The development of clustered regularly interspaced palindromic repeats (CRISPR)-associated protein (Cas) variants with a broader recognition scope is critical for further improvement of CRISPR/Cas systems. The original Cas9 protein from Streptococcus canis (ScCas9) can recognize simple NNG-protospacer adjacent motif (PAM) targets, and therefore possesses a broader range relative to current CRISPR/Cas systems, but its editing efficiency is low in plants. Evolved ScCas9⁺ and ScCas9⁺⁺ variants have been shown to possess higher editing efficiencies in human cells, but their activities in plants are currently unknown. Here, we utilized codon-optimized ScCas9, ScCas9⁺ and ScCas9⁺⁺ and a nickase variant ScCas9n⁺⁺ to systematically investigate genome cleavage activity and cytidine base editing efficiency in rice (Oryza sativa L.). This analysis revealed that ScCas9⁺⁺ has higher editing efficiency than ScCas9 and ScCas9⁺ in rice. Furthermore, we fused the evolved cytidine deaminase PmCDA1 with ScCas9n⁺⁺ to generate a new evoBE4max-type cytidine base editor, termed PevoCDA1-ScCas9n⁺⁺. This base editor achieved stable and efficient multiplex-site base editing at NNG-PAM sites with wider editing windows (C₋₁–C₁₇) and without target sequence context preference. Multiplex-site base editing of the rice genes OsWx (three targets) and OsEui1 (two targets) achieved simultaneous editing and produced new rice germplasm. Taken together, these results demonstrate that ScCas9⁺⁺ represents a crucial new tool for improving plant editing.

Dendrocalamus latiflorus Munro is a woody clumping bamboo with rapid shoot growth. Both genetic transformation and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing techniques are available for D. latiflorus, enabling reverse genetic approaches. Thus, D. latiflorus has the potential to be a model bamboo species. However, the genome sequence of D. latiflorus has remained unreported due to its polyploidy and large genome size. Here, we sequenced the D. latiflorus genome and assembled it into three allele-aware subgenomes (AABBCC), representing the largest genome of a major bamboo species. We assembled 70 allelic chromosomes (2, 737 Mb) for hexaploid D. latiflorus using both single-molecule sequencing from the Pacific Biosciences (PacBio) Sequel platform and chromosome conformation capture sequencing (Hi-C). Repetitive sequences comprised 52.65% of the D. latiflorus genome. We annotated 135 231 protein-coding genes in the genome based on transcriptomes from eight different tissues. Transcriptome sequencing using RNA-Seq and PacBio single-molecule real-time long-read isoform sequencing revealed highly differential alternative splicing (AS) between non-abortive and abortive shoots, suggesting that AS regulates the abortion rate of bamboo shoots. This high-quality hexaploid genome and comprehensive strand-specific transcriptome datasets for this Poaceae family member will pave the way for bamboo research using D. latiflorus as a model species.

Upland cotton is an important global cash crop for its long seed fibers and high edible oil and protein content. Progress in cotton genomics promotes the advancement of cotton genetics, evolutionary studies, functional genetics, and breeding, and has ushered cotton research and breeding into a new era. Here, we summarize high-impact genomics studies for cotton from the last 10 years. The diploid Gossypium arboreum and allotetraploid Gossypium hirsutum are the main focus of most genetic and genomic studies. We next review recent progress in cotton molecular biology and genetics, which builds on cotton genome sequencing efforts, population studies, and functional genomics, to provide insights into the mechanisms shaping abiotic and biotic stress tolerance, plant architecture, seed oil content, and fiber development. We also suggest the application of novel technologies and strategies to facilitate genome-based crop breeding. Explosive growth in the amount of novel genomic data, identified genes, gene modules, and pathways is now enabling researchers to utilize multidisciplinary genomics-enabled breeding strategies to cultivate “super cotton”, synergistically improving multiple traits. These strategies must rise to meet urgent demands for a sustainable cotton industry.

