Insulators are cis-regulatory elements that separate transcriptional units, whereas silencers are elements that repress transcription regardless of their position. In plants, these elements remain largely uncharacterized. Here, we use the massively parallel reporter assay Plant STARR-seq with short fragments of 8 large insulators to identify more than 100 fragments that block enhancer activity. The short fragments can be combined to generate more powerful insulators that abolish the capacity of the strong viral 35S enhancer to activate the 35S minimal promoter. Unexpectedly, when tested upstream of weak enhancers, these fragments act as silencers and repress transcription. Thus, these elements are capable of insulating or repressing transcription, depending on the regulatory context. We validate our findings in stable transgenic Arabidopsis thaliana, maize (Zea mays), and rice (Oryza sativa) plants. The short elements identified here should be useful building blocks for plant biotechnology.

Nitrogen (N) is an essential macronutrient for plant growth and yield, yet optimizing nitrogen use efficiency remains a challenge in agriculture. To better understand the regulatory basis of plant responses to N availability, we constructed a maize-specific nitrogen uptake efficiency gene regulatory network (mNUEGRN) comprising 1625 protein-DNA interactions (PDI) between 70 promoters and 301 transcription factors using enhanced yeast one-hybrid assays. We also projected a sorghum NUE GRN (spNUEGRN) based on maize orthologs and analyzed N-responsive subnetworks in both species using transcriptome profiling under N stress of early deprivation and recovery. Cross-species comparison with an existing Arabidopsis GRN revealed about 18% conserved interaction, corresponding to 11% of the mNUEGRN, particularly within the nitrate assimilation pathways. Notably, bZIP18 and bZIP30 emerged as central regulators in mNUEGRN, forming highly connected feed-forward loops (FFLs). From our time series data, we identified 19 236 and 23 864 differentially expressed genes in maize and sorghum, respectively. Gini correlation analysis uncovered 764 and 638 FFLs in mNUEGRN and spNUEGRN, respectively, of which 22 FFLs in maize and 35 in sorghum were identified in both leaf and root for each species. These FFLs may represent candidate regulatory motifs that contribute to modulating transcriptional responses under fluctuating N conditions, but their potential roles require further investigation. Together, our findings reveal evolutionarily conserved and species-specific regulatory strategies that mediate early N responsiveness, offering a foundation for engineering crops with improved NUE.

Wheat is one of the major staple crops around the world. A transient expression system is crucial for gene functional studies in wheat as stable transfection is still difficult in most cultivars. Protoplasts could serve as a versatile transient expression tool in wheat research. Here, we describe protocols for wheat protoplast isolation and transfection that are enabled by cellulase R-10 and macerozyme R-10 containing enzymatic solution and polyethylene glycol-mediated method, respectively. In addition, we show an example of efficiency evaluation of the emerging base editors in wheat protoplasts. These protocols are of wide use in both conventional gene functional analysis and reagent functionality evaluation of genome editing in wheat.

The majority of plant protein in the world's food supply is derived from soybean (Glycine max). Soybean is a key protein source for global animal feed and is incorporated into plant-based foods for people, including meat alternatives. Soybean protein content is genetically variable and is usually inversely related to seed oil content. ABI3-interacting protein 2 (AIP2) is an E3-RING ubiquitin ligase that targets the seed-specific transcription factor ABI3. Silencing both soybean AIP2 genes (AIP2a and AIP2b) by RNAi enhanced seed protein content by up to seven percentage points, with no significant decrease in seed oil content. The protein content enhancement did not alter the composition of the seed storage proteins. Inactivation of either AIP2a or AIP2b by a CRISPR-Cas9-mediated mutation increased seed protein content, and this effect was greater when both genes were inactivated. Transactivation assays in transfected soybean hypocotyl protoplasts indicated that ABI3 changes the expression of glycinin, conglycinin, 2S albumin, and oleosin genes, indicating that AIP2 depletion increased seed protein content by regulating activity of the ABI3 transcription factor protein. These results provide an example of a gene-editing prototype directed to improve global food security and protein availability in soybean that may also be applicable to other protein-source crops.

