The state of protein phosphorylation can be a key determinant of cellular physiology such as early-stage cancer, but the development of phosphoproteins in biofluids for disease diagnosis remains ...elusive. Here we demonstrate a strategy to isolate and identify phosphoproteins in extracellular vesicles (EVs) from human plasma as potential markers to differentiate disease from healthy states. We identified close to 10,000 unique phosphopeptides in EVs isolated from small volumes of plasma samples. Using label-free quantitative phosphoproteomics, we identified 144 phosphoproteins in plasma EVs that are significantly higher in patients diagnosed with breast cancer compared with healthy controls. Several biomarkers were validated in individual patients using paralleled reaction monitoring for targeted quantitation. This study demonstrates that the development of phosphoproteins in plasma EV as disease biomarkers is highly feasible and may transform cancer screening and monitoring.
Summary
Noncoding RNAs have been extensively described in plant and animal transcriptomes by using high‐throughput sequencing technology. Of these noncoding RNAs, a growing number of long intergenic ...noncoding RNAs (lincRNAs) have been described in multicellular organisms, however the origins and functions of many lincRNAs remain to be explored. In many eukaryotic genomes, transposable elements (TEs) are widely distributed and often account for large fractions of plant and animal genomes yet the contribution of TEs to lincRNAs is largely unknown. By using strand‐specific RNA‐sequencing, we profiled the expression patterns of lincRNAs in Arabidopsis, rice and maize, and identified 47 611 and 398 TE‐associated lincRNAs (TE‐lincRNAs), respectively. TE‐lincRNAs were more often derived from retrotransposons than DNA transposons and as retrotransposon copy number in both rice and maize genomes so did TE‐lincRNAs. We validated the expression of these TE‐lincRNAs by strand‐specific RT‐PCR and also demonstrated tissue‐specific transcription and stress‐induced TE‐lincRNAs either after salt, abscisic acid (ABA) or cold treatments. For Arabidopsis TE‐lincRNA11195, mutants had reduced sensitivity to ABA as demonstrated by longer roots and higher shoot biomass when compared to wild‐type. Finally, by altering the chromatin state in the Arabidopsis chromatin remodelling mutant ddm1, unique lincRNAs including TE‐lincRNAs were generated from the preceding untranscribed regions and interestingly inherited in a wild‐type background in subsequent generations. Our findings not only demonstrate that TE‐associated lincRNAs play important roles in plant abiotic stress responses but lincRNAs and TE‐lincRNAs might act as an adaptive reservoir in eukaryotes.
Significance Statement
Noncoding RNAs have been extensively described, but the origins and functions of many long intergenic noncoding RNAs (lincRNAs) remain to be explored. Here we describe hundreds of transposable element‐associated lincRNAs (TE‐lincRNAs) in rice, maize and Arabidopsis and demonstrate their tissue‐specific transcription. By perturbing chromatin structure, we show that novel TE‐lincRNAs are transcribed and inherited across generations, suggesting that lincRNAs might act as an adaptive reservoir in eukaryotes; indeed we show that loss of function of a TE‐lincRNA attenuates abiotic stress tolerance.
Significance Drought stress induces the accumulation of the plant stress hormone abscisic acid (ABA). ABA then quickly activates the protein kinase OST1/SnRK2.6 to phosphorylate a number of proteins ...in guard cells, resulting in stomatal closure to reduce transpirational water loss. How SnRK2.6 is deactivated and how ABA signaling may be desensitized are unclear. This study found that nitric oxide (NO) resulting from ABA signaling causes S-nitrosylation of SnRK2.6 at a cysteine residue close to the kinase catalytic site, which blocks the kinase activity. Dysfunction of S-nitrosoglutathione (GSNO) reductase causes GSNO overaccumulation in guard cells and ABA insensitivity in stomatal regulation. This work thus reveals how ABA-induced NO functions in guard cells to inactivate SnRK2.6 to negatively feedback regulate ABA signaling.
