Dynamic Light Regulation of Translation Status in Arabidopsis thaliana

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

Noncanonical Alternative Polyadenylation Contributes to Gene Regulation in Response to Hypoxia

Stresses from various environmental challenges continually confront plants, and their responses are important for growth and survival. One molecular response to such challenges involves the alternative polyadenylation of mRNA. In plants, it is unclear how stress affects the production and fate of alternative mRNA isoforms. Using a genome-scale approach, we show that in Arabidopsis thaliana, hypoxia leads to increases in the number of mRNA isoforms with polyadenylated 3′ ends that map to 5′-untranslated regions (UTRs), introns, and protein-coding regions. RNAs with 3′ ends within protein-coding regions and introns were less stable than mRNAs that end at 3′-UTR poly(A) sites. Additionally, these RNA isoforms were underrepresented in polysomes isolated from control and hypoxic plants. By contrast, mRNA isoforms with 3′ ends that lie within annotated 5′-UTRs were overrepresented in polysomes and were as stable as canonical mRNA isoforms. These results indicate that the generation of noncanonical mRNA isoforms is an important feature of the abiotic stress response. The finding that several noncanonical mRNA isoforms are relatively unstable suggests that the production of non-stop and intronic mRNA isoforms may represent a form of negative regulation in plants, providing a conceptual link with mechanisms that generate these isoforms (such as alternative polyadenylation) and RNA surveillance

Arabidopsis translation initiation factor binding protein CBE1 negatively regulates accumulation of the NADPH oxidase respiratory burst oxidase homolog D

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 (RBOHD), the NADPH oxidase responsible for elicitor-induced apoplastic reactive oxygen species (ROS) production. Furthermore, several mRNA decapping and translation initiation factors co-localize with CBE1 and similarly regulate immune signaling. This study thus identifies a novel regulator of immune signaling and provides new insights into ROS regulation, potentially through translational control, during plant stress responses

Genetic strategies for improving crop yields.

The current trajectory for crop yields is insufficient to nourish the world's population by 20501. Greater and more consistent crop production must be achieved against a backdrop of climatic stress that limits yields, owing to shifts in pests and pathogens, precipitation, heat-waves and other weather extremes. Here we consider the potential of plant sciences to address post-Green Revolution challenges in agriculture and explore emerging strategies for enhancing sustainable crop production and resilience in a changing climate. Accelerated crop improvement must leverage naturally evolved traits and transformative engineering driven by mechanistic understanding, to yield the resilient production systems that are needed to ensure future harvests

