogs frequently do not encode identical proteins and are regulated by growth conditions and development, in vivo ribosomes are heterogeneous in their protein content. The regulatory and physiological importance of this heterogeneity is unknown. Here, an improved annotation of the more than two hundred r-protein genes of Arabidopsis is presented that combines proteomic and advanced mRNA expression data. This proteomic investigation and re-annotation of Arabidopsis ribosomes establish a base for future investigations of translational control in plants., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Oxygen deficiency, caused by flooding of all or a portion of a plant, leads to significant gene regulatory and metabolic responses associated with survival. When oxygen-deprived in light, aerial organs and root systems respond in distinct manners because of their respective autotrophy and heterotrophy, as well as intrinsic differences in cell biology and organ function. To better understand organ-specific responses to oxygen deficiency, we monitored changes in the metabolome of roots and shoots of Arabidopsis thaliana seedlings using gas chromatography-mass spectrometry and (1) H-nuclear magnetic resonance spectroscopy. Only roots accumulated high amounts of γ-aminobutyrate (GABA) and lactate, whereas both organs accumulated alanine (Ala) upon hypoxia. Meta-analysis of gene regulation data revealed higher induction of mRNAs coding for fermentative enzymes in roots as compared with shoots. However, the elevation in GABA level was not correlated with changes in transcript abundance, supporting the proposal that post-translational mechanisms are important in metabolic acclimation to hypoxia. The biosynthesis, degradation and function of GABA and Ala during oxygen deprivation and re-aeration is discussed. Finally, a systematic survey of low-oxygen mediated regulation of genes associated with primary metabolism across organs and cell types reveals exciting new avenues for future studies., (© 2014 John Wiley & Sons Ltd.)

Transcriptional activation in response to hypoxia in plants is orchestrated by ethylene-responsive factor group VII (ERF-VII) transcription factors, which are stable during hypoxia but destabilized during normoxia through their targeting to the N-end rule pathway of selective proteolysis. Whereas the conditionally expressed ERF-VII genes enable effective flooding survival strategies in rice, the constitutive accumulation of N-end-rule-insensitive versions of the Arabidopsis thaliana ERF-VII factor RAP2.12 is maladaptive. This suggests that transcriptional activation under hypoxia that leads to anaerobic metabolism may need to be fine-tuned. However, it is presently unknown whether a counterbalance of RAP2.12 exists. Genome-wide transcriptome analyses identified an uncharacterized trihelix transcription factor gene, which we named HYPOXIA RESPONSE ATTENUATOR1 (HRA1), as highly up-regulated by hypoxia. HRA1 counteracts the induction of core low oxygen-responsive genes and transcriptional activation of hypoxia-responsive promoters by RAP2.12. By yeast-two-hybrid assays and chromatin immunoprecipitation we demonstrated that HRA1 interacts with the RAP2.12 protein but with only a few genomic DNA regions from hypoxia-regulated genes, indicating that HRA1 modulates RAP2.12 through protein-protein interaction. Comparison of the low oxygen response of tissues characterized by different levels of metabolic hypoxia (i.e., the shoot apical zone versus mature rosette leaves) revealed that the antagonistic interplay between RAP2.12 and HRA1 enables a flexible response to fluctuating hypoxia and is of importance to stress survival. In Arabidopsis, an effective low oxygen-sensing response requires RAP2.12 stabilization followed by HRA1 induction to modulate the extent of the anaerobic response by negative feedback regulation of RAP2.12. This mechanism is crucial for plant survival under suboptimal oxygenation conditions. The discovery of the feedback loop regulating the oxygen-sensing mechanism in plants opens new perspectives for breeding flood-resistant crops., Competing Interests: The authors have declared that no competing interests exist.