The phytohormone abscisic acid (ABA) plays important roles in plant development and adaptation to environmental stress. ABA induces the production of nitric oxide (NO) in guard cells, but how NO regulates ABA signaling is not understood. Here, we show that NO negatively regulates ABA signaling in guard cells by inhibiting open stomata 1 (OST1)/sucrose nonfermenting 1 (SNF1)-related protein kinase 2.6 (SnRK2.6) through S-nitrosylation. We found that SnRK2.6 is S-nitrosylated at cysteine 137, a residue adjacent to the kinase catalytic site. Dysfunction in the S-nitrosoglutathione (GSNO) reductase (GSNOR) gene in the gsnor1-3 mutant causes NO overaccumulation in guard cells, constitutive S-nitrosylation of SnRK2.6, and impairment of ABA-induced stomatal closure. Introduction of the Cys137 to Ser mutated SnRK2.6 into the gsnor1-3/ost1-3 double-mutant partially suppressed the effect of gsnor1-3 on ABA-induced stomatal closure. A cysteine residue corresponding to Cys137 of SnRK2.6 is present in several yeast and human protein kinases and can be S-nitrosylated, suggesting that the S-nitrosylation may be an evolutionarily conserved mechanism for protein kinase regulation.
Epigenetic variation has been proposed to facilitate adaptation to changing environments, but evidence that natural epialleles contribute to adaptive evolution has been lacking. Here we identify a ...retrotransposon, named "NMR19" (naturally occurring DNA methylation variation region 19), whose methylation and genomic location vary among Arabidopsis thaliana accessions. We classify NMR19 as NMR19-4 and NMR19-16 based on its location, and uncover NMR19-4 as an epiallele that controls leaf senescence by regulating the expression of PHEOPHYTIN PHEOPHORBIDE HYDROLASE (PPH). We find that the DNA methylation status of NMR19-4 is stably inherited and independent of genetic variation. In addition, further analysis indicates that DNA methylation of NMR19-4 correlates with local climates, implying that NMR19-4 is an environmentally associated epiallele. In summary, we discover a novel epiallele, and provide mechanistic insights into its origin and potential function in local climate adaptation.
Dear Editor, Recently, engineered endonucleases, such as Zinc-Finger Nucleases （ZFNs） （Carroll, 2011）, Transcription Activator-Like Effector Nucleases （TALENs） （Mahfouz et al., 2011; Li et al., ...2012）, and Clustered Regularly Interspaced Short Palindromic Repeats （CRISPR）-associated （Cas） systems （Cong et al., 2013） have been successfully used for gene editing in a variety of species. These systems generate double-strand breaks （DSBs） at target loci to drive site-specific DNA sequence modifica- tions. The modifications include sequence insertion and deletion and other mutations in the host genomes via the error-prone non-homologous end joining （NHEJ） pathway or sequence correction or replacement through the error-free homologous recombination （HR） pathway （Symington and Gautier, 2011）. Here, we show that the CRISPR-Cas system can be applied to generate targeted gene mutations and gene corrections in plants, and the system can also be readily engi- neered to achieve deletion of large DNA fragments and for multiplex gene editing in plants.
When under salt stress, plants maintain a high concentration of K
+ and a low concentration of Na
+ in the cytosol. They do this by regulating the expression and activity of K
+ and Na
+ transporters ...and of H
+ pumps that generate the driving force for transport. Although salt-stress sensors remain elusive, some of the intermediary signaling components have been identified. Evidence suggests that a protein kinase complex consisting of the myristoylated calcium-binding protein SOS3 and the serine/threonine protein kinase SOS2 is activated by a salt-stress-elicited calcium signal. The protein kinase complex then phosphorylates and activates various ion transporters, such as the plasma membrane Na
+/H
+ antiporter SOS1.
De novo DNA methylation through the RNA-directed DNA methylation (RdDM) pathway and active DNA demethylation play important roles in controlling genome-wide DNA methylation patterns in plants. Little ...is known about how cells manage the balance between DNA methylation and active demethylation activities. Here, we report the identification of a unique RdDM target sequence, where DNA methylation is required for maintaining proper active DNA demethylation of the Arabidopsis genome. In a genetic screen for cellular antisilencing factors, we isolated several REPRESSOR OF SILENCING 1 ( ros1 ) mutant alleles, as well as many RdDM mutants, which showed drastically reduced ROS1 gene expression and, consequently, transcriptional silencing of two reporter genes. A helitron transposon element (TE) in the ROS1 gene promoter negatively controls ROS1 expression, whereas DNA methylation of an RdDM target sequence between ROS1 5′ UTR and the promoter TE region antagonizes this helitron TE in regulating ROS1 expression. This RdDM target sequence is also targeted by ROS1, and defective DNA demethylation in loss-of-function ros1 mutant alleles causes DNA hypermethylation of this sequence and concomitantly causes increased ROS1 expression. Our results suggest that this sequence in the ROS1 promoter region serves as a DNA methylation monitoring sequence (MEMS) that senses DNA methylation and active DNA demethylation activities. Therefore, the ROS1 promoter functions like a thermostat (i.e., methylstat) to sense DNA methylation levels and regulates DNA methylation by controlling ROS1 expression.
