Michael E. Fromm

Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice

The type II CRISPR/Cas system from Streptococcus pyogenes and its simplified derivative, the Cas9/single guide RNA (sgRNA) system, have emerged as potent new tools for targeted gene knockout in bacteria, yeast, fruit fly, zebrafish and human cells. Here, we describe adaptations of these systems leading to successful expression of the Cas9/sgRNA system in two dicot plant species, Arabidopsis and tobacco, and two monocot crop species, rice and sorghum. Agrobacterium tumefaciens was used for delivery of genes encoding Cas9, sgRNA and a non-fuctional, mutant green fluorescence protein (GFP) to Arabidopsis and tobacco. The mutant GFP gene contained target sites in its 5′ coding regions that were successfully cleaved by a CAS9/sgRNA complex that, along with error-prone DNA repair, resulted in creation of functional GFP genes. DNA sequencing confirmed Cas9/sgRNA-mediated mutagenesis at the target site. Rice protoplast cells transformed with Cas9/sgRNA constructs targeting the promoter region of the bacterial blight susceptibility genes, OsSWEET14 and OsSWEET11, were confirmed by DNA sequencing to contain mutated DNA sequences at the target sites. Successful demonstration of the Cas9/sgRNA system in model plant and crop species bodes well for its near-term use as a facile and powerful means of plant genetic engineering for scientific and agricultural applications.

Multiple exposures to drought 'train' transcriptional responses in Arabidopsis

Pre-exposure to stress may alter plants subsequent responses by producing faster and/or stronger reactions implying that plants exercise a form of stress memory. The mechanisms of plants stress memory responses are poorly understood leaving this fundamental biological question unanswered. Here we show that during recurring dehydration stresses Arabidopsis plants display transcriptional stress memory demonstrated by an increase in the rate of transcription and elevated transcript levels of a subset of the stress-response genes (trainable genes). During recovery (watered) states, trainable genes produce transcripts at basal (preinduced) levels, but remain associated with atypically high H3K4me3 and Ser5P polymerase II levels, indicating that RNA polymerase II is stalled. This is the first example of a stalled RNA polymerase II and its involvement in transcriptional memory in plants. These newly discovered phenomena might be a general feature of plant stress-response systems and could lead to novel approaches for increasing the flexibility of a plants ability to respond to the environment.

Dynamic changes in genome-wide histone H3 lysine 4 methylation patterns in response to dehydration stress in Arabidopsis thaliana

BackgroundThe molecular mechanisms of genome reprogramming during transcriptional responses to stress are associated with specific chromatin modifications. Available data, however, describe histone modifications only at individual plant genes induced by stress. We have no knowledge of chromatin modifications taking place at genes whose transcription has been down-regulated or on the genome-wide chromatin modification patterns that occur during the plants response to dehydration stress.ResultsUsing chromatin immunoprecipitation and deep sequencing (ChIP-Seq) we established the whole-genome distribution patterns of histone H3 lysine 4 mono-, di-, and tri-methylation (H3K4me1, H3K4me2, and H3K4me3, respectively) in Arabidopsis thaliana during watered and dehydration stress conditions. In contrast to the relatively even distribution of H3 throughout the genome, the H3K4me1, H3K4me2, and H3K4me3 marks are predominantly located on genes. About 90% of annotated genes carry one or more of the H3K4 methylation marks. The H3K4me1 and H3K4me2 marks are more widely distributed (80% and 84%, respectively) than the H3K4me3 marks (62%), but the H3K4me2 and H3K4me1 levels changed only modestly during dehydration stress. By contrast, the H3K4me3 abundance changed robustly when transcripts levels from responding genes increased or decreased. In contrast to the prominent H3K4me3 peaks present at the 5-ends of most transcribed genes, genes inducible by dehydration and ABA displayed atypically broader H3K4me3 distribution profiles that were present before and after the stress.ConclusionsA higher number (90%) of annotated Arabidopsis genes carry one or more types of H3K4me marks than previously reported. During the response to dehydration stress the changes in H3K4me1, H3K4me2, and H3K4me3 patterns show different dynamics and specific patterns at up-regulated, down-regulated, and unaffected genes. The different behavior of each methylation mark during the response process illustrates that they have distinct roles in the transcriptional response of implicated genes. The broad H3K4me3 distribution profiles on nucleosomes of stress-induced genes uncovered a specific chromatin pattern associated with many of the genes involved in the dehydration stress response.

