The CRISPR/Cas system became a powerful genome editing tool for basic plant research and crop improvement. Thus far, CRISPR/Cas has been applied to many plants, including Arabidopsis, rice and other ...crop plants. It has been reported that CRISPR/Cas efficiency is generally high in many plants. In this study, we compared the genome editing efficiency of CRISPR/Cas in three different Arabidopsis accessions Col-0, Ler, and C24RDLUC (C24 accession harboring the stress-responsive RD29A promoter-driven luciferase reporter). For the comparison, we chose to target the cold-responsive
(
) genes.
,
, and
genes are tandemly located on Arabidopsis chromosome 4 with redundant functions as the key transcription factors functioning in cold stress signaling and tolerance. Due to the close proximity of these
s on the chromosome, it is impossible to generate
triple mutants (
) by traditional genetic crosses. Therefore, using the CRISPR/Cas tool, we aimed to generate
mutants and compared the genome editing efficiency in different Arabidopsis accessions. Among the accessions, Ler was the most resilient to the CRISPR/Cas deletion with the lowest gene deletion ratio in both T1 and T2 generations. Interestingly, while C24RDLUC showed a high
deletion frequency in T2 only when the gene deletion was observed in T1 generation, Col-0 displayed high ratios of the
deletions in T2 regardless of the presence or absence of the
deletion in T1. Isolated
mutants in C24RDLUC background showed no expression of
,
, and
genes and proteins with reduction in the
target gene expression under cold stress.
Glycine betaine (GB) can confer tolerance to several types of stress at low concentrations, either after application to plants or in transgenics engineered to overproduce GB. Based on earlier studies ...on levels of GB in plants and evidence for effects on gene expression, we hypothesized that at least part of this effect could be ascribed to the activation of the expression of stress tolerance genes. Using a strategy based on high-throughput gene expression analysis with microarrays followed by confirmation with northern blots, we identified Arabidopsis genes upregulated in roots that reinforce intracellular processes protecting cells from oxidative damage and others that appear to be involved in reinforcing a scavenging system for reactive oxygen species (ROS) in cell walls. Upregulated genes in roots include those for the membrane-trafficking RabA4c, the root-specific NADPH-dependent ferric reductase (FRO2) localized to the plasma membrane, mitochondrial catalase 2 and the cell wall peroxidase ATP3a. Comparative studies with wild-type Arabidopsis and knockout mutants for the membrane-trafficking RabA4c gene demonstrated that the mutants respond only slightly to GB, if at all, compared with wild-type in relation to root growth recovery after chilling stress, demonstrating the role of RabA4c in relation to the GB effect. The results point toward links between oxidative stress, gene expression, membrane trafficking and scavenging of ROS such as superoxide and hydrogen peroxide in relation to GB effects on chilling tolerance in plants.
Two Arabidopsis thaliana extragenic mutations that suppress NaCl hypersensitivity of the sos3-1 mutant were identified in a screen of a T-DNA insertion population in the genetic background of Col-0 ...gl1 sos3-1. Analysis of the genome sequence in the region flanking the T-DNA left border indicated that sos3-1 hkt1-1 and sos3-1 hkt1-2 plants have allelic mutations in AtHKT1. AtHKT1 mRNA is more abundant in roots than shoots of wild-type plants but is not detected in plants of either mutant, indicating that this gene is inactivated by the mutations. hkt1-1 and hkt1-2 mutations can suppress to an equivalent extent the Na+sensitivity of sos3-1 seedlings and reduce the intracellular accumulation of this cytotoxic ion. Moreover, sos3-1 hkt1-1 and sos3-1 hkt1-2 seedlings are able to maintain K+
intin medium supplemented with NaCl and exhibit a substantially higher intracellular ratio of K+/NA+than the sos3-1 mutant. Furthermore, the hkt1 mutations abrogate the growth inhibition of the sos3-1 mutant that is caused by K+deficiency on culture medium with low Ca2+(0.15 mM) and <200 µM K+. Interestingly, the capacity of hkt1 mutations to suppress the Na+hypersensitivity of the sos3-1 mutant is reduced substantially when seedlings are grown in medium with low Ca2+(0.15 mM). These results indicate that AtHKT1 is a salt tolerance determinant that controls Na+entry and high affinity K+uptake. The hkt1 mutations have revealed the existence of another Na+influx system(s) whose activity is reduced by high Ca2+ext.
