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. ribosome profiling | uORF | alternative splicing | long intergenic noncoding RNA | translational efficiency

SignificanceNoncoding RNAs are an underexplored reservoir of regulatory molecules in eukaryotes. We analyzed the environmental response of roots to phosphorus (Pi) nutrition to understand how a change in availability of an essential element is managed. Pi availability influenced translational regulation mediated by small upstream ORFs on protein-coding mRNAs. Discovery, classification, and evaluation of long noncoding RNAs (lncRNAs) associated with translating ribosomes uncovered diverse new examples of translational regulation. These included Pi-regulated small peptide synthesis, ribosome-coupled phased small interfering RNA production, and the translational regulation of natural antisense RNAs and other regulatory RNAs. This study demonstrates that translational control contributes to the stability and activity of regulatory RNAs, providing an avenue for manipulation of traits.

Thermopriming induces genome-wide differential gene expression and alternative splicing patterns, and establishes a ‘splicing memory’ that helps plants to survive subsequent and otherwise lethal heat stress.

These authors contributed equally to this work. SUMMARYThe root system is crucial for acquisition of resources from the soil. In legumes, the efficiency of mineral and water uptake by the roots may be reinforced due to establishment of symbiotic relationships with mycorrhizal fungi and interactions with soil rhizobia. Here, we investigated the role of miR396 in regulating the architecture of the root system and in symbiotic interactions in the model legume Medicago truncatula. Analyses with promoter-GUS fusions suggested that the mtr-miR396a and miR396b genes are highly expressed in root tips, preferentially in the transition zone, and display distinct expression profiles during lateral root and nodule development. Transgenic roots of composite plants that over-express the miR396b precursor showed lower expression of six growth-regulating factor genes (MtGRF) and two bHLH79-like target genes, as well as reduced growth and mycorrhizal associations. miR396 inactivation by mimicry caused contrasting tendencies, with increased target expression, higher root biomass and more efficient colonization by arbuscular mycorrhizal fungi. In contrast to MtbHLH79, repression of three GRF targets by RNA interference severely impaired root growth. Early activation of mtr-miR396b, concomitant with posttranscriptional repression of MtGRF5 expression, was also observed in response to exogenous brassinosteroids. Growth limitation in miR396 over-expressing roots correlated with a reduction in cell-cycle gene expression and the number of dividing cells in the root apical meristem. These results link the miR396 network to the regulation of root growth and mycorrhizal associations in plants.

SignificanceFlooding due to extreme weather events can be highly detrimental to plant development and yield. Speedy recovery following stress removal is an important determinant of tolerance, yet mechanisms regulating this remain largely uncharacterized. We identified a regulatory network in Arabidopsis thaliana that controls water loss and senescence to influence recovery from prolonged submergence. Targeted control of the molecular mechanisms facilitating stress recovery identified here could potentially improve performance of crops in flood-prone areas.

34Abiotic stresses in plants are often transient and the recovery phase following stress removal is critical. 35Flooding, a major abiotic stress that negatively impacts plant biodiversity and agriculture, is a 36 sequential stress where tolerance is strongly dependent on viability underwater and during the post-37 flooding period. Here we show that in Arabidopsis thaliana accessions (Bay-0 and Lp2-6), different 38 rates of submergence recovery correlate with submergence tolerance and fecundity. A genome-wide 39 assessment of ribosome-associated transcripts in Bay-0 and Lp2-6 revealed a signaling network 40 regulating recovery processes. Differential recovery between the accessions was related to the activity 41 of three genes: RESPIRATORY BURST OXIDASE HOMOLOG (RBOHD), SENESCENCE-42 ASSOCIATED GENE113 (SAG113) and ORESARA1 (ORE1/NAC6) which function in a regulatory 43 network involving a reactive oxygen species (ROS) burst upon de-submergence and the hormones 44 abscisic acid and ethylene. This regulatory module controls ROS homeostasis, stomatal aperture and 45 chlorophyll degradation during submergence recovery. This work uncovers a signaling network that 46 regulates recovery processes following flooding to hasten the return to pre-stress homeostasis. 48Significance statement 49 Flooding due to extreme weather events can be highly detrimental to plant development and yield. 50Speedy recovery following stress removal is an important determinant of tolerance, yet mechanisms 51 regulating this remain largely uncharacterized. We identified a regulatory network in Arabidopsis 52 thaliana that controls water loss and senescence to influence recovery from prolonged submergence. 53Targeted control of the molecular mechanisms facilitating stress recovery identified here can 54 potentially improve performance of crops in flood-prone areas. 56 \body 57 Introduction 58Plants continuously adjust their metabolism to modulate growth and development within a highly 59 dynamic and often inhospitable environment. Climate change has exacerbated the severity and 60 unpredictability of environmental conditions that are suboptimal for plant growth and survival, including 61 extremes in the availability of water and temperature. Under these conditions, plant resilience to 62 environmental extremes is determined by acclimation not only to the stress itself, but also to recovery 63 following stress removal. This is especially apparent in plants recovering from flooding. Flooding is an 64 abiotic stress that has seen a recent global surge with dramatic consequences for crop yields and 65 plant biodiversity (1-3). Most terrestrial plants, including nearly all major crops, are sensitive to partial 66 to complete submergence of the above ground organs. Inundations that include aerial organs severely 67 reduces gas diffusion rates, and the ensuing impedance to gas exchange compromises both 68 photosynthesis and respiration. Additionally, muddy floodwaters can almost completely block light 69 access thus further hindering photosynthesis. Ul...

Increasing genetic diversity via directed evolution holds great promise to accelerate trait development and crop improvement. We developed a CRISPR/Cas-based directed evolution platform in plants to evolve the rice ( Oryza sativa ) SF3B1 spliceosomal protein for resistance to splicing inhibitors. SF3B1 mutant variants, termed SF3B1-GEX1A-Resistant (SGR), confer variable levels of resistance to splicing inhibitors. Studies of the structural basis of the splicing inhibitor binding to SGRs corroborate the resistance phenotype. This directed evolution platform can be used to interrogate and evolve the molecular functions of key biomolecules and to engineer crop traits for improved performance and adaptation under climate change conditions. Electronic supplementary material The online version of this article (10.1186/s13059-019-1680-9) contains supplementary material, which is available to authorized users.

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