Generation of a high-efficiency adenine base editor with TadA8e for developing wheat dinitroaniline-resistant germplasm

Base editing using CRISPR technologies is an invaluable tool for crop breeding. One of the major base editors, the adenine base editor(ABE), has been successfully used in both model plants and many crops.However, owing to limited editing efficiency, the ABE has been difficult to apply in polyploid crops such as allohexaploid bread wheat that often require simultaneous mutation of multiple alleles for fast breeding. We have designed a wheat high-efficiency ABE(Whie ABE), using the newly developed high-activity adenosine deaminase Tad A8 e. In vivo and in vitro analysis demonstrated the improved applicability of Tad A8 e over the commonly used Tad A7.10. Dinitroaniline is a widely used herbicide with high effectiveness and low toxicity to animals. However, wheat cultivars with tolerance to dinitroaniline are rare, limiting the application of dinitroaniline in wheat planting. Using A-to-G editing with Whie ABE, we found that a Met-to-Thr mutation in wheat tubulin alleles located on chromosomes 1 A, 1 B, 1 D, 4 A, and 4 D increased the resistance of wheat to dinitroaniline, revealing a dosage effect of edited tubulins in resistance. The Whie ABE promises to be a valuable editing tool for accelerating crop improvement and developing herbicide-resistant wheat germplasm.

Creation of aromatic maize by CRISPR/Cas

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.

植物核糖体应激响应机制研究进展

核仁是真核细胞中重要的核结构,核糖体发生最初在核仁中进行,该过程涉及一系列复杂的反应,需要许多核仁相关因子参与。核糖体生物发生出现异常通常引起核仁结构紊乱,并导致细胞周期阻滞、细胞衰老甚至凋亡。核糖体应激响应机制在哺乳动物细胞中研究得较为深入,但在植物细胞中尚不明晰。尽管如此,人们逐渐发现某些植物特有的NAC转录因子家族成员在植物细胞中可能参与包括核糖体应激在内的多种胞内应激响应过程。此外,前期研究发现生长素系统与核糖体生物合成之间存在一种相互协调机制来调控植物发育。该文结合哺乳动物细胞中已知的核糖体应激响应通路,探讨植物细胞潜在的核糖体应激机制。

Perspectives on the Application of Genome-Editing Technologies in Crop Breeding

Most conventional and modern crop-improvement methods exploit natural or artificially induced genetic variations and require laborious characterization of the progenies of multiple generations derived from time-consuming genetic crosses.Genome-editing systems,in contrast,provide the means to rapidly modify genomes in a precise and predictable way,making it possible to introduce improvements directly into elite varieties.Here,we describe the range of applications available to agricultural researchers using existing genome-editing tools.In addition to providing examples of genome-editing applications in crop breeding,we discuss the technical and social challenges faced by breeders using genome-editing tools for crop improvement.

Novel Wx alleles generated by base editing for improvement of rice grain quality

Amylose content(AC),which is regulated by the Waxy(Wx)gene,is a major indicator of eating and cooking quality(ECQ)in rice(Oryza sativa).Thus far,only a limited number of mutations in the N-terminal domain of Wx were found to have a major impact on the AC of rice grains and no mutations with such effects were reported for other regions of the Wx protein.Here,nucleotide substitutions in the middle region of Wx were generated by adenine and cytosine base editors.The nucleotide substitutions led to changes in 15 amino acid residues of Wx,and a series of novel Wx alleles with ACs of 0.3%-29.43%(wild type with AC of 19.87%)were obtained.Importantly,the waxy~(abe2)allele showed a"soft rice"AC,improved ECQ,favorable appearance,and no undesirable agronomic traits.The transgenes were removed from the waxy~(abe2)progeny,generating a promising breeding material for improving rice grain quality.

The Arabidopsis catalase triple mutant reveals important roles of catalases and peroxisome-derived signaling in plant development

Hydrogen peroxide （H2O2） is generated in many metabolic processes. As a signaling molecule, H2O2 plays important roles in plant growth and development, as well as environmental stress response. In Arabidopsis, there are three catalase genes, CAT1, CAT2, and CAT3. The encoded catalases are predominately peroxisomal pro- teins and are critical for scavenging H2O2. Since CAT1 and CAT3 are linked on chromosome 1, it has been almost impossible to generate cat1/3 and cats1/2/3 mutants by traditional genetic tools. In this study, we constructed cat1/3 double mutants and cat1/22/3 triple mutants by CRISPR/Cas9 to investigate the role of catalases. The cat1/ 2/3 triple mutants displayed severe redox disturbance and growth defects under physiological conditionscompared with wild-type and the cat2/3 double mutants. Transcriptome analysis showed a more profound tran- scriptional response in the cat1/2/3 triple mutants compared to the cat2/3 mutants. These differentially expressed genes are involved in plant growth regulation as well as abiotic and biotic stress responses. In addition, expression of OXI1 （OXIDATIVE SIGNAL INDUCIBLE 1） and several MAPK cascade genes were changed dramatically in the catalase triple mutant, suggesting that H2O2 produced in peroxisomes could serve as a peroxisomal retrograde signal.