Low oxygen stress dynamically regulates the translation of cellular mRNAs as a means of energy conservation in seedlings of Arabidopsis thaliana. Most of the highly hypoxia-induced mRNAs are recruited to polysomes and actively translated, whereas other cellular mRNAs become translationally inactive and are either targeted for stabilization or degradation. Here we identify the involvement of OLIGOURIDYLATE BINDING PROTEIN 1 (UBP1), a triple RNA Recognition Motif protein, in dynamic and reversible aggregation of translationally repressed mRNAs during hypoxia. Mutation or down-regulation of UBP1C interferes with seedling establishment and reduces survival of low oxygen stress. By use of messenger ribonucleoprotein (mRNP) immunopurification, we show that UBP1C constitutively binds a subpopulation of mRNAs characterized by uracil-rich 3'-untranslated regions under normoxic conditions. During hypoxia, UBP1C association with non-uracil-rich mRNAs is enhanced concomitant with its aggregation into microscopically visible cytoplasmic foci, referred to as UBP1 stress granules (SGs). This UBP1C-mRNA association occurs as global levels of protein synthesis decline. Upon reoxygenation, rapid UBP1 SG disaggregation coincides with the return of the stabilized mRNAs to polysomes. The mRNAs that are highly induced and translated during hypoxia largely circumvent UBP1C sequestration. Thus, UBP1 is established as a component of dynamically assembled cytoplasmic mRNPs that sequester mRNAs that are poorly translated during a transient low energy stress.

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 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.

Over 13% of all genes in the Arabidopsis thaliana genome encode for proteins classified as having a completely unknown function, with the function of >30% of the Arabidopsis proteome poorly characterized. Although empirical data in the form of mRNA and proteome profiling experiments suggest that many of these proteins play an important role in different biological processes, their functional characterization remains one of the major challenges in modern biology. To expand the annotation of genes with unknown function involved in the response of Arabidopsis to different environmental stress conditions, we selected 1007 such genes and tested the response of their corresponding homozygous T-DNA insertional mutants to salinity, oxidative, osmotic, heat, cold and hypoxia stresses. Depending on the specific abiotic stresses tested, 12-31% of mutants had an altered stress-response phenotype. Interestingly, 832 out of 1007 mutants showed tolerance or sensitivity to more than one abiotic stress treatment, suggesting that genes of unknown function could play an important role in abiotic stress-response signaling, or general acclimation mechanisms. Further analysis of multiple stress-response phenotypes within different populations of mutants revealed interesting links between acclimation to heat, cold and oxidative stresses, as well as between sensitivity to ABA, osmotic, salinity, oxidative and hypoxia stresses. Our findings provide a significant contribution to the biological characterization of genes with unknown function in Arabidopsis and demonstrate that many of these genes play a key role in the response of plants to abiotic stresses., (© 2012 Scandinavian Plant Physiology Society.)

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., (© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.)

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.

Plant-specific group VII Ethylene Response Factor (ERF) transcription factors have emerged as pivotal regulators of flooding and low oxygen responses. In rice (Oryza sativa), these proteins regulate contrasting strategies of flooding survival. Recent studies on Arabidopsis thaliana group VII ERFs show they are stabilized under hypoxia but destabilized under oxygen-replete conditions via the N-end rule pathway of targeted proteolysis. Oxygen-dependent sequestration at the plasma membrane maintains at least one of these proteins, RAP2.12, under normoxia. Remarkably, SUB1A, the rice group VII ERF that enables prolonged submergence tolerance, appears to evade oxygen-regulated N-end rule degradation. We propose that the turnover of group VII ERFs is of ecological relevance in wetland species and might be manipulated to improve flood tolerance of crops., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Mitogen-activated protein kinases (MPKs) are regulated by diverse stresses with a reactive oxygen species (ROS) component. Here, we report the rapid and transient activation of MPK3, MPK4 and MPK6 upon oxygen deprivation as well as reoxygenation in seedlings of Arabidopsis thaliana. MPK activation peaked within 2 h of oxygen deprivation and again at a higher magnitude within 5 min of reoxygenation. MPK6 was the predominant kinase regulated by oxygen availability in both aerial and root tissue, except in mpk6 mutants, which displayed compensatory activation of MPK3. A universal consequence of oxygen deprivation in eukaryotes is inhibition of the terminal step of the mitochondrial electron transport chain (mETC). We demonstrate that treatment of seedlings with the mETC inhibitors antimycin A and potassium cyanide under normoxia promotes transient MPK6 and MPK3 activation. Confocal imaging of seedlings provided evidence that both oxygen deprivation and mETC inhibitors stimulate mitochondria-associated ROS production. We found that seedling survival of prolonged oxygen deprivation was improved in transgenics that ectopically overexpress MPK3, MPK4 and MPK6, but the induction of mRNAs associated with low oxygen acclimation responses were not markedly altered in MPK6 overexpression lines or mpk6 loss-of-function mutants. However, distinctions in MPK6 activation potential were correlated with other differences in mRNAs accumulation. Our findings suggest that oxygen deprivation and reoxygenation trigger mitochondrial ROS production to activate MPK signaling, which in turn regulate reversible processes that aid survival of transient oxygen deprivation.

