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.
Dear Editor, In the past few years, the development of sequence- specific DNA nucleases has progressed rapidly and such nucleases have shown their power in generating efficient targeted mutagenesis ...and other genome editing applica- tions. For zinc finger nucleases （ZFNs） and transcription activator-like effector nucleases （TALENs）, an engi- neered array of sequence-specific DNA binding domains are fused with the DNA nuclease Fokl 1, 2. These nu- cleases have been successful in genome modifications by generating double strand breaks （DSBs）, which are then repaired through non-homologous end joining （NHEJ） or homologous recombination （HR） in different species, including mouse, tobacco and rice 3-5. Recently, an- other breakthrough technology for genome editing, the CRISPR/Cas system, was developed. CRISPR （clustered regulatory interspaced short 12alindromic repeats） loci are variable short spacers separated by short repeats, which are transcribed into non-coding RNAs. The non-coding RNAs form a functional complex with CRISPR-asso- ciated （Cas） proteins and guide the complex to cleave complementary invading DNA 6. After the initial development of a programmable CRISPR/Cas system, it has been rapidly applied to achieve efficient genome editing in human cell lines, zebrafish and mouse 7-10. However, there is still no successful application in plants reported.
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:
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.
Argonautes (AGOs) are conserved proteins that contain an RNA‐binding PAZ domain and an RNase H‐like PIWI domain. In Arabidopsis, except for AGO1, AGO4 and AGO7, the roles of seven other AGOs in gene ...silencing are not known. We found that a mutation in AGO6 partially suppresses transcriptional gene silencing in the DNA demethylase mutant ros1‐1. In ago6‐1ros1‐1 plants, RD29A promoter short interfering RNAs (siRNAs) are less abundant, and cytosine methylation at both transgenic and endogenous RD29A promoters is reduced, compared to that in ros1‐1. Interestingly, the ago4‐1 mutation has a stronger suppression of the transcriptional silencing phenotype of ros1‐1 mutant. Analysis of cytosine methylation at the endogenous MEA‐ISR, AtREP2 and SIMPLEHAT2 loci revealed that the CpNpG and asymmetric methylation levels are lower in either of the ago6‐1 and ago4‐1 single mutants than those in the wild type, and the levels are the lowest in the ago6‐1ago4‐1 double mutant. These results suggest that AGO6 is important for the accumulation of specific heterochromatin‐related siRNAs, and for DNA methylation and transcriptional gene silencing, this function is partly redundant with AGO4.
DNA methylation is a conserved epigenetic mark important for genome integrity, development, and environmental responses in plants and mammals. Active DNA demethylation in plants is initiated by a ...family of 5-mC DNA glycosylases/lyases (i.e., DNA demethylases). Recent reports suggested a role of active DNA demethylation in fruit ripening in tomato. In this study, we generated loss-of-function mutant alleles of a tomato gene, SlDML2, which is a close homolog of the Arabidopsis DNA demethylase gene ROS1. In the fruits of the tomato mutants, increased DNA methylation was found in thousands of genes. These genes included not only hundreds of ripening-induced genes but also many ripening-repressed genes. Our results show that SlDML2 is critical for tomato fruit ripening and suggest that active DNA demethylation is required for both the activation of ripening-induced genes and the inhibition of ripening-repressed genes.
The action of phytohormones in plants requires the spatiotemporal regulation of their accumulation and responses at various levels. Recent studies reveal an emerging relationship between the function ...of phytohormones and epigenetic modifications. In particular, evidence suggests that auxin biosynthesis, transport, and signal transduction is modulated by microRNAs and epigenetic factors such as histone modification, chromatin remodeling, and DNA methylation. Furthermore, some phytohormones have been shown to affect epigenetic modifications. These findings are shedding light on the mode of action of phytohormones and are opening up a new avenue of research on phytohormones as well as on the mech- anisms reaulatino eoioenetic modifications.
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.
Drought stress is an important environmental factor limiting plant productivity. In this study, we screened drought-resistant transgenic plants from 65 promoter-pyrabactin resistance 1-like (PYL) ...abscisic acid (ABA) receptor gene combinations and discovered that pRD29A::PYL9 transgenic lines showed dramatically increased drought resistance and drought-induced leaf senescence in both Arabidopsis and rice. Previous studies suggested that ABA promotes senescence by causing ethylene production. However, we found that ABA promotes leaf senescence in an ethylene-independent manner by activating sucrose nonfermenting 1-related protein kinase 2s (SnRK2s), which subsequently phosphorylate ABA-responsive element-binding factors (ABFs) and Related to ABA-Insensitive 3/VP1 (RAV1) transcription factors. The phosphorylated ABFs and RAV1 up-regulate the expression of senescence-associated genes, partly by up-regulating the expression of Oresara 1. The pyl9 and ABA-insensitive 1-1 single mutants, pyl8-1pyl9 doublemutant, and snrk2.2/3/6 triple mutant showed reduced ABA-induced leaf senescence relative to the WT, whereas pRD29A::PYL9 transgenic plants showed enhanced ABA-induced leaf senescence. We found that leaf senescence may benefit drought resistance by helping to generate an osmotic potential gradient, which is increased in pRD29A::PYL9 transgenic plants and causes water to preferentially flow to developing tissues. Our results uncover the molecular mechanism of ABA-induced leaf senescence and suggest an important role of PYL9 and leaf senescence in promoting resistance to extreme drought stress.
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.
PDF