Transcription activator-like effectors (TALEs) from Xanthomonas sp. have been used as customizable DNA-binding modules for genome-engineering applications. Ralstonia solanacearum TALE-like proteins ...(RTLs) exhibit similar structural features to TALEs, including a central DNA-binding domain composed of 35 amino acid-long repeats. Here, we characterize the RTLs and show that they localize in the plant cell nucleus, mediate DNA binding, and might function as transcriptional activators. RTLs have a unique DNA-binding architecture and are enriched in repeat variable di-residues (RVDs), which determine repeat DNA-binding specificities. We determined the DNA-binding specificities for the RVD sequences ND, HN, NP, and NT. The RVD ND mediates highly specific interactions with C nucleotide, HN interacts specifically with A and G nucleotides, and NP binds to C, A, and G nucleotides. Moreover, we developed a highly efficient repeat assembly approach for engineering RTL effectors. Taken together, our data demonstrate that RTLs are unique DNA-targeting modules that are excellent alternatives to be tailored to bind to user-selected DNA sequences for targeted genomic and epigenomic modifications. These findings will facilitate research concerning RTL molecular biology and RTL roles in the pathogenicity of Ralstonia spp.
We report the characterization of three Ralstonia TAL-like effectors, which mediate DNA binding and can be used as customizable architectures for DNA targeting. We determined DNA-binding specificities of novel repeat variable di-residues (RVDs) and devised a repeat assembly approach for engineering Ralstonia solanacearum TALE-like proteins (RTLs).
Modifications of RNA bases have been found in some mRNAs and non-coding RNAs including rRNAs, tRNAs, and snRNAs, where modified bases are important for RNA function. Little is known about RNA base ...modifications in Arabidopsis thaliana.
In the current work, we carried out a bioinformatics analysis of RNA base modifications in tRNAs and miRNAs using large numbers of cDNA sequences of small RNAs (sRNAs) generated with the 454 technology and the massively parallel signature sequencing (MPSS) method. We looked for sRNAs that map to the genome sequence with one-base mismatch (OMM), which indicate candidate modified nucleotides. We obtained 1,187 sites with possible RNA base modifications supported by both 454 and MPSS sequences. Seven hundred and three of these sites were within tRNA loci. Nucleotide substitutions were frequently located in the T arm (substitutions from A to U or G), upstream of the D arm (from G to C, U, or A), and downstream of the D arm (from G to U). The positions of major substitution sites corresponded with the following known RNA base modifications in tRNAs: N1-methyladenosine (m1A), N2-methylguanosine (m2G), and N2-N2-methylguanosine (m22G).
These results indicate that our bioinformatics method successfully detected modified nucleotides in tRNAs. Using this method, we also found 147 substitution sites in miRNA loci. As with tRNAs, substitutions from A to U or G and from G to C, U, or A were common, suggesting that base modifications might be similar in tRNAs and miRNAs. We suggest that miRNAs contain modified bases and such modifications might be important for miRNA maturation and/or function.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
A novel quad-band bandpass filter with individually controllable centre frequencies and bandwidths using a folded stepped-impedance resonator (SIR) is proposed. The new filter that centres at 2.4, ...3.3, 4.1 and 5.2 GHz has a measured passband insertion loss of no more than 1.6 dB, and has a miniature circuit size of 0.448λg × 0.027λg. The presented filter is fabricated and tested, and the experiment demonstrates the new design. The new quad-band bandpass filter has compact and simple circuit topology, good performance of low passband insertion loss and transmission zeros, and greatly reduced circuit size compared with the relative reports.
Pentatricopeptide repeat (PPR) proteins are sequence-specific RNA-binding proteins that form a pervasive family of proteins conserved in yeast, plants, and humans. The plant PPR proteins are grouped ...mainly into the P and PLS classes. Here, we report the crystal structure of a PLS-class PPR protein from Arabidopsis thaliana called THA8L (THA8-like) at 2.0 Å. THA8L resembles THA8 (thylakoid assembly 8), a protein that is required for the splicing of specific group II introns of genes involved in biogenesis of chloroplast thylakoid membranes. The THA8L structure contains three P-type PPR motifs flanked by one L-type motif and one S-type motif. We identified several putative THA8L-binding sites, enriched with purine sequences, in the group II introns. Importantly, THA8L has strong binding preference for single-stranded RNA over single-stranded DNA or double-stranded RNA. Structural analysis revealed that THA8L contains two extensive patches of positively charged residues next to the residues that are proposed to comprise the RNA-binding codes. Mutations in these two positively charged patches greatly reduced THA8L RNA-binding activity. On the basis of these data, we constructed a model of THA8L-RNA binding that is dependent on two forces: one is the interaction between nucleotide bases and specific amino acids in the PPR motifs (codes), and the other is the interaction between the negatively charged RNA backbone and positively charged residues of PPR motifs. Together, these results further our understanding of the mechanism of PPR protein-RNA interactions.
Background: Pentatricopeptide repeat (PPR) proteins are sequence-specific RNA-binding proteins involved in organelle RNA processing.
