Summary
We developed simplified single transcriptional unit (SSTU) CRISPR systems for multiplex gene editing in rice using FnCpf1, LbCpf1 or Cas9, in which the nuclease and its crRNA array are ...co‐expressed from a single Pol II promoter, without any additional processing machinery. Our SSTU systems are easy to construct and effective in mediating multiplex genome editing.
We developed the simplified single transcriptional unit (SSTU) CRISPR systems for multiplex gene editing in rice using FnCpf1, LbCpf1 or Cas9, in which the nuclease and its crRNA array are co‐expressed from single Pol II promoter, without any additional processing machinery. SSTU systems are simple and effective in multiplex genome editing.
TAL (transcription activator-like) effectors, secreted by phytopathogenic bacteria, recognize host DNA sequences through a central domain of tandem repeats. Each repeat comprises 33 to 35 conserved ...amino acids and targets a specific base pair by using two hypervariable residues known as repeat variable diresidues (RVDs) at positions 12 and 13. Here, we report the crystal structures of an 11.5-repeat TAL effector in both DNA-free and DNA-bound states. Each TAL repeat comprises two helices connected by a short RVD-containing loop. The 11.5 repeats form a right-handed, superhelical structure that tracks along the sense strand of DNA duplex, with RVDs contacting the major groove. The 12th residue stabilizes the RVD loop, whereas the 13th residue makes a base-specific contact. Understanding DNA recognition by TAL effectors may facilitate rational design of DNA-binding proteins with biotechnological applications.
Dear Editor, Rice （Oryza sativa） is the staple food for more than half of the world＇s population. Technologies enabling precise and efficient DNA knock-in or replacement, hereinafter referred to as ...KI, have the potential to revolutionize the generation of crops by precision molecular breeding.
Recent studies have revealed that microRNAs (miRNAs) regulate plant adaptive responses to nutrient deprivation. However, the functional significance of miRNAs in adaptive responses to nitrogen (N) ...limitation remains to be explored. The Arabidopsis miR169 was strongly down-regulated, whereas its targets, NFYA (Nuclear Factor Y, subunit A) family members, were strongly induced by nitrogen N starvation. Analysis of the expression of miR169 precursors showed that MIR169a was substantially down-regulated in both roots and shoots by N starvation. Accumulation of the NFYA family members was suppressed in transgenic Arabidopsis with constitutive expression of MIR169a. Transgenic Arabidopsis plants overexpressing MIR169a accumulated less N and were more sensitive to N stress than the wild type. N sensitivity of 35S::MIR169a might be attributable to impaired uptake systems. These results provide evidence that miRNAs have functional roles in helping plants to cope with fluctuations in N availability in the soil.
Maintaining proper DNA methylation levels in the genome requires active demethylation of DNA. However, removing the methyl group from a modified cytosine is chemically difficult and therefore, the ...underlying mechanism of demethylation had remained unclear for many years. The discovery of the first eukaryotic DNA demethylase, Arabidopsis thaliana REPRESSOR OF SILENCING 1 (ROS1), led to elucidation of the 5‐methylcytosine base excision repair mechanism of active DNA demethylation. In the 20 years since ROS1 was discovered, our understanding of this active DNA demethylation pathway, as well as its regulation and biological functions in plants, has greatly expanded. These exciting developments have laid the groundwork for further dissecting the regulatory mechanisms of active DNA demethylation, with potential applications in epigenome editing to facilitate crop breeding and gene therapy.
Active DNA demethylation is critical for maintaining correct DNA methylation patterns in eukaryotic genomes. This review summarizes the main advances in plant active DNA demethylation, focusing on the base excision repair mechanism, regulatory pathways, and the function of active DNA demethylation during plant growth and development, and plant‐environment interactions
Creation of aromatic maize by CRISPR/Cas Wang, Yanxiao; Liu, Xiaoqin; Zheng, Xiuxiu ...
Journal of integrative plant biology,
September 2021, Letnik:
63, Številka:
9
Journal Article
Recenzirano
Odprti dostop
Aroma is an important quality parameter for breeding in rice (Oryza sativa). For example, the aromatic rice varieties basmati and jasmine rice, with a popcorn‐like scent, are popular worldwide and ...routinely command a price premium. 2‐acetyl‐1‐pyrroline (2AP) is a key flavor compound among over 200 volatiles identified in fragrant rice. A naturally fragrant germplasm exists in multiple plant species besides rice, which all exhibit lower activity of BETAINE ALDEHYDE DEHYDROGENASE 2 (BADH2). However, no equivalent aromatic germplasm has been described in maize (Zea mays). Here, we characterized the two maize BADH2 homologs, ZmBADH2a and ZmBADH2b. We generated zmbadh2a and zmbadh2b single mutants and the zmbadh2a‐zmbadh2b double mutant by CRISPR/Cas in four inbred lines. A popcorn‐like scent was only noticeable in seeds from the double mutant, but not from either single mutant or in wild type. In agreement, we only detected 2AP in fresh kernels and dried mature seeds from the double mutant, which accumulated between 0.028 and 0.723 mg/kg 2AP. These results suggest that ZmBADH2a and ZmBADH2b redundantly participate in 2AP biosynthesis in maize, and represent the creation of the world's first aromatic maize by simultaneous genome editing of the two BADH2 genes.
