Innovation, conservation, and repurposing of gene function in root cell type development.

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.

Flexible Ion Barrier.

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.

Burning questions for a warming and changing world: 15 unknowns in plant abiotic stress.

We present unresolved questions in plant abiotic stress biology as posed by 15 research groups with expertise spanning eco-physiology to cell and molecular biology. Common themes of these questions include the need to better understand how plants detect water availability, temperature, salinity, and rising carbon dioxide (CO2) levels; how environmental signals interface with endogenous signaling and development (e.g. circadian clock and flowering time); and how this integrated signaling controls downstream responses (e.g. stomatal regulation, proline metabolism, and growth versus defense balance). The plasma membrane comes up frequently as a site of key signaling and transport events (e.g. mechanosensing and lipid-derived signaling, aquaporins). Adaptation to water extremes and rising CO2 affects hydraulic architecture and transpiration, as well as root and shoot growth and morphology, in ways not fully understood. Environmental adaptation involves tradeoffs that limit ecological distribution and crop resilience in the face of changing and increasingly unpredictable environments. Exploration of plant diversity within and among species can help us know which of these tradeoffs represent fundamental limits and which ones can be circumvented by bringing new trait combinations together. Better defining what constitutes beneficial stress resistance in different contexts and making connections between genes and phenotypes, and between laboratory and field observations, are overarching challenges.

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.

Variation in upstream open reading frames contributes to allelic diversity in maize protein abundance.

SignificanceProteins are the machinery which execute essential cellular functions. However, measuring their abundance within an organism can be difficult and resource-intensive. Cells use a variety of mechanisms to control protein synthesis from mRNA, including short open reading frames (uORFs) that lie upstream of the main coding sequence. Ribosomes can preferentially translate uORFs instead of the main coding sequence, leading to reduced translation of the main protein. In this study, we show that uORF sequence variation between individuals can lead to different rates of protein translation and thus variable protein abundances. We also demonstrate that natural variation in uORFs occurs frequently and can be linked to whole-plant phenotypes, indicating that uORF sequence variation likely contributes to plant adaptation.

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, 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.

Ethylene augments root hypoxia tolerance via growth cessation and reactive oxygen species amelioration.

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.

PHOSPHORUS-STARVATION TOLERANCE 1 (OsPSTOL1) is prevalent in upland rice and enhances root growth and hastens low phosphate signaling in wheat.

PHOSPHORUS-STARVATION TOLERANCE 1 (OsPSTOL1) is a variably present gene that benefits crown root growth and phosphorus (P) sufficiency in rice (Oryza sativa). To explore the ecophysiological importance of this gene, we performed a biogeographic survey of landraces and cultivars, confirming that functional OsPSTOL1 alleles prevail in low nutrient and drought-prone rainfed ecosystems, whereas loss-of-function and absence haplotypes predominate in control-irrigated paddy varieties of east Asia. An evolutionary history analysis of OsPSTOL1 and related genes in cereal, determined it and other genes are kinase-only domain derivatives of membrane-associated receptor like kinases. Finally, to evaluate the potential value of this kinase of unknown function in another Gramineae, wheat (Triticum aestivum) lines overexpressing OsPSTOL1 were evaluated under field and controlled low P conditions. OsPSTOL1 enhances growth, crown root number, and overall root plasticity under low P in wheat. Survey of root and shoot crown transcriptomes at two developmental stages identifies transcription factors that are differentially regulated in OsPSTOL1 wheat that are similarly controlled by the gene in rice. In wheat, OsPSTOL1 alters the timing and amplitude of regulators of root development in dry soils and hastens induction of the core P-starvation response. OsPSTOL1 and related genes may aid more sustainable cultivation of cereal crops.

Reprogramming of Root Cells during Nitrogen-Fixing Symbiosis Involves Dynamic Polysome Association of Coding and Noncoding RNAs.

Translational control is a widespread mechanism that allows the cell to rapidly modulate gene expression in order to provide flexibility and adaptability to eukaryotic organisms. We applied translating ribosome affinity purification combined with RNA sequencing to characterize translational regulation of mRNAs at early stages of the nitrogen-fixing symbiosis established between Medicago truncatula and Sinorhizobium meliloti Our analysis revealed a poor correlation between transcriptional and translational changes and identified hundreds of regulated protein-coding and long noncoding RNAs (lncRNAs), some of which are regulated in specific cell types. We demonstrated that a short variant of the lncRNA Trans-acting small interference RNA3 (TAS3) increased its association to the translational machinery in response to rhizobia. Functional analysis revealed that this short variant of TAS3 might act as a target mimic that captures microRNA390, contributing to reduce trans acting small interference Auxin Response Factor production and modulating nodule formation and rhizobial infection. The analysis of alternative transcript variants identified a translationally upregulated mRNA encoding subunit 3 of the SUPERKILLER complex (SKI3), which participates in mRNA decay. Knockdown of SKI3 decreased nodule initiation and development, as well as the survival of bacteria within nodules. Our results highlight the importance of translational control and mRNA decay pathways for the successful establishment of the nitrogen-fixing symbiosis.