Plants and animals are obligate aerobes, requiring oxygen for mitochondrial respiration and energy production. In plants, an unanticipated decline in oxygen availability (hypoxia), as caused by roots becoming waterlogged or foliage submergence, triggers changes in gene transcription and messenger RNA translation that promote anaerobic metabolism and thus sustain substrate-level ATP production. In contrast to animals, oxygen sensing has not been ascribed to a mechanism of gene regulation in response to oxygen deprivation in plants. Here we show that the N-end rule pathway of targeted proteolysis acts as a homeostatic sensor of severe low oxygen levels in Arabidopsis, through its regulation of key hypoxia-response transcription factors. We found that plants lacking components of the N-end rule pathway constitutively express core hypoxia-response genes and are more tolerant of hypoxic stress. We identify the hypoxia-associated ethylene response factor group VII transcription factors of Arabidopsis as substrates of this pathway. Regulation of these proteins by the N-end rule pathway occurs through a characteristic conserved motif at the amino terminus initiating with Met-Cys. Enhanced stability of one of these proteins, HRE2, under low oxygen conditions improves hypoxia survival and reveals a molecular mechanism for oxygen sensing in plants via the evolutionarily conserved N-end rule pathway. SUB1A-1, a major determinant of submergence tolerance in rice, was shown not to be a substrate for the N-end rule pathway despite containing the N-terminal motif, indicating that it is uncoupled from N-end rule pathway regulation, and that enhanced stability may relate to the superior tolerance of Sub1 rice varieties to multiple abiotic stresses., (© 2011 Macmillan Publishers Limited. All rights reserved)

Submergence of plant organs perturbs homeostasis by limiting diffusion of oxygen, carbon dioxide and ethylene. In rice (Oryza sativa L.), the haplotype at the multigenic SUBMERGENCE1 (SUB1) locus determines whether plants survive prolonged submergence. SUB1 encodes two or three transcription factors of the group VII ethylene response factor family: SUB1A, SUB1B and SUB1C. The presence of SUB1A-1 and its strong submergence-triggered ethylene-mediated induction confers submergence tolerance through a quiescence survival strategy that inhibits gibberellin (GA)-induced carbohydrate consumption and elongation growth. SUB1C is invariably present and acts downstream of the enhancement of GA responsiveness during submergence. In this study, heterologous ectopic expression of rice SUB1A and SUB1C in Arabidopsis thaliana was used to explore conserved mechanisms of action associated with these genes using developmental, physiological and molecular metrics. As in rice transgenic plants that ectopically express SUB1A-1, Arabidopsis transgenic plants that constitutively express SUB1A displayed GA insensitivity and abscisic acid hypersensitivity. Ectopic SUB1C expression had more limited effects on development, stress responses and the transcriptome. Observation of a delayed flowering phenotype in lines over-expressing SUB1A led to the finding that inhibition of floral initiation is a component of the quiescence survival strategy in rice. Together, these analyses demonstrate conserved as well as specific roles for group VII ethylene response factors in integration of abiotic responses with development., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

