The rice LATE ELONGATED HYPOCOTYL enhances salt tolerance by regulating Na+/K+ homeostasis and ABA signalling.

The circadian clock plays multiple functions in the regulation of plant growth, development and response to various abiotic stress. Here, we showed that the core oscillator component late elongated hypocotyl (LHY) was involved in rice response to salt stress. The mutations of OsLHY gene led to reduced salt tolerance in rice. Transcriptomic analyses revealed that the OsLHY gene regulates the expression of genes related to ion homeostasis and the abscisic acid (ABA) signalling pathway, including genes encoded High-affinity K+ transporters (OsHKTs) and the stress-activated protein kinases (OsSAPKs). We demonstrated that OsLHY directly binds the promoters of OsHKT1;1, OsHKT1;4 and OsSAPK9 to regulate their expression. Moreover, the ossapk9 mutants exhibited salt tolerance under salt stress. Taken together, our findings revealed that OsLHY integrates ion homeostasis and the ABA pathway to regulate salt tolerance in rice, providing insights into our understanding of how the circadian clock controls rice response to salt stress.

Sustainable Aerobic Bromination with Controllable Chemoselectivity.

The formation of C-Br(s) is one of the most fundamental reactions in organic synthesis. Oxidative bromination is a "green" way to achieve it. Aerobic bromination has drawn great interest in the past decades, while the poor substrate scope and selectivity, low efficiency, and the use of metal catalyst still confine its application. In this article, we establish a transition-metal-free aerobic bromination promoted by ionic liquid in a catalytic amount with controllable chemoselectivity toward numbers of C-Br(s) formed, and both NaBr/AcOH and HBr(aq) could be used as the bromine source. This methodology shows high efficiency and has a broad substrate scope for various kinds of C-H(s). We also validate this system by the gram-scale (one-pot) synthesis of functional molecules and direct recycle of the catalyst. The possible radical pathway of this catalysis is also presented with evidence.

Molecular Research for Cereal Grain Quality.

Cereals such as wheat (Triticum aestivum L [...].

Effect of Crop Rotation on Fusarium Mycotoxins and Fusarium Species in Cereals in Sichuan Province (China).

The present study was performed to evaluate the effect of crop rotation on Fusarium mycotoxins and species in cereals in Sichuan Province. A total of 311 cereal samples were randomly collected and analyzed from 2018 to 2019 in Sichuan Province. The results of mycotoxin analysis showed that the major trichothecene mycotoxins in Sichuan Province were nivalenol (NIV) and deoxynivalenol (DON), and the mean concentration of total trichothecenes (including NIV, fusarenone X [4ANIV], DON, 3-acetyldeoxynivalenol [3ADON], and 15-acetyldeoxynivalenol [15ADON]) in wheat was significantly higher than that in maize and rice. The concentration of total trichothecenes in the succeeding crops was significantly higher than that in the previous crops. In addition, wheat grown after maize had reduced incidence and concentration of trichothecene mycotoxins compared with that grown after rice, and ratooning rice grown after rice had increased incidence and concentration of trichothecene mycotoxins. Our data indicated that Fusarium asiaticum with the NIV chemotype was predominant in wheat and rice samples, while the number of the NIV chemotypes of F. asiaticum and Fusarium meridionale and the 15ADON chemotype of Fusarium graminearum in maize were almost the same. Although the composition of Fusarium species was affected by crop rotations, there were no differences when comparing the same crop rotation except for the maize-wheat rotation. Moreover, the same species and chemotype of Fusarium strains originated from different crops in various rotations, but there were no significant differences in pathogenicity in wheat and rice. These results contribute to the knowledge of the effect of crop rotation on Fusarium mycotoxins and species affecting cereals in Sichuan Province, which may lead to improved strategies for control of Fusarium mycotoxins and fungal disease in China.

Loss-of-function mutations of OsbHLH044 transcription factor lead to salinity sensitivity and a greater chalkiness in rice (Oryza sativa L.).