Significance DNA methylation is critical for transposon silencing and gene regulation. DNA methylation levels are determined by the combined activities of DNA methyltransferases and demethylases. This study found a 39-bp DNA methylation monitoring sequence (MEMS) in the promoter of the DNA demethylase REPRESSOR OF SILENCING 1 ( ROS1 ) gene of Arabidopsis plants. DNA methylation of MEMS is responsive to both RNA-directed DNA methylation and ROS1-dependent active demethylation. Thus, MEMS can sense DNA methylation and demethylation activities, and it regulates genomic DNA methylation by adjusting ROS1 expression levels. Our results suggest that the ROS1 promoter, with the MEMS and an adjacent helitron transposon element, functions as a “methylstat” that senses and regulates genomic DNA methylation levels.
Defense against stress and active suppression of growth are two complementary strategies by which plants respond to adverse environments. Although beneficial for plant survival, active growth ...inhibition is often undesirable for crop productivity. Compared with the knowledge on how plants defend against stress-caused cellular impairment, much less is known about how stress signaling regulates plant growth and vice versa. Here, we review recent progress in this area and discuss recent studies suggesting that reciprocal regulation between stress-response and growth-control pathways occurs at multiple levels. Understanding this regulatory network will be critical for resetting the balance between stress resistance and growth in order to engineer stress-resistant and high-yielding crops.
Defense against stress and suppression of growth are complementary strategies by which plants respond to adverse environments. Although beneficial for plant survival, active growth inhibition is often undesirable for crop productivity. Zhang et al. review recent progress in this area and discuss the reciprocal regulation between stress-response and growth-control pathways.
Abscisic acid (ABA) regulates diverse plant processes, growth and development under non-stress conditions and plays a pivotal role in abiotic stress tolerance. Although ABA-regulated genetic ...processes are well known, recent discoveries reveal that epigenetic processes are an integral part of ABA-regulated processes. Epigenetic mechanisms, namely, histone modifications and cytosine DNA methylation-induced modification of genome give rise to epigenomes, which add diversity and complexity to the genome of organisms. Histone monoubiquitination appears to regulate ABA levels in developing seeds through histone H2B monoubiquitination. ABA and H2B ubiquitination dependent chromatin remodeling regulate seed dormancy. Transcription factor networks necessary for seed maturation are repressed by histone deacetylases (HDACs)-dependent and PICKLE chromatin remodeling complexes (CRCs), whereas ABA induces the expression of these genes directly or through repression of HDACs. Abiotic stress-induced ABA regulates stomatal response and stress-responsive gene expression through HDACs and HOS15-dependent histone deacetylation, as well as through the ATP-dependent SWITCH/SUCROSE NONFERMENTING CRC. ABA also probably regulates the abiotic stress response through DNA methylation and short interfering RNA pathways. Further studies on ABA-regulated epigenome will be of immense use to understand the plant development, stress adaptation and stress memory.
The CRISPR/Cas9 system, composed of a single-guide RNA for target recognition and a Cas9 protein for DNA cleavage, has the potential to revolutionize agriculture as well as medicine. Even though ...extensive work has been done to improve the gene editing activity of CRISPR/Cas9, little is known about the regulation of this bacterial system in eukaryotic host cells, especially at the post-transcriptional level.
Here, we evaluate the expression levels of the two CRISPR/Cas9 components and the gene editing efficiency in a set of Arabidopsis mutants involved in RNA silencing. We find that mutants defective in the post-transcriptional gene-silencing pathway display significantly higher Cas9 and sgRNA transcript levels, resulting in higher mutagenesis frequencies than wild-type controls. Accordingly, silencing of AGO1 by introduction of an AGO1-RNAi cassette into the CRISPR/Cas9 vector provides an increase in gene editing efficiency. Co-expression of the viral suppressor p19 from the tomato bushy stunt virus to suppress the plant RNA-silencing pathway shows a strong correlation between the severity of the phenotypic effects caused by p19 and the gene editing efficiency of the CRISPR/Cas9 system for two different target genes, AP1 and TT4.
This system has useful practical applications in facilitating the detection of CRISPR/Cas9-induced mutations in T1 plants as well as the identification of transgene-free T2 plants by simple visual observation of the symptom severity caused by p19. Our study shows that CRISPR/Cas9 gene editing efficiency can be improved by reducing RNA silencing in plants.
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