• The exploitation of natural variation in Arabidopsis thaliana (Arabidopsis) provides a huge potential for the identification of the molecular mechanisms underlying this variation as a result of the availability of a vast array of genetic and genomic resources for this species. Eighty-six Arabidopsis accessions were screened for natural variation in flooding tolerance. This forms the first step towards the identification and characterization of the role of candidate genes contributing to flooding tolerance. • Arabidopsis accessions at the 10-leaf stage were subjected to complete submergence in the dark. Survival curves were plotted to estimate median lethal times as a measure of tolerance. Flooding-associated survival parameters, such as root and shoot oxygen content, initial carbohydrate content and petiole elongation under water, were also measured. • There was a significant variation in submergence tolerance among Arabidopsis accessions. However, the order of tolerance did not correlate with root and shoot oxygen content or initial amounts of shoot starch and total soluble sugars. A negative correlation was observed between submergence tolerance and underwater petiole elongation. • Arabidopsis accessions show considerable variation in the ability to tolerate complete submergence, making it a good species in which to identify and characterize genes and to study mechanisms that contribute to survival under water., (© 2010 The Authors. New Phytologist © 2010 New Phytologist Trust.)

• A detailed description of the molecular response of Arabidopsis thaliana to submergence can aid the identification of genes that are critical to flooding survival. • Rosette-stage plants were fully submerged in complete darkness and shoot and root tissue was harvested separately after the O(2) partial pressure of the petiole and root had stabilized at c. 6 and 0.1 kPa, respectively. As controls, plants were untreated or exposed to darkness. Following quantitative profiling of cellular mRNAs with the Affymetrix ATH1 platform, changes in the transcriptome in response to submergence, early darkness, and O(2)-deprivation were evaluated by fuzzy k-means clustering. This identified genes co-regulated at the conditional, developmental or organ-specific level. Mutants for 10 differentially expressed HYPOXIA-RESPONSIVE UNKNOWN PROTEIN (HUP) genes were screened for altered submergence tolerance. • The analysis identified 34 genes that were ubiquitously co-regulated by submergence and O(2) deprivation. The biological functions of these include signaling, transcription, and anaerobic energy metabolism. HUPs comprised 40% of the co-regulated transcripts and mutants of seven of these genes were significantly altered in submergence tolerance. • The results define transcriptomic adjustments in response to submergence in the dark and demonstrate that the manipulation of HUPs can alter submergence tolerance., (© 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.)

High-throughput technology has facilitated genome-scale analyses of transcriptomic adjustments in response to environmental perturbations with an oxygen deprivation component, such as transient hypoxia or anoxia, root waterlogging, or complete submergence. We showed previously that Arabidopsis (Arabidopsis thaliana) seedlings elevate the levels of hundreds of transcripts, including a core group of 49 genes that are prioritized for translation across cell types of both shoots and roots. To recognize low-oxygen responses that are evolutionarily conserved versus species specific, we compared the transcriptomic reconfiguration in 21 organisms from four kingdoms (Plantae, Animalia, Fungi, and Bacteria). Sorting of organism proteomes into clusters of putative orthologs identified broadly conserved responses associated with glycolysis, fermentation, alternative respiration, metabolite transport, reactive oxygen species amelioration, chaperone activity, and ribosome biogenesis. Differentially regulated genes involved in signaling and transcriptional regulation were poorly conserved across kingdoms. Strikingly, nearly half of the induced mRNAs of Arabidopsis seedlings encode proteins of unknown function, of which over 40% had up-regulated orthologs in poplar (Populus trichocarpa), rice (Oryza sativa), or Chlamydomonas reinhardtii. Sixteen HYPOXIA-RESPONSIVE UNKNOWN PROTEIN (HUP) genes, including four that are Arabidopsis specific, were ectopically overexpressed and evaluated for their effect on seedling tolerance to oxygen deprivation. This allowed the identification of HUPs coregulated with genes associated with anaerobic metabolism and other processes that significantly enhance or reduce stress survival when ectopically overexpressed. These findings illuminate both broadly conserved and plant-specific low-oxygen stress responses and confirm that plant-specific HUPs with limited phylogenetic distribution influence low-oxygen stress endurance.