The most hazardous abiotic stress, salinity, restricted the world crop production, and grain chalkiness affected the grain quality to limit consumers' acceptance. The basic helix-loop-helix (bHLH) proteins modulate massive biological processes in plants. Here the CRISPR/Cas9 gene editing mutants were obtained to detect the function of OsbHLH044. The loss-of-function of OsbHLH044 mutants showed numerous altered plant phenotypes. Notably, the osbhlh044 mutants resulted in prominently reduced morphological and physiological parameters under salt stress. Lower antioxidant activities and higher lipid peroxidation and hydrogen peroxide (H2O2) accumulation in the osbhlh044 mutants caused salinity sensitivity due to elevated reactive oxygen species (ROS). Under salt stress, both shoots and roots of the osbhlh044 mutants acquired higher Na+. Moreover, the expression of ion homeostasis-related genes (OsHKTs, OsHAK, OsSOSs, and OsNHX) and ABA-responsive gene (OsLEA3) was significantly altered in the osbhlh044 mutants after salt stress. The expression levels of genes coding for starch (OsAGPL1, OsSSIIa, OsWx, and OsFLO2) and seed storage proteins (GluA1 and Globulin 1) were significantly decreased, indicating that they synthesize less store starch and proteins, resulting in grain chalkiness in the osbhlh044 mutants. Yeast one Hybrid (Y1H) showed that OsbHLH044 could activate salt- (OsHKT1;3, OsHAK7, OsSOS1, OsSOS2, OsNHX2, and OsLEA3 but not OsHKT2;1), and starch-related genes (OsSSIIa, OsWx, and OsFLO2) by binding to the G-boxes of their promoters. Therefore, the OsbHLH044 gene editing mutants revealed multiple functions, specifically a positive regulator of salt stress and grain quality, which might bring new insights into the breeding of rice varieties.

Loss of Function of the RRMF Domain in OsROS1a Causes Sterility in Rice (Oryza sativa L.).

For crop seed production, the development of anthers and male fertility are the main agronomic traits and key biological processes for flowering plants. Active DNA demethylation regulates many plant developmental processes and is ensured by 5-meC DNA glycosylase enzymes. To find out the role of OsROS1a, OsROS1a gene editing mutants were generated using the CRISPR/Cas9 system. The osros1a mutants had shrink spikelets, smaller anthers and pollen grains, and were not stained by iodine staining showing a significant reduction in total soluble sugar and starch contents as compared to wildtype (WT), which caused complete male sterility. Similarly, the expression of genes involved in pollen and anther development was decreased in osros1a mutants as compared to WT. Furthermore, bisulfite sequencing showed that the CG and CHG methylation of the OsPKS2 gene promoter was significantly increased in the osros1a mutant, which caused a reduced expression of OsPKS2 in osros1a mutants. DNA methylation of the TDR gene promoter was similar between WT and osros1a mutants, indicating that the DNA methylation effect by OsROS1a was gene specific. The expression of OsROS1a in the mutants was not changed, but it produced a frame-shift mutation to truncate the Pem-CXXC and RRMF domains. Combined with previous studies, our findings suggested that the RRMF domain in OsROS1a is the functional domain and loss of RRMF for OsROS1a causes sterility in rice.

A new demethylase gene, OsDML4, is involved in high temperature-increased grain chalkiness in rice.

High temperature (HT) can affect the accumulation of seed storage materials and cause adverse effects on the yield and quality of rice. DNA methylation plays an important role in plant growth and development. Here, we identified a new demethylase gene OsDML4 and discovered its function in cytosine demethylation to affect endosperm formation. Loss of function of OsDML4 induced chalky endosperm only under HT and dramatically reduced the transcription and accumulation of glutelins and 16 kDa prolamin. The expression of two transcription factor genes RISBZ1 and RPBF was significantly decreased in the osdml4 mutants, which caused adverse effects on the formation of protein bodies (PBs) with greatly decreased PB-II number, and incomplete and abnormally shaped PB-IIs. Whole-genome bisulfite sequencing analysis of seeds at 15 d after pollination revealed much higher global methylation levels of CG, CHG, and CHH contexts in the osdml4 mutants compared with the wild type. Moreover, the RISBZ1 promoter was hypermethylated but the RPBF promoter was almost unchanged under HT. No significant difference was detected between the wild type and osdml4 mutants under normal temperature. Our study demonstrated a novel OsDML4-mediated DNA methylation involved in the formation of chalky endosperm only under HT and provided a new perspective in regulating endosperm development and the accumulation of seed storage proteins in rice.