The Arabidopsis trithorax‐like factor ATX1 functions in dehydration stress responses via ABA‐dependent and ABA‐independent pathways

Emerging evidence suggests that the molecular mechanisms driving the responses of plants to environmental stresses are associated with specific chromatin modifications. Here, we demonstrate that the Arabidopsis trithorax-like factor ATX1, which trimethylates histone H3 at lysineu20034 (H3K4me3), is involved in dehydration stress signaling in both abscisic acid (ABA)-dependent and ABA-independent pathways. The loss of function of ATX1 results in decreased germination rates, larger stomatal apertures, more rapid transpiration and decreased tolerance to dehydration stress in atx1 plants. This deficiency is caused in part by reduced ABA biosynthesis in atx1 plants resulting from decreased transcript levels from NCED3, which encodes a key enzyme controlling ABA production. Dehydration stress increased ATX1 binding to NCED3, and ATX1 was required for the increased levels of NCED3 transcripts and nucleosomal H3K4me3 that occurred during dehydration stress. Mechanistically, ATX1 affected the quantity of RNA polymeraseu2003II bound to NCED3. By upregulating NCED3 transcription and ABA production, ATX1 influenced ABA-regulated pathways and genes. ATX1 also affected the expression of ABA-independent genes, implicating ATX1 in diverse dehydration stress-response mechanisms in Arabidopsis.

Four distinct types of dehydration stress memory genes in Arabidopsis thaliana

BackgroundHow plants respond to dehydration stress has been extensively researched. However, how plants respond to multiple consecutive stresses is virtually unknown. Pre-exposure to various abiotic stresses (including dehydration) may alter plants’ subsequent responses by improving resistance to future exposures. These observations have led to the concept of ‘stress memory’ implying that during subsequent exposures plants provide responses that are different from those during their first encounter with the stress. Genes that provide altered responses in a subsequent stress define the ‘memory genes’ category; genes responding similarly to each stress form the ‘non-memory’ category.ResultsUsing a genome-wide RNA-Seq approach we determine the transcriptional responses of Arabidopsis plants that have experienced multiple exposures to dehydration stress and compare them with the transcriptional behavior of plants encountering the stress for the first time. The major contribution of this study is the revealed existence of four distinct, previously unknown, transcription memory response patterns of dehydration stress genes in A.thaliana. The biological relevance for each of the four memory types is considered in the context of four overlapping strategies employed by a plant to improve its stress tolerance and/or survival: 1) increased synthesis of protective, damage-repairing, and detoxifying functions; 2) coordinating photosynthesis and growth under repetitive stress; 3) re-adjusting osmotic and ionic equilibrium to maintain homeostasis; and 4) re-adjusting interactions between dehydration and other stress/hormone regulated pathways.ConclusionsThe results reveal the unknown, hitherto, existence of four distinct transcription memory response types in a plant and provide genome-wide characterization of memory and non-memory dehydration stress response genes in A.thaliana. The transcriptional responses during repeated exposures to stress are different from known responses occurring during a single exposure. GO analyses of encoded proteins suggested implications for the cellular/organismal protective, adaptive, and survival functions encoded by the memory genes. The results add a new dimension to our understanding of plants’ responses to dehydration stress and to current models for interactions between different signaling systems when adjusting to repeated spells of water deficits.

Two Distinct Roles of ARABIDOPSIS HOMOLOG OF TRITHORAX1 (ATX1) at Promoters and within Transcribed Regions of ATX1-Regulated Genes

This work shows that the H3K4 trimethylase ATX1 regulates gene expression via mechanisms different from those reported for its yeast or mammalian homologs. It finds that ATX1 affects transcription at the promoters of target genes by a mechanism distinct from its ability to trimethylate lysine 4 of histone H3 within genes. The Arabidopsis thaliana trithorax-like protein, ATX1, shares common structural domains, has similar histone methyltransferase (HMT) activity, and belongs in the same phylogenetic subgroup as its animal counterparts. Most of our knowledge of the role of HMTs in trimethylating lysine 4 of histone H3 (H3K4me3) in transcriptional regulation comes from studies of yeast and mammalian homologs. Little is known about the mechanism by which ATX1, or any other HMT of plant origin, affects transcription. Here, we provide insights into how ATX1 influences transcription at regulated genes, playing two distinct roles. At promoters, ATX1 is required for TATA binding protein (TBP) and RNA Polymerase II (Pol II) recruitment. In a subsequent event, ATX1 is recruited by a phosphorylated form of Pol II to the +300-bp region of transcribed sequences, where it trimethylates nucleosomes. In support of this model, inhibition of phosphorylation of the C-terminal domain of Pol II reduced the amounts of H3K4me3 and ATX1 bound at the +300-nucleotide region. Importantly, these changes did not reduce the occupancy of ATX1, TBP, or Pol II at promoters. Our results indicate that ATX1 affects transcription at target genes by a mechanism distinct from its ability to trimethylate H3K4 within genes.

