Breeding exceptionally fragrant soybeans for soy milk with strong aroma.

Knockout of the soybean (Glycine max) betaine aldehyde dehydrogenase genes GmBADH1 and GmBADH2 using CRISPR/Cas12i3 enhances the aroma of soybeans. Soy milk made from the gmbadh1/2 double mutant seeds exhibits a much stronger aroma, which consumers prefer; this mutant has potential for enhancing quality in soy-based products.

Genome editing in rice using CRISPR/Cas12i3.

The CRISPR/Cas type V-I is a family of programmable nuclease systems that prefers a T-rich protospacer adjacent motif (PAM) and is guided by a short crRNA. In this study, the genome-editing application of Cas12i3, a type V-I family endonuclease, was characterized in rice. We developed a CRIPSR/Cas12i3-based Multiplex direct repeats (DR)-spacer Array Genome Editing (iMAGE) system that was efficient in editing various genes in rice. Interestingly, iMAGE produced chromosomal structural variations with a higher frequency than CRISPR/Cas9. In addition, we developed base editors using deactivated Cas12i3 and generated herbicide-resistant rice plants using the base editors. These CRIPSR/Cas12i3-based genome editing systems will facilitate precision molecular breeding in plants.

Cas12a-based on-site, rapid detection of genetically modified crops.

A CRISPR/LbCas12a-based nucleic acid detection method that uses crude leaf extracts as samples and is rapid (≤40 min for a full run) and highly sensitive (0.01%) can be used to monitor genetically modified organisms in the field.

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.

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 waxyabe2 allele showed a "soft rice" AC, improved ECQ, favorable appearance, and no undesirable agronomic traits. The transgenes were removed from the waxyabe2 progeny, generating a promising breeding material for improving rice grain quality.

Generating Novel Male Sterile Tomatoes by Editing Respiratory Burst Oxidase Homolog Genes.

Hybrid breeding of tomatoes (Solanum lycopersicum), an important vegetable crop, is an effective way to improve yield and enhance disease and stress resistance. However, the efficiency of tomato hybridization is hindered by self-fertilization, which can be overcome using male sterile lines. It has been reported that reactive oxygen species (ROS) act as a key regulator for anther development, mediated by RBOH (Respiratory Burst Oxidase Homolog) genes. Here, two tomato anther-expressed genes, LeRBOH (Solyc01g099620) and LeRBOHE (Solyc07g042460), were selected to cultivate novel tomato male sterile strains. By using a CRISPR/Cas9 system with a two-sgRNA module, the lerboh, lerbohe, and lerboh lerbohe mutant lines were generated, among which the lerbohe and lerboh lerbohe mutants displayed complete male sterility but could accept wild-type pollens and produce fruits normally. Further analysis uncovered significantly decreased ROS levels and abnormal programmed cell death in lerboh lerbohe anthers, indicating a key role of ROS metabolism in tomato pollen development. Taken together, our work demonstrates a successful application of gene editing via CRISPR/Cas9 in generating male sterile tomatoes and afforded helpful information for understanding how RBOH genes regulating tomato reproduction process.

Fertility restoration of maize CMS-C altered by a single amino acid substitution within the Rf4 bHLH transcription factor.

Type C cytoplasmic male sterility (CMS-C) is the most commonly used form of CMS in maize hybrid seed production. Restorer of fertility 4 (Rf4), the major fertility restorer gene of CMS-C, is located on chromosome 8S. To positionally clone Rf4, a large F3 population derived from a cross between a non-restorer and restorer (n = 5104) was screened for recombinants and then phenotyped for tassel fertility, resulting in a final map-based cloning interval of 12 kb. Within this 12-kb interval, the only likely candidate for Rf4 was GRMZM2G021276, a basic helix-loop-helix (bHLH) transcription factor with tassel-specific expression. The Rf4 gene product contains a nuclear localization signal and is likely to not interact directly with the mitochondria. Sequence analysis of Rf4 revealed four encoded amino acid substitutions between restoring and non-restoring inbreds, however only one substitution, F187Y, was within the highly conserved bHLH domain. The hypothesis that Rf4 restoration is altered by a single amino acid was tested by using clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated protein 9 (Cas9) homology directed repair (HDR) to create isogenic lines that varied for the F187Y substitution. In a population of these CRISPR-Cas9 edited plants (n = 780) that was phenotyped for tassel fertility, plants containing F187 were completely fertile, indicating fertility restoration, and plants containing Y187 were sterile, indicating lack of fertility restoration. Structural modeling shows that this amino acid residue 187 is located within the four helix bundle core, a critical region for stabilizing dimer conformation and affecting interaction partner selection.