Life in the balance: a signaling network controlling survival of flooding

Recent reports on responses to flooding, submergence, and low-oxygen stress have connected components in an essential regulatory network that underlies plasticity in growth and metabolism essential for the survival of distinct flooding regimes. Here, we discuss growth under severe oxygen-limited conditions (anaerobic growth) and less oxygen-deficient underwater conditions (ethylene-driven underwater growth). Low-oxygen stress causes an energy and carbohydrate crisis that must be controlled through regulated consumption of carbohydrates and energy reserves. In rice (Oryza sativa L.), lowoxygen stress, energy homeostasis and growth are connected by a calcineurin B-like interacting binding kinase (CIPK) in seeds germinated under water. In shoots, two opposing adaptive strategies to submergence, elongation (escape) and inhibition of elongation (quiescence), are controlled by related ethylene response factor (ERF) DNA binding proteins that act downstream of ethylene and modulate gibberellin-mediated shoot growth. Increased resolution of the flooding signaling network will require more precise investigation of the interactions between oxygen tension and cellular energy status in regulation of anaerobic metabolism and ethylene-driven growth, both essential to survival in variable flooding environments. Introduction Global warming is associated with increased flooding events adversarial to most plant species and thus affects both crop yield and plant distribution in natural ecosystems [1]. There is an urgent need to increase crop production, particularly rice, in flood-prone regions. Major steps toward breeding tolerant varieties have been made through the characterization of two multigenic loci that control the capacity to endure complete submergence (SUBMERGENCE 1, SUB1) or rapid outgrowth of adverse partial submergence (SNORKEL, SK) [2 ,3 , 4 ,5 ]. New SUB1 rice varieties, produced by markerassisted breeding, are high yielding even following two weeks of complete submergence [6 ,7]. Similarly, better yields of deepwater rice are anticipated, with other improvements to follow [8]. For example, rice and some other species have the remarkable capacity to germinate and elongate coleoptiles and stems under severe oxygen constraints [9,10] (i.e. under anoxia, See glossary). The recognition of rice CIPK15 as a regulator of underwater germination and early shoot elongation [11 ] provides a critical link between sugar sensing, starch utilization and coleoptile elongation under anoxia. The identification CIPK15 and other genes that regulate underwater seed establishment may enable breeding to reduce the practice of seedling transplantation and overall herbicide use. These findings in rice complement data from other genera (i.e. Arabidopsis, Lotus, Poplar, Potamogeton, and Rumex) suggesting there exists a conserved flooding response network in plants that includes ethylene-triggered alterations in gene expression leading to growth and stress-induced catabolism of stored or soluble carbohydrates for energy-efficient production and utilization of ATP. A key challenge is to decipher the interplay between hormones (i.e. ethylene, abscisic acid (ABA), and gibberellic acids (GA)), oxygen availability, and specific metabolites (i.e. ATP, sugars, and pyruvate) that drives a dynamic network balancing growth and quiescence to facilitate survival Ethylene-controlled growth Recently, a quantitative trait locus was discovered encoding two genes that trigger rapid internode elongation in rice varieties when cultivated under partially submerged 'deepwater' conditions. These genes, SNORKEL1 (SK1) and SNORKEL2 (SK2) encode nuclear-localized DNA binding proteins with a single APETALA2/ethylene response factor (ERF) domain [2 ,12]. The SKs are absent in non-deepwater varieties, including all japonicas; however when backcrossed into a japonica, 30% of the internode elongation capacity of the deepwater variety was transferred. Moreover, constitutive overexpression of SK1 or SK2 in a japonica increases the number of elongated internodes, even under non-submerged The presence or absence of submergence-induced shoot elongation relates to the selection pressures of different flooding regimes. Long lasting, relatively shallow floods strongly favor enhanced underwater elongation, whereas short, deep floods restrict underwater elongation and thus 490 Cell signalling and gene regulation Glossary anaerobic growth: growth in the absence of oxygen underwater growth: growth of tissues under water, often under conditions of reduced oxygen availability normoxia: oxygen levels at 1 atm, typically 20.6% anoxia: no oxygen available hypoxia: oxygen levels below the critical oxygen pressure for mitochondrial oxidative phosphorylation for the cell (or organ) Figure 1 Overview of the submergence regulatory network in rice. The network involves three key factors: increased cellular ethylene content, depletion of ATP and consumption of readily available sucrose reserves. Submergence of aerial organs results in an accumulation of ethylene that triggers GApromoted cell elongation. In deepwater rice, ethylene promotes induction of the ERF genes SK1 and SK2 and elevation of GA, driving rapid internode elongation and escape of leaves near the water surface [2 ]. In lines tolerant of deep submergence, ethylene activates the ERF SUB1A-1, which promotes a rise in SLR1 and SLRL1, two transcription factors that directly inhibit GA-mediated activation of gene expression. This enhances survival by a quiescence strategy that limits carbohydrate consumption and elongation growth [3 ,5 ]. In japonica rice, which has neither SK1/2 nor SUB1A-1, submergence promotes underwater elongation of shoots until energy reserves are exhausted. Ethylene-triggered submergence-induced GAresponsiveness promotes expression of SUB1C, which acts upstream of a subset of a-amylases that convert starch into glucose for ATP production. Submergence includes the deprivation of oxygen, which leads to a deficiency in ATP. Under low-oxygen stress, ATP is produced at the substrate-level via glycolysis coupled with NAD + regeneration through ethanolic fermentation [1]. The production of alanine, GABA and succinate also contribute to ATP production [1, Natural variation in ethylene-driven submergenceinduced shoot elongation is also observed in other semi-wetland plants. In the wild species Rumex palustris, this natural variation includes both distinctions in final petiole length following submergence and the timing of elongation [16]. Recently, Manzur et al. [19] presented evidence that shoots of Lotus tenuis elongate upon partial shoot submergence, but not when the entire shoot is submerged; thus, both antithetical escape and quiescence survival strategies can exist within a single species. This could reflect the requirement for a threshold in ethylene or another metabolite to be reached to trigger quiescence. Under water shoot elongation acts synergistically with other leaf traits such as aerenchyma development and efficient underwater photosynthesis [14]. Pierik et al. [20] showed that partial de-submergence leads to an increase in biomass in R. palustris, which displays submergence escape, but not in R. acetosa which invokes quiescence. The drawback of extending leaves above water in R. acetosa was associated with low petiole porosity, highlighting interdependency between adaptive traits. Another collaboration between traits is observed in shoots that elongate underwater toward better illumination and ultimately, the water surface. Improvements in carbon gain needed to sustain elongation growth are achieved near the water surface where higher light levels act synergistically with traits that improve gas exchange (e.g. thin leaves, thin cuticles, thin cell walls, mesophyll chloroplasts that re-orientate toward the epidermis [21], and leaf surface gas films [22 ]). Anaerobic germination and early shoot elongation Rice produces one of the few plant seeds that can germinate under strict anoxia. This so-called anaerobic germination and early growth capacity is accomplished by harnessing reserves to fuel shoot elongation at the expense of root development [10]. This growth strategy ultimately mediates oxygen diffusion via a porous coleoptile/stem to submerged tissues and promotes the transition to autotrophic growth. Work on several species has shown that coleoptile elongation under anoxia or severe hypoxia involves accelerated glycolysis (Pasteur Effect), intensive ethanolic fermentation, limited cytoplasmic acidification, and regulation of cell wall loosening proteins [10,23]. Under anoxia, this elongation growth is not driven by ethylene, which requires oxygen for its biosynthesis [23]. Recently, rice's capacity to germinate and extend its coleoptile under water was shown to involve sensing of cellular energy resources, most likely ATP or soluble sugars [11 ]. It requires SnRK1s, the plant's Snf1/ AMP kinases, shown to sense and adjust cellular homeostasis in response to limitations in cellular energy imposed by hypoxia in mesophyll protoplasts of Arabidopsis [24 ] and CIPK15, which phosphorylates SnRK1A, thereby activating the transcriptional activator MYBS1, which initiates production of a-amylases responsible for starch catabolism to glucose. Seeds with a cipk15 loss-of-function mutation germinate in air but not underwater unless provided sucrose. This implies that a change in energy homeostasis, most likely depletion of sugars, drives consumption of endosperm reserves for shoot growth. Indeed, rice cultivars with early and vigorous coleoptile underwater display higher amylase activity The data indicate that the catabolism of starch during anaerobic germination and in submerged shoots of established plants is triggered by distinct mechanisms. In the case of anaerobic germination, an energy deficiency promotes activation of a-amylase transcription. By contrast in submerged plants, ethylene-promoted GA-responsiveness regulates a-amylase expression. If the signaling that drives anaerobic germination and early shoot elongation and SUB1A-mediated submergence tolerance are indeed independent, then it should be feasible to combine these traits to benefit farmers. Adaptive energy management The reduced efficiency in ATP synthesized per mol of glucose during oxygen deprivation leads to a cellular energy crisis. Plants generally respond by elevating sucrose catabolism, glycolysis and ethanolic fermentation to increase substrate-level production of ATP [1]. Transcriptomic and metabolomic studies of diverse angiosperms report the elevation of mRNAs and enzymes that enable sucrose catabolism and the fermentation of pyruvate to ethanol as well as lactate, alanine and GABA during oxygen deprivation [18 ,24 ,27-34,35 ,36 ,37-40]. Anaerobic substrate-level ATP production may be further augmented though alanine metabolism to succinate via a bifurcation of the TCA pathway Oxygen availability varies in organs and cells both during normal development and periods of external oxygen deprivation because of diffusion barriers and distinctions in metabolic activities. Consistent with this, electrode measurements have resolved oxygen gradients across roots, tubers, and stems Conclusion and perspectives The depth, duration, frequency and seasonal timing of floods impose distinct selection pressures on adaptive traits that enable survival of flooding [14]. Recent advances have provided new insights regarding regulation in three interacting networks of response: firstly, ethylene-driven shoot elongation; secondly, anaerobic seed germination and coleoptile growth; and thirdly, maximized conservation of carbohydrates and energy when oxidative phosphorylation is limited. Natural selection favors traits in particular environments when benefits outweigh costs. Shoot elongation is therefore mainly relevant in relatively shallow but prolonged floods, whereas mobilization of seed reserves for rapid shoot elongation aids establishment of seeds buried in anaerobic mud. When either of these two energydemanding escape strategies is too costly, such as in environments with ephemeral and/or very deep floods, the energy conserving quiescence strategy proves more effective. A general network describing the key submergence-induced pathways in rice is presented in Our current challenges include elucidating downstream targets of relevant transcription factors and unraveling essential cell to whole-plant survival strategies. Additionally, it is of utmost importance to precisely link dynamics in oxygen and ATP to metabolic adjustments and survival strategies. The molecular characterization of genetic variation in flooding response strategies is guaranteed to further enable the breeding of crops that can endure or outgrow flooding, as achieved for rice [2 ,4 ,6 ,7]. This is not only essential for crops cultivated in flood-prone farmlands, but is generally relevant because hypoxia exists also in bulky tubers, meristems, and maturing seeds