Multicellular organs are composed of distinct cell types with unique assemblages of translated mRNAs. Here, ribosome-associated mRNAs were immunopurified from specific cell populations of intact seedlings using Arabidopsis thaliana lines expressing a FLAG-epitope tagged ribosomal protein L18 (FLAG-RPL18) via developmentally regulated promoters. The profiling of mRNAs in ribosome complexes, referred to as the translatome, identified differentially expressed mRNAs in 21 cell populations defined by cell-specific expression of FLAG-RPL18. Phloem companion cells of the root and shoot had the most distinctive translatomes. When seedlings were exposed to a brief period of hypoxia, a pronounced reprioritization of mRNA enrichment in the cell-specific translatomes occurred, including a ubiquitous rise in 49 mRNAs encoding transcription factors, signaling proteins, anaerobic metabolism enzymes, and uncharacterized proteins. Translatome profiling also exposed an intricate molecular signature of transcription factor (TF) family member mRNAs that was markedly reconfigured by hypoxia at global and cell-specific levels. In addition to the demonstration of the complexity and plasticity of cell-specific populations of ribosome-associated mRNAs, this study provides an in silico dataset for recognition of differentially expressed genes at the cell-, region-, and organ-specific levels.

Cellular oxygen deprivation (hypoxia/anoxia) requires an acclimation response that enables survival during an energy crisis. To gain new insights into the processes that facilitate the endurance of transient oxygen deprivation, the dynamics of the mRNA translation state and metabolites were quantitatively monitored in Arabidopsis thaliana seedlings exposed to a short (2 h) or prolonged (9 h) period of oxygen and carbon dioxide deprivation and following 1 h of re-aeration. Hypoxia stress and reoxygenation promoted adjustments in the levels of polyribosomes (polysomes) that were highly coordinated with cellular ATP content. A quantitative comparison of steady-state and polysomal mRNA populations revealed that over half of the cellular mRNAs were restricted from polysome complexes during the stress, with little or no change in abundance. This selective repression of translation was rapidly reversed upon reoxygenation. Comparison of the adjustment in gene transcripts and metabolites demonstrated that profiling of polysomal mRNAs strongly augments the prediction of cellular processes that are altered during cellular oxygen deprivation. The selective translation of a subset of mRNAs promotes the conservation of ATP and facilitates the transition to anaerobic metabolism during low-oxygen stress.

About 40% of the proteins encoded in eukaryotic genomes are proteins of unknown function (PUFs). Their functional characterization remains one of the main challenges in modern biology. In this study we identified the PUF encoding genes from Arabidopsis (Arabidopsis thaliana) using a combination of sequence similarity, domain-based, and empirical approaches. Large-scale gene expression analyses of 1,310 publicly available Affymetrix chips were performed to associate the identified PUF genes with regulatory networks and biological processes of known function. To generate quality results, the study was restricted to expression sets with replicated samples. First, genome-wide clustering and gene function enrichment analysis of clusters allowed us to associate 1,541 PUF genes with tightly coexpressed genes for proteins of known function (PKFs). Over 70% of them could be assigned to more specific biological process annotations than the ones available in the current Gene Ontology release. The most highly overrepresented functional categories in the obtained clusters were ribosome assembly, photosynthesis, and cell wall pathways. Interestingly, the majority of the PUF genes appeared to be controlled by the same regulatory networks as most PKF genes, because clusters enriched in PUF genes were extremely rare. Second, large-scale analysis of differentially expressed genes was applied to identify a comprehensive set of abiotic stress-response genes. This analysis resulted in the identification of 269 PKF and 104 PUF genes that responded to a wide variety of abiotic stresses, whereas 608 PKF and 206 PUF genes responded predominantly to specific stress treatments. The provided coexpression and differentially expressed gene data represent an important resource for guiding future functional characterization experiments of PUF and PKF genes. Finally, the public Plant Gene Expression Database (http://bioweb.ucr.edu/PED) was developed as part of this project to provide efficient access and mining tools for the vast gene expression data of this study.