Results: We identified RNA-binding sites of a small PPR protein (THA8L) from Arabidopsis thaliana and solved its crystal structure.
Conclusion: THA8L-RNA binding is dependent on a combination of specific nucleotide base interactions and nonspecific backbone interactions.
Significance: This work advances our understanding of the mechanism of PPR protein-RNA interaction.
In terms of the overall diffusion sound field, the inherent diffusion state of the space and the scattering coefficient of indoor surfaces work jointly to diffuse sound energy. This study has ...investigated the impact of surface scattering on reverberation time in differently shaped spaces. First, 10 spaces with the same volume but different shapes were calculated using a computer simulation; Next, two typical spaces were selected to calculate 10 states, in which the volume was multiplied. The calculations results show that the impact of surface scattering on reverberation time in differently shaped spaces follows three laws: in a group of spaces with the same variation pattern, T20 varies with scattering coefficients at a similar rate. In a group of spaces with different variation patterns, there is a difference of more than 5% in the change rate of T20 with scattering coefficients; in imperfect diffusion spaces, decay curves vary in accordance with scattering coefficients. If the scattering coefficients are the same, T20 varies with spatial shapes; when the volume of rectangular spaces and trapezoidal space ranges from 3000 to 30,000 m3, the change rate of T20 is less than 5%. In the present study, spaces were classified by the position combination of shape surfaces. On this basis, we proposed and then graded the concept of “morphology diffusivity”.
Mass spectrometry has played a significant role in the identification of unknown phosphoproteins and sites of phosphorylation in biological samples. Analyses of protein phosphorylation, particularly ...large scale phosphoproteomic experiments, have recently been enhanced by efficient enrichment, fast and accurate instrumentation, and better software, but challenges remain because of the low stoichiometry of phosphorylation and poor phosphopeptide ionization efficiency and fragmentation due to neutral loss. Phosphoproteomics has become an important dimension in systems biology studies, and it is essential to have efficient analytical tools to cover a broad range of signaling events. To evaluate current mass spectrometric performance, we present here a novel method to estimate the efficiency of phosphopeptide identification by tandem mass spectrometry. Phosphopeptides were directly isolated from whole plant cell extracts, dephosphorylated, and then incubated with one of three purified kinases—casein kinase II, mitogen-activated protein kinase 6, and SNF-related protein kinase 2.6—along with
16
O
4
- and
18
O
4
-ATP separately for in vitro kinase reactions. Phosphopeptides were enriched and analyzed by LC-MS. The phosphopeptide identification rate was estimated by comparing phosphopeptides identified by tandem mass spectrometry with phosphopeptide pairs generated by stable isotope labeled kinase reactions. Overall, we found that current high speed and high accuracy mass spectrometers can only identify 20%–40% of total phosphopeptides primarily due to relatively poor fragmentation, additional modifications, and low abundance, highlighting the urgent need for continuous efforts to improve phosphopeptide identification efficiency.
Graphical Abstract
ᅟ
Lysine-ε-acetylation (Kac) is a post-translational modification (PTM) that is critical for metabolic regulation and cell signaling in mammals. However, its prevalence and importance in plants remain ...to be determined. Employing high-resolution tandem mass spectrometry, we analyzed protein lysine acetylation in five representative Arabidopsis organs with 2 ~ 3 biological replicates per organ. A total of 2887 Kac proteins and 5929 Kac sites were identified. This comprehensive catalog allows us to analyze proteome-wide features of lysine acetylation. We found that Kac proteins tend to be more uniformly expressed in different organs, and the acetylation status exhibits little correlation with the gene expression level, indicating that acetylation is unlikely caused by stochastic processes. Kac preferentially targets evolutionarily conserved proteins and lysine residues, but only a small percentage of Kac proteins are orthologous between rat and Arabidopsis. A large portion of Kac proteins overlap with proteins modified by other PTMs including ubiquitination, SUMOylation and phosphorylation. Although acetylation, ubiquitination and SUMOylation all modify lysine residues, our analyses show that they rarely target the same sites. In addition, we found that “reader” proteins for acetylation and phosphorylation, i.e., bromodomain-containing proteins and GRF (General Regulatory Factor)/14-3-3 proteins, are intensively modified by the two PTMs, suggesting that they are main crosstalk nodes between acetylation and phosphorylation signaling. Analyses of GRF6/14-3-3λ reveal that the Kac level of GRF6 is decreased under alkaline stress, suggesting that acetylation represses plant alkaline response. Indeed, K56ac of GRF6 inhibits its binding to and subsequent activation of the plasma membrane H
+
-ATPase AHA2, leading to hypersensitivity to alkaline stress. These results provide valuable resources for protein acetylation studies in plants and reveal that protein acetylation suppresses phosphorylation output by acetylating GRF/14-3-3 proteins.
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