ATX1-Generated H3K4me3 Is Required for Efficient Elongation of Transcription, Not Initiation, at ATX1-Regulated Genes

Tri-methylated H3 lysine 4 (H3K4me3) is associated with transcriptionally active genes, but its function in the transcription process is still unclear. Point mutations in the catalytic domain of ATX1 (ARABIDOPSIS TRITHORAX1), a H3K4 methyltransferase, and RNAi knockdowns of subunits of the AtCOMPASS–like (Arabidopsis Complex Proteins Associated with Set) were used to address this question. We demonstrate that both ATX1 and AtCOMPASS–like are required for high level accumulation of TBP (TATA-binding protein) and Pol II at promoters and that this requirement is independent of the catalytic histone modifying activity. However, the catalytic function is critically required for transcription as H3K4me3 levels determine the efficiency of transcription elongation. The roles of H3K4me3, ATX1, and AtCOMPASS–like may be of a general relevance for transcription of Trithorax-activated eukaryotic genes.

H3K27me3 and H3K4me3 Chromatin Environment at Super-Induced Dehydration Stress Memory Genes of Arabidopsis thaliana

Pre-exposure to a stress may alter the plants cellular, biochemical, and/or transcriptional responses during future encounters as a memory from the previous stress. Genes increasing transcription in response to a first dehydration stress, but producing much higher transcript levels in a subsequent stress, represent the super-induced transcription memory genes in Arabidopsis thaliana. The chromatin environment (histone H3 tri-methylations of Lys 4 and Lys 27, H3K4me3, and H3K27me3) studied at five dehydration stress memory genes revealed existence of distinct memory-response subclasses that responded differently to CLF deficiency and displayed different transcriptional activities during the watered recovery periods. Among the most important findings is the novel aspect of the H3K27me3 function observed at specific dehydration stress memory genes. In contrast to its well-known role as a chromatin repressive mechanism at developmentally regulated genes, H3K27me3 did not prevent transcription from the dehydration stress-responding genes. The high H3K27me3 levels present during transcriptionally inactive states did not interfere with the transition to active transcription and with H3K4me3 accumulation. H3K4me3 and H3K27me3 marks function independently and are not mutually exclusive at the dehydration stress-responding memory genes.

Physiological and transcriptional memory in guard cells during repetitive dehydration stress

Arabidopsis plants subjected to a daily dehydration stress and watered recovery cycle display physiological and transcriptional stress memory. Previously stressed plants have stomatal apertures that remain partially closed during a watered recovery period, facilitating reduced transpiration during a subsequent dehydration stress. Guard cells (GCs) display transcriptional memory that is similar to that in leaf tissues for some genes, but display GC-specific transcriptional memory for other genes. The rate-limiting abscisic acid (ABA) biosynthetic genes NINE-CIS-EPOXYCAROTENOID DIOXYGENASE 3 (NCED3) and ALDEHYDE OXIDASE 3 (AAO3) are expressed at much higher levels in GCs, particularly during the watered recovery interval, relative to their low levels in leaves. A genetic analysis using mutants in the ABA signaling pathway indicated that GC stomatal memory is ABA-dependent, and that ABA-dependent SNF1-RELATED PROTEIN KINASE 2.2 (SnRK2.2), SnRK2.3 and SnRK2.6 have distinguishable roles in the process. SnRK2.6 is more important for overall stomatal control, while SnRK2.2 and SnRK2.3 are more important for implementing GC stress memory in the subsequent dehydration response. Collectively, our results support a model of altered ABA production in GCs that maintains a partially closed stomatal aperture during an overnight watered recovery period.

The Arabidopsis chromatin modifier ATX1, the myotubularin-like AtMTM and the response to drought.

Plants respond to environmental stresses by altering transcription of genes involved in the response. The chromatin modifier ATX1 regulates expression of a large number of genes; consequently, factors that affect ATX1 activity would also influence expression from ATX1-regulated genes. Here, we demonstrate that dehydration is such a factor implicating ATX1 in the plants response to drought. In addition, we report that a hitherto unknown Arabidopsis gene, At3g10550, encodes a phosphoinositide 3-phosphatase related to the animal myotubularins (AtMTM1). Myotubularin activities in plants have not been described and herein, we identify an overlapping set of genes co-regulated by ATX1 and AtMTM under drought conditions. We propose that these shared genes represent the ultimate targets of partially overlapping branches of the pathways of the nuclear ATX1 and the cytoplasmic AtMTM1. Our analyses offer first genome-wide insights into the relationship of an epigenetic factor and a lipid phosphatase from the other end of a shared drought responding pathway in Arabidopsis.