Shared and genetically distinct Zea mays transcriptome responses to ongoing and past low temperature exposure.

BACKGROUND: Cold temperatures and their alleviation affect many plant traits including the abundance of protein coding gene transcripts. Transcript level changes that occur in response to cold temperatures and their alleviation are shared or vary across genotypes. In this study we identify individual transcripts and groups of functionally related transcripts that consistently respond to cold and its alleviation. Genes that respond differently to temperature changes across genotypes may have limited functional importance. We investigate if these genes share functions, and if their genotype-specific gene expression levels change in magnitude or rank across temperatures. RESULTS: We estimate transcript abundances from over 22,000 genes in two unrelated Zea mays inbred lines during and after cold temperature exposure. Genotype and temperature contribute to many genes' abundances. Past cold exposure affects many fewer genes. Genes up-regulated in cold encode many cytokinin glucoside biosynthesis enzymes, transcription factors, signalling molecules, and proteins involved in diverse environmental responses. After cold exposure, protease inhibitors and cuticular wax genes are newly up-regulated, and environmentally responsive genes continue to be up-regulated. Genes down-regulated in response to cold include many photosynthesis, translation, and DNA replication associated genes. After cold exposure, DNA replication and translation genes are still preferentially downregulated. Lignin and suberin biosynthesis are newly down-regulated. DNA replication, reactive oxygen species response, and anthocyanin biosynthesis genes have strong, genotype-specific temperature responses. The ranks of genotypes' transcript abundances often change across temperatures. CONCLUSIONS: We report a large, core transcriptome response to cold and the alleviation of cold. In cold, many of the core suite of genes are up or downregulated to control plant growth and photosynthesis and limit cellular damage. In recovery, core responses are in part to prepare for future stress. Functionally related genes are consistently and greatly up-regulated in a single genotype in response to cold or its alleviation, suggesting positive selection has driven genotype-specific temperature responses in maize.

DIACYLGLYCEROL ACYLTRANSFERASE1 Contributes to Freezing Tolerance.

Freezing limits plant growth and crop productivity, and plant species in temperate zones have the capacity to develop freezing tolerance through complex modulation of gene expression affecting various aspects of metabolism and physiology. While many components of freezing tolerance have been identified in model species under controlled laboratory conditions, little is known about the mechanisms that impart freezing tolerance in natural populations of wild species. Here, we performed a quantitative trait locus (QTL) study of acclimated freezing tolerance in seedlings of Boechera stricta, a highly adapted relative of Arabidopsis (Arabidopsis thaliana) native to the Rocky Mountains. A single QTL was identified that contained the gene encoding ACYL-COENZYME A:DIACYLGLYCEROL ACYLTRANSFERASE1 (BstDGAT1), whose expression is highly cold responsive. The primary metabolic enzyme DGAT1 catalyzes the final step in assembly of triacylglycerol (TAG) by acyl transfer from acyl-CoA to diacylglycerol. Freezing tolerant plants showed higher DGAT1 expression during cold acclimation than more sensitive plants, and this resulted in increased accumulation of TAG in response to subsequent freezing. Levels of oligogalactolipids that are produced by SFR2 (SENSITIVE TO FREEZING2), an indispensable element of freezing tolerance in Arabidopsis, were also higher in freezing-tolerant plants. Furthermore, overexpression of AtDGAT1 led to increased freezing tolerance. We propose that DGAT1 confers freezing tolerance in plants by supporting SFR2-mediated remodeling of chloroplast membranes.