Background and Aims: DNA microarrays allow comprehensive estimation of total cellular mRNA levels but are also amenable to studies of other mRNA populations, such as mRNAs in translation complexes (polysomes). The aim of this study was to evaluate the role of translational regulation in response to oxygen deprivation (hypoxia)., Methods: Alterations in total cellular and large polysome (>or=five ribosomes per mRNA) mRNA levels were monitored in response to 12 h of hypoxia stress in seedlings of Arabidopsis thaliana with a full-genome oligonucleotide microarray., Key Results: Comparison of two mRNA populations revealed considerable modulation of mRNA accumulation and diversity in translation in response to hypoxia. Consistent with the global decrease in protein synthesis, hypoxia reduced the average proportion of individual mRNA species in large polysome complexes from 56.1% to 32.1%. A significant decrease in the association with translational complexes was observed for 77% of the mRNAs, including a subset of known hypoxia-induced gene transcripts. The examination of mRNA levels of nine genes in polysomes fractionated through sucrose density gradients corroborated the microarray data. Gene cluster analysis was used to identify mRNAs that displayed co-ordinated regulation. Fewer than half of the highly induced mRNAs circumvented the global depression of translation. Moreover, a large number of mRNAs displayed a significant decrease in polysome association without a concomitant decrease in steady-state accumulation. The abundant mRNAs that encode the ribosomal proteins behaved in this manner. By contrast, a small group of abiotic and biotic stress-induced mRNAs showed a significant increase in polysome association, without a change in abundance. Evaluation of quantitative features of mRNA sequences demonstrated that a low GC nucleotide content of the 5'-untranslated region provides a selective advantage for translation under hypoxia., Conclusions: Alterations in transcript abundance and translation contribute to the differential regulation of gene expression in response to oxygen deprivation.

Immunoaffinity purification of polyribosomes (polysomes) from crude leaf extracts of Arabidopsis (Arabidopsis thaliana) was achieved with transgenic genotypes that overexpress a translational fusion of a ribosomal protein (RP) with a His(6)-FLAG dual epitope tag. In plants with a cauliflower mosaic virus 35S:HF-RPL18 transgene immunopurification with anti-FLAG agarose beads yielded 60-Svedberg ribosomal subunits, intact 80-Svedberg monosomes and polysomes. Sucrose density gradient fractionation of the purified complexes demonstrated that the distribution of polysome size was similar in crude cell extracts and the purified complexes. The immunopurified complexes included putative cytosolic RPs of Arabidopsis and ribosome-associated proteins, as well as full-length transcripts of high and low abundance. Whole-genome profiling using long DNA oligonucleotide-based microarrays provided a high level of reproducibility between polysomal mRNA samples immunopurified from two independent biological replicates (r approximately 0.90). Comparison of immunopurified and total cellular RNA samples revealed that for most of the genes, the mRNAs were associated with the epitope-tagged polysomal complexes, with an average relative level of association of 62.06% +/- 4.39%. The results demonstrate that the immunopurification of polysomes can be a valuable tool for the quantification of mRNAs present in translation complexes in plant cells. This technology can be extended to evaluation of mRNA populations at the cell- or tissue-specific level by regulation of the tagged RP with distinct promoters.

The genome-wide protein sequences from Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) spp. japonica were clustered into families using sequence similarity and domain-based clustering. The two fundamentally different methods resulted in separate cluster sets with complementary properties to compensate the limitations for accurate family analysis. Functional names for the identified families were assigned with an efficient computational approach that uses the description of the most common molecular function gene ontology node within each cluster. Subsequently, multiple alignments and phylogenetic trees were calculated for the assembled families. All clustering results and their underlying sequences were organized in the Web-accessible Genome Cluster Database (http://bioinfo.ucr.edu/projects/GCD) with rich interactive and user-friendly sequence family mining tools to facilitate the analysis of any given family of interest for the plant science community. An automated clustering pipeline ensures current information for future updates in the annotations of the two genomes and clustering improvements. The analysis allowed the first systematic identification of family and singlet proteins present in both organisms as well as those restricted to one of them. In addition, the established Web resources for mining these data provide a road map for future studies of the composition and structure of protein families between the two species.