Summary The Zea Mays BIG GRAIN 1 HOMOLOG 1 (ZM‐BG1H1) was ectopically expressed in maize. Elite commercial hybrid germplasm was yield tested in diverse field environment locations representing commercial models. Yield was measured in 101 tests across all 4 events, 26 locations over 2 years, for an average yield gain of 355 kg/ha (5.65 bu/ac) above control, with 83% tests broadly showing yield gains (range +2272 kg/ha to −1240 kg/ha), with seven tests gaining more than one metric ton per hectare. Plant and ear height were slightly elevated, and ear and tassel flowering time were delayed one day, but ASI was unchanged, and these traits did not correlate to yield gain. ZM‐BG1H1 overexpression is associated with increased ear kernel row number and total ear kernel number and mass, but individual kernels trended slightly smaller and less dense. The ZM‐BG1H1 protein is detected in the plasma membrane like rice OS‐BG1. Five predominant native ZM‐BG1H1 alleles exhibit little structural and expression variation compared to the large increased expression conferred by these ectopic alleles.

Nitrogen is an essential macronutrient for plant growth and basic metabolic processes. The application of nitrogen-containing fertilizer increases yield, which has been a substantial factor in the green revolution1. Ecologically, however, excessive application of fertilizer has disastrous effects such as eutrophication2. A better understanding of how plants regulate nitrogen metabolism is critical to increase plant yield and reduce fertilizer overuse. Here we present a transcriptional regulatory network and twenty-one transcription factors that regulate the architecture of root and shoot systems in response to changes in nitrogen availability. Genetic perturbation of a subset of these transcription factors revealed coordinate transcriptional regulation of enzymes involved in nitrogen metabolism. Transcriptional regulators in the network are transcriptionally modified by feedback via genetic perturbation of nitrogen metabolism. The network, genes and gene-regulatory modules identified here will prove critical to increasing agricultural productivity.

DNA amplification fingerprinting (DAF) was used to evaluate the genetic relationships among 11 cultivars of poinsettia ( Euphorbia pulcherrima Willd.). Amplification was with 10 octamer oligonucleotide primers that generated 336 DNA bands. Thirty-one percent of the bands were polymorphic and distinguished among cultivars. Genetic relationships were evaluated by cluster analysis, and the resulting dendrogram closely agreed with published cultivar relationships. Arbitrary signatures from amplification profiles (ASAP) were further used to characterize two cultivars, `Nutcracker Red' and `Peterstar Red', that were previously found to be genetically and morphologically similar, as well as five cultivars in the “Freedom” series. The DAF products generated with arbitrary octamer primers were reamplified with mini-hairpin decamer primers in these experiments. The ASAP profiles were complex and yielded a total of 231 bands, 38% of which were polymorphic and capable of distinguishing each Freedom cultivar. Five of the eight primer combinations distinguished `Nutcracker Red' from `Peterstar Red'. Thus, closely related cultivars of poinsettia can be separated using new and improved molecular fingerprinting protocols.

The objective was to distinguish between cultivars and evaluate genetic relatedness of poinsettia (Euphorbia pulcherrima) using two methods of DNA fingerprinting—DNA Amplification Fingerprinting (DAF) and Arbitrary Signatures from Amplification Profiles (ASAP). Eleven red poinsettia cultivars were studied, including `Celebrate 2', `Darlyne', `Freedom Red', `Lilo', `Nutcracker Red', `Peterstar Red', `Petoy', `Red Sails', `Supjibi', `V-14 Glory', and `V-17 Angelika'. Amplification was with 10 octamer primers. Gels were visually scored for presence or absence of bands. The 10 primers generated 336 bands. The average number of bands (≈1000 bp) per primer was 34 ranging from 19 to 43. Thirty-one percent of bands were polymorphic and distinguished between each cultivar. The number of unique profiles varied from two to nine. Genetic relationships were evaluated by SAHN cluster analysis based on the distance estimator of Jaccard using the NTSYS-pc program (Numerical taxonomy and multivariate analysis system, version 1.8). The resulting dendrogram closely agreed with known pedigree data. ASAP analysis was used to further assess cultivar identification of two cultivars that were genetically and morphologically similar. Markers were found that separated `Nutcracker Red' and `Peterstar Red'. ASAP analysis separated cultivars within the Freedom series that DAF failed to distinguish. Two cultivars in the Freedom series, `Jingle Bells' and `Marble', were characterized from other cultivars in the series with ASAP.