The Function of DNA Demethylase Gene ROS1a Null Mutant on Seed Development in Rice (Oryza Sativa) Using the CRISPR/CAS9 System.

The endosperm is the main nutrient source in cereals for humans, as it is a highly specialized storage organ for starch, lipids, and proteins, and plays an essential role in seed growth and development. Active DNA demethylation regulates plant developmental processes and is ensured by cytosine methylation (5-meC) DNA glycosylase enzymes. To find out the role of OsROS1a in seed development, the null mutant of OsROS1a was generated using the CRISPR/Cas9 system. The null mutant of OsROS1a was stable and heritable, which affects the major agronomic traits, particularly in rice seeds. The null mutant of OsROS1a showed longer and narrower grains, and seeds were deformed containing an underdeveloped and less-starch-producing endosperm with slightly irregularly shaped embryos. In contrast to the transparent grains of the wild type, the grains of the null mutant of OsROS1a were slightly opaque and rounded starch granules, with uneven shapes, sizes, and surfaces. A total of 723 differential expression genes (DEGs) were detected in the null mutant of OsROS1a by RNA-Seq, of which 290 were downregulated and 433 were upregulated. The gene ontology (GO) terms with the top 20 enrichment factors were visualized for cellular components, biological processes, and molecular functions. The key genes that are enriched for these GO terms include starch synthesis genes (OsSSIIa and OsSSIIIa) and cellulose synthesis genes (CESA2, CESA3, CESA6, and CESA8). Genes encoding polysaccharides and glutelin were found to be downregulated in the mutant endosperm. The glutelins were further verified by SDS-PAGE, suggesting that glutelin genes could be involved in the null mutant of OsROS1a seed phenotype and OsROS1a could have the key role in the regulation of glutelins. Furthermore, 378 differentially alternative splicing (AS) genes were identified in the null mutant of OsROS1a, suggesting that the OsROS1a gene has an impact on AS events. Our findings indicated that the function on rice endosperm development in the null mutant of OsROS1a could be influenced through regulating gene expression and AS, which could provide the base to properly understand the molecular mechanism related to the OsROS1a gene in the regulation of rice seed development.

CRISPR/Cas9 Mediated Knockout of the OsbHLH024 Transcription Factor Improves Salt Stress Resistance in Rice (Oryza sativa L.).

Salinity stress is one of the most prominent abiotic stresses that negatively affect crop production. Transcription factors (TFs) are involved in the absorption, transport, or compartmentation of sodium (Na+) or potassium (K+) to resist salt stress. The basic helix-loop-helix (bHLH) is a TF gene family critical for plant growth and stress responses, including salinity. Herein, we used the CRISPR/Cas9 strategy to generate the gene editing mutant to investigate the role of OsbHLH024 in rice under salt stress. The A nucleotide base deletion was identified in the osbhlh024 mutant (A91). Exposure of the A91 under salt stress resulted in a significant increase in the shoot weight, the total chlorophyll content, and the chlorophyll fluorescence. Moreover, high antioxidant activities coincided with less reactive oxygen species (ROS) and stabilized levels of MDA in the A91. This better control of oxidative stress was accompanied by fewer Na+ but more K+, and a balanced level of Ca2+, Zn2+, and Mg2+ in the shoot and root of the A91, allowing it to withstand salt stress. Furthermore, the A91 also presented a significantly up-regulated expression of the ion transporter genes (OsHKT1;3, OsHAK7, and OsSOS1) in the shoot when exposed to salt stress. These findings imply that the OsbHLH024 might play the role of a negative regulator of salt stress, which will help to understand better the molecular basis of rice production improvement under salt stress.

OsLHY is involved in regulating flowering through the Hd1- and Ehd1- mediated pathways in rice (Oryza sativa L.).

