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.

Detection of Favorable Alleles for Quality- and Yield-related Traits in Wheat Using a Backcross Population

Development of wheat varieties with high yield and good quality has been a major objective in wheat breeding.A BC 1 F 2-3 population was used to detect QTLs for wheat quality related traits: SDS-sedimentation value (Ssd),grain protein content (GPC),grain hardness (GH) and 11 mixograph parameters,as well as five agronomic traits: spike length (SL),spikelet number per spike (SPN),grain number per spike (GN),thousand-grain weight (TGW),and plant height (PH).A total of 44 putative QTLs were detected in the present study,31 for quality parameters and 13 for important agronomic traits,including three important major QTLs.One major QTL for Ssd QSsd.saas-1B.1,linked to barc137,explained on average 21.1% of the phenotypic variation in three environments.The allele increasing Ssd at this locus also significantly increased GN.The second locus on chromosome 1B with the linked marker Barc 61 was a major locus for mixograph parameters.It explained 21.3%-32.5%,24.3%-30.6%,30.6%-37% and 20.1%-22.7% of phenotypic variation for mixing tolerance (MT),weakening slope (WS),midline peak time (MPTi) and midline time x =8 value (MTxW),respectively.The third major QTL,explaining above 40% of plant height variation,close to Rht-B 1 on the short arm of chromosome 4BS,co-located with QTL for quality and yield-related traits.

Dissecting conserved cis-regulatory modules of Glu-1 promoters which confer the highly active endosperm-specific expression via stable wheat transformation

Wheat high-molecular-weight glutenin subunits(HMW-GS) determine dough elasticity and play an essential role in processing quality. HMW-GS are encoded by Glu-1 genes and controlled primarily at transcriptional level, implemented through the interactions between cis-acting elements and trans-acting factors. However, transcriptional mechanism of Glu-1 genes remains elusive. Here we made a comprehensive analysis of cis-regulatory elements within 1-kb upstream of the Glu-1 start codon(-1000 to-1) and identified 30 conserved motifs. Based on motif distribution pattern, three conserved cis-regulatory modules(CCRMs), CCRM1(-300 to-101), CCRM2(-650 to-400), and CCRM3(-950 to-750), were defined, and their functions were characterized in wheat stable transgenic lines transformed with progressive 5′ deletion promoter::GUS fusion constructs. GUS staining, qP CR and enzyme activity assays indicated that CCRM2 and CCRM3 could enhance the expression level of Glu-1, whereas the 300-bp promoter(-300 to-1), spanning CCRM1 and core region(-100 to-1), was enough to ensure accurate Glu-1 initiation at 7 days after flowering(DAF) and shape its spatiotemporal expression pattern during seed development. Further transgenic assays demonstrated that CCRM1-2(-300 to-209) containing Complete HMW Enhancer(-246 to-209) was important for expression level but had no effect on expression specificity in the endosperm. In contrast, CCRM1-1(-208 to-101) was critical for both expression specificity and level of Glu-1. Our findings not only provide new insights to uncover Glu-1 transcription regulatory machinery but also lay foundations for modifying Glu-1 expression.

Mechanisms of autophagy function and regulation in plant growth,development,and response to abiotic stress

Autophagy is an evolutionarily conserved degradation pathway of lysosomes(in mammals)and vacuoles(in yeasts and plants)from lower yeasts to higher mammals.It wraps unwanted organelles and damaged proteins in a double-membrane structure to transport them to vacuoles for degradation and recycling.In plants,autophagy functions in adaptation to the environment and maintenance of growth and development.This review systematically describes the autophagy process,biological functions,and regulatory mechanisms occurring during plant growth and development and in response to abiotic stresses.It provides a basis for further theoretical research and guidance of agricultural production.

TaRLK-6A promotes Fusarium crown rot resistance in wheat

Fusariumcrown rot(FCR),mainly caused by the soil‐borne fungus Fusarium pseudograminearum,is a devastatingdisease of wheat(Triticum aestivum(Ta)).Fusariumcrown rotcauses substantial yield losses and generates mycotoxins inwheat grains that can cause serious health problems in hu-mans and livestock(Powell et al.,2017).Identifying genes thatprovide resistance toF.pseudograminearumand character-izing the molecular mechanisms underlying such resistance arekey to breeding wheat varieties that are resistant to this dev-astating fungus.