Flowering time and stem growth habit determine inflorescence architecture in soybean, which in turn influences seed yield. Dt1, a homolog of Arabidopsis TERMINAL FLOWER 1 (TFL1), is a major controller of stem growth habit, but its underlying molecular mechanisms remain unclear. Here, we demonstrate that Dt1 affects node number and plant height, as well as flowering time, in soybean under long-day conditions. The bZIP transcription factor FDc1 physically interacts with Dt1, and the FDc1-Dt1 complex directly represses the expression of APETALA1 (AP1). We propose that FT5a inhibits Dt1 activity via a competitive interaction with FDc1 and directly upregulates AP1. Moreover, AP1 represses Dt1 expression by directly binding to the Dt1 promoter, suggesting that AP1 and Dt1 form a suppressive regulatory feedback loop to determine the fate of the shoot apical meristem. These findings provide novel insights into the roles of Dt1 and FT5a in controlling the stem growth habit and flowering time in soybean, which determine the adaptability and grain yield of this important crop.

Endogenous circadian clock integrates cyclic signals of environment and daily and seasonal behaviors of organisms to achieve spatiotemporal synchronization, which greatly improves genetic diversity and fitness of species. This review addresses recent studies on the plant circadian system in the field of chronobiology, covering topics on molecular mechanisms, internal and external Zeitgebers, and hierarchical regulation of physiological outputs. The architecture of the circadian clock involves the autoregulatory transcriptional feedback loops, post-translational modifications of core oscillators, and epigenetic modifications of DNA and histones. Here, light, temperature, humidity, and internal elemental nutrients are summarized to illustrate the sensitivity of the circadian clock to timing cues. In addition, the circadian clock runs cell-autonomously, driving independent circadian rhythms in various tissues. The core oscillators responds to each other with biochemical factors including calcium ions, mineral nutrients, photosynthetic products, and hormones. We describe clock components sequentially expressed during a 24-h day that regulate rhythmic growth, aging, immune response, and resistance to biotic and abiotic stresses. Notably, more data have suggested the circadian clock links chrono-culture to key agronomic traits in crops.

Ammonium (NH₄⁺) and nitrate (NO₃⁻) are major inorganic nitrogen (N) sources for plants. When serving as the sole or dominant N supply, NH₄⁺ often causes root inhibition and shoot chlorosis in plants, known as ammonium toxicity. NO₃⁻ usually causes no toxicity and can mitigate ammonium toxicity even at low concentrations, referred to as nitrate-dependent alleviation of ammonium toxicity. Our previous studies indicated a NO₃⁻ efflux channel SLAH3 is involved in this process. However, whether additional components contribute to NO₃⁻-mediated NH₄⁺ detoxification is unknown. Previously, mutations in NO₃⁻ transporter NRT1.1 were shown to cause enhanced resistance to high concentrations of NH₄⁺. Whereas, in this study, we found when the high-NH₄⁺ medium was supplemented with low concentrations of NO₃⁻, nrt1.1 mutant plants showed hyper-sensitive phenotype instead. Furthermore, mutation in NRT1.1 caused enhanced medium acidification under high-NH₄⁺/low-NO₃⁻ condition, suggesting NRT1.1 regulates ammonium toxicity by facilitating H⁺ uptake. Moreover, NRT1.1 was shown to interact with SLAH3 to form a transporter-channel complex. Interestingly, SLAH3 appeared to affect NO₃⁻ influx while NRT1.1 influenced NO₃⁻ efflux, suggesting NRT1.1 and SLAH3 regulate each other at protein and/or gene expression levels. Our study thus revealed NRT1.1 and SLAH3 form a functional unit to regulate nitrate-dependent alleviation of ammonium toxicity through regulating NO₃⁻ transport and balancing rhizosphere acidification.

Photoperiodic flowering is one of the most important factors affecting regional adaptation and yield in soybean (Glycine max). Plant adaptation to long-day conditions at higher latitudes requires early flowering and a reduction or loss of photoperiod sensitivity; adaptation to short-day conditions at lower latitudes involves delayed flowering, which prolongs vegetative growth for maximum yield potential. Due to the influence of numerous major loci and quantitative trait loci (QTLs), soybean has broad adaptability across latitudes. Forward genetic approaches have uncovered the molecular basis for several of these major maturity genes and QTLs. Moreover, the molecular characterization of orthologs of Arabidopsis thaliana flowering genes has enriched our understanding of the photoperiodic flowering pathway in soybean. Building on early insights into the importance of the photoreceptor phytochrome A, several circadian clock components have been integrated into the genetic network controlling flowering in soybean: E1, a repressor of FLOWERING LOCUS T orthologs, plays a central role in this network. Here, we provide an overview of recent progress in elucidating photoperiodic flowering in soybean, how it contributes to our fundamental understanding of flowering time control, and how this information could be used for molecular design and breeding of high-yielding soybean cultivars.