Our objective was to distinguish between eight cultivars of two geranium species, Pelargonium × hortorum L.H. Bailey (cutting and seed geranium) and Pelargonium peltatum (L.) L'Hér. ex Ait. (ivy geranium), and evaluate their genetic relationships using the nucleic acid scanning techniques of DNA amplification fingerprinting (DAF) and/or arbitrary signatures from amplification profiles (ASAP). Cultivars used in the study represented three commercial types: cutting, seed, and ivy geranium. Two seed geranium cultivars from each of the Dynamo and Orbit series were included. Cutting geranium cultivars were `Designer Lilac Chiffon' and `Starburst Red' and the ivy geraniums were `Bernardo Guiber' and `Vinco Guivin'. The ASAP amplification protocol used one of two arbitrary octamer primers, followed by reamplification with one of four different minihairpin primers. ASAP profiles were complex, with 66% of bands being polymorphic and useful in distinguishing between cultivars. Genetic relationships were evaluated by principal coordinate analysis and cluster analysis based on the Jaccard distance estimator. This analysis grouped cultivars by species according to commercial type, i.e., seed geraniums were in one large group, the cutting geraniums were grouped together, and the ivy geraniums were a separate branch.

The objective was to study the flowering response of garden cultivars of Dendranthemum × grandiflorum (Ramat.) Kitamura to temperature and photoperiod. Fifteen garden mum cultivars were grown in ten temperature (18 and 24°C constant day and night greenhouse temperatures) and photoperiod (8, 10, 12, 14, and 16 h) combinations. Rooted cuttings were pinched above the fifth node and placed in the temperature/photoperiod treatments. When axillary shoots developed, all but one shoot was removed to produce a single stemmed plant. Photoperiods were provided by delivering 8 h sunlight, then pulling black cloth and providing daylength extension with incandescent bulbs. Days to visible bud, days to first bud color, days to flower, node number, and stem length were measured. By 11 weeks after the start of photoperiod treatments, no difference was measured in days to flower in the 8-, 10-, and 12-h photoperiods at 18°C. Days to flower increased as photoperiod increased from 12 to 14 h. At 18°C, five cultivars flowered in the 16-h photoperiod, while 10 cultivars developed crown buds, i.e., flower buds that initiated but had not developed. At 24°C, there was no difference in days to flower in the 8and 10-h photoperiod, while days to flower increased as photoperiod increased from 10to 12-h treatment. Cultivars formed crown buds but had not reached flowering in the 14and 16-h photoperiods at 24°C. Regardless of temperature, stem length increased as photoperiod increased above 10 h.

The objective was to distinguish between series of cultivars of Pelargonium x hortorum (zonal geranium), Pelargonium hybrids (seed geranium), and Pelargonium peltatum (ivy leaf geranium) using DNA amplification fingerprinting (DAF) demonstrating the utility of DAF for patent protection to prevent infringement of inventor's rights. Leaf tissue of 10 plants of each cultivar of seedling geranium was bulked for DNA extraction, and cutting and ivy geranium cultivars were bulks of five plants of each cultivar. Isolated DNA from different cultivars of a series were bulked together in their respective series. Seedling geranium series included Dynamo, Glamour, Multibloom, Orbit, Pinto, and Ringo 2000. Cutting geranium series included Designer and Showcase. Ivy geraniums were from the Guillou group. Amplification was with one of two octamer primers, followed by reamplifying with one of four different mini hairpin primers. Gels were visually scored for presence or absence of bands. The four primers generated 336 bands. The average number of bands (_1000 bp) per primer was 40. Twenty percent of bands were polymorphic and distinguished between each series of cultivars. Genetic relationships were evaluated by SAHN cluster analysis based on the distance estimator of Dice using the NTSYS-pc program (Numerical taxonomy and multivariate analysis system, version 1.8). Series were grouped according to species. Seedling geraniums were in one large group, the two cutting geraniums were grouped together and the ivy leaf geraniums were a separate branch.