Flowering time (or heading date in crops) is a critical agronomic trait for rice reproduction and adaptation. The circadian clock is an endogenous oscillator that is involved in controlling photoperiodic flowering. The rice LATE ELONGATED HYPOCOTYL (OsLHY), the core oscillator component of circadian clock, is a homolog of the LHY/CCA1 in Arabidopsis. Here we showed that CRISPR/Cas9-engineered mutations in OsLHY caused late flowering in rice only under natural long-day (nLD) and short-day (nSD) conditions, but not artificial SD (10 h light/14 h dark) conditions. In the oslhy mutant, the diurnal expression of circadian clock-related genes was seriously affected under both LD and SD conditions. Furthermore, the expression of the flowering activators Ehd1, Hd3a and RFT1 was down-regulated and flowering repressors Hd1 and Ghd7 was up-regulated in the oslhy mutant under LD conditions. While the transcripts of flowering-related genes were not dramatically influenced under SD conditions. Dual-luciferase assays showed that OsLHY repressed the transcription of OsGI, Hd1, Ghd7, Hd3a, RFT1 and OsELF3, and activated the transcription of Ehd1. Moreover, the yeast one hybrid assay and electrophoretic mobility shift assay confirmed that OsLHY directly repressed OsGI, RFT1 and OsELF3 by binding to their promoters, which is consistent with that in Arabidopsis. These results suggested that the OsLHY can promote rice flowering mainly through regulating Hd1 and Ehd1.

Extensively Current Activity of Transposable Elements in Natural Rice Accessions Revealed by Singleton Insertions.

Active transposable elements (TEs) have drawn more attention as they continue to create new insertions and contribute to genetic diversity of the genome. However, only a few have been discovered in rice up to now, and their activities are mostly induced by artificial treatments (e.g., tissue culture, hybridization etc.) rather than under normal growth conditions. To systematically survey the current activity of TEs in natural rice accessions and identify rice accessions carrying highly active TEs, the transposon insertion polymorphisms (TIPs) profile was used to identify singleton insertions, which were unique to a single accession and represented the new insertion of TEs in the genome. As a result, 10,924 high-confidence singletons from 251 TE families were obtained, covering all investigated TE types. The number of singletons varied substantially among different superfamilies/families, perhaps reflecting distinct current activity. Particularly, eight TE families maintained potentially higher activity in 3,000 natural rice accessions. Sixty percent of rice accessions were detected to contain singletons, indicating the extensive activity of TEs in natural rice accessions. Thirty-five TE families exhibited potentially high activity in at least one rice accession, and the majority of them showed variable activity among different rice groups/subgroups. These naturally active TEs would be ideal candidates for elucidating the molecular mechanisms underlying the transposition and activation of TEs, as well as investigating the interactions between TEs and the host genome.

Analysis of the Fusarium graminearum Species Complex from Gramineous Weeds Near Wheat Fields in Jiangsu Province, China.

Several weed species are known as alternative hosts of the Fusarium graminearum species complex (FGSC), and their epidemiological significance in Fusarium head blight (FHB) has been investigated; however, scant information is available regarding FGSC occurrence in weeds near Chinese wheat fields. To evaluate the potential role of gramineous weeds surrounding wheat fields in FHB, 306 FGSC isolates were obtained from 210 gramineous weed samples in 2018 in Jiangsu Province. Among them, 289 were Fusarium asiaticum, and the remainder were F. graminearum. Trichothecene genotype and mycotoxin analyses revealed that 74.3% of the F. asiaticum isolates were the 3-acetyldeoxynivalenol (3ADON) chemotype, and the remainder were the nivalenol (NIV) chemotype. Additionally, 82.4% of F. graminearum isolates were the 15-acetyldeoxynivalenol (15ADON) chemotype, and the remainder were the NIV chemotype. FHB severity and trichothecene analysis indicated that F. asiaticum isolates with the 3ADON chemotype were more aggressive than those with the NIV chemotype in wheat. 3ADON and NIV chemotypes of F. asiaticum isolated from weeds and wheat showed no significant differences in pathogenicity in wheat. All selected F. asiaticum isolates produced perithecia, with little difference between the 3ADON and NIV chemotypes. These results highlight the epidemiology of the FGSC isolated from weeds near wheat fields, with implications for reducing FHB inoculum in China.

An efficient and practical aerobic oxidation of benzylic methylenes by recyclable N-hydroxyimide.

An efficient and practical benzylic aerobic oxidation catalyzed by cheap and simple N-hydroxyimide organocatalyst has been achieved with high yields and broad substrate scope. The organocatalyst used can be recycled and reused by simple workup and only minute amount (1 mol% in most cases) of simple iron salt is used as promoter. Phenyl substrates with mild and strong electron-withdrawing group could also be oxygenated in high yields as well as other benzylic methylenes. Influence of substituents, gram-scale application, catalysts decay and general mechanism of this methodology has also been discussed.