Current gene delivery methods for maize are limited to specific genotypes and depend on time-consuming and labor-intensive tissue culture techniques. Here, we report a new method to transfect maize that is culture-free and genotype independent. To enhance efficiency of DNA entry and maintain high pollen viability of 32%-55%, transfection was performed at cool temperature using pollen pretreated to open the germination aperture (40%–55%). Magnetic nanoparticles (MNPs) coated with DNA encoding either red fluorescent protein (RFP), β-glucuronidase gene (GUS), enhanced green fluorescent protein (EGFP) or bialaphos resistance (bar) was delivered into pollen grains, and female florets of maize inbred lines were pollinated. Red fluorescence was detected in 22% transfected pollen grains, and GUS stained 55% embryos at 18 d after pollination. Green fluorescence was detected in both silk filaments and immature kernels. The T1 generation of six inbred lines showed considerable EGFP or GUS transcripts (29%–74%) quantitated by polymerase chain reaction, and 5%–16% of the T1 seedlings showed immunologically active EGFP or GUS protein. Moreover, 1.41% of the bar transfected T1 plants were glufosinate resistant, and heritable bar gene was integrated into the maize genome effectively as verified by DNA hybridization. These results demonstrate that exogenous DNA could be delivered efficiently into elite maize inbred lines recalcitrant to tissue culture-mediated transformation and expressed normally through our genotype-independent pollen transfection system.

Crop breeding during the Green Revolution resulted in high yields largely due to the creation of plants with semi-dwarf architectures that could tolerate high-density planting. Although semi-dwarf varieties have been developed in rice, wheat and maize, none was reported in soybean (Glycine max), and few genes controlling plant architecture have been characterized in soybean. Here, we demonstrate that the auxin efflux transporter PINFORMED1 (GmPIN1), which determines polar auxin transport, regulates the leaf petiole angle in soybean. CRISPR-Cas9-induced Gmpin1abc and Gmpin1bc multiple mutants displayed a compact architecture with a smaller petiole angle than wild-type plants. GmPIN1 transcripts and auxin were distributed asymmetrically in the petiole base, with high levels of GmPIN1a/c transcript and auxin in the lower cells, which resulted in asymmetric cell expansion. By contrast, the (iso)flavonoid content was greater in the upper petiole cells than in the lower cells. Our results suggest that (iso)flavonoids inhibit GmPIN1a/c expression to regulate the petiole angle. Overall, our study demonstrates that a signal cascade that integrates (iso)flavonoid biosynthesis, GmPIN1a/c expression, auxin accumulation, and cell expansion in an asymmetric manner creates a desirable petiole curvature in soybean. This study provides a genetic resource for improving soybean plant architecture.

A new deaminase, TadA8e, was recently evolved in the laboratory. TadA8e catalyzes DNA deamination over 1,000 times faster than ABE7.10. We developed a high-efficiency adenine base editor, rABE8e (rice ABE8e), combining monomeric TadA8e, bis-bpNLS and codon optimization. rABE8e had substantially increased editing efficiencies at NG-protospacer adjacent motif (PAM) and NGG-PAM target sequences compared with ABEmax. For most targets, rABE8e exhibited nearly 100% editing efficiency and high homozygous substitution rates in the specific editing window, especially at Positions A5 and A6. The ability to rapidly generate plant materials with homozygous base substitutions will benefit gene function research and precision molecular breeding.