Analysis of 80S ribosomes of Arabidopsis (Arabidopsis thaliana) by use of high-speed centrifugation, sucrose gradient fractionation, one- and two-dimensional gel electrophoresis, liquid chromatography purification, and mass spectrometry (matrix-assisted laser desorption/ionization time-of-flight and electrospray ionization) identified 74 ribosomal proteins (r-proteins), of which 73 are orthologs of rat r-proteins and one is the plant-specific r-protein P3. Thirty small (40S) subunit and 44 large (60S) subunit r-proteins were confirmed. In addition, an ortholog of the mammalian receptor for activated protein kinase C, a tryptophan-aspartic acid-domain repeat protein, was found to be associated with the 40S subunit and polysomes. Based on the prediction that each r-protein is present in a single copy, the mass of the Arabidopsis 80S ribosome was estimated as 3.2 MD (1,159 kD 40S; 2,010 kD 60S), with the 4 single-copy rRNAs (18S, 26S, 5.8S, and 5S) contributing 53% of the mass. Despite strong evolutionary conservation in r-protein composition among eukaryotes, Arabidopsis 80S ribosomes are variable in composition due to distinctions in mass or charge of approximately 25% of the r-proteins. This is a consequence of amino acid sequence divergence within r-protein gene families and posttranslational modification of individual r-proteins (e.g. amino-terminal acetylation, phosphorylation). For example, distinct types of r-proteins S15a and P2 accumulate in ribosomes due to evolutionarily divergence of r-protein genes. Ribosome variation is also due to amino acid sequence divergence and differential phosphorylation of the carboxy terminus of r-protein S6. The role of ribosome heterogeneity in differential mRNA translation is discussed.

DNA microarrays were used to evaluate the regulation of the proportion of individual mRNA species in polysomal complexes in leaves of Arabidopsis thaliana under control growth conditions and following a mild dehydration stress (DS). The analysis determined that the percentage of an individual gene transcript in polysomes (ribosome loading) ranged from over 95 to <5%. DS caused a decrease in ribosome loading from 82 to 72%, with maintained polysome association for over 60% of the mRNAs with an increased abundance. To identify sequence features responsible for translational regulation, ribosome loading values and features of full-length mRNA sequences were compared. mRNAs with extreme length or high GU content in the 5'-untranslated regions (5'-UTRs) were generally poorly translated. Under DS, mRNAs with both a high GC content in the 5'-UTR and long open reading frame showed a significant impairment in ribosome loading. Evaluation of initiation A+1UG codon context revealed distinctions in the frequency of adenine in nucleotides -10 to -1 (especially at -4 and -3) in mRNAs with different ribosome loading values. Notably, the mRNA features that contribute to translational regulation could not fully explain the variation in ribosome loading, indicating that additional factors contribute to translational regulation in Arabidopsis.

Translational regulation was evaluated for over 2000 genes by measurement of the proportion of individual mRNA species in polysomal (PS) complexes in leaves of non-stressed and moderately dehydration-stressed Arabidopsis. The amount of each mRNA in polysomes ranged from 23 to 97% in non-stressed leaves and was significantly reduced for a large portion of the genes (71%) in response to dehydration. The effect of dehydration on translational status varied extensively between mRNA species. Sixty per cent of the dehydration-inducible mRNAs with twofold or greater increase in abundance maintained PS levels in response to water-deficit stress, while 40% showed impaired ribosome loading (RL). PS association declined significantly for 92% of the mRNAs that displayed a strong decrease in abundance, indicating a relationship between translation and decreased gene transcription and/or mRNA stability. Interestingly, many mRNAs that encode proteins of similar biological function displayed coordinate translational regulation. Thus, the abundance of PS mRNA may provide a more accurate estimate of gene expression than total cellular mRNA because of extensive differential translational regulation.