Orthologous genesPm12andPm21from twowild relatives of wheat show evolutionary conservation but divergent powdery mildew resistance

Wheat powdery mildew,caused by Blumeria graminis f.sp.tritici(Bgt),is a devastating disease that threatens wheat production worldwide.Pm12,which originated from Aegilops speltoides,a wild relative of wheat,confers strong resistance to powdery mildew and therefore has potential use in wheat breeding.Using susceptible mutants induced by gamma irradiation,we physically mapped and isolated Pm12 and showed it to be orthologous to Pm21 from Dasypyrum villosum,also a wild relative of wheat.The resistance function of Pm12 was validated via ethyl methanesulfonatemutagenesis,virus-induced gene silencing,and stable genetic transformation.Evolutionary analysis indicates that the Pm12/Pm21 loci in wheat species are relatively conserved but dynamic.Here,we demonstrated that the two orthologous genes,Pm12 and Pm21,possess differential resistance against the same set of Bgt isolates.Overexpression of the coiledcoil domains of both PM12 and PM21 induces cell death in Nicotiana benthamiana leaves.However,their full-length forms display different cell death-inducing activities caused by their distinct intramolecular interactions.Cloning of Pm12 will facilitate its application in wheat breeding programs.This study also gives new insight into two orthologous resistance genes,Pm12 and Pm21,which show different race specificities and intramolecular interaction patterns.

FED:a web tool for foreign element detection of genome-edited organism

Dear Editor,Genome editing,especially the newly developed CRISPR technology,is now widely implemented for diverse medical and agricultural applications(Puchta,2018).However,for genome editing,the DNA cassettes encoding the editing components are usually assembled and delivered into the cells of organisms(Cong et al.,2013).

Comparative proteomic analysis of cold responsive proteins in two wheat cultivars with different tolerance to spring radiation frost

Spring radiation frost(SRF)is a severe environmental stress which impairs wheat yield and productivity worldwide.To better understand the mechanism of wheat(Triticum aestivum)responding to SRF,a comparative proteomic analysis was performed to analyze the changes of the key proteins in two wheat cultivars Jimai22 and Luyuan301 with high and low tolerance to SRF respectively.A total of 43 differentially expressed proteins(DEPs)which mainly involved in carbohydrate metabolism,amino acid metabolism,resistance proteins and antioxidant enzymes,photosynthesis and cellular respiration proteins,cell-wall related proteins,protein translation/processing/degradation and signal transduction were isolated and identified by two-dimensional electrophoresis and MALDI-TOF-TOF MS.The results revealed that of the 21 DEPs in Jimai22 responding to the SRF,13 DEPs were upregulated and 8 DEPs were downregulated,and that of the 22 DEPs in Luyuan301,9 DEPs were upregulated and 13 DEPs were downregulated.These DEPs might be responsible for the stronger cold resistance of Jimai22 compared to Luyuan301.The expression pattern and function analysis of these DEPs were very significant to understanding the mechanism of the SRF responses in wheat.

HBI1-TCP20 interaction positively regulates the CEPs-mediated systemic nitrate acquisition

Nitrate is the main source of nitrogen for plants but often distributed heterogeneously in soil.Plants have evolved sophisticated strategies to achieve adequate nitrate by modulating the root system architecture. The nitrate acquisition system is triggered by the short mobile peptides C-TERMINALLY ENCODED PEPTIDES(CEPs)that are synthesized on the nitrate-starved roots,but induce the expression of nitrate transporters on the other nitrate-rich roots through an unclear signal transduction pathway. Here,we demonstrate that the transcription factors HBI1 and TCP20 play important roles in plant growth and development in response to fluctuating nitrate supply. HBI1 physically interacts with TCP20, and this interaction was enhanced by the nitrate starvation. HBI1 and TCP20 directly bind to the promoters of CEPs and cooperatively induce their expression. Mutation in HBIs and/or TCP20 resulted in impaired systemic nitrate acquisition response. Our solid genetic and molecular evidence strongly indicate that the HBI1-TCP20 module positively regulates the CEPs-mediated systemic nitrate acquisition.