Root-associated microbes are critical for plant growth and nutrient acquisition. However, scant information exists on optimizing communities of beneficial root-associated microbes or the mechanisms underlying their interactions with host plants. In this report, we demonstrate that root-associated microbes are critical influencers of host plant growth and nutrient acquisition. Three synthetic communities (SynComs) were constructed based on functional screening of 1,893 microbial strains isolated from root-associated compartments of soybean plants. Functional assemblage of SynComs promoted significant plant growth and nutrient acquisition under both N/P nutrient deficiency and sufficiency conditions. Field trials further revealed that application of SynComs stably and significantly promoted plant growth, facilitated N and P acquisition, and subsequently increased soybean yield. Among the tested communities, SynCom1 exhibited the greatest promotion effect, with yield increases of up to 36.1% observed in two field sites. Further RNA-seq implied that SynCom application systemically regulates N and P signaling networks at the transcriptional level, which leads to increased representation of important growth pathways, especially those related to auxin responses. Overall, this study details a promising strategy for constructing SynComs based on functional screening, which are capable of enhancing nutrient acquisition and crop yield through the activities of beneficial root-associated microbes.

As two of the most important environmental factors, light and temperature regulate almost all aspects of plant growth and development. Under natural conditions, light is accompanied by warm temperatures and darkness by cooler temperatures, suggesting that light and temperature are tightly associated signals for plants. Indeed, accumulating evidence shows that plants have evolved a wide range of mechanisms to simultaneously perceive and respond to dynamic changes in light and temperature. Notably, the photoreceptor phytochrome B (phyB) was recently shown to function as a thermosensor, thus reinforcing the notion that light and temperature signaling pathways are tightly associated in plants. In this review, we summarize and discuss the current understanding of the molecular mechanisms integrating light and temperature signaling pathways in plants, with the emphasis on recent progress in temperature sensing, light control of plant freezing tolerance, and thermomorphogenesis. We also discuss the questions that are crucial for a further understanding of the interactions between light and temperature signaling pathways in plants.

The root microbiome refers to the community of microbes living in association with a plant's roots, and includes mutualists, pathogens, and commensals. Here we focus on recent advances in the study of root commensal community which is the major research object of microbiome-related researches. With the rapid development of new technologies, plant–commensal interactions can be explored with unprecedented breadth and depth. Both the soil environment and the host plant drive commensal community assembly. The bulk soil is the seed bank of potential commensals, and plants use root exudates and immune responses to build healthy microbial communities from the available microbes. The plant microbiome extends the functional system of plants by participating in a variety of processes, including nutrient absorption, growth promotion, and resistance to biotic and abiotic stresses. Plants and their microbiomes have evolved adaptation strategies over time. However, there is still a huge gap in our understanding of the regulatory mechanisms of plant–commensal interactions. In this review, we summarize recent research on the assembly of root microbial communities and the effects of these communities on plant growth and development, and look at the prospects for promoting sustainable agricultural development through the study of the root microbiome.

The plant hormone abscisic acid (ABA) is crucial for plant seed germination and abiotic stress tolerance. However, the association between ABA sensitivity and plant abiotic stress tolerance remains largely unknown. In this study, 436 rice accessions were assessed for their sensitivity to ABA during seed germination. The considerable diversity in ABA sensitivity among rice germplasm accessions was primarily reflected by the differentiation between the Xian (indica) and Geng (japonica) subspecies and between the upland-Geng and lowland-Geng ecotypes. The upland-Geng accessions were most sensitive to ABA. Genome-wide association analyses identified four major quantitative trait loci containing 21 candidate genes associated with ABA sensitivity of which a basic helix-loop-helix transcription factor gene, OsbHLH38, was the most important for ABA sensitivity. Comprehensive functional analyses using knockout and overexpression transgenic lines revealed that OsbHLH38 expression was responsive to multiple abiotic stresses. Overexpression of OsbHLH38 increased seedling salt tolerance, while knockout of OsbHLH38 increased sensitivity to salt stress. A salt-responsive transcription factor, OsDREB2A, interacted with OsbHLH38 and was directly regulated by OsbHLH38. Moreover, OsbHLH38 affected rice abiotic stress tolerance by mediating the expression of a large set of transporter genes of phytohormones, transcription factor genes, and many downstream genes with diverse functions, including photosynthesis, redox homeostasis, and abiotic stress responsiveness. These results demonstrated that OsbHLH38 is a key regulator in plant abiotic stress tolerance.