MiR529a controls plant height, tiller number, panicle architecture and grain size by regulating SPL target genes in rice (Oryza sativa L.).

Rice is one of the most important food crops in the world. Breeding high-yield, multi-resistant and high-quality varieties has always been the goal of rice breeding. Rice tiller, panicle architecture and grain size are the constituent factors of yield, which are regulated by both genetic and environmental factors, including miRNAs, transcription factors, and downstream target genes. Previous studies have shown that SPL (SQUAMOSA PROMOTER BINDING-LIKE) transcription factors can control rice tiller, panicle architecture and grain size, which were regulated by miR156, miR529 and miR535. In this study, we obtained miR529a target mimicry (miR529a-MIMIC) transgenic plants to investigate plant phenotypes, physiological and molecular characteristics together with miR529a overexpression (miR529a-OE) and wild type (WT) to explore the function of miR529a and its SPL target genes in rice. We found that OsSPL2, OsSPL17 and OsSPL18 at seedling stage were regulated by miR529a, but there had complicated mechanism to control plant height. OsSPL2, OsSPL16, OsSPL17 and SPL18 at tillering stage were regulated by miR529a to control plant height and tiller number. And panicle architecture and grain size were controlled by miR529a through altering the expression of all five target genes OsSPL2, OsSPL7, OsSPL14, OsSPL16, OsSPL17 and OsSPL18. Our study suggested that miR529a might control rice growth and development by regulating different SPL target genes at different stages, which could provide a new method to improve rice yield by regulating miR529a and its SPL target genes.

Natural variation and evolutionary dynamics of transposable elements in Brassica oleracea based on next-generation sequencing data.

Brassica oleracea comprises various economically important vegetables and presents extremely diverse morphological variations. They provide a rich source of nutrition for human health and have been used as a model system for studying polyploidization. Transposable elements (TEs) account for nearly 40% of the B. oleracea genome and contribute greatly to genetic diversity and genome evolution. Although the proliferation of TEs has led to a large expansion of the B. oleracea genome, little is known about the population dynamics and evolutionary activity of TEs. A comprehensive mobilome profile of 45,737 TE loci was obtained from resequencing data from 121 diverse accessions across nine B. oleracea morphotypes. Approximately 70% (32,195) of the loci showed insertion polymorphisms between or within morphotypes. In particular, up to 1221 loci were differentially fixed among morphotypes. Further analysis revealed that the distribution of the population frequency of TE loci was highly variable across different TE superfamilies and families, implying a diverse expansion history during host genome evolution. These findings provide better insight into the evolutionary dynamics and genetic diversity of B. oleracea genomes and will potentially serve as a valuable resource for molecular markers and association studies between TE-based genomic variations and morphotype-specific phenotypic differentiation.

Analysis of Fusarium graminearum Species Complex from Freshly Harvested Rice in Jiangsu Province (China).

Members of Fusarium graminearum species complex (FGSC) are the major pathogens that cause Fusarium head blight (FHB) in cereals worldwide. Symptoms of FHB on rice, including dark staining or browning of rice glumes, were recently observed in Jiangsu Province, China. To improve our understanding of the pathogens involved, 201 FGSC isolates were obtained from freshly harvested rice samples and identified by phylogenetic analyses. Among the 201 FGSC isolates, 196 were F. asiaticum and the remaining 5 were F. graminearum. Trichothecene chemotype and chemical analyses showed that 68.4% of the F. asiaticum isolates were the 3-acetyldeoxynivalenol (3ADON) chemotype and the remainder were the nivalenol (NIV) chemotype. All of the F. graminearum isolates were the 15-acetyldeoxynivalenol chemotype. Pathogenicity assays showed that both the 3ADON and NIV chemotypes of F. asiaticum could infect wheat and rice spikes. FHB severity and trichothecene toxin analysis revealed that F. asiaticum with the NIV chemotype was less aggressive than that with the 3ADON chemotype in wheat, while the NIV-producing strains were more virulent than the 3ADON-producing strains in rice. F. asiaticum isolates with different chemotypes did not show significant differences in mycelial growth, sporulation, conidial dimensions, or perithecial production. These findings would provide useful information for developing management strategies for the control of FHB in China.