Dysfunction of BETAINE ALDEHYDE DEHYDROGENASE 2 (BADH2) results in the accumulation of 2‐acetyl‐1‐pyrroline, a key fragrant compound of aromatic rice. Mutation of the two maize paralogs of BADH2 by CRISPR/Cas resulted in the creation of aromatic maize.
The capability to maintain cell wall integrity is critical for plants to adapt to unfavourable conditions. L-Arabinose (Ara) is a constituent of several cell wall polysaccharides and many cell ...wall-localised glycoproteins, but so far the contribution of Ara metabolism to abiotic stress tolerance is still poorly understood.
Here, we report that mutations in the MUR4 (also known as HSR8) gene, which is required for the biosynthesis of UDP-Arap in Arabidopsis, led to reduced root elongation under high concentrations of NaCl, KCl, NaNO₃, or KNO₃.
The short root phenotype of the mur4/hsr8 mutants under high salinity is rescued by exogenous Ara or gum arabic, a commercial product of arabinogalactan proteins (AGPs) from Acacia senegal. Mutation of the MUR4 gene led to abnormal cell–cell adhesion under salt stress. MUR4 forms either a homodimer or heterodimers with its isoforms. Analysis of the higher order mutants of MUR4 with its three paralogues, MURL, DUR, MEE25, reveals that the paralogues of MUR4 also contribute to the biosynthesis of UDP-Ara and are critical for root elongation.
Taken together, our work revealed the importance of the Ara metabolism in salt stress tolerance and also provides new insights into the enzymes involved in the UDP-Ara biosynthesis in plants.
Abscisic acid (ABA) is an important phytohormone regulating various plant processes, including seed germination. Although phosphorylation has been suggested to be important, the protein kinases ...required for ABA signaling during seed germination and seedling growth remain elusive. Here, we show that two protein kinases, SNF1-RELATED PROTEIN KINASE2.2 (SnRK2.2) and SnRK2.3, control responses to ABA in seed germination, dormancy, and seedling growth in Arabidopsis thaliana. A snrk2.2 snrk2.3 double mutant, but not snrk2.2 or snrk2.3 single mutants, showed strong ABA-insensitive phenotypes in seed germination and root growth inhibition. Changes in seed dormancy and ABA-induced Pro accumulation consistent with ABA insensitivity were also observed. The snrk2.2 snrk2.3 double mutant had a greatly reduced level of a 42-kD kinase activity capable of phosphorylating peptides from ABF (for ABA Response Element Binding Factor) transcription factors. ABA-induced expression of several genes whose promoters contain an ABA response element (ABRE) was reduced in snrk2.2 snrk2.3, suggesting that the mechanism of SnRK2.2 and SnRK2.3 action in ABA signaling involves the activation of ABRE-driven gene expression through the phosphorylation of ABFs. Together, these results demonstrate that SnRK2.2 and SnRK2.3 are redundant but key protein kinases that mediate a major part of ABA signaling in ARABIDOPSIS:
Broomcorn millet (Panicum miliaceum L.) is the most water-efficient cereal and one of the earliest domesticated plants. Here we report its high-quality, chromosome-scale genome assembly using a ...combination of short-read sequencing, single-molecule real-time sequencing, Hi-C, and a high-density genetic map. Phylogenetic analyses reveal two sets of homologous chromosomes that may have merged ~5.6 million years ago, both of which exhibit strong synteny with other grass species. Broomcorn millet contains 55,930 protein-coding genes and 339 microRNA genes. We find Paniceae-specific expansion in several subfamilies of the BTB (broad complex/tramtrack/bric-a-brac) subunit of ubiquitin E3 ligases, suggesting enhanced regulation of protein dynamics may have contributed to the evolution of broomcorn millet. In addition, we identify the coexistence of all three C
subtypes of carbon fixation candidate genes. The genome sequence is a valuable resource for breeders and will provide the foundation for studying the exceptional stress tolerance as well as C
biology.
Optimized ABE7.10 refers to the GenScript codon‐optimized sequence of adenine deaminase of ABE7.10; this sequence and Anc689APOBEC were directly derived from Koblan et al. (). (c,g) Frequencies of ...base substitutions at the target sites of NRT1.1B (c) and SLR1 (g); the PAM motif is marked in box. (d,h,l) Distribution of the genotypes from transgenic rice plantlets edited at the NRT1.1B (d), SLR1 (h) and ALS (l) target sites. The PAM motif is marked in box, and the red triangles indicate the intended base for conversion. (n) Phenotype of the transgenic rice plantlets treated by herbicide. 0.03% Imazethapyr (Shandong CYNDA) was sprayed on the plantlets, and the photograph was taken 25 days after treatment. The quantity of each genotype from transgenic plantlets is indicated by ×. s1, single nucleotide substitution mutation; s2, two nucleotides substitution mutation, WT, wild type. (r) Frequencies of base substitutions at the target site of ALS‐sg3 edited by ABE and ABEmax. Recently, Nishimasu et al. ( ) reported that a rationally engineered SpCas9 variant, SpCas9‐NG, containing the R1335A/L1111R/D1135V/G1218R/E1219F/A1322R/T1337R seven amino acid alteration, can recognize relaxed NG PAMs in human cells.
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