What makes species unique? The contribution of proteins with obscure features

BACKGROUND: Proteins with obscure features (POFs), which lack currently defined motifs or domains, represent between 18% and 38% of a typical eukaryotic proteome. To evaluate the contribution of this class of proteins to the diversity of eukaryotes, we performed a comparative analysis of the predicted proteomes derived from 10 different sequenced genomes, including budding and fission yeast, worm, fly, mosquito, Arabidopsis, rice, mouse, rat, and human. RESULTS: Only 1,650 protein groups were found to be conserved among these proteomes (BLAST E-value threshold of 10(-6)). Of these, only three were designated as POFs. Surprisingly, we found that, on average, 60% of the POFs identified in these 10 proteomes (44,236 in total) were species specific. In contrast, only 7.5% of the proteins with defined features (PDFs) were species specific (17,554 in total). As a group, POFs appear similar to PDFs in their relative contribution to biological functions, as indicated by their expression, participation in protein-protein interactions and association with mutant phenotypes. However, POF have more predicted disordered structure than PDFs, implying that they may exhibit preferential involvement in species-specific regulatory and signaling networks. CONCLUSION: Because the majority of eukaryotic POFs are not well conserved, and by definition do not have defined domains or motifs upon which to formulate a functional working hypothesis, understanding their biochemical and biological functions will require species-specific investigations

Profiling translatomes of discrete cell populations resolves altered cellular priorities during hypoxia in Arabidopsis

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.Instituto de Biotecnologia y Biologia Molecula