Interactions between CO2-Responsive Switchable Emulsion Droplets Determined by Using Optical Tweezers.

CO2-responsive switchable emulsions have been of great interest in some industrial processes where the stability of the emulsion is only required temporarily, such as oil transport, drug delivery, and fossil fuel production. The good understanding of the stability and instability mechanism is vital to the switchable behavior between emulsification and demulsification. Herein, a novel approach was developed to determine the interactions between two switchable emulsion droplets directly by a dual-laser optical tweezers instrument. The repulsive force between a couple of tetradecane droplets occurs to increase progressively with the increasing concentration of switchable surfactant in solutions. However, the repulsive force appears to decrease progressively in turn when the switchable surfactant concentration is far higher than the critical micelle concentration (CMC). Moreover, the depletion effect starts to emerge in the higher surfactant concentration which is attributed to the switchable surfactant micelles generated in solutions. In addition, according to the measurements of interaction forces, a mechanism of the switchable behavior is well proposed, which is established by the principle of self-assembly/detachment of the switchable surfactant, resulting in the weakening and re-enhancing of the electrostatic double-layer (EDL) repulsive forces between tetradecane droplets, upon selective introduction and removal of CO2. Based on this work, a novel perspective was provided to study the switchable emulsion, which can contribute instructive messages for the understanding of stability and instability mechanisms of switchable emulsions.

In Situ Measurements of Interactions between Switchable Surface-Active Colloid Particles Using Optical Tweezers.

Switchable surface-active colloid particles are critical to the preparation of switchable Pickering emulsions, which are widely involved in multitudinous fundamental and practical fields, such as biomedical, food products, and spinning cosmetics. The stability of switchable surface-active particles relies on the full understanding of interaction forces between individual colloid particles quantitatively. In this work, a dual-laser optical tweezers instrument was applied to measure the interaction forces between silica particles coated with a common cationic surfactant (cetyltrimethylammonium bromide, CTAB) in water, and all of the measured forces can be well fitted with the theoretical model derived from the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory. It was revealed that the minimum surface distance to engender the interaction forces between silica particles was closer progressively with the increase of CTAB concentrations, suggesting that the introduction of CTAB molecules in the solution thinned the electric double layer. In addition, the minimum surface distance between surface-inactive silica particles further decreased compared to surface-active states, although the ζ-potential has returned to the initial value of bare silica in pure water when the molecular ratio of 1:1 anionic surfactant (sodium dodecyl sulfate, SDS) was added into the solution to switch the surface-active silica particles to surface-inactive states. Our results provide a considerate methodology for quantifying the interaction forces and investigating the switchable behaviors of CTAB molecules from the adsorption to desorption at the particle-water interfaces, which provide vital foresights into the stabilization mechanism of switchable surface-active colloid particles and the further development of switchable Pickering emulsions.

Identification, characterization, and functional prediction of circular RNAs in maize.

Circular RNAs (circRNAs) are a new type of intracellular regulator that have been widely identified in animals and plants by high-throughput sequencing. However, there are still few functional studies on circRNAs in plants. To better understand maize circRNAs and their potential functions, we identified 1199 circRNAs in maize from RiboMinus RNA-Seq transcriptome data, and found distinct features of splicing site selection bias, longer flanking introns, and miniature inverted-repeat transposable element (MITE) insertions in flanking introns in maize circRNAs compared to other plant circRNAs. In total, 31 and 36 orthologous circRNAs were identified in rice and maize, respectively, but the orthologous parental genes could not produce orthologous circRNAs, mostly because of long-sequence insertions/deletions at flanking introns and approximately 24.3% of them contained MITE sequences. The majority of maize circRNAs showed high diversity of expression under different treatments and/or in different genetic backgrounds, implying that circRNAs could be involved in various regulatory networks. Twenty-six ecircRNAs were predicted to contain one or more target mimics, and 229 circRNAs had high coding potential, indicating that circRNAs could perform peptide-encoding functions in plants. These results will broaden understanding of the roles of circRNAs in plants and support further functional work on maize.