Identification of quantitative trait loci and a candidate locus for freezing tolerance in controlled and outdoor environments in the overwintering crucifer Boechera stricta.

Development of chilling and freezing tolerance is complex and can be affected by photoperiod, temperature and photosynthetic performance; however, there has been limited research on the interaction of these three factors. We evaluated 108 recombinant inbred lines of Boechera stricta, derived from a cross between lines originating from Montana and Colorado, under controlled long day (LD), short-day (SD) and in an outdoor environment (OE). We measured maximum quantum yield of photosystem II, lethal temperature for 50% survival and electrolyte leakage of leaves. Our results revealed significant variation for chilling and freezing tolerance and photosynthetic performance in different environments. Using both single- and multi-trait analyses, three main-effect quantitative trait loci (QTL) were identified. QTL on linkage group (LG)3 were SD specific, whereas QTL on LG4 were found under both LD and SD. Under all conditions, QTL on LG7 were identified, but were particularly predictive for the outdoor experiment. The co-localization of photosynthetic performance and freezing tolerance effects supports these traits being co-regulated. Finally, the major QTL on LG7 is syntenic to the Arabidopsis C-repeat binding factor locus, known regulators of chilling and freezing responses in Arabidopsis thaliana and other species.

Rootless with undetectable meristem 1 encodes a monocot-specific AUX/IAA protein that controls embryonic seminal and post-embryonic lateral root initiation in maize.

The maize (Zea mays L.) rum1-R (rootless with undetectable meristems 1-Reference) mutant does not initiate embryonic seminal roots and post-embryonic lateral roots at the primary root. Map-based cloning revealed that Rum1 encodes a 269 amino acid (aa) monocot-specific Aux/IAA protein. The rum1-R protein lacks 26 amino acids including the GWPPV degron sequence in domain II and part of the bipartite NLS (nuclear localization sequence). Significantly reduced lateral root density (approximately 35%) in heterozygous plants suggests that the rum1-R is a semi-dominant mutant. Overexpression of rum1-R under the control of the maize MSY (Methionine SYnthase) promoter supports this notion by displaying a reduced number of lateral roots (31-37%). Functional characterization suggests that Rum1 is auxin-inducible and encodes a protein that localizes to the nucleus. Moreover, RUM1 is unstable with a half life time of approximately 22 min while the mutant rum1-R protein is very stable. In vitro and in vivo experiments demonstrated an interaction of RUM1 with ZmARF25 and ZmARF34 (Z. mays AUXIN RESPONSE FACTOR 25 and 34). In summary, the presented data suggest that Rum1 encodes a canonical Aux/IAA protein that is required for the initiation of embryonic seminal and post-embryonic lateral root initiation in primary roots of maize.

Conserved noncoding genomic sequences associated with a flowering-time quantitative trait locus in maize.

Flowering time is a fundamental trait of maize adaptation to different agricultural environments. Although a large body of information is available on the map position of quantitative trait loci for flowering time, little is known about the molecular basis of quantitative trait loci. Through positional cloning and association mapping, we resolved the major flowering-time quantitative trait locus, Vegetative to generative transition 1 (Vgt1), to an approximately 2-kb noncoding region positioned 70 kb upstream of an Ap2-like transcription factor that we have shown to be involved in flowering-time control. Vgt1 functions as a cis-acting regulatory element as indicated by the correlation of the Vgt1 alleles with the transcript expression levels of the downstream gene. Additionally, within Vgt1, we identified evolutionarily conserved noncoding sequences across the maize-sorghum-rice lineages. Our results support the notion that changes in distant cis-acting regulatory regions are a key component of plant genetic adaptation throughout breeding and evolution.

The maize (Zea mays L.) RTCS gene encodes a LOB domain protein that is a key regulator of embryonic seminal and post-embryonic shoot-borne root initiation.