Integrative Analysis from the Epigenome to Translatome Uncovers Patterns of Dominant Nuclear Regulation during Transient Stress.

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.

Conserved and nuanced hierarchy of gene regulatory response to hypoxia.

A dynamic assembly of nuclear and cytoplasmic processes regulate gene activity. Hypoxic stress and the associated energy crisis activate a plurality of regulatory mechanisms including modulation of chromatin structure, transcriptional activation and post-transcriptional processes. Temporal control of genes is associated with specific chromatin modifications and transcription factors. Genome-scale technologies that resolve transcript subpopulations in the nucleus and cytoplasm indicate post-transcriptional processes enable cells to conserve energy, prepare for prolonged stress and accelerate recovery. Moreover, the harboring of gene transcripts associated with growth in the nucleus and macromolecular RNA-protein complexes contributes to the preferential translation of stress-responsive gene transcripts during hypoxia. We discuss evidence of evolutionary variation in integration of nuclear and cytoplasmic processes that may contribute to variations in flooding resilience.

Profiling of Accessible Chromatin Regions across Multiple Plant Species and Cell Types Reveals Common Gene Regulatory Principles and New Control Modules.

The transcriptional regulatory structure of plant genomes remains poorly defined relative to animals. It is unclear how many cis-regulatory elements exist, where these elements lie relative to promoters, and how these features are conserved across plant species. We employed the assay for transposase-accessible chromatin (ATAC-seq) in four plant species (Arabidopsis thaliana, Medicago truncatula, Solanum lycopersicum, and Oryza sativa) to delineate open chromatin regions and transcription factor (TF) binding sites across each genome. Despite 10-fold variation in intergenic space among species, the majority of open chromatin regions lie within 3 kb upstream of a transcription start site in all species. We find a common set of four TFs that appear to regulate conserved gene sets in the root tips of all four species, suggesting that TF-gene networks are generally conserved. Comparative ATAC-seq profiling of Arabidopsis root hair and non-hair cell types revealed extensive similarity as well as many cell-type-specific differences. Analyzing TF binding sites in differentially accessible regions identified a MYB-driven regulatory module unique to the hair cell, which appears to control both cell fate regulators and abiotic stress responses. Our analyses revealed common regulatory principles among species and shed light on the mechanisms producing cell-type-specific transcriptomes during development.

Genetic mechanisms of abiotic stress tolerance that translate to crop yield stability.

Crop yield reduction as a consequence of increasingly severe climatic events threatens global food security. Genetic loci that ensure productivity in challenging environments exist within the germplasm of crops, their wild relatives and species that are adapted to extreme environments. Selective breeding for the combination of beneficial loci in germplasm has improved yields in diverse environments throughout the history of agriculture. An effective new paradigm is the targeted identification of specific genetic determinants of stress adaptation that have evolved in nature and their precise introgression into elite varieties. These loci are often associated with distinct regulation or function, duplication and/or neofunctionalization of genes that maintain plant homeostasis.

A stress recovery signaling network for enhanced flooding tolerance in Arabidopsis thaliana.

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.

Improved Transformation and Regeneration of Indica Rice: Disruption of SUB1A as a Test Case via CRISPR-Cas9.

Gene editing by use of clustered regularly interspaced short palindromic repeats (CRISPR) has become a powerful tool for crop improvement. However, a common bottleneck in the application of this approach to grain crops, including rice (Oryza sativa), is efficient vector delivery and calli regeneration, which can be hampered by genotype-dependent requirements for plant regeneration. Here, methods for Agrobacterium-mediated and biolistic transformation and regeneration of indica rice were optimized using CRISPR-Cas9 gene-editing of the submergence tolerance regulator SUBMERGENCE 1A-1 gene of the cultivar Ciherang-Sub1. Callus induction and plantlet regeneration methods were optimized for embryogenic calli derived from immature embryos and mature seed-derived calli. Optimized regeneration (95%) and maximal editing efficiency (100%) were obtained from the immature embryo-derived calli. Phenotyping of T1 seeds derived from the edited T0 plants under submergence stress demonstrated inferior phenotype compared to their controls, which phenotypically validates the disruption of SUB1A-1 function. The methods pave the way for rapid CRISPR-Cas9 gene editing of recalcitrant indica rice cultivars.

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.