Genome editing by clustered regularly interspaced short palindromic sequences (CRISPR)/CRISPR‐associated protein 9 (Cas9) has revolutionized functional gene analysis and genetic improvement. While reporter‐assisted CRISPR/Cas systems can greatly facilitate the selection of genome‐edited plants produced via stable transformation, this approach has not been well established in seed crops. Here, we established the seed fluorescence reporter (SFR)‐assisted CRISPR/Cas9 systems in maize (Zea mays L.), using the red fluorescent DsRED protein expressed in the endosperm (En‐SFR/Cas9), embryos (Em‐SFR/Cas9), or both tissues (Em/En‐SFR/Cas9). All three SFRs showed distinct fluorescent patterns in the seed endosperm and embryo that allowed the selection of seeds carrying the transgene of having segregated the transgene out. We describe several case studies of the implementation of En‐SFR/Cas9, Em‐SFR/Cas9, and Em/En‐ SFR/Cas9 to identify plants not harboring the genome‐editing cassette but carrying the desired mutations at target genes in single genes or in small‐scale mutant libraries, and report on the successful generation of single‐target mutants and/or mutant libraries with En‐SFR/Cas9, Em‐SFR/Cas9, and Em/En‐SFR/Cas9. SFR‐assisted genome editing may have particular value for application scenarios with a low transformation frequency and may be extended to other important monocot seed crops.

Mitogen-activated protein kinase (MAPK) cascades are key signaling modules downstream of receptors/sensors that perceive either endogenously produced stimuli such as peptide ligands and damage-associated molecular patterns (DAMPs) or exogenously originated stimuli such as pathogen/microbe-associated molecular patterns (P/MAMPs), pathogen-derived effectors, and environmental factors. In this review, we provide a historic view of plant MAPK research and summarize recent advances in the establishment of MAPK cascades as essential components in plant immunity, response to environmental stresses, and normal growth and development. Each tier of the MAPK cascades is encoded by a small gene family, and multiple members can function redundantly in an MAPK cascade. Yet, they carry out a diverse array of biological functions in plants. How the signaling specificity is achieved has become an interesting topic of MAPK research. Future investigations into the molecular mechanism(s) underlying the regulation of MAPK activation including the activation kinetics and magnitude in response to a stimulus, the spatiotemporal expression patterns of all the components in the signaling pathway, and functional characterization of novel MAPK substrates are central to our understanding of MAPK functions and signaling specificity in plants.

Sorghum, the fifth largest cereal crop, has high value as a staple food and raw material for liquor and vinegar brewing. Due to its high biomass and quality, it is also used as the second most planted silage resource. No fragrant sorghums are currently on the market. Through CRISPR/Cas9-mediated knockout of SbBADH2, we obtained sorghum lines with extraordinary aromatic smell in both seeds and leaves. Animal feeding experiments showed that fragrant sorghum leaves were attractable. We believe this advantage will produce great value in the sorghum market for both grain and whole biomass forage.

Cotton (Gossypium spp.) is one of the most important fiber crops worldwide. In the last two decades, transgenesis and genome editing have played important roles in cotton improvement. However, genotype dependence is one of the key bottlenecks in generating transgenic and gene‐edited cotton plants through either particle bombardment or Agrobacterium‐mediated transformation. Here, we developed a shoot apical meristem (SAM) cell‐ mediated transformation system (SAMT) that allowed the transformation of recalcitrant cotton genotypes including widely grown upland cotton (Gossypium hirsutum), Sea island cotton (Gossypium barbadense), and Asiatic cotton (Gossypium arboreum). Through SAMT, we successfully introduced two foreign genes, GFP and RUBY, into SAM cells of some recalcitrant cotton genotypes. Within 2–3 months, transgenic adventitious shoots generated from the axillary meristem zone could be recovered and grown into whole cotton plants. The GFP fluorescent signal and betalain accumulation could be observed in various tissues in GFP‐ and RUBY‐positive plants, as well as in their progenies, indicating that the transgenes were stably integrated into the genome and transmitted to the next generation. Furthermore, using SAMT, we successfully generated edited cotton plants with inheritable targeted mutagenesis in the GhPGF and GhRCD1 genes through CRISPR/Cas9‐mediated genome editing. In summary, the established SAMT transformation system here in this study bypasses the embryogenesis process during tissue culture in a conventional transformation procedure and significantly accelerates the generation of transgenic and gene‐edited plants for genetic improvement of recalcitrant cotton varieties.