Transposon tagging with modified maize Ds-GUS constructs was used to isolate genes induced by oxygen deprivation in Arabidopsis thaliana. Seedlings of 800 gene-trap (DsG) and 600 enhancer-trap (DsE) lines were grown on vertically positioned plates for 1 week, oxygen deprived for up to 24 h and stained for GUS activity. Oxygen deprivation induced intricate patterns of gene expression in seedlings of 65 lines. The insertion site and phenotypes of 15 lines were examined. Surprisingly, none of the insertions were into genes that encode known anaerobic polypeptides. Insertions were identified within or adjacent to genes encoding proteins of regulatory, enzymatic, mitochondrial protein import and unknown function, as well as adjacent to genes encoding a putative receptor-like kinase and putative sensor-histidine kinase. Four lines had significantly lower ADH activity after 24 h of oxygen deprivation and three of these showed reduced stress tolerance. Two lines with wild-type levels of ADH were low-oxygen intolerant. Paradoxically, several lines had significantly higher ADH activity after 12 h of oxygen deprivation but reduced stress tolerance. Caffeine treatment, which increased ADH specific activity in wild-type seedlings under aerobic conditions, was sufficient to increase GUS staining in seven of the 15 lines, providing evidence that these genes may be regulated by cytosolic calcium levels. These results demonstrate the effectiveness of the Ds-GUS tagging system in the identification of genes that are regulated in response to oxygen deprivation and a calcium second messenger.

Transient soil flooding limits cellular oxygen to roots and reduces crop yield. Plant response to oxygen deprivation involves increased expression of the alcohol dehydrogenase gene (ADH) and ethanolic fermentation. Disruption of the Arabidopsis gene that encodes Rop (RHO-like small G protein of plants) guanosine triphosphatase (GTPase) activating protein 4 (ROPGAP4), a Rop deactivator, elevates ADH expression in response to oxygen deprivation but decreases tolerance to stress. Rop-dependent production of hydrogen peroxide via a diphenylene iodonium chloride-sensitive calcium-dependent reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is necessary for induction of both ADH and RopGAP4 expression. Tolerance to oxygen deprivation requires Rop activation and RopGAP4-dependent negative feedback regulation. This Rop signal transduction rheostat balances the ability to increase ethanolic fermentation with survival.

Eukaryotic ribosomes are made of two components, four ribosomal RNAs, and approximately 80 ribosomal proteins (r-proteins). The exact number of r-proteins and r-protein genes in higher plants is not known. The strong conservation in eukaryotic r-protein primary sequence allowed us to use the well-characterized rat (Rattus norvegicus) r-protein set to identify orthologues on the five haploid chromosomes of Arabidopsis. By use of the numerous expressed sequence tag (EST) accessions and the complete genomic sequence of this species, we identified 249 genes (including some pseudogenes) corresponding to 80 (32 small subunit and 48 large subunit) cytoplasmic r-protein types. None of the r-protein genes are single copy and most are encoded by three or four expressed genes, indicative of the internal duplication of the Arabidopsis genome. The r-proteins are distributed throughout the genome. Inspection of genes in the vicinity of r-protein gene family members confirms extensive duplications of large chromosome fragments and sheds light on the evolutionary history of the Arabidopsis genome. Examination of large duplicated regions indicated that a significant fraction of the r-protein genes have been either lost from one of the duplicated fragments or inserted after the initial duplication event. Only 52 r-protein genes lack a matching EST accession, and 19 of these contain incomplete open reading frames, confirming that most genes are expressed. Assessment of cognate EST numbers suggests that r-protein gene family members are differentially expressed.

An Arabidopsis cDNA clone that defines a new class of plant serine/threonine receptor kinases was found to be a member of a family of four clustered genes (lecRK-a1-a4) which have been cloned, sequenced and mapped on chromosome 3. This family belongs to a large superfamily encoding putative receptors with an extracellular domain homologous to legume lectins and appears to be conserved at least among dicots. In the Columbia ecotype only the lecRK-a1 and perhaps the lecRK-a3 gene is functional, since lecRK-a2 is disrupted by a Ty-copia retroelement and lecRK-a4 contains a frameshift mutation. Structural analysis of the lecRK-al and lecRK-a3 deduced amino-acid sequences suggests that the lectin domain is unlikely to be involved in binding monosaccharides but could interact with complex glycans and/or with hydrophobic ligands. Immunodetection of lecRK gene products in plasma membranes purified by free-flow electrophoresis showed that the lecRK-a proteins are probably highly glycosylated integral plasma membrane components.