Maize has a complex root system composed of different root types formed during different stages of development. The rtcs (rootless concerning crown and seminal roots) mutant is impaired in the initiation of the embryonic seminal roots and the post-embryonic shoot-borne root system. The primary root of the mutant shows a reduced gravitropic response, while its elongation, lateral root density and reaction to exogenously applied auxin is not affected. We report here the map-based cloning of the RTCS gene which encodes a 25.5 kDa LOB domain protein located on chromosome 1S. The RTCS gene has been duplicated during evolution. The RTCS-LIKE (RTCL) gene displays 72% sequence identity on the protein level. Both genes are preferentially expressed in roots. Expression of RTCS in coleoptilar nodes is confined to emerging shoot-borne root primordia. Sequence analyses of the RTCS and RTCL upstream genomic regions identified auxin response elements. Reverse transcriptase-PCR revealed that both genes are auxin induced. Microsynteny analyses between maize and rice genomes revealed co-linearity of 14 genes in the RTCS region. We conclude from our data that RTCS and RTCL are auxin-responsive genes involved in the early events that lead to the initiation and maintenance of seminal and shoot-borne root primordia formation.

Cytokinin oxidase gene expression in maize is localized to the vasculature, and is induced by cytokinins, abscisic acid, and abiotic stress.

Cytokinins are hormones that play an essential role in plant growth and development. The irreversible degradation of cytokinins, catalyzed by cytokinin oxidase, is an important mechanism by which plants modulate their cytokinin levels. Cytokinin oxidase has been well characterized biochemically, but its regulation at the molecular level is not well understood. We isolated a cytokinin oxidase open reading frame from maize (Zea mays), called Ckx1, and we used it as a probe in northern and in situ hybridization experiments. We found that the gene is expressed in a developmental manner in the kernel, which correlates with cytokinin levels and cytokinin oxidase activity. In situ hybridization with Ckx1 and transgenic expression of a transcriptional fusion of the Ckx1 promoter to the Escherichia coli beta-glucuronidase reporter gene revealed that the gene is expressed in the vascular bundles of kernels, seedling roots, and coleoptiles. We show that Ckx1 gene expression is inducible in various organs by synthetic and natural cytokinins. Ckx1 is also induced by abscisic acid, which may control cytokinin oxidase expression in the kernel under abiotic stress. We hypothesize that under non-stress conditions, cytokinin oxidase in maize plays a role in controlling growth and development via regulation of cytokinin levels transiting in the xylem. In addition, we suggest that under environmental stress conditions, cytokinin oxidase gene induction by abscisic acid results in aberrant degradation of cytokinins therefore impairing normal development.

The defective kernel 1 (dek1) gene required for aleurone cell development in the endosperm of maize grains encodes a membrane protein of the calpain gene superfamily.

Endosperm of cereal grains is one of the most important renewable resources for food, feed, and industrial raw material. It consists of four triploid cell types, i.e., aleurone, starchy endosperm, transfer cells, and cells of the embryo surrounding region. In maize, the aleurone layer is one cell layer thick and covers most of the perimeter of the endosperm. Specification of maize aleurone cell fate is proposed to occur through activation of the tumor necrosis factor receptor-like receptor kinase CRINKLY4. A second maize gene essential for aleurone cell development is defective kernel 1 (dek1). Here we show that DEK1 shares high homology with animal calpains. The predicted 2,159-aa DEK1 protein has 21 transmembrane regions, an extracellular loop, and a cysteine proteinase domain that shares high homology with domain II of m-calpain from animals. We propose that DEK1 functions to maintain and restrict the aleurone cell fate imposed by CR4 through activation of its cysteine proteinase by contact with the outer endosperm surface. DEK1 seems to be the only member of the calpain superfamily in plants, Arabidopsis DEK1 sharing 70% overall identity with maize DEK1. The expression of dek1 in most plant tissues in maize and Arabidopsis, as well as its presence in a variety of higher plants, including angiosperms and gymnosperms, suggests that DEK1 plays a conserved role in plant signal transduction.