Root architecture is one of the most important agronomic traits that determines rice crop yield. The primary root (PR) absorbs mineral nutrients and provides mechanical support; however, the molecular mechanisms of PR elongation remain unclear in rice. Here, the two loss-of-function T-DNA insertion mutants of root length regulator 4 (OsRLR4), osrlr4-1 and osrlr4-2 with longer PR, and three OsRLR4 overexpression lines, OE-OsRLR4-1/-2/-3 with shorter PR compared to the wild type/Hwayoung (WT/HY), were identified. OsRLR4 is one of five members of the PRAF subfamily of the regulator chromosome condensation 1 (RCC1) family. Phylogenetic analysis of OsRLR4 from wild and cultivated rice indicated that it is under selective sweeps, suggesting its potential role in domestication. OsRLR4 controls PR development by regulating auxin accumulation in the PR tip and thus the root apical meristem activity. A series of biochemical and genetic analyses demonstrated that OsRLR4 functions directly upstream of the auxin transporter OsAUX1. Moreover, OsRLR4 interacts with the TRITHORAX-like protein OsTrx1 to promote H3K4me3 deposition at the OsAUX1 promoter, thus altering its transcription level. This work provides insight into the cooperation of auxin and epigenetic modifications in regulating root architecture and provides a genetic resource for plant architecture breeding.

Japonica/geng and indica/xian are two major rice (Oryza sativa) subspecies with multiple divergent traits, but how these traits are related and interact within each subspecies remains elusive. Brassinosteroids (BRs) are a class of steroid phytohormones that modulate many important agronomic traits in rice. Here, using different physiological assays, we revealed that japonica rice exhibits an overall lower BR sensitivity than indica. Extensive screening of BR signaling genes led to the identification of a set of genes distributed throughout the primary BR signaling pathway with divergent polymorphisms. Among these, we demonstrate that the C38/T variant in BR Signaling Kinase2 (OsBSK2), causing the amino acid change P13L, plays a central role in mediating differential BR signaling in japonica and indica rice. OsBSK2^L13 in indica plays a greater role in BR signaling than OsBSK2^P13 in japonica by affecting the auto-binding and protein accumulation of OsBSK2. Finally, we determined that OsBSK2 is involved in a number of divergent traits in japonica relative to indica rice, including grain shape, tiller number, cold adaptation, and nitrogen-use efficiency. Our study suggests that the natural variation in OsBSK2 plays a key role in the divergence of BR signaling, which underlies multiple divergent traits between japonica and indica.

Heterosis is a fundamental biological phenomenon characterized by the superior performance of hybrids over their parents. Although tremendous progress has been reported in seed crops, the molecular mechanisms underlying heterosis in clonally propagated crops are largely unknown. Potato (Solanum tuberosum L.) is the most important tuber crop and an ongoing revolution is transforming potato from a clonally propagated tetraploid crop into a seed-propagated diploid hybrid potato. In our previous study, we developed the first generation of highly homozygous inbred lines of potato and hybrids with strong heterosis. Here, we integrated transcriptome, metabolome, and DNA methylation data to explore the genetic and molecular basis of potato heterosis at three developmental stages. We found that the initial establishment of heterosis in diploid potato was mainly due to dominant complementation. Flower color, male fertility, and starch and sucrose metabolism showed obvious gene dominant complementation in hybrids, and hybrids devoted more energy to primary metabolism for rapid growth. In addition, we identified ~2 700 allele-specific expression genes at each stage, which likely function in potato heterosis and might be regulated by CHH allele-specific methylation level. Our multi-omics analysis provides insight into heterosis in potato and facilitates the exploitation of heterosis in potato breeding.